L. Hansen

Studies of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) gene in relation to insulin sensitivity among glucose tolerant Caucasians

Abstract.Aims/hypothesis: We examined whether the Pro12-Ala polymorphism of the human peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) gene was related to altered insulin sensitivity among glucose-tolerant subjects or a lower accumulated incidence or prevalence of IGT and Type II (non-insulin-dependent) diabetes mellitus among Scandinavian Caucasians. Methods: The Pro12Ala polymorphism was examined using PCR-RFLP. Whole-body insulin sensitivity measured under hyperinsulinaemic-euglycaemic conditions was estimated in a population-based sample of 616 glucose tolerant Swedish Caucasian men at age 70. In addition, insulin sensitivity index was measured using IVGTT and Bergman minimal modelling in a population-based sample of 364 young healthy Danish Caucasians. Finally, we evaluated whether the polymorphism predicted Type II diabetes and IGT in 841 seventy-year-old Swedish men. A case-control study was carried out in 654 unrelated Danish Type II diabetic patients and 742 Danish glucose tolerant subjects matched for age and sex. Results: Whole-body insulin sensitivity was significantly improved in carriers compared with non-carriers of the Ala-allele of the codon 12 polymorphism in Swedish Caucasian men (6.0 ± 2.5 vs 5.6 ± 2.5 mg · kg–1· min–1· [mU/l]–1· 100, p = 0.044). The same tendency, but not significant, was observed in the insulin sensitivity index among the group of young healthy Danish Caucasians. The incidence of Type II diabetes and IGT among the Swedish subjects at the age of 70 was similar in the three genotype-groups of the Pro12Ala variant and the Ala-allele was not related to a lower prevalence of Type II diabetes in Danish Caucasians. Conclusion/interpretation: The Ala-allele of the PPAR-γ2 polymorphism is associated with improved whole body insulin sensitivity among Swedish Caucasians. [Diabetologia (2001) 44: 1170–1176]

Genetics of type 2 diabetes mellitus: status and perspectives

Throughout the last decade, molecular genetic studies of non‐autoimmune diabetes mellitus have contributed significantly to our present understanding of this diseases complex aetiopathogenesis. Monogenic forms of diabetes (maturity‐onset diabetes of the young, MODY) have been identified and classified into MODY1–6 according to the mutated genes that by being expressed in the pancreatic β‐cells confirm at the molecular level the clinical presentation of MODY as a predominantly insulin secretory deficient form of diabetes mellitus. Genomewide linkage studies of presumed polygenic type 2 diabetic populations indicate that loci on chromosomes 1q, 5q, 8p, 10q, 12q and 20q contain susceptibility genes. Yet, so far, the only susceptibility gene, calpain‐10 (CAPN10), which has been identified using genomewide linkage studies, is located on chromosome 2q37. Mutation analyses of selected ‘candidate’ susceptibility genes in various populations have also identified the widespread Pro12Ala variant of the peroxisome proliferator‐activated receptor‐γ and the common Glu23Lys variant of the ATP‐sensitive potassium channel, Kir6.2 (KCNJ11). These variants may contribute significantly to the risk type 2 diabetes conferring insulin resistance of liver, muscle and fat (Pro12Ala) and a relative insulin secretory deficiency (Glu23Lys). It is likely that, in the near future, the recent more detailed knowledge of the human genome and insights into its haploblocks together with the developments of high‐throughput and cheap genotyping will facilitate the discovery of many more type 2 diabetes gene variants in study materials, which are statistically powered and phenotypically well characterized. The results of these efforts are likely to be the platform for major progress in the development of personalized antidiabetic drugs with higher efficacy and few side effects.

Transcriptional profiling of myotubes from patients with type 2 diabetes: no evidence for a primary defect in oxidative phosphorylation genes

Aims/hypothesisMicroarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independently of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes.MethodsUsing cDNA microarray technology and global pathway analysis with the Gene Map Annotator and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes.ResultsNo single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either method of global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis (PPARGC1A and NRF1) or in mitochondrial mass between diabetic and control myotubes.Conclusions/interpretationThese results support the hypothesis that impaired mitochondrial biogenesis is not a primary defect in the sequence of events leading to insulin resistance and type 2 diabetes.

Genetic variation in the hepatocyte nuclear factor-1a gene in Danish Caucasians with late-onset NIDDM

Summary Non-insulin-dependent diabetes mellitus (NIDDM) is a phenotypically and genetically heterogeneous disorder. A recent random genome mapping study has localized a locus termed NIDDM2 that maps to the region of chromosome 12 that includes MODY3, one of the three genes responsible for maturity-onset diabetes of the young, a monogenic form of NIDDM characterized by early age of onset and autosomal dominant inheritance. These findings suggest that NIDDM2 and MODY3 may represent different alleles of the same gene. MODY3 has recently been shown to be the gene encoding the transcription factor hepatocyte nuclear factor-1α (HNF-1α) thereby allowing us to determine whether mutations in the HNF-1α gene are present in subjects with late-onset NIDDM. We screened 84 white NIDDM patients of Danish ancestry and found four nucleotide substitutions that changed the sequence of HNF-1α, Ile27→Leu, Ala98→Val, Ser487→Asn and Arg583 →Gln, five nucleotide substitutions that were silent and did not change the amino acid, Leu17, Gly288, Leu459 and Thr515, and five substitutions in the intron regions. The frequencies of the codon 27, 98 and 487 amino acid variants were similar in 245 unrelated NIDDM patients and 242 age-matched control subjects. The Arg583→Gln mutation was found in 2 of 245 NIDDM patients and in none of the control subjects. Thus, genetic variation in the HNF-1α gene is not a common factor contributing to NIDDM susceptibility in white subjects of Danish ancestry. [Diabetologia (1997) 40: 473–475]

Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3α and β isoforms in patients with NIDDM

Summary Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active dephosphorylated state. In a search for genetic defects responsible for the decreased insulin stimulated glycogen synthesis seen in patients with non-insulin-dependent diabetes mellitus (NIDDM) and their glucose-tolerant first-degree relatives we have performed mutational analysis of the coding region of the 2 isoforms of GSK-3α and GSK-3β in 72 NIDDM patients and 12 control subjects. No structural changes were detected apart from a few silent mutations. Mapping of the GSK-3α to chromosome 19q13.1–13.2 and the GSK-3β to chromosome 3q13.3-q21 outside known genetic loci linked to NIDDM further makes it unlikely that these genes are involved in the pathogenesis of common forms of NIDDM. [Diabetologia (1997) 40: 940–946]

Genetic variability of the SUR1 promoter in relation to beta-cell function and Type II diabetes mellitus.

Abstract.Aims/hypothesis: We aimed to examine the promoter of SUR1 for genetic variation and to determine if variants were associated with Type II (non-insulin-dependent) diabetes mellitus or measures of beta-cell function. Methods: We examined 465 bp upstream of the ATG site in 46 Type II diabetic patients and 15 glucose tolerant control subjects by SSCP-heteroduplex analysis. Results: We identified an a → t substitution 437 bp upstream of the ATG site. The allelic frequency was similar in 455 unrelated Type II diabetic patients and in 203 glucose tolerant control subjects matched for age (0.036, [95 % CI 0.019–0.053] vs 0.034 [95 % CI 0.009–0.059]; p = 0.92). Among the glucose tolerant subjects there were no differences between non-carriers (n = 189) and carriers (n = 14) of the variant in fasting values or 30 min values of plasma glucose and serum insulin during an oral glucose tolerance test. In a study of 233 glucose tolerant offspring of and spouses to Danish Caucasian Type II diabetic patients, non-carriers (n = 193) and carriers (n = 37) of the –437 a/t polymorphism did not differ in glucose or tolbutamide stimulated insulin response during an intravenous glucose tolerance test with intravenous tolbutamide injection [AUCs-insulin (0–8) min, 2290 ± 1660 vs 2308 ± 1935 pmol/l · min and AUCs-insulin(20–30 min), 3113 ± 2033 vs. 3393 ± 2830 pmol/l · min, respectively]. Conclusion/interpretation: We have identified a novel a/t polymorphism of the SUR1 gene promoter which is not associated with Type II diabetes mellitus or measures of beta-cell function. Previous reported non-functional variants of SUR1 associated with Type II diabetes mellitus still need to be accounted for. [Diabetologia (2001) 44: 1330–1334]

Correspondence analysis of microarray time-course data in case-control design

Although different statistical approaches have been proposed for analyzing microarray time-course data, method for analyzing such data collected using the popular case-control design in clinical investigations has not been proposed perhaps due to the increased complexity for the existing parametric or non-parametric approaches. In this paper, we introduce a new multivariate data analyzing technique, the correspondence analysis, to analyze the high dimensional microarray time-course data in case-control design. We show, through an example on type 2 diabetes, how the nice features of the correspondence analysis can be use to explore the various time-course gene expression profiles that exist in the data. By coordinating and examining the projections on the reduced dimensions by both the genes and the time-course experiments, we are able to identify important genes and time-course patterns and make inferences on their biological relevance. Using the sample replicates, we propose a bootstrap procedure for inferring the significance of contributions on the leading dimensions by both the time-course experiments and the genes. Striking differences in the time-course patterns in the normal controls and diabetes patients have been revealed. In addition, the method also identifies genes that display similar or comparable time-course expression patterns shared by both the cases and the controls. We conclude that our correspondence analysis based approach can be a useful tool for analyzing high dimensional microarray data collected in clinical investigations.

Studies of variability in the PTEN gene among Danish caucasian patients with Type II diabetes mellitus.

Aim/hypothesis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN) has recently been characterized as a novel member in the expanding network of proteins regulating the intracellular effects of insulin. By dephosphorylation of phosphatidyl-inositol-(3, 4, 5)-trisphosphate (PIP3) the PTEN protein regulates the insulin-dependent phosphoinositide 3-kinase (PI3K) signalling cassette and accordingly might function as a regulator of insulin sensitivity in skeletal muscle and adipose tissue. In this study we tested PTEN as a candidate gene for insulin resistance and late-onset Type II (non-insulin-dependent) diabetes mellitus in a Danish Caucasian population. Methods. The nine exons of the PTEN, including intronic flanking regions were analysed by PCR-SSCP and heteroduplex analysis in 62 patients with insulin-resistant Type II diabetes. Results. No mutations predicted to influence the expression or biological function of the PTEN protein but four intronic polymorphisms were identified: IVS1–96 A→G (allelic frequency 0.22, 95 % CI: 0.12–0.32), IVS3 + 99 C→T (0.01, CI: 0–0.03), IVS7–3 TT→T (0.10, CI: 0.03–0.18) and IVS8 + 32 G→T (0.35, CI: 0.23–0.47). The IVS8 + 32 G→T polymorphism was used as a bi-allelic marker for the PTEN locus and examined in 379 patients with Type II diabetes and in 224 control subjects with normal glucose tolerance. The IVS8 + 32 G→T polymorphism in the PTEN was not associated with Type II diabetes and it did not have any effect on body-mass index, blood pressure, HOMA insulin resistance index, or concentrations of plasma glucose, serum insulin or serum C peptide obtained during an oral glucose tolerance test (OGTT). Conlusion/interpretation. Variability in the PTEN is not a common cause of Type II diabetes in the Danish Caucasian population. [Diabetologia (2001) 44: 237–240]

Polymorphisms in the neurogenin 3 gene (NEUROG) and their relation to altered insulin secretion and diabetes in the Danish Caucasian population

Aim/hypothesis. Neurogenin 3 (NEUROG3) is a member of the subfamily of basic-helix-loop-helix (bHLH) transcription factors involved in differentiation of the endocrine pancreas and therefore a possible candidate gene for maturity-onset diabetes of the young (MODY) and Type II (non-insulin-dependent) diabetes mellitus.¶Methods. Using Polymerase-chain-reaction single-stranded-conformation polymorphism, we examined the coding region including the 5-untranslated and 3- untranslated regions of the NEUROG3 in a group of 133 diabetic patients comprising 19 MODY patients, 19 patients with dominant Type I diabetes, and 31 early-onset and 64 late-onset Type II diabetic patients.¶Results. We found two missense mutations, Gly167Arg and Ser199Phe, as well as two non-coding variants in the 5 UTR, a c → t nucleotide variant at position –10 upstream of the start codon in one MODY patient and a 2 base pair (CA) deletion polymorphism at position –44/-45. Allele frequencies measured in 377 diabetic patients and in 217 glucose-tolerant control subjects were: Gly167Arg, 0.04 (95 % CI: 0.02–0.06) and 0.04 (0.02–0.06); Ser199Phe, 0.31 (0.26–0.36) and 0.30 (0.24–0.36); –44–45delCA, 0.33 (0.31–0.35) and 0.35 (0.32–0.38), respectively. Both Ser199Phe and the –44–45delCA were in linkage disequilibrium (χ2 > 60) but the Ser199Phe and the –44–45delCA polymorphism were not associated with consistent changes in fasting- or glucose-induced insulin secretion in 249 glucose-tolerant offspring (first-degree relatives) of Type II diabetic parents or in 217 unrelated middle-aged glucose tolerant subjects.¶Conclusion/interpretation. Genetic variability in NEUROG3 is not associated with dominant Type I diabetes, MODY, Type II diabetes or changes in insulin secretion in the Danish Caucasians examined subjects. [Diabetologia (2001) 44: 123–126]

Adenovirus-mediated expression of a naturally occurring Asp905Tyr variant of the glycogen-associated regulatory subunit of protein phosphatase-1 in L6 myotubes

Aims/hypothesis. The glycogen-associated protein phosphatase-1 (PP1G) is thought to play an important part in the regulation of skeletal muscle glycogen content. We have previously identified an Asp905Tyr polymorphism of the glycogen-associated regulatory subunit of the protein phosphatase 1 (PPP1R3) gene which among healthy subjects was associated with decreased insulin stimulated non-oxidative glucose metabolism, i. e. primary glycogen synthesis. In this study, the functional effect of the polymorphism was examined in vitro.¶Methods. Wild type (PPP1R3-Asp905) and mutant (PPP1R3-Tyr905) PPP1R3 were expressed in L6 myotubes using adenovirus-mediated gene transfer. Basal and insulin-stimulated glucose uptake and glycogen synthesis were measured. Furthermore, the sensitivity of glycogen synthesis to a cyclic AMP agonist was measured.¶Results. Compared with green fluorescent protein-transduced myotubes and non-transduced myotubes, overexpression of PPP1R3-Asp905 and PPP1R3-Tyr905 increased both basal and insulin-stimulated glycogen synthesis approximately twofold. Treatment of both non-transduced and PPP1R3-transduced L6 myotubes with a cAMP agonist decreased both basal and insulin-stimulated glycogen synthesis by about 40 %. Overexpression of PPP1R3 did not affect either basal or insulin-stimulated 2-deoxy-d-glucose uptake compared with green fluorescent protein-transduced cells.¶Conclusion/interpretation. Results obtained from L6 myotubes transduced with PPP1R3-Asp905 or PPP1R3-Tyr905 showed no statistically significant difference. Therefore, the Asp905Tyr variant alone is unlikely to account for the decreased insulin stimulated non-oxidative glucose metabolism observed in the human study reported previously. [Diabetologia (2000) 43: 718–722]