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International Textbook of Diabetes Mellitus, 4th Ed., Excerpt #70: Regulation of Glucose Metabolism in Liver Part 6 of 11

Acute regulation of gluconeogenesis

Prior to discovery of the transcriptional mechanisms described earlier, the control of hepatic glucose metabolism was examined in great detail on the basis of substrate, allosteric, and posttranslational modification. These factors alter gluconeogenic flux rapidly (seconds to minutes) and are a critical first response to increased glucose demand. Much of the acute regulation of gluconeogenesis is based on the antithetic regulation of several gluconeogenic/glycolytic enzyme pairs that catalyze opposing pathways. The metabolic intersections of PC/PDH, PEPCK/PK, G6Pase/glucokinase and fructose-1,6-bisphosphatase/phosphofructokinase are critical sites for the regulation of gluconeogenesis. Logically, alterations that inhibit the regulatory glycolytic enzymes (PDH, GK, PFK, or PK) will promote gluconeogenesis, while alterations that inhibit regulatory gluconeogenic enzymes (PC PEPCK, G6Pase, or fructose-1,6-bisphosphatase will promote glycolysis.

Allosteric and covalent modification

Allosteric modification of enzyme activity occurs when an enzyme binds to a molecule, causing either an increase or decrease in the enzyme’s activity. Allosteric binding sites are separate from the catalytic domains and interact with their effector molecules by noncovalent bonding. Typically, electrostatic or hydrogen bonding between the effector molecule and amino acid residues of the allosteric binding pocket cause a change in the quaternary structure of the protein or otherwise change the binding constant of the catalytic site of the enzyme. Importantly, the effect on conformation and activity is essentially instan- taneous and independent of the transcriptional mechanisms described earlier. In contrast, covalent modification occurs when new functional groups are added to amino acids of the enzyme. The effect of covalent modification also changes the conformation and therefore the activity of the enzyme. The most common types of covalent modifications of metabolic enzymes are phosphorylation and acetylation, though many others can also occur. Covalent modification requires the activity of other proteins such as kinases and/or phosphorylases to add or cleave covalently bound functional groups.

Pyruvate dehydrogenase

Mitochondrial pyruvate can either be decarboxylated by PDH to yield acetyl-CoA (lipogenesis/oxidation) or carboxylated by PC to yield oxaloacetate (gluconeogenesis) (Figure 13.5). These divergent fates of pyruvate are influenced by nutritional state and are reciprocally regulated by allosteric and or covalent modification. PDH is part of a very large complex of proteins known as the pyruvate dehydrogenase complex (PDC). Inasmuch as glucose production is required for survival, hepatic PDH is normally kept in an inactive state by allosteric inhibition and phosphorylation. PDH activity is allosterically inhibited by acetyl-CoA, NADH, and ATP, factors that are replete in the fasted liver. PDH is also inactivated by a family of pyruvate dehydrogenase kinases (PDK) that phosphorylate PDH [33]. PDK-4 expression is decreased by insulin action, increased by counterregulatory hormones and its activity is stimulated by acetyl-CoA, NADH, and ATP [33]. PDH activity is de-repressed during feeding, allowing glucose to be converted to acetyl-CoA for oxidation or lipogenesis (see hepatic glucose disposal later).

Chapter13Fig13.5Pyruvate carboxylase

Suppression of PDH during starvation preserves pyruvate for pyruvate carboxylase (PC) and gluconeogenesis (Figure 13.5). PC is a mitochondrial enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate and exerts more powerful control over gluconeogenesis than all of the other enzymes of the path- way combined [34]. Although its transcriptional regulation is weak, the allosteric control of PC has been thoroughly examined and provides a glimpse into the elegant regulation possible by acute metabolic feedback. In contrast to PDH, PC is allosterically activated by acetyl-CoA [35]. Thus, during fasting, when fat oxidation is increased, PC is activated and provides ample oxaloacetate for gluconeogenesis. Isotope tracer studies indicate that the in vivo activity of PC in the human postabsorptive liver is roughly up to 100 times higher than PDH [36,37].

The rapid regulation of PC activity by allostery is made possible by its tetrameric structure [35]. PC has two biotin domains on each face of a tetrahedral. One domain facilitates the MgATP/HCO3− dependent carboxylation of a biotin, and the other is a transferase domain where the carboxyl group is transferred to pyruvate. Activity of the PC complex requires that the carboxylated biotin domain access the transferase domain of the adjacent monomer. Acetyl-CoA binds amino acid residues in an allosteric binding site and induces a conformation that is favorable for the interaction of these domains. In the absence of acetyl-CoA, these domains cannot interact and PC activity approaches zero. The Ka for acetyl-CoA binding is 20 – 60 uM, a value within the biologic range of acetyl-CoA concentration in liver mitochondria [35]. Intermediates associated with the TCA cycle such as α-ketoglutarate and glutamate reduce the Ka for acetyl-CoA, thereby effectively inhibiting PC activity. This may provide a metabolic mechanism allowing oxaloacetate production to match TCA cycle capacity. Despite the elegant metabolic regulation of PC, its role as a rate-controlling step in gluconeogenesis has received little attention in the study of mechanism leading to the elevation of hepatic glucose production during diabetes.

Pyruvate kinase

Liver pyruvate kinase (L-PK) catalyzes the irreversible conversion of PEP to pyruvate and ADP to ATP. L-PK contributes to the positive regulation of glycolysis and lipogenesis and is discussed more thoroughly in these contexts in the later section on glucose disposal. However, it is worth noting that by opposing the combined actions of PC and PEPCK, L-PK acts as an important negative regulator of gluconeogenesis [15,38]. Experimentally, the generation of pyruvate from gluconeogenic PEP is an active substrate cycle in liver (Figure 13.6). Tracer experiments in hepatocytes [39], isolated liver [40], animal models [41] and humans [37] indicate that roughly half of PC flux is cycled back to pyruvate. Collectively these pathways are referred to as pyruvate cycling, and ostensibly confer greater metabolic flexibility by directing an already active flux towards or away from gluconeogenesis on fast time scales [15]. L-PK is suppressed by glucagon (and to a lesser extent epinephrine) via cAMP-mediated PKA action and as with other examples of actions of counterregulatory hormones, insulin opposes this process. L-PK activity is also allosterically activated by F-1,6-P2 and inhibited by alanine and ATP. Phosphorylation of L-PK by PKA decreases the maximal activation of L-PK by its substrates and potentiates the effectiveness of alanine and ATP to allosterically suppress its activity. Moreover, L-PK is a better substrate for phosphorylation by PKA when it is allosterically inhibited by alanine or ATP than when it is allosterically activated by F-1,6-P2. Thus allosteric regulation not only mediates L-PK activity directly, but also deters phosphorylation of the enzyme [15].

Chapter 13Fig13.6

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