Martin Ridderstråle

PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.

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-1α 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.

Prediction of Non-Alcoholic Fatty Liver Disease and Liver Fat Using Metabolic and Genetic Factors

BACKGROUND & AIMS Our aims were to develop a method to accurately predict non-alcoholic fatty liver disease (NAFLD) and liver fat content based on routinely available clinical and laboratory data and to test whether knowledge of the recently discovered genetic variant in the PNPLA3 gene (rs738409) increases accuracy of the prediction. METHODS Liver fat content was measured using proton magnetic resonance spectroscopy in 470 subjects, who were randomly divided into estimation (two thirds of the subjects, n = 313) and validation (one third of the subjects, n = 157) groups. Multivariate logistic and linear regression analyses were used to create an NAFLD liver fat score to diagnose NAFLD and liver fat equation to estimate liver fat percentage in each individual. RESULTS The presence of the metabolic syndrome and type 2 diabetes, fasting serum (fS) insulin, fS-aspartate aminotransferase (AST), and the AST/alanine aminotransferase ratio were independent predictors of NAFLD. The score had an area under the receiver operating characteristic curve of 0.87 in the estimation and 0.86 in the validation group. The optimal cut-off point of -0.640 predicted increased liver fat content with sensitivity of 86% and specificity of 71%. Addition of the genetic information to the score improved the accuracy of the prediction by only <1%. Using the same variables, we developed a liver fat equation from which liver fat percentage of each individual could be estimated. CONCLUSIONS The NAFLD liver fat score and liver fat equation provide simple and noninvasive tools to predict NAFLD and liver fat content.

Comparison of empagliflozin and glimepiride as add-on to metformin in patients with type 2 diabetes: a 104-week randomised, active-controlled, double-blind, phase 3 trial

BACKGROUND Metformin is the recommended first-line pharmacotherapy for patients with type 2 diabetes. There is no consensus on the optimum second-line pharmacotherapy. We compared the efficacy and safety of the sodium glucose cotransporter 2 inhibitor empagliflozin and the sulfonylurea glimepiride as add-on to metformin in patients with type 2 diabetes. METHODS In this double-blind phase 3 trial, patients (aged ≥18 years) with type 2 diabetes and HbA1c concentrations of 7-10%, despite metformin treatment and diet and exercise counselling, were randomly assigned in a 1:1 ratio with a computer-generated random sequence, stratified by HbA1c, estimated glomerular filtration rate (eGFR), and region, to empagliflozin (25 mg once daily, orally) or glimepiride (1-4 mg once daily, orally) as add-on to metformin for 104 weeks. Patients and investigators were masked to treatment assignment. The primary endpoint was change from baseline in HbA1c levels at weeks 52 and 104. Differences in the primary endpoint were first tested for non-inferiority (based on a margin of 0·3%). If non-inferiority was shown, differences in the primary endpoint at week 104 were then tested for superiority. Analysis was done on the full-analysis set-ie, patients who were treated with at least one dose of study drug and had a baseline HbA1c value. This study is registered with ClinicalTrials.gov, number NCT01167881. A 104-week extension is ongoing. FINDINGS Between August, 2010, and June, 2011, 1549 patients were randomly assigned to receive empagliflozin (n=769) or glimepiride (n=780); four patients in the empagliflozin group did not receive the assigned treatment. Empagliflozin was non-inferior to glimepiride at both timepoints. At week 104, adjusted mean difference in change from baseline in HbA1c with empagliflozin versus glimepiride was -0·11% (95% CI -0·19 to -0·02; p=0·0153 for superiority). Adverse events were reported in 661 (86%) patients treated with empagliflozin and 673 (86%) patients treated with glimepiride. Severe adverse events were reported in 72 (9%) patients in the empagliflozin group and 68 (9%) in the glimepiride group. Serious adverse events were reported in 119 (16%) patients in the empagliflozin group and 89 (11%) in the glimepiride group. Confirmed hypoglycaemic adverse events (plasma glucose ≤3·9 mmol/L or requiring assistance) at week 104 were reported in 19 (2%) patients treated with empagliflozin and 189 (24%) patients treated with glimepiride. INTERPRETATION Empagliflozin might be an effective and a well tolerated second-line treatment option for patients with type 2 diabetes who have not achieved good glycaemic control on metformin. FUNDING Boehringer Ingelheim and Eli Lilly.

Genetic dissection of type 2 diabetes.

Compared to the successful probing of genetic causes of monogenic disorders, dissecting the genetics of complex polygenic diseases has until recently been a fairly slow and cumbersome process. With the introduction of whole genome wide association studies (WGAS) the situation dramatically changed in 2007. The results from several recent WGAS on type 2 diabetes (T2D) and obesity have identified at least eighteen genes consistently associated with T2D. Many of the genes implicate pancreatic beta-cell function in the pathogenesis of T2D whereas only one clearly associate with insulin resistance. The identified genes most likely merely represent the tip of the iceberg in the explanation behind T2D. Refined tools will have to provide a more complete picture of the genetic complexity of T2D over the next few years. In addition to common variants increasing susceptibility for the disease, rare variants with stronger effects, copy number variations, and epigenetic effects like DNA methylation and histone acetylation will become important. Nevertheless, today we are able for the first time to anticipate that the genetics of a complex disease like T2D really can be dissected.

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes. Studies using the selective inhibitor wortmannin.

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC50≈ 100 nM), the insulin‐induced activation and phosphorylation of cGMP‐inhibited cAMP phosphodiesterase (cGI‐PDE) as well as the activation of the insulin‐stimulated cGI‐PDE kinase (IC50≈ 10–30 nM). No direct effects of the inhibitor on the insulin‐stimulated cGI‐PDE kinase, the cGI‐PDE and the hormone‐sensitive lipase were observed. These data suggest that activation of PI 3‐kinase upstream of the insulin‐stimulated cGI‐PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin‐induced cGI‐PDE activation/ phosphorylation and anti‐lipolysis.

Loss of function of the melanocortin 2 receptor accessory protein 2 is associated with mammalian obesity.

Accessory to Obesity? Melanocortin receptors are a family of cell membrane receptors that control diverse physiological functions. Mutations in the gene encoding melanocortin 4 receptor (MC4R) are a cause of familial early-onset obesity. Asai et al. (p. 275) studied the function of an accessory protein for MC4R signaling, MRAP2, and found that mice genetically deficient in MRAP2 develop severe obesity. Sequencing of MRAP2 in unrelated, severely obese humans revealed one individual with a clearly disruptive genetic variant, suggesting that MRAP2 mutations might also be a rare cause of human obesity. In a zebrafish model, Sebag et al. (p. 278) studied two paralogs of the MRAP2 accessory protein, one of which enhanced MC4R responsiveness to α–melanocyte-stimulating hormone, which regulates feeding and growth. Disruption of a protein required for effective signaling by a melanocortin receptor causes severe obesity in mice. Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed melanocortin 2 receptor accessory protein 2 (MRAP2), we characterized mice with whole-body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with melanocortin 4 receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated generation of the second messenger cyclic adenosine monophosphate, suggesting that alterations in Mc4r signaling may be one mechanism underlying the association between Mrap2 disruption and obesity. In a study of humans with severe, early-onset obesity, we found four rare, potentially pathogenic genetic variants in MRAP2, suggesting that the gene may also contribute to body weight regulation in humans.

Obesity and cardiovascular risk factors in type 2 diabetes: results from the Swedish National Diabetes Register.

Objectives.  To compare obese with normal and overweight type 2 diabetic patients regarding body mass index (BMI) and cardiovascular risk factors, and to analyse changes in weight versus risk factors.

Polymorphisms in the adiponutrin gene are associated with increased insulin secretion and obesity

OBJECTIVE The insulin responsive adiponutrin or patatin-like phospholipase 3 (PNPLA3, previously ADPN) gene shows association with obesity and in vitro adipocyte lipolysis. This study aimed to replicate the association between PNPLA3 variants and obesity, and to investigate their effect on insulin resistance and beta-cell function. METHODS rs738409 (Met148Ile) and rs2072907 (C to G) were genotyped using TaqMan allelic discrimination assay in a Swedish population-based sample (n=1811). Oral glucose tolerance test (OGTT) with data from three time points (0, 30, and 120 min) were available from individuals under the age of 50 years (n=973). RESULTS Both variant alleles were associated with decreased prevalence of obesity (P<0.05); odds ratio 0.75 (0.61-0.93) per carried Ile-allele for rs738409 and 0.80 (0.64-1.00) per carried G-allele for rs2072907. For obesity as a quantitative trait, there was no association in the whole population, but in obese subjects body mass index (BMI; P=0.023) and waist (P=0.0098) were higher in carriers of the Ile-allele. The Ile-carriers also displayed decreased insulin secretion in response to OGTT (30 min insulin; P=0.007, insulinogenic index; P=0.0051) with no significant differences in fasting plasma glucose (P=0.31), beta-cell function (disposition index; P=0.17) or homeostasis model of assessment insulin resistance (HOMA-IR; P=0.063). The correlation between BMI and HOMA-IR differed (Met/X versus Ile/Ile, P=0.028), Met-allele carriers were seemingly more insulin resistant at a lower BMI. The rs2072907 variant shows similar results for insulin secretion. The significance of this finding remained after adjusting for age, gender, and level of self-reported leisure-time physical activity. CONCLUSION We confirm the association between PNPLA3 and obesity. In addition, the rs738409 variant was associated with insulin secretion. There seems to be a differential effect of the Ile-allele depending on the degree of obesity, possibly as a consequence of insulin resistance.

Differential gene expression in adipose tissue from obese human subjects during weight loss and weight maintenance.

BACKGROUND Differential gene expression in adipose tissue during diet-induced weight loss followed by a weight stability period is poorly characterized. Markers of these processes may provide a deeper understanding of underlying mechanisms. OBJECTIVE We aimed to identify differentially expressed genes in human adipose tissue during weight loss and weight maintenance after weight loss. DESIGN RNA from subcutaneous abdominal adipose tissue from 9 obese subjects was analyzed by using a complementary DNA microarray at baseline after weight loss on a low-calorie diet and after weight maintenance. RESULTS Subjects lost 18.8 ± 1.8% of weight and maintained this loss during weight maintenance (1.1 ± 2.1%; range: -9.3 to 10.6%). Most differentially expressed genes exhibited a reciprocal regulation and returned to baseline after weight loss (2163 genes) and weight maintenance (3175 genes). CETP and ABCG1, both of which participate in the HDL-mediated reverse cholesterol transport (RCT), were among the most upregulated of the 750 genes that were differentially expressed after both processes. Several genes involved in inflammation were downregulated. The use of real-time polymerase chain reaction confirmed or partially confirmed the previously implicated genes TNMD and MMP9 (both downregulated), PNPLA3 (upregulated), and CIDEA and SCD (both reciprocally regulated). CONCLUSIONS The beneficial effects of weight loss should be investigated after long-term weight maintenance. The processes of weight loss and weight maintenance should be viewed as biologically distinct. CETP and ABCG1 may be important mediators of these effects through HDL-mediated RCT.

Willingness to pay for health improvements associated with anti-diabetes treatments for people with type 2 diabetes

Abstract Objectives: This study aimed to investigate the most important consequences of diabetes medication, as measured by the patients’ willingness to pay (WTP). Research design and methods: People in Sweden were recruited using existing nationwide e-mail panels if they were adults (≥18 years) with type 2 diabetes and were receiving pharmacological anti-diabetes treatment(s). Data were collected electronically and results were analysed using a standard statistical model designed for choice games (conditional logit). Six characteristics relating to treatment of diabetes were examined: weight (gain or loss), mean glycated haemoglobin level (HbA1c), hypoglycaemic events, nausea, need for injections (with or independently of meals), and blood glucose testing. Results: A total of 461 people with type 2 diabetes (291 males; 170 females) completed an internet questionnaire and were eligible for inclusion. Participants placed high value on weight loss and nausea avoidance; they would pay 176 Swedish Krona (SEK)/€15.61 per month to lose 1 kg, and would pay SEK 560 (€49.67) per month to avoid nausea completely. Patients wanting to reduce the number of hypoglycaemic events from three per month to none were willing to pay SEK 419 (€37.17) per month. Patients valued a 1 percentage point reduction in HbA1c at SEK 414 (€36.72) per month. Participants preferred taking tablets to injections and required a compensation of SEK 376 (€33.35) to accept one injection/day. Injections independent of meals were preferred to injections with meals (WTP: SEK 140/€12.42 per month). Potential limitations of this study are that the preferences expressed may not match preferences in real-life situations, and bias through the use of electronic questionnaire, which restricted participation to those with access to, and experience with, the internet. Conclusion: People with type 2 diabetes were willing to pay a considerable amount of money each month to lose weight, reduce or avoid hypoglycaemic events and reduce HbA1C.