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Young breast cancer patients aged 35 years and younger are a small group of women who tend to present at high-risk form of the disease. More analysis of the data on tumor characteristics, treatment, and survival is necessary to help improving treatment and outcome.In this retrospective study, we compared the clinical and tumor characteristics, the treatments, and the survival of 257 women aged ≤ 35 years, with 6566 women aged 50-69 years. We used a registry-based data of patients with invasive, non-metastatic breast cancer diagnosed between 2000 and 2015.Young women showed lower rate of hormone receptor (HR) positivity. Their tumors were more often HER2-positive, which showed lower rate of differentiation and higher rate of Ki-67 expression compared to their older counterparts. Women aged 35 years and younger were more likely to undergo neoadjuvant therapy and mastectomy. Endocrine therapy was underrepresented in young patients. 5-Year disease-free survival (DFS) was significantly lower in the younger patient group (81.7% vs. 91.3%, p 0.001), while 5-year overall survival (OS) was not impaired (91.4% vs. 91.1%, p = 0.847).The unfavorable disease-free survival in the group of younger patients might be explained by their unfavorable tumor characteristics. The surgical treatment appears to be more aggressive in young breast cancer patients and is more frequently combined with chemotherapy and immunotherapy, either in a neoadjuvant or in an adjuvant setting.

Skeletal muscle insulin resistance is the hallmark of type 2 diabetes and develops long before the onset of the disease. It is well accepted that physical activity improves glycemic control, but the knowledge on underlying mechanisms mediating the beneficial effects remains incomplete. Exercise is accompanied by a decrease in intramuscular oxygen levels, resulting in induction of HIF-1α. HIF-1α is a master regulator of gene expression and might play an important role in skeletal muscle function and metabolism. Here we show that HIF-1α is important for glucose metabolism and insulin action in skeletal muscle. By using a genome-wide gene expression profiling approach, we identified RAB20 and TXNIP as two novel exercise/HIF-1α–regulated genes in skeletal muscle. Loss of Rab20 impairs insulin-stimulated glucose uptake in human and mouse skeletal muscle by blocking the translocation of GLUT4 to the cell surface. In addition, exercise/HIF-1α downregulates the expression of TXNIP, a well-known negative regulator of insulin action. In conclusion, we are the first to demonstrate that HIF-1α is a key regulator of glucose metabolism in skeletal muscle by directly controlling the transcription of RAB20 and TXNIP. These results hint toward a novel function of HIF-1α as a potential pharmacological target to improve skeletal muscle insulin sensitivity.

Objective Skeletal muscle is an important secretory organ, producing and releasing numerous myokines, which may be involved in mediating beneficial health effects of physical activity. More than 100 myokines have been identified by different proteomics approaches, but these techniques may not detect all myokines. We used mRNA sequencing as an untargeted approach to study gene expression of secreted proteins in skeletal muscle upon acute as well as long-term exercise. Methods Twenty-six middle-aged, sedentary men underwent combined endurance and strength training for 12 weeks. Skeletal muscle biopsies from m. vastus lateralis and blood samples were taken before and after an acute bicycle test, performed at baseline as well as after 12 weeks of training intervention. We identified transcripts encoding secretory proteins that were changed more than 1.5-fold in muscle after exercise. Secretory proteins were defined based on either curated UniProt annotations or predictions made by multiple bioinformatics methods. Results This approach led to the identification of 161 candidate secretory transcripts that were up-regulated after acute exercise and 99 that where increased after 12 weeks exercise training. Furthermore, 92 secretory transcripts were decreased after acute and/or long-term physical activity. From these responsive transcripts, we selected 17 candidate myokines sensitive to short- and/or long-term exercise that have not been described as myokines before. The expression of these transcripts was confirmed in primary human skeletal muscle cells during in vitro differentiation and electrical pulse stimulation (EPS). One of the candidates we identified was macrophage colony-stimulating factor-1 (CSF1), which influences macrophage homeostasis. CSF1 mRNA increased in skeletal muscle after acute and long-term exercise, which was accompanied by a rise in circulating CSF1 protein. In cultured muscle cells, EPS promoted a significant increase in the expression and secretion of CSF1. Conclusion We identified 17 new, exercise-responsive transcripts encoding secretory proteins. We further identified CSF1 as a novel myokine, which is secreted from cultured muscle cells and up-regulated in muscle and plasma after acute exercise., Highlights • Numerous transcripts were identified that were regulated in human skeletal muscle after acute and/or long-term exercise. • These transcripts encode potential myokines, which may play key roles in local and systemic adaptations to exercise. • CSF1 was identified as a novel myokine. CSF1 was increased after acute exercise, and secreted from cultured human myotubes in response to EPS.

The beneficial health-related effects of exercise are well recognized, and numerous studies have investigated underlying mechanism using various in vivo and in vitro models. Although electrical pulse stimulation (EPS) for the induction of muscle contraction has been used for quite some time, its application on cultured skeletal muscle cells of animal or human origin as a model of in vitro exercise is a more recent development. In this review, we compare in vivo exercise and in vitro EPS with regard to effects on signalling, expression level and metabolism. We provide a comprehensive overview of different EPS protocols and their applications, discuss technical aspects of this model including critical controls and the importance of a proper maintenance procedure and finally discuss the limitations of the EPS model.

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.

CHI3L1 (chitinase-3-like protein 1) is a glycoprotein consisting of 383 amino acids with a molecular mass of 40 kDa, and its serum level is elevated in inflammatory diseases. Although CHI3L1 is described as a biomarker of inflammation, the function of this protein is not completely understood. In the present study, we examined the regulation of CHI3L1 in primary human skeletal muscle cells. Moreover, we analysed potential autocrine effects of CHI3L1. We show that myotubes express CHI3L1 in a differentiation-dependent manner. Furthermore, pro-inflammatory cytokines up-regulate CHI3L1 expression (6-fold) and release (3-fold). Importantly, CHI3L1 treatment blocked TNFα (tumour necrosis factor α)-induced inflammation by inhibiting NF-κB (nuclear factor κB) activation in skeletal muscle cells. We show that this effect is mediated via PAR2 (protease-activated receptor 2). In addition, CHI3L1 treatment diminished the TNFα-induced expression and secretion of IL (interleukin)-8, MCP1 (monocyte chemoattractant protein 1) and IL-6. In addition, impaired insulin action at the level of Akt and GSK3α/β (glycogen synthase kinase 3α/β) phosphoryl-ation and insulin-stimulated glucose uptake was normalized by CHI3L1. In conclusion, the novel myokine CHI3L1, which is induced by pro-inflammatory cytokines, can counteract TNFα-mediated inflammation and insulin resistance in human skeletal muscle cells, potentially involving an auto- and/or para-crine mechanism.

Follistatin-like protein 1 (Fstl1) is a secreted glycoprotein of the follistatin family. Fstl1 is secreted by C2C12 cells, and Akt1 over-expression in skeletal muscle leads to its induction in muscle and increased circulating levels. So far, secretion of Fstl1 by human myotubes and the effect of exercise on its circulating levels have not been investigated. Here, we examined both the regulation of Fstl1 expression and secretion in primary human skeletal muscle cells and the effect of acute exercise on Fstl1 serum concentrations in humans. We show that human myotubes express and secrete Fstl1 in a differentiation-dependent manner. Furthermore, IFNγ and IL-1β significantly increase Fstl1 secretion. Electrical pulse stimulation (EPS)-induced contractile activity of myotubes did not regulate Fstl1. Interestingly, we observed that 60 min cycling increased serum Fstl1 level by 22%. In conclusion, we demonstrate that Fstl1 is expressed and secreted by human myotubes and plasma Fstl1 levels are increased after exercise.

Obesity is associated with many severe chronic diseases and deciphering its development and molecular mechanisms is necessary for promoting treatment. Previous studies have revealed that mitochondrial content is down-regulated in obesity, diabetes, and nonalcoholic fatty liver disease (NAFLD) and proposed that NAFLD and diabetes are mitochondrial diseases. However, the exact mechanisms underlying these processes remain unclear. In this study, we discovered that resistin down-regulated the content and activities of mitochondria, enhanced hepatic steatosis, and induced insulin resistance (IR) in mice. The time course indicated that the change in mitochondrial content was before the change in fat accumulation and development of insulin resistance. When the mitochondrial content was maintained, resistin did not stimulate hepatic fat accumulation. The present mutation study found that the residue Thr464 of the p65 subunit of nuclear factor kappa B was essential for regulating mitochondria. A proximity ligation assay revealed that resistin inactivated peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α) and diminished the mitochondrial content by promoting the interaction of p65 and PGC-1α. Signaling-transduction analysis demonstrated that resistin down-regulated mitochondria by a novel protein kinase C/protein kinase G/p65/PGC-1α-signaling pathway. Conclusion: Resistin induces hepatic steatosis through diminishing mitochondrial content. This reveals a novel pathway for mitochondrial regulation, and suggests that the maintenance of normal mitochondrial content could be a new strategy for treatment of obesity-associated diseases. (HEPATOLOGY 2013)

Studies have shown the implication of specific adipokines or fatty acids (FA) in the pathogenesis of insulin resistance. However, the interplay of adipokines with FA remains poorly understood. This study aimed to investigate the combined effects of adipokines and low concentrations of palmitic acid (PA, 100 µmol/l) on skeletal muscle metabolism. Human skeletal muscle cells were incubated with adipocyte-conditioned medium (CM), PA or PA+CM, and FA transporter and FA metabolism were analysed. CM-incubation increased CD36 level (1.8 fold) and PA-uptake (1.4 fold). However, only co-application of PA+CM resulted in profound lipid accumulation (5.3 fold), 60% reduction of PA-oxidation and 3.5 fold increased diacylglycerol content. Our results support a novel role for adipokines in the pathogenesis of T2D by increasing the lipotoxic potential of PA, notably of low concentrations. This implies an increased lipotoxic risk already at an early stage of weight gain, when lipolysis has not yet contributed to increased plasma free FA levels.

Abdominal obesity is a major risk factor for cardiovascular disease, and recent studies highlight a key role of adipose tissue dysfunction, inflammation, and aberrant adipokine release in this process. An increased demand for lipid storage results in both hyperplasia and hypertrophy, finally leading to chronic inflammation, hypoxia, and a phenotypic change of the cellular components of adipose tissue, collectively leading to a substantially altered secretory output of adipose tissue. In this review we have assessed the adipo-vascular axis, and an overview of adipokines associated with cardiovascular disease is provided. This resulted in a first list of more than 30 adipokines. A deeper analysis only considered adipokines that have been reported to impact on inflammation and NF-κB activation in the vasculature. Out of these, the most prominent link to cardiovascular disease was found for leptin, TNF-α, adipocyte fatty acid-binding protein, interleukins, and several novel adipokines such as lipocalin-2 and pigment epithelium-derived factor. Future work will need to address the potential role of these molecules as biomarkers and/or drug targets.

Context: Zinc-α2-glycoprotein (ZAG) induces lipid mobilization in adipose tissue (AT) and stimulates energy utilization in AT and skeletal muscle by up-regulation of UCP isoforms and GLUT4.Objective: Our study aimed to investigate whether ZAG activates AMPKα, an important regulator of energy metabolism, in human skeletal muscle cells (SkMc).Materials and Methods: SkMc were treated with recombinant ZAG, and activation of AMPKα and ACC, protein abundance of GLUT4, and UCP2 and UCP3 gene expression were analysed.Results: Treatment of SkMc with ZAG induced short-time phosphorylation of AMPKα and ACC. Furthermore, AMPKα phosphorylation was elevated after 24 h, while for ACC no activation was observed. GLUT4 level was increased by 1.3-fold. However, UCP2 and UCP3 expression remained unaltered.Discussion and Conclusion: These results show that ZAG leads to phosphorylation of AMPKα and ACC, thereby activating a pathway central to the regulation of energy metabolism. This mechanism may be involved in mediating the...

OBJECTIVE Comprehensive proteomic profiling of the human adipocyte secretome identified dipeptidyl peptidase 4 (DPP4) as a novel adipokine. This study assessed the functional implications of the adipokine DPP4 and its association to the metabolic syndrome. RESEARCH DESIGN AND METHODS Human adipocytes and skeletal and smooth muscle cells were used to monitor DPP4 release and assess the effects of soluble DPP4 on insulin signaling. In lean and obese subjects, depot-specific expression of DPP4 and its release from adipose tissue explants were determined and correlated to parameters of the metabolic syndrome. RESULTS Fully differentiated adipocytes exhibit a substantially higher release of DPP4 compared with preadipocytes or macrophages. Direct addition of DPP4 to fat and skeletal and smooth muscle cells impairs insulin signaling. A fivefold higher level of DPP4 protein expression was seen in visceral compared with subcutaneous fat of obese patients, with no regional difference in lean subjects. DPP4 serum concentrations significantly correlated with adipocyte size. By using adipose tissue explants from lean and obese subjects, we observed a twofold increase in DPP4 release that strongly correlated with adipocyte volume and parameters of the metabolic syndrome and was decreased to the lean level after weight reduction. DPP4 released from adipose tissue correlated positively with an increasing risk score for the metabolic syndrome. CONCLUSIONS DPP4 is a novel adipokine that may impair insulin sensitivity in an autocrine and paracrine fashion. Furthermore, DPP4 release strongly correlates with adipocyte size, potentially representing an important source of DPP4 in obesity. Therefore, we suggest that DPP4 may be involved in linking adipose tissue and the metabolic syndrome.

OBJECTIVE Chemerin is an adipokine that affects adipogenesis and glucose homeostasis in adipocytes and increases with BMI in humans. This study was aimed at investigating the regulation of chemerin release and its effects on glucose metabolism in skeletal muscle cells. RESEARCH DESIGN AND METHODS Human skeletal muscle cells were treated with chemerin to study insulin signaling, glucose uptake, and activation of stress kinases. The release of chemerin was analyzed from in vitro differentiated human adipocytes and adipose tissue explants from 27 lean and 26 obese patients. RESULTS Human adipocytes express chemerin and chemokine-like receptor 1 (CMKLR1) differentiation dependently and secrete chemerin (15 ng/ml from 106 cells). This process is slightly but significantly increased by tumor necrosis factor-α and markedly inhibited by >80% by peroxisome proliferator–activated receptor-γ activation. Adipose tissue explants from obese patients are characterized by significantly higher chemerin secretion compared with lean control subjects (21 and 8 ng from 107 cells, respectively). Chemerin release is correlated with BMI, waist-to-hip ratio, and adipocyte volume. Furthermore, higher chemerin release is associated with insulin resistance at the level of lipogenesis and insulin-induced antilipolysis in adipocytes. Chemerin induces insulin resistance in human skeletal muscle cells at the level of insulin receptor substrate 1, Akt and glycogen synthase kinase 3 phosphorylation, and glucose uptake. Furthermore, chemerin activates p38 mitogen-activated protein kinase, nuclear factor-κB, and extracellular signal–regulated kinase (ERK)-1/2. Inhibition of ERK prevents chemerin-induced insulin resistance, pointing to participation of this pathway in chemerin action. CONCLUSIONS Adipocyte-derived secretion of chemerin may be involved in the negative cross talk between adipose tissue and skeletal muscle contributing to the negative relationship between obesity and insulin sensitivity.

Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent a novel approach to treat obesity and related metabolic disorders. Recent data suggest that endocannabinoids are also produced by human adipocytes. Here we studied the potential involvement of endocannabinoids in the negative crosstalk between fat and muscle.The protein level of CB1R in human skeletal muscle cells (SkM) during differentiation was analysed using western blotting. SkM were treated with adipocyte-conditioned medium (CM) or anandamide (AEA) in combination with the CB1R antagonists Rim or AM251, and insulin-stimulated Akt phosphorylation and glucose uptake were determined. Furthermore, signalling pathways of CB1R were investigated.We revealed an increase of CB1R protein in SkM during differentiation. Twenty-four hour incubation of SkM with CM or AEA impaired insulin-stimulated Akt(Ser473) phosphorylation by 60% and up to 40%, respectively. Pretreatment of cells with Rim or AM251 reduced the effect of CM by about one-half, while the effect of AEA could be prevented completely. The reduction of insulin-stimulated glucose uptake by CM was completely prevented by Rim. Short-time incubation with AEA activated extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase, and impaired insulin-stimulated Akt(Ser473) phosphorylation, but had no effect on Akt(Thr308) and glycogen synthase kinase 3alpha/beta phosphorylation. In addition, enhanced IRS-1 (Ser307) phosphorylation was observed.Our results show that the CB1R system may play a role in the development of insulin resistance in human SkM. The results obtained with CM support the notion that adipocytes may secrete factors which are able to activate the CB1R. Furthermore, we identified two stress kinases in the signalling pathway of AEA and enhanced IRS-1(Ser307) phosphorylation, potentially underlying the development of insulin resistance.

Insulin resistance in skeletal muscle is an early event in the development of diabetes, with obesity being one of the major contributing factors. In vitro, conditioned medium (CM) from differentiated human adipocytes impairs insulin signaling in human skeletal muscle cells, but it is not known whether insulin resistance is reversible and which mechanisms may underlie this process. CM induced insulin resistance in human myotubes at the level of insulin-stimulated Akt and GSK-3 phosphorylation. In addition, insulin-resistant skeletal muscle cells exhibit enhanced production of reactive oxygen species and ceramide as well as a downregulation of myogenic transcription factors such as myogenin and MyoD. However, insulin resistance was not paralleled by increased apopotosis. Regeneration of myotubes for 24 or 48 h after induction of insulin resistance restored normal insulin signaling. However, the expression level of myogenin could not be reestablished. In addition to decreasing myogenin expression, CM also decreased the release of IL-6 and IL-8 and increased monocyte chemotactic protein-1 (MCP-1) secretion from skeletal muscle cells. Although regeneration of myotubes reestablished normal secretion of IL-6, the release of IL-8 and MCP-1 remained impaired for 48 h after withdrawal of CM. In conclusion, our data show that insulin resistance in skeletal muscle cells is only partially reversible. Although some characteristic features of insulin-resistant myotubes normalize in parallel to insulin signaling after withdrawal of CM, others such as IL-8 and MCP-1 secretion and myogenin expression remain impaired over a longer period. Thus, we propose that the induction of insulin resistance may cause irreversible changes of protein expression and secretion in skeletal muscle cells.

Insulin resistance in skeletal muscle is an early event in the development of diabetes with obesity being one of the major contributing factors. Conditioned medium (CM) from differentiated human adipocytes impairs insulin signalling in human skeletal muscle cells. Recent data on adipocyte-induced insulin resistance in skeletal muscle cells describes underlying mechanisms of this process. Skeletal muscle insulin resistance involves multiple pathways and irreversible changes in the expression level of critical proteins. Furthermore, the reversibility of insulin resistance could be demonstrated. Several strategies to combat insulin resistance have been developed. One recent approach to treat obesity and the metabolic syndrome is the use of endocannabinoid receptor antagonists such as rimonabant. These compounds might also reduce insulin resistance in type 2 diabetes with effects on adipose tissue and liver and possibly skeletal muscle.

Skeletal muscle and white adipose tissue are the largest organs in the human body and both tissues act as endocrine organs capable of secreting many bioactive molecules. There has been some confusion about nomenclature and we suggest that the name myokine should be restricted to a protein or molecule secreted from myocytes, whereas the term adipokine should be used to describe proteins and molecules secreted from adipocytes. In fact, many myokines are also produced by adipocytes and we propose to name them adipo-myokines. Many adipo-myokines produced by skeletal muscle or adipose tissue are influenced by exercise. Therefore, it is likely that adipo-myokines may contribute in the mediation of the health benefits of exercise and physical inactivity probably leads to an altered adipo-myokine profile, which could provide a potential mechanism for the association between sedentary behavior and many chronic diseases. Within this review, we evaluate the effects of acute and chronic exercise on myokine, adipokine, and adipo-myokine production. By using the adipo-myokine concept and including both skeletal muscle and adipose tissue, an attempt is made to gain a global view on the beneficial effects of different exercise programs and the underlying pathways.

Perilipins (PLINs) coat the surface of lipid droplets and are important for the regulation of lipid turnover. Knowledge about the physiological role of the individual PLINs in skeletal muscle is limited although lipid metabolism is very important for muscle contraction. To determine the effect of long-term exercise on PLINs expression, 26 middle-aged, sedentary men underwent 12 weeks combined endurance and strength training intervention. Muscle biopsies from m. vastus lateralis and subcutaneous adipose tissue were taken before and after the intervention and total gene expression was measured with deep mRNA sequencing. PLIN4 mRNA exhibited the highest expression of all five PLINs in both tissues, and the expression was significantly reduced after long-term exercise in skeletal muscle. Moreover, PLIN4 mRNA expression levels in muscle correlated with the expression of genes involved in de novo phospholipid biosynthesis, with muscular content of phosphatidylethanolamine and phosphatidylcholine, and with the content of subsarcolemmal lipid droplets. The PLIN4 protein was mainly located at the periphery of skeletal muscle fibers, with higher levels in slow-twitch as compared to fast-twitch skeletal muscle fibers. In summary, we report reduced expression of PLIN4 after long-term physical activity, and preferential slow-twitch skeletal muscle fibers and plasma membrane-associated PLIN4 location.

Aim Chitinase-3-like protein 1 (CHI3L1) is involved in tissue remodelling and inflammatory processes. Plasma levels are elevated in patients with insulin resistance and T2DM. We recently showed that CHI3L1 and its receptor protease-activated receptor 2 (PAR-2) are expressed in skeletal muscle. Activation of PAR-2 by CHI3L1 protects against TNF-α-induced inflammation and insulin resistance. However, the effect of exercise on CHI3L1 and PAR-2 signalling remains unknown. The aim of this work was to study the impact of exercise on CHI3L1 production and the effect of CHI3L1/PAR-2 signalling on skeletal muscle growth and repair. Methods Three human exercise studies were used to measure CHI3L1 plasma levels (n = 32). In addition, muscle and adipose tissue CHI3L1 mRNA expression was measured in response to acute and long-term exercise (n = 24). Primary human skeletal muscle cells were differentiated in vitro, and electrical pulse stimulation was applied. In addition, myoblasts were incubated with CHI3L1 protein and activation of MAP kinase signalling as well as proliferation was measured. Results Circulating CHI3L1 levels and muscle CHI3L1 mRNA were increased after acute exercise. In addition, CHI3L1 mRNA expression as well as CHI3L1 secretion was enhanced in electrically stimulated cultured myotubes. Incubation of cultured human myoblasts with CHI3L1 protein leads to a strong activation of p44/42, p38 MAPK and Akt as well as enhanced myoblast proliferation. Conclusion Our findings suggest that CHI3L1 is induced by acute exercise and that CHI3L1/PAR-2 signalling activates myocyte proliferation, which is important for restructuring of skeletal muscle in the response to exercise training.

Skeletal muscle and white adipose tissue are the largest organs in the human body and both tissues act as endocrine organs capable of secreting many bioactive molecules. There has been some confusion about nomenclature and we suggest that the name myokine should be restricted to a protein or molecule secreted from myocytes, whereas the term adipokine should be used to describe proteins and molecules secreted from adipocytes. In fact, many myokines are also produced by adipocytes and we propose to name them adipo-myokines. Many adipo-myokines produced by skeletal muscle or adipose tissue are influenced by exercise. Therefore, it is likely that adipo-myokines may contribute in the mediation of the health benefits of exercise and physical inactivity probably leads to an altered adipo-myokine profile, which could provide a potential mechanism for the association between sedentary behavior and many chronic diseases. Within this review, we evaluate the effects of acute and chronic exercise on myokine, adipokine, and adipo-myokine production. By using the adipo-myokine concept and including both skeletal muscle and adipose tissue, an attempt is made to gain a global view on the beneficial effects of different exercise programs and the underlying pathways.

Adipose tissue is an active endocrine organ producing a variety of cytokines and chemokines, which may be involved in the deregulation of glucose and lipid homeostasis as well as in the inflammatory state observed in obesity. We have shown previously that differentiated human adipocytes secrete a variety of cytokines which are able to induce skeletal muscle insulin resistance. However, the regulation of these factors by anti-diabetic drugs has remained mainly undefined. Secretion of IL-6, IL-8, MIP-1alpha/beta, and MCP-1 by adipocytes was found to be downregulated by adiponectin. In parallel to adiponectin, the AMPK activator AICAR also decreased the secretion of most of the measured cytokines including IL-6 and MIP-1alpha/beta but not IL-8. In contrast, the thiazolidinedione troglitazone only slightly reduced cytokine secretion despite increasing the phosphorylation of AMPK. In conclusion, we show that adipocyte secretion is strongly inhibited by the anti-diabetic adipocyte hormone adiponectin, an effect that can also be mimicked by the AMPK activator AICAR. However, the PPARgamma agonist troglitazone is much less effective in reducing cytokine secretion.

The health-promoting effects of regular exercise are well known, and myokines may mediate some of these effects. The small leucine-rich proteoglycan decorin has been described as a myokine for some time. However, its regulation and impact on skeletal muscle has not been investigated in detail. In this study, we report decorin to be differentially expressed and released in response to muscle contraction using different approaches. Decorin is released from contracting human myotubes, and circulating decorin levels are increased in response to acute resistance exercise in humans. Moreover, decorin expression in skeletal muscle is increased in humans and mice after chronic training. Because decorin directly binds myostatin, a potent inhibitor of muscle growth, we investigated a potential function of decorin in the regulation of skeletal muscle growth. In vivo overexpression of decorin in murine skeletal muscle promoted expression of the pro-myogenic factor Mighty, which is negatively regulated by myostatin. We also found Myod1 and follistatin to be increased in response to decorin overexpression. Moreover, muscle-specific ubiquitin ligases atrogin1 and MuRF1, which are involved in atrophic pathways, were reduced by decorin overexpression. In summary, our findings suggest that decorin secreted from myotubes in response to exercise is involved in the regulation of muscle hypertrophy and hence could play a role in exercise-related restructuring processes of skeletal muscle.

Skeletal muscle represents the largest organ of the body in non-obese individuals and is now considered to be an active endocrine organ releasing a host of so-called myokines. These myokines are part of a complex network that mediates communication between muscle, the liver, adipose tissue, the brain and other organs. Recent data suggest that myokines regulated by muscle contraction may play a key role in mediating the health-promoting effects of regular physical activity. Although hundreds of myokines have recently been described in proteomic studies, we currently have a rather limited knowledge of the specific role these myokines play in the prevention of insulin resistance, inflammation and associated metabolic dysfunction. Several myokines are known to have both local and endocrine functions, but in many cases the contribution of physical activity to the systemic level of these molecules remains as yet unexplored. Very recently, novel myokines such as irisin, which is thought to induce a white to brown shift in adipocytes, have gained considerable interest as potential therapeutic targets. In this review, we summarise the most recent findings on the role of myokines in the regulation of substrate metabolism and insulin sensitivity. We further explore the role of myokines in the regulation of inflammation and provide a critical assessment of irisin and other myokines regarding their potential as therapeutic targets.

Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO2max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines.

Adipose tissue is a major endocrine organ, releasing signaling and mediator proteins, termed adipokines, via which adipose tissue communicates with other organs. Expansion of adipose tissue in obesity alters adipokine secretion, which may contribute to the development of metabolic diseases. Although recent profiling studies have identified numerous adipokines, the amount of overlap from these studies indicates that the adipokinome is still incompletely characterized. Therefore, we conducted a complementary protein profiling on concentrated conditioned medium derived from primary human adipocytes. SDS-PAGE/liquid chromatography-electrospray ionization tandem MS and two-dimensional SDS-PAGE/matrix-assisted laser desorption ionization/time of flight MS identified 347 proteins, 263 of which were predicted to be secreted. Fourty-four proteins were identified as novel adipokines. Furthermore, we validated the regulation and release of selected adipokines in primary human adipocytes and in serum and adipose tissue biopsies from morbidly obese patients and normal-weight controls. Validation experiments conducted for complement factor H, αB-crystallin, cartilage intermediate-layer protein, and heme oxygenase-1 show that the release and expression of these factors in adipocytes is regulated by differentiation and stimuli, which affect insulin sensitivity, as well as by obesity. Heme oxygenase-1 especially reveals to be a novel adipokine of interest. In vivo, circulating levels and adipose tissue expression of heme oxygenase-1 are significantly increased in obese subjects compared with lean controls. Collectively, our profiling study of the human adipokinome expands the list of adipokines and further highlights the pivotal role of adipokines in the regulation of multiple biological processes within adipose tissue and their potential dysregulation in obesity.

Obesity is closely associated with muscle insulin resistance and is a major risk factor for the pathogenesis of type 2 diabetes. Regular physical activity not only prevents obesity, but also considerably improves insulin sensitivity and skeletal muscle metabolism. We sought to establish and characterise an in vitro model of human skeletal muscle contraction, with a view to directly studying the signalling pathways and mechanisms that are involved in the beneficial effects of muscle activity. Contracting human skeletal muscle cell cultures were established by applying electrical pulse stimulation. To induce insulin resistance, skeletal muscle cells were incubated with human adipocyte-derived conditioned medium, monocyte chemotactic protein (MCP)-1 and chemerin. Similarly to in exercising skeletal muscle in vivo, electrical pulse stimulation induced contractile activity in human skeletal muscle cells, combined with the formation of sarcomeres, activation of AMP-activated protein kinase (AMPK) and increased IL-6 secretion. Insulin-stimulated glucose uptake was substantially elevated in contracting cells compared with control. The incubation of skeletal muscle cells with adipocyte-conditioned media, chemerin and MCP-1 significantly reduced the insulin-stimulated phosphorylation of Akt. This effect was abrogated by concomitant pulse stimulation of the cells. Additionally, pro-inflammatory signalling by adipocyte-derived factors was completely prevented by electrical pulse stimulation of the myotubes. We showed that the effects of electrical pulse stimulation on skeletal muscle cells were similar to the effect of exercise on skeletal muscle in vivo in terms of enhanced AMPK activation and IL-6 secretion. In our model, muscle contractile activity eliminates insulin resistance by blocking pro-inflammatory signalling pathways. This novel model therefore provides a unique tool for investigating the molecular mechanisms that mediate the beneficial effects of muscle contraction.

An alarming increase in the prevalence of obesity, type 2 diabetes mellitus, and associated diseases can be observed world-wide during the past 20 years. In obesity, profound alterations in the secretion profile of adipokines and inflammatory markers as well as increased lipolysis occur, leading besides other events to elevated levels of free fatty acids, which in turn are distributed to nonadipose tissue such as skeletal muscle. While the amount of intramyocellular lipids can be used as a marker of insulin resistance in physical inactive individuals, these neutral triglycerides themselves are not thought to be harmful. However, they provide a source for the generation of harmful lipid metabolites such as diacylglycerol and ceramide, which are implicated in insulin resistance by perturbing insulin signaling pathways. In this review, we will discuss the role of lipid metabolites in insulin resistance and potential mechanism involved in accumulation of intramyocellular lipids. Furthermore, we will highlight the key role of PGC-1α, which is a master regulator of mitochondrial biogenesis and coordinates the activation of genes involved in oxidative energy production as well as genes involved in fiber type transformation. Finally, the role of exercise in stimulating PGC-1α activity and expression as well as the release of contraction-induced myokines is discussed.

Imbalance between nutritional intake and energy expenditure has been described to culminate in obesity, which predisposes to insulin resistance and type 2 diabetes mellitus. In such states of energy oversupply, excess amounts of lipids are available in tissues and circulation. Over the past years, an increasingly important role in development of skeletal muscle (SkM) insulin resistance has been attributed to lipids and impaired fatty acid metabolism. In this review, we reflect the current state of knowledge about the effects of various lipid-derived mediators on SkM insulin sensitivity. Furthermore, potential mechanisms underlying the biogenesis of intramyocellular ectopic lipid stores are discussed. Previously, a pivotal role was attributed to mitochondrial dysfunction. However, results of recent studies have suggested an important role for exercise deficiency, accompanied by decreased expression levels of peroxisome proliferator-activated receptor-γ coactivator-1α and subsequent, incomplete β-oxidation. Additionally, we summarize the implications of increased levels of lipid-derived endocannabinoids (ECs) for metabolic control in peripheral tissue and highlight the benefits of targeting the EC system.

A variety of studies have documented significant improvements in the treatment of type 1 and 2 diabetes after the introduction of artificial insulins. This review gives an overview of insulin analogues which are currently approved for therapeutical use. Clinical data regarding the efficiency to control blood glucose level as well as improving HbA1c level in comparison to conventional insulin preparations in type 1 and 2 diabetic patients are summarized. Furthermore, special features of insulin analogues regarding their signalling properties are discussed with focus on the proliferative effects of insulin glargine as well as some recent data of insulin detemir.

Mitogenic activity of insulin and insulin analogues and the involvement of the IGF-1 receptor (IGF-1R) is still a controversial issue. We compared levels of the proteins IGF-1R and insulin receptor (InsR) in fibroblasts and smooth muscle cells from healthy donors and assessed the downstream signalling and growth-promoting activity of insulin and insulin analogues.DNA synthesis was monitored in human fibroblasts and coronary artery smooth muscle cells. Using small interfering RNAs, the levels of IGF-1 and InsR were reduced by 95 and 75%, respectively.Enhanced mitogenic potency of insulin and insulin analogues was observed which correlated with increased levels of IGF-1R and/or IRS-1. A reduction in the IGF-1R level significantly blunted stimulation of Akt phosphorylation by IGF-1, AspB10 and glargine by 72, 58 and 40%, respectively. Akt phosphorylation in response to insulin remained unaffected. Silencing of InsR did not significantly alter Akt phosphorylation in response to IGF-1, AspB10 and glargine. IGF-1R knockdown reduced the stimulation of DNA synthesis in response to IGF-1 and glargine to a level identical to that produced by insulin.These data show a prominent role of IGF-1R/Akt signalling in mediating the mitogenic effects of insulin analogues. Regular insulin stimulates DNA synthesis by exclusively activating InsR, whereas insulin analogues mainly signal through IGF-1R. It is suggested that inter-individual differences in the levels of proteins of the IGF-1R system may function as a critical determinant of the mitogenic potency of insulin analogues.

Adipocyte-derived factors might play a role in the development of hepatic insulin resistance. Resistin was identified as an adipokine linking obesity and insulin resistance. Resistin is secreted from adipocytes in rodents but in humans it was proposed to originate from macrophages and its impact for insulin resistance has remained elusive. To analyze the role of adipokines in general and resistin as a special adipokine, we cultured the human liver cell line HepG2 with adipocyte-conditioned medium (CM) containing various adipokines such as IL-6 and MCP-1, and resistin. CM and resistin both induce insulin resistance with a robust decrease in insulin-stimulated phosphorylation of Akt and GSK3. Insulin resistance could be prevented by co-treatment with troglitazone but not by co-stimulation with adiponectin. As human adipocytes do not secrete resistin, HepG2 cells were also treated with resistin added into CM. CM with resistin addition induced stronger insulin resistance than CM alone pointing to a specific role of resistin in the initiation of hepatic insulin resistance in humans.

Remodeling of extracellular matrix (ECM), including regulation of proteoglycans in skeletal muscle can be important for physiological adaptation to exercise. To investigate the effects of acute and long-term exercise on the expression of ECM-related genes and proteoglycans in particular, 26 middle-aged, sedentary men underwent a 12 weeks supervised endurance and strength training intervention and two acute, 45 min bicycle tests (70% VO2max), one at baseline and one after 12 weeks of training. Total gene expression in biopsies from m. vastus lateralis was measured with deep mRNA sequencing. After 45 min of bicycling approximately 550 gene transcripts were >50% upregulated. Of these, 28 genes (5%) were directly related to ECM. In response to long-term exercise of 12 weeks 289 genes exhibited enhanced expression (>50%) and 20% of them were ECM related. Further analyses of proteoglycan mRNA expression revealed that more than half of the proteoglycans expressed in muscle were significantly enhanced after 12 weeks intervention. The proteoglycan serglycin (SRGN) has not been studied in skeletal muscle and was one of few proteoglycans that showed increased expression after acute (2.2-fold, P

Physical activity promotes specific adaptations in most tissues including skeletal muscle. Acute exercise activates numerous signaling cascades including pathways involving mitogen‐activated protein kinases (MAPKs) such as extracellular signal‐regulated kinase (ERK)1/2, which returns to pre‐exercise level after exercise. The expression of MAPK phosphatases (MKPs) in human skeletal muscle and their regulation by exercise have not been investigated before. In this study, we used mRNA sequencing to monitor regulation of MKPs in human skeletal muscle after acute cycling. In addition, primary human myotubes were used to gain more insights into the regulation of MKPs. The two ERK1/2‐specific MKPs, dual specificity phosphatase 5 (DUSP5) and DUSP6, were the most regulated MKPs in skeletal muscle after acute exercise. DUSP5 expression was ninefold higher immediately after exercise and returned to pre‐exercise level within 2 h, whereas DUSP6 expression was reduced by 43% just after exercise and remained below pre‐exercise level after 2 h recovery. Cultured myotubes express both MKPs, and incubation with dexamethasone (Dex) mimicked the in vivo expression pattern of DUSP5 and DUSP6 caused by exercise. Using a MAPK kinase inhibitor, we showed that stimulation of ERK1/2 activity by Dex was required for induction of DUSP5. However, maintaining basal ERK1/2 activity was required for basal DUSP6 expression suggesting that the effect of Dex on DUSP6 might involve an ERK1/2‐independent mechanism. We conclude that the altered expression of DUSP5 and DUSP6 in skeletal muscle after acute endurance exercise might affect ERK1/2 signaling of importance for adaptations in skeletal muscle during exercise.

Brown adipose tissue has gained interest as a potential target to treat obesity and metabolic diseases. Irisin is a newly identified hormone secreted from skeletal muscle enhancing browning of white fat cells, which improves systemic metabolism by increasing energy expenditure in mice. The discovery of irisin raised expectations of its therapeutic potential to treat metabolic diseases. However, the effect of irisin in humans is unclear. Analyses of genomic DNA, mRNA and expressed sequence tags revealed that FNDC5, the gene encoding the precursor of irisin, is present in rodents and most primates, but shows in humans a mutation in the conserved start codon ATG to ATA. HEK293 cells transfected with a human FNDC5 construct with ATA as start codon resulted in only 1% full-length protein compared to human FNDC5 with ATG. Additionally, in vitro contraction of primary human myotubes by electrical pulse stimulation induced a significant increase in PGC1a mRNA expression. However, FNDC5 mRNA level was not altered. FNDC5 mRNA expression in muscle biopsies from two different human exercise studies was not changed by endurance or strength training. Preadipocytes isolated from human subcutaneous adipose tissue exhibited differentiation to brite human adipocytes when incubated with bone morphogenetic protein (BMP) 7, but neither recombinant FNDC5 nor irisin were effective. In conclusion, our findings suggest that it is rather unlikely that the beneficial effect of irisin observed in mice can be translated to humans. © 2013 Raschke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background High-intensity exercise induces many metabolic responses. In is unknown whether the response in the peripheral blood mononuclear cells (PBMCs) reflects the response in skeletal muscle and whether mRNA expression after exercise can be modulated by nutritional intake. The aims were to (i) investigate the effect of dairy proteins on acute responses to exercise in skeletal muscle and PBMCs measuring gene expression and (ii) compare this response in young and older subjects. Methods We performed two separate studies in young (20–40 years) and older subjects (≥70 years). Subjects were randomly allocated to a milk group or a whey group. Supplements were provided immediately after a standardized exercise session. We measured mRNA expression of selected genes after a standardized breakfast and 60/120 min after finishing the exercise, using RT-qPCR. Results We observed no significant differences in mRNA expression between the milk and the whey group; thus, we merged both groups for further analysis. The mRNA expression of IL6, TNF, and CCL2 in skeletal muscle increased significantly after exercise compared with smaller or no increase, in mRNA expression in PBMCs in all participants. The mRNA expression of IL1RN, IL8, and IL10 increased significantly in skeletal muscle and PBMCs. Some mRNA transcripts were differently regulated in older compared to younger participants in PBMCs. Conclusions An acute bout of heavy-load strength exercise, followed by protein supplementation, caused overlapping, but also unique, responses in skeletal muscle and PBMCs, suggesting tissue-specific functions in response to exercise. However, no different effects of the different protein supplements were observed. Altered mRNA expressions in PBMCs of older participants may affect regenerative mechanisms.