Insulin mRNA Expression Research Articles

A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.

Silibinin protects β cells from glucotoxicity through regulation of the Insig-1/SREBP-1c pathway.

Exposure to high glucose may cause glucotoxicity, leading to pancreatic β cell dysfunction including cell apoptosis, impaired glucose‑stimulated insulin secretion (GSIS) and intracellular lipid accumulation. Sterol regulatory element binding protein-1c (SREBP-1c), a key nuclear transcription factor that regulates lipid metabolism, has been proven to play a role in insulin secretion. Insulin induced gene-1 (Insig-1) is an upstream regulatory factor of SREBP-1c. The overexpression of Insig-1 significantly inhibits SREBP-1c expression and thereby blocks the expression of downstream genes. It has been proven that silibinin, a natural flavanone, is involved in a variety of biological functions. In the present study, we examined whether silibinin protects high glucose-induced β cell dysfunction through the Insig-1/SREBP-1c pathway. Our data demonstrated that 30.0 µM of silibinin significantly improved cell viability (P<0.05) after rat insulinoma INS-1 cells were exposed to high glucose for 72 h. Silibinin partially attenuated GSIS following exposure to high glucose for either 24 or 72 h (both P<0.05). As shown by reverse transcription quantitative PCR, silibinin upregulated the mRNA expression of insulin secretion‑related genes [insulin receptor substrate 2 (IRS-2), pancreatic and duodenal homeobox 1 (PDX-1) and insulin], but downregulated uncoupling protein‑2 (UCP-2) expression. Silibinin inhibited intracellular lipid accumulation and free fatty acid (FFA) synthesis. Further experiments revealed that silibinin improved β cell function through the regulation of the Insig-1/SREBP-1c pathway. In conclusion, these results clearly suggest that the protection of β cells from glucotoxicity can be significantly enhanced through the regulation of the Insig-1/SREBP-1c pathway. Thus, silibinin may be a novel therapeutic agent for β cell dysfunction.

Isolation, characterization and potential role in beta cell-endothelium cross-talk of extracellular vesicles released from human pancreatic islets.

The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets.

TGFβ activates insulin‐like growth factor 1‐mediated signaling pathway in human liver cancer cells (144.9)

Transforming growth factor β (TGFβ) potently inhibits the growth of human epithelial cells. However, neoplastic epithelial cells become resistant to TGFβ‐mediated mitoinhibition, and the mechanisms for this alteration during tumorigenesis are not fully understood. In this study, we demonstrate that TGFβ sensitises PLC/PRF/5 cells (Human hepatocellular carcinoma cell line) to insulin‐like growth factor 1 (IGF1), and enhances cell survival in the presence of sorafenib, a therapeutic agent used to treat advanced stage HCC. Firstly, we observed that TGFβ significantly increased mRNA and protein expression of insulin like growth factor 1 receptor (IGF1R) in PLC cells. To test how this result would affect IGF1 signaling, PLC cells were treated with TGFβ for up to 72 hrs, then treated with IGF1 for 30 min. PLC cells pretreated with TGFβ had a much greater response to IGF1 treatment, exhibiting significantly increased levels of pIGF1R and pAkt compared with IGF1 treatment only. Since IGF1 is a pro‐survival mitogen, we further tested whether an increase in IGF1 sensitivity would reduce apoptosis in the presence of sorafenib. The cells were treated with TGFβ for 48 hrs before the incubation with sorafenib for up to 24 hrs. We found that TGFβ pretreatment led to a significant reduction in both cleaved caspase 7 and cleaved PARP, and a significant increase in cell viability compared with sorafenib treatment only. Taken together, our data suggest that TGFβ can regulate the survival of HCC cells via the activation of IGF1 pro‐survival pathway and prevent Sorfafenib‐induced apoptosis.Grant Funding Source: NIH/NCI

CNX-011-67, a novel GPR40 agonist, enhances glucose responsiveness, insulin secretion and islet insulin content in n-STZ rats and in islets from type 2 diabetic patients

BackgroundGPR40 is a G-protein coupled receptor regulating free fatty acid induced and also glucose induced insulin secretion. We generated neonatally-streptozotocin-treated female rats (n-STZ) and treated them with CNX-011-67, a GPR40 agonist to examine the role of GPR40 in modulation of glucose metabolism, insulin secretion and content.MethodsFemale n-STZ animals were orally administered with CNX-011-67 (15 mg/kg body weight, twice daily) or with vehicle for 8 weeks (n = 8 per group). Glucose tolerance in treated animals and insulin secretion, islet insulin content and gene expression in isolated islets were determined. Islets from type 2 diabetic mellitus (T2DM) patients were treated with different concentrations of glucose in presence or absence of CNX-011-67 and insulin secretion was measured.ResultsTreatment of n-STZ rats with GPR40 agonist CNX-011-67 enhanced insulin secretion in response to oral glucose load on day 0 and this response persisted during the treatment period. The treatment also produced a ‘memory effect’ during which insulin secretion in response to oral glucose load remained enhanced, for a week, even in absence of the agonist. Activation of GPR40 enhanced responsiveness of islets to glucose and increased glucose induced insulin secretion and islet insulin content. An increase in islet mRNA expression of GCK, PDX1, insulin and PC was also observed. Acute treatment of islets from n-STZ rats with GPR40 agonist enhanced cellular ATP content. Activation of GPR40 enhanced mitochondrial calcium level in NIT-1 insulinoma cells. CNX-011-67 increased insulin secretion in islets from T2DM patients which were non-responsive to increased glucose concentrationConclusionsOur data provide evidence that activation of GPR40 with CNX-011-67 stimulates glucose metabolism, enhances glucose responsiveness, increases insulin secretion and content and that pharmacological activation of GPR40 will prove beneficial for treatment of T2DM.

Compliant 3D Microenvironment Improves β-Cell Cluster Insulin Expression Through Mechanosensing and β-Catenin Signaling

Type 1 diabetes is chronic disease with numerous complications and currently no cure. Tissue engineering strategies have shown promise in providing a therapeutic solution, but maintenance of islet function and survival within these therapies represents a formidable challenge. The islet microenvironment may hold the key for proper islet maintenance. To elucidate the microenvironmental conditions necessary for improved islet function and survival, three-dimensional (3D) polyacrylamide cell scaffolds were fabricated with stiffnesses of 0.1 and 10 kPa to regulate the spatial and mechanical control of biosignals. Specifically, we show a significant increase in insulin mRNA expression of 3D primary mouse islet-derived and Min6-derived β-cell clusters grown on compliant 0.1 kPa scaffolds. Moreover, these compliant 0.1 kPa scaffolds also increase glucose sensitivity in Min6-derived β-cell clusters as demonstrated by the increased glucose stimulation index. Our data suggest that stiffness-specific insulin processing is regulated through the myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) mechanosensing pathways. Additionally, β-catenin is required for regulation of stiffness-dependent insulin expression. Through activation or inhibition of β-catenin signaling, reversible control of insulin expression is achieved on the compliant 0.1 kPa and overly stiff 10 kPa substrates. Understanding the role of the microenvironment on islet function can enhance the therapeutic approaches necessary to treat diabetes for improving insulin sensitivity and response.

Genome-wide Analysis of DNA Methylation Variations Caused by Chronic Glucolipotoxicity in Beta-Cells

There is a growing body of literature suggesting the role of interactions between genes and the environment in development of type 2 diabetes mellitus (T2DM). However, the interplay between environment and genetic in developing and progressing T2MD is not fully understood. To determine the effects of high-glucose-lipid on the status of DNA methylation in beta cells, and clarify the mechanism of glucolipotoxicity on beta-cell deterioration, the DNA methylation profile was detected in beta-cells cultured with high-glucose-lipid medium.We utilized a high throughput NimbleGen RN34 CpG Island & Promoter Microarray to investigate the DNA methylation profile in beta-cells cultured with high-glucose-lipid medium. To validate the results of microarray, the immunoprecipitation (MeDIP) PCR was used to test the methylation status of some selected genes. The mRNA and protein expression of insulin and Tcf7l2 in these cells were quantified by RT-PCR and western blot, respectively.We have identified a lot of loci which experienced aberrant DNA methylation in beta-cells cultured with high-glucose-lipid medium. The results of MeDIP PCR were consistency to the microarray. An opposite regulation in transcription and translation of Tcf7l2 gene was found. Furthermore, the insulin mRNA and protein expression in beta-cells also decreased after cultured with high-glucose-lipid medium compared with the control cells.We conclude that chronic glucolipotoxicity could induce aberrant DNA methylation of some genes and may affect these genes expression in beta-cells, which might contribute to beta-cell function failure in T2DM and be helpful to explain, at least partially, the mechanism of glucolipotoxicity on beta-cells deterioration.

Murine Insulinoma Cell-Conditioned Medium with ΒETA2/Neurod1 Transduction Efficiently Induces the Differentiation of Adipose-Derived Mesenchymal Stem Cells into β-Like Cells both In Vitro and In Vivo

Background: Mesenchymal stem cells (MSCs), including adipose tissue-derived mesenchymal stem cells (ADSCs), are multipotent and can differentiate into various cell types, including pancreatic β cells. Therefore, ADSCs present a potential cell source for the treatment of type 1 diabetes mellitus (T1DM). However, current in vitro protocols are insufficient to induce fully matured insulin-producing β cells. In this study, we assessed the effectiveness of overexpression of ΒETA2 (NeuroD1), a member of the basic helix–loop–helix transcription factor family, with murine insulinoma cell line-derived conditioned medium (MIN6-CM) to improve the differentiation capacity of ADSCs into insulin-producing cells. Method: Murine ADSCs were isolated from C57BL/6 mice, transduced with several transcriptional factors (TFs), and stable transfectants were established. MIN6-CM was prepared. Syngeneic recipient mice were rendered diabetic by a single injection of streptozotocin, and differentiated cells were transplanted under the kidney capsule of recipient mice. Next, blood glucose levels were monitored. Results: CM alone was sufficient to induce insulin mRNA expression in vitro. However, other TFs were not detected. ADSCs cultured with MIN6-CM induced insulin expressions in vitro, but other β cell-related TFs were been detected. However, BETA2 transduction in MIN6-CM resulted in robust expression of multiple β cell phenotypic markers. Moreover, insulin content analysis revealed insulin protein expression in vitro. Furthermore, in vivo transplant studies revealed the effectiveness of the simultaneous use of BETA2 transduction with the CM. Conclusion: These results suggest that the balance of cytokines and growth factors in addition to gene manipulation would benefit the efficient differentiation of ADSCs into pancreatic β cells. Our technology could provide a path to β cell differentiation and novel cell replacement-based therapies for T1DM.

New insights concerning insulin synthesis and its secretion in rat hippocampus and cerebral cortex: Amyloid-β1–42-induced reduction of proinsulin level via glycogen synthase kinase-3β

The reduction of insulin levels in hippocampal areas is associated with Alzheimer's disease. The present study using rat brain explores the mechanisms of insulin synthesis and secretion, as well as amyloid-β1–42 (Aβ1–42)-induced reduction of proinsulin expression. After confirming the expression of insulin mRNA and proinsulin in rat brain, we visualized and analyzed the motion of insulin secretion in rat hippocampal neurons using pH-sensitive green fluorescent protein (pHluorin) fused to the insulin. In the rat hippocampal neurons expressing insulin–pHluorin, time-lapse confocal laser scanning microscopy revealed the appearance of fluorescent spots induced by depolarization after stimulation with 50mM KCl. In these fluorescent spots, Ca2+-dependent activator protein for secretion 2 (CAPS2), which is the regulator of the dense-core vesicle involving neuronal peptides, was co-localized with insulin–pHluorin. However, Aβ1–42-induced reduction of proinsulin in rat hippocampal neurons was inhibited by treatment with lithium and transfection with glycogen synthase kinase-3β (GSK-3β) siRNA. These results demonstrate that synthesized insulin is secreted from rat hippocampal and cortical neuron's dense-core vesicles, and that activation of GSK-3β in Aβ1–42-induced Alzheimer's model hippocampal neurons decreases the insulin synthesis.

Insulin Secreted From Genetically Engineered Intestinal Cells Reduces Blood Glucose Levels in Diabetic Mice

Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate β-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cell-specific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy.

Enhanced transcription of pancreatic peptide YY by 1α-hydroxyvitamin D3 administration in streptozotocin-induced diabetic mice

Peptide YY (PYY) is a peptide hormone secreted from L cells in the intestine in response to food intake that regulates appetite and gastrointestinal function. PYY is also produced in the pancreatic islets. The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D3 that regulates numerous physiological processes. VDR is expressed in the pancreatic islets and pharmacological VDR activation increases PYY expression in mouse peripheral islet cells. Although VDR is present in insulin-producing β cells as well as non-β cells, the role of β cell VDR in Pyy transcription remains unknown. We treated mice with streptozotocin to ablate β cells in the pancreas. Pancreatic Vdr mRNA expression was decreased in streptozotocin-induced diabetic mice. Interestingly, streptozotocin-treated mice exhibited increased basal Pyy expression and 1α-hydroxyvitamin D3 treatment further increased expression. Moreover, 1α-hydroxyvitamin D3 increased mRNA expression of pancreatic polypeptide and decreased that of neuropeptide Y in streptozotocin-induced diabetic mice but not in control mice. 1α-Hydroxyvitamin D3 slightly increased mRNA expression of insulin but transcript levels were nearly undetectable in the pancreas of streptozotocin-treated mice. Thus, VDR in non-β islet cells is involved in Pyy expression in the mouse pancreas. The findings from this β cell ablation study suggest a hormone transcription regulatory network composed of β cells and non-β cells.

Genetic and biochemical evidence for a functional role of BACE1 in the regulation of insulin mRNA expression

Beta-site amyloid precursor protein cleaving enzyme (BACE1) is highly expressed in pancreatic β-cells. The BACE1 gene is located in a region associated with a high diabetes risk in PIMA Indians. INS-1E cells were used to study the impact of siRNA-mediated BACE1 knockdown and glucose metabolism was characterized in Bace1(-/-) mice. BACE1 gene was sequenced in DNA samples from 48 subjects and 13 representative single nucleotide polymorphisms (SNPs) were then genotyped for association studies in 1,527 Caucasians. Reduction of Bace1 expression results in a significant decrease in insulin mRNA expression in INS-1E cells. Bace1(-/-) mice display significantly lower body weight, lower plasma insulin concentrations, but normal glucose tolerance and insulin sensitivity. In a case-control study including 538 healthy controls and 989 patients with type 2 diabetes (T2D), one SNP (rs535860) was significantly associated with T2D (P < 3.5 × 10(-5) , adjusted for age, sex, and BMI). Reduced Bace1 expression causes impaired insulin expression in pancreatic β-cells of Bace1(-/-) mice, suggesting that BACE1 plays a role in the regulation of insulin biogenesis. The functionally relevant rs535860 SNP may decrease BACE1 expression by creating a new miR-661 binding site and could therefore contribute to T2D development.

Overexpression of intraislet ghrelin enhances β-cell proliferation after streptozotocin-induced β-cell injury in mice

Previously, we reported that exogenous administration of ghrelin ameliorates glucose metabolism in a neonate streptozotocin (STZ)-induced diabetic rat model through enhancement of β-cell proliferation. However, it was not clear whether the observed β-cell proliferation was a direct or indirect effect (e.g., via orexigenic or growth hormone-stimulated pathways) of ghrelin activity. Here, we aimed to investigate whether ghrelin directly impacts β-cell proliferation after STZ-induced injury in mice. Seven-week-old male rat insulin II promoter-ghrelin internal ribosomal sequence ghrelin O-acyltransferase transgenic (RIP-GG Tg) mice, which have elevated pancreatic ghrelin levels, but only minor changes in plasma ghrelin levels when fed a medium-chain triglyceride-rich diet, were treated with STZ. Then, serum insulin, pancreatic insulin mRNA expression, and islet histology were evaluated. We found that the serum insulin levels, but not blood glucose levels, of RIP-GG Tg mice were significantly ameliorated 14 days post-STZ treatment. Pancreatic insulin mRNA expression was significantly elevated in RIP-GG Tg mice, and β-cell numbers in islets were increased. Furthermore, the number of phospho-histone H3⁺ or Ki67⁺ proliferating β-cells was significantly elevated in RIP-GG Tg mice, whereas the apoptotic indexes within the islets, as determined by TUNEL assay, were not changed. These results indicate that ghrelin can directly stimulate β-cell proliferation in vivo after β-cell injury even without its orexigenic or GH-stimulating activities, although it did not have enough impact to normalize the glucose tolerance in adult mice.

Su1788 Pancreatic Fibrosis in Alcohol-Fed LPS Challenged Mice Is Regulated by the TLR4 Receptor

Background: Pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis in chronic pancreatitis (CP) and pancreatic cancer. Secondary diabetes (type 3c diabetes) frequently occurs in advanced CP and pancreatic cancer. In addition, patients with type 2 diabetes also have variable intraand peri-islet fibrosis. Loss of β-cell masses and β-cell dysfunction might play a role in these clinical settings, but the molecular mechanisms linking islet fibrosis, β-cell dysfunction, and β-cell loss remain unknown. We hypothesized that PSCs might play a role in islet fibrosis and alter cell fate in β-cells. Aim: To clarify the role of PSCs in islet fibrosis and the effects of PSCs on β-cell functions. Methods: Expression of α-smooth muscle actin (α-SMA; a marker of activated PSCs) was examined by immunohistochemical staining in the resected specimen of the pancreas from patients undergoing operation for CP or pancreatic cancer. Rat PSCs were isolated from male Wistar rats after perfusion with collagenase P. The RIN-5F rat pancreatic β-cells were mono-cultured or indirectly co-cultured with rat PSCs using culture inserts. Insulin secretion and mRNA expression were examined by the ELISA and real-time PCR, respectively. Cell viability was examined by the MTT assay. Induction of apoptosis was examined by the cytoplasmic histone-associated DNA fragments ELISA and Apoptag® in situ apoptosis detection kit. Activation of caspases 3 and 9 was examined by Western blotting and immunofluorescent staining, respectively. Results: α-SMA-positive activated PSCs were found around and within the fibrotic islets. Expression of insulin was decreased in co-cultured RIN-5F cells than mono-cultured cells. PSCs decreased cell viability and induced apoptosis in RIN-5F cells. These changes were associated with the activation of caspases 3 and 9. Conclusions: PSCs might play a role in the development of islet fibrosis. PSCs decreased insulin expression and induced apoptosis in pancreatic β-cells, suggesting a novel mechanism of diabetes mellitus in diseased pancreas.

Mechanism of insulin production in canine bone marrow derived mesenchymal stem cells

Insulin is a critical hormone in the regulation of blood glucose levels and is produced exclusively by pancreatic islet beta-cells. Insulin deficiency due to reduced pancreatic islet beta-cell number underlies the progression of diabetes mellitus, prompting efforts to develop beta-cell replacement therapies. However, precise information on beta-cell replacement and differentiation in canines is limited. In this study, we established insulin-producing cells from bone marrow derived mesenchymal stem cells transiently expressing canine pancreatic and duodenal homeobox 1 (Pdx1), beta cell transactivator 2 (Beta2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) using a gene transfer technique. Real-time PCR analysis revealed an increase in insulin mRNA expression of transfected cells. And ELISA revealed that insulin protein expressed was detected in cytoplasmic fraction. Insulin immunostaining analysis was performed and observed in cytoplasmic fraction. These results suggest that co-transfection of Pdx1, Beta2 and Mafa induce insulin production in canine BMSCs. Our findings provide a clue to basic research into the mechanisms underlying insulin production in the canines.

Oral l-glutamine increases active GLP-1 (7-36) amide secretion and improves glycemic control in stretpozotocin–nicotinamide induced diabetic rats

l-glutamine is a non-essential amino acid. It decreased blood sugar, stimulated insulin secretion in type 2 diabetic patients. The objective of the present investigation was to evaluate l-glutamine increases glucagon like peptide-1 (GLP-1) (7-36) amide secretion in streptozotocin–nicotinamide (STZ–NTM) induced diabetic Sprague Dawley rats. Molecular docking study was performed to elucidate the molecular basis for GLP-1 receptor agonistic activity. Type 2 diabetes was induced in overnight fasted Sprague Dawley rats pre-treated with nicotinamide (100mg/kg, i.p.) followed by 20min after administration of streptozotocin (55mg/kg, i.p.). The rats were divided into; I – nondiabetic, II – diabetic control, III – sitagliptin (5mg/kg, p.o.), IV –l-glutamine (250mg/kg, p.o.), V –l-glutamine (500mg/kg, p.o.) and VI –l-glutamine (1000mg/kg, p.o.). The l-glutamine and sitagliptin treatment was 8week. Plasma glucose was estimated every week. Body weight, food and water intake were recorded daily. Glycosylated haemoglobin, lipid profile, plasma and colonic active (GLP-1) (7-36) amide, mRNA expression of proglucagon GLP-1, plasma and pancreatic insulin, histology of pancreata and biomarkers of oxidative stress (superoxidase dismutase, reduced glutathione, malondialdehyde, glutathione peroxidase, glutathione S transferase) were measured after 8week. In acute study, the rats were divided into I – glucose (2.5g/kg, p.o.), II – sitagliptin (5mg/kg, p.o.), III –l-glutamine (250mg/kg, p.o.), IV –l-glutamine (500mg/kg, p.o.) and V –l-glutamine (1000mg/kg, p.o.). Plasma glucose, active GLP-1 (7-36) amide concentration and insulin levels were measured after glucose loading. The docking data indicated that l-glutamine bind to the GLP-1 receptor. l-glutamine decreased plasma glucose, increased plasma and pancreatic insulin, increased plasma and colonic active GLP-1 (7-36) amide secretion as well as decreased oxidative stress in streptozotocin–nicotinamide induced diabetic rats.

Embryoid Body Formation is Required for Differentiation of Insulin-Producing Cell Clusters from Mouse Embryonic Stem Cells

In Type 1 diabetes, insulin-producing pancreatic cells, or beta cells, are destroyed by an autoimmune response. Current clinical treatments are indefinite insulin replacement therapy or transplantation of the pancreas or beta islets. The latter two treatments are limited in available donors; a potential alternative is the use of insulin-producing cell clusters (IPCCs) differentiated from embryonic stem cells (ESCs). We hypothesize that IPCCs will reproduce the insulin-producing capacity of healthy beta cells of an adult mouse, and we are testing the efficiency of distinct IPCC culture methods to achieve this goal. Among several existing ESC-IPCC differentiation protocols, Blyszczuk et al. developed the most successful method to date in producing IPCCs that showed similarities to pancreatic beta cells. However, this method is time-intensive, requiring approximately 41 days. We attempted to streamline the protocol by bypassing the formation of embryoid bodies (EBs), reducing the differentiation timeline to 27 days. At several time points during this protocol, IPCC cultures were analyzed by RT-PCR and immunofluorescence for genes and proteins expressed in pancreatic beta islet cells. The mRNA and protein expression of insulin was not observed. Furthermore, ELISA analysis detected low intracellular insulin response after challenging IPCCs with different glucose concentrations. These results reject the hypothesis that EB formation is not required for ESC-IPCC differentiation in vitro. However, it is possible that alpha cells can be differentiated, as glucagon was detected.

Isolation, Characterization and Pro-Angiogenic Activity of Microvesicles (MVs) Derived from Human Pancreatic Islets

Islet transplantation is a therapeutic option for the treatment of type I diabetes. However, pancreata from multiple donors are still needed to guarantee a sufficient islet mass to obtain insulin independence, since a substantial number of transplanted islets fails to engraft into liver or suffers for poor vascular engraftment or for the immune attack. The molecular events involved in the revascularization of transplanted islets are far to be fully elucidated. Microvesicles (MVs) are small particles that play a key role in inter-cellular communication through the transfer of proteins and RNAs. MVs are enriched for microRNAs (miRNAs), small non coding RNAs able to modulate protein transduction. The aims of this study were: 1) isolation and characterization of MVs from purified human islets; 2) evaluation of the pro-angiogenic effect of MVs on human islet-derived endothelial cells (hIECs). MVs were isolated by ultracentrifugation from conditioned culture medium of purified human islets with high parameters of purity, viability and function (insulin stimulation index) to avoid the contamination of exocrine tissue or apoptotic cells. MVs were characterized by Nanosight, FACS, western blot, bioanalyzer, RT-PCR for specific islet-associated genes and miRNAs. We evaluated on isolated hIECs MV-induced: a) transfer of insulin mRNA and protein; b) proliferation (BrdU); c) resistance to apoptosis (TUNEL); d) angiogenesis on Matrigel; e) migration; f) transcriptional profile (mRNA) and g) protein profile of several pro-angiogenic and anti-apoptotic genes. Islet-derived MVs had a mean size of 155±73 nm and expressed different membrane molecules (integrins, CD44, ICAM 1, L-selectin, HLA I). MVs also expressed insulin, c-peptide, AKT, p-AKT, AGO 2 and CD63 proteins, shuttled several mRNAs, among which some involved in insulin secretion and signal transduction (insulin receptor, IRS2, AKT2, PDX1) and several miRNAs, including the beta-cell specific miR-7 and miR-375 and the pro-angiogenic miR-126, miR-296 and miR-130a. MVs were internalized into hIECs inducing an insulin mRNA expression and an early (3 hr) and late (12 hr) insulin protein expression. Moreover, MVs induced cell proliferation, resistance to apoptosis, migration and enhanced angiogenesis in hIECs by the up-regulation of pro-angiogenic, proliferative and anti-apoptotic genes and by the concomitant down-regulation of anti-angiogenic genes. At mRNA level, pro-angiogenic genes up-regulated by MVs were: Angiopoietin 1, CXCL1 FGFR3, VEGFR1, HAND, Jagged 1, Leptin, Midkine, Neurophillin 2 and Tie-2; instead down-regulated anti-angiogenic genes were: BAI1, endostatin, tumstatin, CXCL10, Notch 4, Thrombospondin 1 and Tissue inhibitor of metalloproteinases 1 and 2. Moreover, MVs induced up-regulation of VEGFR1, VEGFR2, VEGF A, Angiopoietin 1, p-AKT, p-ERK, p-eNOS, Bcl-2 at protein level. All these effects were significantly decreased by pre-treatment of MVs with 1U/ml RNase, suggesting a key role of RNA transfer from MVs to target hIECs. In this study, we demonstrated that human islets release biologically active MVs able to shuttle specific proteins, mRNAs and miRNAs into target cells. MVs exert a pro-angiogenic effect on hIECs, suggesting a putative role for islet-derived MVs in beta cell-endothelium cross-talk and in the enhancement of neoangiogenesis processes following islet transplantation.

Cycloart-23-ene-3β, 25-diol stimulates GLP-1 (7–36) amide secretion in streptozotocin–nicotinamide induced diabetic Sprague Dawley rats: A mechanistic approach

In previous study, we have reported cycloart-23-ene-3β, 25-diol is an active antidiabetic constituent isolated from stem bark of Pongamia pinnata (Linn.) Pierre. The objective of the present investigation was to evaluate cycloart-23-ene-3β, 25-diol stimulates glucagon like peptide-1 (GLP-1) (7–36) amide secretion in streptozotocin–nicotinamide induced diabetic Sprague Dawley rats. Molecular docking studies were performed to elucidate the molecular basis for GLP-1 receptor agonistic activity. Type 2 diabetes was induced in overnight fasted Sprague Dawley rats pre-treated with nicotinamide (100mg/kg, i.p.) followed by administration of streptozotocin (55mg/kg, i.p.) 20min after. The rats were divided into following groups; I- non-diabetic, II- diabetic control, III- sitagliptin (5mg/kg, p.o.), IV- cycloart-23-ene-3β, 25-diol (1mg/kg, p.o.). The cycloart-23-ene-3β, 25-diol and sitagliptin treatment was 8 week. Plasma glucose was estimated every week (week 0 to week 8). Body weight, food and water intake were recorded daily. Glycosylated haemoglobin, lipid profile, plasma and colonic active (GLP-1) (7–36) amide, mRNA expression of proglucagnon GLP-1, plasma and pancreatic insulin, histology of pancreata as well as biomarkers of oxidative stress (superoxidase dismutase, reduced glutathione, malondialdehyde, glutathione peroxidase, glutathione S transferase) were measured after 8th week treatment. In acute study, active GLP-1 (7–36) amide release, plasma glucose and insulin were measured during oral glucose tolerance test. The docking data clearly indicated cycloart-23-ene-3β, 25-diol bind to the GLP-1 receptor. It decreased plasma glucose level, increased plasma and pancreatic insulin level as well as increased plasma and colonic active GLP-1 (7–36) amide secretion in streptozotocin–nicotinamide induced diabetic Sprague Dawley rats.

Impaired Insulin/IGF Signaling in Experimental Alcohol-Related Myopathy

Alcohol-related myopathy (Alc-M) is highly prevalent among heavy drinkers, although its pathogenesis is not well understood. We hypothesize that Alc-M is mediated by combined effects of insulin/IGF resistance and oxidative stress, similar to the effects of ethanol on liver and brain. We tested this hypothesis using an established model in which adult rats were pair-fed for 8 weeks with isocaloric diets containing 0% (N = 8) or 35.5% (N = 13) ethanol by caloric content. Gastrocnemius muscles were examined by histology, morphometrics, qRT-PCR analysis, and ELISAs. Chronic ethanol feeding reduced myofiber size and mRNA expression of IGF-1 polypeptide, insulin, IGF-1, and IGF-2 receptors, IRS-1, and IRS-2. Multiplex ELISAs demonstrated ethanol-associated inhibition of insulin, IRS-1, Akt, and p70S6K signaling, and increased activation of GSK-3β. In addition, ethanol-exposed muscles had increased 4-hydroxy-2-nonenal immunoreactivity, reflecting lipid peroxidation, and reduced levels of mitochondrial Complex IV, Complex V, and acetylcholinesterase. These results demonstrate that experimental Alc-M is associated with inhibition of insulin/IGF/IRS and downstream signaling that mediates metabolism and cell survival, similar to findings in alcoholic liver and brain degeneration. Moreover, the increased oxidative stress, which could be mediated by mitochondrial dysfunction, may have led to inhibition of acetylcholinesterase, which itself is sufficient to cause myofiber atrophy and degeneration.