Palmitic acid follows a different metabolic pathway than oleic acid in human skeletal muscle cells; lower lipolysis rate despite an increased level of adipose triglyceride lipase.

Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100µM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid ß-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance.

Glutathione and amino acid concentrations in human liver during short warm ischaemia and reperfusion: a pilot study.

Glutathione is a major antioxidant, and, in the present study, we investigated whether a clinical model of short warm ischaemia and reperfusion of the human liver during surgery would influence glutathione and amino acid metabolism. Previous studies in humans have demonstrated that ischaemia and reperfusion in skeletal muscle for up to 120 min have no major effect on muscle glutathione concentrations. Liver ischaemia and reperfusion in animals have demonstrated diverging results concerning glutathione metabolism. In the present study, six patients with liver malignancies, undergoing liver resection during warm ischaemia, were included. Liver biopsies were obtained from healthy appearing liver tissue from both lobes before ischaemia and at maximal ischaemia, and from the remaining liver lobe after 5, 10, 15, 20, 25 and 30 min of reperfusion. The biopsies were analysed for glutathione, amino acids and lactate. Median ischaemia time was 28 (range, 15-36) min. Lactate increased 266% at maximal ischaemia (P<0.05). No alterations in glutathione concentrations or the redox status of glutathione (GSH/total glutathione) were observed. Glutamate decreased 22% (P<0.05) at maximal ischaemia and increased thereafter 72% at 30 min of reperfusion (P<0.05). Alanine increased 105% at maximal ischaemia (P<0.05) and was normalized during reperfusion. BCAAs (branched-chain amino acids) increased 67% at maximal ischaemia (P<0.05). In conclusion, short-time ischaemia and reperfusion in the human liver did not affect glutathione concentrations, whereas changes were observed in amino acid concentrations during both ischaemia and reperfusion.

Systematic analysis of adaptations in aerobic capacity and submaximal energy metabolism provides a unique insight into determinants of human aerobic performance.

It has not been established which physiological processes contribute to endurance training-related changes (Delta) in aerobic performance. For example, the relationship between intramuscular metabolic responses at the intensity used during training and improved human functional capacity has not been examined in a longitudinal study. In the present study we hypothesized that improvements in aerobic capacity (Vo(2max)) and metabolic control would combine equally to explain enhanced aerobic performance. Twenty-four sedentary males (24 +/- 2 yr; 1.81 +/- 0.08 m; 76.6 +/- 11.3 kg) undertook supervised cycling training (45 min at 70% of pretraining Vo(2max)) 4 times/wk for 6 wk. Performance was determined using a 15-min cycling time trial, and muscle biopsies were taken before and after a 10-min cycle at 70% of pretraining Vo(2max) to quantify substrate metabolism. Substantial interindividual variability in training-induced adaptations was observed for most parameters, yet "low responders" for DeltaVo(2max) were not consistently low responders for other variables. While Vo(2max) and time trial performance were related at baseline (r(2) = 0.80, P < 0.001), the change in Vo(2max) was completely unrelated to the change in aerobic performance. The maximal parameters DeltaVe(max) and DeltaVeq(max) (DeltaVe/Vo(2max)) accounted for 64% of the variance in DeltaVo(2max) (P < 0.001), whereas Deltaperformance was related to changes in the submaximal parameters Veq(submax) (r(2) = 0.33; P < 0.01), muscle Deltalactate (r(2) = 0.32; P < 0.01), and Deltaacetyl-carnitine (r(2) = 0.29; P < 0.05). This study demonstrates that improvements in high-intensity aerobic performance in humans are not related to altered maximal oxygen transport capacity. Altered muscle metabolism may provide the link between training stimulus and improved performance, but metabolic parameters do not change in a manner that relates to aerobic capacity changes.

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

BACKGROUND: Septic patients treated in the intensive care unit (ICU) often develop multiple organ failure including persistent skeletal muscle dysfunction which results in the patient's protracted recovery process. We have demonstrated that muscle mitochondrial enzyme activities are impaired in septic ICU patients impairing cellular energy balance, which will interfere with muscle function and metabolism. Here we use detailed phenotyping and genomics to elucidate mechanisms leading to these impairments and the molecular consequences. METHODOLOGY/PRINCIPAL FINDINGS: Utilising biopsy material from seventeen patients and ten age-matched controls we demonstrate that neither mitochondrial in vivo protein synthesis nor expression of mitochondrial genes are compromised. Indeed, there was partial activation of the mitochondrial biogenesis pathway involving NRF2alpha/GABP and its target genes TFAM, TFB1M and TFB2M yet clearly this failed to maintain mitochondrial function. We therefore utilised transcript profiling and pathway analysis of ICU patient skeletal muscle to generate insight into the molecular defects driving loss of muscle function and metabolic homeostasis. Gene ontology analysis of Affymetrix analysis demonstrated substantial loss of muscle specific genes, a global oxidative stress response related to most probably cytokine signalling, altered insulin related signalling and a substantial overlap between patients and muscle wasting/inflammatory animal models. MicroRNA 21 processing appeared defective suggesting that post-transcriptional protein synthesis regulation is altered by disruption of tissue microRNA expression. Finally, we were able to demonstrate that the phenotype of skeletal muscle in ICU patients is not merely one of inactivity, it appears to be an actively remodelling tissue, influenced by several mediators, all of which may be open to manipulation with the aim to improve clinical outcome. CONCLUSIONS/SIGNIFICANCE: This first combined protein and transcriptome based analysis of human skeletal muscle obtained from septic patients demonstrated that losses of mitochondria and muscle mass are accompanied by sustained protein synthesis (anabolic process) while dysregulation of transcription programmes appears to fail to compensate for increased damage and proteolysis. Our analysis identified both validated and novel clinically tractable targets to manipulate these failing processes and pursuit of these could lead to new potential treatments.

Mitochondrial function in sepsis: respiratory versus leg muscle.

Patients with sepsis-induced multiple organ failure often experience muscle fatigue in both locomotive and respiratory muscles. Muscle fatigue extends intensive care unit stay, mostly in the form of prolonged weaning from the ventilator, and the recovery period after intensive care unit treatment due to general muscle fatigue. Muscle mitochondria are the main determinant of muscle fatigue and fatigability. Derangements in mitochondrial function in locomotive muscles have been described extensively both in animal models and patients with sepsis. Also, in respiratory muscle, mitochondrial function and content are impaired during sepsis. However, in septic patients with multiple organ failure, in locomotive muscle, lower levels of energy-rich compounds accompany the decreased mitochondrial content, whereas in respiratory muscle, the decreased mitochondrial content has no effect on cellular energy metabolism.

Knee replacement surgery as a human clinical model of the effects of ischaemia/reperfusion upon skeletal muscle.

The temporal pattern of metabolic alterations in muscle tissue during total ischaemia and reperfusion are not well-characterized in humans with respect to glutathione, amino acids and energy-rich compounds. In the present study, knee replacement surgery was used as a clinical model to elucidate this pattern of metabolic alterations. Patients (n=15) undergoing elective knee replacement surgery employing tourniquet ischaemia were studied. Muscle biopsies were taken from the quadriceps femoris muscle on the operated side preoperatively, at maximal ischaemia and after 24 h of reperfusion. The biopsies were analysed for glutathione, amino acids and energy-rich compounds. In addition the patients were randomized to receive either glucose or a mannitol infusion in the 24 h following tourniquet ischaemia. During ischaemia, muscle lactate increased by 400% (P<0.05) and phosphocreatine decreased by 70% (P<0.05). During the subsequent 24 h of reperfusion, muscle-reduced glutathione and total glutathione decreased by 27% and 22% (P<0.05) respectively. The muscle amino acid pattern changed during ischaemia with an increase in alanine by 65% (P<0.001) and a decrease in glutamate by 29% (P<0.001). During the reperfusion part of the study, no differences attributable to the infusion of mannitol or glucose were observed. During tourniquet ischaemia and subsequent reperfusion, changes in glutathione metabolism developed, indicating oxidative stress. Knee replacement surgery as a clinical model was useful during the ischaemia period, whereas the reperfusion period was dominated by the general changes seen postoperatively.

The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function.

BACKGROUND: Mutations in the PTEN induced putative kinase 1 (PINK1) are implicated in early-onset Parkinson's disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila. RESULTS: Herein we characterize a novel splice variant of PINK1 (svPINK1) that is homologous to the C-terminus regulatory domain of the protein kinase. Naturally occurring non-coding antisense provides sophisticated mechanisms for diversifying genomes and we describe a human specific non-coding antisense expressed at the PINK1 locus (naPINK1). We further demonstrate that PINK1 varies in vivo when human skeletal muscle mitochondrial content is enhanced, supporting the idea that PINK1 has a physiological role in mitochondrion. The observation of concordant regulation of svPINK1 and naPINK1 during in vivo mitochondrial biogenesis was confirmed using RNAi, where selective targeting of naPINK1 results in loss of the PINK1 splice variant in neuronal cell lines. CONCLUSION: Our data presents the first direct observation that a mammalian non-coding antisense molecule can positively influence the abundance of a cis-transcribed mRNA under physiological abundance conditions. While our analysis implies a possible human specific and dsRNA-mediated mechanism for stabilizing the expression of svPINK1, it also points to a broader genomic strategy for regulating a human disease locus and increases the complexity through which alterations in the regulation of the PINK1 locus could occur.

Proteasome proteolytic activity in skeletal muscle is increased in patients with sepsis.

Patients with sepsis in the ICU (intensive care unit) are characterized by skeletal muscle wasting. This leads to muscle dysfunction that also influences the respiratory capacity, resulting in prolonged mechanical ventilation. Catabolic conditions are associated with a general activation of the ubiquitin-proteasome pathway in skeletal muscle. The aim of the present study was to measure the proteasome proteolytic activity in both respiratory and leg muscles from ICU patients with sepsis and, in addition, to assess the variation of proteasome activity between individuals and between duplicate leg muscle biopsy specimens. When compared with a control group (n=10), patients with sepsis (n=10) had a 30% (P<0.05) and 45% (P<0.05) higher proteasome activity in the respiratory and leg muscles respectively. In a second experiment, ICU patients with sepsis (n=17) had a 55% (P<0.01) higher proteasome activity in the leg muscle compared with a control group (n=10). The inter-individual scatter of proteasome activity was larger between the patients with sepsis than the controls. We also observed a substantial intra-individual difference in activity between duplicate biopsies in several of the subjects. In conclusion, the proteolytic activity of the proteasome was higher in skeletal muscle from patients with sepsis and multiple organ failure compared with healthy controls. It was shown for the first time that respiratory and leg muscles were affected similarly. Furthermore, the variation in proteasome activity between individuals was more pronounced in the ICU patients for both muscle types, whereas the intra-individual variation between biopsies was similar for ICU patients and controls.

Derangements in mitochondrial metabolism in intercostal and leg muscle of critically ill patients with sepsis-induced multiple organ failure.

Critically ill patients treated for multiple organ failure often develop muscle dysfunction. Here we test the hypothesis that mitochondrial and energy metabolism are deranged in leg and intercostal muscle of critically ill patients with sepsis-induced multiple organ failure. Ten critically ill patients suffering from sepsis-induced multiple organ failure and requiring mechanical ventilation were included in the study. A group (n = 10) of metabolically healthy age- and sex-matched patients undergoing elective surgery were used as controls. Muscle biopsies were obtained from the vastus lateralis (leg) and intercostal muscle. The activities of citrate synthase and mitochondrial respiratory chain complexes I and IV and concentrations of ATP, creatine phosphate, and lactate were analyzed. Morphological evaluation of mitochondria was performed by electron microscopy. Activities of citrate synthase and complex I were 53 and 60% lower, respectively, in intercostal muscle of the patients but not in leg muscle compared with controls. The activity of complex IV was 30% lower in leg muscle but not in intercostal muscle. Concentrations of ATP and creatine phosphate were, respectively, 40 and 34% lower, and lactate concentrations were 43% higher in leg muscle but not in intercostal muscle. We conclude that both leg and intercostal muscle show a twofold decrease in mitochondrial content in intensive care unit patients with multiple organ failure, which is associated with lower concentrations of energy-rich phosphates and an increased anaerobic energy production in leg muscle but not in intercostal muscle.

Similar calcification process in acute and chronic human brain pathologies.

Cellular microcalcification observed in a diversity of human pathologies, such as vascular dementia, Alzheimer's disease, Parkinson's disease, astrogliomas, and posttraumatic epilepsy, also develops in rodent experimental models of central nervous system (CNS) neurodegeneration. Central to the neurodegenerative process is the inability of neurons to regulate intracellular calcium levels properly, and this is extensible to fine regulation of the CNS. This study provides evidence of a common pattern of brain calcification taking place in several human pathologies, and in the rat with glutamate-derived CNS lesions, regarding the chemical composition, physical characteristics, and histological environment of the precipitates. Furthermore, a common physical mechanism of deposit formation through nucleation, lineal growth, and aggregation is presented, under the modulation of protein deposition and elemental composition factors. Insofar as calcium precipitation reduces activity signals at no energy expense, the presence in human and rodent cerebral brain lesions of a common pattern of calcification may reflect an imbalance between cellular signals of activity and energy availability for its execution. If this is true, this new step of calcium homeostasis can be viewed as a general cellular adaptative mechanism to reduce further brain damage.

Separation effect and perception of pain and discomfort from two types of orthodontic separators.

AIM: To examine two types of orthodontic separators, focusing on the separating effect and patients' perception of pain and discomfort. METHODS: The separators tested were spring-type and elastomeric separators. Thirty teenagers participated, and all were scheduled for treatment with a fixed orthodontic appliance. Two spring-type and two elastomeric separators were placed alternately in the left or the right quadrant. After a separation period of 5 days, the amount of separation was measured with a leaf gauge. Nine questionnaires with visual analogue scales and questions with fixed answers were used to register the patient perceptions. RESULTS: The mean separation was 0.3 mm for the spring-type and 0.4 mm for the elastomeric separators (P < .05). The springs were considered less painful than the elastomerics, but the difference was not statistically significant. For both separators, the pain was worst at day 2 and subsided almost completely by day 5. Due to pain, 14 of the 30 patients changed their food habits, and 13 took analgesics. CONCLUSIONS: The separation effect of the two separators was considered clinically equivalent and since pain of moderate intensity occurs during the separation period, analgesics and soft food can be recommended.