GLUCONEOGENESIS Summary of handout: Ferchmin 2014 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 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?

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. 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 be synthesized in glycolysis. But lipids do not substitute 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 liver and a small fraction in 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.

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

Gluconeogenesis takes place in the cytosol and in the mitochondria Gluconeogenesis takes place in the cytosol and in the mitochondria. There are two pathways to generate PEPA (phosphoenolpyruvic acid). In both pathways NADH must be generated to allow the activity of glyceraldehyde-3-phosphate dehydrogenase in the reduction of 3-phosphoglyceric acid. 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.

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

Integration of gluconeogenesis and glycolysis

This sketch is for you to review the coordinated regulation of both pathways.

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!!! 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. Galactose, fructose, etc are not glucogenic. They are monosaccharides in equilibrium with glucose! Warning: whoever says that fatty acids with even number of carbons are glucogenic will be decapitated!

Warning: whoever says that fatty acids with even number of carbons are glucogenic will be decapitated!

The many fates of glucose-6-P Free glucose The many fates of glucose-6-P

Any suggestion that I am lecturing about the pentose shunt is a dirty lie!

PENTOSE PHOSPHATE SHUNT or HEXOSE MONOPHOSPHATE PATHWAY In glycolysis there was no net oxidation/reduction only “reshuffling” of the redox state of the carbons Ferchmin 2014 Pentose phosphate pathway or shunt (PPP). 2) Oxidative and isomerization parts. 3) Regulation 4) Metabolic roles of PPP 5) Reduction of glutathione. PENTOSE PHOSPHATE SHUNT or HEXOSE MONOPHOSPHATE PATHWAY This pathway consists of two parts: 1) Glucose-6-P undergoes two oxidations by NADP+, the second is an oxidative decarboxylation that forms a pentose-P. 2) The P-pentoses that are formed during the first part are transformed into glucose-6-P.

2) Nonoxidative steps of pentose phosphate shunt Positive regulator of lipid synthesis

Ferchmin 2014

Several nucleotides can activate sugars Several nucleotides can activate sugars. In the case of glucose for glycogen synthesis it is UTP. See next page The first reaction a), is reversible but it is coupled to b) that releases -5 kcal/mol and favors reaction a).

Sugar nucleotides of glucose or of any other carbohydrate are called activated because sugars bound to nucleotides can be transferred by specific enzymes to proteins and other molecules or can be subjected to enzymatic modifications possible only with activated sugars. UDP-glucuronic is important for detoxification of many drugs and metabolites. Below is shown the formula of conjugated bilirubin: We, primates, lost the ability to make ascorbate. Therefore we are genetically deficient.

Synthesis of uridinediphosphoglucose or UDPGlu Synthesis of Glycogen Synthesis of uridinediphosphoglucose or UDPGlu Do you remember phosphoglyceromutase? Any similarities with phosphoglucomutase? PPi is hydrolyzed by a pyrophosphorylase in a reaction coupled with the pyrophosphorylase to dissipate energy as heat thus making the synthesis of UDP-Glu thermodynamically favorable.

Why do we need to waste 2 ATPs and make glycogen? Deficiency of branching enzyme gives long branches. Causes death at about to years of age. Andersen’s disease The content of glycogen is about 10 % of the wet weight of the liver and 2% of muscle.

Glycogen synthase only adds glucoses to an existing chain of at least 4 glucose residues. Glycogenin acts by catalyzing the addition of glucose to itself (autocatalysis) by first binding glucose from UDP-glucose to the hydroxyl of Tyr-194 from UDP-glucose, by glycogenin's glucosyltransferase. Once sufficient residues have been added, glycogen synthase takes over extending the chain. Glycogenin remains covalently attached to the reducing end of glycogen.

Glycogen degradation The breakdown of glycogen and entry into glycolysis as glucose-6-P is achieved by three enzymes: glycogen phosphorylase, debranching enzyme and phosphoglucomutase. Glycogen phosphorylase produces glucose-1-P plus limit dextrin. The debranching enzyme has a transferase and glycosidase (hydrolase) activities. Hexokinase is bypassed when glucose comes from glycogen! Deficiency of phosphorylase (Mc Adler’s) causes muscle cramps and no lactate formation during exercise. Deficiency of debranching enzyme causes accumulation of limit dextrin In the next lecture we will begin with regulatory mechanisms involved in glycogen metabolism