Maren Carstensen

Novel biomarkers for pre-diabetes identified by metabolomics

Type 2 diabetes (T2D) can be prevented in pre‐diabetic individuals with impaired glucose tolerance (IGT). Here, we have used a metabolomics approach to identify candidate biomarkers of pre‐diabetes. We quantified 140 metabolites for 4297 fasting serum samples in the population‐based Cooperative Health Research in the Region of Augsburg (KORA) cohort. Our study revealed significant metabolic variation in pre‐diabetic individuals that are distinct from known diabetes risk indicators, such as glycosylated hemoglobin levels, fasting glucose and insulin. We identified three metabolites (glycine, lysophosphatidylcholine (LPC) (18:2) and acetylcarnitine) that had significantly altered levels in IGT individuals as compared to those with normal glucose tolerance, with P‐values ranging from 2.4 × 10−4 to 2.1 × 10−13. Lower levels of glycine and LPC were found to be predictors not only for IGT but also for T2D, and were independently confirmed in the European Prospective Investigation into Cancer and Nutrition (EPIC)‐Potsdam cohort. Using metabolite–protein network analysis, we identified seven T2D‐related genes that are associated with these three IGT‐specific metabolites by multiple interactions with four enzymes. The expression levels of these enzymes correlate with changes in the metabolite concentrations linked to diabetes. Our results may help developing novel strategies to prevent T2D.

Effects of coffee consumption on subclinical inflammation and other risk factors for type 2 diabetes: a clinical trial

BACKGROUND Coffee consumption is associated with a decreased risk of type 2 diabetes. Suggested mechanisms underlying the association have included attenuation of subclinical inflammation and a reduction in oxidative stress. OBJECTIVE The aim was to investigate the effects of daily coffee consumption on biomarkers of coffee intake, subclinical inflammation, oxidative stress, glucose, and lipid metabolism. DESIGN Habitual coffee drinkers (n = 47) refrained for 1 mo from coffee drinking; in the second month they consumed 4 cups of filtered coffee/d and in the third month 8 cups of filtered coffee/d (150 mL/cup). Blood samples were analyzed by gas chromatography-mass spectrometry, bead-based multiplex technology, enzyme-linked immunosorbent assay, or immunonephelometry. RESULTS Coffee consumption led to an increase in coffee-derived compounds, mainly serum caffeine, chlorogenic acid, and caffeic acid metabolites. Significant changes were also observed for serum concentrations of interleukin-18, 8-isoprostane, and adiponectin (medians: -8%, -16%, and 6%, respectively; consumption of 8 compared with 0 cups coffee/d). Serum concentrations of total cholesterol, HDL cholesterol, and apolipoprotein A-I increased significantly by 12%, 7%, and 4%, respectively, whereas the ratios of LDL to HDL cholesterol and of apolipoprotein B to apolipoprotein A-I decreased significantly by 8% and 9%, respectively (8 compared with 0 cups coffee/d). No changes were seen for markers of glucose metabolism in an oral-glucose-tolerance test. CONCLUSIONS Coffee consumption appears to have beneficial effects on subclinical inflammation and HDL cholesterol, whereas no changes in glucose metabolism were found in our study. Furthermore, many coffee-derived methylxanthines and caffeic acid metabolites appear to be useful as biomarkers of coffee intake.

Accelerated Increase in Serum Interleukin-1 Receptor Antagonist Starts 6 Years Before Diagnosis of Type 2 Diabetes Whitehall II Prospective Cohort Study

OBJECTIVE Although interleukin-1 receptor antagonist (IL-1Ra) treatment is associated with improved β-cell function and glycemic control in patients with type 2 diabetes, its role in the development of type 2 diabetes remains unclear. We used repeated measurements to characterize IL-1Ra trajectories in individuals who developed type 2 diabetes. RESEARCH DESIGN AND METHODS This case-cohort study, nested within the Whitehall II cohort, was based on 335 incident type 2 diabetes cases and 2,475 noncases. We measured serum IL-1Ra levels at up to three time points per individual and estimated retrospective trajectories of IL-1Ra before diabetes diagnosis (case subjects) or end of follow-up (control subjects) using multilevel analysis. Models were adjusted for age, sex, and ethnicity. RESULTS IL-1Ra levels were already higher in the case than control subjects 13 years before diabetes diagnosis/end of follow-up (mean [95% CI] 302 [290–314] vs. 244 [238–249] pg/ml). In control subjects, IL-1Ra levels showed a modest linear increase throughout the study period. In case subjects, IL-1Ra trajectories were parallel to those in control subjects until 6 years (95% CI 7.5–4.5) before diagnosis and then rose steeply to 399 (379–420) pg/ml at the time of diagnosis (P < 0.0001 for slope difference). Adjustment for BMI and waist circumference as time-varying covariates had little impact on these trajectories. CONCLUSIONS We show elevated IL-1Ra levels for 13 years and an accelerated increase during the last 6 years before type 2 diabetes diagnosis, indicating the presence of an anti-inflammatory response that may act to counterbalance the metabolic and immunologic disturbances that precede type 2 diabetes.

Anti‐inflammatory cytokines and risk of type 2 diabetes

Proinflammatory processes have been investigated extensively in the development of type 2 diabetes, but our knowledge on anti‐inflammatory proteins is rather limited. This article summarizes studies that investigated associations between circulating levels of anti‐inflammatory cytokines and incident type 2 diabetes preferably in prospective epidemiological studies. Adiponectin is the only known anti‐inflammatory protein whose circulating levels are decreased before type 2 diabetes. In contrast, concentrations of interleukin‐1 receptor antagonist (IL‐1RA), transforming growth factor‐β1 (TGF‐β1) and growth differentiation factor‐15 (GDF‐15) are increased and indicate the presence of a compensatory, but eventually futile, counter‐regulation of proinflammatory stimuli. Importantly, a proof‐of‐principle study using recombinant IL‐1RA to improve metabolic control in patients with type 2 diabetes demonstrated that a more pronounced upregulation of this protein than that found in the natural course of diabetes development may have clinical relevance. Other interesting candidates like omentin (which shows similar associations with metabolic parameters as adiponectin), interleukin‐10 (IL‐10) and secreted frizzled‐related protein‐5 (Sfrp5) are currently less well studied with sometimes conflicting results regarding their association with type 2 diabetes. Thus, further research is required to better understand the causal role of proinflammatory cytokines, hypoadiponectinaemia and the upregulation of anti‐inflammatory proteins before the onset of type 2 diabetes.

Mechanisms Underlying the Onset of Oral Lipid–Induced Skeletal Muscle Insulin Resistance in Humans

Several mechanisms, such as innate immune responses via Toll-like receptor-4, accumulation of diacylglycerols (DAG)/ceramides, and activation of protein kinase C (PKC), are considered to underlie skeletal muscle insulin resistance. In this study, we examined initial events occurring during the onset of insulin resistance upon oral high-fat loading compared with lipid and low-dose endotoxin infusion. Sixteen lean insulin-sensitive volunteers received intravenous fat (iv fat), oral fat (po fat), intravenous endotoxin (lipopolysaccharide [LPS]), and intravenous glycerol as control. After 6 h, whole-body insulin sensitivity was reduced by iv fat, po fat, and LPS to 60, 67, and 48%, respectively (all P < 0.01), which was due to decreased nonoxidative glucose utilization, while hepatic insulin sensitivity was unaffected. Muscle PKCθ activation increased by 50% after iv and po fat, membrane Di-C18:2 DAG species doubled after iv fat and correlated with PKCθ activation after po fat, whereas ceramides were unchanged. Only after LPS, circulating inflammatory markers (tumor necrosis factor-α, interleukin-6, and interleukin-1 receptor antagonist), their mRNA expression in subcutaneous adipose tissue, and circulating cortisol were elevated. Po fat ingestion rapidly induces insulin resistance by reducing nonoxidative glucose disposal, which associates with PKCθ activation and a rise in distinct myocellular membrane DAG, while endotoxin-induced insulin resistance is exclusively associated with stimulation of inflammatory pathways.

A Meta-analysis of Gene Expression Signatures of Blood Pressure and Hypertension

Genome-wide association studies (GWAS) have uncovered numerous genetic variants (SNPs) that are associated with blood pressure (BP). Genetic variants may lead to BP changes by acting on intermediate molecular phenotypes such as coded protein sequence or gene expression, which in turn affect BP variability. Therefore, characterizing genes whose expression is associated with BP may reveal cellular processes involved in BP regulation and uncover how transcripts mediate genetic and environmental effects on BP variability. A meta-analysis of results from six studies of global gene expression profiles of BP and hypertension in whole blood was performed in 7017 individuals who were not receiving antihypertensive drug treatment. We identified 34 genes that were differentially expressed in relation to BP (Bonferroni-corrected p<0.05). Among these genes, FOS and PTGS2 have been previously reported to be involved in BP-related processes; the others are novel. The top BP signature genes in aggregate explain 5%–9% of inter-individual variance in BP. Of note, rs3184504 in SH2B3, which was also reported in GWAS to be associated with BP, was found to be a trans regulator of the expression of 6 of the transcripts we found to be associated with BP (FOS, MYADM, PP1R15A, TAGAP, S100A10, and FGBP2). Gene set enrichment analysis suggested that the BP-related global gene expression changes include genes involved in inflammatory response and apoptosis pathways. Our study provides new insights into molecular mechanisms underlying BP regulation, and suggests novel transcriptomic markers for the treatment and prevention of hypertension.

Analyzing Illumina Gene Expression Microarray Data from Different Tissues: Methodological Aspects of Data Analysis in the MetaXpress Consortium

Microarray profiling of gene expression is widely applied in molecular biology and functional genomics. Experimental and technical variations make meta-analysis of different studies challenging. In a total of 3358 samples, all from German population-based cohorts, we investigated the effect of data preprocessing and the variability due to sample processing in whole blood cell and blood monocyte gene expression data, measured on the Illumina HumanHT-12 v3 BeadChip array. Gene expression signal intensities were similar after applying the log2 or the variance-stabilizing transformation. In all cohorts, the first principal component (PC) explained more than 95% of the total variation. Technical factors substantially influenced signal intensity values, especially the Illumina chip assignment (33–48% of the variance), the RNA amplification batch (12–24%), the RNA isolation batch (16%), and the sample storage time, in particular the time between blood donation and RNA isolation for the whole blood cell samples (2–3%), and the time between RNA isolation and amplification for the monocyte samples (2%). White blood cell composition parameters were the strongest biological factors influencing the expression signal intensities in the whole blood cell samples (3%), followed by sex (1–2%) in both sample types. Known single nucleotide polymorphisms (SNPs) were located in 38% of the analyzed probe sequences and 4% of them included common SNPs (minor allele frequency >5%). Out of the tested SNPs, 1.4% significantly modified the probe-specific expression signals (Bonferroni corrected p-value<0.05), but in almost half of these events the signal intensities were even increased despite the occurrence of the mismatch. Thus, the vast majority of SNPs within probes had no significant effect on hybridization efficiency. In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta-analyses.

Impact of common regulatory single-nucleotide variants on gene expression profiles in whole blood

Genome-wide association studies (GWASs) have uncovered susceptibility loci for a large number of complex traits. Functional interpretation of candidate genes identified by GWAS and confident assignment of the causal variant still remains a major challenge. Expression quantitative trait (eQTL) mapping has facilitated identification of risk loci for quantitative traits and might allow prioritization of GWAS candidate genes. One major challenge of eQTL studies is the need for larger sample numbers and replication. The aim of this study was to evaluate the robustness and reproducibility of whole-blood eQTLs in humans and test their value in the identification of putative functional variants involved in the etiology of complex traits. In the current study, we performed comphrehensive eQTL mapping from whole blood. The discovery sample included 322 Caucasians from a general population sample (KORA F3). We identified 363 cis and 8 trans eQTLs after stringent Bonferroni correction for multiple testing. Of these, 98.6% and 50% of cis and trans eQTLs, respectively, could be replicated in two independent populations (KORA F4 (n=740) and SHIP-TREND (n=653)). Furthermore, we identified evidence of regulatory variation for SNPs previously reported to be associated with disease loci (n=59) or quantitative trait loci (n=20), indicating a possible functional mechanism for these eSNPs. Our data demonstrate that eQTLs in whole blood are highly robust and reproducible across studies and highlight the relevance of whole-blood eQTL mapping in prioritization of GWAS candidate genes in humans.

The Human Blood Metabolome-Transcriptome Interface

Biological systems consist of multiple organizational levels all densely interacting with each other to ensure function and flexibility of the system. Simultaneous analysis of cross-sectional multi-omics data from large population studies is a powerful tool to comprehensively characterize the underlying molecular mechanisms on a physiological scale. In this study, we systematically analyzed the relationship between fasting serum metabolomics and whole blood transcriptomics data from 712 individuals of the German KORA F4 cohort. Correlation-based analysis identified 1,109 significant associations between 522 transcripts and 114 metabolites summarized in an integrated network, the ‘human blood metabolome-transcriptome interface’ (BMTI). Bidirectional causality analysis using Mendelian randomization did not yield any statistically significant causal associations between transcripts and metabolites. A knowledge-based interpretation and integration with a genome-scale human metabolic reconstruction revealed systematic signatures of signaling, transport and metabolic processes, i.e. metabolic reactions mainly belonging to lipid, energy and amino acid metabolism. Moreover, the construction of a network based on functional categories illustrated the cross-talk between the biological layers at a pathway level. Using a transcription factor binding site enrichment analysis, this pathway cross-talk was further confirmed at a regulatory level. Finally, we demonstrated how the constructed networks can be used to gain novel insights into molecular mechanisms associated to intermediate clinical traits. Overall, our results demonstrate the utility of a multi-omics integrative approach to understand the molecular mechanisms underlying both normal physiology and disease.

Sfrp5 correlates with insulin resistance and oxidative stress

Secreted frizzled‐related protein 5 (Sfrp5) has been described as novel adipokine in mice with insulin‐sensitising and anti‐inflammatory properties similar to adiponectin. The aim of this study was to compare serum concentrations and determinants of Sfrp5, its pro‐inflammatory antagonist wingless‐type MMTV integration site family member (Wnt)5a and adiponectin in humans and their regulation by coffee.