CHAPTER OUTLINE
Critical Bypass Reactions of Gluconeogenesis
Pyruvate to Phosphoenolpyruvate, Bypass 1
Fructose 1,6-Bisphosphate to Fructose 6-Phosphate, Bypass 2
Glucose 6-Phosphate (G6P) to Glucose (or Glycogen), Bypass 3
Substrates for Gluconeogenesis
Intestinal Gluconeogenesis: Glucose Homeostasis and Control of Feeding Behavior
High-Yield Terms
Cori cycle: describes the interrelationship between lactate production during anaerobic glycolysis and the use of lactate carbons to produce glucose via hepatic gluconeogenesis
Endogenous glucose production: designated EGP, refers to the process of glucose production via gluconeogenesis
Glucose-alanine cycle: describes the interrelationship between pyruvate transamination and alanine during skeletal muscle glycolysis and delivery to the liver where the alanine is deaminated back to pyruvate, which is then diverted into hepatic gluconeogenesis
Critical Bypass Reactions of Gluconeogenesis
Gluconeogenesis is the biosynthesis of new glucose, (ie, not glucose from glycogen). The production of glucose from other carbon skeletons is necessary during periods of fasting and starvation. This is acutely true for the testes, erythrocytes, and kidney medullary cells since each is exclusively dependent upon glucose oxidation for ATP production. The brain, although not restricted solely to glucose, requires adequate rates of gluconeogenesis since it is the organ of highest daily glucose consumption. In addition to glucose, the brain can derive energy from ketone bodies (Chapter 25). The primary carbon skeletons used for gluconeogenesis are derived from pyruvate, lactate, glycerol, and the amino acids alanine and glutamine. The liver is the major site of gluconeogenesis; however, as discussed below, the kidney and the small intestine also have important roles to play in this pathway (Figure 13-1).
FIGURE 13-1: Major pathways and regulation of gluconeogenesis and glycolysis in the liver. Entry points of glucogenic amino acids after transamination are indicated by arrows extended from circles. The key gluconeogenic enzymes are enclosed in double-bordered boxes. The ATP required for gluconeogenesis is supplied by the oxidation of fatty acids. Propionate is of quantitative importance only in ruminants. Arrows with wavy shafts signify allosteric effects; dash-shafted arrows, covalent modification by reversible phosphorylation. High concentrations of alanine act as a “gluconeogenic signal” by inhibiting glycolysis at the pyruvate kinase step. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Pyruvate to Phosphoenolpyruvate, Bypass 1
Conversion of pyruvate to phosphoenolpyruvate (PEP) requires the action of 2 enzymes. The first is an ATP-requiring reaction catalyzed by pyruvate carboxylase (PC), which catalyzes the carboxylation of pyruvate to the TCA cycle intermediate, oxaloacetic acid (OAA). The second enzyme of bypass 1 is the GTP-dependent PEP carboxykinase (PEPCK), which converts OAA to PEP. Since PC incorporated CO2 into pyruvate and it is subsequently released in the PEPCK reaction, no net fixation of carbon occurs. Human cells contain almost equal amounts of mitochondrial and cytosolic PEPCK (designated PEPCK-m and PEPCK-c, respectively), so this second reaction can occur in either cellular compartment.
If OAA is converted to PEP by PEPCK-m, it is transported to the cytosol where it is a direct substrate for gluconeogenesis and nothing further is required. However, the OAA produced by PC (or produced via the TCA cycle) can serve as a gluconeogenic substrate within the cytosol. This occurs by transamination of OAA to aspartate or reduction to malate and coupled transport via a pathway called the malate-aspartate shuttle (see Figure 10-2). The malate-aspartate shuttle is also a major mechanism for transferring electrons from cytosolic NADH into mitochondrial NADH where they can be shunted into oxidative phosphorylation (Chapter 17).
Transamination of OAA to aspartate allows the aspartate to be transported to the cytosol where the reverse transamination takes place yielding cytosolic OAA. This transamination reaction requires continuous transport of glutamate into, and α-ketoglutarate out of, the mitochondrion. Therefore, this process is limited by the availability of these other substrates. Either of these latter 2 reactions will predominate when the substrate for gluconeogenesis is lactate. The reduction of OAA to malate requires NADH, which will be accumulating in the mitochondrion as the energy charge increases. The increased energy charge will allow cells to carry out the ATP costly process of gluconeogenesis. The resultant malate is transported to the cytosol where it is oxidized to OAA by cytosolic malate dehydrogenase (MDH), which requires NAD+ and yields NADH.
The NADH produced during the cytosolic oxidation of malate to OAA is utilized during the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reaction of glycolysis. The coupling of these 2 oxidation-reduction reactions is required to keep gluconeogenesis functional when pyruvate is the principal source of carbon atoms. The conversion of OAA to malate predominates when pyruvate (derived from glycolysis or amino acid catabolism) is the source of carbon atoms for gluconeogenesis. When in the cytoplasm, OAA is converted to PEP by PEPCK-c.
The net result of the PC and PEPCK reactions is:
Pyruvate + ATP + GTP + H2O → PEP + ADP + GDP + Pi + 2H+
High-Yield Concept
The 3 reactions of glycolysis that proceed with a large negative free-energy change are bypassed during gluconeogenesis by using different enzymes. The bypass reactions represent the reversal of the pyruvate kinase, phosphofructokinase-1 (PFK1), and hexokinase/glucokinase-catalyzed reactions.
Fructose 1,6-Bisphosphate to Fructose 6-Phosphate, Bypass 2
Fructose-1,6-bisphosphate (F1,6BP) conversion to fructose 6-phosphate (F6P) is the reverse of the rate-limiting step of glycolysis. The reaction, a simple hydrolysis, is catalyzed by fructose 1,6-bisphosphatase (F1,6BPase). Like the regulation of glycolysis occurring at the PFK1 reaction, the F1,6BPase reaction is a major point of control of gluconeogenesis (see Figure 10-4).
Glucose 6-Phosphate (G6P) to Glucose (or Glycogen), Bypass 3
G6P is converted to glucose through the action of glucose 6-phosphatase (G6Pase). This reaction is also a simple hydrolysis reaction like that of F1,6BPase. In the kidney, muscle, and, especially, the liver, G6P can be shunted toward glycogen if blood glucose levels are adequate. The reactions necessary for glycogen synthesis (Chapter 14) are an alternate bypass series of reactions.
Substrates for Gluconeogenesis
Lactate
Lactate is a major source of carbon atoms for glucose synthesis by gluconeogenesis in the liver. In erythrocytes and during anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). This reaction serves 2 critical functions during anaerobic glycolysis. First, in the direction of lactate formation the LDH reaction requires NADH and yields NAD+ which is then available for use by the GAPDH reaction of glycolysis. These 2 reactions are, therefore, intimately coupled during anaerobic glycolysis. Secondly, the lactate produced by the LDH reaction is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned to the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle (Figure 13-2).
FIGURE 13-2: The lactic acid (Cori cycle) and glucose-alanine cycles. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Pyruvate
Pyruvate, generated in muscle and other peripheral tissues, can be transaminated to alanine which is returned to the liver for gluconeogenesis. This pathway is termed the glucose-alanine cycle (see Figure 13-2). The glucose-alanine cycle serves a critical mechanism for muscle to eliminate waste nitrogen from amino acid catabolism while replenishing its energy supply as glucose. Within the liver the alanine is converted back to pyruvate and used as a gluconeogenic substrate or oxidized in the TCA cycle. The amino nitrogen is converted to urea in the urea cycle and excreted by the kidneys.
Amino Acids
All the amino acids present in proteins, excepting leucine and lysine, can be degraded to TCA cycle intermediates as discussed in Chapter 30. This allows the carbon skeletons of the amino acids to be converted to those in oxaloacetate and subsequently into pyruvate. The pyruvate thus formed can be utilized by the gluconeogenic pathway. When glycogen stores are depleted, in muscle during exertion and liver during fasting, catabolism of muscle proteins to amino acids contributes the major source of carbon for maintenance of blood glucose levels. Of all the amino acids utilized for gluconeogenesis, glutamine is the most important as this amino acid is critical for glucose production by the kidneys and small intestine.
Glycerol
The glycerol backbone of triglycerides can be used for gluconeogenesis. Indeed, the glycerol released from adipose tissue during periods of fasting provides a major source of carbon atom for hepatic gluconeogenesis. The glycerol is first phosphorylated to glycerol 3-phosphate by glycerol kinase followed by dehydrogenation to dihydroxyacetone phosphate (DHAP) by glycerol-3-phosphate dehydrogenase (GPD). The GPD reaction is the same as that used in the transport of cytosolic reducing equivalents into the mitochondrion for use in oxidative phosphorylation. This transport pathway is called the glycerol-phosphate shuttle (see Figure 10-3).
The glycerol backbone of adipose tissue stored triacylglycerols is ensured of being used as a gluconeogenic substrate since adipose cells lack glycerol kinase. In fact, adipocytes require a basal level of glycolysis in order to provide them with DHAP as an intermediate in the synthesis of triacylglycerols (Chapter 20).
Propionate
Oxidation of fatty acids with an odd number of carbon atoms and the oxidation of some amino acids generates as the terminal oxidation product, propionyl-CoA. Propionyl-CoA is converted to the TCA intermediate, succinyl-CoA. This conversion is carried out by the ATP-requiring enzyme, propionyl-CoA carboxylase, then methylmalonyl-CoA epimerase, and finally the vitamin B12 requiring enzyme, methylmalonyl-CoA mutase. The utilization of propionate in gluconeogenesis only has quantitative significance in ruminants (Figure 13-3).
FIGURE 13-3: Metabolism of propionate. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York: McGraw-Hill; 2012.
Intestinal Gluconeogenesis: Glucose Homeostasis and Control of Feeding Behavior
The gut, in particular the small intestine, plays a critical role in the uptake and delivery of glucose from the diet. In addition, the small intestine participates in gluconeogenesis and thus, contributes to endogenous glucose production, EGP (Figure 13-4). As such, the gut plays a central role in the overall regulation of glucose homeostasis. Only recently (a little more than 10 years ago) the expression of glucose 6-phosphatase (G6Pase) within enterocytes of the small intestine was characterized. Expression of G6Pase thus confers upon the intestine the ability to carry out gluconeogenesis. Glutamine serves as the major precursor of glucose formed within the small intestine. The genes for both G6Pase and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-c) are controlled by insulin in the small intestine, similarly to the regulation of these genes in the liver (Figure 13-4).
FIGURE 13-4: Pathways by which the small intestine contributes to maintenance of endogenous glucose production. Reproduced with permission of themedicalbiochemistrypage, LLC.
The importance of intestinal gluconeogenesis, to overall EGP, has been demonstrated both in experimental animals (mice with specific knockout of PEPCK-c in the liver) and in humans in the anhepatic phase during liver transplantation. Even with loss of hepatic PEPCK-c, there is an efficient adaptation to fasting conditions such that blood glucose levels decrease by only 30%. Simultaneously there occurs a significant increase in plasma glutamine concentration. These observations stressed the likely role of the kidney and/or intestine in glucose production, because glutamine is a major glucose precursor in the kidney and the small intestine, but not in the liver. The role of the intestine in plasma glucose control is demonstrated by the fact there are no observable differences in glucose concentration between arterial and portal blood. During periods of fasting, the small intestine accounts for approximately 20% of EGP by 48 hours and up to 35% by 72 hours.
In addition, the gut releases glucose to the portal circulation following the intake of a protein-rich, carbohydrate-free diet. The rate of glucose release by the gut can be 15% to 20% of total EGP when eating a protein-rich diet. Under these dietary conditions there is a demonstrable decrease in food intake in both humans and experimental animals. A similar decrease in food intake is observed in animals with an equivalent amount of glucose infusion directly into the portal vein. Under conditions where intestinal G6Pase is specifically abolished, protein-rich, carbohydrate-free diets do not lead to decreased. Chemical or surgical ablation of portal afferent nerve connections also results in loss of satiety induction by protein-rich diets or portal glucose infusion. Afferent nerves send nerve signals from various body locations to the brain. Brain areas involved in the control of feeding behaviors include the brain stem and the hypothalamus. Detailed discussion of the role of the hypothalamus in the control of feeding behaviors is presented in Chapter 44. Consuming a protein-rich diet (or glucose infusions into the portal vein in experimental animals) results in neuronal activation in several hypothalamic nuclei involved in feeding behavior including the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial nucleus (VMN), and paraventricular nucleus (PVN). When intestinal afferent connections are destroyed, there is no increase in hypothalamic activity upon consumption of a protein-rich diet. These results demonstrate that portal glucose influences feeding behavior via afferent connections to the brain, in particular to the hypothalamus.
