Gluconeogenesis Some tissues, such as brain, RBCs, kidney medulla, testes, embrionic tissues and exercising muscle require a continuing supply of glucose as a metabolic energy. The human brain requires over 120 gm of glucose per day. Mammalian cells make glucose from simpler precursors. Liver glycogen can meet these needs only for 10 to 18 hours without dietary carbohydrate.

During a prolonged fast, Hepatic liver stores are depleted, glc is formed from other molecules such as Lactate Pyruvate Glycerol Alpha keto acids The formation of glc from nonhexose precursors is called gluconeogenesis (formation of new sugar).

Glycolysis and Gluconeogenesis in the liver

Gluconeogenesis

Pyruvate precursors n The direct Glc reserves are sufficient to meet Glc needs for about a day! n Gluconeogenic pathway makes Glc from pyruvate precursors. n Triacyl glycerol > Glycerol + Fatty acids n Glycerol is a precursor of glc, glycerol enters glycolytic pathway as dihydroxyacetone phosphate.

Gluconeogenesis is NOT a reversal of glycolysis n Several reactions MUST differ because of the irreversible steps. HK (hexokinase) PFK (phosphofructokinase) PK (pyruvate kinase)

Let’s make Glc from pyruvate 1. Carboxylation of pyruvate –Pyruvate+CO 2 +ATP+H 2 O-----> OA+ADP+Pi+2H Enzyme:Pyruvate carboxylase OA+GTP----->PEP+GDP+CO 2 Enzyme: PEP-carboxykinase

Domain structure of pyruvate carboxylase ATP grasp: activate bicarbonate ions and transfers CO 2 to the biotin domain. From there CO 2 is transferred to pyruvate..

Carboxylation of pyruvate n Pyruvate carboxylase contains BIOTIN, which is covalently bound to the enzyme through lysine Enzyme + CO 2 + ATP-----> Carboxybiotin-enzyme +ADP +Pi Carboxybiotin-enzyme + pyruvate >OA + Enzyme –BIOTIN carries CO 2...

Biotin is covalently attached group u Biotin serves as a carrier for activated CO 2.   -amino group and carboxylate group of biotin are linked. u CO 2 is found mainly as HCO 3 in our system. u When Acetyl CoA is high then biotin is carboxylated. u The activated carboxyl group is transferred from carboxybiotin to pyruvate to form oxaloacetate.

2. Transport of OA to the cytoplasm Pyuvate carboxylase is a mitochondrial enzyme, whereas the other enzymes in gluconeogenesis are cytoplasmic. OA should be transported to the cytoplasm. How? It is reduced to MALATE first then transferred to the cytoplasm. In the cytoplasm, it is reoxidized to OA.

3. Decarboxylation of cytoplasmic OA n OA is decarboxylated and P-lated by PEP carboxykinase in the cytosol (PEP is made then!) n The overall reaction catalyzed by the combined action of pyruvate carboxylase and PEP carboxykinase provides a pathway from –Pyruvate >PEP –Therefore, once PEP is formed, it enters the reversed reactions of glycolysis until it reaches F-1,6 Bisphosphate!

4. Dephosphorylation of F-1,6BP n Fructose 1,6-bisphosphate + H 2 O------> F-6-P + Pi Enzyme: Fructose1,6-bisphosphatase This enzyme plays an important role in regulation. It is inhibited by F 2,6 BisP, an allosteric modifier whose concentration is influenced by the levels of circulating glucagon. This enzyme is found in liver and kidney.

5. Generation of free Glc n Dephospharylation of Glc 6-P n Glc 6-P + H 2 O------> D-Glc + Pi Enzyme: Glc 6-phosphatase It is found in liver and kidney but not in muscle and brain. Thus, muscle and brain cannot make Glc by gluconeogenesis Type I glycogen storage disease results from an inherited deficiency of glc 6-phosphatase.

Freeing Glc u The final step, freeing Glc takes place in ER lumen where it is hydrolyzed to Glc by Glc 6-Phosphatase which is a membrane bound enzyme. u Calcium binding protein (SP) is necessary for phosphatase activity. u Glc and Pi are shuttled back to the cytosol by a pair of transporters. u The glucose transporter in the ER membrane is like those found in the plasma membrane.

Gluconeogenesis is energetically costly! n The stoichiometry of gluconeogenesis is: 2pruvate + 4ATP + 2GTP + 2NADH + 6H 2 O >Glc 4ADP + 2GDP + 6Pi + 2NAD + + 2H + n In contrast, the stoichiometry of reversal of glycolysis is: 2 pyruvate + 2ATP + 2NADH + 2H2O > Glc + 2ADP + 2Pi + 2NAD + The difference is 4ATP, this is needed to turn energetically unfavorable process to a favorable one!

Gluconeogenesis and glycolysis are reciprocally regulated u Both glycolysis and gluconeogenesis are highly exorgonic under cellular conditions so there is no thermodynamic barrier. u But, amounts and activities of the distinctive enzymes of each pathway are controlled so that both pathways are not highly active at the same time.

Substrate cycles u F-6-P >F 1,6BisP u < u A pair of reactions such as the above one is called “substrate cycle” u There is also some cycling in irreversible reactions. u “Imperfection” in metabolism? u They are sometimes referred as “futile cycles” Futile cycles amplify metabolic signals! The other potential biological role of substrate cycles is the generation of heat produced by the hydrolysis of ATP.

Lactate and alanine formed by contracting muscle are used by other organs u Lactate is a dead end in metabolism. u Lactate should be converted to pyruvate. u The plasma membranes of most cells are highly permeable to lactate and pyruvate, therefore they easily diffuse to go to liver! u Excess lactate enters the liver and is converted pyruvate first then glucose. Thus, the liver restores the level of glucose necessary for active muscle cells, which derive ATP from the glycolytic conversion of glucose into lactate. Contracting skeletal muscle supplies lactate to the liver, which uses it to make glucose. These reactions constitute CORI CYCLE.

LDH enzyme u Lactate > Pyruvate by LDH (lactate dehydrogenase). u The interconversion of pyruvate and lactate are done by different subunits of LDH. LDH is a tetramer. u H---> in he heart u M---> in the muscle

The Cori Cycle

Cooperation between glycolysis and gluconeogenesis

REGULATION u 1. Control point: Pyruvate carboxylase, Acetyl CoA is a + allosteric modulator for pyruvate carboxylase enzyme. –Glc is made from pyruvate when there is a lot Acetyl CoA(more Acetyl CoA than TCA cycle can handle) –Acetyl CoA inhibits pyruvate dehydrogenase enzyme but stimulates pyruvate carboxylase. u 2. Control point: F 1,6 bisphoshatase reaction u 3.Control point: Hormonal control: F-2,6 bisphosphate

Two alternative fates of pyruvate

2 nd control point

Hormonal Control u The special role of liver to maintain constant blood glucose level requires additional control mechanisms. u When blood glucose decreases, glycogen increases and glucose is released. u This hormonal regulation in liver is mediated by fructose- 2,6-bisphosphate, which is a allosteric effector for PFK-1, and F-1,6-bisphosphate

Role of F2,6BP in regulation of Glycolysis and Gluconeogenesis

What is F-2,6-BP? u It is structurally related to F-1,6-BP. u It is not an intermediate. u It is a “regulator” u F-2,6-BP activates PFK-1 and glycolysis. u FBPase and PFK-2 are part of the same enzyme! u An increase in glucagon (during starvation) leads to a decrease in F-2,6-BP overall which goes to a decrease in glycolysis, an increase in glucone ogenesis u A decrease in glucagon (after carbohydrate rich diet) leads to an increase in F-2,6-BP and an increase in glycolysis. u Therefore, F-2,6-BP acts as an intracellular signal indicating “glucose abundant”.