Metabolomic Study of Aging in fa/fa Rats: Multiplatform Urine and Serum Analysis.

Zucker fatty (fa/fa) rats represent a well-established and widely used model of genetic obesity. Because previous metabolomic studies have only been published for young fa/fa rats up to 20 weeks of age, which can be considered early maturity in male fa/fa rats, the aim of our work was to extend the metabolomic characterization to significantly older animals. Therefore, the urinary profiles of obese fa/fa rats and their lean controls were monitored using untargeted NMR metabolomics between 12 and 40 weeks of age. At the end of the experiment, the rats were also characterized by NMR and LC-MS serum analysis, which was supplemented by a targeted LC-MS analysis of serum bile acids and neurotransmitters. The urine analysis showed that most of the characteristic differences detected in young obese fa/fa rats persisted throughout the experiment, primarily through a decrease in microbial co-metabolite levels, the upregulation of the citrate cycle, and changes in nicotinamide metabolism compared with the age-related controls. The serum of 40-week-old obese rats showed a reduction in several bile acid conjugates and an increase in serotonin. Our study demonstrated that the fa/fa model of genetic obesity is stable up to 40 weeks of age and is therefore suitable for long-term experiments.

Reduced phosphatidylcholine level in the intestinal mucus layer of prediabetic NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease with rising incidence. Pre- and manifest T1D is associated with intestinal barrier dysfunction, skewed microbiota composition, and serum dyslipidemia. The intestinal mucus layer protects against pathogens and its structure and phosphatidylcholine (PC) lipid composition may be compromised in T1D, potentially contributing to barrier dysfunction. This study compared prediabetic Non-Obese Diabetic (NOD) mice to healthy C57BL/6 mice by analyzing the intestinal mucus PC profile by shotgun lipidomics, plasma metabolomics by mass spectrometry and nuclear magnetic resonance, intestinal mucus production by histology, and cecal microbiota composition by 16 S rRNA sequencing. Jejunal mucus PC class levels were decreased in early prediabetic NOD vs C57BL/6 mice. In colonic mucus of NOD mice, the level of several PC species was reduced throughout prediabetes. In plasma, similar reductions of PC species were observed in early prediabetic NOD mice, where also increased beta-oxidation was prominent. No histological alterations were found in jejunal nor colonic mucus between the mouse strains. However, the ß-diversity of the cecal microbiota composition differed between prediabetic NOD and C57BL/6 mice, and the bacterial species driving this difference were related to decreased short-chain fatty acid (SCFA)-production in the NOD mice. This study reports reduced levels of PCs in the intestinal mucus layer and plasma of prediabetic NOD mice as well as reduced proportions of SCFA-producing bacteria in cecal content at early prediabetes, possibly contributing to intestinal barrier dysfunction and T1D.

NMR- and MS-Based Untargeted Metabolomic Study of Stool and Serum Samples from Patients with Anorexia Nervosa.

Anorexia nervosa (AN), a pathological restriction of food intake, leads to metabolic dysregulation. We conducted a metabolomics study to reveal changes caused by AN and the effect of hospital realimentation on metabolism. Both stool and serum from patients with AN and healthy controls were analyzed by NMR and MS. Statistical analysis revealed several altered biochemical and anthropometric parameters and 50 changed metabolites, including phospholipids, acylcarnitines, amino acids, derivatives of nicotinic acid, nucleotides, and energy metabolism intermediates. Biochemical and anthropometric parameters were correlated with metabolomic data. Metabolic changes in patients with AN described in our study imply serious system disruption defects, such as the development of inflammation and oxidative stress, changed free thyroxine (fT4) and thyroid-stimulating hormone (TSH) levels, a deficit of vitamins, muscle mass breakdown, and a decrease in ketone bodies as an important source of energy for the brain and heart. Furthermore, our data indicate only a very slight improvement after treatment. However, correlations of metabolomic results with body weight, interleukin 6, tumor necrosis factor α, fT4, and TSH might entail better prognoses and treatment effectiveness in patients with better system parameter status. Data sets are deposited in MassIVE: MSV000087713, DOI: 10.25345/C57R7X.

Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment.

Metabolic transformation of cancer cells leads to the accumulation of lactate and significant acidification in the tumor microenvironment. Both lactate and acidosis have a well-documented impact on cancer progression and negative patient prognosis. Here, we report that cancer cells adapted to acidosis are significantly more sensitive to oxidative damage induced by hydrogen peroxide, high-dose ascorbate, and photodynamic therapy. Higher lactate concentrations abrogate the sensitization. Mechanistically, acidosis leads to a drop in antioxidant capacity caused by a compromised supply of nicotinamide adenine dinucleotide phosphate (NADPH) derived from glucose metabolism. However, lactate metabolism in the Krebs cycle restores NADPH supply and antioxidant capacity. CPI-613 (devimistat), an anticancer drug candidate, selectively eradicates the cells adapted to acidosis through inhibition of the Krebs cycle and induction of oxidative stress while completely abrogating the protective effect of lactate. Simultaneous cell treatment with tetracycline, an inhibitor of the mitochondrial proteosynthesis, further enhances the cytotoxic effect of CPI-613 under acidosis and in tumor spheroids. While there have been numerous attempts to treat cancer by neutralizing the pH of the tumor microenvironment, we alternatively suggest considering tumor acidosis as the Achilles' heel of cancer as it enables selective therapeutic induction of lethal oxidative stress.

The intestinal microbiota and metabolites in patients with anorexia nervosa.

Brain-gut microbiota interactions are intensively studied in connection with various neurological and psychiatric diseases. While anorexia nervosa (AN) pathophysiology is not entirely clear, it is presumably linked to microbiome dysbiosis. We aimed to elucidate the gut microbiota contribution in AN disease pathophysiology. We analyzed the composition and diversity of the gut microbiome of patients with AN (bacteriome and mycobiome) from stool samples before and after renourishment, and compared them to healthy controls. Further, levels of assorted neurotransmitters and short-chain fatty acids (SCFA) were analyzed in stool samples by MS and NMR, respectively. Biochemical, anthropometric, and psychometric profiles were assessed. The bacterial alpha-diversity parameter analyses revealed only increased Chao 1 index in patients with AN before the realimentation, reflecting their interindividual variation. Subsequently, core microbiota depletion signs were observed in patients with AN. Overrepresented OTUs (operation taxonomic units) in patients with AN taxonomically belonged to Alistipes, Clostridiales, Christensenellaceae, and Ruminococcaceae. Underrepresented OTUs in patients with AN were Faecalibacterium, Agathobacter, Bacteroides, Blautia, and Lachnospira. Patients exhibited greater interindividual variation in the gut bacteriome, as well as in metagenome content compared to controls, suggesting altered bacteriome functions. Patients had decreased levels of serotonin, GABA, dopamine, butyrate, and acetate in their stool samples compared to controls. Mycobiome analysis did not reveal significant differences in alpha diversity and fungal profile composition between patients with AN and healthy controls, nor any correlation of the fungal composition with the bacterial profile. Our results show the changed profile of the gut microbiome and its metabolites in patients with severe AN. Although therapeutic partial renourishment led to increased body mass index and improved psychometric parameters, SCFA, and neurotransmitter profiles, as well as microbial community compositions, did not change substantially during the hospitalization period, which can be potentially caused by only partial weight recovery.

Lipid Profiling in Epicardial and Subcutaneous Adipose Tissue of Patients with Coronary Artery Disease.

Coronary artery disease is one of the most frequent causes of morbidity and mortality worldwide. It is even more prevalent in patients with type 2 diabetes mellitus who suffer from obesity and increased accumulation of epicardial fat with a possible contributing role in the development of coronary artery disease. We performed an MS-based lipidomic analysis of subcutaneous and epicardial adipose tissue in 23 patients with coronary artery disease stratified for the presence/absence of type 2 diabetes mellitus and a control group of 13 subjects aiming at identification of factors from epicardial fat contributing to the development of coronary artery disease. The samples of adipose tissues were obtained during elective cardiac surgery. They were extracted and analyzed with and without previous triacylglycerols separation by high-pressure liquid chromatography-mass spectrometry (HPLC-MS). Multivariate and univariate analyses were performed. Lipidomics data were correlated with biochemical parameters. We identified multiple changes in monoacylglycerols, diacylglycerols, triacylglycerols, glycerophosphatidylserines, glycerophosphatidylethanolamines, glycerophosphatidylcholines, ceramides, sphingomyelins, and derivatives of cholesterol. Observed changes included molecules with fatty acids with odd (15:0, 15:1, 17:0, 17:1) and even (10:0, 12:0, 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 20:4, 20:1, 22:0) fatty acids in both types of adipose tissue. More pronounced changes were detected in epicardial adipose tissue compared to subcutaneous adipose tissue of patients with coronary artery disease and type 2 diabetes. Lipidomic analysis of subcutaneous and epicardial adipose tissue revealed different profiles for patients with coronary artery disease and type 2 diabetes, which might be related to coronary artery disease and the presence of type 2 diabetes.

Microbiota, Microbial Metabolites, and Barrier Function in A Patient with Anorexia Nervosa after Fecal Microbiota Transplantation.

The change in the gut microbiome and microbial metabolites in a patient suffering from severe and enduring anorexia nervosa (AN) and diagnosed with small intestinal bacterial overgrowth syndrome (SIBO) was investigated. Microbial gut dysbiosis is associated with both AN and SIBO, and therefore gut microbiome changes by serial fecal microbiota transplantation (FMT) is a possible therapeutic modality. This study assessed the effects of FMT on gut barrier function, microbiota composition, and the levels of bacterial metabolic products. The patient treatment with FMT led to the improvement of gut barrier function, which was altered prior to FMT. Very low bacterial alpha diversity, a lack of beneficial bacteria, together with a great abundance of fungal species were observed in the patient stool sample before FMT. After FMT, both bacterial species richness and gut microbiome evenness increased in the patient, while the fungal alpha diversity decreased. The total short-chain fatty acids (SCFAs) levels (molecules presenting an important source of energy for epithelial gut cells) gradually increased after FMT. Contrarily, one of the most abundant intestinal neurotransmitters, serotonin, tended to decrease throughout the observation period. Overall, gut microbial dysbiosis improvement after FMT was considered. However, there were no signs of patient clinical improvement. The need for an in-depth analysis of the donor´s stool and correct selection pre-FMT is evident.

Minor lipids profiling in subcutaneous and epicardial fat tissue using LC/MS with an optimized preanalytical phase.

Analysis of bioactive lipids in adipose tissue could lead to better understanding of the pathogenesis of obesity and its complications. However, current MS methods are limited by a high content of triacylglycerols (TAGs), which markedly surpasses the amount of other lipids and suppresses their ionization. The aim of our study was thus to optimize the preanalytical phase of lipid analysis in adipose tissue, focusing in particular on less-abundant lipids. Next, the optimized method was used to describe the differences between epicardial and subcutaneous adipose tissues obtained from patients undergoing cardiac surgery. Lipids were extracted using a modified Folch method with subsequent detachment of TAGs by thin layer chromatography (TLC). The extracts with/without TAGs were analyzed by tandem LC/MS. The repeatability of the presented method expressed by the median of the coefficients of variation was 12/5% for analysis with/without TAGs separation, respectively. The difference in the relative abundance of TAGs gained with/without TLC was, on average, 19% and did not reach significance (p value > 0.05) for any identified TAG. The novel preanalytical step allowed us to detect 37 lipids, which could not have been detected without TAG separation, because their signal to noise ratio is <5 in current methods of untargeted lipidomics. These lipids belong predominately to ceramides, glycerophosphatidylserines, glycerophosphatidylinsitols, sphingomyelins, glycerophosphatidylcholines, glycerophosphatidylethanolamines, diacylglycerols. The two adipose tissue depots differed mainly in the following lipid classes: glycerophosphatidylcholines, glycerophosphatidylinositols, glycerophosphatidylethanolamine, and sphingomyelins. Moreover, other major lipids showed distinctly different distributions between the two adipose tissues. Among these, the changes in TAGs were the most striking, which correspond to previously published data describing the differences between omental and subcutaneous adipose tissue. Implementation of the TLC step for the elimination of TAGs was crucial for enhancing the MS detection limit of minor lipids in adipose tissue. The differences between the overall lipid profiles of subcutaneous and epicardial tissue reflect their different functions arising from their location.

Metabolomic Study of Obesity and Its Treatment with Palmitoylated Prolactin-Releasing Peptide Analog in Spontaneously Hypertensive and Normotensive Rats.

In this study, the combination of metabolomics and standard biochemical and biometric parameters was used to describe the metabolic effects of diet-induced obesity and its treatment with the novel antiobesity compound palm11-PrRP31 (palmitoylated prolactin-releasing peptide) in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). The results showed that SHR on a high-fat (HF) diet were normoglycemic with obesity and hypertension, while WKY on the HF diet were normotensive and obese with prediabetes. NMR-based metabolomics revealed mainly several microbial cometabolites altered by the HF diet, particularly in urine. The HF diet induced similar changes in both models. However, two groups of genotype-specific metabolites were defined: metabolites specific to the genotype at baseline (e.g., 1-methylnicotinamide, phenylacetylglycine, taurine, methylamine) and metabolites reacting specifically to the HF diet in individual genotypes (2-oxoglutarate, dimethylamine, N-butyrylglycine, p-cresyl sulfate). The palm11-PrRP31 lowered body weight and improved biochemical and biometric parameters in both strains, and it improved glucose tolerance in WKY rats on the HF diet. In urine, the therapy induced significant decrease of formate and 1-methylnicotinamide in SHR and alanine, allantoin, dimethylamine, and N-butyrylglycine in WKY. Altogether, our study confirms the effectiveness of palm11-PrRP31 for antiobesity treatment.

Metabolomics Based on MS in Mice with Diet-Induced Obesity and Type 2 Diabetes Mellitus: the Effect of Vildagliptin, Metformin, and Their Combination.

Type 2 diabetes mellitus (T2DM) is a major epidemiological problem. Metformin and vildagliptin are well-established antidiabetic drugs. The aim of the study was to evaluate the changes of plasma metabolic profile induced by a high-fat diet (HFD) and subsequent oral administration of metformin, vildagliptin, and their combination in a mouse model of diet-induced obesity (DIO)/T2DM analyzed using quadrupole-time-of-flight mass spectrometry (qTOF-MS). Metformin treatment increased the levels of butyrylcarnitine and acylcarnitine C18:1 concentrations and decreased the levels of isoleucine concentrations compared to untreated HFD mice. Vildagliptin treatment increased levels of butyrylcarnitine and acetylcarnitine. In summary, our metabolomics study revealed multiple differences between obese diabetic HFD mice and lean standard chow diet (SCD) mice, which were partially modifiable by subsequent metformin and vildagliptin treatment.