GLUCONEOGENESIS Summary of handout: Ferchmin 2017 GLUCONEOGENESIS Summary of handout: Comparison with glycolysis, unique and shared enzymes Role of biotin in gluconeogenesis (and comparison with vitamin K which is not involved in gluconeogenesis) "Reversal" of pyruvate kinase. Participation of the mitochondria "Reversal" of Phosphofructokinase "Reversal" of hexokinase The Cori and alanine cycles Regulation. Role of insulin and glucagon in glycolysis and gluconeogenesis. Glycogenic and ketogenic compounds Metabolic role of gluconeogenesis

COMPARISON BETWEEN GLYCOLYSIS AND GLUCONEOGENESIS The overall reaction of gluconeogenesis is: COOH | 2 CO + 4 ATP + 2 GTP + 2 NADH + 2H+ + 2 H2O ➔ glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ CH3 ΔG°'= -9 Kcal/mole The overall reaction of glycolysis is: COOH | Glucose + 2 ADP + 2 Pi + 2 NAD+ ➔ 2 CO + 2 ATP + 2 NADH + 2H+ + 2 H2O CH3 ΔG°'= -20 Kcal/mole Glycolysis yields 2 ATP/glucose plus - 9 Kcal/mole dissipated. Gluconeogenesis is really bad news, it consumes the equivalent of 6 ATP/glucose synthesized. Why would be a need for such a wasteful metabolic pathway?

Gluconeogenesis is the synthesis of glucose from precursors that are not sugars, like lactate, pyruvate, glycerol or glycogenic amino acids. The synthesis of glucose from other sugars simply is not gluconeogenesis. The neo means de novo from non-carbohydrate molecules. (By the way, what was a carbohydrate?) There is no gluconeogenesis from fatty acids except the rare ones with odd number of carbons that have a minute contribution to the synthesis of glucose. Fatty acids contribute to the fasting organism with ATP through β-oxidation and oxidation of ketone bodies in the Krebs cycle. Ketone bodies only partially substitute for glucose and are synthesized by a pathway different from gluconeogenesis. Ketone bodies are potentially dangerous in the absence of glucose (cause metabolic acidosis). In conclusion: lipids can spare glucose because they provide for ATP that otherwise would have being synthesized from glucose. However, lipids do not substitute for glucose. We need about l60 grams of glucose per day, 120 grams are needed for the brain and 40 grams for muscle, erythrocytes, eye lens cells, kidneys medulla, etc. Approximately 200 grams are stored in hepatic glycogen. Gluconeogenesis provides the necessary glucose during fast. The complete gluconeogenesis occurs in the liver and renal cortex. Glycolysis is irreversible therefor gluconeogenesis cannot be the reversal of glycolysis. The enzymes that catalyze the irreversible reactions in glycolysis are overridden in various ingenious ways in gluconeogenesis.

We will study gluconeogenesis by comparing it with glycolysis Left gluconeogenesis------------Right glycolysis How do your reverse an irreversible metabolic step? By using an enzyme that catalyzes the opposite also irreversible step!!! By using an enzyme that catalyzes the opposite also irreversible step!!! How do your reverse an irreversible metabolic step?

Why alcoholic drinks can cause hypoglycemia Why alcoholic drinks can cause hypoglycemia? (including hypoglycemic death). And why sweet alcoholic drinks are even more intoxicating than dry or “bruit”. The last glycolytic step catalyzed by pyruvate kinase is irreversible, the free energy change is high, -7.5 Kcal/mole. To reverse this step in gluconeogenesis two enzymes are used and the process takes place in two cellular compartments. The first enzyme is pyruvate carboxylase, the second is phosphoenolpyruvate carboxykinase. The postulation that the increased NADH2/NAD ratio generated in the liver cell during ethanol metabolism causes the suppression of hepatic gluconeogenesis has been tested in several ways in twenty-eight fasted glycogen depleted dogs in whom hepatic gluconeogenesis was inhibited by infusions of ethanol. First, it was shown that fructose, a non-NAD-dependent precursor of glucose, produced a rapid restoration of hepatic glucose output during ethanol-induced suppression of hepatic gluconeogenesis. Second, in contrast, the infusion of glutamate and α-ketoglutarate, both NAD-dependent precursors of glucose, failed to augment the depressed rate of hepatic gluconeogenesis induced by ethanol. Finally, the administration of methylene blue, a redox dye which oxidizes NADH2 to NAD, not only prevented the expected fall in hepatic glucose output when infused simultaneously with ethanol, but also produced a rapid restoration of hepatic glucose output previously depressed by ethanol administration in fasting dogs. These data are consonant with the thesis that the increased NADH2/NAD ratio, which characterizes ethanol oxidation by the liver cell, causes a partial block at several points in the gluconeogenic pathway and is responsible for the ethanol-induced suppression of hepatic gluconeogenesis. Copyright © 1967 by the American Diabetes Association

We will analyze some reaction in detail First, we will consider the exergonic glycolytic reaction catalyzed by pyruvate kinase and its reversal in gluconeogenesis The above exergonic reaction is overcome by an input of energy and of two complex reactions that regenerate phosphoenolpyruvate. The two enzymes involved are: a) Pyruvate carboxylase b) Phosphoenolpyruvate carboxykinase However, before considering the enzymes we will look at the coenzyme of pyruvate carboxylase

PYRUVATE CARBOXYLASE is exclusively hepatic. So, after leaving the detour of biotin we return to pyruvate carboxylase and gluconeogenesis PYRUVATE CARBOXYLASE is exclusively hepatic. The reaction catalyzed by pyruvate carboxylase takes place in 2 steps: STEP 1: Enz-Biotin + ATP + CO2 ➔ Enz-Carboxybiotin + ADP + Pi This first step requires CH3-CO-CoA (acetyl~S-CoA) STEP 2: Enz-Carboxybiotin + pyruvate ➔ Enz-Biotin + oxaloacetate This is an anaplerotic reaction (re-supplying). It provides oxaloacetate for the Krebs cycle and for gluconeogenesis. Beware, there are cataplerotic steps in the Krebs cycle. The requirement for CH3-CO~S-CoA is a manifestation of the need of oxaloacetate for the TCA cycle or the abundance of CH3-CO-CoA produced by a lipid rich diet that calls for storage of glucogenic intermediaries.

The next step is the synthesis of phosphoenolpyruvic acid from oxaloacetate The synthesis of PEPA reverses the effect of pyruvate kinase

From PEPA to fructose-1,6-bisphosphate all the steps are shared by glycolysis and gluconeogenesis and are reversible. Most steps of gluconeogenesis take place in the cytosol but the synthesis of phosphoenolpyruvic acid (PEPA) requires the mitochondria. PEPA can be synthesized from pyruvate or lactate. In both cases, NADH +H+ must be generated to allow the reduction of 3-phosphoglyceric acid by glyceraldehyde-3-phosphate dehydrogenase. The figure illustrates both cases.

This graph represents the relationship between the activity of both enzymes and the energy status of a muscle cell.

From previous page, we can see the relationship between phosphofructokinase and fructose-1,6-phosphatase In this point we have a metabolic cycle or futile cycle that “wastes” energy but provides more leverage for regulation Metabolic cycles are regulated by the circadian rhythm

__________________________________________________________________ Summary of the enzymatic differences between glycolysis and gluconeogenesis a) Regulatory enzymes __________________________________________________________________Glycolysis Gluconeogenesis __________________________________________________________________ Hexokinase Glucose 6-phosphatase Phosphofructokinase Fructose 1,6-bisphosphatase Pyruvate Pyruvate kinase carboxylase Phosphoenolpyruvate carboxykinase __________________________________________________________________b) The remaining enzymes are shared by both pathways Essential concept: Pathways for breakdown and synthesis of a particular metabolite are always different, utilizing unique enzymes in one or more steps. The difference usually is in the regulatory enzymes. Pyruvate carboxylase is located in liver mitochondrias

Integration of gluconeogenesis and glycolysis

There is a fundamental difference between the role of glycolysis in the “peripheral” organs and the liver. In liver the role of glycolysis is to make you FAT!!!! In muscle is to make you run!!! Galactose, fructose, etc are not glucogenic. They are monosaccharides in equilibrium with glucose! Ethanol and fatty acids are not glucogenic (odd number fatty acids contribute insignificantly to gluconeogenesis). Glycerol, the ketoacids of most amino acids, lactate and pyruvate ARE glucogenic. Although the carbons from fatty acids can end up in glucose it is by reshuffling of carbons without a net synthesis of glucose. This will be further explained in the class about Krebs’s cycle.

Inhibition of gluconeogenesis by high NADH/NAD ratio During the metabolism of ethanol, there is elevated levels of NADH, which affect a number of critical dehydrogenases in the liver required for gluconeogenesis.   The high NADH inhibits conversion of lactate to pyruvate by lactate dehydrogenase, malate to oxaloacetate by malate dehydrogenase, which decreases the availability of pyruvate and oxaloacetate for gluconeogenesis. Excess NADH production because of alcohol consumption inhibits oxidation of lactate to pyruvate via lactate dehydrogenase. What are the repercussions? Less pyruvate created leads to less oxaloacetate. Oxaloacetate under normal conditions would traverse across the mitochondrial membrane into the cytoplasm to be converted to PEP via PEPCK. Less oxaloacetate because of less pyruvate available to gluconeogenesis pathway ultimately leads to hypoglycemia. The postulation that the increased NADH2/NAD ratio generated in the liver cell during ethanol metabolism causes the suppression of hepatic gluconeogenesis has been tested in several ways in twenty-eight fasted glycogendepleted dogs in whom hepatic gluconeogenesis was inhibited by infusions of ethanol. First, it was shown that fructose, a non-NAD-dependent precursor of glucose, produced a rapid restoration of hepatic glucose output during ethanol-induced suppression of hepatic gluconeogenesis. Second, in contrast, the infusion of glutamate and α-ketoglutarate, both NAD-dependent precursors of glucose, failed to augment the depressed rate of hepatic gluconeogenesis induced by ethanol. Finally, the administration of methylene blue, a redox dye which oxidizes NADH2 to NAD, not only prevented the expected fall in hepatic glucose output when infused simultaneously with ethanol, but also produced a rapid restoration of hepatic glucose output previously depressed by ethanol administration in fasting dogs. These data are consonant with the thesis that the increased NADH2/NAD ratio, which characterizes ethanol oxidation by the liver cell, causes a partial block at several points in the gluconeo genic pathway and is responsible for the ethanol-induced suppression of hepatic gluconeogenesis. To make thinks more complicated, hypoglycemia induced by alcohol ingestion is a well-known problem in diabetic patients. However, the mechanisms underlying this phenomenon have largely remained elusive. Because insulin secretion in vivo can be rapidly tuned by changes in pancreatic microcirculation, we evaluated the influence of acute alcohol administration on pancreatic islet blood flow (IBF), and dynamic changes in insulin secretion and glycemia in the rat. Ethanol (10%) or saline was iv injected as a bolus into Wistar rats, yielding serum ethanol concentrations of approximately 8 mmol/liter. Measurements of pancreatic blood flow (PBF) were performed by a microsphere technique in combination with a freeze-thawing technique after 10-min injection. Ethanol preferentially and significantly increased pancreatic IBF approximately 4-fold, whereas not influencing whole PBF. The alcohol also augmented late-phase insulin secretion and induced late hypoglycemia upon ip glucose tolerance tests. The nitric oxide synthase inhibitor N-w-nitro-L-arginine methyl ester and atropine prevented the increased pancreatic IBF, enhanced insulin secretion, and hypoglycemia evoked by ethanol. Thus, our findings demonstrate that ethanol acutely exerts substantial influences on pancreatic microcirculation by evoking a massive redistribution of PBF from the exocrine into the endocrine part via mechanisms mediated by nitric oxide and vagal stimuli, augmenting late-phase insulin secretion, and thereby evoking hypoglycemia. This effect may in part underlie the well-known hypoglycemic properties of alcohol in diabetic patients or in alcoholics with hepatic failure.