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).

Project description:Previously, we described a mouse model where the well-known reproductive carcinogen, diethylstilbestrol (DES), caused uterine adenocarcinoma following neonatal treatment. Tumor incidence was dose-dependent reaching >90% by 18 mo. following 1000 µg/kg/day of DES. These tumors followed the initiation/promotion model of hormonal carcinogenesis with developmental exposure as the initiator, and exposure to ovarian hormones at puberty as the promoter. To identify molecular pathways involved in DES-initiation events, uterine gene expression profiles were examined in prepubertal mice exposed to DES (1, 10 or 1000 µg/kg/day) on days 1-5 and compared to age-matched controls. Of more than 20,000 transcripts, approximately 3% were differentially expressed in at least one DES treatment group compared to controls; several transcripts demonstrated dose-responsiveness. Assessment of gene ontology annotation revealed alterations in genes associated with cell growth, differentiation, and adhesion. When expression profiles were compared to published studies of uteri from 5 day old DES-treated mice, or adult mice treated with 17β estradiol, similarities were seen suggesting persistent differential expression of estrogen responsive genes following developmental DES exposure. Moreover, several significantly altered genes have been identified in human uterine adenocarcinomas. Four altered genes [Lactotransferrin (Ltf), Transforming growth factor beta inducible (Tgfβ1), Cyclin D1 (Ccnd1), and Secreted frizzled-related protein 4 (Sfrp4)], selected for real time RT-PCR analysis, correlated well with the directionality of the microarray data. These data suggest altered gene expression profiles observed two weeks after treatment ceased, were imprinted at the time of developmental exposure and maybe related to the initiation events resulting in carcinogenesis. Experiment Overall Design: There were 3 DES doses (1, 10 and 1000 ug/kg/day) administered to neonates on days 1-5. RNA was pooled for 10 mice, with each dose having its own matching control (corn oil). Dye-flipped hybridizations were performed for each paired comparison.

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