Pernille Keller

Searching for the exercise factor: is IL-6 a candidate?

For years the search for the stimulus that initiates and maintains the change of excitability or sensibility of the regulating centers in exercise has been progressing. For lack of more precise knowledge, it has been called the ‘work stimulus’, ‘the work factor’ or ‘the exercise factor’. In other terms, one big challenge for muscle and exercise physiologists has been to determine how muscles signal to central and peripheral organs. Here we discuss the possibility that interleukin-6 (IL-6) could mediate some of the health beneficial effects of exercise. In resting muscle, the IL-6 gene is silent, but it is rapidly activated by contractions. The transcription rate is very fast and the fold changes of IL-6 mRNA is marked. IL-6 is released from working muscles into the circulation in high amounts. The IL-6 production is modulated by the glycogen content in muscles, and IL-6 thus works as an energy sensor. IL-6 exerts its effect on adipose tissue, inducing lipolysis and gene transcription in abdominal subcutaneous fat and increases whole body lipid oxidation. Furthermore, IL-6 inhibits low-grade TNF-α-production and may thereby inhibit TNF-α-induced insulin resistance and atherosclerosis development. We propose that IL-6 and other cytokines, which are produced and released by skeletal muscles, exerting their effects in other organs of the body, should be named ‘myokines’.

Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes

BackgroundSkeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers.MethodsWe profiled >47,000 mRNA sequences and >500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes.ResultsThe muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P < 7.4 × 10-8), post-translational modification (P < 9.7 × 10-5) and developmental (P < 1.3 × 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation.ConclusionsWe provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions.

Muscle-derived interleukin-6: Lipolytic, anti-inflammatory and immune regulatory effects

Interleukin-6 (IL-6) is produced locally in working skeletal muscle and can account for the exercise-induced increase in plasma IL-6. The transcription rate for IL-6 in muscle nuclei isolated from muscle biopsies during exercise is very high and is enhanced further when muscle glycogen content is low. Furthermore, cultured human primary muscle cells can increase IL-6 mRNA when incubated with the calcium ionophore ionomycin and it is likely that myocytes produce IL-6 in response to muscle contraction. The biological roles of muscle-derived IL-6 have been investigated in studies in which human recombinant IL-6 was infused in healthy volunteers to mimic closely the IL-6 concentrations observed during prolonged exercise. Using stable isotopes, we have demonstrated that physiological concentrations of IL-6 induce lipolysis. Although we have yet to determine the precise biological action of muscle-derived IL-6, our data support the hypothesis that the role of IL-6 released from contracting muscle during exercise is to act in a hormone-like manner to mobilize extracellular substrates and/or augment substrate delivery during exercise. In addition, IL-6 inhibits low-level TNF-α production, and IL-6 produced during exercise probably inhibits TNF-α-induced insulin resistance in peripheral tissues. Hence, IL-6 produced by skeletal muscle during contraction may play an important role in the beneficial health effects of exercise

A transcriptional map of the impact of endurance exercise training on skeletal muscle phenotype

The molecular pathways that are activated and contribute to physiological remodeling of skeletal muscle in response to endurance exercise have not been fully characterized. We previously reported that ∼800 gene transcripts are regulated following 6 wk of supervised endurance training in young sedentary males, referred to as the training-responsive transcriptome (TRT) (Timmons JA et al. J Appl Physiol 108: 1487-1496, 2010). Here we utilized this database together with data on biological variation in muscle adaptation to aerobic endurance training in both humans and a novel out-bred rodent model to study the potential regulatory molecules that coordinate this complex network of genes. We identified three DNA sequences representing RUNX1, SOX9, and PAX3 transcription factor binding sites as overrepresented in the TRT. In turn, miRNA profiling indicated that several miRNAs targeting RUNX1, SOX9, and PAX3 were downregulated by endurance training. The TRT was then examined by contrasting subjects who demonstrated the least vs. the greatest improvement in aerobic capacity (low vs. high responders), and at least 100 of the 800 TRT genes were differentially regulated, thus suggesting regulation of these genes may be important for improving aerobic capacity. In high responders, proangiogenic and tissue developmental networks emerged as key candidates for coordinating tissue aerobic adaptation. Beyond RNA-level validation there were several DNA variants that associated with maximal aerobic capacity (Vo(₂max)) trainability in the HERITAGE Family Study but these did not pass conservative Bonferroni adjustment. In addition, in a rat model selected across 10 generations for high aerobic training responsiveness, we found that both the TRT and a homologous subset of the human high responder genes were regulated to a greater degree in high responder rodent skeletal muscle. This analysis provides a comprehensive map of the transcriptomic features important for aerobic exercise-induced improvements in maximal oxygen consumption.

Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise

Interleukin‐6 (IL‐6) is produced by many different cell types. Human skeletal muscles produce and release high amounts of IL‐6 during exercise; however, the cell source of origin in the muscle is not known. Therefore, we studied the protein expression of IL‐6 by immunohistochemistry in human muscle tissue from biopsies obtained at time points 0, 3, 4.5, 6, 9, and 24 h in relation to 3 h of bicycle exercise performed by healthy young males (n=12) and in resting controls (n=6). The IL‐6 expression was clearly increased after exercise and remained high even by 24 h, relative to pre‐exercise or resting individuals. The IL‐6 immunostainings of skeletal muscle cells were homogeneous and without difference between muscle fiber types. The IL‐6 mRNA peaked immediately after the exercise, and, in accordance, the IL‐6 protein expression within muscle cells was most pronounced around 3 h post‐exercise. However, the finding that plasma IL‐6 concentration peaked in the end of exercise indicates a high turnover of muscle‐derived IL‐6. In conclusion, the finding of marked IL‐6 protein expression exclusively within skeletal muscle fibers following exercise demonstrates that skeletal muscle fibers of all types are the dominant cell source of exercise‐induced release of IL‐6 from working muscle.

Fat-specific Protein 27 Regulates Storage of Triacylglycerol

FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins. In this study, we investigated the function of another member of this family, FSP27 (Cidec), in apoptosis and adipocyte metabolism. Although overexpression of FSP27 is sufficient to increase apoptosis of 293T and 3T3-L1 cells, more physiological levels of expression stimulate spontaneous lipid accumulation in several cell types without induction of adipocyte genes. Increased triacylglycerol is likely due to decreased β-oxidation of nonesterified fatty acids. Altered flux of fatty acids into triacylglycerol may be a direct effect of FSP27 function, which is localized to lipid droplets in 293T cells and 3T3-L1 adipocytes. Stable knockdown of FSP27 during adipogenesis of 3T3-L1 cells substantially decreases lipid droplet size, increases mitochondrial and lipid droplet number, and modestly increases glucose uptake and lipolysis. Expression of FSP27 in subcutaneous adipose tissue of a human diabetes cohort decreases with total fat mass but is not associated with measures of insulin resistance (e.g. homeostasis model assessment). Together, these data indicate that FSP27 binds to lipid droplets and regulates their enlargement.

Distinct expression of muscle-specific microRNAs (myomirs) in brown adipocytes.

MicroRNAs, a novel class of post‐transcriptional gene regulators, have been demonstrated to be involved in several cellular processes regulating the expression of protein‐coding genes. Here we examine murine white and brown primary cell cultures for differential expression of miRNAs. The adipogenesis‐related miRNA miR‐143 was highly expressed in mature white adipocytes but was low in mature brown adipocytes. Three classical “myogenic” miRNAs miR‐1, miR‐133a and miR‐206 were absent from white adipocytes but were specifically expressed both in brown pre‐ and mature adipocytes, reinforcing the concept that brown adipocytes and myocytes derive from a common cell lineage that specifies energy‐dissipating cells. Augmentation of adipocyte differentiation status with norepinephrine or rosiglitazone did not affect the expression of the above miRNAs, the expression levels of which were thus innately regulated. However, expression of the miRNA miR‐455 was enhanced during brown adipocyte differentiation, similarly to the expression pattern of the brown adipocyte differentiation marker UCP1. In conclusion, miRNAs are differentially expressed in white and brown adipocytes and may be important in defining the common precursor cell for myocytes and brown adipocytes and thus have distinct roles in energy‐storing versus energy‐dissipating cells. J. Cell. Physiol. 218: 444–449, 2009.

Dysregulation of mitochondrial dynamics and the muscle transcriptome in ICU patients suffering from sepsis induced multiple organ failure.

Background Septic patients treated in the intensive care unit (ICU) often develop multiple organ failure including persistent skeletal muscle dysfunction which results in the patients protracted recovery process. We have demonstrated that muscle mitochondrial enzyme activities are impaired in septic ICU patients impairing cellular energy balance, which will interfere with muscle function and metabolism. Here we use detailed phenotyping and genomics to elucidate mechanisms leading to these impairments and the molecular consequences. Methodology/Principal Findings Utilising biopsy material from seventeen patients and ten age-matched controls we demonstrate that neither mitochondrial in vivo protein synthesis nor expression of mitochondrial genes are compromised. Indeed, there was partial activation of the mitochondrial biogenesis pathway involving NRF2α/GABP and its target genes TFAM, TFB1M and TFB2M yet clearly this failed to maintain mitochondrial function. We therefore utilised transcript profiling and pathway analysis of ICU patient skeletal muscle to generate insight into the molecular defects driving loss of muscle function and metabolic homeostasis. Gene ontology analysis of Affymetrix analysis demonstrated substantial loss of muscle specific genes, a global oxidative stress response related to most probably cytokine signalling, altered insulin related signalling and a substantial overlap between patients and muscle wasting/inflammatory animal models. MicroRNA 21 processing appeared defective suggesting that post-transcriptional protein synthesis regulation is altered by disruption of tissue microRNA expression. Finally, we were able to demonstrate that the phenotype of skeletal muscle in ICU patients is not merely one of inactivity, it appears to be an actively remodelling tissue, influenced by several mediators, all of which may be open to manipulation with the aim to improve clinical outcome. Conclusions/Significance This first combined protein and transcriptome based analysis of human skeletal muscle obtained from septic patients demonstrated that losses of mitochondria and muscle mass are accompanied by sustained protein synthesis (anabolic process) while dysregulation of transcription programmes appears to fail to compensate for increased damage and proteolysis. Our analysis identified both validated and novel clinically tractable targets to manipulate these failing processes and pursuit of these could lead to new potential treatments.

Exercise induces interleukin-8 expression in human skeletal muscle

Skeletal muscle has been recognized as an endocrine organ, and muscle cell cultures express several cytokines with potential hormonal effects. Interleukin‐8 (IL‐8), a chemokine, which induces angiogenesis, is expressed in working muscles; however, the cell source of origin has not been identified. We aimed to elucidate if IL‐8 protein is: (1) expressed in contracting muscle fibres and (2) whether there is a release of IL‐8 from exercising muscle. Seventeen healthy male volunteers were included in two independent protocols: 3 h of ergometer bicycle exercise at 60% of (n= 6) or rest (n= 5), and 3 h of two‐legged knee‐extensor exercise at 60% of maximal workload (n= 6). Repetitive muscle biopsy samples were obtained from the vastus lateralis in all experiments. A marked increase in IL‐8 mRNA was found in muscle biopsy samples obtained after exercise. A marked IL‐8 protein expression was demonstrated within the cytoplasm of muscle fibres in biopsy samples obtained in the recovery phase following 3 h of bicycle exercise, and the peak occurred 3–6 h postexercise. A small transient net release of IL‐8 from working muscle was found at 1.5 h of knee‐extensor exercise. However, the small release of IL‐8 from muscle did not result in an increase in the systemic plasma concentration of IL‐8, suggesting that muscle‐derived IL‐8 may play a local role, e.g. in angiogenesis.

IL‐6 Gene Expression in Human Adipose Tissue in Response to Exercise – Effect of Carbohydrate Ingestion

Interleukin‐6 (IL‐6) is a cytokine involved in a number of immunological processes, but it is also linked to exercise and possibly energy status. During exercise, muscle IL‐6 mRNA levels and plasma IL‐6 levels are increased and further augmented when intramuscular glycogen levels are low. In contrast, the increase in plasma IL‐6 is blunted if carbohydrate is administered, indicating a substrate‐regulated induction of IL‐6 in human skeletal muscle. Recent studies have demonstrated that IL‐6 is also released from adipose tissue in response to an exercise bout. Furthermore, IL‐6 has been demonstrated to have a lipolytic effect, thus possibly playing a role in mobilisation of energy as free fatty acids (FFA) in response to exercise. The purpose of the present study was to investigate the gene expression pattern of IL‐6 in adipose tissue in response to exercise, and to determine whether gene expression was affected by the ingestion of carbohydrate. Eight male subjects performed 3 h of bicycling with ingestion of a carbohydrate drink or placebo. Fat biopsy samples and blood samples were obtained before, during and in the recovery phase of exercise. Both plasma IL‐6 and adipose IL‐6 mRNA levels increased in response to exercise. IL‐6 gene expression was lower (P < 0.05) in the CHO trial (1.98‐fold increase, confidence interval (CI) 1.16–3.83) compared with the control (6.49‐fold increase, CI 3.57–13.91) at end of exercise. Furthermore, CHO ingestion blunted the increase in plasma IL‐6 levels (P < 0.05) at end of exercise (26.0 ± 3.7 pg ml−1 in the control vs. 15.6 ± 2.4 pg ml−1 in the CHO trial). In conclusion, exercise results in an increase in IL‐6 gene expression in adipose tissue in response to exercise, an effect that is significantly blunted by ingestion of carbohydrate.