Amy Deik

Metabolite Profiling Identifies Pathways Associated With Metabolic Risk in Humans

Background— Although metabolic risk factors are known to cluster in individuals who are prone to developing diabetes mellitus and cardiovascular disease, the underlying biological mechanisms remain poorly understood. Methods and Results— To identify pathways associated with cardiometabolic risk, we used liquid chromatography/mass spectrometry to determine the plasma concentrations of 45 distinct metabolites and to examine their relation to cardiometabolic risk in the Framingham Heart Study (FHS; n=1015) and the Malmö Diet and Cancer Study (MDC; n=746). We then interrogated significant findings in experimental models of cardiovascular and metabolic disease. We observed that metabolic risk factors (obesity, insulin resistance, high blood pressure, and dyslipidemia) were associated with multiple metabolites, including branched-chain amino acids, other hydrophobic amino acids, tryptophan breakdown products, and nucleotide metabolites. We observed strong associations of insulin resistance traits with glutamine (standardized regression coefficients, −0.04 to −0.22 per 1-SD change in log-glutamine; P<0.001), glutamate (0.05 to 0.14; P<0.001), and the glutamine-to-glutamate ratio (−0.05 to −0.20; P<0.001) in the discovery sample (FHS); similar associations were observed in the replication sample (MDC). High glutamine-to-glutamate ratio was associated with lower risk of incident diabetes mellitus in FHS (odds ratio, 0.79; adjusted P=0.03) but not in MDC. In experimental models, administration of glutamine in mice led to both increased glucose tolerance (P=0.01) and decreased blood pressure (P<0.05). Conclusions— Biochemical profiling identified circulating metabolites not previously associated with metabolic traits. Experimentally interrogating one of these pathways demonstrated that excess glutamine relative to glutamate, resulting from exogenous administration, is associated with reduced metabolic risk in mice.

Circulating branched-chain amino acid concentrations are associated with obesity and future insulin resistance in children and adolescents.

What is already known about this subject Circulating concentrations of branched‐chain amino acids (BCAAs) can affect carbohydrate metabolism in skeletal muscle, and therefore may alter insulin sensitivity. BCAAs are elevated in adults with diet‐induced obesity, and are associated with their future risk of type 2 diabetes even after accounting for baseline clinical risk factors.

A Genome-wide Association Study of the Human Metabolome in a Community-Based Cohort

Because metabolites are hypothesized to play key roles as markers and effectors of cardiometabolic diseases, recent studies have sought to annotate the genetic determinants of circulating metabolite levels. We report a genome-wide association study (GWAS) of 217 plasma metabolites, including >100 not measured in prior GWAS, in 2076 participants of the Framingham Heart Study (FHS). For the majority of analytes, we find that estimated heritability explains >20% of interindividual variation, and that variation attributable to heritable factors is greater than that attributable to clinical factors. Further, we identify 31 genetic loci associated with plasma metabolites, including 23 that have not previously been reported. Importantly, we include GWAS results for all surveyed metabolites and demonstrate how this information highlights a role for AGXT2 in cholesterol ester and triacylglycerol metabolism. Thus, our study outlines the relative contributions of inherited and clinical factors on the plasma metabolome and provides a resource for metabolism research.

A Combined Epidemiologic and Metabolomic Approach Improves CKD Prediction

Metabolomic approaches have begun to catalog the metabolic disturbances that accompany CKD, but whether metabolite alterations can predict future CKD is unknown. We performed liquid chromatography/mass spectrometry-based metabolite profiling on plasma from 1434 participants in the Framingham Heart Study (FHS) who did not have CKD at baseline. During the following 8 years, 123 individuals developed CKD, defined by an estimated GFR of <60 ml/min per 1.73 m(2). Numerous metabolites were associated with incident CKD, including 16 that achieved the Bonferroni-adjusted significance threshold of P≤0.00023. To explore how the human kidney modulates these metabolites, we profiled arterial and renal venous plasma from nine individuals. Nine metabolites that predicted CKD in the FHS cohort decreased more than creatinine across the renal circulation, suggesting that they may reflect non-GFR-dependent functions, such as renal metabolism and secretion. Urine isotope dilution studies identified citrulline and choline as markers of renal metabolism and kynurenic acid as a marker of renal secretion. In turn, these analytes remained associated with incident CKD in the FHS cohort, even after adjustment for eGFR, age, sex, diabetes, hypertension, and proteinuria at baseline. Addition of a multimarker metabolite panel to clinical variables significantly increased the c-statistic (0.77-0.83, P<0.0001); net reclassification improvement was 0.78 (95% confidence interval, 0.60 to 0.95; P<0.0001). Thus, the addition of metabolite profiling to clinical data may significantly improve the ability to predict whether an individual will develop CKD by identifying predictors of renal risk that are independent of estimated GFR.

Metabolic consequences of mitochondrial coenzyme A deficiency in patients with PANK2 mutations.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, inborn error of metabolism characterized by iron accumulation in the basal ganglia and by the presence of dystonia, dysarthria, and retinal degeneration. Mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis, represent the most common genetic cause of this disorder. How mutations in this core metabolic enzyme give rise to such a broad clinical spectrum of pathology remains a mystery. To systematically explore its pathogenesis, we performed global metabolic profiling on plasma from a cohort of 14 genetically defined patients and 18 controls. Notably, lactate is elevated in PKAN patients, suggesting dysfunctional mitochondrial metabolism. As predicted, but never previously reported, pantothenate levels are higher in patients with premature stop mutations in PANK2. Global metabolic profiling and follow-up studies in patient-derived fibroblasts also reveal defects in bile acid conjugation and lipid metabolism, pathways that require coenzyme A. These findings raise a novel therapeutic hypothesis, namely, that dietary fats and bile acid supplements may hold potential as disease-modifying interventions. Our study illustrates the value of metabolic profiling as a tool for systematically exploring the biochemical basis of inherited metabolic diseases.

A Plasma Long-Chain Acylcarnitine Predicts Cardiovascular Mortality in Incident Dialysis Patients

Background The marked excess in cardiovascular mortality that results from uremia remains poorly understood. Methods and Results In 2 independent, nested case‐control studies, we applied liquid chromatography‐mass spectrometry‐based metabolite profiling to plasma obtained from participants of a large cohort of incident hemodialysis patients. First, 100 individuals who died of a cardiovascular cause within 1 year of initiating hemodialysis (cases) were randomly selected along with 100 individuals who survived for at least 1 year (controls), matched for age, sex, and race. Four highly intercorrelated long‐chain acylcarnitines achieved the significance threshold adjusted for multiple testing (P<0.0003). Oleoylcarnitine, the long‐chain acylcarnitine with the strongest association with cardiovascular mortality in unadjusted analysis, remained associated with 1‐year cardiovascular death after multivariable adjustment (odds ratio per SD 2.3 [95% confidence interval, 1.4 to 3.8]; P=0.001). The association between oleoylcarnitine and 1‐year cardiovascular death was then replicated in an independent sample (n=300, odds ratio per SD 1.4 [95% confidence interval, 1.1 to 1.9]; P=0.008). Addition of oleoylcarnitine to clinical variables improved cardiovascular risk prediction using net reclassification (NRI, 0.38 [95% confidence interval, 0.20 to 0.56]; P<0.0001). In physiologic profiling studies, we demonstrate that the fold change in plasma acylcarnitine levels from the aorta to renal vein and from pre‐ to post hemodialysis samples exclude renal or dialytic clearance of long‐chain acylcarnitines as confounders in our analysis. Conclusions Our data highlight clinically meaningful alterations in acylcarnitine homeostasis at the time of dialysis initiation, which may represent an early marker, effector, or both of uremic cardiovascular risk.

Blood-Brain Barrier Permeability Is Regulated by Lipid Transport-Dependent Suppression of Caveolae-Mediated Transcytosis

The blood-brain barrier (BBB) provides a constant homeostatic brain environment that is essential for proper neural function. An unusually low rate of vesicular transport (transcytosis) has been identified as one of the two unique properties of CNS endothelial cells, relative to peripheral endothelial cells, that maintain the restrictive quality of the BBB. However, it is not known how this low rate of transcytosis is achieved. Here we provide a mechanism whereby the regulation of CNS endothelial cell lipid composition specifically inhibits the caveolae-mediated transcytotic route readily used in the periphery. An unbiased lipidomic analysis reveals significant differences in endothelial cell lipid signatures from the CNS and periphery, which underlie a suppression of caveolae vesicle formation and trafficking in brain endothelial cells. Furthermore, lipids transported by Mfsd2a establish a unique lipid environment that inhibits caveolae vesicle formation in CNS endothelial cells to suppress transcytosis and ensure BBB integrity.

Branched chain and aromatic amino acids change acutely following two medical therapies for type 2 diabetes mellitus

OBJECTIVE Elevated circulating levels of branched chain and aromatic amino acids (BCAA/AAAs) are associated with insulin resistance and incident type 2 diabetes (T2D). BCAA/AAAs decrease acutely during an oral glucose tolerance test (OGTT), a diagnostic test for T2D. It is unknown whether changes in BCAA/AAAs also signal an early response to commonly used medical therapies for T2D. MATERIALS AND METHODS A liquid chromatography-mass spectrometry approach was used to measure BCAA/AAAs in 30 insulin sensitive (IS) and 30 insulin resistant (IR) subjects before and after: (1) one dose of a sulfonylurea medication, glipizide, 5 mg orally; (2) two days of twice daily metformin 500 mg orally; and (3) a 75-g OGTT. Percent change in BCAA/AAAs was determined after each intervention. RESULTS Following glipizide, which increased insulin and decreased glucose in both subject groups, BCAA/AAAs decreased in the IS subjects only (all P<0.05). Following metformin, which decreased glucose and insulin in only the IR subjects, 4 BCAA/AAAs increased in the IR subjects at or below P=0.05, and none changed in the IS subjects. Following OGTT, which increased glucose and insulin in all subjects, BCAA/AAAs decreased in all subjects (P<0.05). CONCLUSIONS BCAA/AAAs changed acutely during glipizide and metformin administration, and the magnitude and direction of change differed by the insulin resistance status of the individual and the intervention. These results indicate that BCAA/AAAs may be useful biomarkers for monitoring the early response to therapeutic interventions for T2D.

Distinct metabolomic signatures are associated with longevity in humans.

Alterations in metabolism influence lifespan in experimental models, but data in humans are lacking. Here we use liquid chromatography/mass spectrometry to quantify 217 plasma metabolites and examine their relation to longevity in a large cohort of men and women. In 647 individuals followed for up to 20 years, higher concentrations of the citric acid cycle intermediate, isocitrate, and the bile acid, taurocholate, are associated with lower odds of longevity, defined as attaining 80 years of age. In a larger cohort of 2,327 individuals with metabolite data available, higher concentrations of isocitrate but not taurocholate are also associated with worse cardiovascular health at baseline, as well as risk of future cardiovascular disease and death. None of the metabolites identified are associated with cancer risk. Our findings suggest that some, but not all, metabolic pathways to human longevity are dependent on modifying risk for the two most common causes of death.

Effects of sodium benzoate, a widely used food preservative, on glucose homeostasis and metabolic profiles in humans

Sodium benzoate is a widely used preservative found in many foods and soft drinks. It is metabolized within mitochondria to produce hippurate, which is then cleared by the kidneys. We previously reported that ingestion of sodium benzoate at the generally regarded as safe (GRAS) dose leads to a robust excursion in the plasma hippurate level [1]. Since previous reports demonstrated adverse effects of benzoate and hippurate on glucose homeostasis in cells and in animal models, we hypothesized that benzoate might represent a widespread and underappreciated diabetogenic dietary exposure in humans. Here, we evaluated whether acute exposure to GRAS levels of sodium benzoate alters insulin and glucose homeostasis through a randomized, controlled, cross-over study of 14 overweight subjects. Serial blood samples were collected following an oral glucose challenge, in the presence or absence of sodium benzoate. Outcome measurements included glucose, insulin, glucagon, as well as temporal mass spectrometry-based metabolic profiles. We did not find a statistically significant effect of an acute oral exposure to sodium benzoate on glucose homeostasis. Of the 146 metabolites targeted, four changed significantly in response to benzoate, including the expected rise in benzoate and hippurate. In addition, anthranilic acid, a tryptophan metabolite, exhibited a robust rise, while acetylglycine dropped. Although our study shows that GRAS doses of benzoate do not have an acute, adverse effect on glucose homeostasis, future studies will be necessary to explore the metabolic impact of chronic benzoate exposure.