Insulin deprivation decreases insulin degrading enzyme levels in primary cultured cortical neurons and in the cerebral cortex of rats with streptozotocin-induced diabetes.

BACKGROUND: Many studies have indicated a relationship between diabetes and Alzheimer's disease (AD). However, the molecular mechanism underlying this association has not been clarified. Among several factors, insulin degrading enzyme (IDE), which plays roles in the degradation of both insulin and amyloid ß (Aß), has gained interest as a potential target in efforts to solve this puzzle. This study sought to examine the effects of varying insulin and/or glucose concentrations on IDE expression. METHODS: Experiments were performed on primary cultured rat neurons and cortices of rats with streptozotocin (STZ)-induced diabetes. IDE protein and mRNA expression levels were measured by western blot and RT-PCR, respectively. RESULTS: In primary cultured cortical neurons, removal of insulin for 5days reduced the expression of IDE. A five-day treatment with a high concentration of glucose in insulin-free media reduced IDE levels, while a high concentration of glucose in the presence of insulin had no effect. In groups treated with glucose or insulin intermittently, the reduction in IDE levels was observed only in neurons exposed to high glucose together with no insulin for 5days. Shorter incubation periods (48h), either continuously or intermittently, did not affect IDE levels. IDE expression in the cortex of rats with STZ-induced diabetes was found to be decreased. CONCLUSION: Our data suggest that insulin deprivation, rather than high glucose, is a significant determinant of IDE regulation. As evidence indicates potential roles for IDE in diabetes and AD, understanding the mechanisms regulating IDE expression may be important in developing new treatment strategies.

Lack of insulin results in reduced seladin-1 expression in primary cultured neurons and in cerebral cortex of STZ-induced diabetic rats.

Several studies demonstrated that Diabetes mellitus (DM) enhances the risk for Alzheimer's disease (AD). Although hyperglycemia and perturbed function of insulin signaling have been proposed to contribute to AD pathogenesis, the molecular mechanisms behind this association is not clear yet. Seladin-1 is an enzyme catalyzing the last step in cholesterol biosynthesis converting desmosterol to cholesterol. The neuroprotective function of seladin-1 has gained interest in AD research recently. Seladin-1 has anti-apoptotic properties and regulates the expression of ß-secretase (BACE-1). Here we measured seladin-1 mRNA and protein expressions in rat primary cultured neurons under diabetic conditions and also in the brains of rats with streptozotocine (STZ)-induced diabetes. We show that constant lack of insulin for 5days decreased seladin-1 levels in cultured rat primary neurons. Similarly, a decrease in seladin-1 was found in the brains of rats with STZ-induced diabetes. However, if the lack of insulin and/or high glucose treatment was intermittent, neuronal seladin-1 levels were not affected in vitro. On the other hand, treatment of neurons with metformin resulted in a significant increase in seladin-1. Constant lack of insulin for 5days, as well as high glucose treatment, increased the neuronal expression of BACE-1 in vitro, but not in the in vivo model. Our study defines insulin as a regulator of seladin-1 expression for the first time. The relevance of these findings for the association of DM with AD is discussed.

Effects of experimental diabetes on C/EBP proteins in rat hippocampus, sciatic nerve and ganglia.

Neurodegeneration is one of the most important complications of diabetes mellitus (DM). The exact mechanisms underlying neurodegeneration related to diabetic complications such as cognitive deficits and peripheral neuropathy are not clarified yet. Due to the fact that CCAAT/enhancer binding proteins (C/EBPs) have roles in cognitive functions, memory, synaptic plasticity, inflammation, lipid storage, and response to neurotrophic factors, it is possible to suggest that these transcription factors could have roles in neurodegeneration. Hence, in this study, the effects of experimental diabetes on C/EBPs in the hippocampus, sciatic nerve, and ganglia tissues were examined. After experimentally induced diabetes, immunoreactivity of related proteins was measured by western blotting. C/EBPα immunoreactivity in the hippocampus was not altered at 4-weeks but significantly decreased at 12-weeks of diabetes. C/EBPß immunoreactivity was not altered at 4-weeks whereas significantly increased at 12-weeks of diabetes. In the ganglion, C/EBPα immunoreactivity was significantly decreased in diabetes, but C/EBPß immunoreactivity was not affected. In the sciatic nerve, C/EBPα and ß immunoreactivities were significantly decreased in diabetic rats. Furthermore, insulin therapy prevented diabetes-induced alterations in C/EBPα and ß immunoreactivities. This study indicated, for the first time, that DM altered the immunoreactivity of C/EBPs in the nervous system. C/EBPs might be one of the important molecular targets which are responsible for neurodegeneration seen in diabetes.

Megestrol acetate inhibits the expression of cytoplasmic aromatase through nuclear C/EBPß in reperfusion injury-induced ischemic rat hippocampus.

Global ischemia after cardiac arrest, intraoperative hypoxia/hypotension, and hemorrhagic shock causes brain injury resulting in severe neurological and neurobehavioral deficits. Neurodegeneration can be prevented by local aromatase expression, and estrogen synthesis can be neuroprotective in ischemia/reperfusion. Therefore, aromatase, the enzyme that transforms androgens to estrogens, may be a potential target for the study of reperfusion injury after brain ischemia. We investigated the expression of aromatase and C/EBPß using western blotting in rat hippocampus after transient global ischemia plus hypotension. Immunohistochemical analysis was performed for aromatase. After 10min of ischemia, aromatase and C/EBPß expression in cytosolic extracts were observed after 10min and 24h of reperfusion. The expression of both proteins was similar in control and damaged tissues. Immunoblot analysis demonstrated that the highest aromatase expression appeared in damaged hippocampi after 1week and was gradually reduced after 2-10weeks. C/EBPß expression increased at 1week in nuclear extracts of damaged hippocampi. The aromatase inhibitor megestrol acetate (20mg/kg/day) suppressed aromatase and nuclear C/EBPß levels in ischemic hippocampi. Our findings indicate that ischemia as well as chronic neurodegenerative processes leads to an increase in cytoplasmic aromatase and nuclear C/EBPß. Thus, it is possible to hypothesize an interaction between this enzyme gene and transcription factor.

Diabetes alters aromatase enzyme levels in sciatic nerve and hippocampus tissues of rats.

Diabetes mellitus (DM) is associated with increased risk of impaired cognitive function. Diabetic neuropathy is one of the most common and important complications of DM. Estrogens prevent neuronal loss in experimental models of neurodegeneration and accelerate nerve regeneration. Aromatase catalyzes the conversion of androgens to estrogens and expressed in a variety of tissues including neurons. Although insulin is known to regulate the activity of aromatase there is no study about the effects of diabetes on this enzyme. Present study was designed to investigate the effects of experimental diabetes on aromatase expression in nervous system. Gender-based differences were also investigated. Rats were injected with streptozotocin to induce diabetes. At the end of 4 and 12 weeks sciatic nerve and hippocampus homogenates were prepared and evaluated for aromatase proteins. Aromatase expressions in sciatic nerves of both genders were decreased in 4 weeks of diabetes, but in 12 weeks the enzyme levels were increased in females and reached to control levels in male animals. Aromatase levels were not altered in hippocampus at 4 weeks but increased at 12 weeks in female diabetic rats. No significant differences were observed at enzyme levels of hippocampus in male diabetic rats. Insulin therapy prevented all diabetes-induced changes. In conclusion, these results indicated for the first time that, DM altered the expression of aromatase both in central and peripheral nervous systems. Peripheral nervous system is more vulnerable to damage than central nervous system in diabetes. These effects of diabetes differ with gender and compensatory neuroprotective mechanisms are more efficient in female rats.

Does tourniquet application alter the nitrergic responses of rabbit corpus cavernosum penis? A functional study.

OBJECTIVE: To investigate the effects of short and long periods of tourniquet application on corporal nerves, endothelium and smooth muscle responses. METHODS: After the rabbits were anesthetized with xylazine (5 mg/kg) and ketamine hydrochloride (35 mg/kg), a standard rubber circular band was applied to the base of the penis. After waiting for 20, 40 and 60 min, the tourniquets were removed and the penil tissue was reperfused for 5 min. In all groups, relaxation [carbachol, sodium nitroprusside (SNP) and electrical field stimulation (EFS) and contraction (phenylephrine and EFS)] responses were examined. In another set of experiments, the rabbits were killed 24 h after the tourniquet period of 60 min and carbachol-induced relaxation responses were obtained. RESULTS: SNP- and EFS-induced relaxation responses were similar in all groups. Carbachol-induced relaxation responses were not altered in tissues from 20 min tourniquet group, but they were significantly reduced in tissues from 40 and 60 min tourniquet group compared to that from control group. The impaired endothelium-mediated relaxation responses did not return to control levels after 24 h of reperfusion period. Neither phenylephrine nor EFS-mediated contraction responses were altered with tourniquet application. CONCLUSIONS: The results suggest that long period of tourniquet application altered endothelium-dependent muscarinic receptor-mediated relaxation responses. This is the first functional study that examined the effects of tourniquet application on corpus cavernosum tissue. In conclusion, it can be suggested that if tourniquet is necessary in penile surgery the application time of up to 20 min is more appropriate instead of prolonged usage.

Nicotine potentiates the nitrergic relaxation responses of rabbit corpus cavernosum tissue via nicotinic acetylcholine receptors.

The presence of neuronal nicotinic acetylcholine receptors in rabbit corpus cavernosum tissue and possible mechanisms underlying the potentiation of electrical field stimulation induced relaxation by nicotine were analyzed. In corpus cavernosum tissue strips nicotine (3 x 10(-5) M) and acetylcholine (10(-3) M) produced potentiation on electrical field stimulation (amplitude 50 V; frequency 4 Hz; width 0.8 ms) induced relaxation responses. This nicotine-induced potentiation was not altered by atropine (10(-6) M), guanethidine (5 x 10(-6) M) and indomethacin (10(-5) M), but abolished by hexamethonium chloride (10(-5) M) and L-nitro arginine methyl ester (10(-5) M). Nicotine did not cause any alteration on a single dose of carbachol (3 x 10(-5) M) and sodium nitroprusside (10(-5) M) induced relaxation responses. The results suggest that, nicotine-induced potentiation is NO and nicotinic acetylcholine receptor dependent but independent from prostaglandin synthesis, activation of muscarinic receptors and does not require intact adrenergic neurons. Nicotine did not affect smooth muscle and endothelium directly. In conclusion, in this study we showed for the first time that, nicotine acts on the nicotinic acetylcholine receptors located on the nitrergic nerves, thereby evoking the release of NO from these nerve terminals inducing relaxation response in rabbit corpus cavernosum tissue.

Impairment of endothelium- and nerve-mediated relaxation responses in the cavernosal smooth muscle of experimentally diabetic rabbits: role of weight loss and duration of diabetes.

The effects of short- and long-term experimental diabetes on corporal nerve, endothelium and smooth-muscle responses were investigated, and the reasons for possible alterations in corporal smooth muscle responses such as hyperglycaemia, duration of experimental diabetes and/or altered tissue weight were evaluated. Rabbits were injected with alloxan (125 mg/kg) to induce diabetes. Age-matched non-diabetic and diabetic (3 and 9 weeks) and weight-matched non-diabetic groups (9 weeks) were used as control. In all groups, relaxation (carbachol, electrical field stimulation and sodiumnitroprusside) responses were examined. The relaxation responses were expressed as percentage of the precontraction to phenylephrine and as g response/g tissue weight. The effects of elevated glucose were also examined by incubating cavernosal strips in Krebs-Henseleit solution containing 44.4 mM glucose for 6 h. Cavernosal tissues of non-diabetic and 9-week diabetic rabbits were evaluated histologically. Sodiumnitroprusside (10(-7)-10(-4) M) responses were similar in all groups. Relaxation responses to electrical field stimulation (10 s train; amplitude 50 V; frequency 0.5-32 Hz; width 0.8 ms) were only attenuated in the 9-week diabetic group compared to the non-diabetic group. Carbachol (10(-8)-3 x 10(-5) M) responses were attenuated in both diabetic groups. When the relaxation responses expressed as g response/g tissue weight were evaluated, results were similar compared to those expressed as percentage of phenylephrine (10(-5) M). Neither carbachol nor electrical field stimulation mediated responses were impaired with glucose incubation. No morphological degenerations were observed in the endothelium. Diabetes may interfere with the synthesis and/or release of nitric oxide from both nerves and endothelium in corpus cavernosum, and alterations in endothelium-derived responses occur earlier than neurological disturbances. The sensitivity of cavernosal smooth muscle to nitric oxide did not alter in diabetes. Attenuation of responses was not due to decreased tissue weight caused by diabetes.

Two new nitric oxide synthase inhibitors: pyridoxal aminoguanidine and 8-quinolinecarboxylic hydrazide selectively inhibit basal but not agonist-stimulated release of nitric oxide in rat aorta.

Structural modification at one of the guanidine nitrogens of L-arginine has led to the development of a number of compounds N(G)-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine (L-NOARG), N(G)-nitro-L-arginine methyl ester (L-NAME) that competitively inhibit nitric oxide synthase (NOS). It was reported that another chemically related compound known as a glycation inhibitor, aminoguanidine also inhibits NOS. Recently, two new glycation inhibitors, structurally related to aminoguanidine (AG), pyridoxal aminoguanidine (PLAG) and 8-quinoline carboxylic hydrazide (8Q) were synthesized. In this study, the effects of these two inhibitors on responses mediated by constitutive nitric oxide (NO) were investigated in vitro. For this purpose, in the present study vascular responses to phenylephrine and acetylcholine in isolated aortas were evaluated. Incubation (15 min) with PLAG and 8Q (10(-4)M for each) induced potentiation of phenylephrine-induced contraction in endothelium intact but not in endothelium denuded rings of rat aorta. The ability of PLAG or 8Q to augment phenylephrine-induced tone in endothelium containing rings was completely prevented by preincubation with L-arginine (1mM), but not with D-arginine. Both compounds (PLAG, 8Q) did not affect acetylcholine-induced relaxation. These results suggest that both of the new compounds produced a selective inhibition of basal but not agonist stimulated production of nitric oxide in rat aorta.

Omeprazole-induced relaxation in rat aorta is partly dependent on endothelium.

We investigated the effect of omeprazole (1 x 10(-5)-3 x 10(-4)M), an inhibitor of H(+),K(+)-ATPase, on rat aortic rings pre-contracted with phenylephrine (10(-6)M). Omeprazole relaxed the tissue in a concentration-dependent manner. Either removal of the endothelium or incubation with nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5)M) significantly attenuated the relaxations. Pre-treatment with L-arginine (10(-3)M), but not with D-arginine, reversed the inhibitory action of L-NAME. Indomethacin (10(-6)M) and tetraethylammonium (TEA, 10(-2)M) did not affect the relaxant responses to omeprazole indicating the lack of involvement of cyclooxygenase products and K(+) channels, respectively. These results suggest a role of NO in the mechanism of action of omeprazole.

The spin trapping agent PBN stimulates H2 O2 -induced Erk and Src kinase activity in human neuroblastoma cells.

The spin-trap, alpha-phenyl-N-tert-butylnitrone (PBN) has been shown to have neuroprotective properties and may prevent oxidative injury in vivo and in cultured cells. Although PBN quenches reactive oxygen species, the direct mechanism of neuroprotective action is unknown. In the present study, we examined the effects of PBN on the regulation of the mitogen activated kinase Erk and as well as Src family tyrosine kinases, enzymes known to be activated by oxygen species such as H2O2. In SH-SY5Y human neuroblastoma cells, H2O2 induced activation of Erk and Src kinases was markedly potentiated by treatment with PBN. The potentiation by PBN of the Erk and Src kinase activation by H2O2 required extracellular Ca2+ and appeared dependent on voltage sensitive Ca2+ channels. In contrast, PBN did not affect depolarization-dependent or growth factor-dependent Erk and Src kinase phosphorylation. Our results suggest that PBN might have a protective effect on cells by potentiating the anti-apoptotic Erk and Src kinase pathways responding to H2O2, an effect apparently distinct from its ability to trap oxygen free radicals.