The Yucatan minipig model: A new preclinical model of malnutrition in obese patients with acute or chronic diseases.

BACKGROUND & AIMS: Malnutrition can develop in patients with obesity suffering from acute or chronic illness or after obesity surgery, promoting sarcopenic obesity. A better understanding of this pathophysiology and the development of new therapeutics for chronic diseases, that are often complicated with malnutrition and obesity, justify the development of new animal experimental models close to the human physiology. This study aims to characterize the effects of obesity and underfeeding on Yucatan obese minipigs, assessing its validity as a preclinical model for obesity-related malnutrition. METHODS: Sixteen 30-month-old Yucatan minipigs were divided into two groups for 8 weeks: a standard diet group (ST, n = 5) and an obesogenic diet group (OB, n = 11). After 8 weeks, the OB group was further divided into two sub-groups: a standard diet group (OB-ST, n = 5) and a low-calorie/low-protein diet group (OB-LC/LP, n = 6) for 8 weeks. Body composition by CT-Scan and blood parameters were monitored, and trapezius muscle biopsies were collected to analyse signaling pathways involved in protein turnover and energy metabolism. RESULTS: At W8, OB-ST animals exhibited significantly higher body weight (+37.7%, p = 0.03), muscle mass (+24.9%, p = 0.02), and visceral fat (+192.0%, p = 0.03) compared to ST. Trapezius cross sectional area (CSA) normalized to body weight was lower in OB-ST animals (-15.02%, p = 0.017). At W16, no significant changes were observed in protein turnover markers, although REDD1 increased in OB-ST (96.4%, p = 0.02). After 8 weeks of low-caloric/low protein diet, OB-LC/LP showed decreased body weight (-9.8%, p = 0.03), muscle mass (-6.5%, p = 0.03), and visceral fat (-41.5%, p = 0.03) compared to OB-ST animals. Trapezius fiber CSA significantly decreased in OB-LC/LP (-36.1%, p < 0.0001) and normalized to body weight (-25.4%, p < 0.0001), combined to higher ubiquitinated protein content (+38.3%, p = 0.02). CONCLUSION: Our data support that the Yucatan minipig model mimics nutritional and skeletal muscle phenotypes observed in obese patients, with or without protein-energy malnutrition. It also reproduces muscle atrophy observed in chronic diseases or post-obesity surgery, making it a promising preclinical model for obesity-related malnutrition.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM: The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS: We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS: Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 µM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION: Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

The Yucatan minipig model: A new preclinical model of malnutrition induced by a low-calorie/low-protein diet.

BACKGROUND & AIMS: Severe malnutrition exposes patients to adverse outcomes and a higher mortality risk. The Yucatan minipig, closer to human physiology than murine models could be a pertinent and innovative experimental model for studying the physiopathology and consequences of severe malnutrition. The present study aimed to determine whether a low calorie/low protein diet (LC/LP) can reproduce marasmus malnutrition in minipigs, and to characterize body composition, gut microbiota, malnutrition-related blood parameters, and histological and molecular skeletal muscle patterns. METHODS: Eleven Yucatan minipigs were subjected to two different diets: a standard control diet (ST) (n = 5) and a LC/LP diet (n = 6). LC/LP animals daily received 50% of an isocaloric low-protein diet (10.37 MJ/kg, 8.6% protein). Body composition was measured by computed tomography (CT-scan) before (T0) and after 8 weeks of diet (T8). Trapezius and biceps femoris muscles were sampled at the end of protocol to perform histological and molecular analyses. Gut microbiota composition were was also analyzed at T0 and T8 in fecal samples. RESULTS: Eight weeks of LC/LP diet significantly reduced body weight (-12.3 ± 9.5%, P = 0.03) and gut microbiota richness (i.e. number of observed species) (-10.4 ± 8.3%, P = 0.014) compared to baseline. After 8 weeks, LC/LP animals exhibited a significant reduction of retroperitoneal fat and skeletal muscle surface areas (P = 0.03 and P = 0.047, respectively), whereas these parameters remained unchanged in ST animals. These reductions were associated with lower muscle fiber cross-sectional area (CSA) in trapezius (P < 0.001) and biceps femoris (P = 0.003) in LC/LP animals compared to ST. LC/LP diet promoted an increase of AMP kinase phosphorylation in trapezius and biceps femoris (P = 0.05), but did not affect cytochrome c and COX IV protein content, markers of mitochondrial content. Gene and proteins involved in ubiquitin-proteasome system and apoptosis remained unchanged after 8 weeks of LC/LP diet both in trapezius and biceps femoris. CONCLUSION: All these findings support that this experimental minipig model of severe malnutrition is valid to mimic pathophysiological changes occurring in human protein-energy marasmus malnutrition and muscle atrophy associated with malnutrition, as observed in patients with secondary sarcopenia.

Iron metabolism regulation in females and males exposed to simulated microgravity: results from the randomized trial Artificial Gravity Bed Rest-European Space Agency (AGBRESA).

BACKGROUND: Iron metabolism imbalance could contribute to physical deconditioning experienced by astronauts due to its essential role in energy metabolism, cellular respiration, and oxygen transport. OBJECTIVES: In this clinical exploratory study, we wanted to determine whether artificial gravity (AG) training modulated iron metabolism, RBC indices, and body lean mass in healthy male and female participants exposed to head-down tilt (HDT) bed rest, the reference ground-based model of microgravity. METHODS: We recruited 8 healthy female and 16 healthy male participants who were all exposed to HDT bed rest for 60 d. In addition, they were assigned to 3 experimental groups (n = 8/each): controls, continuous AG training in a short-arm centrifuge (1 × 30 min/d), and intermittent AG training (6 × 5 min/d). RESULTS: The iron metabolism responses to simulated microgravity of the AG training groups did not differ significantly from the responses of controls. Independently from AG, we found that both serum iron concentrations (+31.3%, P = 0.027) and transferrin saturation levels (+28.4%, P = 0.009) increased in males after 6 d of HDT bed rest, as well as serum hepcidin concentrations (+36.9%, P = 0.005). The increase of transferrin saturation levels persisted after 57 d of HDT bed rest (+13.5%, P = 0.026), suggesting that long-term exposure to microgravity sustainably increases serum iron availability in males, and consequently the risk of iron excess or misdistribution. In females, 6 and 57 d of HDT bed rest did not significantly change serum iron, transferrin saturation, or hepcidin levels. CONCLUSIONS: The data from this exploratory study suggest that 1) AG training does not influence the iron metabolism responses to microgravity; and 2) iron metabolism parameters, especially iron availability for cells, are significantly increased in males, but not in females, exposed to long-term simulated microgravity. Because of the small sample size of females, we nevertheless must be cautious before concluding that iron metabolism could differently respond to microgravity in females. This trial was registered at https://www.drks.de as DRKS00015677.

Oxidative and glycolytic skeletal muscles deploy protective mechanisms to avoid atrophy under pathophysiological iron overload.

BACKGROUND: Iron excess has been proposed as an essential factor in skeletal muscle wasting. Studies have reported correlations between muscle iron accumulation and atrophy, either through ageing or by using experimental models of secondary iron overload. However, iron treatments performed in most of these studies induced an extra-pathophysiological iron overload, more representative of intoxication or poisoning. The main objective of this study was to determine the impact of iron excess closer to pathophysiological conditions on structural and metabolic adaptations (i) in differentiated myotubes and (ii) in skeletal muscle exhibiting oxidative (i.e. the soleus) or glycolytic (i.e. the gastrocnemius) metabolic phenotypes. METHODS: The impact of iron excess was assessed in both in vitro and in vivo models. Murine differentiated myotubes were exposed to ferric ammonium citrate (FAC) (i.e. 10 and 50 µM) for the in vitro component. The in vivo model was achieved by a single iron dextran subcutaneous injection (1 g/kg) in mice. Four months after the injection, soleus and gastrocnemius muscles were harvested for analysis. RESULTS: In vitro, iron exposure caused dose-dependent increases of iron storage protein ferritin (P < 0.01) and dose-dependent decreases of mRNA TfR1 levels (P < 0.001), which support cellular adaptations to iron excess. Extra-physiological iron treatment (50 µM FAC) promoted myotube atrophy (P = 0.018), whereas myotube size remained unchanged under pathophysiological treatment (10 µM FAC). FAC treatments, whatever the doses tested, did not affect the expression of proteolytic markers (i.e. NF-κB, MurF1, and ubiquitinated proteins). In vivo, basal iron content and mRNA TfR1 levels were significantly higher in the soleus compared with the gastrocnemius (+130% and +127%; P < 0.001, respectively), supporting higher iron needs in oxidative skeletal muscle. Iron supplementation induced muscle iron accumulation in the soleus and gastrocnemius muscles (+79%, P < 0.001 and +34%, P = 0.002, respectively), but ferritin protein expression only increased in the gastrocnemius (+36%, P = 0.06). Despite iron accumulation, muscle weight, fibre diameter, and myosin heavy chain distribution remained unchanged in either skeletal muscle. CONCLUSIONS: Together, these data support that under pathophysiological conditions, skeletal muscle can protect itself from the related deleterious effects of excess iron.

Does Physical Inactivity Induce Significant Changes in Human Gut Microbiota? New Answers Using the Dry Immersion Hypoactivity Model.

Gut microbiota, a major contributor to human health, is influenced by physical activity and diet, and displays a functional cross-talk with skeletal muscle. Conversely, few data are available on the impact of hypoactivity, although sedentary lifestyles are widespread and associated with negative health and socio-economic impacts. The study aim was to determine the effect of Dry Immersion (DI), a severe hypoactivity model, on the human gut microbiota composition. Stool samples were collected from 14 healthy men before and after 5 days of DI to determine the gut microbiota taxonomic profiles by 16S metagenomic sequencing in strictly controlled dietary conditions. The α and ß diversities indices were unchanged. However, the operational taxonomic units associated with the Clostridiales order and the Lachnospiraceae family, belonging to the Firmicutes phylum, were significantly increased after DI. Propionate, a short-chain fatty acid metabolized by skeletal muscle, was significantly reduced in post-DI stool samples. The finding that intestine bacteria are sensitive to hypoactivity raises questions about their impact and role in chronic sedentary lifestyles.

Intermittent reloading does not prevent reduction in iron availability and hepcidin upregulation caused by hindlimb unloading.

NEW FINDINGS: What is the central question of this study? Could skeletal muscle be involved in microgravity-induced iron misdistribution by modulating expression of hepcidin, the master regulator of iron metabolism? What is the main finding and its importance? We demonstrate, in rats, that hepcidin upregulation is not a transient adaptation associated with early exposure to microgravity and that intermittent reloading does not limit microgravity-induced iron misdistribution despite having a beneficial effect on soleus muscle wasting. ABSTRACT: In humans, exposure to microgravity during spaceflight causes muscle atrophy, changes in iron storage and a reduction in iron availability. We previously observed that during 7 days of simulated microgravity in rats, hepcidin plays a key role in iron misdistribution, and we suggested that a crosstalk between skeletal muscle and liver could regulate hepcidin synthesis in this context. In the present study in rats, we investigated the medium-term effects of simulated microgravity on iron metabolism. We also tested whether intermittent reloading (IR) to target skeletal muscle atrophy limits iron misdistribution efficiently. For this purpose, Wistar rats underwent 14 days of hindlimb unloading (HU) combined or not combined with daily IR. At the end of this period, the serum iron concentration and transferrin saturation were significantly reduced, whereas hepatic hepcidin mRNA was upregulated. However, the main signalling pathways involved in hepcidin synthesis in the liver (BMP-small mothers against decapentaplegic (SMAD), interleukin-6-STAT3 and ERK1/2) were unaffected. Unlike what was observed after 7 days of HU, the iron concentration in the spleen, liver and skeletal muscle was comparable between control animals and those that underwent HU or HU plus IR for 14 days. Despite its beneficial effect on soleus muscle atrophy and slow-to-fast myosin heavy chain distribution, IR did not significantly prevent a reduction in iron availability and hepcidin upregulation. Altogether, these results highlight that iron availability is durably reduced during longer exposure to simulated microgravity and that the related hepcidin upregulation is not a transient adaptation to these conditions. The results also suggest that skeletal muscle does not necessarily play a key role in the iron misdistribution that occurs during simulated microgravity.

Simulated microgravity disturbs iron metabolism and distribution in humans: Lessons from dry immersion, an innovative ground-based human model.

The objective of the present study was to determine the effects of dry immersion, an innovative ground-based human model of simulated microgravity and extreme physical inactivity, on iron homeostasis and distribution. Twenty young healthy men were recruited and submitted to 5 days of dry immersion (DI). Fasting blood samples and MRI were performed before and after DI exposure to assess iron status, as well as hematological responses. DI increased spleen iron concentrations (SIC), whereas hepatic iron store (HIC) was not affected. Spleen iron sequestration could be due to the concomitant increase in serum hepcidin levels (P < .001). Increased serum unconjugated bilirubin, as well as the rise of serum myoglobin levels support that DI may promote hemolysis and myolysis. These phenomena could contribute to the concomitant increase of serum iron and transferrin saturation levels (P < .001). As HIC remained unchanged, increased serum hepcidin levels could be due both to higher transferrin saturation level, and to low-grade pro-inflammatory as suggested by the significant rise of serum ferritin and haptoglobin levels after DI (P = .003 and P = .003, respectively). These observations highlight the need for better assessment of iron metabolism in bedridden patients, and an optimization of the diet currently proposed to astronauts.

Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.

Hereditary aceruloplasminemia (HA), related to mutations in the ceruloplasmin (Cp) gene, leads to iron accumulation. Ceruloplasmin ferroxidase activity being considered essential for macrophage iron release, macrophage iron overload is expected, but it is not found in hepatic and splenic macrophages in humans. Our objective was to get a better understanding of the mechanisms leading to iron excess in HA. A clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) knockout of the Cp gene was performed on Sprague-Dawley rats. We evaluated the iron status in plasma, the expression of iron metabolism genes, and the status of other metals whose interactions with iron are increasingly recognized. In Cp-/- rats, plasma ceruloplasmin and ferroxidase activity were absent, together with decreased iron concentration and transferrin saturation. Similarly as in humans, the hepatocytes were iron overloaded conversely to hepatic and splenic macrophages. Despite a relative hepcidin deficiency in Cp-/- rats and the loss of ferroxidase activity, potentially expected to limit the interaction of iron with transferrin, no increase of plasma non-transferrin-bound iron level was found. Copper was decreased in the spleen, whereas manganese was increased in the plasma. These data suggest that the reported role of ceruloplasmin cannot fully explain the iron hepatosplenic phenotype in HA, encouraging the search for additional mechanisms.-Kenawi, M., Rouger, E., Island, M.-L., Leroyer, P., Robin, F., Remy, S., Tesson, L., Anegon, I., Nay, K., Derbré, F., Brissot, P., Ropert, M., Cavey, T., Loréal, O. Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.

Gut bacteria are critical for optimal muscle function: a potential link with glucose homeostasis.

Gut microbiota is involved in the development of several chronic diseases, including diabetes, obesity, and cancer, through its interactions with the host organs. It has been suggested that the cross talk between gut microbiota and skeletal muscle plays a role in different pathological conditions, such as intestinal chronic inflammation and cachexia. However, it remains unclear whether gut microbiota directly influences skeletal muscle function. In this work, we studied the impact of gut microbiota modulation on mice skeletal muscle function and investigated the underlying mechanisms. We determined the consequences of gut microbiota depletion after treatment with a mixture of a broad spectrum of antibiotics for 21 days and after 10 days of natural reseeding. We found that, in gut microbiota-depleted mice, running endurance was decreased, as well as the extensor digitorum longus muscle fatigue index in an ex vivo contractile test. Importantly, the muscle endurance capacity was efficiently normalized by natural reseeding. These endurance changes were not related to variation in muscle mass, fiber typology, or mitochondrial function. However, several pertinent glucose metabolism markers, such as ileum gene expression of short fatty acid chain and glucose transporters G protein-coupled receptor 41 and sodium-glucose cotransporter 1 and muscle glycogen level, paralleled the muscle endurance changes observed after treatment with antibiotics for 21 days and reseeding. Because glycogen is a key energetic substrate for prolonged exercise, modulating its muscle availability via gut microbiota represents one potent mechanism that can contribute to the gut microbiota-skeletal muscle axis. Taken together, our results strongly support the hypothesis that gut bacteria are required for host optimal skeletal muscle function.

Skeletal muscle ceramides do not contribute to physical-inactivity-induced insulin resistance.

Physical inactivity increases the risk to develop type 2 diabetes, a disease characterized by a state of insulin resistance. By promoting inflammatory state, ceramides are especially recognized to alter insulin sensitivity in skeletal muscle. The present study was designed to analyze, in mice, whether muscle ceramides contribute to physical-inactivity-induced insulin resistance. For this purpose, we used the wheel lock model to induce a sudden reduction of physical activity, in combination with myriocin treatment, an inhibitor of de novo ceramide synthesis. Mice were assigned to 3 experimental groups: voluntary wheel access group (Active), a wheel lock group (Inactive), and wheel lock group treated with myriocin (Inactive-Myr). We observed that 10 days of physical inactivity induces hyperinsulinemia and increases basal insulin resistance (HOMA-IR). The muscle ceramide content was not modified by physical inactivity and myriocin. Thus, muscle ceramides do not play a role in physical-inactivity-induced insulin resistance. In skeletal muscle, insulin-stimulated protein kinase B phosphorylation and inflammatory pathway were not affected by physical inactivity, whereas a reduction of glucose transporter type 4 content was observed. Based on these results, physical-inactivity-induced insulin resistance seems related to a reduction in glucose transporter type 4 content rather than defects in insulin signaling. We observed in inactive mice that myriocin treatment improves glucose tolerance, insulin-stimulated protein kinase B, adenosine-monophosphate-activated protein kinase activation, and glucose transporter type 4 content in skeletal muscle. Such effects occur regardless of changes in muscle ceramide content. These findings open promising research perspectives to identify new mechanisms of action for myriocin on insulin sensitivity and glucose metabolism.

Allopurinol partially prevents disuse muscle atrophy in mice and humans.

Disuse muscle wasting will likely affect everyone in his or her lifetime in response to pathologies such as joint immobilization, inactivity or bed rest. There are no good therapies to treat it. We previously found that allopurinol, a drug widely used to treat gout, protects muscle damage after exhaustive exercise and results in functional gains in old individuals. Thus, we decided to test its effect in the prevention of soleus muscle atrophy after two weeks of hindlimb unloading in mice, and lower leg immobilization following ankle sprain in humans (EudraCT: 2011-003541-17). Our results show that allopurinol partially protects against muscle atrophy in both mice and humans. The protective effect of allopurinol is similar to that of resistance exercise which is the best-known way to prevent muscle mass loss in disuse human models. We report that allopurinol protects against the loss of muscle mass by inhibiting the expression of ubiquitin ligases. Our results suggest that the ubiquitin-proteasome pathway is an appropriate therapeutic target to inhibit muscle wasting and emphasizes the role of allopurinol as a non-hormonal intervention to treat disuse muscle atrophy.

The Oxygen Paradox, the French Paradox, and age-related diseases.

A paradox is a seemingly absurd or impossible concept, proposition, or theory that is often difficult to understand or explain, sometimes apparently self-contradictory, and yet ultimately correct or true. How is it possible, for example, that oxygen "a toxic environmental poison" could be also indispensable for life (Beckman and Ames Physiol Rev 78(2):547-81, 1998; Stadtman and Berlett Chem Res Toxicol 10(5):485-94, 1997)?: the so-called Oxygen Paradox (Davies and Ursini 1995; Davies Biochem Soc Symp 61:1-31, 1995). How can French people apparently disregard the rule that high dietary intakes of cholesterol and saturated fats (e.g., cheese and paté) will result in an early death from cardiovascular diseases (Renaud and de Lorgeril Lancet 339(8808):1523-6, 1992; Catalgol et al. Front Pharmacol 3:141, 2012; Eisenberg et al. Nat Med 22(12):1428-1438, 2016)?: the so-called, French Paradox. Doubtless, the truth is not a duality and epistemological bias probably generates apparently self-contradictory conclusions. Perhaps nowhere in biology are there so many apparently contradictory views, and even experimental results, affecting human physiology and pathology as in the fields of free radicals and oxidative stress, antioxidants, foods and drinks, and dietary recommendations; this is particularly true when issues such as disease-susceptibility or avoidance, "healthspan," "lifespan," and ageing are involved. Consider, for example, the apparently paradoxical observation that treatment with low doses of a substance that is toxic at high concentrations may actually induce transient adaptations that protect against a subsequent exposure to the same (or similar) toxin. This particular paradox is now mechanistically explained as "Adaptive Homeostasis" (Davies Mol Asp Med 49:1-7, 2016; Pomatto et al. 2017a; Lomeli et al. Clin Sci (Lond) 131(21):2573-2599, 2017; Pomatto and Davies 2017); the non-damaging process by which an apparent toxicant can activate biological signal transduction pathways to increase expression of protective genes, by mechanisms that are completely different from those by which the same agent induces toxicity at high concentrations. In this review, we explore the influences and effects of paradoxes such as the Oxygen Paradox and the French Paradox on the etiology, progression, and outcomes of many of the major human age-related diseases, as well as the basic biological phenomenon of ageing itself.

Maintaining a regular physical activity aggravates intramuscular tumor growth in an orthotopic liposarcoma model.

Today, care teams within cancer centers encourage patients to be physically active, after diagnosis, based on data obtained mainly from breast, colon and prostate cancer. Intriguingly, the impact of physical activity (PA) on intramuscular tumors (e.g. sarcomas) has not been specifically addressed and, thus, could be mistakenly confounded with other cancers. In this preclinical study we assessed the impact of PA on intramuscular liposarcoma (LS) evolution. Four-week-old nude male mice were active by voluntary running on wheels, for six weeks. Then, mice were divided into four groups with open or restricted access to wheels, which have received an orthotopic intramuscular injection of either vehicle or human LS, SW872, cells. Active mice presented ~1.5 fold increase in tumor mass, which was mainly due to higher cellular mitosis and proliferation. This bulging intramuscular tumor mass altered muscle function, as evidence by overall muscle strength and maximum running capacity. From a molecular point of view, active mice exhibited poor levels of Phospho-p38Thr180/Tyr182 and p21 content in tumors and also displayed low amounts of circulating insulin comparing to inactive counterparts. Insulin induced Phospho-p38Thr180/Tyr182 and p21 expression in SW872 cells, in vitro. The expression of p21 was regulated in a p38-dependent fashion, since inhibition of p38 activity abolished the up-regulation of p21. Our data suggest that insulin-dependent activation of p38 MAPK-p21 pathway is a possible mechanism responsible for delaying tumor growth in inactive mice. Clinically, patients with lower-extremities LS could be advised to reduce or minimize their levels of PA during the preoperative period.

Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation.

NEW FINDINGS: What is the central question of this study? Although microgravity is well known to reduce circulating iron in astronauts, the underlying mechanism is still unknown. We investigated whether hepcidin, a key hormone regulating iron metabolism, could be involved in this deleterious effect. What is the main finding and its importance? We show that hindlimb suspension, a model of microgravity, stimulates the production of hepcidin in liver of rats. In agreement with the biological role of hepcidin, we found a decrease of circulating iron and an increase of spleen iron content in hindlimb-unloaded rats. Consequently, our study supports the idea that hepcidin could play a role in the alteration of iron metabolism parameters observed during spaceflight. During spaceflight, humans exposed to microgravity exhibit an increase of iron storage and a reduction of circulating iron. Such perturbations could promote oxidative stress and anaemia in astronauts. The mechanism by which microgravity modulates iron metabolism is still unknown. Herein, we hypothesized that microgravity upregulates hepcidin, a hormone produced by the liver that is the main controller of iron homeostasis. To test this hypothesis, rats were submitted to hindlimb unloading (HU), the reference model to mimic the effects of microgravity in rodents. After 7 days, the mRNA level of hepcidin was increased in the liver of HU rats (+74%, P = 0.001). In agreement with the biological role of hepcidin, we found an increase of spleen iron content (+78%, P = 0.030) and a decrease of serum iron concentration (-35%, P = 0.002) and transferrin saturation (-25%, P = 0.011) in HU rats. These findings support a role of hepcidin in microgravity-induced iron metabolism alteration. Furthermore, among the signalling pathways inducing hepcidin mRNA expression, we found that only the interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) axis was activated by HU, as shown by the increase of phospho-STAT3 (+193%, P < 0.001) and of the hepatic mRNA level of haptoglobin (+167%, P < 0.001), a STAT3-inducible gene, in HU rats. Taken together, these data support the idea that microgravity may alter iron metabolism through an inflammatory process upregulating hepcidin.

The diaphragm is better protected from oxidative stress than hindlimb skeletal muscle during CLP-induced sepsis.

OBJECTIVES: The aim of this study was to determine whether non-lethal sepsis induced by cecal ligation and puncture (CLP) modulates oxidative damage and enzymatic antioxidant defenses in diaphragm and hindlimb skeletal muscles (soleus and Extensor Digitorus Longus (EDL)). METHODS: Female Wistar rats were divided into four experimental groups: (1) control animals, (2) animals sacrificed 2 hours or (3) 7 days after CLP, and (4) sham-operated animals. At the end of the experimental procedure, EDL, soleus, and diaphragm muscles were harvested and 4-hydroxynonenal (HNE)-protein adducts and protein carbonyl contents were examined in relation to superoxide dismutase and catalase expression and activities. RESULTS: We observed that both non-respiratory oxidative (i.e. soleus) and glycolytic skeletal muscles (i.e. EDL) are more susceptible to sepsis-induced oxidative stress than diaphragm, as attested by an increase in 4-HNE protein adducts and carbonylated proteins after 2 hours of CLP only in soleus and EDL. DISCUSSION: These differences could be explained by higher basal enzymatic antioxidant activities in diaphragm compared to hindlimb skeletal muscles. Together, these results demonstrate that diaphragm is better protected from oxidative stress than hindlimb skeletal muscles during CLP-induced sepsis.

Single Muscle Immobilization Decreases Single-Fibre Myosin Heavy Chain Polymorphism: Possible Involvement of p38 and JNK MAP Kinases.

PURPOSE: Muscle contractile phenotype is affected during immobilization. Myosin heavy chain (MHC) isoforms are the major determinant of the muscle contractile phenotype. We therefore sought to evaluate the effects of muscle immobilization on both the MHC composition at single-fibre level and the mitogen-activated protein kinases (MAPK), a family of intracellular signaling pathways involved in the stress-induced muscle plasticity. METHODS: The distal tendon of female Wistar rat Peroneus Longus (PL) was cut and fixed to the adjacent bone at neutral muscle length. Four weeks after the surgery, immobilized and contralateral PL were dissociated and the isolated fibres were sampled to determine MHC composition. Protein kinase 38 (p38), extracellular signal-regulated kinases (ERK1/2), and c-Jun- NH2-terminal kinase (JNK) phosphorylations were measured in 6- and 15-day immobilized and contralateral PL. RESULTS: MHC distribution in immobilized PL was as follows: I = 0%, IIa = 11.8 ± 2.8%, IIx = 53.0 ± 6.1%, IIb = 35.3 ± 7.3% and I = 6.1 ± 3.9%, IIa = 22.1 ± 3.4%, IIx = 46.6 ± 4.5%, IIb = 25.2 ± 6.6% in contralateral muscle. The MHC composition in immobilized muscle is consistent with a faster contractile phenotype according to the Hill's model of the force-velocity relationship. Immobilized and contralateral muscles displayed a polymorphism index of 31.1% (95% CI 26.1-36.0) and 39.3% (95% CI 37.0-41.5), respectively. Significant increases in p38 and JNK phosphorylation were observed following 6 and 15 days of immobilization. CONCLUSIONS: Single muscle immobilization at neutral length induces a shift of MHC composition toward a faster contractile phenotype and decreases the polymorphic profile of single fibres. Activation of p38 and JNK could be a potential mechanism involved in these contractile phenotype modifications during muscle immobilization.

From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy.

In the literature, the terms physical inactivity and immobilization are largely used as synonyms. The present review emphasizes the need to establish a clear distinction between these two situations. Physical inactivity is a behavior characterized by a lack of physical activity, whereas immobilization is a deprivation of movement for medical purpose. In agreement with these definitions, appropriate models exist to study either physical inactivity or immobilization, leading thereby to distinct conclusions. In this review, we examine the involvement of oxidative stress in skeletal muscle insulin resistance and atrophy induced by, respectively, physical inactivity and immobilization. A large body of evidence demonstrates that immobilization-induced atrophy depends on the chronic overproduction of reactive oxygen and nitrogen species (RONS). On the other hand, the involvement of RONS in physical inactivity-induced insulin resistance has not been investigated. This observation outlines the need to elucidate the mechanism by which physical inactivity promotes insulin resistance.

Myriocin prevents muscle ceramide accumulation but not muscle fiber atrophy during short-term mechanical unloading.

Bedridden patients in intensive care unit or after surgery intervention commonly develop skeletal muscle weakness. The latter is promoted by a variety of prolonged hospitalization-associated conditions. Muscle disuse is the most ubiquitous and contributes to rapid skeletal muscle atrophy and progressive functional strength reduction. Disuse causes a reduction in fatty acid oxidation, leading to its accumulation in skeletal muscle. We hypothesized that muscle fatty acid accumulation could stimulate ceramide synthesis and promote skeletal muscle weakness. Therefore, the present study was designed to determine the effects of sphingolipid metabolism on skeletal muscle atrophy induced by 7 days of disuse. For this purpose, male Wistar rats were treated with myriocin, an inhibitor of de novo synthesis of ceramides, and subjected to hindlimb unloading (HU) for 7 days. Soleus muscles were assayed for fiber diameter, ceramide levels, protein degradation, and apoptosis signaling. Serum and liver were removed to evaluate the potential hepatoxicity of myriocin treatment. We found that HU increases content of saturated C16:0 and C18:0 ceramides and decreases soleus muscle weight and fiber diameter. HU increased the level of polyubiquitinated proteins and induced apoptosis in skeletal muscle. Despite a prevention of C16:0 and C18:0 muscle accumulation, myriocin treatment did not prevent skeletal muscle atrophy and concomitant induction of apoptosis and proteolysis. Moreover, myriocin treatment increased serum transaminases and induced hepatocyte necrosis. These data highlight that inhibition of de novo synthesis of ceramides during immobilization is not an efficient strategy to prevent skeletal muscle atrophy and exerts adverse effects like hepatotoxicity.