The regulation of glucose entry into fat and muscle cells is one of the primary acute effects of insulin essential for the maintenance of whole body glucose homeostasis. This regulation is perturbed in insulin resistance syndromes, including type 2 diabetes, and therefore a better understanding of how insulin controls glucose flux into these cells will ultimately have a significant impact on human health and disease. Insulin regulates glucose flux into fat and muscle cells by controlling the amount of the Glut4 glucose transporter in the plasma membrane. Efforts of numerous labs over the past decade have established that this control is by a surprisingly complex, multi-step process. Recent applications of advanced microscopy methods and molecular cell biological approaches (including gene silencing strategies) have led to the formulation of a detailed model for Glut4 trafficking. In unstimulated cells it is important to limit the amount of Glut4 in the plasma membrane, and thereby restrict glucose flux into fat and muscle, which preserves blood glucose for use by the brain. Limiting Glut4 in the plasma membrane is accomplished by two distinct intracellular transport cycles. One involves traffic of Glut4 between endosomes and a specialized peri-nuclear compartment. The second involves the targeting of Glut4 from endosomes to Glut4-specialized vesicles (GSVs) that inefficiently dock and fuse with the plasma membrane in the absence of insulin-stimulation. Insulin action intersects Glut4 trafficking at a couple of steps that alter Glut4 transport through these pathways. Full understanding of insulin control of glucose transport requires an understanding of each step of this multi-step process. Here I propose to address some of the key outstanding questions in the field. Throughout the work I propose the use of quantitative microscopy methods and molecular cell biological approaches that we have used in our past studies, many of which I have had a hand in introducing to the study of Glut4.
In Aim1 we will analyze insulin control of pre- fusion steps of GSV association with the PM, with a specific emphasis on regulation by rab10. We will also create an adipose-specific rab10 knockout mouse to probe the physiologic role of adipose rab10 in the control of whole body glucose homeostasis.
In Aim2 we will further characterize the role of IRAP in Glut4 sorting to GSVs, and we will use a membrane-tethered yeast 2-hybrid screen to identify proteins that bind to IRAP and Glut4 cytoplasmic sequences. In secondary analyses we will determine the roles of these proteins in the control of Glut4 and IRAP trafficking.
In aim3 we will use SILAC mass spectrometry approaches to identify proteins of the peri-nuclear compartment, and in secondary analyses determine their contributions to the control of Glut4 trafficking. The results of this ambitious and comprehensive study will significantly advance our understanding of the molecular mechanisms controlling the trafficking of Glut4 between the plasma membrane and endomembrane compartments of adipocytes.

Public Health Relevance

Insulin controls blood glucose levels, in part, by controlling the transport of glucose from the blood into muscle and fat cells. This regulation is defective in type 2 diabetes, contributing to elevated blood glucose of that disease. Insulin regulation of glucose transport is by a complex mechanism that controls the amount of a glucose transporter (known as Glut4) on the surface of cells. The details of this mechanism are not understood. In this project I propose studies to better understand how insulin regulates the amount of Glut4 on the surface of adipocytes. The long term objective of this work is to identify the proteins required for normal regulation of Glut4, use this information to identify what is defective in type 2 diabetes and thereby identify potential targets for the development of pharmacologic agents that mimics insulin's effects on glucose transport as a treatment for type 2 diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
4R01DK052852-18
Application #
8963461
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Haft, Carol R
Project Start
1997-09-01
Project End
2017-11-30
Budget Start
2015-12-01
Budget End
2017-11-30
Support Year
18
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee et al. (2016) Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes. Cell Rep 17:3305-3318
Bruno, Joanne; Brumfield, Alexandria; Chaudhary, Natasha et al. (2016) SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes. J Cell Biol 214:61-76
Vazirani, Reema P; Verma, Akanksha; Sadacca, L Amanda et al. (2016) Disruption of Adipose Rab10-Dependent Insulin Signaling Causes Hepatic Insulin Resistance. Diabetes 65:1577-89
Kajno, Esi; McGraw, Timothy E; Gonzalez, Eva (2015) Development of a new model system to dissect isoform specific Akt signalling in adipocytes. Biochem J 468:425-34
Antonescu, Costin N; McGraw, Timothy E; Klip, Amira (2014) Reciprocal regulation of endocytosis and metabolism. Cold Spring Harb Perspect Biol 6:a016964
Sadacca, L Amanda; Bruno, Joanne; Wen, Jennifer et al. (2013) Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs. Mol Biol Cell 24:2544-57
Wu, Ning; Zheng, Bin; Shaywitz, Adam et al. (2013) AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1. Mol Cell 49:1167-75
Gan, Lixia; Guo, Kaiying; Cremona, Maria Laura et al. (2012) TNF-α up-regulates protein level and cell surface expression of the leptin receptor by stimulating its export via a PKC-dependent mechanism. Endocrinology 153:5821-33
Gonzalez, Eva; Flier, Emily; Molle, Dorothee et al. (2011) Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor. Proc Natl Acad Sci U S A 108:10162-7
Mohammad, Sameer; Ramos, Lavoisier S; Buck, Jochen et al. (2011) Gastric inhibitory peptide controls adipose insulin sensitivity via activation of cAMP-response element-binding protein and p110β isoform of phosphatidylinositol 3-kinase. J Biol Chem 286:43062-70

Showing the most recent 10 out of 34 publications