The effect of chronic ethanol ingestion on protein metabolism in type-I- and type-II-fibre-rich skeletal muscles of the rat.

1. The effects of chronic ethanol feeding on muscles containing a predominance of either Type I (aerobic, slow-twitch) or Type II (anaerobic, fast-twitch) fibres were studied. Male Wistar rats, weighing approx. 90 g or 280 g, were pair-fed on a nutritionally complete liquid diet containing 36% of total energy as ethanol, or isovolumetric amounts of the same diet in which ethanol was replaced by isoenergetic glucose. After 6 weeks feeding, fractional rates of protein synthesis were measured with a flooding dose of L-[4-(3)H]-phenylalanine and muscles were analysed for protein, RNA and DNA. 2. Ethanol feeding decreased muscle weight, protein, RNA and DNA contents in both small and large rats. Type-II-fibre-rich muscles showed greater changes than did Type-I-fibre-rich muscles. Changes in protein paralleled decreases in DNA. 3. The capacity for protein synthesis (RNA/protein), fractional rates of protein synthesis and absolute rates of protein synthesis were decreased by ethanol feeding in both small and large rats. The amounts of protein synthesized relative to RNA and DNA were also decreased. Changes were less marked in Type-I than in Type-II-fibre-rich muscles. Loss of protein, RNA and DNA was greater in small rats, but protein synthesis was more markedly affected in large rats. 4. It was concluded that chronic ethanol feeding adversely affects protein metabolism in skeletal muscle. Fibre composition and animal size are also important factors in determining the pattern of response.     

Differential effect of lentil feeding on proteosynthesis rates in the large intestine, liver and muscle of rats

The aim of this work was to test the hypothesis that the trophic effect of lentil feeding on large intestine results from a stimulation of protein synthesis and to determine whether it interferes with protein metabolism in other splanchnic or peripheral organs. Two groups of growing Sprague Dawley male rats were pair-fed iso-caloric iso-nitrogenous balanced diets containing either cooked lentils (Lens esculenta puyensis) or casein as unique protein source. Protein synthesis rates were measured in vivo, in large intestine, liver and gastrocnemius at the postprandial state. In large intestine, protein and ribonucleic acid contents were higher in the lentil-fed group than in the control group, and the amount of proteins synthesized was also higher (+57%). By contrast, liver protein and ribonucleic acid contents as well as protein synthesis rates were significantly lower in the lentil-fed group than in the control group. In the gastrocnemius muscle protein and ribonucleic acid contents were significantly lower and the amount of protein synthesized was also lower (-18%) in the lentil fed group than in the control group. This study suggests that stimulation of protein synthesis in the large intestine is compensated for by a decrease in liver and muscle.     

Protein synthesis during the developmental growth of the small and large intestine of the rat.

The developmental growth and associated changes in protein synthesis were measured (in vivo) in the combined small and large intestine from 18 days in utero to 105 weeks post partum. Similar post-natal (3-105 weeks) changes were also studied in the separated large and small intestine, and in the mucosal and muscularis externa + serosal layers of the small intestine. Although the protein and nucleic acid contents of the whole intestine increased throughout both pre- and post-natal life, the maximal (11%) intestinal contribution to whole-body growth occurred 3 weeks after birth; this value declined to only 2.5-3.5% at both extremes of the age range studied. Between the 18-day foetus and old age the fractional rate of protein synthesis decreased from 107 to 61% per day. This developmental decline (43%) was, however, much smaller than that found in most other body tissues over the same period. Similar developmental trends (between weaning and senility) were found in both the small and the large intestine when studied separately, the small intestine in all respects contributing proportionately more than the large intestine to both the combined intestinal and whole-body values. At each age the large intestine possessed significantly lower fractional rates of synthesis and associated ribosomal activities. For the most part, the fractional synthesis rates in the mucosa and serosa of the small intestine were very similar, with each declining slightly with increasing age. These developmental changes are discussed with respect to functional aspects within the gastrointestinal tract.     

The effect of chronic and acute dietary restriction on the growth and protein turnover of fast and slow types of rat skeletal muscle

Changes in the growth and protein turnover of the anterior tibialis and soleus muscles were studied in response to acute and chronic dietary restriction (50% of ad libitum intake) between 3 and 149 weeks post partum. The effect of long-term dietary restriction from weaning to senescence was to retard the growth and normal developmental of the two types of skeletal muscle. This was evident from measurements of various parameters of growth, i.e. total protein, RNA and DNA and protein/DNA-P, which were reduced by approximately 50% when compared with age-matched controls. These decreases, however, were not accompanied by a decline in the fractional rate of synthesis (%/day) or ribosomal activity (mg protein/day per mg RNAP). The slowing down of the age-related decline in muscle growth has been attributed to a reduction in RNA capacity (RNA/protein), with similar responses in the fast- and slow-twitch skeletal muscles. The initial effects of piecemeal feeding of this restricted diet on the two types of muscle were also monitored. Short term starvation effects, i.e. 24 hr after feeding a reduced ration, were measured on the protein content and RNA/protein of both the anterior tibialis and soleus muscles; both parameters were unchanged within 24 hr. In contrast, a rapid and significant decline in the ribosomal synthetic activity (mg/d per mg RNAP), and a corresponding fall in the fractional rate of synthesis, occurred within 24 hr of feeding.     

Effect of chronic ethanol ingestion on pancreatic protein synthesis

The effect of chronic ethanol feeding on pancreatic protein synthesis was assessed by studying the rate of incorporation of [3H]leucine into proteins in isolated rat pancreatic acini in vitro. Chronic ethanol feeding increased the rate of protein synthesis (2-3-fold) compared to controls fed an isocaloric diet. The onset of the increase in protein synthesis was detectable 2 days after the beginning of ethanol feeding, reached a maximum after 7 days and remained constant for up to 4 months. The increased incorporation of [3H]leucine was not due to an increased turnover of proteins as measured in pulse-chase experiments. After separation of individual digestive enzymes by SDS-polyacrylamide gel electrophoresis and determination of the distribution of radioactivity in different proteins, a general increase in the rate of incorporation of the label into all of the proteins was observed. In contrast to the observations made with isolated acini, there was no significant difference between the control and ethanol-fed groups when the rate of pancreatic protein synthesis was measured in vivo. However, overnight withdrawal of ethanol led to an increase of approx. 70% in protein synthesis in the ethanol-fed group. These results suggest that chronic ethanol ingestion modifies the control of pancreatic protein synthesis; the enhanced protein synthesis is expressed in isolated acini, i.e., in the absence of physiological factors present during chronic ethanol ingestion and in vivo after ethanol withdrawal.     

Intestinal protein synthesis in guinea pigs infected with Trichostrongylus colubriformis

The fractional synthesis rate (FSR) and daily synthesis of protein were measured in the small and large intestines of infected guinea pigs and uninfected animals fed ad libitum or quantitatively reduced rations. The FSR of the infected and parasite-free parts of the small intestine was unchanged but was increased by about 40% in the large intestine. Daily protein synthesis (mg/g body wt.) by infected guinea pig was greater by about 24% in the entire small intestine and by over 70% in the large intestine. These increases were not due to anorexia since the FSR and daily protein synthesis by the small and large intestines of the reduced ration animals were less than those of the infected group. Greater weight of the small intestine may explain increases in daily protein synthesis in the small, but not in the large intestine where weight was unchanged. Responses which may affect protein synthesis in the infected and parasite-free intestines are discussed.     

Protein metabolism in the small intestine of the ethanol-fed rat

The effects of chronic ethanol feeding on the small intestine were investigated in young rats. Rats were fed a nutritionally-adequate liquid diet, containing 36 per cent of total energy as ethanol (treated, n = 7), or isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose (controls, n = 7). After six weeks the wet weight and total tissue contents of protein, RNA and DNA were significantly reduced by 21 per cent, 23 per cent, 16 per cent and 28 per cent respectively, (p less than 0.014). Rates of protein synthesis were measured with L[4(3H)]phenylalanine and fractional rates (defined as the percentage of constituent tissue protein synthesised each hour, i.e. ks, % h-1) were calculated from the specific radioactivity of free phenylalanine in both tissue homogenates and plasma. Ethanol-feeding reduced ks by approx 10 per cent (p less than 0.181). The amount of protein synthesized unit-1 RNA was also reduced by approx 15 per cent (p less than 0.059) but the amount of protein synthesis unit-1 DNA was unaffected by ethanol-feeding (p less than 1.000). In contrast, the absolute rates of protein synthesis were reduced by approximately 30 per cent (p less than 0.022). It was concluded that, as the small intestine contributes to approx. 20-25 per cent of whole body synthesis these results may have an important effect on whole body nitrogen homeostasis and may have implications for the gastrointestinal effects of ethanol seen during chronic alcoholic abuse.     

Chronic ethanol feeding causes depression of mitochondrial elongation factor Tu in the rat liver: implications for the mitochondrial ribosome

Chronic ethanol feeding is known to negatively impact hepatic energy metabolism. Previous studies have indicated that the underlying lesion responsible for this may lie at the level of the mitoribosome. The aim of this study was to characterize the structure of the hepatic mitoribosome in alcoholic male rats and their isocalorically paired controls. Our experiments revealed that chronic ethanol feeding resulted in a significant depletion of both structural (death-associated protein 3) and functional [elongation factor thermo unstable (EF-Tu)] mitoribosomal proteins. In addition, significant increases were found in nucleotide elongation factor thermo stable (EF-Ts) and structural mitochondrial ribosomal protein L12 (MRPL12). The increase in MRPL12 was found to correlate with an increase in the levels of the 39S large mitoribosomal subunit. These changes were accompanied by decreased levels of nuclear- and mitochondrially encoded respiratory subunits, decreased amounts of intact respiratory complexes, decreased hepatic ATP levels, and depressed mitochondrial translation. Mathematical modeling of ethanol-mediated changes in EF-Tu and EF-Ts using prederived kinetic data predicted that the ethanol-mediated decrease in EF-Tu levels could completely account for the impaired mitochondrial protein synthesis. In conclusion, chronic ethanol feeding results in a depletion of mitochondrial EF-Tu levels within the liver that is mathematically predicted to be responsible for the impaired mitochondrial protein synthesis seen in alcoholic animals.     

3-Methylhistidine turnover in the whole body, and the contribution of skeletal muscle and intestine to urinary 3-methylhistidine excretion in the adult rat.

The tissue origin of 3-methylhistidine (N tau-methylhistidine) was investigated in adult female rats. The decay of labelling of urinary 3-methylhistidine was compared with the labelling of protein-bound 3-methylhistidine in skeletal muscle and intestine after the injection of [methyl-14C]methionine. The decay curve for urinary 3-methylhistidine was much steeper than that in muscle or intestine, falling to values lower than those in either tissue after 30 days. The lack of decay of labelling in muscle during the first 30 days is shown to result from the persistence of label in the precursor S-adenosylmethionine. The relative labelling of urinary, skeletal-muscle and intestinal 3-methylhistidine cannot be explained in terms of skeletal muscle accounting for a major proportion of urinary 3-methylhistidine. Measurements were also made of the steady-state synthesis rate of protein-bound 3-methylhistidine in intestinal smooth muscle in vivo in adult female rats. This involved measurement of the overall rate of protein synthesis and measurement of the relative rates of synthesis of 3-methylhistidine and of mixed protein. The synthesis rate of 3-methylhistidine was 29.1%/day, compared with the overall rate of 77.1%/day for mixed, non-mucosal intestinal protein. Measurement of the amount of 3-methylhistidine in skeletal muscle (0.632 +/- 0.024 mumol/g) and in the whole body (0.332 +/- 0.013 mumol/g) indicate that, although the muscle pool is 86% of the total, because of its slow turnover rate of 1.1-1.6%/day, it only accounts for 38-52% of the observed excretion. Measurements of the mass of the intestine (9.95 g/250 g body wt.) and protein-bound 3-methylhistidine content (0.160 mumol/g of tissue) indicate a pool size of 1.59 mumol/250 micrograms rat. Thus 463 nmol of the urinary excretion/day would originate from the intestine, 22% of the total. The tissue source of the remaining urinary excretion is not identified, but other non-muscle sources constituting about 10% of the whole-body pool could account for this with turnover rates of only 6%/day, a much lower value than the turnover rate in the intestine.     

The effect of food restriction and low protein diet upon collagen type I and III ratio in rat skin

It has been demonstrated that the content of the collagen type I is more affected by both chronic low protein diet feeding and chronic food deprivation (50% food intake) than the content of collagen type III. By introducing these dietary regimes the proportion of collagen type I to collagen type III ratio drops from 2.1 to 1.3 indicating the higher proportion of collagen type III in the skin at the end of the experiment (after 18 months of chronic feeding). It was also observed that the total concentration of hydroxyproline (hyp) in the skin decreases considerably in both food restricted animals and those fed a low protein diet. It is suggested that, under the present experimental conditions, the balance between collagen break-down and synthesis is shifted and, furthermore, that this shift is different for collagen type I and III and results in an altered ratio of these two collagen species in the skin. Refeeding of animals leads to a higher than normal collagen type I to III ratio indicating thus a relatively higher proportion of collagen type I in this tissue.     

The effect of metabolic acidosis on the synthesis and turnover of rat renal phosphate-dependent glutaminase.

Regulation of the mitochondrial phosphate-dependent glutaminase activity is an essential component in the control of renal ammoniagenesis. Alterations in acid-base balance significantly affect the amount of the glutaminase that is present in rat kidney, but not in brain or small intestine. The relative rates of glutaminase synthesis were determined by comparing the amount of [35S]methionine incorporated into specific immunoprecipitates with that incorporated into total protein. In a normal animal, the rate of glutaminase synthesis constitutes 0.04% of the total protein synthesis. After 7 days of metabolic acidosis, the renal glutaminase activity is increased to a value that is 5-fold greater than normal. During onset of acidosis, the relative rate of synthesis increases more rapidly than the appearance of increased glutaminase activity. The increased rate of synthesis reaches a plateau within 5 days at a value that is 5.3-fold greater than normal. Recovery from chronic acidosis causes a rapid decrease in the relative rate of glutaminase synthesis, but a gradual decrease in glutaminase activity. The former returns to normal within 2 days, whereas the latter requires 11 days. The apparent half-time for glutaminase degradation was found to be 5.1 days and 4.7 days for normal and acidotic rats respectively. These results indicate that the increase in renal glutaminase activity associated with metabolic acidosis is due primarily to an increase in its rate of synthesis. From the decrease in activity that occurs upon recovery from acidosis, the true half-life for the glutaminase was estimated to be 3 days.     

Prenatal blockage of lymphotoxin beta receptor and TNF receptor p55 signaling cascade resulted in the acceleration of tissue genesis for isolated lymphoid follicles in the large intestine

Signaling by lymphotoxin (LT) and TNF is essential for the organogenesis of secondary lymphoid tissues in systemic and mucosal compartments. In this study, we demonstrated that the progeny of mice treated with fusion protein of LTbetaR and IgGFc (LTbetaR-Ig) or LTbetaR-Ig plus TNFR55-Ig (double Ig) showed significantly increased numbers of isolated lymphoid follicles (ILF) in the large intestine. Interestingly, double Ig treatment accelerated the maturation of large intestinal ILF. Three-week-old progeny of double Ig-treated mice showed increased numbers of ILF in the large intestine, but not in the small intestine. Furthermore, alteration of intestinal microflora by feeding of antibiotic water did not affect the increased numbers of ILF in the large intestine of double Ig-treated mice. Most interestingly, mice that developed numerous ILF also had increased levels of activation-induced cytidine deaminase expression and numbers of IgA-expressing cells in the lamina propria of the large intestine. Taken together, these results suggest that ILF formation in the large intestine is accelerated by blockage of LTbetaR and TNFR55 signals in utero, and ILF, like colonic patches, might play a role in the induction of IgA response in the large intestine.     

IGF-I/IGFBP-3 ameliorates alterations in protein synthesis, eIF4E availability, and myostatin in alcohol-fed rats

Chronic alcohol consumption decreases the concentration of the anabolic hormone IGF-I, and this change is associated with impaired muscle protein synthesis. The present study evaluated the ability of IGF-I complexed with IGF-binding protein (IGFBP)-3 to modulate the alcohol-induced inhibition of muscle protein synthesis in gastrocnemius. After 16 wk on an alcohol-containing diet, either the IGF-I/IGFBP-3 binary complex (BC) or saline was injected two times daily for three consecutive days. After the final injection of BC (3 h), plasma IGF-I concentrations were elevated in alcohol-fed rats to values not different from those of similarly treated control animals. Alcohol feeding decreased the basal rate of muscle protein synthesis by limiting translational efficiency. BC treatment of alcohol-fed rats increased protein synthesis back to basal control values, but the rate remained lower than that of BC-injected control rats. The BC partially reversed the alcohol-induced decrease in the binding of eukaryotic initiation factor (eIF)4E with eIF4G. This change was associated with reversal of the alcohol-induced dephosphorylation of eIF4G but was independent of changes in the phosphorylation of either 4E-BP1 or eIF4E. However, BC reversed the alcohol-induced increase in IGFBP-1 and muscle myostatin, known negative regulators of IGF-I action and muscle mass. Hence, exogenous IGF-I, administered as part of a BC to increase its circulating half-life, can in part reverse the decreased protein synthesis observed in muscle from chronic alcohol-fed rats by stimulating selected components of translation initiation. The data support the role of IGF-I as a mediator of chronic alcohol myopathy in rats.     

Quantification of the main digestive processes in ruminants: the equations involved in the renewed energy and protein feed evaluation systems

The evolution of feeding systems for ruminants towards evaluation of diets in terms of multiple responses requires the updating of the calculation of nutrient supply to the animals to make it more accurate on aggregated units (feed unit, or UF, for energy and protein digestible in the intestine, or PDI, for metabolizable protein) and to allow prediction of absorbed nutrients. The present update of the French system is based on the building and interpretation through meta-analysis of large databases on digestion and nutrition of ruminants. Equations involved in the calculation of UF and PDI have been updated, allowing: (1) prediction of the out flow rate of particles and liquid depending on the level of intake and the proportion of concentrate, and the use of this in the calculation of ruminal digestion of protein and starch from in situ data; (2) the system to take into account the effects of the main factors of digestive interactions (level of intake, proportion of concentrate, rumen protein balance) on organic matter digestibility, energy losses in methane and in urine; (3) more accurate calculation of the energy available in the rumen and the efficiency of its use for the microbial protein synthesis. In this renewed model UF and PDI values of feedstuffs vary depending on diet composition, and intake level. Consequently, standard feed table values can be considered as being only indicative. It is thus possible to predict the nutrient supply on a wider range of diets more accurately and in particular to better integrate energy×protein interactions occurring in the gut.     

The effect of starvation on the rate of protein synthesis in rat liver and small intestine.

1. A method is described that allows for measurement of protein synthesis in liver and intestine in the rat. By injecting a massive amount of [14C]leucine (100 mumol/100 g body wt.) an attempt has been made to over come problems of precursor specific radioactivity and problems arising from the breakdown of labelled protein that are encountered when tracer amounts of amino acids are used. 2. Starvation for 2 days resulted in decline in the rate of total liver protein synthesis from 87%/day to 62%/day. 3. In jejunal mucosa the rate of protein synthesis was 136%/day. This declined to 105%/day after 2 days of starvation.     

Effects of chronic alcohol consumption on regulation of myocardial protein synthesis

Heart disease represents an important etiology of mortality in chronic alcoholics. The purpose of the present study was to examine potential mechanisms for the inhibitory effect of chronic alcohol exposure (16 wk) on the regulation of myocardial protein metabolism. Chronic alcohol feeding resulted in a lower heart weight and 25% loss of cardiac protein per heart compared with pair-fed controls. The loss of protein mass resulted in part from a diminished (30%) rate of protein synthesis. Ethanol exerted its inhibition of protein synthesis through diminished translational efficiency rather than lower RNA content. Chronic ethanol administration decreased the abundance of eukaryotic initiation factor (eIF)4G associated with eIF4E in the myocardium by 36% and increased the abundance of the translation response protein (4E-BP1) associated with eIF4E. In addition, chronic alcohol feeding significantly reduced the extent of p70S6 kinase (p70(S6K)) phosphorylation. The decreases in the phosphorylation of 4E-BP1 and p70(S6K) did not result from a reduced abundance of mammalian target of rapamycin (mTOR). These data suggest that a chronic alcohol-induced impairment in myocardial protein synthesis results in part from inhibition in peptide chain initiation secondary to marked changes in eIF4E availability and p70(S6K) phosphorylation.     

Dietary conjugated linoleic acid has limited effects on tissue protein anabolism in sedentary and exercising adult rats

The effects of conjugated linoleic acid isomers (CLA) and endurance training on lean body mass are expected to result from their action on tissue protein metabolism. The aim of this study was to analyze their effects on protein metabolism in 2 muscles, the small intestine and liver of adult rats. Four-month-old male Wistar rats were fed diets containing either no CLA, cis-9, trans-11 CLA isomer (1 g.100 g(-1)), trans-10, cis-12 CLA isomer (1 g.100 g(-1)) or both isomers (1 g.100 g(-1) each) for 6 weeks. Half of the rats were subjected to endurance training by running on a treadmill. At the end of this period, the rats were injected with a flooding dose of (13)C-valine to determine protein synthesis rates in the post-absorptive (experiment 1) and in the post-prandial (experiment 2) states. No effect of CLA or endurance training were detected in the small intestine. Training reduced food intake and protein synthesis rates in the liver but no effect was found on the protein synthesis rates in muscles. In the post-absorptive state, protein synthesis rate was increased by feeding the trans-10, cis-12 CLA isomer alone in the liver (+9%) or in combination with the cis-9, trans-11 isomer in the gastrocnemius (+30%), mostly in sedentary rats. In the post-prandial state, the cis-9, trans-11 CLA isomer tended to reduce the protein synthesis rate in the gastrocnemius muscle. However, no effect of CLA was found on muscle protein amounts. In conclusion, CLA isomers would have limited but differential effects on tissue protein metabolism in adult rats.     

Effect of Exercise on Tissue Protein Synthesis in Rats

The effect of exercise on the protein metabolism in skeletal muscles (gastrocnemius and soleus), liver and small intestine was investigated in rats. Treadmill treatment for 7 d resulted in atrophy of the liver and small intestine, which was associated with a reduction in protein content. The rates of protein synthesis in the liver and small intestine were significantly suppressed in rats subjected to exercise. The change in protein synthesis in the visceral organs was mediated by the change in RNA activity (protein synthesis per unit RNA) but not by the change in RNA concentration. The tissue weight and the rate of protein synthesis in the gastrocnemius and soleus muscles were not affected by exercise. The results suggest that these changes in protein synthesis in the liver and small intestine may explain, at least partly, the atrophy of these organs which was observed after 7 d of exercise.