FOXO1 Gene Expression Research Articles

Diverse inhibition of forkhead box O1 activity by linoleic acid isomers - potential role in lipid metabolism in HepG2 cells and livers of C57BL/6J mice.

Conjugated dienes of linoleic acid (CLA) are constitutional and geometric isomers of linoleic acid that are commonly used as dietary supplements during body mass reduction. Their role in the reduction of lipid deposits in liver tissue is not unequivocal. CLA contain an equimolar mixture of two isomers of linoleic acid: trans-10,cis-12 CLA and cis-9,trans-11. Only one isomer - trans-10,cis-12 CLA exhibits fat-reducing properties, cis-9,trans-11 CLA does not. The main goal of this study was to determine if CLA isomers affect the activation of forkhead box O1 (FoxO1) in liver cells and tissue. FoxO1 is a protein that plays a crucial role in regulation of lipid and carbohydrates metabolism. In vitro and in vivo models of our study were HepG2 cells and C57BL/6J mice. Methods used in the study were qPCR - quantification of FoxO1 gene expression, Western blot - posttranslational phosphorylation of FoxO1, Oil Red O (ORO) - lipid staining and ELISA - quantification of apoB100. In both models trans-10,cis-12 CLA diminished FoxO1 gene expression: decrease by 44.1 ± 20.9% SD in the cells and 65.4 ± 29.8% SD in mice. The lowest accumulation of lipids (drop of 37.2 ± 1.7% SD) and the highest increase of apoB100 protein (74 ± 12.8% SD) were detected in the medium of cells cultured with trans-10,cis-12 CLA. Both isomers of linoleic acid have different effects on lipid metabolism. Isomer c9,t11 CLA accelerates lipogenesis, whereas isomer t10,c12 CLA activates secretion of lipids in HepG2 cells. In contrast to the in vitro study, unfortunately this pro-health property of t10,c12 CLA was not confirmed in the in vivo model. This casts a shadow on CLA dietary supplements that are commonly used among people with type 2 diabetes, NAFLD (non-alcoholic liver disease) or a metabolic syndrome in order to lose weight.

Effects of acute resistance exercise on proteolytic and myogenic markers in skeletal muscles of former weightlifters and age-matched sedentary controls.

Former athletes who continue a regular, performance-oriented training throughout life provide a unique model for studying successful aging. With this in mind, the current study aimed to compare the effects of an acute resistance exercise on proteolytic and myogenic markers in older weightlifters and untrained participants. Sixteen older men (8 former weightlifters, 8 age-matched untrained controls) with an age of 61.2±8.2 years volunteered to participate in the study. Two days after assessing 1-RM, an acute exercise protocol (3 sets, 70-75% of one-repetition maximum until voluntary fatigue) was applied unilaterally on the dominant leg while the other leg served as control. Three hours after termination of the exercise, skeletal muscle tissue was obtained from m. vastus lateralis of both legs. Acute resistance exercise led to an up-regulation (>1.5-fold) of 14 genes in controls and of 13 genes in weightlifters. The transcription factors FOS and early growth response 1 (EGR1), as well as the E3 protein ligase TRIM63 comprised the most responsive genes to resistance exercise (EGR1:15.7-fold increase, P=0.003, FOS: 36.3-fold increase, P<0.001; TRIM63: 2.9-fold increase, P<0.001). In addition, myostatin levels were decreased in the exercised leg (0.6-fold, P<0.001). FOXO3 gene expression was significantly higher in weightlifters than in untrained controls (1.5-fold, P=0.042). Trained and untrained older adults respond to an acute bout of resistance exercise in a very similar way irrespective of training status, although some differences exist in FOXO3, potentially reflecting the superior capacity of trained persons in regulating cellular homeostasis.

CFTR and FOXO1 gene expression are reduced and high mobility group box 1 (HMGB1) is increased in the ovaries and serum of women with polycystic ovarian syndrome

We previously described increased HMGB1 and reduced FOXO1 dependent on CFTR loss of function in cystic fibrosis (CF) and we showed in vitro that HMGB1 was lowered by insulin. Reduced CFTR gene expression has been described in granulosa cells (GC) from PCOS-induced rats. We aimed at studying CFTR and FOXO1 gene expression in GC, HMGB1 concentrations in serum and follicular fluids (FF), and insulin and IL-6 in FF in PCOS women. Thirty PCOS and 36 non-PCOS women (CTRL) undergoing in vitro fertilization were enrolled. CFTR and FOXO1 gene expression were downregulated in PCOS (p ≤ .05). HMGB1 was higher in PCOS both in FF (p ≤ .05) and in serum (p < .005) whereas insulin was lower, and IL-6 was unchanged with respect to controls. 17-β estradiol was higher in PCOS than in CTRL (p ≤ .005). HMGB1 correlated negatively with insulin in FF (p ≤ .005). The increase in HMGB1 both in FF and in serum, likely reflects both low grade inflammation and insulin sensitivity. IL-6 was unchanged possibly reflecting functions other than inflammation.

Effect of oral magnesium sulfate administration on blood glucose hemostasis via inhibition of gluconeogenesis and FOXO1 gene expression in liver and muscle in diabetic rats

The present study was designed to investigate the possible role of Mg2+ in suppression of phosphoenolpyruvate carboxy kinase (PEPCK) enzyme via inhibition of FOXO1gene expression in liver and we also examined whether Mg contributes to decrease blood glucose in muscle via inhibiting FOXO1 gene and protein expression. Fifty rats in five groups of experiment were considered as; non-diabetic control (NDC), Mg2+-treated non-diabetic control (Mg2+-NDC), chronic diabetic (CD), Mg2+-treated chronic diabetic (Mg2+-CD), and insulin-treated chronic diabetic (Ins-CD). Streptozotocin (STZ) was used for diabetes induction. The Mg2+-CD and Mg2+-NDC groups received 10 g/l of magnesium sulfate (MgSO4) added to drinking water, and Ins-CD group received 2.5 U/kg of insulin. The blood glucose level and body weight were measured weekly. After 16 weeks, intraperitoneal glucose tolerance test (IPGTT) was done and blood samples were taken to determine the plasma levels of Mg and gastrocnemius muscle legs, and liver were isolated for both Forkhead transcription factor (FOXO1) and PEPCK enzyme genes and proteins expression. Administration of MgSO4 improved IPGTT, lowered blood glucose levels and decreased FOXO1 and PEPCK genes and proteins expression in muscle and liver, while insulin just could decrease FOXO1 gene and protein expression in the muscle. These findings illustrated that MgSO4 improved hyperglycemia via inhibition of FOXO1 gene and protein level in the muscle and liver, and it also decreased blood glucose level by prohibition of gluconeogenesis pathway in the liver. However, long time administration of insulin did not have any effect on liver.

Dendrobium mixture regulates hepatic gluconeogenesis in diabetic rats via the phosphoinositide-3-kinase/protein kinase B signaling pathway.

The present study aimed to evaluate the impact of dendrobium mixture (DMix) on the gene and protein expression of insulin signaling pathway-associated factors in the livers of diabetic rats. The molecular mechanisms by which DMix inhibits gluconeogenesis were also investigated. A total of 47 female Wistar rats were used in the present study. Of these, 11 rats were randomly selected as healthy controls and diabetes was induced in the remaining 36 rats by administering a high-fat and high-sugar diet for 6 weeks, followed by two intraperitoneal injections of streptozotocin. The 36 rats were screened for diabetes and then randomly divided into three groups: Model, metformin and DMix groups. Following 12 weeks of treatment, the fasting blood glucose (FBG), glycosylated serum protein (GSP), serum insulin, blood lipids [total cholesterol (Tch) and triglycerides (TG)], alanine transaminase (ALT) and aspartate transaminase (AST) were assessed. In addition, hematoxylin and eosin staining was used for histomorphological examination of the liver tissues. The mRNA expression of insulin receptor (InsR), forkhead box protein O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in the liver was measured with reverse transcription-quantitative polymerase chain reaction and the protein expression of InsR, phosphoinositide-3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt), p-Akt, FoxO1, PEPCK and G6Pase in the liver was measured by western blot analysis. The FBG, GSP, InsR, Tch, TG, ALT and AST levels were significantly lower in the DMix-treated group compared with the model group (P<0.05). In addition, DMix treatment notably improved liver histopathology and significantly increased the gene and protein expression of InsR, PI3K and Akt (P<0.05). DMix treatment also significantly reduced the gene and protein expression of FoxO1, PEPCK and G6Pase (P<0.05). DMix effectively reduced FBG and blood lipids and significantly improved liver function and insulin resistance in diabetic rats, possibly by regulating the gene and protein expression of molecules associated with the PI3K/Akt signaling pathway.

FOXO3 Expression in the Human Ovary [6OP

INTRODUCTION: To assess FOXO3 gene expression levels and members of the KL-PI3K-AKT-FOXO3 pathway in pre- and postmenopausal human ovaries. To evaluate in a temporal manner protein localization of FOXO3 in pre- and postmenopausal human ovaries by immunohistochemistry (IHC). METHODS: Total RNA was isolated from human ovary collected from pre- and postmenopausal patients undergoing oophorectomy and used for real-time qPCR for members of the KL-PTEN-PI3K-FOXO3 pathway, as well as other genes. IHC was performed on archived paraffin embedded ovaries to visualize the presence and localization of FOXO3 in human ovaries ranging from fetal to menopausal. RESULTS: FOXO3 is expressed in premenopausal human ovary and to a significantly reduced level in postmenopausal ovary. Nine additional genes in the KL-PTEN-PI3K-FOXO3 pathway were also expressed in premenopausal ovaries but like FOXO3 displayed significantly decreased levels in the postmenopausal cohort. IHC analysis revealed strong nuclear FOXO3 staining in primordial follicles from pre-pubertal ovaries through menarche/post-pubertal. A dominant population of primordial follicles were identified in older post-pubertal ovaries and in the reproductive/premenopausal groups that displayed light to negative nuclear staining for FOXO3, with a second much less abundant population of primordial follicles retaining strong positive nuclear FOXO3 staining. Postmenopausal ovaries were devoid of follicles but had strong stromal FOXO3 staining. CONCLUSION: As predicted from mouse genetic models, FOXO3 mRNA and protein is present in premenopausal human ovary as are nine members of the KL- PI3K-AKT-FOXO3 pathway. FOXO3 localization in the human ovary undergoes an age-dependent transition from strongly positive nuclei in young ovaries to predominantly light to negative staining in reproductive/premenopausal. Given FOXO3's importance to genome stability, primordial follicles with high nuclear FOXO3 levels may define high quality oocytes lacking detrimental levels of DNA damage, so that the ratio of FOXO3 positive to negative oocytes may explain the consequences of decreasing ovarian reserve.

Vitamin D Attenuates FOXO1-Target Atrophy Gene Expression in C2C12 Muscle Cells.

Vitamin D is known to be effective for the prevention of muscle atrophy, such as age-related sarcopenia. However, vitamin D action in skeletal muscle tissue and muscle cells is largely unknown. We previously found that a transcription factor, FOXO1 gene expression, was induced in various muscle atrophy conditions causing muscle atrophy by upregulating atrophy-related genes, including atrogin 1 (ubiquitin ligase) and cathepsin L (lysosomal proteinase). In this study, we found that vitamin D inhibited FOXO1-mediated transcriptional activity in a reporter gene assay. Moreover, vitamin D suppressed the glucocorticoid-induced gene expression of atrogin 1 and cathepsin L in C2C12 myoblasts. Thus, vitamin D may prevent muscle atrophy via the FOXO1-mediated pathway in muscle cells.

MiR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1.

Objective: To investigate the role of FOXO1 and miR-183-96-182 clusters in ox-LDL induced endothelial cell apoptosis. Methods: FOXO1 overexpression (OE) and knockdown (KD) as well as AKT1 OE in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were achieved by lentiviral transduction. Upregulation of miR-183-5p, miR-182-5p or miR-96-5p was mimicked by agomir treatment. FOXO1 gene transcription was monitored by FOXO1 promotor reporter assay. Cell apoptosis in culture was monitored by TiterTACS in situ detection. Regulation of FOXO1 gene expression by an miRNA targeting mechanism was monitored by AGO2-RNA immunoprecipitation assay. Results: FOXO1 mRNA and protein expression levels in ox-LDL treated HUVECs or HAECs were significantly upregulated due to transcriptional and miRNA targeting mechanisms. MiR-183-5p, miR-182-5p and miR-96-5p expression levels in HUVECs or HAECs were significantly reduced by ox-LDL treatment, the overexpression of which by agomir treatment partially reduced the FOXO1 mRNA/protein expression levels and cell apoptosis which was upregulated by ox-LDL treatment. FOXO1 overexpression antagonized the effect of the agomir treatment indicated above. MiR-183-5p, miR-182-5p and miR-96-5p agomir treatment partially rescued the FOXO1 pSer256/total FOXO1 protein ratio and the AKT1 pSer473 level that were reduced by ox-LDL treatment in the HUVECs or HAECs. AKT1 overexpression significantly reduced FOXO1 protein expression, increased miR-182-5p and miR-183-5p expression, and partially alleviated ox-LDL induced HUVEC or HAEC apoptosis in an miR-183-5p and miR-182-5p-dependent manner. Conclusion: miR-183-96-182 clusters could partially alleviate ox-LDL-induced apoptosis in HUVECs or HAECs by targeting FOXO1.

Lack of Ikaros cripples expression of Foxo1 and its targets in naive T cells.

Ikaros is a transcription factor that regulates lymphocyte development from the level of the haematopoietic stem cell. Lack of Ikaros reduces the ability of progenitor cells to commit to the T-cell lineage, resulting in reduced numbers of early thymic T-cell progenitors and mature T cells. Mature CD4 T cells that lack Ikaros have defects in proliferation, T helper cell differentiation, cytokine expression and the ability to become anergic. A role for Ikaros in the naive T cell has not yet been identified. The receptors interleukin-7 receptor α (IL-7Rα) and l-selectin are important for ensuring survival and proper homing of naive T cells, respectively. Here we show that lack of Ikaros leads to reduced expression of these receptors in naive T cells, which impacts their ability to home and survive in response to IL-7. We define the mechanism underlying this phenotype as a requirement for Ikaros in maintenance of expression of Foxo1, a transcriptional regulator that is required for their expression. We also demonstrate that CD4 T cells lacking Ikaros are significantly crippled in their ability to become induced regulatory T cells, a phenotype also linked to reduced Foxo1 expression. Finally, we show that restoring Ikaros function to Ikaros-deficient CD4 T cells increases levels of Foxo1 message. Together, these studies define, for the first time, a role for Ikaros in naive T cells and establish it as the first transcriptional regulator required for maintaining levels of Foxo1 gene expression in these cells.

Mutations in 5-methylcytosine oxidase TET2 and RhoA cooperatively disrupt T cell homeostasis.

Angioimmunoblastic T cell lymphoma (AITL) represents a distinct, aggressive form of peripheral T cell lymphoma with a dismal prognosis. Recent exome sequencing in patients with AITL has revealed the frequent coexistence of somatic mutations in the Rho GTPase RhoA (RhoAG17V) and loss-of-function mutations in the 5-methylcytosine oxidase TET2. Here, we have demonstrated that TET2 loss and RhoAG17V expression in mature murine T cells cooperatively cause abnormal CD4+ T cell proliferation and differentiation by perturbing FoxO1 gene expression, phosphorylation, and subcellular localization, an abnormality that is also detected in human primary AITL tumor samples. Reexpression of FoxO1 attenuated aberrant immune responses induced in mouse models adoptively transferred with T cells and bearing genetic lesions in both TET2 and RhoA. Our findings suggest a mutational cooperativity between epigenetic factors and GTPases in adult CD4+ T cells that may account for immunoinflammatory responses associated with AITL patients.

Foxo3 gene expression and oxidative status in beta-thalassemia minor subjects

BackgroundOxidative stress may aggravate symptoms of hemolytic anemias such as beta-thalassemia. FoxO3 activation results in resistance to oxidative stress in fibroblasts and neuronal cell cultures.ObjectiveThe purpose of this research was to study FoxO3 gene expression and oxidative status in beta-thalassemia minor individuals.MethodsSixty-three subjects (42 apparently healthy individuals and 21 with beta-thalassemia minor) were analyzed at the Universidad Nacional de Tucumán, Argentina, between September 2013 and June 2014. A complete blood count, hemoglobin electrophoresis in alkaline pH and hemoglobin A2 levels were quantified. Moreover, thiobarbituric acid reactive species, erythrocyte catalase activity and iron status were evaluated. Beta-thalassemia mutations were determined by real-time polymerase chain reaction. FoxO3 gene expression was investigated by real-time reverse transcription-polymerase chain reaction using mononuclear cells from peripheral blood.ResultsSubjects were grouped as children (≤12 years), and adult women and men. The analysis of erythrocyte catalase activity/hemoglobin ratio revealed a significant difference (p-value <0.05) between healthy and beta-thalassemia minor adults, but no significant difference was observed in the thiobarbituric acid reactive species levels and FoxO3 gene expression (p-value >0.05). Thiobarbituric acid reactive species and the erythrocyte catalase activity/hemoglobin ratio were not significantly different on comparing the type of beta-thalassemia mutation (β0 or β+) present in carriers.ConclusionsThe lack of systemic oxidative imbalance demonstrated by thiobarbituric acid reactive species is correlated to the observation of normal FoxO3 gene expression in mononuclear cells of peripheral blood. However, an imbalanced antioxidant state was shown by the erythrocyte catalase activity/hemoglobin ratio in beta-thalassemia minor carriers. It would be necessary to study FoxO3 gene expression in reticulocytes to elucidate the role of FoxO3 in this pathology.

MiR-181c restrains nitration stress of endothelial cells in diabetic db/db mice through inhibiting the expression of FoxO1

Endothelial dysfunction played an important role in the progression of diabetes mellitus (DM). miR-181c has been implicated in many diseases, including DM. However, the molecular mechanisms of miR-181c regulate this process remained poorly understood. Healthy ICR mice were divided into control group (n = 10) and db/db DM group (n = 10). The expression of miR-181c and FoxO1 were both investigated in diabetic db/db mice or high glucose-induced endothelial cells (MAECs and END-D). Here we found that down-regulation of miR-181c and the activation of FoxO1/iNOS were observed in mice and endothelial cells. Furthermore, we verified that miR-181c directly targeted and inhibited FoxO1 gene expression by targeting its 3′-UTR through luciferase reporter assay. Knockdown of FoxO1 reversed the up-regulation of iNOS, nitrotyrosine and the down-regulation of p-eNOSSer1177/eNOS in high glucose (30 mM)-induced MAECs cells. In addition, over-expression of miR-181c could reverse the enhanced nitration stress induced by high glucose, while this effect could be attenuated by pcDNA-FoxO1 in MAECs. These results shown that miR-181c attenuated nitration stress through regulating FoxO1 expression and affecting endothelial cell function, which offering a new target for the development of preventive or therapeutic agents against DM.

Does Resveratrol Improve Insulin Signalling in HepG2 Cells?

Diabetes mellitus is a common metabolic disorder with high global prevalence. It is characterized by a decrease in insulin secretion or a decrease in insulin sensitivity or both. The aim of the present study was to investigate the effects of resveratrol treatment on the expression of the genes involved in insulin signalling cascade, such as Forkhead box protein O1 (FoxO1), 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and mammalian target of rapamycin (mTOR). HepG2 cells were cultured in serum-free medium with high concentrations of glucose and insulin and then were treated with resveratrol (5, 10 and 20 µM) for 24 and 48 hours. Complementary deoxyribonucleic acids (cDNAs) were synthesized followed by RNA extraction. Real-time quantitative reverse transcription polymerase chain reaction was used to analyze the expression of FoxO1, PDPK1 and mTOR. Resveratrol increased the expression of PDPK1, mTOR and FoxO1. No significant difference was seen among differing dosages of resveratrol, but treatments for 48 hours exerted the greatest effectiveness. Our results were consistent with other studies showing the beneficial effects of resveratrol on diabetes. However, considering the effects of resveratrol in increasing FoxO1 and gluconeogenic gene expression, long-term usage of resveratrol should be investigated in greater depth in future studies.

Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1. Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n=11) and control group (n=11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy. The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047±0.108, FoxF1 Ctrl 1.419±0.01, p=0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74±8.925, FoxC2 Ctrl 27.408±7.487, p=0.776; FoxO1 CDH 0.011±0.002, FoxO1 Ctrl 0.011±0.001, p=0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls. Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression during embryogenesis may participate in vascular maldevelopment resulting in PH in this animal model.

Restoring miR122 in human stem-like hepatocarcinoma cells, prompts tumor dormancy through Smad-independent TGF-β pathway.

miR122 is the prevalent miRNA in adult healthy liver and it is responsible for liver stem cell differentiation towards hepatocyte lineage. Its expression is frequently lost in hepatocellular carcinoma (HCC). We studied the effects of restoring miR122 expression in a distinctive cell line derived from human HCC-BCLC9 cells-with a solid stem-like cell profile, high tumor initiating ability and undetectable miR122 expression. We generated a stable BCLC9 cell line that expresses miR122 (BCLC9-miR122). Restitution of miR122 in BCLC9 cells, decreases cell proliferation rate and reduces significantly tumor size in vivo. BCLC9-miR122 cells down-regulate expression of MYC, KLF4, FOXM1, AKT2 and AKT3 genes and up-regulate FOXO1 and FOXO3A gene expression. In addition, miR122 transfected cells decreased AKT2 kinase activation while decreased FOXO1 and FOXO3A protein inactivation. Reduction in tumor size in BCLC9-miR122 associated with an increase in p38MAPK protein expression and activation leading to a low phospho-ERK1/2 to phospho-p38 ratio. Treatment of miR122 positive cells with an inhibitor of TGFBR1 activation, abolished tumor dormancy program and recovered cell proliferation rate through a Smad-independent TGF-β response.HCC stem-like cells can be directed towards cell differentiation and tumor dormancy by restoring miR122 expression. We demonstrate, for the first time, that dormancy program is achieved through a Smad-independent TGF-β pathway. Reestablishing miR122 expression is a promising therapeutic strategy that would work concurrently reducing tumor aggressiveness and decreasing disease recurrence.

Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata).

Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA) to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs). This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs), MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates the importance of an adequate level of Lysine in fishmeal diet formulation for optimum growth.

Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes.

Glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, both in vitro and in vivo. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene, we identified three glucocorticoid receptor (GR) binding regions (GBRs): one being upstream of the transcription start site, -17kbGBR; and two in introns, +45kbGBR and +71kbGBR. Together, these three GBRs contain four 15-bp glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay revealed that Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR and +71kbGBR upon 30- and 60-min Dex treatment, respectively. Conversely, Dex treatment did not affect the chromatin structure near the -17kbGBR, in which the GRE is located in the linker region. Dex treatment also increased histone H3 and/or H4 acetylation in genomic regions near all three GBRs. Moreover, using chromatin conformation capture (3C) assay, we showed that Dex treatment increased the interaction between the -17kbGBR and two genomic regions: one located around +500 bp and the other around +73 kb. Finally, the transcriptional coregulator p300 was recruited to all three GBRs upon Dex treatment. The reduction of p300 expression decreased FoxO3 gene expression and Dex-stimulated interaction between distinct genomic regions of FoxO3 gene identified by 3C. Overall, our results demonstrate that glucocorticoids activated FoxO3 gene transcription through multiple GREs by chromatin structural change and DNA looping.

High-throughput sequencing reveals key genes and immune homeostatic pathways activated in myeloid dendritic cells by Porphyromonas gingivalis 381 and its fimbrial mutants.

The human microbiome consists of highly diverse microbial communities that colonize our skin and mucosal surfaces, aiding in maintenance of immune homeostasis. The keystone pathogen Porphyromonas gingivalis induces a dysbiosis and disrupts immune homeostasis through as yet unclear mechanisms. The fimbrial adhesins of P. gingivalis facilitate biofilm formation, invasion of and dissemination by blood dendritic cells; hence, fimbriae may be key factors in disruption of immune homeostasis. In this study we employed RNA-sequencing transcriptome profiling to identify differentially expressed genes (DEGs) in human monocyte-derived dendritic cells (MoDCs) in response to in vitro infection/exposure by Pg381 or its isogenic mutant strains that solely express minor-Mfa1 fimbriae (DPG3), major-FimA fimbriae (MFI) or are deficient in both fimbriae (MFB) relative to uninfected control. Our results yielded a total of 479 DEGs that were at least two-fold upregulated and downregulated in MoDCs significantly (P ≤ 0.05) by all four strains and certain DEGs that were strain-specific. Interestingly, the gene ontology biological and functional analysis shows that the upregulated genes in DPG3-induced MoDCs were more significant than other strains and associated with inflammation, immune response, anti-apoptosis, cell proliferation, and other homeostatic functions. Both transcriptome and quantitative polymerase chain reaction results show that DPG3, which solely expresses Mfa1, increased ZNF366, CD209, LOX1, IDO1, IL-10, CCL2, SOCS3, STAT3 and FOXO1 gene expression. In conclusion, we have identified key DC-mediated immune homeostatic pathways that could contribute to dysbiosis in periodontal infection with P. gingivalis.

Influenza infection results in upregulation of inflammatory and atrophy genes in murine skeletal muscle (VIR5P.1146)

Abstract Though influenza is a respiratory infection, myalgia is a common symptom and associations with acute myositis and rhabdomyolysis have been noted. It is currently unclear if these symptoms are secondary due to the systemic response or if muscle may contribute to inflammation. Further, the effect of influenza on muscle damage and potential repair is not well established. We aimed to determine the expression level of select myokines, atrophy, and myogenic markers in skeletal muscle during influenza infection. Young C57BL/6 mice were inoculated with Pr8 influenza virus (IN, 500 EID50) and sacrificed 7 or 15 days post infection (DPI) or were uninfected (n=4/group). Gastrocnemius gene expression was analyzed via RT-qPCR. Myostatin, IL4, IL6, IL6 receptor alpha, Ubiquitin B, Ubiquitin C, Foxo1, Atrogin-1, and MuRF-1 gene expression was upregulated 7 DPI indicating increased inflammation, protein degradation, and atrophy. Expression of positive regulators of muscle growth and maintenance (Igf-1, MyoD, and Mef2C) was downregulated at this time as well. At 15 DPI Atrogin-1 and MuRF-1 were downregulated though upregulation of myogenic markers did not occur. Our results indicate influenza infection is associated with muscle-localized inflammation, protein degradation, and atrophy that is resolved by day 15 post infection. It is known that influenza infection in the elderly leads to increased disability and loss of independence; it is likely muscle atrophy is a contributing factor.

Lycopene-rich tomato oleoresin modulates plasma adiponectin concentration and mRNA levels of adiponectin, SIRT1, and FoxO1 in adipose tissue of obese rats.

To investigate whether lycopene can modulate adiponectin levels and SIRT1 and FoxO1 gene expression in the adipose tissue of diet-induced obese rats. Male Wistar rats were first fed with hypercaloric diet (HD, n = 12) for 6 weeks, and afterward, these rats were randomly assigned to receive HD (n = 6) or HD with lycopene-rich tomato oleoresin (equivalent to lycopene 10 mg/kg body weight (BW)/day, HD + L, n = 6) by gavage for additional 6 weeks. Plasma lycopene and adiponectin levels were analyzed by high-performance liquid chromatography and immunoassay, respectively. The messenger RNA (mRNA) expressions of adiponectin, Sirtuin 1 (SIRT1), Forkhead box O 1 (FoxO1), fatty acid translocase/cluster of differentiation 36 (FAT/CD36), and PPARγ in adipose tissues were determined by quantitative polymerase chain reaction. Lycopene was detected in the plasma of rats in HD + L group but not in the HD group. Although both BW and adiposity were not different between the two groups, there was a significant increase in both plasma concentration and mRNA expression of adiponectin in the adipose tissue of the HD + L group. In addition, the lycopene supplementation upregulated mRNA expressions of SIRT1, FoxO1, and FAT/CD36 but downregulated PPARγ in adipose tissue of obese rats. These data suggest that lycopene, in the concentration used, is not toxic and also its health benefits in adipose tissue may play a role against obesity-related complications.