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PKB Is Required for Adipose Differentiation of Mouse Embryonic

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PKB Is Required for Adipose Differentiation of Mouse Embryonic Research Article 889 PKB␣ is required for adipose differentiation of mouse embryonic fibroblasts Anne Baudry, Zhong-Zhou Yang and Brian A. Hemmings* Friedrich Miescher Institute for Biomedical Research, Maulbeerstr. 66, CH-4058, Basel, Switzerland *Author for correspondence (e-mail: [email protected]) Accepted 14 November 2005 Journal of Cell Science 119, 889-897 Published by The Company of Biologists 2006 doi:10.1242/jcs.02792 Summary Protein kinase B␣ (PKB␣) is a key regulator of metabolism, downregulated in PKB␣-deficient MEFs but could be proliferation and differentiation. We have explored the role restored by expressing an active PKB␣ in the deficient cells. of PKB␣ in adipogenesis using wild-type and PKB␣- The level of lipocalin 2, renin 1 and receptor-activity- knockout mouse embryonic fibroblasts (MEFs) and show modifying protein 3 genes expressed by adipose cells was that lack of PKB␣ prevents MEF differentiation into also decreased in PKB␣-deficient MEFs, and are inhibited adipocytes. Expression of ectopic PKB␣ in PKB␣-deficient by LY294002 treatment during early adipocyte cells restores adipogenesis. We identified 80 genes whose differentiation of 3T3-L1 cells. The results underscore an expression was upregulated in wild-type MEFs during essential role for PKB␣ in the transcriptional program adipogenesis but whose expression was significantly required for adipogenesis. reduced in PKB␣-deficient MEFs under the same conditions. Significantly, the regulator of adipogenesis Key words: PKB␣, Adipocyte differentiation, Mouse embryonic Krüppel-like transcription factor 15 gene expression was fibroblasts, Microarray analysis Introduction many cellular processes stimulated by insulin and growth Adipogenesis is a complex process with multiple steps factors (Brazil and Hemmings, 2001; Lawlor and Alessi, 2001; including growth arrest, clonal expansion, withdrawal from the Whiteman et al., 2002). The three members of the PKB family, cell cycle and terminal differentiation (Gregoire et al., 1998; PKB␣ (Akt1), PKB␤ (Akt2), and PKB␥ (Akt3), are encoded Rosen et al., 2000). In parallel, adipose conversion is by distinct genes but share a similar structural organisation Journal of Cell Science accompanied by temporally regulated expression of numerous (Datta et al., 1999; Scheid and Woodgett, 2001; Hill and genes. Some of these genes encode transcription factors, such Hemmings, 2002). It remains a matter of debate whether these as members of the CCAAT/enhancer binding protein (C/EBP) isoforms have specific or redundant functions in vivo. This family and peroxisome proliferator-activated receptor ␥ issue was addressed recently by disruption of different PKB (PPAR␥). These are key regulators of this transcriptional genes in mice (Yang et al., 2004). PKB␤-deficient mice exhibit program and promote the expression of most genes insulin resistance and a diabetes-like syndrome (Cho et al., characterising fat cells, such as the adipocyte-specific fatty- 2001a; Garofalo et al., 2003). Depending on the genetic acid-binding protein aP2 (Spiegelman et al., 1993). background, PKB␤-knockout mice are also characterised by Evidence has accumulated that insulin and insulin-like moderate growth deficiency as well as age-dependent decrease growth factor-1 (IGF-1) signalling pathways play a significant in adipose tissue mass (Garofalo et al., 2003). However, MEFs role in adipogenesis. Both are essential inducers of adipocyte derived from these mutants are not significantly different to differentiation (Smith et al., 1988). Insulin receptor-deficient wild-type MEFs in their ability to differentiate into adipocytes mice display underdeveloped adipose tissue (Cinti et al., 1998) (Peng et al., 2003). Unlike PKB␤-deficient mice, PKB␣- and mouse embryonic fibroblasts (MEFs) derived from these knockout mice have normal glucose homeostasis but display mutants fail to differentiate into adipocytes (Nakae et al., deficient placental development, which results in growth 2003). Furthermore, a double-knockout deficiency of insulin retardation and enhanced neonatal morbidity (Chen et al., receptor substrate-1 (IRS-1) and IRS-2 results in failure of the 2001; Cho et al., 2001b; Yang et al., 2003). Subcutaneous fat adipocyte developmental program (Miki et al., 2001). Finally, is also moderately reduced in the absence of PKB␣, phosphatidylinositol 3-kinase (PI 3-kinase) activity transiently implicating this isoform in adipogenesis (Yang et al., 2003). increases during conversion of preadipocytes into adipocytes Finally, the major characteristic of PKB␥-knockout mice is (Sakaue et al., 1998), and the presence of PI 3-kinase inhibitors reduction in brain size (Easton et al., 2005; Tschopp et al., completely blocks the differentiation process (Tomiyama et al., 2005). The phenotypic differences between these PKB- 1995; Christoffersen et al., 1998; Xia and Serrero, 1999), knockout mice suggest that PKB isoforms participate jointly in indicating that the PI 3-kinase pathway is also involved in the regulation of some cellular processes, such as growth, but adipogenesis. also exert specific roles in vivo, for example in glucose The protein kinase B (PKB, also named Akt) is a metabolism. downstream target of PI 3-kinase and plays a critical role in Previous studies have suggested a role for PKB in 890 Journal of Cell Science 119 (5) adipogenesis, because expression of a constitutively activated form of PKB in 3T3-L1 pre-adipose cells caused spontaneous differentiation into adipocytes (Kohn et al., 1996; Magun et al., 1996). In the current study, we investigated the role of the ␣ isoform of PKB in adipose differentiation. We show that MEFs derived from PKB␣-deficient mice fail to differentiate into adipocytes in vitro and identified target genes regulated by PKB␣ during the early steps of adipocyte conversion. Results Defective adipogenesis in the absence of PKB␣ To investigate the role of PKB␣ in adipocyte differentiation, MEFs were derived from wild-type and PKB␣–/– embryos from the same litter and thus have an identical genetic background. They were induced to differentiate into adipocytes in vitro using a standard adipogenic induction cocktail of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), the synthetic glucocorticoid dexamethasone and insulin, followed by addition of insulin every 2 days (see Materials and Methods for details). After 8 days of differentiation, Oil Red Fig. 1. Inhibition of adipogenesis in PKB␣–/– MEF cells. (A) Two O staining was performed to monitor intracellular lipid days after confluence, MEF cells were induced to differentiate into accumulation. Wild-type MEFs cultured in the presence of adipocytes with IBMX, dexamethasone and insulin for 48 hours differentiating agents accumulated fat droplets but no obvious followed by insulin alone every 2 days. Cells were stained with Oil- lipid accumulation was observed in PKB␣–/– MEFs (Fig. 1A). Red O at 8 days post-induction. (B) Expression of adipocyte-specific ␣–/– genes after stimulation of the adipose differentiation process in wild- This failure to support lipid storage in PKB MEFs persisted ␣–/– for at least 16 days following induction of differentiation (data type and PKB MEFs. Total RNA was extracted from cells at the times indicated and analysed by RT-PCR as described in the not shown). As the adipogenic process is characterised by Materials and Methods. expression of specific adipocyte markers such as PPAR␥ and aP2, we analysed expression of these two transcripts in both cell types by semi-quantitative RT-PCR. Whereas treatment of wild-type MEFs with adipogenic inducers led to the induction of Pparg and aP2 mRNA, no change in mRNA levels for either marker was detected in the absence of PKB␣ (Fig. 1B). The three PKB isoforms (␣, ␤, ␥) are expressed in wild-type Journal of Cell Science MEFs (Peng et al., 2003). Western blot analysis using isoform- specific antibodies was performed to determine the expression profile of the three isoforms during differentiation in wild-type and PKB␣–/– MEFs. The data presented in Fig. 2A confirm the absence of PKB␣ protein in PKB␣–/– MEFs and also showed that the amounts of PKB␤ and ␥ were not significantly different in PKB␣–/– and wild-type MEFs, precluding any compensatory upregulation of PKB␤ and/or ␥ in the absence of the ␣ isoform. In addition, the activation status of PKB was monitored during adipose conversion using phospho-specific PKB antibodies that recognise the two crucial phosphorylation sites of PKB present in all three isoforms: Thr308 (in PKB␣) in the activation loop of the kinase domain and Ser473 (in PKB␣) in the hydrophobic motif (Alessi et al., 1996; Meier et al., 1997; Brodbeck et al., 1999). PKB activity increased during differentiation of wild-type MEFs whereas this activity is reduced in the absence of PKB␣ expression (Fig. 2B). Taken Fig. 2. Levels of the three PKB isoforms and the phosphorylation together, these results show that the PKB␣ isoform is necessary status of PKB in wild-type and PKB␣–/– MEFs during adipose for the adipogenic differentiation of MEFs. conversion. Total cellular extracts were prepared from cells before (day 0) or at 2, 5, 8 and/or 12 days after adipose induction treatment ␣ ␤ ␥ Ectopic expression of PKB␣ restores adipogenesis in and analysed by western blotting with PKB -, PKB - and PKB - ␣–/– specific antibodies (A) or with phospho-PKB specific antibodies PKB MEFs (pSer473 and pThr308) (B). Blots were reprobed with anti-actin To examine whether the impairment of adipocyte antibodies to control for equal loading. differentiation observed
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