Isolation and characterization of muscle stem cells, fibro-adipogenic progenitors, and macrophages from human skeletal muscle biopsies.

Animal models clearly illustrate that the maintenance of skeletal muscle mass depends on the function and interaction of a heterogeneous population of resident and infiltrating mononuclear cells. Several lines of evidence suggest that mononuclear cells also play a role in muscle wasting in humans, and targeting these cells may open new treatment options for intervention or prevention in sarcopenia. Methodological and ethical constraints have perturbed exploration of the cellular characteristics and function of mononuclear cells in human skeletal muscle. Thus, investigations of cellular phenotypes often depend on immunohistochemical analysis of small tissue samples obtained by needle biopsies, which do not match the deep phenotyping of mononuclear cells obtained from animal models. Here, we have developed a protocol for fluorescence-activated cell sorting (FACS), based on single-cell RNA-sequencing data, for quantifying and characterizing mononuclear cell populations in human skeletal muscle. Muscle stem cells, fibro-adipogenic progenitors, and two subsets of macrophages (CD11c+/-) are present in needle biopsies in comparable quantities per milligram tissue to open surgical biopsies. We find that direct cell isolation is preferable due to a substantial shift in transcriptome when using preculture before the FACS procedure. Finally, in vitro validation of the cellular phenotype of muscle stem cells, fibro-adipogenic progenitors, and macrophages confirms population-specific traits. This study demonstrates that mononuclear cell populations can be quantified and subsequently analyzed from needle biopsy material and opens the perspective for future clinical studies of cellular mechanisms in muscle wasting.

Insulin resistance induced by growth hormone is linked to lipolysis and associated with suppressed pyruvate dehydrogenase activity in skeletal muscle: a 2 × 2 factorial, randomised, crossover study in human individuals.

AIMS/HYPOTHESIS: Growth hormone (GH) causes insulin resistance that is linked to lipolysis, but the underlying mechanisms are unclear. We investigated if GH-induced insulin resistance in skeletal muscle involves accumulation of diacylglycerol (DAG) and ceramide as well as impaired insulin signalling, or substrate competition between fatty acids and glucose. METHODS: Nine GH-deficient male participants were randomised and examined in a 2 × 2 factorial design with and without administration of GH and acipimox (an anti-lipolytic compound). As-treated analyses were performed, wherefore data from three visits from two patients were excluded due to incorrect GH administration. The primary outcome was insulin sensitivity, expressed as the AUC of the glucose infusion rate (GIRAUC), and furthermore, the levels of DAGs and ceramides, insulin signalling and the activity of the active form of pyruvate dehydrogenase (PDHa) were assessed in skeletal muscle biopsies obtained in the basal state and during a hyperinsulinaemic-euglycaemic clamp (HEC). RESULTS: Co-administration of acipimox completely suppressed the GH-induced elevation in serum levels of NEFA (GH versus GH+acipimox, p < 0.0001) and abrogated GH-induced insulin resistance (mean GIRAUC [95% CI] [mg min-1 kg-1] during the HEC: control, 595 [493, 718]; GH, 468 [382, 573]; GH+acipimox, 654 [539, 794]; acipimox, 754 [618, 921]; GH vs GH+acipimox: p = 0.004). GH did not significantly change either the accumulation of DAGs and ceramides or insulin signalling in skeletal muscle, but GH antagonised the insulin-stimulated increase in PDHa activity (mean ± SEM [% from the basal state to the HEC]: control, 47 ± 19; GH, -15 ± 21; GH+acipimox, 3 ± 21; acipimox, 57 ± 22; main effect: p = 0.02). CONCLUSIONS/INTERPRETATION: GH-induced insulin resistance in skeletal muscle is: (1) causally linked to lipolysis; (2) not associated with either accumulation of DAGs and ceramides or impaired insulin signalling; (3) likely to involve substrate competition between glucose and lipid intermediates. TRIAL REGISTRATION: ClinicalTrials.gov NCT02782208 FUNDING: The work was supported by the Grant for Growth Innovation (GGI), which was funded by Merck KGaA, Darmstadt, Germany. Graphical abstract.

Substrate metabolism, hormone and cytokine levels and adipose tissue signalling in individuals with type 1 diabetes after insulin withdrawal and subsequent insulin therapy to model the initiating steps of ketoacidosis.

AIMS/HYPOTHESIS: Lack of insulin and infection/inflammation are the two most common causes of diabetic ketoacidosis (DKA). We used insulin withdrawal followed by insulin administration as a clinical model to define effects on substrate metabolism and to test whether increased levels of counter-regulatory hormones and cytokines and altered adipose tissue signalling participate in the early phases of DKA. METHODS: Nine individuals with type 1 diabetes, without complications, were randomly studied twice, in a crossover design, for 5 h followed by 2.5 h high-dose insulin clamp: (1) insulin-controlled euglycaemia (control) and (2) after 14 h of insulin withdrawal in a university hospital setting. RESULTS: Insulin withdrawal increased levels of glucose (6.1 ± 0.5 vs 18.6 ± 0.5 mmol/l), NEFA, 3-OHB (127 ± 18 vs 1837 ± 298 µmol/l), glucagon, cortisol and growth hormone and decreased HCO3- and pH, without affecting catecholamine or cytokine levels. Whole-body energy expenditure, endogenous glucose production (1.55 ± 0.13 vs 2.70 ± 0.31 mg kg-1 min-1), glucose turnover, non-oxidative glucose disposal, lipid oxidation, palmitate flux (73 [range 39-104] vs 239 [151-474] µmol/min), protein oxidation and phenylalanine flux all increased, whereas glucose oxidation decreased. In adipose tissue, Ser473 phosphorylation of Akt and mRNA levels of G0S2 decreased, whereas CGI-58 (also known as ABHD5) mRNA increased. Protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase phosphorylations were unaltered. Insulin therapy decreased plasma glucose concentrations dramatically after insulin withdrawal, without any detectable effect on net forearm glucose uptake. CONCLUSIONS/INTERPRETATION: Release of counter-regulatory hormones and overall increased catabolism, including lipolysis, are prominent features of preacidotic ketosis induced by insulin withdrawal, and dampening of Akt insulin signalling and transcriptional modulation of ATGL activity are involved. The lack of any increase in net forearm glucose uptake during insulin therapy after insulin withdrawal indicates muscle insulin resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT02077348 FUNDING: This study was supported by Aarhus University and the KETO Study Group/Danish Agency for Science Technology and Innovation.

No effect of resveratrol on VLDL-TG kinetics and insulin sensitivity in obese men with nonalcoholic fatty liver disease.

The present study (NCT01446276, ClinicalTrials.gov) assessed long-term effects of high-dose Resveratrol (RSV) on basal and insulin-mediated very low-density lipoprotein triglyceride (VLDL-TG), palmitate and glucose kinetics, and liver fat content in men with nonalcoholic fatty liver disease (NAFLD). Participants (n = 16) were non-diabetic, upper-body obese (BMI > 28 kg/m2 , WHR > 0.9) men with NAFLD who were randomized (1:1) in a double-blinded, placebo-controlled clinical trial to either RSV or placebo (500 mg 3 times daily) for 6 months. Magnetic resonance (MR) spectroscopy, dual-X-ray absorptiometry and MR imaging assessed liver fat content and body composition, respectively. 14 C-labeled VLDL-TG and 3 H-labeled glucose and palmitate tracers, in combination with indirect calorimetry and breath samples, were used to assess kinetics and substrate oxidations during basal and hyperinsulinaemic euglycaemic clamp conditions. RSV did not improve either basal or insulin-mediated VLDL-TG secretion, oxidation or clearance rates, nor did it affect palmitate or glucose turnover. Likewise, no changes in body composition or liver fat content occurred following RSV compared with placebo treatment. Therefore, RSV cannot be recommended for treatment of metabolic abnormalities in NAFLD.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

Effects of insulin-induced hypoglycaemia on lipolysis rate, lipid oxidation and adipose tissue signalling in human volunteers: a randomised clinical study.

AIMS/HYPOTHESIS: The aims of this study were to determine the role of lipolysis in hypoglycaemia and define the underlying intracellular mechanisms. METHODS: Nine healthy volunteers were randomised to treatment order of three different treatments (crossover design). Treatments were: (1) saline control; (2) hyperinsulinaemic hypoglycaemia (HH; i.v. bolus of 0.1 U/kg insulin); and (3) hyperinsulinaemic euglycaemia (HE; i.v. bolus of 0.1 U/kg insulin and 20% glucose). Inclusion criteria were that volunteers were healthy, aged >18 years, had a BMI between 19 and 26 kg/m2, and provided both written and oral informed consent. Exclusion criteria were the presence of a known chronic disease (including diabetes mellitus, epilepsy, ischaemic heart disease and cardiac arrhythmias) and regular use of prescription medication. The data was collected at the medical research facilities at Aarhus University Hospital, Denmark. The primary outcome was palmitic acid flux. Participants were blinded to intervention order, but caregivers were not. RESULTS: Adrenaline (epinephrine) and glucagon concentrations were higher during HH than during both HE and control treatments. NEFA levels and lipid oxidation rates (determined by indirect calorimetry) returned to control levels after 105 min. Palmitate flux was increased to control levels during HH (p = NS) and was more than twofold higher than during HE (overall mean difference between HH vs HE, 114 [95% CI 64, 165 µmol/min]; p < 0.001). In subcutaneous adipose tissue biopsies, we found elevated levels of hormone-sensitive lipase (HSL) and perilipin-1 phosphorylation 30 min after insulin injection during HH compared with both control and HE. There were no changes in the levels of adipose triglyceride lipase (ATGL), comparative gene identification-58 (CGI-58) or G0/G1 switch gene 2 (G0S2) proteins. Insulin-stimulated phosphorylation of Akt and mTOR were unaffected by hypoglycaemia. Expression of the G0S2 gene increased during HE and HH compared with control, without changes in ATGL (also known as PNPLA2) or CGI-58 (also known as ABHD5) mRNA levels. CONCLUSIONS/INTERPRETATION: These findings suggest that NEFAs become a major fuel source during insulin-induced hypoglycaemia and that lipolysis may be an important component of the counter-regulatory response. These effects appear to be mediated by rapid stimulation of protein kinase A (PKA) and HSL, compatible with activation of the ß-adrenergic catecholamine signalling pathway. TRIAL REGISTRATION: ClinicalTrials.gov NCT01919788 FUNDING: : The study was funded by Aarhus University, the Novo Nordisk Foundation and the KETO Study Group/Danish Agency for Science Technology and Innovation (grant no. 0603-00479, to NM).

Inflammation Downregulates UCP1 Expression in Brown Adipocytes Potentially via SIRT1 and DBC1 Interaction.

Brown adipose tissue thermogenesis at the cost of energy is not only important for the development of obesity, but also possesses great promise in anti-obesity treatment. Uncoupling protein 1 (UCP1) expression has been reported to be under control of the intracellular deacetylase SIRT1. Here, we investigated the effect and mechanism of inflammation and sirtuin-1 (SIRT1) activation on the induction of thermogenic genes in immortalized brown adipocytes incubated with LPS or IL1ß and mice with elevated inflammatory tone. In vitro stimulation of brown adipocytes with dibutyryl cyclic adenosine monophosthate (dbcAMP) reduced the expression of deleted in breast cancer-1 (Dbc1) (SIRT1 inhibitor) and increased the Ucp1 expression. Silencing of SIRT1 attenuated dbcAMP induction of Ucp1. In contrast, IL1ß increased the expression of Dbc1 and greatly reduced the induction of Ucp1. Similarly, in vivo studies revealed decreased expression of Ucp1 in brown adipose tissue (BAT) in mice chronically infused with LPS. Resveratrol, a known SIRT1 activator, partly rescued the Ucp1 downregulation by inflammation in both the cell cultures and mice. Here, we describe how the expression of Ucp1 in BAT is controlled via SIRT1 and is reduced under inflammation and can be rescued by SIRT1 activation by resveratrol. We suggest the reduced UCP1 expression under inflammation is mediated by the increased expression of DBC1, which inhibits SIRT1 activity.

Reduced fat mass and increased lean mass in response to 1 year of melatonin treatment in postmenopausal women: A randomized placebo-controlled trial.

OBJECTIVE: Apart from regulating the circadian rhythm, melatonin exerts a variety of actions in the living organism. Among these functions, melatonin is believed to have a positive effect on body weight and energy metabolism. So far, the evidence for this relies mainly on animal models. In this study, we aimed to determine the effects of melatonin on body composition, lipid and glucose metabolism in humans. DESIGN/METHODS: In a double-blind, placebo-controlled study, we randomized 81 postmenopausal women to 1 year of treatment with melatonin (1 or 3 mg nightly) or placebo. Body composition was measured by DXA. Measures were obtained at baseline and after 1 year of treatment along with leptin, adiponectin and insulin. Markers of glucose homeostasis were measured at the end of the study. RESULTS: In response to treatment, fat mass decreased in the melatonin group by 6·9% (95% CI: 1·4%; 12·4%, P = 0·02) compared to placebo. A borderline significant increase in lean mass of 5·2% was found in the melatonin group compared to placebo (3·3%, (IQR:-1·7; 6·2) vs -1·9%, (IQR: -5·7; 5·8), P = 0·08). After adjusting for BMI, lean mass increased by 2·6% (95% CI: 0·1; 5·0, P = 0·04) in the melatonin group. Changes in body weight and BMI did not differ between groups. Adiponectin increased borderline significantly by 21% in the melatonin group compared to placebo (P = 0·08). No significant changes were observed for leptin, insulin or markers of glucose homeostasis. CONCLUSION: Our results suggest a possibly beneficial effect of melatonin on body composition and lipid metabolism as 1 year of treatment reduces fat mass, increases lean mass and is associated with a trend towards an increase in adiponectin.

Erythropoietin does not activate erythropoietin receptor signaling or lipolytic pathways in human subcutaneous white adipose tissue in vivo.

BACKGROUND: Erythropoietin (Epo) exerts direct effects on white adipose tissue (WAT) in mice in addition to its erythropoietic effects, and in humans Epo increases resting energy expenditure and affect serum lipid levels, but direct effects of Epo in human WAT have not been documented. We therefore investigated the effects of acute and prolonged Epo exposure on human WAT in vivo. METHOD: Data were obtained from two clinical trials: 1) acute Epo exposure (rHuEpo, 400 IU/kg) followed by WAT biopsies after 1 h and 2) 10 weeks treatment with the erythropoiesis-stimulating agent (ESA) Darbepoietin-alpha. Biopsies were analyzed by PCR for Epo receptor (Epo-R) mRNA. A new and highly specific antibody (A82, Amgen) was used to evaluate the presence of Epo-R by western blot analysis in addition to Epo-R signaling proteins (Akt, STAT5, p70s6k, LYN, and p38MAPK), activation of lipolytic pathways (ATGL, HSL, CGI-58, G0S2, Perilipin, Cidea, Cidec, AMPK, and ACC), and mitochondrial biogenesis (VDAC, HSP90, PDH, and SDHA). RESULTS: No evidence of in vivo activation of the Epo-R in WAT could be documented despite detectable levels of Epo-R mRNA. CONCLUSION: Thus, in contradiction to animal studies, Epo treatment within a physiological relevant range in humans does not exert direct effects in a subcutaneous WAT.

Prolonged erythropoietin treatment does not impact gene expression in human skeletal muscle.

INTRODUCTION: We tested for the presence of erythropoietin receptor (Epo-R) in human skeletal muscle and alterations in gene expression after prolonged use of an erythropoiesis-stimulating agent (ESA). METHODS: Nine healthy men were treated with ESA for 10 weeks (darbepoietin alfa). Muscle biopsies were collected before and after treatment. Alterations in gene expression were evaluated by gene array. Western blot and PCR analysis were used to test for Epo-R presence in human skeletal muscle. RESULTS: Very low Epo-R mRNA levels were found, but a new and sensitive antibody did not identify Epo-R protein in human skeletal muscle. The between-subject variation in skeletal muscle gene expression was greater than that observed in response to prolonged ESA treatment. CONCLUSIONS: Erythropoietin is unlikely to exert direct effects in human skeletal muscle due to a lack of Epo-R protein. Furthermore, prolonged ESA treatment does not seem to exert either direct or indirect effects on skeletal muscle gene expression.

Gene expression in skeletal muscle after an acute intravenous GH bolus in human subjects: identification of a mechanism regulating ANGPTL4.

Growth hormone (GH) acutely stimulates lipolysis and fat oxidation, a process that operates postabsorptively and involves activation of the JAK-STAT pathway in the target tissue; no in vivo data exist regarding subsequent GH-regulated gene transcription. We obtained serum samples and muscle biopsies in human subjects before and 2 h after administration of a GH bolus. A significant (~75%) elevation in serum FFA levels was recorded post GH. Microarray identified 79 GH-regulated genes in muscle. With qRT-PCR, we then examined the expression of selected genes in the presence and absence of glucose-induced suppression of lipolysis. Four genes involved in the JAK-STAT5 signaling pathway were regulated by GH, including SOCS1-3 and CISH, in addition to three genes associated with insulin action: NFκB1A, PIK3C2B, and PRKAG2. The gene encoding ANGPTL4, a protein involved in lipolysis and suppression of LPL activity, exhibited the most pronounced upregulation (5.6-fold) after GH, which was abrogated by concomitant suppression of lipolysis. Therefore, the GH-induced stimulation of ANGPTL4 gene expression seems secondary to induction of lipolysis. This new concept implies that abundant supply of circulating FFA decreases the need for alternative triglyceride-derived FFA through distinct inhibition of LPL mediated by increased ANGPTL4 gene expression in human muscle.

Whole body metabolic effects of prolonged endurance training in combination with erythropoietin treatment in humans: a randomized placebo controlled trial.

UNLABELLED: Erythropoietin (Epo) administration improves aerobic exercise capacity and insulin sensitivity in renal patients and also increases resting energy expenditure (REE). Similar effects are observed in response to endurance training. The aim was to compare the effects of endurance training with erythropoiesis-stimulating agent (ESA) treatment in healthy humans. Thirty-six healthy untrained men were randomized to 10 wk of either: 1) placebo (n = 9), 2) ESA (n = 9), 3) endurance training (n = 10), or 4) ESA and endurance training (n = 8). In a single-blinded design, ESA/placebo was injected one time weekly. Training consisted of biking for 1 h at 65% of wattmax three times per week. Measurements performed before and after the intervention were as follows: body composition, maximal oxygen uptake, insulin sensitivity, REE, and palmitate turnover. Uncoupling protein 2 (UCP2) mRNA levels were assessed in skeletal muscle. Fat mass decreased after training (P = 0.003), whereas ESA induced a small but significant increase in intrahepatic fat (P = 0.025). Serum free fatty acid (FFA) levels and palmitate turnover decreased significantly in response to training, whereas the opposite pattern was found after ESA. REE corrected for lean body mass increased in response to ESA and training, and muscle UCP2 mRNA levels increased after ESA (P = 0.035). Insulin sensitivity increased only after training (P = 0.011). IN CONCLUSION: 1) insulin sensitivity is not improved after ESA treatment despite improved exercise capacity, 2) the calorigenic effects of ESA may be related to increased UCP2 gene expression in skeletal muscle, and 3) training and ESA exert opposite effects on lipolysis under basal conditions, increased FFA levels and liver fat fraction was observed after ESA treatment.

The effect of high-dose vitamin D supplementation on calciotropic hormones and bone mineral density in obese subjects with low levels of circulating 25-hydroxyvitamin d: results from a randomized controlled study.

Low levels of 25-hydroxyvitamin D (25OHD) are associated with increased bone turnover and risk of fractures. Plasma 25OHD is inversely related to body mass index, and vitamin D deficiency is common in obesity. We aimed to determine whether vitamin D supplementation affects bone turnover and bone mineral density (BMD) in obese subjects. Fifty-two healthy obese men and women aged 18-50 years with plasma 25OHD levels below 50 nmol/L were randomized to 7,000 IU of cholecalciferol daily or placebo for 26 weeks. We measured plasma levels of 25OHD, parathyroid hormone (PTH), and markers of bone turnover, as well as BMD at the hip, spine, forearm, and whole body. Compared with placebo, treatment with cholecalciferol increased mean plasma 25OHD from 35 to 110 nmol/L (p < 0.00001) and significantly decreased PTH (p < 0.05). BMD increased significantly at the forearm by 1.6 ± 0.7 % (p = 0.03). The bone resorption marker C-terminal telopetide of type 1 collagen (CTX) decreased borderline significantly in the cholecalciferol group compared with the placebo group (p = 0.07). Changes in plasma 25OHD correlated inversely with changes in plasma levels of bone-specific alkaline phosphatase (r = -0.38, p = 0.01) and CTX (r = -0.33, p = 0.03). Changes in CTX correlated inversely with changes in spine BMD (r = -0.45, p = 0.04). Increasing circulating 25OHD levels by cholecalciferol treatment is of importance to bone health in young obese subjects as increased levels of 25OHD are associated with a decrease in both PTH and bone turnover and with an increase in BMD at the forearm.

Acute exercise increases circulating inflammatory markers in overweight and obese compared with lean subjects.

The primary aim of the present study was to investigate if overweight and obese compared to lean individuals displayed differences in levels of inflammatory markers in circulation, skeletal muscle (SM) and adipose tissue (AT) after acute exercise. Fifteen lean (BMI: 22.4 ± 2 kg/m(2)) and 16 overweight or obese (BMI 31.8 ± 3 kg/m(2)) individuals were included in the study. They completed 120 min of ergometer bicycling at 55-60 % of maximal heart rate. Blood samples were obtained at baseline (T = 0), after 60 (T = 60) and 120 min of exercise (T = 120), and analyzed using an ELISA method. SM and AT biopsies were obtained at T0 and T120, and mRNA expression was investigated using a Real-time RT-PCR method. Circulating IL-6, TNF-α, IL-8, and IL-15 all increased at T = 120 min (p < 0.01). Circulating IL-6 and IL-15 increased in all subjects at T = 120 min (p < 0.01), but only the increase of IL-6 was significantly higher in overweight and obese subjects (p < 0.05), and was positively correlated with body fat percentage (p < 0.01). Circulating IL-8 and TNF-α were increased in overweight and obese (p < 0.05) but not in lean subjects. Acute exercise induced an increase in IL-6 mRNA expression in SM biopsies (p < 0.05). IL-6 as well as adiponectin mRNA expression was increased in AT biopsies (p < 0.05); however, no effect of body weight was found. The findings suggest that the systemic inflammatory response to acute exercise is different in lean compared to overweight and obese subjects, with a more pronounced increase in inflammatory markers (e.g., IL-6, IL-8, and TNF-α) in overweight and obese individuals.

Whole-Body and Forearm Muscle Protein Metabolism in Patients With Acromegaly Before and After Treatment.

BACKGROUND: Active acromegaly is characterized by increased lean body mass, but the mechanisms underlying the protein anabolic effect are unclear. AIM: To study if active acromegaly induces reversible changes in whole-body and skeletal muscle protein kinetics. PATIENTS AND METHODS: Eighteen patients with acromegaly were investigated before and 47 ± 10 weeks after disease control by surgery (n = 8) and/or medical treatment (n = 10). Labeled phenylalanine and tyrosine tracers were employed to assess whole-body and regional forearm muscle protein kinetics. Intramyocellular protein signaling was assessed in skeletal muscle biopsies, and whole-body dual-energy X-ray absorptiometry scan and indirect calorimetry assessed lean body mass (LBM) and resting energy expenditure, respectively. RESULTS: Disease control induced a 7% decrease in lean body mass (P < .000) and a 14% decrease in LBM-adjusted energy expenditure. Whole-body phenylalanine breakdown decreased after disease control (P = .005) accompanied by a decrease in the degradation of phenylalanine to tyrosine (P = .005) and a decrease in whole-body phenylalanine synthesis (P = .030). Skeletal muscle protein synthesis tended to decrease after disease control (P = .122), whereas the muscle protein breakdown (P = .437) and muscle protein loss were unaltered (P = .371). Unc-51 like autophagy activating kinase 1 phosphorylation, an activator of protein breakdown, increased after disease control (P = .042). CONCLUSIONS: Active acromegaly represents a reversible high flux state in which both whole-body protein breakdown and synthesis are increased, whereas forearm muscle protein kinetics are unaltered. Future studies are needed to decipher the link between protein kinetics and the structure and function of the associated growth hormone-induced increase in lean body mass.

Sex chromosome aneuploidies give rise to changes in the circular RNA profile: A circular transcriptome-wide study of Turner and Klinefelter syndrome across different tissues.

Purpose: The landscape of circular RNAs (circRNAs), an important class of non-coding RNAs that regulate gene expression, has never been described in human disorders of sex chromosome aneuploidies. We profiled circRNAs in Turner syndrome females (45,X; TS) and Klinefelter syndrome males (47,XXY; KS) to investigate how circRNAs respond to a missing or an extra X chromosome. Methods: Samples of blood, muscle and fat were collected from individuals with TS (n = 33) and KS (n = 22) and from male (n = 16) and female (n = 44) controls. CircRNAs were identified using a combination of circRNA identification pipelines (CIRI2, CIRCexplorer2 and circRNA_finder). Results: Differential expression of circRNAs was observed throughout the genome in TS and KS, in all tissues. The host-genes from which several of these circRNAs were derived, were associated with known phenotypic traits. Furthermore, several differentially expressed circRNAs had the potential to capture micro RNAs that targeted protein-coding genes with altered expression in TS and KS. Conclusion: Sex chromosome aneuploidies introduce changes in the circRNA transcriptome, demonstrating that the genomic changes in these syndromes are more complex than hitherto thought. CircRNAs may help explain some of the genomic and phenotypic traits observed in these syndromes.

Reversible insulin resistance in muscle and fat unrelated to the metabolic syndrome in patients with acromegaly.

BACKGROUND: Patients with active acromegaly exhibit insulin resistance despite a lean phenotype whereas controlled disease improves insulin sensitivity and increases fat mass. The mechanisms underlying this paradox remain elusive, but growth hormone (GH)-induced lipolysis plays a central role. The aim of the study was to investigative the molecular mechanisms of insulin resistance dissociated from obesity in patients with acromegaly. METHODS: In a prospective study, twenty-one patients with newly diagnosed acromegaly were studied at diagnosis and after disease control obtained by either surgery alone (n=10) or somatostatin analogue (SA) treatment (n=11) with assessment of body composition (DXA scan), whole body and tissue-specific insulin sensitivity and GH and insulin signalling in adipose tissue and skeletal muscle. FINDINGS: Disease control of acromegaly significantly reduced lean body mass (p<0.001) and increased fat mass (p<0.001). At diagnosis, GH signalling (pSTAT5) was constitutively activated in fat and enhanced expression of GH-regulated genes (CISH and IGF-I) were detected in muscle and fat. Insulin sensitivity in skeletal muscle, liver and adipose tissue increased after disease control regardless of treatment modality. This was associated with enhanced insulin signalling in both muscle and fat including downregulation of phosphatase and tensin homolog (PTEN) together with reduced signalling of GH and lipolytic activators in fat. INTERPRETATION: In conclusion, the study support that uncontrolled lipolysis is a major feature of insulin resistance in active acromegaly, and is characterized by upregulation of PTEN and suppression of insulin signalling in both muscle and fat. FUNDING: This work was supported by a grant from the Independent Research Fund, Denmark (7016-00303A) and from the Alfred Benzon Foundation, Denmark.

ADRA1A-Gαq signalling potentiates adipocyte thermogenesis through CKB and TNAP.

Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis1. Aside from cAMP signalling downstream of ß-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α1-adrenergic receptor (AR) and ß3-AR signalling induces the expression of thermogenic genes of the futile creatine cycle2,3, and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α1-AR subtype (ADRA1A) and Gαq to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gαq and Gαs signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A-Gαq-futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis.

Effect of industrially produced trans fat on markers of systemic inflammation: evidence from a randomized trial in women.

Consumption of industrially produced trans fatty acids (IP-TFA) has been positively associated with systemic markers of low-grade inflammation and endothelial dysfunction in cross-sectional studies, but results from intervention studies are inconclusive. Therefore, we conducted a 16 week double-blind parallel intervention study with the objective to examine the effect of IP-TFA intake on biomarkers of inflammation, oxidative stress, and endothelial dysfunction. Fifty-two healthy overweight postmenopausal women (49 completers) were randomly assigned to receive either partially hydrogenated soybean oil (15.7 g/day IP-TFA) or control oil without IP-TFA. After 16 weeks, IP-TFA intake increased baseline-adjusted serum tumor necrosis factor (TNF) α by 12% [95% confidence interval (CI): 5-20; P = 0.002] more in the IP-TFA group compared with controls. Plasma soluble TNF receptors 1 and 2 were also increased by IP-TFA [155 pg/ml (CI: 63-247); P < 0.001 and 480 pg/ml (CI: 72-887); P = 0.02, respectively]. Serum C-reactive protein, interleukin (IL) 6 and adiponectin and subcutaneous abdominal adipose tissue mRNA expression of IL6, IL8, TNFα, and adiponectin as well as ceramide content were not affected by IP-TFA, nor was urinary 8-iso-prostaglandin-F(2α). In conclusion, this dietary trial indicates that the mechanisms linking dietary IP-TFA to cardiovascular disease may involve activation of the TNFα system.

Differential effects of dietary protein sources on postprandial low-grade inflammation after a single high fat meal in obese non-diabetic subjects.

BACKGROUND: Obesity is a state of chronic low-grade inflammation. Chronic low-grade inflammation is associated with the pathophysiology of both type-2 diabetes and atherosclerosis. Prevention or reduction of chronic low-grade inflammation may be advantageous in relation to obesity related co-morbidity. In this study we investigated the acute effect of dietary protein sources on postprandial low-grade inflammatory markers after a high-fat meal in obese non-diabetic subjects. METHODS: We conducted a randomized, acute clinical intervention study in a crossover design. We supplemented a fat rich mixed meal with one of four dietary proteins - cod protein, whey isolate, gluten or casein. 11 obese non-diabetic subjects (age: 40-68, BMI: 30.3-42.0 kg/m2) participated and blood samples were drawn in the 4 h postprandial period. Adiponectin was estimated by ELISA methods and cytokines were analyzed by multiplex assay. RESULTS: MCP-1 and CCL5/RANTES displayed significant postprandial dynamics. CCL5/RANTES initially increased after all meals, but overall CCL5/RANTES incremental area under the curve (iAUC) was significantly lower after the whey meal compared with the cod and casein meals (P = 0.0053). MCP-1 was initially suppressed after all protein meals. However, the iAUC was significantly higher after whey meal compared to the cod and gluten meals (P = 0.04). CONCLUSION: We have demonstrated acute differential effects on postprandial low grade inflammation of four dietary proteins in obese non-diabetic subjects. CCL5/RANTES initially increased after all meals but the smallest overall postprandial increase was observed after the whey meal. MCP-1 was initially suppressed after all 4 protein meals and the whey meal caused the smallest overall postprandial suppression. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT00863564.