S4 Glycolysis Gluconeogenesis_da9f8fb54ed1cd011679d9a61f3bb666.pdf

Glycolysis & Gluconeogenesis Richard L. Sabina, PhD Suggested Reading: Mark’s 6th ed. Chapters 22, 28 Session Objectives: 1. Summarize opposing pathways of glycolysis and gluconeogenesis, discuss which reactions are reversible or irrever...

Glycolysis & Gluconeogenesis Richard L. Sabina, PhD Suggested Reading: Mark’s 6th ed. Chapters 22, 28 Session Objectives: 1. Summarize opposing pathways of glycolysis and gluconeogenesis, discuss which reactions are reversible or irreversible, and the purpose served by each in either catabolizing (glycolysis) or participating in the de novo synthesis (gluconeogenesis) of glucose. 2. Distinguish between aerobic and anaerobic glycolysis. 3. Identify key regulatory points in glycolysis & gluconeogenesis and predict how changes in cellular conditions will affect the metabolic rate of these two pathways. 4. Compare and contrast the metabolic roles of glycolysis and control mechanisms in liver and skeletal muscle. 5. Identify select clinical connections of glycolysis, such as lactic acidosis, arsenic poisoning, and inherited deficiencies, and gluconeogenesis, such as alcohol, anemia, and diabetes. 6. Identify various substrates used by the gluconeogenic pathway for the synthesis of glucose, including adipose triglycerides, TCA cycle intermediates, glucogenic amino acids, and discuss the roles of the alanine-lactate and Cori cycles.  The BIG Picture w glycolysis: pathway that metabolizes the most common sugar, glucose, to pyruvate (aerobic glycolysis) or lactate (anaerobic glycolysis). - net energy yield is modest (2 ATP/glucose), but pathway able to run at high speed and in the absence of oxygen (anaerobic glycolysis). - when glucose supply is good (e.g., from glycogen or blood), pathway can quickly make a lot of ATP (at expense of flooding tissue & plasma with lactic acid [potential acidosis from anaerobic glycolysis]; example - ischemic tissue [e.g., stroke, myocardial infarction] runs out of oxygen long before glucose) w gluconeogenesis: essentially the reverse of glycolysis and serves the purpose of replenishing glucose consumed by glycolysis. - liver (kidney [minor; ñ role in T2DM]); serves to maintain blood glucose during fasting - muscle (and other energy-utilizing tissues); process incomplete - pathway ends at G-6-P (no glucose-6-phosphatase!!), which is used to replenish glycogen w irreversible enzymes of glycolysis are replaced with different irreversible enzymes in gluconeogenesis (reversible steps are generally shared) - control takes place at irreversible steps and is affected by both metabolites and hormones (insulin, epinephrine & glucagon) Session Objective #1: Glycolysis Summary note reversible vs. w catabolizes one 6-C irreversible steps molecule of glucose to two 3-C molecules of pyruvate (or lactate) - yields modest net production of ATP and reducing equivalents (NADH) Ÿ overall yield: 2 pyruvate, 2 NADH, 2 ATP/glucose w important reactions catalyzed by: 1) hexokinase/glucokinase1,2 2) phosphofructokinase-1 (PFK-1)1,2 3) glyceraldehyde 3-phosphate dehydrogenase4 4) 3-phosphoglycerate kinase3 5) pyruvate kinase1,2,3 6) lactate dehydrogenase5 (shown on next slide) 1 - regulatory step 2 - irreversible step 3 - produces ATP 4 - produces NADH 5- re-generates NAD+ NOTE: these intermediates & products are x2  Session Objective #2: Aerobic vs Anerobic Glycolysis Oxygen Supply Determines End Products of Glycolysis w aerobic conditions; end product, pyruvate - NADH re-oxidized to NAD+ by shuttles; reducing equivalents transferred into mitochondria for electron transport chain (requires O2 & mitochondria) - NAD+ used for continued glycolysis - pyruvate used for acetyl CoA synthesis - examples; liver, normoxia; muscle, distance walking w anaerobic conditions; end product, lactate - NADH re-oxidized to NAD+ by lactate dehydrogenase (reduces pyruvate to lactate) - NAD+ used for continued glycolysis - examples; liver, hypoxia; muscle, sprint running - lactate production is the only fate of pyruvate in cells without mitochondria, e.g., red blood cells Session Objective #3: Regulation of Glycolysis Key Enzymes of Glycolysis w hexokinase/glucokinase; - important regulatory step (review Glycogen Metabolism, slide #6); historically, included in the glycolytic pathway (formally, pathway begins with phosphofructokinase-1 [PFK-1]) - requires ATP & Mg2+ Enzyme Tissue Km (mM) Inhibition by G-6-P hexokinase all 0.1 Yes enables glucose to be phosphorylated at high intracellular glucokinase liver, 10.0 No concentrations in pancreas these tissues!! w phosphofructokinase-1 (PFK-1): - catalyzes the rate-limiting step in glycolysis (also committed & first irreversible step of glycolysis) - requires ATP & Mg2+ - reaction is irreversible & regulated (Fructose 1,6-diphosphate)  Key Enzymes of Glycolysis (cont.) w phosphoglycerate kinase: - uses high energy of 1,3-bisphosphoglycerate acyl-phosphate bond and generates ATP (example of substrate level phosphorylation) - reversible reaction; not regulated; requires ADP and Mg2+ w pyruvate kinase: - couples high energy of phosphoenolpyruvatye acyl-phosphate bond and generates ATP (another example of substrate level phosphorylation) - requires ADP, K+, Mg2+ - reaction is irreversible & regulated Ÿ feed-forward activated by fructose 1,6-bisphosphate (enables coupled regulation of the two irreversible steps of glycolysis) Ÿ protein kinase A (PKA) target (phosphorylated & less active during fasted state) w lactate dehydrogenase (LDH): - produces lactate and re-generates NAD+ - reaction is reversible Ÿ operates in opposite direction in liver during exercise (component of Cori cycle; more later)  Regulation of Phosphofructokinase-1 (PFK-1) w allosteric regulation - AMP, ADP, fructose-2,6-BP (liver) stimulate activity Ÿ ADP and AMP increase (in muscle) during exercise - ATP, citrate, inhibit activity Ÿ ATP decreases (in muscle) during exercise Ÿ relative citrate levels indicator of TCA cycle intermediates w fructose 2,6-bisphosphate (fructose 2,6-BP; liver only): - metabolized by phosphofructokinase-2 (PFK-2 protein; bi-functional enzyme; PKA target) Ÿ PFK-2 activity produces F-2,6-BP (from small amount of F-6-P) Ÿ FBP-2 (fructose 2,6-bisphosphatase) activity degrades F-2,6-BP (back to F-6-P) - opposing activities controlled by LIVER ONLY!! phosphorylation state of PFK-2 Ÿ fed (high insulin:glucagon) ° ↓ enzyme phosphorylation (↑PFK-2 activity = ↑F-2,6-BP) Ÿ fasted (low insulin:glucagon) ° ↑ enzyme phosphorylation (↑FBP-2 activity = ↓F-2,6-BP) PKA phosphorylation of PFK-2 = ê small molecule activator of PFK-1  Regulation of Pyruvate Kinase (PK) w short-term control: isoforms expressed in liver, kidney, RBC allosterically-regulated - feed-forward activated by fructose-1,6-BP - inhibited by ATP w medium-term control: phosphorylation (PKA target; liver & intestine) - fed (high insulin:glucagon); more active, as insulin signal stimulates protein phosphatase-1 (PK dephosphorylated) - fasted (low insulin:glucagon); less active, as glucagon stimulates PKA (PK phosphorylated) Note: reinforces regulation at PFK-1; i.e., PKA also phosphorylates bifunctional PFK-2, resulting in F-2,6-BP catabolism (F-2,6-BP activates PFK-1, so its catabolism also slows glycolysis)  Regulation of Glycolysis Summary w Overview - key factors: Ÿ glucose supply (substrate) Ÿ energy supply; ATP/ADP/AMP (allosteric regulators) Ÿ fed-fasted state (insulin/glucagon ratio) Ÿ oxygen supply (aerobic vs anaerobic metabolism) Ÿ other allosteric regulators (citrate, glucose-6-phosphate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate) - key regulatory enzymes: Ÿ hexokinase (regulated by glucose-6-phosphate) Ÿ glucokinase (gene transcriptionñ by high insulin:glucagon ratio) Ÿ phosphofructokinase-1 (regulated by allosteric effectors) Ÿ phosphofructokinase-2 (metabolizes allosteric effector & regulated by PKA phosphorylation) Ÿ pyruvate kinase (regulated by allosteric effectors & PKA phosphorylation) Session Objective #4: Regulation of Glycolysis in Muscle Regulation of Glycolysis in Muscle by Energy Supply Session Objective #5: Clinical Aspects of Glycolysis Select Clinical Aspects of Glycolysis w lactic acidosis - excess lactate released by cells during anaerobic glycolysis Ÿ lactate: acid that can lower blood pH to dangerous levels (lactate [5 mM] → blood pH