FoxO1 Acetylation Research Articles

Pyrrolidine dithiocarbamate protects pancreatic β-cells from oxidative damage through regulation of FoxO1 activity in type 2 diabetes rats.

Pyrrolidine dithiocarbamate (PDTC) can lower the blood glucose level and improve the insulin sensitivity in diabetic rats. However, the mechanisms underlying this effect of PDTC treatment in diabetic rats remained uncertain. In this study, we evaluated the mechanisms by which PDTC conferred protection against oxidative damage to pancreatic islet β-cells in rats with experimental type 2 diabetes mellitus (DM). DM in the rats was elicited by long-term high-fat diet accompanied with a single intraperitoneal (i.p.) injection of a low dose of streptozotocin. After a 7-day administration of PDTC (50 mg/kg/day i.p.), blood glucose levels were measured and pancreatic tissues were collected for the determination of various biochemical and enzymatic activities using immunohistochemistry, immunofluorescence, and western blot techniques. The percentage of apoptotic pancreatic islet β-cells was detected by flow cytometry. The results showed that diabetic rats had elevated blood glucose levels and insulin resistance, accompanied with an increase in malondialdehyde content, nitrotyrosine production, and inducible nitric oxide synthase expression. A decrease in superoxide dismutase and glutathione peroxidase activities was also observed in DM rats, culminating with elevated β-cell apoptosis. PDTC treatment significantly reduced the oxidative damage and the β-cell apoptosis, and also increased the insulin production through down-regulating FoxO1 acetylation and up-regulating nuclear PDX-1 level. These data suggested that PDTC can protect islet β-cells from oxidative damage and improve insulin production through regulation of PDX-1 and FoxO1 in a DM rat model.

Inhibition of FoxO1 acetylation by INHAT subunit SET/TAF-Iβ induces p21 transcription

Post-translational modification of forkhead family transcription factor, FoxO1, is an important regulatory mode for its diverse activities. FoxO1 is acetylated by HAT coactivators and its transcriptional activity is decreased via reduced DNA binding affinity. Here, we report that SET/TAF-Iβ inhibited p300-mediated FoxO1 acetylation in an INHAT domain-dependent manner. SET/TAF-Iβ interacted with FoxO1 and activated transcription of FoxO1 target gene, p21. Moreover, SET/TAF-Iβ inhibited acetylation of FoxO1 and increased p21 transcription induced by oxidative stress. Our results suggest that SET/TAF-Iβ inhibits FoxO1 acetylation and activates its transcriptional activity toward p21.

Acetylated FoxO1 mediates high-glucose induced autophagy in H9c2 cardiomyoblasts: Regulation by a polyphenol -(−)-epigallocatechin-3-gallate

ObjectiveFoxO1 acts as a pivotal transcription factor in insulin signaling. However, in hyperglycemia induced cardiac complications, whether FoxO1 is involved remains unclear. The goal of this study was to delineate the potential role of FoxO1 under high-glucose condition. Materials/methodsWe investigated insulin resistance and reactive oxygen species (ROS) generation in H9c2 cardiomyoblasts after high-glucose exposure. A series of autophagy biomarkers were measured and further confirmed by LC3 turnover assay. Using gene silencing and overexpression experiments we dissected the molecular mechanisms of FoxO1 regulated autophagy. We also tested the protective effect of (−)-epigallocatechin-3-gallate (EGCG, a green tea-derived polyphenol) in high-glucose treated H9c2 cardiomyoblasts. ResultsHigh-glucose elicited elevated ROS, autophagy and FoxO1 abundance in cultured H9c2 cardiomyoblasts. Specifically, high-glucose significantly augmented the acetylated FoxO1 in cytosol. In line, compared with 3A-FoxO1 (majorly localized in nuclei with a strong transcriptional activity), overexpression of WT-FoxO1 led to more intense elevated autophagy with enhanced acetylation of FoxO1. In addition, FoxO1 RNAi brought down autophagy induced by high-glucose. Intriguingly, EGCG successfully reversed ROS, autophagy and acetylated FoxO1 in high-glucose treated H9c2 cells. ConclusionOur findings suggest that FoxO1, especially the acetylated form, regulates high-glucose induced autophagy in H9c2 cardiomyoblasts, which can be prevented by EGCG via a possible ROS-FoxO1 pathway.

Nicotinamide mononucleotide, an intermediate of NAD+ synthesis, protects the heart from ischemia and reperfusion.

Nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD+) synthesis, and Sirt1, an NAD+-dependent histone deacetylase, protect the heart against ischemia/reperfusion (I/R). It remains unknown whether Nampt mediates the protective effect of ischemic preconditioning (IPC), whether nicotinamide mononucleotide (NMN, 500 mg/kg), a product of Nampt in the NAD+ salvage pathway, mimics the effect of IPC, or whether caloric restriction (CR) upregulates Nampt and protects the heart through a Sirt1-dependent mechanism. IPC upregulated Nampt protein, and the protective effect of IPC against ischemia (30 minutes) and reperfusion (24 hours) was attenuated at both early and late phases in Nampt +/− mice, suggesting that Nampt plays an essential role in mediating the protective effect of IPC. In order to mimic the effect of Nampt, NMN was administered by intraperitoneal injection. NMN significantly increased the level of NAD+ in the heart at baseline and prevented a decrease in NAD+ during ischemia. NMN protected the heart from I/R injury when it was applied once 30 minutes before ischemia or 4 times just before and during reperfusion, suggesting that exogenous NMN protects the heart from I/R injury in both ischemic and reperfusion phases. The protective effect of NMN was accompanied by decreases in acetylation of FoxO1, but it was not obvious in Sirt1 KO mice, suggesting that the effect of NMN is mediated through activation of Sirt1. Compared to control diet (90% calories), CR (60% calories for 6 weeks) in mice led to a significant reduction in I/R injury, accompanied by upregulation of Nampt. The protective effect of CR against I/R injury was not significant in cardiac-specific Sirt1 KO mice, suggesting that the protective effect of CR is in part mediated through the Nampt-Sirt1 pathway. In conclusion, exogenous application of NMN and CR protects the heart by both mimicking IPC and activating Sirt1.

Modulating effect of SIRT1 activation induced by resveratrol on Foxo1‐associated apoptotic signalling in senescent heart

Elevations of cardiomyocyte apoptosis and fibrotic deposition are major characteristics of the ageing heart. Resveratrol, a polyphenol in grapes and red wine, is known to improve insulin resistance and increase mitochondrial biogenesis through the SIRT1-PGC-1α signalling axis. Recent studies attempted to relate SIRT1 activation by resveratrol to the regulation of apoptosis in various disease models of cardiac muscle. In the present study, we tested the hypothesis that long-term (8-month) treatment of resveratrol would activate SIRT1 and improve the cardiac function of senescent mice through suppression of Foxo1-associated pro-apoptotic signalling. Our echocardiographic measurements indicated that the cardiac systolic function measured as fractional shortening and ejection fraction was significantly reduced in aged mice when compared with the young mice. These reductions, however, were not observed in resveratrol-treated hearts. Ageing significantly reduced the deacetylase activity, but not the protein abundance of SIRT1 in the heart. This reduction was accompanied by increased acetylation of the Foxo1 transcription factor and transactivation of its target, pro-apoptotic Bim. Subsequent analyses indicated that pro-apoptotic signalling measured as p53, Bax and apoptotic DNA fragmentation was up-regulated in the heart of aged mice. In contrast, resveratrol restored SIRT1 activity and suppressed elevations of Foxo1 acetylation, Bim and pro-apoptotic signalling in the aged heart. In parallel, resveratrol also attenuated the ageing-induced elevations of fibrotic collagen deposition and markers of oxidative damage including 4HNE and nitrotyrosine. In conclusion, these novel data demonstrate that resveratrol mitigates pro-apoptotic signalling in senescent heart through a deacetylation mechanism of SIRT1 that represses the Foxo1-Bim-associated pro-apoptotic signalling axis.

CBP-Mediated FOXO-1 Acetylation Inhibits Pancreatic Tumor Growth by Targeting SirT

Here, we investigated the potential mechanism of capsaicin-mediated apoptosis in pancreatic cancer cells. Capsaicin treatment phosphorylated c-jun-NH2-kinase (JNK); forkhead box transcription factor, class O (FOXO1); and BIM in BxPC-3, AsPC-1, and L3.6PL cells. The expression of BIM increased in response to capsaicin treatment. Capsaicin treatment caused cleavage of caspase-3 and PARP, indicating apoptosis. Antioxidants tiron and PEG-catalase blocked capsaicin-mediated JNK/FOXO/BIM activation and protected the cells from apoptosis. Furthermore, capsaicin treatment caused a steady increase in the nuclear expression of FOXO-1, leading to increased DNA binding. Capsaicin-mediated expression of BIM was found to be directly dependent on the acetylation of FOXO-1. The expression of CREB-binding protein (CBP) was increased, whereas SirT-1 was reduced by capsaicin treatment. Using acetylation mimic or defective mutants, our result demonstrated that phosphorylation of FOXO-1 was mediated through acetylation by capsaicin treatment. JNK inhibitor attenuated the phosphorylation of FOXO-1, activation of BIM, and abrogated capsaicin-induced apoptosis. Moreover, silencing FOXO1 by siRNA blocked capsaicin-mediated activation of BIM and apoptosis, whereas overexpression of FOXO-1 augmented its effects. Silencing Bim drastically reduced capsaicin-mediated cleavage of caspase-3 and PARP, indicating the role of BIM in apoptosis. Oral administration of 5 mg/kg capsaicin substantially suppressed the growth of BxPC-3 tumor xenografts in athymic nude mice. Tumors from capsaicin-treated mice showed an increase in the phosphorylation of JNK, FOXO-1, BIM, and levels of CBP, cleavage of caspase-3, PARP, and decreased SirT-1 expression. Taken together, our results suggest that capsaicin activated JNK and FOXO-1, leading to the acetylation of FOXO-1 through CBP and SirT-1. Acetylated FOXO1 induced apoptosis in pancreatic cancer cells through BIM activation.

MTOR Complex 2 Controls Glycolytic Metabolism in Glioblastoma through FoxO Acetylation and Upregulation of c-Myc

Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.

Ginsenoside Rc modulates Akt/FoxO1 pathways and suppresses oxidative stress

Ginsenoside Rc (Rc), a protopanaxadiol type ginsenoside, is the active component mainly responsible for the therapeutic and pharmacologic properties of ginseng, which are derived from its suppression of superoxide-induced free radicals. Forkhead box O (FoxO1) regulates various genes involved in cellular metabolism related to cell death and response to oxidative stress, and Rc is known to prevent FoxO1 phosphorylation by activation of PI3K/Akt and subsequent inhibition of AMP-activated protein kinase (AMPK) in cells exposed to tert-butylhydroperoxide (t-BHP). In the current study, we attempted the mechanism of increased catalase expression by Rc through inhibition of FoxO1 activation resulting from t-BHP-induced production of reactive species (RS). We found that overexpression of catalase induced by Rc resulted in suppression of RS production in kidney human embryo kidney 293T cells (HEK293T) cells, and that oxidative stress induced activation of PI3K/Akt and inhibition of the AMPK pathway and FoxO1 phosphorylation, leading to down-regulation of catalase, a FoxO1-targeting gene. In addition, treatment of HEK293T cells with Rc resulted in cAMP-response element-binding protein (CREB)-binding protein (CBP) regulated FoxO1 acetylation. Our results suggest that Rc modulates FoxO1 phosphorylation through activation of PI3K/Akt and inhibition of AMPK and FoxO1 acetylation through interaction with CBP and SIRT1, and that this leads to upregulation of catalase under conditions of oxidative stress.

Prenatal Ethanol Exposure Causes Glucose Intolerance with Increased Hepatic Gluconeogenesis and Histone Deacetylases in Adult Rat Offspring: Reversal by Tauroursodeoxycholic Acid

Prenatal ethanol exposure results in increased glucose production in adult rat offspring and this may involve modulation of protein acetylation by cellular stress. We used adult male offspring of dams given ethanol during gestation days 1–7 (early), 8–14 (mid) and 15–21 (late) compared with those from control dams. A group of ethanol offspring was treated with tauroursodeoxycholic acid (TUDCA) for 3 weeks. We determined gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase, hepatic free radicals, histone deacetylases (HDAC), acetylated foxo1, acetylated PEPCK, and C/EBP homologous protein as a marker of endoplasmic reticulum stress. Prenatal ethanol during either of the 3 weeks of pregnancy increased gluconeogenesis, gluconeogenic genes, oxidative and endoplasmic reticulum stresses, sirtuin-2 and HDAC3, 4, 5, and 7 in adult offspring. Conversely, prenatal ethanol reduced acetylation of foxo1 and PEPCK. Treatment of adult ethanol offspring with TUDCA reversed all these abnormalities. Thus, prenatal exposure of rats to ethanol results in long lasting oxidative and endoplasmic reticulum stresses explaining increased expression of gluconeogenic genes and HDAC proteins which, by deacetylating foxo1 and PEPCK, contribute to increased gluconeogenesis. These anomalies occurred regardless of the time of ethanol exposure during pregnancy, including early embryogenesis. As these anomalies were reversed by treatment of the adult offspring with TUDCA, this compound has therapeutic potentials in the treatment of glucose intolerance associated with prenatal ethanol exposure.

The novel growth factor, progranulin, stimulates mouse cholangiocyte proliferation via sirtuin-1-mediated inactivation of FOXO1

Progranulin (PGRN), a secreted growth factor, regulates the proliferation of various epithelial cells. Its mechanism of action is largely unknown. Sirtuin 1 (Sirt1) is a protein deacetylase that is known to regulate the transcriptional activity of the forkhead receptor FOXO1, thereby modulating the balance between proapoptotic and cell cycle-arresting genes. We have shown that PGRN is overexpressed in cholangiocarcinoma and stimulates proliferation. However, its effects on hyperplastic cholangiocyte proliferation are unknown. In the present study, the expression of PGRN and its downstream targets was determined after bile duct ligation (BDL) in mice and in a mouse cholangiocyte cell line after stimulation with PGRN. The effects of PGRN on cholangiocyte proliferation were assessed in sham-operated (sham) and BDL mice treated with PGRN or by specifically knocking down endogenous PGRN expression using Vivo-Morpholinos or short hairpin RNA. PGRN expression and secretion were upregulated in proliferating cholangiocytes isolated after BDL. Treatment of mice with PGRN increased biliary mass and cholangiocyte proliferation in vivo and in vitro and enhanced cholangiocyte proliferation observed after BDL. PGRN treatment decreased Sirt1 expression and increased the acetylation of FOXO1, resulting in the cytoplasmic accumulation of FOXO1 in cholangiocytes. Overexpression of Sirt1 in vitro prevented the proliferative effects of PGRN. Conversely, knocking down PGRN expression in vitro or in vivo inhibited cholangiocyte proliferation. In conclusion, these data suggest that the upregulation of PGRN may be a key feature stimulating cholangiocyte proliferation. Modulating PGRN levels may be a viable technique for regulating the balance between ductal proliferation and ductopenia observed in a variety of cholangiopathies.

Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice

Leucine supplementation has been shown to prevent high-fat diet (HFD)-induced obesity, hyperglycemia, and dyslipidemia in animal models, but the underlying mechanisms are not fully understood. Recent studies suggest that activation of Sirtuin 1 (SIRT1) is an important mechanism to maintain energy and metabolic homeostasis. We therefore examined the involvement of SIRT1 in leucine supplementation-prevented obesity and insulin resistance. To accomplish this goal, male C57BL/6J mice were fed normal diet or HFD, supplemented with or without leucine. After 2 mo of treatment, alterations in SIRT1 expression, insulin signaling, and energy metabolism were analyzed. Eight weeks of HFD induced obesity, fatty liver, mitochondrial dysfunction, hyperglycemia, and insulin resistance in mice. Addition of leucine to HFD correlated with increased expression of SIRT1 and NAMPT (nicotinamide phosphoribosyltransferase) as well as higher intracellular NAD(+) levels, which decreased acetylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and forkhead box O1 (FoxO1). The deacetylation of PGC1α may contribute to upregulation of genes controlling mitochondrial biogenesis and fatty acid oxidation, thereby improving mitochondrial function and preventing HFD-induced obesity in mice. Moreover, decreased acetylation of FoxO1 was accompanied by decreased expression of pseudokinase tribble 3 (TRB3) and reduced the association between TRB3 and Akt, which enhanced insulin sensitivity and improved glucose metabolism. Finally, transfection of dominant negative AMPK prevented activation of SIRT1 signaling in HFD-Leu mice. These data suggest that increased expression of SIRT1 after leucine supplementation may lead to reduced acetylation of PGC1α and FoxO1, which is associated with attenuation of HFD-induced mitochondrial dysfunction, insulin resistance, and obesity.

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H2O2-induced viability loss, which specifically evokes an autophagic response. Exposed to H2O2, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.

Necdin Controls Foxo1 Acetylation in Hypothalamic Arcuate Neurons to Modulate the Thyroid Axis

The forkhead transcription factor Foxo1 regulates energy homeostasis by modulating gene expression in the hypothalamus. Foxo1 undergoes post-translational modifications such as phosphorylation and acetylation, which modulate its functional activities. Sirtuin1 (Sirt1), a nicotinamide adenine dinucleotide-dependent protein deacetylase, regulates the acetylation status of Foxo1 in mammalian cells. Necdin, a pleiotropic protein required for neuronal development and survival, interacts with both Sirt1 and p53 to facilitate p53 deacetylation. The necdin gene (Ndn), an imprinted gene transcribed only from the paternal allele, is strongly expressed in hypothalamic neurons. Here, we demonstrate that necdin controls the acetylation status of Foxo1 in vivo in hypothalamic arcuate neurons to modulate the thyroid function. Necdin forms a stable ternary complex with Sirt1 and Foxo1, diminishes Foxo1 acetylation, and suppresses the transcriptional activity of Foxo1 in vitro. Paternal Ndn mutant mice express high levels of acetylated Foxo1 and mRNAs encoding agouti-related protein and neuropeptide Y in the hypothalamus in vivo during the juvenile period. The mutant mice exhibit endocrine dysfunction characteristic of hypothalamic hypothyroidism. Chemically induced hyperthyroidism and hypothyroidism lead to hypothalamic responses similar to those under necdin-deficient and excessive conditions, respectively, suggesting that thyroid hormone serves as a negative regulator of this system. These results suggest that necdin regulates Foxo1 acetylation and neuropeptide gene expression in the arcuate neurons to modulate the hypothalamic-pituitary-thyroid axis during development.

Putting the brakes on FOXO1 in fat

The Foxo1 forkhead transcription factor is a critical negative regulator of insulin action. In this issue of The EMBO Journal , Nakae and colleagues (Nakae et al , 2012) describe a novel Foxo1 Co‐Repressor (FCoR) protein in adipocytes, showing that FCoR enhances the acetylation of Foxo1 to inhibit Foxo1 activity, promote adipocyte differentiation, and improve insulin sensitivity.

Hepatic foxO1 acetylation is regulated by menin and influenced by insulin signaling

ObjectiveTo elucidate the relationship between hepatic menin and FoxO1 with respect to protein interaction and post‐translational modification of a metabolically relevant protein.MethodNIH‐3T3 cells were stably transfected with human insulin receptor, serum starved, and protein levels were measured. HepG2 cells were serum starved and treated with 100nM insulin. Animals were fasted 18h, re‐fed 4h, 7h, and random fed.ResultsMenin levels were significantly decreased by 24h exposure to insulin in 3T3 cells stably transfected with the human insulin receptor, however acute effects of insulin show increases in menin mRNA expression. In the liver and HepG2 cells menin interacts with the metabolic protein FoxO1 in an insulin dependent manner. Menin siRNA results in an insulin dependent decrease in FoxO1 acetylation.ConclusionsInsulin regulates menin expression both acutely and chronically in a biphasic manner. Furthermore, insulin mediates FoxO1 and menin interaction after 2h of exposure in HepG2 cells and with fasting and refeeding in C57/BLK6 mice. Loss of menin expression down‐regulate the acetylation state of FoxO1 in an insulin dependent manner. This data implicates menin in the regulation of a metabolic pathway via post‐translational modification of FoxO1 which regulates its cellular localization.Institutional Funding from the University of Toledo College of Medicine was used for these studies.

Benzyl Isothiocyanate Causes FoxO1-Mediated Autophagic Death in Human Breast Cancer Cells

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.

Modulation of FoxO1 phosphorylation/acetylation by baicalin during aging

Baicalin is a flavonoid known to modify various redox-related biological activities. Included is its ability to suppress reactive species (RS) producing activity and modulate nuclear factor-κB through cellular redox regulation with enhanced thiol ability.FoxO regulates various genes that are known to be involved in cellular metabolism related to cell death and the oxidative stress response. One such case is the prevention of FoxO1 expression by activated insulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt, which leads to increased oxidative stress and aging processes.In the present study, we attempted to elucidate the molecular modulation of antioxidant baicalin on the insulin-induced FoxO1 inactivation. We used HEK293T cultured cells and kidney tissue isolated from 24-month-old rats treated with baicalin at a dose of 10 or 20 mg/kg/day for 10 days.We found that baicalin enhanced catalase and suppressed RS production in cell system and in isolated kidney tissue in contrast to the nontreated aged rats. Results also showed activation of insulin signaling (PI3K/Akt), FoxO1 phosphorylation/acetylation and the down-regulation of catalase and manganese superoxide dismutase, both of which are FoxO1-targeting genes. Furthermore, baicalin-treated rats showed a decreased FoxO1 phosphorylation via PI3K/Akt cascade and FoxO1 acetylation by the cAMP-response element-binding protein binding protein (CBP). These results strongly suggest that treatment with baicalin influenced phosphorylation/acetylation of FoxO1 by up-regulating PI3K/Akt signaling through insulin in aged rats. Our results further reveal that baicalin regulated FoxO1 phosphorylation via PI3K/Akt by insulin and FoxO1 acetylation by the interaction of CBP and SIRT1, leading to changes in catalase gene expression during aging.

Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism

Resveratrol (3,5,4′-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.

Glucagon-Like Peptide 1 Inhibits the Sirtuin Deacetylase SirT1 to Stimulate Pancreatic β-Cell Mass Expansion

OBJECTIVEThe glucoincretin hormone glucagon-like peptide 1 (GLP-1) enhances glucose-stimulated insulin secretion and stimulates pancreatic β-cell mass expansion. We have previously shown that the forkhead transcription factor FoxO1 is a prominent transcriptional effector of GLP-1 signaling in the β-cell. FoxO1 activity is subject to a complex regulation by Akt-dependent phosphorylation and SirT1-mediated deacetylation. In this study, we aimed at investigating the potential role of SirT1 in GLP-1 action.RESEARCH DESIGN AND METHODSFoxO1 acetylation levels and binding to SirT1 were studied by Western immunoblot analysis in INS832/13 cells. SirT1 activity was evaluated using an in vitro deacetylation assay and correlated with the NAD+-to-NADH ratio. The implication of SirT1 in GLP-1–induced proliferation was investigated by BrdU incorporation assay. Furthermore, we determined β-cell replication and mass in wild-type and transgenic mice with SirT1 gain of function after daily administration of exendin-4 for 1 week.RESULTSOur data show that GLP-1 increases FoxO1 acetylation, decreases the binding of SirT1 to FoxO1, and stunts SirT1 activity in β-INS832/13 cells. GLP-1 decreases both the NAD+-to-NADH ratio and SirT1 expression in INS cells and isolated islets, thereby providing possible mechanisms by which GLP-1 could modulate SirT1 activity. Finally, the action of GLP-1 on β-cell mass expansion is abolished in both transgenic mice and cultured β-cells with increased dosage of SirT1.CONCLUSIONSOur study shows for the first time that the glucoincretin hormone GLP-1 modulates SirT1 activity and FoxO1 acetylation in β-cells. We also identify SirT1 as a negative regulator of β-cell proliferation.

Dissociation of the Glucose and Lipid Regulatory Functions of FoxO1 by Targeted Knockin of Acetylation-Defective Alleles in Mice

FoxO1 integrates multiple metabolic pathways. Nutrient levels modulate FoxO1 acetylation, but the functional consequences of this posttranslational modification are unclear. To answer this question, we generated mice bearing alleles that encode constitutively acetylated and acetylation-defective FoxO1 proteins. Homozygosity for an allele mimicking constitutive acetylation (Foxo1(KQ/KQ)) results in embryonic lethality due to cardiac and angiogenesis defects. In contrast, mice homozygous fora constitutively deacetylated Foxo1 allele (Foxo1(KR/KR)) display a unique metabolic phenotype of impaired insulin action on hepatic glucose metabolism but decreased plasma lipid levels and low respiratory quotient that are consistent with a state of preferential lipid usage. Moreover, Foxo1(KR/KR) mice showa dissociation between weight gain and insulin resistance in predisposing conditions (high fat diet, diabetes, and insulin receptor mutations), possibly due to decreased cytokine production in adipose tissue. Thus, acetylation inactivates FoxO1 during nutrient excess whereas deacetylation selectively potentiates FoxO1 activity, protecting against excessive catabolism during nutrient deprivation.