Project description:Recent discovery reveals HFD insult can cause insulin resistance very rapidly, but the underlying mechanism is still not well understood. We performed a short term experiment in a Diet Induced Insulin resistance mouse model. Objective: Insulin resistance (IR) is one of the earliest predictors of type 2 diabetes. However, diagnosis of IR is limited. High fat fed mouse models provide key insights into IR. We hypothesized that early features of IR are associated with persistent changes in gene expression (GE) and endeavoured to (a) develop novel methods for improving signal:noise in analysis of human GE using mouse models; (b) identify a GE motif that accurately diagnoses IR in humans; and (c) identify novel biology associated with IR in humans. Methods: We integrated human muscle GE data with longitudinal mouse GE data and developed an unbiased three-level cross-species analysis platform (single-gene, gene-set and networks) to generate a gene expression motif (GEM) indicative of IR. A logistic regression classification model validated GEM in 3 independent human datasets (n =115). Results: This GEM of 93 genes substantially improved diagnosis of IR compared to routine clinical measures across multiple independent datasets. Individuals misclassified by GEM possessed other metabolic features raising the possibility that they represent a separate metabolic subclass. The GEM was enriched in pathways previously implicated in insulin action and revealed novel associations between Î²-catenin and Jak1 and IR. Functional analyses using small molecule inhibitors showed an important role for these proteins in insulin action. Conclusions: This study shows that systems approaches for identifying molecular signatures provides a powerful way to stratify individuals into discrete metabolic groups. Moreover, we speculate that the Î²-catenin pathway may represent a novel biomarker for IR in humans that warrant future investigation. Comparison of gene expression in muscle tissue during High Fat Diet (HFD) time course (day 5 and day 42). Chow diet will serve as control for HFD. 4 samples per group serve as experimental replicates.

Project description:In obesity an increase in β-cell mass occurs to cope with the rise in insulin demand. This β -cell plasticity is essential to avoid the onset of hyperglycemia, although the molecular mechanisms that regulate this process remain unclear. This study analyzed the role of adipose tissue in the control of β -cell replication. Using a diet-induced model of obesity, we obtained conditioned media from three different white adipose tissue depots. Only in the adipose tissue depot surrounding the pancreas did the diet induce changes that led to an increase in INS1E cells and the islet replication rate. To identify the factors responsible for this proliferative effect, adipose tissue gene expression analysis was conducted by microarrays and quantitative RT-PCR. Of all the differentially expressed proteins, only the secreted ones were studied. IGF binding protein 3 (Igfbp3) was identified as the candidate for this effect. Furthermore, in the conditioned media, although the blockage of IGFBP3 led to an increase in the proliferation rate, the blockage of IGF-I receptor decreased it. Taken together, these data show that obesity induces specific changes in the expression profile of the adipose tissue depot surrounding the pancreas, leading to a decrease in IGFBP3 secretion. This decrease acts in a paracrine manner, stimulating the β -cell proliferation rate, probably through an IGF-I-dependent mechanism. This cross talk between the visceral-pancreatic adipose tissue and β -cells is a novel mechanism that participates in the control of β -cell plasticity. (Endocrinology 153: 177–187, 2012) Adult male Wistar rats (Charles River Laboratories, Wilmington, MA), 7 wk old (weighing 225–250 g), were caged individually in a 12-h light, 12-h dark cycle in a temperature- and humidity-controlled environment. Animals were divided into two dietary sets for 30 days. One group was fed with standard chow diet (supplying 8% of calories as fat; type AO4 from Panlab, Barcelona, Spain). The second group was fed with a cafeteria diet (66% of calories as fat), as previously described (Endocrinology 146:4362–4369, 2005). Adipose tissue from the mesenteric surrounding the pancreas (pMES), was excised, weighed, cut, and rapidly frozen in liquid nitrogen for RNA isolation. Ten micrograms of total RNA from pMES adipose tissue were converted into cRNA, biotinylated, fragmented, and hybridized to GeneChip Rat Genome 230 2.0 (Affymetrix, Santa Clara, CA). Five microarrays were hybridized, three with independent samples coming from rats fed with standard chow (lean group) and two with independent samples coming from rats fed with the cafeteria diet (obese group).

Project description:Diet-induced obesity is reported to induce a phenotypic switch in adipose tissue macrophages from an antiinflammatory M2 state to a proinflammatory M1 state. Telmisartan, an angiotensin II type 1 receptor antagonist and a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, reportedly has beneficial effects on insulin sensitivity. We studied the effects of telmisartan on the adipose tissue macrophage phenotype in high fat-fed mice. Telmisartan was administered for 5 weeks to high fat-fed C57BL/6 mice. Insulin sensitivity, macrophage infiltration, and the gene expressions of M1 and M2 markers in epididymal fat tissues were examined. Insulin- or a glucose-tolerance test showed that telmisartan treatment improved insulin resistance, decreasing the body weight gain, visceral fat weight and adipocyte size without affecting the amount of food intake. Telmisartan treatment reduced the number of CD11c-positive cells and crown-like structures. Telmisartan reduced the mRNA expressions of M1 macrophage markers, such as TNF-alpha and IL-6, and increased the expression of M2 markers, such as IL-10 and Mgl2. The reduction of M1 macrophage markers, as well as the increased gene expression of M2 markers especially IL-10, is a possible mechanism for the improvement of insulin sensitivity by telmisartan. Six-week-old male C57BL/6J mice were purchased from CLEA Japan. The mice were fed a chow that contained 10% of its calories from fat (control) or a high-fat diet (HFD) that contained 30% of its calories from fat for 24 weeks. The high fat-fed mice were randomized to 3 groups. Either telmisartan (~3 mg/kg/day) in drinking water (HFD+Tel), candesartan (~3 mg/kg/day) in drinking water (HFD+Can), or a HFD without any drugs (HFD) was administered for the next 5 weeks. Two mice were treated per group. Epididymal adipose tissues were rapidly removed from each mouse. Gene expression in epididymal fat tissue was analyzed using a GeneChip® system with the Mouse Genome 430 2.0 Array, which was spotted with 45,101 probe sets (Affymetrix, Santa Clara, CA, USA). Sample preparation for the array hybridization was performed according to the manufacturer’s instructions. In short, 5 μg of total RNA was used to synthesize double-stranded cDNA using the GeneChip® Expression 3′-Amplification Reagents One-Cycle cDNA Synthesis Kit (Affymetrix). Biotin-labeled cRNA was then synthesized from the cDNA using GeneChip® Expression 3′-Amplification Reagents for IVT Labeling (Affymetrix). After fragmentation, the biotinylated cRNA was hybridized to arrays at 45 °C for 16 h. The arrays were washed, stained with streptavidin-phycoerythrin, and scanned using a probe array scanner. The scanned chip was analyzed using the GeneChip Analysis Suite software (Affymetrix). Hybridization intensity data were converted into a presence/absence call for each gene, and changes in gene expression between experiments were detected by a comparison analysis. Data was shown as the fold change relative to the expression level of normal chow-fed mice.

Project description:Obesity is associated with an increase in β-cell mass in response to the rising demand for insulin. β-cell plasticity is essential to maintaining glucose homeostasis, however, the cellular and molecular mechanisms by which β-cell mass is regulated remain poorly understood. Recently, we described the existence of a crosstalk between the peripancreatic adipose tissue and β-cells as a novel mechanism that participates in the regulation of β-cell plasticity. Here, we identify the secreted frizzled-related protein (Sfrp) 5 as down-regulated in the pancreatic islets of obese rats as well as in the pancreatic islets of human obese patients. Our results demonstrate that the silencing of Sfrp5 induces an increase in β-cell proliferation, which we correlate with the activation of Wnt signaling and of the MAPK and PI3 kinase pathways. Together, these findings expand our understanding of the mechanisms underlying β-cell proliferation under conditions of obesity. Furthermore, this study opens new insights into the specific targeting of Sfrp5 as a novel therapeutic strategy for balancing β-cell mass. Adult male Wistar rats (Charles River Laboratories, Wilmington, MA), 7 wk old (weighing 225–250 g), were caged individually in a 12-h light, 12-h dark cycle in a temperature- and humidity-controlled environment. Animals were divided into two dietary sets for 10 days. One group was fed with standard chow diet (supplying 8% of calories as fat; type AO4 from Panlab, Barcelona, Spain). The second group was fed with a cafeteria diet (66% of calories as fat), as previously described (Endocrinology 2012;153:177-87). Pancreatic islets were isolated through collagenase perfusion of the pancreas, Histopaque gradient and hand-picking under microscopic guidance. Ten micrograms of total RNA from islets were converted into cRNA, biotinylated, fragmented, and hybridized to GeneChip Rat Genome 230 2.0 (Affymetrix, Santa Clara, CA). Ten microarrays were hybridized, five with independent samples coming from rats fed with standard chow (lean group) and five with independent samples coming from rats fed with the cafeteria diet (obese group).

Project description:Obesity is associated with an increase in ?-cell mass in response to the rising demand for insulin. ?-cell plasticity is essential to maintaining glucose homeostasis, however, the cellular and molecular mechanisms by which ?-cell mass is regulated remain poorly understood. Recently, we described the existence of a crosstalk between the peripancreatic adipose tissue and ?-cells as a novel mechanism that participates in the regulation of ?-cell plasticity. Here, we identify the secreted frizzled-related protein (Sfrp) 5 as down-regulated in the pancreatic islets of obese rats as well as in the pancreatic islets of human obese patients. Our results demonstrate that the silencing of Sfrp5 induces an increase in ?-cell proliferation, which we correlate with the activation of Wnt signaling and of the MAPK and PI3 kinase pathways. Together, these findings expand our understanding of the mechanisms underlying ?-cell proliferation under conditions of obesity. Furthermore, this study opens new insights into the specific targeting of Sfrp5 as a novel therapeutic strategy for balancing ?-cell mass. Adult male Wistar rats (Charles River Laboratories, Wilmington, MA), 7 wk old (weighing 225–250 g), were caged individually in a 12-h light, 12-h dark cycle in a temperature- and humidity-controlled environment. Animals were divided into two dietary sets for 30 days. One group was fed with standard chow diet (supplying 8% of calories as fat; type AO4 from Panlab, Barcelona, Spain). The second group was fed with a cafeteria diet (66% of calories as fat), as previously described (Endocrinology 2012;153:177-87). Pancreatic islets were isolated through collagenase perfusion of the pancreas, Histopaque gradient and hand-picking under microscopic guidance. Ten micrograms of total RNA from islets were converted into cRNA, biotinylated, fragmented, and hybridized to GeneChip Rat Genome 230 2.0 (Affymetrix, Santa Clara, CA). Ten microarrays were hybridized, five with independent samples coming from rats fed with standard chow (lean group) and five with independent samples coming from rats fed with the cafeteria diet (obese group).

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Cross-species Gene Expression Analysis Identifies a Novel Set of Genes Implicated in Human Insulin Sensitivity

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