How Does Glycation Affect Binding Parameters of the Albumin-Gliclazide System in the Presence of Drugs Commonly Used in Diabetes? In Vitro Spectroscopic Study.

In this research, the selected drugs commonly used in diabetes and its comorbidities (gliclazide, cilazapril, atorvastatin, and acetylsalicylic acid) were studied for their interactions with bovine serum albumin-native and glycated. Two different spectroscopic methods, fluorescence quenching and circular dichroism, were utilized to elucidate the binding interactions of the investigational drugs. The glycation process was induced in BSA by glucose and was confirmed by the presence of advanced glycosylation end products (AGEs). The interaction between albumin and gliclazide, with the presence of another drug, was confirmed by calculation of association constants (0.11-1.07 × 104 M-1). The nature of changes in the secondary structure of a protein depends on the drug used and the degree of glycation. Therefore, these interactions may have an influence on pharmacokinetic parameters.

Anemarrhenae asphodeloides rhizoma Extract Enriched in Mangiferin Protects PC12 Cells against a Neurotoxic Agent-3-Nitropropionic Acid.

The rhizome of Anemarrhena asphodeloides Bunge, used in Traditional Chinese Medicine as a brain function-improving herb, is a promising source of neuroprotective substances. The aim of this study was to evaluate the protective action of xanthones from A. asphodeloides rhizomes on the PC12 cell line exposed to the neurotoxic agent-3-nitropropionic acid (3-NP). The xanthone-enriched fraction of the ethanolic extract of A. asphodeloides (abbreviated from now on as XF, for the Xanthone Fraction), rich in polyphenolic xanthone glycosides, in concentrations from 5 to 100 µg/mL, and 3-NP in concentrations from 2.5 to 15 mM, were examined. After 8, 16, 24, 48, and 72 h of exposure of cells to various combinations of 3-NP and XF, the MTT viability assay was performed and morphological changes were estimated by confocal fluorescence microscopy. The obtained results showed a significant increase in the number of cells surviving after treatment with XF with exposure to neurotoxic 3-NP and decreased morphological changes in PC12 cells in a dose and time dependent manner. The most effective protective action was observed when PC12 cells were pre-incubated with the XF. This effect may contribute to the traditional indications of this herb for neurological and cognitive complaints. However, a significant cytotoxicity observed at higher XF concentrations (over 10 µg/mL) and longer incubation time (48 h) requires caution in future research and thorough investigation into potential adverse effects.

The Dephosphorylation of p70S6 (Thr389) Kinase as a Marker of L-Glutamate-Induced Excitotoxicity Related to Diabetes Disturbances-an Unconventional In Vitro Model.

Excitotoxicity is a modern clinical condition included in the pathogenesis of Alzheimer's disease. It is connected with diabetic disturbance, and it is still being analyzed in the context of the participation of the PI3K/mTOR pathway. A very important protein belonging to this pathway is p70S6K, whose activation promotes the pathogenesis of type 2 diabetes by the induction of insulin resistance. The study model was based on a PC12 cell line, derived from the pheochromocytoma of a rat adrenal medulla, cultured in RPMI 1640. The three reagents were used in different concentrations to create the model of excitotoxicity related to diabetes disturbances: L-glutamate (2.5 mM; 10 mM), glucose (150 mM; 200 mM), and insulin (0.093 mM; 0.371 mM). The aim of our study was to examine and evaluate the levels of phosphorylation of proteins involved in signal transduction controlled by MAPK, PI3K/Akt, and mTOR signaling pathways in L-glutamate-induced excitotoxicity with comorbid hyperglycemia and hyperinsulinemia imitating diabetic disturbances in in vitro conditions on PC12 cells. The results we obtained demonstrated the increased phosphorylation of p70S6K in Thr389 residue in almost all combinations of reagents, except for those including the highest concentration of L-glutamate, in which dephosphorylation was confirmed. This confirms the inhibition of mTOR kinase and suggests that p70S6K (Thr389) plays a functional role in the regulation of the signaling pathway in excitotoxicity related to diabetic disturbances.

Neurometabolic Evidence Supporting the Hypothesis of Increased Incidence of Type 3 Diabetes Mellitus in the 21st Century.

The most recent evidence supports the existence of a link between type 2 diabetes (T2DM) and Alzheimer's Disease (AD), described by the new term: type 3 diabetes (T3D). The increasing incidence of T2DM in the 21st century and accompanying reports on the higher risk of AD in diabetic patients prompts the search for pathways linking glycemia disturbances and neurodegeneration. It is suggested that hyperglycemia may lead to glutamate-induced excitotoxicity, a pathological process resulting from excessive depolarization of membrane and uncontrolled calcium ion influx into neuronal cells. On the other hand, it has been confirmed that peripheral insulin resistance triggers insulin resistance in the brain, which may consequently contribute to AD by amyloid beta accumulation, tau phosphorylation, oxidative stress, advanced glycation end products, and apoptosis. Some literature sources suggest significant amylin involvement in additional amyloid formation in the central nervous system, especially under hyperamylinemic conditions. It is particularly important to provide early diagnostics in people with metabolic disturbances, especially including fasting insulin and HOMA-IR, which are necessary to reveal insulin resistance. The present review reveals the most recent and important evidence associated with the phenomenon of T3D and discusses the potential lacks of prevention and diagnostics for diabetes which might result in neurometabolic disorders, from a pharmacotherapy perspective.

Ex vivo model for the assessment of the cytotoxicity of biological materialfrom patients with carbohydrate metabolism disturbances.

BACKGROUND: Carbohydrate metabolism disturbances have long been considered the cause of civilisation diseases, such as type 2 diabetes, obesity, or cardiovascular diseases. Currently an increasing number of theses also link impaired glucose and/or insulin metabolism to neurodegenerative diseases, calling them neurometabolic diseases. AIM OF THE STUDY: Aim of the study was to assess the cytotoxic influence of multicompound biological material (blood serum) from people with different carbohydrate metabolism disturbances to the viability of PC12 cell line. MATERIAL AND METHODS: Undifferentiated PC12 cell line were incubated for 48 hours in standard conditions with the addition of human serum from individuals with diffrent (low and high) levels of hyperglycaemia (LGL and HGL) and hyperinsulinaemia (LIL and HIL). The cytotoxicity was estimated by the MTT test, and the viability percentage (SP%) was calculated in relation to control samples (cells incubated only with RPMI). RESULTS: The obtained results indicate cytotoxic activity and decreased viability of the PC12 cells after 48 hours of incubation with human serum with different degrees of hyperglycaemia and insulinaemia. Cell viability increased slightly with the increase in glucose level but decreased with the increase in insulin concentration in individual groups, but without statistical significance. CONCLUSIONS: Blood serum, as multicompound biological material, influences negatively PC12 cell line but in a variety of ways. Increasing hyperinsulinaemia has a higher cytotoxic effect on the cells than hyperglycaemia, which probably results from the fact that it is compensated by other components of biological material; however, further studies are necessary to obtain more detailed characteristics of these processes.

The Antiglycoxidative Ability of Selected Phenolic Compounds-An In Vitro Study.

Hyperglycemia and oxidative stress may be observed in different diseases as important factors connected with their development. They often occur simultaneously and are considered together as one process: Glycoxidation. This can influence the function or structure of many macromolecules, for example albumin, by changing their physiological properties. This disturbs the homeostasis of the organism, so the search for natural compounds able to inhibit the glycoxidation process is a current and important issue. The aim of this study was the examination of the antiglycoxidative capacity of 16 selected phenolic compounds, belonging to three phenolic groups, as potential therapeutic agents. Their antiglycoxidative ability, in two concentrations (2 and 20 µM), were examined by in vitro study. The inhibition of the formation of both glycoxidative products (advanced glycation end products (AGEs) and advanced oxidation protein products (AOPPs)) were assayed. Stronger antiglycoxidative action toward the formation of both AOPPs and AGEs was observed for homoprotocatechuic and ferulic acids in lower concentrations, as well as catechin, quercetin, and 8-O-methylurolithin A in higher concentrations. Homoprotocatechuic acid demonstrated the highest antiglycoxidative capacity in both examined concentrations and amongst all of them. A strong, significant correlation between the percentage of AOPPs and AGEs inhibition by compounds from all phenolic groups, in both examined concentrations, was observed. The obtained results give an insight into the antiglycoxidative potential of phenolic compounds and indicate homoprotocatechuic acid to be the most promising antiglycoxidative agent, but further biological and pharmacological studies are needed.

The effect of glycation on bovine serum albumin conformation and ligand binding properties with regard to gliclazide.

Albumin, the major serum protein, plays a variety of functions, including binding and transporting endogenous and exogenous ligands. Its molecular structure is sensitive to different environmental modifiers, among which glucose is one of the most significant. In vivo albumin glycation occurs under physiological conditions, but it is increased in diabetes. Since bovine serum albumin (BSA) may serve as a model protein in in vitro experiments, we aimed to investigate the impact of glucose-mediated BSA glycation on the binding capacity towards gliclazide, as well as the ability of this drug to prevent glycation of the BSA molecule. To reflect normo- and hyperglycemia, the conditions of the glycation process were established. Structural changes of albumin after interaction with gliclazide (0-14µM) were determined using fluorescence quenching and circular dichroism spectroscopy. Moreover, thermodynamic parameters as well as energy transfer parameters were determined. Calculated Stern-Volmer quenching constants, as well as binding constants for the BSA-gliclazide complex, were lower for the glycated form of albumin than for the unmodified protein. The largest, over 2-fold, decrease in values of binding parameters was observed for the sample with 30mM of glucose, reflecting the poorly controlled diabetic state, which indicates that the degree of glycation had a critical influence on binding with gliclazide. In contrast to significant changes in the tertiary structure of BSA upon binding with gliclazide, only slight changes in the secondary structure were observed, which was reflected by about a 3% decrease of the α-helix content of glycated BSA (regardless of glucose concentration) in comparison to unmodified BSA. The presence of gliclazide during glycation did not affect its progress. The results of this study indicate that glycation significantly changed the binding ability of BSA towards gliclazide and the scale of these changes depended on glucose concentration. It may have a direct impact on the free drug fraction and its pharmacokinetic behavior, including the risk of hypoglycemic episodes or unexpected interactions with other ligands. The use of BSA in examining binding effects upon glycation seems to be good model for preliminary research and may be used to identify a potential drug response in a diabetic state.

Epigenetic modifications: An important mechanism in diabetic disturbances.

In the search for explanations of diabetes pathomechanisms, especially the development of its vascular complications (micro- and macrovascular ), although current, good metabolic control of diabetes, attention was drawn to the role of epigenetic inheritance associated with epigenetic modifications of histone proteins and DNA in hyperglycemia conditions. This study showed the significant role of DNA methylation and histone epigenetic modifications (a different nature and a different degree) in the transmission of information that is not connected with gene inheritance but concerns the persistent changes induced by hyperglycemia..Attention was paid to the role of DNA methylation of pancreatic cells in the pathogenesis of type 1 diabetes, but also type 2. The important role of DNA methylation changes in a so-called intrauterine growth restriction (IUGR) as reason of subsequent development of diabetes was particularly emphasized. In the pathogenesis of type 2 diabetes and its complications, especially microvascular complications, the greatest share and importance of epigenetic modifications on mitochondrial DNA metylation are the most important. The multidirectionality Complicaand complexity of epigenetic modifications of histone proteins indicate their importance in the development of diabetic disturbances. An especially important role is attributed to methylation and acetylation of histone proteins, in particular on arginine and lysine, whose changes occur most frequently. Moreover, epigenetic modifications of the enzymes, especially methylases, responsible for these processes are the underlying. It has been indicated that the identification of epigenetic differences within the DNA or histone proteins may be a useful prognostic biomarker of susceptibility to the disease development in the future. Moreover, they may become a potential target for future therapeutic interventions for clinical disorders in diabetes.

Adverse effect of the disturbances of glycemia and insulimemia on model PC12 cells - preliminary report.

BACKGROUND: One of the most important worldwide health problems of the 21st century is an increasing incidence of diabetes and insulin resistance. Morover, it is indicated that both these disturbances are connected with an increased incidence of Alzheimer's Disease. The literature data indicate that not only disturbed glucose concentration, especially hyperglycemia, is a crucial factor of the development of dementia but those data also emphasize that hyperphysiological concentrations of insulin and insulin resistance of brain tissue is an increasingly significant factor. The aim of this study was to evaluate the influence of glucose and insulin concentration reached in human carbohydrate metabolism disorders such as i.e. impaired fasting glucose, impaired glucose tolerance and diabetes state as well as averageand high degree hyperinsulinemia, on the survival of PC12 cell line. MATERIAL AND METHODS: Because of the close association indicated between diabetes and neurodegenerative diseases, in the experiment we used PC12 cell line derived from a transplantable rat pheochromocytoma, commonly used as an neurotoxicity and neuroprotection model. These cells were incubated in RPMI 1640 with addition of fetal bovine serum, horse serum, antibiotics and appropriate concentrations of glucose(from 84 to 240 mg/mL) and insulin(0.5 to 7 mg/mL) at 37°C in a humidified atmosphere containing 5% of CO2for 24 h and 48 h. Cell viability was expressed as a percentage of survival (PS [%]) against the negative control after MTT assay execution. RESULTS: The highest mortality was demonstrated for PC12 lines incubated for 24 h with the glucose level reflecting the condition of diabetes mellitus (DM), while after an incubation period of 48 h, the highest mortality was demonstrated for the incubation with insulin concentration corresponding to high levels of hyperinsulinemia (HH). Conclutions. The results suggest the greater susceptibility of PC12 cells to extended hyperinsulinaemia incubation than hyperglycemia,which indicates the increasing importance of insulin disorders in the induction of cell death.The results demonstrated in our experiment are particularly important for the development of a study model for testing the substances with hypoglycaemic, hypoinsulinemic and neuroprotective action.

Increased chitotriosidase activity in plasma of patients with type 2 diabetes.

INTRODUCTION: Chitotriosidase (CHIT1) is a chitinolytic enzyme involved mainly in the immune and inflammatory response. It shows increased activity in many pathologies, including in newly diagnosed type 2 diabetes (T2D). This study aimed to investigate this enzyme's activity in plasma of patients with ongoing T2D and indicate factors related to the increased activity of this enzyme. MATERIAL AND METHODS: Ninety-one patients and 46 control subjects without abnormalities in carbohydrate metabolism and inflammatory states were enrolled in the study. Plasma CHIT1 activity was measured by a spectrofluorometric method. Routine laboratory parameters such as blood glucose, total cholesterol and HDL fraction, triglyceride, glycated hemoglobin, white blood cell count and C-reactive protein were measured by standard methods. RESULTS: We found that the chitotriosidase activity was significantly higher (p < 0.001) in type 2 diabetic patients and positively associated with parameters of glycemic control (levels of glucose and glycated hemoglobin) and blood pressure. Plasma glucose level and systolic blood pressure were independent determinants of increased CHIT1 activity in T2D patients, even after adjustment for disease duration, body mass index, parameters of inflammation and lipid metabolism. We also found that increased CHIT1 activity was associated with occurrence of diabetic angiopathies. CONCLUSIONS: This investigation indicates a possible role of chitotriosidase in the course of T2D, especially in relation to development of diabetic angiopathies.

[The biochemical carcinogenesis of selected heavy metals in bladder cancer]. / Biochemiczna kancerogeneza wybranych metali ciezkich w raku pecherza moczowego.

Bladder cancer takes the second place in the classification of morbidity of urinary system cancers. Many chemical factors take part in cancerogenesis. It is suggested that exposure to heavy metals such as arsenic, chromium, nickel and cadmium as well as its metabolites may trigger the bladder cancer through inducing excessive reactive oxygen species production and oxidative stress formation which are responsible for DNA damage. In patients with bladder cancer is observed the disorder of processes regulated by p-53, including apoptosis. There are many patients with bladder cancer with confirmed absence of retinoblastoma protein, which is responsible of holding on the process of coming up the cells with mutation into synthesis, where the replication process undergoes. It is mentioned that excessive expression of proto-oncogenes may also cause the bladder cancer. The article concerns biochemical effects of exposure to chosen heavy metals and their potential role in bladder cancer progression.