Gluconeogenesis Notes

Glycogen

• Storage form of glucose.

Glycogenolysis

• Breakdown of glycogen to glucose.

Gluconeogenesis

• Synthesis of glucose from non-carbohydrate precursors.

Oxidation

• Process of losing electrons.

NEFAS

• Non-esterified fatty acids.

Ketone Bodies

• Alternative fuel source produced during prolonged fasting.

Glycerol

• A precursor for gluconeogenesis, derived from triglycerides.

Lipolysis

• Breakdown of triglycerides into glycerol and fatty acids.

Triglycerides

• Storage form of fat in adipose tissue.

Liver

• Major site of gluconeogenesis.

Lactate

• A carbon source for gluconeogenesis, produced in muscle and red blood cells.

Adipose Tissue

• Tissue that stores triglycerides.

Gut

• Site of dietary carbohydrate absorption.

Pyruvate

• A key intermediate in both glycolysis and gluconeogenesis.

Alanine

• An amino acid that can be converted to pyruvate for gluconeogenesis.

Protein

• Source of amino acids for gluconeogenesis.

Proteolysis

• Breakdown of protein to amino acids.

Glutamine

• An amino acid involved in gluconeogenesis.

Muscle

• Site of protein breakdown to provide amino acids for gluconeogenesis.

Topics

• Maintaining blood glucose concentration within narrow limits in the fed and fasting states.
• Steps of gluconeogenesis, how irreversible enzymes of glycolysis are bypassed, and how glycolysis and gluconeogenesis are reciprocally regulated.
• Carbon sources for gluconeogenesis. Evaluate the relative importance of different precursors for gluconeogenesis in feeding, fasting, and exercise.
• Enzyme defects in Von Gierke disease.
• Multiple carboxylase deficiency diseases and their impact on gluconeogenesis.

Gluconeogenesis vs. Glycolysis

• Glycolysis
  • Glucose is catabolized.
  • ATP is produced.
  • $NAD^+$ is reduced to NADH.
• Gluconeogenesis
  • Glucose is synthesized.
  • ATP is consumed.
  • NADH is oxidized to generate $NAD^+$.
• The reactions in the gluconeogenesis pathway CANNOT be the exact reverse of the reactions in the glycolysis pathway.

Location of Gluconeogenesis

• Gluconeogenesis occurs primarily in the LIVER.
• During an overnight fast, 90% of gluconeogenesis occurs in the liver and 10% in the renal cortex.
• In prolonged fasting (starvation), the renal cortex contributes to ~40% to total glucose production.
• Insulin and glucagon levels regulate gluconeogenesis:
  • $\uparrow$insulin $\downarrow$glucagon: favors glycolysis
  • $\downarrow$insulin $\uparrow$glucagon: favors gluconeogenesis
  • $\uparrow$FA: Increased Fatty Acids.

Sources of Blood Glucose

• Fed State
  • Source: Dietary carbohydrate
  • Fate: Increased glycogen and FA synthesis
• Fasting State
  • Sources: Glycogen, Glycerol, Lactate, Amino acids
  • Fate: Glycogenolysis, Gluconeogenesis
• Starved State
  • Sources: Glycerol, Lactate, Amino acids
  • Fate: Gluconeogenesis
  • Brain adapts to using ketones, sparing glucose.
• Glucose oxidized (g/h):
  • Fed: 40
  • Fasting: 20
  • Starved: Decreased further

Carbon Substrates for Gluconeogenesis: Lactate

• Lactate can be converted back to glucose via gluconeogenesis.
• Lactate
  • Background: Oxidoreductase enzyme that catalyzes the reversible conversion of pyruvate to lactate.
  • $Pyruvate + NADH \rightarrow Lactate + NAD^+$
  • Non-specific indication of tissue damage.
  • Causes:
    • Oxygen absent or limited (anaerobic conditions)
    • Exercise or strenuous activity
    • Cancer (Warburg effect)
    • Respiratory conditions
    • Hematologic, cardiac, GI, or kidney pathologies
    • Infectious disease
    • Medications
    • Alcohol
    • Trauma
  • Diagnosis: LDH test (blood from vein, CSF, pleural effusion, abdominal fluid).
  • Treatment: Additional testing (CBC, CMP, LDH isoenzyme test), Vitamin C (ascorbic acid).

Cori Cycle

• Lactate produced by exercising muscles (O2 limited) and cells lacking mitochondria (RBCs, lens of eye) diffuses into the blood and is taken up by the LIVER where it is converted back to glucose via gluconeogenesis.
• OXYGEN LIMITED When else does this happen?
  • Collapse of circulatory system
    • heart attack
    • pulmonary embolism
    • hemorrhage
    • shock
  • Hypoxia
• Less O2 -> ATP from Glycolysis -> Lactate

Carbon Substrates for Gluconeogenesis: Amino Acids (AAs)

• Amino acids from muscle protein degradation can be used for gluconeogenesis.

• Alanine is converted to pyruvate via alanine aminotransferase.

  • $Alanine + \alpha-ketoglutarate \rightarrow Pyruvate + Glutamate$

  • Figure 18-4 Lehninger Principles of Biochemistry, Sixth Edition

Amino Acids and Gluconeogenesis

• Not all amino acids can be used for gluconeogenesis.
• Glucogenic: can be converted to glucose.
• Ketogenic: can be converted to ketone bodies.
• Both glucogenic and ketogenic.
• You don't need to know where each A.A. goes
• Glucogenic:
  • Alanine, Glycine, Threonine
  • Cysteine, Serine
  • Asparagine Aspartate
  • Arginine Proline
  • Histidine Glutamine
  • Glutamate
  • Methionine Threonine
  • Isoleucine Valine
• Ketogenic
  • Leucine
  • Lysine
• Both:
  • Isoleucine
  • Tyrosine
  • Phenylalanine
  • Tryptophan

Carbon Substrates for Gluconeogenesis: Glycerol

• Glycerol is derived from the degradation of triacylglycerides in adipose tissue.
• Hormone-sensitive lipase is activated by glucagon, epinephrine, and cortisol.
• Adipose tissue lacks glycerol kinase.

Conversion of Glycerol to Glucose

• In Liver
  • Glycerol + ATP -> Glycerol-3-P + ADP(Glycerol kinase)
  • Glycerol-3-P + NAD+ -> Dihydroxyacetone phosphate + NADH(Glycerol 3-phosphate dehydrogenase)
• Adipose cell
  • FA -> To beta-oxidation
• Low insulin/high glucagon

Inhibition of Pyruvate Dehydrogenase Complex (PDC)

• Inhibition of Pyruvate dehydrogenase complex (PDC) in gluconeogenesis prevents futile cycling.
• PDC is inactive during gluconeogenesis.
• PDC is active during glycolysis.
• Entry of pyruvate into the TCA/Citric acid cycle
• Pyruvate is directed towards gluconeogenesis because high levels of acetyl-CoA and NADH from FA oxidation act to inhibit the (PDC)

Gluconeogenesis Bypasses Irreversible Steps of Glycolysis

• (1 of 3)
• In gluconeogenesis, pyruvate is converted to PEP in 2 steps (PEPCK)
  • The pairing of carboxylation with decarboxylation helps drive reactions that are otherwise energetically unfavorable.
• Levels:
  • $\downarrow$insulin $\uparrow$glucagon $\uparrow$FA

Generation of PEP from Gluconeogenic Precursors

• The presence of acetyl CoA (from increased β-oxidation due to increased glucagon) is a signal that there is an excess of energy.
• While acetyl CoA itself cannot be used to generate glucose, its presence activates pyruvate carboxylase which is needed to start gluconeogenesis.
• Step 1: $CO_2$ is activated and transferred by pyruvate carboxylase to its biotin prosthetic group.
• Step 2: The enzyme then transfers the $CO_2$ to pyruvate, generating oxaloacetate.
• Step 3: Oxaloacetate cannot cross the mitochondrial membrane so it is reduced to malate that can.
  • $Oxaloacetate + NADH + H^+ \rightarrow Malate + NAD^+$; $\Delta G$ is -20 kJ mol-1
• Step 4: In the cytosol, malate is reoxidized to oxaloacetate, which is oxidatively decarboxylated to phosphoenolpyruvate by PEP carboxykinase.
  • $Oxaloacetate + GTP \rightarrow Phosphoenolpyruvate + GDP + CO_2$

Role of Biotin in Pyruvate Carboxylase Reaction

• Biotin is first deprotonated.
• Pyruvate deprotonated.
• BIOTIN – A cofactor covalently attached to the enzyme through an amide linkage to the ε-amino group of a Lys residue, à biotinyl-enzyme.
• The reaction occurs in two phases, which occur at two different sites in the enzyme.
  • At catalytic site 1, bicarbonate ion is converted to $CO_2$ at the expense of ATP.
    • Forms Carboxyphosphate CO2 reacts with biotin à carboxy-biotinyl-enzyme.
  • Biotin-Lysine: A long arm that carries $CO_2$ of carboxy-biotinyl-enzyme to catalytic site 2
    • $CO_2$ is released and reacts with pyruvate à oxalo acetate

Pyruvate Carboxylase Structure

• Pyruvate Carboxylase: A homo-tetramer (4 identical copies)
• The biotin carboxylation domain catalyzes the formation of carboxyphosphate
• attachment of $CO_2$ to the biotin carboxyl carrier protein (BCCP), the site of the covalently attached biotin.
• BCCP-$CO<em>2$ leaves the biotin carboxylase active site and swings to the pyruvate carboxylase domain, which transfers the $CO</em>2$ to pyruvate to form oxaloacetate.

Biotin (Vitamin B7)

• A coenzyme used in $CO_2$ activation and transfer in carboxylation reactions
• The biotin-lysine (biocytin) complex
• Biotin is involved in the synthesis of fatty acids (acetyl-CoA carboxylase), gluconeogenesis (pyruvate carboxylase), and the degradation of valine, isoleucine, and odd-chained fatty acids.
• Biotin is synthesized by microflora in the intestinal tract and can be obtained in the diet from liver, soybeans, nuts, and egg yolks (but NOT egg white. Egg white contains Avidin which binds biotin).
• Multiple carboxylase deficiency (~1/60,000) is a form of metabolic disorder involving failures of carboxylation enzymes.
• MCD results from biotinase deficiency which cleaves biotin from the lysine. Prevents recycling of biotin and it is lost to the urine as biocytin.
• Symptoms are muscle pain, lethargy, anorexia, and depression. Treatment for biotinase deficiency is biotin supplementation.

Phosphoenolpyruvate Carboxykinase (PEPCK)

• A gluconeogenic enzyme that is not regulated allosterically (instead, it's regulated at the DNA transcription level).
• Deficiency leads to symptoms that include:
  • Fasting-induced hypoglycemia
  • lactic acidemia
  • loss of muscle tone (hypotonia)
  • abnormal enlargement of the liver (hepatomegaly)
  • failure to thrive

Decarboxylation

• “The decarboxylation of otherwise unreactive molecules can be achieved by introducing electron sinks β to the carboxylate, such as ketone, thial, or p-quinone methide, which stabilize the carbanion- formed upon decarboxylation by conjugation.” Nguyen et al. (2024) JACS Au. Decarboxylation in Natural Products Biosynthesis

Prevention of Futile Cycle

• Prevention of the futile cycle during glycolysis and gluconeogenesis
• In fasting, FA are the major source of energy

Summary: Conversion of Pyruvate to PEP in the Liver

• Glucagon via cAMP.
• Pyruvate to PEP occurs in the mitochondria and cytosol.
• Pyruvate can be converted to OAA via pyruvate carboxylase(requires Biotin).
• Alanine can be converted to pyruvate via transamination.
• Lactate can be converted to pyruvate.
• OAA exits from the mitochondrion either as aspartate or malate.
• 图Inducible enzyme
• 区Inactive enzyme

Gluconeogenesis Bypasses Irreversible Steps (2 of 3)

• Need two molecules of pyruvate to generate one glucose.
• Aldolase makesF-1,6-BP fromDHAP and G3P.

Glucagon and Gluconeogenesis

• Glucagon reduces concentration of fructose 2, 6 bisphosphate and enhances rate of gluconeogenesis in the liver
• High glucagon/insulin ratio causes elevated cAMP and increased levels of active protein kinase A.
• Increased protein kinase A activity favors the phosphorylated form of the bifunctional PFK-2/FBP-2.

Fructose 2,6-Bisphosphate Regulation

• Fructose 2,6-Bisphosphate reciprocally regulates Glycolysis and Gluconeogenesis
• High in well-fed state.
• Low in fasting.

Gluconeogenesis Bypasses Irreversible Steps (3 of 3)

• Glucose 6-phosphatase
  • Only expressed in the liver and renal cortex (also used for release of glucose into the blood in glycogenolysis).
  • Insulin decreases the expression of glucose 6-phosphatase, whereas glucagon increases its expression.
  • The Km of glucokinase (10 mM) is too high to reconvert Glucose (4 mM) back to G-6-P during gluconeogenesis.

Location and Function of Glucose 6-Phosphatase

• Glucose 6-phosphatase deficiency causes Von Gierke's disease (also a Glycogen Storage Disease).
• Characterized by severe hypoglycemia, enlarged liver, lipidemia, and lactic acidosis.

Energy Yield/Cost

• Glycolysis: Glucose + 2 ADP + 2 Pi + 2 NAD+ ➞ 2 Pyruvate + 2 ATP + 2 NADH
• Gluconeogenesis: 2 Pyruvate + 6 ATP (2 GTP) + 2 NADH ➞ Glucose + 6 ADP (2 GDP) + 6 Pi + 2 NAD+
• 2 Glycerol + 2 ATP + 2 NAD+ ➞ Glucose + 2 ADP + 2 Pi + 2 NADH

Regulation of Glycolysis & Gluconeogenesis in the Liver

• Reciprocal regulation by F-2,6-BP, AMP, ATP, Citrate, and Alanine.

Glucose Metabolism

• Glucose metabolism in various tissues (effects of insulin)

Tissue Interrelationships During Fasting

• Glycogenolysis in liver is induced due to low insulin/glucagon ratio. The brain and RBCs use the glucose released by the liver.
• Adipose releases FFAs and glycerol from stored TGs.
• The liver converts FA-derived Ac-CoA to make ketone bodies that are used as energy by the brain and muscles.
• Protein in the muscle is broken down and amino acids travel to the liver to be used as gluconeogenic precursors.
• Amino acid metabolism in liver generates urea that travels to the kidneys for excretion.
• Lactate produced in the RBCs and glycerol made in the adipose return to the liver for gluconeogenesis.

Hypoglycemia and Alcohol Intoxication

• Alcohol impairs gluconeogenesis because pyruvate is not available as a substrate for glucose production (hypoglycemia).
• Alcohol impairs conversion of lactate to pyruvate and increases serum lactic acid levels.
• Alcoholic hyperlipidemia is caused due to excess glucagon secretion, induced by starvation and counter-regulatory hormones.

Insulin Resistance in Type 2 Diabetes

• However, in type 2 diabetes(non-insulin-dependent diabetes), Insulin fails to turn OFF FoxO1
• Expression of Pck1, G6Pc and other gluconeogenic enzymes.
• This“Insulin resistance” isa defining feature of type 2 diabetes.
• The higher-than-normal levelsof Pck1 and G6Pc result in an increased output of glucose by the liver even when glucose from the diet ispresent.
• Blood glucose risesto abnormally high levels, which causeshyperglycemia.
• High blood glucose: pancreas releases insulin into bloodstream.
• Insulin àactivation of PI3-KààphosphorylatesAkt.
• Akt phosphorylatesFoxO1 ànuclear exclusion.
• Reduced transcriptionof glucose6-phosphatase(G6Pc)and phosphoenolpyruvate carboxykinase(Pck1)
• lowersthe ratesof gluconeogenesis.

Gluconeogenesis Concept Map

• Substrates for gluconeogenesis: Lactate, Pyruvate, Glycerol, Amino acids
• Regulation of gluconeogenesis during fasting
  • Fasting state
  • Release of fatty acids from adipose tissue
  • Fatty acid oxidation in the liver
  • ↑ Acetyl CoA in liver
  • ↑ Blood glucose
  • Release of glucagon
  • ↑cAMP
  • ↑ Protein kinase A activity
  • ↓Fructose 2,6-bis-phosphate
  • ↓Pyruvate kinase activity
  • ↓Conversion of PEP to pyruvate
  • ↑PEP is diverted to the synthesis of glucose