DNA microarrays can be used to identify gene expression changes characteristic of human disease. This is challenging, however, when relevant differences are subtle at the level of individual genes. We introduce an analytical strategy, Gene Set Enrichment Analysis, designed to detect modest but coordinate changes in the expression of groups of functionally related genes. Using this approach, we identify a set of genes involved in oxidative phosphorylation whose expression is coordinately decreased in human diabetic muscle. Expression of these genes is high at sites of insulin-mediated glucose disposal, activated by PGC-1alpha and correlated with total-body aerobic capacity. Our results associate this gene set with clinically important variation in human metabolism and illustrate the value of pathway relationships in the analysis of genomic profiling experiments.

Genetic variants in the gene encoding for transcription factor-7-like 2 (TCF7L2) have been associated with type 2 diabetes (T2D) and impaired β cell function, but the mechanisms have remained unknown. We therefore studied prospectively the ability of common variants in TCF7L2 to predict future T2D and explored the mechanisms by which they would do this. Scandinavian subjects followed for up to 22 years were genotyped for 3 SNPs (rs7903146, rs12255372, and rs10885406) in TCF7L2, and a subset of them underwent extensive metabolic studies. Expression of TCF7L2 was related to genotype and metabolic parameters in human islets. The CT/TT genotypes of SNP rs7903146 strongly predicted future T2D in 2 independent cohorts (Swedish and Finnish). The risk T allele was associated with impaired insulin secretion, incretin effects, and enhanced rate of hepatic glucose production. TCF7L2 expression in human islets was increased 5-fold in T2D, particularly in carriers of the TT genotype. Overexpression of TCF7L2 in human islets reduced glucose-stimulated insulin secretion. In conclusion, the increased risk of T2D conferred by variants in TCF7L2 involves the enteroinsular axis, enhanced expression of the gene in islets, and impaired insulin secretion.

Summary. From a previously reported 5-year screening programme of 6,956 47-49-year-old Malm6 males, a series of 41 subjects with early-stage Type 2 (non-insulin-dependent) diabetes mellitus and 181 subjects with impaired glucose tolerance were selected for prospective study and to test the feasibility aspect of long-term intervention with an emphasis on life-style changes. A 5-year protocol, including an initial 6-months (randomised) pilot study, consisting of dietary treatment and/or increase of physical activity or training with annual check-ups, was completed by 90% of subjects. Body weight was reduced by 2.3-3.7 % among participants, whereas values increased by 0.5-1.7 % in non-intervened subjects with impaired glucose tolerance and in normal control subjects (p < 0.0001); maximal oxygen uptake (ml. min -1. kg -1) was increased by 10-14 % vs decreased by 5-9 %, respectively (p < 0.0001). Glucose tolerance was normalized in > 50% of subjects with impaired glucose tolerance, the accumulated incidence of diabetes was 10.6 %, and more than 50 % of the diabetic patients were in remission after a mean follow-up of 6 years. Blood pressure, lipids, and hyperinsulinaemia were reduced and early insulin responsiveness to glucose loading preserved. Improvement in glucose tolerance was correlated to weight reduction (r --0.19, p < 0.02) and increased fitness (r = 0.22, p < 0.02). Treatment was safe, and mortality was low (in fact 33 % lower than in the remainder of the cohort). We conclude that long-term intervention in the form of diet and physical exercise is feasible even on a large scale, and that substantial metabolic improvement can be achieved which may contribute to prevent or postpone manifest diabetes.Key words: Impaired glucose tolerance, Type 2 (non-insulindependent) diabetes mellitus, oral glucose tolerance test, insulin, long-term intervention, diet, physical exercise.Diabetes mellitus and its complications constitute a major health problem in modern societies [1,2]. Type 2 (non-insulin-dependent) diabetes is associated with an increased frequency of cardiovascular risk factors [3] and subsequent cardiovascular disease and mortality [4][5][6]; and in the agegroup under consideration here, the expected reduction in lifespanis 5-7 years [4]. Inpreviousstudies, annualprogression rates from impaired glucose tolerance (IGT) to Type 2 diabetes of 2-3 % have been found [7][8][9], though the causal link with known risk factors remains unclear [10]. Further preventive studies, with an emphasis on life-style changes, e. g., diet and physical exercise, are needed [11][12][13]. Apart from the fact that the efficacy of oral agents in preventing Type 2 diabetes and its sequelae has yet to be established [13], non-pharmacological treatment is an approach of great importance, not onlyvis-fi-vis glucosemetabolismbut also vis-a-vis the overallmetabolic profile [14,15].We have previously reported on studies where 6-12-month periods of dietary treatment or physical training (or both) were found to be beneficial in cases of IGT...

Epigenetic mechanisms are implicated in gene regulation and the development of different diseases. The epigenome differs between cell types and has until now only been characterized for a few human tissues. Environmental factors potentially alter the epigenome. Here we describe the genome-wide pattern of DNA methylation in human adipose tissue from 23 healthy men, with a previous low level of physical activity, before and after a six months exercise intervention. We also investigate the differences in adipose tissue DNA methylation between 31 individuals with or without a family history of type 2 diabetes. DNA methylation was analyzed using Infinium HumanMethylation450 BeadChip, an array containing 485,577 probes covering 99% RefSeq genes. Global DNA methylation changed and 17,975 individual CpG sites in 7,663 unique genes showed altered levels of DNA methylation after the exercise intervention (q<0.05). Differential mRNA expression was present in 1/3 of gene regions with altered DNA methylation, including RALBP1, HDAC4 and NCOR2 (q<0.05). Using a luciferase assay, we could show that increased DNA methylation in vitro of the RALBP1 promoter suppressed the transcriptional activity (p = 0.03). Moreover, 18 obesity and 21 type 2 diabetes candidate genes had CpG sites with differences in adipose tissue DNA methylation in response to exercise (q<0.05), including TCF7L2 (6 CpG sites) and KCNQ1 (10 CpG sites). A simultaneous change in mRNA expression was seen for 6 of those genes. To understand if genes that exhibit differential DNA methylation and mRNA expression in human adipose tissue in vivo affect adipocyte metabolism, we silenced Hdac4 and Ncor2 respectively in 3T3-L1 adipocytes, which resulted in increased lipogenesis both in the basal and insulin stimulated state. In conclusion, exercise induces genome-wide changes in DNA methylation in human adipose tissue, potentially affecting adipocyte metabolism.

Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 males and 94 females and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1050 genes (e.g. FHL2, NOX4 and PLG). Interestingly, many reported epigenetic biomarkers of aging in blood, including ELOVL2, FHL2, KLF14 and GLRA1, also showed significant correlations between adipose tissue DNA methylation and age in our study. The most significant association between age and adipose tissue DNA methylation was found upstream of ELOVL2. We identified 2825 genes (e.g. FTO, ITIH5, CCL18, MTCH2, IRS1 and SPP1) where both DNA methylation and expression correlated with BMI. Methylation at previously reported HIF3A sites correlated significantly with BMI in females only. HbA1c (range 28-46 mmol/mol) correlated significantly with the methylation of 711 sites, annotated to, for example, RAB37, TICAM1 and HLA-DPB1. Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation of candidate genes for obesity, type 2 diabetes and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue.

Oxidative stress has been ascribed a role in the pathogenesis of diabetes and its complications, and stress proteins have been shown to protect organisms in vitro and in vivo against oxidative stress. To study the putative role of one of the most abundant cytoprotective stress proteins, inducible cytoplasmic 72-kDa-mass heat shock protein (Hsp-72), in the pathogenesis of diabetes, we measured its mRNA concentration in muscle biopsies from six type 2 diabetic patients and six healthy control subjects (protocol 1) as well as in 12 twin pairs discordant for type 2 diabetes and 12 control subjects undergoing a euglycemic-hyperinsulinemic clamp in combination with indirect calorimetry (protocol 2). The amount of Hsp-72 mRNA in muscle was significantly lower in type 2 diabetic patients than in healthy control subjects (in protocol 1: 5.2 ؎ 2.2 vs. 53 ؎ 32 million copies of Hsp-72 mRNA/g total RNA, n ‫؍‬ 6, P ‫؍‬ 0.0039; in protocol 2: 3.2 ؎ 3.3 vs. 43 ؎ 31 million copies of Hsp-72 mRNA/g total RNA, n ‫؍‬ 12, P ‫؍‬ 0.0001). Hsp-72 mRNA levels were also markedly reduced in the nondiabetic co-twins compared with healthy control subjects (5.8 ؎ 5.0 vs. 43 ؎ 31, n ‫؍‬ 12, P ‫؍‬ 0.0001), but they were also statistically significantly different from their diabetic co-twins when the difference between the pairs was compared (P ‫؍‬ 0.0280). Heat shock protein mRNA content in muscle of examined patients correlated with the rate of glucose uptake and other measures of insulin-stimulated carbohydrate and lipid metabolism. In conclusion, the finding of decreased levels of Hsp-72 mRNA in skeletal muscle of patients with type 2 diabetes and its relationship with insulin resistance raises the question of whether heat shock proteins are involved in the pathogenesis of skeletal muscle insulin resistance in type 2 diabetes.

Cross-sectional studies have reported strong correlations between plasma levels of complement C3, insulin, and glucose. This prospective study explored whether elevated levels of C3, C4, and other inflammationsensitive plasma proteins (ISPs; fibrinogen, orosomucoid, ␣1-antitrypsin, haptoglobin, and ceruloplasmin) are associated with the development of diabetes. Plasma proteins were measured in 2,815 nondiabetic healthy men, age 38 -50 years, who were reexamined after a mean follow-up of 6.1 years. Diabetes development (n ‫؍‬ 123) was studied in relation to baseline levels of plasma proteins. After adjusting for age, screening year, and glucose at baseline, the odds ratio (95% CI) for developing diabetes was 1.00, 2.4 (1.1-5.3), 2.9 (1.4 -6.0), and 5.6 (2.8 -10.9), respectively, for men with C3 in the 1st, 2nd, 3rd, and 4th quartiles (trend: P < 0.00001). Fibrinogen, haptoglobin, C4, and the number of elevated ISPs were also related to future diabetes in this model. Only C3 was significantly associated with diabetes development after further adjustments for potential confounders, including BMI, insulin, and other inflammatory markers. We concluded that the risk of developing diabetes is related to levels of complement C3. Diabetes 54:570 -575, 2005 C omplement C3 and C4 are the major plasma proteins of the immune system complement pathways. The synthesis of these proteins is increased in response to inflammation and infection but at a slower rate than for traditional acute phase proteins (1,2). Both C3 and C4 have shown substantial correlations with obesity (3-6), and high gene expression of these complement components has been reported in omental adipose tissue in obese men (3). High C3 levels have been reported in subjects with diabetes and insulin resistance (6 -9).It has been shown that the cleavage product of C3, acylation-stimulating protein (ASP), is a paracrine metabolic factor that stimulates the uptake of glucose and fat storage in human adipose tissue (10 -12). ASP deficiency in mice has been associated with resistance to weight gain on a high-fat diet, despite increased food intake (13). However, whether C3 is associated with an increased risk of developing diabetes is unknown.Several inflammatory markers have been associated with the incidence of diabetes, including C-reactive protein (CRP) (14 -17), orosomucoid (18), sialic acid (18), white blood cells (18,19), and interleukin (IL)-6 (20). It has been proposed that diabetes is a disease of the innate immune system (21). However, several studies have reported a nonsignificant relation between inflammation and incidence of diabetes. Negative or inconclusive results have been reported for CRP (17,22), haptoglobin (18), fibrinogen (16,18), white blood cells (16), and ␣1-antitrypsin (18). It is unclear whether the discrepancies among studies are related to differences with respect to inflammatory markers, study populations, or other factors.Previous substudies from the Malmö Preventive Study have shown that the risk of developing cardiovascular diseases,...

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