Twenty-four-hour changes of S-adenosylmethionine, S-adenosylhomocysteine adenosine and their metabolizing enzymes in rat liver; possible physiological significance in phospholipid methylation.

1. The metabolic control of adenosine concentration in the rat liver through the 24-hr cycle is related to the activity of adenosine-metabolizing enzymes [5'-nucleotidase (5'N), adenosine deaminase (A.D.), adenosine kinase (A.K.) and S-adenosylhomocysteine hydrolase (SAH-H)]. 2. Two peaks of adenosine were observed, one at 12:00 hr caused by high activity of 5'N and SAH-H, and the other at 02:00 hr, caused by a decrease in purine catabolism and purine utilization, low activity of SAH-H and de novo purine formation. 3. The similarity of the adenosine and S-adenosylmethionine (SAM) profiles through the 24-hr cycle suggests a role of adenosine in transmethylation reactions, because, during the night (02:00 hr), the metabolic conditions favor the formation and accumulation of S-adenosylhomocysteine (SAH), with consequent inhibition of transmethylation reactions. 4. In the 24-hr variation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the lowest ratio of PC/PE was observed at 24:00-02:00 hr when SAH concentration is high, whereas the highest PC/PE ratio occurs at the same time as one of the SAM/SAH ratio maxima.     

Comparative study of the heterophil phagocytic function in young and old ring doves (<Emphasis Type="Italic">Streptopelia risoria</Emphasis>) and its relationship with melatonin levels

A functional connection between the pineal gland (via the hormone melatonin) and the immune system has been suggested. In our previous results in the ring dove, we observed diurnal oscillations in the levels of this neurohormone in young animals and a decline in its plasma levels with advancing age (which is accompanied by the absence of diurnal rhythm). We also noted enhanced phagocytic activity of heterophils from old animals after in vitro incubation with both physiological and pharmacological doses of melatonin. Here, we evaluate the functional capacity of ring dove (Streptopelia risoria) heterophils in young (2 years of age) and old (8 years and more) animals at different times of day (0:00, 10:00 and 16:00, the times when the maximum, minimum, and mean values, respectively, of melatonin levels are observed in young animals). The phagocytic capacities for the ingestion of latex beads and Candida albicans were evaluated, as well as the oxidative metabolism which accompanies phagocytosis. At all three times of day studied, the heterophil phagocytic function with both latex and C. albicans was significantly greater in the young than in the old animals, and in the young animal cells it was significantly higher at 0:00. In addition, in the presence of latex beads, there was a significant decline at 10:00 and 0:00 of superoxide anion levels in the young animals relative to the old. In the young animals, there was a decline at 0:00 in comparison with both 10:00 and 16:00, and in the old animals there was a decline at both 0:00 and 16:00 compared with 10:00. These results could be due, at least in part, to the absence of a diurnal rhythm of melatonin in old animals, and to an enhancing effect of that hormone on young animals heterophil phagocytic function, which would also neutralize the oxidative stress deriving from this immune function.Abbreviations PBS phosphate-buffered saline - MIF macrophage migration-inhibition factor - PI phagocytosis index - PP phagocytosis percentage - PE phagocytosis efficiencyCommunicated by G. Heldmaier     

Time-Dependent Variations in the Coagulation Factors II,VII, IX,and X in Young and Elderly Volunteers

The existence of circadian (24-h) rhythms in the coagulation activity of vitamin K-dependent coagulation factors (Factors II, VII, IX, and X) were studied in six healthy young (18–30 years old) and six healthy elderly (69–75 years old) men. Aliquots of 5 ml of blood were obtained from each of the 12 subjects at six different time points over a 24-h period. Factors II, VII, and X were quantified by the prothrombin time test, whereas Factor IX was analyzed by the activated partial thromboplastin time test. Significant circadian variations were found for Factors II and VII in both age groups. The peak and trough values for Factor II were observed at 16: 00 and 00: 00 in young men and at 12: 00 and 16: 00 in elderly men. The amplitude of the rhythmic variation of Factor II was 3.3 ± 1.0 and 4.2 ± 0.9% in young and elderly volunteers, respectively. For Factor VII, the highest values were found during the activity period (08: 00–16: 00), while the lowest values occurred at night (00: 00) for both groups of subjects. The amplitude of the rhythms was twice as large in the young (6.2 ± 2.3%) as in the elderly (3.7 ± 0.8%). The data suggest that age does not alter significantly the chronobiology of Factors II and VII.     

Time-Dependent Variations in the Coagulation Factors II, VII, IX, and X in Young and Elderly Volunteers

The existence of circadian (24-h) rhythms in the coagulation activity of vitamin K-dependent coagulation factors (Factors II, VII, IX, and X) were studied in six healthy young (18-30 years old) and six healthy elderly (69-75 years old) men. Aliquots of 5 ml of blood were obtained from each of the 12 subjects at six different time points over a 24-h period. Factors II, VII, and X were quantified by the prothrombin time test, whereas Factor IX was analyzed by the activated partial thromboplastin time test. Significant circadian variations were found for Factors II and VII in both age groups. The peak and trough values for Factor II were observed at 16: 00 and 00: 00 in young men and at 12: 00 and 16: 00 in elderly men. The amplitude of the rhythmic variation of Factor II was 3.3 ± 1.0 and 4.2 ± 0.9% in young and elderly volunteers, respectively. For Factor VII, the highest values were found during the activity period (08: 00-16: 00), while the lowest values occurred at night (00: 00) for both groups of subjects. The amplitude of the rhythms was twice as large in the young (6.2 ± 2.3%) as in the elderly (3.7 ± 0.8%). The data suggest that age does not alter significantly the chronobiology of Factors II and VII.     

Comparative study of the activity/rest rhythms in young and old ringdove (Streptopelia risoria): correlation with serum levels of melatonin and serotonin

Aging is characterized by changes in the circadian rhythms of melatonin, serotonin, and sleep/wakefulness, alterations that affect sleep quality. The authors studied the circadian rhythms of serotonin and melatonin in young and old ringdoves (Streptopelia risoria) (2-3 and 10-12 yrs old, respectively), animals that are characterized by being monophasic and active by day, like humans. The aim was to correlate the indole rhythms with the animals' activity/rest periods. The animals were kept under a 12:12 h light/dark cycle, fed ad libitum, and housed in separate cages equipped for activity recording. Activity pulses were recorded with one actometer per animal (two perpendicular infrared transmitters) and were logged every 15 min by a computer program (DAS 16) throughout the experiment. Melatonin was measured by radioimmunoassay and serotonin by ELISA at intervals of 3 h (from 09:00 to 18:00 h) and 1 h (from 21:00 to 06:00 h), respectively. The results showed a reduction in nocturnal vs. diurnal activity of 89% and 61% in the young and old animals, respectively, with 100% considered to be the diurnal activity of each group. The amplitude of a cosine function fit to the melatonin concentrations of the old animals was half that of the young birds. The acrophase and nadir were at 02:00 and 14:00 h in the young and 01:00 and 13:00 h in the old animals, respectively. The amplitude of the corresponding cosine function fit to the serotonin concentrations in the old birds was one-third that of the young animals. The acrophase and nadir were at 15:00 and 03:00 h in the young and 16:00 and 04:00 h in the old animals, respectively. For both melatonin and serotonin, the concentrations in the young animals were significantly higher than in the old at most of the measurement times. There was a clear negative correlation between the circadian rhythms of activity and the serum melatonin levels in both young and old animals. The equivalent correlation for serotonin was positive, and stronger in the case of the young animals. The results suggest a possible relationship between the observed decline in the amplitude of the old animals' melatonin and serotonin rhythms and the lower percentage reduction in their nocturnal relative to diurnal activity pulses compared to the young animals. In conclusion, the circadian rhythms of melatonin and serotonin undergo alterations with age that could be involved in the changes in age-associated sleep.     

Circadian rhythm in circulating CD16-positive natural killer (NK) cells in macaque monkeys,implication of plasma cortisol levels

The daily change in both percentage and absolute number of circulating major lymphocyte subset was determined with young Japanese monkeys and rhesus monkeys. The blood sample was collected at four hour-intervals beginning at 16:00 for 24 hours under the condition of applying tethering system by which blood samples could be collected without restraint. During the dark period (from 20:00 to 08:00), the number of peripheral lymphocytes increased and that of granulocytes decreased, resulting in no significant change in the number of total peripheral white blood cells. The absolute number of CD4 + T, CD8 + T, and CD20 + B cells showed the significant daily change similar to that in number of peripheral lymphocytes, indicating no proportional change in these subsets. The typical proportional change was observed in CD16 + natural killer (NK) cells and the percentage of CD16 + cells decreased during dark period (from 20:00 to 04:00) and increased in the morning (from 08:00 to 12:00). The NK activity determined by killing K562 target cells showed the same changing pattern as that of percentage in CD16+ NK cells. The changing pattern of both percentage and activity of NK cells was consistent with that of plasma cortisol levels. In addition, the intravenous injection of 300 μg/kg of cortisol induced increase in plasma cortisol levels and decrease in percentage of CD16 + NK cells during the first 60 min after cortisol injection. These results strongly suggest that the levels of peripheral functional CD16 + NK cells might be directly regulated by plasma cortisol level in macaque monkeys.     

G2 restriction point within populations of hepatocytes and tongue keratinocytes in young, growing mice

The authors studied the effect of either extracts from liver (LE) or the malignant tumour ES2 (TE) or plasma from intact mice (PI) or tumour-bearing animals (PT) on the mitotic activity of the hepatocytes and tongue keratinocytes in young, growing C3H/s male mice (28+/-1 days old). Animals standardized for periodicity analysis were injected intraperitoneally with either TE, LE, PI, PT, or saline (S) at 16:00 h with 0.01 ml of sample/g of body weight and were then killed at (time of day/h post-injection) 20:00/04, 00:00/08, and 04:00/12. Colchicine (2 microg/g) was injected 4 h before death. Samples of the liver and tongue from each animal were processed for histology and assessment of mitotic index. The results were expressed as colchicine-arrested metaphases/1000 nuclei. The TE and LE stimulated the mitotic activity of hepatocytes and tongue keratinocytes. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.     

Control of birth in rats by RU 486, an antiprogesterone compound

Rats, isolated at mating (Day 1 of pregnancy), were submitted to either 8 h (8L:16D, Exp. I) or 14 h (14L:10D, Exp. II) of light daily with lights on from 12:00 h to 20:00 h and from 06:00 to 20:00 h respectively. In Exp. I, a single dose of RU 486 (10 mg in 0.2 ml ethanol) was given cutaneously at 08:00 h (Group A1), 12:00 h (Group B1), 19:00 h (Group C1) on Day 21 and at 08:00 h (Group D1) and 12:00 h (Group E1) on Day 22. In Exp. II, the same dose of RU 486 was given at 08:00 h (Group A2), 12:00 h (Group B2) and 19:00 h (Group C2) on Day 21. The solvent was given once at each of the preceding times to the control groups (T1 and T2) in both experiments. Groups T1 and T2 gave birth at two periods, the first on Day 22, the second on Day 23; the proportion of births during each of these periods depended on the light regimen (66.3% in 8L:16D; 50% in 14L:10D on Day 22). The distribution of births in Groups D1 and E1 treated on Day 22 were similar to their controls (T1). Rats treated on Day 21 (Groups A1, A2, B1, B2, C1, C2) gave birth over single periods on Day 22 after an interval correlated with the time of RU 486 administration. The earlier the treatment was given, the higher was the number of dead young and the lower the weight of live young 1 day after birth. These effects of prematurity did not impair further survival rates or weight at weaning.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative Study of the Activity/Rest Rhythms in Young and Old Ringdove (Streptopelia Risoria): Correlation with Serum Levels of Melatonin and Serotonin

Aging is characterized by changes in the circadian rhythms of melatonin, serotonin, and sleep/wakefulness, alterations that affect sleep quality. The authors studied the circadian rhythms of serotonin and melatonin in young and old ringdoves (Streptopelia risoria) (2–3 and 10–12 yrs old, respectively), animals that are characterized by being monophasic and active by day, like humans. The aim was to correlate the indole rhythms with the animals' activity/rest periods. The animals were kept under a 12∶12 h light/dark cycle, fed ad libitum, and housed in separate cages equipped for activity recording. Activity pulses were recorded with one actometer per animal (two perpendicular infrared transmitters) and were logged every 15 min by a computer program (DAS 16) throughout the experiment. Melatonin was measured by radioimmunoassay and serotonin by ELISA at intervals of 3 h (from 09∶00 to 18∶00 h) and 1 h (from 21∶00 to 06∶00 h), respectively. The results showed a reduction in nocturnal vs. diurnal activity of 89% and 61% in the young and old animals, respectively, with 100% considered to be the diurnal activity of each group. The amplitude of a cosine function fit to the melatonin concentrations of the old animals was half that of the young birds. The acrophase and nadir were at 02∶00 and 14∶00 h in the young and 01∶00 and 13∶00 h in the old animals, respectively. The amplitude of the corresponding cosine function fit to the serotonin concentrations in the old birds was one‐third that of the young animals. The acrophase and nadir were at 15∶00 and 03∶00 h in the young and 16∶00 and 04∶00 h in the old animals, respectively. For both melatonin and serotonin, the concentrations in the young animals were significantly higher than in the old at most of the measurement times. There was a clear negative correlation between the circadian rhythms of activity and the serum melatonin levels in both young and old animals. The equivalent correlation for serotonin was positive, and stronger in the case of the young animals. The results suggest a possible relationship between the observed decline in the amplitude of the old animals' melatonin and serotonin rhythms and the lower percentage reduction in their nocturnal relative to diurnal activity pulses compared to the young animals. In conclusion, the circadian rhythms of melatonin and serotonin undergo alterations with age that could be involved in the changes in age‐associated sleep.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

The effects of feeding and housing on the behaviour of the laboratory rabbit

The effects of housing, feeding time and diet composition on the behaviour of the laboratory rabbit were examined. The animals were caged individually in single or double metal cages with perforated metal floors, metal walls, and bars in the front, or kept as a group in floor pens. The light/dark cycle was 12/12 h with light from 04:00 to 16:00 h and 30 min twilight. One experiment compared feeding equal energy levels of a high energy diet (10.1 MJ/kg) and with a low energy diet (7.0 MJ/kg) at 08:00 h. The second experiment compared feeding the high energy diet at 08:00 h and at 14:00 h. In both studies the behaviour of the rabbits was recorded between 08:00 and 14:00 h and between 16:00 and 22:00 h. Feeding the animals at 14:00 h reduced abnormal behaviour during the dark period compared to feeding at 08:00 h, whereas no difference in behaviour could be detected between feeding a high-energy and a low-energy diet at 08:00 h. Animals in floor pens generally showed less abnormal behaviour than caged animals. The results indicate that the welfare for caged rabbits can be improved by feeding the animals in the afternoon rather than in the morning.     

Acute exposure to 50 Hz magnetic field does not affect hematologic or immunologic functions in healthy young men: A circadian study

Some epidemiological studies report a relationship between magnetic field exposure and such human diseases as leukemia and immune system disturbances. The few published studies on animals do not demonstrate field exposure-related alterations in hematologic and immune systems. The data presented here are part of a broader study designed to investigate the possible effects of acute exposure to a 50 Hz linearly polarized magnetic field (10 μT) on hematologic and immunologic functions. Thirty-two young men (20–30 years old) were divided into two groups (control group, i.e., sham-exposed, 16 subjects; exposed group, 16 subjects). All subjects participated in two 24 h experiments to evaluate the effects of both continuous and intermittent (1 h “off” and 1 h with the field switched “on” and “off” every 15 s) exposure to linearly polarized magnetic fields. The subjects were exposed to the magnetic field (generated by three Helmholtz coils per bed) from 23:00 to 08:00 while lying down. Blood samples were collected during each session at 3 h intervals from 11:00 to 20:00 and hourly from 22:00 to 08:00. No significant differences were observed between sham-exposed (control) and exposed men for hemoglobin concentration, hematocrit, red blood cells, platelets, total leukocytes, monocytes, lymphocytes, eosinophils, or neutrophils. Immunologic variables [CD3, CD4, CD8, natural killer (NK) cells and B cells] were unaltered. To our knowledge, this study is the first to document the effects of a 50 Hz magnetic field on the circadian rhythm of human hematologic and immune functions, and it suggests that acute exposure to either a continuous or an intermittent 50 Hz linearly polarized magnetic field of 10 μT, at least under the conditions of our experiment, does not affect either these functions or their circadian rhythms in healthy young men. © 1996 Wiley-Liss, Inc.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.     

Regulation of glucose utilization and lipogenesis in adipose tissue of diabetic and fat fed animals: Effects of insulin and manganese

In order to evaluate the modulatory effects of manganese, high fat diet fed and alloxan diabetic rats were taken and the changes in the glucose oxidation, glycerol release and effects of manganese on these parameters were measured from adipose tissue. An insulin-mimetic effect of manganese was observed in the adipose tissue in the controls and an additive effect of insulin and manganese on glucose oxidation was seen when Mn²⁺ was addedin vitro. The flux of glucose through the pentose phosphate pathway and glycolysis was significantly decreased in high fat fed animals. Although thein vitro addition of Mn²⁺ was additive with insulin when¹⁴CO2 was measured from control animals, it was found neither in young diabetic animals (6–8 weeks old) nor in the old (16 weeks old). Both insulin and manganese caused an increased oxidation of carbon-1 of glucose and an increase of its incorporation into¹⁴C-lipids in the young control animals; the additive effect of insulin and manganese suggests separate site of action. This effect was decreased in fat fed animals, diabetic animals and old animals. Manganese alone was found to decrease glycerol in both the control and diabetic adipose tissue inin vitro incubations. The results of the effects of glucose oxidation, lipogenesis, and glycerol release in adipose tissue of control and diabetic animals of different ages are presented together with the effect of manganese on adipose tissue from high fat milk diet fed animals.     

Function of 5'-nucleotidase in the uptake of adenosine from AMP by human lymphocytes.

The function of 5'-nucleotidase in nucleoside uptake from AMP was investigated in human lymphocytes by comparing the transport in cells containing this enzyme (5'N+) with that in cells deficient in the activity (5'N-). The rate of adenosine and Pi uptake from AMP was 3.9-fold greater in the 5'N+ then in the 5'N- lymphocytes. There was no difference in transport between these cells when incubated with adenosine or Pi. These results indicate that phosphorylytic cleavage of AMP by 5'-nucleotidase is necessary for the uptake of the nucleotide and Pi moieties by the human lymphocyte.     

The effect of indomethacin on the stimulation of protein synthesis by insulin in young post-absorptive rats.

Groups of young rats (100 g body wt.) were starved from 23:00 to 11:00 h. The animals were then infused intravenously with diluent or insulin at three different doses to achieve plasma insulin concentrations of 20, 50 and 150 microunits/ml. Before the start of the infusion, animals received a single intravenous injection of indomethacin (250 micrograms) or diluent. After 20 min of infusion, the rats were injected with a large amount of labelled phenylalanine and were killed 10 min later. Insulin produced a dose-dependent decrease in plasma glucose and a dose-dependent rise in protein synthesis in cardiac, gastrocnemius, plantaris and soleus muscles. Protein synthesis in the liver was unaffected by insulin. Indomethacin had no effect on plasma glucose concentrations, but blocked the insulin-induced rise in protein synthesis in cardiac, gastrocnemius and plantaris, but not in soleus muscle. The hormone also increased the plasma concentration of prostaglandin E2 and of prostaglandins F2 alpha and E2 in gastrocnemius and plantaris muscle. The results show close similarities to previous observations with isolated rabbit muscles in vitro and suggest that the involvement of arachidonic acid metabolism in the action of insulin on protein synthesis is of physiological significance.     

Reduced levels of thyroid hormones, insulin, and glucose, and lower body core temperature in the growth hormone receptor/binding protein knockout mouse

The mechanisms that are responsible for the extension of lifespan in the mouse with targeted disruption (knockout [KO]) of the growth hormone (GH) receptor/binding protein (GHR-KO) are unknown. However, in the long-living Ames dwarf mouse, blood glucose and body core temperature (Tco) are consistently lower than in normal mice. In addition, insulin levels are reduced and corticosterone levels are elevated in male dwarfs. These functional alterations, similar to those seen in animals under caloric restriction, have not been proven to be causally related to the extension of lifespan, but they do provide some insight into what traits may be necessary for long life. Therefore, to investigate which of these parameters are similarly affected in two genetically unrelated, yet similarly long-living mouse models, we measured Tco, thyroid hormones (triiodothyronine [T3] and thyroxine [T4]), and insulin, in addition to morning and afternoon levels of glucose and corticosterone, in young adult male and/or female GHR-KO mice and their normal siblings. Tco in GHR-KO mice was numerically reduced throughout the 24-hr period; however, these differences were only significant 4 hr prior to lights-off (14:00 hr), immediately after lights-off (18:00 hr), and during the 3 hr preceding lights on (03:00 to 06:00 hr). GHR-KO mice had significantly reduced levels of T3 and T4, while the ratio of these hormones was similar to that in normal mice. Insulin levels in GHR-KO mice were lower than in normal mice; levels in male GHR-KO mice were below the detectable limits of the assay used. Glucose levels in GHR-KO mice (male and females) were lower than in normal mice in measurements taken in both morning and afternoon; however, these differences arose from consistent reductions in males, as morning glucose levels in GHR-KO females were similar to those of normal mice. Corticosterone levels measured in blood plasma collected under basal (nonstressed) conditions showed sex-related alterations. Basal corticosterone levels in female GHR-KO mice were similar to normal females, while those in male GHR-KO mice were higher than in normal males in the afternoon. Corticosterone levels in stressed GHR-KO females were similar to those measured in stressed normal females. These data show that the long-living GHR-KO mouse shares a reduction in glucose, insulin, thyroid hormones, and Tco with the Ames dwarf mouse. Reductions in these parameters may be important to the underlying mechanisms of delayed aging in these animals.     

Differential effect of insulin and elevated glucose level on adenosine handling in rat T lymphocytes

Reduced proliferation potential is among other T cell functional defects long known feature of diabetes. However, the mechanism responsible for this impairment is still unknown. Our study was undertaken to investigate the effect of changes in glucose and insulin concentrations on adenosine metabolism, transport and receptor-mediated action in rat T lymphocytes. Presented results indicate that vulnerability of T cells to metabolic stress is determined by insulin but not by glucose concentration. However, glucose and insulin differentially affected the activities of adenosine metabolizing enzymes in resting and proliferating T cells. The Con A-induced proliferation of cultured T lymphocytes did not depended on expression level and functional state of nucleoside transporters. Inhibition of adenosine kinase (AK) with 5-iodotubercidin lowers the proliferation potential of T cells to the level observed for insulin-deprived cells. Moreover, insulin-deprived T lymphocytes but not cells cultured in the presence of insulin released significant quantities of adenosine. Under resting conditions, the cAMP level was fivefold higher in cells deprived of insulin comparing to cells cultured in the presence of insulin. Exposition of insulin-deprived T lymphocytes to specific antagonist (ZM241385) of A2a receptor but not to specific antagonist (Alloxazine) of A2b receptor suppressed cAMP elevation and completely restored the proliferation potential of T cells. Concluding, adenosine released by insulin-deprived T cells due to suppressed AK activity by acting on A2a receptors leads to increases in cAMP level and suppression of T cell proliferation. We assume that this mechanism may significantly contribute to immune impairment observed in diabetes.     

Delta5-3beta-hydroxysteroid dehydrogenase and estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos

Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. Δ⁵-3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00–09:00 hr on day 3, the activity then increased, peaked at 01:00–03:00 hr on day 4, considerably declined by 08:00–09:00 hr (day 4), and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, Dickmann and Dey, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster.In addition to 3βHSD, estradiol-17β-hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen.     

Insulin resistance in fat cells from obese Zucker rats - Evidence for an impaired activation and translocation of protein kinase B and glucose transporter 4

The effect of insulin on glucose transport, glucose transporter 4 (Glut4) translocation, and intracellular signaling were measured in fat cells from lean and obese Zucker rats of different ages. Insulin-stimulated glucose transport was markedly reduced in adipocytes from old and obese animals. The protein content of Glut4 and insulin receptor substrates (IRS) 1 and 2 were also reduced while other proteins, including the p85 subunit of PI3-kinase, Shc and the MAP kinases (ERK1 and 2) were essentially unchanged. There was a marked impairment in the insulin stimulated tyrosine phosphorylation of IRS-1 and 2 as well as activation of PI3-kinase and PKB in cells from old and obese animals. Furthermore, insulin-stimulated translocation of both Glut4 and PKB to the plasma membrane was virtually abolished. The phosphotyrosine phosphatase inhibitor, vanadate, increased the insulin- stimulated upstream signaling including PI3-kinase and PKB activities as well as rate of glucose transport. Thus, the insulin resistance in cells from old and obese Zucker rats can be accounted for by an impaired translocation process, due to signaling defects leading to a reduced activation of PI3-kinase and PKB, as well as an attenuated Glut4 protein content.