Contribution of the Long Noncoding RNA H19 to ß-Cell Mass Expansion in Neonatal and Adult Rodents.

Pancreatic ß-cell expansion throughout the neonatal period is essential to generate the appropriate mass of insulin-secreting cells required to maintain blood glucose homeostasis later in life. Hence, defects in this process can predispose to diabetes development during adulthood. Global profiling of transcripts in pancreatic islets of newborn and adult rats revealed that the transcription factor E2F1 controls expression of the long noncoding RNA H19, which is profoundly downregulated during the postnatal period. H19 silencing decreased ß-cell expansion in newborns, whereas its re-expression promoted proliferation of ß-cells in adults via a mechanism involving the microRNA let-7 and the activation of Akt. The offspring of rats fed a low-protein diet during gestation and lactation display a small ß-cell mass and an increased risk of developing diabetes during adulthood. We found that the islets of newborn rats born to dams fed a low-protein diet express lower levels of H19 than those born to dams that did not eat a low-protein diet. Moreover, we observed that H19 expression increases in islets of obese mice under conditions of increased insulin demand. Our data suggest that the long noncoding RNA H19 plays an important role in postnatal ß-cell mass expansion in rats and contributes to the mechanisms compensating for insulin resistance in obesity.

Metabolic fate of glucose and candidate signaling and excess-fuel detoxification pathways in pancreatic ß-cells.

Glucose metabolism promotes insulin secretion in ß-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes ß-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the ß-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in ß-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in ß-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.

Contribution of Intronic miR-338-3p and Its Hosting Gene AATK to Compensatory ß-Cell Mass Expansion.

The elucidation of the mechanisms directing ß-cell mass regeneration and maintenance is of interest, because the deficit of ß-cell mass contributes to diabetes onset and progression. We previously found that the level of the microRNA (miRNA) miR-338-3p is decreased in pancreatic islets from rodent models displaying insulin resistance and compensatory ß-cell mass expansion, including pregnant rats, diet-induced obese mice, and db/db mice. Transfection of rat islet cells with oligonucleotides that specifically block miR-338-3p activity increased the fraction of proliferating ß-cells in vitro and promoted survival under proapoptotic conditions without affecting the capacity of ß-cells to release insulin in response to glucose. Here, we evaluated the role of miR-338-3p in vivo by injecting mice with an adeno-associated viral vector permitting specific sequestration of this miRNA in ß-cells. We found that the adeno-associated viral construct increased the fraction of proliferating ß-cells confirming the data obtained in vitro. miR-338-3p is generated from an intron of the gene coding for apoptosis-associated tyrosine kinase (AATK). Similarly to miR-338-3p, we found that AATK is down-regulated in rat and human islets and INS832/13 ß-cells in the presence of the cAMP-raising agents exendin-4, estradiol, and a G-protein-coupled Receptor 30 agonist. Moreover, AATK expression is reduced in islets of insulin resistant animal models and selective silencing of AATK in INS832/13 cells by RNA interference promoted ß-cell proliferation. The results point to a coordinated reduction of miR-338-3p and AATK under insulin resistance conditions and provide evidence for a cooperative action of the miRNA and its hosting gene in compensatory ß-cell mass expansion.

Epidermal growth factor receptor signaling promotes pancreatic ß-cell proliferation in response to nutrient excess in rats through mTOR and FOXM1.

The cellular and molecular mechanisms underpinning the compensatory increase in ß-cell mass in response to insulin resistance are essentially unknown. We previously reported that a 72-h coinfusion of glucose and Intralipid (GLU+IL) induces insulin resistance and a marked increase in ß-cell proliferation in 6-month-old, but not in 2-month-old, Wistar rats. The aim of the current study was to identify the mechanisms underlying nutrient-induced ß-cell proliferation in this model. A transcriptomic analysis identified a central role for the forkhead transcription factor FOXM1 and its targets, and for heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a ligand of the EGF receptor (EGFR), in nutrient-induced ß-cell proliferation. Phosphorylation of ribosomal S6 kinase, a mammalian target of rapamycin (mTOR) target, was increased in islets from GLU+IL-infused 6-month-old rats. HB-EGF induced proliferation of insulin-secreting MIN6 cells and isolated rat islets, and this effect was blocked in MIN6 cells by the EGFR inhibitor AG1478 or the mTOR inhibitor rapamycin. Coinfusion of either AG1478 or rapamycin blocked the increase in FOXM1 signaling, ß-cell proliferation, and ß-cell mass and size in response to GLU+IL infusion in 6-month-old rats. We conclude that chronic nutrient excess promotes ß-cell mass expansion via a pathway that involves EGFR signaling, mTOR activation, and FOXM1-mediated cell proliferation.

MicroRNAs contribute to compensatory ß cell expansion during pregnancy and obesity.

Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional ß cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory ß cell mass expansion is largely unknown. We found in rodents that ß cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in ß cells using specific anti-miR molecules mimicked gene expression changes occurring during ß cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory ß cell mass expansion occurring under different insulin resistance states.

Beta-cell failure in diet-induced obese mice stratified according to body weight gain: secretory dysfunction and altered islet lipid metabolism without steatosis or reduced beta-cell mass.

OBJECTIVE: C57Bl/6 mice develop obesity and mild hyperglycemia when fed a high-fat diet (HFD). Although diet-induced obesity (DIO) is a widely studied model of type 2 diabetes, little is known about beta-cell failure in these mice. RESEARCH DESIGN AND METHODS: DIO mice were separated in two groups according to body weight gain: low- and high-HFD responders (LDR and HDR). We examined whether mild hyperglycemia in HDR mice is due to reduced beta-cell mass or function and studied islet metabolism and signaling. RESULTS: HDR mice were more obese, hyperinsulinemic, insulin resistant, and hyperglycemic and showed a more altered plasma lipid profile than LDR. LDR mice largely compensated insulin resistance, whereas HDR showed perturbed glucose homeostasis. Neither LDR nor HDR mice showed reduced beta-cell mass, altered islet glucose metabolism, and triglyceride deposition. Insulin secretion in response to glucose, KCl, and arginine was impaired in LDR and almost abolished in HDR islets. Palmitate partially restored glucose- and KCl-stimulated secretion. The glucose-induced rise in ATP was reduced in both DIO groups, and the glucose-induced rise in Ca(2+) was reduced in HDR islets relatively to LDR. Glucose-stimulated lipolysis was decreased in LDR and HDR islets, whereas fat oxidation was increased in HDR islets only. Fatty acid esterification processes were markedly diminished, and free cholesterol accumulated in HDR islets. CONCLUSIONS: beta-Cell failure in HDR mice is not due to reduced beta-cell mass and glucose metabolism or steatosis but to a secretory dysfunction that is possibly due to altered ATP/Ca(2+) and lipid signaling, as well as free cholesterol deposition.

Glucagon-like peptide-1 induced signaling and insulin secretion do not drive fuel and energy metabolism in primary rodent pancreatic beta-cells.

BACKGROUND: Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic beta-cells, in particular cAMP, Ca(2+) and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors. METHODOLOGY/PRINICIPAL FINDINGS: GLP-1 or Ex-4 at high glucose caused release (approximately 20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on beta-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca(2+)](i) and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered. CONCLUSIONS/SIGNIFICANCE: The results indicate that GLP-1 barely affects beta-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the beta-cell, and that the beta-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a "push" (fuel substrate driven) process, rather than a "pull" mechanism secondary to enhanced insulin release as well as to Ca(2+), cAMP and PKB signaling.

Pioglitazone acutely reduces insulin secretion and causes metabolic deceleration of the pancreatic beta-cell at submaximal glucose concentrations.

Thiazolidinediones (TZDs) have beneficial effects on glucose homeostasis via enhancement of insulin sensitivity and preservation of beta-cell function. How TZDs preserve beta-cells is uncertain, but it might involve direct effects via both peroxisome proliferator-activated receptor-gamma-dependent and -independent pathways. To gain insight into the independent pathway(s), we assessed the effects of short-term (

Glucose represses PPARα gene expression via AMP-activated protein kinase but not via p38 mitogen-activated protein kinase in the pancreatic ß-cell.

BACKGROUND: Peroxisome proliferator-activated receptor α (PPARα) regulates the expression of fatty acid metabolism genes and is thought to play a role in the regulation of insulin secretion and lipid detoxification. We have examined the mechanism whereby glucose decreases PPARα gene expression in the pancreatic ß-cell. METHODS: INS832/13 ß-cell and isolated rat islets were incubated at 3 and 20 mM glucose for 18 h in the absence or presence of adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators and inhibitors, as well as p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. In another set of experiments, INS832/13 were infected with an adenovirus expressing a dominant-negative form of AMPK. PPARα expression levels were measured by reverse transcription polymerase chain reaction and Western blot. RESULTS: Elevated glucose reduced the abundance of the PPARα transcript and protein, and its target genes acyl-coenzyme A (CoA) oxidase (ACO) and uncoupling protein 2 (UCP-2) in INS832/13 ß-cell and isolated rat islets. Glucose reduced AMPK activity, while the AMPK activators 5-amino-4-imidazolecarboxamide riboside and metformin increased PPARα expression and suppressed the action of glucose. By contrast, the AMPK inhibitor compound C mimicked the glucose effect. A dominant negative form of AMPKα reduced the PPARα, ACO and UCP-2 transcripts to the same extent as elevated glucose. Pharmacological evidence indicated that glucose-regulated PPARα expression does not involve p38 MAPK, a target of AMPK in several cell types. CONCLUSIONS: The results indicate that glucose represses PPARα gene expression via AMPK, but not via p38 MAPK in the ß-cell.