Huntingtin-Associated Protein 1 (Hap1) is Involved in Insulin Secretion From Pancreatic Beta Cells Open Access

Cape, Austin David (2010)

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Huntingtin-Associated Protein 1 (Hap1) is Involved in Insulin Secretion From
Pancreatic Beta Cells
Hap1 was identified as the first binding partner with huntingtin, the Huntington disease
(HD) protein. Studies have revealed Hap1 participates in intracellular trafficking in
neuronal cells, which is adversely affected by the mutant version of huntingtin (mHtt).
Recently, Hap1 was found in pancreatic beta (β) cells and other endocrine cells that
secrete hormones via similar exocytic mechanisms. However, the role of Hap1 in these
endocrine cells is unknown. We generated a mouse model selectively depleted of Hap1 in
pancreatic β cells using the Cre-loxP genetic manipulation system. These Hap1 knockout
(KO) mice displayed impairment of glucose tolerance without affecting insulin
sensitivity. We validated that decreased Hap1, via its shRNA, reduced glucose-stimulated
insulin release in Min6 cells, a well-studied pancreatic β cell line. In addition, ELISA
measurement of insulin levels in blood confirmed Hap1 KO mice are defective for insulin
release in response to glucose treatment. Electron microscopy revealed decreased
numbers of insulin-containing vesicles at the docking sites in β cell membranes of Hap1
KO mice. Glucose-stimulated insulin release also reduced the phosphorylation of
pHap1A in both cell and mouse models. Moreover, this glucose-induced
dephosphorylation caused more Hap1 to associate with kinesin light chain and dynactin
p150, both of which are involved in microtubule-dependent trafficking. These results
suggest Hap1-associated intracellular trafficking is important for vesicular release of
insulin from pancreatic β cells, providing a potential target for treating the impairment of
β cell insulin release seen in HD and other metabolic disorders.

Table of Contents

Table of Contents

Chapter1: General Introduction

1.1 Huntingtin Associated Protein 1 (Hap1) 2

1.2 Hap1 Function In Molecular Trafficking 4

1.3 Neuroendocrine System 9

1.4 The Pancreas 10

1.5 b Cell Dysfunction in HD 14

1.6 Dissertation Goals 14

Chapter 2: Creation of Hap1 Conditional b Cell Knockout Mice

Introduction 21

Results 23

Discussion 26

Materials and Methods 28

Chapter 3: Hap1 Depletion Reduces Insulin Secretion From Pancreas

Introduction 40

Results 43

Discussion 45

Materials and Methods 48

Chapter 4: Biochemical and Mechanistic Studies of Hap1 in b Cells

Introduction 58

Results 60

Discussion 63

Materials and Methods 66

Chapter 5: Summary and Discussion

Summary of Results 78

Future Directions 79

References 85

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