The stimulation of insulin secretion by glucose can be modulated by multiple nutritive, hormonal, and pharmacological inputs. Fatty acids potentiate insulin secretion through the generation of intracellular signaling molecules and through the activation of cell surface receptors. The G-protein–coupled receptor 40 (GPR40), also known as free fatty acid receptor 1 (we will use GPR40 in this review), has emerged as an important component in the fatty acid augmentation of insulin secretion. By signaling predominantly through Gαq/11, GPR40 increases intracellular calcium and activates phospholipases to generate diacylglycerols resulting in increased insulin secretion. Synthetic small-molecule agonists of GPR40 enhance insulin secretion in a glucose-dependent manner in vitro and in vivo with a mechanism similar to that found with fatty acids. GPR40 agonists have shown efficacy in increasing insulin secretion and lowering blood glucose in rodent models of type 2 diabetes. Recent phase I and phase II clinical trials in humans have shown that the GPR40 agonist TAK-875 reduces fasting and postprandial blood glucose and lowers HbA1c with efficacy equal to that of the sulfonylurea glimepiride without inducing hypoglycemia or evidence of tachyphylaxis. These data suggest that targeting the GPR40 receptor can be a viable therapeutic option for the treatment of type 2 diabetes.
Congenital generalized lipodystrophy (CGL), secondary to AGPAT2 mutation is characterized by the absence of adipocytes and development of severe insulin resistance. In the current study, we investigated the adipogenic defect associated with AGPAT2 mutations. Adipogenesis was studied in muscle-derived multipotent cells (MDMCs) isolated from vastus lateralis biopsies obtained from controls and subjects harboring AGPAT2 mutations and in 3T3-L1 preadipocytes after knockdown or overexpression of AGPAT2. We demonstrate an adipogenic defect using MDMCs from control and CGL human subjects with mutated AGPAT2. This defect was rescued in CGL MDMCs with a retrovirus expressing AGPAT2. Both CGL-derived MDMCs and 3T3-L1 cells with knockdown of AGPAT2 demonstrated an increase in cell death after induction of adipogenesis. Lack of AGPAT2 activity reduces Akt activation, and overexpression of constitutively active Akt can partially restore lipogenesis. AGPAT2 modulated the levels of phosphatidic acid, lysophosphatidic acid, phosphatidylinositol species, as well as the peroxisome proliferator–activated receptor γ (PPARγ) inhibitor cyclic phosphatidic acid. The PPARγ agonist pioglitazone partially rescued the adipogenic defect in CGL cells. We conclude that AGPAT2 regulates adipogenesis through the modulation of the lipome, altering normal activation of phosphatidylinositol 3-kinase (PI3K)/Akt and PPARγ pathways in the early stages of adipogenesis.
Acute respiratory distress syndrome (ARDS) remains a significant hazard to human health and is clinically challenging because there are no prognostic biomarkers and no effective pharmacotherapy. The lung compartment metabolome may detail the status of the local environment that could be useful in ARDS biomarker discovery and the identification of drug target opportunities. However, neither the utility of bronchoalveolar lavage fluid (BALF) as a biofluid for metabolomics nor the optimal analytical platform for metabolite identification are established. To address this, we undertook a study to compare metabolites in BALF samples from patients with ARDS and healthy controls using a newly developed liquid chromatography (LC)-mass spectroscopy (MS) platform for untargeted metabolomics. Following initial testing of three different high performance liquid chromatography (HPLC) columns, we determined that reversed phase (RP)-LC and hydrophilic interaction chromatography (HILIC), were the most informative chromatographic methods because they yielded the most and highest quality data. Following confirmation of metabolite identification, statistical analysis resulted in 37 differentiating metabolites in the BALF of ARDS compared with health across both analytical platforms. Pathway analysis revealed networks associated with amino acid metabolism, glycolysis and gluconeogenesis, fatty acid biosynthesis, phospholipids and purine metabolism in the ARDS BALF. The complementary analytical platforms of RPLC and HILIC-LC generated informative, insightful metabolomics data of the ARDS lung environment.
biomarkers; critical illness; metabolomics; lung injury; bioinformatics; phospholipids; lactate; xanthine oxidase; hippurate; pharmacotherapy
A critical application of metabolomics is the evaluation of tissues, which are often the primary sites of metabolic dysregulation in disease. Laboratory rodents have been widely used for metabolomics studies involving tissues due to their facile handing, genetic manipulability and similarity to most aspects of human metabolism. However, the necessary step of administration of anesthesia in preparation for tissue sampling is not often given careful consideration, in spite of its potential for causing alterations in the metabolome. We examined, for the first time using untargeted and targeted metabolomics, the effect of several commonly used methods of anesthesia and euthanasia for collection of skeletal muscle, liver, heart, adipose and serum of C57BL/6J mice. The data revealed dramatic, tissue-specific impacts of tissue collection strategy. Among many differences observed, post-euthanasia samples showed elevated levels of glucose 6-phosphate and other glycolytic intermediates in skeletal muscle. In heart and liver, multiple nucleotide and purine degradation metabolites accumulated in tissues of euthanized compared to anesthetized animals. Adipose tissue was comparatively less affected by collection strategy, although accumulation of lactate and succinate in euthanized animals was observed in all tissues. Among methods of tissue collection performed pre-euthanasia, ketamine showed more variability compared to isoflurane and pentobarbital. Isoflurane induced elevated liver aspartate but allowed more rapid initiation of tissue collection. Based on these findings, we present a more optimal collection strategy mammalian tissues and recommend that rodent tissues intended for metabolomics studies be collected under anesthesia rather than post-euthanasia.
The formation of hepatocyte Mallory-Denk bodies (MDBs), which are aggregates of keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin-binding protein p62, has a genetic predisposition component in humans and mice. We tested the hypothesis that metabolomic profiling of the MDB-susceptible C57BL and the MDB-resistant C3H mouse strains can illuminate MDB-associated pathways. Using both targeted and unbiased metabolomic analyses we demonstrated significant differences in intermediates of purine metabolism. Further analysis revealed that C3H and C57BL livers differ significantly in the mRNA level, protein expression, and enzymatic activity of the adenosine-generating enzyme ecto-5′-nucleotidase (CD73), which was significantly lower in C57BL livers. CD73 mRNA levels were also dramatically decreased in human liver biopsies from hepatitis C and non-alcoholic fatty liver disease patients. Feeding mice with a diet containing the MDB-inducing agent 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) significantly decreased CD73 protein and activity in C57BL livers and resulted in loss of plasma membrane CD73 expression and activity in isolated mouse hepatocytes. To further examine the role of CD73 in MDB formation in vivo, we fed wild-type and CD73−/− mice a DDC-containing diet. Liver enlargement, p62 induction, and disappearance of the K8/K18 cytoskeleton were attenuated in CD73−/− compared to wild-type livers. MDB formation, as assessed by biochemical and immunofluorescence detection of keratin and ubiquitin complexes, was also absent in CD73−/− mice. CONCLUSION: Purine metabolism and CD73 expression are linked to susceptibility to MDB formation in livers of different mouse strains. The expression of the adenosine-generating enzyme CD73 contributes to experimental MDB induction and is highly regulated in MDB-associated liver injury in mice and in chronic human liver disease.
metabolomics; purines; adenosine; protein aggregation
To evaluate the impact of a managed care obesity intervention that requires enrollment in an intensive medical weight management program, a commercial weight loss program, or a commercial pedometer-based walking program to maintain enhanced benefits.
Design and Methods
Prospective observational study involving 1,138 adults with BMI ≥32 kg/m2 with one or more comorbidities or BMI ≥35 kg/m2 enrolled in a commercial, independent practice association-model health maintenance organization. Body mass index, blood pressure, lipids, HbA1c or fasting glucose, and per-member per-month costs were assessed one year before and one year after program implementation.
Program uptake (90%) and one year adherence (79%) were excellent. Enrollees in all three programs exhibited improved clinical outcomes and reduced rates of increase in direct medical costs compared to members who did not enroll in any program.
A managed care obesity intervention that offered financial incentives for participation and a variety of programs was associated with excellent program uptake and adherence, improvements in cardiovascular risk factors, and a lower rate of increase in direct medical costs over one year.
weight loss; cost analysis; managed care
Motifs are repeating patterns that determine the local properties of networks. In this work, we characterized all 3-node motifs using enzyme commission numbers of the International Union of Biochemistry and Molecular Biology to show that motif abundance is related to biochemical function. Further, we present a comparative analysis of motif distributions in the metabolic networks of 21 species across six kingdoms of life. We found the distribution of motif abundances to be similar between species, but unique across cellular organelles. Finally, we show that motifs are able to capture inter-species differences in metabolic networks and that molecular differences between some biological species are reflected by the distribution of motif abundances in metabolic networks.
Heightened impulsivity and inefficient inhibitory control are increasingly recognized as risk factors for unhealthy eating and obesity but the underlying processes are not fully understood. We used structural equation modeling to investigate the relationships between impulsivity, inhibitory control, eating behavior, and body mass index (BMI) in 210 undergraduates who ranged from underweight to obese. We demonstrate that impulsivity and inhibitory control deficits are positively associated with several facets of unhealthy eating, including overeating in response to external food cues and in response to negative emotional states, and making food choices based on taste preferences without consideration of health value. We further show that such unhealthy eating is, for the most part, associated with increased BMI, with the exception of Restraint Eating, which is negatively associated with BMI. These results add to our understanding of the impact of individual differences in impulsivity and inhibitory control on key aspects of unhealthy eating and may have implications for the treatment and prevention of obesity.
cognitive control; executive function; response inhibition; decision making; eating; obesity; structural equation modeling
Omega-3 (n-3) fatty acids have been implicated in mood disorders, yet clinical trials supplementing n-3 fats have shown mixed results. However, the predominant focus of this research has been on the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). We used an unbiased approach to assay plasma n-3 and omega-6 (n-6) species that interact at the level of biosynthesis and down-stream processing, to affect brain function and, potentially, mood. We used lipomic technology to assay plasma levels of n-3 and n-6 fatty acids from 40 bipolar and 18 control subjects to investigate differences in plasma levels and associations with the burden of disease markers, neuroticism and global assessment of function (GAF) and mood state (Hamilton Depression Scale (HAM-D)). Most significantly, we found the levels of dihomo-gamma-linolenic acid (DGLA) to positively correlate with neuroticism and HAM-D scores and negatively correlate with GAF scores; and HAM-D to negatively correlate with linoleic acid (LA) and positively correlate with fatty acid desaturase 2 (FADS2) activity, an enzyme responsible for converting LA to gamma-linolenic acid (GLA). These associations remained significant following Bonferroni multiple testing correction. These data suggest that specific n-6 fatty acids and the enzymes that control their biosynthesis may be useful biomarkers in measurements of depressive disorders and burden of disease, and that they should be considered when investigating the roles of n-3s.
omega-3 fatty acids; omega-6 fatty acids; dihomogamma linolenic acid; fatty acid desaturase; neuroticism; global assessment of function
Aerobic capacity is a strong predictor of all-cause mortality and can influence many complex traits. To explore the biological basis underlying this connection, we developed via artificial selection two rat lines that diverge for intrinsic (i.e. inborn) aerobic capacity and differ in risk for complex disease traits. Here we conduct the first in-depth pedigree and molecular genetic analysis of these lines, the high capacity runners (HCR) and low capacity runners (LCR). Our results show that both HCR and LCR lines maintain considerable narrow-sense heritability (h2) for the running capacity phenotype over 28 generations (h2 = 0.47 ± 0.02 and 0.43 ± 0.02, respectively). To minimize inbreeding, the lines were maintained by rotational mating. Pedigree records predict that the inbreeding coefficient increases at a rate of <1% per generation, ~37-38% slower than expected for random mating. Genome-wide 10K SNP genotype data for generations 5, 14, and 26 demonstrate substantial genomic evolution: between-line differentiation increased progressively, while within-line diversity deceased. Genome-wide average heterozygosity decreased at a rate of <1% per generation, consistent with pedigree-based predictions and confirming the effectiveness of rotational breeding. Linkage disequilibrium index r2 decreases to 0.3 at ~3 Mb, suggesting that the resolution for mapping quantitative trait loci (QTL) can be as high as 2-3 cM. To establish a test population for QTL mapping, we conducted an HCR-LCR intercross. Running capacity of the F1 population (n=176) was intermediate of the HCR and LCR parentals (28 pairs); and the F2 population (n=645) showed a wider range of phenotypic distribution. Importantly, heritability in the F0-F2 pedigree remained high (h2~0.6). These results suggest that the HCR-LCR lines can serve as a valuable system for studying genomic evolution, and a powerful resource for mapping QTL for a host of characters relevant to human health.
Progress in developing treatments for diabetic neuropathy is slowed by our limited understanding of how disturbances in metabolic substrates- glucose and fatty acids- produce nerve injury. In this review, we present the current oxidative stress hypothesis and experimental data that support it. We identify weaknesses in our understanding of diabetes-disordered metabolism in the neurovascular unit; i.e. in critical cell types of the microvascular endothelium, peripheral sensory neurons, and supporting Schwann cells. Greater understanding of peripheral nervous system bioenergetics may provide insight into new drug therapies or improvements in dietary interventions in diabetes or even pre-diabetes.
Background: The biochemical pathways underlying glucose-stimulated insulin secretion have not been fully elucidated.
Results: Mass spectrometry analysis revealed rapid and substantial metabolic reprogramming evoked by glucose in INS-1 cells.
Conclusion: Metabolomics allowed testing and generation of multiple hypotheses regarding glucose effects in insulin-secreting cells.
Significance: Insights into the biochemical basis of glucose-stimulated insulin secretion are critical for understanding root causes of type 2 diabetes.
Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mm glucose and in response to increasing concentrations of glucose in the INS-1 832/13 β-cell line. U-[13C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.
Beta Cell; Insulin Secretion; Intermediary Metabolism; Metabolism; Metabolomics
Low aerobic exercise capacity is a powerful predictor of premature morbidity and mortality for healthy adults as well as those with cardiovascular disease For aged populations, poor performance on treadmill or extended walking tests indicates closer proximity to future health declines. Together, these findings suggest a fundamental connection between aerobic capacity and longevity.
Through artificial selective breeding, we developed an animal model system to prospectively test the association between aerobic exercise capacity and survivability (aerobic hypothesis).
Methods and Results
Laboratory rats of widely diverse genetic backgrounds (N:NIH stock) were selectively bred for low or high intrinsic (inborn) treadmill running capacity. Cohorts of male and female rats from generations 14, 15 and 17 of selection were followed for survivability and assessed for age-related declines in cardiovascular fitness including maximal oxygen uptake (VO2max), myocardial function, endurance performance, and change in body mass. Median lifespan for low exercise capacity rats was 28-45% shorter than high capacity rats (hazard ratio, 0.06; P<.001). VO2max, measured across adulthood was a reliable predictor of lifespan (P<.001). During progression from adult to old age, left ventricular myocardial and cardiomyocyte morphology, contractility, and intracellular Ca2+ handling in both systole and diastole, as well as mean blood pressure, were more compromised in rats bred for low aerobic capacity. Physical activity levels, energy expenditure (VO2), and lean body mass were all better sustained with age in rats bred for high aerobic capacity.
These data obtained from a contrasting heterogeneous model system provide strong evidence that genetic segregation for aerobic exercise capacity can be linked with longevity and useful for deeper mechanistic exploration.
Exercise capacity; longevity; cardiomyocyte function; rat models; aging
AGPAT isoforms catalyze the acylation of lysophosphatidic acid (LPA) to form phosphatidic acid (PA). AGPAT2 mutations are associated with defective adipogenesis. Muscle and adipose tissue share common precursor cells. We investigated the role of AGPAT isoforms in skeletal muscle development. We demonstrate that small interference RNA-mediated knockdown of AGPAT1 expression prevents the induction of myogenin, a key transcriptional activator of the myogenic program, and inhibits the expression of myosin heavy chain. This effect is rescued by transfection with AGPAT1 but not AGPAT2. Knockdown of AGPAT2 has no effect. The regulation of myogenesis by AGPAT1 is associated with alterations on actin cytoskeleton. The role of AGPAT1 on actin cytoskeleton is further supported by colocalization of AGPAT1 to areas of active actin polymerization. AGPAT1 overexpression was not associated with an increase in PA levels. Our observations strongly implicate AGPAT1 in the development of skeletal muscle, specifically to terminal differentiation. These findings are linked to the regulation of actin cytoskeleton.
Cytoskeleton; Phosphatidic acid; AGPAT2; C2C12; Skeletal muscle; Actin
Immune cells are somewhat unique in that activation responses can alter quantitative phenotypes upwards of 100,000-fold. To date little is known about the metabolic adaptations necessary to mount such dramatic phenotypic shifts. Screening for novel regulators of macrophage activation, we found nonprotein kinases of glucose metabolism among the most enriched classes of candidate immune modulators. We find that one of these, the carbohydrate kinase-like protein CARKL, is rapidly downregulated in vitro and in vivo upon LPS stimulation in both mice and humans. Interestingly, CARKL catalyzes an orphan reaction in the pentose phosphate pathway, refocusing cellular metabolism to a high-redox state upon physiological or artificial downregulation. We find that CARKL-dependent metabolic reprogramming is required for proper M1- and M2-like macrophage polarization and uncover a rate-limiting requirement for appropriate glucose flux in macrophage polarization.
► Screened 199 human kinases for their immunoregulatory potential ► CARKL bridges glycolysis, the pentose phosphate pathway, and immune function ► CARKL focuses cellular metabolism toward a “high-redox” state ► CARKL regulation is required for macrophage polarization
A simple, fast, and reproducible sample preparation procedure was developed for relative quantification of metabolites in adherent mammalian cells using the clonal β-cell line INS-1 as a model sample. The method was developed by evaluating the effect of different sample preparation procedures on high performance liquid chromatography- mass spectrometry quantification of 27 metabolites involved in glycolysis and the tricarboxylic acid cycle on a directed basis as well as for all detectable chromatographic features on an undirected basis. We demonstrate that a rapid water rinse step prior to quenching of metabolism reduces components that suppress electrospray ionization thereby increasing signal for 26 of 27 targeted metabolites and increasing total number of detected features from 237 to 452 with no detectable change of metabolite content. A novel quenching technique is employed which involves addition of liquid nitrogen directly to the culture dish and allows for samples to be stored at −80 °C for at least 7 d before extraction. Separation of quenching and extraction steps provides the benefit of increased experimental convenience and sample stability while maintaining metabolite content similar to techniques that employ simultaneous quenching and extraction with cold organic solvent. The extraction solvent 9:1 methanol: chloroform was found to provide superior performance over acetonitrile, ethanol, and methanol with respect to metabolite recovery and extract stability. Maximal recovery was achieved using a single rapid (~1 min) extraction step. The utility of this rapid preparation method (~5 min) was demonstrated through precise metabolite measurements (11% average relative standard deviation without internal standards) associated with step changes in glucose concentration that evoke insulin secretion in the clonal β-cell line INS-1.
metabolomics; liquid chromatography-mass spectrometry; adherent mammalian cell sample preparation; INS-1; β-cells; HILIC; solvent; extraction time
Selection on running capacity has created rat phenotypes of high capacity runners (HCR) that have enhanced cardiac function and low capacity runners (LCR) that exhibit risk factors of metabolic syndrome. We analysed hearts of HCR and LCR from generation 22 of selection using DIGE and identified proteins from MS database searches. The running capacity of HCR was 6-fold greater than LCR. DIGE resolved 957 spots and proteins were unambiguously identified in 369 spots. Protein expression profiling detected 67 statistically significant (P<0.05; false discovery rate <10 %, calculated using q-values) differences between HCR and LCR. Hearts of HCR rats exhibited robust increases in the abundance of each enzyme of the beta-oxidation pathway. In contrast, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCR also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of alpha B-crystallin at serine 59, which is a common point of convergence in cardiac stress signalling. Thus proteomic analysis revealed selection on low running capacity is associated with perturbations in cardiac energy metabolism and provided the first evidence that the LCR cardiac proteome is exposed to greater oxidative stress.
2D Gel Electrophoresis; Mass Spectrometry; Animal Selection Model
Polyunsaturated fatty acids (PUFA) have shown efficacy in the treatment of bipolar disorder, however their specific role in treating the illness is unclear. Serum PUFA and dietary intakes of PUFA associate with suicidal behavior in epidemiological studies. The objective of this study was to assess serum n-3 and n-6 PUFA levels in bipolar subjects and determine possible associations with suicidal risk, including suicidal history and relevant personality factors that have been associated with suicidality. We studied 27 bipolar subjects using the NEO-PI to assess the big five personality factors, structured interviews to verify diagnosis and assess suicidal history, and lipomics to quantify n-3 and n-6 PUFA in serum. We found positive associations between personality factors and ratios of n-3 PUFA, suggesting that conversion of short chain to long chain n-3s and the activity of enzymes in this pathway may associate with measures of personality. Thus, ratios of docosahexaenoic acid (DHA) to alpha linolenic acid (ALA) and the activity of fatty acid desaturase 2 (FADS2) involved in the conversion of ALA to DHA were positively associated with openness factor scores. Ratios of eicosapentaenoic acid (EPA) to ALA and ratios of EPA to DHA were positively associated with agreeableness factor scores. Finally, serum concentrations of the n-6, arachidonic acid (AA), were significantly lower in subjects with a history of suicide attempt compared to non-attempters. The data suggest that specific lipid profiles, which are controlled by an interaction between diet and genetics, correlate with suicidal history and personality factors related to suicidal risk. This study provides preliminary data for future studies to determine whether manipulation of PUFA profiles (through diet or supplementation) can affect personality measures and disease outcome in bipolar subjects and supports the need for further investigations into individualized specific modulations of lipid profiles to add adjunctive value to treatment paradigms.
Summary: Metscape is a plug-in for Cytoscape, used to visualize and interpret metabolomic data in the context of human metabolic networks. We have developed a metabolite database by extracting and integrating information from several public sources. By querying this database, Metscape allows users to trace the connections between metabolites and genes, visualize compound networks and display compound structures as well as information for reactions, enzymes, genes and pathways. Applying the pathway filter, users can create subnetworks that consist of compounds and reactions from a given pathway. Metscape allows users to upload experimental data, and visualize and explore compound networks over time, or experimental conditions. Color and size of the nodes are used to visualize these dynamic changes. Metscape can display the entire metabolic network or any of the pathway-specific networks that exist in the database.
Availability: Metscape can be installed from within Cytoscape 2.6.x under ‘Network and Attribute I/O’ category. For more information, please visit http://metscape.ncibi.org/tryplugin.html
Supplementary information: Supplementary data are available at Bioinformatics online.
Metabolite profiles can be used for identifying molecular signatures and mechanisms underlying diseases since they reflect the outcome of complex upstream genomic, transcriptomic, proteomic and environmental events. The scarcity of publicly accessible large scale metabolome datasets related to human disease has been a major obstacle for assessing the potential of metabolites as biomarkers as well as understanding the molecular events underlying disease-related metabolic changes. The availability of metabolite and gene expression profiles for the NCI-60 cell lines offers the possibility of identifying significant metabolome and transcriptome features and discovering unique molecular processes related to different cancer types.
We utilized a combination of analytical methods in the R statistical package to evaluate metabolic features associated with cancer cell lines from different tissue origins, identify metabolite-gene correlations and detect outliers cell lines based on metabolome and transcriptome data. Statistical analysis results are integrated with metabolic pathway annotations as well as COSMIC and Tumorscape databases to explore associated molecular mechanisms.
Our analysis reveals that although the NCI-60 metabolome dataset is quite noisy comparing with microarray-based transcriptome data, it does contain tissue origin specific signatures. We also identified biologically meaningful gene-metabolite associations. Most remarkably, several abnormal gene-metabolite relationships identified by our approach can be directly linked to known gene mutations and copy number variations in the corresponding cell lines.
Our results suggest that integrative metabolome and transcriptome analysis is a powerful method for understanding molecular machinery underlying various pathophysiological processes. We expect the availability of large scale metabolome data in the coming years will significantly promote the discovery of novel biomarkers, which will in turn improve the understanding of molecular mechanism underlying diseases.
The molecular mechanisms underlying the sex differences in human muscle morphology and function remain to be elucidated. The sex differences in the skeletal muscle transcriptome in both the resting state and following anabolic stimuli, such as resistance exercise (RE), might provide insight to the contributors of sexual dimorphism of muscle phenotypes. We used microarrays to profile the transcriptome of the biceps brachii of young men and women who underwent an acute unilateral RE session following 12 weeks of progressive training. Bilateral muscle biopsies were obtained either at an early (4 h post-exercise) or late recovery (24 h post-exercise) time point. Muscle transcription profiles were compared in the resting state between men (n = 6) and women (n = 8), and in response to acute RE in trained exercised vs. untrained non-exercised control muscle for each sex and time point separately (4 h post-exercise, n = 3 males, n = 4 females; 24 h post-exercise, n = 3 males, n = 4 females). A logistic regression-based method (LRpath), following Bayesian moderated t-statistic (IMBT), was used to test gene functional groups and biological pathways enriched with differentially expressed genes.
This investigation identified extensive sex differences present in the muscle transcriptome at baseline and following acute RE. In the resting state, female muscle had a greater transcript abundance of genes involved in fatty acid oxidation and gene transcription/translation processes. After strenuous RE at the same relative intensity, the time course of the transcriptional modulation was sex-dependent. Males experienced prolonged changes while females exhibited a rapid restoration. Most of the biological processes involved in the RE-induced transcriptional regulation were observed in both males and females, but sex specificity was suggested for several signaling pathways including activation of notch signaling and TGF-beta signaling in females. Sex differences in skeletal muscle transcriptional regulation might implicate a mechanism behind disproportional muscle growth in males as compared with female counterparts after RE training at the same relative intensity.
Sex differences exist in skeletal muscle gene transcription both at rest and following acute RE, suggesting that sex is a significant modifier of the transcriptional regulation in skeletal muscle. The findings from the present study provide insight into the molecular mechanisms for sex differences in muscle phenotypes and for muscle transcriptional regulation associated with training adaptations to resistance exercise.
Forebrain neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Several strategies have been employed to eliminate adult neurogenesis and thereby determine whether depleting adult-born neurons disrupts specific brain functions, but some approaches do not specifically target neural progenitors. We have developed a transgenic mouse line to reversibly ablate adult neural stem cells and suppress neurogenesis. The nestin-tk mouse expresses herpes simplex virus thymidine kinase (tk) under the control of the nestin 2nd intronic enhancer, which drives expression in neural progenitors. Administration of ganciclovir (GCV) kills actively dividing cells expressing this transgene. We found that peripheral GCV administration suppressed SVZ-olfactory bulb and DG neurogenesis within two weeks but caused systemic toxicity. Intracerebroventricular GCV infusion for 28 days nearly completely depleted proliferating cells and immature neurons in both the SVZ and DG without systemic toxicity. Reversibility of the effects after prolonged GCV infusion was slow and partial. Neurogenesis did not recover 2 weeks after cessation of GCV administration, but showed limited recovery 6 weeks after GCV that differed between the SVZ and DG. Suppression of neurogenesis did not inhibit antidepressant responsiveness of mice in the tail suspension test. These findings indicate that SVZ and DG neural stem cells differ in their capacity for repopulation, and that adult-born neurons are not required for antidepressant responses in a common behavioral test of antidepressant efficacy. The nestin-tk mouse should be useful for studying how reversible depletion of adult neurogenesis influences neurophysiology, other behaviors, and neural progenitor dynamics.
subventricular zone; dentate gyrus; neural stem cell; antidepressant; depression; tail suspension test
NAD+ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD+-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD+ to nicotinamide ratio and the NAD+ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD+ metabolism requires analytical definition and quantification of the key metabolites.
Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD+ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD+ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD+ or nicotinamide levels in ways that would increase Sir2 activity.
The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.
Reversed-phase, packed capillary liquid chromatography interfaced by electrospray ionization to mass spectrometry was explored as an analytical method for determination of metabolites in microscale tissue samples using single islets of Langerhans as a model system. Using a 75 μm inner diameter column coupled to a quadrupole ion trap mass spectrometer in full scan mode, detection limits of 0.1 to 33 fmol were achieved for glycoloytic and tricarboxylic acid cycle metabolites. Reproducible processing of islets for analysis with little loss of metabolites was performed by rapid freezing followed by methanol: water extraction. The method yielded 20 μL of extract of which just 15 nL was injected suggesting the potential for performing multiple assays on the same islet. Approximately 200 presumed metabolites could be detected, of which 22 were identified by matching retention times and MS/MS spectra to standards. Relative standard deviations for peak detection was from 7 to 18% and was unaffected by storage for up to 11 days. The method was used to detect changes in metabolism associated increasing extracellular islet glucose concentration from 3 to 20 mM yielding results largely consistent with known metabolism of islets. Because most previous studies of islet metabolism have only observed a few compounds at once and require far more tissue, this measurement method represents a significant advance for studies of metabolism of islets and other microscale samples.
KATP channels couple the intracellular energy state to membrane excitability and regulate a wide array of biologic activities. KATP channels contain a pore-forming inwardly rectifying potassium channel and a sulfonylurea receptor regulatory subunit (SUR1 or SUR2). To clarify the role of KATP channels in vascular smooth muscle, we studied Sur2 gene-targeted mice (Sur2–/–) and found significantly elevated resting blood pressures and sudden death. Using in vivo monitoring, we detected transient, repeated episodes of coronary artery vasospasm in Sur2–/– mice. Focal narrowings in the coronary arteries were present in Sur2–/– mice consistent with vascular spasm. We treated Sur2–/– mice with a calcium channel antagonist and successfully reduced vasospastic episodes.