1H NMR-based metabolomic study on resistance to diet-induced obesity in AHNAK knock-out mice

AHNAK is a giant protein of approximately 700 kDa identified in human neuroblastomas and skin epithelial cells. Recently, we found that AHNAK knock-out (AHNAK(-/-)) mice have a strong resistance to high-fat diet-induced obesity. In this study, we applied (1)H NMR-based metabolomics with multivariate statistical analysis to compare the altered metabolic patterns detected in urine from high-fat diet (HFD) fed wild-type and AHNAK(-/-) mice and investigate the mechanisms underlying the resistance to high-fat diet-induced obesity in AHNAK(-/-) mice. In global profiling, principal components analysis showed a clear separation between the chow diet and HFD groups; wild-type and AHNAK(-/-) mice were more distinctly separated in the HFD group compared to the chow diet group. Based on target profiling, the urinary metabolites of HFD-fed AHNAK(-/-) mice gave higher levels of methionine, putrescine, tartrate, urocanate, sucrose, glucose, threonine, and 3-hydroxyisovalerate. Furthermore, two-way ANOVAs indicated that diet type, genetic type, and their interaction (gene × diet) affect the metabolite changes differently. Most metabolites were affected by diet type, and putrescine, threonine, urocanate, and tartrate were also affected by genetic type. In addition, cis-aconitate, succinate, glycine, histidine, methylamine (MA), phenylacetylglycine (PAG), methionine, putrescine, uroconate, and tartrate showed interaction effects. Through the pattern changes in urinary metabolites of HFD-fed AHNAK(-/-) mice, our data suggest that the strong resistance to HFD-induced obesity in AHNAK(-/-) mice comes from perturbations of amino acids, such as methionine, putrescine, threonine, and histidine, which are related to fat metabolism. The changes in metabolites affected by microflora such as PAG and MA were also observed. In addition, resistance to obesity in HFD-fed AHNAK(-/-) mice was not related to an activated tricarboxylic acid cycle. These findings demonstrate that (1)H NMR-based metabolic profiling of urine is suitable for elucidating possible biological pathways perturbed by functional loss of AHNAK on HFD feeding and could elucidate the mechanism underlying the resistance to high-fat diet-induced obesity in AHNAK(-/-) mice.     

(1)H NMR-based metabolomic profiling in mice infected with Mycobacterium tuberculosis

Tuberculosis (TB) is one of three major infectious diseases, and the control of TB is becoming more difficult because of the emergence of multidrug-resistant and extensively drug-resistant strains. In this study, we explored the (1)H NMR-based metabolomics of TB using an aerobic TB infection model. Global profiling was applied to characterize the responses of C57Bl/6 mice to an aerobic infection with virulent Mycobacterium tuberculosis (MTB). The metabolic changes in organs (i.e., the lung, the target organ of TB, and the spleen and liver, remote systemic organs) and in serum from control and MTB-infected rats were investigated to clarify the host-pathogen interactions in MTB-infected host systems. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showed distinct separation between control and MTB-infected rats for all tissue and serum samples. Several tissue and serum metabolites were changed in MTB-infected rats, as compared to control rats. The precursors of membrane phospholipids, phosphocholine, and phosphoethanolamine, as well as glycolysis, amino acid metabolism, nucleotide metabolism, and the antioxidative stress response were altered based on the presence of MTB infection. This study suggests that NMR-based global metabolite profiling of organ tissues and serum could provide insight into the metabolic changes in host infected aerobically with virulent Mycobacterium tuberculosis.     

1H NMR-based metabolic profiling reveals inherent biological variation in yeast and nematode model systems

The application of metabolomics to human and animal model systems is poised to provide great insight into our understanding of disease etiology and the metabolic changes that are associated with these conditions. However, metabolomic studies have also revealed that there is significant, inherent biological variation in human samples and even in samples from animal model systems where the animals are housed under carefully controlled conditions. This inherent biological variability is an important consideration for all metabolomics analyses. In this study, we examined the biological variation in (1)H NMR-based metabolic profiling of two model systems, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Using relative standard deviations (RSD) as a measure of variability, our results reveal that both model systems have significant amounts of biological variation. The C. elegans metabolome possesses greater metabolic variance with average RSD values of 29 and 39%, depending on the food source that was used. The S. cerevisiae exometabolome RSD values ranged from 8% to 12% for the four strains examined. We also determined whether biological variation occurs between pairs of phenotypically identical yeast strains. Multivariate statistical analysis allowed us to discriminate between pair members based on their metabolic phenotypes. Our results highlight the variability of the metabolome that exists even for less complex model systems cultured under defined conditions. We also highlight the efficacy of metabolic profiling for defining these subtle metabolic alterations.     

Impact of diet-induced obesity on the mouse brain phosphoproteome

Obesity is closely associated to several diseases such as type 2 diabetes, cardiovascular disease, hepatic steatosis, airway disease, neurodegeneration, biliary diseases and certain cancers. It is, therefore, of importance to assess the role of nutrition in disease prevention as well as its effect in the course of such pathologies. In the present study, we addressed the impact of the exposure to different obesogenic diets in the mice brains phosphoproteome. To analyze if the obesity could be able to modify the protein pattern expression of brain neurons, obesity was induced in two different groups of mice. One group of mice was fed with hyperglycemic diet (HGD) and the other one was fed with high-fat diet (HFD), both for 12 weeks. A control group of lean mice was fed with a standard diet (SD). Metabolic parameters were measured before sacrifice, and brains were harvested for label-free phosphoproteomic analysis. Mice brains were analyzed to find differences, if any, in protein phosphorylation. Interestingly, the changes were independent of the obesogenic diet as no changes were detected between the two obese groups. Dephosphorylation of proteins involved in neuronal development (among others SYNGAP1 and PPP1R9B), in vesicle trafficking (for example SNAP91 and AMPH) and in cytoskeletal functions (for example, CLASP2 and GSK3B) was identified, while increased phosphorylation was detected for microtubule proteins (such as MAP2 and MAPT). Phospho site analysis of the mouse brain proteome reveals important changes that point to a connection between diet-induced obesity and impairment of neuronal functions and signaling.     

Beta-arrestin-1 protein represses diet-induced obesity

Diet-related obesity is a major metabolic disorder. Excessive fat mass is associated with type 2 diabetes, hepatic steatosis, and arteriosclerosis. Dysregulation of lipid metabolism and adipose tissue function contributes to diet-induced obesity. Here, we report that β-arrestin-1 knock-out mice are susceptible to diet-induced obesity. Knock-out of the gene encoding β-arrestin-1 caused increased fat mass accumulation and decreased whole-body insulin sensitivity in mice fed a high-fat diet. In β-arrestin-1 knock-out mice, we observed disrupted food intake and energy expenditure and increased macrophage infiltration in white adipose tissue. At the molecular level, β-arrestin-1 deficiency affected the expression of many lipid metabolic genes and inflammatory genes in adipose tissue. Consistently, transgenic overexpression of β-arrestin-1 repressed diet-induced obesity and improved glucose tolerance and systemic insulin sensitivity. Thus, our findings reveal that β-arrestin-1 plays a role in metabolism regulation.     

1H NMR-based metabolic profiling of urinary tract infection: combining multiple statistical models and clinical data

Urinary tract infection (UTI) encompasses a variety of clinical syndromes ranging from mild to life-threatening conditions. As such, it represents an interesting model for the development of an analytically based scoring system of disease severity and/or host response. Here we test the feasibility of this concept using ¹H NMR based metabolomics as the analytical platform. Using an exhaustively clinically characterized cohort and taking advantage of the multi-level study design, which opens possibilities for case?Ccontrol and longitudinal modeling, we were able to identify molecular discriminators that characterize UTI patients. Among those discriminators a number (e.g. acetate, trimethylamine and others) showed association with the degree of bacterial contamination of urine, whereas others, such as, for instance, scyllo-inositol and para-aminohippuric acid, are more likely to be the markers of morbidity.     

High resolution 1H NMR-based metabolomics indicates a neurotransmitter cycling deficit in cerebral tissue from a mouse model of Batten disease

The neuronal ceroid lipofuscinoses (NCLs) constitute a range of progressive neurological disorders primarily affecting children. Although six of the causative genes have been characterized, the underlying disease pathogenesis for this family of disorders is unknown. Using a metabolomics approach based on high resolution 1H NMR spectroscopy of the cortex, cerebellum, and remaining regions of the brain in conjunction with statistical pattern recognition, we report metabolic deficits associated with juvenile NCL in a Cln3 knock-out mouse model. Tissue from Cln3 null mutant mice aged 1-6 months was characterized by an increased glutamate concentration and a decrease in -amino butyric acid (GABA) concentration in aqueous extracts from the three regions of the brain. These changes are consistent with the reported altered expression of genes involved in glutamate metabolism in older mice and imply a change in neurotransmitter cycling between glutamate/glutamine and the production of GABA. Further variations in myo-inositol, creatine, and N-acetyl-aspartate were also identified. These metabolic changes were distinct from the normal aging/developmental process. Together, these changes represent the first documented pre-symptomatic symptoms of the Cln3 mouse at 1 month of age and demonstrate the versatility of 1H NMR spectroscopy as a tool for phenotyping mouse models of disease.     

1H NMR based metabolite profiling for optimizing the ethanol extraction of Wolfiporia cocos

Metabolite profiling of Wolfiporia cocos (family: Polyporaceae) had been much advancement in recent days, and its analysis by nuclear magnetic resonance (NMR) spectroscopy has become well established. However, the highly important trait of W. cocos still needs advanced protocols despite some standardization. Partial least squares discriminant analysis (PLS-DA) was used as the multivariate statistical analysis of the ¹H NMR data set. The PLS-DA model was validated, and the key metabolites contributing to the separation in the score plots of different ethanol W. cocos extract. ¹H NMR spectroscopy of W. cocos identified 33 chemically diverse metabolites in D₂O, consisting of 13 amino acids, 11 organic acids 2 sugars, 3 sugar alcohols, 1 nucleoside, and 3 others. Among these metabolites, the levels of tyrosine, proline, methionine, sarcosine, choline, acetoacetate, citrate, 4-aminobutyrate, aspartate, maltose, malate, lysine, xylitol, lactate threonine, leucine, valine, isoleucine, uridine, guanidoacetate, arabitol, mannitol, glucose, and betaine were increased in the 95% ethanol extraction sample compared with the levels in other samples, whereas level of acetate, phenylalanine, alanine, succinate, and fumarate were significantly increased in the 0% ethanol extraction sample. A biological triterpenoid, namely pachymic acid, was detected from different ethanol P. cocos extract using ¹H-NMR spectra were found in CDCl₃. This is the first report to perform the metabolomics profiling of different ethanol W. cocos extract. These researches suggest that W. cocos can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.     

Changes in brain activity after a diet-induced obesity

Compared to lean subjects, obese men have less activation in the dorsolateral prefrontal cortex, a brain area implicated in the inhibition of inappropriate behavior, satiety, and meal termination. Whether this deficit precedes weight gain or is an acquired feature of obesity remains unknown. An adult animal model of obesity may provide insight to this question since brain imaging can be performed in lean vs. obese conditions in a controlled study. Seven diet-induced obese adult minipigs were compared to nine lean adult minipigs housed in the same conditions. Brain activation after an overnight fasting was mapped in lean and obese subjects by single photon emission computed tomography. Cerebral blood flow, a marker of brain activity, was measured in isoflurane-anesthetized animals after the intravenous injection of 99mTc-HMPAO (750 MBq). Statistical analysis was performed using statistical parametric mapping (SPM) software and cerebral blood flow differences were determined using co-registered T1 magnetic resonance imaging (MRI) and histological atlases. Deactivations were observed in the dorsolateral and anterior prefrontal cortices in obese compared to lean subjects. They were also observed in several other structures, including the ventral tegmental area, the nucleus accumbens, and nucleus pontis. On the contrary, activations were found in four different regions, including the ventral posterior nucleus of the thalamus and middle temporal gyrus. Moreover, the anterior and dorsolateral prefrontal cortices as well as the insular cortex activity was negatively associated with the body weight. We suggested that the reduced activation of prefrontal cortex observed in obese humans is probably an acquired feature of obesity since it is also found in minipigs with a diet-induced obesity.     

1H NMR-based metabonomic profiling of rat serum and urine to characterize the subacute effects of carbamate insecticide propoxur

Carbamate insecticide propoxur is widely used in agriculture and public health programs. To prevent adverse health effects arising from exposure to this insecticide, sensitive methods for detection of early stage organismal changes are necessary. We present here an integrative metabonomic approach to investigate toxic effects of pesticide in experimental animals. Results showed that propoxur even at low dose levels can induce oxidative stress, impair liver function, enhance ketogenesis and fatty acid β-oxidation, and increase glycolysis, which contribute to the hepatotoxocity. These findings highlight the applicability of ¹H NMR spectroscopy and multivariate statistics in elucidating the toxic effects of propoxur.     

Genetic algorithm for shift-uncertainty correction in 1-D NMR-based metabolite identifications and quantifications

MOTIVATION: The analysis of metabolic processes is becoming increasingly important to our understanding of complex biological systems and disease states. Nuclear magnetic resonance spectroscopy (NMR) is a particularly relevant technology in this respect, since the NMR signals provide a quantitative measure of the metabolite concentrations. However, due to the complexity of the spectra typical of biological samples, the demands of clinical and high-throughput analysis will only be fully met by a system capable of reliable, automatic processing of the spectra. An initial step in this direction has been taken by Targeted Profiling (TP), employing a set of known and predicted metabolite signatures fitted against the signal. However, an accurate fitting procedure for (1)H NMR data is complicated by shift uncertainties in the peak systems caused by measurement imperfections. These uncertainties have a large impact on the accuracy of identification and quantification and currently require compensation by very time consuming manual interactions. Here, we present an approach, termed Extended Targeted Profiling (ETP), that estimates shift uncertainties based on a genetic algorithm (GA) combined with a least squares optimization (LSQO). The estimated shifts are used to correct the known metabolite signatures leading to significantly improved identification and quantification. In this way, use of the automated system significantly reduces the effort normally associated with manual processing and paves the way for reliable, high-throughput analysis of complex NMR spectra. RESULTS: The results indicate that using simultaneous shift uncertainty correction and least squares fitting significantly improves the identification and quantification results for (1)H NMR data in comparison to the standard targeted profiling approach and compares favorably with the results obtained by manual expert analysis. Preservation of the functional structure of the NMR spectra makes this approach more realistic than simple binning strategies.     

Metabolic profiling of Staphylococcus aureus cultivated under aerobic and anaerobic conditions with (1)H NMR-based nontargeted analysis

Staphylococcus aureus is a major pathogen in the medical area and food-producing sector. Detailed analyses of its basic cell physiology will help comprehensively understand this pathogen, which will be useful for developing novel diagnostic and treatment tools. Oxygen is one of the most crucial growth-limiting factors for S. aureus. In this study, to characterize and distinguish metabolic profiles of S. aureus cultivated under aerobic and anaerobic conditions, nontargeted analyses of both types of cultures were carried out using (1)H nuclear magnetic resonance spectroscopy. Fifty compounds were identified by Chenomx software. Characteristics of metabolic profiles were achieved by using principal components analysis. During aerobic growth, S. aureus mainly consumed glucose, alanine, arginine, glycine, isoleucine, leucine, phenylalanine, and acetate. Meanwhile, it accumulated 17 metabolites, mainly 2-oxoglutarate, isobutyrate, isovalerate, succinate, and ethanol. Under anaerobic condition, S. aureus mainly consumed glucose, arginine, and threonine. Meanwhile, it accumulated 13 metabolites, mainly ethanol, lactate, and ornithine. The representative metabolites that could most significantly differentiate metabolic profiles of S. aureus were isobutyrate, isovalerate, and succinate in aerobic cultivation; and lactate, ethanol, and ornithine in anaerobic cultivation. Among these metabolites, isobutyrate and ornithine were present only in aerobic and anaerobic culture, respectively.     

TRPV1-null mice are protected from diet-induced obesity

We explored a role for the capsaicin receptor, transient receptor potential channel vanilloid type 1 (TRPV1), in the regulation of feeding and body mass. On a 4.5% fat diet, wild-type and TRPV1-null mice gained equivalent body mass. On an 11% fat diet, however, TRPV1-null mice gained significantly less mass and adiposity; at 44 weeks the mean body weights of wild-type and TRPV1-null mice were approximately 51 and 34g, respectively. Both groups of mice consumed equivalent energy and absorbed similar amounts of lipids. TRPV1-null mice, however, exhibited a significantly greater thermogenic capacity. Interestingly, we found that 3T3-L1 preadipocytes expressed functional calcitonin gene-related peptide receptors. Thus, these data support a potential neurogenic mechanism by which TRPV1-sensitive sensory nerves may regulate energy and fat metabolism.     

NMR-based metabolic profiling of children with premature adrenarche

Metabolomics - The effects of lipopolysaccharides (i.e., endotoxin; LPS) on metabolism are poorly defined in lactating dairy cattle experiencing hyperlipidemia. Our objective was to explore the...     

Okara, soybean residue, prevents obesity in a diet-induced murine obesity model

We examined the effect of okara on the prevention of obesity in mice. A modified AIN-76 diet with a high fat content (14.1% of crude fat) was used as a basal diet. Male ICR mice were fed ad libitum with the basal diet or a dried okara-supplemented basal diet (10, 20, or 40%) for 10 weeks. The okara intake dose-dependently suppressed the development of body weight and epididymal white adipose tissue (EWAT), and prevented an increase of plasma lipids, including total cholesterol, LDL cholesterol, and non-esterified fatty acid. The okara intake also prevented steatosis in the liver. Real-time RT-PCR revealed that the okara intake induced down-regulation of the fatty acid synthetase gene and up-regulation of the cholesterol 7 alpha-hydroxylase (CYP7A1) gene in the liver. We also found that the okara intake caused a marked reduction in the expression of leptin and TNF-alpha genes in EWAT. Our results suggest that okara is beneficial in preventing obesity.     

1H NMR-based metabolite profiling as a potential selection tool for breeding passive resistance against Fusarium head blight (FHB) in wheat

¹H NMR measurements on protein-free extracts from wheat leaf and stem tissue were used to investigate the biochemical correlates of partial resistance to fungal species implicated in the Fusarium head blight (FHB) disease complex. The wheat genotypes included five commercial wheat cultivars and 116 wheat genotypes, from the CIMMYT international FHB breeding programme in Mexico, that had been bred for FHB disease resistance, utilizing exotic, typically Chinese, resistance sources. Principal component analysis of the nuclear magentic resonance (NMR)-derived metabolite profiles revealed distinct separation of the commercial wheat cultivars from the majority of the CIMMYT wheat genotypes with the commercial cultivars exhibiting markedly higher carbohydrate concentrations. A cross-validated partial least squares (PLS) regression model of the metabolite profile against the partial disease resistance component latent period (delay in sporulation of the fungus on the plant tissue) predicted latent periods that were significantly correlated with the experimentally determined values ( R ² = 0.34, P  < 0.001). Identified metabolites that were found in plants with shorter latent periods (higher disease susceptibility) included choline, the single most influential metabolite in the PLS model, betaine, the amino acids glutamine, glutamate and alanine, trans -aconitate and sucrose. Metabolites related to increased disease resistance included glucose and unassigned resonances in the carbohydrate and aromatic regions of the NMR spectra. The current study has demonstrated the potential of metabolite profiling as a tool for marker-assisted selection in commercial breeding for resistance to FHB in wheat.     

(1)H NMR-based metabonomic profiling of rat serum and urine to characterize the subacute effects of carbamate insecticide propoxur

Carbamate insecticide propoxur is widely used in agriculture and public health programs. To prevent adverse health effects arising from exposure to this insecticide, sensitive methods for detection of early stage organismal changes are necessary. We present here an integrative metabonomic approach to investigate toxic effects of pesticide in experimental animals. Results showed that propoxur even at low dose levels can induce oxidative stress, impair liver function, enhance ketogenesis and fatty acid β-oxidation, and increase glycolysis, which contribute to the hepatotoxocity. These findings highlight the applicability of (1)H NMR spectroscopy and multivariate statistics in elucidating the toxic effects of propoxur.