Lovisa E. Johansson

Polymorphisms in the adiponutrin gene are associated with increased insulin secretion and obesity

OBJECTIVE The insulin responsive adiponutrin or patatin-like phospholipase 3 (PNPLA3, previously ADPN) gene shows association with obesity and in vitro adipocyte lipolysis. This study aimed to replicate the association between PNPLA3 variants and obesity, and to investigate their effect on insulin resistance and beta-cell function. METHODS rs738409 (Met148Ile) and rs2072907 (C to G) were genotyped using TaqMan allelic discrimination assay in a Swedish population-based sample (n=1811). Oral glucose tolerance test (OGTT) with data from three time points (0, 30, and 120 min) were available from individuals under the age of 50 years (n=973). RESULTS Both variant alleles were associated with decreased prevalence of obesity (P<0.05); odds ratio 0.75 (0.61-0.93) per carried Ile-allele for rs738409 and 0.80 (0.64-1.00) per carried G-allele for rs2072907. For obesity as a quantitative trait, there was no association in the whole population, but in obese subjects body mass index (BMI; P=0.023) and waist (P=0.0098) were higher in carriers of the Ile-allele. The Ile-carriers also displayed decreased insulin secretion in response to OGTT (30 min insulin; P=0.007, insulinogenic index; P=0.0051) with no significant differences in fasting plasma glucose (P=0.31), beta-cell function (disposition index; P=0.17) or homeostasis model of assessment insulin resistance (HOMA-IR; P=0.063). The correlation between BMI and HOMA-IR differed (Met/X versus Ile/Ile, P=0.028), Met-allele carriers were seemingly more insulin resistant at a lower BMI. The rs2072907 variant shows similar results for insulin secretion. The significance of this finding remained after adjusting for age, gender, and level of self-reported leisure-time physical activity. CONCLUSION We confirm the association between PNPLA3 and obesity. In addition, the rs738409 variant was associated with insulin secretion. There seems to be a differential effect of the Ile-allele depending on the degree of obesity, possibly as a consequence of insulin resistance.

Differential gene expression in adipose tissue from obese human subjects during weight loss and weight maintenance.

BACKGROUND Differential gene expression in adipose tissue during diet-induced weight loss followed by a weight stability period is poorly characterized. Markers of these processes may provide a deeper understanding of underlying mechanisms. OBJECTIVE We aimed to identify differentially expressed genes in human adipose tissue during weight loss and weight maintenance after weight loss. DESIGN RNA from subcutaneous abdominal adipose tissue from 9 obese subjects was analyzed by using a complementary DNA microarray at baseline after weight loss on a low-calorie diet and after weight maintenance. RESULTS Subjects lost 18.8 ± 1.8% of weight and maintained this loss during weight maintenance (1.1 ± 2.1%; range: -9.3 to 10.6%). Most differentially expressed genes exhibited a reciprocal regulation and returned to baseline after weight loss (2163 genes) and weight maintenance (3175 genes). CETP and ABCG1, both of which participate in the HDL-mediated reverse cholesterol transport (RCT), were among the most upregulated of the 750 genes that were differentially expressed after both processes. Several genes involved in inflammation were downregulated. The use of real-time polymerase chain reaction confirmed or partially confirmed the previously implicated genes TNMD and MMP9 (both downregulated), PNPLA3 (upregulated), and CIDEA and SCD (both reciprocally regulated). CONCLUSIONS The beneficial effects of weight loss should be investigated after long-term weight maintenance. The processes of weight loss and weight maintenance should be viewed as biologically distinct. CETP and ABCG1 may be important mediators of these effects through HDL-mediated RCT.

The Swedish CArdioPulmonary BioImage Study: objectives and design.

Cardiopulmonary diseases are major causes of death worldwide, but currently recommended strategies for diagnosis and prevention may be outdated because of recent changes in risk factor patterns. The Swedish CArdioPulmonarybioImage Study (SCAPIS) combines the use of new imaging technologies, advances in large‐scale ‘omics’ and epidemiological analyses to extensively characterize a Swedish cohort of 30 000 men and women aged between 50 and 64 years. The information obtained will be used to improve risk prediction of cardiopulmonary diseases and optimize the ability to study disease mechanisms. A comprehensive pilot study in 1111 individuals, which was completed in 2012, demonstrated the feasibility and financial and ethical consequences of SCAPIS. Recruitment to the national, multicentre study has recently started.

Genetic variance in the adiponutrin gene family and childhood obesity.

Aim The adiponutrin gene family consists of five genes (PNPLA1-5) coding for proteins with both lipolytic and lipogenic properties. PNPLA3 has previously been associated with adult obesity. Here we investigated the possible association between genetic variants in these genes and childhood and adolescent obesity. Methods/Results Polymorphisms in the five genes of the adiponutrin gene family were selected and genotyped using the Sequenom platform in a childhood and adolescent obesity case-control study. Six variants in PNPLA1 showed association with obesity (rs9380559, rs12212459, rs1467912, rs4713951, rs10947600, and rs12199580, p<0.05 after adjustment for age and gender). Three variants in PNPLA3 showed association with obesity before, but not after, adjustment for age and gender (rs139051, rs12483959, and rs2072907, p>0.05). When analyzing these SNPs in relation to phenotypes, two SNPs in the PNPLA3 gene showed association with insulin sensitivity (rs12483959: β = −0.053, p = 0.016, and rs2072907: β = −0.049, p = 0.024). No associations were seen for PNPLA2, PNPLA4, and PNPLA5. Conclusions Genetic variation in the adiponutrin gene family does not seem to contribute strongly to obesity in children and adolescents. PNPLA1 exhibited a modest effect on obesity and PNPLA3 on insulin sensitivity. These data, however, require confirmation in other cohorts and ethnic groups.

Bayesian methods for meta-analysis of causal relationships estimated using genetic instrumental variables.

Genetic markers can be used as instrumental variables, in an analogous way to randomization in a clinical trial, to estimate the causal relationship between a phenotype and an outcome variable. Our purpose is to extend the existing methods for such Mendelian randomization studies to the context of multiple genetic markers measured in multiple studies, based on the analysis of individual participant data. First, for a single genetic marker in one study, we show that the usual ratio of coefficients approach can be reformulated as a regression with heterogeneous error in the explanatory variable. This can be implemented using a Bayesian approach, which is next extended to include multiple genetic markers. We then propose a hierarchical model for undertaking a meta-analysis of multiple studies, in which it is not necessary that the same genetic markers are measured in each study. This provides an overall estimate of the causal relationship between the phenotype and the outcome, and an assessment of its heterogeneity across studies. As an example, we estimate the causal relationship of blood concentrations of C-reactive protein on fibrinogen levels using data from 11 studies. These methods provide a flexible framework for efficient estimation of causal relationships derived from multiple studies. Issues discussed include weak instrument bias, analysis of binary outcome data such as disease risk, missing genetic data, and the use of haplotypes.

Expression of the transcription factor 7-like 2 gene (TCF7L2) in human adipocytes is down regulated by insulin.

Variants in the TCF7L2 gene (transcription factor 7-like 2) have shown strong association with type 2 diabetes with two defined risk haplotypes, HapA and HapB(T2D). TCF7L2 may play a role in both glucose homeostasis and adipogenesis. Our aim was to characterize the TCF7L2 mRNA expression and regulation in human adipose tissue. We quantified TCF7L2 mRNA levels in cultured human adipocytes and in biopsies from visceral (VAT) and subcutaneous (SAT) adipose tissue from 38 obese non-diabetic subjects, using real-time PCR. The influence of haplotype and clinical traits on TCF7L2 mRNA levels were investigated. In vitro, insulin decreased TCF7L2 mRNA expression. This effect was attenuated in cells incubated with the free fatty acids palmitate or oleate. In vivo, we found significantly higher expression in SAT from more insulin resistant subjects. No correlations between TCF7L2 mRNA expression and obesity measures were observed. TCF7L2 expression was higher in VAT than in SAT and when stratifying for haplotype, this difference was seen in HapA carriers but not in non-HapA carriers. In conclusion, TCF7L2 mRNA levels in adipocytes are decreased by insulin and seem to increase in insulin resistant subjects and in HapA carriers.

The visfatin (PBEF1) G-948T gene polymorphism is associated with increased high-density lipoprotein cholesterol in obese subjects.

The newly discovered adipokine visfatin has been hypothesized to be related to obesity and insulin resistance. In this study, we investigate if the 2 single nucleotide polymorphisms rs4730153 and G-948T are associated with obesity and/or related traits and whether they influence the messenger RNA (mRNA) levels of PBEF1 (originally the abbreviation for pre-B-cell colony-enhancing factor 1) in visceral and subcutaneous adipose tissue (VAT and SAT). We found that obese carriers of the PBEF1 G-948T variant allele had significantly higher levels of high-density lipoprotein cholesterol (GG, 1.1 [0.97-1.3] mmol/L; GT + TT, 1.3 [1.0-1.5] mmol/L; P = .02). Other than that, neither rs4730153 nor G-948T had any major impact on any of the obesity-related phenotypes. There was no difference in mRNA expression between VAT and SAT (2.08 +/- 0.17 and 2.09 +/- 0.14, respectively; P = .26), but there was a nonsignificant trend toward higher PBEF1 mRNA levels in the variant allele carriers concerning both VAT and SAT for both single nucleotide polymorphisms. A significant correlation was observed between body mass index and PBEF1 mRNA expression in SAT (R = 0.37, P = .03) but not in VAT (R = 0.26, P = .12). In conclusion, PBEF1 G-948T is associated with increased high-density lipoprotein cholesterol; but genetic variation in PBEF1 does not seem to have a major impact on the development of obesity or on the expression of the gene.

Interaction between PPARG Pro12Ala and ADIPOQ G276T concerning cholesterol levels in childhood obesity

AIM The aim of this study was to investigate the role of two candidate gene polymorphisms for insulin resistance and lipid levels in obese children and adolescents. METHODS Two markers of insulin resistance and lipid levels, Pro12Ala in peroxisome proliferator-activated receptor-gamma2 (PPARG) and G276T in adiponectin (ADIPOQ), were genotyped in 285 obese children and adolescents. As the apolipoprotein E (APOE) polymorphisms C112R and R158C are known to have a profound impact on lipid levels in both children and adults, results were adjusted for APOE genotype. RESULTS We found no association for PPARG or ADIPOQ polymorphisms with Body Mass Index (BMI), High Density Lipoprotein (HDL)-cholesterol, triglycerides or Insulin Resistance estimated by Homeostasis Model of Assessment (HOMA-IR). Wild type carriers of PPARG Pro12Ala (p<0.05), homozygous carriers of the variant allele of ADIPOQ G276T (p<0.001) and epsilon4 carriers of APOE (p<0.001) had higher total and low density lipoprotein (LDL)-cholesterol levels adjusted for age, gender, BMI and insulin sensitivity. A PPARG/ADIPOQ risk genotype combination was identified by analysis of covariance (ANCOVA) comparing all existing combinations. Carriers of PPARG Pro/Pro and ADIPOQ 276 T/T had higher total (5.0 [4.1-5.8] vs. 4.1 [3.6-4.6] mmol/l) and LDL-cholesterol levels (3.7 [2.9-4.5] vs. 3.0 [2.5-3.5] mmol/l) compared with carriers of other combinations (p<0.001). Importantly, the PPARG and ADIPOQ associations were unaffected when adjusting for APOE genotype. CONCLUSIONS Genetic variants in candidate genes for insulin resistance are associated with cholesterol levels in obese children and adolescents, and may offer additional information in the risk assessment of obese children.

The hormone-sensitive lipase C–60G promoter polymorphism is associated with increased waist circumference in normal-weight subjects

Objective:Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from triglyceride stores in adipocytes. The aim of the present study was to investigate the role of the HSL gene promoter variant C-60G, a polymorphism which previously has been associated with reduced promoter activity in vitro, in obesity and type 2 diabetes.Design:We genotyped two materials consisting of obese subjects and non-obese controls, one material with offspring-parents trios, where the offspring was abdominally obese and one material with trios, where the offspring had type 2 diabetes or impaired glucose homeostasis. HSL promoter containing the HSL C-60G G-allele was generated and tested against a construct with the C-allele in HeLa cells and primary rat adipocytes. HSL mRNA levels were quantified in subcutaneous and visceral fat from 33 obese subjects.Results:We found that the common C-allele was associated with increased waist circumference and WHR in lean controls, but there was no difference in genotype frequency between obese and non-obese subjects. There was a significant increased transmission of C-alleles to the abdominally obese offspring but no increased transmission of C-alleles was observed to offspring with impaired glucose homeostasis. The G-allele showed reduced transcription in HeLa cells and primary rat adipocytes. HSL mRNA levels were significantly higher in subcutaneous compared to visceral fat from obese subjects.Conclusion:The HSL C-60G polymorphism is associated with increased waist circumference in non-obese subjects.

The P2Y(13) Met-158-Thr Polymorphism, Which Is in Linkage Disequilibrium with the P2Y(12) Locus, Is Not Associated with Acute Myocardial Infarction.

Background and Aims The aims of this study were to investigate (1) if P2Y12 polymorphisms defining the P2Y12 H2 allele are associated with any other SNPs that may explain the previously reported association with increased ADP induced platelet activation and association with peripheral arterial disease and coronary artery disease and (2) if such variants are associated with acute myocardial infarction (AMI) or classical risk factors for AMI. Methods and Results The P2Y13 Met-158-Thr polymorphism was found to be in linkage disequilibrium (LD) with the P2Y12 H2 haplotype (all examined SNPs: D′ = 1.0, r2 = 0.936–1.0), defining a novel P2Y12 H2/P2Y13 Thr-158 haplotype. Genotyping of an AMI case control population (n = 1244 cases, 2488 controls) revealed no association of the P2Y13 Thr-158 allele with AMI (OR = 0.96, 95% C.I. 0.82–1.12, P = 0.63). Also, no differences between the genotype frequencies of P2Y13 Met-158-Met and Met-158-Thr/Thr-158-Thr were seen in AMI case-control subpopulations (early onset AMI OR = 1.06, 95% C.I. 0.85–1.31, P = 0.62); family history of AMI (OR = 0.98, 95% C.I. 0.78–1.22, P = 0.83) nor in early onset AMIs with family history of AMI (OR = 1.0, 95% C.I. 0.74–1.36, P = 1.0). Genotyping of the P2Y13 Met-158-Thr polymorphism in a population based sample (n = 6055) revealed no association with cardiovascular risk factors. In addition, the P2Y13 Met-158-Thr polymorphism was genotyped in a diabetes case-control population, and associations were found neither with DM nor with any examined DM risk factors. Conclusion Genotyping The P2Y13 Met-158-Thr polymorphism is in tight LD with the P2Y12 locus but is not associated with AMI or classical cardiovascular risk factors.