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

Ca2+ Entry via TRPC1 is Essential for Cellular Differentiation and Modulates Secretion via the SNARE Complex

Adipocyte functionality, including adipocyte differentiation and adipokine secretion, is essential in obesity-associated metabolic syndrome. Here, we provide evidence that Ca2+ influx in primary adipocytes, especially upon store-depletion, plays an important role in adipocyte differentiation, functionality, and subsequently metabolic regulation. The endogenous Ca2+ entry channel in both subcutaneous and visceral adipocytes was dependent on TRPC1-STIM1 and blocking Ca2+ entry with SKF-96365 or TRPC1-/- derived adipocytes inhibited adipocyte differentiation. Additionally, TRPC1-/- mice have decreased organ weight, but increased adipose deposition and reduced serum adiponectin and leptin concentrations, without affecting total adipokine expression. Mechanistically, TRPC1- mediated Ca2+ entry regulated SNARE complex formation and agonist –mediated secretion of adipokine loaded vesicles was inhibited in TRPC1-/- adipose. These results suggest an unequivocal role of TRPC1 in adipocytes differentiation and adiponectin secretion, and loss of TRPC1 disturbs metabolic homeostasis

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

Effects of prenatal low protein and postnatal high fat diets on visceral adipose tissue macrophage phenotypes and IL-6 expression in Sprague Dawley rat offspring

Adipose tissue macrophages (ATM) are implicated in adipose tissue inflammation and obesity-related insulin resistance. Maternal low protein models result in fetal programming of obesity. The study aims to answer whether maternal undernutrition by protein restriction affects the ATM M1 or M2 phenotype under postnatal high fat diet in F1 offspring. Using a rat model of prenatal low protein (LP, 8% protein) diet followed by a postnatal high fat energy diet (HE, 45% fat) or low fat normal energy diet (NE, 10% fat) for 12 weeks, we investigated the effects of these diets on adiposity, programming of the offspring ATM phenotype, and the associated inflammatory response in adipose tissue. Fat mass in newborn and 12-week old LP fed offspring was lower than that of normal protein (20%; NP) fed offspring; however, the adipose tissue growth rate was higher compared to the NP fed offspring. While LP did not affect the number of CD68+ or CD206+ cells in adipose tissue of NE offspring, it attenuated the number of these cells in offspring fed HE. In offspring fed HE, LP offspring had a lower percentage of CD11c+CD206+ ATMs, whose abundancy was correlated with the size of the adipocytes. Noteworthy, similar to HE treatment, LP increased gene expression of IL-6 within ATMs. Two-way ANOVA showed an interaction of prenatal LP and postnatal HE on IL-6 and IL-1β transcription. Overall, both LP and HE diets impact ATM phenotype by affecting the ratio of CD11c+CD206+ ATMs and the expression of IL-6

Additional file 2: of Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

Distribution of markers of white adipocytes and preadipocytes between the adipocyte fraction and SVF. The transcripts for ASC1 and Wdnm1-like (A) or PDGFR-alpha (B) were evaluated in the adipocyte fraction and SVF of 4 month and 10 days old mice kept at room temperature (22 °C). Transcripts were normalized to SVF. Values are means ± SEM (n = 3–5). (*) different from SVF, p ≤ 0.05, two-tailed t-test. (PPTX 97 kb

Additional file 1: of Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

Western blots of UCP1, beta-actin, FGF21, P2RX5, and CD137 in WAT of control mice (22 °C) and mice exposed to cold (5 °C). (PPTX 332 kb

Additional file 3: of Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

Thermoregulatory markers in cells from the SVF and beige adipocytes in vitro. The transcripts for CIDEA, Cox8b, and UCP1 were evaluated in SVF cells and beige adipocytes in culture. Transcripts were normalized to SVF cells. Values are means ± SEM (n = 3). (*) different from SVF cells, p ≤ 0.05, two-tailed t-test. (PPTX 71 kb

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