calpastatin and Weight-Gain

The objective of this study was to determine whether the improvements in growth and efficiency of gain achieved by recombinant porcine somatotropin (pST) are associated with altered expression of the p94, calpastatin, or alpha-actin genes in porcine longissimus (LD) muscle. Forty-eight barrows (initial 64.2 to 67.4 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial in a randomized complete block design. Factors were duration of treatment (3 or 6 wk) and pST administration (0 or 3 mg x pig(-1) x d(-1)). Plasma samples were obtained 24 h after the first pST injection and at the end of the each treatment period for assays of selected variables. The LD samples were obtained at 3 and 6 wk of pST treatment. Northern blot analysis of calpastatin expression in LD muscle revealed three distinct transcription products of approximately 8.5 (CPST I), 5.5 (CPST II), and 2.5 (CPST III) kb; CPST II was reduced (P < .02) 33 and 61% by pST at 3 and 6 wk, respectively, whereas CPST I and III were not influenced (P > .12). Neither alpha-actin nor p94 was responsive to pST injection. As expected, pST resulted in higher (50%, P < .02) plasma insulin within 24 h and one- and twofold higher (P < .01) concentrations at 3 and 6 wk, respectively. Glucose was increased (P < .01) at 3 (15%) and 6 (10%) wk, whereas urea nitrogen was reduced (32 to 36%, P < .01). The efficacy of pST was evident in that ADG was improved (P < .01) 11 to 13% independent of time. Likewise, feed intake was reduced (P < .01) 10 to 11% and gain: feed improved (P < .01) approximately 26% for pigs receiving pST independent of time. These data indicate that the enhanced muscle growth achieved by pST is not associated with altered expression of p94 or alpha-actin, or an increase in the abundance of any calpastatin transcription product.

Topics: Actins; Animals; Blood Glucose; Blood Urea Nitrogen; Blotting, Northern; Calcium-Binding Proteins; Calpain; Gene Expression; Growth Hormone; Insulin; Male; Recombinant Proteins; RNA, Messenger; Swine; Transcription, Genetic; Weight Gain

Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Topics: 3T3 Cells; Adipocytes; Adipose Tissue; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Collagen; Diet, High-Fat; Disease Models, Animal; Fibrosis; Inflammation; Liver; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Obesity; Weight Gain

This experiment was conducted to investigate the effects of feeding a protein-free diet on mRNA levels of the calpain system in skeletal muscle of growing pigs during a 15-d feeding trial. Twenty crossbred barrows were divided into two dietary treatments: control or protein-free diet (mean initial weight for both groups: 38.3 kg). Daily diets were provided at 2.5 times energy for maintenance (twice a day). On d 0, 3, and 14, biopsies were taken from longissimus muscle between the third and fourth ribs (d 0 and 3) and between the fourth and fifth rib (d 14). On d 15, animals were slaughtered and longissimus muscles were dissected and analyzed for calpastatin, and mu- and m-calpain activity. From biopsies, mRNA level of skeletal muscle calpain, mu- and m-calpain, and calpastatin were measured using reversed transcription PCR. Subsequently, PCR products were quantified using ELISA. Feeding the protein-free diet lowered growth rate to almost zero. Only total level of mRNA of mu-calpain on d 14 was influenced by dietary treatments, being lower for the protein-free group than for the control group (P < .05). However, proteolytic activities were not different between treatments. Total RNA concentration in longissimus muscle decreased during the experiment for both treatments, but on d 14 this was more pronounced for the protein-free than for the control group (P < .05). If mRNA levels were corrected for this change, specific mRNA level on d 14 of skeletal muscle calpain and mu-calpain were lower (P < .05) for the protein-free than for the control group. These data suggest that activity of the components of the calpain system are differentially regulated.

Topics: Aging; Animals; Base Sequence; Biopsy; Body Weight; Calcium-Binding Proteins; Calpain; Diet; Diet, Protein-Restricted; DNA Primers; DNA Probes; DNA, Complementary; Eating; Enzyme-Linked Immunosorbent Assay; Male; Muscle, Skeletal; Polymerase Chain Reaction; RNA, Messenger; Swine; Weight Gain

The objective of this study was to determine the effects of dietary administration of a beta-adrenergic agonist (BAA; L-644,969) and male sex condition (ram vs wether) on muscle growth and meat quality of Dorset x Romonov lambs believed to be heterozygous for the callipyge gene. At approximately 17 wk of age, lambs were blocked by weight within each sex condition and randomly assigned to BAA treatment group. The interaction of BAA and male sex condition was not significant for any of the traits measured. Rams had greater initial and final live weights, average daily gain, and hot carcass weight (P < .01). Rams did not differ (P > .05) from wethers with respect to any of the carcass traits, possibly because the wethers were so lean and heavily-muscled that there was little room for improvement. Kidney-pelvic fat weight was reduced 26% by BAA (P < .05). Knife separable lean weight and whole carcass proximate composition were not affected (P > .05) by BAA or male sex condition. Administration of BAA increased calpastatin activity at 20 d (1.1 vs 1.5 units/g), but not at 0 h (3.9 vs 4.8 units/g) postmortem, decreased myofibril fragmentation index (60.7 vs 44.9), and increased shear force (8.2 vs 10.9 kg) at 20 d postmortem (P < .05). These data suggest that muscle growth rates are near maximum in lambs expressing the callipyge gene, regardless of male sex condition or BAA treatment. Therefore, it seems that the callipyge gene exerts most, but not all, of its effect through intracellular events similar to those initiated by administering BAA.

Topics: Adrenergic beta-Agonists; Animals; Body Composition; Body Weight; Calcium-Binding Proteins; Diet; Heterozygote; Hydrogen-Ion Concentration; Male; Meat; Muscle Development; Muscle, Skeletal; Orchiectomy; Pyridines; Random Allocation; Sheep; Temperature; Weight Gain

To estimate the heritability (h2) of postrigor calpastatin activity (CA), 555 steers were reared and processed conventionally. Breed-types included purebreds (Angus [A], Braunvieh [B], Charolais [C], Gelbvieh [G], Hereford [H], Limousin [L], Pinzgauer [P], Red Poll [RP], and Simmental [S]), composite populations (MARC I [1/4 C, 1/4 B, 1/4 L, 1/8 H, 1/8 A], MARC II [1/4 S, 1/4 G, 1/4 H, 1/4 A], and MARC III [1/4 RP, 1/4 H, 1/4 P, 1/4 A]), and F1 crosses (H, A, C, G, P, Shorthorn, Galloway, Longhorn, Nellore, Piedmontese, or Salers x H or A). Steers were serially slaughtered on an age-constant (across breed groups) basis. Heritability estimates for CA, i.m. fat content (IMF), Warner-Bratzler shear (WBS) force, retail product yield (RPY), and ADG were .65 +/- .19, .93 +/- .02, .53 +/- .15, .45 +/- .18, and .32 +/- .26, respectively. The genetic correlations (rg) of CA with WBS, RPY, and ADG were .50 +/- .22, .44 +/- .25, and -.52 +/- .37, respectively. The rg of IMF with WBS, RPY, and ADG were -.57 +/- .16, -.63 +/- .15, and -.04 +/- .11, respectively. These h2 and rg estimates indicate that it should be possible to select for improvements in CA, IMF, and WBS. However, selection against CA may be a more suitable approach for improving meat tenderness than selection for increased IMF because the level of genetic antagonism between CA and RPY was not as great as that between IMF and RPY.

Topics: Adipose Tissue; Animals; Body Composition; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Least-Squares Analysis; Male; Meat; Muscles; Phenotype; Postmortem Changes; Weight Gain

The effect of castration on endogenous proteinase activity and myofibrillar protein turnover was investigated in cattle. Six each of MARC III composite bulls and steers weighing approximately 210 kg were given ad libitum access to a typical growing diet. At 0, 42, 84, 126, and 168 d, two consecutive 24-h urine samples were obtained. Urine was analyzed for N tau-methylhistidine (N tau MH) and creatinine. Following slaughter after 170 d on feed, a longissimus muscle sample was removed immediately from each carcass for quantification of mu-calpain, m-calpain, calpastatin, cystatin(s), cathepsin B, and cathepsin B + L activities. Bulls were heavier (P < .05) at 126 and 168 d and more efficient (P < .05) in conversion of feed to gain at 84 and 168 d than were steers. Compared with steers, bulls excreted less (P < .05) N tau MH at 84, 126, and 168 d and displayed lower (P < .05) fractional degradation rates (FDR) at all sample times. No differences (P > .05) in calpain or cathepsin activities were observed between bulls and steers. However, muscle from bulls had greater (P < .05) activities of calpastatin and cystatin(s) than that from steers. A negative relationship existed between d-168 FDR and calpastatin (r = -.72; P < .05) and cystatin (r = -.62; P < .05) activities. These results indicate that decreased FDR of skeletal muscle from growing bulls contributes to their greater efficiency of growth and could be related partially to cystatin-mediated cathepsin activity and(or) calpastatin-mediated calpain activity.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Cattle; Creatinine; Cystatins; DNA; Endopeptidases; Male; Methylhistidines; Muscle Development; Muscle Proteins; Muscles; Orchiectomy; Random Allocation; RNA; Weight Gain

Forty wether lambs were used in a 2 x 4 factorial arrangement to determine the response of animal performance, muscle growth, proteinase activity, and meat tenderness to beta-adrenergic agonist (BAA) supplementation. Lambs were fed a finishing diet with or without 4 ppm of L644,969 and slaughtered after 0, 2, 4, and 6 wk of treatment. The ADG was higher (P < .05) in the treated than in the control lambs after 2 wk and returned to control levels thereafter. Semitendinosus weight and calpastatin activity were higher and mu-calpain activity was lower in the treated than in the control lambs after 2, 4, and 6 wk. Cathepsin B activity was higher (P < .01) and cystatin-like activity was lower (P < .05) after 2 wk in treated than in control lambs but returned to control levels thereafter. Longissimus protein:DNA was higher after 4 (P < .05) and 6 (P < .01) wk in the treated lambs than in the controls. The concentration of RNA and RNA:DNA ratio were higher (P < .01) in the longissimus and semitendinosus muscles in the treated lambs after 2 wk and remained higher throughout the study. Semitendinosus protein and RNA content were higher after 2, 4, and 6 wk and DNA content was higher after 2 and 6 wk in the treated than in the control lambs. Longissimus shear-force values were higher (P < .001) in the treated than in the control lambs at all slaughter end points. These data indicate a rapid alteration of muscle growth, activity of the calpain-calpastatin system, and meat tenderness during BAA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic beta-Agonists; Aging; Animal Feed; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Cystatins; DNA; Endopeptidases; Male; Meat; Muscle Development; Muscle Proteins; Muscles; Pyridines; Random Allocation; RNA; Sheep; Weight Gain

Eight MARC III composite (1/4 Hereford, 1/4 Angus, 1/4 Pinzgauer, and 1/4 Red Poll) steers weighing approximately 350 kg were fed 0 or 3 ppm of the beta-adrenergic agonist L644,969 (Merck Sharp and Dohme Laboratories, Rahway, NJ) for 6 wk in a high-concentrate diet. Feed efficiency was higher (P less than .05) in beta-adrenergic agonist-fed steers at 1, 3, 5, and 6 wk on trial. Average daily gain was greater (P less than .05) in beta-adrenergic agonist-fed steers at 3, 5, and 6 wk on treatment. Fractional degradation rate (percentage/day) of skeletal muscle myofibrillar protein was 27.1% lower (P less than .05) in beta-adrenergic agonist-fed steers at 3 wk on trial. Fractional accretion rate (percentage/day) of skeletal muscle myofibrillar protein in beta-adrenergic agonist-fed steers was higher (P less than .05) at 1, 3, 5, and 6 wk on trial. The beta-adrenergic agonist-fed steers had heavier (P less than .05) carcasses (9.6%), larger (P less than .05) longissimus muscle areas (24.3%), and lower (P less than .05) USDA yield grades (43.8%). Marbling degree, USDA quality grade, kidney, pelvic, and heart fat percentage, and 12th rib fat thickness were not different (P greater than .05). Calpastatin activity was higher (P less than .05) in muscle from the beta-adrenergic agonist-fed steers at 0 and 7 d postmortem. There were no differences (P greater than .05) in mu- or m-calpain or in cathepsins B or B+L or cystatin(s) between beta-adrenergic agonist-fed and control steers.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Cattle; Crosses, Genetic; Endopeptidases; Male; Meat; Muscle Development; Muscle Proteins; Muscles; Pyridines; Random Allocation; Weight Gain

The objectives of this experiment were to assess effects of animal age and castration on activities of calpain I, calpain II, and calpastatin in sheep skeletal muscle. Ten newborn male lambs (2.9 kg), six weaned wethers (23.2 kg), six weaned rams (22.2 kg), six market wethers (55.4 kg), and six market rams (60.2 kg) were slaughtered and samples of biceps femoris were taken for assay of calpain I (micromolar calcium-dependent proteinase), calpain II (millimolar calcium-dependent proteinase), and calpastatin. Preweaning weight gain was similar for rams and wethers; however, postweaning ram growth exceeded (P less than .05) that of wethers. Ram biceps femoris weights at market were greater than those of wethers (P less than .05). Irrespective of age or gender, activity of calpain II was two- to threefold greater than that of calpain I. Muscle calpastatin activity was severa fold higher than calpain I and II activities. Activities of calpains and calpastatin declined (P less than .05) between birth and weaning. A portion of these losses were due to a dilution effect caused by accumulation of muscle proteins. Neonatal attenuation of calpain activities may underlie age-related attenuation of fractional rates of muscle protein degradation. Although ram muscle growth exceeded that of wethers, no differences (P greater than .05) in activities of muscle calpains or calpastatin were detected between these two groups at weaning or at market weight. Hence, castration did not influence lamb muscle growth by altering muscle calpain or calpastatin activities.

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Male; Muscle Development; Muscle Proteins; Muscles; Orchiectomy; RNA; Sheep; Weight Gain

To examine the effect of a beta-adrenergic agonist (BAA) on muscle growth, proteinase activities, and postmortem proteolysis, 16 wether lambs were randomly assigned to receive 0 or 4 ppm of L644,969 in a completely mixed high-concentrate diet for 6 wk. Weight of the biceps femoris was 18.6% heavier in treated lambs. At 0 h after slaughter, treated lambs had higher cathepsin B (35.6%), cathepsins B + L (19.1%), calpastatin (62.8%), and m-calpain (24.6%) than control lambs, but both groups had similar mu-calpain activities. In both longissimus and biceps femoris muscles, treated lambs had higher protein and RNA and lower DNA concentrations. However, total DNA was not affected, indicating that the increase in muscle mass was probably due to muscle hypertrophy rather than to hyperplasia. The pattern of postmortem proteolysis was significantly altered by BAA feeding. In treated lambs, postmortem storage had no effect on the myofibril fragmentation index and degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with BAA feeding. Similar proteolytic systems are thought to be involved in antemortem and postmortem degradation of myofibrillar proteins, so BAA-mediated protein accretion is probably due, at least in part, to reduced protein degradation. To examine whether protein synthesis was altered with BAA feeding, the level of skeletal muscle alpha-actin mRNA was quantified. Longissimus muscle alpha-actin mRNA abundance was 30% greater in BAA-fed lambs. Collectively, these results indicate that dietary administration of BAA increases muscle mass through hypertrophy and that the increase in muscle protein accretion is due to reduced degradation and possibly to increased synthesis of muscle proteins.

Topics: Actins; Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; DNA; Endopeptidases; Hypertrophy; Least-Squares Analysis; Male; Meat; Muscle Development; Muscle Proteins; Muscles; Postmortem Changes; Pyridines; Random Allocation; RNA; RNA, Messenger; Sheep; Weight Gain

The objectives of this study were to examine effects of a beta-adrenergic agonist (cimaterol) on growth and muscle development in rabbits and to examine cimaterol's effects on myofibrillar protein degradation (MPD) and on activities of several proteolytic enzymes including the calcium-dependent proteinases (CDP). Twelve New Zealand White rabbits were assigned to either control diets or to diets containing cimaterol for 35 d, after which they were killed and effects on performance and tissue weight gains were determined. Urine was collected from d 21 through 28 from each rabbit for assessment of N tau-methylhistidine (NMH) excretion. Cimaterol increased rates of gain, efficiency of gain and skeletal muscle weights. Enhancement in muscle weight was associated with an increase in total DNA and with a reduction in NMH. Cimaterol did not affect activities of cathepsin B, cathepsin D or neutral serine proteinase, but it reduced activities of the millimolar and micromolar forms of the CDP by 58 and 57%, respectively, and it reduced activity of the inhibitor of the CDP (calpastatin) by 52%. Cimaterol-dependent myofibrillar protein accretion was likely mediated, at least in part, by a reduction in MPD. The change in MPD was associated with a reduction in muscle CDP activities. Cimaterol-dependent muscle hypertrophy therefore may involve changes in calcium-dependent proteolysis of myofibrillar proteins. The significance of the effects of cimaterol on calpastatin activity is not known.

Topics: Animals; Calcium-Binding Proteins; Calpain; DNA; Ethanolamines; Liver; Male; Methylhistidines; Muscle Development; Muscle Proteins; Muscles; Myofibrils; Rabbits; Random Allocation; RNA; Weight Gain