Effects And Mechanisms Of TBHQ-improved Pressure Overload-induced Cardiac Remodeling

BackgroundHeart failure is a debilitating disease associated with high morbidity and mortality, and with increased healthcare costs. Under stress conditions such chronic overload or myocardial infarction, cardiac myocytes initiate a hypertrophic response, which is thought to be an adaptive reaction and have compensatory effects on cardiac pumping functions; however, the presence of persistent stress stimuli will eventually result in myocardial decompensation, which is associated with ventricular dilatation, myocardial fibrosis, oxidative stress, contractile dysfunction and finally heart failure. Mechanisms underlying the transition from adaptive hypertrophy to heart failure are poorly understood. Several lines of evidence have suggested that loss of cardiac myocytes in the form of apoptosis and/or necrosis, abnormalities in intracellular calcium homeostasis, and uncontrolled fibrosis in the myocardium may all have essential roles in this pathological process, and these may represent potential therapeutic targets to prevent the development heart failure. Despite the intense research in this area, currently we still lack an effective pharmacological approach that can block the progression of myocyte decompensation and heart failure.Tert-butylhydroquinone (TBHQ) is a phenolic chain-breaking antioxidant used to prevent lipid peroxidation. TBHQ has been identified as an activator of nuclear factor erythroid 2-related factor 2 (Nrf2), a redox-sensitive transcription factor involved in Phase Ⅱ detoxification response in mammalian cells. In response to cellular stress, activated Nrf2 mediates expression of an array of cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione peroxidase, heme oxygenase-1 (HO-1), and superoxide dismutase. In line with these effects of Nrf2, several in vivo studies showed that TBHQ treatment elicited significant cytoprotective actions in different organs under pathological conditions. For example, TBHQ showed prominent neuro-protective actions in experimental traumatic brain injury, ischemic stroke and subarachnoid hemorrhage. Similarly, there is evidence that TBHQ treatment can inhibit ischemia-reperfusion injury and oxidative stress in kidneys. The neuro-and renal-protective actions are mediated by the Nrf2 antioxidant system.A recent study indicates that TBHQ may have potential cardioprotective effects. However, the effects of TBHQ on pathological cardiac remodeling, and the underlying mechanisms have not been systematically evaluated. Therefore, in the present study, we examined the effects of TBHQ on cardiac remodeling induced by pressure overload, and explored the potential mechanisms of the TBHQ effects.Objectives1. To investigate the effects of TBHQ on pressure overload-induced ventricular remodeling.2. To explore the mechanisms of the effects of TBHQ on cardiac remodeling.Materials and Methods1. Aniamals and treatment groupsMale C57BL/6 mice were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). All animal studies were in accordance with the Animal Management Guidelines of the Chinese Ministry of Health and approved by the Ethics Committee of the Qilu Hospital of the Shandong University. Male C57BL/6 mice at the age of 10 to 12-wk were randomly divided into eight groups:sham group; TAC group; TBHQ group; TAC+TBHQ group; TAC+Rapamycin group; TAC+Rapamycin+TBHQ group; TAC+Ly294002 group; TAC+Ly294002+TBHQ group.2. Transverse aortic constrictionThe cardiac pressure overload model was created by transverse aortic constriction (TAC) using a modified surgical technique that did not require thoracotomy. Mice were anesthetized with intraperitoneal (i.p.) injection of 0.8% pentobarbital sodium (60 mg/kg). A horizontal incision was made at the level of suprasternal notch. Once the trachea was located, a longitudinal cut was made down under the sternum and the aortic arch exposed. Then the aortic arch was partially ligated against a 27-gauge needle using a 7-0 silk suture between the origins of innominate and left carotid arteries. The needle was then retrieved and the incision closed. Sham operation was performed with the same procedure except for the step of aortic ligation.3. Echocardiographic measurementsUltrasound echocardiography was performed before and at 2 and 4 weeks after TAC. Under general anesthesia with 1-2% isoflurane, the procedure was conducted using a high resolution echocardiography system (Vevo 770, Visual Sonics, Canada) with a 35-MHz transducer. Two-dimensional parasternal long-and short-axis images of the LV were recorded. Using the M-mode function, the LV end-diastolic diameter (LVDd), LV end-systolic diameter (LVDs), inter-ventricular septum end-diastolic thickness (IVSTd) and LV posterior wall end-diastolic thickness (PWTd) were measured. LV fractional shortening (FS) ejection fraction (FS) and corrected LV mass were automatically calculated by the echocardiographic system.4. Histology and immunohistochemistry analysesHearts were perfuse-fixed in situ for 5 min and further fixed in 4% paraformaldehyde overnight, dehydrated, and embedded in paraffin. Short-axis sections of 5μm thickness were all cut at the horizontal plane at the level of the papillary muscle. Sections were stained with hematoxylin and eosin (H&E), picrosirius red, or Masson’s trichrome. Specific immunohistochemical staining for 4-HNE was performed using a rabbit polyclonal antibody.5. Hydroxyproline assayHydroxyproline assay was performed to quantify the collagen content in LV, using a commercial kit from Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions.6. Western blottingProteins from left ventricular tissues were extracted in lysis buffer, then protein samples were separated by SDS-PAGE and electro-transferred to nitrocellulose membrane. The membrane was blocked with 5% nonfat milk and incubated with specific primary antibodies at 4℃ overnight. Secondary HRP-conjugated antibodies were developed with ECL Prime reagents and detected with a LAS-4000 luminescent image analyzer.7. Assessment of protein carbonylationThe level of protein carbonylation was assessed using a method described previously with some modifications.8. TUNEL assayApoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique. TUNEL positive cells were surveyed in 10 random 400x fields for each section.9. Real-time quantitative PCRReal-time PCR was carried out using Taqman Gene Expression primer-probe sets. The 18S RNA was used as the house-keeping gene. The relative quantification method was used for data analysis.10. Akt kinase activity assay and Nrf2 activation analysisAkt kinase activity was detected using an Akt Kinase Assay Kit (CST) according to the manufacturer’s instructions. The Nrf2 DNA binding activity were measured in nuclear proteins using a Trans AM Nrf2 Kit according to the protocol provided by the manufacturer.11. Hepatic content of hemoglobinHepatic content of hemoglobin were detected using Drabkin’s reagent (Sigma) according to the manufacturer’s instructions.12. Statistical analysisData were expressed as mean±standard error of the mean (SEM). For statistical analysis, unpaired t-test or one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparisons as appropriate were performed using SPSS 13.0 software. A value of P<0.05 was considered statistically significant.Results1. TBHQ reduced TAC induced mortality in miceTBHQ treatment reduced TAC-induced mortality in mice.2. TBHQ attenuated TAC-induced ventricular dilatation and heart failureThen we characterized the time course of pressure overload-induced cardiac remodeling under the present experimental settings. We found that at 2 weeks, there was a significant LV wall thickening, but no LV dilatation. At 4 weeks, however, there was a notable LV dilatation, which was accompanied by thinning of the ventricular wall, indicating a status of myocardial decompensation. We observed that TBHQ treatment prevented the occurrence of LV dilatation and the accompanying ventricular wall thinning induced by TAC. Consistent with the above findings, we demonstrated that the EF and FS were not different between sham and TAC groups at 2 weeks, whereas EF and FS were significantly decreased in TAC animals at 4 weeks. TBHQ treatment restored the EF and FS values in TAC animals.Moreover, we found that the weight of lungs was significantly elevated in TAC animals at 4 weeks, indicating a sign of congestive heart failure. This change was similarly prevented by TBHQ treatment. We also measured the hepatic content of hemoglobin (passive liver congestion) as an additional sign of congestive heart failure, using Drabkin’s reagent (Sigma). We showed that the hepatic hemoglobin content was significantly elevated in the TAC group as compared to sham, and this change was attenuated by TBHQ.3. Effects of TBHQ on myocardial hypertrophy and fibrosisMorphometric analysis revealed that TAC resulted in typical myocyte hypertrophy. However, treatment with TBHQ showed no significant effect on TAC-induced myocyte hypertrophy. Consistently, the echocardiography data also showed that TBHQ treatment maintained a hypertrophic phenotype of the ventricular wall throughout the period of TAC. The heart to body weight ratio, the heart weight to tibia length ratio and the echo-derived LV mass to tibia length ratio significantly increased in the TAC group at 4 weeks. However, TBHQ treatment had no significant effects on the heart weight to tibia length ratio or the echo-derived LV mass to tibia length ratio, while it partially reduced the heart to body weight ratio. TAC significantly upregulated the expression levels of atrial natriuretic peptide and B-type natriuretic peptide, markers of pathological hypertrophy; however, treatment with TBHQ further increased the natriuretic peptide levels. We also assessed changes in myocardial fibrosis using Masson’s trichrome staining, picrosirius red staining and hydroxyproline assay. We found that TBHQ had no significant effects on the severity of TAC-induced fibrosis.4. TBHQ did not activate Nrf2 or AMPK in the myocardiumWe measured the endogenous levels of Nrf2 in LV homogenates. Surprisingly, under current experimental setting, TBHQ failed to stimulate Nrf2 accumulation in the myocardium. To confirm this finding, we directly measured the Nrf2 DNA binding activity of the nuclear protein extracts of LV. We showed that there were no significant changes in Nrf2 activity in different groups. In addition, we measured expression levels of the Nrf2 target genes NQO1, HO-1, glutathione peroxidase-1 and thioredoxin; we found that TBHQ did not significantly change the mRNA levels of these genes. We showed that in the liver and kidney, the expressions of NQO1 and HO-1 were all significantly elevated in TBHQ-treated animals. Also, we demonstrated that the phosphorylation level of AMPKa was not affected by TBHQ. Moreover, TBHQ exhibited no significant effects on expression levels of the ER stress markers CHOP and GRP78.5. TBHQ inhibited myocardium apoptosisTUNEL assays in LV sections demonstrated that TAC induced-10-fold increase in myocyte apoptosis as compared to sham animals. TBHQ treatment significantly suppressed TAC-induced apoptosis by about 60%.6. TBHQ increased Akt phosphorylation in the myocardiumWe found that in TBHQ-treated hearts, there was a significant increase in the phosphorylation level of Akt as compared to sham and TAC animals. In accordance with the enhanced Akt phosphorylation, we observed that the phosphorylation level of Bad was also increased in TBHQ-treated hearts. In contrast, phosphorylation of ERK1/2 was not affected by TBHQ. We directly measured the Akt kinase activity using an assay kit from Cell Signaling Technology. Akt activity was significantly increased in TBHQ-treated hearts. Moreover, we showed that the phosphorylation level of the endogenous Akt substrates glycogen synthase kinase-3β and mammalian target of rapamycin (mTOR) were also increased by TBHQ. To clarify whether the effect of TBHQ on Akt activation was isoform specific, we measured the phosphorylation levels of Akt1 and Akt2, and found that phosphorylation of both of Aktl and Akt2 was increased in TBHQ-treated hearts.7. Effects of TBHQ on cardiac remodeling after blockade of the Akt pathwaySignificant decreases in LV contractile functions and reduction of hypertrophy were detected at 2 weeks in TAC+LY294002 group. In the presence of LY294002 co-treatment, TBHQ showed no significant beneficial effects on TAC-induced cardiac dysfunctions. Compared to Sham group, the endogenous Akt phosphorylation level was enhanced in TAC group. In addition, there was no significant difference in the level of apoptosis between Sham and TAC group at 2 weeks. Compared to TAC group, concomitant LY294002 treatment abrogated TAC-induced Akt activation, and significantly increased the level of apoptosis. Moreover, we showed that TBHQ had no significant effects on either Akt phosphorylation or apoptosis in the presence of LY294002.Significant decreases in hypertrophy were detected at 2 weeks in TAC+ Rapamycin group compared to TAC group. Rapamycin co-treatment improved the FS and EF values in TAC group at 4 weeks. TBHQ still showed further beneficial effects on LV contractile functions in the presence of rapamycin.8. TBHQ reduced reactive aldehyde production and protein carbonylation in myocardiumTo clarify whether TBHQ exerted any antioxidant effects in the cardiac tissue, we performed immunohistochemistry for 4-HNE. TAC significantly increased 4-HNE production in the myocardium, which was reduced by TBHQ treatment. Using SDS-PAGE and western blotting, we demonstrated that the abundance of carbonylated proteins in the myocardium was significantly raised by TAC, and TBHQ treatment suppressed TAC-induced accumulation of protein carbonyls.Conclusion1. TBHQ treatment significantly improves pressure overload-induced cardiac remodeling.2. TBHQ exerts beneficial effects on the progression of heart failure by inhibiting cardiomyocyte apoptosis and formation of reactive aldehyde species, but not associated with Nrf2.3. TBHQ-induced inhibition of apoptosis was mediated by enhancing the activation of the Akt pathway.BackgroundOur in vivo experiments suggest that Tert-butylhydroquinone (TBHQ) has important protective effects on cardiac remodeling by inhibiting apoptosis, but the corresponding cellular mechanisms of the action are not totally clear. TBHQ, a synthetic phenolic antioxidant, is a commonly used food preservative because of its potent anti-lipid peroxidation activity. Several lines of evidence have demonstrated that TBHQ has antioxidant, anti-infammatory, and anti-apoptosis effects, and displays potent cell-protective properties.4-Hydroxynonenal (4-HNE) is one of the major end products of lipid peroxidation and is thought to be an inducer of oxidative stress, it may be involved in the pathogenesis of a number of oxidative stress-related diseases such as Alzheimer’s disease, atherosclerosis, ischemia/reperfusion, obesity, and diabetes. Studies indicate that 4-HNE may be involved in modulating intracellular signaling pathways related to cell cycle control and regulation of gene expression. Recent studies have shown that HNE can induce cell apoptosis via multiple signaling pathways.Receptor Tyrosine Kinases (RTKs) are cell surface receptors for various growth factors. They transmit signals from the extracellular environment to the interior of a cell, initiating signaling cascades that lead to diverse phenotypic responses ranging from adhesion, migration, proliferation, differentiation, survival to apoptosis. Furthermore, numerous diseases result from abnormalities of the activity, abundance, cellular distribution, or regulation of RTKs. Insulin-like growth factor-1 receptor (IGF-1R) is a conventional RTK. Upon ligand binding, IGF-1R induces a number of cellular phosphorylation cascades including the PI3K/AKT signaling pathway. Protein tyrosine phosphatases (PTPs) catalyze dephosphorylation of tyrosine residues:these enzymes are widely expressed in the body. Together with RTKs, PTPs are responsible for fine tuning of the cellular protein tyrosine phosphorylation level. PTP1B is a classic PTP, which has been demonstrated to negatively regulate the insulin-PI3K/AKT signaling pathway. PTEN is another negative regulator of the Akt pathway, which is involved in modulating a variety of cellular regulation processes. In the present study, we aimed to clarify whether TBHQ could inhibit HNE-induced cardiac myocyte apoptosis by activating the PI3K/AKT signaling pathway, and whether this action was related to PTP inhibition and augmentation of IGF-1R signaling.Objectives1. To test the effects of TBHQ on HNE-induced apoptosis in cultured cardiac myocytes2. To test whether PI3K/Akt signaling pathway mediates the anti-apoptotic effect of TBHQ in cardiac myocytes3. To explore the mechanisms of TBHQ-induced Akt activation and test whether the PTP1B/PTEN and IGF-1R pathways may be involvedMaterials and Methods1. Cell cultureH9c2 rat myocyte-like cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum in a humidified incubator with 5% CO2 at 37℃. H9c2 cells used for the experiments were between passages 3 and 6.Primary cultures of neonatal myocytes from 1-2 days old Sprague-Dawley rats were prepared according to previously published protocols. In brief, heart tissues were digested with collagenase. Cells were recovered by centrigugation, resuspended in plating medium. After incubation for 2 hours at 37℃, the attached fibroblasts on the dish were removed, and cardiomyocytes in the supernatants were resuspended in plating medium and plated on collagen-coated cell culture flasks. After 2-3 days, cardiomyocytes were washed and cultured in maintenance medium.2. Cell viability assayMTT assay was employed to study the effects of TBHQ and 4-HNE on the viability of H9c2 cells. The cells were plated in lml of complete culture medium containing TBHQ and 4-HNE in 24-well plates. After incubation for 24h,200μl of MTT (5mg/ml) was added to each well and incubated for two hours, after which 200μl of DMSO was added. The plates were then centrifuged (1800×g,5min at 4℃). The absorbance at 540nm was recorded using a microplate reader. The effects of TBHQ and 4-HNE were calculated as% cell viability where DMSO-treated cells were taken as 100% control.3. Western blot analysisCells were homogenized in lysis buffer after stimulation, then protein samples were separated by SDS-PAGE and electro-transferred onto nitrocellulose membranes. The membrane was blocked with 5% nonfat milk and incubated with specific primary antibodies at 4℃ overnight. Secondary HRP-conjugated antibodies were developed with ECL Prime reagents and then detected with a LAS-4000 luminescent image analyzer.4. Caspase3/7 activity assayCells seeded in 96-well plates were incubated with the Caspase-Glo 3/7 substrate reagent to test Caspase3/7 activity. The samples were transferred to a white-walled plate and the luminescence signal was measured in a Varioskan Flash plate reader.5. TUNEL labelingApoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique. TUNEL positive cells were surveyed in 10 random fields for each section.6. Statistical analysisData were expressed as mean±standard error of the mean (SEM). For statistical analysis, unpaired t-est or one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparisons as appropriate were performed using SPSS 13.0 software. A value of P< 0.05 was considered statistically significant.Results1. TBHQ had no significant effect on myocardial cell viabilityWe demonstrated that treatment of H9c2 cells with 5、10、20、40μM of TBHQ had no significant effect on myocyte viability.2. TBHQ induced Akt phosphorylation in H9c2 cellsWe found that TBHQ from 10μM to 40μM significantly increased Akt phosphorylation, while TBHQ at 1μM showed no effect. We further demonstrated that the effects of TBHQ on Akt activition were time-dependent, with the strongest effects being observed at 1 hr. Further extension of the time of drug treatment, did not increase Akt activition additionally.3.4-HNE inhibited cell viability and induced apoptosisWe found that 4-HNE at 10 or 20μshowed no significant effects on cell viability. Above 40μM,4-HNE showed a significant dose-dependent inhibition of H9c2 cells viability as compared to untreated controls.We observed that 4-HNE at 10μM showed no significant effect on caspase 3/7 activity. Above 20μM,4-HNE induced a dose-dependent increase in caspase 3/7 activity in H9c2 cells as compared to untreated controls.4. TBHQ inhibited 4-HNE induced apoptosisWe performed TUNEL staining in cultured H9c2 cells and demonstrated significant anti-apoptotic actions of TBHQ.5. TBHQ inhibited 4-HNE induced apoptosis by activating PI3K/Akt signaling pathwayWe demonstrated that TBHQ suppressed 4-HNE-induced caspase 3 cleavage, which was accompanied by concomitant augmentation in Akt phosphorylation. We further demonstrated that the anti-apoptotic effect of TBHQ was abolished in the presence of wortmannin or Akti. To confirm the results of caspase 3 western blotting, we directly measured the activity of caspase 3/7 and revealed that TBHQ treatment suppressed 4-HNE-elicited caspase 3/7 activation, while this effect of TBHQ was reversed by wortmannin or Akti.6. TBHQ inhibited 4-HNE induced apoptosis in primary neonatal cardiomyocytesWe observed that 4-HNE in the presence or absence of TBHQ does not affect the phosphrylation of Akt in primary neonatal cardiomyocyte. TBHQ treatment suppressed 4-HNE-elicited caspase 3/7 activation in primary neonatal cardiomyocytes. In the presence of endothelin, TBHQ still showed anti-apoptotic effects in primary neonatal cardiomyocyte.7. Effects of PTP1B and PTEN inhibitors on TBHQ-induced Akt phosphorylationWe demonstrated that treatment with a PTP1B inhibitor significantly increased Akt phosphorylation in H9c2 cells, while TBHQ did not further increase Akt phosphorylation in the presence of the PTP1B inhibitor.We demonstrated that treatment with a PTEN inhibitor significantly increased Akt phosphorylation in H9c2 cells, while in the presence of the PTEN inhibitor, TBHQ still showed increaseing effect on Akt phosphorylation.8. Effects of TBHQ on the level of protein tyrosine phosphorylationWe found that TBHQ from 5μM to 40μM significantly increased the level of protein tyrosine phosphorylation in H9c2 cells.9. Effects of TBHQ on the level of IGF-1 receptor phosphorylationWe found that TBHQ from 10 μM to 40 μM significantly increased the level of IGF-1 receptor phosphorylation in H9c2 cells.Conclusions1. TBHQ may protects against reactive aldehyde-induced cardiac myocyte apoptosis by activating the PI3K/AKT pathway2. Our results suggest that TBHQ-induced activation of Akt may involved decreased PTP1B function and augmented IGF-1R signalingBackgroundIn the above chapters, we showed that TBHQ significantly increased Akt phosphorylation in heart tissues and in cultured cardiomyocytes; and this effect might play an important cardioprotective role in cardiac remodeling induced by pressure overload. Akt is a multifuctional serine/threonine protein kinase, also known as protein kinase B, which implicated in a diverse range of cellular functions including cell metabolism, survival, migration, and gene expression. Dysregulation of Akt is associated with a variety of human diseases such as cancer, diabetes, cardiovascular and neurological diseases. Akt activation may cause divergent outcomes in different tissues. In some circumstances, Akt activation may play a protective role; on the other hand, excessive or prolonged activation of Akt may also result in the formation of tumor cells. Therefore, it is necessary to clarify the pharmacological effects of TBHQ on Akt phosphorylation in different tissues. Therefore, in the following study, we applied TBHQ intervention in mice and compared the pharmacological effects of TBHQ on Akt in different tissues. We had found that the pharmacological effects of TBHQ organizational differences. Meanwhile, we tested the effects of TBHQ on Akt phosphorylation in nonmyocytes in vitro.Objectives1. To examine the effects of TBHQ on Akt phosphorylation in HUVECs and 3T3 cells in vitro2. To examine the effects of TBHQ on Akt phosphorylation in the normal liver, kidney, brain and aortic tissues in vivoMaterials and Methods1. Cell cultureHUVECs and 3T3 cells were obtained from ATCC and cultured in complete endothelial cell medium (ECM) and Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum in a humidified incubator with 5% CO2 at 37℃. HUVECs and 3T3 cells used for the experiments were between passages 3 and 6.2. Aniamal groupingMale C57BL/6 mice at the age of 8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). All animal studies were in accordance with the Animal Management Guidelines of the Chinese Ministry of Health and approved by the Ethics Committee of the Qilu Hospital of the Shandong University. Mice were randomly divided into two groups:control group; TBHQ group.3. Western blot analysisTissues and cells were homogenized in lysis buffer, then total protein samples were separated by SDS-PAGE and electro-transferred to nitrocellulose membranes. The membrane was blocked with 5% nonfat milk and incubated with specific primary antibodies. Secondary HRP-conjugated antibodies were developed with ECL Prime reagents and detected with a LAS-4000 luminescent image analyzer.4. Statistical analysisData were expressed as mean±standard error of the mean (SEM). For statistical analysis, unpaired t-test or one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparisons as appropriate were performed using SPSS 13.0 software. A value of P< 0.05 was considered statistically significant.Results1. Effects of TBHQ on Akt phosphorylation in HUVECsHUVECs were treated with TBHQ at 1,10,50μM for 24 hours, and DMSO as a control. We demonstrated that TBHQ significantly increased the level of phosphorylated Akt in HUVECs in a concentration-dependent manner.2. Effects of TBHQ on Akt phosphorylation in 3T3 cells3T3 cells were treated with TBHQ at 1,10,50μM for 24 hours, and DMSO as a control. We demonstrated that TBHQ significantly increased the level of phosphorylated Akt in 3T3 cells in a concentration-dependent manner.3. Effects of TBHQ on Akt phosphorylation in liver tissueWe demonstrated that in vivo treatment with TBHQ significantly increased the level of phosphorylated Akt in the liver tissue.4. Effects of TBHQ on Akt phosphorylation in kidney tissueWe demonstrated that in vivo treatment with TBHQ significantly increased the level of phosphorylated Akt in the kidney tissue.5. Effects of TBHQ on Akt phosphorylation in aortic tissueWe demonstrated that in vivo treatment with TBHQ significantly increased the level of phosphorylated Akt in the aortic tissue.6. Effects of TBHQ on Akt phosphorylation in brain tissueWe demonstrated that in vivo treatment with TBHQ had no effects on the level of phosphorylated Akt in the brain tissue.Conclusions1. TBHQ can activate Akt in both HUVECs and 3T3 cells2. TBHQ treatment in mice in vivo can activate Akt in the liver, kidney and aortic tissues3. TBHQ treatment in mice can not activate Akt in the brain tissue...