2 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Kita 21-Nishi 11, Kita-ku, Sapporo 001-0021, Japan; and 3 Core Research for Evaluational Science and Technology Program (CREST), Japan Science and Technology Corporation (JST), Graduate School of Pharmaceutical Sciences, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Kita 21-Nishi 11, Kita-ku, Sapporo 001-0021, Japan
Received on May 26, 2004; revised on August 2, 2004; accepted on August 4, 2004
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Abstract |
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Key words: detergent-resistant microdomain (DRM) / ganglioside GM3 / insulin receptor / insulin resistance / lipid rafts
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Introduction |
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Insulin resistance, defined as the decreased ability of cells or tissues to respond to physiological levels of insulin, is thought to be the primary defect in the pathophysiology of type 2 diabetes (Virkamaki et al., 1999). Numerous studies have implicated tumor necrosis factor alpha (TNF
) as having a role in insulin resistance, both in cultured adipocyte and whole-animal models (Hotamisligil et al., 1993
, 1995
; Uysal et al., 1997
). In adipocytes cultured in relatively low concentrations of TNF
(which does not cause a generalized suppression of gene expression), interference with insulin action occurs. This effect requires prolonged treatment (at least 72 h), unlike many acute responses to this cytokine (Guo and Donner, 1996
). This protracted effect suggests that TNF
induces the synthesis of an inhibitor that is the actual effector.
One clue as to the mechanism of this hormone's unique actions may lie in the compartmentalization of the signaling molecules themselves. Cellular membranes contain subdomains called detergent-resistant microdomains (DRMs), because they are detergent-insoluble and highly enriched in cholesterol and glycosphingolipids (GSLs), but lacking in phospholipids (Hakomori, 2000; Simons and Toomre, 2000
). Within the past decade, data have emerged from many laboratories implicating these lipid microdomains as critical for proper compartmentalization of insulin signaling in adipocytes (reviewed in Bickel, 2002
, and Cohen et al., 2003).
Gangliosides, a family of sialic acidcontaining GSLs, are an important component of DRMs. In adipose tissues from various species, including human and mouse, GM3 is the most abundant ganglioside (Ohashi, 1979). Recently, we reported that in mouse 3T3-L1 adipocytes insulin resistance induced by TNF
was accompanied by increased GM3 expression. Indeed, we demonstrated that a chronic state of insulin resistance in adipocytes, induced by 100 pM TNF
, was accompanied by an up-regulation of GM3 synthesis at the transcriptional level. Moreover, the pharmacological depletion of GM3 prevented a TNF
-induced defect in insulin-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), providing evidence that GM3 functions as an inhibitor of insulin metabolic signaling during chronic exposure to TNF
(Tagami et al., 2002
). We were able to extend these in vitro observations to living animals using obese Zucker fa/fa rats and ob/ob mice, in which the GM3 synthase mRNA levels in the white adipose tissues are significantly higher than in their lean controls (Tagami et al., 2002
).
In the present study, we examine the effect of TNF on the composition and function of DRMs in adipocytes and demonstrate that increased GM3 levels result in the elimination of insulin receptors (IRs) from the DRMs, whereas caveolin and flotillin remain in the DRMs. Thus we present a new pathological feature of insulin resistance in adipocytes induced by TNF
.
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Results |
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GSL depletion attenuates the TNF-induced inhibition of both insulin-stimulated IR internalization and elimination of IR from DRMs
To investigate whether the inhibition of insulin-dependent IR internalization and the elimination of IR from the microdomains in TNF-treated cells were due to increases in GM3, we employed an inhibitor of glucosylceramide synthase, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). After GM3 depletion by D-PDMP, the suppression of IR internalization was indeed partially recovered (Figure 6A). Additionally, the elimination of IRs from the DRMs was effectively blocked (Figure 6B). There was no obvious change in the accumulation of IR in the DRMs after insulin stimulation (data not shown). That D-PDMP treatment was able to counteract the TNF
-induced inhibition of both insulin-stimulated IR internalization and elimination of IRs from the DRMs indicates direct involvement of GM3 in the chronic state of insulin resistance in adipocytes.
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Discussion |
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In a previous study of insulin resistance induced in adipocytes by TNF, we presented evidence that the transformation to a resistant state may depend on increased ganglioside GM3 biosynthesis following up-regulated SAT-1 gene expression. Additionally, GM3 may function as an inhibitor of insulin signaling during chronic exposure to TNF
(Tagami et al., 2002
). These findings are further supported by the recent report that mice lacking SAT-1 exhibit enhanced insulin signaling (Yamashita et al., 2003
). Because GSLs, including GM3, are important components of DRMs/caveolae, we pursued the possibility that increased GM3 levels in DRMs confer insulin resistance on TNF
-treated 3T3-L1 adipocytes.
Evidence suggesting that caveolae and caveolins play a major role in insulin signaling initially came from experiments using rat adipocytes, in which gold-labeled insulin was endocytosed by a mechanism involving clathrin-independent, uncoated invaginations (Goldberg et al., 1987). Immunogold electron (Gustavsson et al., 1999
) and immunofluorescence microscopy (Kimura et al., 2002
) further demonstrated that IRs are highly concentrated in caveolae. Additionally, Couet et al. (1997)
demonstrated the presence of a caveolin binding motif (
XXXX
XX
) in the ß subunit of IRs that could bind to the scaffold domain of caveolin. Moreover, mutation of this motif resulted in the inhibition of insulin signaling (Nystrom et al., 1999
). Indeed, mutations of the IRß subunit have been found in type 2 diabetes patients (Imamura et al., 1994
, 1998
; Iwanishi et al., 1993
). Recently, Lisanti's laboratory reported that caveolin-1-null mice developed insulin resistance when placed on a high-fat diet (Cohen et al., 2003a
). Interestingly, insulin signaling, as measured by IR phosphorylation and its downstream targets, was selectively decreased in the adipocytes of these animals while signaling in both muscle and liver cells was normal (Cohen et al., 2003a
). This signaling defect was attributed to a 90% decrease in IR protein content in the adipocytes, with no changes in mRNA levels, indicating that caveolin-1 serves to stabilize the IR protein (Cohen et al., 2003a
,b
). These studies clearly indicate the critical importance of the interaction between caveolin and IR in executing successful insulin signaling in adipocytes.
Saltiel and colleagues (Mastick et al., 1995) found that insulin stimulation of 3T3-L1 adipocytes was associated with tyrosine phosphorylation of caveolin-1. However, because only trace levels of IR were recovered in the caveolae microdomains in assays with a buffer of 1% Triton X-100, they speculated on the presence of intermediate molecule(s) bridging IR and caveolin (Mastick and Saltiel, 1997
). Gustavsson et al. (1999)
also observed the dissociation of IRs from caveolin-containing DRMs after treatments of 0.3 and 0.1% Triton X-100. It has been reported that comparison of protein and lipid contents of DRMs prepared with a variety of detergents exhibited the considerable differences in their ability to selectively solubilize membrane proteins and to enrich sphingolipids and cholesterol over glycerophospholipids, and Triton was the most reliable detergent (Schuck et al., 2003
). Therefore we performed a flotation assay with a wide range of Triton X-100 concentrations to identify the protein of interest that might weakly associate with DRMs.
In an assay system containing less than 0.08% Triton X-100, we were able to show that in normal adipocyte IRs can localize to DRMs., However, in the presence of TNF, IR was selectively eliminated from the DRMs, whereas caveolin-1 remained (Figure 3C). Thus by employing low detergent concentrations we were able to demonstrate for the first time the presence of IR in DRMs. We currently believe that elimination of IR from the DRMs by TNF
treatment is due to an excessive accumulation of GM3 in these microdomains, especially because preventing GM3 biosynthesis using D-PDMP attenuated the elimination of IR from the DRMs (Figure 6B). Reportedly, the localization in the DRMs of several proteins (including receptor protein tyrosine kinases) can be affected by changes in the expression levels of GSLs. For example, overexpression of the ganglioside GM1 in Swiss 3T3 cells results in the dispersion of ß type platelet-derived growth factor receptor from the DRMs (Mitsuda et al., 2002
). Similarly, the genetically enhanced accumulation of endogenous GM3 in keratinocytes caused the dissociation of caveolin-1 from the DRMs, thereby changing the signaling of the epidermal growth factor receptor (Wang et al., 2002
). In HuH7 hepatoma cells, which lack caveolin, IRs associate with DRMs in response to insulin stimulation, but cross-linking of GM2 by its antibody results in a loss of this association (Vainio et al., 2002
). Such results support the likelihood that localization of IRs to the DRMs is affected by the presence of not only caveolin but also GSLs, especially gangliosides.
Studies in adipocytes have implicated the endosomal apparatus as the site of insulin-stimulated IRS-1 tyrosine phosphorylation by activated IR kinase and associated PI-3 kinase activation (Kublaoui et al., 1995). Likewise, in isolated adipocytes treated with insulin, tyrosine-phosphorylated IRS-1 levels and PI-3 kinase activity were 10-fold greater in microsomes than at the plasma membrane (Kelly and Ruderman, 1993
). The time course for the accumulation of internalized IR kinase closely paralleled the time course of the IRS-1 phosphorylation (Kublaoui et al., 1995
). Additionally, the C860S mutation in the extracellular domain of the ß subunit of IR was found to reduce insulin-stimulated IR internalization, as well as IRS-1 tyrosine phosphorylation, without changing the autophosphorylation of IR and MAP kinase activation (Maggi et al., 1998
). Collectively, all these data strongly suggest that the autophosphorylation of IR in response to insulin stimulation will not be affected by the IR localization in membranes such as caveolin-rich microdomains (raft) or nonraft membranes in adipocytes but the successful internalization of the IR through microdomains is necessary for tyrosine phosphorylation of IRS-1.
There have been no reports on IR internalization in a state of insulin resistance induced by TNF. We found that in TNF
-treated 3T3-L1 adipocytes the insulin-dependent IR internalization and the intracellular movement of IRS-1 were greatly suppressed, leading to an uncoupling of the IRIRS-1 signaling (Figure 5). Additionally, tyrosine phosphorylation of IRS-1 in response to insulin was selectively impaired without affecting the activation of IR and MAP kinase (Figure 4). The observed impairment of IR internalization by TNF
may be attributed to the elimination of IR from microdomains due to the excess accumulation of GM3 (Figures 3A and 3C). Although the localization of IRs to DRMs may be maintained by the association with caveolin-1 as mentioned, the excess accumulation of GM3 in the DRMs may weaken IR-caveolin interaction. Indeed, IR but not caveolin-1 was coimmunoprecipitated with anti-GM3 antibody (unpublished data). Further work is needed to elucidate the mechanisms for the interactions of the ganglioside GM3, IR, and caveolin in the microdomains.
The reason for the complete interruption of the IRS-1 movement in the TNF-treated adipocytes on insulin stimulation is worthy of further studies, including those into the role of intracellular GM3 (Figure 5D). Indeed, GSLs are also known to be internalized via a clathrin-independent mechanism (Marks and Pagano, 2002
; Puri et al., 2001
). We are in the process of expanding our studies regarding the functional and structural changes of microdomains in the state of insulin resistance and type 2 diabetes.
We employed an inhibitor of glucosylceramide synthase, D-PDMP (Inokuchi and Radin, 1987; Radin et al., 1993
), to deplete cellular GSLs derived from glucosylceramide. This inhibitor is able to reduce the ganglioside content with minimum effect on phospholipids, neutral lipids, and glycoproteins (Barbour et al., 1992
). D-PDMP was able to counteract the TNF
-induced increase in GM3 content in adipocytes and to normalize the TNF
-induced defect in the tyrosine phosphorylation of IRS-1 in response to insulin stimulation, as reported previously (Tagami et al., 2002
). Moreover, this inhibitor was able to counteract the TNF
-induced suppression of IR internalization (Figure 6A) and IR elimination from DRMs (Figure 6B) required for insulin metabolic signaling, inspiring a new therapeutic strategy we have termed microdomain ortho-signaling therapy. Thus we were encouraged to measure the effect of D-PDMP on the TNF
-induced defect of glucose uptake, but so far have not observed any recovery (data not shown). Nevertheless, the possible therapeutic implication for insulin resistance will be pursued more extensively. Toward this end, we have recently succeeded in developing a new potent inhibitor of glucosyl ceramide synthase (D-PDMP) and its analogs, which have no general cytotoxic effect (Jimbo et al., 2000
).
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Materials and methods |
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Immunoprecipitation and immunoblotting
Cell extracts from adipocytes were prepared as described previously (Tagami et al., 2002). IR and IRS-1 were immunopurified with specific antibodies preadsorbed to protein A/G-Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA) then submitted to sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) under reducing conditions (Tagami et al., 2002
). Anti-Fyn (FYN3) and anti-phospho-ERK (E-4) antibodies and peroxidase-conjugated anti-rabbit IgG and peroxidase-conjugated anti-mouse IgG were from the same supplier. Western blot analysis was performed using the ECL western blot kit (Amersham Biosciences, Buckinghamshire, UK) and the Lumi-Light Plus western blotting substrate (Roche, Mannheim, Germany). Anti-p44/42 MAP kinase (ERK) rabbit polyclonal antibody was purchased from Cell Signaling (Beverly, MA). Anti-IRS-1 rabbit antiserum was from Upstate Biotechnology (Lake Placid, NY). Anti-caveolin-1, anti-flotillin-1, and anti-phosphotyrosine monoclonal PY20 antibodies were all purchased from BD Transduction Laboratories (Lexington, KY). For use in protein determination assays, bicinchoninic acid reagent was obtained from Pierce Chemical (Rockford, IL).
Lipid and cholesterol analyses
Total lipids were extracted from cells with chloroform:methanol (1:1 and 1:2, v/v, successively), and purified as described elsewhere (Inokuchi et al., 1989) to obtain the acidic glycolipid fraction. GM3, the primary ganglioside in these cells (Reed et al., 1980
), was quantified with a dual-wavelength flying spot scanner (CS9000; Shimadzu, Kyoto, Japan) at a reflectance mode of 500 nm, with area integration. Standard GM3 was kindly provided by Snow Brand Foods (Saitama, Japan). Cholesterol levels were determined using the Cholesterol CII assay kit (Wako, Osaka, Japan). Ceramide levels in total lipid extracts of 3T3-L1 cells were measured using a modified version of the diacylglycerol kinase (DGK) assay of Preiss et al. (1986)
Briefly, 20 µl micellar lipids were added to 0.2 µl dithiothreitol (1 M), 2 µl Escherichia coli DGK (7.1 U/ml), 1 µl [
-32P]ATP (10 mCi/ml in tricine buffer, pH 6.7), 50 µl reaction buffer (100 mM imidazole, pH 6.6, 100 mM LiCl, 25 mM MgCl2, and 2 mM ethylene glycol bis(2-aminoethyl ether)-tetra acetic acid, pH 6.6), and 17.8 µl dilution buffer (100 mM imidazole, pH 6.6, with 1 mM diethylenetriamine penta-acetic acid). After incubating for 30 min at 37°C, lipids were extracted with 0.6 ml chloroform:methanol (1:1, v/v). After vortexing, 265 µl of 1 M KCl was added, and the phases were separated by centrifugation. An aliquot of the organic phase was dried, resuspended with 20 µl chloroform, and spotted on a high-performance thin-layer chromatography plate. Lipids were separated in chloroform:acetone:methanol:acetic acid:water (10:4:3:2:1). Radioactive bands were visualized with an imaging analyzer (BAS-2000, Fuji Film), and 32P-ceramide-1-phosphate was quantified.
Cell fractionation
IRs and IRS-1s were fractionated from 3T3-L1 adipocytes as described previously (Clark et al., 2000) with slight modifications as illustrated in Figure 2. All procedures were performed at 4°C. Briefly, cell homogenates were centrifuged at 700 x g for 10 min to remove nuclei and large cellular debris. The supernatant was centrifuged at 13,000 x g for 20 min to pellet the plasma membrane and mitochondria. This supernatant was subjected to further centrifugation at 30,000 x g for 30 min to pellet the high-density microsomal fraction. The resultant supernatant was subjected to a final centrifugation at 175,000 x g for 75 min to obtain the high-speed pellet, and the supernatant from this centrifugation was designated the cytosol fraction. The high-speed pellet was solubilized in 1% SDS in PBS.
Sucrose gradient centrifugation
All steps were carried out at 4°C. Differentiated 3T3-L1 adipocytes were washed with PBS and lysed in 2 ml TNE buffer (10 mM TrisHCl, pH 7.5, 150 mM NaCl, 5 mM ethylenediamine tetra-acetic acid), containing protease inhibitors and 2 mM Na3VO4 and various concentrations of Triton X-100. Lysates were centrifuged for 5 min at 1300 x g to remove nuclei and large cellular debris, and the supernatants were diluted with equal volumes of 85% (w/v) sucrose in TNE buffer. In an ultracentrifuge tube the diluted lysates were overlaid with 4 ml 30% sucrose (w/v) in TNE buffer, then with 4 ml 5% sucrose (w/v) in TNE buffer. The samples were centrifuged at 39,000 rpm for 18 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA), and 1-ml fractions were collected from the top for immunoblotting and lipid analysis.
For the nondetergent sucrose density gradient, 3T3-L1 adipocytes were washed with PBS and rapidly scraped into 2 ml 500 mM sodium carbonate buffer (pH 11), then homogenized using a Polytron tissue grinder (three 10-s bursts; Kinematica GmbH, Brinkmann Instruments, Westbury, NY). The homogenates were then sonicated three times for 20 s. All homogenates were centrifuged for 5 min at 1300 x g to remove nuclei and large cellular debris. Each homogenate was then adjusted to 42.5% sucrose by the addition of 2 ml 85% sucrose prepared in MBS (25 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5, with 150 mM NaCl). The solution (4 ml) was placed at the bottom of an ultracentrifuge tube. Above this, a 535% discontinuous sucrose gradient was formed (4 ml 5% sucrose/4 ml 35% sucrose, both in MBS containing 250 mM sodium carbonate). The tube was centrifuged at 39,000 rpm for 18 h in an SW41 rotor and the total solutions was fractionated as already described.
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Acknowledgements |
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Abbreviations |
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References |
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