CARBOHYDRATE METABOLISM😊

General

Rate Limiting Enzyme

Glycolysis

• Only oxidation pathway without Oxygen.

• Only pathway generating ATP without Oxygen.

• Only oxidation in Cytoplasm.

Glycolysis	ㅤ
Aerobic process	7 ATPs / Glucose (+ 2 Pyruvate )
↳ Hexokinase	Glucose → Glucose 6 Phosphate • [- 1 ATP] • Irriversible
↳ Phosphofructokinase 1	Fructose 6 P → Fructose 1, 6 Bisphosphate • [- 1 ATP] • Irriversible
↳ Glyceraldehyde 3 P dehydrogenase	Glyceraldehyde 3 P → 1, 3 biphosphoglycerate • [+ 2 NADH = + 5 ATP]
↳ Phosphoglycerate kinase	1, 3 biphosphoglycerate → 3 phosphoglycerate • [+ 2 ATP] • Substrate level Phosphorylation
↳ Pyruvate kinase	Phosphoenolpyruvate → Pyruvate • [+ 2 ATP] • Substrate level Phosphorylation • Irriversible
Anaerobic process	2 ATPs / Glucose (+ 2 Lactate)
ㅤ	In Red Blood Cells • RBCs lack mitochondria • Cannot perform aerobic respiration
Link Reaction	Gain 5 ATP / Glucose
ㅤ	Oxidative decarboxylation • Pyruvate (3C) + NAD + CoA →Acetyl CoA (2C) + NADH + CO2
Total ATP at this stage:	7 ATPs + 5 ATPs = 12 ATPs.
TCA Cycle	• Always active (fed/fasting) • Amphibolic Pathway
1. Rate-limiting enzyme / Regulatory Enzymes:	• Irreversible
↳ Citrate synthase	ㅤ
↳ Isocitrate dehydrogenase	1st oxidative decarboxylation
↳ α-Ketoglutarate dehydrogenase	2nd oxidative decarboxylation
2. Substrate-level phosphorylation	• Gain 1 GTP (1 ATP) per Acetyl CoA
↳ Succinate thiokinase	Succinyl-CoA → Succinate
3. NADH Yielding	• Gain 3 NADH (7.5 ATP) per Acetyl CoA
↳ Isocitrate dehydrogenase	Isocitrate → α-Ketoglutarate
↳ α-Ketoglutarate dehydrogenase	α-Ketoglutarate → Succinyl-CoA
↳ Malate dehydrogenase	Malate → Oxaloacetate
4. FADH Yielding	• Gain 1 FADH (1.5 ATP) per Acetyl CoA
↳ Succinate dehydrogenase	Succinate → Fumarate
TCA Cycle Energetics	ㅤ
• Per Acetyl-CoA	10 ATP (Per Cycle x 2 = 20 ATP)
• Per Pyruvate ↳ (PDH + TCA) ↳ Add 2.5 ATP from Link reaction	12.5 ATP
• Per Glucose ↳ (Glycolysis + PDH + TCA)	32 ATP

Aerobic Glycolysis

ATP-Utilizing Steps:

ATP-Producing Steps:

Step-by-Step Pathway

Cori Cycle and Cahill Cycle

• Function
• Provide Glucose to Muscle
• Transport of ammonia to Liver

Muscle protein breakdown

• Release Alanine → Substrate for gluconeogenesis

• During prolonged fasting, the liver uses alanine.

• Emaciation & ↓ in muscle mass.

Transport of NH₃

• 1st-line defence in hyperammonaemia
• Cahill cycle
• Alanine = Transport form from skeletal muscles

• NOTE
• Glutamate + NH3 → Glutamine
• Glutamate = Transport form from other parts
• Enzyme: Glutamine synthetase (in mitochondria)
• Glutamate from
• Amino acids
• NH3 from
• Amino sugars
• Pyrimidine
• Purine
• Porphyrins

Hexokinase vs Glucokinase

Gluconeogenesis

• Reversal of glycolysis.

• Synthesis of glucose from non-carbohydrate precursors.

• It is an anabolic pathway.

• Requirements for gluconeogenesis:
1. Substrates: GLA

ㅤ	Substrates	Source
1	Glycerol	• Peripheral Lipolysis • DHAP
2	Lactate	• Cori cycle
3	Alanine (Prolonged starvation)	• Cahill cycle
4	Propionyl CoA → Succinyl-CoA	• Odd chain FA
5	Glucogenic amino acids	• All except Lys, Leu

• NOTE: Acetyl CoA
• NOT SUBSTRATE OF TCA
• But Strongest activator of Gluconeogenesis

Odd chain FA:



2. Energy
• Require 6 ATPs
• for 2 lactate → 1 glucose.
• Require 11 ATPs
• For 2 pyruvate → 1 glucose

Clinical Relevance:

• Muscle cannot perform gluconeogenesis
• lacks Glucose-6-phosphatase

• Liver cannot use G6P released for gluconeogenesis
• G6Pase is in ER

Energy Requirement:

• To convert 2 Lactate → 1 Glucose:
• Pyruvate carboxylase: 2 ATP
• PEP carboxykinase: 2 GTP
• 1,3-BPG kinase: 2 ATP
• Total: 6 ATP equivalents

• To convert 2 Pyruvate → 1 Glucose:
• Total: 6 + 5 (2 x NADH for link reaction reversal) = 11 ATP equivalents

Regulation of Glycolysis and Gluconeogenesis

Tandem/Bifunctional Enzyme

• Involved in Glucose Regulation during Starvation

• Both pathways are reversible.

• Allosteric regulators
• When one pathway is active, other is inactive.
• Prevents a futile cycle (vas cycle)

• In starvation
• ↓ Blood glucose → Glucagon release.
• Glucagon → Gs receptor → ↑ cAMP.
• cAMP → activates Protein Kinase A (PKA).
• PKA phosphorylates tandem enzyme → activates F-2,6-bisphosphatase.
• F-2,6-BP is cleaved → PFK-1 not stimulated
• Note: F-2,6-BP stimulate PFK-1
• Glycolysis stops
• Gluconeogenesis starts → Plasma glucose increases

Q. Identify A

• (Image showing regulation of glycolysis and gluconeogenesis, 'A' points to Fructose 2,6 bisphosphate).
• A. Fructose 1,6 bisphosphate.
• B. Fructose 2,6 bisphosphate.
• C. PFK-1.
• D. PFK-2.
Explanation:
• Upper half: glycolysis.
• Rate-limiting enzyme: Phosphofructokinase-1 (PFK-1).
• Lower half: gluconeogenesis.
• Rate-limiting enzyme: Fructose 1,6 bisphosphatase.
• A is a common regulator of both enzymes.
• A stimulates glycolysis and inhibits gluconeogenesis.
• A is Fructose 2,6 bisphosphate.

Fates of Pyruvate

4 main fates:

Steps in Starvation and High Energy

Fate of Glucose 6 Phosphate (G6P)

Fate of Acetyl CoA → Anabolism

• Explains why a Carbohydrate diet is Lipogenic.
• High energy → ⛔ Glycolysis.

Difference between NADH and NADPH

• Note: Only NADH/FADH2 give ATP, never NADPH.

HMP Pathway sites

• Oxidative stress sites
• RBC / Lens

• Steroid / FA synthesis sites
• Liver
• Adipose
• Adrenal cortex
• Gonads

• Never the sites for HMP:
• Non lactating mammary glands, Skin.

NADPH Uses

• Regeneration of Glutathione:
• Glutathione (GSH)
• γ-glutamyl-cysteinyl-glycine (GSH)
• Tripeptide composed of:
• Glutamic acid + Cysteine + Glycine
• GSH + H2O2 → water + GS-SG (oxidised)
• via Glutathione peroxidase (dep on Selenocysteine)
• GS-SG (oxidised) → GSH (reduced)
• Regenerating Enzyme: Glutathione reductase (dep on Vit B2)
• Hydrogen source: NADPH.
• Peroxide Protects and Reductase replenishes/regenerates
• Functions
• Important antioxidant for RBCs.
• Free radical scavenger (Antioxidant role)
• Glutathione peroxidase
• Transport of ammonia
• via Meister’s cycle / Gamma-glutamyl cycle
• Conjugation of unconjugated bilirubin (detoxification reactions)??
• Coenzyme role

• G6PD deficiency
• Most are asymptomatic.
• ↓ G6PD enzyme + Exposure to oxidants or infection
• NADPH not regenerated → ⛔ Glutathione reductase
• ↓ Glutathione → H2O2 is not detoxified.
• H2O2 causes oxidative damage to RBC membrane → Hemolytic anemia
• Hemolysis type
• Extravascular >> intravascular
• Intermittent, symptoms with precipitating factors.
• So no HSM

Use of Ribose-5-Phosphate:

• Used for Purine and Pyrimidine nucleotide synthesis.

• Examples:
• Adenosine + RSP → Adenosine monophosphate.
• Guanine + RSP → GMP.
• Cytosine + RSP → CMP.

Transamination

Amino Acid	Converted To	Enzyme
Amino acids	Keto acids	• Transaminase • Require Pyridoxal phosphate (active form of Vitamin B6)
Alanine	Pyruvate	ALT / SGPT ↳ ALT → PT → Pyruvate
Aspartate	OAA	AST / SGOT ↳ AST → OT → OAA

• Site: Cytoplasm of all organs

• Reversible reaction

• Ping-pong mechanism (Bi-bi):
• 2 substrate
• 2 product reaction

Exceptions to Transamination

1. Proline

2. Hydroxyproline

3. Lysine

4. Threonine

---

• Pro (Proline, hydroxyproline) doesn’t lyse (Lysine) through transamination (threonine)

Which one of these amino acids does not enter the Krebs cycle by forming Acetyl CoA via pyruvate?

1. Glycine

2. Tyrosine

3. Hydroxyproline

4. Alanine
ANS
• Tyrosine:
• Tyrosine → fumarate + acetoacetate.
• Tyre acid ozhikkumbho fumes avum
• These products enter the Krebs cycle
• not through pyruvate or Acetyl CoA.
• Does not use the pyruvate pathway.

Applied Biochemistry

• Gyrate atrophy (retina & choroid)
• Defect in: δ-ornithine aminotransferase
• Enzyme which undergo Non α amino acid Transamination
• Treatment:
• Restrict ornithine & arginine
• Supplement PLP (B₆)

Tricarboxylic Acid (TCA) Cycle / Krebs Cycle

Regulation

• Always active

Anaplerotic Reactions

• Definition: Reactions that replenish TCA cycle intermediates.

• Key Reactions:

Substrates	Source	Other Uses of substrates
Oxaloacetate	Pyruvate	ㅤ
Succinyl-CoA	Propionyl-CoA	Heme Synthesis
α-Ketoglutarate (B6)	Glutamate	GABA
Oxaloacetate	Aspartate	ㅤ
Citrate	Fatty acid synthesis	ㅤ

• Acteyl CoA is not a substrate of Krebs cycle
• So not an anaplerotic reaction

• Where is NADH released? → I (ID) Know (AKG) Mam (Malate D)

• Where is CO2 released? → I Know (ID, AKG)

Location:

• Mitochondrial matrix
• Exception:
• Succinate dehydrogenase – located on inner mitochondrial membrane

Functions:

• Final oxidative pathway for:
• Lipids
• Carbohydrates
• Proteins

• Produces:
• NADH
• FADH₂
• GTP (→ ATP via oxidative phosphorylation)

• Provides biosynthetic intermediates

• Amphibolic: both catabolic and anabolic roles

Significance

• Irreversible step

• Acetyl-CoA is never gluconeogenic.

• Explains why fat cannot be converted to glucose

• Therefore, all even-chain fatty acids are never glucogenic.
• They catabolize to n/2 acetyl-CoA.

• Exceptions:
• glycerol, odd-chain fatty acids → Propionyl-CoA → Succinyl-CoA

Oxidative decarboxylation

• Multienzyme complexes

Enzyme complex

Preferred Fuel

Anaerobic Cells

• Must use only glucose.
• Lactate → Substrate for gluconeogenesis

• These cells include:
• RBCs, Retinal cells, Corneal cells (lack mitochondria).
• White muscle fibres (store glycogen, no myoglobin).
• Renal medulla.

• Anaerobic glycolysis generates 2 ATPs.

Aerobic Cells

• Can use fatty acids > glucose
• Reason:
• Glucose → 32 ATPs;
• fatty acid (Palmitic acid) → 106 ATPs.

• Examples using fatty acids:
• Cardiac muscle fibres.
• Red muscle fibres.
• They store fatty acids as triacylglycerol and Cholesterol ester.

Exception:

• Neurons
• Use glucose as preferred fuel.
• Reason: Fatty acids attached to albumin cannot cross blood-brain barrier (BBB).

• Fuel Use Comparison:
• RBCs use glucose anaerobically:
• 1 glucose → 2 Lactate + 2 ATPs.
• Neurons use glucose aerobically:
• 1 glucose → CO2 + 32 ATPs.

• Hepatocytes
• Preferred fuel: Amino acid.
• Enzyme: Glucokinase, with lower affinity for glucose.
• Encounters dietary glucose first (via portal vein).
• Low enzyme affinity allows glucose to pass to peripheral tissues,
• preventing hypoglycemia.
• Cannot use dietary fatty acids directly.
• They are absorbed as Chylomicrons via lymphatics,
• entering systemic circulation first.

Fasting and Starvation Stages

• Starvation = Fuel deficiency
• A deficiency of glucose, fatty acids, and amino acids.
• Aim during starvation
• Maintain body glucose levels.
• Bcz neurons and RBCs mainly depend on glucose.

• Energy deficiency = ATP deficiency

• Major sources of plasma glucose during starvation:
• Dietary glucose
• 2 to 2 and half hours
• Liver glycogenolysis
• Major source of Glucose for 1st 16 hrs of starvation
• Gluconeogenesis
• Occurs from 6 hours up to 2-3 weeks of starvation.

Stage	Duration After Food Intake	Primary Energy Source
Early Fasting	4–16 hours	Hepatic glycogenolysis
Fasting	16–48 hours	Gluconeogenesis
Prolonged Fasting/ Starvation	48 hours – 5 days	Ketone body synthesis
Prolonged Starvation	>5 days	Muscle proteolysis

• Fed → Insulin → Dephosphorylation

• Mnemonic: Insulin Hate HSL

• so prick with PIN (PGE, Niacin, Insulin)

ㅤ	Fed state	Fasting state
Hormone	• Insulin (anabolic hormone)	• Glucagon • Hypoglycemia in CAMPil • give Glucagon injection
↳ MOA	• Activates phosphodiesterase • ↓ cAMP	• Activates adenyl cyclase → ATP • ↑ cAMP → activates protein kinase A ↳ Glycogen phosphorylase (activated) ↳ Glycogen synthase (inhibited)
ㅤ	ㅤ	Other Counter-regulatory hormones • Epinephrine/norepinephrine • Growth hormone • Glucocorticoids • Thyroid hormones
State	Dephosphorylated	Phosphorylated
Lipase activated	Lipoprotein Lipase (LPL)	Hormone Sensitive Lipase (HSL) Inhibited by • Insulin • PGE1 • Niacin • Insulin hate HSL • so prick with PIN (PGE, Niacin, Insulin)
↳ Function	• Chylomicron TGA → FA + Glycerol • to enter Adipose cells in fed state	• Adipose TGA → FA + Glycerol • for transport to liver in fasting state
ㅤ	Enzymes & Pathways activated	Enzymes & Pathways activated
Pathways activated	• Glycolysis + • Link Reaction + All anabolic pathways • Glycogen synthesis • Cholesterol synthesis • FA synthesis • Protein synthesis	• Gluconeogenesis + All Catabolic pathways • Glycogenolysis • KB synthesis / breakdown • FA (β) oxidation • Peripheral lipolysis • Amino acid oxidation
Enzymes activated	All anabolic + • Glycogen synthase • Acetyl CoA carboxylase • HMG CoA reductase Glycolysis enzymes • Phosphofructokinase • Pyruvate DH Exception ATP Citrate Lyase ↳ FA synthesis ↳ Citrate → Acetyl CoA ↳ activated by insulin ↳ Active in phosphorylated state	All catabolic + Gluconeogenesis enzymes • Fructose 1 , 6 bisphosphate • Glycogen phosphorylase NOTE: • Gluconeogenesis is anabolic • Glycolysis is catabolic
Compartmentalisation	Cytoplasm	Mitochondria
ㅤ	All above pathways + • Glycogenolysis • HMP shunt	All above except Glycogenolysis + • TCA • ETC • PDH
Others	Cholesterol synthesis (Steroids) ↳ Cytoplasm + SER Bile acid synthesis (Steroids) ↳ Smooth Endoplasmic Reticulum	Oxidised in Peroxisomes ↳ Very long chain fatty acid + ↳ Branched chain Fatty acids
Both	ㅤ	ㅤ
ㅤ	• Start in mitochondria • Finish in cytoplasm	PUBG • Pyrimidine Synthesis • Urea cycle • Haem synthesis (blood) • Gluconeogenesis ↳ Oxaloacetate reaches Cyp for gluconeogenesis

Phosphocreatine

• During resting → ATP + Creatine → Phosphocreatine

• Provide high energy phosphate to muscle

• rapidly available

• Replenish ATP

Metabolic Fuel for RBCs During Starvation

• A. Aminoacids

• B. Ketone bodies

• C. Glucose

• D. Fatty acids
Answer:
C. Glucose

Peripheral lipolysis

Adipose TGA → FA + Glycerol

• By Hormone-sensitive lipase
• Catabolic enzyme
• Insulin → Anabolic
• ⛔ hormone-sensitive lipase.
• Insulin ↓ in starvation
• All counter-regulatory hormones → Catabolic.
• They ↑ in starvation
• They stimulate hormone-sensitive lipase.
• Examples:
1. Glucagon,
2. Growth hormone,
3. Norepinephrine,
4. Cortisol.

• Products → Both enter Liver
• Glycerol
• Substrate for gluconeogenesis
• FA
• Undergoes β-oxidation → releases Acetyl-CoA.
• Acetyl-CoA :

ㅤ	Sequence of Utilization	Notes
1	Used in TCA cycle	• Energy for ketone body synthesis
2	Enters KB synthesis	• Liver don’t use KB ↳ d/t absent thiophorase
3	Activates Gluconeogenesis	• Liver don’t use Glucose ↳ Glucokinase has low affinity • Glucose-6-phosphate is present in SER ↳ Hence Glucose not released immediately

• This results in loss of weight.

Applied Biochemistry:

• Defect in liver β-oxidation
• Non ketotic hypoglycemia
• due to no KB formation or gluconeogenesis.

• Insulin Resistance:
• ↑ TAG hydrolysis → ↑ Free fatty acids in blood → Fatty liver disease

• Niacin:
• Inhibit HSL
• Locks TAG in adipose tissue → Used for hypertriglyceridemia treatment

Diabetes as a Fasting/Starvation-like State

• Absence of insulin.
• ↑ TAG hydrolysis → ↑ Free fatty acids in blood → Fatty liver disease

• Effects:
• GLUT-4 is inactive
• Blood glucose remains high
• Cells behave as if in a fasting state

• Catabolic state:
• All catabolic pathways are activated.

• Only Exception Anabolic process in Chronic Diabetes :
• Fat synthesis in liver.

• Results:
• ↑ VLDL synthesis
• ↑ Cholesterol synthesis

Electron Transport Chain (ETC)

• 2 Mobile Complexes:
• Ubiquinone (Q): Between Complex I and Complex III.
• Cytochrome C: Between Complex III and Complex IV.

• Direction of Electron Transport:
• NADH electron: Enters through Complex I.
• FADH2 electron: Enters through Complex II.

Energetics

• 1 NADH = 10 Protons pumped = 2.5 ATP

• 1 FADH = 6 Protons pumped = 1.5 ATP

NOTE

• Mechanical energy → Chemical Energy
• When they cross Fo (rolling gate) → mechanical energy → transferred to F1 subunit → converts ADP to ATP

• 4 “C”s of Complex 4
• Remember Oxygen next to C4 → Oxidase, Heme, Copper (instead of iron)
• Cytochrome oxidase
• Copper/heme protein
• Carbon monoxide inhibits it
• Cyanide inhibits it

Inhibitors of ETC

Uncouplers

• Function:
• They uncouple fuel oxidation
• Oxidation ✔
• Phosphorylation ✗ → ATP is not produced.

• Effects:
1. ATP is not produced
2. High heat production
3. Increased rate of oxidation

• They are the only ETC inhibitors that do not affect oxidation.

Physiological Uncouplers:

• Vaishna → Brown girl (Brown fat) → From gramam (Gramicidin) → has a Long (Long chain FA) Vaalu (Valinomycin) → always hot (thermogenin) → thyroid ↑↑ (Thyroxine)

• Thermogenin (Uncoupling protein 1)

• Long chain fatty acid

• Ionophores (Valinomycin, gramicidin)

• Bilirubin

• Thyroxine
• Upregulation of uncoupler proteins in mitochondrial membrane
• Allows hydrogen ions to bypass ATP production.
• Leads to heat generation, heat intolerance in thyrotoxicosis, and increased BMR.

• Brown Adipose Tissue (BAT)
• Brown due to abundant mitochondria with uncoupler proteins.
• Bypasses ATP production, leading to heat generation (non-shivering thermogenesis).

• Aids neonates and hibernating animals.

Most important mechanism for protection against hypothermia in neonates:

• Non shivering thermogenesis

• Due to the presence of brown fat
• Around scapula, axilla
• lipid deposits rich in mitochondria
• Release of norepinephrine and uncoupling of beta oxidation of fat → heat production

• Mnemonic: AD ThyLI (adi thyli)

• Newborns: Interscapular, perirenal, mediastinal, perivascular, supraclavicular.

• Adults: Less, but persists in supraclavicular and paravertebral regions.

Artificial Uncouplers:

• Examples:
• 2,4 Dinitrophenol
• Dinitrocresol
• Valinomycin
• Nalinomycin
• Nigericin
• Aspirin overdone
• FCCP

• Once tried as anti-obesity drugs.
• Caused all fuels to be oxidized and energy liberated as heat.
• No ATP production and no anabolism occurred.

• Side effects:
• Intolerable heat production
• Posterior subcapsular cataracts

Rossmann fold

• Rossmann fold is a protein structural motif.

• Located at the N-terminal end of the enzyme.

• Function:
• Binds NAD⁺ or NADP⁺.
• In dehydrogenases.