Glucose-6-phosphate not used for direct glycogen synthesis can be metabolized in the glycolytic pathway. About half of the glucose taken up by liver during duodenal glucose absorption will undergo glycolysis to pyruvate. The resulting pyruvate can be used for lipogenesis and amino acid synthesis, lactate production, or cycled back to glucose-6-phosphate for indirect glycogen synthesis. There are 10 steps in the glycolytic pathway (Figure 13.3). Seven are catalyzed by enzymes with equilibrium constants that allow the reaction to proceed in either the “forward” (glycolytic) or “reverse” (gluconeogenic) direction dependent upon physiologic changes in the relative concentrations of substrates and products of the reactions. Three enzymatic steps, glucose phosphorylation by glucokinase (see earlier), the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate by phosphofructokinase, and the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase are considered essentially irreversible due to a large release of free energy. Free energy is provided by the hydrolysis of ATP in the case of the glucokinase and phosphofructokinase reactions, and via the favorable thermodynamics inherent in the conversion of an enol phosphate to a ketone in the pyruvate kinase reaction. In the previous section of this chapter, we discussed distinct enzymes that have evolved to circumvent the otherwise irreversible glycolytic steps and allow gluconeogenesis to occur. Thus, the forward reactions of glycolysis serve to produce pyruvate and also moderate gluconeogenesis during feeding. However, despite the elegance of the regulatory mechanisms of glycolysis, liver is, first and foremost, a gluconeogenic organ and gluconeogenesis remains modestly active even during the unfavorable conditions of a glucose load . This residual activity provides a mechanism to prevent lactate build-up, and is the basis for indirect glycogen synthesis.
Regulation of phosphofructokinase (PFK)
Glucose-6-phosphate is rapidly isomerized to fructose-6- phosphate, and then undergoes phosphorylation to fructose-1,6- bisphosphate by PFK with ATP serving as the phosphate donor. PFK displays sigmoidal kinetics, typical of enzymes that are regulated by allosteric ligands. Its activity is regulated by hepatic energy charge, such that increases in the ratio of ATP:ADP inhibits enzyme activity. Citrate, the product of the first committed step of the TCA cycle, is also a potent inhibitor of PFK activity. This interaction reduces glycolysis and increases gluconeogenesis when TCA cycle intermediates are replete and energy production is high. However, the most important allosteric regulator of PFK activity in liver is fructose-2,6-bisphosphate (F-2,6-P2) [84 – 86]. F-2,6-P2 activates PFK at low micromolar concentrations via two main actions. First, it converts the kinetic profile of PFK from sigmoidal to hyperbolic with respect to the concentration of the PFK substrate fructose-6-phosphate. This change in activity increases the affinity (lowers the Km) of PFK for fructose-6-phosphate. Second, F-2,6-P2 relieves inhibition of PFK by ATP, such that higher concentrations of the nucleotide are required to suppress enzyme activity.
Biochemical and molecular studies revealed that the synthesis and degradation of F-2,6-P2 is catalyzed by a single protein with two distinct catalytic sites . This protein was named 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase but is also known more simply as “the bifunctional enzyme.” The bifunctional enzyme found in liver is regulated by fasting and glucagon action through cAMP-dependent PKA phosphorylation of a serine residue near its N-terminus . Phosphorylation at this site inhibits the 2-kinase activity of the enzyme, and increases its bisphosphatase activity, resulting in a net decrease in F-2,6-P2 concentration within cells. This in turn leads to reduced affinity of PFK for its substrate fructose-6-phosphate and an increase in sensitivity of the enzyme to the inhibitory effects of ATP, leading to a net decrease in enzyme activity. Under these conditions, glycolysis is inhibited and gluconeogenesis is promoted.
In the transition from the fasted to the fed state, this regulation is reversed. Following a mixed meal, insulin and glucose levels rise in the blood while glucagon is decreased. These conditions cause dephosphorylation of the bifunctional enzyme, restoring its 2-kinase activity and causing a rapid increase in F-2,6-P2 levels. The dephosphorylation of the bifunctional enzyme appears to be mediated by a hexose-phosphate-sensitive protein phosphatase (PP2A). Xylulose-5-phosphate, an intermediate of the pentose phosphate pathway, was first discovered to activate PP2A . Thus, carbohydrate-regulated PP2A provides a direct link between the postprandial change in intracellular glucose concentration and the reactivation of the 2-kinase activity of the bifunctional enzyme. In addition to the effect of glucose to activate PP2A, the increase in insulin levels in the postprandial state serves to stimulate degradation of cAMP to AMP via phosphodiesterases, thus decreasing the activity of PKA and contributing to maintenance of the bifunctional enzyme in a nonphosphorylated state.
Hepatic glycolysis as a therapeutic target for diabetes
Genetic modification of the bifunctional enzyme, so that it favors formation of F-2,6-P2 and activation of PFK, induces hepatic glycolysis, reduces glucose production and improves glycemia in diabetic models, without the deleterious effects on circulating FFA and TG levels that occurs with glucokinase activation . Although both glucokinase and PFK activation stimulate hepatic glycolysis, unlike glucokinase activation, PFK activation appears to avoid induction of lipogenesis by preventing the accumulation of mono-phosphorylated glycolytic intermediates (i.e., xylulose-5P and glucose-6P) that activate ChREBP . However, studies have been limited to genetic activation in rodent models and as was also the case for glucokinase, a more detailed understanding of the metabolic consequences of activation of the bifunctional enzyme/PFK is required, including better understanding of changes in lipid metabolism and the potential for hypoglycemia.
Regulation of pyruvate kinase (PK)
PK catalyzes the conversion of PEP to pyruvate, the last of the irreversible steps of glycolysis in liver. As described in the discussion of its role in gluconeogenesis (see earlier), pyruvate kinase is subject to both allosteric and transcriptional regulation (Figure 13.6). For example, liver isoform of pyruvate kinase (L-PK) is activated by the product of the PFK reaction, fructose-1,6-bisphosphate . This represents a “feed-forward” mechanism by which flux through an early step in glycolysis activates a later step. Similar to PFK, L-PK is sensitive to the energy charge of the cell, and its activity is decreased in the presence of a high ATP:ADP + AMP ratio. Similar to the bifunctional enzyme, L-PK is inhibited by PKA-mediated serine phosphorylation . Phosphorylation increases the apparent Km of the enzyme for it substrate PEP, and renders the enzyme more sensitive to inhibition by a high energy charge (elevated ATP:ADP + AMP ratio).
Like the other key glycolytic enzymes, transcription of the L-PK gene is decreased by fasting or insulinopenic diabetes, and is increased in the fed state or by insulin injection. Glucose appears to be a more direct and potent regulator of L-PK transcription than insulin, which may be acting to antagonize the effects of glucagon by activating phosphodiesterases and lowering cellular cAMP levels. In addition, the same protein phosphatase that dephosphorylates the bifunctional enzyme when sugar phosphate levels are elevated also dephosphorylates and activates ChREBP. ChREBP stimulates transcription of L-PK, as well as lipogenic genes such as acetyl-CoA carboxylase and fatty acid synthase. Thus, a rise in circulating glucose concentration and a proportional increase in the rate of hepatic glycolysis and pentose monophosphate shunt activity in the fasted-to-fed transition is translated into an increase in 2-kinase activity of the bifunctional enzyme via activation of PP2A, resulting in an increase in F-2,6-P2 levels and increased flux through PFK to generate fructose-1,6-bisphosphate. This intermediate then serves as a feed-forward activator of a later step in glycolysis, L-PK. Finally, allosteric activation of pyruvate kinase is supplemented by another action of the Xu5P-regulated protein phosphatase, the dephosphorylation and activation of the ChREBP transcription factor which stimulates L-PK gene transcription.