Differential effects of OATP2B1 on statin accumulation and toxicity in a beta cell model.

An increased risk of new-onset diabetes mellitus has been recently reported for statin therapy, and experimental studies have shown reduced glucose-stimulated insulin secretion (GSIS) and mitochondrial dysfunction in beta cells with effects differing among agents. Organic anion transporting polypeptide (OATP) 2B1 contributes to hepatic uptake of rosuvastatin, atorvastatin and pravastatin, three known substrates. Since OATP2B1 is present in beta cells of the human pancreas, we investigated if OATP2B1 facilitates the local accumulation of statins in a rat beta cell model INS-1 832/13 (INS-1) thereby amplifying statin-induced toxicity. OATP2B1 overexpression in INS-1 cells via adenoviral transduction showed 2.5-, 1.8- and 1.4-fold higher cellular retention of rosuvastatin, atorvastatin and pravastatin, respectively, relative to LacZ control, while absolute intracellular concentration was about twice as high for the lipophilic atorvastatin compared to the more hydrophilic rosuvastatin and pravastatin. After 24 h statin treatment at high concentrations, OATP2B1 enhanced statin toxicity involving activation of intrinsic apoptosis (caspase 3/7 activation) and mitochondrial dysfunction (NADH dehydrogenase activity) following rosuvastatin and atorvastatin, which was partly reversed by isoprenoids. OATP2B1 had no effect on statin-induced reduction in GSIS, mitochondrial electron transport chain complex expression or caspase 9 activation. We confirmed a dose-dependent reduction in insulin secretion by rosuvastatin and atorvastatin in native INS-1 with a modest change in cellular ATP. Collectively, our results indicate a role of OATP2B1, which is abundant in human beta cells, in statin accumulation and statin-induced toxicity but not insulin secretion of rosuvastatin and atorvastatin in INS-1 cells.

Beta-cell ß1 integrin deficiency affects in utero development of islet growth and vascularization.

The ß1 integrin subunit contributes to pancreatic beta cell growth and function through communication with the extracellular matrix (ECM). The effects of in vitro and in vivo ß1 integrin knockout have been extensively studied in mature islets, yet no study to date has examined how the loss of ß1 integrin during specific stages of pancreatic development impacts beta cell maturation. Beta-cell-specific tamoxifen-inducible Cre recombinase (MIP-CreERT) mice were crossed with mice containing floxed Itgb1 (ß1 integrin) to create an inducible mouse model (MIPß1KO) at the second transition stage (e13.5) of pancreas development. By e19.5-20.5, the expression of beta-cell ß1 integrin in fetal MIPß1KO mice was significantly reduced and these mice displayed decreased beta cell mass, density and proliferation. Morphologically, fetal MIPß1KO pancreata exhibited reduced islet vascularization and nascent endocrine cells in the ductal region. In addition, decreased ERK phosphorylation was observed in fetal MIPß1KO pancreata. The expression of transcription factors needed for beta-cell development was unchanged in fetal MIPß1KO pancreata. The findings from this study demonstrate that ß1 integrin signaling is required during a transition-specific window in the developing beta-cell to maintain islet mass and vascularization.

The marine compound and elongation factor 1A1 inhibitor, didemnin B, provides benefit in western diet-induced non-alcoholic fatty liver disease.

Inhibition of eukaryotic elongation factor 1A1 (EEF1A1) with the marine compound didemnin B decreases lipotoxic HepG2 cell death in vitro and improves early stage non-alcoholic fatty liver disease (NAFLD) in young genetically obese mice. However, the effects of didemnin B on NAFLD in a model of long-term diet-induced obesity are not known. We investigated the effects of didemnin B on NAFLD severity and metabolic parameters in western diet-induced obese mice, and on the cell types that contribute to liver inflammation and fibrosis in vitro. Male 129S6 mice were fed either standard chow or western diet for 26 weeks, followed by intervention with didemnin B (50 µg/kg) or vehicle by intraperitoneal (i.p.) injection once every 3 days for 14 days. Didemnin B decreased liver and plasma triglycerides, improved oral glucose tolerance, and decreased NAFLD severity. Moreover, didemnin B moderately increased hepatic expression of genes involved in ER stress response (Perk, Chop), and fatty acid oxidation (Fgf21, Cpt1a). In vitro, didemnin B decreased THP-1 monocyte proliferation, disrupted THP-1 monocyte-macrophage differentiation, decreased THP-1 macrophage IL-1ß secretion, and decreased hepatic stellate cell (HSteC) proliferation and collagen secretion under both basal and lipotoxic (high fatty acid) conditions. Thus, didemnin B improves hepatic steatosis, glucose tolerance, and blood lipids in obesity, in association with moderate, possibly hormetic, upregulation of pathways involved in cell stress response and energy balance in the liver. Furthermore, it decreases the activity of the cell types implicated in liver inflammation and fibrosis in vitro. These findings highlight the therapeutic potential of partial protein synthesis inhibition in the treatment of NAFLD.

Two-Week Isocaloric Time-Restricted Feeding Decreases Liver Inflammation without Significant Weight Loss in Obese Mice with Non-Alcoholic Fatty Liver Disease.

Prolonged, isocaloric, time-restricted feeding (TRF) protocols can promote weight loss, improve metabolic dysregulation, and mitigate non-alcoholic fatty liver disease (NAFLD). In addition, 3-day, severe caloric restriction can improve liver metabolism and glucose homeostasis prior to significant weight loss. Thus, we hypothesized that short-term, isocaloric TRF would improve NAFLD and characteristics of metabolic syndrome in diet-induced obese male mice. After 26 weeks of ad libitum access to western diet, mice either continued feeding ad libitum or were provided with access to the same quantity of western diet for 8 h daily, over the course of two weeks. Remarkably, this short-term TRF protocol modestly decreased liver tissue inflammation in the absence of changes in body weight or epidydimal fat mass. There were no changes in hepatic lipid accumulation or other characteristics of NAFLD. We observed no changes in liver lipid metabolism-related gene expression, despite increased plasma free fatty acids and decreased plasma triglycerides in the TRF group. However, liver Grp78 and Txnip expression were decreased with TRF suggesting hepatic endoplasmic reticulum (ER) stress and activation of inflammatory pathways may have been diminished. We conclude that two-week, isocaloric TRF can potentially decrease liver inflammation, without significant weight loss or reductions in hepatic steatosis, in obese mice with NAFLD.

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-KitßTg) ageing mice (~60 weeks) to determine the effect of its activation on beta cell dysfunction and insulin secretion. METHODS: Wild-type and c-KitßTg 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-KitßTg mice, we generated beta cell-specific inducible insulin receptor knockout in ageing c-KitßTg mice (c-KitßTg;ßIRKO mice) and examined the ageing mice for glucose tolerance and islet histology. RESULTS: Ageing c-KitßTg 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-KitßTg mouse islets. Protein analyses of isolated islets showed increased insulin receptor, phosphorylated IRS-1Ser612 and cleaved poly(ADP-ribose) polymerase levels in ageing c-KitßTg mice. Ageing c-KitßTg mouse islets treated ex vivo with insulin demonstrated reduced Akt phosphorylation, indicating that prolonged c-Kit induced beta cell insulin insensitivity. Ageing c-KitßTg;ßIRKO mice displayed improved glucose tolerance and beta cell function compared with ageing c-KitßTg 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.

Characterization of OATP1B3 and OATP2B1 transporter expression in the islet of the adult human pancreas.

Organic anion-transporting polypeptides (OATPs) are membrane proteins that mediate cellular uptake of structurally diverse endogenous and exogenous compounds, including bile salts, thyroid and sex hormones, pharmacological agents, and toxins. Roles of OATPs in human liver are well established. Our recent report suggested the presence of the hepatic transporter OATP1B3 in human ß cells. The aim of this study was to better characterize cellular localization and interindividual variation in OATP1B3 expression in human adult islets as a function of age, sex, and pancreatic disease, and to assess the expression of other OATPs. High transcript levels of OATP1B3, OATP2B1, OATP1A2, but not OATP1B1 were observed in isolated human adult islets. While OATP1B3 protein expression was variable, the carrier co-localized more frequently with glucagon-positive α cells than insulin-positive ß cells in islets of normal pancreatic tissues from ten subjects using dual immunostaining. Moreover, OATP1B3 co-staining with endocrine cells was two- to three-fold higher in older (≥60 years) than younger (<60 years) subjects. In comparison, in a subset of three individuals, OATP2B1 was primarily found in ß cells, suggesting a distinct expression pattern for OATP1B3 and OATP2B1 in islets. Abundant OATP1B3 staining was also observed in islet as well as ductal cells of diseased tissues of patients with pancreatitis or pancreatic adenocarcinoma. Considering the abundance of key OATP carriers in ß and α cells, potential implications of OATP transport in islet cell function may be suggested. Future studies are needed to gain insights into their specific endocrine roles as well as pharmacological relevance.

Rfx6 directs islet formation and insulin production in mice and humans.

Insulin from the beta-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the beta-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate beta-cells for patients with diabetes.

A survival Kit for pancreatic beta cells: stem cell factor and c-Kit receptor tyrosine kinase.

The interactions between c-Kit and its ligand, stem cell factor (SCF), play an important role in haematopoiesis, pigmentation and gametogenesis. c-Kit is also found in the pancreas, and recent studies have revealed that c-Kit marks a subpopulation of highly proliferative pancreatic endocrine cells that may harbour islet precursors. c-Kit governs and maintains pancreatic endocrine cell maturation and function via multiple signalling pathways. In this review we address the importance of c-Kit signalling within the pancreas, including its profound role in islet morphogenesis, islet vascularisation, and beta cell survival and function. We also discuss the impact of c-Kit signalling in pancreatic disease and the use of c-Kit as a potential target for the development of cell-based and novel drug therapies in the treatment of diabetes.

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 ß 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 αVß3. Fibrin-cultured human fetal pancreatic cells had significantly increased expression of PDX-1, glucagon, insulin, and VEGF-A, along with increased integrin αVß3 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.

Aldehyde dehydrogenase 1 activity in the developing human pancreas modulates retinoic acid signalling in mediating islet differentiation and survival.

AIMS/HYPOTHESIS: Aldehyde dehydrogenase 1 (ALDH1), a human stem-cell marker, is an enzyme responsible for converting retinaldehydes to retinoic acids (RAs) to modulate cell differentiation. However, data on expression levels and functional roles of ALDH1 during human fetal pancreatic development are limited. The focus of this study was to characterise ALDH1 expression patterns and to determine its functional role in islet cell differentiation. METHODS: The presence of ALDH1 in the human fetal pancreas (8-22 weeks) was characterised by microarray, quantitative RT-PCR, western blotting and immunohistological approaches. Isolated human fetal islet-epithelial cell clusters were treated with ALDH1 inhibitors, retinoic acid receptor (RAR) agonists and ALDH1A1 small interfering (si)RNA. RESULTS: In the developing human pancreatic cells, high ALDH1 activity frequently co-localised with key stem-cell markers as well as endocrine transcription factors. A high level of ALDH1 was expressed in newly differentiated insulin(+) cells and this decreased as development progressed. Pharmacological inhibition of ALDH1 activity in human fetal islet-epithelial cell clusters resulted in reduced endocrine cell differentiation and increased cell apoptosis, and was reversed with co-treatment of RAR/RXR agonists. Furthermore, siRNA knockdown of ALDH1A1 significantly decreased RAR expression and induced cell apoptosis via suppression of the phosphoinositide 3-kinase (PI3K) pathway and activation of caspase signals. CONCLUSIONS/INTERPRETATION: Our findings indicate that ALDH1(+) cells represent a pool of endocrine precursors in the developing human pancreas and that ALDH1 activity is required during endocrine cell differentiation. Inhibition of ALDH1-mediated retinoid signalling impairs human fetal islet cell differentiation and survival.

A fusion protein derived from plants holds promising potential as a new oral therapy for type 2 diabetes.

The incretin hormone glucagon-like peptide-1 (GLP-1) is recognized as a promising candidate for the treatment of type 2 diabetes (T2D), with one of its mimetics, exenatide (synthetic exendin-4) having already been licensed for clinical use. We seek to further improve the therapeutic efficacy of exendin-4 (Ex-4) using innovative fusion protein technology. Here, we report the production in plants a fusion protein containing Ex-4 coupled with human transferrin (Ex-4-Tf) and its characterization. We demonstrated that plant-made Ex-4-Tf retained the activity of both proteins. In particular, the fusion protein stimulated insulin release from pancreatic ß-cells, promoted ß-cell proliferation, stimulated differentiation of pancreatic precursor cells into insulin-producing cells, retained the ability to internalize into human intestinal cells and resisted stomach acid and proteolytic enzymes. Importantly, oral administration of partially purified Ex-4-Tf significantly improved glucose tolerance, whereas commercial Ex-4 administered by the same oral route failed to show any significant improvement in glucose tolerance in mice. Furthermore, intraperitoneal (IP) injection of Ex-4-Tf showed a beneficial effect in mice similar to IP-injected Ex-4. We also showed that plants provide a robust system for the expression of Ex-4-Tf, producing up to 37 µg prEx-4-Tf/g fresh leaf weight in transgenic tobacco and 137 µg prEx-4-Tf/g freshweight in transiently transformed leaves of N. benthamiana. These results indicate that Ex-4-Tf holds substantial promise as a new oral therapy for type 2 diabetes. The production of prEx-4-Tf in plants may offer a convenient and cost-effective method to deliver the antidiabetic medicine in partially processed plant food products.

Design and Mechanism Study of High-Safety and Long-Life Electrolyte for High-Energy-Density Lithium-Ion Batteries.

Although nonflammable electrolytes are beneficial for battery safety, they often adversely affect the electrochemical performance of lithium-ion batteries due to their poor compatibility with electrodes. Herein, we design a nonflammable electrolyte consisting of cyclic carbonate and 2,2-difluoroethyl acetate (DFEA) solvents paired with several surface-film-forming additives, significantly improving the safety and cycling performance of NMC811||SiOx/graphite pouch cells. The DFEA solvent exhibits not only good flame retardancy but also lower lowest unoccupied molecular orbital (LUMO) energy, promoting the formation of a robust inorganic-rich and gradient-architecture hybrid interface between the SiOx/graphite anode and electrolyte. The double insurance of good flame retardancy of the DFEA solvent and decreased exothermic effects of both bulk electrolyte and DFEA-derived solid electrolyte interphase (SEI) can ensure the high safety of the pouch cell. Moreover, the highly robust SEI can prevent the excessive reduction decomposition of the electrolyte and alleviate the structural decay of the anode, which can restrain the formation of lithium deposition on the anode surface and further suppress the structural decay of NMC materials. This contributes to the unprecedented cycling performance of the NMC811||SiOx/graphite pouch cells with a capacity retention of 80% after 1000 cycles at a 0.33C rate.

Downregulation of Fas activity rescues early onset of diabetes in c-Kit(Wv/+) mice.

c-Kit and its ligand stem cell factor (SCF) are important for ß-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering ß-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of ß-cell mass and function with upregulation of Fas expression in c-Kit(Wv/+) mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in ß-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates ß-cell survival and function. We generated double mutant, c-Kit(Wv/+);Fas(lpr/lpr) (Wv(-/-)), mice to study the physiological and functional role of Fas with respect to ß-cell function in c-Kit(Wv/+) mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-Kit(Wv/+) mice showed a significant increase in ß-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv(-/-) mice displayed improved ß-cell function, with significantly enhanced insulin secretion and increased ß-cell mass and proliferation compared with Wv(+/+) mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-κB pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of ß-cell survival and function.

ß1 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, ß1 integrin, has a role in cell function, survival and differentiation. Recently, we demonstrated a profound in vivo role of ß1 integrin expression in the pancreas on glucose homeostasis and islet function. Here, we extend these results by examining the role of ß1 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-ß1 integrin were injected with tamoxifen or corn oil to generate mice deleted or not for ß1 integrin. Pancreata derived from these male mice were analyzed by quantitative reverse transcriptase-polymerase chain reaction, western blot and immunofluorescence. Our results showed that ß1 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<0.05-0.01). Compared with control pancreata, ß1 integrin-deficient pancreata showed reduced mRNA expression of extracellular matrix (collagen type Iα2, fibronectin and laminin) genes (P<0.05), detached acini clusters and lost focal adhesion structure. Moreover, ß1 integrin-deficient pancreatic acinar cells displayed decreased proliferation (P<0.05) and increased apoptosis (P<0.001). 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 ß1 integrin as an essential component for maintaining exocrine pancreatic structure and function.

Activation of protein kinase Cδ leads to increased pancreatic acinar cell dedifferentiation in the absence of MIST1.

Pancreatic ductal adenocarcinoma (PDAC) has a 5 year survival rate post-diagnosis of < 5%. Individuals with chronic pancreatitis (CP) are 20-fold more likely to develop PDAC, making it a significant risk factor for PDAC. While the relationship for the increased susceptibility to PDAC is unknown, loss of the acinar cell phenotype is common to both pathologies. Pancreatic acinar cells can dedifferentiate or trans-differentiate into a number of cell types including duct cells, ß cells, hepatocytes and adipocytes. Knowledge of the molecular pathways that regulate this plasticity should provide insight into PDAC and CP. MIST1 (encoded by Bhlha15 in mice) is a transcription factor required for complete acinar cell maturation. The goal of this study was to examine the plasticity of acinar cells that do not express MIST1 (Mist1(-/-) ). The fate of acinar cells from C57Bl6 or congenic Mist1(-/-) mice expressing an acinar specific, tamoxifen-inducible Cre recombinase mated to Rosa26 reporter LacZ mice (Mist1(CreERT/-) R26r) was determined following culture in a three-dimensional collagen matrix. Mist1(CreERT/-) R26r acini showed increased acinar dedifferentiation, formation of ductal cysts and transient increases in PDX1 expression compared to wild-type acinar cells. Other progenitor cell markers, including Foxa1, Sox9, Sca1 and Hes1, were elevated only in Mist1(-/-) cultures. Analysis of protein kinase C (PKC) isoforms by western blot and immunofluorescence identified increased PKCε accumulation and nuclear localization of PKCδ that correlated with increased duct formation. Treatment with rottlerin, a PKCδ-specific inhibitor, but not the PKCε-specific antagonist εV1-2, reduced acinar dedifferentiation, progenitor gene expression and ductal cyst formation. Immunocytochemistry on CP or PDAC tissue samples showed reduced MIST1 expression combined with increased nuclear PKCδ accumulation. These results suggest that the loss of MIST1 is a common event during PDAC and CP and events that affect MIST1 function and expression may increase susceptibility to these pathologies.

Isocyanate Additives Improve the Low-Temperature Performance of LiNi0.8Mn0.1Co0.1O2||SiOx@Graphite Lithium-Ion Batteries.

LiNi0.8Mn0.1Co0.1O2||SiOx@graphite (NCM811||SiOx@G)-based lithium-ion batteries (LIBs) exhibit high energy density and have found wide applications in various fields, including electric vehicles. Nonetheless, its low-temperature performance remains a challenge. One of the most efficacious strategies to enhance the low-temperature functionality of battery is the development of appropriate electrolytes with low-temperature suitability. Herein, p-tolyl isocyanate (PTI) and 4-fluorophenyl isocyanate (4-FI) are used as additive substances to integrate into the electrolytes to improve the low-temperature performance of the battery. Theoretical calculations and experimental results indicate that PTI and 4-FI can both preferentially generate a stable SEI on the electrode surface, which is beneficial to reduce the interfacial impedance. As a result, the additive, i.e. 4-FI, is superior to PTI in improving the low-temperature performance of the battery due to the optimization of F in the SEI membrane components. At room temperature, the cyclic stability of the NCM811/SiOx@G pouch cell increases from 92.5% (without additive) to 94.2% (with 1% 4-FI) after 200 cycles at 0.5 C. Under the operating temperature of -20 °C, the cyclic stability of the NCM811/SiOx@G pouch cell increases from 83.2% (without additive) to 88.6% (with 1% 4-FI) after 100 cycles at 0.33 C. Therefore, a rational interphase design involving the modification of the additive structure is a cost-effective way to improve the performance of LIBs.

Inhibition of Gsk3ß activity improves ß-cell function in c-KitWv/+ male mice.

Previous studies have shown that the stem cell marker, c-Kit, is involved in glucose homeostasis. We recently reported that c-Kit(Wv/+) male mice displayed the onset of diabetes at 8 weeks of age; however, the mechanisms by which c-Kit regulates ß-cell proliferation and function are unknown. The purpose of this study is to examine if c-Kit(Wv/+) mutation-induced ß-cell dysfunction is associated with downregulation of the phospho-Akt/Gsk3ß pathway in c-Kit(Wv/+) male mice. Histology and cell signaling were examined in C57BL/6J/Kit(Wv/+) (c-Kit(Wv/+)) and wild-type (c-Kit(+/+)) mice using immunofluorescence and western blotting approaches. The Gsk3ß inhibitor, 1-azakenpaullone (1-AKP), was administered to c-Kit(Wv/+) 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-Kit(Wv/+) mice (P<0.05) along with a decrease in phosphorylated Gsk3ß (P<0.05), and cyclin D1 protein level (P<0.01) when compared with c-Kit(+/+) mice. However, c-Kit(Wv/+) mice that received 1-AKP treatment demonstrated normal fasting blood glucose with significantly improved glucose tolerance. 1-AKP-treated c-Kit(Wv/+) mice also showed increased ß-catenin, cyclin D1 and Pdx-1 levels in islets, demonstrating that inhibition of Gsk3ß activity led to increased ß-cell proliferation and insulin secretion. These data suggest that c-Kit(Wv/+) male mice had alterations in the Akt/Gsk3ß signaling pathway, which lead to ß-cell dysfunction by decreasing Pdx-1 and cyclin D1 levels. Inhibition of Gsk3ß could prevent the onset of diabetes by improving glucose tolerance and ß-cell function.

ß1-Integrin-A Key Player in Controlling Pancreatic Beta-Cell Insulin Secretion via Interplay With SNARE Proteins.

Shortcomings in cell-based therapies for patients with diabetes have been revealed to be, in part, a result of an improper extracellular matrix (ECM) environment. In vivo, pancreatic islets are emersed in a diverse ECM that provides physical support and is crucial for healthy function. ß1-Integrin receptors have been determined to be responsible for modulation of beneficial interactions with ECM proteins influencing beta-cell development, proliferation, maturation, and function. ß1-Integrin signaling has been demonstrated to augment insulin secretion by impacting the actin cytoskeleton via activation of focal adhesion kinase and downstream signaling pathways. In other secretory cells, evidence of a bidirectional relationship between integrins and exocytotic machinery has been demonstrated, and, thus, this relationship could be present in pancreatic beta cells. In this review, we will discuss the role of ECM-ß1-integrin interplay with exocytotic proteins in controlling pancreatic beta-cell insulin secretion through their dynamic and unique signaling pathway.

Collagen IV-ß1-Integrin Influences INS-1 Cell Insulin Secretion via Enhanced SNARE Protein Expression.

ß1-integrin is a key receptor that regulates cell-ECM interactions and is important in maintaining mature beta-cell functions, including insulin secretion. However, there is little reported about the relationship between ECM-ß1-integrin interactions and exocytotic proteins involved in glucose-stimulated insulin secretion (GSIS). This study examined the effect of collagen IV-ß1-integrin on exocytotic proteins (Munc18-1, Snap25, and Vamp2) involved in insulin secretion using rat insulinoma (INS-1) cell line. Cells cultured on collagen IV (COL IV) had promoted INS-1 cell focal adhesions and GSIS. These cells also displayed changes in levels and localization of ß1-integrin associated downstream signals and exocytotic proteins involved in insulin secretion. Antibody blocking of ß1-integrin on INS-1 cells cultured on COL IV showed significantly reduced cell adhesion, spreading and insulin secretion along with reduced exocytotic protein levels. Blocking of ß1-integrin additionally influenced the cellular localization of exocytotic proteins during the time of GSIS. These results indicate that specific collagen IV-ß1-integrin interactions are critical for proper beta-cell insulin secretion.

N-acetyl-L-cysteine treatment reduces beta-cell oxidative stress and pancreatic stellate cell activity in a high fat diet-induced diabetic mouse model.

Obesity plays a major role in type II diabetes (T2DM) progression because it applies metabolic and oxidative stress resulting in dysfunctional beta-cells and activation of intra-islet pancreatic stellate cells (PaSCs) which cause islet fibrosis. Administration of antioxidant N-acetyl-L-cysteine (NAC) in vivo improves metabolic outcomes in diet-induced obese diabetic mice, and in vitro inhibits PaSCs activation. However, the effects of NAC on diabetic islets in vivo are unknown. This study examined if dosage and length of NAC treatment in HFD-induced diabetic mice effect metabolic outcomes associated with maintaining healthy beta-cells and quiescent PaSCs, in vivo. Male C57BL/6N mice were fed normal chow (ND) or high-fat (HFD) diet up to 30 weeks. NAC was administered in drinking water to HFD mice in preventative treatment (HFDpNAC) for 23 weeks or intervention treatment for 10 (HFDiNAC) or 18 (HFDiNAC+) weeks, respectively. HFDpNAC and HFDiNAC+, but not HFDiNAC, mice showed significantly improved glucose tolerance and insulin sensitivity. Hyperinsulinemia led by beta-cell overcompensation in HFD mice was significantly rescued in NAC treated mice. A reduction of beta-cell nuclear Pdx-1 localization in HFD mice was significantly improved in NAC treated islets along with significantly reduced beta-cell oxidative stress. HFD-induced intra-islet PaSCs activation, labeled by αSMA, was significantly diminished in NAC treated mice along with lesser intra-islet collagen deposition. This study determined that efficiency of NAC treatment is beneficial at maintaining healthy beta-cells and quiescent intra-islet PaSCs in HFD-induced obese T2DM mouse model. These findings highlight an adjuvant therapeutic potential in NAC for controlling T2DM progression in humans.