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.
Jordens,
I., Molle, D., Xiong, W., Keller, S.R., and McGraw, T.E. (2010). Insulin-regulated aminopeptidase is a key
regulator of GLUT4 trafficking by controlling the sorting of GLUT4 from
endosomes to specialized insulin-regulated vesicles. Mol Biol Cell 21, 2034-2044.
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.
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.
