Pathways to adipogenesis and probing of a key metabolic regulator: PDHx

Adipocyte differentiation is a complex process involving up-regulation of many genes accompanied by triglyceride accumulation and various cellular morphological changes. In the present study, cultures of a mouse preadipocyte cell line (3T3-L I) were treated with long chain fatty acids (LCFA), oleic acid (OA) or linoleic acid (LA), in the presence or absence of insulin. The effects of LCFA and insulin on triglyceride accumulation and changes in expression of adipocyte differentiation specific genes were studied. In addition, these changes were compared to changes in adipocyte differentiation induced by a standard hormonal differentiation medium (DM1). Triglyceride accumulation and gene expression levels were induced in the presence of LCFA and insulin and not in the presence of LCFA alone. This study demonstrated that LCFA in the presence of insulin are potent inducers of adipogenesis. Further, mechanisms regulating adipogenesis induction in the presence of insulin and LCFA were studied. The data show that the insulin signaling pathway is essential in the adipogenesis process. Interestingly, genistein, an estrogenic compound known to be an inhibitor of adipogenesis, synergistically stimulated adipogenesis in the presence of LCFA plus insulin and not in the presence of insulin alone.;Molecular beacons (MBs) are short sequences of oligonucleotides also containing fluorophores that emit fluorescence only upon MB binding to a complementary nucleotide target sequence. Novel MBs containing 2'-N-(pyren-1-yl) carbonyl-2'-amino-LNA monomers were designed to target the mouse pyruvate dehydrogenase complex, component X (Pdhx) mRNA sequence. Specificity and sensitivity of MBs were tested using antisense and sense RNA strands of mouse Pdhx synthesized by in vitro transcription. Incubation of antisense mRNA with the MB showed a dose-response relationship and linear increase of fluorescence emission. Similarly, MBs incubated with target complementary DNA sequences demonstrated an exponential increase in fluorescence emission with increasing amount of MBs. Furthermore, fluorescence of MBs delivered into 3T3-L1 cells was observed to increase in a dose-dependent manner suggesting that pyrenes are able to fluoresce in cellular conditions. In addition, MB fluorescence was found to he stable in the cells for up to 48 h post transfection. However, delivery of MBs into cells did not show down-regulation of Pdhx mRNA expression. Finally, to test the effect of the nucleotide backbone structure of MB on the resulting fluorescent signal intensity, MB having phosphorothioate (PS-DNA) or 2'-O-Me RNA as backbone elements were compared. MBs with the 2'-O-Me RNA backbone structure provide enhanced fluorescence, compared to MB containing the PS-DNA backbone.