1. Metabolic fates of glucose.pdf

METABOLISM AND CELL BIOLOGY (PHRD 515) 1 Course ID PhrD515 Course Title Metabolism and Cell Biology Course Meets Mondays 09:00 AM – 12:00 PM (Synchronous Sessions) Location PSC108 Coordinator Enrique Cadenas Office PSC6...

METABOLISM AND CELL BIOLOGY (PHRD 515) 1 Course ID PhrD515 Course Title Metabolism and Cell Biology Course Meets Mondays 09:00 AM – 12:00 PM (Synchronous Sessions) Location PSC108 Coordinator Enrique Cadenas Office PSC614 Contact Info [email protected] Office Hours By e-mail appointment Wednesdays 3:00-4:00 PM 2  METABOLISM AND CELL BIOLOGY (PHRD 515) WEEKLY SCHEDULE WEEK 1 WEEK 5 1. Carbohydrate Metabolism I 11. Mitochondrial Therapeutics 2. Carbohydrate Metabolism II 12. Cell Signaling I 3. Bioenergetics WEEK 6 WEEK 2 13. Cell Signaling II 4. Biochemistry of Fat and NSAIDs 14. Cell Signaling III-IV 5. Lipid Metabolism WEEK 7 WEEK 3 15. Redox Biology 6. Dietary Lipids and Lipoproteins 16. Inflammation 7. Obesity WEEK 8 8. Atherosclerosis 17. Alzheimer’s I WEEK 4 18. Alzheimer’s II 9. Starve/Feed Cycle 10. Diabetes 3  PhrD515 – Metabolism and Cell Biology – Fall 2024 Modules Topics Asynchronous Sessions Synchronous Sessions 26-Aug-24 Metabolic Fate of Carbohydrates 1 Carbohydrate Metabolism I Week 1 09:00AM 12:00 PM Mitochondrial Bioenergetics Module 1 2 Carbohydrate Metabolism II Group Project 3 Bioenergetics 5-Sep-24 NSAIDs Week 2 1:00 PM 3:00 PM Lipid Metabolism hormone regulation Module 2 4 NSAIDs Recordings on NSAIDs and Quizzes - Module 1 5 Lipid Metabolism lipid metabolism Group Project 9-Sep-24 Lipoproteins Obesity Atherosclerosis 6 Lipoproteins Week 3 09:00AM 12:00 PM Quizzes - Module 2 Module 3 7 Obesity Group Project 8 Atherosclerosis 16-Sep-24 Week 3 09:00AM 12:00 PM Quizzes - Module 3 Module 4 9 Starve-Feed cycle Group Project 10 Diabetes Midterm Exam 20-Sept-24 1–3 PM (cover material from Week 1 to Week 4 included) 23-Sep-24 Therapeutics/Cell Signaling 11 Mitochondrial Therapeutics Week 5 09:00AM 12:00 PM Quizzes - Modules 4 Module 5 12 Cell Signaling I Group Projects 13 Cell Signaling II 30-Sep-23 Cell Signaling Redox Biology 14 Cell Signaling III-IV Week 6 09:00AM 12:00 PM Quizzes - Modules 5 Module 6 15 Redox Biology Group Projects 7-Oct-24 Inflammation I/Neurodegeneration 16 Inflammation Week 7 09:00AM 12:00 PM Quizzes - Modules 6 Module 7 17 Ineurodegeneration I 9-Oct-23 14-Oct-24 Neurodegeneration II – Revision Recordings on Week 8 09:00AM 12:00 PM Module 8 18 Neurodegeneration II Neurodegeneration I and II Reading Material Revision Final Exam 16-Oct-24 1–3 PM (covers material from Week 5 to Week 8 included)  METABOLISM AND CELL BIOLOGY (PHRD 515) REQUIRED READINGS Primary didactic materials can be found in the detailed handouts. Asynchronous sessions (Lecture recordings and corresponding handouts) will be available only for Module 2 and Module 8. Synchronous sessions consist of formative lectures. Exams, quizzes, and group projects will adhere strictly to the material in the handouts No supplementary materials are required unless provided for group projects. These supplementary materials require mastering the concepts in the handouts 5  METABOLISM AND CELL BIOLOGY (PHRD 515) ASSESSMENT METHODS Grading Breakdown Grading Scale A 95-100 Assignment % A- 90-94 _______________________________________ B+ 87-89 B 83-86 Midterm Exam….................. 40 B- 80-82 Group Projects/Quizzes........ 20 C+ 77-79 C 73-76 Final Exam........................... 40 C- 70-72 D+ 67-69 D 63-66 D- 60-62 F 59 and below Midterm- and Final Exam: Multiple Choice/ExamSoft In Class: Weekly Quizzes (Multiple Choice) Group Projects/Reports 6  AI GENERATORS AI NOT PERMITTED Since creating, analytical, and critical thinking skills are part of the learning outcomes of this course, all assignments should be prepared by the student working individually or in groups. Students may not have another person or entity complete any substantive portion of the assignment. Developing strong competencies in these areas will prepare you for a competitive workplace. Therefore, using AI- generated tools is prohibited in this course, will be identified as plagiarism, and will be reported to the Office of Academic Integrity. 7  MODULE 1 AUGUST 26, 2024 1. METABOLIC FATES OF GLUCOSE 2. ENDOCRINE REGULATION 3. BIOENERGETICS 8 1 1. CARBOHYDRATE METABOLISM I 9  WHY METABOLISM OF CARBOHYDRATES AND LIPIDS? SIGNIFICANCE: Balance of insulin and glucagon secretin important for maintaining balance STARVE-FEED CYCLE DIABETES OBESITY ATHEROSCLEROSIS CELL SIGNALING ALZHEIMER’S DISEASE [lactate] insulin [pyruvate] glucagon HYPOXIA b cells a cells Pancreas 10  1. CARBOHYDRATE METABOLISM I – CONTENT – I. Glycemia Values Regulatory steps II. How does glucose enter the cells? Significance of fructose-2,6-bisphosphate GLUT1 The Cori Cycle GLUT2 Clinical correlations GLUT4 Hypoglycemia and premature infants III. Overview of glucose metabolism Hypoglycemia and alcohol intoxication IV. Glycolysis Stages of glycolysis VI. Pentose Phosphate Pathway Regulatory steps Purpose and functions Fates of pyruvate Phase I and Phase II Clinical correlations Regulatory step Lactic acidosis Clinical correlations Hemolytic anemia Wernicke-Korsakoff syndrome V. Gluconeogenesis Drug hemolytic anemia Gluconeogenic and glycolytic tissues Pernicious anemia 11  1. CARBOHYDRATE METABOLISM I – LEARNING OBJECTIVES – Discuss the allosteric regulation of glycolysis and gluconeogenesis Identify insulin-sensitive glucose transporters and explain why they are insulin-sensitive Recognize the function of high-capacity, bidirectional glucose transporters and in which tissue they are highly expressed Remember the only regulatory step in the pentose phosphate pathway Analyze and identify the genetic deficiencies in hemolytic anemia, drug hemolytic anemia, and pernicious anemia Distinguish between galactosemia and lactose intolerance and their respective genetic deficiencies Identify the genetic deficiency that occurs in hypoglycemia of premature infants and evaluate the function of the Cori cycle 12 Remember the fasting values I. GLYCEMIA VALUES Fasting State Post-Prandial Glucose Glucose Glucose (minimum) (mg/dl) (maximum) (mg/dl) 2-3 hours after (eating (mg/dl) Hypoglycemia – < 59 < 60 Early hypoglycemia 60 79 60 – 70 Normal 80 100 < 140 Early diabetes 101 126 140 – 200 Diabetes > 126 – > 200 13  I. HbA1c Hemoglobin A1c (HbA1c or A1C) is used to diagnose type 1 and type 2 diabetes, to identify pre-diabetes, and to monitor management of diabetes. Non-enzymic glycation of HbA1c is facilitated by hyper- glycemia and it offers accurate values of glycemia. The higher HbA1c levels, the poorer the glycemia control. Normal HbA1c range: 4.4-5.6%. HOW TO COMPARE Blood Sugar HbA1c 4% 60 (mg/dL) 5% 90 6% 120 7% 150 Normal Prediabetes Diabetes 8% 180 < or = to 5.6 5.7-6.4 6.5+ 210 180 210 120 150 Blood Glucose 240 270 9% (mg/dL) 90 300 10% 240 11% 270 12% 300 13% 330 14  II. HOW DOES GLUCOSE ENTER THE CELLS? Glucose Transporters (GLUTs) The transport of monosaccharides and other small carbon compounds across membranes is mediated by the GLUT family of glucose integral membrane proteins. There are 14 human GLUT proteins that possess various substrate specificities and are divided in three classes: Class I includes GLUT1, GLUT2, GLUT3, GLUT4, and GLUT 14 Class II includes GLUT5, GLUT7, GLUT9, and GLUT11 Class III includes GLUT6, GLUT8, GLUT10, GLUT12 glucose _______________________________________________________________________________________________________ GLUT Tissue Distribution Link to disease ________________________________________________________________________________________________________ GLUT1 erythrocytes, brain, brain-blood barrier GLUT1 Deficiency syndrome blood tissue barriers, fetal tissues ataxia, dyskinesia GLUT2 Liver, islet of Langerhans, kidney Fanconi-Bickel syndrome brain, intestine (type II diabetes) GLUT3 brain (neurons), testis GLUT4 adipose tissue, skeletal and cardiac Type II diabetes muscle, brain (neurons) GLUT6 brain, spleen, leukocytes 15  GLUT1 GLUT1 is expressed in many cells and at its highest levels in the human erythrocyte membrane. GLUT1 plays a critical role in brain glucose uptake as the major GLUT isoform expressed in brain endothelial cells and astrocytes: under normal physiological conditions the brain is absolutely dependent on glucose as a fuel source and transport across the blood-brain barrier is limiting for brain glucose metabolism. GLUT1 is responsible for mediating materno-placental transfer of glucose in humans. Altered placental GLUT1 levels affect the fetus. Patients with GLUT1 deficiency syndrome (GDS) experience seizures in early infancy and exhibit developmental delay, ataxia, and neuro- behavioral symptoms. Upregulation of GLUT1 expression is observed in a wide variety of tumors and is likely to be an essential process for tumor progression. 16  GLUT2 GLUT2 is a high-capacity, bidirectional glucose glucose transporter highly expressed in liver. High expression also occurs in the islet of Langerhans, kidney, and intestine. Mutations in the gene encoding GLUT2 in humans give rise to Fanconi-Bickel Syndrome glucose (FBS), a rare autosomal-recessive inborn error of metabolism, which resembles type I glycogen storage disease 17  GLUT4 GLUT4, prominently expressed in adipo- glucose GLUT4 cytes, skeletal muscle, and cardiomyo- cytes, functions as an insulin-responsive glucose transporter. GLUT4 plays an exocytosis endocytosis important role in the regulation of whole body glucose homeostasis. Inhibition of GLUT4 causes acute peripheral insulin resistance. GLUT4 is also expressed in Insulin receptor— cholinergic neurons (often co-expressed | with GLUT3). The intracellular pool of Insulin plasma membrane ! GLUT4 proteins can be mobilized when insulin binds to the insulin receptor: this triggers an intracellular signaling pathway that causes rapid mobilization or translocation of glucose transporters into the plasma membrane of a fat or muscle cell, thus greatly increasing glucose uptake. 18  GLUT4 glucose GLUT4, prominently expressed in adipocytes, GLUT4 skeletal muscle, and cardiomyocytes, functions as exocytosis endocytosis an insulin-responsive glucose transporter. GLUT4 plays an important role in the regulation of whole body glucose homeostasis. Inhibition of GLUT4 Insulin causes acute peripheral insulin resistance. receptor— GLUT4 is also expressed in cholinergic neurons | Insulin plasma er u in membrane ! (often co-expressed with GLUT3). The intra- snI c etp snI l glucose ro | lu — n i cellular pool of GLUT4 proteins can be mobiliz- GLUT4 ed when insulin binds to the insulin receptor: this ceox exocytosis ty s endocytosis triggers an intracellular signaling pathway that is o causes rapid mobilization or translocation of m e asm a lp marb en glucose transporters into the plasma membrane of X ! Insulin lg c receptor— a fat or muscle cells, thus greatly increasing so u e 4TUL coedn G | glucose uptake. ty s Insulin plasma is o membrane ! 19 GLUCOSE TRANSPORTERS DISTRIBUTION IN DIFFERENT ORGANS BRAIN GLUT1 GLUT3 GLUT4 PANCREAS GLUT1 GLUT2 GUT GLUT2 GLUT5 BLOOD ADIPOSE GLUCOSE TISSUE 4-10 nM GLUT4 LIVER GLUT2 KIDNEY GLUT2 GLUT9 MUSCLE GLUT4 renal excretion < 0.5 g/day 20  III. OVERVIEW OF GLUCOSE METABOLISM Glycogen AAnabolism NABOLISM Glycogenolysis Glycogenesis Pentose Phosphate GLUCOSE Pathway Ribose-5-P Pathway CatCaCatabolism ATABOLISM Glycolysis Gluconeogenesis bolism Pyruvate 21  III. OVERVIEW OF GLUCOSE METABOLISM Glycogen AAnabolism NABOLISM Glycogenolysis Glycogenesis Pentose Phosphate GLUCOSE Pathway Ribose-5-P Pathway CatCaCatabolism ATABOLISM Glycolysis Gluconeogenesis bolism Pyruvate 22  IV. GLYCOLYSIS Glycolysis is a sequence of reactions glucose that converts the six-carbon glucose (C6H12O6) into the three-carbon mono- GLYCOLYSIS Anaerobic process saccharide pyruvate (C3H4O3) with for- Modest generation of ATP mation of modest amounts of ATP. glucose The enzymic steps of glycolysis occur in GLYCOLYSIS the cytosol in anaerobiosis (absence of Anaerobic process pyruvate lactate glucoseModest generation of ATP CYTOSOL oxygen). Pyruvate formed can have two fates: TRICARBOXILIC ACID CYCLE MITOCHONDRIA further reduced in cytosol to lactate GLYCOLYSIS Anaerobic process pyruvate RESPIRATORY CHAIN (anaerobic process). This pathway Modest generation of ATP pyruvate lactate CYTOSOL occurs under intense physical Aerobic process exercise OXILIC ACID CYCLE Large generation of ATP MITOCHONDRIA further metabolized in mitochondria acetyl-CoA pyruvate pyruvate lactate CYCLE CHAIN to acetyl-CoA (aerobic process) and CYTOSOL generating large amounts of ATP Aerobic process IRATORY CO2 + Energy Large generation of ATP MITOCHONDRI pyruvate acetyl-CoA AIN 23  glucose ATP Consumption IV. GLYCOLYSIS modest amount of energy created The conversion of glucose into Stage I ATP pyruvate (glycolysis) entails two stages: glyceraldehyde- STAGE I: conversion of glucose 3-phosphate into glyceraldehyde-3-phosphate. ATP is consumed in this stage. STAGE II: conversion of glyceral- ATP Generation ATP dehyde-3-phosphate into pyruvate Stage II or lactate with conservation of energy as ATP. pyruvate lactate 24  IV. GLYCOLYSIS STAGE I of glycolysis is the conversion of glucose glucose-6P (6 C) to two 3-C units: dihydroxy- ATP hexokinase acetone-P and glyceraldehyde-3P. ADP In STAGE I, ATP is consumed in two reactions: glucose-6P The phosphorylation of glucose to glucose-6P by hexokinase (HK), the first regulatory point fructose-6P in glycolysis ( ). phosphofructo-kinase ATP The phosphorylation of fructose-6P to fruc- ADP fructose-1,6-bisP tose-1,6–bisP by phosphofructokinase (PFK), the second, most important regulatory point in glycolysis ( ). Dihydroxyacetone-P is converted to glyceral- dihydroxy- acetone-P glyceraldehyde-3P dehyde-3-P by an isomerase. Hence, the products of STAGE I of glycolysis are two triose P isomerase molecules of glyceraldehyde-3P. 25  IV. GLYCOLYSIS glyceraldehyde-3P dehydrogenase Two molecules of glyceraldehyde-3P enter STAGE II of glycolysis where they are converted into pyruvate by a series of oxido-reductases and one kinase. The latter, pyruvate kinase (PK), constitutes the regulatory point in STAGE II of glycolysis ( ). This reaction generates ATP as well as another upstream kinase (phospho- pyruvate kinase glycerate kinase). 26  IV. GLYCOLYSIS The allosteric and hormonal regulation of the three key steps of glycolysis is as follows: First control step: hexokinase hexokinase Allosteric regulator: (–) glucose-6-P Second control step: phosphofructokinase phosphofructo- Allosteric regulators: (–) ATP, citrate, H+ kinase (+) AMP, fructose-2,6-P pyruvate kinase Third control step: pyruvate kinase Allosteric regulators: (–) ATP, alanine 27  ATP consumption IV. GLYCOLYSIS (–) glucose-6P glucose ATP consumption 2Pi + 2ADP + 2 NAD+ (–) ATP, citrate Stage I (+) AMP, fructose-2,6-P 2 ATP + 2 NADH pyruvate ATP formation Stage II (–) ATP, alanine ATP formation 28  IV. GLYCOLYSIS Fates of Pyruvate Pyruvate formed during metabolism of glucose can have two fates: If it remains in cytosol, it can be reduced to lactate by the action of lactic dehydrogenase (LDH) If it enters mitochondria, it can be oxidized to LACTATE the two-carbon compound acetyl-CoA by the action of the pyruvate dehydrogenase complex LDH (PDH) GLYCOLYSIS GLUCOSE PYRUVATE PDH ACETYL-COA 29  IV. GLYCOLYSIS LACTATE FORMATION glucose 2Pi + 2ADP + 2 NAD+ Stage I 2 ATP + 2 NADH pyruvate glucose 2Pi + 2ADP 1,3-P-glycerate 2 ATP Stage II lactate lactate LDH 30  IV. GLYCOLYSIS – CLINICAL CORRELATIONS – LACTIC ACIDOSIS Lactic acid levels in blood are around 1.2 mM. Lactic acidosis, most commonly encountered form of metabolic acidosis, is characterized by blood lactate levels higher than 5 mM as a consequence of overproduction of lactate, underutilization of lactate, or both. Anaerobic glycolysis (all cytosolic processes are anaerobic) generates lactate; however, most tissues do no produce large quantities of lactate because pyruvate enters mitochondria where it is oxidized –in an O2-dependent metabolic pathway– to acetyl-CoA (all mitochondrial processes are aerobic). CYTOSOL CYTOSOL MITOCHONDRIA MITOCHONDRIA (anaerobiosis) (anaerobiosis) (aerobiosis) (aerobiosis) NoOO22requirements No requirement OO2 2required is required for the for oxidative for glycolysis for glycolysis oxidative reactions reactions glycolysis pyruvate dehydrogenase glucose 2 pyruvate 2 acetyl-CoA 2 CO2 2 lactate TCA 31  IV. GLYCOLYSIS – CLINICAL CORRELATIONS – LACTIC ACIDOSIS Lactic acidosis occurs when tissue oxygenation is inadequate; tissues respond with increased lactate generation, thus reverting to anaerobic glycolysis. An example is muscle exercise, which can deplete the tissue of O2 and cause overproduction of lactic acid. Tissue hypoxia occurs in all forms of shock, convulsions, and in circulatory and pulmonary failures. Under all these conditions, lactate cytosol production is increased and utilization mitochondria is decreased. Lactate (aerobiosis) (anaerobiosis) utilization is decreased in liver diseases, O2 requirement for ethanol, and certain of No O2 requirement pyruvate dehydrogenase and [lactate] [pyruvate] drugs.for glycolysis TCA cycle CYTOSOL MITOCHONDRIA (anaerobiosis) (aerobiosis) HYPOXIA pyruvate pyruvate glycolysis pyruvate dehydrogenase glycolysis glycolysis dehydrogenase glucose Glucose 2 pyruvate dehydrogenase22Acetyl 2 Pyruvate acetyl-CoA CoA glucose 2 pyruvate 2 acetyl-CoA 2 CO2 22CO CO22 Muscle exercise 2 lactate TCA 2 lactate TCA ¯[pyruvate] Shock 2 lactate TCA ­[lactate] Liver Diseases Ethanol Intoxication Circulatory failure HYPOXIA HYPOXIA Pulmonary failure 32  IV. GLYCOLYSIS – CLINICAL CORRELATIONS – Pyruvate kinase deficiency and hemolytic anemia Mature erythrocytes are dependent on the glycolytic (anaerobic) pathway for ATP production, which is required to maintain ion pumps, such as the Na,K–ATPase. Without ATP, erythrocytes swell and lyse (excessive erythrocyte lysis is known as hemolytic anemia). Pyruvate kinase deficiency is the most common genetic defect of glycolysis that is known to lead to anemia (because cells cannot be replaced rapidly enough by erythropoiesis). 33  III. OVERVIEW OF GLUCOSE METABOLISM Glycogen AAnabolism NABOLISM Glycogenolysis Glycogenesis Pentose Phosphate GLUCOSE Pathway Ribose-5-P Pathway CatCaCatabolism ATABOLISM Glycolysis Gluconeogenesis bolism Pyruvate 34  V. GLUCONEOGENESIS Gluconeogenesis is the sequence of reactions involved in the conversion of pyruvate to glucose. The major site of gluconeogenesis is in liver and secondarily in the cortex of the kidney. These two gluconeogenic tissues help maintain the glucose blood level sufficiently high, so that brain, skeletal muscle, and heart muscle (which have very low rates of gluconeogenesis or no gluconeogenesis at all) can incorporate glucose for their metabolic demands. GLUCOSE BRAIN GLUCONEOGENESIS LIVER GLYCOLYSIS HEART GLUCOSE KIDNEY SKELETAL MUSCLE GLUCONEOGENIC GLYCOLYTIC PYRUVATE TISSUES TISSUES 35  V. GLUCONEOGENESIS Glucose-6-Phosphatase Some of the reactions of glycolysis are freely reversible and operative in the pyruvate → glucose conversion. The three Phospho Fructo Phosphatase regulatory reactions in glycolysis catalyzed by kinases are irrevers- ible: 1 Hexokinase 2 Phosphofructokinase 3 Pyruvate kinase These reactions are bypassed in gluconeogenesis by 3 Pyruvate carboxylase P-enolpyruvate-carboxykinase 2 Phospho-fructo phosphatase Pyruvate carboxylase 1 Glucose 6-phosphatase P-enol-pyruvate-carboxykinase 36  V. GLUCONEOGENESIS Conversion of pyruvate into phospho-enol-pyruvate a In mitochondria: Pyruvate carboxylase (PC): stimulated by acetyl-CoA b In cytosol: Phosphoenol-pyruvate carboxykinase (PEP-CK): inhibited by ADP (–) ADP P-ENOL PYRUVATE malate CARBOXYKINASE dehydrogenase P-enol oxaloacetate malate pyruvate NAD+ GTP GDP NADH + CO2 (+) acetyl-CoA PYRUVATE malate CARBOXYLASE dehydrogenase pyruvate oxaloacetate malate Biotin NADH ATP + ADP NAD+ CO2 + Pi 37  V. GLUCONEOGENESIS Conversion of fructose-1,6-bisP to fructose-6-P In cytosol: Phosphofructo-phosphatase (PFPase) (–) AMP, fructose-2,6-bisP (+) citrate Phosphofructo- phosphofructo- phosphatase phosphatase fructose-1,6-bisP fructose-6-P (+) citrate (–) H2OAMP, fructose-2,6-bisP Pi Conversion of glucose-6P to glucose In cytosol: Glucose-6-phosphatase glucose-6- phosphatase glucose-6P glucose H2O Pi 38  4 V. GLUCONEOGENESIS Step 1 catalyzed by pyruvate carboxylase in mitochondria, (+) regulated by acetyl- 3 (+) citrate (–) AMP, fructose-2,6-P CoA Step 2 catalyzed by phosphoenol-pyruvate carboxykinase (PEP-CK) in cytosol, (–) regulated by ADP Step 3 catalyzed by phosphofructophosphat- (–) ADP ase in cytosol, (+) regulated by citrate; (–) regulated by AMP, fruct- 2 ose-2,6-bisphoshate Step 4 catalyzed by glucose-6-phosphatase in cytosol (+) acetyl-CoA 1 39  V. GLUCONEOGENESIS Fructose-2,6-bisP co-regulates both glycolysis and gluconeogenesis Fructose-2,6-bisphosphate is not a substrate fructose-6-P GLUCONEOGENESIS but an allosteric modulator of both Pi fructose-1,6-bisphosphatase glycolysis and gluconeogenesis. This is a H2O concerted activity resulting in inhibition of fructose-1,6- gluconeogenesis (by inhibition of phospho- bisphosphate fructose-2,6- bisphosphate fructo-phosphatase) and stimulation of fructose-1,6- glycolysis (by activation of phosphofructo- bisphosphate GLYCOLYSIS kinase). ADP phosphofructokinase ATP fructose-6-P 40  V. GLUCONEOGENESIS Tissue-specific fate of glucose-6-P derived from gluconeogenesis Liver is the major gluconeogenic tissue serving the demands for glucose of peripheral tissues. This is because liver contains glucose-6-phosphatase and, hence, can send glucose to blood and thereby regulate glycemia. In contrast, skeletal muscle is a glycolytic tissue, does not contain glucose-6-phosphatase, and is not involved in regulation of glycemia. Upon metabolism of glucose, skeletal muscle is involved in the export of lactic acid to blood. PYRUVATE GLUCOSE GLUCOSE GLUCOSE PYRUVATE LACTATE LACTATE 41  CORTISOL INCREASES GLUCONEOGENESIS UPON BINDING TO THE GLUCOCORTICOID-RESPONSIVE GENE cortisol Cortisol, the primary stress hormone, is cholesterol synthesized in the adrenal cortex. Synthesis cortisol mitochondria ❶ of cortisol occurs in mitochondria from ❺ ❺ pregnenolone 11-deoxy- cortisol cholesterol, which is converted to pregnen- olone ❶. In a second step, pregnenolone is pregnenolone ❷ converted to progesterone ❷ in the ER and progesterone to 17a-hydroxy-progesterone ❸and 11-de- 11-deoxy- ❹ 17a-hydroxy- ❸ oxycortisol ❹. 11-deoxycortisol in mito- cortisol progesterone chondria is meta-bolized to cortisol ❺and endoplasmic reticulum released from the mitochondrion. 38  CORTISOL INCREASES GLUCONEOGENESIS UPON BINDING TO THE GLUCOCORTICOID-RESPONSIVE GENE P-EnolPyruvate Carboxykinase (PEPCK) is the second control point and rate-limiting step in gluconeogenesis (synthesis of glucose). It catalyzes the conversion of oxaloacetate to P-enol pyruvate in the cytosol. When cortisol translocates to the nucleus it binds to the GRG (GlucocorticoidResponsiveGene) leading to the transcriptional activa- tion of PEPCK, thereby increasing hepatic glucose production CORTISOL PEPCK GLUCONEOGENESIS GRG 39  V. GLUCONEOGENESIS The Cori Cycle The CORI CYCLE describes a situation in which lactate formed by the contracting skeletal muscle under conditions of intense physical exercise is converted to glucose in the liver; the liver furnishes glucose back to the contracting skeletal muscle, which obtain ATP from the glycolytic breakdown of the six-carbon sugar. glucose liver glucose glucose Gluconeogenesis Glycolysis pyruvate pyruvate lactate lactate muscle lactate 44  V. GLUCONEOGENESIS – CLINICAL CORRELATIONS – HYPOGLYCEMIA AND PREMATURE INFANTS Newborn infants are almost completely dependent on glucose obtained from liver gluconeogenesis because other metabolic pathways which provide energy to the brain are not well developed. The hepatic gluconeogenic capacity is limited in newborn infants because the enzyme phosphoenolpyruvatecarboxykinase (PEP-CK) is present in low amounts during the first hours after birth; induction of the enzyme to the level required to prevent hypoglycemia during the stress of fasting requires several hours. ¯glucose LIVER BRAIN ¯glucose ¯glucose P-enol pyruvate acetyl-CoA ¯PEP-CK CO2, ATP pyruvate lactate alanine 45  V. GLUCONEOGENESIS – CLINICAL CORRELATIONS – HYPOGLYCEMIA AND ALCOHOL INTOXICATION Ethanol inhibits gluconeogenesis in liver upon its oxidation to acetaldehyde with production of NADH; the large load of reducing equivalents (NADH) forces the equilibrium of the lactate dehydrogenase-catalyzed reactions in the direction of lactate, thus resulting in inhibition of gluconeo- ethanol acetaldehyde genesis by limiting amounts of pyruvate available for the reaction catalyzed by PEP NAD+ NADH carboxykinase. The considerable lactate accumulation in lactate pyruvate blood leads to lactic acidosis. Children are highly dependent on gluconeogenesis while fasting, and accidental ingestion of alcohol glucose by a child can produce severe hypoglycemia. 46  III. OVERVIEW OF GLUCOSE METABOLISM Glycogen AAnabolism NABOLISM Glycogenolysis Glycogenesis Pentose Phosphate GLUCOSE Pathway Ribose-5-P Pathway CatCaCatabolism ATABOLISM Glycolysis Gluconeogenesis bolism Pyruvate 47  VI. PENTOSE PHOSPHATE PATHWAY The pentose phosphate pathway is not a main pathway for obtaining energy from the oxidation of glucose: instead it is a multifunctional pathway specialized to carry out three main activities (depending on the organism and its metabolic rate): 1 Generation of reducing power in the form of NADPH in the cytosol. This activity is prominent in tissues such as liver, mammary gland, adipose tissue, and adrenal cortex that actively carry out the reductive syntheses of fatty acids and steroids, which require NADPH. Conversely, the pentose phosphate pathway activity is very low in skeletal muscle. 2 Generation of D-ribose-5-phosphate for nucleic acid synthesis 3 Oxidative degradation of pentoses to hexoses, which enter the glycolytic sequence 48  VI. PENTOSE PHOSPHATE PATHWAY The biochemical reactions of the pentose phosphate pathway are divided into two sequential phases that occur in cytosol: Phase I or Oxidative Phase includes oxidation-reduction reactions, the main purpose of which is to furnish NADPH for synthesis of fatty acids and cholesterol. The key enzymes in this stage are dehydrogenases. Phase II or Non-Oxidative Phase includes changes in the carbon skeleton of pentoses and serves as a link to the glycolytic pathway. The key enzymes in this stage are transketolases and transaldolases. STAGE MAIN ENZYMES PURPOSE STAGE I Glucose-6P Generation of Dehydrogenases reducing power as NADPH Ribulose-5P STAGE II Ribulose-5P Generation of Transketolases pentoses and Transaldolases branching to glycolysis C3, C4, C6, C7 49  VI. PENTOSE PHOSPHATE PATHWAY Phase I or Oxidative Phase: Generation of NADPH This stage involves 3 reactions, 2 of which reduce NADP+ to NADPH. The first reaction is the conversion of glucose-6-P to 6-P-gluconolactone catalyzed by glucose-6-P dehydrogenase with production of NADPH. This reaction is the control point in the pentose phosphate pathway. Glucose-6-P 6C 6Cglucose-6-P 6C 6C glucose-6-P glucose-6P glucose-6-P dehydrogenase is activated by NADP+. 6C glucose-6-P NADP+ glucose-6-P NADP+ glucose-6-P glucose-6-P dehydrogenase NADP+ NADP + The second reaction in Phase I converts dehydrogenase glucose-6-P NA dehydrogenase NADPH dehydrogenase NADPH 6-P-gluconolactone to 6-P-gluconate, 6-P-Gluconolactone N 6-P-Gluconolactone 6-P-Gluconolactone 6-P-Gluconolactone Phase I which is decarboxylated by 6P-gluconate laconase Phase I Phase I laconase laconase Phase I dehydrogenase to ribulose-5-P. Phase I laconase 6P-Gluconate generates two NADPH molecules during 6P-Gluconate6P-Gluconate NADP+ 6-P-gluconate NADP6P-Gluconate + 6-P-gluconate dehydrogenase 6-P-gluconate NADP+ + the course of two dehydrogenase- dehydrogenase 6-P-gluconate NADPNA dehydrogenase NADPH + CO NADPH dehydrogenase 2 catalyzed reactions. 5C ribulose-5-P N 5C ribulose-5P ribulose-5-P ribulose-5-P 5C 5C ribulose-5-P 5C 50  VI. PENTOSE PHOSPHATE PATHWAY Phase II or Non-Oxidative Phase: Generation of Pentoses and Branching to Glycolysis This non-oxidative phase generates a series of pentoses, tetroses, and hexoses; pentoses are used for the synthesis of RNA and DNA and hexoses (fructose-6-P) branch into glycolysis. The key enzymes involved in this phase are transketolases and trans- aldolases: Transketolases catalyze the transfer of two-carbon units and they require thiamine pyrophosphate (TPP) –a derivative of vitamin B1– to transfer activated aldehydes. Transaldolases transfer three-carbon units. Fructose-6-P (6C) and glyceraldehydes-3-P (3C) are generated during this phase and they branch into glycolysis. 51  VI. PENTOSE PHOSPHATE PATHWAY glucose-6-phosphate regulatory GLUCOSE-6-P NADPH point DEHYDROGENASE 6-phosphoglucono- lactone STAGE I REDUCTIVE LACONASE BIOSYNTHETIC PATHWAYS 6-phosphogluconate 6-PHOSPHOGLUCONATE NADPH + CO2 DEHYDROGENASE ribulose-5-phosphate ISOMERASE EPIMERASE NUCLEIC ribose-5-phosphate xylulose-5-phosphate ACID SYNTHESIS TRANSKETOLASE TPP (B1) glyceraldehyde- sedoheptulose GLYCOLYSIS 3-phosphate 7-phosphate STAGE II TRANSALDOLASE erythrose-4 fructose-6- -phosphate phosphate TRANSKETOLASE TPP (B1) Remember this part glyceraldehyde fructose-6- GLYCOLYSIS 3-phosphate phosphate GLYCOLYSIS 52  VI. PENTOSE PHOSPHATE PATHWAY – CLINICAL CORRELATIONS – Wernicke-Korsakoff syndrome The levels of thiamine or vitamin B1 in the diet, along with some genetic factors affecting the binding of thiamine to transketolase, are important determinants in a neuropsychiatric disorder, the Wernicke-Korsakoff syndrome, characterized by paralysis of eye movements, impairment of memory, and markedly deranged mental function. A diet low or deficient in vitamin B1 is not the only determinant of the Wernicke-Korsakoff syndrome; its development seems to be also related to a low affinity of transketolase for its prostetic group, vitamin B1 or thiamine. TRANS- ENVIRONMENTAL GENETIC KETOLASE TPP B1 FACTORS FACTORS DIET DEFICIENT WEAK BINDING OF OR LOW IN TRANSKETOLASE TO THIAMINE (VITAMIN B1) THIAMINE (VITAMIN B1) ? TRANS- …… WERNICKE-KORSAKOFF SYNDROME KETOLASE …… TPP 53  VI. PENTOSE PHOSPHATE PATHWAY – CLINICAL CORRELATIONS – Drug Hemolytic Anemia The pentose phosphate pathway in the erythrocyte is the main source of NADPH for the reduction of glutathione disulfide (GSSG) to glutathione (GSH). GSH is a tripeptide consisting of glycine (gly) + cysteine (cys) + g- glutamic acid (g-glu): g-glu—cys—gly g-glu—cys—gly | | SH S | disulfide bond S | g-glu—cys—gly GSH GSSG Glutathione (GSH) occurs in cells in the mM range, whereas glutathione disulfide (GSSG) occurs in the µM range. Usually, the ratio GSH/GSSG is about 100-1000. 54  VI. PENTOSE PHOSPHATE PATHWAY – CLINICAL CORRELATIONS – Drug Hemolytic Anemia The reduction of GSSG to GSH is catalyzed by glutathione reductase; the reduction occurs at expense of NADPH GSSG + NADPH 2GSH + NADP+ glutathione reductase In turn, glutathione (GSH) is used to remove hydrogen peroxide (H2O2) from the erythrocyte in a reaction catalyzed by glutathione peroxidase. This enzyme is dependent on selenium –as a prosthetic group– for its activity. 2GSH + H2O2 GSSG + H2O glutathione peroxidase This reaction is important, for accumulation of H2O2, an oxidant, may decrease the life span of the erythrocyte by increasing the rate of oxidation of hemoglobin to met-hemoglobin. 55  VI. PENTOSE PHOSPHATE PATHWAY – CLINICAL CORRELATIONS – Drug Hemolytic Anemia Genetic deficiency of glucose-6-P dehydrogenase (Glu6P DH) is relatively benign until certain drugs, which undergo metabolic modifications with formation of peroxides, are administered. Deficiency of glucose-6P-dehydrogenase (drug hemolytic anemia) impairs the ability of red blood cells to generate NADPH and this is manifested as red cell hemolysis when the susceptible individual is subjected to oxidants, such as the antimalarial primaquine, aspirin, or sulfonamides. pentose phosphate pathway 6Pglucono NADPH lactone GSSG H2O drug hemolytic Glu6P GR GPx anemia DH Primaquine glucose-6P NADP+ 2 GSH H2O2 Aspirin Sulfonamides 56  VI. PENTOSE PHOSPHATE PATHWAY – CLINICAL CORRELATIONS – DRUG HEMOLYTIC ANEMIA However, the genetic deficiency in glucose-6P-dehydrogenase becomes a protective device against plasmodial parasites that attack erythrocytes: for example, those causing malaria. These parasites required GSH (the reduced form); because patients afflicted with a deficiency of glucose-6P-dehydrogenase have less GSH, the parasite has less chance to replicate and continue infection. Hence, this mutation offers some type of protection against malaria. The antimalarial agent (primaquine) increases the level of toxic peroxides and causes hemolysis and anemia. Certain foods (for example, fava beans) may also produce the same effects. If unchecked the symptoms may become severe and lead to death. pentose phosphate pathway 6Pglucono NADPH lactone GSSG H2O drug hemolytic Glu6P GR GPx anemia DH Primaquine glucose-6P NADP+ 2 GSH H2O2 Aspirin Sulfonamides 57  GLYCOLYSIS AND PENTOSE PHOSPHATE PATHWAY – CLINICAL CORRELATIONS – HEMOLYTIC ANEMIA entails a deficiency of pyruvate kinase (a key enzyme in the glycolytic pathway) whereas DRUG HEMOLYTIC ANEMIA entails a deficiency of glucose-6P-dehydrogenase (the only control point in the pentose phosphate pathway). Hemolytic Anemia Drug Hemolytic Anemia 58  PERNICIOUS ANEMIA VITAMIN B12 DEFICIENCY AND PERNICIOUS ANEMIA Vitamin B12 deficiency is common in the United states and pernicious anemia is one of the most common causes of vitamin B12 deficiency due to atrophic gastritis. The normal process requires an intrinsic factor secreted in the stomach, which binds vitamin B12. The complex is recognized by a specific receptor in the ileum and a releasing factor facilitates the splitting of the complex and absorption of vitamin B12. Deficiency in the intrinsic factor leads to impaired absorption of vitamin B12 as observed during pernicious anemia. STOMACH ILEUM intrisic factor B12a B12a B12a +++ (Co+++)) (CO Cobalamine Cobalamine or or B12a Vitamin Vitamin BB12 specific releasing 12 receptor factor 59 Significance for Pernicious Anemia and Acidosis Vitamin deficiency and genetic errors – In humans there are two major symptoms of vitamin B12 deficiency: hematopoietic and neurological. The basis for these deficits can be twofold: Formation of coenzyme B12 requires reaction of B12s – Co+ with ATP, which leads to coenzyme B12 formation in a reaction catalyzed by transferase. When the transferase is absent, as in some inherited disorders, methylmalonyl CoA metabolism is impaired. This condition becomes evident in the first year of life, when the main symptom is aciduria (methylmalonic aciduria). Although this form of methylmalonic aciduria could be lethal, some improvements can be observed upon treatment with vitamin B12 or cobalamin. Pernicious Anemia and Acidosis Significance for Pernicious Anemia and Acidosis The neurological disorders associated with vitamin B12 deficiency may be related to the functionality of methylmalonyl-CoA mutase and the accumulation of methylmalonyl: the progressive demyelination of nervous tissue observed in vitamin B12 deficiency was suggested to be a consequence of the accumulated methylmalonyl-CoA that interferes with myelin sheath formation by means of inhibition of fatty acid biosynthesis and/or stimulation of branched-chain fatty acids (which disrupt the membrane structure). Hence the biochemical defects accounting for deficiency syndromes can be due to: Deficiency of intrinsic factor Deficiency of transferase Interference with demyelination Diagnosis is made by the levels of methylmalonic acid and homocysteine levels. Conversion of methyl- pernicious anemia malonyl-CoA to succinyl-CoA methylmalonic aciduria Vitamin B12 Coenzyme B12 Methylation of homocysteine peripheral neuropathy to methionine  Hemolytic Anemia Drug Hemolytic Anemia intrisic factor Pernicious Anemia B12a B12a B12a (CO +++ (Co+++)) Cobalamine Cobalamine or or B12a Vitamin Vitamin B B12 specific releasing 12 receptor factor 63  VII. DISACCHARIDES There are three common disaccharides: glycogen Sucrose, or common table sugar, is widely found in plants and is composed of glucose galactose glucose-1P + fructose. LACTOSE glucose glucose-6P Lactose, is present in milk (approx. 5% concentration) and is composed of MALTOSE galactose + glucose. SUCROSE fructose fructose-6P Maltose is obtained from the breakdown if starch and is composed of glucose + fructose-1P dihydroxy- acetone-P glucose. The breakdown products of these disacchar- glyceraldehyde-3-P ides can be channeled into glycolysis by different pathways: 64  VII. DISACCHARIDES Metabolism of Galactose Galactose, obtained from lactose hydrolysis, is converted to glucose-1P and enters the glycolytic pathway as glucose-6-P. Conversion of galactose to glucose requires the action of galactokinase followed by the reaction catalyzed by galactose-1P-uridyl transferase. UPD-galactose is recovered to UDP-glucose by the action of an epimerase. The new UDP-glucose is used again to transform galacose-1P lactoseinto glucose-1P. lactose gut UDP-galactose lactase galactose 1P-uridyl galactokinase transferase galactose galactose-1P UDP-glucose glucose-1P UDP-galactose epimerase 65  VII. DISACCHARIDES – CLINICAL CORRELATIONS – GALACTOSEMIA Galactosemia is a genetic disease due to the absence of galactose-1P-uridyl transferase; this blocks the conversion of galactose-1P to glucose-1P, thus resulting in an accumulation of galactose in blood. Heterozygotes usually express enough enzyme to accommodate ordinary dietary intake of galactose. In homozygous, the buildup of un- metabolized galactose causes liver damage, mental retardation, and cataracts. Treatment of galactosemic individuals is relatively simple: exclude galactose (dairy products) from the diet. lactose 66