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OBJECTIVE--The purpose of this study was to evaluate the role of the S6K arm of mammalian target of rapamycin complex 1 (mTORC1) signaling in regulation of β-cell mass and function. Additionally, we aimed to delineate the importance of in vivo S6K activation in the regulation of insulin signaling and the extent to which alteration of insulin receptor substrate (IRS) signaling modulates β-cell mass and function. RESEARCH DESIGN AND METHODS--The current experiments describe the phenotype of transgenic mice overexpressing a constitutively active form of S6K under the control of the rat insulin promoter. RESULTS--Activation of S6K signaling in these mice improved insulin secretion in the absence of changes in β-cell mass. The lack of β-cell mass expansion resulted from decreased [G.sub.1]-S progression and increased apoptosis. This phenotype was associated with increased p16 and p27 and decreased Cdk2 levels. The changes in cell cycle were accompanied by diminished survival signals because of impaired IRS/Akt signaling. CONCLUSIONS--This work defines the importance of S6K in regulation of β-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces β-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in β-cell failure and diabetes in conditions of nutrient overload. Diabetes 59:2390-2399, 2010, Pancreatic β-cells expand their function and mass in both physiologic and pathologic states of nutrient excess and increased insulin demand. Failure of β-cells to expand adequately in settings of increased [...]

OBJECTIVE--β-Cells are particularly susceptible to fatty acid-induced apoptosis associated with decreased insulin receptor/ phosphatidylinositol-3 kinase/Akt signaling and the activation of stress kinases. We examined the mechanism of fatty acid-induced apoptosis of mouse β-cells especially as related to the role played by endoplasmic reticulum (ER) stress-induced Foxo1 activation and whether decreasing Foxo1 activity could enhance cell survival. RESEARCH DESIGN AND METHODS--Mouse insulinoma (MIN6) cells were administered with fatty acids, and the role of Foxo1 in mediating effects on signaling pathways and apoptosis was examined by measuring Foxo1 activity and using dominant-negative Foxo1. RESULTS--Increasing fatty acid concentrations (100-400 µmol/1 palmitate or oleate) led to early Jun N[H.sub.2]-terminal kinase (JNK) activation that preceded induction of ER stress markers and apoptosis. Foxo1 activity was increased with fatty acid administration and by pharmacological inducers of ER stress, and this increase was prevented by JNK inhibition. Fatty acids induced nuclear localization of Foxo1 at 4 h when Akt activity was increased, indicating that FoxO1 activation was not mediated by JNK inhibition of Akt. In contrast, fatty acid administration for 24 h was associated with decreased insulin signaling. A dominant-negative Foxo1 adenovirus (Adv-DNFoxo) conferred cells with protection from ER stress and fatty acid-mediated apoptosis. Microarray analysis revealed that fatty acid induction of gene expression was in most cases reversed by Adv-DNFoxo, including the proapoptotic transcription factor CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein). CONCLUSIONS--Early induction of JNK and Foxo1 activation plays an important role in fatty acid-induced apoptosis. Expressing a dominant-negative allele of Foxo1 reduces expression of apoptotic and ER stress markers and promotes B-cell survival from fatty acid and ER stress, identifying a potential therapeutic target for preserving β-cells in type 2 diabetes., Insulin-resistant states are characterized by an initial adaptive expansion of β-cell mass to increase insulin production for maintaining euglycemia. However, a significant number of insulin-resistant individuals develop impaired insulin secretion [...]

Glucose controls islet β-cell mass and function at least in part through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway downstream of insulin signaling. The Foxo proteins, transcription factors known in other tissues to be negatively regulated by Akt activation, affect proliferation and metabolism. In this study, we tested the hypothesis that glucose regulates Foxo1 activity in the β-cell via an autocrine/paracrine effect of released insulin on its receptor. Mouse insulinoma cells (MIN6) were starved overnight for glucose (5 mmol/l) then refed with glucose (25 mmol/l), resulting in rapid Foxo1 phosphorylation (30 min, P < 0.05 vs. untreated). This glucose response was demonstrated to be time (0.5-2 h) and dose (5-30 mmol/l) dependent. The use of inhibitors demonstrated that glucose-induced Foxo1 phosphorylation was dependent upon depolarization, calcium influx, and PI3K signaling. Additionally, increases in glucose concentration over a physiological range (2.5-20 mmol/l) resulted in nuclear to cytoplasmic translocation of Foxo1. Phosphorylation and translocation of Foxo1 following glucose refeeding were eliminated in an insulin receptor knockdown cell line, indicating that the glucose effects are mediated primarily through the insulin receptor. Activity of Foxo1 was observed to increase with decreased glucose concentrations, assessed by an IGF binding protein-1 promoter luciferase assay. Starvation of MIN6 cells identified a putative Foxo1 target, Chop, and a Choppromoter luciferase assay in the presence of cotransfected Foxo1 supported this hypothesis. The importance of these observations was that nutritional alterations in the β-cell are associated with changes in Foxo1 transcriptional activity and that these changes are predominantly mediated through glucose-stimulated insulin secretion acting through its own receptor. Diabetes 55:1581-1591, 2006, Nutrients have major effects on pancreatic β-cell function and growth. The β-cells adapt to high concentrations of extracellular glucose and growth factors with concomitant enhanced secretion of insulin and an [...]

Proliferation is the major component for maintenance of β-cell mass in adult animals. Activation of phosphoinositide 3-kinase/Akt-kinase pathway is a critical regulator of β-cell mass. Pancreatic β-cell overexpression of constitutively active Akt in mice (ca[Akt.sup.Tg]) resulted in marked expansion of β-cell mass by increase in β-cell proliferation and size. The current studies provide new insights into the molecular mechanisms involved in β-Cell proliferation by Akt. Proliferation of β-cells in ca[Akt.sup.Tg] was associated with increased cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 (cdk4) activity. To determine the role of cdk4 in fl-cell proliferation induced by Akt, we generated ca[Akt.sup.Tg] mice that were homozygous, heterozygous, or nullizygous for cdk4. The results of these studies showed that deletion of one cdk4 allele significantly reduced β-cell expansion in ca[Akt.sup.Tg] mice by decreased proliferation. ca[Akt.sup.Tg] mice deficient in cdk4 developed β-cell failure and diabetes. These experiments suggest that Akt induces β-cell proliferation in a cdk4-dependent manner by regulation of cyclin D1, cyclin D2, and p21 levels. These data also indicate that alteration in levels of these cell cycle components could affect the maintenance of β-cell mass in basal states and the adaptation of β-cells to pathological states resulting in diabetes., The defects that result in diabetes are diverse, but pancreatic β-cell failure is a major component of this disease (1,2). Pancreatic β-cell mass results from a dynamic balance of neogenesis, [...]

Glucose and insulin are important regulators of islet β-cell growth and function by activating signaling pathways resulting in transcriptional changes that lead to adaptive responses. Several immediate early genes have been shown to be rapidly induced by glucose-activated depolarization in islet β-cells. The current studies address aspects of glucose-regulated transcription: 1) the number and characteristics of these genes, 2) if depolarization is the major mechanism, and 3) if glucose-stimulated insulin secretion is responsible, because insulin per se can activate transcription. Here, the expression profiles of glucose-responsive insulinoma cells 45 min after the addition of glucose, KCl to induce depolarization, or insulin were assessed by endocrine pancreas cDNA microarrays. Glucose activated more than 90 genes, representing diverse gene ontology functions, and most were not previously known to be glucose responsive. KCl activated 80% of these same glucose-regulated genes and, along with the effects of pretreatment with diazoxide, suggested that glucose signaling is mediated primarily via depolarization. There were > 150 genes activated by insulin, and remarkably 71% were also regulated by glucose. Preincubation with a phosphatidylinositol (PI) 3-kinase inhibitor resulted in almost total inhibition of depolarization and insulin-activated transcriptional responses. Thus, through gene expression profiling, these data demonstrate that glucose and insulin rapidly activate a PI 3-kinase pathway, resulting in transcription of a common set of genes. This is consistent with glucose activation of gene transcription either directly or indirectly through a paracrine/ autocrine effect via insulin release. These results illustrate that expression gene profiling can contribute to the elucidation of important β-cell biological functions., Pancreatic islet β-cells can be regulated by multiple stimuli, including nutrients and growth factors. β-Cell proliferation and function are controlled by plasma glucose concentration and by growth factors acting via [...]

The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed 'PancChip 4.0,' which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research., Despite recent progress in [beta]-cell biology and diabetes research, tools for the treatment of diabetes have not changed fundamentally. Although it is now clear that islet transplantation is a valuable [...]

During embryonic life, the development of a proper mass of mature pancreatic tissue is thought to require the proliferation of precursor cells, followed by their differentiation into endocrine or acinar [...]

Activins are members of the transforming growth factor-β superfamily. They have a wide range of biological effects on cell growth and differentiation. For transmembrane signaling, activins bind directly to activin [...]