Comparative profiling of gut microbiota and metabolome in diet-induced obese and insulin-resistant C57BL/6J mice.

Diet-based models are commonly used to investigate obesity and related disorders. We conducted a comparative profiling of three obesogenic diets HFD, high fat diet; HFHF, high fat high fructose diet; and HFCD, high fat choline deficient diet to assess their impact on the gut microbiome and metabolome. After 20 weeks, we analyzed the gut microbiota and metabolomes of liver, plasma, cecal, and fecal samples. Fecal and plasma bile acids (BAs) and fecal short-chain fatty acids (SCFAs) were also examined. Significant changes were observed in fecal and cecal metabolites, with increased Firmicutes and decreased Bacteroidetes in the HFD, HFHF, and HFCD-fed mice compared to chow and LFD (low fat diet)-fed mice. Most BAs were reduced in plasma and fecal samples of obese groups, except taurocholic acid, which increased in HFCD mice's plasma. SCFAs like acetate and butyrate significantly decreased in obesogenic diet groups, while propionic acid specifically decreased in the HFCD group. Pathway analysis revealed significant alterations in amino acid, carbohydrate metabolism, and nucleic acid biosynthesis pathways in obese mice. Surprisingly, even LFD-fed mice showed distinct changes in microbiome and metabolite profiles compared to the chow group. This study provides insights into gut microbiome dysbiosis and metabolite alterations induced by obesogenic and LFD diets in various tissues. These findings aid in selecting suitable diet models to study the role of the gut microbiome and metabolites in obesity and associated disorders, with potential implications for understanding similar pathologies in humans.

Characterization of lipid signatures in the plasma and insulin-sensitive tissues of the C57BL/6J mice fed on obesogenic diets.

Diet-induced obesity mouse models are widely utilized to investigate the underlying mechanisms of dyslipidemia, glucose intolerance, insulin resistance, hepatic steatosis, and type 2 diabetes mellitus (T2DM), as well as for screening potential drug compounds. However, there is limited knowledge regarding specific signature lipids that accurately reflect dietary disorders. In this study, we aimed to identify key lipid signatures using LC/MS-based untargeted lipidomics in the plasma, liver, adipose tissue (AT), and skeletal muscle tissues (SKM) of male C57BL/6J mice that were fed chow, LFD, or obesogenic diets (HFD, HFHF, and HFCD) for a duration of 20 weeks. Furthermore, we conducted a comprehensive lipid analysis to assess similarities and differences with human lipid profiles. The mice fed obesogenic diets exhibited weight gain, glucose intolerance, elevated BMI, glucose and insulin levels, and a fatty liver, resembling characteristics of T2DM and obesity in humans. In total, we identified approximately 368 lipids in plasma, 433 in the liver, 493 in AT, and 624 in SKM. Glycerolipids displayed distinct patterns across the tissues, differing from human findings. However, changes in sphingolipids, phospholipids, and the expression of inflammatory and fibrotic genes showed similarities to reported human findings. Significantly modulated pathways in the obesogenic diet-fed groups included ceramide de novo synthesis, sphingolipid remodeling, and the carboxylesterase pathway, while lipoprotein-mediated pathways were minimally affected. This study provides a tissue-specific comparison of lipid composition, highlighting the usefulness of DIO models in preclinical research. However, caution is warranted when extrapolating findings from these models to dyslipidemia-associated pathologies and their complications in humans.

Feeding enhances fibronectin adherence of quiescent lymphocytes through non-canonical insulin signalling.

Nutritional availability during fasting and refeeding affects the temporal redistribution of lymphoid and myeloid immune cells among the circulating and tissue-resident pools. Conversely, nutritional imbalance and impaired glucose metabolism are associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent immune cell function and trafficking are scanty. Here, we report that oral glucose load in mice and healthy men enhances the adherence of circulating peripheral blood mononuclear cells (PBMCs) and lymphocytes to fibronectin. Adherence to fibronectin is also observed upon regular intake of breakfast following overnight fasting in healthy subjects. This glucose load-induced phenomenon is abrogated in streptozotocin-injected mice that lack insulin. Intra-vital microscopy in mice demonstrated that oral glucose feeding enhances the homing of PBMCs to injured blood vessels in vivo. Furthermore, employing flow cytometry, Western blotting and adhesion assays for PBMCs and Jurkat-T cells, we elucidate that insulin enhances fibronectin adherence of quiescent lymphocytes through non-canonical signalling involving insulin-like growth factor-1 receptor (IGF-1R) autophosphorylation, phospholipase C gamma-1 (PLCγ-1) Tyr783 phosphorylation and inside-out activation of ß-integrins respectively. Our findings uncover the physiological relevance of post-prandial insulin spikes in regulating the adherence and trafficking of circulating quiescent T-cells through fibronectin-integrin interaction.

Modulations in human neutrophil metabolome and S-glutathionylation of glycolytic pathway enzymes during the course of extracellular trap formation.

Neutrophil extracellular trap formation (NETosis) has been irrefutably referred to as a distinct and unique form of active cell death with the purpose to counteract invading pathogens or augmenting the inflammatory cascade. Since the discovery, consistent efforts have been made to understand the various aspects of the initiation and sustenance of NETosis. In this study, using a global metabolomics approach during the phorbol 12-myristate 13-acetate (PMA) induced NETosis in human neutrophils, various metabolic pathways were found to be altered which includes intermediates related to, carbohydrate metabolism, and redox related metabolites, nucleic acid metabolism, and amino acids metabolism. Enrichment analysis of the metabolite sets highlighted the importance of the pentose phosphate pathway (PPP) and glutathione metabolism PMA-induced NETotic neutrophils. Further, analysis of the glutathyniolation status of neutrophil proteins by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) indicated six different glutathionylated proteins: among them, two metabolically important proteins were α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with MALDI score 166 and 70 respectively. Other proteins were lactoferrin, ß-actin, c-myc promoter-binding protein, and uracil DNA glycosylase with MALDI scores of 96, 167, 104, and 68 respectively. Besides, activation of signalling proteins involved in metabolic regulation is also correlated with NETosis. Altogether, a balance between reactive oxygen species-glutathione metabolism seems to regulate the activity of glycolytic enzymes such as GAPDH and α-enolase during PMA-induced NETosis in a time-dependent manner.

Serum and cecal metabolic profile of the insulin resistant and dyslipidemic p47phox knockout mice.

Involvement of NOX-dependent oxidative stress in the pathophysiology of metabolic disorders as well as in the maintenance of metabolic homeostasis has been demonstrated previously. In the present study, the metabolic profile in p47phox-/- and WT mice fed on a chow diet was evaluated to assess the role of metabolites in glucose intolerance and dyslipidemia under altered oxidative stress conditions. p47phox-/- mice displayed glucose intolerance, dyslipidemia, hyperglycemia, insulin resistance (IR), hyperinsulinemia, and altered energy homeostasis without any significant change in gluconeogenesis. The expression of genes involved in lipid synthesis and uptake was enhanced in the liver, adipose tissue, and intestine tissues. Similarly, the expression of genes associated with lipid efflux in the liver and intestine was also enhanced. Enhanced gut permeability, inflammation, and shortening of the gut was evident in p47phox-/- mice. Circulating levels of pyrimidines, phosphatidylglycerol lipids, and 3-methyl-2-oxindole were augmented, while level of purine was reduced in the serum. Moreover, the cecal metabolome was also altered, as was evident with the increase in indole-3-acetamide, N-acetyl galactosamine, glycocholate, and a decrease in hippurate, indoxyl sulfate, and indigestible sugars (raffinose and melezitose). Treatment of p47phox-/- mice with pioglitazone, marginally improved glucose intolerance, and dyslipidemia, with an increase in PUFAs (linoleate, docosahexaenoic acid, and arachidonic acid). Overall, the results obtained in p47phox-/- mice indicate an association of IR and dyslipidemia with altered serum and cecal metabolites (both host and bacterial-derived), implying a critical role of NOX-derived ROS in metabolic homeostasis.

Vancomycin-Induced Modulation of Gram-Positive Gut Bacteria and Metabolites Remediates Insulin Resistance in iNOS Knockout Mice.

The role of oxidative and nitrosative stress has been implied in both physiology and pathophysiology of metabolic disorders. Inducible nitric oxide synthase (iNOS) has emerged as a crucial regulator of host metabolism and gut microbiota activity. The present study examines the role of the gut microbiome in determining host metabolic functions in the absence of iNOS. Insulin-resistant and dyslipidemic iNOS-/- mice displayed reduced microbial diversity, with a higher relative abundance of Allobaculum and Bifidobacterium, gram-positive bacteria, and altered serum metabolites along with metabolic dysregulation. Vancomycin, which largely depletes gram-positive bacteria, reversed the insulin resistance (IR), dyslipidemia, and related metabolic anomalies in iNOS-/- mice. Such improvements in metabolic markers were accompanied by alterations in the expression of genes involved in fatty acid synthesis in the liver and adipose tissue, lipid uptake in adipose tissue, and lipid efflux in the liver and intestine tissue. The rescue of IR in vancomycin-treated iNOS-/- mice was accompanied with the changes in select serum metabolites such as 10-hydroxydecanoate, indole-3-ethanol, allantoin, hippurate, sebacic acid, aminoadipate, and ophthalmate, along with improvement in phosphatidylethanolamine to phosphatidylcholine (PE/PC) ratio. In the present study, we demonstrate that vancomycin-mediated depletion of gram-positive bacteria in iNOS-/- mice reversed the metabolic perturbations, dyslipidemia, and insulin resistance.

Modulation of Insulin Resistance, Dyslipidemia and Serum Metabolome in iNOS Knockout Mice following Treatment with Nitrite, Metformin, Pioglitazone, and a Combination of Ampicillin and Neomycin.

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.

Systemic Insulin Resistance and Metabolic Perturbations in Chow Fed Inducible Nitric Oxide Synthase Knockout Male Mice: Partial Reversal by Nitrite Supplementation.

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.

Vasoreactivity of isolated aortic rings from dyslipidemic and insulin resistant inducible nitric oxide synthase knockout mice.

Recent study from this lab indicated enhanced susceptibility of iNOS KO mice for diet induced obesity (DIO) and systemic insulin resistance (IR) as compared to C57BL/6 (WT) mice. The present study investigates aortic vasoreactivity in high fat diet (HFD) induced insulin resistant iNOS KO mice. WT and iNOS KO mice were fed with 45% HFD/10% LFD for ten weeks. Systemic IR was assessed via measurement of circulating lipids, glucose, and insulin; while phenylephrine (PE)/acetylcholine (ACh) induced responses were monitored in the isolated aortic rings. To understand the mechanism, qPCR or Western blotting experiments were performed in aorta and Ea.hy926 cells. After 10 weeks of HFD feeding, significant increase in the body weight/fat mass, augmented circulating lipids, glucose, insulin and inflammatory cytokines along with impaired acetylcholine induced aortic vasorelaxation and enhanced iNOS expression was observed in the aortic tissue of WT mice. In the aminoguanidine (AG, 20 mg/kg for 4 weeks) treated WT mice and also in iNOS KO mice, acetylcholine induced vasorelaxation was significantly preserved. Further, acetylcholine mediated vasorelaxation correlated with increased eNOS phosphorylation at Ser1177 residue in the iNOS KO mice and same was also observed in the iNOS silenced Ea.hy926 cells. Moreover, treatment of Ea.hy926 cells with palmitic acid or TNFα also caused a significant decrease in eNOS activity, which was reversed in iNOS silenced Ea.hy926 cells suggesting the role of iNOS in the reduction of eNOS activity. The study thus implies a critical role of iNOS in vascular diseases associated with dyslipidemia/IR.

Anti-thrombotic efficacy of S007-867: Pre-clinical evaluation in experimental models of thrombosis in vivo and in vitro.

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 ZV50 (53.4 ±â€¯5.7%). Mechanistically, S007-867 (10-300 µM) inhibited collagen-induced ATP release, thromboxane A2 (TxA2) generation, intra-platelet [Ca+2] 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.

Standardized fraction of Xylocarpus moluccensis fruits improve vascular relaxation and plaque stability in dyslipidemic models of atherosclerosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Xylocarpus moluccensis (Lamk.) M. Roem of family Meliaceae has triterpenoids rich fruits. Triterpenoids have been known to possess cardioprotection and anti-atherosclerotic activities (Han and Bakovic, 2015; Wu et al., 2009). Standardized fraction of these fruits exhibited anti-dyslipidemic (Srivastava et al., 2015), anti-inflammatory (Ravangpai et al., 2011) and CNS depressant activity (Sarker et al., 2007). However, there is no report in the literature on its cardiovascular effects. AIM OF THE STUDY: The present study was undertaken to assess vasoprotective, anti-atherosclerotic and further examine the anti-dyslipidemic effect of the standardized fraction of Xylocarpus moluccensis (F018) fruits in the mechanical injury and high fat diet (HFD) induced dyslipidemic/ atherosclerosis models. MATERIALS AND METHODS: Guinea pigs were fed 0.08% cholesterol + 15% fat diet for 3 weeks, while ApoE KO mice were fed high fat diet for 18 weeks to induce dyslipidemia and atherosclerosis. A combination of balloon injury and high fat diet (1% cholesterol, 6% peanut oil) for 5 weeks was used to accelerate atherosclerosis in NZW rabbits. F018 was administered once daily by oral route in guinea pigs (10, 25 or 50mg/kg/day for 3 weeks), ApoE KO mice (50mg/kg/day for 6 weeks) and in NZW rabbit (25mg/kg/day for 5 weeks) to monitor its effect on dyslipidemia, vasoreactivity and plaque composition by using standard methodologies. RESULTS: F018 treatment in guinea pigs (25 and 50mg/kg/day), ApoE mice (50mg/kg/day) and rabbits (25mg/kg/day) significantly reduced plasma lipids and improved ACh induced vasorelaxation. Anti-dyslipidemic effect of F018 seems to be due to the modulation of enterohepatic genes involved in the cholesterol absorption and excretion. Moreover, significant improvement in the acetylcholine (ACh) induced vasorelaxation was accompanied with reduced inflammatory burden and enhanced activation of eNOS in ApoE mice aortic tissue. Similarly inflammatory cytokines, immunolabeling of macrophage marker (CD68) and MMP-9 were reduced along with augmentation in vascular smooth muscle cells and collagen type I and III in the mechanically injured iliac artery segment in the rabbits. CONCLUSIONS: Altogether, F018 preserved vasoreactivity, reduced atherosclerotic plaque progression and enhanced plaque stability by reducing lipids, inflammatory cytokines, improving endothelial function and collagen content.