IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following day 6. Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level.NEW & NOTEWORTHY Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.

Redox state and altered pyruvate metabolism contribute to a dose-dependent metformin-induced lactate production of human myotubes.

Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases in blood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metformin increases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to produce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potential contribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treatment (16-776 µM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78 µM, metformin induced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respirometry, we found metformin to inhibit the respiratory chain complex I (776 µM, P < 0.01) along with decreasing the [NAD+]:[NADH] ratio (776 µM, P < 0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation mediated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39 µM, P < 0.01). On the other hand, in human skeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased under metformin treatment (P < 0.05). In conclusion, the findings suggest that metformin-induced inhibition of pyruvate oxidation combined with altered cellular redox state shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-dependent lactate production in primary human myotubes.NEW & NOTEWORTHY Metformin shifts the equilibrium of lactate dehydrogenase (LDH) reaction by low dose-induced phosphorylation of pyruvate dehydrogenase (PDH) resulting in inhibition of pyruvate oxidation and high dose-induced increase in NADH, which explains the dose-dependent lactate production of differentiated human skeletal muscle cells.

Acute and long-term exercise adaptation of adipose tissue and skeletal muscle in humans: a matched transcriptomics approach after 8-week training-intervention.

BACKGROUND: Exercise exerts many health benefits by directly inducing molecular alterations in physically utilized skeletal muscle. Molecular adaptations of subcutaneous adipose tissue (SCAT) might also contribute to the prevention of metabolic diseases. AIM: To characterize the response of human SCAT based on changes in transcripts and mitochondrial respiration to acute and repeated bouts of exercise in comparison to skeletal muscle. METHODS: Sedentary participants (27 ± 4 yrs) with overweight or obesity underwent 8-week supervised endurance exercise 3×1h/week at 80% VO2peak. Before, 60 min after the first and last exercise bout and 5 days post intervention, biopsies were taken for transcriptomic analyses and high-resolution respirometry (n = 14, 8 female/6 male). RESULTS: In SCAT, we found 37 acutely regulated transcripts (FC > 1.2, FDR < 10%) after the first exercise bout compared to 394, respectively, in skeletal muscle. Regulation of only 5 transcripts overlapped between tissues highlighting their differential response. Upstream and enrichment analyses revealed reduced transcripts of lipid uptake, storage and lipogenesis directly after exercise in SCAT and point to ß-adrenergic regulation as potential major driver. The data also suggest an exercise-induced modulation of the circadian clock in SCAT. Neither term was associated with transcriptomic changes in skeletal muscle. No evidence for beigeing/browning was found in SCAT along with unchanged respiration. CONCLUSIONS: Adipose tissue responds completely distinct from adaptations of skeletal muscle to exercise. The acute and repeated reduction in transcripts of lipid storage and lipogenesis, interconnected with a modulated circadian rhythm, can counteract metabolic syndrome progression toward diabetes.

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.

DEUS: an R package for accurate small RNA profiling based on differential expression of unique sequences.

SUMMARY: Despite their fundamental role in various biological processes, the analysis of small RNA sequencing data remains a challenging task. Major obstacles arise when short RNA sequences map to multiple locations in the genome, align to regions that are not annotated or underwent post-transcriptional changes which hamper accurate mapping. In order to tackle these issues, we present a novel profiling strategy that circumvents the need for read mapping to a reference genome by utilizing the actual read sequences to determine expression intensities. After differential expression analysis of individual sequence counts, significant sequences are annotated against user defined feature databases and clustered by sequence similarity. This strategy enables a more comprehensive and concise representation of small RNA populations without any data loss or data distortion. AVAILABILITY AND IMPLEMENTATION: Code and documentation of our R package at http://ibis.helmholtz-muenchen.de/deus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hepatokines-a novel group of exercise factors.

Regular physical activity not only improves the exercise capacity of the skeletal muscle performing the contractions, but it is beneficial for the whole body. An extensive search for "exercise factors" mediating these beneficial effects has been going on for decades. Particularly skeletal muscle tissue has been investigated as a source of circulating exercise factors, and several myokines have been identified. However, exercise also has an impact on other tissues. The liver is interposed between energy storing and energy utilising tissues and is highly active during exercise, maintaining energy homeostasis. Recently, a novel group of exercise factors-termed hepatokines-has emerged. These proteins (fibroblast growth factor 21, follistatin, angiopoietin-like protein 4, heat shock protein 72, insulin-like growth factor binding protein 1) are released from the liver and increased in the bloodstream during or in the recovery after an exercise bout. In this narrative review, we evaluate this new group of exercise factors focusing on the regulation and potential function in exercise metabolism and adaptations. These hepatokines may convey some of the beneficial whole-body effects of exercise that could ameliorate metabolic diseases, such as obesity or type 2 diabetes.

Linking bioenergetic function of mitochondria to tissue-specific molecular fingerprints.

Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.

Comprehensive Profiling by Non-targeted Stable Isotope Tracing Capillary Electrophoresis-Mass Spectrometry: A New Tool Complementing Metabolomic Analyses of Polar Metabolites.

Mass spectrometry (MS) driven metabolomics is a frequently used tool in various areas of life sciences; however, the analysis of polar metabolites is less commonly included. In general, metabolomic analyses lead to the detection of the total amount of all covered metabolites. This is currently a major limitation with respect to metabolites showing high turnover rates, but no changes in their concentration. Such metabolites and pathways could be crucial metabolic nodes (e.g., potential drug targets in cancer metabolism). A stable-isotope tracing capillary electrophoresis-mass spectrometry (CE-MS) metabolomic approach was developed to cover both polar metabolites and isotopologues in a non-targeted way. An in-house developed software enables high throughput processing of complex multidimensional data. The practicability is demonstrated analyzing [U-13 C]-glucose exposed prostate cancer and non-cancer cells. This CE-MS-driven analytical strategy complements polar metabolite profiles through isotopologue labeling patterns, thereby improving not only the metabolomic coverage, but also the understanding of metabolism.

Nasal insulin administration does not affect hepatic glucose production at systemic fasting insulin levels.

AIMS: To evaluate the effects of brain insulin on endogenous glucose production in fasting humans, with a focus on hepatic glucose release by performing a randomized, placebo-controlled, blinded, crossover experiment. MATERIALS AND METHODS: On two separate days, 2 H2 -glucose was infused to nine healthy lean men, and blood was sampled from the hepatic vein and a radial artery. On day 1, participants received 160 U human insulin through nasal spray, and on day 2 they received placebo spray, together with an intravenous insulin bolus to mimic spillover of nasal insulin to the circulation. Hepatic glucose fluxes and endogenous glucose production were calculated. RESULTS: Plasma insulin concentrations were similar on the two study days, and no differences in whole-body endogenous glucose production or hepato-splanchnic glucose turnover were detected. CONCLUSIONS: Nasal administration of insulin does not influence whole-body or hepatic glucose production in fasting humans. By contrast, pharmacological delivery of insulin to the brain might modulate insulin effectiveness in glucose-producing tissue when circulating insulin levels are elevated; therefore, the metabolic consequences of brain insulin action appear to be dependent on metabolic prandial status.

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.

Quality Control of Serum and Plasma by Quantification of (4E,14Z)-Sphingadienine-C18-1-Phosphate Uncovers Common Preanalytical Errors During Handling of Whole Blood.

BACKGROUND: Nonadherence to standard operating procedures (SOPs) during handling and processing of whole blood is one of the most frequent causes affecting the quality of serum and plasma. Yet, the quality of blood samples is of the utmost importance for reliable, conclusive research findings, valid diagnostics, and appropriate therapeutic decisions. METHODS: UHPLC-MS-driven nontargeted metabolomics was applied to identify biomarkers that reflected time to processing of blood samples, and a targeted UHPLC-MS analysis was used to quantify and validate these biomarkers. RESULTS: We found that (4E,14Z)-sphingadienine-C18-1-phosphate (S1P-d18:2) was suitable for the reliable assessment of the pronounced changes in the quality of serum and plasma caused by errors in the phase between collection and centrifugation of whole blood samples. We rigorously validated S1P-d18:2, which included the use of practicality tests on >1400 randomly selected serum and plasma samples that were originally collected during single- and multicenter trials and then stored in 11 biobanks in 3 countries. Neither life-threatening disease states nor strenuous metabolic challenges (i.e., high-intensity exercise) affected the concentration of S1P-d18:2. Cutoff values for sample assessment were defined (plasma, ≤0.085 µg/mL; serum, ≤0.154 µg/mL). CONCLUSIONS: Unbiased valid monitoring to check for adherence to SOP-dictated time for processing to plasma or serum and/or time to storage of whole blood at 4 °C is now feasible. This novel quality assessment step could enable scientists to uncover common preanalytical errors, allowing for identification of serum and plasma samples that should be excluded from certain investigations. It should also allow control of samples before long-term storage in biobanks.

GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

Lysophosphatidylcholines activate PPARδ and protect human skeletal muscle cells from lipotoxicity.

Metabolomics studies of human plasma demonstrate a correlation of lower plasma lysophosphatidylcholines (LPC) concentrations with insulin resistance, obesity, and inflammation. This relationship is not unraveled on a molecular level. Here we investigated the effects of the abundant LPC(16:0) and LPC(18:1) on human skeletal muscle cells differentiated to myotubes. Transcriptome analysis of human myotubes treated with 10µM LPC for 24h revealed enrichment of up-regulated peroxisome proliferator-activated receptor (PPAR) target transcripts, including ANGPTL4, PDK4, PLIN2, and CPT1A. The increase in both PDK4 and ANGPTL4 RNA expression was abolished in the presence of either PPARδ antagonist GSK0660 or GSK3787. The induction of PDK4 by LPCs was blocked with siRNA against PPARD. The activation of PPARδ transcriptional activity by LPC was shown as PPARδ-dependent luciferase reporter gene expression and enhanced DNA binding of the PPARδ/RXR dimer. On a functional level, further results show that the LPC-mediated activation of PPARδ can reduce fatty acid-induced inflammation and ER stress in human skeletal muscle cells. The protective effect of LPC was prevented in the presence of the PPARδ antagonist GSK0660. Taking together, LPCs can activate PPARδ, which is consistent with the association of high plasma LPC levels and PPARδ-dependent anti-diabetic and anti-inflammatory effects.

Do diabetes and obesity affect the metabolic response to exercise?

PURPOSE OF REVIEW: Exercise is recommended as therapeutic intervention for people at risk to develop type 2 diabetes to prevent or treat the disease. Recent studies on the influence of obesity and type 2 diabetes on the outcome of exercise programs are discussed. RECENT FINDINGS: Poor glycemic control before an intervention can be a risk factor of reduced therapeutic benefit from exercise. But the acute metabolic response to exercise and the transcriptional profile of the working muscle is similar in healthy controls and type 2 diabetic patients, including but not limited to intact activation of skeletal muscle AMP-activated kinase signaling, glucose uptake and expression of peroxisome proliferator-activated receptor gamma coactivator 1α. The increase in plasma acylcarnitines during exercise is not influenced by type 2 diabetes or obesity. The hepatic response to exercise is dependent on the glucagon/insulin ratio and the exercise-induced increase in hepatokines such as fibroblast growth factor 21 and follistatin is impaired in type 2 diabetes and obesity, but consequences for the benefit from exercise are unknown yet. SUMMARY: Severe metabolic dysregulation can reduce the benefit from exercise, but the intact response of key metabolic regulators in exercising skeletal muscle of diabetic patients demonstrates the effectiveness of exercise programs to treat the disease.

Type 2 diabetes alters metabolic and transcriptional signatures of glucose and amino acid metabolism during exercise and recovery.

AIMS/HYPOTHESIS: The therapeutic benefit of physical activity to prevent and treat type 2 diabetes is commonly accepted. However, the impact of the disease on the acute metabolic response is less clear. To this end, we investigated the effect of type 2 diabetes on exercise-induced plasma metabolite changes and the muscular transcriptional response using a complementary metabolomics/transcriptomics approach. METHODS: We analysed 139 plasma metabolites and hormones at nine time points, and whole genome expression in skeletal muscle at three time points, during a 60 min bicycle ergometer exercise and a 180 min recovery phase in type 2 diabetic patients and healthy controls matched for age, percentage body fat and maximal oxygen consumption (VO2). RESULTS: Pathway analysis of differentially regulated genes upon exercise revealed upregulation of regulators of GLUT4 (SLC2A4RG, FLOT1, EXOC7, RAB13, RABGAP1 and CBLB), glycolysis (HK2, PFKFB1, PFKFB3, PFKM, FBP2 and LDHA) and insulin signal mediators in diabetic participants compared with controls. Notably, diabetic participants had normalised rates of lactate and insulin levels, and of glucose appearance and disappearance, after exercise. They also showed an exercise-induced compensatory regulation of genes involved in biosynthesis and metabolism of amino acids (PSPH, GATM, NOS1 and GLDC), which responded to differences in the amino acid profile (consistently lower plasma levels of glycine, cysteine and arginine). Markers of fat oxidation (acylcarnitines) and lipolysis (glycerol) did not indicate impaired metabolic flexibility during exercise in diabetic participants. CONCLUSIONS/INTERPRETATION: Type 2 diabetic individuals showed specific exercise-regulated gene expression. These data provide novel insight into potential mechanisms to ameliorate the disturbed glucose and amino acid metabolism associated with type 2 diabetes.

The lipid profile of brown adipose tissue is sex-specific in mice.

Brown adipose tissue (BAT) is a thermogenic organ with a vital function in small mammals and potential as metabolic drug target in humans. By using high-resolution LC-tandem-mass spectrometry, we quantified 329 lipid species from 17 (sub)classes and identified the fatty acid composition of all phospholipids from BAT and subcutaneous and gonadal white adipose tissue (WAT) from female and male mice. Phospholipids and free fatty acids were higher in BAT, while DAG and TAG levels were higher in WAT. A set of phospholipids dominated by the residue docosahexaenoic acid, which influences membrane fluidity, showed the highest specificity for BAT. We additionally detected major sex-specific differences between the BAT lipid profiles, while samples from the different WAT depots were comparatively similar. Female BAT contained less triacylglycerol and more phospholipids rich in arachidonic and stearic acid whereas another set of fatty acid residues that included linoleic and palmitic acid prevailed in males. These differences in phospholipid fatty acid composition could greatly affect mitochondrial membranes and other cellular organelles and thereby regulate the function of BAT in a sex-specific manner.

Secretome profiling of primary human skeletal muscle cells.

The skeletal muscle is a metabolically active tissue that secretes various proteins. These so-called myokines have been proposed to affect muscle physiology and to exert systemic effects on other tissues and organs. Yet, changes in the secretory profile may participate in the pathophysiology of metabolic diseases. The present study aimed at characterizing the secretome of differentiated primary human skeletal muscle cells (hSkMC) derived from healthy, adult donors combining three different mass spectrometry based non-targeted approaches as well as one antibody based method. This led to the identification of 548 non-redundant proteins in conditioned media from hSkmc. For 501 proteins, significant mRNA expression could be demonstrated. Applying stringent consecutive filtering using SignalP, SecretomeP and ER_retention signal databases, 305 proteins were assigned as potential myokines of which 12 proteins containing a secretory signal peptide were not previously described. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and may contribute to our understanding of the role of myokines in multiple biological processes. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Mice Lacking Free Fatty Acid Receptor 1 (GPR40/FFAR1) are Protected Against Conjugated Linoleic Acid-Induced Fatty Liver but Develop Inflammation and Insulin Resistance in the Brain.

BACKGROUND/AIMS: Conjugated linoleic acids (CLAs) affect body fat distribution, induce insulin resistance and stimulate insulin secretion. The latter effect is mediated through the free fatty acid receptor-1 (GPR40/FFAR1). This study examines whether GPR40/FFAR1 interacts with tissue specific metabolic changes induced by CLAs. METHODS AND RESULTS: After chronic application of CLAs C57BL/6J wild type (WT) and GPR40/FFAR1 (Ffar1(-/-)) knockout mice developed insulin resistance. Although CLAs accumulated in liver up to 46-fold genotype-independently, hepatic triglycerides augmented only in WT mice. This triglyceride deposition was not associated with increased inflammation. In contrast, in brain of CLA fed Ffar1(-/-) mice mRNA levels of TNF-α were 2-fold higher than in brain of WT mice although CLAs accumulated genotype-independently in brain up to 4-fold. Concomitantly, Ffar1(-/-) mice did not respond to intracerebroventricular (i.c.v.) insulin injection with an increase in cortical activity while WT mice reacted as assessed by radiotelemetric electrocorticography (ECoG) measurements. In vitro incubation of primary murine astrocytes confirmed that CLAs stimulate neuronal inflammation independent of GPR40/FFAR1. CONCLUSION: This study discloses that GPR40/FFAR1 indirectly modulates organ-specific effects of CLAs: the expression of functional GPR40/FFAR1 counteracts CLA-induced inflammation and insulin resistance in the brain, but favors the development of fatty liver.

Exercise, skeletal muscle and inflammation: ARE-binding proteins as key regulators in inflammatory and adaptive networks.

The role of inflammation in skeletal muscle adaptation to exercise is complex and has hardly been elucidated so far. While the acute inflammatory response to exercise seems to promote skeletal muscle training adaptation and regeneration, persistent, low-grade inflammation, as seen in a multitude of chronic diseases, is obviously detrimental. The regulation of cytokine production in skeletal muscle cells has been relatively well studied, yet little is known about the compensatory and anti-inflammatory mechanisms that resolve inflammation and restore tissue homeostasis. One important strategy to ensure sequential, timely and controlled resolution of inflammation relies on the regulated stability of mRNAs encoding pro-inflammatory mediators. Many key transcripts in early immune responses are characterized by the presence of AU-rich elements (AREs) in the 3'-untranslated regions of their mRNAs, allowing efficient fine-tuning of gene expression patterns at the post-transcriptional level. AREs exert their function by recruiting particular RNA-binding proteins, resulting, in most cases, in de-stabilization of the target transcripts. The best-characterized ARE-binding proteins are HuR, CUGBP1, KSRP, AUF1, and the three ZFP36 proteins, especially TTP/ZFP36. Here, we give a general introduction into the role of inflammation in the adaptation of skeletal muscle to exercise. Subsequently, we focus on potential roles of ARE-binding proteins in skeletal muscle tissue in general and specifically exercise-induced skeletal muscle remodeling. Finally, we present novel data suggesting a specific function of TTP/ZFP36 in exercise-induced skeletal muscle plasticity.

Fetuin-A influences vascular cell growth and production of proinflammatory and angiogenic proteins by human perivascular fat cells.

AIMS/HYPOTHESIS: Fetuin-A (alpha2-Heremans-Schmid glycoprotein), a liver-derived circulating glycoprotein, contributes to lipid disorders, diabetes and cardiovascular diseases. In a previous study we found that perivascular fat cells (PVFCs) have a higher angiogenic potential than other fat cell types. The aim was to examine whether fetuin-A influences PVFC and vascular cell growth and the expression and secretion of proinflammatory and angiogenic proteins, and whether TLR4-independent pathways are involved. METHODS: Mono- and co-cultures of human PVFCs and endothelial cells were treated with fetuin-A and/or palmitate for 6-72 h. Proteins were quantified by ELISA and Luminex, mRNA expression by real-time PCR, and cell growth by BrDU-ELISA. Some PVFCs were preincubated with a nuclear factor κB NFκBp65 inhibitor, or the toll-like receptor 4 (TLR4) inhibitor CLI-095, or phosphoinositide 3-kinase (PI3K)/Akt inhibitors and/or stimulated with insulin. Intracellular forkhead box protein O1 (FoxO1), NFκBp65 and inhibitor of κB kinase ß (IKKß) localisation was visualised by immunostaining. RESULTS: PVFCs expressed and secreted IL-6, IL-8, plasminogen activator inhibitor 1 (PAI-1), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-BB, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), placental growth factor (PLGF) and hepatocyte growth factor (HGF). Fetuin-A upregulated IL-6 and IL-8, and this was potentiated by palmitate and blocked by CLI-095. Immunostaining and electrophoretic mobility shift assay (EMSA) showed partial NFκBp65 activation. MCP-1 was upregulated and blocked by CLI-095, but not by palmitate. However, HGF was downregulated, which was slightly potentiated by palmitate. This effect persisted after TLR4 pathway blockade. Stimulation of insulin-PI3K-Akt signalling by insulin resulted in nuclear FoxO1 extrusion and HGF upregulation. Fetuin-A counteracted these insulin effects. CONCLUSIONS/INTERPRETATION: Fetuin-A and/or palmitate influence the expression of proinflammatory and angiogenic proteins only partially via TLR4 signalling. HGF downregulation seems to be mediated by interference with the insulin-dependent receptor tyrosine kinase pathway. Fetuin-A may also influence angiogenic and proinflammatory proteins involved in atherosclerosis.