Identification and regulation of the xenometabolite derivatives cis- and trans-3,4-methylene-heptanoylcarnitine in plasma and skeletal muscle of exercising humans.

Little is known about xenometabolites in human metabolism, particularly under exercising conditions. Previously, an exercise-modifiable, likely xenometabolite derivative, cis-3,4-methylene-heptanoylcarnitine, was reported in human plasma. Here, we identified trans-3,4-methylene-heptanoylcarnitine, and its cis-isomer, in plasma and skeletal muscle by liquid chromatography-mass spectrometry. We analyzed the regulation by exercise and the arterial-to-venous differences of these cyclopropane ring-containing carnitine esters over the hepatosplanchnic bed and the exercising leg in plasma samples obtained in three separate studies from young, lean and healthy males. Compared with other medium-chain acylcarnitines, the plasma concentrations of the 3,4-methylene-heptanoylcarnitine isomers only marginally increased with exercise. Both isomers showed a more than twofold increase in the skeletal muscle tissue of the exercising leg; this may have been due to the net effect of fatty acid oxidation in the exercising muscle and uptake from blood. The latter idea is supported by a more than twofold increased net uptake in the exercising leg only. Both isomers showed a constant release from the hepatosplanchnic bed, with an increased release of the trans-isomer after exercise. The isomers differ in their plasma concentration, with a four times higher concentration of the cis-isomer regardless of the exercise state. This is the first approach studying kinetics and fluxes of xenolipid isomers from tissues under exercise conditions, supporting the hypothesis that hepatic metabolism of cyclopropane ring-containing fatty acids is one source of these acylcarnitines in plasma. The data also provide clear evidence for an exercise-dependent regulation of xenometabolites, opening perspectives for future studies about the physiological role of this largely unknown class of metabolites.

PI3K-resistant GSK3 controls adiponectin formation and protects from metabolic syndrome.

Metabolic syndrome is characterized by insulin resistance, obesity, and dyslipidemia. It is the consequence of an imbalance between caloric intake and energy consumption. Adiponectin protects against metabolic syndrome. Insulin-induced signaling includes activation of PI3 kinase and protein kinase B (PKB)/Akt. PKB/Akt in turn inactivates glycogen synthase kinase (GSK) 3, a major regulator of metabolism. Here, we studied the significance of PI3K-dependent GSK3 inactivation for adiponectin formation in diet-induced metabolic syndrome. Mice expressing PI3K-insensitive GSK3 (gsk3(KI)) and wild-type mice (gsk3(WT)) were fed a high-fat diet. Compared with gsk3(WT) mice, gsk3(KI) mice were protected against the development of metabolic syndrome as evident from a markedly lower weight gain, lower total body and liver fat accumulation, better glucose tolerance, stronger hepatic insulin-dependent PKB/Akt phosphorylation, lower serum insulin, cholesterol, and triglyceride levels, as well as higher energy expenditure. Serum adiponectin concentration and the activity of transcription factor C/EBPα controlling the expression of adiponectin in adipose tissue was significantly higher in gsk3(KI) mice than in gsk3(WT) mice. Treatment with GSK3 inhibitor lithium significantly decreased the serum adiponectin concentration of gsk3(KI) mice and abrogated the difference in C/EBPα activity between the genotypes. Taken together, our data demonstrate that the expression of PI3K-insensitive GSK3 stimulates the production of adiponectin and protects from diet-induced metabolic syndrome.

Differential effects of intranasal insulin and caffeine on cerebral blood flow.

Insulin is an important modulator of brain functions such as memory and appetite regulation. Besides the effect on neuronal activity, it is also possible that insulin has a direct vasodilatory effect on cerebral blood flow (CBF). We investigated the impact of increased insulin levels in the central nervous system on basal and task-induced CBF as well as blood oxygenation level-dependent (BOLD) response in the visual cortex using pulsed arterial spin-labeling MRI. An intranasal insulin application was used to avoid peripheral hyperinsulinaemia, which would lead to a cascade of hormonal changes. In a control experiment, caffeine was applied due to its well-known impact on the vasculature of the brain leading to a reliable reduction of CBF. Eight lean subjects were included in the study. On 2 separate days, intranasal human insulin or caffeine tablets were given to the subjects after fasting over night. On each day, basal CBF and task-induced CBF were measured before and 30 min after application of insulin or caffeine in each subject. During the task condition, a flickering checkerboard was presented. Insulin had no effect on basal CBF and task-induced CBF in comparison with drug-free baseline measurement in the visual cortex and control regions. After caffeine application, however, there was a significant decrease of CBF during stimulation in the visual cortex. The BOLD response was not altered by insulin or caffeine between pre- and postdose measurements. In conclusion, we found no evidence for a direct vasodilatory effect of intranasal insulin on the cerebral vascular system in this study.

Effects of 4-week very-high-fructose/glucose diets on insulin sensitivity, visceral fat and intrahepatic lipids: an exploratory trial.

An increasing amount of fructose in the diet is suggested to play a causal role in the pathogenesis of the metabolic syndrome, type 2 diabetes and fatty liver. Our aim was to investigate and compare the effects of very high fructose and very high glucose in hyperenergetic diets on glucose and lipid metabolism and on fat depots in healthy humans. We conducted an exploratory, prospective, randomised, single-blinded, intervention trial. Participants in addition to a balanced weight-maintaining diet received 150 g of fructose or glucose/d for 4 weeks. Insulin sensitivity was estimated from oral glucose tolerance tests. Visceral and subcutaneous abdominal fat was determined with MRI. Liver fat and intramyocellular lipids of the tibialis anterior muscle were measured with (1)H magnetic resonance spectroscopy. A total of twenty healthy subjects (fructose group n 10 and glucose group n 10; twelve males and eight females) completed the study. They had a mean age of 30·5 (SEM 2·0) years and a mean BMI of 25·9 (SEM 0·5) kg/m(2). Insulin sensitivity appeared to decrease both in the fructose and glucose groups. TAG markedly increased in the fructose group. No strong alterations or treatment effects were found for liver fat, visceral fat, subcutaneous abdominal fat and intramyocellular lipids of the tibialis anterior muscle. In conclusion, the effects of very high fructose and very high glucose in hyperenergetic diets on glucose metabolism and body fat composition were not different in the healthy participants of the present study. However, elevation of plasma TAG seemed to be fructose-specific.

Muscle-Liver Substrate Fluxes in Exercising Humans and Potential Effects on Hepatic Metabolism.

CONTEXT: The liver is crucial to maintain energy homeostasis during exercise. Skeletal muscle-derived metabolites can contribute to the regulation of hepatic metabolism. OBJECTIVE: We aim to elucidate which metabolites are released from the working muscles and taken up by the liver in exercising humans and their potential influence on hepatic function. METHODS: In two separate studies, young healthy men fasted overnight and then performed an acute bout of exercise. Arterial-to-venous differences of metabolites over the hepato-splanchnic bed and over the exercising and resting leg were investigated by capillary electrophoresis- and liquid chromatography-mass spectrometry metabolomics platforms. Liver transcriptome data of exercising mice were analyzed by pathway analysis to find a potential overlap between exercise-regulated metabolites and activators of hepatic transcription. RESULTS: During exercise, hepatic O2 uptake and CO2 delivery were increased two-fold. In contrast to all other free fatty acids (FFA), those FFA with 18 or more carbon atoms and a high degree of saturation showed a constant release in the liver vein and only minor changes by exercise. FFA 6:0 and 8:0 were released from the working leg and taken up by the hepato-splanchnic bed. Succinate and malate showed a pronounced hepatic uptake during exercise and were also released from the exercising leg. The transcriptional response in the liver of exercising mice indicates the activation of HIF-, NRF2-, and cAMP-dependent gene transcription. These pathways can also be activated by succinate. CONCLUSION: Metabolites circulate between working muscles and the liver and may support the metabolic adaption to exercise by acting both as substrates and as signaling molecules.

Acute regulation of metabolic genes and insulin receptor substrates in the liver of mice by one single bout of treadmill exercise.

Acute exercise performance represents a major metabolic challenge for the skeletal muscle, but also for the liver as the most important source of energy. However the molecular adaptation of the liver to one single bout of exercise is largely unknown. C57BL/6 mice performed a 60 min treadmill run at high aerobic intensity. Liver, soleus and white gastrocnemius muscle were removed immediately after exercise. The single bout of exercise resulted in a very rapid and pronounced induction of hepatic metabolic enzymes and regulators of metabolism or transcription: glucose-6-phosphatase (G6Pase; 3-fold), pyruvate dehydrogenase kinase-4 (PDK4; 4.8-fold), angiopoietin-like 4 (2.1-fold), insulin receptor substrate (IRS)-2 (5.1-fold), peroxisome proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha; 3-fold). In soleus and white gastrocnemius muscle the up-regulation of IRS-2 and PDK4 was less pronounced compared with the liver and no significant induction of PGC-1alpha could be detected at this early time point. Activation of AMPK was found in both liver and white gastrocnemius muscle as phosphorylation of Thr-172. The induction of endogenous insulin secretion by a glucose load directly after the exercise bout resulted in a significantly higher PKB/Akt phosphorylation in the liver of exercised mice. The markedly enhanced IRS-2 protein amount, and presumably reduced serine/threonine phosphorylation of the IRS proteins induced by the acute exercise could be responsible for this enhanced action of insulin. In conclusion, acute exercise induced a rapid and pronounced transcriptional adaptation in the liver, and regulated hepatic IRS proteins leading to improved cellular insulin signal transduction.

Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: a 5-year follow-up study.

BACKGROUND: Cluster analyses have proposed different diabetes phenotypes using age, BMI, glycaemia, homoeostasis model estimates, and islet autoantibodies. We tested whether comprehensive phenotyping validates and further characterises these clusters at diagnosis and whether relevant diabetes-related complications differ among these clusters, during 5-years of follow-up. METHODS: Patients with newly diagnosed type 1 or type 2 diabetes in the German Diabetes Study underwent comprehensive phenotyping and assessment of laboratory variables. Insulin sensitivity was assessed using hyperinsulinaemic-euglycaemic clamps, hepatocellular lipid content using magnetic resonance spectroscopy, hepatic fibrosis using non-invasive scores, and peripheral and autonomic neuropathy using functional and clinical criteria. Patients were reassessed after 5 years. The German Diabetes Study is registered with ClinicalTrials.gov, number NCT01055093, and is ongoing. FINDINGS: 1105 patients were classified at baseline into five clusters, with 386 (35%) assigned to mild age-related diabetes (MARD), 323 (29%) to mild obesity-related diabetes (MOD), 247 (22%) to severe autoimmune diabetes (SAID), 121 (11%) to severe insulin-resistant diabetes (SIRD), and 28 (3%) to severe insulin-deficient diabetes (SIDD). At 5-year follow-up, 367 patients were reassessed, 128 (35%) with MARD, 106 (29%) with MOD, 88 (24%) with SAID, 35 (10%) with SIRD, and ten (3%) with SIDD. Whole-body insulin sensitivity was lowest in patients with SIRD at baseline (mean 4·3 mg/kg per min [SD 2·0]) compared with those with SAID (8·4 mg/kg per min [3·2]; p<0·0001), MARD (7·5 mg/kg per min [2·5]; p<0·0001), MOD (6·6 mg/kg per min [2·6]; p=0·0011), and SIDD (5·5 mg/kg per min [2·4]; p=0·0035). The fasting adipose-tissue insulin resistance index at baseline was highest in patients with SIRD (median 15·6 [IQR 9·3-20·9]) and MOD (11·6 [7·4-17·9]) compared with those with MARD (6·0 [3·9-10·3]; both p<0·0001) and SAID (6·0 [3·0-9·5]; both p<0·0001). In patients with newly diagnosed diabetes, hepatocellular lipid content was highest at baseline in patients assigned to the SIRD cluster (median 19% [IQR 11-22]) compared with all other clusters (7% [2-15] for MOD, p=0·00052; 5% [2-11] for MARD, p<0·0001; 2% [0-13] for SIDD, p=0·0083; and 1% [0-3] for SAID, p<0·0001), even after adjustments for baseline medication. Accordingly, hepatic fibrosis at 5-year follow-up was more prevalent in patients with SIRD (n=7 [26%]) than in patients with SAID (n=5 [7%], p=0·0011), MARD (n=12 [12%], p=0·012), MOD (n=13 [15%], p=0·050), and SIDD (n=0 [0%], p value not available). Confirmed diabetic sensorimotor polyneuropathy was more prevalent at baseline in patients with SIDD (n=9 [36%]) compared with patients with SAID (n=10 [5%], p<0·0001), MARD (n=39 [15%], p=0·00066), MOD (n=26 [11%], p<0·0001), and SIRD (n=10 [17%], p<0·0001). INTERPRETATION: Cluster analysis can characterise cohorts with different degrees of whole-body and adipose-tissue insulin resistance. Specific diabetes clusters show different prevalence of diabetes complications at early stages of non-alcoholic fatty liver disease and diabetic neuropathy. These findings could help improve targeted prevention and treatment and enable precision medicine for diabetes and its comorbidities. FUNDING: German Diabetes Center, German Federal Ministry of Health, Ministry of Culture and Science of the state of North Rhine-Westphalia, German Federal Ministry of Education and Research, German Diabetes Association, German Center for Diabetes Research, Research Network SFB 1116 of the German Research Foundation, and Schmutzler Stiftung.

Angiopoietin-like protein 4 is an exercise-induced hepatokine in humans, regulated by glucagon and cAMP.

OBJECTIVE: Angiopoietin-like protein-4 (ANGPTL4) is a circulating protein that is highly expressed in liver and implicated in regulation of plasma triglyceride levels. Systemic ANGPTL4 increases during prolonged fasting and is suggested to be secreted from skeletal muscle following exercise. METHODS: We investigated the origin of exercise-induced ANGPTL4 in humans by measuring the arterial-to-venous difference over the leg and the hepato-splanchnic bed during an acute bout of exercise. Furthermore, the impact of the glucagon-to-insulin ratio on plasma ANGPTL4 was studied in healthy individuals. The regulation of ANGPTL4 was investigated in both hepatic and muscle cells. RESULTS: The hepato-splanchnic bed, but not the leg, contributed to exercise-induced plasma ANGPTL4. Further studies using hormone infusions revealed that the glucagon-to-insulin ratio is an important regulator of plasma ANGPTL4 as elevated glucagon in the absence of elevated insulin increased plasma ANGPTL4 in resting subjects, whereas infusion of somatostatin during exercise blunted the increase of both glucagon and ANGPTL4. Moreover, activation of the cAMP/PKA signaling cascade let to an increase in ANGPTL4 mRNA levels in hepatic cells, which was prevented by inhibition of PKA. In humans, muscle ANGPTL4 mRNA increased during fasting, with only a marginal further induction by exercise. In human muscle cells, no inhibitory effect of AMPK activation could be demonstrated on ANGPTL4 expression. CONCLUSIONS: The data suggest that exercise-induced ANGPTL4 is secreted from the liver and driven by a glucagon-cAMP-PKA pathway in humans. These findings link the liver, insulin/glucagon, and lipid metabolism together, which could implicate a role of ANGPTL4 in metabolic diseases.

Partial characterisation of the human GFAT promoter: effect of single nucleotide polymorphisms on promoter function.

The 5'-flanking region of the human glutamine:fructose-6-phosphate amidotransferase (GFAT) gene was characterised as a functional active promoter and the GFAT gene contained multiple transcription start sites. A novel single nucleotide polymorphism identified at position -1412 (G to C) had a functional effect on promoter activity and EMSA revealed specific binding of nuclear proteins to this region.

The -913 G/A glutamine:fructose-6-phosphate aminotransferase gene polymorphism is associated with measures of obesity and intramyocellular lipid content in nondiabetic subjects.

Increases in glutamine:fructose-6-phosphate aminotransferase (GFAT) protein levels directly activate flux through the hexosamine biosynthetic pathway. This pathway has been involved as a fuel sensor in energy metabolism and development of insulin resistance. We screened the 5'-flanking region of the human GFAT gene for polymorphisms and subsequently genotyped 412 nondiabetic, metabolically characterized Caucasians for the two single-nucleotide polymorphisms (SNP) at positions -913 (G/A) and -1412 (C/G) with rare allele frequencies of 42% and 16%, respectively. The -913 G SNP was associated with significantly higher body mass index and percent body fat in men (P = 0.02 and 0.004, respectively), but not in women (P = 0.47 and 0.26, respectively). In the subgroup of individuals (n = 193) who underwent hyperinsulinemic-euglycemic clamp, an association of the -913 G SNP with insulin sensitivity independent of body mass index was not detected. Moreover, the -913 G allele in a group of 71 individuals who had undergone magnetic resonance spectroscopy was associated with higher intramyocellular lipid content (IMCL) in tibialis anterior muscle (4.21 +/- 0.31 vs. 3.36 +/- 0.35; P = 0.04) independent of percent body fat and maximal aerobic power. The -1412 SNP had no effect on percent body fat, insulin sensitivity, or IMCL. In conclusion, we identified two polymorphisms in the 5'-flanking region of GFAT, of which the -913 SNP seems to alter the risk for obesity and IMCL accumulation in male subjects.

Palmitate-induced activation of the hexosamine pathway in human myotubes: increased expression of glutamine:fructose-6-phosphate aminotransferase.

The nutrient sensing capacity of the hexosamine biosynthetic pathway (HBP) has been implicated in the development of insulin resistance of skeletal muscle. To study the molecular mechanism of the free fatty acid (FFA)-induced activation of the HBP myotubes obtained from muscle biopsies of metabolically characterized, subjects were stimulated with different fatty acids for 20 h. Incubation with the saturated fatty acids palmitate and stearate (0.5 mmol/l) resulted in a three- to fourfold increase in mRNA expression of glutamine:fructose-6-phosphate aminotransferase (GFAT), the key and rate-limiting enzyme of the hexosamine pathway. Unsaturated fatty acids or 30 mmol/l glucose had little or no effect. Palmitate increased the amount of GFAT protein nearly two-fold, and subsequently, the concentration of UDP-N-acetylglucosamine, the end product of the HBP, was 1.3-fold enhanced in the palmitate-stimulated myotubes. The nonmetabolized fatty acid bromopalmitate had no effect. The DNA binding activity of the transcription factor Sp1, a target downstream of the HBP, was increased by palmitate and completely lost after enzymatic removal of O-GlcNAc. No correlation was found between the palmitate-induced increase in GFAT protein and the insulin resistance in the respective subjects. The findings reveal a new mechanism for how FFAs induce the activation of the HBP.

Evidence for a novel TGF-beta1-independent mechanism of fibronectin production in mesangial cells overexpressing glucose transporters.

Recent experimental work indicates that the hyperglycemia-induced increase in mesangial matrix production, which is a hallmark in the development of diabetic nephropathy, is mediated by increased expression of GLUT1. Mesangial cells stably transfected with human GLUT1 mimic the effect of hyperglycemia on the production of the extracellular matrix proteins, particularly fibronectin, when cultured under normoglycemic conditions. Our investigation of the molecular mechanism of this effect has revealed that the enhanced fibronectin production was not mediated by the prosclerotic cytokine transforming growth factor (TGF)-beta1. We found markedly increased nuclear content in Jun proteins, leading to enhanced DNA-binding activity of activating protein 1 (AP-1). AP-1 inhibition reduced fibronectin production in a dosage-dependent manner. Moreover, inhibition of classic protein kinase C (PKC) isoforms prevented both the activation of AP-1 and the enhanced fibronectin production. In contrast to mesangial cells exposed to high glucose, no activation of the hexosamine biosynthetic, p38, or extracellular signal-related kinase 1 and 2 mitogen-activated protein kinase pathways nor any increase in TGF-beta1 synthesis could be detected, which could be explained by the absence of oxidative stress in cells transfected with the human GLUT1 gene. Our data indicate that increased glucose uptake and metabolism induce PKC-dependent AP-1 activation that is sufficient for enhanced fibronectin production, but not for increased TGF-beta1 expression.

Genetic factors and insulin secretion: gene variants in the IGF genes.

IGFs are important regulators of pancreatic beta-cell development, growth, and maintenance. Mutations in the IGF genes have been found to be associated with type 2 diabetes, myocardial infarction, birth weight, and obesity. These associations could result from changes in insulin secretion. We have analyzed glucose-stimulated insulin secretion using hyperglycemic clamps in carriers of a CA repeat in the IGF-I promoter and an ApaI polymorphism in the IGF-II gene. Normal and impaired glucose-tolerant subjects (n = 237) were independently recruited from three different populations in the Netherlands and Germany to allow independent replication of associations. Both first- and second-phase insulin secretion were not significantly different between the various IGF-I or IGF-II genotypes. Remarkably, noncarriers of the IGF-I CA repeat allele had both a reduced insulin sensitivity index (ISI) and disposition index (DI), suggesting an altered balance between insulin secretion and insulin action. Other diabetes-related parameters were not significantly different for both the IGF-I and IGF-II gene variant. We conclude that gene variants in the IGF-I and IGF-II genes are not associated with detectable variations in glucose-stimulated insulin secretion in these three independent populations. Further studies are needed to examine the exact contributions of the IGF-I CA repeat alleles to variations in ISI and DI.

Effect of glimepiride on insulin-stimulated glycogen synthesis in cultured human skeletal muscle cells: a comparison to glibenclamide.

OBJECTIVE: To examine the effect of glimepiride on insulin-stimulated glycogen synthesis in cultured human skeletal muscle cells in comparison with glibenclamide. RESEARCH DESIGN AND METHODS: Myotubes derived from glucose-tolerant subjects were incubated with glimepiride or glibenclamide (0-100 micro mol/l) for 4 h and with or without insulin (100 nmol/l) for 2 h, and subsequently glycogen synthesis was determined. RESULTS: Glimepiride had no significant effect on basal glycogen synthesis; in contrast, glimepiride caused a dose-dependent increase of insulin-stimulated glycogen synthesis, with a maximal effect of 39.97 +/- 8.4% (mean +/- SEM, n = 4, P < 0,02). The time course of this glimepiride effect on insulin-stimulated glycogen synthesis showed a peak after 12 h incubation with a half maximal effect after 4 h. Preincubation of the myotubes with wortmannin (100 nmol/l), an inhibitor of phosphatidylinositol (PI)- 3 kinase, caused an inhibition of this glimepiride effect on insulin-stimulated glycogen synthesis. In contrast to glimepiride, incubation of myotubes with glibenclamide (0-100nmol/l), a second generation sulfonylurea, had no significant effect on basal or insulin-stimulated glycogen synthesis. CONCLUSIONS: Incubation of cultured human skeletal muscle cells derived from glucose-tolerant subjects with glimepiride caused a dose-dependent increase of insulin-stimulated glycogen synthesis using therapeutic glimepiride concentrations. This glimepiride effect seems to be mediated via the PI3 kinase pathway. In contrast to glimepiride, glibenclamide had no significant effect on basal or insulin-stimulated glycogen synthesis. These results suggest that glimepiride, beside its well-known effect to stimulate insulin secretion, possess an insulin-sensitizing action in cultured human skeletal muscle cells in support of the concept of an extrapancreatic action of glimepiride.

Acute effect of glimepiride on insulin-stimulated glucose metabolism in glucose-tolerant insulin-resistant offspring of patients with type 2 diabetes.

OBJECTIVE: This study addressed whether acute infusion of glimepiride influences glucose metabolism independent of its effect on insulin secretion. RESEARCH DESIGN AND METHODS: Ten healthy, glucose-tolerant but insulin-resistant probands were subjected to a placebo-controlled, double-blind, cross-over study. Each individual received infusions of either 0.15 mol/l saline or glimepiride in randomized order on two separate occasions. A three-step hyperinsulinemic (0.5, 1.0, and 1.5 mU. kg(-1). min(-1))-euglycemic glucose clamp was performed on both occasions to determine insulin sensitivity. Glimepiride-induced insulin secretion was inhibited by octreotide. Endogenous glucose production and glucose elimination were measured with the "hot" glucose infusion method using U-[(13)C]glucose as tracer. Glucose oxidation was determined from indirect calorimetry. Lipolysis was evaluated by measurements of nonesterified fatty acid (NEFA) and glycerol concentration and measurement of glycerol production. RESULTS: Plasma glucose and insulin concentrations were not significantly different between glimepiride or saline infusions. There was a significant increase in the rate of glucose infusion necessary to maintain euglycemia during infusion of glimepiride during the low- (12.2 +/- 1.1 vs. 16.1 +/- 1.7 micro mol. kg(-1). min(-1)) and intermediate-dose insulin infusion (24.4 +/- 1.7 vs. 30.0 +/- 2.8 micro mol. kg(-1). min(-1)). This was explained by an increased rate of glucose elimination and to a lesser degree by a decrease in glucose production. Glucose oxidation rate was not different. NEFA and glycerol concentration and glycerol production were equally suppressed. CONCLUSIONS: Glimepiride improves peripheral glucose uptake and decreases endogenous glucose production independent of its insulin secretagogue action. The effects shown in this acute study are, however, too small to be considered therapeutically beneficial for the individual patient.

Insulin resistant young subjects at risk of accelerated atherosclerosis exhibit a marked reduction in peripheral endothelial function early in life but not differences in intima-media thickness.

Reduced bio-availability of nitric oxide leading to disturbed flow mediated (endothelial dependent) vasodilation (FMD) has been shown to be an early functional abnormality of the vascular system in insulin resistant individuals and other subjects at high risk for accelerated atherosclerosis. In addition, an increase of the intima-media thickness (IMT) is regarded as an early marker of morphological alterations of the vessel wall. Whether endothelial dysfunction (ED) is evident already at an early stage when morphological changes of the vessel wall are not apparent is still an open question. We, therefore, examined IMT and peripheral endothelial function in a group of young insulin resistant subjects in a cross-sectional study and compared these results with a metabolically healthy (insulin sensitive) control group. We measured IMT (distal common carotid arteries), endothelium-dependent and endothelium-independent vasodilation (flow mediated and glyceroltrinitrate induced vasodilation of the brachial artery) non-invasively with high resolution ultrasound (13 MHz) in 91 young normoglycemic subjects (40/51 M/F, median: 31 years, range 18-50 years). Insulin sensitivity was measured with a euglycemic, hyperinsulinemic glucose clamp. Despite a marked reduction in flow-mediated vasodilation in insulin resistant (IR) subjects (FMD: median 3.4%, range -4.0 to 12.5 in IR versus 6.6%, range -1.2 to 20.1% in insulin sensitive subjects; P = 0.017), there was no difference in endothelial independent vasodilation (16.3%, range 5.7-41.0% versus 16.1%, range 0.5-39.2%) and in IMT (0.50 mm, range 0.39-0.66 and 0.51, 0.40-0.70 mm, respectively). These data suggest that ED can be detected very early in the life of insulin resistant subjects whereas no significant structural changes, indicated by a thickening of the intima-media layer, could be found. We therefore conclude that for identification of subjects with a high risk for accelerated atherosclerosis at an early stage, measurement of flow mediated vasodilation of the brachial artery may be more helpful than measuring thickness of the vascular wall.

[Urinary albumin excretion and noninvasive assessment of peripheral endothelial dysfunction with high-resolution ultrasound in type 2 diabetic subjects and nondiabetic controls]. / Renale Albuminausscheidung und nichtinvasive Endothelfunktionstestung mit hochauflösendem Ultraschall bei Typ-2-Diabetikern und Nichtdiabetikern.

BACKGROUND: Recent theory in pathogenesis of atherosclerosis has focused on the pathobiology of the artery wall including the emerging influence of the nitric oxide (NO) system on thrombogenicity and trigger mechanisms leading to morphologic changes culminating in the stenotic plaque. Therefore, diagnostic evaluation of disturbances in NO bioavailability might be of prognostic relevance regarding primary prevention of cardiovascular disease. Disturbances in NO production can be measured noninvasively with conventional high-resolution ultrasound. On the other hand, particularly in individuals with diabetes, microalbuminuria is thought to be associated with an increased risk of cardiovascular events. Thereby it is still unknown, whether an increase in renal albumin excretion can be regarded as an indicator of global endothelial dysfunction, or whether other partial functions such as the nitric oxide system might be disturbed earlier. PROBANDS AND METHODS: Therefore, the NO system and renal albumin excretion were examined in 129 subjects (56 with type 2 diabetes and 73 nondiabetics). Nitric oxide production was assessed by measuring flow-mediated vasodilatation (FMD) of the brachial artery using a 13-MHz linear array. Comparison was done between subjects with disturbed endothelial NO production (FMD < 5%) and subjects with normal regulation of the vascular tone (FMD > 5%). RESULTS: In normoalbuminuric individuals (< 20 microg/min, and < 20 mg/l, respectively), neither for the group of subjects with type 2 diabetes nor in the group of nondiabetics, relevant differences could be found in renal albumin excretion (RAE) rate between subjects with disturbed and normal FMD (RAE in diabetics 4.8 +/- 5.5 vs. 4.6 +/- 5.1 mg/l and in nondiabetics 5.1 +/- 2.6 vs. 4.9 +/- 2.7 microg/min). Both groups were well balanced regarding other risk factors of the metabolic syndrome (systolic/diastolic blood pressure, glucose and lipid metabolism). Furthermore, comparison of FMD in subjects with microalbuminuria (20-200 microg/min and 20-200 mg/l, respectively, n = 18) versus normoalbuminuric individuals (n = 111) again did not reveal a significant difference for the diabetic group (FMD median 4.3% [range 1.8-7.6%] vs. 5.0% [range 1.1-9.1%]) nor for the nondiabetic group (FMD median 4.7% [range 3.1-13.3%] vs. 5.2% [range -1.2-31.6%]). However, this analysis underlined the considerable influence of the classic cardiovascular risk factors. Particularly in the nondiabetic group, individuals with microalbuminuria showed higher blood pressure (p = 0.05) and a higher body mass index (p < 0.01). CONCLUSION: From these results, it is concluded that both procedures (renal albumin excretion rate and the measurement of endothelium-dependent vasodilatation) investigate two independent disturbances of the vascular wall. Furthermore, these results lead to the hypothesis that disturbances in endothelial NO production occur early and may already be operative before renal albumin excretion increases. Thus, for the purpose of actually identifying cardiovascular high-risk subjects early, peripheral endothelial dysfunction should be measured in addition to renal albumin excretion rate.

Lipidomics analysis reveals efficient storage of hepatic triacylglycerides enriched in unsaturated fatty acids after one bout of exercise in mice.

BACKGROUND: Endurance exercise induces lipolysis, increases circulating concentrations of free fatty acids (FFA) and the uptake and oxidation of fatty acids in the working muscle. Less is known about the regulation of lipid metabolism in the liver during and post-exercise. METHODOLOGY/PRINCIPAL FINDINGS: We performed an ultra fast liquid chromatography-mass spectrometry (UFLC-MS) based lipidomics analysis of liver tissue samples obtained from C57Bl/6J mice immediately after a 60 min treadmill run of moderate intensity, and after 3 h of recovery. The PLS-DA scores plot for 115 quantified lipid molecular species revealed a clear separation of the hepatic lipid profile of sedentary from recovering mice, but not from mice immediately after running. 21 lipid species were considered to be most responsible for the difference in the hepatic lipid profiles, including 17 triacylglycerides (TG), one lysophosphatidylcholine (LPC) and three phosphatidylcholines (PC). TG species were found to be more abundant in the recovery phase, while PC species were decreased. The degree of accumulation of individual TG species correlated well with the amount of theoretical energy stored whereas no increase was found for TG species containing only saturated or one monounsaturated fatty acid. Total liver TG content as assayed by an enzymatic method was increased to 163% in the recovery phase, while it was significantly decreased in skeletal muscle by the exercise bout and remained less in the recovery phase. Results from fasted and refed mice indicate that fasting-induced lipolysis was associated with a pronounced accumulation of hepatic TG, which is reversed by refeeding for 5 h. Thus food intake per se did not elevate hepatic TG. CONCLUSION: These data indicate that high availability of FFA induced by endurance exercise or fasting resulted in a transient hepatic TG accumulation, while muscle TG content was decreased during exercise presumably due to increased muscle fatty acid oxidation.

Upregulation of IL-6 mRNA by IL-6 in skeletal muscle cells: role of IL-6 mRNA stabilization and Ca2+-dependent mechanisms.

Skeletal muscle cells have been established as significant producers of IL-6 during exercise. This IL-6 production is discussed as one possible mediator of the beneficial effects of physical activity on glucose and fatty acid metabolism. IL-6 itself could be the exercise-related factor that upregulates and maintains its own production. We investigated this hypothesis and the underlying molecular mechanism in cultured C(2)C(12) cells. IL-6 led to a rapid and prolonged increase in IL-6 mRNA, which was also found in human myotubes. Because IL-6 has been shown to activate AMP-activated kinase (AMPK), we studied whether, in turn, activated AMPK induces IL-6 expression. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside upregulated IL-6 mRNA expression, which was blocked by knockdown of AMPK alpha(1) and alpha(2) using small, interfering RNA (siRNA) oligonucleotides. However, the effect of IL-6 was shown to be independent of AMPK, since the siRNA approach silencing the AMPK alpha-subunits did not reduce the upregulation of IL-6 induced by IL-6 stimulation. The self-stimulatory effect of IL-6 partly involves a Ca(2+)-dependent pathway: IL-6 increased intracellular Ca(2+), and intracellular blockade of Ca(2+) with a Ca(2+) chelator reduced the IL-6-mediated increase in IL-6 mRNA levels. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase kinase with STO-609 or the siRNA approach decreased IL-6 mRNA levels of control and IL-6-stimulated cells. A major, STO-609-independent mechanism is the IL-6-mediated stabilization of its mRNA. The data suggest that IL-6 could act as autocrine factor upregulating its mRNA levels, thereby supporting its function as an exercise-activated factor in skeletal muscle cells.