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

Dominant mutations at the agouti locus such as viable yellow (Avy) 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 (Ca2+)-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

n3 and n6 polyunsaturated fatty acids differentially modulate prostaglandin E secretion but not markers of lipogenesis in adipocytes

A dramatic rise in the incidence of obesity in the U.S. has accelerated the search for interventions that may impact this epidemic. One recently recognized target for such intervention is adipose tissue, which secretes a variety of bioactive substances including prostaglandins. Prostaglandin E2 (PGE2) has been shown to decrease lipolysis in adipocytes, but limited studies have explored alternative mechanisms by which PGE2 might impact obesity, such as adipogenesis or lipogenesis. Studies conducted on ApcMin/+ mice indicated that selective inhibition of the cyclooxygenase (COX)-2 enzyme led to significant reductions in fatty acid synthase (FAS) activity in adipose tissue suggesting lipogenic effects of PGE2. To further investigate whether these lipid mediators directly regulate lipogenesis, we used 3T3-L1 adipocytes to determine the impact of eicosapentaenoic acid (EPA) and celecoxib on PGE2 formation and FAS used as a lipogenic marker. Both arachidonic acid (AA) and EPA dose-dependently increased PGE secretion from adipocytes. AA was expectedly more potent and exhibiting at 150 uM dose a 5-fold increase in PGE2 secretion over EPA. Despite higher secretion of PGE by EPA and AA compared to control, neither PUFA significantly altered FAS activity. By contrast both AA and EPA significantly decreased FAS mRNA levels. Addition of celecoxib, a selective COX-2 inhibitor, significantly decreased PGE2 secretion (p < 0.05) versus control, and also significantly decreased FAS activity (p < 0.05). Unexpectedly, the combination of exogenous PGE2 and celecoxib further decreased the FAS activity compared to PGE2 alone or untreated controls. In conclusion, EPA-mediated inhibition of AA metabolism did not significantly alter FAS activity while both AA and EPA significantly decreased FAS mRNA expression. COX-2 inhibition significantly decreased PGE2 production resulting in a decrease in FAS activity and expression that was not reversed with the addition of exogenous PGE2, suggesting an additional mechanism that is independent of COX-2

Nutritional epigenomics: a portal to disease prevention

Epigenetics can be defined as inheritable and reversible phenomena that affect gene expression without altering the underlying base pair sequence. Epigenomics is the study of genome-wide epigenetic modifications. Because gene expression changes are critical in both normal development and disease progression, epigenetics is widely applicable to many aspects of biological research. The influences of nutrients and bioactive food components on epigenetic phenomena such as DNA methylation and various types of histone modifications have been extensively investigated. Because an individual's epigenetic patterns are established during early gestation and are changed and personalized by environmental factors during our lifetime, epigenetic mechanisms are quite important in the development of transgenerational and adult obesity as well as in the development of diabetes mellitus. Aging and cancer demonstrate profound genome-wide DNA methylation changes, suggesting that nutrition may affect the aging process and cancer development through epigenetic mechanisms

Tart Cherry Reduces Inflammation in Adipose Tissue of Zucker Fatty Rats and Cultured 3T3-L1 Adipocytes

Obesity increases adipose tissue inflammation and secretion of pro-inflammatory adipokines, which have systemic effects on the organism&#8217;s health status. Our objective was to dissect mechanisms of anti-inflammatory effects of tart cherry (TC) in adipose tissue of Zucker fatty rats, and cultured 3T3-L1 adipocytes. Rats were fed either a control diet, or 4% TC powder diets for eight weeks. Body and epididymal fat pad weights were not significantly different between control and TC groups. However, rats fed the TC diet had significantly reduced adipose tissue inflammation (p &lt; 0.05), as determined by reduced mRNA levels of pro-inflammatory markers including interleukin-6 (IL-6), tumor necrosis factor alpha (TNF&#945;), interleukin-1beta (IL-1&#946;), monocyte chemoattractant protein 1 (MCP-1), inducible nitric oxide synthase (iNOS), and CD-11b, and increased mRNA levels of type-1 arginase (Arg-1) anti-inflammatory marker. Consistent with these in vivo results, TC significantly decreased expression of IL-6 mRNA and protein levels in lipopolysaccharide (LPS) stimulated adipocytes compared to those stimulated with LPS, but no TC. Moreover, both in vivo (rat adipose tissue) and in vitro (3T3-L1 adipocytes), phosphorylation of p65-NF-&#954;B subunit was significantly reduced by TC. Additionally, TC decreased mRNA expression of fatty acid synthase (FASN), and increased expression of peroxisome proliferator-activated receptor alpha (PPAR&#945;), master regulator of lipid oxidation, and anti-oxidant markers nuclear factor erythroid-derived 2-related factor (NRFs) in both models. In conclusion, our findings indicate that TC downregulates inflammation in part via the nuclear factor kappa B (NF-&#954;B) pathway in adipose tissue. Thus, TC may serve as a potential intervention to reduce obesity-associated inflammation

Leptin differentially regulate STAT3 activation in <it>ob/ob</it> mouse adipose mesenchymal stem cells

<p>Abstract</p> <p>Background</p> <p>Leptin-deficient <it>ob/ob</it> mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes <it>ex vivo</it> and thereby contribute toward increased adipocyte cell numbers, obesity, and inflamm ation. Currently, information is lacking regarding regulation of adipose stem cell numbers as well as leptin-induced inflammation and its signaling pathway in <it>ob/ob</it> mice.</p> <p>Methods</p> <p>Using leptin deficient <it>ob/ob</it> mice, we investigated whether leptin injection into <it>ob/ob</it> mice increases adipose stem cell numbers and adipose tissue inflammatory marker MCP-1 mRNA and secretion levels. We also determined leptin mediated signaling pathways in the adipose stem cells.</p> <p>Results</p> <p>We report here that adipose stem cell number is significantly increased following leptin injection in <it>ob/ob</it> mice and with treatment of isolated stem cells with leptin <it>in vitro</it>. Leptin also up-regulated MCP-1 secretion in a dose- and time-dependent manner. We further showed that increased MCP-1 mRNA levels were due to increased phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) Ser727 but not STAT3 Tyr705 phosphorylation, suggesting differential regulation of MCP-1 gene expression under basal and leptin-stimulated conditions in adipose stem cells.</p> <p>Conclusions</p> <p>Taken together, these studies demonstrate that leptin increases adipose stem cell number and differentially activates STAT3 protein resulting in up-regulation of MCP-1 gene expression. Further studies of mechanisms mediating adipose stem cell hyperplasia and leptin signaling in obesity are warranted and may help identify novel anti-obesity target strategies.</p

ob/ob mouse adipose mesenchymal stem cells

Background: Leptin-deficient ob/ob mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes ex vivo and thereby contribute toward increased adipocyte cell numbers, obesity, and inflamm ation. Currently, information is lacking regarding regulation of adipose stem cell numbers as well as leptin-induced inflammation and its signaling pathway in ob/ob mice. Methods: Using leptin deficient ob/ob mice, we investigated whether leptin injection into ob/ob mice increases adipose stem cell numbers and adipose tissue inflammatory marker MCP-1 mRNA and secretion levels. We also determined leptin mediated signaling pathways in the adipose stem cells. Results: We report here that adipose stem cell number is significantly increased following leptin injection in ob/ob mice and with treatment of isolated stem cells with leptin in vitro. Leptin also up-regulated MCP-1 secretion in a dose- and time-dependent manner. We further showed that increased MCP-1 mRNA levels were due to increased phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) Ser727 but not STAT3 Tyr705 phosphorylation, suggesting differential regulation of MCP-1 gene expression under basal and leptin-stimulated conditions in adipose stem cells. Conclusions: Taken together, these studies demonstrate that leptin increases adipose stem cell number and differentially activates STAT3 protein resulting in up-regulation of MCP-1 gene expression. Further studies of mechanisms mediating adipose stem cell hyperplasia and leptin signaling in obesity are warranted and may help identify novel anti-obesity target strategies