Mechanisms of agouti-induced obesity : effects on adipocyte metabolism and interaction with insulin

Dominant mutations at the agouti locus such as viable yellow (A^vy) cause a syndrome of marked obesity and diabetes in addition to a characteristic yellow coat color. Recent studies indicate that agouti acts both centrally and peripherally to induce obesity.We hypothesized that agouti modulation of adipocyte metabolism may account for part of the yellow mouse obesity. Studies from our laboratory indicated that agouti increases adipocyte de novo lipogenesis and triglyceride levels in a calcium (Ca^{2+)-dependent manner. However, the precise molecular mechanisms that are involved in agouti regulation of adipocyte metabolism, have not been determined.}

The objective of this work is to determine the mechanisms of agouti action on adipocyte metabolism using two specific markers of adiposity: (1) leptin, the product of the obesity gene, ob which is secreted by fat cells in amounts that are positively correlated with adiposity, and (2) fatty acid synthase (FAS), a key de novo lipogenic enzyme which is highly responsive to hormonal and nutritional changes.

We investigated effects of agouti and its interaction with insulin on leptin synthesis and secretion in cultured adipocytes as well as in transgenic mice overexpressing agouti in adipose tissue (under the control of adipocyte specific promoter aP2). We also investigated whether transcription rate of the FAS gene in cultured adipocytes is altered via agouti specific response elements in the FAS promoter.

Results from this study demonstrated that agouti significantly increase intracellular and plasma leptin levels in aP2 transgenic mice relative to control mice. Further,administration of insulin (1 unit/day) increased intracellular leptin levels without any significant effect on plasma leptin. The lack of insulin effect on plasma leptin levels were further confirmed by in vitro assays; media collected from 3T3-L1 adipocytes that were treated with 100 nM insulin showed no effect on leptin secreted into the culture media. These results suggest that agouti increases leptin synthesis and secretion while insulin only modulates leptin synthesis. Agouti may increase leptin levels as a result of its effect on triglyceride storage. Alternatively, agouti may directly regulate leptin synthesis and secretion by altering ob gene expression. However, further studies are required to determine mechanisms of agouti and insulin regulation of leptin.

In addition to agouti regulation of leptin, our study demonstrate that both agouti and insulin upregulate FAS gene transcription. Furthermore, agouti and insulin exert additive effects on FAS gene transcription. Using transfection assays, we demonstrated that transcriptional regulation of the FAS gene by agouti was mediated by novel agouti response elements within the FAS promoter. This agouti responsive region mapped to a region distinct from the previously identified insulin responsive region. We confirmed the specificity of adipocyte nuclear factor(s) binding to this response region by electrophoretic gel mobility-shift assays. Interestingly, agouti response elements appeared to be also responsive to intracellular calcium.In summary, the results from this study indicate that agouti effects on adipocyte may contribute to yellow mouse obesity. We demonstrated that agouti effects adipocyte metabolism by (1) inducing synthesis and secretion of leptin levels, and (2) by increasingFAS transcription rate via novel agouti responsive elements that are distinct from the previously mapped insulin response element.

Results from this investigation are relevant to human obesity. Unlike mice, humans normally express agouti in adipose tissue. As shown in aP2 transgenic mice, high levels of insulin (especially in hyperinsulinemic type II diabetic patients) combined with agouti expression in adipose tissue may contribute to increased adiposity. Therefore, modulation of agouti expression/action may be a potential target in therapeutic intervention to treat obesity and diabetes.