metabolic regulation

Integration of metabolism

PSBCTC302 – Intermediary metabolismM.Sc. Biochemistry

Biochemistry is connected to medicine

Nucleic acids

Genetic diseases

Proteins

Sickle cellAnemia,

PKU

Lipids

Atherosclerosis

Carbohydrates

Diabetesmellitus

From www.genome.ad.jp/kegg/pathway/map/map01100.html

CYTOPLASM

GlycolysisFatty acid synthesisPentose phosphate pathway

TCA cycleOxidative phosphorylationβ-oxidation of fatsKetone body formation

MITOCHONDRIA

Metabolic Compartmentalization

GluconeogenesisUrea Cycle

Glucose 6-P

Pyruvate

Metabolic Fates of Glucose 6-P, Pyruvate and Acetyl CoA

Ribose 5-P

Glucose 1-P Glycogen

Fructose 6-P Pyruvate

6-Phosphogluconate

Malonyl CoA TCA cycle Glucose

Lactate

Acetyl CoA OxaloacetateAlanine

Ethanol

Ketone bodies CO2 + H2OFatty acids

Cholesterol

CitrateHMG CoA

GLUCONEOGENESISGLYCOLYSIS

β-D-Glucose

ATP ADP

Mg2+

Hexokinase

β-D-Glucose 6-phosphate

β-D-Glucose 1-phosphate

Phospho glucomutase

Fructose 6-phosphate

Phospho glucoisomerase

TO GLYCOLYSIS

6-Phosphogluconolactone

TO PENTOSEPHOSPHATE

PATHWAY

NADP + NADPH + H + G 6-Phosphate dehydrogenase

Galactose 1-phosphate

UDP-galactose

Galactose 1-phosphate

uridyltransferase UDP-glucose pyrophosphorylase

UDP-glucose

UTP

PPi

TO GLYCOGENESIS

Metabolic Fates of Glucose 6-Phosphate

Metabolic Fates Of Pyruvate

Pyruvate

Lactatedehydrogenase

Lactate 1. CONVERSION TO LACTATE

NADH + H

+ N

AD+

Acetyl-CoA

NADH + H+ + CO2Pyruvate dehydrogenase

(TPP, Lipoic acid, FAD, Mg2+)3. CONVERSION TO ACETYL CoA

NAD+ + CoA-SH

Alcohol dehydrogenase Ethanol

H+

Pyruvate decarboxylase Acetaldehyde

2. CONVERSION TO ETHANOL

NADH + H+ NAD+CO2

Pyruvate

Metabolic Fates Of Pyruvate

4. CONVERSION TO ALANINE

Glutamate

⍺-Ketoglutarate

Alanine

Alanine

transaminase

Oxaloacetate

ATP + HCO3-

ADP + Pi

Mg2+Pyruvate

carboxylase

5. CONVERSION TO OXALOACETATE

Acetyl-CoA

TO GLUCONEOGENESIS

TO TCA CYCLE

Malic enzyme

Aspartate

Phosphoenolpyruvate

GTP

GDP + CO2

PEP carboxykinase

Citrate synthase

Aspartatetransaminase

Glutamate

⍺-Ketoglutarate

Citrate

NADPH + H+

NADP+

Metabolic Fates Of Acetyl CoA

Acetyl CoA

AcetoacetateHMG CoA lyase

Acetyl CoA

CoA-SHAcetoacetyl CoA thiolase

Acetoacetyl CoAHMG CoAsynthase

CoA

3-Hydroxy-3-methylglutaryl CoA (HMG-CoA)

Acetyl CoA

HMG CoA reductase2 NADPH + H+

2 NADP+ + CoA

Mevalonate

1. CONVERSION TO CARBON DIOXIDE2. CONVERSION TO FATS3. CONVERSION TO STEROLS4. CONVERSION TO KETONE BODIES

TCA cycleCitrate

Oxaloacetate

CoA-SH

H2O Citrate synthase

CO2

β-Hydroxybutyrate

3-hydroxybutyratedehydrogenase

NAD+

NADH + H+

Spontaneous

Acetone

Cholesterol

Squalene

Isopentenyl pyrophosphate

Malonyl CoA

Acetyl CoA

carboxylase

Pyruvate pump

Citrate/Malateexchanger

Malate/α-ketoglutarate

exchanger

Citrate

Pyruvate

Pyruvate

Oxaloacetate

Malate

Citrate

Oxaloacetate

Pyruvate carboxylaseATP + CO2

ADP + Pi

CoA

α-Ketoglutarate

Malate

Citrate synthase

Malate

Malate dehydrogenaseNADH + H+

NAD+

Malic enzymeNADP+

NADPH + H+ + CO2

ATP-citrate lyase ATP + CoA

ADP + Pi + Acetyl CoA

CYTOPLASM

Citrate-Pyruvate Shuttle

Inner Mitochondrial Membrane

MITOCHONDRIAL MATRIX

Movement of Acetyl CoA from mitochondria to cytoplasm

Acetyl CoA

α-Ketoglutarate

Aspartate/Glutamateexchanger

MITOCHONDRIAL MATRIX

Oxaloacetate

Malate

Aspartate

Oxaloacetate

Malate

Aspartate

Aminotransferase

CYTOPLASM

NADH + H+

NAD+NAD+

Glutamate

⍺- Ketoglutarate

Glutamate

⍺- Ketoglutarate

Inner Mitochondrial Membrane

NADH + H+ Malate dehydrogenase

Transport of NADH from mitochondria to cytoplasm

Aminotransferase

⍺-Ketoglutarate/Malateexchanger

Malate-Aspartate Shuttle

Mitochondrial Shuttles

Malate dehydrogenase

Ketone Body Formation

LIVER MITOCHONDRIA BLOODCIRCULATION

CARDIAC MUSCLE, RENAL CORTEX AND BRAIN

MITOCHONDRIAAcetyl CoA

Acetyl CoA

β-Hydroxy-β-methylglutaryl CoA

HMG CoA synthaseCoA

Acetyl CoA3-Hydroxy-3-methylglutaryl

CoA (HMG-CoA)

Acetoacetate

HMG CoA lyaseAcetyl CoA

Acetoacetate

Acetoacetyl CoA

CoAAcetoacetyl CoA thiolase

Acetyl CoA Acetoacetyl CoA

β-HydroxybutyrateAcetone

β-hydroxybutyrate DHSpontaneous /Acetoacetatedecarboxylase

NAD+

NADH+ + H+CO2

β-HydroxybutyrateAcetone

Acetyl CoA

Acetoacetyl CoA thiolaseAcetyl CoA

CoAAcetoacetyl CoA

3-oxoacyl CoA transferaseSuccinyl CoA

Succinate

Acetoacetate

β-hydroxybutyrate dehydrogenaseNAD+

NADH+ + H+

β-Hydroxybutyrate

Metabolic Profiles Of Muscle• Muscle uses glucose, fatty acids and ketone bodies as fuel. In resting muscle, fuel of choice is fatty acids which

meets 85% of energy need. Skeletal muscle has stores of glycogen comprising 1-2% of its mass (lasts about 10’), for ready availability of glucose during bursts of activity. Muscle accounts for 50% of total oxygen consumption in resting and 90% in vigorous exercise state. During intense exercise, oxygen supply is limited and rate of glycolysis exceeds that of citric acid cycle by 100 times. So, NAD+ required to keep glycolysis going is regenerated by lactate fermentation. However, the accumulation of lactate may lower pH, in turn inhibiting phosphofructokinase activity, thus slowing down glycolytic flux. Besides, depletion of glycogen and phosphocreatine levels may also lead to muscular fatigue. Oxygen is required to restore ATP levels by oxidative phosphorylation to 5.2 mM. ATP, inturn, is used in the liver to convert lactate to glucose by Cori cycle. During recovery phase, ATP is required to maintain skeletal muscle phosphocreatine levels (between 10-30 mM)

Phosphocreatine Creatine

ADP ATP

Creatine kinase

Mg2+

Substrate level phosphorylation

𝚫G°’ = -13 KJ mol-1

• While a marathoner uses aerobic respiration for slow release of energy from fatty acids and glycogen, a sprinter uses phosphocreatine, ATP and anaerobic respiration for quick release of energy. In general, the order of preference of substrate used for ATP generation during muscular activity are : phosphocreatine (lasts less than a minute) > glucose from muscle glycogen > glucose from liver (anaerobic boost) > aerobic metabolism. At times, skeletal muscle is broadly classified into type I and type II or red and white muscle or slow-twitch and fast-twitch, based on the mitochondrial density and consequent aerobic (oxidative) or anaerobic (glycolytic) respiration, respectively. Unlike skeletal muscle, heart muscle has less variable workload, lacks anaerobic respiration, and stores no fuel reserves. Heart muscle respires aerobically, using fatty acids as fuel of choice. Besides, ketone bodies are used preferentially over glucose as fuel. Lactate can also serve as fuel for heart

• Lactate dehydrogenase (LDH) is a tetrameric enzyme involved in anaerobic glucose metabolism. It has two isoforms, 75% identical in amino acid composition, one primarily found in heart muscle (H) and other in skeletal muscle (M). These M and H subunits can combine in all possible combinations, giving five different isozymes (M4,

H4, M3H, M2H2 or MH3). An increase in LDH in blood predicts tissue damage, e.g., raised H4 relative to H3M

indicates myocardial infarction. H4 isozyme (LDH 1) has higher affinity for lactate. Also, M4 isozyme (LDH 5) is

allosterically inhibited by pyruvate, but H4 is not. These differences reflect heart and skeletal muscle

requirements

Metabolic Profiles Of Muscle

• Gluconeogenesis• High [NAD+]/[NADH]

Glucose Lactate

Glycogen

LIVER

6 ATP

Cori Cycle

Glucose

Glycogen

Lactate• Lactic fermentation• Low [NAD+]/[NADH]

MUSCLE

2 ATP

BLOOD

Glucose LactateR.B.C’s2 ATP

Pyruvate

Pyruvate

LDH

LDH

NADH NAD+

NADH NAD+

• Liver is central to intermediary metabolism. It is biochemically versatile

• Liver stores enough glycogen to last a day long supply of glucose. Liver regulates blood glucose levels and

clears blood of toxic metabolites. Also, liver has glucokinase (Km for glucose 10 mM) besides hexokinase ≅(Km for glucose 0.1 mM). This promotes utilization of glucose for glycogen and fatty acid synthesis, and ≅ensures nutrition to brain and muscle

• During fed-state, liver converts excess blood sugar to fat while during starvation, it produces ketone bodies, preserving glucose for the brain. Liver lacks 3-oxoacyl CoA transferase required to use ketone bodies as fuel. Also, anaerobic respiration doesn't occur exclusively in heart and liver

• Fatty acid synthesis occurs mainly in liver. Esterification of free fatty acids with glycerol forms triacylglycerols (TAG’s) which are packaged for transport as very low density lipoproteins

• Only liver has the enzyme CPS-I and the other enzymes required for urea cycle. Majority of amino acids are catabolized in the liver and the ammonia produced is fixed into urea

Metabolic Profiles Of Liver

• Kidney, like gut, has glutaminase enzyme that produces ammonia by deamination of glutamine, which was produced in liver by action of glutamine synthetase, to glutamate. Ammonia is toxic and is excreted as ammonium

• Main role of kidneys is to filter wastes like creatinine and urea from blood into urine and maintain blood pH to 7.4 by excreting HCO3

- and NH4+. Kidneys express aquaporins, proteins that permit passage of water at high rate

but not solutes or ions, including hydronium ions

• During starvation, kidney, like liver, can perform gluconeogenesis (glucose synthesis from lactate, glycerol, pyruvate and amino acids) and may produce up to half of the blood’s glucose

• Only liver and kidney have glucose 6-phosphatase enzyme. These organs maintain the blood glucose levels by converting G6P to glucose for export to other tissues via the bloodstream. G6P activates glycogen synthase, so absence of G-6-phosphatase (von-Gierke’s disease) leads to large accumulation of glycogen in the liver and kidney

Metabolic Profiles Of Kidney

• Extracellular lipoprotein lipases, activated by insulin, hydrolyze the TAG’s in plasma to glycerol and free fatty acids (FFA’s). FFA’s can be re-esterified with glycerol 3-phosphate for adipose storage as TAG’s. Adipose tissue requires a steady supply of glucose to generate glycerol 3-phosphate

• When blood glucose concentration drops, glucagon activates the hormone sensitive lipase for hydrolysis of stored triacylglycerols and release of free fatty acids and glycerol into the blood stream. Serum albumin carries the free fatty acids to tissues while glycerol is phosphorylated and dehydrogenated to DiHAP for glycolysis, gluconeogenesis or lipogenesis in liver

• Leptin is a 16 kDa protein encoded by ob gene which shows adipocyte specific expression. Leptin has receptors in hypothalamus. Mutations in ob gene cause leptin deficiency resulting in obesity, a condition where BMI (Weight in kg/height in meter2) is above 30. Likewise, ob-/ob- mice can’t synthesize leptin, and are obese. Their obesity can be corrected by leptin injection

Metabolic Profiles Of Adipose Tissue

• Glucose is the primary brain fuel. Since brain stores very little glycogen, it requires continuous supply of glucose. Fatty acids cannot traverse the blood brain barrier as they are bound to protein. Only under prolonged starvation, ketone bodies can partially replace glucose as energy source

• Though an average brain constitutes only 2% adult body mass, it consumes 120 g glucose per day, and amounts to 20% of resting oxygen consumption. About 70% of this energy is utilized to power the Na+-K+ ATPase which maintain the membrane potential for nerve transmission

• The steady concentration of glucose in brain is about 1 mM. Glucose is transported into the brain by the GLUT3 glucose transporter. Under normal conditions, GLUT3 remains saturated with glucose because the blood glucose concentration is around 5 mM while the Km of GLUT3 for glucose is 1.6 mM. However, if blood

glucose concentration drops below 2.2 mM, which is around the Km of GLUT3, brain becomes dysfunctional

which may lead to coma, irreversible damage and death

Metabolic Profiles Of Brain

Metabolic Regulation Of Energy Fed State (Post-prandial):

• Food with high glycemic index stimulates

pancreas to release insulin and shuts down

glucagon release

• Insulin stimulates glucose utilization for

energy generation or storage, first as

glycogen and subsequently as TAG’s. TAG’s

move from liver as VLDL to tissue and

majority get stored in adipose. Remnant

chylomicrons are reabsorbed by liver

• Remaining glucose travels via blood for

uptake by peripheral tissue including muscle

and adipose. Adipose uses glucose to

generate glycerol 3-P for TAG synthesis

Fasting State:

• Fall in blood sugar lowers insulin and raises

glucagon level, triggering release of stored fuel

• Glucagon stimulates glycogenolysis and

gluconeogenesis, and protein catabolism

• As glycogen stores start to deplete with

prolonged fasting, TAG’s and ketone bodies

serve as major energy source for muscle and

heart, saving glucose for brain and RBCs

• After 3-4 days, body begins to starve. Except

RBCs and brain, tissues starts using fat.

Decreased rate of gluconeogenesis spares

muscle protein till fat stores run-out when

proteins are used as fuel untill organ-failure

Hormonal Regulation Of Metabolism

• Insulin: Signals high blood glucose. Promotes uptake of glucose and fatty acids, and synthesis of glycogen and TAG’s

• Glucagon: Signals low blood glucose. Stimulates glycogenolysis and gluconeogenesis; inhibits glycogenesis and

glycolysis. Also mobilizes fatty acids and glycerol by activating lipases

• Epinephrine and norepinephrine: Released by adrenal medulla during exertion and anxiety, increase the heart rate

and blood pressure to meet the raised energy/oxygen demand (fight or flight response). Epinephrine targets: a)

adipose to mobilize fat, b) muscle to increase F 2,6-bisP which in-turn activates PFK-1, and c) liver to produce glucose

• Cortisol: Produced slowly by adrenal cortex to meet energy demand during long term stress. Cortisol targets: a)

adipose tissue to promote release of fatty acids from TAG’s, b) muscle to promote protein breakdown and amino acid

export to liver for gluconeogenesis, and c) liver to promote gluconeogenesis by increasing pyruvate carboxylase levels

• Ghrelin: A gastrointestinal peptide that enhances appetite

• Leptin: Released by adipose and acts on hypothalamus. It decreases hunger. Expression de-regulation causes obesity

• Role of AMP-activated protein kinase (AMPK) in carbohydrate and fat metabolism

Regulation Of Major Metabolic Pathways

ATP cAMP + PPi

Glucagon Epinephrine

Adenylate

cyclase

cAMP dependent protein kinase

ATP ADP

Phosphorylase kinase

Phosphorylase kinase

OH

(Active)(Inactive)

Phosphorylase b Phosphorylase aPhosphorylase b kinase

2ATP 2ADP Glycogen

Glucose 1-P

GLYCOGENOLYSIS

(Less active) (Active)

OH OH

Glycogen synthase a

(Inactive)

(Active)

ATP

ADP

Glycogen synthase b

GLYCOGENESIS

Protein Kinase A

Protein Kinase A

cAMP

Proteinphosphophatase I

Insulin,

PP

P P

Proteinphosphophatase I

P

Glucose, Glucose 6-P

Glucose 6-Phosphatase

Gluconeogensis

GlycolysisGlu G 6-P

Hexokinase

𝚫G°’ = -4 Kcal mol-1

G 6-P

PEP-carboxykinase

ADP

F 6-P F 1,6-bisP

PFK 1

ATP; Citrate; Low pH, Glucagon

𝚫G°’ = -3.4 Kcal mol-1

AMP; Insulin;F 2,6-bisP

F 1,6-bisphosphatase

AMP; F 2,6-bisP Citrate, ATP

Oxaloacetate

Acetyl CoAADP

Pyruvate carboxylase

PyruvatePEP

Pyruvate Kinase

𝚫G°’ = -7.5 Kcal mol-1

ATP, Alanine AMP, F 1,6-bisP

Several steps

RECIPROCAL REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS IN THE LIVER


⍺-Ketoglutarate

Oxaloacetate

Acetyl CoA

Fumarate

Succinyl CoA

Citrate

Isocitrate

Succinate

Malate

Cis-aconitate

Steps involved:1. Condensation: Citrate synthase2a. Dehydration: Aconitase2b. Hydration: Aconitase3. Oxidative decarboxylation: Isocitrate dehydrogenase4. Oxidative decarboxylation: -ketoglutarate dehydrogenase⍺5. Substrate level phosphorylation: Succinyl-CoA synthetase6. Dehydrogenation: Succinate dehydrogenase7. Hydration: Fumarase8. Dehydrogenation: Malate dehydrogenase

5

6

7

8

4

3

2b

2a1

3 NADH

1 GTP

1 FADH2

CoA-SH

CO2

H2O

H2O

CO2H2O

CoA-SH

CoA-SH

H2O

Regulation Of Kreb’s cycle

Pyruvate

Pyruvate dehydrogenase complex

CoA, NAD+

TPP, Mg2+, NAD+, FAD, LipS2

Mg2+

Fe2+

Fe2+𝚫G°’ = -7.7 Kcal mol-1




NADH + H+

(TPP, Mg2+, FAD, LipS2)


Acetyl CoA Malonyl CoA

Acetyl CoA

carboxylase

Palmitoyl CoA, AMPK

Citrate

FATTY ACID BIOSYNTHESIS

HCO3-

• Fatty acid metabolism is regulated by Acetyl CoA carboxylase by end product inhibition and reversible adenylation. It is also regulated by carnitine acyl transferase I, which limits the transport of fatty acids into mitochondrial matrix for β-oxidation.


AMINO ACID BIOSYNTHESIS

Glutamine synthetase

Glutamate

Glutamine

Adenylated

(Inactive)

Glutamine synthetase

ATP PPiAdenylyl

transferas

e

ADP Pi

AMP

Glutaminase

Alanine, Glycine, AMP, Tryptophan, Carbamoyl phosphate, Glucosamine 6-phosphate

ATP + NH4+

ADP + Pi

Deadenylated

(Active)

PA

PB

UMP

UTP

PPi

UMP

H2O

Uridylyl transferase

ATP, α-Ketoglutarate

Glutamine

Cumulative

feedback inhibition


5-Phosphoribosyl 1-pyrophosphate

5-Phosphoribosyl amine

Inosinate

Adenylosuccinate

AMPGMP

Xanthylate

Glu-PRPP amidotransferase

Ribose 5-phosphate

PRPP synthetase

IMP dehydrogenase

Adenylosuccinate dehydrogenase

PURINE BIOSYNTHESIS PYRIMIDINE BIOSYNTHESIS

Glutamine + HCO3- + ATP Glutamine + HCO3

- + ATP

Carbamoyl phosphateCarbamoyl phosphate

Orotidylate

UMP

UDP

UTP

CTP

PRPP

Carbamoyl synthetase II

Carbamoyl synthetase II

Aspartate transcarbamoylase

Carbamoyl aspartate

Aspartate transcarbamoylase

Carbamoyl aspartate

Orotidylate

UMP

UDP

UTP

CTP

PRPP

ADP, GDP

Orotate Orotate

Asp Asp

ADP, GDPADP, GDP

ADP

ATP

GDP

GTP

BACTERIAL ANIMAL

RESOURCES

Biochemistry by: Lehninger, Nelson and Cox

Biochemistry by: Voet and Voet

Biochemistry by: Lubert Stryer

Biochemistry by: Campbell and Farrell

Lippincotts Illustrated Biochemistry

Biochemistry and Molecular Biology by Elliott and Elliot

Biochemistry with Clinical Correlations by Thomas Devlin

metabolic regulation

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