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.

Chitinase-3-like protein 1 protects skeletal muscle from TNFα-induced inflammation and insulin resistance.

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/ß (glycogen synthase kinase 3α/ß) 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.

Myokines in insulin resistance and type 2 diabetes.

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.

The myokine decorin is regulated by contraction and involved in muscle hypertrophy.

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.

Resistin reduces mitochondria and induces hepatic steatosis in mice by the protein kinase C/protein kinase G/p65/PPAR gamma coactivator 1 alpha pathway.

UNLABELLED: 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.

Identification and validation of novel adipokines released from primary human adipocytes.

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.

Questionnaire Study to Investigate the Preferences of Children, Parents, and Healthcare Professionals for Different Formulations of Oral Medicinal Products.

Since the acceptability of a medicine can significantly impact therapeutic outcomes, this study aimed to determine and compare the preferences of children, parents, and healthcare professionals for the most commonly used pediatric oral medicine formulations (syrup, mini-tablets, oblong tablets, round tablets) addressing all pediatric age groups, 0-<18 years (y). This survey study employed sex-, age-, and participant group-adapted questionnaires for eight cohorts of participants, i.e., children 6-<12 y, adolescents 12-<18 y, parents of children in four age groups (0-<2 y, 2-<6 y, 6-<12 y, and 12-<18 y), nurses, and pediatricians. Descriptive statistics were used for data analysis. In the age groups 0-<2 y and 2-<6 y, mini-tablets were preferred over syrup by all participants. In the age group 6-12 y, solid dosage forms were also preferred over syrup by all participants. In the age group 12-<18 y, healthcare professionals preferred solid dosage forms over syrup. Parents preferred higher amounts of mini-tablets and syrup compared to round and oblong tablets, while adolescents' preferences did not differentiate between these formulations. Based on the study results and in contrast to current practice, it is suggested to consider solid dosage forms for future age-appropriate medicinal products already for younger age groups.

Inflammation and metabolic dysfunction: links to cardiovascular diseases.

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.

Obesity-associated insulin resistance in skeletal muscle: role of lipid accumulation and physical inactivity.

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.

Role of lipid-derived mediators in skeletal muscle insulin resistance.

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 ß-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.

Insulin analogues: action profiles beyond glycaemic control.

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.

Conditioned medium obtained from in vitro differentiated adipocytes and resistin induce insulin resistance in human hepatocytes.

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.

Gene expression is differentially regulated in skeletal muscle and circulating immune cells in response to an acute bout of high-load strength exercise.

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.

Dual specificity phosphatase 5 and 6 are oppositely regulated in human skeletal muscle by acute exercise.

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.

Hypoxia in Combination With Muscle Contraction Improves Insulin Action and Glucose Metabolism in Human Skeletal Muscle via the HIF-1α Pathway.

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.

Exercise and Regulation of Adipokine and Myokine Production.

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.

Perilipin 4 in human skeletal muscle: localization and effect of physical activity.

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.

The effect of acute and long-term physical activity on extracellular matrix and serglycin in human skeletal muscle.

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 < 0.001) as well as long-term exercise (1.4-fold, P < 0.001). Cultured, primary human skeletal muscle cells expressed and secreted SRGN. When the expression of SRGN was knocked down, the expression and secretion of serpin E1 (SERPINE1) increased. In conclusion, acute and especially long-term exercise promotes enhanced expression of several ECM components and proteoglycans. SRGN is a novel exercise-regulated proteoglycan in skeletal muscle with a potential role in exercise adaptation.

Regulation of follistatin-like protein 1 expression and secretion in primary human skeletal muscle cells.

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-1ß 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.

Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells.

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.