The overall objective of this research proposal is to further our understanding of the basic molecular mechanisms accounting for the intracellular trafficking, insulin-stimulated translocation and fusion of the insulin-responsive glucose transporter (GLUT4) from intracellular storage sites to the plasma membrane in adipocytes. Although significant progress has been made in terms of the insulin signaling pathways responsible for GLUT4 translocation, there is little information with regard to the regulatory events and proteins involved in specific intracellular trafficking steps. Moreover, the basic biophysical mechanism of biological membrane fusion for this, or any other system, has not been elucidated. Recently, we have found that newly synthesized GLUT4 protein traffics through the Golgi complex and directly enters the insulin-responsive storage compartment without first transiting the plasma membrane and undergoing endocytosis. Based upon the temporal properties of GLUT4 biosynthesis and intracellular sorting coupled with the use of siRNA gene silencing, we have devised a novel paradigm for examining the targeting machinery and vesicle trafficking events that function at different membrane transport steps in the GLUT4 lifecycle. Based upon these data we propose to use siRNA coupled with the temporal expression of GLUT4 reporter constructs to functionally map proteins involved in the biosynthetic sorting to the insulin-responsive compartment (Class 1) from those required for the exit from this compartment (Class 2), those required for endocytosis (Class 3) and those required for recycling back to the insulin-responsive compartment (Class 4). We propose to use this approach to identify and classify the specific requirements for the SNARE family of proteins required for biological membrane fusion. In parallel, we have begun to unravel the biochemical nature of the SNARE-dependent fusion process itself using a combination of GLUT4 trafficking cell biological approach coupled with biophysical analysis of phospholipid-SNARE protein interactions. Our preliminary data has demonstrated that the juxtamembrane domain (JMD) of both plasma membrane Syntaxins (Stx) and VAMP are required for this process through electrostatic interactions with acidic phospholipids. To address the mechanism of fusion pore formation, we will use a series of biophysical and cell biological approaches to determine the role of membrane electrostatics and the tandem VAMP2 tryptophans in this key biological process.

Public Health Relevance

Diabetes, obesity and insulin resistant states can all be characterized as defects in the body's ability to adjust for differences in energy consumption and output. One of the key biological responses to insulin action is the translocation and fusion of the facilitative GLUT4 glucose transporter isoform from intracellular storage sites to the plasma membrane in skeletal muscle and adipose tissue. This process results in an approximate 10-fold increase in the number of cell surface GLUT4 molecules that accounts for the post-prandial increase in peripheral tissue glucose uptake. Thus, understanding the mechanism for this translocation process and the ability of insulin to increase glucose uptake are essentially process for the maintenance of normal glucose homeostasis. 1

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK082694-03
Application #
8249162
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Haft, Carol R
Project Start
2010-03-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
3
Fiscal Year
2012
Total Cost
$341,006
Indirect Cost
$135,581
Name
Albert Einstein College of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Zong, Haihong; Armoni, Michal; Harel, Chava et al. (2012) Cytochrome P-450 CYP2E1 knockout mice are protected against high-fat diet-induced obesity and insulin resistance. Am J Physiol Endocrinol Metab 302:E532-9
Vatish, Manu; Tesfa, Lydia; Grammatopoulos, Dimitris et al. (2012) Inhibition of Akt activity and calcium channel function coordinately drive cell-cell fusion in the BeWO choriocarcinoma placental cell line. PLoS One 7:e29353
Zong, Haihong; Wang, Cheng-Chun; Vaitheesvaran, Bhavapriya et al. (2011) Enhanced energy expenditure, glucose utilization, and insulin sensitivity in VAMP8 null mice. Diabetes 60:30-8