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Elevated ceramide concentrations in adipocytes and skeletal muscle impair PKB (protein kinase B; also known as Akt)-directed insulin signalling to key hormonal end points. An important feature of this inhibition involves the ceramide-induced activation of atypical PKCzeta (protein kinase C-zeta), which associates with and negatively regulates PKB. In the present study, we demonstrate that this inhibition is critically dependent on the targeting and subsequent retention of PKCzeta-PKB within CEM (caveolin-enriched microdomains), which is facilitated by kinase interactions with caveolin. Ceramide also recruits PTEN (phosphatase and tensin homologue detected on chromosome 10), a 3'-phosphoinositide phosphatase, thereby creating a repressive membrane microenvironment from which PKB cannot signal. Disrupting the structural integrity of caveolae by cholesterol depletion prevented caveolar targeting of PKCzeta and PKB and suppressed kinase-caveolin association, but, importantly, also ameliorated ceramide-induced inhibition of PKB. Consistent with this, adipocytes from caveolin-1-/- mice, which lack functional caveolae, exhibit greater resistance to ceramide compared with caveolin-1+/+ adipocytes. We conclude that the recruitment and retention of PKB within CEM contribute significantly to ceramide-induced inhibition of PKB-directed signalling.

We have previously shown that angiotensin II (Ang II) increases the expression of the gene encoding adipocyte fatty acid synthase (FAS). Here we investigate the mechanism responsible for increased FAS gene transcription by Ang II. We demonstrate that Ang II increased luciferase activity by 3-fold in 3T3-L1 adipocytes transfected with fusion constructs linking the FAS promoter to the luciferase reporter gene. Interestingly, we mapped the Ang II regulatory sequences to the insulin-responsive region (E box) in the proximal FAS promoter. The E box alone was able to mediate Ang II responsiveness when linked to a heterologous promoter. However, this response was lost when mutations that abolished the binding of the E box to its transcription factors were introduced. Using adenoviral overexpression of a dominant-negative form of adipocyte determination and differentiation factor 1 (ADD1), a transcription factor that binds to the insulin-responsive E box, we demonstrated that ADD1 was required for Ang II regulation of the FAS gene in 3T3-L1 adipocytes. Furthermore, ADD1 expression was also up-regulated by Ang II. With the use of transfections as well as glucose transport assays, we further demonstrated that Ang II stimulation of the FAS gene was dependent on glucose. In conclusion, this is the first report that Ang II regulates adipocyte FAS gene transcription via insulin response sequences in a glucose-dependent manner and that this regulation is mediated at least in part via the ADD1 transcription factor.

We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA. Using the FAS gene as a model of an ADD1-regulated promoter in transiently transfected isolated rat adipocytes or mature 3T3-L1 adipocytes, a potent inhibition of the activity of the FAS-chloramphenicol acetyltransferase reporter gene was observed by overexpression of Id2 or Id3. Reciprocally, co-transfection of Id3 antisense and ADD1 expression vectors in preadipocytes potentiated the ADD1/SREBP-1c effect on the FAS promoter activity. Finally, in the non adipogenic NIH-3T3 cell line, most of the ADD1-mediated trans-activation of the FAS promoter was counteracted by co-transfection of Id2 or Id3 expression vectors. Previous studies have indicated Id gene expression to be down-regulated during adipogenesis [Moldes, Lasnier, Fève, Pairault and Djian (1997) Mol. Cell. Biol. 17, 1796-1804]. We here demonstrated that there was a dramatic rise of Id2 and Id3 mRNA levels when 3T3-L1 adipocytes or isolated rat fat cells were exposed to lipolytic and anti-lipogenic agents, forskolin and isoproterenol. Taken together, our data show that Id products are functionally involved in modulating ADD1/SREBP-1c transcriptional activity, and thus lipogenesis in adipocytes.