Changes in protein abundance in skeletal muscle are central to a large number of metabolic and ot... more Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including...
Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated ... more Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes. Studies have shown that thiazolidinediones (TZDs) improve metabolic disturbances in PCOS patients. We hypothesized that the effect of TZDs in PCOS is, in part, mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity. Using Affymetrix microarrays, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene expression in skeletal muscle of 10 obese women with PCOS metabolically characterized by a euglycemic-hyperinsulinemic clamp. Moreover, we explored gene expression changes between these PCOS patients before treatment and 13 healthy women. Treatment with pioglitazone improved insulin-stimulated glucose metabolism and plasma adiponectin, and reduced fasting serum insulin (all P,0.05). Global pathway analysis using Gene Map Annotator and Pathway Profiler (GenMAPP 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1) revealed a significant upregulation of genes representing mitochondrial oxidative phosphorylation (OXPHOS), ribosomal proteins, mRNA processing reactome, translation factors, and proteasome degradation in PCOS after pioglitazone therapy. Quantitative real-time PCR suggested that upregulation of OXPHOS genes was mediated by an increase in PGC-1a expression (P,0.05). Pretreatment expression of genes representing OXPHOS and ribosomal proteins was down-regulated in PCOS patients compared to healthy women. These data indicate that pioglitazone therapy restores insulin sensitivity, in part, by a coordinated upregulation of genes involved in mitochondrial OXPHOS and ribosomal protein biosynthesis in muscle in PCOS. These transcriptional effects of pioglitazone may contribute to prevent the onset of type 2 diabetes in these women.
Exercise benefits a variety of organ systems in mammals, and some of the best-recognized effects ... more 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 coactivator PGC1α Here we show that PGC1α expression in muscle stimulates an increase in expression of Fndc5, a membrane protein that is cleaved and secreted as a new 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.
Liver injury linked to insulin resistance is characterized by mild to moderate increases in amino... more Liver injury linked to insulin resistance is characterized by mild to moderate increases in aminotransferase activity. A soluble form of CD36 (sCD36) was recently identified in human plasma. The aim of this study was to evaluate the relationships among plasma sCD36, insulin sensitivity (SI) and indicators of liver health. We evaluated a cohort of men from the general population (n=117). As expected, serum (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were associated positively with body mass index (BMI) and age and negatively with SI (minimal model method). Circulating sCD36 was positively associated with ALT, AST and GGT in subjects with altered glucose tolerance, but not in those with normal glucose tolerance. The difference in the slope of the relationships was significant (P=.01). Age, BMI and triglycerides (but not sCD36) contributed independently to 29% of ALT variance in subjects with normal glucose tolerance. In contrast, SI and sCD36 contributed independently to 39% of ALT variance in subjects with altered glucose tolerance. The correlation between ALT activity and sCD36 was confirmed in an independent, replication study. In summary, circulating sCD36 could represent a novel marker of liver injury in subjects with altered glucose tolerance.
The Journal of Clinical Endocrinology & Metabolism, 2005
Administration of ghrelin, the endogenous ligand for the GH secretagogue receptor, stimulates not... more Administration of ghrelin, the endogenous ligand for the GH secretagogue receptor, stimulates not only GH secretion but also appetite and food intake in humans. Endogenous ghrelin levels display a distinct circadian rhythm, which is reciprocal to that of insulin and presumed to be meal dependent and not associated with GH secretion. We tested the hypothesis that food deprivation could impact circadian serum ghrelin levels and unmask meal-independent regulatory mechanisms.
Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing num... more Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.
Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioe... more Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small ...
Protein phosphorylation plays an essential role in signal transduction pathways that regulate sub... more Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin-resistant muscle using phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO(2). The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines, and 13 phosphotyrosines in at least 2 out of 3 ...
Protein phosphatase 2A (PP2A) acts on a number of enzymes involved in the insulin regulation of g... more Protein phosphatase 2A (PP2A) acts on a number of enzymes involved in the insulin regulation of glucose uptake and glycogen synthesis. This study was carried out to investigate the effect of insulin on PP2A expression in skeletal muscles of type 2 diabetic and control subjects. Ten type 2 diabetic and 10 matched, control subjects were studied using the euglycaemic-hyperinsulinaemic clamp technique combined with indirect calorimetry. Immunoreactive protein levels of the catalytic alpha subunit of PP2A (PP2A-C alpha) were measured in biopsies from the vastus lateralis muscle obtained in the basal and insulin-stimulated state. In type 2 diabetic subjects insulin-mediated glucose disposal, glucose oxidation and nonoxidative glucose metabolism were reduced, whereas lipid oxidation was increased (all P < 0.05). Insulin down-regulated PP2A-C alpha expression in skeletal muscle of the control subjects (P < 0.05) but not in the type 2 diabetic subjects. In the control subjects, the insulin-mediated decrease in PP2A-C alpha correlated with the insulin-mediated increase in glucose disposal, glucose oxidation, nonoxidative glucose metabolism (all P < 0.05) and decrease in lipid oxidation (P < 0.01). In the type 2 diabetic subjects these relationships were absent. Down-regulation of PP2A-C alpha expression by insulin in skeletal muscle seems to be associated with a normal insulin action on glucose storage, glucose and lipid oxidation. Impaired down-regulation of PP2A-C alpha expression by insulin may be a marker for insulin resistance and contribute to the pathogenesis of type 2 diabetes.
Endocrinology and Metabolism Clinics of North America, 2008
Type 2 diabetes mellitus (T2D) is the most common metabolic disease, currently affecting approxim... more Type 2 diabetes mellitus (T2D) is the most common metabolic disease, currently affecting approximately 170 million people worldwide . The development of T2D is the result of a complex interaction between genetic and environmental factors [2], the latter being evidenced by a rapid decrease in age at onset under the influence of a Western lifestyle and marked by physical inactivity, increased caloric intake, and obesity. T2D and obesity are characterized by insulin resistance in major metabolic tissues such as skeletal muscle, liver, and fat cells. In T2D, failure of the pancreatic b-cells to compensate for this abnormality causes hyperglycemia . In T2D and obesity, an elevated risk of cardiovascular disease causes increased morbidity and mortality and decreased quality of life, and places a substantial economic burden on our health system. A better understanding of the complex pathogenesis of insulin resistance in T2D and obesity is therefore of major importance to improve preventive and therapeutic strategies.
Aim/hypothesis Studies have suggested a link between insulin resistance and mitochondrial dysfunc... more Aim/hypothesis Studies have suggested a link between insulin resistance and mitochondrial dysfunction in skeletal muscles. Our primary aim was to investigate the effect of aerobic training on mitochondrial respiration and mitochondrial reactive oxygen species (ROS) release in skeletal muscle of obese participants with and without type 2 diabetes. Methods Type 2 diabetic men (n=13) and control (n=14) participants matched for age, BMI and physical activity completed 10 weeks of aerobic training. Pre-and posttraining muscle biopsies were obtained before a euglycaemichyperinsulinaemic clamp and used for measurement of respiratory function and ROS release in isolated mitochondria. Results Training significantly increased insulin sensitivity, maximal oxygen consumption and muscle mitochondrial respiration with no difference between groups. When expressed in relation to a marker of mitochondrial density (intrinsic mitochondrial respiration), training resulted in increased mitochondrial ADP-stimulated respiration (with NADH-generating substrates) and decreased respiration without ADP. Intrinsic mitochondrial respiration was not different between groups despite lower insulin sensitivity in type 2 diabetic participants. Mitochondrial ROS release tended to be higher in participants with type 2 diabetes. Conclusions/interpretation Aerobic training improves muscle respiration and intrinsic mitochondrial respiration in untrained obese participants with and without type 2 diabetes. These adaptations demonstrate an increased metabolic fitness, but do not seem to be directly related to training-induced changes in insulin sensitivity.
Aims/hypothesis Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolis... more Aims/hypothesis Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolism. The purpose of the present study was to evaluate effects of the endogenous LXR agonist 22-R-hydroxycholesterol (22-R-HC) and its stereoisomer 22-S-hydroxycholesterol (22-S-HC), in comparison with the synthetic agonist T0901317 on lipid and glucose metabolism in human skeletal muscle cells (myotubes). Methods Myotubes established from lean and obese control volunteers and from obese type 2 diabetic volunteers were treated with LXR ligands for 4 days. Lipid and glucose metabolisms were studied with labelled precursors, and gene expression was analysed using real-time PCR. Results Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers. Furthermore, 22-S-HC efficiently counteracted the T0901317-induced enhancement of lipid formation. Moreover, synthesis of diacylglycerol, cholesteryl ester and free cholesterol from acetate was reduced below baseline by 22-S-HC, whereas glucose uptake and oxidation were increased. Both 22-S-HC and 22-R-HC, in contrast to T0901317, decreased the expression of genes involved in cholesterol synthesis, whereas only 22-R-HC, like T0901317, increased the expression of the gene encoding the reverse cholesterol transporter ATP-binding cassette subfamily A1 (ABCA1). Conclusions/interpretation T0901317-induced lipogenesis and lipid formation was more pronounced in myotubes from type 2 diabetic patients than from lean individuals. 22-S-HC counteracted these effects and reduced de novo lipogenesis below baseline, while glucose uptake and oxidation were increased. Keywords Glucose metabolism . Human myotubes . Hydroxycholesterols . Lipid metabolism . LXR antagonist . Type 2 diabetes . Liver X receptor Abbreviations ChREBP carbohydrate responsive element-binding protein FASN fatty acid synthase HMGC hydroxymethylglutaryl-coenzyme A LXR liver X receptor 22-R-HC 22-R-hydroxycholesterol 22-S-HC 22-S-hydroxycholesterol SREBP sterol regulatory element-binding protein SCD1 stearoyl-coenzyme A desaturase 1 Diabetologia (
Multiple defects in the regulation of glucose metabolism are believed to play a role in the patho... more Multiple defects in the regulation of glucose metabolism are believed to play a role in the pathophysiology of Type 2 diabetes mellitus . Thus, basal rates of glucose production are often elevated , and suppression of glucose production (GP) as well as stimulation of glucose disappearance ( R d) by insulin, is impaired in Type 2 diabetes . In the study of this issue, the primed-constant tracer infusion method plays a major role. This method is combined with the euglycaemic hyperinsulinaemic clamp technique to assess the effects of insulin on glucose metabolism, independent of the effects of hyperglycaemia per se .
Aim: Insulin resistance in subjects with type 2 diabetes (T2D) and obesity is associated with an ... more Aim: Insulin resistance in subjects with type 2 diabetes (T2D) and obesity is associated with an imbalance between the availability and the oxidation of lipids. We hypothesized that maximal whole-body lipid oxidation during exercise (FATmax) is reduced and that training-induced metabolic adaptation is attenuated in T2D. Methods: Obese T2D (n ¼ 12) and control (n ¼ 11) subjects matched for age, sex, physical activity and body mass index completed 10 weeks of aerobic training. Subjects were investigated before and after training with maximal and submaximal exercise tests and euglycaemic-hyperinsulinaemic clamps combined with muscle biopsies. Results: Training increased maximal oxygen consumption (VO 2max ) and muscle citrate synthase activity and decreased blood lactate concentrations during submaximal exercise in both groups (all p < 0.01). FATmax increased markedly (40-50%) in both T2D and control subjects after training (all p < 0.001). There were no significant differences in these variables and lactate threshold (%VO 2max ) between groups before or after training. Insulinstimulated glucose disappearance rate (Rd) was lower in T2D vs. control subjects both before and after training. Rd increased in response to training in both groups (all p < 0.01). There was no correlation between Rd and measures of oxidative capacity or lipid oxidation during exercise or the training-induced changes in these parameters. Conclusions: FATmax was not reduced in T2D, and muscle oxidative capacity increased adequately in response to aerobic training in obese subjects with and without T2D. These metabolic adaptations to training seem to be unrelated to changes in insulin sensitivity and indicate that an impaired capacity for lipid oxidation is not a major cause of insulin resistance in T2D.
Accumulating evidence suggests an association between insulin sensitivity and albuminuria, which,... more Accumulating evidence suggests an association between insulin sensitivity and albuminuria, which, even in the normal range, is a risk factor for cardiovascular diseases. We evaluated whether insulin sensitivity is associated with albuminuria in healthy subjects.
Aim. -This study explored the association of depressive symptoms with indices of insulin sensitiv... more Aim. -This study explored the association of depressive symptoms with indices of insulin sensitivity and insulin secretion in a cohort of non-diabetic men and women aged 30 to 64 years.
The importance of reducing sedentary time is increasingly being recognized in the prevention of d... more The importance of reducing sedentary time is increasingly being recognized in the prevention of diabetes and cardiovascular disease. Despite this, the prospective association between sedentary time and physical activity with insulin sensitivity and cardiometabolic risk factors has been little studied. In an analysis of data from the European RISC study, sedentary time and time spent in activity of moderate or vigorous intensity were assessed by accelerometry at baseline in 313 men and 414 women, aged 30-60 years, with insulin sensitivity as measured by euglycaemic-hyperinsulinaemic clamp. Three years later, cardiometabolic risk factors (anthropometry, glucose, insulin, lipids) were available for 549 participants. In cross-sectional analyses using baseline data, after adjusting for age, gender, recruitment centre and time spent in activity of moderate or vigorous intensity, significant unfavourable associations were observed between higher sedentary time with body weight, HDL cholesterol, triglycerides, clamp-measured insulin sensitivity and insulin secretion (all P(trend)&lt;0.002). Sedentary time remained significantly associated with insulin secretion after adjusting for insulin sensitivity (P(trend)=0.02). In longitudinal analyses, higher baseline sedentary time was associated with 3-year increases in fasting glucose, fasting insulin and the HOMA insulin-resistance index score for the 50% of the study population who increased their BMI by at least 0.3 kg/m(2) (all P(trend)&lt;0.01); these relationships remained significant after adjusting for time spent in activity of moderate or vigorous intensity. The 3-year increase in insulin secretion was lower in those spending more time doing activity of moderate or vigorous intensity (P(trend)=0.03). These prospective data suggest that less sedentary behaviour may partly counteract some of the negative effects of increasing body weight on glucose-insulin homoeostasis.
Changes in protein abundance in skeletal muscle are central to a large number of metabolic and ot... more Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including...
Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated ... more Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes. Studies have shown that thiazolidinediones (TZDs) improve metabolic disturbances in PCOS patients. We hypothesized that the effect of TZDs in PCOS is, in part, mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity. Using Affymetrix microarrays, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene expression in skeletal muscle of 10 obese women with PCOS metabolically characterized by a euglycemic-hyperinsulinemic clamp. Moreover, we explored gene expression changes between these PCOS patients before treatment and 13 healthy women. Treatment with pioglitazone improved insulin-stimulated glucose metabolism and plasma adiponectin, and reduced fasting serum insulin (all P,0.05). Global pathway analysis using Gene Map Annotator and Pathway Profiler (GenMAPP 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1) revealed a significant upregulation of genes representing mitochondrial oxidative phosphorylation (OXPHOS), ribosomal proteins, mRNA processing reactome, translation factors, and proteasome degradation in PCOS after pioglitazone therapy. Quantitative real-time PCR suggested that upregulation of OXPHOS genes was mediated by an increase in PGC-1a expression (P,0.05). Pretreatment expression of genes representing OXPHOS and ribosomal proteins was down-regulated in PCOS patients compared to healthy women. These data indicate that pioglitazone therapy restores insulin sensitivity, in part, by a coordinated upregulation of genes involved in mitochondrial OXPHOS and ribosomal protein biosynthesis in muscle in PCOS. These transcriptional effects of pioglitazone may contribute to prevent the onset of type 2 diabetes in these women.
Exercise benefits a variety of organ systems in mammals, and some of the best-recognized effects ... more 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 coactivator PGC1α Here we show that PGC1α expression in muscle stimulates an increase in expression of Fndc5, a membrane protein that is cleaved and secreted as a new 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.
Liver injury linked to insulin resistance is characterized by mild to moderate increases in amino... more Liver injury linked to insulin resistance is characterized by mild to moderate increases in aminotransferase activity. A soluble form of CD36 (sCD36) was recently identified in human plasma. The aim of this study was to evaluate the relationships among plasma sCD36, insulin sensitivity (SI) and indicators of liver health. We evaluated a cohort of men from the general population (n=117). As expected, serum (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were associated positively with body mass index (BMI) and age and negatively with SI (minimal model method). Circulating sCD36 was positively associated with ALT, AST and GGT in subjects with altered glucose tolerance, but not in those with normal glucose tolerance. The difference in the slope of the relationships was significant (P=.01). Age, BMI and triglycerides (but not sCD36) contributed independently to 29% of ALT variance in subjects with normal glucose tolerance. In contrast, SI and sCD36 contributed independently to 39% of ALT variance in subjects with altered glucose tolerance. The correlation between ALT activity and sCD36 was confirmed in an independent, replication study. In summary, circulating sCD36 could represent a novel marker of liver injury in subjects with altered glucose tolerance.
The Journal of Clinical Endocrinology & Metabolism, 2005
Administration of ghrelin, the endogenous ligand for the GH secretagogue receptor, stimulates not... more Administration of ghrelin, the endogenous ligand for the GH secretagogue receptor, stimulates not only GH secretion but also appetite and food intake in humans. Endogenous ghrelin levels display a distinct circadian rhythm, which is reciprocal to that of insulin and presumed to be meal dependent and not associated with GH secretion. We tested the hypothesis that food deprivation could impact circadian serum ghrelin levels and unmask meal-independent regulatory mechanisms.
Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing num... more Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.
Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioe... more Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small ...
Protein phosphorylation plays an essential role in signal transduction pathways that regulate sub... more Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin-resistant muscle using phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO(2). The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines, and 13 phosphotyrosines in at least 2 out of 3 ...
Protein phosphatase 2A (PP2A) acts on a number of enzymes involved in the insulin regulation of g... more Protein phosphatase 2A (PP2A) acts on a number of enzymes involved in the insulin regulation of glucose uptake and glycogen synthesis. This study was carried out to investigate the effect of insulin on PP2A expression in skeletal muscles of type 2 diabetic and control subjects. Ten type 2 diabetic and 10 matched, control subjects were studied using the euglycaemic-hyperinsulinaemic clamp technique combined with indirect calorimetry. Immunoreactive protein levels of the catalytic alpha subunit of PP2A (PP2A-C alpha) were measured in biopsies from the vastus lateralis muscle obtained in the basal and insulin-stimulated state. In type 2 diabetic subjects insulin-mediated glucose disposal, glucose oxidation and nonoxidative glucose metabolism were reduced, whereas lipid oxidation was increased (all P &lt; 0.05). Insulin down-regulated PP2A-C alpha expression in skeletal muscle of the control subjects (P &lt; 0.05) but not in the type 2 diabetic subjects. In the control subjects, the insulin-mediated decrease in PP2A-C alpha correlated with the insulin-mediated increase in glucose disposal, glucose oxidation, nonoxidative glucose metabolism (all P &lt; 0.05) and decrease in lipid oxidation (P &lt; 0.01). In the type 2 diabetic subjects these relationships were absent. Down-regulation of PP2A-C alpha expression by insulin in skeletal muscle seems to be associated with a normal insulin action on glucose storage, glucose and lipid oxidation. Impaired down-regulation of PP2A-C alpha expression by insulin may be a marker for insulin resistance and contribute to the pathogenesis of type 2 diabetes.
Endocrinology and Metabolism Clinics of North America, 2008
Type 2 diabetes mellitus (T2D) is the most common metabolic disease, currently affecting approxim... more Type 2 diabetes mellitus (T2D) is the most common metabolic disease, currently affecting approximately 170 million people worldwide . The development of T2D is the result of a complex interaction between genetic and environmental factors [2], the latter being evidenced by a rapid decrease in age at onset under the influence of a Western lifestyle and marked by physical inactivity, increased caloric intake, and obesity. T2D and obesity are characterized by insulin resistance in major metabolic tissues such as skeletal muscle, liver, and fat cells. In T2D, failure of the pancreatic b-cells to compensate for this abnormality causes hyperglycemia . In T2D and obesity, an elevated risk of cardiovascular disease causes increased morbidity and mortality and decreased quality of life, and places a substantial economic burden on our health system. A better understanding of the complex pathogenesis of insulin resistance in T2D and obesity is therefore of major importance to improve preventive and therapeutic strategies.
Aim/hypothesis Studies have suggested a link between insulin resistance and mitochondrial dysfunc... more Aim/hypothesis Studies have suggested a link between insulin resistance and mitochondrial dysfunction in skeletal muscles. Our primary aim was to investigate the effect of aerobic training on mitochondrial respiration and mitochondrial reactive oxygen species (ROS) release in skeletal muscle of obese participants with and without type 2 diabetes. Methods Type 2 diabetic men (n=13) and control (n=14) participants matched for age, BMI and physical activity completed 10 weeks of aerobic training. Pre-and posttraining muscle biopsies were obtained before a euglycaemichyperinsulinaemic clamp and used for measurement of respiratory function and ROS release in isolated mitochondria. Results Training significantly increased insulin sensitivity, maximal oxygen consumption and muscle mitochondrial respiration with no difference between groups. When expressed in relation to a marker of mitochondrial density (intrinsic mitochondrial respiration), training resulted in increased mitochondrial ADP-stimulated respiration (with NADH-generating substrates) and decreased respiration without ADP. Intrinsic mitochondrial respiration was not different between groups despite lower insulin sensitivity in type 2 diabetic participants. Mitochondrial ROS release tended to be higher in participants with type 2 diabetes. Conclusions/interpretation Aerobic training improves muscle respiration and intrinsic mitochondrial respiration in untrained obese participants with and without type 2 diabetes. These adaptations demonstrate an increased metabolic fitness, but do not seem to be directly related to training-induced changes in insulin sensitivity.
Aims/hypothesis Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolis... more Aims/hypothesis Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolism. The purpose of the present study was to evaluate effects of the endogenous LXR agonist 22-R-hydroxycholesterol (22-R-HC) and its stereoisomer 22-S-hydroxycholesterol (22-S-HC), in comparison with the synthetic agonist T0901317 on lipid and glucose metabolism in human skeletal muscle cells (myotubes). Methods Myotubes established from lean and obese control volunteers and from obese type 2 diabetic volunteers were treated with LXR ligands for 4 days. Lipid and glucose metabolisms were studied with labelled precursors, and gene expression was analysed using real-time PCR. Results Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers. Furthermore, 22-S-HC efficiently counteracted the T0901317-induced enhancement of lipid formation. Moreover, synthesis of diacylglycerol, cholesteryl ester and free cholesterol from acetate was reduced below baseline by 22-S-HC, whereas glucose uptake and oxidation were increased. Both 22-S-HC and 22-R-HC, in contrast to T0901317, decreased the expression of genes involved in cholesterol synthesis, whereas only 22-R-HC, like T0901317, increased the expression of the gene encoding the reverse cholesterol transporter ATP-binding cassette subfamily A1 (ABCA1). Conclusions/interpretation T0901317-induced lipogenesis and lipid formation was more pronounced in myotubes from type 2 diabetic patients than from lean individuals. 22-S-HC counteracted these effects and reduced de novo lipogenesis below baseline, while glucose uptake and oxidation were increased. Keywords Glucose metabolism . Human myotubes . Hydroxycholesterols . Lipid metabolism . LXR antagonist . Type 2 diabetes . Liver X receptor Abbreviations ChREBP carbohydrate responsive element-binding protein FASN fatty acid synthase HMGC hydroxymethylglutaryl-coenzyme A LXR liver X receptor 22-R-HC 22-R-hydroxycholesterol 22-S-HC 22-S-hydroxycholesterol SREBP sterol regulatory element-binding protein SCD1 stearoyl-coenzyme A desaturase 1 Diabetologia (
Multiple defects in the regulation of glucose metabolism are believed to play a role in the patho... more Multiple defects in the regulation of glucose metabolism are believed to play a role in the pathophysiology of Type 2 diabetes mellitus . Thus, basal rates of glucose production are often elevated , and suppression of glucose production (GP) as well as stimulation of glucose disappearance ( R d) by insulin, is impaired in Type 2 diabetes . In the study of this issue, the primed-constant tracer infusion method plays a major role. This method is combined with the euglycaemic hyperinsulinaemic clamp technique to assess the effects of insulin on glucose metabolism, independent of the effects of hyperglycaemia per se .
Aim: Insulin resistance in subjects with type 2 diabetes (T2D) and obesity is associated with an ... more Aim: Insulin resistance in subjects with type 2 diabetes (T2D) and obesity is associated with an imbalance between the availability and the oxidation of lipids. We hypothesized that maximal whole-body lipid oxidation during exercise (FATmax) is reduced and that training-induced metabolic adaptation is attenuated in T2D. Methods: Obese T2D (n ¼ 12) and control (n ¼ 11) subjects matched for age, sex, physical activity and body mass index completed 10 weeks of aerobic training. Subjects were investigated before and after training with maximal and submaximal exercise tests and euglycaemic-hyperinsulinaemic clamps combined with muscle biopsies. Results: Training increased maximal oxygen consumption (VO 2max ) and muscle citrate synthase activity and decreased blood lactate concentrations during submaximal exercise in both groups (all p < 0.01). FATmax increased markedly (40-50%) in both T2D and control subjects after training (all p < 0.001). There were no significant differences in these variables and lactate threshold (%VO 2max ) between groups before or after training. Insulinstimulated glucose disappearance rate (Rd) was lower in T2D vs. control subjects both before and after training. Rd increased in response to training in both groups (all p < 0.01). There was no correlation between Rd and measures of oxidative capacity or lipid oxidation during exercise or the training-induced changes in these parameters. Conclusions: FATmax was not reduced in T2D, and muscle oxidative capacity increased adequately in response to aerobic training in obese subjects with and without T2D. These metabolic adaptations to training seem to be unrelated to changes in insulin sensitivity and indicate that an impaired capacity for lipid oxidation is not a major cause of insulin resistance in T2D.
Accumulating evidence suggests an association between insulin sensitivity and albuminuria, which,... more Accumulating evidence suggests an association between insulin sensitivity and albuminuria, which, even in the normal range, is a risk factor for cardiovascular diseases. We evaluated whether insulin sensitivity is associated with albuminuria in healthy subjects.
Aim. -This study explored the association of depressive symptoms with indices of insulin sensitiv... more Aim. -This study explored the association of depressive symptoms with indices of insulin sensitivity and insulin secretion in a cohort of non-diabetic men and women aged 30 to 64 years.
The importance of reducing sedentary time is increasingly being recognized in the prevention of d... more The importance of reducing sedentary time is increasingly being recognized in the prevention of diabetes and cardiovascular disease. Despite this, the prospective association between sedentary time and physical activity with insulin sensitivity and cardiometabolic risk factors has been little studied. In an analysis of data from the European RISC study, sedentary time and time spent in activity of moderate or vigorous intensity were assessed by accelerometry at baseline in 313 men and 414 women, aged 30-60 years, with insulin sensitivity as measured by euglycaemic-hyperinsulinaemic clamp. Three years later, cardiometabolic risk factors (anthropometry, glucose, insulin, lipids) were available for 549 participants. In cross-sectional analyses using baseline data, after adjusting for age, gender, recruitment centre and time spent in activity of moderate or vigorous intensity, significant unfavourable associations were observed between higher sedentary time with body weight, HDL cholesterol, triglycerides, clamp-measured insulin sensitivity and insulin secretion (all P(trend)&lt;0.002). Sedentary time remained significantly associated with insulin secretion after adjusting for insulin sensitivity (P(trend)=0.02). In longitudinal analyses, higher baseline sedentary time was associated with 3-year increases in fasting glucose, fasting insulin and the HOMA insulin-resistance index score for the 50% of the study population who increased their BMI by at least 0.3 kg/m(2) (all P(trend)&lt;0.01); these relationships remained significant after adjusting for time spent in activity of moderate or vigorous intensity. The 3-year increase in insulin secretion was lower in those spending more time doing activity of moderate or vigorous intensity (P(trend)=0.03). These prospective data suggest that less sedentary behaviour may partly counteract some of the negative effects of increasing body weight on glucose-insulin homoeostasis.
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Papers by Kurt Højlund