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.

Genome wide meta-analysis highlights the role of genetic variation in RARRES2 in the regulation of circulating serum chemerin.

Chemerin is an adipokine proposed to link obesity and chronic inflammation of adipose tissue. Genetic factors determining chemerin release from adipose tissue are yet unknown. We conducted a meta-analysis of genome-wide association studies (GWAS) for serum chemerin in three independent cohorts from Europe: Sorbs and KORA from Germany and PPP-Botnia from Finland (total N = 2,791). In addition, we measured mRNA expression of genes within the associated loci in peripheral mononuclear cells by micro-arrays, and within adipose tissue by quantitative RT-PCR and performed mRNA expression quantitative trait and expression-chemerin association studies to functionally substantiate our loci. Heritability estimate of circulating chemerin levels was 16.2% in the Sorbs cohort. Thirty single nucleotide polymorphisms (SNPs) at chromosome 7 within the retinoic acid receptor responder 2 (RARRES2)/Leucine Rich Repeat Containing (LRRC61) locus reached genome-wide significance (p<5.0×10-8) in the meta-analysis (the strongest evidence for association at rs7806429 with p = 7.8×10-14, beta = -0.067, explained variance 2.0%). All other SNPs within the cluster were in linkage disequilibrium with rs7806429 (minimum r2 = 0.43 in the Sorbs cohort). The results of the subgroup analyses of males and females were consistent with the results found in the total cohort. No significant SNP-sex interaction was observed. rs7806429 was associated with mRNA expression of RARRES2 in visceral adipose tissue in women (p<0.05 after adjusting for age and body mass index). In conclusion, the present meta-GWAS combined with mRNA expression studies highlights the role of genetic variation in the RARRES2 locus in the regulation of circulating chemerin concentrations.

PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.

DNA microarrays can be used to identify gene expression changes characteristic of human disease. This is challenging, however, when relevant differences are subtle at the level of individual genes. We introduce an analytical strategy, Gene Set Enrichment Analysis, designed to detect modest but coordinate changes in the expression of groups of functionally related genes. Using this approach, we identify a set of genes involved in oxidative phosphorylation whose expression is coordinately decreased in human diabetic muscle. Expression of these genes is high at sites of insulin-mediated glucose disposal, activated by PGC-1alpha and correlated with total-body aerobic capacity. Our results associate this gene set with clinically important variation in human metabolism and illustrate the value of pathway relationships in the analysis of genomic profiling experiments.

Increasing circulating levels of Tenascin C in response to the Wingate anaerobic test.

AIM: Tenascin C (TNC) is a large extracellular matrix glycoprotein. It is involved in development and upregulated both during tissue repair and in several pathological conditions, including cardiovascular disease. Extracellular matrix proteins play a role in promoting exercise responses, leading to adaptation, regeneration, and repair. The main goal of this study was to investigate whether a short anaerobic effort leads to increased levels of TNC in serum. METHODS: Thirty-nine healthy men performed a Wingate test followed by a muscle biopsy. Myoblasts were isolated from the muscle biopsies and differentiated to myotubes ex vivo. TNC RNA was quantified in the biopsies, myotubes and myoblasts using RNA sequencing. Blood samples were drawn before and 5 min after the Wingate test. Serum TNC levels were measured using enzyme-linked immunosorbent assay. RESULTS: After the Wingate test, serum TNC increased on average by 23% [15-33], median [interquartile range]; PWilcoxon < 0.0001. This increase is correlated with peak power output and power drop, but not with VO2max . TNC RNA expression is higher in myoblasts and myotubes compared to skeletal muscle tissue. CONCLUSION: TNC is secreted systemically as a response to the Wingate anaerobic test in healthy males. The response was positively correlated with peak power and power drop, but not with VO2max which implicates a relation to mechanical strain and/or blood flow. With higher expression in undifferentiated myoblast cells than muscle tissue, it is likely that TNC plays a role in muscle tissue remodelling in humans. Our findings open for research on how TNC contributes to exercise adaptation.

Genetic determinants of circulating GIP and GLP-1 concentrations.

The secretion of insulin and glucagon from the pancreas and the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) from the gastrointestinal tract is essential for glucose homeostasis. Several novel treatment strategies for type 2 diabetes (T2D) mimic GLP-1 actions or inhibit incretin degradation (DPP4 inhibitors), but none is thus far aimed at increasing the secretion of endogenous incretins. In order to identify new potential therapeutic targets for treatment of T2D, we performed a meta-analysis of a GWAS and an exome-wide association study of circulating insulin, glucagon, GIP, and GLP-1 concentrations measured during an oral glucose tolerance test in up to 7,828 individuals. We identified 6 genome-wide significant functional loci associated with plasma incretin concentrations in or near the SLC5A1 (encoding SGLT1), GIPR, ABO, GLP2R, F13A1, and HOXD1 genes and studied the effect of these variants on mRNA expression in pancreatic islet and on metabolic phenotypes. Immunohistochemistry showed expression of GIPR, ABO, and HOXD1 in human enteroendocrine cells and expression of ABO in pancreatic islets, supporting a role in hormone secretion. This study thus provides candidate genes and insight into mechanisms by which secretion and breakdown of GIP and GLP-1 are regulated.

Growth hormone replacement therapy induces insulin resistance by activating the glucose-fatty acid cycle.

The effects of GH replacement therapy on energy metabolism are still uncertain, and long-term benefits of increased muscle mass are thought to outweigh short-term negative metabolic effects. This study was designed to address this issue by examining both short-term (1 wk) and long-term (6 months) effects of a low-dose (9.6 micro g/kg body weight.d) GH replacement therapy or placebo on whole-body glucose and lipid metabolism (oral glucose tolerance test and euglycemic hyperinsulinemic clamp combined with indirect calorimetry and infusion of 3-[(3)H]glucose) and on muscle composition and muscle enzymes/metabolites, as determined from biopsies obtained at the end of the clamp in 19 GH-deficient adult subjects. GH therapy resulted in impaired insulin-stimulated glucose uptake at 1 wk (-52%; P = 0.008) and 6 months (-39%; P = 0.008), which correlated with deterioration of glucose tolerance (r = -0.481; P = 0.003). The decrease in glucose uptake was associated with an increase in lipid oxidation at 1 wk (60%; P = 0.008) and 6 months (60%; P = 0.008) and a concomitant decrease in glucose oxidation. The deterioration of glucose metabolism during GH therapy also correlated with the enhanced rate of lipid oxidation (r = -0.508; P = 0.0002). In addition, there was a shift toward more glycolytic type II fibers during GH therapy. In conclusion, replacement therapy with a low-dose GH in GH-deficient adult subjects is associated with a sustained deterioration of glucose metabolism as a consequence of the lipolytic effect of GH, resulting in enhanced oxidation of lipid substrates. Also, a shift toward more insulin-resistant type II X fibers is seen in muscle. Glucose metabolism should be carefully monitored during long-term GH replacement therapy.

Inhibition of lipolysis during acute GH exposure increases insulin sensitivity in previously untreated GH-deficient adults.

OBJECTIVE: Previous studies evaluating the lipolytic effect of GH have in general been performed in subjects on chronic GH therapy. In this study we assessed the lipolytic effect of GH in previously untreated patients and examined whether the negative effect of enhanced lipolysis on glucose metabolism could be counteracted by acute antilipolysis achieved with acipimox. METHODS: Ten GH-deficient (GHD) adults participated in four experiments each, during which they received in a double-blind manner: placebo (A); GH (0.88+/-0.13 mg) (B); GH+acipimox 250 mg b.i.d. (C); and acipimox b.i.d. (no GH) (D), where GH was given the night before a 2 h euglycemic, hyperinsulinemic clamp combined with infusion of [3-(3)H]glucose and indirect calorimetry. RESULTS: GH increased basal free fatty acid (FFA) levels by 74% (P=0.0051) and insulin levels by 93% (P=0.0051). This resulted in a non-significant decrease in insulin-stimulated glucose uptakes (16.61+/-8.03 vs 12.74+/-5.50 micromol/kg per min (s.d.), P=0.07 for A vs B). The rates of insulin-stimulated glucose uptake correlated negatively with the FFA concentrations (r=-0.638, P<0.0001). However, acipimox caused a significant improvement in insulin-stimulated glucose uptake in the GH-treated patients (17.35+/-5.65 vs 12.74+/-5.50 micromol/kg per min, P=0.012 for C vs B). The acipimox-induced enhancement of insulin-stimulated glucose uptake was mainly due to an enhanced rate of glucose oxidation (8.32+/-3.00 vs 5.88+/-2.39 micromol/kg per min, P=0.07 for C vs B). The enhanced rates of glucose oxidation induced by acipimox correlated negatively with the rate of lipid oxidation in GH-treated subjects both in basal (r=-0.867, P=0.0093) and during insulin-stimulated (r=-0.927, P=0.0054) conditions. GH did not significantly impair non-oxidative glucose metabolism (6.86+/-5.22 vs 8.67+/-6.65 micromol/kg per min, P=NS for B vs A). The fasting rate of endogenous glucose production was unaffected by GH and acipimox administration (10.99+/-1.98 vs 11.73+/-2.38 micromol/kg per min, P=NS for B vs A and 11.55+/-2.7 vs 10.99+/-1.98 micromol/kg per min, P=NS for C vs B). On the other hand, acipimox alone improved glucose uptake in the untreated GHD patients (24.14+/-8.74 vs 16.61+/-8.03 micromol/kg per min, P=0.0077 for D vs A) and this was again due to enhanced fasting (7.90+/-2.68 vs 5.16+/-2.28 micromol/kg per min, P=0.01 for D vs A) and insulin-stimulated (9.78+/-3.68 vs 7.95+/-2.64 micromol/kg per min, P=0.07 for D vs A) glucose oxidation. CONCLUSION: The study of acute administration of GH to previously untreated GHD patients provides compelling evidence that (i) GH-induced insulin resistance is mainly due to induction of lipolysis by GH; and (ii) inhibition of lipolysis can prevent the deterioration of insulin sensitivity. The question remains whether GH replacement therapy should, at least at the beginning of therapy, be combined with means to prevent an excessive stimulation of lipolysis by GH.

Effects of morning cortisol replacement on glucose and lipid metabolism in GH-treated subjects.

OBJECTIVE: Insulin resistance is a frequent consequence of GH replacement therapy but patients on GH replacement therapy often also have replacement of other hormone deficiencies which theoretically could modify the metabolic effects of GH. In particular, cortisol replacement if given in supra physiologic doses immediately before the evaluation of insulin sensitivity could influence insulin sensitivity. The aim of this study was thus to evaluate the effect of morning cortisol replacement given prior to a euglycaemic clamp combined with infusion of [3-(3)H]glucose and indirect calorimetry on glucose and lipid metabolism. METHODS: Ten GH/ACTH-deficient adults received, in a double-blind manner, either cortisol (A) or placebo (B) before the clamp whereas five GH-deficient-ACTH-sufficient adults participated in a control (C) clamp experiment. All subjects received GH replacement therapy. RESULTS: Serum cortisol levels were significantly higher after cortisol than after placebo (324+/-156 vs 132+/-136 mmol/l; P=0.006) and similar to controls (177+/-104 mmol/l). As a measure of the biological effect of cortisol, eosinophil leukocyte counts in peripheral blood decreased (164+/-91 x 10(9)/l vs 216+/-94 x 10(9)/l; P=0.04). Cortisol replacement had no significant effect on insulin-stimulated glucose uptake (11.8+/-1.8 vs 13.2+/-3.9 micromol/kg min), either on glucose oxidation or on glucose storage. There was also no significant effect of cortisol on fasting endogenous glucose production and no effect was seen on serum free fatty acid concentrations. CONCLUSION: Administration of cortisol in the morning before a clamp cannot explain the insulin resistance seen with GH replacement therapy.

Fat feeding impairs glycogen synthase activity in mice without effects on its gene expression.

To examine whether the effects of high-fat feeding on glycogen synthase (GS) activity and mRNA levels differ between diabetes-prone (C57BL/6J) and diabetes-resistant mice (NMRI), we measured GS activity and mRNA levels in muscle from C57BL/6J and NMRI mice fed a high-fat or normal chow diet for 3, 6, and 15 months. As compared with chow feeding, fat feeding increased plasma insulin levels in C57BL/6J mice at 15 months (464 +/- 29 v 267 +/- 47 pmol/L, P =.005), which was associated with elevated plasma glucose levels at 15 months (5.3 +/- 0.3 v 3.8 +/- 0.2 mmol/L, P =.001). Fat feeding increased plasma insulin levels also in NMRI mice at 15 months (705 +/- 145 v 275 +/- 64 pmol/L, P =.01) without, however, a rise of plasma glucose levels. In parallel with increased insulin levels, decreased muscle GS fractional velocity (FV) was observed at 6 (49.0% +/- 2.6% v 69.1% +/- 7.3%, P =.04) and 15 (45.8% +/- 1.8% v 53.4% +/- 1.6 %, P <.01) months but not at 3 months in the fat-fed C57BL/6J mice. Similarly, there was a significant decrease in GS fractional activity at 3 (57.9% +/- 4.3% v 70.4% +/- 2.6 %, P <.03) and 15 (47.3% +/- 2.4% v 56.4% +/- 2.1%, P =.02) but not at 6 months in the fat-fed NMRI mice. The decrease in GS activity was not associated with changes in mRNA levels at any time points. We conclude that (1) fat feeding results in similar elevation of plasma insulin levels and impairs GS activity in C57BL/6J and NMRI mice, and (2) the changes in GS activity do not involve effects on gene expression.

The triglyceride content in skeletal muscle is associated with hepatic but not peripheral insulin resistance in elderly twins.

CONTEXT AND OBJECTIVE: Total muscle triglyceride (MT) content has been associated with insulin resistance. We investigated the predictors and impact of MT on relevant metabolic parameters including peripheral and hepatic insulin resistance in elderly twins. DESIGN AND PARTICIPANTS: Seventy-four elderly same-sex twins underwent hyperinsulinemic euglycemic clamps preceded by an iv glucose tolerance test. Aerobic capacity (VO2max) and body composition (dual-energy x-ray absorptiometry scan) were determined in all twins. A biopsy from the vastus lateralis muscle was excised in the fasting state. The muscle triacylglycerol content was analyzed by biochemical extraction from these biopsies. RESULTS: The percentage of total body fat was the only independent predictor of MT content. After adjustment for trunk fat percentages and sex, MT level was significantly associated to fasting plasma levels of glucose and insulin as well as hepatic insulin resistance. However, the association was weakened after adjustment for total fat percentages. A 1 SD (34.5 mmol/kg dry weight) increase in MT content was associated with a 24% increase of hepatic insulin resistance. No association between MT content and peripheral insulin sensitivity was observed. CONCLUSION: MT content is associated with hepatic but not peripheral insulin resistance in elderly twins. We speculate that MT content may reflect the general ectopic accumulation of triglycerides, including fat in the liver.

GAD antibody positivity predicts type 2 diabetes in an adult population.

OBJECTIVE: To evaluate the significance of GAD antibodies (GADAs) and family history for type 1 diabetes (FH(T1)) or type 2 diabetes (FH(T2)) in nondiabetic subjects. RESEARCH DESIGN AND METHODS: GADAs were analyzed in 4,976 nondiabetic relatives of type 2 diabetic patients or control subjects from Finland. Altogether, 289 (5.9%) were GADA(+)-a total of 253 GADA(+) and 2,511 GADA(-) subjects participated in repeated oral glucose tolerance tests during a median time of 8.1 years. The risk of progression to diabetes was assessed using Cox regression analysis. RESULTS: Subjects within the highest quartile of GADA(+) (GADA(+)(high)) had more often first-degree FH(T1) (29.2 vs. 7.9%, P < 0.00001) and GADA(+) type 2 diabetic (21.3 vs. 13.7%, P = 0.002) or nondiabetic (26.4 vs. 13.3%, P = 0.010) relatives than GADA(-) subjects. During the follow-up, the GADA(+) subjects developed diabetes significantly more often than the GADA(-) subjects (36/253 [14.2%] vs. 134/2,511 [5.3%], P < 0.00001). GADA(+)(high) conferred a 4.9-fold increased risk of diabetes (95% CI 2.8-8.5) compared with GADA(-)-seroconversion to positive during the follow-up was associated with 6.5-fold (2.8-15.2) and first-degree FH(T1) with 2.2-fold (1.2-4.1) risk of diabetes. Only three subjects developed type 1 diabetes, and others had a non-insulin-dependent phenotype 1 year after diagnosis. GADA(+) and GADA(-) subjects did not clinically differ at baseline, but they were leaner and less insulin resistant after the diagnosis of diabetes. CONCLUSIONS: GADA positivity clusters in families with type 1 diabetes or latent autoimmune diabetes in adults. GADA positivity predicts diabetes independently of family history of diabetes, and this risk was further increased with high GADA concentrations.

Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels.

New strategies for prevention and treatment of type 2 diabetes (T2D) require improved insight into disease etiology. We analyzed 386,731 common single-nucleotide polymorphisms (SNPs) in 1464 patients with T2D and 1467 matched controls, each characterized for measures of glucose metabolism, lipids, obesity, and blood pressure. With collaborators (FUSION and WTCCC/UKT2D), we identified and confirmed three loci associated with T2D-in a noncoding region near CDKN2A and CDKN2B, in an intron of IGF2BP2, and an intron of CDKAL1-and replicated associations near HHEX and in SLC30A8 found by a recent whole-genome association study. We identified and confirmed association of a SNP in an intron of glucokinase regulatory protein (GCKR) with serum triglycerides. The discovery of associated variants in unsuspected genes and outside coding regions illustrates the ability of genome-wide association studies to provide potentially important clues to the pathogenesis of common diseases.