GLUT4 expression in 3T3-L1 adipocytes is repressed by proteasome inhibition, but not by inhibition of calpains

Molecular and Cellular Endocrinology 232 (2005) 37–45

GLUT4 expression in 3T3-L1 adipocytes is repressed by proteasome inhibition, but not by inhibition of calpains David W. Cooke a, ∗ , Yashomati M. Patel b a

Department of Pediatrics and the Ilyssa Center for Molecular and Cellular Endocrinology, Johns Hopkins University School of Medicine, Park 211, 600 North Wolfe Street, Baltimore, MD 21287, USA b Department of Biology, University of North Carolina at Greensboro, Greensboro, NC 27402, USA Received 2 November 2004; accepted 8 December 2004

Abstract Because of recent studies showing linkage of type 2 diabetes with the calpain 10 gene, we investigated the ability of calpains to regulate GLUT4 expression in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the calpain inhibitor ALLN significantly decreased the mRNA and protein expression of GLUT4. GLUT4 expression was not affected by treatment with the more selective calpain inhibitors PD150606, calpeptin, or a calpastatin peptide. In contrast, treatment with the proteasome inhibitors lactacystin or MG132 repressed GLUT4 mRNA level to 35% (10 ␮M lactacystin) and 12% (10 ␮M MG132) of control levels. Therefore, the expression of GLUT4 in 3T3-L1 adipocytes was repressed by proteasome inhibition, but not by inhibition of calpains; the effect of ALLN was due to its ability to inhibit proteasome function, rather than its action to inhibit calpains. Concomitant with the repression of GLUT4 mRNA levels, proteasome inhibition decreased GLUT4 protein levels in 3T3-L1 adipocytes. The decrease in GLUT4 expression occurred at the transcriptional level, as treatment with proteasome inhibitors decreased GLUT4 transcription measured by a nuclear run-on assay. Thus, these data demonstrate a new pathway for the regulation of GLUT4 expression that involves proteasomal degradation of factors that regulate GLUT4 expression. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: GLUT4; Adipocyte; 3T3-L1 cells; Calpain; Proteasome; Insulin resistance

1. Introduction Glucose transporters are a family of proteins whose presence at the cell surface allows glucose to cross the plasma membrane (Olson and Pessin, 1996; Watson et al., 2004). These integral membrane proteins allow glucose to diffuse down its concentration gradient in a non-energy dependent process. The rate of glucose entry into a cell is dependent on the quantity of glucose transporters present at the cell surface. GLUT4 is one of these glucose transporters, and is expressed in high levels only in white and brown adipoctyes and in cardiac and skeletal myocytes. In the basal state, most of the GLUT4 is sequestered in vesicular membranes inside these cell; with exposure of these cells to insulin, these vesicles ∗

Corresponding author. Tel.: +1 410 955 6463; fax: +1 410 955 9773. E-mail address: [email protected] (D.W. Cooke).

0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.12.008

are translocated to the plasma membrane where they fuse, resulting in an increase in GLUT4 content at the cell surface, allowing an increase in the rate of glucose entry (Watson et al., 2004). Thus, GLUT4 is the insulin-responsive glucose transporter, and is responsible for the insulin-stimulated increase in glucose uptake that occurs in fat and muscle. Therefore, the insulin sensitivity of fat and muscle, with respect to the ability of insulin to stimulate glucose uptake into these tissues, is proportional to the GLUT4 content in the cells of these tissues. In type 2 diabetes, one of the main pathophysiologic defects is insulin resistance, a decrease in cellular responses to insulin. This decreased insulin sensitivity includes a blunting of the insulin-stimulated increase in glucose uptake into fat and muscle. In muscle cells, this is due to a defect in the ability of insulin to stimulate translocation of GLUT4 to the plasma membrane. While this same defect is also

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present in adipocytes, the decreased insulin sensitivity of adipocytes is also due to decreased expression of GLUT4 in these cells (Minokoshi et al., 2003; Shepherd and Kahn, 1999). Indeed, GLUT4 protein and mRNA levels are decreased in adipocytes in most cases of insulin resistance, including that present in human obesity and type 2 diabetes, as well as in numerous rodent models of insulin resistance and non-insulin dependent (NIDDM) (Shepherd and Kahn, 1999). Adipose tissue accounts for only a small fraction of glucose disposal after a meal, with the majority of glucose taken up by muscle (Minokoshi et al., 2003). On this basis, it would not be expected that diminished glucose uptake into adipocytes would have a significant impact on glucose homeostasis. However, experimental evidence has now demonstrated conclusively that decreased GLUT4 expression in adipocytes decreases insulin sensitivity in muscle and liver, resulting in a change in glucose homeostasis (Minokoshi et al., 2003). Notably, mice that have been genetically engineered so that expression of GLUT4 is selectively eliminated only in adipocytes display evidence of insulin resistance in muscle and liver, resulting in abnormal glucose control and overall impaired insulin sensitivity (Abel et al., 2001). In addition, when GLUT4 expression is selectively increased in adipose tissue of mice, the animals show increased insulin sensitivity and enhanced glucose tolerance (Shepherd et al., 1993). The ability of alterations in GLUT4 expression in adipocytes to alter insulin sensitivity suggests this as a potential therapeutic target for the correction of the insulin resistance that occurs in obesity and is a primary abnormality in type 2 diabetes. Therefore, it will be important to understand the molecular control of GLUT4 expression in adipocytes. Recent data has demonstrated that the risk for the development of type 2 diabetes is linked to the calpain 10 gene (Cox et al., 2004; Horikawa et al., 2000). While some studies have been unable to confirm the association found in the initial report (Fingerlin et al., 2002; Hegele et al., 2001; Horikawa et al., 2000; Rasmussen et al., 2002; Tsai et al., 2001), a number of reports in various populations have demonstrated linkage to the calpain 10 gene (see references within, Cox et al., 2004). The association appears to be quite complex, however, as the linkage is strongest to an extended haplotype combination within the calpain 10 gene (Horikawa et al., 2000), and the high-risk extended haplotype combination has differed across some of the studies (Elbein et al., 2002; Malecki et al., 2002; Orho-Melander et al., 2002). Because the genetic risk for type 2 diabetes is felt to be due to the contribution of numerous genes, these differences could be due to the contribution of calpain 10 (and other genes) to the risk of type 2 diabetes varying among different populations. Another aspect that has made it difficult to understand the association of calpain 10 with type 2 diabetes risk is that the DNA sequence variations that contribute to this association are in regions of the calpain 10 gene that do not encode the amino acid sequence: the highest association was with a sequence change in the middle of the third intron (Horikawa et al., 2000). Thus,

it is unclear how these sequence changes that do not alter the amino acid sequence of calpain 10 could lead to insulin resistance. It has been suggested that the calpain 10 genotype might alter calpain 10 expression level, and indeed, one report demonstrated that calpain 10 expression in muscle differed based on genotype (Baier et al., 2000). With regard to the adipocyte, one study found that calpain inhibition decreased insulin-stimulated glucose uptake in adipocytes (Sreenan et al., 2001), while another study found that glucose uptake in adipocytes differed based on calpain 10 genotype (Hoffstedt et al., 2002). The calpain 10 gene product is a member of the calpain family of cysteine proteases (Suzuki et al., 2004). Thus, one potential mechanism for calpain 10 to influence insulin sensitivity, and thus, associate with a risk for type 2 diabetes, would be if the level of GLUT4 protein in adipocytes were controlled directly or indirectly by the action of this protease. Therefore, we investigated whether calpains might regulate GLUT4 expression in 3T3-L1 adipocytes, a well established model of tissue adipocytes.

2. Materials and methods 2.1. Cell culture 3T3-L1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose, supplemented with 10% calf serum, and maintained at 37 ◦ C and 10% carbon dioxide. Two days after the cells had reached confluence they were induced to differentiate by treatment with DMEM media containing 0.5 mM isobutylmethylxanthine, 1 ␮M dexamethasone, 167 nM insulin and 10% fetal bovine serum for two days, followed by treatment with DMEM media containing 167 nM insulin and 10% fetal bovine serum for two days. The cells were then maintained in DMEM with 10% fetal bovine serum and fed every other day. All cell culture medium was supplemented throughout with 62.5 ␮g/ml penicillin, 100 ␮g/ml streptomycin and 8 ␮g/ml biotin. 2.2. Treatments Differentiated 3T3-L1 adipocytes were treated with protease inhibitors or vehicle beginning 8–12 days after differentiation was induced. N-acetyl-l-leucinyl-lleucinal-l-norleucinal (ALLN, Sigma, St. Louis, MO) is a tripeptide aldehydes that is a competitive inhibitor of calpain proteases (Sasaki et al., 1990). Lactacystin, PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)2-propenoic acid), calpeptin (benzyloxycarbonylleucylnorleucinal), and MG132 (carbobenzoxy-l-leucyl-l-leucyll-leucinal) were obtained from Sigma (St. Louis, MO). These inhibitors were dissolved in dimethylsulfoxide (DMSO, St. Louis, MO) with the final concentration of DMSO that cells were exposed to being no greater than 0.2%. The cell-

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permeable calpastatin peptide and the negative control peptide were obtained from Calbiochem (San Diego, CA), and were dissolved in water. 2.3. Northern analysis After treatment with inhibitors, RNA was purified from cells using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA was quantitated by UV absorbance at 260 nm; in all cases the ratio of the UV absorbance at 260 nm to that at 280 nm was greater than 1.9. Ten micrograms of total RNA was separated by electrophoresis through a 1.2% agarose gel containing 6.5% formaldehyde. The RNA was transferred to a nylon membrane (Hybond-N; Amersham Corp., Piscataway, NJ) and fixed by UV irradiation. 18S RNA was visualized by methylene blue staining of the membrane to confirm equivalence of loading and integrity of the RNA. The membrane was then hybridized with a 1.7 kb murine GLUT4 cDNA (2 × 106 cpm/ml) that was labeled by random hexamer priming (Stratagene, La Jolla, CA). Hybridization was performed in Ultrahyb (Ambion Inc., Austin, TX) at 42 ◦ C for 16 h. The filter was washed at high stringency (0.1 × SSC, 0.1% sodium dodecyl sulfate (SDS) at 42 ◦ C) for 40 min. Band intensity of the 2.7 kb GLUT4 mRNA was quantitated using the Molecular Imager FX (BioRad, Hercules, California) bio-imaging autoanalyzer. For the indicated experiments, the membrane was then stripped in a boiling solution of 0.1% SDS. The membrane was analyzed on the imager to confirm the absence of residual signal, then re-probed with a radiolabeled 2.4 kb murine GLUT1 cDNA. The 2.7 kb GLUT1 mRNA was then quantitated on the imager. Finally, the membrane was re-probed with a radiolabeled 0.1 kb murine 18S cDNA. GLUT4 and GLUT1 mRNA results were normalized to the 18S content of the sample based on quantitation of the 18S rRNA signal on the imager. 2.4. Western immunoblot analysis After treatment with the inhibitors, the cells were washed twice with phosphate-buffered saline (PBS), then lysed in RIPA buffer (1% Igepal CA-630, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA). Protein quantitation was by the Bradford assay (Biorad, Hercules, CA). SDS sample loading buffer was added to 20 ␮g of cellular protein to a final concentration of 2% SDS, 10% glycerol, 100 mM dithiothreitol, 50 mM Tris–HCl, pH 6.8 and the samples were loaded on a 10% denaturing SDS-polyacrylamide gel and processed as described previously (Cooke and Lane, 1999b). Blots were probed with a rabbit polyclonal GLUT4 antibody (Chemicon Int., Temecula, CA) and developed with an enhanced chemiluminescent reagent (Amersham, Piscataway, NJ). The GLUT4 signal was quantitated using the NIH image software.

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2.5. Nuclear run-on assay Three 100 mm dishes of control or treated 3T3-L1 adipocytes were pooled and nuclei were isolated by lysis with Igepal CA-630 (Greenberg and Ziff, 1984). The DNA content of the nuclei was quantitated by lysing an aliquot in 1% SDS, 40 mM Tris, pH 8.0 and measuring the UV absorbance at 260 nm. Nascent RNA transcripts from aliquots of nuclei with equivalent DNA content were extended, then purified using Trizol (Invitrogen, Carlsbad, CA) (O’Conner and Wade, 1992). The precipitated 32 P-labeled transcripts were resuspended in hybridization buffer, and an aliquot was quantitated on a scintillation counter. The RNA was denatured at 65 ◦ C for 10 min prior to addition to the cDNA-containing membranes. Linearized plasmid, containing the cDNA for murine GLUT4 was denatured and spotted onto nylon membrane (Hybond-N; Amersham, Piscataway, NJ). Linearized Bluescript plasmid was used as a negative control, and EcoR1digested genomic DNA purified from 3T3-L1 cells was used as a hybridization and loading control. The DNA was fixed by UV irradiation and the membranes were incubated in hybridization solution (50% formamide, 4 × SSC, 5 × Denhardt’s solution, 50 mM sodium phosphate buffer, pH 7.0, 100 ␮g/ml yeast tRNA, 0.5 mg/ml sodium pyrophosphate, and 1% SDS) at 42 ◦ C for 4 h. Equal amounts (based on the radioactivity) of the 32 P-labeled run-on transcripts were incubated with individual membranes at 42 ◦ C for 48 h. The membranes were then washed twice in 2 × SSC, 0.1% SDS at 42 ◦ C for 20 min, and twice in 0.1 × SSC. 0.1% SDS at 42 ◦ C for 30 min. The hybridized radioactivity was quantitated on a Molecular Imager FX (BioRad, Hercules, CA).

3. Results 3.1. Protease inhibition decreases GLUT4 mRNA levels To investigate the ability of calpains to regulate the level of GLUT4 expression in adipocytes, fully differentiated 3T3-L1 adipocytes were treated with the calpain inhibitor ALLN and GLUT4 mRNA level was quantitated by Northern analysis. As seen in Fig. 1, treatment of 3T3-L1 adipocytes with ALLN for 6 h decreased the level of GLUT4 mRNA: concentrations of ALLN as low as 1–5 ␮M were able to repress GLUT4 expression, with 100 ␮M ALLN decreasing the GLUT4 mRNA level over 80%. This effect was not the result of a generalized decrease in gene expression, as expression of GLUT1 mRNA was not decreasd by treatment with 15 or 100 ␮M ALLN (Fig. 2). This effect of ALLN on GLUT4 expression suggested a role of calpains in the regulation of GLUT4 expression, However, as with many chemical inhibitors, ALLN is not absolutely specific for inhibition of calpains; at higher concentrations it can also inhibit proteasome-mediated protein degradation (Rock et al., 1994). To determine if proteasome

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Fig. 1. The effect of the calpain inhibitor ALLN on GLUT4 expression in 3T3-L1 adipocytes. RNA was purified from 3T3-L1 adipocytes that had been treated with the indicated concentration of ALLN for 6 h. GLUT4 mRNA level was assayed by Northern analysis as shown at the top; also shown is the 18S RNA band detected by methylene blue staining of the membrane. The bar graph shows the mean ± S.D. for four experiments based on quantitation of the GLUT4 mRNA on a Molecular Imager FX (BioRad, Hercules, CA). * P < 0.025 versus control (t-test).

inhibition would alter GLUT4 expression, 3T3-L1 adipocytes were treated with the proteasome inhibitor lactacystin. As seen in Fig. 2, treatment of 3T3-L1 adipocytes with 10 ␮M lactacystin decreased GLUT4 expression to a similar degree as 100 ␮M ALLN (in these and some later experiments, cells were also treated with insulin as a control, as it has previously been demonstrated that insulin decreases GLUT4 expression and increases GLUT1 expression in 3T3-L1 adipocytes.) As was seen with ALLN, lactacystin did not repress GLUT1 expression. The effect of lactacystin suggests that inhibiting proteasomal protein degradation in 3T3-L1 adipocytes results in a decrease in GLUT4 mRNA level. The effect of ALLN is more difficult to interpret. It was possible that the effects seen were merely due to the ability of ALLN to inhibit proteasomal activity at higher concentrations. However, particularly given the ability of relatively low concentrations of ALLN to repress GLUT4 expression, it remained possible that GLUT4 expression could be repressed by inhibition of either proteasome function or calpain activity. To further investigate this, the ability of additional proteasome and calpain inhibitors to repress GLUT4 expression was tested. 3.2. Proteasome inhibitors decrease GLUT4 expression while selective calpain inhibitors do not To confirm that inhibition of proteasome activity in 3T3L1 adipocytes decreased GLUT4 expression, cells were

Fig. 2. The effect of insulin, ALLN and lactacystin on GLUT4 and GLUT1 expression in 3T3-L1 adipocytes. RNA was purified from 3T3-L1 adipocytes that had been treated with 1 ␮M insulin (I), 15 ␮M ALLN (A15), 100 ␮M ALLN (A100), or 10 ␮M lactacystin (Lact), for 6 h and from diluent-treated control cells (C). GLUT4 and GLUT1 mRNA levels were assayed by Northern analysis as shown at the top; also shown is the 18S RNA band detected by methylene blue staining of the membrane. The bar graph shows the mean ± S.D. for four or more experiments based on quantitation of the GLUT4 and GLUT1 mRNA on a Molecular Imager FX (BioRad, Hercules, CA). * P < 0.025 versus control (t-test).

treated with MG132. MG132 has a different mechanism of action than lactacystin, with MG132 functioning as a competitive inhibitor of proteasome function. As seen in Fig. 3, even low concentrations of MG132 resulted in marked suppression of GLUT4 expression. To further investigate whether inhibition of calpain activity in 3T3-L1 adipocytes decreases GLUT4 expression, cells were treated with a number of additional, more selective calpain inhibitors. Calpeptin is similar to ALLN in being a small, cell-permeable peptide-analog competitive inhibitor of calpain. However, unlike ALLN, calpeptin does not inhibit proteasome function (Katagiri et al., 1999). PD150606 is a cell-permeable, non-competitive, non-peptide calpain inhibitor that is directed at the calcium-binding sites of calpain (rather than at the active site, where ALLN and calpeptin act) Calpeptin and PD150606 inhibited calpain activity in 3T3-L1 adipocytes to the same degree as did ALLN (not shown). However, as seen in Fig. 4, neither calpeptin nor PD150606 repressed GLUT4 expression even at 100 ␮M.

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Fig. 3. The effect of MG132 on GLUT4 expression in 3T3-L1 adipocytes. RNA was purified from 3T3-L1 adipocytes that had been treated with the indicated concentration of MG132 for 6 h. GLUT4 mRNA level was assayed by Northern analysis as shown at the top; also shown is the 18S RNA band detected by methylene blue staining of the membrane. The bar graph shows the mean ± S.D. for four experiments based on quantitation of the GLUT4 mRNA on a Molecular Imager FX (BioRad, Hercules, CA). t-Tests indicated that both treatments were different from control (P < 0.0001).

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Fig. 5. The effect of ALLN and lactacystin on GLUT4 protein expression in 3T3-L1 adipocytes. Whole cell extract was prepared from 3T3-L1 adipocytes that had been treated with 100 ␮M ALLN, or 10 ␮M lactacystin (Lact) for 24 h and from diluent-treated control cells (C). 20 ␮g total cellular protein was separated on a 10% SDS–PAGE gel and transferred to PVDF membrane. Membranes were probed with polyclonal anti-GLUT4 antibody, then developed using a chemiluminescent assay and exposure to X-ray film. The bar graph shows the mean from five or more experiments based on quantitation of the GLUT4 band on the X-ray film using the NIH Image software * P < 0.01 versus control (t-test).

Calpastatin is a protein that is an endogenous inhibitor of calpain activity. As a final test of whether inhibiting calpain activity could repress GLUT4 expression, 3T3-L1 adipocytes were treated with a peptide fragment of calpastatin. This calpastatin peptide fragment has been demonstrated to be a cell-permeable inhibitor of calpain activity. A peptide containing the same amino acid composition as the calpastatin peptide but in a scrambled order was used as a negative control. Treatment of 3T3-L1 adipocytes with the calpastatin peptide in concentrations from 30 to 2000 nM for 8, 16, or 24 h did not demonstrate any ability to specifically repress GLUT4 expression (not shown). Thus, the ability of ALLN to repress GLUT4 expression appears to be due to its ability to repress proteasome function at higher concentrations, rather than its ability to inhibit calpain activity. 3.3. Proteasome inhibition decreases GLUT4 protein levels

Fig. 4. The effect of ALLN, PD150606 and calpeptin on GLUT4 expression in 3T3-L1 adipocytes. RNA was purified from 3T3-L1 adipocytes that had been treated with 15 ␮M or 100 ␮M ALLN (A15, A100), 25 ␮M or 100 ␮M PD150606 (P25, P100), or 25 ␮M or 100 ␮M calpeptin (C25, C100) for 6 hours and from diluent-treated control cells (CONT). GLUT4 mRNA levels were assayed by Northern analysis as shown at the top; also shown is the 18S RNA band detected by methylene blue staining of the membrane. The bar graph shows the mean ± S.D. from three or four independent experiments based on quantitation of the GLUT4 mRNA on a Molecular Imager FX (BioRad, Hercules, CA). * P < 0.05 versus control (t-test).

The direct effect of inhibition of protease function might be expected to increase protein levels. However, since proteasome inhibition in 3T3-L1 adipocytes caused a marked reduction in GLUT4 mRNA level, it was of interest to determine the effect of proteasome inhibition on GLUT4 protein levels in 3T3-L1 adipocytes. As seen in Fig. 5, treatment of 3T3-L1 adipocytes with either a high concentration of ALLN or lactacystin resulted in a decrease in GLUT4 protein level after 24 h. In contrast, levels of GLUT1 protein were not changed by ALLN or lactacystin treatment (not shown).

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Fig. 6. The effect of ALLN and lactacystin on GLUT4 transcription in 3T3L1 adipocytes. Nuclei were prepared from 3T3-L1 adipocytes that had been treated with 15 ␮M ALLN or 10 ␮M lactacystin (Lact) for 3 h and from diluent-treated control cells (C). Radiolabeled run-on transcripts were prepared and were hybridized to nylon membranes containing immobilized GLUT4 cDNA, Bluescript plasmid DNA (as a negative control) and EcoR1digested 3T3-L1 genomic DNA (as a loading and hybridization control). The bar graph shows the mean ± S.D. (N = 4 for ALLN, N = 3 for lactacystin) based on quantitation on a Molecular Imager FX (Biorad, Hercules, CA). t-Tests indicated that both treatments were different from control (P < 0.05).

3.4. Proteasome inhibition decreases the rate of GLUT4 transcription To begin to investigate the mechanism through which inhibition of proteasome activity leads to repression of GLUT4 mRNA level, the effect of proteasome inhibition on the rate of GLUT4 transcription was investigated. Treatment of 3T3L1 adipocytes with either 15 ␮M ALLN or 10 ␮M lactacystin decreased the rate of GLUT4 gene transcription by over 60%, as quantitated by a nuclear run-on assay (Fig. 6).

4. Discussion The finding that the risk for type 2 diabetes is linked in some populations to the calpain 10 gene has sparked an interest in determining a mechanism that could explain this association. A report by Sreenan et al. (2001) found that treatment with calpain inhibitors decreased the insulin-stimulated glucose uptake in adipocytes. Insulin-stimulated glucose uptake in adipocytes is highly correlated with the expression level of GLUT4, and altered expression of GLUT4 in adipocytes may play a role in the insulin resistance of type 2 diabetes (Shepherd and Kahn, 1999). Because of this, we have been interested in identifying mechanisms of GLUT4 regulation in adipocytes. Therefore, we sought to determine if the expression of GLUT4 in adipocytes was regulated by calpains.

A number of cell-permeable agents have been used to inhibit calpain activity. As yet, however, no specific calpain 10 inhibitor has been identified. ALLN and calpeptin interact with the active site of calpains. Therefore, these compounds would have the potential to inhibit all calpain isoforms, including calpain 10, as homology in the protease domain is what defines them as calpain family members. PD150606 inhibits calpains by interacting with the calcium-binding sites contained in the calmodulin-like EF-hand domain of the typical calpains (Suzuki et al., 2004). This domain is not present in calpain 10. Calpeptin and PD150606 are much more selective for inhibition of calpains compared to ALLN (Carafoli and Molinari, 1998; Katagiri et al., 1999; Wang et al., 1996). Calpastatin is an endogenous calpain inhibitor with activity only against calpains (Suzuki et al., 2004). As noted above, calpain 10 lacks a calmodulin-like domain; therefore, it should not be inhibited by calpastatin, as interaction with this domain is necessary for calpastatin inhibition of calpains. However, the calpastatin peptide fragment used in the experiments reported here interacts directly with the active site of calpains, without a requirement for a calmodulin-like domain. Therefore, the calpastatin peptide fragment, like ALLN and calpeptin, also has the potential to inhibit all calpain isoforms, including calpain 10. The effect of the various calpain inhibitors used in this study on GLUT4 expression in 3T3-L1 adipocytes was not consistent with regulation of GLUT4 by calpain. While treatment with ALLN decreased expression of GLUT4, relatively high concentrations were required for a large effect. In addition, GLUT4 expression was not decreased by treatment with the more selective calpain inhibitors calpeptin and PD15606 (at concentrations that inhibited calpain activity in 3T3-L1 adipocytes to the same degree as ALLN), nor with the calpastatin peptide. This difference between the action of ALLN and the other inhibitors can not be explained based on their mechanisms of action and the classes of calpains that these inhibitors would therefore be predicted to inhibit. PD15606, which has a different mechanism of action than ALLN, may not inhibit the same calpains as ALLN; however, calpeptin and the calpastatin peptide act on the active site of calpains, as does ALLN, and so would be expected to have similar efficacy against each of the calpains. Thus, the most likely explanation for the effect of ALLN on GLUT4 expression is that it is acting through a mechanism other than its ability to inhibit calpains. Based on the experiments presented, the ability of calpain inhibitors to decrease insulin-stimulated glucose uptake in adipocytes, as well as the association of calpain 10 with type 2 diabetes, is not related to regulation of GLUT4 expression in adipocytes by calpain. A recent report by Otani et al. (2004) demonstrated that overexpression of calpastatin in muscle resulted in an increase in GLUT4 protein, but a decrease in GLUT4 mRNA, with no change in insulin-stimulated glucose uptake. The relative insulin resistance of these muscles (based on the lack of an increase in insulin-stimulated glucose uptake in spite of a more than three-fold increase in GLUT4

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protein level) is consistent with our prior observation that inhibition of calpain in 3T3-L1 adipocytes decreased insulinstimulated glucose uptake by inhibiting actin reorganization and GLUT4 translocation to the plasma membrane (Paul et al., 2003). The findings of Otani et al. that inhibition of calpain by the overexpression of calpastatin in muscle results in a decrease in GLUT4 mRNA level differs from our observation that calpain inhibition does not alter GLUT4 mRNA level in 3T3-L1 adipocytes. It is possible that the observed differences are due to the use of different calpain inhibitors in the two studies, such that the overexpressed calpastatin inhibited a calpain isoform not inhibited by the calpastatin peptide or other inhibitors used in these experiments. However, this seems unlikely, as there is no evidence that calpastatin can inhibit calpain isoforms that the calpastatin peptide cannot (and in fact the calpastatin peptide is expected to have activity against a broader range of isoforms, as discussed above.) There are a number of more likely explanations for this difference. The first is that regulation of GLUT4 expression in muscle may differ from that in adipose tissue (Shepherd and Kahn, 1999). This is certainly true in insulin resistant states, where GLUT4 expression (based on both protein and mRNA level) is decreased in adipocytes but is not altered in muscle (Shepherd and Kahn, 1999). Another difference in the two experiments is that the studies of Otani et al. were performed in vivo, so that compensatory factors elicited by the overexpression of calpastatin in muscle may have led to the repression of GLUT4 mRNA level, rather than the repression being a direct result of calpastatin overexpression. Finally, it is also notable that calpastatin overexpression led to more than a three-fold increase in GLUT4 protein level in muscle, as well as a smaller increase in GLUT1 protein level. The decreased GLUT4 mRNA expression may be a response to this increase in protein levels of these transporters. The increased GLUT4 protein level observed by Otani et al. (2004) in the muscles overexpressing calpastatin also differs markedly from our findings using calpain inhibitors in 3T3-L1 adipocytes. While we did not examine GLUT4 protein levels for the inhibitors that did not alter GLUT4 mRNA expression, the effect of 100 ␮M ALLN or 10 ␮M lactacystin was to decrease GLUT4 protein levels in parallel to the decrease in GLUT4 message levels. Consistent with our interpretation that the effect of ALLN was due to its ability to inhibit proteasome activity at higher concentrations, 15 ␮M ALLN had no effect on GLUT4 protein level (data not shown). This difference in the potential role of calpains on GLUT4 protein levels may also be due to differences in the control of GLUT4 expression in muscle and adipose tissue. In addition to being a calpain inhibitor, at higher concentrations ALLN is also known to inhibit other cysteine proteases, as well as to inhibit proteasome function (Rock et al., 1994). We therefore, investigated whether inhibition of proteasome function could alter GLUT4 expression. Indeed, both of the proteasome inhibitors that were tested, lactacystin and MG132, were highly effective in repressing GLUT4 expression in 3T3-L1 adipocytes. In addition,

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not only did proteasome inhibition lead to a decrease in the GLUT4 mRNA level, but it also suppressed transcription of the GLUT4 gene, as measured by the nuclear run-on assay. The most direct explanation for this effect is that proteasome inhibition stabilized an inhibitor of GLUT4 expression. The transcription factors that have been implicated in the regulation of GLUT4 expression include MEF2 (Olson and Knight, 2003; Thai et al., 1998), O/E proteins (Dowell and Cooke, 2002), C/EBP (Hernandez et al., 2003; Kaestner et al., 1990), NF1 (Cooke and Lane, 1999a,b), KLF15 (Gray et al., 2002), PPAR␥ (Armoni et al., 2003), LXR (Dalen et al., 2003), and a novel zinc-finger transcription factor termed GEF (Knight et al., 2003; Oshel et al., 2000). MEF2, C/EBP, KLF15, LXR and GEF have all been characterized as activators of GLUT4 expression. O/E proteins and NF1 have not been directly demonstrated to either activate or repress GLUT4 expression, but have been shown to participate in the down-regulation of GLUT4 expression in 3T3-L1 adipocytes when these cells are treated with insulin or cyclic-AMP analogs (Cooke and Lane, 1998, 1999a,b). Thus, the O/E proteins and NF1, as well as PPAR␥ and its coactivator PGC-1␣, which have been shown to inhibit GLUT4 expression (Armoni et al., 2003; Miura et al., 2003), may be more likely candidates as factors that mediate the repression of GLUT4 expression with proteasome inhibition. Our demonstration that inhibition of proteasome function leads to decreased expression of GLUT4 raises at least two questions for future investigations. The first is whether alterations in proteasomal degradation of factors that regulate GLUT4 expression play a role in the decreased expression of GLUT4 in adipocytes in insulin resistant states. If so, this would be another aspect of insulin resistance that may involve the proteasome pathway, as it has been hypothesized that insulin-induced proteasomal degradation of IRS-1 may contribute to insulin resistance (Zhande et al., 2002). The second question is whether this pathway of regulation of GLUT4 expression could be used as a pharmacologic target to correct the repressed GLUT4 expression in adipocytes in insulin resistant states. The fact that 80% of all cellular proteins are degraded by the proteasome (Adams, 2004) would appear to make broad proteasomal inhibition an unlikely therapeutic target. Nonetheless, there is good evidence that this approach has merit in the treatment of cancer, due at least in part to the increased sensitivity of neoplastic cells to proteasome inhibition (Adams, 2003, 2004). It seems more likely, however, that more focused inhibition of proteasome activity will be needed to utilized this as a means of manipulating GLUT4 expression. Fortunately, there is specificity in the proteasomal degradation pathway that makes this feasible. Proteins only undergo degradation by the proteasome after they have been ubiquinated. Ubiquitination is an enzymatic process that results in the covalent attachment of a chain of ubiquitins, a small peptide, to the target protein. The third step in ubiquitination, the actual addition of ubiquitin to the target protein, is catalyzed by a family of E3 ubiquitinprotein ligases, whose members may number in the hundreds

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(Adams, 2003). The E3 enzymes have specificity as to which proteins they will ubiquitinate, making them attractive targets for focused proteasomal inhibition (Finley et al., 2004; Pickart, 2004). Another level of regulation of proteasomal degradation is that in many cases ubiquitination is dependent on other covalent modification of the target proteins. For instance, ubiquitination occurs after phosphorylation of some target protein, while in other cases, phosphorylation suppresses ubiquitination and degradation (Pickart, 2004; Schwartz and Ciechanover, 1999). Such a process would also be a potential target for pharmacologic manipulation. Further studies will be needed to determine the role of the ubiquitin-proteasome pathway in the in vivo regulation of GLUT4 expression in normal and insulin resistant states. However, the observation that the proteasomal degradation pathway is a potential regulator of GLUT4 expression broadens the possible avenues through which GLUT4 might be manipulated. This is likely to be a useful tool for dissecting the factors that are important in regulating GLUT4 expression, and may identify targets for pharmacologic manipulation.

Acknowledgments We thank D. Diaczok for expert technical assistance and R. Kohanski for helpful discussions and critical review of this manuscript. This work was supported by the Ilyssa Center for Molecular and Cellular Endocrinology and NIH Grant DK55831 (D.W.C.).

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