Xanthine oxidase modulates Glutamic Acid Decarboxylase 65 isoform (GAD65) protein expression in rat pancreatic islets

Xanthine oxidase modulates Glutamic Acid Decarboxylase 65 isoform (GAD65) protein expression in rat pancreatic islets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Xanthine oxidase modulates Glutamic Acid Decarboxylase 65 isoform (GAD65) protein expression in rat pancreatic islets KONIKA RAZDAN, VARSHIESH RAINA This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3829266/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: It is widely presumed that aberrant expression of GAD65 can sensitize beta cells to autoimmunity and moreover, several studies indicate Xanthine Oxidase (XO) plays significant role in Type-1 diabetes (T1D) pathology. The present study aimed to investigate whether XO modulates GAD65 protein expression in rat islets/pancreas in presence or absence of STZ. Methods and results: Female Wistar rats were used for pancreatic islet isolation and T1D STZ model by injecting single dose of STZ (40 mg/kg body weight). GAD65 and XO protein expression was studied by immunoblot in islets/pancreas and STZ/T1D cytokines (such as IL-1 beta, TNF-α and IFN-γ)/XO siRNA/Allopurinol (XO inhibitor)/1400W (iNOS inhibitor) were used in treatment groups. Enzymatic assay was done to measure XO activity in islets/serum/pancreas and iNOS activity in culture supernatant. STZ led to a significant increase in GAD65 expression and XO activity in islets which was reduced in presence of Allopurinol and XO siRNA. In pancreas of T1D rat model, expression of GAD65 decreased and XO increased; XO activity decreased; whereas in sera XO activity increased. Treatment of islets with T1D cytokines revealed that cytokine cocktail declined GAD65 protein levels and XO activity and intervention with iNOS inhibitor reversed the effect. During ontogeny of pancreas GAD65 expression was more in the fetal/infantile pancreas compared to adult pancreas and Allopurinol challenged pregnant rats confirmed that GAD65 expression is dependent on XO activity. Conclusion: This study for the first time clearly demonstrates that XO is positive regulator of GAD65 expression in rat islets and STZ-T1D model. GAD65 xanthine oxidase p53 Allopurinol Type-1 diabetes Figures Figure 1 Figure 2 Figure 3 Figure 4 INTODUCTION Type-1 Diabetes (T1D) is a multifactorial endocrine pancreatic auto-immune disorder which is clinically diagnosed by the presence of circulating auto-antibodies against some antigens, of which prominent one includes glutamic acid decarboxylase isoform 65 (GAD65) [ 1 , 2 ]. As per estimates, nearly 70–80% of newly diagnosed T1D patients are diagnosed with GAD65 autoantibodies [ 3 ]. GAD65 is an apo-enzyme that catalyzes the formation of γ-amino butyric acid (GABA), predominantly in GABA-ergic nerve cells and pancreatic islets [ 4 ]. The exact physiological function of GAD65 in pancreas is still poorly understood but few studies show that GABA - promotes beta-cell growth and survival; suppresses glucagon secretion from alpha cells; and suppresses inflammation and increases regulatory T-cell numbers [ 5 ]. Among mammalian species, GAD65 is predominantly expressed in human and rat pancreas [ 6 ]. The recognition of GAD65 as auto-antigen in T1D instigated in 1982 from immunoprecipitation studies which showed a 64 Kd protein was precipitated by diabetic sera in Type-1 diabetic patients [ 7 ]. Since then, several studies in spontaneous animal models of autoimmune diabetes have strongly supported that GAD65 is highly immunogenic and potential biomarker for T1D [ 8 ]. Despite, these evidences, it is still unclear, why GAD65 auto-antibodies are generated? Notably, GAD65 specific auto-antibodies are present in the serum before the destruction of beta-cells of pancreas. Moreover, a study showed that aberrant accumulation of GAD65 in pancreas during endoplasmic stress can sensitize beta cells for auto-immune assault [ 9 ]. Such abnormal accumulation of proteins is commonly encountered in neurological disorders, and among several factors, over-expression of proteins has been found to be a major reason. Interestingly, pancreatic beta cells and neurons share some similarities like both are highly prone to physiological stress due to scarcity of free radical scavenging enzymes. So, it is quite likely that similar mechanism might operate in beta cells that leads to aberrant expression of GAD65. So far, studies have demonstrated that GAD65 expression gets altered under high glucose, blocking mitochondrial electron transport system, hypoxia, glutamate, Streptozotocin (STZ) and transcriptional factors (like sp-1, NF-kB and SMAR1 to be putative positive regulatory while as cytokines like IL-1 beta act as a negative regulator) [10, 11, 12, 13] . Thus, finding molecules that influence cell stress can be an important candidate molecule that can regulate GAD65 expression. Xanthine oxidase (XO), is prototype of the molybdenum hydroxylase family of enzymes that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid. Under normal conditions XO exists majorly in an inactive form namely xanthine dehydrogenase (XDH), thus given a name of XDH / XO system. XO has broad range of activity for endogenous / exogenous substrate’s, thus aids in drug-metabolism and detoxification [14] . It generates free radicals (.OH, O 2− and H 2 O 2 ) which are involved in variety of pathophysiological conditions such as atherosclerosis, endothelial dysfunction, hemolytic diseases, viral infections, hepatotoxicity, acute pancreatitis, respiratory distress syndrome, ischemia-reperfusion injury, inflammatory and autoimmune diseases [15, 16, 17] . Moreover, XO derived free radicals can affect various transcriptional regulators like AP-1, NFkB and p53 which are involved in dysfunction of target cells or tissues [18, 19] . In both Type-1 and Type-2 diabetes, there are several reports which show XO causes oxidative damage as well as its associated complications, and the role of XO was also proposed in experimental model of diabetes [20, 21] . Despite its strong association with the pathology of Type-1 or Type-2 diabetes; however, no study till date has investigated the role of XO in regulating GAD65 expression. Streptozotocin (STZ) is a natural compound derived from Streptomyces achromogenes that is selectively toxic to the cells expressing Glut-2 receptor [22] . Because beta cells express high level of Glut-2 receptor, STZ has been widely used to study beta cell function in in-vitro and animal models of Type-1 and Type-2 diabetes. STZ induced beta cell death occurs by many mechanisms such as DNA alkylation, protein glycosylation, over-stimulation of poly (ADP-ribose) polymerase (PARP) and depletion of energy stores (like NAD + and ATP) [23] . Here we report for the first time, a dramatic effect of XO on GAD65 protein expression in isolated rat pancreatic islets / STZ induced experimental Type-1 diabetic rat model, and further demonstrate the association of XO activity with GAD65 protein expression during ontogeny of pancreas. MATERIAL AND METHODS Islet isolation from fetal, neonatal and adult pancreas The female Sprague Dawley rats (150 to 200 g, 8 to 12 weeks old) were obtained from experimental animal facility of National Centre for Cell Science, Pune, India. During the period of experiments, the animals were housed at 21 0 C with 12 hours’ light and 12 hours’ darkness cycle. Food and water were available to animal’s ad libitum. Fetal pancreata (from pregnant Sprague Dawley rats at E21.5 of gestation) neonatal and adult pancreases were surgically dissected under sterile conditions in compliance with bioethics legislation in India. Islets were isolated according to the procedure described previously [24] . Briefly, under halothane anesthesia, the abdomen was cut open and the pancreas was distended by infusion of 10 ml sterile Krebs-Ringer-Hepes solution (Sigma Aldrich, USA) containing 10% bovine serum albumin (BSA fraction V, Sigma Aldrich) through cannulated common bile duct. Afterwards, the pancreas was excised, chopped and digested using a two-stage incubation of 20 min at 37 o C with successively 1.0 and 0.7 mg/ml collagenase P (Sigma Aldrich, USA). Islets were separated from exocrine tissue by centrifugation over a discontinuous dextran (Sigma Aldrich) gradient and further purified by handpicking. Approximately, 100 Islets per 25 cm 2 were cultured in Petri dishes in CMRL 1066 (Sigma Aldrich), containing 10% fetal calf serum (Sigma Aldrich), 8.3 mmol/l glucose (Sigma Aldrich), 10 mmol/l HEPES (Sigma Aldrich, USA) and 1% penicillin/streptomycin (Sigma Aldrich, USA), at 37 o C in humidified air containing 5% CO2. STZ and Cytokine treatment to islets 500 mM Stock solution of Streptozotocin (Sigma Aldrich) was reconstituted in 0.01 M citrate buffer (pH 4.5) just before treatment. 100 ml of 0.1 M sodium citrate buffer was prepared by mixing 55.5ml of 0.1M trisodium citrate (Sigma Aldrich, USA) and 44.5ml of 0.1M citric acid monohydrate (Sigma Aldrich). pH was adjusted with 1 N NaOH and measured by pH meter (Thermo Fischer Scientific, USA). The buffer was filter sterilized prior to islet treatment at working concentrations of 0.5 mM, 1.0 mM and 2.0 mM for a period of 24 hrs. Intact islets were treated with a mixture of TNF-α (6000 UI ml − 1 ), IL1-β (600 UI ml − 1 ) and IFN-γ (6000 UI ml − 1 ) (Merck Millipore, Burlington, MA, USA) and STZ for 48 hrs. to assess the effect [25] . Induction of Type-1 Diabetes and measurement of blood glucose : Type-1 diabetes in female Sprague Dawley rats (150 to 200 g, 8 to 12 weeks old) was induced by injecting intra-peritoneally single dose of STZ at concentration of 40 mg/kg body weight as described previously [26] . Briefly, 10 rats were used per group and housed in cages for 5 days at 21 0 C with 12 hours’ light and 12 hours’ darkness cycle. Before the start of experiment, all rats were fasted for 6 to 8 hr prior to STZ treatment. Glucose level in serum were measured as per the instructions mentioned in “Glucose Calorimetric Detection Kit” (EIAGLUC; Thermo Fisher Scientific, USA). siRNA transfection Around 500 islets were seeded in culture dishes containing CMRL 1066 (Sigma Aldrich) containing 10% fetal calf serum (Sigma Aldrich), 8.3 mmol/l glucose (Sigma Aldrich), 10 mmol/l HEPES (Thermo Fisher Scientific, USA). A final transfection volume of 2.5 mL per dish contained 50 nmol/L scrambled (sc-37007; Santa Cruz Biotechnology, Santa Cruz, CA and XO siRNA (sc-270202; Santa Cruz Biotechnology, Santa Cruz, CA) in Opti-MEM (31985062; Thermo Fischer Scientific, USA) reduced serum media and 6.25 mL Lipofectamine RNAiMAX (Thermo Fischer Scientific, USA). A second transfection was performed 24 h after the first transfection and all functional experiments were done 72 h after the first transfection. Immunoblotting and antibodies : Protein isolation was done in radio-immunoprecipitation assay (RIPA) buffer (R0278; Sigma Aldrich). Prior to lysis, islets were washed in PBS and pelleted by centrifugation at 800 × g for 5 min; whereas dissected pancreata were directly put in lysis buffer, homogenized, sonicated for 15 s and centrifuged at 14000 x g for 10 minutes. About 50 µg of protein was loaded for immunoblotting and detected with primary antibodies against rat p53 (A-1; sc-393031; Santa Cruz Biotechnology, Santa Cruz, CA), Xanthine Oxidase (A-3; sc-398548; Santa Cruz Biotechnology, Santa Cruz, CA), ser15 p53 (#9284; Cell Signaling Technology), GAD65 (sc-377145; Santa Cruz Biotechnology, Santa Cruz, CA) and beta actin (C-4; sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA) at 1: 500. (Sigma-Aldrich). The rabbit (mouse (#7074; Cell Signaling Technology) and mouse (#7076; Cell Signaling Technology) HRP conjugated secondary antibodies were used at 1: 3000. Xanthine oxidase Assay : Islets and pancreata were lysed with potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and protease inhibitors. The homogenates were centrifuged at 12,000 g for 15 min at 4°C, and supernatants were collected. XO activity in islet / pancreata and serum was detected by the method as described previously [27] . Briefly, 60 µl of 0.1 M Tris containing 40 mg/l horseradish peroxidase (HRP) buffer (pH 7.5) was mixed with 20 µl 2 mM xanthine (see above) and 10 µl of ortho-phenylendiamine water solution. Reaction was initiated by adding 50 µl of sample. Control samples (blank) did not contain xanthine. Samples were incubated for 30 min at 40°C followed by termination of the reaction with 100 µl of 1.88 M H 2 SO 4 solution. H 2 O 2 standards were analysed to calculate XOD activity (concentrations – 0.1–100 nmol/ml). The absorbance was measured at 492 nm. XOD activity was expressed in nmol H 2 O 2 produced per 1 min per mg protein. Controls were normalised as 100%. XOD activities in other samples were expressed as % control. Nitrite determination. Aliquots of the culture medium were deproteinized by adding 35% sulfosalicylic acid. Samples were incubated for 15 min at 4 o C and then centrifuged for 15 min at 12,000 g. One part of 0.1% naphthylenediamine dihydrochloride was added to the supernatant, together with 1% sulfonilamide and 5% concentrated H3PO4. The reaction was carried out at 60 o C for 1 min, and the absorbance was measured at 546 nm in a spectrophotometer (DU-62; Beckman Instruments, Inc., Palo Alto, CA) against a standard curve. Statistical analysis All data are expressed as means ± SD derived from at least three independent experiments unless otherwise specified. Statistical significance of results was evaluated on raw values prior to conversion to percent of control, by a repeated measure one-way analysis of variance (ANOVA) test with Tukey’s post-hoc pairwise comparison, or t-test, using IBM SPSS Statistics (Version 21). P values 0.05 were considered non-significant. RESULTS Effect of STZ on protein expression of Glutamic acid decarboxylase isoform 65 (GAD65) and Xanthine oxidase (XO) in isolated rat islets Upon treatment of the isolated rat islets with different concentrations of STZ (i.e., 0.5 mM, 1.0 mM and 2.0 mM), for a time period of 24 hours, the immunoblot showed that in comparison to control (without STZ), 1.0 mM and 2.0 mM STZ concentration led to a significant increase in GAD65 protein expression, while as, XO protein expression displayed no change (Fig. 1 a ) . Quantitative analysis shown by error bar graph revealed that STZ led to a maximum ≃4.0-fold relative increase in GAD65 protein expression when compared to control and 1.0 mM STZ was the minimal effective concentration to display such change ( Fig. 1 b ). [MTT assay was also done to determine the effect of STZ on cell viability and it was observed that islet viability was – 99.0% at 0.5mm STZ; 98.0% at 1.0 mm STZ and 76.0% at 2.0 mm STZ (data not shown here)]. We further performed time kinetic for duration of 6hr, 12hr and 24 hrs., to study the protein expression of XO and GAD65 in presence of 1.0 mM STZ. In immunoblot, we found that at 1.0 mM STZ, only the protein expression of GAD65 displayed a significant increase which was evident from 12 hrs. onwards and, on the other hand, XO protein expression displayed no change at any time point (Fig. 1 c ) . Quantitative analysis shown by error bar graph revealed ≃4.0-fold increase in XO protein expression at 12 and 24 hrs. ( Fig. 1 d ) . XO activity and effect of XO inhibitor (Allopurinol) / XO siRNA on GAD65 protein levels in isolated rat islets upon STZ treatment. To investigate whether, XO enzymatic activity in rat islets is affected upon STZ treatment, we measured XO activity in isolated rat islets in presence of 1.0 mM STZ for time duration of 6 hrs., 12 hrs. and 24 hrs. We observed that 1.0 mM STZ led to a significant increase in XO activity which was 27.5% at 6 hrs., 137.75% at 12 hrs., and 303.0% at 12 hrs. when compared to control ( Table 1 ; Panel 1) . Moreover, the data also depicts a robust increase in XO activity from 12 hrs. onwards. Alongside, when the islets were co-incubated with 500 µM Allopurinol (well-known XO inhibitor) in presence or absence of STZ for 24 hrs., the XO activity was significantly reduced i.e., allopurinol vs control (96%) and STZ + Allopurinol vs control (97.34%) ( Table 1 ; Panel 2) . In this context, we investigated whether the STZ induced increase in XO activity modulates GAD65 protein expression in these rat islets by employing two approaches: one, which involved downregulating XO protein expression by transient transfection of islets with XO siRNA and second, which involved inhibiting XO enzymatic activity by co-incubation of islets with Allopurinol. As shown in immunoblot ( Fig. 2 a ) , XO siRNA transfection in islets significantly decreased XO protein expression, and when these XO siRNA transfected islets were treated with STZ, no significant increase in GAD65 protein expression was observed compared to STZ treated islets. The relative fold change in protein expression is shown in error bar graph which shows ≃2.66-fold decrease in GAD65 protein expression in XO siRNA + STZ vs STZ and ≃1.26-fold increase in GAD65 protein expression in XO siRNA + STZ / scrambled XO siRNA / XO siRNA vs control ( Fig. 2 b ) . In case of Allopurinol (400 µM) treated islets, XO protein expression showed no significant change whereas GAD65 protein expression showed a significant increase in Allopurinol / Allopurinol + STZ vs control and significant decrease in Allopurinol + STZ group vs STZ group ( Fig. 2 c ) . The relative fold change in protein expression is shown in error bar graph which shows that Allopurinol led to ≃ 2.2-fold increase in GAD65 protein expression in Allopurinol / Allopurinol + STZ vs control and ≃ 2.66-fold decrease in GAD65 protein expression in STZ + XO siRNA group vs STZ group ( Fig. 2 d ) . GAD65 protein expression in STZ induced Type-1 diabetic rat model and rat islets treated with diabetogenic cytokines such as IL-1 β, TNF-α and IFN-γ Type-1 diabetes is in female Wistar rats was induced by injecting intra-peritoneally single dose of STZ at concentration of 40 mg/kg body weight (as in material and methods) and diabetes was monitored by measuring rise in blood glucose levels at day 0, day 1, day, day 6, day 12 and day 18. As shown in Table 2 , STZ induced a significant increase in blood glucose levels which was ≃ 2.0-fold at day 1 vs control; ≃ 2.6-fold at day 6 vs control; ≃3.0-fold at day 12 vs control and ≃ 3.65-fold at day 18 vs control. Immunoblot for XO and GAD65 in dissected pancreata of these wistar female rats was performed at same days and it was observed that GAD65 protein expression significantly decreased from day 1, whereas, XO protein expression significantly increased from day 6 ( Fig. 3 a ) . The relative fold change in protein expression is shown in error bar graph which shows a significant decrease of protein expression in GAD65 by ≃ 2.0-fold at day 1 vs control; ≃ 2.0-fold at day 6 vs control; ≃4.0-fold at day 12 vs control; ≃ 10.0-fold at day 18 vs control, and significant increase of protein expression in XO by ≃ 1.15-fold at day 1 vs control; ≃ 1.2-fold at day 6 vs control; ≃1.5-fold at day 12 vs control; ≃ 1.5-fold at day 18 vs control ( Fig. 3 b ) . We next measured the XO activity in the pancreata and sera of these STZ induced Type-1 diabetic rats which is represented in table as % decrease/increase in XO activity. It was observed that there occurred a significant decrease in pancreatic XO activity from day 1 onwards in comparison to control ( Table 3 ) . However, in serum of these animals, XO activity was significantly increased from day 1 onwards in comparison to control ( Table 4 ) . During the pathophysiology of Type-1 diabetes, cytokines such as IL-1β, TNF-α and IFN-γ are known to play a major role. We thus treated isolated rat islets with these cytokines to investigate their effect on the protein expression of XO and GAD65. In immunoblot, we found that neither of the cytokines either alone or in cocktail changed the protein expression of XO; however, GAD65 protein expression was significantly reduced in presence of IL-1 β beta and cocktail of these cytokines (IL-1β + TNF-α + IFN-γ) ( Fig. 3 c ) . The relative fold change in protein expression is shown in error bar graph which shows that GAD65 levels decreased to ≃ 1.3-fold in presence of IL-1 β and ≃ 2.0-fold in presence of cocktail of these cytokines ( Fig. 3 d ) . XO activity was also measured as represented by % decrease in XO activity and we observed a significant decline in XO activity in islets treated with IL-1 beta (about 28%) and cocktail of cytokines (about 75%) in comparison to control ( Table 5 ). Interestingly, we came across a study wherein nitric oxide has been shown to inhibit the XO activity. We thus performed an immunoblot for inducible nitric oxide synthase enzyme (iNOS) and also measured nitrite levels in the islets treated with these cytokines. As shown in Fig. 3 c, none of the cytokines alone was capable to alter iNOS protein expression; however, in presence of the cocktail of these cytokines there occurred robust expression of iNOS protein. On the other hand, measurement of nitrite levels in the supernatants of islets revealed significant increase in presence of IL-1 β (about 32%); IFN-γ (about 7.7%), TNF-α (about 52%) and TNF-α + IFN-γ + IL-1 β (about 392.3%), in comparison to control ( Table 6 ) . On comparison of data from Fig. 3 c and Table 6 , we also observed that cocktail of cytokines is more effective to induce the changes in GAD65 protein expression or XO activity. So, we treated islets with 1400W (a nitric oxide synthase inhibitor) either alone or along-with cytokine cocktail and in the immunoblot, we observed comparable protein expression of GAD65 among control vs 1400W vs cocktail of cytokines + 1400W ( Fig. 3 f ) . Relation of GAD65 and XO in fetal, neonatal and adult rat pancreas We also studied the association between XO and GAD65 during the course of pancreatic ontogeny from fetal stage – neonatal stage (at 6 days, 12 days and 24 days) - adult stage. Immunoblot revealed no significant change in protein expression of XO from fetal to adult stage; however, protein expression of GAD65 started to show significant decline from neonatal (24 days) onwards and the decline in protein expression was more evident in adult pancreas ( Fig. 4 a ) . The relative fold change in protein expression is shown in error bar graph which shows decrease in GAD65 expression of about ≃1.15-fold at neonatal day 24 and ≃2.5-fold in adult pancreas vs fetal / neonatal (6 days) / neonatal (12 days) ( Fig. 4 b ) . Alongside, XO activity in fetal, neonatal and adult pancreas was measured and we observed a significant decline in XO activity from fetal to adult stage of pancreas ( Table 8 ) . To further substantiate our findings, we performed our study on fetal pancreas for reasons mainly due to - high protein expression of both the molecules (GAD65 & XO) and high XO activity; an ideal dynamic environment which involves continuous cellular remodeling process and an ideal therapeutic strategy to mitigate T1D before birth. Female pregnant rats were injected intraperitoneally with Allopurinol at concentration of 30 mg kg − 1 at fetal 20.0 days and fetal pancreas were dissected to study protein expression of GAD65. Immunoblot revealed that GAD65 protein expression was drastically reduced in fetal pancreas of Allopurinol injected pregnant rat. The relative fold change in protein expression is shown in error bar graph which shows ≃2.6-fold decrease in protein expression of GAD65 with Allopurinol in comparison to control. DISCUSSION Various factors can contribute towards aberrant accumulation of proteins and among them cellular defects in transcriptional regulation are well described in neurological diseases [28] . Xanthine oxidase (XO) is well known molecule which have been extensively studied for their pathological role in various diseases, including diabetes [14, 16] . In this study, we underpin for the first time the role of XO in regulating GAD65 expression in pancreatic islets and T1D rat model. We choose rat islets as they predominantly express GAD65 and moreover, islets represent a suitable in-vitro experimental model that closely mimics in-vivo system [ 6 ]. STZ is a well-known diabetogenic agent which specifically causes beta cell dysfunction by various mechanisms; and has been previously demonstrated in our study to upregulate GAD65 in mouse pancreatic islets [23, 12] . In this study also, we report a significant increase in GAD65 protein expression upon STZ treatment in rat pancreatic islets which further substantiates the fact that indeed STZ is a potent regulator of GAD65 in pancreatic islets. While as, STZ had no effect on XO protein levels; however, XO activity was significantly enhanced in rat pancreatic islets. This was not surprising as previous studies have also demonstrated that XO induced cellular stress is not strictly dependent on XO levels but on XO activity in various cells [29, 30] . Elevated XO activity has also been observed with increased risk of developing Type-1 and Type-2 diabetes, diabetic vascular complications and muscular oxidative stress in STZ induced experimental diabetes [31, 32, 33, 34] . Remarkably, when the endogenous XO protein expression was silenced by XO siRNA or its activity was inhibited by Allopurinol (a well-known inhibitor of XO), STZ induced GAD65 protein expression was substantially abolished, reinforcing the fact XO activity is required for GAD65 protein expression. The role of Allopurinol is well documented to reduce severity of proteinuria, ameliorate oxidative stress in Type-1 diabetic patients, improve endothelial dysfunction in Type-2 diabetic patients and so on [35, 36]. Thus, our results clearly indicate that XO activity can play a crucial role in pathogenesis of TID by modulating GAD65 expression. Having established the fact that STZ induced upregulation of XO activity is responsible for GAD65 overexpression, we expanded our study to STZ induced experimental animal model of T1D (which is induced by single dose of STZ in rats) [26]. Diabetes was confirmed by rise in glucose levels and assessment of GAD65 and XO protein levels in pancreas at different days’ post STZ injection revealed a significant decrease in GAD65 and significant increase in XO protein levels. Alongside, it was observed that XO activity was significantly increased in serum and significantly decreased in pancreas as the diabetes progressed. To the best of our knowledge, this is the first study which demonstrates differential activity of XO in STZ induced experimental T1D. However, our study is in concordance with the earlier studies which demonstrate high serum XO activity both human and animal models of Type-2 diabetes [37, 38]. Notably, few studies have also found negative correlation or no correlation of XO activity with diabetes duration [39, 40] . Our study also demonstrates negative regulation of XO activity in pancreas of rat model of Type-1 diabetes, but we do believe that further investigation is needed to gain more insight in other animal models and Type-1 diabetic patients. In addition, we also suggest that the difference of XO activity among Type-1 and Type-2 diabetic groups can possibly happen because the mode of pathology is different in both diseases. T1D is an immune mediated disease and the proinflammatory cytokines such as IL-1 β, TNF-α and IFN-γ have been found to be major players in beta cell destruction [ 1 ]. In order to establish the fact that indeed change in the expression of these molecules is caused by auto-immune assault, we treated the rat islets with IL-1 β, TNF-α and IFN-γ cytokines. We observed that only IL-1 beta led to a significant decrease in GAD65 levels and the effect was more prominent in presence of cocktail of these cytokines. However, XO protein levels were not altered in any of these treatments, implying that there may be other factors that control the expression of XO in STZ induced Type-1 diabetes. Pertinent to mention here that similar kind of observation was published in which authors observed that IL-1 β dramatically reduces GAD65 expression whereas TNF-α and IFN-γ had no effect on GAD65 expression [41] . Besides, other studies also showed that cytokines have negative effect on islet cell antigens like IA-2, insulin and Zinc transporter-8 (ZnT8) [42, 43] . Thus, our finding further supports the earlier findings which demonstrate that proinflammatory cytokines have inhibitory effect on GAD65 expression. We next investigated whether the above-mentioned cytokines modulate the XO activity in islets. We observed a dramatic decline in XO activity and major effect was observed when islets were treated with cocktail of cytokines. This decline in XO activity coincided with XO activity as observed in pancreas of T1D rats. Interestingly, we came across studies which show negative effect of nitric oxide synthase on XO activity [44, 45] . Moreover, a study showed that treatment of rat Insulinoma INS-1 cells with either of these cytokines led significant downregulation of IA-2 and insulin mRNA in nitric oxide dependent manner [46] . We also got interested to find out whether nitric oxide plays any role in modulating XO activity and thus GAD65 expression. We also observed a robust increase in nitrite levels of islet culture supernatant treated with cocktail of cytokines. We further show that cytokine mixture led to a significant over-expression of inducible nitric oxide synthase (iNOS) and co-incubation with 1400W (a potent inhibitor of INOS) not only restored the GAD65 protein levels but enhanced XO activity. Thus, our study provides additional evidence that nitric oxide is negative regulator of XO activity and opens new window of opportunity for its implication in T1D. To further corroborate our findings, we examined the expression of these molecules during ontogeny of pancreas from fetal -neonatal-adult stage. During the ontogeny while as XO protein levels remained unaltered but GAD65 protein levels showed a significant decline after 24th week of gestation. In agreement with previous studies, our study also demonstrates that GAD65 is expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas [47] . Parallely, we measured XO activity in pancreas which was found to decrease from fetal to adult stage. Remarkably, when pregnant females were challenged with Allopurinol a significant decline in GAD65 protein expression occurred which clearly demonstrates that XO activity is indeed one of the major factors that regulates GAD65 expression during pancreatic development. This study also indicates that aberrant activity of XO might be one of the predisposing factors for aberrant accumulation of GAD65 which is implicated in beta cell dysfunction and loss during pathogenesis of T1D [48] . Furthermore, the idea behind application of Allopurinol in pregnant rats in our study was to find whether GAD65 protein expression can be reduced during fetal pancreas development which might open up new window of opportunity to mitigate Type-1 diabetes. In conclusion, our study for the first time demonstrates substantial role of XO in regulating expression of GAD65 in pancreatic islets and further demonstrate their positive association during ontogeny of pancreas. Declarations Ethical Approval for animal study: No ethical approval was required for this study as per the law applicable in our country. However, the study was carried out in in strict accordance with the Ethics Committee for Animal studies of the National Centre for Cell Science and international guidelines. Human ethics: Not applicable Consent for publication: Not applicable Availability of supporting data: Not applicable Competing Interests: Authors declare no competing interests. Funding source: No funding source. The work was supported by the NCCS, GMC Jammu and EIPL. Author’s Contribution: Dr Varshiesh Raina designed, performed experiments, wrote and modified the paper. Dr Konika Razdan analyzed data, prepared figures and performed experiments. Both authors reviewed the manuscript, and the manuscript is approved by both authors for publication. Acknowledgment: The work was supported by NCCS Pune, EIPL and Department of Microbiology, GMC, Jammu. We would like to thank Dr G.C.Mishra (Ex-director NCCS) and Late Dr P.B.Parab (Scientist G) for all the support and facility for conducting animal experiments. References DiMeglio LA, Evans-Molina C, Oram RA (2018) Type 1 diabetes. Lancet 391:2449-2462. https://doi.org/10.1016/S0140-6736(18)31320-5 Basu M, Pandit K, Banerjee M, et al (2020) Profile of Auto-antibodies (Disease Related and Other) in Children with Type 1 Diabetes. Indian J Endocrinol Metab 24:256-259. https://doi.org/10.4103/ijem.IJEM_63_20 Fenalti G, Buckle AM (2010) Structural biology of the GAD autoantigen. Autoimmun Rev 9:148-152. https://doi.org/10.1016/j.autrev.2009.05.003 Hagan DW, Ferreira SM, Santos GJ, et al (2022) The role of GABA in islet function. Front Endocrinol (Lausanne) 13:972115-972133. https://doi.org/10.3389/fendo.2022.972115 Al-Kuraishy HM, Hussian NR, Al-Naimi MS, et al (2021) The Potential Role of Pancreatic γ-Aminobutyric Acid (GABA) in Diabetes Mellitus. Int J Prev Med24:12-19. https://doi.org/ 10.4103/ijpvm.IJPVM_278_19 Kim J, Richter W, Aanstoot HJ, et al (1993) Differentialexpression of GAD65 and GAD67 in human, rat, and mouse pancreatic islets. Diabetes 42:1799-1808. https://doi.org/10.2337/diab.42.12.1799 Baekkeskov S, Nielsen JH, Marner B, et al (1982) Autoantibodies in newly diagnosed diabetic children immunoprecipitate specific human islet cell proteins. Nature 298:167-169. https://doi.org/10.1038/298167a0 Capitani G, Biase DD, Gut H, et al (2005) Structural model of human GAD65: prediction and interpretation of biochemical and immunogenic features. Proteins 59:7-14. https://doi.org/10.1002/prot.20372 Kim YK, Sussel L, Davidson HW (2021) Inherent Beta Cell Dysfunction Contributes to Autoimmune Susceptibility. Biomolecules 11:512-522. https://doi.org/3390/biom11040512 Bjork E, Kampe O, Karlsson FA (1992) Glucose regulation of the autoantigen GAD65 in human pancreatic islets. J Clin Endocrinol Metab 75:1574-1576.https://doi.org/10.1210/jcem.75.6.1464667 Liu H, Li S, Zhang Y (2009) Dynamic regulation of glutamic acid decarboxylase 65 gene expression in rat testis. Acta Biochim Biophys Sin (Shanghai) 41:545-553. https://doi.org/10.1093/abbs/gmp043 Singh S, Raina V, Chaval, PL, et al (2012) Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment. BMC Mol Biol 14:13-28. https://doi.org/10.1186/1471-2199-13-28 Kajita Y, Mushiake H (2021) Heterogeneous GAD65 Expression in Subtypes of GABAergic Neurons Across Layers of the Cerebral Cortex and Hippocampus. Front Behav Neurosci 15:750869-750883. https://doi.org/10.3389/fnbeh.2021.750869 Battelli MG, Polito L, Bortolotti M, et al (2016) Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a Detoxifying Enzyme. Curr Med Chem 23:4027-4036. https://doi.org/10.2174/0929867323666160725091915 Polito L,Bortolotti M, Battelli MG, et al (2021) Xanthine oxidoreductase: A leading actor in cardiovascular disease drama. Redox Biol 48:102195-102205. https://doi.org/10.1016/j.redox.2021.102195 Cheh C, Lu JM, et al (2016) Hyperuricemia-Related Diseases and Xanthine Oxidoreductase (XOR) Inhibitors: An Overview. Med Sci Monit 22:2501-2512. https://doi.org/10.12659/msm.899852 Ichida K, Amaya Y, Okamoto K, et al (2012) Mutations Associated with Functional Disorder of Xanthine Oxidoreductase and Hereditary Xanthinuria in Humans. Int J Mol Sci 13:15475-15495. https://doi.org/10.3390/ijms131115475 Morgan MJ, Liu ZG (2011) Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res 21:103-115.https://doi.org/10.1038/cr.2010.178 Battelli MG, Bortolotti M, Bolognesi A, et al (2020) Pro-Aging Effects of Xanthine Oxidoreductase Products. Antioxidants (Basel) 9:839-855. https://doi.org/10.3390/antiox9090839 Sunagawa S, Shirakura T, Hokama N, et al (2019) Activityof xanthine oxidase in plasma correlates with indices of insulin resistance and liver dysfunction in patients with type 2 diabetes mellitus and metabolic syndrome: A pilot exploratory study. J Diabetes Investig 10:94-103. https://doi.org/10.1111/jdi.12870 Slobodnick A., Toprover M, Greenberg J, Crittenden DB, et al (2020) Allopurinol use and type2 diabetes incidence among patients with gout: A VA retrospective cohort study. Medicine (Baltimore) 99:e21675-e21682. https://doi.org/10.1097/MD.0000000000021675 Al-Nahdi AMT, John A, Raza H (2017) Elucidation of Molecular Mechanisms of Streptozotocin-Induced Oxidative Stress, Apoptosis, and Mitochondrial Dysfunction in Rin-5F Pancreatic β -Cells. Oxid Med Cell Longev 2017:7054272-7054287. https://doi.org/10.1155/2017/7054272 Szkudelski T. The Mechanism of Alloxan and Streptozotocin Action in B Cells of the Rat Pancreas. Physiol Res, 2001,50(6):537-546. Van-Suylichem PT, Wolters GH, van Schilfgaarde R (1990) The efficacy of density gradients for islet purification: a comparison of seven density gradients, Transpl Int 3:156-161. https://doi.org/10.1007/BF00355463 Laporte C, Tubbs E, Cristante J, et al (2019) Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin. Stem Cell Res Ther 10:85-97. https://doi.org/10.1186/s13287-019-1190-4 Ghasemi A,Jeddi S (2023) Streptozotocin as a tool for induction of rat models of diabetes: a practical guide. EXCLI J 22:274-294. https://doi.org/10.17179/excli2022-5720 Nicholas SA, Bubnov VV, Yasinska IM, et al (2011) Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1beta.Cell Mol Life Sci 68:151-158. https://doi.org/10.1007/s00018-010-0450-3 Chung GC, Hyosang L, Sung BL (2018) Mechanisms of protein toxicity in neurodegenerative diseases. Cell Mol Life Sci 75:3159-3180. https://doi.org/10.1007/s00018-018-2854-4 Bortolotti M, Polito L, Battelli MG, et al (2021) Xanthine oxidoreductase: One enzyme for multiple physiological tasks. Redox Biol 41:101882-101888. https://doi.org/10.1016/j.redox.2021.101882 Battelli MG,Polito L, Bortolotti M, et al (2016) Xanthine oxidoreductase in cancer: more than a differentiation marker. Cancer Med 5:546-557. https://doi.org/10.1002/cam4.601 Bravard A, Bonnard C, Durand A, et al (2011) Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice. Am J Physiol Endocrinol Metab 300: E581-E91.https://doi.org/10.1152/ajpendo.00455.2010 Kim SM, Choi YW, Seok HY, et al (2012) Reducing serum uric acid attenuates TGF-beta1-induced profibrogenic progression in type 2 diabetic nephropathy. Nephron Exp Nephrol 121:e109-121.https://doi.org/10.1159/000343567 Hernandez-Hernandez ME, Torres-Rasgado E, Pulido-Perez P, et al (2022) Disordered Glucose Levels Are Associated with Xanthine Oxidase Activity in Overweight Type 2 Diabetic Women. Int J Mol Sci 23:11177-11188. https://doi.org/10.3390/ijms231911177 Hasan M, Fariha KA, Barman Z, et al (2022) Assessment of the relationship between serum xanthine oxidase levels and type 2 diabetes: a cross-sectional study. Sci Rep 12:20816-20825. https://doi.org/10.1038/s41598-022-25413-w Slobodnick A, Toprover M, Greenberg J, et al (2020) Allopurinol use and type 2 diabetes incidence among patients with gout. Medicine (Baltimore) 99: e21675-e21682. https://doi.org/10.1097/MD.0000000000021675 Yang Y, Zhao J, Qiu J, et al (2018) Xanthine oxidaseinhibitor allopurinol prevents oxidative stress‐mediated atrial remodeling in alloxan‐induced diabetes mellitus rabbits. J Am Heart Assoc 7: e008807-e00880722. https://doi.org/10.1161/JAHA.118.008807 Desco M, Asensi M, Marquez R, et al (2002) Xanthine Oxidase Is Involved in Free Radical Production in Type 1 Diabetes. Diabetes 51:1118-1124. https://doi.org/10.2337/diabetes.51.4.1118 Miric DJ, , Kisic BM, Filipovic-Danic S, et al (2016) Xanthine Oxidase Activity in Type 2 Diabetes Mellitus Patients with and without Diabetic Peripheral Neuropathy. J Diabetes Res 2016:4370490-4370497. https://doi.org/10.1155/2016/4370490 Okuyama T,Shirakawa J, Nakamura T, et al (2021) Association of the plasma xanthine oxidoreductase activity with the metabolic parameters and vascular complications in patients with type 2 diabetes. Sci Rep 11:3768-3781. https://doi.org/10.1038/s41598-021-83234-9 Aliciguzel Y, Ozen I, Aslan M, et al (2003) Activities of xanthine oxidoreductase and antioxidant enzymes in different tissues of diabetic rats. J Lab Clin Med 142:172-177. https://doi.org/10.1016/S0022-2143(03)00110-0 Hao W, Palmer JP (1995) The effect of cytokines on expression of glutamic acid decarboxylase-65 in cultured islets. Autoimmunity 22:209-218. https://doi.org/10.1089/jir.1995.15.1075 Gu Y, Merriman C,Guo Z, et al (2021) Novel autoantibodies to the β-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes. J Autoimmun 122:102677-102697. https://doi.org/10.1016/j.jaut.2021.102677 Russell MA, Morgan NG (2014) The impact of anti-inflammatory cytokines on the pancreatic β-cell, Islets 6: e950547-e950557. https://doi.org/10.4161/19382014.2014.950547 Fukahori M,Ichimori K, Ishida H, et al (1994) Nitric oxide reversibly suppresses xanthine oxidase activity. Free Radic Res 21:203-212. https://doi.org/10.3109/10715769409056572 Ichimori K, Fukahori M, Nakazawa H, et al (1999) Inhibition of Xanthine Oxidase and Xanthine Dehydrogenase by Nitric Oxide. J Biol Chem 274:7763-7768. https://doi.org/10.1074/jbc.274.12.7763 Steinbrenner H,Nguyen T, Wohlrab U, et al (2002) Effect of Proinflammatory Cytokines on Gene Expression of the Diabetes-Associated Autoantigen IA-2 in INS-1 Cells. Endocrinology 143:3839-3845. https://doi.org/10.1210/en.2002-220583 Mally MI, Cirulli V, Otonkoski T, et al (1996) Ontogeny and Tissue Distribution of Human GAD Expression. Diabetes 45:496-501. https://doi.org/10.2337/diab.45.4.496 Phelps EA,Cianciaruso C, Michael IP, et al (2016) Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity. Diabetes 65:2686-2699. https://doi.org/10.2337/db16-0180 Tables Table 1 XO activity in rat islets (n = 500 islets / group) treated with 1.0mM STZ at different time points and effect of Allopurinol. Group 1.0 mM STZ XO activity (nmol H 2 O 2 /min mg protein) in islets % increase in XO activity % decrease in XO activity Control - 0.98 ± 0.18 - - Panel 1 6 hr + 1.25 ± 0.45 * 27.5% - 12 hr + 2.33 ± 0.29 *$ 137.75% - 24 hr + 3.95 ± 0.30 *$ 303.0% - Panel 2 Allopurinol (500 µM) 0.042 ± 0.0051 * - 96.0% Allopurinol (500 µM) + 1.0 mM STZ for 24 hrs 0.026 ± 0.0065 *$ - 97.34% Table 2 Blood glucose levels at different days in female Wistar rats (n = 5/group) injected with 40 mg/kg body weight of STZ. Group Treatment Blood Glucose Levels (mg/dl) Day 0 Day 1 Day 6 Day 12 Day 18 Control - 100.7 ± 3.8 103.39 ± 4.2 112 ± 3.1 110 ± 2.8 109 ± 5.4 STZ (40 mg/kg body weight) + 109.38 ± 4.18 220.66 ± 15.34 # 280.62 ± 40.9 # 333.14 ± 62.19 # 400 ± 59.42 # Table 3 XO activity in pancreas of female Wistar rats (n = 5/group) at different days injected with 40 mg/kg body weight of STZ. Group STZ (40 mg/kg body weight) XO activity (nmol H 2 O 2 /min mg protein) in pancreas % decrease in XO activity Control - 1.58 ± 0.78 - Day 1 + 1.07 ± 0.75 * 32.3% Day 6 + 0.71 ± 0.59 * 55.1% Day 12 + 0.43 ± 0.27 * 72.8% Day 18 + 0.18 ± 0.11 * 88.6% Table 4 XO activity in serum of female Wistar rats (n = 5/group) at different days injected with 40 mg/kg body weight of STZ. Group STZ (40 mg/kg body weight) XO activity (nmol H 2 O 2 /min mg protein) in serum % increase in XO activity Control - 3.03 ± 1.0 - Day 1 + 5.5 ± 1.75 # 81.5% Day 6 + 9.05 ± 2.47 # 198.7% Day 12 + 12.22 ± 2.9 # 919.0% Day 18 + 18.0 ± 3.41 # 1497.0% Table 5 XO activity in rat islets (n = 500/group) treated with IL-1β, TNF-α and IFN-γ cytokines. Rat islets were treated with IL-1β (1 ng/ml), TNF-α (200 units/ml), IFN-γ (500 units/ml) and cocktail cytokines for a period of 48 hours. XO activity (nmol H 2 O 2 /min mg protein) was analyzed as described in materials and methods. Results are shown as mean values ± S.D. of three independent experiments; # p < 0.05 vs control. Controls were normalised as 100%. XOD activities in other samples were expressed as % control. Group XO activity (nmol H 2 O 2 /min mg protein) in islets % Increase in XO activity % Decrease in XO activity Control 0.98 ± 0.18 - - IL-1 β 0.7 ± 0.25 # - 28.0% IFN-γ 1.23 ± 0.43 # 25.5% - TNF-α 1.16 ± 0.35 # 18.4% - IL-1 β / IFN-γ / TNF-α 0.245 ± 0.11 # - 75.0% Table 6 Nitrite concentration in medium containing islets (n = 50/group) treated with IL-1β, TNF-α and IFN-γ cytokines. Rat islets were treated with IL-1β (1 ng/ml), TNF-α (200 units/ml), IFN-γ (500 units/ml) and cocktail cytokines for a period of 48 hours. Nitrite concentration (pmol/islet) was analyzed as described in materials and methods. Results are shown as mean values ± S.D. of three independent experiments; # p < 0.05 vs control. Controls were normalised as 100%. XOD activities in other samples were expressed as % control. Group Medium nitrite (pmol/islet) % Increase in nitrite levels Control 6.5 ± 1.87 - IL-1 β 8.6 ± 1.5 # 32% IFN-γ 7.0 ± 2.21 # 7.7% TNF-α 9.9 ± .35 # 52% IL-1 β / IFN-γ / TNF-α 32 ± 5.1 # 392.3% Table 7 XO activity in rat islets (n = 500/group) treated with cocktail of cytokines (IL-1β, TNF-α and IFN-γ) and effect of 1400W (iNOS inhibitor). Rat islets were treated with cocktail of cytokines (IL-1β, 1 ng/ml; TNF-α, 200 units/ml and IFN-γ, 500 units/ml) in presence or absence of 1400W; 80 µM) for a period of 48 hours. XO activity (nmol H 2 O 2 /min mg protein) was analyzed as described in materials and methods. Results are shown as mean values ± S.D. of three independent experiments; # p < 0.05 vs control; * P < 0.01 vs 1400W. Controls were normalized as 100%. XOD activities in other samples were expressed as % control. Group XO activity (nmol H 2 O 2 /min mg protein) in islets % Increase in XO activity % Decrease in XO activity Control 0.98 ± 0.18 - - 1400W 1.1 ± 0.25 # 12.2% - IL-1 β / IFN-γ / TNF-α 0.245 ± 0.11 #* - 75.0% 1400W + IL-1 β / IFN-γ / TNF-α 14.3 ± 1.28 #* 1359.0% - Table 8 XO activity in fetal, neonatal and adult pancreas during ontogeny in female Wistar rats. XO activity (nmol H 2 O 2 /min mg protein) was measured in fetal (24 gestation weeks), neonatal (6–24 days’ post birth) and adult pancreas as described in materials and methods. Results are shown as mean values ± S.D. of three independent experiments; number of animals in each group was 5 (n = 5 / group); # P < 0.05 vs fetal pancreas. Controls were normalised as 100%. XOD activities in other samples were expressed as % control. Group XO activity (nmol H 2 O 2 /min mg protein) in pancreas % decrease in XO activity (compared to adult pancreas Fetal 21.5d 6.01 ± 2.33 - Neonatal 6d 5.5 ± 1.99 # 8.5% Neonatal 12d 3.11 ± 1.65 # 48.25% Neonatal 24d 2.23 ± 0.46 # 63.0% Adult pancreas 1.58 ± 0.783 # 73.0% Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3829266","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265353797,"identity":"b8345578-2f88-48f0-aa88-cd86d3baf981","order_by":0,"name":"KONIKA RAZDAN","email":"","orcid":"","institution":"Government Medical College","correspondingAuthor":false,"prefix":"","firstName":"KONIKA","middleName":"","lastName":"RAZDAN","suffix":""},{"id":265353798,"identity":"70d7ebe1-9c41-4256-8bf7-7960982a09ea","order_by":1,"name":"VARSHIESH RAINA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYJACZgYDBgYD9gYg08CCFC08B0BaJIjVAlacAKKI0GJw/OzjzwUFNvbmks+vbvhRIMHA396dgF/LmXQz6RkGaYk7Z+eU3ewBOkzizNkNeLVINqSxMfMYHE4wuJ2TdoMHqMVAIpeAlv5nzJ95DP7bG9w8k3bzDzFa+CXSGKR5DA4wbrjBfuw2UbbwSzxjA2pJTtxwJofttoyBBA9Bv7DxpwEd9sfO3uD48Wc33/yxkeNv78WvBQnwGIBJYpWDAPsDUlSPglEwCkbBCAIAHU1CY16KU8kAAAAASUVORK5CYII=","orcid":"","institution":"Government Medical College","correspondingAuthor":true,"prefix":"","firstName":"VARSHIESH","middleName":"","lastName":"RAINA","suffix":""}],"badges":[],"createdAt":"2024-01-02 10:44:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3829266/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3829266/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49292614,"identity":"03b7d59e-6921-4722-9f74-c1d197aadd6e","added_by":"auto","created_at":"2024-01-08 05:49:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":201268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of STZ on protein expression of Xanthine Oxidase (XO) and GAD65 in rat islets (n = 500/group).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eImmunoblot showing protein expression of GAD65 and XO in islets at different concentrations of STZ (0.5 mM STZ, 1.0 Mm STZ and 2.0 mM STZ) for a period of 24 hrs. GAD65 protein levels showing a significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) increase at 1.0 Mm STZ / 2.0 mM STZ and no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change at 0.5 mM STZ in comparison to control. XO protein levels showing no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change at any concentration of STZ in comparison to control. \u003cstrong\u003ec)\u003c/strong\u003e Immunoblot showing protein expression of GAD65 and XO in islets at different time points (6, 12 and 24 hrs.) in presence of1.0 Mm STZ. GAD65 protein levels showing a significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) increase at 12 / 24 hrs and no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change at 6 hrs. in comparison to control. XO protein levels showing no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change till 24 hrs compared to control. in comparison to control. \u0026nbsp;\u003cstrong\u003eb, d)\u003c/strong\u003e Error bar graphs displaying mean values and SD of three densitometry readings per band of GAD65 and XO from three independent western blots adjusted for β-actin in independent blots (as depicted by relative fold change). \u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3829266/v1/c5cdd5899eb5c36c774e358f.jpg"},{"id":49292612,"identity":"48a6abfb-54c2-407b-abd2-61711fbdbe45","added_by":"auto","created_at":"2024-01-08 05:49:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of XO siRNA and Allopurinol on expression of GAD65 and XO in rat islets (n = 500 islets / group) in presence or absence of STZ.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eImmunoblot showing protein expression of GAD65 and XO in rat islets transiently transfected with XO siRNA and scrambled siRNA in presence or absence of 1.0 mM STZ. In presence of XO siRNA, GAD65 protein levels showing no significant (\u003csup\u003e$\u003c/sup\u003ep \u0026gt; 0.05) change in protein expression upon STZ treatment when compared to scrambled or XO siRNA group; significant (\u003csup\u003e@\u003c/sup\u003ep \u0026lt; 0.05) decrease when compared to STZ group. XO protein levels showing significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) decrease in XO siRNA or STZ+XO siRNA group compared to control. No significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change observed in STZ and scrambled siRNA group compared to control. Scrambled siRNA is used as a negative control. \u0026nbsp;Both siRNA was used at concentration of 100 nmol/L.\u003cstrong\u003e c)\u003c/strong\u003e Immunoblot showing protein expression of GAD65 and XO in rat islets co-incubated with 500 µM Allopurinol (XO inhibitor) in presence or absence of 1.0 mM STZ. GAD65 protein levels showing a significant (\u003csup\u003e@\u003c/sup\u003ep \u0026lt; 0.05) decrease in control / Allopurinol alone / STZ+Allopurinol group vs STZ group. XO protein levels displaying no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change compared to control.\u0026nbsp; \u003cstrong\u003eb, d)\u003c/strong\u003e Error bar graphs displaying mean values and SD of three densitometry readings per band of GAD65 and XO from three independent western blots adjusted for β-actin in independent blots (as depicted by relative fold change).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3829266/v1/1fe63483485f60d5233561cb.jpg"},{"id":49292613,"identity":"18e7da95-c894-4137-9cec-0a3da3e25a31","added_by":"auto","created_at":"2024-01-08 05:49:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of XO and GAD65 in Type-1 diabetic rats and cytokine treated islets (n = 500 islets / group).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea)\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eImmunoblot showing protein expression of XO and GAD65 in pancreas of Type-1 diabetic wistar female rats at different days’ post STZ injection. Type-1 diabetes was induced by intraperitoneal injection of single dose of STZ at 40 mg/kg body weight. GAD65 protein levels showing significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) decrease post 1-day STZ compared to control (without STZ). XO protein levels showing significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) increase post 1 day compared to control. \u003cstrong\u003ec)\u003c/strong\u003e Immunoblot showing protein expression XO and GAD65 in islets (n = 500) treated with cytokines TNF-α (6000 UI\u0026nbsp;ml\u003csup\u003e− 1\u003c/sup\u003e), IL1-β (600 UI\u0026nbsp;ml\u003csup\u003e− 1\u003c/sup\u003e) and IFN-γ (6000 UI\u0026nbsp;ml\u003csup\u003e− 1\u003c/sup\u003e) and cocktail of cytokines (TNF-α + IL1-β + IFN-γ) for a period of 48 hrs. In comparison to control, IL1-β and cocktail of cytokines showing a significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) decrease in protein expression of GAD65 whereas no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change in GAD65 protein expression of islets treated with TNF-α or IFN-γ. XO protein levels displaying no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change in protein expression compared to control. \u003cstrong\u003eb, d)\u003c/strong\u003e Error bar graphs displaying mean values and SD of three densitometry readings per band of GAD65 and XO from three independent western blots adjusted for β-actin in independent blots (as depicted by relative fold change). \u003cstrong\u003ee)\u003c/strong\u003e Immunoblot showing protein expression of inducible nitric oxide synthase (iNOS) in rat islets treated with cytokines TNF-α (6000 UI\u0026nbsp;ml\u003csup\u003e− 1\u003c/sup\u003e), IL1-β (600 UI\u0026nbsp;ml\u003csup\u003e− 1\u003c/sup\u003e) and IFN-γ (6000 UI\u0026nbsp;ml\u003csup\u003e− 1\u003c/sup\u003e) and cocktail of cytokines (TNF-α + IL1-β + IFN-γ) for a period of 48 hrs. A robust increase in iNOS protein levels observed in cocktail of cytokines \u003cstrong\u003ef)\u003c/strong\u003e Immunoblot showing protein levels of GAD65 and XO in rat islets treated with cytokine cocktail or 1400W (an iNOS inhibitor). While as 1400W shows no effect on GAD65 or XO protein levels but GAD65 protein levels are fully restored in 1400 + cytokine cocktail group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3829266/v1/ef616efcf7241495fdfbd050.jpg"},{"id":49292615,"identity":"0e94a204-e2c4-4d53-bd93-662b0a39a7c7","added_by":"auto","created_at":"2024-01-08 05:49:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":100029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of XO and GAD65 during ontogeny of pancreas and effect of Allopurinol on these molecules in fetus (n = 5 animals/group). a) \u003c/strong\u003eImmunoblot showing protein levels of GAD65 and XO in fetus (25.1 gestational week), neonatal (6-, 12- and 24-days’ post birth) and adult (18 weeks). Compared to fetal pancreas, GAD65 protein expression shows no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) difference in neonatal pancreas but a significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) decline in adult pancreas. XO displays no significant (\u003csup\u003e#\u003c/sup\u003ep \u0026gt; 0.05) change in protein levels. XO protein levels showing no significant change \u003cstrong\u003ec)\u003c/strong\u003e Immunoblot showing significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05) decrease in GAD65 protein levels in fetal pancreas after injecting Allopurinol at concentration of 30 mg kg\u003csup\u003e−1\u003c/sup\u003e at fetal 20.0 days. \u003cstrong\u003eb, d)\u003c/strong\u003e Error bar graphs displaying mean values and SD of three densitometry readings per band of GAD65 and XO from three independent western blots adjusted for β-actin in independent blots (as depicted by relative fold change).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3829266/v1/b4723017b2ede98f4c25a2b5.jpg"},{"id":64859320,"identity":"9e485335-5860-403e-8195-855868660ccf","added_by":"auto","created_at":"2024-09-19 16:02:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1616913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3829266/v1/c95559cd-25c4-4057-87fb-926b614ca015.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Xanthine oxidase modulates Glutamic Acid Decarboxylase 65 isoform (GAD65) protein expression in rat pancreatic islets","fulltext":[{"header":"INTODUCTION","content":"\u003cp\u003eType-1 Diabetes (T1D) is a multifactorial endocrine pancreatic auto-immune disorder which is clinically diagnosed by the presence of circulating auto-antibodies against some antigens, of which prominent one includes glutamic acid decarboxylase isoform 65 (GAD65) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As per estimates, nearly 70\u0026ndash;80% of newly diagnosed T1D patients are diagnosed with GAD65 autoantibodies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. GAD65 is an apo-enzyme that catalyzes the formation of γ-amino butyric acid (GABA), predominantly in GABA-ergic nerve cells and pancreatic islets [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The exact physiological function of GAD65 in pancreas is still poorly understood but few studies show that GABA - promotes beta-cell growth and survival; suppresses glucagon secretion from alpha cells; and suppresses inflammation and increases regulatory T-cell numbers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among mammalian species, GAD65 is predominantly expressed in human and rat pancreas [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe recognition of GAD65 as auto-antigen in T1D instigated in 1982 from immunoprecipitation studies which showed a 64 Kd protein was precipitated by diabetic sera in Type-1 diabetic patients [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Since then, several studies in spontaneous animal models of autoimmune diabetes have strongly supported that GAD65 is highly immunogenic and potential biomarker for T1D [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Despite, these evidences, it is still unclear, why GAD65 auto-antibodies are generated? Notably, GAD65 specific auto-antibodies are present in the serum before the destruction of beta-cells of pancreas. Moreover, a study showed that aberrant accumulation of GAD65 in pancreas during endoplasmic stress can sensitize beta cells for auto-immune assault [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Such abnormal accumulation of proteins is commonly encountered in neurological disorders, and among several factors, over-expression of proteins has been found to be a major reason. Interestingly, pancreatic beta cells and neurons share some similarities like both are highly prone to physiological stress due to scarcity of free radical scavenging enzymes. So, it is quite likely that similar mechanism might operate in beta cells that leads to aberrant expression of GAD65. So far, studies have demonstrated that GAD65 expression gets altered under high glucose, blocking mitochondrial electron transport system, hypoxia, glutamate, Streptozotocin (STZ) and transcriptional factors (like sp-1, NF-kB and SMAR1 to be putative positive regulatory while as cytokines like IL-1 beta act as a negative regulator) \u003cb\u003e[10, 11, 12, 13]\u003c/b\u003e. Thus, finding molecules that influence cell stress can be an important candidate molecule that can regulate GAD65 expression.\u003c/p\u003e \u003cp\u003eXanthine oxidase (XO), is prototype of the molybdenum hydroxylase family of enzymes that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid. Under normal conditions XO exists majorly in an inactive form namely xanthine dehydrogenase (XDH), thus given a name of XDH / XO system. XO has broad range of activity for endogenous / exogenous substrate\u0026rsquo;s, thus aids in drug-metabolism and detoxification \u003cb\u003e[14]\u003c/b\u003e. It generates free radicals (.OH, O\u003csup\u003e2\u0026minus;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) which are involved in variety of pathophysiological conditions such as atherosclerosis, endothelial dysfunction, hemolytic diseases, viral infections, hepatotoxicity, acute pancreatitis, respiratory distress syndrome, ischemia-reperfusion injury, inflammatory and autoimmune diseases \u003cb\u003e[15, 16, 17]\u003c/b\u003e. Moreover, XO derived free radicals can affect various transcriptional regulators like AP-1, NFkB and p53 which are involved in dysfunction of target cells or tissues \u003cb\u003e[18, 19]\u003c/b\u003e. In both Type-1 and Type-2 diabetes, there are several reports which show XO causes oxidative damage as well as its associated complications, and the role of XO was also proposed in experimental model of diabetes \u003cb\u003e[20, 21]\u003c/b\u003e. Despite its strong association with the pathology of Type-1 or Type-2 diabetes; however, no study till date has investigated the role of XO in regulating GAD65 expression.\u003c/p\u003e \u003cp\u003eStreptozotocin (STZ) is a natural compound derived from \u003cem\u003eStreptomyces achromogenes\u003c/em\u003e that is selectively toxic to the cells expressing Glut-2 receptor \u003cb\u003e[22]\u003c/b\u003e. Because beta cells express high level of Glut-2 receptor, STZ has been widely used to study beta cell function in in-vitro and animal models of Type-1 and Type-2 diabetes. STZ induced beta cell death occurs by many mechanisms such as DNA alkylation, protein glycosylation, over-stimulation of poly (ADP-ribose) polymerase (PARP) and depletion of energy stores (like NAD\u003csup\u003e+\u003c/sup\u003e and ATP) \u003cb\u003e[23]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eHere we report for the first time, a dramatic effect of XO on GAD65 protein expression in isolated rat pancreatic islets / STZ induced experimental Type-1 diabetic rat model, and further demonstrate the association of XO activity with GAD65 protein expression during ontogeny of pancreas.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003e \u003cstrong\u003eIslet isolation from fetal, neonatal and adult pancreas\u003c/strong\u003e \u003cp\u003eThe female Sprague Dawley rats (150 to 200 g, 8 to 12 weeks old) were obtained from experimental animal facility of National Centre for Cell Science, Pune, India. During the period of experiments, the animals were housed at 21\u003csup\u003e0\u003c/sup\u003eC with 12 hours\u0026rsquo; light and 12 hours\u0026rsquo; darkness cycle. Food and water were available to animal\u0026rsquo;s ad libitum. Fetal pancreata (from pregnant Sprague Dawley rats at E21.5 of gestation) neonatal and adult pancreases were surgically dissected under sterile conditions in compliance with bioethics legislation in India. Islets were isolated according to the procedure described previously \u003cb\u003e[24]\u003c/b\u003e. Briefly, under halothane anesthesia, the abdomen was cut open and the pancreas was distended by infusion of 10 ml sterile Krebs-Ringer-Hepes solution (Sigma Aldrich, USA) containing 10% bovine serum albumin (BSA fraction V, Sigma Aldrich) through cannulated common bile duct. Afterwards, the pancreas was excised, chopped and digested using a two-stage incubation of 20 min at 37\u003csup\u003eo\u003c/sup\u003eC with successively 1.0 and 0.7 mg/ml collagenase P (Sigma Aldrich, USA). Islets were separated from exocrine tissue by centrifugation over a discontinuous dextran (Sigma Aldrich) gradient and further purified by handpicking. Approximately, 100 Islets per 25 cm\u003csup\u003e2\u003c/sup\u003e were cultured in Petri dishes in CMRL 1066 (Sigma Aldrich), containing 10% fetal calf serum (Sigma Aldrich), 8.3 mmol/l glucose (Sigma Aldrich), 10 mmol/l HEPES (Sigma Aldrich, USA) and 1% penicillin/streptomycin (Sigma Aldrich, USA), at 37\u003csup\u003eo\u003c/sup\u003eC in humidified air containing 5% CO2.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSTZ and Cytokine treatment to islets\u003c/strong\u003e \u003cp\u003e500 mM Stock solution of Streptozotocin (Sigma Aldrich) was reconstituted in 0.01 M citrate buffer (pH 4.5) just before treatment. 100 ml of 0.1 M sodium citrate buffer was prepared by mixing 55.5ml of 0.1M trisodium citrate (Sigma Aldrich, USA) and 44.5ml of 0.1M citric acid monohydrate (Sigma Aldrich). pH was adjusted with 1 N NaOH and measured by pH meter (Thermo Fischer Scientific, USA). The buffer was filter sterilized prior to islet treatment at working concentrations of 0.5 mM, 1.0 mM and 2.0 mM for a period of 24 hrs. Intact islets were treated with a mixture of TNF-α (6000 UI ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), IL1-β (600 UI ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and IFN-γ (6000 UI ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Merck Millipore, Burlington, MA, USA) and STZ for 48 hrs. to assess the effect \u003cb\u003e[25]\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInduction of Type-1 Diabetes and measurement of blood glucose\u003c/b\u003e: Type-1 diabetes in female Sprague Dawley rats (150 to 200 g, 8 to 12 weeks old) was induced by injecting intra-peritoneally single dose of STZ at concentration of 40 mg/kg body weight as described previously \u003cb\u003e[26]\u003c/b\u003e. Briefly, 10 rats were used per group and housed in cages for 5 days at 21\u003csup\u003e0\u003c/sup\u003eC with 12 hours\u0026rsquo; light and 12 hours\u0026rsquo; darkness cycle. Before the start of experiment, all rats were fasted for 6 to 8 hr prior to STZ treatment. Glucose level in serum were measured as per the instructions mentioned in \u0026ldquo;Glucose Calorimetric Detection Kit\u0026rdquo; (EIAGLUC; Thermo Fisher Scientific, USA).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003esiRNA transfection\u003c/strong\u003e \u003cp\u003eAround 500 islets were seeded in culture dishes containing CMRL 1066 (Sigma Aldrich) containing 10% fetal calf serum (Sigma Aldrich), 8.3 mmol/l glucose (Sigma Aldrich), 10 mmol/l HEPES (Thermo Fisher Scientific, USA). A final transfection volume of 2.5 mL per dish contained 50 nmol/L scrambled (sc-37007; Santa Cruz Biotechnology, Santa Cruz, CA and XO siRNA (sc-270202; Santa Cruz Biotechnology, Santa Cruz, CA) in Opti-MEM (31985062; Thermo Fischer Scientific, USA) reduced serum media and 6.25 mL Lipofectamine RNAiMAX (Thermo Fischer Scientific, USA). A second transfection was performed 24 h after the first transfection and all functional experiments were done 72 h after the first transfection.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoblotting and antibodies\u003c/b\u003e: Protein isolation was done in radio-immunoprecipitation assay (RIPA) buffer (R0278; Sigma Aldrich). Prior to lysis, islets were washed in PBS and pelleted by centrifugation at 800 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min; whereas dissected pancreata were directly put in lysis buffer, homogenized, sonicated for 15 s and centrifuged at 14000 x g for 10 minutes. About 50 \u0026micro;g of protein was loaded for immunoblotting and detected with primary antibodies against rat p53 (A-1; sc-393031; Santa Cruz Biotechnology, Santa Cruz, CA), Xanthine Oxidase (A-3; sc-398548; Santa Cruz Biotechnology, Santa Cruz, CA), ser15 p53 (#9284; Cell Signaling Technology), GAD65 (sc-377145; Santa Cruz Biotechnology, Santa Cruz, CA) and beta actin (C-4; sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA) at 1: 500. (Sigma-Aldrich). The rabbit (mouse (#7074; Cell Signaling Technology) and mouse (#7076; Cell Signaling Technology) HRP conjugated secondary antibodies were used at 1: 3000.\u003c/p\u003e \u003cp\u003e \u003cb\u003eXanthine oxidase Assay\u003c/b\u003e: Islets and pancreata were lysed with potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and protease inhibitors. The homogenates were centrifuged at 12,000 g for 15 min at 4\u0026deg;C, and supernatants were collected. XO activity in islet / pancreata and serum was detected by the method as described previously \u003cb\u003e[27]\u003c/b\u003e. Briefly, 60 \u0026micro;l of 0.1 M Tris containing 40 mg/l horseradish peroxidase (HRP) buffer (pH 7.5) was mixed with 20 \u0026micro;l 2 mM xanthine (see above) and 10 \u0026micro;l of ortho-phenylendiamine water solution. Reaction was initiated by adding 50 \u0026micro;l of sample. Control samples (blank) did not contain xanthine. Samples were incubated for 30 min at 40\u0026deg;C followed by termination of the reaction with 100 \u0026micro;l of 1.88 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e standards were analysed to calculate XOD activity (concentrations \u0026ndash; 0.1\u0026ndash;100 nmol/ml). The absorbance was measured at 492 nm. XOD activity was expressed in nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e produced per 1 min per mg protein. Controls were normalised as 100%. XOD activities in other samples were expressed as % control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNitrite determination.\u003c/b\u003e Aliquots of the culture medium were deproteinized by adding 35% sulfosalicylic acid. Samples were incubated for 15 min at 4\u003csup\u003eo\u003c/sup\u003eC and then centrifuged for 15 min at 12,000 g. One part of 0.1% naphthylenediamine dihydrochloride was added to the supernatant, together with 1% sulfonilamide and 5% concentrated H3PO4. The reaction was carried out at 60\u003csup\u003eo\u003c/sup\u003eC for 1 min, and the absorbance was measured at 546 nm in a spectrophotometer (DU-62; Beckman Instruments, Inc., Palo Alto, CA) against a standard curve.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analysis\u003c/strong\u003e \u003cp\u003eAll data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD derived from at least three independent experiments unless otherwise specified. Statistical significance of results was evaluated on raw values prior to conversion to percent of control, by a repeated measure one-way analysis of variance (ANOVA) test with Tukey\u0026rsquo;s post-hoc pairwise comparison, or t-test, using IBM SPSS Statistics (Version 21). P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant and P values\u0026thinsp;\u0026gt;\u0026thinsp;0.05 were considered non-significant.\u003c/p\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEffect of STZ on protein expression of Glutamic acid decarboxylase isoform 65 (GAD65) and Xanthine oxidase (XO) in isolated rat islets\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUpon treatment of the isolated rat islets with different concentrations of STZ (i.e., 0.5 mM, 1.0 mM and 2.0 mM), for a time period of 24 hours, the immunoblot showed that in comparison to control (without STZ), 1.0 mM and 2.0 mM STZ concentration led to a significant increase in GAD65 protein expression, while as, XO protein expression displayed no change (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Quantitative analysis shown by error bar graph revealed that STZ led to a maximum ≃4.0-fold relative increase in GAD65 protein expression when compared to control and 1.0 mM STZ was the minimal effective concentration to display such change \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e [MTT assay was also done to determine the effect of STZ on cell viability and it was observed that islet viability was \u0026ndash; 99.0% at 0.5mm STZ; 98.0% at 1.0 mm STZ and 76.0% at 2.0 mm STZ (data not shown here)].\u003c/p\u003e \u003cp\u003eWe further performed time kinetic for duration of 6hr, 12hr and 24 hrs., to study the protein expression of XO and GAD65 in presence of 1.0 mM STZ. In immunoblot, we found that at 1.0 mM STZ, only the protein expression of GAD65 displayed a significant increase which was evident from 12 hrs. onwards and, on the other hand, XO protein expression displayed no change at any time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Quantitative analysis shown by error bar graph revealed ≃4.0-fold increase in XO protein expression at 12 and 24 hrs. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eXO activity and effect of XO inhibitor (Allopurinol) / XO siRNA on GAD65 protein levels in isolated rat islets upon STZ treatment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether, XO enzymatic activity in rat islets is affected upon STZ treatment, we measured XO activity in isolated rat islets in presence of 1.0 mM STZ for time duration of 6 hrs., 12 hrs. and 24 hrs. We observed that 1.0 mM STZ led to a significant increase in XO activity which was 27.5% at 6 hrs., 137.75% at 12 hrs., and 303.0% at 12 hrs. when compared to control \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; \u003cb\u003ePanel 1)\u003c/b\u003e. Moreover, the data also depicts a robust increase in XO activity from 12 hrs. onwards. Alongside, when the islets were co-incubated with 500 \u0026micro;M Allopurinol (well-known XO inhibitor) in presence or absence of STZ for 24 hrs., the XO activity was significantly reduced i.e., allopurinol vs control (96%) and STZ\u0026thinsp;+\u0026thinsp;Allopurinol vs control (97.34%) \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; \u003cb\u003ePanel 2)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn this context, we investigated whether the STZ induced increase in XO activity modulates GAD65 protein expression in these rat islets by employing two approaches: one, which involved downregulating XO protein expression by transient transfection of islets with XO siRNA and second, which involved inhibiting XO enzymatic activity by co-incubation of islets with Allopurinol. As shown in immunoblot \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, XO siRNA transfection in islets significantly decreased XO protein expression, and when these XO siRNA transfected islets were treated with STZ, no significant increase in GAD65 protein expression was observed compared to STZ treated islets. The relative fold change in protein expression is shown in error bar graph which shows ≃2.66-fold decrease in GAD65 protein expression in XO siRNA\u0026thinsp;+\u0026thinsp;STZ vs STZ and ≃1.26-fold increase in GAD65 protein expression in XO siRNA\u0026thinsp;+\u0026thinsp;STZ / scrambled XO siRNA / XO siRNA vs control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. In case of Allopurinol (400 \u0026micro;M) treated islets, XO protein expression showed no significant change whereas GAD65 protein expression showed a significant increase in Allopurinol / Allopurinol\u0026thinsp;+\u0026thinsp;STZ vs control and significant decrease in Allopurinol\u0026thinsp;+\u0026thinsp;STZ group vs STZ group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. The relative fold change in protein expression is shown in error bar graph which shows that Allopurinol led to ≃ 2.2-fold increase in GAD65 protein expression in Allopurinol / Allopurinol\u0026thinsp;+\u0026thinsp;STZ vs control and ≃ 2.66-fold decrease in GAD65 protein expression in STZ\u0026thinsp;+\u0026thinsp;XO siRNA group vs STZ group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGAD65 protein expression in STZ induced Type-1 diabetic rat model and rat islets treated with diabetogenic cytokines such as IL-1 β, TNF-α and IFN-γ\u003c/b\u003e \u003c/p\u003e \u003cp\u003eType-1 diabetes is in female Wistar rats was induced by injecting intra-peritoneally single dose of STZ at concentration of 40 mg/kg body weight (as in material and methods) and diabetes was monitored by measuring rise in blood glucose levels at day 0, day 1, day, day 6, day 12 and day 18. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, STZ induced a significant increase in blood glucose levels which was ≃ 2.0-fold at day 1 vs control; ≃ 2.6-fold at day 6 vs control; ≃3.0-fold at day 12 vs control and ≃ 3.65-fold at day 18 vs control.\u003c/p\u003e \u003cp\u003eImmunoblot for XO and GAD65 in dissected pancreata of these wistar female rats was performed at same days and it was observed that GAD65 protein expression significantly decreased from day 1, whereas, XO protein expression significantly increased from day 6 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The relative fold change in protein expression is shown in error bar graph which shows a significant decrease of protein expression in GAD65 by ≃ 2.0-fold at day 1 vs control; ≃ 2.0-fold at day 6 vs control; ≃4.0-fold at day 12 vs control; ≃ 10.0-fold at day 18 vs control, and significant increase of protein expression in XO by ≃ 1.15-fold at day 1 vs control; ≃ 1.2-fold at day 6 vs control; ≃1.5-fold at day 12 vs control; ≃ 1.5-fold at day 18 vs control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe next measured the XO activity in the pancreata and sera of these STZ induced Type-1 diabetic rats which is represented in table as % decrease/increase in XO activity. It was observed that there occurred a significant decrease in pancreatic XO activity from day 1 onwards in comparison to control \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. However, in serum of these animals, XO activity was significantly increased from day 1 onwards in comparison to control \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eDuring the pathophysiology of Type-1 diabetes, cytokines such as IL-1β, TNF-α and IFN-γ are known to play a major role. We thus treated isolated rat islets with these cytokines to investigate their effect on the protein expression of XO and GAD65. In immunoblot, we found that neither of the cytokines either alone or in cocktail changed the protein expression of XO; however, GAD65 protein expression was significantly reduced in presence of IL-1 β beta and cocktail of these cytokines (IL-1β\u0026thinsp;+\u0026thinsp;TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. The relative fold change in protein expression is shown in error bar graph which shows that GAD65 levels decreased to ≃ 1.3-fold in presence of IL-1 β and ≃ 2.0-fold in presence of cocktail of these cytokines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. XO activity was also measured as represented by % decrease in XO activity and we observed a significant decline in XO activity in islets treated with IL-1 beta (about 28%) and cocktail of cytokines (about 75%) in comparison to control \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eInterestingly, we came across a study wherein nitric oxide has been shown to inhibit the XO activity. We thus performed an immunoblot for inducible nitric oxide synthase enzyme (iNOS) and also measured nitrite levels in the islets treated with these cytokines. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, none of the cytokines alone was capable to alter iNOS protein expression; however, in presence of the cocktail of these cytokines there occurred robust expression of iNOS protein. On the other hand, measurement of nitrite levels in the supernatants of islets revealed significant increase in presence of IL-1 β (about 32%); IFN-γ (about 7.7%), TNF-α (about 52%) and TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ\u0026thinsp;+\u0026thinsp;IL-1 β (about 392.3%), in comparison to control \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. On comparison of data from Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, we also observed that cocktail of cytokines is more effective to induce the changes in GAD65 protein expression or XO activity. So, we treated islets with 1400W (a nitric oxide synthase inhibitor) either alone or along-with cytokine cocktail and in the immunoblot, we observed comparable protein expression of GAD65 among control vs 1400W vs cocktail of cytokines\u0026thinsp;+\u0026thinsp;1400W \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRelation of GAD65 and XO in fetal, neonatal and adult rat pancreas\u003c/h2\u003e \u003cp\u003eWe also studied the association between XO and GAD65 during the course of pancreatic ontogeny from fetal stage \u0026ndash; neonatal stage (at 6 days, 12 days and 24 days) - adult stage. Immunoblot revealed no significant change in protein expression of XO from fetal to adult stage; however, protein expression of GAD65 started to show significant decline from neonatal (24 days) onwards and the decline in protein expression was more evident in adult pancreas \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The relative fold change in protein expression is shown in error bar graph which shows decrease in GAD65 expression of about ≃1.15-fold at neonatal day 24 and ≃2.5-fold in adult pancreas vs fetal / neonatal (6 days) / neonatal (12 days) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAlongside, XO activity in fetal, neonatal and adult pancreas was measured and we observed a significant decline in XO activity from fetal to adult stage of pancreas \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. To further substantiate our findings, we performed our study on fetal pancreas for reasons mainly due to - high protein expression of both the molecules (GAD65 \u0026amp; XO) and high XO activity; an ideal dynamic environment which involves continuous cellular remodeling process and an ideal therapeutic strategy to mitigate T1D before birth. Female pregnant rats were injected intraperitoneally with Allopurinol at concentration of 30 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at fetal 20.0 days and fetal pancreas were dissected to study protein expression of GAD65. Immunoblot revealed that GAD65 protein expression was drastically reduced in fetal pancreas of Allopurinol injected pregnant rat. The relative fold change in protein expression is shown in error bar graph which shows ≃2.6-fold decrease in protein expression of GAD65 with Allopurinol in comparison to control.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eVarious factors can contribute towards aberrant accumulation of proteins and among them cellular defects in transcriptional regulation are well described in neurological diseases \u003cb\u003e[28]\u003c/b\u003e. Xanthine oxidase (XO) is well known molecule which have been extensively studied for their pathological role in various diseases, including diabetes \u003cb\u003e[14, 16]\u003c/b\u003e. In this study, we underpin for the first time the role of XO in regulating GAD65 expression in pancreatic islets and T1D rat model.\u003c/p\u003e \u003cp\u003eWe choose rat islets as they predominantly express GAD65 and moreover, islets represent a suitable in-vitro experimental model that closely mimics in-vivo system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. STZ is a well-known diabetogenic agent which specifically causes beta cell dysfunction by various mechanisms; and has been previously demonstrated in our study to upregulate GAD65 in mouse pancreatic islets \u003cb\u003e[23, 12]\u003c/b\u003e. In this study also, we report a significant increase in GAD65 protein expression upon STZ treatment in rat pancreatic islets which further substantiates the fact that indeed STZ is a potent regulator of GAD65 in pancreatic islets. While as, STZ had no effect on XO protein levels; however, XO activity was significantly enhanced in rat pancreatic islets. This was not surprising as previous studies have also demonstrated that XO induced cellular stress is not strictly dependent on XO levels but on XO activity in various cells \u003cb\u003e[29, 30]\u003c/b\u003e. Elevated XO activity has also been observed with increased risk of developing Type-1 and Type-2 diabetes, diabetic vascular complications and muscular oxidative stress in STZ induced experimental diabetes \u003cb\u003e[31, 32, 33, 34]\u003c/b\u003e. Remarkably, when the endogenous XO protein expression was silenced by XO siRNA or its activity was inhibited by Allopurinol (a well-known inhibitor of XO), STZ induced GAD65 protein expression was substantially abolished, reinforcing the fact XO activity is required for GAD65 protein expression. The role of Allopurinol is well documented to reduce severity of proteinuria, ameliorate oxidative stress in Type-1 diabetic patients, improve endothelial dysfunction in Type-2 diabetic patients and so on \u003cb\u003e[35, 36].\u003c/b\u003e Thus, our results clearly indicate that XO activity can play a crucial role in pathogenesis of TID by modulating GAD65 expression.\u003c/p\u003e \u003cp\u003eHaving established the fact that STZ induced upregulation of XO activity is responsible for GAD65 overexpression, we expanded our study to STZ induced experimental animal model of T1D (which is induced by single dose of STZ in rats) \u003cb\u003e[26].\u003c/b\u003e Diabetes was confirmed by rise in glucose levels and assessment of GAD65 and XO protein levels in pancreas at different days\u0026rsquo; post STZ injection revealed a significant decrease in GAD65 and significant increase in XO protein levels. Alongside, it was observed that XO activity was significantly increased in serum and significantly decreased in pancreas as the diabetes progressed. To the best of our knowledge, this is the first study which demonstrates differential activity of XO in STZ induced experimental T1D. However, our study is in concordance with the earlier studies which demonstrate high serum XO activity both human and animal models of Type-2 diabetes \u003cb\u003e[37, 38].\u003c/b\u003e Notably, few studies have also found negative correlation or no correlation of XO activity with diabetes duration \u003cb\u003e[39, 40]\u003c/b\u003e. Our study also demonstrates negative regulation of XO activity in pancreas of rat model of Type-1 diabetes, but we do believe that further investigation is needed to gain more insight in other animal models and Type-1 diabetic patients. In addition, we also suggest that the difference of XO activity among Type-1 and Type-2 diabetic groups can possibly happen because the mode of pathology is different in both diseases.\u003c/p\u003e \u003cp\u003eT1D is an immune mediated disease and the proinflammatory cytokines such as IL-1 β, TNF-α and IFN-γ have been found to be major players in beta cell destruction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In order to establish the fact that indeed change in the expression of these molecules is caused by auto-immune assault, we treated the rat islets with IL-1 β, TNF-α and IFN-γ cytokines. We observed that only IL-1 beta led to a significant decrease in GAD65 levels and the effect was more prominent in presence of cocktail of these cytokines. However, XO protein levels were not altered in any of these treatments, implying that there may be other factors that control the expression of XO in STZ induced Type-1 diabetes. Pertinent to mention here that similar kind of observation was published in which authors observed that IL-1 β dramatically reduces GAD65 expression whereas TNF-α and IFN-γ had no effect on GAD65 expression \u003cb\u003e[41]\u003c/b\u003e. Besides, other studies also showed that cytokines have negative effect on islet cell antigens like IA-2, insulin and Zinc transporter-8 (ZnT8) \u003cb\u003e[42, 43]\u003c/b\u003e. Thus, our finding further supports the earlier findings which demonstrate that proinflammatory cytokines have inhibitory effect on GAD65 expression.\u003c/p\u003e \u003cp\u003eWe next investigated whether the above-mentioned cytokines modulate the XO activity in islets. We observed a dramatic decline in XO activity and major effect was observed when islets were treated with cocktail of cytokines. This decline in XO activity coincided with XO activity as observed in pancreas of T1D rats. Interestingly, we came across studies which show negative effect of nitric oxide synthase on XO activity \u003cb\u003e[44, 45]\u003c/b\u003e. Moreover, a study showed that treatment of rat Insulinoma INS-1 cells with either of these cytokines led significant downregulation of IA-2 and insulin mRNA in nitric oxide dependent manner \u003cb\u003e[46]\u003c/b\u003e. We also got interested to find out whether nitric oxide plays any role in modulating XO activity and thus GAD65 expression. We also observed a robust increase in nitrite levels of islet culture supernatant treated with cocktail of cytokines. We further show that cytokine mixture led to a significant over-expression of inducible nitric oxide synthase (iNOS) and co-incubation with 1400W (a potent inhibitor of INOS) not only restored the GAD65 protein levels but enhanced XO activity. Thus, our study provides additional evidence that nitric oxide is negative regulator of XO activity and opens new window of opportunity for its implication in T1D.\u003c/p\u003e \u003cp\u003eTo further corroborate our findings, we examined the expression of these molecules during ontogeny of pancreas from fetal -neonatal-adult stage. During the ontogeny while as XO protein levels remained unaltered but GAD65 protein levels showed a significant decline after 24th week of gestation. In agreement with previous studies, our study also demonstrates that GAD65 is expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas \u003cb\u003e[47]\u003c/b\u003e. Parallely, we measured XO activity in pancreas which was found to decrease from fetal to adult stage. Remarkably, when pregnant females were challenged with Allopurinol a significant decline in GAD65 protein expression occurred which clearly demonstrates that XO activity is indeed one of the major factors that regulates GAD65 expression during pancreatic development. This study also indicates that aberrant activity of XO might be one of the predisposing factors for aberrant accumulation of GAD65 which is implicated in beta cell dysfunction and loss during pathogenesis of T1D \u003cb\u003e[48]\u003c/b\u003e. Furthermore, the idea behind application of Allopurinol in pregnant rats in our study was to find whether GAD65 protein expression can be reduced during fetal pancreas development which might open up new window of opportunity to mitigate Type-1 diabetes.\u003c/p\u003e \u003cp\u003eIn conclusion, our study for the first time demonstrates substantial role of XO in regulating expression of GAD65 in pancreatic islets and further demonstrate their positive association during ontogeny of pancreas.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval for animal study:\u003c/strong\u003e No ethical approval was required for this study as per the law applicable in our country. However, the study was carried out in in strict accordance with the\u0026nbsp;Ethics Committee for Animal studies of the National Centre for Cell Science and\u0026nbsp;international guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman ethics:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e Authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding source:\u003c/strong\u003e No funding source. The work was supported by the NCCS, GMC Jammu and EIPL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contribution:\u003c/strong\u003e Dr Varshiesh Raina designed, performed experiments, wrote and modified the paper. Dr Konika Razdan analyzed data, prepared figures and performed experiments. Both authors reviewed the manuscript, and the manuscript is approved by both authors for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e The work was supported by NCCS Pune, EIPL and Department of Microbiology, GMC, Jammu. We would like to thank Dr G.C.Mishra (Ex-director NCCS) and Late Dr P.B.Parab (Scientist G) for all the support and facility for conducting animal experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDiMeglio LA, Evans-Molina C, Oram RA (2018) Type 1 diabetes. Lancet 391:2449-2462. https://doi.org/10.1016/S0140-6736(18)31320-5\u003c/li\u003e\n\u003cli\u003eBasu M,\u0026nbsp;Pandit K,\u0026nbsp;Banerjee M, et al (2020) Profile of Auto-antibodies (Disease Related and Other) in Children with Type 1 Diabetes. Indian J Endocrinol Metab\u0026nbsp;24:256-259. https://doi.org/10.4103/ijem.IJEM_63_20\u003c/li\u003e\n\u003cli\u003eFenalti G, Buckle AM (2010) Structural biology of the GAD autoantigen. Autoimmun Rev 9:148-152. https://doi.org/10.1016/j.autrev.2009.05.003\u003c/li\u003e\n\u003cli\u003eHagan DW, Ferreira SM, Santos GJ, et al (2022) The role of GABA in islet function. Front Endocrinol (Lausanne) 13:972115-972133. https://doi.org/10.3389/fendo.2022.972115\u003c/li\u003e\n\u003cli\u003eAl-Kuraishy HM, Hussian NR, Al-Naimi MS, et al (2021) The Potential Role of Pancreatic \u0026gamma;-Aminobutyric Acid (GABA) in\u0026nbsp;Diabetes Mellitus. Int J Prev Med24:12-19. https://doi.org/ 10.4103/ijpvm.IJPVM_278_19\u003c/li\u003e\n\u003cli\u003eKim J, Richter W, Aanstoot HJ, et al (1993) Differentialexpression\u0026nbsp;of\u0026nbsp;GAD65\u0026nbsp;and\u0026nbsp;GAD67\u0026nbsp;in human, rat, and mouse pancreatic islets. Diabetes 42:1799-1808. https://doi.org/10.2337/diab.42.12.1799\u003c/li\u003e\n\u003cli\u003eBaekkeskov S, Nielsen JH, Marner B, et al (1982) Autoantibodies in newly diagnosed diabetic children immunoprecipitate specific human islet cell proteins.\u0026nbsp;Nature 298:167-169. https://doi.org/10.1038/298167a0\u003c/li\u003e\n\u003cli\u003eCapitani G, Biase DD, Gut H, et al (2005) Structural model of human GAD65: prediction and interpretation of biochemical and immunogenic features. Proteins 59:7-14. https://doi.org/10.1002/prot.20372\u003c/li\u003e\n\u003cli\u003eKim YK, Sussel L, Davidson HW (2021) Inherent Beta Cell Dysfunction Contributes to Autoimmune Susceptibility. \u003cem\u003eBiomolecules \u003c/em\u003e11:512-522. https://doi.org/3390/biom11040512\u003c/li\u003e\n\u003cli\u003eBjork E,\u0026nbsp;Kampe O,\u0026nbsp;Karlsson FA (1992) Glucose regulation of the autoantigen GAD65 in human pancreatic islets. J Clin Endocrinol Metab 75:1574-1576.https://doi.org/10.1210/jcem.75.6.1464667\u003c/li\u003e\n\u003cli\u003eLiu H, Li S, Zhang Y (2009) Dynamic regulation of glutamic acid decarboxylase 65 gene expression in rat testis. Acta Biochim Biophys Sin (Shanghai) 41:545-553. https://doi.org/10.1093/abbs/gmp043\u003c/li\u003e\n\u003cli\u003eSingh S, Raina V,\u0026nbsp;Chaval, PL, et al (2012) Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment. BMC Mol Biol 14:13-28. https://doi.org/10.1186/1471-2199-13-28\u003c/li\u003e\n\u003cli\u003eKajita Y, Mushiake H (2021) Heterogeneous GAD65 Expression in Subtypes of GABAergic Neurons Across Layers of the Cerebral Cortex and Hippocampus. Front Behav Neurosci 15:750869-750883. https://doi.org/10.3389/fnbeh.2021.750869\u003c/li\u003e\n\u003cli\u003eBattelli MG,\u0026nbsp;Polito L,\u0026nbsp;Bortolotti M, et al (2016) Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a Detoxifying Enzyme. Curr Med Chem 23:4027-4036. https://doi.org/10.2174/0929867323666160725091915\u003c/li\u003e\n\u003cli\u003ePolito L,Bortolotti M,\u0026nbsp;Battelli MG, et al (2021) Xanthine oxidoreductase: A leading actor in cardiovascular disease drama. Redox Biol 48:102195-102205. https://doi.org/10.1016/j.redox.2021.102195\u003c/li\u003e\n\u003cli\u003eCheh C, Lu JM, et al (2016) Hyperuricemia-Related Diseases and Xanthine Oxidoreductase (XOR) Inhibitors: An Overview. Med Sci Monit 22:2501-2512. https://doi.org/10.12659/msm.899852\u003c/li\u003e\n\u003cli\u003eIchida K,\u0026nbsp;Amaya Y,\u0026nbsp;Okamoto K, et al (2012) Mutations Associated with Functional Disorder of Xanthine Oxidoreductase and Hereditary Xanthinuria in Humans. \u003cem\u003eInt J Mol Sci \u003c/em\u003e13:15475-15495. https://doi.org/10.3390/ijms131115475\u003c/li\u003e\n\u003cli\u003eMorgan MJ, Liu ZG (2011) Crosstalk of reactive oxygen species and NF-\u0026kappa;B signaling. Cell Res 21:103-115.https://doi.org/10.1038/cr.2010.178\u003c/li\u003e\n\u003cli\u003eBattelli MG, Bortolotti M, Bolognesi A, et al (2020) Pro-Aging Effects of Xanthine Oxidoreductase Products. Antioxidants (Basel) 9:839-855. https://doi.org/10.3390/antiox9090839\u003c/li\u003e\n\u003cli\u003eSunagawa S, Shirakura T, Hokama N, et al (2019) Activityof\u0026nbsp;xanthine\u0026nbsp;oxidase\u0026nbsp;in plasma correlates with indices of insulin resistance and liver dysfunction in patients with type 2 diabetes mellitus\u0026nbsp;and metabolic syndrome: A pilot exploratory study. J Diabetes Investig 10:94-103. https://doi.org/10.1111/jdi.12870\u003c/li\u003e\n\u003cli\u003eSlobodnick A., Toprover M, Greenberg J, Crittenden DB, et al (2020) Allopurinol use and\u0026nbsp;type2\u0026nbsp;diabetes\u0026nbsp;incidence among patients with gout: A VA retrospective cohort study. Medicine (Baltimore) 99:e21675-e21682. https://doi.org/10.1097/MD.0000000000021675\u003c/li\u003e\n\u003cli\u003eAl-Nahdi AMT, John A, Raza H (2017) Elucidation of Molecular Mechanisms of Streptozotocin-Induced Oxidative Stress, Apoptosis, and Mitochondrial Dysfunction in Rin-5F Pancreatic\u0026nbsp;\u003cem\u003e\u0026beta;\u003c/em\u003e-Cells. Oxid Med Cell Longev 2017:7054272-7054287. https://doi.org/10.1155/2017/7054272\u003c/li\u003e\n\u003cli\u003eSzkudelski T. The Mechanism of Alloxan and Streptozotocin Action in B Cells of the Rat Pancreas. Physiol Res, 2001,50(6):537-546.\u003c/li\u003e\n\u003cli\u003eVan-Suylichem PT, Wolters GH, van Schilfgaarde R (1990) The efficacy of density gradients for islet purification: a comparison of seven density gradients, Transpl Int 3:156-161. https://doi.org/10.1007/BF00355463\u003c/li\u003e\n\u003cli\u003eLaporte C,\u0026nbsp;Tubbs E,\u0026nbsp;Cristante J, et al (2019) Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin. Stem Cell Res Ther 10:85-97. https://doi.org/10.1186/s13287-019-1190-4\u003c/li\u003e\n\u003cli\u003eGhasemi A,Jeddi S (2023) Streptozotocin as a tool for induction of rat models of diabetes: a practical guide. EXCLI J 22:274-294. https://doi.org/10.17179/excli2022-5720\u003c/li\u003e\n\u003cli\u003eNicholas SA, Bubnov VV, Yasinska IM, et al (2011) Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1beta.Cell Mol Life Sci 68:151-158. https://doi.org/10.1007/s00018-010-0450-3\u003c/li\u003e\n\u003cli\u003eChung GC,\u0026nbsp;Hyosang L,\u0026nbsp;Sung BL (2018) Mechanisms of protein toxicity in neurodegenerative diseases. Cell Mol Life Sci 75:3159-3180. https://doi.org/10.1007/s00018-018-2854-4\u003c/li\u003e\n\u003cli\u003eBortolotti M,\u0026nbsp;Polito L,\u0026nbsp;Battelli MG, et al (2021) Xanthine oxidoreductase: One enzyme for multiple physiological tasks. Redox Biol 41:101882-101888. https://doi.org/10.1016/j.redox.2021.101882\u003c/li\u003e\n\u003cli\u003eBattelli MG,Polito L, Bortolotti M, et al (2016) Xanthine oxidoreductase in cancer: more than a\u0026nbsp;differentiation marker. Cancer Med 5:546-557. https://doi.org/10.1002/cam4.601\u003c/li\u003e\n\u003cli\u003eBravard A, Bonnard C, Durand A, et al (2011) Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice.\u0026nbsp;Am J Physiol Endocrinol Metab 300: E581-E91.https://doi.org/10.1152/ajpendo.00455.2010\u003c/li\u003e\n\u003cli\u003eKim SM, Choi YW, Seok HY, et al (2012) Reducing serum uric acid attenuates TGF-beta1-induced profibrogenic progression in type 2 diabetic nephropathy.\u0026nbsp;Nephron Exp Nephrol 121:e109-121.https://doi.org/10.1159/000343567\u003c/li\u003e\n\u003cli\u003eHernandez-Hernandez ME, Torres-Rasgado E, Pulido-Perez P, et al (2022) Disordered Glucose Levels Are Associated with Xanthine Oxidase Activity in Overweight Type 2 Diabetic Women. Int J Mol Sci 23:11177-11188. https://doi.org/10.3390/ijms231911177\u003c/li\u003e\n\u003cli\u003eHasan M,\u0026nbsp;Fariha KA,\u0026nbsp;Barman Z,\u0026nbsp;et al (2022) Assessment of the relationship between serum xanthine oxidase levels and type 2 diabetes: a cross-sectional study. Sci Rep 12:20816-20825. https://doi.org/10.1038/s41598-022-25413-w\u003c/li\u003e\n\u003cli\u003eSlobodnick A,\u0026nbsp;Toprover M, Greenberg J,\u0026nbsp; et al (2020) Allopurinol use and type 2 diabetes incidence among patients with gout. Medicine (Baltimore) 99: e21675-e21682. https://doi.org/10.1097/MD.0000000000021675\u003c/li\u003e\n\u003cli\u003eYang Y, Zhao J, Qiu J, et al (2018) Xanthine oxidaseinhibitor\u0026nbsp;allopurinol\u0026nbsp;prevents\u0026nbsp;oxidative\u0026nbsp;stress‐mediated\u0026nbsp;atrial remodeling\u0026nbsp;in alloxan‐induced\u0026nbsp;diabetes mellitus\u0026nbsp;rabbits. J Am Heart Assoc 7: e008807-e00880722. https://doi.org/10.1161/JAHA.118.008807\u003c/li\u003e\n\u003cli\u003eDesco M, Asensi M, Marquez R, et al (2002) Xanthine Oxidase Is Involved in Free Radical Production in Type 1 Diabetes. \u003cem\u003eDiabetes \u003c/em\u003e51:1118-1124. https://doi.org/10.2337/diabetes.51.4.1118\u003c/li\u003e\n\u003cli\u003eMiric DJ,\u003csup\u003e ,\u0026nbsp;\u0026nbsp;\u003c/sup\u003eKisic BM, Filipovic-Danic S, \u003csup\u003e\u0026nbsp;\u003c/sup\u003eet al (2016) Xanthine Oxidase Activity in Type 2 Diabetes Mellitus Patients with and without Diabetic Peripheral Neuropathy. J Diabetes Res 2016:4370490-4370497. https://doi.org/10.1155/2016/4370490\u003c/li\u003e\n\u003cli\u003eOkuyama T,Shirakawa J,\u0026nbsp;Nakamura T,\u0026nbsp; et al (2021) Association of the plasma xanthine oxidoreductase activity with the metabolic parameters and vascular complications in patients with type 2 diabetes. Sci Rep 11:3768-3781. https://doi.org/10.1038/s41598-021-83234-9\u003c/li\u003e\n\u003cli\u003eAliciguzel Y,\u0026nbsp;Ozen I,\u0026nbsp;Aslan M,\u0026nbsp;et al (2003) Activities of xanthine oxidoreductase and antioxidant enzymes in different tissues of diabetic rats. J Lab Clin Med 142:172-177. https://doi.org/10.1016/S0022-2143(03)00110-0\u003c/li\u003e\n\u003cli\u003eHao W, Palmer JP (1995) The effect of cytokines on expression of glutamic acid decarboxylase-65 in cultured islets. Autoimmunity 22:209-218. https://doi.org/10.1089/jir.1995.15.1075\u003c/li\u003e\n\u003cli\u003eGu Y,\u0026nbsp;Merriman C,Guo Z,\u0026nbsp;et al (2021) Novel autoantibodies to the \u0026beta;-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes. J Autoimmun 122:102677-102697. https://doi.org/10.1016/j.jaut.2021.102677\u003c/li\u003e\n\u003cli\u003eRussell MA, Morgan NG (2014) The impact of anti-inflammatory cytokines on the pancreatic \u0026beta;-cell, Islets 6: e950547-e950557. https://doi.org/10.4161/19382014.2014.950547\u003c/li\u003e\n\u003cli\u003eFukahori M,Ichimori K,\u0026nbsp;Ishida\u0026nbsp;H, et al (1994) Nitric oxide reversibly suppresses xanthine oxidase activity. Free Radic Res 21:203-212. https://doi.org/10.3109/10715769409056572\u003c/li\u003e\n\u003cli\u003eIchimori K, Fukahori M, Nakazawa H, et al (1999) Inhibition of Xanthine Oxidase and Xanthine Dehydrogenase by Nitric Oxide. J Biol Chem 274:7763-7768. https://doi.org/10.1074/jbc.274.12.7763\u003c/li\u003e\n\u003cli\u003eSteinbrenner H,Nguyen T,\u0026nbsp;Wohlrab U,\u0026nbsp;et al (2002) Effect of Proinflammatory Cytokines on Gene Expression of the Diabetes-Associated Autoantigen IA-2 in INS-1 Cells. Endocrinology 143:3839-3845. https://doi.org/10.1210/en.2002-220583\u003c/li\u003e\n\u003cli\u003eMally MI, Cirulli V, Otonkoski T, et al (1996) Ontogeny and Tissue Distribution of Human GAD Expression. \u003cem\u003eDiabetes \u003c/em\u003e45:496-501. https://doi.org/10.2337/diab.45.4.496\u003c/li\u003e\n\u003cli\u003ePhelps EA,Cianciaruso C,\u0026nbsp;Michael IP,\u0026nbsp;et al (2016) Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity. Diabetes 65:2686-2699. https://doi.org/10.2337/db16-0180\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eXO activity in rat islets (n\u0026thinsp;=\u0026thinsp;500 islets / group) treated with 1.0mM STZ at different time points and effect of Allopurinol.\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.0 mM STZ\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eXO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) in islets\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% increase in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% decrease in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003ePanel 1\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e6 hr\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003e*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e27.5%\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e12 hr\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003e*$\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e137.75%\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e24 hr\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e3.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003e*$\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e303.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003ePanel 2\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eAllopurinol (500 \u0026micro;M)\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.042\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0051\u003csup\u003e*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e96.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eAllopurinol (500 \u0026micro;M)\u0026thinsp;+\u0026thinsp;1.0 mM STZ for 24 hrs\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.026\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0065\u003csup\u003e*$\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e97.34%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" style=\"width: 703.759px;\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eBlood glucose levels at different days in female Wistar rats (n\u0026thinsp;=\u0026thinsp;5/group) injected with 40 mg/kg body weight of STZ.\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth style=\"width: 160px;\" rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 64px;\" rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eTreatment\u003c/div\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 431.759px;\" colspan=\"5\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eBlood Glucose Levels (mg/dl)\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth style=\"width: 77px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 0\u003c/div\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 91px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 1\u003c/div\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 84px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 6\u003c/div\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 91px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 12\u003c/div\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 73px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 18\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 160px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e100.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e103.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e112\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e109\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 160px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eSTZ (40 mg/kg body weight)\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e109.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.18\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e220.66\u0026thinsp;\u0026plusmn;\u0026thinsp;15.34\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e280.62\u0026thinsp;\u0026plusmn;\u0026thinsp;40.9\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e333.14\u0026thinsp;\u0026plusmn;\u0026thinsp;62.19\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e400\u0026thinsp;\u0026plusmn;\u0026thinsp;59.42\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eXO activity in pancreas of female Wistar rats (n\u0026thinsp;=\u0026thinsp;5/group) at different days injected with 40 mg/kg body weight of STZ.\u003c/span\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eSTZ\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(40 mg/kg body weight)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eXO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) in pancreas\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% decrease in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 1\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003e*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e32.3%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 6\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003e*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e55.1%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 12\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003e*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e72.8%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 18\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e88.6%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eXO activity in serum of female Wistar rats (n\u0026thinsp;=\u0026thinsp;5/group) at different days injected with 40 mg/kg body weight of STZ.\u003c/span\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eSTZ\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(40 mg/kg body weight)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eXO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) in serum\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% increase in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 1\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e81.5%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 6\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e9.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e198.7%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 12\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e12.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e919.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eDay 18\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1497.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eXO activity in rat islets (n\u0026thinsp;=\u0026thinsp;500/group) treated with IL-1\u0026beta;, TNF-\u0026alpha; and IFN-\u0026gamma; cytokines.\u003c/span\u003e Rat islets were treated with IL-1\u0026beta; (1 ng/ml), TNF-\u0026alpha; (200 units/ml), IFN-\u0026gamma; (500 units/ml) and cocktail cytokines for a period of 48 hours. XO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) was analyzed as described in materials and methods. Results are shown as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. of three independent experiments; \u003csup\u003e#\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs control. Controls were normalised as 100%. XOD activities in other samples were expressed as % control.\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eXO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) in islets\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% Increase in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% Decrease in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eIL-1 \u0026beta;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e28.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eIFN-\u0026gamma;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e25.5%\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eTNF-\u0026alpha;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e18.4%\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eIL-1 \u0026beta; / IFN-\u0026gamma; / TNF-\u0026alpha;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.245\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e75.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\u0026nbsp;\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eNitrite concentration in medium containing islets (n\u0026thinsp;=\u0026thinsp;50/group) treated with IL-1\u0026beta;, TNF-\u0026alpha; and IFN-\u0026gamma; cytokines.\u003c/span\u003e Rat islets were treated with IL-1\u0026beta; (1 ng/ml), TNF-\u0026alpha; (200 units/ml), IFN-\u0026gamma; (500 units/ml) and cocktail cytokines for a period of 48 hours. Nitrite concentration (pmol/islet) was analyzed as described in materials and methods. Results are shown as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. of three independent experiments; \u003csup\u003e#\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs control. Controls were normalised as 100%. XOD activities in other samples were expressed as % control.\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eMedium nitrite (pmol/islet)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% Increase in nitrite levels\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eIL-1 \u0026beta;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e32%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eIFN-\u0026gamma;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e7.7%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eTNF-\u0026alpha;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;.35\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e52%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eIL-1 \u0026beta; / IFN-\u0026gamma; / TNF-\u0026alpha;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e392.3%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eXO activity in rat islets (n\u0026thinsp;=\u0026thinsp;500/group) treated with cocktail of cytokines (IL-1\u0026beta;, TNF-\u0026alpha; and IFN-\u0026gamma;) and effect of 1400W (iNOS inhibitor).\u003c/span\u003e Rat islets were treated with cocktail of cytokines (IL-1\u0026beta;, 1 ng/ml; TNF-\u0026alpha;, 200 units/ml and IFN-\u0026gamma;, 500 units/ml) in presence or absence of 1400W; 80 \u0026micro;M) for a period of 48 hours. XO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) was analyzed as described in materials and methods. Results are shown as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. of three independent experiments; \u003csup\u003e#\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs control; \u003csup\u003e*\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs 1400W. Controls were normalized as 100%. XOD activities in other samples were expressed as % control.\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eXO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) in islets\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% Increase in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% Decrease in XO activity\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1400W\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e12.2%\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eIL-1 \u0026beta; / IFN-\u0026gamma; / TNF-\u0026alpha;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.245\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003e#*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e75.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1400W\u0026thinsp;+\u0026thinsp;IL-1 \u0026beta; / IFN-\u0026gamma; / TNF-\u0026alpha;\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003csup\u003e#*\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1359.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\u0026nbsp;\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eXO activity in fetal, neonatal and adult pancreas during ontogeny in female Wistar rats. XO activity\u003c/span\u003e (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) was measured in fetal (24 gestation weeks), neonatal (6\u0026ndash;24 days\u0026rsquo; post birth) and adult pancreas as described in materials and methods. Results are shown as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. of three independent experiments; number of animals in each group was 5 (n\u0026thinsp;=\u0026thinsp;5 / group); \u003csup\u003e#\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs fetal pancreas. Controls were normalised as 100%. XOD activities in other samples were expressed as % control.\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eXO activity (nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min mg protein) in pancreas\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e% decrease in XO activity (compared to adult pancreas\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eFetal 21.5d\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e6.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eNeonatal 6d\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e8.5%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eNeonatal 12d\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e48.25%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eNeonatal 24d\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e63.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eAdult pancreas\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.783\u003csup\u003e#\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e73.0%\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"GAD65, xanthine oxidase, p53, Allopurinol, Type-1 diabetes","lastPublishedDoi":"10.21203/rs.3.rs-3829266/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3829266/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eIt is widely presumed that aberrant expression of GAD65 can sensitize beta cells to autoimmunity and moreover, several studies indicate Xanthine Oxidase (XO) plays significant role in Type-1 diabetes (T1D) pathology. The present study aimed to investigate whether XO modulates GAD65 protein expression in rat islets/pancreas in presence or absence of STZ.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and results: \u003c/strong\u003eFemale Wistar rats were used for pancreatic islet isolation and T1D STZ model by injecting single dose of STZ (40 mg/kg body weight). GAD65 and XO protein expression was studied by immunoblot in islets/pancreas and STZ/T1D cytokines (such as IL-1 beta, TNF-α and IFN-γ)/XO siRNA/Allopurinol (XO inhibitor)/1400W (iNOS inhibitor) were used in treatment groups. Enzymatic assay was done to measure XO activity in islets/serum/pancreas and iNOS activity in culture supernatant. STZ led to a significant increase in GAD65 expression and XO activity in islets which was reduced in presence of\u003cstrong\u003e \u003c/strong\u003eAllopurinol and XO siRNA.\u003cstrong\u003e \u003c/strong\u003eIn pancreas of T1D rat model, expression of GAD65 decreased and XO increased; XO activity decreased; whereas in sera XO activity increased. Treatment of islets with T1D cytokines revealed that cytokine cocktail declined GAD65 protein levels and XO activity and intervention with iNOS inhibitor reversed the effect. During ontogeny of pancreas GAD65 expression was more in the fetal/infantile pancreas compared to adult pancreas and Allopurinol challenged pregnant rats confirmed that GAD65 expression is dependent on XO activity. \u003cstrong\u003eConclusion: \u003c/strong\u003eThis study for the first time clearly demonstrates that XO is positive regulator of GAD65 expression in rat islets and STZ-T1D model.\u003c/p\u003e","manuscriptTitle":"Xanthine oxidase modulates Glutamic Acid Decarboxylase 65 isoform (GAD65) protein expression in rat pancreatic islets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 05:49:47","doi":"10.21203/rs.3.rs-3829266/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91f409ea-33f1-44e3-aa37-03d1eae55044","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-19T15:54:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 05:49:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3829266","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3829266","identity":"rs-3829266","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}