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Glucagon I

ContributorsT.T. Aoki • T.L. Blundell • C. Bordi • J. Brange • W. W. BromerG. F. Cahill, Jr. • A. D. Chemngton • J.-L. Chiasson • T.H. ClausJ.WEnsinck • S. Falkmer • D.W.Foster • L.G. HedingC. Hellerstrom • B. de Hemptinne • J. J. Hoist • K.S. KochE J. Lad • P. J. Lefebvre • H. L. Leffert • J. D. McGarryR.B. Merrifield • A. J. Moody • S. Mojsov • R. NosadiniL.Orci • C. R. Park • A. Perrelet • S. J. Pilkis • M. PingelM. Rodbell • E. Samols • B. Skelbaek-Pedersen • H. SkellyR.J. Smith • W. Stalmans • L.Thim • A.Tiengo • R.H. UngerI. Valverde • S. Van Noorden

Editor

EJ. Lefebvre

UNIVERSITATS-BIBUOTHEK

Springer-VerlagBerlin Heidelberg New York Tokyo 1983

Contents

Chemistry and Physicochemistry of Glucagon

CHAPTER 1

Chemical Characteristics of Glucagon. W. W. BROMER. With 2 Figures

A. Introduction 1B. Isolation and Purification 1C. Properties 2D. Amino Acid Sequence 3E. Covalent Chemical Modification and Biologic Function 5

I. Limitations of the Approach • • • 5II. General Considerations 7

III. The Question of Several Active Sites 7IV. Amino Terminal and Diamino Modifications 12V. Inhibitors of Glucagon 13

VI. Modifications of Glutamyl, Lysyl, Arginyl, and TryptophylResidues 14

VII. Modifications of Tyrosyl Residues 15VIII. Methionyl Residue and Carboxyl Terminal Modifications . . . 16

IX. Summary of Covalent Modifications and Function 18References . 19

CHAPTER 2

The Chemical Synthesis of Glucagon. R. B. MERRIFIELD and S. MOJSOV

With 8 Figures

A. Introduction 23B. Early Synthetic Efforts 24C. The First Total Synthesis 24D. Further Syntheses by Fragment Condensation in Solution 26E. Solid Phase Fragment Synthesis 28F. Stepwise Solid Phase Synthesis 30G. Conclusions 33References 34

XIV Contents

CHAPTER 3

The Conformation of Glucagon. T. L. BLUNDELL. With 10 Figures

A. Introduction 37B. The Crystal Structure 37

I. Crystals 37II. Protomer Conformation 41

III. Trimer Conformation 42C. The Solution Structure 46

I. Monomer 46II. Trimers 47

III. Fibrils 49D. Conformation of Micelle-Bound Glucagon 50E. Conformation and Storage Granules 51F. Conformation and Receptor Binding 54References . 55

Morphology of the A-cell of Islets of Langerhans,Biosynthesis of Glucagon and Related Peptides

CHAPTER 4

Glucagon- and Glicentin-Producing Cells. L. ORCI, C. BORDI, R. H. UNGER,

and A. PERRELET. With 11 Figures

A. Introduction 57B. Pancreas 57

I. Morphological Features 57II. Distribution of A-cells 59

III. Intracellular Distribution of Secretory Polypeptides 66C. Digestive Tract 68D. Pathology of A- and L-cells 68E. Conclusions 71F. Appendix 73

I. Sampling of the Pancreas 73II. Immunofluorescence Technique 73

III. Quantitative Evaluation . 74References 76

CHAPTER 5

Ontogeny and Phylogeny of the Glucagon CellS. FALKMER and S. VAN NOORDEN. With 15 Figures

A. Introduction 81B. Material, Methods, Nomenclature 82C. Prokaryotes, Eukaryote Protozoa, Coelenterates 82

Contents XV

D. Protostomian Invertebrates 86I. Molluscs 86

II. Arthropods 881. Crustaceans 882. Insects 89

E. Deuterostomian Invertebrates 90I. Echinoderms, Hemichordates 90

II. Protochordates (Tunicates, Amphioxus) 91F. Vertebrates 94

I. Agnatha (Cyclostomes; Jawless Fish) 95II. Gnathostomata 97

1. Cartilaginous Fish 972. Bony Fish 1013. Tetrapods 102

G. Discussion and Summarising Conclusions 110References 112

CHAPTER 6

The Biosynthesis of Glucagon. C. HELLERSTROM. With 6 Figures

A. Introduction 121B. General Aspects of the Formation, Intracellular Conversion, and Storage

of Peptide Hormones 121C. Biosynthesis of Glucagon 125

I. Formation of Preproglucagon 125II. Formation and Conversion of Proglucagon 126

III. Structure and Storage of Proglucagon and Glucagon 132D. Concluding Remarks 134References 136

CHAPTER 7

Glucagon, Glicentin, and Related PeptidcsA. J. MOODY and L. THIM. With 4 Figures

A. Introduction 139B. The Immunochemistry of Gut GLIs 140C. The Chemistry of Crude Gut GLIs 141

I. Distribution 141II. Size 141

III. Charge 142D. The Chemistry of Glicentin, GRPP, and Oxyntomodulin 142

I. Isolation of Glicentin 142II. Sequence of Glicentin 143

III. Relationship of Glicentin to Other Peptides 143IV. Structural Analysis of Glicentin 145V. Isolation of GRPP 146

XVI Contents

VI. Chemistry of GRPP 147VII. Relationship of GRPP to Other Pep tides 147

VIII. Chemistry of Oxyntomodulin 149E. The Immunochemistry of Glicentin 149

I. Reaction of Glicentin with Anti-Glucagon Sera 149II. Anti-Glicentin Sera 149

III. Radioimmunoassay for Glicentin 150IV. Distribution of Immunoreactive Glicentin 150

F. Glicentin Cells 151I. Intestinal Gut GLI Cells .151

II. The Glicentin Cell 1521. Animal Tissue 1522. Normal Human Tissue 1523. Pathologic Human Tissue 153

III. Fine Structure of the A- and L-cell Secretory Granules 153G. Glicentin and Glucagon Biosynthesis 154H. Circulating Gut GLIs 157

I. Radioimmunoassay of Gut GLI 157II. Factors Controlling Gut GLI Release 157

III. Circulating Forms of Gut GLI 158IV. Circulating Gut GLI Levels in Adult Humans 159V. Circulating Gut GLI Levels in the Perinatal Period 161

J. Effects of Gut GLIs 161I. Postulated Effects 161

II. Effects of Partially Purified Gut GLIs 162III. Effects of Pure Gut GLIs 163

1. Synthetic Peptides 1632. Oxyntomodulin 1643. Glicentin 164

K. Discussion 165I. Clinical Significance of Circulating Gut GLIs 165

II. Structure-Function Relationships of Gut GLIs 165III. Role of Gut GLIs 167

References 167

Production and Assay of Glucagon

CHAPTER 8Glucagon Preparations. M. PINGEL, B. SKELBAEK-PEDERSEN, and J. BRANGE

With 3 FiguresA. Introduction 175B. Production 175C. Pharmaceutical Preparations 176D. Assays 179

I. Physicochemical Methods 1791. Ultraviolet Absorption 1792. Electrophoretic Methods 179

Contents XVII

3. Chromatographic Methods 1804. High Pressure Liquid Chromatography 180

II. Bioassay Methods 180E. Stability 181F. Timing of Action 184References 186

CHAPTER 9

The Immunogenicity of Glucagon. L. G. HEDING. With 4 Figures

A. Introduction 189B. Immunogenicity of Glucagon and Glucagon Fragments 190

I. Species Used for Immunization 190II. Immunogen 190

1. Glucagon 1902. Glucagon Fragments 1913. Coupling Procedures 1924. Mode of Administration, Dose, Frequency, Adjuvant 194

C. Characterization of the Glucagon Antibodies 195I. Affinity and Capacity 195

II. Specificity 1951. Reactivity with Glicentin, Gut GLIs, and Glucagon Analogs . 1952. Reactivity with Glucagon Fragments 197

D. Purification of Mixtures of Glucagon Antibodies 198E. Summary 199

I. Production of COOH-Terminal Specific Antibodies 199II. Production of NH2-Terminal Specific Antibodies Giving Linear

Dilution Curves with Gut GLI 199References 200

CHAPTER 10

Immunoassays for Glucagon. J. W. ENSINCK. With 3 Figures

A. Introduction 203B. Method of Radioimmunoassay 204

I. Sources of Peptides 204II. Preparation and Purification of Radiolabeled Ligand 205

III. Preparation of Standard 206IV. Production and Characterization of Antisera 206

1. Antigenic Determinants and Coupling Procedures 2062. Immunization Procedure 2083. Characterization of Antisera 209

V. Assay Procedure 209C. Measurement of Glucagon-Related Peptides 211

I. Collection and Processing of Plasma 212II. Contribution of Different Species to Plasma Levels 213

D. Summary 217References 217

XVIII Contents

CHAPTER 11

Heterogeneity of Circulating Glucagon and Glucagon-Like ImmunoreactivityI. VALVERDE. With 10 Figures

A. Introduction 223B. Types of Glucagon Antibodies 223

I. COOH Terminal-Specific . 224II. NH2 Terminal-Specific 224

C. Plasma COOH Terminal-Specific Antibody-Reacting Components . . 224I. Plasma IRG Components in the Normal Adult 226

1. Basal State 2262. After Intravenous Arginine Administration 2273. After Glucose Administration 228

II. Plasma IRG Components in the Neonatal Period 228III. Plasma IRG in Pathologic States 228

1. Glucagonoma 2282. Diabetes 2303. Pancreatectomy 2314. Chronic Hypoglycemia 2335. Renal Failure 2346. Cirrhosis of the Liver 2357. Other Conditions 236

IV. Origin of Plasma IRG Components 236D. Plasma GLI Components 237

I. Basal State and During Glucose Absorption 2381. Normal Humans 2382. Normal Dogs 2383. Nephrectomized Dogs 239

II. Origin of Plasma GLI Components 239E. Concluding Remarks 240References 241

CHAPTER 12

Radioreceptorassays for Glucagon. J. J. HOLST. With 4 Figures

A. Introduction 245B. Glucagon Radioreceptorassay Methodology 245

I. Receptor Preparations 2451. Isolated Cells 2452. Isolated Membranes 246

II. Radioactive Glucagon 247III. Incubation Conditions 248

1. Temperature and Incubation Time 2482. Reagent Volumes 2483. Assay Buffer 2494. Degradation 2495. Plasma Effects 2506. Separation 250

Contents XIX

C. Performance and Applications of the Glucagon Radioreceptorassay . 250I. Requirements for Ligand-Receptor Interaction 250

1. Coupling of Binding to Biologic Activity 2512. Specificity of the Receptor 252

II. Applications 2551. Assay of Gut Peptides 2552. Assay of Pancreatic Peptides 2573. Glucagonomas 258

References 258

Actions of Glucagon

CHAPTER 13

The Actions of Glucagon at Its Receptor: Regulation of Adenylate CyclaseM. RODBELL. With 3 Figures

A. Introduction 263B. Characteristics of Hormone-Sensitive Adenylate Cyclase Systems . . . 264

I. Role of GTP in Hormone Action 264II. Specificity for Guanine Nucleotides 265

III. Effects of Adenosine 265IV. Role of GTPase 266V. Actions of Cholera Toxin 267

VI. Actions of Guanine Nucleotides on Hormone Receptors . . . . 267VII. Effects of GTP on Hormone Binding Versus Action 268

VIII. Multiple Sites of GTP Action 269IX. Characteristics of the N Unit 270X. Independent Complexes of N with R and C 272

XL Characteristics of the Catalytic Unit 273XII. Regulation by Divalent Cations 273

XIII. The Role of Sulfhydryl Groups in Transduction 274XIV. The Role of Membrane Lipids . 275XV. Desensitization of Glucagon Action 276

XVI. Relationship Between Glucagon Binding and Action 277C. The Glucagon Receptor 278D. Target Analysis of the Glucagon-Sensitive Adenylate Cyclase System . 279E. A Model for Glucagon Action : 280F. The Glucagon "Message" 282G. Summary and Conclusions 285References 285

CHAPTER 14

Glucagon and Liver Glycogen Metabolism. W. STALMANS. With 7 Figures

A. Glucagon, Glycogenolysis, and Glucose Output 291B. The Enzymes Involved in Hepatic Glycogen Metabolism 292

I. The General Pathways 292

XX Contents

II. The Regulated Enzymes 2931. Protein Kinases 2932. Protein Phosphatases 294

C. The Enzymic Mechanism of Glucagon Action 294I. Glucagon Causes Glycogenolysis via Phosphorylase a 295

1. Facts 2952. Questions 295

II. Glycogenolysis Mediated by cAMP 2971. Activation of the Glycogenolytic Cascade 2982. An Effect of cAMP on Phosphorylase Phosphatase 303

III. Can Glucagon Act in a cAMP-Independent Way? 303IV. Glucagon also Causes Inactivation of Glycogen Synthase . . . . 305

D. Expression and Modulation of Glucagon Action 306I. Regulation of Hepatic Glycogen Metabolism by Glucagon . . . . 306

II. Modulation of the Response to Glucagon 3081. Effects of Insulin and Glucose 3082. Glucocorticoid Hormones 308

References 309

CHAPTER 15

Glucagon and Gluconeogenesis. T.H. CLAUS, C.R. PARK, and S.J. PILKIS

With 10 Figures

A. Introduction 315B. Sites of Action of Glucagon on Hepatic Gluconeogenesis 317

I. Action on Mitochondrial Sites 3181. Pyruvate Carboxylase 3182. Pyruvate Dehydrogenase 322

II. Action on Extramitochondrial Sites 3231. Phosphoenolpyruvate - Pyruvate Substrate Cycle 3232. Fructose-6-phosphate-Fructose-l,6-bisphosphate Substrate Cycle 333

III. Other Possible Sites of Glucagon Action 342C. Summary and Overview 344References 348

CHAPTER 16

Glucagon and Liver Glucose Output In VivoJ.-L.CHIASSON and A.D. CHERRINGTON. With 8 Figures

A. Introduction 361B. Effects of Glucagon on Hepatic Glucose Production 361

I. Glycogenolysis 361II. Gluconeogenesis 366

C. Glucagon-Insulin Interaction in the Regulation of Hepatic GlucoseProduction 370

I. Glycogenolysis 370II. Gluconeogenesis 373

Contents XXI

D. Role of Glucagon in the Regulation of Glucose Homeostasis 373I. Feasting 374

II. Fasting 376III. Exercise 376IV. Diabetes Mellitus 377

References 379

CHAPTER 17

Glucagon and Ketogenesis. J.D. MCGARRY and D.W. FOSTER

With 5 Figures

A. Introduction 383B. Intrahepatic Factors in the Regulation of Ketogenesis 383C. Emergence of a Ketogenic Role for Glucagon 387

I. Studies in Animals 387II. Studies in Humans 388

D. Interactions of Insulin and Glucagon on Hepatic Metabolism . . . . 389I. The "Fed to Fasted" Transition 390

II. The "Fasted to Fed" Transition 393E. Overview 395References 396

CHAPTER 18

Glucagon and Amino Acid Metabolism. G.F. CAHILL, JR.,

T.T. AOKI, and R.J. SMITH. With 5 Figures

A. Introduction 399B. Effects of Glucagon on Amino Acid Levels 401C. Glucagon and Liver Amino Acid Metabolism 405D. Glucagon and Muscle Amino Acid Metabolism 408E. Physiologic and Pathologic States in Which Glucagon Affects Amino

Acid Metabolism •'. • • 408I. Glucagon Deficiency 408

II. Glucagon Excess 4091. Diabetes Mellitus 4092. Glucagonoma 4103. Trauma and Sepsis 410

F. Summary .411References 411

CHAPTER 19

Glucagon and Adipose Tissue Lipolysis. P. J. LEFEBVRE

With 5 Figures

A. Introduction 419B. Glucagon and Adipose Tissue 419

I. Species Variations in Glucagon-Induced Lipolysis 419II. Mechanisms Involved in the Lipolytic Action of Glucagon . . . 420

XXII Contents

III. Glucagon-Induced Lipolysis and Glucose Metabolism in theAdipocyte . 421

IV. Factors Affecting Glucagon-Induced Adipose Tissue Lipolysis . . 4221. Size of the Adipocytes, Age and Nutritional Status of the

Animals 4222. Innervation of the Adipose Tissue 4233. Hypophysectomy, Adrenalectomy, Hypothyroidism 4244. Insulin 4245. Prostaglandins and Prostaglandin Synthesis Inhibitors . . . . 4256. Other Factors 427

V. Glucagon-Like Peptides and Adipose Tissue Lipolysis 428VI. Brown Adipose Tissue and Nonshivering Thermogenesis . . . . 428

VII. Adipose Tissue Lipoprotein Lipase Activity 429C. Glucagon and Circulating Free Fatty Acids 429

I. Intravenous, Subcutaneous, or Intramuscular Injection of HighDoses of Glucagon in Mammals 4291. The Early Rise in Plasma FFA 4292. The Secondary Drop in Plasma FFA 4303. The Late Increase in Plasma FFA 431

II. Intraarterial, Intravenous, or Intraportal Infusion of Low Dosesof Glucagon in Mammals 431

III. Intravenous Injection or Infusion of Glucagon in Birds 432D. Physiologic Importance of Glucagon-Induced Lipolysis 432

I. Fasting 433II. Muscular Exercise 433

III. Adaptation to Extrauterine Life 434References 435

CHAPTER 20

Glucagon and Lipoprotein Metabolism. A. TIENGO and R. NOSADINI

With 4 Figures

A. Introduction 441B. Glucagon Effects on Liver Lipid Metabolism 443C. Metabolic Effects of Glucagon In Vivo 446References 448

CHAPTER 21

Glucagon and Liver Regeneration. H.L. LEFFERT, K.S. KOCH,

P.J. LAD, B. DE HEMPTINNE, and H. SKELLY. With 9 Figures

A. Nature of Liver Regeneration 453I. Phenomenology 453

1. Key Questions 4532. Background 4533. Kinetic Aspects 454

Contents XXIII

II. The Endocrine Hypothesis 4561. Concerted Control by Peptide Hormones 4562. Interactions with Nutrients 4573. Two Sequential Rate-Limiting Events 457

B. Regulatory Evidence 458

I. Direct 458II. Indirect 459

C. Physiology 462

I. Extrahepatic 4621. Bloodstream 4622. Responsive Tissues 463

II. Hepatic 4631. Hepatocytes 4632. Nonparenchymal Cells 466

III. Specificity 4661. Mimetics 4662. Nonhepatocyte Targets 466

D. Mechanisms of Action 467

I. Potentiation of Signal 2 4671. Kinetic Evidence 4672. Synergisms with Other Peptide Hormones 4683. Nonrequirement of Glucocorticoids 470

II. Molecular 4701. cAMP-Dependent 4702. cAMP-Independent 472

III. Functional Linkages 4721. Na+ Gradient-Dependent Amino Acid Cotransport 4722. Altered Hepatocyte Lipid Metabolism 4733. Deoxyribonucleoside Triphosphate Production . 474

IV. Inhibitory Aspects 4741. Hepatocyte Loci 4742. Desynchronization of Diurnal Rhythms 475

E. Future Goals 475

I. Role of Other Factors 4751. High Molecular Weight Glucagon 4752. Intrahepatic Loci 476

II. Additional Problems 4761. Complexity of Animal Cell Regulatory Processes 4762. Limitations of Current Experimental Models 477

III. Reasons for Optimism 477

F. Summary 477

References 478

XXIV Contents

CHAPTER 22

Glucagon and Insulin Secretion. E. SAMOLS

A. Introduction and Historical Overview 485B. Experimental Stimulation of Insulin Secretion by Glucagon In Vivo

and In Vitro 487I. Effect of Food and Fasting 488

II. Adrenergic Effects 488III. Insulinotropic Effect of Glucagon in Disease 489IV. Insulinotropic Effect of Glucagon in the Child, Neonate, and

Fetus 490V. Mechanism of Insulinotropic Effect of Glucagon 491

C. Consideration of the Evidence for the Endogenous Insulinotropic Effectof Glucagon 493

I. Inherent Difficulties in Testing the Hypothesis 493II. Anatomy of the Islets of Langerhans 494

III. Dynamic and Magnitudinal Changes in Glucagon and InsulinAfter Secretagogue Administration 496

IV. Augmentation of Insulin Secretion by Protein and/or Fat Meals . 497V. Active and Passive Immunization 497

VI. Diabetes Mellitus 498VII. Glucagonoma-Induced Hyperglucagonemia and Hyperinsulinemia 498

VIII. Evidence Against an Insulinotropic Effect of EndogenousGlucagon 499

D. Potential Physiologic and Pathologic Significance of the InsulinotropicEffect of Glucagon 499

E. Effect of Insulin on Glucagon Secretion 500I. Effect of Exogenous Insulin on Glucagon Release In Vivo . . . 501

II. Effect of endogenous Insulin on Glucagon Release In Vivo . . . 503III. Effect of Exogenous Insulin on Glucagon Secretion In Vitro . 503IV. Mechanism of the Glucagonsuppressive Effect of Insulin . . . . 505

F. Evidence For and Against an Intraislet Negative Insulin-GlucagonFeedback 506

G. New Hypotheses and Concepts in Local Intraislet Regulation ofA- and B-cells 508

I. Compartmentalization: Systemic Versus Local Intraislet Effects . 508II. The Principle of Uncertainty in Studies of Local Islet Interactions . 509

III. Possible Paracrine Effects on Growth and Differentiation . . . 509H. Synopsis and Conclusions 509References 511

Subject Index 519