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.

Obesity and renal disease: not all fat is created equal and not all obesity is harmful to the kidneys.

The prevalence of obesity is increasing worldwide and contributes to many health problems, including kidney disease. Unexpectedly, 10-30% of obese individuals are apparently not at increased risk of metabolic diseases, e.g. type 2 diabetes, cardiovascular disease and risk of renal disease. Their phenotype is labeled 'metabolically healthy obesity'. In the search for mechanisms explaining this unexpected condition, a favourable type of body fat distribution with low insulin resistance and with low subclinical inflammation has been identified. Furthermore, signalling pathways have been found that distinguish between metabolically benign and malignant obesity. In addition, the important roles of fatty acids, adipokines and hepatokines were identified. These factors regulate insulin resistance and subclinical inflammation. Onset and evolution of chronic kidney disease (CKD) are affected by obesity. CKD also increases the risk of insulin resistance and subclinical inflammation, two pathways that play an important role in the pathogenesis of renal malfunction. This brief review summarizes novel insights, specifically how distinct body fat compartments (including perivascular and even renal sinus fat) may have an impact on progression of CKD.

Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle cells: role of the STAT3 pathway.

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6(-/-) skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6(-/-) myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6(-/-) cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6(-/-) cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

Cerebrospinal fluid fatty acids in glucocerebrosidase-associated Parkinson's disease.

BACKGROUND: Heterozygous mutations in the glucocerebrosidase gene lead to an increased risk for and to more severe alpha-synuclein-associated pathology in Parkinson's disease. As both glucocerebrosidase and alpha-synuclein interact with fatty acids, we hypothesized that cerebrospinal fluid fatty acid levels are altered in these Parkinson's disease patients. METHODS: Cerebrospinal fluid levels of 13 fatty acids in 8 Parkinson's disease patients with a heterozygous glucocerebrosidase mutation were compared with those of 41 idiopathic Parkinson's disease patients and 30 controls using gas chromatography. RESULTS: Parkinson's disease patients with a heterozygous glucocerebrosidase mutation had lower levels of palmitoleic (P ≤ .007), oleic (P ≤ .016), linoleic (P ≤ .005), arachidonic (P ≤ .003), eicosapentaenoic (P ≤ .003) and decosahexaenoic (P ≤ .03) acids and lower levels of total fatty acids (P < .005) compared with both idiopathic Parkinson's disease patients and control subjects. CONCLUSIONS: These results suggest that abnormalities of fatty acid metabolism are specifically involved in the pathogenesis of Parkinson's disease associated with a heterozygous glucocerebrosidase mutation.

Inhibitory effects of glycosaminoglycans on basal and stimulated transforming growth factor-ß1 expression in mesangial cells: biochemical and structural considerations.

A number of glycosaminoglycan (GAG) species related to heparin, dermatan sulfate (DeS) and chondroitin sulfate were tested for their ability to interfere with the physiological expression and/or pathological overexpression of the TGF-ß1 gene. The influence of the molecular weight, molecular weight distribution, degree of sulfation and location of the sulfate groups was examined in an attempt to unveil fine relationships between structure and activity. The nature of the polysaccharide plays a major part, heparins proving able to inhibit both basal and stimulated TGF-ß1 gene expression, DeSs being essentially inactive and chondroitin sulfates only inhibiting stimulated TGF-ß1 gene expression. Within this frame, the particular physical and chemical properties of some GAGs appear to further modulate TGF-ß1 gene response. Judging from our investigation, chondroitin sulfates seem the most promising for potential pharmacological applications in disorders characterized by fibrogenic TGF-ß1 overexpression.

Relationships between hepatic stearoyl-CoA desaturase-1 activity and mRNA expression with liver fat content in humans.

Stearoyl-CoA desaturase-1 (SCD1) has gained much interest as a future drug target to treat fatty liver and its consequences. However, there are few and inconsistent human data about expression and activity of this important enzyme. We investigated activity and expression of SCD1 and their relationships with liver fat (LF) content in human liver samples. Fifty subjects undergoing liver surgery were studied. SCD1 activity was estimated from the ratio of oleate (C18:1) to stearate (C18:0) within lipid subfractions. Furthermore, SCD1 mRNA expression and LF content were measured. Similarly to previous studies, we observed a strong positive correlation between LF content and the C18:1/C18:0 ratio in the combined fatty acid (FA) fractions (r = 0.96, P < 0.0001), which could be interpreted as higher SCD1 activity with increasing LF. However, hepatic SCD1 mRNA expression did not correlate with LF (r = 0.16, P = 0.13). To solve these conflicting data, we analyzed the FA composition of hepatic lipid subfractions. With increasing LF content the amount of FAs from the triglyceride (TG) fraction increased (r = 0.96, P < 0.0001), whereas the FAs from the phospholipid (PL) fraction remained unchanged (r = -0.17, P = 0.19). Of these two major lipid fractions, the C18:1/C18:0 ratio in TG was 16-fold higher than in PL. Supporting the SCD1 mRNA expression data, the C18:1/C18:0 ratio of the TG or PL fraction did not correlate with LF (r = 0.26, P = 0.12 and r = 0.08, P = 0.29). We provide novel information that SCD1 activity and mRNA expression appear not to be elevated in subjects with high LF content. We suggest that the FA composition of lipid subclasses, rather than of mixed lipids, should be analyzed to estimate SCD1 activity.

Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser675 and Ser907.

The identity of specific serine phosphorylation residues of insulin receptor substrate (IRS)-2 and their impact on insulin signal transduction are largely unknown. Ser(675) and Ser(907) of mouse IRS-2 are adjacent to PI 3-kinase or Grb2 binding domains, respectively. Using monoclonal phosphosite-specific antibodies, we demonstrated the phosphorylation of both serines after stimulation of Fao hepatoma cells with insulin, anisomycin, or phorbol esters. Phosphorylation of both sites was a late and prolonged event during insulin treatment and was also detected in liver tissue of insulin-treated as well as refed mice. Inhibition and siRNA-mediated knockdown of ERK1/2 indicated that the insulin-induced phosphorylation of Ser(907) was ERK dependent. Phosphorylation of Ser(907) did not prevent the insulin-induced association of IRS-2 with Grb2, but phosphorylation of the adjacent Tyr(911) was proved to be crucial in HEK 293 cells expressing IRS-2 Ala mutants. The insulin-induced phosphorylation of Ser(675) was prevented by inhibition and siRNA-mediated knockdown of mTOR but not of p70(S6K1). Mutation of Ser(675) to Ala did not affect downstream insulin signaling but increased the half-life of the protein, suggesting an involvement of phospho-Ser(675) in an accelerated degradation of IRS-2. Moreover, the insulin-induced degradation of IRS-2 was blocked by inhibition of mTOR. We conclude that the two novel insulin-dependent serine phosphorylation sites of IRS-2 were not involved in the regulation of the adjacent PI 3-kinase and Grb2 binding domains but might be implicated in the ERK- and mTOR-mediated negative feedback control.

A race for RAGE ligands.

In experimental animals a causal involvement of the multiligand receptor for advanced glycation end products (RAGE) in the development of diabetic vascular complications has been demonstrated. However, the nature of RAGE ligands present in patients with diabetic nephropathy has not yet been defined; this leaves open the relevance of the RAGE system to the human disease.

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.

Metabonomics study on the effects of the ginsenoside Rg3 in a beta-cyclodextrin-based formulation on tumor-bearing rats by a fully automatic hydrophilic interaction/reversed-phase column-switching HPLC-ESI-MS approach.

The goal of this study was the application of a novel, fully automatic column-switching approach in a metabonomics study combining the orthogonal selectivities of hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography. The temporal, pharmacodynamic effects of the ginsenoside Rg3 on the metabonome in urine of healthy and liver-tumor-bearing rats have been investigated. Within a total analysis time of 52 min we detected 5686 polar, and on the second column an additional 1808 apolar, urinary metabolite ions. The administration of a single, high dose of Rg3 in a beta-cyclodextrin-based formulation led to a considerable change of the metabolic pattern in cancer rats during 3 days studied. Seventeen biomarker candidates including three apolar metabolites, which were not retained on the HILIC column, were detected. Overall, the results suggest that the developed liquid chromatography-mass spectrometry strategy is a promising tool in metabonomics studies for global analysis of highly complex biosamples. It may not only increase the number of discovered biomarkers but consequently improve the comprehensive information on metabolic changes in a fully automatic manner.

Maillard products as biomarkers in cancer.

Because tumors exert increased glycolysis rates, a high intracellular carbonyl stress with the formation of Maillard products may evolve. Therefore, we studied the presence of N epsilon-(carboxymethyl)lysine (CML) modification in breast cancer tissues from 20 patients and found significant cytoplasmatic staining in tumor cells that was independent of the tumor stage, tumor type, and microanatomic localization. Studying breast cancer cell lines, we also found strong cytoplasmatic CML staining that was again independent of their invasive or metastatic behavior. Our results reveal that tumor cells show a strong cytoplasmatic immunoreactivity to CML without evident association with breast carcinoma type, differentiation, tumor stage, or intratumoral localization. We conclude that CML formation is a general tumor cell-associated process.

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.

Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion muLC/ES-API-CID-MS hybrid scan technique.

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-zeta. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS(3). Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-zeta phosphorylation site in the insulin receptor substrate -1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.

Simultaneous extraction of metabolome and lipidome with methyl tert-butyl ether from a single small tissue sample for ultra-high performance liquid chromatography/mass spectrometry.

A common challenge for scientists working with animal tissue or human biopsy samples is the limitation of material and consequently, the difficulty to perform comprehensive metabolic profiling within one experiment. Here, we present a novel approach to simultaneously perform targeted and non-targeted metabolomics as well as lipidomics from one small piece of liver or muscle tissue by ultra-high performance liquid chromatography/mass spectrometry (UHPLC/MS) following a methyl tert-butyl ether (MTBE)-based extraction. Equal relative amounts of the resulting polar and non-polar fractions were pooled, evaporated and reconstituted in the appropriate solvent for UHPLC/MS analysis. This mix was comparable or superior in yield and reproducibility to a standard 80% methanol extraction for the profiling of polar and lipophilic metabolites (free carnitine, acylcarnitines and FFA). The mix was also suitable for non-targeted metabolomics, an easy measure to increase the metabolite coverage by 30% relative to using the polar fraction alone. Lipidomics was performed from an aliquot of the non-polar fraction. This novel strategy could successfully be applied to one mouse soleus muscle with a dry weight of merely 2.5 mg. By enabling a simultaneous profiling of lipids and metabolites with mixed polarity while saving material for molecular, biochemical or histological analyses, our approach may open up new perspectives toward a comprehensive investigation of small, valuable tissue samples.

Production and release of acylcarnitines by primary myotubes reflect the differences in fasting fat oxidation of the donors.

CONTEXT: Acylcarnitines are biomarkers of incomplete ß-oxidation and mitochondrial lipid overload but indicate also high rates of mitochondrial fatty acid oxidation. It is unknown whether the production of acylcarnitines in primary human myotubes obtained from lean, metabolically healthy subjects reflects the fat oxidation in vivo. OBJECTIVE: Our objective was to quantify the acylcarnitine production in myotubes obtained from subjects with low and high fasting respiratory quotient (RQ). METHODS: Fasting RQ was determined by indirect calorimetry. Muscle biopsies from the vastus lateralis muscle were taken from 6 subjects with low fasting RQ (mean 0.79 ± 0.03) and 6 with high fasting RQ (0.90 ± 0.03), and satellite cells were isolated, cultured, and differentiated to myotubes. Myotubes were cultivated with 125 µM (13)C-labeled palmitate for 30 minutes and 4 and 24 hours. Quantitative profiling of 42 intracellular and 31 extracellular acylcarnitines was performed by stable isotope dilution-based metabolomics analysis by liquid chromatography coupled to mass spectrometry. RESULTS: Myotubes from donors with high fasting RQ produced and released significant higher amounts of medium-chain acylcarnitines. High (13)C8 and (13)C10 acylcarnitine levels in the extracellular compartment correlated with high fasting RQ. The decreased expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD) in these myotubes can explain the higher rate of incomplete fatty acid oxidation. A lower intracellular [(13)C]acetylcarnitine to carnitine and lower intracellular (13)C16/(13)C18 acylcarnitine to carnitine ratio indicate reduced fatty acid oxidation capacity in these myotubes. Mitochondrial DNA content was not different. CONCLUSION: Acylcarnitine production and release from primary human myotubes of donors with high fasting RQ indicate a reduced fatty acid oxidation capacity and a higher rate of incomplete fatty acid oxidation. Thus, quantitative profiling of acylcarnitine production in human myotubes can be a suitable tool to identify muscular determinants of fat oxidation in vivo.