83.Leptin & Growth Hormone Secretion

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83.Leptin & Growth Hormone Secretion

 

 

 

CATEGORY: Anabolic Steroids 100 Courses

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ACKNOWLEDGEMENTS iii

LIST OF FIGURES ix

LIST OF TABLES xi

1 LITERATURE REVIEW 1

Obesity ………………………………………………………………………………………………………… 1

Obesity Models …………………………………………………………………………………………….. 3

Theories of Food Intake …………………………………………………………………………………. 6

Leptin ………………………………………………………………………………………………………….. 9

Roles of Leptin……………………………………………………………………………………………. 12

Regulation of Leptin……………………………………………………………………………………. 15

Leptin and Hormonal Interactions …………………………………………………………………. 16

Leptin Receptors …………………………………………………………………………………………. 19

Leptin Signaling………………………………………………………………………………………….. 23

Leptin Resistance ………………………………………………………………………………………… 25

Human Leptin Mutations ……………………………………………………………………………… 28

Leptin Treatment in Humans and Leptin Gene Therapy …………………………………… 29

Growth Hormone ………………………………………………………………………………………… 30

Excess of Deficiency of Growth Hormone ……………………………………………………… 35

Growth Hormone and Obesity………………………………………………………………………. 36

Leptin and Growth Hormone ………………………………………………………………………… 39

Objectives…………………………………………………………………………………………………… 41

2 GENERAL METHODS 43

Animals ……………………………………………………………………………………………………… 43

Diets ………………………………………………………………………………………………………….. 43

Feeding, Pair-feeding, and Body Weight Measurements ………………………………….. 43

Leptin Challenge Test………………………………………………………………………………….. 44

Alzet Osmotic Minipumps ……………………………………………………………………………. 45

Right Atrial Cannulation………………………………………………………………………………. 45

Anesthesia………………………………………………………………………………………………. 45

Preparation……………………………………………………………………………………………… 45

Surgery…………………………………………………………………………………………………… 46

Recovery ………………………………………………………………………………………………… 46

Blood sampling cages ………………………………………………………………………………….. 46

Blood Sampling and Tissue Collection…………………………………………………………… 47

Blood Collection from Cannulae ……………………………………………………………….. 47

Blood Collection from Tail Vein ……………………………………………………………….. 47

Blood Collection via Cardiac Puncture……………………………………………………….. 47

Trunk Blood Collection ……………………………………………………………………………. 48

Collection of Hypothalamus ……………………………………………………………………… 48

Pituitary and Organ Weights……………………………………………………………………… 48

Cell Culture………………………………………………………………………………………………… 48

GH1 Cells……………………………………………………………………………………………….. 48

Plating Cells ……………………………………………………………………………………………. 49

Five-Day Time-Course …………………………………………………………………………….. 49

Media Experiment……………………………………………………………………………………. 49

Collecting RNA from GH1 Cells……………………………………………………………….. 50

Radioimmunoassays…………………………………………………………………………………….. 50

GH Iodination …………………………………………………………………………………………. 50

Growth Hormone RIA ……………………………………………………………………………… 51

IGF Iodination…………………………………………………………………………………………. 51

IGF RIA …………………………………………………………………………………………………. 52

Leptin RIA ……………………………………………………………………………………………… 52

Insulin RIA……………………………………………………………………………………………… 53

Glucose and Triglyceride Assays…………………………………………………………………… 54

DNA Assay ………………………………………………………………………………………………… 54

RNA extraction, RT-PCR, Southern Blot……………………………………………………….. 55

Protein Extraction and Western Immunoblotting …………………………………………….. 57

Protein Extraction ……………………………………………………………………………………. 57

Micro BCA Protein Assay………………………………………………………………………… 58

Western Immunoblot Analysis for Leptin Receptor ……………………………………… 59

3 CIRCULATING GROWTH HORMONE LEVELS ARE ELEVATED IN

RATS FED A HIGH-FAT DIET 62

Introduction………………………………………………………………………………………………… 62

Methods……………………………………………………………………………………………………… 63

Animals ………………………………………………………………………………………………….. 63

Cannulation Surgery, Blood Sampling, and Tissue Collection ………………………. 64

Radioimmunoassays…………………………………………………………………………………. 65

RT-PCR for Leptin Receptor …………………………………………………………………….. 65

Statistics …………………………………………………………………………………………………….. 66

Results ……………………………………………………………………………………………………….. 66

Body Weight …………………………………………………………………………………………… 66

Food Intake …………………………………………………………………………………………….. 66

Leptin …………………………………………………………………………………………………….. 67

Leptin Receptor mRNA ……………………………………………………………………………. 67

IGF ………………………………………………………………………………………………………… 67

Growth Hormone Profile ………………………………………………………………………….. 67

Discussion………………………………………………………………………………………………….. 72

4 LEPTIN TREATMENT INCREASES GROWTH HORMONE SECRETION

IN CULTURED GH1 CELLS 77

Introduction………………………………………………………………………………………………… 77

Methods……………………………………………………………………………………………………… 79

GH1 Cells……………………………………………………………………………………………….. 79

Plating Cells and Leptin Concentration………………………………………………………. 79

Five-Day Time-Course …………………………………………………………………………….. 79

Media Experiment……………………………………………………………………………………. 80

RT-PCR for Leptin Receptor …………………………………………………………………….. 80

Western Immunoblot for Leptin Receptor …………………………………………………… 80

GH Radioimmunoassay……………………………………………………………………………. 81

Statistics …………………………………………………………………………………………………. 81

Results ……………………………………………………………………………………………………….. 81

Ob-Rb mRNA and Protein Expression ………………………………………………………. 81

Five-Day Time-Course …………………………………………………………………………….. 82

Media Experiment……………………………………………………………………………………. 82

Discussion………………………………………………………………………………………………….. 85

5 LEPTIN RESISTANCE IS ASSOCIATED WITH HYPOTHALAMIC

RECEPTOR mRNA AND PROTEIN DOWNREGULATION 88

Introduction………………………………………………………………………………………………… 88

Methods……………………………………………………………………………………………………… 89

Animals ………………………………………………………………………………………………….. 89

Groups……………………………………………………………………………………………………. 90

Serum Measurements ……………………………………………………………………………….. 90

Leptin Challenge Test Pilot Study……………………………………………………………… 91

Leptin Challenge Test………………………………………………………………………………. 91

RT-PCR for Leptin Receptor …………………………………………………………………….. 91

Western Immunoblot for Leptin Receptor …………………………………………………… 92

Statistics …………………………………………………………………………………………………….. 92

Results ……………………………………………………………………………………………………….. 93

Leptin …………………………………………………………………………………………………….. 93

Body Weight …………………………………………………………………………………………… 93

Food Intake …………………………………………………………………………………………….. 93

Leptin Challenge Test Pilot Studies……………………………………………………………. 94

Leptin Challenge Test………………………………………………………………………………. 94

Leptin Receptor mRNA in Hypothalamus …………………………………………………… 94

Leptin Receptor Protein Expression in Hypothalamus ………………………………….. 94

IGF-1 Values…………………………………………………………………………………………… 95

Hormonal and Metabolic Measures ……………………………………………………………. 95

Discussion………………………………………………………………………………………………… 100

6 LEPTIN’S EFFECTS ON FOOD INTAKE AND BODY WEIGHT ARE

DIFFERENTIALLY ATTENUATED IN RATS FED A LOW-FAT OR HIGHFAT

DIET 107

Introduction………………………………………………………………………………………………. 107

Methods……………………………………………………………………………………………………. 108

Animals and Diets ………………………………………………………………………………….. 108

Osmotic Pumps, Blood Sampling, and Body Temperature ………………………….. 109

Leptin and IGF-1 Radioimmunoassays……………………………………………………… 109

Statistics …………………………………………………………………………………………………… 110

Results ……………………………………………………………………………………………………… 110

Effects of Diet on Food Intake …………………………………………………………………. 110

Leptin …………………………………………………………………………………………………… 110

Effects of Diet and Leptin on Food Intake…………………………………………………. 111

Effects of Diet and Leptin on Body Weight ………………………………………………. 111

Effects of Diet and Leptin on IGF-1 Values ………………………………………………. 112

Effects of Diet and Leptin on Organ Weights…………………………………………….. 112

Effects of Diet and Leptin on Body Temperature……………………………………….. 112

Discussion………………………………………………………………………………………………… 119

7 GENERAL DISCUSSION 125

REFERENCES 134

BIOGRAPHICAL SKETCH 166

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