The Interrelationship between Polyunsaturated Fatty Acids, α-Tocopherol and Glutathione Peroxidase in Chicken and Porcine Skeletal Muscles

Two species of chrysomelid leaf beetles found in Brazil, Diabrotica speciosa and Cerotoma arcuata, are strongly attracted to the root of Ceratosanthes hilariana (Cucurbitaceae). Root extracts stimulate a compulsive feeding response. The major feeding stimulants isolated from these extracts were cucurbitacin B and its 23,24-dihydro derivative.     

Requirement of Plasminogen Binding to Its Cell-Surface Receptor α-Enolase for Efficient Regeneration of Normal and Dystrophic Skeletal Muscle

Adult regenerative myogenesis is central for restoring normal tissue structure and function after muscle damage. In muscle repair after injury, as in severe myopathies, damaged and necrotic fibers are removed by infiltrating inflammatory cells and then replaced by muscle stem cells or satellite cells, which will fuse to form new myofibers. Extracellular proteolysis mediated by uPA-generated plasmin plays a critical role in controlling inflammation and satellite-cell-dependent myogenesis. α-enolase has been described as plasminogen receptor in several cell types, where it acts concentrating plasmin proteolytic activity on the cell surface. In this study, we investigated whether α-enolase plasminogen receptor plays a regulatory role during the muscular repair process. Inhibitors of α-enolase/plasminogen binding: MAb11G1 (a monoclonal antibody against α-enolase) and ε-aminocaproic acid, EACA (a lysine analogue) inhibited the myogenic abilities of satellite cells-derived myoblasts. Furthermore, knockdown of α-enolase decreased myogenic fusion of myoblasts. Injured wild-type mice and dystrophic mdx mice were also treated with MAb11G1 and EACA. These treatments had negative impacts on muscle repair impairing satellite cell functions in vitro in agreement with blunted growth of new myofibers in vivo. Furthermore, both MAb11G1 and EACA treatments impaired adequate inflammatory cell infiltration and promoted extracellular matrix deposition in vivo, which resulted in persistent degeneration. These results demonstrate the novel requirement of α-enolase for restoring homeostasis of injured muscle tissue, by controlling the pericellular localization of plasmin activity.     

Sphingosine Kinase 1 Is Regulated by Peroxisome Proliferator-activated Receptor α in Response to Free Fatty Acids and Is Essential for Skeletal Muscle Interleukin-6 Production and Signaling in Diet-induced Obesity

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3)_. Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1^−/− mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1^−/− mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.     

Effect of Animal and Industrial Trans Fatty Acids on HDL and LDL Cholesterol Levels in Humans – A Quantitative Review

Background

Trans fatty acids are produced either by industrial hydrogenation or by biohydrogenation in the rumens of cows and sheep. Industrial trans fatty acids lower HDL cholesterol, raise LDL cholesterol, and increase the risk of coronary heart disease. The effects of conjugated linoleic acid and trans fatty acids from ruminant animals are less clear. We reviewed the literature, estimated the effects trans fatty acids from ruminant sources and of conjugated trans linoleic acid (CLA) on blood lipoproteins, and compared these with industrial trans fatty acids.

Methodology/Principal Findings

We searched Medline and scanned reference lists for intervention trials that reported effects of industrial trans fatty acids, ruminant trans fatty acids or conjugated linoleic acid on LDL and HDL cholesterol in humans. The 39 studies that met our criteria provided results of 29 treatments with industrial trans fatty acids, 6 with ruminant trans fatty acids and 17 with CLA. Control treatments differed between studies; to enable comparison between studies we recalculated for each study what the effect of trans fatty acids on lipoprotein would be if they isocalorically replaced cis mono unsaturated fatty acids. In linear regression analysis the plasma LDL to HDL cholesterol ratio increased by 0.055 (95%CI 0.044–0.066) for each % of dietary energy from industrial trans fatty acids replacing cis monounsaturated fatty acids The increase in the LDL to HDL ratio for each % of energy was 0.038 (95%CI 0.012–0.065) for ruminant trans fatty acids, and 0.043 (95% CI 0.012–0.074) for conjugated linoleic acid (p = 0.99 for difference between CLA and industrial trans fatty acids; p = 0.37 for ruminant versus industrial trans fatty acids).

Conclusions/Significance

Published data suggest that all fatty acids with a double bond in the trans configuration raise the ratio of plasma LDL to HDL cholesterol.     

Differential Membrane Localization and Intermolecular Associations of α-Dystrobrevin Isoforms in Skeletal Muscle

α-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length α-dystrobrevin-1 (84 kD), and COOH-terminal truncated α-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that α-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. α-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, α-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro–translated proteins, α-dystrobrevin-2 binds dystrophin, whereas α-dystrobrevin-1 binds both dystrophin and utrophin. α-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, α-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of α-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the α-dystrobrevins.     

Inhibition of Autophagic Turnover in β-Cells by Fatty Acids and Glucose Leads to Apoptotic Cell Death

Autophagy, a cellular recycling process responsible for turnover of cytoplasmic contents, is critical for maintenance of health. Defects in this process have been linked to diabetes. Diabetes-associated glucotoxicity/lipotoxicity contribute to impaired β-cell function and have been implicated as contributing factors to this disease. We tested the hypothesis that these two conditions affect β-cell function by modulating autophagy. We report that exposure of β-cell lines and human pancreatic islets to high levels of glucose and lipids blocks autophagic flux and leads to apoptotic cell death. EM analysis showed accumulation of autophagy intermediates (autophagosomes), with abundant engulfed cargo in palmitic acid (PA)- or glucose-treated cells, indicating suppressed autophagic turnover. EM studies also showed accumulation of damaged mitochondria, endoplasmic reticulum distention, and vacuolar changes in PA-treated cells. Pulse-chase experiments indicated decreased protein turnover in β-cells treated with PA/glucose. Expression of mTORC1, an inhibitor of autophagy, was elevated in β-cells treated with PA/glucose. mTORC1 inhibition, by treatment with rapamycin, reversed changes in autophagic flux, and cell death induced by glucose/PA. Our results indicate that nutrient toxicity-induced cell death occurs via impaired autophagy and is mediated by activation of mTORC1 in β-cells, contributing to β-cell failure in the presence of metabolic stress.     

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

Fat Oxidation,Fitness and Skeletal Muscle Expression of Oxidative/Lipid Metabolism Genes in South Asians: Implications for Insulin Resistance?

Background

South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.

Methodology/Principal Findings

Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO_2max (40.6±6.6 vs 52.4±5.7 ml.kg⁻¹.min⁻¹, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg⁻¹.min⁻¹ at 55% VO_2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg⁻¹.min⁻¹ at 25 ml O₂.kg⁻¹.min⁻¹, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO_2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO_2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.

Conclusions/Significance

These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.     

E3 Ubiquitin Ligase,WWP1, Interacts with AMPKα2 and Down-regulates Its Expression in Skeletal Muscle C2C12 Cells

It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway.     

Relationship between signal transduction and PPARα-regulated genes of lipid metabolism in rat hepatic-derived Fao cells

The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of ³²P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, α isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.     

Associations of Whole Blood n-3 and n-6 Polyunsaturated Fatty Acids with Blood Pressure in Children and Adolescents – Results from the IDEFICS/I.Family Cohort

BackgroundPolyunsaturated n-3 and n-6 polyunsaturated fatty acids (PUFA) are precursors of biologically active metabolites that affect blood pressure (BP) regulation. This study investigated the association of n-3 and n-6 PUFA and BP in children and adolescents.MethodsIn a subsample of 1267 children aged 2–9 years at baseline of the European IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) cohort whole blood fatty acids were measured by a validated gas chromatographic method. Systolic and diastolic BP was measured at baseline and after two and six years. Mixed-effects models were used to assess the associations between fatty acids at baseline and BP z-scores over time adjusting for relevant covariables. Models were further estimated stratified by sex and weight status.ResultsThe baseline level of arachidonic acid was positively associated with subsequent systolic BP (β = 0.08, P = 0.002) and diastolic BP (β = 0.07, P<0.001). In thin/normal weight children, baseline alpha-linolenic (β = -1.13, P = 0.003) and eicosapentaenoic acid (β = -0.85, P = 0.003) levels were inversely related to baseline and also to subsequent systolic BP and alpha-linolenic acid to subsequent diastolic BP. In overweight/obese children, baseline eicosapentaenoic acid level was positively associated with baseline diastolic BP (β = 0.54, P = 0.005).ConclusionsLow blood arachidonic acid levels in the whole sample and high n-3 PUFA levels in thin/normal weight children are associated with lower and therefore healthier BP. The beneficial effects of high n-3 PUFA on BP were not observed in overweight/obese children, suggesting that they may have been overlaid by the unfavorable effects of excess weight.     

Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin β-4 in the Dystrophin Deficient Mouse

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tβ4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8–10 weeks old, were treated with 150 µg of Tβ4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tβ4 and amount of fibrosis were quantified using immunohistochemistry and Gomori''s tri-chrome staining, respectively. Mdx mice treated with Tβ4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tβ4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tβ4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.     

Pain in Parkinson′s Disease: A Cross-Sectional Study of Its Prevalence,Types, and Relationship to Depression and Quality of Life

Pain is an important and distressing symptom in Parkinson’s disease (PD). Our aim was to determine the prevalence of pain, its various types and characteristics, as well as its impact on depression and quality of life (QoL) in patients with PD. How pain differs in early- and advanced-stage PD and male and female PD patients was of special interest. One hundred PD patients on dopaminergic medications had a neurological examination and participated in a structured interview on pain characteristics and completed standardized questionnaires. A total of 76% of the patients had pain. The following types of pain were present: musculoskeletal pain accounted for 41% of the total pain, dystonic pain for 17%, central neuropathic pain for 22%, radicular pain for 27%, and other pains (non-radicular low back pain, arthritic, and visceral pain) made up 24%. One type of pain affected 29% of all the subjects, two types 35%, three types 10%, and four types of pain were reported by 2%. All types of pain were more prevalent in advanced-stage PD subjects than in early-stage PD subjects, except for arthritic pain (subclassified under”other pain”). The frequency and intensity of actual, average, and worst experienced pain were significantly more severe in advanced-stage subjects. PD subjects with general pain and in advanced stages were more depressed and had poorer QoL. Depression correlated with worst pain in the last 24 hours and with pain periodicity (the worst depression score in patients with constant pain). QoL correlated with average pain in the last 7 days. Pain is a frequent problem in PD patients, and it worsens during the course of the disease.     

The Effect of Neoadjuvant Chemoradiotherapy on Whole-Body Physical Fitness and Skeletal Muscle Mitochondrial Oxidative Phosphorylation In Vivo in Locally Advanced Rectal Cancer Patients – An Observational Pilot Study

Background

In the United Kingdom, patients with locally advanced rectal cancer routinely receive neoadjuvant chemoradiotherapy. However, the effects of this on physical fitness are unclear. This pilot study is aimed to investigate the effect of neoadjuvant chemoradiotherapy on objectively measured in vivo muscle mitochondrial function and whole-body physical fitness.

Methods

We prospectively studied 12 patients with rectal cancer who completed standardized neoadjuvant chemoradiotherapy, recruited from a large tertiary cancer centre, between October 2012 and July 2013. All patients underwent a cardiopulmonary exercise test and a phosphorus magnetic resonance spectroscopy quadriceps muscle exercise-recovery study before and after neoadjuvant chemoradiotherapy. Data were analysed and reported blind to patient identity and clinical course. Primary variables of interest were the two physical fitness measures; oxygen uptake at estimated anaerobic threshold and oxygen uptake at Peak exercise (ml.kg⁻¹.min⁻¹), and the post-exercise phosphocreatine recovery rate constant (min⁻¹), a measure of muscle mitochondrial capacity in vivo.

Results

Median age was 67 years (IQR 64–75). Differences (95%CI) in all three primary variables were significantly negative post-NACRT: Oxygen uptake at estimated anaerobic threshold −2.4 ml.kg⁻¹.min⁻¹ (−3.8, −0.9), p = 0.004; Oxygen uptake at Peak −4.0 ml.kg⁻¹.min⁻¹ (−6.8, −1.1), p = 0.011; and post-exercise phosphocreatine recovery rate constant −0.34 min⁻¹ (−0.51, −0.17), p<0.001.

Conclusion

The significant decrease in both whole-body physical fitness and in vivo muscle mitochondrial function raises the possibility that muscle mitochondrial mechanisms, no doubt multifactorial, may be important in deterioration of physical fitness following neoadjuvant chemoradiotherapy. This may have implications for targeted interventions to improve physical fitness pre-surgery.

Trial Registration

Clinicaltrials.gov registration {"type":"clinical-trial","attrs":{"text":"NCT01859442","term_id":"NCT01859442"}}NCT01859442     

Secretory Cavity Development and Its Relationship with the Accumulation of Essential Oil in Fruits of Citrus medica L. var. sarcodactylis （Noot.） Swingle

The developmental types of secretory cavities in Citrus remain controversial. The relationship between secretory cavity development and the accumulation of essential oil in fruits of Citrus species is also unknown. In order to develop better insights into these problems, histological, histochemical, and cytochemical methods were used to investigate secretory cavity development and the accumulation of essential oll at different developmental stages of fruits of Citrus medica L. var. sarcodactylis （Noot.） Swingle. The results indicate that the secretory cavity of the variety seemed to originate from an epidermal cell and a subepldermal cell. These two cells underwent successive divisions, resulting In the formation of two parts： （Ⅰ） a conical cap; and （Ⅱ） a globular gland. The formation of the lumen was schlzolysigenous. Regular changes in the size of vacuoles and the accumulation of essential oil were revealed during the process of secretory cavity development. In addition, when fruits were a light yellow or golden color, the structure of secretory cavities was well developed and the content of essential oil in a single fruit reached a maximum. It would be most appropriate to collect the fruit as a medicinal material at this time.