Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106
Submitted 16 December 2002 ; accepted in final form 5 April 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
glucose transporter-4; insulin; G proteins; proline-rich tyrosine kinase 2; p38 mitogen-activated protein kinase
Recent evidence also implicates the action of Gq/11 in
mediating both insulin (13,
15) and
endothelin-1-stimulated (12,
21) glucose uptake in 3T3-L1
adipocytes. G
11, a member of the Gq family of heterotrimeric
GTP-binding proteins, is highly expressed in adipocytes
(5). Activation of endothelin A
receptors by the vasoactive peptide endothelin-1 potently stimulates glucose
transport in 3T3-L1 adipocytes via activation of G
q/11
(12,
36). Most studies find that
endothelin-1-stimulated glucose uptake is independent of PI 3-kinase activity
(5,
16,
36), although one report
indicates that PI 3-kinase is involved
(12). Instead, endothelin-1
increases glucose uptake via a mechanism that requires tyrosine
phosphorylation of a number of effector proteins, including
G
q/11 and the src family tyrosine kinase Yes, as
well as the tyrosine kinase proline-rich tyrosine kinase 2 (PYK2) and the
scaffolding protein paxillin
(11,
21). The GTPase ADP
ribosylation factor 6 and cortical actin polymerization also contribute to
G
q/11-mediated GLUT4 translocation
(5).
Short- and long-term ethanol exposure is associated with impaired glucose
utilization. Ethanol consumption increases circulating glucose concentrations
(8), glucose intolerance, and
insulin resistance (25,
38), and chronic heavy alcohol
consumption is an independent risk factor for the development of type 2
diabetes in some populations
(9,
32,
34). However, the mechanisms
for this disruption of glucose homeostasis by ethanol are not well understood.
Adipocytes isolated from rats fed ethanol as part of the high-fat
Lieber-DeCarli diet for 4 wk have impaired insulin-stimulated glucose uptake
(22,
35). In many conditions, such
as obesity, high-fat diets, or exposure to TNF-, suppression of
insulin-stimulated glucose transport is associated with impaired insulin
receptor-dependent activation of PI 3-kinase
(17,
24). However, chronic ethanol
feeding was not associated with impaired insulin signaling to PI 3-kinase and
Akt in isolated adipocytes
(22). These data suggest that
chronic ethanol targets alternative, PI 3-kinase-independent signaling
pathways to suppress insulin-stimulated glucose transport. Because
endothelin-1 stimulates glucose transport via mechanisms that are independent
of PI 3-kinase (5,
16), we tested the hypothesis
that chronic ethanol feeding impairs endothelin-1-stimulated glucose uptake
and investigated the effects of chronic ethanol exposure on the signaling
pathways contributing to endothelin-1-mediated glucose uptake in
adipocytes.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male Wistar rats (150160 g) were purchased from Harlan Sprague
Dawley (Indianapolis, IN). The Lieber-DeCarli ethanol diet was from Dyets
(Bethlehem, PA). Antibodies were obtained from the following sources: rabbit
polyclonal anti-GLUT4 (Biogenesis, Sandown, NH), anti-Gs
(Calbiochem, La Jolla, CA), anti-G
q/11 (Santa Cruz
Technology, Santa Cruz, CA), anti-PYK2 (BD Transduction Labs, San Diego, CA),
phosphospecific PYK2 antibodies (Biosource International, Camarillo, CA),
phosphospecific Akt and Akt antibodies (Cell Signaling Technology, Beverly,
MA), and monoclonal anti-phosphotyrosine (PY20; BD Transduction Labs). Goat
anti-rabbit or anti-mouse IgG (Fab fragment) coupled to horseradish peroxidase
and adenosine deaminase were from Roche (Indianapolis, IN).
2-Deoxy-[3H]glucose was from Amersham (Arlington Heights, IL).
Insulin and endothelin-1 were from Sigma (St. Louis, MO). All cell culture
reagents were from GIBCO (Grand Island, NY).
Methods
Animal care and feeding. Rats were housed in individual cages in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle. Animals were acclimatized for 3 days after arrival and provided with free access to Purina rat chow and water. Animals were then allowed free access to liquid diet (18) without ethanol for 2 days and then randomly assigned to the ethanol-fed or pair-fed groups. The ethanolfed group was allowed free access to liquid diet with 17% of calories as ethanol for 2 days and then provided with diet containing 35% of calories from ethanol for 4 wk. Controls were pair fed a liquid diet that was identical to the ethanol diet except that maltose dextrins were isocalorically substituted for ethanol. Control diets had 18% of calories as protein, 35% as fat, and 47% as carbohydrates compared with the ethanol diets containing 18% protein, 35% fat, 12% carbohydrate, and 35% ethanol (18). Pair-fed rats were given the same amount of food that their ethanol-fed pair had consumed in the preceding 24 h. Procedures involving animals were approved by the Institutional Animal Care Board at Case Western Reserve University.
Uptake of 2-deoxy-[3H]glucose. After the 4-wk feeding period, animals were anesthetized by intraperitoneal injection with pentothal sodium (0.2 ml/100 g), and epididymal fat pads were removed. Adipocytes were isolated by collagenase digestion as previously described (35), counted, and diluted to 5 x 105 cells/ml in phosphate-buffered saline with 1 mM MgCl2, 0.68 mM CaCl2, pH 7.4, 1 mg/ml BSA, 1 mM pyruvic acid, and 1 U/ml adenosine deaminase (incubation buffer). Adipocytes were stimulated with and without 10 nM endothelin-1 or 10 nM insulin for 30 min, and uptake of 2-deoxy-[1,2-3H]glucose (final concentration 2.5 mM, 0.5 µCi/tube) was measured over 3 min for 2-deoxyglucose (35). In some experiments, adipocytes were pretreated or not with 5 µM SB-203580 for 15 min before being stimulated with endothelin-1. Nonspecific uptake was measured in the presence of 10 mM phloretin.
Insulin- and endothelin-1-stimulated PYK2 and p38 MAPK
phosphorylation. Isolated adipocytes (2 x 106
cells/ml) were treated with or without insulin or endothelin-1 for 210
min. For analysis of PYK2 tyrosine phosphorylation, cells were lysed for 15
min at 4°C in 10x lysis buffer [for a final concentration of 1%
Nonidet P-40 and 1% Triton X-100 in 50 mM Tris · HCl (pH 7.4), 150 mM
NaCl, 2 mM EGTA, and protease inhibitors (Complete; Boehringer Mannheim) and
phosphatase inhibitors (1 mM Na vanadate, 20 mM Na pyrophosphate, 100 mM
NaF)]. Lysates were vortexed briefly and centrifuged for 2 min, and the
infranatant below the fat cake was removed with a syringe. Samples were then
normalized for protein content and separated by SDS-PAGE for Western blotting.
For analysis of p38 MAPK phosphorylation, cells were lysed directly in Laemmli
sample buffer, boiled for 5 min, and separated by SDS-PAGE for Western blot
analysis.
Isolation of subcellular fractions. For the measurement of G
protein quantity, isolated adipocytes were homogenized in 20 mM Tris, pH 7.4,
1 mM EDTA, and 255 mM sucrose with protease inhibitors (homogenizing buffer)
by use of a Wheaton glass homogenizer with a tight-fitting pestle (clearance
0.05 µm). A plasma membrane-enriched fraction was prepared by
centrifugation of homogenates at 16,000 g for 15 min. The protein
content of the 16,000-g pellet was measured, and equal quantities
were separated by either 10% polyacrylamide (for Gs) or
12.5% polyacrylamide (G
q/11) SDS-PAGE for Western blotting.
Recovery of G
q in the pellet compared with supernatant was
followed to ensure complete recovery of plasma membranes; all immunoreactive
G
q was found in the 16,000-g pellet (data not
shown).
To determine the subcellular localization of GLUT4, isolated adipocytes (1 x 106 cells) were incubated with or without 100 nM insulin or 10 nM endothelin-1 for 30 min at 37°C. Reactions were terminated by the addition of 2 mM KCN, and adipocytes were homogenized as described. Purified plasma membrane and low-density microsomes were isolated by differential centrifugation as previously described (22). In some experiments, plasma membrane-enriched fractions, isolated as described for measuring G protein quantity, were used to assess GLUT4 and GLUT1 translocation in response to endothelin-1 and insulin. Recovery of syntaxin 4, a plasma membrane protein, was followed to ensure complete recovery of plasma membranes (data not shown).
Western blotting. PVDF membranes were blocked with 5% nonfat dry milk or 3% BSA in Tris-buffered saline (TBS; 50 mM Tris, 150 mM NaCl) containing 0.1% Tween (TBS-T) for 2 h, washed twice with TBS-T, and then incubated with primary antibody overnight at 4°C. Membranes were washed again and probed with horseradish peroxidase-coupled goat anti-rabbit or anti-mouse IgG Fab fragments for 1 h. Bound antibody was visualized using enhanced chemiluminescence reagent. Immunoreactive protein quantity was assessed by scanning densitometry; film exposure times were in the linear range of detectability. After probing for phosphorylated p38 or phosphospecific PYK2, membranes were stripped and reprobed with antibodies to total p38 or another phosphospecific PYK2 form. Total PYK2 was not well detected in stripped membranes and so was measured on separate membranes rather than on the same membranes as the phosphorylated forms of PYK2.
Statistical Analysis
Because of limitations in the amount of tissue available from each animal, assays were conducted on adipocytes isolated from multiple feeding trials. Each trial involved six rats per dietary treatment; adipocytes from two rats were pooled for isolation of purified plasma membrane and low-density microsomes. Values reported are means ± SE. Data were analyzed by Student's t-test or the general linear models program on the SAS statistical package for personal computer. Differences between groups were determined by least square means. Data were log transformed when necessary to produce a normal distribution.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Both insulin and endothelin-1 increased GLUT4 protein at the plasma membrane in adipocytes isolated from pair-fed rats (Fig. 2). Insulin stimulation resulted in a more robust recruitment of GLUT4 to the plasma membrane compared with endothelin-1 (Fig. 2A), consistent with the greater increase in glucose uptake observed in response to insulin compared with endothelin-1 treatment. Stimulation with endothelin-1 decreased immunoreactive GLUT4 in low-density microsomes in pair-fed rats to 88 ± 8% of basal (Fig. 2B). GLUT4 in high-density microsomes was not affected by endothelin-1 treatment (data not shown). We have previously reported (23) that chronic ethanol feeding decreases total GLUT4 expression in adipocytes by 30% compared with adipocytes from pair-fed rats. After chronic ethanol feeding, GLUT4 at the plasma membrane in nonstimulated cells (basal) was higher (271 ± 51 units of arbitrary density, n = 7) compared with adipocytes from pair-fed rats (194 ± 36, n = 9; Fig. 2A). Moreover, neither insulin nor endothelin-1 increased GLUT4 protein associated with the plasma membrane after ethanol feeding (Fig. 2, A and B). GLUT1 content in the plasma membrane did not change in response to treatment either with hormone or with chronic ethanol feeding (Fig. 2A).
|
Although the signal transduction cascade leading from activation of adipocytes with endothelin-1 to increased glucose transport is not completely understood, data from several groups indicate that endothelin-1-stimulated glucose transport in 3T3-L1 adipocytes is independent of PI 3-kinase (5, 16). Insulin rapidly increased the phosphorylation of Akt in adipocytes from pair-fed rats. However, endothelin-1 did not stimulate the phosphorylation of Akt (Fig. 3). After chronic ethanol feeding, insulin-stimulated Akt phosphorylation was normal (Fig. 3), consistent with our previous data showing that chronic ethanol feeding is not associated with impaired activation of PI 3-kinase or Akt by insulin (22). Taken together, these data suggest that endothelin-1-stimulated glucose uptake is independent of the PI 3-kinase/Akt-signaling pathway and, furthermore, that chronic ethanol feeding does not impair insulin-dependent activation of Akt.
|
The heterotrimeric G protein G11 is a critical mediator
of endothelin-1-stimulated glucose uptake
(12,
21). G
11 and
G
q have also been implicated as modulators of
insulin-stimulated glucose uptake
(13,
15). Therefore, we
investigated the effect of chronic ethanol feeding on expression of
G
q/11 proteins in rat adipocytes. Immunoreactive quantities
of G
s, G
11, and G
q were
measured by Western blot in plasma membrane enriched fractions prepared from
adipocytes isolated from pair- and ethanol-fed rats. Although the quantity of
G
s increased twofold after chronic ethanol feeding
(Fig. 4)
(22,
35), the quantity of
G
11 was decreased to 30% after chronic ethanol feeding
compared with pair-fed animals (Fig.
4). There was no effect of chronic ethanol feeding on
G
q expression (Fig.
4).
|
The downstream elements in the endothelin-1/Gq/11 pathway
leading to glucose uptake in adipocytes are not completely understood.
Tyrosine phosphorylation of G
q/11
(11) as well as that of
tyrosine kinase PYK2 and the scaffold protein paxillin
(21) is increased in response
to endothelin-1 stimulation in 3T3-L1 adipocytes. A dominant negative
construct of PYK2 [calcium-dependent protein kinase-related nonkinase (CRNK)]
inhibits endothelin-1-stimulated GLUT4 translocation in 3T3-L1 adipocytes,
demonstrating a functional role for this kinase in endothelin-1-stimulated
glucose uptake (21). We
hypothesized that, if the chronic ethanol-induced decrease in
G
11 contributes to impaired endothelin-1-stimulated glucose
uptake after chronic ethanol, then endothelin-1-stimulated tyrosine
phosphorylation of PYK2 should also be decreased after chronic ethanol
feeding. We first characterized the effects of insulin and endothelin-1 on
PYK2 tyrosine phosphorylation in adipocytes isolated from control rats.
Insulin rapidly increased the tyrosine phosphorylation of proteins migrating
at the apparent molecular weights of insulin receptor substrate-1 and the
insulin receptor
-subunit (Fig.
5). Endothelin-1 did not stimulate tyrosine phosphorylation of
these peptides (Fig. 5). Using
antibodies specific for Tyr402 and Tyr881 of PYK2, we
show that endothelin-1, but not insulin, increased tyrosine phosphorylation of
PYK2 (Fig. 5). Both insulin and
endothelin-1 stimulated p38 MAPK phosphorylation, but the response to
endothelin-1 was more robust than the response to insulin
(Fig. 5).
|
Adipocytes isolated from pair- and ethanol-fed rats were stimulated or not with 10 nM endothelin-1, and tyrosine phosphorylation of PYK2 was measured using phosphospecific antibodies. Tyr402 is an autophosphorylation site and a target for interaction with the src homology 2 (SH2) domain of Src family kinases. Activated Src, in turn, phosphorylates PYK2-Tyr881, allowing for the association of SH2 domains of adaptor proteins such as Grb2 (4). Phosphorylation of PYK2 at Tyr579 and Tyr 580 is required for maximal PYK2 activity (4). Endothelin-1 rapidly stimulated the tyrosine phosphorylation of all four tyrosine residues in adipocytes isolated from pair-fed rats (Fig. 6). In contrast, endothelin-1 did not increase the tyrosine phosphorylation of PYK2 at any of the four sites in adipocytes isolated from chronic ethanol-fed rats (Fig. 6). Total quantity of PYK2 was decreased to 64 ± 33% (n = 5) in adipocytes from ethanol-fed rats compared with pair-fed rats (Fig. 6).
|
Activation of endothelin-1 receptor or Gq/11 stimulates
p38 MAPK in a variety of cell types
(10,
37). Furthermore, p38 MAPK
activity has been implicated in mediating full activation of glucose transport
in response to insulin (2,
26,
27,
30). Here, we show that
pretreatment of rat adipocytes with 5 µM SB-203580, an inhibitor of p38
MAPK activation, suppressed endothelin-1-stimulated glucose uptake in
adipocytes from pair-fed rats (Fig.
7A). In this experiment, chronic ethanol feeding
decreased endothelin-1-stimulated glucose uptake compared with pair-fed rats
but was not further decreased by pretreatment with SB-203580
(Fig. 7A).
Endothelin-1 stimulated the phosphorylation of p38 MAPK in adipocytes isolated
from pair-fed rats over 515 min
(Fig. 7B). We
hypothesized that, if activation of p38 MAPK by endothelin-1 was suppressed
after chronic ethanol feeding similarly to the decrease in PYK2 activation,
this could also contribute to impaired endothelin-1-stimulated glucose
transport. However, chronic ethanol feeding had no effect on either
endothelin-1-stimulated phosphorylation of p38 or total p38 expression
(Fig. 7B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chronic ethanol feeding also decreases insulin-stimulated glucose uptake
(22,
35). However, impaired insulin
stimulation of glucose transport is not associated with impaired activation of
PI 3-kinase or Akt (22). This
suggests that chronic ethanol impairs a PI 3-kinase-independent signaling
pathway that contributes to insulin-stimulated glucose transport. One such
potential target for chronic ethanol action is the activation of the
heterotrimeric G protein Gq/11. Activation of
G
q/11 in response to insulin or endothelin-1 or by
overexpression of constitutively active G
11 (Q209L) or
G
q (Q209L) increases glucose uptake in 3T3-L1 adipocytes
(5,
12,
13,
15,
16). G
q/11
also contributes to both endothelin-1-
(12) and insulin-mediated
glucose transport (13,
15). Several reports have
found that the contribution of G
q/11 to glucose transport is
independent of activation of PI 3-kinase
(5,
15,
16,
36), although the involvement
of PI 3-kinase remains controversial
(12). We have found that
chronic ethanol exposure specifically decreases expression of
G
11 in isolated rat adipocytes. In contrast to the decrease
in G
11, chronic ethanol feeding increases expression of
G
s (35) and
has no effect on G
q expression. Chronic ethanol exposure
regulates expression of heterotrimeric G proteins; the individual G protein
family members affected by chronic ethanol are cell type specific
(7). Here, we have found that
ethanol feeding results in a specific decrease in expression of
G
11 in adipocytes. Decreased G
11 likely
contributes to impaired insulin- and endothelin-1-stimulated glucose transport
after chronic ethanol exposure.
The downstream elements in the Gq/11 pathway leading to
glucose uptake in adipocytes are not completely understood. The available data
suggest that insulin- or endothelin-1-stimulated G
q/11
activation may function via distinct signaling pathways. For example, the
tyrosine kinase PYK2 is essential for endothelin-1 stimulation of glucose
uptake in 3T3-L1 adipocytes
(21). A dominant negative
construct of PYK2 (CRNK) inhibits endothelin-1-but not insulin-stimulated
GLUT4 translocation in 3T3-L1 adipocytes
(21). In isolated rat
adipocytes, endothelin-1, but not insulin, increased tyrosine phosphorylation
of PYK2 (Fig. 5), consistent
with the response in 3T3-L1 adipocytes
(21). However, after chronic
ethanol feeding, endothelin-1 no longer increased tyrosine phosphorylation of
PYK2 in isolated rat adipocytes. These results suggest that the reduction in
G
11 expression after chronic ethanol impairs endothelin-1
activation of PYK2, a required intermediate in endothelin-1-stimulated glucose
uptake.
In contrast to impaired endothelin-1-stimulated PYK2 activation, chronic
ethanol feeding had no effect on endothelin-1-stimulated p38 MAPK
phosphorylation in isolated rat adipocytes. p38 MAPK is another downstream
kinase activated in response to endothelin-1 and insulin
(10,
26,
30). Activation of p38 MAPK
via G protein-coupled receptors involves activation of Gq/11
(19,
37). PYK2 is an intermediate
in endothelin-1-mediated activation of p38 MAPK in some, but not all, cell
types (28). For example, G
protein-coupled receptor activation of MAPK is similar in mouse embryonic
fibroblasts from wild-type and
pyk2-/- mice
(1). Because chronic ethanol
feeding did not impair endothelin-1-stimulated p38 MAPK activation despite a
suppression in PYK2 activation, PYK2 does not appear to be involved in p38
MAPK activation by endothelin-1 in rat adipocytes. p38 MAPK activity has been
implicated in mediating insulin-stimulated glucose transport activity in
3T3-L1 adipocytes, L6 myotubes
(26,
30), and skeletal muscle
(27). Here, we report that
inhibition of p38 MAPK activity by pretreatment with SB-203580 also suppresses
endothelin-1-stimulated glucose uptake
(Fig. 7A). Studies
utilizing chemical inhibitors of p38 MAPK suggest that p38 MAPK activation
leads to an increase in the catalytic/transport activity of GLUT4 rather than
GLUT4 translocation to the plasma membrane
(26,
30). After chronic ethanol
feeding, GLUT4 content at the plasma membrane was higher in nonstimulated
(basal) adipocytes compared with cells from pair-fed rats. Although
endothelin-1 increased GLUT4 at the plasma membrane in adipocytes from
pair-fed rats, endothelin-1 did not increase plasma membrane GLUT4 in
adipocytes from ethanol-fed rats above baseline. We have previously
demonstrated (22,
35) that chronic ethanol
feeding impairs the accessibility of GLUT4 at the plasma membrane. Thus,
despite the sustained ability of endothelin-1 to activate p38 MAPK, it is
unlikely that p38 MAPK could stimulate glucose transport because of a
decreased quantity and/or accessibility of GLUT4 at the cell surface after
chronic ethanol feeding. Instead, decreased activation of PYK2 after chronic
ethanol is more likely an important contributor to impaired
endothelin-1-stimulated GLUT4 translocation and glucose uptake. PYK2 is
required for the formation of cortical F-actin in response to endothelin-1, a
required step for GLUT4 translocation
(20,
31,
33). Thus it is possible that
impaired PYK2 activation after chronic ethanol may lead to abnormal formation
of cortical F-actin and impaired GLUT4 translocation to the plasma
membrane.
Chronic ethanol feeding impairs both insulin- and endothelin-1-stimulated
glucose transport in rat adipocytes
(22,
35). We have found that
decreased G11 expression in adipocytes after chronic ethanol
feeding may contribute, at least in part, to impaired glucose transport due to
a decreased activation of PYK2 tyrosine phosphorylation, a required
intermediate in endothelin-1-stimulated glucose uptake. However, the
mechanisms by which chronic ethanol feeding lead to decreased
G
11 expression and impaired transport are not clear. Further
experimentation is required to understand the in vivo factors involved in the
development of impaired insulin- and endothelin-1-stimulated glucose uptake in
adipocytes after chronic ethanol feeding.
![]() |
DISCLOSURE |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|