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Endocrinology Vol. 147, No. 10 4811-4819
Copyright © 2006 by The Endocrine Society

Calpain System Regulates the Differentiation of Adult Primitive Mesenchymal ST-13 Adipocytes

Yukiko Yajima, Mayumi Sato, Hiroyuki Sorimachi, Mitsushi Inomata, Masatoshi Maki and Seiichi Kawashima

Departments of Molecular Biology (Y.Y., S.K.) and Enzymatic Regulation for Cell Functions (H.S.) and Pharmaceutical Research and Development Center (M.S.), Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan; Biomembrane Research Groups (M.I.), Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan; and Laboratory of Molecular and Cellular Regulation (M.M.), Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

Address all correspondence and requests for reprints to: Yukiko Yajima, Tokyo Metropolitan Institute of Medical Science, 18-22 Honkomagome 3-chome, Bunkyo-ku, Tokyo 113-8613, Japan. E-mail: yajima{at}rinshoken.or.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-{alpha} (C/EBP{alpha}), peroxisome proliferator-activated receptor (PPAR)-{gamma}, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor {alpha}, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca2+-dependent degradation of PPAR{gamma} and C/EBP{alpha} but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPAR{gamma} and C/EBP{alpha}. Under both conditions, calpain inhibitors almost completely prevented C/EBP{alpha} cleavage but partially blocked the decrease of PPAR{gamma}. Two ubiquitous forms of calpain, µ- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of µ- but not m-calpain was found in the nucleus. Finally, stable dominant-negative µ-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPAR{gamma} and C/EBP{alpha} during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPOCYTES PLAY AN active role in many different physiological and pathological processes that regulate energy metabolism. Recent studies indicate that adipose tissue functions as an endocrine organ, secreting various unrelated bioactive molecules. This finding has expanded the previous notion that adipose tissue plays only a passive role in lipid metabolism. Adipogenesis occurs during both prenatal and postnatal periods and continues throughout the life span of many organisms (1). Adipogenesis of embryonic fibroblasts may be essential for life (2), but the modulation of adult preadipocyte differentiation can have profound effects at extraadipose sites. One example is the association of lipoatrophy with systemic insulin resistance and diabetes in a transgenic mouse model (3). The 3T3-L1 embryonic preadipocyte cell line was clonally isolated from Swiss-3T3 cells derived from disaggregated 17- to 19-d-old mouse embryos. This line has frequently served as a model of prenatal adipogenesis (4). On the other hand, the precise adipogenesis program for primitive adult preadipocyte cells remains unknown. We showed that ST-13 preadipocytes established from adult primitive mesenchymal cell (5) and primary preadipocytes derived from adults possess an adipogenesis program distinct from that of 3T3-L1 (6). ST-13 cells might therefore be representative of the adipogenesis program for adult-specific adipocytes.

Calpain is a calcium-dependent cytosolic cysteine protease that assumes two ubiquitous forms, one of which is highly sensitive to Ca2+ (µ-calpain), whereas the other is less sensitive (m-calpain). Calpain binds to membranes on its activation and proteolyses substrate proteins in a limited manner (7). The ability of calpain to alter the activities or functions of cytoskeletal proteins, enzymes, and receptors by limited proteolysis suggests its involvement in various Ca2+-regulated cellular functions, including developmental events (8, 9). Patel and Lane (10) have shown that calpain is required for the differentiation of 3T3-L1 preadipocytes into adipocytes induced by standard adipocyte inducers such as methylisobutylxanthine, dexamethasone, insulin, and fetal bovine serum. Calpain is expressed in preadipocytes and its level decreases during differentiation. The calpain inhibitor, acetyl-L-leucyl-L-leucyl-L-norleucinal, disrupts the ability of methylisobutylxanthine to induce differentiation. Furthermore, calpain degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation (11). Therefore, calpain positively regulates the adipogenesis of 3T3-L1 embryonic preadipocytes.

The present study investigates the role of calpain in the adipogenic program of adult primitive ST-13 mesenchymal cells using N-benzyloxycarbonyl-L-leucyl-L-leucinal (ZLLal), a specific calpain inhibitor (12), and overexpressing calpastatin, a specific endogenous inhibitor of calpain. We demonstrate that both of these strategies accelerated the differentiation of ST-13 cells into adipocytes. We also show that the calpastatin overexpression in ST-13 adipocytes stimulated peroxisome proliferator-activated receptor (PPAR)-{gamma} and CCAAT/enhancer-binding protein (C/EBP)-{alpha} expression but suppressed insulin-stimulated glucose uptake. We found that calpain cleaved PPAR{gamma} and C/EBP{alpha} in the nucleus. Furthermore, stable dominant-negative µ-calpain transfectants showed accelerated adipogenesis. These results strongly suggest that calpain is involved in the differentiation of adult primitive ST-13 adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Acetyl-calpastatin active fragment (184–210), Ara-C (cytosine ß-D-arabinofuranoside), and m-calpain were purchased from Sigma Chemical Co. (St. Louis, MO). ZLLal was obtained from the Peptide Institute, Inc. (Osaka, Japan), µ-calpain from Calbiochem. Inc. (San Diego, CA) and [3H]2-deoxy-D-glucose from NEN Life Science Products (Boston, MA). LIPOFECTAMINE PLUS reagent and G418 were purchased from Gibco BRL (Gaithersburg, MD). Routine reagents were purchased from Sigma unless otherwise specified. Antibodies raised against insulin receptor (IR)-{alpha}, IRß, insulin receptor substrate (IRS)-1, IRS-2, IRS-3, PPAR{gamma}, C/EBP{alpha}, mouse µ-calpain, mouse m-calpain, human calpastatin, and sterol regulatory element-binding protein 1 (SREBP-1) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). GLUT4 antibody was from GTonline (Minneapolis, MN), ß-actin antibody was from Chemicon Co. (Temecula, CA), FLAG M2 antibody was from Strategene (Garden Grove, CA), and alkaline phosphatase-conjugated secondary antibodies as well as the Vector substrate kit were from Promega Co. (La Jolla, CA). Monoclonal ß-tubulin antibody T-4026 (Sigma) and polyclonal poly-ADP-ribose polymerase antibody (PARP; BIOMOL, Plymouth Meeting, PA) were used to verify the quality of subcellular fractionation. Rabbit antibodies specific to the pre- and postautolytic forms of µ- and m-calpain were raised against synthetic peptides as described (13, 14).

Cell culture.
Adipocytic conversion in vitro (6, 15) has been studied in detail using ST-13 cell lines established from adult primitive mesenchymal cell lines (5). 3T3-L1 embryonic fibroblast cells were obtained from the American Type Culture Collection (Manassas, VA). Both lines were maintained under a 5% CO2 atmosphere in DMEM-Ham’s F-12 nutrient medium (1:1 mixture) supplemented with 10% calf serum. ST-13 and 3T3-L1 preadipocytes were induced to differentiate into adipocytes using a standard hormonal cocktail containing 1 µM dexamethasone, 0.5 mM methylisobutylxanthine, and 1 µg/ml insulin for 2 d. Thereafter the basal medium containing 10% fetal bovine serum was replenished every other day. Differentiated adipocytes were stained with Oil Red O (16). Triacylglycerol was quantified using triglyceride test kits (Wako Co., Tokyo, Japan), and cells were counted as described by Entenmann and Hauner (17). To estimate the effect of an ionophore (A23187) on the degradation of transcription factors, ST-13 adipocytes after 5 d of differentiation were exposed to various manipulations. Where applicable, ZLLal was added to DMEM containing 0.5% fetal bovine serum 60 min before adding A23187. Control cultures were maintained in DMEM containing 0.5% fetal bovine serum.

Isolation of stable transformants expressing calpastatin cDNA and dominant negative µ-calpain
Full-length pcDNA-I-hemagglutinin-tagged human calpastatin cDNA was cloned into the expression vector PMKIT-neo(+) (a gift from Dr. K. Maruyama, Tokyo Medical and Dental University, Tokyo, Japan). Briefly, ST-13 preadipocytes (1 x 105 cells in 1.5 ml of medium) were cultured for 18 h in a 35-mm culture dish, and medium was replaced with MEM serum-free medium (Life Technologies, Inc., Grand Island, NY) before transfection. Plasmid DNA (7.5 µg/dish) was diluted with MEM (0.25 ml), mixed with Lipofectamine 2000 (3 µl diluted with 0.25 ml MEM), and incubated for 30 min. The mixture was added to cells and incubated for 20 h. Cells were selected with 400 µg/ml of G418, and transfectants were analyzed by immunoblotting of hemagglutinin staining. Three clonal cell lines (sense 1, 5, and 8) expressing the high calpastatin levels were further examined. Dominant-negative pcDNA3.1-N-Flag-tagged human µ-calpain cDNA (C115S) (18) was also transfected to ST-13 preadipocytes using the LIPOFECTAMINE PLUS reagent overnight as described above. Cells were selected with 400 µg/ml of G418, and transfectants were analyzed by immunoblotting of FLAG staining. Two clonal cell lines (DN-µCL-4 and -11) expressing the highest dominant-negative µ-calpain levels were further examined. Preadipocytes that overexpressed calpastatin or dominant-negative µ-calpain were induced to differentiate into adipocytes as described above.

Subcellular fractionation and immunoblotting
Cells were fractionated by a modification of the method of Tanaka et al. (19). In brief, the cells were harvested and resuspended in ice-cold buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 µM dithiothreitol, 1 µM phenylmethanesulfonyl fluoride, 10 µg/ml leupeptin, and 2.5 µg/ml aprotinin]. Lysates prepared by shearing the cells through a 25-gauge needle 20 times were clarified by centrifugation at 1000 x g for 5 min. The resulting pellet (nuclear fraction) was washed twice with buffer A. The supernatant was further separated into cytosolic and membrane fractions by ultracentrifugation at 100,000 x g for 30 min. For preparation of a nuclear extract, the pelleted nuclei were resuspended in buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 µM dithiothreitol, 1 µM phenylmethanesulfonyl fluoride, 10 µg/ml leupeptin, and 2.5 µg/ml aprotinin], and rocked vigorously at 4 C for 15 min. The nuclear lysate was centrifuged at 7000 x g for 10 min, and the supernatant was divided into aliquots and stored at –80 C. Cellular fractions were resolved on SDS-polyacrylamide gradient gels (4/20%) and transferred onto Immobilon-P membranes. Nonspecific binding on the membranes was blocked with 5% skim milk in Tris-buffered saline for 1 h at room temperature. The membranes were then incubated with primary antibodies, and then respective proteins were detected using alkaline phosphatase-conjugated secondary antibodies and Vector substrate kits.

Northern blot analysis
Total RNA was isolated using Isogen reagents (Nippon Gene, Inc., Toyama, Japan) according to the manufacturer’s instruction. Ten micrograms of total RNA were analyzed for PPAR{gamma} mRNA and C/EBP{alpha} mRNA as described previously (6).

Degradation of PPAR{gamma} and C/EBP{alpha}by calpain in vitro
The nuclear lysate (40 µg protein) of ST-13 adipocytes after 5 d of differentiation was incubated with 0.1 U of m- and µ-calpain for 0.5 h at 30 C in reaction buffer [50 mM Tris HCl (pH 7.4), 10 mM KCl containing 2% mercaptoethanol]. Thereafter the samples were boiled in SDS sample buffer, resolved by 10% SDS-PAGE, and immunoblotted.

[3H]2-deoxy-D-glucose uptake assay in differentiated adipocytes
We assayed [3H]2-deoxy-D-glucose uptake using a modification of the method described by Wu et al. (20). Preadipocytes overexpressing calpastatin or vector alone were cultured in 24-well plates (Corning, Inc. Costar, Corning, NY) and differentiated into adipocytes using the standard MDI (M, methylisobutylxanthine; D, dexamethasone; I, insulin) protocol. Seven days later, cells were incubated for 3 h in serum-free DMEM and rinsed at room temperature four times with fresh KRPH buffer [5 mM phosphate (NaH2PO4-H2O + Na2HPO4-7H2O) (pH 7.4), 20 mM HEPES (pH 7.4), 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl]. The buffer was removed and the cells were incubated with or without 100 nM insulin in KRPH buffer at 37 C for 20 min. The buffer was replaced with 1 µCi/well [3H]2-deoxy-D-glucose in KRPH buffer containing 100 µM 2-deoxy-D-glucose for 10 min at room temperature. The supernatant was removed and the plates were rinsed four times with ice-cold PBS. The plates were drained and the cells were lysed overnight in 0.5 ml/well of 0.1 N NaOH. Lysates (400 µl) were neutralized with 40 µl of 1 N HCl, mixed with 4 ml of Ecoscint A (National Diagnostics; Atlanta, GA), and then counted in scintillation vials.

Statistical analysis
Data were statistically analyzed using Analyze-It software for Microsoft Excel (Analyze-It Software, Ltd., Leeds, UK). Statistical significance was determined by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clonal expansion is not required for adipocyte differentiation of ST-13 cells
We reported that ST-13 preadipocytes established from adult primitive mesenchymal cells and primary preadipocytes possess an adipogenesis program distinct from that of 3T3-L1 (6). Because calpain activity is required during the clonal expansion phase of 3T3-L1 adipogenesis, we examined whether the adipogenesis of ST-13 cells requires mitotic clonal expansion (Fig. 1Go). Unlike 3T3-L1 adipocyte differentiation, the standard hormonal inducers did not stimulate cell proliferation, suppress the accumulation of triacylglycerol induced by Ara-C, or change the level of p27 during the differentiation of adult primitive mesenchymal preadipocyte ST-13 cells. These results indicated that ST-13 cells do not require clonal expansion for adipocyte differentiation.


Figure 1
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  		FIG. 1. ST-13 preadipocytes do not require clonal expansion for differentiation. A, 3T3-L1 and ST-13 preadipocytes were induced to differentiate using standard MDI protocol. Cells were counted on d 7 and normalized to the values of unstimulated preadipocytes. B, Effect of Ara-C on triglyceride (TG) accumulation. Confluent ST-13 and 3T3-L1 preadipocytes were induced to differentiate for 7 d with or without Ara-C (0.3 µg/ml). Data from three separate experiments were calculated as ratios of triglyceride accumulation determined under standard differentiation conditions (d 0–7) without Ara-C represented as 100%, mean ± SEM. *, P < 0.01, compared with control (Cont) cells. C, Cascade expression of p18, p21, and p27 proteins during differentiation of 3T3-L1 and ST-13 preadipocytes. Cultured preadipocytes underwent differentiation under standard MDI protocol. Whole-cell lysates were collected on indicated days, and 10 µg of protein were examined by Western blotting.

 
Calpain inhibitor, ZLLal, and overexpressed calpastatin stimulate ST-13 preadipocyte differentiation
ST-13 and 3T3-L1 preadipocytes were incubated with the standard adipogenic hormones in the presence or absence of ZLLal for 7 d and fixed and stained with Oil Red O to measure triacylglycerol accumulation. The staining profile of ST-13 preadipocytes incubated with MDI and ZLLal showed significantly accelerated differentiation, compared with the cells exposed only to MDI. However, the accumulation of cytoplasmic triacylglycerol in 3T3-L1 cells incubated with MDI and ZLLal was similar to that of confluent preadipocytes maintained in calf serum for 7 d without differentiation inducers (Fig. 2AGo, left panel). The accumulation of triacylglycerol in MDI-induced 3T3-L1 cells was dose-dependently blocked by ZLLal, whereas that in ST-13 cells was obviously enhanced and peaked at 20 µM (Fig. 2AGo, right panel). We tested the effect of calpastatin, a specific endogenous inhibitor of calpain, on the ability of ST-13 and 3T3-L1 to differentiate into adipocytes (Fig. 2BGo). The preadipocytes were stably transfected with a vector expressing human calpastatin and induced to differentiate as described above. Triacylglycerol accumulation was remarkably reduced in representative 3T3-L1 cells harboring this calpastatin vector. In contrast, ST-13 cells overexpressing human calpastatin predominantly consisted of fully differentiated adipocytes. These cells contained 192 ± 8 (sense 5) and 182 ± 6 (sense 8) mg of triacylglycerol/mg DNA, compared with 81 ± 5 mg/mg DNA in control cells. These results indicate that calpain differently modulates the adipogenesis of embryonic 3T3-L1 preadipocytes and adult mesenchymal ST-13 preadipocytes.


Figure 2
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  		FIG. 2. Effects of calpain inhibitors, ZLLal and calpastatin, on adipocyte differentiation. A (left panel), Two-day postconfluent (d 0) ST-13 and 3T3-L1 preadipocytes were subjected to standard MDI protocol with or without ZLLal (20 µM) and then stained with Oil Red O on d 7. Confluent preadipocytes maintained in 10% calf serum for 7 d [control (Cont)] were also stained with Oil Red O on d 7. A (right panel), 3T3-L1 and ST-13 preadipocytes were induced to differentiate as above with various doses of ZLLal for 7 d. Data from three separate experiments were calculated as ratio of triacylglycerol accumulation determined in the absence of ZLLal represented as 100%, means ± SEM. B (upper panel), Representative ST-13 and 3T3-L1 cells stably transfected with human calpastatin gene were induced to differentiate as above and triglyceride (TG) accumulation was estimated. *, P < 0.01, compared with cells harboring vector alone (V). B (lower panel), Proteins in transfected cells were Western blotted against human calpastatin antiserum to detect human calpastatin transgene expression on d 0.

 
Characterization of ST-13 adipocytes overexpressing calpastatin
The inhibition of calpain activity, either with ZLLal or by calpastatin overexpression in ST-13 preadipocytes, rather activated the entire differentiation pathway. We examined the effect of calpastatin overexpression on the expression of adipogenesis-related proteins. The expressions of PPAR{gamma}, C/EBP{alpha}, and SREBP-1 (key transcriptional regulators of adipogenesis) were up-regulated in ST-13 cells overexpressing calpastatin, compared with cells harboring the vector during the late stage of differentiation (Fig. 3AGo). In addition, various adipocyte marker proteins, including IR, IRSs, and GLUT4, were significantly increased in cells overexpressing calpastatin, compared with the control cells (Fig. 3BGo). The level of IRß did not differ between the cells overexpressing calpastatin and the control cells at d 8. Furthermore, the basal glucose uptake was little affected in cells overexpressing calpastatin, whereas insulin-mediated glucose uptake was decreased to 57 ± 10% for sense 1 and to 52 ± 3% for sense 8 of that of vector adipocytes, respectively (Fig. 3CGo). However, we found that insulin-stimulated AKT Ser/Thr phosphorylation was similar in the calpastatin-overexpressing adipocytes (sense 8) and vector adipocytes (data not shown). It seems that inhibition of calpain did not affect the insulin-mediated proximal steps of the phosphatidylinositol 3-kinase pathway despite increasing the proteins related to insulin-signaling cascade such as IR, IRSs, and GLUT4. Therefore, insulin resistance in the calpastatin-overexpressing cells may be caused by other possibilities such that calpain involved in the GLUT4 vesicle translocation during insulin-stimulated glucose uptake in adipocytes (21). Thus, these data show that calpastatin overexpression in adult mesenchymal ST-13 preadipocytes enhances the differentiation response and induces insulin resistance.


Figure 3
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  		FIG. 3. Enhanced expression of adipocyte-specific proteins and insulin signaling molecules and suppression of insulin-stimulated glucose uptake by calpastatin overexpression. A, Representative ST-13 preadipocyte lines harboring vector alone (vector) or human calpastatin gene (sense 8) were induced to differentiate by standard MDI protocol. Nuclear fractions (to determine PPAR{gamma}1 and -2 and C/EBP{alpha}) and whole-cell lysates (to determine SREBP-1) were prepared from cells induced to differentiate with MDI protocol at indicated days, and then nuclear fraction (10 µg proteins) and cell lysates (20 µg proteins) were Western blotted with antisera raised against PPAR{gamma}, C/EBP{alpha}, and SREBP-1. B, Membrane and cytosolic fractions were prepared from cells harboring vector alone (vector) or human calpastatin gene (sense 8) after induced to differentiate at d 0 and 8, and equal amounts of cell extracts were Western blotted with antisera raised against IR{alpha}, IRß, IRS-1, -2, -3, and GLUT4. V0, vector cells at d 0; V8, vector cells at d 8; S0, sense 8 cells at d 0; S8, sense 8 cells at d 8. Densitometric analysis was performed after normalizing proteins signals to that of ß-actin band, and the data are expressed as arbitrary units. Data points are means ± SEM from three separate experiments. Insets, Results of representative immunoblotting for each protein. C, Effect of insulin on the [3H]2-deoxy-D-glucose uptake into ST-13 adipocytes stably transfected with calpastatin gene. Stable ST-13 preadipocyte lines harboring human calpastatin gene, sense 8 and sense 1, vector, and wild-type cells were induced to differentiate for 7 d. Rates of [3H]2-deoxy-D-glucose uptake were measured after stimulation with 100 nM insulin as described in Materials and Methods. Results are means ± SEM of three separate experiments. *, P < 0.05 and **, P < 0.01, compared with cells harboring vector.

 
Cleavage of PPAR{gamma} and C/EBP{alpha} by calpain
We tested whether endogenous transcription factors could be cleaved by calpains in vitro. We incubated equal amounts of nuclear proteins from ST-13 cells after 5 d of adipocyte differentiation with m- and µ-calpain in the presence or absence of 5 mM CaCl2 for m-calpain and 0.5 mM CaCl2 for µ-calpain, respectively, as a calpain activator. Figure 4Go shows that in the presence of activated calpain, endogenous PPAR{gamma}s, and C/EBP{alpha} but not SREBP-1 almost completely disappeared. This effect was dose dependent on calpains and was prevented by the addition of ZLLal (200 µM) or calpastatin peptide (50 µM) and in the absence of exogenous calcium. However, the addition of ZLLal and calpastatin peptide both completely recovered the level of C/EBP{alpha}, but the levels of PPAR{gamma}s were partially recovered to that of control. Possible involvement, although partial, of ZLLal- and calpastatin-insensitive and Ca2+-activated protease(s) in the degradation of PPAR{gamma}s is suggested.


Figure 4
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  		FIG. 4. Calpains cleave transcriptional factors in vitro. Cell-free calpain assay was used. Nuclear extracts were incubated with m- and µ-calpain (0.1 U) in the presence or absence of exogenous calcium. Then nuclear fraction (10 µg proteins) was Western blotted with antisera raised against PPAR{gamma}, C/EBP{alpha}, and SREBP-1. Data are from one of four experiments producing identical results.

 
We also found that proteolytic fragmentation patterns by m- and µ-calpain were similar for PPAR{gamma}s and C/EBP{alpha}. On the addition of 0.5 mM, but not 5 mM CaCl2, to the nuclear extract, the amounts of PPAR{gamma}s and C/EBP{alpha} were slightly reduced (PPAR{gamma}1, 59.3 ± 5.0%; PPAR{gamma}2, 67.2 ± 8.9%; and C/EBP{alpha}, 81 ± 9.8% of control level), indicating that endogenous µ-calpain-like protease in the nuclear extract may be activated.

Next we analyzed whether endogenous PPAR{gamma} and C/EBP{alpha} in ST-13 adipocytes undergo proteolysis induced by calcium entry into the cells. Five days after the differentiation program, ST-13 adipocytes were exposed to the selective calcium ionophore (A23187) at a final concentration of 5 µM. Nuclear fractions were collected after 30, 60, or 120 min and processed for Western blotting (Fig. 5Go). On exposure to the ionophore, endogenous levels of PPAR{gamma}1, PPAR{gamma}2, and C/EBP{alpha} in the cultures time-dependently decreased to 53.6 ± 6.8, 59.5 ± 4.3, and 59.2 ± 5.5% (n = 5) of control value, respectively, at 120 min, and ZLLal (200 µM) significantly prevented those cleavages to the 70.9 ± 5.2% (PPAR{gamma}1), 75.9 ± 4.9% (PPAR{gamma}2), and 91.6 ± 6.3% (C/EBP{alpha}) of control value. On the other hand, the limited proteolytic fragment of PPAR{gamma} and C/EBP{alpha} were not observed in the experiments of A23187 treatment to ST-13 adipocytes, indicating that the intermediate fragments are unstable and degraded further by any other cellular protease(s) in living cells. To ascertain whether the decrease in PPAR{gamma}s and C/EBP{alpha} protein levels induced by A23187 are not a consequence of changes in the protein synthesis, ST-13 adipocytes were incubated with A23187 and 100 µg/ml cycloheximide to prevent additional protein synthesis. The decay of the PPAR{gamma}1, -2, and C/EBP{alpha} was quicker in the presence of A23187 at 120 min incubation as shown in Fig. 5CGo, suggesting that those decreases by A23187 did not result from inhibition of protein synthesis but from the stimulation of protein degradation. Furthermore, we assessed whether the loss of these protein shown here could be a consequence of mRNA metabolism. Figure 5DGo shows that 120 min of A23187 treatment left PPAR{gamma} and C/EBP{alpha} mRNA largely intact. It has been reported that A23187 did not lead to repression of PPAR{gamma}2 gene expression in primary cultures of brown adipocytes (22).


Figure 5
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  		FIG. 5. A23187 induces degradation of transcriptional factors in ST-13 adipocytes. A, ST-13 adipocytes at 5 d after the differentiation program were exposed to A23187 calcium ionophore (5 µM). Pretreatment of ZLLal was done for 60 min before addition of A23187. The nuclear fraction was extracted. And 10 µg proteins were Western blotted with antisera raised against PPAR{gamma}, C/EBP{alpha}, and PARP. B, Densitometric analysis was performed after normalizing PPAR{gamma}s and C/EBP{alpha} signals to that of the PARP band. Data are from one of five different experiments producing identical results. Results are means ± SEM of five separate experiments. a, P < 0.05 and b, P < 0.01, compared with control cells, and c, P < 0.05, compared with cell-treated A23187 alone for 2 h. C, Differentiated ST-13 cells were treated for the time indicated with 100 µg/ml of cycloheximide in the presence or absence of A23187. Then nuclear fraction (10 µg proteins) was Western blotted with antisera raised against PPAR{gamma}, C/EBP{alpha}, and PARP. D, Total RNA and nuclear proteins were prepared from ST-13 adipocytes treated with A23187 for the time indicated and analyzed in Northern blots with a probe against PPAR{gamma} and C/EBP{alpha} and Western blots with antiserum raised against PPAR{gamma} and C/EBP{alpha}, respectively. Methylene blue staining of the 28S rRNA is shown as a loading control. Immunoblots and Northern blot show representative data from three separate experiments.

 
These data strongly suggest that A23187 has an effect on the amounts PPAR{gamma} and C/EBP{alpha} proteins without affecting their protein synthesis and the mRNA metabolism. Next, A23187-stimulated PPAR{gamma} and C/EBP{alpha} degradation was examined in ST-13 adipocytes stably expressing human calpastatin. Two independent cell lines, ST-13 sense-1 and -8, and ST-13 cells lines expressing empty vector were used. The A23187-stimulated PPAR{gamma} and C/EBP{alpha} degradations were significantly reduced in both sence-1 and -8 cells, compared with that in vector cells (Fig. 6Go). Thus, calpain in ST-13 cells plays a crucial role(s) in A23187-mediated PPAR{gamma} and C/EBP{alpha} degradation.


Figure 6
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  		FIG. 6. Effect of A23187 on the degradation of transcriptional factors in ST-13 adipocytes stably expressing human calpastatin. Two independent cell lines, ST-13 sense-1 and -8, and ST-13 cells lines expressing empty vector were used. Adipocytes at 5 d after the differentiation program were exposed to A23187 (5 µM) for 2 h. The nuclear fraction was extracted, and 10 µg proteins were Western blotted with antisera raised against PPAR{gamma}, C/EBP{alpha}, and PARP. B, Densitometric analysis was performed after normalizing PPAR{gamma} and C/EBP{alpha} signals to that PARP band. Data are from one of three different experiments producing identical results. Results are means ± SEM of three separate experiments. *, P < 0.05 and **, P < 0.01, compared with vector cells treated A23187 for 2 h.

 
µ-Calpain localizes to and is activated within the nuclear compartment and is involved in adipogenesis
Two isoforms, µ- and m-calpain, are ubiquitously expressed, known as conventional calpains, and their activities are suppressed by calpastatin similarly. To further understand which of the calpain isoenzymes cleaves PPAR{gamma}and C/EBP{alpha} and in which subcellular compartment it occurs, Western blots of cytosolic and nuclear fractions from ST-13 cells were probed with relevant calpain antibodies and the subcellular localization of the active and inactive forms of these proteases (pre- and post-calpain, respectively) was determined. Pre-µ-calpain was expressed abundantly in preadipocytes and the levels declined during differentiation in both fractions, whereas the active form of µ-calpain increased in both fractions (Fig. 7Go). Pre-m-calpain levels increased during the differentiation in both fractions, and active form of m-calpain was found in cytosol of the late stage of adipogenesis but was barely detectable in the nuclear compartment.


Figure 7
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  		FIG. 7. Subcellular localization of calpains. A, Western blots of differentiated ST-13 cells show cytoplasmic (C) and nuclear (N) distributions of relevant calpains. Equal loading and verification of fractionation were confirmed by probing Western blots with anti ß-tubulin and anti-PARP antibodies, respectively.

 
These results suggest that µ-calpain, but not m-calpain, is specifically activated in the nucleus of ST-13 cells and might cleave endogenous PPAR{gamma} and C/EBP{alpha}. Therefore, we examined whether µ-calpain is responsible for adipocyte differentiation of ST-13 cells, using the dominant-negative form of human µ-calpain (DN-µCL), which was constructed from pcDNA3.1-N-Flag-tagged human µ-calpain cDNA (C115S) (18). DN-µCL or empty vector was transfected into ST-13 preadipocytes, and stable clones were obtained by neomycin selection. Two transfectants selected expressed endogenous µ-calpain and DN-µCL with the ratio of 1:0.91 (DN-µCL4) and 1:0.82 (DN-µCL11) expression of DN-µCL, which were detected with anti-µ-calpain mAb and calculated from the relative intensity of each band using National Institutes of Health Image 1.63F (Fig. 8AGo). DN-µCL-FLAG was also detected with anti-FLAG mAb at identical sites to those detected with anti-µ-calpain mAb. Adipocyte differentiation was examined in ST-13 preadipocytes stably expressing human DN-µ-CL. Two independent cell lines, ST-13hDN-µ-CL-4 and -11, and ST-13 cell lines expressing empty vector were used. Triacylglycerol accumulation was significantly increased to 9.91 ± 0.53- and 9.61 ± 0.31-fold of that in the controls in ST-13hDN-µ-CL-4 and -11, compared with that in ST-13 vector cells (Fig. 8BGo). Furthermore, we found that levels of PPAR{gamma} and C/EBP{alpha} in ST-13hDN-µ-CL-4 cells at 3, 5, and 7 d after starting the differentiation program were higher than that in ST-13 vector cells. Thus, µ-calpain plays a crucial role(s) in adipocyte differentiation of ST-13 preadipocytes established from adult primitive mesenchymal cells.


Figure 8
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  		FIG. 8. Transfection of dominant-negative µ-calpain-FLAG stimulates adipocyte differentiation of ST-13 cells. A, Representative ST-13 preadipocyte lines harboring vector alone (vector) or dominant-negative µ-calpain-FLAG gene (ST-13hDN-µ-CL-4 and -11) were lysed with SDS sample buffer. The expression level of dominant-negative µ-calpain-FLAG or FLAG-vector in transfected ST-13 preadipocytes was assessed by immunoblot using anti-FLAG or µ-calpain antibody. B, Representative ST-13 preadipocyte lines harboring vector alone (vector) or dominant-negative µ-calpain-FLAG gene (ST-13hDN-µ-CL-4 and -11) were induced to differentiate by standard MDI protocol, and triglyceride (TG) accumulation was estimated on d 6. *, P < 0.01, compared with vector cells. Cont, Control. C, Nuclear fractions were prepared from cells induced to differentiate with MDI protocol at indicated days, and then nuclear fraction (10 µg) was Western blotted with antisera raised against PPAR{gamma}, C/EBP{alpha}, and PARP. The immunoblots show representative data from three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we showed that inhibition of calpain activity by either a synthetic inhibitor ZLLal or overexpression of endogenous inhibitor protein calpastatin enhanced lipid accumulation and induced resistance of the insulin response in adult primitive mesenchymal ST-13 adipocytes. Our results also indicated that µ-calpain rather than m-calpain could be responsible for the control of ST-13 adipocyte differentiation through the regulation of the levels of PPAR{gamma}s and C/EBP{alpha}.

Calpain is required for clonal expansion during embryonic 3T3-L1 adipocyte differentiation. We previously showed that the mechanisms through which ST-13 preadipocytes and 3T3-L1 embryonic fibroblasts differentiate into adipocytes differ (6). We also presented data supporting the notion that the program through which primary preadipocytes from adult rat adipose tissue differentiate into adipocytes is similar to that of ST-13 (6). Human preadipocytes in primary culture do not undergo clonal expansion during differentiation (17, 23), and here we found that clonal expansion was not required for adipocyte differentiation of ST-13 (Fig. 1Go). Therefore, ST-13 preadipocytes might pass through clonal expansion directly to achieve adipogenesis and thus not require calpain activity during the early part of this process not like 3T3-L1 adipocyte differentiation.

Blocking calpain activity by overexpressing human calpastatin stimulated differentiation and up-regulated adipocyte-specific transcriptional factors such as PPAR{gamma}, C/EBP{alpha}, and SREBP-1 proteins in ST-13 cells (Fig. 3Go). Endogenous PPAR{gamma} and C/EBP{alpha} in nuclear extracts of ST-13 adipocytes were cleaved in vitro by calpain and an increase in the intracellular calcium levels of ST-13 adipocytes by A23187 also caused the proteolyses of endogenous PPAR{gamma} and C/EBP{alpha}. The degradation of C/EBP{alpha} under both conditions was almost completely prevented by exogenously added calpain inhibitors, but that of PPAR{gamma}s were partially recovered. However, in calpastatin-overexpressing ST-13 cells, the A23187-induced degradations of PPAR{gamma} and C/EBP{alpha} were significantly reduced. It has been reported that human calpastatin blocks calpain autolysis, and the calpastatin fragment generated by this process inhibits the proteolytic activity of calpain (24, 25). It seemed that human calpastatin overexpressed in cells inhibits m- and µ-calpain autolysis and the activity of postcalpain; thus, PPAR{gamma} and C/EBP{alpha}degradation could be prevented by calpain inactivation in these cells.

The molecular mechanism by which calpastatin overexpression enhances the amount of SREBP-1 remains obscure. In 3T3-L1 adipocytes, PPAR{gamma} is supposedly reduced by the treatment of the specific ligand such as thiazolidinediones or interferon-{gamma} and the proteolytic cleavage is involved via proteasome-dependent pathway (26, 27). Although PPAR{gamma}s appear to be labile proteins, the proteasome inhibitor, lactacystin, did not inhibit the activity of A23187 toward PPAR{gamma} and C/EBP{alpha} degradation (data not shown). Whether these proteolytic pathways act independently or sequentially is currently unknown. Because C/EBP family members, such as C/EBPß in liver and CHOP-10 in 3T3-L1 adipocytes, were reported to be substrates of calpain (28, 29), future experiments should reveal whether C/EBP{alpha} is also cleaved by calpain in adipocytes. Furthermore, the expression of insulin signaling factors, such as IR{alpha}, IRS-1, -2, -3, and GLUT4, was significantly increased in ST-13 cells overexpressing calpastatin (Fig. 3BGo). Consistent with this, calpastatin overexpression results in a 3-fold increase in GLUT4 protein in skeletal muscle (30) and calpain inhibition prevents IRS-1 degradation in 3T3-L1 adipocytes and prostate epithelial cells (31, 32).

Although usually viewed as cytoplasmic enzymes, several calpains localize in the nucleus, and their endogenous inhibitor, calpastatin, localizes not only in the cytoplasm but also in the cell nucleus (33, 34). Because we found in the present study that the activated µ- but not m-calpain localized in the nuclear fraction of ST-13 cells, µ-calpain would be an important inhibitor of the adipocytic differentiation process of ST-13 cells by the continuous proteolytic cleavage of PPAR{gamma} and C/EBP{alpha}. DN-µ-calpain-expressing cells were significantly stimulated to accumulate triacylglycerol and high levels of PPAR{gamma} and C/EBP{alpha}, compared with those in vector cells. Thus, µ-calpain plays a crucial role(s) in adipocyte differentiation of ST-13 preadipocytes established from adult primitive mesenchymal cells.

One key finding from our studies is that calpain inhibitor stimulates adipocyte hypertrophy presumably due to the inhibition of PPAR{gamma} and C/EBP{alpha} degradations. PPAR{gamma} plays a pivotal role in adipocyte differentiation and C/EBP{alpha} has been implicated in maintenance of the terminally differentiated adipocyte phenotype. However, on the high-fat (HF) diet, normal amounts of PPAR{gamma} activity seen in wild-type mice increase triglyceride content in white adipocyte tissue, skeletal muscle, and liver due to a combination of increased fatty acid influx into white adipocyte tissue, skeletal muscle, and liver and HF diet-induced leptin resistance, leading to insulin resistance and obesity. On the other hand, moderate reduction of PPAR{gamma} activity observed in untreated heterozygous PPAR{gamma}-deficient mice is protected from a HF diet or aging-induced adipocyte hypertrophy, obesity, and insulin resistance (35, 36). It seems that calpain could contribute to prevent adipocyte hypertrophy regulating the amount of PPAR{gamma} in adipocytes. On the other hand, adipocytes exposed to calpain inhibitors become insulin resistant as measured by insulin-sensitive glucose uptake (21, 37), and here we found that calpastatin overexpression in ST-13 adipocytes also reduced the amount of insulin-mediated glucose uptake. Moreover, calpain inhibitor stimulates cardiomyocyte and muscle hypertrophy (38, 39). Taken together, these findings suggest that calpain activity is linked to obesity and insulin resistance. Future studies are needed to address whether calpains are required for adipocyte progression and pathology. Overall, the results of this study suggest that calpain activity is necessary for initiating the differentiation of 3T3-L1 embryonic preadipocytes and preventing the hypertrophy of adult primitive mesenchymal ST-13 adipocytes.


   Footnotes
 
Preliminary results of this investigation were presented at the 12th International Congress of Endocrinology, Lisbon, Portugal, August 2004.

Disclosure statement: the authors have nothing to disclose.

First Published Online July 20, 2006

Abbreviations: C/EBP, CCAAT/enhancer-binding protein; HF, high fat; IR, insulin receptor; IRS, insulin receptor substrate; KRPH, phosphate, HEPES, MgSO4, CaCl2, NaCl, and KCl; MDI, medium to induce differentiation; PARP, poly-ADP-ribose polymerase; PPAR, peroxisome proliferator-activated receptor; SREBP-1, sterol regulatory element-binding protein 1; ZLLal, N-benzyloxycarbonyl-L-leucyl-L-leucinal.

Received December 23, 2005.

Accepted for publication July 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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