Protocol to knock in or out target genes in mouse epidydimal white adipose tissue by spot injecting adeno-associated viruses.

Visceral fat obesity is more strongly associated with ectopic fat deposition, lipotoxicity, and metabolic disease compared to generalized obesity. To study the function of visceral fat tissue, we describe steps to knock in or out target genes by spot injecting adeno-associated viruses (AAV) in visceral fat tissue. We provide details on anesthesia, incision, and spot injection into the epidydimal white adipose tissue (eWAT) of live anesthetized mice. Furthermore, we detail an efficient technique for expressing exogenous protein in mouse eWAT. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2022).

A MDM2 inhibitor MX69 inhibits adipocytes adipogenesis and differentiation.

Adipose tissue, a key regulator of systemic energy homeostasis, can synthesize and store triglycerides to meet long-term energy demands. In response to nutrient overload, adipose tissue expands by hypertrophy or hyperplasia. As an oncogene, MDM2 has exerted diverse biological activities including human development, tissue regeneration, and inflammation, in addition to major oncogenic activities. Recently, some studies indicated that MDM2 plays an important role in adipose tissue function. However, the role of MX69, a MDM2 inhibitor, in adipose tissue function has not been fully elucidated. Here, we administered MX69 intraperitoneally to high-fat diet-induced obesity (DIO) wild type C57BL/6 mice and found that MX69 could promote the body weight and white adipose tissue weight of DIO mice. Moreover, MX69 had no effects on glucose tolerance and insulin sensitivity in DIO mice. And MX69 treatment decreased the size of adipocytes and fat deposition in adipose tissue and inhibited 3T3-L1 preadipocytes differentiation. Mechanistically, MX69 inhibited the protein levels of MDM2 and the mRNA levels of genes related to adipogenesis and differentiation. In summary, our results indicated that MDM2 has a crucial and complex role in regulating adipose tissue function.

Murine double minute 2 aggravates adipose tissue dysfunction through ubiquitin-mediated six-transmembrane epithelial antigen of prostate 4 degradation.

Healthy adipose tissue is crucial to maintain normal energy homeostasis. Little is known about the role of murine double minute 2 (MDM2), an E3 ubiquitin ligase and has been highlighted in oncopathology, in adipose tissue. Our results indicated that MDM2 expression was associated with nutritional status. Mdm2 adipocyte-specific knock-in (Mdm2-AKI) mice exhibited exacerbated weight gain, insulin resistance, and decreased energy expenditure. Meanwhile, chronic high-fat diet (HFD) exposure caused obvious epididymal white adipose tissue (eWAT) dysfunction, such as senescence, apoptosis, and chronic inflammation, thereby leading to hepatic steatosis in Mdm2-AKI mice. Mechanically, MDM2 could interact with six-transmembrane epithelial antigen of prostate 4 (STEAP4) and inhibit STEAP4 expression through ubiquitin-mediated STEAP4 degradation. Thereinto, the K18 and K161 sites of STEAP4 were ubiquitin-modificated by MDM2. Finally, STEAP4 restoration in eWAT of Mdm2-AKI mice on a HFD rescued MDM2-induced adipose dysfunction, insulin resistance, and hepatic steatosis. Summary, the MDM2-STEAP4 axis in eWAT plays an important role in maintaining healthy adipose tissue function and improving hepatic steatosis.

Dexamethasone-induced Krüppel-like factor 9 expression promotes hepatic gluconeogenesis and hyperglycemia.

Chronic glucocorticoid therapy has serious side effects, including diabetes and fatty liver. However, the molecular mechanisms responsible for steroid-induced diabetes remain largely enigmatic. Here, we show that hepatic Krüppel-like factor 9 (Klf9) gene expression is induced by dexamethasone and fasting. The overexpression of Klf9 in primary hepatocytes strongly stimulated Pgc1a gene expression through direct binding to its promoter, thereby activating the gluconeogenic program. However, Klf9 mutation abolished the stimulatory effect of dexamethasone on cellular glucose output. Adenovirus-mediated overexpression of KLF9 in the mouse liver markedly increased blood glucose levels and impaired glucose tolerance. Conversely, both global Klf9-mutant mice and liver-specific Klf9-deleted mice displayed fasting hypoglycemia. Moreover, the knockdown of Klf9 in the liver in diabetic mouse models, including ob/ob and db/db mice, markedly lowered fasting blood glucose levels. Notably, hepatic Klf9 deficiency in mice alleviated hyperglycemia induced by chronic dexamethasone treatment. These results suggest a critical role for KLF9 in the regulation of hepatic glucose metabolism and identify hepatic induction of KLF9 as a mechanism underlying glucocorticoid therapy-induced diabetes.

Spermidine ameliorates non-alcoholic fatty liver disease through regulating lipid metabolism via AMPK.

In this study, treatment of high-fat diet-induced obesity (DIO) C57BL/6J mice with spermidine decreased body weight and subcutaneous and visceral fat content, reversed the apparent hepatosteatosis, and reduced hepatic intracellular and serum triglyceride and total cholesterol concentrations. Moreover, spermidine treatment improved glucose tolerance and insulin sensitivity in DIO mice. The mechanism studies indicated that spermidine indeed increased the phosphorylation of hepatic AMP-activated protein kinase (AMPK), and inhibited the expression of lipogenic genes in vivo and in vitro. Moreover, these spermidine-mediated molecular effects were also abolished by compound C, an inhibitor of AMPK, in primary hepatocytes. In summary, spermidine protected against DIO-induced hepatosteatosis by decreasing lipogenic genes expression through an AMPK-mediated mechanism.

SIRT6 regulates metabolic homeostasis in skeletal muscle through activation of AMPK.

Because of the mass and functions in metabolism, skeletal muscle is one of the major organs regulating whole body metabolic homeostasis. SIRT6, a histone deacetylase, has been shown to regulate metabolism in liver and brain; however, its specific role in skeletal muscle is undetermined. In the present study we explored physiological function of SIRT6 in muscle. We generated a muscle-specific SIRT6 knockout mouse model. The mice with SIRT6 deficiency in muscle displayed impaired glucose homeostasis and insulin sensitivity, attenuated whole body energy expenditure, and weakened exercise performance. Mechanistically, deletion of SIRT6 in muscle decreased expression of genes involved in glucose and lipid uptake, fatty acid oxidation, and mitochondrial oxidative phosphorylation in muscle cells because of the reduced AMP-activated protein kinase (AMPK) activity. In contrast, overexpression of SIRT6 in C2C12 myotubes activates AMPK. Our results from both gain- and loss-of-function experiments identify SIRT6 as a physiological regulator of muscle mitochondrial function. These findings indicate that SIRT6 is a potential therapeutic target for treatment of type 2 diabetes mellitus.

KLF10 transcription factor regulates hepatic glucose metabolism in mice.

AIM/HYPOTHESIS: Abnormal activation of hepatic gluconeogenesis leads to hyperglycaemia. However, the molecular mechanisms underlying dysregulated hepatic gluconeogenesis remain to be fully defined. Here, we explored the physiological role of Krüppel-like factor 10 (KLF10) in regulating hepatic glucose metabolism in mice. METHODS: Hepatic KLF10 expression in wild-type C57BL/6J mice, the db/db mouse model of diabetes, the ob/ob mouse model of obesity and high-fat-diet-induced obese (DIO) mice was measured. Adenoviruses expressing Klf10 or Klf10-specific short-hairpin RNA were injected into wild-type C57BL/6J mice, db/db or DIO mice. Expression of gluconeogenic genes in the liver and blood glucose levels were measured. GTTs and pyruvate tolerance tests were performed. The molecular mechanism by which KLF10 regulates hepatic glucose metabolism was explored. RESULTS: Hepatic KLF10 expression was regulated by nutritional status in wild-type mice and upregulated in diabetic, obese and DIO mice. Overexpression of KLF10 in primary hepatocytes increased the expression of gluconeogenic genes and cellular glucose output. C57BL/6J mice with KLF10 overexpression in the liver displayed increased blood glucose levels and impaired glucose tolerance. Conversely, hepatic KLF10 knockdown in db/db and DIO mice decreased blood glucose levels and improved glucose tolerance. Furthermore, luciferase reporter gene assay and chromatin immunoprecipitation analysis indicated that KLF10 activates Pgc-1α (also known as Ppargc1a) gene transcription via directly binding to its promoter region. CONCLUSIONS/INTERPRETATION: KLF10 is an important regulator of hepatic glucose metabolism and modulation of KLF10 expression in the liver may be an attractive approach for the treatment of type 2 diabetes.

Retinoic acid ameliorates high-fat diet-induced liver steatosis through sirt1.

In this study, treatment of C57BL/6J (wild type, WT) mice fed a high-fat diet (HFD) with retinoic acid (RA) decreased body weight and subcutaneous and visceral fat content, reversed the apparent hepatosteatosis, and reduced hepatic intracellular triglyceride and serum alanine transaminase (ALT) and aspartate aminotransferase (AST) concentrations. Moreover, RA treatment improved glucose tolerance and insulin sensitivity in WT mice fed a HFD. However, these RA-induced effects in WT mice fed a HFD were alleviated in liver specific Sirtuin 1 (Sirt1) deficient (LKO) mice fed a HFD. Furthermore, RA also could not improve glucose tolerance and insulin sensitivity in LKO mice fed a HFD. The mechanism studies indicated that RA indeed increased the expression of hepatic Sirt1 and superoxide dismutase 2 (Sod2), and inhibited the expression of sterol regulatory element binding protein 1c (Srebp-1c) in WT mice in vivo and in vitro. RA decreased mitochondrial reactive oxygen species (ROS) production in WT primary hepatocytes and increased mitochondrial DNA (mtDNA) copy number in WT mice liver. However, these RA-mediated molecular effects were also abolished in the liver and primary hepatocytes from LKO mice. In summary, RA protected against HFD-induced hepatosteatosis by decreasing Srebp-1c expression and improving antioxidant capacity through a Sirt1-mediated mechanism.

Cold-Inducible SIRT6 Regulates Thermogenesis of Brown and Beige Fat.

Promoting development and function of brown and beige fat may reduce obesity. Here, we show that fat SIRT6 expression is markedly induced by cold exposure and a ß-adrenergic agonist. Deletion of SIRT6 in adipose tissue impairs the thermogenic function of brown adipocytes, causing a morphological "whitening" of brown fat, reduced oxygen (O2) consumption, obesity, decreased core body temperature, and cold sensitivity. Fat SIRT6-deleted mice exhibit increased blood glucose levels, severe insulin resistance, and hepatic steatosis. Moreover, SIRT6 deficiency inhibits the browning of white adipose tissue (WAT) following cold exposure or ß3-agonist treatment. Depletion of SIRT6 expression in brown adipocytes reduces expression of thermogenic genes, causing a reduction in cellular respiration. Conversely, SIRT6 overexpression in primary fat cells stimulates the thermogenic program. Mechanistically, SIRT6 interacts with and promotes phospho-ATF2 binding to the PGC-1α gene promoter to activate its expression. The present study reveals a critical role for SIRT6 in regulating thermogenesis of fat.

Celastrol ameliorates liver metabolic damage caused by a high-fat diet through Sirt1.

OBJECTIVE: Celastrol was recently identified as a potential novel treatment for obesity. However, the effect of Celastrol on nonalcoholic fatty liver disease (NAFLD) remains elusive. The aim of this study is to evaluate the role of Celastrol in NAFLD. METHODS: Functional studies were performed using wild-type C57BL/6J (WT) mice and liver specific Sirt1-deficient (LKO) mice. The molecular mechanism was explored in primary mouse liver and primary hepatocytes. RESULTS: When WT mice receiving a high-fat diet (HFD) were treated with Celastrol, reductions in body weight, subcutaneous and visceral fat content, and liver lipid droplet formation were observed, along with reduced hepatic intracellular triglyceride and serum triglyceride, free fatty acid, and ALT concentrations. Furthermore, Celastrol decreased hepatic sterol regulatory element binding protein 1c (Srebp-1c) expression, enhanced the phosphorylation of hepatic AMP-activated protein kinase α (AMPKα), and increased the expression of hepatic serine-threonine liver kinase B1 (LKB1). Additionally, Celastrol treatment improved glucose tolerance and insulin sensitivity in WT mice fed the HFD. Celastrol administration also improved the anti-inflammatory and anti-oxidative status by inhibiting nuclear factor kappa B (NFκB) activity and the mRNA levels of proinflammatory cytokines and increasing mitochondrial DNA copy number and anti-oxidative stress genes expression in WT mice liver, in vivo and in vitro. Moreover, Celastrol induced hepatic Sirt1 expression in WT mice, in vivo and in vitro. These Celastrol-mediated protective effects in WT mice fed a HFD were abolished in LKO mice fed a HFD. It was more interesting that Celastrol aggravated HFD-induced liver damage in LKO mice fed a HFD by inhibiting the phosphorylation of AMPKα and boosting the translocation of NFκB into the nucleus, thereby resulting in the increase of Srebp-1c expression and the mRNA levels of liver proinflammatory cytokines. CONCLUSIONS: Celastrol ameliorates NAFLD by decreasing lipid synthesis and improving the anti-oxidative and anti-inflammatory status. And Sirt1 has an important role in Celastrol-ameliorating liver metabolic damage caused by HFD.

Metformin impairs systemic bile acid homeostasis through regulating SIRT1 protein levels.

Metformin is widely used to treat hyperglycemia. However, metformin treatment may induce intrahepatic cholestasis and liver injury in a few patients with type II diabetes through an unknown mechanism. Here we show that metformin decreases SIRT1 protein levels in primary hepatocytes and liver. Both metformin-treated wild-type C57 mice and hepatic SIRT1-mutant mice had increased hepatic and serum bile acid levels. However, metformin failed to change systemic bile acid levels in hepatic SIRT1-mutant mice. Molecular mechanism study indicates that SIRT1 directly interacts with and deacetylates Foxa2 to inhibit its transcriptional activity on expression of genes involved in bile acids synthesis and transport. Hepatic SIRT1 mutation elevates Foxa2 acetylation levels, which promotes Foxa2 binding to and activating genes involved in bile acids metabolism, impairing hepatic and systemic bile acid homeostasis. Our data clearly suggest that hepatic SIRT1 mediates metformin effects on systemic bile acid metabolism and modulation of SIRT1 activity in liver may be an attractive approach for treatment of bile acid-related diseases such as cholestasis.

The hepatic FOXQ1 transcription factor regulates glucose metabolism in mice.

AIM/HYPOTHESIS: Hepatic forkhead box q1 (FOXQ1) expression levels are regulated by nutritional and pathophysiological status. In this study we investigated the role of FOXQ1 in the regulation of hepatic gluconeogenesis. METHODS: We used multiple mouse and cell models to study the role of FOXQ1 in regulating expression of gluconeogenic genes, and cellular and hepatic glucose production. RESULTS: Expression of hepatic FOXQ1 was regulated by fasting in normal mice and was dysregulated in diabetic mice. Overexpression of FOXQ1 in primary hepatocytes inhibited expression of gluconeogenic genes and decreased cellular glucose output. Hepatic FOXQ1 rescue in db/db and high-fat diet-induced obese mice markedly decreased blood glucose level and improved glucose intolerance. In contrast, wild-type C57 mice with hepatic FOXQ1 deficiency displayed increased blood glucose levels and impaired glucose tolerance. Interestingly, studies into molecular mechanisms indicated that FOXQ1 interacts with FOXO1, thereby blocking FOXO1 activity on hepatic gluconeogenesis, preventing it from directly binding to insulin response elements mapped in the promoter region of gluconeogenic genes. CONCLUSIONS/INTERPRETATION: FOXQ1 is a novel factor involved in regulating hepatic gluconeogenesis, and the decreased FOXQ1 expression in liver may contribute to the development of type 2 diabetes.

Mst1 regulates hepatic lipid metabolism by inhibiting Sirt1 ubiquitination in mice.

Previous study showed mammalian Ste20-like kinase (Mst1) may serve as target for the development of new therapies for diabetes. However, the function of Mst1 involved in liver lipid metabolism has remained elusive. In this study, we report that the liver of Mst1 knockout (Mst1(-/-)) mice showed more severe liver metabolic damage under fasting and high-fat diet than that of control mice. And fasting induced hepatic Mst1 expression. Mst1 overexpression inhibited Srebp-1c expression and increased the expression of antioxidant genes in primary hepatocytes. We also found that fasting-induced expression of hepatic Sirt1 was attenuated in Mst1(-/-) mice. Mst1 overexpression promoted Sirt1 expression, probably due to inhibiting Sirt1 ubiquitination. In summary, our study suggests that Mst1 regulates hepatic lipid metabolism by inhibiting Sirt1 ubiquitination in mice.

Forkhead Box P1 (FOXP1) Transcription Factor Regulates Hepatic Glucose Homeostasis.

Dysregulation of hepatic gluconeogenesis contributes to the pathogenesis of diabetes, yet the detailed molecular mechanisms remain to be fully elucidated. Here we show that FOXP1, a transcriptional repressor, plays a key role in the regulation of systemic glucose homeostasis. Hepatic expression levels of FOXP1 are decreased in diabetic mice. Modest hepatic overexpression of FOXP1 in mice inhibited the expression of gluconeogenic genes, such as peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6PC), leading to a decrease in hepatic glucose production and fasting blood glucose levels in normal mice and different mouse models of diabetes, including db/db diabetic and high-fat diet-induced obese mice. FOXP1 physically interacted with FOXO1 in vivo and competed with FOXO1 for binding to the insulin response element in the promoter region of gluconeogenic genes, thereby interfering expression of these genes. These results identify a previously unrecognized role for FOXP1 in the transcriptional control of hepatic glucose homeostasis.

Sirt1 mediates the effect of the heme oxygenase inducer, cobalt protoporphyrin, on ameliorating liver metabolic damage caused by a high-fat diet.

BACKGROUND & AIMS: Heme oxygenase 1 (HO-1)-mediated increases in adiponectin, ameliorate the deleterious effects of obesity and metabolic syndrome; however, the effect of HO-1 on hepatic lipid metabolism remains elusive. The aim of this study is to evaluate the role of HO-1 in hepatic lipid metabolism. METHODS: Functional studies were performed using C57BL/6J (WT) mice and Sirt1 liver specific mutant (Sirt1-deficient) mice. The molecular mechanism was explored in primary hepatocytes and mouse liver. RESULTS: Chronic exposure to high-fat diet (HFD) induced hepatic steatosis in WT mice. Treatment of WT mice on HFD with cobalt protoporphyrin (CoPP), an inducer of HO-1 activity, decreased body weight and visceral fat content, reduced intracellular hepatic triglyceride and serum total cholesterol concentrations, and decreased liver lipid droplet formation. Compared with WT mice, the administration of CoPP to Sirt1-deficient mice on HFD increased visceral fat content, and slightly promoted liver lipid droplet formation. CoPP improved glucose tolerance and insulin sensitivity in WT mice on HFD, but compromised insulin sensitivity in Sirt1-deficient mice on HFD. Furthermore, CoPP-induced Sirt1 expression and decreased sterol regulatory element binding protein 1c (SREBP-1c) expression in WT mice on HFD. However, CoPP promoted SREBP-1c expression in Sirt1-deficient hepatocytes, which was reversed by a protein tyrosine phosphatase 1b inhibitor. Additionally, while the administration of CoPP to WT mice on HFD improved antioxidant and anti-inflammatory states, these CoPP-mediated effects were abolished in Sirt1-deficient mice. CONCLUSIONS: Sirt1 mediates the effect of CoPP on ameliorating liver metabolic damage caused by HFD.

[Preface for special issue on proteomics (2014)].

Proteomics is one of the most important functional genomics research in the post-genomic era, which is closely related to medical biology, chemistry, physics, information science and modern technology. Through review research progress of some important proteomics, a proteomics special issue is published so as to find problems, explore the possible applications and outlook the development prospects of proteomics. The special issue consists of reviews and original papers, mainly involving in the following aspects, i) proteomics about different species such as humans, mammals, prokaryotes and actinobacterial; ii) proteomics methodology and techniques including tandem mass spectrometry analysis, film (urimem) preservation of urine protein, quantitative proteomic analysis and meta analysis; iii) function and application of proteome such as spider (Latrodectus tredecimguttatus) toxins proteome, protein phosphorylation proteome, oocytes and early embryos proteomes, liver fibrosis proteome, drug-resistant mycobacterium tuberculosis proteome, etc.

Hepatic differentiated embryo-chondrocyte-expressed gene 1 (Dec1) inhibits sterol regulatory element-binding protein-1c (Srebp-1c) expression and alleviates fatty liver phenotype.

Hepatic steatosis, characterized by ectopic hepatic triglyceride accumulation, is considered as the early manifestation of non-alcoholic fatty liver diseases (NAFLD). Increased SREBP-1c level and activity contribute to excessive hepatic triglyceride accumulation in NAFLD patients; however, negative regulators of Srebp-1c are not well defined. In this study, we show that Dec1, a critical regulator of circadian rhythm, negatively regulates hepatic Srebp-1c expression. Hepatic Dec1 expression levels are markedly decreased in NAFLD mouse models. Restored Dec1 gene expression levels in NAFLD mouse livers decreased the expression of Srebp-1c and lipogenic genes, subsequently ameliorating the fatty liver phenotype. Conversely, knockdown of Dec1 expression by an adenovirus expressing Dec1-specific shRNA led to an increase in hepatic TG content in normal mouse livers. Correspondingly, expression levels of lipogenic genes, including Srebp-1c, Fas, and Acc, were increased in livers of mice with Dec1 knockdown. Moreover, a functional lipogenesis assay suggested that Dec1 overexpression repressed lipid synthesis in primary hepatocytes. Finally, a luciferase reporter gene assay indicates that DEC1 inhibits Srebp-1c gene transcription via the E-box mapped to the promoter region. Chromatin immunoprecipitation confirmed that DEC1 proteins bound to the identified E-box element. Our studies indicate that DEC1 is an important regulator of Srebp-1c expression and links circadian rhythm to hepatic lipogenesis. Activation of Dec1 can alleviate the nonalcoholic fatty liver phenotype.

Involvement of KLF11 in hepatic glucose metabolism in mice via suppressing of PEPCK-C expression.

BACKGROUND: Abnormal hepatic gluconeogenesis is related to hyperglycemia in mammals with insulin resistance. Despite the strong evidences linking Krüppel-like factor 11 (KLF11) gene mutations to development of Type 2 diabetes, the precise physiological functions of KLF11 in vivo remain largely unknown. RESULTS: In current investigation, we showed that KLF11 is involved in modulating hepatic glucose metabolism in mice. Overexpression of KLF11 in primary mouse hepatocytes could inhibit the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase (cytosolic isoform, PEPCK-C) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), subsequently decreasing the cellular glucose output. Diabetic mice with overexpression of KLF11 gene in livers significantly ameliorated hyperglycemia and glucose intolerance; in contrast, the knockdown of KLF11 expression in db/m and C57BL/6J mice livers impaired glucose tolerance. CONCLUSIONS: Our data strongly indicated the involvement of KLF11 in hepatic glucose homeostasis via modulating the expression of PEPCK-C.

Cobalt protoporphyrin induces HO-1 expression mediated partially by FOXO1 and reduces mitochondria-derived reactive oxygen species production.

BACKGROUND: Reactive oxygen species arise in the mitochondria as byproducts of respiration and oxidase activity and have important roles in many physiological and pathophysiological conditions. The level of reactive oxygen species is regulated by a number of enzymes and physiological antioxidants, including HO-1, Sod2, catalase and COX-2, etc. And HO-1 against oxidative stress requires an increase in stress-responsive genes, such as Sod2 and catalase. Especially for the activity of HO-1, cobalt protoporphyrin is known to be a potent and effective inducer in many tissues. The transcription factor, FOXO1 is resistant to oxidative stress through downregulating reactive oxygen species production. Previous study showed that FOXO1 induces HO-1 expression by binding to HO-1 promoter. The question whether cobalt protoporphyrin induces HO-1 expression mediated by FOXO1 and subsequently lessens reactive oxygen species production remains to be elucidated. RESULTS: Cobalt protoporphyrin enhances the expression of FOXO1 and facilitates FOXO1 binding to HO-1 promoter and increasing its transcriptional activity without influencing the FOXO1 protein stability. CoPP induces HO-1 and other oxidative stress-responsive genes expression, such as catalase, cytochrome c, Sod2, and COX-2, and decreases mitochondria-derived reactive oxygen species production, which are mediated partially by FOXO1. CONCLUSIONS: Cobalt protoporphyrin induces HO-1 and other oxidative stress-responsive genes expression mediated partially by FOXO1, and has an important role in reducing cellular reactive oxygen species level. Cobalt protoporphyrin may be a more promising therapeutic agent to upregulate some antioxidantive genes.