Peirang Cao

The muscle-specific ubiquitin ligase atrogin-1/MAFbx mediates statin-induced muscle toxicity

Statins inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis, and are widely used to treat hypercholesterolemia. These drugs can lead to a number of side effects in muscle, including muscle fiber breakdown; however, the mechanisms of muscle injury by statins are poorly understood. We report that lovastatin induced the expression of atrogin-1, a key gene involved in skeletal muscle atrophy, in humans with statin myopathy, in zebrafish embryos, and in vitro in murine skeletal muscle cells. In cultured mouse myotubes, atrogin-1 induction following lovastatin treatment was accompanied by distinct morphological changes, largely absent in atrogin-1 null cells. In zebrafish embryos, lovastatin promoted muscle fiber damage, an effect that was closely mimicked by knockdown of zebrafish HMG-CoA reductase. Moreover, atrogin-1 knockdown in zebrafish embryos prevented lovastatin-induced muscle injury. Finally, overexpression of PGC-1alpha, a transcriptional coactivator that induces mitochondrial biogenesis and protects against the development of muscle atrophy, dramatically prevented lovastatin-induced muscle damage and abrogated atrogin-1 induction both in fish and in cultured mouse myotubes. Collectively, our human, animal, and in vitro findings shed light on the molecular mechanism of statin-induced myopathy and suggest that atrogin-1 may be a critical mediator of the muscle damage induced by statins.

Expression of the Irisin Precursor FNDC5 in Skeletal Muscle Correlates With Aerobic Exercise Performance in Patients With Heart Failure

Background—Exercise-induced increase in peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr; (PGC-1&agr;) expression has been shown to increase the expression of the fibronectin type III domain containing 5 (FNDC5) gene and thereby its product, irisin, in mice. Given that exercise intolerance is a hallmark characteristic of heart failure (HF), and because PGC-1&agr; and irisin promote exercise benefits in animals, we hypothesized that expression of these genes relates to aerobic performance in patients with HF. Methods and Results—Systolic HF (left ventricular ejection fraction ⩽40%) patients underwent cardiopulmonary exercise testing to evaluate aerobic performance. High versus low aerobic performance was assessed using oxygen consumption (peak VO2 [>14 versus ⩽14 mL O2·kg−1·min−1]) and ventilatory efficiency (VE/VCO2 slope [<34 versus ≥34]). Muscle biopsies of the vastus lateralis and real-time polymerase chain reaction were used to quantify muscle gene expression. Twenty-four patients were studied. FNDC5 (5.7±3.5 versus 3.1±1.2, P<0.05) and PGC-1&agr; (9.9±5.9 versus 4.5±1.9, P<0.01) gene expressions were greater in the high-peak VO2 group; correlation between FNDC5 and PGC-1&agr; was significant (r=0.56, P<0.05) only in the high-peak VO2 group. Similarly, FNDC5 and PGC-1&agr; gene expression was greater in the high-performance group based on lower VE/VCO2 slopes (5.8±3.6 versus 3.3±1.4, P<0.05 and 9.7±6 versus 5.3±3.4, P<0.05); FNDC5 also correlated with PGC-1&agr; (r=0.55, P<0.05) only in the low VE/VCO2 slope group. Conclusions—This is the first study to show that FNDC5 expression relates to functional capacity in a human HF population. Lower FNDC5 expression may underlie reduced aerobic performance in HF patients.

Statin-induced muscle damage and atrogin-1 induction is the result of a geranylgeranylation defect

Statins are widely used to treat hypercholesterolemia but can lead to a number of side effects in muscle, including rhabdomyolysis. Our recent findings implicated the induction of atrogin‐1, a gene required for the development of muscle atrophy, in statin‐induced muscle damage. Since statins inhibit many biochemical reactions besides cholesterol synthesis, we sought to define the statin‐inhibited pathways responsible for atrogin‐1 expression and muscle damage. We report here that lovastatin‐induced atrogin‐1 expression and muscle damage in cultured mouse myotubes and zebrafish can be prevented in the presence of geranylgeranol but not farnesol. Further, inhibitors of the transfer of geranylgeranyl isoprene units to protein targets cause statin muscle damage and atrogin‐1 induction in cultured cells and in fish. These findings support the concept that dysfunction of small GTP‐binding proteins lead to statin‐induced muscle damage since these molecules require modification by geranylgeranyl moieties for their cellular localization and activity. Collectively, our animal and in vitro findings shed light on the molecular mechanism of statin‐induced myopathy and suggest that atrogin‐1 may be regulated by novel signaling pathways.—Cao, P., Hanai, J., Tanksale, P., Imamura, S., Sukhatme, V. P., Lecker, S. H. Statin‐induced muscle damage and atrogin‐1 induction is the result of a geranylgeranylation defect. FASEB J. 23, 2844–2854 (2009). www.fasebj.org

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

AIMS Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. METHODS AND RESULTS Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. CONCLUSION These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Mechanisms Involved in 3,5-Cyclic Adenosine Monophosphate-Mediated Inhibition of the Ubiquitin-Proteasome System in Skeletal Muscle

Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.

Atrogin-1 Affects Muscle Protein Synthesis and Degradation When Energy Metabolism Is Impaired by the Antidiabetes Drug Berberine

OBJECTIVE Defects in insulin/IGF-1 signaling stimulate muscle protein loss by suppressing protein synthesis and increasing protein degradation. Since an herbal compound, berberine, lowers blood levels of glucose and lipids, we proposed that it would improve insulin/IGF-1 signaling, blocking muscle protein losses. RESEARCH DESIGN AND METHODS We evaluated whether berberine ameliorates muscle atrophy in db/db mice, a model of type 2 diabetes, by measuring protein synthesis and degradation in muscles of normal and db/db mice treated with or without berberine. We also examined mechanisms for berberine-induced changes in muscle protein metabolism. RESULTS Berberine administration decreased protein synthesis and increased degradation in muscles of normal and db/db mice. The protein catabolic mechanism depended on berberine-stimulated expression of the E3 ubiquitin ligase, atrogin-1. Atrogin-1 not only increased proteolysis but also reduced protein synthesis by mechanisms that were independent of decreased phosphorylation of Akt or forkhead transcription factors. Impaired protein synthesis was dependent on a reduction in eIF3-f, an essential regulator of protein synthesis. Berberine impaired energy metabolism, activating AMP-activated protein kinase and providing an alternative mechanism for the stimulation of atrogin-1 expression. When we increased mitochondrial biogenesis by expressing peroxisome proliferator–activated receptor γ coactivator-1α, berberine-induced changes in muscle protein metabolism were prevented. CONCLUSIONS Berberine impairs muscle metabolism by two novel mechanisms. It impairs mitochonidrial function stimulating the expression of atrogin-1 without affecting phosphorylation of forkhead transcription factors. The increase in atrogin-1 not only stimulated protein degradation but also suppressed protein synthesis, causing muscle atrophy.

Effect of water content on thermal oxidation of oleic acid investigated by combination of EPR spectroscopy and SPME-GC-MS/MS

Promotion of water to the thermal oxidation of oleic acid was detected by the combination of EPR, SPME-GC-MS/MS and GC. Spin-trapping technique was used to identify and quantify the radical species formed during thermal oxidation of oleic acid by using DMPO as electron spin trap. The most abundant radical species were identified as DMPO-alkyl radical adducts. EPR intensity plateau of the samples with 5% water content was 140% higher than the samples without water. It implies oleic acid samples with high water content had high level of oxidation rates. The proportion of aldehydes of the samples with 2% water content was the maximum about 59.97%. Among the formed products, (E,E)-2,4-decadienal has genotoxic and cytotoxic effects, whose percentage was nearly twice comparing with that of 5-0% water content. This study demonstrated that higher water content in frying systems would contribute to seriously oxidation and degradation of oleic acids.

Digestion fates of different edible oils vary with their composition specificities and interactions with bile salts

The digestion fates of different edible oils are different. The objective of this study was to understand the influences of lipid composition on their digestion fates, and investigate the roles of bile salts (BS) played in emulsified lipid system (whey protein isolate as emulsifier) in the in-vitro small intestine digestion stage. Three typical oils (palm oil (PO), rapeseed oil (RO) and linseed oil (LINO)) were chosen. Results showed that with the BS addition increased from 0.0 to 2.0 mg/mL, the increasing magnitude of the different fatty acid (FA) apparent release rate constants were: PO > RO ≈ LINO. Although the maximum FA release extent changed with BS addition, the order were: PO > RO > LINO. These may probably be attributed to palmitic acids, the most abundant FA in PO, was mostly located on the Sn-1, 3 positions of triacylglycerol (TAG) molecules, which contributed to the pancreatic lipase hydrolysis action. The relatively short chain length and the lower hydrophobicity also favored this process. However, Sn-1, 3 positions of TAGs in RO and LINO were mainly long chain mono- or poly-unsaturated FAs, which restricted the continuous lipid hydrolysis. Furthermore, the lipid composition may also affect the BS behavior on the O/W emulsion droplet surface, thus modulating lipase hydrolysis reaction. These findings can provide some basic understandings of the digestion differences of different oils.

Non-triglyceride components modulate the fat crystal network of palm kernel oil and coconut oil

PKO and CNO are composed of 97-98% triacylglycerols and 2-3% minor non-triglyceride components (FFA, DAG and MAG). Triglycerides were separated from minor components by chromatographic method. The lipid composition, thermal properties, polymorphism, isothermal crystallization behavior, nanostructure and microstructure of PKO, PKO-TAG, CNO and CNO-TAG were evaluated. Removal of minor components had no effect on lipid composition and equilibrium solid fat contents. However, presence of minor components did increase the slip melting point and promoted the onset of crystallization from DSC crystallization profiles. The thickness of the nanoscale crystals increased with no polymorphic transformation after removing the minor components. Crystallization kinetics revealed that minor components decreased crystal growth rate with higher t1/2. Sharp changes in the values of the Avrami constant k and exponent n were observed for all fats around 10°C. Increases in n around 10°C indicated a change from one-dimensional to multi-dimensional growth. From the results of polarized light micrographs, the transformation from the coarser crystal structure to tiny crystal structure occurred in microstructure networks at the action of minor components.

Effects of Polar Compounds Generated from the Deep-Frying Process of Palm Oil on Lipid Metabolism and Glucose Tolerance in Kunming Mice

In the present study, effects of deep-fried palm oil, specifically polar compounds generated during the frying process, on animal health including lipid and glucose metabolism and liver functions were investigated. Kunming mice were fed a high-fat diet containing deep-fried palm oil or purified polar compounds for 12 weeks. Their effects on animal health including hepatic lipid profile, antioxidant enzyme activity, serum biochemistry, and glucose tolerance were analyzed. Our results revealed that the consumption of polar compounds was related to the change of lipid deposition in liver and adipose tissue, as well as glucose tolerance alteration in Kunming mice. Correspondingly, the transcription study of genes involved in lipid metabolism including PPARα, Acox1, and Cpt1α indicated that polar compounds probably facilitated the fatty acid oxidation on peroxisomes, whereas lipid oxidation in mitochondria was suppressed. Furthermore, glucose tolerance test (GTT) revealed that a high amount of polar compound intake impaired glucose tolerance, indicating its effect on glucose metabolism in vivo. Our results provide critical information on the effects of polar compounds generated from the deep-frying process of palm oil on animal health, particularly liver functions and lipid and glucose metabolism, which is important for the evaluation of the biosafety of frying oil.