Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects.

Increased adipose tissue lipogenesis is associated with enhanced insulin sensitivity. Mice overexpressing the Glut4 glucose transporter in adipocytes have elevated lipogenesis and increased glucose tolerance despite being obese with elevated circulating fatty acids. Lipidomic analysis of adipose tissue revealed the existence of branched fatty acid esters of hydroxy fatty acids (FAHFAs) that were elevated 16- to 18-fold in these mice. FAHFA isomers differ by the branched ester position on the hydroxy fatty acid (e.g., palmitic-acid-9-hydroxy-stearic-acid, 9-PAHSA). PAHSAs are synthesized in vivo and regulated by fasting and high-fat feeding. PAHSA levels correlate highly with insulin sensitivity and are reduced in adipose tissue and serum of insulin-resistant humans. PAHSA administration in mice lowers ambient glycemia and improves glucose tolerance while stimulating GLP-1 and insulin secretion. PAHSAs also reduce adipose tissue inflammation. In adipocytes, PAHSAs signal through GPR120 to enhance insulin-stimulated glucose uptake. Thus, FAHFAs are endogenous lipids with the potential to treat type 2 diabetes.

PAHSAs reduce cellular senescence and protect pancreatic beta cells from metabolic stress through regulation of Mdm2/p53.

Senescence in pancreatic beta cells plays a major role in beta cell dysfunction, which leads to impaired glucose homeostasis and diabetes. Therefore, prevention of beta cell senescence could reduce the risk of diabetes. Treatment of nonobese diabetic (NOD) mice, a model of type 1 autoimmune diabetes (T1D), with palmitic acid hydroxy stearic acids (PAHSAs), a novel class of endogenous lipids with antidiabetic and antiinflammatory effects, delays the onset and reduces the incidence of T1D from 82% with vehicle treatment to 35% with PAHSAs. Here, we show that a major mechanism by which PAHSAs protect islets of the NOD mice is by directly preventing and reversing the initial steps of metabolic stress-induced senescence. In vitro PAHSAs increased Mdm2 expression, which decreases the stability of p53, a key inducer of senescence-related genes. In addition, PAHSAs enhanced expression of protective genes, such as those regulating DNA repair and glutathione metabolism and promoting autophagy. We demonstrate the translational relevance by showing that PAHSAs prevent and reverse early stages of senescence in metabolically stressed human islets by the same Mdm2 mechanism. Thus, a major mechanism for the dramatic effect of PAHSAs in reducing the incidence of type 1 diabetes in NOD mice is decreasing cellular senescence; PAHSAs may have a similar benefit in humans.

Human health risk assessment of nitrate in private well waters of shallow quaternary alluvial aquifer.

Excessive nitrate intake via ingestion pathway and dermal absorption exposures has adverse health impacts on human health. This study evaluated groundwater (GW) nitrate concentrations and health risks which focused on ingestion and dermal exposures to residents in Bachok District, Kelantan, Malaysia. Three hundred (300) samples of private wells were collected and it is found that the nitrate concentrations ranging between 0.11 and 64.01 mg/L NO3-N with a mean value of 10.45 ± 12.67 mg/L NO3-N. The possible health hazards of nitrate by ingestion and dermal contact were assessed using USEPA human health risk assessment model for adult males and females. It is observed that the mean Hazard Quotient (HQ) values of adult males and females were 0.305 ± 0.364 and 0.261 ± 0.330, respectively. About 7.3% (n = 10) and 4.9% (n = 8) of adult males and females had HQ values more than 1, respectively. It was also observed that the mean of HQderm was lesser than HQoral for males and females. The spatial distribution of HQ by interpolation method showed high nitrate concentrations (> 10 mg/L NO3-N) were distributed from the centre to the southern part of the study location, which identified as an agricultural area, indicating the used of nitrogenous fertilizers as the main source of GW nitrate contamination in this area. The findings of this study are valuable for establishing private well water protection measures to stop further deterioration of GW quality caused by nitrate.

Factors associated with the spatial accessibility of healthcare services measured by the floating catchment area (FCA)-based method: A scoping review.

INTRODUCTION: The floating catchment area (FCA) method has emerged as the most comprehensive and accurate method for quantifying the spatial accessibility of health care services. There were variants of the FCA-based method that was continuously improvised by the researchers to suit specific local contexts and the different nature of healthcare service delivery. This scoping review identifies factors associated with the spatial accessibility of healthcare services that were specifically measured using the FCAbased method. MATERIALS AND METHODS: This scoping review was performed through electronic databases (PubMed and ScienceDirect) using keywords: 'spatial accessibility', 'floating catchment area' and 'factors'. Google Scholar and Mendeley Network were also used as additional sources to obtain relevant studies. RESULTS: A total of 32 articles were included in this review. Factors identified can be distinguished into two broad categories, which are spatial and non-spatial factors. Spatial factors were remoteness or distance from the urban centre, areas in close proximity to main roads, and some specific geographical characteristics such as mountainous and deltaic regions, whereas non-spatial factors were the degree of urbanisation, population density and various demographic profiles of the population such as socioeconomic status, health need, and minority ethnic composition. CONCLUSION: This study adds to the body of literature pertinent to the factors associated with spatial accessibility to healthcare services. These findings could give insight for researchers to consider and incorporate those additional variables to further improve the FCA-based method calculations.

A Review of Spatial Analysis Application in Childhood Malnutrition Studies.

Approximately 230 million children under 5 years old of age suffer from malnutrition and over half of the children below 5 years old deaths are due to malnutrition nowadays. To gain a better understanding of this problem, the application of spatial analysis has risen exponentially in recent years. In this review, the present state of information on the use of spatial analysis in childhood malnutrition studies was evaluated using four databases of digital scientific journals: ScienceDirect, Scopus, PubMed and CINAHL. We chose 2,278 articles from the search results and a total of 27 articles met our criteria for review. The following information was extracted from each article: objective of study, study area, types of malnutrition, subject, data sources, computer software packages, spatial analysis and factors associated with childhood malnutrition. A total of 10 spatial analysis methods were reported in the reviewed articles and the Bayesian geoadditive regression model was the most common method applied in childhood malnutrition studies. This review highlights the importance of the application of spatial analysis in determining the geographic distribution of malnutrition cases, hotspot areas and risk factors correlated with childhood malnutrition. It also provides implications for strategic initiatives to eradicate all forms of malnutrition.

Distinct biological activities of isomers from several families of branched fatty acid esters of hydroxy fatty acids (FAHFAs).

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous lipids with antidiabetic and anti-inflammatory effects. Each FAHFA family consists of esters with different acyl chains and multiple isomers with branch points at different carbons. Some FAHFAs, including palmitic acid hydroxy stearic acids (PAHSAs), improve insulin sensitivity and glucose tolerance in mice by enhancing glucose-stimulated insulin secretion (GSIS), insulin-stimulated glucose transport, and insulin action to suppress hepatic glucose production and reducing adipose tissue inflammation. However, little is known about the biological effects of other FAHFAs. Here, we investigated whether PAHSAs, oleic acid hydroxy stearic acid, palmitoleic acid hydroxy stearic acid, and stearic acid hydroxy stearic acid potentiate GSIS in ß-cells and human islets, insulin-stimulated glucose uptake in adipocytes, and anti-inflammatory effects in immune cells. We also investigated whether they activate G protein-coupled receptor 40, which mediates the effects of PAHSAs on insulin secretion and sensitivity in vivo. We show that many FAHFAs potentiate GSIS, activate G protein-coupled receptor 40, and attenuate LPS-induced chemokine and cytokine expression and secretion and phagocytosis in immune cells. However, fewer FAHFAs augment insulin-stimulated glucose uptake in adipocytes. S-9-PAHSA, but not R-9-PAHSA, potentiated GSIS and glucose uptake, while both stereoisomers had anti-inflammatory effects. FAHFAs containing unsaturated acyl chains with higher branching from the carboxylate head group are more likely to potentiate GSIS, whereas FAHFAs with lower branching are more likely to be anti-inflammatory. This study provides insight into the specificity of the biological actions of different FAHFAs and could lead to the development of FAHFAs to treat metabolic and immune-mediated diseases.

Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation.

Lipids have fundamental roles in the structure, energetics, and signaling of cells and organisms. The recent discovery of fatty acid esters of hydroxy fatty acids (FAHFAs), lipids with potent antidiabetic and anti-inflammatory activities, indicates that our understanding of the composition of lipidome and the function of lipids is incomplete. The ability to synthesize and test FAHFAs was critical in elucidating the roles of these lipids, but these studies were performed with racemic mixtures, and the role of stereochemistry remains unexplored. Here, we synthesized the R- and S- palmitic acid ester of 9-hydroxystearic acid (R-9-PAHSA, S-9-PAHSA). Access to highly enantioenriched PAHSAs enabled the development of a liquid chromatography-mass spectrometry (LC-MS) method to separate and quantify R- and S-9-PAHSA, and this approach identified R-9-PAHSA as the predominant stereoisomer that accumulates in adipose tissues from transgenic mice where FAHFAs were first discovered. Furthermore, biochemical analysis of 9-PAHSA biosynthesis and degradation indicate that the enzymes and pathways for PAHSA production are stereospecific, with cell lines favoring the production of R-9-PAHSA and carboxyl ester lipase (CEL), a PAHSA degradative enzyme, selectively hydrolyzing S-9-PAHSA. These studies highlight the role of stereochemistry in the production and degradation of PAHSAs and define the endogenous stereochemistry of 9-PAHSA in adipose tissue. This information will be useful in the identification and characterization of the pathway responsible for PAHSA biosynthesis, and access to enantiopure PAHSAs will elucidate the role of stereochemistry in PAHSA activity and metabolism in vivo.

Branched Fatty Acid Esters of Hydroxy Fatty Acids Are Preferred Substrates of the MODY8 Protein Carboxyl Ester Lipase.

A recently discovered class of endogenous mammalian lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), possesses anti-diabetic and anti-inflammatory activities. Here, we identified and validated carboxyl ester lipase (CEL), a pancreatic enzyme hydrolyzing cholesteryl esters and other dietary lipids, as a FAHFA hydrolase. Variants of CEL have been linked to maturity-onset diabetes of the young, type 8 (MODY8), and to chronic pancreatitis. We tested the FAHFA hydrolysis activity of the CEL MODY8 variant and found a modest increase in activity as compared with that of the normal enzyme. Together, the data suggest that CEL might break down dietary FAHFAs.

TIAM1-RAC1 signalling axis-mediated activation of NADPH oxidase-2 initiates mitochondrial damage in the development of diabetic retinopathy.

AIMS/HYPOTHESIS: In diabetes, increased retinal oxidative stress is seen before the mitochondria are damaged. Phagocyte-like NADPH oxidase-2 (NOX2) is the predominant cytosolic source of reactive oxygen species (ROS). Activation of Ras-related C3 botulinum toxin substrate 1 (RAC1), a NOX2 holoenzyme member, is necessary for NOX2 activation and ROS generation. In this study we assessed the role of T cell lymphoma invasion and metastasis (TIAM1), a guanine nucleotide exchange factor for RAC1, in RAC1 and NOX2 activation and the onset of mitochondrial dysfunction in in vitro and in vivo models of glucotoxicity and diabetes. METHODS: RAC1 and NOX2 activation, ROS generation, mitochondrial damage and cell apoptosis were quantified in bovine retinal endothelial cells exposed to high glucose concentrations, in the retina from normal and streptozotocin-induced diabetic rats and mice, and the retina from human donors with diabetic retinopathy. RESULTS: High glucose activated RAC1 and NOX2 (expression and activity) and increased ROS in endothelial cells before increasing mitochondrial ROS and mitochondrial DNA (mtDNA) damage. N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine, trihydrochloride (NSC23766), a known inhibitor of TIAM1-RAC1, markedly attenuated RAC1 activation, total and mitochondrial ROS, mtDNA damage and cell apoptosis. An increase in NOX2 expression and membrane association of RAC1 and p47(phox) were also seen in diabetic rat retina. Administration of NSC23766 to diabetic mice attenuated retinal RAC1 activation and ROS generation. RAC1 activation and p47(phox) expression were also increased in the retinal microvasculature from human donors with diabetic retinopathy. CONCLUSIONS/INTERPRETATION: The TIAM1-RAC1-NOX2 signalling axis is activated in the initial stages of diabetes to increase intracellular ROS leading to mitochondrial damage and accelerated capillary cell apoptosis. Strategies targeting TIAM1-RAC1 signalling could have the potential to halt the progression of diabetic retinopathy in the early stages of the disease.

Data-driven synthesis of proteolysis-resistant peptide hormones.

Peptide hormones are key physiological regulators, and many would make terrific drugs; however, the therapeutic use of peptides is limited by poor metabolism including rapid proteolysis. To develop novel proteolysis-resistant peptide hormone analogs, we utilize a strategy that relies on data from simple mass spectrometry experiments to guide the chemical synthesis of proteolysis-resistant analogs (i.e., data-driven synthesis). Application of this strategy to oxyntomodulin (OXM), a peptide hormone that stimulates insulin secretion from islets and lowers blood glucose in vivo, defined the OXM cleavage site in serum, and this information was used to synthesize a proteolysis-resistant OXM analog (prOXM). prOXM and OXM have similar activity in binding and glucose stimulated-insulin secretion assays. Furthermore, prOXM is also active in vivo. prOXM reduces basal glucose levels and improves glucose tolerance in mice. The discovery of prOXM suggests that proteolysis-resistant variants of other important peptide hormones can also be found using this strategy to increase the number of candidate therapeutic peptides.

Spatial Accessibility of Primary Care in the Dual Public-Private Health System in Rural Areas, Malaysia.

Disparities in access to health services in rural areas represent a global health issue. Various external factors contribute to these disparities and each root requires specific remedial action to alleviate the issue. This study elucidates an approach to assessing the spatial accessibility of primary care, considering Malaysia's dual public-private system specifically in rural areas, and identifies its associated ecological factors. Spatial accessibility was calculated using the Enhance 2-Step Floating Catchment Area (E2SFCA) method, modified as per local context. Data were secondary sourced from Population and Housing Census data and administrative datasets pertaining to health facilities and road network. The spatial pattern of the E2SFCA scores were depicted using Hot spot Analysis. Hierarchical multiple linear regression and geographical weight regression were performed to identify factors that affect E2SFCA scores. Hot spot areas revolved near the urban agglomeration, largely contributed by the private sector. Distance to urban areas, road density, population density dependency ratios and ethnic composition were among the associated factors. Accurate conceptualization and comprehensive assessment of accessibility are crucial for evidence-based decision making by the policymakers and health authorities in identifying areas that need attention for a more specific and localized planning and development.

L- threo -C6-pyridinium-ceramide bromide, a novel cationic ceramide, induces NADPH oxidase activation, mitochondrial dysfunction and loss in cell viability in INS 832/13 ß-cells.

BACKGROUND: Emerging evidence indicates that exposure of isolated pancreatic ß-cells to elevated glucose [glucotoxicity] or saturated fatty acids such as palmitate [lipotoxicity] or both [glucolipotoxicity] results in excessive intracellular oxidative stress mediated by phagocyte-like NADPH oxidase [Nox2]. Pharmacological evidence also implicates the intracellular generation of ceramide [CER] as one of the mediators of palmitate-induced cytotoxicity of the islet ß- cell. Herein, we investigated the effects of L-threo-C6-pyridinium-ceramide bromide, a novel water soluble cationic ceramide [Ws-CER], on mitochondrial function and cell viability in insulin-secreting INS 832/13 cells. METHODS: Ws-CER, was synthesized as we reported earlier. Rac1 activation was quantitated by pull-down assay. Mitochondrial membrane potential was quantitated by JC-1 staining. Nox2 subunit expression and caspase-3 activity were determined by Western blotting. RESULTS: Our findings suggested a marked increase in the Nox2 activation [i.e., ROS generation and subunit expression and activation] in cells exposed to Ws-CER. We also noticed a significant reduction in mitochondrial membrane potential, increased in caspase-3 activity and associated loss in cell viability in Ws-CER-treated cells. CONCLUSION: Based on these data we conclude that ceramide-induced Nox2-mediated oxidative stress couples mitochondrial dysfunction to loss in cell viability in the pancreatic ß-cell.

Matrix metalloproteinase-9, -10, and -12, MDM2 and p53 expression in mouse liver during dimethylnitrosamine-induced oxidative stress and genomic injury.

Treatment during early tumor development has greater success because tissue growth remains largely confined to its original locus. At later stages, malignant cells migrate from their original location, invade surrounding normal areas, and can disseminate widely throughout the body. Remodeling of the extracellular matrix (ECM) serves as a key facilitator of this dissemination. Proteolytic enzymes including plasmin and matrix metalloproteinases (MMPs) play an integral role in degrading the surrounding ECM proteins and clearing a path for tumor cell migration. Specific MMPs are highly expressed late during malignant tumor invasion. It is not understood whether early changes in MMPs influence apoptotic and necrotic cell death, processes known to govern the early stages of carcinogenesis. Similarly, the interaction between MDM2 and p53 is tightly controlled by a complex array of post-translational modifications, which in turn dictates the stability and activity of both p53 and MDM2. The present studies examine the hypothesis that model hepatotoxin dimethylnitrosamine (DMN), which is also a model carcinogen, will induce the MMP family of proteins after administration in hepatotoxic doses. Doses of 25, 50, and 100 mg/kg DMN were administered i.p. to male C3H mice. Changes in parameters associated with apoptotic and necrotic cell death, DNA damage, cell proliferation, and extracellular proteinases were examined in liver at 24 h. Serum ALT activity, oxidative stress [malondialdehyde], and caspase-activated DNAse mediated DNA laddering increased in a dose-dependent manner, as did the level of MDM2 protein. MMP-9, -10 and -12 (gelatinase-B, stromelysin-2, macrophage elastase), and p53 protein levels increased following 25 mg/kg DMN, but were successively decreased after higher DMN doses. The results of this study demonstrate changes in MDM2 and MMPs during DMN-induced acute liver injury and provide a plausible linkage between DMN-induced oxidative stress-mediated genomic injury and its likely involvement in setting the stage for initiating subsequent metastatic disease at later circumstances.

An in vitro evaluation of efficacy of ViaSpan, aloe vera, gatorade solution, and propolis storage media for maintaining the periodontal ligament cell viability.

Background: Replantation is a commonly performed method for avulsed tooth. A vital periodontal membrane (periodontal ligament [PDL]) is significant for the successful healing of replanted teeth. Hence, various storage media are used to preserve the viability of periodontal cells before replantation. Objectives: The present study was conducted to evaluate the efficacy of ViaSpan, Aloe vera, Gatorade solution, and propolis storage media for maintaining the PDL cell viability. Materials and Methods: The present study was conducted on 40 recently extracted teeth which were randomly divided into four study storage groups: Group I: ViaSpan, Group II: Aloe vera, Group III: Gatorade solution, and Group IV: Propolis. Later they were subjected to centrifugation, and the cells from supernatant were colored with 0.4% trypan blue for determination of viability. The obtained data were statistically evaluated with SPSS package (21.0 version, Inc.; Chicago, IL, USA) using analysis of variance, Mann-Whitney test, and Post hoc tests. Results: The mean viable periodontal cell in Group I was 30.2 cumm, in Group II was 24.6 cumm, Group III was 14.5 cumm, and Group IV in 31.4. The difference was significant (P < 0.01). Post hoc test between different groups revealed a significant difference in mean viable periodontal cells (P < 0.001). Propolis, ViaSpan, and Aloe vera had higher pH and osmolality values. Conclusion: This study found that propolis had higher periodontal cell viability followed by ViaSpan solution and Aloe vera and least in Gatorade solution. Propolis, ViaSpan, and Aloe vera media can be used as a storage media.

Résumé Contexte: la Replantation est une méthode couramment utilisée pour la dent avulsée. Une membrane parodontale vitale (ligament parodontal [PDL]) est important pour la guérison réussie des dents replantées. Par conséquent, divers supports de stockage sont utilisés pour préserver la viabilité du parodontal les cellules avant la réimplantation. Objectifs: la présente étude a été menée pour évaluer L'efficacité de ViaSpan, Aloe vera, solution de Gatorade, et des supports de stockage de propolis pour maintenir la viabilité des cellules PDL. Matériaux et Méthodes: la présente étude a été menée sur 40 récemment dents extraites qui ont été divisées au hasard en quatre groupes de stockage d'étude: Groupe I: ViaSpan, Groupe II: aloe vera, Groupe III: Gatorade solution, et groupe IV: Propolis. Plus tard, ils ont été soumis à une centrifugation et les cellules du surnageant ont été colorées avec 0,4% de trypan bleu pour la détermination de la viabilité. Les données obtenues ont été évaluées statistiquement avec le package SPSS (version 21.0, Inc. Chicago, ILLINOIS, états-unis) en utilisant l'analyse de la variance, le test de Mann-Whitney et les tests post hoc. Résultats: la cellule parodontale viable moyenne dans le Groupe I était de 30,2 cumm, dans Le groupe II était de 24,6 cumm, le Groupe III de 14,5 cumm et le Groupe IV de 31,4. La différence était significative (P < 0,01). Essai post hoc entre différents groupes ont révélé une différence significative dans les cellules parodontales viables moyennes (P < 0,001). Propolis, ViaSpan et aloe vera avaient plus valeurs de pH et d'osmolalité. Conclusion: Cette étude a révélé que la propolis avait une viabilité cellulaire parodontale plus élevée suivie D'une solution de ViaSpan et Aloe vera et moins dans la solution de Gatorade. La Propolis, le ViaSpan et L'aloe vera peuvent être utilisés comme support de stockage. Mots-clés: aloe vera, Gatorade, cellules parodontales, propolis, ViaSpan.

Facing flood disaster: A cluster randomized trial assessing communities' knowledge, skills and preparedness utilizing a health model intervention.

Floods occur when a body of water overflows and submerges normally dry terrain. Tropical cyclones or tsunamis cause flooding. Health and safety are jeopardized during a flood. As a result, proactive flood mitigation measures are required. This study aimed to increase flood disaster preparedness among Selangor communities in Malaysia by implementing a Health Belief Model-Based Intervention (HEBI). Selangor's six districts were involved in a single-blinded cluster randomized controlled trial Community-wide implementation of a Health Belief Model-Based Intervention (HEBI). A self-administered questionnaire was used. The intervention group received a HEBI module, while the control group received a health talk on non-communicable disease. The baseline variables were compared. Immediate and six-month post-intervention impacts on outcome indicators were assessed. 284 responses with a 100% response rate. At the baseline, there were no significant differences in ethnicity, monthly household income, or past disaster experience between groups (p>0.05). There were significant differences between-group for intervention on knowledge, skills, preparedness (p<0.001), Perceived Benefit Score (p = 0.02), Perceived Barrier Score (p = 0.03), and Cues to Action (p = 0.04). GEE analysis showed receiving the HEBI module had effectively improved knowledge, skills, preparedness, Perceived Benefit Score, Perceived Barrier Score, and Cues to Action in the intervention group after controlling the covariate. Finally, community flood preparedness ensured that every crisis decision had the least impact on humans. The HEBI module improved community flood preparedness by increasing knowledge, skill, preparedness, perceived benefit, perceived barrier, and action cues. As a result, the community should be aware of this module. Clinical trial registration: The trial registry name is Thai Clinical Trials Registry, trial number TCTR20200202002.

Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic ß-cells: evidence for regulation by Rac1.

Reactive oxygen species (ROS) are important mediators of cellular signal transduction cascades such as proliferation, migration, and apoptosis. Chronic exposure of isolated ß-cells to proinflammatory cytokines elevates intracellular oxidative stress leading to the demise of pancreatic ß-cells culminating in the onset of diabetes. Although the mitochondrial electron transport chain is felt to be the primary source of ROS, several lines of recent evidence suggest that phagocyte-like NADPH oxidase plays a central role in cytokine-mediated ROS generation and apoptosis of ß-cells. However, the precise mechanisms underlying the regulation of NADPH oxidase remain unknown. To address this, insulin-secreting INS 832/13 cells were treated with cytomix (IL-1ß, IFN-γ, and TNF-α; 10 ng/ml each) for different time intervals (0-24 h). A significant, time-dependent increase in NADPH oxidase activation/intracellular ROS production, p47(phox) subunit, but not p67(phox) subunit, expression of the phagocyte-like NADPH oxidase were demonstrable under these conditions. Furthermore, siRNA-p47(phox) transfection or exposure of INS 832/13 cells to apocynin, a selective inhibitor of NADPH oxidase, markedly attenuated cytomix-induced ROS generation in these cells. Cytomix-mediated mitochondrial dysfunction in INS 832/13 cells was evident by a significant loss of mitochondrial membrane potential (MMP) and upregulated caspase 3 activity. Cytomix treatment also caused a transient (within 15 min) activation of Rac1, a component of the NADPH oxidase holoenzyme. Furthermore, GGTI-2147 and NSC23766, known Rac1 inhibitors, not only attenuated the cytomix-induced Rac1 activation but also significantly prevented loss of MMP (NSC23766 > GGTI-2147). However, NSC23766 had no effect on cytomix-induced NO generation or caspase 3 activation, suggesting additional regulatory mechanisms might underlie these signaling steps. Together, these findings suggested that Rac1-mediated regulation of phagocyte-like NADPH oxidase contributes to cytokine-mediated mitochondrial dysfunction in the ß-cell.

Phagocyte-like NADPH oxidase generates ROS in INS 832/13 cells and rat islets: role of protein prenylation.

Recent evidence suggests that an acute increase in the generation of phagocyte-like NADPH-oxidase (Nox)-mediated reactive oxygen species (ROS) may be necessary for glucose-stimulated insulin secretion. Using rat islets and INS 832/13 cells, we tested the hypothesis that activation of specific G proteins is necessary for nutrient-mediated intracellular generation of ROS. Stimulation of ß-cells with glucose or a mixture of mitochondrial fuels (mono-methylsuccinate plus α-ketoisocaproic acid) markedly elevated intracellular accumulation of ROS, which was attenuated by selective inhibitors of Nox (e.g., apocynin or diphenyleneiodonium chloride) or short interfering RNA-mediated knockdown of p47(phox), one of the subunits of Nox. Selective inhibitors of protein prenylation (FTI-277 or GGTI-2147) markedly inhibited nutrient-induced ROS generation, suggesting that activation of one (or more) prenylated small G proteins and/or γ-subunits of trimeric G proteins is involved in this signaling axis. Depletion of endogenous GTP levels with mycophenolic acid significantly reduced glucose-induced activation of Rac1 and ROS generation in these cells. Other immunosuppressants, like cyclosporine A or rapamycin, which do not deplete endogenous GTP levels, failed to affect glucose-induced ROS generation, suggesting that endogenous GTP is necessary for glucose-induced Nox activation and ROS generation. Treatment of INS 832/13 cells or rat islets with pertussis toxin (Ptx), which ADP ribosylates and inhibits inhibitory class of trimeric G proteins (i.e., G(i) or G(o)), significantly attenuated glucose-induced ROS generation in these cells, implicating activation of a Ptx-sensitive G protein in these signaling cascade. Together, our findings suggest a prenylated Ptx-sensitive signaling step couples Rac1 activation in the signaling steps necessary for glucose-mediated generation of ROS in the pancreatic ß-cells.

Global deletion of NTPDase3 protects against diet-induced obesity by increasing basal energy metabolism.

BACKGROUND: Ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), also known as CD39L3, is the dominant ectonucleotidase expressed by beta cells in the islet of Langerhans and on nerves. NTPDase3 catalyzes the conversion of extracellular ATP and ADP to AMP and modulates purinergic signaling. Previous studies have shown that NTPDase3 decreases insulin release from beta-cells in vitro. This study aims to determine the impact of NTPDase3 in diet-induced obesity (DIO) and metabolism in vivo. METHODS: We developed global NTPDase3 deficient (Entpd3-/-) and islet beta-cell-specific NTPDase-3 deficient mice (Entpd3flox/flox,InsCre) using Ins1-Cre targeted gene editing to compare metabolic phenotypes with wildtype (WT) mice on a high-fat diet (HFD). RESULTS: Entpd3-/- mice exhibited similar growth rates compared to WT on chow diet. When fed HFD, Entpd3-/- mice demonstrated significant resistance to DIO. Entpd3-/- mice consumed more calories daily and exhibited less fecal calorie loss. Although Entpd3-/- mice had no increases in locomotor activity, the mice exhibited a significant increase in basal metabolic rate when on the HFD. This beneficial phenotype was associated with improved glucose tolerance, but not higher insulin secretion. In fact, Entpd3flox/flox,InsCre mice demonstrated similar metabolic phenotypes and insulin secretion compared to matched controls, suggesting that the expression of NTPDase3 in beta-cells was not the primary protective factor. Instead, we observed a higher expression of uncoupling protein 1 (UCP-1) in brown adipose tissue and an augmented browning in inguinal white adipose tissue with upregulation of UCP-1 and related genes involved in thermogenesis in Entpd3-/- mice. CONCLUSIONS: Global NTPDase3 deletion in mice is associated with resistance to DIO and obesity-associated glucose intolerance. This outcome is not driven by the expression of NTPDase3 in pancreatic beta-cells, but rather likely mediated through metabolic changes in adipocytes.

Validity and Reliability of the Malay Versions of Bloating Severity (BSQ-M) and Quality of Life (BLQoL-M) Questionnaires.

Abdominal bloating (AB) is a prevalent and bothersome symptom, but there are no specific measures for severity and quality of life (QoL) other than the Bloating Severity Questionnaire (BSQ) and Bloating Quality of Life (BLQoL). We aimed to translate the BSQ and BLQoL into the Malay language and to validate them using exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) approaches. The 12-item BSQ has two components, seven-item severity in general (SevGen) and five-item severity in the past 24 h (Sev24), and BLQOL has five items. Translation to the Malay language (BSQ-M and BLQoL-M) was performed using standard forward and backward processes. EFA followed by CFA were performed in participants with AB due to functional bowel disorders, with the purpose of examining the validity and reliability of the questionnaires translated into Malay. After EFA with 152 participants, all the items of BSQ-M remained in the model. Total variance extracted was 53.26% for BSQ-M and 58.79% for BLQoL-M. The internal consistency based on Cronbach's alpha values was 0.52 for SevGen, 0.86 for Sev24, and 0.81 for BLQoL-M. After performing CFA with another 323 participants, the final measurement model for BSQ-M and BLQoL-M fit the data well in terms of several fit indices (BSQ-M: root mean square error of approximation (RMSEA) = 0.050, Comparative Fit Index (CFI) = 0.966, Tucker-Lewis Fit Index (TLI) = 0.956, and standardized root mean squared residual (SRMR) = 0.051; BLQoL-M: RMSEA = 0.071, CFI = 0.985, TLI = 0.962, SRMR = 0.021). The composite reliability for BSQ-M and BLQoL-M were satisfactory (SevGen = 0.83, Sev24 = 0.89, BLQoL = 0.80). The intraclass correlation (ICC) results showed excellent stability for BSQ-M and BLQoL-M, ranging from 0.74 to 0.93. The Malay language versions of BSQ-M and BLQoL-M are valid and reliable instruments for measuring the severity and QoL of AB for the Asian population with functional bowel disorders.

Regulation of glucose- and mitochondrial fuel-induced insulin secretion by a cytosolic protein histidine phosphatase in pancreatic beta-cells.

We report localization of a cytosolic protein histidine phosphatase (PHP; approximately 16 kDa) in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knockdown of PHP markedly reduced glucose- or mitochondrial fuel-induced but not KCl-induced insulin secretion. siRNA-mediated knockdown of PHP also attenuated mastoparan-induced insulin secretion, suggesting its participation in G protein-sensitive signaling steps, leading to insulin secretion. Functional assays revealed that the beta-cell PHP catalyzes the dephosphorylation of ATP-citrate lyase (ACL). Silencing of PHP expression markedly reduced ACL activity, suggesting functional regulation of ACL by PHP in beta-cells. Coimmunoprecipitation studies revealed modest effects of glucose on the interaction between PHP and ACL. Confocal microscopic evidence indicated that glucose promotes association between ACL and nm23-H1, a known kinase histidine kinase, but not between PHP and ACL. Furthermore, metabolic viability of INS 832/13 cells was resistant to siRNA-PHP, suggesting no regulatory roles of PHP in cell viability. Finally, long-term exposure (24 h) of INS 832/13 cells or rat islets to high glucose (30 mM) increased the expression of PHP. Such increases in PHP expression were also seen in islets derived from the Zucker diabetic fatty rat compared with islets from the lean control animals. Together, these data implicate regulatory roles for PHP in a G protein-sensitive step involved in nutrient-induced insulin secretion. In light of the current debate on putative regulatory roles of ACL in insulin secretion, additional studies are needed to precisely identify the phosphoprotein substrate(s) for PHP in the cascade of events leading to nutrient-induced insulin secretion.