Activity of Lysosomal Enzymes During Protein Malnutrition and Progesterone Supplementation in the Mouse.

This study investigated the effects of protein malnutrition and progesterone supplementation on the activities of a spectrum of lysosomal enzymes in tissue fragments of mouse liver and kidney. The working hypothesis was that the known anti-stress action of progesterone could have to do with the inhibition of lysosomes which are engaged in apoptotic and oxidative stress-induced responses. The study investigated the effects of exogenous progesterone in chronically (3 weeks) protein-malnourished (10% protein) mice on the activities of lysosomal hydrolases in liver and kidney tissues. Progesterone was injected intraperitoneally in a dose of 2 µg/g body mass dissolved in a vehicle volume of 10 µL/g body mass during the final 3 days of exposure to either low 10% or standard 16% protein content in the chow. After euthanizing the animals, tissue fragments of liver and kidney assayed for the content of lysosomal enzymes. The results demonstrated the stimulating effect of protein malnutrition on lysosomal activities. We further found, contrary to our hypothesis, that progesterone supplementation during both standard and low-protein conditions enhanced lysosomal activities, particularly acting in concert with protein malnutrition in kidney tissue. The effects were selective concerning both lysosomal enzymes and tissues and of highly variable magnitude. Nonetheless, we believe we have shown that progesterone assists protein malnutrition in stimulation of lysosomal enzymes, which suggests the possibility of the hormone's engagement in cleansing the cellular milieu in disorders consisting of accumulation of toxic molecules.

[State of the energy-supply system of the liver mitochondria under the conditions of alimentary deficiency of protein].

The NADH-dehydrogenase and succinate dehydrogenase activity of the rats' liver mitochondria under the conditions of alimentary deprivation of protein has been studied. Research was carried out on 65 white non-linear rats divided according to the diet protein content into three groups: 1--rats fed a hypoprotein diet (7% of protein, 10% of fat u 83% of carbohydrates; n = 26); 2--rats fed a protein-free diet (n = 26); 3--rats fed a complete semi-synthetic ration (14% of protein, 10% of fat u 76% of carbohydrates; n = 13). The NADH-dehydrogenase activity was estimated by spectrophotometric method, succinate dehydrogenase activity--by the intensity of reduction of the potassium ferricyanide. It has been estimated that the decrease of NADH-dehydrogenase activity of mitochondria occurred on the 14th day of feeding rats with protein-free diet, and four-week feeding of rats under these conditions lead to the decrease of enzyme activity by 5,5 fold compared with the control group (0.506 +/- 0.040 nmol NADH/min/mg of protein) and by 3,0 fold compared with the previous stage of the experiment. At the same time a hypoprotein diet caused 2-fold decrease of NADH-dehydrogenase activity of liver mitochondria only on the 28th day. It has been shown that the succinate dehydrogenase activity didn't change significantly after two-week maintenance of rats on a protein-free diet as compared with control, while the four-week maintenance on both hypoprotein and protein-free diet lead to the decrease of the succinate dehydrogenase activity. Specifically, under the conditions of the hypoprotein diet succinate dehydrogenase activity of liver mitochondria decreased twofold and under the conditions of the protein free diet-- threefold. Probably, the disorders at the level of Complex I of respiratory chain underlie the realization of the changes in the system of energy biotransformation in mitochondria under the conditions of alimentary protein deficiency.

[L-arginine metabolism enzyme activities in rat liver subcellular fractions under condition of protein deprivation].

The features of arginase and NO-synthase pathways of arginine's metabolism have been studied in rat liver subcellular fractions under condition of protein deprivation. During the experimental period (28 days) albino male rats were kept on semi synthetic casein diet AIN-93. The protein deprivation conditions were designed as total absence of protein in the diet and consumption of the diet partially deprived with 1/2 of the casein amount compared to in the regular diet. Daily diet consumption was regulated according to the pair feeding approach. It has been shown that the changes of enzyme activities, involved in L-arginine metabolism, were characterized by 1.4-1.7 fold decrease in arginase activity, accompanied with unchanged NO-synthase activity in cytosol. In mitochondrial fraction the unchanged arginase activity was accompanied by 3-5 fold increase of NO-synthase activity. At the terminal stages of the experiment the monodirectional dynamics in the studied activities have been observed in the mitochondrial and cytosolfractions in both experimental groups. In the studied subcellular fractions arginase activity decreased (2.4-2.7 fold with no protein in the diet and 1.5 fold with partly supplied protein) and was accompanied by NO-synthase activity increase by 3.8 fold in cytosole fraction, by 7.2 fold in mitochondrial fraction in the group with no protein in the diet and by 2.2 and 3.5 fold in the group partialy supplied with protein respectively. The observed tendency is presumably caused by the switch of L-arginine metabolism from arginase into oxidizing NO-synthase parthway.

[Activity of the marker liver enzymes under the conditions of toxic hepatitis and alimentary deprivation of protein].

The activity of the sorbitoldehydrogenase (SDH), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in the blood serum of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. The animals were divided into 3 groups: 1--rats with acute acetaminophen-induced hepatitis, maintained on the full ration; 2--rats with acute acetaminophen-induced hepatitis, maintained under the conditions of alimentary deprivation of protein; 3--control. The activity of the sorbitol dehydrogenase in blood serum was determined by the kinetic method, activity of the alanine aminotransferase and alkaline phosphatase - photometrically. It is shown, that in animals with the model hepatitis the activity of sorbitol dehydrogenase in blood serum increases 20-fold, wherein statistical significance between animals with hepatitis maintained under the conditions of full ration and those of low-protein diet is not established. In the group of animals with acetaminophen-induced hepatitis the preservation on the control level of the alkaline phosphatase activity on the base of the increase of alanine aminotransferase by 2.2 times and ratio ALT/ALP>5 testifies about hepatocellular liver injury. In the group of animals with drug-induced hepatitis and alimentary deprivation of protein, the increase of the alkaline phosphatase and alanine aminotransferase activity is observed, herewith the ratio ALT/ALP ranges from 2 to 5 and testifies about mixed liver injury. The conclusion was made, that alimentary deprivation of protein is the critical factor for the development of the disturbances of functional and structural liver integrity, and the therapeutic approaches to the correction of the drug-induced liver injury should be different depending on the value of protein ration in the anamnesis, taking into account the different types of liver injury.

[Digestive enzyme activities in rats kept on standard or surplus breast-feeding and on low-protein diet directly after weaning].

Activities of digestive enzymes (maltase, alkaline phosphatase, amino peptidase M, and glycyl-L-leucine dipeptidase) in small and large intestine, liver, and kidney were studied in rats of different ages kept in normal (8) and low (3) amounts of pups per litter. The low-protein diet for 10 days at once after weaning was found to change the mass of the organs and their digestive enzyme activities in all studied rat groups. The revealed changes were more prominent in rats kept under conditions of breast-overfeeding. In adult animals of this group, distribution of the alkaline phosphatase activity along the small intestine differed from that in control rats. The obtained results seem to confirm the fact that any disturbance of the nutrition quality in early ontogenesis leads to disturbance of the "metabolic programming of enzyme systems" of digestive and non-digestive organs.

[Effect of weaning terms and protein deficit in the rat pup nutrition on activities of digestive enzymes].

Restriction of protein in nutrition of rat pups weaned at different terms has been found to produce changes in activities of digestive enzymes (maltase, alkaline phosphatase, aminopeptidase M, and glycyl-L-leucine dipeptidase) in the small and large intestine both at once after cessation of nutrition with low-protein diet for 10 days and 4 months later. In adult animals after the earlier or later weaning there are observed not only a decrease or increase of the enzyme activities, but also a different type of distribution of the alkaline phosphatase activity along the small intestine, which is more pronounced in the lately weaned rats. Thus, disturbance of metabolic programming of enzyme systems of the small and large intestine due to a change of quality of nutrition in early ontogenesis depends on terms of weaning of animals.

[Specificity of activity antioxidative enzymes at protein deficiency and excessive content Cu, Zn, Mn, Se in the food].

The research of kinetic properties (Km u Vmax) of two enzymes: Superoxide Dismutase and Glutathione Peroxidase from rats liver and blood and lipid peroxidation induced by both a low protein diet (8%) and 2-fold concentration Cu, Zn, Mn, Se in diet. There was a change of Km and Vmax: the reduction of Km(GPH) was in liver at 28 d and the increase of Km(SOD) was in liver in group with 2-fold concentration Cu, Zn, Mn, Se in diet. The analysis of Km and Vmax of Superoxide Dismutase and Glutathione Peroxidase in different alimentary influence may be as one of methods for assessment individualization of diet therapy.

[Effect of diarrhea on nutrient utilization in protein deficient or protein-calorie deficient rats]. / Efecto de la diarrea sobre la utilización de nutrientes en ratas con desnutrición proteico-calórica o proteica.

Diarrhea increases the effects of malnutrition. Accordingly, the effect of diarrhea on two types of malnutrition (protein deficiency and protein-calorie deficiency) was studied. The experiment included 42 young Sprague Dawley rats. The rats were distributed into three groups with 14 rats per group. During the first 16 of the experiment, the first group was fed a control diet ad libitum, the second received the same diet but with food intake reduced in 50% whereas the third group was offered a protein deficient diet. Thus, at the end of this period there were well-fed rats (control), as well as protein and protein-calorie malnourished rats. Then one half of the rats in each group were given lactose to produce diarrhea and all rats continued with their previously assigned diet and feeding regime during one more week. Therefore, during this period there were control rats, protein deficient rats and protein-calorie deficient rats with and without diarrhea. The results showed that diarrhea caused a substantial reduction in food intake and growth in the well-fed rats and also in the group fed the protein deficient diet. However, the protein-calorie deficient group did not reduce its intake nor its growth rate. As a result, diarrhea caused malnutrition in the control group and increased malnutrition in the protein deficient but it did not have an additional effect in the protein-calorie deficient rats. The apparent absorption of lipids and nitrogen measured in these rats showed that the absorption reduction caused by diarrhea was more pronounced in the protein deficient group. This group also had the lowest activities of intestinal disaccharidases. These results showed that diarrhea had a more detrimental effect in protein deficient than in protein-calorie deficient rats.

[Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

The expression of matrix metalloproteinases 8 and 9 by neutrophils of Wistar albino rats with severe qualitative and quantitative protein malnutrition.

Neutrophils are the major cellular immune components in response to bacterial infections. Neutrophil enzymes are important in invasion, inflammation, and infection processes. In order to understand the basic effects of protein malnutrition on neutrophils we studied matrix metalloproteinases 8 and 9 (MMP-8 and MMP-9) production in severe quantitative and qualitative protein malnutrition in rats. Wistar rats (2 months old) were divided into four groups each with three subgroups and fed various protein-containing diets (24% protein, 20% gelatin-containing and N-free) for 7, 14, 21, and/or 28 days. Neutrophil enzyme expression was determined by Western blotting. Leukocytes decreased significantly due to malnutrition (p = 0.001 ) whilst the percentage of neutrophils increased (p = 0.02) in protein-deprived groups. Neutrophils of malnourished rats produced lower levels of MMP-8 at early stages of protein deprivation with an increase in the following weeks. MMP-9 production by neutrophils from N-free diet fed animals was highest after one week. Serum MMP-9 levels decreased in the qualitative but not in the quantitative protein malnutrition groups. Results suggest that neutrophils might be important in reuse of body cell proteins during fasting or malnutrition conditions and dietary manipulation might have profound effects on MMP-8 and -9 production in rats.

Liver damage and caspase-dependent apoptosis is related to protein malnutrition in mice: effect of methionine.

This study aimed to determine whether the effects on the mouse liver caused by three periods of feeding a protein-free diet for 5 days followed by a normal complete diet for 5 days (3PFD-CD) are prevented by a constant methionine supply (3PFD+Met-CD). The expressions of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) were assessed by proteomics and reverse transcriptase-polymerase chain reactions. The liver redox status was examined by measuring the activities of superoxide dismutase (SOD) and catalase (CAT), as well as protein carbonylation. Because oxidative stress can result in apoptosis, the activity and content of caspase-3, as well as the x-linked inhibitor of the apoptosis protein (XIAP) and mitochondrial caspase-independent apoptosis inducing factor (AIF) contents were assessed. In addition, the liver histomorphology was examined. Compared to the controls fed a normal complete diet throughout, feeding with 3PFD-CD increased the FAS content, decreased the CAIII content, decreased both the SOD and CAT activities, and increased protein carbonylation. It also activated caspase-3, decreased the XIAP content, decreased the AIF content, increased the number of GSTP1-positive foci and caspase-3-positive cells, and caused fatty livers. Conversely, the changes were lessened to varying degrees in mice fed 3PFD+Met-CD. The present results indicate that a regular Met supply lessens the biochemical changes, damage, and caspase-dependent apoptosis provoked by recurrent dietary amino acid deprivation in the mouse liver.

[NADH:ubiquinone reductase and succinate dehydrogenase activity in the liver of rats with acetaminophen-induced toxic hepatitis on the background of alimentary protein deficiency].

The ratio between the redox forms of the nicotinamide coenzymes and key enzymatic activity of the I and II respiratory chain complexes in the liver cells mitochondria of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. It was estimated, that under the conditions of acute acetaminophen-induced hepatitis of rats kept on a low-protein diet during 4 weeks a significant decrease of the NADH:ubiquinone reductase and succinate dehydrogenase activity with simultaneous increase of the ratio between redox forms of the nicotinamide coenzymes (NAD+/NADH) is observed compared to the same indices in the liver cells of animals with experimental hepatitis kept on the ration balanced by all nutrients. Results of research may become basic ones for the biochemical rationale for the approaches directed to the correction and elimination of the consequences of energy exchange in the toxic hepatitis, induced on the background of protein deficiency.

Ornithine decarboxylase in rat skin.

Ornithine decarboxylase (ODC; E.C.4.1.1.17) activities can be stimulated 2-10 fold in rat epidermis and dermis by hair plucking. Stimulation does not involve the removal of a soluble ODC inhibitor. ODC activity in the dermis and whole skin decreased with aging, while the epidermis showed little change. The apparent Km for ornithine and the heat stability of ODC in plucked and unplucked skin were similar. ODC was assayed in plucked and unplucked skin of rats fed diets containing between 2 and 24% protein. Activities in both plucked and unplucked skin were higher in the animals fed diets with higher protein contents. ODC levels were positively correlated with the weight changes undergone by rats on controlled-protein diets. In animals restricted to 2% protein diets and rehabilitated with 16% protein diets, enzyme levels were increased after 2 days rehabilitation and peaked after 5 days rehabilitation. The responsiveness of ODC to changes in dietary protein may be useful in the diagnosis of protein malnutrition.

Metabolism of lysosomal enzymes in the protein-deficient weanling rat.

We have shown that the protein-deficient weanling rat fed a 3% casein diet, within 2 to 4 wk, exhibits marked changes in serum lysosomal hydrolases similar to those observed in children suffering from protein-calorie malnutrition: serum hexosaminidase, alpha-mannosidase, and beta-glucuronidase activities increase 3-fold, 2-fold, and 50%, respectively, whereas the acid phosphatase levels decrease by 50%. Rehabilitation of the protein-deficient animals with a diet containing 25% protein (i.e., casein) results in a rapid restoration of the plasma lysosomal hydrolase profiles to normal in less than 1 wk. The specific activities of various tissue lysosomal enzymes change significantly in the protein-deficient animals; however, no overall consistent pattern of change is apparent. In general, the greatest number of changes in lysosomal enzymes occurs in the kidney, whereas the brain exhibits the smallest differences between experimental and control animals in this regard. Perfusion experiments have shown that the rate of release of lysosomal enzymes from livers of rats fed the protein-deficient diet is profoundly altered when compared to that of control animals. Studies of the variation of enzyme secretion with time have demonstrated that the rate of secretion of hexosaminidase by the liver remains low and then rises markedly (3-fold) after the animals have been consuming the 3% casein diet for 16 days. In contrast, the secretion of both acid phosphatase and beta-glucuronidase is markedly depressed in the early phase of protein malnutrition (i.e., 7 to 16 days), and then increases greatly by the 3rd wk. These results demonstrate that changes occur in the rate of secretion of lysosomal enzymes by the liver during the course of experimental protein malnutrition.

Metabolic effects of oral contraceptives in monkeys fed on adequate and low protein diets.

PIP: Rhesus monkeys were given either high estrogen, low progesterone or low estrogen-high progesterone oral contraceptive agents (OCAs) and maintained on either a 16% protein diet or a 4% protein diet. Both OCAs created a small but significant fall in hemoglobin and serum protein. On both diets, OCAs led to a gradual elevation of serum alkaline phosphatase and serum glutamic oxalic transaminase (SGOT) activities. There were no histologic liver lesions. On the low protein diet, glucose tolerance was impaired between cycles 10 and 11, and on the high protein diet between cycles 16 and 20. Vitamins B-12, A, and thiamine tended to be lower in OCA animals. The conclusion of these experiments is that the hazard of using OCAs is not exaggerated by protein deficiency.^ieng

A new family of protein kinases--the mitochondrial protein kinases.

Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes. These protein kinases, responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in mitochondrial matrix space and have most likely evolved from genes originally present in respiration-dependent bacteria endocytosed by primitive eukaryotic cells. Long-term regulatory mechanisms involved in the control of the activities of these two kinases are of considerable interest. Dietary protein deficiency increases the activity of branched-chain alpha-ketoacid dehydrogenase kinase associated with the branched-chain alpha-ketoacid dehydrogenase complex. The amount of branched-chain alpha-ketoacid dehydrogenase kinase protein associated with the branched-chain alpha-ketoacid dehydrogenase complex and the message level for branched-chain alpha-ketoacid dehydrogenase kinase are both greatly increased in the liver of rats starved for protein, suggesting increased expression of the gene encoding branched-chain alpha-ketoacid dehydrogenase kinase. The increase in branched-chain alpha-ketoacid dehydrogenase kinase activity results in greater phosphorylation and lower activity of the branched-chain alpha-ketoacid dehydrogenase complex. The metabolic consequence is conservation of branched chain amino acids for protein synthesis during periods of dietary protein deficiency. Two isoforms of pyruvate dehydrogenase kinase have been identified and cloned. Pyruvate dehydrogenase kinase 1, the first isoform cloned, corresponds to the 48 kDa subunit of the pyruvate dehydrogenase kinase isolated from rat heart tissue. Pyruvate dehydrogenase kinase 2, the second isoform cloned, corresponds to the 45 kDa subunit of this enzyme. In addition, it also appears to correspond to a possibly free or soluble form of pyruvate dehydrogenase kinase that was originally named kinase activator protein. Assuming that differences in kinetic and/or regulatory properties of these isoforms exist, tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex. Starvation and the diabetic state are known to greatly increase activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat. This contributes in these states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Nutritional rehabilitation of skeletal muscle in protein-deprived young rats.

The effect of severe protein deprivation and subsequent nutritional rehabilitation on the fibre size and mitochondrial enzyme activity of the extensor digitorum longus (EDL) and soleus muscles of the young rat has been examined. Protein deprived rats showed atrophy of type 2 fibres predominantly, reduced histochemical activity of succinic dehydrogenase (SDH) and reduced biochemical activity of citrate synthase. Nutritional rehabilitation indicated by resumption of the original body weight resulted in complete restitution of the weight of the muscles and the size of type 1 and type 2 fibres, but not of the activity of SDH and citrate synthase. The results indicate that regarding size, type 2 fibres tend to be more influenced than type 1 fibres by the nutritional supply. The mitochondrial enzyme activity which is decreased by protein deprivation does not regain the normal levels as quickly as the muscle fibres resume their normal size.

Studies on liver sulphurylase activity in rats: vitamin-A-deficient and low-protein diets.

Studies on the sulphurylation of phenols by livers from normal and vitamin-A-deficient rats showed a significant reduction in sulphurylating activity in the acute stage of deficiency. At the 'plateau stage' of deficiency, decrease in this activity was marginal and was not significant. Rats consuming a 6% casein diet had a significantly lower liver sulphurylating activity when compared to those fed 12 and 18% casein diets. Comparative data on the sulphurylating activity of rats maintained on diets providing poor-quality protein, i.e. ragi (Eleusine coracana) and maize (Zea mays) revealed a higher sulphurylating activity in the maize-fed group showing thereby that the amino acid composition of the protein apparently influences the enzyme activity associated with sulphurylation of phenols. It is thus shown that liver sulphurylating activity is influenced by quantity and quality of protein consumed.

Adrenalectomy: its effects on systemic tryptophan metabolism in normal and protein malnourished rats.

Rats born to dams fed either a low protein diet (8% casein) or a normal diet (25% casein) started 5 weeks prior to conception and continued through lactation were bilaterally adrenalectomized or received a sham-operation at 30 days of age. At 60 days of age, the systemic tryptophan metabolism of th 8% and 25% adrenalectomized rats was compared to the sham-operated controls of each diet group. While adrenal ablation produced significant decreases in the brain serotonin and metabolite concentrations and marked increases in brain tryptophan concentrations for both diet groups compared to their respective controls, these substances remained significant higher in all malnourished rats than in the well-nourished groups. Also, the major modulator of the peripheral metabolic pathways which regulates the availability of free plasma tryptophan (total tryptophan, albumin, and nonesterified fatty acid concentrations) was the nutritional status of the rats rather than their treatment condition. Only plasma corticosterone concentrations showed changes (significantly decreases) as a consequence of adrenal ablation for either diet group. Overall, the data indicated that under physiological conditions the adrenal cortex has an important function in determining brain tryptophan utilization, whereas its role in regulating peripheral tryptophan metabolism is minimal.

An assessment of the clinical usefulness of plasma ribonuclease assays.

The usefulness of plasma ribonuclease assays was studied in (i) patients with possible protein deficiency, (ii) patients with myelomatosis, (iii) patients with carcinoma of the breast. In each group, the major factor associated with elevation of plasma ribonuclease was impairment of renal function. The assay was therefore of little value in the assessment of patients with myelomatosis or carcinoma of the breast. However, in the patients with possible protein deficiency and normal renal function, an elevation of plasma ribonuclease is, in general, associated with a decrease in serum albumin, transferrin and cholinesterase. Plasma ribonuclease may therefore be a useful parameter in the assessment of protein nutritional status.