RINm5F Cells Research Articles

Apigetrin ameliorates streptozotocin-induced pancreatic β-cell damages via attenuating endoplasmic reticulum stress

The pathogenesis of diabetes is associated with dysfunction of pancreatic β-cells. To ameliorate the β-cell dysfunction, it has propelled great interest to search pharmacological agents from natural plants. This study explored the protective effect of apigetrin, a flavonoid present in natural plants, against streptozotocin (STZ)-induced cell damages in RINm5F cells and the potential mechanisms. Apigetrin was found to inhibit the elevation of intracellular reactive oxygen species levels, restore the impairment of antioxidant enzymes, and recover the disruption of redox homeostasis in the STZ-treated pancreatic β-cells. Moreover, treatment of apigetrin significantly suppressed the STZ-induced apoptosis in the analysis of apoptotic sub-G1 population and the protein expressions of cleaved poly(ADP-ribose) polymerase and caspase-3. Furthermore, apigetrin attenuated STZ-induced endoplasmic reticulum (ER) stress, indicated by the reduction of ER stress biomarkers, including overloading of mitochondrial calcium, increase in glucose-regulated protein 78, phosphorylation of protein kinase RNA-like ER kinase and its downstream eukaryotic initiation factor 2α, cleavage of activating transcription factor 6 and caspase-12, up-regulation of CCAAT/enhancer binding protein homologous protein, and induction of spliced X-box binding protein 1. Additionally, pretreatment with 4-phenylbutyric acid, a classic ER stress inhibitor, augmented these beneficial effects of apigetrin. In conclusion, these results demonstrated that apigetrin could improve the STZ-induced pancreatic β-cell damages via mitigation of oxidative stress and ER stress and supported the application of apigetrin to developing the novel therapeutics of diabetes.

System χc- overexpression prevents 2-deoxy-d-ribose-induced β-cell damage

Pancreatic β-cells are vulnerable to oxidative stress, which promotes β-cell failure in type 2 diabetes. System χc- is a sodium-independent, cystine/glutamate antiporter that mediates the exchange of extracellular l-cystine and intracellular l-glutamate. The import of l-cystine through this transporter is the rate-limiting step in the glutathione (GSH) biosynthesis pathway that plays a significant role in antioxidative defense. Previously, we reported that 2-deoxy-d-ribose (dRib) induces oxidative damage through GSH depletion in pancreatic β-cells. In the current study, we elucidated the mechanism underlying the oxidative stress-induced β-cell damage. We measured the intracellular l-[14C]cystine uptake, GSH content, reactive oxygen species (ROS) levels, cytotoxicity, and apoptosis in rat insulinoma cell line, RINm5F. Treatment of dRib decreased the intracellular l-[14C]cystine uptake and GSH content and increased the intracellular ROS levels, cytotoxicity, and apoptosis in a time- and dose-dependent manner. Conversely, 2-mercaptoethanol (2-ME), a cystine uptake enhancer, recovered the dRib-induced decrease in l-[14C]cystine uptake, GSH content, and cell viability in a Na+-independent manner. In the case of isolated islets, dRib dose-dependently decreased the intracellular l-[14C]cystine uptake and cell viability; however, dRib-induced cytotoxicity was completely recovered by adding N-acetyl cysteine (NAC). To confirm that system χc- mediates the oxidative stress-induced β-cell damage, we overexpressed xCT (the substrate-specific subunit of system χc-) using a lentiviral vector in RINm5F cells. Overexpression of xCT fully recovered the dRib-induced decrease in l-[14C]cystine uptake and GSH content and prevented the dRib-induced increase in ROS levels, cytotoxicity, and apoptosis. The overexpression of xCT showed a protective effect against dRib-induced oxidative damage in RINm5F cells. Our study showed that dRib depletes intracellular GSH content through inhibition of cystine transport via system χc- in β-cells.

Oligomannuronate prevents mitochondrial dysfunction induced by IAPP in RINm5F islet cells by inhibition of JNK activation and cell apoptosis

BackgroundOligomannuronates (OM) are natural products from alginate that is frequently used as food supplement. The aim of this study was to investigate the in vitro protective effects of OM on RINm5F cells against human Islet amyloid polypeptide (IAPP) induced mitochondrial dysfunction, as well as the underlying mechanisms.MethodsIn the present study, we obtained several kinds of OM with different molecular masses, and then we used RINm5F cells as a model to elucidate the involvement of JNK signal pathway in hIAPP-induced mitochondrial dysfunction in pancreatic beta cells, and the protective effects of OM are associated with its ability to attenuate the mitochondrial dysfunction.ResultsOur results demonstrated that human IAPP induced mitochondrial dysfunction, as evidence by loss of ΔΨm and ATP content, and decrease in oxygen consumption and complex activities, was accompanied by JNK activation, changes in the expressions of Bcl-2 and Bax proteins, release of cytochrome c (Cyto-c) and apoptosis inducing factor (AIF) from mitochondria into cytosol. Interestingly, the human IAPP induced damage in RINm5F cells were effectively restored by co-treatment of OM. Moreover, JNK activation was required for the OM mediated changes in RINm5F cells.ConclusionsOM prevented mitochondrial dysfunction induced by human IAPP in RINm5F islet cells through JNK dependent signaling pathways.

Curcumin inhibits alloxan-induced pancreatic islet cell damage via antioxidation and antiapoptosis.

The present study elucidates the possible protective effects of curcumin on β-cells damaged by oxidative stress and its significance in controlling diabetes mellitus in in vitro experiments. Pancreatic islet (RIN-m5F) cells were treated with 25 mmol/L alloxan (AXN) to induce cell damage and the protective effects of curcumin were observed. The results showed that curcumin significantly promoted the cellular activity of AXN-treated RIN-m5F cells, decreased the ratio of apoptosis, downregulated the level of malondialdehyde, upregulated the levels of superoxide dismutase and reactive oxygen species, increased the expression of Bcl-2, cleaved caspase-3, and cleaved PARP1, and decreased the expression of Bax in AXN-treated cells. These results suggest that curcumin inhibits AXN-induced damage in RIN-m5F cells via antioxidative and antiapoptotic mechanisms.

Effects of Sclerocarya birrea Stem-Bark Extracts on Glucose Uptake, Insulin Synthesis and Expression of Selected Genes Involved in the Synthesis and Secretion of Insulin in Rat Insulinoma Pancreatic Beta Cells

The present study reported the effects of Sclerocarya birrea stem-bark (SBSB) extracts on glucose uptake, insulin synthesis and the expression of glucose transporter 2 (GLUT2), glucokinase, pancreatic duodenal homeobox-1 (PDX-1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) and pre-proinsulin genes in rat insulinoma (RIN)-m5F pancreatic beta cells. The amount of glucose takenup by RIN-m5F cells was measured using a glucose oxidase-based assay kit. Intracellular and secreted insulin were measured using an enzyme linked immunoassay kit. Pre-proinsulin gene expression was determined using the conventional polymerase chain reaction (PCR) technique, while the expressions of GLUT2, glucokinase, PDX-1 and MafA genes were evaluated using quantitative real-time PCR technique. Of the four SBSB extracts investigated in the study, only the SBSB hexane extract positively affected all the study variables in RIN-m5F cells compared with the DMSO control. Thus, the SBSB hexane extract contains phytochemicals capable of enhancing insulin synthesis partly through up-regulation of the expression of GLUT2, glucokinase, PDX-1, MafA and pre-proinsulin genes.

Emulsified isoflurane protects beta cells against high glucose-induced apoptosis via inhibiting endoplasmic reticulum stress.

Pancreatic beta cell damage induced by glucose toxicity is an important factor in type 2 diabetes mellitus (T2DM). It has become evident that endoplasmic reticulum stress (ERS)-induced apoptosis was contributed to beta cell dysfunction and insulin resistance. Our previous work showed that emulsified isoflurane (EIso) could alleviate ERS in lung reperfusion injury. This study aimed to elucidate whether EIso could alleviate apoptosis induced by glucose in rat islet RIN-m5F beta cells via inhibiting ERS. RIN-m5F cells were divided into five groups: the control group; the 0.1G group, cultured in 0.1M glucose for 24 h; the 0.3G group, cultured in 0.3M glucose for 24 h; the 0.3G + 57E group, cultured in 0.3M glucose with 57 µM EIso for 24 h, and the 0.3G + 76E group, cultured in 0.3M glucose with 76 µM EIso for 24 h. First, cell proliferation was measured by MTT assay, and the level of insulin secretion was measured with enzyme-linked immunosorbent assay (ELISA) kit. Second, the expression of B cell leukemia/lymphoma 2 (Bcl-2) associated X (Bax) and Bcl-2 were detected by Western blotting. The level of caspase-3 activity was assessed by colorimetric method. Finally, the ERS marker CHOP and GRP78 expression were detected by Western blotting. The levels of activating transcription factor-6 (ATF6), X-box-binding protein 1 (Xbp1), and eukaryotic translation initiation factor-2α (eIF2α) mRNA were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) after being treated with EIso for 24 h. We found that exposure to high glucose reduced RIN-m5F cell viability, stimulated the secretion of insulin, activated caspase-3, improved the expression of Bax, and down-regulated Bcl-2. EIso improved the survival and protected the function of RIN-m5F. Compared to the 0.3G group, treatment with EIso inhibited the activity of caspase-3, and decreased the expression of Bax. The expression of CHOP and GRP78 were significantly suppressed by EIso at 24 h in a dose-dependent manner. The level of ATF6, Xbp1, and eIF2α mRNA of RIN-m5F were enhanced by high glucose, but only eIF2α mRNA was significantly decreased by EIso treatment. The present study suggests that high glucose induces rat islet beta cell RIN-m5F apoptosis and aggravates the function of beta cells. EIso protects beta cells against high glucose through the ERS-dependent apoptotic pathway and might serve as a potential therapy for diabetes.

Mechanism of the Hypoglycemic Activity and Hepatoprotective Effect of the Aqueous Extract of Cecropia obtusifolia Bertol.

In Central and South American traditional medicine, people use Cecropia obtusifolia Bertol (Cecropiaceae) for the treatment of diabetes mellitus. However, its hypoglycemic action mechanism at pancreatic and liver level has been poorly explored. The present research aimed to establish the influence of the aqueous extract of C. obtusifolia, standardized in its content of chlorogenic acid, on insulin secretion in RINm5F cells and over the liver carbohydrates and lipids metabolism, and to determine concomitantly its hepatoprotective effect on mice with streptozotocin-induced diabetes. In RINm5F cells, concentrations 5, 50, 100, and 200 μg/mL of aqueous extract of C. obtusifolia were used to determine [Ca2+]i and insulin secretion. In an acute study, the extract was administered at doses of 500 mg/kg. In another test (subacute), the extract was daily administrated to diabetic mice (200 mg/kg/day) for 30 days. Blood glucose levels and other biochemical parameters were determined, and a liver histological analysis was performed. In RINm5F cells, C. obtusifolia increased [Ca2+]i and insulin secretion, whereas in diabetic mice exhibited acute and subacute hypoglycemic effects. Daily administration of C. obtusifolia to diabetic mice also increased liver glycogen storage and glycogen synthase levels, without apparent changes in gluconeogenesis. Besides, it increased peroxisome proliferator-activated receptor-α (PPAR-α) and long-chain-fatty-acid-CoA ligase 1 (ACSL-1) expression and reduced triglycerides, transaminases (alanine aminotransferase and aspartate aminotransferase), and collagen fibers, modifying anti-inflammatory (adiponectin and interleukin-10) and inflammatory (tumor necrosis factor-α) cytokines in serum. Therefore, the hypoglycemic effect of C. obtusifolia implicates a dual action, promoting insulin secretion, liver glycogen accumulation, and hepatoprotection by decreasing collagen fibers and inflammatory markers, whereas it improves lipid metabolism, due in part to PPAR-α.

Delphinidin-induced autophagy protects pancreatic β cells against apoptosis resulting from high-glucose stress via AMPK signaling pathway.

Hyperglycemia, a diagnostic characteristic of diabetes mellitus, is detrimental to pancreatic β cells. Delphinidin, a member of the anthocyanin family, inhibits glucose absorption, increases glucagon-like peptide-1 (GLP-1) secretion, and improves insulin secretion in diabetes. However, whether delphinidin plays a protective role in pancreatic β-cell mass and function is not clear. In this study, delphinidin was found to decrease the high-glucose-induced apoptosis of RIN-m5F pancreatic β cells. In addition, delphinidin induced autophagy in RIN-m5F cells under the normal and high-glucose conditions, while 3-methyladenine (3-MA) inhibition of autophagy significantly diminished the protective role of delphinidin against high-glucose-induced apoptosis of pancreatic β cells. Delphinidin also decreased the level of cleaved caspase 3 and increased the phosphorylation level of AMP-activated protein kinase α (AMPKα) Thr172. Compound C, an AMPK inhibitor, was found to decrease the ratio of LC3-II/LC3-I, and the apoptotic rate of high-glucose-injured cells was increased after treatment with delphinidin, indicating that delphinidin attenuated the negative effects of high-glucose stress to cells. In conclusion, our data demonstrate that delphinidin protects pancreatic β cells against high-glucose-induced injury by autophagy regulation via the AMPK signaling pathway. These findings might shed light on the underlying mechanisms of diabetes and help improve the prevention and therapy of this common disease.

Hydrogen peroxide permeability of cellular membranes in insulin-producing cells

Hydrogen peroxide (H2O2) plays a central role in redox signalling and in oxidative stress-mediated cell death. It is generated through multiple mechanisms at various intracellular sites. Due to its chemical stability it can reach distant sites of action. However, its hydrophilicity can hamper lipid membrane passage. We therefore studied the kinetics of H2O2 diffusion through subcellular membranes employing the H2O2 biosensor HyPer in insulin-producing RINm5F cells.Plasma- and ER-membrane-bound HyPer sensors facing the cytosolic compartment reacted twice as fast to H2O2 compared to sensors expressed in peroxisomes and mitochondria. Overexpression of the H2O2-inactivating enzyme catalase in the ER-lumen and in the peroxisomes retarded the reaction time of HyPer, both localised within the peroxisomes as well as at the cytosolic surface of the ER. The unsaturated fatty acid oleic acid did not affect the reaction of the peroxisomal HyPer sensor to H2O2, while the saturated fatty acid palmitic acid accelerated its reaction time to H2O2 in this organelle.The results show that the plasma-, peroxisomal, and mitochondrial membrane of insulin-producing RINm5F cells are permeable for H2O2. Nonetheless, the organelle membranes retard H2O2 diffusion due to a barrier function of the lipid membrane, as documented by retarded reaction times of the intraorganellar sensors. Accelerated decomposition of H2O2 by catalase, expressed in the peroxisomes or the ER, further retarded the HyPer sensor reaction time. The results show that redox signalling and oxidative stress-mediated toxicity are crucially dependent on physicochemical membrane properties and antioxidative defence mechanisms in health and disease.

Tinospora cordifolia preserves pancreatic beta cells and enhances glucose uptake in adipocytes to regulate glucose metabolism in diabetic rats

The purpose of this study was to evaluate the pancreatic beta cell protective and glucose uptake enhancing effect of the water extract of Tinospora cordifolia stem (TCSE) by using rat insulinoma (RIN)-m5F cells and 3T3-L1 adipocytes. RIN-m5F cells were stimulated with interleukin-1β and interferon-γ, and the effect of TCSE on insulin secretion and cytokine-induced toxicity was measured by ELISA and MTT assay, respectively. The glucose uptake and protein expression were measured by fluorometry and western blotting. Antidiabetic effect of TCSE was measured using streptozotocin-induced diabetic rats. TCSE dose dependently increased cell viability and insulin secretion in RIN-m5F cells. In addition, TCSE increased both the glucose uptake and glucose transporter 4 translocation in 3T3-L1 adipocytes via PI3K pathway. Finally, TCSE significantly lowered blood glucose and diet intake and increased body weight in streptozotocin-induced diabetic rats. The level of serum insulin and hepatic glycogen was increased, whereas the level of serum triglyceride, total cholesterol, dipeptidyl peptidase-4, and thiobarbituric acid reactive substances was decreased in TCSE-administered rats. TCSE also increased glucose transporter 4 protein expression in the adipose tissue and liver of TCSE-fed diabetic rats. Our results suggested that TCSE preserved RIN-m5F cells from cytokine-induced toxicity and enhanced glucose uptake in 3T3-L1 adipocytes, which may regulate glucose metabolism in diabetic rats.

Cadmium exposure induces pancreatic β-cell death via a Ca2+-triggered JNK/CHOP-related apoptotic signaling pathway

Cadmium (Cd) is known to be ranked the 7th hazardous substance in the Substance Priority List by Agency for Toxic Substances and Disease Registry. The experimental and epidemiological data have suggested that Cd is linked to the development of diabetes mellitus (DM). The molecular mechanism of Cd on the pancreatic β-cell cytotoxicity still remains unclear. Evidence has pointed toward that Ca2+ is an important regulator of toxic insult-induced β-cell cytotoxicity. The role of Ca2+ in the Cd-induced β-cell cytotoxicity is still unknown. In this study, we found that Cd exposure significantly inhibited insulin secretion and cell viability in the pancreatic β-cell-derived RIN-m5F cells. Cd exposure induced apoptotic events, including the increased populations of apoptotic cells and sub-G1 hypodiploid cells, and caspase-3/-7/-9 and poly (ADP-ribose) polymerase (PARP) activation, which largely depended on the activation of c-Jun N-terminal kinase (JNK) and C/EBP homologous protein (CHOP). Transfection with siRNAs for JNK and CHOP or pretreatment with specific pharmacological inhibitor of JNK (SP600125) in β-cells effectively prevented the Cd-induced insulin secretion dysfunction and apoptosis. JNK-specific siRNA dramatically suppressed Cd-induced JNK phosphorylation and CHOP protein expression, but JNK phosphorylation could not be inhibited by CHOP-specific siRNA. Furthermore, Cd exposure significantly increased the intracellular calcium ([Ca2+]i) levels. Buffering the Ca2+ response with BAPTA/AM effectively abrogated the Cd-induced [Ca2+]i elevation, insulin secretion dysfunction, apoptosis, and protein expression of JNK phosphorylation and CHOP activation. Taken together, these findings demonstrated that Cd exposure exerts β-cell death via a [Ca2+]i-dependent JNK activation-activated downstream CHOP-related apoptotic signaling pathway.

2122-P: System x(c)- Is a Key Molecule in Oxidative Stress-Induced ß-Cell Damage

Pancreatic β-cells are vulnerable to oxidative stress, which is related to β-cell failure in type 2 diabetes. System x(c)- is a sodium-independent cystine/glutamate antiporter that mediates the exchange of extracelluar L-cystine and intracellular L-glutamate. The import of L-cystine through this transporter is the limiting state of intracellular glutathione (GSH) synthesis that has a major role in defends against oxidative stress. Our previous study reported that 2-deoxy-D-ribose (dRib) induces oxidative damage through GSH depletion in pancreatic β-cells. The aim of the present study was to elucidate the mechanism underlying oxidative stress-induced β-cell damage. We measured intracellular L-[14C] cysteine uptake, GSH content and reactive oxygen species (ROS) levels, cytotoxicity and apoptosis in rat insulinoma cell line (RINm5F cells). Stimulation with dRib dose- and time-dependently decreased intracellular L-[14C] cystine uptake and GSH content and increased intracellular ROS levels, cytotoxicity and apoptosis. The addition of 2-mercaptoethanol (2-ME), a cystine uptake enhancer, sodium-independently recovered the dRib-induced decreases in L-[14C] cystine uptake, GSH content and cell viability. To determine clearly whether system x(c)- deficiency mediate the oxidative stress-induced β-cell damage, we overexpressed xCT, the substrate-specific subunit of the system x(c)-, by using a lentiviral vector in RINm5F cells. Overexpression of xCT fully recovered the dRib-induced decreases in L-[14C] cystine uptake and GSH content and prevented the dRib-induced ROS rises, cytotoxicity and apoptosis. In summary, overexpression of xCT showed protective effects against oxidative damage in RINm5F cells. It suggests that system x(c)- deficiency play a critical role in oxidative stress-induced β-cell damage. Disclosure S. Yoo: None. J. Bae: None. H. Chin: None. G. Koh: None.

Chemometric analysis of selective polyphenolic groups in Asparagus racemosus (Shatavar) root extracts by traditional and supercritical fluid (CO2) based extractions

ABSTRACTAsparagus racemosus root extracts were prepared by supercritical fluid (CO2), soxhlet, and maceration-based methods also with various pretreatments. Thereafter, these root extracts were analyzed by High-Performance Liquid Chromatography, along with the chemometric study of the disparate phenolic groups. Among these, supercritical fluid (CO2) based extract has a larger number of polar compounds and the antioxidant activity (98.54 ± 0.22 µM Trolox equivalent mg−1). It also has the best cell viability (94.37 ± 1.12%) and insulin release (0.82 ± 1.12 ng mL−1) on β-pancreatic RINm-5F cells whereas, the best extractive yield (75.80 ± 3.44% w/w) was observed for pretreated aqueous soxhlet-based extract.

Viscothionin purified from mistletoe (Viscum album var. coloratum Ohwi) induces insulin secretion from pancreatic beta cells

Ethnopharmacological relevanceMistletoe (Viscum album), an evergreen parasitic plant, has been widely used as an oriental phytomedicine to treat diabetes mellitus. However, it is unknown which mistletoe constituent exerts the beneficial effect against the disease. In this study, we examined the hypoglycemic activity of mistletoe and investigated whether the polypeptide viscothionin, purified from mistletoe, was responsible for the activity. Materials and methodsMistletoe extracts were prepared by heating mistletoe powder made of leaves and twigs in water for 3, 6, 9, and 12 h. Rat insulinoma RINm5F cells were used to test the cytotoxicity of the extracts and their effects on the secretion of insulin and its precursor, C-peptide. The inhibitory effects of a mistletoe extract on glucose absorption were measured using an α-glucosidase inhibition assay. To determine the component of mistletoe responsible for the observed effects, the mistletoe extract was precipitated with ethanol or hydrolyzed with a protease for further testing. A potential active constituent of mistletoe was isolated by chromatography and molecular weight cut-off fractionation, and its ability to induce insulin secretion was investigated. ResultsA 12-h heat-treated mistletoe extract, showing no cytotoxicity, significantly increased the secretion of insulin and C-peptide by RINm5F cells and enhanced the expression of glucose transporter type 4 (GLUT-4), insulin receptor substrate 1 (IRS-1), and protein kinase B (also known as AKT) in differentiated C2C12 cells. The extract also inhibited α-glucosidase activity. After ethanol precipitation, the extract showed much stronger effects on insulin- and C-peptide-secreting activities of cells, whereas the enzyme-hydrolyzed extract was less effective than the original extract, suggesting that the effect was mediated by a proteinaceous constituent of mistletoe. Subsequent analysis showed that viscothionin, a heat-stable 6-kDa polypeptide isolated from mistletoe, increased the level of insulin secretion by more than 20-fold compared to that induced by the extract. ConclusionsOur study indicates that the hypoglycemic effect of mistletoe is mediated by its insulinotropic action and α-glucosidase inhibitory activity, and the effect is due to viscothionin, one of the major bioactive constituents of mistletoe.

Protective effect of Schisandra chinensis extracts against H2O2-induced oxidative damage in RINm5F cells

Objective: To study the protective effect of Schisandra chinensis extracts (SCEs) on the RINm5F cells against H2O2-induced oxidative damage and to provide a basis for the study of the role of SCEs in the prevention and treatment of diabetic oxidative stress. Materials and Methods: We performed ultrasonic preparation of SCEs and verified the presence of 8 lignans by high-performance liquid chromatography (HPLC). An in vitro model of H2O2-induced oxidative damage was established using RINm5F cells and treated with various concentrations of SCEs (high dose, medium dose, and low dose). The antioxidative activity of SCEs was observed and its mechanisms were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, and measuring malondialdehyde (MDA) and superoxide dismutase assay (SOD) according to the instructions of kits. Results: The total lignan content in SCEs was 18.86 mg/mL; the concentration of 8 kinds of lignans were schisandrin, 5.99 mg/mL; schisandrol B, 3.27 mg/mL; schisantherin A, 1.99 mg/mL; schisanhenol, 0.94 mg/mL; anwulignan, below the detection limit; deoxyschisandrin, 1.33 mg/mL; schisandrin B, 3.95 mg/mL; and schisandrin C, 1.39 mg/mL. SCEs promoted proliferation of islet cells and exhibited a protective effect against H2O2-induced oxidative damage in islet cells. The cell survival rates in the high-dose and medium-dose SCEs groups were greater than that in the model group by 63.4% and 45.6%, respectively; the protective effect of SCEs against oxidative damage was dose dependent. The intracellular MDA content was significantly decreased (P Abbreviation used: Schisandra chinensis Extracts (SCEs).

EFEITO INSULINOTRÓPICO E CITOPROTETOR DO EXTRATO BRUTO DE FOLHAS DE EUGENIA UNIFLORA L.

Eugenia uniflora L. leaves (“pitangueira”) are popularly used as an alternative to the treatment of diabetes mellitus. The objective of this work was to evaluate the cytotoxicity, antioxidant, cytoprotection and insulinotropic activity of crude leaf extracts of Eugenia uniflora L. on RINm5f cells. The antioxidant activity was analyzed by the Folin-Ciocalteau method; cytotoxicity and cytoprotection using the methods of MTT reduction and HE staining and the insulinotropic effect by radioimmunoassay. The aqueous extract (AQ) presented higher phenolic content than the alcoholic extract (ALC). Both showed low cytotoxicity and ALC presented a significant insulinotropic effect. AQ and ALC showed a cytoprotective effect, increasing cell viability and preventing changes in cell morphology induced by H2O2. The results obtained in this work are indicative of the potential of these extracts as coadjuvants in the prevention and treatment of diabetes mellitus. Key words: antioxidants, cytotoxicity, cytoprotection, flavonoids, hydrogen peroxide.

Protective effects of \u03b3-aminobutyric acid against H2O2-induced oxidative stress in RIN-m5F pancreatic cells

Backgroundγ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and reported to maintain the redox homeostasis and insulin secretion function of pancreatic β cells. This study tested the hypothesis that GABA maintains cellular redox status, and modulates glycogen synthase kinase (GSK)-3β and antioxidant-related nuclear factor erythroid 2-related factor 2 (NRF2) nuclear mass ratio in the H2O2-injured RINm5F cells.MethodsRINm5F cells were treated with/without GABA (50, 100 and 200 μmol/L) for 48 h and then exposed to 100 μmol/L H2O2 for 30 min. Viable cells were harvested, and dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used to detect reactive oxygen species (ROS) level; cellular redox status and insulin secretion were measured; cell viability was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; mitochondrial membrane potential (MMP) was detected by flow cytometry; relative genes levels were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR); western blotting was used to determine protein expression of GSK-3β and p-GSK-3β (Ser9), and nuclear and cytoplasmic NRF2.ResultsH2O2 increased ROS production, and induced adverse affects in relation to antioxidant defense systems and insulin secretion. These changes were restored by treatment with 100 and 200 μmol/L GABA. In addition, 100 or 200 μmol/L GABA induced membrane depolarization and increased cell viability. These effects were mediated by Caspase-3, Bcl-2 associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) expression. Western blotting indicated that GABA inhibited GSK-3β by increasing p-GSK-3β (Ser9) level, and directed the transcription factor NRF2 to the nucleus.ConclusionIn rat insulin-producing RINm5F cells, GABA exerts its protective effect by regulating GSK-3β and NRF2, which governs redox homeostasis by inhibiting apoptosis and abnormal insulin secretion by exposure to H2O2.

Protective Effect of NecroX-7 on Islets against Oxidative Stress and Inflammation

Oxidative stress generated during islet isolation, culture, and transplantation causes islet cell damage mediated by hypoxia, reactive oxygen species (ROS) and inflammatory responses. We investigated the protective effects of NecroX-7, a novel ROS scavenger, during isolation and/or culture of islets. For ex vivo studies, islets from heterozygote human islet amyloid polypeptide (hIAPP+/-) mice and C57BL/6J mice were isolated by collagenase with and without supplementation with 20 uM of NecroX-7. Supplementation with NecroX-7 provided markedly increased islet viability and ATP contents, and attenuated ROS, transcription of c-Jun N-terminal kinases, high mobility group box-1 (HMGB1) and proinflammatory cytokines including interleukin-1beta (IL-1b), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-a). Transplantation of islets, which is derived from islet isolation method using supplementation with NecroX-7, into syngeneic subrenal subcapsular of hIAPP+/- mice also improved post-transplant blood glucose levels. Also, NecroX-7 protected RINm5F cells from t-BHP-induced and serum deprivation induced toxicity in in vitro culture by dose-dependent manner from 0.1 uM to 20 uM. Supplementation of medium with NecroX-7 during serum-deprived culture condition also improved impaired viability and serum deprivation-induced ROS in islets from hIAPP+/- mice by dose-dependent manner from 0.1 uM to 20 uM. These findings suggest that NecroX-7 supplementation during the islet isolation and/or culture process suppressed inflammatory responses and ROS induced by serum deprivation and provides a potential benefit to improve post-transplantation outcomes. Disclosure Y. Kwon: None. H. Lee: None. H. Kim: None. G. Kim: None. S. Jin: None. M. Lee: None. J. Kim: None.

Effect of High-Glucose Concentration on the E3 Ubiquitin Ligase Activity of Mdm2

The degradation of p53 occurs via ubiquitin-26S proteosome, after Mdm2 (murine double minute 2) marks p53 with ubiquitins. The concentration and phosphorylation of Mdm2 and p53, greatly affect the ubiquitination rate of p53, by avoiding the recognition and physical interaction between p53 and Mdm2. Phosphorylation of Mdm2 in Ser395 by ATM decreases its ability to direct the degradation of p53. Therefore, we aim to study the effects of high glucose concentrations on the RING domain of Mdm2 and its ubiquitin E3 ligase activity. We cultured RINm5F cells in RPMI-1640 medium supplemented with 10% fetal bovine serum (v / v) at two concentrations of glucose 5 and 30 mM for up to 72 h, in a humid atmosphere at 37 ° C and 5% CO2. At the end of the treatment the cells were lysed to separate the nuclear and cytosolic fraction. After quantifying the proteins, they were visualized on SDS-PAGE and transferred to a PVDF membrane. Primary antibodies against Mdm2, Mdm2 p-Ser395, ATM, Arf, c-Abl, and ubiquitin were used and revealed using an amplified fluorescence kit from GE. The results show that the stress caused by high concentrations of glucose stimulates the phosphorylation of Mdm2 in Ser 395 by ATM activation. The phosphorylation of Ser395 alters the RING domain of Mdm2 and decreases its ubiquitin E3 ligase activity, ubiquitination and degradation of p53. Therefore, stress due to increased glucose decreases the ubiquitin E3 ligase activity of Mdm2 by inducing the phosphorylation of this residue, which leads to the stabilization and increase of p53 and the subsequent activation of apoptotic events. The Ser 395 residue of the RING domain is important for the efficient function of ubiquitin ligase of Mdm2 in β cells under conditions of hyperglycemia. Disclosure R. Barzalobre Geronimo: None.

A Rationally Designed Hsp70 Variant Rescues the Aggregation-Associated Toxicity of Human IAPP in Cultured Pancreatic Islet β-Cells.

Molecular chaperones are key components of the protein homeostasis system against protein misfolding and aggregation. It has been recently shown that these molecules can be rationally modified to have an enhanced activity against specific amyloidogenic substrates. The resulting molecular chaperone variants can be effective inhibitors of protein aggregation in vitro, thus suggesting that they may provide novel opportunities in biomedical and biotechnological applications. Before such opportunities can be exploited, however, their effects on cell viability should be better characterised. Here, we employ a rational design method to specifically enhance the activity of the 70-kDa heat shock protein (Hsp70) against the aggregation of the human islet amyloid polypeptide (hIAPP, also known as amylin). We then show that the Hsp70 variant that we designed (grafted heat shock protein 70 kDa-human islet amyloid polypeptide, GHsp70-hIAPP) is significantly more effective than the wild type in recovering the viability of cultured pancreatic islet β-cells RIN-m5F upon hIAPP aggregation. These results indicate that a full recovery of the toxic effects of hIAPP aggregates on cultured pancreatic cells can be achieved by increasing the specificity and activity of Hsp70 towards hIAPP, thus providing evidence that the strategy presented here provides a possible route for rationally tailoring molecular chaperones for enhancing their effects in a target-dependent manner.