Peroxisome proliferator-activated receptor γ (PPARγ) plays indispensable roles in adipogenesis, which is frequently impaired under pathological conditions such as non-alcoholic steatohepatitis (NASH). Thus, a potent PPARγ antagonist, T0070907 is known as a useful tool for understanding such pathological conditions, while T007097 was also suggested to have PPARγ-independent actions. In the present study, we found that T0070907 inhibited adipogenesis concomitantly with the induction of rapid apoptosis of immature adipocytes within 2 h, whereas another PPARγ antagonist, SR-202 did not show such cytotoxicity. However, T0070907 did not affect the viabilities of pre-adipocytes, mature adipocytes, and NIH-3T3 fibroblasts. The cytotoxic effect of T0070907 was not inhibited by GW1929, a PPARγ agonist, but was inhibited by α-tocopherol, which was previously shown to provide clinical benefit to NASH patients. Interestingly, treatment with high amounts of α-tocopherol alone slightly increased the cellular lipid content in mature adipocytes, but did not affect PPARγ-dependent luciferase reporter expression in COS-7 cells. Moreover, other lipophilic antioxidants, such as tocotrienols, tert-butylhydroquinone, and butylated hydroxyanisole, also inhibited T0070907-induced apoptosis like α-tocopherol. Consequently, it is suggested that T0070907 efficiently inhibits adipogenesis, not only via PPARγ-dependent manner, but also through the induction of apoptosis specifically against immature adipocytes via oxidative stress in a PPARγ-independent manner.
Prolyl oligopeptidase (EC 3.4.21.26, prolyl endopeptidase) cDNA from bovine brain was cloned by PCR, and the amplified fragment was used as a probe to screen the cDNA library from bovine brain. The obtained clone contained a 2.7 kb DNA fragment with an open reading frame of 2130 nucleotides, and encoded a protein of 710 amino acids with a deduced molecular weight of 80640 Da. The deduced amino acid sequence is 95, 94, 51 and 48% homologous to those of human T-cell, porcine brain, Aeromonas hydrophila, and Flavobacterium meningosepticum prolyl endopeptidases, respectively. The bovine brain prolyl endopeptidase-encoding cDNA was expressed using an expression vector bearing a tac promoter, with an approximate yield of 20 micrograms/ml of cell culture.
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