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6.3: Some Details of Glycolysis - Biology

6.3: Some Details of Glycolysis - Biology


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A. Glycolysis, Stage 1

Reaction 1: In the first reaction of glycolysis, the enzyme hexokinase rapidly phosphorylates glucose entering the cell, forming glucose-6-phosphate (G-6-P). As shown below, the overall reaction is exergonic; the free energy change for the reaction is -4 Kcal per mole of G-6-P synthesized.

This is a coupled reaction, in which phosphorylation of glucose is coupled to ATP hydrolysis. The free energy of ATP hydrolysis (an energetically favorable reaction) fuels the glucose phosphorylation (an energetically unfavorable reaction). The reaction is also biologically irreversible, as shown by the single vertical arrow. Excess dietary glucose can be stored in most cells (especially liver and kidney cells) as a highly branched polymer of glucose monomers called glycogen. In green algae and plants, glucose made by photosynthesis is stored as polymers of starch. When glucose is necessary for energy, glycogen and starch hydrolysis forms glucose-1- phosphate (G-1-P) which is then converted to G-6-P.

Let’s look at the energetics (free energy flow) of the hexokinase-catalyzed reaction. This reaction can be seen as the sum of two reactions shown below.

Recall that ATP hydrolysis is an exergonic reaction, releasing ~7 Kcal/mole (rounding down!) in a closed system under standard conditions. The condensation reaction of glucose phosphorylation occurs with a DGo of +3 Kcal/mole. This is an endergonic reaction under standard conditions. Summing up the free energy changes of the two reactions, we can calculate the overall DGo of -4 Kcal/mole for the coupled reaction under standard conditions in a closed system.

The reactions above are written as if they are reversible. However, we said that the overall coupled reaction is biologically irreversible. Where’s the contradiction? To explain, we say that an enzyme-catalyzed reaction is biologically irreversible when the products have a relatively low affinity for the enzyme active site, making catalysis (acceleration) of the reverse reaction very inefficient. Enzymes catalyzing biologically irreversible reactions don’t allow going back to reactants, but they are often allosterically regulated. This is the case for hexokinase. Imagine a cell that slows its consumption of G-6-P because its energy needs are being met. What happens when G-6-P levels rise in cells? You might expect the hexokinase reaction to slow down so that the cell doesn’t unnecessarily consume a precious nutrient energy resource. The allosteric regulation of hexokinase is illustrated below.

As G-6-P concentrations rise in the cell, excess G-6-P binds to an allosteric site on hexokinase. The conformational change in the enzyme is then transferred to the active site, inhibiting the reaction.

152 Glycolysis Stage 1, Reaction 1

Reaction 2: In this slightly endergonic and reversible reaction, isomerase catalyzes the isomerization of G-6-P to fructose-6-P (F-6-P), as shown below.

Reaction 3: In this biologically irreversible reaction, 6-phosphofructokinase (6-P- fructokinase) catalyzes the phosphorylation of F-6-P to make fructose 1,6 di- phosphate (F1,6 diP). This is also a coupled reaction, in which ATP provides the second phosphate. The overall reaction is written as the sum of two reactions, as shown below.

Like the hexokinase reaction, the 6-P-fructokinase reaction is a coupled, exergonic and allosterically regulated reaction. Multiple allosteric effectors, including ATP, ADP and AMP and long-chain fatty acids regulate this enzyme.

Reactions 4 and 5: These are the last reactions of the first stage of glycolysis. In reaction 4, F1,6 diP (a 6-C sugar) is reversibly split into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P). In reaction 5 (also reversible), DHAP is converted into another G-3-P. Here are the reactions:

The net result is the formation of two molecules of G-3-P in the last reactions of Stage 1 of glycolysis. The enzymes F-diP aldolase and triose-P-isomerase both catalyze freely reversible reactions. Also, both reactions proceed with a positive free energy change and are therefore endergonic. The sum of the free energy changes for the splitting of F1,6 diP into two G-3-Ps is a whopping +7.5 Kcal per mole, a very energetically unfavorable process.

Summing up, by the end of Stage 1 of glycolysis, we have consumed two ATP molecules, and split one 6C carbohydrate into two 3-C carbohydrates. We have also seen two biologically irreversible and allosterically regulated enzymes.

153 Glycolysis Stage 1; Reactions 2-5

B. Glycolysis, Stage 2

We will follow just one of the two molecules of G-3-P generated by the end of Stage 1 of glycolysis, but remember that both are proceeding through Stage 2 of glycolysis.

Reaction 6: This is a redox reaction. G-3-P is oxidized to 1,3, diphosphoglyceric acid (1,3, diPG) and NAD+ is reduced to NADH. The reaction catalyzed by glyceraldehyde-3-phopsphate dehydrogenase is shown below.

In this freely reversible endergonic reaction, a hydrogen molecule (H2) is removed from G-3-P, leaving behind phosphoglyceric acid. This short-lived oxidation intermediate is phosphorylated to make 1,3 diphosphoglyceric acid (1,3diPG). At the same time, the hydrogen molecule is split into a hydride ion (H-) and a proton (H+). The H- ions reduce NAD+ to NADH, leaving the protons behind in solution. Remember that all of this is happening in the active site of the same enzyme!

Even though it catalyzes a reversible reaction, G-3-P dehydrogenase is allosterically regulated. However, in contrast to the regulation of hexokinase, that of G-3-P dehydrogenase is more complicated! The regulator is NAD+ and the mechanism of allosteric regulation of G-3-P dehydrogenase by NAD+ is called negative cooperativity. It turns out that the higher the [NAD+] in the cell, the lower the affinity of the enzyme for more NAD+ and the faster the reaction in the cell! The mechanism is discussed at the link below.

154 Glycolysis Stage 2; Reaction 6

Reaction 7: The reaction shown below is catalyzed by phosphoglycerate kinase. It is freely reversible and exergonic, yielding ATP and 3-phosphoglyceric acid (3PG).

Catalysis of phosphate group transfer between molecules by kinases is called substrate-level phosphorylation, often the phosphorylation of ADP to make ATP. In this coupled reaction the free energy released by hydrolyzing a phosphate from 1,3 diPG is used to make ATP. Remember that this reaction occurs twice per starting glucose. Two ATPs have been synthesized to this point in glycolysis. We call 1,3 diPG a very high-energy phosphate compound.

Reaction 8: This freely reversible endergonic reaction moves the phosphate from the number 3 carbon of 3PG to the number 2 carbon as shown below.

Mutases like phoshoglycerate mutase catalyze transfer of functional groups within a molecule.

Reaction 9: In this reaction (shown below), enolase catalyzes the conversion of 2PG to phosphoenol pyruvate (PEP).

Reaction 10: This reaction results in the formation of pyruvic acid (pyruvate), as shown below. Remember again, two pyruvates are produced per starting glucose molecule.

The enzyme pyruvate kinase couples the biologically irreversible, exergonic hydrolysis of a phosphate from PEP and transfer of the phosphate to ADP in a coupled reaction. The reaction product, PEP, is another very high-energy phosphate compound.

155 Glycolysis Stage 2; Reactions 7-10

Pyruvate kinase is allosterically regulated by ATP, citric acid, long-chain fatty acids, F1,6 diP, and one of its own substrates, PEP.

In incomplete (aerobic) glycolysis, pyruvate is oxidized in mitochondria during respiration (see the Alternate Fates of Pyruvate above). Fermentations are called complete glycolysis because pyruvate is reduced to one or another end product. Recall that muscle fatigue results when skeletal muscle uses anaerobic fermentation to get energy during vigorous exercise. When pyruvate is reduced to lactic acid (lactate), lactic acid accumulation causes muscle fatigue. The enzyme Lactate Dehydrogenase (LDH) that catalyzes this reaction is regulated, but not allosterically. Instead different muscle tissues regulate LDH by making different versions of the enzyme! Click the Link below for an explanation.

156 Fermentation: Regulation of Pyruvate Reduction is NOT Allosteric!

C. A Chemical and Energy Balance Sheet for Glycolysis

Compare the balance sheets for complete glycolysis (fermentation) to lactic acid and incomplete (aerobic) glycolysis, showing chemical products and energy transfers.

There are two reactions in Stage 2 of glycolysis that each yield a molecule of ATP. Since each of these reactions occurs twice per starting glucose molecule, Stage 2 of glycolysis produces four ATP molecules. Since Stage 1 consumed two ATPs, the net yield of chemical energy as ATP by the end of glycolysis is two ATPs, whether complete to lactate or incomplete to pyruvate! Because they can’t make use of oxygen, anaerobes have to settle for the paltry 15 Kcal worth of ATP that they get from a fermentation. Since there are 687 Kcal potentially available from the complete combustion of a mole of glucose, there is a lot more free energy left to be captured during the rest of respiration.

157 Balance Sheet of Glycolysis

Remember also that the only redox reaction in aerobic glycolysis is in Stage 2. This is the oxidation of G-3-P, a 3C glycolytic intermediate. Now check out the redox reaction a fermentation pathway. Since pyruvate, also a 3C intermediate, was reduced, there has been no net oxidation of glucose (i.e., glycolytic intermediates) in complete glycolysis.

By this time, you will have realized that glycolysis is a net energetically favorable (downhill, spontaneous) reaction pathway in a closed system, with an overall negative ΔGo. Glycolysis is also normally spontaneous in most of our cells, driven by a constant need for energy to do cellular work. Thus the actual free energy of glycolysis, or ΔG’, is also negative. In fact, glycolysis in actively respiring cells proceeds with release of more free energy than it would in a closed system. In other words, the ΔG’ for glycolysis in active cells is more negative than the ΔGo of glycolysis!

Now, for a moment, let’s look at gluconeogenesis, the Atkins Diet and some not-so- normal circumstances when glycolysis essentially goes in reverse, at least in a few cell types. Under these conditions, glycolysis is energetically unfavorable, and those reverse reactions are the ones proceeding with a negative ΔG’!


Insulin: Actions and Regulation | Endocrinology

In this article we will discuss about the action and regulation of insulin.

Actions of Insulin (Figs 6.43, 6.44):

1. On carbohydrate metabolism

Action on Carbohydrate Metabolism:

i. It is a hypoglycemic agent.

ii. Decreases the blood glucose level.

iii. The normal fasting blood glucose level is in the range of 60-90 mg%.

The actions of insulin on carbohydrate metabolism are:

a. Increasing the peripheral utilization of glucose:

In most of the tissues of the body, for transfer of glucose from ECF to ICF and glycolysis to be brought about inside the cell, insulin is essential. Some of the tissues which do not require insulin for peripheral utilization of glucose are whole of brain except satiety center, RBCs, renal tubules and mucosa of gastrointestinal tract.

During the movement of glucose from ECF to ICF, potassium will also be transferred from ECF to ICF. Because of this, plasma potassium level falls. It is for this reason, when insulin is administered in large doses as done in the treatment of diabetic ketoacidotic coma, along with insulin, potassium should be administered to prevent hypokalemia and its deleterious effects.

Insulin can be also administered, if one has to treat cases of severe hyperkalemia but since insulin is a very powerful hypoglycemic agent, in the treatment of hyperkalemia, along with insulin glucose also must be given to prevent patient developing hypoglycemia and its consequences.

b. Utilization of glucose to supply energy spares the proteins from getting catabolized. This is known as protein sparing effect.

c. It also increases the glucose uptake by the liver and enhances the conversion of glucose to glycogen. This is brought about by enhancing the activity of glycogen synthase. This glycogen gets stored in liver.

d. It also decreases glycogenolysis in liver and muscle tissue. So the breakdown of glycogen to glucose will be less.

e. It decreases gluconeogenesis that is formation of glucose from non-carbohydrate sources, like amino acids and fatty acids.

On Protein Metabolism:

It facilitates the transfer of amino acids from ECF to ICF. The amino acids that have entered the ICF will be utilized for protein synthesis. At the same time, it will also decrease the breakdown of proteins. Incorporation of amino acids into proteins leads to retention of nitrogen in the body and brings about positive nitrogen balance.

The proteins synthesized will be used for growth of tissues and organs. This facilitates the growth, repairing of wounds, adequate resistance against infections (because of immunoglobulins) and gain of weight. In case, the child suffers from juvenile diabetes, growth of the child decreases because of loss of aforesaid actions of insulin on protein metabolism.

On Fat Metabolism:

It is a lipogenetic agent. It acts on the adipose tissue and increases the activity of lipoprotein lipase and decreases the hormone sensitive lipase activity. This leads to increased lipogenesis and decreased lipolysis. The fatty acids are transferred from ECF to ICF in adipose tissue. These fatty acids are converted to neutral fats and triglycerides and stored in adipose tissue.

Deposition of fats will increase the weight of the person. Absence of insulin brings about increase in the free fatty acid levels in circulation. When glucose cannot be utilized to supply energy, the fatty acids are metabolized to supply energy. This brings about increased formation of ketone bodies. Such situation is called as ketoacidosis.

Since insulin is involved in transfer of potassium from ECF to ICF, it decreases plasma potassium level.

Regulation of Secretion of Insulin:

1. One of the important factors which regulate insulin secretion is plasma glucose level (Fig. 6.45). More is the plasma glucose level more will be the amount of insulin secreted. The amount of insulin secreted for the same amount of glucose, depends whether glucose is administered orally or intravenously.

Administration of glucose through oral route brings about enhanced insulin secretion than intravenous administration. This is because of the influence of some of the GI tract hormones and vagus nerve influence on the beta cells in islets.

2. Some of the other factors which enhance insulin secretion are amino acids, keto acids, exercise, GI tract hormones, ACh, etc. (Table 6.11).

3. Some of the factors which decrease insulin secretion are somatostatin, potassium depletion, alpha adrenergic stimulation, etc.

i. Administration of toxic substances, like alloxan, streptozotocin, destroys the beta cells of islets of Langerhans and thus lead to insulin lack.

ii. Lack of insulin results in increased blood glucose (hyperglycemia) level.

iii. When blood glucose level exceeds 180 mg% (renal threshold), it leads to glycosuria. That is glucose appears in urine. Hence the condition is known as diabetes mellitus.

iv. When glucose is excreted, since glucose is an osmotically active substance, it drags water also with it. This will give rise to polyuria.

v. In the hypothalamus, there are two centers namely hunger center which is located in the lateral hypothalamic nucleus and ventromedial nucleus which acts as satiety center. Normally, the hunger center activity is under constant inhibitory influence from the satiety center.

The utilization of glucose by satiety center is dependent on insulin. When insulin is absent, the activity of satiety center is depressed. Because of this, the inhibition on the hunger center by satiety center is decreased (disinhibition). Now, the hunger center activity becomes unopposed and gives rise to increased hunger and hence polyphagia.

Pathophysiology of Diabetes Mellitus:

Diabetes mellitus can be of two types namely type I or insulin-dependent diabetes mellitus (IDDM) and type II or non-insulin-dependent diabetes mellitus (NIDDM).

ii. Severe or absolute insulin deficiency.

iii. Less genetic predisposition.

v. More prone to ketoacidosis.

ii. Relative insulin deficiency.

iii. Reduced sensitivity of tissue to insulin.

iv. Genetic predisposition is more.

vi. Non-ketotic hyperosmolar coma.

Whatever may be type of diabetes, there will be fault in glucose metabolism.

Decreased peripheral utilization and increased hepatic glycogenolysis brings about an increase of blood glucose level:

i. Increased blood glucose (above renal threshold of 180 mg%) level leads to glycosuria.

ii. Glycosuria leads to polyuria (increased urine excretion) since glucose is an osmotically active substance. This type of polyuria differs from polyuria of diabetes insipidus because in diabetes insipidus, the diuresis is termed as water diuresis and the specific gravity of urine will be low.

iii. Increased loss of water along with urine leads to dehydration and resulting in stimulation of thirst center and hence there will be polydypsia (increased drinking).

iv. Unopposed activity of hunger center results in polyphagia (increased eating).

a. Polyuria, polydypsia, polyphagia

b. Weightloss inspite of polyphagia

c. 3 polys are very characteristic features of DM.

v. Since the blood glucose level is more and body protein content is less, the person is more susceptible to infection and poor wound healing.

vi. Increased protein catabolism leads to weight loss and poor growth. The amino acids which are the end products of protein catabolism will be used for gluconeogenesis.

vii. In the absence of glucose getting metabolized to supply energy, fats catabolism increases and this gives rise to lipolysis. The beta oxidation of fatty acids brings about increased formation of ketone bodies and lead to ketoacidosis. Ketone bodies can be excreted along with expired air and hence the breath of these patients will have characteristic apple odor.

The cardinal symptoms of diabetes mellitus are shown in Fig. 6.46.

Tests for Diabetes Mellitus:

1. Test for glucose in urine.

2. Test the fasting blood glucose level.

3. Perform oral glucose tolerance test (GTT).

Coma is one of the serious problems of glucose metabolism. The person may get into coma both because of hyperglycemia or hypoglycemia. One of the examples of hypoglycemic coma is improper management of diabetic patients especially when the antidiabetic drug is taken but diet is compromised or drug taken may be more than the required dose. Among the two types of coma, hypoglycemic coma is more dangerous.

The signs and symptoms of hypoglycemic coma are:

1. Dizziness and nervousness due to increased autonomic discharge.

5. Ataxia that is incoordination of movements.

7. Nervousness and apprehension.

How to differentiate whether coma is because of hypo- or hyperglycemia?

When comatose patient is brought into the emergency room, time should not be wasted. Treatment to the patient has to be started as fast as possible. One of the first and foremost things to be done is, when it is not certain that coma is due to hypo- or hyperglycemia, infuse glucose intravenously to the patient.

If coma is because of hypoglycemia, the patient recovers without any further procedure. If coma is due to hyper­glycemia, the patient will not recover and infusion of glucose would not harm the patient anymore. Now the treatment has to be initiated to manage the hyperglycemic coma.

The differences between hyperglycemic and hypoglycemic coma has been detailed in Table 6.12.

Some of the conditions in which glycosuria occurs are:

1. Diabetes mellitus due to problem in glucose metabolism.

2. Renal glycosuria due to problem in function of renal tubules.

3. Alimentary glycosuria occurs in hyperthyroidism. In hyperthyroidism, the rate of absorption of glucose from GI tract is enhanced and this may lead to glycosuria.

4. Picquare glycosuria in which constant stimulation of brainstem area leads to glycosuria.


Vhl deletion in osteoblasts boosts cellular glycolysis and improves global glucose metabolism

1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

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1 Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), KU Leuven, Leuven, Belgium.

2 Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium.

3 Department of Nuclear Medicine, Hospices Civils de Lyon, Lyon, France.

4 INSERM UMR1033 — LYOS, Université de Lyon, Lyon, France.

5 Department of Rheumatology, Hospices Civils de Lyon, Lyon, France.

6 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

7 Baltimore Veterans Administration Medical Center, Baltimore, Maryland, USA.

Address correspondence to: Christa Maes, Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, Gasthuisberg O&N 1, Herestraat 49, Box 813, B-3000 Leuven, Belgium. Phone: 32.16.37.26.56 Email: [email protected]

Published February 12, 2018 - More info

The skeleton has emerged as an important regulator of systemic glucose homeostasis, with osteocalcin and insulin representing prime mediators of the interplay between bone and energy metabolism. However, genetic evidence indicates that osteoblasts can influence global energy metabolism through additional, as yet unknown, mechanisms. Here, we report that constitutive or postnatally induced deletion of the hypoxia signaling pathway component von Hippel–Lindau (VHL) in skeletal osteolineage cells of mice led to high bone mass as well as hypoglycemia and increased glucose tolerance, not accounted for by osteocalcin or insulin. In vitro and in vivo data indicated that Vhl-deficient osteoblasts displayed massively increased glucose uptake and glycolysis associated with upregulated HIF-target gene expression, resembling the Warburg effect that typifies cancer cells. Overall, the glucose consumption by the skeleton was increased in the mutant mice, as revealed by 18 F-FDG radioactive tracer experiments. Moreover, the glycemia levels correlated inversely with the level of skeletal glucose uptake, and pharmacological treatment with the glycolysis inhibitor dichloroacetate (DCA), which restored glucose metabolism in Vhl-deficient osteogenic cells in vitro, prevented the development of the systemic metabolic phenotype in the mutant mice. Altogether, these findings reveal a novel link between cellular glucose metabolism in osteoblasts and whole-body glucose homeostasis, controlled by local hypoxia signaling in the skeleton.

The skeleton serves to provide structural support to the body, allow movement, protect internal organs, regulate calcium homeostasis, and provide niches for hematopoiesis. Additionally, over the past decade the skeleton has emerged as an important endocrine organ, with osteoblast-derived osteocalcin representing a major player in the regulation of glucose and insulin homeostasis ( 1 , 2 ). Osteocalcin, in its undercarboxylated form, is released as a hormone into the circulation and promotes pancreatic β cell proliferation, insulin production, and peripheral insulin sensitivity ( 3 ). Osteoblasts in turn respond to insulin signaling by increasing bone remodeling and the production and bioavailability of undercarboxylated osteocalcin ( 4 , 5 ).

Recent reports also implicated osteocalcin-independent mechanisms in the bone-metabolism interplay. For example, genetically induced ablation of osteoblasts or conditional inactivation of glycogen synthase kinase–3β or β-catenin in osteoblasts led to systemic metabolic alterations, which could not be fully rescued or explained by congruent changes in serum osteocalcin ( 6 – 8 ). These studies indicated that osteoblasts can additionally influence global glucose homeostasis and energy metabolism through mechanisms that are as yet unknown and that may, or may not, be endocrine in nature.

Hypoxia-driven pathways play major roles in pathological conditions such as cancer and metastasis, but are also vital in normal development and tissue homeostasis ( 9 ). Particularly in the bone microenvironment, which is physiologically hypoxic, mechanisms regulating cellular adaptation to oxygen-poor conditions are crucial ( 10 ). The main orchestrator of the responses to hypoxia is HIF, a heterodimer transcription factor comprising a constitutive β subunit (HIF-β) and an oxygen-regulated α subunit (HIF-1α or HIF-2α). In oxygen-rich conditions, HIF prolyl hydroxylases (PHDs) hydroxylate specific residues in the HIF-α protein, rendering it a substrate for the E3 ubiquitin ligase von Hippel–Lindau (VHL) and a target for proteasomal degradation. When oxygen levels drop below a critical level, HIF-α is not hydroxylated and degraded, but instead accumulates in the cell, liaises with HIF-β, and induces a hypoxia-triggered transcriptional program.

The key HIF target genes help cells face the challenges of low tissue oxygen, with 2 major mechanisms standing out. First, HIF-regulated genes such as VEGF and erythropoietin (EPO) increase the tissue oxygen supply by inducing angiogenesis and erythropoiesis ( 9 ). Genetic studies in mice documented that also in osteoblast lineage cells, HIF directly regulates VEGF expression, thereby inducing blood vessel growth and bone formation and rendering the hypoxia signaling pathway the first recognized coupler of angiogenesis and osteogenesis ( 10 , 11 ). The HIF pathway has also been shown to directly regulate EPO expression in osteoblasts, modulating erythropoiesis in the local hematopoietic BM environment ( 12 ).

Second, hypoxia-regulated transcription activates genes and pathways that reduce oxygen consumption and the cellular dependence on oxygen, including by mediating a bioenergetic switch from oxidative phosphorylation to glycolysis as major route of ATP production ( 9 ). This shift involves direct HIF-mediated upregulation of glycolytic enzymes such as pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA), promoting the conversion of pyruvate into lactate, and of glucose transporters (GLUTs, particularly GLUT1) to enhance glucose uptake, thereby compensating for the energy inefficiency of glycolysis. A similar metabolic reprogramming is also prominent in cancer cells, which commonly metabolize glucose by glycolysis even in the presence of oxygen, a phenomenon known as the Warburg effect or aerobic glycolysis ( 13 , 14 ). Correspondingly, HIF-induced regulation of cellular glucose metabolism is being intensely investigated, with promising therapeutic potential in cancer and metastasis ( 15 ).

At present, relatively little is known about the metabolic pathways and substrates preferentially used by osteoblast lineage cells. Moreover, these may change throughout their lifespan and differentiation progress, and are likely context dependent ( 16 , 17 ). Recent in vivo work using genetically modified mouse models has been actively investigating this topic, and revealed a prominent role for glucose metabolism during osteoblast differentiation ( 18 , 19 ). Moreover, aerobic glycolysis has been implicated as an important driver of bone formation and shown to be regulated by osteoanabolic pathways involving Wnt-LRP5 signaling, parathyroid hormone, and HIF ( 20 – 23 ).

In this study, we show that the hypoxia signaling pathway directs cellular metabolism in osteoblast lineage cells, with HIF-target gene expression potently stimulating glycolysis and glucose consumption. Intriguingly, our findings further indicate that the repercussions of this metabolic regulation in osteoblasts extend beyond the bone environment, to the control of whole-body energy metabolism. Specifically, we found that deletion of Vhl in osteoprogenitors, a model of persistent HIF activation, promoted glycolysis and glucose utilization by osteolineage cells. This led to an overall increase in glucose uptake from the circulation by the skeleton, which in turn correlated with reduced blood glucose levels in the mutant mice. The hypoglycemic phenotype was associated with an increased glucose tolerance that could not be explained through the known endocrine actions of osteocalcin and insulin, but which could be pharmacologically rescued by administration of a glycolysis inhibitor. This suggests that increased glucose usage by osteolineage cells can, possibly directly, stimulate systemic glucose clearance and improve glucose tolerance, and even lead to a sustained decrease in the blood glucose levels and to decreased peripheral fat accumulation. Strikingly, a parallel was seen with cancer patients carrying glucose-avid bone metastases, who also showed an inverse correlation between the local glucose uptake in the tumor lesions and global blood glucose levels, further underscoring the power of the Warburg effect.

Altogether, our findings suggest that hypoxia signaling–induced excessive glycolysis in osteolineage cells can lower systemic glucose levels by increasing glucose utilization by the skeleton. This simple yet unexpected concept brings a potential new angle to the sophisticated integration of the skeleton in global nutrient homeostasis, and may have broad clinical impact with regard to bone and metabolic disorders, as well as in cancer pathology and therapy.

Vhl ablation in osteoprogenitors causes increased bone density and cortical porosity, along with hypervascularization and alterations in the BM environment. To investigate the impact of HIF signaling in osteoprogenitors and the osteoblast lineage cells derived thereof, Osx(SP7)-Cre:GFP mice ( 24 ) were crossed with Vhl floxed mice ( 25 ). Osx-GFP:Cre TG/+ Vhl +/+ mice were used as the control group. Genetic targeting of Vhl precludes the oxygen-dependent inactivation of HIF, thus representing a model of constitutive HIF activity and hypoxia signaling pathway responses, as documented previously ( 11 , 12 , 26 ).

Vhl conditional knockout (cKO) mice displayed reduced skeletal growth (Figure 1A and Supplemental Figure 1A supplemental material available online with this article https://doi.org/10.1172/JCI97794DS1) and a marked high bone mass phenotype at postnatal stages, characterized by a progressive accumulation of trabecular bone even at advanced age, as shown by micro-CT of the long bones (Figure 1, B–D). Excessive trabeculae extended far into the diaphyseal bone shaft, associated with thinner, highly porous, trabecularized cortical bone (Figure 1, B–F). High bone volume was also evident in the vertebrae (Figure 1G). Overall, bones in the mutant mice were heavier relative to BW than in controls (Supplemental Figure 1B). In line with previous reports on comparable mouse models ( 11 , 12 ), Vhl cKO bones showed elevated expression of the direct HIF target genes Vegf and Epo (Figure 1H), associated with skeletal hypervascularization, BM fibrosis, and splenomegaly (Figure 1I and Supplemental Figure 1C).

High bone mass and skeletal abnormalities in mice lacking Vhl in osteoblast lineage cells. (A) Tibia length at 12 weeks (n = 4–7 per genotype). (B) Representative 3D micro-CT reconstructions of tibias from 12-week-old mice. (C) Bone volume relative to tissue volume (BV/TV, in %) determined by micro-CT at the indicated ages (n = 3–5/group). (D and E) Micro-CT analysis of the trabecular (D) and cortical (E) tibia regions at 12 weeks (n = 3–5), showing BV/TV trabecular number (Tb.N), separation (Tb.Sp), and thickness (Tb.Th) and cortical thickness (Cort.Th) and porosity (Cort.Por). (F) Representative transverse micro-CT section of the tibia. (G) Vertebral BV/TV determined by micro-CT (n = 4). (H) Relative (Rel.) mRNA levels of Vegf and Epo in full bones of 12-week-old mice (n = 7). (I) Tibia histology showing H&E staining, PECAM-1 IHC for blood vessels (including magnifications), and reticulin-positive fibers (black stain and yellow arrow) indicative of BM fibrosis in Vhl cKO bones. Scale bars: 500 μm (H&E PECAM-1 left), 50 μm (PECAM-1 right reticulin). Graphs represent mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test between genotypes.

Inactivation of Vhl in osteoprogenitors leads to an expanded pool of early osteolineage cells and a low bone remodeling status in adult mice. We further documented the bone remodeling status of the mice by analyzing bone formation and osteoblast differentiation on the one hand and bone resorption by osteoclasts on the other. Static, dynamic, and cellular histomorphometry indicated that by 12 weeks of age, the high bone mass phenotype was associated with substantially decreased active bone formation and mineralization activity as determined by calcein labeling (Supplemental Figure 2 and Figure 2, A and B), along with reduced presence of fully differentiated cuboidal osteoblasts on the bone surfaces of Vhl cKO mice compared with controls (Figure 2C). In line therewith, differentiation and mineralization of Vhl-deficient osteoblasts were reduced in vitro (Supplemental Figure 3). Expansion of the pool of early osteolineage cells in vivo was further supported by the increased presence of Osx-expressing cells (Figure 2D), increased expression of the early osteogenic cell marker runt-related transcription factor 2 (Runx2), and strongly decreased expression of the late osteoblast and osteocyte markers osteocalcin (Ocn) and sclerostin (Sost) (Figure 2E) in bones of Vhl cKO mice. Overall, abundant yet quite disorganized collagen and osteoid deposition was observed in the mutant bones by sirius red and van Gieson staining (Figure 2, F and G). Impaired maturation and turnover of the bone matrix were also indicated by safranin O staining, which revealed abundant remnants of cartilage matrix within the trabecular bone structures of Vhl cKO mice, extending abnormally deep into the diaphysis (Figure 2H). While sustained cartilage remnants are generally indicative of reduced resorptive activity, we could not detect significant alterations in the number of osteoclasts (Figure 2, I and J) or in the expression levels of osteoclast-specific marker genes such as Rank and cathepsin K (CathK), in Vhl cKO bones (Figure 2K). The expression levels of Rankl and osteoprotegerin (Opg), prime regulators of osteoclastogenesis expressed by osteoblast lineage cells, were both significantly increased in Vhl cKO bones compared with controls, yet the overall Rankl/Opg ratio remained indifferent (Figure 2K). Last, the serum procollagen type 1 N-terminal propeptide (P1NP) and C-terminal telopeptide (CTX) levels, respective markers of bone formation and resorption, were not significantly altered in Vhl cKO versus control mice at 12 weeks of age however, when corrected for trabecular bone perimeter, both parameters were significantly decreased in the mutant mice (Figure 2, L and M), in line with the overall presumption of a relatively low bone turnover status in adult Vhl cKO mice.

Inactivation of Vhl in osteoprogenitors leads to an expanded pool of early osteolineage cells and a low bone turnover status in adult mice. (A) BV/TV (%) as quantified on von Kossa–stained sections (n = 8). (B) Mineral apposition rate (MAR) and bone formation rate corrected for bone surface (BFR/BS) quantified in mice injected with calcein twice with a 3-day interval (n = 3–7). (C) Numbers of mature, cuboidal osteoblasts located on the bone trabecular surface, expressed as absolute numbers (N.Ob in the region of interest, per unit of tissue area [T.Ar], left) or relative to bone perimeter (N.Ob/B.Pm, right) (n = 7–8). (D) Co-IHC for PECAM-1 and GFP (left) and number of Osx-Cre:GFP + cells (right) (n = 5). Scale bar: 50 μm. (E) mRNA levels of the osteoblastogenesis markers Runx2, Osx, dentin matrix acidic phosphoprotein 1 (Dmp1), Ocn, and Sost (n = 7–8). (F) Representative sirius red–stained sections showing collagen fibers dispersed in the inter-trabecular BM environment (arrows) in mutant mice (n = 3). Scale bars: 100 μm. (G) von Kossa/van Gieson staining (n = 4), revealing mineralized bone (black) and nonmineralized osteoid (red, arrows). Scale bars: 50 μm. (H) Safranin O staining (n = 5). Scale bars: 50 μm. (I) Tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts (red, see magnification in inset) (n = 6–8). Scale bars: 200 μm. (J) Number of TRAP + osteoclasts (N.Oc) in absolute terms per T.Ar (left) and relative to B.Pm (right) (n = 6–8). (K) mRNA expression of osteoclast-specific markers (Rank, CathK) and osteoclastogenesis regulators (RankL, Opg) (n = 7). (L) Serum CTX values, expressed in absolute terms (left, n = 13–18) or corrected for B.Pm (right, n = 5–7). (M) Serum P1NP values, in absolute terms (left, n = 11–18) and corrected for B.Pm (right, n = 3–6). Graphs represent mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test between genotypes.

Vhl cKO mice are lean, despite reduced physical activity, and display hypoglycemia and increased glucose tolerance. Surprisingly, constitutive deletion of VHL in osteoprogenitors was associated with distinct alterations in whole-body homeostatic processes. Vhl cKO mice showed markedly low BWs compared with littermate control mice from 3 weeks of age onward, and they appeared to be resistant to age-related weight gain, with the deviation in BW between the genotypes increasing throughout postnatal life (Figure 3A). The mutant mice had a lean appearance, displaying greatly reduced abdominal and subcutaneous fat (Figure 3, B and C), associated with reduced circulating levels of leptin but without changes in circulating adiponectin or in serum triglycerides (Supplemental Figure 4, A–C). Indirect calorimetry showed normal food intake, oxygen consumption, and heat production in Vhl cKO mice, in the face of a significant decrease in ambulatory activity, suggesting that the basal metabolic rate was increased in the mutant mice (Figure 3, D and E). Respiratory exchange ratio (RER) values were around 0.8 in both genotypes (Figure 3F). Serum analysis indicated consistently lower blood glucose levels in Vhl cKO mice, both in random-fed and fasted conditions, from 6 weeks of age onward (Figure 3, G and H, and Supplemental Figure 4D). The hypoglycemic phenotype was accompanied by enhanced clearance of glucose from the blood following i.p. glucose injection during a glucose tolerance test (GTT), and an overall increase in glucose tolerance at the age of 6 weeks (Supplemental Figure 4E) and 12 weeks (Figure 3I). Likely as a secondary consequence of the persistently lower glycemia, the rate-limiting gluconeogenesis enzyme phosphoenolpyruvate carboxykinase (PEPCK) was upregulated in the liver and muscle of constitutive Vhl cKO mice (Figure 3J), suggesting compensatory gluconeogenesis, and glycogen stores were virtually abolished in their livers (Figure 3K).

Osteoprogenitor-targeted Vhl cKO mice are lean, despite normal food intake and reduced physical activity, and display a hypoglycemic phenotype with increased systemic glucose tolerance. (A) BW of control and constitutive Vhl cKO mice at the indicated ages (n = 3–10/group). (B) Abdominal fat mass as absolute values, in grams (left), and relative as percentage of BW (right) (n = 10–14) in 12-week-old male mice. (C) Representative H&E-stained skin sections. Scale bar: 100 μm. Arrows point to subcutaneous fat. (D) Food intake over 2 days/nights, in absolute values (left) and corrected for the metabolic BW (BW 0.75 , BW raised to the three-quarter power, as commonly used to normalize energy metabolism data) (right) (n = 6–7). (E) Indirect calorimetry measurements of oxygen consumption, heat production, and ambulatory activity, corrected for BW 0.75 (n = 6–7). (F) RER (n = 6–7). (G) Blood glucose levels in random-fed state from P1.5 to P42 (n = 6–8/group). (H) Blood glucose levels after overnight fasting at 12 weeks (n = 7–9). (I) GTT and its quantification as AUC (n = 7–9). (J) Pepck mRNA levels in liver and muscle (n = 4). (K) PAS staining on liver, revealing glycogen content (n = 4). Scale bar: 100 μm. (L) Serum insulin levels in random-fed (left) and fasted (right) conditions (n = 4–9). (M) GSIS (n = 9). conc, concentration. (N) ITT and AUC quantification (n = 6–9). (O) HOMA-IR (n = 9). All analyses were performed on male control and constitutive Vhl cKO mice at 12 weeks of age, unless indicated otherwise. Graphs represent mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test between genotypes, unless indicated otherwise.

Unexpectedly, in light of the reduced glycemia and increased glucose tolerance, no alterations were observed in the serum insulin levels of Vhl cKO mice compared with controls in random-fed or fasted conditions (Figure 3L). Also, the insulin-producing β cell area in the pancreas (Supplemental Figure 5) and endogenous insulin secretion in response to glucose injection (glucose-stimulated insulin secretion test [GSIS]) (Figure 3M) were normal. Moreover, insulin tolerance tests (ITTs) demonstrated a reduction in insulin sensitivity in Vhl cKO mice (Figure 3N). Yet, even though Vhl cKO mice appeared to be less insulin sensitive than controls, calculation of the homeostatic model assessment of insulin resistance (HOMA-IR) did not classify them as insulin resistant (Figure 3O). Furthermore, no evidence was found for peripheral insulin resistance or altered insulin signaling in the liver or skeletal muscle of Vhl cKO mice, as indicated by gene expression analysis of insulin targets, enzymes involved in carbohydrate metabolism, and markers of muscle fiber composition (Supplemental Figures 6 and 7), and as corroborated by normal glucose uptake in these insulin target tissues (Supplemental Figure 6D and see below, Increased skeletal glucose uptake correlates with reduced glycemia levels).

Altogether, these data indicate that constitutive skeletal-targeted Vhl cKO mice have a high bone mass, improved glucose metabolism, and a lean body, all in the face of normal food intake and reduced physical activity.

Mice with constitutive or induced skeletal Vhl deletion develop high bone mass, low glycemia levels, and enhanced systemic glucose tolerance, not accounted for by insulin or osteocalcin. As described above, constitutive Vhl cKO mice were in a state of persistent low glycemia with increased glucose tolerance from juvenile ages onward, which appeared to be associated with a failure to build up energy stores such as those normally provided by peripheral fat and liver glycogen. This observed lipodystrophy/lipoatrophy consequently contributed to the complexity of interpreting the metabolic phenotype in this constitutive model. Therefore, we additionally generated a postnatally induced Vhl cKO (PN–Vhl cKO) mouse model, by preventing the recombination of the floxed Vhl gene during development and until postnatal week 3 through the administration of doxycycline, which silences the tetracycline-off system–containing (Tet-Off–containing) Osx-Cre:GFP transgene ( 24 ) (Figure 4A). In contrast to the constitutive Vhl cKO mouse, the induced mutant PN–Vhl cKO mice appeared neither smaller nor leaner than control littermates, as evidenced by their normal BW and abdominal fat accumulation (Figure 4, B and C). Circulating levels of adiponectin were also unchanged in PN–Vhl cKO mice (Figure 4D), and their livers showed no alterations in mRNA expression levels of genes involved in glycolysis, gluconeogenesis (Pepck), and glycogen metabolism (Supplemental Figure 7). Interestingly though, from the age of 12 weeks and most evidently by 24 weeks, the PN–Vhl cKO mice recapitulated all other key features of the constitutive Vhl cKO model, as they displayed a marked high bone mass (Figure 4, E–H), reduced fasted blood glucose levels (Figure 4I), and increased glucose tolerance (Figure 4J) compared with control littermates, without alterations in serum insulin levels (Figure 4K) or insulin sensitivity (Figure 4L). This phenotype was documented in both male and female groups of mice (Figure 4 and Supplemental Figure 8). As in the constitutive model, the local alterations in the bones of PN–Vhl cKO mice were associated with increased expression of Vegf and Epo, skeletal hypervascularization, and splenomegaly (Supplemental Figure 9). Indirect calorimetry showed normal oxygen consumption, heat production, and ambulatory activity, but a significant increase in RER in the PN–Vhl cKO mutants, suggesting a slightly increased glucose metabolism (Supplemental Figure 10).

PN–Vhl cKO mice recapitulate the key skeletal and systemic features of the constitutive Vhl cKO model while showing normal BW and fat mass. (A) Scheme of doxycyclin administration to silence Osx-Cre:GFP activity from conception until weaning. (B) BW of control and PN–Vhl cKO mice at the indicated ages (n = 6–9). (C) Abdominal fat weight as percentage of BW at 24 weeks (n = 6–7). (D) Serum adiponectin levels at 24 weeks (n = 7–8). (E) Relative BV/TV change over time analyzed by in vivo micro-CT. (F and G) Bone mineral density (BMD) (F) and BV/TV (%) (G) determined by ex vivo micro-CT at 24 weeks (n = 7–8). (H) Representative sirius red–stained tibia sections (scale bars: 500 μm) (n = 3). (I) Blood glucose levels at 3 weeks of age (overnight fast, n = 6), 12 weeks (overnight fast, n = 6–8), 15 weeks (3-hour fast, n = 6–8), and 24 weeks of age (overnight fast, n = 7–8). (J) GTT at 12 weeks (n = 3–7) and 24 weeks (n = 7–8), with corresponding AUC calculations. (K) Serum insulin levels in random-fed conditions at 24 weeks in females (left, n = 4–5) and males (right, n = 7). (L) ITT and corresponding AUC (n = 4–5). Graphs represent mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test between genotypes, unless indicated otherwise. All data in BJ and L were obtained in male control and induced PN–Vhl cKO mice. Corresponding data from female groups are shown in Supplemental Figure 8.

These data show that the local and systemic repercussions of constitutive Vhl deletion in osteolineage cells were recapitulated in a context of normal development and baseline physiology using a postnatal inducible genetic system. The PN–Vhl cKO mouse thereby provided an additional and simplified model to explore the role of the skeleton in the regulation of global glucose homeostasis.

Since these findings underscored a novel potential link between hypoxia pathway signaling in osteolineage cells and whole-body energy metabolism, we thoroughly checked the skeletal specificity of the genetic targeting strategy. The history of the Osx-Cre:GFP–mediated recombination was evaluated in sections of bone and soft tissues using the IRG transgenic reporter mouse, in which cells switch from red fluorescent protein to GFP expression following Cre-mediated recombination. As expected, the reporter readout in bone revealed Osx-Cre–targeted GFP + cells in the hypertrophic chondrocyte regions of the growth plate, throughout the metaphysis and in the cortical and trabecular bone areas (Supplemental Figure 11A). The GFP signal corresponded with osteolineage cells on the bone surfaces as well as some GFP + reticular cells dispersed within the BM stroma (Supplemental Figure 11A, middle) and the majority of bone-embedded osteocytes (Supplemental Figure 11A, bottom). Abundant Osx-Cre–targeted cells and progeny were observed in bones from Vhl cKO mice (Supplemental Figure 11B). When evaluating the IRG reporter readout of Osx-Cre activity in other tissues, including the spleen, liver, and intestine, we did not observe any GFP + cells (Supplemental Figure 11C). Furthermore, we quantified the mRNA levels of Vhl and a panel of highly responsive HIF target genes (phosphoglycerate kinase 1 [Pgk1], Epo, Glut1, and Vegf) in brain, liver, pancreas, and small intestine of control and constitutive Vhl cKO mice. No detectable upregulation was seen for any of these genes in the soft tissues derived from the mutant mice (Supplemental Figure 12A), whereas they all showed very strong upregulation in Vhl-deficient osteoblasts and in bones derived from Vhl cKO mice (Supplemental Figure 12, B and C). The sensitivity of this approach and significance of this finding is underscored by the fact that these whole-bone samples contain only around 2%–2.5% osteolineage cells, with the majority of the sample representing the nontargeted hematopoietic cells of the BM (data not shown). When the Osx-Cre:GFP + cells were selectively sorted from calvaria or long bones of control and Vhl cKO mice by FACS, they revealed effective inactivation of the Vhl gene. As shown in Supplemental Figure 12D, Vhl mRNA levels were reduced (by up to 80%) specifically in the Osx-Cre:GFP + cell fractions derived from mutant mice and not in the Osx-Cre:GFP – cell fractions. Altogether these data indicate that Vhl recombination was efficient in the osteolineage cells of bone, but absent or marginal in the nonskeletal tissues tested.

Given that osteoblast-derived osteocalcin is the principal known mediator of the interplay between bone and energy metabolism, we next quantified the osteocalcin protein levels in the serum of mice with constitutive or induced conditional Vhl deletion. Interestingly, circulating osteocalcin levels were strongly reduced (up to 80%) in Vhl mutant mice compared with controls (Figure 5, A and B), corresponding to the low bone remodeling status of the mice (see Figure 2). These low serum osteocalcin levels were in line with the substantial downregulation of Ocn mRNA expression in bones of Vhl cKO and PN–Vhl cKO mice (Figure 5, C and D) and with reduced Ocn mRNA expression in Vhl-deficient primary osteoblasts (Figure 5E). However, these results were unpredicted in light of the hypoglycemic phenotype, since serum osteocalcin levels generally inversely relate to plasma glucose ( 3 ).

Vhl-deficient mice develop increased glucose tolerance and low glycemia despite greatly reduced production and levels of osteocalcin in their bones and serum. (A and B) Serum osteocalcin (Ocn) levels in (A) control and constitutive Vhl cKO mice (n = 9–14) and (B) control and PN–Vhl cKO mice (n = 7–8.). (C and D) Ocn mRNA levels in bones of (C) 12-week-old control and constitutive Vhl cKO mice (n = 7) and (D) 24-week-old control and PN–Vhl cKO mice (n = 6–8). (E) Ocn mRNA levels in osteoblasts derived from Vhl-floxed mice and transduced with AdEmpty or AdCre in vitro (also see Figure 6) (n = 5). All data were obtained in male mice. Graphs represent mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test between genotypes, unless indicated otherwise.

Altogether, our data indicate that skeletal-targeted Vhl cKO mice, both the constitutive and the postnatally induced models, showed a high-bone-mass phenotype locally and low glycemia with increased glucose tolerance systemically. Intriguingly, the low glycemia and increased glucose tolerance in Vhl cKO mice could not be simply explained through increased insulin or osteocalcin signaling, indicating that an alternative mechanism must underlie the phenotype.

Excessive glucose uptake and glycolysis in Vhl-deficient osteoblasts. In search of the mechanism underlying the systemic phenotype of the Vhl cKO mice, we reverted to the primary target of our genetic strategy, the osteoblast, and used Vhl fl/fl primary osteoblasts transduced in vitro with adenoviruses expressing Cre (AdCre) or carrying a control vector (AdEmpty/AdGFP) as a model system. The AdCre-treated cells showed over 90% downregulated Vhl mRNA levels and effective HIF-1α protein stabilization (Figure 6, A and B).

Vhl-deficient osteoblasts show increased glucose uptake and glycolysis. (A) Relative mRNA expression levels of Vhl in cultured primary osteoblasts derived from Vhl fl/fl mice and transduced with adenoviruses expressing GFP (AdGFP) or Cre (AdCre) (n = 5 paired independent cell pools). (B) Western blot for HIF-1α confirming its effective stabilization in Vhl-deficient cells (AdCre) compared with control cultures (AdGFP) (n = 3). (C and D) mRNA levels of (C) Pgk1, Pdk1, hexokinase 2 (HkII), and Ldha, and (D) Glut1–4 in AdGFP- versus AdCre-transduced Vhl fl/fl osteoblasts (n = 5 cell pools). (E) Glucose (left) and lactate (right) concentration in unconditioned (black bars) and 24-hour-conditioned medium (n = 5–6). med., medium. (F) 2-NBDG uptake in primary osteoblasts (normalized for DNA content of the well, n = 3). (GI) Extracellular flux analysis of primary osteoblasts (n = 5), showing (G) ECAR, (H) basal oxygen consumption, and (I) OCR during a mitochondrial stress test including the oxygen consumption needed for ATP production and at maximum respiration, all normalized for DNA content. (J) Relative mRNA expression levels of mitochondrial biogenesis genes in control (AdGFP) versus Vhl-deficient (AdCre) cultured osteoblasts (n = 5), showing peroxisome proliferator–activated receptor γ coactivator 1α (Pgc1α), transcription factor A, mitochondrial (Tfam), transcription factor B1, mitochondrial (Tfbm1), nuclear respiratory factor 1 (Nrf1) and 2 (Nrf2). (K) ATP production, normalized for DNA (n = 4). (L) Western blot for phosphorylated and total AMPK, showing representative results of n = 3 paired independent cell pools (AdGFP, AdCre) and quantification of the signal in n = 6 pools. Graphs represent mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test between genotypes.

Consistent with the presence of consensus hypoxia-response elements (HREs, the recognition sites for HIF) in their regulatory sequences, the genes encoding the glycolysis-regulating enzymes PGK1, PDK1, and LDHA were significantly upregulated in Vhl-deficient cells (Figure 6C). AdCre cells also displayed elevated expression of several GLUTs (Figure 6D), particularly GLUT1, the dominant glucose transporter in osteoblasts ( 18 ). In line with these changes at the gene expression level, Vhl-deficient cells showed increased uptake of glucose and production of lactate, as documented by analysis of conditioned culture media (Figure 6E) and quantification of cellular uptake of 2-NBDG, a fluorescently labeled glucose analog (Figure 6F) that is not metabolized. Extracellular flux analysis confirmed that the mutant cells displayed a change in their bioenergetics, as evidenced by increased extracellular acidification rates (ECARs) (Figure 6G), reflecting the strongly increased lactic acid secretion, and decreased basal oxygen consumption rates (OCRs) (Figure 6H), indicative of reduced glucose oxidation. The response of AdCre-treated cells to mitochondrial stress test components (oligomycin, FCCP, and rotenone) indicated trends toward reduced oxygen consumption for ATP production (P = 0.065) and maximum respiration capacity (Figure 6I), yet no significant differences were seen in these circumstances. In line with these results, we documented slightly downregulated expression of mitochondrial biogenesis markers in Vhl-deficient cells (Figure 6J). Yet, despite the lowered mitochondrial respiration, the levels of ATP produced by AdCre-treated cells were normal (Figure 6K), as was the phosphorylation status of the cellular energy sensor AMPK (Figure 6L).

These data provide evidence that the uptake and energy-inefficient glycolytic breakdown of glucose is greatly increased in Vhl-deficient osteoblasts, to the extent that their reduced mitochondrial respiration is compensated for and cellular energy homeostasis is maintained.

Osteoblast lineage cells are prime glucose-consuming cells in the bone environment. The molecular alterations observed in vitro were confirmed in vivo: bones of Vhl cKO and PN–Vhl cKO mice displayed increased mRNA expression of key glycolytic enzymes, most pronouncedly Pgk1 and Pdk1, and of Glut1 (Figure 7, A and B). Immunostaining confirmed an increased presence of GLUT1 in the mutant bones of both the constitutive and the postnatally induced models, particularly in osteoblast lineage cells on and around the bone surfaces (Figure 7, C and D). To visualize the uptake of glucose in situ, we next administered 2-NBDG to control and Vhl cKO mice, and assessed the uptake and accumulation of the compound in the tibia and calvaria, harvested 5 minutes (data not shown) or 45 minutes (Figure 7E and Supplemental Figure 13, A and B) after the injection. These experiments revealed that osteoblasts lining the bone surfaces take up glucose quickly and abundantly, and represent by far the most glucose-avid cells in mouse bones, with relatively sparse uptake being detected in chondrocytes, hematopoietic cells, and osteocytes (Figure 7E, upper panels and Supplemental Figure 13A). The major glucose-consuming cells included presumed immature osteogenic cells in the primary spongiosa (close to the growth plate) and in the trabecular bone microenvironment, flattened bone lining cells, and cuboidal mature osteoblasts on the trabeculae and on the cortical bone, which were overall particularly abundant in the mutant mice (Figure 7E, bottom panels).

Osteoblast lineage cells are the prime glucose-consuming cells in the skeleton. (A) Relative mRNA levels of Pgk1, Pdk1, HkII, Ldha (left), and Glut1–4 (right) in bones of 12-week-old control and constitutive Vhl cKO mice (n = 7). (B) mRNA levels of Pgk1 and Glut1 in bones of control and PN–Vhl cKO mice at 24 weeks (n = 6–8). (C and D) GLUT-1 IHC on tibia sections of (C) Vhl cKO mice (scale bars: 200 μm [top] and 25 μm [bottom, magnified views]) and (D) PN–Vhl cKO mice (scale bars: 500 μm [top] and 100 μm [bottom, magnified views]). (E) Visualization of 2-NBDG uptake in tibias harvested 45 minutes after injection the signal is strongest in the metaphyseal (meta), trabecular (trab), and cortical (cort) bone regions (most evidently localizing in osteolineage cells on and around the bone surfaces [arrows]), and less intense in growth plate (gp) chondrocytes, BM hematopoietic cells, and bone-embedded osteocytes. Scale bars: 50 μm. Data are shown as mean ± SEM *P < 0.05, ***P < 0.001 by Student’s t test. CE show representative sections of n = 3 mice analyzed per genotype.

These data suggest that osteoblast lineage cells, in particular osteoprogenitors and osteoblasts, are responsible for a substantial part of the skeletal glucose uptake. Alterations in their glucose utilization may consequently impact notably on the skeleton’s overall glucose consumption. The increase in glycolytic pathway activation caused by Vhl deficiency in osteolineage cells in vivo may therefore affect total glucose uptake in the bones of the mutant mice.

Increased skeletal glucose uptake correlates with reduced glycemia levels. To quantify the glucose consumption by the skeleton, we next performed micro-PET scans and biodistribution assays using the radioactive tracer 18 F-fluorodeoxyglucose ( 18 F-FDG). In normal adult mice, the skeleton was found to take up a considerable portion of the injected glucose (14.7% of retrieved dose) relative to glucose storage tissues and high energy–demanding organs (e.g., liver 7.4%, brain 6.3%, heart 11.7%) (Figure 8A). Strikingly, the uptake of glucose was significantly and consistently increased in bones (except forelimbs) of Vhl cKO mice compared with control animals, as shown by tissue-specific quantification of the 18 F-FDG uptake (Figure 8B and Supplemental Table 1 showing soft tissues) and by micro-PET images analyzed over a 60-minute time frame (Figure 8, C–E). Time-activity curves of 18 F-FDG uptake revealed increased accumulation of the glucose analog in the metaphyseal regions of the tibias and femurs of Vhl cKO mice (Figure 8, C–E), corresponding to the regions of intense population by glucose-avid osteoblasts (see Figure 7, C–E). Three-compartment model kinetic analyses further substantiated the increased 18 F-FDG uptake in the mutant bones (Supplemental Figure 13C). In contrast to the skeleton, which represents the targeted organ in this Osx-Cre:GFP–driven genetic strategy, the liver showed no differences in 18 F-FDG uptake between the genotypes (Figure 8, B and F). Most interestingly, we found that the 18 F-FDG uptake values in the bones of individual mice negatively correlated with the animal’s glycemic levels (Figure 8G), whereas such correlation was not seen in the liver (Figure 8H).

Increased uptake of glucose in bones of Vhl cKO mice inversely correlates with their blood glucose levels. (A) Uptake of 18 F-FDG in organs harvested from control mice 45 minutes after i.v. injection, expressed as percentage of the total documented uptake (retrieved activity) in the respective individual animals. Mean ± SEM, n = 14. (B) Uptake of 18 F-FDG in the liver and various bones of control and Vhl cKO mice at 12 weeks, calculated as percentage of the total retrieved activity in the respective animal and expressed relative to the controls (n = 4–7). (C) Time-lapse imaging of 18 F-FDG uptake by micro-PET (n = 6–7), showing for each genotype the coronal slice selected for highest 18 F-FDG intensity at the level of the tibia (asterisk) (left image), the sagittal slice displaying the highest intensity signal in the vertebral column (arrow) (upper right), and a coronal maximum intensity projection of the hind limb (HL) (lower right). FL, forelimb. (DF) Kinetic analyses of micro-PET scans over a 60-minute time frame (n = 6–7), showing the quantified plateau activity in the distal femur and proximal tibia (D) and kinetic analyses over the full time frame in these bone regions (E) and in the liver (F). (G and H) Analysis of correlation between glycemia level and relative 18 F-FDG uptake (at 45 minutes) in bone (G) or in liver (H). Individual data points of control (black dots) and Vhl cKO (red dots) mice are shown, and P value and Pearson correlation coefficient (r) values total n = 36. Graphs represent mean ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test between genotypes, unless indicated otherwise.

These findings suggested that manifest uptake of glucose by a selected subset of cells primed toward excessive (HIF-driven) glycolysis, in this case osteoblasts, was able to affect global glucose homeostasis. We next sought to test the conceptual basis of this hypothesis in an independent model and a human setting. Increased glycolysis through activation of the Warburg effect (often even HIF-induced) is a hallmark of malignant cells moreover, the selective high uptake of glucose by tumor cells forms the basis of the diagnostic use of 18 F-FDG PET/CT scans for clinical detection of cancer and metastases ( 15 ). In questioning whether the activity of glucose-avid tumor cells may be linked to serum glycemia, we analyzed the 18 F-FDG PET/CT scans of 10 patients with lung adenocarcinomas (presented in Supplemental Table 2) displaying metastatic cancer lesions in one or several bones ( 27 ). Interestingly, the level of glucose uptake in the metastatic lesions (calculated as average maximum and mean 18 F-FDG standardized uptake values [SUVs]) inversely correlated with the blood glucose levels and glycated hemoglobin values (Hba1C, a measure of the 3-month average plasma glucose concentration) (Figure 9 and Supplemental Table 3). By contrast, no correlation was found between SUV indices and insulin, HOMA-IR, or HOMA-β (denoting β cell functioning), or between blood glucose or HbA1c and total osteocalcin or bone resorption, assessed by serum CTX (Supplemental Table 3).

Higher glucose uptake values in glucose-avid tumor metastases correlate with lower glycemia levels in a cohort of patients with lung adenocarcinoma with bone metastases. (A and B) Analysis of correlation between average maximum 18 F-FDG SUVs (SUV Max Avg) in bone metastases of individual patients with cancer, and their fasted glycemia levels (A) or glycated hemoglobin (HbA1c) levels (B). Individual patient data points and P value and Spearman correlation coefficient (r) are shown n = 10.

These data support the concept that metabolic rewiring resulting in excessive glucose utilization by a selective subset of cells can effectively impact blood glucose levels, and additionally underscore the potential broad-ranging implications and clinical significance of our findings.

Increased glycolysis in Vhl-deficient osteoblast lineage cells can affect systemic glucose homeostasis. The results described above raised the hypothesis that increased glycolysis in osteoblast lineage cells could, possibly directly, lower blood glucose levels and cause disturbances in whole-body glucose homeostasis. If this hypothesis is correct, then pharmacological restoration of the reprogrammed cellular metabolism by administration of a glycolysis inhibitor would be expected to prevent the systemic glucose sensitivity in Vhl cKO mice. To test this, we used dichloroacetate (DCA), a compound whose therapeutic benefits against cancer are being tested in clinical trials ( 15 ). DCA inhibits glycolysis by inhibiting PDK1, an enzyme that inhibits pyruvate dehydrogenase and thereby the flux of pyruvate into the mitochondria and the tricarboxylic acid cycle (Supplemental Figure 14A). HIF-1 directly and potently induces Pdk1 expression in hypoxic cancer cells and conceivably also in our Vhl-deficient osteoblasts (Figure 6C and Figure 7A), thereby promoting the conversion of pyruvate into lactate in the cytosol.

Administration of DCA to control (AdEmpty) and Vhl-deficient (AdCre) osteoblasts indeed restored the increased glycolysis (reflected in the ECARs) and the reduced oxidative respiration (as based on the OCRs) of the mutant cells to the baseline control levels (Figure 10A and Supplemental Figure 14B). Consequently, the increased glucose utilization of cells lacking Vhl was diminished by DCA in a dose-dependent manner (Supplemental Figure 14C). Next, we tested the effect of DCA in vivo (Figure 10B). Interestingly, administration of DCA to PN–Vhl cKO mice prevented the development of the deregulated whole-body glucose metabolism. Specifically, DCA treatment corrected the lower fasted blood glucose levels of PN–Vhl cKO mice (Figure 10C) as well as their increased glucose tolerance (Figure 10D), rendering DCA-treated PN–Vhl cKO mice globally indistinguishable from their age- and sex-matched control littermates (either vehicle- or DCA-treated). No alterations were observed among any of the groups in BW over time or in abdominal fat weight (data not shown). Similar data were obtained in female (Figure 10, C and D) and male (not shown) groups of mice.

Pharmacological inhibition of glycolysis by DCA specifically prevents the global metabolic phenotype of mice with skeletal Vhl deficiency. (A) ECAR in control (AdEmpty) and Vhl-deficient (AdCre) primary osteoblasts, measured over time (left) and averaged (right) in basal conditions or following administration of DCA at the indicated time (n = 7). Data represent mean ± SEM *P < 0.05 Student’s t test between pairs. (B) Protocol outlining the administration of the PDK1 inhibitor DCA from weeks 3 to 24 of postnatal life of control and PN–Vhl cKO mice, in which Vhl inactivation was postponed by suppressing Osx-Cre:GFP activity with doxycycline. (C) Fasting glucose levels (2-way ANOVA, P < 0.05 for interaction between genotype and DCA treatment and *P < 0.05 between control and PN–Vhl cKO mice in vehicle group by Bonferroni’s post test n = 5–6). (D) GTT (*P < 0.05 multiple Student’s t tests between time points) and corresponding AUC calculations (2-way ANOVA, P < 0.05 for interaction between genotype and DCA treatment and *P < 0.05 between control and PN–Vhl cKO mice in vehicle group by Bonferroni’s post test n = 5–6). (E) Tibia BV/TV determined by ex vivo micro-CT in male (n = 6–8 blue bars) and female (n = 4–5 red bars) mice. (F) Vegf and Epo mRNA levels in full bones (n = 7–9 mice/group). (G) Spleen weight as percentage of BW at 24 weeks in male (n = 6–7) and female (n = 5) mice. In EG, graphs represent mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001 2-way ANOVA with Bonferroni’s post test. Blue graphs, male data red graphs, female data.

In contrast to the rescued systemic metabolic phenotype, bone analyses by micro-CT and histology revealed that the development of the high-bone-mass phenotype in PN–Vhl cKO mice remained unaffected by DCA treatment (Figure 10E and Supplemental Figure 14D). Increased expression of the HIF-target genes Vegf and Epo in bones with conditional Vhl deficiency was also maintained upon DCA treatment (Figure 10F), as were the pronounced local alterations in the bone environment, including the hypervascularization (Supplemental Figure 14D). Likely as a consequence of the partial obliteration of the BM cavity by the excessive bone matrix, PN–Vhl cKO mice showed splenomegaly regardless of the DCA treatment, as documented in both male and female animals (Figure 10G).

These data indicate that pharmacological inhibition of glycolysis by DCA, a compound shown to correct the increased glucose utilization by Vhl-deficient osteoblasts, was able to prevent the global metabolic phenotype of the skeletal-targeted Vhl cKO mice and uncouple it from the high bone mass. Although DCA was systemically administered, these findings strongly suggest a link between the metabolic rewiring of the mutant osteoblasts toward excessive glycolysis, so-called hyper-Warburgism, and the systemic phenotype of altered glucose homeostasis and energy metabolism (Figure 11).

Schematic summary. Left: Vhl inactivation in osteoprogenitors and the osteoblast lineage cells derived from them locally leads to excessive HIF stabilization and transcriptional activity, including strong upregulation of glucose transporters (Glut1) and glycolysis-promoting enzymes (Pgk1, Pdk1). These molecular changes are associated with increased glucose uptake and glycolysis in the Vhl-deficient osteolineage cells, and increased glucose consumption by the skeleton as a whole. Right: Systemically, Vhl cKO mice showed consistently reduced blood glucose levels and an increased glucose tolerance that could not be explained through effects on insulin or osteocalcin. Link between local and systemic phenotypes: Since systemic administration of the glycolysis inhibitor DCA rescued the metabolic phenotype, it is possible that the low glycemia was a direct consequence of the increased uptake of glucose in bone, although potential contributions by unknown endocrine-acting osteokines (osteocrine signals) cannot at present be excluded. These new findings strongly suggest that local glucose utilization in the skeleton contributes to systemic glucose clearance and metabolic homeostasis, a concept that may help in the development of therapies beneficial for both bone and metabolic health.

The data presented here provide genetic and pharmacological support for the concept that the cellular metabolism of the osteoblast and the level of glucose consumption in bone might have repercussions on global glucose clearance and energy homeostasis (Figure 11). These results implicate the hypoxia signaling pathway and local (HIF-driven) glycolysis in the bone-metabolism interplay, and extend our insight into the skeletal contribution to the regulation of integrated, whole-body homeostatic balances.

Hypoglycemia and increased glucose tolerance in skeletal-targeted Vhl mutant mice. While previous studies have shown that activation of the HIF pathway in osteolineage cells is anabolic to bone, to our knowledge no study has previously reported a systemic metabolic phenotype in Osx-Cre–driven Vhl cKO mice or in a comparable mouse model. Yet intriguingly, both the constitutive and the postnatally induced skeletal-targeted Vhl cKO mice generated here displayed not only a high bone mass, but also a marked whole-body phenotype characterized by permanently low glycemia levels and increased glucose tolerance. In the constitutive model, the inactivation of Vhl early in life led to an increased metabolic rate and failure to build up energy stores, causing a lean appearance with a marked deficiency in body fat. This lipodystrophy or lipoatrophy may plausibly explain the mildly reduced sensitivity to insulin of the constitutive Vhl cKO mice alternatively, it could represent an adaptive response to the permanently low blood glucose levels, in order to prevent hypoglycemic death. Of note, the reduced BW of the constitutive Vhl cKO mice was already evident at postnatal week 3, the time at which doxycycline was only beginning to be washed out of the system in the PN–Vhl cKO model. The postponement of Vhl deletion in the Osx-expressing target cell population until 3 weeks of age successfully avoided much of the potentially confounding phenomena in key endocrine peripheral tissues that contributed to the complexity of the constitutive model (such as the fat deficiency) thus, the PN–Vhl cKO mouse recapitulated the key features of low glycemia and increased glucose tolerance in a context of normal development, early postnatal growth, and baseline physiology. We presume that, by the time of pervasive Vhl deletion in the skeleton, the PN–Vhl cKO mice were already relatively resilient to some of the systemic effects of increased glucose uptake by the mutant osteolineage cells, and that their bodies were better furnished than those of the constitutive Vhl cKO mice to respond to these changes and safeguard some of the homeostatic balances. The PN–Vhl cKO model may thereby be viewed as a model of reduced stringency on the system (hypomorph) compared with the effects of constitutive skeletal Vhl loss, which provided us with a simplified and supportive additional model for dissecting how genetic, molecular, and cellular changes in the bone environment can affect systemic glucose metabolism.

Initially, we hypothesized that osteocalcin may have been involved in integrating the skeletal mutagenesis with the global repercussions on glucose homeostasis and energy metabolism. However, the unexpectedly low production and strikingly low circulating levels of osteocalcin in the Vhl-deficiency models (that reflect impaired terminal osteoblast differentiation and low bone turnover, as discussed below) are not in correspondence with a dominant role for osteocalcin signaling in the systemic phenotype of our mice. The serum levels of osteocalcin (total and undercarboxylated forms) are generally inversely related to plasma glucose, fat mass, and the degree of insulin resistance in mice and humans ( 3 , 28 , 29 ). Osteocalcin –/– mice show an obese phenotype with hyperglycemia, hypoinsulinemia, and reduced insulin secretion and sensitivity compared with WT mice ( 3 ). The reduced insulin sensitivity of the constitutive Vhl cKO mice may thus possibly be explained through the endocrine actions of osteocalcin, but certainly not their hypoglycemic and lean phenotype. In fact, the low blood glucose levels in both our Vhl-deficient mouse models would be expected to lower insulin production and increase insulin sensitivity yet these responses were not seen, suggesting that counteracting mechanisms may have been operating to increase insulin production and reduce insulin sensitivity. Low osteocalcin would be expected to reduce insulin sensitivity ( 3 ), which could reflect a contribution of the low osteocalcin levels to the global metabolic phenotypes. With regard to the insulin levels, however, low osteocalcin cannot provide such a counteracting mechanism, as low osteocalcin is itself associated with reduced insulin production and secretion. The reasons underlying the aberrantly normal insulin levels in these hypoglycemic mice thus remain enigmatic.

Given that the alterations in global glucose homeostasis in mice with Vhl inactivation could not be simply explained through the known endocrine actions of insulin or osteocalcin, our findings support the notion that osteolineage cells can impact global energy metabolism through other as-yet-uncharacterized mechanisms, as suggested previously ( 6 – 8 ).

Cellular glucose utilization by osteoblasts as a determinant of global energy homeostasis. Intriguingly, while the low blood glucose levels in Vhl-depleted mice could not be explained by increased production of or sensitivity to insulin, nor by increased levels of osteocalcin, they instead correlated significantly with increased skeletal uptake of glucose from the circulation, as evidenced by in vivo 18 F-FDG tracer experiments. While glucose uptake was overall increased in bones of the mutant mice, 18 F-FDG levels were normal in the majority of soft tissues (such as liver, spleen, lungs, heart, stomach, and skeletal muscle, as determined in resting conditions, under general anesthesia [see Supplemental Table 1]). Strikingly, already in a basal (WT) setting, the skeleton presented as a major contributor to the body’s glucose consumption, accounting for almost 15% of the glucose uptake from the blood in anesthetized mice. This uptake is similar to or even higher than that seen in some of the established glucose-avid tissues such as heart, liver, and brain, a finding that is in line with other recent work ( 30 ). Plausible explanations are the large total contribution of the skeleton to the body and the high energetic demands associated with bone formation and maintenance. In accordance with this latter notion, we found that within bone, the most marked glucose-avid cell types were those residing on and in proximity of the trabecular and cortical bone surfaces, corresponding to osteoprogenitors and osteoblasts by location and morphology. Osteocytes appeared to contribute little to glucose consumption in murine bones.

Osteolineage cells thus appear responsible for a great deal of the skeletal glucose consumption. Early in vitro work indicated that osteogenic cells are highly glycolytic ( 31 , 32 ). Recent in vivo studies provided evidence that glucose uptake in osteoblasts stands partly under the control of insulin, is mediated via GLUT1 and GLUT4, and is crucial for osteoblast differentiation and bone formation ( 18 , 19 ). Our data now provide in vivo evidence that increased utilization of glucose by osteoblasts, such as instigated by genetic activation of HIF signaling, can put substantial pressure on global glucose homeostasis. Notably, the number of osteolineage cells in the body, taken cumulatively in the more than 200 bones constituting the skeleton, is likely considerable. In this light, it seems plausible that drastic changes in their glucose consumption could have repercussions on overall systemic glucose homeostasis. Vhl deletion greatly increased the glycolytic flux and glucose utilization by osteogenic cells in vitro and in vivo. Such a shift in the use of glucose as a source of energy from oxidative phosphorylation toward its breakdown via glycolysis, accompanied by increased glucose uptake to maintain energy demands (ATP production), resembles the Warburg effect that typifies cancer cells ( 13 – 15 ). Interestingly, the experimental anticancer drug and glycolysis inhibitor DCA corrected, at least partly, the bioenergetic switch and increased glucose consumption of Vhl-deficient osteolineage cells, and prevented the global metabolic phenotype. Thus, osteolineage cells are largely responsible for the high skeletal demand of glucose, and inducing a state of hyper-Warburgism in these cells can increase the local glycolytic flux and glucose utilization, apparently even to such an extent that it affects the overall glucose clearance from the circulation (Figure 11).

It thus seems that a continual drain of glucose toward the skeleton (as is possibly the case for other tissues) is able to cause an uncompensated imbalance in global glucose homeostasis, leading to an altered overall energy metabolism and even a failure to build up or a depletion of the body’s energy stores. The potential far-reaching therapeutic implications of this concept are reinforced by our finding that patients carrying glucose-avid bone metastatic tumor masses also showed an inverse correlation between the local glucose uptake in the cancer lesions and global glycemia levels. This observation opens an avenue of potential significance in tumor diagnosis, management, and treatment, warranting further investigation. Notable in this regard, for instance, is the rare but documented occurrence of clinically asymptomatic hypoglycemia and lactic acidosis in certain nonpancreatic malignancies, especially lymphomas, which has been attributed to an extreme manifestation of the Warburg effect ( 33 – 35 ).

Possibly, a rewired osteoblastic metabolism may be able to influence systemic glucose clearance directly, although involvement of secondary osteocrine signals cannot be excluded (Figure 11). Yet the finding that DCA could uncouple the high-bone-mass phenotype from the increased glucose tolerance in PN–Vhl cKO mice, by rescuing specifically the whole-body metabolic imbalance while having no discernible impact on the local bone manifestations, may argue against a critical role of endocrine signals emanating from the anomalous bone but in favor of a direct mechanism. Possibly in line with this hypothesis, mice overexpressing GLUT1 in osteoblasts showed systemic metabolic alterations similarly including increased glucose tolerance, although high circulating osteocalcin levels could also explain the phenotype in this model ( 18 ). This may also predict that, conversely, a significant reduction in osteoblast number or a deficiency in osteoblastic glucose uptake may be associated with reduced skeletal glucose utilization and impaired systemic glucose disposal. In this regard, it is interesting to note that mouse models of osteoblast ablation or of osteoblast-specific deletion of GLUT1 or GLUT4 were all associated with reduced glucose clearance and/or hyperglycemia, although these phenotypes were complex and often attributable at least in part to osteocalcin ( 6 , 18 , 19 ).

Combined VHL/HIF downstream effectors mediate the high-bone-mass phenotype in mice lacking Vhl in osteolineage cells. Our data indicate that skeletal-specific Vhl cKO mice have high bone mass and a disrupted BM microenvironment that is heavily vascularized with dilated blood vessels. Surprisingly, the bone formation and mineralization rate was greatly decreased at 12 weeks of age, associated with impaired terminal differentiation of osteoblasts and an expansion of the pool of relatively immature osteolineage cells depositing disorganized, woven bone matrix. No major alterations could be detected in the number of osteoclasts however, the presence of aberrant cartilage remnants within the trabecular bone strongly suggests impaired osteoclast functioning. Inefficient resorption of the matrix could be caused by osteoclastic alterations or, alternatively, by anomalous modifications of the matrix rendering it more resistant to degradation. The latter could result, for instance, from alterations in the biosynthesis of collagens in chondrocytes and osteolineage cells, as HIF signaling is known to improve the efficiency of posttranslational hydroxylation of collagens and collagen crosslinking, thereby determining the conformational stability of collagen triple helices ( 10 , 36 , 37 ). The low bone turnover state at adult ages in Vhl-deficient mice indicates that increased bone matrix deposition and mineralization must have occurred at earlier stages in life, similar to the findings made upon Ocn-Cre–driven Vhl inactivation, targeting mature osteoblasts ( 11 ). These mice showed increased bone formation at 7 days of age, followed by a decline in bone turnover associated with progressively increased bone mass ( 11 ).

Intriguingly, DCA treatment revealed that blocking glycolysis only reversed the low glycemia and increased glucose tolerance, but not the augmented bone mass of Vhl mutants. In other words, the local effect on bone mass and the systemic effects on energy metabolism could be uncoupled by DCA-mediated inhibition of glycolysis. This finding indicates that, on the one hand, the control of glucose homeostasis by osteoblast lineage cells is not a bona fide consequence of the high bone mass (as discussed above), and on the other hand, the high bone mass does not rely solely on the enhanced glycolysis. This latter aspect can be reconciled with the findings by Regan et al. ( 21 ), who found that DCA did restore the increased bone mass of mice conditionally expressing a stabilized form of HIF-1α, by the fact that Vhl deletion also stabilizes HIF-2α, as well as other less-well-characterized factors ( 26 ). The repercussions of enhanced signaling by HIF-1α versus HIF-2α were recently studied in models of Osx-Cre:GFP–driven overexpression of the respective HIFs ( 38 ), confirming and extending earlier studies indicating that the genes regulated by HIF-1α and HIF-2α in osteoblasts are overlapping but nonidentical. While HIF-1α appears to be primarily responsible for meditating the metabolic switch to glycolysis, VEGF upregulation in osteogenic cells is controlled by both HIF-1α and HIF-2α ( 10 , 21 , 38 ). Additionally, HIF-2α has been shown to be the main regulator for EPO production by osteoblasts ( 12 ) and to regulate OPG, the factor that inhibits osteoclastogenesis by counteracting RANKL ( 38 ). All of these downstream effectors of the VHL/HIF axis — whose increased expression fully correlated with the presence of the local bone phenotype in our Vhl-deficient models, including in being unaffected by DCA treatment — could have contributed to or caused the high bone mass and BM alterations. First, VEGF represents a key player in the tight coupling of angiogenesis and osteogenesis. Several studies have shown that inhibiting or increasing angiogenesis by modulation of VEGF expression decreases or stimulates bone formation, respectively ( 10 , 39 – 41 ). Overall, the bone phenotype described here in Vhl cKO mice resembles in many respects the phenotype associated with induced VEGF overexpression in the osteochondro-lineage cells of adult mouse bones, including the observed hypervascularization, BM fibrosis, and unbalanced bone formation and turnover ( 41 ). This suggests that increased VEGF expression by VHL-deficient osteoblasts, and consequent VEGF-mediated angiogenic-osteogenic coupling, constitutes a prime contributor to the local bone alterations in Vhl cKO mice. Second, besides regulating erythropoiesis, EPO has also been shown to stimulate bone formation and repair ( 42 ). Third, OPG has been recognized as a direct transcriptional target of HIF-2α, and at least part of the net bone anabolic effect of HIF-2α has been ascribed to reduced bone resorption ( 38 ).

Altogether, our data on 2 models of Vhl deletion, in light of the available knowledge and published work, suggest that the changes in systemic energy metabolism are a consequence of increased osteolineage cell glycolysis and bone glucose uptake, whereas the high bone mass is presumably due to a combination of mechanisms, including — possibly among others — increased skeletal vascularization and altered angiogenic-osteogenic coupling, excessive glycolytic pathway activation in osteolineage cells, increased activity of early osteolineage cells with unbalanced bone formation and mineralization, and reduced bone resorption. These combined effects are mediated by dysregulation of various genes downstream of both HIF-1α and HIF-2α.

In conclusion, this study reveals that cellular glucose utilization by osteoblasts may represent an important determinant of global glucose homeostasis, with the capacity to override the established endocrine mechanisms involving the osteocalcin-insulin axis. This new link between bone and systemic energy metabolism may be direct, or act via as-yet-uncharacterized osteocrine factors, and stands under the control of the hypoxia signaling pathway in osteolineage cells (Figure 11). These findings may have implications for the future use of bone anabolic therapies for osteoporosis, particularly regarding their potential interplay with metabolic homeostasis and disorders such as obesity and diabetes mellitus.

Animals. Osx-Cre:GFP mice ( 24 ) were crossed with Vhl floxed mice ( 25 ) in a mixed genetic background, as detailed in Supplemental Methods. For suppression of Vhl inactivation, doxycycline (Sigma-Aldrich) was added to the drinking water at 1 mg/ml and refreshed 3 times a week from conception until P21. Next, mice were randomly assigned to DCA (Sigma-Aldrich) or vehicle treatment groups DCA was administered from 3 until 24 weeks of age (2 mg/ml drinking water).

Micro-CT. We used the in vivo Skyscan 1076 micro-CT system (Bruker) with scanning parameters 50 kV, 100 μA, and 9-μm voxel size, and applied a 1-mm aluminum filter. Postmortem micro-CT scans of tibias and vertebrae were made using the ex vivo Skyscan 1172 device (Bruker) at 50 kV, 200 μA, 5-μm voxel size, and a 0.5-mm aluminum filter. Scans were reconstructed and analyzed using NRecon, CTAn, and CTVol software (Bruker), according to standardized protocols as detailed in Supplemental Methods.

Histology, IHC, and histomorphometry. Bones and soft tissues were processed for histology and stained as previously described ( 40 , 41 ). IHC for PECAM-1/CD31 (BD Biosciences, catalog 550274) on paraffin sections was as previously described ( 43 ). For costaining with GFP antibodies (chicken anti-GFP, 10 μg/ml, Abcam, ab13970), frozen sections were used the PECAM-1 signal was amplified using fluorescein-tyramide according to the TSA Cyanine 3 System (PerkinElmer) and GFP was detected using a secondary goat anti-chicken antibody (5 μg/ml, Abcam, ab96951). Paraffin sections were reacted for periodic acid–Schiff (PAS) or reticulin using commercial kits (Sigma-Aldrich). For GLUT-1 IHC, we used rabbit anti-mouse primary antibodies (MilliporeSigma, 07-1401) at 2.5 μg/ml and goat–anti-rabbit polyclonal secondary antibodies (Abcam, ab6720) at 5 μg/ml. Insulin IHC was performed as described in Supplemental Methods. Images were taken with an Olympus IX83 inverted microscope equipped with DP73 camera. Histomorphometry was performed as previously described ( 44 ) and as detailed in Supplemental Methods.

Cell culture studies. Primary osteoblasts were isolated from long bones of 12-week-old Vhl fl/fl mice by enzymatic digestion as outlined in Supplemental Methods, combining the yield from 3 mice to obtain 1 independent cell pool. Each cell pool was split into a pair of an experimental and a control sample, transduced respectively with adenoviruses encoding Cre (AdCre) or carrying a GFP-expressing or empty vector (AdGFP/AdEmpty Viral Vector Core, University of Iowa see Supplemental Methods). Glucose and lactate levels were measured in the conditioned medium. Glucose uptake in the cells was determined by adding 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG ThermoFisher Scientific) to the medium at 100 μM for 8 hours and measuring fluorescence at 485/540 nm. ATP production was quantified using the ATPLite kit (PerkinElmer). Extracellular flux analysis was performed on a Seahorse Bioscience XFp analyzer (Agilent), measuring OCR and ECAR during baseline conditions and after addition of mitochondrial stress test components (1 μM oligomycin, 1 μM FCCP, 0.5 μM rotenone) or DCA (50 mM MilliporeSigma). Results were normalized for DNA content of the corresponding culture well. In vitro osteogenic differentiation was performed as previously described ( 44 ).

Gene expression analysis. Western blot and real-time quantitative RT-PCR were performed as described in Supplemental Methods, using the primers and probes detailed in Supplemental Table 4. Relative mRNA levels were calculated by the ΔΔCt method, using Hprt as housekeeping gene.

Metabolic studies and bioassays. For GTT and GSIS, glucose was injected i.p. after overnight fasting, at 2 g/kg BW and 3 g/kg BW, respectively. For ITT, mice were fasted for 3 hours and injected i.p. with 0.75 U/kg BW insulin. Blood glucose was measured using a OneTouch Verio glucose monitor (LifeScan). ELISA assays were used to determine serum insulin, leptin, adiponectin (all from Crystal Chem), osteocalcin (Immunotopics), CTX, and P1NP (both from ImmunoDiagnostic Systems). Indirect calorimetry was done as described in Supplemental Methods.

Radioactive glucose tracing. 18 F-FDG biodistribution assays and micro-PET imaging were performed in the KU Leuven molecular Small Animal Imaging Centre (moSAIC). 18 F-FDG was prepared through an Ion Beam Applications synthesis module. After overnight fasting, 10- to 12-week-old mice were anesthetized with isoflurane inhalation before tail vein injection with 18 F-FDG (doses given in μCi = BW×16). Mice were sacrificed 45 minutes later, or subjected to small-animal PET imaging for 60 minutes using a lutetium oxyorthosilicate detector–based FOCUS 220 tomograph (Siemens/Concorde Microsystems). Time-activity curves were made using PMODv.3.1 software (PMOD Technologies LLC). For details, see Supplemental Methods.

Fluorescent glucose tracing experiments. Eight-week-old anesthetized mice were injected with 25 mg/kg BW of 2-NBDG via the tail vein, and sacrificed 5 or 45 minutes after injection. Tissues were fixed with 4% paraformaldehyde at 4°C for 4 hours. Calvaria were imaged whole-mount using a MZ165 stereomicroscope (Leica). Tibias were decalcified for 2 weeks in 0.5 M EDTA, embedded in freezing medium (Freeze Gel Q Path, VWR), sectioned, and analyzed and imaged using an Olympus IX83 microscope.

Human data. We investigated data from 10 patients from the noninterventional, prospective POUMOS-TEC cohort that includes patients presenting with first bone metastases from adenocarcinoma lung cancer (stage IV) ( 27 ). As detailed in Supplemental Methods, serum biochemical parameters and FDG SUV parameters for each bone metastatic lesion observed by 18 F-FDG PET/CT scans were determined. Correlations were computed and assessed using 2-tailed nonparametric tests.

Statistics. All data are presented as mean ± SEM. Comparisons between 2 groups were done by 2-tailed Student’s t tests. For multiple comparisons between groups, we performed 2-way ANOVA with Bonferroni post hoc tests (GraphPad Prism 5). Correlations were computed and assessed using Pearson correlation coefficient tests. For the human data, correlations were computed by nonparametric Spearman correlation coefficient tests. Throughout the study, P values below 0.05 were considered significant.

Study approval. The animal experiments were in accordance with the institutional authorities’ guidelines and formally approved by the Animal Ethics Committee of the KU Leuven. The POUMOS-TEC cohort was approved by the local ethics committee (CPP Sud Est IV, Lyon, France) and registered into ClinicalTrials.gov under the ID NCT02810262. Patients provided written informed consent.

ND, TLC, and C. Maes designed the study. ND performed the majority of the experiments, with specific contributions by RJT, EMM, RV, and CMT, and assistance from TB, EN, and RC. C. Mathieu, BVDS, CBC, and TLC contributed essential equipment, expertise, clinical data, and critical suggestions. ND and C. Maes analyzed data and wrote the manuscript. C. Maes supervised the study.

The authors thank R. Kroes, B. Dubois, M.C. Carlier, and L. Chambard for assistance A.M. Böhm and M. Mesnieres for help in optimization of techniques A. Van Santvoort, T. Buelens, C. Casteels, J. Wouters, B. de Laat, and K. Van Laere for sharing small-animal imaging infrastructure and help with tracer assays and P. Agostinis, A. van Vliet, and S. Van Eygen for Seahorse use. We thank A. McMahon and E. Schipani for sharing mouse lines. T.J. Martin, H. Kronenberg, and F. Luyten are acknowledged for critically reading the manuscript and providing valuable input. We also thank F. Giammarile, P. Clézardin, N. Girard, and the members of the SBE for helpful discussions. This work was supported by grants from the European Research Council (ERC Starting Grant 282131 under the European Union’s Seventh Framework Programme, FP/2007–2013), Research Foundation Flanders (FWO, G.094416N), and University of Leuven (OT/14/121) to C. Maes, and by the NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (subaward to C. Maes of R01AR049410 to TLC). CBC is supported by the Hospices Civils de Lyon (Young Investigator Grant 2011), EMM is an FWO fellow, and ND holds a doctoral fellowship of the Agency for Innovation by Science and Technology in Flanders (IWT).

Conflict of interest: The authors have declared that no conflict of interest exists.


Contents

  • About the Authors
  • Preface
  • Acknowledgments
  • Supplements
  • 1. The Dynamic Cell
    • 1.1. Evolution: At the Core of Molecular Change
    • 1.2. The Molecules of Life
    • 1.3. The Architecture of Cells
      • Cells Are Surrounded by Water-Impermeable Membranes
      • Membranes Serve Functions Other Than Segregation
      • Prokaryotes Comprise a Single Membrane-Limited Compartment
      • Eukaryotic Cells Contain Many Organelles and a Complex Cytoskeleton
      • Cellular DNA Is Packaged within Chromosomes
      • The Cell Cycle Follows a Regular Timing Mechanism
      • Mitosis Apportions the Duplicated Chromosomes Equally to Daughter Cells
      • Cell Differentiation Creates New Types of Cells
      • Cells Die by Suicide
      • Multicellularity Requires Extracellular Glues
      • Tissues Are Organized into Organs
      • Body Plan and Rudimentary Tissues Form Early in Embryonic Development
      • 2.1. Covalent Bonds
        • Each Atom Can Make a Defined Number of Covalent Bonds
        • The Making or Breaking of Covalent Bonds Involves Large Energy Changes
        • Covalent Bonds Have Characteristic Geometries
        • Electrons Are Shared Unequally in Polar Covalent Bonds
        • Asymmetric Carbon Atoms Are Present in Most Biological Molecules
        • α and β Glycosidic Bonds Link Monosaccharides
        • SUMMARY
        • The Hydrogen Bond Underlies Water’s Chemical and Biological Properties
        • Ionic Interactions Are Attractions between Oppositely Charged Ions
        • Van der Waals Interactions Are Caused by Transient Dipoles
        • Hydrophobic Bonds Cause Nonpolar Molecules to Adhere to One Another
        • Multiple Noncovalent Bonds Can Confer Binding Specificity
        • Phospholipids Are Amphipathic Molecules
        • The Phospholipid Bilayer Forms the Basic Structure of All Biomembranes
        • SUMMARY
        • Equilibrium Constants Reflect the Extent of a Chemical Reaction
        • The Concentration of Complexes Can Be Estimated from Equilibrium Constants for Binding Reactions
        • Biological Fluids Have Characteristic pH Values
        • Hydrogen Ions Are Released by Acids and Taken Up by Bases
        • The Henderson-Hasselbalch Equation Relates pH and Keq of an Acid-Base System
        • Buffers Maintain the pH of Intracellular and Extracellular Fluids
        • SUMMARY
        • Living Systems Use Various Forms of Energy, Which Are Interconvertible
        • The Change in Free Energy ΔG Determines the Direction of a Chemical Reaction
        • The ΔG of a Reaction Depends on Changes in Enthalpy (Bond Energy) and Entropy
        • Several Parameters Affect the ΔG of a Reaction
        • The ΔG°′ of a Reaction Can Be Calculated from Its Keq
        • Cells Must Expend Energy to Generate Concentration Gradients
        • Many Cellular Processes Involve Oxidation-Reduction Reactions
        • An Unfavorable Chemical Reaction Can Proceed If It Is Coupled with an Energetically Favorable Reaction
        • Hydrolysis of Phosphoanhydride Bonds in ATP Releases Substantial Free Energy
        • ATP Is Used to Fuel Many Cellular Processes
        • SUMMARY
        • Chemical Reactions Proceed through High-Energy Transition States
        • Enzymes Accelerate Biochemical Reactions by Reducing Transition-State Free Energy
        • SUMMARY
        • Key Concept
        • Key Concept
        • Key Concept
        • General References
        • 3.1. Hierarchical Structure of Proteins
          • The Amino Acids Composing Proteins Differ Only in Their Side Chains
          • Peptide Bonds Connect Amino Acids into Linear Chains
          • Four Levels of Structure Determine the Shape of Proteins
          • Graphic Representations of Proteins Highlight Different Features
          • Secondary Structures Are Crucial Elements of Protein Architecture
          • Motifs Are Regular Combinations of Secondary Structures
          • Structural and Functional Domains Are Modules of Tertiary Structure
          • Sequence Homology Suggests Functional and Evolutionary Relationships between Proteins
          • SUMMARY
          • The Information for Protein Folding Is Encoded in the Sequence
          • Folding of Proteins in Vivo Is Promoted by Chaperones
          • Chemical Modifications and Processing Alter the Biological Activity of Proteins
          • Cells Degrade Proteins via Several Pathways
          • Aberrantly Folded Proteins Are Implicated in Slowly Developing Diseases
          • SUMMARY
          • Proteins Are Designed to Bind a Wide Range of Molecules
          • Antibodies Exhibit Precise Ligand-Binding Specificity
          • Enzymes Are Highly Efficient and Specific Catalysts
          • An Enzyme’s Active Site Binds Substrates and Carries Out Catalysis
          • Kinetics of an Enzymatic Reaction Are Described by Vmax and Km
          • Many Proteins Contain Tightly Bound Prosthetic Groups
          • A Variety of Regulatory Mechanisms Control Protein Function
          • SUMMARY
          • Proteins Interact with Membranes in Different Ways
          • Hydrophobic α Helices in Transmembrane Proteins Are Embedded in the Bilayer
          • Many Integral Proteins Contain Multiple Transmembrane α Helices
          • Multiple β Strands in Porins Form Membrane-Spanning �rrels”
          • Covalently Attached Hydrocarbon Chains Anchor Some Proteins to the Membrane
          • Some Peripheral Proteins Are Soluble Enzymes That Act on Membrane Components
          • SUMMARY
          • Proteins Can Be Removed from Membranes by Detergents or High-Salt Solutions
          • Centrifugation Can Separate Particles and Molecules That Differ in Mass or Density
          • Electrophoresis Separates Molecules according to Their Charge:Mass Ratio
          • Liquid Chromatography Resolves Proteins by Mass, Charge, or Binding Affinity
          • Highly Specific Enzyme and Antibody Assays Can Detect Individual Proteins
          • Radioisotopes Are Indispensable Tools for Detecting Biological Molecules
          • Protein Primary Structure Can Be Determined by Chemical Methods and from Gene Sequences
          • Time-of-Flight Mass Spectrometry Measures the Mass of Proteins and Peptides
          • Peptides with a Defined Sequence Can Be Synthesized Chemically
          • Protein Conformation Is Determined by Sophisticated Physical Methods
          • SUMMARY
          • Key Concept
          • Key Experiment
          • Key Application
          • General References
          • Web Sites
          • Hierarchical Structure of Proteins
          • Folding, Modification, and Degradation of Proteins
          • Functional Design of Proteins
          • Purifying, Detecting, and Characterizing Proteins
          • 4.1. Structure of Nucleic Acids
            • Polymerization of Nucleotides Forms Nucleic Acids
            • Native DNA Is a Double Helix of Complementary Antiparallel Chains
            • DNA Can Undergo Reversible Strand Separation
            • Many DNA Molecules Are Circular
            • Local Unwinding of DNA Induces Supercoiling
            • RNA Molecules Exhibit Varied Conformations and Functions
            • SUMMARY
            • Both DNA and RNA Chains Are Produced by Copying of Template DNA Strands
            • Nucleic Acid Strands Grow in the 5′ → 3′ Direction
            • RNA Polymerases Can Initiate Strand Growth but DNA Polymerases Cannot
            • Replication of Duplex DNA Requires Assembly of Many Proteins at a Growing Fork
            • Organization of Genes in DNA Differs in Prokaryotes and Eukaryotes
            • Eukaryotic Primary RNA Transcripts Are Processed to Form Functional mRNAs
            • SUMMARY
            • Messenger RNA Carries Information from DNA in a Three-Letter Genetic Code
            • Experiments with Synthetic mRNAs and Trinucleotides Broke the Genetic Code
            • The Folded Structure of tRNA Promotes Its Decoding Functions
            • Nonstandard Base Pairing Often Occurs between Codons and Anticodons
            • Aminoacyl-tRNA Synthetases Activate Amino Acids by Linking Them to tRNAs
            • Each tRNA Molecule Is Recognized by a Specific Aminoacyl-tRNA Synthetase
            • Ribosomes Are Protein-Synthesizing Machines
            • SUMMARY
            • The AUG Start Codon Is Recognized by Methionyl-tRNAi Met
            • Bacterial Initiation of Protein Synthesis Begins Near a Shine-Dalgarno Sequence in mRNA
            • Eukaryotic Initiation of Protein Synthesis Occurs at the 5′ End and Internal Sites in mRNA
            • During Chain Elongation Each Incoming Aminoacyl-tRNA Moves through Three Ribosomal Sites
            • Protein Synthesis Is Terminated by Release Factors When a Stop Codon Is Reached
            • Simultaneous Translation by Multiple Ribosomes and Their Rapid Recycling Increase the Efficiency of Protein Synthesis
            • SUMMARY
            • References
            • Key Concept
            • Key Experiment
            • Key Application
            • General References
            • Nucleic Acids: Structure and General Properties
            • Nucleic Acid Synthesis
            • The Genetic Code
            • Initiation
            • Transfer RNA and Amino Acids
            • Ribosomes
            • The Steps in Protein Synthesis
            • 5.1. Microscopy and Cell Architecture
              • Light Microscopy Can Distinguish Objects Separated by 0.2 μm or More
              • Samples for Light Microscopy Usually Are Fixed, Sectioned, and Stained
              • Fluorescence Microscopy Can Localize and Quantify Specific Molecules in Cells
              • Confocal Scanning and Deconvolution Microscopy Provide Sharper Images of Three-Dimensional Objects
              • Phase-Contrast and Nomarski Interference Microscopy Visualize Unstained Living Cells
              • Transmission Electron Microscopy Has a Limit of Resolution of 0.1 nm
              • Scanning Electron Microscopy Visualizes Details on the Surfaces of Cells and Particles
              • SUMMARY
              • Flow Cytometry Separates Different Cell Types
              • Disruption of Cells Releases Their Organelles and Other Contents
              • Different Organelles Can Be Separated by Centrifugation
              • Organelle-Specific Antibodies Are Useful in Preparing Highly Purified Organelles
              • SUMMARY
              • Phospholipids Are the Main Lipid Constituents of Most Biomembranes
              • Every Cellular Membrane Forms a Closed Compartment and Has a Cytosolic and an Exoplasmic Face
              • Several Types of Evidence Point to the Universality of the Phospholipid Bilayer
              • All Integral Proteins and Glycolipids Bind Asymmetrically to the Lipid Bilayer
              • The Phospholipid Composition Differs in Two Membrane Leaflets
              • Most Lipids and Integral Proteins Are Laterally Mobile in Biomembranes
              • Fluidity of Membranes Depends on Temperature and Composition
              • Membrane Leaflets Can Be Separated and Each Face Viewed Individually
              • The Plasma Membrane Has Many Common Functions in All Cells
              • SUMMARY
              • Lysosomes Are Acidic Organelles That Contain a Battery of Degradative Enzymes
              • Plant Vacuoles Store Small Molecules and Enable the Cell to Elongate Rapidly
              • Peroxisomes Degrade Fatty Acids and Toxic Compounds
              • Mitochondria Are the Principal Sites of ATP Production in Aerobic Cells
              • Chloroplasts, the Sites of Photosynthesis, Contain Three Membrane-Limited Compartments
              • The Endoplasmic Reticulum Is a Network of Interconnected Internal Membranes
              • Golgi Vesicles Process and Sort Secretory and Membrane Proteins
              • The Double-Membraned Nucleus Contains the Nucleolus and a Fibrous Matrix
              • The Cytosol Contains Many Particles and Cytoskeletal Fibers
              • SUMMARY
              • References
              • Key Concept
              • Key Experiment
              • Key Application
              • Light Microscopy
              • Electron Microscopy
              • Cell Structure: Histology Texts and Atlases
              • Purification of Cells and Their Parts
              • Biomembranes: Structural Organization and Basic Functions
              • Organelles of the Eukaryotic Cell
              • 6.1. Growth of Microorganisms in Culture
                • Many Microorganisms Can Be Grown in Minimal Medium
                • Mutant Strains of Bacteria and Yeast Can Be Isolated by Replica Plating
                • SUMMARY
                • Rich Media Are Required for Culture of Animal Cells
                • Most Cultured Animal Cells Grow Only on Special Solid Surfaces
                • Primary Cell Cultures Are Useful, but Have a Finite Life Span
                • Transformed Cells Can Grow Indefinitely in Culture
                • Fusion of Cultured Animal Cells Can Yield Interspecific Hybrids Useful in Somatic-Cell Genetics
                • Hybrid Cells Often Are Selected on HAT Medium
                • Hybridomas Are Used to Produce Monoclonal Antibodies
                • SUMMARY
                • Viral Capsids Are Regular Arrays of One or a Few Types of Protein
                • Most Viral Host Ranges Are Narrow
                • Viruses Can Be Cloned and Counted in Plaque Assays
                • Viral Growth Cycles Are Classified as Lytic or Lysogenic
                • Four Types of Bacterial Viruses Are Widely Used in Biochemical and Genetic Research
                • Animal Viruses Are Classified by Genome Type and mRNA Synthesis Pathway
                • Viral Vectors Can Be Used to Introduce Specific Genes into Cells
                • SUMMARY
                • References
                • Key Concept
                • Key Experiment
                • Key Application
                • Growth of Microorganisms in Culture
                • Growth of Animal Cells in Culture
                • Viruses: Structure, Function, and Uses
                • 7.1. DNA Cloning with Plasmid Vectors
                  • Plasmids Are Extrachromosomal Self-Replicating DNA Molecules
                  • E. Coli Plasmids Can Be Engineered for Use as Cloning Vectors
                  • Plasmid Cloning Permits Isolation of DNA Fragments from Complex Mixtures
                  • Restriction Enzymes Cut DNA Molecules at Specific Sequences
                  • Restriction Fragments with Complementary “Sticky Ends” Are Ligated Easily
                  • Polylinkers Facilitate Insertion of Restriction Fragments into Plasmid Vectors
                  • Small DNA Molecules Can Be Chemically Synthesized
                  • SUMMARY
                  • Bacteriophage λ Can Be Modified for Use as a Cloning Vector and Assembled in Vitro
                  • Nearly Complete Genomic Libraries of Higher Organisms Can Be Prepared by λ Cloning
                  • cDNA Libraries Are Prepared from Isolated mRNAs
                  • Larger DNA Fragments Can Be Cloned in Cosmids and Other Vectors
                  • SUMMARY
                  • Libraries Can Be Screened with Membrane-Hybridization Assay
                  • Oligonucleotide Probes Are Designed Based on Partial Protein Sequences
                  • Specific Clones Can Be Identified Based on Properties of the Encoded Proteins
                  • Gel Electrophoresis Resolves DNA Fragments of Different Size
                  • Multiple Restriction Sites Can Be Mapped on a Cloned DNA Fragment
                  • Pulsed-Field Gel Electrophoresis Separates Large DNA Molecules
                  • Purified DNA Molecules Can Be Sequenced Rapidly by Two Methods
                  • SUMMARY
                  • Stored Sequences Suggest Functions of Newly Identified Genes and Proteins
                  • Comparative Analysis of Genomes Reveals Much about an Organism’s Biology
                  • Homologous Proteins Involved in Genetic Information Processing Are Widely Distributed
                  • Many Yeast Genes Function in Intracellular Protein Targeting and Secretion
                  • The C. elegans Genome Encodes Numerous Proteins Specific to Multicellular Organisms
                  • SUMMARY
                  • Southern Blotting Detects Specific DNA Fragments
                  • Northern Blotting Detects Specific RNAs
                  • Specific RNAs Can Be Quantitated and Mapped on DNA by Nuclease Protection
                  • Transcription Start Sites Can Be Mapped by S1 Protection and Primer Extension
                  • SUMMARY
                  • E. coli Expression Systems Can Produce Full-Length Proteins
                  • Eukaryotic Expression Systems Can Produce Proteins with Post-Translational Modifications
                  • Cloned cDNAs Can Be Translated in Vitro to Yield Labeled Proteins
                  • SUMMARY
                  • PCR Amplification of Mutant Alleles Permits Their Detection in Small Samples
                  • DNA Sequences Can Be Amplified for Use in Cloning and as Probes
                  • SUMMARY
                  • SUMMARY
                  • References
                  • Key Experiment
                  • Key Application
                  • Key Experiment
                  • DNA Cloning with Plasmid Vectors
                  • Constructing DNA Libraries with λ Phage and Other Cloning Vectors
                  • Identifying, Analyzing, and Sequencing Cloned DNA
                  • Bioinformatics
                  • Analyzing Specific Nucleic Acids in Complex Mixtures
                  • Producing High Levels of Proteins from Cloned cDNAs
                  • Polymerase Chain Reaction: An Alternative to Cloning
                  • DNA Microarrays: Analyzing Genome-wide Expression
                  • 8.1. Mutations: Types and Causes
                    • Mutations Are Recessive or Dominant
                    • Inheritance Patterns of Recessive and Dominant Mutations Differ
                    • Mutations Involve Large or Small DNA Alterations
                    • Mutations Occur Spontaneously and Can Be Induced
                    • Some Human Diseases Are Caused by Spontaneous Mutations
                    • SUMMARY
                    • Temperature-Sensitive Screens Can Isolate Lethal Mutations in Haploids
                    • Recessive Lethal Mutations in Diploids Can Be Screened by Use of Visible Markers
                    • Complementation Analysis Determines If Different Mutations Are in the Same Gene
                    • Metabolic and Other Pathways Can Be Genetically Dissected
                    • Suppressor Mutations Can Identify Genes Encoding Interacting Proteins
                    • SUMMARY
                    • Segregation Patterns Indicate Whether Mutations Are on the Same or Different Chromosomes
                    • Chromosomal Mapping Locates Mutations on Particular Chromosomes
                    • Recombinational Analysis Can Map Genes Relative to Each Other on a Chromosome
                    • DNA Polymorphisms Are Used to Map Human Mutations
                    • Some Chromosomal Abnormalities Can Be Mapped by Banding Analysis
                    • SUMMARY
                    • Cloned DNA Segments Near a Gene of Interest Are Identified by Various Methods
                    • Chromosome Walking Is Used to Isolate a Limited Region of Contiguous DNA
                    • Physical Maps of Entire Chromosomes Can Be Constructed by Screening YAC Clones for Sequence-Tagged Sites
                    • Physical and Genetic Maps Can Be Correlated with the Aid of Known Markers
                    • Further Analysis Is Needed to Locate a Mutation-Defined Gene in Cloned DNA
                    • Protein Structure Is Deduced from cDNA Sequence
                    • SUMMARY
                    • Specific Sites in Cloned Genes Can Be Altered in Vitro
                    • DNA Is Transferred into Eukaryotic Cells in Various Ways
                    • Normal Genes Can Be Replaced with Mutant Alleles in Yeast and Mice
                    • Foreign Genes Can Be Introduced into Plants and Animals
                    • SUMMARY
                    • References
                    • Key Concept
                    • Key Experiment
                    • Key Application
                    • Mutations: Types and Causes
                    • Isolation and Analysis of Mutants
                    • Genetic Mapping of Mutations
                    • Molecular Cloning of Genes Defined by Mutations
                    • Gene Replacement and Transgenic Animals
                    • 9.1. Molecular Definition of a Gene
                      • Bacterial Operons Produce Polycistronic mRNAs
                      • Most Eukaryotic mRNAs Are Monocistronic and Contain Introns
                      • Simple and Complex Transcription Units Are Found in Eukaryotic Genomes
                      • SUMMARY
                      • Genomes of Higher Eukaryotes Contain Much Nonfunctional DNA
                      • Cellular DNA Content Does Not Correlate with Phylogeny
                      • Protein-Coding Genes May Be Solitary or Belong to a Gene Family
                      • Tandemly Repeated Genes Encode rRNAs, tRNAs, and Histones
                      • Reassociation Experiments Reveal Three Major Fractions of Eukaryotic DNA
                      • Simple-Sequence DNAs Are Concentrated in Specific Chromosomal Locations
                      • DNA Fingerprinting Depends on Differences in Length of Simple-Sequence DNAs
                      • SUMMARY
                      • Movement of Mobile Elements Involves a DNA or RNA Intermediate
                      • Mobile Elements That Move as DNA Are Present in Prokaryotes and Eukaryotes
                      • Viral Retrotransposons Contain LTRs and Behave Like Retroviruses in the Genome
                      • Nonviral Retrotransposons Lack LTRs and Move by an Unusual Mechanism
                      • Retrotransposed Copies of Cellular RNAs Occur in Eukaryotic Chromosomes
                      • Mobile DNA Elements Probably Had a Significant Influence on Evolution
                      • SUMMARY
                      • Inversion of a Transcription-Control Region Switches Salmonella Flagellar Antigens
                      • Antibody Genes Are Assembled by Rearrangements of Germ-Line DNA
                      • Generalized DNA Amplification Produces Polytene Chromosomes
                      • SUMMARY
                      • Most Bacterial Chromosomes Are Circular with One Replication Origin
                      • Eukaryotic Nuclear DNA Associates with Histone Proteins to Form Chromatin
                      • Chromatin Exists in Extended and Condensed Forms
                      • Acetylation of Histone N-Termini Reduces Chromatin Condensation
                      • Eukaryotic Chromosomes Contain One Linear DNA Molecule
                      • SUMMARY
                      • Chromosome Number, Size, and Shape at Metaphase Are Species Specific
                      • Nonhistone Proteins Provide a Structural Scaffold for Long Chromatin Loops
                      • Chromatin Contains Small Amounts of Other Proteins in Addition to Histones and Scaffold Proteins
                      • Stained Chromosomes Have Characteristic Banding Patterns
                      • Chromosome Painting Distinguishes Each Homologous Pair by Color
                      • Heterochromatin Consists of Chromosome Regions That Do Not Uncoil
                      • Three Functional Elements Are Required for Replication and Stable Inheritance of Chromosomes
                      • Yeast Artificial Chromosomes Can Be Used to Clone Megabase DNA Fragments
                      • SUMMARY
                      • Mitochondria Contain Multiple mtDNA Molecules
                      • Genes in mtDNA Exhibit Cytoplasmic Inheritance and Encode rRNAs, tRNAs, and Some Mitochondrial Proteins
                      • The Size and Coding Capacity of mtDNA Vary Considerably in Different Organisms
                      • Products of Mitochondrial Genes Are Not Exported
                      • Mitochondrial Genetic Codes Differ from the Standard Nuclear Code
                      • Mutations in Mitochondrial DNA Cause Several Genetic Diseases in Man
                      • Chloroplasts Contain Large Circular DNAs Encoding More Than a Hundred Proteins
                      • SUMMARY
                      • References
                      • Key Experiment
                      • Key Application
                      • Key Concept
                      • Chromosomal Organization of Genes and Noncoding DNA
                      • Mobile DNA
                      • Functional Rearrangements in Chromosomal DNA
                      • Organizing Cellular DNA into Chromosomes
                      • Morphology and Functional Elements of Eukaryotic Chromosomes
                      • Organelle DNAs
                      • 10.1. Bacterial Gene Control: The Jacob-Monod Model
                        • Enzymes Encoded at the lac Operon Can Be Induced and Repressed
                        • Mutations in lacI Cause Constitutive Expression of lac Operon
                        • Isolation of Operator Constitutive and Promoter Mutants Support Jacob-Monod Model
                        • Regulation of lac Operon Depends on Cis-Acting DNA Sequences and Trans-Acting Proteins
                        • Biochemical Experiments Confirm That Induction of the lac Operon Leads to Increased Synthesis of lac mRNA
                        • SUMMARY
                        • Footprinting and Gel-Shift Assays Identify Protein-DNA Interactions
                        • The lac Control Region Contains Three Critical Cis-Acting Sites
                        • RNA Polymerase Binds to Specific Promoter Sequences to Initiate Transcription
                        • Differences in E. coli Promoter Sequences Affect Frequency of Transcription Initiation
                        • Binding of lac Repressor to the lac Operator Blocks Transcription Initiation
                        • Most Bacterial Repressors Are Dimers Containing α Helices That Insert into Adjacent Major Grooves of Operator DNA
                        • Ligand-Induced Conformational Changes Alter Affinity of Many Repressors for DNA
                        • Positive Control of the lac Operon Is Exerted by cAMP-CAP
                        • Cooperative Binding of cAMP-CAP and RNA Polymerase to lac Control Region Activates Transcription
                        • Transcription Control at All Bacterial Promoters Involves Similar but Distinct Mechanisms
                        • Transcription from Some Promoters Is Initiated by Alternative Sigma (σ) Factors
                        • Many Bacterial Responses Are Controlled by Two-Component Regulatory Systems
                        • SUMMARY
                        • Most Genes in Higher Eukaryotes Are Regulated by Controlling Their Transcription
                        • Regulatory Elements in Eukaryotic DNA Often Are Many Kilobases from Start Sites
                        • Three Eukaryotic Polymerases Catalyze Formation of Different RNAs
                        • The Largest Subunit in RNA Polymerase II Has an Essential Carboxyl-Terminal Repeat
                        • RNA Polymerase II Initiates Transcription at DNA Sequences Corresponding to the 5′ Cap of mRNAs
                        • SUMMARY
                        • TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA
                        • Promoter-Proximal Elements Help Regulate Eukaryotic Genes
                        • Transcription by RNA Polymerase II Often Is Stimulated by Distant Enhancer Sites
                        • Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements
                        • SUMMARY
                        • Biochemical and Genetic Techniques Have Been Used to Identify Transcription Factors
                        • Transcription Activators Are Modular Proteins Composed of Distinct Functional Domains
                        • DNA-Binding Domains Can Be Classified into Numerous Structural Types
                        • Heterodimeric Transcription Factors Increase Gene-Control Options
                        • Activation Domains Exhibit Considerable Structural Diversity
                        • Multiprotein Complexes Form on Enhancers
                        • Many Repressors Are the Functional Converse of Activators
                        • SUMMARY
                        • Initiation by Pol II Requires General Transcription Factors
                        • Proteins Comprising the Pol II Transcription-Initiation Complex Assemble in a Specific Order in Vitro
                        • A Pol II Holoenzyme Multiprotein Complex Functions in Vivo
                        • SUMMARY
                        • N-Termini of Histones in Chromatin Can Be Modified
                        • Formation of Heterochromatin Silences Gene Expression at Telomeres and Other Regions
                        • Repressors Can Direct Histone Deacetylation at Specific Genes
                        • Activators Can Direct Histone Acetylation at Specific Genes
                        • Chromatin-Remodeling Factors Participate in Activation at Some Promoters
                        • Activators Stimulate the Highly Cooperative Assembly of Initiation Complexes
                        • Repressors Interfere Directly with Transcription Initiation in Several Ways
                        • Regulation of Transcription-Factor Expression Contributes to Gene Control
                        • Lipid-Soluble Hormones Control the Activities of Nuclear Receptors
                        • Polypeptide Hormones Signal Phosphorylation of Some Transcription Factors
                        • SUMMARY
                        • Transcription Initiation by Pol I and Pol III Is Analogous to That by Pol II
                        • T7 and Related Bacteriophages Express Monomeric, Largely Unregulated RNA Polymerases
                        • Mitochondrial DNA Is Transcribed by RNA Polymerases with Similarities to Bacteriophage and Bacterial Enzymes
                        • Transcription of Chloroplast DNA Resembles Bacterial Transcription
                        • Transcription by Archaeans Is Closer to Eukaryotic Than to Bacterial Transcription
                        • SUMMARY
                        • References
                        • Key Concept
                        • Key Experiment
                        • Key Application
                        • Bacterial Gene Control: The Jacob-Monod Model
                        • Bacterial Transcription Initiation
                        • Eukaryotic Gene Control: Purposes and General Principles
                        • Regulatory Sequences in Eukaryotic Protein-Coding Genes
                        • Eukaryotic Transcription Activators and Repressor
                        • RNA Polymerase II Transcription-Initiation Complex
                        • Molecular Mechanisms of Eukaryotic Transcription Control
                        • Other Transcription Systems
                        • 11.1. Transcription Termination
                          • Rho-Independent Termination Occurs at Characteristic Sequences in E. coli DNA
                          • Premature Termination by Attenuation Helps Regulate Expression of Some Bacterial Operons
                          • Rho-Dependent Termination Sites Are Present in Some λ-Phage and E. coli Genes
                          • Sequence-Specific RNA-Binding Proteins Can Regulate Termination by E. coli RNA Polymerase
                          • Three Eukaryotic RNA Polymerases Employ Different Termination Mechanisms
                          • Transcription of HIV Genome Is Regulated by an Antitermination Mechanism
                          • Promoter-Proximal Pausing of RNA Polymerase II Occurs in Some Rapidly Induced Genes
                          • SUMMARY
                          • The 5′-Cap Is Added to Nascent RNAs Shortly after Initiation by RNA Polymerase II
                          • Pre-mRNAs Are Associated with hnRNP Proteins Containing Conserved RNA-Binding Domains
                          • hnRNP Proteins May Assist in Processing and Transport of mRNAs
                          • Pre-mRNAs Are Cleaved at Specific 3′ Sites and Rapidly Polyadenylated
                          • Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions
                          • Spliceosomes, Assembled from snRNPs and a Pre-mRNA, Carry Out Splicing
                          • Portions of Two Different RNAs Are Trans-Spliced in Some Organisms
                          • Self-Splicing Group II Introns Provide Clues to the Evolution of snRNAs
                          • Most Transcription and RNA Processing Occur in a Limited Number of Domains in Mammalian Cell Nuclei
                          • SUMMARY
                          • U1A Protein Inhibits Polyadenylation of Its Pre-mRNA
                          • Tissue-Specific RNA Splicing Controls Expression of Alternative Fibronectins
                          • A Cascade of Regulated RNA Splicing Controls Drosophila Sexual Differentiation
                          • Multiple Protein Isoforms Are Common in the Vertebrate Nervous System
                          • SUMMARY
                          • Nuclear Pore Complexes Actively Transport Macromolecules between the Nucleus and Cytoplasm
                          • Receptors for Nuclear-Export Signals Transport Proteins and mRNPs out of the Nucleus
                          • Pre-mRNAs in Spliceosomes Are Not Exported from the Nucleus
                          • Receptors for Nuclear-Localization Signals Transport Proteins into the Nucleus
                          • Various Nuclear-Transport Systems Utilize Similar Proteins
                          • HIV Rev Protein Regulates the Transport of Unspliced Viral mRNAs
                          • SUMMARY
                          • RNA Editing Alters the Sequences of Pre-mRNAs
                          • Some mRNAs Are Associated with Cytoplasmic Structures or Localized to Specific Regions
                          • Stability of Cytoplasmic mRNAs Varies Widely
                          • Degradation Rate of Some Eukaryotic mRNAs Is Regulated
                          • Translation of Some mRNAs Is Regulated by Specific RNA-Binding Proteins
                          • Antisense RNA Regulates Translation of Transposase mRNA in Bacteria
                          • SUMMARY
                          • Pre-rRNA Genes Are Similar in All Eukaryotes and Function as Nucleolar Organizers
                          • Small Nucleolar RNAs (snoRNAs) Assist in Processing rRNAs and Assembling Ribosome Subunits
                          • Self-Splicing Group I Introns Were the First Examples of Catalytic RNA
                          • All Pre-tRNAs Undergo Cleavage and Base Modification
                          • Splicing of Pre-tRNAs Differs from Other Splicing Mechanisms
                          • SUMMARY
                          • References
                          • Key Application
                          • Key Experiment
                          • Key Concept
                          • Transcription Termination
                          • Processing of Eukaryotic mRNA
                          • Regulation of mRNA Processing
                          • Signal-Mediated Transport through Nuclear Pore Complexes
                          • Other Mechanisms of Post-Transcriptional Control
                          • Processing of rRNA and tRNA
                          • 12.1. General Features of Chromosomal Replication
                            • DNA Replication Is Semiconservative
                            • Most DNA Replication Is Bidirectional
                            • DNA Replication Begins at Specific Chromosomal Sites
                            • SUMMARY
                            • DnaA Protein Initiates Replication in E. coli
                            • DnaB Is an E. coli Helicase That Melts Duplex DNA
                            • E. coli Primase Catalyzes Formation of RNA Primers for DNA Synthesis
                            • At a Growing Fork One Strand Is Synthesized Discontinuously from Multiple Primers
                            • E. coli DNA Polymerase III Catalyzes Nucleotide Addition at the Growing Fork
                            • The Leading and Lagging Strands Are Synthesized Concurrently
                            • Eukaryotic Replication Machinery Is Generally Similar to That of E. coli
                            • Telomerase Prevents Progressive Shortening of Lagging Strands during Eukaryotic DNA Replication
                            • SUMMARY
                            • Type I Topoisomerases Relax DNA by Nicking and Then Closing One Strand of Duplex DNA
                            • Type II Topoisomerases Change DNA Topology by Breaking and Rejoining Double-Stranded DNA
                            • Replicated Circular DNA Molecules Are Separated by Type II Topoisomerases
                            • Linear Daughter Chromatids Also Are Separated by Type II Topoisomerases
                            • SUMMARY
                            • Proofreading by DNA Polymerase Corrects Copying Errors
                            • Chemical Carcinogens React with DNA Directly or after Activation
                            • The Carcinogenic Effect of Chemicals Correlates with Their Mutagenicity
                            • DNA Damage Can Be Repaired by Several Mechanisms
                            • Eukaryotes Have DNA-Repair Systems Analogous to Those of E. coli
                            • Inducible DNA-Repair Systems Are Error-Prone
                            • SUMMARY
                            • The Crossed-Strand Holliday Structure Is an Intermediate in Recombination
                            • Double-Strand Breaks in DNA Initiate Recombination
                            • The Activities of E. coli Recombination Proteins Have Been Determined
                            • Cre Protein and Other Recombinases Catalyze Site-Specific Recombination
                            • SUMMARY
                            • References
                            • Key Application
                            • Key Concept
                            • Key Experiment
                            • General Features of Chromosomal Replication
                            • The DNA Replication Machinery
                            • Role of Topoisomerases in DNA Replication
                            • DNA Damage and Repair and Their Role in Carcinogenesis
                            • Recombination between Homologous DNA Sites
                            • 13.1. Overview of the Cell Cycle and Its Control
                              • The Cell Cycle Is an Ordered Series of Events Leading to Replication of Cells
                              • Regulated Protein Phosphorylation and Degradation Control Passage through the Cell Cycle
                              • Diverse Experimental Systems Have Been Used to Identify and Isolate Cell-Cycle Control Proteins
                              • SUMMARY
                              • MPF Promotes Maturation of Oocytes and Mitosis in Somatic Cells
                              • Mitotic Cyclin Was First Identified in Early Sea Urchin Embryos
                              • Cyclin B Levels and MPF Activity Change Together in Cycling Xenopus Egg Extracts
                              • Ubiquitin-Mediated Degradation of Mitotic Cyclins Promotes Exit from Mitosis
                              • Regulation of APC Activity Controls Degradation of Cyclin B
                              • SUMMARY
                              • Two Classes of Mutations in S. pombe Produce Either Elongated or Very Small Cells
                              • S. pombe Cdc2 -� Heterodimer Is Equivalent to Xenopus MPF
                              • Phosphorylation of the Catalytic Subunit Regulates MPF Kinase Activity
                              • Conformational Changes Induced by Cyclin Binding and Phosphorylation Increase MPF Activity
                              • Other Mechanisms Also Control Entry into Mitosis by Regulating MPF Activity
                              • SUMMARY
                              • Phosphorylation of Nuclear Lamins by MPF Leads to Nuclear-Envelope Breakdown
                              • Other Early Mitotic Events May Be Controlled Directly or Indirectly by MPF
                              • APC-Dependent Unlinking of Sister Chromatids Initiates Anaphase
                              • Phosphatase Activity Is Required for Reassembly of the Nuclear Envelope and Cytokinesis
                              • SUMMARY
                              • S. cerevisiae Cdc28 Is Functionally Equivalent to S. pombe Cdc2
                              • Three G1 Cyclins Associate with Cdc28 to form S Phase – Promoting Factors
                              • Kinase Activity of Cdc28 – G1 Cyclin Complexes Prepares Cells for the S Phase
                              • Degradation of the S-Phase Inhibitor Sic1 Triggers DNA Replication
                              • Multiple Cyclins Direct Kinase Activity of Cdc28 during Different Cell-Cycle Phases
                              • Replication at Each Origin Is Initiated Only Once during the Cell Cycle
                              • SUMMARY
                              • Mammalian Restriction Point is Analogous to start in Yeast Cells
                              • Multiple Cdks and Cyclins Regulate Passage of Mammalian Cells through the Cell Cycle
                              • Regulated Expression of Two Classes of Genes Returns G0 Mammalian Cells to the Cell Cycle
                              • Passage through the Restriction Point Depends on Activation of E2F Transcription Factors
                              • Cyclin A Is Required for DNA Synthesis and Cdk1 for Entry into Mitosis
                              • Mammalian Cyclin-Kinase Inhibitors Contribute to Cell-Cycle Control
                              • SUMMARY
                              • The Presence of Unreplicated DNA Prevents Entry into Mitosis
                              • Improper Assembly of the Mitotic Spindle Leads to Arrest in Anaphase
                              • G1 and G2 Arrest in Cells with Damaged DNA Depends on a Tumor Suppressor and Cyclin-Kinase Inhibitor
                              • SUMMARY
                              • References
                              • Key Concept
                              • Key Experiment
                              • Key Application
                              • Overview of the Cell Cycle and Its Control
                              • Biochemical Studies with Oocytes, Eggs, and Early Embryos
                              • Genetic Studies with S. pombe
                              • Molecular Mechanisms for Regulating Mitotic Events
                              • Genetic Studies with S. cerevisiae
                              • Cell-Cycle Control in Mammalian Cells
                              • Checkpoints in Cell-Cycle Regulation
                              • 14.1. Cell-Type Specification and Mating-Type Conversion in Yeast
                                • Combinations of DNA-Binding Proteins Regulate Cell-Type Specification in Yeast
                                • Mating of α and a Cells Is Induced by Pheromone-Stimulated Gene Expression
                                • Multiple Regulation of HO Transcription Controls Mating-Type Conversion
                                • Silencer Elements Repress Expression at HML and HMR
                                • SUMMARY
                                • Embryonic Somites Give Rise to Myoblasts, the Precursors of Skeletal Muscle Cells
                                • Myogenic Genes Were First Identified in Studies with Cultured Fibroblasts
                                • Myogenic Proteins Are Transcription Factors Containing a Common bHLH Domain
                                • MEFs Function in Concert with MRFs to Confer Myogenic Specificity
                                • Myogenic Stages at Which MRFs and MEFs Function in Vivo Have Been Identified
                                • Multiple MRFs Exhibit Functional Diversity and Permit Flexibility in Regulating Development
                                • Terminal Differentiation of Myoblasts Is under Positive and Negative Control
                                • A Network of Cross-Regulatory Interactions Maintains the Myogenic Program
                                • Neurogenesis Requires Regulatory Proteins Analogous to bHLH Myogenic Proteins
                                • Progressive Restriction of Neural Potential Requires Inhibitory HLH Proteins and Local Cell-Cell Interactions
                                • bHLH Regulatory Circuitry May Operate to Specify Other Cell Types
                                • SUMMARY
                                • Drosophila Has Two Life Forms
                                • Patterning Information Is Generated during Oogenesis and Early Embryogenesis
                                • Four Maternal Gene Systems Control Early Patterning in Fly Embryos
                                • Morphogens Regulate Development as a Function of Their Concentration
                                • Maternal bicoid Gene Specifies Anterior Region in Drosophila
                                • Maternally Derived Inhibitors of Translation Contribute to Early Drosophila Patterning
                                • Graded Expression of Several Gap Genes Further Subdivides the Fly Embryo into Unique Spatial Domains
                                • Expression of Three Groups of Zygotic Genes Completes Early Patterning in Drosophila
                                • Selector (Hox) Genes Occur in Clusters in the Genome
                                • Combinations of Different Hox Proteins Contribute to Specifying Parasegment Identity in Drosophila
                                • Specificity of Drosophila Hox-Protein Function Is Mediated by Exd Protein
                                • Hox-Gene Expression Is Maintained by Autoregulation and Changes in Chromatin Structure
                                • Mammalian Homologs of Drosophila ANT-C and BX-C Genes Occur in Four Hox Complexes
                                • Mutations in Hox Genes Result in Homeotic Transformations in the Developing Mouse
                                • SUMMARY
                                • Flowers Contain Four Different Organs
                                • Three Classes of Genes Control Floral-Organ Identity
                                • Many Floral Organ–Identity Genes Encode MADS Family Transcription Factors
                                • SUMMARY
                                • References
                                • Key Concept
                                • Key Experiment
                                • Key Application
                                • Cell-Type Specification and Mating-Type Conversion in Yeast
                                • Cell-Type Specification in Animals
                                • Anteroposterior Specification during Embryogenesis
                                • Specification of Floral-Organ Identity in Arabidopsis
                                • 15.1. Diffusion of Small Molecules across Phospholipid Bilayers
                                • 15.2. Overview of Membrane Transport Proteins
                                  • SUMMARY
                                  • Three Main Features Distinguish Uniport Transport from Passive Diffusion
                                  • GLUT1 Transports Glucose into Most Mammalian Cells
                                  • SUMMARY
                                  • Ionic Gradients and an Electric Potential Are Maintained across the Plasma Membrane
                                  • The Membrane Potential in Animal Cells Depends Largely on Resting K + Channels
                                  • Na + Entry into Mammalian Cells Has a Negative ΔG
                                  • SUMMARY
                                  • Plasma-Membrane Ca 2+ ATPase Exports Ca 2+ Ions from Cells
                                  • Muscle Ca 2+ ATPase Pumps Ca 2+ Ions from the Cytosol into the Sarcoplasmic Reticulum
                                  • Na + /K + ATPase Maintains the Intracellular Na + and K + Concentrations in Animal Cells
                                  • V-Class H + ATPases Pump Protons across Lysosomal and Vacuolar Membranes
                                  • The ABC Superfamily Transports a Wide Variety of Substrates
                                  • SUMMARY
                                  • Na + -Linked Symporters Import Amino Acids and Glucose into Many Animal Cells
                                  • Na + -Linked Antiporter Exports Ca 2+ from Cardiac Muscle Cells
                                  • AE1 Protein, a Cl − /HCO3 − Antiporter, Is Crucial to CO2 Transport by Erythrocytes
                                  • Several Cotransporters Regulate Cytosolic pH
                                  • Numerous Transport Proteins Enable Plant Vacuoles to Accumulate Metabolites and Ions
                                  • SUMMARY
                                  • The Intestinal Epithelium Is Highly Polarized
                                  • Transepithelial Movement of Glucose and Amino Acids Requires Multiple Transport Proteins
                                  • Parietal Cells Acidify the Stomach Contents While Maintaining a Neutral Cytosolic pH
                                  • Tight Junctions Seal Off Body Cavities and Restrict Diffusion of Membrane Components
                                  • Other Junctions Interconnect Epithelial Cells and Control Passage of Molecules between Them
                                  • SUMMARY
                                  • Osmotic Pressure Causes Water to Move across Membranes
                                  • Different Cells Have Various Mechanisms for Controlling Cell Volume
                                  • Water Channels Are Necessary for Bulk Flow of Water across Cell Membranes
                                  • Simple Rehydration Therapy Depends on Osmotic Gradient Created by Absorption of Glucose and Na +
                                  • Changes in Intracellular Osmotic Pressure Cause Leaf Stomata to Open
                                  • SUMMARY
                                  • References
                                  • Key Concept
                                  • Key Experiment
                                  • Key Application
                                  • Uniporter-Catalyzed Transport of Specific Molecules
                                  • Ion Channels, Intracellular Ion Environment, and Membrane Electric Potential
                                  • Active Transport and ATP Hydrolysis
                                  • Cotransport Catalyzed by Symporters and Antiporters
                                  • Transport across Epithelia
                                  • Osmosis, Water Channels, and the Regulation of Cell Volume
                                  • 16.1. Oxidation of Glucose and Fatty Acids to CO2
                                    • Cytosolic Enzymes Convert Glucose to Pyruvate
                                    • Substrate-Level Phosphorylation Generates ATP during Glycolysis
                                    • Anaerobic Metabolism of Each Glucose Molecule Yields Only Two ATP Molecules
                                    • Mitochondria Possess Two Structurally and Functionally Distinct Membranes
                                    • Mitochondrial Oxidation of Pyruvate Begins with the Formation of Acetyl CoA
                                    • Oxidation of the Acetyl Group of Acetyl CoA in the Citric Acid Cycle Yields CO2 and Reduced Coenzymes
                                    • Inner-Membrane Proteins Allow the Uptake of Electrons from Cytosolic NADH
                                    • Mitochondrial Oxidation of Fatty Acids Is Coupled to ATP Formation
                                    • Oxidation of Fatty Acids in Peroxisomes Generates No ATP
                                    • The Rate of Glucose Oxidation Is Adjusted to Meet the Cell’s Need for ATP
                                    • SUMMARY
                                    • The Proton-Motive Force in Mitochondria Is Due Largely to a Voltage Gradient across the Inner Membrane
                                    • Electron Transport in Mitochondria Is Coupled to Proton Translocation
                                    • Electrons Flow from FADH2 and NADH to O2 via a Series of Multiprotein Complexes
                                    • CoQ and Cytochrome c Shuttle Electrons from One Electron Transport Complex to Another
                                    • Reduction Potentials of Electron Carriers Favor Electron Flow from NADH to O2
                                    • CoQ and Three Electron Transport Complexes Pump Protons out of the Mitochondrial Matrix
                                    • Experiments with Membrane Vesicles Support the Chemiosmotic Mechanism of ATP Formation
                                    • Bacterial Plasma-Membrane Proteins Catalyze Electron Transport and Coupled ATP Synthesis
                                    • ATP Synthase Comprises a Proton Channel (F0) and ATPase (F1)
                                    • The F0F1 Complex Harnesses the Proton-Motive Force to Power ATP Synthesis
                                    • Transporters in the Inner Mitochondrial Membrane Are Powered by the Proton-Motive Force
                                    • Rate of Mitochondrial Oxidation Normally Depends on ADP Levels
                                    • Brown-Fat Mitochondria Contain an Uncoupler of Oxidative Phosphorylation
                                    • SUMMARY
                                    • Photosynthesis Occurs on Thylakoid Membranes
                                    • Three of the Four Stages in Photosynthesis Occur Only during Illumination
                                    • Each Photon of Light Has a Defined Amount of Energy
                                    • Chlorophyll a Is Present in Both Components of a Photosystem
                                    • Light Absorption by Reaction-Center Chlorophylls Causes a Charge Separation across the Thylakoid Membrane
                                    • Light-Harvesting Complexes Increase the Efficiency of Photosynthesis
                                    • SUMMARY
                                    • Photoelectron Transport in Purple Bacteria Produces a Charge Separation
                                    • Both Cyclic and Noncyclic Electron Transport Occur in Bacterial Photosynthesis
                                    • Chloroplasts Contain Two Functionally and Spatially Distinct Photosystems
                                    • An Oxygen-Evolving Complex in PSII Regenerates P680
                                    • Cyclic Electron Flow in PSI Generates ATP but No NADPH
                                    • PSI and PSII Are Functionally Coupled
                                    • Both Plant Photosystems Are Essential for Formation of NADPH and O2
                                    • SUMMARY
                                    • CO2 Fixation Occurs in the Chloroplast Stroma
                                    • Synthesis of Sucrose Incorporating Fixed CO2 Is Completed in the Cytosol
                                    • Light Stimulates CO2 Fixation by Several Mechanisms
                                    • Photorespiration, Which Consumes O2 and Liberates CO2, Competes with Photosynthesis
                                    • The C4 Pathway for CO2 Fixation Is Used by Many Tropical Plants
                                    • Sucrose Is Transported from Leaves through the Phloem to All Plant Tissues
                                    • SUMMARY
                                    • References
                                    • Key Concept
                                    • Key Experiment
                                    • Key Concept
                                    • Oxidation of Glucose and Fatty Acids to CO2
                                    • Electron Transport and Oxidative Phosphorylation
                                    • Photosynthetic Stages and Light Absorbing Pigments
                                    • Molecular Analysis of Photosystems
                                    • CO2 Metabolism During Photosynthesis
                                    • 17.1. Synthesis and Targeting of Mitochondrial and Chloroplast Proteins
                                      • Most Mitochondrial Proteins Are Synthesized as Cytosolic Precursors Containing Uptake-Targeting Sequences
                                      • Cytosolic Chaperones Deliver Proteins to Channel-Linked Receptors in the Mitochondrial Membrane
                                      • Matrix Chaperones and Chaperonins Are Essential for the Import and Folding of Mitochondrial Proteins
                                      • Studies with Chimeric Proteins Confirm Major Features of Mitochondrial Import
                                      • The Uptake of Mitochondrial Proteins Requires Energy
                                      • Proteins Are Targeted to Submitochondrial Compartments by Multiple Signals and Several Pathways
                                      • The Synthesis of Mitochondrial Proteins Is Coordinated
                                      • Several Uptake-Targeting Sequences Direct Proteins Synthesized in the Cytosol to the Appropriate Chloroplast Compartment
                                      • SUMMARY
                                      • C- and N-Terminal Targeting Sequences Direct Entry of Folded Proteins into the Peroxisomal Matrix
                                      • Peroxisomal Protein Import Is Defective in Some Genetic Diseases
                                      • SUMMARY
                                      • Secretory Proteins Move from the Rough ER Lumen through the Golgi Complex and Then to the Cell Surface
                                      • Analysis of Yeast Mutants Defined Major Steps in the Secretory Pathway
                                      • Anterograde Transport through the Golgi Occurs by Cisternal Progression
                                      • Plasma-Membrane Glycoproteins Mature via the Same Pathway as Continuously Secreted Proteins
                                      • SUMMARY
                                      • A Signal Sequence on Nascent Secretory Proteins Targets Them to the ER and Is Then Cleaved Off
                                      • Two Proteins Initiate the Interaction of Signal Sequences with the ER Membrane
                                      • Polypeptides Move through the Translocon into the ER Lumen
                                      • GTP Hydrolysis Powers Protein Transport into the ER in Mammalian Cells
                                      • SUMMARY
                                      • Most Nominal Cytosolic Transmembrane Proteins Have an N-Terminal Signal Sequence and an Internal Topogenic Sequence
                                      • A Single Internal Topogenic Sequence Directs Insertion of Some Single-Pass Transmembrane Proteins
                                      • Multipass Transmembrane Proteins Have Multiple Topogenic Sequences
                                      • After Insertion in the ER Membrane, Some Proteins Are Transferred to a GPI Anchor
                                      • SUMMARY
                                      • Disulfide Bonds Are Formed and Rearranged in the ER Lumen
                                      • Correct Folding of Newly Made Proteins Is Facilitated by Several ER Proteins
                                      • Assembly of Subunits into Multimeric Proteins Occurs in the ER
                                      • Only Properly Folded Proteins Are Transported from the Rough ER to the Golgi Complex
                                      • Many Unassembled or Misfolded Proteins in the ER Are Transported to the Cytosol and Degraded
                                      • ER-Resident Proteins Often Are Retrieved from the Cis-Golgi
                                      • SUMMARY
                                      • Different Structures Characterize N- and O-Linked Oligosaccharides
                                      • O-Linked Oligosaccharides Are Formed by the Sequential Transfer of Sugars from Nucleotide Precursors
                                      • ABO Blood Type Is Determined by Two Glycosyltransferases
                                      • A Common Preformed N-Linked Oligosaccharide Is Added to Many Proteins in the Rough ER
                                      • Modifications to N-Linked Oligosaccharides Are Completed in the Golgi Complex
                                      • Oligosaccharides May Promote Folding and Stability of Glycoproteins
                                      • Mannose 6-Phosphate Residues Target Proteins to Lysosomes
                                      • Lysosomal Storage Diseases Provided Clues to Sorting of Lysosomal Enzymes
                                      • SUMMARY
                                      • Sequences in the Membrane-Spanning Domain Cause the Retention of Proteins in the Golgi
                                      • Different Vesicles Are Used for Continuous and Regulated Protein Secretion
                                      • Proproteins Undergo Proteolytic Processing Late in Maturation
                                      • Some Proteins Are Sorted from the Golgi Complex to the Apical or Basolateral Plasma Membrane
                                      • SUMMARY
                                      • The LDL Receptor Binds and Internalizes Cholesterol-Containing Particles
                                      • Cytosolic Sequences in Some Cell-Surface Receptors Target Them for Endocytosis
                                      • The Acidic pH of Late Endosomes Causes Most Receptors and Ligands to Dissociate
                                      • The Endocytic Pathway Delivers Transferrin-Bound Iron to Cells
                                      • Some Endocytosed Proteins Remain within the Cell
                                      • Transcytosis Moves Some Ligands across Cells
                                      • SUMMARY
                                      • At Least Three Types of Coated Vesicles Transport Proteins from Organelle to Organelle
                                      • Clathrin Vesicles Mediate Several Types of Intracellular Transport
                                      • COP I Vesicles Mediate Retrograde Transport within the Golgi and from the Golgi Back to the ER
                                      • COP II Vesicles Mediate Transport from the ER to the Golgi
                                      • Specific Fusion of Intracellular Vesicles Involves a Conserved Set of Fusion Proteins
                                      • Conformational Changes in Influenza HA Protein Trigger Membrane Fusion
                                      • SUMMARY
                                      • References
                                      • Key Concept
                                      • Key Experiment
                                      • Key Application
                                      • Synthesis and Targeting of Mitochondrial and Chloroplast Proteins
                                      • Synthesis and Targeting of Peroxisomal Proteins
                                      • Translocation of Secretory Proteins across the ER Membrane
                                      • Insertion of Membrane Proteins into the ER Membrane
                                      • Post-Translational Modifications and Quality Control in the Rough ER
                                      • Protein Glycosylation in the ER and Golgi Complex
                                      • Golgi and Post-Golgi Protein Sorting and Proteolytic Processing
                                      • Receptor-Mediated Endocytosis and the Sorting of Internalized Proteins
                                      • Molecular Mechanisms of Vesicular Traffic
                                      • 18.1. The Actin Cytoskeleton
                                        • Eukaryotic Cells Contain Abundant Amounts of Highly Conserved Actin
                                        • ATP Holds Together the Two Lobes of the Actin Monomer
                                        • G-Actin Assembles into Long, Helical F-Actin Polymers
                                        • F-Actin Has Structural and Functional Polarity
                                        • The Actin Cytoskeleton Is Organized into Bundles and Networks of Filaments
                                        • Cortical Actin Networks Are Connected to the Membrane
                                        • Actin Bundles Support Projecting Fingers of Membrane
                                        • SUMMARY
                                        • Actin Polymerization in Vitro Proceeds in Three Steps
                                        • Actin Filaments Grow Faster at One End Than at the Other
                                        • Toxins Disrupt the Actin Monomer-Polymer Equilibrium
                                        • Actin Polymerization Is Regulated by Proteins That Bind G-Actin
                                        • Some Proteins Control the Lengths of Actin Filaments by Severing Them
                                        • Actin Filaments Are Stabilized by Actin-Capping Proteins
                                        • Many Movements Are Driven by Actin Polymerization
                                        • SUMMARY
                                        • All Myosins Have Head, Neck, and Tail Domains with Distinct Functions
                                        • Myosin Heads Walk along Actin Filaments
                                        • Myosin Heads Move in Discrete Steps, Each Coupled to Hydrolysis of One ATP
                                        • Myosin and Kinesin Share the Ras Fold with Certain Signaling Proteins
                                        • Conformational Changes in the Myosin Head Couple ATP Hydrolysis to Movement
                                        • SUMMARY
                                        • Some Muscles Contract, Others Generate Tension
                                        • Skeletal Muscles Contain a Regular Array of Actin and Myosin
                                        • Smooth Muscles Contain Loosely Organized Thick and Thin Filaments
                                        • Thick and Thin Filaments Slide Past One Another during Contraction
                                        • Titin and Nebulin Filaments Organize the Sarcomere
                                        • A Rise in Cytosolic Ca 2+ Triggers Muscle Contraction
                                        • Actin-Binding Proteins Regulate Contraction in Both Skeletal and Smooth Muscle
                                        • Myosin-Dependent Mechanisms Also Control Contraction in Some Muscles
                                        • SUMMARY
                                        • Actin and Myosin II Are Arranged in Contractile Bundles That Function in Cell Adhesion
                                        • Myosin II Stiffens Cortical Membranes
                                        • Actin and Myosin II Have Essential Roles in Cytokinesis
                                        • Membrane-Bound Myosins Power Movement of Some Vesicles
                                        • SUMMARY
                                        • Controlled Polymerization and Rearrangements of Actin Filaments Occur during Keratinocyte Movement
                                        • Ameboid Movement Involves Reversible Gel-Sol Transitions of Actin Networks
                                        • Myosin I and Myosin II Have Important Roles in Cell Migration
                                        • Migration of Cells Is Coordinated by Various Second Messengers and Signal-Transduction Pathways
                                        • SUMMARY
                                        • References
                                        • Key Concept
                                        • Key Experiment
                                        • Key Concept
                                        • General References
                                        • Web Sites
                                        • Actin Cytoskeleton
                                        • Dynamics of Actin Assembly
                                        • Myosin: The Actin Motor Protein
                                        • Muscle: A Specialized Contractile Machine
                                        • Actin and Myosin in Nonmuscle Cells
                                        • Cell Locomotion
                                        • 19.1. Microtubule Structures
                                          • Heterodimeric Tubulin Subunits Compose the Wall of a Microtubule
                                          • Microtubules Form a Diverse Array of Both Permanent and Transient Structures
                                          • Microtubules Assemble from Organizing Centers
                                          • Most Microtubules Have a Constant Orientation Relative to MTOCs
                                          • The γ-Tubulin Ring Complex Nucleates Polymerization of Tubulin Subunits
                                          • SUMMARY
                                          • Microtubule Assembly and Disassembly Occur Preferentially at the (+) End
                                          • Dynamic Instability Is an Intrinsic Property of Microtubules
                                          • Colchicine and Other Drugs Disrupt Microtubule Dynamics
                                          • Assembly MAPs Cross-Link Microtubules to One Another and Other Structures
                                          • Bound MAPs Alter Microtubule Dynamics
                                          • SUMMARY
                                          • Fast Axonal Transport Occurs along Microtubules
                                          • Microtubules Provide Tracks for the Movement of Pigment Granules
                                          • Intracellular Membrane Vesicles Travel along Microtubules
                                          • Kinesin Is a (+) End𠄽irected Microtubule Motor Protein
                                          • Each Member of the Kinesin Family Transports a Specific Cargo
                                          • Dynein Is a (−) End –𠁝irected Motor Protein
                                          • Dynein-Associated MBPs Tether Cargo to Microtubules
                                          • Multiple Motor Proteins Are Associated with Membrane Vesicles
                                          • SUMMARY
                                          • All Eukaryotic Cilia and Flagella Contain Bundles of Doublet Microtubules
                                          • Ciliary and Flagellar Beating Are Produced by Controlled Sliding of Outer Doublet Microtubules
                                          • Dynein Arms Generate the Sliding Forces in Axonemes
                                          • Axonemal Dyneins Are Multiheaded Motor Proteins
                                          • Conversion of Microtubule Sliding into Axonemal Bending Depends on Inner-Arm Dyneins
                                          • Proteins Associated with Radial Spokes May Control Flagellar Beat
                                          • Axonemal Microtubules Are Dynamic and Stable
                                          • SUMMARY
                                          • The Mitotic Apparatus Is a Microtubule Machine for Separating Chromosomes
                                          • The Kinetochore Is a Specialized Attachment Site at the Chromosome Centromere
                                          • Centrosome Duplication Precedes and Is Required for Mitosis
                                          • Dynamic Instability of Microtubules Increases during Mitosis
                                          • Organization of the Spindle Poles Orients the Assembly of the Mitotic Apparatus
                                          • Formation of Poles and Capture of Chromosomes Are Key Events in Spindle Assembly
                                          • Kinetochores Generate the Force for Poleward Chromosome Movement
                                          • During Anaphase Chromosomes Separate and the Spindle Elongates
                                          • Astral Microtubules Determine Where Cytokinesis Takes Place
                                          • Plant Cells Reorganize Their Microtubules and Build a New Cell Wall during Mitosis
                                          • SUMMARY
                                          • Functions and Structure of Intermediate Filaments Distinguish Them from Other Cytoskeletal Fibers
                                          • IF Proteins Are Classified into Six Types
                                          • Intermediate Filaments Can Identify the Cellular Origin of Certain Tumors
                                          • All IF Proteins Have a Conserved Core Domain and Are Organized Similarly into Filaments
                                          • Intermediate Filaments Are Dynamic Polymers in the Cell
                                          • Various Proteins Cross-Link Intermediate Filaments and Connect Them to Other Cell Structures
                                          • IF Networks Support Cellular Membranes
                                          • Intermediate Filaments Are Anchored in Cell Junctions
                                          • Desmin and Associated Proteins Stabilize Sarcomeres in Muscle
                                          • Disruption of Keratin Networks Causes Blistering
                                          • SUMMARY
                                          • References
                                          • Key Concept
                                          • Key Experiment
                                          • Key Application
                                          • Microtubule Structures
                                          • Microtubule Dynamics and Associated Proteins
                                          • Kinesin, Dynein, and Intracellular Transport
                                          • Cilia and Flagella: Structure and Movement
                                          • Microtubule Dynamics and Motor Proteins during Mitosis
                                          • Intermediate Filaments
                                          • 20.1. Overview of Extracellular Signaling
                                            • Signaling Molecules Operate over Various Distances in Animals
                                            • Receptor Proteins Exhibit Ligand-Binding and Effector Specificity
                                            • Hormones Can Be Classified Based on Their Solubility and Receptor Location
                                            • Cell-Surface Receptors Belong to Four Major Classes
                                            • Effects of Many Hormones Are Mediated by Second Messengers
                                            • Other Conserved Proteins Function in Signal Transduction
                                            • Common Signaling Pathways Are Initiated by Different Receptors in a Class
                                            • The Synthesis, Release, and Degradation of Hormones Are Regulated
                                            • SUMMARY
                                            • Hormone Receptors Are Detected by Binding Assays
                                            • KD Values for Cell-Surface Hormone Receptors Approximate the Concentrations of Circulating Hormones
                                            • Affinity Techniques Permit Purification of Receptor Proteins
                                            • Many Receptors Can Be Cloned without Prior Purification
                                            • SUMMARY
                                            • Binding of Epinephrine to Adrenergic Receptors Induces Tissue-Specific Responses
                                            • Stimulation of β-Adrenergic Receptors Leads to a Rise in cAMP
                                            • Critical Features of Catecholamines and Their Receptors Have Been Identified
                                            • Trimeric Gs Protein Links β-Adrenergic Receptors and Adenylyl Cyclase
                                            • Some Bacterial Toxins Irreversibly Modify G Proteins
                                            • Adenylyl Cyclase Is Stimulated and Inhibited by Different Receptor-Ligand Complexes
                                            • GTP-Induced Changes in G Favor Its Dissociation from Gβγ and Association with Adenylyl Cyclase
                                            • G and G Interact with Different Regions of Adenylyl Cyclase
                                            • Degradation of cAMP Also Is Regulated
                                            • SUMMARY
                                            • Ligand Binding Leads to Autophosphorylation of RTKs
                                            • Ras and Gα Subunits Belong to the GTPase Superfamily of Intracellular Switch Proteins
                                            • An Adapter Protein and GEF Link Most Activated RTKs to Ras
                                            • SH2 Domain in GRB2 Adapter Protein Binds to a Specific Phosphotyrosine in an Activated RTK
                                            • Sos, a Guanine Nucleotide –𠁞xchange Factor, Binds to the SH3 Domains in GRB2
                                            • SUMMARY
                                            • Signals Pass from Activated Ras to a Cascade of Protein Kinases
                                            • Ksr May Function as a Scaffold for the MAP Kinase Cascade Linked to Ras
                                            • Phosphorylation of a Tyrosine and a Threonine Activates MAP Kinase
                                            • Various Types of Receptors Transmit Signals to MAP Kinase
                                            • Multiple MAP Kinase Pathways Are Found in Eukaryotic Cells
                                            • Specificity of MAP Kinase Pathways Depends on Several Mechanisms
                                            • SUMMARY
                                            • cAMP and Other Second Messengers Activate Specific Protein Kinases
                                            • cAPKs Activated by Epinephrine Stimulation Regulate Glycogen Metabolism
                                            • Kinase Cascades Permit Multienzyme Regulation and Amplify Hormone Signals
                                            • Cellular Responses to cAMP Vary among Different Cell Types
                                            • Anchoring Proteins Localize Effects of cAMP to Specific Subcellular Regions
                                            • Modification of a Common Phospholipid Precursor Generates Several Second Messengers
                                            • Hormone-Induced Release of Ca 2+ from the ER Is Mediated by IP3
                                            • Opening of Ryanodine Receptors Releases Ca 2+ Stores in Muscle and Nerve Cells
                                            • Ca 2+ -Calmodulin Complex Mediates Many Cellular Responses
                                            • DAG Activates Protein Kinase C, Which Regulates Many Other Proteins
                                            • Synthesis of cGMP Is Induced by Both Peptide Hormones and Nitric Oxide
                                            • SUMMARY
                                            • The Same RTK Can Be Linked to Different Signaling Pathways
                                            • Multiple G Proteins Transduce Signals to Different Effector Proteins
                                            • Gβγ Acts Directly on Some Effectors in Mammalian Cells
                                            • Glycogenolysis Is Promoted by Multiple Second Messengers
                                            • Insulin Stimulation Activates MAP Kinase and Protein Kinase B
                                            • Insulin and Glucagon Work Together to Maintain a Stable Blood Glucose Level
                                            • Receptors for Many Peptide Hormones Are Down-Regulated by Endocytosis
                                            • Phosphorylation of Cell-Surface Receptors Modulates Their Activity
                                            • Arrestins Have Two Roles in Regulating G Protein –𠁜oupled Receptors
                                            • SUMMARY
                                            • CREB Links cAMP Signals to Transcription
                                            • MAP Kinase Regulates the Activity of Many Transcription Factors
                                            • Phosphorylation-Dependent Protein Degradation Regulates NF-㮫
                                            • SUMMARY
                                            • References
                                            • Key Application
                                            • Key Concept
                                            • Key Experiment
                                            • Overview of Extracellular Signaling
                                            • Identification and Purification of Cell-Surface Receptors
                                            • G Protein𠄼oupled Receptors and Their Effectors
                                            • Receptor Tyrosine Kinases and Ras
                                            • MAP Kinase Pathways
                                            • Second Messengers
                                            • Interaction and Regulation of Signaling Pathway
                                            • From Plasma Membrane to Nucleus
                                            • 21.1. Overview of Neuron Structure and Function
                                              • Specialized Regions of Neurons Carry Out Different Functions
                                              • Synapses Are Specialized Sites Where Neurons Communicate with Other Cells
                                              • Neurons Are Organized into Circuits
                                              • SUMMARY
                                              • The Resting Potential, Generated Mainly by Open “Resting” K + Channels, Is Near EK
                                              • Opening and Closing of Ion Channels Cause Predictable Changes in the Membrane Potential
                                              • Membrane Depolarizations Spread Passively Only Short Distances
                                              • Voltage-Gated Cation Channels Generate Action Potentials
                                              • Action Potentials Are Propagated Unidirectionally without Diminution
                                              • Movements of Only a Few Na + and K + Ions Generate the Action Potential
                                              • Myelination Increases the Velocity of Impulse Conduction
                                              • SUMMARY
                                              • Patch Clamps Permit Measurement of Ion Movements through Single Channels
                                              • Voltage-Gated K + Channels Have Four Subunits Each Containing Six Transmembrane α Helices
                                              • P Segments Form the Ion-Selectivity Filter
                                              • The S4 Transmembrane α Helix Acts as a Voltage Sensor
                                              • Movement of One N-Terminal Segment Inactivates Shaker K + Channels
                                              • All Pore-Forming Ion Channels Are Similar in Structure to the Shaker K + Channel
                                              • Voltage-Gated Channel Proteins Probably Evolved from a Common Ancestral Gene
                                              • SUMMARY
                                              • Many Small Molecules Transmit Impulses at Chemical Synapses
                                              • Influx of Ca 2+ Triggers Release of Neurotransmitters
                                              • Synaptic Vesicles Can Be Filled, Exocytosed, and Recycled within a Minute
                                              • Multiple Proteins Participate in Docking and Fusion of Synaptic Vesicles
                                              • Chemical Synapses Can Be Excitatory or Inhibitory
                                              • Two Classes of Neurotransmitter Receptors Operate at Vastly Different Speeds
                                              • Acetylcholine and Other Transmitters Can Activate Multiple Receptors
                                              • Transmitter-Mediated Signaling Is Terminated by Several Mechanisms
                                              • Impulses Transmitted across Chemical Synapses Can be Amplified and Computed
                                              • Impulse Transmission across Electric Synapses Is Nearly Instantaneous
                                              • SUMMARY
                                              • Opening of Acetylcholine-Gated Cation Channels Leads to Muscle Contraction
                                              • All Five Subunits in the Nicotinic Acetylcholine Receptor Contribute to the Ion Channel
                                              • Two Types of Glutamate-Gated Cation Channels May Function in a Type of �llular Memory”
                                              • GABA- and Glycine-Gated Cl − Channels Are Found at Many Inhibitory Synapses
                                              • Cardiac Muscarinic Acetylcholine Receptors Activate a G Protein That Opens K + Channels
                                              • Catecholamine Receptors Induce Changes in Second-Messenger Levels That Affect Ion-Channel Activity
                                              • A Serotonin Receptor Indirectly Modulates K + Channel Function by Activating Adenylate Cyclase
                                              • Some Neuropeptides Function as Both Transmitters and Hormones
                                              • SUMMARY
                                              • Mechanoreceptors and Some Other Sensory Receptors Are Gated Cation Channels
                                              • Visual Signals Are Processed at Multiple Levels
                                              • The Light-Triggered Closing of Na + Channels Hyperpolarizes Rod Cells
                                              • Absorption of a Photon Triggers Isomerization of Retinal and Activation of Opsin
                                              • Cyclic GMP Is a Key Transducing Molecule in Rod Cells
                                              • Rod Cells Adapt to Varying Levels of Ambient Light
                                              • Color Vision Utilizes Three Opsin Pigments
                                              • A Thousand Different G Protein –𠁜oupled Receptors Detect Odors
                                              • SUMMARY
                                              • Repeated Conditioned Stimuli Cause Decrease in Aplysia Withdrawal Response
                                              • Facilitator Neurons Mediate Sensitization of Aplysia Withdrawal Reflex
                                              • Coincidence Detectors Participate in Classical Conditioning and Sensitization
                                              • Long-Term Memory Requires Protein Synthesis
                                              • SUMMARY
                                              • References
                                              • Key Concept
                                              • Key Experiment
                                              • Key Application
                                              • Overview of Neuron Structure and Function
                                              • The Action Potential and Conduction of Electric Impulses
                                              • Molecular Properties of Voltage-Gated Ion Channels
                                              • Neurotransmitters, Synapses, and Impulse Transmission
                                              • Neurotransmitter Receptors
                                              • Sensory Transduction
                                              • Learning and Memory
                                              • 22.1. Cell-Cell Adhesion and Communication
                                                • Cadherins Mediate Ca 2+ -Dependent Homophilic Cell-Cell Adhesion
                                                • N-CAMs Mediate Ca 2+ -Independent Homophilic Cell-Cell Adhesion
                                                • Selectins and Other CAMs Participate in Leukocyte Extravasation
                                                • Cadherin-Containing Junctions Connect Cells to One Another
                                                • Gap Junctions Allow Small Molecules to Pass between Adjacent Cells
                                                • Connexin, a Transmembrane Protein, Forms Cylindrical Channels in Gap Junctions
                                                • SUMMARY
                                                • Integrins Mediate Weak Cell-Matrix and Cell-Cell Interactions
                                                • Cell-Matrix Adhesion Is Modulated by Changes in the Activity and Number of Integrins
                                                • De-adhesion Factors Promote Cell Migration and Can Remodel the Cell Surface
                                                • Integrin-Containing Junctions Connect Cells to the Substratum
                                                • SUMMARY
                                                • The Basic Structural Unit of Collagen Is a Triple Helix
                                                • Collagen Fibrils Form by Lateral Interactions of Triple Helices
                                                • Assembly of Collagen Fibers Begins in the ER and Is Completed outside the Cell
                                                • Mutations in Collagen Reveal Aspects of Its Structure and Biosynthesis
                                                • Collagens Form Diverse Structures
                                                • SUMMARY
                                                • Laminin and Type IV Collagen Form the Two-Dimensional Reticulum of the Basal Lamina
                                                • Fibronectins Bind Many Cells to Fibrous Collagens and Other Matrix Components
                                                • Proteoglycans Consist of Multiple Glycosaminoglycans Linked to a Core Protein
                                                • Many Growth Factors Are Sequestered and Presented to Cells by Proteoglycans
                                                • Hyaluronan Resists Compression and Facilitates Cell Migration
                                                • SUMMARY
                                                • The Cell Wall Is a Laminate of Cellulose Fibrils in a Pectin and Hemicellulose Matrix
                                                • Cell Walls Contain Lignin and an Extended Hydroxyproline-Rich Glycoprotein
                                                • A Plant Hormone, Auxin, Signals Cell Expansion
                                                • Cellulose Fibrils Are Synthesized and Oriented at the Plant Cortex
                                                • Plasmodesmata Directly Connect the Cytosol of Adjacent Cells in Higher Plants
                                                • SUMMARY
                                                • Key Concept
                                                • Key Application
                                                • Key Experiment
                                                • General References
                                                • Cell-Cell Adhesion and Communication
                                                • Cell-Matrix Adhesion
                                                • Collagen
                                                • Noncollagen Components of the Extracellular Matrix
                                                • The Dynamic Plant Cell Wall
                                                • 23.1. Dorsoventral Patterning by TGFβ-Superfamily Proteins
                                                  • TGFβ Proteins Bind to Receptors That Have Serine/Threonine Kinase Activity
                                                  • Activated TGFβ Receptors Phosphorylate Smad Transcription Factors
                                                  • Dpp Protein, a TGFβ Homolog, Controls Dorsoventral Patterning in Drosophila Embryos
                                                  • Sequential Inductive Events Regulate Early Xenopus Development
                                                  • Inductive Effect of TGFβ Homologs Is Regulated Post-Translationally
                                                  • A Highly Conserved Pathway Determines Dorsoventral Patterning in Invertebrates and Vertebrates
                                                  • SUMMARY
                                                  • Modification of Secreted Hedgehog Precursor Yields a Cell-Tethered Inductive Signal
                                                  • Binding of Hedgehog to the Patch Receptor Relieves Inhibition of Smo
                                                  • Hedgehog Organizes Pattern in the Chick Limb and Drosophila Wing
                                                  • Hedgehog Induces Wingless, Which Triggers a Highly Conserved Signaling Pathway
                                                  • SUMMARY
                                                  • Hedgehog Gradient Elicits Different Cell Fates in the Vertebrate Neural Tube
                                                  • Cells Can Detect the Number of Ligand-Occupied Receptors
                                                  • Target Genes That Respond Differentially to Morphogens Have Different Control Regions
                                                  • SUMMARY
                                                  • Reciprocal Epithelial-Mesenchymal Interactions Regulate Kidney Development
                                                  • Activation of the Ret Receptor Promotes Growth and Branching of the Ureteric Bud
                                                  • The Basal Lamina Is Essential for Differentiation of Many Epithelial Cells
                                                  • Cell-Surface Ephrin Ligands and Receptors Mediate Reciprocal Induction during Angiogenesis
                                                  • The Conserved Notch Pathway Mediates Lateral Interactions
                                                  • Interactions between Two Equivalent Cells Give Rise to AC and VU cells in C. elegans
                                                  • Neuronal Development in Drosophila and Vertebrates Depends on Lateral Interactions
                                                  • SUMMARY
                                                  • Individual Neurons Can Be Identified Reproducibly and Studied
                                                  • Growth Cones Guide the Migration and Elongation of Developing Axons
                                                  • Different Neurons Navigate along Different Outgrowth Pathways
                                                  • Various Extracellular-Matrix Components Support Neuronal Outgrowth
                                                  • Growth Cones Navigate along Specific Axon Tracts
                                                  • Soluble Graded Signals Can Attract and Repel Growth Cones
                                                  • SUMMARY
                                                  • Three Genes Control Dorsoventral Outgrowth of Neurons in C. elegans
                                                  • Vertebrate Homologs of C. elegans UNC-6 Both Attract and Repel Growth Cones
                                                  • UNC-40 Mediates Chemoattraction in Response to Netrin in Vertebrates
                                                  • UNC-5 and UNC-40 Together Mediate Chemorepulsion in Response to Netrin
                                                  • Prior Experience Modulates Growth-Cone Response to Netrin
                                                  • Other Signaling Systems Can Both Attract and Repel Growth Cones
                                                  • SUMMARY
                                                  • Visual Stimuli Are Mapped onto the Tectum
                                                  • Temporal Retinal Axons Are Repelled by Posterior Tectal Membranes
                                                  • Ephrin A Ligands Are Expressed as a Gradient along the Anteroposterior Tectal Axis
                                                  • The EphA3 Receptor Is Expressed in a Nasal-Temporal Gradient in the Retina
                                                  • Motor Neurons Induce Assembly of the Neuromuscular Junction
                                                  • SUMMARY
                                                  • Programmed Cell Death Occurs through Apoptosis
                                                  • Neutrophins Promote Survival of Neurons
                                                  • Three Classes of Proteins Function in the Apoptotic Pathway
                                                  • Pro-Apoptotic Regulators Promote Caspase Activation
                                                  • Some Trophic Factors Prevent Apoptosis by Inducing Inactivation of a Pro-Apoptotic Regulator
                                                  • SUMMARY
                                                  • References
                                                  • Key Concept
                                                  • Key Application
                                                  • Key Experiment
                                                  • Dorsoventral Patterning by TGFβ-Superfamily Proteins
                                                  • Tissue Patterning by Hedgehog and Wingless
                                                  • Molecular Mechanisms of Responses to Morphogens
                                                  • Reciprocal and Lateral Inductive Interactions
                                                  • Overview of Neuronal Outgrowth
                                                  • Directional Control of Neuronal Outgrowth
                                                  • Formation of Topographic Maps and Synapses
                                                  • Cell Death and Its Regulation
                                                  • 24.1. Tumor Cells and the Onset of Cancer
                                                    • Metastatic Tumor Cells Are Invasive and Can Spread
                                                    • Alterations in Cell-to-Cell Interactions Are Associated with Malignancy
                                                    • Tumor Growth Requires Formation of New Blood Vessels
                                                    • DNA from Tumor Cells Can Transform Normal Cultured Cells
                                                    • Development of a Cancer Requires Several Mutations
                                                    • Cancers Originate in Proliferating Cells
                                                    • SUMMARY
                                                    • Gain-of-Function Mutations Convert Proto-Oncogenes into Oncogenes
                                                    • Oncogenes Were First Identified in Cancer-Causing Retroviruses
                                                    • Slow-Acting Carcinogenic Retroviruses Can Activate Cellular Proto-Oncogenes
                                                    • Many DNA Viruses Also Contain Oncogenes
                                                    • Loss-of-Function Mutations in Tumor-Suppressor Genes Are Oncogenic
                                                    • The First Tumor-Suppressor Gene Was Identified in Patients with Inherited Retinoblastoma
                                                    • Loss of Heterozygosity of Tumor-Suppressor Genes Occurs by Mitotic Recombination or Chromosome Mis-segregation
                                                    • SUMMARY
                                                    • Misexpressed Growth-Factor Genes Can Autostimulate Cell Proliferation
                                                    • Virus-Encoded Activators of Growth-Factor Receptors Act as Oncoproteins
                                                    • Activating Mutations or Overexpression of Growth-Factor Receptors Can Transform Cells
                                                    • Constitutively Active Signal-Transduction Proteins Are Encoded by Many Oncogenes
                                                    • Deletion of the PTEN Phosphatase Is a Frequent Occurrence in Human Tumors
                                                    • Inappropriate Expression of Nuclear Transcription Factors Can Induce Transformation
                                                    • SUMMARY
                                                    • Passage from G1 to S Phase Is Controlled by Proto-Oncogenes and Tumor-Suppressor Genes
                                                    • Loss of TGFβ Signaling Contributes to Abnormal Cell Proliferation and Malignancy
                                                    • SUMMARY
                                                    • Mutations in p53 Abolish G1 Checkpoint Control
                                                    • Proteins Encoded by DNA Tumor Viruses Can Inhibit p53 Activity
                                                    • Some Human Carcinogens Cause Inactivating Mutations in the p53 Gene
                                                    • Defects in DNA-Repair Systems Perpetuate Mutations and Are Associated with Certain Cancers
                                                    • Chromosomal Abnormalities Are Common in Human Tumors
                                                    • Telomerase Expression May Contribute to Immortalization of Cancer Cells
                                                    • SUMMARY
                                                    • References
                                                    • Key Concept
                                                    • Key Experiment
                                                    • Key Concept
                                                    • Tumor Cells and the Onset of Cancer
                                                    • Oncogenic Mutations Affecting Cell Proliferation
                                                    • Mutations Causing Loss of Cell-Cycle Control
                                                    • Mutations Affecting Genome Stability

                                                    By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.


                                                    Intermediates Produced During the Calvin Cycle

                                                    The Calvin Cycle involves the process of carbon fixation to produce organic compounds necessary for metabolic processes.

                                                    Learning Objectives

                                                    Outline the function of the intermediates produced in the major phases of the Calvin Cycle

                                                    Key Takeaways

                                                    Key Points

                                                    • The Calvin Cycle can be divided into three major phases: Phase 1: carbon fixation Phase 2: reduction Phase 3: regeneration.
                                                    • The intermediates of the Calvin Cycle include ADP, NADP+, inorganic phosphate, and 3-phosphoglycerate.
                                                    • Many of the intermediates or products of the Calvin Cycle are regenerated back into earlier stages of the process.

                                                    Key Terms

                                                    • autotroph: Any organism that can synthesize its food from inorganic substances, using heat or light as a source of energy.
                                                    • coenzyme: Any small molecule that is necessary for the functioning of an enzyme.
                                                    • phosphorylation: The process of transferring a phosphate group from a donor to an acceptor often catalysed by enzymes

                                                    The Calvin Cycle is characterized as a carbon fixation pathway. The Calvin Cycle is also referred to as the reductive pentose phosphate cycle or the Calvin-Benson-Bassham cycle. The process of carbon fixation involves the reduction of carbon dioxide to organic compounds by living organisms. The Calvin cycle is most often associated with carbon fixation in autotrophic organisms, such as plants, and is recognized as a dark reaction. In organisms that require carbon fixation, the Calvin cycle is a means to obtain energy and necessary components for growth. Some examples of microorganisms that utilize the Calvin cycle include cyanobacteria, purple bacteria, and nitrifying bacteria. Specifically, the Calvin cycle involves reducing carbon dioxide to the sugar triose phosphate, most commonly known as glyceraldehyde 3-phosphate (GAP). Throughout the Calvin Cycle, there are numerous intermediate molecules made which are consistently withdrawn and utilized to create cellular material and participate in cellular processes. The Calvin cycle can be divided into three major phases which include: Phase 1: carbon fixation Phase 2: reduction and Phase 3: regeneration of ribulose. The following is a brief overview of the intermediates created during the Calvin cycle.

                                                    The Calvin Cycle: An overview of the Calvin Cycle.

                                                    Phase 1: Carbon Fixation Intermediates

                                                    During phase 1 of this cycle, the CO2 molecule is incorporated into one of two 3-phosphoglycerate molecules (3-PGA). This process requires the enzyme RuBisCO and both ATP and NADPH. Once 3-PGA is formed, one of two molecules formed continues into the reduction phase (phase 2). The additional 3-PGA is utilized in additional metabolic pathways such as glycolysis and gluconeogenesis. The structure of 3-PGA allows it to be combined and rearranged to form sugars which can be transported to additional cells or stored for energy.

                                                    Phase 2: Reduction

                                                    During phase 2 of this cycle, the newly formed 3-PGA undergoes phosphorylation by the enzyme phosphoglycerate kinase which utilizes ATP. The result of this phosphorylation is the production of 1,3-bisphosphoglycerates and ADP products. The ADP product that is produced via the breakdown of ATP will be utilized in additional pathways and be converted back into ATP. The inter conversions of ATP to ADP and ADP to ATP is a key process in supplying energy in numerous processes. This energy is necessary for cellular growth and metabolic processes.

                                                    Once the bisphosphoglycerate molecules are formed, they must be converted and further reduced to GAP by NADPH. The intermediate of this product is the conversion of NADPH to NADP+ and an inorganic phosphate ion. NADP+ is a coenzyme which is necessary for the function of NADPH. The functions that require NADP+ include anabolic reactions such as lipid and nucleic acid synthesis. The inorganic phosphate ion is often a result of regulatory metabolic processes. The phosphate ions are used in processes such as buffering cells, conversions of AMP/ADP to ATP and production of materials involved in structure such as bone and teeth. It is important to note that these intermediates or products (inorganic phosphate, NADP+ and ADP) processed by phase 2 are often regenerated back into the cycle.

                                                    Phase 3: Regeneration of Ribulose

                                                    The GAP molecules at this point are the end product of the Calvin cycle, which is responsible for reducing carbon to a sugar form. However, additional GAP molecules that are formed will be converted to ribulose-1,5-bisphosphate (RuBP), which is responsible for the conversion of CO2 to 3-PGA in phase 1, via numerous steps. The G3P, which is destined to exit the cycle, will be used for carbohydrate synthesis and additional pathways.


                                                    Related Testing

                                                    Arterial blood gas (ABG) sampling, is a test often performed in an inpatient setting to assess the acid-base status of a patient. A needle is used to draw blood from an artery, often the radial, and the blood is analyzed to determine parameters such as the pH, pC02, pO2, HCO3, oxygen saturation, and more. This allows the physician to understand the status of the patient better. ABGs are especially important in the critically ill. They are the main tool utilized in adjusting to the needs of a patient on a ventilator. The following are the most important normal values on an ABG:

                                                    The ability to quickly and efficiently read an ABG, especially in reference to inpatient medicine, is paramount to quality patient care.

                                                    Other tests that are important to perform when analyzing the acid-base status of a patient include those that measure electrolyte levels and renal function. This helps the clinician gather information that can be used to determine the exact mechanism of the acid-base imbalance as well as the factors contributing to the disorders.[6][3]


                                                    Availability of data and materials

                                                    Database S1: E. coli. Excel file containing GEM-PRO related information for E. coli (Additional file 3).

                                                    Table 01: GEM-PRO master dataframe. All reactions, genes, sequence and structure ID mappings.

                                                    Table 02: Enzyme complex information for the associated reaction.

                                                    Table 02a: Updates to the previous complex information available in 2013.

                                                    Table 03: 3D structural properties of all representative structures per gene.

                                                    Table 03a: 3D structural properties of all homology models.

                                                    Table 04: PFAM retrieved and computed properties.

                                                    Table 05: Structural quality of PDB structures, including PSQS and PROCHECK scores.

                                                    Table 06: Structural quality of homology, including TM-scores, C-scores, PSQS, and PROCHECK scores.

                                                    Database S2: T. maritima. Excel file containing GEM-PRO related information for T. maritima (Additional file 2).

                                                    Table 01: GEM-PRO master dataframe. All reactions, genes, sequence and structure ID mappings.

                                                    Table 02: Enzyme complex information for the associated reaction.

                                                    Table 03: 3D structural properties of all representative structures per gene.

                                                    Table 04: PFAM retrieved and computed properties.

                                                    Table 05: Structural quality of PDB structures, including PSQS and PROCHECK scores.

                                                    Table 06: Structural quality of homology, including TM-scores, C-scores, PSQS, and PROCHECK scores.

                                                    Dataframes, mapping files, analysis scripts, tutorials and other documentation have been uploaded to a public Github repository and are available at: https://github.com/SBRG/GEMPro/tree/master/GEMPro_recon/.

                                                    Four iPython tutorial notebooks are hosted in the same Git repository and are available for viewing:


                                                    Availability of data and materials

                                                    My Personal Mutanome is available at https://mutanome.lerner.ccf.org to all users without any login or registration restrictions. The code for all mutation mapping and analysis can be found in https://github.com/ChengF-Lab/mutanome under the MIT License [66] and on Zenodo [67]. Gene and protein information was retrieved from HGNC (https://www.genenames.org/) [21] and UniProt (https://www.uniprot.org/uniprot/) [22]. PPI interface information was combined from three sources: PDB (http://www.rcsb.org/) [16], ECLAIR (http://interactomeinsider.yulab.org/) [17], and Interactome3D (https://interactome3d.irbbarcelona.org/) [18]. Protein functional sites were downloaded from dbPTM (http://dbptm.mbc.nctu.edu.tw/) [24], PhosphoSitePlus (https://www.phosphosite.org/homeAction.action) [25], Phospho.ELM (http://phospho.elm.eu.org/) [26], PTMD (http://ptmd.biocuckoo.org/) [27], and BioLiP (https://zhanglab.ccmb.med.umich.edu/BioLiP/) [28]. Somatic mutation and cancer patient information was downloaded from TCGA GDC Data Portal (https://portal.gdc.cancer.gov/). Mutation scores were retrieved from https://cadd.gs.washington.edu/ [30] and http://www.mutfunc.com/ [31]. Drug response data were retrieved from GDSC (http://www.cancerrxgene.org/) [32].


                                                    Diversity of metabolism: pathways in plants and bacteria

                                                    Pathway Organisms
                                                    photosynthesis plants and cyanobacteria
                                                    nitrogen fixation specialized soil bacteria
                                                    oxidation or reduction of inorganic minerals archaebacteria
                                                    acid- and gas-producing fermentations anaerobic bacteria

                                                    While the scope of this text is mostly restricted to human metabolism, it is useful to take a brief look beyond these confines. There are several mainstream metabolic pathways that occur in all classes of living organisms. A good example is glycolysis, the main pathway of glucose degradation, which is found all the way up from Escherichia coli to Homo sapiens . On the other hand, some of the metabolic processes in plants or in distinct groups of microbes are quite different from those found in man or animals.

                                                    Photosynthesis enables plants to create glucose—and from it, the carbon skeletons of all their other metabolites—from nothing but CO2 and water. The same is true of blue-green algae or cyanobacteria. 1 Organisms incapable of photosynthesis are heterotrophic , which means that they must feed on other organisms. In contrast, photosynthesis makes organisms autotrophic , that is, capable of feeding themselves. 2 Note, however, that plant life also depends on pathways other than photosynthesis. An example is the degradation of starch, which is stored in large amounts in plant seeds such as wheat and rice as well as in bulbs such as potatoes. The pathways of starch utilization employed by plants are analogous to those found in animals.

                                                    Nitrogen fixation, that is, the reduction of atmospheric nitrogen ( N2 ) to ammonia ( NH3 ), is performed by the bacterium Sinorhizobium meliloti and related soil bacteria. All other living organisms require nitrogen in already reduced form, and therefore depend on these bacteria. The word Rhizobium —the former first name of this bacterium—means “living on roots”. Sinorhizobium meliloti thrives on the roots of plants, which take up the surplus ammonia supplied by the bacteria and utilize it for synthesizing their own amino acids the plants, in turn, provide the bacteria with a nutrient-rich environment. This symbiotic process is common with legumes such as alfalfa, soy beans, and peas. Including these plants in crop rotation schemes helps to keep the soil supplied with reduced nitrogen. Alternatively, the nitrogen fixation bottleneck can be bypassed altogether by supplying reduced nitrogen with chemical fertilizers. 3

                                                    Some archaebacteria , which live in exotic environments such as submarine volcanic hot springs, have developed correspondingly exotic metabolic pathways. For example, some of these organisms are capable of extracting energy from the oxidation of iron or the reduction of sulfur.

                                                    While all these pathways are certainly very interesting, we will not consider them any further in these notes. Instead, we will confine the discussion to the major metabolic pathways that occur in the human body. We will also relate these pathways to human health and disease, and to some of the therapeutic strategies that have been developed for metabolic diseases.

                                                    In the remainder of this chapter, we will start with a broad overview of foodstuffs and their digestion and uptake in the intestinal organs. This will provide some important context for the detailed discussion of metabolic pathways in the subsequent chapters.


                                                    Pharmacogenomics and Precision Medicine

                                                    Pharmacodynamic Pharmacogenes

                                                    G6PD—Rasburicase—G6PD is necessary to help protect red blood cells from oxidative stress and prevent excessive formation of methemoglobin. 55 Increased oxidative stress can lead to hemolytic anemia, resulting in cardiovascular, renal, liver, and other organ system complications. 55–57 Additionally, red blood cell hemolysis in G6PD deficiency can lead to methemoglobinemia. 58 In this setting, neither oxygen nor carbon dioxide is carried as iron in hemoglobin is in the ferric state rather than the ferrous state. 57,58 Methemoglobinemia may result in death in severe cases. 57–59 There are well over 150 G6PD variants and, unlike the previous examples, G6PD variants are not designated using the star “∗” nomenclature. Individuals with a normal phenotype are not at increased risk of hemolytic anemia, whereas those with a deficient phenotype of any kind are at risk of acute hemolytic anemia. A “variable” phenotype may be identified in some individuals and requires further testing to ascertain risk. 60

                                                    Numerous drugs and chemicals can elicit complications in patients with G6PD deficiency due to a G6PD variant or variants. 60 Rasburicase is indicated for use in managing plasma uric acid concentrations in patients with hematologic or solid tumor cancers who are receiving drugs that can cause lysis of cells. 61 Tumor lysis can result in elevated plasma uric acid concentrations, which can cause acute kidney injury. 61 Rasburicase administration in the presence of G6PD variants can result in hemolytic anemia and methemoglobinemia. Rasburicase is contraindicated in persons with a phenotype of “deficient” (of any kind). There are many potential diplotype combinations, which can result in the various phenotype designations. Table 31.8 presents examples of the rasburicase–G6PD interaction. Additionally, tables related to drugs to be avoided or monitored in patients who are G6PD deficient are provided in the literature. 60

                                                    Table 31.8 . Example G6PD Gene Diplotypes, Related Phenotype, and Potential Rasburicase Therapeutic Consequence

                                                    Example DiplotypePhenotypeRasburicase Therapeutic Consequence
                                                    B/Sao BoriaNormalUtilize drug as indicated
                                                    Chatham/ChathamDeficientContraindication to the use of the drug—consider alternative
                                                    Villeurbanne/VilleurbanneDeficientContraindication to the use of the drug—consider alternative

                                                    VKORC1—Warfarin—There are a number of genes that have been found to affect warfarin dosage requirements. These include variants of CYP2C9, and CYP4F2 and VKORC1, the former of which is a PK-related pharmacogene and the latter two of which are PD-related pharmacogenes. The CYP2C9 variants, ∗2 and ∗3 are the most common, result in decreased clearance of warfarin, potentially leading to decreased dose requirements. 62 Here, considering the vitamin K epoxide reductase complex subunit 1 gene (VKORC1), individuals with adenine (A) replacing guanine (G) at c.-1639 (c.-1639G>A rs9923231) are more sensitive to warfarin. According to the product package labeling, individuals who are homozygous for the A allele, regardless of CYP2C9 diplotype, have a reduced warfarin dose requirement. 63 Dosing algorithms can be applied in this case, along with other information, including genetics to calculate the appropriate dose. 64–66 Table 31.9 shows the impact of both VKORC1 and CYP2C9 on initial warfarin dose (mg/day) recommendations. Algorithms can refine initial dosing and have been recommended for various populations. 64,67

                                                    Table 31.9 . VKORC1 and CYP2C9 Genotypes and Diplotypes, Related to Predicted Daily Warfarin Dose Requirements

                                                    Numerous other drug–gene interactions have been identified beyond the examples here. Groups such as CPIC and the DPWG continue to develop and publish evidence-based guidelines that provide clinicians with clear ways forward when considering PGx as a component of clinical decision-making. Pharmacists continue to lead the way in the application of PGx across practice settings.