Regulation of glycolysis and gluconeogenesis

Dr. Vér Ágota

Gluconeogenesis (Synthesis of glucose) from pyruvate utilizes many of the same enzymes as Glycolysis.

Three Glycolysis reactions have such a large negative G that they are essentially irreversible. These steps must be bypassed in Gluconeogenesis.Glycolysis occurs in all tissues

Gluconeogenesis occurs mainly in liver (to a more limited extent in kidney & small intestine under some conditions). 

Irreversible glycolytic stepsbypassed

1. Glucokinase (GK)

2. Phosphofructokinase-1 (PFK-1)

3. Pyruvate kinase (PK)

by Glucose-6-phosphatase

by Fructose 1,6-bisphosphatase (FBP-1)

by Pyruvate Carboxylase (PC) & Phosphoenolpyruvate carboxykinase (PEPCK)

glycolysis gluconeogenesis

Production: gluconeogenesis (6 high energy bound is required)

Utilization: glycolysis ( net output 2 ATP)

Futile cycling very wastefulDirection and magnitude of substrate

movements controlled

Glucose production vs utilisation

Control of glycolysis and gluconeogenesis

Regulated steps of glycolysis(glucose transportand the irreversiblereactions)

glucose

glucose

G6P

F6P

F1,6BP

PEP

pyruvate

GK

GLUT

PK

PFK1

pla

sma m

em

bra

ne

cytosolECspace

oxalacetate

PEPCK

FBPase

G6Pase

Regulated stepsIn gluconeogenesis

PC

Reaction of glucokinase and glucose 6-P phosphatase

glucose

Glucose-6-P

GK G-6-Pase

Properties of Glucokinase and Hexokinase

Substrate specificity Glucose Hexose

GK regulation

Protein-protein interaction (F-6-P, F-1-P, glucose)

Gene transcription: insulin, feeding + glucagon, fasting -

glucose 6-phosphate

Regulation of activity of Glucokinase (GK ) regulatory protein (RP)

fructose 6-phosphate

fructose 1,6-bisphosphate

ATP

ADPPFK1

glucoseGK active

glucose

+

+

fructose

fructoseFructose 1-phosphateFK

-

GK-RPinactive

RP

Glucokinase – glucose sensor function

bloodglucose

cellInsulin secretion

cell glucokinase

hepatocyteglucokinase

- -

+

+

+ +

SP=stabilizing proteinT1= glucose 6-phosphate transporterT2= phosphate transporterT3= glucose transporter

Glucose -6-phosphatase system

Glucose -6-phosphatase system Liver, kidney, pancreatic beta cells, gall bladder,

testis, spleen, intestines Km : 2 mM G-6-P (0.2 mM) the flux though this step is

proportional to i.c. G-6-P At 5 mM level GK activity is balanced by

opposing activity of G-6-P phosphatase

G-6-P phosphatase is activeted in starvation and diabetes

Gene transcription occurs similarly as PEPCK (insulin inhibits)

Induced after birth

Regulation of glycolysis (PFK1) and gluconeogenesis (Fructose1,6-Bisphosphatase)

The opposite effects of adenine nucleotides on Phosphofructokinase (Glycolysis) Fructose-1,6-bisphosphatase (Gluconeogenesis)

ensures that when cellular ATP is high (AMP would then be low), glucose is not degraded to make ATP.

When ATP is high it is more useful to the cell to store glucose as glycogen.

When ATP is low (AMP would then be high), the cell does not expend energy in synthesizing glucose.

Phosphofructokinase 1

Allosteric inhibitors: ATP, citrate, fatty acidsActivators: AMP, F-2,6-P Irreversible, main place of regulation

„committed step”Citrate increases the inhibitory effect of ATPF-2,6-P –inhibition of the inhibitory effect of ATP T – R transitionTetramer structure (370 kD) – sigmoidal curveM:muscle, P:platelet, L:liver isoenzymes

PFK1 is inhibited by ATP P

FK

act

ivit

y

0

10

20

30

40

50

60

0 0.5 1 1.5 2[Fructose-6-phosphate] mM

PFK

Activ

ity

high [ATP]

low [ATP]

At high concentration, ATP binds at a low-affinity regulatory site, promoting

the tense conformation.

Sigmoidal dependence of reaction rate on [fructose-6-phosphate] is observed at high

[ATP ]

Phosphofructokinase, the rate-limiting step of Glycolysis, is allosterically inhibited by ATP.

At high concentration, ATP binds at a low-affinity regulatory site, promoting the tense conformation.

Sigmoidal dependence of reaction rate on [fructose-6-phosphate is observed at high [ATP ]

PFK activity in the presence of the globally controlled allosteric regulator fructose-2,6-bisphosphate is similar to that at low ATP.

Fructose-2,6-bisphosphate promotes the relaxed state, activating Phosphofructokinase even at high [ATP].

Thus activation by fructose-2,6-bisphosphate, whose concentration fluctuates in response to external hormonal signals, supersedes local control by [ATP].

The effect of F2,6BP on F1,6BP phosphatase

PFK1 – F1,6bPase co-ordinated regulation

fructose 6-phosphate

PFK1

ATP

ADP

Pi

H2O

F1,6-bPase

fructose 1,6-bisphosphate

GLIC

OLY

SY

SG

LUC

ON

EO

GE

NE

SIS

F 2,6-bP

AMP, ADP

ATP, citrate

tandem enzymeinsulin glucagon

ATP

ADP

Pi

H2O

Reciprocal control

FA, H+

Fructose 2,6 –bisphosphate is not a glycolyses intermediate

Phosphorylated form:phosphataseDephosphorylated form:kinase

PFK2/FBPase2

Pyruvate kinase

Tissue-specific isoenzymes.

PK-L (in liver) is regulated allostericallyFeedforward activation by F-1,6 BP +, alanin, ATP -. and hormonally (cAMP dependent phosphorylation = inactivation)

PK-M (in skeletal muscle) is not regulated.

Regulation of pyruvate kinase in liver

fructose 1,6-bisphosphate

phosphoenolpyruvate

pyruvate

ADP

ATPPK-L

ATPalanine

feed-forward activation

INSULIN

GLUCAGONphosphorylation

dephosphorylation

When gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose. Oxaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The increase in [acetyl CoA] activates Pyruvate Carboxylase to make oxaloacetate.

Pyruvate Carboxylase (pyruvate oxaloacetate) is allosterically activated by acetyl CoA.

[Oxaloacetate] tends to be limiting for Krebs cycle.

Glucose-6-phosphatase glucose-6-P glucose

Gluconeogenesis Glycolysis

pyruvate fatty acids

acetyl CoA ketone bodies oxaloacetate citrate

Krebs Cycle

PEPCK

Not an allosteric enzymeInduced by glucagon

Coordinated Regulation of Gluconeogenesisand GlycolysisSUMMARY

Regulation of enzyme quantity

Fasting: glucagon, cortisol induces gluconeogenic

enzymes represses glycolytic enzymes liver making glucose

Feeding: insulin induces glycolytic enzymes represses gluconeogenic

enzymes liver using glucose

Coordinated Regulation of Glycolysis and Gluconeogenesis

Allosteric EffectsPyruvate kinase vs Pyruvate

carboxylase PK - Inhibited by ATP and alanine PC - Activated by acetyl CoA

PFK-1 vs FBPase-1 PFK-1 activated by AMP and & F2,6P2

FBPase-1 inhibited by AMP & F2,6P2

Coordinated Regulation of Gluconeogenesis and Glycolysis

Short-term Hormonal Effects Glucagon, Insulin

cAMP & F2,6P2

PFK-2 & FBPase-2 A Bifunctional enzyme cAMP

Inactivates PFK-2 Activates FBPase-2 Decreases F2,6P2

• Reduces activation of PFK-1• Reduces inhibition of FBPase-1

Low blood sugar results in High gluconeogenesis Low glycolysis

 

glycolysis and gluconeogenesis Summary

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

Digestion of carbohydrate

GI Tract Functions

•Digestion -breakdown of complex macromolecules to di-& monomeric molecules.

•Absorption-fuels traverse GI track to cells & tissues of the body.

•Fuel sources.–Carbohydrates.–Lipids.–Proteins.

Dietary carbohydrate from which humans gain energy enter the body in complex forms, such as disaccharides and the polymers starch (amylose and amylopectin) and glycogen.

The polymer cellulose is also consumed but not digested.

The first step in the metabolism of digestible carbohydrate is the conversion of the higher polymers to simpler, soluble forms that can be transported across the intestinal wall and delivered to the tissues.

Digestion of carbohydrates

The breakdown of polymeric sugars begins in the mouth. Saliva has a slightly acidic pH of 6.8 and contains lingual amylase that begins the digestion of carbohydrates. The action of lingual amylase is limited to the area of the mouth and the esophagus; it is virtually inactivated by the much stronger acid pH of the stomach. Once the food has arrived in the stomach, acid hydrolysis contributes to its degradation; specific gastric proteases and lipases aid this process for proteins and fats, respectively. The mixture of gastric secretions, saliva, and food, known collectively as chyme, moves to the small intestine.

Amylase

The α-amylases are calcium metalloenzymes, completely unable to function in the absence of calcium. By acting at random locations along the starch chain, α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin.

In animals, it is a major digestive enzyme and its optimum pH is 6.7-7.0.

In human physiology, both the salivary and pancreatic amylases are α-Amylases.

Sucrase

Sucrase is the name given to a number of enzymes that catalyse the hydrolysis of sucrose to fructose and glucose

Sucrose intolerance (also known as Congenital Sucrase-Isomaltase Deficiency (CSID) or Sucrase-isomaltase deficiency) occurs when sucrase is not secreted in the small intestine. With sucrose intolerance, the result of consuming sucrose is excess gas production and often diarrhea and malabsorption.

Sucrase is secreted by the tips of the villi of the epithelium in the small intestine. Its levels are reduced in response to villi-blunting events such as celiac sprue and the inflammation associated with the disorder. The levels increase in Pregnancy/Lactation and Diabetes as the villi hypertrophy.

Lactase

Lactase (LCT), a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharide lactose into constituent galactose and glucose monomers.

In humans, lactase is present predominantly along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine.

Lactase is essential for digestive hydrolysis of lactose in milk. Deficiency of the enzyme causes lactose intolerance.

Carbohydrate Digestion & Absorption

Carrier mechanisms for monosaccharides(glucose, fructose and galactose).

–Na+-independent facilitated diffusion. Fructose transport. In conjunction with glucose transporter (GLUT-5). –Located on serosalside of enterocytemembrane. –Moves glucose into capillaries. –Fructose moves down its concentration gradient.

5Glut5

Sodium-dependent glucose cotransporters

Sodium-dependent glucose cotransporters are a family of glucose transporter found in the intestinal mucosa of the small intestine (SGLT1) and the proximal tubule of the nephron (SGLT2 and SGLT1). They contribute to renal glucose reabsorption.

These proteins use the energy from a downhill sodium gradient to transport glucose across the apical membrane against an uphill glucose gradient. Therefore, these co-transporters are an example of secondary active transport. Both SGLT1 and SGLT2 are known as symporters since both sodium and glucose are transported in the same direction across the membrane.

Passive transport - GLUTs

Facilitated diffusion of glucose through the cellular membrane is catalyzed by glucose carriers (protein symbol GLUT, gene symbol SLC2 for Solute Carrier Family 2) that belong to a superfamily of transport facilitators (major facilitator superfamily).

Molecule movement by such transporter proteins occurs by facilitated diffusion. This makes them energy independent.

GLUT transporters

Plasma membrane carriers of glucose.Catalyze facilitated diffusion (passive, bi-directional trp.)12 transmembrane helices.More than 5 isoforms with different function and characteristics.

GLUT transporters

GLUT transportersGLUT1 and GLUT3high affinity (KM ≈ 1 mM)Expressed in every cell except hepatocytes (liver) and pancreatic β-cells.Ensures steady glucose uptake in RBC, CNS, kidney medulla, testis(glucose-dependent cells). Blood- brain, blood placenta- barrier

GLUT2low affinity (KM ≈ 15 mM)Expressed in hepatocytes and pancreatic β-cells (glucose sensor cells).Makes glucose uptake proportional with blood glucose concentration.

GLUT4Intermediate affinity (KM ≈ 5 mM)Insulin-dependent expression in skeletal muscle and adipocytes (facultative glucose consuming cells).Adjusts glucose consumption to availability.

GLUT5Expressed in intestinal epithelial cells and kidney tubular epithelial cells.Participates in glucose absorption and re-absorption.