Streptozotocin-induced Type-1 Diabetes Research Articles

Blockade of the colony-stimulating factor-1 receptor reverses bone loss in osteoporosis mouse models.

Mice lacking either colony-stimulating factor-1 (CSF-1) or its receptor, CSF-1R, display osteopetrosis. Accordingly, genetic deletion or pharmacological blockade of CSF-1 prevents the bone loss associated with estrogen deficiency. However, the role of CSF-1R in osteoporosis models of type-1 diabetes (T1D) and ovariectomy (OVX) has not been examined. Thus, we evaluated whether CSF-1R blockade would relieve the bone loss in a model of primary osteoporosis (female mice with OVX) and a model of secondary osteoporosis (female with T1D) using micro-computed tomography. Female ICR mice at 10weeks underwent OVX or received five daily administrations of streptozotocin (ip, 50mg/kg) to induce T1D. Four weeks after OVX and 14weeks after first injection of streptozotocin, mice received an anti-CSF-1R (2G2) antibody (10mg/kg, ip; once/week for 6weeks) or vehicle. At the last day of antibody administration, mice were sacrificed and femur and tibia were harvested for micro-computed tomographyanalysis. Mice with OVX had a significant loss of trabecular bone at the distal femoral and proximal tibial metaphysis. Chronic treatment with anti-CSF-1R significantly reversed the trabecular bone loss at these anatomical sites. Streptozotocin-induced T1D resulted in significant loss of trabecular bone at the femoral neck and cortical bone at the femoral mid-diaphysis. Chronic treatment with anti-CSF-1R antibody significantly reversed the bone loss observed in mice with T1D. Our results demonstrate that blockade of CSF-1R signaling reverses bone loss in two different mouse models of osteoporosis.

Histological evidence of chitosan-encapsulated curcumin suppresses heart and kidney damages on streptozotocin-induced type-1 diabetes in mice model

High blood glucose in diabetic patients often causes cardiovascular diseases (CVDs) that threats to human life. Curcumin (Cur) is known as an antioxidant agent, possesses anti-inflammatory activity, and prevents CVDs. However, the clinical application of curcumin was limited due to its low bioavailability. This study aimed to investigate the ameliorative effects of chitosan-encapsulated curcumin (CEC) on heart and kidney damages in streptozotocin-induced type-1 diabetes C57BL/6 mice model. The results showed that Cur- and CEC-treatments downregulated the blood sugar and total cholesterol level as well as enhanced insulin secretion. However, blood pressure, triglycerides content, and very low-density lipoprotein-cholesterol content were not changed. Histochemistry analysis revealed that both curcumin and chitosan-encapsulated curcumin ameliorated cell hypertrophy and nucleus enlargement in the left ventricular of heart and reduced fibrosis in the kidney, especially after the chitosan-encapsulated curcumin treatment. Our study suggested that chitosan can effectively enhance the protective effect of curcumin on the heart and kidney damages in type-1 diabetes mice model.

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice.

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1-3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.

Leukotriene-B4 modulates macrophage metabolism and fat loss in type 1 diabetic mice.

Serum levels of leukotriene-B4 (LTB4) are increased in type 1 diabetes (T1D) and it mediates systemic inflammation and macrophage reprogramming associated with this condition. Herein, we investigated the involvement of LTB4 in adiposity loss, hyperlipidemia, and changes in macrophage metabolism in a mouse model of streptozotocin-induced T1D. LTB4 receptor (BLT1) antagonist u75302 was employed to block LTB4 effects. As expected, hypoinsulinemia in T1D was associated with hyperglycemia, low levels of glucagon, hyperlipidemia, significant body fat loss, and increased white adipose tissue expression of Fgf21, a marker for lipolysis. With the exception of hyperglycemia and hypoglucagonemia, blockade of LTB4 signaling reverted these parameters in T1D mice. Along with hyperlipidemia, macrophages from T1D mice exhibited higher lipid uptake and accumulation. These cells also had enhanced glycolysis and oxidative metabolism and these parameters were dependent on the mitochondrial uncoupling respiration, as evidenced by elevated expression of oxidation markers carnitine palmitoyltransferase and uncoupling protein 1. Interestingly, all these parameters were at least partially reverted in T1D mice treated with u75302. Altogether, these findings suggest that in T1D mice LTB4/BLT1 is involved in the fat loss, hyperlipidemia, and increased macrophage lipid uptake and metabolism with an important involvement of mitochondrial uncoupling activity. These previously unrecognized LTB4/BLT1 functions may be explored in future to therapeutically alleviate severity of hyperlipidemia and systemic inflammation in T1D.

396-P: SGLT2 Inhibition Suppresses Glucagon Response to Hypoglycemia in Male but Not Female Rats with Streptozotocin-Induced Type 1 Diabetes

Sodium glucose co-transporter 2 inhibitors (SGLT2i) show promise as an add-on to insulin therapy in patients with type 1 diabetes (T1D), however their effect on glucagon secretion in T1D is not yet known. Increases in glucagon have the potential to protect against hypoglycemia that may develop from insulin overtreatment or exercise. We investigated whether 8 days of SGLT2i treatment (10mg/kg empagliflozin, 2x/day) altered plasma glucagon concentrations in response to voluntary running wheel activity and insulin-induced hypoglycemia in male and female Wistar rats with streptozotocin-induced T1D. SGLT2 inhibition did not alter basal or post-exercise glucagon concentrations and did not affect exercise-associated changes in blood glucose (BG) levels. Additionally, SGLT2 inhibition did not alter voluntary activity levels. However, in males, SGLT2 inhibition lowered plasma glucagon response during an insulin tolerance test (+22.7±30.8 pg/mL vs. +40.4±48.6 pg/mL, p=0.02) and decreased the time taken to reach hypoglycemia (30±13.4 min vs. 63.8±17.2 min, p<0.0001), as compared to placebo. We also observed novel sex differences in the response of BG and glucagon concentrations to voluntary exercise. In female rats alone, voluntary exercise decreased BG concentrations (-2.9±2.6 mM vs. +1.7±1.8 mM, p=0.0003) and led to lower post-exercise glucagon concentrations (37.3±47.9 pg/mL vs. 117.9±86.0 pg/mL, p=0.01), as compared to inactive animals. On average, voluntary activity levels were higher in females than in males (539.2±199.4 m vs. 308.4±215.6 m, p=0.02), however running distances were not correlated with changes in BG levels during exercise (r2=0.02). Our finding of increased hypoglycemia risk with SGLT2i has concerning implications for its use as an adjunct to insulin therapy in T1D. The novel sex differences that we observed should be investigated in individuals with T1D to determine their human applicability and potential mechanisms. Disclosure C.A. Greenberg: None. E.R. Mandel: None. A.M. Pasieka: None. M. Jahangiriesmaili: None. J. Tremblay: None. M. Riddell: Advisory Panel; Self; Xeris Pharmaceuticals, Inc. Research Support; Self; Dexcom, Inc. Speaker's Bureau; Self; Insulet Corporation, Medtronic MiniMed, Inc. Stock/Shareholder; Self; Zucara Therapeutics Inc.

Sodium Butyrate Ameliorates Streptozotocin-Induced Type 1 Diabetes in Mice by Inhibiting the HMGB1 Expression.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the immune cell-mediated progressive destruction of pancreatic β-cells. High-mobility group box 1 protein (HMGB1) has been recognized as a potential immune mediator to enhance the development of T1D. So we speculated that HMGB1 inhibitors could have anti-diabetic effect. Sodium butyrate is a short fatty acid derivative possessing anti-inflammatory activity by inhibiting HMGB1. In the current study, we evaluated the effects of sodium butyrate in streptozotocin (STZ)-induced T1D mice model. Diabetes was induced by multiple low-dose injections of STZ (40 mg/kg/day for 5 consecutive days), and then sodium butyrate (500 mg/kg/day) was administered by intraperitoneal injection for 7 consecutive days after STZ treatment. Blood glucose, incidence of diabetes, body weight, pancreatic histopathology, the amounts of CD4+T cell subsets, IL-1β level in serum and pancreatic expressions levels of HMGB1, and NF-κB p65 protein were analyzed. The results showed that sodium butyrate treatment decreased blood glucose and serum IL-1β, improved the islet morphology and decreased inflammatory cell infiltration, restored the unbalanced Th1/Th2 ratio, and down-regulated Th17 to normal level. In addition, sodium butyrate treatment can inhibit the pancreatic HMGB1 and NF-κB p65 protein expression. Therefore, we proposed that sodium butyrate should ameliorate STZ-induced T1D by down-regulating NF-κB mediated inflammatory signal pathway through inhibiting HMGB1.

Evaluation of anti-diabetic attributes of Lactobacillus rhamnosus MTCC: 5957, Lactobacillus rhamnosus MTCC: 5897 and Lactobacillus fermentum MTCC: 5898 in streptozotocin induced diabetic rats

Interest in probiotics has grown significantly in the last decades due to their reported nutritional and health promoting effects. The aim of this study is to investigate the therapeutic potential of probiotic fermented milk (PFM) prepared using three different probiotic strains i.e. Lactobacillus rhamnosus MTCC: 5957, Lactobacillus rhamnosus MTCC: 5897 and Lactobacillus fermentum MTCC: 5898; independently or in combination, for treating streptozotocin induced type-1 diabetes in male Wistar rats. Diabetic rats were fed with PFM preparations for 6 weeks and then analyzed for the various biochemical parameters associated. The results indicated that feeding of PFM significantly improved glucose metabolism (fasting blood glucose, glycated hemoglobin, serum insulin), serum inflammation status (tumor necrosis factor-α, and serum interleukin-6), oxidative stress (thiobarbituric acid reactive substance, catalase, superoxide dismutase and glutathione peroxidase activities in liver and kidney), serum lipid profile (total cholesterol, low density lipoprotein-cholesterol, very low density lipoprotein-cholesterol, triglycerides) in diabetic rats. In addition, feeding of PFM has significantly reduced mRNA expression of pepck and g6pase genes that code the key enzymes of gluconeogenesis pathway. The results of this study showed that daily consumption of PFM can be effective in combating of type −1 diabetes and its complications.

Attenuation of type-1 diabetes-induced cardiovascular dysfunctions by direct thrombin inhibitor in rats: a mechanistic study.

Chronic diabetes is associated with ventricular dysfunctions in the absence of hypertension and coronary artery diseases. This condition is termed as diabetic cardiomyopathy (DCM). There is no favourable treatment available for the management of diabetic cardiomyopathy. Recent studies have reported increase in circulating thrombin level among diabetic patients which is responsible for hypercoagulability of blood. Thrombin induces inflammation and fibrosis, and enhances cardiac cell growth and contractility in vitro. In this study, we have investigated the effects of argatroban; a direct thrombin inhibitor against DCM in streptozotocin-induced type-1 diabetes. Diabetes was induced by single dose of streptozotocin (STZ; 50mg/kg, i.p.) in male Sprague-Dawley rats. After 4 weeks of diabetes induction, the animals were treated with argatroban (0.3 and 1mg/kg, i.p. daily) for the next 4 weeks. The effect of argatroban was evaluated against diabetes-associated cardiac dysfunction, structural alteration and protein expression. STZ-induced diabetic rats exhibited significant decline in left ventricular functions. Four weeks of treatments with argatroban significantly improved ventricular functions without affecting heart rate. Further, it also protected heart against structural changes induced by diabetes as shown by reduction in fibrosis, hypertrophy and apoptosis. The improvement in cardiac functions and structural changes was associated with significant reduction in left ventricular expression of thrombin receptor also termed as protease-activated receptor-1 or PAR1, p-AKT (ser-473), p-50 NFκB and caspase-3 proteins. This study demonstrates beneficial effects of argatroban via improvement in cardiac functions and structural changes in STZ-induced DCM. These effects may be attributed through reduction in cardiac inflammation, fibrosis and apoptosis.

Saccharomyces Boulardii Attenuates Autonomic Cardiovascular Dysfunction and Modulates Inflammatory Cytokines in Diabetic Mice

The effects of probiotics on blood glucose and cardiovascular complications induced by diabetes have been inconsistent. Therefore, the aim was to assess the effect of Saccharomyces boulardii (Sb) on metabolic and cardiovascular dysfunction in streptozotocin (STZ)-induced type-1-diabetes (STZ-T1D). Male C57BL/6 mice were randomized into: control (C), diabetes (D), control+Sb (CSb) and diabetes+Sb (DSb) (n=6-12/group). Mice were made diabetic with STZ (150mg/kg) and daily treated with Sb (0,5x108 CFU) by oral gavage. After 8 weeks of treatment, blood pressure (BP) and heart rate (HR) signals obtained from conscious mice were recorded. Spectral analysis of systolic BP (BPV) and HR variabilities (HRV) was performed to estimate autonomic modulation to the heart and vessels. Plasma was collected and heart was excised for biochemical analyses. D mice presented metabolic dysfunction, with increased blood glucose, triglycerides and cholesterol, as compared with C animals. On the other hand, treatment with Sb reduced blood glucose (DSb=238±39 vs. D=378±17 mg/dl) and triglycerides (DSb=121±11 vs. D=169±10 mg/dl), with no effect on cholesterol levels (DSb=119±4 vs. D=106±9 mg/dl). Probiotic treatment also reduced cardiac IL-6 (DSb=80±8 vs. D=113±13 pg/mg) and increased IL-10 (DSb=251±16 vs. D=185±11 pg/mg), leading to a beneficial impact on heart nitric oxide levels (DSb=12.4±1.3 vs. D=7.6±0.9 pg/mg). D mice presented a significant reduction in HRV, BPV and also in low-frequency component of systolic BP (SBP). Treatment of D mice with Sb increased HRV (DSb=20±0.9 vs. D=10±0.9 ms²), BPV (DSb=8±0.4 vs. D=5±0.3 mmHg²) and low-frequency component of SBP (DSb=98±8 vs. D=55±8 mmHg²). Our study shows, for the first time, that administration of Sb is strongly associated with glycemic control, cardiovascular protection and improvement of inflammatory profile in STZ-T1D. Disclosure A.B.P. Brandão: None. I.C. de Abreu: None. F. Aimbire: None. E.M. Higa: None. A. Casali: None. F.G. Ferreira: None. R.M. Albuquerque: None. L.B. Santos: None. M.C. Irigoyen: None. K.R. Casali: None. T.S. Cunha: None.

Abstract 615: Absence of RIP3 Reduces Atherosclerotic Progression in Type 1 Diabetes

Introduction: Patients suffering from Type 1 diabetes (T1D) have increased risk of developing atherosclerosis. T1D is a chronic autoimmune metabolic disease characterized by a deficiency in insulin secretion and hyperglycemia, promoting oxidative stress and low-grade inflammation in part via advanced glycation end products (AGE). Recently, we and others showed that necroptosis, a newly defined pro-inflammatory programmed cell death, drives advanced atherosclerosis. Given that diabetes accelerates inflammation and atherosclerosis, we hypothesized that hyperglycemia may exacerbate macrophage necroptosis to drive atherosclerotic lesion formation. Methods/Results: Male Ldlr-/- mice were lethally irradiated, transplanted with bone-marrow from WT or Rip3 -/- mice and then administered streptozotocin (STZ) or vehicle daily for 5 days to induce T1D, prior to being fed a western diet for 6 wks. As expected, STZ-treated WT BMT Ldlr -/- mice had a marked increased in atherosclerotic aortic lesion area compared to non-diabetic mice (1.08%±0.30 vs 2.13%±0.45). which was blunted in Rip3 -/- BMT Ldr -/- mice (arch: 49% reduction, sinus: 54% reduction, p<0.05). Bone marrow derived macrophages (BMDMs) cultured in high glucose (HG) increased cell death in response to necroptotic stimuli (oxidized LDL ± caspase inhibitor zVAD) relative to those cultured in low glucose (LG). Further, the addition of recombinant s100A8/9, a soluble AGE, markedly amplified oxLDL-induced death in HG cultured BMDMs with minimal effects on LG cultured BMDMs, suggesting that pro-inflammatory AGEs released in diabetes can augment macrophage cell death. Further, the increased cell death induced by oxLDL+S100A8/9 was inhibited by a necroptotic inhibitor, Nec-1, or in Rip3 -/- BMDMs cultured in HG. Mechanistically, treatment of BMDMs cultured in HG with oxLDL+s100A8/9 increased the expression of Rip3 and Mlkl (~5-fold and ~2-fold respectively), which may drive necroptotic cell death during hyperglycemia. Conclusion: Deletion of Rip3 markedly halts atherosclerotic progression in a STZ-induced T1D mouse model by inhibiting macrophage necroptosis induced by both atherogenic ligands (oxLDL) and soluble AGE products (s100A8/9).

Honokiol Ameliorates Myocardial Ischemia/Reperfusion Injury in Type 1 Diabetic Rats by Reducing Oxidative Stress and Apoptosis through Activating the SIRT1-Nrf2 Signaling Pathway.

Reducing oxidative stress is a crucial therapeutic strategy for ameliorating diabetic myocardial ischemia/reperfusion (MI/R) injury. Honokiol (HKL) acts as an effective cardioprotective agent for its strong antioxidative activity. However, its roles and underlying mechanisms against MI/R injury in type 1 diabetes (T1D) remain unknown. Since SIRT1 and Nrf2 are pivotal regulators in diabetes mellitus patients suffering from MI/R injury, we hypothesized that HKL ameliorates diabetic MI/R injury via the SIRT1-Nrf2 signaling pathway. Streptozotocin-induced T1D rats and high-glucose-treated H9c2 cells were exposed to HKL, with or without administration of the SIRT1 inhibitor EX527, SIRT1 siRNA, or Nrf2 siRNA, and then subjected to I/R operation. We found that HKL markedly improved the postischemic cardiac function, decreased the infarct size, reduced the myocardial apoptosis, and diminished the reactive oxygen species generation. Intriguingly, HKL remarkably activated SIRT1 signaling, enhanced Nrf2 nuclear translocation, increased antioxidative signaling, and decreased apoptotic signaling. However, these effects were largely abolished by EX527 or SIRT1 siRNA. Additionally, our cellular experiments showed that Nrf2 siRNA blunted the cytoprotective effects of HKL, without affecting SIRT1 expression and activity. Collectively, these novel findings indicate that HKL abates MI/R injury in T1D by ameliorating myocardial oxidative damage and apoptosis via the SIRT1-Nrf2 signaling pathway.

In vivo administration of urolithin A and B prevents the occurrence of cardiac dysfunction in streptozotocin-induced diabetic rats

BackgroundEmerging evidence suggests that specific (poly)phenols may constitute new preventative strategies to counteract cell oxidative stress and myocardial tissue inflammation, which have a key role in the patho-physiology of diabetic cardiomyopathy. In a rat model of early diabetes, we evaluated whether in vivo administration of urolithin A (UA) or urolithin B (UB), the main gut microbiota phenolic metabolites of ellagitannin-rich foods, can reduce diabetes-induced microenvironmental changes in myocardial tissue, preventing cardiac functional impairment.MethodsAdult Wistar rats with streptozotocin-induced type-1 diabetes (n = 29) were studied in comparison with 10 control animals. Diabetic rats were either untreated (n = 9) or subjected to daily i.p. injection of UA (n = 10) or UB (n = 10). After 3 weeks of hyperglycaemia, hemodynamics, cardiomyocyte contractile properties and calcium transients were measured to assess cardiac performance. The myocardial expression of the pro-inflammatory cytokine fractalkine and proteins involved in calcium dynamics (sarcoplasmic reticulum calcium ATPase, phospholamban and phosphorylated phospholamban) were evaluated by immunoblotting. Plasma, urine and tissue distribution of UA, UB and their phase II metabolites were determined.ResultsIn vivo urolithin treatment reduced by approximately 30% the myocardial expression of the pro-inflammatory cytokine fractalkine, preventing the early inflammatory response of cardiac cells to hyperglycaemia. The improvement in myocardial microenvironment had a functional counterpart, as documented by the increase in the maximal rate of ventricular pressure rise compared to diabetic group (+18% and +31% in UA and UB treated rats, respectively), and the parallel reduction in the isovolumic contraction time (−12%). In line with hemodynamic data, both urolithins induced a recovery of cardiomyocyte contractility and calcium dynamics, leading to a higher re-lengthening rate (+21%, on average), lower re-lengthening times (−56%), and a more efficient cytosolic calcium clearing (−32% in tau values). UB treatment also increased the velocity of shortening (+27%). Urolithin metabolites accumulated in the myocardium, with a higher concentration of UB and UB-sulphate, potentially explaining the slightly higher efficacy of UB administration.ConclusionsIn vivo urolithin administration may be able to prevent the initial inflammatory response of myocardial tissue to hyperglycaemia and the negative impact of the altered diabetic milieu on cardiac performance.

Pomegranate peel extract ameliorates autoimmunity in animal models of multiple sclerosis and type 1 diabetes

Pomegranate peel extract (PPE) was obtained by aqueous-ethanol extraction and was abundant in punicalin, punicalagin and ellagic acid. As these compounds are known to act against inflammation and oxidative stress in synergistic fashion, effects of PPE were tested in vitro on immune cells and in animal models of multiple sclerosis and type 1 diabetes (T1D). In vitro, PPE inhibited interleukin-17 (IL-17) production from gut-associated lymphoid tissue (GALT) cells. PPE also reduced production of IL-17 in activated T cells isolated from animals with experimental autoimmune encephalomyelitis (EAE). It efficiently down-regulated clinical symptoms of EAE in DA rats and of streptozotocin-induced T1D in C57BL/6 mice. The effect of PPE in T1D was mediated by inhibition of immune cell infiltration into pancreatic islets and through interference with IL-17 and IFN-γ production in GALT. Our results suggest that PPE has the potential to be used as phytopharmaceutical for certain autoimmune and chronic inflammatory disorders.

Inhibitors of Poly(ADP-Ribose)Polymerase-1 as Agents Providing Correction of Brain Dysfunctions Induced by Experimental Diabetes

The effects of 1,5-isoquinolinediol (IQD) and nicotinamide (NAm), inhibitors of poly-(ADP-ribose) polymerase-1 (PARP-1), on inflammatory processes and activation of PARP-1 under conditions of the development of experimental diabetic neuropathy, DN (a complication of streptozotocin-induced type-1 diabetes) in rats were studied. The content of IL-4 in blood serum in the case of DN was 50% higher, while that of monocyte-chemotactic protein-1 was 90% higher than those in the control. The content of gamma-interferon also increased, while the content of the granulocyte-macrophage colony-stimulating factor did not change. Against the background of activation of PARP-1 and a decrease in the content of the substrate of this enzyme nicotinamide adenine dinucleotide (NAD) in the brain, fragmentation of PARP-1 was intensified; an increase in the ratio of the contents of a 89 kDa fragment/intact enzyme molecules proved this fact. The mentioned two structurally dissimilar PARP-1 inhibitors partly or entirely normalized the above parameters under DN conditions. These results demonstrate that PARP-1 is one of the main functional targets in realization of the effects of IQD and NAm. At the same time, the spectrum of action of these inhibitors is wider. In particular, they affect the level of proinflammatory cytokines. The ability of the investigated PARP-1 inhibitors to prevent cell death in the brain by suppressing activation and fragmentation of the above-mentioned enzyme shows that other types of action of these agents at the molecular level are possible; these may be the maintenance of the genome integrity in the brain structures under DN conditions and preventing the development of inflammatory processes. Thus, the examined inhibitors can be used in the future in the treatment of brain dysfunctions that are complications of type-1 diabetes mellitus.

FoxO1 Promotes Mitophagy in the Podocytes of Diabetic Male Mice via the PINK1/Parkin Pathway.

We recently showed that forkhead-box class O1 (FoxO1) activation protects against high glucose-induced injury by preventing mitochondrial dysfunction in the rat kidney cortex. In addition, FoxO1 has been reported to mediate putative kinase 1 (PINK1) transcription and promote autophagy in response to mitochondrial oxidative stress in murine cardiomyocytes. In this study, we ascertained whether overexpressing FoxO1 in the kidney cortex reverses preestablished diabetic nephropathy in animal models. The effect of FoxO1 on mitophagy signaling pathways was evaluated in mouse podocytes. In vivo experiments were performed in male KM mice. A mouse model of streptozotocin (STZ)-induced type 1 diabetes (T1D) was used, and lentiviral vectors were injected into the kidney cortex to overexpress FoxO1. A mouse podocyte cell line was treated with high concentrations of glucose and genetically modified using lentiviral vectors. We found aberrant mitochondrial morphology and reduced adenosine triphosphate production. These mitochondrial abnormalities were due to decreased mitophagy via reduced phosphatase/tensin homolog on chromosome 10-induced PINK1/Parkin-dependent signaling. FoxO1 upregulation and PINK1/Parkin pathway activation can individually restore injured podocytes in STZ-induced T1D mice. Our results link the antioxidative activity of FoxO1 with PINK1/Parkin-induced mitophagy, indicating a novel role of FoxO1 in diabetic nephropathy.

Polyphenolic enriched extract of Cassia glauca Lamk, improves streptozotocin-induced type-1 diabetes linked with partial insulin resistance in rats

Ethnopharmacological relevanceTraditionally Cassia glauca (CG) has been used to treat diabetes. Aim of the studyThe study was undertaken to evaluate anti-diabetic and antioxidant activity of polyphenolic enriched extract of CG in standardized streptozotocin (STZ)-induced diabetic rats. Materials and methodsThe effect of ethanol (CGE) and water (CGW) extracts of CG (200 and 400mg/kg) treatment were evaluated in STZ (50mg/kg, iv) induced diabetic rats. On 10th day, oral glucose tolerance test and degree of insulin resistance was calculated. On 13th day, insulin tolerance test was performed to know the peripheral utilization of glucose. On 15th day, blood glucose, lipid profiles and endogenous antioxidant levels were estimated. In addition, the effects on oral glucose/sucrose tolerance test in normal rats. Further, HPLC fingerprinting profile of CGE and simultaneous quantification of biomarkers were carried out. ResultsSupplementation with CGE and CGW significantly reduced STZ-induced deleterious effects and improved glucose tolerance, and insulin tolerance. In addition, supplementation also decreased oxidative stress by improving endogenous antioxidant levels. Furthermore, administration significantly improves sucrose tolerance suggesting that extract possess inhibition of α-glucosidase enzyme. Further, HPLC studies revealed that CGE contains three bioactive polyphenolic compounds viz., rutin (0.10±0.01mg/g), luteolin-7-glucoside (0.06±0.01mg/g) and isorhoifolin (0.7±0.05mg/g). ConclusionObserved beneficial outcome of CG might be attributed to the presence of polyphenolic compounds and mediated by interacting with multiple targets of diabetes and oxidative stress. Taken together, this study provided the scientific evidence for the traditional use of CG.

Gut microbiota translocation to the pancreatic lymph nodes triggers NOD2 activation and contributes to T1D onset.

Type 1 diabetes (T1D) is an autoimmune disease that is triggered by both genetic and environmental factors, resulting in the destruction of pancreatic β cells. The disruption of the intestinal epithelial barrier and consequent escape of microbial products may be one of these environmental triggers. However, the immune receptors that are activated in this context remain elusive. We show here that during streptozotocin (STZ)-induced T1D, the nucleotide-binding oligomerization domain containing 2 (NOD2), but not NOD1, participates in the pathogenesis of the disease by inducing T helper 1 (Th1) and Th17 cells in the pancreatic LNs (PLNs) and pancreas. Additionally, STZ-injected wild-type (WT) diabetic mice displayed an altered gut microbiota compared with vehicle-injected WT mice, together with the translocation of bacteria to the PLNs. Interestingly, WT mice treated with broad-spectrum antibiotics (Abx) were fully protected from STZ-induced T1D, which correlated with the abrogation of bacterial translocation to the PLNs. Notably, when Abx-treated STZ-injected WT mice received the NOD2 ligand muramyl dipeptide, both hyperglycemia and the proinflammatory immune response were restored. Our results demonstrate that the recognition of bacterial products by NOD2 inside the PLNs contributes to T1D development, establishing a new putative target for intervention during the early stages of the disease.

The IFIH1 A946T autoimmune risk variant exhibits enhanced signaling and promotes T1D pathogenesis

Abstract Genome wide association studies have linked SNPs in innate signaling programs to an increased risk for autoimmune disease. One SNP, rs1990760, within the IFIH1 viral sensor (leading to an A to T amino acid change at codon 946) is strongly associated with multiple autoimmune diseases including type 1 diabetes (T1D). The impact of this variant on viral sensing and autoimmune pathogenesis, however, has not yet been determined. Using an overexpression system, we demonstrate that IFIH1-A946T exhibits heightened basal and post-stimulation signaling and increased viral control. Consistent with this finding, primary PBMCs from healthy subjects expressing the variant exhibit heightened signaling. To study IFIH1-A946T in vivo, we generated a novel knock-in murine model. Both heterozygous and homozygous variant mice display enhanced basal Type I IFN mRNA expression and splenomegaly. Moreover, homozygous pups manifest an embryonic survival defect. Further, IFIH1-T946 mice exhibit increased frequency of STZ-induced T1D. Finally, intercross of IFIH1-T946 mice with animals expressing the T1D risk variant in PTPN22 lead to a synergistic increase in T1D development. Together, our data demonstrate that IFIH1-A946T alters antiviral signaling and strongly supports the model that increased Type I IFN production driven by the variant leads to an inflammatory state that promotes autoimmunity, particularly in association with other autoimmune risk alleles.

Brugia malayi soluble and excretory‐secretory proteins attenuate development of streptozotocin‐induced type 1 diabetes in mice

Understanding the modulation of the host-immune system by pathogens-like filarial parasites offers an alternate approach to prevent autoimmune diseases. In this study, we have shown that treatment with filarial proteins prior to or after the clinical onset of streptozotocin-induced type-1 diabetes (T1D) can ameliorate the severity of disease in BALB/c mice. Pre-treatment with Brugia malayi adult soluble (Bm A S) or microfilarial excretory-secretory (Bm mf ES) or microfilarial soluble (Bm mf S) antigens followed by induction of diabetes led to lowering of fasting blood glucose levels with as many as 57.5-62.5% of mice remaining nondiabetic. These proteins were more effective when they were used to treat the mice with established T1D as 62.5-71.5% of the mice turned to be nondiabetic. Histopathological examination of pancreas of treated mice showed minor inflammatory changes in pancreatic islet cell architecture. The therapeutic effect was found to be associated with the decreased production of cytokines TNF-α & IFN-γ and increased production of IL-10 in the culture supernatants of splenocytes of treated mice. A switch in the production of anti-insulin antibodies from IgG2a to IgG1 isotype was also seen. Together these results provide a proof towards utilizing the filarial derived proteins as novel anti-diabetic therapeutics.

Therapeutic inhibition of mitochondrial reactive oxygen species with mito-TEMPO reduces diabetic cardiomyopathy

AimsThe mitochondria are important sources of reactive oxygen species (ROS) in the heart. Mitochondrial ROS production has been implicated in the pathogenesis of diabetic cardiomyopathy, suggesting that therapeutic strategies specifically targeting mitochondrial ROS may have benefit in this disease. We investigated the therapeutic effects of mitochondria-targeted antioxidant mito-TEMPO on diabetic cardiomyopathy. MethodsThe mitochondria-targeted antioxidant mito-TEMPO was administrated after diabetes onset in a mouse model of streptozotocin-induced type-1 diabetes and type-2 diabetic db/db mice. Cardiac adverse changes were analyzed and myocardial function assessed. Cultured adult cardiomyocytes were stimulated with high glucose, and mitochondrial superoxide generation and cell death were measured. ResultsIncubation with high glucose increased mitochondria superoxide generation in cultured cardiomyocytes, which was prevented by mito-TEMPO. Co-incubation with mito-TEMPO abrogated high glucose-induced cell death. Mitochondrial ROS generation, and intracellular oxidative stress levels were induced in both type-1 and type-2 diabetic mouse hearts. Daily injection of mito-TEMPO for 30 days inhibited mitochondrial ROS generation, prevented intracellular oxidative stress levels, decreased apoptosis and reduced myocardial hypertrophy in diabetic hearts, leading to improvement of myocardial function in both type-1 and type-2 diabetic mice. Incubation with mito-TEMPO or inhibition of Nox2-containing NADPH oxidase prevented oxidative stress levels and cell death in high glucose-stimulated cardiomyocytes. Mechanistic study revealed that the protective effects of mito-TEMPO were associated with down-regulation of ERK1/2 phosphorylation. ConclusionsTherapeutic inhibition of mitochondrial ROS by mito-TEMPO reduced adverse cardiac changes and mitigated myocardial dysfunction in diabetic mice. Thus, mitochondria-targeted antioxidants may be an effective therapy for diabetic cardiac complications.