CHEM 1050 Flashcards

Metabolic Regulation

• Chemical Messengers

  • Endocrine:
    • Hormones secreted into the blood, affecting distant target cells.
    • React with cells throughout the body due to high concentration (e.g., insulin, glucagon, cortisol).
  • Paracrine:
    • Substances secreted by non-endocrine cells (e.g., liver or muscle cells).
    • Act on nearby cells; specificity depends on cell location due to low concentrations.
  • Autocrine:
    • Act on the secreting cell itself or nearby same-type cells.
    • Most autocrine cells also function as paracrine cells.
    • Some signals can function in multiple manners.

• Cell Signaling Overview

  • Begins with the release of a chemical messenger.
  • Messenger diffuses or is transported in blood/extracellular fluids.
  • Binds to a specific receptor on a target cell (membrane-bound) or diffuses through the lipid bilayer (steroid hormones).
  • Receptor binding elicits a response that can be terminated.

Intracellular Receptors

• Bind hydrophobic chemical messengers.
  • Usually elicit a transcriptional response (slow change in cellular phenotype).
  • Example: Cortisol binds an intracellular receptor.

Membrane Receptors

• Bind hydrophilic chemical messengers.
  • Directly change enzyme activity through protein-protein interactions (fast change in cellular phenotype).
  • Examples:
    • Insulin binds tyrosine kinases.
    • Glucagon binds G-protein coupled receptors.

Cortisol Signaling

• Cortisol is released from the adrenal cortex and diffuses into the bloodstream.
  • Being hydrophobic, it's transported bound to serum albumin and steroid hormone binding globulin.
• It diffuses through the plasma membrane of the cell and binds to intracellular cortisol receptors in the cytosol.
• Ligand binding induces a conformational change:
  • Dimerization of receptors occurs.
  • Exposes a nuclear translocation signal, allowing the hormone-receptor complex to enter the nucleus.
• In the nucleus, the complex acts as a transcription factor.
  • Binds to the hormone response element (HRE) or glucocorticoid response element (GRE) on the DNA.
  • This binding either increases (induction) or decreases (repression) gene transcription, depending on the GRE location.
• The signal is terminated by the liver destroying cortisol, thus lowering its concentration.

G-Protein-Coupled Receptor (GPCR) Cascade (Glucagon Signaling)

• Hormone binds to an extracellular domain of a 7-helix receptor (GPCR), causing a conformational change transmitted to a G-protein on the cytosolic side.

  • The nucleotide-binding site on Gα becomes more accessible to the cytosol, where GTP concentration is higher than GDP concentration.
  • Gα releases GDP and binds GTP in exchange.

• GTP substitution causes another conformational change in Gα.

• Gα-GTP dissociates from the inhibitory subunit complex.

  • Activates adenylyl cyclase (or an effector enzyme).

• Adenylyl cyclase catalyzes the synthesis of cAMP (second messenger).

• cAMP activates Protein Kinase A.

• Phosphodiesterase degrades cAMP to terminate the signal.

• Several types of GPCRs exist, depending on the regulatory complex:

  • Gαs → increases cAMP.
  • Gαi → decreases cAMP.
  • Gαq → Increases phospholipase C activity.

Insulin Signaling

• Amplifies signals through kinase cascades, with Receptor Tyrosine Kinase receptors located in the cell membrane.
  • These receptors dimerize upon ligand binding and autophosphorylation occurs on the inner side of the membrane.
• The active receptor phosphorylates Insulin Receptor Substrate (IRS).
  • Phosphorylated IRS binds other proteins to amplify the signal:
    1. PI3Kinase
    2. Phosphoinositol Kinase 1 (PDK1) which activates Protein Kinase B (PKB).
    3. Grb2 → activates a MAPK cascade.
       • These proteins (Grb2 and PI3Kinase) have an SH2 domain but bind to different sites on the IRS protein.

Phosphatidylinositide Metabolism

• Provides a connection between the hormone receptor and intracellular calcium.
• Gsq activates Phospholipase C
  • Cleavage of PIP2 by Phospholipase C yields two second messengers:
    • inositol-1,4,5-trisphosphate (IP3) → enhances $Ca^{2+}$ release from the endoplasmic reticulum
    • diacylglycerol (DG) → activates Protein kinase C
• Kinases catalyze sequential transfer Pi from ATP to hydroxyl groups at positions 5 & 4 of the inositol ring of phosphatidylinositol, to yield phosphatidylinositol-4,5-bisphosphate (PIP2).

Metabolism in the FED State

Metabolism in the FASTED State

Pathways Activated /Enhanced by Each Hormone

• Insulin (anabolic)
  • Liver
    • Glycolysis
    • TCA
    • Glycogen synthesis
    • Fatty acid synthesis
    • Protein synthesis
  • Skeletal muscle
    • Glucose uptake through GLUT4
    • Protein synthesis
    • Glycogen synthesis
  • Adipose
    • Triacylglycerol synthesis
    • Glucose uptake through GLUT4
• Glucagon (catabolic)
  • Liver
    • Glycogenolysis
    • Gluconeogenesis
• Epinephrine (catabolic)
  • Liver
    • Glycogenolysis (via alpha-agonist pathway)
    • Enhances gluconeogenesis
  • Skeletal muscle
    • Glycogenolysis Via cAMP pathway
• Cortisol (catabolic)
  • Liver
    • Lipolysis
    • β-oxidation of fatty acids through enhanced transcription of the proteins required for the process (PEPCK)
  • Skeletal muscle
    • Promotes protein catabolism to provide amino acids for gluconeogenesis occurring in the liver
  • Adipose
    • Lipolysis

Glycolysis – Cytosolic Pathway

• Glycolysis takes place in the cytosol of a cell, and it can be broken down into two main phases:
  1. Energy-requiring phase (uses 2 ATP). In this phase, the starting molecule of glucose is rearranged, and two phosphate groups are attached. The phosphate groups make the modified sugar—now called fructose 1,6-bisphosphate—unstable, allowing it to split in half and form two phosphate-bearing three-carbon sugars. The three-carbon sugars formed when the sugar is cleaved by aldolase B are: glyceraldehyde-3- phosphate and DHAP. Only glyceraldehyde-3-phosphate is directly oxidized; DHAP is readily converted into glyceraldehyde-3-phosphate.
  2. Energy-releasing phase (produces 4 ATP and 2 NADH). In this phase, each three-carbon sugar is converted into another three-carbon molecule, pyruvate, through a series of reactions. In these reactions, two ATP molecules and one NADH molecule are made. Because this phase takes place twice, it makes four ATP and two NADH overall.
     • Aerobic pathway: Glucose is oxidized to pyruvate and oxidation continues in the mitochondria to generate acetyl-CoA -NAD+ is regenerated by transport of reducing equivalents across the mitochondrial membrane using one of two shuttles
       • Glycerolphosphate shuttle (week 3)
       • Malate shuttle (week 3)
     • Anaerobic pathway: Glucose is oxidized to lactate
       • This will occur either in the absence of oxygen
       • In cells that lack mitochondria (red blood cells)
       • Skeletal muscle oxidation of glycogen under anaerobic conditions
       • Impaired activity of the pyruvate dehydrogenase complex

Regulatory Steps in Glycolysis

1.  Conversion of Glucose to Glucose 6-phosphate. Uses ATP.
    *   **Enzymes:** Glucokinase or Hexokinase
        *   Glucokinase vs. hexokinase
            *   Glucokinase and hexokinase are isozymes. They catalyze the same enzymatic reaction. Isozymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction.
            *   Hexokinase has a low Km for glucose and is found in tissues other than the liver. It is rapidly saturated.
            *   Glucokinase has a high Km for glucose and is found liver or pancreas. It is not rapidly saturated and can remove glucose steadily from the bloodstream as a means of allowing for excess glucose to be stored as glycogen (triacylglycerol).
2.  Conversion of Fructose 6-phosphate to Fructose 1,6-bisphosphate. Uses ATP.
    *   **Enzyme:** Phosphofructokinase-1 (PFK1)
        *   Fructose 6-phosphate can also be converted to fructose 2,6-bisphosphate by Phosphofructokinase-2 (PFK2) – NOTE: this is not considered part of glycolysis but is a shunt of the pathway.
            *   PFK2 is regulated through phosphorylation.
            *   When PFK2 is dephosphorylated, it is ACTIVE as a kinase
                *   Fructose 2,6-bisphosphate enhances the activity of PFK1 (allosteric activator)
                *    Fructose 1,6-bisphosphate is a FEEDFORWARD activator of Pyruvate kinase.
3.  Conversion of phosphoenolpyurvate (PEP) to pyruvate
    *   **Enzyme:** Pyruvate Kinase (PK)
        *   Catalyzes the irreversible reaction of PEP to pyruvate
        *   Regulated by feedforward regulation
            *   Allosterically activated by fructose 1,6-bisphosphate

Pyruvate Fate

• Pyruvate generated through this process can either:
  • Enter the TCA cycle through the pyruvate dehydrogenase complex
  • Be converted to lactate under anaerobic conditions
  • Be transaminated to alanine

Regulation Summary of Glycolysis

Summary of TCA Regulation

Connections to Other Pathways

• Fatty acid synthesis
  • Citrate is shuttled out of the TCA
    • In the cytosol it is cleaved by citrate lyase to generate: acetyl-CoA and oxaloacetate (OAA)
    • Acetyl-CoA is used as a substrate for fatty acid synthesis
    • OAA is reduced to malate → malate is decarboxylated back to pyruvate which can reenter the TCA
      • This conversion by malic enzyme produces NADPH
• Anaplerotic reactions
  • Reactions that transfer net carbon into the TCA cycle
    • Carboxylation of pyruvate → oxaloacetate (by pyruvate carboxylases)
    • Transamination reactions
      • Glutamate → alpha-ketoglutarate
      • Alanine → pyruvate
      • Aspartate → oxaloacetate

Review Shuttles

• Shuttle systems
  1. Glycerophosphate shuttle (Glycerol 3-phosphate shuttle)
     • Moves reducing equivalents of NADH from the cytosol to an FAD in the mitochondrion
     • FAD is a required cofactor glycerol-3 phosphate dehydrogenase
     • FAD is tightly or covalently bound to the dehydrogenase.
     • Cytosolic dihydroxyacetone phosphate is reduced to glycerol 3-phosphate -->glycerol 3-phosphate moves into the mitochondria and transfers the e- to FAD bound to the dehydrogenase
  2. Malate-Aspartate shuttle
     • Oxaloacetate DOES not move across the mitochondrial membrane!
     • Starting in the cytosol:
       • Oxaloacetate is REDUCED to malate by cytosolic malate dehydrogenase
       • The shuttle moves malate (reduced form of oxaloacetate) into the mitochondria
     • Once in the mitochondria:
       • Malate can be OXIDIZED back to oxaloacetate by mitochondrial malate dehydrogenase
         • The resulting oxaloacetate can undergo a TRANSAMINATION REACTION (See week 1) with glutamate
           • The reaction transfers the $NH_3^+$ from glutamate TO oxaloacetate to generate
           • → alpha-ketoglutarate and aspartate
           • Aspartate can move in / out of the mitochondria
             • Aspartate can be converted to oxaloacetate
             • For each NADH equivalent → 2.5 molecules of ATP are generated

Glycogen Synthesis and Degradation

• Glycogen is stored primarily in the:

  • Liver
  • Skeletal muscle

• Pathway for synthesis and degradation are identical:

  • Synthesis
    • Phosphoglucomutase: Glucose 6-P is isomerized → Glucose 1-P
    • UDPGlc pyrophosphorylase: Glucose 1-P + UTP → UDPGlc
    • Glycogen synthase:→ adds UDPGlc →terminal end of glycogen (1→4 linkage)
      • Glycogenin primer is required
  • Degradation
    • Glycogen phosphorylase: releases glucose 1-P from glycogen
    • Glucose 6-Phosphatase: Glucose 6-P → Free Glucose
      • Enzyme is required to de-phosphorylate glucose to be released from the liver
      • Key difference in liver vs. muscle glycogenolysis

Regulation of Glycogen Metabolism in Liver

• In the liver glycogen synthesis and degradation are regulated through two main mechanisms:
  • Glucagon activation of GPCR
    • Activation of Adenylate cyclase → increases cAMP
    • cAMP activates Protein kinase A (PKA) →phosphorylates phosphorylase kinase
      • phosphorylated phosphorylase kinase → phosphorylates glycogen phosphorylase→ glycogen degradation
    • cAMP activates PKA → phosphorylates glycogen synthase → inactivates
  • Epinephrine binds α-adrenergic
    • Cleavage of PIP → IP3 and DAG
    • IP3 → stimulates $Ca^{2+}$ release from ER
      • $Ca^{2+}$ stimulates two responses
        • 1) $Ca^{2+}$ calmodulin
          • → activates phosphorylase kinase→ phosphorylates glycogen phosphorylase →glycogen degradation
          • calmodulin dependent kinase →phosphorylates glycogen synthase →inactivates
        • DAG → activates protein kinase C (PKC)

Regulation of Glycogen Metabolism in Skeletal Muscle

• In the skeletal muscle glycogen synthesis and degradation are regulated through three main mechanisms:
  • NOTE: skeletal muscle is NOT impacted by GLUCAGON
    • Epinephrine activation of GPCR
      • Activation of Adenylate cyclase →increases cAMP
      • cAMP activates Protein kinase A (PKA) → phosphorylates phosphorylase kinase
        • phosphorylated phosphorylase kinase → phosphorylates glycogen phosphorylase→ glycogen degradation
      • cAMP activates PKA →phosphorylates glycogen synthase →inactivates
    • $Ca^{2+}$ mediated muscle contraction
      • 1) $Ca^{2+}$ calmodulin
        • →activates phosphorylase kinase → phosphorylates glycogen phosphorylase→glycogen degradation
    • Elevated AMP → allosterically activates glycogen phosphorylase

Regulation Summary of Glycogen Metabolism

Gluconeogenesis

• Predominantly occurs in the liver
  • Substrates
    • Lactate – predominantly from the Cori cycle
    • Glycerol – predominantly released during lipolysis
    • Amino acids: primarily alanine from skeletal muscle
      • Lactate and alanine are shuttle from skeletal muscle
    • NOTE: FATTY ACIDS or KETOGENIC amino acids ARE NOT A SUBSTRATE
      • These are metabolized to acetyl-CoA and are fully oxidized in the TCA cycle

Unique Steps in Gluconeogenesis

• There are 4 steps that are unique to gluconeogenesis. These steps overcome the 3 regulatory steps in glycolysis.

  • Pyruvate carboxylase (PC; mitochondrial enzyme): carboxylates pyruvate to OAA

    • OAA is reduced to malate and malate can leave the mitochondria
    • Requires acetyl-CoA for allosteric activation; biotin as a cofactor; CO2 is used

  • Phosphoenol pyruvate carboxykinase (PEPCK; cytosolic enzyme): converts cytosolic OAA → phosphoenol pyruvate

    • Transcription of this enzyme is enhanced by high levels of cortisol
    • NOTE: Pyruvate carboxylase and PEPCK are both required to overcome the glycolytic reaction catalyzed by pyruvate kinase

  • Fructose 1,6-bisphosphatase (FBP-1; cytosolic): converts fructose 1,6-bisphosphate to fructose 6-phosphate

    • Activity is inhibited by AMP and fructose 2,6-bisphosphate
    • Enzyme overcomes the irreversible glycolytic reaction catalyzed by phosphofructokinase-1
    • NOTE: Fructose 2,6-bisphosphatase (FBP-2) is active and reduces the amount of fructose 2,6 bisphosphate as a means of reducing the allosteric activation of PFK1 which enhances the reverse reaction catalyzed by fructose 1,6- bisphosphatase
      • FBP-2 is a bifunctional enzyme
        • In the FED state the enzyme has kinase activity and is termed Phosphofructokinase-2 (PFK2)
          • Generates fructose 2,6-bisphosphate that allosterically activates PFK-1
            • PFK-2 is dephosphorylated and the kinase portion is active and the phosphatase portion is inactive
        • In the FASTED state the enzyme has phosphatase activity – Fructose 2, 6-bisphosphatase
          • Generates fructose 6-phosphate
            • FBP-2 is phosphorylated and the phosphatase portion is active; kinase portion is inactive

  • Glucose 6-phosphatase: dephosphorylates glucose 6-phosphate to free glucose that can be released from the liver.

    • This enzyme is used by both gluconeogenesis and glycogenolysis.

  • Maintaining blood glucose

    • BOTH glycogenolysis and GNG provide glucose for this.

Regulation Summary of Gluconeogenesis

Alanine-Glucose Cycle and Cori Cycle

• Cori cycle (Lactic acid cycle)
  • Lactate formed in skeletal muscle or red blood cells → transported to the liver
    • Can be used as a substrate for GNG to make glucose
    • Glucose can enter circulation as a substrate for tissues
• Alanine-Glucose cycle
  • Alanine released from muscle during fasted state → transported to the liver
    • Alanine → transaminated to pyruvate and used as a substrate for GNG
    • Glucose can enter circulation as a substrate for tissues

Pentose Phosphate Pathway

• The pentose phosphate pathway has two distinct segments: irreversible oxidative and reversible non-oxidative
  • These portions of the pathway can operate independent of one another
  • Deficiencies in the oxidative portion can result in hemolytic anemia
  • Two primary products: ribulose 5-phosphate and NADPH

Irreversible Oxidation Portion of the Pathway

• Irreversible Oxidation portion of the pathway converts Glucose 6 –phosphate → ribulose 5-phosphate
  • Two enzymes are required:
    • Glucose 6-phosphate dehydrogenase (G6PDH)
    • 6-phosphogluconate dehydrogenase
      • NADPH is produced during each reaction
  • Regulatory enzyme: Glucose 6-phosphate dehydrogenase (G6PDH)
    • NADPH will inhibit the activity of G6PDH

NADPH Uses

• NADPH is used as/for:
  • Reducing agent in many tissues
  • Reduction of glutathione in RBC
  • Also used for fatty acid synthesis in other tissues
  • NADPH is not oxidized through the electron transport

Reversible Non-Oxidative Portion of the Pathway

• Reversible non-oxidative of the pathway converts ribulose 5-phosphate back to intermediates of glycolysis (glyceraldehyde 3-phosphate and fructose 6-phosphate)
  • Consists of near-equilibrium reactions
  • Two primary enzymes:
    • Transketolase: Moves 2-carbon units
      • Transketolase requires thiamine pyrophosphate (TPP) a cofactor
      • Measurement of erythrocyte transketolase activity is a measure of thiamine nutritional status
    • Transaldolase: Moves 3-carbon carbon units depending on the cellular needs
  • Ribose 5-phosphate produce functions as a 5-carbon sugar required for nucleotide synthesis

Pentose Phosphate Pathway and Red Blood Cells (RBCs)

• The pentose phosphate pathway is the only source of NADPH in RBCs
  • Note: NADPH can also be produced by malic enzyme in other tissues
  • NADPH produced in RBCs is required for the reduction of oxidized glutathione
  • Glutathione (GSH) is a tri-peptide needed to oxidized hydrogen peroxide
    • Glutathione peroxidase and glutathione reductase regenerate reduced glutathione through a series of reactions using NADPH

Glucose 6 Phosphate Dehydrogenase Deficiency

• Glucose 6 phosphate dehydrogenase deficiency Loss of the enzyme in red blood cells can lead to hemolysis/jaundice due to a decrease in NADPH production
  • Presentation of hemolytic anemia
  • Decrease in reduced GSH

Fructose Metabolism

• Fructokinase: Fructose is taken up and phosphorylated to fructose 1-phosphate
  • Deficiency in fructokinase → fructosuria
    • Fructose is not trapped in the cell so this will not produce fasting hypoglycemia
    • Benign deficiency
• Aldolase B: Cleaves fructose 1-phosphate to cleave fructose into dihydroxyacetone phosphate (DHAP) and glyceraldehyde
  • These products can enter glycolysis
    • Note: glyceraldehyde is phosphorylated to glyceraldehyde 3-phosphate first
  • Fructose metabolism bypasses PFK-1 → Oxidation of fructose is very rapid
  • Deficiency of aldolase B: Hereditary fructose intolerance
    • Fructose 1-phosphate trapped in the liver → hepatomegaly
    • Other clinical manifestations include:
      • Reducing sugar (fructose) in urine
      • Fasting hypoglycemia and vomiting
    • Elevated fructose consumption → can contribute to:
      • Hypertriglyceridemia due to increased VLDL synthesis
      • Gout due to increased purine synthesis

Galactose Metabolism

• Galactokinase: Galactose is taken up by the liver and phosphorylated to galactose 1-phosphate
• Galactose 1-phosphate uridyl transferase:
  • Requires UDP-Glucose
  • Generates glucose 1-phosphate which enters glycolysis
  • Deficiency in galactose 1-phosphate uridyl transferase:
    • Can result in liver failure due to accumulation of galactose 1-phosphate