RESUMEN
Relative quantitation RT-PCR was used to investigate the regulation of leptin expression in 3T3-F442A adipocytes by glucose and insulin. The results showed that glucose and insulin stimulated the expression of leptin in 3T3-F442A adipocytes, but they did not act synergically. Over-high concentration of glucose suppressed the expression of leptin and the effect of insulin. The elevation of the expression of leptin was characterized with saturation. The concentration of glucose is very important for the regulation of leptin expression.
RESUMEN
Glucose and insulin stimulate leptin gene expression in vitro and in vivo. To identify cis-elements that are responsible for the glucose and insulin effects, mouse 3T3-L1 adipocytes were transiently transfected with reporter constructs with serial deletions in mouse ob gene promoter. The cis-elements were identified with Gel mobility shift assays (GMSA), DNase I footprint assays and PCR mediated site-directed mutation assays. Transient transfections detected a negative cis-acting element, a glucose-responsive element (GLRE), and an insulin-responsive element (IRE) in the region from -1 719 bp to -1 452 bp of mouse ob gene. This region does not contain any known GLRE or IRE. GMSA identified a DNA binding protein which specifically binds a native probe prepared from mouse ob gene promoter (-1 719 bp/-1 452 bp), and the binding was repressed by glucose or insulin. DNase I footprint assays and PCR mediated site-directed mutations assays identified that the binding motif AGCAAAA, spanning -1 698 bp to -1 692 bp of the mouse ob gene promoter, was responsible for the effects of glucose and insulin on ob gene expression. These studies suggest that a negative cis-acting element is located between -1 719 bp and -1 452 bp of the mouse ob gene promoter, and glucose and insulin simulate mouse ob gene expression by repressing the binding of a transcription factor to this element. This element, AGCAAAA, spanning -1 698 bp to -1 692 bp is a novel GLRE and IRE.