Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine.

Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.

Leptin treatment has vasculo-protective effects in lipodystrophic mice.

Lipodystrophy syndromes (LDs) are characterized by loss of adipose tissue, metabolic complications such as dyslipidemia, insulin resistance, and fatty liver disease, as well as accelerated atherosclerosis. As a result of adipose tissue deficiency, the systemic concentration of the adipokine leptin is reduced. A current promising therapeutic option for patients with LD is treatment with recombinant leptin (metreleptin), resulting in reduced risk of mortality. Here, we investigate the effects of leptin on endothelial to mesenchymal transition (EndMT), which impair the functional properties of endothelial cells and promotes atherogenesis in LD. Leptin treatment reduced inflammation and TGF-ß2-induced expression of mesenchymal genes and prevented impairment of endothelial barrier function. Treatment of lipodystrophic- and atherosclerosis-prone animals (Ldlr-/-; aP2-nSrebp1c-Tg) with leptin reduced macrophage accumulation in atherosclerotic lesions, vascular plaque protrusion, and the number of endothelial cells with mesenchymal gene expression, confirming a reduction in EndMT in LD after leptin treatment. Treatment with leptin inhibited LD-mediated induction of the proatherosclerotic cytokine growth/differentiation factor 15 (GDF15). Inhibition of GDF15 reduced EndMT induction triggered by plasma from patients with LD. Our study reveals that in addition to the effects on adipose tissue function, leptin treatment exerts beneficial effects protecting endothelial function and identity in LD by reducing GDF15.

Glucose tolerance and insulin resistance/sensitivity associate with retinal layer characteristics: the LIFE-Adult-Study.

AIMS/HYPOTHESIS: As the prevalence of insulin resistance and glucose intolerance is increasing throughout the world, diabetes-induced eye diseases are a global health burden. We aim to identify distinct optical bands which are closely related to insulin and glucose metabolism, using non-invasive, high-resolution spectral domain optical coherence tomography (SD-OCT) in a large, population-based dataset. METHODS: The LIFE-Adult-Study randomly selected 10,000 participants from the population registry of Leipzig, Germany. Cross-sectional, standardised phenotyping included the assessment of various metabolic risk markers and ocular imaging, such as SD-OCT-derived thicknesses of ten optical bands of the retina. Global and Early Treatment Diabetic Retinopathy Study (ETDRS) subfield-specific optical retinal layer thicknesses were investigated in 7384 healthy eyes of 7384 participants from the LIFE-Adult-Study stratified by normal glucose tolerance, prediabetes (impaired fasting glucose and/or impaired glucose tolerance and/or HbA1c 5.7-6.4% [39-47 mmol/mol]) and diabetes. The association of optical retinal band characteristics with different indices of glucose tolerance (e.g. fasting glucose, area under the glucose curve), insulin resistance (e.g. HOMA2-IR, triglyceride glucose index), or insulin sensitivity (e.g. estimated glucose disposal rate [eGDR], Stumvoll metabolic clearance rate) was determined using multivariable linear regression analyses for the individual markers adjusted for age, sex and refraction. Various sensitivity analyses were performed to validate the observed findings. RESULTS: In the study cohort, nine out of ten optical bands of the retina showed significant sex- and glucose tolerance-dependent differences in band thicknesses. Multivariable linear regression analyses revealed a significant, independent, and inverse association between markers of glucose intolerance and insulin resistance (e.g. HOMA2-IR) with the thickness of the optical bands representing the anatomical retinal outer nuclear layer (ONL, standardised ß=-0.096; p<0.001 for HOMA2-IR) and myoid zone (MZ; ß=-0.096; p<0.001 for HOMA2-IR) of the photoreceptors. Conversely, markers of insulin sensitivity (e.g. eGDR) positively and independently associated with ONL (ß=0.090; p<0.001 for eGDR) and MZ (ß=0.133; p<0.001 for eGDR) band thicknesses. These global associations were confirmed in ETDRS subfield-specific analyses. Sensitivity analyses further validated our findings when physical activity, neuroanatomical cell/tissue types and ETDRS subfield categories were investigated after stratifying the cohort by glucose homeostasis. CONCLUSIONS/INTERPRETATION: An impaired glucose homeostasis associates with a thinning of the optical bands of retinal ONL and photoreceptor MZ. Changes in ONL and MZ thicknesses might predict early metabolic retinal alterations in diabetes.

Pregnancy and acromegaly: clinical outcomes of retrospectively analysed data from the German acromegaly registry.

CONTEXT: Acromegaly is a rare disease caused by excessive growth hormone (GH) secretion, mostly induced by pituitary adenomas. The care of pregnant women with acromegaly is challenging, in part due to existing clinical data being limited and not entirely consistent with regard to potential risks for mother and child. OBJECTIVE: To retrospectively examine data on pregnancy and maternal as well as neonatal outcomes in patients with acromegaly. DESIGN & METHODS: Retrospective data analysis from 47 pregnancies of 31 women treated in centers of the German Acromegaly Registry. RESULTS: 87.1% of the studied women underwent transsphenoidal surgery before pregnancy. In 51.1% a combination of dopamine agonists and somatostatin analogs were used before pregnancy. Three women did not receive any therapy for acromegaly. During pregnancy only 6.4% received either somatostatin analogs or dopamine agonists. In total, 70.2% of all documented pregnancies emerged spontaneously. Gestational diabetes was diagnosed in 10.6% and gravid hypertension in 6.4%. Overall, no preterm birth was detected. Indeed, 87% of acromegalic women experienced a delivery without complications. CONCLUSION: Pregnancies in women with acromegaly are possible and the course of pregnancy is in general safe for mother and child both with and without specific treatment for acromegaly. The prevalence of concomitant metabolic diseases such as gestational diabetes is comparable to the prevalence in healthy pregnant women. Nevertheless, larger studies with more data in pregnant patients with acromegaly are needed to provide safe and effective care for pregnant women with this condition.

Circulating acyl-CoA-binding protein/diazepam-binding inhibitor in gestational diabetes mellitus.

BACKGROUND: Acyl-CoA-binding protein (ACBP)/diazepam-binding inhibitor has recently been characterized as an endocrine factor affecting energy balance and lipid metabolism. However, regulation of ACBP in women with gestational diabetes mellitus (GDM) during pregnancy, as well as postpartum, has not been investigated, so far. METHODS: ACBP was quantified in 74 women with GDM and 74 healthy, gestational age-matched, pregnant controls using an enzyme-linked immunosorbent assay. Furthermore, ACBP was quantified post-partum in 82 women (i.e. 41 women with previous GDM vs. 41 previous control women). ACBP was related to measures of obesity, hypertension, glucose and lipid metabolism, renal function, and inflammation during pregnancy and postpartum. RESULTS: During pregnancy, median [interquartile range] ACBP levels were not significantly different in women with GDM (40.9 [40.0] µg/l) compared to healthy, pregnant controls (29.1 [32.3] µg/l) (p = 0.215). ACBP serum concentrations increased from 30.3 [40.5] µg/l during pregnancy to 59.7 [33.2] µg/l after pregnancy in the entire cohort (p < 0.001). This observed elevation was consistent across both subgroups of women, those with prior GDM and those without. Multivariate analysis revealed that homeostasis model assessment of beta cell function (HOMA2-B) and creatinine positively and independently correlated with serum ACBP after pregnancy, while multivariate analysis during pregnancy showed no significant correlations. CONCLUSIONS: Circulating ACBP is not a marker of GDM status, but ACBP is decreased during pregnancy, irrespective of GDM status. Furthermore, ACBP is related to beta cell function and renal markers in women after pregnancy.

Evidence-based cutoffs for total and adjusted calcium: a major factor in detecting severe hypo- and hypercalcemia.

OBJECTIVES: Severe hypo- and hypercalcemia are common and urgent treatment is recommended. Free calcium (fCa) is the gold standard but needs blood gas tests with challenging preanalytics. Total calcium (tCa) and calculated adjusted calcium (aCa) are readily available, but their interpretation is hampered by identical tCa and aCa cutoffs, laborious local aCa calculation and difficult comparability of calcium biomarkers. METHODS: Laboratory results from University Medicine Leipzig were evaluated over a five-year period (236,274 patients). A local aCa equation was derived by linear least squares regression, the agreement between fCa, tCa and aCa assessed with Cohen's κ and decision thresholds derived by this indirect method. RESULTS: The local aCa equation was created from data of 9,756 patients, each with one paired measurement of tCa, fCa and albumin. Derived aCa cutoffs (1.95/3.15 mmol/L) differ markedly from derived tCa cutoffs (1.6/2.9 mmol/L) and severe hypo- and hypercalcemia can be more accurately assessed by aCa (κ=0.489, 0.812) than by tCa (κ=0.445, 0.744). Comparing our approach to standard care (tCa, literature cutoff), a total 3,250 of 3,680 (88.3 %) misclassified measurements were correctly classified when using aCa with evidence-based cutoffs. CONCLUSIONS: Optimized cutoffs for aCa and tCa hold great potential for improved patient care. Locally derived aCa equations differ mostly in the chosen mean normal calcium and provide minimal overall improvement, but entail a close examination of the used cutoffs before application.

Antifibrotic Soluble Thy-1 Correlates with Renal Dysfunction in Chronic Kidney Disease.

Kidney fibrosis is a major culprit in the development and progression of chronic kidney disease (CKD), ultimately leading to the irreversible loss of organ function. Thymocyte differentiation antigen-1 (Thy-1) controls many core functions of fibroblasts relevant to fibrogenesis but is also found in a soluble form (sThy-1) in serum and urine. We investigated the association of sThy-1 with clinical parameters in patients with CKD receiving hemodialysis treatment compared to individuals with a preserved renal function. Furthermore, Thy-1 tissue expression was detected in a mouse model of diabetic CKD (eNOS-/-; db/db) and non-diabetic control mice (eNOS-/-). Serum and urinary sThy-1 concentrations significantly increased with deteriorating renal function, independent of the presence of diabetes. Serum creatinine is the major, independent, and inverse predictor of serum sThy-1 levels. Moreover, sThy-1 is not only predicted by markers of renal function but is also itself an independent and strong predictor of markers of renal function, i.e., serum creatinine. Mice with severe diabetic CKD show increased Thy-1 mRNA and protein expression in the kidney compared to control animals, as well as elevated urinary sThy-1 levels. Pro-fibrotic mediators, such as interleukin (IL)-4, IL-13, IL-6 and transforming growth factor ß, increase Thy-1 gene expression and release of sThy-1 from fibroblasts. Our data underline the role of Thy-1 in the control of kidney fibrosis in CKD and raise the opportunity that Thy-1 may function as a renal antifibrotic factor.

Interplay between adipose tissue secreted proteins, eating behavior and obesity.

PURPOSE: Adipokines may play an important role in the complex etiology of human obesity and its metabolic complications. Here, we analyzed the relationship between 15 adipokines, eating behavior and body-mass index (BMI). METHODS: The study included 557 participants of the Sorbs (62.1% women, 37.9% men) and 3101 participants of the population-based LIFE-Adult cohorts (53.4% women, 46.4% men) who completed the German version of the Three-Factor-Eating Questionnaire to assess the eating behavior types cognitive restraint, disinhibition and hunger. Serum levels of 15 adipokines, including adiponectin, adipocyte fatty acid-binding protein (AFABP), angiopoietin-related growth factor (AGF), chemerin, fibroblast growth factor (FGF)-19, FGF-21, FGF-23, insulin-like growth factor (IGF)-1, interleukin (IL) 10, irisin, progranulin, vaspin, pro-neurotensin (pro-NT), pro-enkephalin (PENK) and leptin were measured. Based on significant correlations between several adipokines with different eating behavior items and BMI, we conducted mediation analyses, considering the eating behavior items as potential mediation variable towards BMI. RESULTS: Here, we found that the positive association between chemerin, AFABP or leptin and BMI in Sorbian women was mediated by higher restraint or disinhibited eating, respectively. Additionally, in Sorbian women, the negative relation between IGF-1 and BMI was mediated by higher disinhibition and the positive link between AGF and BMI by lower disinhibition. In Sorbian men, the negative relationship between PENK and BMI was mediated by lower disinhibition and hunger, whereas the negative relation between IGF-1 and BMI was mediated by higher hunger. In the LIFE-Adult women´s cohort, associations between chemerin and BMI were mediated by decreased hunger or disinhibition, respectively, whereas relations between PENK and BMI were fully mediated by decreased disinhibition. CONCLUSION: Our study suggests that adipokines such as PENK, IGF-1, chemerin, AGF, AFABP and leptin might affect the development of obesity by directly modifying individual eating behavior. Given the observational nature of the study, future experimental or mechanistic work is warranted.

Genomic insights into the origin of farming in the ancient Near East.

We report genome-wide ancient DNA from 44 ancient Near Easterners ranging in time between ~12,000 and 1,400 bc, from Natufian hunter-gatherers to Bronze Age farmers. We show that the earliest populations of the Near East derived around half their ancestry from a 'Basal Eurasian' lineage that had little if any Neanderthal admixture and that separated from other non-African lineages before their separation from each other. The first farmers of the southern Levant (Israel and Jordan) and Zagros Mountains (Iran) were strongly genetically differentiated, and each descended from local hunter-gatherers. By the time of the Bronze Age, these two populations and Anatolian-related farmers had mixed with each other and with the hunter-gatherers of Europe to greatly reduce genetic differentiation. The impact of the Near Eastern farmers extended beyond the Near East: farmers related to those of Anatolia spread westward into Europe; farmers related to those of the Levant spread southward into East Africa; farmers related to those of Iran spread northward into the Eurasian steppe; and people related to both the early farmers of Iran and to the pastoralists of the Eurasian steppe spread eastward into South Asia.

A Copeptin-Based Approach in the Diagnosis of Diabetes Insipidus.

BACKGROUND: The indirect water-deprivation test is the current reference standard for the diagnosis of diabetes insipidus. However, it is technically cumbersome to administer, and the results are often inaccurate. The current study compared the indirect water-deprivation test with direct detection of plasma copeptin, a precursor-derived surrogate of arginine vasopressin. METHODS: From 2013 to 2017, we recruited 156 patients with hypotonic polyuria at 11 medical centers to undergo both water-deprivation and hypertonic saline infusion tests. In the latter test, plasma copeptin was measured when the plasma sodium level had increased to at least 150 mmol per liter after infusion of hypertonic saline. The primary outcome was the overall diagnostic accuracy of each test as compared with the final reference diagnosis, which was determined on the basis of medical history, test results, and treatment response, with copeptin levels masked. RESULTS: A total of 144 patients underwent both tests. The final diagnosis was primary polydipsia in 82 patients (57%), central diabetes insipidus in 59 (41%), and nephrogenic diabetes insipidus in 3 (2%). Overall, among the 141 patients included in the analysis, the indirect water-deprivation test determined the correct diagnosis in 108 patients (diagnostic accuracy, 76.6%; 95% confidence interval [CI], 68.9 to 83.2), and the hypertonic saline infusion test (with a copeptin cutoff level of >4.9 pmol per liter) determined the correct diagnosis in 136 patients (96.5%; 95% CI, 92.1 to 98.6; P<0.001). The indirect water-deprivation test correctly distinguished primary polydipsia from partial central diabetes insipidus in 77 of 105 patients (73.3%; 95% CI, 63.9 to 81.2), and the hypertonic saline infusion test distinguished between the two conditions in 99 of 104 patients (95.2%; 95% CI, 89.4 to 98.1; adjusted P<0.001). One serious adverse event (desmopressin-induced hyponatremia that resulted in hospitalization) occurred during the water-deprivation test. CONCLUSIONS: The direct measurement of hypertonic saline-stimulated plasma copeptin had greater diagnostic accuracy than the water-deprivation test in patients with hypotonic polyuria. (Funded by the Swiss National Foundation and others; ClinicalTrials.gov number, NCT01940614 .).

Renal function and lipid metabolism are major predictors of circumpapillary retinal nerve fiber layer thickness-the LIFE-Adult Study.

BACKGROUND: Circumpapillary retinal nerve fiber layer thickness (cpRNFLT) as assessed by spectral domain optical coherence tomography (SD-OCT) is a new technique used for the detection and evaluation of glaucoma and other optic neuropathies. Before translating cpRNFLT into clinics, it is crucially important to investigate anthropometric, biochemical, and clinical parameters potentially affecting cpRNFLT in a large population-based dataset. METHODS: The population-based LIFE-Adult Study randomly selected 10,000 participants from the population registry of Leipzig, Germany. All participants underwent standardized systemic assessment of various cardiometabolic risk markers and ocular imaging, including cpRNFLT measurement using SD-OCT (Spectralis, Heidelberg Engineering). After employing strict SD-OCT quality criteria, 8952 individuals were analyzed. Multivariable linear regression analyses were used to evaluate the independent associations of various cardiometabolic risk markers with sector-specific cpRNFLT. For significant markers, the relative strength of the observed associations was compared to each other to identify the most relevant factors influencing cpRNFLT. In all analyses, the false discovery rate method for multiple comparisons was applied. RESULTS: In the entire cohort, female subjects had significantly thicker global and also sectoral cpRNFLT compared to male subjects (p < 0.05). Multivariable linear regression analyses revealed a significant and independent association between global and sectoral cpRNFLT with biomarkers of renal function and lipid profile. Thus, thinner cpRNFLT was associated with worse renal function as assessed by cystatin C and estimated glomerular filtration rate. Furthermore, an adverse lipid profile (i.e., low high-density lipoprotein (HDL) cholesterol, as well as high total, high non-HDL, high low-density lipoprotein cholesterol, and high apolipoprotein B) was independently and statistically significantly related to thicker cpRNFLT. In contrast, we do not observe a significant association between cpRNFLT and markers of inflammation, glucose homeostasis, liver function, blood pressure, or obesity in our sector-specific analysis and globally. CONCLUSIONS: Markers of renal function and lipid metabolism are predictors of sectoral cpRNFLT in a large and deeply phenotyped population-based study independently of previously established covariates. Future studies on cpRNFLT should include these biomarkers and need to investigate whether incorporation will improve the diagnosis of early eye diseases based on cpRNFLT.

Circulating cell adhesion molecules in metabolically healthy obesity.

BACKGROUND/OBJECTIVES: People with metabolically healthy obesity (MHO) may still have an increased risk for cardiovascular mortality compared to metabolically healthy lean (MHL) individuals. However, the mechanisms linking obesity to cardiovascular diseases are not entirely understood. We therefore tested the hypothesis that circulating cell adhesion molecules (CAMs) are higher in MHO compared to MHL individuals. SUBJECTS/METHODS: Serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), E-selectin and P-selectin were measured in age- and sex-matched groups of MHL (n = 32), MHO categorized into BMI-matched insulin sensitive (IS, n = 32) or insulin resistant (IR) obesity (n = 32) and people with metabolically unhealthy obesity (MUO, n = 32). RESULTS: Indeed, individuals with MHO have significantly higher sICAM-1, E-selectin, and P-selectin serum concentrations compared to MHL people. However, these CAMs are still significantly lower in IS compared to IR MHO. There was no difference between the groups in sVCAM-1 serum concentrations. Compared to all other groups, circulating adhesion molecules were significantly higher in individuals with MUO. CONCLUSIONS: These findings suggest that obesity-related increased cardiovascular risk is reflected and may be mediated by significantly higher CAMs. The mechanisms causing elevated adhesion molecules even in the absence of overt cardio-metabolic risk factors and whether circulating CAMs could predict cardiovascular events need to be explored.

Genome-wide meta-analysis identifies novel determinants of circulating serum progranulin.

Progranulin is a secreted protein with important functions in processes including immune and inflammatory response, metabolism and embryonic development. The present study aimed at identification of genetic factors determining progranulin concentrations. We conducted a genome-wide association meta-analysis for serum progranulin in three independent cohorts from Europe: Sorbs (N = 848) and KORA (N = 1628) from Germany and PPP-Botnia (N = 335) from Finland (total N = 2811). Single nucleotide polymorphisms (SNPs) associated with progranulin levels were replicated in two additional German cohorts: LIFE-Heart Study (Leipzig; N = 967) and Metabolic Syndrome Berlin Potsdam (Berlin cohort; N = 833). We measured mRNA expression of genes in peripheral blood mononuclear cells (PBMC) by micro-arrays and performed mRNA expression quantitative trait and expression-progranulin association studies to functionally substantiate identified loci. Finally, we conducted siRNA silencing experiments in vitro to validate potential candidate genes within the associated loci. Heritability of circulating progranulin levels was estimated at 31.8% and 26.1% in the Sorbs and LIFE-Heart cohort, respectively. SNPs at three loci reached study-wide significance (rs660240 in CELSR2-PSRC1-MYBPHL-SORT1, rs4747197 in CDH23-PSAP and rs5848 in GRN) explaining 19.4%/15.0% of the variance and 61%/57% of total heritability in the Sorbs/LIFE-Heart Study. The strongest evidence for association was at rs660240 (P = 5.75 × 10-50), which was also associated with mRNA expression of PSRC1 in PBMC (P = 1.51 × 10-21). Psrc1 knockdown in murine preadipocytes led to a consecutive 30% reduction in progranulin secretion. In conclusion, the present meta-GWAS combined with mRNA expression identified three loci associated with progranulin and supports the role of PSRC1 in the regulation of progranulin secretion.

Adipocytokines are not associated with gestational diabetes mellitus but with pregnancy status.

AIMS: Adipose tissue-secreted proteins, i.e. adipocytokines, have been identified as potential mediators linking fat mass and adipose tissue dysfunction with impaired glucose homeostasis, alterations in the inflammatory status, and risk of diabetes. The aim of this study was to determine whether seven circulating adipocytokines are associated with gestational diabetes mellitus (GDM) or are altered by metabolic and weight changes during pregnancy itself. METHODS: A panel of seven adipocytokines (i.e. adiponectin, adipocyte fatty acid-binding protein, chemerin, leptin, Pro-Enkephalin, progranulin, and Pro-Neurotensin) was quantified in serum in a cross-sectional cohort of 222 women with the following three groups matched for age and body mass index: (i) 74 pregnant women with GDM; (ii) 74 pregnant women without GDM; and (iii) 74 non-pregnant and healthy women. A stepwise statistical approach was used by performing pairwise comparisons, principal component analysis (PCA), and partial least square discriminant analysis (PLS-DA). RESULTS: Five out of seven adipocytokines were dysregulated between pregnant and non-pregnant women, i.e. adiponectin, chemerin, leptin, Pro-Enkephalin, and progranulin. None of the adipocytokines significantly differed between GDM and non-GDM status during pregnancy. The same five adipocytokines clustered in a principal component representing pregnancy-induced effects. Fasting insulin was the most relevant parameter in the discrimination of GDM as compared to pregnant women without GDM, whereas chemerin and adiponectin were most relevant factors to discriminate pregnancy status. CONCLUSIONS: Pregnancy status but not presence of GDM can be distinguished by the seven investigated adipocytokines in discrimination analyses.

Increased Growth Differentiation Factor 15 in Patients with Hypoleptinemia-Associated Lipodystrophy.

Objective. Similar to obesity, lipodystrophy (LD) causes adipose tissue dysfunction and severe metabolic complications. Growth differentiation factor 15 (GDF15) belongs to the transforming growth factor ß superfamily and is dysregulated in metabolic disease including obesity and diabetes mellitus. Circulating levels in LD and the impact of leptin treatment have not been investigated so far. Material and Methods. GDF15 serum levels were quantified in 60 LD patients without human immunodeficiency virus infection and 60 controls matched for age, gender, and body mass index. The impact of metreleptin treatment on circulating GDF15 was assessed in a subgroup of patients. GDF15 mRNA expression was determined in metabolic tissues of leptin-deficient lipodystrophic aP2-nSREBP1c-Tg mice, obese ob/ob mice, and control C57Bl6 mice. Results. Median GDF15 serum concentrations were significantly higher in LD patients (819 ng/L) as compared to the control group (415 ng/L) (p < 0.001). In multiple linear regression analysis, an independent and positive association remained between GDF15 on one hand and age, patient group, hemoglobin A1c, triglycerides, and C-reactive protein on the other hand. Moreover, there was an independent negative association between GFD15 and estimated glomerular filtration rate. Circulating GDF15 was not significantly affected by metreleptin treatment in LD patients. Gdf15 was upregulated in leptin-deficient lipodystrophic mice as compared to controls. Moreover, Gdf15 mRNA expression was downregulated by leptin treatment in lipodystrophic and obese animals. Conclusions. Serum concentrations of GDF15 are elevated in LD patients and independently associated with markers of metabolic dysfunction. Gdf15 expression is higher in lipodystrophic mice and downregulated by leptin treatment.

Metabolic effects of genetic variation in the human REPIN1 gene.

BACKGROUND: Replication initiator 1 (Repin1) is a zinc finger protein highly expressed in liver and adipose tissue. The Repin1 resides within a quantitative trait locus (QTL) for body weight and triglyceride levels in the rat, and its hepatic deletion in mice results in improved insulin sensitivity and lower body weight. Here, we analyzed whether genetic variation within the Repin1 affects parameters of glucose and lipid metabolism. METHODS: We sequenced REPIN1 in 48 non-related Caucasian subjects. We discovered a 12 base pair deletion (12 bp del; rs3832490), which was subsequently genotyped in two well-characterized cohorts (N = 3013) to test for associations with metabolic traits. Functional consequences of the variant were investigated in HepG2 cells in vitro. RESULTS: In human cohorts, we show that the 12 bp del associates with improved glucose metabolism (lower fasting plasma glucose, fasting plasma insulin, and HOMA IR). Cells transfected with the plasmid carrying the 12 bp del variant are characterized by increased GLUT2 and fatty acid translocase CD36 expression and more lipid droplets. CONCLUSION: Our data suggest that genetic variation in human REPIN1 plays a role in glucose and lipid metabolism by differentially affecting the expression of REPIN1 target genes including glucose and fatty acid transporters.

Six Novel Loci Associated with Circulating VEGF Levels Identified by a Meta-analysis of Genome-Wide Association Studies.

Vascular endothelial growth factor (VEGF) is an angiogenic and neurotrophic factor, secreted by endothelial cells, known to impact various physiological and disease processes from cancer to cardiovascular disease and to be pharmacologically modifiable. We sought to identify novel loci associated with circulating VEGF levels through a genome-wide association meta-analysis combining data from European-ancestry individuals and using a dense variant map from 1000 genomes imputation panel. Six discovery cohorts including 13,312 samples were analyzed, followed by in-silico and de-novo replication studies including an additional 2,800 individuals. A total of 10 genome-wide significant variants were identified at 7 loci. Four were novel loci (5q14.3, 10q21.3, 16q24.2 and 18q22.3) and the leading variants at these loci were rs114694170 (MEF2C, P = 6.79 x 10(-13)), rs74506613 (JMJD1C, P = 1.17 x 10(-19)), rs4782371 (ZFPM1, P = 1.59 x 10(-9)) and rs2639990 (ZADH2, P = 1.72 x 10(-8)), respectively. We also identified two new independent variants (rs34528081, VEGFA, P = 1.52 x 10(-18); rs7043199, VLDLR-AS1, P = 5.12 x 10(-14)) at the 3 previously identified loci and strengthened the evidence for the four previously identified SNPs (rs6921438, LOC100132354, P = 7.39 x 10(-1467); rs1740073, C6orf223, P = 2.34 x 10(-17); rs6993770, ZFPM2, P = 2.44 x 10(-60); rs2375981, KCNV2, P = 1.48 x 10(-100)). These variants collectively explained up to 52% of the VEGF phenotypic variance. We explored biological links between genes in the associated loci using Ingenuity Pathway Analysis that emphasized their roles in embryonic development and function. Gene set enrichment analysis identified the ERK5 pathway as enriched in genes containing VEGF associated variants. eQTL analysis showed, in three of the identified regions, variants acting as both cis and trans eQTLs for multiple genes. Most of these genes, as well as some of those in the associated loci, were involved in platelet biogenesis and functionality, suggesting the importance of this process in regulation of VEGF levels. This work also provided new insights into the involvement of genes implicated in various angiogenesis related pathologies in determining circulating VEGF levels. The understanding of the molecular mechanisms by which the identified genes affect circulating VEGF levels could be important in the development of novel VEGF-related therapies for such diseases.

Integration of Genome-Wide SNP Data and Gene-Expression Profiles Reveals Six Novel Loci and Regulatory Mechanisms for Amino Acids and Acylcarnitines in Whole Blood.

Profiling amino acids and acylcarnitines in whole blood spots is a powerful tool in the laboratory diagnosis of several inborn errors of metabolism. Emerging data suggests that altered blood levels of amino acids and acylcarnitines are also associated with common metabolic diseases in adults. Thus, the identification of common genetic determinants for blood metabolites might shed light on pathways contributing to human physiology and common diseases. We applied a targeted mass-spectrometry-based method to analyze whole blood concentrations of 96 amino acids, acylcarnitines and pathway associated metabolite ratios in a Central European cohort of 2,107 adults and performed genome-wide association (GWA) to identify genetic modifiers of metabolite concentrations. We discovered and replicated six novel loci associated with blood levels of total acylcarnitine, arginine (both on chromosome 6; rs12210538, rs17657775), propionylcarnitine (chromosome 10; rs12779637), 2-hydroxyisovalerylcarnitine (chromosome 21; rs1571700), stearoylcarnitine (chromosome 1; rs3811444), and aspartic acid traits (chromosome 8; rs750472). Based on an integrative analysis of expression quantitative trait loci in blood mononuclear cells and correlations between gene expressions and metabolite levels, we provide evidence for putative causative genes: SLC22A16 for total acylcarnitines, ARG1 for arginine, HLCS for 2-hydroxyisovalerylcarnitine, JAM3 for stearoylcarnitine via a trans-effect at chromosome 1, and PPP1R16A for aspartic acid traits. Further, we report replication and provide additional functional evidence for ten loci that have previously been published for metabolites measured in plasma, serum or urine. In conclusion, our integrative analysis of SNP, gene-expression and metabolite data points to novel genetic factors that may be involved in the regulation of human metabolism. At several loci, we provide evidence for metabolite regulation via gene-expression and observed overlaps with GWAS loci for common diseases. These results form a strong rationale for subsequent functional and disease-related studies.

On the impact of relatedness on SNP association analysis.

BACKGROUND: When testing for SNP (single nucleotide polymorphism) associations in related individuals, observations are not independent. Simple linear regression assuming independent normally distributed residuals results in an increased type I error and the power of the test is also affected in a more complicate manner. Inflation of type I error is often successfully corrected by genomic control. However, this reduces the power of the test when relatedness is of concern. In the present paper, we derive explicit formulae to investigate how heritability and strength of relatedness contribute to variance inflation of the effect estimate of the linear model. Further, we study the consequences of variance inflation on hypothesis testing and compare the results with those of genomic control correction. We apply the developed theory to the publicly available HapMap trio data (N=129), the Sorbs (a self-contained population with N=977 characterised by a cryptic relatedness structure) and synthetic family studies with different sample sizes (ranging from N=129 to N=999) and different degrees of relatedness. RESULTS: We derive explicit and easily to apply approximation formulae to estimate the impact of relatedness on the variance of the effect estimate of the linear regression model. Variance inflation increases with increasing heritability. Relatedness structure also impacts the degree of variance inflation as shown for example family structures. Variance inflation is smallest for HapMap trios, followed by a synthetic family study corresponding to the trio data but with larger sample size than HapMap. Next strongest inflation is observed for the Sorbs, and finally, for a synthetic family study with a more extreme relatedness structure but with similar sample size as the Sorbs. Type I error increases rapidly with increasing inflation. However, for smaller significance levels, power increases with increasing inflation while the opposite holds for larger significance levels. When genomic control is applied, type I error is preserved while power decreases rapidly with increasing variance inflation. CONCLUSIONS: Stronger relatedness as well as higher heritability result in increased variance of the effect estimate of simple linear regression analysis. While type I error rates are generally inflated, the behaviour of power is more complex since power can be increased or reduced in dependence on relatedness and the heritability of the phenotype. Genomic control cannot be recommended to deal with inflation due to relatedness. Although it preserves type I error, the loss in power can be considerable. We provide a simple formula for estimating variance inflation given the relatedness structure and the heritability of a trait of interest. As a rule of thumb, variance inflation below 1.05 does not require correction and simple linear regression analysis is still appropriate.