No effect of 10 weeks erythropoietin treatment on lipid oxidation in healthy men.

Studies indicate that erythropoietin (EPO) has effect on lipid and energy metabolism; however, the impact of EPO on lipid oxidation in vivo has not been well documented. Here, we evaluate whether long-term erythropoiesis-stimulating agent (ESA) treatment affects the oxidation of plasma very low-density lipoprotein triglycerides (VLDL-TG) fatty acids (FA), plasma free fatty acids (FFA) and non-plasma (residual) FA in healthy, young, sedentary men. Infusion of [1-14C]VLDL-TG and [9,10-3H]palmitate was used in combination with indirect calorimetry to assess resting lipid fuel utilization and kinetics, and resting energy expenditure (REE) before and after 10 weeks of ESA exposure compared with placebo. REE increased significantly during ESA compared with placebo (P = 0.023, RM-ANOVA). Oxidation rates of VLDL-TG FA, FFA, and residual FA remained unchanged during ESA compared with placebo. The relative contribution of the lipid stores was greatest for FFA (47.1%) and the total lipid oxidation rate and was not significantly different between ESA and placebo-treated subjects. We conclude that long-term ESA treatment of healthy young men increases REE but does not alter the oxidation rates of plasma and non-plasma FA sources.

Effect of a Low-Carbohydrate High-Fat Diet and a Single Bout of Exercise on Glucose Tolerance, Lipid Profile and Endothelial Function in Normal Weight Young Healthy Females.

Low-carbohydrate-high-fat (LCHF) diets are efficient for weight loss, and are also used by healthy people to maintain bodyweight. The main aim of this study was to investigate the effect of 3-week energy-balanced LCHF-diet, with >75 percentage energy (E%) from fat, on glucose tolerance and lipid profile in normal weight, young, healthy women. The second aim of the study was to investigate if a bout of exercise would prevent any negative effect of LCHF-diet on glucose tolerance. Seventeen females participated, age 23.5 ± 0.5 years; body mass index 21.0 ± 0.4 kg/m2, with a mean dietary intake of 78 ± 1 E% fat, 19 ± 1 E% protein and 3 ± 0 E% carbohydrates. Measurements were performed at baseline and post-intervention. Fasting glucose decreased from 4.7 ± 0.1 to 4.4 mmol/L (p < 0.001) during the dietary intervention whereas fasting insulin was unaffected. Glucose area under the curve (AUC) and insulin AUC did not change during an OGTT after the intervention. Before the intervention, a bout of aerobic exercise reduced fasting glucose (4.4 ± 0.1 mmol/L, p < 0.001) and glucose AUC (739 ± 41 to 661 ± 25, p = 0.008) during OGTT the following morning. After the intervention, exercise did not reduce fasting glucose the following morning, and glucose AUC during an OGTT increased compared to the day before (789 ± 43 to 889 ± 40 mmol/L∙120min-1, p = 0.001). AUC for insulin was unaffected. The dietary intervention increased total cholesterol (p < 0.001), low-density lipoprotein (p ≤ 0.001), high-density lipoprotein (p = 0.011), triglycerides (p = 0.035), and free fatty acids (p = 0.021). In conclusion, 3-week LCHF-diet reduced fasting glucose, while glucose tolerance was unaffected. A bout of exercise post-intervention did not decrease AUC glucose as it did at baseline. Total cholesterol increased, mainly due to increments in low-density lipoprotein. LCHF-diets should be further evaluated and carefully considered for healthy individuals.

Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes.

The lipolytic effects of growth hormone (GH) have been known for half a century and play an important physiological role for substrate metabolism during fasting. In addition, sustained GH-induced lipolysis is causally linked to insulin resistance. However, the underlying molecular mechanisms remain elusive. In the present study, we obtained experimental data in human subjects and used human adipose-derived stromal vascular cells (hADSCs) as a model system to elucidate GH-triggered molecular signaling that stimulates adipose tissue lipolysis and insulin resistance in human adipocytes. We discovered that GH downregulates the expression of fat-specific protein (FSP27), a negative regulator of lipolysis, by impairing the transcriptional ability of the master transcriptional regulator, peroxisome proliferator-activated receptor-γ (PPARγ) via MEK/ERK activation. Ultimately, GH treatment promotes phosphorylation of PPARγ at Ser273 and causes its translocation from nucleus to the cytosol. Surprisingly, FSP27 overexpression inhibited PPARγ Ser273 phosphorylation and promoted its nuclear retention. GH antagonist treatment had similar effects. Our study identifies a novel signaling mechanism by which GH transcriptionally induces lipolysis via the MEK/ERK pathway that acts along PPARγ-FSP27 in human adipose tissue.

Molecular adaptations in human subcutaneous adipose tissue after ten weeks of endurance exercise training in healthy males.

Endurance exercise training induces adaptations in metabolically active organs, but adaptations in human subcutaneous adipose tissue (scAT) remains incompletely understood. On the basis of animal studies, we hypothesized that endurance exercise training would increase the expression of proteins involved in lipolysis and glucose uptake in scAT. To test these hypotheses, 19 young and healthy males were randomized to either endurance exercise training (TR; age 18-24 yr; BMI 19.0-25.4 kg/m2) or a nonexercising control group (CON; age 21-35 yr; BMI 20.5-28.8 kg/m2). Abdominal subcutaneous fat biopsies and blood were obtained at rest before and after intervention. By using Western blotting and PCR, we determined expression of lipid droplet-associated proteins, various proteins involved in substrate metabolism, and mRNA abundance of cell surface G protein-coupled receptors (GPCRs). Adipose tissue insulin sensitivity was determined from fasting plasma insulin and nonesterified fatty acids (adipose tissue insulin resistance index; Adipo-IR). Adipo-IR improved in TR compared with CON ( P = 0.03). This was accompanied by increased insulin receptor (IR) protein expression in scAT with a 1.54-fold (SD 0.79) change from baseline in TR vs. 0.85 (SD 0.30) in CON ( P = 0.007). Additionally, hexokinase II (HKII) and succinate dehydrogenase complex subunit A (SDHA) protein increased in TR compared with CON ( P = 0.006 and P = 0.04, respectively). We did not observe changes in lipid droplet-associated proteins or mRNA abundance of GPCRs. Collectively, 10 weeks of endurance exercise training improved adipose tissue insulin sensitivity, which was accompanied by increased IR, HKII, and SDHA protein expression in scAT. We suggest that these adaptations contribute to an improved metabolic flexibility. NEW & NOTEWORTHY This study is the first to investigate the molecular adaptations in human subcutaneous adipose tissue (scAT) after endurance exercise training compared with a nonexercising control group. We show that endurance exercise training improves insulin sensitivity in human scAT, and this is accompanied by increased expression of insulin receptor, hexokinase II, and succinate dehydrogenase complex subunit A. Collectively, our data suggest that endurance exercise training induces molecular adaptations in human scAT, which may contribute to an improved metabolic flexibility.

Protein intake in the early recovery period after exhaustive exercise improves performance the following day.

The aim of the present study was to investigate the effect of protein and carbohydrate ingestion during early recovery from exhaustive exercise on performance after 18-h recovery. Eight elite cyclists (V̇o2max: 74.0 ± 1.6 ml·kg-1·min-1) completed two exercise and diet interventions in a double-blinded, randomized, crossover design. Participants cycled first at 73% of V̇o2max (W73%) followed by 1-min intervals at 90% of V̇o2max until exhaustion. During the first 2 h of recovery, participants ingested either 1.2 g carbohydrate·kg-1·h-1 (CHO) or 0.8 g carbohydrate + 0.4 g protein·kg-1·h-1 (CHO + PROT). The diet during the remaining recovery period was similar for both interventions and adjusted to body weight. After an 18-h recovery, cycling performance was assessed with a 10-s sprint test, 30 min of cycling at W73%, and a cycling time trial (TT). The TT was 8.5% faster (41:53 ± 1:51 vs. 45:26 ± 1:32 min; P < 0.03) after CHO + PROT compared with CHO. Mean power output during the sprints was 3.7% higher in CHO + PROT compared with CHO (1,063 ± 54 vs. 1,026 ± 53 W; P = 0.01). Nitrogen balance in the recovery period was negative in CHO and neutral in CHO + PROT (-82.4 ± 11.5 vs. 7.0 ± 15.4 mg/kg; P < 0.01). In conclusion, TT and sprint performances were improved 18 h after exhaustive cycling by CHO + PROT supplementation during the first 2 h of recovery compared with isoenergetic CHO supplementation. Our results indicate that intake of carbohydrate plus protein after exhaustive endurance exercise more rapidly converts the body from a catabolic to an anabolic state than carbohydrate alone, thus speeding recovery and improving subsequent cycling performance.NEW & NOTEWORTHY Prolonged high intensity endurance exercise depends on glycogen utilization and high oxidative capacity. Still, exhaustion develops and effective recovery strategies are required to compete in multiday stage races. We show that coingestion of protein and carbohydrate during the first 2 h of recovery is superior to isoenergetic intake of carbohydrate to stimulate recovery, and improves both endurance time-trial and 10-s sprint performance the following day in elite cyclists.

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.

Lipid droplet size and location in human skeletal muscle fibers are associated with insulin sensitivity.

In skeletal muscle, an accumulation of lipid droplets (LDs) in the subsarcolemmal space is associated with insulin resistance, but the underlying mechanism is not clear. We aimed to investigate how the size, number, and location of LDs are associated with insulin sensitivity and muscle fiber types and are regulated by aerobic training and treatment with an erythropoiesis-stimulating agent (ESA) in healthy young untrained men. LD analyses were performed by quantitative transmission electron microscopy, and insulin sensitivity was assessed by a hyperinsulinemic-euglycemic clamp. At baseline, we found that only the diameter (and not the number) of individual subsarcolemmal LDs was negatively associated with insulin sensitivity (R2 = 0.20, P = 0.03, n = 29). Despite 34% (P = 0.004) fewer LDs, the diameter of individual subsarcolemmal LDs was 20% (P = 0.0004) larger in type 2 fibers than in type 1 fibers. Furthermore, aerobic training decreased the size of subsarcolemmal LDs in the type 2 fibers, and ESA treatment lowered the number of both intermyofibrillar and subsarcolemmal LDs in the type 1 fibers. In conclusion, the size of individual subsarcolemmal LDs may be involved in the mechanism by which LDs are associated with insulin resistance in skeletal muscle.

Increased VLDL-TG Fatty Acid Storage in Skeletal Muscle in Men With Type 2 Diabetes.

Context: Lipoprotein lipase (LPL) activity is considered the rate-limiting step of very-low-density-lipoprotein triglycerides (VLDL-TG) tissue storage, and has been suggested to relate to the development of obesity as well as insulin resistance and type 2 diabetes. Objective: The objective of the study was to assess the relationship between the quantitative storage of VLDL-TG fatty acids and LPL activity and other storage factors in muscle and adipose tissue. In addition, we examine whether such relations were influenced by type 2 diabetes. Design: We recruited 23 men (12 with type 2 diabetes, 11 nondiabetic) matched for age and body mass index. Postabsorptive VLDL-TG muscle and subcutaneous adipose tissue (abdominal and leg) quantitative storage was measured using tissue biopsies in combination with a primed-constant infusion of ex vivo triolein labeled [1-14C]VLDL-TG and a bolus infusion of ex vivo triolein labeled [9,10-3H]VLDL-TG. Biopsies were analyzed for LPL activity and cellular storage factors. Results: VLDL-TG storage rate was significantly greater in men with type 2 diabetes compared with nondiabetic men in muscle tissue (P = 0.02). We found no significant relationship between VLDL-TG storage rate and LPL activity or other storage factors in muscle or adipose tissue. However, LPL activity correlated with fractional VLDL-TG storage in abdominal fat (P = 0.04). Conclusions: Men with type 2 diabetes have increased VLDL-TG storage in muscle tissue, potentially contributing to increased intramyocellular triglyceride and ectopic lipid deposition. Neither muscle nor adipose tissue storage rates were related to LPL activity. This argues against LPL as a rate-limiting step in the postabsorptive quantitative storage of VLDL-TG.

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.

Insulin sensitivity, body composition and adipose depots following 12 w combined endurance and strength training in dysglycemic and normoglycemic sedentary men.

CONTEXT: Insulin resistance and dysglycemia are associated with physical inactivity and adiposity, and may be improved by exercise. OBJECTIVE: Investigate the effect of exercise on insulin sensitivity, body composition and adipose depots in sedentary men with (n = 11) or without (n = 11) overweight and dysglycemia. MATERIAL AND METHODS: Euglycemic-hyperinsulinemic clamp, ankle-to-neck MRI, MRS, muscle and adipose tissue biopsies before and after 12 weeks combined strength and endurance exercise. RESULTS: Insulin sensitivity, VO2max, strength, whole-body and muscle fat content, and abdominal adipose depots were improved without obvious differences between normo- and dysglycemic men. Hepatic fat, waist circumference and subcutaneous adipose tissue were reduced in the dysglycemic group. For both groups plasma adiponectin was reduced, whereas IL-6 was unchanged. Visceral fat was preferentially lost compared with other adipose depots. DISCUSSION AND CONCLUSION: Body composition, fat distribution and insulin sensitivity improved following training in sedentary middle-aged men with and without dysglycemia.

Impaired Insulin Suppression of VLDL-Triglyceride Kinetics in Nonalcoholic Fatty Liver Disease.

CONTEXT: Nonalcoholic fatty liver disease (NAFLD) is associated with glucose and lipid metabolic abnormalities. However, insulin suppression of very low-density lipoprotein-triglyceride (VLDL-TG) kinetics is not fully understood. OBJECTIVE: The objective of the study was to determine VLDL-TG, glucose, and palmitate kinetics during fasting and hyperinsulinemia in men with (NAFLD+) and without NAFLD (NAFLD−). DESIGN: Twenty-seven nondiabetic, upper-body obese (waist to hip ratio > 0.9, body mass index > 28 kg/m2) men, 18 NAFLD+, and nine NAFLD− determined by magnetic resonance spectroscopy were enrolled.14C-labeled VLDL-TG and 3H-labeled glucose and palmitate tracers were applied in combination with indirect calorimetry and breath samples to assess kinetics and substrate oxidations postabsorptively and during a hyperinsulinemic-euglycemic clamp. Dual-X-ray absorptiometry and magnetic resonance imaging assessed body composition. RESULTS: Liver fat content was greater in NAFLD+ than NAFLD− men (21.0% vs 3.7%), even though body composition, metabolites (except triglycerides), and insulin were similar in the groups. Insulin suppression of VLDL-TG secretion (P = .0001), oxidation (P = .0003), and concentration (P= .008) as well as percentage decreases were lower in NAFLD+ than NAFLD− men (secretion: 31.9% ± 17.2% vs 64.7% ± 19.9%; oxidation: −9.0% ± 24.7% vs 46.5% ± 36.6%; concentration: 11.9% ± 20.7% vs 56.2% ± 22.9%, all P < .001). Likewise, lower insulin suppression of very low-density lipoprotein particle size was present in NAFLD+ than NAFLD− men (P = .0002). Conversely, insulin suppression of endogenous glucose production was similar in the groups. CONCLUSIONS: Compared with endogenous glucose production, the inability of NAFLD+ men to suppress VLDL-TG kinetics to compensate for the increased liver fat content seems to be an early pathophysiological manifestation of male NAFLD+. These data suggest therapeutic targets reducing liver fat content may ameliorate metabolic abnormalities associated with NAFLD and presumably diabetes.

Satellite cell response to erythropoietin treatment and endurance training in healthy young men.

KEY POINT: Erythropoietin (Epo) treatment may induce myogenic differentiation factor (MyoD) expression and prevent apoptosis in satellite cells (SCs) in murine and in vitro models. Endurance training stimulates SC proliferation in vivo in murine and human skeletal muscle. In the present study, we show, in human skeletal muscle, that treatment with an Epo-stimulating agent (darbepoetin-α) in vivo increases the content of MyoD(+) SCs in healthy young men. Moreover, we report that Epo receptor mRNA is expressed in adult human SCs, suggesting that Epo may directly target SCs through ligand-receptor interaction. Moreover, endurance training, but not Epo treatment, increases the SC content in type II myofibres, as well as the content of MyoD(+) SCs. Collectively, our results suggest that Epo treatment can regulate human SCs in vivo, supported by Epo receptor mRNA expression in human SCs. In effect, long-term Epo treatment during disease conditions involving anaemia may impact SCs and warrants further investigation. Satellite cell (SC) proliferation is observed following erythropoitin treatment in vitro in murine myoblasts and endurance training in vivo in human skeletal muscle. The present study aimed to investigate the effects of prolonged erythropoiesis-stimulating agent (ESA; darbepoetin-α) treatment and endurance training, separately and combined, on SC quantity and commitment in human skeletal muscle. Thirty-five healthy, untrained men were randomized into four groups: sedentary-placebo (SP, n = 9), sedentary-ESA (SE, n = 9), training-placebo (TP, n = 9) or training-ESA (TE, n = 8). ESA/placebo was injected once weekly and training consisted of ergometer cycling three times a week for 10 weeks. Prior to and following the intervention period, blood samples and muscle biopsies were obtained and maximal oxygen uptake (V̇O2, max) was measured. Immunohistochemical analyses were used to quantify fibre type specific SCs (Pax7(+)), myonuclei and active SCs (Pax7(+)/MyoD(+)). ESA treatment led to elevated haematocrit, whereas endurance training increased V̇O2, max. Endurance training led to an increase in SCs associated with type II fibres (P < 0.05), whereas type I fibres showed no changes. Both ESA treatment and endurance training increased Pax7(+)/MyoD(+) cells, whereas only ESA treatment increased the total content of MyoD(+) cells. Epo-R mRNA presence in adult SC was tested with real-time RT-PCR using fluorescence-activated cell sorting (CD56(+)/CD45(-)/CD31(-)) to isolate cells from a human rectus abdominis muscle and was found to be considerably higher than in whole muscle. In conclusion, endurance training and ESA treatment may separately stimulate SC commitment to the myogenic program. Furthermore, ESA-treatment may alter SC activity by direct interaction with the Epo-R expressed on SCs.

Comparative Effects of Aerobic Training and Erythropoietin on Oxygen Uptake in Untrained Humans.

Sieljacks, P, Thams, L, Nellemann, B, Larsen, MS, Vissing, K, and Christensen, B. Comparative effects of aerobic training and erythropoietin on oxygen uptake in untrained humans. J Strength Cond Res 30(8): 2307-2317, 2016-The present study examines responses to 10 weeks of aerobic training and/or erythropoiesis-stimulating agent (ESA) treatment on maximal oxygen uptake (V[Combining Dot Above]O2max). Thirty-six healthy, untrained men were randomly assigned to sedentary-placebo (n = 9), sedentary-ESA (SE) (n = 9), training-placebo (TP) (n = 10), or training-ESA (TE) (n = 8). The participants were treated subcutaneously once weekly with ESA (darbepoietin-α, week 1-3; 40 µg and week 4-10; 20 µg) or a placebo for 10 weeks. The training consisted of supervised cycling 3 times per week for 1 hour at an average of 65% of maximal watt, with a progressive overload during the intervention period. V[Combining Dot Above]O2max, wattmax, and hematological values were measured throughout the study. In addition, the total training workload and estimated energy consumption were recorded after each training session. ESA treatment increased hemoglobin (∼11 and ∼14%, p < 0.001) and hematocrit (∼12 and ∼13%, p < 0.001) in the SE and TE groups, respectively. The relative (but not absolute) increases in V[Combining Dot Above]O2max were more pronounced (p < 0.01) in TE (27 ± 6%), compared with SE (15 ± 4%) but not TP (19 ± 4%), indicating that training is superior to ESA in stimulating V[Combining Dot Above]O2max in untrained men. The increased oxygen uptake in the TE group did not result in higher absolute training workloads than in the TP group. In untrained men, training exhibits a greater stimulus for improvements in V[Combining Dot Above]O2max than ESA treatment, without pronounced additive effects, which is supported by similar average training workloads and energy consumption in TP and TE. Thus, in untrained men, training alone seems sufficient to induce improvement in V[Combining Dot Above]O2max.

Lean body mass, not FFA, predicts VLDL-TG secretion rate in healthy men.

OBJECTIVE: Triglyceride is a risk factor for cardiovascular disease. However, the impact of body composition and free fatty acid (FFA) levels on very-low-density-lipoprotein triglyceride (VLDL-TG) secretion remains controversial. The aim was to identify predictors of VLDL-TG secretion in a data set compiled from seven previously published studies. METHODS: VLDL-TG kinetics was studied in 96 healthy men covering a wide span in body composition. A primed-constant infusion of ex vivo labeled [1-(14)C]-triolein VLDL-TG was used. Body composition was determined by dual X-ray absorptiometry and computed tomography scanning. Energy expenditure was measured by indirect calorimetry. Palmitate flux was measured by a [9,10-(3)H]-palmitate infusion. RESULTS: VLDL-TG secretion rate correlated significantly with body mass index (BMI), lean body mass (LBM), total fat mass, resting energy expenditure (REE), and insulin. A trend toward an inverse relationship between VLDL-TG secretion rate and FFA concentration was observed. In mixed model linear regression analysis, VLDL-TG secretion rate was positively associated with LBM (P = 0.03), and VLDL-TG clearance rate was inversely related to total fat mass (P < 0.01). CONCLUSIONS: LBM is a predictor of VLDL-TG secretion in healthy men, whereas FFA availability is not associated with VLDL-TG secretion. The work suggests reporting VLDL-TG secretion rates normalized for LBM when comparing subjects with differences in body composition.

Serum proteomic changes after randomized prolonged erythropoietin treatment and/or endurance training: detection of novel biomarkers.

INTRODUCTION: Despite implementation of the biological passport to detect erythropoietin abuse, a need for additional biomarkers remains. We used a proteomic approach to identify novel serum biomarkers of prolonged erythropoiesis-stimulating agent (ESA) exposure (Darbepoietin-α) and/or aerobic training. TRIAL DESIGN: Thirty-six healthy young males were randomly assigned to the following groups: Sedentary-placebo (n = 9), Sedentary-ESA (n = 9), Training-placebo (n = 10), or Training-ESA (n = 8). They were treated with placebo/Darbepoietin-α subcutaneously once/week for 10 weeks followed by a 3-week washout period. Training consisted of supervised biking 3/week for 13 weeks at the highest possible intensity. Serum was collected at baseline, week 3 (high dose Darbepoietin-α), week 10 (reduced dose Darbepoietin-α), and after a 3-week washout period. METHODS: Serum proteins were separated according to charge and molecular mass (2D-gel electrophoresis). The identity of proteins from spots exhibiting altered intensity was determined by mass spectrometry. RESULTS: Six protein spots changed in response to Darbepoietin-α treatment. Comparing all 4 experimental groups, two protein spots (serotransferrin and haptoglobin/haptoglobin related protein) showed a significant response to Darbepoietin-α treatment. The haptoglobin/haptoglobin related protein spot showed a significantly lower intensity in all subjects in the training-ESA group during the treatment period and increased during the washout period. CONCLUSION: An isoform of haptoglobin/haptoglobin related protein could be a new anti-doping marker and merits further research. TRIAL REGISTRATION: ClinicalTrials.gov NCT01320449.

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.

Isolated hyperglycaemia does not increase VLDL-triacylglycerol secretion in type 1 diabetic men.

AIMS/HYPOTHESIS: In type 1 diabetes, abnormalities of both glucose and lipoprotein metabolism are seen. The relationship between these factors is not understood, but studies indicate that hyperglycaemia may increase hepatic VLDL-triacylglycerol (VLDL-TG) secretion and reduce VLDL-TG fatty acid oxidation, which could lead to the development of dyslipidaemia. The aim of this study was to determine the isolated effect of hyperglycaemia on VLDL-TG and NEFA kinetics in men with type 1 diabetes. METHODS: VLDL-TG and palmitate kinetics were measured in eight men with type 1 diabetes using ex vivo labelled VLDL-TG and palmitate tracers. A 2.5 h basal period (plasma glucose 5 mmol/l) was followed by a 4 h hyperglycaemic period (plasma glucose 16 mmol/l). Steady-state VLDL-TG kinetics (VLDL-TG secretion, clearance and oxidation rates) were assessed by an isotope dilution technique using an intravenous primed-constant infusion of ex vivo labelled [1-(14)C]VLDL-TG in combination with sampling of blood and expired air. Palmitate turnover was measured using [9,10-(3)H]palmitate. RESULTS: The VLDL-TG secretion rate (36.0 ± 9.6 vs 30.8 ± 6.1 µmol/min, NS) and clearance rate (209 ± 30.4 vs 197 ± 41.7 ml/min, NS) were unchanged during the basal and hyperglycaemic periods, resulting in unchanged VLDL-TG concentrations (0.25 ± 0.11 µmol/l vs 0.28 ± 0.10 µmol/l, NS). In addition, VLDL-TG fatty acid oxidation and palmitate flux were not changed during hyperglycaemia. CONCLUSIONS/INTERPRETATION: Four hours of acute hyperglycaemia (16 mmol/l) without a concomitant increase in insulin does not alter VLDL-TG and NEFA kinetics in men with type 1 diabetes. CLINICAL TRIAL REGISTRY NUMBER: NCT01178957.

Erythropoietin administration alone or in combination with endurance training affects neither skeletal muscle morphology nor angiogenesis in healthy young men.

The aim was to investigate the ability of an erythropoiesis-stimulating agent (ESA), alone or in combination with endurance training, to induce changes in human skeletal muscle fibre and vascular morphology. In a comparative study, 36 healthy untrained men were randomly dispersed into the following four groups: sedentary-placebo (SP, n = 9); sedentary-ESA (SE, n = 9); training-placebo (TP, n = 10); or training-ESA (TE, n = 8). The ESA or placebo was injected once weekly. Training consisted of progressive bicycling three times per week for 10 weeks. Before and after the intervention period, muscle biopsies and magnetic resonance images were collected from the thigh muscles, blood was collected, body composition measured and endurance exercise performance evaluated. The ESA treatment (SE and TE) led to elevated haematocrit, and both ESA treatment and training (SE, TP and TE) increased maximal O2 uptake. With regard to skeletal muscle morphology, TP alone exhibited increases in whole-muscle cross-sectional area and fibre diameter of all fibre types. Also exclusively for TP was an increase in type IIa fibres and a corresponding decrease in type IIx fibres. Furthermore, an overall training effect (TP and TE) was statistically demonstrated in whole-muscle cross-sectional area, muscle fibre diameter and type IIa and type IIx fibre distribution. With regard to muscle vascular morphology, TP and TE both promoted a rise in capillary to muscle fibre ratio, with no differences between the two groups. There were no effects of ESA treatment on any of the muscle morphological parameters. Despite the haematopoietic effects of ESA, we provide novel evidence that endurance training rather than ESA treatment induces adaptational changes in angiogenesis and muscle morphology.

Ten weeks of aerobic training does not result in persistent changes in VLDL triglyceride turnover or oxidation in healthy men.

OBJECTIVE: Very low density lipoprotein triglyceride (VLDL-TG) and free fatty acids (FFA) constitute a substantial proportion of human energy supply both at rest and during exercise. Exercise acutely decreases VLDL-TG concentration, and VLDL-TG clearance is increased after an exercise bout. However, the effects of long-term training are not clear. DESIGN: The aim was to investigate long-term effects of training by direct assessments of VLDL-TG and palmitate kinetics and oxidation in healthy lean men (n=9) at rest, before and after a 10-week training program, compared with a non-training control group (n=9). METHODS: VLDL-TG kinetics were assessed by a primed constant infusion of [1-14C]VLDL-TG, and VLDL-TG oxidation by specific activity (14CO2) in expired air. The metabolic study days were placed 60-72 h after the last exercise bout. RESULTS: Palmitate kinetics and oxidation were assessed by a 2 h constant infusion of [9,10-(3)H]palmitate. In the training group (n=9), maximal oxygen uptake increased significantly by ≈20% (P<0.05), and the insulin sensitivity (assessed by the hyperinsulinemic-euglycemic clamp) improved significantly (P<0.05). Despite these metabolic improvements, no changes were observed in VLDL-TG secretion, clearance, or oxidation or in palmitate kinetics. CONCLUSION: We conclude that 10 weeks of exercise training did not induce changes in VLDL-TG and palmitate kinetics in healthy lean men.

Fasting increases human skeletal muscle net phenylalanine release and this is associated with decreased mTOR signaling.

AIM: Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR), a key regulator of cell growth. METHODS: Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days. RESULTS: Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation. CONCLUSIONS: Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth.