A Partial Loss-of-Function Variant in AKT2 Is Associated With Reduced Insulin-Mediated Glucose Uptake in Multiple Insulin-Sensitive Tissues: A Genotype-Based Callback Positron Emission Tomography Study.

Rare fully penetrant mutations in AKT2 are an established cause of monogenic disorders of glucose metabolism. Recently, a novel partial loss-of-function AKT2 coding variant (p.Pro50Thr) was identified that is nearly specific to Finns (frequency 1.1%), with the low-frequency allele associated with an increase in fasting plasma insulin level and risk of type 2 diabetes. The effects of the p.Pro50Thr AKT2 variant (p.P50T/AKT2) on insulin-stimulated glucose uptake (GU) in the whole body and in different tissues have not previously been investigated. We identified carriers (N = 20) and matched noncarriers (N = 25) for this allele in the population-based Metabolic Syndrome in Men (METSIM)study and invited these individuals back for positron emission tomography study with [18F]-fluorodeoxyglucose during euglycemic hyperinsulinemia. When we compared p.P50T/AKT2 carriers to noncarriers, we found a 39.4% reduction in whole-body GU (P = 0.006) and a 55.6% increase in the rate of endogenous glucose production (P = 0.038). We found significant reductions in GU in multiple tissues-skeletal muscle (36.4%), liver (16.1%), brown adipose (29.7%), and bone marrow (32.9%)-and increases of 16.8-19.1% in seven tested brain regions. These data demonstrate that the p.P50T substitution of AKT2 influences insulin-mediated GU in multiple insulin-sensitive tissues and may explain, at least in part, the increased risk of type 2 diabetes in p.P50T/AKT2 carriers.

Heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.

Finding new causes of monogenic diabetes helps understand glycaemic regulation in humans. To find novel genetic causes of maturity-onset diabetes of the young (MODY), we sequenced MODY cases with unknown aetiology and compared variant frequencies to large public databases. From 36 European patients, we identify two probands with novel RFX6 heterozygous nonsense variants. RFX6 protein truncating variants are enriched in the MODY discovery cohort compared to the European control population within ExAC (odds ratio = 131, P = 1 × 10-4). We find similar results in non-Finnish European (n = 348, odds ratio = 43, P = 5 × 10-5) and Finnish (n = 80, odds ratio = 22, P = 1 × 10-6) replication cohorts. RFX6 heterozygotes have reduced penetrance of diabetes compared to common HNF1A and HNF4A-MODY mutations (27, 70 and 55% at 25 years of age, respectively). The hyperglycaemia results from beta-cell dysfunction and is associated with lower fasting and stimulated gastric inhibitory polypeptide (GIP) levels. Our study demonstrates that heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.Maturity-onset diabetes of the young (MODY) is the most common subtype of familial diabetes. Here, Patel et al. use targeted DNA sequencing of MODY patients and large-scale publically available data to show that RFX6 heterozygous protein truncating variants cause reduced penetrance MODY.

The Metabolic Syndrome in Men study: a resource for studies of metabolic and cardiovascular diseases.

The Metabolic Syndrome in Men (METSIM) study is a population-based study including 10,197 Finnish men examined in 2005-2010. The aim of the study is to investigate nongenetic and genetic factors associated with the risk of T2D and CVD, and with cardiovascular risk factors. The protocol includes a detailed phenotyping of the participants, an oral glucose tolerance test, fasting laboratory measurements including proton NMR measurements, mass spectometry metabolomics, adipose tissue biopsies from 1,400 participants, and a stool sample. In our ongoing follow-up study, we have, to date, reexamined 6,496 participants. Extensive genotyping and exome sequencing have been performed for essentially all METSIM participants, and >2,000 METSIM participants have been whole-genome sequenced. We have identified several nongenetic markers associated with the development of diabetes and cardiovascular events, and participated in several genetic association studies to identify gene variants associated with diabetes, hyperglycemia, and cardiovascular risk factors. The generation of a phenotype and genotype resource in the METSIM study allows us to proceed toward a "systems genetics" approach, which includes statistical methods to quantitate and integrate intermediate phenotypes, such as transcript, protein, or metabolite levels, to provide a global view of the molecular architecture of complex traits.

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.

A genomic approach to therapeutic target validation identifies a glucose-lowering GLP1R variant protective for coronary heart disease.

Regulatory authorities have indicated that new drugs to treat type 2 diabetes (T2D) should not be associated with an unacceptable increase in cardiovascular risk. Human genetics may be able to guide development of antidiabetic therapies by predicting cardiovascular and other health endpoints. We therefore investigated the association of variants in six genes that encode drug targets for obesity or T2D with a range of metabolic traits in up to 11,806 individuals by targeted exome sequencing and follow-up in 39,979 individuals by targeted genotyping, with additional in silico follow-up in consortia. We used these data to first compare associations of variants in genes encoding drug targets with the effects of pharmacological manipulation of those targets in clinical trials. We then tested the association of those variants with disease outcomes, including coronary heart disease, to predict cardiovascular safety of these agents. A low-frequency missense variant (Ala316Thr; rs10305492) in the gene encoding glucagon-like peptide-1 receptor (GLP1R), the target of GLP1R agonists, was associated with lower fasting glucose and T2D risk, consistent with GLP1R agonist therapies. The minor allele was also associated with protection against heart disease, thus providing evidence that GLP1R agonists are not likely to be associated with an unacceptable increase in cardiovascular risk. Our results provide an encouraging signal that these agents may be associated with benefit, a question currently being addressed in randomized controlled trials. Genetic variants associated with metabolic traits and multiple disease outcomes can be used to validate therapeutic targets at an early stage in the drug development process.

Glucose-Dependent Insulinotropic Polypeptide Stimulates Osteopontin Expression in the Vasculature via Endothelin-1 and CREB.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with extrapancreatic effects beyond glycemic control. Here we demonstrate unexpected effects of GIP signaling in the vasculature. GIP induces the expression of the proatherogenic cytokine osteopontin (OPN) in mouse arteries via local release of endothelin-1 and activation of CREB. Infusion of GIP increases plasma OPN concentrations in healthy individuals. Plasma endothelin-1 and OPN concentrations are positively correlated in patients with critical limb ischemia. Fasting GIP concentrations are higher in individuals with a history of cardiovascular disease (myocardial infarction, stroke) when compared with control subjects. GIP receptor (GIPR) and OPN mRNA levels are higher in carotid endarterectomies from patients with symptoms (stroke, transient ischemic attacks, amaurosis fugax) than in asymptomatic patients, and expression associates with parameters that are characteristic of unstable and inflammatory plaques (increased lipid accumulation, macrophage infiltration, and reduced smooth muscle cell content). While GIPR expression is predominantly endothelial in healthy arteries from humans, mice, rats, and pigs, remarkable upregulation is observed in endothelial and smooth muscle cells upon culture conditions, yielding a "vascular disease-like" phenotype. Moreover, the common variant rs10423928 in the GIPR gene is associated with increased risk of stroke in patients with type 2 diabetes.

Simvastatin Impairs Insulin Secretion by Multiple Mechanisms in MIN6 Cells.

Statins are widely used in the treatment of hypercholesterolemia and are efficient in the prevention of cardiovascular disease. Molecular mechanisms explaining statin-induced impairment in insulin secretion remain largely unknown. In the current study, we show that simvastatin decreased glucose-stimulated insulin secretion in mouse pancreatic MIN6 ß-cells by 59% and 79% (p<0.01) at glucose concentration of 5.5 mmol/l and 16.7 mmol/l, respectively, compared to control, whereas pravastatin did not impair insulin secretion. Simvastatin induced decrease in insulin secretion occurred through multiple targets. In addition to its established effects on ATP-sensitive potassium channels (p = 0.004) and voltage-gated calcium channels (p = 0.004), simvastatin suppressed insulin secretion stimulated by muscarinic M3 or GPR40 receptor agonists (Tak875 by 33%, p = 0.002; GW9508 by 77%, p = 0.01) at glucose level of 5.5 mmol/l, and inhibited calcium release from the endoplasmic reticulum. Impaired insulin secretion caused by simvastatin treatment were efficiently restored by GPR119 or GLP-1 receptor stimulation and by direct activation of cAMP-dependent signaling pathways with forskolin. The effects of simvastatin treatment on insulin secretion were not affected by the presence of hyperglycemia. Our observation of the opposite effects of simvastatin and pravastatin on glucose-stimulated insulin secretion is in agreement with previous reports showing that simvastatin, but not pravastatin, was associated with increased risk of incident diabetes.

The cancer-associated FGFR4-G388R polymorphism enhances pancreatic insulin secretion and modifies the risk of diabetes.

The fibroblast growth factor receptor 4 (FGFR4)-R388 single-nucleotide polymorphism has been associated with cancer risk and prognosis. Here we show that the FGFR4-R388 allele yields a receptor variant that preferentially promotes STAT3/5 signaling. This STAT activation transcriptionally induces Grb14 in pancreatic endocrine cells to promote insulin secretion. Knockin mice with the FGFR4 variant allele develop pancreatic islets that secrete more insulin, a feature that is reversed through Grb14 deletion and enhanced with FGF19 administration. We also show in humans that the FGFR4-R388 allele enhances islet function and may protect against type 2 diabetes. These data support a common genetic link underlying cancer and hyperinsulinemia.

Link between GIP and osteopontin in adipose tissue and insulin resistance.

Low-grade inflammation in obesity is associated with accumulation of the macrophage-derived cytokine osteopontin (OPN) in adipose tissue and induction of local as well as systemic insulin resistance. Since glucose-dependent insulinotropic polypeptide (GIP) is a strong stimulator of adipogenesis and may play a role in the development of obesity, we explored whether GIP directly would stimulate OPN expression in adipose tissue and thereby induce insulin resistance. GIP stimulated OPN protein expression in a dose-dependent fashion in rat primary adipocytes. The level of OPN mRNA was higher in adipose tissue of obese individuals (0.13 ± 0.04 vs. 0.04 ± 0.01, P < 0.05) and correlated inversely with measures of insulin sensitivity (r = -0.24, P = 0.001). A common variant of the GIP receptor (GIPR) (rs10423928) gene was associated with a lower amount of the exon 9-containing isoform required for transmembrane activity. Carriers of the A allele with a reduced receptor function showed lower adipose tissue OPN mRNA levels and better insulin sensitivity. Together, these data suggest a role for GIP not only as an incretin hormone but also as a trigger of inflammation and insulin resistance in adipose tissue. Carriers of the GIPR rs10423928 A allele showed protective properties via reduced GIP effects. Identification of this unprecedented link between GIP and OPN in adipose tissue might open new avenues for therapeutic interventions.

Exome array analysis identifies new loci and low-frequency variants influencing insulin processing and secretion.

Insulin secretion has a crucial role in glucose homeostasis, and failure to secrete sufficient insulin is a hallmark of type 2 diabetes. Genome-wide association studies (GWAS) have identified loci contributing to insulin processing and secretion; however, a substantial fraction of the genetic contribution remains undefined. To examine low-frequency (minor allele frequency (MAF) 0.5-5%) and rare (MAF < 0.5%) nonsynonymous variants, we analyzed exome array data in 8,229 nondiabetic Finnish males using the Illumina HumanExome Beadchip. We identified low-frequency coding variants associated with fasting proinsulin concentrations at the SGSM2 and MADD GWAS loci and three new genes with low-frequency variants associated with fasting proinsulin or insulinogenic index: TBC1D30, KANK1 and PAM. We also show that the interpretation of single-variant and gene-based tests needs to consider the effects of noncoding SNPs both nearby and megabases away. This study demonstrates that exome array genotyping is a valuable approach to identify low-frequency variants that contribute to complex traits.

Pleiotropic effects of GIP on islet function involve osteopontin.

OBJECTIVE: The incretin hormone GIP (glucose-dependent insulinotropic polypeptide) promotes pancreatic ß-cell function by potentiating insulin secretion and ß-cell proliferation. Recently, a combined analysis of several genome-wide association studies (Meta-analysis of Glucose and Insulin-Related Traits Consortium [MAGIC]) showed association to postprandial insulin at the GIP receptor (GIPR) locus. Here we explored mechanisms that could explain the protective effects of GIP on islet function. RESEARCH DESIGN AND METHODS: Associations of GIPR rs10423928 with metabolic and anthropometric phenotypes in both nondiabetic (N = 53,730) and type 2 diabetic individuals (N = 2,731) were explored by combining data from 11 studies. Insulin secretion was measured both in vivo in nondiabetic subjects and in vitro in islets from cadaver donors. Insulin secretion was also measured in response to exogenous GIP. The in vitro measurements included protein and gene expression as well as measurements of ß-cell viability and proliferation. RESULTS: The A allele of GIPR rs10423928 was associated with impaired glucose- and GIP-stimulated insulin secretion and a decrease in BMI, lean body mass, and waist circumference. The decrease in BMI almost completely neutralized the effect of impaired insulin secretion on risk of type 2 diabetes. Expression of GIPR mRNA was decreased in human islets from carriers of the A allele or patients with type 2 diabetes. GIP stimulated osteopontin (OPN) mRNA and protein expression. OPN expression was lower in carriers of the A allele. Both GIP and OPN prevented cytokine-induced reduction in cell viability (apoptosis). In addition, OPN stimulated cell proliferation in insulin-secreting cells. CONCLUSIONS: These findings support ß-cell proliferative and antiapoptotic roles for GIP in addition to its action as an incretin hormone. Identification of a link between GIP and OPN may shed new light on the role of GIP in preservation of functional ß-cell mass in humans.

Impact of positive family history and genetic risk variants on the incidence of diabetes: the Finnish Diabetes Prevention Study.

OBJECTIVE: We aimed to investigate the influence of positive family history (FH+) of diabetes and 19 known genetic risk loci on the effectiveness of lifestyle changes and their predictive value on the incidence of type 2 diabetes in the Finnish Diabetes Prevention Study (DPS). RESEARCH DESIGN AND METHODS: A total of 522 subjects with impaired glucose tolerance (IGT) were randomized into the control (n = 257) and intervention (n = 265) groups. The mean follow-up was 6.2 years (median 7 years), and the lifestyle intervention, aimed at weight reduction, healthy diet, and increased physical activity, lasted for 4 years (range 1-6 years). An oral glucose tolerance test (OGTT) and assessment of basic clinical variables were performed annually. RESULTS: The effect of intervention on the incidence of diabetes was almost similar in subjects with FH+ compared with subjects with a negative family history (FH-) of diabetes during the entire follow-up. In the Cox model, including FH, genetic risk SNPs, and randomization group, and adjusted for the effects of age, sex, BMI, and study center, only lifestyle intervention had a significant effect (hazard ratio 0.55, 95% CI 0.41-0.75, P < 0.001) on the incidence of diabetes. Further analyses showed that in addition to the baseline glucose and insulin values, 1-year changes in 2-h glucose and 2-h insulin achieved by lifestyle intervention had a significant effect on the incidence of diabetes. CONCLUSIONS: These results emphasize the effectiveness of lifestyle intervention in reducing the risk of diabetes in high-risk individuals independently of genetic or familial risk of type 2 diabetes.

Association of common genetic variation in the FOXO1 gene with beta-cell dysfunction, impaired glucose tolerance, and type 2 diabetes.

CONTEXT: The transcription factor forkhead box protein (FOX) O1A plays a crucial role in regulation of beta-cell function and metabolic effects of insulin in the liver. OBJECTIVE: The objective of the study was to investigate whether common genetic variation within the FOXO1 gene encoding FOXO1A contributes to prediabetic phenotypes, such as insulin resistance or beta-cell dysfunction, and to risk of type 2 diabetes. DESIGN AND SETTINGS: Study I was a study enrolling thoroughly phenotyped subjects from Germany at increased risk for type 2 diabetes. Study II was a population-based study of Finnish men for the assessment of the prevalence of type 2 diabetes and metabolic syndrome. PARTICIPANTS: Study I included 941 nondiabetic subjects (353 males, 588 females, aged 39 +/- 1 yr, body mass index 29.2 +/- 0.3 kg/m(2)). Study II included 5957 middle-aged men (870 type 2 diabetic and 5087 nondiabetic subjects). INTERVENTIONS: Genotyping for 10 single-nucleotide polymorphisms (SNPs) covering 100% of common genetic variation (minor allele frequency >or=10%) within the FOXO1 gene (r(2) >or= 0.8) based on HapMap data, oral glucose tolerance test, and in a subset additionally a hyperinsulinemic-euglycemic clamp. MAIN OUTCOME MEASUREMENTS: Parameters of insulin secretion, insulin resistance, and glucose tolerance status were measured. RESULTS: In the German subjects at increased risk for type 2 diabetes, SNPs rs2721068 and rs17446614 were significantly (P = 0.0045 and P = 0.0018, respectively) and SNPs rs17446593 and rs2297627 were nominally (P = 0.0091 and P = 0.0387, respectively) associated with beta-cell dysfunction. rs2721068, rs17446614, and rs2297627 were also nominally associated with impaired glucose tolerance (P = 0.0264, P = 0.0162, and P = 0.0221, respectively). Minor allele carriers showed reduced insulin secretion and elevated glucose levels during an oral glucose tolerance test. Investigating the relevance of our findings in a separate cohort, we found that SNP rs2721068 was significantly associated with type 2 diabetes in the additive (P = 0.002) and dominant model (P = 0.009) in Finnish men. CONCLUSIONS: Common genetic variation within the FOXO1 gene affects insulin secretion and glucose tolerance and associates with an increased risk of type 2 diabetes.