Publications

Isotopic ratio outlier analysis (IROA) is a (13)C metabolomics profiling method that eliminates sample to sample variance, discriminates against noise and artifacts, and improves identification of compounds, previously done with accurate mass liquid chromatography/mass spectrometry (LC/MS). This is the first report using IROA technology in combination with accurate mass gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS), here used to examine the S. cerevisiae metabolome. S. cerevisiae was grown in YNB media, containing randomized 95% (13)C, or 5%(13)C glucose as the single carbon source, in order that the isotopomer pattern of all metabolites would mirror the labeled glucose. When these IROA experiments are combined, the abundance of the heavy isotopologues in the 5%(13)C extracts, or light isotopologues in the 95%(13)C extracts, follows the binomial distribution, showing mirrored peak pairs for the molecular ion. The mass difference between the (12)C monoisotopic and the (13)C monoisotopic equals the number of carbons in the molecules. The IROA-GC/MS protocol developed, using both chemical and electron ionization, extends the information acquired from the isotopic peak patterns for formulas generation. The process that can be formulated as an algorithm, in which the number of carbons, as well as the number of methoximations and silylations are used as search constraints. In electron impact (EI/IROA) spectra, the artifactual peaks are identified and easily removed, which has the potential to generate "clean" EI libraries. The combination of chemical ionization (CI) IROA and EI/IROA affords a metabolite identification procedure that enables the identification of coeluting metabolites, and allowed us to characterize 126 metabolites in the current study.

BACKGROUND: Radiation-induced liver disease (RILD) is a dose-limiting factor in curative radiation therapy (RT) for liver cancers, making early detection of radiation-associated liver injury absolutely essential for medical intervention. A metabolomic approach was used to determine metabolic signatures that could serve as biomarkers for early detection of RILD in mice.

METHODS: Anesthetized C57BL/6 mice received 0, 10 or 50 Gy Whole Liver Irradiation (WLI) and were contrasted to mice, which received 10 Gy whole body irradiation (WBI). Liver and plasma samples were collected at 24 hours after irradiation. The samples were processed using Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry.

RESULTS: Twenty four hours after WLI, 407 metabolites were detected in liver samples while 347 metabolites were detected in plasma. Plasma metabolites associated with 50 Gy WLI included several amino acids, purine and pyrimidine metabolites, microbial metabolites, and most prominently bradykinin and 3-indoxyl-sulfate. Liver metabolites associated with 50 Gy WLI included pentose phosphate, purine, and pyrimidine metabolites in liver. Plasma biomarkers in common between WLI and WBI were enriched in microbial metabolites such as 3 indoxyl sulfate, indole-3-lactic acid, phenyllactic acid, pipecolic acid, hippuric acid, and markers of DNA damage such as 2-deoxyuridine. Metabolites associated with tryptophan and indoles may reflect radiation-induced gut microbiome effects. Predominant liver biomarkers in common between WBI and WLI were amino acids, sugars, TCA metabolites (fumarate), fatty acids (lineolate, n-hexadecanoic acid) and DNA damage markers (uridine).

CONCLUSIONS: We identified a set of metabolomic markers that may prove useful as plasma biomarkers of RILD and WBI. Pathway analysis also suggested that the unique metabolic changes observed after liver irradiation was an integrative response of the intestine, liver and kidney.

PURPOSE: Assessing whole-body radiation injury and absorbed dose is essential for remediation efforts following accidental or deliberate exposure in medical, industrial, military, or terrorist incidents. We hypothesize that variations in specific metabolite concentrations extracted from blood plasma would correlate with whole-body radiation injury and dose.

METHODS AND MATERIALS: Groups of C57BL/6 mice (n=12 per group) were exposed to 0, 2, 4, 8, and 10.4 Gy of whole-body gamma radiation. At 24 hours after treatment, all animals were euthanized, and both plasma and liver biopsy samples were obtained, the latter being used to identify a distinct hepatic radiation injury response within plasma. A semiquantitative, untargeted metabolite/lipid profile was developed using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, which identified 354 biochemical compounds. A second set of C57BL/6 mice (n=6 per group) were used to assess a subset of identified plasma markers beyond 24 hours.

RESULTS: We identified a cohort of 37 biochemical compounds in plasma that yielded the optimal separation of the irradiated sample groups, with the most correlated metabolites associated with pyrimidine (positively correlated) and tryptophan (negatively correlated) metabolism. The latter were predominantly associated with indole compounds, and there was evidence that these were also correlated between liver and plasma. No evidence of saturation as a function of dose was observed, as has been noted for studies involving metabolite analysis of urine.

CONCLUSIONS: Plasma profiling of specific metabolites related to pyrimidine and tryptophan pathways can be used to differentiate whole-body radiation injury and dose response. As the tryptophan-associated indole compounds have their origin in the intestinal microbiome and subsequently the liver, these metabolites particularly represent an attractive marker for radiation injury within blood plasma.

CONTEXT: Intrauterine HIV/antiretroviral (ARV) and postnatal ARVs are known to perturb energy metabolism and could have permanent effects on future metabolic health. Such maladaptive effects could be mediated by changes in mitochondrial function and intermediary metabolism due to fetal and early-life ARV exposure in HIV/ARV-exposed uninfected (HEU) infants.

OBJECTIVE: The objective of the study was to understand the relationship(s) between mitochondrial fuel use (assessed via acylcarnitines and branched chain amino acids) and preprandial insulin in infants exposed to in utero HIV/ARV plus postnatal zidovudine or nevirapine compared with HIV/ARV-unexposed uninfected (HUU) infants.

DESIGN: This was a prospective cohort study with the following three groups: 1) intrauterine HIV/ARV/postnatal zidovudine-exposed (HEU-A), 2) intrauterine HIV/ARV/postnatal nevirapine-exposed (HEU-N), and 3) HUU infants. Principal component analysis and linear regression modeling were performed to assess the association between in utero HIV/ARV exposure and infant insulin.

SETTING: The study was conducted at Cameroonian urban antenatal centers.

PARTICIPANTS: HIV-infected and -uninfected pregnant woman/infant dyads participated in the study.

MAIN OUTCOME: Preprandial insulin was the main outcome measured.

RESULTS: Of 366 infants, 38 were HEU-A, 118 HEU-N. Forty intermediary metabolites were consolidated into seven principal components. In a multivariate analysis, both HEU-A (β = -.116, P= .012) and HEU-N (β = -.070, P= .022) demonstrated lower insulin compared with HUU infants. However, at high levels of plasma metabolites, HEU-A (β = .027, P= .050) exhibited higher insulin levels than HEU-N or HUU infants. A unique array of short-chain acylcarnitines (β = .044, P= .001) and branched-chain amino acids (β = .033, P= .012) was associated with insulin.

CONCLUSION: HEU-A and HEU-N infants have lower preprandial insulin levels at 6 weeks of age and appear to use metabolic fuel substrates differently than HUU infants. Future studies are warranted to determine whether observed differences have lasting metabolic implications, such as later insulin resistance.

BACKGROUND: Infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, results in chronic infection that leads to cardiomyopathy with increased mortality and morbidity in endemic regions. In a companion study, our group found that a high-fat diet (HFD) protected mice from T. cruzi-induced myocardial damage and significantly reduced post-infection mortality during acute T. cruzi infection.

METHODS: In the present study metabolic syndrome was induced prior to T. cruzi infection by feeding a high fat diet. Also, mice were treated with anti-diabetic drug metformin.

RESULTS: In the present study, the lethality of T. cruzi (Brazil strain) infection in CD-1 mice was reduced from 55% to 20% by an 8-week pre-feeding of an HFD to induce obesity and metabolic syndrome. The addition of metformin reduced mortality to 3%.

CONCLUSIONS: It is an interesting observation that both the high fat diet and the metformin, which are known to differentially attenuate host metabolism, effectively modified mortality in T. cruzi-infected mice. In humans, the metabolic syndrome, as presently construed, produces immune activation and metabolic alterations that promote complications of obesity and diseases of later life, such as myocardial infarction, stroke, diabetes, Alzheimer's disease and cancer. Using an evolutionary approach, we hypothesized that for millions of years, the channeling of host resources into immune defences starting early in life ameliorated the effects of infectious diseases, especially chronic infections, such as tuberculosis and Chagas disease. In economically developed countries in recent times, with control of the common devastating infections, epidemic obesity and lengthening of lifespan, the dwindling benefits of the immune activation in the first half of life have been overshadowed by the explosion of the syndrome's negative effects in later life.

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

KDM5 family proteins are critically important transcriptional regulators whose physiological functions in the context of a whole animal remain largely unknown. Using genome-wide gene expression and binding analyses in Drosophila adults, we demonstrate that KDM5 (Lid) is a direct regulator of genes required for mitochondrial structure and function. Significantly, this occurs independently of KDM5's well-described JmjC domain-encoded histone demethylase activity. Instead, it requires the PHD motif of KDM5 that binds to histone H3 that is di- or trimethylated on lysine 4 (H3K4me2/3). Genome-wide, KDM5 binding overlaps with the active chromatin mark H3K4me3, and a fly strain specifically lacking H3K4me2/3 binding shows defective KDM5 promoter recruitment and gene activation. KDM5 therefore plays a central role in regulating mitochondrial function by utilizing its ability to recognize specific chromatin contexts. Importantly, KDM5-mediated regulation of mitochondrial activity is likely to be key in human diseases caused by dysfunction of this family of proteins.

Insulin integrates hepatic glucose and lipid metabolism, directing nutrients to storage as glycogen and triglyceride. In type 2 diabetes, levels of the former are low and the latter are exaggerated, posing a pathophysiologic and therapeutic conundrum. A branching model of insulin signalling, with FoxO1 presiding over glucose production and Srebp-1c regulating lipogenesis, provides a potential explanation. Here we illustrate an alternative mechanism that integrates glucose production and lipogenesis under the unifying control of FoxO. Liver-specific ablation of three FoxOs (L-FoxO1,3,4) prevents the induction of glucose-6-phosphatase and the repression of glucokinase during fasting, thus increasing lipogenesis at the expense of glucose production. We document a similar pattern in the early phases of diet-induced insulin resistance, and propose that FoxOs are required to enable the liver to direct nutritionally derived carbons to glucose versus lipid metabolism. Our data underscore the heterogeneity of hepatic insulin resistance during progression from the metabolic syndrome to overt diabetes, and the conceptual challenge of designing therapies that curtail glucose production without promoting hepatic lipid accumulation.

Deficiency of IQGAP2, a scaffolding protein expressed primarily in liver leads to rearrangements of hepatic protein compartmentalization and altered regulation of enzyme functions predisposing development of hepatocellular carcinoma and diabetes. Employing a systems approach with proteomics, metabolomics and fluxes characterizations, we examined the effects of IQGAP2 deficient proteomic changes on cellular metabolism and the overall metabolic phenotype. Iqgap2-/- mice demonstrated metabolic inflexibility, fasting hyperglycemia and obesity. Such phenotypic characteristics were associated with aberrant hepatic regulations of glycolysis/gluconeogenesis, glycogenolysis, lipid homeostasis and futile cycling corroborated with corresponding proteomic changes in cytosolic and mitochondrial compartments. IQGAP2 deficiency also led to truncated TCA-cycle, increased anaplerosis, increased supply of acetyl-CoA for de novo lipogenesis, and increased mitochondrial methyl-donor metabolism necessary for nucleotides synthesis. Our results suggest that changes in metabolic networks in IQGAP2 deficiency create a hepatic environment of a 'pre-diabetic' phenotype and a predisposition to non-alcoholic fatty liver disease (NAFLD) which has been linked to the development of hepatocellular carcinoma.

The liver plays a central role in metabolism and mediating insulin action. To dissect the effects of insulin on the liver in vivo, we have studied liver insulin receptor knockout (LIRKO) mice. Because LIRKO livers lack insulin receptors, they are unable to respond to insulin. Surprisingly, the most profound derangement observed in LIRKO livers by microarray analysis is a suppression of the cholesterologenic genes. Sterol regulatory element binding protein (SREBP)-2 promotes cholesterologenic gene transcription, and is inhibited by intracellular cholesterol. LIRKO livers show a slight increase in hepatic cholesterol, a 40% decrease in Srebp-2, and a 50-90% decrease in the cholesterologenic genes at the mRNA and protein levels. In control mice, SREBP-2 and cholesterologenic gene expression are suppressed by fasting and restored by refeeding; in LIRKO mice, this response is abolished. Similarly, the ability of statins to induce Srebp-2 and the cholesterologenic genes is lost in LIRKO livers. In contrast, ezetimibe treatment robustly induces Srepb-2 and its targets in LIRKO livers, raising the possibility that insulin may regulate SREBP-2 indirectly, by altering the accumulation or distribution of cholesterol within the hepatocyte. Taken together, these data indicate that cholesterol synthesis is a key target of insulin action in the liver.