· Identification of insulin-mediated signaling cascades
· Regulation of intracellular GLUT4 vesicle trafficking and biogenesis

Jeffrey E. Pessin, Ph.D.
Chairman and Professor, Pharmacological Sciences

Chairman and Professor of the Department of Pharmacology
Ph.D., The University of Illinois
Postdoctoral Fellow, Worcester MA

It is well established that the glucose transporter isoform GLUT4 is sequestered into specialized intracellular storage compartments and following insulin stimulation these compartments rapidly redistribute to the plasma membrane. The increased number of cell surface transporters then facilitates the uptake of glucose that is essential for the maintenance of normal glucose homeostasis. Defects in this process lead to states of insulin resistance and impaired glucose tolerance ultimately leading to diabetes.

Thus, my laboratory is focused onto dissecting the molecular basis of insulin action in order to develop a mechanistic understanding of the normal and pathophysiology primarily associated with insulin resistance and Type II diabetes. To address the complicated molecular issues involved in insulin action and signal transduction, we are currently utilizing a three-pronged approach. The first major research area is to examine the proximal molecular targets that are responsible for the insulin-stimulated translocation of the GLUT4 protein from intracellular storage sites to the plasma membrane. We and others have previously demonstrated that PI 3-kinase activity is required for insulin-stimulated glucose uptake and GLUT4 translocation. However, the activation of this pathway, although necessary, is not sufficient to mediate the full extent of insulin-stimulated glucose uptake or GLUT4 translocation. Recently, we have identified a novel signaling pathway that functions in parallel to, but in concert with, the PI 3-kinase pathway in the regulation of GLUT4 translocation. This pathway involves the insulin receptor mediated tyrosine phosphorylation of Cbl and its recruitment to lipid raft microdomains in the plasma membrane. We have also begun to identify several of the downstream targets of Cbl, the mechanism by which Cbl is recruited to these plasma membrane lipid raft subdomains and the function of these domains in insulin-stimulated metabolic actions.

The second major research focus in my laboratory is to understand the insulin-dependent regulation of SNARE protein interactions that are responsible for the docking and fusion of the GLUT4 containing vesicles with the plasma membrane. Previously, we and others have demonstrated that the VAMP2 protein functions as the GLUT4 vesicle SNARE (v-SNARE), while syntaxin 4 and SNAP23 function as the plasma membrane target or t-SNARE. Disruption of any of these interactions inhibits insulin-stimulated GLUT4 translocation. These findings have established a paradigm for understanding GLUT4 vesicle trafficking functionally relating to synaptic vesicle trafficking. During the past period of time, we have also demonstrated that Synip and Munc18c are SNARE accessory proteins that modulate the interaction of syntaxin 4 with the GLUT4 vesicle VAMP2 protein. Synip appears to behave as an inhibitor of VAMP2 binding which is repressed following insulin stimulation. In contrast, Munc18c is a positive fusogenic protein required to interconvert syntaxin 4 between an open to a closed conformational state. This transition between the open and closed state is required for VAMP2 binding.

Another key issue is to determine how the GLUT4 vesicles are actually moved to the plasma membrane. Is this a random event or is the traffic directed through actin/tubulin motors? Once these vesicles arrive at the plasma membrane, do they undergo a priming or tethering step before converting to a SNARE bound complex, in analogy to that observed for rapid first phase of synaptic transmission. Finally, what are the specific insulin regulated conformational changes that allow for the GLUT4 vesicle fusion process to occur? To address these issues, we are determining the role of the cytoskeleton in the trafficking of GLUT4 vesicles by expression of various mutants, blocking antibodies (microinjection) and analysis of GFP tagged fusion proteins. Standard biochemical and genetic approaches (GST-pull downs and two-hybrid screens) are also be employed to identify the presence of tethering proteins associated with GLUT4 vesicles. Finally, temperature sensitive mutants of several SNARE and SNARE accessory proteins are being used to determine the precise stages of these different molecular events. These findings will be correlated with protein structural analysis of the various complexes at the permissive and non-permissive temperatures.

Finally, one of the most powerful and unambiguous approaches to understanding the specific role of a molecule in signaling is to completely prevent the expression of the protein followed by determination of the resultant phenotype. To demonstrate that the effect observed is specific to the given protein, it can then be re-introduced by transfection and should restore the cells to a normal phenotype. This is being accomplished by using homologous recombination of embryo stem (ES) cells, followed by introduction of these toti-potent cells in early stage mouse embryos resulting in chimeric knockout mice. Following several rounds of breeding, the ES cells are represented in the germ line and both heterozygotic and homozygotic knockout animals are being produced. In addition, tissue-specific knockout mice are generated by the use of the Cre/Lox system. Furthermore, primary mouse embryo fibroblasts are prepared from these animals and induced to differentiate into a fully functional adipocyte phenotype in culture. Currently, we have successfully generated, or obtained from collaborators, mouse knockouts for the two Cbl isoforms (c-Cbl, Cbl-b), syntaxin 4, Munc18c, and VAMP3, and we are in the process of preparing VAMP2, Synip and TC10 knockouts.

Peer-Reviewed Publications (2000-2002)

Thurmond, D.C., Kanzaki, M., Khan, A. and Pessin, J.E. Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive aminopeptidase storage vesicles. Mol. Cell. Biol., 20,379-388, 2000.

Watson, R.T. and Pessin, J.E. Functional cooperation of two independent targeting domains in syntaxin 6 is required for its efficient localization in the trans-Golgi network of 3T3L1 adipocytes. J. Biol. Chem., 275:1261-1268, 2000.

Kanzaki, M., Watson, R.T., Artemyev, N. and Pessin, J.E. The trimeric GTP-binding protein (G(q)/G(11)) alpha subunit is required for insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes. J. Biol. Chem., 275:7167-7175, 2000.

Yang, C., Watson, R.T., Elmendorf, J.S., Sacks, D.B. and Pessin, J.E. Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol(3,4,5)trisphosphate in 3T3L1 adipocytes. Mol. Endo., 14:317-326, 2000.

Kao, A.W. and Pessin, J.E. Functional comparison of the role dynamin 2 splice variants on GLUT4 endocytosis in 3T3L1 adipocytes. Am. J. Physiol., 278:E825-E831, 2000.

Mora, S. and Pessin, J.E. The MEF2A isoform is required for striated muscle-specific expression of the insulin-responsive GLUT4 glucose transporter. J. Biol. Chem, 275:16323-16328, 2000.

Baumann, C.A., Ribon, V., Kanzaki, M., Thurmond, D.C., Mora, S., Shigematsu, S., Bickel, P.E., Pessin, J.E. and Saltiel, A.R. CAP defines a second signaling pathway required for insulin-stimulated glucose transport. Nature, 407:202-207, 2000.

Thurmond, D.C. and Pessin, J.E. Discrimination of GLUT4 vesicle trafficking from fusion using a temperature-sensitive Munc18c mutant. EMBO J., 19: 3565-3575, 2000.

Khan, A.H, Thurmond, D.C., Yang, C., Ceresa, B.P., Sigmund C.D. and Pessin, J.E. Munc18c regulates insulin-stimulated GLUT4 translocation to the transverse-tubules in skeletal muscle. J. Biol. Chem., 276:4063-4069, 2001.

Yang, Mora, S., Ryder, J.W., Coker, K.J., Hansen, P., Allen, L.-A. and Pessin, J.E. VAMP3 null mice display normal constitutive, insulin- and exercise-regulated vesicle trafficking. Mol. Cell. Biol., 21:1573-1580, 2001.

Mora, S., Yang, C., Ryder, J.W. Boeglin, D. and Pessin, J.E. The MEF2A and MEF2D isoforms are differentially regulated in muscle and adipose tissue during states of insulin deficiency. Endocrinology, 142:1999-2004, 2001.

Shigematsu, S., Miller, S.L. and Pessin, J.E. Differentiated 3T3L1 adipocytes are composed of heterogenous cell populations with distinct receptor tryosine kinase signaling properties. J. Biol. Chem., 276:15292-15297, 2001.

Watson, R.T. and Pessin, J.E. Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5. Am. J. Physiol. 281:C215-C223, 2001.

Chiang, S.-H., Bauman, C.A., Kanzaki, M., Thurmond, D.C., Watson, R.T., Neudauer, C.L., Macara, I.G., Pessin, J.E. and Saltiel A.R. Insulin-stimulated GLUT4 translocation requires the CAP-dependent but PI 3-kinase-independent activation of the small GTP binding protein TC10. Nature, 410:944-948, 2001.

Yang, C., Coker, K.J., Kim, J.K., Mora, S., Thurmond, D.C., Davis, A.C., Yang, B., Williamson, R.A., Shulman, G.I. and Pessin J.E. Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance. J. Clin. Invest., 107:1311-1318, 2001.

Watson, R.T., Shigematsu, S., Chiang, S.-H., Mora, S. Kanzaki, M. Macara, I.G., Saltiel, A.R. and Pessin, J.E. Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation. J. Cell Biol., 154:829-840, 2001.

Kanzaki, M. and Pessin, J.E. Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling. J. Biol. Chem., 276:42436-42444, 2001.

Kanzaki, M., Watson, R.T., Khan, A. and Pessin, J.E. Insulin stimulates actin comet tails on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes. J. Biol. Chem., 276:49331-49336, 2001.

Ikonomov, O.C., Sbrissa, D., Mlak, K., Kanzaki, K., Pessin, J.E. and Shisheva, A. Functional dissection of lipid and protein kinase signals of PIKfyve reveal the role of PtdIns 3,5-P2 production for endomembrane integrity. J. Biol. Chem., 277:9206-9211, 2002.

Kanzaki, M., Watson, R.T., Hou, J.C., Stamnes, M., Saltiel, A.R. and Pessin, J.E. The small GTP binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes. Mol. Biol. Cell. In Press, 2002.

Chiang, S.-H., Hou, J.C., Pessin, J.E. and Saltiel, A.R. Cloning and functional characterization of related TC10 isoforms, a subfamily of Rho proteins involved in insulin-stimulated glucose transport. J. Biol. Chem., 277:13067-13073, 2002..

Shigematsu, S., Khan, A.H., Kanzaki, M. and Pessin, J.E. Intracellular GLUT4 distribution but not insulin-stimulated GLUT4 exocytosis and recycling are microtubule-dependent. Mol. Endo., 16:1060-1068, 2002.

Watson, R.T., Furukawa, M., Chiang, S.-H., Boeglin, D., Kanzaki, M., Saltiel, A.R. and Pessin, J.E. The carboxyl terminal targeting motif specifies TC10 activation, endomembrane trafficking, plasma membrane microdomain localization and regulation of GLUT4 translocation. Submitted.

Allen, L.A.H., Yang, C. and Pessin, J.E. Rate and Extent of Phagocytosis in Macrophages Lacking Vamp3. J. Leukoc. Bio. In Press, 2002.

Chapters/Reviews (2000-2002)

Thurmond, D., Elmendorf, J., Coker, K.J., Okada, S. and Pessin, J.E. Vesicular trafficking and GLUT4 translocation. In: Diabetes Mellitus: A fundamental and clinical text. D. LeRoith, S. Taylor and J. Olefsky, Eds., Lippincott Publishers, 2000.

Pessin, J.E. and Saltiel, A.R. Insulin-stimulated GLUT4 trafficking: Lessons learned from synaptic transmission. In: Adipocyte Biology and Hormone Signaling. J. Ntambi, Ed., IOS Press, The Netherlands, pp. 1-10, 2000.

Pessin, J.E. and Saltiel, A.R. Signaling pathways in insulin action: Molecular targets of insulin resistance. J. Clin. Invest., 106:165-169, 2000.

Watson, R.T. and Pessin, J.E. Intracellular organization of insulin signaling and GLUT4 translocation. In: Recent Progress in Hormone Research. A.R. Means, Ed. Vol. 56, 175-193, 2001.

Thurmond, D.C. and Pessin, J.E. Molecular basis for insulin-stimulated GLUT4 translocation. Current Opinion in Endocrinology & Diabetes. 8:67-73, 2001.

Watson, R.T. and Pessin, J.E. Subcellular compartmentalization and trafficking of the insulin-responsive glucose transporter, GLUT4. Experimental Cell Research. 271:75-83, 2001.

Thurmond, D.C. and Pessin, J.E. Molecular machinery involved in the insulin-regulated fusion of GLUT4-containing vesicles with the plasma membrane. Molecular Membrane Biology. 18:237-245, 2001.

Mora, S. and Pessin, J.E. Current status of transgenic and genetic approaches to understanding metabolic defects. In: Frontiers in Animal Diabetes Research: Muscle Metabolism. H. Wallberg-Henriksson and J.R. Zierath, Eds. Vol. 4, 227-252, 2002.

Kanzaki, M. and Pessin, J.E. Signal integration and the specificity of insulin action. Cell Biochemistry and Biophysics. In press, 2002.

Mora, S. and Pessin, J.E. Insulin regulation of facilitative glucose transport. In: 2002 Yearbook of Science & Technology. McGraw-Hill, In press, 2002.

Saltiel, A.R. and Pessin, J.E. Insulin action in time and space. Trends in Cell Biology. 12:65-71, 2002.

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