Metabolomic associations of impaired awareness of hypoglycaemia in type 1 diabetes.

This study investigates impaired awareness of hypoglycaemia (IAH), a complication of insulin therapy affecting 20-40% of individuals with type 1 diabetes. The exact pathophysiology is unclear, therefore we sought to identify metabolic signatures in IAH to elucidate potential pathophysiological pathways. Plasma samples from 578 individuals of the Dutch type 1 diabetes biomarker cohort, 67 with IAH and 108 without IAH (NAH) were analysed using the targeted metabolomics Biocrates AbsoluteIDQ p180 assay. Eleven metabolites were significantly associated with IAH. Genome-wide association studies of these 11 metabolites identified significant single nucleotide polymorphisms (SNPs) in C22:1-OH and phosphatidylcholine diacyl C36:6. After adjusting for the SNPs, 11 sphingomyelins and phosphatidylcholines were significantly higher in the IAH group in comparison to NAH. These metabolites are important components of the cell membrane and have been implicated to play a role in cell signalling in diabetes. These findings demonstrate the potential role of phosphatidylcholine and sphingomyelins in IAH.

Blood and adipose tissue steroid metabolomics and mRNA expression of steroidogenic enzymes in periparturient dairy cows differing in body condition.

In high-yielding dairy cows, the rapidly increasing milk production after parturition can result in a negative nutrient balance, since feed intake is insufficient to cover the needs for lactation. Mobilizing body reserves, mainly adipose tissue (AT), might affect steroid metabolism. We hypothesized, that cows differing in the extent of periparturient lipomobilization, will have divergent steroid profiles measured in serum and subcutaneous (sc)AT by a targeted metabolomics approach and steroidogenic enzyme profiles in scAT and liver. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to a high (HBCS) or normal body condition (NBCS) group fed differently until week 7 antepartum to either increase (HBCS BCS: 3.8 ± 0.1 and BFT: 2.0 ± 0.1 cm; mean ± SEM) or maintain BCS (NBCS BCS: 3.0 ± 0.1 and BFT: 0.9 ± 0.1 cm). Blood samples, liver, and scAT biopsies were collected at week -7, 1, 3, and 12 relative to parturition. Greater serum concentrations of progesterone, androsterone, and aldosterone in HBCS compared to NBCS cows after parturition, might be attributed to the increased mobilization of AT. Greater glucocorticoid concentrations in scAT after parturition in NBCS cows might either influence local lipogenesis by differentiation of preadipocytes into mature adipocytes and/or inflammatory response.

Targeted assessment of the metabolome in skeletal muscle and in serum of dairy cows supplemented with conjugated linoleic acid during early lactation.

In the dairy cow, late gestation and early lactation are characterized by a complexity of metabolic processes required for the homeorhetic adaptation to the needs of fetal growth and milk production. Skeletal muscle plays an important role in this adaptation. The objective of this study was to characterize the metabolome in skeletal muscle (semitendinosus muscle) and in serum of dairy cows in the context of the physiological changes occurring in early lactation and to test the effects of dietary supplementation (from d 1 in milk onwards) with conjugated linoleic acids (sCLA; 100 g/d; supplying 7.6 g of cis-9,trans-11 CLA and 7.6 g of trans-10,cis-12 CLA per cow/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). The metabolome was characterized in skeletal muscle samples collected on d 21 and 70 after calving in conjunction with their serum counterpart using a targeted metabolomics approach (AbsoluteIDQ p180 kit; Biocrates Life Sciences AG, Innsbruck, Austria). Thereby 188 metabolites from 6 different compound classes (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids, and hexoses) were quantified in both sample types. In both groups, dry matter intake increased after calving. It was lower in sCLA than in CTR on d 21, which resulted in reduced calculated net energy and metabolizable protein balances. On d 21, the concentrations of dopamine, Ala, and hexoses in the skeletal muscle were higher in sCLA than in CTR. On d 21, the changed metabolites in serum were mainly long-chain (>C24) diacyl phosphatidylcholine PC (PC-aa) and acyl-alkyl phosphatidylcholine (PC-ae), along with lysophosphatidylcholine acyl (lysoPC-a) C26:1 that were all lower in sCLA than in CTR. Supplementation with CLA affected the muscle concentrations of 22 metabolites on d 70 including 10 long-chain (>C22) sphingomyelin (SM), hydroxysphingomyelin [SM(OH)], PC-aa, and PC-ae along with 9 long-chain (>C16) lysoPC-a and 3 metabolites related to amino acids (spermine, citrulline, and Asp). On d 70, the concentrations of lysoPC-a C18:2 and C26:0 in serum were higher in the sCLA cows than in the CTR cows. Regardless of treatment, the concentrations of Ile, Leu, Phe, Lys, His, Met, Trp, and hydroxybutyrylcarnitine (C4-OH) decreased, whereas those of ornithine, Gln, and trans-4-hydroxyproline (t4-OH-Pro) increased from d 21 to 70 in muscle. The significantly changed metabolites in serum with time of lactation were 28 long-chain (>C30) PC-ae and PC-aa, 7 long-chain (>C16) SM and SM(OH), along with lysoPC-a C20:3 that were all increased. In conclusion, in addition to other significantly changed metabolites, CLA supplementation mainly led to changes in muscle and serum concentrations of glycerophospholipids and sphingolipids that might reflect the phospholipid compositional changes in muscle. The metabolome changes observed in sCLA on d 21 seem to be, at least in part, due to the lower DMI in these cows. The changes in the muscle concentrations of AA from d 21 to 70, which coincided with the steady energy and MP balances, might reflect a shift of protein synthesis/degradation balance toward synthesis.

Metabolome profiling in skeletal muscle to characterize metabolic alterations in over-conditioned cows during the periparturient period.

The transition from late gestation to early lactation is associated with extensive changes in metabolic, endocrine, and immune functions in dairy cows. Skeletal muscle plays an important role in maintaining the homeorhetic adaptation to the metabolic needs of lactation. The objective of this study was to characterize the skeletal muscle metabolome in the context of the metabolic changes that occur during the transition period in dairy cows with high (HBCS) versus normal body condition (NBCS). Fifteen weeks antepartum, 38 pregnant multiparous Holstein cows were assigned to 1 of 2 groups, which were fed differently to reach the targeted BCS and back fat thickness (BFT) until dry-off at -49 d before calving (HBCS: >3.75 and >1.4 cm; NBCS: <3.5 and <1.2 cm). During the dry period and the subsequent lactation, both groups were fed identical diets. The differences in both BCS and BFT were maintained throughout the study. The metabolome was characterized in skeletal muscle samples (semitendinosus muscle) collected on d -49, 3, 21, and 84 relative to calving using a targeted metabolomics approach (AbsoluteIDQ p180 kit; Biocrates Life Sciences AG, Innsbruck, Austria), which allowed for the quantification of up to 188 metabolites from 6 different compound classes (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids, and hexoses). On d -49, the concentrations of citrulline and hydroxytetradecadienyl-l-carnitine in muscle were higher in HBCS cows than in NBCS cows, but those of carnosine were lower. Over-conditioning did not affect the muscle concentrations of any of the metabolites on d 3. On d 21, the concentrations of phenylethylamine and linoleylcarnitine in muscle were lower in HBCS cows than in NBCS cows, and the opposite was true for lysophosphatidylcholine acyl C20:4. On d 84, the significantly changed metabolites were mainly long-chain (>C32) acyl-alkyl phosphatidylcholine and di-acyl phosphatidylcholine, along with 3 long-chain (>C16) sphingomyelin that were all lower in HBCS cows than in NBCS cows. These data contribute to a better understanding of the metabolic adaptation in skeletal muscle of dairy cows during the transition period, although the physiological significance and underlying molecular mechanisms responsible for the regulation of citrulline, hydroxytetradecadienyl-l-carnitine, carnosine, and phenylethylamine associated with over-conditioning are still elusive and warrant further investigation. The changes observed in muscle lysophosphatidylcholine and phosphatidylcholine concentrations may point to an alteration in phosphatidylcholine metabolism, probably resulting in an increase in membrane stiffness, which may lead to abnormalities in insulin signaling in the muscle of over-conditioned cows.

Proteasome activity and expression of mammalian target of rapamycin signaling factors in skeletal muscle of dairy cows supplemented with conjugated linoleic acids during early lactation.

The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis via its main downstream effectors, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein (4EBP1). The ubiquitin-proteasome system (UPS) is the main proteolytic pathway in muscle, and the muscle-specific ligases tripartite motif containing 63 (TRIM63; also called muscle-specific ring-finger protein 1, MuRF-1) and F-box only protein 32 (FBXO32; also called atrogin-1) are important components of the UPS. We investigated 20S proteasome activity and mRNA expression of key components of mTOR signaling and UPS in skeletal muscle of dairy cows during late gestation and early lactation and tested the effects of dietary supplementation (from d 1 in milk) with conjugated linoleic acids (sCLA; 100 g/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). Blood and muscle tissue (semitendinosus) samples were collected on d -21, 1, 21, and 70 relative to parturition. Dry matter intake increased with time of lactation in both groups. It was lower in sCLA than in CTR on d 21, which resulted in a reduced calculated metabolizable protein balance. Most serum and muscle concentrations of AA followed time-related changes but were unaffected by CLA supplementation. In both groups, serum and muscle 3-methylhistidine (3-MH) concentrations and the ratio of 3-MH:creatinine increased from d -21 to d 1, followed by a decline on d 21. The mRNA abundance of MTOR on d 21 and 70 was greater in sCLA than in CTR. The abundance of 4EBP1 mRNA did not differ between groups but was upregulated in both on d 1. The mRNA abundance of S6K1 on d 70 was greater in CTR than in sCLA, but remained unchanged over time in both groups. The mRNA abundance of FBXO32 (encoding atrogin-1) on d 21 was greater in sCLA than in CTR. The mRNA abundance of TRIM63 (also known as MuRF1) showed a similar pattern as FBXO32 in both groups: an increase from d -21 to d 1, followed by a decline. The mRNA for the α (BCKDHA) and ß (BCKDHB) polypeptide of branched-chain α-keto acid dehydrogenase was elevated in sCLA and CTR cows on d 21, respectively, suggesting a role of CLA in determining the metabolic fate of branched-chain AA. For the mTOR protein, no group differences were observed. The abundance of S6K1 protein was greater across all time points in sCLA versus CTR. The antepartum 20S proteasome activity in muscle was elevated in both groups compared with postpartum, probably reflecting the start of protein mobilization before parturition. Plasma insulin concentrations decreased in both groups postpartum but to a greater extent in CTR than in sCLA, resulting in greater insulin concentrations in sCLA than in CTR. Thus, the greater abundance of MTOR mRNA and S6K1 protein in sCLA compared with CTR might be mediated by the greater plasma insulin postpartum. The upregulation of MTOR mRNA in sCLA cows on d 21, despite greater FBXO32 mRNA abundance, may reflect a simultaneous activation of both anabolic and catabolic signaling pathways, likely resulting in greater protein turnover.

Mammalian target of rapamycin signaling and ubiquitin-proteasome-related gene expression in skeletal muscle of dairy cows with high or normal body condition score around calving.

The objective of the current study was to investigate the effects of overconditioning around calving on gene expression of key components of the mammalian target of rapamycin (mTOR) pathway and ubiquitin-proteasome system (UPS) in skeletal muscle as well as the AA profiles in both serum and muscle of periparturient cows. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to either a high body condition group (HBCS; n = 19) or a normal body condition group (NBCS; n = 19) and were fed different diets until dry-off (d -49 relative to calving) to amplify the difference. The groups were also stratified for comparable milk yields (NBCS: 10,361 ± 302 kg; HBCS: 10,315 ± 437 kg). At dry-off, the NBCS cows (parity: 2.42 ± 1.84; body weight: 665 ± 64 kg) had a body condition score (BCS) <3.5 and backfat thickness (BFT) <1.2 cm, whereas the HBCS cows (parity: 3.37 ± 1.67; body weight: 720 ± 57 kg) had a BCS >3.75 and BFT >1.4 cm. During the dry period and the subsequent lactation, both groups were fed identical diets but maintained the BCS and BFT differences. Blood samples and skeletal muscle biopsies (semitendinosus) were repeatedly (d -49, +3, +21, and +84 relative to calving) collected for assessing the concentrations of free AA and the mRNA abundance of various components of mTOR and UPS. The differences in BCS and BFT were maintained throughout the study. The circulating concentrations of most AA with the exception of Gly, Gln, Met, and Phe increased in early lactation in both groups. The serum concentrations of Ala (d +21 and +84) and Orn (d +84) were lower in HBCS cows than in NBCS cows, but those of Gly, His, Leu, Val, Lys, Met, and Orn on d -49 and Ile on d +21 were greater in HBCS cows than in NBCS cows. The serum concentrations of 3-methylhistidine, creatinine, and 3-methylhistidine:creatinine ratio increased after calving (d +3) but did not differ between the groups. The muscle concentrations of all AA (except for Cys) remained unchanged over time and did not differ between groups. The muscle concentrations of Cys were greater on d -49 but tended to be lower on d +21 in HBCS cows than in NBCS cows. On d +21, mTOR and eukaryotic translation initiation factor 4E binding protein 1 mRNA abundance was greater in HBCS cows than in NBCS cows, whereas ribosomal protein S6 kinase 1 was not different between the groups. The mRNA abundance of ubiquitin-activating enzyme 1 (d +21), ubiquitin-conjugating enzyme 1 (d +21), atrogin-1 (d +21), and ring finger protein-1 (d +3) enzymes was greater in HBCS cows than in NBCS cows, whereas ubiquitin-conjugating enzyme 2 was not different between the groups. The increased mRNA abundance of key components of mTOR signaling and of muscle-specific ligases of HBCS cows may indicate a simultaneous activation of anabolic and catabolic processes and thus increased muscle protein turnover, likely as a part of the adaptive response to prevent excessive loss of skeletal muscle mass during early lactation.

Biogenic amines: Concentrations in serum and skeletal muscle from late pregnancy until early lactation in dairy cows with high versus normal body condition score.

Biogenic amines (BA) are a class of nitrogenous compounds that are involved in a wide variety of physiological processes, but their role in transition cows is poorly understood. Our objectives were to describe the longitudinal changes of BA in serum and in skeletal muscle during the transition period and to characterize temporal responses of BA in relation to body condition score (BCS) of periparturient dairy cows. Fifteen weeks before calving, 36 multiparous Holstein cows were assigned to 2 groups (n = 18 per group) that were fed differently to reach either high [HBCS; net energy for lactation (NEL) = 7.2 MJ/kg of dry matter (DM)] or normal BCS (NBCS; NEL = 6.8 MJ/kg of DM) at dry-off. The targeted BCS and back fat thickness (BFT) at dry-off (HBCS, >3.75 and >1.4 cm; NBCS, <3.5 and <1.2 cm) were reached. Thereafter, both groups were fed identical diets. Blood samples and muscle (semitendinosus) biopsies were collected at d -49, +3, +21, and +84 relative to parturition. In serum and skeletal muscle, BA concentrations were measured using a targeted metabolomics assay. The data were analyzed as a repeated measure using the MIXED procedure of SAS. The serum concentrations of most BA (i.e., creatinine, taurine, carnosine putrescine, spermine, α-aminoadipic acid, acetylornithine, kynurenine, serotonin, hydroxyproline, asymmetric dimethylarginine, and symmetric dimethylarginine) fluctuated during the transition period, while others (i.e., spermidine, phenylethylamine) did not change with time. The muscle concentrations of BA remained unchanged over time. Creatinine had the highest concentrations in the serum, while carnosine had the highest concentration among the muscle BA. The serum concentrations of creatinine (d +21), putrescine (d +84), α-aminoadipic acid (d +3), and hydroxyproline (d +21) were or tended to be higher for HBCS compared with NBCS postpartum. The serum concentrations of symmetric dimethylarginine (d -49) and acetylornithine (d +84) were or tended to be lower for HBCS compared with NBCS, respectively. The serum kynurenine/tryptophan ratio was greater with HBCS than with NBCS (d +84). Compared with NBCS, HBCS was associated with lower muscle concentrations of carnosine, but those of hydroxyproline were higher (d -49). In both serum and muscle, the asymmetric dimethylarginine concentrations were greater with HBCS than with NBCS (d -49). No correlation was found between serum and skeletal muscle BA. This study indicates that overconditioning of dairy cows may influence serum and muscle BA concentrations in the periparturient period.

Acylcarnitine profiles in serum and muscle of dairy cows receiving conjugated linoleic acids or a control fat supplement during early lactation.

Acylcarnitines (ACC) are formed when fatty acid (FA)-coenzyme A enters the mitochondria for ß-oxidation and the tricarboxylic acid cycle through the carnitine shuttle. Concentrations of ACC may vary depending on the metabolic conditions, but can accumulate when rates of ß-oxidation exceed those of tricarboxylic acid. This study aimed to characterize muscle and blood serum acylcarnitine profiles, to determine the mRNA abundance of muscle carnitine acyltransferases, and to test whether dietary supplementation (from d 1 in milk) with conjugated linoleic acids (CLA; 100 g/d; each 12% of trans-10,cis-12 and cis-9,trans-11 CLA; n = 11) altered these compared with control fat-supplemented cows (CTR; n = 10). Blood samples and biopsies from the semitendinosus musclewere collected on d -21, 1, 21, and 70 relative to parturition. Serum and muscle ACC profiles were quantified using a targeted metabolomics approach. The CLA supplement did not affect the variables examined. The serum concentration of free carnitine decreased with the onset of lactation. The concentrations of acetylcarnitine, hydroxybutyrylcarnitine, and the sum of short-chain ACC in serum were greater from d -21 to 21 than thereafter. The serum concentrations of long-chain ACC tetradecenoylcarnitine (C14:1) and octadecenoylcarnitine (C18:1) concentrations were greater on d 1 and 21 compared with d -21. Muscle carnitine remained unchanged, whereas short- and medium-chain ACC, including propenoylcarnitine (C3:1), hydroxybutyrylcarnitine, hydroxyhexanoylcarnitine, hexenoylcarnitine (C6:1), and pimelylcarnitine were increased on d 21 compared with d -21 and decreased thereafter. In muscle, the concentrations of long-chain ACC (from C14 to C18) were elevated on d 1. The mRNA abundance of carnitine palmitoyltransferase 1, muscle isoform (CPT1B) increased 2.8-fold from d -21 to 1, followed by a decline to nearly prepartum values by d 70, whereas that of CPT2 did not change over time. The majority of serum and muscle short- and long-chain ACC were positively correlated with the FA concentrations in serum, whereas serum carnitine and C5 were negatively correlated with FA. Time-related changes in the serum and muscle ACC profiles were demonstrated that were not affected by the CLA supplement at the dosage used in the present study. The elevated concentrations of long-chain ACC species in muscle and of serum acetylcarnitine around parturition point to incomplete FA oxidation were likely due to insufficient metabolic adaptation in response to the load of FA around parturition.

A network-based conditional genetic association analysis of the human metabolome.

Background: Genome-wide association studies have identified hundreds of loci that influence a wide variety of complex human traits; however, little is known regarding the biological mechanism of action of these loci. The recent accumulation of functional genomics ("omics"), including metabolomics data, has created new opportunities for studying the functional role of specific changes in the genome. Functional genomic data are characterized by their high dimensionality, the presence of (strong) statistical dependency between traits, and, potentially, complex genetic control. Therefore, the analysis of such data requires specific statistical genetics methods. Results: To facilitate our understanding of the genetic control of omics phenotypes, we propose a trait-centered, network-based conditional genetic association (cGAS) approach for identifying the direct effects of genetic variants on omics-based traits. For each trait of interest, we selected from a biological network a set of other traits to be used as covariates in the cGAS. The network can be reconstructed either from biological pathway databases (a mechanistic approach) or directly from the data, using a Gaussian graphical model applied to the metabolome (a data-driven approach). We derived mathematical expressions that allow comparison of the power of univariate analyses with conditional genetic association analyses. We then tested our approach using data from a population-based Cooperative Health Research in the region of Augsburg (KORA) study (n = 1,784 subjects, 1.7 million single-nucleotide polymorphisms) with measured data for 151 metabolites. Conclusions: We found that compared to single-trait analysis, performing a genetic association analysis that includes biologically relevant covariates can either gain or lose power, depending on specific pleiotropic scenarios, for which we provide empirical examples. In the context of analyzed metabolomics data, the mechanistic network approach had more power compared to the data-driven approach. Nevertheless, we believe that our analysis shows that neither a prior-knowledge-only approach nor a phenotypic-data-only approach is optimal, and we discuss possibilities for improvement.

The human metabolic profile reflects macro- and micronutrient intake distinctly according to fasting time.

Although the impact of dietary patterns on human serum metabolites has been examined, the fasting effect on the metabolic profile has not yet been considered. The aim of this cross-sectional study is to investigate the influence of fasting regarding the association between dietary patterns, reflected by macro- and micronutrient intake, and human serum metabolites in a population-based cohort. A total 1197 non-diabetic German adults aged 45 to 83 years, who participated in baseline of the CARLA study 2002-2006 and had metabolite quantification were selected for this study. Macro- and micronutrient intakes were estimated from a food frequency questionnaire (FFQ). Concentrations of 134 serum metabolites were measured by targeted metabolomics AbsoluteIDQ p150 Kit. The association of dietary patterns with serum metabolites was calculated by means of linear regression and the influence of the fasting status was considered by including interaction terms with each macro- and micronutrient. Higher self-reported intake of alcohol and lower self-reported intake of organic acids were associated with higher concentrations of acylcarnitines and phosphatidylcholines. Mainly the associations between dietary patterns and acylcarnitines and hexose were altered after including interaction terms, suggesting effect modification by fasting status. No effect from fasting time was seen for amino acids and saturated, mono- and polyunsaturated phosphatidylcholines.

Instability of personal human metabotype is linked to all-cause mortality.

Disruption of metabolic homeostasis is an important factor in many diseases. Various metabolites have been linked to higher risk of morbidity and all-cause mortality using metabolomics in large population-based cohorts. In these studies, baseline metabolite levels were compared across subjects to identify associations with health outcomes, implying the existence of 'healthy' concentration ranges that are equally applicable to all individuals. Here, we focused on intra-individual changes in metabolite levels over time and their link to mortality, potentially allowing more personalized risk assessment. We analysed targeted metabolomics data for 134 blood metabolites from 1409 participants in the population-based CARLA cohort at baseline and after four years. Metabotypes of the majority of participants (59%) were extremely stable over time indicated by high correlation between the subjects' metabolite profiles at the two time points. Metabotype instability and, in particular, decrease of valine were associated with higher risk of all-cause mortality in 7.9 years of follow-up (hazard ratio (HR) = 1.5(95%CI = 1.0-2.3) and 0.2(95%CI = 0.1-0.3)) after multifactorial adjustment. Excluding deaths that occurred in the first year after metabolite profiling showed similar results (HR = 1.8(95%CI = 1.1-2.8)). Lower metabotype stability was also associated with incident cardiovascular disease (OR = 1.2(95%CI = 1.0-1.3)). Therefore, changes in the personal metabotype might be a valuable indicator of pre-clinical disease.

Serum and plasma amino acids as markers of prediabetes, insulin resistance, and incident diabetes.

Presently, routine screening misses many cases of prediabetes and early type 2 diabetes (T2D). Therefore, better biomarkers are needed for a simple and early detection of abnormalities of glucose metabolism and prediction of future T2D. Possible candidates for this include plasma or serum amino acids because glucose and amino acid metabolism are closely connected. This review presents the available evidence of this connectivity and discusses its clinical implications. First, we examine the underlying physiological, pre-analytical, and analytical issues. Then, we summarize results of human studies that evaluate amino acid levels as markers for insulin resistance, prediabetes, and future incident T2D. Finally, we illustrate the interconnection of amino acid levels and metabolic syndrome with our own data from a deeply phenotyped human cohort. We also discuss how amino acids may contribute to the pathophysiology of T2D. We conclude that elevated branched-chain amino acids and reduced glycine are currently the most robust and consistent amino acid markers for prediabetes, insulin resistance, and future T2D. Yet, we are cautious regarding the clinical potential even of these parameters because their discriminatory power is insufficient and their levels depend not only on glycemia, but also on other components of the metabolic syndrome. The identification of more precise intermediates of amino acid metabolism or combinations with other biomarkers will, therefore, be necessary to obtain in order to develop laboratory tests that can improve T2D screening.

Associations of anthropometric markers with serum metabolites using a targeted metabolomics approach: results of the EPIC-potsdam study.

BACKGROUND/OBJECTIVES: The metabolic consequences of type of body shape need further exploration. Whereas accumulation of body mass in the abdominal area is a well-established metabolic risk factor, accumulation in the gluteofemoral area is controversially debated. We evaluated the associations of anthropometric markers of overall body mass and body shape with 127 serum metabolites within a sub-sample of the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. SUBJECTS/METHODS: The cross-sectional analysis was conducted in 2270 participants, randomly drawn from the EPIC-Potsdam cohort. Metabolites were measured by targeted metabolomics. To select metabolites related with both waist circumference (WC) (abdominal subcutaneous and visceral fat) and hip circumference (HC) (gluteofemoral fat, muscles and bone structure) correlations (r) with body mass index (BMI) as aggregating marker of body mass (lean and fat mass) were calculated. Relations with body shape were assessed by median metabolite concentrations across tertiles of WC and HC, mutually adjusted to each other. RESULTS: Correlations revealed 23 metabolites related to BMI (r⩾I0.20 I). Metabolites showing relations with BMI were showing similar relations with HC adjusted WC (WCHC). In contrast, relations with WC adjusted HC (HCWC) were less concordant with relations of BMI and WCHC. In both sexes, metabolites with concordant relations regarding WCHC and HCWC included tyrosine, diacyl-phosphatidylcholine C38:3, C38:4, lyso-phosphatidylcholine C18:1, C18:2 and sphingomyelin C18:1; metabolites with opposite relations included isoleucine, diacyl-phosphatidylcholine C42:0, acyl-alkyl-phosphatidylcholine C34:3, C42:4, C42:5, C44:4 and C44:6. Metabolites specifically related to HCWC included acyl-alkyl-phosphatidylcholine C34:2, C36:2, C38:2 and C40:4, and were solely observed in men. Other metabolites were related to WCHC only. CONCLUSIONS: The study revealed specific metabolic profiles for HCWC as marker of gluteofemoral body mass differing from those for BMI and WCHC as markers of overall body mass and abdominal fat, respectively. Thus, the study suggests that gluteofemoral mass may have less-adverse metabolic implications than abdominal fat.

Increased amino acids levels and the risk of developing of hypertriglyceridemia in a 7-year follow-up.

BACKGROUND: Recently, five branched-chain and aromatic amino acids were shown to be associated with the risk of developing type 2 diabetes (T2D). AIM: We set out to examine whether amino acids are also associated with the development of hypertriglyceridemia. MATERIALS AND METHODS: We determined the serum amino acids concentrations of 1,125 individuals of the KORA S4 baseline study, for which follow-up data were available also at the KORA F4 7 years later. After exclusion for hypertriglyceridemia (defined as having a fasting triglyceride level above 1.70 mmol/L) and diabetes at baseline, 755 subjects remained for analyses. RESULTS: Increased levels of leucine, arginine, valine, proline, phenylalanine, isoleucine and lysine were significantly associated with an increased risk of hypertriglyceridemia. These associations remained significant when restricting to those individuals who did not develop T2D in the 7-year follow-up. The increase per standard deviation of amino acid level was between 26 and 40 %. CONCLUSIONS: Seven amino acids were associated with an increased risk of developing hypertriglyceridemia after 7 years. Further studies are necessary to elucidate the complex role of these amino acids in the pathogenesis of metabolic disorders.

Linking diet, physical activity, cardiorespiratory fitness and obesity to serum metabolite networks: findings from a population-based study.

OBJECTIVE: It is not yet resolved how lifestyle factors and intermediate phenotypes interrelate with metabolic pathways. We aimed to investigate the associations between diet, physical activity, cardiorespiratory fitness and obesity with serum metabolite networks in a population-based study. METHODS: The present study included 2380 participants of a randomly drawn subcohort of the European Prospective Investigation into Cancer and Nutrition-Potsdam. Targeted metabolomics was used to measure 127 serum metabolites. Additional data were available including anthropometric measurements, dietary assessment including intake of whole-grain bread, coffee and cake and cookies by food frequency questionnaire, and objectively measured physical activity energy expenditure and cardiorespiratory fitness in a subsample of 100 participants. In a data-driven approach, Gaussian graphical modeling was used to draw metabolite networks and depict relevant associations between exposures and serum metabolites. In addition, the relationship of different exposure metabolite networks was estimated. RESULTS: In the serum metabolite network, the different metabolite classes could be separated. There was a big group of phospholipids and acylcarnitines, a group of amino acids and C6-sugar. Amino acids were particularly positively associated with cardiorespiratory fitness and physical activity. C6-sugar and acylcarnitines were positively associated with obesity and inversely with intake of whole-grain bread. Phospholipids showed opposite associations with obesity and coffee intake. Metabolite networks of coffee intake and obesity were strongly inversely correlated (body mass index (BMI): r = -0.57 and waist circumference: r = -0.59). A strong positive correlation was observed between metabolite networks of BMI and waist circumference (r = 0.99), as well as the metabolite networks of cake and cookie intake with cardiorespiratory fitness and intake of whole-grain bread (r = 0.52 and r = 0.50; respectively). CONCLUSIONS: Lifestyle factors and phenotypes seem to interrelate in various metabolic pathways. A possible protective effect of coffee could be mediated via counterbalance of pathways of obesity involving hepatic phospholipids. Experimental studies should validate the biological mechanisms.

Variation of serum metabolites related to habitual diet: a targeted metabolomic approach in EPIC-Potsdam.

BACKGROUND/OBJECTIVE: Serum metabolites have been linked to higher risk of chronic diseases but determinants of serum metabolites are not clear. We aimed to investigate the association between habitual diet as a modifiable risk factor and relevant serum metabolites. SUBJECTS/METHODS: This cross-sectional study comprised 2380 EPIC-Potsdam participants. Intake of 45 food groups was assessed by food frequency questionnaire and concentrations of 127 serum metabolites were measured by targeted metabolomics. Reduced rank regression was used to find dietary patterns that explain the maximum variation of metabolites. RESULTS: In the multivariable-adjusted model, the proportion of explained variation by habitual diet was ranked as follows: acyl-alkyl-phosphatidylcholines (5.7%), sphingomyelins (5.1%), diacyl-phosphatidylcholines (4.4%), lyso-phosphatidylcholines (4.1%), acylcarnitines (3.5%), amino acids (2.2%) and hexose (1.6%). A pattern with high intake of butter and low intake of margarine was related to acylcarnitines, acyl-alkyl-phosphatidylcholines, lyso-phosphatidylcholines and hydroxy-sphingomyelins, particularly with saturated and monounsaturated fatty acid side chains. A pattern with high intake of red meat and fish and low intake of whole-grain bread and tea was related to hexose and phosphatidylcholines. A pattern consisting of high intake of potatoes, dairy products and cornflakes particularly explained methionine and branched chain amino acids. Dietary patterns related to type 2 diabetes-relevant metabolites included high intake of red meat and low intake of whole-grain bread, tea, coffee, cake and cookies, canned fruits and fish. CONCLUSIONS: Dietary patterns characterized by intakes of red meat, whole-grain bread, tea and coffee were linked to relevant metabolites and could be potential targets for chronic disease prevention.

Alcohol-induced metabolomic differences in humans.

Alcohol consumption is one of the world's major risk factors for disease development. But underlying mechanisms by which moderate-to-heavy alcohol intake causes damage are poorly understood and biomarkers are sub-optimal. Here, we investigated metabolite concentration differences in relation to alcohol intake in 2090 individuals of the KORA F4 and replicated results in 261 KORA F3 and up to 629 females of the TwinsUK adult bioresource. Using logistic regression analysis adjusted for age, body mass index, smoking, high-density lipoproteins and triglycerides, we identified 40/18 significant metabolites in males/females with P-values <3.8E-04 (Bonferroni corrected) that differed in concentrations between moderate-to-heavy drinkers (MHD) and light drinkers (LD) in the KORA F4 study. We further identified specific profiles of the 10/5 metabolites in males/females that clearly separated LD from MHD in the KORA F4 cohort. For those metabolites, the respective area under the receiver operating characteristic curves were 0.812/0.679, respectively, thus providing moderate-to-high sensitivity and specificity for the discrimination of LD to MHD. A number of alcohol-related metabolites could be replicated in the KORA F3 and TwinsUK studies. Our data suggests that metabolomic profiles based on diacylphosphatidylcholines, lysophosphatidylcholines, ether lipids and sphingolipids form a new class of biomarkers for excess alcohol intake and have potential for future epidemiological and clinical studies.