Circulating glucose levels are tightly regulated. To identify novel glycemic loci, we performed meta-analyses of 21 genome-wide associations studies informative for fasting glucose (FG), fasting insulin (FI) and indices of β-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 non-diabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with FG/HOMA-B and two associated with FI/HOMA-IR. These include nine new FG loci (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and FAM148B) and one influencing FI/HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB/TMEM195 with type 2 diabetes (T2D). Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify T2D risk loci, as well as loci that elevate FG modestly, but do not cause overt diabetes.

To extend understanding of the genetic architecture and molecular basis of type 2 diabetes (T2D), we conducted a meta-analysis of genetic variants on the Metabochip involving 34,840 cases and 114,981 controls, overwhelmingly of European descent. We identified ten previously unreported T2D susceptibility loci, including two demonstrating sex-differentiated association. Genome-wide analyses of these data are consistent with a long tail of further common variant loci explaining much of the variation in susceptibility to T2D. Exploration of the enlarged set of susceptibility loci implicates several processes, including CREBBP-related transcription, adipocytokine signalling and cell cycle regulation, in diabetes pathogenesis.

To examine the mechanism by which lipids cause insulin resistance in humans, skeletal muscle glycogen and glucose-6-phosphate concentrations were measured every 15 min by simultaneous 13 C and 31 P nuclear magnetic resonance spectroscopy in nine healthy subjects in the presence of low (0.18 Ϯ 0.02 mM [mean Ϯ SEM]; control) or high (1.93 Ϯ 0.04 mM; lipid infusion) plasma free fatty acid levels under euglycemic ( ‫ف‬ 5.2 mM) hyperinsulinemic ( ‫ف‬ 400 pM) clamp conditions for 6 h. During the initial 3.5 h of the clamp the rate of whole-body glucose uptake was not affected by lipid infusion, but it then decreased continuously to be ‫ف‬ 46% of control values after 6 h ( P Ͻ 0.00001). Augmented lipid oxidation was accompanied by a ‫ف‬ 40% reduction of oxidative glucose metabolism starting during the third hour of lipid infusion ( P Ͻ 0.05). Rates of muscle glycogen synthesis were similar during the first 3 h of lipid and control infusion, but thereafter decreased to ‫ف‬ 50% of control values (4.0 Ϯ 1.0 vs. 9.3 Ϯ 1.6 mol/[kg и min], P Ͻ 0.05). Reduction of muscle glycogen synthesis by elevated plasma free fatty acids was preceded by a fall of muscle glucose-6-phosphate concentrations starting at ‫ف‬ 1.5 h (195 Ϯ 25 vs. control: 237 Ϯ 26 M; P Ͻ 0.01). Therefore in contrast to the originally postulated mechanism in which free fatty acids were thought to inhibit insulin-stimulated glucose uptake in muscle through initial inhibition of pyruvate dehydrogenase these results demonstrate that free fatty acids induce insulin resistance in humans by initial inhibition of glucose transport/phosphorylation which is then followed by an ‫ف‬ 50% reduction in both the rate of muscle glycogen synthesis and glucose oxidation. ( J. Clin. Invest. 1996. 97:2859-2865.) Key words: free fatty acids • muscle glycogen • glucose transport • nuclear magnetic resonance spectroscopy • glucose-6-phosphate

SummaryOverweight and obesity affect ~1.5 billion people worldwide, and are major risk factors for type-2 diabetes (T2D), cardiovascular disease and related metabolic and inflammatory disturbances.1,2 Although the mechanisms linking adiposity to its clinical sequelae are poorly understood, recent studies suggest that adiposity may influence DNA methylation,3–6 a key regulator of gene expression and molecular phenotype.7 Here we use epigenome-wide association to show that body mass index (BMI, a key measure of adiposity) is associated with widespread changes in DNA methylation (187 genetic loci at P<1x10-7, range P=9.2x10-8 to 6.0x10-46; N=10,261 samples). Genetic association analyses demonstrate that the alterations in DNA methylation are predominantly the consequence of adiposity, rather than the cause. We find the methylation loci are enriched for functional genomic features in multiple tissues (P<0.05), and show that sentinel methylation markers identify gene expression signatures at 38 loci (P<9.0x10-6, range P=5.5x10-6 to 6.1x10-35, N=1,785 samples). The methylation loci identified highlight genes involved in lipid and lipoprotein metabolism, substrate transport, and inflammatory pathways. Finally, we show that the disturbances in DNA methylation predict future type-2 diabetes (relative risk per 1SD increase in Methylation Risk Score: 2.3 [2.07-2.56]; P=1.1x10-54). Our results provide new insights into the biologic pathways influenced by adiposity, and may enable development of new strategies for prediction and prevention of type-2 diabetes and other adverse clinical consequences of obesity.

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of heritability. To test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole genome sequencing in 2,657 Europeans with and without diabetes, and exome sequencing in a total of 12,940 subjects from five ancestral groups. To increase statistical power, we expanded sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support a major role for lower-frequency variants in predisposition to type 2 diabetes.

Skeletal muscle insulin resistance is a common feature of obesity, dyslipidaemia and arterial hypertension, and it is an important predisposing factor for Type II (non-insulin-dependent) diabetes mellitus and premature cardiovascular disease [1]. Evidence has been provided that lipids could have an important role in insulin resistance: i) lipid oxidation is increased in insulin resistant states [2] and ii) increase of plasma concentrations of non-esterified fatty acids (NEFA) decreases skeletal muscle glucose uptake and glycogen synthesis [3]. The impact of intramyocellular lipid (IMCL) content on insulin sensitivity has previously been examined from muscle biopsies. The results showed that IMCL are an important source of energy within the muscle [4] and that increased IMCL content is associated with impaired insulin-stimulated glucose uptake in rats [5] as well as in healthy humans [6,7] and in those with Type I (insulin-dependent) diabetes mellitus [8].Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy now enables non-invasive quantification of the IMCL content in humans [9±11]. This study was designed: i) to examine the cross-sectional relation between IMCL and whole body insulin sensitivity in non-diabetic humans by using non-invasive localized proton NMR spectroscopy and ii) to compare the relative contributions of IMCL, BMI and Diabetologia (1999) Summary Recent muscle biopsy studies have shown a relation between intramuscular lipid content and insulin resistance. The aim of this study was to test this relation in humans by using a novel proton nuclear magnetic resonance ( 1 H NMR) spectroscopy technique, which enables non-invasive and rapid (~45 min) determination of intramyocellular lipid (IMCL) content. Normal weight non-diabetic adults (n = 23, age 29 2 years, BMI = 24.1 0.5 kg/m 2 ) were studied using cross-sectional analysis. Insulin sensitivity was assessed by a 2-h hyperinsulinaemic (~450 pmol/l)-euglycaemic (~5 mmol/l) clamp test. Intramyocellular lipid concentrations were determined by using localized 1 H NMR spectroscopy of soleus muscle. Simple linear regression analysis showed an inverse correlation (r = ±0.692, p = 0.0017) between intramyocellular lipid content and M-value (100±120 min of clamp) as well as between fasting plasma non-esterified fatty acid concentration and M-value (r = ±0.54, p= 0.0267). Intramyocellular lipid content was not related to BMI, age and fasting plasma concentrations of triglycerides, non-esterified fatty acids, glucose or insulin. These results show that intramyocellular lipid concentration, as assessed non invasively by localized 1 H NMR spectroscopy, is a good indicator of whole body insulin sensitivity in non-diabetic, non-obese humans. [Diabetologia (1999) 42: 113±116]

The association of hepatic mitochondrial function with insulin resistance and non-alcoholic fatty liver (NAFL) or steatohepatitis (NASH) remains unclear. This study applied high-resolution respirometry to directly quantify mitochondrial respiration in liver biopsies of obese insulin-resistant humans without (n = 18) or with (n = 16) histologically proven NAFL or with NASH (n = 7) compared to lean individuals (n = 12). Despite similar mitochondrial content, obese humans with or without NAFL had 4.3- to 5.0-fold higher maximal respiration rates in isolated mitochondria than lean persons. NASH patients featured higher mitochondrial mass, but 31%-40% lower maximal respiration, which associated with greater hepatic insulin resistance, mitochondrial uncoupling, and leaking activity. In NASH, augmented hepatic oxidative stress (H2O2, lipid peroxides) and oxidative DNA damage (8-OH-deoxyguanosine) was paralleled by reduced anti-oxidant defense capacity and increased inflammatory response. These data suggest adaptation of the liver ("hepatic mitochondrial flexibility") at early stages of obesity-related insulin resistance, which is subsequently lost in NASH.

The liver constitutes a key organ in systemic metabolism, contributing substantially to the development of insulin resistance and type 2 diabetes mellitus (T2DM). The mechanisms underlying these processes are not entirely understood, but involve hepatic fat accumulation, alterations of energy metabolism and inflammatory signals derived from various cell types including immune cells. Lipotoxins, mitochondrial function, cytokines and adipocytokines have been proposed to play a major part in both NAFLD and T2DM. Patients with NAFLD are commonly insulin resistant. On the other hand, a large number of patients with T2DM develop NAFLD with its inflammatory complication, NASH. The high incidence of NASH in patients with T2DM leads to further complications, such as liver cirrhosis and hepatocellular carcinoma, which are increasingly recognized. Therapeutic concepts such as thiazolidinediones (glitazones) for treating T2DM also show some efficacy in the treatment of NASH. This Review will describe the multifaceted and complex interactions between the liver and T2DM.