Age-related changes of NA(+), K(+) - ATPase, Ca(+2) - ATPase and Mg(+2) - ATPase activities in rat brain synaptosomes.

Cerebral metabolism of glucose, one of the determinants of tissue ATP level, is crucial for central nervous system function. The activity of P-type pumps, namely Na(+), K(+) - ATPase, Ca(+2) - ATPase and Mg (+2) - ATPase were examined in brain synaptosomes of 5 - day, 3 - month and 18 - month - old rats to determine if changes in enzyme activity related to aging are potentially associated with alterations in glucose homeostasis. Activities of all the ATPases studied in isolated brain synaptosomes were expressed in micromol of Pi liberated from ATP by 1 mg of synaptosome protein during one hour. Serum glucose concentration was measured by the glucose oxidase method and insulin level was estimated by the RIA. Our results demonstrate that 18 - month - old rats are characterized by hyperglycemia and hyperinsulinemia. Their serum glucose concentration was significantly increased approx. 62.3% and 135.8 % as compared to 3 - month - old rats and 5 - day, newborn rats, respectively. An enormous increase in serum insulin concentration in the old, hyperglycemic rats was observed concomitantly. As a result of these changes the insulin - to - glucose ratio in the old rats was greatly increased approx. (270% and 230%) compared to young, mature and newborn rats. Hyperglycemia and hyperinsulinemia occurring in the old rats, had a different impact on activities of the ATPases tested. Our results have revealed that Na(+), K(+) - ATPase activity remains almost unchanged with age, the activity of Ca(+2) - ATPase decreases, whereas that of Mg(+2) - ATPase increases significantly in old, insulin resistant rats. In conclusion it seems that changes in activity of different P - type pumps may differ with aging and that adaptation of specific ATPases to internal environment alterations is not identical.

Effect of hypothermia on insulin-receptor interaction in different rat tissues.

Experimental hypothermia caused extensive changes in the number of both classes of insulin receptors in different rat tissues. In the liver, the number of high affinity insulin receptors (HAIRs) decreased by 50% (from 25.3 to 12.6 fmol/mg membrane protein), whereas number of low affinity insulin receptors (LAIRs) was almost unchanged in comparison to normothermic animals (5.63 and 4.39 pmol/mg, respectively). In the adipose tissue, number of both classes was reduced--HAIRs by 81% (from 24.0 to 4.50 fmol/mg) and LAIRs by 92% (from 16.0 to 1.29 pmol/mg). In the skeletal muscle, capacity of HAIRs was not changed (16.2 and 19.3 fmol/mg in normo- and hypothermic animals, respectively), whereas number of LAIRs increased by 150% (from 6.65 to 16.6 pmol/mg). Hypothermic rats also showed lower amount (by 85%) of LAIRs in the heart muscle (9.37 and 1.43 pmol/mg in control and experimental animals, respectively). Simultaneously, no significant changes were found in HAIRs (16.3 and 11.9 fmol/mg, respectively) and LAIRs (4.43 and 3.88 pmol/mg, respectively) in the brain. These differences in insulin receptors responses to hypothermia may reflect different physiological role of insulin in the regulation of target cell metabolism and/or the differences in tissue distribution of the insulin receptor isoforms.

Insulin receptor--structural and functional characteristics.

The insulin receptor is a complex multifunctional protein located on the cell surface within the fluid mosaic matrix of the plasma membrane. Its ability to bind insulin is determined by structural and spatial specificity, high affinity and reversibility. Insulin action at the cellular level can be considered in three stages. The first stage includes the insulin receptor itself, the insulin substrate (IRS-1) and the molecules that interact with IRS-1. The second stage is that of cascade phosphorylation and dephosphorylation. The third stage concerns the final biological effects of the insulin cascade. The biological specificity of insulin binding and heterogeneity of insulin action in different cells has been well documented. This review intends to summarize the available data of structural and functional characteristics of insulin receptor.

Age dependent changes of insulin receptors in rat tissues.

Aging is associated with insulin resistance but the exact molecular mechanism is still unknown. Tissue insulin resistance can be evoked by the decreased sensitivity to insulin, the decreased responsiveness to hormone or both. As the first step in insulin action is its binding to alfa subunits of the receptor we, therefore, studied the insulin binding kinetics in plasma membranes of the liver, heart and skeletal muscle in order to establish whether their ability to bind the hormone is altered with aging. Plasma membranes were prepared and purified according to Havrankowa and binding assay was performed using (125I)-iodoinsulin. The kinetic parameters of the hormone-receptor interaction were analysed by the method of Scatchard using the LIGAND-Pc v.3.1. computer program. The binding potency of insulin was calculated as IC50 using ALLFIT-Pc v.2.7. computer program. We have shown that there are striking differences in insulin binding kinetics in newborn and old rats, depending on kind of tissue tested. The liver plasma membranes ability for insulin binding, number of high (HAIR) and low (LAIR) affinity insulin receptors, values of the dissociation constants and products of association constants and number of insulin receptors, were almost the same, being not dependent on age of the rats. By contrast, there is less high affinity insulin receptors in skeletal muscle of the old animals. The most dramatic changes in insulin binding occur in the heart where both high and low affinity insulin receptors are greatly affected by aging. Our results indicate that the response of the three tissues tested to hyperglycemia and hyperinsulinemia, observed in the old rats, has not been identical and probably can be accounted for by the different distribution of insulin receptor isoforms in the liver, heart and skeletal muscles as shown recently by Vidal et al.

Characteristics of insulin receptor binding to various rat tissues.

The first step in insulin action is its specific binding to alpha-subunits of the receptor in the plasma membrane. Rats of Wistar strain are commonly used as laboratory animals but there are no data comparing insulin binding by various rat tissues. We studied the insulin binding kinetics in plasma membranes isolated from hearts, livers, brains, skeletal muscles, adipose tissue, thymus and testes in order to compare their ability to bind 125I-insulin and to test which membrane preparation is most useful and convenient for such a study. The dissociation constant (Kd) and the quantity of receptors measured as a binding capacity were determined by the Scatchard method using the LIGAND computer program whereas the binding potency of insulin was calculated as IC50 using the ALLFIT computer program. We also introduced the product of Ka x R50 (affinity constant multiplied by binding capacity) as an index which describes the functional features of insulin receptors taking into account both number of insulin receptors and their affinity. Taking all the parameters of insulin binding tested together we can conclude that the liver and, to some extent, adipose tissue may provide a useful model for studying hormone-receptor interaction. By contrast, to the group of rat tissues responding rather poorly to insulin belong thymus and testis.

Effect of hypothermia on the insulin-receptor interaction in skeletal muscle plasma membranes.

The aim of the study was to investigate the effect of hypothermia on (125J)-insulin binding to rat skeletal muscle membranes and to determine whether the decrease in blood insulin concentration could be related to changes in the number or in the affinity of insulin receptor sites according to the down-regulation theory. Rat skeletal muscle membranes were prepared from control, normothermic rats (Tr = 35.6 +/- 0.3 degree C) and hypothermic rats (Tr = 26.0 +/- 0.5 degree C) and purified according to Havrankowa. In order to determine the kinetic parameters of the hormone-receptor interaction the data from the competition binding studies were analysed by the method of Scatchard using the LIGAND Pc.v.3.1. computer program of Munson and Rodbard. We have shown that under hypothermic conditions insulin receptors number is significantly increased in specific hindlimb skeletal muscles but the changes take place mainly in the low affinity receptors class. The phenomenon probably results from the lack of spare high affinity insulin receptors in skeletal muscle as shown recently by Camps et al.

Effect of hypothermia on the insulin-receptor interaction in adipose plasma membranes.

The aim of the study was to compare the number and affinity of insulin receptors in adipose tissue of both normothermic and hypothermic rats. Plasma membranes from epididymal adipose tissue were prepared and purified according to Havrankowa and binding assay was performed using (125I)-iodoinsulin. The kinetic parameters of the hormone-receptor interaction were analysed by the method of Scatchard using the LIGAND--Pc v.3.1. computer program of Munson and Rodbard. The binding potency of insulin was calculated as IC50 using the ALLFIT--Pc v. 2.7. computer program. Maximum specific insulin binding to plasma membranes in adipose tissue was decreased in the hypothermic rats but half-maximum displacement of tracer insulin was similar in the normothermic and hypothermic group suggesting that reduced receptor numbers rather than reduced affinity accounted for the difference.

[Structure and function of F0F1-ATP-ase in procaryotes and eucaryotes]. / Budowa i funkcja F0F1-ATP-azy u Procaryota i Eucaryota.

The paper presents recent data on the structure and the mechanism of the action of F0F1-ATP-ase at the cellular level both in Procaryota and Eucaryota. The regulation of the synthesis and hydrolysis of ATP has also been discussed.

Effect of hypothermia on the insulin--receptor interaction in liver plasma membranes.

The aim of the study was to estimate the effect of hypothermia on (125J)-iodoinsulin binding to liver plasma membranes. Rat liver membranes were prepared from control, normothermic rats (Tr = 35.6 +/- 0.3 degrees C) and hypothermic rats (Tr = 26.5 +/- 0.9 degrees C) and purified according to Havrankowa. In addition, serum insulin and glucagon levels by means of RIA and glucose concentration using the glucose oxidase method were measured. Scatchard analysis was used to determine the kinetic parameters of the hormone receptor interaction. The data showed no significant differences in the affinity of the binding sites but indicated a decrease in receptor concentrations in liver plasma membranes from hypothermic rats. In contrast to changes in serum insulin level which was decreased by about 50% in hypothermic rats blood glucose concentrations did not significantly differ between the hypothermic and normothermic ones. Our results show that in hypothermic rats the hormonal adaptation operates on the level of the number of liver receptors whereas the insulin receptor affinity remains unaffected.

[Effect of insulin on temperature and metabolic responses in rats during normothermia and hypothermia]. / Wplyw insuliny na odpowiedz temperaturowa i metaboliczna u szczurów w przebiegu normotermii i hipotermi.

As shown in our previous study, hypothermia provokes a variety of hormonal changes including inhibition of insulin secretion and increase in blood serum glucagon level. According to Therminarias et al. the administration of exogenous insulin to dogs subjected to hypothermia causes a calorigenic effect by enhancing oxygen consumption and rising the intensity of shivering thermogenesis. The study was aimed at establishing whether exogenous insulin administered to rats subjected to brief hypothermia and having the shivering thermogenesis blocked by thiobutabarbital anesthesia can influence rectal temperature, the levels of some hormones and energy metabolism. The results obtained suggest that 1) insulin administration causes an increase in the energy charge potential (ECP) both in the liver and in skeletal muscle of the rat, indicating the domination of anabolic processes both in normothermic and hypothermic conditions, 2) there is a negative correlation between the levels of insulin and free fatty acids and the activity of isocitric dehydrogenase in rat liver mitochondria, and 3) the administration of insulin at a dose provoking metabolic response both in normothermic and hypothermic conditions was ineffective in provoking temperature response, indicating the existence of a functional dissociation between the various effects of the same dose of exogenous insulin.

[Effect of tolbutamide on lipolytic processes in blood and fat tissue of rats maintained in normothermic and hypothermic conditions]. / Wplyw tolbutamidu na procesy lipolityczne we krwi i tkance tluszczowej szczurów przebywajacych w warunkach normotermii i hipotermii.

The effect of tolbutamide administered in vivo or added to the incubation medium on lipolysis in adipose tissue and in blood has been studied in rats. Tissue lipolysis was lowered by 118 to 156% in groups of rats receiving tolbutamide in vivo. The addition of tolbutamide directly to the incubation medium caused an increase in the lipid mobilizing activity, while the addition of insulin inhibited lipolysis in each case. The effect of tolbutamide administered in vivo may be indirect through an increase in insulin binding and/or the drug may influence directly the permeability of cell membrane for ions and glucose. An increase in the lipid mobilizing activity observed in experiments consisting in the addition of tolbutamide to the incubation medium may be linked to the low glucose level and greater demand for free fatty acids or to the low level of insulin in the incubation medium.

In vivo effect of 2-deoxy-D-glucose on glucose-6-phosphate dehydrogenase activity in the cytosol of liver, heart and skeletal muscle of rats.

2-deoxy-D-glucose (2-DG), the unmetabolizable analogue of glucose induces a series of metabolic, hormonal and behavioral responses, causing cellular glucoprivation. According to in vitro studies, 2-DG inhibits phosphofructokinase in cultured human cells. The present investigations deal with changes in the cytosolic glucose-6-phosphate dehydrogenase activity following in vivo 2-DG administration. A single dose of 2-DG (600 mg/kg) has no influence on the activity of glucose-6-phosphate dehydrogenase in the cytosol of liver, heart and skeletal muscle of the rat. The concomitant increase in serum glucose, lactate and FFA concentrations observed in the study indicates indirectly a stimulation of adrenergic system. After three days of successive administration of 2-DG to rats, dehydrogenase activity decreased in the liver by approx 57% and in the skeletal muscle by approx 82% in comparison with control animals. Moreover the in vivo effect of 2-DG was found to be fully reversible, probably when the total amount of the inhibitor was excreted.

In vivo effect of 2-deoxy-D-glucose on adenine nucleotide levels in the liver and skeletal muscle of rats.

The present report indicates that 2-deoxy-D-glucose (2-DG) at a single dose causing reduction of Tre has no influence on liver and skeletal muscle content of ATP, ADP and AMP, the ATP/ADP ratio, energy charge potential (ECP) and total adenine nucleotides (TAN). After administration of 2-DG for 3) successive days, the level of ATP, ATP/ADP ratio, the values of ECP and TAN are decreased both in the liver and skeletal muscle. However, 72 hours after the last injection of 2-DG adenine nucleotide contents returned to the values observed in control group, indicating that the in vivo effect of this glucose analogue is fully reversible.

[Mechanism of action and applications for glucose analogs]. / Mechanizm dzialania i zastosowanie analogów glukozy.

The paper presents recent problems of the mechanism of the action of glucose analogs (especially 2-deoxy-D-glucose and 5-thio-D-glucose) at the cellular level as well as their application in experimental and clinical medicine. It has been discussed, whether 2-DG and 5-TG could be assumed to represent nonmetabolizable antimetabolites of glucose.

The effect of 4-aminopyrazolo (3,4-d) pyrimidine (4-APP) on some parameters of carbohydrate metabolism in the rat.

The study was designed to evaluate some parameters of carbohydrate metabolism in the rat as influenced by 4-APP, an adenine analogue. Adult female rats were injected with 1 mg 4-APP/100 g body weight/day for 3 days. 4-APP evoked a marked enlargement of the liver with lipid droplets accumulation in hepatocytes. This was accompanied by a marked lowering of the liver glycogen content. Within 3 days 4-APP did not change serum glucose, insulin and free fatty acid concentration. Serum glycogenolytic activity studied in an in vitro system showed 7 times as high glucose releasing ability in 4-APP treated rats as that of the serum of control animals. 4-APP resulted also in a marked enlargement of the adrenal medulla and lowered adrenaline and noradrenaline concentrations in the gland. The possibility of an activation of glycogenolysis in the liver of 4-APP treated rats has been discussed.

Triiodothyronine effect on some parameters of carbohydrate metabolism in rat blood and liver. I. In vivo effect of triiodothyronine on liver glycogen content, blood metabolites and serum glycogenolytic activity.

Short (7 days) T3-treatment has no influence on the plasma glucose and FFA concentration and blood lactate level, as well as on the hepatic glycogen content in the rats. The rise in T3 levels, observed in our study, was accompanied by a fall in T4 concentrations, indicating suppression of the endogenous T4 production. On the other hand, glycogenolytic activity of rat serum in vitro is the highest in the system: control liver slices-serum of rats pretreated with T3 as compared to the system: control liver slices-control serum. These observations may lead to conclusions, that serum of rats pretreated with T3 contains factors exhibiting the greater ability to mobilize glucose from liver slices than the control serum (euthyroid). The possibility, that pretreatment with T3 may cause a decrease in the number and/or affinity of beta-adrenergic receptors in the rat liver, is discussed.

Hyperthermia effect on lipolytic processes in rat blood and adipose tissue.

Rats anaesthetized with Brevinarcon were placed in a high-temperature chamber (air temperature 50 degrees C, relative humidity 50%) for induction of hyperthermia (rectal temperature 41.0 +/- +/- 0.5 degrees C). The control group comprised rats anaesthetized in the same way but kept at room temperature. In the serum in both groups glucose, free fatty acids, immunoreactive insulin, lipolytic activity and ability to mobilize lipids in vitro were determined. It was shown that the glucose and free fatty acid levels and the activity mobilizing serum lipids in vitro in the rats subjected to hyperthermia were lower than in the control group by 12%, 23% and 150% respectively. The lipolytic activity of the serum of rats subjected to hyperthermia was 42% higher, and the level of immunoreactive insulin rose by about 224% in relation to the control group. These results point to inhibition of lipolysis in the adipose tissue with simultaneous activation of intravascular lipolysis during hyperthermia in rats.

Adrenocortical function in 4-APP treated rats: a coupled stereological and biochemical study.

Adrenocortical function in 4-APP-induced (4-aminopyrazolo[3,4-d]pyrymidine) lipoprotein-deficient rats was studied in relation to quantitative morphologic changes in the gland. 4-APP treatment results in enlargement of the adrenal cortex and its zona fasciculata and reticularis cells. In enlarged livers, cholesterol and free fatty acid concentrations were similar to that of control rats, however a marked accumulation of triglycerides with a concomitant drop in hepatic delta 4-steroid hydrogenase activity was found. A profound drop in serum cholesterol in both, high and low density lipoproteins, as well as triglycerides and plasma corticosterone concentrations was accompanied by a marked lowering of cholesterol and corticosterone concentration in the adrenal gland. Corticosterone output by adrenal homogenates was higher in 4-APP treated rats than in control animals. Such a treatment did not change cholesterol side-chain cleavage, 11 beta-hydroxylase, 3 beta-ol dehydrogenase-isomerase, steroid 5 alpha-reductase and neutral lipase activities when expressing results per unit weight of tissue or protein. However, when calculating per adrenocortical cell, adenine analogue applied increased 11 beta-hydroxylase, steroid 5 alpha-reductase and neutral lipase activities. Thus, coupled biochemical and stereologic studies revealed a complex and multidirectional effect of 4-APP on the rat adrenal cortex. This effect may be caused by serum lipoprotein deficiency and by toxic and stressful action of the adenine analogue on the rat. Also a direct effect of 4-APP on rat adrenal cortex may not be excluded.