TNF{alpha}-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3

Kazuya Kabayama2,3, Takashige Sato2, Futoshi Kitamura2, Satoshi Uemura2, Byoung Won Kang2, Yasuyuki Igarashi2 and Jin-ichi Inokuchi1,2,3

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


1 To whom correspondence should be addressed; e-mail: inokuchi{at}kinou02.pharm.hokudai.ac.jp

Received on May 26, 2004; revised on August 2, 2004; accepted on August 4, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low-density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. Tumor necrosis factor alpha (TNF{alpha}) induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. In other studies we have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNF{alpha}, suggesting a specific function for GM3. In the DRMs from TNF{alpha}-treated 3T3-L1 adipocytes, GM3 levels were doubled compared with results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, whereas caveolin and flotillin levels were unchanged. Furthermore, insulin-dependent IR internalization and intracellular movement of the IR substrate 1(IRS-1) were both greatly suppressed in the treated cells, leading to an uncoupling of IR–IRS-1 signaling. GM3 depletion was able to counteract the TNF{alpha}-induced inhibitions of IR internalization and accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.

Key words: detergent-resistant microdomain (DRM) / ganglioside GM3 / insulin receptor / insulin resistance / lipid rafts


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Insulin has multiple, tissue-specific effects on cells, which are elicited through both metabolic and mitogenic signaling. In culture, insulin can act as a growth factor for cells, sharing many mitogenic signaling pathways with other growth factors. However, the metabolic effects of insulin are unique and cannot be reproduced with other cellular stimuli.

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., 1999Go). Numerous studies have implicated tumor necrosis factor alpha (TNF{alpha}) as having a role in insulin resistance, both in cultured adipocyte and whole-animal models (Hotamisligil et al., 1993Go, 1995Go; Uysal et al., 1997Go). In adipocytes cultured in relatively low concentrations of TNF{alpha} (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, 1996Go). This protracted effect suggests that TNF{alpha} 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, 2000Go; Simons and Toomre, 2000Go). 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, 2002Go, and Cohen et al., 2003).

Gangliosides, a family of sialic acid–containing GSLs, are an important component of DRMs. In adipose tissues from various species, including human and mouse, GM3 is the most abundant ganglioside (Ohashi, 1979Go). Recently, we reported that in mouse 3T3-L1 adipocytes insulin resistance induced by TNF{alpha} was accompanied by increased GM3 expression. Indeed, we demonstrated that a chronic state of insulin resistance in adipocytes, induced by 100 pM TNF{alpha}, was accompanied by an up-regulation of GM3 synthesis at the transcriptional level. Moreover, the pharmacological depletion of GM3 prevented a TNF{alpha}-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{alpha} (Tagami et al., 2002Go). 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., 2002Go).

In the present study, we examine the effect of TNF{alpha} 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{alpha}.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
GM3 but not ceramide is increased in adipocytes in a chronic state of insulin resistance
We reported previously that the state of insulin resistance in 3T3-L1 adipocytes induced by TNF{alpha} is accompanied by increased GM3 ganglioside expression through up-regulation of GM3 synthase (SAT-I) gene expression (Tagami et al., 2002Go). TNF{alpha} activates several signaling cascades, including the stimulation of sphingomyelinase and consequent ceramide production (Hannun, 1994Go). Exogenous sphingomyelinase and cell-permeable (short-chain) ceramides have been shown to mimic some effects of TNF{alpha}, including decreases in the insulin-dependent tyrosine phosphorylation of IRS-1 (Kanety et al., 1996Go; Peraldi et al., 1996Go) and glucose uptake (Brindley et al., 1999Go). Therefore we examined endogenous ceramide production in TNF{alpha}-treated adipocytes. After TNF{alpha} treatment, ceramide levels doubled throughout the first 6 h of treatment, then returned to control levels by 24 h (Figure 1A). At 96 h, though, there was no increase, suggesting that ceramide has no role in insulin signaling during the chronic state of TNF{alpha}-induced insulin resistance. Using the same lipid extracts, we observed a prolonged increase in GM3 levels as reported previously (Tagami et al., 2002Go). The increase in GM3 appeared as early as 1 h, reaching a plateau at 48 h, and remained at a high level throughout the chronic TNF{alpha} treatment (96 h) (Figure 1B). These findings led us to investigate the pathophysiological role(s) of GM3 in adipocytes having a state of insulin resistance.



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Fig. 1. GM3, but not ceramide, increases in adipocytes in a chronic state of insulin resistance. After treatment with 100 pM TNF{alpha} for various times (0–96 h), 3T3-L1 adipocytes were scraped, and total lipids were extracted. (A) Lipid samples were assayed for ceramide content using a DGK assay system as described under Materials and methods. (B) Ganglioside fractions were prepared from the extracted lipids and separated by TLC. GM3 content was quantified as described under Materials and methods. Data shown are means ± SD (n = 3). *p < 0.05.

 
IR is a component of the DRMs and is selectively eliminated from the DRMs during a state of insulin resistance induced by TNF{alpha}
Studies of the presence of IRs in DRMs/caveolae have provided conflicting data (Gustavsson et al., 1999Go; Kimura et al., 2002Go; Mastick and Saltiel, 1997Go; Muller et al., 2001Go). We evaluated the localization of IRs in a flotation assay following extraction with increasing concentrations of Triton X-100 or under hypertonic alkaline conditions (500 mM sodium carbonate) (Figure 2). In the carbonate buffer, IRs from normal 3T3-L1 cells were found exclusively in the low-density, insoluble fractions 4 and 5, which are known to carry DRMs, whereas a small portion of the IRs in TNF{alpha}-treated cells shifted to fractions 6–8. On the other hand, there was no accumulation in the DRMs in cells, untreated or treated with TNF{alpha}, when examined using an extraction buffer containing 1% (data not shown) or 0.1% Triton X-100, concurring with a previous report using high levels of detergent (Gustavsson et al., 1999Go). However, when the flotation assay was performed using buffer with lower concentrations of Triton X-100 (0.08% or 0.05%), the DRMs were able to hold the IRs, although in the TNF{alpha}-treated cells the IRs tended to shift to higher-density fractions.



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Fig. 2. IR can localize to DRMs in normal adipocytes but tends to shift to high-density fractions on TNF{alpha} treatment. TNF{alpha}-treated (96 h) or untreated adipocytes were homogenized with 500 mM sodium carbonate nondetergent buffer, or lysed with buffer containing detergent (0.05%, 0.08%, or 0.1% Triton X-100). Samples were then subjected to a sucrose density gradient flotation assay as described under Materials and methods. The gradient fractions from low (1) to high (12) density were subjected to SDS–PAGE and immunoblotted with an anti-IRß antibody. This figure is a representative of three independent experiments.

 
Next, using membranes extracted with the 0.08% Triton X-100 buffer, we analyzed the distribution of GM3 and cholesterol and of proteins normally associated with DRMs (e.g., caveolin, flotillin, fyn), in each fraction of the sucrose density gradient (Figure 3). GM3 was preferentially localized at the DRMs in both normal and TNF{alpha}-treated 3T3-L1 adipocytes. Remarkably, though, the accumulation of GM3 observed in the DRMs was twofold higher in the TNF{alpha}-treated cells (Figure 3A). There were no distinct differences in the expression levels or distribution of cholesterol (Figure 3B), caveolin, flotillin, or fyn (Figure 3C). Taken together, these results clearly demonstrate the selective elimination of IRs from the DRMs and the accumulation of GM3 in adipocytes under a chronic state of TNF{alpha}-induced insulin resistance.



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Fig. 3. Accumulation of GM3 in the DRMs and selective dissociation of IRs from DRMs in a state of insulin resistance. Adipocytes, untreated (open bars) or treated with 100 pM TNF{alpha} for 96 h (solid bars), were lysed with buffer containing 0.08% Triton X-100 and subjected to a sucrose density gradient floatation assay. (A) GM3 levels in the sucrose gradient fractions were examined by high-performance TLC analysis. (B) Cholesterol levels in the sucrose gradient fractions were measured by a commercial assay. (C) The samples were subjected to SDS–PAGE and immunoblotted with antibodies against IRß, caveolin-1, flotillin, or fyn. Data in (A) and (B) are shown as means ± SD (n = 3). *p < 0.05. (C) is a representative of three independent experiments.

 
TNF{alpha} selectively attenuates insulin-dependent IRS-1 tyrosine phosphorylation without affecting MAP kinase activation
Insulin signaling can be classified into mitogenic and metabolic activities. Mitogenic signaling was found to be normal in cells with impaired insulin-dependent internalization (Maggi et al., 1998Go; Parpal et al., 2001Go). Additionally, the prolonged treatment (at least 72 h) of 3T3-L1 adipocytes with low concentrations of TNF{alpha} (<0.1 nM) reportedly resulted in a pronounced inhibition of tyrosine phosphorylation of IRS-1 with little effect on IR autophosphorylation (Guo and Donner, 1996Go). We obtained similar results (Tagami et al., 2002Go). Under these conditions, we examined insulin-dependent mitogen-activated protein (MAP) kinase activation in TNF{alpha}-treated 3T3-L1 cells but found no suppression (Figure 4B), despite the selective inhibition of the insulin-dependent tyrosine phosphorylation of IRS-1 in these cells (Figure 4A).



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Fig. 4. TNF{alpha} selectively attenuates insulin-dependent IRS-1 tyrosine phosphorylation without affecting MAP kinase activation. 3T3-L1 adipocytes were cultured for 96 h in maintenance medium without or with 100 pM TNF{alpha}, then incubated for 6 h in serum-free medium containing 0.5% BSA in the absence or presence of TNF{alpha}. Cells were then stimulated with 100 nM insulin for 5 min. (A) Proteins in cell lysates were immunoprecipitated with an antiserum to IR or IRS-1, subjected to SDS–PAGE, and transferred to PVDF membrane. Western blotting was then performed with anti-phosphotyrosine (pY), anti-IRß, or anti-IRS-1 antibodies. (B) For detection of insulin-dependent MAP kinase activation, the proteins in the cell lysates were subjected to SDS–PAGE and immunoblotted with anti-ERK and anti-phospho-ERK antibodies.

 
TNF{alpha} inhibits insulin-dependent IR internalization
The internalization of IR into endosomes and the subsequent intracellular movement of IRS-1 to the endosomes appear to be important steps in the metabolic signaling of insulin in adipocytes (Clark et al., 2000Go; Klein et al., 1987Go; Kublaoui et al., 1995Go; Parpal et al., 2001Go). Therefore, we examined the subcellular localization of IR, IRS-1, and GM3 in 3T3-L1 adipocytes, untreated or treated with insulin and/or TNF{alpha} (Figure 5). Cell fractions were obtained as illustrated in Figure 5A. Insulin-dependent IR internalization from P1 to P2 (Figure 5B, lanes 1 and 2) and insulin-dependent colocalization of IR (Figure 5B, lane 2) and IRS-1 (Figure 5C, lane 2) at P2 were evident. This movement allows the formation of an intracellular signaling complex that appears to be important for the metabolic signaling of insulin (glucose uptake). Under steady-state conditions, the major concentrations of IR and IRS-1 were localized at P1 and P3, respectively (Figure 5B, lane 1 versus Figure 5C, lane 1), but small amounts of IR and IRS-1 were colocalized in the P2 fraction, suggesting basal insulin signaling. However, TNF{alpha} treatment interfered with this colocalization (Figure 5B, lane 3 versus Figure 5C, lane 3). Furthermore, TNF{alpha} treatment resulted in the marked inhibition of insulin-dependent IR internalization (Figure 5B, lane 4) and translocation of IRS-1 from P3 to P2 and cytosol (Figure 5C, lane 4). Such suppression during TNF{alpha}-induced insulin resistance would essentially result in the inhibition of efficient intracellular signalosome formation.



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Fig. 5. TNF{alpha} inhibits insulin-dependent IR endocytosis. (A) Method of fractionation. 3T3-L1 adipocytes were cultured for 96 h in maintenance medium without or with TNF{alpha}, then incubated for 6 h in serum-free medium, containing 0.5% BSA, in the absence or presence of TNF{alpha}. Cells were stimulated with 100 nM insulin for 15 min. Homogenized cells were subjected to differential centrifugation to yield fractions enriched in plasma membranes (P1) and high-density microsomes (P2), then a high-speed pellet (P3) and a cytosol (S3) fraction. (B and C) Cell fractions (20 µg protein) were resolved by SDS–PAGE and immunoblotted with an anti-IRß (B) or anti-IRS-1 (C) antibody. (D) Relative amounts of GM3 in the fractions of TNF{alpha}-treated or untreated adipocytes were determined as described under Materials and methods. Data in (B) and (C) are shown as means ± SD (n = 3). *p < 0.05.

 
To examine the subcellular distribution of GM3, we also analyzed the GM3 levels in the P1, P2, and P3 fractions of the adipocytes treated with or without TNF{alpha}. We found that GM3 was distributed throughout every fraction. Additionally, TNF{alpha} treatment increased GM3 levels almost equally in each fraction (Figure 5D).

GSL depletion attenuates the TNF{alpha}-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{alpha}-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{alpha}-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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Fig. 6. GM3 depletion attenuates TNF{alpha}-induced inhibition of both insulin-stimulated IR internalization and elimination of IR from the DRMs. (A) 3T3-L1 adipocytes were cultured in maintenance medium with or without TNF{alpha} for 96 h in the presence or absence of 20 µM D-PDMP. Cells were further incubated for 8 h in serum-free medium containing 0.5% BSA in the absence or presence of TNF{alpha} and D-PDMP as descirbed, then stimulated with 100 nM insulin for 15 min. The cells were subjected to differential centrifugation as in Figure 2, and the fractions were assayed by SDS–PAGE and immunoblotting with an anti-IRß antibody. (B) 3T3-L1 cells were cultured in maintenance medium with or without TNF{alpha} for 96 h in the presence or absence of 20 µM D-PDMP. The cells were lysed with buffer containing 0.08% Triton X-100, and the lysates were subjected to a sucrose density gradient floatation assay as described under Materials and methods. The gradient fractions, from low (1) to high (12) density, were subjected to SDS–PAGE and immunoblotted with an anti-IRß antibody. Data shown are means ± SD (n = 3). *p < 0.05.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Caveolae are a subset of membrane microdomains particularly abundant in adipocytes (Fan et al., 1983Go). Critical dependence of the insulin metabolic signal transduction on caveolae/microdomains in adipocytes has been demonstrated. Disruption of microdomains by cholesterol extraction with ß-cyclodextrin resulted in progressive inhibition of tyrosine phosphorylation of IRS-1 and activation of glucose transport in response to insulin although autophosphorylation of IR and activation of MAP kinase were not impaired (Parpal et al., 2001Go). Similarities between these cell culture results and the findings in many cases of clinical insulin resistance (Virkamaki et al., 1999Go), thereby suggesting a potential role for microdomains in the pathogenesis of this disorder. Gangliosides are also known as structurally and functionally important components in microdomains, however, there has been no report on their role(s) in microdomains in adipocytes.

In a previous study of insulin resistance induced in adipocytes by TNF{alpha}, 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{alpha} (Tagami et al., 2002Go). These findings are further supported by the recent report that mice lacking SAT-1 exhibit enhanced insulin signaling (Yamashita et al., 2003Go). 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{alpha}-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., 1987Go). Immunogold electron (Gustavsson et al., 1999Go) and immunofluorescence microscopy (Kimura et al., 2002Go) further demonstrated that IRs are highly concentrated in caveolae. Additionally, Couet et al. (1997)Go demonstrated the presence of a caveolin binding motif ({phi}XXXX {phi}XX{phi}) 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., 1999Go). Indeed, mutations of the IRß subunit have been found in type 2 diabetes patients (Imamura et al., 1994Go, 1998Go; Iwanishi et al., 1993Go). Recently, Lisanti's laboratory reported that caveolin-1-null mice developed insulin resistance when placed on a high-fat diet (Cohen et al., 2003aGo). 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., 2003aGo). 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., 2003aGo,bGo). 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., 1995Go) 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, 1997Go). Gustavsson et al. (1999)Go 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., 2003Go). 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{alpha}, 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{alpha} 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., 2002Go). 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., 2002Go). 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., 2002Go). 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., 1995Go). 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, 1993Go). The time course for the accumulation of internalized IR kinase closely paralleled the time course of the IRS-1 phosphorylation (Kublaoui et al., 1995Go). 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., 1998Go). 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{alpha}. We found that in TNF{alpha}-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 IR–IRS-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{alpha} 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{alpha}-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, 2002Go; Puri et al., 2001Go). 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, 1987Go; Radin et al., 1993Go), 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., 1992Go). D-PDMP was able to counteract the TNF{alpha}-induced increase in GM3 content in adipocytes and to normalize the TNF{alpha}-induced defect in the tyrosine phosphorylation of IRS-1 in response to insulin stimulation, as reported previously (Tagami et al., 2002Go). Moreover, this inhibitor was able to counteract the TNF{alpha}-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{alpha}-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., 2000Go).


    Materials and methods
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 Abstract
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 Results
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 Materials and methods
 References
 
Cell line and culture conditions
Murine 3T3-L1 preadipocytes were cultured, maintained, and differentiated as described previously (Tagami et al., 2002Go). Recombinant human TNF{alpha} (Genzyme-Techne, Cambridge, MA) was dissolved in phosphate-buffered saline (PBS) containing 0.1% fatty acid–free, growth factor–depleted bovine serum albumin (BSA; Sigma-Aldrich, Japan) and then added directly to the cell culture media at a concentration of 100 pM. After culturing for 96 h, the media on the treated cells and untreated controls were replaced with serum-free Dulbecco's modified Eagle medium containing 0.5% BSA, with or without 100 pM TNF{alpha}, for 6 h. For insulin studies, the cells were stimulated with 100 nM insulin for 5 or 15 min as specified. Cellular GSLs were depleted using 20 µM D-PDMP, an inhibitor of glucosylceramide synthase, synthesized as described previously (Inokuchi and Radin, 1987Go).

Immunoprecipitation and immunoblotting
Cell extracts from adipocytes were prepared as described previously (Tagami et al., 2002Go). 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., 2002Go). 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., 1989Go) to obtain the acidic glycolipid fraction. GM3, the primary ganglioside in these cells (Reed et al., 1980Go), 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)Go 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 [{gamma}-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., 2000Go) 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 Tris–HCl, 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 5–35% 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.


    Acknowledgements
 
We thank Dr. Norman S. Radin (University of Michigan) for valuable comments. This study was supported in part by grants-in aid 15390019 (to J.I.) and 15040202 (Scientific Research on Priority Area to J.I.) from the Ministry of Education, Culture, Sports, Science and Technology. This work was also supported by the Mizutani Foundation for Glycoscience (to J.I.).


    Abbreviations
 
BSA, bovine serum albumin; DGK, diacylglycerol kinase; D-PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; DRM, detergent-resistant membrane microdomain; GSL, glycosphingolipid; IR, insulin receptor; IRS-1, insulin receptor substrate-1; MAP, mitogen-activated protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SAT-I, GM3 synthase; SDS, sodium dodecyl sulfate; TNF{alpha}, tumor necrosis factor alpha


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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