An adequate blood glucose level must be maintained at all times

Some cells and tissues, including brain and erythrocytes, depend on glucose because they cannot oxidize alternative fuels. The brain alone consumes nearly 120 g of glucose per day. Therefore the body maintains a blood glucose level of 4.0 to 5.5 mmol/L (70–100 mg/dl) at all times. Dietary carbohydrates provide glucose for a few hours after a meal. During these hours, the blood glucose concentration can rise as high as 8.5 mmol/L (150 mg/dl). In the fasting state, however, the liver has to produce glucose by two pathways:

Gluconeogenesis bypasses the three irreversible reactions of glycolysis

The easiest strategy for glucose synthesis would be to reverse glycolysis by making glucose from pyruvate and lactate. To do so, however, the gluconeogenic pathway has to bypass the three irreversible reactions of glycolysis: those catalyzed by hexokinase, phosphofructokinase (PFK), and pyruvate kinase (Fig. 22.1).

Figure 22.1 Reactions of glycolysis and gluconeogenesis. NAD, Nicotinamide adenine dinucleotide; PEP, phosphoenolpyruvate; P_i, inorganic phosphate.

The pyruvate kinase reaction of glycolysis is irreversible despite ATP synthesis. Reversing this reaction by going back from pyruvate to phosphoenolpyruvate (PEP) requires 14.8 kcal/mol, or at least two high-energy phosphate bonds.

This feat is accomplished in a sequence of two reactions (Fig. 22.2). First, pyruvate is carboxylated to oxaloacetate by pyruvate carboxylase. This ATP-dependent carboxylation reaction was described as an anaplerotic reaction of the tricarboxylic acid (TCA) cycle in Chapter 21. The second reaction, catalyzed by PEP-carboxykinase, converts oxaloacetate into PEP. It requires GTP, which supplies the phosphate group in PEP. The two reactions combined have a standard free energy change (ΔG^0′) of +0.2 kcal/mol, but in the cell they proceed only from pyruvate to PEP because of the high cellular concentration ratios of [ATP]/[ADP], [GTP]/[GDP], and [pyruvate]/[PEP].

Figure 22.2 First bypass of gluconeogenesis, from pyruvate to phosphoenolpyruvate (PEP).

Whereas pyruvate carboxylase is strictly mitochondrial, PEP-carboxykinase is both mitochondrial and cytoplasmic. PEP is transported across the inner mitochondrial membrane, whereas oxaloacetate is shuttled into the cytoplasm after being reduced to malate or transaminated to aspartate (see Fig. 21.21).

The remaining irreversible reactions of glycolysis, catalyzed by PFK and hexokinase, are bypassed by the hydrolytic removal of phosphate. Fructose-1,6-bisphosphatase hydrolyzes the phosphate from carbon 1 of fructose-1,6-bisphosphate (Fig. 22.3), and glucose-6-phosphatase removes the phosphate from glucose-6-phosphate. Both reactions are irreversible. Unlike the other gluconeogenic enzymes, which are cytoplasmic (except pyruvate carboxylase), glucose-6-phosphatase resides on the inner surface of the endoplasmic reticulum membrane.

Figure 22.3 Second and third bypasses of gluconeogenesis.

Gluconeogenesis requires six phosphoanhydride bonds for the synthesis of one glucose molecule from two molecules of pyruvate or lactate. Pyruvate carboxylase consumes two ATP molecules, PEP-carboxykinase consumes two GTP molecules, and phosphoglycerate kinase consumes two ATP molecules in the reversal of substrate-level phosphorylation.

Fatty acids cannot be converted into glucose

Lactate and alanine are convenient substrates of gluconeogenesis because they are readily converted to pyruvate by lactate dehydrogenase and by transamination, respectively (Fig. 22.4). Oxaloacetate is not only a gluconeogenic intermediate but also a member of the TCA cycle. This is important because most amino acids are degraded to TCA cycle intermediates. Through the TCA cycle, these “glucogenic” amino acids feed into gluconeogenesis.

Figure 22.4 Most important substrates of gluconeogenesis. Although acetyl-coenzyme A enters the tricarboxylic acid (TCA) cycle, it is not a substrate of gluconeogenesis because the citrate synthase reaction does not involve the net synthesis of a TCA cycle intermediate.

Glycerol is another substrate of gluconeogenesis. It enters the pathway at the level of the triose phosphates (Fig. 22.5).

Figure 22.5 Glycerol enters gluconeogenesis (and glycolysis) at the level of the triose phosphates. The glycerol for gluconeogenesis in the liver is derived from triglyceride hydrolysis in adipose tissue. RBC, Red blood cell.

Acetyl-coenzyme A (acetyl–CoA) cannot be converted to glucose. The pyruvate dehydrogenase reaction is irreversible, and there are no alternative reactions to channel acetyl-CoA into gluconeogenesis. Fatty acids are degraded to acetyl-CoA. Therefore the fatty acids that are released from adipose tissue during fasting cannot be turned into glucose. Gluconeogenesis depends on amino acids and, to a lesser extent, on lactic acid and glycerol.

Glycolysis and gluconeogenesis are regulated by hormones

Simultaneous activity of glycolysis and gluconeogenesis would achieve nothing but ATP hydrolysis. To minimize such a futile cycle, it is mandatory to control the irreversible reactions at all three bypasses to ensure that only the glycolytic reactions or only the gluconeogenic reactions take place, but not both.

The following hormones participate in the control of hepatic glycolysis and gluconeogenesis:

The hormones regulate the synthesis of the distinctive glycolytic and gluconeogenic enzymes at the level of transcription (Fig. 22.6, A). Because this involves the synthesis of new enzyme protein and most of the enzymes have lifespans of a few days in the cell, regulation of enzyme synthesis works on a time scale of days rather than minutes.

Figure 22.6 Reciprocal regulation of glycolysis and gluconeogenesis in the liver. , Glucokinase; , phosphofructokinase; , pyruvate kinase; , pyruvate carboxylase; , phosphoenolpyruvate (PEP)-carboxykinase; , fructose-1,6-bisphosphatase; , glucose-6-phosphatase. , Stimulation; , inhibition. A, Substrate flow during fasting and in the well-fed state, and the effects of hormones on the amounts of glycolytic and gluconeogenic enzymes. Regulation of enzyme synthesis and degradation is the most important long-term (hours to days) control mechanism. In most cases, the hormone acts by changing the rate of transcription or by affecting the stability of the messenger RNA. Some of the insulin effects shown here require the presence of glucose. B, Short-term regulation of glycolysis and gluconeogenesis by reversibly binding effectors and by phosphorylation/dephosphorylation. , Allosteric and competitive effects; , phosphorylation. Only pyruvate kinase and phosphofructo-2-kinase/fructose-2,6-bisphosphatase are regulated by cyclic AMP (cAMP)-dependent phosphorylation. CoA, Coenzyme A.

Glycolysis and gluconeogenesis are fine tuned by allosteric effectors and hormone-induced enzyme phosphorylations

The short-term control of glycolysis and gluconeogenesis is shown in Figure 22.6, B.

The glycolytic enzyme pyruvate kinase is the most important regulated enzyme in the PEP-pyruvate cycle. It is allosterically inhibited by ATP and alanine and activated by fructose-1,6-bisphosphate. The concentration of fructose-1,6-bisphosphate is high when PFK is activated and fructose-1,6-bisphosphatase is inhibited. Its effect on pyruvate kinase is an example of feedforwardstimulation. Pyruvate kinase is also inhibited by cAMP-induced phosphorylation.

PEP-carboxykinase is not known to be subject to short-term regulation, but pyruvate carboxylase is allosterically activated by acetyl-CoA and competitively inhibited by ADP. This ensures that gluconeogenesis is launched only when sufficient metabolic energy is available.

ATP and citrate stimulate fructose-1-6-bisphosphatase but inhibit PFK. Therefore high energy charge and the availability of metabolites favor gluconeogenesis over glycolysis. The most potent modulator of these two enzymes, however, is fructose-2,6-bisphosphate. This regulatory metabolite, not to be confused with the glycolytic intermediate fructose-1,6-bisphosphate, is an activator of PFK and an inhibitor of fructose-1,6-bisphosphatase.

Fructose-2,6-bisphosphate is both synthesized from and degraded to fructose-6-phosphate by a unique bifunctional enzyme that combines the activities of a 6-phosphofructo-2-kinase (PFK-2) and a fructose-2,6-bisphosphatase on the same polypeptide.

This bifunctional enzyme is phosphorylated by the cAMP-activated protein kinase A in response to glucagon and is dephosphorylated in the presence of insulin. The dephosphorylated enzyme acts as a kinase that makes fructose-2,6-bisphosphate, whereas the phosphorylated form acts as a phosphatase that breaks it down (Fig. 22.7). Therefore the level of fructose-2,6-bisphosphate in the liver is high when insulin is high and glucagon is low, and the level of fructose-2,6-bisphosphate is low when insulin is low and glucagon is high. As a consequence, glycolysis is turned on when insulin is high, and gluconeogenesis is turned on when glucagon is high.

Figure 22.7 Synthesis and degradation of fructose-2,6-bisphosphate, the most important regulator of phosphofructokinase and fructose-1,6-bisphosphatase. This regulatory metabolite is synthesized and degraded by a bifunctional enzyme that combines the kinase and phosphatase activities on the same polypeptide. Cyclic AMP (cAMP)-induced phosphorylation inhibits the kinase activity and stimulates the phosphatase activity of the bifunctional enzyme. , Phosphorylation; , dephosphorylation; , allosteric effect; , stimulation; , inhibition.

Through fructose-2,6-bisphosphate, insulin and glucagon regulate glycolysis and gluconeogenesis on a minute-to-minute time scale. In addition to this hormonal control, fructose-6-phosphate stimulates the kinase activity and inhibits the phosphatase activity of the bifunctional enzyme by an allosteric mechanism.

The glucose-phosphorylating enzyme in the liver is isoenzyme 4 of hexokinase, better known as glucokinase. The most important kinetic difference between glucokinase and the other isoenzymes of hexokinase is the Michaelis constant (K_m) for glucose. Whereas the other forms of hexokinase have K_m values near 0.1 mmol/L (2 mg/dl), glucokinase has a K_m near 10 mmol/L (200 mg/dl). Glucokinase also shows a sigmoidal rather than hyperbolic relationship between glucose concentration and reaction rate (Fig. 22.8).

Figure 22.8 Approximate reaction rates of hexokinase (isoenzymes 1, 2, and 3) and glucokinase at different substrate concentrations. The sigmoidal relationship between reaction rate and substrate concentration for glucokinase accentuates the increase of the reaction rate with increased glucose level. V_max, Maximal reaction rate.

Because the steep part of the curve is in the range of physiological glucose concentrations, the reaction rate rises substantially with rising glucose level. Glucose rapidly equilibrates across the hepatocyte membrane with the help of the GLUT2 transporter; therefore, the intracellular glucose concentration rises in parallel with the blood glucose concentration after a carbohydrate-rich meal.

Glucokinase is inhibited by the CoA thioesters of long-chain fatty acids. These products are most abundant during fasting, when the liver metabolizes large amounts of fatty acids from adipose tissue. Glucokinase is also regulated by a glucokinase regulatory protein that inhibits its activity and sequesters it in the nucleus in the presence of fructose-6-phosphate. Glucose and fructose-1-phosphate (a product of fructose metabolism) activate glucokinase by binding to this regulatory protein.

Like glucokinase, glucose-6-phosphatase is affected by substrate availability. With a K_m of 3 mmol/L for glucose-6-phosphate, it is not saturated under ordinary conditions.

The stimulation of gluconeogenesis by high energy charge and high concentrations of citrate and acetyl-CoA is counterintuitive. Gluconeogenesis is active in the fasting state. Why would the levels of ATP and metabolites be increased rather than decreased in a starving organism?

The reason is that gluconeogenesis takes place mainly in the liver, and the liver receives large quantities of fatty acids from adipose tissue during fasting. Fatty acid oxidation is less tightly controlled by feedback inhibition than is glucose oxidation, and the levels of ATP and acetyl-CoA in the liver actually are elevated during fasting. Thus the energy for gluconeogenesis is supplied by fatty acid oxidation.

Carbohydrate is stored as glycogen

Glycogen granules are seen in many cell types, but the most important stores are in liver and skeletal muscle. In the well-fed state, the glycogen content of the liver is up to 8% of the fresh weight: 100 to 120 g in the adult. The glycogen concentration in skeletal muscle is 1% or a bit less, but because most people have more muscle than liver, the total amount of muscle glycogen exceeds that in the liver.

Glycogen is a branched polymer of between 10,000 and 40,000 glucose residues held together by α-1,4 glycosidic bonds. Approximately one in 12 glucose residues serves as a branch point by forming an α-1,6 glycosidic bond with another glucose residue (Fig. 22.9). With a molecular weight between 10⁶ and 10⁷ D, it is as big as a complete human ribosome (4.2 × 10⁶ D).

Figure 22.9 Structure of glycogen. Left, Overall structure. Note the large number of nonreducing ends, which are required as substrates for the enzymes of glycogen metabolism. Right, Structure around a branch point.

Theoretically, the molecule has only one reducing end with a free hydroxyl group at carbon 1 but a large number of nonreducing ends with a free hydroxyl group at carbon 4. The enzymes of glycogen synthesis and glycogen degradation are nested between the outer branches of the molecule and act only on the nonreducing ends. Therefore the many nonreducing end branches of glycogen facilitate its rapid synthesis and degradation.

Glycogen is readily synthesized from glucose

The steps in the synthesis of glycogen from glucose are outlined in Figures 22.10 and 22.11. Glucose-6-phosphate is isomerized to glucose-1-phosphate by phosphoglucomutase. There are about 20 molecules of glucose-6-phosphate for every molecule of glucose-1-phosphate at equilibrium. Glucose-1-phosphate then reacts with uridine triphosphate (UTP) to form UDP–glucose. This otherwise reversible reaction is driven to completion by the subsequent hydrolysis of pyrophosphate. UDP-glucose is the activated form of glucose for biosynthetic reactions.

Figure 22.10 Synthesis of uridine diphosphate (UDP)-glucose. UDP-glucose is the activated form of glucose for glycogen synthesis but also for the synthesis of other complex carbohydrates (see Table 8.3).

Figure 22.11 Glycogen synthase reaction. UDP, Uridine diphosphate.

UDP is attached to C-1 of glucose; therefore, it is this carbon that forms the glycosidic bond. The bond between glucose and UDP is energy rich. With a free energy content of 7.3 kcal/mol, it rivals the phosphoanhydride bonds in ATP. The free energy content of an α-1,4 glycosidic bond in glycogen is only 4.5 kcal/mol.

Glycogen synthase forms the α-1,4 glycosidic bonds in glycogen by transferring the glucose residue from UDP-glucose to the 4-hydroxyl group at the nonreducing end of the glycogen molecule, elongating the outer branches of glycogen by one glucose residue at a time (see Fig. 22.11).

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