Mouse skeletal muscle adaptations to different durations of treadmill exercise after the cessation of FOLFOX chemotherapy.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is a treatment for colorectal cancer that can induce persistent fatigue and metabolic dysfunction. Regular exercise after chemotherapy cessation is widely recommended for cancer patients and has been shown to improve fatigue resistance in mice. However, gaps remain in understanding whether the early systemic and skeletal muscle adaptations to regular exercise are altered by prior FOLFOX chemotherapy treatment. Furthermore, the effects of exercise duration on early metabolic and skeletal muscle transcriptional adaptations are not fully established. Purpose: Investigate the effects of prior FOLFOX chemotherapy treatment on the early adaptations to repeated short- or long-duration treadmill exercise, including the fasting regulation of circulating metabolic regulators, skeletal muscle COXIV activity and myokine/exerkine gene expression in male mice. Methods: Male C57BL6/J mice completed 4 cycles of FOLFOX or PBS and were allowed to recover for 4-weeks. Subsets of mice performed 14 sessions (6 d/wk, 18 m/min, 5% grade) of short- (10 min/d) or long-duration (55 min/d) treadmill exercise. Blood plasma and muscle tissues were collected 48-72 h after the last exercise bout for biochemical analyses. Results: Long-duration exercise increased fasting plasma osteocalcin, LIF, and IL-6 in healthy PBS mice, and these changes were ablated by prior FOLFOX treatment. Slow-oxidative soleus muscle COXIV activity increased in response to long-duration exercise in PBS mice, which was blocked by prior FOLFOX treatment. Fast-glycolytic plantaris muscle COXIV activity increased with short-duration exercise independent of FOLFOX administration. There was a main effect for long-duration exercise to increase fasting muscle IL-6 and COXIV mRNA expression independent of FOLFOX. FOLFOX administration reduced muscle IL-6, LIF, and BDNF mRNA expression irrespective of long-duration exercise. Interestingly, short-duration exercise suppressed the FOLXOX induction of muscle myostatin mRNA expression. Conclusion: FOLFOX attenuated early exercise adaptations related to fasting circulating osteocalcin, LIF, and IL-6. However, prior FOLFOX treatment did not alter the exercise adaptations of plantaris muscle COXIV activity and plasma adiponectin. An improved understanding of mechanisms underlying exercise adaptations after chemotherapy will provide the basis for successfully treating fatigue and metabolic dysfunction in cancer survivors.

Increasing Trunk Mass Evokes Lower Extremity Biomechanical Plasticity during Stair Descent.

The purpose of this study was to investigate the influence of simulated changes in body mass on lower extremity joint work and relative joint contributions during stair descent. Ten healthy recreationally active college-age participants performed five stair descent trials in each of five loading conditions: no added load and with an additional 5%, 10%, 15% and 20% of their body weight. Three-dimensional ankle, knee and hip joint powers were calculated using a six degree-of-freedom model in Visual3D (C-Motion Inc., Germantown, MD, USA). Sagittal plane joint work was calculated as the joint power curve integrated with respect to time during the period between initial contact and toe off. Prism 9.0 (GraphPad Inc., San Diego, CA) was used to perform univariate 1 × 5 repeated measures analyses of variance to determine the effect of added mass on absolute and relative joint work values for total and for each lower extremity joint independently. Increasing added mass was associated with greater total lower extremity negative work during the stair descent task (p < 0.001). At the ankle, increasing added mass was associated with increasing magnitudes of negative joint work. Increasing added mass was associated with greater relative contributions of the ankle and reduced knee contributions to total negative lower extremity joint work (p = 0.014 and p = 0.006). The current findings demonstrated increases in ankle joint contributions to total lower extremity work while knee joint contributions to total lower extremity work were reduced in response to increasing added mass.

Recovery from FOLFOX chemotherapy-induced systemic and skeletal muscle metabolic dysfunction in mice.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is used to treat colorectal cancer and can acutely induce metabolic dysfunction. However, the lasting effects on systemic and skeletal muscle metabolism after treatment cessation are poorly understood. Therefore, we investigated the acute and lasting effects of FOLFOX chemotherapy on systemic and skeletal muscle metabolism in mice. Direct effects of FOLFOX in cultured myotubes were also investigated. Male C57BL/6J mice completed four cycles (acute) of FOLFOX or PBS. Subsets were allowed to recover for 4 wk or 10 wk. Comprehensive Laboratory Animal Monitoring System (CLAMS) metabolic measurements were performed for 5 days before study endpoint. C2C12 myotubes were treated with FOLFOX for 24 hr. Acute FOLFOX attenuated body mass and body fat accretion independent of food intake or cage activity. Acute FOLFOX decreased blood glucose, oxygen consumption (V̇o2), carbon dioxide production (V̇co2), energy expenditure, and carbohydrate (CHO) oxidation. Deficits in V̇o2 and energy expenditure remained at 10 wk. CHO oxidation remained disrupted at 4 wk but returned to control levels after 10 wk. Acute FOLFOX reduced muscle COXIV enzyme activity, AMPK(T172), ULK1(S555), and LC3BII protein expression. Muscle LC3BII/I ratio was associated with altered CHO oxidation (r = 0.75, P = 0.03). In vitro, FOLFOX suppressed myotube AMPK(T172), ULK1(S555), and autophagy flux. Recovery for 4 wk normalized skeletal muscle AMPK and ULK1 phosphorylation. Our results provide evidence that FOLFOX disrupts systemic metabolism, which is not readily recoverable after treatment cessation. FOLFOX effects on skeletal muscle metabolic signaling did recover. Further investigations are warranted to prevent and treat FOLFOX-induced metabolic toxicities that negatively impact survival and life quality of patients with cancer.NEW & NOTEWORTHY The present study demonstrates that FOLFOX chemotherapy induces long-lasting deficits in systemic metabolism. Interestingly, FOLFOX modestly suppressed skeletal muscle AMPK and autophagy signaling in vivo and in vitro. The FOLFOX-induced suppression of muscle metabolic signaling recovered after treatment cessation, independent of systemic metabolic dysfunction. Future research should investigate if activating AMPK during treatment can prevent long-term toxicities to improve health and quality of life of patients with cancer and survivors.

Effects of Feeding Time on Markers of Muscle Metabolic Flexibility Following Acute Aerobic Exercise in Trained Mice Undergoing Time Restricted Feeding.

Time-restricted feeding (TRF) is becoming a popular way of eating in physically active populations, despite a lack of research on metabolic and performance outcomes as they relate to the timing of food consumption in relation to the time of exercise. The purpose of this study was to determine if the timing of feeding/fasting after exercise training differently affects muscle metabolic flexibility and response to an acute bout of exercise. Male C57BL/6 mice were randomized to one of three groups for 8 weeks. The control had ad libitum access to food before and after exercise training. TRF-immediate had immediate access to food for 6 h following exercise training and the TRF-delayed group had access to food 5-h post exercise for 6 h. The timing of fasting did not impact performance in a run to fatigue despite TRF groups having lower hindlimb muscle mass. TRF-delayed had lower levels of muscle HSL mRNA expression and lower levels of PGC-1α expression but displayed no changes in electron transport chain enzymes. These results suggest that in young populations consuming a healthy diet and exercising, the timing of fasting may not substantially impact metabolic flexibility and running performance.

A high fat western diet attenuates phasic dopamine release.

Natural rewards, such as food and social interaction, as well as drugs of abuse elicit increased mesolimbic dopamine release in the nucleus accumbens (NAc). Drugs of abuse, however, increase NAc dopamine release to a greater extent and are known to induce lasting changes on the functioning of the mesolimbic dopamine pathway. Less is known about the long-term effects of diet composition on this reward pathway. In the present study, two diets were compared: a higher-fat diet (Western Diet: WD) and a control diet (standard lab chow) on their effect on the mesolimbic dopamine system. Twenty male C57BL/6 J mice were placed on one of these diets at 7 weeks old. After twelve weeks on the diet, in vivo fixed potential amperometry was used to measure real-time stimulation-evoked dopamine release in the NAc of anesthetized mice before and after an i.p. injection of the dopamine transporter (DAT) inhibitor nomifensine. Results indicated that diet altered mesolimbic dopamine functioning. Mice that consumed the WD demonstrated a hypodopaminergic profile, specifically reduced baseline dopamine release and an attenuated dopaminergic response to DAT inhibition compared to the control diet group. Thus, diet may play a role in mediating dopamine-related behavior, disorders associated with dopamine dysfunction, and pharmacological treatments aimed at altering dopamine transmission.

High-Frequency Stimulation on Skeletal Muscle Maintenance in Female Cachectic Mice.

Cancer cachexia, an unintentional body weight loss due to cancer, affects patients' survival, quality of life, and response to chemotherapy. Although exercise training is a promising intervention to prevent and treat cancer cachexia, our mechanistic understanding of cachexia's effect on contraction-induced muscle adaptation has been limited to the examination of male mice. Because sex can affect muscle regeneration and response to contraction in humans and mice, the effect of cachexia on the female response to eccentric contraction warrants further investigation. PURPOSE: The purpose of this study was to determine whether high-frequency electric stimulation (HFES) could attenuate muscle mass loss during the progression of cancer cachexia in female tumor-bearing mice. METHODS: Female wild-type (WT) and Apc (Min) mice (16-18 wk old) performed either repeated bouts or a single bout of HFES (10 sets of 6 repetitions, ~22 min), which eccentrically contracts the tibialis anterior (TA) muscle. TA myofiber size, oxidative capacity, anabolic signaling, and catabolic signaling were examined. RESULTS: Min had reduced TA muscle mass and type IIa and type IIb fiber sizes compared with WT. HFES increased the muscle weight and the mean cross-sectional area of type IIa and type IIb fibers in WT and Min mice. HFES increased mTOR signaling and myofibrillar protein synthesis and attenuated cachexia-induced AMPK activity. HFES attenuated the cachexia-associated decrease in skeletal muscle oxidative capacity. CONCLUSION: HFES in female mice can activate muscle protein synthesis through mTOR signaling and repeated bouts of contraction can attenuate cancer-induced muscle mass loss.

Delta-6-desaturase (FADS2) inhibition and omega-3 fatty acids in skeletal muscle protein turnover.

Polyunsaturated fatty acids (PUFAs) are essential dietary components. They are not only used for energy, but also act as signaling molecules. The delta-6 desaturase (D6D) enzyme, encoded by the FADS2 gene, is one of two rate limiting enzymes that convert the PUFA precursors - α-linolenic (n-3) and linoleic acid (n-6) to their respective metabolites. Alterations in the D6D enzyme activity alters fatty acid profiles and are associated with metabolic and inflammatory diseases including cardiovascular disease and type 2 diabetes. Omega-3 PUFAs, specifically its constituent fatty acids DHA and EPA, are known for their anti-inflammatory ability and are also beneficial in the prevention of skeletal muscle wasting, however the mechanism for muscle preservation is not well understood. Moreover, little is known of the effects of altering the n-6/n-3 ratio in the context of a high-fat diet, which is known to downregulate protein synthesis. Twenty C57BL6 male mice were fed a high-fat lard (HFL, 45% fat (mostly lard), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 13:1) diet for 6 weeks. Mice were then divided into 4 groups (n = 5 per group): HFL- , high-fat oil- (HFO, 45% fat (mostly Menhaden oil), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 1:3), HFL+ (HFL diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day), and HFO+ (HFO diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day). After 2 weeks on their respective diets and treatments, animals were sacrificed and gastrocnemius muscle harvested. Protein turnover signaling were analyzed via Western Blot. 4-EBP1 and ribosomal protein S6 expression were measured. A two-way ANOVA revealed no significant change in the phosphorylation of both 4EBP-1 and ribosomal protein S6 with diet or inhibitor. There was a significant reduction in STAT3 phosphorylation with the inhibition of FADS2 (p = 0.03). Additionally, we measured markers of protein degradation through levels of FOXO phosphorylation, ubiquitin, and LC3B expression; there was a trend towards increased phosphorylation of FOXO (p = 0.08) and ubiquitinated proteins (p = 0.05) with FADS2 inhibition. LC3B expression, a marker of autophagy, was significantly higher in the HFL plus FADS2 inhibition group from all other comparisons. Lastly, we analyzed activation of mitochondrial biogenesis which is closely linked with protein synthesis through PGC1-α and Cytochrome-C expression, however no significant differences were associated with either marker across all groups. Collectively, these data suggest that the protective effects of muscle mass by omega-3 fatty acids are from inhibition of protein degradation. Our aim was to determine the role of PUFA metabolites, DHA and EPA, in skeletal muscle protein turnover and assess the effects of n-3s independently. We observed that by inhibiting the FADS2 enzyme, the protective effect of n-3s on protein synthesis and proliferation was lost; concomitantly, protein degradation was increased with FADS2 inhibition regardless of diet.

Short-term pyrrolidine dithiocarbamate administration attenuates cachexia-induced alterations to muscle and liver in ApcMin/+ mice.

Cancer cachexia is a complex wasting condition characterized by chronic inflammation, disrupted energy metabolism, and severe muscle wasting. While evidence in pre-clinical cancer cachexia models have determined that different systemic inflammatory inhibitors can attenuate several characteristics of cachexia, there is a limited understanding of their effects after cachexia has developed, and whether short-term administration is sufficient to reverse cachexia-induced signaling in distinctive target tissues. Pyrrolidine dithiocarbamate (PDTC) is a thiol compound having anti-inflammatory and antioxidant properties which can inhibit STAT3 and nuclear factor κB (NF-κB) signaling in mice. This study examined the effect of short-term PDTC administration to ApcMin/+ mice on cachexia-induced disruption of skeletal muscle protein turnover and liver metabolic function. At 16 weeks of age ApcMin/+ mice initiating cachexia (7% BW loss) were administered PDTC (10mg/kg bw/d) for 2 weeks. Control ApcMin/+ mice continued to lose body weight during the treatment period, while mice receiving PDTC had no further body weight decrease. PDTC had no effect on either intestinal tumor burden or circulating IL-6. In muscle, PDTC rescued signaling disrupting protein turnover regulation. PDTC suppressed the cachexia induction of STAT3, increased mTORC1 signaling and protein synthesis, and suppressed the induction of Atrogin-1 protein expression. Related to cachectic liver metabolic function, PDTC treatment attenuated glycogen and lipid content depletion independent to the activation of STAT3 and mTORC1 signaling. Overall, these results demonstrate short-term PDTC treatment to cachectic mice attenuated cancer-induced disruptions to muscle and liver signaling, and these changes were independent to altered tumor burden and circulating IL-6.

Pseudouridine synthase 1 deficient mice, a model for Mitochondrial Myopathy with Sideroblastic Anemia, exhibit muscle morphology and physiology alterations.

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.

Differential Bone Loss in Mouse Models of Colon Cancer Cachexia.

Cachexia is a distinctive feature of colorectal cancer associated with body weight loss and progressive muscle wasting. Several mechanisms responsible for muscle and fat wasting have been identified, however it is not known whether the physiologic and molecular crosstalk between muscle and bone tissue may also contribute to the cachectic phenotype in cancer patients. The purpose of this study was to clarify whether tumor growth associates with bone loss using several experimental models of colorectal cancer cachexia, namely C26, HT-29, and ApcMin/+. The effects of cachexia on bone structure and strength were evaluated with dual energy X-ray absorptiometry (DXA), micro computed tomography (µCT), and three-point bending test. We found that all models showed tumor growth consistent with severe cachexia. While muscle wasting in C26 hosts was accompanied by moderate bone depletion, no loss of bone strength was observed. However, HT-29 tumor bearing mice showed bone abnormalities including significant reductions in whole-body bone mineral density (BMD), bone mineral content (BMC), femoral trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), but no declines in strength. Similarly, cachexia in the ApcMin/+ mice was associated with significant decreases in BMD, BMC, BV/TV, Tb.N, and Tb.Th as well as decreased strength. Our data suggest that colorectal cancer is associated with muscle wasting and may be accompanied by bone loss dependent upon tumor type, burden, stage and duration of the disease. It is clear that preserving muscle mass promotes survival in cancer cachexia. Future studies will determine whether strategies aimed at preventing bone loss can also improve outcomes and survival in colorectal cancer cachexia.

Eccentric contraction-induced myofiber growth in tumor-bearing mice.

Cancer cachexia is characterized by the progressive loss of skeletal muscle mass. While mouse skeletal muscle's response to an acute bout of stimulated low-frequency concentric muscle contractions is disrupted by cachexia, gaps remain in our understanding of cachexia's effects on eccentric contraction-induced muscle growth. The purpose of this study was to determine whether repeated bouts of stimulated high-frequency eccentric muscle contractions [high-frequency electrical muscle stimulation (HFES)] could stimulate myofiber growth during cancer cachexia progression, and whether this training disrupted muscle signaling associated with wasting. Male Apc(Min/+) mice initiating cachexia (N = 9) performed seven bouts of HFES-induced eccentric contractions of the left tibialis anterior muscle over 2 wk. The right tibialis anterior served as the control, and mice were killed 48 h after the last stimulation. Age-matched C57BL/6 mice (N = 9) served as wild-type controls. Apc(Min/+) mice lost body weight, muscle mass, and type IIA, IIX, and IIB myofiber cross-sectional area. HFES increased myofiber cross-sectional area of all fiber types, regardless of cachexia. Cachexia increased muscle noncontractile tissue, which was attenuated by HFES. Cachexia decreased the percentage of high succinate dehydrogenase activity myofibers, which was increased by HFES, regardless of cachexia. While cachexia activated AMP kinase, STAT3, and ERK1/2 signaling, HFES decreased AMP kinase phosphorylation, independent of the suppression of STAT3. These results demonstrate that cachectic skeletal muscle can initiate a growth response to repeated eccentric muscle contractions, despite the presence of a systemic cachectic environment.

Liver inflammation and metabolic signaling in ApcMin/+ mice: the role of cachexia progression.

The ApcMin/+ mouse exhibits an intestinal tumor associated loss of muscle and fat that is accompanied by chronic inflammation, insulin resistance and hyperlipidemia. Since the liver governs systemic energy demands through regulation of glucose and lipid metabolism, it is likely that the liver is a pathological target of cachexia progression in the ApcMin/+ mouse. The purpose of this study was to determine if cancer and the progression of cachexia affected liver endoplasmic reticulum (ER)-stress, inflammation, metabolism, and protein synthesis signaling. The effect of cancer (without cachexia) was examined in wild-type and weight-stable ApcMin/+ mice. Cachexia progression was examined in weight-stable, pre-cachectic, and severely-cachectic ApcMin/+ mice. Livers were analyzed for morphology, glycogen content, ER-stress, inflammation, and metabolic changes. Cancer induced hepatic expression of ER-stress markers BiP (binding immunoglobulin protein), IRE-1α (endoplasmic reticulum to nucleus signaling 1), and inflammatory intermediate STAT-3 (signal transducer and activator of transcription 3). While gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was suppressed by cancer, glycogen content or protein synthesis signaling remained unaffected. Cachexia progression depleted liver glycogen content and increased mRNA expression of glycolytic enzyme PFK (phosphofrucktokinase) and gluconeogenic enzyme PEPCK. Cachexia progression further increased pSTAT-3 but suppressed p-65 and JNK (c-Jun NH2-terminal kinase) activation. Interestingly, progression of cachexia suppressed upstream ER-stress markers BiP and IRE-1α, while inducing its downstream target CHOP (DNA-damage inducible transcript 3). Cachectic mice exhibited a dysregulation of protein synthesis signaling, with an induction of p-mTOR (mechanistic target of rapamycin), despite a suppression of Akt (thymoma viral proto-oncogene 1) and S6 (ribosomal protein S6) phosphorylation. Thus, cancer induced ER-stress markers in the liver, however cachexia progression further deteriorated liver ER-stress, disrupted protein synthesis regulation and caused a differential inflammatory response related to STAT-3 and NF-κB (Nuclear factor-κB) signaling.

Sex differences in the relationship of IL-6 signaling to cancer cachexia progression.

A devastating aspect of cancer cachexia is severe loss of muscle and fat mass. Though cachexia occurs in both sexes, it is not well-defined in the female. The Apc(Min/+) mouse is genetically predisposed to develop intestinal tumors; circulating IL-6 is a critical regulator of cancer cachexia in the male Apc(Min/+) mouse. The purpose of this study was to examine the relationship between IL-6 signaling and cachexia progression in the female Apc(Min/+) mouse. Male and female Apc(Min/+) mice were examined during the initiation and progression of cachexia. Another group of females had IL-6 overexpressed between 12 and 14 weeks or 15-18 weeks of age to determine whether IL-6 could induce cachexia. Cachectic female Apc(Min/+) mice lost body weight, muscle mass, and fat mass; increased muscle IL-6 mRNA expression was associated with these changes, but circulating IL-6 levels were not. Circulating IL-6 levels did not correlate with downstream signaling in muscle in the female. Muscle IL-6r mRNA expression and SOCS3 mRNA expression as well as muscle IL-6r protein and STAT3 phosphorylation increased with severe cachexia in both sexes. Muscle SOCS3 protein increased in cachectic females but decreased in cachectic males. IL-6 overexpression did not affect cachexia progression in female Apc(Min/+) mice. Our results indicate that female Apc(Min/+) mice undergo cachexia progression that is at least initially IL-6-independent. Future studies in the female will need to determine mechanisms underlying regulation of IL-6 response and cachexia induction.

The effect of radiation dose on mouse skeletal muscle remodeling.

BACKGROUND: The purpose of this study was to determine the effect of two clinically relevant radiation doses on the susceptibility of mouse skeletal muscle to remodeling. MATERIALS AND METHODS: Alterations in muscle morphology and regulatory signaling were examined in tibialis anterior and gastrocnemius muscles after radiation doses that differed in total biological effective dose (BED). Female C57BL/6 (8-wk) mice were randomly assigned to non-irradiated control, four fractionated doses of 4 Gy (4x4 Gy; BED 37 Gy), or a single 16 Gy dose (16 Gy; BED 100 Gy). Mice were sacrificed 2 weeks after the initial radiation exposure. RESULTS: The 16 Gy, but not 4x4 Gy, decreased total muscle protein and RNA content. Related to muscle regeneration, both 16 Gy and 4x4 Gy increased the incidence of central nuclei containing myofibers, but only 16 Gy increased the extracellular matrix volume. However, only 4x4 Gy increased muscle 4-hydroxynonenal expression. While both 16 Gy and 4x4 Gy decreased IIB myofiber mean cross-sectional area (CSA), only 16 Gy decreased IIA myofiber CSA. 16 Gy increased the incidence of small diameter IIA and IIB myofibers, while 4x4 Gy only increased the incidence of small diameter IIB myofibers. Both treatments decreased the frequency and CSA of low succinate dehydrogenase activity (SDH) fibers. Only 16 Gy increased the incidence of small diameter myofibers having high SDH activity. Neither treatment altered muscle signaling related to protein turnover or oxidative metabolism. CONCLUSIONS: Collectively, these results demonstrate that radiation dose differentially affects muscle remodeling, and these effects appear to be related to fiber type and oxidative metabolism.

Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice.

Although there are currently no approved treatments for cancer cachexia, there is an intensified interest in developing therapies because of the high mortality index associated with muscle wasting diseases. Successful treatment of the cachectic patient focuses on improving or maintaining body weight and musculoskeletal function. Nutraceutical compounds, including the natural phytochemical quercetin, are being examined as potential treatments because of their anti-inflammatory, antioxidant, and anticarcinogenic properties. The purpose of this study was to determine the effect of quercetin supplementation on the progression of cachexia in the adenomatous polyposis coli (Apc)(Min/+) mouse model of colorectal cancer. At 15 wk of age, C57BL/6 and male Apc(Min/+) mice were supplemented with 25 mg/kg of quercetin or vehicle solution mix of Tang juice and water (V) daily for 3 wk. Body weight, strength, neuromuscular performance, and fatigue were assessed before and after quercetin or V interventions. Indicators of metabolic dysfunction and inflammatory signaling were also assessed. During the treatment period, the relative decrease in body weight in the Apc(Min/+) mice gavaged with V (Apc(Min/+)V; -14% ± 2.3) was higher than in control mice gavaged with V (+0.6% ± 1.0), control mice gavaged with quercetin (-2% ± 1.0), and Apc(Min/+) mice gavaged with quercetin (Apc(Min/+)Q; -9% ± 1.3). At 18 wk of age, the loss of grip strength and muscle mass shown in Apc(Min/+)V mice was significantly attenuated (P < 0.05) in Apc(Min/+)Q mice. Furthermore, Apc(Min/+)V mice had an induction of plasma interleukin-6 and muscle signal transducer and activator of transcription 3 phosphorylation, which were significantly (P < 0.05) mitigated in Apc(Min/+)Q mice, despite having a similar tumor burden. Quercetin treatment did not improve treadmill run-time-to-fatigue, hyperglycemia, or hyperlipidemia in cachectic Apc(Min/+) mice. Overall, quercetin supplementation positively affected several aspects of cachexia progression in mice and warrants further exploration as a potential anticachectic therapeutic.

Cachectic skeletal muscle response to a novel bout of low-frequency stimulation.

While exercise benefits have been well documented in patients with chronic diseases, the mechanistic understanding of cachectic muscle's response to contraction is essentially unknown. We previously demonstrated that treadmill exercise training attenuates the initiation of cancer cachexia and the development of metabolic syndrome symptoms (Puppa MJ, White JP, Velazquez KT, Baltgalvis KA, Sato S, Baynes JW, Carson JA. J Cachexia Sarcopenia Muscle 3: 117-137, 2012). However, cachectic muscle's metabolic signaling response to a novel, acute bout of low-frequency contraction has not been determined. The purpose of this study was to determine whether severe cancer cachexia disrupts the acute contraction-induced response to low-frequency muscle contraction [low-frequency stimulation (LoFS)]. Metabolic gene expression and signaling was examined 3 h after a novel 30-min bout of contraction (10 Hz) in cachectic Apc(Min/+) (Min) and C57BL/6 (BL-6) mice. Pyrrolidine dithiocarbamate, a STAT/NF-κB inhibitor and free radical scavenger, was administered systemically to a subset of mice to determine whether this altered the muscle contraction response. Although glucose transporter-4 mRNA was decreased by cachexia, LoFS increased muscle glucose transporter-4 mRNA in both BL-6 and Min mice. LoFS also induced muscle peroxisome proliferator-activated receptor-γ and peroxisome proliferator-activated receptor-α coactivator-1 mRNA. However, in Min mice, LoFS was not able to induce muscle proliferator-activated receptor-α coactivator-1 targets nuclear respiratory factor-1 and mitochondrial transcription factor A mRNA. LoFS induced phosphorylated-S6 in BL-6 mice, but this induction was blocked by cachexia. Administration of pyrrolidine dithiocarbamate for 24 h rescued LoFS-induced phosphorylated-S6 in cachectic muscle. LoFS increased muscle phosphorylated-AMP-activated protein kinase and p38 in BL-6 and Min mice. These data demonstrate that cachexia alters the muscle metabolic response to acute LoFS, and combination therapies in concert with muscle contraction may be beneficial for improving muscle mass and function during cachexia.

Skeletal muscle glycoprotein 130's role in Lewis lung carcinoma-induced cachexia.

Chronic inflammation is associated with cachexia-induced skeletal muscle mass loss in cancer. Levels of IL-6 cytokine family members are increased during cancer-related cachexia and induce intracellular signaling through glycoprotein130 (gp130). Although muscle STAT3 and circulating IL-6 are implicated in cancer-induced muscle wasting, there is limited understanding of muscle gp130's role in this process. Therefore, we investigated the role of skeletal muscle gp130 (skm-gp130) in cancer-induced alterations in the regulation of muscle protein turnover. Lewis lung carcinoma (LLC) cells were injected into 8-wk-old skm-gp130-knockout (KO) mice or wild-type mice. Skeletal muscle loss was attenuated by 16% in gp130-KO mice, which coincided with attenuated LLC-induced phosphorylation of muscle STAT3, p38, and FOXO3. gp130 KO did not restore mTOR inhibition or alter AMP-activated protein kinase (AMPK) expression. The induction of atrogin expression and p38 phosphorylation in C2C12 myotubes exposed to LLC-treated medium was attenuated by gp130 inhibition, but mTOR inhibition was not restored. STAT signaling inhibition in LLC-treated myotubes did not attenuate the induction of p38 or AMPK phosphorylation. We concluded that, during LLC-induced cachexia, skm-gp130 regulates muscle mass signaling through STAT3 and p38 for the activation of FOXO3 and atrogin, but does not directly regulate the suppression of mTOR.

Characterization of the male ApcMin/+ mouse as a hypogonadism model related to cancer cachexia.

Cancer cachexia, the unintentional loss of lean body mass, is associated with decreased quality of life and poor patient survival. Hypogonadism, involving a reduction in circulating testosterone, is associated with the cachectic condition. At this time there is a very limited understanding of the role of hypogonadism in cancer cachexia progression. This gap in our knowledge is related to a lack of functional hypogonadal models associated with cancer cachexia. The Apc(Min/+) mouse is an established colorectal cancer model that develops an IL-6 dependent cachexia which is physiologically related to human disease due to the gradual progression of tumor development and cachexia. The purpose of this study was to assess the utility of the Apc(Min/+) mouse for the examination of hypogonadism during cancer cachexia and to investigate if IL-6 has a role in this process. We report that Apc(Min/+) mice that are weight stable have comparable testosterone levels and gonad size compared to wild type mice. Cachectic Apc(Min/+) mice exhibit a reduction in circulating testosterone and gonad size, which has a significant association with the degree of muscle mass and functional strength loss. Circulating testosterone levels were also significantly associated with the suppression of myofibrillar protein synthesis. Skeletal muscle and testes androgen receptor expression were decreased with severe cachexia. Although testes STAT3 phosphorylation increased with severe cachexia, systemic IL-6 over-expression for 2 weeks was not sufficient to reduce either testes weight or circulating testosterone. Inhibition of systemic IL-6 signaling by an IL-6 receptor antibody to Apc(Min/+) mice that had already initiated weight loss was sufficient to attenuate a reduction in testes size and circulating testosterone. In summary, the Apc(Min/+) mouse becomes hypogonadal with the progression of cachexia severity and elevated circulating IL-6 levels may have a role in the development of hypogonadism during cancer cachexia.

Muscle mTORC1 suppression by IL-6 during cancer cachexia: a role for AMPK.

Although catabolic signaling has a well-established role in muscle wasting during cancer cachexia, the suppression of anabolic signaling also warrants further investigation. In cachectic tumor-bearing mice, circulating IL-6 levels are associated with suppressed muscle protein synthesis and mTORC1 signaling. We have found AMPK and IGF-I/insulin signaling, two well-known regulators of the mammalian target of rapamycin (mTOR), are altered with the progression of cachexia. How IL-6 can induce suppression of mTORC1 signaling remains to be established. The purpose of this study was to examine mTOR complex 1 (mTORC1) activation and regulation by IL-6 during cancer cachexia. IL-6 effects on mTOR activation were examined in Apc(Min/+) mouse skeletal muscle and C2C12 myotubes. Systemic IL-6 overexpression in Apc(Min/+) mice produced a dose-dependent suppression of mTOR signaling that corresponded to induction of STAT3 and AMPK phosphorylation. This result was also evident in IL-6-treated myotubes. Basal mTOR activation and mTOR responsiveness to glucose administration were suppressed in cachectic skeletal muscle. However, insulin induction of mTOR activity was maintained in IL-6-treated myotubes. Whereas IL-6 suppression of myotube mTOR activity was rescued by AMPK inhibition, inhibition of STAT3 signaling was not sufficient to rescue IL-6 suppression of mTOR activity. Last, treadmill exercise training was able to prevent IL-6-induced inhibition of mTOR signaling in Apc(Min/+) mice independently of activated STAT. In conclusion, we report dose-dependent suppression of mTOR activity by IL-6 and suppressed mTOR responsiveness to glucose administration in Apc(Min/+) mice. IL-6 suppression of mTOR activity was dependent on AMPK activation and independent of STAT signaling in myotubes.

Testosterone regulation of Akt/mTORC1/FoxO3a signaling in skeletal muscle.

Low endogenous testosterone production, known as hypogonadism is commonly associated with conditions inducing muscle wasting. Akt signaling can control skeletal muscle mass through mTOR regulation of protein synthesis and FoxO regulation of protein degradation, and this pathway has been previously identified as a target of androgen signaling. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C(2)C(12) myotubes. C57BL/6 mice were either castrated for 42 days or castrated and treated with the nandrolone decanoate (ND) (6 mg/kg bw/wk). Testosterone loss (TL) significantly decreased volitional grip strength, body weight, and gastrocnemius (GAS) muscle mass, and ND reversed these changes. Related to muscle mass regulation, TL decreased muscle IGF-1 mRNA, the rate of myofibrillar protein synthesis, Akt phosphorylation, and the phosphorylation of Akt targets, GSK3ß, PRAS40 and FoxO3a. TL induced expression of FoxO transcriptional targets, MuRF1, atrogin1 and REDD1. Muscle AMPK and raptor phosphorylation, mTOR inhibitors, were not altered by low testosterone. ND restored IGF-1 expression and Akt/mTORC1 signaling while repressing expression of FoxO transcriptional targets. Testosterone (T) sensitivity of Akt/mTORC1 signaling was examined in C(2)C(12) myotubes, and mTOR phosphorylation was induced independent of Akt activation at low T concentrations, while a higher T concentration was required to activate Akt signaling. Interestingly, low concentration T was sufficient to amplify myotube mTOR and Akt signaling after 24 h of T withdrawal, demonstrating the potential in cultured myotubes for a T initiated positive feedback mechanism to amplify Akt/mTOR signaling. In summary, androgen withdrawal decreases muscle myofibrillar protein synthesis through Akt/mTORC1 signaling, which is independent of AMPK activation, and readily reversible by anabolic steroid administration. Acute Akt activation in C(2)C(12) myotubes is sensitive to a high concentration of testosterone, and low concentrations of testosterone can activate mTOR signaling independent of Akt.