Common variant in the HMGA2 gene increases susceptibility to nephropathy in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is a chronic metabolic disorder associated with devastating microvascular complications. Genome-wide association studies have identified more than 60 genetic variants associated with type 2 diabetes and/or glucose and insulin traits, but their role in the progression of diabetes is not established. The aim of this study was to explore whether these variants were also associated with the development of nephropathy in patients with type 2 diabetes. METHODS: We studied 28 genetic variants in 2,229 patients with type 2 diabetes from the local Malmö Scania Diabetes Registry (SDR) published during 2007-2010. Diabetic nephropathy (DN) was defined as micro- or macroalbuminuria and/or end-stage renal disease. Estimated glomerular filtration rate (eGFR) was assessed using the MDRD-4 formula. Replication genotyping of rs1531343 was performed in diabetic (Steno type 2 diabetes [n = 345], Genetics of Diabetes Audit and Research in Tayside Scotland [Go-DARTS] [n = 784]) and non-diabetic (Malmö Preventive Project [n = 2,523], Botnia study [n = 2,247]) cohorts. RESULTS: In the SDR, HMGA2 single-nucleotide polymorphism rs1531343 was associated with DN (OR 1.50, 95% CI 1.20, 1.87, p = 0.00035). In the combined analysis totalling 3,358 patients with type 2 diabetes (n = 1,233 cases, n = 2,125 controls), carriers of the C-allele had a 1.45-fold increased risk of developing nephropathy (95% CI 1.20, 1.75, p = 0.00010). Furthermore, the risk C-allele was associated with lower eGFR in patients with type 2 diabetes (n = 2,499, ß ± SEM, -3.7 ± 1.2 ml/min, p = 0.002) and also in non-diabetic individuals (n = 17,602, ß ± SEM, -0.008 ± 0.003 ml/min (log( e )), p = 0.006). CONCLUSIONS/INTERPRETATION: These data demonstrate that the HMGA2 variant seems to be associated with increased risk of developing nephropathy in patients with type 2 diabetes and lower eGFR in both diabetic and non-diabetic individuals and could thus be a common denominator in the pathogenesis of type 2 diabetes and kidney complications.

The human L-type calcium channel Cav1.3 regulates insulin release and polymorphisms in CACNA1D associate with type 2 diabetes.

AIMS/HYPOTHESIS: Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes. METHODS: Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Cav)1.2 and Cav1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Cav1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes. RESULTS: In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Cav1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes. CONCLUSION/INTERPRETATION: We conclude that the L-type calcium channel Cav1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.

Family history of Type 1 diabetes affects insulin secretion in patients with 'Type 2' diabetes.

AIMS: The aim was to evaluate the impact of family history of diabetes on the phenotype of patients diagnosed with Type 2 diabetes and the frequency of susceptibility genotypes. METHODS: Patients with Type 2 diabetes with family history for both Type 1 and Type 2 diabetes (FH(MIX, n) = 196) or Type 2 diabetes only (FH(T2), n = 139) matched for age, sex, BMI and age at diagnosis, underwent an oral glucose tolerance test and a combined glucagon test and insulin tolerance test. Glutamic acid decarboxylase (GAD) antibodies and major Type 1 and Type 2 diabetes susceptibility gene variants were analysed. Patients were stratified into groups according to family history or GAD antibody positivity (GADA+, GADA-) or a combination of these (GADA+/FH(MIX), GADA+/FH(T2), GADA-/FH(MIX), GADA-/FH(T2)). RESULTS: Compared with other patients, those with FH(MIX) more often had GAD antibodies (14.3 vs. 4.3%, P = 0.003), and those with both FH(MIX) and GAD antibodies had the highest frequency of insulin deficiency (stimulated serum C-peptide < 0.7 nmol/l, GADA+/FH(MIX) 46.4% vs. GADA-/FH(MIX) 9.5% (P < 0.00001), GADA-/FH(T2) 4.5% (P < 0.00001), GADA+/FH(T2) 0%). Patients with GADA+/FH(MIX) more often had HLA-DQB1 risk genotypes compared with patients with GADA-/FH(MIX) or GADA-/FH(T2D) (47 vs. 23 or 14%, P = 0.05 and P < 0.00001, respectively). In logistic regression analyses, FH(MIX), GAD antibody positivity and HLA risk genotypes were independently associated with insulin deficiency. CONCLUSION: A family history for both type 1 and type 2 diabetes was associated with higher prevalence of GAD antibodies and HLA-DQB1 risk genotypes than a family history of type 2 diabetes only, and was associated with earlier and more severe development of insulin deficiency, which was only partially explained by GAD antibodies and HLA.

Effect of a common variant of the PCSK2 gene on reduced insulin secretion.

AIM/HYPOTHESIS: Individuals at risk of developing type 2 diabetes show a progressive decline in insulin secretion and increased insulin resistance over time. However, inability of the beta cells to compensate for the increased insulin resistance represents a key defect leading to overt type 2 diabetes. The aims of the present study were to replicate the association between genetic variants of the PCSK2 gene and insulin secretion, and to explore the effect on risk of type 2 diabetes. METHODS: Replication of PCSK2 variants against insulin secretion included 7,682 non-diabetic Scandinavian individuals. Insulin secretion was measured as the corrected insulin response or disposition index, i.e. insulin secretion adjusted for the degree of insulin resistance. Risk of type 2 diabetes was studied in 28,287 Scandinavian individuals. RESULTS: The C-allele of PCSK2 rs2208203 was associated with reduced insulin secretion measured as the corrected insulin response (n = 8,151; ß = -0.112, p = 1.3 × 10(-6)) as well as disposition index (n = 8,078, ß = -0.128, p = 1.6 × 10(-7)). The variant was also associated with lower fasting glucagon levels (ß = -0.084, p = 0.005) in non-diabetic individuals with a fasting plasma glucose of over 5.5 mmol/l. In human pancreatic islets, PCSK2 expression correlated negatively with HbA(1c) (n = 133, r = -0.196, p = 0.038), and showed a tendency to be lower in hyperglycaemic (HbA(1c) ≥6.0% or type 2 diabetes; n = 47, p = 0.13) than normoglycaemic (HbA(1c) >6.0%; n = 66) donors. The presence of the PCSK2 rs2208203 risk allele did not influence gene expression, nor did it show an apparent risk in terms of type 2 diabetes. CONCLUSIONS/INTERPRETATION: A variant of the PCSK2 gene was associated with reduced glucose-stimulated insulin secretion, but also with lower glucagon levels, which could potentially counteract the effects of decreased insulin secretion on the risk of type 2 diabetes.

Telomere length in blood and skeletal muscle in relation to measures of glycaemia and insulinaemia.

AIMS: Skeletal muscle is a major metabolic organ and plays important roles in glucose metabolism, insulin sensitivity and insulin action. Muscle telomere length reflects the myocyte's exposure to harmful environmental factors. Leukocyte telomere length is considered a marker of muscle telomere length and is used in epidemiologic studies to assess associations with ageing-related diseases where muscle physiology is important. However, the extent to which leucocyte and muscle telomere length are correlated is unknown, as are their relative correlations with glucose and insulin concentrations. The purpose of this study was to determine the extent of these relationships. METHODS: Leucocyte and muscle telomere length were measured by quantitative real-time polymerase chain reaction in participants from the Malmö Exercise Intervention (n = 27) and the Prevalence, Prediction and Prevention of Diabetes-Botnia studies (n = 31). Participants in both studies were free from Type 2 diabetes. We assessed the association between leucocyte telomere length, muscle telomere length and metabolic traits using Spearmen correlations and multivariate linear regression. Bland-Altman analysis was used to assess agreement between leucocyte and muscle telomere length. RESULTS: In age-, study-, diabetes family history- and sex-adjusted models, leucocyte and muscle telomere length were positively correlated (r = 0.39, 95% CI 0.15-0.59). Leucocyte telomere length was inversely associated with 2-h glucose concentrations (r = -0.58, 95% CI -1.0 to -0.16), but there was no correlation between muscle telomere length and 2-h glucose concentrations (r = 0.05, 95% CI -0.35 to 0.46) or between leucocyte or muscle telomere length with other metabolic traits. CONCLUSIONS: In summary, the current study supports the use of leucocyte telomere length as a proxy for muscle telomere length in epidemiological studies of Type 2 diabetes aetiology.

A common variant upstream of the PAX6 gene influences islet function in man.

AIMS/HYPOTHESIS: Impaired glucose tolerance and impaired insulin secretion have been reported in families with PAX6 mutations and it is suggested that they result from defective proinsulin processing due to lack of prohormone convertase 1/3, encoded by PCSK1. We investigated whether a common PAX6 variant would mimic these findings and explored in detail its effect on islet function in man. METHODS: A PAX6 candidate single nucleotide polymorphism (rs685428) was associated with fasting insulin levels in the Diabetes Genetics Initiative genome-wide association study. We explored its potential association with glucose tolerance and insulin processing and secretion in three Scandinavian cohorts (N = 8,897 individuals). In addition, insulin secretion and the expression of PAX6 and transcriptional target genes were studied in human pancreatic islets. RESULTS: rs685428 G allele carriers had lower islet mRNA expression of PAX6 (p = 0.01) and PCSK1 (p = 0.001) than AA homozygotes. The G allele was associated with increased fasting insulin (p (replication) = 0.02, p (all) = 0.0008) and HOMA-insulin resistance (p (replication) = 0.02, p (all) = 0.001) as well as a lower fasting proinsulin/insulin ratio (p (all) = 0.008) and lower fasting glucagon (p = 0.04) and gastric inhibitory peptide (GIP) (p = 0.05) concentrations. Arginine-stimulated (p = 0.02) insulin secretion was reduced in vivo, which was further reflected by a reduction of glucose- and potassium-stimulated insulin secretion (p = 0.002 and p = 0.04, respectively) in human islets in vitro. CONCLUSIONS/INTERPRETATION: A common variant in PAX6 is associated with reduced PAX6 and PCSK1 expression in human islets and reduced insulin response, as well as decreased glucagon and GIP concentrations and decreased insulin sensitivity. These findings emphasise the central role of PAX6 in the regulation of islet function and glucose metabolism in man.

Heritability and familiality of type 2 diabetes and related quantitative traits in the Botnia Study.

AIMS/HYPOTHESIS: To study the heritability and familiality of type 2 diabetes and related quantitative traits in families from the Botnia Study in Finland. METHODS: Heritability estimates for type 2 diabetes adjusted for sex, age and BMI are provided for different age groups of type 2 diabetes and for 34 clinical and metabolic traits in 5,810 individuals from 942 families using a variance component model (SOLAR). In addition, family means of these traits and their distribution across families are calculated. RESULTS: The strongest heritability for type 2 diabetes was seen in patients with age at onset 35-60 years (h (2) = 0.69). However, including patients with onset up to 75 years dropped the h (2) estimates to 0.31. Among quantitative traits, the highest h (2) estimates in all individuals and in non-diabetic individuals were seen for lean body mass (h (2) = 0.53-0.65), HDL-cholesterol (0.52-0.61) and suppression of NEFA during OGTT (0.63-0.76) followed by measures of insulin secretion (insulinogenic index [IG(30)] = 0.41-0.50) and insulin action (insulin sensitivity index [ISI] = 0.37-0.40). In contrast, physical activity showed rather low heritability (0.16-0.18), whereas smoking showed strong heritability (0.57-0.59). Family means of these traits differed two- to fivefold between families belonging to the lowest and highest quartile of the trait (p < 0.00001). CONCLUSIONS/INTERPRETATION: To detect stronger genetic effects in type 2 diabetes, it seems reasonable to restrict inclusion of patients to those with age at onset 35-60 years. Sequencing of families with extreme quantitative traits could be an important next step in the dissection of the genetics of type 2 diabetes.

A large multi-centre European study validates high-sensitivity C-reactive protein (hsCRP) as a clinical biomarker for the diagnosis of diabetes subtypes.

AIMS/HYPOTHESIS: An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed. Biomarkers could help to prioritise patients for genetic investigation. We recently demonstrated that high-sensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype-phenotype relationship and compare different hsCRP assays. METHODS: High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY (n = 457), glucokinase (GCK)-MODY (n = 404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY (n = 54) and type 2 diabetes (n = 582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values >10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curve-derived C-statistic. RESULTS: In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies (z score -21.8, p < 5 × 10(-105)). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l, p < 5 × 10(-5)). High-sensitivity CRP values between assays were strongly correlated (r (2) ≥ 0.91, p ≤ 1 × 10(-5)). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy. CONCLUSIONS/INTERPRETATION: In the largest study to date, we have established that hsCRP is a clinically valid biomarker for HNF1A-MODY in European populations. Given the modest costs and wide availability, hsCRP could translate rapidly into clinical practice, considerably improving diagnosis rates in monogenic diabetes.

A family history of diabetes is associated with reduced physical fitness in the Prevalence, Prediction and Prevention of Diabetes (PPP)-Botnia study.

AIMS/HYPOTHESIS: We studied the impact of a family history of type 2 diabetes on physical fitness, lifestyle factors and diabetes-related metabolic factors. METHODS: The Prevalence, Prediction and Prevention of Diabetes (PPP)-Botnia study is a population-based study in Western Finland, which includes a random sample of 5,208 individuals aged 18 to 75 years identified through the national Finnish Population Registry. Physical activity, dietary habits and family history of type 2 diabetes were assessed by questionnaires and physical fitness by a validated 2 km walking test. Insulin secretion and action were assessed based upon OGTT measurements of insulin and glucose. RESULTS: A family history of type 2 diabetes was associated with a 2.4-fold risk of diabetes and lower physical fitness (maximal aerobic capacity 29.2 +/- 7.2 vs 32.1 +/- 7.0, p = 0.01) despite having similar reported physical activity to that of individuals with no family history. The same individuals also had reduced insulin secretion adjusted for insulin resistance, i.e. disposition index (p < 0.001) despite having higher BMI (27.4 +/- 4.6 vs 26.0 +/- 4.3 kg/m(2), p < 0.001). CONCLUSIONS/INTERPRETATION: Individuals with a family history of type 2 diabetes are characterised by lower physical fitness, which cannot solely be explained by lower physical activity. They also have an impaired capacity of beta cells to compensate for an increase in insulin resistance imposed by an increase in BMI. These defects should be important targets for interventions aiming at preventing type 2 diabetes in individuals with inherited susceptibility to the disease.

Assessing the effect of interaction between an FTO variant (rs9939609) and physical activity on obesity in 15,925 Swedish and 2,511 Finnish adults.

AIMS/HYPOTHESIS: Recent reports have suggested that genotypes at the FTO locus interact with physical activity to modify levels of obesity-related traits. We tested this hypothesis in two non-diabetic population-based cohorts, the first from southern Sweden and the second from the Botnia region of western Finland. METHODS: In total 2,511 Finnish and 15,925 Swedish non-diabetic middle-aged adults were genotyped for the FTO rs9939609 variant. Physical activity was assessed by questionnaires and standard clinical procedures were conducted, including measures of height and weight and glucose regulation. Tests of gene x physical activity interaction were performed using linear interaction effects to determine whether the effect of this variant on BMI is modified by physical activity. RESULTS: The minor A allele at rs9939609 was associated with higher BMI in both cohorts, with the per allele difference in BMI being about 0.13 and 0.43 kg/m(2) in the Swedish and Finnish cohorts, respectively (p < 0.0001). The test of interaction between physical activity and the rs9939609 variant on BMI was not statistically significant after controlling for age and sex in either cohort (Sweden: p = 0.71, Finland: p = 0.18). CONCLUSIONS/INTERPRETATION: The present report does not support the notion that physical activity modifies the effects of the FTO rs9939609 variant on obesity risk in the non-diabetic Swedish or Finnish adults studied here.

Age influences DNA methylation and gene expression of COX7A1 in human skeletal muscle.

AIMS/HYPOTHESIS: Reduced oxidative capacity of the mitochondria in skeletal muscle has been suggested to contribute to insulin resistance and type 2 diabetes. Moreover, a set of genes influencing oxidative phosphorylation (OXPHOS) is downregulated in diabetic muscle. Here we studied whether genetic, epigenetic and non-genetic factors influence a component of the respiratory chain, COX7A1, previously shown to be downregulated in skeletal muscle from patients with type 2 diabetes. The specific aims were to: (1) evaluate the impact of genetic (single nucleotide polymorphisms [SNPs]), epigenetic (DNA methylation) and non-genetic (age) factors on the expression of COX7A1 in human skeletal muscle; and (2) investigate whether common variants in the COX7A1 gene are associated with increased risk of type 2 diabetes. METHODS: COX7A1 mRNA expression was analysed in muscle biopsies from young (n = 110) and elderly (n = 86) non-diabetic twins and related to measures of in vivo metabolism. Genetic variants (three SNPs) from the COX7A1 locus were genotyped in the twins and in two independent type 2 diabetes case-control cohorts (n = 1466 and 6380, respectively). DNA methylation of the COX7A1 promoter was analysed in a subset of twins (ten young, ten elderly) using bisulphite sequencing. RESULTS: While DNA methylation of the COX7A1 promoter was increased in muscle from elderly compared with young twins (19.9 +/- 8.3% vs 1.8 +/- 2.7%; p = 0.035), the opposite was found for COX7A1 mRNA expression (elderly 1.00 +/- 0.05 vs young 1.68 +/- 0.06; p = 0.0005). The heritability of COX7A1 expression was estimated to be 50% in young and 72% in elderly twins. One of the polymorphisms investigated, rs753420, influenced basal COX7A1 expression in muscle of young (p = 0.0001) but not of elderly twins. The transcript level of COX7A1 was associated with increased in vivo glucose uptake and VO(2max) (p = 0.009 and p = 0.001, respectively). We did not observe any genetic association between COX7A1 polymorphisms and type 2 diabetes after correcting for multiple testing. CONCLUSIONS/INTERPRETATION: Our results provide further evidence for age as a factor influencing DNA methylation and expression of OXPHOS genes, and thereby in vivo metabolism.

The use of primary hepatocytes from brown trout (Salmo trutta lacustris) and the fish cell lines RTH-149 and ZF-L for in vitro screening of (anti)estrogenic activity of wood extractives.

Wood extractives are constituents of wood present in pulp and paper mill effluents, which may cause reproductive disturbances in fish. In the present study, we examined three cellular in vitro bioassays in order to assess (anti)estrogenic potencies of the wood extractives dehydroabietic acid (DHAA), isopimaric acid (IPA), betulinol (BET), hydroxymatairesinol (HMR), a phytosterol preparation (ULT), an oxidized phytosterol preparation (OX) and the model estrogen 17beta-estradiol (E2). The test systems used were primary hepatocyte cultures from brown trout and two piscine liver cell lines, RTH-149 and ZF-L. Estrogenicity was measured as vitellogenin (Vtg) secretion in cell culture medium. The primary hepatocytes cultures responded to E2 in a dose-dependent way. Vtg induction was inhibited with a simultaneous exposure to 4-hydroxytamoxifen (4-HT) indicating an estrogen receptor mediated response. DHAA and ULT induced a weak statistically non-significant Vtg production, and weak additive effects were found in some combination treatments of wood extractives and E2. Additionally, a pulp mill effluent tested on primary hepatocytes induced Vtg production when exposed at a 1% dilution. The cell lines secreted negligible amounts of Vtg upon E2 stimulation, which was neither dose-dependent nor inhibited by 4-HT. In conclusion, trout primary hepatocytes could be useful for assessing (anti)estrogenic potencies of compounds, and the wood extractives and a pulp mill effluent showed only weak or no estrogenic activity in this model system.

Polymorphisms in the gene encoding the voltage-dependent Ca(2+) channel Ca (V)2.3 (CACNA1E) are associated with type 2 diabetes and impaired insulin secretion.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion is dependent on the electrical activity of beta cells; hence, genes encoding beta cell ion channels are potential candidate genes for type 2 diabetes. The gene encoding the voltage-dependent Ca(2+) channel Ca(V)2.3 (CACNA1E), telomeric to a region that has shown suggestive linkage to type 2 diabetes (1q21-q25), has been ascribed a role for second-phase insulin secretion. METHODS: Based upon the genotyping of 52 haplotype tagging single nucleotide polymorphisms (SNPs) in a type 2 diabetes case-control sample (n = 1,467), we selected five SNPs that were nominally associated with type 2 diabetes and genotyped them in the following groups (1) a new case-control sample of 6,570 individuals from Sweden; (2) 2,293 individuals from the Botnia prospective cohort; and (3) 935 individuals with insulin secretion data from an IVGTT. RESULTS: The rs679931 TT genotype was associated with (1) an increased risk of type 2 diabetes in the Botnia case-control sample [odds ratio (OR) 1.4, 95% CI 1.0-2.0, p = 0.06] and in the replication sample (OR 1.2, 95% CI 1.0-1.5, p = 0.01 one-tailed), with a combined OR of 1.3 (95% CI 1.1-1.5, p = 0.004 two-tailed); (2) reduced insulin secretion [insulinogenic index at 30 min p = 0.02, disposition index (D (I)) p = 0.03] in control participants during an OGTT; (3) reduced second-phase insulin secretion at 30 min (p = 0.04) and 60 min (p = 0.02) during an IVGTT; and (4) reduced D (I) over time in the Botnia prospective cohort (p = 0.05). CONCLUSIONS/INTERPRETATION: We conclude that genetic variation in the CACNA1E gene contributes to an increased risk of the development of type 2 diabetes by reducing insulin secretion.

Genetic testing for maturity onset diabetes of the young: uptake, attitudes and comparison with hereditary non-polyposis colorectal cancer.

AIMS/HYPOTHESIS: Mutations in hepatic nuclear factor 1alpha cause a monogenic form of diabetes, maturity onset diabetes of the young type 3 (MODY3). Our aim was (1) to assess the uptake of genetic testing for MODY3 and to determine factors affecting it, and (2) to compare attitudes to predictive genetic testing between families with MODY3 and a previously studied group at risk of hereditary non-polyposis colorectal cancer (HNPCC). METHODS: Adult members of two extended MODY3 pedigrees, either with diabetes or a 50% risk of having inherited the mutation (n=144, age 18-60 years), were invited to an educational counselling session followed by a possibility to obtain the gene test result. Data were collected through questionnaires before counselling and 1 month after the test disclosure. RESULTS: Eighty-nine out of 144 (62%) participated in counselling, and all but one wanted the test result disclosed. No significant sociodemographic differences were observed between the participants and non-participants. The counselling uptake was similar among diabetic and non-diabetic subjects. Uncertainty about the future and the risk for the children were the most common reasons to take the gene test. At follow-up, most subjects in both MODY3 (100%) and HNPCC (99%) families were satisfied with their decision to take the test and trusted the result. The majority of both diabetic and non-diabetic subjects considered that the MODY3 gene test should be offered either in childhood (50 and 37%) or as a teenager (30 and 37%). CONCLUSIONS: Genetic testing for MODY3 was well accepted among both diabetic and non-diabetic participants. The subjects found the gene test reliable and they were satisfied with their decision regarding the predictive test.

Effects of the wood extractive betulinol and 17beta-oestradiol on reproduction in zebrafish, Danio rerio (Hamilton)--complications due to a bacterial infection.

Zebrafish were exposed to the wood extractive betulinol (5 microg L(-1)) and to 17beta-oestradiol (E2, 0.27 microg L(-1)) for 8 weeks in an attempt to study the possible endocrine-disrupting activity of betulinol. Females exposed to betulinol showed increased spawning intensity, while males exposed to betulinol and E2 had increased incidences of structural alterations in the testes. However, histological examination of the fish revealed that they were infected by acid-fast bacteria suspected to be Mycobacterium sp. despite a careful examination of their health state prior to the onset of the experiment. Fish exposed to betulinol and E2 showed more serious consequences of the bacterial infection than control fish indicating that the test chemicals had weakened the immune defence of the fish. When the exposure was repeated with healthy fish, an increase in the proportion of spermatogonia was seen in the testes of betulinol-treated males. A similar alteration, although not statistically significant, was also seen in the first experiment. However, no increase in the incidences of structural alterations in the testes was seen in betulinol- and E2-treated fish in the second experiment. Our study indicates that betulinol might have an endocrine-disrupting effect in zebrafish, but the increase in incidences of structural alterations in the testes might have been caused by a synergistic action between the test compounds and the bacterial infection. Our study stresses the importance of carefully checking the health of experimental fish, not only prior to the onset of an experiment but also upon termination of the experiment, in order to avoid misinterpretation of the results.

Familiality of metabolic abnormalities is dependent on age at onset and phenotype of the type 2 diabetic proband.

To determine the impact of a family history of the common form of type 2 diabetes and the phenotype of the proband on anthropometric and metabolic variables in normoglycemic first-degree relatives, we studied 2,100 first-degree relatives of patients with the common form of type 2 diabetes (FH+) and 388 subjects without a family history of diabetes (FH-). All subjects participated in an oral glucose tolerance test to allow measurement of insulin secretion [30-min incremental insulin/glucose (I/G 30)] and insulin sensitivity [homeostasis model assessment (HOMA) of insulin resistance (IR)]. A subset participated in a euglycemic clamp (n = 75) and an intravenous glucose tolerance test (n = 300). To study the effect of a particular phenotype of the proband, insulin secretion and sensitivity were also compared between first-degree relatives of diabetic probands with high and low waist-to-hip ratio (WHR) and probands with early and late onset of diabetes. FH+ subjects were more insulin resistant, as seen from a higher HOMA-IR index (P = 0.006) and a lower rate of insulin-stimulated glucose uptake (P = 0.001) and had more features of the metabolic syndrome (P = 0.02, P = 0.0002) compared with FH- subjects. Insulin secretion adjusted for insulin resistance (disposition index, DI) was also lower in the FH+ vs. FH- subjects (P = 0.04). Relatives of diabetic probands with a high WHR had reduced insulin-mediated glucose uptake compared with relatives of probands with a low WHR (P = 0.04). Relatives of diabetic patients with age at onset <44 yr had higher HOMA IR (P < 0.005) and lower DI (P < 0.005) than relatives of patients with age at onset >65 yr (highest quartile). We conclude that early age at onset of type 2 diabetes and abdominal obesity have a significant influence on the metabolic phenotype in the nondiabetic first-degree relative.

Culturing and characterization of astrocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss).

An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly-L-lysine (PLL), poly-D-lysin (PDL) or poly-L-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1 x 10(7) cells/cm(2) confluence was obtained within 3-4 weeks at an incubation temperature of 18 degrees C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+](i), 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and gamma-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture.

The metabolic syndrome influences the risk of chronic complications in patients with type II diabetes.

AIMS/HYPOTHESIS: We examined features of the metabolic syndrome to see if they modified the risk of chronic diabetic complications in patients with Type II (non-insulin-dependent) diabetes mellitus. METHODS: A total of 85 randomly selected patients with the metabolic syndrome (WHO definition) were compared with 85 Type II diabetic patients matched for age, sex, duration of diabetes, glycaemic control and without the syndrome to assess the microvascular and macrovascular complications. RESULTS: The patients with the metabolic syndrome had a higher prevalence of cardiovascular disease (52 vs 21%, p < 0.001), microalbuminuria or macroalbuminuria (23 vs 7%, p = 0.003) and distal neuropathy (16 vs 6%, p = 0.048) than patients without the syndrome. The patients with the metabolic syndrome had smaller LDL particle size (25.4+/-1.4 vs 26.4+/-1.1 nm; p < 0.001), which correlated with the ratio of serum triglycerides to HDL cholesterol (r = -0.64, p < 0.001). In a multiple logistic regression analysis the metabolic syndrome was associated with coronary heart disease (RR 3.84, p < 0.001) and microalbuminuria (RR 3.99, p = 0.01). Small LDL particle size was independently associated with neuropathy (RR 0.58; p = 0.04), whereas a high HbA1c was related to neuropathy (RR 1.69, p = 0.04), retinopathy (RR 1.53, p = 0.002) and microalbuminuria (RR 1.54, p = 0.01). CONCLUSION/INTERPRETATION: Although chronic hyperglycaemia is the main predictor of microvascular complications in patients with Type II diabetes, this risk is modified by some of the components of the metabolic syndrome.

Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions.

Aged HS erythrocytes with a defined primary defect in band 3 protein or ankyrin were incubated with amphiphiles (detergents) at sublytic concentrations (37 C, 60 min) or glucose-starved (37 C, 24 h). In line with previous studies, the release of AChE (exovesicles) from HS erythrocytes during glucose-starvation was significantly higher (11%) compared to that from control erythrocytes (1%). Control and HS cells responded, however, similarly to amphiphile-treatment (non-starving conditions). Amphiphiles induced similar types of shape alterations and a similar amount of AChE release (14-15%). Furthermore, the size and shape of amphiphile-induced exo- and endovesicles released from control and HS erythrocytes were similar. The results suggest that the stability properties of the membrane are not seriously disturbed in aged HS erythrocytes under non-starving conditions.

Cardiovascular morbidity and mortality associated with the metabolic syndrome.

OBJECTIVE: To estimate the prevalence of and the cardiovascular risk associated with the metabolic syndrome using the new definition proposed by the World Health Organization RESEARCH DESIGN AND METHODS: A total of 4,483 subjects aged 35-70 years participating in a large family study of type 2 diabetes in Finland and Sweden (the Botnia study) were included in the analysis of cardiovascular risk associated with the metabolic syndrome. In subjects who had type 2 diabetes (n = 1,697), impaired fasting glucose (IFG)/impaired glucose tolerance (IGT) (n = 798) or insulin-resistance with normal glucose tolerance (NGT) (n = 1,988), the metabolic syndrome was defined as presence of at least two of the following risk factors: obesity, hypertension, dyslipidemia, or microalbuminuria. Cardiovascular mortality was assessed in 3,606 subjects with a median follow-up of 6.9 years. RESULTS: In women and men, respectively, the metabolic syndrome was seen in 10 and 15% of subjects with NGT, 42 and 64% of those with IFG/IGT, and 78 and 84% of those with type 2 diabetes. The risk for coronary heart disease and stroke was increased threefold in subjects with the syndrome (P < 0.001). Cardiovascular mortality was markedly increased in subjects with the metabolic syndrome (12.0 vs. 2.2%, P < 0.001). Of the individual components of the metabolic syndrome, microalbuminuria conferred the strongest risk of cardiovascular death (RR 2.80; P = 0.002). CONCLUSIONS: The WHO definition of the metabolic syndrome identifies subjects with increased cardiovascular morbidity and mortality and offers a tool for comparison of results from diferent studies.