Spontaneously hypertensive and Wistar Kyoto rats are genetically disparate.

Spontaneously hypertensive rats (SHR) are one of the most common animal models used to study essential hypertension in humans. Because SHR and normotensive Wistar Kyoto (WKY) rats were both established from the same parental, normotensive Wistar stock, WKY animals have been used almost exclusively as control animals in studies of SHR. Recently, the suitability of WKY rats as normotensive controls for SHR has been challenged. To establish whether or not SHR and WKY rats share the same immunologic backgrounds, we initially performed a series of skin grafting experiments on these animals. In all cases, grafts of SHR donor skin to WKY recipients and of WKY donor skin to SHR recipients resulted in complete rejection within 7 to 10 days. In addition, grafts of WKY donor skin to other WKY recipients resulted in graft rejection. By contrast, skin grafts between SHRs were always accepted. To further characterize the genetic distinctions between SHR and WKY rats, allelic profiles based on a series of immunologic and biochemical markers were established for each strain. These findings clearly establish that SHR and WKY rats differ at the major histocompatibility complex, in specific blood group antigens, and in a panel of isozymic markers. Moreover, whereas SHRs have the same genetic profiles irrespective of source, some colonies of WKY rats are outbred, as judged by their variant allelic profiles.     

Brain arachidonic acid incorporation is decreased in heart fatty acid binding protein gene-ablated mice

Heart fatty acid binding protein (H-FABP) is expressed in neurons, but its role in brain fatty acid incorporation and metabolism is poorly defined. We examined the effect of H-FABP gene ablation on brain incorporation of arachidonic ([1-(14)C]20:4n-6) or palmitic ([1-(14)C]16:0) acid in vivo. Analysis of brain mRNA confirmed gene ablation and demonstrated no compensatory changes in the levels of other FABP mRNA in the gene-ablated mice. In brains from H-FABP gene-ablated mice, the incorporation coefficient for [1-(14)C]20:4n-6 was reduced 24%, while that for [1-(14)C]16:0 was unaffected. Within the organic and aqueous fractions, significantly more [1-(14)C]20:4n-6 was distributed into the aqueous fraction, suggesting a disruption in the metabolic targeting of 20:4n-6 in these mice. There was less incorporation of [1-(14)C]20:4n-6 into total phospholipids and a marked reduction (51%) in the level of incorporation into the choline glycerophospholipids (ChoGpl). Because FABP can influence steady-state lipid mass, brain individual lipid masses were measured. The brain total phospholipid mass was reduced 17% by gene ablation, ascribed to a 27% and 32% reduction in the masses of ChoGpl and sphingomyelin, respectively. Plasmalogen subclass masses were also reduced, suggesting that H-FABP may augment brain plasmalogen synthesis. In gene-ablated mice, the phosphatidylinositol 20:4n-6 level was reduced 25%, while the proportion of total n-6 fatty acids was reduced in the major phospholipid classes. Thus, these results demonstrate for the first time that H-FABP expression influences brain 20:4n-6 uptake and trafficking as well as steady-state brain lipid levels.     

Dysfunction of supramedullary alpha-adrenergic mechanisms following sino-aortic denervation in Kyoto Wistar rats

Central α-adrenergic mechanisms of blood pressure regulation were investigated by injecting norepinephrine or bradykinin into the carotid input of the cross-circulated head preparations of normotensive Wistar Kyoto rats (WKY). Rats were divided into three groups: sham-operated (sham), carotid sinuses denervated (SD) and carotid sinuses and aortic nerves debuffered (SAD). Norepinephrine, 5 μg, produced vasodepression in all rats, accompanied by corresponding decreases in sympathetic nerve activity recorded in some rats. Magnitude of vasodepression was largest in SAD rats. In sham rats, bradykinin, 1 μg, produced a biphasic response:initial vasodepression followed by a sustained pressor phase. This was accompanied by corresponding changes in peripheral sympathetic nerve activity recorded in some rats. In both SAD and SD rats bradykinin-induced vasodepression was abolished, while the magnitude of the pressor phase became more prominent. The increase in the pressor phase was greater in SAD than in SD rats. In similar studies of spontaneously hypertensive rats (SHR), responses to both α-adrenergic agonist and bradykinin are augmented, suggesting a dysfunction of hypothalamic α-adrenergic mechanisms. Since in the present study it has been shown that sino-aortic denervation produces effects similar to those seen in SHR, dysfunction of buffer nerves may account for the deficient central α-adrenergic mechanisms in SHR.     

Metabolic performance and insulin binding of mammary cells isolated from Wistar and Zucker rats

A method for the isolation of cells from lactating mammary gland tissue of rats is described. Metabolic properties of mammary cells isolated by the described method either from Wistar of Zucker rats as well as insulin binding were studied. Whereas synthesis of protein and lipid showed no significant differences, the rate of glucose oxidation is slightly increased in cells isolated from Zucker rats. Incorporation of (14)C-uridine in the RNA-fraction is stimulated by insulin; the half-maximal concentration is given by 5 x 10(-10) mol/1). Specific binding of insulin to both types of cells can be described by a nonlinear Scatchard-plot. The apparent affinity constants for the high affinity receptors (1 x 10(-10) mol/1) correlate well with the described metabolic effect of insulin on RNA-synthesis. The total capacity for insulin binding is reduced in cells isolated from Zucker rats when compared to those from Wistar rats.     

Inhalation of Roman chamomile essential oil attenuates depressive-like behaviors in Wistar Kyoto rats

The idea of aromatherapy, using essential oils, has been considered as an alternative antidepressant treatment. In the present study, we investigated the effect of Roman chamomile essential oil inhalation for two weeks on depressive-like behaviors in Wistar-Kyoto(WKY) rats. We found that inhalation of either Roman chamomile or one of its main components α-pinene,attenuated depressive-like behavior in WKY rats in the forced swim test. Using isobaric tags for relative and absolute quantitation analysis(iTRAQ), we found that inhalation of α-pinene increased expression of proteins that are involved in oxidative phosphorylation, such as cytochrome c oxidase subunit 6C-2, cytochrome c oxidase subunit 7A2, ATPase inhibitor in the hippocampus, and cytochrome c oxidase subunit 6C-2, ATP synthase subunit e, Acyl carrier protein, and Cytochrome b-c1 complex subunit 6 in the PFC(prefrontal cortex). In addition, using the quantitative real-time polymerase chain reaction technique, we confirmed an increase of parvalbumin mRNA expression in the hippocampus, which was shown to be upregulated by 2.8-fold in iTRAQ analysis, in α-pinene treated WKY rats. These findings collectively suggest the involvement of mitochondrial functions and parvalbumin-related signaling in the antidepressant effect of α-pinene inhalation.     

Hepatic biotransformation in lean and obese Wistar Kyoto rats: comparison to that in streptozotocin-pretreated Sprague-Dawley rats

1. Phase I and phase II biotransformation was compared in streptozotocin-induced hypoinsulinemic (STZ) and genetic hyperinsulinemic (WKY-fatty) rats. 2. Total cytochrome P-450 concentrations were reduced in both STZ and WKY, whereas styrene oxide hydrolase and benzphetamine N-demethylase activities were normal in STZ and reduced in WKY. 3. UDP-glucuronosyltransferase activity was decreased toward testosterone and 1-naphthol in STZ and WKY, and was increased toward estrone in the obese female WKY. 4. Glutathione S-transferase activity was decreased in STZ toward 1-chloro-2,4-dinitrobenzene, ethacrynic acid and sulfobromophthalein, but was similar to that in normal rats for WKY.     

Hypothesis with abnormal amino acid metabolism in depression and stress vulnerability in Wistar Kyoto rats

While abnormalities in monoamine metabolism have been investigated heavily per potential roles in the mechanisms of depression, the contribution of amino acid metabolism in the brain remains not well understood. In additional, roles of the hypothalamus–pituitary–adrenal axis in stress-regulation mechanisms have been of much focus, while the contribution of central amino acid metabolism to these mechanisms has not been well appreciated. Therefore, whether depression-like states affect amino acid metabolism and their potential roles on stress-regulatory mechanisms were investigated by comparing Wistar Kyoto rats, which display depression-like behaviors and stress vulnerability, to control Wistar rats. Brain amino acid metabolism in Wistar Kyoto rats was greatly different from normal Wistar rats, with special reference to lower cystathionine and serine levels. In addition, Wistar Kyoto rats demonstrated abnormality in dopamine metabolism compared with Wistar rats. In the case of stress response, amino acid levels having a sedative and/or hypnotic effect were constant in the brain of Wistar Kyoto rats, though these amino acid levels were reduced in Wistar rats under a stressful condition. These results suggest that the abnormal amino acid metabolism may induce depression-like behaviors and stress vulnerability in Wistar Kyoto rats. Therefore, we hypothesized that abnormalities in amino acid and monoamine metabolism may induce depression, and amino acid metabolism in the brain may be related to stress vulnerability.     

Insulin binding to erythroblastic leukemic cells is decreased by insulin and increased by amino acids

Erythroblastic leukemic cells incubated in media supplemented with high amino acid concentrations subsequently bound 55.5% more [¹²⁵I]insulin than cells incubated in low amino acid media. Cycloheximide (1 μg/ml) abolished this effect. Incubation of cells with physiological (100 μU/ml) doses of insulin in low amino acid media decreased insulin binding by 34.7%. In high amino acid media the same dose of insulin decreased insulin binding by 33% compared to supplemented media without insulin but increased binding by 22.5% compared to basal media without insulin. The data suggest that amino acids or specific amino acids may exert a regulatory influence on insulin receptor homeostasis.     

Brain hydrogen sulfide is severely decreased in Alzheimer's disease

Although hydrogen sulfide (H2S) is generally thought of in terms of a poisonous gas, it is endogenously produced in the brain from cysteine by cystathionine beta-synthase (CBS). H2S functions as a neuromodulator as well as a smooth muscle relaxant. Here we show that the levels of H2S are severely decreased in the brains of Alzheimer's disease (AD) patients compared with the brains of the age matched normal individuals. In addition to H2S production CBS also catalyzes another metabolic pathway in which cystathionine is produced from the substrate homocysteine. Previous findings, which showed that S-adenosyl-l-methionine (SAM), a CBS activator, is much reduced in AD brain and that homocysteine accumulates in the serum of AD patients, were confirmed. These observations suggest that CBS activity is reduced in AD brains and the decrease in H2S may be involved in some aspects of the cognitive decline in AD.     

Sensitivity of spontaneously hypertensive and of Wistar Kyoto rats to the antidipsogenic action of eledoisin

This study investigated the sensitivity of spontaneously hypertensive rats (SHR) and of Wistar Kyoto rats (WKR) to the antidipsogenic action of the tachykinin eledoisin (ELE). Drinking was evoked by: (a) intracerebroventricular (i.c.v.) injection of angiotensin II, (b) subcutaneous (s.c.) administration of hypertonic NaCl (1.5 M; 1 ml/100 g b.wt.) or (c) 18 h of water deprivation with free access to food. In accordance with previous studies, the dipsogenic effect of all three treatments was exaggerated in the SHR. And when treated with i.c.v. ELE (12.5-25 ng/rat) they were far less sensitive than WKR to its antidipsogenic action on angiotensin-induced drinking. Smaller differences in strain sensitivity were also observed for the effect of ELE on cell dehydration- and on water deprivation-induced drinking, but only at the dose of 200 and 50 ng/rat, respectively. The different sensitivity of the SHR to the antidipsogenic effect of ELE supports the idea that tachykininergic mechanisms for control of water intake are differently regulated in the SHR than they are in the normotensive WKR.     

Production of lysophosphatidic acid by lysophospholipase D in incubated plasma of spontaneously hypertensive rats and Wistar Kyoto rats.

Lysophosphatidic acid has been identified as a vasopressor principle in incubated mammalian plasma and sera, and shown to be generated extracellulary by lysophospholipase D-like activity. In this study, we monitored the time course of changes in the major phospholipid fractions during incubation of plasma, and found that polyunsaturated lysophosphatidic acids accumulate more rapidly than saturated lysophosphatidic acids at expense of the corresponding lysophosphatidylcholines. We compared the phospholipase activities for producing bioactive LPA in age-matched spontaneously hypertensive rats and Wistar Kyoto rats. The lysophospholipase D activity in rat plasma was found to be independent of strain and age. We suggest that lysophospholipase D functions in rat for persistent production of bioactive LPA in the circulation throughout life. However, our finding that production of LPA in spontaneously hypertensive rats was not greater than that in Wistar Kyoto rats does not seem to support the idea that increased production of LPA is involved in the pathogenesis of hypertension.     

Ghrelin infused into the portal vein inhibits glucose-stimulated insulin secretion in Wistar rats

Although accumulating evidence has shown crucial roles of ghrelin and insulin in food intake and energy metabolism, the exact relationship between these hormones remains unclear. In this study, we determined the in vivo effect of ghrelin on insulin secretion. We demonstrated that ghrelin inhibited the glucose-stimulated release of insulin when infused into the portal vein of Wistar rats. However, ghrelin infusion into the femoral vein did not induce such an inhibitory effect. Hepatic vagotomy or coinfusion with atropine methyl bromide diminished the inhibitory effect of ghrelin on glucose-stimulated insulin secretion. In conclusion, ghrelin exerts an inhibitory effect on glucose-stimulated insulin secretion via the hepatic portal system and the vagus nerve. The decrease in ghrelin level after a meal is important for the occurrence of the incretin effect in rats.     

Hyperinsulinemia in transgenic mice carrying multiple copies of the human insulin gene

We are investigating human insulin gene expression in transgenic mice. An 8.8 kilobase (kb) human genomic DNA fragment, including the insulin gene (1.4 kb) and 2 kb of 5' human flanking sequences, was introduced into mouse embryos by pronuclear microinjection. Two lines of transgenic mice have been established, both of which carry the intact human gene in multiple copies. Animals from both lines have significantly higher insulin levels than control mice, and the degree of hyperinsulinemia shows a positive correlation with human gene copy number in the two lines. Expression of the human gene is confirmed by the detection of human C-peptide in plasma. Tissue specificity of expression is maintained, with human insulin mRNA detectable only in the pancreas. The transgenics maintain normal fasting blood glucose in spite of their high insulin levels, but preliminary studies show them to be glucose intolerant when given a glucose load. These mice provide a model system for further studies on the regulation of insulin gene expression and on the effects of chronic hyperinsulinemia on glucose homeostasis.     

Ascorbate uptake is decreased in the hippocampus of ageing rats

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H(2)O(2)-induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H(2)O(2)) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells.     

Effect of angiotensin AT2 receptor stimulation on vascular cyclic GMP production in normotensive Wistar Kyoto rats

In the present study in normotensive Wistar Kyoto rats (WKY), we investigated whether any angiotensin II (ANG II) increases in vascular cyclic GMP production were via stimulation of AT(2) receptors. Adult WKY were infused for 4h with ANG II (30 ng/kg per min, i.v.) or vehicle (0.9% NaCl, i.v.) after pretreatment with (1) vehicle, (2) losartan (100 mg/kg p.o.), (3) PD 123319 (30 mg/kg i.v.), (4) losartan+PD 123319, (5) icatibant (500 microg/kg i.v.), (6) L-NAME (1 mg/kg i.v.), (7) minoxidil (3 mg/kg i.v.). Mean arterial blood pressure (MAP) was continuously monitored, and plasma ANG II and aortic cyclic GMP were measured at the end of the study. ANG II infusion over 4h raised MAP by a mean of 13 mmHg. This effect was completely prevented by AT(1) receptor blockade. PD 123319 slightly attenuated the pressor effect induced by ANG II alone (123.4+/-0.8 versus 130.6+/-0.6) but did not alter MAP in rats treated simultaneously with ANG II + losartan (113+/-0.6 versus 114.3+/-0.8). Plasma levels of ANG II were increased 2.2-3.7-fold by ANG II infusion alone or ANG II in combination with the various drugs. The increase in plasma ANG II levels was most pronounced after ANG II+losartan treatment but absent in rats treated with losartan alone. Aortic cyclic GMP levels were not significantly changed by either treatment. Our results demonstrate that the AT(2) receptor did not contribute to the cyclic GMP production in the vascular wall of normotensive WKY.     

Concentrated loss of insulin secretion in Wistar rats with normal glucose tolerance

Rats with decreased insulin response and with normal glucose tolerance were concentrated by repeated selective breeding of normal Wistar rats with low insulinogenic index. In general, the mean insulinogenic index of the inbred offsprings showed a tendency to decrease more than their parents generation. Thus mean insulinogenic indices in second (F2), third (F3) and fourth (F4) generations were significantly reduced more than the normal rats without glucose intolerance. Pancreatic islets from the F3 and F4 rats lost partially their ability to release insulin at 20 mM glucose in vitro. It is suggested that a defect responsible for the decreased insulin response in the F2, F3 and F4 rats resulted from a loss of the ability to secrete insulin in each islet, and that this defect was concentrated by repeated selective breeding of normal Wistar rats.     

Protein kinase C activity in platelets from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY)

Calcium-activated phospholipid dependent protein kinase (protein kinase C) activity in platelets was measured in 4, 12, and 20-week-old SHR and WKY. At age 4-weeks, there was no significant difference in protein kinase C activity and systolic blood pressure between SHR and WKY. In 12 and 20-week-old SHR, both protein kinase C activity and systolic blood pressure were significantly higher than in the age-matched WKY. These results suggest that protein kinase C may be involved in the control of blood pressure in SHR and WKY.     

Mechanism of decreased insulinogenesis in manganese-deficient rats. Decreased insulin mRNA levels

Manganese-deficient rats exhibited seven-fold lower preproinsulin mRNA levels compared to control, as detected by dot blot hybridization of both total and poly(A)+ RNA using a preproinsulin cDNA probe. No differences in the size of the insulin mRNA were observed. Thus, decreased mRNA levels may be a major contributing factor to the decreased insulinogenesis observed in manganese-deficient rats.