GLUCONEOGENESIS Index of slides 1) Index Ferchmin 2018 Index of slides 1) Index 2) Description of gluconeogenesis 2) 3-6 comparison between glycolysis and gluconeogenesis 3) 7-11 reversal in gluconeogenesis of pyruvate kinase 4) 11-13 steps shared by glycolysis and gluconeogenesis and release of free glucose. 5) 14 Cori and alanine cycles 6) 15-17 Regulation of gluconeogenesis 7) 18 Additional concepts and glucogenic and not glucogenic metabolites.

Gluconeogenesis is the synthesis of glucose from precursors that are not sugars, for example, lactate, pyruvate, glycerol or glycogenic amino acids. The synthesis of glucose from other sugars is not gluconeogenesis. The neo means de novo from non-carbohydrate molecules. There is no gluconeogenesis from fatty acids except the rare ones with the odd number of carbons which have a minute contribution to the synthesis of glucose, and its role is of rather academic value.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 (you will study this later). In conclusion: lipids can spare glucose because they provide for ATP that otherwise would be synthesized in glycolysis. But lipids do not substitute glucose. We need about l60 grams of glucose per day, 120 grams are required 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 a small fraction in the kidney. Since glycolysis is irreversible gluconeogenesis cannot be the reversal of glycolysis. The enzymes that catalyze the irreversible reactions in glycolysis are overridden in various ingenious ways in 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 yield 2 ATP/glucose plus a net dissipated - 9 Kcal/mole. Gluconeogenesis is really bad news, it consumes the equivalent of 6 ATP/glucose synthesized. Why would be a need for such a metabolic pathway?

We will study gluconeogenesis by comparing it with 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?

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.

__________________________________________________________________ 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 mitochondrial and hepatic

The above exergonic reaction is overcome by an input of energy and First we will consider the reversal in gluconeogenesis of the exergonic glycolytic reaction catalyzed by pyruvate kinase. The reaction is shown below: 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

With reference to pyruvate carboxylase This part is a “pop up” about a different subject marginally related to gluconeogenesis but of practical importance (NBE). There are two vitamins involved in CO2 incorporation in mammalian tissues: biotin and vitamin K. In gluconeogenesis biotin is a cofactor for pyruvate carboxylase. Biotin binds avidly to AVIDIN. Vitamin K is involved in the posttranslational synthesis of γ-carboxyglutamate involved prominently in blood coagulation. Biotin is attached to the amino of lysine in carbon ε. Biotin is a cofactor for 1) Pyruvate carboxylase 2) β-methylcrotonylCoA carboxylase 3) Propionyl CoA carboxylase 4) Acetyl CoA carboxylase Biotin Vitamin K

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. 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 The framed figure on this slide summarizes the reactions in which pyruvate is transformed into PEPA and does not represent an actual single step of gluconeogenesis.

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 to glyceraldehyde-3-phosphate by the enzyme glyceraldehyde-3-phosphate dehydrogenase. Ethanol consumption causes the accumulation of NADH and leads to a depletion of NAD+. The low concentration of NAD+ inhibits gluconeogenesis by preventing the oxidation of lactate to pyruvate. When instead of lactate, pyruvate, enters the mitochondria where it is transformed into malate. In the next step, malate is transported out of the mitochondria where cytosolic malate dehydrogenase uses NAD+ to generate oxaloacetate a precursor of PEPA in gluconeogenesis. Consider the effect of drinking ethanol on athletic performance when muscles generate lactate and liver supports muscle activity by regenerating glucose through the Cori cycle. Do not drink alcohol when you want to excel in sports performance. Notice that lactate dehydrogenase and cytosolic malate dehydrogenase supply the NADH necessary for the reversal of the glycolytic step catalyzed by the glyceraldehyde-3-P dehydrogenase. From PEPA to fructose-1,6-bisphosphate all the steps are shared by glycolysis and gluconeogenesis and are reversible.

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

Integration of gluconeogenesis and glycolysis The lactate produced by anaerobic glycolysis in muscle is released into the bloodstream and taken up by the liver where gluconeogenesis occurs. The glucose is then released into the bloodstream and supplied to the muscles to sustain further glycolysis. If muscle activity has stopped, the glucose is used to replenish the supplies of glycogen through glycogenesis.

The issue of phosphofructokinase-2 will be revisited when the interaction between carbohydrates and lipid synthesis will be discussed. Gluconeogenesis is mediated by glucagon released from the pancreas when blood glucose is low. Glucagon activates Protein Kinase A (a cyclic AMP-regulated kinase) which triggers phosphorylation of regulatory proteins resulting in inhibition of liver glycolysis and stimulation of gluconeogenesis. Insulin counteracts glucagon by inhibiting gluconeogenesis. In type 2 diabetes there is an excess of glucagon and insulin resistance. Insulin can no longer inhibit the gene expression of enzymes such as PEPCK which leads to increased activity of gluconeogenesis. The anti-diabetic drug metformin reduces blood glucose primarily through inhibition of gluconeogenesis, overcoming the failure of insulin to inhibit gluconeogenesis due to insulin resistance.

__________________________________________________________________ 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

This slide is a repetition of the two previous slides and could be useful for reviewing the coordination of the regulation of glycolysis and gluconeogenesis. Notice that the level of fructose 2,6-bisphosphate is high in the fed state and low in starvation. Another important control is the inhibition of pyruvate kinase by phosphorylation during starvation.

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 reshuffling takes place in the Krebs’s cycle.