OBJECTIVE: In this study, the combined effect of two stressors, namely, electromagnetic fields (EMFs) from mobile phones and fructose consumption, on hypothalamic and hepatic master metabolic regulators of the AMPK/SIRT1-UCP2/FOXO1 pathway were elucidated to delineate the underlying molecular mechanisms of insulin resistance. METHODS: Weaned Wistar rats (28 days old) were divided into 4 groups: Normal, Exposure Only (ExpO), Fructose Only (FruO), and Exposure and Fructose (EF). Each group was provided standard laboratory chow ad libitum for 8 weeks . Additionally, the control groups, namely, the Normal and FruO groups, had unrestricted access to drinking water and fructose solution (15%), respectively. Furthermore, the respective treatment groups, namely, the ExpO and EF groups, received EMF exposure (1,760 MHz, 2 h/day x 8 weeks). In early adulthood, mitochondrial function, insulin receptor signaling, and oxidative stress signals in hypothalamic and hepatic tissues were assessed using western blotting and biochemical analysis. RESULT: In the hypothalamic tissue of EF, SIRT1, FOXO 1, p-PI3K, p-AKT, Complex III, UCP2, MnSOD, and catalase expressions and OXPHOS and GSH activities were significantly decreased ( P < 0.05) compared to the Normal, ExpO, and FruO groups. In hepatic tissue of EF, the p-AMPKα, SIRT1, FOXO1, IRS1, p-PI3K, Complex I, II, III, IV, V, UCP2, and MnSOD expressions and the activity of OXPHOS, SOD, catalase, and GSH were significantly reduced compared to the Normal group ( P < 0.05). CONCLUSION The findings suggest that the combination of EMF exposure and fructose consumption during childhood and adolescence in Wistar rats disrupts the closely interlinked and multi-regulated crosstalk of insulin receptor signals, mitochondrial OXPHOS, and the antioxidant defense system in the hypothalamus and liver.
Humans ; Rats ; Animals ; Adult ; Rats, Wistar ; Fructose/metabolism* ; Catalase ; Receptor, Insulin/metabolism* ; AMP-Activated Protein Kinases/metabolism* ; Electromagnetic Fields/adverse effects* ; Sirtuin 1/metabolism* ; Cell Phone ; Phosphatidylinositol 3-Kinases/metabolism* ; Forkhead Box Protein O1/metabolism* ; Uncoupling Protein 2

Objective: To investigate the effect and mechanism of miR-96-5p on apoptosis of PC12 cells induced by maltol aluminum. Methods: In January 2021, PC12 cells at logarithmic growth phase were divided into blank control group and low, medium and high dose group. Cells in each group were treated with 0, 100, 200 and 400 μmol/L maltol aluminum for 24 hours respectively. Cells were collected and cell apoptosis rates were detected by flow cytometry, miR-96-5p and insulin receptor substrate 1 (IRS1) mRNA expressions were detected by qRT-PCR, and the protein expression levels of cysteine protease 3 (Caspase3) 、activated cysteine protease 3 (Cleaved-caspase3) 、IRS1、phosphorylated protein kinase B (p-AKT) and phosphorylated glucose synthesis kinase 3β (p-GSK3β) were detected by western blotting. The target binding relationship between miR-96-5p and IRS1 was detected by double luciferase reporter gene experiment. The miR-96-5p inhibitor cells and negative control cells were constructed after transfecting PC12 cells with miR-96-5p inhibitor for 24 hours. The cells were divided into blank control group, negative control group, aluminum exposure group, aluminum exposure+negative control group, aluminum exposure+miR-96-5p inhibition group, and miR-96-5p inhibition group. After transfecting PC12 cells with miR-96-5p inhibition and IRS1 siRNA for 24 h, the cells were divided into aluminum exposure+miR-96-5p inhibition+negative control group and aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group. The control group was cultured in complete culture medium, and cells in the aluminum exposure group were treated with 200 μmol/L maltol aluminum for 24 hours. Cells in each group were collected and the apoptosis rate, miR-96-5p and IRS1 mRNA expression levels, as well as protein expression levels of Caspase3, Cleaved-caspase3, IRS1, p-AKT, and p-GSK3β were measured. Results: After 24 hours of exposure, compared with blank control group and low-dose group, the apoptosis rates, relative expressions of Caspase3 and Cleaved-caspase3 proteins, and relative expressions of miR-96-5p in the medium and high-dose groups of PC12 cells were significantly increased, while the relative expression levels of IRS1 mRNA, IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05). Targetscan prediction and double luciferase report experiment both proved that IRS1 was a direct target gene of miR-96-5p. In the transfection experiment, compared with the aluminum exposure group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins, the relative expression of miR-96-5p in the aluminum exposure+miR-96-5p inhibition group were significantly decreased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly increased (P<0.05). In the IRS1 low expression experiment, compared with the aluminum exposure+miR-96-5p inhibition+negative control group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins in the aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group were significantly increased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05) . Conclusion: The increased expression of miR-96-5p and the targeted inhibition of IRS1 may be one of the mechanisms of apoptosis of PC12 cells induced by maltol aluminum exposure.
Animals ; Rats ; Aluminum/toxicity* ; Apoptosis ; Cell Proliferation ; Glycogen Synthase Kinase 3 beta/metabolism* ; Insulin Receptor Substrate Proteins/metabolism* ; MicroRNAs/metabolism* ; PC12 Cells ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger

OBJECTIVE: To observe the effect of electroacupuncture (EA) on protein expressions of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate-1 (IRS-1) in hypothalamus and morphology of pancreas islet in Zucker diabetic fatty (ZDF) rats, and to explore its possible mechanism on improving plasma glucose and insulin resistance of type 2 diabetes mellitus (T2DM). METHODS: Twelve SPF male ZDF rats were selected and fed with high-fat diet for 4 weeks to establish the T2DM model, after modeling, the rats were randomly divided into a model group and an EA group, 6 rats in each one. Besides, 6 SPF male Zucker lean rats were selected as a blank group. In the EA group, EA was applied at "Pishu" (BL 20), "Weiwanxiashu" (EX-B 3), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), with continuous wave, 15 Hz in frequency, 2 mA in intensity, once a day, 20 min each time, 6 times a week for 4 weeks. The fasting plasma glucose (FPG) was measured before and after intervention. The serum level of fasting insulin (FINS) was measured by radioimmunoassay, and the homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated; the morphological change of pancreas islets was observed by HE staining; the protein expressions of SOCS3 and IRS-1 in hypothalamus were detected by Western blot. RESULTS: Before intervention, compared with the blank group, FPG in the model group and the EA group was increased (P<0.01). After intervention, compared with the blank group, FPG, serum level of FINS and HOMA-IR were increased (P<0.01), the protein expression of SOCS3 was increased while IRS-1 was decreased in the hypothalamus in the model group (P<0.01). Compared with the model group, FPG, serum level of FINS and HOMA-IR were decreased (P<0.01), the protein expression of SOCS3 was decreased while IRS-1 was increased in the hypothalamus in the EA group (P<0.01). In the model group, the shape of pancreas islets was irregular, the area of pancreas islets and the number of islet β cell nuclei were decreased, the nuclei of islet β cell was compensatory enlargement. In the EA group, the shape and the area of pancreas islets and the number of islet β cell nuclei were improved, the compensatory increase of islet β cell nuclei was alleviated compared with the model group. CONCLUSION Electroacupuncture can reduce the fasting plasma glucose, improve the morphology of pancreas islets, and alleviate the insulin resistance in ZDF rats. The mechanism may relate to the down-regulation of SOCS3 and up-regulation of IRS-1 in the hypothalamus, and improving the function of hypothalamus in regulating peripheral glucose metabolism.
Acupuncture Points ; Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Type 2/therapy* ; Electroacupuncture ; Hypothalamus/metabolism* ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance ; Male ; Pancreas/metabolism* ; Rats ; Rats, Zucker ; Suppressor of Cytokine Signaling 3 Protein/metabolism*

OBJECTIVE: To explore the effect of curcumin on the insulin receptor substrate 1 (IRS1)/phosphatidylinositol-3-kinase (PI3K)/endometrial expression of glucose 4 (GLUT4) signalling pathway and its regulator, phosphatase and tensin homolog (PTEN), in a rat model of polycystic ovarian syndrome (PCOS). METHODS: PCOS model was induced by letrozole intragastric administration. Sprague-Dawley rats were randomized into 4 groups according to a random number table: (1) control group; (2) PCOS group, which was subjected to PCOS and received vehicle; (3) curcumin group, which was subjected to PCOS and treated with curcumin (200 mg/kg for 2 weeks); and (4) curcumin+LY294002 group, which was subjected to PCOS, and treated with curcumin and LY294002 (a specific PI3K inhibitor). Serum hormone levels (17 β-estradiol, follicle stimulating hormone, luteinizing hormone, progesterone, and testosterone) were measured by enzyme linked immunosorbent assay, and insulin resistance (IR) was assessed using the homeostasis model assessment of IR. Ovarian tissues were stained with haematoxylin and eosin for pathological and apoptosis examination. Expression levels of key transcriptional regulators and downstream targets, including IRS1, PI3K, protein kinase B (AKT), GLUT4, and PTEN, were measured via reverse transcription polymerase chain reaction and Western blot, respectively. RESULTS: The PCOS group showed impaired ovarian morphology and function. Compared with the PCOS group, curcumin treatment exerted ovarioprotective effects, down-regulated serum testosterone, restored IR, inhibited inflammatory cell infiltration in ovarian tissues, decreased IRS1, PI3K, and AKT expressions, and up-regulated GLUT4 and PTEN expressions in PCOS rats (P<0.05 or P<0.01). In contrast, IRS1, PI3K, AKT, and PTEN expression levels were not significantly different between PCOS and curcumin+LY294002 groups (P>0.05). CONCLUSION The beneficial effects of curcumin on PCOS rats included the alteration of serum hormone levels and recovery of morphological ovarian lesions, in which, PTEN, a new target, may play a role in regulating the IRS1/PI3K/GLUT4 pathway.
Animals ; Female ; Humans ; Rats ; Cell Proliferation ; Curcumin/therapeutic use* ; Follicle Stimulating Hormone ; Glucose ; Hyperandrogenism ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance ; Ovarian Cysts ; Ovarian Neoplasms ; Phosphatidylinositol 3-Kinase/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Polycystic Ovary Syndrome/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Testosterone

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

A stable hepatoma cell line(Hep G2 cell) insulin resistance model was established and used to analyze the effect of effective components of Mori Folium in alleviating insulin resistance,and preliminary explore the mechanism for alleviating insulin resistance. The Hep G2 insulin action concentration and the duration of action were investigated using the glucose oxidase method(GOD-POD method) to establish a stable Hep G2 insulin resistance model. Normal control group,model group,Mori Folium polysaccharide group,Mori Folium flavonoid group and rosiglitazone group were divided to determine the glucose consumption. The effect of Mori Folium effective components on Hep G2 insulin resistance was analyzed. The mRNA expressions of JNK,IRS-1 and PDX-1 in each group were detected by Real-time quantitative PCR(qRT-PCR). The protein expressions of p-JNK,IRS-1 and PDX-1 were detected by Western blot. And the mechanism of effective components of Mori Folium in alleviating insulin resistance was investigated. The results showed that the glucose consumption was significantly decreased in the insulin resistance cells after incubation with 25. 0 mg·L-1 insulin for 36 h(P<0. 01),and the model was relatively stable within 36 h. Mori Folium polysaccharides and flavonoids all alleviated insulin resistance,among which Mori Folium flavonoids had better effect in alleviating Hep G2 insulin resistance(P<0. 05). The qRT-PCR analysis showed that Mori Folium polysaccharides and flavonoids could inhibit JNK and IRS-1 mRNA expressions,while enhancing PDX-1 mRNA expression. Western blot analysis displayed that Mori Folium polysaccharides and flavonoids could inhibit p-JNK and IRS-1 protein expressions,while enhancing PDX-1 protein expression. Mori Folium polysaccharides and flavonoids can alleviate insulin resistance in Hep G2 cells,and its mechanism may be the alleviation of insulin resistance by inhibiting JNK signaling pathway.
Drugs, Chinese Herbal ; pharmacology ; Glucose ; Hep G2 Cells ; Homeodomain Proteins ; metabolism ; Humans ; Insulin ; Insulin Receptor Substrate Proteins ; metabolism ; Insulin Resistance ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; Morus ; chemistry ; Plant Leaves ; chemistry ; Trans-Activators ; metabolism

Insulin resistance,as the main link in the pathogenesis of type 2 diabetes mellitus( T2 DM),runs through the whole process of occurrence and development of T2 DM and is closely related to the insulin receptor signaling pathway. Insulin stimulation causes autophosphorylation of the insulin receptor( IR),which then activates tyrosine phosphorylation of insulin receptor substrate( IRS).Phosphorylation of IRS can induce and activate phosphatidylinositol 3-kinase( PI3 K),subsequently activate downstream 3-phosphoinositide-dependent protease 1( PDK1) and Akt/PKB,and finally promote expression and translocation of glucose transporter 4 to increase glucose uptake of insulin-sensitive tissues and alleviate insulin resistance. Currently,oral hypoglycemic agents for clinical treatment of T2 DM have different side effects on the human body. Traditional Chinese medicine not only has a wide range of sources and abundant types,but also has comprehensive multi-component,multi-link and multi-target effects,showing unique advantages in the treatment of diabetes. In recent years,more and more researchers at home and abroad pay attention to the active ingredients in traditional Chinese medicine for alleviating insulin resistance. In this paper,we would summarize the active hypoglycemic ingredients of traditional Chinese medicine associated with the insulin receptor signaling pathway,which may provide some theoretical guidance for the development of traditional Chinese medicine in the treatment of diabetes.
Diabetes Mellitus, Type 2 ; Humans ; Hypoglycemic Agents/therapeutic use* ; Insulin ; Insulin Receptor Substrate Proteins ; Insulin Resistance ; Medicine, Chinese Traditional ; Phosphatidylinositol 3-Kinases ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; Receptor, Insulin/metabolism* ; Signal Transduction

Angiogenesis is a crucial process in the development of inflammatory diseases, including cancer, psoriasis and rheumatoid arthritis. Recently, several alkaloids from Picrasma quassioides had been screened for angiogenic activity in the zebrafish model, and the results indicated that 1-methoxycarbony-β-carboline (MCC) could effectively inhibit blood vessel formation. In this study, we further confirmed that MCC can inhibit, in a concentration-dependent manner, the viability, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as the regenerative vascular outgrowth of zebrafish caudal fin in vivo. In the zebrafish xenograft assay, MCC inhibited the growth of tumor masses and the metastatic transplanted DU145 tumor cells. The proteome profile array of the MCC-treated HUVECs showed that MCC could down-regulate several angiogenesis-related self-secreted proteins, including ANG, EGF, bFGF, GRO, IGF-1, PLG and MMP-1. In addition, the expression of two key membrane receptor proteins in angiogenesis, TIE-2 and uPAR, were also down-regulated after MCC treatment. Taken together, these results shed light on the potential therapeutic application of MCC as a potent natural angiogenesis inhibitor via multiple molecular targets.
Angiogenesis Inhibitors ; chemistry ; pharmacology ; Animals ; Carbolines ; chemistry ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Epidermal Growth Factor ; genetics ; metabolism ; Fibroblast Growth Factors ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Neovascularization, Physiologic ; drug effects ; Picrasma ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Receptor, TIE-2 ; genetics ; metabolism ; Zebrafish ; embryology

Catalpol, a major bioactive component from Rehmannia glutinosa, which has been used to treat diabetes. The present study was designed to elucidate the anti-diabetic effect and mechanism of action for catalpol in db/db mice. The db/db mice were randomly divided into six groups (10/group) according to their blood glucose levels: db/db control, metformin (positive control), and four dose levels of catalpol treatment (25, 50, 100, and 200 mg·kg), and 10 db/m mice were used as the normal control. All the groups were administered orally for 8 weeks. The levels of fasting blood glucose (FBG), random blood glucose (RBG), glucose tolerance, insulin tolerance, and glycated serum protein (GSP) and the globe gene expression in liver tissues were analyzed. Our results showed that catalpol treatment obviously reduced water intake and food intake in a dose-dependent manner. Catalpol treatment also remarkably reduce fasting blood glucose (FBG) and random blood glucose (RBG) in a dose-dependent manner. The RBG-lowering effect of catalpol was better than that of metformin. Furthermore, catalpol significantly improved glucose tolerance and insulin tolerance via increasing insulin sensitivity. Catalpol treatment significantly decreased GSP level. The comparisons of gene expression in liver tissues among normal control mice, db/db mice and catalpol treated mice (200 and 100 mg·kg) indicated that there were significant increases in the expressions of 287 genes, whichwere mainly involved in lipid metabolism, response to stress, energy metabolism, and cellular processes, and significant decreases in the expressions of 520 genes, which were mainly involved in cell growth, death, immune system, and response to stress. Four genes expressed differentially were linked to glucose metabolism or insulin signaling pathways, including Irs1 (insulin receptor substrate 1), Idh2 (isocitrate dehydrogenase 2 (NADP), mitochondrial), G6pd2 (glucose-6-phosphate dehydrogenase 2), and SOCS3 (suppressor of cytokine signaling 3). In conclusion, catalpol ecerted significant hypoglycemic effect and remarkable therapeutic effect in db/db mice via modulating various gene expressions.
Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; genetics ; metabolism ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; analysis ; Gene Expression ; drug effects ; Glucosephosphate Dehydrogenase ; genetics ; metabolism ; Humans ; Hypoglycemic Agents ; administration & dosage ; Insulin ; metabolism ; Insulin Receptor Substrate Proteins ; genetics ; metabolism ; Iridoid Glucosides ; administration & dosage ; analysis ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Rehmannia ; chemistry ; Suppressor of Cytokine Signaling 3 Protein ; genetics ; metabolism

BACKGROUND: Epidemiological studies have suggested that noise exposure may increase the risk of type 2 diabetes mellitus (T2DM), and experimental studies have demonstrated that noise exposure can induce insulin resistance in rodents. The aim of the present study was to explore noise-induced processes underlying impaired insulin sensitivity in mice. METHODS: Male ICR mice were randomly divided into four groups: a control group without noise exposure and three noise groups exposed to white noise at a 95-dB sound pressure level for 4 h/day for 1, 10, or 20 days (N1D, N10D, and N20D, respectively). Systemic insulin sensitivity was evaluated at 1 day, 1 week, and 1 month post-noise exposure (1DPN, 1WPN, and 1MPN) via insulin tolerance tests (ITTs). Several insulin-related processes, including the phosphorylation of Akt, IRS1, and JNK in the animals' skeletal muscles, were examined using standard immunoblots. Biomarkers of inflammation (circulating levels of TNF-α and IL-6) and oxidative stress (SOD and CAT activities and MDA levels in skeletal muscles) were measured via chemical analyses. RESULTS: The data obtained in this study showed the following: (1) The impairment of systemic insulin sensitivity was transient in the N1D group but prolonged in the N10D and N20D groups. (2) Noise exposure led to enhanced JNK phosphorylation and IRS1 serine phosphorylation as well as reduced Akt phosphorylation in skeletal muscles in response to exogenous insulin stimulation. (3) Plasma levels of TNF-α and IL-6, CAT activity, and MDA concentrations in skeletal muscles were elevated after 20 days of noise exposure. CONCLUSIONS Impaired insulin sensitivity in noise-exposed mice might be mediated by an enhancement of the JNK/IRS1 pathway. Inflammation and oxidative stress might contribute to insulin resistance after chronic noise exposure.
Animals ; Biomarkers ; metabolism ; Inflammation ; physiopathology ; Insulin Receptor Substrate Proteins ; genetics ; metabolism ; Insulin Resistance ; genetics ; immunology ; MAP Kinase Signaling System ; physiology ; Male ; Mice ; Mice, Inbred ICR ; Mitogen-Activated Protein Kinase 8 ; genetics ; metabolism ; Noise ; adverse effects ; Oxidative Stress ; physiology ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Random Allocation ; Time Factors