iNOS, an important mediator of inflammation, has emerged as an important metabolic regulator. There are conflicting observations on the incidence of insulin resistance (IR) due to hyperglycemia/dyslipidemia in iNOS−/− mice. There are reports that high fat diet (HFD) fed mice exhibited no change, protection, or enhanced susceptibility to IR. Similar observations were also reported for low fat diet (LFD) fed KO mice. In the present study chow fed iNOS−/− mice were examined for the incidence of IR, and metabolic perturbations, and also for the effect of sodium nitrite supplementation (50 mg/L). In IR-iNOS−/− mice, we observed significantly higher body weight, BMI, adiposity, blood glucose, HOMA-IR, serum/tissue lipids, glucose intolerance, enhanced gluconeogenesis, and disrupted insulin signaling. Expression of genes involved in hepatic and adipose tissue lipid uptake, synthesis, oxidation, and gluconeogenesis was upregulated with concomitant downregulation of genes for hepatic lipid excretion. Nitrite supplementation restored NO levels, significantly improved systemic IR, glucose tolerance, and also reduced lipid accumulation by rescuing hepatic insulin sensitivity, glucose, and lipid homeostasis. Obesity, gluconeogenesis, and adipose tissue insulin signaling were only partially reversed in nitrite supplemented iNOS−/− mice. Our results thus demonstrate that nitrite supplementation to iNOS−/− mice improves insulin sensitivity and metabolic homeostasis, thus further highlighting the metabolic role of iNOS.

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Oxidative and nitrosative stress plays a pivotal role in the incidence of metabolic disorders. Studies from this lab and others in iNOS-/- mice have demonstrated occurrence of insulin resistance (IR), hyperglycemia and dyslipidemia highlighting the importance of optimal redox balance. The present study evaluates role of nitrite, L-arginine, antidiabetics (metformin, pioglitazone) and antibiotics (ampicillin-neomycin combination, metronidazole) on metabolic perturbations observed in iNOS-/- mice. The animals were monitored for glucose tolerance (IPGTT), IR (insulin, HOMA-IR, QUICKI), circulating lipids and serum metabolomics (LC-MS). Hyperglycemia, hyperinsulinemia and IR were rescued by nitrite, antidiabetics, and antibiotics treatments in iNOS-/- mice. Glucose intolerance was improved with nitrite, metformin and pioglitazone treatment, while ampicillin-neomycin combination normalised the glucose utilization in iNOS-/- mice. Increased serum phosphatidylethanolamine lipids in iNOS-/- mice were reversed by metformin, pioglitazone and ampicillin-neomycin; dyslipidemia was however marginally improved by nitrite treatment. The metabolic improvements were associated with changes in selected serum metabolites-purines, ceramide, 10-hydroxydecanoate, glucosaminate, diosmetin, sebacic acid, 3-nitrotyrosine and cysteamine. Bacterial metabolites-hippurate, indole-3-ethanol; IR marker-aminoadipate and oxidative stress marker-ophthalmate were reduced by pioglitazone and ampicillin-neomycin, but not by nitrite and metformin treatment. Results obtained in the present study suggest a crucial role of gut microbiota in the metabolic perturbations observed in iNOS-/- mice.

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Fructose consumption is responsible for the onset of insulin resistance (IR), and metabolic syndrome. It possesses no functional utility in body and its detrimental effects on hepatic metabolic milieu are beyond those produced by glucose. The need of the hour is to identify fructose-induced IR as an unique pathological state to be managed differentially. The effect of aqueous leaf extract of Aegle marmelos (AM) on hepatic markers of insulin resistance using HepG2 cells cultured in either fructose or glucose-rich environment is investigated. Human hepatocellular carcinoma cells (HepG2) were grown under standard conditions in either-DMEM without glucose (NC), DMEM with high glucose 25 mM (Glu), DMEM-glucose+0.55 mM fructose (FC1), DMEM-glucose+1 mM fructose (FC2) or DMEM-glucose+1 mM fructose+0.1 µM insulin (FC3). The cells were treated with either AM, rutin, quercetin, metformin or pioglitazone and assessed for levels of hexokinase, phosphofructokinase (PFK), aldehyde dehydrogenase, phosphatidylinositol kinase (PI3K), signal transducer and activator of transcription-3 (STAT-3), mitochondrial target of rapamycin (mTOR), hypoxia-induced factor (HIF-1α), vascular endothelial growth factor (VEGF) and tumour necrosis factor (TNF-α). Summarily, when results from fructose- and glucose-rich environment were compared, then (1) IR was more pronounced in former; (2) AM performed better in former; (3) metformin and pioglitazone were equivocal in either; (4) rutin and quercetin showed deviant effects from AM; and lastly (5) effects of rutin were closer to AM than quercetin. We hypothesize that AM ameliorates fructose-induced IR through a mechanism which is distinct from standard drugs and not shared by individual phytoconstituents in toto.

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Pharmacological inhibition of platelet collagen interaction is a promising therapeutic strategy to treat intra-vascular thrombosis. S007-867 is a novel synthetic inhibitor of collagen-induced platelet aggregation. It has shown better antithrombotic protection than aspirin and clopidogrel with minimal bleeding tendency in mice. The present study is aimed to systematically investigate the antithrombotic efficacy of S007-867 in comparison to aspirin and clopidogrel in vivo and to delineate its mechanism of action in vitro. Aspirin, clopidogrel, and S007-867 significantly reduced thrombus weight in arterio-venous (AV) shunt model in rats. In mice, following ferric chloride induced thrombosis in either carotid or mesenteric artery; S007-867 significantly prolonged the vessel occlusion time (1.2-fold) and maintained a sustained blood flow velocity for >30 min. Comparatively, clopidogrel showed significant prolongation in TTO (1.3-fold) while aspirin remained ineffective. Both S007-867 and aspirin did not alter bleeding time in either kidney or spleen injury models, and thus maintained hemostasis, while clopidogrel showed significant increase in spleen bleeding time (1.7-fold). The coagulation parameters namely thrombin time, prothrombin time or activated partial thromboplastin time remained unaffected even at high concentration of S007-867 (300 µM), thus implying its antithrombotic effect to be primarily platelet mediated. S007-867 significantly inhibited collagen-mediated platelet adhesion and aggregation in mice ex-vivo. Moreover, when blood was perfused over a highly thrombogenic combination of collagen mimicking peptides like CRP-GFOGER-VWF-III, S007-867 significantly reduced total thrombus volume or ZV (53.4 ± 5.7%). Mechanistically, S007-867 (10-300 μM) inhibited collagen-induced ATP release, thromboxane A (TxA) generation, intra-platelet [Ca] flux and global tyrosine phosphorylation including PLCγ2. Collectively the present study highlights that S007-867 is a novel synthetic inhibitor of collagen induced platelet activation, that effectively maintains blood flow velocity and delays vascular occlusion. It inhibits thrombogenesis without compromising hemostasis. Therefore, S007-867 may be further developed for the treatment of thrombotic disorders in clinical settings.