Beta TC3 Research Articles

Antioxidant and Antidiabetic Activity of Cornus mas L. and Crataegus monogyna Fruit Extracts.

The present study evaluated three green extraction methods, accelerated solvent extraction (ASE), ultrasound-assisted extraction (UAE), and laser irradiation extraction (LE), for the polyphenolic compounds and vitamin C extraction of Cornus mas L. and Crataegus monogyna fruit extracts. The polyphenols and vitamin C of extracts were quantified using HPLC-DAD, and the total phenolic content, flavonoid content, antioxidant activity (DPPH and reducing power), and antidiabetic activity were also studied. The antidiabetic activity was examined by the inhibition of α-amylase and α-glucosidase, and in vitro on a beta TC cell line (β-TC-6). The results showed significant differentiation in the extraction yield between the methods used, with the ASE and LE presenting the highest values. The C. mas fruit extract obtained by ASE exhibited the best antioxidant activity, reaching an IC50 value of 31.82 ± 0.10 µg/mL in the DPPH assay and 33.95 ± 0.20 µg/mL in the reducing power assay. The C. mas fruit extracts obtained by ASE and LE also have the highest inhibitory activity on enzymes associated with metabolic disorders: α-amylase (IC50 = 0.44 ± 0.02 µg/mL for the extract obtained by ASE, and 0.11 ± 0.01 µg/mL for the extract obtained by LE at combined wavelengths of 1270 + 1550 nm) and α-glucosidase (IC50 of 77.1 ± 3.1 µg/mL for the extract obtained by ASE, and 98.2 ± 4.7 µg/mL for the extract obtained by LE at combined wavelengths of 1270 + 1550 nm). The evaluation of in vitro antidiabetic activity demonstrated that the treatment with C. mas and C. monogyna fruit extracts obtained using ASE stimulated the insulin secretion of β-TC-6 cells, both under normal conditions and hyperglycemic conditions, as well. All results suggest that C. mas and C. monogyna fruit extracts are good sources of bioactive molecules with antioxidant and antidiabetic activity.

Liposomal formulations of Oleae europaea L.: analyzing the antioxidant and antidiabetic activities along with toxicity profile in pancreatic beta TC6 cell line

ABSTRACT Olea europaea L. (Oleaceae), is rich in phenolic content and has powerful antioxidant and antidiabetic activity. However, there are no medicinal products prepared due to this feature. Therefore, this study aims to characterize an O. europaea extract with strong antioxidant and antidiabetic properties and to prepare nanoformulations containing this extract. To determine the activities of the extracts prepared from the leaves of the plant, DPPH• and ABTS•+ scavenging, Fe+3 reducing activity, α-amylase, and α-glucosidase inhibition assays were performed. The oleuropein content of the absolute ethanol extract with the highest activity was analysed by HPLC. The characterized extract was loaded into liposomes and chitosan coated liposomes, and the long-term sustainability of their activity was investigated. The encapsulation efficiency was 65.2% for the liposome and 66.8% for the chitosan-coated liposome formulation. The amounts of the extracts released from the formulations were evaluated to exhibit antioxidant and antidiabetic activity.

In vitro oxygen imaging of acellular and cell-loaded beta cell replacement devices

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices’ full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices’ in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO2) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin–lattice relaxation rate (R1) of oxygen-sensitive spin probes such as trityl OX071 and pO2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the “Oxygen Measurement Core” was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members’ laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D.

Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction

We previously reported a depletion of murine regenerating islet-derived protein 2 (REG2) in pancreatic islets of glutathione peroxidase-1 (Gpx1) overexpressing (OE) mice. The present study was to explore if and how the REG2 depletion contributed to an augmented glucose stimulated insulin secretion (GSIS) in OE islets. After we verified a consistent depletion (90%, p < 0.05) of REG2 mRNA, transcript, and protein in OE islets compared with wild-type (WT) controls, we treated cultured and perifused OE islets (70 islets/sample) with REG2 (1 μg/ml or ml · min) and observed 30–40% (p < 0.05) inhibitions of GSIS by REG2. Subsequently, we obtained evidences of co-immunoprecipitation, cell surface ligand binding, and co-immunofluorescence for a ligand-receptor binding between REG2 and transmembrane, L-type voltage-dependent Ca2+ channel (CaV1.2) in beta TC3 cells. Mutating the C-type lectin binding domain of REG2 or deglycosylating CaV1.2 removed the inhibition of REG2 on GSIS and(or) the putative binding between the two proteins. Treating cultured OE and perifused WT islets with REG2 (1 μg/ml or ml · min) decreased (p < 0.05) Ca2+ influx triggered by glucose or KCl. An intraperitoneal (ip) injection of REG2 (2 μg/g) to OE mice (6-month old, n = 10) decreased their plasma insulin concentration (46%, p < 0.05) and elevated their plasma glucose concentration (25%, p < 0.05) over a 60 min period after glucose challenge (ip, 1 g/kg). In conclusion, our study identifies REG2 as a novel regulator of Ca2+ influx and insulin secretion, and reveals a new cascade of GPX1/REG2/CaV1.2 to explain how REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca2+ influx and augmented GSIS. These findings create new links to bridge redox biology, tissue regeneration, and insulin secretion.

MTORC1 and mTORC2 Complexes Regulate the Untargeted Metabolomics and Amino Acid Metabolites Profile through Mitochondrial Bioenergetic Functions in Pancreatic Beta Cells.

Background: Pancreatic beta cells regulate bioenergetics efficiency and secret insulin in response to glucose and nutrient availability. The mechanistic Target of Rapamycin (mTOR) network orchestrates pancreatic progenitor cell growth and metabolism by nucleating two complexes, mTORC1 and mTORC2. Objective: To determine the impact of mTORC1/mTORC2 inhibition on amino acid metabolism in mouse pancreatic beta cells (Beta-TC-6 cells, ATCC-CRL-11506) using high-resolution metabolomics (HRM) and live-mitochondrial functions. Methods: Pancreatic beta TC-6 cells were incubated for 24 h with either: RapaLink-1 (RL); Torin-2 (T); rapamycin (R); metformin (M); a combination of RapaLink-1 and metformin (RLM); Torin-2 and metformin (TM); compared to the control. We applied high-resolution mass spectrometry (HRMS) LC-MS/MS untargeted metabolomics to compare the twenty natural amino acid profiles to the control. In addition, we quantified the bioenergetics dynamics and cellular metabolism by live-cell imaging and the MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed using ANOVA on Ranks and post-hoc Welch t-Tests. Results: RapaLink-1, Torin-2, and rapamycin decreased L-aspartate levels compared to the control (p = 0.006). Metformin alone did not affect L-aspartate levels. However, L-asparagine levels decreased with all treatment groups compared to the control (p = 0.03). On the contrary, L-glutamate and glycine levels were reduced only by mTORC1/mTORC2 inhibitors RapaLink-1 and Torin-2, but not by rapamycin or metformin. The metabolic activity network model predicted that L-aspartate and AMP interact within the same activity network. Live-cell bioenergetics revealed that ATP production was significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and the vehicle control (274.23 ± 38.17), p < 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink-1 (67.17 ± 3.52), Torin-2 (55.93 ± 8.76), or rapamycin (80.01 ± 4.36, p = 0.006). Conclusions: Dual mTORC1/mTORC2 inhibition by RapaLink-1 and Torin-2 differentially altered the amino acid profile and decreased mitochondrial respiration compared to rapamycin treatment which only blocks the FRB domain on mTOR. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a bioenergetics profile that could be targeted to reduce mitochondrial stress.

Natural COA water inhibits mitochondrial ROS-mediated apoptosis through Plk3 downregulation under STZ diabetic stress in pancreatic β-cell lines

Diabetes from pancreatic β cell death and insulin resistance is a serious metabolic disease in the world. Although the overproduction of mitochondrial reactive oxygen species (ROS) plays an important role in the pathogenesis of diabetes, its specific molecular mechanism remains unclear. Here, we show that the natural Charisma of Aqua (COA) water plays a role in Streptozotocin (STZ) diabetic stress-induced cell death inhibition. STZ induces mitochondrial ROS by increasing Polo-like kinase 3 (Plk3), a major mitotic regulator, in both Beta TC-6 and Beta TC-tet mouse islet cells and leads to apoptosis. Overexpression of Plk3 regulates an increase in mitochondrial ROS as well as cell death, also these events were inhibited by Plk3 gene knockdown in STZ diabetic stimulated-Beta TC-6 cells. Interestingly, we found that natural COA water blocks mitochondrial ROS generation through the reduction of Plk3 and prevents apoptosis in STZ-treated beta cells. Furthermore, using the 3D organoid (ex vivo) system, we confirmed that the insulin secretion of the supernatant medium under STZ treated pancreatic β-cells is protected by the natural COA water. These findings demonstrate that the natural water COA has a beneficial role in maintaining β cell function through the inhibition of mitochondrial ROS-mediated cell death, and it might be introduced as a potential insulin stabilizer.

Mitochondrial Bioenergetics Profile With Different Mechanistic Target of Rapamycin Complexes (mTORC1/mTORC2) Inhibitors in Pancreatic Beta-Cell Lines (Beta-TC-6)

Abstract Objectives We investigated the impact of mTOR complexes inhibition with RapaLink-1, a third-generation bivalent mTORC1/mTORC2 inhibitor that links rapamycin with a mTOR-kinase inhibitor, or Torin-2 (ATP-competitive) on mitochondrial dynamics, bioenergetics, and extracellular flux. Methods We used insulin-secreting, glucose-responsive pancreatic beta cells derived from transgenic mice expressing SV40 (Beta-TC-6 cells, ATCC-11506). Cells were treated for 24 hours with either: RapaLink; Torin-2; rapamycin; metformin; a combination of metformin and RapaLink, or Torin-2; compared to vehicle control. Bioenergetic dynamics and cellular metabolism were quantified using MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed by ANOVA followed by an unpaired t-test. Results Rapalink-1 and Torin-2 incubation decreased basal OCR compared to rapamycin alone or vehicle control (189.58 ± 48.58, 116.85 ± 26.55 versus 405.01 ± 21.57, 444.19 ± 58.59, p &amp;lt; 0.01) in Beta TC-6 pancreatic cell lines. ATP production was also significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and vehicle control (274.23 ± 38.17), p &amp;lt; 0.01. Similarly, proton leak decreased in RapaLink-1 (67.34 ± 15.40) and Torin-2 (44.48 ± 9.23) treated beta cells, compared to rapamycin (154.56 ± 12.53) or control (169.96 ± 20.52), p &amp;lt; 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink (67.17 ± 3.52) Torin-2 (55.93 ± 8.76) or rapamycin (80.01 ± 4.36), but was less than the control group (108.80 ± 7.19), p = 0.006. Conclusions The combination of mTORC1 and mTORC2 inhibition by RapaLink-1 or Torin-2 decreased mitochondrial respiration compared to rapamycin treatment. Decreased OCR suggests reduced initial energy requirements, while decreased ATP production indicates decreased energy demands. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a mitochondrial bioenergetics profile that could be targeted to reduce mitochondrial stress. Funding Sources City University of New York, GC Advanced Science Research Center Seed Grant Award

Novel Approach to the Treatment of Diabetes: Embryonic Stem Cell and Insulin-Loaded Liposomes and Nanocochleates.

This study aims to investigate and compare the effects of insulin and embryonic stem-cell (ESC) loaded liposomes (LPs) and nanocochleate formulations and their PEGylated forms on the glucose levels. All formulations were characterized considering particle size, zeta potential, polydispersity index and encapsulation efficiencies. In-vitro insulin that releases from the formulations was determined using Franz-type diffusion cells. A cytotoxicity test revealed that none of the formulations was toxic to cells in any concentrations. The effects of the formulations on diabetic cells induced with glucose and streptozotocin (STZ) were then investigated in cell culture studies. Although glucose levels were decreased by the formulations after incubation, the liposomal formulations were found to be better. In experiments that were conducted on mice, it was observed again that blood glucose levels decreased successfully when diabetic pancreatic beta TC cells were incubated with the formulations, and all formulations were found to be effective in decreasing blood glucose levels in diabetic mice. Although ESC-loaded LPs were found to be the most effective formulation, LPs and nanocochleate formulations may also be used for the repair of pancreatic cells. This proposed ESC treatment is considered to be an attractive approach and a potential source for cell replacement therapy in the treatment of diabetes.

Effects of Insulin and Embryonic Stem Cells loaded PLGA Nanoparticles on Pancreatic Beta TC Cells

Effects of Insulin and Embryonic Stem Cells loaded PLGA Nanoparticles on Pancreatic Beta TC Cells

Nanoliposomal Resveratrol as a Novel Approach to Treatment of Diabetes Mellitus.

Diabetes mellitus (DM) is a chronic metabolic disease and the subgroup of DM is called type II which is the most common form. The incidence of type II is increasing worldwide and it focuses on several new approaches to efficiently treatment of diabetes. Resveratrol (RSV) is known to be strong antioxidant and has an insulin-like effect in streptozotocin (STZ)-induced diabetic cells. It plays an active role at treatment of diabetes with reducing the oxidative stress, lowering glucose levels and protection of beta cells which are responsible for insulin secretion. In our study, we prepared two different RSV-loaded nanoliposomes (LPs), characterized in vitro and evaluated efficiencies of LPs on diabetes and related oxidative stress. Release and transport studies of RSV through dialyse membrane and pancreatic beta TC (β TC) cells were investigated from its solution and LPs. Stability studies were performed at two different conditions (4 °C and 25 °C ± 60% relative humidity) for 3 months. Particle size (PS), zeta potential (ZP), polydispersity index (PDI), encapsulation efficiency (EE) and type of the formulations were determined. β TC cell line was used in cell culture studies and cell viability was measured with using 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) cytotoxicity test. The antidiabetic effects of RSV LPs were investigated on β TC cell induced with glucose and STZ and we evaluated relationship between glucose and insulin concentration before and after incubation with LPs containing RSV. Antioxidant and preventive effects of RSV-loaded LPs against diabetes-associated oxidative stress were determined with superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme assay. When all results were evaluated together, these new developed liposomal formulations significantly decreased high glucose levels in diabetic cell groups synchronous with increasing insulin levels and they showed prolonged antioxidant activity against oxidative stress for 24 hours compared to RSV solution.

Development and Characterization of Insulin-loaded Liposome-chitosan-Nanoparticle (LCS-NP) Complex and Investigation of Transport Properties Through a Pancreatic Beta Tc Cell Line.

In recent years, studies on oral use have increased rapidly due to the restrictive aspects of parenteral administration of indispensable peptide-structured insulin in the rapidly growing worldwide treatment of diabetes. The aim of the study was to examine the development of a novel insulin-loaded LCS-NP complex, and its characterization and efficacy on pancreatic cells responsible for insulin release. Blank liposomes and insulin-loaded LCS-NPs were prepared using dry film hydration and ionotropic gelation methods, respectively. The LCS-NP complex was prepared by mixing liposomes/NPs in a 2:1 (w/w) ratio. The cytotoxic effects of the various concentrations of insulin and formulation components on the pancreatic cell line were determined using a 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay and quantities to be used in the formulation were determined. Particle size, zeta potential, encapsulation efficiency, in vitro release profile and release kinetics, and transport properties of the prepared complex were investigated. The newly developed insulin-loaded LCS-NP complex had a particle size of 2.85±0.035 μm and zeta potential of 8.11±1.025 mV. The encapsulation yield was found as 48±1.1%. In vitro insulin release from the complex was 80.9±2.71%. Insulin transport from β Tc cells was 30.50%. Permeability coefficients (log k) were calculated as -1.280±0.070 for the insulin solution and -1.020±0.062 for the insulin-loaded complex. This study suggests that insulin could be successfully loaded into the newly developed LCS-NP complex, and it is thought that this complex carries an effective formulation potential for long-term efficacy in the treatment of diabetes.

Microfabrication of scaffold-free tissue strands for three-dimensional tissue engineering

In this note, we report a practical and efficient method based on a coaxial extrusion and microinjection technique for biofabrication of scaffold-free tissue strands. Tissue strands were obtained using tubular alginate conduits as mini-capsules with well-defined permeability and mechanical properties, where their removal by ionic decrosslinking allowed the formation of scaffold-free cell aggregates in the form of cylindrical strands with well-defined morphology and geometry. Rat dermal fibroblasts and mouse insulinoma beta TC3 cells were used to fabricate both single-cellular and heterocellular tissue strands with high cell viability, self-assembling capability and the ability to express cell-specific functional markers. By taking advantage of tissue self-assembly, we succeeded in guiding the fusion of tissue strands to fabricate larger tissue patches. The presented approach enables fabrication of cell aggregates with controlled dimensions allowing highly long strands, which can be used for various applications, including fabrication of scale-up complex tissues and of tissue models for drug screening and cancer studies.

Tissue strands as "bioink" for scale-up organ printing.

Organ printing, takes tissue spheroids as building blocks together with additive manufacturing technique to engineer tissue or organ replacement parts. Although a wide array of cell aggregation techniques has been investigated, and gained noticeable success, the application of tissue spheroids for scale-up tissue fabrication is still worth investigation. In this paper, we introduce a new micro-fabrication technique to create tissue strands at the scale of 500-700μm as a "bioink" for future robotic tissue printing. Printable alginate micro-conduits are used as semi-permeable capsules for tissue strand fabrication. Mouse insulinoma beta TC3 cell tissue strands were formed upon 4 days post fabrication with reasonable mechanical strength, high cell viability close to 90%, and tissue specific markers expression. Fusion was readily observed between strands when placing them together as early as 24h. Also, tissue strands were deposited with human umbilical vein smooth muscle cells (HUVSMCs) vascular conduits together to fabricated miniature pancreatic tissue analog. Our study provided a novel technique using tissue strands as "bioink" for scale-up bioprinting of tissues or organs.

Synthesis and biological evaluation of thienopyrimidine derivatives as GPR119 agonists

A series of thienopyrimidine derivatives was synthesized and evaluated for their GPR119 agonistic ability. Several thienopyrimidine derivatives containing R1 and R2 substituents displayed potent GPR119 agonistic activity. Among them, compound 5d, which is a prototype, showed good in vitro activity with an EC50 value of 3nM and human and rat liver microsomal stability. Compound 5d exhibited no CYP inhibition and induction, Herg binding, or mutagenic potential. Compound 5d showed increase insulin secretion in beta TC-6 cell and lowered the glucose excursion in mice in an oral glucose-tolerance test.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5′-phosphatase interaction with c-Abl

It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. Human EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

Effect of Environmental Contaminants on Beta Cell Function

There is an increasing concern that chemicals in the environment are contributing to the global rise in the prevalence of type 2 diabetes (T2D). However, there is limited evidence for direct effects of these chemicals on beta cell function. Therefore, the goals of this study were (1) to test the hypothesis that environmental contaminants can directly affect beta cell function and (2) examine mechanistic pathways by which these contaminants could affect beta cell function. Using mouse beta TC-6 cells, we examined the acute effects of 6 substances (benzo[a]pyrene, bisphenol A [BPA], propylparaben, methylparaben, perfluorooctanoic acid, and perfluorooctyl sulfone) on insulin secretion. Only BPA treatment directly affected insulin secretion. Furthermore, chronic exposure to BPA altered the expression of key proteins in the cellular and endoplasmic reticulum stress response. These data suggest that long-term BPA exposure may be detrimental to beta cell function and ultimately be an important contributor to the etiology of T2D.

Mixed Lineage Kinase-3 Stabilizes and Functionally Cooperates with TRIBBLES-3 to Compromise Mitochondrial Integrity in Cytokine-induced Death of Pancreatic Beta Cells

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of non-obese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.

Murine Pancreatic Beta TC3 Cells Show Greater2′,5′-Oligoadenylate Synthetase (2′5′AS) Antiviral Enzyme Activity and Apoptosis Following IFN-αor Poly(I:C) Treatment than Pancreatic Alpha TC3 Cells

Type 1 diabetes is caused by autoimmune destruction of pancreatic beta cells, possibly virus initiated. Virus infection induces alpha-interferon (IFN-α), leading to upregulation of genes encoding double-stranded (ds) RNA-dependent antiviral enzymes 2′, 5′-oligoadenylate synthetase (2′5′AS) and PKR (p68). To investigate whether beta cell specificity could be due to antiviral differences between beta and alpha cells, we treated beta and alpha TC3 cell lines with IFN-α and/or poly(I:C) (a synthetic dsRNA). Results showed that, following IFN-α stimulation, increases in 2′5′AS levels and activities were significantly higher in beta than alpha cells (P < .001), whereas increases in PKR level and activity were comparable in the two cell types. Poly(I:C) stimulated 2′5′AS activity in beta but not alpha cells, and co-transfection IFN-α plus poly(I:C) induced apoptosis in beta but not alpha cells. These findings suggest that the elevated 2′5′AS response of pancreatic beta cells could render them particularly vulnerable to damage and/or apoptosis during virus infection.

Proinflammatory Cytokine Gene Polymorphisms among Iranian Patients with Asthma

Asthma is one of the most common respiratory diseases caused by acute and chronic inflammation of airways. Proinflammatory cytokines could contribute to this inflammatory process. This study was performed in order to analyze the genetic profile of proinflammatory cytokines in Iranian asthmatic patients. The allele and genotype frequencies of a number polymorphic genes coding for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor (IL-1R), IL-1RA, and IL-6 were investigated in 60 patients with asthma in comparison with 140 controls using polymerase chain reaction with sequence-specific primers. The most frequent genotypes in our patients were TNF-alpha GA at position -308 (P = 0.001), TNF-alpha AA at position -238 (P = 0.01), IL-1 alpha TC at position -889 (P = 0.0001), IL-1 beta TC at position -511 (P = 0.0001), and IL-1RA TC at position Mspa-I 11100 (P = 0.001). In contrast, the frequencies of the genotypes TNF-alpha GG at position -308 (P = 0.001), IL-1 alpha CC at position -889 (P = 0.005), IL-1 beta CC at position -511 (P = 0.0001), and IL-1RA TT at position Mspa-I 11100 (P = 0.0001) in the patient group were significantly lower than controls. The most frequent haplotypes for TNF-alpha (positions 308, -238) was A/A in the patient group in comparison with controls (P = 0.0001). While environmental factors are important in the development of asthma, genetic factors could have a critical role in the expression of the disease. Considering the high frequency of presence of TNF-alpha AG genotype (-308), it seems that the production of TNF-alpha in the asthmatic patients could be higher than normal subjects.

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Regulation of blood glucose is a fundamental homeostasis in the body. Insulin is released from pancreatic beta-cells in response to changes in blood glucose, the defect of which leads to impaired insulin secretion and diabetes mellitus. Pancreatic beta-cells that release insulin occupy approximately 70% of the islet cells, while alpha, delta, PP-cells are also present in islets. Therefore, analysis of beta-cells is a direct approach for studying mechanisms of insulin secretion. Insulin secretion is regulated by cytosolic Ca(2+) in beta-cells, and its concentration and localization can be measured in real time by fluorescence imaging using indicators. Glucose metabolism is assessed by measurements of NAD(P)H by its autofluorescence. Furthermore, beta-cells are equipped with ion channels, which transduce glucose-evoked metabolic signals to electric signals. Electrophysiological analysis by patch clamp techniques detects the activity of various ion channels and membrane potential. Several beta-cell lines including HIT, MIN6, INS1, RIN, and beta TC are used; however, they do not necessarily retain normal responsiveness to glucose. Therefore, analysis of physiologic functions of beta-cells requires the use of acutely isolated or primary cultured beta-cells from normal animals. The methods for preparation of islets and single beta-cells using collagenase can be applied to a variety of animals of small to large sizes, which can produce islets and beta-cells with physiological responsiveness to glucose. These primary cultured beta-cells can be used for elucidating signal transduction mechanisms, and evaluating effects of drugs, providing excellent tools for physiological, pharmacological, and disease-oriented studies.