Aims/hypothesis Morphological changes that occur during pancreatic endocrine cell differentiation have been shown in rodent systems to be dependent on sequential alterations in transcription factor expression. However, similar data for humans have been limited. The aim of the present study was to provide a connection between pancreatic morphology, transcription factor gene expression and protein localisation during human fetal development. Methods Human fetal pancreases were examined at early (8-12 weeks of fetal age), middle (14-16 weeks) and late (19-21 weeks) stages, using immunohistological, microarray and qRT-PCR analyses. Results We observed a significant decrease in pancreatic duodenal homeobox 1 (PDX-1) + /cytokeratin 19 + cells (p<0.001), with a simultaneous increase in PDX-1 + /insulin + cells from 8 to 21 weeks (p<0.05). Increased PDX-1/ insulin co-localisation within islet clusters was noted, while no co-expression of PDX-1 with glucagon was found, suggesting that loss of PDX-1 is essential for alpha cell formation. Given that neurogenin 3 (NGN3) expression is critical for establishing the endocrine cell programme in the rodent pancreas, we examined its expression pattern and colocalisation in PDX-1 + , insulin + and glucagon + cells. Colocalisation of NGN3 with PDX-1, insulin and glucagon was noted during early development, with significant decreases in middle and late stages (p<0.001). Our microarray and co-localisation analyses of transcription factors linked to NGN3 demonstrated that ISL1 transcription factor (ISL1), neurogenic differentiation 1 (NEUROD1), NK2 related transcription factor related, locus 2 (NKX2-2) and paired box gene 6 (PAX6) were upregulated during development and present in all four endocrine cell types, while NK6 related transcription factor related, locus 1 (NKX6-1) was expressed exclusively in beta cells. Conclusions/interpretation This study is an important step towards identifying key molecular factors involved in development of the human fetal endocrine pancreas.

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectininduced signaling and other molecules involved is not clearly defined. Therefore, we investigated the role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular (2.5 g/ml) or full-length (5 g/ml) adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression, and secretion were measured. Lysates were subjected to Western blot analysis to determine phosphorylation of AMP-activated protein kinase (AMPK), Akt, or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min or 1-and 24-h stimulation. ERK was significantly phosphorylated after 24-h treatment with adiponectin, whereas Akt was activated at all time points examined. 24-h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis, and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor), and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, the data also suggest adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.Type 2 diabetes is characterized by both a loss of insulin sensitivity and beta cell dysfunction in the pancreas. Adiponectin is an adipocyte-derived hormone that shows a strong negative correlation with insulin resistance and obesity (1, 2). Studies show that whereas adiponectin knock-out mice develop insulin resistance and glucose intolerance when challenged with a high fat diet, adiponectin-overexpressing mice are highly insulin-sensitive and are resistant to diet-induced diabetes (3-6). Adiponectin is reported to stimulate fatty acid oxidation and glucose uptake and reduce gluconeogenesis in myocytes and hepatocytes, thus increasing peripheral insulin sensitivity (7). Reduced adiponectin levels are also correlated with reduced vascular function and an increase in coronary artery disease (8).Adiponectin forms trimers or higher order complexes including hexamers and oligomers (9). These higher order complexes of full-length adiponectin (fAd) 4 are the primary circulating forms, and localized proteolytic cleavage of fAd has been shown to produce globular adiponectin (gAd) (10). Two proposed homologous adiponectin receptors (AdipoR1 and AdipoR2) were cloned and shown to be wi...

c-Kit tyrosine receptor kinase, a well-established stem cell marker, is expressed in a variety of tissues including the pancreas. The involvement of c-Kit in fetal rat and human endocrine pancreatic development, survival, and function has been well characterized but primarily using in vitro experimental approaches. Therefore, the aim of the current study was to examine whether deficiency of a functional c-Kit receptor would have physiological and functional implications in vivo. We characterized the c-Kit mutant mouse, c-Kit(W-v/+), to evaluate the in vivo role of c-Kit in beta-cell growth and function. Here we report that male c-Kit(W-v/+) mice, at 8 wk of age, showed high fasting blood glucose levels and impaired glucose tolerance, which was associated with low levels of insulin secretion after glucose stimulation in vivo and in isolated islets. Morphometric analysis revealed that beta-cell mass was significantly reduced (50%) in male c-Kit(W-v/+) mice when compared with controls (c-Kit(+/+)) (P < 0.05). In parallel, a reduction in pancreatic duodenal homeobox-1 and insulin gene expression in whole pancreas as well as isolated islets of c-Kit(W-v/+) male mice was noted along with a decrease in pancreatic insulin content. Furthermore, the reduction in beta-cell mass in male c-Kit(W-v/+) mice was associated with a decrease in beta-cell proliferation. Interestingly, these changes were not observed in female c-Kit(W-v/+) mice until 40 wk of age. Our results clearly demonstrate that the c-Kit receptor is involved in the regulation of glucose metabolism, likely through an important role in beta-cell development and function.

β1-Integrin, a critical regulator of β cell survival and function, has been shown to protect against cell death and promote insulin expression and secretion in rat and human islet cells in vitro. The aim of the present study was to examine whether the knockout of β1-integrin in collagen I-producing cells would have physiological and functional implications in pancreatic endocrine cells in vivo. Using adult mice with a conditional knockout of β1-integrin in collagen I-producing cells, the effects of β1-integrin deficiency on glucose metabolism and pancreatic endocrine cells were examined. Male β1-integrin-deficient mice display impaired glucose tolerance, with a significant reduction in pancreatic insulin content (p < 0.01). Morphometric analysis revealed a significant reduction in β cell mass (p < 0.001) in β1-integrin-deficient mice, along with a significant decrease in β cell proliferation, Pdx-1 and Nkx6.1 expression when compared with controls. Interestingly, these physiological and morphometric alterations in female β1-integrin-deficient mice were less significant. Furthermore, β1-integrin-deficient mice displayed decreased FAK (p < 0.05) and ERK1/2 (p < 0.001) phosphorylation, reduced cyclin D1 levels (p < 0.001) and increased caspase 3 cleavage (p < 0.01), while no changes in Akt phosphorylation were observed, indicating that the β1-integrin signals through the FAK-MAPK-ERK pathway in vivo. Our results demonstrate that β1-integrin is involved in the regulation of glucose metabolism and contributes to the maintenance of β cell survival and function in vivo.

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β1-integrin is a well-established regulator of β-cell activities; however, the role of its associated α-subunits is relatively unknown. Previously, we have shown that human fetal islet and INS-1 cells highly express α3β1-integrin and that collagens I and IV significantly enhance their survival and function; in addition, blocking β1 function in the fetal islet cells decreased adhesion on collagen I and increased apoptosis. The present study investigates the effect of blocking α3. Using α3 blocking antibody or small interfering RNA, the effects of α3-integrin blockade were examined in isolated human fetal or adult islet cells or INS-1 cells, cultured on collagens I or IV. In parallel, β1 blockade was analyzed in INS-1 cells. Perturbing α3 function in human islet or INS-1 cells resulted in significant decreases in cell function (adhesion, spreading, proliferation and Pdx1 and insulin expression/secretion), primarily on collagen IV. A significant decrease in focal adhesion kinase and ERK1/2 phosphorylation and increased caspase3 cleavage were observed on both collagens. These effects were similar to changes after β1 blockade. Interestingly, only α3 blockade reduced expression of phospho-Akt and members of its downstream signaling cascades (glycogen synthase kinase β and X-linked inhibitor of apoptosis), demonstrating a specific effect of α3 on the phosphatidylinositol 3-kinase/Akt pathway. These results suggest that α3- as well as β1-integrin-extracellular matrix interactions are critical for modulating β-cell survival and function through specialized signaling cascades and enhance our understanding of how to improve islet microenvironments for cell-based treatments of diabetes.

Focal adhesion kinase (FAK) plays a central role in integrin signalling, which regulates growth and survival of tumours. Here we show that FAK protein levels are increased in adipose tissue of insulin-resistant obese mice and humans. Disruption of adipocyte FAK in mice or in 3T3 L1 cells decreases adipocyte survival. Adipocyte-specific FAK knockout mice display impaired adipose tissue expansion and insulin resistance on prolonged metabolic stress from a high-fat diet or when crossed on an obese db/db or ob/ob genetic background. Treatment of these mice with a PPARγ agonist does not restore adiposity or improve insulin sensitivity. In contrast, inhibition of apoptosis, either genetically or pharmacologically, attenuates adipocyte death, restores normal adiposity and improves insulin sensitivity. Together, these results demonstrate that FAK is required for adipocyte survival and maintenance of insulin sensitivity, particularly in the context of adipose tissue expansion as a result of caloric excess.

Integrin-extracellular matrix interactions are important determinants of beta cell behaviours. The β1 integrin is a well-known regulator of beta cell activities; however, little is known of its associated α subunits. In the present study, αβ1 integrin expression was examined in the rat insulinoma cell line (INS-1) to identify their role in beta cell survival and function. Seven α subunits associated with β1 integrin were identified, including α1-6 and αV. Among these heterodimers, α3β1 was most highly expressed. Common ligands for the α3β1 integrin, including fibronectin, laminin, collagen I and collagen IV were tested to identify the most suitable matrix for INS-1 cell proliferation and function. Cells exposed to collagen I and IV demonstrated significant increases in adhesion, spreading, cell viability, proliferation, and FAK phosphorylation when compared to cells cultured on fibronectin, laminin and controls. Integrin-dependent attachment also had a beneficial effect on beta cell function, increasing Pdx-1 and insulin gene and protein expression on collagens I and IV, in parallel with increased basal insulin release and enhanced insulin secretion upon high glucose challenge. Furthermore, functional blockade of α3β1 integrin decreased cell adhesion, spreading and viability on both collagens and reduced Pdx-1 and insulin expression, indicating that its interactions with collagen matrices are important for beta cell survival and function. These results demonstrate that specific αβ1 integrin-ECM interactions are critical regulators of INS-1 beta cell survival and function and will be important in designing optimal conditions for cell-based therapies for diabetes treatment.