Identification of distinct transcriptome signatures of human adipose tissue from fifteen depots.

The functional and metabolic characteristics of specific adipose tissue (AT) depots seem to be determined by intrinsic mechanisms. We performed a comprehensive transcriptome profiling of human AT from distinct fat depots to unravel their unique features potentially explaining molecular mechanisms underlying AT distribution and their contribution to health and disease. Post-mortem AT samples of five body donors from 15 anatomical locations were collected. Global mRNA expression was measured by Illumina® Human HT-12 v4 Expression BeadChips. Data were validated using qPCR and Western Blot in a subset of ATs from seven additional body donors. Buccal and heel AT clearly separated from the "classical" subcutaneous AT depots, and perirenal and epicardial AT were distinct from visceral depots. Gene-set enrichment analyses pointed to an inflammatory environment and insulin resistance particularly in the carotid sheath AT depot. Moreover, the epicardial fat transcriptome was enriched for genes involved in extracellular matrix remodeling, inflammation, immune signaling, coagulation, thrombosis, beigeing, and apoptosis. Interestingly, a striking downregulation of the expression of leptin receptor was found in AT from heel compared with all other AT depots. The distinct gene expression patterns are likely to define fat depot specific AT functions in metabolism, energy storage, immunity, body insulation or as cushions. Improved knowledge of the gene expression profiles of various fat depots may strongly benefit studies aimed at better understanding of the genetics and the pathophysiology of obesity and adverse body fat composition.

Genetically programmed changes in transcription of the novel progranulin regulator.

Progranulin is a glycoprotein marking chronic inflammation in obesity and type 2 diabetes. Previous studies suggested PSRC1 (proline and serine rich coiled-coil 1) to be a target of genetic variants associated with serum progranulin levels. We aimed to identify potentially functional variants and characterize their role in regulation of PSRC1. Phylogenetic module complexity analysis (PMCA) prioritized four polymorphisms (rs12740374, rs629301, rs660240, rs7528419) altering transcription factor binding sites with an overall score for potential regulatory function of Sall > 7.0. The effects of these variants on transcriptional activity and binding of transcription factors were tested by luciferase reporter and electrophoretic mobility shift assays (EMSA). In parallel, blood DNA promoter methylation of two regions was tested in subjects with a very high (N = 100) or a very low (N = 100) serum progranulin. Luciferase assays revealed lower activities in vectors carrying the rs629301-A compared with the C allele. Moreover, EMSA indicated a different binding pattern for the two rs629301 alleles, with an additional prominent band for the A allele, which was finally confirmed with the supershift for the Yin Yang 1 transcription factor (YY1). Subjects with high progranulin levels manifested a significantly higher mean DNA methylation (P < 1 × 10-7) in one promoter region, which was in line with a significantly lower PSRC1 mRNA expression levels in blood (P = 1 × 10-3). Consistently, rs629301-A allele was associated with lower PSRC1 mRNA expression (P < 1 × 10-7). Our data suggest that the progranulin-associated variant rs629301 modifies the transcription of PSRC1 through alteration of YY1 binding capacity. DNA methylation studies further support the role of PSRC1 in regulation of progranulin serum levels. KEY MESSAGES: PSRC1 (proline and serine rich coiled-coil 1) SNPs are associated with serum progranulin levels. rs629301 regulates PSRC1 expression by affecting Yin Yang 1 transcription factor (YY1) binding. PSRC1 is also epigenetically regulated in subjects with high progranulin levels.

Pro-neurotensin depends on renal function and is related to all-cause mortality in chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) have a high risk of premature cardiovascular diseases (CVD) and show increased mortality. Pro-neurotensin (Pro-NT) was associated with metabolic diseases and predicted incident CVD and mortality. However, Pro-NT regulation in CKD and its potential role linking CKD and mortality have not been investigated, so far. METHODS: In a central lab, circulating Pro-NT was quantified in three independent cohorts comprising 4715 participants (cohort 1: patients with CKD; cohort 2: general population study; and cohort 3: non-diabetic population study). Urinary Pro-NT was assessed in part of the patients from cohort 1. In a 4th independent cohort, serum Pro-NT was further related to mortality in patients with advanced CKD. Tissue-specific Nts expression was further investigated in two mouse models of diabetic CKD and compared to non-diabetic control mice. RESULTS: Pro-NT significantly increased with deteriorating renal function (P < 0.001). In meta-analysis of cohorts 1-3, Pro-NT was significantly and independently associated with estimated glomerular filtration rate (P ≤ 0.002). Patients in the middle/high Pro-NT tertiles at baseline had a higher all-cause mortality compared to the low Pro-NT tertile (Hazard ratio: 2.11, P = 0.046). Mice with severe diabetic CKD did not show increased Nts mRNA expression in different tissues compared to control animals. CONCLUSIONS: Circulating Pro-NT is associated with impaired renal function in independent cohorts comprising 4715 subjects and is related to all-cause mortality in patients with end-stage kidney disease. Our human and rodent data are in accordance with the hypotheses that Pro-NT is eliminated by the kidneys and could potentially contribute to increased mortality observed in patients with CKD.

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.

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.

Novel Mutations in the Asparagine Synthetase Gene (ASNS) Associated With Microcephaly.

Microcephaly is a devastating condition defined by a small head and small brain compared to the age- and sex-matched population. Mutations in a number of different genes causative for microcephaly have been identified, e.g., MCPH1, WDR62, and ASPM. Recently, mutations in the gene encoding the enzyme asparagine synthetase (ASNS) were associated to microcephaly and so far 24 different mutations in ASNS causing microcephaly have been described. In a family with two affected girls, we identified novel compound heterozygous variants in ASNS (c.1165G > C, p.E389Q and c.601delA, p.M201Wfs∗28). The first mutation (E389Q) is a missense mutation resulting in the replacement of a glutamate residue evolutionary conserved from Escherichia coli to Homo sapiens by glutamine. Protein modeling based on the known crystal structure of ASNS of E. coli predicted a destabilization of the protein by E389Q. The second mutation (p.M201Wfs∗28) results in a premature stop codon after amino acid 227, thereby truncating more than half of the protein. The novel variants expand the growing list of microcephaly causing mutations in ASNS.

Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family.

BACKGROUND: There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity. METHODS: Serum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. RESULTS: As zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry, and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family. CONCLUSION: Our study suggests that the zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

Identification of Disease-Related Genes Using a Genome-Wide Association Study Approach.

Genome-wide association studies (GWAS) provide a hypothesis-free approach to discover genetic variants contributing to the risk of a certain disease or disease-related trait. Ongoing efforts to annotate the human genome have helped to localize disease-causing variants and point to mechanisms by which genetic variants might exert functional effects. By integrating bioinformatics approaches with in vivo and in vitro genomic strategies to predict and subsequently validate the functional roles of GWAS-identified variants, disease-related pathways can be characterized, providing new possibilities for therapeutic intervention. Here, we describe a basic workflow, from sample preparation to data analysis, for performing a GWAS to identify disease genes. We also discuss resources for the annotation and interpretation of GWAS results.

Relationship Between 12 Adipocytokines and Distinct Components of the Metabolic Syndrome.

Objective: Adipose tissue-derived signals potentially link obesity and adipose tissue dysfunction with metabolic and cardiovascular diseases. Although some adipocytokines have been closely related to metabolic and cardiovascular traits, it is unknown which adipocytokine or adipocytokine clusters serve as meaningful markers of metabolic syndrome (MS) components. Therefore, this study investigated the associations of 12 adipocytokines with components of the MS to identify the most relevant cytokines potentially related to specific metabolic profiles. Research Design and Methods: Twelve cytokines [adiponectin, adipocyte fatty acid-binding protein (AFABP), angiopoietin-related growth factor, chemerin, fibroblast growth factor (FGF) 19, FGF21, FGF23, insulin-like growth factor-1, interleukin 10, irisin, progranulin, and vaspin] were quantified in a cross-sectional cohort of 1046 subjects. Hypothesis-free cluster analysis, multivariate regression analyses with parameters of the MS, and discriminant analysis were performed to assess associations and the relative importance of each cytokine for reflecting MS and its components. Results: Among the studied adipocytokines, adiponectin, AFABP, chemerin, and FGF21 showed the strongest associations with MS and several MS components in discriminant analyses and multiple regression models. For certain metabolic components, these adipocytokines were better discriminators than routine metabolic markers. Other cytokines investigated in the present cohort are less able to distinguish between metabolically healthy and unhealthy subjects. Conclusions: Adiponectin, AFABP, chemerin, and FGF21 showed the strongest associations with MS components in a general population, suggesting that adverse adipose tissue function is a major contributor to these metabolic abnormalities. Future prospective studies should address the question whether these adipocytokines can predict the development of metabolic disease states.