Body composition is the main determinant for the difference in type 2 diabetes pathophysiology between Japanese and Caucasians.

OBJECTIVE This cross-sectional clinical study compared the pathophysiology of type 2 diabetes in Japanese and Caucasians and investigated the role of demographic, genetic, and lifestyle-related risk factors for insulin resistance and ß-cell response. RESEARCH DESIGN AND METHODS A total of 120 Japanese and 150 Caucasians were enrolled to obtain comparable distributions of high/low BMI values across glucose tolerance states (normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes), which were assessed by oral glucose tolerance tests. BMI in the two cohorts was distributed around the two regional cutoff values for obesity. RESULTS Insulin sensitivity was higher in Japanese compared with Caucasians, as indicated by the homeostatic model assessment of insulin resistance and Matsuda indices, whereas ß-cell response was higher in Caucasians, as measured by homeostatic model assessment of ß-cell function, the insulinogenic indices, and insulin secretion ratios. Disposition indices were similar for Japanese and Caucasians at all glucose tolerance states, indicating similar ß-cell response relative to the degree of insulin resistance. The main determinants for differences in metabolic indices were measures of body composition, such as BMI and distribution of adipose tissue. Differences in ß-cell response between Japanese and Caucasians were not statistically significant following adjustment by differences in BMI. CONCLUSIONS Our study showed similar disposition indices in Japanese and Caucasians and that the major part of the differences in insulin sensitivity and ß-cell response between Japanese and Caucasians can be explained by differences in body composition.

Studies of metabolic phenotypic correlates of 15 obesity associated gene variants.

AIMS: Genome-wide association studies have identified novel BMI/obesity associated susceptibility loci. The purpose of this study is to determine associations with overweight, obesity, morbid obesity and/or general adiposity in a Danish population. Moreover, we want to investigate if these loci associate with type 2 diabetes and to elucidate potential underlying metabolic mechanisms. METHODS: 15 gene variants in 14 loci including TMEM18 (rs7561317), SH2B1 (rs7498665), KCTD15 (rs29941), NEGR1 (rs2568958), ETV5 (rs7647305), BDNF (rs4923461, rs925946), SEC16B (rs10913469), FAIM2 (rs7138803), GNPDA2 (rs10938397), MTCH2 (rs10838738), BAT2 (rs2260000), NPC1 (rs1805081), MAF (rs1424233), and PTER (rs10508503) were genotyped in 18,014 middle-aged Danes. RESULTS: Five of the 15 gene variants associated with overweight, obesity and/or morbid obesity. Per allele ORs ranged from 1.15-1.20 for overweight, 1.10-1.25 for obesity, and 1.41-1.46 for morbid obesity. Five of the 15 variants moreover associated with increased measures of adiposity. BDNF rs4923461 displayed a borderline BMI-dependent protective effect on type 2 diabetes (0.87 (0.78-0.96, p = 0.008)), whereas SH2B1 rs7498665 associated with nominally BMI-independent increased risk of type 2 diabetes (1.16 (1.07-1.27, p = 7.8×10(-4))). CONCLUSIONS: Associations with overweight and/or obesity and measures of obesity were confirmed for seven out of the 15 gene variants. The obesity risk allele of BDNF rs4923461 protected against type 2 diabetes, which could suggest neuronal and peripheral distinctive ways of actions for the protein. SH2B1 rs7498665 associated with type 2 diabetes independently of BMI.

A systems biology approach identifies inflammatory abnormalities between mouse strains prior to development of metabolic disease.

OBJECTIVE: Type 2 diabetes and obesity are increasingly affecting human populations around the world. Our goal was to identify early molecular signatures predicting genetic risk to these metabolic diseases using two strains of mice that differ greatly in disease susceptibility. RESEARCH DESIGN AND METHODS: We integrated metabolic characterization, gene expression, protein-protein interaction networks, RT-PCR, and flow cytometry analyses of adipose, skeletal muscle, and liver tissue of diabetes-prone C57BL/6NTac (B6) mice and diabetes-resistant 129S6/SvEvTac (129) mice at 6 weeks and 6 months of age. RESULTS: At 6 weeks of age, B6 mice were metabolically indistinguishable from 129 mice, however, adipose tissue showed a consistent gene expression signature that differentiated between the strains. In particular, immune system gene networks and inflammatory biomarkers were upregulated in adipose tissue of B6 mice, despite a low normal fat mass. This was accompanied by increased T-cell and macrophage infiltration. The expression of the same networks and biomarkers, particularly those related to T-cells, further increased in adipose tissue of B6 mice, but only minimally in 129 mice, in response to weight gain promoted by age or high-fat diet, further exacerbating the differences between strains. CONCLUSIONS: Insulin resistance in mice with differential susceptibility to diabetes and metabolic syndrome is preceded by differences in the inflammatory response of adipose tissue. This phenomenon may serve as an early indicator of disease and contribute to disease susceptibility and progression.

Combined analyses of 20 common obesity susceptibility variants.

OBJECTIVE: Genome-wide association studies and linkage studies have identified 20 validated genetic variants associated with obesity and/or related phenotypes. The variants are common, and they individually exhibit small-to-modest effect sizes. RESEARCH DESIGN AND METHODS: In this study we investigate the combined effect of these variants and their ability to discriminate between normal weight and overweight/obese individuals. We applied receiver operating characteristics (ROC) curves, and estimated the area under the ROC curve (AUC) as a measure of the discriminatory ability. The analyses were performed cross-sectionally in the population-based Inter99 cohort where 1,725 normal weight, 1,519 overweight, and 681 obese individuals were successfully genotyped for all 20 variants. RESULTS: When combining all variants, the 10% of the study participants who carried more than 22 risk-alleles showed a significant increase in probability of being both overweight with an odds ratio of 2.00 (1.47-2.72), P = 4.0 x 10(-5), and obese with an OR of 2.62 (1.76-3.92), P = 6.4 x 10(-7), compared with the 10% of the study participants who carried less than 14 risk-alleles. Discrimination ability for overweight and obesity, using the 20 single nucleotide polymorphisms (SNPs), was determined to AUCs of 0.53 and 0.58, respectively. When combining SNP data with conventional nongenetic risk factors of obesity, the discrimination ability increased to 0.64 for overweight and 0.69 for obesity. The latter is significantly higher (P < 0.001) than for the nongenetic factors alone (AUC = 0.67). CONCLUSIONS: The discriminative value of the 20 validated common obesity variants is at present time sparse and too weak for clinical utility, however, they add to increase the discrimination ability of conventional nongenetic risk factors.

Studies of CTNNBL1 and FDFT1 variants and measures of obesity: analyses of quantitative traits and case-control studies in 18,014 Danes.

BACKGROUND: A genome-wide scan in unrelated US Caucasians identified rs7001819 upstream of farnesyl-diphosphate farnesyltransferase 1 (FDFT1) and multiple variants within catenin (cadherin-associated protein), beta-like 1 (CTNNBL1) to associate strongly with body mass index (BMI). The most significantly associating variants within CTNNBL1 including rs6013029 and rs6020846 were additionally confirmed to associate with morbid obesity in a French Caucasian case-control sample. The aim of this study was to investigate the impact of these three variants on obesity, through analyses of obesity-related quantitative traits, and case-control studies in large study samples of Danes. METHODS: The FDFT1 rs7001819, CTNNBL1 rs6013029 and rs6020846 were genotyped, using TaqMan allelic discrimination, in a combined study sample comprising 18,014 participants ascertained from; the population-based Inter99 cohort (n = 6,514), the ADDITION Denmark screening study cohort (n = 8,662), and a population-based sample (n = 680) and a type 2 diabetic patients group (n = 2,158) from Steno Diabetes Center. RESULTS: Both CTNNBL1 variants associated with body weight and height with per allele effect sizes of 1.0 [0.3-0.8] kg and 0.6 [0.2-0.9] cm, respectively, for the rs6020846 G-allele. No association was observed with BMI and waist circumference. In case-control studies neither of the CTNNBL1 variants showed association with overweight, obesity or morbid obesity (rs6013029: Odds Ratio (OR)(overweight) = 1.02 [0.90-1.16], OR(obesity) = 1.09 [0.95-1.25], OR(morbidobesity) = 1.26 [0.91-1.74]; rs6020846: OR(overweight) = 1.05 [0.93-1.18], OR(obesity) = 1.13 [1.00-1.28], OR(morbidobesity) = 1.17 [0.86-1.61]). However, in meta-analyses of the present and the previous study, both the rs6013029 T-allele and the rs6020846 G-allele increased the risk of developing morbid obesity (rs6013029: OR(combined) = 1.36 [1.12-1.64], p = 0.002; rs6020846: OR(combined) = 1.26 [1.06-1.51], p = 0.01), and obesity (rs6013029: OR(combined) = 1.17 [1.04-1.31], p = 0.007; rs6020846: OR(combined) = 1.17 [1.05-1.30], p = 0.004). The FDFT1 rs7001819 C-allele showed no association with obesity-related quantitative measures or dichotomous measures of overweight, obesity and morbid obesity. CONCLUSION: CTNNBL1 variants associated with body weight and height, and confer the risk of developing obesity in meta-analyses combining the present and a previous study. FDFT1 rs7001819 showed no association with obesity, neither when analysing quantitative traits nor when performing case-control studies of obesity.

Lack of association between PKLR rs3020781 and NOS1AP rs7538490 and type 2 diabetes, overweight, obesity and related metabolic phenotypes in a Danish large-scale study: case-control studies and analyses of quantitative traits.

BACKGROUND: Several studies in multiple ethnicities have reported linkage to type 2 diabetes on chromosome 1q21-25. Both PKLR encoding the liver pyruvate kinase and NOS1AP encoding the nitric oxide synthase 1 (neuronal) adaptor protein (CAPON) are positioned within this chromosomal region and are thus positional candidates for the observed linkage peak. The C-allele of PKLR rs3020781 and the T-allele of NOS1AP rs7538490 are reported to strongly associate with type 2 diabetes in various European-descent populations comprising a total of 2,198 individuals with a combined odds ratio (OR) of 1.33 [1.16-1.54] and 1.53 [1.28-1.81], respectively. Our aim was to validate these findings by investigating the impact of the two variants on type 2 diabetes and related quantitative metabolic phenotypes in a large study sample of Danes. Further, we intended to expand the analyses by examining the effect of the variants in relation to overweight and obesity. METHODS: PKLR rs3020781 and NOS1AP rs7538490 were genotyped, using TaqMan allelic discrimination, in a combined study sample comprising a total of 16,801 and 16,913 individuals, respectively. The participants were ascertained from four different study groups; the population-based Inter99 cohort (nPKLR = 5,962, nNOS1AP = 6,008), a type 2 diabetic patient group (nPKLR = 1,873, nNOS1AP = 1,874) from Steno Diabetes Center, a population-based study sample (nPKLR = 599, nNOS1AP = 596) from Steno Diabetes Center and the ADDITION Denmark screening study cohort (nPKLR = 8,367, nNOS1AP = 8,435). RESULTS: In case-control studies we evaluated the potential association between rs3020781 and rs7538490 and type 2 diabetes and obesity. No significant associations were observed for type 2 diabetes (rs3020781: pAF = 0.49, OR = 1.02 [0.96-1.10]; rs7538490: pAF = 0.84, OR = 0.99 [0.93-1.06]). Neither did we show association with overweight or obesity. Additionally, the PKLR and the NOS1AP genotypes were demonstrated not to have a major influence on diabetes-related quantitative metabolic phenotypes. CONCLUSION: We failed to provide evidence of an association between PKLR rs3020781 and NOS1AP rs7538490 and type 2 diabetes, overweight, obesity or related quantitative metabolic phenotypes in large-scale studies of Danes.

Non-replication of genome-wide based associations between common variants in INSIG2 and PFKP and obesity in studies of 18,014 Danes.

BACKGROUND: The INSIG2 rs7566605 and PFKP rs6602024 polymorphisms have been identified as obesity gene variants in genome-wide association (GWA) studies. However, replication has been contradictory for both variants. The aims of this study were to validate these obesity-associations through case-control studies and analyses of obesity-related quantitative traits. Moreover, since environmental and genetic factors may modulate the impact of a genetic variant, we wanted to perform such interaction analyses. We focused on physical activity as an environmental risk factor, and on the GWA identified obesity variants in FTO (rs9939609) and near MC4R (rs17782313) as genetic risk factors. MATERIALS AND METHODS: The four variants were genotyped in a combined study sample comprising a total of 18,014 subject ascertained from, the population-based Inter99 cohort (n = 6,514), the ADDITION screening cohort (n = 8,662), a population-based study sample (n = 680) and a type 2 diabetic patient group (n = 2,158) from Steno Diabetes Center. RESULTS: No association with overweight, obesity or obesity-related measures was shown for either the INSIG2 rs7566605 or the PFKP rs6602024 variants. However, an interaction between the INSIG2 rs7566605 variant and the level of self-reported physical activity (p(Int) = 0.004) was observed. A BMI difference of 0.53 (SE 0.42) kg/m(2) was found when comparing physically passive homozygous C-allele carriers with physically passive G-allele carriers. No interactions between the two variants and FTO rs9939609 and MC4R rs17782313 were observed. CONCLUSIONS: The INSIG2 rs7566605 and PFKP rs6602024 polymorphisms play no apparent role in the development of common forms of obesity in the Danish population. However, if replicated, the INSIG2 rs7566605 may influence the level of BMI in combination with the level of physical activity.

Mitochondrial proton leak in obesity-resistant and obesity-prone mice.

We quantified uncoupling proteins (UCPs) in molar amounts and assessed proton conductance in mitochondria isolated from interscapular brown adipose tissue (IBAT) and hindlimb muscle [known from prior work to contain ectopic brown adipose tissue (BAT) interspersed between muscle fibers] of obesity-resistant 129S6/SvEvTac (129) and obesity-prone C57BL/6 (B6) mice under conditions of low (LF) and high-fat (HF) feeding. With usual feeding, IBAT mitochondrial UCP1 content and proton conductance were greater in 129 mice than B6. However, with HF feeding, UCP1 and proton conductance increased more in B6 mice. Moreover, with HF feeding GDP-inhibitable proton conductance, specific for UCP1, equaled that seen in the 129 strain. UCP1 expression was substantial in mitochondria from hindlimb muscle tissue (ectopic BAT) of 129 mice as opposed to B6 but did not increase with HF feeding in either strain. As expected, muscle UCP3 expression increased with HF feeding in both strains but did not differ by strain. Moreover, the proton conductance of mitochondria isolated from hindlimb muscle tissue did not differ by strain or diet. Our data uncover a response to weight gain in obesity-prone (compared to resistant) mice unrecognized in prior studies that examined only UCP1 mRNA. Obesity-prone mice have the capacity to increase both IBAT UCP1 protein and mitochondrial proton conductance as much or more than obesity-resistant mice. But, this is only achieved only at a higher body mass and, therefore, may be adaptive rather than preventative. Neither obesity-prone nor resistant mice respond to HF feeding by expressing more UCP1 in ectopic BAT within muscle tissue.

Ectopic brown adipose tissue in muscle provides a mechanism for differences in risk of metabolic syndrome in mice.

C57BL/6 (B6) mice subjected to a high-fat diet develop metabolic syndrome with obesity, hyperglycemia, and insulin resistance, whereas 129S6/SvEvTac (129) mice are relatively protected from this disorder because of differences in higher basal energy expenditure in 129 mice, leading to lower weight gain. At a molecular level, this difference correlates with a marked higher expression of uncoupling protein 1 (UCP1) and a higher degree of uncoupling in vitro in mitochondria isolated from muscle of 129 versus B6 mice. Detailed histological examination, however, reveals that this UCP1 is in mitochondria of brown adipocytes interspersed between muscle bundles. Indeed, the number of UCP1-positive brown fat cells in intermuscular fat in 129 mice is >700-fold higher than in B6 mice. These brown fat cells are subject to further up-regulation of UCP1 after stimulation with a beta3-adrenergic receptor agonist. Thus, ectopic deposits of brown adipose tissue in intermuscular depots with regulatable expression of UCP1 provide a genetically based mechanism of protection from weight gain and metabolic syndrome between strains of mice.

Effects of diet and genetic background on sterol regulatory element-binding protein-1c, stearoyl-CoA desaturase 1, and the development of the metabolic syndrome.

Both environmental and genetic factors play important roles in the development of the metabolic syndrome. To elucidate how these factors interact under normal conditions, C57Bl/6 (B6) and 129S6/SvEvTac (129) mice were placed on a low-fat or high-fat diet. Over 18 weeks, the 129 strain developed features of the metabolic syndrome, notably obesity, hyperinsulinemia, and glucose intolerance only on the high-fat diet; the B6 strain on the other hand developed these features on both diets. High-fat feeding of both strains led to decreased serum triglycerides, hepatic steatosis, and hypercholesterolemia; however, B6 mice developed worse steatosis and a larger increase in LDL cholesterol. Both B6 background and high-fat feeding increased sterol regulatory element-binding protein-1c (SREBP-1c), a key regulator of lipogenic gene transcription, and its downstream targets. Stearoyl-CoA desaturase 1 (SCD1), an enzyme that regulates monounsaturated fatty acid (MUFA) synthesis, was also increased at the mRNA and enzyme activity levels by both high-fat feeding and B6 background. Furthermore, lipid analysis revealed increased hepatic triglycerides and MUFAs in B6 and high-fat-fed mice. Thus, dietary fat and genetic background act through SREBP-1c and SCD1 to affect hepatic lipid metabolism contributing to the development of the metabolic syndrome.

Genetic determinants of energy expenditure and insulin resistance in diet-induced obesity in mice.

Diet-induced obesity is the primary determinant of the current epidemic of diabetes. We have explored the role of genetics in this phenomenon, using C57Bl/6 (B6), 129S6/SvEvTac (129), and intercross (B6 x 129)F2 mice on a low- or high-fat diet. Over an 18-week period, B6 and F2 mice gained more weight, had higher levels of insulin and leptin, and showed greater glucose intolerance than 129 mice, despite lower food intake. By contrast, metabolic rate and diet-induced thermogenesis were significantly higher in the 129 mice. Genome-wide scans identified several quantitative trait loci, including a quantitative trait locus that was linked with hyperinsulinemia/insulin resistance on chromosome 14 in a region similar to that seen in mice with genetically induced insulin resistance. Microarray analysis indicated significant changes in expression levels between B6 and 129 mice in the identified chromosomal area of Wnt5a and protein kinase Cdelta (PKCdelta). Thus, caloric efficiency, i.e., the "thrifty gene," is a dominant-acting genetic determinant of diet-induced obesity in mice and can be linked to a locus on chromosome 14, including genes linked to adipose development and insulin sensitivity.

Impact of genetic background on development of hyperinsulinemia and diabetes in insulin receptor/insulin receptor substrate-1 double heterozygous mice.

Type 2 diabetes is a complex disease in which genetic and environmental factors interact to produce alterations in insulin action and insulin secretion, leading to hyperglycemia. To evaluate the influence of genetic background on development of diabetes in a genetically susceptible host, we generated mice that are double heterozygous (DH) for knockout of the insulin receptor and insulin receptor substrate-1 on three genetic backgrounds (C57BL/6 [B6], 129Sv, and DBA). Although DH mice on all backgrounds showed insulin resistance, their phenotypes were dramatically different. B6 DH mice exhibited marked hyperinsulinemia and massive islet hyperplasia and developed early hyperglycemia, with 85% overtly diabetic by 6 months. By contrast, 129Sv DH mice showed mild hyperinsulinemia and minimal islet hyperplasia, and < 2% developed diabetes. DBA mice had slower development of hyperglycemia, intermediate insulin levels, and evidence of islet degeneration, with 64% developing diabetes. Thus, mice carrying the same genetic defects on different backgrounds exhibited the full spectrum of abnormalities observed in humans with type 2 diabetes, which allowed for identification of potential loci that promote development of the diabetic phenotype.

Identification of interactive loci linked to insulin and leptin in mice with genetic insulin resistance.

Mice double heterozygous (DH) for deletion of insulin receptor and insulin receptor substrate-1 are lean, insulin resistant, and have a phenotype that strongly depends on the genetic background of the mouse. On the C57BL/6 (B6) background, DH mice develop marked hyperinsulinemia and diabetes, whereas on the 129S6 background, DH mice exhibit only mild elevations of insulin and remain free of diabetes. F2 male mice created by an intercross between these two strains exhibit a 60% incidence of diabetes and a bell-shaped distribution of insulin levels as related to glucose, reminiscent of that in humans with type 2 diabetes. These mice also exhibit a wide range of leptin levels as related to body weight. A genome-wide scan of F2 mice reveals a quantitative trait locus (QTL) related to hyperinsulinemia on chromosome 14 (D14Mit55) with a peak logarithm of odds (LOD) score of 5.6, accounting for up to 69% of this trait. A QTL with a LOD score of 3.7 related to hyperleptinemia is present on chromosome 7 at D12Mit38 (a marker previously assigned to chromosome 12) in the area of the uncoupling protein 2/3 gene cluster. This locus also interacts synergistically with D14Mit55 in development of hyperinsulinemia and with a QTL on chromosome 12 (D12Mit231) related to hyperglycemia. These data demonstrate how multiple genetic modifiers can interact and influence the development of diabetes and the phenotype of animals with genetically programmed insulin resistance and provide evidence as to the location and nature of these genes.

[Genetic defects in insulin signalling proteins. Implications for the pathogenesis of type 2 diabetes]. / Genetiske defekter i insulinsignaleringsproteiner. Implikationer for patogenesen for type 2-diabetes.

The pathogenesis of type 2 diabetes mellitus is complex and involves abnormalities in both the action and secretion of insulin. These abnormalities are caused by a complicated interplay between genes and environment. A determination of the genetic defects that predispose to either insulin resistance or decreased insulin secretion is important, as an improved understanding of the underlying molecular mechanisms may be essential for the development of the most effective treatment. This paper focuses on genetic variants identified in genes encoding proteins in the early insulin signalling cascade. Variations frequently occur in these genes, but their effect varies in different populations. This may suggest that the genetic background is a considerable factor and that the synergistic effect of several variants plays a major role. Future, genetic-epidemiological studies of large populations are therefore important in order to obtain sufficient statistical power. The paper also discusses recent results that suggests, that insulin itself has an effect on insulin secretion by the beta-cell and that insulin signalling in the CNS plays an important role in the regulation of energy disposal, fuel metabolism, and reproduction.

Characterization of the Met326Ile variant of phosphatidylinositol 3-kinase p85alpha.

Phosphatidylinositol 3-kinase is a key step in the metabolic actions of insulin. Two amino acid substitutions have been identified in the gene for the regulatory subunit of human p85alpha, Met-326Ile, and Asn-330Asp, and the former has been associated with alterations in glucose/insulin homeostasis. When the four human p85alpha proteins were expressed in yeast, a 27% decrease occurred in the level of protein expression of p85alpha(Ile/Asp) (P = 0.03) and a 43% decrease in p85alpha(Ile/Asn) (P = 0.08) as compared with p85alpha(Met/Asp). Both p85alpha(Ile/Asp) and p85alpha(Ile/Asn) also exhibited increased binding to phospho-insulin receptor substrate-1 by 41% and 83%, respectively (P < 0.001), as compared with p85alpha(Met/Asp). The expression of p85alpha(Ile) was also slightly decreased and the binding to insulin receptor substrate-1 slightly increased in brown preadipocytes derived from p85alpha knockout mice. Both p85alpha(Met) and p85alpha(Ile) had similar effects on AKT activity and were able to reconstitute differentiation of the preadipocytes, although the triglyceride concentration in fully differentiated adipocytes and insulin-stimulated 2-deoxyglucose uptake were slightly lower than in adipocytes expressing p85alpha(Met). Thus, the Met-326Ile variant of p85alpha is functional for intracellular signaling and adipocyte differentiation but has small alterations in protein expression and activity that could play a role in modifying insulin action.