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activity after cervical artificial insemination in the ewe
Eric Houdeau, Pierre Raynal, Pierre-Guy Marnet, Guy Germain, Pierre Mormède, Bernadette Rossano, Régine Monnerie, Marie-Jeanne Prud’Homme
To cite this version:
Eric Houdeau, Pierre Raynal, Pierre-Guy Marnet, Guy Germain, Pierre Mormède, et al.. Plasma levels of cortisol and oxytocin, and uterine activity after cervical artificial insemination in the ewe.
Reproduction Nutrition Development, EDP Sciences, 2002, 42 (4), pp.381-392. �10.1051/rnd:2002033�.
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Regulation of muscle growth and lipid metabolism
Dr. J. Grizard, Dr. A. Orzechowski
Skeletal muscle is a tissue of major economic importance for meat production. It also plays a major role in other physiological functions, especially in the adaptation to injury and stress. Many attempts have been made to improve meat quality and muscle growth, and to reduce muscle wasting. Central to these strategies is a comprehensive knowledge of the regulation of muscle development and metabolism in various species.
Muscle growth results from hypertrophying myofibers, which increase their myonuclear number by recruitment of satellite cells. Determined myoblasts, under conditions that favour differentiation, align and fuse to form multinucleated myotubes, however, the mechanisms underlying the recruitment of satellite cells to hyperplastic or hypertrophic growth have not been established. Ontogenesis of skele- tal muscle includes many steps regulated by cytokines and hormones acting through transcription of muscle regulatory factors (MRFs). Recently discovered MRF called myostatin focuses special atten- tion since it represses muscle growth. The glucocorticoid action and the oxidative stress has also to be considered especially face to muscle cell death. Other hormonal factors such as leptin also need special concern due to their impact on body gains and tissue repartitioning.
Skeletal muscle proteins are continuously synthesised and degraded. Muscle protein deposition and growth depend on the relative rates of these pathways which undergo complex regulation. Muscle, as a proportion of total protein, increases from approximately 30 to 45% for most species during the postnatal life. Due to its slow rate of protein turnover, compared with liver and the gastroin- testinal tract, muscle accounts for only 20–35% of whole body protein synthesis. The increase in protein synthesis and the decrease in proteolysis both accounted for postprandial muscle anabolism.
Insulin, p70S6 kinase and amino acids appear as the main mediators of these pathways. Substrate avail- ability is therefore major in the control of muscle protein disposition.
It has been suggested that longitudinal bone growth may be the primary target for overall somatic growth regulation since skeletal muscle growth could be dependant on a potentiating stimulus of bone growth possibly exerted by passive stretch. The intact nervous system seems to be indispens- able for normal growth of the bones. Specific micronutrients (e.g. minerals and flavonoids) are potential tools to improve bone mineralisation and prevent osteoporosis.
Finally, crucial metabolic link between muscle and adipose tissue concerns free fatty acids. There- fore, the relation between muscle function and lipid metabolism focuses special attention. Adipocytes are decisive for the deposition of body fat. Hence, the mechanisms leading to the mobilisation, redis- tribution and utilisation of body fat seem to play a key role in body composition and metabolic reg- ulations. Studies on lipid metabolism were essentially performed in an attempt to meet the require- ments of consumers (conjugated linoleic acid effects, supplementation with polyunsaturated fatty acids).
Net nutrient metabolism in the hindlimb of growing lambs supplemented with propionate.
L. Majdoub, M. Vermorel, I. Ortigues-Marty.
(INRA, URH, Équipe Nutriments et Métabolisme, Theix, 63122 Saint-Genès-Champanelle, France) Muscle growth and characteristics, involved in meat quality, are influenced by muscle energy metabolism (Hocquette et al.). These authors hypothesise that the amount and the nature of nutrients available to the muscle can orient mus- cle energy metabolism towards a glycolitic or an oxidative type. The objectives of this study were to determine the effects of propionate supple- mentation on the net hindlimb fluxes of some energy and nitrogen substrates in growing lambs.
Six male lambs (32 kg ± 2.2) were surgically equipped with a rumen cannula and blood catheters (external iliac vein and artery). They were fed with frozen rye-grass, at an estimated level of 2 220 kcal ME.d–1. Rumen infusions of a saline solution (C), or of propionate solutions at 199 (P1) and 361 kcal EM.d–1(P2) were applied as described previously (Majdoub L. et al., EAAP publication 103 Snekkersten, Danemark (2001) 285). Net hindlimb fluxes of blood glucose, L-lactate and plasma non esterified fatty acids (NEFA), ammonia (NH3) and a-amino-N were determined. Because of the lack of catheter patency, the results for the hindlimb are based on four animals only. The iliac blood flow was not modified by propionate infusion and averaged 6.83 ± 0.13 L.h–1. Animals in the control treatment were characterised by a slight release of a-amino-N (351.6 mmol.h–1), NH3 (253.2 mmol.h–1), lactate (246.0 mmol.h–1) and NEFA (15.6 mmol.h–1) and an uptake of glucose (–13.8 mmol.h–1). Compared to the control, pro- pionate supplementation increased the net uptake of glucose by 17.4% (P1) and 34.8% (P2) (P < 0.05) and switched lactate and AGNE fluxes from a net release to a net uptake (305.1 mmol.h–1 and 17.1 mmol.h–1on average for lactate and AGNE respectively, P < 0.05). A switch to a net uptake of a-amino-N was also noted, amount- ing to 1 075.8 mmol.h–1(P2) (P < 0.06) and the release of NH3tended to decrease (NS). These results suggest that propionate supplementation probably enhanced muscle protein synthesis as well as the storage of energy substrates into hindlimb glycogen and fat, thereby reducing lipolysis. The effects of propionate infusion on hindlimb metabolism are comparable to those of insulin (Hocquette J.F. et al., Livest. Prod. Sci. 56
(1998) 115). In conclusion, propionate supple- mentation increased the uptake of energy and nitrogen substrates by the hindlimb, sug- gesting changes in muscle energy metabolism and increases in protein, glycogen and fat depo- sition.
Effects of the myostatin growth factor on the proliferation and differentiation of bovine myoblasts. V. Deveaux, J. Bouley, I. Cassar-Malek, B. Picard. (INRA, Unité de Recherche sur les Herbivores, Équipe Croissance et Métabolisme du Muscle, 63122 Saint-Genès- Champanelle, France)
Myostatin, a member of the TGF-bsuperfam- ily, is a negative regulator of muscle develop- ment. In double-muscled cattle, inactivation of this growth factor due to gene mutation is respon- sible for a higher total number of fibres. The aim of this study was to describe myostatin expression during muscle fibre formation in bovine foetal muscles. The expression pattern was followed by RT-PCR and northern-blot techniques using total RNA extracted from Semitendinosus (ST), Biceps femoris (BF) and Masseter (Ma) of 110, 180, 230, 260 day-old foetuses. In situ hybridis- ation and cytoimmunochemistry analyses were also performed to assign myostatin expression to muscle fibres of the first or second genera- tions. The level of expression was different according to the muscle and varied with devel- opment. A strong expression was detected during the first two thirds of gestation. It declined dur- ing the last trimester concomitantly with terminal differentiation in the three muscles. These data were further supported by our observations on primary myoblast cultures. Expression of myo- statin mRNA was weak during proliferation, peaked at the onset of fusion and then declined with terminal differentiation (Deveaux V. et al., Comp. Biochem. Physiol., 126/A, Suppl. 1 (2000) S23). In situ hybridisation performed with foetal muscle (from 90 to 260 days) indicated that myo- statin mRNA were located in muscle bundles.
In ST muscle, co-localisation with different myosin heavy chain (MHC) isoforms, showed that (1) only few secondary fibres expressed myostatin, (2) these cells were positively stained for developmental MHC. Interestingly, fibres expressing myostatin were exclusively of type IIA at 260 days pc. This suggests that fibres
having achieved their differentiation no longer expressed myostatin. In situ hybridisation on pri- mary cultures corroborated these observations, since terminally differentiated myotubes did not express myostatin. The present data show that myostatin expression is regulated during foetal muscle development, decreasing during termi- nal differentiation. At the end of gestation, it is restricted to fibres delayed in their differentia- tion. Our previous results (Deveaux V. et al., Comp. Biochem. Physiol., 126/A, Suppl. 1 (2000) S23) and data from the literature (Thomas M. et al., J. Biol. Chem. 275 (2000) 40235–40243) demonstrate that myostatin has an antiprolifera- tive influence. The present results further sug- gest that this factor could also regulate early events of muscle differentiation.
Effects of grass feeding on muscle character- istics of finishing charolais steers. A. Listrat, C. Jurie, I. Cassar-Malek, L. Bouraoua, B. Picard, D. Micol, J.F. Hocquette (INRA, Unité de Recherches sur les Herbivores, Équipe Crois- sance et Métabolisme du Muscle, Theix, 63122 Saint-Genès-Champanelle, France)
Extensive beef production systems on grass are promoted to improve both animal welfare and the image of beef meat for consumers. However, the effects of consuming pasture grass on muscle characteristics and, hence beef meat quality need to be addressed. This study was aimed at com- paring the influence of either pasture grass or maize silage feeding on the muscular character- istics of growing and finishing charolais steers.
To achieve this goal, 38 and 46 charolais steers of 20 and 30 months of age respectively were used. For the two ages, the animals were fed the two diets from weaning onwards: grass at pasture or maize silage indoors. Samples of two skeletal muscles with different metabolic properties were taken at slaughter: Semitendinosus (ST) which is a glycolytic muscle, and Rectus abdominis (RA) which is more oxidative. The study of the muscular characteristics focused on content and solubility of collagen and metabolic enzyme activities. Determination of lactate dehydroge- nase (LDH) activity characterised muscle gly- colytic metabolism and isocitrate dehydrogenase (ICDH) and citrate synthase (CS) activities, char- acterised muscle oxidative metabolism. In addi- tion, plasma concentrations of thyroid hormones
(T3and T4) were measured by radio immunoas- say throughout the growth period. Similar results of enzyme activity, collagen content and plasma hormone concentrations were found in both age groups. The ST muscle of animals fed grass con- tained less insoluble collagen than the maize silage fed animals (–15%, P < 0.05), but there was no significant differences in RA. The activ- ity of oxidative metabolic enzymes were higher in RA for the pasture fed animals than for those fed corn silage (CS: +19–46%, P < 0.05, ICDH:
+25–30%, P < 0.05). At 30 months of age, the plasma concentrations of T3and T4were higher in the pasture fed animals (P < 0.001) than those fed maize silage. In conclusion, the grass fin- ished animals were characterised by a more oxidative metabolism in RA muscle which may have been induced by the higher plasma levels of T3and T4. They were also characterised by less insoluble collagen in ST, but not in RA. These modifications are likely to be favourable for meat tenderness and flavour. A strict effect of grass feeding on muscular characteristics cannot be ascertained because other factors, such as phys- ical activity at pasture, may influence muscle characteristics.
Leptin fully suppresses acetylcholine-induced insulin secretion in perfused chicken pancreas.
Y. Benomar, M. Taouis, N. Rideau (INRA, Station de Recherches Avicoles, 37380 Nouzilly, France)
In mammals, leptin is produced primarily in the adipose tissues and secreted into the blood. Cir- culating leptin acts through specific receptors (Ob-Rb) located in the region of the hypothala- mus that regulates feeding behavior and energy expenditure (Campfield L.A. et al., Horm. Metab.
Res. 28 (1996) 619–632), Tartaglia L.A. et al., Cell 83 (1995) 1263–1270). However, Ob-Rb have been identified in the pancreatic b-cells that produce insulin, supporting evidence that leptin directly regulates insulin release (Kieffer T.J.
et al., Biochem. Biophys. Res. Commun. 224 (1996) 522–527). The chicken leptin cDNA has been cloned and sequenced (AF012727, AF082500) (Taouis M. et al., Gene 208 (1998) 239–242; Ashwell C.M. et al., Am. J. Physiol.
276 (1999) R226–R232) and in this species, the leptin messenger is expressed not only in the adipose tissue but also in the liver. The effect of recombinant leptin on the central nervous
system was clearly demonstrated following intracerebroventricular injection leading to a decrease of food intake in chickens (Denbow D.M. et al., Physiol. Behav. 69 (2000) 359–362, Dridi S. et al., Am. J. Physiol. Endocrinol. Metab.
279 (2000) E116–E123). A direct effect of leptin on the peripheral target tissues has not yet been demonstrated in this species. This study was designed to study the effect of recombinant chicken leptin on acetylcholine-induced insulin secretion by an isolated perfused chicken pan- creas. Recombinant chicken leptin (10 nM) or diazoxide (100 mM) were introduced at 20 min after the beginning of the stimulation with acetyl- choline (1 mM) + D-glucose (14 mM) and con- comitantly perfused for 20 min. Leptin rapidly (within 2 min) and significantly (p < 0.0001) suppressed acetylcholine-induced insulin secre- tion with no change in the volume outflow rate.
Diazoxide had a similar effect. Mean insulin output (ng.min–1) was 14.6 ± 1.9; 1.0 ± 0.1 and 2.3 ± 0.2 in the presence of glucose + acetyl- choline, glucose + acetylcholine + leptin and glu- cose + acetylcholine + diazoxide, respectively (n = 3, p < 0.05). In subsequent experiments, tolbutamide (100 mM) was introduced at 15 min after leptin and concomitantly perfused for 10 min; it immediately and fully reversed the suppressive effect of leptin on acetylcholine- induced insulin release. Insulin output was restored to a value significantly higher than the pre-leptin level and remained at this level dur- ing the 10 min tolbutamide perfusion. Mean insulin output (ng.min–1) was 13.0 ± 1.6; 3.5 ± 1.1 and 18.5 ± 1.0 (mean ± SE, n = 3, p < 0.05) in the presence of glucose + acetylcholine; glu- cose + acetylcholine + leptin and glucose + acetylcholine + leptin + tolbutamide, respec- tively. Our findings suggest that the chicken b-cells are very sensitive to leptin. The inver- sion by tolbutamide of the leptin suppressive effect on insulin secretion suggests that KATP channels may be a target for leptin to inhibit insulin secretion in the chicken pancreas.
Preconditioning with milimolar sodium ascor- bate protects against ROS/RNS-induced insulin resistance and cell death in cultures of rat L6 muscle cells. M. Lokociejewska, P. Muras, A. Orzechowski, M.M. Godlewski (Department of Physiology, Biochemistry, Phar- macology and Toxicology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 War- saw, Poland)
A growing body of evidence suggests that oxi- dation and nitrosation modulate growth perfor- mance by an impact on cell proteins and gene expression (Marshall H.E. et al., FASEB J. 14 (2000) 1889–1900; Elsasser T.H. et al., Dom. Anim. Endocrinol. 19 (2000) 75–84). It is thought that the cellular response to oxidative and nitrosative stress, including desensitisation to insulin, might be reversed by the action of antiox- idants. One day effects of hydrogen peroxide (H2O2; 0.1, 0.5, 1 mM), the superoxide anion radical •O2– (0.1, 0.5, 1 mM), nitric oxide (•NO;
0.5, 2.5, 5 mM), and peroxynitrite (ONOO–; 0.1, 0.5, 1 mM) on insulin mitogenic action was stud- ied in L6 satellite cells. An insulin stimulating effect on cell mitogenicity (DNA synthesis) was inhibited by 0.1 mM H2O2Similarly, the appli- cation of an •O2–donor (0.1–1 mM) moderately inhibited insulin-enhanced mitogenicity. The ONOO–donor 3-morpholinosydnonimine (SIN-1;
0.1–0.5 mM), and the •NO donor, sodium nitro- prusside (SNP; 0.5–2.5 mM), also inhibited insulin-stimulated synthesis of DNA. Altogether, these results show that H2O2, •O2–, •NO, ONOO– may decrease the growth promoting activity of insulin in myoblasts under oxidative or nitrosative stress (Orzechowski A. and Grzelkowska K., Pol. J. Vet. Sci. 3 (2000) 3–12). Cell viability showed that neither H2O2, •O2–, SIN-1, nor SNP induced cell death when present below 0.5 mM however they became deadly cytotoxic at mil- imolar concentrations. Symptoms of cell shrink- ing with nuclear chromatin condensation, col- lapse of chromatin into patches along the nuclear membrane and formation of apoptotic bodies were detected after 24 hours. Apoptosis was eval- uated in situ on the basis of the apoptotic index and oligonucleosomal fragmentation of nuclear DNA. Cell viability was monitored and the above-mentioned index confirmed the lack of cell respiration in dead myoblasts, especially those treated with SNP. Our results indicate that massive apoptosis is induced by H2O2and SIN-1 and that •NO induces necrotic cell death (Orzechowski A. and Grzelkowska K., Pol. J.
Vet. Sci. 3 (2000) 193–195). Cells were exposed to various antioxidants: sodium ascorbate (10 mM); tocopherol (10 mM); N-acetyl-cysteine (0.2; 1 mM), deferoxamine (100 mM), hemo- globin (50 mM), quercetin (10 mM); catalase (100 U.mL–1) or SOD-1 (10 U.mL–1) 24 h prior to the subsequent addition of ROS/RNS plus insulin (100 nM). The above-mentioned
antioxidants at low doses did not abrogate the dose-dependent deleterious effects of ROS/RNS (cell viability and apoptosis, or resistance to insulin-dependent mitogenicity). In contrast, pre- treatment with sodium ascorbate (1 mM) per- mitted to maintain an elevated cell mitogenicity in response to insulin regardless of the type of ROS/RNS used. These results were confirmed by an in situ study of fixed cells using laser scan- ning cytometry. Therefore, ROS/RNS-induced growth retardation of L6 myoblasts could not be reversed by pretreatment with various antioxi- dants at low doses, but it responded to milimolar concentrations of vitamin C which could be a good candidate for clinical studies in prophy- laxis for the prevention of ROS/RNS-induced insulin resistance and growth retardation.
Combination of amino acids and insulin is required for regulating postprandial protein synthesis in rat skeletal muscle.
M. Prod’Hommea, M. Balagea, S. Sinauda, D.
Dardeveta, T. Varyb, S. Kimballb, L. Jeffersonb, J. Grizarda (a INRA, Unité de Nutrition et Métabolisme Protéique, Theix 63122 Saint- Genès-Champanelle, France; bDepartment of Cellular Molecular Physiology, Pennsylvania State University, College of Medecine, Hershey, Pennsylvania, USA)
Many studies suggest that amino acids and insulin play major roles in promoting postprandial pro- tein anabolism. However, their respective and relative contributions, as well as the signals involved in the regulation of protein synthesis in response to feeding remain unclear. Although in vitro studies clearly show that insulin and amino acids can independently increase the skele- tal muscle protein synthesis rate, the assessment of their respective roles in vivo is complicated by the concomitant changes in their concentra- tions that are generally induced by experimental changes in plasma insulin and aminoacid con- centrations. In the present study, the respective roles of insulin and amino acids in the regula- tion of skeletal muscle protein after feeding were examined in 41 growing rats fasted 17 h and refed 1 h with either 25% or 0% aminoacid/pro- tein meal. In each nutritional condition, post- prandial insulin secretion was either maintained (Control groups: C25and C0groups) or blocked by injection of a specific inhibitor of insulin
secretion, the diazoxide (Diazoxide groups: DZ25 and DZ0groups) (Sinaud S. et al., Am. J. Phys- iol. Endocrinol. Metab. 276 (1999) E50–E61).
Muscle protein synthesis rate was examined in vitro in Epitrochlearis taken in the postabsorptive state or 2 h after food intake. The signalling path- way was investigated at the same time in Gas- trocnemius. Only feeding 25% amino acid/
protein meal in the presence of increased plasma insulin concentration (C25 group) stimulated protein synthesis in the skeletal muscle when compared to the postabsorptive state (+20%, P < 0.05). The stimulation of protein synthesis resulted from an improvement of the translation initiation step. It was indeed associated with increased phosphorylation of 4E-BP1, reduced binding of eIF-4E to 4E-BP1 and increased assembly of the active eIF4E.eIF4G complex.
The p70S6k was also hyperphosphorylated in response to the 25% amino acid/protein meal (appearance on Sodium Dodecyl Sulfate poly- acrylamide gels of bands with the slowest elec- trophoretic mobility). Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency was not able to stimulate the muscle protein synthesis rate or regulate initiation factors and p70S6k compared with fasted rats.
Taken together, our results suggest that both insulin and amino acids are required to stimu- late protein synthesis, inhibit protein degrada- tion and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.
Leucine supplementation reverses the aging- induced defect in postprandial inhibition of skeletal muscle proteasome-dependent prote- olysis. L. Combaret, D. Dardevet, D. Attaix, J. Grizard (Nutrition and Protein Metabolism Unit, Human Nutrition Research Center of Cler- mont-Ferrand and INRA, 63122 Theix, France) In the young and the adult, an increased skeletal muscle protein synthesis rate and inhibition of proteolysis result in a positive nitrogen balance in the postprandial state. Recent studies suggest that muscle wasting in old animals results in part from a reduction in postprandial protein depo- sition, due to defects in both protein synthesis and ubiquitin-proteasome-dependent proteoly- sis. In vitro studies have shown that muscle
protein synthesis is much less stimulated by the branched-chain amino acid, leucine, in the old rat than in the young animal. Moreover, leucine is known to inhibit the lysosomal pathway in incubated rat skeletal muscles, and possibly the ubiquitin-proteasome-dependent process. This study was undertaken (i) to explore the in vivo mechanisms responsible for the regulation of muscle proteolysis in the postprandial state and (ii) to determine whether an oral leucine supplementation may regulate the ubiquitin- proteasome-dependent proteolysis in old rats.
Rats (n = 9–11 animals per group) were studied in the postabsorptive state (PA) and in the post- prandial state (PP). A third group corresponded to rats in the postprandial state, following leucine supplementation in the diet (PP LEU), in order to increase by 2-fold the plasma leucine postpran- dial concentration. Rates of protein breakdown were measured in incubated epitrochlearis mus- cles from 8-month- and 22-month-old (adult and old respectively, n = 9–10 in the PA, PP and PP LEU groups for each age) rats with appro- priate inhibitors of the lysosomal/Ca2+-depen- dent, and proteasome-dependent pathways. The chymotrypsin-like activity of muscle protea- somes (partially purified using a glycerol gradi- ent) was also measured. Feeding was inhibited by 57% (P < 0.05) ubiquitin-proteasome-dependent proteolysis in adult animals, but not in old rats. In adult rats where this pathway is down-regulated by feeding, there is no additional effect induced by leucine supplementation in the diet. By con- trast, leucine supplementation in old animals completely restored the inhibition of the protea- some-dependent proteolysis by feeding. Accord- ingly, in old animals from the PP LEU group, the leucine-induced regulation of proteasome- dependent proteolysis correlated with concomi- tant changes in the chymotrypsin-like activities of muscle proteasomes (–124% and –55% respec- tively, P < 0.05 compared to animals from the PP group). In conclusion, this work showed that during the postprandial state there is a defect in the regulation of proteasome-dependent prote- olysis in skeletal muscles from old rats, and that this defect may be corrected by leucine supple- mentation in the diet. The underlying supple- mentation as well as the impact of chronic leucine supplementation on muscle wasting in old sub- jects, are under investigation.
The outline of glucocorticoid-dependent myo- genesis and caspase-3-independent cell death in cultures of rat L6 muscle cells. M. Janka, A. Orzechowskia, B. Gajkowskab, T. Sadkowskia, M.M. Godlewskia(aDepartment of Physiology, Biochemistry, Pharmacology and Toxicology, Warsaw Agricultural University, Nowoursyn- owska 166, 02–787 Warsaw, Poland; b Labora- tory of Cell Ultrastructure, M.R.C. Polish Academy of Sciences, Warsaw, Poland) We observed in vivo cell death of muscle pre- cursor cells (MPC) in the soleus muscle of catabolic rats overloaded for 5 days with dexamethasone (2 mg.kg–1body weight; Dex) (Orzechowski A. et al., Horm. Met. Res. 32 (2000) 174–180). Elimination of MPC was asso- ciated with an increased activity of m-calpain (Jank et al., Pol. J. Vet. Sci. 3 (2000) 213–218).
Our current study was therefore directed at delin- eating the signalling pathway involved in glu- cocorticoid-induced death of rat L6 myoblasts. To our knowledge, this is the first report that describes the cascade of events that lead to Dex- dependent complex biological rearrangements resulting in accelerated myogenesis or muscle cell death. Confluent cultures of L6 rat myoblasts conditioned to favour myogenesis were studied every other day during either 10 days or on day 4. Cells were treated with one of the increased doses of Dex (2, 20, 200 nM) and/or blockers of particular targets in the intracellular survival sig- nal transduction pathways. Protein synthesis by [3H]leucine incorporation into acid precipitable proteins, cell viability by an MTT formazan assay, DNA oligonucleosomal fragmentation, ultrastructural studies, laser scanning cytometry (LSC) and confocal microscopy were used to determine indices of metabolic and structural modifications within the muscle cells. At low doses (2 nM), Dex apparently accelerated the fusion of muscle cells evidenced by the higher number of myotubes. At higher doses, muscle cells died on day 6. (20 nM) or day 4. (200 nM).
Every observed Dex-induced effect was depen- dent upon gene regulation as proven by a blockade at the genomic level (transcription/
translation) with 7-actinomycin D (7-AAD) (50 ng.mL–1). Dex-induced changes in cell phys- iology and necrobiology were mediated by a reactive oxygen species, most likely H2O2since cell death induced by 200 nM of Dex was efficiently blocked either by sodium ascorbate
(0.1 and 1 mM), N-acetylcysteine (NAC) (1 mM) or the specific H2O2 scavenger catalase (1 000 U.mL–1). Reduced cell viability was also dependent on the influx of Ca2+from the extra- cellular pool as shown by the reversal of cell death by 1 mM of EGTA. Muscle cell viability, which was neither synergic nor additive to Dex but was a ROS-dependent reduction, was observed after a phosphatidylinositol 3-kinase (PI-3K) blockade with LY 294002 (10 mM). In contrast, cell viability reduced by inhibiting ERK1/ERK2 MAPK with PD 98059 (25 mM) appeared to stay in line (additive effect) with the Dex-dependent signalling pathway. m-Calpain entirely confers muscle cell death machinery, since Dex-induced cell death was reversed by specific ALLN (5 or 25 mM) or non-specific (ALLM 25 mM) m-calpain blockers, respectively.
Dex-induced cell death occurred even in the pres- ence of the caspase-3 inhibitor Ac DEVD CHO (100 mM) – protease of the execution phase of apoptosis. Neither the inhibitor of phospho- lipase C and phospholipase A2, namely U 73122 (1 mM), nor cAMP (0.1; 1 mM) could rescue cells from Dex-induced cell death. However, Dex-induced low cell viability was completely restored by the PKC inhibitor H7 (30 mM) but not TPA (100 ng.mL–1). Immunohistochemical studies revealed higher expression of both cal- pastatin and m-calpain in muscle cells treated with Dex. Death of muscle cells was charac- terised by extensive cytosolic vacuolisation, cell blabbing, chromatin margination but not DNA oligonucleosomal fragmentation. We conclude that the following cascade might represent a sim- plified version of Dex-induced cell death of mus- cle cells: Dex > Genome > ROS > PKC > Ca2+
> m-Calpains > Cell Death.
The effect of MyoD family gene polymorphism on meat deposition in pigs. D. Cieslaku a, W. Kapelanskiu b, S. Grajewskab, M. Bocianb (a Institute of Genetics and Animal Breeding, Jastrzebiec, 05-552 Wólka Kosowska, Poland;
b University of Technology and Agriculture, Mazowiecka 28, 85-084 Bydgoszcz, Poland) The family of MyoD genes controls muscle development. Polymorphism of three MyoD genes have been well described: MYOG (the MspI polymorphism in the 3’ end – Soumillion et al., Mamm. Genome 8 (1997) 564–568), MYF3
(the DdeI polymorphism in intron 1 – Knoll H.
et al., Anim. Genet. 28 (1997) 321) and MYF5 (the HinfI polymorhphism in intron 1 – Te Pas M.F. et al., Mamm. Genome 10 (1999) 123–127);
the DdeI polymorphism in intron 2 – Stratil A.
and Cnepica S. , Anim. Genet. 30 (1999) 79–80.
In the present study, the relationships between these polymorphisms and lean tissue deposition of pig carcasses was studied. The population composed of Zlotnicka Spotted (ZS), Polish Lan- drace (PL), Pietrain (PI) and [PI´ (PI´ ZS)]
and [PI´ (LW´ PL)] pigs was used. Each breed consisted of 30 pigs except the [PI´ (LW´ PL)]
cross, consisting of 115 animals. The rearing and nutritional conditions were similar and all pigs were slaughtered at 105 kg body weight. Fre- quencies of MYOG, MYF3 and MYF5 alleles were determined by PCR-RFLP analysis and turned out to be breed specific. Variance analy- sis to study the associations between MyoD geno- types and lean carcass traits was performed using all 235 pigs as one group. The statistical model included the influence of the breed, the RYR1 genotype, sex and slaughter weight as the covari- ate. Half carcass meatiness, loin mass and meati- ness, loin “eye” area, tenderloin mass and meati- ness, and ham mass and meatiness were compared for the respective genotypes of the examined genes. Additionally skin with subcu- taneous fat of the loin and ham and mean back- fat thickness from 5 measurements were consid- ered. With respect to the MYOG polymorphism, a total of 23 AA, 95 AB, and 117 BB genotypes were observed. Loin and ham meat content was significantly lower in BB compared to AB (p£ 0.05) animals. Backfat thickness of BB was higher than that of both AB and AA (p£ 0.05) pigs. Also the skin with subcutaneous fat of loin and ham of the BB animals was significantly higher (p£ 0.05) than in the heterozygotes. The pigs of the AA genotype did not differ signifi- cantly from AB or BB. With respect to MYF3, polymorphism analysis revealed 60 of AA, 96 of AC and 79 of CC genotypes. Loin mass was significantly higher in heterozygotes (p£ 0.05 compared to CC animals). Tenderloin and ham meat contents were significantly lower in the AC animals (p£ 0.05) than in both types of homozygotes. These values were similar when AA and CC animals were compared. In turn fat- ness characteristics (skin with subcutaneous fat of ham and mean backfat thickness) appeared to be significantly higher in the heterozygous animals compared to AA only. Analysis of the
,
MYF5/HinfI polymorphism revealed 88 AA, 92 AB and 55 BB genotypes. The study of the DdeI polymorphism revealed 119 of AA, 99 of AB and 17 of BB animals. In both cases, no associ- ations with carcass characteristics were found.
In conclusion, we assume that the MspI poly- morphism in the 3’ end of the MYOG gene and in intron 1 of the MYF3 gene showed some asso- ciations with carcass muscularity. Because MyoD genes are known to be expressed only in the mus- cle tissue, the observed differences in fat depo- sition may indicate different rates of muscle maturing dependent on the genotype. As a post- natal process, it is related to satellite cell activity controlled by the MyoD family genes. It is there- fore possible that the mutations described here may change the functions of the MYOG and MYF3 genes in the regulation of the satellite cell activity.
Sequence analysis of the cytb gene in Bison bison and Bison bonasus sp. B. Prusak, G.
Grzybowski (Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrerbiec, 05-552 Wólka Kosowska, Poland) Cytochrome b is one of the best known of the 9–10 proteins that make up complex III of the mitochondrial oxidative phosphorylation system and it is the only one of them encoded by the mitochondrial genome. The nucleotide sequence of the cytb gene contains species – specific infor- mation and has been used as a tool in a number of studies on species identification, phylogenetic relationships and species evolution. The aim of this work was to analyse the cytb gene sequence in two wild-living specimens: Bison bison and Bison bonasus. The next step was to compare the derived sequences with adequate sequences existing in GenBank. Biological material (hair) was obtained from 4 individuals of both investi- gated specimens. DNA from hair bulbs was extracted according to an organic procedure.
Prior to extraction the hairs were sonicated in a 5% solution of Alconox for 30 min, and were then washed in sterile water. The PCR reaction was performed in a 9600 GeneAmp (PE Biosys- tems) using primers given by Parson et al. The sequences of the forward and reverse primers were modified in such a way that one oligonu- cleotide in a pair contained a universal primer sequence –21(M13). The PCR products (of 313 base pair length) were purified through ultra-
filtration using Microcon 100 microconcentra- tors (AMICON). The quantity and quality of products was tested in a 3% NuSieve 3:1 agarose gel (FMC) in relation to the DNA mass standard.
Purified products were sequenced with the ABI Prism Big Dye Primer Cycle Sequencing Ready Reaction Kit (PE Biosystems) according to the user’s manual. The sequencing products were separated in a DNA sequencer ABI PRISM 377 (PE Biosystems). The electrophoretic data were analysed by the sequence Navigator v.1.0.1 soft- ware (PE Biosystems). For all investigated indi- viduals of Bison bonasus one type of the cytb gene sequence was obtained. For individuals of Bison bison, we found two types of the cytb gene sequence differing in one nucleotide position (transition C (type I) > T (type II). A comparison of cytb gene sequences from Bison bison (type I and II) and Bison bonasus revealed 91.3% iden- tity (27 variable nucleotide positions) and 91.6%
identity (26 variable nucleotide positions) respec- tively. These data shows that although both spec- imens belong to the same genus, geographic iso- lation led to divergence of the phylogenetic lines of these specimens. A database search revealed the presence of the cytb gene sequence for Bison bonasus (accession no. Y15005) of 100% simi- larity with our sequence. Both types of cytb gene sequences of Bison bison (type I and II) did not have their identical representation in the Gen- Bank. We found a sequence of 99% and 98%
identity respectively for the type I and type II cytb gene sequence of Bison bison (accession no. AF 036273.1).
Estimation of Myostatin mRNA expression in chicken muscle by real time RT-PCR: effects of nutritional state and genotype. A. Guernec, B. Chevalier, M.J. Duclos (Station de Recherches Avicoles, INRA, Nouzilly, France)
Muscle development is regulated by positive and negative factors, like Myostatin (MSTN), a mem- ber of the Tranforming Growth Factor-bsuper- family, which acts as a paracrine inhibitor of muscle cell proliferation (Thomas M. et al., J.
Biol. Chem. 275 (2000) 40235–40243). The pre- sent study was conducted to evaluate if variations in muscular MSTN mRNA levels could be linked to quantitative differences in muscle development
,
induced by selection for growth rate or by the nutritional status in chickens. Male chickens from genotypes divergently selected for high (HG) or low (LW) growth rate were raised in cages and fed ad libitum until 6 weeks of age, when they were submitted to ad libitum feeding or fasting for 48 h (n = 6 chickens per nutritional state and genotype), (Beccavin C. et al., J. Endocrinol.
168 (2001) 297–306). Total RNA were prepared from the Pectoralis major muscle and submit- ted to reverse transcription using random hex- amers. The cMSTN and the cTATA box Binding Protein (TBP) cDNAs were quantified by real time PCR using specific primers deduced from the chicken cDNA sequences. All experiments were performed using a Light Cycler apparatus and the Fast start DNA Master SYBR green I kit (Roche Diagnostics, Meylan, France). Relative quantification was made in reference to a stan- dard curve constituted of serial dilutions of a pool of muscle cDNA from HG chickens. Based on a previous study conducted in human tissues, the TBP gene was chosen as a reference (Bièche I. et al., Clin. Chem. 45 (1999) 1148–1156) and the results were finally expressed as the ratio of MSTN/TBP and were analysed by 2 way ANOVA followed by comparisons of means between groups. The levels of TBP cDNA fluc- tuated slightly between samples but were not significantly altered by the genotype (P = 0.5) or the nutritional state (P = 0.5), validating them as a reference in chicken muscle as in human tis- sues. The absolute levels of MSTN mRNA and the MSTN/TBP ratio were significantly altered by the nutritional state (P = 0.0001), but not by genotype (P = 0.08). The ratio was markedly higher (about 7 fold) in the fed than in the fasted state in chickens from both genotypes, suggest- ing a selective down-regulation of MSTN mRNA levels during fasting. Further experiments are needed to asses if co-ordinated MSTN protein variations are observed. In conclusion, a proce- dure was validated using real time PCR to mea- sure MSTN mRNA levels in the chicken mus- cle and revealed an unexpected down-regulation of this parameter during fasting.
Characterisation of p70 S6kinase in chicken muscle. K. Bigot, M. Taouis, M. Derouet, S. Crochet, S. Tesseraud (Station de recherches avicoles, INRA, Nouzilly, France)
In mammals, the p70 S6 kinase regulates the ini- tiation phase of RNA translation, a limiting step in protein synthesis (Dufner A. and Thomas G., Exp. Cell. Res. 253 (1999) 100–109). This kinase is also a key element of the insulin signalling pathway (Kimball S.R. et al., Am. J. Physiol.
274 (1998) 221–228; Patti M.E. et al., J. Clin.
Invest. 101 (1998) 1519–1529). The present study was aimed at characterising the chicken p70 S6 kinase in two metabolic types of muscles (Pectoralis major and Gastrocnemius muscles) and to examine their regulation by the nutritional state or by insulin. Three-week-old male chicks were divided into 3 groups: (1) fasted for 16 h, (2) refed for 30 min after a 16 h-fast, (3) sub- mitted to an intravenous injection of insulin (1 UI.kg–1of Body Weight) after a 16 h-fast then removed 7 or 15 min after the injection. The expression of p70 S6 kinase mRNA was assessed by RT-PCR and the 130 pb amplified fragment was cloned and sequenced. The p70 S6 kinase activation was determined by western blot and its specific activity by immune complex kinase assay. As demonstrated by RT-PCR and the west- ern blot, the chicken p70 S6kinase gene and pro- tein were expressed in the two muscles. Refeed- ing resulted in a decreased mobility corresponding to the activation of p70 S6 kinase, irrespective of the muscle type. The p70S6 kinase specific activ- ity measured by the incorporation of ATPg[33P]
into its substrate increased by 2-fold (P < 0.01, n = 4) in the Pectoralis major and by 3-fold (P < 0.05, n = 5) in the Gastrocnemius muscle.
Insulin injection activated muscle p70 S6 kinase but this activation appeared delayed in the Pec- toralis major compared to the Gastrocnemius muscle (15 vs. 7 min). The p70 S6 kinase activity, measured 15 min after insulin treatment, was increased by 9-fold (P < 0.001, n = 5) in the Pec- toralis major and by 12-fold (P < 0.001, n = 5) in the Gastrocnemius muscles. In conclusion, these results show that p70 S6 kinase is expressed in the chicken muscle and is activated by both refeeding and insulin. p70 S6 kinase expression and activation are more pronounced in the Gas- trocnemius than in the Pectoralis major muscle.
The signalling pathways involved in chicken tis- sue p70 S6 kinase activation by nutrients and insulin remain undetermined and the underlying mechanisms responsible for the regulation of muscle protein synthesis await clarification.
Compared metabolism of cis-9, trans-11 Con- jugated Linoleic Acid (CLA) in the liver of rat and bovine animals. A. De La Torrea, D. Moutya, J.M. Chardignyb, J.P. Noëlc, O.
Loreauc, D. Duranda, D. Baucharta(a INRA Theix, URH, 63122 Saint-Genès-Champanelle, France; b INRA Dijon, UNL, 21034 Dijon Cedex, France; c CEA Saclay, Service des Molécules Marquées, 91191 Gif-sur-Yvette Cedex, France) Conjugated linoleic acid (CLA) represents a group of positional and geometric conjugated dienoic isomers of linoleic acid. At low concen- trations, CLA can exhibit beneficial effects on human pathologies such as anticarcinogenic and hypocholesterolemic activities. CLA isomers, such as cis-9, trans-11 CLA, occur mainly in ruminant products as a result of bacterial activi- ties in the rumen but it appears that they are also synthesised from trans vaccenic acid in D9 desat- urase-rich tissues. However, the metabolism of CLA in tissues and organs is poorly documented.
This work compares the in vitro hepatic metabolism of cis-9,trans-11 CLA of animal models for monogastric (rat) and ruminant (bovine) animals by using the in vitro method of incubated liver slices. Liver samples were taken from six 2 year-old steers and six 4 month- old male rats given standard diets and immedi- ately cut into slices. Liver slices were incubated for 17 h in an RPMI medium containing 0.75 mM of a mixture of FA (comparable to plasma bovine non esterified FA, (Chilliard Y.
et al., Reprod. Nutr. Dev. 24 (1984) 469–482) and 3 mCi.mL–1 (0.146 mM) of [1-14C]
cis-9,trans-11 CLA according to Gruffat-Monty D. et al. (J. Biochem. 126 (1999) 188-193). The intensity of CLA oxidation was determined by measuring the production of CO2trapped in hyamine hydroxide (complete oxidation) and perchloric acid soluble products (ASP, partial oxidation). Products of CLA desaturation were analysed by gas-liquid chromatography coupled with a radiodetector and those of CLA esterifi- cation (neutral lipids, NL, and phospholipids, PL) were separated by liquid chromatography and counted for radioactivity. CLA secretion as part of very low density lipoprotein (VLDL) was determined from VLDL particles isolated by ultracentrifugal flotation and counted for radioac- tivity. Statistical analyses were carried out by using the Student t-test. Incorporation of cis- 9,trans-11 CLA into liver cells was 2.5-fold lower in bovines (P < 0.001) than in rats. Similarly,
complete and partial oxidation of CLA were 10-fold lower (P < 0.001) and 1.7-fold lower (P < 0.01) respectively in bovines than in rats.
However, the oxidation rate of CLA averaged 65 and 49% (P < 0.01) of CLA incorporated by hepatocytes of bovines and rats, respectively.
CLA was desaturated into conjugated C18:3 for 12.7% and 26.9% of total non oxidised CLA (P < 0.001) in bovine and rat liver cells, respec- tively. Esterification of CLA into NL was 8.2-fold lower (P < 0.001) in the bovine than in the rat liver, but esterification into PL was sim- ilar for the 2 animal species. CLA was secreted as part of the VLDL at a low and similar extent (0.024 and 0.016 nmol.g–1liver) in bovines and rats respectively. When expressed as a percent of CLA incorporated into cells, the secretion rate of VLDL was 1.65-fold higher (P < 0.01) for bovine than for rat hepatocytes. In conclusion, rat hepatocytes exhibit a greater ability for the incorporation of cis-9,trans-11 CLA as usual FA such as oleate (Graulet B. et al., J. Biochem. 124 (1998) 1212–1219) than bovine hepatocytes.
However, the liver of both animal species rapidly uses a large part of CLA as energy fuel (around 50% of incorporated CLA) while oleate is only oxidised by 20% in both species. An important part of cis-9,trans-11 CLA escaping from oxi- dation in bovines and in rats was converted into conjugated C18:3 whose metabolism in extra- hepatic tissues and physiological effects in man have to be specifically analysed.
Are peroxidation and fluidification of lipopro- teins favoured by n-6 PUFA in steers? V.
Scislowskia, D. Duranda, D. Moutya, M.
Laplaudb, C. Mottac, D. Baucharta(a INRA, Cen- tre de Recherches de Clermont-Ferrand/Theix, URH, 63122, Saint-Genès-Champanelle, France;
b Inserm U551, CHU, 87000 Limoges, France;
c Laboratoire de Biochimie, CHU, 63000 Cler- mont-Ferrand, France)
Supplementation of diets with vegetable oils rich in polyunsaturated fatty acids (PUFA) for fat- tening cattle can improve the nutritional value of meat for the consumer (Clinquart et al., INRA Prod. Anim. 8 (1995) 29). However, such dietary supplementation with PUFA, which are very sen- sitive to peroxydation, can have a negative effect on the health of animals by production of cyto- toxic peroxidation products. The aim of our study
was to determine the degree of susceptibility to lipoperoxidation and the consequences on flu- idity of lipoprotein particles in steers given a diet enriched in n-6 PUFA protected or not from bac- terial hydrogenation in the rumen. Eighteen Charolais´ Salers steers (BW: 502 kg) were fed, for 70 days, a control diet consisting of dry for- age (54%) and concentrate (46%) (diet C, n = 6) or the same diet supplemented with 4% of lipids rich in C18:2 n-6 (150 g.d–1) provided by sun- flower seeds (“unprotected” PUFA) (diet S, n = 6) or sunflower oil continuously infused in the proximal duodenum (“protected” PUFA) (diet O, n = 6). The chemical analysis of low density lipoproteins (LDL, 1.019 < d < 1.060 g.mL–1), and of two forms of high density lipoproteins (light form, HDL l, 1.060 < d < 1.091 g.mL–1; heavy form, HDL h, 1.091 < d < 1.180 g.mL–1), purified by gradient density ultracentrifugation (Bauchart D. et al., J. Lipid Res. 30 (1989) 1499), showed that duodenal oil infusion (diet O) sig- nificantly increased the ratio of PUFA/saturated fatty acids (FA) in phospholipids (PL) and in cholesteryl esters (CE) in the three classes of lipoproteins when compared with the diets C and S (´1.5 in PL;´ 1.5 to 2.8 in CE, P < 0.06). The resistance of lipoprotein particles to CuCl2- induced lipoperoxidation (Esterbauer M. et al., Free Radic. Res. Commun. 6 (1989) 67), which is the highest in LDL compared to HDL l and especially HDL h, was not modified significantly by lipid supplements (diets S and O) as shown by the lack of an effect on the lag phase. This was probably the consequence of a higher activity of antioxidants (such as vitamin E) provided by the oil supplements. However, the maximal amount of oxidised FA (determined by the production of conjugated dienes) strongly increased with diet O compared to those in diets C and S (´1.6, P < 0.005). In spite of the PUFA enrichment of lipids in the three lipoprotein classes with lipid supplemented diets, the fluidity of the lipopro- teins (measured by fluorescence polarisation according to Shinitzky M. and Inbar M., Biochem.
Biophys. Acta 433 (1976) 133), was not signif- icantly modified (8.5 and 9.1 vs. 9.5 poises for diets S, O and C respectively). These unexpected results could be explained by the concominant enrichment of lipoparticles with cholesterol, the main lipid rigidifier of natural membranes, observed in lipid supplemented diets. In conclu- sion, lipid supplementation of diets with sun- flower oil in steers improves the dietetic value of meat, with no apparent alteration of health of
the animals, probably because antioxidants pro- tect PUFA and therefore prevent the production of cytotoxic products. Indeed, these dietary treat- ments did not increase the lipoperoxidation pro- cesses nor the fluidity of lipoproteins in normal husbandry conditions but it cannot be excluded that such dietary treatments could play a negative role in situations of strong stress such as extreme climate conditions, restrictive feeding, or stress during slaughtering.
Long term effects of flavonoids on bone metabolism in ovariectomised rats. M.N.
Horcajada, B. Chanteranne, C. Puel, J. Mathey, M.J. Davicco, C. Rémésy, J.P. Barlet, V. Coxam (INRA, Unité des Maladies Métaboliques et Micronutriments, Centre de Clermont/Theix, 63122 Saint-Genès-Champanelle, France) In the Mediterranean area, a very low incidence of osteoporosis has been reported as compared to other European countries. A major characteristic of the Mediterranean diet is high fruit and veg- etable consumption. Since such foods are rich in polyphenols of which some exhibit protective properties towards osteoporosis, these com- pounds could be involved in bone protection.
We thus compared the long-term effect of four polyphenols (flavonoids) on bone loss in ovariec- tomised rats, an animal model for osteoporosis.
Ten control 3-month old female Wistar rats (298 ± 7 g) were sham-operated (SH), while thirty were ovariectomised (OVX). SH and ten OVX rats were then fed a standard diet containing 0.8%
diet dry matter (DM) in calcium and 0.6% in phosphorus. Among the 20 other OVX, half were fed a standard diet supplemented with isoflavones (OVXI) (genistein: 159, daidzein: 156, glycitine:
33 mg/g; i.e. 0.25% diet DM), and half with rutine (OVXR), a flavanol (0.25% diet DM), for 3 months. None of the tested flavonoids exhibited a uterotrophic effect, the uterine weight being decreased by 90% both in OVXI and OVXR.
With regards to bone parameters, the decrease in metaphyseal femoral density (M-BMD, g/cm2) (0.2281 vs. 0.2497 in OVX and SH respectively;
P < 0.05) was prevented by isoflavones (0.2433) or rutine (0.2419) supplements. As far as total femoral density was concerned, the same pat- tern was observed. In contrast, ovariectomy, ingestion of isoflavones, or rutin had no signifi- cant effect upon diaphyseal femoral density. On
