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.

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.

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.

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.

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.

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.

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.

Direct cross-talk of interleukin-6 and insulin signal transduction via insulin receptor substrate-1 in skeletal muscle cells.

The exercise-induced interleukin (IL)-6 production and secretion within skeletal muscle fibers has raised the question of a putative tissue-specific function of IL-6 in the energy metabolism of the muscle during and after the exercise. In the present study, we followed the hypothesis that IL-6 signaling may directly interact with insulin receptor substrate (IRS)-1, a keystone in the insulin signaling cascade. We showed that IL-6 induces a rapid recruitment of IRS-1 to the IL-6 receptor complex in cultured skeletal muscle cells. Moreover, IL-6 induced a rapid and transient phosphorylation of Ser-318 of IRS-1 in muscle cells and in muscle tissue, but not in the liver of IL-6-treated mice, probably via the IL-6-induced co-recruitment of protein kinase C-delta. This Ser-318 phosphorylation improved insulin-stimulated Akt phosphorylation and glucose uptake in myotubes since transfection with an IRS-1/Glu-318 mutant simulating a permanent phospho-Ser-318 modification increased Akt phosphorylation and glucose uptake. Noteworthily, two inhibitory mechanisms of IL-6 on insulin action, phosphorylation of the inhibitory Ser-307 residue of IRS-1 and induction of SOCS-3 expression, were only found in liver but not in muscle of IL-6-treated mice. Thus, the data provided evidence for a possible molecular mechanism of the physiological metabolic effects of IL-6 in skeletal muscle, thereby exerting short term beneficial effects on insulin action.

Disseminated epitheloid hemangioendothelioma mimicking symptoms of systemic vasculitis.

BACKGROUND: Epitheloid hemangioendothelioma is a rare malignant tumor which can involve bones, liver, lungs, kidneys, deep soft tissue, muscles, dermis, and central nervous system. Multifocal disease occurs in 10% of the cases. The clinical presentation results from occlusion of small blood vessels due to the disease itself or as a paraneoplastic syndrome. CASE REPORT: We present a patient with symptoms suggesting systemic vasculitis (ESR and CRP elevated, weight loss, arthralgia, livedoid rash, and skin ulcerations) who finally was diagnosed having a disseminated epitheloid hemangioendothelioma when PET scan revealed hypermetabolic multifocal skeletal and soft tissue lesions. DISCUSSION: Diseases mimicking systemic vasculitis and the value of PET in this setting are discussed.

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.

Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt.

Previous studies showed an insulin-"desensitizing" action of IL-6 on glycogen synthesis in hepatocytes. We recently found no inhibition of the proximal steps of the insulin signal cascade in human skeletal muscle cells. Because these data indicate a possible tissue-specific effect of IL-6, we investigated the influence of IL-6 on insulin-stimulated glycogen synthesis in these cells. At first, we found that incubation of the cells with 20 ng/ml IL-6 alone induced phosphorylation of Ser473 of Akt, but not of Thr308 time dependently and we observed that IL-6 augments insulin-induced Ser473 and Thr308 phosphorylation in the low nanomolar range of insulin. Moreover, IL-6 increased insulin-stimulated phosphorylation of glycogen synthase kinase-3. Accordingly, IL-6 enhanced glycogen synthesis in the presence of 3 and 10 nM insulin, whereas IL-6 alone had only a marginal effect. IL-6 treatment of C57Bl/6 mice readily stimulated phosphorylation of Ser473 in skeletal muscle. Our result that IL-6 did not induce Ser473 phosphorylation in the liver of these mice suggests a tissue-specific effect. Together, our data demonstrate a novel insulin-sensitizing function of IL-6 on glycogen synthesis in skeletal muscle cells and indicate that IL-6 exerts cell/tissue-specific effects on insulin action.

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.

C825T polymorphism of the G protein beta3 subunit is associated with obesity but not with insulin sensitivity.

OBJECTIVE: The common C825T polymorphism of the gene that encodes the G protein beta3 subunit has been shown to influence lipolysis in human adipocytes and to be associated with hypertension, body fat distribution, and obesity. In addition, it has been shown to be associated with insulin resistance in a small group of hypertensive subjects. We investigated whether this polymorphism contributed to the variability in obesity in our population from southern Germany and whether it was associated with insulin sensitivity of lipolysis and/or glucose disposal. RESEARCH METHODS AND PROCEDURES: We determined percentage body fat, body fat distribution, glucose tolerance [oral glucose-tolerance test (OGTT)], insulin sensitivity, and serum free fatty acids using data from OGTTs (N = 774) and clamp (euglycemic hyperinsulinemic clamp, N = 216) in normal and impaired glucose tolerant subjects who were genotyped for this polymorphism. RESULTS: Compared with noncarriers of the C825T mutation, subjects with the C825T variant (prevalence approximately 32%) had higher percentage body fat (p = 0.02) and higher BMI (p = 0.03). No conclusive effect was seen on serum free fatty acids measured either during fasting or at the end of a 2-hour OGTT. Insulin sensitivity determined during the OGTT and during the clamp, both adjusted for age, gender, and percentage body fat, was not different between the genotypes (p = 0.33 and p = 0.48, respectively). DISCUSSION: We have concluded that the C825T polymorphism in the G protein beta3 subunit played an important role in the determination of obesity in this German population. However, it probably had no direct effects on insulin sensitivity of lipolysis and glucose disposal.

Palmitate, but not unsaturated fatty acids, induces the expression of interleukin-6 in human myotubes through proteasome-dependent activation of nuclear factor-kappaB.

Circulating interleukin-6 (IL-6), insulin, and free fatty acid (FFA) concentrations are associated with impaired insulin action in obese and type 2 diabetic individuals. However, a causal relationship between elevated plasma FFAs and IL-6 has not been shown. Because skeletal muscle represents a major target of impaired insulin action, we studied whether FFAs may affect IL-6 expression in human myotubes. We demonstrate that specifically saturated FFAs, e.g. palmitate (0.25 mm), induce IL-6 mRNA expression and protein secretion by a proteasome-dependent mechanism that leads to a rapid and chronic activation of nuclear factor-kappaB. Insulin, high glucose concentrations, or unsaturated FFAs did not activate IL-6 expression. In fact, the unsaturated FFA linoleate inhibited palmitate-induced IL-6 production. Because inhibition of palmitate metabolism by the acyl-CoA synthetase inhibitor triacsin C did not abolish IL-6 expression, it appears that the palmitate molecule per se exerts the observed effects. Furthermore, we show that in human myotubes, IL-6 activates the phosphorylation of signal transducer and activator of transcription 3 in concentrations similar to hepatocytes. However, no inhibitory effect of IL-6 on insulin action, determined as phosphatidylinositol 3-kinase association with insulin receptor substrate-1, Akt phosphorylation, and glycogen synthesis, was detected. We conclude that IL-6 expression may be modulated by the composition of circulating FFA, e.g. by diet, and that skeletal muscle cells could be target cells for IL-6.

Upstream stimulatory factor (USF) proteins induce human TGF-beta1 gene activation via the glucose-response element-1013/-1002 in mesangial cells: up-regulation of USF activity by the hexosamine biosynthetic pathway.

The hyperglycemia-enhanced flux through the hexosamine biosynthetic pathway (HBP) has been implicated in the up-regulated gene expression of transforming growth factor-beta1 (TGF-beta1) in mesangial cells, thus leading to mesangial matrix expansion and diabetic glomerulosclerosis. Since the -1013 to -1002 region of the TGF-beta1 promoter shows high homology to glucose-response elements (GlRE) formerly described in genes involved in glucose metabolism, we studied the function of the GlRE in the high glucose-induced TGF-beta1 gene activation in mesangial cells. We found that high glucose concentrations enhanced the nuclear amount of upstream stimulatory factors (USF) and their binding to this sequence. Fusion of the GlRE to the thymidine kinase promoter resulted in glucose responsiveness of this promoter construct. Overexpression of either USF-1 or USF-2 increased TGF-beta1 promoter activity 2-fold, which was prevented by mutation or deletion of the GlRE. The high glucose-induced activation of the GlRE is mediated by the HBP; increased flux through the HBP induced by high glucose concentrations, by glutamine, or by overexpression of the rate-limiting enzyme glutamine:fructose-6-phosphate aminotransferase (GFAT) particularly activated USF-2 expression. GFAT-overexpressing cells showed higher USF binding activity to the GlRE and enhanced promoter activation via the GlRE. Increasing O-GlcNAc modification of proteins by streptozotocin, thereby mimicking HBP activation, also resulted in increased mRNA and nuclear protein levels of USF-2, leading to enhanced DNA binding activity to the GlRE. USF proteins themselves were not found to be O-GlcNAc-modified. Thus, we have provided evidence for a new molecular mechanism linking high glucose-enhanced HBP activity with increased nuclear USF protein levels and DNA binding activity and with up-regulated TGF-beta1 promoter activity.

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.