Small RNA sequencing reveals snoRNAs and piRNA-019825 as novel players in diabetic kidney disease.

INTRODUCTION: Micro- and macrovascular complications are common among persons with type 2 diabetes. Recently there has been growing interest to investigate the potential of circulating small non-coding RNAs (sncRNAs) as contributors to the development of diabetic complications. In this study we investigate to what extent circulating sncRNAs levels associate with prevalent diabetic kidney disease (DKD) in persons with type 2 diabetes. METHODS: Plasma sncRNAs levels were determined using small RNA-seq, allowing detection of miRNAs, snoRNAs, piRNAs, tRNA fragments, and various other sncRNA classes. We tested for differentially expressed sncRNAs in persons with type 2 diabetes, with DKD (n = 69) or without DKD (n = 405). In secondary analyses, we also tested the association with eGFR, albuminuria (UACR), and the plasma proteome. RESULTS: In total seven sncRNAs were negatively associated with prevalent DKD (all PFDR ≤ 0.05). Including one microRNA (miR-143-5p), five snoRNAs (U8, SNORD118, SNORD24, SNORD107, SNORD87) and a piRNA (piR-019825 | DQ597218). Proteomic analyses showed that the seven sncRNAs, and especially the piRNA piR-019825, were associated with plasma levels of 24 proteins of which several have known associations with kidney function including TNF sR-I (TNFRFS1A), DAN (NBL1) and cystatin C (CST3). CONCLUSION: We have identified novel small non-coding RNAs, primarily from classes other than microRNAs, that are associated with diabetic kidney disease. Our results show that the involvement of small non-coding RNAs in DKD goes beyond the already known microRNAs and also involves other classes of sncRNA, in particular snoRNAs and the piRNA piR-019825, that have never been studied before in relation to kidney function.

Clinical profiles of post-load glucose subgroups and their association with glycaemic traits over time: An IMI-DIRECT study.

AIM: To examine the hypothesis that, based on their glucose curves during a seven-point oral glucose tolerance test, people at elevated type 2 diabetes risk can be divided into subgroups with different clinical profiles at baseline and different degrees of subsequent glycaemic deterioration. METHODS: We included 2126 participants at elevated type 2 diabetes risk from the Diabetes Research on Patient Stratification (IMI-DIRECT) study. Latent class trajectory analysis was used to identify subgroups from a seven-point oral glucose tolerance test at baseline and follow-up. Linear models quantified the associations between the subgroups with glycaemic traits at baseline and 18 months. RESULTS: At baseline, we identified four glucose curve subgroups, labelled in order of increasing peak levels as 1-4. Participants in Subgroups 2-4, were more likely to have higher insulin resistance (homeostatic model assessment) and a lower Matsuda index, than those in Subgroup 1. Overall, participants in Subgroups 3 and 4, had higher glycaemic trait values, with the exception of the Matsuda and insulinogenic indices. At 18 months, change in homeostatic model assessment of insulin resistance was higher in Subgroup 4 (ß = 0.36, 95% CI 0.13-0.58), Subgroup 3 (ß = 0.30; 95% CI 0.10-0.50) and Subgroup 2 (ß = 0.18; 95% CI 0.04-0.32), compared to Subgroup 1. The same was observed for C-peptide and insulin. Five subgroups were identified at follow-up, and the majority of participants remained in the same subgroup or progressed to higher peak subgroups after 18 months. CONCLUSIONS: Using data from a frequently sampled oral glucose tolerance test, glucose curve patterns associated with different clinical characteristics and different rates of subsequent glycaemic deterioration can be identified.

Variants in Pharmacokinetic Transporters and Glycemic Response to Metformin: A Metgen Meta-Analysis.

Therapeutic response to metformin, a first-line drug for type 2 diabetes (T2D), is highly variable, in part likely due to genetic factors. To date, metformin pharmacogenetic studies have mainly focused on the impact of variants in metformin transporter genes, with inconsistent results. To clarify the significance of these variants in glycemic response to metformin in T2D, we performed a large-scale meta-analysis across the cohorts of the Metformin Genetics Consortium (MetGen). Nine candidate polymorphisms in five transporter genes (organic cation transporter [OCT]1, OCT2, multidrug and toxin extrusion transporter [MATE]1, MATE2-K, and OCTN1) were analyzed in up to 7,968 individuals. None of the variants showed a significant effect on metformin response in the primary analysis, or in the exploratory secondary analyses, when patients were stratified according to possible confounding genotypes or prescribed a daily dose of metformin. Our results suggest that candidate transporter gene variants have little contribution to variability in glycemic response to metformin in T2D.

Sex-specific effects of naturally occurring variants in the dopamine receptor D2 locus on insulin secretion and type 2 diabetes susceptibility.

AIMS: Modulation of dopamine receptor D2 (DRD2) activity affects insulin secretion in both rodents and isolated pancreatic ß-cells. We hypothesized that single nucleotide polymorphisms in the DRD2/ANKK1 locus may affect susceptibility to type 2 diabetes in humans. METHODS: Four potentially functional variants in the coding region of the DRD2/ANKK1 locus (rs1079597, rs6275, rs6277, rs1800497) were genotyped and analysed for type 2 diabetes susceptibility in up to 25 000 people (8148 with type 2 diabetes and 17687 control subjects) from two large independent Dutch cohorts and one Danish cohort. In addition, 340 Dutch subjects underwent a 2-h hyperglycaemic clamp to investigate insulin secretion. Since sexual dimorphic associations related to DRD2 polymorphisms have been previously reported, we also performed a gender-stratified analysis. RESULTS: rs1800497 at the DRD2/ANKK1 locus was associated with a significantly increased risk for type 2 diabetes in women (odds ratio 1.14 (1.06-1.23); P = 4.1*104) but not in men (odds ratio 1.00 (95% CI 0.93-1.07); P = 0.92) or the combined group. Although rs1800497 was not associated with insulin secretion, we did find another single nucleotide polymorphism in this locus, rs6275, to be associated with increased first-phase glucose-stimulated insulin secretion in women (P = 5.5*104) but again not in men (P = 0.34). CONCLUSION: The present data identify DRD2/ANKK1 as a potential sex-specific type 2 diabetes susceptibility gene.

Low mitochondrial DNA content associates with familial longevity: the Leiden Longevity Study.

Long-lived individuals delay aging and age-related diseases like diabetes, hypertension, and cardiovascular disease. The exact underlying mechanisms are largely unknown, but enhanced mitochondrial biogenesis and preservation of mitochondrial function have been suggested to explain healthy ageing. We investigated whether individuals belonging to long-lived families have altered mitochondrial DNA (mtDNA) content, as a biomarker of mitochondrial biogenesis and measured expression of genes regulating mitochondrial biogenesis. mtDNA and nuclear DNA (nDNA) levels were measured in blood samples from 2,734 participants from the Leiden Longevity Study: 704 nonagenarian siblings, 1,388 of their middle-aged offspring and 642 controls. We confirmed a negative correlation of mtDNA content in blood with age and a higher content in females. The middle-aged offspring had, on average, lower levels of mtDNA than controls and the nonagenarian siblings had an even lower mtDNA content (mtDNA/nDNA ratio = 0.744 ± 0.065, 0.767 ± 0.058 and 0.698 ± 0.074, respectively; p controls-offspring = 3.4 × 10(-12), p controls-nonagenarians = 6.5 × 10(-6)), which was independent of the confounding effects of age and gender. Subsequently, we examined in a subset of the study the expression in blood of two genes regulating mitochondrial biogenesis, YY1 and PGC-1α. We found a positive association of YY1 expression and mtDNA content in controls. The observed absence of such an association in the offspring suggests an altered regulation of mitochondrial biogenesis in the members of long-lived families. In conclusion, in this study, we show that mtDNA content decreases with age and that low mtDNA content is associated with familial longevity. Our data suggest that preservation of mitochondrial function rather than enhancing mitochondrial biogenesis is a characteristic of long-lived families.

The role of pharmacogenetics in drug disposition and response of oral glucose-lowering drugs.

The primary goal of type 2 diabetes mellitus (T2DM) disease management is improvement of quality of life and prevention of complications. One way to achieve these goals is improving glycemic control by using different types of oral glucose-lowering medications. Currently seven different pharmacological oral glucose-lowering drug classes are available, each with its own mechanism of action and characteristics regarding absorption, distribution, metabolism, and elimination. Unfortunately, the response to the different types of glucose-lowering medication is highly variable between individuals resulting in unnecessary treatment failure. Genetic factors are thought to contribute to the variability in response and may present an opportunity to improve treatment outcome. In recent years, many efforts were taken to identify genetic variants that influence the pharmacokinetics, pharmacodynamics, and ultimately the therapeutic response of the different oral glucose-lowering drugs. Indeed several genetic variants are associated with the response to oral glucose-lowering drugs. This review comprises current knowledge on genetic variants affecting both pharmacokinetics and pharmacodynamics of oral glucose-lowering drugs. Included variants are located in genes coding for drug transporters, i.e., the organic anion-transporting family and the organic cation transporter family; genes involved in metabolism, i.e., cytochrome P450 superfamily; genes coding for drug receptors; T2DM-associated genes; and genes identified by genome-wide association studies (GWASs). Furthermore, this review provides insight into current status and future directions for personalized medicine in T2DM.

BclI glucocorticoid receptor polymorphism is associated with greater body fatness: the Hoorn and CODAM studies.

CONTEXT: The BclI polymorphism in the glucocorticoid receptor (GR) gene is associated with enhanced glucocorticoid (GC) sensitivity. OBJECTIVE: Our objective was to investigate the association of the BclI polymorphism with body fatness and insulin resistance. DESIGN AND SETTING: We conducted an observational cohort study, combining data from 2 cohort studies enriched with individuals with impaired glucose metabolism and/or diabetes mellitus type 2 (DM2). PATIENTS AND METHODS: We examined 1228 participants (mean age 64.7 years, 45% women) from the Cohort Study on Diabetes and Atherosclerosis Maastricht (CODAM, n = 543) and the Hoorn Study (n = 685). Body mass index (BMI), waist and hip circumferences, and waist-to-hip ratio (WHR) were obtained; insulin resistance was estimated using the homeostasis model assessment for insulin resistance (HOMA2-IR). RESULTS: We identified 519 noncarriers (CC), 540 heterozygous (CG) carriers, and 169 homozygous (GG) carriers of the G-allele of the BclI polymorphism. Homozygous carriers had a higher BMI (28.9 vs 27.9 kg/m(2)) and waist (99.6 vs 97.2 cm) and hip (105.5 vs 103.2 cm) circumference compared with noncarriers, also after adjustment for age, sex, cohort, glucose tolerance, and lifestyle risk factors: ß = 0.94 kg/m(2) (95% confidence interval, 0.24-1.63), ß = 2.84 cm (0.95;4.73) and ß = 2.38 cm (0.88-3.87), respectively. Similar results were obtained when comparing homozygous carriers with heterozygous carriers: ß = 1.03 kg/m(2) (0.34-1.72), ß = 2.20 cm (0.31-4.08) and ß = 1.99 cm (0.51-3.48), respectively. There were no differences in WHR. Ln-HOMA2-IR was higher in GG carriers compared with CG carriers; 0.29 vs 0.17 [ß = 0.09 (0.01-0.17)], but this effect was attenuated after adjustment for BMI [ß = 0.04 (-0.04 to 0.11)]. CONCLUSION: Homozygous carriers of the BclI polymorphism of the GR gene have significantly greater total body fatness, contributing to higher HOMA2-IR, compared with heterozygous carriers and noncarriers.

Effects of induced hyperinsulinaemia with and without hyperglycaemia on measures of cardiac vagal control.

AIMS/HYPOTHESIS: We examined the effects of serum insulin levels on vagal control over the heart and tested the hypothesis that higher fasting insulin levels are associated with lower vagal control. We also examined whether experimentally induced increases in insulin by beta cell secretagogues, including glucagon-like peptide-1 (GLP-1), will decrease vagal control. METHODS: Respiration and ECGs were recorded for 130 healthy participants undergoing clamps. Three variables of cardiac vagal effects (the root mean square of successive differences [rMSSD] in the interbeat interval of the heart rate [IBI], heart-rate variability [HRV] caused by peak-valley respiratory sinus arrhythmia [pvRSA], and high-frequency power [HF]) and heart rate (HR) were obtained at seven time points during the clamps, characterised by increasing levels of insulin (achieved by administering insulin plus glucose, glucose only, glucose and GLP-1, and glucose and GLP-1 combined with arginine). RESULTS: Serum insulin level was positively associated with HR at all time points during the clamps except the first-phase hyperglycaemic clamp. Insulin levels were negatively correlated with variables of vagal control, reaching significance for rMSSD and log10HF, but not for pvRSA, during the last four phases of the hyperglycaemic clamp (hyperglycaemic second phase, GLP-1 first and second phases, and arginine). These associations disappeared when adjusted for age, BMI and insulin sensitivity. Administration of the beta cell secretagogues GLP-1 and arginine led to a significant increase in HR, but this was not paired with a significant reduction in HRV measures. CONCLUSION/INTERPRETATION: Experimentally induced hyperinsulinaemia is not correlated with cardiac vagal control or HR when adjusting for age, BMI and insulin sensitivity index. Our findings suggest that exposure to a GLP-1 during hyperglycaemia leads to a small acute increase in HR but not to an acute decrease in cardiac vagal control.

Glucocorticoid receptor gene polymorphisms are associated with reduced first-phase glucose-stimulated insulin secretion and disposition index in women, but not in men.

AIM: Glucocorticoids are efficacious anti-inflammatory agents, but, in susceptible individuals, these drugs may induce glucose intolerance and diabetes by affecting ß-cell function and insulin sensitivity. We assessed whether polymorphisms in the glucocorticoid receptor gene NR3C1 associate with measures of ß-cell function and insulin sensitivity derived from hyperglycaemic clamps in subjects with normal or impaired glucose tolerance. METHODS: A cross-sectional cohort study was conducted in four academic medical centres in the Netherlands and Germany. Four hundred and forty-nine volunteers (188 men; 261 women) were recruited with normal glucose tolerance (n=261) and impaired glucose tolerance (n=188). From 2-h hyperglycaemic clamps, first- and second-phase glucose-stimulated insulin secretion, as well as insulin sensitivity index and disposition index, were calculated. All participants were genotyped for the functional NR3C1 polymorphisms N363S (rs6195), BclI (rs41423247), ER22/23EK (rs6189/6190), 9ß A/G (rs6198) and ThtIIII (rs10052957). Associations between these polymorphisms and ß-cell function parameters were assessed. RESULTS: In women, but not in men, the N363S polymorphism was associated with reduced disposition index (P=1.06 10(-4) ). Also only in women, the ER22/23EK polymorphism was associated with reduced first-phase glucose-stimulated insulin secretion (P=0.011) and disposition index (P=0.003). The other single-nucleotide polymorphisms were not associated with ß-cell function. Finally, none of the polymorphisms was related to insulin sensitivity. CONCLUSION: The N363S and ER22/23EK polymorphisms of the NR3C1 gene are negatively associated with parameters of ß-cell function in women, but not in men.

A gene variant near ATM is significantly associated with metformin treatment response in type 2 diabetes: a replication and meta-analysis of five cohorts.

AIMS/HYPOTHESIS: In this study we aimed to replicate the previously reported association between the glycaemic response to metformin and the SNP rs11212617 at a locus that includes the ataxia telangiectasia mutated (ATM) gene in multiple additional populations. METHODS: Incident users of metformin selected from the Diabetes Care System West-Friesland (DCS, n = 929) and the Rotterdam Study (n = 182) from the Netherlands, and the CARDS Trial (n = 254) from the UK were genotyped for rs11212617 and tested for an association with both HbA(1c) reduction and treatment success, defined as the ability to reach the treatment target of an HbA(1c) ≤ 7 % (53 mmol/mol). Finally, a meta-analysis including data from literature was performed. RESULTS: In the DCS cohort, we observed an association between rs11212617 genotype and treatment success on metformin (OR 1.27, 95% CI 1.03, 1.58, p = 0.028); in the smaller Rotterdam Study cohort, a numerically similar but non-significant trend was observed (OR 1.45, 95% CI 0.87, 2.39, p = 0.15); while in the CARDS cohort there was no significant association. In meta-analyses of these three cohorts separately or combined with the previously published cohorts, rs11212617 genotype is associated with metformin treatment success (OR 1.24, 95% CI 1.04, 1.49, p = 0.016 and OR 1.25, 95% CI 1.33, 1.38, p = 7.8 × 10(-6), respectively). CONCLUSIONS/INTERPRETATION: A gene variant near ATM is significantly associated with metformin treatment response in type 2 diabetic patients from the Netherlands and the UK. This is the first robustly replicated common susceptibility locus found to be associated with metformin treatment response.

The heritability of beta cell function parameters in a mixed meal test design.

AIMS/HYPOTHESIS: We estimated the heritability of individual differences in beta cell function after a mixed meal test designed to assess a wide range of classical and model-derived beta cell function parameters. METHODS: A total of 183 healthy participants (77 men), recruited from the Netherlands Twin Register, took part in a 4 h protocol, which included a mixed meal test. Participants were Dutch twin pairs and their siblings, aged 20 to 49 years. All members within a family were of the same sex. Insulin sensitivity, insulinogenic index, insulin response and postprandial glycaemia were assessed, as well as model-derived parameters of beta cell function, in particular beta cell glucose sensitivity and insulin secretion rates. Genetic modelling provided the heritability of all traits. Multivariate genetic analyses were performed to test for overlap in the genetic factors influencing beta cell function, waist circumference and insulin sensitivity. RESULTS: Significant heritabilities were found for insulinogenic index (63%), beta cell glucose sensitivity (50%), insulin secretion during the first 2 h postprandial (42-47%) and postprandial glycaemia (43-52%). Genetic factors influencing beta cell glucose sensitivity and insulin secretion during the first 30 postprandial min showed only negligible overlap with the genetic factors that influence waist circumference and insulin sensitivity. CONCLUSIONS/INTERPRETATION: The highest heritability for postprandial beta cell function was found for the insulinogenic index, but the most specific indices of heritability of beta cell function appeared to be beta cell glucose sensitivity and the insulin secretion rate during the first 30 min after a mixed meal.

Genetic association analysis of LARS2 with type 2 diabetes.

AIMS/HYPOTHESIS: LARS2 has been previously identified as a potential type 2 diabetes susceptibility gene through the low-frequency H324Q (rs71645922) variant (minor allele frequency [MAF] 3.0%). However, this association did not achieve genome-wide levels of significance. The aim of this study was to establish the true contribution of this variant and common variants in LARS2 (MAF > 5%) to type 2 diabetes risk. METHODS: We combined genome-wide association data (n = 10,128) from the DIAGRAM consortium with independent data derived from a tagging single nucleotide polymorphism (SNP) approach in Dutch individuals (n = 999) and took forward two SNPs of interest to replication in up to 11,163 Dutch participants (rs17637703 and rs952621). In addition, because inspection of genome-wide association study data identified a cluster of low-frequency variants with evidence of type 2 diabetes association, we attempted replication of rs9825041 (a proxy for this group) and the previously identified H324Q variant in up to 35,715 participants of European descent. RESULTS: No association between the common SNPs in LARS2 and type 2 diabetes was found. Our replication studies for the two low-frequency variants, rs9825041 and H324Q, failed to confirm an association with type 2 diabetes in Dutch, Scandinavian and UK samples (OR 1.03 [95% CI 0.95-1.12], p = 0.45, n = 31,962 and OR 0.99 [0.90-1.08], p = 0.78, n = 35,715 respectively). CONCLUSIONS/INTERPRETATION: In this study, the largest study examining the role of sequence variants in LARS2 in type 2 diabetes susceptibility, we found no evidence to support previous data indicating a role in type 2 diabetes susceptibility.

Genetic influences on the insulin response of the beta cell to different secretagogues.

AIMS/HYPOTHESIS: The aim of the present study was to estimate the heritability of the beta cell insulin response to glucose and to glucose combined with glucagon-like peptide-1 (GLP-1) or with GLP-1 plus arginine. METHODS: This was a twin-family study that included 54 families from the Netherlands Twin Register. The participants were healthy twin pairs and their siblings of the same sex, aged 20 to 50 years. Insulin response of the beta cell was assessed by a modified hyperglycaemic clamp with additional GLP-1 and arginine. Insulin sensitivity index (ISI) was assessed by the euglycaemic-hyperinsulinaemic clamp. Multivariate structural equation modelling was used to obtain heritabilities and the genetic factors underlying individual differences in BMI, ISI and secretory responses of the beta cell. RESULTS: The heritability of insulin levels in response to glucose was 52% and 77% for the first and second phase, respectively, 53% in response to glucose + GLP-1 and 80% in response to an additional arginine bolus. Insulin responses to the administration of glucose, glucose + GLP-1 and glucose + GLP-1 + arginine were highly correlated (0.62< r <0.79). Heritability of BMI and ISI was 74% and 60% respectively. The genetic factors that influenced BMI and ISI explained about half of the heritability of insulin levels in response to the three secretagogues. The other half was due to genetic factors specific to the beta cell. CONCLUSIONS/INTERPRETATION: In healthy adults, genetic factors explain most of the individual differences in the secretory capacity of the beta cell. These genetic influences are partly independent from the genes that influence BMI and ISI.

Combined effects of single-nucleotide polymorphisms in GCK, GCKR, G6PC2 and MTNR1B on fasting plasma glucose and type 2 diabetes risk.

AIMS/HYPOTHESIS: Variation in fasting plasma glucose (FPG) within the normal range is a known risk factor for the development of type 2 diabetes. Several reports have shown that genetic variation in the genes for glucokinase (GCK), glucokinase regulatory protein (GCKR), islet-specific glucose 6 phosphatase catalytic subunit-related protein (G6PC2) and melatonin receptor type 1B (MTNR1B) is associated with FPG. In this study we examined whether these loci also contribute to type 2 diabetes susceptibility. METHODS: A random selection from the Dutch New Hoorn Study was used for replication of the association with FGP (2,361 non-diabetic participants). For the genetic association study we extended the study sample with 2,628 participants with type 2 diabetes. Risk allele counting was used to calculate a four-gene risk allele score for each individual. RESULTS: Variants of the GCK, G6PC2 and MTNR1B genes but not GCKR were associated with FPG (all, p

Neuropeptide Y polymorphism significantly magnifies diabetes and cardiovascular disease risk in obesity: the Hoorn Study.

The leucine7 to proline7 (Leu7Pro) polymorphism in preproneuropeptide Y (preproNPY) has been associated with accelerated atherosclerosis and type II diabetes, both of which are obesity-related diseases. The current study evaluated the impact of obesity on the disease risk linked to the Leu7Pro polymorphism of preproNPY in 393 elderly subjects. In 6 years follow-up, the polymorphism alone did not change the risk for abnormal glucose regulation, while obesity was associated with a significant 3-fold risk (odds ratio (OR) 2.95; 95% confidence interval (CI) 1.81-4.81, P<0.001) and the Leu7Pro polymorphism-obesity interaction, with a remarkable 12-fold risk (OR 12.33; 95% CI 1.18-128.35, P<0.05). The Leu7Pro polymorphism modified significantly the 10-year incidence of cardiovascular events, causing a 7.6-fold increase in the hazard ratio (HR 7.58; 95% CI 2.87-20.03, P<0.001) in the obese but not in the nonobese subjects. The results indicate that obesity may be a pivotal factor in multiplying the disease risk associated with the Leu7Pro polymorphism in preproNPY.

Variants of CDKAL1 and IGF2BP2 affect first-phase insulin secretion during hyperglycaemic clamps.

AIMS/HYPOTHESIS: Genome-wide association studies have recently identified novel type 2 diabetes susceptibility gene regions. We assessed the effects of six of these regions on insulin secretion as determined by a hyperglycaemic clamp. METHODS: Variants of the HHEX/IDE, CDKAL1, SLC30A8, IGF2BP2 and CDKN2A/CDKN2B genes were genotyped in a cohort of 146 participants with NGT and 126 with IGT from the Netherlands and Germany, who all underwent a hyperglycaemic clamp at 10 mmol/l glucose. RESULTS: Variants of CDKAL1 and IGF2BP2 were associated with reductions in first-phase insulin secretion (34% and 28%, respectively). The disposition index was also significantly reduced. For gene regions near HHEX/IDE, SLC30A8 and CDKN2A/CDKN2B we did not find significant associations with first-phase insulin secretion (7-18% difference between genotypes; all p > 0.3). None of the variants showed a significant effect on second-phase insulin secretion in our cohorts (2-8% difference between genotypes, all p > 0.3). Furthermore, the gene variants were not associated with the insulin sensitivity index. CONCLUSIONS: Variants of CDKAL1 and IGF2BP2 attenuate the first phase of glucose-stimulated insulin secretion but show no effect on the second phase of insulin secretion. Our results, based on hyperglycaemic clamps, provide further insight into the pathogenic mechanism behind the association of these gene variants with type 2 diabetes.

Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms.

AIMS/HYPOTHESIS: Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with type 2 diabetes and reduced insulin secretion. The transcription factor TCF7L2 is an essential factor for glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells. We studied whether a defect in the enteroinsular axis contributes to impaired insulin secretion in carriers of TCF7L2 polymorphisms. METHODS: We genotyped 1,110 non-diabetic German participants for five single nucleotide polymorphisms in TCF7L2. All participants underwent an OGTT; GLP-1 secretion was measured in 155 participants. In 210 participants, an IVGTT combined with a hyperinsulinaemic-euglycaemic clamp was performed. In another 160 participants from the Netherlands and 73 from Germany, a hyperglycaemic clamp (10 mmol/l) was performed. In 73 German participants this clamp was combined with a GLP-1 infusion and an arginine bolus. RESULTS: The OGTT data confirmed that variants in TCF7L2 are associated with reduced insulin secretion. In contrast, insulin secretion induced by an i.v. glucose challenge in the IVGTT and hyperglycaemic clamp was not different between the genotypes. GLP-1 concentrations during the OGTT were not influenced by the TCF7L2 variants. However, GLP-1-infusion combined with a hyperglycaemic clamp showed a significant reduction in GLP-1-induced insulin secretion in carriers of the risk allele in two variants (rs7903146, rs12255372, p < 0.02). CONCLUSIONS/INTERPRETATION: Variants of TCF7L2 specifically impair GLP-1-induced insulin secretion. This seems to be rather the result of a functional defect in the GLP-1 signalling in beta cells than a reduction in GLP-1 secretion. This defect might explain the impaired insulin secretion in carriers of the risk alleles and confers the increased risk of type 2 diabetes.

Mitochondrial diabetes and its lessons for common Type 2 diabetes.

Multiple pathogenic pathways are able to deregulate glucose homoeostasis leading to diabetes. The 3243A>G mutation in the mtDNA (mitochondrial DNA)-encoded tRNALeu,UUR gene was found by us to be associated with a particular diabetic subtype, designated MIDD (maternally inherited diabetes and deafness). This mutation causes an imbalance in the mitochondrion between proteins encoded by the nuclear and mitochondrial genomes, resulting in a gradual deterioration of glucose homoeostasis during life. Remarkably, carriers of the 3243A>G mutation are generally not obese. The mutation also results in enhanced radical production by mitochondria. We propose that this mutation leads to the development of diabetes due to an inappropriate storage of triacylglycerols within adipocytes. The result is a fatty acid-induced deterioration of pancreatic beta-cell function. In combination with an enhanced radical production in the beta-cell due to the mutation, this leads to an age-dependent, accelerated decline in insulin production. In common Type 2 (non-insulin-dependent) diabetes, which is generally associated with obesity, a decline in mitochondrial function in adipose cells seems to result in an inappropriate scavenging of fatty acids by beta-oxidation. As a consequence, a systemic overload with fatty acids occurs, leading to an enhanced decline in beta-cell function due to lipotoxicity.

Novel mitochondrial DNA length variants and genetic instability in a family with diabetes and deafness.

We have identified two locations with novel multiplasmic length variants in the mitochondrial DNA of a family with diabetes and deafness. At nt568 in the D-loop, the 6-bp polycytidine tract was found to be variable in length up to a total of 12 residues. A second region with length variants was found at nt8281 in the intergenic COII-tRNA(Lys) region, which consists of two copies of the 9-bp repeat CCCCCTCTA. Only the second repeat occurs in a heteroplasmic C(9-14)A form with both T residues largely deleted. In addition, the mtDNA contained a number of new homoplasmic point mutations. Both length variants are stably inherited in a maternal way with no major changes in their length distribution. In contrast, during culture of fibroblasts from the proband the average length of the polycytidine tracts is increased at both locations indicating a fibroblast-specific genetic instability. Cybrid cells containing mtDNA from the proband proliferate less efficient than cybrids with wild-type mtDNA in co-culture experiments, suggesting functional consequences of the mtDNA length variants or the additional homoplasmic point mutations. Since oxygen consumption was not severely affected, these mutation seem less detrimental for mitochondrial function than the A3243G diabetogenic mutation and most other pathogenic mtDNA mutations. The contribution of mtDNA length variants to the phenotype of members of this family is discussed.

The Gly482Ser variant in the peroxisome proliferator-activated receptor gamma coactivator-1 is not associated with diabetes-related traits in non-diabetic German and Dutch populations.

The peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) is involved in regulation of fatty acid oxidation, skeletal muscle fiber type specificity, and gluconeogenesis. The prevalent Gly482Ser variant in PGC-1 was shown to be associated with type 2 diabetes in some but not all studies. Moreover, it is unclear whether it influences prediabetic subphenotypes in non-diabetic populations. We studied the association of this variant with glucose tolerance (oral glucose tolerance test), insulin sensitivity (euglycemic hyperinsulinemic clamp) of glucose disposal and antilipolysis, insulin secretion (hyperglycemic clamp, 10 mM), maximal oxygen consumption (VO(2)max, bicycle ergometry), and intramyocellular lipids (magnetic resonance spectroscopy, tibialis and soleus muscle) in a normal glucose tolerant German cohort (n = 423) and a normal (n = 65) and impaired glucose tolerant (n = 94) cohort from the Netherlands. No statistically significant association with an examined phenotype was detected in any of the study cohorts. Specifically, VO(2)max and the soleus-to-tibialis ratio of intramyocellular lipid contents as a surrogate parameter of fiber type specificity was not different between the genotype groups. We conclude, that the Gly482Ser variant in PGC-1 is not associated with diabetes-related traits or skeletal muscle fiber type composition in a non-diabetic German and Dutch population.