Plasma and stool metabolomic biomarkers of non-alcoholic fatty liver disease in Argentina

Background: Non-invasive biomarkers are urgently needed to identify patients with non-alcoholic fatty liver disease (NAFLD) at risk of disease progression, particularly in high prevalence areas such as Latin America. In this regard, targeted metabolomics is a powerful technology for discovering new gut microbiome-derived metabolites. Thus, we aimed to identify potential metabolomic biomarkers related to NAFLD stage in Argentina, and to assess their relationship with clinical and host genetic factors. Methods: Adult healthy volunteers (HV) and biopsy-proven simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) patients were recruited. Demographic, clinical and food frequency consumption data, as well as plasma and stool samples were collected. SNP rs738409 (PNPLA3 gene) was determined in all volunteers. HPLC and flow injection analysis with MS/MS in tandem was applied for metabolomic studies using the MxP Quant 500 Kit (Biocrates Life Sciences AG, Austria). Significantly different metabolites among groups were identified with MetaboAnalyst v4.0. Bivariate and multivariate analyses were used to identify variables that were independently related to NAFLD stage. Forward stepwise logistic regression models were constructed to design the best feature combination that could distinguish between study groups. Receiver Operating Characteristic (ROC) curves were used to evaluate models? accuracy.Results: 19 HV, 12 SS and 22 NASH patients were recruited. Diet was similar between groups. The concentration of 33 out of 424 detected metabolites (25 in plasma and 8 in stool) was significantly different among study groups. Levels of triglycerides (TG) were higher among NAFLD patients, whereas levels of phosphatidylcholines (PC) and lysoPC were higher among HV. The PNPLA3 risk genotype for NAFLD and NASH (GG) was related to higher plasma levels of eicosenoic acid FA(20:1) (p<0.001). Plasma metabolites showed a higher accuracy for diagnosis of NAFLD and NASH when compared to stool metabolites (Table 1). Body mass index (BMI) and plasma levels of PC aa C24:0, FA(20:1) and TG(16:1_34:1) showed high accuracy for diagnosis of NAFLD; whereas the best AUROC for discriminating NASH from SS was that of plasma levels of PC aa C24:0 and PC ae C40:1 (Table 1).Conclusions: Gut microbiome-derived metabolomic biomarkers were identified in plasma and stool, but plasma metabolites were better diagnostic biomarkers of NAFLD and NASH in Argentina. Further validation studies are needed.Fil: Mazzini, Flavia Noelia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Cook, Frank. Novartis Institutes For Biomedical Research; Estados UnidosFil: Gounarides, John. Novartis Institutes For Biomedical Research; Estados UnidosFil: Marciano, Sebastian. Hospital Italiano; ArgentinaFil: Haddad, Leila. Hospital Italiano; ArgentinaFil: Tamaroff, Ana Jesica. Hospital Italiano; ArgentinaFil: Casciato, Paola. Hospital Italiano; ArgentinaFil: Narvaez, Adriana Haydée. Hospital Italiano; ArgentinaFil: Mascardi, María Florencia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Anders, Margarita. Hospital Alemán; ArgentinaFil: Orozco, Federico. Hospital Alemán; ArgentinaFil: Quiroz, Nicolas. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Risk, Marcelo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Gutt, Susana. Hospital Italiano; ArgentinaFil: Gadano, Adrián Carlos. Hospital Italiano; ArgentinaFil: Mendez Garcia, Celia. Hospital Italiano; ArgentinaFil: Marro, Martin. Novartis Institutes For Biomedical Research; Estados UnidosFil: Penas Steinhardt, Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Trinks, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaThe Liver Meeting Digital ExperienceEstados UnidosAmerican Association for the Study of the Liver Diseas

Direct Bacterial Killing In Vitro by Recombinant Nod2 Is Compromised by Crohn's Disease-Associated Mutations

Background: A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn’s disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations. Methodology/Principal Findings: In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn’s-associated mutations. Wildtype, but not Crohn’s-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity. Conclusions/Significance: The lack of anti-bacterial activity demonstrated with Crohn’s-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphism

Sample Preparation and LC-MS methods for the measurement of NAD, NADP, NADPH. NADH and NMN

No Abstract - This is a Analytical Method that we would share and potentially transfer to a third party CR

Effects of cevoglitazar, a dual PPARalpha/gamma agonist, on ectopic fat deposition in fatty Zucker rats.

AIM: By acting as both insulin sensitizers and lipid-lowering agents, dual-acting peroxisome proliferator-activated receptors alpha/gamma (PPARalpha/gamma) agonists may be used to improve glucose tolerance in type 2 diabetic patients without inducing adiposity and body weight gain. Here, in an animal model of obesity and insulin resistance, the metabolic response to cevoglitazar, a dual PPARalpha/gamma, was characterized using a combination of in vivo and ex vivo magnetic resonance methodologies and compared to treatment effects of fenofibrate, a PPARalpha agonist, and pioglitazone, a PPARgamma agonist. METHODS: Four groups of fatty Zucker rats: (i) Vehicle; (ii) fenofibrate 150 mg/kg; (iii) pioglitazone 30 mg/kg; and (iv) cevoglitazar 5 mg/kg were investigated before and after treatment. Animals were fed a fat-enriched (54% kcal fat) diet for 6 weeks, 2 weeks high of fat-exposure alone followed by a 4-week dosing period. RESULTS AND CONCLUSIONS: Cevoglitazar was as effective as pioglitazone at improving glucose tolerance. However, unlike pioglitazone, both fenofibrate and cevoglitazar reduced BW gain and adiposity, independent of food intake. All three treatment regimens normalized intramyocellular lipids. Metabolic profiling showed that in the muscle cevoglitazar improves the lipid profile via both PPARalpha- and PPARgamma-mediated mechanisms. Pioglitazone reduced hepatic lipid accumulation, while cevoglitazar and fenofibrate reduced hepatic lipid concentration below baseline levels (p < 0.05). Metabolic profiling showed that in the liver, cevoglitazar functions largely through PPARalpha agonism resulting in increased beta-oxidation. Cevoglitazar only induced small changes to the lipid composition of visceral fat. In subcutaneous fat, however, cevoglitazar induced changes similar to those observed with fenofibrate suggesting export of fatty acids from this depot

Full Utilization of the Mass Spectrometer by On-Demand sharing with Multiple LC Units

The concept and implementation of full utilization of mass spectrometer by on-demand sharing with many LC units is reported. Sharing mass spectrometer with multiple LC’s is not new; however, to fully utilize the MS duty cycle, the number of LC units that a MS will need to recruit is application dependent and could be significantly larger than the current commercial or published implementations. For the example of a single analyte the number can be close to the peak capacity at a first degree of approximation. It remains to be seen whether a MS can be configured to have such a flexibility and scalability. Here we construct a prototypical system using commercially available software and hardware and demonstrate its performance with real applications. The ultimate goal is to have a system that allows the ‘plug & play’ of LC modules on-demand to fully utilize the mass spectrometer for any applications

Relationship between visceral adiposity and intramyocellular lipid content in two rat models of insulin resistance.

High visceral adiposity and intramyocellular lipid levels (IMCL) are both associated with the development of type 2 diabetes. The relationship between visceral adiposity and IMCL levels was explored in diet- and glucocorticoid-induced models of insulin resistance. In the diet-induced model, lean and fa/fa Zucker rats were fed either normal or high-fat (HF) chow over 4 wk. Fat distribution, IMCL content in the tibialis anterior (TA) muscle (IMCL(TA)), and whole body insulin resistance were measured before and after the 4-wk period. The HF diet-induced increase in IMCL(TA) was strongly correlated with visceral fat accumulation and greater glucose intolerance in both groups. The increase in IMCL(TA) to visceral fat accumulation was threefold greater for fa/fa rats. In the glucocorticoid-induced model, insulin sensitivity was impaired with dexamethasone. In vivo adiposity and IMCL(TA) content measurements were combined with ex vivo analysis of plasma and muscle tissue. Dexamethasone treatment had minimal effects on visceral fat accumulation while increasing IMCL(TA) levels approximately 30% (P < 0.05) compared with controls. Dexamethasone increased plasma glucose by twofold and increased the saturated fatty acid content of plasma lipids [fatty acid (CH2)n/omegaCH3 ratio +15%, P < 0.05]. The lipid composition of the TA muscle was unchanged by dexamethasone treatment, indicating that the relative increase in IMCL(TA) observed in vivo resulted from a decrease in lipid oxidation. Visceral adiposity may influence IMCL accumulation in the context of dietary manipulations; however, a "causal" relationship still remains to be determined. Dexamethasone-induced insulin resistance likely operates under a different mechanism, i.e., independently of visceral adiposity

Hepatic glycogen cycling contributes to glucose lowering effects of the glucokinase activator LCZ960

Aims/Hypothesis Glucokinase (GK) acts as a glucose sensor by facilitating glucose phosphorylation into glucose-6-phosphate (G6P) in the liver and pancreas, the two key target tissues. LCZ960, a small molecule glucokinase activator (GKA) exerts a stimulatory effect on GK activity in hepatocyte in vitro. The current study was aimed to verify in vivo that LCZ960 stimulates glucose uptake primarily through a mechanism involving hepatic GK activation. Methods Acute and chronic LCZ960 treatment-induced changes in glycemia and hepatic glucose turnover were measured in high fat diet-induced obese (DIO) mice and rats, respectively. G6P production and glycogen cycling were quantified by 13C-MR spectroscopy during a 120-min [1-13C]glucose infusion, followed by a 60-min pulse-chase with [12C]glucose to mimic postprandial conditions in rats. Results Acute treatment with LCZ960 dose-dependently reduced blood glucose without hypoglycemic effect in DIO mice. Chronic LCZ960 treatment maintained normoglycemia and improved glucose tolerance without increased insulin secretion in DIO mice and rats. In rats, LCZ960 stimulated a 240% increase in the glycogen synthase flux. However, due to a much higher glycogen breakdown (LCZ960: 47.8±15.2 vs control: 3.9±1.0 mol/kg/min), this translated to a much smaller increase (46%) in glycogen storage (Vsyn net 64.3±8.6 mol/kg/min). Despite a 4-fold increase in hepatic glycogen turnover (LCZ960: 36.0±5.5% vs. control: 8.3±2.0%), LCZ960 did not impact glucose-stimulated G6P accumulation. Conclusions/Interpretation LCZ960 maintains normoglycemia without causing hypoglycemia in rodents. Under hyperglycemic conditions, LCZ960 caused a robust increase in hepatic glycogen cycling. Because net hepatic glycogen storage is diminished in poorly controlled patients with type 2 diabetes, stimulation of glycogen synthesis may contribute to antihyperglycemic properties of GKA

Age and muscle-type modulated role of intramyocellular lipids in the progression of insulin resistance in nondiabetic Zucker rats.

The effect of muscle fiber type and maturation on intramyocellular lipid (IMCL) content and its relationship to insulin resistance was investigated. Intramyocellular lipid content in slow-twitch (soleus) and fast-twitch (tibialis anterior, TA) muscles of fa/fa (Zucker fatty rat, ZFR) and age-matched lean (Zucker lean rat, ZLR) Zucker rats were repeatedly measured over 3 months. Intramyocellular lipid levels in both the soleus and the TA were significantly higher in the ZFR relative to the ZLR. For the ZFR, IMCL TA increased by approximately 2-fold from 5.3 to 8.4 weeks of age. No subsequent accumulation of IMCL TA occurred in ZFR from 8.4 up to 13.1 weeks of age. For ZLR, IMCL TA contents steadily decreased from 6.6 to 13.1 weeks of age (-77%, P<.05). In contrast, IMCL levels in the soleus were not significantly altered in either rat strain over the course of the study. Maximum impairment in whole-body insulin sensitivity in ZFR was observed at 9-weeks of age, concomitant with peak IMCL TA accumulation. Insulin-stimulated 2-deoxy-D-glucose (2DG) transport in the TA muscle of 10.2- and 14.1-week-old ZFR was significantly impaired relative to age-matched ZLR. Insulin-stimulated glucose uptake in the soleus of ZFR and ZLR decreased (P<.05) as the animals matured (ZFR, -49%; ZLR, -69%). Overall, these results support the hypothesis that fast-twitch glycolytic muscles play a major role during the onset of insulin resistance. In addition, proper timing may govern the success of a pharmacological studies aimed at measuring the impact of insulin-sensitizing drugs on IMCL