Forkhead Transcription Factor FoxO1 Research Articles

FOXO1 induced fatty acid oxidation in hepatic cells by targeting ALDH1L2.

Lipid metabolism disorder is the primary feature of numerous refractory chronic diseases. Fatty acid oxidation, an essential aerobic biological process, is closely related to the progression of NAFLD. The forkhead transcription factor FOXO1 has been reported to play an important role in lipid metabolism. However, the molecular mechanism through which FOXO1 regulates fatty acid oxidation remains unclear. Transcriptomic analysis was performed to examine the cellular expression profile to determine the functional role of FOXO1 in HepG2 cells with palmitic acid (PA)-induced lipid accumulation. FOXO1-binding motifs at the promoter region of aldehyde dehydrogenase 1 family member L2 (ALDH1L2) were predicted via bioinformatic analysis and confirmed via luciferase reporter assay. Overexpression of ALDH1L2 was induced to recover the impaired fatty acid oxidation in FOXO1-knockout cells. Knockout of FOXO1 aggravated lipid deposition in hepatic cells. Transcriptomic profiling revealed that knockout of FOXO1 increased the expression of genes associated with fatty acid synthesis but decreased the expression of carnitine palmitoyltransferase1a (CPT1α) and adipose triglyceride lipase (ATGL), which contribute to fatty acid oxidation. Mechanistically, FOXO1 was identified as a transcription factor of ALDH1L2. Knockout of FOXO1 significantly decreased the protein expression of ALDH1L2 and CPT1α in vitro and in vivo. Furthermore, overexpression of ALDH1L2 restored fatty acid oxidation in FOXO1-knockout cells. The findings of this study indicate that FOXO1 modulates fatty acid oxidation by targeting ALDH1L2.

Hepatocyte FoxO1 Deficiency Protects From Liver Fibrosis via Reducing Inflammation and TGF-β1-mediated HSC Activation

The O-class of the forkhead transcription factor FoxO1 is a crucial factor mediating insulin→PI3K→Akt signaling and governs diverse cellular processes. However, the role of hepatocyte FoxO1 in liver fibrosis has not been well-established. In his study, we investigated the role of hepatocyte FoxO1 in liver fibrosis and uncovered the underlying mechanisms. Liver fibrosis was established by carbon tetrachloride (CCL4) administration and compared between liver-specific deletion of FoxO1 deletion (F1KO) and control (CNTR) mice. Using genetic and bioinformatic strategies invitro and invivo, the role of hepatic FoxO1 in liver fibrosis and associated mechanisms was established. Increased FoxO1 expression and FoxO1 signalingactivation were observed in CCL4-induced fibrosis. Hepatic FoxO1 deletion largely attenuated CCL4-induced liver injury and fibrosis compared with CNTR mice. F1KOmice showed ameliorated CCL4-induced hepatic inflammation and decreased TGF-β1 mRNA and protein levels compared with those of CNTR mice. In primary hepatocytes, FoxO1 deficiency reduced TGF-β1 expression andsecretion. Conditioned medium (CM) collected from wild-type hepatocytes treated with CCL4 activated humanHSC cell line (LX-2); such effect was attenuated byFoxO1 deletion in primary hepatocytes or neutralization of TGF-β1 in the CM using TGF-β1 antibody. Hepatic FoxO1overexpression in CNTR mice promoted CCL4-induced HSC activation; such effect was blocked in L-TGF-β1KO mice. Hepatic FoxO1 mediates CCL4-inducled liver fibrosis via upregulating hepatocyte TGF-β1 expression, stimulating hepatic inflammation and TGF-β1-mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.

The APC/C E3 ligase subunit ANAPC11 mediates FOXO3 protein degradation to promote cell proliferation and lymph node metastasis in urothelial bladder cancer

Urothelial bladder cancer (UBC) is one of the most prevalent malignancies worldwide, with striking tumor heterogeneity. Elucidating the molecular mechanisms that can be exploited for the treatment of aggressive UBC is a particularly relevant goal. Protein ubiquitination is a critical post-translational modification (PTM) that mediates the degradation of target protein via the proteasome. However, the roles of aberrant protein ubiquitination in UBC development and the underlying mechanisms by which it drives tumor progression remain unclear. In this study, taking advantage of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 technology, we identified the ubiquitin E3 ligase ANAPC11, a critical subunit of the anaphase-promoting complex/cyclosome (APC/C), as a potential oncogenic molecule in UBC cells. Our clinical analysis showed that elevated expression of ANAPC11 was significantly correlated with high T stage, positive lymph node (LN) metastasis, and poor outcomes in UBC patients. By employing a series of in vitro experiments, we demonstrated that ANAPC11 enhanced the proliferation and invasiveness of UBC cells, while knockout of ANAPC11 inhibited the growth and LN metastasis of UBC cells in vivo. By conducting immunoprecipitation coupled with mass spectrometry, we confirmed that ANAPC11 increased the ubiquitination level of the Forkhead transcription factor FOXO3. The resulting decrease in FOXO3 protein stability led to the downregulation of the cell cycle regulator p21 and decreased expression of GULP1, a downstream effector of androgen receptor signaling. Taken together, these findings indicated that ANAPC11 plays an oncogenic role in UBC by modulating FOXO3 protein degradation. The ANAPC11–FOXO3 regulatory axis might serve as a novel therapeutic target for UBC.

O-246 The molecular mechanism of miR-96-5p in the pathogenesis and treatment of polycystic ovary syndrome

Abstract Study question Polycystic ovary syndrome, characterized by the androgen excess and arrest of antral follicles, is lacking novel specific diagnostic biomarkers and therapeutic targets. Summary answer Study of the molecular mechanisms of microRNAs in the etiology and its potential applications in PCOS provides the insight. What is known already Some of molecular mechanisms of microRNAs have been demonstrated involved in the etiology of PCOS Study design, size, duration Clinically, we collected the samples of serum, follicular fluid (FF) and primary human granulosa cells (hGCs) of PCOS patients (n = 70) vs. non-PCOS women (n = 60). Experimentally, we carried out studies with 3-types of induced PCOS-like mice. Participants/materials, setting, methods By analysing the expression levels of miR-96-5p in serum, follicular fluid (FF) and primary human granulosa cells (hGCs) of PCOS patients (n = 70) vs. non-PCOS women (n = 60). as well as in ovaries from 3-types of induced PCOS-like mice. Main results and the role of chance Clinically, we demonstrated that the elevated circulating miR-96-5p levels were significantly correlated with the PCOS disordered endocrine clinical features, and the area under the curve of receiver operating characteristic was 0.8344, with 75.71% specificity and 80% sensitivity. Mechanically, we identified miR-96-5p as an androgen-regulated miRNA that directly targets the forkhead transcription factor FOXO1. Inhibition of miR-96-5p decreased estrogen synthesis, while decreasing the cell proliferation index of KGN via regulating the expression of FOXO1 and its downstream genes. Inversely, inhibition of FOXO1 abrogated the effect of miR-96-5p on estrogen synthesis and proliferation index. Of note, ovarian intra-bursal injection of miR-96-5p agomir rescued the phenotypes of dehydroepiandrosterone-induced PCOS like mice. Limitations, reasons for caution This study needs a large-scale clinical investigation to confirm the specificity and sensitivity of miR-96-5p as a clinical biomaker. Wider implications of the findings In conclusion, our results clarified a vital role of miR-96-5p in the pathogenesis of PCOS and might serve as a novel diagnostic biomarker and therapeutic target for PCOS. Trial registration number no

Resveratrol Improves the Progression of Osteoarthritis by Regulating the SIRT1-FoxO1 Pathway-Mediated Cholesterol Metabolism.

Osteoarthritis (OA) is considered a metabolic disorder. This study investigated the effect of resveratrol (RES) on cholesterol accumulation in osteoarthritic articular cartilage via the silent information regulator 1 (SIRT1)/forkhead transcription factor (FoxO1) pathway. Interleukin (IL)-1β-treated chondrocytes that mimic OA chondrocytes were used in in vitro experiments. The optimal RES concentration was selected based on the results of chondrocyte proliferation in the Cell Counting Kit-8 assay. Western blotting, immunofluorescence, and reverse transcription-quantitative polymerase chain reaction were performed. For the animal experiments, mice were randomly divided into the RES group (n = 15), medial meniscus destabilization group (n = 15), and sham group (n = 15), and each group received the same dose of RES or saline. Articular cartilage tissue was obtained eight weeks after surgery for relevant histological analysis. Clinical tissue test results suggest that downregulation of the SIRT1/FoxO1 pathway is associated with cholesterol buildup in OA chondrocytes. For the in vitro studies, RES increased the expression of SIRT1 and phosphorylation of FoxO1 in IL-1β-treated chondrocytes, promoted the expression of cholesterol efflux factor liver X receptor alpha (LXRα), and inhibited the expression of cholesterol synthesis-associated factor sterol-regulatory element binding proteins 2 (SREBP2). This reduced IL-1β-induced chondrocytes cholesterol accumulation. SIRT1 inhibition prevented the RES-mediated reduction in cholesterol buildup. Inhibiting FoxO1 but not SIRT1 reduced FoxO1 phosphorylation and increased cholesterol buildup in cultured chondrocytes. Additionally, in vivo experiments have shown that RES can alleviate cholesterol buildup and pathological changes in OA cartilage. Our findings suggest that RES regulates cholesterol buildup in osteoarthritic articular cartilage via the SIRT1/FoxO1 pathway, thereby improving the progression of OA.

The molecular mechanism of miR-96-5p in the pathogenesis and treatment of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS), characterized by the androgen excess and arrest of antral follicles, is a common endocrine disorder among women lacking specific diagnostic biomarkers and therapeutic targets. Herein, we studied the molecular mechanism of miR-96-5p in the process of PCOS and its potential applications in PCOS. Clinically, we found that miR-96-5p significantly decreased in serum, follicular fluid and primary human granulosa cells (hGCs) of PCOS patients (n = 70) vs non-PCOS women (n = 60), as well as in the ovaries of 3-types of induced PCOS-like mice. Furthermore, we demonstrated that the elevated circulating miR-96-5p levels were significantly correlated with the PCOS disordered endocrine clinical features, and the area under the curve of receiver operating characteristic was 0.8344, with 75.71% specificity and 80% sensitivity. Mechanically, we identified miR-96-5p as an androgen-regulated miRNA that directly targets the forkhead transcription factor FOXO1. Inhibition of miR-96-5p decreased estrogen synthesis, while decreasing the cell proliferation index of KGN via regulating the expression of FOXO1 and its downstream genes. Inversely, inhibition of FOXO1 abrogated the effect of miR-96-5p on estrogen synthesis and proliferation index. Of note, ovarian intra-bursal injection of miR-96-5p agomir rescued the phenotypes of dehydroepiandrosterone-induced PCOS like mice. In conclusion, our results clarified a vital role of miR-96-5p in the pathogenesis of PCOS and might serve as a novel diagnostic biomarker and therapeutic target for PCOS.

Exercise postconditioning reduces ischemic injury via suppression of cerebral gluconeogenesis in rats.

Pre-stroke exercise conditioning reduces neurovascular injury and improves functional outcomes after stroke. The goal of this study was to explore if post-stroke exercise conditioning (PostE) reduced brain injury and whether it was associated with the regulation of gluconeogenesis. Adult rats received 2h of middle cerebral artery (MCA) occlusion, followed by 24h of reperfusion. Treadmill activity was then initiated 24h after reperfusion for PostE. The severity of the brain damage was determined by infarct volume, apoptotic cell death, and neurological deficit at one and three days after reperfusion. We measured gluconeogenesis including oxaloacetate (OAA), phosphoenolpyruvate (PEP), pyruvic acid, lactate, ROS, and glucose via ELISA, as well as the location and expression of the key enzyme phosphoenolpyruvate carboxykinase (PCK)-1/2 via immunofluorescence. We also determined upstream pathways including forkhead transcription factor (FoxO1), p-FoxO1, 3-kinase (PI3K)/Akt, and p-PI3K/Akt via Western blot. Additionally, the cytoplasmic expression of p-FoxO1 was detected by immunofluorescence. Compared to non-exercise control, PostE (*p<.05) decreased brain infarct volumes, neurological deficits, and cell death at one and three days. PostE groups (*p<.05) saw increases in OAA and decreases in PEP, pyruvic acid, lactate, ROS, glucose levels, and tissue PCKs expression on both days. PCK-1/2 expressions were also significantly (*p<.05) suppressed by the exercise setting. Additionally, phosphorylated PI3K, AKT, and FoxO1 protein expression were significantly induced by PostE at one and three days (*p<.05). In this study, PostE reduced brain injury after stroke, in association with activated PI3K/AKT/FoxO1 signaling, and inhibited gluconeogenesis. These results suggest the involvement of FoxO1 regulation of gluconeogenesis underlying post-stroke neuroprotection.

FoxO1 Promotes Liver Fibrosis through TGF‐β1 mediated Hepatic Stellate Cell Activation

The O‐class of the forkhead transcription factor FoxO1 is a crucial factor mediating the action of insulin→PI3K→Akt and governs diverse cellular processes. To investigate the role of FoxO1 in control of liver fibrosis, we generated liver‐specific deletion of FoxO1 gene in mice (L‐F1KO). Using the mouse model, we injected intraperitoneally (i.p.) corn oil or 20% solution of carbon tetrachloride (CCL4) twice per week for 4 weeks (with 2.5 ul/g body weight) in WT and L‐F1KO mice. H&amp;E staining and Sirius Red staining revealed that CCl4‐induced liver injury and fibrosis were significantly appeared, but the effects were largely attenuated in L‐F1KO mice. Moreover, we found that both mRNA and protein levels of TGF‐β1, a cytokine that activates hepatic stellate cell (HSC) for fibrogenesis, significantly decreased in the liver L‐F1KO mice compared to WT mice in response to CCL4 treatment. In primary hepatocytes, with FoxO1‐loss of function and gain‐of function, we confirmed the regulation of TGF‐β1 expression by FoxO1. Moreover, conditional medium (CM) collected from WT hepatocytes treated with CCL4 activated human HSC cell line (LX‐2), such effect was attenuated by FoxO1 deletion in primary hepatocytes or neutralization of TGF‐β1 in the CM using TGF‐β1 antibody. These results indicate a crosstalk between hepatocytes and HSC via FoxO1‐regualted TGF‐β1. To further confirm this concept in vivo, we overexpressed FoxO1 in the liver of WT and liver‐specific TGF‐β1 deficient (L‐TGF‐β1KO) mice with adenovirus expressing FoxO1 (adFoxO1), and injected (i.p.) these mice with CCL4. We found that FoxO1 overexpression in WT mice promoted CCL4‐induced liver fibrosis and HSC activation, and such effect was blocked in L‐TGF‐β1KO mice. Taken together, our data indicate that hepatic FoxO1 promotes CCL4‐inducled liver fibrosis via upregulating TGF‐β1 expression in hepatocytes and stimulating TGF‐β1‐mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.

FOXO1 represses lymphatic valve formation and maintenance via PRDM1.

Intraluminal lymphatic valves (LVs) contribute to the prevention of lymph backflow and maintain circulatory homeostasis. Several reports have investigated the molecular mechanisms which promote LV formation; however, the way in which they are suppressed is not completely clear. We show that the forkhead transcription factor FOXO1 is a suppressor of LV formation and maintenance in lymphatic endothelial cells. Oscillatory shear stress by bidirectional flow inactivates FOXO1 via Akt phosphorylation, resulting in the upregulation of a subset of LV-specific genes mediated by downregulation of a transcriptional repressor, PRDM1. Mice with an endothelial-specific Foxo1 deletion have an increase in LVs, and overexpression of Foxo1 in mice produces a decrease in LVs. Genetic reduction of PRDM1 rescues the decrease in LV by Foxo1 overexpression. In conclusion, FOXO1 plays a critical role in lymph flow homeostasis by preventing excess LV formation. This gene might be a therapeutic target for lymphatic circulatory abnormalities.

Effect of water caltrop (Trapa bispinosaRoxb.) pericarp extract on improving insulin resistance in STZ-induced diabetic mice

BackgroundThe treatment of diabetes with plant ingredients such as in Traditional Chinese Medicine (TCM) is an alternative to classical chemotherapy. PurposeTo explore the ingredients of total dried water caltrop (Trapa bispinosaRoxb.) pericarp powder and a dried ethanol extract for their capacity to reduce insulin resistance. MethodsA treatment group received water caltrop (Trapa bispinosaRoxb.) pericarp powder (at one dose) or extract (at three doses) for four weeks with inclusion of proper control groups. The different indexes were measured four weeks after administration. ResultsTreatment could significantly improve the health index of type 2 diabetic mice. The mRNA and protein expression of SIRT1/PGC1α and PI3K/Akt signaling pathways were significantly increased, while the mRNA expression of forkhead transcription factor (FoxO1) was decreased in the treatment group. Conclusionsoral administration of water caltrop (Trapa bispinosaRoxb.) pericarp powder in the treatment of diabetes may be achieved by restoring pancreatic function, improving oxidative stress and liver gluconeogenesis.

Advanced Glycation End-Products and Hyperglycemia Increase Angiopoietin-2 Production by Impairing Angiopoietin-1-Tie-2 System.

The angiopoietin-Tie-2 system plays a crucial role in the maintenance of endothelial integrity. Hyperglycemia and advanced glycation end-products (AGEs) are involved in endothelial cell dysfunction responsible of the pathogenesis of microvascular complications of diabetes. Here, we investigated whether glycated serum (GS) or hyperglycemia (HG) affect the angiopoietin-Tie-2 system in the microvascular endothelial cells HMEC-1. We found that culture for 5 days in the presence of AGEs and HG (alone or in combination) decreased cell proliferation, increased reactive oxygen species (ROS) production, and reduced ratio between the oxidized and the reduced form of glutathione. Since angiopoietin-1 (Ang-1) signaling regulates angiopoietin-2 (Ang-2) expression through inactivation of the forkhead transcription factor FoxO1, we investigated intracellular signaling of Ang-1 and expression of Ang-2. HG and AGEs reduced phosphorylation of Akt and abrogated phosphorylation of FoxO1 induced by Ang-1 without affecting neither Tie-2 expression nor its activation. Furthermore, AGEs and/or HG induced nuclear translocation of FoxO1 and increased Ang-2 production. In conclusion, we demonstrated that both hyperglycemia and AGEs affect the angiopoietin-Tie-2 system by impairing Ang-1/Tie-2 signaling and by increasing Ang-2 expression. These results suggest that therapeutic strategies useful in preventing or delaying the onset of diabetic vascular complications should be aimed to preserve Ang-1 signaling.

Endothelial specific deletion of FOXO1 alters pericyte coverage in the developing retina

Pericytes are mural cells that cover small blood vessels. While defects in pericyte coverage are known to be involved in various vessel related pathologies, including diabetic retinopathy, the molecular mechanisms underlying pericyte coverage are not fully understood. In this study, we investigated the contribution of the forkhead transcription factor FOXO1 in endothelial cells to pericyte coverage in the developing retina. We observed retinal pericytes in tamoxifen-inducible endothelium-specific Foxo1 deletion mice. Tamoxifen was injected at postnatal day 1–3 and the retinas were harvested at P21. Our results demonstrated that Foxo1 deletion in the endothelium affected arteriole pericyte morphology without altering pericyte number, proliferation, and apoptosis. We hypothesized that abnormal pericyte morphogenesis in the knockout retina was caused by impaired pericyte differentiation. FOXO1 silencing by siRNA in the primary artery endothelium further revealed that THBS1 (thrombospondin 1), which promotes pericyte differentiation via TGFβ activation, was reduced in the FOXO1-deficient endothelium. Immunohistochemistry of FOXO1 knockout mice showed reduced numbers of phospho-Smad3+ arteriole pericytes compared with wild-type mice. In addition, endothelium-pericyte co-culture analysis revealed that pericytes cultured with FOXO1-deficient endothelial cells failed to differentiate sufficiently; this failure was partially rescued by the addition of recombinant THBS1 to the supernatant.The findings suggest that endothelial FOXO1 contributes to pericyte differentiation via regulation of THBS1 expression. This study provides new insights into the molecular mechanism of pericyte coverage in the context of endothelium-derived regulation and highlights a new therapeutic target for pericyte-related pathology.

Protective effect of silencing Stat1 on high glucose-induced podocytes injury via Forkhead transcription factor O1-regulated the oxidative stress response

BackgroundPodocyte plays an important role in maintaining the integrity and function of the glomerular filtration barrier. Various studies reported that forkhead transcription factor (Fox) O1 played a key role in anti-oxidative signaling. This study aimed to investigate the role of Stat1 in high glucose (HG) -induced podocyte injury.MethodsUnder normal glucose, hypertonic and HG stimulated podocyte conditions, cell counting kit-8 (CCK-8) assay, flow cytometry and western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were respectively carried out to determine cell viability, apoptosis, reactive oxygen species (ROS) production and related genes expressions. We then respectively used silent Stat1, simultaneous silencing Stat1 and FoxO1 and over-expression of FoxO1, to observe whether they/it could reverse the damage of podocytes induced by HG.ResultsHigh glucose attenuated cell survival and promoted cell apoptosis in MPC-5 cells at the same time, and it was also observed to promote the protein expression of Stat1 and the FoxO1 expression inhibition. Silencing Stat1 could reverse HG-induced podocytes injury. Specifically, siStat1 increased cell viability, inhibited cell apoptosis and attenuated ROS level in a high-glucose environment. Cleaved caspase-3 and pro-apoptosis protein Bax was significantly down-regulated, and anti-apoptosis protein Bcl-2 was up-regulated by siStat1. The antioxidant genes Catalase, MnSOD, NQO1 and HO1 were up-regulated by siStat1. We found that silencing FoxO1 reversed the protective effect of siStat1 on the HG-induced podocytes injury.ConclusionsSilencing Stat1 could reverse the effects of high glucose-triggered low cell viability, cell apoptosis and ROS release and the functions of Stat1 might be involved in FoxO1 mediated-oxidative stress in nucleus.

Heparin prevents oxidative stress-induced apoptosis in human decidualized endometrial stromal cells.

Clinical trials have shown that administering heparin during the luteal phase has beneficial effects on implantation and live birth rates. Heparin exerts direct effects on decidual human endometrial stromal cells (HESCs), which are independent of its anticoagulant effect. However, the accurate effects of heparin on the decidualization process remain unidentified. Here, we demonstrate that HESCs become dramatically resistant to oxidative stress upon decidualization, and we hypothesize a possible direct action of heparin on the decidualization of HESCs, which would lead to improved implantation. To test this hypothesis, we established primary HESC cultures and propagated them, and then we decidualized confluent cultures with 8-bromo-cAMP, with medroxyprogesterone acetate, and with or without heparin. We treated the cells with hydrogen peroxide (H2O2) as a source of reactive oxygen species (ROS). Adding heparin to decidualized HESCs induced prolactin secretion. Decidualized HESCs treated with heparin were prevented from undergoing apoptosis induced by oxidative stress. Heparin induced nuclear accumulation of the forkhead transcription factor FOXO1 and expression of its downstream target, the ROS scavenger superoxide dismutase 2. These results demonstrate that heparin-treated decidualized HESCs acquired further resistance to oxidative stress, suggesting that heparin may improve the implantation environment.

Role of PTP/PTK trans activated insulin-like signalling pathway in regulation of grasshopper (Oedaleus asiaticus) development.

Protein tyrosine phosphatase (PTPs) and protein tyrosine kinase (PTKs) genes are responsible for the regulation of insect insulin-like pathway (ILP), cells growth, metabolism initiation, gene transcription and observing immune response. Signal transduction in insect cell is also associated with PTPs and PTKs. The grasshopper (Oedaleus asiaticus) 'Bey-Bienko' were treated withdsRNA of protein tyrosine non-receptor type 4 (PTPN4) and protein tyrosine kinase 5 (PTK5) along with control (water). Applying dsPTK5 treatments in 5th instar of Oedaleus asiaticus, significant reduction was recorded in body dry mass, growth rate and overall performance except survival rate. Whereas with PTPN4, no such significant impact on all of these growth parameters was recorded. Expression of genes in ILP 5th instar of Oedaleus asiaticus by the application of dsPTPN4 and dsPTK5 revealed that PTK, INSR (insulin receptor), IRS (insulin receptor substrate), PI3K (phosphoinositide 3-kinase), PDK (3-phosphoinositide-dependent protein kinase), Akt (protein kinase B) and FOXO (forkhead transcription factor) significantly expressed with downregulation except PTPN4, which remained non-significant. On the other hand, the phosphorylation level of ILP four proteins in O. asiaticus with the treatment of dsPTPN4 and dsPTK5 significantly affected P-IRS and P-FOXO, while P-INSR and P-AKT remained stable at the probability level of 5%. This indicated that the stress response in the O. asiaticus insulin-like signalling pathway (ILP) reduced. Regarding association of protective enzymatic activities, ROS (relative oxygen species), CAT (catalase) and PO (phenol oxidase) increased significantly with exposure to dsPTK5 as compared to dsPTPN4 and control, while exposure of 5th instar of O. asiaticus to dsPTPN4 treatment slightly raised CAT and PO activities with but significant contribution. No such significant effect on MFO and POD was seen using dsPTPN4 and dsPTK5. This showed that in the ILP of O. asiaticus, PTK5 was detrimental to growth, body mass and overall performance, which ultimately benefited insect detoxification with high-energy cost.

Novel Mechanism of Foxo1 Phosphorylation in Glucagon Signaling in Control of Glucose Homeostasis

Dysregulation of hepatic glucose production (HGP) serves as a major underlying mechanism for the pathogenesis of type 2 diabetes. The pancreatic hormone glucagon increases and insulin suppresses HGP, controlling blood glucose homeostasis. The forkhead transcription factor Foxo1 promotes HGP through increasing expression of genes encoding the rate-limiting enzymes responsible for gluconeogenesis. We previously established that insulin suppresses Foxo1 by Akt-mediated phosphorylation of Foxo1 at Ser256 in human hepatocytes. In this study, we found a novel Foxo1 regulatory mechanism by glucagon, which promotes Foxo1 nuclear translocation and stability via cAMP- and protein kinase A-dependent phosphorylation of Foxo1 at Ser276 Replacing Foxo1-S276 with alanine (A) or aspartate (D) to block or mimic phosphorylation, respectively, markedly regulates Foxo1 stability and nuclear localization in human hepatocytes. To establish in vivo function of Foxo1-Ser276 phosphorylation in glucose metabolism, we generated Foxo1-S273A and Foxo1-S273D knock-in (KI) mice. The KI mice displayed impaired blood glucose homeostasis, as well as the basal and glucagon-mediated HGP in hepatocytes. Thus, Foxo1-Ser276 is a new target site identified in the control of Foxo1 bioactivity and associated metabolic diseases.

Foxo1-Ser253 Phosphorylation Regulates Glucose Homeostasis in Mice

The forkhead transcription factor Foxo1 is a key mediator in insulin signaling pathway that controls hepatic glucose production (HGP) and pancreatic beta-cell function. Upon activation of the protein kinase Akt, insulin promotes glycogen synthesis and inhibits gluconeogenesis in the liver, reducing blood glucose. We previously demonstrated human Foxo1-Ser256, an equivalent to mouse Foxo1-Ser253, is a key phosphorylation site for insulin and Akt suppression, promoting Foxo1 nuclear export and suppressing the expression of the target gene for liver gluconeogenesis. Here we investigated the role of Foxo1-Ser253 phosphorylation in control of glucose hemostasis in vivo, by generating Foxo1-S253A/A knock-in (KI) mice, in which Foxo1-Ser253 replaced by alanine (A mutation) that blocks phosphorylation. Foxo1-S253A/A mice displayed mild increases in feeding blood glucose and insulin level, but reductions in fasting blood glucose and glucagon concentration, as well as a decrease in the ratio of the number of pancreatic α-cells/β-cells per islet. Foxo1-S253A/A mice exhibited slight increases in insulin sensitivity but barely changed glucose uptake among tissues, and an enhanced energy expenditure. Further analyses indicate that Foxo1-S253A/A enhanced Foxo1 ability and promoted the effect of glucagon on HGP. This is the first report demonstrating that Foxo1-Ser253 phosphorylation status itself is sufficient to affect HGP and glucose homeostasis, as well as regulate the synthesis of insulin and glucagon in in vivo. Disclosure H. Yan: None. Y. Wu: None. Q. Pan: None. Z. Shen: None. H. Zheng: None. M.F. White: Advisory Panel; Self; Housey Pharmaceutical Research Laboratories. Y. Sun: None. S. Guo: None.

A Novel Mechanism by Foxo1 Phosphorylation Mediates Glucagon Signaling in Control of Glucose Homeostasis

Glucagon increases hepatic glucose production (HGP) to maintain blood glucose homeostasis. The forkhead transcription factor Foxo1 promotes HGP through increasing expression of genes encoding the rate-limiting metabolic enzymes for gluconeogenesis. We previously established that insulin suppresses Foxo1 by Akt-mediated phosphorylation at Ser256 in human hepatocytes. Here we demonstrated glucagon promotes Foxo1 nuclear translocation and stability via protein kinase A (PKA)-dependent phosphorylation of Foxo1 at Ser276, serving as a novel mechanism of glucagon action in control of glucose homeostasis. In vitro protein kinase assay showed a direct phosphorylation of Foxo1 at Ser276 by PKA. Replacing Foxo1-Ser276 with alanine (S276A) or aspartate (S276D) reduced or increased Foxo1 stability in human hepatocytes, respectively. To establish the in vivo function of Foxo1-Ser276 phosphorylation in glucose metabolism, we generated Foxo1-S273A and Foxo1-S273D knock-in mice, revealing the novel mechanism by which glucagon, via PKA-dependent phosphorylation of Foxo1 at Ser276, promotes Foxo1 nuclear localization, stability, and HGP. Thus, Foxo1-Ser276 is a potential target site for the control of Foxo1 bioactivity and associated metabolic diseases. Disclosure Y. Wu: None. H. Yan: None. Q. Pan: None. H. Zheng: None. S.A. Dahanayaka: None. Y. Sun: None. A. Zhou: None. L. Zhang: None. S. Guo: None.

Drosophila Kruppel homolog 1 represses lipolysis through interaction with dFOXO

Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. We found that Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.

HMGA1 is a novel transcriptional regulator of the FoxO1 gene

PurposeThe forkhead transcription factor (FoxO1) is a master transcriptional regulator of fundamental cellular processes ranging from cell proliferation and differentiation to inflammation and metabolism. However, despite its relevance, the mechanism(s) underlying FoxO1 gene regulation are largely unknown. We have previously shown that the chromatin factor high-mobility group A1 (HMGA1) plays a key role in the transcriptional regulation of glucose-responsive genes, including some that are involved in FoxO1-mediated glucose metabolism. Here we investigated the impact of HMGA1 on FoxO1 gene expression.MethodsFoxO1 protein and gene expression studies were performed by Western blot analysis combined with qRT-PCR of material from human cultured cells and EBV-transformed lymphoblasts, and from primary cultured hepatocytes from wild-type and Hmga1–/– mice. Reporter gene assays and chromatin immunoprecipitation for binding of HMGA1 to the endogenous FoxoO1 locus were performed in cells overexpressing HMGA1 and in cells pretreated with siRNA targeting HMGA1.ResultsHMGA1 increased FoxO1 mRNA and protein expression in vitro, in cultured HepG2 and HEK-293 cells by binding FoxO1 gene promoter, thereby activating FoxO1 gene transcription. Forced expression of HMGA1 in primary cultured hepatocytes from Hmga1–/– mice and in EBV-transformed lymphoblasts from subjects with reduced expression of endogenous HMGA1 increased FoxO1 mRNA and protein levels.ConclusionThese findings may contribute to the understanding of FoxO1 gene regulation and its role in metabolism.