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Programme in Cell Biology (G.S., J.K., R.S., D.K., R.G., A.K.), The Hospital for Sick Children, Department of Biochemistry (R.S., A.K.) and Institute of Medical Science (D.K.), University of Toronto, Toronto, Ontario, M5G 1X8, Canada
Address all correspondence and requests for reprints to: Amira Klip, Program in Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada. E-mail: amira{at}sickkids.on.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Insulin regulates blood glucose levels principally by stimulating the uptake of glucose into muscle and fat cells and curbing hepatic glucose output (7, 8). Glucose transport occurs via a family of glucose transporter proteins (GLUTs), of which GLUT4 is the major insulin responsive isoform in muscle and fat tissues. Intracellular GLUT4 is translocated to the cell surface in response to insulin, by a mechanism requiring the activation of PI3K (9, 10). However, we have shown that a membrane permeable acetoxymethylester derivative of the PI3K product PI (3, 4, 5) P3 caused translocation of GLUT4 (Sweeney, G., R. Somwar, L. Foster, T. Jiang, G. Prestwich, P. Nielsen, and A. Klip, submitted work) without increasing glucose uptake (11). Moreover, activation of PI3K via stimuli other than insulin does not lead to elevations in glucose uptake (12). Collectively, these results suggest that PI3K activation is necessary but not sufficient to stimulate glucose uptake.
We have also shown that several inhibitors of p38 MAP kinase reduce insulin-stimulated glucose uptake without altering GLUT4 arrival at the cell surface (13, 14). Therefore, it is possible that insulin stimulates glucose uptake by increasing both the amount of glucose transporters at the cell surface and their activity. The above studies raise the hypothesis that the pathophysiology of insulin resistance may involve alterations in the ability of insulin to stimulate p38 MAP kinase and thus activate glucose transporters. Here we examine the ability of leptin to directly reduce insulin-stimulated glucose uptake and GLUT4 translocation in skeletal muscle cells and explore the molecular mechanisms underlying this effect.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Determination of 2-deoxyglucose uptake
L6 myotubes transfected with myc-tagged GLUT4 (L6-GLUT4myc) were
grown and differentiated as previously characterized (15).
Myotubes (12-well plates) were deprived of serum for 5 h with
-MEM and 25 mM glucose before experimental
manipulations. 2-Deoxyglucose uptake measurements were carried out for
5 min as described previously (16).
Measurement of GLUT4myc translocation in L6 myotubes
The content of myc-tagged GLUT4 at the cell surface was measured
by an antibody-coupled densitometric assay (15) as
follows: Quiescent L6 GLUT4myc myotubes, after treatment with insulin
and/or leptin, were washed once with PBS then fixed with 3%
paraformaldehyde in PBS for 3 min at room temperature. The fixative was
immediately neutralized by incubation with 1% (wt/vol) glycine in PBS
at 4 C for 10 min. The cell monolayer was then blocked with 10%
(wt/vol) goat serum and 3% (wt/vol) BSA in PBS at 4 C for at least 30
min. Primary antibody (anti-c-myc, 9E10) was then added into
the cultures at a dilution of 1:100 and maintained for 30 min at 4 C.
The cells were washed with PBS before introducing peroxidase-conjugated
rabbit antimouse IgG (1:1000). After 30 min at 4 C, the cells were
washed and 1 ml OPD reagent (0.4 mg/ml O-phenylenediamine
dihydrochloride and 0.4 mg/ml urea hydrogen peroxide in 0.05
M phosphate-citrate buffer) was added to each
well for 10 min at room temperature. The reaction was stopped by
addition of 0.25 ml of 3 N HCl. The supernatant
was collected and the optical absorbance was measured at 492 nm.
Detection of tyrosine phosphorylation of insulin receptor
ß-subunit, IRS-1, IRS-2, and PI3K activity associated with
antiphosphotyrosine
Myotubes were incubated in the presence or absence of leptin
(100 nM, 30 min) then treated with 100 nM
insulin for 5 min and immunoprecipitation using antiphosphotyrosine
antibody performed essentially as described previously
(16). Immunoprecipitated proteins were resolved by 7.5%
SDS-PAGE and then electrotransferred onto polyvinylidenedifluoride
membranes and immunoblotted with antiinsulin receptor ß-subunit
antibody (1:500) or anti-IRS-1 or -IRS-2 antibodies (1:500). Detection
was by the enhanced chemiluminescence method. To determine PI3K
activity, cell extracts were prepared as above for immunoprecipitation
with antiphosphotyrosine antibody (16), and PI3K activity
was measured as described previously (13).
Detection of Akt, p38 MAPK, ERK, and CREB phosphorylation using
phosphospecific antibodies
To detect phosphorylation of Akt, p38 MAPK, ERK, and CREB using
phosphospecific antibodies, cell lysates were prepared from myotubes
grown on 6-well plates using 300 µl 2x Laemmli sample buffer
containing 1 mM
Na3VO4, 100 nM
okadaic acid, protease inhibitors (1 mM benzamidine, 10
µM E-64, 1 µM leupeptin, 1 µM
pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride), and
7.5% ß-mercaptoethanol. Samples were vortexed for 1 min, passed 5
times through a 25-gauge syringe and heated for 15 min at 65 C. Lysates
were then centrifuged for 5 min at 1,000 rpm. Approximately 60 µg (30
µl) were resolved by 10% SDS-PAGE then immunoblotted with
phospho-antibodies (1:500 dilution). HRP-coupled goat antirabbit
secondary (1:7500) was used in each case and detected by the enhanced
chemiluminescence method.
Statistical analysis
Statistical analysis was performed using either paired
t test or ANOVA test (Fischer, multiple comparisons) where
appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The results shown confirm
that leptin receptor polypeptide is expressed in these cells. Based
upon observations on the apparent molecular weight of leptin receptor
isoforms in other tissues (23, 24) our results suggest the
presence of both long (
200 kDa) and short (100–120 kDa) leptin
receptor isoforms in these cells. Furthermore, since the antibody used
recognizes an epitope common to all isoforms and should therefore
recognize all isoforms equally, the results also suggest that the short
leptin receptor isoform(s) is the predominant form expressed in these
cells. This contrasts with the ratio of leptin receptor isoforms seen
in hypothalamus and macrophages (23, 25) and is suggestive
of potential direct effects of leptin in muscle tissue that may not be
seen in other tissues. Figure 1B shows that pretreatment of L6 myotubes
for 30 min with leptin (100 nM) reduced the stimulation of
2-deoxyglucose uptake by insulin. The maximum inhibition produced was
approximately 50–60% of the normal insulin-stimulated response. A
leptin preincubation time of 5 min was not sufficient for the
inhibitory action to develop. This observation suggests that
intracellular changes induced by leptin which result in inhibition of
insulin action require longer than 5 min to be established. Therefore,
in subsequent experiments examining the mechanism of leptin action a
preincubation time of 30 min was used. Figure 1C shows that the
stimulation of 2-deoxyglucose uptake was dependent on the concentration
of leptin in the preincubation period. A leptin concentration of 0.1
nM had no significant effect, while at 2 nM (32
ng/ml) and 100 nM (1600 ng/ml) significant inhibition of
40–60% of the insulin response was observed.
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, although leptin reduced
insulin-stimulated glucose uptake, it had no effect on the 2-fold
increase in cell surface GLUT4myc induced by insulin. This result
suggests that inhibition of GLUT4 translocation is not the mechanism
whereby leptin inhibits insulin-stimulated 2-deoxyglucose uptake.
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we examine the effect of leptin
on early insulin signaling. The earliest detectable event subsequent to
insulin binding is autophosphorylation of its receptor ß-subunit. The
increase in tyrosine phosphorylation of the insulin receptor after 5
min of insulin stimulation was not altered after 30 min preincubation
with leptin (Fig. 3A). Insulin-stimulated tyrosine phosphorylation of
two immediate insulin receptor substrates, IRS-1 and IRS-2, was also
unaffected by leptin (Fig. 3A). PI3K has been shown to play a
necessary, albeit insufficient role, in the stimulation of glucose
uptake (10, 11). We examined the activity of PI3K
associated with phosphotyrosine immunoprecipitates from control and
insulin-stimulated cells and found an increase of approximately 7-fold.
Neither this response to insulin nor basal PI3K activity were affected
by preincubation with leptin for 30 min (Fig. 3B). An important
downstream target of PI3K products contributing to GLUT4 translocation
is the serine/threonine kinase Akt/PKB (9). The mechanism
of activation of Akt involves phosphorylation of the kinase on two
residues, Thr308 and Ser473. Insulin caused a robust phosphorylation of
Akt on both Thr308 and Ser473 after 10 min that was not altered by
preincubation with leptin (Fig. 3C). These results are consistent with
the observation that similar conditions had no effect on the ability of
insulin to induce GLUT4myc translocation (Fig. 2
).
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shows that insulin caused a
significant increase in p38 MAP kinase phosphorylation. However, a
similar increase was not detected in lysates prepared from cells which
had been pretreated with leptin before stimulation with insulin. Leptin
alone had no effect on p38 MAP kinase phosphorylation. We also examined
the activity of p38 MAP kinase in muscle cells by measuring
phosphorylation of CREB, a known downstream effector of p38 MAP kinase.
Treatment of cells with insulin resulted in increased phosphorylation
of CREB, assessed using a phosphospecific antibody (Fig. 4). This
increase in response to insulin was no longer evident when cells were
pretreated with leptin, again reflecting an ability of leptin to
prevent insulin-stimulated p38 MAP kinase activity. Finally, to explore
any possible generalized effects of leptin we examined the effect of
insulin on another member of the MAP kinase family (ERK) under similar
conditions. As shown in Fig. 4, and in contrast to p38 MAPK, the
increased phosphorylation of ERK in response to insulin remained intact
when cells were pretreated with leptin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(27), FFA (28) and resistin (29)
influence the insulin sensitivity of muscle and possibly other tissues.
Here we focus on the role of leptin, the product of the obesity gene,
the plasma levels of which correlate positively with body mass index
(5). Physiological control of blood glucose levels is primarily regulated by increased glucose clearance in response to insulin (7). Skeletal muscle, and to a lesser extent fat tissue, are quantitatively the most important target tissues for insulin-stimulated glucose disposal (7). Given the established association between obesity and type 2 diabetes, an important question to address is whether leptin may have a direct effect to modulate basal or insulin-stimulated glucose uptake. The detection of leptin receptor mRNAs in skeletal muscle and adipocytes suggested such a possibility (17). Here we confirmed leptin receptor protein expression (long and short forms) in L6 myotubes by Western blot analysis. It has been reported previously that leptin has no effect on basal glucose uptake in human (30), rat (31), or mouse (31, 32) skeletal muscle nor in rat (30, 32, 33) and mouse (30) adipocytes. In accordance with these reports, we show here that leptin does not alter basal glucose uptake in L6 myotubes. However, long-term leptin treatment of rodents increased 2-deoxyglucose uptake in EDL and soleus muscles, albeit to a smaller extent than that seen with insulin (34).
Importantly, we show that acute preincubation of L6 myotubes with 2 nM or 100 nM leptin reduces the ability of insulin to increase glucose uptake by up to 60%. This direct inhibitory effect of leptin required preincubation of cells with leptin for 30 min. Consistent with our observations, Friedman and colleagues (17) have shown that 1 h preincubation with leptin, again in the nanomolar range, directly suppressed insulin-stimulated glucose uptake (by 50%) in rat skeletal muscle in vitro. Interestingly, that study also reported that leptin could not affect insulin action in muscle from leptin-resistant obese Koletsky rats (17). Similar results to ours have also been reported for cultured rat adipocytes where 16 h preincubation with 2 nM leptin reduced insulin-stimulated glucose uptake, whereas lower leptin concentrations reduced insulin sensitivity (35). The effect of leptin is reversible as the normal response to insulin returns after leptin removal. Leptin also has inhibitory effects on other insulin-stimulated responses in vitro, such as conversion of fatty acids to triglycerides (36). Therefore, our studies and others suggest that leptin has a potential capacity to reduce insulin action. The concentrations of hormones used in in vitro studies preclude direct correlation to physiologically relevant responses, yet it is tantalizing to propose that under certain circumstances there is potential for leptin to be an insulin resistance-inducing agent in obese individuals.
The physiological role of leptin in controlling insulin-stimulated glucose uptake and metabolism remains controversial as other studies have shown that leptin may also improve insulin sensitivity (4). Thus, there are paradoxical reports that sustained excess or lack of leptin can both cause insulin resistance (37, 38, 39, 40). In C2C12 muscle cells leptin (30 min, 100 ng/ml) caused a small increase in glucose uptake and movement of GLUT4 to the plasma membrane (19, 41). Increased glucose metabolism and insulin sensitivity were observed in transgenic skinny mice overexpressing leptin to levels similar to those observed in obese individuals (37). In addition, leptin infusion into rats for 48 h resulted in a 30% increase in insulin sensitivity as determined by whole body glucose utilization during hyperinsulinemic glucose clamps (38). Conversely, lipodystrophy and leptin deficiency cause hyperinsulinemia and insulin resistance in mice (40), and it has been proposed that leptin could act as an antidiabetic hormone in these animals (39). Furthermore, treatment of streptozotocin-diabetic rats with leptin for 12–14 d normalized blood glucose levels and improve insulin sensitivity (42). Clearly, the effect of leptin in vivo is complex, and it has been proposed that the effects of leptin on glucose metabolism in vivo are heavily dependent on a central nervous system-regulated (43, 44). Indeed, microinjection of leptin into the ventromedial hypothalamus increased glucose uptake in peripheral tissues in rats (45). Subcutaneous infusion of leptin (4 mg/kg/d) for 7 d caused tissue-specific effects with a stimulation of glucose uptake in rat skeletal muscle and brown adipose tissue and a decrease in white adipose tissue (34).
Experimental conditions, including route of administration and duration of leptin treatment, age of animals/humans and the use of ex vivo tissue or cell lines may partly explain the conflicting reports on the effect of leptin on basal and insulin-stimulated glucose uptake (46, 47). In particular, the time of leptin treatment before analysis of insulin action is likely to be of great importance. Indeed, one study that reports no effect of leptin on insulin-stimulated glucose transport added both hormones together with no preincubation period (32). We have shown in this study that a short (5 min) preincubation period with leptin is not sufficient to impinge upon insulin action. Thus, the conclusion of that study is in accordance with the findings shown here that 5 min preincubation with leptin has no significant effect on insulin-stimulated glucose uptake. A slightly longer preincubation (30 min) was needed to observe the reduction in insulin action. Therefore, the variable temporal parameters employed in published literature may explain the discrepancies regarding the effect of leptin on glucose transport. However, the acute inhibitory effect of high leptin levels on insulin action seen by us and others (17, 36) in muscle cells, in fat cells (35), and liver (48, 49) or liver cells (50) uncovers an intriguing action of leptin directly on peripheral tissues, which may have been overlooked by the whole body in vivo studies quoted above.
Signals engaged by leptin
Our results show that leptin has no direct effect on GLUT4myc
content at the surface of unstimulated L6 myotubes nor on the
insulin-stimulated increase in GLUT4myc translocation. In keeping with
this observation, insulin-stimulated signaling events thought to
participate in mediating GLUT4 translocation were also unaffected.
These included tyrosine phosphorylation of the insulin receptor, IRS-1
and IRS-2, PI3K activity and phosphorylation of Akt on Thr308 and
Ser473. Thus, leptin reduced insulin-stimulated glucose uptake without
any effect on incorporation of GLUT4 to the plasma membrane or in the
signals mediating this event.
Until recently, translocation of GLUT4 to the cell surface was thought to be sufficient for insulin-stimulated glucose uptake (51). However, several studies have now suggested that full stimulation of glucose uptake by insulin requires both translocation and activation of GLUT4 (11, 13, 14). We have shown that inhibitors of p38 MAP kinase reduce insulin-stimulated glucose uptake in muscle and fat cells without any effect on GLUT4 translocation (13, 14). Because the effects of leptin on L6 muscle cells resembled those found upon inhibition of p38 MAP kinase activation, we examined the possibility that leptin may prevent the increase in insulin-stimulated p38 MAP kinase and GLUT4 activity in response to insulin. Insulin-stimulated phosphorylation (activation) of p38 MAP kinase and of a known effector of p38 MAP kinase, CREB, were inhibited by leptin pretreatment. Importantly, whereas insulin-stimulated p38 MAP kinase phosphorylation was inhibited, leptin had no effect on the phosphorylation of another member of the MAP kinase family, ERK, in response to insulin.
The above results suggest that the reduction in insulin-stimulated glucose uptake by leptin may be caused by preventing p38 MAP kinase activation and subsequently GLUT4 activation in response to insulin. The precise mechanism whereby leptin can cross-talk with selective insulin-stimulated signaling events is unknown. Leptin can stimulate a diverse range of intracellular signaling pathways and recent studies have begun to demonstrate an ability of leptin to affect selective insulin signaling pathways. For example, in Fao cells insulin-stimulated IRS-1 tyrosine phosphorylation and associated PI3K activity were enhanced by leptin pretreatment, whereas signaling via IRS-2 was inhibited (24). Together with our results, these reports suggest that the intracellular cross-talk between leptin and insulin is cell type-specific and requires further investigation.
In summary, preincubation of cultured rat skeletal muscle cells with leptin can reduce the ability of insulin to increase glucose uptake. However, insulin-stimulated GLUT4 translocation, insulin receptor, IRS-1 and IRS-2 tyrosine phosphorylation, PI3-kianase activity and phosphorylation of Akt on Thr308 and Ser473 were unaffected by leptin. Instead, the molecular mechanism of reduced glucose uptake may involve decreased insulin-stimulated p38 MAP kinase activation and consequently reduced activation of GLUT4. These results suggest that insulin-stimulated GLUT4 activity can be regulated by another hormone, leptin. While other factors undoubtedly contribute to the development of insulin-resistance associated with obesity in vivo, this study highlights a potentially important direct effect of leptin to reduce insulin-stimulated glucose uptake in muscle cells.
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Acknowledgments |
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Footnotes |
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1 Current address: Department of Biology, York University, Toronto M3J
1P3, Canada. 
Abbreviations: CREB, cAMP response element binding protein; GLUT, glucose transporter; IRS-1 or -2, insulin receptor substrates.
Received April 5, 2001.
Accepted for publication July 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and insulin
resistance: summary and future prospects. Mol Cell Biochem 182:169–175[CrossRef][Medline]
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