Medical Physiology, 3rd Edition

Energy Interconversion From Cycling between 6-Carbon and 3-Carbon Molecules

Some metabolic reactions are neither uniquely anabolic nor catabolic, but they serve to interconvert the carbon skeletons of the building blocks of the three major energy forms—carbohydrates, proteins, and lipids. In this subchapter, we focus on two major pathways of interconversion: glycolysis and gluconeogenesis.

Glycolysis converts the 6-carbon glucose molecule to two 3-carbon pyruvate molecules

The breakdown of glucose to pyruvate (Fig. 58-6A) can occur in the presence of O2 (aerobic glycolysis) or the absence of O2 (anaerobic glycolysis). This process yields 47 kcal of free energy per mole of glucose. Of this energy, the cell can trap enough to yield directly 2 moles of ATP per mole of glucose (Table 58-3), even under the relatively inefficient anaerobic conditions. Under aerobic conditions, the mitochondria can generate an additional three or five ATP molecules per glucose molecule from two reduced nicotinamide adenine dinucleotide (NADH) molecules. imageN58-5 Cells that contain few mitochondria (e.g., fast-twitch muscle fibers; see pp. 249–250) or no mitochondria (i.e., erythrocytes imageN58-6) rely exclusively on anaerobic glycolysis for energy.

image

FIGURE 58-6 Glycolysis and gluconeogenesis. A, Highlighted in green are the three enzymes that catalyze reactions in glycolysis that are essentially irreversible. These enzymes are subject to allosteric regulation. B, Highlighted in blue are the three gluconeogenic bypasses, which circumvent the three irreversible steps of glycolysis. The precursors for gluconeogenesis include amino acids (illustrated here for alanine), pyruvate, and lactate. Green arrow indicates stimulation. NAD+/NADH, oxidized and reduced forms of nicotinamide adenine dinucleotide, respectively.

TABLE 58-3

Generation of ATP from Glycolysis*

REACTION

ATP CHANGE PER GLUCOSE

Glucose → G6P

−1

Fructose-6-phosphate → Fructose-1,6-bisphosphate

−1

2 × (1,3-Bisphosphoglycerate) → 2 × (3-Phosphoglycerate)

+2

2 × Phosphoenolpyruvate → 2 × Pyruvate

+2

 

Net +2

*Under anaerobic conditions, glycolysis—which takes place in the cytosol—yields two lactate molecules plus two ATP molecules per glucose molecule. Under aerobic conditions, the metabolism of glucose does not proceed to lactate and thus yields a net gain of two NADH molecules per glucose molecule. The oxidative phosphorylation of each NADH molecule will yield 1.5 or 2.5 ATP molecules, depending on which shuttle system the cell uses to transfer the reducing equivalents from the cytosol into the mitochondria. imageN58-15 Therefore, under aerobic conditions, glycolysis of one glucose molecule generates two ATP molecules directly plus three or five ATP molecules via the oxidative phosphorylation of the two NADH molecules, for a total of five or seven ATP molecules per glucose molecule. This yield is summarized in the first two rows of Table 58-4.

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NADH/NAD+ versus NADPH/NADP+

Contributed by Alisha Bouzaher

NADH and NAD+ are, respectively, the reduced and oxidized forms of nicotinamide adenine dinucleotide (NAD) and their close analogs are NADPH and NADP+, the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate (NADP). The coenzymes NADH and NADPH each consist of two nucleotides joined at their phosphate groups by a phosphoanhydride bond. NADPH is structurally distinguishable from NADH by the additional phosphate group residing on the ribose ring of the nucleotide, which allows enzymes to preferentially interact with either molecule.

Total concentrations of NAD+/NADH (10−5M) are higher in the cell by ~10-fold compared to NADP+/NADPH (10−6M). Ratios of the oxidized and reduced forms of these coenzymes offer perspective into the metabolic activity of the cell. The high NAD+/NADH ratio favors the transfer of a hydride from a substrate to NAD+ to form NADH, the reduced form of the molecule and oxidizing agent. Therefore, NAD+ is highly prevalent within catabolic reaction pathways where reducing equivalents (carbohydrate, fats, and proteins) transfer protons and electrons to NAD+. NADH acts as an energy carrier, transferring electrons from one reaction to another. Conversely, the NADP+/NADPH ratio is low, favoring the transfer of a hydride to a substrate oxidizing NADPH to NADP+. Thus, NADPH is utilized as a reducing agent within anabolic reactions, particularly the biosynthesis of fatty acids.

References

Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 6th ed. WH Freeman: New York; 2012.

Wikipedia. s.v. Nicotinamide adenine dinucleotide. [Last modified May 8, 2015]  http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide [Accessed May 15, 2015].

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Red Blood Cells

Contributed by Emile Boulpaep, Walter Boron

As noted on pages 434–435 of the text, mature erythrocytes lack all organelles, including mitochondria. Of the glucose that they take up, 90% undergoes glycolysis to provide ATP. The remaining 10% follows the pentose phosphate pathway (see Fig. 58-1)—also known as the hexose monophosphate shunt or the phosphogluconate pathway—to produce NADPH (i.e., reducing equivalents) as well as 5-carbon sugars.

About a century and a half ago, Pasteur recognized that glycolysis by yeast occurs faster in anaerobic conditions than in aerobic conditions. imageN58-7 This Pasteur effect reflects the cell's attempt to maintain a constant [ATP]i by controlling the rate at which glycolysis breaks down glucose to generate ATP. The key is the allosteric regulation of enzymes that catalyze the three reactions in the glycolytic pathway that are essentially irreversible: hexokinase (or glucokinase in liver and pancreas), phosphofructokinase (PFK), and pyruvate kinase (highlighted in green in Fig. 58-6A). In each case, either the direct reaction product (i.e., G6P in the case of hexokinase) or downstream metabolic products (e.g., ATP in the case of the other two) inhibits the enzyme. If glycolysis should temporarily outstrip the cell's need for ATP, the buildup of products slows glycolysis. Thus, introducing O2 activates the citric acid cycle (see p. 1185), which raises [ATP]i, inhibits PFK and pyruvate kinase, and slows glycolysis.

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Allosteric Regulation of Glycolytic Enzymes by Metabolic Products (Pasteur Effect)

Contributed by Ethan Nadel, Emile Boulpaep, Walter Boron

As pointed out on page 1176, the three enzymes of the glycolytic pathway that catalyze reactions that are essentially irreversible are also all under all allosteric control.

Hexokinase in muscle (the comparable enzyme in liver is glucokinase) catalyzes the essentially irreversible conversion of free glucose to G6P (see Fig. 58-6A) and thereby traps the sugar inside the cell and enables it to enter the glycolytic pathway. Hexokinase is inhibited by increased concentrations of its reaction product, G6P. Thus, when the concentration of G6P increases excessively, hexokinase is temporarily inhibited (negative feedback), which reduces the rate of G6P production and enables it to match once again the rate of consumption in the steady state.

The enzyme phosphofructokinase (PFK) catalyzes the essentially irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate (see Fig. 58-6A). The activity of PFK is allosterically regulated by the cellular energy status, increasing with decreases in [ATP]i (or with increases in ATP breakdown products), and vice versa. Further, an increased concentration of citrate—which indicates that pyruvate is being provided to the citric acid cycle faster than it can be used—increases the inhibitory influence of ATP on PFK, attenuating the rate of glycolysis further and thus signaling that the cell's energy needs are being met.

Pyruvate kinase catalyzes the final step of glycolysis, the essentially irreversible transfer of the phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding ATP and pyruvate (see Fig. 58-6A). A high [ATP]i decreases the affinity of pyruvate kinase for PEP, reducing the rate of reaction at the [PEP]i normally prevailing. Pyruvate kinase is also inhibited by acetyl CoA, which is the entry molecule to the citric acid cycle, a source of high ATP production.

The allosteric inhibitors (indicated above in bold italics) of the aforementioned three enzymes are responsible for the observation by Pasteur (the so-called Pasteur effect) that glycolysis is slower under aerobic conditions, when these allosteric inhibitors are present at higher concentrations.

Under anaerobic conditions, cells convert pyruvate to lactate, which is accompanied by the accumulation of H+lactic acidosis. This acidosis, in turn, can impede muscle contraction by decreasing muscle cell pH, which can result in muscle cramps and inhibition of key glycolytic enzymes needed for ATP synthesis. Thus, sustained skeletal muscle activity depends on the aerobic metabolism of pyruvate as well as FAs.

Gluconeogenesis converts nonhexose precursors to the 6-carbon glucose molecule

Gluconeogenesis is essential for life because the brain and anaerobic tissues—formed elements of blood (erythrocytes, leukocytes), bone marrow, and the renal medulla—normally depend on glucose as the primary fuel source. The daily glucose requirement of the brain in an adult is ~120 g, which accounts for most of the 180 g of glucose produced by the liver. The major site for gluconeogenesis is the liver, with the renal cortex making a much smaller contribution. During prolonged fasting (2 to 3 months), the kidney can account for up to 40% of total glucose production.

Although glycolysis converts glucose to pyruvate (see Fig. 58-6A) and gluconeogenesis converts pyruvate to glucose (see Fig. 58-6B), gluconeogenesis is not simply glycolysis in reverse. The thermodynamic equilibrium of glycolysis lies strongly on the side of pyruvate formation (i.e., the ΔG is very negative). Thus, in contrast to glycolysis, gluconeogenesis requires energy, consuming four ATP, two GTP, and two NADH molecules for every glucose molecule formed. Most of the ΔG decrease in glycolysis occurs in the three essentially irreversible steps indicated by single arrows in Figure 58-6A. Gluconeogenesis bypasses these three irreversible, high-ΔG glycolytic reactions by using four enzymes: pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and G6Pase (see Fig. 58-6B). The enzymes of gluconeogenesis and glycolysis are present in separate cellular compartments to minimize futile cycling of substrates between glycolysis and gluconeogenesis; the glycolytic enzymes reside in the cytosolic compartment, whereas the gluconeogenic enzymes are present in the mitochondria (pyruvate carboxylase) or in the lumen of the endoplasmic reticulum (G6P).

The liver accomplishes gluconeogenesis by taking up, and converting to glucose, several nonhexose precursors (see Fig. 58-6B). These include two breakdown products of glycolysis (lactate and pyruvate), all the intermediates of the citric acid cycle, 18 of the 20 amino acids, and glycerol. Regardless of the precursor—except for glycerol—all pathways go through oxaloacetate (OA). Thus, the liver can convert lactate to pyruvate, and then convert pyruvate to OA via pyruvate carboxylase, consuming one ATP. Similarly, the citric acid cycle can convert all its intermediates to OA. Finally, the liver can deaminate all amino acids—except leucine and lysine—to form pyruvate, OA, or three other intermediates of the citric acid cycle (α-ketoglutarate, succinyl coenzyme A [CoA], or fumarate). The major gluconeogenic amino acids are alanine and glutamine.imageN58-8 Leucine and lysine are not gluconeogenic because their deamination leads to acetyl CoA, which cannot generate net OA (see p. 1185). Similarly, FAs are not gluconeogenic because their breakdown products are almost exclusively acetyl CoA. In contrast, leucine and lysine are ketogenic because cells can convert acetyl CoA to FAs (Fig. 58-7) or ketone bodies (see p. 1185).

image

FIGURE 58-7 FA synthesis. The left side of the figure shows how the cell effectively transports acetyl CoA from the inside of the mitochondrion to the cytoplasm—exporting citrate and then taking up pyruvate. The key enzyme is citrate lyase. The right side of the figure shows how the cell generates FAs, two carbons at a time. Glucose can feed into the system via pyruvate. Amino acids can feed into the system via acetyl CoA (ketogenic amino acids), pyruvate, or intermediates of the citric acid cycle. NAD+/NADH, oxidized and reduced forms of nicotinamide adenine dinucleotide, respectively; NADP+/NADPH, oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate, respectively. imageN58-5

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Gluconeogenic and Ketogenic Amino Acids

Contributed by Emile Boulpaep, Walter Boron

image

Once the liver has converted the precursor to OA, the next step is the conversion of OA to phosphoenolpyruvate (PEP) by PEPCK, which consumes one GTP molecule (see Fig. 58-6B). The liver can convert PEP to fructose-1,6-bisphosphate (F-1,6-BP) by using the glycolytic enzymes in reverse. The gluconeogenic precursor glycerol enters the pathway at dihydroxyacetone phosphate. FBPase converts F-1,6-BP to fructose-6-phosphate, and G6Pase completes gluconeogenesis by converting G6P to glucose.

The major gluconeogenic precursors are (1) lactate, which is derived from glycolysis in muscle and anaerobic tissues; (2) alanine, which is mostly derived from glycolysis and transamination of pyruvate in skeletal muscle; and (3) glycerol, which is derived from lipolysis in adipocytes.

Reciprocal regulation of glycolysis and gluconeogenesis minimizes futile cycling

We already noted that key glycolytic and gluconeogenic enzymes are located in separate compartments. The liver also reciprocally and coordinately regulates these processes so that when one pathway is active the other pathway is relatively inactive. This regulation is important because both glycolysis and gluconeogenesis are highly exergonic, and therefore no thermodynamic barrier prevents futile cycling of substrates between these two pathways. Because glycolysis creates two ATP molecules and gluconeogenesis consumes four ATP and two GTP molecules, a full cycle from one glucose to two pyruvates and back again would have a net cost of two ATP and two GTP molecules. The liver regulates flux through these pathways in the short term mostly by allosteric regulation of enzyme activity, and in the long term by transcriptional regulation of gene expression.

Allosteric Regulation

PFK (glycolysis) is stimulated by AMP, whereas it is inhibited by citrate and ATP (see Fig. 58-6A). FBPase (gluconeogenesis) is inhibited by AMP and is activated by citrate (see Fig. 58-6B). In addition, fructose-2,6-bisphosphate (F-2,6-BP), imageN58-9 which is under the reciprocal control of glucagon and insulin, reciprocally regulates these two enzymes, stimulating PFK and inhibiting FBPase. In the fed state, when the glucagon level is low (see pp. 1050–1051) and the insulin level is high (see p. 1041), [F-2,6-BP] is high, which promotes consumption of glucose. Conversely, in the fasted state, [F-2,6-BP] is low, which promotes gluconeogenesis.

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Fructose-2,6-Bisphosphate

Contributed by Emile Boulpaep, Walter Boron

Although structurally very similar to fructose-1,6-bisphosphate, fructose-2,6-bisphosphate (F-2,6-BP) is neither a substrate nor a product in either glycolysis or gluconeogenesis. However, it is an allosteric regulator of both glycogenolysis and gluconeogenesis.

The enzyme phosphofructokinase 2 (PFK-2) phosphorylates fructose-6-phosphate to yield F-2,6-BP. Conversely, the enzyme fructose-2,6,-bisphosphatase 2 (FBPase-2) removes one phosphate from F-2,6-BP to yield fructose-6-phosphate. Interestingly, a single bifunctional protein mediates both the PFK-2 and FBPase-2 activities. These two enzymes are distinct from phosphofructokinase 1 (PFK in Fig. 58-6A) and fructose-1,6-bisphosphatase (FBPase-1; see Fig. 58-6B).

Fructose-6-phosphate is an important allosteric regulator of both glycolysis and gluconeogenesis. By modulating the two enzymes noted above, insulin raises but glucagon lowers cytosolic [F-2,6-BP]. Thus, in the fed state (high insulin/low glucagon), [F-2,6-BP] rises, thereby stimulating glycolysis but inhibiting gluconeogenesis.

Reference

Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 3rd ed. Worth Publishers: New York; 2000 [pp; 732; 733].

Similarly, the liver reciprocally regulates pyruvate kinase (glycolysis) and pyruvate carboxylase/PEPCK (gluconeogenesis). High concentrations of ATP and alanine inhibit pyruvate kinase, whereas ADP inhibits pyruvate carboxylase. Furthermore, acetyl CoA inhibits pyruvate kinase but activates pyruvate carboxylase.

In this way, high concentrations of biosynthetic precursors and ATP favor gluconeogenesis and suppress glycolysis. Conversely, high concentrations of AMP, reflecting a low energy charge of the liver, suppress gluconeogenesis and favor glycolysis.

Transcriptional Regulation

More long-term regulation of gluconeogenesis and glycolysis occurs by hormonal regulation of gene expression. The major hormones involved in this process are insulin, glucagon, epinephrine, and cortisol. In contrast to allosteric regulation, which occurs in seconds to minutes, transcriptional regulation occurs over hours to days. Insulin, which increases following a meal (see p. 1041), stimulates the expression of the glycolytic enzymes PFK and pyruvate kinase, as well as the enzyme that makes F-2,6-BP. imageN58-9 In addition, as noted in Figure 51-8, insulin suppresses the expression of the key gluconeogenic enzymes PEPCK, FBPase, and G6Pase. Insulin leads to the phosphorylation of the FOXO1 transcription factor, which prevents FOXO1 from entering the nucleus and activating transcription of genes that encode these enzymes.

Conversely, glucagon, the levels of which increase during starvation, inhibits the expression of the glycolytic enzymes PFK and pyruvate kinase, as well as the enzyme that makes F-2,6-BP. Epinephrine and norepinephrine, released under conditions of stress, have actions similar to those of glucagon. At the same time, these hormones stimulate the expression of the gluconeogenic enzymes PEPCK and G6Pase via cAMP and protein kinase A (PKA). Phosphorylation of the transcriptional factor CREB (cAMP response element–binding protein; see p. 89) directly promotes both increased transcription of gluconeogenic genes (e.g., PEPCK) and increased transcription of the transcriptional cofactor PGC-1α (peroxisome proliferator–activated receptor-γ coactivator-1α). PGC-1α then binds and activates the transcription factors HNF4 and FOXO1, which further promote the transcription of these key gluconeogenic enzymes.

Cells can convert glucose or amino acids into FAs

The body—principally the liver—can convert glucose to FAs. As shown in Figure 58-6A, glycolysis converts glucose to pyruvate, which can enter the mitochondrion via the mitochondrial pyruvate carrier, or MPC (see Fig. 58-7). When ATP demand is low, high levels of ATP, acetyl CoA, and NADH inside the mitochondria inhibit pyruvate dehydrogenase, which converts pyruvate to acetyl CoA—the normal entry point into the citric acid cycle (see p. 1185). Conversely, high levels of ATP and acetyl CoA stimulate pyruvate carboxylase, which instead converts pyruvate to OA, the last element in the citric acid cycle. The mitochondrion then converts the OA and acetyl CoA to citrate, which it exports to the cytosol via an exchanger called the citrate carrier, or CIC (SLC25A1). A cytosolic enzyme called citrate lyase converts the citrate back to OA and acetyl CoA. The hepatocyte converts the cytosolic OA to malate or pyruvate, each of which can re-enter the mitochondrion. Thus, the net effect is to make acetyl CoA disappear from the mitochondrion and appear in the cytosol for FA synthesis.

As noted above, the breakdown of ketogenic amino acids imageN58-8 yields acetyl CoA (see p. 1176). This acetyl CoA can also contribute to FA synthesis. In addition, the breakdown of other amino acids yields pyruvate or intermediates of the citric acid cycle, which again can contribute to FA synthesis.

The synthesis of FAs from acetyl CoA takes place in the cytosol, whereas the oxidation of FAs to acetyl CoA occurs in the mitochondrion (see below, pp. 1183–1185). The first committed step—and the rate-limiting step—in FA synthesis is the ATP-dependent carboxylation of acetyl CoA (2 carbons) to form malonyl CoA (3 carbons), catalyzed by acetyl CoA carboxylase (ACC).imageN58-10 The next step is the sequential addition of 2-carbon units to a growing acyl chain, shown as –CO–(CH2)n–CH3 in Figure 58-7, to produce an FA. With each round of elongation, a malonyl CoA molecule reacts with FA synthase, which then decarboxylates the malonyl moiety and condenses the growing acyl chain to the remaining 2-carbon malonyl fragment. The subsequent reduction, dehydration, and reduction steps—all catalyzed by the same multifunctional peptide—complete one round of elongation. Starting from a priming acetyl group (which comes from acetyl CoA), seven rounds of addition—in addition to a hydrolysis step to remove the acyl chain from the enzyme—are required to produce palmitate:

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Acetyl CoA Carboxylase Isoforms

Contributed by Emile Boulpaep, Walter Boron

The two isoforms of ACC, encoded by different genes, are the following:

ACC1:

265 kDa

Cytosolic

Highly expressed in lipogenic tissues: liver, adipocytes, lung, mammary gland

Involved in FA synthesis

ACC2:

280 kDa

Has a unique 144-amino-acid sequence at the amino terminus; amino acids 1 to 20 may be a leader sequence

Membrane protein, associated with mitochondria, thought to face the cytosol

Expressed in heart, skeletal muscle more than liver

Regulates β-oxidation by producing malonyl CoA; carnitine acyltransferase I is the committed step

image

(58-3)

where NADPH and NADP+ are the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate, respectively. The cell esterifies the FAs to glycerol to make TAGs (see Fig. 58-3A). imageN58-11 The liver can package TAGs as very-low-density lipoproteins (VLDLs; see p. 968) for export to the blood.

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Digestion, Absorption, and Storage of Triacylglycerols

Contributed by Ethan Nadel

image

EFIGURE 58-1 A, Pancreatic lipase breaks down TAGs into FAs and 2-MAGs in the lumen of the small intestine. These products enter the enterocyte, which uses either the MAG or the phosphatidic acid pathway to re-esterify them. The resulting TAGs are packaged into chylomicrons, which enter the lymph and, ultimately, the blood. B, Lipoprotein lipase (LPL) in capillaries hydrolyzes TAGs in chylomicrons to FA and glycerol. Adipocytes re-esterify the FAs to TAGs for storage in fat droplets. Hepatocytes take up remnant chylomicrons (by receptor-mediated endocytosis), glycerol, and some FA.

The body permits only certain energy interconversions

The body has a hierarchy for energy interconversion. As discussed above, the body can convert amino acids to glucose (gluconeogenesis) and fat, glucose to fat, and glucose to certain amino acids. However, the body cannot convert fat to either glucose or amino acids. Fats can only be stored or oxidized. The reason is that cells oxidize FAs two carbons at a time to acetyl CoA (a 2-carbon molecule; see pp. 1183–1185), which they cannot convert into pyruvate (a 3-carbon molecule) or OA (a 4-carbon molecule). The only exceptions are the uncommon FAs that have an odd number of carbon atoms, and even with these, only the terminal 3-carbon unit escapes oxidation to acetyl CoA. Thus, almost all carbon atoms in FAs end up as acetyl CoA, which enters the citric acid cycle (see p. 1185). There, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase release the two carbon atoms of acetyl CoA as two CO2 molecules, which thus yields no net production of OA or pyruvate. In contrast, plants have two additional enzymes (the glyoxylate cycle) that allow them to convert two molecules of acetyl CoA to OA and glucose.