Phosphorylation Of Forkhead Box O1 Research Articles

Impact of Prospero Homeobox-1 (PROX-1) οn the Oncogenic Phenotypes of Hepatocellular Carcinoma Cells.

Transcriptional factor prospero homeobox-1 (PROX-1) is crucial for the embryonic development of various organs and cell fate specification. It exhibits either an oncogenic or tumor suppressive activity depending on cancer types. However, the relationship between PROX-1 and hepatocellular carcinoma (HCC) remains obscure. This study was conducted to investigate the effect of PROX-1 on the invasive and oncogenic phenotypes of human HCC cells. The effect of PROX-1 on tumor cell behavior was investigated by using a pcDNA-myc vector and a small interfering RNA in HepG2 and Huh7 human HCC cell lines. Flow cytometry, migration, invasion, proliferation, and tube formation assays were performed. PROX-1 expression in human HCC cells was explored by western blotting. PROX-1 overexpression enhanced tumor cell proliferation and inhibited apoptosis and cell cycle arrest by modulating the activities of caspase-3, PARP, and cyclin-dependent kinase inhibitors, including p21, p27, and p57 in HCC cells. After PROX-1 overexpression, the number of migrating and invading HCC cells significantly increased, and the expression levels of N-cadherin and Snail increased in HCC cells. PROX-1 overexpression enhanced angiogenesis through increased VEGF-A and VEGF-C expression and decreased angiostatin expression. PROX-1 overexpression also increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and forkhead box O1 (FOXO1) in HCC cells. After PROX-1 knockdown, their phosphorylation was reversed. PROX-1 overexpression is associated with the invasive and oncogenic phenotypes of human HCC cells via GSK-3β and FOXO1 phosphorylation.

The regulation of AMPK pathway in liver abnormal lipid deposition caused by high carbohydrate diet in rice field eel

AMP-activated protein kinase-alpha 1 (AMPKα1) plays a vital role in glucose and lipid metabolism. Five isonitrogenous and isolipid diets with starch levels (15%, 20%, 25%, 30% and 35%) were formulated to study the modulation of AMPKα1 in hepatic lipid deposition induced by high gelatinized starch diet in rice field eel (Monopterus albus) (initial body weight, 30.00 ± 0.10 g) and the growth trial lasted for 8 weeks. The results showed that dietary gelatinized starch did not affect the growth performance of rice filed eel compared with S15 group. High gelatinized starch could induce AMPKα1 expression and glucose catabolism, and inhibit hepatic glycogen synthesis without effecting glucose content. Dietary ≥ 30% gelatinized starch began to dramatically suppress gluconeogenesis and promote the lipid synthesis, followed by a higher triglyceride (TG) and hepatic crude lipid. Dietary 20–30% gelatinized starch induced phosphorylation of AMPKα compared to 15% gelatinized starch diet, whereas 35% starch dramatically suppressed AMPKα phosphorylation together with unnormal increase in phosphorylation of forkhead box O1 A (FOXO1A) and acetyl-CoA carboxylase (ACC), TG content compared to 30% starch. In short, high gelatinized starch could induce AMPKα1 expression and promoted systemic metabolism, whereas dietary ≥ 25% gelatinized starch initially suppressed AMPKα1 activation and caused abnormal lipid deposition in liver of rice field eel.

PA YEAST SC-1, Polyamine-Rich Saccharomyces cerevisiae, Induces Muscle Hypertrophy in C2C12 Myotubes.

Maintenance of appropriate muscle mass is necessary for good quality of life as skeletal muscles play critical roles in locomotion, metabolic homeostasis, and thermogenesis. Polyamines are essential metabolites that regulate several important cellular functions. In C57BL6 mice who underwent sciatic nerve transection of the hind limb, compensatory muscle hypertrophy is enhanced by the administration of polyamines. However, the action mechanisms of polyamines in muscle hypertrophy remain unclear. Here, we isolated PA YEAST SC-1, a polyamine-rich Saccharomyces cerevisiae, from Baker's yeast. We examined whether PA YEAST SC-1 induces muscle hypertrophy and elucidated the underlying action mechanisms of polyamines and the active ingredients in PA YEAST SC-1 using C2C12 myotubes. PA YEAST SC-1 at 1 mg/mL increased myosin heavy chain expression in C2C12 myotubes. Mechanistically, PA YEAST SC-1 induced the activation of Akt/mechanistic target of rapamycin kinase/p70S6K signaling. Furthermore, PA YEAST SC-1 decreased the expression levels of the ubiquitin ligases, atrogin-1 and muscle RING finger-1, via forkhead box O1 phosphorylation. These findings suggest PA YEAST SC-1 as an effective food ingredient for the treatment of muscle hypertrophy.

Calpain7 negatively regulates human endometrial stromal cell decidualization in EMs by promoting FoxO1 nuclear exclusion via hydrolyzing AKT1.

Endometrial receptivity damage caused by impaired decidualization may be one of the mechanisms of infertility in endometriosis (EMs). Our previous study demonstrated that Calpain-7 (CAPN7) is abnormally overexpressed in EMs. Whether CAPN7 affects the regulation of decidualization and by what mechanism CAPN7 regulates decidualization remains to be determined. In this study, we found CAPN7 expression decreased during human endometrial stromal cell (HESC) decidualization in vitro. CAPN7 negatively regulated decidualization in vitro and in vivo. We also identified one conserved potential PEST sequence in the AKT1 protein and found that CAPN7 was able to hydrolyse AKT1 and enhance AKT1's phosphorylation. Correspondingly, CAPN7 notably promoted the phosphorylation of Forkhead Box O1 (FoxO1), the downstream of AKT1 protein, at Ser319, leading to increased FoxO1 exclusion from nuclei and attenuated FoxO1 transcriptional activity in decidualized HESC. In addition, we detected endometrium CAPN7, p-AKT1, and p-FoxO1 expressions were increased in EMs. These data demonstrate that CAPN7 negatively regulates HESC decidualization in EMs probably by promoting FoxO1's phosphorylation and FoxO1 nuclear exclusion via hydrolyzing AKT1. The dysregulation of CAPN7 may be a novel cause of EMs.

Transcriptional regulation of LGALS9 by HAND2 and FOXO1 in human endometrial stromal cells in women with regular cycles.

Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was upregulated by heart and neural crest derivatives expressed 2 (HAND2) and downregulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on Day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after a 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.

HnRNP-F promotes the proliferation of bladder cancer cells mediated by PI3K/AKT/FOXO1.

Our previous study showed that heterogeneous nuclear ribonucleoprotein F (hnRNP-F) could induce epithelial-mesenchymal transition and metastasis in bladder cancer (BC), however, the role and mechanism of hnRNP-F in mediating the proliferative ability of BC cells remain unclear. HnRNP-F promoted the proliferation of BC cells by using BC cell lines and cell counting kit-8 (CCK8), colony formation and flow cytometry assays in vitro. Furthermore, the association of hnRNP-F with the phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT) signalling pathway was confirmed by western blotting after bioinformatic analysis. HnRNP-F expression was significantly decreased by treatment with the PI3K/AKT signalling pathway inhibitor LY294002, whereas hnRNP-F knockdown did not significantly affect PI3K or AKT expression, suggesting that hnRNP-F is likely a downstream target of the PI3K/AKT pathway. Forkhead box O1 (FOXO1) is a molecule downstream of PI3K/AKT and can be inhibited by phosphorylation. In addition, chromatin immunoprecipitation (ChIP) and luciferase reporter assays indicated that FOXO1 expression was negatively correlated with hnRNP-F expression as FOXO1 was found to bind to the promoter region of hnRNP-F mRNA and inhibit its transcription. To sum up, our findings suggest that hnRNP-F expression is regulated by the PI3K/AKT-mediated phosphorylation of FOXO1, with phosphorylation inhibiting FOXO1, which subsequently allows hnRNP-F to promote proliferation. This finding is a novel discovery in BC and could help reveal the mechanism of BC progression.

Abstract MP131: The CDK2-FOXO1 Axis Mediates Doxorubicin-induced Cardiomyocyte Apoptosis and Atrophy

Recent clinical investigations indicate that anthracycline-based chemotherapy induces early declines in heart mass in cancer patients. A decline in heart mass can be caused by a decrease in cardiac cell number due to exaggerated cell death, and/or a reduction in cell size via atrophy. Our previous study reveals that the anthracycline doxorubicin (DOX) induces apoptotic death of cardiomyocytes through activation of cyclin-dependent kinase 2 (CDK2). However, the signaling pathway downstream of CDK2 remains to be characterized. In addition, it is unclear whether the same pathway also mediates cardiac atrophy. Here we demonstrated that DOX exposure induced CDK2-dependent phosphorylation of forkhead box O1 (FOXO1) at S249, leading to transcription of its pro-apoptotic target gene Bim. In cultured cardiomyocytes, treatment with the small molecule FOXO1 inhibitor AS1842856, or transfection with FOXO1-specific siRNAs protected against DOX-induced apoptosis and mitochondrial damage. Oral administration of AS1842856 in mice hindered apoptosis and prevented DOX-induced decline in left ventricular systolic function. Intriguingly, pharmacologic inhibition of FOXO1 also attenuated DOX-induced cardiac atrophy, likely due to repression of MuRF1, a pro-atrophic FOXO1 target gene. In conclusion, DOX exposure induces CDK2-dependent FOXO1 activation, resulting in cardiomyocyte apoptosis and atrophy. Our results identify FOXO1 as a promising drug target for DOX-induced cardiotoxicity. FOXO1 inhibitors may represent a novel class of therapeutics for cardioprotection during chemotherapy.

Reduced methylation of PP2Ac promotes ethanol–induced lipid accumulation through FOXO1 phosphorylation in vitro and in vivo

Although disturbance of the methionine cycle and sequent decrease in hepatic methylation capacity are known to be important factors in the development of alcoholic liver injury, the underlying mechanisms are not fully understood. Here, we investigated the importance of the methylation of protein phosphatase 2A (PP2A) in alcoholic liver disease (ALD). We found that the severity of ethanol-induced liver injury and the extent of demethylation of PP2A catalytic C subunit (PP2Ac) were reduced after treatment with betaine, a methyl donor involved in the methionine-homocysteine cycle. These results suggest that PP2Ac methylation is decreased due to a broad decrease in hepatic methylation capacity after exposure to ethanol. Moreover, we found that the reduction in PP2Ac methylation led to increased degradation of the regulatory Bα subunit, thus promoting the phosphorylation and nuclear exclusion of Forkhead box O1 (FOXO1) and reducing FOXO1 transcriptional activity. Ultimately, the reduced activity of FOXO1 led to increased expression of TXNIP, which caused hepatic lipid accumulation. Our findings suggest that the reduction of PP2A methylation, a result of decrease hepatic methylation capacity, played an important role in ethanol–induced lipid accumulation via down-regulation of PP2A/Bα and FOXO1 phosphorylation.

Low shear stress induces endothelial cell apoptosis and monocyte adhesion by upregulating PECAM‑1 expression.

Low shear stress serves an important role in the initiation and progression of atherosclerotic lesions, with an impact on progression, but its detailed mechanisms are .not yet fully known. The present study aimed to investigate endothelial cell (EC) apoptosis, as well as monocyte adhesion induced by low shear stress and the potential underlying mechanisms. The expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) was demonstrated to be enhanced in human umbilical vascular ECs with a trend that was associated with time when stimulated by low shear stress compared with unstimulated cells. EC apoptosis was increased under low shear stress compared with unstimulated cells, and knockdown of PECAM-1 inhibited this process. Furthermore, downregulation of PECAM-1 reduced monocyte adhesion induced by low shear stress compared with that in the negative control cells. Mechanistically, PECAM-1 small interfering RNA transfection increased Akt and forkhead box O1 phosphorylation under low shear stress conditions compared with that in the negative control cells. Collectively, the findings of the present study revealed that low shear stress induced EC apoptosis and monocyte adhesion by upregulating PECAM-1 expression, which suggested that PECAM-1 may be a potential therapeutic target for atherosclerosis.

Overexpressed ITGA2 contributes to paclitaxel resistance by ovarian cancer cells through the activation of the AKT/FoxO1 pathway.

Ovarian cancer is one of the most malignant tumors of the female reproductive system, with high invasiveness. The disease is a severe threat to women's health. The ITGA2 gene, which codes for integrin subunit α2, is involved in the proliferation, invasion, and metastasis of cancer cells. Although previous studies have shown that ITGA2 increases in ovarian cancer, the specific molecular mechanism of how ITGA2 promotes ovarian cancer proliferation and metastasis is still unclear. In this study, we confirmed that ITGA2 was elevated in ovarian cancer, which led to poor prognosis and survival. Overexpressed ITGA2 promoted the proliferation of ovarian cancer cells. We also found that ITGA2 regulated the phosphorylation of forkhead box O1 (FoxO1) by mediating AKT phosphorylation, which provided a reasonable explanation for ITGA2’s role in ovarian cancer’s resistance to albumin paclitaxel. In summary, ITGA2 could be used as a new therapeutic target and prognostic indicator in ovarian cancer.

Effect of dapagliflozin, a sodium-glucose co-transporter-2 inhibitor, on gluconeogenesis in proximal renal tubules.

To investigate the effect of dapagliflozin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, on renal gluconeogenesis in vitro, ex vivo and in vivo. We treated HK-2 cells (human renal proximal tubule cells) and mouse primary renal proximal tubule cells with dapagliflozin, and evaluated the process of renal gluconeogenesis. We also examined the effect of dapagliflozin on renal gluconeogenesis in normoglycaemic and hyperglycaemic mice. Dapagliflozin enhanced renal gluconeogenesis in vitro, ex vivo and in vivo. It increased phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), peroxisome proliferative activated receptor-gamma co-activator 1α (PGC-1α) and phosphorylated cyclic-AMP response element binding protein (CREB) expression and decreased phosphorylated Forkhead Box O1 (FOXO1) expression in HK-2 cells, mouse primary renal proximal tubule cells, and the mouse renal cortex. Glutamine enhanced the gluconeogenic effect of dapagliflozin in HK-2 cells. Also, dapagliflozin increased 14 C-glutamine utilization in HK-2 cells. Glucagon did not affect dapagliflozin-induced enhancement in renal gluconeogenesis in HK-2 cells. SGLT2 gene knockdown with siRNA resulted in an increase of gluconeogenic gene expression and associated transcription factors in HK-2 cells. Dapagliflozin reduced fasting plasma glucose levels and improved oral glucose tolerance and insulin tolerance in high-fat diet-fed hyperglycaemic mice, although renal gluconeogenesis was enhanced. Dapagliflozin increased levels of gluconeogenic enzyme in the renal cortex and consequently increased renal gluconeogenesis, which is mediated by SGLT2 inhibition.

Central administration of insulin and refeeding lead to the phosphorylation of AKT, but not FOXO1, in the hypothalamus of broiler chicks

Several studies in rodents and layer chickens have demonstrated that insulin upregulates hypothalamic AKT-mediated signaling and expression of proopiomelanocortin (POMC, the precursor of alpha-melanocyte stimulating hormone, an anorexigenic peptide) and suppresses appetite in these animals. However, a previous study has also reported that insulin fails to suppress food intake in broiler chicks. In the present study, no significant differences were observed in hypothalamic AKT and forkhead box O1 (FOXO1) phosphorylation levels between broiler and layer chicks. The phosphorylation rate of AKT, but not that of FOXO1, increased in the hypothalami of broilers refed for 1 h after a 24-h fast, with a corresponding increase in plasma insulin concentration. Intracerebroventricular (ICV) administration of 50 pmol insulin, which could decrease food intake in broiler chicks, significantly increased the AKT phosphorylation rate, whereas no significant change was observed in FOXO1 phosphorylation or POMC expression after ICV insulin administration. These findings suggest that hypothalamic AKT responds to insulin in broiler chicks, but FOXO1-mediated regulation of POMC expression is not induced by insulin, which may be one of the causes of excessive food intake in broiler chickens.

The mTORC2-Akt1 Cascade Is Crucial for c-Myc to Promote Hepatocarcinogenesis in Mice and Humans.

Hepatocellular carcinoma (HCC) is a deadly form of liver cancer with limited treatment options. The c-Myc transcription factor is a pivotal player in hepatocarcinogenesis, but the mechanisms underlying c-Myc oncogenic activity in the liver remain poorly delineated. Mammalian target of rapamycin complex 2 (mTORC2) has been implicated in cancer by regulating multiple AGC kinases, especially AKT proteins. In the liver, AKT1 and AKT2 are widely expressed. While AKT2 is the major isoform downstream of activated phosphoinositide 3-kinase and loss of phosphatase and tensin homolog-induced HCC, the precise function of AKT1 in hepatocarcinogenesis is largely unknown. In the present study, we demonstrate that mTORC2 is activated in c-Myc-driven mouse HCC, leading to phosphorylation/activation of Akt1 but not Akt2. Ablation of Rictor inhibited c-Myc-induced HCC formation in vivo. Mechanistically, we discovered that loss of Akt1, but not Akt2, completely prevented c-Myc HCC formation in mice. Silencing of Rictor or Akt1 in c-Myc HCC cell lines inhibited phosphorylated forkhead box o1 expression and strongly suppressed cell growth in vitro. In human HCC samples, c-MYC activation is strongly correlated with phosphorylated AKT1 expression. Higher expression of RICTOR and AKT1, but not AKT2, is associated with poor survival of patients with HCC. In c-Myc mice, while rapamycin, an mTORC1 inhibitor, had limited efficacy at preventing c-Myc-driven HCC progression, the dual mTORC1 and mTORC2 inhibitor MLN0128 effectively promoted tumor regression by inducing apoptosis and necrosis. Conclusion: Our study indicates the functional contribution of mTORC2/Akt1 along c-Myc-induced hepatocarcinogenesis, with AKT1 and AKT2 having distinct roles in HCC development and progression; targeting both mTORC1 and mTORC2 may be required for effective treatment of human HCC displaying c-Myc amplification or overexpression.

Role of Insulin-like Growth Factor-1 in the Central Regulation of Feeding Behavior in Chicks.

Insulin-like growth factor-1 (IGF-1) is a key regulator of muscle development and metabolism in chickens. Recently, we have demonstrated that intracerebroventricular administration of IGF-1 significantly decreased food intake in broiler chicks. However, the molecular mechanisms underlying the IGF-1-induced anorexia and the anorexigenic effect of IGF-1 in different strains of commercial chicks have not been investigated. Neuropeptide Y (NPY, a hypothalamic orexigenic neuropeptide), agouti-related protein (AgRP, a hypothalamic orexigenic neuropeptide), and proopiomelanocortin (POMC, the precursor of hypothalamic anorexigenic neuropeptides) play important roles in the regulation of food intake in both mammals and chickens. Evidence shows that several cell signaling pathways in the hypothalamus are involved in regulating the feeding behavior of mammals. In the present study, we first investigated the effects of IGF-1 on the expression of appetite-regulating neuropeptides and phosphorylation of signaling molecules in the hypothalamus of broiler chicks. Intracerebroventricular administration of IGF-1 significantly increased the mRNA levels of POMC, whereas the mRNA levels of NPY and AgRP were not significantly altered. IGF-1 also significantly induced the phosphorylation of v-Akt murine thymoma viral oncogene homolog 1 (AKT) in the hypothalamus of chicks, but did not influence the phosphorylation of forkhead box O1, S6 protein, AMP-activated protein kinase, and extracellular signal-regulated kinase 1/2. We also compared the effect of IGF-1 on food intake in broiler chicks (a hyperphagic strain of chickens) and layer chicks. Results demonstrated that the threshold of IGF-1-induced anorexia in broiler chicks was higher than that in layer chicks. Our observations suggest that hypothalamic POMC and AKT may be involved in the IGF-1-induced anorexigenic pathway and that high threshold of IGF-1-induced anorexia in broiler chicks might be one of the causes of hyperphagia in broiler chicks. Overall, it appears that IGF-1 plays important roles in the central regulation of feeding behavior in chicks.

Role of the mTOR‑FOXO1 pathway in obesity‑associated renal tubulointerstitial inflammation.

Since obesity is largely responsible for the growing incidence of renal tubulointerstitial inflammation, exploration into the mechanisms of obesity‑associated tubulointerstitial inflammation is essential. Studies have demonstrated that mammalian target of rapamycin(mTOR) is a crucial molecule in the pathogenesis of renal inflammation, including regulating the expression of inflammatory factors. The purpose of the present study was to further elucidate the role of mTOR in obesity‑associated tubulointerstitial inflammation. In the clinical study, obese and healthy subjects were recruited for physical examination, as well as the collection of blood and urine samples. Further study was performed on a high fat diet(HFD)‑induced obese rat model and a cultured human renal tubular epithelial cell line(HK‑2). The clinical study demonstrated that the participants with obesity had increased serum lipids, creatinine(Cr), urinary albumin to creatinine ratio(UACR) and urinary neutrophil gelatinase‑associated lipocalin(u‑NGAL). Moreover, the level of urinary monocyte chemoattractant protein‑1(u‑MCP‑1) was increased in the participants with obesity, and it was positively correlated with free fatty acid(FFA), UACR and u‑NGAL. In the invivo study, the results indicated that the levels of serum lipids, Cr and blood urea nitrogen(BUN), as well as 24h urine protein and u‑NGAL, were significantly increased in the HFD‑fed obese rats. In addition, the infiltration of CD68+ cells into the renal interstitial area and the release of interleukin‑1β(IL‑1β) was observed in the kidneys of obese rats. Meanwhile, the supernatant from HK‑2 cells treated with palmitic acid stimulated THP‑1 monocyte migration. The upregulation of MCP‑1, phosphorylated forkhead boxO1 (p‑FOXO1), and phosphorylated mTOR (p‑mTOR) was observed invivo and invitro. However, inhibition of mTOR was able to alleviate the above effects. Overall, these results demonstrated that activated mTOR induced FOXO1 phosphorylation, which mediates renal MCP‑1 release, causes tubulointerstitial inflammation and ultimately leads to pathological renal changes and dysfunction. However, inhibition of mTOR may play a renoprotective role during the progression of obesity‑associated tubulointerstitial inflammation.

MiR‑338‑3p mediates gluconeogenesis via targeting of PP4R1 in hepatocytes.

Hyperglycaemia is a characteristic of type2 diabetes. In hepatocytes, impaired insulin sensitivity leads to increased gluconeogenesis and decreased glycogenesis. MicroRNA (miR)‑338‑3p is associated with tumour necrosis factor (TNF)‑α‑induced suppression of hepatic glycogenesis via regulation of protein phosphatase 4 regulatory subunit 1 (PP4R1). However, the effect of miR‑338‑3p on gluconeogenesis in hepatocytes remains unknown. In a previous study, it was demonstrated that miR‑338‑3p is downregulated in the livers of mice and in mouse HEPA1‑6 hepatocytes following treatment with TNF‑α. In the present study, the effect of miR‑338‑3p on TNF‑α‑induced gluconeogenesis in hepatocytes was investigated. The levels of phosphorylated‑FOXO1/FOXO1, phosphoenolpyruvate carboxykinase (PEPCK), peroxisome proliferator‑activated receptor γ coactivator (PGC‑1α) and glucose‑6‑phosphatase (G6Pase) were measured by western blotting. The mRNA levels of PEPCK, PGC‑1α and G6Pase were determined by quantitative polymerase chain reaction. Pyruvate tolerance testing was used to determine the gluconeogenesis of mouse livers. The results demonstrated that treatment with TNF‑α resulted in increased levels of gluconeogenesis in the livers of mice and decreased miR‑338‑3p expression levels in HEPA1‑6 cells. Overexpression of miR‑338‑3p reversed TNF‑α‑induced glucose production via enhancement of phosphorylated forkhead box O1 levels and downregulation of the expression levels of genes associated with gluconeogenesis, including peroxisome proliferator‑activated receptor γ coactivator‑1α, phosphoenolpyruvate carboxykinase and glucose‑6‑phosphatase. However, inhibition of miR‑338‑3p expression was revealed to enhance gluconeogenesis in the livers of mice and in HEPA1‑6 cells. Furthermore, downregulation of PP4R1 was revealed to attenuate the effect on glucose production following treatment with miR‑338‑3p inhibitors. In conclusion, the results of the present study revealed that miR‑338‑3p may be involved in TNF‑α‑mediated gluconeogenesis via targeting of PP4R1 in hepatocytes.

NaF Reduces KLK4 Gene Expression by Decreasing Foxo1 in LS8 Cells

Decreased expression and increased phosphorylation of Forkhead box o1 (Foxo1) in ameloblasts were observed both in vivo and in vitro when treated by fluoride. The present study aims to investigate the possible relationship between Foxo1 and enamel matrix proteinases, matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), in NaF-treated ameloblasts. Ameloblast-like cells (LS8 cells) were exposed to NaF at selected concentration (0/2mM) for 24h. Gene overexpression and silencing experiments were used to up- and down-regulate Foxo1 expression. The expression levels of Foxo1, MMP20, and KLK4 were detected by quantitative real-time PCR and western blot. Dual luciferase reporter assay was performed to evaluate the regulation of Foxo1 on the transcriptional activity of KLK4 promoter. The results showed that KLK4 expression was decreased in LS8 cells treated by NaF, while MMP20 expression was not changed. Foxo1 activation led to significantly up-regulation of KLK4 in LS8 cells under NaF condition. Knockout of Foxo1 markedly decreased klk4 expression in mRNA level, and intensified inhibition occurred in LS8 cells when combined with NaF treatment. However, the variation trend of MMP20 was not clear. Dual luciferase reporter assay showed that Foxo1 activation enhanced the transcriptional activity of KLK4 promoter. These findings suggest that the decrease of Foxo1 expression induced by high fluoride was a cause for low KLK4 expression.

Upregulating the Expression of Survivin‐HBXIP Complex Contributes to the Protective Role of IMM‐H004 in Transient Global Cerebral Ischemia/Reperfusion

IMM-H004, a 3-piperazinylcoumarin compound derived from coumarin, has been proved effective against CA1 cell loss and spatial learning impairments resulting from transient global ischemia/reperfusion (TGCI/R), while the mechanism is still largely unknown. Here, we confirmed that treatment of rats with IMM-H004 immediately after TGCI/R ameliorated delayed neuronal death (DND) in the CA1 of hippocampus and cortex. Further study suggested that IMM-H004 contributed to the expression of antiapoptotic protein survivin through the activation of PI3K-dependent protein kinase B (PKB/Akt), which led to the phosphorylation of forkhead box O1 (FoxO1), then relieved the inhibiting effect on survivin promoter. Additionally, IMM-H004 also enhanced the expression of hepatitis B X-interacting protein (HBXIP), which formed a complex with survivin to prevent the activation of caspase death cascade, thereby halting apoptotic cell death. Finally, we injected a HBXIP siRNA into hippocampus and performed microelectroporation before ischemia/reperfusion, which abolished the protective effect of IMM-H004. Further study revealed that HBXIP maintained the high expression of Akt and survivin. Collectively, our findings demonstrated that DND after TGCI/R was alleviated by IMM-H004 through promoting the formation of survivin-HBXIP complex, which further emphasized the importance of endogenous protein involved in self-repair after stroke. Support or Funding Information This work was supported by the National Natural Science Foundation of China (81730096, U1402221), CAMS Innovation Fund for Medical Sciences (CIFMS) ( 2016-I2M-1-004), the Scientific Research Foundation of the Higher Education Institutions of Hunan Province (15K091), Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (BZ0150), PUMC Graduate Education and Teaching Reform Project (10023201600801). Schematic diagram of the neural protective role of IMMH004 after TGCI/R This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leucine promotes differentiation of porcine myoblasts through the protein kinase B (Akt)/Forkhead box O1 signalling pathway.

Leucine, one of the branched-chain amino acids, is the only amino acid to regulate protein turnover in skeletal muscle. Leucine not only increases muscle protein synthesis, but also decreases muscle protein degradation. It is well documented that leucine plays a positive role in differentiation of murine muscle cells. However, the role of leucine on porcine myoblast differentiation and its mechanism remains unclear. In this study, porcine myoblasts were induced to differentiate with differentiation medium containing different concentrations of leucine, and wortmannin was used to interdict the activity of protein kinase B (Akt). We found that leucine increased the number of myosin heavy chain-positive cells and creatine kinase activity. Moreover, leucine increased the mRNA and protein levels of myogenin and myogenic determining factor (MyoD). In addition, leucine increased the levels of phosphorylated Akt/Akt and phosphorylated Forkhead box O1 (P-FoxO1)/FoxO1, as well as decreased the protein level of FoxO1. However, wortmannin, a specific repressor of PI3K/Akt signalling pathway, attenuated the positive role of leucine on porcine myoblast differentiation. Our results suggest that leucine promotes porcine myoblast differentiation through the Akt/FoxO1 signalling pathway.

Liraglutide improves hepatic insulin resistance via the canonical Wnt signaling pathway.

Liraglutide, a modified form of glucagon‑like peptide‑1 (GLP‑1), is used in the treatment of diabetes mellitus. However, the underlying mechanism by which liraglutide improves liver insulin resistance remains to be elucidated. The proto‑oncogene Wnt (Wnt) signaling pathway has been reported to be associated with glucose and lipid metabolism. Using invivo and invitro models of diabetes and insulin resistance, it was investigated whether the beneficial effects of liraglutide on liver glucose metabolism are mediated by the Wnt signaling pathway. The results of the present study demonstrate that body weight, fasting blood glucose, insulin levels and the homeostasis model assessment for insulin resistance were markedly decreased in db/db mice treated with liraglutide compared with control mice. Liraglutide also improved liver morphology and reduced the accumulation of lipid droplets. Furthermore, the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was downregulated, whereas the expression of phosphorylated forkhead boxO1, Wnt signaling pathway‑associated molecules, β‑catenin, transcription factor7‑like2 and phosphorylated glycogen synthase kinase-3β was upregulated in the liver of mice treated with liraglutide. In the invitro study, increased gluconeogenesis and decreased glucose uptake rates were observed in insulin resistant hepatocytes; treatment with liraglutide significantly reversed this effect. Furthermore, transfection of insulin resistant hepatocytes with β‑catenin small interfering RNA attenuated the effects of liraglutide, suggesting that liraglutide improves insulin resistance via activating the β‑catenin/Wnt signaling pathway. The results of the present study suggest a novel mechanism underlying liraglutide‑mediated improvements in insulin resistance in the liver. The Wnt signaling pathway may be a potential therapeutic target for the treatment of altered hepatic physiology in insulin resistance.