Influence of protein malnutrition followed by a balanced refeeding on the synthesis level of pancreatic hydrolases in growing rats

The intake of 5% gluten + casein (4:1, W:W) (PM) (E group) diet compared to 20% (16:4, W:W) (control group) caused an overall decreased synthesis. With the duration of PM, Tg2 and Chtg1 synthesis remained steady, while lipase synthesis decreased and amylase synthesis increased. At the beginning of refeeding (two days), in E group Tg2 and amylase synthesis was increased with rebound effect compared to controls, then decreased, Chtg1 synthesis was higher than control synthesis and remained constant, lipase synthesis was increased and reached the control value only after 9 days.     

Spermatogenesis in the prepuberty Wistar rat during protein-energy malnutrition followed by balanced refeeding

Protein-calorie malnutrition was induced by giving a 2% casein diet to a strain of post-weaning male Wistar rats from 26 to 56 days of age. They were then rehabilitated by a 15% casein diet till 86 days of age. Body and testis weights, diameter of seminiferous tubules and spermatogenetic score were compared to those of controls fed with the 15% casein diet. The unbalanced diet retarded growth by 30 days. Although rehabilitation of body and testis weights was incomplete, the spermatogenetic function equalled that of the controls at the end of the experiment. This aspect has been discussed.     

Glycosylation and sorting pathways of lysosomal enzymes in mussel digestive cells

Our aim was to contribute to the understanding of the synthesis, maturation and activation of lysosomal enzymes in an invertebrate cellular model: the endo-lysosomal system (ELS) of mussel digestive cells. The activities of 5′–nucleotidase (AMPase), arylsulphatase (ASase) and acid phosphatase (AcPase), which are transported towards acidic compartments as membrane proteins, were localised by enzyme cytochemistry. AcPase activity was found within large heterolysosomes and residual bodies. ASase was located in endosomes, endolysosomes and heterolysosomes. AcPase and ASase activities were recorded within small vesicles and cisterns of the trans-Golgi network. Conversely, AMPase activity was primarily found in microvilli and apical vesicles and, less conspicuously, in lysosomes and the cis-side of the Golgi and the cis-Golgi network (CGN). In order to understand the processes of synthesis and maturation of these lysosomal enzymes, selected glycoconjugates were localised after lectin cytochemistry. N-acetylglucosamine, mannose and fucose residues were almost ubiquitous in the ELS, as were galactose residues, which were apparently less abundant. N-acetylglucosamine residues occurred in the inner membrane co-localised with mannose residues within the lysosomal and pre-lysosomal acidic compartments. Based on these results, glycosylation and sorting pathways are proposed for both soluble and membrane enzymes. Unlike in mammalian cells, O-glycosylation is fully completed in the CGN, mannose addition in N-glycosylation extends beyond the CGN and galactose addition is fully achieved at the intermediate side. Sorting of soluble lysosomal enzymes, as in crustaceans, is mediated by the indirect transport of membrane-linked proteins with GlcNAc1-P6Man residues that are removed in endolysosomes and heterolysosomes.This work was funded by projects UPV 075.327–EA033/92 and UPV 075.327–EA053/93 of the University of the Basque Country and by a grant to Consolidated Research Groups (UPV/EHU). Y.R. was the recipient of a MEC–DGCYT pre-doctoral fellowship.     

Course of minimal oxygen consumption in the young rat during protein energy malnutrition followed by return to a balanced diet

Growth and resting oxygen consumption (VO2 minimal) were studied on two strains of six post-weaning male rats during 60 days. The first strain was fed with a balanced diet (15% casein) for 60 days, the second received a 2% casein diet for 30 days, and then the balanced diet for the remaining 30 days. The 2% casein diet completely stopped the body weight and VO2 minimal increases. When return to a balanced diet, these parameters show an evolution similar to that of controls of the same body weight. The VO2 minimal had the same kind of evolution as body weight and was well correlated with growth speed. The protein restriction involves a strong and temporary reduction of the VO2 minimal, which will correspond to a nutritional stress.     

Comparative study of the effects of 2 types of protein-energy malnutrition followed by a balanced refeeding, on the lipase, phospholipase A2 and amylase activities of the pancreas and pancreatic juice in the growing rat

The intake of 2 p. 100 casein diet as only protein source decreased overall phospholipase A2 activity. Amylase activity was more affected than lipase one. The effects of intake of 5 p. 100 gluten diet as only protein source were less important than 2 p. 100 casein diet. Refeeding on 20 p. 100 or 15 p. 100 casein diet caused a considerable increase of phospholipase A2, lipase and amylase activities.     

Levels of mRNAs coding for lipogenic enzymes in rat lung upon fasting and refeeding and during perinatal development

The relative amounts of mRNAs coding for fatty-acid synthase (EC 2.3.1.85), acetyl-CoA carboxylase (EC 6.4.1.2), ATP citrate lyase (EC 4.1.3.8) and malic enzyme (EC 1.1.1.40) were determined in lungs and livers of adult rats that were normally fed, starved for 48 h or starved for 48 h and subsequently refed for 72 h with a carbohydrate-rich, fat-free diet. In the liver, starvation caused a small decrease in the relative abundance of the mRNAs which was not statistically significant. Subsequent refeeding caused a statistically significant increase in mRNAs for all of the enzymes studied. In the lung, no significant changes were found, indicating that the regulation of the abundance of mRNAs encoding the lipogenic enzymes in the lung differs from that in the liver. In the developing rat lung, mRNA for fatty-acid synthase increased 3-fold in abundance between fetal days 18 and 20 and decreased directly after birth (at day 22 of gestation). A similar pattern was observed for ATP citrate lyase mRNA. The level of acetyl-CoA carboxylase mRNA decreased significantly after birth. These observations indicate that in perinatal rat lungs, pretranslational regulation is involved in the control of the synthesis of these enzymes. The abundance of acetyl-CoA carboxylase mRNA did not change in the prenatal period, a time during which the specific activity of this enzyme increases. This lack of correlation between the specific activity of acetyl-CoA carboxylase and the abundance of its mRNA may indicate that translational regulation of the synthesis of the enzyme or post-synthetic regulatory effects on enzyme molecules are involved in the control of this enzyme in the prenatal period. No changes in the abundance of lung malic enzyme mRNAs were observed throughout the perinatal period.     

Evidence for translational control of storage protein biosynthesis during embryogenesis ofAvena sativa L. (oat endosperm)

Oat polysomes direct the synthesisin vitro of a large number of products, the majority of which are the salt-soluble globulins (1,3,10,11,21). Total RNA or poly A⁺ RNA isolated from these polysomes directs the synthesis of the same number and types of products; however, the amount of globulins synthesized no longer represents the major products; rather, there is a decreased level of globulins and an increased amount of the other products synthesizedin vitro (6, 18). These results imply that the translational control can dictate final product levels. Reconstruction experiments using oat poly A⁺ mRNA and polysomal factors that are made free of endogenous RNA by nuclease digestion demonstrate that these factors do influence the translational specificity of oat globulin mRNA relative to other mRNAs. It is suggested that translational control is partially responsible for the levels of globulin in the mature grain.     

Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non-PKC-delta-dependent mechanism

Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.     

The digestive enzymes and acidity of the pellets regurgitated by raptors

The activity of six digestive enzymes (amylase, chitinase, trypsin, chymotrypsin, carboxypeptidase A, pepsin) was examined in the water-soluble contents of the pellets egested by ten species of raptors (kestrel, saker, lanner, goshawk, barn owl, tawny owl, little owl, long-eared owl, African great-owl and steppe eagle). All the enzymes studied were present in the pellets from these birds, except for chitinase which was not detected in the pellets of the goshawk and the steppe eagle, and amylase and carboxypeptidase absent in the material egested by the lanner. The origin of the enzymes studied was examined. Pancreatic enzymes, which are present in the pellets, arise from a reflux of intestinal fluid into the stomach. The importance of this phenomenon is discussed. The acidity of the pellets was measured. Relations existing between the type of food, characteristics of the pellet and the digestive process in raptors are analysed. The evolutionary advantage of pellet egestion is discussed.     

Protein malnutrition and response of pancreatic acinar cells to stimulation by cholecystokinin

Pancreatic lobules were isolated from 2 groups of male Wistar rats after 23 days of diet. A control group (C) fed on a 20% protein diet (16% gluten + 4% casein) and an experimental group (E) on a 5% protein diet (4% gluten + 1% casein). After isolation, lobules were preincubated 10 min with 10 muCi [3H]-leucine, washed, then incubate within Krebs Ringer bicarbonate Hepes. Basal secretion, then stimulated secretion (50 pM of cholecystokinin (CCK] of radioactive and non-radioactive protein and amylase outputs were measured. During basal secretion, in (E) group, lobules secreted more proteins than (C) one, the same outputs of amylase and radioactive protein were observed in both groups. The stimulated secretion by CCK increased the outputs of non-radioactive protein and amylase of lobules (T) (2-3 fold), but was without effect on lobule (E) outputs. Therefore, a low-protein diet involved a decrease of CCK sensibility on acinar cells, this fact might be mediated by a decreasing number and/or affinity of their CCK receptors.     

Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

Influence of a 36 h fast followed by refeeding with glucose, glycerol or placebo on metabolism and performance during prolonged exercise in man

Five men were studied during exercise to exhaustion on an electrically braked cycle ergometer at 70% of VO2max. The four experimental treatments were as follows: fasted for 36 h (A); fasted (36 h) and refed with glucose (B) or glycerol (C); postabsorptive (overnight fast, D). In B and C the subjects were given a drink containing glucose or glycerol (1g per kg body weight) 45 min before starting exercise. A placebo drink was given 45 min before exercise on treatments A and D. Despite an increased availability of circulating free fatty acids, beta-hydroxybutyrate and glycerol exercise time to exhaustion was significantly lower after fasting (treatment A 77.7 +/- 6.8 min) compared with treatment D (119.5 +/- 5.8 min). Refeeding with glucose or glycerol did not significantly improve performance (92.4 +/- 11.8 min and 80.8 +/- 3.6 min respectively) compared with treatment A and lowered circulating levels of FFA and beta-HB during exercise compared with A. Despite the probability of low liver glycogen levels after fasting, none of the subjects became hypoglycaemic (blood glucose less than 4 mmol.l-1) during exercise and their blood lactate concentrations were not high at exhaustion. Plasma levels of branched chain amino acids (BCAA) decreased progressively during exercise on treatments A, B and C and were considerably lower at exhaustion compared with treatment D. Falling plasma concentrations of BCAA during prolonged exercise may be implicated in the generation of central fatigue.     

RNA polymerase activities and chromatin protein composition of rat liver during methionine deprivation and refeeding

The reversible effect of dietary methionine deficiency was studied in young adult rats. The sensitivity of nuclear chromatin to micrococcal nuclease (EC3.1.4.7) digestion and the composition of the chromatin proteins were unaffected by the dietary regimens. The specific chromatin-bound RNA polymerase II activity decreased during methionine deficiency. Refeeding of methionine for 2 days restored the activity in the nuclease-released chromatin. RNA polymerase I plus III activity remained unchanged. Total RNA polymerase activity changed with the liver wet weight which was reduced during methionine deficiency and was not restored to control level after 2 days of methionine refeeding. RNA polymerase activity was altered by methionine deficiency. The recovery was independent of major modifications of the chromatin structure and protein composition.