Solid-liquid Two-phase Flow Numerical Simulation around Guide Vanes of Mixed-flow Water Turbine

AbstractBased on the N-S control equation and κ-ɛ-Ap turbulent model, the solid-liquid two-phase flow field around the guide vanes of a mixed-flow water turbine was simulated. The solid-liquid two-phase flow characters around the guide vanes were analyzed on the design work condition with different volume fraction and particles diameter of solid phase. The effects of the solid particles diameter and volume fraction on the flow around the guide vane are discussed, based on which the prediction and analysis of abrasion and cavitation properties on the guiding device were given. The prediction results were identical with the test date, which can provide reference on the abrasion prediction and optimal design of guide vane

β1 integrin-extracellular matrix interactions are essential for maintaining exocrine pancreas architecture and function

Integrin receptors are responsible for integrating extracellular matrix signals inside the cell. The most prominent integrin receptor, β1 integrin, has a role in cell function, survival and differentiation. Recently, we demonstrated a profound in vivo role of β1 integrin expression in the pancreas on glucose homeostasis and islet function. Here, we extend these results by examining the role of β1 integrin in exocrine pancreatic structure and function. Adult C57Bl/6 mice hemizygous for a collagen type Iα2 (Col1a2) promoter-controlled tamoxifen-inducible Cre recombinase gene and homozygous for loxP-β1 integrin were injected with tamoxifen or corn oil to generate mice deleted or not for β1 integrin. Pancreata derived from these male mice were analyzed by quantitative reverse transcriptase-polymerase chain reaction, western blot and immunofluorescence. Our results showed that β1 integrin-deficient mice displayed a significant decrease in pancreas weight with a significant reduction of amylase, regenerating islet-derived protein II and carboxypeptidase-A expression (P\u3c0.05-0.01). Compared with control pancreata, β1 integrin-deficient pancreata showed reduced mRNA expression of extracellular matrix (collagen type Iα2, fibronectin and laminin) genes (P\u3c0.05), detached acini clusters and lost focal adhesion structure. Moreover, β1 integrin-deficient pancreatic acinar cells displayed decreased proliferation (P\u3c0.05) and increased apoptosis (P\u3c0.001). Apoptosis was reduced to that of controls when isolated exocrine clusters were cultured in media supplemented with extracellular matrix proteins. Taken together, these results implicate β1 integrin as an essential component for maintaining exocrine pancreatic structure and function. © 2013 USCAP, Inc All rights reserved

Maintaining human fetal pancreatic stellate cell function and proliferation require β1 integrin and collagen I matrix interactions

Pancreatic stellate cells (PaSCs) are cells that are located around the acinar, ductal, and vasculature tissue of the rodent and human pancreas, and are responsible for regulating extracellular matrix (ECM) turnover and maintaining the architecture of pancreatic tissue. This study examines the contributions of integrin receptor signaling in human PaSC function and survival. Human PaSCs were isolated from pancreata collected during the 2nd trimester of pregnancy and identified by expression of stellate cell markers, ECM proteins and associated growth factors. Multiple integrins are found in isolated human PaSCs, with high levels of β1, a3 and a5. Cell adhesion and migration assays demonstrated that human PaSCs favour collagen I matrix, which enhanced PaSC proliferation and increased TGFβ1, CTGF and a3β1 integrin. Significant activation of FAK/ERK and AKT signaling pathways, and up-regulation of cyclin D1 protein levels, were observed within PaSCs cultured on collagen I matrix. Blocking β1 integrin significantly decreased PaSC adhesion, migration and proliferation, further complementing the aforementioned findings. This study demonstrates that interaction of β1 integrin with collagen I is required for the proliferation and function of human fetal PaSCs, which may contribute to the biomedical engineering of the ECM microenvironment needed for the efficient regulation of pancreatic development

Fibrin supports human fetal islet-epithelial cell differentiation via p70 s6k and promotes vascular formation during transplantation

The human fetal pancreas expresses a variety of extracellular matrix (ECM) binding receptors known as integrins. A provisional ECM protein found in blood clots that can bind to integrin receptors and promote β cell function and survival is fibrin. However, its role in support of human fetal pancreatic cells is unknown. We investigated how fibrin promotes human fetal pancreatic cell differentiation in vitro and in vivo. Human fetal pancreata were collected from 15 to 21 weeks of gestation and collagenase digested. Cells were then plated on tissue-culture polystyrene, or with 2D or 3D fibrin gels up to 2 weeks, or subcutaneously transplanted in 3D fibrin gels. The human fetal pancreas contained rich ECM proteins and expressed integrin αVβ3. Fibrin-cultured human fetal pancreatic cells had significantly increased expression of PDX-1, glucagon, insulin, and VEGF-A, along with increased integrin αVβ3 and phosphorylated FAK and p70 s6k. Fibrin-cultured cells treated with rapamycin, the mTOR pathway inhibitor, had significantly decreased phospho-p70 s6k and PDX-1 expression. Transplanting fibrin-mixed cells into nude mice improved vascularization compared with collagen controls. These results suggest that fibrin supports islet cell differentiation via p70 s6k and promotes vascularization in human fetal islet-epithelial clusters in vivo

Critical role of β1 integrin in postnatal beta-cell function and expansion

β1 integrin is essential for pancreatic beta-cell development and maintenance in rodents and humans. However, the effects of a temporal beta-cell specific β1 integrin knockout on adult islet function are unknown. We utilized a mouse insulin 1 promoter driven tamoxifen-inducible Cre-recombinase β1 integrin knockout mouse model (MIPβ1KO) to investigate β1 integrin function in adult pancreatic beta-cells. Adult male MIPβ1KO mice were significantly glucose intolerant due to impaired glucose-stimulated insulin secretion in vivo and ex vivo at 8 weeks post-tamoxifen. The expression of Insulin and Pancreatic and duodenal homeobox-1 mRNA was significantly reduced in MIPβ1KO islets, along with reductions in insulin exocytotic proteins. Morphological analyses demonstrated that beta-cell mass, islet density, and the number of large-sized islets was significantly reduced in male MIPβ1KO mice. Significant reductions in the phosphorylation of signaling molecules focal adhesion kinase, extracellular signal-regulated kinases 1 and 2, and v-Akt murine thymoma viral oncogene were observed in male MIPβ1KO islets when compared to controls. MIPβ1KO islets displayed a significant increase in protein levels of the apoptotic marker cleaved-Poly (ADP-ribose) polymerase and a reduction of the cell cycle marker cyclin D1. Female MIPβ1KO mice did not develop glucose intolerance or reduced beta-cell mass until 16 weeks post-tamoxifen. Glucose intolerance remained in both genders of aged MIPβ1KO mice. This data demonstrates that β1 integrin is required for the maintenance of glucose homeostasis through postnatal beta-cell function and expansion

Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8–20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8–15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14–20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/ exocrine granules

Inhibition of Gsk3β activity improves β-cell function in c-Kit\u3csup\u3eWv/+\u3c/sup\u3e male mice

Previous studies have shown that the stem cell marker, c-Kit, is involved in glucose homeostasis. We recently reported that c-KitWv/+ male mice displayed the onset of diabetes at 8 weeks of age; however, the mechanisms by which c-Kit regulates β-cell proliferation and function are unknown. The purpose of this study is to examine if c-KitWv/+ mutation-induced β-cell dysfunction is associated with downregulation of the phospho-Akt/Gsk3β pathway in c-KitWv/+ male mice. Histology and cell signaling were examined in C57BL/6J/KitWv/+ (c-Kit Wv/+) and wild-type (c-Kit+/+) mice using immunofluorescence and western blotting approaches. The Gsk3β inhibitor, 1-azakenpaullone (1-AKP), was administered to c-KitWv/+ and c-Kit+/+ mice for 2 weeks, whereby alterations in glucose metabolism were examined and morphometric analyses were performed. A significant reduction in phosphorylated Akt was observed in the islets of c-KitWv/+ mice (P\u3c0.05) along with a decrease in phosphorylated Gsk3β (P\u3c0.05), and cyclin D1 protein level (P\u3c0.01) when compared with c-Kit+/+ mice. However, c-KitWv/+ mice that received 1-AKP treatment demonstrated normal fasting blood glucose with significantly improved glucose tolerance. 1-AKP-treated c-KitWv/+ mice also showed increased β-catenin, cyclin D1 and Pdx-1 levels in islets, demonstrating that inhibition of Gsk3β activity led to increased β-cell proliferation and insulin secretion. These data suggest that c-KitWv/+ male mice had alterations in the Akt/Gsk3β signaling pathway, which lead to β-cell dysfunction by decreasing Pdx-1 and cyclin D1 levels. Inhibition of Gsk3β could prevent the onset of diabetes by improving glucose tolerance and β-cell function. © 2012 USCAP, Inc All rights reserved

Long-term c-Kit overexpression in beta cells compromises their function in ageing mice

Aims/hypothesis: c-Kit signalling regulates intracellular pathways that enhance beta cell proliferation, insulin secretion and islet vascularisation in mice up to 28 weeks of age and on short-term high-fat diet. However, long-term c-Kit activation in ageing mouse islets has yet to be examined. This study utilises beta cell-specific c-Kit-overexpressing transgenic (c-KitβTg) ageing mice (~60 weeks) to determine the effect of its activation on beta cell dysfunction and insulin secretion. Methods: Wild-type and c-KitβTg mice, aged 60 weeks, were examined using metabolic tests to determine glucose tolerance and insulin secretion. Pancreas histology and proteins in isolated islets were examined to determine the expression of beta cell transcription factors, proliferation and intracellular signalling. To determine the role of insulin receptor signalling in ageing c-KitβTg mice, we generated beta cell-specific inducible insulin receptor knockout in ageing c-KitβTg mice (c-KitβTg;βIRKO mice) and examined the ageing mice for glucose tolerance and islet histology. Results: Ageing c-KitβTg mice progressively developed glucose intolerance, compared with age-matched wild-type littermates, due to impaired insulin secretion. Increased beta cell mass, proliferation and nuclear forkhead box transcription factor O1 (FOXO1) expression and reduced exocytotic protein levels were detected in ageing c-KitβTg mouse islets. Protein analyses of isolated islets showed increased insulin receptor, phosphorylated IRS-1Ser612 and cleaved poly(ADP-ribose) polymerase levels in ageing c-KitβTg mice. Ageing c-KitβTg mouse islets treated ex vivo with insulin demonstrated reduced Akt phosphorylation, indicating that prolonged c-Kit induced beta cell insulin insensitivity. Ageing c-KitβTg;βIRKO mice displayed improved glucose tolerance and beta cell function compared with ageing c-KitβTg mice. Conclusions/interpretation: These findings indicate that long-term c-Kit overexpression in beta cells has a negative impact on insulin exocytosis and that temporally dependent regulation of c-Kit–insulin receptor signalling is important for optimal beta cell function

C-Kit and stem cell factor regulate PANC-1 cell differentiation into insulin-and glucagon-producing cells

Recent evidence has shown that stem cell factor (SCF) and its receptor, c-Kit, have an important role in pancreatic islet development by promoting islet cell differentiation and proliferation. In this study, we examined the role of c-Kit and SCF in the differentiation and proliferation of insulin- and glucagon-producing cells using a human pancreatic duct cell line (PANC-1). Our study showed that increased expression of endocrine cell markers (such as insulin and glucagon) and transcription factors (such as PDX-1 and PAX-6) coincided with a decrease in CK19 and c-Kit cells (P0.001) during PANC-1 cell differentiation, determined by immunofluorescence and qRT-PCR. Cells cultured with exogenous SCF showed an increase in insulin (26%) and glucagon (35%) cell differentiation (P0.01), an increase in cell proliferation (P0.05) and a decrease in cell apoptosis (P0.01). siRNA knockdown of c-Kit resulted in a decrease in endocrine cell differentiation with a reduction in PDX-1 and insulin mRNA, as well as the number of cells immunostaining for PDX-1 and insulin. Taken together, these results show that c-Kit/SCF interactions are involved in mediating islet-like cluster formation and islet-like cell differentiation in a human pancreatic duct cell line. © 2010 USCAP, Inc All rights reserved

Expression and function of αβ1 integrins in pancretic beta (INS-1) cells

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