Recent progress in elucidating signalling proteolytic pathways in muscle wasting: potential clinical implications.

AIMS: Muscle wasting prevails with disuse (bedrest and immobilisation) and is associated with many diseases (cancer, sepsis, diabetes, kidney failure, trauma, etc.). This results first in prolonged hospitalisation with associated high health-care costs and second and ultimately in increased morbidity and mortality. The precise characterisation of the signalling pathways leading to muscle atrophy is therefore particularly relevant in clinical settings. DATA SYNTHESIS: Recent major papers have identified highly complex intricate pathways of signalling molecules, which induce the transcription of the muscle-specific ubiquitin protein ligases MAFbx/Atrogin-1 and MuRF1 that are overexpressed in nearly all muscle wasting diseases. These signalling pathways have been targeted with success in animal models of muscle wasting. In particular, these findings have revealed a finely tuned crosstalk between both anabolic and catabolic processes. CONCLUSIONS: Whether or not such strategies may be useful for blocking or at least limiting muscle wasting in weight losing and cachectic patients is becoming nowadays a very exciting clinical challenge.

Coordinate expression of the 19S regulatory complex and evidence for ubiquitin-dependent telethonin degradation in the unloaded soleus muscle.

Catabolic stimuli induce a coordinate expression of the 20S proteasome subunits in skeletal muscles. However, contradictory data have been obtained for the 19S regulatory complex (RC) subunits, which could reflect differential regulation at the transcriptional and/or translational level. To address this point we used a well-established model of muscle atrophy (hindlimb suspension) and determined the mRNA levels for 19S subunits belonging to both the base (non-ATPase S1, ATPases S7 and S8) and the lid (S14) of the 19S RC. Concomitant increased mRNA levels were observed for all studied subunits in rat soleus muscles after 9 days of unloading. In addition, analysis of polysome profiles showed a similar proportion of actively translated mRNA (50%) in unloaded and control soleus muscle. Furthermore, the repressed pool of messenger ribonucleoparticles (mRNPs) was low in both control (14%) and unloaded (15%) animals. Our data show that representative 19S subunits (S7 and S8) were efficiently translated, suggesting a coordinate production of 19S RC subunits. The 19S RC is responsible for the binding of polyubiquitin conjugates that are subsequently degraded inside the 20S proteasome core particle. We observed that soleus muscle atrophy was accompanied by an accumulation of ubiquitin conjugates. Purification of ubiquitin conjugates using the S5a 19S subunit followed by deubiquitination identified telethonin as a 26S proteasome substrate. In conclusion, muscle atrophy induces a concomitant expression of 26S proteasome subunits. Substrates to be degraded include a protein required for maintaining the structural integrity of sarcomeres.

Myostatin gene deletion prevents glucocorticoid-induced muscle atrophy.

Glucocorticoids mediate muscle atrophy in many catabolic states. Myostatin expression, a negative regulator of muscle growth, is increased by glucocorticoids and myostatin overexpression is associated with lower muscle mass. This suggests that myostatin is required for the catabolic effects of glucocorticoids. We therefore investigated whether myostatin gene disruption could prevent muscle atrophy caused by glucocorticoids. Male myostatin knockout (KO) and wild-type mice were subjected to dexamethasone treatment (1 mg/kg.d for 10 d or 5 mg/kg.d for 4 d). In wild-type mice, daily administration of low-dose dexamethasone for 10 d resulted in muscle atrophy (tibialis anterior: -15%; gastrocnemius: -13%; P < 0.01) due to 15% decrease in the muscle fiber cross-sectional area (1621 +/- 31 vs. 1918 +/- 64 microm(2), P < 0.01). In KO mice, there was no reduction of muscle mass nor fiber cross-sectional area after dexamethasone treatment. Muscle atrophy after 4 d of high-dose dexamethasone was associated with increased mRNA of enzymes involved in proteolytic pathways (atrogin-1, muscle ring finger 1, and cathepsin L) and increased chymotrypsin-like proteasomal activity. In contrast, the mRNA of these enzymes and the proteasomal activity were not significantly affected by dexamethasone in KO mice. Muscle IGF-I mRNA was paradoxically decreased in KO mice (-35%, P < 0.05); this was associated with a potentially compensatory increase of IGF-II expression in both saline and dexamethasone-treated KO mice (2-fold, P < 0.01). In conclusion, our results show that myostatin deletion prevents muscle atrophy in glucocorticoid-treated mice, by blunting the glucocorticoid-induced enhanced proteolysis, and suggest an important role of myostatin in muscle atrophy caused by glucocorticoids.

Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging.

We studied glucocorticoid-induced muscle wasting and subsequent recovery in adult (7-mo-old) and old (22-mo-old) rats, since the increased incidence of various disease states may result in glucocorticoids hypersecretion in aging. Adult and old rats received dexamethasone in their drinking water and were then allowed to recover. Muscle wasting occurred more rapidly in old rats and the recovery of muscle mass was impaired, suggesting that glucocorticoids may be involved in the emergence of muscle atrophy with advancing age. According to measurements in incubated epitrochlearis muscles, dexamethasone-induced muscle wasting mainly resulted from increased protein breakdown in the adult, but from depressed protein synthesis in the aged animal. Increased expression of cathepsin D, m-calpain, and ubiquitin was observed in the muscles from both dexamethasone-treated adult and old rats. By contrast, the disappearance of the stimulatory effect of glucocorticoids on protein break-down in aging occurred along with a loss of ability of steroids to enhance the expression of the 14-kD ubiquitin carrier protein E2, which is involved in protein substrates ubiquitinylation, and of subunits of the 20 S proteasome (the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates). Thus, if glucocorticoids play any role in the progressive muscle atrophy seen in aging, this is unlikely to result from an activation of the ubiquitin-proteasome proteolytic pathway.

Muscle wasting in a rat model of long-lasting sepsis results from the activation of lysosomal, Ca2+ -activated, and ubiquitin-proteasome proteolytic pathways.

We studied the alterations in skeletal muscle protein breakdown in long lasting sepsis using a rat model that reproduces a sustained and reversible catabolic state, as observed in humans. Rats were injected intravenously with live Escherichia coli; control rats were pair-fed to the intake of infected rats. Rats were studied in an acute septic phase (day 2 postinfection), in a chronic septic phase (day 6), and in a late septic phase (day 10). The importance of the lysosomal, Ca2+ -dependent, and ubiquitin-proteasome proteolytic processes was investigated using proteolytic inhibitors in incubated epitrochlearis muscles and by measuring mRNA levels for critical components of these pathways. Protein breakdown was elevated during the acute and chronic septic phases (when significant muscle wasting occurred) and returned to control values in the late septic phase (when wasting was stopped). A nonlysosomal and Ca2+ -independent process accounted for the enhanced proteolysis, and only mRNA levels for ubiquitin and subunits of the 20 S proteasome, the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates, paralleled the increased and decreased rates of proteolysis throughout. However, increased mRNA levels for the 14-kD ubiquitin conjugating enzyme E2, involved in substrate ubiquitylation, and for cathepsin B and m-calpain were observed in chronic sepsis. These data clearly support a major role for the ubiquitin-proteasome dependent proteolytic process during sepsis but also suggest that the activation of lysosomal and Ca2+ -dependent proteolysis may be important in the chronic phase.

Protein metabolism in the small intestine during cancer cachexia and chemotherapy in mice.

The impact of cancer cachexia and chemotherapy on small intestinal protein metabolism and its subsequent recovery was investigated. Cancer cachexia was induced in mice with colon 26 adenocarcinoma, which is a small and slow-growing tumor characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C6H12ClN3O4S). Both healthy mice and tumor-bearing mice were given a single i.p. injection of cystemustine (20 mg/kg) 3 days after the onset of cachexia. Cancer cachexia led to a reduced in vivo rate of protein synthesis in the small intestine relative to healthy mice (-13 to -34%; P < 0.05), resulting in a 25% loss of protein mass (P < 0.05), and decreased villus width and crypt depth (P < 0.05). In treated mice, acute cytotoxicity of chemotherapy did not promote further wasting of small intestinal protein mass, nor did it result in further damage to intestinal morphology. In contrast, mucosal damage and a 17% reduction in small intestinal protein mass (P < 0.05) were evident in healthy mice treated with cystemustine, suggesting that the effects of chemotherapy on the small intestine in a state of cancer cachexia are not additive, which was an unexpected finding. Complete and rapid recovery of small intestinal protein mass in cured mice resulted from an increase in the rate of protein synthesis compared with healthy mice (23-34%; P < 0.05). Northern hybridizations of mRNA encoding components of the major proteolytic systems suggested that proteolysis may not have mediated intestinal wasting or recovery. A major clinical goal should be to design methods to improve small intestinal protein metabolism before the initiation of chemotherapy.

Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats.

Little information is available on proteolytic pathways responsible for muscle wasting in cancer cachexia. Experiments were carried out in young rats to demonstrate whether a small (< 0.3% body weight) tumor may activate the lysosomal, Ca(2+)-dependent, and/or ATP-ubiquitin-dependent proteolytic pathway(s) in skeletal muscle. Five days after tumor implantation, protein mass of extensor digitorum longus and tibialis anterior muscles close to a Yoshida sarcoma was significantly reduced compared to the contralateral muscles. According to in vitro measurements, protein loss totally resulted from increased proteolysis and not from depressed protein synthesis. Inhibitors of lysosomal and Ca(2+)-dependent proteases did not attenuate increased rates of proteolysis in the atrophying extensor digitorum longus. Accordingly, cathepsin B and B+L activities, and mRNA levels for cathepsin B were unchanged. By contrast, ATP depletion almost totally suppressed the increased protein breakdown. Furthermore, mRNA levels for ubiquitin, 14 kDa ubiquitin carrier protein E2, and the C8 or C9 proteasome subunits increased in the atrophying muscles. Similar adaptations occurred in the muscles from cachectic animals 12 days after tumor implantation. These data strongly suggest that the activation of the ATP-ubiquitin-dependent proteolytic pathway is mainly responsible for muscle atrophy in Yoshida sarcoma-bearing rats.

Assessment of in vivo protein synthesis in lamb tissues with [3H]valine flooding doses.

Week-old lambs received an intravenous injection of 4.3, 8.5, 12.8 or 17.1 mmol [3H]valine/5 kg body weight, i.e., 3.6-14.4-times the whole-body free valine content. To ensure that protein synthesis measurements in lambs are reliable within a 30-min period, these large amounts of valine must account for at least around 11-times the total free pool of valine. This amounted to 12.8 mmol valine/5 kg body weight. There were no significant variations in plasma insulin and plasma glucagon levels 5, 13 and 30 min after the injection of so much valine. The fractional rates of protein synthesis were determined in tissues of animals receiving either 12.8 or 17.1 mmol valine/5 kg body weight. The rates of protein synthesis in the jejunum (87.5%/day), liver (106.6%/day) and tensor fasciae latae muscle (18.8%/day) of lambs injected with the 12.8 mmol [3H]valine flooding dose, were in the range of data obtained in immature rats. Increasing the flooding amount of valine up to 17.1 mmol/5 kg body weight did not significantly alter protein synthesis rates in the jejunum, liver or skeletal muscle. This suggested that both the flooding-dose method in itself and valine had no effect on in vivo protein synthesis.

Ubiquitin-proteasome-dependent proteolytic activity remains elevated after zymosan-induced sepsis in rats while muscle mass recovers.

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.

Insulin-like growth factor-1 and insulin resistance in skeletal muscles of adult and old rats.

A study was designed to compare the effects, in vitro, of insulin-like growth factor-1 (IGF-1) and insulin on rat epitrochlearis muscle metabolism during aging (1, 6-8, or 18-20 months). Our results showed that in young epitrochlearis, IGF-1 was equipotent to insulin in stimulating 2-deoxy-glucose and aminoisobutyric acid transport but more potent in increasing tyrosine incorporation into protein. Both insulin and IGF-1 action on glucose transport was decreased in adult compared with young muscle. Whereas an insulin resistance of amino acid transport and protein synthesis was also recorded in adult rat muscle, the stimulatory effect of IGF-1 on these processes was abolished. Thus the degree of resistance observed varied both with the agonist and with the subsequent metabolic process observed. Whereas modifications of IGF-1 action in mature animals may be correlated in part to the dramatic decrease of IGF-1 receptors (80%), no similar observations were recorded for the insulin receptor. Since muscle IGF-1 receptor gene expression did not decrease in parallel with receptor number, an alteration in IGF-1 receptor messenger RNA (mRNA) translation or receptor degradation may be hypothetized. We concluded that: 1) In contrast to glucose transport, intracellular IGF-1 and insulin postreceptor pathways leading to amino acid uptake and protein metabolism differ. 2) Modification in postbinding events might be involved in decreased insulin- and IGF-1-stimulated muscle metabolism during aging.

Glucocorticoids do not regulate the expression of proteolytic genes in skeletal muscle from Cushing's syndrome patients.

Glucocorticoids signal enhanced proteolysis in various instances of muscle atrophy and increased gene expression of components of the lysosomal, Ca(2+)-dependent, and/or ubiquitin-proteasome proteolytic pathways in both rat skeletal muscle and myotubes. Cushing's syndrome is characterized by chronic excessive glucocorticoid production, which results in muscle wasting. We report here no change in messenger RNA levels for cathepsin D (a lysosomal proteinase), m-calpain (a Ca(2+)-activated proteinase), ubiquitin, 14-kDa ubiquitin-activating enzyme E2, and 20S proteasome subunits (i.e. critical components of the ubiquitin-proteasome proteolytic process) in skeletal muscle from such patients. Thus, in striking contrast with animal studies, glucocorticoids did not regulate the expression of muscle proteolytic genes in Cushing's syndrome. In humans, messenger RNA levels, for at least ubiquitin and proteasome subunits, are elevated in acute situations of muscle wasting, such as head trauma or sepsis. Because Cushing's syndrome is a chronic catabolic condition, we suggest that the lack of regulation of proteolytic genes in such patients may represent an adaptive regulatory mechanisms, preventing sustained increased protein breakdown and avoiding rapid muscle wasting.

No alteration in gene expression of components of the ubiquitin-proteasome proteolytic pathway in dystrophin-deficient muscles.

Increased expression of critical components of the ubiquitin-dependent proteolytic pathway occurs in any muscle wasting condition so far studied in rodents where proteolysis rises. We have recently reported similar adaptations in head trauma patients [Mansoor et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2714-2718]. We demonstrate here that the increased muscle protein breakdown seen in mdx mice only correlated with enhanced expression of m-calpain, a Ca(2+)-activated proteinase. By contrast, no change in mRNA levels for components of the ubiquitin-proteasome proteolytic process was seen in muscles from both mdx mice and Duchenne muscular dystrophy patients. Thus, gene expression of components of this pathway is not regulated in the chronic wasting that characterizes muscular dystrophy.

Nutritional and hormonal control of protein breakdown.

Multiple lines of evidence suggest that the ubiquitin-proteasome-dependent proteolytic pathway is the major degradative process responsible for the loss of muscle proteins seen in various pathological states and following food deprivation. The first step in this pathway is the covalent attachment of polyubiquitin chains to protein substrates. This signal targets the substrates for subsequent hydrolysis into peptides by the 26S proteasome. Several metabolic abnormalities (reduced food intake, impaired mobility, and perturbations in the production or responsiveness of catabolic and anabolic hormones, cytokines and/or proteolysis inducing factors) act in concert to contribute to muscle wasting in disease states. We cite recent evidence that insulin, glucocorticoids, thyroid hormones, and nutrients regulate the rates of ubiquitinylation of protein substrates and of proteasome-dependent proteolysis in skeletal muscle.

Role of protein intake on protein synthesis and fiber distribution in the unweighted soleus muscle.

Protein turnover in skeletal muscle is very sensitive to protein intake. To examine whether protein intake is able to affect protein synthesis in the atrophied soleus muscle, the effects of a high-protein (30%, HP) and a medium-protein (15%, MP) diet were studied in rats after 21 days of hindlimb unweighting. Three weeks of unweighting induced a sharp decrease in food intake (30%). The fractional rate of protein synthesis (ks) was determined in vivo in the slow-twitch soleus muscle by use of a flooding-dose method. With respect to pair-fed animals, a significant reduction in ks occurred (33%) in MP non-weight-bearing rats, whereas it was of lesser magnitude and not significant in HP rats. In the atrophied soleus muscle of non-weight-bearing MP rats, a large decrease (42%) in type I fiber distribution was accompanied by an increase in intermediate and type IIa fibers. By contrast, a higher percentage of type I fiber was maintained with the HP diet. However, the HP diet had no beneficial effect in preventing the decrease in either type I fiber cross-sectional area (65%) or the average decrease in absolute myofibrillar and mitochondrial volumes (69 and 52%, respectively). These results demonstrate that an HP intake did not prevent soleus muscle atrophy but may sustain protein synthesis and partly preserve fiber type distribution without affecting the ultrastructural composition of fibers. Because the circulating level of free 3,5,3'-triiodothyronine was reduced by 14% with the HP diet, this effect on fiber type distribution, and possibly protein synthesis, may involve thyroid hormones.

Fasting does not increase mRNA levels of proteolytic systems in small intestinal mucosa of the rat.

Fasting results in rapid and profound wasting of the small intestine. mRNA levels of genes encoding critical components of proteolytic systems were measured in small intestinal mucosa to indirectly assess the possible role that proteolysis plays in mediating this wasting. Male Sprague-Dawley rats (120 g; n = 6 per group) were either fed or fasted for 1 or 2 days. Small intestinal mucosal mass decreased by 19% and 31% after 1 and 2 days of fasting, respectively (P < 0.05). Fasting did not significantly change mRNA levels for lysosomal (cathepsin B) or ubiquitin-proteasome-dependent (ubiquitin, 14-kDa ubiquitin-conjugating-enzyme E2, and the C8 and C9 proteasome subunits) systems. Northern hybridizations were also performed using membranes made with poly A(+) mRNA instead of total RNA. mRNA levels for these proteolytic systems and m-calpain did not significantly change with fasting. These data clearly demonstrated that fasting does not increase expression of genes encoding critical components of proteolytic systems in the small intestinal mucosa, suggesting that increased proteolysis cannot explain wasting of the small intestinal mucosa during brief fasting in young rats.

Effects of alimentary whole proteins versus their small peptide hydrolysates on liver and skeletal muscle during the acute inflammation phase in the rat.

Acute inflammation induces changes in liver proteins with an increase in synthesis of positive acute-phase proteins such as alpha1-acid glycoprotein (alpha1-AGP) and a decrease in synthesis of negative acute-phase proteins such as albumin. This is associated with muscle wasting, mediated by increased proteolysis and impaired protein synthesis. As protein metabolism can be altered in other situations (malnutrition, growth) by the form of the dietary nitrogen, we studied the effects of the molecular form of nitrogen on liver and skeletal muscle adaptation, looking at gene expression for two acute-phase proteins (albumin and alpha1-AGP) and a number of muscle proteins (alpha1-actin, ubiquitin and C9 proteasome subunit). Two groups of 24 Wistar rats (250 g) were injected S/C with 0.125 ml turpentine/rat and were fed one of two liquid diets. These diets had caloric, nitrogen, carbohydrate and lipid content but differed in the molecular form of the nitrogen source (whole protein [WP] versus peptide hydrolysate [PH]). Liver and muscle adaptation were studied at 18, 42 or 66 h after turpentine injection. Weight, deoxyribonucleic acid and protein content of the liver were significantly higher with the WP diet than with the PH diet at 42 h and 66 h. There was more alpha1-AGP messenger ribonucleic acid (mRNA) at 18 h and less albumin mRNA at 42 h. Thus, the PH diet causes a more rapid increase in alpha1-AGP mRNA content and a smaller decrease in albumin mRNA content after turpentine injection than the WP diet. However, the changes in plasma acute-phase proteins (albumin and alpha1-AGP) were similar with the two diets. In skeletal muscle, there was no change in mRNA levels for the C9 proteasome subunit at any time point with both diets compared to the controls. However, there were greater ubiquitin mRNA levels at 18|h and less alpha-actin mRNA levels at 18 h, 42 h and 66 h following turpentine injection in the two dietary groups than in the controls. These results suggest that the molecular form of nitrogen ingested regulates hepatic gene transcription or mRNA stability of acute-phase proteins, during the early period of inflammation, but did not affect the expression of muscle proteins, which was altered by turpentine injection. Post-transcriptional control of acute-phase protein genes may contribute to the maintenance of similar plasma levels.

The role of adrenal hormones in the response of glutamine synthetase to fasting in adult and old rats.

BACKGROUND & AIMS: During fasting, skeletal muscle exports increased amounts of glutamine (Gln) while increasing the production of this amino acid by glutamine synthetase (GS) in order to maintain the intramuscular Gln pool. Glucocorticoid hormones are believed to be the principal mediators of GS induction during stress conditions. The aim of this study was to evaluate (1) the effect of fasting on GS activity and expression in skeletal muscle during aging and consequently, (2) the role of glucocorticoids in fasting-induced GS activity. METHODS: Male Wistar rats (6-, 22-month old) were fasted for 5 days and both the activity and expression of GS were measured in tibialis anterior muscle. To better demonstrate the role of glucocorticoids in the response of GS to fasting, we suppressed their action by RU38486 administration (a potent glucocorticoid antagonist) and their production by adrenalectomy in fed and fasted rats. RESULTS: An increase in fasting-induced GS activity was observed in skeletal muscles from both adult and aged rats. Adrenalectomy, but surprisingly not RU38486, suppressed the fasting-induced increase in GS activity and expression. CONCLUSION: The data clearly show that the GS responsiveness to fasting was not modified by aging in skeletal muscle.

Effects of underfeeding and refeeding on weight and cellularity of splanchnic organs in ewes.

We assessed the effects of a long and severe period of underfeeding, followed by a rapid refeeding with a high-concentrate diet, on weight, protein mass, and cellularity of the splanchnic organs in adult ewes. Twenty-four ewes, allocated to four groups of six, were fed a forage diet (50% regrowth of natural grassland hay and 50% wheat straw) either at maintenance (groups M and MO) or at 40% maintenance (groups U and UO) for 78 d. Groups M and U were then slaughtered, and groups MO and UO were subsequently overfed a high-concentrate diet (52% hay, 20% barley, 16% rapeseed meal, 4% fish meal, and 8% Megalac) at 236% maintenance for 26 d before being slaughtered. During the experiment, feed was adjusted to maintain feed supply at a constant percentage of animal requirements. After slaughter, fresh weight, dry weight, and protein mass of the reticulorumen, omasum, abomasum, small intestine, large intestine, and liver were measured. Cellularity was assessed from nucleic acids and protein contents for both ruminal mucosa and muscular-serosa layers, jejunum, and liver. The concentrations of ubiquitin and cathepsin D mRNA were measured in ruminal mucosa and muscular-serosa layers and in jejunum. Underfeeding decreased protein mass of splanchnic organs, especially in liver (-29%) and reticulorumen (-39%). Refeeding previously underfed animals increased protein mass of liver (+102%) and small intestine (+59%). No carry-over effect of the previous level of intake (UO vs. MO) was observed on the protein mass of splanchnic tissues after 26 d of refeeding. Variations in liver mass were mainly due to hypertrophy, as determined by the protein:DNA ratio, whereas variations in small intestinal mass were mainly due to hyperplasia, as determined by the amount of DNA. By contrast, changes in rumen mass associated with increasing ME intake seemed to be related to hypertrophy in the muscular-serosal component and hyperplasia in the epithelial component. The concentrations of ubiquitin and cathepsin D mRNA in the rumen and jejunum were not modified by feeding level, demonstrating that the expression of these genes for proteolytic enzymes was unchanged under these conditions.

Protein synthesis and growth in the gastrointestinal tract of the young preruminant lamb.

1. In Expt 1, fractional synthesis rates (FSR) of tissue protein were measured along the gastrointestinal tract (GIT) of six 1-week-old, milk-fed lambs by using a large amount of L-[3,4(n)-3H]valine. 2. In Expt 2, eighteen lambs were used to determine the fractional growth rate (FGR) of gastrointestinal tissue protein. 3. FSRMinimum (Min) and FSRMaximum (Max) were calculated assuming plasma or tissue homogenate free valine specific radioactivity was representative of the valine precursor pool for protein synthesis. There were no significant differences between FSRMin and FSRMax in any gastrointestinal tissue of lambs used in Expt 1 (P greater than 0.05). FSR gradually and significantly (P less than 0.05) increased from the oesophagus (FSRMax 26.5%/d), reticulo-rumen (30.1%/d), omasum (41.0%/d) and abomasum (56.1%/d) to small intestine (87.5%/d), and then declined significantly (P less than 0.05) towards the caecum (45.2%/d) and the colon (38.4%/d). No significant differences were observed between FSR in the duodenum, jejunum or ileum (P greater than 0.05). 4. FGR ranged from 2.6%/d in the oesophagus to 8.7%/d in the omasum. The ratio, FGR:FSR, which reflected the efficiency of protein deposition, was at a maximum in the stomachs and caecum and at a minimum in the small intestine. 5. The relative contribution of the oesophagus, stomachs, small intestine and large intestine to GIT protein synthesis was 1, 13, 76 and 10% respectively. The GIT accounted for approximately 11.5% of whole-body protein synthesis.