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Department of Pathology (M.V.E-F., J.W., A.W., R.W.H.), University of Alabama at Birmingham, and Birmingham VAMC (M.V.E.-F., A.W.), Birmingham, Alabama 35294-0007
Address all correspondence and requests for reprints to: Robert W. Hardy, Ph.D., Department of Pathology, University of Alabama at Birmingham, LHRB Room 506, Birmingham, Alabama 35294. E-mail: hardy{at}lh.path.uab.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Explanted rat adipocytes and 3T3-L1 adipocytes are established in vitro models for studying the signaling mechanism required for insulin-inducible glucose uptake and storage. We have previously demonstrated that exposure of rat adipocytes to certain fatty acids, specifically long-chain saturated fatty acids (LCSFA), resulted in a biphasic response over time (14, 15). Initially, palmitate treatment of rat adipocytes leads to an increase in GLUT4-mediated glucose transport, which is followed by insulin insensitivity for glucose transport following a 4-h exposure to palmitate. Furthermore, we have previously been able to demonstrate a separation of signaling pathways for GLUT4-mediated glucose transport from those mediating glycogen and lipid synthesis by expressing various signaling-restricted epidermal growth factor (EGF) receptors in 3T3-L1 adipocytes (16). Our goals, by extending our studies in 3T3-L1 adipocytes and rat adipocytes, are to determine the following: 1) whether fatty acid induced insulin resistance affects only specific actions of insulin; 2) whether EGF receptor-mediated glucose transport and storage are resistant to LCSFA treatment; and 3) how insulin-induced GLUT4-mediated glucose transport is specifically impaired by LCSFA treatment. Herein, we present results that both rat adipocytes and 3T3-L1 adipocytes are most sensitive to palmitate-induced insulin resistance. Furthermore, palmitate-induced insulin resistance in adipocytes is limited only to insulin- and EGF-stimulated glucose transport and does not affect the ability of either insulin or EGF to stimulate glycogen synthesis. Finally, we present evidence that the early mechanism by which elevated FFA levels inhibit glucose transport is by impairment of GLUT4 activation and not GLUT4 translocation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3T3-L1 Cell culture and differentiation
A subclone of 3T3-L1 fibroblasts (ATCC) that does not present
detectable EGFR after conversion to adipocytes (18) was maintained in
DMEM (25 mmol/liter glucose) supplemented with 7.5% BSA and
antibiotics (penicillin and streptomycin, 200 kU/liter each) in 5%
CO2, 90% humidity, at 37 C. The cells were differentiated
into adipocytes by standard procedures (19). The cells used for
experimentation were >80% differentiated (as determined visually).
Generation of 3T3-L1 adipocytes stably expressing the full length human
epidermal growth factor receptor (EGFR) was described previously
(18).
Addition of LCSFA to buffers and media
Fatty acids were added to aqueous solutions using albumin as a
physiologic carrier to introduce hydrophobic fatty acids to cells (20).
Fatty acid-free (FAF) BSA was prepared by the acid charcoal method of
Chen (21) and specified fatty acids were added back to the FAF BSA via
celite by the method of Spector and Hoak (22). Fatty acid
concentrations were measured using an enzymatic colorimetric kit (Wako
Pure Chemical Industries, Osaka, Japan).
Fatty-acid induced insulin resistance
Adipocytes were made insulin resistant by pretreatment with
fatty acids as previously described (15). Briefly, cells were incubated
with in Krebs-Ringer phosphate buffer (KRP) (pH 7.6) containing 10
nM glucose, 1 mM fatty acid, and 1% BSA at 37
C. Control cells were treated identically as treatments except FAF BSA
was added to the KRP buffer instead of fatty-acid loaded BSA. The
duration of incubation was either 4 h for rat adipocytes or
overnight for 3T3-L1 adipocytes. The incubation buffer was removed and
cells were resuspended for another hour at 37 C, with KRP (pH 7.0)
containing 1 mM pyruvate and 1% FAF BSA. Cells were then
washed two times with KRP (pH 7.6) containing 1% FAF BSA and
resuspended in appropriate buffers for experimentation.
2-Deoxyglucose transport
2-Deoxyglucose transport in rat adipocytes were performed as
previously described (15). Briefly, control and fatty-acid treated
cells were incubated for 15 min at 37 C with or without 1
nM insulin, after which 3H-2-deoxyglucose was
added for 3 min. Cells were separated by centrifugation through dinonyl
pthalate oil and counted by liquid scintillation. Nonspecific glucose
transport was determined in the presence of 50 µM
cytochalasin B.
2-Deoxyglucose transport in 3T3-L1 adipocytes were performed as previously described (18). Briefly, control and fatty-acid treated cells were washed once with KRP buffer and incubated with hormone (100 nM insulin or 25 nM EGF) for 20 min at 37 C. 2-Deoxy-D-[1-3H]glucose was added and the incubation was continued for 10 min. Assays were terminated with two rapid washes of iced PBS buffer. Cells were solubilized with NaOH and label detected by scintillation counting.
Rat adipocyte plasma membranes isolation
Plasma membrane enriched fractions were prepared by differential
centrifugation and separation on a Ficoll concentration gradient
according to the method of McKeel and Jarett (23). Briefly cells were
homogenized, centrifuged at 20,000 x g for 15 min at 4
C. The pellet was resuspended, centrifuged at 1000 x g
for 10 min at 4 C, and the supernatant was then centrifuged at
21,000 x g for 20 min at 4 C. The pellet was
resuspended and centrifuged at 58,000 x g for 30 min
at 4 C on a Ficoll gradient. The plasma membrane containing band was
removed and pelleted by centrifugation at 21,000 x g
for 30 min at 4 C. The pellet was resuspended and frozen at -70 C for
immunoblotting and glucose transport assays. The purity of plasma
membrane fractions was verified by measuring 5' nucleotidase as
previously described (24). Our results indicate that the low density
microsomal fractions contain only low levels of 5' nucleotidase (10%,
6%, 6%, and 8% of the corresponding plasma membrane fractions in
control untreated, control plus insulin, palmitate and palmitate plus
insulin, respectively).
Plasma membrane associated glucose transport
D-Glucose transport was measured in plasma membranes
according to the filter assay method of Weber et al. (25).
Briefly, filters (Type GS, 0.22 µM, Millipore, Bedford,
MA) were soaked in ice cold stopping solution (0.1 mM
phloretin, 0.1 mM D- and L-glucose
in TES (0.25 M sucrose, 10 mM Tris-HCl, pH 7.6,
1 mM EDTA) for 30 min. Plasma membranes were sonicated for
10 sec and 30–50 µg of plasma membranes were added per sample in
30–50 µl TES buffer and equilibrated with an equal volume of 0.2
mM D- and L-glucose in TES at room
temperature for 30 min. The membranes are then pulsed with an equal
volume of pulsing solution ([3H]-L-glucose
and [14C]-D-glucose, 1.0 µCi per sample of
each sugar, 0.1 mM final concentration for each). Initial
rates of glucose transport were measured by stopping transport after 3
sec with ice-cold stopping solution. The stopped sample was placed on
ice and filtered within 3 min. The filter was immediately washed three
times with ice-cold stopping solution. The filters were dried, treated
with 1 M NaOH, and counted by liquid scintillation.
Nonspecific glucose transport is determined by the
[3H]-L-glucose counts.
Immunodetection of GLUT4 translocation and insulin receptor
autophosphorylation
Plasma membrane enriched fractions were separated by SDS-PAGE
and transferred onto Immobilon-P membrane (Millipore). For detection of
GLUT4, the membrane was probed with rabbit polyclonal antibodies
against GLUT4 (East Acres Biologicals, Southbridge, MA) and visualized
with antirabbit antibody conjugated to alkaline phosphatase (BioRad,
Richmond, CA). For determining insulin receptor autophosphorylation in
rat adipocytes, the membrane was probed with mouse monoclonal
antiphosphotyrosine antibodies (UBI, Lake Placid, NY) and visualized
with antirabbit antibody conjugated to alkaline phosphatase. The
insulin receptor was identified on the immunoblots on the basis of its
molecular weight. To determine insulin receptor autophosphorylation in
3T3-L1 adipocytes, the insulin receptor was immunoprecipitated using
mouse monoclonal antiinsulin receptor antibodies (Oncogene Research
Products, Uniondale, NY) as described previously (18) before SDS-PAGE
and subsequent immunoblotting, as above.
Glycogen synthesis assay
Incorporation of glucose into glycogen was measured essentially
as described previously (26). After cells were serum starved in DMEM
supplemented with 0.2% BSA (2 h), the medium was replaced with Buffer
A (25 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1.7
mM KCl, 1 mM CaCl2, 1.47 mMK2HPO4,
0.8 mM MgSO4, 0.2% BSA). Insulin (100
nM) or EGF (25 nM) was added and the incubation
continued for 1 h at 37 C.
[U14C]D-glucose was added, and the incubation
was continued for 2 h. Assays were terminated by washing with
ice-cold PBS and immediate addition of 0.5 M NaOH to lyse
the cells. Lysates were spotted onto filter paper and were washed three
times with ice-cold 67% ethanol and once with acetone. The remaining
label on the filter paper was detected by scintillation counting.
Trypsin digestion of cell surface GLUT4
Adipocytes were trypsinized according to the method of Czech and
Buxton (27). Briefly, control and palmitate-treated cells were treated
with or without 10 nM insulin for 15 min at 37 C, then 2
mM potassium cyanide for 20 min TPCK-treated trypsin (Sigma
Chemical Co., St. Louis, MO) was added (1 mg/ml final concentration)
for 30 min at 37 C. At the end of the digestion period, soybean trypsin
inhibitor was added (2 mg/ml final concentration) and the cells were
quickly washed three times with KRP containing 20 mM HEPES
(pH 7.4), 1 mM pyruvate, 1 mg/ml trypsin inhibitor, and 2%
albumin before homogenization and membrane preparation.
Statistical analysis
Results are expressed as means ± SEM.
Comparisons between groups were assessed using a paired, Student’s
t test. P < 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In 3T3-L1 adipocytes
palmitate, myristate, and stearate each inhibited insulin-induced
glucose transport by 60%, 43%, and 46%, respectively (Fig. 1B). This
inhibition by palmitate was noted for insulin concentrations down to 1
nM, below concentrations at which insulin interacts with
the IGF-1 receptor. The increased length of incubation from 4 h
for rat adipocytes to overnight (
16 h) for 3T3-L1 adipocytes likely
accounts for the differences between the effects of LCSFA observed for
rat adipocytes vs. 3T3-L1 adipocytes.
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), a concentration in the range noted
in diabetic and obese individuals (15). The inhibitory effect of
prolonged palmitate exposure on insulin-induced glucose transport was
detectable after 4 h for both rat adipocytes and 3T3-L1 adipocytes
(Fig. 2, A and B). The differences in
insulin stimulation of glucose transport in rat adipocytes under fatty
acid free (control) and palmitate treatment conditions over time were
determined to be statistically significant by using pairwise analysis
(P < 0.05). The time of onset of insulin resistance
was comparable to those found in whole-body lipid infusion studies (12, 13).
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). Palmitate also did not impair insulin
stimulation of glycogen synthase in rat adipocytes (data not shown).
This suggests that the initial direct effects of palmitate-induced
insulin resistance is restricted to insulin stimulation of glucose
transport and not induction of glucose storage.
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). Furthermore,
palmitate treatment appeared to be specific for EGF-induced glucose
transport, similarly to insulin-induced responses, as EGF-induced
glycogen synthesis was only marginally affected with palmitate
treatment (11.11 ± 2.12% inhibition relative to FAF treatment,
n = 2 in quadruplicate) compared with glucose transport. As our
previous findings had demonstrated a divergence of the signaling
pathways for glucose transport from glycogen and lipid synthesis at the
level of the EGF receptor (18), these findings would suggest that
palmitate negatively affects events further down the signaling
pathway.
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, top panel). This has been confirmed by specific
immunoprecipitation of insulin receptors from palmitate-treated 3T3-L1
adipocytes (Fig. 5, bottom panel) and rat adipocytes (data
not shown). Densitometric analyses of the immunoblots of insulin
receptors indicated that insulin stimulated 4.6 ± 1.8-fold and
2.7 ± 1.3-fold increases in insulin receptor tyrosyl
autophosphorylation for palmitate and control treated rat adipocytes,
respectively (n = 3), and 8.1 ± 2.4-fold and 7.3 ±
0.6-fold increases for palmitate and control treated 3T3-L1 adipocytes,
respectively (n = 2).
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).
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). Densitometric
analyses of these immunoblots demonstrated no significant difference
between control and palmitate treated cells with respect to insulin
stimulated GLUT4 translocation to the plasma membrane [9 ±
1.8-fold and a 8.2 ± 1.8-fold, for control and palmitate-treated
respectively (n = 3)]. However, the amount of GLUT4, as
determined by densitometry of plasma membrane-GLUT4 immunoblots, under
basal and insulin-stimulating conditions were actually greater in
palmitate-treated vs. untreated controls [1.8 ±
0.4-fold and 1.4 ± 0.1-fold increases for basal and
insulin-stimulated cells, respectively (n = 3)], further
supporting the contention that LCSFA treatment did not impair GLUT4
translocation.
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). This was confirmed with densitometric analysis
of the immunoblots demonstrating a 26 ± 2 percent increase in
GLUT4 accessible to trypsin cleavage in palmitate-treated cells (n
= 3). Therefore, palmitate treatment actually increases the amount of
GLUT4 that is accessible to the extracellular space.
Finally, insulin may increase the intrinsic transport rate of GLUT4 at
the cell surface, which is manifested in an increased susceptibility of
GLUT4 to photoaffinity labeling (31, 32). The intrinsic glucose
transport activities of the plasma membranes of these cells were
measured to determine whether palmitate was affecting the activation of
GLUT4 by insulin stimulation (Fig. 8).
The intrinsic glucose transport activity of plasma membranes were lower
for palmitate-treated cells than for control cells despite a slight
increase in the levels of GLUT4 translocated to the plasma membrane
with insulin stimulation of palmitate-treated cells (Fig. 7A). These
results suggest palmitate inhibits activation of GLUT4 by insulin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) at physiologic concentrations (Fig. 1C). This effect of LCSFA
on insulin signaling is most pronounced with the fatty acid, palmitate
(Figs. 1A and B), and occurs after 4 h of exposure, which is
comparable to in vivo lipid infusion studies (Fig. 2A and B)
(12, 13).
Using 3T3-L1 adipocytes, we also demonstrate that the effects of fatty
acids on adipocytes are specific for glucose transport and do not
directly affect insulin stimulation of either glycogen synthesis (Fig. 3). This further supports previous in vitro findings, which
demonstrate that glucose storage is not dependent on hormone-induced
glucose transport in adipocytes (16, 35). Previously, we have
demonstrated that overexpression of EGF receptors in 3T3-L1 adipocytes
confer an EGF-dependent capability to transport glucose in a
GLUT4-dependent manner (18). Stimulation of glucose transport by EGF in
these adipocytes failed to stimulate a corresponding increase in IRS-1
phosphorylation, suggesting that the mechanism of EGF receptor-mediated
stimulation of glucose transport may be different than that of the
insulin receptor. However, palmitate treatment of 3T3-L1 adipocytes
that overexpressed the EGF receptor also inhibited EGF-induced glucose
transport (Fig. 4). Furthermore, the inhibitory effect of palmitate was
specific for EGF-induced glucose transport as the glycogen synthesis
pathway was not impaired.
The ability of glycogen synthesis to be independent of fatty-acid
impaired glucose transport in 3T3-L1 adipocytes suggests that glucose
transport is not rate limiting for glycogen synthesis in
vitro, and that the impairment of glycogen synthesis and glucose
oxidation in vivo, by elevated FFA is via the generation of
a distinct extracellular signal such as TNF-
(36, 37, 38). This would
argue for a further level of complexity in the body’s response to high
levels of FFA, one being direct negative modulation of glucose
transport, the other being an indirect inhibition of glycogen
synthesis, which leads to further deterioration of the insulin
response.
We have previously demonstrated that initial exposure to palmitate,
like insulin, stimulates basal glucose transport in rat adipocytes by a
mechanism that involves both the activation of the insulin receptor
kinase and translocation of GLUT4 to the plasma membrane (14). This
initial effect of palmitate exposure over long term might lead to
decreased insulin sensitivity as a consequence of glucose toxicity
through the intracellular accumulation of glucosamine pathway
metabolites (reviewed in 28 . However, we believe this mechanism
of fatty acid-induced insulin resistance is unlikely as inhibition of
glutamine:fructose-6-phosphate amidotransferase, the rate-limiting
enzyme action in glucosamine induced insulin resistance, by azaserine
had no effect on the ability of long-term palmitate treatment to induce
insulin resistance (Fig. 6).
A major question is, how do LCSFA impair glucose uptake? The failure of
the EGF receptor signaling pathway to bypass fatty acid induced
resistance (Fig. 4) suggests that the site of action for the fatty
acids is at a common component in the stimulation of glucose transport
by insulin and EGF. We have previously demonstrated that GLUT4 is a
common endpoint for both EGF and insulin-induced glucose transport
(18). Insulin stimulation of glucose transport requires several steps:
translocation of GLUT4-containing vesicles to the plasma membrane;
fusion of vesicle membranes to the plasma membrane to present GLUT4 to
the exterior of the cell; and activation of GLUT4 (reviewed in 30 . Palmitate did not impair insulin-stimulated translocation of GLUT4
to the plasma membrane (Fig. 7A). Furthermore, it did not prevent the
exposure of GLUT4 on the cell exterior and actually led to a slight
increase in insulin-resistant adipocytes (Fig. 7B). However, palmitate
did impair the ability of GLUT4 containing plasma membrane fractions to
transport glucose (Fig. 8). This finding is not surprising as studies
have suggested a separate activation step that modulates the intrinsic
transporter activity of GLUT4 (31, 32). This finding is consistent with
a recent in vivo study that demonstrated decreased
photoaffinity labeling (ATB-BMPA) of cell surface GLUT4 from skeletal
muscle of high fat fed mice (39), as this reagent labels those cell
surface glucose transporters present in a catalytically active state
rather than total cell surface glucose transporters (32). Thus,
palmitate exposure likely leads to an initial, direct impairment of
glucose transport in adipocytes by inhibiting activation of intrinsic
GLUT4 transporter activity at the plasma membrane.
By studying the in vitro effects of long-term exposure to fatty acids on adipocytes, we have begun to dissect the intracellular mechanism through which elevated fatty acids may lead to insulin resistance in vivo. The effect of LCSFA, particularly palmitate, is specific for the process of glucose transport and does not reduce the insulin-sensitivity of glycogen synthesis. Furthermore, the ability of glycogen synthesis to be independent of fatty acid-impaired glucose transport in 3T3-L1 adipocytes is suggestive of a separate mechanism by which elevated FFA may lead to impairment of glycogen synthesis in vivo. Herein we have provided mechanistic insight for the initial, FFA-dependent aspect of adipocyte insulin resistance, in that LCSFA appears to alter glucose uptake by directly impairing hormone-stimulated uptake by inhibiting activation of GLUT4. This suggests that FFA levels are a potential therapeutic target for ameliorating insulin resistance.
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Footnotes |
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Received February 20, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in human obesity and insulin resistance. J
Clin Invest 95:2409–2415
on insulin action in cultured rat skeletal muscle cells.
Endocrinology 137:2441–2446[Abstract]
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