Gene x Gene Interactions Highlight the Role of Incretin Resistance for Insulin Secretion.

Introduction: Genetic polymorphisms in TCF7L2 are the strongest common risk variants for type 2 diabetes mellitus (T2D). We and others have shown that genetic variation in TCF7L2 and WFS1 affect incretin-stimulated insulin secretion. A recent genome-wide association study discovered genetic variants associated with incretin levels. We hypothesized that these SNPs (single nucleotide polymorphisms) interact with the well-known TCF7L2 variant rs7903146 on insulin secretion due to their incretin altering effect. Methods: In this retrospective analysis, we used data from the cross-sectional TUEF-cohort (n = 2929) and a hyperglycemic clamp study using additional GLP-1 infusion at the end of the clamp (n = 76). Insulin secretion was measured by evaluating OGTT-derived indexes of insulin secretion and insulin/C-peptide levels during clamp. We genotyped rs7903146 in TCF7L2, rs10010131 in WFS1, and six SNPs associated with GLP-1 and GIP levels. Results: One of the six incretin-associated SNPs, rs17681684 in GLP2R, exhibited significant SNP x SNP interactions with rs7903146 in TCF7L2 on insulin secretion (p = 0.0024) after correction for multiple testing. Three further SNP's showed nominally significant interactions (p < 0.05). In the hyperglycemic clamp study, rs7903146 in TCF7L2 also interacted with rs17681684 on AUC C-peptide during the GLP-1 stimulation phase, thereby replicating the above finding. Conclusion: The findings exemplify the role of SNP x SNP interactions in the genetics of type 2 diabetes mellitus and corroborate the existence of clinically relevant differences in incretin sensitivity.

A Polygenic Risk Score of Lipolysis-Increasing Alleles Determines Visceral Fat Mass and Proinsulin Conversion.

CONTEXT: Primary dysregulation of adipose tissue lipolysis caused by genetic variation and independent of insulin resistance could explain unhealthy body fat distribution and its metabolic consequences. OBJECTIVE: To analyze common single nucleotide polymorphisms (SNPs) in 48 lipolysis-, but not insulin-signaling-related genes, to form polygenic risk scores of lipolysis-associated SNPs, and to investigate their effects on body fat distribution, glycemia, insulin sensitivity, insulin secretion, and proinsulin conversion. STUDY DESIGN, PARTICIPANTS, AND METHODS: SNP array, anthropometric, and metabolic data were available from up to 2789 participants without diabetes of the Tübingen Family study of type 2 diabetes characterized by oral glucose tolerance tests. In a subgroup (n = 942), magnetic resonance measurements of body fat stores were available. RESULTS: We identified insulin-sensitivity-independent nominal associations (P < 0.05) of SNPs in 10 genes with plasma free fatty acids (FFAs), in 7 genes with plasma glycerol and in 6 genes with both, plasma FFAs and glycerol. A score formed of the latter SNPs (in ADCY4, CIDEA, GNAS, PDE8B, PRKAA1, PRKAG2) was associated with plasma FFA and glycerol measurements (1.4*10-9 ≤ P ≤ 1.2*10-5), visceral adipose tissue mass (P = 0.0326), and proinsulin conversion (P ≤ 0.0272). The more lipolysis-increasing alleles a subject had, the lower was the visceral fat mass and the lower the proinsulin conversion. CONCLUSIONS: We found evidence for a genetic basis of adipose tissue lipolysis resulting from common SNPs in CIDEA, AMP-activated protein kinase subunits, and cAMP signaling components. A genetic score of lipolysis-increasing alleles determined lower visceral fat mass and lower proinsulin conversion.

Single Nucleotide Polymorphisms in the G-Protein Coupled Receptor Kinase 5 (GRK5) Gene are associated with Plasma LDL-Cholesterol Levels in Humans.

Genetically modified mice models suggest an important role for G-protein-coupled receptor kinase 5 (GRK5) in the pathophysiology of obesity and related disorders. We investigated whether single nucleotide polymorphisms (SNPs) in the gene encoding GRK5 affect cardiometabolic traits in humans. We genotyped 3 common SNPs in intron 1 (rs1980030, rs10466210, rs9325562) and one SNP in intron 3 (rs10886471) of GRK5 in 2332 subjects at risk for type 2 diabetes. Total- and visceral fat mass were measured by magnetic resonance (MR) tomography and liver fat content by 1H-MR spectroscopy. Insulin secretion and sensitivity were estimated during an OGTT and measured during the euglycemic, hyperinsulinemic clamp (n = 498). Carriers of the minor allele of rs10466210 and rs1980030 had higher total- and LDL-cholesterol levels (p = 0.0018 and p = 0.0031, respectively, for rs10466210; p = 0.0035 and p = 0.0081, respectively, for rs1980030), independently of gender, age, BMI and lipid-lowering drugs. The effects of rs10466210 withstood Bonferroni correction. Similar associations were observed with apolipoprotein B levels (p = 0.0034 and p = 0.0122, respectively). Carriers of the minor allele of rs10466210 additionally displayed a trend for higher intima-media thickness of the carotid artery (p = 0.075). GRK5 may represent a novel target for strategies aiming at lowering LDL-cholesterol levels and at modifying cardiovascular risk.

Genome-wide analysis of PDX1 target genes in human pancreatic progenitors.

OBJECTIVE: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing ß-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult ß-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. METHODS: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. RESULTS: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult ß-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. CONCLUSIONS: Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a ß-cell developmental program.

Excessive fuel availability amplifies the FTO-mediated obesity risk: results from the TUEF and Whitehall II studies.

Variation in FTO is the most important common genetic determinant of body weight. Altered energy metabolism could underlie this association. We hypothesized that higher circulating glucose or triglycerides can amplify the FTO impact on BMI. In 2671 subjects of the TUEF study, we investigated the interaction effect of fasting glucose and triglyceride levels with rs9939609 in FTO on BMI. We analysed the same interaction effect by longitudinally utilizing mixed effect models in the prospective Whitehall II study. In TUEF, we detected an interaction effect between fasting glucose and fasting triglycerides with rs9939609 on BMI (p = 0.0005 and p = 5 × 10-7, respectively). The effect size of one risk allele was 1.4 ± 0.3 vs. 2.2 ± 0.44 kg/m² in persons with fasting glucose levels below and above the median, respectively. Fasting triglycerides above the median increased the per-allele effect from 1.4 ± 0.3 to 1.7 ± 0.4 kg/m2. In the Whitehall II study, body weight increased by 2.96 ± 6.5 kg during a follow-up of 13.5 ± 4.6 yrs. Baseline fasting glucose and rs9939609 interacted on weight change (p = 0.009). Higher fasting glucose levels may amplify obesity-risk in FTO carriers and lead to an exaggerated weight gain over time. Since weight gain perpetuates metabolic alterations, this interplay may trigger a vicious circle that leads to obesity and diabetes.

DPP4 gene variation affects GLP-1 secretion, insulin secretion, and glucose tolerance in humans with high body adiposity.

OBJECTIVE: Dipeptidyl-peptidase 4 (DPP-4) cleaves and inactivates the insulinotropic hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide, collectively termed incretins. DPP-4 inhibitors entered clinical practice as approved therapeutics for type-2 diabetes in 2006. However, inter-individual variance in the responsiveness to DPP-4 inhibitors was reported. Thus, we asked whether genetic variation in the DPP4 gene affects incretin levels, insulin secretion, and glucose tolerance in participants of the TÜbingen Family study for type-2 diabetes (TÜF). RESEARCH DESIGN AND METHODS: Fourteen common (minor allele frequencies ≥0.05) DPP4 tagging single nucleotide polymorphisms (SNPs) were genotyped in 1,976 non-diabetic TÜF participants characterized by oral glucose tolerance tests and bioimpedance measurements. In a subgroup of 168 subjects, plasma incretin levels were determined. RESULTS: We identified a variant, i.e., SNP rs6741949, in intron 2 of the DPP4 gene that, after correction for multiple comparisons and appropriate adjustment, revealed a significant genotype-body fat interaction effect on glucose-stimulated plasma GLP-1 levels (p = 0.0021). Notably, no genotype-BMI interaction effects were detected (p = 0.8). After stratification for body fat content, the SNP negatively affected glucose-stimulated GLP-1 levels (p = 0.0229), insulin secretion (p = 0.0061), and glucose tolerance (p = 0.0208) in subjects with high body fat content only. CONCLUSIONS: A common variant, i.e., SNP rs6741949, in the DPP4 gene interacts with body adiposity and negatively affects glucose-stimulated GLP-1 levels, insulin secretion, and glucose tolerance. Whether this SNP underlies the reported inter-individual variance in responsiveness to DPP-4 inhibitors, at least in subjects with high body fat content, remains to be shown.

Common variation in the sodium/glucose cotransporter 2 gene SLC5A2 does neither affect fasting nor glucose-suppressed plasma glucagon concentrations.

AIM: Inhibition of sodium/glucose cotransporter 2 (SGLT2), the key transport protein in renal glucose reabsorption, promotes glucose excretion and represents a new concept in the therapy of type-2 diabetes. In addition, SGLT2 inhibition elevates circulating glucagon concentrations and enhances hepatic glucose production. Since SGLT2 is expressed in human pancreatic α-cells and regulates glucagon release, we tested whether common variants of the SGLT2 gene SLC5A2 associate with altered plasma glucagon concentrations in the fasting state and upon glucose challenge. METHODS: A study population of 375 healthy subjects at increased risk for type-2 diabetes, phenotyped by a 5-point oral glucose tolerance test (OGTT) and genotyped for recently described SLC5A2 tagging single nucleotide polymorphisms (SNPs), was selected for plasma glucagon measurements. RESULTS: After adjustment for gender, age, body mass index, and insulin sensitivity, the four tagging SNPs (rs9924771, rs3116150, rs3813008, rs9934336), tested separately or as genetic score, were neither significantly nor nominally associated with plasma glucagon concentrations at any time during the OGTT, with the inverse AUC of glucagon or the glucagon fold-change during the OGTT (p ≥ 0.2, all). Testing for genotype-related differences in the time course of the glucagon response using MANOVA did also not reveal any significant or nominal associations (p ≥ 0.5, all). CONCLUSION: We could not obtain statistically significant evidence for a role of common SLC5A2 variants in the regulation of glucagon release in the fasting state or upon glucose challenge. Moreover, the reported nominal effects of individual SLC5A2 variants on fasting and post-challenge glucose levels may probably not be mediated by altered glucagon release.

Genetic determination of body fat distribution and the attributive influence on metabolism.

OBJECTIVE: Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with estimates of body fat distribution. Using predefined risk allele scores, the correlation of these scores with precisely quantified body fat distribution assessed by magnetic resonance (MR) imaging techniques and with metabolic traits was investigated. METHODS: Data from 4,944 MR scans from 915 subjects of European ancestry were analyzed. Body fat distribution was determined by MR imaging and liver fat content by 1 H-MR spectroscopy. All subjects underwent a five-point 75-g oral glucose tolerance test. A total of 65 SNPs with reported genome-wide significant associations regarding estimates of body fat distribution were genotyped. Four genetic risk scores were created by summation of risk alleles. RESULTS: A higher allelic load of waist-to-hip ratio SNPs was associated with lower insulin sensitivity, higher postchallenge glucose levels, and more visceral and less subcutaneous fat mass. CONCLUSIONS: GWAS-derived polymorphisms estimating body fat distribution are associated with distinct patterns of body fat distribution exactly measured by MR. Only the risk score associated with the waist-to-hip ratio in GWAS showed an unhealthy pattern of metabolism and body fat distribution. This score might be useful for predicting diseases associated with genetically determined, unhealthy obesity.

Influence of common polymorphisms in the SLC5A2 gene on metabolic traits in subjects at increased risk of diabetes and on response to empagliflozin treatment in patients with diabetes.

OBJECTIVE: Inhibition of the renal sodium-glucose cotransporter 2 (SGLT2) is a novel concept in the therapy of diabetes mellitus. In this study, we first assessed whether common single nucleotide polymorphisms (SNPs) in the SGLT2-encoding gene SLC5A2 affect diabetes-related metabolic traits in subjects at risk for type 2 diabetes and, second, whether these have pharmacogenetic relevance by interfering with the response to empagliflozin treatment in patients with type 2 diabetes. PATIENTS AND METHODS: Samples from a metabolically well-phenotyped cross-sectional study population (total N=2600) at increased risk for type 2 diabetes and pooled pharmacogenetic samples from patients from four phase III trials of empagliflozin (in total: 603 receiving empagliflozin, 305 receiving placebo) were genotyped for five common SNPs (minor allele frequencies ≥5%) present in the SLC5A2 gene locus. RESULTS: In the cross-sectional study, none of the SLC5A2 SNPs significantly influenced metabolic traits such as body fat, insulin sensitivity/resistance, insulin release, HbA1c, plasma glucose, or systolic blood pressure when multiple testing was taken into account (all P≥0.0083). Further, no relevant effect on response to treatment with empagliflozin on HbA1c, fasting glucose, weight, or systolic blood pressure was observed for the SNPs tested in the pharmacogenetic study. CONCLUSION: Common genetic variants in the SLC5A2 gene neither affects diabetes-related metabolic traits nor have a clinically relevant impact on response to treatment with the SGLT2 inhibitor empagliflozin.

Generation of a human induced pluripotent stem cell (iPSC) line from a patient carrying a P33T mutation in the PDX1 gene.

Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4) and Type 2 Diabetes Mellitus (T2DM). To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a P33T heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 P33T iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms.

Generation of a human induced pluripotent stem cell (iPSC) line from a patient with family history of diabetes carrying a C18R mutation in the PDX1 gene.

Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4) and Type 2 Diabetes Mellitus (T2DM). To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a C18R heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 C18R iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms.

Genetic Variation in the 11ß-hydroxysteroid-dehydrogenase 1 Gene Determines NAFLD and Visceral Obesity.

CONTEXT/OBJECTIVE: Acute pharmacological inhibition of 11ß-hydroxysteroid-dehydrogenase 1 (11ß-HSD1), which converts cortisone into the much more potent cortisol in peripheral tissues, results in reduction of total, visceral, and liver fat but not insulin resistance. We now investigated whether lifelong alterations of 11ß-HSD1 activity similarly affect these cardiometabolic risk parameters by studying single-nucleotide polymorphisms (SNPs) in the 11ß-HSD1-coding gene (HSD11B1). DESIGN/METHODS: Liver fat content was measured by 1H-magnetic resonance spectroscopy and total and visceral fat mass by 1H-magnetic resonance tomography in 327 subjects. Insulin sensitivity (IS) was estimated during an oral glucose tolerance test and the euglycemic, hyperinsulinemic clamp (n = 219). Nine SNPs covering the whole HSD11B1 gene were genotyped. RESULTS: After correction for multiple testing, liver fat content strongly correlated with three SNPs, rs2235543, rs12565406, and rs4844880 (P = .0002, P = .001, and P = .0009, respectively), independently of gender and age. There was a nominal association of these SNPs with hepatic IS but only of rs4844880 with whole-body IS. Subjects homozygous for the major allele had an adjusted odds ratio of 2.16 (95% confidence interval [CI] 1.23-3.90) for rs2235543, 2.06 (95% CI 1.08-4.13) for rs12565406, and 1.95 (95% CI 1.13-3.49) for rs4844880 for having nonalcoholic fatty liver disease compared with carriers of the minor allele. Less strong associations of these SNPs with visceral fat mass were observed. In liver biopsies, carriers of the minor alleles of rs2235543 and rs12565406 had significantly lower HSD11B1 mRNA expression (n = 105, P = .034 and P = .0086, respectively). CONCLUSIONS: 11ß-HSD1 may be an important enzyme in the pathogenesis of fatty liver and visceral obesity and a promising target for their treatment.

Interaction between the obesity-risk gene FTO and the dopamine D2 receptor gene ANKK1/TaqIA on insulin sensitivity.

AIMS/HYPOTHESIS: Variations in FTO are the strongest common genetic determinants of adiposity, and may partly act by influencing dopaminergic signalling in the brain leading to altered reward processing that promotes increased food intake. Therefore, we investigated the impact of such an interaction on body composition, and peripheral and brain insulin sensitivity. METHODS: Participants from the Tübingen Family study (n = 2245) and the Malmö Diet and Cancer study (n = 2921) were genotyped for FTO SNP rs8050136 and ANKK1 SNP rs1800497. Insulin sensitivity in the caudate nucleus, an important reward area in the brain, was assessed by fMRI in 45 participants combined with intranasal insulin administration. RESULTS: We found evidence of an interaction between variations in FTO and an ANKK1 polymorphism that associates with dopamine (D2) receptor density. In cases of reduced D2 receptor availability, as indicated by the ANKK1 polymorphism, FTO variation was associated with increased body fat and waist circumference and reduced peripheral insulin sensitivity. Similarly, altered central insulin sensitivity was observed in the caudate nucleus in individuals with the FTO obesity-risk allele and diminished D2 receptors. CONCLUSIONS/INTERPRETATION: The effects of variations in FTO are dependent on dopamine D2 receptor density (determined by the ANKK1 polymorphism). Carriers of both risk alleles might, therefore, be at increased risk of obesity and diabetes.

[A rare cause of diabetes mellitus]. / Eine seltene Ursache für einen Diabetes mellitus - Fall 5 / 2016.

UNLABELLED: Medical history and clinical examination: We report on a 52-year-old lean female patient with diabetes mellitus, treated with various glucose-lowering medications including insulin for 6 years. Both her mother and great-grandmother were also diagnosed with diabetes. At admission, she presents with good glycemic control and no signs of diabetes-related comorbidities. LABORATORY TESTS: HbA1c is 6 %. On the basis of positive family history for diabetes, normal body weight and negative antibodies against islet-cell antigens, we perform blood molecular genetic analyses with the suspicion of a genetic form of diabetes. Genetic tests reveal a monogenic form of diabetes (MODY, Maturity Onset Diabetes of the Young) due to an heterozygous mutation in exon 6 of the glucokinase (GCK) gene. The same mutation is subsequently detected in the patient's mother. DIAGNOSIS, TREATMENT AND COURSE: We diagnose MODY2 diabetes due to a glucokinase gene mutation. Repaglinide therapy is withdrawn due to lack of solid literature data showing beneficial cardiometabolic outcomes in patients with diabetes. Based on the MODY diagnosis, we recommend sulfonylurea treatment, which is however rejected by the patient due to a history of hypoglycemic episodes under the lowest dose. Thus, sitagliptin-treatment is continued under regular visits in our outpatient clinic. CONCLUSIONS: The joint presence of a positive family history, age under 25 years at diagnosis of diabetes, normal bodyweight, negative diabetes-specific autoantibodies and mild hyperglycaemia should raise the suspicion of a monogenic form of diabetes such as MODY and result in molecular-genetic testing.

Identification of proliferative and mature ß-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of ß-cells. Pancreatic ß-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature ß-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger ß-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for ß-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional ß-cell heterogeneity and induce ß-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional ß-cell mass in diabetic patients.

Genome-Wide Association Study of the Modified Stumvoll Insulin Sensitivity Index Identifies BCL2 and FAM19A2 as Novel Insulin Sensitivity Loci.

Genome-wide association studies (GWAS) have found few common variants that influence fasting measures of insulin sensitivity. We hypothesized that a GWAS of an integrated assessment of fasting and dynamic measures of insulin sensitivity would detect novel common variants. We performed a GWAS of the modified Stumvoll Insulin Sensitivity Index (ISI) within the Meta-Analyses of Glucose and Insulin-Related Traits Consortium. Discovery for genetic association was performed in 16,753 individuals, and replication was attempted for the 23 most significant novel loci in 13,354 independent individuals. Association with ISI was tested in models adjusted for age, sex, and BMI and in a model analyzing the combined influence of the genotype effect adjusted for BMI and the interaction effect between the genotype and BMI on ISI (model 3). In model 3, three variants reached genome-wide significance: rs13422522 (NYAP2; P = 8.87 × 10(-11)), rs12454712 (BCL2; P = 2.7 × 10(-8)), and rs10506418 (FAM19A2; P = 1.9 × 10(-8)). The association at NYAP2 was eliminated by conditioning on the known IRS1 insulin sensitivity locus; the BCL2 and FAM19A2 associations were independent of known cardiometabolic loci. In conclusion, we identified two novel loci and replicated known variants associated with insulin sensitivity. Further studies are needed to clarify the causal variant and function at the BCL2 and FAM19A2 loci.

FTO Genotype Interacts with Improvement in Aerobic Fitness on Body Weight Loss During Lifestyle Intervention.

OBJECTIVE: Not every participant responds with a comparable body weight loss to lifestyle intervention, despite the same compliance. Genetic factors may explain parts of this difference. Variation in fat mass and obesity-associated gene (FTO) is the strongest common genetic determinant of body weight. The aim of the present study was to evaluate the impact of FTO genotype differences in the link between improvement of fitness and reduction of body weight during a lifestyle intervention. METHODS: We genotyped 292 healthy subjects for FTO rs8050136. Participants underwent a 9-month lifestyle intervention. Before and after intervention, aerobic fitness was tested by bicycle (VO2max) and treadmill spiroergometry (individual anaerobic threshold (IAT), subgroup of N = 192). RESULTS: Participants lost body weight (p < 0.0001) independent of FTO genotype (p = 0.5). There was a significant correlation between improvement in VO2max and decrease in body weight (p < 0.0001). FTO genotype interacted with this relationship (p = 0.0042 for VO2max, p = 0.0049 for IAT). When stratifying the cohort according to their improvement in VO2max, FTO obesity-risk A-allele carriers in the higher quartiles of improvement in fitness lost significantly less body weight. CONCLUSIONS: Our data reveal that genetic variation in FTO impacts on body weight reduction during lifestyle intervention only in subjects with marked improvement in aerobic fitness.

Granulocyte colony-stimulating factor (G-CSF): A saturated fatty acid-induced myokine with insulin-desensitizing properties in humans.

OBJECTIVE: Circulating long-chain free fatty acids (FFAs) are important metabolic signals that acutely enhance fatty acid oxidation, thermogenesis, energy expenditure, and insulin secretion. However, if chronically elevated, they provoke inflammation, insulin resistance, and ß-cell failure. Moreover, FFAs act via multiple signaling pathways as very potent regulators of gene expression. In human skeletal muscle cells differentiated in vitro (myotubes), we have shown in previous studies that the expression of CSF3, the gene encoding granulocyte colony-stimulating factor (G-CSF), is markedly induced upon FFA treatment and exercise. METHODS AND RESULTS: We now report that CSF3 is induced in human myotubes by saturated, but not unsaturated, FFAs via Toll-like receptor 4-dependent and -independent pathways including activation of Rel-A, AP-1, C/EBPα, Src, and stress kinases. Furthermore, we show that human adipocytes and myotubes treated with G-CSF become insulin-resistant. In line with this, a functional polymorphism in the CSF3 gene affects adipose tissue- and whole-body insulin sensitivity and glucose tolerance in human subjects with elevated plasma FFA concentrations. CONCLUSION: G-CSF emerges as a new player in FFA-induced insulin resistance and thus may be of interest as a target for prevention and treatment of type 2 diabetes.

Glucose-Raising Polymorphisms in the Human Clock Gene Cryptochrome 2 (CRY2) Affect Hepatic Lipid Content.

Circadian rhythms govern vital functions. Their disruption provokes metabolic imbalance favouring obesity and type-2 diabetes. The aim of the study was to assess the role of clock genes in human prediabetes. To this end, genotype-phenotype associations of 121 common single nucleotide polymorphisms (SNPs) tagging ARNTL, ARNTL2, CLOCK, CRY1, CRY2, PER1, PER2, PER3, and TIMELESS were assessed in a study population of 1,715 non-diabetic individuals metabolically phenotyped by 5-point oral glucose tolerance tests. In subgroups, hyperinsulinaemic-euglycaemic clamps, intravenous glucose tolerance tests, and magnetic resonance imaging/spectroscopy were performed. None of the tested SNPs was associated with body fat content, insulin sensitivity, or insulin secretion. Four CRY2 SNPs were associated with fasting glycaemia, as reported earlier. Importantly, carriers of these SNPs' minor alleles revealed elevated fasting glycaemia and, concomitantly, reduced liver fat content. In human liver tissue samples, CRY2 mRNA expression was directly associated with hepatic triglyceride content. Our data may point to CRY2 as a novel switch in hepatic fuel metabolism promoting triglyceride storage and, concomitantly, limiting glucose production. The anti-steatotic effects of the glucose-raising CRY2 alleles may explain why these alleles do not increase type-2 diabetes risk.

Variation in the Phosphoinositide 3-Kinase Gamma Gene Affects Plasma HDL-Cholesterol without Modification of Metabolic or Inflammatory Markers.

OBJECTIVE: Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic ß-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis. STUDY DESIGN: Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis. RESULTS: After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs' in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs. CONCLUSIONS: We could demonstrate that common genetic variation in the PIK3CG locus, possibly via altered PIK3CG gene expression, determines plasma HDL-cholesterol concentrations. Since HDL2-, but not HDL3-, cholesterol is influenced by PIK3CG variants, PI3Kγ may play a role in HDL clearance rather than in HDL biogenesis. Even though the molecular pathways connecting PI3Kγ and HDL metabolism remain to be further elucidated, this finding could add a novel aspect to the pathophysiological role of PI3Kγ in atherogenesis.