-- GLUCONEOGENESIS

--

Definition: Definition the biosynthesis of glucose primarily from pyruvate and its precursors.

The gluconeogenesis pathway is similar to the reverse of glycolysis but differs at critical sites. control of these opposing pathways is reciprocal so that physiological conditions favoring one disfavor the other and vice versa. General principles of metabolic control -- a) pathways are not simple reversals of each other and b) under reciprocal control

Why do we produce glucose?
a) Need to maintain glucose levels within a narrow range in blood. b) Some tissue-- brain, erythrocytes, and muscles in exertion use glucose at a rapid rate and sometimes require glucose in addition to dietary glucose. c) The brain uses mostly glucose and erythrocytes can use only glucose as a source of energy.

Where is glucose synthesized?  The liver comes to rescue.  The major precursor for glucose biosynthesis is pyruvate. . The liver is the major location for gluconeogenesis.

 glycerol from certain lipids.most important is alanine  TCA cycle intermediates  propionate from breakdown of certsin fatty acids and amino acids. . forms pyruvate  some amino acid carbon skeletonsfrom diet or breakdown of muscle protein during starvation.What are the sources of pyruvate precursor?  lactate²from muscle.

-. Lactate is the primary source for pyruvate. (Notice glucose cannot be made from acetyl CoA . ultimately to glucose.This excess lactate cannot be further oxidized in muscle.In muscle. -.Lactate is released from the muscles to the blood and travels to the liver for conversion to pyruvate and. -. lactate is produced in great quantities during exertion.

other amino acids . alanine.beginning GDP + CO2 phosphoenolpyruvate GTP phosphoenolpyruvate carboxykinase BYPASS 1 oxaloacetate Some amino acids ADP + Pi ATP + CO2 pyruvate carboxylase (‚pyruvate kinase) PYRUVATE (3C) »lactate.Gluconeogenesis .

fructose 1.6-bisphosphate aldolase triose phosphate isomerase dihydroxyacetone phosphate glyceraldehyde 3-phosphate dehydrogenase glyceraldehyde 3-phosphate NAD+ +Pi NADH +H+ 1.3-bisphosphoglycerate ADP phosphoglycerate kinase ATP 3-phosphoglycerate phosphoglyceromutase 2-phosphoglycerate enolase phosphoenolpyruvate .

GLUCOSE glucose Pi (‚hexokinase) BYPASS 3 6-phosphatase Glucose 6-phosphate phosphoglucoisomerase Fructose 6-phosphate BYPASS 2 fructose 1.6-bisphosphate .6 bisphosphatase Pi (‚phosphofructokinase) Fructose 1.

Input: 2 pyruvate + 4 ATP + 2 GTP + 2 NADH Output: glucose + 4 ADP + 2 GDP + 2 NAD++ 6 Pi .Cost: The production of glucose is energy expensive.

In glycolysis. and pyruvate kinase . there are three irreversible kinase reactions at control points involving: hexokinase. phosphofructokinase. phosphofructokinase. hexokinase.

 The control points are the same for gluconeogenesis and for glycolysis.  The rest of the steps use the same enzymes as glycolysis. .In gluconeogenesis. these reactions must be forced the other way.  Four unique enzymes are used to bypass these irreversible steps.

Complex scheme. This bypasses pyruvate kinase.Bypass number 1. phosphoenolpyruvate. . Pyruvate to phosphoenolpyruvate.

NOTE: Pyruvate has transport system to enter mitochondria . Only this enzyme of the gluconeogenesis pathway is mitochondrial.a) pyruvate to oxaloacetate Enzyme = pyruvate carboxylase  located inside mitochondria.

.Carboxylations involving CO2 almost always use the vitamin biotin as a cofactor. Reaction: Reaction: pyruvate + CO2 + ATP + H2O ' oxaloacetate + ADP + Pi -.

-.Subreactions: Subreactions: Enz-biotin + ATP + CO2 + H2O ' Enz-carboxybiotin + ADP + Pi Enz-carboxybiotin + pyruvate ' Enz-biotin + oxaloacetate .

.pyruvate carboxylase - acetyl-CoA is positive modulator  absolutely required for activity  higher acetyl-CoA indicates that adequate carbon levels available for TCA cycle to provide energy glucose can be synthesized and exported from liver.  oxaloacetate important in the citric acid cycle. which is entirely mitochondrial.-.

 mitochondrial membranes are nearly impermeable to oxaloacetate. For gluconeogenesis. So how does it get out? . oxaloacetate must leave the mitochondria because all the rest of the gluconeogenesis enzymes are in the cytosol.

three steps malate dehydrogenase mitochondrial enzyme Oxaloacetate + NADH + H ' malate + (mitochondria) + NAD malate can be malate oxaloacetate transported through HO O the mitochondrial C-COO membrane HCH COO + .b) transport .

cytosolic enzyme + malate + NAD ' oxaloacetate + + (cytosolic) NADH + H .malate dehydrogenase -.

NET so far: pyruvate + ATP + GTP ¼ PEP + ADP + GDP + Pi .c) The GTP-dependent decarboxylation of oxaloacetate Enzyme = PEP carboxykinase cytosolic enzyme.  CO2 added is lost in this step. as all others oxaloacetate + GTP¼phosphoenolpyruvate + CO2 + GDP  uses GTP. not ATP.

High cost = two energy rich phosphates  so a total of four high energy bonds are already utilized here per glucose to be synthesized. Then uses glycolytic enzymes in steps to fructose-1.6-bisphosphate .

irreversible  enzyme is highly regulated F6P isomerizes to glucose-6-phosphate via phosphoglucoisomerase .6Fructose-1.6-bisphosphate + H2O ¼ fructose-6-P + Pi  bypasses phosphofructokinase  a simple hydrolysis.Bypass number 2. Fructose-1.6-bisphosphatase Reaction: fructose-1.  highly exergonic.6bisphosphate bisphosphate to fructose-6-phosphate fructoseEnzyme = fructose-1.

Glucose-6-phosphate Glucoseto glucose. irreversible  not present in muscle . Enzyme = glucose-6-phosphatase glucoseReaction: glucose-6-phosphate + H2O ¼ glucose + Pi  bypasses hexokinase & glucokinase  highly exergonic. glucose.Bypass number 3.

*************************************** Total Energy Cost = 6 high energy bonds used per glucose synthesized. four more than produced in glycolysis. These four are needed to convert pyruvate to PEP. ************************************** .

-.key enzymes targeted.CONTROL: -.gluconeogenesis serves as an alternative source of glucose when supplies are low and is largely controlled by diet.gluconeogenesis and glycolysis are controlled in reciprocal fashion. .high carbohydrate in meal reduce gluconeogenesis and fasting increases. -. -.

. inhibited by acetyl CoA.  pyruvate kinase is also inhibited by ATP and the liver form by alanine.  activated by acetyl-CoA (required) vs. PYRUVATE CARBOXYLASE.  high levels of acetyl-CoA signals that enough carbon substrate available for citric acid cycle.REGULATION OF GLUCONEOGENESIS: Key enzymes: 1. pyruvate kinase. CARBOXYLASE.

6-bisphosphate. FRUCTOSE 1.6-bisphosphate. high glucose    Recall that the reciprocal enzyme. . low energy  Strongly inhibited by fructose-2. in glycolysis.6 BISPHOSPHATASE BISPHOSPHATASE  Strongly inhibited by AMP.2. is strongly activated by AMP and fructose-2. phosphofructokinase.

Fructose-2. .6-bisphosphatase and phosphofructokinase.6-bisphosphate is the most important regulator of glycolysis and gluconeogenesis through its reciprocal effects on fructose 1.6Fructose-2.

PHOSPHOFRUCTOKINASE-2/ PHOSPHOFRUCTOKINASEFRUCTOSE BISPHOSPHATASE-2 BISPHOSPHATASEBifunctional enzyme fructose-6-phosphate + ATP ¼ fructose-2.6-bisphosphate ¼ fructose-6-phosphate + Pi Enzyme highly regulated to control levels of F 2.6-P .6-bisphosphate fructose-2.3.

6 bisphosphate Pi The next slides show how these activities are controlled .2 FBPase-2 ADP F2.PHOSPHOFRUCTOKINASE-2/ PHOSPHOFRUCTOKINASEFRUCTOSE BISPHOSPHATASE-2 BISPHOSPHATASEATP F-6-P PFK.

6 BP PFK. µgluconeogenesis increases F 2.FBPasePFK-2 FBPase-2 active inactive .FBPasePFK-2 FBPase-2 inactive active PKA = protein decreases F 2.6 BP kinase A ¶glycolysis.single proteintwo functions ATP glucagon PKA + (µcAMP) P ADP PFK.

Glucagon: hormone released when glucose levels are low.  signal to elevate blood glucose levels  increases intracellular levels of cAMP in liver and elsewhere  cAMP activates protein kinase A = cAMP-dependent protein kinase  stimulating gluconeogenesis and glycogenolysis .

Consequences A. If glucose¶ & ATPµ ¼ µcAMP ¼ bisP µFBPase-2 ¼ ¶F2. If glucoseµ & AMPµ ¼ µPFK-2¼ µF2.6 BPase¼µgluconeogenesis Also ¶PFK-1¼ ¶glycolysis .6bisP¼ µPFK-1¼ µglycolysis Also ¶F1.6bisP F2.6 bisP¼ µF1.6 BPase ¼¶gluconeogenesis B.

lactate.Summary of Gluconeogenesis Ø purpose.alternative source of glucose rather than dietary carbohydrates or glycogen breakdown Øprimary precursors are pyruvate. glycerol. part of fatty acids and certain amino acids (glucogenic) Ø 3 essentially irreversible steps of glycolysis are bypassed .

and phosphofructokinase-2/fructose bisphosphatase-2 .6 bisphosphatase.Ø regulated via pyruvate carboxylase. fructose 1.

Branches are linked 1´6.GLYCOGEN METABOLISM Glycogen: a highly branched polymer of glucose. . Chains have glycosidic links 1´4.

= glycogenesis .Glucose stored in polymeric form as glycogen mostly in the liver and skeletal muscle. = glycogenolysis  Enough glucose and energy triggers synthesis of glycogen.  Glucose can be rapidly delivered to the blood stream when needed upon degradation of glycogen.

.Glycogenesis = GLYCOGEN BIOSYNTHESIS ============================== Glucose is transported into the liver cell by a specific glucose transporter and immediately phosphorylated.Most of the glucose in a cell is in the form of glucose-6-phosphate. ============================== -.

glucose-1phosphate phosphate  reversible reaction allows G1P conversion to G6P in glycogenolysis .1.Conversion of glucose-6-phosphate to glucose-1-phosphate Enzyme = phosphoglucomutase -D-glucose-6-D.

2.Synthesis of Uridine Diphosphoglucose Enzyme = UDP-glucose pyrophosphorylase Reaction: glucose-1-phosphate + UTP ¼ + PPi Then PPi ¼ 2 Pi pyrophosphorylase UDP-glucose .

energy is used to activate glucose -. the commiting step  Phosphoryl transfer -.UTP is the energy equivalent of ATP -.two phosphates from UTP are lost as PPi  PPi is broken down by an enzyme PPi ¼ 2 Pi driving the reaction to the right .

3.Glycogen synthesis Enzyme = glycogen synthase UDP-glucose + (glucose)n¼UDP+(glucose)n+1 + glycogen .

 glucose always added to nonreducing end.  glycogen synthase is inhibited by phosphorylation. regulated by glucagon ( discussed later) . The glycosidic bond formed is (1 ´ 4).

Branching Enzyme = branching enzyme  introduces branching by transferring a teminal fragment of 6-7 residues from a growing chain to a 6-position farther back in a chain. .  branching accelerates the rate of glucose release during degradation.  makes a branch with an (1´6) link creating two ends to add glucose.4.

6   branching  bond   enzyme                   .      new    1.

Release of glucose-1-phosphate Enzyme = glycogen phosphorylase non+ Pi reducing zzz ends  glucose-1.GLYCOGENOLYSIS-DEGRADATION OF GLYCOGEN 1.-.+ zzz phosphate  .

4 glycosidic link is cleaved by phosphorylysis with retention of energy potential in the phosphate ester of glucose-1-phosphate. always acts at nonreducing end  1. .

 stops at fourth glucose from a 1,6 branch point  contrast with enzymes acting on starch and glycogen in the gut, which yield sugars, not sugar phosphates, as products.  activated by phosphorylation, regulated by glucagon and epinephrine

2. Debranching - two parts Enzyme = debranching enzyme (both) zzz  E (1´6) link  transferase  zzz Transfers chain of three glucoses to any nonreducing end

 E (1´6) link zzz debranching enzyme (glucosidase) zzz

+ 
= glucose

1,6 linkage cleaved

phosphoglucomutase then yields glucose-6-phosphate. which can be dephosphorylated or enter glycolysis.zzz glycogen phosphorylase or phosphorylase for short glucose-1-phosphate one at a time as previously shown -. .

Metabolic Regulation of Mammalian Glycogen Levels -. . -.Glycogen reserves are the most immediately available large source of metabolic energy for mammals.Storage and utilization are under dietary and hormonal control.

glucagon -.insulin  Primary enzyme targets in glycogen metabolism= glycogen phosphorylase and glycogen synthase.epinephrine (adrenaline) = ´fight-or-flightµ -. Primary hormones = -. . The actions of the hormones are indirect.

mashed potatoes.Example. wine Sleep immediately. CH2O ¼ high blood glucose levels ¼ higher insulin ¼ glycogenesis .hormones and diet Dinner at 9:00 pm -. sherbert for dessert.steak. sleep late During sleep: amino acids.

Wake late for class ¼adrenaline rush ¼ run to class glycogen ¼ glucose ¼ lactate epinephrine (= adrenaline) ¼ glycogenolysis .

A polypeptide hormone produced in -cells of the islets of Langerhan of the pancreas pancreas.Acts primarily on liver cells. -. -.Receptors on surface of liver cells.Stimulates glycogen breakdown & inhibits glycogenesis. -. -.Glucagon also blocks glycolysis & stimulates gluconeogenesis.low glucose levels -.======== HORMONES ========= Glucagon . .

Receptors on surface of cells.Stimulates glycogen breakdown & inhibits glycogenesis. Glucagon and epinephrine both stimulate intracellular pathway via increasing levels of cAMP.Epinephrine . -.Acts primarily on skeletal muscle.low glucose levels -. . -.

-. -.Increases glycogenesis in muscle. -.High levels of glucose induce release of insulin from -cells of islets of Langerhan in the pancreas. -.Intracellular signal pathway involves complex sequential phosphorylations and dephosphorylations.Insulin is polypeptide hormone. .Insulin -.Detected by receptors at surface of muscle cells.

-.A cascade is a mechanism in which enzymes activate other enzymes sequentially usually leading to an amplification of an initial signal.A cyclic AMP cascade is used by both epinephrine and glucagon. .cAMP Cascade -.

Epinephrine/Glucagon Cascade Regulating Glycogen Metabolism Outside Epinephrine AC Inside Receptor GProtein PM cAMP+PPi ATP AC = adenylate cyclase PM = plasma membrane .

activating it RR cAMP RR + C C cAMP cAMP CC tetramer dimer 2 active monomers R2C2 inactive R2 + 2C active Dissociation caused by conformational change induced by binding of cAMP Active kinase C subunits add phosphate to proteins using ATP .cAMP binds to protein kinase A.

inactivating it . activating it C monomer ATP + phosphorylase kinase Ä phosphorylase kinase-P + ADP active Note: subunit C also phosphorylates glycogen synthase. kinase.PKA catalytic C subunits phosphorylate phosphorylase kinase.

Active phosphorylase kinase transfers phosphate to glycogen phosphorylase.P Glycogen phosphorylase a (active form) then degrades glycogen: glycogen + Pi Ä glucose-1-P+(glucose)n-1 . activating this enzyme. ATP + phosphorylase b Ä ADP + phosphorylase a. phosphorylase.

-. glycogenolysis is inhibited.How is Glycogenolysis Inhibited? -.when need for glucose is over.a phosphoprotein phosphatase hydrolyzes the protein phosphates to reconvert reconvert: phosphorylase a ¼ phosphorylase b (inactive) and phosphorylase kinase (active) ¼ phosphorylase kinase (inactive) .

How is Glycogenesis Activated? Activated? -.Complex process stimulated by insulin.Insulin indirectly activates a phosphoprotein phosphatase: glycogen synthase(D) glycogen synthase(I) synthase(D)Äglycogen synthase(I) phosphorylated dephosphorylated less active active active dependent on G-6-P independent of G-6-P . -.

-. The less active phosphorylated glycogen synthase can be activated by high levels of glucose-6-phosphate. -.Another way: In active muscle.bypasses hormonal regulation . there may still be high glucose-6-phosphate. -.dephosphorylation is the major pathway for stimulation of glycogenesis in liver and resting muscles.

synthase decreasing its activity.epinephrine and glucagon inhibit glycogen synthesis. inactivating it.] . a) protein kinase A subunit C phosphorylates glycogen synthase. b) also phosphorylase kinase can phosphorylate glycogen synthase.How is Glycogenesis Inhibited? Inhibited? -. [It·s called synthase phosphorylase kinase because of its dual function.

Epinephrine and glucagon stimulate glycogenolysis and inhibit glycogenesis via a cAMP and a phosphorylation cascade.Glycogenesis is stimulated by insulin in a pathway ending in the dephosphorylation of glycogen synthase.Glycogenolysis is also inhibited via dephosphorylation. -.SIMPLISTIC SUMMARY: -. Ä releases glucose -. Ä takes up glucose .

Glycogen Storage Diseases: -.A family of serious. diseases caused by mutations in the enzymes involving in glycogen storage and breakdown. although not necessarily fatal. .

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