M. Julia Brosnan

An atlas of genetic influences on human blood metabolites.

Genome-wide association scans with high-throughput metabolic profiling provide unprecedented insights into how genetic variation influences metabolism and complex disease. Here we report the most comprehensive exploration of genetic loci influencing human metabolism thus far, comprising 7,824 adult individuals from 2 European population studies. We report genome-wide significant associations at 145 metabolic loci and their biochemical connectivity with more than 400 metabolites in human blood. We extensively characterize the resulting in vivo blueprint of metabolism in human blood by integrating it with information on gene expression, heritability and overlap with known loci for complex disorders, inborn errors of metabolism and pharmacological targets. We further developed a database and web-based resources for data mining and results visualization. Our findings provide new insights into the role of inherited variation in blood metabolic diversity and identify potential new opportunities for drug development and for understanding disease.

Metabolomic markers reveal novel pathways of ageing and early development in human populations

Background Human ageing is a complex, multifactorial process and early developmental factors affect health outcomes in old age. Methods Metabolomic profiling on fasting blood was carried out in 6055 individuals from the UK. Stepwise regression was performed to identify a panel of independent metabolites which could be used as a surrogate for age. We also investigated the association with birthweight overall and within identical discordant twins and with genome-wide methylation levels. Results We identified a panel of 22 metabolites which combined are strongly correlated with age (R2 = 59%) and with age-related clinical traits independently of age. One particular metabolite, C-glycosyl tryptophan (C-glyTrp), correlated strongly with age (beta = 0.03, SE = 0.001, P = 7.0 × 10−157) and lung function (FEV1 beta = −0.04, SE = 0.008, P = 1.8 × 10−8 adjusted for age and confounders) and was replicated in an independent population (n = 887). C-glyTrp was also associated with bone mineral density (beta = −0.01, SE = 0.002, P = 1.9 × 10−6) and birthweight (beta = −0.06, SE = 0.01, P = 2.5 × 10−9). The difference in C-glyTrp levels explained 9.4% of the variance in the difference in birthweight between monozygotic twins. An epigenome-wide association study in 172 individuals identified three CpG-sites, associated with levels of C-glyTrp (P < 2 × 10−6). We replicated one CpG site in the promoter of the WDR85 gene in an independent sample of 350 individuals (beta = −0.20, SE = 0.04, P = 2.9 × 10−8). WDR85 is a regulator of translation elongation factor 2, essential for protein synthesis in eukaryotes. Conclusions Our data illustrate how metabolomic profiling linked with epigenetic studies can identify some key molecular mechanisms potentially determined in early development that produce long-term physiological changes influencing human health and ageing.

Assessing the phenotypic effects in the general population of rare variants in genes for a dominant Mendelian form of diabetes.

Genome sequencing can identify individuals in the general population who harbor rare coding variants in genes for Mendelian disorders and who may consequently have increased disease risk. Previous studies of rare variants in phenotypically extreme individuals display ascertainment bias and may demonstrate inflated effect-size estimates. We sequenced seven genes for maturity-onset diabetes of the young (MODY) in well-phenotyped population samples (n = 4,003). We filtered rare variants according to two prediction criteria for disease-causing mutations: reported previously in MODY or satisfying stringent de novo thresholds (rare, conserved and protein damaging). Approximately 1.5% and 0.5% of randomly selected individuals from the Framingham and Jackson Heart Studies, respectively, carry variants from these two classes. However, the vast majority of carriers remain euglycemic through middle age. Accurate estimates of variant effect sizes from population-based sequencing are needed to avoid falsely predicting a substantial fraction of individuals as being at risk for MODY or other Mendelian diseases.

An integrated epigenomic analysis for type 2 diabetes susceptibility loci in monozygotic twins

DNA methylation has a great potential for understanding the aetiology of common complex traits such as Type 2 diabetes (T2D). Here we perform genome-wide methylated DNA immunoprecipitation sequencing (MeDIP-seq) in whole-blood-derived DNA from 27 monozygotic twin pairs and follow up results with replication and integrated omics analyses. We identify predominately hypermethylated T2D-related differentially methylated regions (DMRs) and replicate the top signals in 42 unrelated T2D cases and 221 controls. The strongest signal is in the promoter of the MALT1 gene, involved in insulin and glycaemic pathways, and related to taurocholate levels in blood. Integrating the DNA methylome findings with T2D GWAS meta-analysis results reveals a strong enrichment for DMRs in T2D-susceptibility loci. We also detect signals specific to T2D-discordant twins in the GPR61 and PRKCB genes. These replicated T2D associations reflect both likely causal and consequential pathways of the disease. The analysis indicates how an integrated genomics and epigenomics approach, utilizing an MZ twin design, can provide pathogenic insights as well as potential drug targets and biomarkers for T2D and other complex traits.

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export

Objective A branched-chain amino acid (BCAA)-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition. Methods Zucker-lean rats (ZLR) and Zucker-fatty rats (ZFR) were fed either a custom control, low fat (LF) diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val) were reduced by 45% (LF-RES). We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism. Results LF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH), was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp. Conclusions Our data are consistent with a model wherein elevated circulating BCAA contribute to development of obesity-related insulin resistance by interfering with lipid oxidation in skeletal muscle. BCAA-dependent lowering of the skeletal muscle glycine pool appears to contribute to this effect by slowing acyl-glycine export to the urine.

The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins

Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

Apolipoprotein CIII and N-terminal prohormone b-type natriuretic peptide as independent predictors for cardiovascular disease in type 2 diabetes

BACKGROUND AND AIMS Developing sparse panels of biomarkers for cardiovascular disease in type 2 diabetes would enable risk stratification for clinical decision making and selection into clinical trials. We examined the individual and joint performance of five candidate biomarkers for incident cardiovascular disease (CVD) in type 2 diabetes that an earlier discovery study had yielded. METHODS Apolipoprotein CIII (apoCIII), N-terminal prohormone B-type natriuretic peptide (NT-proBNP), high sensitivity Troponin T (hsTnT), Interleukin-6, and Interleukin-15 were measured in baseline serum samples from the Collaborative Atorvastatin Diabetes trial (CARDS) of atorvastatin versus placebo. Among 2105 persons with type 2 diabetes and median age of 62.9 years (range 39.2-77.3), there were 144 incident CVD (acute coronary heart disease or stroke) cases during the maximum 5-year follow up. We used Cox Proportional Hazards models to identify biomarkers associated with incident CVD and the area under the receiver operating characteristic curves (AUROC) to assess overall model prediction. RESULTS Three of the biomarkers were singly associated with incident CVD independently of other risk factors; NT-proBNP (Hazard Ratio per standardised unit 2.02, 95% Confidence Interval [CI] 1.63, 2.50), apoCIII (1.34, 95% CI 1.12, 1.60) and hsTnT (1.40, 95% CI 1.16, 1.69). When combined in a single model, only NT-proBNP and apoCIII were independent predictors of CVD, together increasing the AUROC using Framingham risk variables from 0.661 to 0.745. CONCLUSIONS The biomarkers NT-proBNP and apoCIII substantially increment the prediction of CVD in type 2 diabetes beyond that obtained with the variables used in the Framingham risk score.