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

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.

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- (C/EBP), peroxisome proliferator-activated receptor (PPAR)-, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor , 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 and C/EBP but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPAR and C/EBP. Under both conditions, calpain inhibitors almost completely prevented C/EBP cleavage but partially blocked the decrease of PPAR. 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 and C/EBP during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.

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