Research Projects

A. Insulin Action in Adipocytes
Insulin stimulates glucose transport into adipose and muscle by promoting the redistribution of the Glut4 glucose transporter from intracellular compartments to the plasma membrane (PM). In the fasted state intracellular sequestration of Glut4 defends against hypoglycemia by limiting glucose flux into muscle and fat, whereas insulin-stimulated translocation of Glut4 to the PM promotes postprandial glucose disposal. The regulation of Glut4 trafficking in muscle and adipose is critical for the maintenance of glucose homeostasis.  Insulin does not properly induce Glut4 translocation in insulin resistant syndromes (e.g., type 2-diabetes), contributing to the pathophysiologies of those conditions. The molecular lesion(s) responsible for the disruption of Glut4 trafficking in insulin resistance and T2D have not been elucidated. A more complete understanding of the Glut4 trafficking mechanism will contribute significantly to our understanding of the control of Glut4 by identifying and characterizing the proteins that determine Glut4 distribution in unstimulated and insulin-stimulated adipocytes. A more comprehensive understanding of Glut4 trafficking is required for the rationale development of therapeutic interventions that treat insulin resistance by mimicking insulin’s effects.

The recent application of optical microscopy methods and siRNA technologies have provided for major advances in the understanding of Glut4 trafficking in physiologically relevant model cell systems. The results of these studies, which represent the efforts of many labs, demonstrate that the regulation of Glut4 expression in the PM is by the surprisingly complex, multi-step process illustrated in Fig.1. In both unstimulated and insulin-stimulated cells, Glut4 is transported among the PM, endosomes, a peri-nuclear storage compartment and transport vesicles (referred to as "GLUT4 specialized vesicles" or GSVs). Basal intracellular retention involves 2 Glut4 transport cycles: 1) between endosomes and the peri-nuclear storage compartment, and 2) between GSVs and endosomes. The peri-nuclear compartment has a major role in the intracellular sequestration of Glut4 in unstimulated cells, and the GSVs are the specialized vesicles that ferry Glut4 to the PM.

Insulin achieves translocation by specifically altering the Glut4 trafficking kinetics of a number of steps. Insulin signaling, via distinct effectors, intersects Glut4 trafficking at multiple steps (Fig.1). Since the effect of insulin on the expression of Glut4 in the PM is the sum of these steps, it is important to have a comprehensive understanding of each of the regulated steps. Work in the lab currently focuses on 3 aspects of Glut4 behavior:

Project 1: Functional analysis of rab10.
Insulin activation of rab10 controls the pre-fusion engagement of GSVs with the PM of adipocytes. Perturbation of rab10 severely inhibits translocation of Glut4 to the PM. The aims of this work are to identify the rab10 effectors, and to probe how the rab10-regulated step integrates with the other controlled steps of Glut4 trafficking. These studies include the use of biochemical, molecular cell biological and proteomic approaches in studies of cultured adipocytes and physiologic studies a rab10 adipose-specific KO mouse we have recently generated.

Project 2: Characterize the Glut4 sorting machinery and define the role of IRAP in this process.
Significant progress has been made in deciphering the Glut4 motifs responsible for specialized trafficking (3, 6-8), yet nothing is known about the proteins that bind these motifs. We have shown that IRAP is an essential component of the machinery that sorts Glut4 from endosomes to the GSVs (1). The objectives of the studies are to: a) define the role of IRAP in Glut4 sorting using IRAP structure-function studies and to use moelcualr cell biological and proteomic methods to identify proteins that regulate Glut4 and IRAP sorting.

Project 3: Characterize the peri‐nuclear Glut4 storage compartments of adipocytes.
The peri-nuclear compartment has a pivotal role in the intracellular sequestration of Glut4, yet little is known about this compartment (3, 5). To address this gap in knowledge, we have developed a method to specifically immuno-isolate this compartment using Glut4 mutants that are differentially distributed among the Glut4-containing compartments. The protein composition of the peri-nuclear compartment is being determined by comparative SILAC mass spectrometry. These data will identify the compartment and identify the proteins that potentially regulate transport to and from this site, thereby providing a significant advance for the field. The functional roles of the proteins identified in the proteomic analysis will be examined in secondary studies.

Select recent related publications
Gonzalez, E., Flier, E., Molle, D., Accili, D., and McGraw, T.E. (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, 10162-10167.

Xiong, W., Jordens, I., Gonzalez, E., and McGraw, T.E. (2010). GLUT4 is sorted to vesicles whose accumulation beneath and insertion into the plasma membrane are differentially regulated by insulin and selectively affected by insulin resistance. Mol Biol Cell 21, 1375-1386.

Kahn, B.B., and McGraw, T.E. (2010). Rosiglitazone, PPARgamma, and type 2 diabetes. N Engl J Med 363, 2667-2669.

Gonzalez E & McGraw TE (2009) Insulin-modulated Akt subcellular localization determines Akt isoform-specific signaling. Proc Natl Acad Sci U S A 106(17):7004-7009.

Sano, H., Eguez, L., Teruel, M.N., Fukuda, M., Chuang, T.D., Chavez, J.A., Lienhard, G.E., and McGraw, T.E. (2007). Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane. Cell Metab 5, 293-303.

Blot, V., and McGraw, T.E. (2006). GLUT4 is internalized by a cholesterol-dependent nystatin-sensitive mechanism inhibited by insulin. Embo J 25, 5648-5658.

Gonzalez, E., and McGraw, T.E. (2006). Insulin signaling diverges into Akt-dependent and -independent signals to regulate the recruitment/docking and the fusion of GLUT4 vesicles to the plasma membrane. Mol Biol Cell 17, 4484-4493..

Eguez, L., Lee, A., Chavez, J.A., Miinea, C.P., Kane, S., Lienhard, G.E., and McGraw, T.E. (2005). Full intracellular retention of GLUT4 requires AS160 Rab GTPase activating protein. Cell Metab 2, 263-272.

Project 4:  Studies of GIPR trafficking in adipocytes: regulation and signaling.
GIP action in adipocytes.
The Glucose-dependent Insulinotropic Polypeptide (GIP), a gut hormone secreted in response to nutrients, has a major role in overall metabolic regulation. Although most studies have focused on its role to promote glucose-stimulated insulin secretion from beta-cells, GIP is known to have important functions in other tissues. Regarding the extra-pancreatic effects of GIP, we have recently shown GIP to be a potent sensitizer of adipocytes to insulin. Conceptually, this insulin-sensitizing effect is similar to the glucose-sensitizing effects of GIP on beta-cells; in both cases GIP sets the tone of the response of the target cells to physiologic stimuli: glucose in the case of beta-cells and insulin in adipocytes.  Adipose has an important endocrine role in the regulation of whole body metabolism; therefore, GIP setting the tone of insulin response of adipocytes will have wide-ranging effects on metabolism, underscoring the importance of understanding GIP and its actions, particularly in adipocytes.  A more comprehensive knowledge of the mechanism of GIP action in adipocytes might lead to the identification of strategies to pharmacologically modulate insulin action as a treatment for diabetes and insulin resistance. A first step towards that goal is to achieve a better understanding of the biology of GIP receptor (GIPR) in adipocytes.

Despite the great deal that is known from human data and studies of genetically engineered mice about the role of GIP in physiology, little is known about the biology of the GIPR in adipocytes. The objective of this work is to address this gap in our knowledge by providing a comprehensive description of the biology of GIPR, a member of the GPCR family of receptors, in cultured adipocytes (Figure 2). The work in this proposal will establish a foundation for understanding GIP’s functions at a molecular level and provide a framework for ongoing studies of GIP’s role in metabolism. These cellular studies address an immediate need in field, and these results will have an important and lasting impact.

A better understanding of the function of GIP in adipocytes might lead to the development of strategies to pharmacologically modulate insulin action as a treatment for insulin resistance. A first step in this regard would be a more complete description of the biology of the GIPR. Despite the great deal that is known about the role of GIP in physiology from human data and studies of genetically engineered mice, little is known about the biology of the GIPR, a G-Protein Coupled Receptor (GPCR) biology in adipocytes. The objective of this proposal is to address this gap in our knowledge by providing a clear and comprehensive description of the biology of GIPR in cultured adipocytes.

In this project we address three areas of GIPR biology:
                1. GIPR trafficking. Regulated trafficking is key for the function and regulation of GPCRs. We are developing a system for studying GIPR trafficking in adipocytes for use in studies to: a) characterize the trafficking of GIPR in basal adipocytes and following physiologic stimuli; b) link GIPR trafficking to signal transduction and the biological effects of GIP; c) investigate the impact of a naturally occurring GIPR mutation linked to obesity on GIPR biology; d) establish the impact of insulin-resistance on GIPR behavior; e) perform structure-function studies identifying the sequences of GIPR responsible for its trafficking.

                2. GIPR phosphorylation: roles in trafficking and signaling. Ligand-induced phosphorylation has a major role in the regulation of the functions of GPCRs. We are identifing the residues of GIPR that are phosphorylated by GIP stimulation, defining the role of the phosphorylation(s) in GIPR trafficking and signal transduction, and generating GIPR phospho-specific antibodies.

                3. Role of GPCR kinases (GRKs) and b-arrestins in GIP signaling and GIPR traffic. The GRKs and b-arrestins control the internalization/down regulation of activated GPCR’s, and therefore control signaling of the GPCRs. We are using siRNA knockdown to identify the GRK(s) responsible for GIPR phosphorylation, characterizing the effect of GRK knockdown on GIPR function, and determining the effect of b-arrestin knockdown on GIPR receptor trafficking and signaling.

Select recent related publications
Mohammad, S., Ramos, L.S., Buck, J., Levin, L.R., Rubino, F., and McGraw, T.E. (2011). Gastric inhibitory peptide controls adipose insulin sensitivity via activation of CREB and p110beta isoform of PI3 kinase. J. Biol. Chem. 2011 286: 43062-43070

B. Pro-tumorigenic functions of fibroblasts in Lung Cancer
Despite improvements in the diagnosis, staging and treatment of lung cancer, the disease remains the leading cause of cancer deaths for both men and women worldwide. Lung cancer research efforts have mainly focused on understanding the biology of the epithelial component of the tumor mass (the cancer cell) such as activation or inactivation of relevant genes and or alterations in key signaling pathways within the cancer cell. Consequently, less is known about the contribution of non-epithelial cellular elements of the tumor, such as stromal fibroblasts, to carcinogenesis. The overarching hypothesis of this work is that the tumorigenic activity of fibroblasts is determined by specific alterations in their gene  expression profiles that are either tumor induced or imparted by exogenous factors, and that a pro-tumorigenic alteration in the transcriptome can occur in fibroblasts within or beyond the tumor mass.

In addition to transformed cancer cells, the tumor mass contains a variety of cell types collectively referred to as the tumor microenvironment. The tumor microenvironment cells provide essential support for the cancer cells. Specifically targeting cells of the tumor microenvironment holds significant clinical promise since those strategies are synergistic with approaches that target the cancer cells. Fibroblasts resident within the tumor, referred to as cancer-associated fibroblasts (CAFs), have profound pro-tumorigenic activities and therefore these cells are prime therapeutic targets in the tumor microenvironment. It is believed that fibroblasts from non-neoplastic tissues are not pro-tumorigenic, leading to the hypothesis that fibroblasts within the tumor are specifically and stably reprogrammed to provide support for the cancer cells.

A wealth of clinical information justifies the significance of focusing on the roles of fibroblasts on tumorigenesis. Clinical observations by surgeons and pathologists suggest that tumoral desmoplasia is a poor prognostic indicator since such tumors are highly invasive locally and metastasize with higher frequency. For example, a fibrous stroma is a known poor prognostic marker in early stage squamous and adenocarcinoma of the lung. Furthermore, podoplanin-expressing fibroblasts are independent predictors of survival and recurrence in adenocarcinoma of the lung, colon and breast. In advanced lung cancer (stage 4) treatment targeting genes activated in cancer cells by mutations or rearrangements is associated with emergence of drug resistance either due secondary mutations or alternatively due to activation of alternate survival pathways mediated by fibroblasts in the tumor microenvironment.  From a therapeutic point of view, fibroblasts are genetically stable and therefore targeting fibroblasts may enhance the therapeutic benefit of existing treatments and or overcome stromal-mediated drug resistance mechanisms. These observations suggest that that targeting fibroblasts may be of therapeutic value in both early and advanced stage disease and possibly cancer prevention.

In collaboration with Nasser Altorki, MD (Professor of Cardiothoracic Surgery, Weill-Cornell College of Cornell University) we have begun a project to discover how and why CAFs are pro-tumorigenic. In these studies we use RNA seq, metabolomic and proteomic methods to profile primary human CAFs isolated from lung tumors, lung fibroblasts isolated from non-neoplastic lung tissue adjacent to the tumor, and dermal fibroblasts.  The results of these profiling studies will be used to generate hypothesis for the pro-tumorigenic activities of CAFs.  These hypotheses will be tested in functional studies using molecular cell biological methods in vitro studies and in vivo tumorigenesis studies.

Select recent related publications
Metabolic alterations in lung cancer-associated fibroblasts correlated with increased glycolytic metabolism of the tumor Virendra K. Chaudhri, Gregory G. Salzler, Salihah A.Dick, Melanie S. Buckman, Raffaella Sordella, Edward D. Karoly, Robert Mohney, Brendon M. Stiles, Olivier Elemento, Nasser K. Altorki, Timothy E. McGraw. Molecular Cancer Research, In press.

Wu, N., Zheng, A Shaywitz, Y Dagon, C Tower, G Bellinger, C-H Shen, J Wen, J Asara, TE McGraw, BB Kahn, LC Cantley. AMPK-dependent degradation of TXNIP in response to energy stress results in enhanced glucose uptake via GLUT1. 2013. Molecular Cell 49:1-9.

Stiles, B.M., Nasar, A., Mirza, F., Paul, S., Lee, P.C., Port, J.L., McGraw, T.E., and Altorki, N.K. (2013). Ratio of positron emission tomography uptake to tumor size in surgically resected non-small cell lung cancer. The Annals of thoracic surgery 95, 397-404.