Dataset Information


Real-time quantitative PCR analysis of microRNAS in islets of mice exposed to low-protein diet during gestation

ABSTRACT: Maternal low-protein diet increases the susceptibility of offspring to type 2 diabetes. An insult to the pancreas during development can have long-term consequences on β-cell mass and function. Because nutrients and growth factors signaling converge on mTOR, we hypothesized that mTOR plays a central role in β-cell programming during fetal development. In this study, we revealed that newborns of dams exposed to low-protein diet (LP0.5) throughout pregnancy exhibited decreased insulin levels, β-cell fraction, and mTOR signaling. Adult LP0.5 offspring demonstrated enhanced insulin sensitivity but remained glucose intolerant due to an insulin secretory defect and not reduced β-cell mass. The insulin secretory defect was distal to Ca2+ influx, at the level of proinsulin biosynthesis and insulin content. LP0.5 islets exhibited reduced mTOR protein and increased expression of specific microRNAs. The reductions in mTOR protein and insulin secretion in LP0.5 islets were restored to normal by blockade of microRNAs 199a-3p and 342. Finally, transient activation of mTORC1 signaling in β-cells during the last week of pregnancy rescued the neonatal β-cell fraction defect and metabolic abnormalities in LP0.5 mice. These findings identify a major role of microRNAs and mTOR in β-cell programing by maternal low-protein diet. To investigate which microRNAs are altered in islets isolated from 2-3 months old male offspring of dams fed low-protein diet (LP0.5) or control diet throughout pregnancy Pancreatic islets were isolated from 2-3 months old male offspring of dams fed low-protein diet (LP0.5) or control diet throughout pregnancy. Islets were isolated by collagenase digestion. The pancreas was perfused via the common duct with 1 mg/ml collagenase XI (Sigma-Aldrich) in HBSS (Life Technologies). Pancreatic digestion was carried out at 37 °C for 15 min, after which cold HBSS with 2.5% FBS (Life Technologies) was added. The suspension was centrifuged at 1000 rpm for 30s, washed 3 times with HBSS with 2.5% FBS, resuspended in RPMI complete media (RPMI 1640 with 5 mM Glucose, 10% FBS,100 IU/ml penicillin, 100 g/ml streptomycin) and poured onto a 70 µm cell strainer (BD Falcon, BD Biosciences). Islets were rinsed and handpicked. Islets were allowed to recover overnight in RPMI complete media before RNA isolation using MirVana Kit (Life Technologies).

ORGANISM(S): Mus musculus  

SUBMITTER: Emilyn Alejandro   Emilyn U Alejandro  Ernesto Bernal-Mizrachi 

PROVIDER: E-GEOD-59279 | ArrayExpress | 2014-07-11



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