I. Insulin-regulated movement of GLUT4
Insulin-regulated
membrane trafficking and whole body glucose homeostasis. One of the main
acute effects of insulin is to regulate the disposal and storage of dietary glucose by stimulating the uptake of glucose into muscle and fat (Figure
1). Insulin regulates glucose
uptake into these cells by recruiting membrane vesicles containing the GLUT4 glucose transporters from the interior of cells to the cell
surface, where it allows glucose to enter cells by facultative diffusion. Once in the cytoplasm, the glucose is
phosphorylated and thereby trapped inside cells. The effect of insulin on GLUT4 distribution is
reversible. Within an hour of
insulin removal, GLUT4 is removed from the membrane and restored intracellular
in vesicles ready to be re-recruited to the surface by insulin. Thus, glucose uptake by muscle and fat
cells is regulated by modulating the number of GLUT4 glucose transporters on
the surface of cells.
Understanding
how insulin regulates trafficking of GLUT4 is key for understanding the
molecular changes underlying type II diabetes. Type II diabetes, sometimes referred to as late onset
diabetes, is a disease in which individuals develop a resistance to insulin,
such that insulin is no longer able to stimulate uptake of glucose into fat and
muscle. This gives rise to
hyperglycemia and a number of vascular and cardiovascular problems. Therefore, understanding how
insulin regulates the movement of GLUT4 at a molecular level may ultimately
lead to the development of new ways to treat type II diabetes. Go to the American Diabetes Association Homepage for more information on diabetes: http://www.diabetes.org/main/homepage.jsp
Translocation Model
The basic
translocation model for insulin regulation of glucose uptake was first proposed
20 years ago. In the ensuing years
the work of a large number of laboratories have tested and refined this
hypothesis. An overview of the
current model for insulin-regulation of GLUT4 trafficking is provided below.
Key for the
regulation of glucose uptake is the sequestering of GLUT4 intracellularly in
the absence of insulin (often referred to as the basal state). The basal state retention is achieved
by a kinetic mechanism in which GLUT4-containing vesicles very slowly fuse with the plasma membrane and any GLUT4
on the cell surface is rapidly internalized and redelivered to the storage
pool. In this kinetic retention,
the amount of GLUT4 on the surface is dependent on the rates of internalization
and return to the plasma membrane (exocytosis). Less than 5% of GLUT4 is on the surface in the basal state,
indicating that GLUT4 is internalized 20 times faster than it is returned to
the cell surface.
Insulin
stimulates the exocytic rate of GLUT4 without significantly altering its
internalization, and it is the increase in GLUT4 exocytosis that results in the
net redistribution (translocation) of GLUT4 to the surface. Consequently, as in the basal state,
GLUT4 in the presence of insulin cycles between the cell surface and
intracellular compartments.
However, only the GLUT4 on the surface provides for increase glucose
uptake.
In addition
to GLUT4, a protein called IRAP is the only other protein known to traffic
through the insulin-regulated pathway.
Although the function of IRAP is not currently known, studies of IRAP
have proved to be useful for understanding insulin-regulated membrane
trafficking.
The
GLUT4/IRAP trafficking pathway, continual traffic between intracellular
compartments and the plasma membrane, is conceptually similar to the general endocytic trafficking pathway. Endocytosis is a mechanism by which many membrane
proteins are internalized from the cell surface, transported through endosomes
and returned to the cell surface. The most commonly used marker for this
pathway is the transferrin receptor.
A receptor involved in the uptake of the transferrin, an iron carrying
protein. Insulin does not alter
the distributions of proteins that traffic through the general endocytic
pathway. There is physical overlap
between the general and specialized insulin-regulated trafficking pathways since GLUT4 is internalized
through clathrin-coated pits, the major endocytic uptake system in cells. Therefore, a key to understanding
how insulin regulates GLUT4 trafficking is to understand how this specialized
pathway differs from the general endocytic pathway.
Some of the
major unanswered questions to be addressed in the field are to:
1)
Identify and characterize the intracellular membrane compartments through which
GLUT4 and IRAP traffics.
2)
Characterize how GLUT4 and IRAP are sorted away from the general endocytic
recycling pathway.
3)
Identify the mechanism responsible for the slow exocytosis of GLUT4/IRAP in the
basal state.
4)
Identify how the insulin signal transduction pathway intersects with the IRAP
trafficking pathway.
A large
number of labs are currently working on these important questions. In my lab we have developed biochemical
and quantitative optical microscopy methods for the analysis of
insulin-regulated trafficking in cultured cells. We are currently studying two
cell models. One is 3T3-L1
adipocytes, the standard model for these studies, and the other is fibroblast
cells.
Although fat
and muscle are classic insulin-responsive tissues responsible for glucose
disposal, we have found that other cell types (e.g., fibroblasts) also have an
insulin-regulated trafficking pathway.
One major difference between the pathways in fibroblasts and adipocytes
is the magnitude of the response, with insulin stimulating an ~10 fold
translocation of GLUT4 in adipocytes and a 2 to 3 fold translocation in
fibroblasts. Although the
magnitude is smaller, the pathways in the differentiated and undifferentiated
cells share a number of similarities and I anticipate that understanding the
pathway in the more experimentally amenable fibroblasts will a provide a
context for analyzing the more complex pathway adipocytes.
In our
recent work we have mapped out the overlap between the general endosomal system
and the specialized pathway in both adipocytes and fibroblasts. Using quantitative single cell assays
we are now further testing this model as well as using dominant inhibitory
constructs and siRNA approaches to identify the functions of specific proteins
in insulin-regulated membrane trafficking.
Some recent
papers from the lab on insulin-regulated GLUT4 trafficking:
Create reporters to
study dynamics of insulin-regulated trafficking:
Lampson, M.A., A.
Racz, S.W. Cushman, and T.E. McGraw.
2001. Demonstration of insulin-responsive trafficking of GLUT4 and
vpTR in fibroblasts. J.
Cell Science 113:4065-4076.
Charcaterzie Insulin-regulated
traffic in fibroblasts:
Lampson, M. A., J.
Schmoranzer, A. Ziegerer, S. Simon and T.E. McGraw. 2001. Retention
in the endosomal recycling compartment is regulated by specialized vesicle
budding. Mol. Biol. Cell.12:3489-3501.
Characterize dynamics
of GLUT4 traffic in adipocytes
Zeigerer, A., M. A.
Lampson, D. D. Sabatini, M. Adesnik, M. Ren, and T. E. McGraw. 2002. GLUT4 retention
in adipocytes requires two intracellular insulin-regulated transport steps. Mol. Biol. Cell.
13:2421-2435.
Insulin-regulated
movement of GLUT4
Links
to affiliated graduate programs
Presentations
(Teaching)