Effect of a high fat intake on protein utilization during ontogenetic development

Male rats (Wistar strain, Velaz, Prague) aged 30, 90 and 150 days were fed 14 days ad libitum on a high fat diet (containing 40% margarine) and their growth (PER, NPR) and utilization (NPU, LPU) parameters of protein (casein) biological value were compared with those of animals given a standard gel containing 10% margarine (the diets were isoenergetic). The course of gluconeogenesis in their liver was also determined by measuring phosphoenolpyruvate carboxykinase (PEPCK) activity. A high fat intake had a negative effect on the growth parameters of protein biological value, PER and NPR, in all three age groups and on the utilization parameters NPU and LPU in 30- and 90-day-old rats. The nonsignificant increase in NPU and LPU in the oldest animals was evidently related to the equal protein intake compared with the control, indicating that proteins need to be utilized more economically in the presence of a raised fat intake; owing to their far lower fat intake compared with 90-day-old animals on a high fat diet, the fat had a less negative effect on their protein utilization than in the younger age group. The negative effect of a high fat intake was confirmed by high activation of gluconeogenesis in 30- and 90-day-old animals and by raised phosphoenolpyruvate carboxykinase activity in 150-day-old rats, in which the supposition that gluconeogenesis would be highly activated if the diet were administered for a period other than 14 days cannot be ignored. The biological and biochemical methods employed in this study can be used with a wider perceptual range of dietary nutrients to determine optimum nutrient values under different conditions.     

Determination of the optimum proportion of saccharides in the diet of adult rats

SPF male Wistar rats weighing 250-260 g and aged 90 days were fed 14 days on diets with a constant 10% protein (casein) content, a constant 11% fat (margarine) content and mounting saccharide (rice starch: sugar: potato starch - 6.4: 1.2: 1) contents of 31, 36, 41, 46, 51, 56, 61 and 66%. Protein intake and the body and liver nitrogen values were used to determine the utilization parameters of protein biological value, i.e. NPU (body) and LPU (liver), for the individual diets. Liver gluconeogenesis was also studied by measuring specific phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1.6-diphosphatase (FDP-ase) activity. On the basis of linearity between the growth parameter NPR and protein and saccharide intake we determined the reciprocal relationship of the intake of the two nutrients and used it to compute the optimum saccharide concentration for the diet. The 51% saccharide diet gave the best protein utilization (the maximum (net) protein utilization value) in the 90-day-old rat organism. This was confirmed by the course of gluconeogenesis, which was significantly activated in the presence of 31-46% saccharide diets. By substituting the optimum protein intake in the reciprocal saccharide-protein intake relationship we obtained the optimum saccharide intake, which corresponded to a 49% concentration in the diet. With its use of a biological, biochemical and computation method, the study is a contribution to the determination of optimum nutrient values.     

Influence of the time of intake of a high fat diet on gluconeogenesis

Male Wistar rats aged 75 and 150 days were given high fat diet (36.5 weight % and 30 weight % fat) over a period of 14 days. The growth (PER, NPR) and utilization (NPU, LPU) parameters of protein biological value and liver phosphoenolpyruvate carboxykinase (PEPCK) activity were determined. In another experiment, the time dependence of liver gluconeogenesis enzyme (PEPCK and fructose-1,6-diphosphatase /FDP-ase/) and transaminase (alanine and aspartate aminotransferase /ALT, AST/) activities during 24 days' administration of the diet were determined. A 14 days' high fat intake had a negative effect on protein utilization in the organism of 75- and 150-day-old animals, which was more pronounced in the younger age group (a bigger drop in net protein utilization /NPU/ and greater stimulation of PEPCK activity). In 150-day-old animals the negative effect of a high fat intake was already manifested on the 6th to 10th day of the diet to the same degree as in the younger animals on the 14th day, as seen from the increase in all the enzyme activities. The paper presents findings on differences in the degree of the negative effect of a high fat intake on protein utilization with reference to age.     

The relationship between protein retention and energy requirements in rats of different ages

Male Wistar rats aged 30, 75 and 150 days were fed for 14 days ad libitum on diets with an optimum protein content (15% for 30-day-old, 12.5% for 75-day-old and 10% for 150-day-old animals) and a mounting fat content (from 5 to 40%), supplemented by saccharides (from 76 to 41%). Net protein utilization was determined for each of the diets from the body nitrogen and protein intake values. Protein retention values were determined from protein intake on the basis of net protein utilization (NPU). Energy intake was computed from fat and saccharide intake, using energy coefficients. The optimum fat content of the diet, evaluated from the maximum protein retention value per day and the minimum amount of energy needed for the retention of 1 g protein, is 30% at 30 days, 15% at 75 days and 10% at 150 days. Protein retention per kg body weight falls with advancing age--mildly at 75 days compared with 30 days, but markedly at 150 days. From their smaller weight increments and NPU values and also from their lower protein retention, 150-day-old animals are characterized by slower growth and higher protein requirements for maintenance of their organism likewise demonstrated by the growth parameter net protein ratio (NPR). Energy requirements for total protein retention/day per kg body weight diminish with age. In old age a small amount of energy is needed only for the maintenance of body functions. This study contributes to the expression of the interrelationship of energy requirements and protein retention.     

Determination of the optimum dose of fats in the food of rats of different ages

Over a 14-day period, male SPF Wistar rats with an initial weight of 60, 200 and 250 g (ages 30, 75 and 90 days) were given diets with a constant protein content (10% casein) and a mounting fat content (10, 15, 20, 25, 30, 40 and 50% margarine, at the expense of the saccharides in the standard diet). The utilization parameter of protein biological value (NPU) for the various diets was determined from protein intake and the body nitrogen values. The reciprocal relation of the intake of the two nutrients was determined on the basis of linearity between th growth parameter NPR and protein and fat intake and, after substituting the optimum protein intake, was used to compute the optimum fat content of the diet. From the aspect of the maximum NPU value the optimum fat contents were 30% for 30-day-old, 15% for 75-day-old and 10% for 90-day-old animals. In all three age groups (according to the growth curve for the standard Larsen diet animals with an equal growth rate of 3 g/day), protein utilization under optimum nutritional conditions was the same. The optimum fat content of the diet computed from the reciprocal relationship of fat and protein intake is in agreement with the value obtained by the biological method - 29.4% (49.5 energy %) for 30-day-old animals, 15,8% (30.4% of the total energy value of the diet) for 75-day-old animals and 11.83% (23.7% of total energy) for 90-day-old rats. By using a biological and a mathematical method, this study contributes to the determination of optimum physiological doses of nutrients.     

Determination of the optimum doses of nutrients in a diet.

For 14 days, SPF male Wistar rats with an initial weight of 60 g were given isocaloric diets (1.7 MJ/100 g diet) containing 10% protein (casein) and 5, 10, 25 and 40% fat (margarine). Two utilization parameters of the protein biological value--net protein utilization (NPU) and liver protein utilization (LPU)--were determined from protein intake and body and liver nitrogen. These results were supplemented by a study of the course of the antithetical processes of gluconeogenesis and glycolysis, of the citric acid cycle and transamination processes and of the liver and muscle amino acid spectrum. A high (40%) fat diet significantly reduced the protein biological value parameters NPU and LPU and liver and muscle amino acid values, stimulated gluconeogenesis and inhibited glycolysis and the citric acid cycle, together with associated transamination processes in the liver. Activation of these processes in the muscles provided substrates for increased gluconeogenesis. The negative effect of a low fat + high carbohydrate diet was less marked. The optimum diet for weaned rats is thus a diet containing 10% protein and 10--25% fat. The study, which submits several possible ways of determining optimum nutrient intakes under different physiological conditions shows that diets with more detailed nutrient concentrations should be used.     

Age dependence of the utilization of different quality proteins in animal experiments.

Male rats aged 45, 85, 145 and 270 days (daily body mass increments on an optimal diet containing casein were 6.73, 2.88, 0.53 and 0.31 g respectively) were fed 15 days ad libitum on a diet with a nutrient content physiological for their age, in which the protein source was milk casein (ratio of essential to nonessential amino acids E/N = 0.79, compensation coefficient K = 14) or wheat gluten (E/N = 0.30, K = -8). In the case of gluten, net protein utilization (NPU) fell markedly in rapidly growing animals aged 45 and 85 days (33 and 30% more than with casein), indicating that without essential amino acid compensation, gluten is inadequate for animals of this age, whose organism requires fully ensured proteosynthesis for growth and development. In adolescence and adulthood (145 and 270 days), the utilization of proteins is not dependent on their quality (the decrease in NPU 13 and 12%--is nonsignificant). That means that a smaller amount of essential amino acids, including the limiting amino acid in uncompensated protein, is sufficient for the maintenance and renewal of organs and tissues, i.e. for proteosynthesis. The activation of gluconeogenesis (phosphoenolpyruvate carboxykinase activity in the liver) after the intake of plant protein confirms the effect of proteins on catabolic processes.     

Erythrocytes and age. Disintegration of erythrocytes by brilliant cresyl blue in correlation to age

The author describes changes in the disintegration of erythrocytes by brilliant cresyl blue in correlation to age, in rats aged 21, 42, 90-105, 340-360 and 690-720 days. The erythrocytes were incubated for 4 hours in an isotonic NaCl solution, in Krebs-Ringer solution and in each of these solutions plus brilliant cresyl blue. Disintegration in plain NaCl solution was found to be the greatest in the case of erythrocytes from 690- to 720-day-old rats. In the same solution plus brilliant cresyl blue, the rate of disintegration was very high in 21-day-old, 42-day-old and 690- to 720-day-old animals; at 90-105 days it was lower and at 340-360 days it was the lowest. Disintegration of erythrocytes in plain Krebs-Ringer solution was the lowest at 21 and 42 days; in the other age groups it was slightly higher. On adding brilliant cresyl blue, the rate of disintegration rose significantly in 21-, 42- and 690- to 720-day-old animals; at 90-105 days and 340-360 days it was no different from disintegration in plain Krebs-Ringer solution. It can be seen from the results that the rate of brilliant cresyl blue-induced erythrocyte disintegration is dependent on the age of the animals from which the erythrocytes are taken.     

Gluconeogenesis and P-enolpyruvate carboxykinase in liver and kidney of long-term fasted quails

The activity of cytoplasmic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) in kidney and liver, and in vivo gluconeogenic activity, were determined during different phases of prolonged fasting in quails. The fasting-induced changes in the activity of kidney cytoplasmic PEPCK were positively correlated with the changes in gluconeogenesis. Both activities increased at the initial phase (I) of fasting to levels 65% to 100% higher than fed values, and decreased during the protein-sparing period (phase II), although remaining higher than in fed birds. At the catabolic final phase (III) both kidney cytoplasmic PEPCK activity and gluconeogenesis increased markedly, attaining levels 115% to 150% higher than fed values. The activity of liver cytoplasmic PEPCK, present in appreciable amounts in quails, did not change during phases I and II of fasting, but increased to levels 60% higher than fed values at the final phase (III). Plasma glucose levels at phase III did not differ significantly from those at phases I and II. In both kidney and liver the activity of the mitochondrial PEPCK was not significantly affected by fasting. The data suggest that the kidney cytoplasmic PEPCK is the main enzyme responsible for gluconeogenesis adjustments during food deprivation in quails, and that this function is complemented at the final phase by enzyme present in liver cytosol. Accepted: 14 April 2000     

Effects of dieldrin on hepatic carbohydrate metabolism in the suckling and adult rat

Hepatic carbohydrate metabolism was studied in adult and suckling rats given age-specific LD50 doses of dieldrin po. These doses in 5-, 10-, and 60-day-old Wistar rats were 38, 28, and 63 mg/kg, respectively. Plasma glucose and free fatty acids (FFA), and hepatic glycogen, phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-diphosphatase (FDP), and glucose-6-phosphatase (G6P) were measured 1 and 3 h after administration of the insecticide. Plasma glucose concentrations were elevated (17%) in some 5-day-old rats after 1 h and in all adults after 1 and 3 h (45 and 30%, respectively). Plasma FFA concentrations were decreased (9%) in the 5-day-old rat 1 h after dieldrin. Hepatic glycogen content was reduced in both 5- and 10-day-old pups at 1 hour (22 and 17%, respectively). Hepatic FDP activity was elevated in the 5-day-old rat at 1 h (17%) and was decreased (10%) in the 10-day-old rat at 3 h. Hepatic PEPCK activity was increased in adult animals by 30% 1 h after dieldrin. Furthermore, PEPCK activity was increased at 3 h in rats of all ages (76%, 5-day-old pup; 115%, 10-day-old pup; 56%, 60-day-old adult). Hepatic G6P activity was unaltered by dieldrin. Thus only the activity of hepatic PEPCK is consistently elevated by dieldrin exposure. However, this enhanced PEPCK activity is associated with dieldrin-induced hyperglycemia only in the adult rat.     

Determination of optimum fat intake

In a 14-day experiment, adult rats weighting 250-260 g were given ad libitum diets with a constant protein content [casein - 10 %] and a mounting fat content [margarine - 0, 10, 20, 30 and 40 % - added at the expense of carbohydrates]. The growth parameters of protein biological value - PER and NPR - were determined and together with weight increments in correlation to protein an fat intake [a linear correlation] they were used on the basis of their regression equations to calculate the reciprocal relationships of fat and protein intake [for equality y]. By substituting the optimum protein intake [43.3 g/14 days - according to NPU], the optimum fat intake for adult rats was found to correspond to dietary fat concentrations of 9.72 % [y = weight increments], 12,82 % [y = PER] and 11.83 % [y = NPR]. These results wee verified in a biological experiment in which adult animals, for 14 days, were given a limited amount [31 g/animal per day] of diets containing 10 % protein [casein] an d9, 10, 11, 12, and 13 % fat [margarine - at the expense of carbohydrates] with the aim of studying the effect of a mounting fat intake on optimum protein intake [43.4 g/14 days]. The correctness of the mathematical method for determining the optimum fat intake was verified by a biological method [the optimum net protein utilization , NPU, which is highest in diets containing 11 % fats, i.e. 22.5 % of the total energy value of the diet]. It was also found that this method, both along and in conjunction with other methods [especially the one using the NPR parameter], was suitable for the determination of optimum nutrient values.     

Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic energy metabolism

We used an allelogenic Cre/loxP gene targeting strategy in mice to determine the role of cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in hepatic energy metabolism. Mice that lack this enzyme die within 3 days of birth, while mice with at least a 90% global reduction of PEPCK, or a liver-specific knockout of PEPCK, are viable. Surprisingly, in both cases these animals remain euglycemic after a 24-h fast. However, mice without hepatic PEPCK develop hepatic steatosis after fasting despite up-regulation of a variety of genes encoding free fatty acid-oxidizing enzymes. Also, marked alterations in the expression of hepatic genes involved in energy metabolism occur in the absence of any changes in plasma hormone concentrations. Given that a ninefold elevation of the hepatic malate concentration occurs in the liver-specific PEPCK knockout mice, we suggest that one or more intermediary metabolites may directly regulate expression of the affected genes. Thus, hepatic PEPCK may function more as an integrator of hepatic energy metabolism than as a determinant of gluconeogenesis.     

Cytosolic phosphoenolpyruvate carboxykinase does not solely control the rate of hepatic gluconeogenesis in the intact mouse liver

When dietary carbohydrate is unavailable, glucose required to support metabolism in vital tissues is generated via gluconeogenesis in the liver. Expression of phosphoenolpyruvate carboxykinase (PEPCK), commonly considered the control point for liver gluconeogenesis, is normally regulated by circulating hormones to match systemic glucose demand. However, this regulation fails in diabetes. Because other molecular and metabolic factors can also influence gluconeogenesis, the explicit role of PEPCK protein content in the control of gluconeogenesis was unclear. In this study, metabolic control of liver gluconeogenesis was quantified in groups of mice with varying PEPCK protein content. Surprisingly, livers with a 90% reduction in PEPCK content showed only a approximately 40% reduction in gluconeogenic flux, indicating a lower than expected capacity for PEPCK protein content to control gluconeogenesis. However, PEPCK flux correlated tightly with TCA cycle activity, suggesting that under some conditions in mice, PEPCK expression must coordinate with hepatic energy metabolism to control gluconeogenesis.     

The development of the acinar heterotopic pattern of phosphoenolpyruvate carboxykinase activity in the newborn rat

The postnatal appearance of phosphoenolpyruvate carboxykinase activity (PEPCK) and acinar heterotopy was investigated in newborn rats aged 2 h, 12 h, 24 h and 3 days, as well as in juvenile rats aged 25 days. The livers showed an almost homogeneous distribution of activity along the sinusoidal length at the beginning of extrauterine life where energy needs are greatest. Compared to rats aged 2 h, the PEPCK activity was higher in the livers from rats aged 12 h. The increase in activity was most pronounced in the intermediary zone. After 24 h of extrauterine life the activity decreased again creating a homogeneous acinar activity pattern. By day 3 activity had increased in the periportal zone, while decreasing in the perivenous zone, resulting in a periportal to perivenous gradient. By day 25 total activity had reached highest values both in males and females, due to a relatively high perivenous activity. The more prominent acinar gradient corresponded approximately to the one seen in adult animals.     

The development of the acinar heterotopic pattern of phosphoenolpyruvate carboxykinase activity in the newborn rat

Summary The postnatal appearance of phosphoenolpyruvate carboxykinase activity (PEPCK) and acinar heterotopy was investigated in newborn rats aged 2 h, 12 h, 24 h and 3 days, as well as in juvenile rats aged 25 days. The livers showed an almost homogeneous distribution of activity along the sinusoidal length at the beginning of extrauterine life where energy needs are greatest. Compared to rats aged 2 h, the PEPCK activity was higher in the livers from rats aged 12 h. The increase in activity was most pronounced in the intermediary zone. After 24 h of extrauterine life the activity decreased again creating a homogeneous acinar activity pattern. By day 3 activity had increased in the periportal zone, while decreasing in the perivenous zone, resulting in a periportal to perivenous gradient. By day 25 total activity had reached highest values both in males and females, due to a relatively high perivenous activity. The more prominent acinar gradient corresponded approximately to the one seen in adult animals.     

Osteocalcin is vitamin D-dependent during the perinatal period in the rat

The vitamin D-dependence of renal calbindin D-28K and osteocalcin during the perinatal period was studied in fetuses (days 18 and 21) and neonates (days 2, 12, 17 and 22) of rats fed either a standard diet (0.85% Ca-0.7% P; "high Ca-P diet" rats) or a mildly Ca-P restricted diet (0.2% Ca-0.2% P; "low Ca-P diet" rats). Body weight and plasma calcium levels were identical in both groups. Plasma 1,25(OH)2D concentrations were markedly higher in the low Ca-P diet rats at all stages of fetal and neonatal life (in 22-day-old neonates: 536 +/- 58 pg/ml versus 126 +/- 12 pg/ml). 1,25(OH)2D concentrations increased between day 18 and 21 of fetal life, remained constant between day 21 of fetal and day 12 of neonatal life, and increased sharply between day 12 and 17 in both groups; after day 17, 1,25(OH)2D concentrations increased further in pups fed the low Ca-P diet. Renal calbindin D-28K reached peak concentrations on day 12 of neonatal life; calbindin D-28K levels were similar in the high and low Ca-P diet rats at all stages of perinatal development. Plasma osteocalcin levels increased steadily during the perinatal period; at most stages of perinatal life, and already from the fetal period was osteocalcin higher in the low Ca-P diet rats than in the high Ca-P diet rats (in 22-day-old pups: 1106 +/- 47 ng/ml versus 429 +/- 14 ng/ml). Femoral osteocalcin concentrations were also increased in fetal and early neonatal (days 2 and 12) low Ca-P diet rats, while the femoral calcium content and concentration of these rats were decreased in the late neonatal period (days 12, 17 and 22). These studies indicate that osteocalcin is vitamin D-dependent in the fetal and neonatal rat.     

Developmental changes in audiogenic epilepsy and myoclonus in KM rats

The reaction to a single sound stimulation (a bell) applied daily and the time of the first appearance of myoclonus were recorded in Krushinsky-Molodkina (KM) rats of different ages (30, 60, and 150-200 days). The audiogenic seizures in adult animals were of short latency and always resulted in tonic convulsions, which were stable in their patten. In contrast, in young animals (in particular, in 30-day-old rats), the seizures were less pronounced and unstable. The daily sound stimulation produced a gradual development of myoclonic seizures in young and adult rats. In old rats, the stable myoclonus appeared on the 17th day of stimulation, and in 30-day-old animals the myoclonus was recorded on the 26th day. In 60-day-old rats the myoclonus appeared for the first time on the 20th day of stimulation. It is suggested that these age differences can be explained by the btain immaturity (in particular, of neurotransmitter system) in young animals.     

Moderate prenatal carbon monoxide exposure produces persistent, and apparently permanent, memory deficits in rats

The effects of moderate (150 +/- 2 ppm) prenatal carbon monoxide (CO) exposure (maternal HbCO concentrations of 15.6 +/- 1.1%) on learning and memory were assessed in young and aged adult rats using a two-way active avoidance paradigm. In experiment 1, the prenatal CO-exposed rats at 120 days of age acquired a conditioned avoidance response equally well as control animals in a 100-trial session. However, following a 24-hr interval the CO-exposed rats failed to demonstrate significant retention of the task as indicated by the absence of significant improvement in performance over the indicated by the absence of significant improvement in performance over the previous day; control subjects did show significant retention. In experiment 2, in which 120-day-old animals received 50 training trials per day until a criterion of ten consecutive avoidance responses was met, the prenatal CO-exposed subjects again acquired the task as well as control animals. When tested for retention 28 days later, a significant memory impairment was again observed in terms of trials required to reattain the avoidance criterion as well as in total percent avoidance responding. In neither experiment did an analysis of initial or average latency to escape the footshock stimulus reveal any significant alterations. These latter results suggest that the observed performance impairment reflected a memory deficit and not a disruption of sensory, motor, or motivational factors. In experiment 3, prenatal CO-exposed rats approximately 1 year of age (300-360 days of age) showed impairment relative to air-exposed controls in both the original learning and retention of the two-way avoidance response. Again, however, there was no evidence for alterations in performance factors per se. Collectively these data indicate that while young adult rats prenatally exposed to 150 ppm CO demonstrate an associative deficit restricted to memory impairment, aged adults similarly exposed during the prenatal period display a more pronounced deficit similar to that recently reported for animals tested as juveniles. The importance of parametric manipulations in uncovering long-term toxicity is also discussed.     

Assessment of toxicological effects of mancozeb in male rats after chronic exposure

Mancozeb, an ethylenebisdithiocarbamate fungicide was administered orally to male rats at doses 0, 500, 1000 and 1500 mg/kg/day for 90, 180 and 360 days produced dose dependent signs of poisoning, loss in body weight gain and mortality. However the signs of toxicity and mortality were more pronounced initially at 0-90 days as compared to 90-360 days of treatment period. A significant increase in the relative weight of liver and slight decrease in the kidney weight were observed in animals exposed to mancozeb (1000 and 1500 mg/kg/day) for 180 and 360 days associated with pathomorphological changes in liver, brain and kidney. Mancozeb has produced significant enzymatic changes in the activities of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) throughout the period of study in a dose dependent manner. The alterations in the activity of enzymes associated with pathomorphological changes suggest that the chronic exposure of mancozeb produced significant toxicological effects in rats.