We’ve reported a high expression of IGF-I in pancreatic islet β-cells

We’ve reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. In freshly isolated islets the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence 11 was observed exclusively in glucagon-producing islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced Quetiapine fumarate enzymatic activities. Dexamethasone (DEX) and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice 48 pre-incubation of DEX caused a significant decrease in insulin release while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells which together with DHC caused apoptosis and a significant decrease in Quetiapine fumarate proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets which may mediate part of the IGF-I effects on cell proliferation survival and insulin secretion. Introduction Insulin-like growth Quetiapine fumarate factor I Quetiapine fumarate (IGF-I) stimulates proliferation of pancreatic islet cells in a Quetiapine fumarate glucose-dependent manner protects the β-cells against the development of diabetes mellitus and exerts insulin-like effects on insulin target tissues [1 2 It activates IGF-I receptor through tyrosine phosphorylation and recruits intracellular substrates such as insulin receptor substrates (IRS) 1-4 and Shc which trigger activations of PI3K/Akt Ras/MAPK Quetiapine fumarate (Erk) and c-Jun N-terminal kinase (JNK) pathways [3] leading to stimulation on protein synthesis cell survival and proliferation. We have previously reported that MT-IGF mice exhibit both highly concentrated IGF-I overexpression in the β-cells of the pancreas and significant level of resistance to streptozotocin-induced diabetes [4]. To be able to explore book molecular goals that mediate IGF-I activities we lately performed a whole-genome cDNA microarray evaluation on total RNA ready from isolated islets of MT-IGF mice and discovered 82 genes particularly up- or down-regulated [5]. HSD11B1 encoding 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) was among the prominent goals previously not been shown to be normally portrayed in the pancreatic islets nor governed by IGF-I. It’s been known that glucocorticoids at pharmacological concentrations straight influence β-cell integrity and function [1 6 7 Either reduced or elevated insulin secretion continues to be reported in response to both severe (in mins) and extended (hours to times) exposure. Nevertheless the function of intracrine creation of energetic glucocorticoids inside the islets is certainly poorly understood. As a reductase 11 catalyzes the conversion of inert cortisone in humans (11-dehydrocorticosterone DHC in rodents) to active cortisol (corticosterone) in the liver and adipose tissues while the isozyme 11β-HSD2 (and 11β-HSD1 direct stimulation by IGF-I Comparable to our previous report [5] freshly isolated pancreatic islets pooled from 3 wild-type mice were allowed to recover overnight in culture medium made up of 11 mM glucose and 10% fetal bovine serum [13]. In a 24-well plate islets were distributed in triplicates of 20 for each condition and were cultured for 0 to 72 h in the same medium but contained only 1% fetal bovine serum upon treatment with or without 10?8 M of recombinant human IGF-I Long R3 (I1271 Sigma-Aldrich). Total protein lysate was used for Western blots against 11β-HSD1 and β-actin. To study its effect on protein degradation MIN6 cells overexpressing 11β-HSD1 (MIN6-HSD1 see below) were treated with cyclohexmide (CYC003 Bioshop; 10 mg/L 2 h) to block protein synthesis before IGF-I Hes2 treatment for 12-48 h. Dual-labeled immunofluorescence and immunohistochemistry Paraffin sections of the pancreas taken from 3-5 month aged male MT-IGF and wild-type littermates were dewaxed rehydrated and blocked with 10% donkey serum followed by incubation with rabbit anti-11β-HSD1 antibodies (1:100; H-10 sc-20175 Santa Cruz and ab83522 Abcam Cambridge MA) at 4°C overnight. After washing with PBS sections were independently stained with anti-glucagon (C-18 sc-7779 Santa Cruz) and guinea pig polyclonal anti-insulin (ab7842 Abcam) followed by Alexa Fluor 594 conjugated donkey anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-guinea pig IgG (Life technologies Carlsbad CA) [14 15 The images were analyzed using Axioshop 2 plus.