Protein tyrosine phosphatase profiling studies during brown adipogenic differentiation of mouse primary brown preadipocytes

Abstract

There is a correlation between obesity and the amount of brown adipose tissue; however, the molecular mechanism of brown adipogenic differentiation has not been as extensively studied. In this study, we performed a protein tyrosine phosphatase (PTP) profiling analysis during the brown adipogenic differentiation of mouse primary brown preadipocytes. Several PTPs, including PTPRF, PTPRZ, and DUSP12 showing differential expression patterns were identified. In the case of DUSP12, the expression level is dramatically downregulated during brown adipogenesis. The ectopic expression of DUSP12 using a retroviral expression system induces the suppression of adipogenic differentiation, whereas a catalytic inactive DUSP12 mutant showed no effect on differentiation. These results suggest that DUSP12 is involved in brown adipogenic differentiation and may be used as a target protein for the treatment or prevention of obesity by the regulation of brown adipogenic differentiation.

Fig. 1.. Brown adipogenic differentiation of mouse primary preadipocytes. (A) The accumulation of lipid droplets in the cells was measured using Oil-red O staining. (B) The mRNA expression levels of brown adipocyte-specific markers, in this case UCP-1, PGC-1α, PRDM16 and PPARγ, were analyzed in a RT-PCR analysis. The total RNA was extracted on the indicated days of differentiation. β-actin was used as a loading control.

Fig. 2.. PTP mRNA profiling analysis during adipogenesis. (A) PTP mRNA profiling analysis during the brown adipogenesis of primary mouse brown preadipocytes. We performed the experiments three times using independent samples. The PTPs showing differential expression patterns are displayed. (B) mRNA profiling analysis of PTPRF, PTPRN, PTPRV, PTPRZ, and DUSP12 during the white adipogenesis of 3T3-L1 preadipocytes. The mRNA expression levels of PTPs were measured by RT-PCR.

Fig. 3.. Overexpression of DUSP12 suppresses brown adipogenic differentiation. (A) The protein level of DUSP12 was checked by western blot analysis. (B) The expression of DUSP12 was confirmed by western blot analysis using the anti-DUSP12 antibody. (C) Brown preadipocytes expressing DUSP12 or a DUSP12-CS mutant were induced to differentiate into mature brown adipocytes for six days after culturing with a differentiation medium. Then, the samples were stained with Oil-red O to visualize the lipid droplets. (D) Quantification of the stained cells was performed using a dye extraction buffer. Data represent the mean percentage levels ± s.d. values compared with a control vector (n = 3; *P ＜ 0.05). (E) The expression levels of brown-specific adipogenic markers (UCP-1 and PGC-1α) were analyzed using RT-PCR after DUSP12 overexpression.