Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin

Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) +/-leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.

Expression profiling of type 2 diabetes susceptibility genes in the pancreatic islets, adipose tissue and liver of obese mice

Type 2 diabetes (T2D) is characterized by impaired beta cell function and insulin resistance. T2D susceptibility genes identified by Genome-wide association studies (GWAS) are likely to have roles in both impaired insulin secretion from the beta cell as well as insulin resistance. The aim of this study was to use gene expression profiling to assess the effect of the diabetic milieu on the expression of genes involved in both insulin secretion and insulin resistance. We measured the expression of 43 T2D susceptibility genes in the islets, adipose and liver of leptin-deficient Ob/Ob mice compared with Ob/+ littermates. The same panel of genes were also profiled in cultured rodent adipocytes, hepatocytes and beta cells in response to high glucose conditions, to distinguish expression effects due to elevated glycemia from those on the causal pathway to diabetes or induced by other factors in the diabetic microenviroment. We found widespread deregulation of these genes in tissues from Ob/Ob mice, with differential regulation of 23 genes in adipose, 18 genes in liver and one gene (Tcf7l2) in islets of diabetic animals (Ob/Ob) compared to control (Ob/+) animals. However, these expression changes were in most cases not noted in glucose-treated adipocyte, hepatocyte or beta cell lines, indicating that they may not be an effect of hyperglycemia alone. This study indicates that expression changes are apparent with diabetes in both the insulin producing beta cells, but also in peripheral tissues involved in insulin resistance. This suggests that incidence or progression of diabetic phenotypes in a mouse model of diabetes is driven by both secretory and peripheral defects. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart New York.

Effects of metformin on BRIN-BD11 beta-cell insulin secretory desensitization induced by prolonged exposure to sulphonylureas

Aims: Prolonged exposure of pancreatic beta-cells in vitro to the sulphonylureas tolbutamide and glibenclamide induces subsequent desensitization of insulinotropic pathways. Clinically, the insulin-sensitizing biguanide drug metformin is often administered alongside sulphonylurea as antidiabetic therapy. The present study examines the functional effects of metformin (200 µM) on tolbutamide- and glibenclamide-induced desensitisation. Methods: Acute and prolonged (18 h) effects of exposure to tolbutamide and glibenclamide alone, or in the presence of metformin, were examined in insulin-secreting BRIN-BD11 cells. Results: In acute 20 min incubations at 1.1 mM glucose, metformin increased (1.2-1.7-fold; p <0.001) the insulin-releasing actions of tolbutamide and glibenclamide. At 16.7 mM glucose, metformin significantly enhanced glibenclamide-induced insulin release at all concentrations (50-400 µM) examined, but tolbutamide-stimulated insulin secretion was only augmented at higher concentrations (300-400 µM). Exposure for 18 h to 100 µM tolbutamide or glibenclamide significantly impaired insulin release in response to glucose and a broad range of insulin secretagogues. Concomitant culture with metformin (200 µM) prevented or partially reversed many of the adverse effects on K channel dependent and independent insulinotropic pathways. Beneficial effects of metformin were also observed in cells exposed to glibenclamide for 18 h with significant improvements in the insulin secretory responsiveness to alanine, GLP-1 and sulphonylureas. The decrease of viable cell numbers observed with glibenclamide was reversed by co-culture with metformin, but cellular insulin content was depressed. Conclusions: The results suggest that metformin can prevent the aspects of sulphonylurea-induced beta-cell desensitization. © 2010 Blackwell Publishing Ltd.

Leptin decreases apoptosis and alters BCL-2:bax ratio in clonal rodent pancreatic beta-cells

The adipocyte derived peptide hormone leptin is known to regulate apoptosis and cell viability in several cells and tissues, as well as having several pancreatic islet beta-cell specific effects such as inhibition of glucose-stimulated insulin secretion. This study investigated the effects of leptin upon apoptosis induced by serum depletion and on expression of the apoptotic regulators B-cell leukaemia 2 gene product (BCL-2) and BCL2-associated X protein (Bax) in the glucose-responsive BRIN-BD11 beta-cell line.

β-cell-specific glucocorticoid reactivation attenuates inflammatory β-cell destruction

Progression and severity of type 1 diabetes is dependent upon inflammatory induction of nitric oxide production and consequent pancreatic β-cell damage. Glucocorticoids (GCs) are highly effective anti-inflammatory agents but have been precluded in type 1 diabetes and in islet transplantation protocols because they exacerbated insulin resistance and suppressed β-cell insulin secretion at the high-doses employed clinically. In contrast, physiological-range elevation of GC action within β-cells ameliorated lipotoxic β-cell failure in transgenic mice overexpressing the intracellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (MIP-HSD1^tg/+ mice). Here, we tested the hypothesis that elevated β-cell 11beta-HSD1 protects against the β-cell destruction elicited by streptozotocin (STZ), a toxin that dose-dependently mimics aspects of inflammatory and autoimmune β-cell destruction. MIP-HSD1^tg/+ mice exhibited an episodic protection from the severe hyperglycemia caused by a single high dose of STZ associated with higher and sustained β-cell survival, maintained β-cell replicative potential, higher plasma and islet insulin levels, reduced inflammatory macrophage infiltration and increased anti-inflammatory T regulatory cell content. MIP-HSD1^tg/+ mice also completely resisted mild hyperglycemia and insulitis induced by multiple low-dose STZ administration. In vitro, MIP-HSD1^tg/+ islets exhibited attenuated STZ-induced nitric oxide production, an effect reversed with a specific 11beta-HSD1 inhibitor. GC regeneration selectively within β-cells protects against inflammatory β-cell destruction, suggesting therapeutic targeting of 11beta-HSD1 may ameliorate processes that exacerbate type 1 diabetes and that hinder islet transplantation.

Regenerative Cues of Myeloid Cells on Pancreatic Epithelium

Thesis (Ph.D.)--University of Washington, 2016-07

Olfactomedin-4 is a candidate biomarker of solid gastric, colorectal, pancreatic, head and neck, and prostate cancers

International audience

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Autologous Platelet Gel: Five Cases Illustrating Use On Sickle Cell Disease Ulcers.

Leg ulcers represent a particularly disabling complication in patients with sickle cell disease (SCD). Platelet gel (PG) is a novel therapeutic strategy used for accelerating wound healing of a wide range of tissues through the continuous release of platelet growth factors. Here, we describe the use of PG preparation according to Anitua's PRGF (preparations rich in growth factors) protocol for treating chronic nonhealing ulcers in patients with SCD. A positive response occurred in 3 patients with an area reduction of 85.7% to 100%, which occurred within 7 to 10 weeks, and a 35.2% and 20.5% of area reduction in 2 other patients, who however, had large ulcers. After calcium chloride addition, the platelet-rich plasmas demonstrated enhanced platelet-derived growth factors-BB (P < .001), transforming growth factor-β1 (P = .015), vascular endothelial growth factors (P = .03), and hepatocyte growth factors (nonsignificant) secretion. Furthermore, calcium chloride addition induced a significant decrease in platelet number (P = .0134) and there was no leukocyte detection in the PG product. These results demonstrate that PG treatment might impact the healing of leg ulcers in sickle cell disease, especially in patients with small ulcers.

Integrated Proteomics Identified Up-regulated Focal Adhesion-mediated Proteins In Human Squamous Cell Carcinoma In An Orthotopic Murine Model.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Desquamation Is A Novel Phenomenon For Collective Prostate Epithelial Cell Deletion After Castration.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Metabolic Memory Of ß-cells Controls Insulin Secretion And Is Mediated By Camkii.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of metabolic memory, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca(2+)-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.