Jussi Pihlajamäki

A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity

The peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that has a pivotal role in adipocyte differentiation and expression of adipocyte-specific genes. The PPARγ1 and γ2 isoforms result from alternative splicing and have ligand-dependent and -independent activation domains. PPARγ2 has an additional 28 amino acids at its amino terminus that renders its ligand-independent activation domain 5-10-fold more effective than that of PPARγ1. Insulin stimulates the ligand-independent activation of PPARγ1 and γ2 (ref. 5), however, obesity and nutritional factors only influence the expression of PPARγ2 in human adipocytes. Here, we report that a relatively common Pro12Ala substitution in PPARγ2 is associated with lower body mass index (BMI; P=0.027; 0.015) and improved insulin sensitivity among middle-aged and elderly Finns. A significant odds ratio (4.35, P=0.028) for the association of the Pro/Pro genotype with type 2 diabetes was observed among Japanese Americans. The PPARγ2 Ala allele showed decreased binding affinity to the cognate promoter element and reduced ability to transactivate responsive promoters. These findings suggest that the PPARγ2 Pro12Ala variant may contribute to the observed variability in BMI and insulin sensitivity in the general population.

Serum Bile Acids Are Higher in Humans With Prior Gastric Bypass: Potential Contribution to Improved Glucose and Lipid Metabolism

The multifactorial mechanisms promoting weight loss and improved metabolism following Roux‐en‐Y gastric bypass (GB) surgery remain incompletely understood. Recent rodent studies suggest that bile acids can mediate energy homeostasis by activating the G‐protein coupled receptor TGR5 and the type 2 thyroid hormone deiodinase. Altered gastrointestinal anatomy following GB could affect enterohepatic recirculation of bile acids. We assessed whether circulating bile acid concentrations differ in patients who previously underwent GB, which might then contribute to improved metabolic homeostasis. We performed cross‐sectional analysis of fasting serum bile acid composition and both fasting and post‐meal metabolic variables, in three subject groups: (i) post‐GB surgery (n = 9), (ii) without GB matched to preoperative BMI of the index cohort (n = 5), and (iii) without GB matched to current BMI of the index cohort (n = 10). Total serum bile acid concentrations were higher in GB (8.90 ± 4.84 µmol/l) than in both overweight (3.59 ± 1.95, P = 0.005, Ov) and severely obese (3.86 ± 1.51, P = 0.045, MOb). Bile acid subfractions taurochenodeoxycholic, taurodeoxycholic, glycocholic, glycochenodeoxycholic, and glycodeoxycholic acids were all significantly higher in GB compared to Ov (P < 0.05). Total bile acids were inversely correlated with 2‐h post‐meal glucose (r = −0.59, P < 0.003) and fasting triglycerides (r = −0.40, P = 0.05), and positively correlated with adiponectin (r = −0.48, P < 0.02) and peak glucagon‐like peptide‐1 (GLP‐1) (r = 0.58, P < 0.003). Total bile acids strongly correlated inversely with thyrotropic hormone (TSH) (r = −0.57, P = 0.004). Together, our data suggest that altered bile acid levels and composition may contribute to improved glucose and lipid metabolism in patients who have had GB.

Multiple Abnormalities in Glucose and Energy Metabolism and Coordinated Changes in Levels of Adiponectin, Cytokines, and Adhesion Molecules in Subjects With Metabolic Syndrome

Background—Detailed metabolic defects in glucose and energy metabolism and abnormalities in a variety of cardiovascular risk factors are largely unknown in subjects with the metabolic syndrome. Methods and Results—We characterized the metabolic syndrome in 119 nondiabetic offspring of diabetic probands. Cardiovascular risk factors, including cytokines and adhesion molecules, were measured. Insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp and indirect calorimetry; intra-abdominal fat and subcutaneous fat were assessed by CT; and maximal oxygen consumption was measured with a bicycle ergometer test. By applying factor analysis, we identified a single factor, the metabolic syndrome factor, from the following variables: 2-hour glucose, fasting insulin, body mass index, waist, HDL cholesterol, triglycerides, and mean blood pressure. Subjects with the highest factor score were defined as having the metabolic syndrome. During hyperinsulinemia, the highest factor score was associated with decreased rates of glucose oxidation and nonoxidative glucose disposal, high rates of lipid oxidation, low energy expenditure, and impaired suppression of free fatty acids during hyperinsulinemia. Furthermore, the metabolic syndrome was associated with a high amount of visceral fat, hypoadiponectinemia, a low maximum oxygen uptake, and high levels of C-reactive protein, proinflammatory cytokines, and adhesion molecules. Conclusions—The metabolic syndrome is characterized by an excess of intra-abdominal fat, hypoadiponectinemia, insulin resistance in skeletal muscle and adipose tissue, multiple defects in glucose and energy metabolism, and elevated levels of cytokines and adhesion molecules.

Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease

Excess hepatic storage of triglycerides is considered a benign condition, but nonalcoholic steatohepatitis (NASH) may progress to fibrosis and promote atherosclerosis. Carriers of the TM6SF2 E167K variant have fatty liver as a result of reduced secretion of very‐low‐density lipoproteins (VLDLs). As a result, they have lower circulating lipids and reduced risk of myocardial infarction. In this study, we aimed to assess whether TM6SF2 E167K affects liver damage and cardiovascular outcomes in subjects at risk of NASH. Liver damage was evaluated in 1,201 patients who underwent liver biopsy for suspected NASH; 427 were evaluated for carotid atherosclerosis. Cardiovascular outcomes were assessed in 1,819 controls from the Swedish Obese Subjects (SOS) cohort. Presence of the inherited TM6SF2 E167K variant was determined by TaqMan assays. In the liver biopsy cohort, 188 subjects (13%) were carriers of the E167K variant. They had lower serum lipid levels than noncarriers (P < 0.05), had more‐severe steatosis, necroinflammation, ballooning, and fibrosis (P < 0.05), and were more likely to have NASH (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.23‐2.79) and advanced fibrosis (OR, 2.08; 95% CI: 1.20‐3.55), after adjustment for age, sex, body mass index, fasting hyperglycemia, and the I148M PNPLA3 risk variant. However, E167K carriers had lower risk of developing carotid plaques (OR, 0.49; 95% CI: 0.25‐0.94). In the SOS cohort, E167K carriers had higher alanine aminotransferase ALT and lower lipid levels (P < 0.05), as well as a lower incidence of cardiovascular events (hazard ratio: 0.61; 95% CI: 0.39‐0.95). Conclusions: Carriers of the TM6SF2 E167K variant are more susceptible to progressive NASH, but are protected against cardiovascular disease. Our findings suggest that reduced ability to export VLDLs is deleterious for the liver. (Hepatology 2015;61:506‐514)

Genomewide Scan for Familial Combined Hyperlipidemia Genes in Finnish Families, Suggesting Multiple Susceptibility Loci Influencing Triglyceride, Cholesterol, and Apolipoprotein B Levels

Familial combined hyperlipidemia (FCHL) is a common dyslipidemia predisposing to premature coronary heart disease (CHD). The disease is characterized by increased levels of serum total cholesterol (TC), triglycerides (TGs), or both. We recently localized the first locus for FCHL, on chromosome 1q21-q23. In the present study, a genomewide screen for additional FCHL loci was performed. In stage 1, we genotyped 368 polymorphic markers in 35 carefully characterized Finnish FCHL families. We identified six chromosomal regions with markers showing LOD score (Z) values >1.0, by using a dominant mode of inheritance for the FCHL trait. In addition, two more regions emerged showing Z>2.0 with a TG trait. In stage 2, we genotyped 26 more markers and seven additional FCHL families for these interesting regions. Two chromosomal regions revealed Z>2.0 in the linkage analysis: 10p11.2, Z=3.20 (theta=.00), with the TG trait; and 21q21, Z=2.24 (theta=.10), with the apoB trait. Furthermore, two more chromosomal regions produced Z>2.0 in the affected-sib-pair analysis: 10q11.2-10qter produced Z=2.59 with the TC trait and Z=2.29 with FCHL, and 2q31 produced Z=2.25 with the TG trait. Our results suggest additional putative loci influencing FCHL in Finnish families, some potentially affecting TG levels and some potentially affecting TC or apoB levels.

Hyperglycemia and a Common Variant of GCKR Are Associated With the Levels of Eight Amino Acids in 9,369 Finnish Men

We investigated the association of glycemia and 43 genetic risk variants for hyperglycemia/type 2 diabetes with amino acid levels in the population-based Metabolic Syndrome in Men (METSIM) Study, including 9,369 nondiabetic or newly diagnosed type 2 diabetic Finnish men. Plasma levels of eight amino acids were measured with proton nuclear magnetic resonance spectroscopy. Increasing fasting and 2-h plasma glucose levels were associated with increasing levels of several amino acids and decreasing levels of histidine and glutamine. Alanine, leucine, isoleucine, tyrosine, and glutamine predicted incident type 2 diabetes in a 4.7-year follow-up of the METSIM Study, and their effects were largely mediated by insulin resistance (except for glutamine). We also found significant correlations between insulin sensitivity (Matsuda insulin sensitivity index) and mRNA expression of genes regulating amino acid degradation in 200 subcutaneous adipose tissue samples. Only 1 of 43 risk single nucleotide polymorphisms for type 2 diabetes or hyperglycemia, the glucose-increasing major C allele of rs780094 of GCKR, was significantly associated with decreased levels of alanine and isoleucine and elevated levels of glutamine. In conclusion, the levels of branched-chain, aromatic amino acids and alanine increased and the levels of glutamine and histidine decreased with increasing glycemia, reflecting, at least in part, insulin resistance. Only one single nucleotide polymorphism regulating hyperglycemia was significantly associated with amino acid levels.

Statin use and non-alcoholic steatohepatitis in at risk individuals

BACKGROUND & AIMS Excess hepatic free cholesterol contributes to the pathogenesis of non-alcoholic steatohepatitis, and statins reduce cholesterol synthesis. Aim of this study was to assess whether statin use is associated with histological liver damage related to steatohepatitis. METHODS The relationship between statin use, genetic risk factors, and liver damage was assessed in a multi-center cohort of 1201 European individuals, who underwent liver biopsy for suspected non-alcoholic steatohepatitis. RESULTS Statin use was recorded in 107 subjects, and was associated with protection from steatosis, NASH, and fibrosis stage F2-F4, in a dose-dependent manner (adjusted p<0.05 for all). In 100 treated patients matched 1:1 for modality of recruitment, gender, presence of IFG or type 2 diabetes, PNPLA3 I148M risk alleles, TM6SF2 E167K variant, age, and BMI, statin use remained associated with protection from steatosis (OR 0.09, 95% C.I. 0.01-0.32; p=0.004), steatohepatitis (OR 0.25, 95% C.I. 0.13-0.47; p<0.001), and fibrosis stage F2-F4 (OR 0.42, 95% C.I. 0.20-0.8; p=0.017). Results were confirmed in a second analysis, where individuals were matched within recruitment center (p<0.05 for all). The protective effect of statins on steatohepatitis was stronger in subjects not carrying the I148M PNPLA3 risk variant (p=0.02 for interaction), as statins were negatively associated with steatohepatitis in patients negative (p<0.001), but not in those positive for the I148M variant (p=n.s.). CONCLUSIONS Statin use was associated with protection towards the full spectrum of liver damage in individuals at risk of non-alcoholic steatohepatitis. However, the I148M PNPLA3 risk variant limited this beneficial effect.

The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent.

BACKGROUND & AIMS Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

Diverse associations of 25-hydroxyvitamin D and 1,25-dihydroxy-vitamin D with dyslipidaemias.

Abstract.  Karhapää P, Pihlajamäki J, Pörsti I, Kastarinen M, Mustonen J, Niemelä O, Kuusisto J (University of Eastern Finland, Kuopio; University of Tampere, Tampere; and Laboratory and Medical Research Unit, University of Tampere, Tampere; Finland). Diverse associations of 25‐hydroxyvitamin D and 1,25‐dihydroxyvitamin D with dyslipidaemias. J Intern Med 2010; 268: 604–610.

SIRT1 mRNA Expression May Be Associated With Energy Expenditure and Insulin Sensitivity

OBJECTIVE Sirtuin 1 (SIRT1) is implicated in the regulation of mitochondrial function, energy metabolism, and insulin sensitivity in rodents. No studies are available in humans to demonstrate that SIRT1 expression in insulin-sensitive tissues is associated with energy expenditure and insulin sensitivity. RESEARCH DESIGN AND METHODS Energy expenditure (EE), insulin sensitivity, and SIRT1 mRNA adipose tissue expression (n = 81) were measured by indirect calorimetry, hyperinsulinemic-euglycemic clamp, and quantitative RT-PCR in 247 nondiabetic offspring of type 2 diabetic patients. RESULTS High EE during the clamp (r = 0.375, P = 2.8 × 10−9) and high ΔEE (EE during the clamp − EE in the fasting state) (r = 0.602, P = 2.5 × 10−24) were associated with high insulin sensitivity. Adipose tissue SIRT1 mRNA expression was significantly associated with EE (r = 0.289, P = 0.010) and with insulin sensitivity (r = 0.334, P = 0.002) during hyperinsulinemic-euglycemic clamp. Furthermore, SIRT1 mRNA expression correlated significantly with the expression of several genes regulating mitochondrial function and energy metabolism (e.g., peroxisome proliferator–activated receptor γ coactivator-1β, estrogen-related receptor α, nuclear respiratory factor-1, and mitochondrial transcription factor A), and with several genes of the respiratory chain (e.g., including NADH dehydrogenase [ubiquinone] 1α subcomplex 2, cytochrome c, cytochrome c oxidase subunit IV, and ATP synthase). CONCLUSIONS Impaired stimulation of EE by insulin and low SIRT1 expression in insulin-sensitive tissues is likely to reflect impaired regulation of mitochondrial function associated with insulin resistance in humans.