Bankhead, Katharine Ruth; (2002) Effects of insulin on murine Lep expression in adipocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Leptin is a recently discovered hormone, encoded by the obese (ob/Lep) gene, which is expressed predominantly in adipose tissue. It is involved in regulating the size of adipose tissue mass and signalling nutritional status. Insulin has been demonstrated to be among a number of factors that regulate Lep mRNA levels in vivo, although it remains unclear whether it exerts its effects by a direct, or indirect mechanism(s). An RNase protection assay was designed to gain further insight into the effects of insulin on the steady-state levels of Lep mRNA in adipose tissue, both in vivo and in vitro. As expected, a subcutaneous injection of insulin into overnight fasted mice significantly increased Lep mRNA levels within four hours. However, in an insulin-responsive adipose tissue explant model, a significant change in Lep mRNA levels could only be demonstrated at supraphysiological concentrations of insulin. As an alternative model, reporter-gene plasmids were constructed containing 0.5kb, or 1.0kb of the murine Lep gene 5' flanking region, with or without a 200bp segment containing exon 1 and part of intron 1, (p0.5lep-Luc, p0.7lep-Luc, p1.0lep-Luc and p1.2lep-Luc). Transient transfection of p0.5lep-Luc or p0.7lep-Luc, in both 3T3-F442A preadipocytes and adipocytes, significantly repressed luciferase activity compared to a promoterless control, whereas p1.0lep-Luc and p1.2lep-Luc significantly increased luciferase activity. Transient transfection with p1.2lep-Luc elicited a concentration-dependent down-regulation of luciferase activity in response to chronic insulin treatment in preadipocytes, and initial studies showed no significant effect in adipocytes, although a trend towards a concentration-dependent increase was seen. In conclusion, insulin, administered acutely or chronically, has little or no direct effect on Lep mRNA levels, in the murine adipocyte cell models used in this study.

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