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Human thermogenic adipose tissue mitigates metabolic disease, raising much interest in understanding its development and function. Here, we show that human thermogenic adipocytes specifically express a primate-specific long non-coding RNA, LINC00473 which is highly correlated with UCP1 expression and decreased in obesity and type-2 diabetes. LINC00473 is detected in progenitor cells, and increases upon differentiation and in response to cAMP. In contrast to other known adipocyte LincRNAs, LINC00473 shuttles out of the nucleus, colocalizes and can be crosslinked to mitochondrial and lipid droplet proteins. Up- or down- regulation of LINC00473 results in reciprocal alterations in lipolysis, respiration and transcription of genes associated with mitochondrial oxidative metabolism. Depletion of PLIN1 results in impaired cAMP-responsive LINC00473 expression and lipolysis, indicating bidirectional interactions between PLIN1, LINC00473 and mitochondrial oxidative functions. Thus, we suggest that LINC00473 is a key regulator of human thermogenic adipocyte function, and reveals a role for a LincRNA in inter-organelle communication and human energy metabolism., Competing Interests: Competing Interests Statement The authors declare no competing interests.

Background : Being born with low birth weight (LBW) is a risk factor for muscle insulin resistance and type 2 diabetes (T2D), which may be mediated by epigenetic mechanisms programmed by the intrauterine environment. Epigenetic mechanisms exert their prime effects in developing cells. We hypothesized that muscle insulin resistance in LBW subjects may be due to early differential epigenomic and transcriptomic alterations in their immature muscle progenitor cells. Results : Muscle progenitor cells were obtained from 23 healthy young adult men born at term with LBW, and 15 BMI-matched normal birth weight (NBW) controls. The cells were subsequently cultured and differentiated into myotubes. DNA and RNA were harvested before and after differentiation for genome-wide DNA methylation and RNA expression measurements.After correcting for multiple comparisons (q ≤ 0.05), 56 CpG sites were found to be significantly, differentially methylated in myoblasts from LBW compared with NBW men, of which the top five gene-annotated CpG sites ( SKI, ARMCX3, NR5A2, NEUROG, ESRRG ) previously have been associated to regulation of cholesterol, fatty acid and glucose metabolism and muscle development or hypertrophy. LBW men displayed markedly decreased myotube gene expression levels of the AMPK-repressing tyrosine kinase gene FYN and the histone deacetylase gene HDAC7 . Silencing of FYN and HDAC7 was associated with impaired myotube formation, which for HDAC7 reduced muscle glucose uptake. Conclusions : The data provides evidence of impaired muscle development predisposing LBW individuals to T2D is linked to and potentially caused by distinct DNA methylation and transcriptional changes including down regulation of HDAC7 and FYN in their immature myoblast stem cells.

Infants rely on brown adipose tissue (BAT) as a primary source of thermogenesis. In some adult humans, residuals of brown adipose tissue are adjacent to the central nervous system and acute activation increases metabolic rate. Brown adipose tissue (BAT) recruitment occurs during cold acclimation and includes secretion of factors, known as batokines, which target several different cell types within BAT, and promote adipogenesis, angiogenesis, immune cell interactions, and neurite outgrowth. All these processes seem to act in concert to promote an adapted BAT. Recent studies have also provided exciting data on whole body metabolic regulation with a broad spectrum of mechanisms involving BAT crosstalk with liver, skeletal muscle, and gut as well as the central nervous system. These widespread interactions might reflect the property of BAT of switching between an active thermogenic state where energy is highly consumed and drained from the circulation, and the passive thermoneutral state, where energy consumption is turned off. (Endocrine Reviews 41: XXX - XXX, 2020)., (© Endocrine Society 2019.)

Adipokines secreted from white adipose tissue play a role in metabolic crosstalk and homeostasis, whereas the brown adipose secretome is less explored. We performed high-sensitivity mass-spectrometry-based proteomics on the cell media of human adipocytes derived from the supraclavicular brown adipose and from the subcutaneous white adipose depots of adult humans. We identified 471 potentially secreted proteins covering interesting categories such as hormones, growth factors, extracellular matrix proteins, and proteins of the complement system, which were differentially regulated between brown and white adipocytes. A total of 101 proteins were exclusively quantified in brown adipocytes, and among these was ependymin-related protein 1 (EPDR1). EPDR1 was detected in human plasma, and functional studies suggested a role for EPDR1 in thermogenic determination during adipogenesis. In conclusion, we report substantial differences between the secretomes of brown and white human adipocytes and identify novel candidate batokines that can be important regulators of human metabolism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of "brite/beige" thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Human and rodent brown adipose tissues (BAT) appear morphologically and molecularly different. Here we compare human BAT with both classical brown and brite/beige adipose tissues of 'physiologically humanized' mice: middle-aged mice living under conditions approaching human thermal and nutritional conditions, that is, prolonged exposure to thermoneutral temperature (approximately 30 °C) and to an energy-rich (high-fat, high-sugar) diet. We find that the morphological, cellular and molecular characteristics (both marker and adipose-selective gene expression) of classical brown fat, but not of brite/beige fat, of these physiologically humanized mice are notably similar to human BAT. We also demonstrate, both in silico and experimentally, that in physiologically humanized mice only classical BAT possesses a high thermogenic potential. These observations suggest that classical rodent BAT is the tissue of choice for translational studies aimed at recruiting human BAT to counteract the development of obesity and its comorbidities.

Objective: Increasing the amounts of functionally competent brown adipose tissue (BAT) in adult humans has the potential to restore dysfunctional metabolism and counteract obesity. In this study, we aimed to characterize the human perirenal fat depot, and we hypothesized that there would be regional, within-depot differences in the adipose signature depending on local sympathetic activity., Methods: We characterized fat specimens from four different perirenal regions of adult kidney donors, through a combination of qPCR mapping, immunohistochemical staining, RNA-sequencing, and pre-adipocyte isolation. Candidate gene signatures, separated by adipocyte morphology, were recapitulated in a murine model of unilocular brown fat induced by thermoneutrality and high fat diet., Results: We identified widespread amounts of dormant brown adipose tissue throughout the perirenal depot, which was contrasted by multilocular BAT, primarily found near the adrenal gland. Dormant BAT was characterized by a unilocular morphology and a distinct gene expression profile, which partly overlapped with that of subcutaneous white adipose tissue (WAT). Brown fat precursor cells, which differentiated into functional brown adipocytes were present in the entire perirenal fat depot, regardless of state. We identified SPARC as a candidate adipokine contributing to a dormant BAT state, and CLSTN3 as a novel marker for multilocular BAT., Conclusions: We propose that perirenal adipose tissue in adult humans consists mainly of dormant BAT and provide a data set for future research on factors which can reactivate dormant BAT into active BAT, a potential strategy for combatting obesity and metabolic disease., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background/aim: The aim of this study was to analyze survivorship and functional outcome of cementless spongy metal structured total hip arthroplasty (THA) after ultra-long-term follow-up., Patients and Methods: A total of 100 THAs in patients under 65 years at initial surgery were performed between 1985 and 1989 at our Department. Twenty patients (23 hips) were available for final follow-up. Implant survivorship and functional results (Merle d'Aubigné, SF-12) were assessed., Results: At a mean follow-up of 30 years (range=27.7-31.7 years), the overall stem survival rate was 82.6% (19/23 stems) and the overall cup survival rate was 52.2% (12/23 cups). Revision surgeries were performed for aseptic loosening in all cases. Functional evaluation revealed good to excellent results in 16 patients (80%) Conclusion: Spongy metal structured cementless THA provides remarkable survivorship and excellent functional results even after ultra-long-term follow-up. Further research regarding modern implants, bearing surfaces etc. is required to assess survivorship and clinical outcomes of different implant designs., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

In the version of this article initially published, in the graph keys in Fig. 1i, the colors indicating 'Ob' and 'Ad' were red and blue, respectively, but should have been blue and red, respectively; the shapes indicating 'MUS' and 'BM' were a triangle and a square, respectively, but should have been a square and a triangle, respectively. The errors have been corrected in the HTML and PDF versions of the article.

Mesenchymal (stromal) stem cells (MSCs) constitute populations of mesodermal multipotent cells involved in tissue regeneration and homeostasis in many different organs. Here we performed comprehensive characterization of the transcriptional and epigenomic changes associated with osteoblast and adipocyte differentiation of human MSCs. We demonstrate that adipogenesis is driven by considerable remodeling of the chromatin landscape and de novo activation of enhancers, whereas osteogenesis involves activation of preestablished enhancers. Using machine learning algorithms for in silico modeling of transcriptional regulation, we identify a large and diverse transcriptional network of pro-osteogenic and antiadipogenic transcription factors. Intriguingly, binding motifs for these factors overlap with SNPs related to bone and fat formation in humans, and knockdown of single members of this network is sufficient to modulate differentiation in both directions, thus indicating that lineage determination is a delicate balance between the activities of many different transcription factors.

Background: Sex differences are known to impact muscle phenotypes, metabolism, and disease risk. Skeletal muscle stem cells (satellite cells) are important for muscle repair and to maintain functional skeletal muscle. Here we studied, for the first time, effects of sex on DNA methylation and gene expression in primary human myoblasts (activated satellite cells) before and after differentiation into myotubes., Method: We used an array-based approach to analyse genome-wide DNA methylation and gene expression in myoblasts and myotubes from 13 women and 13 men. The results were followed up with a reporter gene assay., Results: Genome-wide DNA methylation and gene expression differences between the sexes were detected in both myoblasts and myotubes, on the autosomes as well as the X-chromosome, despite lack of exposure to sex hormones and other factors that differ between sexes. Pathway analysis revealed higher expression of oxidative phosphorylation and other metabolic pathways in myoblasts from women compared to men. Oxidative phosphorylation was also enriched among genes with higher expression in myotubes from women. Forty genes in myoblasts and 9 in myotubes had differences in both DNA methylation and gene expression between the sexes, including LAMP2 and SIRT1 in myoblasts and KDM6A in myotubes. Furthermore, increased DNA methylation of LAMP2 promoter had negative effects on reporter gene expression. Five genes (CREB5, RPS4X, SYAP1, XIST, and ZRSR2) showed differential DNA methylation and gene expression between the sexes in both myoblasts and myotubes. Interestingly, differences in DNA methylation and expression between women and men were also found during differentiation (myoblasts versus myotubes), e.g., in genes involved in energy metabolism. Interestingly, more DNA methylation changes occur in women compared to men on autosomes., Conclusion: All together, we show that epigenetic and transcriptional differences exist in human myoblasts and myotubes as well as during differentiation between women and men. We believe that these intrinsic differences might contribute to sex dependent differences in muscular phenotypes.

Obesity involves a contrasting expansion of the energy-storing white fat and loss of functionally competent brown fat, an energy-consuming thermogenic adipose. Leveraging our understanding of white and brown adipocyte recruitment and investigating factors that regulate these processes might reveal novel targets for counteracting obesity. In vitro differentiation of primary preadipocytes mimics many of the morphological and transcriptional events occurring during adipogenesis in vivo. Moreover, preadipocytes isolated from a specific depot maintain features of their originating niche. This makes in vitro adipogenesis a valuable model for identifying differential regulation patterns between brown and white adipogenesis. In this chapter, we describe step-by-step how to isolate brown and white preadipocytes from human tissue biopsies and how to culture and differentiate them in vitro. We discuss this process, what to consider, and how this in vitro system can be used to model in vivo adipogenesis.

In addition to their outstanding significance in the field of tumor orthopaedics, megaprostheses are becoming increasingly important for revision arthroplasty. Despite their wide application, the reported complication and failure rates remain high. The analysis of failure mechanisms roughly allows a breakdown into mechanical and non-mechanical causes; soft-tissue failure, loosening and periprosthetic infection play outstanding roles. Each type of failure can be associated to a specific prosthetic component: To avoid soft tissue failure, muscular connection to the prosthesis is essential; to avoid loosening, the anchoring technique on the stem is crucial; to prevent infection, the surface condition or possibly the coating of the prosthesis seem to play major roles. Some fully-fledged proximal femoral replacement systems have become established on the market and have similar clinical outcomes. However, there are significant differences in anchoring techniques, modularity or module connections and soft tissue connection. In any case, the high failure rates show that there is great potential for future developments. Innovative suggestions for each component are certainly possible through cross-linking to already functioning systems. Together with new findings from basic research, future systems should be able to significantly reduce the rate of prosthetic failures., Competing Interests: Die Autoren geben an, dass kein Interessenkonflikt besteht., (Georg Thieme Verlag KG Stuttgart · New York.)

Brown adipose tissue (BAT) is a metabolically active organ that contributes to the maintenance of systemic metabolism. The sympathetic nervous system plays important roles in the homeostasis of BAT and promotes its browning and activation. However, the role of other neurotransmitters in BAT homeostasis remains largely unknown. Our metabolomic analyses reveal that gamma-aminobutyric acid (GABA) levels are increased in the interscapular BAT of mice with dietary obesity. We also found a significant increase in GABA-type B receptor subunit 1 (GABA-BR1) in the cell membranes of brown adipocytes of dietary obese mice. When administered to obese mice, GABA induces BAT dysfunction together with systemic metabolic disorder. Conversely, the genetic inactivation or inhibition of GABA-BR1 leads to the re-browning of BAT under conditions of metabolic stress and ameliorated systemic glucose intolerance. These results indicate that the constitutive activation of GABA/GABA-BR1 signaling in obesity promotes BAT dysfunction and systemic metabolic derangement., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Activation of energy expenditure in thermogenic fat is a promising strategy to improve metabolic health, yet the dynamic processes that evoke this response are poorly understood. Here we show that synthesis of the mitochondrial phospholipid cardiolipin is indispensable for stimulating and sustaining thermogenic fat function. Cardiolipin biosynthesis is robustly induced in brown and beige adipose upon cold exposure. Mimicking this response through overexpression of cardiolipin synthase (Crls1) enhances energy consumption in mouse and human adipocytes. Crls1 deficiency in thermogenic adipocytes diminishes inducible mitochondrial uncoupling and elicits a nuclear transcriptional response through endoplasmic reticulum stress-mediated retrograde communication. Cardiolipin depletion in brown and beige fat abolishes adipose thermogenesis and glucose uptake, which renders animals insulin resistant. We further identify a rare human CRLS1 variant associated with insulin resistance and show that adipose CRLS1 levels positively correlate with insulin sensitivity. Thus, adipose cardiolipin has a powerful impact on organismal energy homeostasis through thermogenic fat bioenergetics., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brown adipose tissue (BAT) is a therapeutic target for metabolic diseases; thus, understanding its metabolic circuitry is clinically important. Many studies of BAT compare rodents mildly cold to those severely cold. Here, we compared BAT remodeling between thermoneutral and mild-cold-adapted mice, conditions more relevant to humans. Although BAT is renowned for catabolic β-oxidative capacity, we find paradoxically that the anabolic de novo lipogenesis (DNL) genes encoding ACLY, ACSS2, ACC, and FASN were among the most upregulated by mild cold and that, in humans, DNL correlates with Ucp1 expression. The regulation and function of adipocyte DNL and its association with thermogenesis are not understood. We provide evidence suggesting that AKT2 drives DNL in adipocytes by stimulating ChREBPβ transcriptional activity and that cold induces the AKT2-ChREBP pathway in BAT to optimize fuel storage and thermogenesis. These data provide insight into adipocyte DNL regulation and function and illustrate the metabolic flexibility of thermogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Brown fat dissipates energy as heat and protects against obesity. Here, we identified nuclear factor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide open chromatin analysis of murine brown and white fat followed by motif analysis of brown-fat-specific open chromatin regions. NFIA and the master transcriptional regulator of adipogenesis, PPARγ, co-localize at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARγ, leading to increased chromatin accessibility and active transcription. Introduction of NFIA into myoblasts results in brown adipocyte differentiation. Conversely, the brown fat of NFIA-knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. Finally, expression of NFIA and the brown-fat-specific genes is positively correlated in human brown fat. These results indicate that NFIA activates the cell-type-specific enhancers and facilitates the binding of PPARγ to control the brown fat gene program.

Activation of brown adipose tissue (BAT) in adult humans increase glucose and fatty acid clearance as well as resting metabolic rate, whereas a prolonged elevation of BAT activity improves insulin sensitivity. However, substantial reductions in body weight following BAT activation has not yet been shown in humans. This observation raise the possibility for feedback mechanisms in adult humans in terms of a brown fat-brain crosstalk, possibly mediated by batokines, factors produced by and secreted from brown fat. Batokines also seems to be involved in BAT recruitment by stimulating proliferation and differentiation of brown fat progenitors. Increasing human BAT capacity could thus include inducing brown fat biogenesis as well as identifying novel batokines. Another attractive approach would be to induce a brown fat phenotype, the so-called brite or beige fat, within the white fat depots. In adult humans, white fat tissue transformation into beige has been observed in patients with pheochromocytoma, a norepinephrine-producing tumor. Interestingly, human beige fat is predominantly induced in regions that were BAT during early childhood, possibly reflecting that a presence of human beige progenitors is depot specific and originating from BAT. In conclusion, to utilize the anti-obesity potential of human BAT focus should be directed towards identifying novel regulators of brown and beige fat progenitor cells, as well as feedback mechanisms of BAT activation. This would allow for identification of novel anti-obesity targets., (Copyright © 2017. Published by Elsevier B.V.)

Background: Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize intrinsic properties of myocytes, associated independently with T2D or obesity., Methods: We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor on genome-wide transcription. This setup enabled us to identify intrinsic properties, originating from muscle precursor cells and retained in the corresponding myocytes. Bioinformatic and statistical methods, including differential expression analysis, gene-set analysis, and metabolic network analysis, were used to characterize the different myocytes., Results: We found that the transcriptional program associated with obesity alone was strikingly similar to that induced specifically by T2D. We identified a candidate epigenetic mechanism, H3K27me3 histone methylation, mediating these transcriptional signatures. T2D and obesity were independently associated with dysregulated myogenesis, down-regulated muscle function, and up-regulation of inflammation and extracellular matrix components. Metabolic network analysis identified that in T2D but not obesity a specific metabolite subnetwork involved in sphingolipid metabolism was transcriptionally regulated., Conclusions: Our findings identify inherent characteristics in myocytes, as a memory of the in vivo phenotype, without the influence from a diabetic or obese extracellular environment, highlighting their importance in the development of T2D.

The liking and selective ingestion of palatable foods-including sweets-is biologically controlled, and dysfunction of this regulation may promote unhealthy eating, obesity, and disease. The hepatokine fibroblast growth factor 21 (FGF21) reduces sweet consumption in rodents and primates, whereas knockout of Fgf21 increases sugar consumption in mice. To investigate the relevance of these findings in humans, we genotyped variants in the FGF21 locus in participants from the Danish Inter99 cohort (n = 6,514) and examined their relationship with a detailed range of food and ingestive behaviors. This revealed statistically significant associations between FGF21 rs838133 and increased consumption of candy, as well as nominal associations with increased alcohol intake and daily smoking. Moreover, in a separate clinical study, plasma FGF21 levels increased acutely after oral sucrose ingestion and were elevated in fasted sweet-disliking individuals. These data suggest the liver may secrete hormones that influence eating behavior., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: MicroRNAs (miRNAs) are increasingly recognized as fine-tuning regulators of metabolism, and are dysregulated in several disease conditions. With their capacity to rapidly change gene expression, miRNAs are also important regulators of development and cell differentiation. In the current study, we describe an impaired myogenic capacity of muscle stem cells isolated from humans with type 2 diabetes (T2DM) and assess whether this phenotype is regulated by miRNAs., Methods: We measured global miRNA expression during in vitro differentiation of muscle stem cells derived from T2DM patients and healthy controls., Results: The mir-23b/27b cluster was downregulated in the cells of the patients, and a pro-myogenic effect of these miRNAs was mediated through the p53 pathway, which was concordantly dysregulated in the muscle cells derived from humans with T2DM., Conclusions: Our results indicate that we have identified a novel pathway for coordination of myogenesis, the miR-23b/27b-p53 axis that, when dysregulated, potentially contributes to a sustained muscular dysfunction in T2DM.

Context: Offspring of women with gestational diabetes (O-GDM) or type 1 diabetes mellitus (O-T1DM) have been exposed to hyperglycemia in utero and have an increased risk of developing metabolic disease in adulthood., Design: In total, we recruited 206 adult offspring comprising the two fetal hyperglycemic groups, O-GDM and O-T1DM, and, as a control group, offspring from the background population (O-BP). Subcutaneous fat biopsies were obtained and preadipocyte cell cultures were established from adult male O-GDM (n = 18, age 30.1 ± 2.5 years), O-T1DM (n = 18, age 31.6 ± 2.2 years), and O-BP (n = 16; age, 31.5 ± 2.7 years) and cultured in vitro., Main Outcome Measures: First, we studied in vivo adipocyte histology. Second, we studied in vitro preadipocyte leptin secretion, gene expression, and LEP DNA methylation. This was studied in combination with in vitro preadipocyte lipogenesis, lipolysis, and mitochondrial respiration., Results: We show that subcutaneous adipocytes from O-GDM are enlarged compared with O-BP adipocytes. Preadipocytes isolated from male O-GDM and O-T1DM and cultured in vitro displayed decreased LEP promoter methylation, increased leptin gene expression, and elevated leptin secretion throughout differentiation, compared with adipocytes established from male O-BP. In addition, the preadipocytes demonstrated functional defects including decreased maximal mitochondrial capacity with increased lipolysis and decreased ability to store fatty acids when challenged with 3 days of extra fatty acid supply., Conclusions: Taken together, these findings show that intrinsic epigenetic and functional changes exist in preadipocyte cultures from individuals exposed to fetal hyperglycemia who are at increased risk of developing metabolic disease., (Copyright © 2017 Endocrine Society)

Background: Unicompartmental total knee arthroplasty (UKA) is a well-established treatment option for unicondylar osteoarthritis, and generally leads to better functional results than tricompartimental total knee arthroplasty (TKA). However, revision rates of UKAs are reported as being higher; a major reason for this is aseptic loosening of the tibial component due to implant-cement-bone interface fatigue. The objective of this study was to determine the effects of trabecular bone preparation, prior to implantation of tibial UKAs, on morphological and biomechanical outcomes in a cadaver study., Methods: Cemented UKAs were performed in 18 human cadaver knees after the bone bed was cleaned using pulsed lavage (Group A), conventional brush (Group B) or no cleaning at all (Group C, control). Morphologic cement penetration and primary stability were measured., Results: The area proportion under the tibial component without visible cement penetration was significantly higher in Group C (21.9%, SD 11.9) than in both Group A (7.1%, SD 5.8), and Group B (6.5%, SD 4.2) (P=0.007). The overall cement penetration depth did not differ between groups. However, in the posterior part, cement penetration depth was significantly higher in Group B (1.9mm, SD 0.3) than in both Group A (1.3mm, SD 0.3) and Group C (1.4mm, SD 0.3) (P=0.015). The mode of preparation did not show a substantial effect on primary stability tested under dynamic compression-shear test conditions (P=0.910)., Conclusion: Bone preparation significantly enhances cement interdigitation. The application of a brush shows similar results compared with the application of pulsed lavage., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Human skeletal muscle stem cells are important for muscle regeneration. However, the combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Additionally, obesity is associated with low relative muscle mass and diminished metabolism. Epigenetic alterations taking place during myogenesis might contribute to these defects., Methods: We used Infinium HumanMethylation450 BeadChip Kit (Illumina) and HumanHT-12 Expression BeadChip (Illumina) to analyze genome-wide DNA methylation and transcription before versus after differentiation of primary human myoblasts from 14 non-obese and 14 obese individuals. Functional follow-up experiments were performed using siRNA mediated gene silencing in primary human myoblasts and a transgenic mouse model., Results: We observed genome-wide changes in DNA methylation and expression patterns during differentiation of primary human muscle stem cells (myoblasts). We identified epigenetic and transcriptional changes of myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6, PAX7, MEF2A, MEF2C, and MEF2D), cell cycle regulators, metabolic enzymes and genes previously not linked to myogenesis, including IL32, metallothioneins, and pregnancy-specific beta-1-glycoproteins. Functional studies demonstrated IL-32 as a novel target that regulates human myogenesis, insulin sensitivity and ATP levels in muscle cells. Furthermore, IL32 transgenic mice had reduced insulin response and muscle weight. Remarkably, approximately 3.7 times more methylation changes (147,161 versus 39,572) were observed during differentiation of myoblasts from obese versus non-obese subjects. In accordance, DNMT1 expression increased during myogenesis only in obese subjects. Interestingly, numerous genes implicated in metabolic diseases and epigenetic regulation showed differential methylation and expression during differentiation only in obese subjects., Conclusions: Our study identifies IL-32 as a novel myogenic regulator, provides a comprehensive map of the dynamic epigenome during differentiation of human muscle stem cells and reveals abnormal epigenetic changes in obesity.

Aims/hypothesis: Low birthweight (LBW) is associated with dysfunctions of adipose tissue and metabolic disease in adult life. We hypothesised that altered epigenetic and transcriptional regulation of adipose-derived stem cells (ADSCs) could play a role in programming adipose tissue dysfunction in LBW individuals., Methods: ADSCs were isolated from the subcutaneous adipose tissue of 13 normal birthweight (NBW) and 13 LBW adult men. The adipocytes were cultured in vitro, and genome-wide differences in RNA expression and DNA methylation profiles were analysed in ADSCs and differentiated adipocytes., Results: We demonstrated that ADSCs from LBW individuals exhibit multiple expression changes as well as genome-wide alterations in methylation pattern. Reduced expression of the transcription factor cyclin T2 encoded by CCNT2 may play a key role in orchestrating several of the gene expression changes in ADSCs from LBW individuals. Indeed, silencing of CCNT2 in human adipocytes decreased leptin secretion as well as the mRNA expression of several genes involved in adipogenesis, including MGLL, LIPE, PPARG, LEP and ADIPOQ. Only subtle genome-wide mRNA expression and DNA methylation changes were seen in mature cultured adipocytes from LBW individuals., Conclusions/interpretation: Epigenetic and transcriptional changes in LBW individuals are most pronounced in immature ADSCs that in turn may programme physiological characteristics of the mature adipocytes that influence the risk of metabolic diseases. Reduced expression of CCNT2 may play a key role in the developmental programming of adipose tissue.

Brown (BAT) and white (WAT) adipose tissues play distinct roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes. The AMP-activated protein kinase (AMPK) is a cellular energy sensor, but its role in regulating BAT and WAT metabolism is unclear. We generated an inducible model for deletion of the two AMPK β subunits in adipocytes (iβ1β2AKO) and found that iβ1β2AKO mice were cold intolerant and resistant to β-adrenergic activation of BAT and beiging of WAT. BAT from iβ1β2AKO mice had impairments in mitochondrial structure, function, and markers of mitophagy. In response to a high-fat diet, iβ1β2AKO mice more rapidly developed liver steatosis as well as glucose and insulin intolerance. Thus, AMPK in adipocytes is vital for maintaining mitochondrial integrity, responding to pharmacological agents and thermal stress, and protecting against nutrient-overload-induced NAFLD and insulin resistance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Skeletal myocytes are metabolically active and susceptible to insulin resistance and are thus implicated in type 2 diabetes (T2D). This complex disease involves systemic metabolic changes, and their elucidation at the systems level requires genome-wide data and biological networks. Genome-scale metabolic models (GEMs) provide a network context for the integration of high-throughput data. We generated myocyte-specific RNA-sequencing data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism, connected through the downregulated dihydrolipoamide dehydrogenase. Strikingly, the gene signature underlying this metabolic regulation successfully classifies the disease state of individual samples, suggesting that regulation of these pathways is a ubiquitous feature of myocytes in response to T2D., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims/hypothesis: We aimed to identify microRNAs (miRNAs) associated with type 2 diabetes and risk of developing the disease in skeletal muscle biopsies from phenotypically well-characterised twins., Methods: We measured muscle miRNA levels in monozygotic (MZ) twins discordant for type 2 diabetes using arrays. Further investigations of selected miRNAs included target prediction, pathway analysis, silencing in cells and association analyses in a separate cohort of 164 non-diabetic MZ and dizygotic twins. The effects of elevated glucose and insulin levels on miRNA expression were examined, and the effect of low birthweight (LBW) was studied in rats., Results: We identified 20 miRNAs that were downregulated in MZ twins with diabetes compared with their non-diabetic co-twins. Differences for members of the miR-15 family (miR-15b and miR-16) were the most statistically significant, and these miRNAs were predicted to influence insulin signalling. Indeed, miR-15b and miR-16 levels were associated with levels of key insulin signalling proteins, miR-15b was associated with the insulin receptor in non-diabetic twins and knockdown of miR-15b/miR-16 in myocytes changed the levels of insulin signalling proteins. LBW in twins and undernutrition during pregnancy in rats were, in contrast to overt type 2 diabetes, associated with increased expression of miR-15b and/or miR-16. Elevated glucose and insulin suppressed miR-16 expression in vitro., Conclusions: Type 2 diabetes is associated with non-genetic downregulation of several miRNAs in skeletal muscle including miR-15b and miR-16, potentially targeting insulin signalling. The paradoxical findings in twins with overt diabetes and twins at increased risk of the disease underscore the complexity of the regulation of muscle insulin signalling in glucose homeostasis.

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Genetics, epigenetics, and environment may together affect the susceptibility for type 2 diabetes (T2D). Our aim was to dissect molecular mechanisms underlying T2D using genome-wide expression and DNA methylation data in adipose tissue from monozygotic twin pairs discordant for T2D and independent case-control cohorts. In adipose tissue from diabetic twins, we found decreased expression of genes involved in oxidative phosphorylation; carbohydrate, amino acid, and lipid metabolism; and increased expression of genes involved in inflammation and glycan degradation. The most differentially expressed genes included ELOVL6, GYS2, FADS1, SPP1 (OPN), CCL18, and IL1RN. We replicated these results in adipose tissue from an independent case-control cohort. Several candidate genes for obesity and T2D (e.g., IRS1 and VEGFA) were differentially expressed in discordant twins. We found a heritable contribution to the genome-wide DNA methylation variability in twins. Differences in methylation between monozygotic twin pairs discordant for T2D were subsequently modest. However, 15,627 sites, representing 7,046 genes including PPARG, KCNQ1, TCF7L2, and IRS1, showed differential DNA methylation in adipose tissue from unrelated subjects with T2D compared with control subjects. A total of 1,410 of these sites also showed differential DNA methylation in the twins discordant for T2D. For the differentially methylated sites, the heritability estimate was 0.28. We also identified copy number variants (CNVs) in monozygotic twin pairs discordant for T2D. Taken together, subjects with T2D exhibit multiple transcriptional and epigenetic changes in adipose tissue relevant to the development of the disease., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Menopause is associated with an increased incidence of insulin resistance and metabolic diseases. In a chronic palmitate treatment model, we investigated the role of skeletal muscle fatty acid exposure in relation to the metabolic deterioration observed with menopause. Human skeletal muscle satellite cells were isolated from premenopausal (n = 6) and postmenopausal (n = 5) women. In an in vitro model, the myotubes were treated with palmitate (300 µM) for one-, two- or three days during differentiation. Effects on lipid accumulation, inflammation and insulin signaling were studied. Palmitate treatment led to a 108% (CI 95%: 50%; 267%) increase in intramyocellular ceramide in the myotubes from the postmenopausal women (post-myotubes) compared with a 26% (CI 95%: -57%; 96%) increase in myotubes from the premenopausal women (pre-myotubes), (p<0.05). Furthermore, post-myotubes had a 22% (CI 95%: 4%; 34%) increase in pJNK (p = 0.04) and a 114% (CI 95%: 50%; 177%) increase in Hsp70 protein expression (p = 0.03) after three days of palmitate treatment, compared with pre-myotubes, in which no increase in either pJNK (-12% (CI 95: -26%; 2%)) or Hsp70 (7% (CI 95: -78%; 91%)) was detected. Furthermore, post-myotubes showed a blunted insulin stimulated phosphorylation of AS160 in response to chronic palmitate treatment compared with pre-myotubes (p = 0.02). The increased intramyocellular ceramide content in the post-myotubes was associated with a significantly higher mRNA expression of Serine Palmitoyltransferase1 (SPT1) after one day of palmitate treatment (p = 0.03) in post-myotubes compared with pre-myotubes. Our findings indicate that post-myotubes are more prone to develop lipid accumulation and defective insulin signaling following chronic saturated fatty acid exposure as compared to pre-myotubes.

MiRNAs are potent intracellular posttranscriptional regulators and are also selectively secreted into the circulation in a cell-specific fashion. Global changes in miRNA expression in skeletal muscle in response to endurance exercise training have been reported. Therefore, our aim was to establish the miRNA signature in human plasma in response to acute exercise and chronic endurance training by utilizing a novel methodological approach. RNA was isolated from human plasma collected from young healthy men before and after an acute endurance exercise bout and following 12 weeks of endurance training. Global miRNA (742 miRNAs) measurements were performed as a screening to identify detectable miRNAs in plasma. Using customized qPCR panels we quantified the expression levels of miRNAs detected in the screening procedure (188 miRNAs). We demonstrate a dynamic regulation of circulating miRNA (ci-miRNA) levels following 0 hour (miR-106a, miR-221, miR-30b, miR-151-5p, let-7i, miR-146, miR-652 and miR-151-3p), 1 hour (miR-338-3p, miR-330-3p, miR-223, miR-139-5p and miR-143) and 3 hours (miR-1) after an acute exercise bout (P<0.00032). Where ci-miRNAs were all downregulated immediately after an acute exercise bout (0 hour) the 1 and 3 hour post exercise timepoints were followed by upregulations. In response to chronic training, we identified seven ci-miRNAs with decreased levels in plasma (miR-342-3p, let-7d, miR-766, miR-25, miR-148a, miR-185 and miR-21) and two miRNAs that were present at higher levels after the training period (miR-103 and miR-107) (P<0.00032). In conclusion, acute exercise and chronic endurance training, likely through specific mechanisms unique to each stimulus, robustly modify the miRNA signature of human plasma.

Age dependent decline in skeletal muscle function leads to impaired metabolic flexibility in elderly individuals. Physical activity and testosterone treatment have proven efficient strategies for delaying this condition. However, a common molecular pathway has not been identified. Muscle specific miRNAs (myomiRs) regulate metabolic pathways in skeletal muscle, are regulated by physical activity, and have response elements for testosterone in their promoter region. We therefore hypothesized that myomiRs would be regulated in skeletal muscle during aging. We further investigated any potential gender-dependent regulation of these miRNAs. We found that the myomiRs miR-1, miR-133a, and miR-133b were increased in skeletal muscle of elderly men compared to younger men. In addition, miR-133a/133b expression was markedly higher in women compared to men. Elimination of circulating testosterone in men was associated with lower levels of miR-133a and miR-133b. A positive regulatory effect of testosterone on miR-133a/133b expression was confirmed in castrated male C57BL/6J mice and in a model of primary human myocytes. Yet, an improvement of fitness level in the testosterone depleted men resulted in a down-regulation of miR133a/b. In conclusion, alterations in fitness level and circulating testosterone seem to represent two independent regulatory events where testosterone is a specific regulator of miR-133a/b expression.

Low birth weight (LBW) is associated with increased risk of the development of type 2 diabetes (T2D). The appetite-regulating hormone leptin is released from mature adipocytes, and its production may be decreased in immature preadipocytes from LBW individuals. We recruited 14 men born with LBW and 13 controls born with normal birth weight (NBW). Biopsy samples were obtained from subcutaneous abdominal fat depots, and preadipocytes were isolated and cultured. Gene expression of leptin and selected differentiation markers were analyzed during preadipocyte differentiation, and cell culture media were collected to analyze leptin secretion. DNA methylation of CpG sites in the leptin promoter was measured using pyrosequencing. We found that differentiating preadipocytes from LBW individuals showed reduced leptin gene expression and a corresponding reduced leptin release compared with NBW individuals. Mean DNA methylation of the proximal LEP promoter was increased in LBW compared with NBW individuals. The notion of impaired adipocyte maturation in LBW individuals was supported by a lower mRNA expression of the differentiation markers; fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, and GLUT4. Our findings are consistent with impaired preadipocyte maturation, contributing to an increased risk of the development of T2D in LBW subjects.

There is a current debate in the literature on whether human fat derived from the supraclavicular region should be classified as brown, or as the white fat-derived less potent, brite/beige. This commentary addresses whether the existing classification defined in mice is sufficient to describe the types of thermogenic adipocytes in humans. We recently published a contradictory mRNA expression signature of human supraclavicular fat defined by an upregulation of the brite marker TBX1 along with the classical brown markers ZIC1 and LHX8, as well as genes indicating brown fat activity including UCP1, PGC-1α, and PRDM16; and, finally, a downregulation of the white/brite markers HOXC8 and HOXC9. Subcutaneous fat was used as reference material. Another recent study presents a higher expression of ZIC1 and a lower expression of TBX1 in interscapular compared with supraclavicular fat. Here, however, there was no difference in UCP1, PGC-1α, PRDM16, suggesting both depots had equal brown fat potency. Taken together, supraclavicular brown fat derived from adult humans seems to represent a type of brown fat with distinct features from both subcutaneous white/brite and interscapular brown fat. Therefore, the classification of adipocyte subtypes defined in mice may need reconsideration when applying to humans.

Background: The objective of our study was to evaluate the impact of a single- ("implant only") versus a double-layer ("implant & bone") cementing technique on the primary stability of unicompartmental tibial plateaus under dynamic compression-shear loading conditions in human tibiae., Methods: Twelve fresh-frozen human knees of a mean donor age of 72.3 years were used to perform medial UKA under a less invasive parapatellar surgical approach. The tibiae were divided into two groups of matched pairs based on comparable trabecular bone mineral density. To assess the primary stability, a new method based on a combination of dynamic compression-shear testing, kinematic analysis of the tibial plateau migration relative to the bone and evaluation of the cement layer by CT-scans and fragments cut through the implant-cement-bone interface in the frontal plane was introduced., Findings: For the "implant only" cementation technique the mean load to failure was 2600 (SD 675) N and for "implant & bone" it was 2820 (SD 915) N. Between the final load level at failure and the bone mineral density a significant correlation was found for the groups "implant only" (r(s) = 0.875) and "implant & bone" (r(s) = 0.907)., Interpretation: From our observations, we conclude that there is no significant difference between a single- ("implant only") and double-layer ("implant & bone") cementing technique in the effect on the primary stability of unicompartmental tibia plateaus, in terms of failure load, correlation between final load at failure and bone mineral density, migration characteristics, cement layer thickness and penetration depth., (© 2013.)

Interleukin (IL)-6 is a cytokine with pleiotropic functions in different tissues and organs. Skeletal muscle produces and releases significant levels of IL-6 after prolonged exercise and is therefore considered as a myokine. Muscle is also an important target of the cytokine. IL-6 signaling has been associated with stimulation of hypertrophic muscle growth and myogenesis through regulation of the proliferative capacity of muscle stem cells. Additional beneficial effects of IL-6 include regulation of energy metabolism, which is related to the capacity of actively contracting muscle to synthesize and release IL-6. Paradoxically, deleterious actions for IL-6 have also been proposed, such as promotion of atrophy and muscle wasting. We review the current evidence for these apparently contradictory effects, the mechanisms involved and discuss their possible biological implications., (© 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.)

Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.

Human brown adipose tissue (BAT) has been detected in adults but was recently suggested to be of brite/beige origin. We collected BAT from the supraclavicular region in 21 patients undergoing surgery for suspected cancer in the neck area and assessed the gene expression of established murine markers for brown, brite/beige, and white adipocytes. We demonstrate that a classical brown expression signature, including upregulation of miR-206, miR-133b, LHX8, and ZIC1 and downregulation of HOXC8 and HOXC9, coexists with an upregulation of two newly established brite/beige markers, TBX1 and TMEM26. A similar mRNA expression profile was observed when comparing isolated human adipocytes from BAT and white adipose tissue (WAT) depots, differentiated in vitro. In conclusion, our data suggest that human BAT might consist of both classical brown and recruitable brite adipocytes, an observation important for future considerations on how to induce human BAT., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response to LIF. The mRNA and protein expressions of LIF and its receptor (LIFR) were measured in skeletal muscle biopsies from healthy individuals and patients with type 2 diabetes by use of qPCR and Western blot. LIF signaling and response were studied following administration of recombinant LIF and siRNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts from diabetic patients. Nonetheless, in the diabetic myoblasts, LIF-induced phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3 was impaired. The deficient response to LIF administration in the diabetic myoblasts was further emphasized by a lack of increase in LIF-stimulated cell proliferation and a decreased LIF-stimulated induction of the proliferation-promoting factors cyclin D1, JunB, and c-myc. SOCS3 protein was upregulated in diabetic myoblasts, and knockdown of SOCS3 rescued LIF-induced gene expression in diabetic myoblasts, whereas neither STAT1 or STAT3 signaling nor proliferation rate was affected. In conclusion, although LIF and LIFR proteins were increased in muscle tissue and myoblasts from diabetic patients, LIF signaling and LIF-stimulated cell proliferation were impaired in diabetic myoblasts, suggesting a novel mechanism by which muscle function is compromised in diabetes.

Obesity and type 2 diabetes are associated with chronically elevated systemic levels of IL-6, a pro-inflammatory cytokine with a role in skeletal muscle metabolism that signals through the IL-6 receptor (IL-6Rα). We hypothesized that skeletal muscle in obesity-associated type 2 diabetes develops a resistance to IL-6. By utilizing western blot analysis, we demonstrate that IL-6Rα protein was down regulated in skeletal muscle biopsies from obese persons with and without type 2 diabetes. To further investigate the status of IL-6 signaling in skeletal muscle in obesity-associated type 2 diabetes, we isolated satellite cells from skeletal muscle of people that were healthy (He), obese (Ob) or were obese and had type 2 diabetes (DM), and differentiated them in vitro into myocytes. Down-regulation of IL-6Rα was conserved in Ob myocytes. In addition, acute IL-6 administration for 30, 60 and 120 minutes, resulted in a down-regulation of IL-6Rα protein in Ob myocytes compared to both He myocytes (P<0.05) and DM myocytes (P<0.05). Interestingly, there was a strong time-dependent regulation of IL-6Rα protein in response to IL-6 (P<0.001) in He myocytes, not present in the other groups. Assessing downstream signaling, DM, but not Ob myocytes demonstrated a trend towards an increased protein phosphorylation of STAT3 in DM myocytes (P = 0.067) accompanied by a reduced SOCS3 protein induction (P<0.05), in response to IL-6 administration. Despite this loss of negative control, IL-6 failed to increase AMPKα2 activity and IL-6 mRNA expression in DM myocytes. There was no difference in fusion capacity of myocytes between cell groups. Our data suggest that negative control of IL-6 signaling is increased in myocytes in obesity, whereas a dysfunctional IL-6 signaling is established further downstream of IL-6Rα in DM myocytes, possibly representing a novel mechanism by which skeletal muscle function is compromised in type 2 diabetes.

The cytokine leukemia inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of myoblasts. We hypothesized that LIF is a contraction-induced myokine functioning in an autocrine fashion to activate gene regulation of human muscle satellite cell proliferation. Skeletal muscle LIF expression, regulation, and action were examined in two models: 1) young men performing a bout of heavy resistance exercise of the quadriceps muscle and 2) cultured primary human satellite cells. Resistance exercise induced a ninefold increase in LIF mRNA content in skeletal muscle, but LIF was not detectable in plasma of the subjects. However, electrically stimulated cultured human myotubes produced and secreted LIF, suggesting that LIF is a myokine with local effects. The well established exercise-induced signaling molecules PI3K, Akt, and mTor contributed to the regulation of LIF in cultured human myotubes as chemical inhibition of PI3K and mTor and siRNA knockdown of Akt1 were independently sufficient to downregulate LIF. Human myoblast proliferation was increased by recombinant exogenous LIF and decreased by siRNA knockdown of the endogenous LIF receptor. Finally, the transcription factors JunB and c-Myc, which promote myoblast proliferation, were induced by LIF in cultured human myotubes. Indeed, both JunB and c-Myc were also increased in skeletal muscle following resistance exercise. Our data suggest that LIF is a contraction-induced myokine, potentially acting in an autocrine or paracrine fashion to promote satellite cell proliferation.

Muscle specific miRNAs, myomiRs, have been shown to control muscle development in vitro and are differentially expressed at rest in diabetic skeletal muscle. Therefore, we investigated the expression of these myomiRs, including miR-1, miR-133a, miR-133b and miR-206 in muscle biopsies from vastus lateralis of healthy young males (n = 10) in relation to a hyperinsulinaemic–euglycaemic clamp as well as acute endurance exercise before and after 12 weeks of endurance training. The subjects increased their endurance capacity, VO2max (l min−1) by 17.4% (P < 0.001), and improved insulin sensitivity by 19% (P < 0.01). While myomiR expression remained stable during a hyperinsulinaemic–euglycaemic clamp, an acute bout of exercise increased mir-1 (P < 0.05) and mir-133a (P < 0.05) expression before, but not after, training. In resting biopsies, endurance training for 12 weeks decreased basal expression of all four myomiRs (P < 0.05). Interestingly, all myomiRs reverted to their pre-training expression levels 14 days after ceasing the training programme. Components of major pathways involved in endurance adaptation such as MAPK and TGF-β were predicted to be targeted by the myomiRs examined. Tested predicted target proteins included Cdc42 and ERK 1/2. Although these proteins were downregulated between post-training period and 2 weeks of cessation, an inverse correlation between myomiR and target proteins was not found. In conclusion, our data suggest myomiRs respond to physiological stimuli, but their role in regulating human skeletal muscle adaptation remains unknown.

A low maximal oxygen consumption (VO2max) is a strong risk factor for premature mortality. Supervised endurance exercise training increases VO2max with a very wide range of effectiveness in humans. Discovering the DNA variants that contribute to this heterogeneity typically requires substantial sample sizes. In the present study, we first use RNA expression profiling to produce a molecular classifier that predicts VO2max training response. We then hypothesized that the classifier genes would harbor DNA variants that contributed to the heterogeneous VO2max response. Two independent preintervention RNA expression data sets were generated (n=41 gene chips) from subjects that underwent supervised endurance training: one identified and the second blindly validated an RNA expression signature that predicted change in VO2max ("predictor" genes). The HERITAGE Family Study (n=473) was used for genotyping. We discovered a 29-RNA signature that predicted VO2max training response on a continuous scale; these genes contained approximately 6 new single-nucleotide polymorphisms associated with gains in VO2max in the HERITAGE Family Study. Three of four novel candidate genes from the HERITAGE Family Study were confirmed as RNA predictor genes (i.e., "reciprocal" RNA validation of a quantitative trait locus genotype), enhancing the performance of the 29-RNA-based predictor. Notably, RNA abundance for the predictor genes was unchanged by exercise training, supporting the idea that expression was preset by genetic variation. Regression analysis yielded a model where 11 single-nucleotide polymorphisms explained 23% of the variance in gains in VO2max, corresponding to approximately 50% of the estimated genetic variance for VO2max. In conclusion, combining RNA profiling with single-gene DNA marker association analysis yields a strongly validated molecular predictor with meaningful explanatory power. VO2max responses to endurance training can be predicted by measuring a approximately 30-gene RNA expression signature in muscle prior to training. The general approach taken could accelerate the discovery of genetic biomarkers, sufficiently discrete for diagnostic purposes, for a range of physiological and pharmacological phenotypes in humans.

Background: Skeletal 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., Methods: We 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., Results: The 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 x 10-8), post-translational modification (P < 9.7 x 10-5) and developmental (P < 1.3 x 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., Conclusions: We 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.

Physical exercise induces a network of alterations in the transcriptome and proteome of the skeletal muscle, resulting in modifications of the muscle physiology. Intriguingly, exercise also transiently induces the production of both reactive oxygen species (ROS) and some inflammatory cytokines in skeletal muscle. In fact, it seems that exercise-induced ROS are able to stimulate cytokine production from skeletal muscle. Despite the initial view that ROS were potentially cell damaging, it now seems possible that these substances have important roles in the regulation of cell signaling. Muscle-derived cytokines, so-called 'myokines', are distinguished from inflammation and instead possess important anti-inflammatory and metabolic properties. In this opinion piece, we suggest that both ROS and myokines are important players in muscle adaptation to exercise.

Genome sequencing projects have provided the substrate for an unimaginable number of biological experiments. Further, genomic technologies such as microarrays and quantitative and exquisitely sensitive techniques such as real-time quantitative polymerase chain reaction have made it possible to reliably generate millions of data points per experiment. The data can be high quality and yield entirely new insights into how gene expression is coordinated under complex physiological situations. It can also be that the data and interpretation are meaningless because of a lack of physiological context or experimental control. Thus, functional genomics is now being applied to study metabolic physiology with varying degrees of success. From the genome sequencing projects we also have the information needed to design chemical tools that can knock down a gene transcript, even distinguishing between splice variants in mammalian cells. Use of such technologies, inspired by nature's endogenous RNAi mechanism-microRNA targeting, comes with significant caveats. While the discipline of Pharmacology taught us last century that inhibitor action specificity is dependent on the concentration used, these experiences have been ignored by users of siRNA technologies. What we provide in this chapter is some considerations and observations from functional genomic studies. We are largely concerned with the phase that follows a microarray study, where a candidate gene is selected for manipulation in a system that is considered to be simpler than the in vivo mammalian tissue and thus the methods discussed largely apply to this cell biology phase. We apologize for not referring to all relevant publications and for any technical considerations we have also failed to factor into our discussion.