Cooperativity between the preproinsulin mRNA untranslated regions Is necessary for glucose-stimulated translation

Glucose regulates proinsulin biosynthesis via stimulation of the translation of the preproinsulin mRNA in pancreatic β-cells. However, the mechanism by which this occurs has remained unclear. Using recombinant adenoviruses that express the preproinsulin mRNA with defined alterations, the untranslated regions (UTRs) of the preproinsulin mRNA were examined for elements that specifically control translation of the mRNA in rat pancreatic islets. These studies revealed that the preproinsulin 5′-UTR was necessary for glucose stimulation of preproinsulin mRNA translation, whereas the 3′-UTR appeared to suppress translation. However, together the 5′- and 3′-UTRs acted cooperatively to markedly increase glucose-induced proinsulin biosynthesis. In primary hepatocytes the presence of the preproinsulin 3′-UTR led to reduced mRNA levels compared with the presence of the SV40 3′-UTR, consistent with the presence of mRNA stability determinants in the 3′-UTR that stabilize the preproinsulin mRNA in a pancreatic β-cell-specific manner. Translation of these mRNAs in primary hepatocytes was not stimulated by glucose, indicating that regulated translation of the preproinsulin mRNA occurs in a pancreatic β-cell-specific manner. Thus, the untranslated regions of the preproinsulin mRNA play crucial roles in regulating insulin production and therefore glucose homeostasis by regulating the translation and the stability of the preproinsulin mRNA

Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

Objective: Glucagon-like peptide 1 (GLP-1) enhances insulin secretion and protects β-cell mass. Diabetes therapies targeting the GLP-1 receptor (GLP-1R), expressed in numerous tissues, have diminished dose-response in patients with type 2 diabetes compared with healthy human controls. The aim of this study was to determine the mechanistic causes underlying the reduced efficacy of GLP-1R ligands. Methods: Using primary mouse islets and the β-cell line MIN6, outcomes downstream of the GLP-1R were analyzed: Insulin secretion; phosphorylation of the cAMP-response element binding protein (CREB); cAMP responses. Signaling systems were studied by immunoblotting and qRT-PCR, and PKA activity was assayed. Cell surface localization of the GLP-1R was studied by confocal microscopy using a fluorescein-tagged exendin-4 and GFP-tagged GLP-1R. Results: Rodent β-cells chronically exposed to high glucose had diminished responses to GLP-1R agonists including: diminished insulin secretory response; reduced phosphorylation of (CREB); impaired cAMP response, attributable to chronically increased cAMP levels. GLP-1R signaling systems were affected by hyperglycemia with increased expression of mRNAs encoding the inducible cAMP early repressor (ICER) and adenylyl cyclase 8, reduced PKA activity due to increased expression of the PKA-RIα subunit, reduced GLP-1R mRNA expression and loss of GLP-1R from the cell surface. To specifically examine the loss of GLP-1R from the plasma membrane a GLP-1R-GFP fusion protein was employed to visualize subcellular localization. Under low glucose conditions or when PKA activity was inhibited, GLP-1R-GFP was found at the plasma membrane. Conversely high glucose, expression of a constitutively active PKA subunit, or exposure to exendin-4 or forskolin led to GLP-1R-GFP internalization. Mutation of serine residue 301 of the GLP-1R abolished the glucose-dependent loss of the receptor from the plasma membrane. This was associated with a loss of an interaction between the receptor and the small ubiquitin-related modifier (SUMO), an interaction that was found to be necessary for internalization of the receptor. Conclusions: These data show that glucose acting, at least in part, via PKA leads to the loss of the GLP-1R from the cell surface and an impairment of GLP-1R signaling, which may underlie the reduced clinical efficacy of GLP-1R based therapies in individuals with poorly controlled hyperglycemia

FFA2 Contribution to Gestational Glucose Tolerance Is Not Disrupted by Antibiotics

<div><p>During the insulin resistant phase of pregnancy, the mRNA expression of free fatty acid 2 receptor (<i>Ffar2</i>) is upregulated and as we recently reported, this receptor contributes to insulin secretion and pancreatic beta cell mass expansion in order to maintain normal glucose homeostasis during pregnancy. As impaired gestational glucose levels can affect metabolic health of offspring, we aimed to explore the role of maternal <i>Ffar2</i> expression during pregnancy on the metabolic health of offspring and also the effects of antibiotics, which have been shown to disrupt gut microbiota fermentative activity (the source of the FFA2 ligands) on gestational glucose homeostasis. We found that maternal <i>Ffar2</i> expression and impaired glucose tolerance during pregnancy had no effect on the growth rates, <i>ad lib</i> glucose and glucose tolerance in the offspring between 3 and 6 weeks of age. To disrupt short chain fatty acid production, we chronically treated WT mice and <i>Ffar2</i>^<i>-/-</i> mice with broad range antibiotics and further compared their glucose tolerance prior to pregnancy and at gestational day 15, and also quantified cecum and plasma SCFAs. We found that during pregnancy antibiotic treatment reduced the levels of SCFAs in the cecum of the mice, but resulted in elevated levels of plasma SCFAs and altered concentrations of individual SCFAs. Along with these changes, gestational glucose tolerance in WT mice, but not <i>Ffar2</i>^<i>-/-</i> mice improved while on antibiotics. Additional data showed that gestational glucose tolerance worsened in <i>Ffar2</i>^<i>-/-</i> mice during a second pregnancy. Together, these results indicate that antibiotic treatment alone is inadequate to deplete plasma SCFA concentrations, and that modulation of gut microbiota by antibiotics does not disrupt the contribution of FFA2 to gestational glucose tolerance.</p></div

Antibiotics alter the relative abundance of individual SCFAs in circulation during pregnancy.

<p>Total plasma SCFA levels which includes acetate, propionate, and butyrate measured in WT and <i>Ffar2</i>^-/- mice at G0 <b>(a)</b> and G15 <b>(b)</b> under control vs antibiotic-treated conditions. (<b>b-h</b>) Relative abundance of individual SCFAs (acetate, <b>c-d</b>; propionate, <b>e-f</b>; and butyrate, <b>g-h</b>) in WT and <i>Ffar2</i>^-/- mice at G0 (<b>c, e</b> and <b>g</b>) and G15 (<b>d, f</b> and <b>h</b>) under control vs antibiotic-treated conditions. WT, white bars; <i>Ffar2</i>^-/-, black bars. Data are represented as mean ± SEM n = 6–15, and were analyzed by Student’s t-test (*p ≤ 0.05).</p

Litter size and morbidity rate

<p>Litter size and morbidity rate</p