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Silvia Corvera, M.D.Academic Role: Professor
Faculty Appointment(s) In:
Other Affiliation(s):
Cell Biology of Insulin Action and Type 2 Diabetes
1. What are the cellular and molecular mechanisms that underlie the effect of insulin on glucose transport. PI 3-kinase and the control of Endosomal Traffic. It is known that insulin causes an increase in the steady state concentration of a facilitative glucose carrier, GLUT4, at the plasma membrane and this leads to a stimulation in the rate of glucose uptake by fat and muscle cells. Thus, understanding insulin action entails elucidating how the trafficking of transmembrane proteins such as GLUT4 among endosomal membranes is regulated by the signal transduction pathways that occur upon activation of the insulin receptor. One of the key components of the insulin signaling cascade is the enzyme PI 3-kinase. PI 3-kinases are a family of enzymes that phosphorylate the 3’ position of the inositol ring of phosphatidylinositol. The products of PI 3-kinases range from the monophosphorylated PI(3)P, to the tri-phosphorylated PI(3,4,5)P3. To better understand the specific functions of these phospholipids we used a proteomics approach to identify proteins that interact with PI 3-kinase products. Work by Varsha Patki led to the discovery that EEA1, a soluble protein that associates with early endosomes, interacts specifically with PI(3)P (1). EEA1 contains a C-terminal motif called the FYVE domain, that directly interacts with the lipid head group (2). Work by Deirdre Lawe and Akira Hayakawa has defined the role of the FYVE domain in EEA1, and the structural basis for its interaction with PI(3)P on the membrane of the endosome (3,4,5). Their work is now focused on clarifying the precise role of EEA1 in endosomal dynamics and cell function, using RNA interference, live cell imaging and construction of protein interaction maps. To better understand the relationship between EEA1, endosome function and GLUT4 trafficking, we have utilized imaging technology to delineate the cellular trafficking pathways used by GLUT4. Varsha Patki has analyzed the dynamics of eGFP-GLUT4 in live cells in quasi-real time using technology developed by the Biomedical Imaging Group at UMASS (6). More recent developments in this imaging technology have enabled us to follow two fluorephores simultaneously. With this we have been able to visualize the traffic of transferrin relative to EEA1 (Figure 1), and will be able to image GLUT4 movements relative to EEA1 and to other transmembrane proteins that traffic in the endocytic pathway such as the transferrin receptor. We expect to obtain novel important information on the cell biology of insulin action on GLUT4, that will necessary for understanding the role of specific regulatory proteins in this pathway. EEA1 is a relatively abundant protein likely to be involved in key aspects of endosome function including protein sorting and membrane fusion. However, other less abundant proteins contain FYVE domains. One of them is the protein SARA, which facilitates signaling through TGFb receptors. Work by Susan Hayes, a graduate student in the laboratory has shown that SARA co-localizes with EEA1, and that TGFb receptors internalize and remain in the endosome while catalyzing the phosphorylation of Smad-2, a transcription factor that conveys the TGFb signal to the nucleus. Impairment of TGFb receptor entry into the endosomes also impairs Smad-2 phosphorylation and nuclear translocation (7), suggesting the exciting hypothesis that important aspects of signal transduction occur at the endosome (7). It also indicates that the FYVE domain is used by proteins that are primarily involved in the control of membrane traffic, such as EEA1, and by proteins primarily involved in signal transduction, such as SARA. We are currently conducting a genetic screen using RNA interference in C. elegans to catalogue FYVE domain containing proteins involved in the regulation of endosome traffic. Mechanism of Action of Insulin Sensitizers Currently we are exploiting the dramatic improvements in sensitivity of mass spectroscopy that have occurred in the last few years to identify more proteins involved in insulin action. To this end we have used an approach that combines subcellular fractionation, 2-dimensional protein separation (first dimension consists of separation based on So , second dimension SDS-PAGE), and mass spectroscopy (8). This work is part of a collaborative network of researchers working to provide genomic and proteomic information relevant to research on Type 2 Diabetes to the scientific community at large(www.diabetesgenome.org). Our work is done in close collaboration with the Proteomic Mass Spectrometry lab headed by Dr. John Leszyk (www.umassmed.edu/proteomic). In conjunction with our efforts to identify novel proteins involved in insulin action, we have explored the effects of drugs that are able to enhance insulin sensitivity in Diabetic patients. In studying the effects of one of these drugs, rosiglitazone, two undergraduate students, Karen Mendelson and Greg Bell, discovered that fat cell differentiation is accompanied by a massive enhancement of mitochondrial biogenesis, and that rosiglitazone enhances this process. These results were further characterized by Leanne Wilson-Fritch and Alison Burkart, graduate students in the lab, who determined that rosiglitazone also caused alterations in the morphology of mitochondria in treated adipose cells (8). These findings suggest the hypothesis that the metabolism of the white adipose cell impacts upon insulin sensitivity. Leanne, Alison and My Chouinard are currently exploring this new research avenue by analyzing the actions of this drug in animal models of obesity and insulin resistance, and by delving in more detail into the mechanisms of energy control in white fat. Links to References
(2) Patki et al (1998) (3) Lawe, et al.(2002) (4) Lawe et al.(2003)
(6) Patki et al. (2001) www.molbiolcell.org/content/vol12/issue1/images/data/129/F1/DC1/demo1.mov
(8) Wilson-Fritch, et al.(2003)
Effects of CaM inhibitors on the localization of GFP-EEA1 and Alexa 594-transferrin in COS-7 cells. Cells expressing GFP-EEA1 were incubated with Alexa 594-transferrin for 10 min and washed once before addition of 100 µM W7. Stacks of 21 optical sections were collected every 30 s imaging alternatively with lasers tuned to 488- and 594-nm wavelengths. After image restoration, stacks were projected into a single 2D image. Projections for the times indicated are shown. The arrow indicates the concentration of transferrin in the recycling endosome after 15 min of treatment with W7.
TGFß receptor internalization into to EEA1-positive endosomes is blocked by dominant-negative dynamin. (A) Cos-7 cells cotransfected with HA-tagged type I receptor (RI) and myc-tagged type II TGFß receptor, and either wild-type (WtDyn) or K44E dynamin (left, WT; right, K44E) were incubated for 1 h at 4°C with 100 pM TGFß and antibodies against the HA epitope, and then for 60 min at 37°C. Cells were fixed and stained with a human antiserum against EEA1 (indicated as EEA1, red) and a mouse antiserum against dynamin I (blue), and secondary antibodies to the anti-HA antibody (RI, green). The overlap between the three signals is displayed in the panel labeled Dyn + EEA1 + RI. The overlap between EEA1 + RI is shown as indicated, and the large bottom panel represents an enlarged area of this panel (EEA1+RI (wtDyn or DynK44E). From Reference 8
Mitochondria in live rosiglitazone-treated cells. On day 7 of differentiation, 3T3-L1 adipocytes were treated with trypsin and then seeded on coverslips. On day 8, the coverslips were either left untreated (control) or treated with 1 µM rosiglitazone for 24 or 48 h. Cells were incubated with 100 nM MitoTracker Green FM and imaged as described in Fig. 4. Images represent 10 optical sections (2.5 µm) projected into a single plane (top panels) or a single optical section through the middle of the cell (middle panels). Images were pseudocolored, and higher-intensity pixel values are displayed in red. Higher magnifications of the areas delineated by the squares are shown in the bottom panels. Arrows point to reticular structures representing mitochondria, which tend to decrease in length in response to rosiglitazone.
Phone: 508 856 6898
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Our laboratory utilizes imaging, proteomics and genomics to gain understanding of cellular processes that, when impaired, lead to human disease. We have focused our efforts on understanding Type 2 Diabetes, a metabolic disease that currently affects 12 million adults in the USA alone. Type 2 Diabetes is defined as a higher than normal level of blood glucose under fasting conditions, and is characterized by an impaired response of fat and muscle cells to the hormone insulin. This impairment in insulin action is manifested by a decreased ability of fat and muscle cells to take up glucose in response to insulin. We are addressing two broad questions in the laboratory:
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