Article

Whole body 3-methylhistidine production and proteinase activities in porcine muscle after protein-free feeding and realimentation

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  • MTI BioTech Inc.
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Abstract

Whole body 3-methylhistidine (3MH) production rates and proteinase activities in porcine skeletal muscles were studied during a protein-free feeding period and subsequent realimentation. Out of 54 castrated male pigs (35 kg on day 0), six pigs were slaughtered on day 0, and 48 were randomly divided between six dietary treatment groups. During the 14 day protein-free feeding period, three isocaloric diets were provided on a restricted basis: Control (C), protein-free, carbohydrate-rich (PF/CH) or protein-free, fat-rich (PF/FAT). On day 14, eight pigs per treatment group were slaughtered. During the seven day realimentation period, all remaining pigs received the control diet at a restricted level and this formed the other three treatment groups: C–C, PF/CH–C and PF/FAT–C. On day 21, these pigs were slaughtered. Measurement of the 3MH production rate was performed during the last three days of each period. Feeding either one of the PF diets stopped the growth rate and increased the 3MH production rate, whereas proteinase and inhibitor activities in skeletal muscles were not influenced. During realimentation, the growth rate and feed efficiency were higher for both PF–C treatments than for the C–C treatment only during the first three days. The 3MH production rate, as well as proteinase and inhibitor activities in muscles, were not different between treatment groups. These data suggest that compensatory growth occurred only during the first three days of the realimentation period, but pigs did not fully compensate.

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... Several studies report short term alterations in plasma creatinine after transport at weaning (Wittish et al. 2014) or in response to different regimen in weaned or growing pigs (He et al. 2011;Devi et Kim. 2014;Park et al. 2014). ...
Thesis
The concept of robustness can be defined as the ability to maintain performances and health whatever environmental conditions. Weaning is the step where the biggest part of antibiotics is used because it is the source of multiple perturbations for the piglet. The identification of robust pigs could allow settling specific care and/or genetic selection on this criteria. The objectives of this thesis were to identify physiological parameters associated with the robustness of piglet at weaning and to predict this robustness by biological variables describing those measured responses before and after weaning. To answer to this objective, physiological variables were first measured in very different environments and, then, those ones associated with the robustness were identified. A first experiment was realized in experimental unit, aiming to study the effects of age, weaning conditions and health on the evolution of blood variables describing immune and metabolic status, stress and oxidative stress around weaning. In a second study, some physiological markers were measured on piglets coming from 16 commercial farms around weaning. Growth performances and health status were the two controlled factors of variations of farms. The analysis of data allowed us to show a high influence of health status on physiological variables around weaning. Some variables describing oxidative status, metabolic status and the activation of immune system were associated with the robustness of piglet at weaning. Thus, the most robust piglets are those ones who, in favorable or unfavorable environments, have a capacity to limit their oxidative stress, to mobilize less body reserves and to activate quickly their immune system. Those variables will have to be validated with other datasets.
Article
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Article
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Article
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This experiment was conducted to determine the relationship between 3-methylhistidine (3MH) production and proteinase activity in skeletal muscles of growing barrows. Barrows at 13 wk of age were randomly assigned to either control diet available on an ad libitum basis (21% of ME consisted of protein; control group), control diet fed restricted (pair-fed with barrows in protein-free group; intake-restricted group), or protein-free diet available on an ad libitum basis (protein-free group) for 14 d. During the last 3 d, blood samples were collected for determination of 3MH production rate, which is a measure of myofibrillar protein breakdown. At slaughter, two muscles were taken: masseter (M) and longissimus (L) muscles. The muscle samples were analyzed for calpastatin, mu-calpain, m-calpain, multicatalytic proteinase (MCP), cathepsin B, cathepsins B+L, and cystatins activities. Both muscles were also analyzed for amounts of DNA, RNA, total protein, and myofibrillar and sarcoplasmic proteins. Growth rate (kilograms/day) was influenced by dietary treatments (P < .05). Fractional breakdown rate (FBR, percentage/day) of skeletal muscle, as calculated from 3MH production rate (micromoles.kilogram-1.day-1), was 27% higher for the protein-free group than for the control group. However, no differences in proteinase activities were observed, except for lower MCP activity in the M muscle of the protein-free group than in that of the other groups (P < .05). In the present study, no direct relation was observed between myofibrillar protein degradation rate and proteinase activities in skeletal muscle during a protein-free feeding strategy.
Article
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Direct in vivo methodology is not available to accurately evaluate muscle turnover in pigs. Urinary 3-methylhistidine (3MH) excretion, which is used as an in vivo marker of muscle protein breakdown in humans and cattle, is not a valid indicator for pigs. The present study proposes that data from a single bolus dose of 3-[methyl-2H3]methylhistidine tracer can mathematically describe 3MH metabolism in pigs. Plasma concentration of the tracer is described by a linear time-invariant three-compartment model by using the SAAM/CONSAM computer modeling program. The model defines masses and fluxes of 3MH within the pigs and, in particular, the intracellular de novo production of 3MH, which should reflect muscle proteolysis. The de novo production of 3MH as calculated by the model was 621 mumol/d, corresponding to a fractional breakdown rate of 2.28%/d, which is similar to values reported by using indirect methodology. These data also suggest that certain model compartments may be indicators of body muscle mass (mass of compartment 3, r = .59, P = .006). The mathematical model developed does not depend on urine collections and can be used to assess changes in muscle proteolysis in vivo.
Book
The biotechnological advances of recent years have put us on the brink of unprecedented gains in animal productivity. Manipulation of animal growth rate and composition of gain is now possible by a variety of techniques. Ex­ amples include ingestion of beta-adrenergic agonists, injection of somatotropin, castration, immunization, and gene insertion. Animal Growth Regulation ad­ dresses modem concepts of growth regulation with an emphasis on agricul­ turally important animals. This emphasis is not exclusive, as many situations exist in which the only information available was generated in other species, and this information has been included for the sake of clarity and completeness. However, because of the overall orientation of this volume, particular attention has been given to the regulation of skeletal muscle, adipose tissue, and bone growth. Certain hormones and growth factors have a profound influence on growth regulation and this basic physiological knowledge is being harnessed to maniplilate growth. Thus, considerable emphasis has been given to growth hor­ mone-somatomedinlinsulinlike growth factor regulation of cell and tissue growth. The involvement of peptides coded by protooncogenes and of negative growth regulators, such as transforming growth factor-l3, represents an emerging area of molecular biology wherein basic knowledge offers potential exploitation for growth manipulation. Opportunities also exist for regulation of protein turn­ over, especially from the standpoint of protein degradation. Therefore, a place was reserved for these topics in order to provide relevant basic knowledge.
Chapter
The dynamic nature of cellular proteins was demonstrated over 40 years ago (Schoenheimer et al., 1939). But it was not until 1967–1972 that the measurement of protein turnover in vivo received systematic attention, notably by Wa-terlow’s group (Waterlow and Stephen, 1967; Picou and Taylor-Roberts, 1969; Garlick, 1969; Garlick and Marshall, 1972). It is now generally accepted that cellular proteins are always subject to continual degradation even when protein is neither gained nor lost from the body.
Chapter
Schoenheimer and Rittenberg’s paper published nearly 50 years ago (Schoen-heimer and Rittenberg, 1940) established that accumulation of muscle tissue or muscle growth must depend on both the rate of muscle protein synthesis and the rate of muscle protein degradation. Despite this axiom, most of the attention of animal scientists during the period from 1940 to 1980 focused on increasing the rate of muscle growth by increasing the rate of muscle protein synthesis. Because of this, a great deal is known about the mechanism of muscle protein synthesis and how it is controlled. Little is known, however, about the mechanism of muscle protein degradation. It is clear that muscle proteins turn over metabolically with half lives ranging from 2 to 20 days (Low and Goldberg, 1973; Koizumi, 1974; Rubenstein et al., 1976; Martin et al., 1977; Zak et al., 1977; Millward et al., 1978; Martin, 1981; Wolitsky et al., 1984), but the nature of the proteolytic enzymes responsible for this turnover remains unknown. It was learned in 1969 that the rate of muscle protein degradation can vary over a wide range in response to physiological demand (Goldberg, 1969a,b).
Article
Molecular mechanisms underlying changes in muscle protein turnover are not fully understood. In this study, effects of fasting on mRNA concentrations encoding several proteinases in skeletal muscle were investigated. Proteinases included calpains I and II, cathepsin D, and proteasome. We also examined effects of fasting on the calpain small subunit, calpastatin, and on β-actin mRNAs for comparative purposes. Fasting increased mRNAs encoding all proteinases, calpain small subunit, and calpastatin in skeletal muscle but reduced β-actin mRNA. This effect was most pronounced for cathepsin D. To determine whether the changes observed in skeletal muscle occurred in other tissues, we examined effects of fasting on proteinase mRNA concentrations in liver, lung, and kidney. Fasting either had no effect or reduced proteinase, calpastatin, and calpain small subunit mRNA concentrations in these tissues. Fasting reduced β-actin mRNA concentration in all tissues. Therefore, proteinase gene expression in skeletal muscle differs from other tissues during fasting. Despite the changes in calpain mRNA concentrations in muscle, changes in calpain activities were not detected. This suggests that either calpain synthesis was concomitantly reduced or calpain turnover was increased during fasting. Differences in calpain mRNA concentrations were detected among tissues and these were related to differences in calpain concentrations and activities among tissues. We conclude that calpain and other proteinase genes are co-regulated in muscle in a manner that differs from other tissues, and that fasting-dependent changes in muscle calpain mRNA serve to maintain calpain concentrations at fixed levels at a time when muscle protein synthesis is reduced or calpain stability is reduced. Finally, we conclude that differences in calpain mRNAs among tissues underlie the differences in tissue calpain concentrations and activities.
Article
In pig breeding it is quite common to select for bodyweight gain, feed conversion and slaughter quality. Various values have been found by different research- workers for the relationships between these traits. These differences in values have mainly been caused by differences between feeding levels and by those between chemical composition of carcases. Protein and fat deposition can be calculated from bodyweight gain and feed intake; these traits would take into account differences in feeding level and. chemical composition of carcases better than bodyweight gain and feed conversion do.Therefore an investigation was done- to find out how precisely protein and fat deposition could be predicted from bodyweight gain, bodyweight and feed intake, and- to study the variation in protein and fat deposition at restricted and ad libitum feeding and the relation between these factors, growth and carcase traits.For the calculation of protein and fat deposition three models were obtained from data in the literature (EBC, EBK and MEK models - Section 2.3). These models were based on physiological data connected with bodyweight and feed utilization (maintenance, protein and fat deposition).In the MEK model it was assumed that all ME was used for maintenance, protein and fat deposition according to the following equation:ME = ME M + c ΔP + d ΔF.The EBC and EBK models started from the energy balance:EB = 5.7 ΔP + 9.46 ΔF.In order to estimate the amount of ME P or EB that was used for protein and fat deposition, respectively, the research-workers calculated from literature or their own data the relationships between the components of growth. EB was calculated from ME using the equationEB = (ME - ME M ) x efficiency.The equations that were used to calculate protein (ΔP) and fat (ΔF) deposition in the EBC, EM and MEK models, were:In these models 4 factors were varied:- maintenance requirement: 80, 100 or 120 kcal ME/kg3/4;- efficiency for synthesizing protein and fat from ME P . For the EBC and EBK models the same figure was assumed for protein as for fat: 0. 55, 0.65 or 0. 75. In the MEK model the ME costs (kcal/g) for synthesizing protein and fat were assumed to be: 16 and 13, 13 and 13, or 11.4 and 12.6;- the ratio protein to protein + water in the EBC model: a constant value was assumed or a value was calculated from the amount of protein and water at each bodyweight. These amounts have been estimated using allometric equations;- the amounts of protein, water and fat gain (in the EBC model) or the amount of bodyweight gain minus gut fill (in the EBK and MEK models). The alternative values of this factor have been obtained using linear or allometric equations between bodyweight and the amounts of ash and gut fill or gut fill.The equations or values used for calculating the values of the 4 factors are shown in Table 3.4.To judge the precision of the prediction of protein and fat deposition the following 4 traits were calculated:- the level of protein and fat deposition;- the values of correlation coefficients between calculated and found protein and fat deposition.The data used consisted of energy and N balances from 6 different investigations, and results of chemical analysis of the empty body of pigs. In addition, bodyweight gain was estimated using a cubic curve in 3 sets of data mentioned above. Thus, totally 10 sets of data were available for the computations. For the computations the values of correlation coefficients between calculated and found protein and fat deposition were transformed by the Z transformation of FISHER.With the statistical Model 3.1, the following effects were tested for each trait and each model:- differences between sets of data,- differences between factors, and- differences between models.The level of the four traits differed considerably between the various sets of data (Tables 3.5 and 3.7). In addition the interaction between data and factors was always significant. These interactions are also shown in Table 3.6 by the great differences between sets of data for the values of regression coefficients of the traits on the factors. The differences between sets of data might be caused by:- differences between feeding level, feed composition, sex or breed;- systematic differences between experimental procedures used by the research workers for energy and N balances, weighings etc.Computation of bodyweight gain from a cubic curve doubled the value of the correlation coefficient between calculated and found protein deposition in JUST NIELSEN's data, compared with those, found by using the weight gain obtained from linear interpolation between 2 weighings. Using a cubic curve, in LUDVIGSEN and THORBEK's data the same value of correlation coefficient was found, and in BREIREM's data a lower value, compared with those obtained by calculating bodyweight gain from linear interpolation. The value of the correlation coefficient between calculated and found fat deposition in JUST NIELSEN's data was 0.970, using data from energy balances, compared with 0.257 using chemical analysis of the empty body. With reference to JUST NIELSEN (1970), it has been stated that more data are necessary to be sure about the value and precision of results from energy and N balances or from comparative slaughter techniques.It has been indicated that the interactions between factors and sets of data have been partly caused by systematic differences in the various sets of data between bodyweights of the pigs (Table 3.14).The relative contribution of the variance in calculated protein and fat deposition to differences between sets of data was 60 to 70 % in the three models; the relative contribution to this effect by the variance in the value of correlation coefficients between calculated and found protein and fat deposition was 95 to 99 % (Table 3.8). The relative contribution of the variance in protein and fat deposition to the factors, maintenance and efficiency was - excluding protein deposition in the MEK Model - considerably higher than their contribution to the other two factors.If a higher maintenance requirement, a lower efficiency or a greater amount of water in the bodyweight gain were considered, the protein deposition and the value of the correlation coefficient between calculated and found protein deposition increased; however, then the fat deposition and the value of correlation coefficient between calculated and found fat deposition decreased (Tables 3.6 and 3.15). These changes in protein and fat deposition follow from the equations in Section 2.3, but they can also be explained by the difference between energy content of protein and fat. The changes in the values of correlation coefficients between calculated and found protein and fat deposition has been explained by a non-linear relationship between calculated and found protein and fat deposition.It is doubtful, whether the highest values of the correlation coefficients between calculated and found protein deposition also gave the best prediction of these traits.There were only small differences between the 3 models (Table 3.10). The value of 4.6838 for LBM /protein in the EBK and MEK models was lower than it should be, if based on the average bodyweight of the pigs in the various sets of data. Therefore with this value, protein deposition was overestimated and fat deposition was underestimated. The values of correlation coefficients between calculated and found protein deposition were significantly lower in the MEK model than in the EBC and EBK models.The equations used for the calculation of protein and fat deposition in Chapter 4 were based on the EBK model. To make a choice between the different combinations of the 4 factors it was assumed that:- maintenance requirement is 100 kcal ME/kg3/4, - protein deposition using N balances was overestimated by 15.5 %, and- 'real' EB was: 9.46 Δ F (found) + 5.7 (1-0.845) Δ P (found).If this 'real' EB was taken into account, a value of 0.62 for the factor efficiency was obtained. For the factor LBM /protein the value was used, that was obtained at each bodyweight from the bodyweight and the amount of fat, gut fill and protein, using allometric equations (Table 3.3). Δ L E was calculated from the bodyweight and amount of gut fill changing linearly in relation to weight.The variation in protein and fat deposition and the relation between these traits, growth and carcase traits were investigated in pigs fed restrictedly and ad libitum. The data of the restrictedly fed pigs were obtained from 356 males, 540 castrated males and 770 females of the DL and DY breeds (Table 4.2). The data of the ad libitum fed pigs were obtained from 29 progeny groups (4 to 9 DL females per group) of sires (Table 4.4). The restrictedly fed pigs were tested from 25 to 100 kg bodyweight, and the feed was adjusted according to liveweight (Table 4.1). The ad libitum fed pigs got the same ration. They were fattened - starting at an age of about 9 weeks - for 4 or 6 months. The pigs were divided at random between treatments within litters and within pens. The bodyweight at each day was computed using a cubic curve. For the restrictedly fed pigs it was assumed that the feeding level was proportional to the feeding schedule advised (Table 4.1). For the ad libitum fed pigs the daily feed intake was computed from the weekly intakes by the method of 'parabolic splines'.The course of protein and fat deposition of restrictedly fed males, females and castrated males in relation to bodyweight was in rather good agreement with the data in literature (Figure 4. 1). The protein deposition in castrated males and females of the DL breed was significantly lower than those in the DY breed from about 45 kg onwards; in males of DL breed the protein deposition was lower during the whole testing period. At the end of the testing period the difference between males and females was about +40 g protein per day. At that moment the difference between females and castrated males was +7 g protein in the DL breed and + 16 g in the DY breed. The average difference between breeds and between sexes for the whole testing period are given in Table 4.9. The average daily protein deposition was 7.53 g higher in the DY breed than it was in the DL breed, and the fat deposition was 4.90 g lower in the DY breed. The differences between sexes were smaller than those mentioned in the literature. Probably, these smaller differences were partly caused by an underestimation of maintenance in pigs with a higher protein deposition. In ad libitum fed pigs the average daily protein deposition was about 25 % lower than in restrictedly fed pigs, and the daily fat deposition was about 25 % higher (Tables 4.8 and 4.11). These differences could be partly attributed to wastage of feed with self-feeders, by differences in activity between restrictedly fed pigs (housed individually) and the ad libitum fed pigs (housed in groups) and the possible influence of the feeding level on the ratio LBM to protein.The coefficients of variation of daily protein deposition was 1.6 times higher than those of bodyweight gain and feeding level; the coefficients of variation of daily fat deposition were 30% lower. The values of the heritabilities of protein and fat deposition were 0.176 and 0.080. These values computed for the traits bodyweight gain, feed conversion and backfat thickness were 0.249, 0.231 and 0.545, respectively.The values of correlation coefficients between protein deposition and bodyweight gain in restrictedly fed pigs were nearly +1. It was argued that these values were to be expected when the feeding schedule was according to bodyweight. The values of correlation coefficients between bodyweight gain and feed conversion, and carcase traits were nearly the same compared with those between protein and fat deposition, and carcase traits (Tables 4.12 and 4.13). The values of correlation coefficients between protein deposition, fat deposition, bodyweight gain and feed conversion in ad libitum fed pigs were significantly lower than those in restrictedly fed pigs (Table 4.15). The value of the correlation coefficient between bodyweight gain and protein deposition in ad libitum fed pigs was in rather good agreement with the value found in literature (Table 4.17). The value of correlation coefficient between protein deposition and lean cuts (0.743) and this between fat deposition and backfat thickness (0.796) in ad libitum fed pigs were significantly higher than those in restrictedly fed pigs. However one should keep in mind that only 29 groups of pigs were fattened ad libitum. To make clear the variation in protein and fat deposition and the relationships between these traits and bodyweight gain, feed conversion and carcase traits, it is necessary to collect more energy and N balances of pigs fed individually and at various feeding levels.
Article
Summary Skeletal muscle growth of swine differing in muscularity was studied by analysis of DNA, RNA and protein in the biceps femoris muscle of "light muscled" (LM) and "heavy muscled" (HM) Duroc pigs. Three I-1M litters and 3 LM litters were produced by mating HM gilts to HM boars and LM gilts to LM boars, respectively. Boars and gilts were selected based on visual appraisal and on carcass data of littermate barrows and gilts. One pig from each litter was slaughtered at I, 15, 30, 62, 105, 145 and 210 days of age (42 pigs total). Growth rate did not differ among HM and LM pigs. HM pigs had less backfat, larger loin eye areas and greater semitendinosus and biceps femoris weights. These differences were accentuated later in the growth period. DNA and RNA concentrations in the biceps femoris muscle decreased significantly with age (P'< .01) but there were no significant differences between groups. Protein concentrations increased between 1 and 30 days but declined between 30 and 210 days of age and were slightly higher in I-IM pigs. Total DNA, RNA, and protein in the biceps femoris muscle increased with age (P< .01). Total DNA and total protein was greater in the HM pigs when differences over all age groups were analyzed 0 a < .05) but group differences were not significant for total RNA even though the HM pigs had higher total RNA. RNA to DNA and protein to DNA ratios tended to increase with age indicating hypertrophic growth. RNA to DNA and protein to DNA ratios were significantly different between HM and LM pigs in a few age groups but no regular pattern of differences was present. It was concluded that the greater biceps femoris muscle mass in I-IM pigs was accompanied by a proportional increase in the number of nuclei present in the muscle since protein to DNA ratios were similar between the two groups of animals.
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A simple and rapid method for measuring 3-methylhistidine (3MH) in plasma and urine is described. Internal standard, 1-methylhistidine (1MH), was added to plasma, acidified and absorbed onto cation-exchange columns. It was then eluted from columns, dried, and derivatized for gas chromatography/mass spectrometry. A major fragment of 3MH was monitored at 238 u and 3-methyl-(methyl-2H3)histidine (d3-3MH) (used for in vivo kinetics) at 241 u, whereas 1MH was monitored at 340 u and eluted 0.5 min later than 3MH. Standard curves for plasma analysis were linear and nanamole amounts of 3MH in plasma were determined with a precision of 3.5%. 3MH was also quantitated in urine; however, because of substantial amounts of 1MH, (18O2)1MH was used as the internal standard. Nanamole amounts of 3MH were determined in urine with a precision of 2.7%. Application of the 3MH analytical method was used to develop a kinetic compartmental model by using the stable isotope of 3MH, d3-3MH. Cattle, like humans, quantitatively excrete 3MH in the urine. A young bovine was injected with d3-3MH and the enrichment curve in plasma was evaluated in order to obtain a steady-state production rate of 3MH. The decay curve was modeled through the use of NIH-SAAM modeling program. The kinetics of d3-3MH from plasma were adequately described by a three-pool compartmental model. The de novo production rate of 3MH estimated in the calf was 665 mumol per day. This corresponded to an estimated fractional turnover rate of 1.56% per day, which was similar to estimates obtained from urine collections.(ABSTRACT TRUNCATED AT 250 WORDS)
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This manuscript summarizes research results from our laboratory regarding the role of endogenous proteases in post mortem proteolysis resulting in meat tenderization. Proteolysis of key myofibrillar proteins is the principal reason for ultrastructural changes in skeletal muscle associated with meat tenderization. Proteases should have the following characteristics to be considered as possible candidates for bringing about post mortem changes: i) to be located within skeletal muscle cells; ii) to have access to the substrate ie, myofibrils); and iii) to be able to hydrolyze the same proteins that are degraded during post mortem storage. Of the proteases located within skeletal muscle cells and thus far characterized, only calpains have all of the above characteristics. Numerous experiments conducted in our laboratory have indicated that the calcium-dependent proteolytic system (calpains) is responsible for post mortem proteolysis. Some of this evidence includes: 1) incubation of muscle slices with buffer containing Ca2+ accelerates post mortem proteolysis; 2) incubation of muscle slices with Ca2+ chelators inhibits post mortem proteolysis; 3) infusion or injection of carcasses with a solution of calcium chloride accelerates post mortem proteolysis and the tenderization process such that post mortem storage beyond 24 h to ensure meat tenderness is no longer necessary; 4) infusion of carcasses with zinc chloride, a potent inhibitor of calpains, blocks post mortem proteolysis and the tenderization process; and 5) feeding a beta-adrenergic agonist to lambs results in a reduction of the proteolytic capacity of the calpain system, which leads to a decreased rate of post mortem proteolysis and produces tough meat.(ABSTRACT TRUNCATED AT 250 WORDS)
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Muscle protein degradation has an important role in rate of muscle growth. It has been difficult to develop procedures for measuring rate of muscle protein degradation in living animals, and most studies have used in vitro systems and muscle strips to determine rate of protein degradation. The relationship between results obtained by using muscle strips and rate of muscle protein turnover in living animals is unclear because these strips are in negative nitrogen balance and often develop hypoxic cores. Also, rate of protein degradation is usually estimated by release of labeled amino acids, which reflects an average rate of degradation of all cellular proteins and does not distinguish between rates of degradation of different groups of proteins such as the sarcoplasmic and the myofibrillar proteins in muscle. A number of studies have suggested that the calpain system initiates turnover of myofibrillar proteins, which are the major group of proteins in striated muscle, by making specific cleavages that release thick and thin filaments from the surface of the myofibril and large polypeptide fragments from some of the other myofibrillar proteins. The calpains do not degrade myofibrillar proteins to small peptides or to amino acids, and they cause no bulk degradation of sarcoplasmic proteins. Hence, the calpains are not directly responsible for release of amino acids during muscle protein turnover. Activity of the calpains in living cells is regulated by calpastatin and Ca2+, but the nature of this regulation is still unclear.
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The aim of this review is to assess the current understanding of the regulation protein synthesis and degradation in striated muscle, a tissue which constitutes the largest single element of the protein-bound nitrogen pool in the mammalian organism. Numerous experimental interventions alter protein turnover rates in vitro and we will discuss whether or not these might fulfil regulatory functions in vivo. We will also consider the molecular mechanisms that may be involved in the regulation of protein turnover.
Article
In order to examine the effects of streptozotocin-induced diabetes, dietary protein, and starvation on protein degradation in skeletal muscle of perfused rat hindquarters, rates of myofibrillar and total protein degradation were estimated from the release of 3-methylhistidine (N tau-methylhistidine, 3-MH) and tyrosine, respectively. In rats fed a 20% protein diet (controls), the fractional degradation rate of myofibrillar protein was approximately 56% of the total muscle protein. In streptozotocin-induced diabetic rats, 3-MH release by perfused muscle increased significantly on d 1 of treatment and sustained a high level thereafter. By contrast, tyrosine release did not change. Feeding a 50% protein diet for 1 wk altered neither 3-MH nor tyrosine release. Protein-free feeding, though, suppressed tyrosine release to 49% of controls, but did not affect 3-MH release. Starvation for 3 d did not affect tyrosine release, but did increase 3-MH release to 203% of controls. These results indicate that in diabetic and starved rats myofibrillar protein is preferentially degraded, while in protein-deficient rats, non-myofibrillar protein degradation is selectively suppressed. From these observations, we conclude that the degradation of myofibrillar and non-myofibrillar proteins in skeletal muscle can be differentially regulated.
Article
Previous studies have demonstrated that brief fasting augments and refeeding a complete diet diminishes the breakdown of myofibrillar proteins in rat skeletal muscle. The purpose of the present study was to determine which dietary component(s) was responsible for this effect and to determine the role of insulin and amino acids. Myofibrillar proteolysis was evaluated by measuring the release of 3-methylhistidine by perfused rat muscle of 1-day fasted rats and 1-day fasted rats refed for 4-24 h with a complete, protein-free, or lipid meal. For comparison, tyrosine release by perfused muscle was measured in the absence and presence of cycloheximide to evaluate net and total proteolysis, respectively. Refeeding of either diet increased plasma insulin. Despite this, myofibrillar proteolysis decreased only when protein or amino acids was included in the test meal. On the other hand, the complete or protein-free meal decreased tyrosine release in the absence but not in the presence of cycloheximide, suggesting that either diet enhanced muscle protein synthesis. Most amino acids in plasma and muscle decreased after refeeding the protein-free meal, whereas after the complete meal some amino acids in plasma and muscle increased, whereas other decreased or changed little. These results indicate that decreased myofibrillar proteolysis in muscle after refeeding of food-deprived rats requires dietary protein or amino acids. They also suggest that hormonal and/or nutritional factors other than insulin and amino acids may orchestrate this response. However, a role of amino acids cannot yet be excluded, because it is conceivable that changes in specific amino acids in plasma instead of muscle may signal diminished proteolysis.
Article
Young pigs were fed a low protein diet for varying lengths of time, followed by repletion, to evaluate responses of selected constituents of the longissimus dorsi muscle. Protein restriction was initiated at 2 wk of age during suckling, and continued to weaning at 5 wk, to 7 wk and to 9 wk (restriction periods 1,2 and 3 respectively). A group of pigs was repleted for 3 wk after each restriction period (repletion 1, 2 and 3). Age controls were fed a diet with adequate protein throughout the 12 wk study. Body weight, muscle weight and protein, muscle content and concentrations of DNA, RNA and hydroxyproline did not differ from age controls at weaning. The dam, therefore, was able to furnish strong nutritional support even though her diet was inadequate. Restriction periods 2 and 3 resulted in reduced body and muscle weight, and alterations in protein, DNA, RNA and hydroxyproline. Response to repletion was a function of age rather than length of time consuming the low protein diet. Repletion period 1 resulted in a weak general response. Significant changes toward normal in all constituents altered by the low protein diet occurred during repletion periods 2 and 3. Muscle cytochrome oxidase was unaffected by dietary protein level whereas cathepsin D was elevated by the low protein diet. This study suggests an ultimate return to normal muscle composition, but also the unlikelihood that normal size would be obtained by repleted pigs unless there was an extension of the growth period.
Article
A simple and rapid assay for quantitative determinations of DNA in crude homogenates is described. The method is based on the enhancement of fluorescence seen when bisbenzimidazole (Hoechst 33258) binds to DNA. Crude homogenates in which chromatin has been dissociated with high salt buffer can be assayed directly and reliably in a few minutes. The dissociation of chromatin is critical to accurate determinations of DNA in biological materials using this method. The assay can detect as little as 10 ng of DNA with rather unsophisticated instrumentation.
Article
Total protein and actomyosin degradation rates were determined in perfused rat hemicorpus preparations. By simultaneously measuring the release of two nonmetabolizable amino acids phenylalanine and N tau-methylhistidine from the hemicorpus, the respective rates of total protein and actomyosin degradation could be calculated. When rats were deprived of food for 48 h, the rate of total protein degradation increased to 148% of the fed controls. If rats were food deprived and then refed for 24 h, the degradation rate decreased to only 79% of the rate of fed controls. Measurement of N tau-methylhistidine release indicated that food deprivation led to a dramatic increase in the rate of actomyosin degradation (427% of fed), whereas refeeding decreased the actomyosin degradation rate to that of the fed controls. Calculations of the fractional degradation rates show that actomyosin breaks down at a much slower rate than the nonactomyosin proteins (1.5 vs. 20.8%/day in preparations from fed rats, and 6.2 vs. 28.2%/day in preparations from food-deprived rats). Therefore, the contribution of actomyosin breakdown to total muscle protein breakdown is small in the fed state (11%) and increased threefold after food deprivation. The addition of insulin to the perfusion medium decreased the rate of total protein degradation by 18% in preparations from food-deprived rats with no significant effect on actomyosin degradation. Thus, in vitro, insulin's major effect may be to decrease the degradation of more rapidly turning over, nonactomyosin proteins. Protein degradation, as well as protein synthesis, contributes to the adaptation of muscle to starvation and refeeding.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Summary Two experiments involving 288 pigs were conducted to determine the effects of short- term feed restriction during the growing period on average daily gain (ADG), average daily feed consumption, feed required/unit of gain (F/G), age at 100 kg and average backfat depth at 100 kg. Pigs were fed 100, 85 or 70% of ad libitum consumption for 2- or 4-wk periods in Exp. 1 and 100 or 85% for 2- or 4-wk periods in Exp. 2, and then allowed ad libitum consumption during the postrestriction period to 100 kg body weight. During the restriction period, ADG was reduced by feed restriction, but F/G was not affected. In the postrestriction period, daily feed consumption was similar for all treat- ments, while ADG tended to be higher for previously restricted than for control pigs, and pigs fed 85% ad libitum tended to gain faster than those fed 70%. In Exp. 1, pigs restricted to 85% for 4 wk gained 8% faster than control pigs and F/G was reduced by 5.6% after ad libitum consumption was resumed. For the total test period (including restriction), ADG was not affected by treatment in either experiment. In Exp. 1, restricted pigs consumed less feed (P
Article
1. The validity of the urinary excretion of N τ -methyl histidine (N τ -MH) by pigs as an index of muscle protein breakdown in vivo was tested using the criterion of the rate of recovery of radioactivity in urine following an intravenous dose of N τ -[ ¹⁴ CH 3 ]methyl histidine. 2. Urinary recoveries of radioactivity from five animals were less than 21% of dose in 7 d after which the daily recovery was less than 0.3% per day. 3. The incomplete recoveries of radioactivity were associated with the presence in muscle of a large. pool of non-protein-bound N τ -MH, the concentration of which increased with age. 4. The N τ -MH in this pool was present as free N τ -MH and in a dipeptide which constituted more than 90% of the total non-protein-bound N τ -MH. The contribution of the peptide increased with age, reaching 99.8% in older animals. 5. The pool of non-protein-bound N τ -MH was maintained and increased in both established and newly accreted tissue by retention of some of the N τ -MH released by muscle protein breakdown, only a proportion of which was therefore available for excretion. Hence, the urinary excretion of N τ -MH is not a valid index of muscle protein breakdown in pig.
Article
Infection in young growing animals is manifested by poor tissue protein accretion; during subsequent catch-up growth this is reversed. To account for these changes, protein synthesis and degradation were measured in vivo in skeletal muscle, skin, liver and small intestine in weanling rats during catch-up growth after Escherichia coli infection. Observations were made at d 4, 6, 8, 11 and 14, when infected rats had elevated nitrogen balance. Liver protein mass and turnover were not affected by treatment. Although protein mass of small intestine fell during infection, catch-up was achieved before d 4, suggesting a high priority for protein repletion in this tissue. On d 4, protein mass was lower (P < 0.05) in muscle (-19%) and skin (-23%) in infected vs. control rats. Thereafter growth rates of skeletal muscle and skin were higher (P < 0.001) in infected rats compared with controls. Catch-up growth was most pronounced early, but continued throughout the study. During catch-up growth, protein synthesis (mg/d) in muscle and skin was not different between control and infected animals. Protein synthesis was maintained in muscle because RNA mass was maintained. During catch-up growth in muscle and skin of infected rats, there was lower protein degradation (mg/d) than in controls (P < 0.05). We conclude that alterations in protein turnover during catch-up growth are tissue and time dependent and are different from those described in other hyperanabolic states.
Article
Within 1 h after slaughter, two 10-g samples of longissimus muscle were obtained from four crossbred beef cattle. Samples were homogenized in three or six volumes of extraction solution that consisted of 50 mM Tris base, 10 mM EDTA, and 10 mM 2-mercaptoethanol, pH adjusted to 8.3 with 6 N HCl. After centrifugation the supernatant from the three-volume extract was fractionated by addition of solid (NH4) 2SO4. Proteins that precipitate between 40 and 65% (NH4) 2SO4 were dialyzed and then loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 100 to 400 mM (125 mL of each; Method A). The six-volume extract was loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 0 to 350 mM (250 mL of each; Method B). Total peptidase activity eluted from the column was determined using the synthetic peptide N-CBZ-Gly-Gly-Leu-p-nitroanilide. Method B yielded greater multicatalytic proteinase complex (MCP) activities (picomoles of p-nitroaniline released/hour-1) per gram of muscle (1,538.25 +/- 105.15) than did Method A (1,195.05 +/- 86.55; P < .05). In addition, Method B permitted the quantification of calpain activity from the same fractions eluted. The relationship between enzyme activity and assay time (up to 45 min) and protein concentration (up to 10 micrograms) in the assay was linear. Studies indicated that the optimum temperature is in the range of 50 to 60 degrees C and the optimum pH in the range of 7.5 to 8.5.(ABSTRACT TRUNCATED AT 250 WORDS)
A new catheterization method was developed to repeatedly take blood samples from young pigs, with minimal disturbance to the animal. The entire procedure takes about 15 minutes, requires a light plane of surgical anesthesia, and causes only a small insertion wound. An additional advantage of this catheterization method is the possibility of replacing the catheter swiftly during the process of blood sample collection if necessary.
sures of skeletal muscle breakdown in protein-deficient grow
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Gene expression of calpains and breath and Trout (1973) fed two week-old pigs a low their specific endogenous inhibitor, calpastatin, in skeletal (5%) protein diet for three, five or seven weeks, muscle of fed and fasted rabbits
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Ilian, M.A., Forsberg, N.E., 1992. Gene expression of calpains and breath and Trout (1973) fed two week-old pigs a low their specific endogenous inhibitor, calpastatin, in skeletal (5%) protein diet for three, five or seven weeks, muscle of fed and fasted rabbits. Biochem. J. 287, 163–171.
Cellular growth of skeletal infection in weanling rats
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altered during compensatory growth following Escherichia coli Powell, S.E., Aberle, E.D., 1975. Cellular growth of skeletal infection in weanling rats. J. Nutr. 125, 520–530.