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.

Determination of albendazole sulfoxide in human plasma by using liquid chromatography-tandem mass spectrometry.

A rapid, simple and sensitive method was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for determination of albendazole sulfoxide (ABZOX) in human plasma. The plasma samples were extracted by protein precipitation using albendazole sulfoxide-d3 as internal standard (IS). The chromatographic separation was performed on Waters Xbridge C18Column (100×4.6mm, 3.5µm) with a mobile phase consisting of ammonia solution, water and methanol at a flow rate of 0.70mL/min. ABZOX was detected and identified by mass spectrometry with electrospray ionization (ESI) in positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 3-1500ng/mL for ABZOX. This method was successfully applied to the bioequivalence study in human plasma samples.

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.

Preparation and in vitro/in vivo evaluation of microparticle formulations containing meloxicam.

In this study, we have formulated chitosan-coated sodium alginate microparticles containing meloxicam (MLX) and aimed to investigate the correlation between in vitro release and in vivo absorbed percentages of meloxicam. The microparticle formulations were prepared by orifice ionic gelation method with two different sodium alginate concentrations, as 1% and 2% (w/v), in order to provide different release rates. Additionally, an oral solution containing 15 mg of meloxicam was administered as the reference solution for evaluation of in vitro/in vivo correlation (ivivc). Following in vitro characterization, plasma levels of MLX and pharmacokinetic parameters [elimination half-life (t(1/2)), maximum plasma concentration (C(max)), time for C(max) (t(max))] after oral administration to New Zealand rabbits were determined. Area under plasma concentration-time curve (AUC(0-∞)) was calculated by using trapezoidal method. A linear regression was investigated between released% (in vitro) and absorbed% (in vivo) with a model-independent deconvolution approach. As a result, increase in sodium alginate content lengthened in vitro release time and in vivo t(max) value. In addition, for ivivc, linear regression equations with r(2) values of 0.8563 and 0.9402 were obtained for microparticles containing 1% and 2% (w/v) sodium alginate, respectively. Lower prediction error for 2% sodium alginate formulations (7.419 ± 4.068) compared to 1% sodium alginate formulations (9.458 ± 5.106) indicated a more precise ivivc for 2% sodium alginate formulation.

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.