A Signature of Exaggerated Adipose Tissue Dysfunction in Type 2 Diabetes Is Linked to Low Plasma Adiponectin and Increased Transcriptional Activation of Proteasomal Degradation in Muscle.

Insulin resistance in skeletal muscle in type 2 diabetes (T2D) is characterized by more pronounced metabolic and molecular defects than in obesity per se. There is increasing evidence that adipose tissue dysfunction contributes to obesity-induced insulin resistance in skeletal muscle. Here, we used an unbiased approach to examine if adipose tissue dysfunction is exaggerated in T2D and linked to diabetes-related mechanisms of insulin resistance in skeletal muscle. Transcriptional profiling and biological pathways analysis were performed in subcutaneous adipose tissue (SAT) and skeletal muscle biopsies from 17 patients with T2D and 19 glucose-tolerant, age and weight-matched obese controls. Findings were validated by qRT-PCR and western blotting of selected genes and proteins. Patients with T2D were more insulin resistant and had lower plasma adiponectin than obese controls. Transcriptional profiling showed downregulation of genes involved in mitochondrial oxidative phosphorylation and the tricarboxylic-acid cycle and increased expression of extracellular matrix (ECM) genes in SAT in T2D, whereas genes involved in proteasomal degradation were upregulated in the skeletal muscle in T2D. qRT-PCR confirmed most of these findings and showed lower expression of adiponectin in SAT and higher expression of myostatin in muscle in T2D. Interestingly, muscle expression of proteasomal genes correlated positively with SAT expression of ECM genes but inversely with the expression of ADIPOQ in SAT and plasma adiponectin. Protein content of proteasomal subunits and major ubiquitin ligases were unaltered in the skeletal muscle of patients with T2D. A transcriptional signature of exaggerated adipose tissue dysfunction in T2D, compared with obesity alone, is linked to low plasma adiponectin and increased transcriptional activation of proteasomal degradation in skeletal muscle.

Circulating Follistatin and Activin A and Their Regulation by Insulin in Obesity and Type 2 Diabetes.

BACKGROUND: Circulating follistatin (Fst) binds activin A and thereby regulates biological functions such as muscle growth and ß-cell survival. However, Fst and activin A's implication in metabolic regulation is unclear. OBJECTIVE: To investigate circulating Fst and activin A in obesity and type 2 diabetes (T2D) and determine their association with metabolic parameters. Further, to examine regulation of Fst and activin A by insulin and the influence of obesity and T2D hereon. METHODS: Plasma Fst and activin A levels were analyzed in obese T2D patients (N = 10) closely matched to glucose-tolerant lean (N = 12) and obese (N = 10) individuals in the fasted state and following a 4-h hyperinsulinemic-euglycemic clamp (40 mU·m-2·min-1) combined with indirect calorimetry. RESULTS: Circulating Fst was ~30% higher in patients with T2D compared with both lean and obese nondiabetic individuals (P < .001), while plasma activin A was unaltered. In the total cohort, fasting plasma Fst correlated positively with fasting plasma glucose, serum insulin and C-peptide levels, homeostasis model assessment of insulin resistance, and hepatic and adipose tissue insulin resistance after adjusting for age, gender and group (all r > 0.47; P < .05). However, in the individual groups these correlations only achieved significance in patients with T2D (not plasma glucose). Acute hyperinsulinemia at euglycemia reduced circulating Fst by ~30% (P < .001) and this response was intact in patients with T2D. Insulin inhibited FST expression in human hepatocytes after 2 h and even further after 48 h. CONCLUSIONS: Elevated circulating Fst, but not activin A, is strongly associated with measures of insulin resistance in patients with T2D. However, the ability of insulin to suppress circulating Fst is preserved in T2D.

Translation and cultural adaptation of the Communication Assessment Tool (CAT), developing a Danish and Norwegian version.

OBJECTIVE: To translate and cultural adapt the 14-item Communication Assessment Tool (CAT) into Norwegian and Danish, making them as similar as possible. DESIGN: This was a translation and validation study including individual interviews for content and face validity and a patient survey for internal consistency and floor-ceiling effect. SETTING: Outpatient clinic at the Department of Internal Medicine, Lillebaelt Hospital, Denmark and a Norwegian general practice. PARTICIPANTS: Ten patients were included for individual interviews and 440 participants completed the survey. MAIN OUTCOME MEASURE: Translation and validation of the CAT. RESULTS: Despite minor differences in the use of words in the translated versions of CAT, the final versions were very similar. Based on the content and face validation and after agreement with the developers, it was decided to include a 'non-applicable' answering option, not a part of the original version. The use of 'non-applicable' for each item ranged from 0% to 30% in Norway and from 0% to 6.1% in Denmark. The overall CAT score, i.e. items rated excellent, were 55.5% in Norway and 50.3% in Denmark. For each item, the CAT score ranged between 31.3% and 69.8% in Norway and 33.7% and 57.4% in Denmark. CONCLUSION: The translated and validated CAT can be used to measure patients' perspectives on clinicians' communication skills in Denmark and Norway.

Effects of insulin and exercise training on FGF21, its receptors and target genes in obesity and type 2 diabetes.

AIMS/HYPOTHESIS: Pharmacological doses of FGF21 improve glucose tolerance, lipid metabolism and energy expenditure in rodents. Induced expression and secretion of FGF21 from muscle may increase browning of white adipose tissue (WAT) in a myokine-like manner. Recent studies have reported that insulin and exercise increase FGF21 in plasma. Obesity and type 2 diabetes are potentially FGF21-resistant states, but to what extent FGF21 responses to insulin and exercise training are preserved, and whether FGF21, its receptors and target genes are altered, remains to be established. METHODS: The effects of insulin during euglycaemic-hyperinsulinaemic clamps and 10 week endurance training on serum FGF21 were examined in individuals with type 2 diabetes and in glucose tolerant overweight/obese and lean individuals. Gene expression of FGF21, its receptors and target genes in muscle and WAT biopsies was evaluated by quantitative real-time PCR (qPCR). RESULTS: Insulin increased serum and muscle FGF21 independent of overweight/obesity or type 2 diabetes, and there were no effects associated with exercise training. The insulin-induced increases in serum FGF21 and muscle FGF21 expression correlated tightly (p < 0.001). In WAT, overweight/obesity with and without type 2 diabetes led to reduced expression of KLB, but increased FGFR1c expression. However, the expression of most FGF21 target genes was unaltered except for reduced CIDEA expression in individuals with type 2 diabetes. CONCLUSIONS/INTERPRETATION: Insulin-induced expression of muscle FGF21 correlates strongly with a rise in serum FGF21, and this response appears intact in overweight/obesity and type 2 diabetes. FGF21 resistance may involve reduced KLB expression in WAT. However, increased FGFR1c expression or other mechanisms seem to ensure adequate expression of most FGF21 target genes in WAT.

Markers of autophagy are adapted to hyperglycaemia in skeletal muscle in type 2 diabetes.

AIMS/HYPOTHESIS: Autophagy is a catabolic process that maintains cellular homeostasis by degradation of protein aggregates and selective removal of damaged organelles, e.g. mitochondria (mitophagy). Insulin resistance in skeletal muscle has been linked to mitochondrial dysfunction and altered protein metabolism. Here, we investigated whether abnormalities in autophagy are present in human muscle in obesity and type 2 diabetes. METHODS: Using a case-control design, skeletal muscle biopsies obtained in the basal and insulin-stimulated states from patients with type 2 diabetes during both euglycaemia and hyperglycaemia, and from glucose-tolerant lean and obese individuals during euglycaemia, were used for analysis of mRNA levels, protein abundance and phosphorylation of autophagy-related proteins. RESULTS: Muscle transcript levels of autophagy-related genes (ULK1, BECN1, PIK3C3, ATG5, ATG7, ATG12, GABARAPL1, MAP1LC3B, SQSTM1, TP53INP2 and FOXO3A [also known as FOXO3]), including some specific for mitophagy (BNIP3, BNIP3L and MUL1), and protein abundance of autophagy-related gene (ATG)7 and Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), as well as content and phosphorylation of forkhead box O3A (FOXO3A) were similar among the groups. Insulin reduced lipidation of microtubule-associated protein light chain 3 (LC3)B-I to LC3B-II, a marker of autophagosome formation, with no effect on p62/sequestosome 1 (SQSTM1) content in muscle of lean and obese individuals. In diabetic patients, insulin action on LC3B was absent and p62/SQSTM1 content increased when studied under euglycaemia, whereas the responses of LC3B and p62/SQSTM1 to insulin were normalised during hyperglycaemia. CONCLUSIONS/INTERPRETATION: Our results demonstrate that the levels of autophagy-related genes and proteins in muscle are normal in obesity and type 2 diabetes. This suggests that muscle autophagy in type 2 diabetes has adapted to hyperglycaemia, which may contribute to preserve muscle mass.

Glucose tolerance is associated with differential expression of microRNAs in skeletal muscle: results from studies of twins with and without type 2 diabetes.

AIMS/HYPOTHESIS: We aimed to identify microRNAs (miRNAs) associated with type 2 diabetes and risk of developing the disease in skeletal muscle biopsies from phenotypically well-characterised twins. METHODS: We measured muscle miRNA levels in monozygotic (MZ) twins discordant for type 2 diabetes using arrays. Further investigations of selected miRNAs included target prediction, pathway analysis, silencing in cells and association analyses in a separate cohort of 164 non-diabetic MZ and dizygotic twins. The effects of elevated glucose and insulin levels on miRNA expression were examined, and the effect of low birthweight (LBW) was studied in rats. RESULTS: We identified 20 miRNAs that were downregulated in MZ twins with diabetes compared with their non-diabetic co-twins. Differences for members of the miR-15 family (miR-15b and miR-16) were the most statistically significant, and these miRNAs were predicted to influence insulin signalling. Indeed, miR-15b and miR-16 levels were associated with levels of key insulin signalling proteins, miR-15b was associated with the insulin receptor in non-diabetic twins and knockdown of miR-15b/miR-16 in myocytes changed the levels of insulin signalling proteins. LBW in twins and undernutrition during pregnancy in rats were, in contrast to overt type 2 diabetes, associated with increased expression of miR-15b and/or miR-16. Elevated glucose and insulin suppressed miR-16 expression in vitro. CONCLUSIONS: Type 2 diabetes is associated with non-genetic downregulation of several miRNAs in skeletal muscle including miR-15b and miR-16, potentially targeting insulin signalling. The paradoxical findings in twins with overt diabetes and twins at increased risk of the disease underscore the complexity of the regulation of muscle insulin signalling in glucose homeostasis.

Human muscle fiber type-specific insulin signaling: impact of obesity and type 2 diabetes.

Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that insulin-mediated glucose metabolism is different between muscle fiber types. We hypothesized that differences are due to fiber type-specific expression/regulation of insulin signaling elements and/or metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis muscles from lean, obese, and type 2 diabetic subjects before and after a hyperinsulinemic-euglycemic clamp. Type I fibers compared with type II fibers have higher protein levels of the insulin receptor, GLUT4, hexokinase II, glycogen synthase (GS), and pyruvate dehydrogenase-E1α (PDH-E1α) and a lower protein content of Akt2, TBC1 domain family member 4 (TBC1D4), and TBC1D1. In type I fibers compared with type II fibers, the phosphorylation response to insulin was similar (TBC1D4, TBC1D1, and GS) or decreased (Akt and PDH-E1α). Phosphorylation responses to insulin adjusted for protein level were not different between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS, and PDH-E1α) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared with lean and obese subjects. We conclude that human type I muscle fibers compared with type II fibers have a higher glucose-handling capacity but a similar sensitivity for phosphoregulation by insulin.

Genome-wide analysis of DNA methylation differences in muscle and fat from monozygotic twins discordant for type 2 diabetes.

BACKGROUND: Monozygotic twins discordant for type 2 diabetes constitute an ideal model to study environmental contributions to type 2 diabetic traits. We aimed to examine whether global DNA methylation differences exist in major glucose metabolic tissues from these twins. METHODOLOGY/PRINCIPAL FINDINGS: Skeletal muscle (n = 11 pairs) and subcutaneous adipose tissue (n = 5 pairs) biopsies were collected from 53-80 year-old monozygotic twin pairs discordant for type 2 diabetes. DNA methylation was measured by microarrays at 26,850 cytosine-guanine dinucleotide (CpG) sites in the promoters of 14,279 genes. Bisulfite sequencing was applied to validate array data and to quantify methylation of intergenic repetitive DNA sequences. The overall intra-pair variation in DNA methylation was large in repetitive (LINE1, D4Z4 and NBL2) regions compared to gene promoters (standard deviation of intra-pair differences: 10% points vs. 4% points, P<0.001). Increased variation of LINE1 sequence methylation was associated with more phenotypic dissimilarity measured as body mass index (r = 0.77, P = 0.007) and 2-hour plasma glucose (r = 0.66, P = 0.03) whereas the variation in promoter methylation did not associate with phenotypic differences. Validated methylation changes were identified in the promoters of known type 2 diabetes-related genes, including PPARGC1A in muscle (13.9±6.2% vs. 9.0±4.5%, P = 0.03) and HNF4A in adipose tissue (75.2±3.8% vs. 70.5±3.7%, P<0.001) which had increased methylation in type 2 diabetic individuals. A hypothesis-free genome-wide exploration of differential methylation without correction for multiple testing identified 789 and 1,458 CpG sites in skeletal muscle and adipose tissue, respectively. These methylation changes only reached some percentage points, and few sites passed correction for multiple testing. CONCLUSIONS/SIGNIFICANCE: Our study suggests that likely acquired DNA methylation changes in skeletal muscle or adipose tissue gene promoters are quantitatively small between type 2 diabetic and non-diabetic twins. The importance of methylation changes in candidate genes such as PPARGC1A and HNF4A should be examined further by replication in larger samples.

Increased serum concentrations of persistent organic pollutants among prediabetic individuals: potential role of altered substrate oxidation patterns.

CONTEXT: There is a need for a better understanding of the potential role of persistent organic pollutants (POPs) in the pathogenesis of type 2 diabetes. OBJECTIVE: Our objective was to determine the association of serum concentrations of POPs with early signs of type 2 diabetes in regard to glucose and lipid metabolism. RESEARCH DESIGN AND METHODS: In this cross-sectional study, we used recent studies of 148 Danish middle-aged normoglycemic, prediabetic, and diabetic individuals examined by the euglycemic-hyperinsulinemic clamp technique with indirect calorimetry; 66 of these individuals also had an i.v. glucose tolerance test. Concentrations of POPs were analyzed in banked serum from the participants. Associations with basal and insulin-stimulated glucose and lipid metabolism were assessed after adjustment for age, sex, and body fat percentage. RESULTS: Individuals with prediabetes and diabetes had higher serum concentrations of several POPs compared with normoglycemic individuals. In the nondiabetic population, higher POPs levels were associated with elevated fasting plasma glucose concentrations as well as reduced glucose oxidation, elevated lipid oxidation, and elevated serum concentrations of free fatty acids (P < 0.05). We found no associations of POPs with first-phase insulin secretion, hepatic or peripheral insulin sensitivity, or nonoxidative glucose metabolism. CONCLUSIONS: Diabetic and prediabetic individuals have elevated serum concentrations of POPs. In nondiabetic individuals, POPs exposure is related to altered substrate oxidation patterns with lower glucose oxidation and higher lipid oxidation rates. These findings indicate that POPs may affect peripheral glucose metabolism by modifying pathways involved in substrate partitioning rather than decreasing insulin-dependent glucose uptake.

A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.

Exercise benefits a variety of organ systems in mammals, and some of the best-recognized effects of exercise on muscle are mediated by the transcriptional co-activator PPAR-γ co-activator-1 α (PGC1-α). Here we show in mouse that PGC1-α expression in muscle stimulates an increase in expression of FNDC5, a membrane protein that is cleaved and secreted as a newly identified hormone, irisin. Irisin acts on white adipose cells in culture and in vivo to stimulate UCP1 expression and a broad program of brown-fat-like development. Irisin is induced with exercise in mice and humans, and mildly increased irisin levels in the blood cause an increase in energy expenditure in mice with no changes in movement or food intake. This results in improvements in obesity and glucose homeostasis. Irisin could be therapeutic for human metabolic disease and other disorders that are improved with exercise.

Acute hyperinsulinemia is followed by increased serum concentrations of fibroblast growth factor 23 in type 2 diabetes patients.

BACKGROUND: Phosphate homeostasis is connected to glucose metabolism and is influenced by insulin, but the role of fibroblast growth factor 23 (FGF23) is unknown in this relation. Therefore, the levels of FGF23 and phosphate were investigated during a euglycemic-hyperinsulinemic clamp in healthy and type 2 diabetic individuals. METHODS: The study population consisted of ten type 2 diabetic patients, ten weight-matched glucose-tolerant obese subjects, and ten healthy lean subjects. All subjects underwent a 4-h euglycemic-hyperinsulinemic clamp using physiological hyperinsulinemia (40 mU/min per m(2)) to determine glucose disposal rates. Blood samples for measurement of serum FGF23 and insulin, plasma phosphate and glucose, and HbA(1c) were drawn before and after insulin infusion. RESULTS: The three groups did not differ in baseline levels of serum FGF23. Insulin infusion increased serum FGF23 in the diabetic group (p = 0.009), but not in the other groups. The increase in serum FGF23 correlated strongly with increase in insulin levels in the diabetic group (r = 0.83; p = 0.003). In the overall group insulin infusion suppressed plasma phosphate concentrations (p = 0.006), but with no differences between the groups. CONCLUSIONS: Physiological hyperinsulinemia is under euglycemic conditions followed by a significant increase in serum FGF23 concentrations in diabetic individuals, which correlated with change in insulin level. The interplay between insulin effects and FGF23 may be important for the understanding of phosphate metabolism in relation to type 2 diabetes and its vascular complications.

The SNARE protein SNAP23 and the SNARE-interacting protein Munc18c in human skeletal muscle are implicated in insulin resistance/type 2 diabetes.

OBJECTIVE: Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS: We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp. Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6. RESULTS: We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects. Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS: We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23.

Increased subsarcolemmal lipids in type 2 diabetes: effect of training on localization of lipids, mitochondria, and glycogen in sedentary human skeletal muscle.

The purpose of the study was to investigate the effect of aerobic training and type 2 diabetes on intramyocellular localization of lipids, mitochondria, and glycogen. Obese type 2 diabetic patients (n = 12) and matched obese controls (n = 12) participated in aerobic cycling training for 10 wk. Endurance-trained athletes (n = 15) were included for comparison. Insulin action was determined by euglycemic-hyperinsulinemic clamp. Intramyocellular contents of lipids, mitochondria, and glycogen at different subcellular compartments were assessed by transmission electron microscopy in biopsies obtained from vastus lateralis muscle. Type 2 diabetic patients were more insulin resistant than obese controls and had threefold higher volume of subsarcolemmal (SS) lipids compared with obese controls and endurance-trained subjects. No difference was found in intermyofibrillar lipids. Importantly, following aerobic training, this excess SS lipid volume was lowered by approximately 50%, approaching the levels observed in the nondiabetic subjects. A strong inverse association between insulin sensitivity and SS lipid volume was found (r(2)=0.62, P = 0.002). The volume density and localization of mitochondria and glycogen were the same in type 2 diabetic patients and control subjects, and showed in parallel with improved insulin sensitivity a similar increase in response to training, however, with a more pronounced increase in SS mitochondria and SS glycogen than in other localizations. In conclusion, this study, estimating intramyocellular localization of lipids, mitochondria, and glycogen, indicates that type 2 diabetic patients may be exposed to increased levels of SS lipids. Thus consideration of cell compartmentation may advance the understanding of the role of lipids in muscle function and type 2 diabetes.

Acute hyperinsulinemia decreases plasma osteoprotegerin with diminished effect in type 2 diabetes and obesity.

OBJECTIVE: Osteoprotegerin (OPG) is a soluble tumour necrosis factor-receptor-like molecule present in connective tissues, especially bone and vasculature. It is known to accumulate in the arterial wall in diabetes. As its synthesis in vascular cells is decreased by insulin, we wanted to elucidate the acute effects of insulin on plasma OPG concentrations in individuals with type 2 diabetes and obese individuals compared with lean controls. DESIGN: The study population consisted of ten type 2 diabetic, ten obese subjects, and ten lean subjects with no family history of diabetes. METHODS: All subjects underwent a 4-h euglycemic-hyperinsulinemic clamp. Plasma OPG, insulin, lactate, HbA1c, cholesterol, triglycerides, free fatty acids (FFA), and glucose disposal rate were measured before and at the end of the clamp. RESULTS: Baseline OPG concentrations did not differ significantly between groups. Insulin infusion decreased plasma OPG concentrations in all groups (P<0.01); however, the fall in OPG was 50% less in obese and type 2 diabetic individuals (P=0.007). Baseline OPG correlated with fasting insulin, baseline lactate, and low density lipoprotein cholesterol in the diabetic group, and with baseline FFA in the lean group. The relative change of OPG in response to insulin correlated inversely with HbA1c and baseline FFA in the lean group. CONCLUSIONS: Acute hyperinsulinemia decreases plasma OPG, but with diminished effect in individuals with type 2 diabetes and obesity. Increased levels of OPG in arteries and plasma in diabetes together with the capability of plasma OPG as a cardiovascular risk predictor may be related to the described effects of insulin.

Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes.

We tested the hypothesis of a lower respiratory capacity per mitochondrion in skeletal muscle of type 2 diabetic patients compared with obese subjects. Muscle biopsies obtained from 10 obese type 2 diabetic and 8 obese nondiabetic male subjects were used for assessment of 3-hydroxy-Acyl-CoA-dehydrogenase (HAD) and citrate synthase activity, uncoupling protein (UCP)3 content, oxidative stress measured as 4-hydroxy-2-nonenal (HNE), fiber type distribution, and respiration in isolated mitochondria. Respiration was normalized to citrate synthase activity (mitochondrial content) in isolated mitochondria. Maximal ADP-stimulated respiration (state 3) with pyruvate plus malate and respiration through the electron transport chain (ETC) were reduced in type 2 diabetic patients, and the proportion of type 2X fibers were higher in type 2 diabetic patients compared with obese subjects (all P < 0.05). There were no differences in respiration with palmitoyl-l-carnitine plus malate, citrate synthase activity, HAD activity, UCP3 content, or oxidative stress measured as HNE between the groups. In the whole group, state 3 respiration with pyruvate plus malate and respiration through ETC were negatively associated with A1C, and the proportion of type 2X fibers correlated with markers of insulin resistance (P < 0.05). In conclusion, we provide evidence for a functional impairment in mitochondrial respiration and increased amount of type 2X fibers in muscle of type 2 diabetic patients. These alterations may contribute to the development of type 2 diabetes in humans with obesity.