L4 Gluconeogenesis

* Carbohydrate metabolism Intermediary Metabolism

Dr. ABDULHUSSEIN AlGENABI
Suggested reference:Champe, Harvey and Ferrier, Lippincott’s Illustrated Reviews – Biochemistry, 3rd Edition

* Kinetic properties of glucose transporters

Uptake in liver and pancreas b-cells is proportional to plasma concentration
GLUT-2
GLUT-3
GLUT-1
Uptake in brain is independent of plasma concentration over physiological range
Km = concentration at which half maximum rate of transport occurs (1/2 Vmax)


* Intracellular pool of GLUT4 in membranous vesicles translocate to the cell membrane when insulin binds to its receptor. The presence of more receptors increases the Vmax for glucose uptake (does not affect Km). When insulin signal is withdrawn, GLUT4 proteins return to their intracellular pool. GLUT4 is present in muscle and adipose tissue.
GLUT4 activity is regulated by insulin-dependent translocation

* Glucose action in the b-cell

Glucose enters the b-cell as blood glucose concentration rises. Glycolysis to generate ATP closes K+ channels in the cell membrane, stopping outward transport, and opening Ca+ channels. Inward flux of Ca+ causes exocytosis of insulin-containing secretory vesicles. Glucose also stimulates synthesis of new insulin.

* Fate of glucose in muscle

GLUT4
Glucose
Glucose
Glucose-6-P
Hexokinase
Glycogen synthesis
Glycolysis
Insulin
+

* Glycogen accumulation in muscle

* Fate of glucose in adipocytes
GLUT4
Glucose
Glucose
Insulin
+
Glucose-6-P
Hexokinase
LPL
Insulin
+
Glycerol-3-P
Triglycerides
Fatty acids
Insulin
-
Lipoproteins

* How is metabolism regulated?

Fast mechanisms, for immediate changes
Substrate concentration Allosteric regulation (feedback, feed forward) Phosphorylation-dephosphorylation Signals emanating from hormone action
Slow mechanisms, for long-term changes
Genetic regulation Response to diet and other environmental variables

* long term effects

How is metabolism regulated?
Rapid effect
Rapid effects

* Overview of glucose metabolic pathways

Glycolysis: from G6P to pyruvate Gluconeogenesis: from oxaloacetate to G6P Glycogen synthesis: from G6P to glycogen Glycogenolysis: from glycogen to G6P TCA cycle
The pathways must be carefully regulated to keep pathways going in opposite directions from proceeding simultaneously.

* Control points in glycolysis

hexokinase
Glucose-6-P
-
*

* Why is phosphofructokinase, rather than hexokinase, the key control point of glycolysis?

Because glucose-6-phosphate is not only an intermediate in glycolysis. It is also involved in glycogen synthesis and the pentose phosphate pathway. PFK catalyzes the first unique and irreversible reaction in glycolysis.

* Hormonal control of F-2,6-P2 levels and glycolysis

Hormonal regulation of bifunctional enzyme Glucagon (liver) or epinephrine (muscle) increase cAMP levels, activate cAMP-dependent protein kinase. In liver, this leads to decreased F-2,6-P and inhibits glycolysis. The effect is opposite in muscle; epinephrine stimulates glycolysis. Insulin decreases cAMP, increases F-2,6-P stimulates glycolysis. Phosphorylation of PFK2 by protein kinase activates its phosphatase activity on F2,6P in liver.

* Gluconeogenesis

Mechanism to maintain adequate glucose levels in tissues, especially in brain (brain uses 120 g of the 160g of glucose needed daily). Erythrocytes also require glucose. Occurs exclusively in liver (90%) and kidney (10%) Glucose is synthesized from non-carbohydrate precursors derived from muscle, adipose tissue: pyruvate and lactate (60%), amino acids (20%), glycerol (20%)

* Gluconeogenesis takes energy and is regulated

Converts pyruvate to glucose Gluconeogenesis is not simply the reverse of glycolysis; it utilizes unique enzymes (pyruvate carboxylase, PEPCK, fructose-1,6-bisphosphatase, and glucose-6-phosphatase) for irreversible reactions. 6 ATP equivalents are consumed in synthesizing 1 glucose from pyruvate in this pathway
hexokinase
Glucose-6-P
-
Glucose 6-phosphatase

* Irreversible steps in gluconeogenesis

First step by a gluconeogenic-specific enzyme occurs in the mitochondria
pyruvate oxaloacetate
Pyruvate carboxylase
Once oxaloacetate is produced, it is reduced to malate so that it can be transported to the cytosol. In the cytosol, oxaloacetate is subsequently dexcarboxylated/phosphorylated by PEPCK (phosphoenolpyruvate carboxykinase), a second enzyme unique to gluconeogenesis.
The resulting phosphoenol pyruvate is metabolized by glycolysis enzymes in reverse, until the next irreversible step

* Irreversible steps in gluconeogenesis (continued)

Fructose 1,6-bisphosphate + H2O
fructose-6-phosphate + Pi
Fructose 1,6- Bisphosphatase (FBPase)
In liver, glucose-6-phosphate can be dephosphorylated to glucose, which is released and transported to other tissues. This reaction occurs in the lumen of the endoplasmic reticulum.
Requires 5 proteins!
2) Ca-binding stabilizing protein (SP)
1) G-6-P transporter
3) G-6-Pase
4) Glucose transporter 5) Pi transporter

* Gluconeogenesis and Glycolysis are reciprocally regulated

Fructose 1,6-bisphosphatase is main regulatory step in gluconeogenesis. Corresponding step in glycolysis is 6-phosphofructo-1-kinase (PFK-1). These two enzymes are regulated in a reciprocal manner by several metabolites.
Fructose-6-phosphate
Fructose 1,6-bisphosphate
6-phosphofructo -1-kinase
Fructose 1,6-bisphosphatase
+ Citrate - AMP - F 2,6-BP
Citrate - AMP + F 2,6-BP +
Reciprocal control—prevents simultaneous reactions in same cell.


* GLUCOSE
G-6-Pase
GK
G-6-P
F-6-P
P-ENOLPYRUVATE
PEPCK
PK
PYRUVATE
OXALOACETATE
FBPase 1
PFK 1
F-1,6-P2
Glycolysis
Gluconeogenesis
Increase Hepatic Glucose Utilization
Decrease Hepatic Glucose Output

* GLUCOSE

G-6-Pase
GK
G-6-P
F-6-P
P-ENOLPYRUVATE
PEPCK
PK
PYRUVATE
OXALOACETATE
FBPase 1
PFK 1
F-1,6-P2
Glycolysis
Gluconeogenesis
Decrease Hepatic Glucose Utilization
Increase Hepatic Glucose Output



During anaerobic glycolysis, there is a net production in the cytosol of lactate and NAD+ NADH CO2 ATP FADH2
QUIZ-1


During glycolysis, when each molecule of glucose is catabolized to two molecules of pyruvate, most of the potential energy contained in glucose is: A) transferred to ADP, forming ATP. B) transferred directly to ATP. C) retained in the two pyruvates. D) stored in the NADH produced. E) used to phosphorylate fructose to form fructose 6-phosphate.
QUIZ-2