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doi: 10.1242/10.1242/jcs.00044

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Reduced IRS-2 and GLUT4 expression in PPAR{gamma}2-induced adipocytes derived from C/EBPß and C/EBP{delta}-deficient mouse embryonic fibroblasts

Hiroyasu Yamamoto1, Shogo Kurebayashi1, Takahisa Hirose1,2, Haruhiko Kouhara1 and Soji Kasayama1,*

1 Department of Molecular Medicine (C-4), Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
2 Department of Internal Medicine, Nishinomiya Municipal Central Hospital, Nishinomiya, Hyogo 663-8014, Japan


Figure 1
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  		Fig. 1. Morphological differentiation of wild-type mouse embryonic fibroblasts (MEFs) and C/EBPß/{delta}-deficient MEFs into adipocytes. Wild-type and C/EBPß/{delta}-deficient MEFs were infected with empty adenovirus (pAdex) or with PPAR{gamma}2-expressing adenovirus (pAdex-mPPAR{gamma}2) followed by incubation with 10 µM troglitazone or 3 µM 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2). On day 8, the cells were stained with Oil Red O, and morphological changes were monitored by light microscopy.

 

Figure 2
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  		Fig. 2. Intracellular triglyceride content in wild-type and C/EBPß/{delta}-deficient MEFs. Intracellular triglyceride was extracted from wild-type and C/EBPß/{delta}-deficient cells on day 8 after infection with pAdex ({square}) or with pAdex-mPPAR{gamma}2 ({blacksquare}) followed by stimulation with 10 µM troglitazone. Values represent the mean±s.e.m. obtained from nine samples in each analysis.

 

Figure 3
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  		Fig. 3. Gene expression in wild-type and C/EBPß/{delta}-deficient cells during PPAR{gamma}2-induced adipogenesis. Total RNA was extracted from wild-type and C/EBPß/{delta}-deficient cells on the indicated day after the pAdex-mPPAR{gamma}2 infection. (A) 10 µg of RNA was used for northern blot analyses. (B) Densitometrical data from three separate experiments are plotted as relative abundance of mRNA normalized to GAPDH mRNA (mean±s.e.m.).

 

Figure 4
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  		Fig. 4. Insulin-stimulated 2-deoxyglucose (2-DG) uptake in wild-type and C/EBPß/{delta}-deficient MEFs as well as wild-type and C/EBPß/{delta}-deficient adipocytes. Wild-type and C/EBPß/{delta}-deficient MEFs were infected with pAdex or with pAdex-mPPAR{gamma}2 followed by stimulation with 10 µM troglitazone. In the presence ({blacksquare}) or absence ({square}) of 100 nM insulin, [3H]-2-DG uptake was determined. Results show the mean±s.e.m. obtained from triplicate assays typical of three separate experiments.

 

Figure 5
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  		Fig. 5. Protein and gene expression of insulin receptor (IR), insulin receptor substrate 1 (IRS-1) and IRS-2 in wild-type and C/EBPß/{delta}-deficient cells during PPAR{gamma}2-induced adipogenesis. Wild-type and C/EBPß/{delta}-deficient MEFs were infected with pAdex-mPPAR{gamma}2, followed by stimulation with 10 µM troglitazone. (A) Before and 8 days after the induction, whole cell lysates were collected for western blot analyses (top panels). Densitometrical data from seven separate experiments are plotted as relative abundance (mean±s.e.m.; bottom panels). (B) Ten µg of RNA collected before and 8 days after the induction was used for northern blot analyses. The experiment was repeated five times with similar results, and the results of a representative experiment are shown.

 




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