Reaction 3: Glucose 6-phosphate à Glucose
4. Propionate
Propionate is a product of lipid digestion in ruminants, and of β-oxidation of fatty acids (with an odd number of carbon atoms). Propionate is activated to propionyl CoA by acyl-CoA synthetase and then carboxylated to a 4-C intermediate, methylmalonyl CoA. This compound can be isomerised to succinyl CoA, an intermediate of the TCA cycle. Subsequent entry into gluconeogenesis is via malate.
Fig 5.6.8. Route for conversion of propionate to succinyl-CoA which enters the gluconeogenic pathway via malate (Source: Murray et al, 2003, p155 fig 19-2)
There is no net conversion of fatty acids to glucose in mammals even though oxidation of fatty acids directly provides acetyl-CoA to the TCA cycle. The PDH reaction, which converts pyruvate to acetyl-CoA, cannot be reversed by any “by-pass” step. The enzymes that release pyruvate from citrate, viz. “malic” enzyme and citrate lyase, are more involved in lipogenesis than in gluconeogenesis.
REGULATION OF GLUCONEOGENESIS
The rate of gluconeogenesis depends on the activities of its key enzymes and the availability of its precursors.
The key regulatory enzymes in gluconeogenesis are:
• pyruvate carboxylase
• phosphoenolpyruvate carboxykinase
• fructose 1,6-bisphosphatase-1
• glucose 6-phosphatase.
The activities of these enzymes are low when blood glucose is high (as in carbohydrate-feeding) and high when blood glucose is low (as in starvation and diabetes). Their regulation of these enzymes is by both allosteric and hormonal mechanisms.
Acetyl-CoA is an allosteric activator of pyruvate carboxylase, The concentration of acetyl CoA increases on consumption of a high fat diet (owing to increased β-oxidation of fatty acids); hence such a nutritional state favors gluconeogenesis.
Citrate, which is a product of the condensation of acetyl-CoA with OAA, stimulates FBPase-1 allosterically.
ADP and AMP increase in concentration when the energy state of the cell is low and this is not conducive to gluconeogenesis. ADP inhibits both pyruvate carboxylase and PEP-CK, while AMP and fructose 2,6-bisphosphate inhibit FBPase-1.
The hormones, glucagon, epinephrine and glucocorticoids, are inducers of the synthesis of all the gluconeogenic enzymes, while insulin is a repressor.
The availability of precursors also affects gluconeogenesis. Conditions in extrahepatic tissues that increase the production of lactate and alanine are conducive to gluconeogenesis in the liver.
A high rate of anaerobic glycolysis in active muscle increases lactate production, while starvation increases breakdown of muscle proteins to release alanine. High levels of Ala also inhibit pyruvate kinase in the liver so that glycolysis is decreased. Glucagon increases lipolysis in adipose tissue and makes glycerol available for gluconeogenesis while insulin has the opposite effect.
Ethanol inhibits gluconeogenesis. The oxidation of ethanol in the liver increases [NADH] and shifts the LDH/MDH reactions to favor lactate/malate over pyruvate/OAA respectively. It is therefore unwise to consume liquor on an empty stomach!
Section 5.6B
RECIPROCAL REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS The hepatic cells have a triple access to glucose supply viz from the blood, by glycogenolysis and by gluconeogenesis. Since the liver provides glucose as energy fuel to other tissues in preference to its own self, a tight regulation is required on its ability to degrade glucose by glycolysis and synthesize glucose by gluconeogenesis. At any point in time, the pathway favored depends on the levels of blood glucose and on the energy state of the hepatic cells. Conditions that favor gluconeogensis concomittantly suppress glycolysis, and vice- versa.
Both pathways have many common and reversible steps. Hence, regulation is excercised on the enzymes catalyzing those reactions which are distinct for each pathway.
The key enzymes for glycolysis are pyruvate kinase (PK), phosphofructokinase-1 (PFK-1) and glucokinase/hexokinase (GK/HK). The key enzymes for gluconeogenesis are pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEP-CK), fructose 1,6-bisphosphatase (FBPase-1) and glucose 6-phosphatase (Glu 6-Pase). They are regulated reciprocally by allosteric or hormonal control of their activity or transcriptional control of their quantity.
(It would be advisable here to revise the regulatory mechanisms explained for glycolysis in Section 1 of this chapter. In the discussion below, we have used the color blue for glycolytic enzymes and orange for gluconeogenic enzymes).
Allosteric regulation
Concentrations of critical local molecules cause allosteric modulation of the activities of PFK-1 vs FBPase-1 and PK vs PC and they play a primary role in determining whether glycolysis or gluconeogensis will take place.
The allosteric modulators affect the pathways as follows:
a. [ATP]/[AMP]
When the energy state of the cell is low, [AMP] is high and the synthesis of ATP is required.
Since AMP activates PFK-1 and inhibits FBPase-1, and ADP inhibits PC, hence glycolysis is favored and gluconeogenesis is suppressed to make more ATP available to the cell.
Conversely, when the energy state of the cell is high, increased levels of ATP inhibit PFK-1 and PK, while low [AMP] does not inhibit FBPase-1; hence gluconeogenesis is favored over glycolysis. During processes that consume energy, a small change in [ATP] causes a large change in [AMP]. This enables PFK-1 to respond even to small changes in the energy status of the cell and thereby control glycolysis.
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Fig 5.6.9. Allosteric modulators of the glycolytic and gluconegenic enzymes involved in reciprocal regulation of the two pathways. (Source: Berg et al, 2002, fig 16.30)
b. Acetyl-CoA
Increased β-oxidation of fatty acids occurs under conditions of starvation, high-fat diet or diabetes.
The consequent increase in [acetyl-CoA] activates both PC and FBPase-1 allosterically, stimulating gluconeogenesis but suppressing glycolysis:
• ↑ β-oxidation of fatty acids à ↑ [acetyl-CoA] à ↑ PC activity à ↑ OAA formation à ↑ [citrate] à stimulates FBPase-1 activity à gluconeogeneis ↑
• ↑ [citrate] à inhibits PFK-1 à glycolysis ↓
Increased [acetyl-CoA] provides more ATP as energy to facilitate gluconeogenesis, but the same high cytosolic levels of ATP inhibit PFK-1 and also PK (by decreased formation of Fru 1,6 BP) to suppress glycolysis. High [acetyl -CoA] also inhibits PDH, reducing pyruvate oxidation i.e. the metabolic fate of pyruvate changes in the liver and kidney on transition from “well-fed” to starved state.
c. Fructose 2,6-bisphosphate
Fru 2,6-BP is the most potent allosteric effector of PFK-1 and inhibitor of FBPase-1 in liver. It relieves inhibition of PFK-1 by ATP and increases affinity for Fru 6-P. It inhibits FBPase-1 by increasing Km for Fru 1,6-BP.
Fig 5.6.10. Reciprocal regulation of glycolysis and gluconeogenesis by Fructose 2,6-bisphosphate . (Source: Nelson and Cox, 2003, p 582 fig 15-23)
The concentration of Fru 2,6-BP in the cell depends on the activity of the bifunctional enzyme, (PFK-2 + FBPase-2) which we encountered earlier in the regulation of glycolysis.
When [blood glucose] is highà ↑ [Fru 6-P]à inhibits FBPase-2 à ↑ [Fru 2,6-BP] à stimulates PFK-1 and inhibits FBPase-1 à hence glycolysis ↑ and gluconeogenesis ↓
The dominance of kinase vs phosphatase activity of the bifunctional enzyme depends on allosteric and hormonal factors.
d. Glucose 6-phosphate
Accumulation of glucose 6-P in cells (by decreased activity of PFK-1) causes allosteric inhibition of hexokinase and decreased uptake of glucose in extrahepatic tissues like muscle. The situation is different in the liver where. glucokinase is inhibited by reversible binding of a regulatory protein specific to the liver (refer Fig 5.1.16).
When blood glucose is high, efficiency of GLUT2 transporter ensures that cytosolic glucose levels in the hepatic cells equilibrate with blood levels, glucokinase is freed from inhibition, and Glc 6-P continues to be formed. However, if [blood glucose] falls below optimal levels, Fru 6-P causes binding of the regulatory protein to glucokinase and formation of Glc 6-P is inhibited. This ensures that liver does not compete with other tissues for glucose.