The Effect of Cancer Cachexia Progression on the Feeding Regulation of Skeletal Muscle Protein Turnover

Cancer cachexia is defined as the unintentional loss of skeletal muscle mass with or without fat loss that cannot be reversed by conventional nutritional support. Cachexia occurs in ~20% of cancer patients. More specifically, 50% of lung cancer patients, the most common cancer worldwide, develop cachexia. Cachexia occurs most often in lung and gastrointestinal cancers, whereas breast and prostate have the lowest rate of cachexia. Cancer-induced cachexia disrupts skeletal muscle protein turnover (decreasing protein synthesis and increasing protein degradation). Skeletal muscle’s capacity for protein synthesis is highly sensitive to local and systemic stimuli that are controlled by mTORC1 and AMPK signaling. During cachexia, altered protein turnover is thought to occur through suppressed anabolic signaling via mTORC1, coinciding with the chronic activation of AMPK. While progress has been made in understanding some of the mechanisms underlying the suppressed anabolic signaling in cachectic muscle, gaps still remain in our understanding of muscle’s ability to respond to anabolic stimulus prior to cachexia development. The purpose of this study was to determine if cachexia progression disrupts the feeding regulation of AMPK signaling and if gp130 signaling and muscle contraction could regulate this process. Specific aim 1 examined the feeding regulation of skeletal muscle protein synthesis in pre-cachectic tumor bearing mice. Feeding increased muscle protein synthesis, while lowering AMPK signaling in pre-cachectic tumor bearing mice. Importantly, pre-cachectic tumor bearing mice have overall suppressed muscle protein synthesis independent of the fast or fed condition. Muscle specific AMPK loss was sufficient to improve the fasting suppression of muscle mTORC1 and protein synthesis in pre-cachectic tumor bearing mice. Specific aim 2 examined if muscle gp130 signaling regulates the feeding regulation of AMPK during cancer cachexia progression. Muscle gp130 loss lowered the fasting induction of AMPK in pre-cachectic tumor bearing mice without improving protein synthesis. Muscle gp130 loss did not alter the feeding regulation of muscle Akt/mTORC1 signaling and protein synthesis. Specific Aim 3 examined if an acute bout of muscle contractions could improve the muscle protein synthesis response to feeding during the progression of cachexia. Pre-cachectic tumor bearing mice exhibit suppressed protein synthesis in response low frequency electrical stimulation, and the inability to synergistically induce protein synthesis in response to feeding and contraction. In summary, pre-cachectic tumor bearing mice have lowered Akt/mTORC1 signaling and protein synthesis. Feeding can induce Akt/mTORC1 and protein synthesis and AMPK regulates the fasting suppression of protein synthesis in pre-cachectic tumor bearing mice. While gp130 loss reduces AMPK it is not sufficient to improve protein synthesis in pre-cachectic tumor bearing mice. The added protein synthesis response to feeding and contraction is blunted in pre-cachectic tumor bearing mice. These findings provide novel insight into the regulation of Akt/mTORC1 signaling and protein synthesis in response to feeding. Additionally, these studies highlight gp130’s regulation of AMPK prior to cachexia development, and the blunted anabolic muscle response to feeding and contraction in pre-cachectic tumor bearing mice. By understanding these intracellular signaling processes and perturbations prior to cachexia development, we will be able to elucidate potential therapeutic targets and treatment options to manipulate and prevent cancer cachexia.

PSVII-7 Prematurity alters the regulation of Akt signaling in skeletal muscle of piglets

Objectives: Postnatal growth faltering is common after preterm birth. Recently we showed that premature birth in piglets impairs normal postnatal weight gain and skeletal muscle protein synthesis compared to piglets born at term. This response is associated with a reduction in the feeding-induced activation of Akt and subsequent downstream signaling, despite no change in insulin receptor activation. The aim of this study was to identify key regulators of Akt responsible for the blunted anabolic response in preterm muscle. Methods: Piglets delivered by cesarean section 11 d (preterm/PT) or 2 d (term/T) before term birth were fed by total parenteral nutrition. On day 3, after 4 h fasting, piglets were fasted one additional h or fed orally a sow milk replacer (per kg body weight: 31.5 kcal, 1.3 g carbohydrate, 2.7 g amino acids BW, 1.6 g lipid). Positive and negative regulators of Akt activity in longissimus dorsi muscle were determined by Western blot. Results: Phosphorylation of Akt1 and Akt2, but not Akt3, was lower in PT than in T pigs (P < 0.05). Phosphorylation of Akt activators, PDK1 and mTORC2, and the abundance of Ubl4A, a positive regulator of Akt, were lower in PT than in T (P < 0.05). Abundance of Akt inhibitors, PHLPP and SHIP2, but not PTEN, was higher in PT than in T (P < 0.05). Activation of the Akt phosphatase, PP2A, was unaffected by feeding in PT but inhibited by feeding in T pigs (P < 0.05). Conclusions: These results show that the feeding-induced activation of positive regulators of Akt is reduced by preterm birth, whereas the activation of negative regulators is enhanced. Our findings suggest that premature birth impairs the activation of Akt that is essential for channeling dietary nutrients for anabolism and likely contributes to the postnatal growth faltering of prematurity. Research Support: NIH and USDA.

The Regulatory Roles of PPARs in Skeletal Muscle Fuel Metabolism and Inflammation: Impact of PPAR Agonism on Muscle in Chronic Disease, Contraction and Sepsis

The peroxisome proliferator-activated receptor (PPAR) family of transcription factors has been demonstrated to play critical roles in regulating fuel selection, energy expenditure and inflammation in skeletal muscle and other tissues. Activation of PPARs, through endogenous fatty acids and fatty acid metabolites or synthetic compounds, has been demonstrated to have lipid-lowering and anti-diabetic actions. This review will aim to provide a comprehensive overview of the functions of PPARs in energy homeostasis, with a focus on the impacts of PPAR agonism on muscle metabolism and function. The dysregulation of energy homeostasis in skeletal muscle is a frequent underlying characteristic of inflammation-related conditions such as sepsis. However, the potential benefits of PPAR agonism on skeletal muscle protein and fuel metabolism under these conditions remains under-investigated and is an area of research opportunity. Thus, the effects of PPARγ agonism on muscle inflammation and protein and carbohydrate metabolism will be highlighted, particularly with its potential relevance in sepsis-related metabolic dysfunction. The impact of PPARδ agonism on muscle mitochondrial function, substrate metabolism and contractile function will also be described.

α-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFα/IFNγ Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

A Moderate Serving of a Lower-Quality, Incomplete Protein Does Not Stimulate Skeletal Muscle Protein Synthesis

Objectives Dietary proteins can be broadly characterized by their origin (animal-or plant-based) and amino acid composition (complete vs. incomplete). Meals containing > 20 g of high-quality, complete protein have repeatedly been shown to robustly stimulate skeletal muscle protein synthesis. However, breakfast in many Western countries is dominated by wheat-based products. Wheat and bread are considered a “lower-quality” incomplete source of protein, containing relatively low amounts of lysine and threonine. We hypothesized that a meal containing > 20 g of wheat-based protein would offer no anabolic advantage over a control meal containing only 5 g of plant-based protein. Methods In a subset of healthy, middle-aged women from our recently completed trial (n = 6/17, 53 ± 7 y, 27 ± 2 kg/m2), we measured post-prandial skeletal muscle protein synthesis, blood glucose, insulin and appetite for 3 h following the ingestion of: i) a wheat-based protein meal (INCOMPLETE: 717 kcal, 23 g protein, 120 g carbohydrate, 16 g fat) or ii) a low protein, plant-based, control meal (CONTROL: 542 kcal, 5 g protein, 86 g carbohydrate and 23 g fat). Venous blood samples and vastus lateralis muscle biopsy samples were obtained during a primed (2.0 mmol/kg) constant infusion (0.08 mmol/(kg/min)) of L-[ring-13C6]phenylalanine. All analyses were performed using established, standard techniques. Results Preliminary results indicate post-prandial skeletal muscle protein synthesis was similar in both cohorts (INCOMPLETE: 0.050 ± 0.012%/h vs. CONTROL: 0.054 ± 0.025%/h; p = 0.83) and consistent with fasting values historically measured by our lab. Blood glucose area under the curve (AUC; p = 0.82), insulin AUC (p = 0.85) and hunger AUC were similar in both cohorts. Conclusions A moderate serving of incomplete protein failed to robustly stimulate skeletal muscle protein synthesis. Consumption of a higher-quality, completeprotein meal is likely required to acutely increase muscle protein anabolism. Funding Sources National Cattlemen's Beef Association

Regulation of Akt Signaling in Skeletal Muscle Is Altered by Prematurity in a Neonatal Piglet Model

Objectives Preterm birth is a risk factor for growth faltering. We recently showed in a neonatal piglet model of prematurity that premature birth decreases weight gain and skeletal muscle protein synthesis. This is associated with reduced insulin-induced activation of signaling components downstream of Akt but with no change in the activation of the IR/IRS-1/PI3K axis upstream of Akt. The aim of this study was to identify key regulatory molecules involved in Akt activation that are responsible for the differential response of skeletal muscle to insulin in preterm compared to term pigs. Methods Piglets were delivered by cesarean section 11 d (preterm/PT) or 2 d (term/T) before term birth and a jugular vein catheter was placed for delivery of total parenteral nutrition. On day 3, after 4 h fasting, piglets were fasted one additional h or fed an elemental diet (31.5 kcal/kg, 1.3 g/kg carbohydrate, 2.7 g/kg amino acids, and 1.6 g/kg lipid), similar in composition to sow milk, via oral gavage. Longissimus dorsi muscle was collected following euthanasia (fasted state or 60 min after feeding). Positive and negative regulators of Akt activity were determined by immunoprecipitation and immunoblotting assays. Results Akt1 and Akt2 phosphorylation were lower in PT than in T pigs (P < 0.05), whereas Akt3 phosphorylation was not affected by prematurity. The phosphorylation of Akt activators PDK1 and mTORC2, but not FAK, was lower in PT than in T pigs (P < 0.05). The abundance of Ubl4A, a positive regulator of Akt, was lower in PT than in T pigs (P < 0.05). The abundance Akt inhibitors PHLPP and SHIP2, but not PTEN and IP6K1, was significantly higher in PT than in T pigs (P < 0.05). Activation of the phosphatase PP2A was lower in PT than in T pigs (P < 0.05), but its activation was not affected by feeding. However, PP2A activation was inhibited by feeding in T pigs (P < 0.05). Conclusions These results showed that following preterm birth, the postprandial activation of positive regulators of Akt is reduced whereas the activation of negative regulators of Akt is enhanced. Our findings suggest that premature birth impairs the activation of Akt that is essential for channeling dietary nutrients for anabolism and likely contributes to the extrauterine growth faltering of prematurity. Funding Sources NIH and USDA.

Intermittent Leucine Pulses Enhance Skeletal Muscle mTOR Signaling and Protein Synthesis in Continuously Fed Preterm Pigs

Objectives Extrauterine growth restriction in premature infants is associated with reduced lean mass and long-term morbidities. We have reported previously that intermittent parenteral pulses of Leu promote skeletal muscle mTOR signaling and protein synthesis of continuously fed neonatal pigs born at term. The objective of this study was to determine the effect of prematurity on the response of skeletal muscle anabolic pathways to intermittent parenteral Leu pulses in continuously fed pigs. Methods Pigs delivered 10 d preterm by C-section were fitted with jugular vein and carotid artery catheters and an orogastric feeding tube. Pigs were advanced from parenteral to enteral feeding over 4 d (195 kcal · kg−1 · d−1; 13.5 g protein · kg−1 · d−1). On day 4, pigs were assigned to 1 of 4 treatments: 1) ALA (continuous feeding, 7.5 mL · kg−1 · h−1; 800 μmol Ala · kg−1 · h−1 every 4 h; n = 7); 2) L1 × (continuous feeding; 800 μmol Leu · kg−1 · h−1 every 4 h; n = 6); 3) L2 × (continuous feeding; 1600 μmol Leu · kg−1 · h−1 every 4 h; n = 6); and 4) INT (intermittent feeding; 30 mL · kg−1 fed over 15 min every 4 h; n = 5). On day 5, pigs received L-[ring-2H5]-Phe 30 min after starting the pulse (groups 1, 2, and 3) or meal feeding (group 4). Pigs were euthanized 30 min after isotope injection and longissimus dorsi muscle was collected. Protein synthesis was determined by LC/MS-MS. Indices of amino acid signaling and mTOR activation were determined by immunoprecipitation and immunoblot assays. Results Skeletal muscle protein synthesis was higher in L2 × (+37%) and INT (+31%) compared to ALA (P < 0.05), but was not different between L2 × and INT; protein synthesis in L1 × was intermediate and not different from all other groups. The phosphorylation of 4EBP1, downstream of mTOR, was higher in L2 × and INT compared to ALA (P < 0.05), whereas 4EBP1 phosphorylation in L1 × was lower compared to INT (P < 0.05) but not different compared to ALA and L2 × . The abundance of mTOR · RagA complex, upstream of mTOR and activated in response to Leu, was higher in L2 × and INT compared to ALA and L1 × (P < 0.05). Conclusions These results show that parenteral Leu can enhance anabolic signaling and protein synthesis in skeletal muscle during continuous feeding in preterm pigs, but the dose required is higher than in pigs born at term. Funding Sources Research was supported by NIH and USDA.

Amino Acid Trafficking and Skeletal Muscle Protein Synthesis: A Case of Supply and Demand

Skeletal muscle protein synthesis is a highly complex process, influenced by nutritional status, mechanical stimuli, repair programs, hormones, and growth factors. The molecular aspects of protein synthesis are centered around the mTORC1 complex. However, the intricacies of mTORC1 regulation, both up and downstream, have expanded overtime. Moreover, the plastic nature of skeletal muscle makes it a unique tissue, having to coordinate between temporal changes in myofiber metabolism and hypertrophy/atrophy stimuli within a tissue with considerable protein content. Skeletal muscle manages the push and pull between anabolic and catabolic pathways through key regulatory proteins to promote energy production in times of nutrient deprivation or activate anabolic pathways in times of nutrient availability and anabolic stimuli. Branched-chain amino acids (BCAAs) can be used for both energy production and signaling to induce protein synthesis. The metabolism of BCAAs occur in tandem with energetic and anabolic processes, converging at several points along their respective pathways. The fate of intramuscular BCAAs adds another layer of regulation, which has consequences to promote or inhibit muscle fiber protein anabolism. This review will outline the general mechanisms of muscle protein synthesis and describe how metabolic pathways can regulate this process. Lastly, we will discuss how BCAA availability and demand coordinate with synthesis mechanisms and identify key factors involved in intramuscular BCAA trafficking.

Profiling Serum Antibodies Against Muscle Antigens in Facioscapulohumeral Muscular Dystrophy Finds No Disease-Specific Autoantibodies

Background: FSHD is caused by specific genetic mutations resulting in activation of the Double Homeobox 4 gene (DUX4). DUX4 targets hundreds of downstream genes eventually leading to muscle atrophy, oxidative stress, abnormal myogenesis, and muscle inflammation. We hypothesized that DUX4-induced aberrant expression of genes triggers a sustained autoimmune response against skeletal muscle cells. Objective: This study aimed at the identification of autoantibodies directed against muscle antigens in FSHD. Moreover, a possible relationship between serum antibody reactivity and DUX4 expression was also investigated. Methods: FSHD sera (N = 138, 48±16 years, 48%male) and healthy control sera (N = 20, 47±14 years, 50%male) were analyzed by immunoblotting for antibodies against several skeletal muscle protein extracts: healthy muscle, FSHD muscle, healthy and FSHD myotubes, and inducible DUX4 expressing myoblasts. In addition, DUX4 expressing myoblasts were analyzed by immunofluorescence with FSHD and healthy control sera. Results: The results showed that the reactivity of FSHD sera did not significantly differ from that of healthy controls, with all the tested muscle antigen extracts. Besides, the immunofluorescent staining of DUX4-expressing myoblasts was not different when incubated with either FSHD or healthy control sera. Conclusion: Since the methodology used did not lead to the identification of disease-specific autoantibodies in the FSHD cohort, we suggest that autoantibody-mediated pathology may not be an important disease mechanism in FSHD. Nevertheless, it is crucial to further unravel if and which role the immune system plays in FSHD pathogenesis. Other innate as well as adaptive immune players could be involved in the complex DUX4 cascade of events and could become appealing druggable targets.