Changes In Metabolic Phenotype Research Articles

Phenotypic and Transcriptomic Response of Auxotrophic Mycobacterium avium Subsp. paratuberculosis leuD Mutant under Environmental Stress

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.

The ATP-P2X7 Signaling Axis Is Dispensable for Obesity-Associated Inflammasome Activation in Adipose Tissue

Inflammasome activation in adipose tissue has been implicated in obesity-associated insulin resistance and type 2 diabetes. However, when and how inflammasome is activated in adipose tissue remains speculative. Here we test the hypothesis that extracellular ATP, a potent stimulus of inflammasome in macrophages via purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7), may play a role in inflammasome activation in adipose tissue in obesity. Our data show that inflammasome is activated in adipose tissue upon 8-week feeding of 60% high-fat diet (HFD), coinciding with the onset of hyperglycemia and hyperinsulinemia as well as the induction of P2X7 in adipose tissue. Unexpectedly, P2X7-deficient animals on HFD exhibit no changes in metabolic phenotypes, inflammatory responses, or inflammasome activation when compared with the wild-type controls. Similar observations have been obtained in hematopoietic cell–specific P2X7-deficient animals generated by bone marrow transplantation. Thus, we conclude that inflammasome activation in adipose tissue in obesity coincides with the onset of hyperglycemia and hyperinsulinemia but, unexpectedly, is not mediated by the ATP-P2X7 signaling axis. The nature of the inflammasome-activating danger signal(s) in adipose tissue in obesity remains to be characterized.

Serum Metabolite Concentrations and Decreased GFR in the General Population

Metabolites such as creatinine and urea are established kidney function markers. High-throughput metabolomic studies have not been reported in large general population samples spanning normal kidney function and chronic kidney disease (CKD). Cross-sectional observational studies of the general population. 2 independent samples: KORA F4 (discovery sample, n = 3,011) and Twins UK (validation sample, n = 984). EXPOSURE FACTORS: 151 serum metabolites, quantified by targeted mass spectrometry. Metabolites and their 22,650 ratios were analyzed by multivariable-adjusted linear regression for their association with glomerular filtration rate (eGFR), estimated separately from creatinine and cystatin C levels by CKD-EPI (CKD Epidemiology Collaboration) equations. After correction for multiple testing, significant metabolites (P < 3.3 × 10(-4) for single metabolites; P < 2.2 × 10(-6) for ratios) were meta-analyzed with independent data from the TwinsUK Study. Replicated associations with eGFR were observed for 22 metabolites and 516 metabolite ratios. Pooled P values ranged from 7.1 × 10(-7) to 1.8 × 10(-69) for the replicated single metabolites. Acylcarnitines such as glutarylcarnitine were associated inversely with eGFR (-3.73 mL/min/1.73 m(2) per standard deviation [SD] increase, pooled P = 1.8 × 10(-69)). The replicated ratio with the strongest association was the ratio of serine to glutarylcarnitine (P = 3.6 × 10(-81)). Almost all replicated phenotypes associated with decreased eGFR (<60 mL/min/1.73 m(2); n = 172 cases) in KORA F4: per 1-SD increment, ORs ranged from 0.29-2.06. Across categories of a metabolic score consisting of 3 uncorrelated metabolites, the prevalence of decreased eGFR increased from 3% to 53%. Cross-sectional study design, GFR was estimated, limited number of metabolites. Distinct metabolic phenotypes were reproducibly associated with eGFR in 2 separate population studies. They may provide novel insights into renal metabolite handling, improve understanding of pathophysiology, or aid in the diagnosis of kidney disease. Longitudinal studies are needed to clarify whether changes in metabolic phenotypes precede or result from kidney function impairment.

Abstract P137: Metabolomic Analysis of the Early and Late Hypertrophic Heart

The metabolic adaptations to acute myocardial pressure overload are characterized by alterations in metabolism that drive the hypertrophic response and that balance workload with energy demand. Under conditions of chronic pressure overload, it is known that substrate utilization becomes less flexible and that the heart shifts energy preference from fatty acids to glucose. Nevertheless, the metabolic changes that underlie the progression of compensated hypertrophy to heart failure are incompletely understood and attempts to correct the known metabolic defects to delay decompensation have been largely unsuccessful. To identify key changes in metabolic phenotype that could underlie progression to heart failure, we measured metabolites in a transverse aortic constriction (TAC) mouse model using an unbiased metabolomic approach. Hearts were harvested 1 d, 1 wk and 8 wks after sham or TAC operation, and metabolites were extracted from the hearts and analyzed via GC/MS and LC/MS/MS. The signal intensities of 288 named metabolites were re-scaled to median values. Welch’s t-test and two-way ANOVA were used to identify metabolites that changed significantly with pressure overload and progression to heart failure. Echocardiographic measurements showed a significant decrease in ejection fraction after 1 d (65±2% vs. 49±5%) and 8 wks (61±2% vs. 34±8%) of TAC; 1 wk of TAC showed a compensated phenotype characterized by a largely preserved ejection fraction. One day after TAC, only 1.7% of the metabolites changed significantly; however, nearly all amino acids measured were increased by 1 wk. By 8 wks, amino acids returned to near sham levels and a significant and robust decrease in phospholipid, carnitine, inositol, sterol, and fatty acid metabolites occurred. These findings demonstrate that the temporal changes in metabolic phenotype are more complex than previously thought. The preservation of pathways involved in lipid and amino acid metabolism may be important for maintaining myocardial energetics and preventing pump failure under conditions of chronic pressure overload.

Metabolomics Analysis of the Responses to Partial Hepatectomy in Hepatocellular Carcinoma Patients

In this study, liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOFMS) was employed for investigating the metabolome of the sera collected from hepatocellular carcinoma (HCC) patients before and 3 to 5 months after partial hepatectomy. To investigate the changes in metabolic phenotypes after the hepatic resection, principal components analysis (PCA) and support vector machine (SVM) were performed for the data grouping and classification. Based on the obtained SVM model, mass spectrometry spectra, database searching as well as the confirmation from authentic standards, several differentiating metabolites were tentatively identified. To improve visualization, z-score plot and heat map display were performed, which exhibited the changes in concentration of the metabolites. As a result, depletion of circulating carnitine, reduced amino acid biosynthesis and increased rate of lipid peroxidation were observed. Meanwhile, up-regulation of hypoxanthine indicated that purine metabolism might serve as the salvage pathway. Collectively, the results reflected metabolic responses to surgical operation in HCC patients, suggesting perturbation of energy metabolism may occur in 3 to 5 months after the partial hepatectomy.

Change in Vascular Adhesion Protein‐1 and Metabolic Phenotypes after Vertical Banded Gastroplasty for Morbid Obesity

The association between circulating vascular adhesion protein-1 (VAP-1) and metabolic phenotypes has been shown to be inconsistent. The current study explored whether the changes in serum VAP-1 levels correlate with the changes in metabolic phenotypes after weight reduction surgery. Clinical characteristics and serum VAP-1 levels in 20 morbidly obese subjects (mean BMI 38.84 kg/m(2)) were measured before and after vertical banded gastroplasty. Before surgery, serum VAP-1 levels correlated positively with fasting plasma glucose (gamma = 0.56, p = 0.01) and negatively with insulin levels (gamma = -0.51, p = 0.021). After surgery, the changes in serum VAP-1 levels were negatively correlated with the changes in waist circumference (gamma = -0.57, p = 0.011), diastolic blood pressure (DBP) (gamma = -0.56, p = 0.015), and mean arterial pressure (gamma = -0.46, p = 0.055). In multivariate regression, serum VAP-1 levels were negatively correlated with waist circumference (beta = -2.36, p = 0.014) and DBP (beta = -3.02, p = 0.017) after adjusting for age and gender. The change in DBP was negatively correlated with the change in VAP-1 levels after adjusting for age, gender, and steady-state plasma glucose. The results suggest that VAP-1 levels are correlated with fasting glucose and insulin levels in morbidly obese subjects. After surgery, the changes in VAP-1 levels were associated with changes in visceral adiposity and DBP. Serum VAP-1 might modulate DBP independently from the changes in insulin resistance in morbidly obese people.

A metabolic hypothesis of cell growth and death in pancreatic cancer.

Tumor cells, just as other living cells, possess the potential for proliferation, differentiation, cell cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these conditions, characterized by the production of both energy and special substrates necessary for the cells to function in that particular state. Unlike that of normal living cells, the metabolic phenotype of tumor cells supports the proliferative state. To present the metabolic hypothesis that (1) cell transformation and tumor growth are associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, while enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition, and (2) phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another. Evidence is presented that demonstrates opposite changes in metabolic phenotypes induced by TGF-beta, a cell-transforming agent, and tumor growth-inhibiting phytochemicals such as genistein and Avemar, or novel synthetic anti-leukemic drugs such as STI571 (Gleevec). Intermediary metabolic enzymes that mediate the growth signaling pathways and promote malignant cell transformation may serve as high-efficacy nongenetic novel targets for cancer therapies.

Gleevec (STI571) Influences Metabolic Enzyme Activities and Glucose Carbon Flow toward Nucleic Acid and Fatty Acid Synthesis in Myeloid Tumor Cells

Chronic myeloid leukemia cells contain a constitutively active Bcr-Abl tyrosine kinase, the target protein of Gleevec (STI571) phenylaminopyrimidine class protein kinase inhibitor. Here we provide evidence for metabolic phenotypic changes in cultured K562 human myeloid blast cells after treatment with increasing doses of STI571 using [1,2-13C2]glucose as the single tracer and biological mass spectrometry. In response to 0.68 and 6.8 microm STI571, proliferation of Bcr-Abl-positive K562 cells showed a 57% and 74% decrease, respectively, whereas glucose label incorporation into RNA decreased by 13.4% and 30.1%, respectively, through direct glucose oxidation, as indicated by the decrease in the m1/Sigma(m)n ratio in RNA. Based on the in vitro proliferation data, the IC50 of STI571 in K562 cultures is 0.56 microm. The decrease in 13C label incorporation into RNA ribose was accompanied by a significant fall in hexokinase and glucose-6-phosphate 1-dehydrogenase activities. The activity of transketolase, the enzyme responsible for nonoxidative ribose synthesis in the pentose cycle, was less affected, and there was a relative increase in glucose carbon incorporation into RNA through nonoxidative synthesis as indicated by the increase in the m2/Sigma(m)n ratio in RNA. The restricted use of glucose carbons for de novo nucleic acid and fatty acid synthesis by altering metabolic enzyme activities and pathway carbon flux of the pentose cycle constitutes the underlying mechanism by which STI571 inhibits leukemia cell glucose substrate utilization and growth. The administration of specific hexokinase/glucose-6-phosphate 1-dehydrogenase inhibitor anti-metabolite substrates or competitive enzyme inhibitor compounds, alone or in combination, should be explored for the treatment of STI571-resistant advanced leukemias as well as that of Bcr-Abl-negative human malignancies.