Low Intracellular Free Glucose Is Low Compared with Extracellular Glucose

1.

- low intracellular free [glucose] is low compared with extracellular [glucose]

- glucose enters cells via carrier-mediated facilitated diffusion (GLUT-4 transporter) that is enhanced in adipose and muscle cells by insulin

- Vmax increased à increase in number of transporters; not a Km change (increased binding affinity)

- glucose transporters associated with membrane fragments stored in Golgi

- transporters translocated to plasma membrane à fusion; increase in GLUT-4 transporters

- insulin receptor inactivated à excess GLUT-4 returns to Golgi

- Liver doesn’t promote transport of glucose into hepatocytes (no transporters stored in Golgi)

- Liver uses GLUT-2 transporter à high capacity to handle increased blood [glucose] after a meal

- Insulin indirectly enhanced net inward flux of glucose by converting intracellular glucose to glucose-6-P through induction of glucokinase

2. Regulation of glucose phosphorylation

- allosteric control of hexokinase (low Km, high affinity); feedback inhibition but its product glucose-6-P à prevents accumulation of phosphorylated glycolytic intermediates that would trap phosphate needed for ATP synthesis (excess glucose-g-6-P promotes the storage of glycogen for the same reason)

- hormonal control of glucokinase (high Km, low affinity); liver and pancreas use glucokinase ensuring that when glucose is in excess it continues to be metabolized in these tissues

- in liver à insulin induces synthesis of glucokinase in the fed state; involves increased synthesis of a specific protein by signaling DNA to generate mRNA from the region that encodes for that protein; induction important for the prevention of hyperglycemia after a large meal containing many carbs

3. Regulation of phosphofructokinase-1 (PFK-1)

Reaction that opposes PFK-1

- PFK-1 is rate-determining step of glycolysis

- fructose-1,6-BPase catalyzes conversion of fructose-1,6-BP to fructose-6-P; gluconeogenic pathway in liver; also present in muscle to permit conversion of lactate à glycogen

- no regulation of opposing reaction à energy consumed because fructose-1,6-BPase does not regenerate ATP consumed by PFK-1

Activation of PFK-1 by AMP/inhibition of PFK-1 by ATP

- energy low in muscle à glycolysis increases

- AMP regulates PFK-1; it signal this need for energy in the muscle cell

- ATP declines à [AMP] increases 100-fold à signal that energy availability is low

- muscle in resting state à [AMP] is low

- AMP also activates glycogenolysis (glycogen à glucose-6-P)

- AMP is an allosteric activator of PFK-1

- when [ATP] is high, [AMP] is low; ATP inhibits PFK-1

- AMP inactivates fructose-1,6-BPase to prevent wasteful use of ATP

Inhibition by acidic conditions

- during anaerobic carbohydrate metabolism in muscle, lactic acid is a product

- excess production of acid à lower intracellular pH; to prevent further decline of cell pH through production of more acid via metabolism à elevated [H+] concentration inhibits PFK-1 to slow acid production

Inhibition by citrate

- citrate is a feedback inhibitor of glycolysis by allosterically inactivating PFK-1

- fed state à citrate derived from glucose carbons for fat production; excess accumulation of citrate in cytoplasm à glycolysis inhibited to reduce this supply of carbons

- excess citrate serves as a signal for carbon accumulation; activates F-1,6-BPase

Activation by fructose-2,6-BP

- most important regulator of glycolysis in liver is fructose-2,6-BP

- intracellular [fructose-2,6-BP] related to amount of glucose in blood

- blood glucose levels increase after a meal à amount of fructose-2,6-BP increases in parallel

- hepatic glycolysis generates pyruvate that is converted to acetyl CoA (can be used for fatty acid synthesis à provides a means of storing excess dietary carbohydrates that isn’t glycogen)

- excess glucose in blood after a meal à intracellular liver [fructose-2,6-BP] rises to activate PFK-1 à glycolysis

- fructose-2,6-BP inhibits fructose-1,6-BPase (FBPase-1) à gluconeogenesis

4. Metabolism and regulation fructose-2,6-BP pathway

Pathway

- formation of fructose-2,6-BP catalyzed by PFK-2

- breakdown of fructose-2,6-BP catalyzed by fructose-2,6-BPase (FBPase-2)

- PFK-2 uses fructose-6-P from glycolytic pathway à product of kinase reaction (fructose-2,6-BP) is only a regulator of PFK-1 and fructose-1,6-BPase

Regulation under high glucose conditions

- PFK-2 and FBPase-2 involved in fructose-2,6-BP metabolism for a single protein complex called a bifunctional enzyme

- high glucose in blood after meal à circulating insulin increases à signal in cell that causes PFK-2 activity to be expressed and FBPase-2 to be suppressed

- fructose-6-P (increased during metabolism of dietary glucose) activates PFK-2; allosteric effects on bifunctional enzyme

- fructose-6-P inhibits FBPase-2

- glucose and insulin increase in blood à fructose-2,6-BP produced in liver in response to both hormonal and allosteric regulation à increase in glycolytic rate by allosterically activating PFK-1 to maximally process dietary glucose in liver

Regulation under low glucose conditions

- when blood glucose falls à increased glucagon in circulation à phosphatase activity (FBPase-2) is expressed, kinase activity (PFK-2) is suppressed

- active fructose-2,6-BPase (FBPase-2) degrades fructose-2,6-BP so that stimulation of glycolysis is lost à hepatic use of glucose ceases under conditions when blood glucose is low

5. Regulation of pyruvate kinase

Inhibition by citrate and ATP

- liver glycolysis is regulated by pyruvate kinase; regulation of this enzyme coordinates with control of PFK-1

- citrate and ATP are allosteric inhibitors of both enzymes

- prevent accumulation of phosphorylated intermediates

Allosteric activation

- pyruvate kinase activated by fructose-1,6-BP and phosphoenolpyruvate (PEP) ensuring that glycolytic intermediates between PFK-1 and pyruvate kinase are kept at minimal concentration

- flux through PFK-1 increases à [fructose-1,6-BP] increases à pyruvate kinase activated

Inhibition by alanine

- alanine is the primary amino acid precursor for glucose synthesis

- essential that glycolysis be shut off when liver is synthesizing glucose

- alanine allosterically inhibits pyruvate kinase

6. Regulation of glucagon and insulin of pyruvate kinase via covalent modification

Hormonal regulation

- liver pyruvate kinase activity decreases following its phosphorylation in response to glucagon

- pyruvate kinase activity increases under high blood glucose conditions because insulin promotes dephosphorylation of the enzyme

- insulin/high blood glucose à increased activity of liver glycolysis by causing activation of both PFK-1 (by raising fructose-2,6-BP levels by activating PFK-2) and pyruvate kinase (by dephosphorylation)

- starvation/low blood glucose à glucagon reverses effects on both enzymes: decreases PFK-1 activity (by decreasing fructose-2,6-BP via activating FBPase-2) and decreases pyruvate kinase activity (phosphorylation)