24h withdrawal following repeated administration of caffeine attenuates brain serotonin but not tryptophan in rat brain: Implications for caffeine-induced depression

Caffeine injected at doses of 20, 40 and 80 mg/kg increased brain levels of tryptophan, 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat brain. In view of a possible role of 5-HT in caffeine-induced depression the effects of repeated administration of high doses of caffeine on brain 5-HT metabolism are investigated in rats. Caffeine was injected at doses of 80 mg/kg daily for five days. Control animals were injected with sahne daily for five days. On the 6th day caffeine (80 mg/kg) injected to 5 day sahne injected rats increased brain levels of tryptophan, 5-HT and 5-HIAA. Plasma total tryptophan levels were not affected and free tryptophan increased. Brain levels of 5-HT and 5-HIAA but not tryptophan decreased in 5 day caffeine injected rats injected with sahne on the 6th day. Plasma total and free tryptophan were not altered hi these rats. Caffeine-induced increases of brain tryptophan but not 5-HT and 5-HIAA were greater in 5 day caffeine than 5 day sahne injected rats. The findings are discussed as repeated caffeine administration producing adaptive changes in the serotonergic neurons to decrease the conversion of tryptophan to 5-HT and this may precipitate depression particularly in conditions of caffeine withdrawal.     

Serotonin enhances beta-endorphin secretion to lower plasma glucose in streptozotocin-induced diabetic rats

Although serotonin, serotonin uptake inhibitors and serotonin precursors (including tryptophan or 5-hydroxytryptophan) are known to have hypoglycemic action in rodents or human, it is not clear whether serotonin has hypoglycemic effect in streptozotocin-induced diabetic rats (STZ-diabetic rats). The aim of this study was to investigate the action of serotonin in regulating the plasma glucose STZ-diabetic rats. Plasma glucose, insulin, beta-endorphin and adrenaline were assessed after intraperitoneal administration of serotonin. Serotonin produced hypoglycemic effects without altering plasma insulin and adrenaline levels but increasing beta-endorphin level in STZ-diabetic rats. The glycogen content in soleus muscle was increased at 90 min after application of serotonin (0.3 mg/kg) in STZ-diabetic rats. Dihydroergotamine (non-selective 5-HT receptor blocker) and pimozide (5-HT(7) receptor blocker) abolished the hypoglycemic effect of serotonin in STZ-diabetic rats. Serotonin-induced hypoglycemic effect in association with the increase of beta-endorphin release was abolished in bilaterally adrenalectomized STZ-diabetic rats. In isolated adrenal gland of STZ-diabetic rats, the increase of beta-endorphin secretion in response to serotonin was reduced by either dihydroergotamine or pimozide. Pretreatment with naloxone (1.0 mg/kg, i.p.) prevented serotonin-induced plasma glucose lowering effect in STZ-diabetic rats. The results demonstrated that serotonin may activate 5-HT(7) receptor on rat adrenal gland to enhance of beta-endorphin secretion, which then stimulates the opioid receptor to increase peripheral glucose utilization, resulting in decreased plasma glucose levels in STZ-diabetic rats.     

Effects of clofibrate on plasma tryptophan, growth hormone, and prolactin and on brain tryptophan and serotonin in prepubertal rats

The effects of clofibrate administration (200 mg/kg, po) on somatic growth, plasma levels of lipids, tryptophan, growth hormone (GH), and prolactin (PRL), as well as on brain concentrations of tryptophan and 5-hydroxytryptamine (5-HT) were studied in prepubertal male rats. The drug did not significantly alter ponderal growth, but an appreciable reduction of tail length was observed in rats treated for 30 days. Triglyceride concentrations in plasma showed a 43% diminution after 30 days of treatment, whereas free fatty acid (FFA) levels were not modified. Clofibrate administration for 7, 15, or 30 days caused a fall in total tryptophan and a significant increase of the free fraction in plasma with no change in brain tryptophan levels. Brain 5-HT was generally unaffected but a marked elevation of this parameter was noted in rats treated for 15 days. Plasma GH and PRL concentrations remained unaltered. It may be concluded from these findings that the slight reduction of somatic growth, the diminution of triglycerides, and the increase of free tryptophan in plasma, induced by chronic clofibrate treatment, are not associated with variations in brain tryptophan and 5-HT levels or with modifications of plasma GH and PRL titers.     

Injected tryptophan increases brain but not plasma tryptophan levels more in ethanol treated rats

In previous studies, long term treatment with ethanol has been shown to enhance brain 5-hydroxytryptamine 5-(HT) metabolism by increasing the activity of the regulatory enzyme tryptophan hydroxylase and or availability of circulating tryptophan secondarily to an inhibition of hepatic tryptophan pyrrolase. In the present study ethanol treatment given for two weeks decreased hepatic apo-tryptophan pyrrolase but not total tryptophan pyrrolase activity in rats. Tryptophan levels in plasma and brain did not increase significantly. But there was a marked increase of 5-HT but not 5-hydroxyindoleacetic acid (5-HIAA) concentration in brain, suggesting a possible increase in the activity of tryptophan hydroxylase. The effect of a tryptophan load on brain 5-HT metabolism was therefore compared in controls and ethanol treated rats. One hour after tryptophan injection (50 mg/kg i.p.) plasma concentrations of total and free tryptophan were identical in controls and ethanol treated rats, but the increases of brain tryptophan 5-HT and 5-HIAA were considerably greater in the latter group. The results are consistent with long term ethanol treatment enhancing brain serotonin metabolism and show that brain uptake/utilization of exogenous tryptophan is increased in ethanol treated rats and may be useful to understand the role and possible mechanism of tryptophan/serotonin involvement in mood regulation.     

Injected tryptophan increases brain but not plasma tryptophan levels more in ethanol treated rats

In previous studies, long term treatment with ethanol has been shown to enhance brain 5-hydroxytryptamine 5-(HT) metabolism by increasing the activity of the regulatory enzyme tryptophan hydroxylase and or availability of circulating tryptophan secondarily to an inhibition of hepatic tryptophan pyrrolase. In the present study ethanol treatment given for two weeks decreased hepatic apo-tryptophan pyrrolase but not total tryptophan pyrrolase activity in rats. Tryptophan levels in plasma and brain did not increase significantly. But there was a marked increase of 5-HT but not 5-hydroxyindoleacetic acid (5-HIAA) concentration in brain, suggesting a possible increase in the activity of tryptophan hydroxylase. The effect of a tryptophan load on brain 5-HT metabolism was therefore compared in controls and ethanol treated rats. One hour after tryptophan injection (50 mg/kg i.p.) plasma concentrations of total and free tryptophan were identical in controls and ethanol treated rats, but the increases of brain tryptophan 5-HT and 5-HIAA were considerably greater in the latter group. The results are consistent with long term ethanol treatment enhancing brain serotonin metabolism and show that brain uptake/utilization of exogenous tryptophan is increased in ethanol treated rats and may be useful to understand the role and possible mechanism of tryptophan/serotonin involvement in mood regulation.     

A STUDY OF PROPOSED DETERMINANTS OF BRAIN TRYPTOPHAN CONCENTRATION IN RATS AFTER PORTOCAVAL ANASTOMOSIS OR SHAM OPERATION

Liver dysfunction was produced in rats by surgical portocaval anastomosis (PCA), and the time-course of changes in brain tryptophan and 5-HT metabolism studied in relation to plasma changes possibly influencing brain tryptophan concentration. Brain tryptophan and 5-hydroxyindolylacetic acid (5-HIAA) levels were increased greatly and maximally on the day after PCA and remained high. 5-HT changes were less marked but had a similar time-course. Plasma total tryptophan was little changed but plasma free tryptophan was raised. The latter change showed a similar time-course to that of brain tryptophan but was not large enough to account completely for it. Sham operation was followed by significant but transient increases in plasma free tryptophan, brain tryptophan and 5-HIAA but these were much smaller than after PCA. Brain tryptophan did not correlate with plasma total tryptophan either in control or PCA rats but it correlated significantly with plasma free tryptophan in both groups. However brain levels were much higher in PCA rats than in controls with similar plasma free tryptophan levels at all times from the first day after operation. The increase of brain tryptophan in anastomosed rats not accounted for by plasma free tryptophan was explained neither by insulin changes nor by an increase of the insulin/glucagon ratio nor by changes in plasma concentrations of those amino acids which compete with tryptophan for entry into brain. The results therefore indicate an unknown influence on brain tryptophan concentration in PCA rats. As tyrosine changes in brain and plasma after PCA were very similar to those of tryptophan this influence may not be specific to tryptophan. Results suggest that under the conditions used brain tryptophan concentrations of both PCA and control rats are more influenced by changes of plasma free tryptophan concentration than by changes of plasma concentrations of competing amino acids.     

REGIONAL ACCUMULATION OF TRYPTOPHAN AND SEROTONIN METABOLISM FOLLOWING TRYPTOPHAN LOADING IN DIABETIC RATS

Abstract— Streptozotocin-induced diabetes in rats reduces brain tryptophan but is without effect on the central levels of 5-hydroxytryptamine (5-HT) or 5-hydroxyindoleacetic acid (5-HIAA). The present work investigates the effect of diabetes on the accumulation of brain tryptophan, 5-HT and 5-HIAA in various brain regions following a systemic tryptophan load. The results indicate that diabetes severely restricts the uptake of tryptophan by brain but that the tryptophan that is accumulated is normally converted to 5-HT and 5-HIAA. Possible mechanisms which might explain the apparent resistance of 5-HT metabolism to decreased precursor levels in diabetics are discussed.     

THE EFFECT OF A LOW PROTEIN DIET AND EXOGENOUS INSULIN ON BRAIN TRYPTOPHAN AND ITS METABOLITES IN THE WEANLING RAT

—Male Wistar rats aged 24 days were divided into three groups. Two groups were given a high protein (250 g/kg casein) and a low protein (30 g/kg casein) diet respectively. The third group was given an amount of the high protein diet containing the same amount of energy as that consumed by the low protein diet rats. The plasma of the animals on low protein contained 20% of the concentration of tryptophan of animals on the other two diets. In these animals the concentration of tryptophan was reduced in the forebrain, cerebellum and brain stem, and the concentrations of 5-HT and 5-hydroxyindoleacetic acid were reduced in the forebrain and brain stem. The low protein diet decreased the total uptake of l -[G-³H]tryptophan into the brain and its incorporation into brain protein. Plasma insulin concentrations were reduced in the low protein and ‘restricted high protein’ animals and the plasma corticosterone concentration was raised in the low protein animals. Exogenous insulin did not raise the plasma tryptophan concentration in the low protein animals but it increased the uptake of l -[G-³H]tryptophan into the brain and its incorporation into protein. Rehabilitation for 7 days restored the plasma and brain tryptophan concentrations and those of brain 5-HT and 5-hydroxyindoleacetic acid to control values.     

Rapid repletion of brain serotonin in malnourished corn-fed rats following L-tryptophan injection.

The concentration of tryptophan in serum, and the levels of tryptophan, serotonin (5-HT), and 5-hydroxyindole-acetic acid (5-HIAA) in brain are substantially reduced in rats that consume for 6 weeks a diet in which corn is the only source of protein. Single injections of L-tryptophan (25, 50, or 100 mg/kg) cause dose-related increases in brain tryptophan, 5-HT, and 5-HIAA in corn-fed animals. At each dose, brain tryptophan content rises to a proportionately greater extent in corn-fed rats than in well-nourished controls, even though serum tryptophan concentrations attain higher levels in controls. This difference may reflect the greatly reduced serum concentrations in corn-fed rats of other large neutral amino acids that compete with tryptophan for uptake into the brain (tyrosine, phenylalanine, leucine, isoleucine, and valine). However, the substantial decrease in serum albumin levels also diminishes the binding of tryptophan to serum albumin; thus it is not yet possible to state which of these changes is responsible for the much greater increments in brain tryptophan observed in corn-fed rats after tryptophan injection. The fact that tryptophan administration rapidly restores brain 5-hydroxyindole levels in corn-fed animals suggests that the reductions in 5-HT and 5-HIAA levels associated with this type of malnutrition may be largely caused by inadequate availability of substrate.     

TRYPTOPHAN LOADING: CONSEQUENT EFFECTS ON THE SYNTHESIS OF KYNURENINE AND 5-HYDROXYINDOLES IN RAT BRAIN

Abstract— Tryptophan loading of rats resulted in a continuous non-linear uptake of l -tryptophan from plasma into the brain. The optimum tryptophan load for increasing cerebral 5-hydroxytryptamine (5-HT) level was 25 mg/kg. Above this, there was a gradual decrease both in the levels and synthesis of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) as assessed from simultaneous intraperitoneal or intraventricular injections of l [¹⁴C]tryptophan. A 5–10 fold increase in cerebral tryptophan produced a limited stimulation of 5-HT synthesis. When the cerebral tryptophan level reached 1 ± 10 -4 , substrate inhibition in vivo of the tryptophan monooxygenase (tryptophan-5-hydroxylase) but not of the indoleamine-2,3-dioxygenase occurred. Cerebral synthesis of kynurenine increased linearly with increasing tryptophan load. At a plasma ratio of 50:1 tryptophan to kynurenine, tryptophan loading interfered with the entry of peripheral kynurenine. Tryptophan loading also increased the efflux of 5-hydroxyindoles from the brain. One hour after intraperitoneal injection of l -kynurenine sulfate (5 mg/kg) into rats, there was a shift in the plasma ratio of l -tryptophan to l -kynurenine to 4:1. In these rats, a 20% reduction of cerebral tryptophan was noted.     

Acute effects of caffeine injection on neutral amino acids and brain monoamine levels in rats

The injection of caffeine (100 mg/kg, i.p.) into male rats acutely increased brain levels of trytophan, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). Blood levels of glucose, nonesterified fatty acids (NEFA) and insulin also increased, while those of the aromatic and branched-chain amino acids fell. Serum tryptophan levels either did not fall, or increased. Consequently, the serum ratio of trypthopahn to the sum of other large neutral amino acids (LNAA) increased. Less consistently noted were increases in serum free tryptophan levels. Brain tyrosine levels were not appreciably altered by caffeine, nor was the serum tyrosine ratio. In dose-response studies, 25 mg/kg of caffeine was the minimal effective dose needed to raise brain tryptophan, but only the 100 mg/kg dose elevated all three indoles in brain. In no experiments did caffeine, at any time or dose, alter brain levels of dopamine or norepinephrine. Caffeine thus probably raises brain tryptophan levels by causing insulin secretion, and thereby changing plasma amino acid levels to favor increased tryptophan uptake into brain. The rises in brain 5-HT and 5-HIAA may follow from the increase in brain tryptophan, although further data are required clearly to establish such a mechanism.     

Effect of Tryptophan on Extracellular Concentrations of Tryptophan and 5-Hydroxyindoleacetic Acid in the Striatum and Cerebellum

The effects of L-tryptophan (50 mg/kg i.p.) on extracellular concentrations of tryptophan and the 5-hydroxytryptamine (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in the rat striatum and cerebellum, regions with rich and poor 5-HT innervation, respectively. Determinations were on perfusates from dialysis probes in the brains of conscious, freely moving rats. The pharmacokinetic profiles of dialysate tryptophan after tryptophan load (peak concentration, time to peak concentration, area under curve, and half-life) in the two regions did not differ significantly. The dialysate 5-HIAA concentration in the striatum rose two- to threefold after the administration of tryptophan. Therefore, as 5-HIAA was undetectable in the cerebellum either before or after the administration of tryptophan, the increase of 5-HIAA in the striatum is unlikely to depend appreciably on its production within the cerebral vasculature or outside the brain or on its entering the striatum through a blood-brain barrier damaged by placement of the dialysis probe. Overall, the findings strengthen previous evidence that extracellular 5-HIAA concentrations determined by cerebral dialysis are a valid measure of the metabolism of 5-HT of brain neuronal origin.     

Brain catecholaminergic and tryptophan responses to restraint are attenuated by nitric oxide synthase inhibition

Increases in the brain concentrations of tryptophan and in serotonin (5-HT) metabolism are commonly observed in animals under stress. Previous experiments indicated that the increase in brain tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) observed in response to administration of endotoxin (lipopolysaccharide, LPS) and interleukin-1 (IL-1) were largely prevented by pretreatment with N-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase (NOS). Therefore we tested whether the increases in tryptophan and 5-HT metabolism observed following restraint and footsthock were similarly affected. Mice were injected with L-NAME (30 mg/kg) or saline and restrained for 40 min. Restraint caused increases in concentrations of tryptophan and the catabolites of dopamine (DA), norepinephrine (NE) and 5-HT in the medial prefrontal cortex, hypothalamus, and brain stem. The L-NAME pretreatment significantly attenuated, but did not prevent, the changes in tryptophan and catecholamine metabolism, with a very small effect on the increase in plasma corticosterone. When mice pretreated with L-NAME were subjected to 30 min footshock, the NOS inhibitor had no statistically significant effects on the increases in DA, NE and 5-HT metabolism, but tended to attenuate the increases in tryptophan. We interpret these results to indicate that NOS plays a relatively small role in the cerebral neurochemical responses to restraint and footshock, but the role in the restraint-induced changes was greater than that in the footshock-induced ones. The attenuation of the restraint-related effects on the catecholamines most probably reflects a contribution to the CNS responses from peripheral vascular changes which are likely to be limited by the inhibition of NOS.     

STUDIES IN VIVO ON THE RELATIONSHIP BETWEEN BRAIN TRYPTOPHAN, BRAIN 5-HT SYNTHESIS AND HYPERACTIVITY IN RATS TREATED WITH A MONOAMINE OXIDASE INHIBITOR AND L-TRYPTOPHAN

Abstract— The effect of l -tryptophan loading upon the amount of 5-HT accumulating in the brains of rats pretreated with a monoamine oxidase inhibitor was studied. The amount of brain 5-HT accumulated increased with increasing tryptophan dosages and brain tryptophan concentrations up to a tryptophan dose of 120 mg/kg body wt. and a brain tryptophan of about 70 μg/g brain. Above this dose and concentration no further increase in brain 5-HT accumulation occurred. After monoamine oxidase inhibition and tryptophan loading gross hyperactivity and hyperpyrexia occurred. Monoamine oxidase inhibition, tryptophan administration and intact aromatic amino acid decarboxylase activity were all collectively essential for the production of hyperactivity and hyperpyrexia. DL-Parachlorophenyl-alanine prevented both the occurrence of hyperactivity and the increased accumulation of, brain 5-HT. Indices of hyperactivity correlated with the amount of brain 5-HT accumulating in 1 h after tryptophan loading but not with the overall concentration of brain 5-HT, suggesting that hyperactivity was dependent upon the rate of 5-HT synthesis. Reserpine and tetra-benazine pretreatment speeded the onset and rate of development of the hyperactive state without altering the synthesis of brain 5-HT. It is suggested that when monoamine oxidase is inhibited and the rate of 5-HT synthesis is increased, granular uptake and storage of 5-HT and other rate-limiting mechanisms for 5-HT inactivation are unable to prevent 5-HT 'spilling over’to produce hyperactivity. The crucial dependence of 5-HT synthesis upon brain tryptophan concentration and the ability of intraneuronal metabolism, when monoamine oxidase activity is intact, to cope with increased 5-HT synthesis and prevent ‘spillover’, raise the possibility that brain 5-HT synthesis is normally in excess of functional needs, and suggest that intraneuronal metabolism and the intraneuronal organization of 5-HT pools are of more importance than synthesis in regulating the amount of 5-HT available for functional activity.     

PLASMA TRYPTOPHAN AND 5-HT METABOLISM IN THE CNS OF THE NEWBORN RAT

—The relationships between plasma tryptophan and 5-HT metabolism in the CNS were studied in newborn rats and compared with adults. Both the concentration of free tryptophan in plasma and that of the amino-acid in brain were much higher immediately after birth than later on. Drugs such as salicylate and chlordiazepoxide, which increased brain tryptophan concentrations in adults by displacing the plasma amino acid bound to serum albumin, were ineffective in newborn rats: most of the amino acid being already free in their plasma. The study of 5-HT metabolism in brain stem slices revealed that the affinity of the uptake process for tryptophan was higher in newborn than in adult animals, whereas the reverse situation was observed for the enzyme complex involved in 5-HT synthesis (lower apparent K_m in adults). In addition, the catabolism of newly synthesized 5-HT was more rapid in newborn than in adult tissues. Finally, the free state of tryptophan in plasma of newborn animals induced in brain both a high amino acid concentration and, in contrast to the situation observed in adults, a synthesis rate of 5-HT very near its maximal value.     

Amino acids and central fatigue

Summary. There is an increasing interest in the mechanisms behind central fatigue, particularly in relation to changes in brain monoamine metabolism and the influence of specific amino acids on fatigue. Several studies in experimental animals have shown that physical exercise increases the synthesis and metabolism of brain 5-hydroxytryptamine (5-HT). Support for the involvement of 5-HT in fatigue can be found in studies where the brain concentration of 5-HT has been altered by means of pharmacological agents. When the 5-HT level was elevated in this way the performance was impaired in both rats and human subjects, and in accordance with this a decrease in the 5-HT level caused an improvement in running performance in rats. The precursor of 5-HT is the amino acid tryptophan and the synthesis of 5-HT in the brain is thought to be regulated by the blood supply of free tryptophan in relation to other large neutral amino acids (including the branched-chain amino acids, BCAA) since these compete with tryptophan for transport into the brain. Studies in human subjects have shown that the plasma ratio of free tryptophan/BCAA increases during and, particularly, after sustained exercise. This would favour the transport of tryptophan into the brain and also the synthesis and release of 5-HT which may lead to central fatigue. Attempts have been made to influence the 5-HT level by giving BCAA to human subjects during different types of sustained heavy exercise. The results indicate that ingestion of BCAA reduces the perceived exertion and mental fatigue during exercise and improves cognitive performance after the exercise. In addition, in some situations ingestion of BCAA might also improve physical performance; during exercise in the heat or in a competitive race when the central component of fatigue is assumed to be more pronounced than in a laboratory experiment. However, more experiments are needed to further clarify the effect of BCAA and also of tryptophan ingestion on physical performance and mental fatigue. Received January 3, 2000 / Accepted February 1, 2000     

Somatostatin concentration and binding in the rat hypothalamus and striatum during severe insulin-induced hypoglycemia

Hypoglycemia was induced by administration of insulin (40 I.U./kg) to 24 h fasted rats. Somatostatinlike immunoreactivity (SLI) and¹²⁵I-Tyr¹¹-somatostatin binding were measured in the striatum and hypothalamus at the onset of hypoglycemic coma (5–10 min). No significant changes in SLI concentration were detected in either site although the total number of specific somatostatin receptors in the striatum membranes, but not in the hypothalamus, decreased in insulin-injected rats when compared with the control group.     

Stimulatory role for brain serotoninergic system on prolactin secretion in the male rat.

Systemic administration of parachlorophenylalanine (PCPA, 100 mg/kg sc on alternate days X two times), a blocker of serotonin (5-HT) synthesis, considerably decreased brain 5-HT and plasma prolactin (PRL) levels in young male rats. Intraventricular (IVT) administration of 5,7-dihydroxytryptamine (5,7-DHT, 200 mug/20 mul), a neurotoxic drug which destroys 5-HT nerve terminals, induced, 3, 12, and 30 days after treatment, a marked depletion of brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) and considerably reduced plasma PRL levels at each time interval. Feeding of rat for up to 4 days with a tryptophan (TP)-deficient diet, caused a depletion of brain 5-HT and 5-HIAA contents and did not modify plasma PRL levels. Addition of TP (2 g/kg of diet) to the TP-deficient diet resulted in increased brain 5-HT and 5-HIAA contents and significantly increased PRL levels. These data provide evidence for the role of the 5-HT system in the maintenance of tonic PRL secretion.     

Simultaneous measurement of nitric oxide,blood glucose,and monoamines in the hippocampus of diabetic rat: an in vivo microdialysis study

The present study was undertaken to examine the relationships among the levels of nitric oxide (NO), monoamines, and blood glucose in the diabetic hippocampus. The levels of NO and monoamines (serotonin, 5-hydroxytryptamine [5-HT] and dopamine [DA]) were simultaneously measured in several experiments, using in vivo microdialysis techniques. We used both experimentally and spontaneously diabetic rats as the diabetic animal model, and compared the findings with those obtained from non-diabetic rats. The effects of the changed level of blood glucose due to insulin administration on the levels of NO, 5-HT, and DA were assessed. Total NO metabolite levels (NOx) were calculated as the sum of nitrite (NO2-) and nitrate (NO3-) levels. The results in the present study showed that: (1) the plasma levels of NOx in both diabetic rats were low compared to those in control rats, (2) the hippocampal NOx levels in both diabetic rats were almost the same as those in control rats, while the levels of 5-HT and DA were low in the diabetics, and (3) a sudden decrease in the plasma glucose level due to insulin administration reduced the NOx level as well as enhanced the 5-HT level in the diabetic hippocampus, a finding consistent with the results of 7 days administration of insulin. Taken together, these findings suggest that changes in the plasma glucose level cause, at least in part, the changes in the levels of NOx and monoamines in the diabetic brain.     

Plasma constituents and mortality in rat pups given chronic insulin via injection, pellet, or osmotic minipump

Two studies compared the glucose responses of 9-day-old rats given subcutaneous insulin, either continuously or via daily injection, for 10 days. In Experiment 1, implanted pellets released a total of 0, 1.9, or 5.7 U insulin/kg the first 24 h. Injected doses were larger, 0 or 8 U/kg. Injections caused no deaths, but insulin-releasing pellets caused high mortality within 24 h. Pups surviving the pellets were normoglycemic by treatment day 8. In Experiment 2, pups received 0.184 U of insulin daily, approximately 8 U/kg at 9 days, via either injection or osmotic minipump. All pups survived. Injected pups were hypoglycemic 2 h postinjection through treatment day 10, whereas pups with insulin minipumps were normoglycemic by day 5. Insulin injections, but not minipumps, lowered plasma triglycerides on day 10. To examine age differences in response to insulin, additional pups and adults received daily injections of 0 or 8 U/kg for 10 days. All survived. Insulin lowered plasma glucose more in pups than in adults and reduced triglycerides in pups but not in adults. The rapid development of normoglycemia in pups with insulin minipumps, compared with pups injected daily with the same dose, suggests that continuous early insulin may produce insulin resistance.