Amy E. Larsen

The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy

Skeletal muscle atrophy occurs in many chronic diseases and disuse conditions. Its severity reduces patient recovery, independence and quality of life. The discovery of two muscle-specific E3 ubiquitin ligases, MAFbx/atrogin-1 and Muscle RING Finger-1 (MuRF1), promoted an expectation of these molecules as targets for therapeutic development. While numerous studies have determined the conditions in which MAFbx/atrogin-1 and MuRF1 mRNA levels are regulated, few studies have investigated their functional role in skeletal muscle. Recently, studies identifying new target substrates for MAFbx/atrogin-1 and MuRF1, outside of their response to the initiation of muscle atrophy, suggest that there is more to these proteins than previously appreciated. This review will highlight our present knowledge of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy, the impact of potential therapeutics and their known regulators and substrates. Finally, we will comment on new approaches that may expand our knowledge of these two molecules in their control of skeletal muscle function.

Actions of short-term fasting on human skeletal muscle myogenic and atrogenic gene expression

Background: Skeletal muscle mass is governed by multiple IGF-1-sensitive positive regulators of muscle-specific protein synthesis (myogenic regulatory factors which includes myoD, myogenin and Myf5) and negative regulators, including the atrogenic proteins myostatin, atrogin-1 and muscle ring finger 1 (MuRF-1). The coordinated control of these myogenic and atrogenic factors in human skeletal muscle following short-term fasting is currently unknown. Method: Healthy adults (n = 6, age 27.6 years) undertook a 40-hour fast. Skeletal muscle biopsy (vastus lateralis) and venous blood samples were taken 3, 15 and 40 h into the fast after an initial standard high-carbohydrate meal. Gene expression of the myogenic regulator factors (myoD, myogenin and Myf5) and the atrogenic factors (myostatin, atrogin-1 and MuRF-1) were determined by real-time PCR analysis. Plasma myostatin and IGF-1 were determined by ELISA. Results: There were no significant alterations in either the positive or negative regulators of muscle mass at either 15 or 40 h, when compared to gene expression measured 3 h after a meal. Similarly, plasma myostatin and IGF-1 were also unaltered at these times. Conclusions: Unlike previous observations in catabolic and cachexic diseased states, short-term fasting (40 h) fails to elicit marked alteration of the genes regulating both muscle-specific protein synthesis or atrophy. Greater periods of fasting may be required to initiate coordinated inhibition of myogenic and atrogenic gene expression.

Resistance exercise increases NF-κB activity in human skeletal muscle

Intense resistance exercise causes a significant inflammatory response. NF-κB has been identified as a prospective key transcription factor mediating the postexercise inflammatory response. The purpose of this study was to determine whether a single bout of intense resistance exercise regulates NF-κB signaling in human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis of five recreationally active, but not strength-trained, males (21.9 ± 1.3 yr) prior to, and at 2 and 4 h following, a single bout of intense resistance exercise. A further five subjects (4 males, 1 female) (23 ± 0.89 yr) were recruited as a nonexercise control group to examine the effect of the muscle biopsy protocol on key markers of skeletal muscle inflammation. Protein levels of IκBα and phosphorylated NF-κB (p65), as well as the mRNA expression of inflammatory myokines monocyte chemoattractant protein 1 (MCP-1), IL-6, and IL-8 were measured. Additionally, NF-κB (p65) DNA binding to the promoter regions of MCP-1, IL-6, and IL-8 was investigated. IκBα protein levels decreased, while p-NF-κB (p65) protein levels increased 2 h postexercise and returned to near-baseline levels by 4-h postexercise. Immunohistochemical data verified these findings, illustrating an increase in p-NF-κB (p65) protein levels, and nuclear localization at 2 h postexercise. Furthermore, NF-κB DNA binding to MCP-1, IL-6, and IL-8 promoter regions increased significantly 2 h postexercise as did mRNA levels of these myokines. No significant change was observed in the nonexercise control group. These novel data provide evidence that intense resistance exercise transiently activates NF-κB signaling in human skeletal muscle during the first few hours postexercise. These findings implicate NF-κB in the transcriptional control of myokines known to be central to the postexercise inflammatory response.

Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid.

In contrast to the well‐characterized effects of specialized proresolving lipid mediators (SPMs) derived fromeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), little is known about themetabolic fate of the intermediary long‐chain (LC) n‐3 polyunsaturated fatty acid (PUFA) docosapentaenoic acid (DPA). In this double blind crossover study, shifts in circulating levels of n‐3 and n‐6 PUFA‐derived bioactive lipid mediators were quantified by an unbiased liquid chromatography‐tandem mass spectrometry lipidomic approach. Plasma was obtainedfrom humansubjects before andafter 7 d of supplementationwith pure n‐3DPA, n‐3 EPA or placebo (olive oil). DPA supplementation increased the SPM resolvin D5n‐3DPA (RvD5n‐3DPA) and maresin (MaR)‐1, the DHA vicinal diol 19, 20‐dihydroxy‐DPA and n‐6 PUFA derived 15‐keto‐PG E2 (15‐keto‐PGE2). EPA supplementation had no effect on any plasma DPA or DHA derived mediators, but markedly elevated monohydroxy‐eicosapentaenoic acids (HEPEs), including the e‐series resolvin (RvE) precursor 18‐HEPE; effects not observed with DPA supplementation. These data show that dietary n‐3 DPA and EPA have highly divergent effects on human lipid mediator profile, with no overlap in PUFA metabolites formed. The recently uncovered biologic activity of n‐3 DPA docosanoids and their marked modulation by dietary DPA intake reveals a unique and specific role of n‐3 DPA in human physiology.—Markworth, J. F., Kaur, G., Miller, E.G., Larsen, A. E., Sinclair, A. J., Maddipati, K.R., Cameron‐Smith, D. Divergent shifts in lipid mediator profile following supplementation with n‐3 docosapentaenoic acid and eicosapentaenoic acid. FASEB J. 30, 3714–3725 (2016) www.fasebj.org

Postprandial metabolism of docosapentaenoic acid (DPA, 22:5n−3) and eicosapentaenoic acid (EPA, 20:5n−3) in humans

The study of the metabolism of docosapentaenoic acid (DPA, 22:5n-3) in humans has been limited by the unavailability of pure DPA and the fact that DPA is found in combination with eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in natural products. In this double blind cross over study, pure DPA and EPA were incorporated in meals served to healthy female volunteers. Mass spectrometric methods were used to study the chylomicron lipidomics. Plasma chylomicronemia was significantly reduced after the meal containing DPA compared with the meal containing EPA or olive oil only. Both EPA and DPA were incorporated into chylomicron TAGs, while there was less incorporation into chylomicron phospholipids. Lipidomic analysis of the chylomicron TAGs revealed the dynamic nature of chylomicron TAGs. The main TAG species that EPA and DPA were incorporated into were EPA/18:1/18:1, DPA/18:1/16:0 and DPA/18:1/18:1. There was very limited conversion of DPA and EPA to DHA and there were no increases in EPA levels during the 5h postprandial period after the DPA meal. In conclusion, EPA and DPA showed different metabolic fates, and DPA hindered the digestion, ingestion or incorporation into chylomicrons of the olive oil present in the meal.

Postprandial Plasma Phospholipids in Men Are Influenced by the Source of Dietary Fat

BACKGROUND Postprandial lipemia represents a risk factor for chronic diseases, including type 2 diabetes. Little is known about the effect of dietary fat on the plasma lipidome in the postprandial period. OBJECTIVE The objective of this study was to assess the effect of dairy fat and soy oil on circulating postprandial lipids in men. METHODS Men (40-60 y old, nonsmokers; n = 16) were randomly assigned in a crossover design to consume 2 breakfast meals of dairy-based or soy oil-based foods. The changes in the plasma lipidome during the 4-h postprandial period were analyzed with electrospray ionization tandem mass spectrometry and included 316 lipid species in 23 classes and subclasses, representing sphingolipids, phospholipids, glycerolipids, and sterols. RESULTS Nonparametric Friedman tests showed significant changes in multiple plasma lipid classes, subclasses, and species in the postprandial period after both dairy and soy meals. No difference was found in triglyceridemia after each meal. However, 6 endogenous lipid classes increased after dairy but decreased after soy (P < 0.05), including ether-linked phospholipids and plasmalogens and sphingomyelin (not present in soy), dihexosylceramide, and GM3 ganglioside. Phosphatidylcholine and phosphatidylinositol were not affected by the soy meal but were significantly elevated after the dairy meal (8.3% and 16%, respectively; P < 0.05). CONCLUSIONS The changes in postprandial plasma phospholipids in men relate to the diet composition and the relative size of the endogenous phospholipid pools. Despite similar lipemic responses as measured by changes in triglyceride concentrations, the differential responses to dairy and soy meals derived through lipidomic analysis of phospholipids suggest differences in the metabolism of soybean oil and dairy fat. The increased concentrations of plasmalogens, with potential antioxidant capacity, in the postprandial period after dairy but not soy meals may represent a further important difference in the response to these sources of fat. The trial was registered at www.anzctr.org.au as ACTRN12610000562077.

Lipidomic Profiling of Chylomicron Triacylglycerols in Response to High Fat Meals

Using lipidomic methodologies the impact that meal lipid composition and metabolic syndrome (MetS) exerts on the postprandial chylomicron triacylglycerol (TAG) response was examined. Males (9 control; 11 MetS) participated in a randomised crossover trial ingesting two high fat breakfast meals composed of either dairy-based foods or vegetable oil-based foods. The postprandial lipidomic molecular composition of the TAG in the chylomicron-rich (CM) fraction was analysed with tandem mass spectrometry coupled with liquid chromatography to profile CM TAG species and targeted TAG regioisomers. Postprandial CM TAG concentrations were significantly lower after the dairy-based foods compared with the vegetable oil-based foods for both control and MetS subjects. The CM TAG response to the ingested meals involved both significant and differential depletion of TAG species containing shorter- and medium-chain fatty acids (FA) and enrichment of TAG molecular species containing C16 and C18 saturated, monounsaturated and diunsaturated FA. Furthermore, there were significant changes in the TAG species between the food TAG and CM TAG and between the 3- and 5-h postprandial samples for the CM TAG regioisomers. Unexpectedly, the postprandial CM TAG concentration and CM TAG lipidomic responses did not differ between the control and MetS subjects. Lipidomic analysing of CM TAG molecular species revealed dynamic changes in the molecular species of CM TAG during the postprandial phase suggesting either preferential CM TAG species formation and/or clearance.

Effects of Conjugated Linoleic Acid on Myogenic and Inflammatory Responses in a Human Primary Muscle and Tumor Coculture Model

The antiproliferative and anti-inflammatory properties of conjugated linoleic acid (CLA) make it a potentially novel treatment in chronic inflammatory muscle wasting disease, particularly cancer cachexia. Human primary muscle cells were grown in coculture with MIA PaCa-2 pancreatic tumor cells and exposed to varying concentrations of c9,t11 and t10,c12 CLA. Expression of myogenic (Myf5, MyoD, myogenin, and myostatin) and inflammatory genes (CCL-2, COX-2, IL-8, and TNF-α) were measured by real-time PCR. The t10,c12 CLA isomer, but not the c9,t11 isomer, significantly decreased MIA PaCa-2 proliferation by between 15% and 19%. There was a marked decrease in muscle MyoD and myogenin expression (78% and 62%, respectively), but no change in either Myf5 or myostatin, in myotubes grown in coculture with MIA PaCa-2 cells. CLA had limited influence on these responses. A similar pattern of myogenic gene expression changes was observed in myotubes treated with TNF-α alone. Several-fold significant increases in CCL-2, COX-2, IL-8, and TNF-α expression in myotubes were observed with MIA PaCa-2 coculture. The c9,t11 CLA isomer significantly decreased basal expression of TNF-α in myotubes and could ameliorate its tumor-induced rise. The study provides insight into the anti-inflammatory and antiproliferative actions of CLA and its application as a therapeutic agent in inflammatory disease states.

Postprandial Responses to Lipid and Carbohydrate Ingestion in Repeated Subcutaneous Adipose Tissue Biopsies in Healthy Adults

Adipose tissue is a primary site of meta-inflammation. Diet composition influences adipose tissue metabolism and a single meal can drive an inflammatory response in postprandial period. This study aimed to examine the effect lipid and carbohydrate ingestion compared with a non-caloric placebo on adipose tissue response. Thirty-three healthy adults (age 24.5 ± 3.3 year (mean ± standard deviation (SD)); body mass index (BMI) 24.1 ± 3.2 kg/m2, were randomised into one of three parallel beverage groups; placebo (water), carbohydrate (maltodextrin) or lipid (dairy-cream). Subcutaneous, abdominal adipose tissue biopsies and serum samples were collected prior to (0 h), as well as 2 h and 4 h after consumption of the beverage. Adipose tissue gene expression levels of monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) increased in all three groups, without an increase in circulating TNF-α. Serum leptin (0.6-fold, p = 0.03) and adipose tissue leptin gene expression levels (0.6-fold, p = 0.001) decreased in the hours following the placebo beverage, but not the nutrient beverages. Despite increased inflammatory cytokine gene expression in adipose tissue with all beverages, suggesting a confounding effect of the repeated biopsy method, differences in metabolic responses of adipose tissue and circulating adipokines to ingestion of lipid and carbohydrate beverages were observed.

Resistance exercise increases NF-kB activity in human skeletal muscle

Intense resistance exercise causes a significant inflammatory response. NF-κB has been identified as a prospective key transcription factor mediating the postexercise inflammatory response. The purpose of this study was to determine whether a single bout of intense resistance exercise regulates NF-κB signaling in human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis of five recreationally active, but not strength-trained, males (21.9 ± 1.3 yr) prior to, and at 2 and 4 h following, a single bout of intense resistance exercise. A further five subjects (4 males, 1 female) (23 ± 0.89 yr) were recruited as a nonexercise control group to examine the effect of the muscle biopsy protocol on key markers of skeletal muscle inflammation. Protein levels of IκBα and phosphorylated NF-κB (p65), as well as the mRNA expression of inflammatory myokines monocyte chemoattractant protein 1 (MCP-1), IL-6, and IL-8 were measured. Additionally, NF-κB (p65) DNA binding to the promoter regions of MCP-1, IL-6, and IL-8 was investigated. IκBα protein levels decreased, while p-NF-κB (p65) protein levels increased 2 h postexercise and returned to near-baseline levels by 4-h postexercise. Immunohistochemical data verified these findings, illustrating an increase in p-NF-κB (p65) protein levels, and nuclear localization at 2 h postexercise. Furthermore, NF-κB DNA binding to MCP-1, IL-6, and IL-8 promoter regions increased significantly 2 h postexercise as did mRNA levels of these myokines. No significant change was observed in the nonexercise control group. These novel data provide evidence that intense resistance exercise transiently activates NF-κB signaling in human skeletal muscle during the first few hours postexercise. These findings implicate NF-κB in the transcriptional control of myokines known to be central to the postexercise inflammatory response.