Effects of dietary medium-chain triacylglycerol on mRNA level of gluconeogenic enzymes in malnourished rats.

We have reported previously that dietary medium-chain triacylglycerol (MCT) improved serum albumin concentration and protein balance in malnourished rats. To clarify the mechanisms for this effect of MCT, hepatic messenger RNA levels of gluconeogenic enzymes, pyruvate dehydrogenase (PDH) and alanine aminotransferase (ALT) were measured in rats fed low-protein diets containing either MCT or isocaloric long-chain triacylglycerol (LCT) for 2 wk. The serum albumin concentration in rats fed the MCT diet was significantly higher compared with those fed the LCT diet. Serum free fatty acids and ketone body fraction were higher in rats fed MCT compared with those fed the LCT diet. The hepatic mRNA level of PDH was significantly lower in rats fed MCT than those fed LCT. But, there was no significant difference between the two groups in mRNA of gluconeogenic enzymes or ALT. These results suggest that ketone bodies, which are an alternative energy source and might spare blood glucose, increase by MCT feeding, and the reason for the PEM (protein-energy malnutrition)-improving effect of MCT is not caused by suppression of gluconeogenesis.

Effects of cysteine on the pharmacokinetic parameters of omeprazole in rats with protein-calorie malnutrition: partial restoration of some parameters to control levels by oral cysteine supplementation.

BACKGROUND: It has been reported that omeprazole is mainly metabolized via the hepatic cytochrome (CYP) 1A1/2, 3A1/2, and 2D1, and the expressions and mRNA levels of CYP1A2, 2C11, and 3A1/2 decreased in protein-calorie malnutrition (PCM) rats compared with controls. Interestingly, the decreased CYP1A2, 2C11, and 3A1/2 in PCM rats returned fully or partially to control levels by oral cysteine supplementation (PCMC rats). Hence, it could be expected that some pharmacokinetic parameters of omeprazole might change in PCM rats and partially restore to control levels in PCMC rats. The purpose of this study is to investigate the pharmacokinetic changes of omeprazole in PCM rats and restoration of the parameters in PCMC rats to control levels. METHODS: Omeprazole was administered intravenously (20 mg/kg) and orally (40 mg/kg) to control, PCM, and PCMC rats. RESULTS: The following pharmacokinetic parameters were changed in PCM rats and partially returned to control levels in PCMC rats: the area under the plasma concentration-time curve (AUC; 387, 762, and 539 microg min/mL for control, PCM, and PCMC rats, respectively, after intravenous [IV] administration, and the corresponding values after oral administration: 115, 304, and 201 microg min/mL), total body clearance (51.7, 25.5, and 37.1 mL/min/kg, respectively), nonrenal clearance (51.5, 25.4, and 36.1 mL/min/kg, respectively), and in vitro intrinsic clearance (0.158, 0.118, and 0.138 mL/min/mg protein). CONCLUSIONS: PCM was associated with significant changes in some omeprazole pharmacokinetics and the pharmacokinetic parameters restored to control levels by oral cysteine.

Effect of protein and calorie malnutrition on drug metabolism in rat - in vitro.

PURPOSE: To study the effect of protein and calorie malnutrition on in vitro drug metabolism of protein and calorie malnourished juvenile and adult rats. METHOD: Microsomal incubation was used as a means of monitoring drug metabolism changes, HPLC was employed to quantify metabolites and enzyme immunoassay (EIA) was used for rat growth hormone (rGH) monitoring. RESULTS: Protein and calorie malnutrition significantly decreased levels of microsomal protein and total P450. Microsome of protein and calorie malnourished rats showed impaired testosterone 16alpha- and 2alpha- hydroxylation (CYP2C11), testosterone 6beta-hydroxylation (CYP3A), and testosterone 7alpha-hydroxylation (CYP2A1). Testosterone 16beta-hydroxylation (CYP2B1) did not show any significant change, neither in capacity nor affinity. The quantity and the secretion pattern of rGH were not altered in protein and calorie malnourished rats compared to those in healthy animals. CONCLUSIONS: Serum albumin is not a good indicator of malnutrition. The capacity and affinity of CYP2C11, CYP3A and CYP2A1 were compromised by protein and calorie malnutrition. The impairment of drug metabolism in protein and calorie malnourished rats was not caused by the alteration of rGH.

Intestinal enzymes during malnutrition & infection in rabbits.

BACKGROUND & OBJECTIVES: Malnutrition plays an important role in the intestinal absorption of nutrients. However, reports are not consistent whether intestinal enzymes are decreased in the presence of malnutrition. It is also not clear whether simultaneous presence of malnutrition and infection adds to the problem of malabsorption of nutrients. The aim of the present study was to determine intestinal functions in terms of concentrations of disaccharidase enzymes during diarrhoea and protein energy malnutrition. METHODS: Concentrations of three disaccharidase enzymes, namely maltase, sucrase and lactase were measured in nine energy-restricted and five control rabbits during diarrhoea induced by rabbit diarrhoeagenic Escherichia coli (RDEC-1). Malnutrition was achieved in the rabbit model by feeding the animals for 30 days with half the amount of food fed to well-nourished control rabbits. Both the energy-restricted and the control groups were challenged by RDEC-1. Diarrhoea occurred on day 1-7 after administration of the strain. After onset of diarrhoea, both groups of rabbits were sacrificed and their intestinal mucosa was examined to determine the concentration of lactase, maltase and sucrase. RESULTS: The energy-restricted animals and controls did not differ significantly for concentrations (units/mg proteins) of lactase (0.65 +/- 0.28 vs 0.56 +/- 0.17 ), maltase (6.20 +/- 2.70 vs 6.47 +/- 1.90) and sucrase (5.42 +/- 2.30 vs 5.13 +/- 1.40) measured during acute infectious diarrhoea. INTERPRETATION & CONCLUSION: The results suggested that the enzymatic functions of the intestinal brush border were not statistically different during diarrhoea among malnourished rabbits compared with their well-nourished counterparts.

[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.

PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation.

Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model.

Selenium and zinc protect brain mitochondrial antioxidants and electron transport chain enzymes following postnatal protein malnutrition.

AIMS: Selenium (Se) and zinc (Zn) are trace elements required for optimal brain functions. Thus, the role of Se and Zn against protein malnutrition induced oxidative stress on mitochondrial antioxidants and electron transport chain (ETC) enzymes from rats' brain were investigated. MAIN METHODS: Normal protein (NP) and low protein (LP) rats were fed with diets containing 16% and 5% casein respectively for a period of 10weeks. Then the rats were supplemented with Se and Zn at a concentration of 0.15mgL(-1) and 227mgL(-1) in drinking water for 3weeks after which the rats were sacrificed. KEY FINDINGS: The results obtained from the study showed significant (p<0.05) increase in lipid peroxidation (LPO), ROS production, oxidized glutathione (GSSG) levels and mitochondrial swelling and significant (p<0.05) reductions in catalase (CAT) and Mn-superoxide dismutase (Mn-SOD) activities, glutathione (GSH) levels, GSH/GSSG ratio and MTT reduction as a result of LP ingestion. The activities of mitochondrial ETC enzymes were also significantly inhibited in both the cortex and cerebellum of LP-fed rats. Supplementation with either Se or Zn restored the alterations in all the parameters. SIGNIFICANCE: The study showed that Se and Zn might be beneficial in protecting mitochondrial antioxidants and ETC enzymes against protein malnutrition induced oxidative stress.

Turnover of rat liver malic enzyme during induction by protein deprivation.

The half-lives of hepatic malic enzyme and total liver soluble proteins were determined in protein-sufficient and protein-deficient rats after injection of tracer doses of radioactively labeled leucine. The results obtained in these experiments have demonstrated that the increased levels of malic enzyme obtained under conditions of severe protein restriction are due to elevated rates of synthesis of the enzyme protein, with no apparent change in the rate of its degradation.

The effect of cysteine on the altered expression of class alpha and mu glutathione S-transferase genes in the rat liver during protein-calorie malnutrition.

Protein-calorie malnutrition (PCM) represents a global health problem. The breakdown rate of glutathione S-transferase (GST) subunits determines their differential contents during protein depletion. Hepatic GST expression and the underlying mechanistic basis were investigated in PCM rats. PCM caused no change in rGSTA1/2 subunit. In contrast, rGSTA3/5 subunit was 2.4-fold induced during PCM, while the levels for rGSTM1 and M2 subunits were 30% and 70% suppressed. Increased GSTA3/5 expression was significantly prevented by cysteine or methionine treatment, although such treatment failed to restore the rGSTM2 level. In contrast to differential GST protein expression, PCM caused a 5-10-fold increase in rGSTA2/A3/A5 and M1 mRNAs, whereas rGSTM2 mRNA was 70% decreased. The elevations in rGSTA2/A3/A5 and M1 mRNAs were completely abolished by cysteine or methionine treatment during PCM, although the rGSTM2 mRNA level was not restored. PCM induced oxidative stress in the liver, as evidenced by protein carbonylation. Antioxidant response element (ARE)-binding activity of nuclear extracts from PCM rats was increased, which was immunodepleted with anti-Nrf-1/2 antibodies. Activation of nuclear ARE-binding proteins was inhibited by cysteine. Data showed that hepatic GSTs were differentially expressed during PCM, that certain GST mRNAs were increased with the ARE activation, and that cysteine was active in preventing increases in GST mRNAs and ARE activation.

Studies on hepatic and extrahepatic lipoprotein lipases in protein-calorie malnutrition.

Postheparin serum lipolytic activities (hepatic and extrahepatic), serum free fatty acid, and triglyceride levels were measured in 16 kwashiorkor, 14 marasmic, and 14 control children. The results showed that the reduction in total postheparin lipolytic activity in kwashiorkor was in the activity of hepatic origin. In marasmus, the total postheparin lipolytic activity, hepatic and extrahepatic activities, were within normal range. The was no evidence for the presence of inhibitors of postheparin lipolytic activity in the serum of kwashiorkor or marasmic children. Fasting serum-free fatty acid level was significantly elevated in kwashiorkor, while the level in marasmus was not significantly different from control value. The serum triglyceride levels in both conditions showed no significant differences from the control value. These findings suggest that the defective production of hepatic lipoprotein lipase, as well as increased influx of free fatty acid into the liver, could account for the accumulation of fat in the liver of kwashiorkor and not in that of marasmic children.

Intestinal disaccharidases in malnourished infant rats.

In infants suffering from protein-calorie malnutrition, the decreased intestinal mucosal lactase specific activity could be due either to the protein-calorie malnutrition or to the commonly associated enteritis (viral or bacterial) and intestinal parasites. We studied intestinal mucosal disaccharidase (lactase, sucrase, and maltase) specific activity in suckling (1 and 2 wk old), weanling (3 wk old), and postweaning (4 and 6 wk old) control and growth-retarded (malnourished) rats. Growth retardation was induced by feeding mother rats and postweaning rats a diet deficient in protein. In the malnourished rats, with few exceptions, specific activity of the disaccharidases in the intestinal mucosa were similar to those in the corresponding control groups of rats. However, because of marked mucosal atrophy total intestinal mucosal disaccharidase activities were more than 50% lower in the malnourished rats. These findings suggest that the specific activity of the intestinal mucosal disaccharidases is not affected by malnutrition per se.

Biotin supplementation affects lymphocyte carboxylases and plasma biotin in severe protein-energy malnutrition.

We studied the effect of a supplement of biotin (10 mg/d) or a placebo under double-blind conditions on plasma biotin concentrations and lymphocyte propionyl CoA carboxylase (PCC) and pyruvate carboxylase (PC) in 22 children with severe protein-energy malnutrition (PEM) (5 with kwashiorkor, 10 with marasmus, and 7 "sugar babies"). There were significant differences between the malnourished and control subjects only for PCC, although not among the three PEM types. Six of the patients had both PC and PCC activities below the lowest of the normal control subjects; there was no correlation between biotin concentrations and carboxylase activities in individual patients. In response to biotin supplementation, the greatest change in lymphocyte carboxylase activities was detected in patients who had abnormally decreased initial carboxylase activities, but the response was not related to initial plasma biotin concentration. These results indicate that these enzyme deficiencies are the result of a nutritionally determined biotin deficiency, that carboxylases and especially PCC are better indicators of the biotin status in individual patients than is the plasma biotin concentration, and that in some malnourished patients biotin deficiency may be rate-limiting in their nutritional homeostasis.

Erythrocyte membrane Na+ and K+ activated adenosine triphosphatase in protein-calorie malnutrition.

Total, ouabain insensitive and ouabain sensitive Na+ and K+ adenosine triphosphatase activity in the erythrocyte membrane of protein-calorie malnourished children with marasmus and kwashiorkor was compared with the enzyme activity in apparently healthy children (normals). Na+ and K+ contents of erythrocyte and plasma were also determined in these patients. Specific activity (units per milligram of membrane protein) of ouabain sensitive Na+ and K+ adenosine triphosphatase was significantly higher in erythrocyte membrane preparations from children with kwashiorkor but not from children with marasmus. After 4 to 5 weeks of treatment with diets sufficient in protein and calories the specific activity of the enzyme was lower as compared to that on admission. Erythrocyte and plasma Na+ content (microgram/10(6) cells and microgram per milliliter of plasma) in children with kwashiorkor were not different from those in normal children, however, there was reduction in K+ content of erythrocytes and plasma of these children. After treatment, erythrocyte Na+ and K+ and plasma K+ in children with kwashiorkor increased significantly. In marasmic children erythrocyte Na+ was higher as compared to normal but there were no differences in K+ content of either eyrthrocytes or plasma in these children.

Effect of hypoenergetic feeding and refeeding on muscle and mononuclear cell activities of mitochondrial complexes I--IV in enterally fed rats.

BACKGROUND: Previous studies suggested that cell energetics are altered by malnutrition. OBJECTIVE: We hypothesized that nutritional manipulations influence mitochondrial enzyme activities of the electron transport chain in both skeletal muscle and blood mononuclear cells. DESIGN: After a gastrostomy tube was inserted, 44 rats were randomly assigned to 1 of 4 experimental groups: control fed (CF; 364 kJ/d for 7 d), hypoenergetic fed (HF; 92 kJ/d for 7 d), hypoenergetic protein refed (HPR; 92 kJ/d for 7 d and then 129 kJ/d for 1 d), and hypoenergetic glucose refed (HGR; 92 kJ/d for 7 d and then 129 kJ/d for 1 d). The protein and glucose contents of the liquid formulas were different for the HPR and HGR groups. After mitochondria were isolated from the soleus muscle, the activities of complexes I--IV were measured spectrophotometrically. Because of the lack of available tissue, only the activity of complex I was measured in the mononuclear cell extract. RESULTS: The recovery of complex activities in the CF and HF groups was not significantly different in the mitochondrial fraction of the soleus muscle. Compared with that in the CF group, the activities of complexes I--III in the mitochondrial fraction of the soleus muscle and the activity of complex I in mononuclear cells were significantly lower in the HF group. The activities of complexes I--III in the mitochondrial fraction of the soleus muscle and the activity of complex I in mononuclear cells were significantly higher in the HPR than in the HF group. The activity of complex IV was generally not affected by nutritional manipulations. CONCLUSION: Malnutrition decreases activities of mitochondrial complexes, which are restored by protein but not glucose refeeding.

Prevention of c-Jun/activator protein-1 activation and microsomal epoxide hydrolase induction in the rat liver by cysteine during protein-calorie malnutrition.

Protein-calorie malnutrition (PCM), a major global health problem, arises during protein and/or energy deficit due to disease and nutritional inadequacy. To date, cellular adaptive responses and gene expression associated with PCM remain poorly understood. In view of the primary role of the liver in energy conversion, the present study was designed to investigate changes in hepatic morphology and molecular alterations during PCM. PCM caused marked decreases in the cytoplasmic eosinophilic content and nuclear shrinkage in the hepatocytes with a decrease in glutathione content. The nuclear activator protein-1 (AP-1) complex was activated in the liver of PCM rats. AP-1-binding activity of nuclear extracts produced from PCM rats was reduced by the presence of anti-c-Jun antibody. Microsomal epoxide hydrolase (mEH), a phase II detoxifying enzyme, was 4-fold induced, with a 20-fold increase in the mRNA level during PCM. In contrast to the PCM-induced changes in hepatic morphology, PCM rats supplemented with cysteine showed an increase in the GSH level and well-preserved hepatic structures with mild fat degeneration. Cysteine supplementation inhibited the activation of AP-1 and the induction of mEH in PCM rats. These results provided evidence: (i) that PCM alters liver morphology with a decrease in the glutathione level; (ii) that cysteine may serve as a key element responsible for preserving hepatic morphology and maintaining the glutathione level; and (iii) that cysteine was active in preventing the activation of AP-1 and mEH induction in the liver during PCM.

Rat liver mitochondrial cytochrome c oxidase and cytochromes in experimental protein-energy malnutrition.

Cytochrome c oxidase activity and cytochromes b, (c+c1) and a(+a3) concentrations were determined in liver mitochondria from rats fed the following diets: controls (group 1) fed ad libitum, energy-restricted (group 2) and protein-deficient (group 3). The animals were fed for two time intervals, 3--5 and 7--9 weeks. At 3--5 weeks, the cytochrome oxidase specific activity (nmol cytochrome c oxidized/mg protein/min) and cytochrome concentrations (nmol/mg protein) were not different in groups 2 and 3 as compared to group 1. At 7--9 weeks, the cytochrome oxidase specific activity and concentrations of cytochromes b, (c+c1) and a(+a3) were significantly reduced in group 2 rats as compared to well-fed controls. The Michaelis-Menten constant, Km apparent for ferrocytochrome c, was significantly higher in group 2 as compared to group 1. In group 3 rats, cytochrome oxidase specific activity and cytochrome b, a(+a3) concentrations were not different from group 1 at 7-9 weeks. However, cytochrome (c+c1) concentration was higher in group 3, resulting in an elevated ratio of cytochrome (c+c1) to cytochrome a(+a3) as compared to groups 1 and 2.

The activity of serum ribonuclease in protein-energy malnutrition.

Serum alkaline ribonuclease activity and serum albumin concentration were determined in 25 normal children and 59 children with protein-energy malnutrition. The increase in serum ribonuclease was marked in marasmus and marasmic kwashiorkor. The ribonuclease activity dropped significantly after two weeks of treatment and returned to normal by four weeks. In kwashiorkor, serum ribonuclease activity was significantly lower than control and returned to normal after four weeks of treatment. These findings support previous observations that the serum ribonuclease is a good criterion of the nutritional status and indicates that the enzyme activity, particularly when related to serum albumin, is a good prognostic index in this respect.

Changes in serum lysosomal hydrolases in marasmus.

The activities of several lysosomal hydrolases including beta-glucuronidase, acid phosphatase and hexosaminidase were compared in serum from 19 well-nourished subjects and 13 children (age 5--24 months) who were suffering from marasmus. The marasmic children exhibited growth retardation and muscle wastage but had normal serum protein values and absence of psychomotor retardation or oedema. Significant changes were observed in serum beta-glucuronidase and acid phosphatase activities. Compared to the control group, serum beta-glucuronidase (determined at pH 4.5 using the fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucuronide) was 2.3-fold higher (p less than 0.001) in the marasmic children. In contrast, serum acid phosphatase values were approximately 50% lower (p less than 0.01) in the marasmic population. Serum hexosaminidase values in the two groups under study were not significantly different. Determination of the beta-glucuronidase to acid phosphatase ratio permitted effective discrimination (p less than 0.001) of serum from normal and protein-calorie malnourished children. The finding that the elevated value of the beta-glucuronidase : acid phosphatase ratio (0.64--1.37) decreased to within the normal range of values (0.10--0.43) after nutritional rehabilitation of several marasmic cases indicates that the determination of serum lysosomal hydrolases using fluorogenic substrates might provide a rapid and sensitive quantitative method for objectively evaluating the status of protein-calorie malnourished children and their responsiveness to nutritional therapy.

Ribonuclease activity in plasma and leucocytes of malnourished children.

RNAase activity was measured in plasma and leucocytes of manourished children. In subjects suffering from kwashiorkor, alk. RNAase levels in plasma and leucocytes were markedly raised and they returned to normal after therapy. However, the enzyme activity was not altered in undernourished children. These data indicate that circulating level of alkaline. RNAase is not a useful parameter for detecting milder grades of protein-calorie malnutrition (PCM).

Protein-calorie malnutrition: a study of red blood cell and serum enzymes during and after crisis.

This paper reports a study of changes in red blood cell enzymes and some serum parameters during and after treatment of protein-calorie malnutrition. The red cell GSH levels were low during the crisis, together with the levels of GSSG:NADPH reductase, GSH:H2O2 peroxidase, aspartate aminotransferase and alanine aminotransferase. After treatment the levels of all these enzymes increased significantly to normal values. Of the serum parameters investigated, significant reduction in the activity of the enzymes cholinesterase, catecholamine oxidase, total proteins, albumin, urea and electrolytes were obvious, and returned to normal values after treatment. Ceruloplasmin activity remained low even after three weeks' treatment and could not be related to copper levels. The results are discussed in relation to anemia and liver damage that may accompany the syndrome.