Impaired glycogen breakdown and synthesis in phosphoglucomutase 1 deficiency.

OBJECTIVE: We investigated metabolism and physiological responses to exercise in an 18-year-old woman with multiple congenital abnormalities and exertional muscle fatigue, tightness, and rhabdomyolysis. METHODS: We studied biochemistry in muscle and fibroblasts, performed mutation analysis, assessed physiological responses to forearm and cycle-ergometer exercise combined with stable-isotope techniques and indirect calorimetry, and evaluated the effect of IV glucose infusion and oral sucrose ingestion on the exercise response. RESULTS: Phosphoglucomutase type 1 (PGM1) activity in muscle and fibroblasts was severely deficient and PGM1 in muscle was undetectable by Western blot. The patient was compound heterozygous for missense (R422W) and nonsense (Q530X) mutations in PGM1. Forearm exercise elicited no increase in lactate, but an exaggerated increase in ammonia, and provoked a forearm contracture. Comparable to patients with McArdle disease, the patient developed a 'second wind' with a spontaneous fall in exercise heart rate and perceived exertion. Like in McArdle disease, this was attributable to an increase in muscle oxidative capacity. Carbohydrate oxidation was blocked during exercise, and the patient had exaggerated oxidation of fat to fuel exercise. Exercise heart rate and perceived exertion were lower after IV glucose and oral sucrose. Muscle glycogen level was low normal. CONCLUSIONS: The second wind phenomenon has been considered to be pathognomonic for McArdle disease, but we demonstrate that it can also be present in PGM1 deficiency. We show that severe loss of PGM1 activity causes blocked muscle glycogenolysis that mimics McArdle disease, but may also limit glycogen synthesis, which broadens the phenotypic spectrum of this disorder.

Gene expression profiling in patients with polymyalgia rheumatica before and after symptom-abolishing glucocorticoid treatment.

BACKGROUND: The pathophysiology, including the impact of gene expression, of polymyalgia rheumatica (PMR) remains elusive. We profiled the gene expression in muscle tissue in PMR patients before and after glucocorticoid treatment. METHODS: Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 arrays in muscle biopsies from 8 glucocorticoid-naive patients with PMR and 10 controls before and after prednisolone-treatment for 14 days. For 14 genes, quantitative real-time PCR (qRT-PCR, n = 9 in both groups) was used to validate the microarray findings and to further investigate the expression of genes of particular interest. RESULTS: Prednisolone normalized erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in PMR patients. A total of 165 putatively clinically relevant, differentially expressed genes were identified (cut-off: fold difference > ±1.2, difference of mean > 30, and p < 0.05); of these, 78 genes differed between patients and controls before treatment, 131 genes responded to treatment in a given direction only in patients, and 44 fulfilled both these criteria. In 43 of the 44 genes, treatment counteracted the initial difference. Functional clustering identified themes of biological function, including regulation of protein biosynthesis, and regulation of transcription and of extracellular matrix processes. Overall, qRT-PCR confirmed the microarray findings: Microarray-detected group differences were confirmed for 9 genes in 17 of 18 comparisons (same magnitude and direction of change); lack of group differences in microarray testing was confirmed for 5 genes in 8 of 10 comparisons. Before treatment, using qRT-PCR, expression of interleukin 6 (IL-6) was found to be 4-fold higher in patients (p < 0.05). CONCLUSIONS: This study identifies genes in muscle, the expression of which may impact the pathophysiology of PMR. Moreover, the study adds further evidence of the importance of IL-6 in the disease. Follow-up studies are needed to establish the exact pathophysiological relevance of the identified genes. The study was retrospectively listed on the ISRCTN registry with study ID ISRCTN69503018 and date of registration the 26th of July 2017.

Contraction-induced lipolysis is not impaired by inhibition of hormone-sensitive lipase in skeletal muscle.

In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.

Effects of interferon ß-1a and interferon ß-1b monotherapies on selected serum cytokines and nitrite levels in patients with relapsing-remitting multiple sclerosis: a 3-year longitudinal study.

OBJECTIVE: Interferon (IFN)ß treatment is a mainstay of relapsing-remitting multiple sclerosis (RRMS) immunotherapy. Its efficacy is supposedly a consequence of impaired trafficking of inflammatory cells into the central nervous system and modification of the proinflammatory/antiinflammatory cytokine balance. However, the effects of long-term monotherapy using various IFNß preparations on cytokine profiles and the relevance of these effects for the therapy outcome have not yet been elucidated. METHODS: Changes were compared in serum levels of TNFα, IFNγ, interleukin (IL)-6, IL-10 and nitrite between RRMS patients given 3-year treatment with intramuscular IFNß-1a (30 µg once a week) or subcutaneous IFNß-1b (250 µg every other day). Only the data from patients who completed the 3-year study (n = 20 and n = 18, respectively) were analyzed. RESULTS: Three-year IFNß-1a or IFNß-1b monotherapy reduced serum nitrite levels by 77 and 71%, respectively, lowered multiple sclerosis relapse annual rate by 70 and 71%, respectively, and significantly and similarly lowered Expanded Disability Status Scale scores in both study groups (by 0.9 on average). The two monotherapies showed little if any effect on cytokine levels and cytokine level ratios after the first year, but exerted diverging effects on these indices later on; the only exception was the IFNγ/IL-6 ratio that showed a monotonous rise in both study groups over the entire study period. CONCLUSION: During long-term IFNß monotherapy, the levels of the studied cytokines show no relevance to the course of RRMS and neurological status of patients, whereas there seems to be a link between these clinical indices and the activity of nitric oxide-mediated pathways.

Studies in Rats of Combined Muscle and Liver Perfusion and of Muscle Extract Indicate That Contractions Release a Muscle Hormone Directly Enhancing Hepatic Glycogenolysis.

Background: Established neuroendocrine signals do not sufficiently account for the exercise-induced increase in glucose production. Using an innovative, yet classical cross-circulation procedure, we studied whether contracting muscle produces a factor that directly stimulates hepatic glycogenolysis. Methods: Isolated rat hindquarters were perfused in series with isolated livers. Results: Stimulation of the sciatic nerve of one or both legs resulted in an increase in force, which rapidly waned. During one-legged contractions, hepatic glucose production increased initially (from −0.9 ± 0.5 (mean ± SE) to 3.3 ± 0.7 µmol/min, p < 0.05). The peak did not differ significantly from that seen after 20 nM of epinephrine (5.1 ± 1.2 µmol/min, p > 0.05). In response to two-legged contractions, the increase in hepatic glucose production (to 5.4 ± 1.3 µmol/min) was higher (p < 0.05) and lasted longer than that seen during one-legged contractions. During contractions, peak hepatic glucose output exceeded concomitant hepatic lactate uptake (p < 0.05), and glucose output decreased to basal levels, while lactate uptake rose to a plateau. Furthermore, in separate experiments an increase in lactate supply to isolated perfused livers increased lactate uptake, but not glucose output. In intact rats, intra-arterial injection of extract made from mixed leg muscle elicited a prolonged increase (p < 0.05) in plasma glucose concentration (from 5.2 ± 0.1 mM to 8.3 ± 1.5 mM). In perfused livers, muscle extract increased glucose output dose dependently. Fractionation by chromatography of the extract showed that the active substance had a MW below 2000. Conclusion: This study provides evidence that contracting skeletal muscle may produce a hormone with a MW below 2000, which enhances hepatic glycogenolysis according to energy needs. Further chemical characterization is warranted.

Increased muscle interstitial levels of inflammatory cytokines in polymyalgia rheumatica.

OBJECTIVE: Polymyalgia rheumatica (PMR) is characterized by aching of the proximal muscles and increased blood levels of markers of inflammation. Despite the muscle complaints, the current view is that symptoms are caused by inflammation in synovial structures. The purpose of this study was to elucidate the disease mechanisms in symptomatic muscles by measuring interstitial levels of cytokines before and after prednisolone treatment. METHODS: Twenty glucocorticoid-naive patients newly diagnosed as having PMR and 20 control subjects were studied before and after 14 days of prednisolone therapy (20 mg/day). Interstitial concentrations of interleukin-1α/ß (IL-1α/ß), IL-1 receptor antagonist, IL-6, IL-8, tumor necrosis factor α (TNFα), and monocyte chemoattractant protein 1 were measured in symptomatic vastus lateralis and trapezius muscles using the microdialysis technique. Plasma levels were measured simultaneously. RESULTS: Prednisolone abolished symptoms in all of the PMR patients within 1-2 days; the erythrocyte sedimentation rate and C-reactive protein levels were normalized on day 14. In both muscles, interstitial concentrations of all cytokines were markedly higher (P < 0.05) in the PMR patients than in the controls before treatment. In both patients and controls, interstitial levels of most cytokines were higher than plasma levels, with the exception of IL-1α and TNFα, which were lower in both groups. In the PMR patients, interstitial concentrations were normalized after prednisolone treatment. CONCLUSION: This study introduces a novel view of PMR, indicating that increased interstitial levels of inflammatory cytokines in symptomatic muscles play a role in the pathophysiology of the disease and that cytokines may be released locally. To explore the disease specificity, similar studies in other primary inflammatory conditions are warranted.

Testosterone affects hormone-sensitive lipase (HSL) activity and lipid metabolism in the left ventricle.

Fatty acids, which are the major cardiac fuel, are derived from lipid droplets stored in cardiomyocytes, among other sources. The heart expresses hormone-sensitive lipase (HSL), which regulates triglycerides (TG) breakdown, and the enzyme is under hormonal control. Evidence obtained from adipose tissue suggests that testosterone regulates HSL activity. To test whether this is also true in the heart, we measured HSL activity in the left ventricle of sedentary male rats that had been treated with testosterone supplementation or orchidectomy with or without testosterone substitution. Left ventricle HSL activity against TG was significantly elevated in intact rats supplemented with testosterone. HSL activity against both TG and diacylglyceride was reduced by orchidectomy, whereas testosterone replacement fully reversed this effect. Moreover, testosterone increased left ventricle free fatty acid levels, caused an inhibitory effect on carbohydrate metabolism in the heart, and elevated left ventricular phosphocreatine and ATP levels as compared to control rats. These data indicate that testosterone is involved in cardiac HSL activity regulation which, in turn, may affect cardiac lipid and carbohydrate metabolism.

Imaging of insulin signaling in skeletal muscle of living mice shows major role of T-tubules.

Insulin stimulates glucose transport in skeletal muscle by glucose transporter GLUT4 translocation to sarcolemma and membrane invaginations, the t-tubules. Although muscle glucose uptake plays a key role in insulin resistance and type 2 diabetes, the dynamics of GLUT4 translocation and the signaling involved are not well described. We have now developed a confocal imaging technique to follow trafficking of green fluorescent protein-labeled proteins in living muscle fibers in situ in anesthetized mice. Using this technique, by imaging the dynamics of GLUT4 translocation and phosphatidylinositol 3,4,5 P(3) (PIP(3)) production in response to insulin, here, for the first time, we delineate the temporal and spatial distribution of these processes in a living animal. We find a 10-min delay of maximal GLUT4 recruitment and translocation to t-tubules compared with sarcolemma. Time-lapse imaging of a fluorescent dye after intravenous injection shows that this delay is similar to the time needed for insulin diffusion into the t-tubule system. Correspondingly, immunostaining of muscle fibers shows that insulin receptors are present throughout the t-tubule system. Finally, PIP(3) production, an early event in insulin signaling, progresses slowly along the t-tubules with a 10-min delay between maximal PIP(3) production at sarcolemma compared with deep t-tubules following the appearance of dye-labeled insulin. Our findings in living mice indicate a major role of the t-tubules in insulin signaling in skeletal muscle and show a diffusion-associated delay in insulin action between sarcolemma and inner t-tubules.

Skeletal muscle metabolism during prolonged exercise in Pompe disease.

OBJECTIVE: Pompe disease (glycogenosis type II) is caused by lysosomal alpha-glucosidase deficiency, which leads to a block in intra-lysosomal glycogen breakdown. In spite of enzyme replacement therapy, Pompe disease continues to be a progressive metabolic myopathy. Considering the health benefits of exercise, it is important in Pompe disease to acquire more information about muscle substrate use during exercise. METHODS: Seven adults with Pompe disease were matched to a healthy control group (1:1). We determined (1) peak oxidative capacity (VO2peak) and (2) carbohydrate and fatty acid metabolism during submaximal exercise (33 W) for 1 h, using cycle-ergometer exercise, indirect calorimetry and stable isotopes. RESULTS: In the patients, VO2peak was less than half of average control values; mean difference -1659 mL/min (CI: -2450 to -867, P = 0.001). However, the respiratory exchange ratio increased to >1.0 and lactate levels rose 5-fold in the patients, indicating significant glycolytic flux. In line with this, during submaximal exercise, the rates of oxidation (ROX) of carbohydrates and palmitate were similar between patients and controls (mean difference 0.226 g/min (CI: 0.611 to -0.078, P = 0.318) and mean difference 0.016 µmol/kg/min (CI: 1.287 to -1.255, P = 0.710), respectively). CONCLUSION: Reflecting muscle weakness and wasting, Pompe disease is associated with markedly reduced maximal exercise capacity. However, glycogenolysis is not impaired in exercise. Unlike in other metabolic myopathies, skeletal muscle substrate use during exercise is normal in Pompe disease rendering exercise less complicated for e.g. medical or recreational purposes.

Noradrenaline-induced increases in calcium and tension in skeletal muscle conductance and resistance arteries from rats with post-infarction heart failure.

We tested the hypothesis that arterial reactivity to noradrenaline is augmented in congestive heart failure (CHF), which could contribute to the deleterious changes in peripheral vascular resistance and compliance in this condition. From male Wistar rats with post-infarction CHF and sham-operated rats, skeletal muscle conductance and resistance arteries (mean lumen diameters: 514 and 186 microm) were isolated and mounted on wire myographs, and wall tension was recorded in response to cumulative application of acetylcholine and noradrenaline to the vessel segments. In a subset of experiments, wall tension and cytosolic free calcium ion concentration [Ca(2+)](i) were recorded simultaneously during noradrenaline application, using wire myography and the FURA-2 technique. No significant differences were found in the arterial baseline levels of [Ca(2+)](i) or tension between CHF and sham rats. In the resistance arteries of CHF rats, the noradrenaline-induced increases in [Ca(2+)](i) were significantly enhanced (P=0.003). Despite the augmented [Ca(2+)](i) levels, the tension responses to noradrenaline were unaltered in these arteries. In the conductance arteries, there were no significant differences in noradrenaline-induced [Ca(2+)](i) or tension responses between CHF and control rats. CHF did not alter vascular morphology or change vascular relaxations to acetylcholine in either type of artery. In conclusion, these results do not support the contention that arterial reactivity to noradrenaline is augmented in the skeletal muscle vascular bed in CHF. On the contrary, the unchanged contractile responsiveness in the resistance arteries despite the enhanced levels of [Ca(2+)](i) during noradrenaline application suggests that the contractile function of these vessels is compromised in CHF. Neither vascular remodeling, endothelial dysfunction nor changes in baseline vascular tone could be demonstrated in the skeletal muscle vascular bed of this animal model of heart failure.

Ca(2+) sensitisation of force production by noradrenaline in femoral conductance and resistance arteries from rats with postinfarction congestive heart failure.

In this study we tested the hypothesis that arterial myofilament Ca(2+) sensitivity and/or the Ca(2+) sensitising effect of noradrenaline (NA) is enhanced in post-infarction congestive heart failure (CHF), which could contribute to the high peripheral vascular resistance in this condition. Femoral skeletal muscle resistance and conductance arteries (mean lumen diameters of 159 and 519 microm) from rats with CHF and sham-operated control rats were used. Isometric tension development and intracellular free calcium concentration ([Ca(2+)](i)) were measured simultaneously in isolated vessel segments using wire myography and the FURA-2 fluorescence technique. In conductance and resistance arteries, the resting levels of [Ca(2+)](i) and tension in physiological saline solution (PSS) and active tension in response to single doses of 125 mM K(+) (KPSS) were unaffected by CHF. During cumulative application of extracellular Ca(2+) to arteries depolarised with 125 mM K(+) or activated with 30 microM NA, [Ca(2+)](i) and vessel wall tension were similar in CHF and control rats. However, the conductance arteries showed significantly higher calcium sensitivity than resistance arteries in these experiments. We conclude that an abnormality in the sensitivity of the contractile apparatus to Ca(2+), or in NA-induced Ca(2+) sensitisation in arterial vascular smooth muscle cells is unlikely to contribute to the ubiquitously elevated vascular resistance associated with CHF. However, our data demonstrate significant differences in vascular Ca(2+) handling, myofilament Ca(2+) sensitivity and tension development between resistance and conductance arteries, regardless of CHF.

Circadian variations in clinical symptoms and concentrations of inflammatory cytokines, melatonin, and cortisol in polymyalgia rheumatica before and during prednisolone treatment: a controlled, observational, clinical experimental study.

BACKGROUND: In contrast to rheumatoid arthritis (RA), no systematic investigation of diurnal variation has been carried out in polymyalgia rheumatica (PMR). The aim of the study was to provide the often-requested documentation of the 24-h time course of clinical symptoms in PMR and relate them to concentrations during the day of melatonin, inflammatory cytokines, and cortisol. Furthermore, the effects of 14 days of prednisolone treatment were studied. METHODS: Ten glucocorticoid-naive patients newly diagnosed with PMR and seven non-PMR control subjects were studied for 24 h before treatment and during the 14th day of treatment with 20 mg/day of prednisolone. Global pain and generalized muscle stiffness were monitored by using visual analogue scales, and blood was drawn repeatedly. RESULTS: In untreated patients, pain and stiffness peaked in the early morning, showing a plateau between 04:00 and 08:00, and then declined to a nadir at 16:00 (2P < 0.05). Plasma concentrations of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, IL-1ß, and IL-4 varied with time in both groups (2P < 0.05) and peaked between 04:00 and 08:00. Furthermore, except for IL-1ß, concentrations of these cytokines and of IL-10 were higher throughout the 24-h observation period in patients than in control subjects (2P < 0.05). Also, melatonin and cortisol were consistently higher in patients (2P < 0.05) and varied with time (2P < 0.05), peaking around 02:00 and 08:00, respectively. In patients, prednisolone abolished symptoms, normalized C-reactive protein, and reduced melatonin, IL-6, IL-8, and TNF-α concentrations (2P < 0.05), while IL-10 increased between 10:00 and 14:00. CONCLUSIONS: In PMR, key symptoms show diurnal variation. Furthermore, in PMR, concentrations of melatonin, several pro- and anti-inflammatory cytokines, and cortisol are increased throughout the day and show diurnal variation, as also seen in healthy subjects. The time courses and the inhibitory effects of prednisolone indicate that in PMR, as proposed for RA, melatonin stimulates cytokine production, which in turn accounts at least partly for the symptoms. Furthermore, overall, cortisol may downregulate cytokine production and symptoms. Stimulation of IL-10 secretion may participate in the anti-inflammatory effects of prednisolone. These findings support use of chronotherapy in PMR and encourage study of circadian variations in other inflammatory autoimmune diseases.

Expression profiling reveals differences in metabolic gene expression between exercise-induced cardiac effects and maladaptive cardiac hypertrophy.

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise-induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise-induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in beta-oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise-induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise-induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.

Prevention of hyperglycemia in Zucker diabetic fatty rats by exercise training: effects on gene expression in insulin-sensitive tissues determined by high-density oligonucleotide microarray analysis.

Exercise training (ET) causes metabolic improvement in the prediabetic and diabetic states. However, only little information exists on the changes to ET at the transcriptional level in insulin-sensitive tissues. We have investigated the gene expression changes in skeletal muscle, liver, fat, and pancreatic islets after ET in male Zucker diabetic fatty (ZDF) rats. Eighteen ZDF rats (7 weeks old) were divided in a control and ET group. Exercise was performed using a motorized treadmill (20 m/min 1 hour daily for 6 days a week). Blood glucose, weight, and food intake were measured weekly. After 5 weeks, blood samples, soleus muscle, liver, visceral fat (epididymal fat pads), and islet tissue were collected. Gene expression was quantified with Affymetrix RG-U34A array (16 chips). Exercise training ameliorates the development of hyperglycemia and reduces plasma free fatty acid and the level of glucagon-insulin ratio (P < .05). In skeletal muscle, the expression of 302 genes increased, whereas that of 119 genes decreased. These changes involved genes related to skeletal muscle plasticity, Ca(2+) signals, energy metabolism (eg, glucose transporter 1, phosphorylase kinase), and other signaling pathways as well as genes with unknown functions (expressed sequence tags). In the liver, expression of 148 genes increased, whereas that of 199 genes decreased. These were primarily genes involved in lipogenesis and detoxification. Genes coding for transcription factors were changed in parallel in skeletal muscle and liver tissue. Training did not markedly influence the gene expression in islets. In conclusion, ET changes the expression of multiple genes in the soleus muscle and liver tissue and counteracts the development of diabetes, indicating that ET-induced changes in gene transcription may play an important role en the prevention of diabetes.

Skeletal muscle metabolism is impaired during exercise in glycogen storage disease type III.

OBJECTIVE: Glycogen storage disease type IIIa (GSDIIIa) is classically regarded as a glycogenosis with fixed weakness, but we hypothesized that exercise intolerance in GSDIIIa is related to muscle energy failure and that oral fructose ingestion could improve exercise tolerance in this metabolic myopathy. METHODS: We challenged metabolism with cycle-ergometer exercise and measured substrate turnover and oxidation rates using stable isotope methodology and indirect calorimetry in 3 patients and 6 age-matched controls on 1 day, and examined the effect of fructose ingestion on exercise tolerance in the patients on another day. RESULTS: Total fatty acid oxidation rates during exercise were higher in patients than controls, 32.1 (SE 1.2) vs 20.7 (SE 0.5; range 15.8-29.3) µmol/kg/min (p = 0.048), and oxidation of carbohydrates was lower in patients, 1.0 (SE 5.4) vs 38.4 (SE 8.0; range 23.0-77.1) µmol/kg/min (p = 0.024). Fructose ingestion improved exercise tolerance in the patients. CONCLUSION: Similar to patients with McArdle disease, in whom muscle glycogenolysis is also impaired, GSDIIIa is associated with a reduced skeletal muscle oxidation of carbohydrates and a compensatory increase in fatty acid oxidation, and fructose ingestion improves exercise tolerance. Our results indicate that GSDIIIa should not only be viewed as a glycogenosis with fixed skeletal muscle weakness, but should also be considered among the glycogenoses presenting with exercise-related dynamic symptoms caused by muscular energy deficiency. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that ingestion of fructose improves exercise tolerance in patients with GSDIIIa.

Insulin resistance and increased muscle cytokine levels in patients with mitochondrial myopathy.

CONTEXT: Mitochondrial dysfunction has been proposed to cause insulin resistance and that might stimulate cytokine production. OBJECTIVE: The objective of the study was to elucidate the association between mitochondrial myopathy, insulin sensitivity, and cytokine levels in muscle. DESIGN AND SETTING: This was an experimental, controlled study in outpatients. PARTICIPANTS: Eight overnight-fasted patients (P) with various inherited mitochondrial myopathies and eight healthy subjects (C) matched for sex, age, weight, height, and physical activity participated in the study. INTERVENTIONS: The intervention included a 120-minute hyperinsulinemic, euglycemic clamp. Another morning, microdialysis of both vastus lateralis muscles for 4 hours, including one-legged, knee extension exercise for 30 minutes, was performed. MAIN OUTCOME MEASURES: Glucose infusion rate during 90-120 minutes of insulin infusion was measured. Cytokine concentrations in dialysate were also measured. RESULTS: Muscle strength, percentage fat mass, and creatine kinase in plasma did not differ between groups. The maximal oxygen uptake was 21 ± 3 (SE) (P) and 36 ± 3(C) mL/kg·min (2P < .05). Basal insulin, C-peptide, and glucagon were higher in P (55 ± 10, 980 ± 92, and 102 ± 13 pM) than in C (36 ± 12, 712 ± 98, and 44 ± 10 pM) (two-sided significance testing [2P ]< .05). The homeostasis model assessment insulin sensitivity index and glucose infusion rate (6.8 ± 1.0 vs 9.4 ± 1.3 mg/min·kg) were lower, and free fatty acids and glycerol at 120 minutes were higher in P vs C (2P < .05). Dialysate concentrations of TNF-α, IL-6, IL-8, IL-10, and monocyte chemoattractant protein-1 were higher in P vs C (2P < .05). Dialysate concentrations of these cytokines and of IL-1 receptor antagonist increased during exercise (2P < .05), identically in P and C. No differences existed in plasma cytokine concentrations. CONCLUSIONS: In patients with a variety of mitochondrial myopathies, insulin sensitivity of muscle, adipose tissue, and pancreatic A cells is reduced, supporting that mitochondrial function influences insulin action. Furthermore, a local, low-grade inflammation of potential clinical importance exists in the muscle of these patients.

Contraction and AICAR stimulate IL-6 vesicle depletion from skeletal muscle fibers in vivo.

Recent studies suggest that interleukin 6 (IL-6) is released from contracting skeletal muscles; however, the cellular origin, secretion kinetics, and signaling mechanisms regulating IL-6 secretion are unknown. To address these questions, we developed imaging methodology to study IL-6 in fixed mouse muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP-containing vesicles and protein by 62% (P < 0.05), occurring rapidly and progressively over 25 min of contraction. However, contraction-mediated IL-6-EGFP reduction was normal in muscle-specific AMP-activated protein kinase (AMPK) α2-inactive transgenic mice. In contrast, the AMPK activator AICAR decreased IL-6-EGFP vesicles, an effect that was inhibited in the transgenic mice. In conclusion, resting skeletal muscles contain IL-6-positive vesicles that are expressed throughout myofibers. Contractions stimulate the rapid reduction of IL-6 in myofibers, occurring through an AMPKα2-independent mechanism. This novel imaging methodology clearly establishes IL-6 as a contraction-stimulated myokine and can be used to characterize the secretion kinetics of other putative myokines.

Activity of the neuroendocrine axes in patients with polymyalgia rheumatica before and after TNF-α blocking etanercept treatment.

INTRODUCTION: In this study, we evaluated the activity of the neuroendocrine axes in patients with polymyalgia rheumatica (PMR) before and after tumor necrosis factor (TNF)-α-blocking etanercept treatment, which previously has been shown to reduce interleukin 6 (IL-6) and C-reactive protein (CRP) markedly in PMR. METHODS: Plasma samples were collected from 10 glucocorticoid-naïve patients with PMR and 10 matched controls before and after etanercept treatment (25 mg biweekly for 2 weeks). The primary end points were pre- and posttreatment levels of adrenocorticotropic hormone (ACTH), cortisol, adrenaline, thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), prolactin, and insulin-like growth factor 1 (IGF-1). RESULTS: Before TNF-α-blocking treatment, plasma TNF-α, ACTH, and cortisol levels were higher in patients versus controls (P < 0.05 and P < 0.001, respectively); during TNF-α blockade in patients, levels of both hormones decreased (P < 0.05 and P < 0.01, respectively), whereas levels in controls increased (P < 0.05), abolishing the pretreatment differences. Pretreatment adrenaline levels were more than twice as high in patients than in controls (P < 0.01); after treatment in patients, levels had decreased (P < 0.05) but remained higher versus controls (P < 0.05). Levels of the other hormones never differed significantly between groups (P > 0.05). CONCLUSIONS: In PMR, TNF-α may increase the activities of the hypothalamic-pituitary-adrenal and the hypothalamic-sympthoadrenomedullary axes. Secretion of TSH, FSH, prolactin, and IGF-1 is not clearly changed in PMR. TRIAL REGISTRATION: ClinicalTrials.gov (NCT00524381).

Elevated muscle interstitial levels of pain-inducing substances in symptomatic muscles in patients with polymyalgia rheumatica.

Polymyalgia rheumatica (PMR) is characterized by aching proximal muscles and systemic inflammation. We explored the pain-eliciting mechanisms by measuring interstitial levels in muscle of potentially pain-inducing substances as well as local blood flow. Twenty glucocorticoid-naive patients with newly diagnosed PMR and 20 controls were examined before and after 14 days of prednisolone (20 mg/day). Concentrations of glutamate, prostaglandin E(2) (PGE(2)), bradykinin, serotonin, adenosine triphosphate, lactate, pyruvate, and potassium as well as extraction of (3)H(2)O were measured in symptomatic vastus lateralis and trapezius muscles using microdialysis. Plasma levels were measured simultaneously. To be considered potentially pain inducing, interstitial concentrations of candidates should be higher in patients vs. controls, be normalized by prednisolone, and be higher in muscle vs. plasma. Prednisolone abolished symptoms in all patients within 2 days. Before treatment glutamate in both muscles (vastus: 60±7 vs. 38±7 µmol/L; trapezius: 60±6 vs. 43±7 µmol/L) and PGE(2) in vastus (911±200 vs. 496±122 pg/mL) were higher in patients than in controls (P<0.05), and higher in muscle than in plasma (P<0.05). Prednisolone abolished the differences between patients and controls. No other candidate completely fulfilled the predefined requirements for pain-inducing substances in PMR. (3)H(2)O extraction was identical between groups. In conclusion, local release of glutamate and PGE(2), but not ischemia, may contribute to the muscle pain in PMR. This supports the view that intramuscular mechanisms are important in PMR.

Effect of etanercept in polymyalgia rheumatica: a randomized controlled trial.

INTRODUCTION: To elucidate in polymyalgia rheumatica (PMR) the role of tumor necrosis factor (TNF) α and the therapeutic potential of blockade with soluble TNF-α receptor, we carried out the first randomized controlled trial with etanercept in PMR. METHODS: Twenty newly diagnosed, glucocorticoid (GC) naïve patients with PMR and 20 matched non-PMR control subjects completed the trial. Subjects were randomized in a 1:1 ratio to monotherapy with etanercept (25 mg s.c. biweekly) or placebo (saline) for 14 days. Study outcomes were assessed at baseline and after 14 days. The primary outcome was the change in PMR activity score (PMR-AS). Secondary outcomes were: changes in erythrocyte sedimentation rate (ESR) and plasma levels of TNF-α and interleukin (IL) 6; patients' functional status (health assessment questionnaire) and cumulative tramadol intake during the trial. RESULTS: At baseline, plasma TNF-α was higher in patients than in controls (P < 0.05). The concentration always increased with etanercept treatment (P < 0.05). In patients, etanercept decreased PMR-AS by 24% (P = 0.011), reflecting significant improvements in shoulder mobility, physician's global assessment and C-reactive protein, and insignificant (P > 0.05) improvements in duration of morning stiffness and patient's assessment of pain. In parallel, ESR and IL-6 were reduced (P < 0.05). Placebo treatment did not change PMR-AS, ESR and IL-6 (P > 0.05). Functional status did not change and tramadol intake did not differ between patient groups. In controls, no changes occurred in both groups. CONCLUSIONS: Etanercept monotherapy ameliorates disease activity in GC naïve patients with PMR. However, the effect is modest, indicating a minor role of TNF-α in PMR. TRIAL REGISTRATION: ClinicalTrials.gov (NCT00524381).