Early posthatch starvation decreases satellite cell proliferation and skeletal muscle growth in chicks

The effect of posthatch starvation on skeletal muscle growth and satellite cell proliferation was examined in chicks. Chicks were either fed or starved for 48 h posthatch (d 0-d 2, d 2-d 4 or d 4-d 6) and then refed for 41 d. Body and breast muscle weights were significantly lower in starved chicks than in fed controls throughout the experiment. Histochemical staining revealed that skeletal muscle fiber development in the starved group lagged behind that of the fed group. Starvation from d 2 to 4 and d 4 to 6 posthatch had a progressively lesser effect than did immediate posthatch starvation (P < 0.05). In vitro culturing of breast muscle satellite cells revealed that DNA synthesis and number of cells per gram of muscle in the fed chicks peaked on d 2 and d 3, and then declined. In contrast, DNA synthesis in the cells of starved chicks declined on d 2 and increased on d 3 when chicks were refed. A similar pattern was seen for the number of cells per gram muscle; however, in general cell numbers tended to be higher in the starved group than in controls (P < 0.1). The results obtained with cultured cells were parallel with in situ immunostaining with 5-bromo-2'-deoxyuridine and proliferating cell nuclear antigen in breast muscle from experimental chicks, and with growth hormone receptor expression. These results suggest that satellite cell cultures are a reliable tool for evaluating muscle growth in postnatal chickens. We conclude that sufficient feed in the immediate postnatal period is critical for satellite cell proliferation and skeletal muscle development and is thus important for optimal muscle growth.     

Protein hydrolysate vs free amino acid-based diets on the nutritional recovery of the starved rat

Summary Background and Aims To test the hypothesis that a peptide-based enteral product was equivalent to a low-fat, free amino acid-based formula in the nutritional and functional recovery of the starved rat. Methods Sixteen male Wistar rats were starved for 3 days. Then, rats were randomised to a whey protein hydrolysate-based diet or a free amino acid-based diet and refed for 3 days. The experiment was designed to provide the same energy intake in both groups. The parameters studied included body weight gain, nitrogen retention, plasma free amino acid concentrations, muscle glutamine concentrations and glutathione levels in gut mucosa and liver. Results Weight gain was statistically higher on the peptide-based diet than on the elemental diet after rhe refeeding period. This difference in weight gain was associated with a statistically higher nitrogen retention. Plasma and muscle free glutamine concentrations were higher in rats fed the whey protein hydrolysate-based diet than those in rat refed the free amino acid-based diet, even though the glutamine intake was higher in the latter group. Glutathione concentrations in liver and gut mucosa were similar in the groups. Conclusion We conclude that enteral diets containing peptides were more effective than a diet containing free amino acids in the nutritional recovery of the starved rat. Received: 17 May 2000, Accepted: 1 September 2000     

Day length has a major effect on the response of protein synthesis rates to feeding in growing Japanese quail

We investigated the effect of day length on mixed protein fractional synthesis rates (K(S)) in 14- and 21-d-old Japanese quail (Coturnix c. japonica) habituated to either a long day length, 18 h light/6 h dark (LDL), or short day length, 6 h light/18 h dark (SDL), with free access to food during the light period. Rates of protein synthesis were measured by a flooding dose of L-[1-(13)C]leucine. In both groups, we measured K(S) of pectoral muscle, liver and heart after an overnight period of food deprivation and after 2-h food access at dawn. Rates of protein synthesis were also measured in LDL quail starved for 18 h and refed for 2 h. SDL chicks were smaller and had lower tissue weights at 2 wk of age than did LDL chicks (P<0.05). Starvation led to a lower rate of protein synthesis in those animals starved for 18 h. Food availability after starvation for 18 h induced a significant rise in tissue protein synthesis in both SDL and LDL quail (P<0.05). This increase was absent in LDL quail after a 6-h starvation period. There was an increase in K(S) to ad hoc changes in food supply. By determining the daily period in which feeding can occur, day length has a major effect on protein synthesis rates. This effect will determine the overall growth chicks are able to achieve that have been subjected to different day lengths.     

Refeeding of rats fasted 36 hours with five different carbohydrates and with malt extract: differential effects on glycogen deposition in liver and muscle, on plasma insulin and on plasma triglyceride levels

Rats fasted for 36 h were refed for 1, 2, 4 or 6 h with a diet containing 12 g/100 g casein, 2 g/100 g NaCl and 86 g/100 g glucose, fructose, maltose, sucrose, starch or malt extract. Blood glucose reached constant levels after 1 to 2 h of refeeding. The increase in plasma insulin paralleled food intake rather than the increase in blood glucose. Plasma triglycerides decreased upon refeeding starch, maltose and malt extract and increased with sucrose and fructose. Recovery of absorbed carbohydrates was highest in rats refed malt extract. Glycogen deposition in muscle was highest in rats fed malt extract and lowest in those fed fructose; sucrose yielded intermediate values. Glucose, maltose and starch resulted in muscle glycogen depositions slightly lower than those obtained with malt extract. In liver, sucrose and fructose were better precursors for glycogen than glucose and starch. With carbohydrates containing only glucose units, much more glycogen was found to be deposited in total muscle than in liver. This asymmetry was less notable or even was reversed with sucrose and fructose. Glycogen deposition in muscle and in liver is influenced by the carbohydrate used for refeeding, and muscle, rather than liver, is the main glycogen storing tissue.     

Food deprivation and refeeding influence growth, nutrient retention and functional recovery of rats.

The objective of this work was to determine the effects of starvation and refeeding on growth, nutritional recovery and intestinal repair in starved rats. Male Wistar rats, weighing 200 g, were starved for 3 d, then refed a soy-based diet for another 3 d. Normally fed rats were given the same diet and used as controls. The variables assessed were as follows: body weight gain and nitrogen retention during recovery after starvation; muscle glutamine concentration; tissue protein content; gut mucosa and liver glutathione levels; intestinal permeability to ovalbumin, lactulose and mannitol; and intestinal tissue apoptosis. Starvation was associated with lower muscle glutamine levels and intestinal mucosa impairment, including a lower content of mucosal protein, a higher level of oxidized glutathione, enhanced permeability to macromolecules and greater numbers of apoptotic cells. Refeeding for 3 d resulted in rapid repair of gut atrophy and normalization of not only intestinal permeability but also of the majority of metabolic markers assessed in other tissues. In conclusion, with the use of severely starved rats, we have established a reversible experimental animal model of malnutrition that might prove useful in comparing the effectiveness of different enteral diets.     

Absence of a generalized disaccharide effect in adult female rats

Adult female Sprague-Dawley rats were either prefed ground nonpurified diet, starved 48 h, then refed a purified carbohydrate diet for 72 h or shifted from ground nonpurified diet directly to a purified carbohydrate diet for 72 h. Diets were formulated to contain 65% carbohydrate either as the disaccharides maltose or sucrose or as their respective monosaccharide equivalents glucose and invert sugar (glucose: fructose, 1:1). Alternations in hepatic glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and malic enzyme (ME) activities, relative liver size and food efficiency were determined. Rats starved and refed invert sugar had higher levels of G6PDH and ME than those red glucose, indicating a positive fructose effect. The greatest changes in hepatic enzyme activities were observed in rats consuming diets containing sucrose. Positive fructose and disaccharide effects were obtained with sucrose for all enzymes studied in both dietary shift and starve-refeed studies. No disaccharide effect was observed with maltose. In conclusion, females did not display a generalized disaccharide effect with either dietary shifting or starvation refeeding.     

CHANGES IN LIPOGENESIS DURING REFEEDING OF FASTED MICE

Rates of fatty acid and cholesterol synthesis during refeeding after fasting in mice were affected by prestarvation body weight. composition of the prestarvation diet, duration of fasting and refeeding, and composition of the diet refed. The increases were much greater in liver than in carcass or epididymal fat.     

Protein synthesis and translation initiation factor activation in neonatal pigs fed increasing levels of dietary protein

Limited data suggest that the growth of low-birth-weight infants is enhanced by feeding a high-protein diet; however, the mechanisms involved in the effect have not been delineated. To identify these mechanisms, 34 pigs were fed from 2 to 7 d of age [60 g dry matter/(kg body weight . d)] isocaloric milk diets that contained levels of dietary protein that were marginal, adequate, and in excess of the piglets protein requirement (21, 33, and 45% of dry matter, respectively). Dietary protein replaced lactose and fat on an isocaloric basis. Fractional protein synthesis rates, various biomarkers of translational regulation, and plasma glucose and insulin levels were measured in overnight food-deprived and fed pigs. Mean daily weight gain of pigs fed the 33 and 45% protein diets was greater than that of pigs fed the 21% protein diet (P < 0.01). Plasma glucose (P = 0.07) and insulin (P < 0.01) levels decreased as dietary protein increased 60 min after feeding. Protein synthesis rates in longissimus dorsi, gastrocnemius, masseter, heart, liver, kidney, jejunum, and pancreas were greater in the fed than in the food-deprived state (P < 0.01). Protein synthesis in skeletal muscle did not change with protein intake in the fed state, but decreased quadratically (P < 0.01) with increasing dietary protein in the food-deprived state. Protein kinase B, ribosomal protein S6 kinase 1(S6K1), and eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) were more phosphorylated, and assembly of the inactive eukaryotic initiation factor 4E . 4E-BP1 complex in muscle and liver was reduced in the fed state (P < 0.001) and were not consistently affected by dietary protein level. The results suggest that feeding stimulates protein synthesis, and this is modulated by the activation of initiation factors that regulate mRNA binding to the ribosomal complex. However, the provision of a high-protein diet that exceeds the protein requirement does not further enhance protein synthesis or translation initiation factor activation.     

Stoppage of glycogenesis and "over-shoot" of induction of lipogenesis and its related enzyme activities in the liver of fasted-refed rats.

To elucidate the causes of changes of carbohydrate metabolic pathways, the time course of utilization of dietary [U-14C]sucrose and induction of enzyme activities in the livers of rats were investigated. Adult male rats of BHE strain were refed after a fast of 2 days. The nutritionally complete refeeding diet contained 60% sucrose as the only source of carbohydrate. [U-14C]Sucrose was included in the diet on either day 1 or day 2, or both of refeeding. During the first day of refeeding, the radioactivity was incorporated mainly into liver glycogen which rose to over 100 mg/g. During the second day, little 14C appeared in the liver glycogen, which decreased sharply while glucose-6-phosphatase activity increased. The glycogenic pathway thus appeared to be blocked. On the other hand, 14C incorporation in the liver fat was minimal during the first day, but was quite extensive during the second day of refeeding. The enhanced lipogenesis was accompanied by large increases of activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-malic dehydrogenase. Results clearly indicate that the carbohydrate load in the liver of intact animals was initially metabolized by the glycogenic pathway. When glycogenesis stopped, carbohydrate was metabolized differently. The enhanced incorporation of [U-14C]sucrose into liver lipids indicates an increased formation of acetyl CoA and an accelerated formation and use of NADPH, probably from increasing dehydrogenase activities. Our data suggest that the blockage of synthesis of glycogen with the continuation of carbohydrate load was a primary cause in over-shooting induction of hepatic dehydrogenase activities and lipogenesis.     

Metabolic differences between genetically lean and fat chickens are partly attributed to the alteration of insulin signaling in liver.

Insulin signaling [tyrosine phosphorylation of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Src homology and collagen protein (Shc) and phosphatidyl inositol 3'-kinase activity (PI 3'-kinase)] was studied in the liver and thigh muscles of fat (FL) and lean (LL) chickens. These lines result from a divergent selection on abdominal fat pad size. The divergence is of metabolic origin. Extreme nutritional states were studied (fed, 48-h starved and 30-min refed). Such conditions significantly altered insulin signaling in chicken liver, but surprisingly not in the muscle (except the phosphorylation of Shc in the refed state). No major differences that could account for this divergence were found in muscle. Liver IR number and Shc protein did not differ between genotypes. Liver IRS-1 (protein and messenger) was lower in the fed state and higher in the starved state in FL compared to that in LL chickens. In the fed state, tyrosine phosphorylation of liver IR, IRS-1 and Shc action was higher in FL than in LL chickens that in the absence of insulin resistance rely on higher plasma insulin levels. In the starved state, phosphorylation of liver IR was lower, but the phosphorylation of IR and IRS-1 were higher in LL than in FL chickens, most likely in response to higher plasma glucose and insulin in the lean genotype. In the refed state, the phosphorylation of liver IR and IRS-1 did not differ between genotypes despite significantly lower plasma insulin in FL chickens. Finally, PI 3'-kinase was not affected by the genotype. A significant activation of early steps of insulin signaling in liver of fed FL chickens may at least partly account for their increased liver lipogenesis and ultimately their fattening.     

The drug LY79771 affects fat regain by starved and refed BHE rats

The effect of the drug LY79771 on the fat rebound response of BHE rats to starvation-refeeding was studied. Three experiments were conducted. Experiment 1 determined the effect of the drug on the composition of the regained weight following a period of starvation. The drug-treated rats had significantly less body fat after refeeding than did the control rats. Experiment 2 measured the liver and fat pad lipid levels and the activities of two NADP-linked enzymes after starvation-refeeding. The classic two- to threefold hepatic glucose-6-phosphate dehydrogenase and malic enzyme overshoot and increase in liver and fat pad lipid levels were seen in refed controls but not in refed LY79771-treated rats. Experiment 3 measured de novo fatty acid synthesis in LY79771-treated and control rats. Treatment with LY79771 resulted in lower hepatic fatty acid synthesis in starved and refed rats. These observations suggest that LY79771 can be effective in preventing fat regain following energy deprivation.     

Early effects of refeeding a complete diet on the translational initiation activity in rat liver slices

A complete diet containing 23% protein was fed to two-day-fasted rats. The effects of this diet on protein biosynthesis, through the examination of liver slices, was investigated. (1) Refeeding enhanced the protein-synthesizing activity with better preservation of heavy polysomes in the liver slices. Protein in the diet was necessary to induce, through refeeding, the activation of protein synthesis in the liver. (2) The incorporation of [3H]methionine into 40S subunits and 80S subunits, where "run-off" 40S and 60S subunits had been accumulated by preincubation with 2 x 10(-6) M pactamycin, was much higher in the liver slices of refed rats than in the liver slices of fasted rats. By using chain initiation inhibitors, it was shown that labeled 40S and 80S subunits were 40S and 80S initiation complexes, respectively.     

Leucine supplementation enhances skeletal muscle recovery in rats following exercise.

This study was designed to determine the ability of leucine to enhance muscle recovery after exercise. Male rats (200 g) were divided into five groups: sedentary, food-deprived (SF); exercised, food-deprived (EF); exercised, fed a carbohydrate meal (EC); exercised, fed a leucine meal (EL); and exercised, fed a combination of carbohydrate and leucine (ECL). All meals were administered by oral gavage immediately following exercise. EC and ECL meals were isocaloric and provided 15% of daily energy intake. EL and ECL meals each provided 270 mg leucine. Rats ran on a motor-driven treadmill for 2 h at 36 m/min and were killed 1 h postexercise. Plasma glucose and insulin were measured, and the gastrocnemius and plantaris muscles were excised as a unit to determine glycogen levels and the fractional rate of skeletal muscle protein synthesis (Ks). Exercise did not alter plasma glucose or insulin. In contrast, prolonged exercise reduced muscle glycogen (-51%) and Ks (-18%). Refeeding a combination of carbohydrate and leucine increased plasma insulin relative to the EF and SF groups and produced complete recovery of muscle Ks and glycogen to values not different from those in SF rats. Feeding leucine alone restored Ks to that in the SF group without affecting plasma glucose or insulin concentrations. Feeding carbohydrate alone enhanced the rate of glycogen repletion compared to the EF group, concomitant with increases in plasma glucose and insulin. The degree of glycogen recovery correlated with plasma insulin concentrations (r = 0.58, P < 0.05). These data suggest that leucine stimulates muscle protein synthesis following exercise, independent of increased plasma insulin. This is the first demonstration that orally administered leucine stimulates recovery of skeletal muscle protein synthesis after exercise.     

Early posthatch feeding stimulates satellite cell proliferation and skeletal muscle growth in turkey poults

The effect of early posthatch feeding on skeletal muscle growth and satellite cell myogenesis was studied in turkey poults. Poults were either fed immediately posthatch or food-deprived for the first 48 h and then refed for the rest of the experiment. Body and breast muscle weights were lower in the starved poults than in fed controls throughout the experiment (P < 0.05). Cultures of breast muscle satellite cells revealed significantly higher DNA synthesis in the fed group than in the starved group as early as d 1 (P < 0.05). These levels continued to rise, reaching approximately 500-fold those of feed-deprived poults on d 4. In the latter group, thymidine incorporation peaked only on d 6, and then declined. Thereafter, it decreased to the same levels as those in the fed group. Satellite cell number per gram muscle increased until d 4, and was higher in the fed group than in the starved group (P < 0.05). Pax7 levels in cell cultures derived from the fed group were markedly higher than in the starved group on d 2 (P < 0.05). Myogenin levels in both culture and muscle were higher in the fed than in the starved groups until d 4 (P < 0.05). Phosphorylation of the survival factor Akt and cyclin-dependent kinase inhibitor p21 levels were higher in cells derived from the fed group relative to those from the starved group 48 h posthatch (P < 0.05). Similarly, Akt phosphorylation and insulin-like growth factor I (IGF-I) levels were significantly higher in the muscles of the fed group (P < 0.05). Together, these results suggest that immediate posthatch feeding of poults is critical for satellite cell survival and myogenesis probably via IGF-I.     

Dietary protein concentration regulates the mRNA expression of chicken hepatic malic enzyme

Chicken hepatic malic enzyme activity varies with dietary protein content. The mechanisms responsible for this alteration in activity are unclear. In a series of four experiments, broiler chicks were allowed free access for 1.5, 3, 6 or 24 h to a low (13 g/100 g diet), basal (22 g/100 g diet) or high (40 g/100 g diet) protein diet. The diets were isocaloric and had equal concentrations of dietary fat. Hepatic malic enzyme mRNA expression and enzyme activity as well as total liver lipid concentration were examined for each experimental duration. There were no differences in the expression of the mRNA for malic enzyme at 1.5 h, but at 3, 6 and 24 h, malic enzyme mRNA expression was significantly (P < 0.05) reduced in chicks fed the high protein diet and significantly enhanced in chicks fed the low protein diet compared with chicks fed the basal diet. Hepatic malic enzyme activities and total lipid concentration were not different among the chicks fed the different diets at 1.5 and 3 h. At 6 and 24 h, malic enzyme activity and total liver lipid concentration were both significantly greater in birds fed the low protein diet compared with levels in the birds fed the other two diets. In birds fed the high protein diet, malic enzyme activity and total liver lipid concentration were significantly reduced at 24 h compared with birds fed the basal diet. In a final experiment, the observed differences in malic enzyme mRNA expression at 6 h were confirmed when chicks were given access to isocaloric diets with the same protein levels as the initial 4 experiments, but with the dietary concentration of carbohydrate held constant. The results suggest that previously observed alterations in the activity of malic enzyme, which were correlated with dietary protein intake, are due to rapid changes in the mRNA expression of this enzyme.     

Refeeding after fasting in rats: effects of duration of starvation and refeeding on food efficiency in diet-induced obesity

Rats with diet-induced obesity starved for 8, 15, and 25 d lost liver and muscle glycogen, excess protein, and fat in proportion to duration of starvation. Fat-cell size decreased but fat-cell number did not. Upon refeeding, body fat was only partly restored, with further increase in adipocyte hyperplasia occurring in the starved obese rats. In contrast, fat-cell size was restored to near that of the prefasting value in the starved controls (dry-food-fed, fasted 4 d) after refeeding. With refeeding, food efficiency increased only if starvation had caused a reduction of adipocyte size below normal. Change in food efficiency was not associated with decreases in total carcass protein, specific tissue proteins, or glycogen stores but was correlated with degree of adipocyte filling. It is possible that adipose tissue status somehow modulates energy-dissipating mechanisms.     

Dietary protein level alters oxidative phosphorylation in heart and liver mitochondria of chicks.

To determine the effects of dietary protein level on cardiac and hepatic mitochondrial oxidative phosphorylation, chicks were fed on semi-purified diets of different protein levels (7, 25, 43 and 61% of metabolizable energy content) for 7, 14 and 21 d. All diets were formulated to contain equivalent fat, mineral and vitamin contents on a gross energy basis. Cardiac and hepatic mitochondrial oxidative phosphorylation rates were assessed polarographically with pyruvate and malate as substrates. Cardiac mitochondria isolated from chicks fed on a 43 or 61% protein-energy diet for 7 d exhibited significantly reduced ADP:oxygen (ADP:O) ratios when compared with mitochondria isolated from chicks fed on a lower-protein-energy diet. Feeding low- (7%) protein-energy diets for 14 d resulted in a relatively increased ADP:O ratio in the heart. Responses of ADP:O ratios to protein level in hepatic mitochondria showed more dependency on protein level than in heart muscle; at all feeding periods the ADP:O ratio decreased with an increase in protein level. As a result, ATP synthesized in the liver, expressed as nmol/mg mitochondrial protein per min, significantly decreased with increased dietary protein level. A parallel correlation was observed, in chicks fed on diets with different levels of protein, between ADP:O ratio for liver mitochondria and body fat. These results suggest that the reduction in oxidative phosphorylation in the heart and liver of animals fed on a higher protein-energy diet may partly contribute to the depression of body fat.     

Effect of diet type on enhanced intestinal protein synthesis by the gut microflora in the chick

An experiment was conducted to investigate the effect of diet type on enhanced protein synthesis by the gut microflora in the chick intestine. Both germ-free (GF) and conventional (CV) White Leghorn chicks were fed ad libitum from 4 to 14 d of age either a nonpurified diet or a purified diet having 19% crude protein and 12.1 kJ/g metabolizable energy value. At 14 d of age, protein synthesis was measured in duodenum, jejunoileum and ceca after a large dose of L-[4-3H]phenylalanine injected intravenously via a wing vein. The results showed that wet tissue weights for all intestinal sections were significantly greater in CV birds than in GF counterparts with consistently larger differences between the two environments in chicks fed the nonpurified diet than in those fed the purified diet. Protein synthesis in all intestinal sections was significantly enhanced by the presence of gut microflora in terms of both fractional rate (%/d) and absolute rate [mg/(100 g BW.d)]. The effect of diet type on fractional and absolute rates of protein synthesis was most remarkable in ceca where the significant difference between GF and CV states was found only in chicks fed the nonpurified diet but not in those fed the purified diet.     

Effect of insulin administration on cardiac glycogen synthase and synthase phosphatase activity in rats fed diets high in protein, fat or carbohydrate

Rats were fed a 70% carbohydrate, 70% protein, 70% fat, or a standard purified diet for 7 d to determine the effect of the diet on heart glycogen synthase response to an acute insulin challenge. Rats fed the high protein or the high fat diets, i.e., the carbohydrate-free diets, exhibited insulin resistance as evidenced by higher plasma glucose levels following insulin administration when compared to rats fed high carbohydrate or standard diet. The diets had no effect on the initial proportion of synthase in the active or I form. Insulin injection resulted in an increase in the proportion of synthase in the active form in rats fed the standard, high carbohydrate or high protein diets, but not in rats fed the high fat diet. Synthase phosphatase activity was similar in rats fed one of the four diets compared to rats fed a nonpurified diet. Thus the lack of synthase response to insulin in fat-fed rats was not due to diminished synthase phosphatase activity. Neither the diets nor insulin administration had any effect on the proportion of phosphorylase in the active or a form. Cardiac glycogen was significantly lower in rats fed the high fat diet than in those fed the standard diet. The latter was a surprising observation since the high fat diet was used to simulate a starved state and cardiac glycogen concentrations increase with starvation.