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Proteomics has been leveraged to study type 2 diabetes (T2D), but the majority of available data are from White participants. Here, we extend prior work by analyzing a large cohort of self-identified African Americans (AAs) in the Jackson Heart Study (JHS, n=1,313). We found 325 proteins associated with incident diabetes after adjusting for age, sex, and batch (FDR-q

In addition to their fundamental role in clearance, the kidneys release select molecules into the circulation, but whether any of these anabolic functions provides insight on kidney health is unknown. Using aptamer-based proteomics, we characterized arterial (A)-to-renal venous (V) gradients for >1,300 proteins in 22 individuals who underwent invasive sampling. Although most of the proteins that changed significantly decreased from A to V, consistent with renal clearance, several were found to increase, the most significant of which was testican-2. To assess the clinical implications of these physiologic findings, we examined proteomic data in the Jackson Heart Study (JHS), an African-American cohort (n = 1,928), with replication in the Framingham Heart Study (FHS), a White cohort (n = 1,621). In both populations, testican-2 had a strong, positive correlation with estimated glomerular filtration rate (eGFR). In addition, higher baseline testican-2 levels were associated with a lower rate of eGFR decline in models adjusted for age, gender, hypertension, type 2 diabetes, body mass index, baseline eGFR, and albuminuria. Glomerular expression of testican-2 in human kidneys was demonstrated by immunohistochemistry, immunofluorescence, and electron microscopy, while single-cell RNA sequencing of human kidneys showed expression of the cognate gene, SPOCK2, exclusively in podocytes. In vitro, testican-2 increased glomerular endothelial tube formation and motility, raising the possibility that its secretion has a functional role within the glomerulus. Taken together, our findings identify testican-2 as a podocyte-derived biomarker of kidney health and prognosis.

Heart failure (HF) is a major public health problem characterized by inability of the heart to maintain sufficient output of blood. The systematic characterization of circulating proteins across different stages of HF may provide pathophysiological insights and identify therapeutic targets. Here we report application of aptamer-based proteomics to identify proteins associated with prospective HF incidence in a population-based cohort, implicating modulation of immunological, complement, coagulation, natriuretic and matrix remodeling pathways up to two decades prior to overt disease onset. We observe further divergence of these proteins from the general population in advanced HF, and regression after heart transplantation. By leveraging coronary sinus samples and transcriptomic tools, we describe likely cardiac and specific cellular origins for several of the proteins, including Nt-proBNP, thrombospondin-2, interleukin-18 receptor, gelsolin, and activated C5. Our findings provide a broad perspective on both cardiac and systemic factors associated with HF development., Heart failure is a major health issue worldwide. Here, Egerstedt et al. perform proteomic profiling of human plasma at different stages of heart failure, providing a comprehensive analysis of changes in the plasma proteome during disease progression.

Anthracyclines and HER2-targeted antibodies are very effective for the treatment of breast cancer, but their use is limited by cardiotoxicity. In this nested case-control study, we assessed the role of intermediary metabolism in 38 women with breast cancer treated with anthracyclines and trastuzumab. Using targeted mass spectrometry to measure 71 metabolites in the plasma, we identified changes in citric acid and aconitic acid that differentiated patients who developed cardiotoxicity from those who did not. In patients with cardiotoxicity, the magnitude of change in citric acid at three months correlated with the change in left ventricular ejection fraction (LVEF) and absolute LVEF at nine months. Patients with cardiotoxicity also demonstrated more pronounced changes in purine and pyrimidine metabolism. Early metabolic changes may therefore provide insight into the mechanisms associated with the development of chemotherapy-associated cardiotoxicity.

Background: Single-stranded DNA aptamers are oligonucleotides of ≈50 base pairs in length selected for their ability to bind proteins with high specificity and affinity. Emerging DNA aptamer-based technologies may address limitations of existing proteomic techniques, including low sample throughput, which have hindered proteomic analyses of large cohorts. Methods: To identify early biomarkers of myocardial injury, we applied an aptamer-based proteomic platform that measures 1129 proteins to a clinically relevant perturbational model of planned myocardial infarction (PMI), patients undergoing septal ablation for hypertrophic cardiomyopathy. Blood samples were obtained before and at 10 and 60 minutes after PMI, and protein changes were assessed by repeated-measures analysis of variance. The generalizability of our PMI findings was evaluated in a spontaneous myocardial infarction cohort (Wilcoxon rank-sum). We then tested the platform’s ability to detect associations between proteins and Framingham Risk Score components in the Framingham Heart Study, performing regression analyses for each protein versus each clinical trait. Results: We found 217 proteins that significantly changed in the peripheral vein blood after PMI in a derivation cohort (n=15; P P 85% were directionally consistent and reached nominal significance. We detected many protein changes that are novel in the context of myocardial injury, including Dickkopf-related protein 4, a WNT pathway inhibitor (peak increase 124%, P =1.29E-15) and cripto, a growth factor important in cardiac development (peak increase 64%, P =1.74E-4). Among the 40 validated proteins that increased within 1 hour after PMI, 23 were also elevated in patients with spontaneous myocardial infarction (n=46; P P =2.54E-22) and trigger factor 2 (β=0.2846, P =5.71E-17). Furthermore, we developed a novel workflow integrating DNA-based immunoaffinity with mass spectrometry to analytically validate aptamer specificity. Conclusions: Our results highlight an emerging proteomics tool capable of profiling >1000 low-abundance analytes with high sensitivity and high precision, applicable both to well-phenotyped perturbational studies and large human cohorts, as well.

Elevated levels of aldosterone are a modifiable contributor to clinical worsening in heart failure with reduced ejection fraction (HFrEF). Endothelin-1 (ET-1), which is increased in HFrEF, induces pulmonary endothelial aldosterone synthesis in vitro. However, whether transpulmonary aldosterone release occurs in humans or aldosterone relates to functional capacity in HFrEF is not known. Therefore, we aimed to characterize ET-1 and transpulmonary aldosterone levels in HFrEF and determine if aldosterone levels relate to peak volume of oxygen uptake (pVO2).Data from 42 consecutive HFrEF patients and 18 controls referred for invasive cardiopulmonary exercise testing were analyzed retrospectively.Radial ET-1 levels (median [interquartile range]) were higher in HFrEF patients compared with controls (17.5 [11.5-31.4] vs 11.5 [4.4-19.0] pg/ml, p = 0.04). A significant ET-1 transpulmonary gradient (pulmonary arterial [PA] - radial arterial levels) was present in HFrEF (p0.001) but not in controls (p = 0.24). Compared with controls, aldosterone levels (median [interquartile range]) were increased in HFrEF patients in the PA (364 [250-489] vs 581 [400-914] ng/dl, p0.01) and radial compartments (366 [273-466] vs 702 [443-1223] ng/dl, p0.001). Akin to ET-1, a transpulmonary increase (median [interquartile range]) in aldosterone concentration was also observed between controls and HFrEF patients at rest (7.5 [-54 to 40] vs 61.6 [-13.6 to 165] ng/dl, p = 0.01) and peak exercise (-20.7 [-39.6 to 79.1] vs 25.8 [-29.2 to 109.3] ng/dl, p = 0.02). The adjusted pVO2 correlated inversely with aldosterone levels at peak activity in the PA (r = -0.31, p = 0.01) and radial artery (r = -0.32, p = 0.01).These data provide preliminary evidence in support of increased transpulmonary aldosterone levels in HFrEF and suggest an inverse relationship between circulating aldosterone and pVO2. Future prospective studies are needed to characterize the functional effects of transpulmonary and circulating aldosterone on cardiac reserve capacity in HFrEF.

Background Pulmonary hypertension and associated right ventricular (RV) dysfunction are important determinants of morbidity and mortality, which are optimally characterized by invasive hemodynamic measurements. Objectives This study sought to determine whether metabolite profiling could identify plasma signatures of right ventricular-pulmonary vascular (RV-PV) dysfunction. Methods We measured plasma concentrations of 105 metabolites using targeted mass spectrometry in 71 individuals (discovery cohort) who underwent comprehensive physiological assessment with right-sided heart catheterization and radionuclide ventriculography at rest and during exercise. Our findings were validated in a second cohort undergoing invasive hemodynamic evaluations (n = 71), as well as in an independent cohort with or without known pulmonary arterial (PA) hypertension (n = 30). Results In the discovery cohort, 21 metabolites were associated with 2 or more hemodynamic indicators of RV-PV function (i.e., resting right atrial pressure, mean PA pressure, pulmonary vascular resistance [PVR], and PVR and PA pressure-flow response [ΔPQ] during exercise). We identified novel associations of RV-PV dysfunction with circulating indoleamine 2,3-dioxygenase (IDO)–dependent tryptophan metabolites (TMs), tricarboxylic acid intermediates, and purine metabolites and confirmed previously described associations with arginine–nitric oxide metabolic pathway constituents. IDO-TM levels were inversely related to RV ejection fraction and were particularly well correlated with exercise PVR and ΔPQ. Multisite sampling demonstrated transpulmonary release of IDO-TMs. IDO-TMs also identified RV-PV dysfunction in a validation cohort with known risk factors for pulmonary hypertension and in patients with established PA hypertension. Conclusions Metabolic profiling identified reproducible signatures of RV-PV dysfunction, highlighting both new biomarkers and pathways for further functional characterization.

We have developed a novel plasma protein analysis platform with optimized sample preparation, chromatography, and MS analysis protocols. The workflow, which utilizes chemical isobaric mass tag labeling for relative quantification of plasma proteins, achieves far greater depth of proteome detection and quantification while simultaneously having increased sample throughput than prior methods. We applied the new workflow to a time series of plasma samples from patients undergoing a therapeutic, "planned" myocardial infarction for hypertrophic cardiomyopathy, a unique human model in which each person serves as their own biologic control. Over 5300 proteins were confidently identified in our experiments with an average of 4600 proteins identified per sample (with two or more distinct peptides identified per protein) using iTRAQ four-plex labeling. Nearly 3400 proteins were quantified in common across all 16 patient samples. Compared with a previously published label-free approach, the new method quantified almost fivefold more proteins/sample and provided a six- to nine-fold increase in sample analysis throughput. Moreover, this study provides the largest high-confidence plasma proteome dataset available to date. The reliability of relative quantification was also greatly improved relative to the label-free approach, with measured iTRAQ ratios and temporal trends correlating well with results from a 23-plex immunoMRM (iMRM) assay containing a subset of the candidate proteins applied to the same patient samples. The functional importance of improved detection and quantification was reflected in a markedly expanded list of significantly regulated proteins that provided many new candidate biomarker proteins. Preliminary evaluation of plasma sample labeling with TMT six-plex and ten-plex reagents suggests that even further increases in multiplexing of plasma analysis are practically achievable without significant losses in depth of detection relative to iTRAQ four-plex. These results obtained with our novel platform provide clear demonstration of the value of using isobaric mass tag reagents in plasma-based biomarker discovery experiments.

One of the main challenges within clinical practice today involves attaining the knowledge necessary to treat patients safely and effectively. The explosion of scientific breakthroughs within the health-care setting has created a new challenge for today's practitioners: staying informed. In turn, the pharmaceutical industry has been challenged with providing information that is accurate, meaningful, and compliant with US Food and Drug Administration guidelines. In this article, we review how the pharmaceutical industry has tried to fill this need through the role of the pharmaceutical clinical educator (PCE). We describe the PCE role and the different forms of education and support that can be provided to advanced practice providers (APPs). We also address the conflict of interest issues surrounding a collaborative relationship between APPs and pharmaceutical industry APPs.

Clinical practice guidelines currently endorse a reliance on clinical symptoms of overt left ventricular (LV) failure to time aortic valve replacement for severe aortic stenosis; however, delayed aortic valve replacement can result in irreversible LV injury and adverse outcomes. Blood metabolomic signatures possess prognostic value in heart failure; this study assesses whether they are informative in aortic stenosis.To evaluate the value of metabolomic signatures in reflecting the extent of maladaptive LV remodeling in patients with end-stage aortic stenosis undergoing transcatheter aortic valve replacement, and to assess whether this procedure reverses metabolomic aberrations.This study of 44 patients with symptomatic severe aortic stenosis who underwent transfemoral transcatheter aortic valve replacement at a single-center tertiary care hospital. Liquid chromatography-mass spectrometry-based metabolomic profiling was performed on blood samples collected before and 24 hours after the procedure, and analyses were conducted to identify metabolites related to the measures of LV remodeling.We evaluated LV ejection fraction, LV mass index, and relative wall thickness, as well as levels of the acylcarnitines C16, C18:1, C18:2, C18, C26, choline, and kynurenine.We enrolled 44 patients with severe aortic stenosis with a mean (SD) age of 81.9 (8.5) years, of whom 23 (52%) were women. The mean (SD) LV ejection fraction was 56.7% (18.2%), mean (SD) LV mass index was 117.3 (41.4) g/m2, and relative wall thickness was 0.53 (0.14). The mean β values of acylcarnitines C16, C18:1, C18:2, C18, and C26 were independently associated with LV mass index (C16: mean, 19.24; 95% CI, 5.48-33.01; P = .008; C18:1: mean, 26.18; 95% CI, 14.04-38.32; P 1.0 × 10-4; C18:2: mean, 17.42; 95% CI, 3.40-31.43; P = .02; C18: mean, 25.25; 95% CI, 10.91-39.58; P = .001; C26: mean, 19.93; 95% CI, 4.41-35.45; P = .01), even after adjustments for age, sex, diabetes status, renal function, and B-type natriuretic peptide (BNP). Circulating levels of C18:2 acylcarnitine were associated with LV ejection fraction before and after multivariable adjustment (mean, -6.11; 95% CI, -10.88 to 1.34; P = .01). Blood metabolite levels did not independently relate to relative wall thickness. Within 24 hours of transcatheter aortic valve replacement, circulating levels of C16 decreased by 30.2% (P = 7.3 × 10-6), C18:1 by 42.7% (P = 3.7 × 10-8), C18:2 by 37.3% (P = 5.1 × 10-6), and C18 by 38.3% (P = 3.4 × 10-5).In symptomatic patients with severe aortic stenosis undergoing transcatheter aortic valve replacement, circulating levels of long-chain acylcarnitines were independently associated with measures of maladaptive LV remodeling, and metabolic perturbations lessened after procedure completion. Further efforts are needed to determine the clinical applicability of these novel biomarkers.

Background: Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury. Methods: We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls. Results: We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; P P 90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; P 0 subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers. Conclusions: Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury.

Background: We recently identified 156 proteins in human plasma that were each associated with the net Framingham Cardiovascular Disease Risk Score using an aptamer-based proteomic platform in Framingham Heart Study Offspring participants. Here we hypothesized that performing genome-wide association studies and exome array analyses on the levels of each of these 156 proteins might identify genetic determinants of risk-associated circulating factors and provide insights into early cardiovascular pathophysiology. Methods: We studied the association of genetic variants with the plasma levels of each of the 156 Framingham Cardiovascular Disease Risk Score–associated proteins using linear mixed-effects models in 2 population-based cohorts. We performed discovery analyses on plasma samples from 759 participants of the Framingham Heart Study Offspring cohort, an observational study of the offspring of the original Framingham Heart Study and their spouses, and validated these findings in plasma samples from 1421 participants of the MDCS (Malmö Diet and Cancer Study). To evaluate the utility of this strategy in identifying new biological pathways relevant to cardiovascular disease pathophysiology, we performed studies in a cell-model system to experimentally validate the functional significance of an especially novel genetic association with circulating apolipoprotein E levels. Results: We identified 120 locus-protein associations in genome-wide analyses and 41 associations in exome array analyses, the majority of which have not been described previously. These loci explained up to 66% of interindividual plasma protein-level variation and, on average, accounted for 3 times the amount of variation explained by common clinical factors, such as age, sex, and diabetes mellitus status. We described overlap among many of these loci and cardiovascular disease genetic risk variants. Finally, we experimentally validated a novel association between circulating apolipoprotein E levels and the transcription factor phosphatase 1G. Knockdown of phosphatase 1G in a human liver cell model resulted in decreased apolipoprotein E transcription and apolipoprotein E protein levels in cultured supernatants. Conclusions: We identified dozens of novel genetic determinants of proteins associated with the Framingham Cardiovascular Disease Risk Score and experimentally validated a new role for phosphatase 1G in lipoprotein biology. Further, genome-wide and exome array data for each protein have been made publicly available as a resource for cardiovascular disease research.

A yeast artificial chromosome (YAC) mouse model of Huntington's disease (YAC128) develops motor abnormalities, age-dependent striatal atrophy and neuronal loss. Alteration of neurotransmitter receptors, particularly glutamate and dopamine receptors, is a pathological hallmark of Huntington's disease. We therefore analyzed neurotransmitter receptors in symptomatic YAC128 Huntington's disease mice. We found significant increases in N-methyl-d-aspartate, AMPA and metabotropic glutamate receptor binding, which were not due to increases in receptor subunit mRNA expression levels. Subcellular fractionation analysis revealed increased levels of glutamate receptor subunits in synaptic membrane fractions from YAC128 mice. We found no changes in dopamine, GABA or adenosine receptor binding, nor did we see alterations in dopamine D1, D2 or adenosine A2a receptor mRNA levels. The receptor abnormalities in YAC128 transgenic mice thus appear limited to glutamate receptors. We also found a significant decrease in preproenkephalin mRNA in the striatum of YAC128 mice, which contrasts with the lack of change in levels of mRNA encoding neurotransmitter receptors. Taken together, the abnormal and selective increases in glutamate receptor subunit expression and binding are not due to increases in receptor subunit expression and may exert detrimental effects. The decrease in preproenkephalin mRNA suggests a selective transcriptional deficit, as opposed to neuronal loss, and could additionally contribute to the abnormal motor symptoms in YAC128 mice.

We analyzed NMDA receptor subunit mRNAs, proteins, and anchoring proteins in mice transgenic for exon 1 of the HD gene. R6/2 mice had decreased levels of mRNAs encoding epsilon1 and epsilon2 NMDA receptor subunits (mouse orthologs of rat NR2A and NR2B subunits), but not the zeta1 subunit (mouse ortholog of NR1), as assessed by gene expression profiling and Northern blotting. In situ hybridization resolved mRNA decreases spatially to the CA1 field of hippocampus. Western blotting revealed decreases in plasma membrane-associated epsilon1 and epsilon2 subunits in hippocampus, and decreases in plasma membrane-associated zeta1 subunit in cortex and hippocampus. In addition, PSD-95 and alpha-actinin-2, proteins essential for anchoring NMDA receptors, were decreased. Finally, we found a decreased level of tyrosine-phosphorylated epsilon1 subunit, another determinant of NMDA receptor trafficking, in R6/2 hippocampus. Taken together, these data demonstrate multiple levels of NMDA receptor dysregulation, including abnormalities in mRNA expression levels, receptor stoichiometry, protein phosphorylation, and receptor trafficking.

Background: It is sometimes challenging to distinguish hypertrophic cardiomyopathy (HCM) from other cardiac conditions such as hypertensive heart disease. The present study utilized metabolite profiling, a novel technology that measures serum concentrations of biochemical substances, to distinguish

Elevated pulmonary arterial aldosterone levels (PA-ALDO) are associated with decreased exercise capacity in pulmonary hypertension patients despite normal left ventricular (LV) function. Pulmonary endothelial ALDO synthesis occurs in vitro under experimental conditions akin to LV systolic heart failure (HFrEF); however, the relevance of PA-ALDO to the clinical profile of HFrEF is not known. Therefore, we analyzed PA and radial arterial plasma in control and HFrEF patients to assess for transpulmonary ALDO release and determine if circulating ALDO is related to hemodynamic indices of cardiac reserve capacity during exercise. Data from 20 controls (51 ± 13 y, 13 females, LVEF=0.66 ± 0.9, volume of oxygen uptake at peak exercise [pVO2]=94.1 ± 11.5 % predicted) and 42 consecutive HFrEF patients (57 ± 13 y, 20 females, LVEF=0.36 ± 0.1, pVO2=47.1 ± 2.5 % predicted) undergoing invasive cardiopulmonary exercise testing were analyzed. At rest, HFrEF patients had increased PA-ALDO (714 ± 450 vs. 380 ± 187 ng/dl, P

Huntington disease is an autosomal dominant neurodegenerative disease with no effective treatment. Minocycline is a tetracycline derivative with proven safety. After ischemia, minocycline inhibits caspase-1 and inducible nitric oxide synthetase upregulation, and reduces infarction. As caspase-1 and nitric oxide seem to play a role in Huntington disease, we evaluated the therapeutic efficacy of minocycline in the R6/2 mouse model of Huntington disease. We report that minocycline delays disease progression, inhibits caspase-1 and caspase-3 mRNA upregulation, and decreases inducible nitric oxide synthetase activity. In addition, effective pharmacotherapy in R6/2 mice requires caspase-1 and caspase-3 inhibition. This is the first demonstration of caspase-1 and caspase-3 transcriptional regulation in a Huntington disease model.

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

We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.

Dyslipidemia is an independent risk factor for type 2 diabetes, although exactly which of the many plasma lipids contribute to this remains unclear. We therefore investigated whether lipid profiling can inform diabetes prediction by performing liquid chromatography/mass spectrometry-based lipid profiling in 189 individuals who developed type 2 diabetes and 189 matched disease-free individuals, with over 12 years of follow up in the Framingham Heart Study. We found that lipids of lower carbon number and double bond content were associated with an increased risk of diabetes, whereas lipids of higher carbon number and double bond content were associated with decreased risk. This pattern was strongest for triacylglycerols (TAGs) and persisted after multivariable adjustment for age, sex, BMI, fasting glucose, fasting insulin, total triglycerides, and HDL cholesterol. A combination of 2 TAGs further improved diabetes prediction. To explore potential mechanisms that modulate the distribution of plasma lipids, we performed lipid profiling during oral glucose tolerance testing, pharmacologic interventions, and acute exercise testing. Levels of TAGs associated with increased risk for diabetes decreased in response to insulin action and were elevated in the setting of insulin resistance. Conversely, levels of TAGs associated with decreased diabetes risk rose in response to insulin and were poorly correlated with insulin resistance. These studies identify a relationship between lipid acyl chain content and diabetes risk and demonstrate how lipid profiling could aid in clinical risk assessment.

Exercise provides numerous salutary effects, but our understanding of how these occur is limited. To gain a clearer picture of exercise-induced metabolic responses, we have developed comprehensive plasma metabolite signatures by using mass spectrometry to measure >200 metabolites before and after exercise. We identified plasma indicators of glycogenolysis (glucose-6-phosphate), tricarboxylic acid cycle span 2 expansion (succinate, malate, and fumarate), and lipolysis (glycerol), as well as modulators of insulin sensitivity (niacinamide) and fatty acid oxidation (pantothenic acid). Metabolites that were highly correlated with fitness parameters were found in subjects undergoing acute exercise testing and marathon running and in 302 subjects from a longitudinal cohort study. Exercise-induced increases in glycerol were strongly related to fitness levels in normal individuals and were attenuated in subjects with myocardial ischemia. A combination of metabolites that increased in plasma in response to exercise (glycerol, niacinamide, glucose-6-phosphate, pantothenate, and succinate) up-regulated the expression of nur77, a transcriptional regulator of glucose utilization and lipid metabolism genes in skeletal muscle in vitro. Plasma metabolic profiles obtained during exercise provide signatures of exercise performance and cardiovascular disease susceptibility, in addition to highlighting molecular pathways that may modulate the salutary effects of exercise.

ESRD is a state of small-molecule disarray. We applied liquid chromatography/tandem mass spectrometry-based metabolite profiling to survey350 small molecules in 44 fasting subjects with ESRD, before and after hemodialysis, and in 10 age-matched, at-risk fasting control subjects. At baseline, increased levels of polar analytes and decreased levels of lipid analytes characterized uremic plasma. In addition to confirming the elevation of numerous previously identified uremic toxins, we identified several additional markers of ESRD, including dicarboxylic acids (adipate, malonate, methylmalonate, and maleate), biogenic amines, nucleotide derivatives, phenols, and sphingomyelins. The pattern of lipids was notable for a universal decrease in lower-molecular-weight triacylglycerols, and an increase in several intermediate-molecular-weight triacylglycerols in ESRD compared with controls; standard measurement of total triglycerides obscured this heterogeneity. These observations suggest disturbed triglyceride catabolism and/or beta-oxidation in ESRD. As expected, the hemodialysis procedure was associated with significant decreases in most polar analytes. Unexpected increases in several metabolites, however, indicated activation of a broad catabolic program, including glycolysis, lipolysis, ketosis, and nucleotide breakdown. In summary, this study demonstrates the application of metabolite profiling to identify markers of ESRD, provide perspective on uremic dyslipidemia, and broaden our understanding of the biochemical effects of hemodialysis.

Emerging metabolomic tools have created the opportunity to establish metabolic signatures of myocardial injury. We applied a mass spectrometry–based metabolite profiling platform to 36 patients undergoing alco-hol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of planned myo-cardial infarction (PMI). Serial blood samples were obtained before and at various intervals after PMI, with patients undergoing elective diagnostic coronary angiography and patients with spontaneous myocardial infarction (SMI) serving as negative and positive controls, respectively. We identified changes in circulating levels of metabolites participating in pyrimidine metabolism, the tricarboxylic acid cycle and its upstream con-tributors, and the pentose phosphate pathway. Alterations in levels of multiple metabolites were detected as early as 10 minutes after PMI in an initial derivation group and were validated in a second, independent group of PMI patients. A PMI-derived metabolic signature consisting of aconitic acid, hypoxanthine, trimethylamine N-oxide, and threonine differentiated patients with SMI from those undergoing diagnostic coronary angiogra-phy with high accuracy, and coronary sinus sampling distinguished cardiac-derived from peripheral metabolic changes. Our results identify a role for metabolic profiling in the early detection of myocardial injury and sug-gest that similar approaches may be used for detection or prediction of other disease states.

Transcriptional dysregulation is a central pathogenic mechanism in Huntington's disease (HD); HD and transgenic mouse models of HD demonstrate down-regulation of specific genes at the level of mRNA expression. Furthermore, neuronal intranuclear inclusions (NIIs) have been identified in the brains of R6/2 mice and HD patients. One possibility is that NIIs contribute to transcriptional dysregulation by sequestering transcription factors. We therefore assessed the relationship between NIIs and transcriptional dysregulation in the R6/2 mouse, using double-label in situ hybridization combined with immunohistochemistry, and laser capture microdissection combined with quantitative real-time PCR. There was no difference in transcript levels of specific genes between NII-positive and NII-negative neurons. These results demonstrate that NIIs do not cause decreases in D2, PPE and PSS mRNA levels in R6/2 striatum and therefore are not involved in the down-regulation of these specific genes in this HD model. In addition, these observations argue against the notion that NIIs protect against transcriptional dysregulation in HD.

One of the characteristic findings in human Huntington's disease (HD) is the alteration of neurotransmitter receptors. To a remarkable degree, transgenic HD mouse models recapitulate neurotransmitter receptor alterations. Neurotransmitter receptors can be assessed at the protein level by using receptor-binding autoradiography. One can also measure levels of receptor messenger RNA with in situ hybridization (ISH), employing either oligonucleotide or ribonucleotide probes. Both of these techniques-receptor-binding autoradiography and in situ hybridization-yield quantitative and regionally specific information regarding neurotransmitter receptors. We describe techniques for performing receptor-binding autoradiography and two types of in situ hybridization using oligonucleotide and ribonucleotide probes. With receptor binding and ISH, one can obtain quantitative region-specific assessments of neurotransmitter receptor alteration, a key pathologic event in HD pathogenesis.

One of the characteristic findings in human Huntington's disease (HD) is the alteration of neurotransmitter receptors. To a remarkable degree, transgenic HD mouse models recapitulate neurotransmitter receptor alterations. Neurotransmitter receptors can be assessed at the protein level by using receptor-binding autoradiography. One can also measure levels of receptor messenger RNA with in situ hybridization (ISH), employing either oligonucleotide or ribonucleotide probes. Both of these techniques-receptor-binding autoradiography and in situ hybridization-yield quantitative and regionally specific information regarding neurotransmitter receptors. We describe techniques for performing receptor-binding autoradiography and two types of in situ hybridization using oligonucleotide and ribonucleotide probes. With receptor binding and ISH, one can obtain quantitative region-specific assessments of neurotransmitter receptor alteration, a key pathologic event in HD pathogenesis.

There is increasing evidence that transcriptional dysregulation is important in Huntington's disease pathogenesis. The transcriptional protein, nuclear corepressor (NCoR), acts to repress transcription of nuclear hormone receptors, such as the thyroid hormone receptor (TR) and retinoic acid receptor, in the absence of their appropriate ligand. NCoR has been shown to bind to the mutated huntingtin protein in a yeast two-hybrid screen. This aberrant interaction may have profound effects on both the function of the NCoR protein and on its control of nuclear hormone receptor-mediated transcription. To test this hypothesis, reporter gene assays were performed in inducible PC12 cell lines expressing exon 1 of the human huntingtin protein (Htt) with either a 25 or 103 polyglutamine (Q) repeat. Expression of mutant 103Q protein appears to enhance the ability of NCoR to repress TR-mediated transcription in the absence of ligand. Western analyses indicated that the expression of the mutant 103Q Htt protein did not change the endogenous NCoR levels in the HD103Q PC12 cells when compared to uninduced cells. Interestingly, using GST pull-down assays we found that a mutant Htt exon 1 construct with 53 polyglutamine (HD53Q) did not bind to NCoR in a polyglutamine-dependent fashion. These findings suggest that an aberrant NCoR-Htt interaction does not exist in vitro. Expression of the mutant 103Q protein was also found to enhance ligand-dependent activation of TR and retinoic acid receptor. In vitro binding data shows that TR binds to HD53Q in the presence of ligand. Taken together these data suggest that Htt may function as a transcriptional coactivator of nuclear hormone receptors.