Phosphorylation Of FoxO1 Research Articles

Eicosapentaenoic acid-mediated activation of PGAM2 regulates skeletal muscle growth and development via the PI3K/AKT pathway

Eicosapentaenoic acid regulates glucose uptake in skeletal muscle and significantly affects whole-body energy metabolism. However, the underlying molecular mechanism remains unclear. Here we report that eicosapentaenoic acid activates phosphoglycerate mutase 2, which mediates the conversion of 2-phosphoglycerate into 3-phosphoglycerate. This enzyme plays a pivotal role in glycerol degradation, thereby facilitating the proliferation and differentiation of satellite cells in skeletal muscle. Interestingly, phosphoglycerate mutase 2 inhibits mitochondrial metabolism, promoting the formation of fast-type muscle fibers. Treatment with eicosapentaenoic acid and phosphoglycerate mutase 2 knockdown induced opposite transcriptomic changes, most of which were enriched in the PI3K-AKT signaling pathway. Phosphoglycerate mutase 2 activated the PI3K-AKT signaling pathway, which inhibited the phosphorylation of FOXO1, and, in turn, inhibited mitochondrial function and promoted the formation of fast-type muscle fibers. Our results suggest that eicosapentaenoic acid promotes skeletal muscle growth and regulates glucose metabolism by targeting phosphoglycerate mutase 2 and activating the PI3K/AKT signaling pathway.

The regulation of MFG-E8 on the mitophagy in diabetic sarcopenia via the HSPA1L-Parkin pathway and the effect of D-pinitol.

Diabetic sarcopenia is a disease-related skeletal muscle disorder that causes progressive symptoms. The complete understanding of its pathogenesis is yet to be unravelled, which makes it difficult to develop effective therapeutic strategies. This study investigates how MFG-E8 affects mitophagy and the protective role of D-pinitol (DP) in diabetic sarcopenia. In vivo, streptozotocin-induced diabetic SAM-R1 (STZ-R1) and SAM-P8 (STZ-P8) mice (16-week-old) were used, and STZ-P8 mice were administrated of DP (150mg/kg per day) for 6weeks. Gastrocnemius muscles were harvested for histological analysis including transmission electron microscopy. Proteins were evaluated via immunohistochemistry (IHC), immunofluorescence (IF), and western blotting (WB) assay. In vitro, advanced glycation end products (AGEs) induced diabetic and D-galactose (DG) induced senescent C2C12 models were established and received DP, MFG-E8 plasmid (Mover)/siRNA (MsiRNA), or 3-MA/Torin-1 intervention. Proteins were evaluated by IF and WB assay. Immunoprecipitation (IP) and co-immunoprecipitation (CO-IP) were used for hunting the interacted proteins of MFG-E8. In vivo, sarcopenia, mitophagy deficiency, and up-regulated MFG-E8 were confirmed in the STZ-P8 group. DP exerted protective effects on sarcopenia and mitophagy (DP+STZ-P8 vs. STZ-P8; all P<0.01), such as increased lean mass (8.47±0.81g vs. 7.08±1.64g), grip strength (208.62±39.45g vs. 160.87±26.95g), rotarod tests (109.7±11.81s vs. 59.3±20.97s), muscle cross-sectional area (CSA) (1912.17±535.61μm2 vs. 1557.19±588.38μm2), autophagosomes (0.07±0.02 per μm2 vs. 0.02±0.01 per μm2), and cytolysosome (0.07±0.03 per μm2 vs. 0.03±0.01 per μm2). DP down-regulated MFG-E8 in both serum (DP+STZ-P8: 253.19±34.75pg/mL vs. STZ-P8: 404.69±78.97pg/mL; P<0.001) and gastrocnemius muscle (WB assay. DP+STZ-P8: 0.39±0.04 vs. STZ-P8: 0.55±0.08; P<0.01). DP also up-regulated PINK1, Parkin and LC3B-II/I ratio, and down-regulated P62 in gastrocnemius muscles (all P<0.01). In vitro, mitophagy deficiency and MFG-E8 up-regulation were confirmed in diabetic and senescent models (all P<0.05). DP and MsiRNA down-regulated MFG-E8 and P62, and up-regulated PINK1, Parkin and LC3B-II/I ratio to promote mitophagy as Torin-1 does (all P<0.05). HSPA1L was confirmed as an interacted protein of MFG-E8 in IP and CO-IP assay. Mover down-regulated the expression of Parkin via the HSPA1L-Parkin pathway, leading to mitophagy inhibition. MsiRNA up-regulated the expression of PINK1 via SGK1, FOXO1, and STAT3 phosphorylation pathways, leading to mitophagy stimulation. MFG-E8 is a crucial target protein of DP and plays a distinct role in mitophagy regulation. DP down-regulates the expression of MFG-E8, reduces mitophagy deficiency, and alleviates the symptoms of diabetic sarcopenia, which could be considered a novel therapeutic strategy for diabetic sarcopenia.

SIRT3: A Potential Target of Different Types of Osteoporosis.

Osteoporosis (OP) is a common age-related disease. OP is mainly a decrease in bone density and mass caused by the destruction of bone microstructure, which leads to an increase in bone fragility. SIRT3 is a mitochondrial deacetylase that plays critical roles in mitochondrial homeostasis, metabolic regulation, gene transcription, stress response, and gene stability. Studies have shown that the higher expression levels of SIRT3 are associated with decreased levels of oxidative stress in the body and may play important roles in the prevention of age-related diseases. SIRTs can enhance the osteogenic potential and osteoblastic activity of bone marrow mesenchymal stromal cells not only by enhancing PGC-1α, FOXO3, SOD2, and oxidative phosphorylation, but also by anti-aging and reducing mitochondrial autophagy. SIRT3 is able to upregulate antioxidant enzymes to exert an inhibitory effect on osteoclasts, however, it has been shown that the inflammatory cascade response can in turn increase SIRT3 and inhibit osteoclast differentiation through the AMPK-PGC-1β pathway. SIRT3 plays an important role in different types of osteoporosis by affecting osteoblasts, osteoclasts, and bone marrow mesenchymal cells. In this review, we discuss the classification and physiological functions of SIRTs, the effects of SIRT3 on OCs osteoblasts, and BMSCs, and the roles and mechanisms of SIRT3 in different types of OP, such as diabetic OP, glucocorticoid-induced OP, postmenopausal OP, and senile OP.

Inhibition of the RPS6KA1/FoxO1 signaling axis by hydroxycitric acid attenuates HFD-induced obesity through MCE suppression

BackgroundBecause obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (–)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PurposeThis study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. MethodsPreadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. ResultsHere, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. ConclusionsThese findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.

Sinularin stabilizes FOXO3 protein to trigger prostate cancer cell intrinsic apoptosis

Sinularin, a natural product that purified from soft coral, exhibits anti-tumor effects against various human cancers. However, the mechanisms are not well understood. In this study, we demonstrated that Sinularin inhibited the viability of human prostate cancer cells in a dose-dependent manner and displayed significant cytotoxicity only at high concentration against normal prostate epithelial cell RWPE-1. Flow cytometry assay demonstrated that Sinularin induced tumor cell apoptosis. Further investigations revealed that Sinularin exerted anti-tumor activity through intrinsic apoptotic pathway along with up-regulation of pro-apoptotic protein Bax and PUMA, inhibition of anti-apoptotic protein Bcl-2, mitochondrial membrane potential collapses, and release of mitochondrial proteins. Furthermore, we illustrated that Sinularin induced cell apoptosis via up-regulating PUMA through inhibition of FOXO3 degradation by the ubiquitin–proteasome pathway. To explore how Sinularin suppress FOXO3 ubiquitin–proteasome degradation, we tested two important protein kinases AKT and ERK that regulate FOXO3 stabilization. The results revealed that Sinularin stabilized and up-regulated FOXO3 via inhibition of AKT- and ERK1/2-mediated FOXO3 phosphorylation and subsequent ubiquitin–proteasome degradation. Our findings illustrated the potential mechanisms by which Sinularin induced cell apoptosis and Sinularin may be applied as a therapeutic agent for human prostate cancer.

The Hexane Extract of Citrus sphaerocarpa Ameliorates Visceral Adiposity by Regulating the PI3K/AKT/FoxO1 and AMPK/ACC Signaling Pathways in High-Fat-Diet-Induced Obese Mice.

Obesity is an emerging global health issue with an increasing risk of disease linked to lifestyle choices. Previously, we reported that the hexane extract of Citrus sphaerocarpa (CSHE) suppressed lipid accumulation in differentiated 3T3-L1 adipocytes. In this study, we conducted in vivo experiments to assess whether CSHE suppressed obesity in zebrafish and mouse models. We administered 10 and 20 μg/mL CSHE to obese zebrafish juveniles. CSHE significantly inhibited visceral fat accumulation compared to untreated obese fish. Moreover, the oral administration (100 μg/g body weight/day) of CSHE to high-fat-diet-induced obese mice significantly reduced their body weight, visceral fat volume, and hepatic lipid accumulation. The expression analyses of key regulatory genes involved in lipid metabolism revealed that CSHE upregulated the mRNA expression of lipolysis-related genes in the mouse liver (Pparα and Acox1) and downregulated lipogenesis-related gene (Fasn) expression in epididymal white adipose tissue (eWAT). Fluorescence immunostaining demonstrated the CSHE-mediated enhanced phosphorylation of AKT, AMPK, ACC, and FoxO1, which are crucial factors regulating adipogenesis. CSHE-treated differentiated 3T3L1 adipocytes also exhibited an increased phosphorylation of ACC. Therefore, we propose that CSHE suppresses adipogenesis and enhances lipolysis by regulating the PI3K/AKT/FoxO1 and AMPK/ACC signaling pathways. These findings suggested that CSHE is a promising novel preventive and therapeutic agent for managing obesity.

CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy

ABSTRACT CARM1 (coactivator associated arginine methyltransferase 1) has recently emerged as a powerful regulator of skeletal muscle biology. However, the molecular mechanisms by which the methyltransferase remodels muscle remain to be fully understood. In this study, carm1 skeletal muscle-specific knockout (mKO) mice exhibited lower muscle mass with dysregulated macroautophagic/autophagic and atrophic signaling, including depressed AMP-activated protein kinase (AMPK) site-specific phosphorylation of ULK1 (unc-51 like autophagy activating kinase 1; Ser555) and FOXO3 (forkhead box O3; Ser588), as well as MTOR (mechanistic target of rapamycin kinase)-induced inhibition of ULK1 (Ser757), along with AKT/protein kinase B site-specific suppression of FOXO1 (Ser256) and FOXO3 (Ser253). In addition to lower mitophagy and autophagy flux in skeletal muscle, carm1 mKO led to increased mitochondrial PRKN/parkin accumulation, which suggests that CARM1 is required for basal mitochondrial turnover and autophagic clearance. carm1 deletion also elicited PPARGC1A (PPARG coactivator 1 alpha) activity and a slower, more oxidative muscle phenotype. As such, these carm1 mKO-evoked adaptations disrupted mitophagy and autophagy induction during food deprivation and collectively served to mitigate fasting-induced muscle atrophy. Furthermore, at the threshold of muscle atrophy during food deprivation experiments in humans, skeletal muscle CARM1 activity decreased similarly to our observations in mice, and was accompanied by site-specific activation of ULK1 (Ser757), highlighting the translational impact of the methyltransferase in human skeletal muscle. Taken together, our results indicate that CARM1 governs mitophagic, autophagic, and atrophic processes fundamental to the maintenance and remodeling of muscle mass. Targeting the enzyme may provide new therapeutic approaches for mitigating skeletal muscle atrophy. Abbreviation: ADMA: asymmetric dimethylarginine; AKT/protein kinase B: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ATG: autophagy related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; CARM1: coactivator associated arginine methyltransferase 1; Col: colchicine; CSA: cross-sectional area; CTNS: cystinosin, lysosomal cystine transporter; EDL: extensor digitorum longus; FBXO32/MAFbx: F-box protein 32; FOXO: forkhead box O; GAST: gastrocnemius; H2O2: hydrogen peroxide; IMF: intermyofibrillar; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; mKO: skeletal muscle-specific knockout; MMA: monomethylarginine; MTOR: mechanistic target of rapamycin kinase; MYH: myosin heavy chain; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; OXPHOS: oxidative phosphorylation; PABPC1/PABP1: poly(A) binding protein cytoplasmic 1; PPARGC1A/PGC-1α: PPARG coactivator 1 alpha; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PRMT: protein arginine methyltransferase; Sal: saline; SDMA: symmetric dimethylarginine; SIRT1: sirtuin 1; SKP2: S-phase kinase associated protein 2; SMARCC1/BAF155: SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily c member 1; SOL: soleus; SQSTM1/p62: sequestosome 1; SS: subsarcolemmal; TA: tibialis anterior; TFAM: transcription factor A, mitochondrial; TFEB: transcription factor EB; TOMM20: translocase of outer mitochondrial membrane 20; TRIM63/MuRF1: tripartite motif containing 63; ULK1: unc-51 like autophagy activating kinase 1; VPS11: VPS11 core subunit of CORVET and HOPS complexes; WT: wild-type.

Abstract A115: mTOR targeting in STK11 deficient Non-Small Cell Lung Cancer (NSCLC): final results, pre-clinical rationale and biomarker analysis of a phase II trial of the mTORC1/2 inhibitor vistusertib in STK11 deficient lung adenocarcinoma (NLMT B2)

Abstract Background: STK11 loss of function is common in lung adenocarcinoma (LUAD). STK11 deficient LUAD demonstrates mTOR activation, increased HIF-1a expression and GLUT1 up-regulation, resulting in increased glucose metabolism. STK11 loss often co-occurs with KRAS mutation. KRAS and STK11 co-mutations synergise to drive metabolic re-programming and heightened glucose dependency, reversible with mTORC1/mTORC2 inhibition. mTORC2 is a central node driving enhanced glycolysis independent of Raptor and HIF-1α. mTORC1/2 inhibition suppresses pS6K, p4EBP1, HIF1a and GLUT1 in STK11/KRAS double mutant NSCLC cells and suppresses growth in vivo. Vistusertib (AZD2014) is a selective mTORC1/2 inhibitor. We report the results of outcomes of LUAD patients with STK11 loss+/-KRAS mutation treated with vistusertib, the first study to investigate the impact of mTOR inhibition in STK11 deficient LUAD. Results: 19 patients (17 per-protocol) had STK11 loss without concomitant KRAS mutation (cohort B2S). 30 patients had STK11 loss with concomitant KRAS mutation (26 per-protocol) (cohort B2D). The confirmed objective response rate (95% credible interval) was 3.8% (0.1 – 18.5) for B2S, 9.8% (2.4 – 24.3) for B2D. Durable clinical benefit ((DCB), defined as no progression at 4th 6 weekly on treatment CT assessment) rate was 14.6% (3.6 – 34.7) for B2S, 24.4% (11.1 – 42.3) for B2D. Tumour growth inhibition (median % change from baseline (range)) in those with DCB was -18.5% (-48.0, 3.0) and in those with progression &amp;lt;3 months/death +10.5% (-6.0, 42.0). Median PFS was 2.29 months (1.47 – 3.85) for B2S, 2.77 months (1.91 – 4.22) for B2D. Median OS was 9.84 months (6.23 – 16.85) for B2S, 6.08 months (4.20 – 9.26) for B2D. Clinically achievable concentrations of vistusertib achieve dose-dependent inhibition of STK11 deficient LUAD cell line viability in vitro. In contrast, IC50 for everolimus (mTORC1-only inhibition) exceeds pharmacologically achievable doses in all tested STK11 deficient cell lines. Rebound FOXO and Akt phosphorylation is observed after 48 hours of vistusertib treatment, with parallel activation of ERK. Longitudinal ctDNA from B2S/B2D were analysed using the Illumina TSO500 ctDNA platform. Using conventional p&amp;lt;0.05 for significance, 2 genes were enriched at progression versus baseline: SMARCA4 (3/36 pre, 11/27 post, p=0.005) and FOXP1 (0/36 pre, 4/27 post, p=0.029). TRAF2 mutations were non-significantly enriched post-treatment (1/36 pre, 5/27 post, P = 0.076). Conclusions: Despite strong rationale for mTORC1/2 inhibition in STK11 deficient LUAD, activity of vistusertib is modest. Pre-clinical work suggests that STK11 mutant LUAD cell lines are not exempt from relief of feedback inhibition during prolonged mTORC1/2 inhibition, driving Akt and FOXO phosphorylation. Further, longitudinal ctDNA analysis suggests that vistusertib treatment may drive selection for SMARCA4 mutations, which appears to be a common mechanism of resistance to targeted therapies. HLR and PF contributed equally. LB and GM contributed equally. Citation Format: Helen L Robbins, Peter Fletcher, Joshua Savage, Manita Mehmi, Yvonne Summers, Alastair Greystoke, Noelle O’Rourke, Sanjay Popat, Pooja Jain, James Spicer, Judith Cave, Paul Shaw, David Gilligan, Danielle Power, Dean Fennel, Andrew D Beggs, Lucinda Billingham, Gary Middleton. mTOR targeting in STK11 deficient Non-Small Cell Lung Cancer (NSCLC): final results, pre-clinical rationale and biomarker analysis of a phase II trial of the mTORC1/2 inhibitor vistusertib in STK11 deficient lung adenocarcinoma (NLMT B2) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A115.

Gandouling alleviates nerve injury through PI3K/Akt/FoxO1 and Sirt1/FoxO1 signaling pathway to inhibit autophagy in the rats model of Wilson's disease.

Previous studies have shown that Gandouling (GDL) may alleviate the nerve damage caused by Wilson's disease (WD) by inhibiting the autophagy of nerve cell mitochondria. However, its mechanisms are still unclear. Revealing the therapeutic mechanism of GDL is beneficial for its clinical application and provides theoretical support for the development of new formulations for treating WD. This time we found that the oxidative stress level in the body of the copper-overloaded WD rates increased, neurons in the hippocampus were damaged, and autophagy occurred. GDL reversed these situations and significantly improved the learning, memory, and spatial cognitive abilities of the high-copper-loaded WD rates. After GDL intervention, the expression of phosphatidylinositol-3 kinase (PI3K), phosphorylated serine-threonine protein kinase (AKT), and phosphorylated forkhead box protein O1 (FoxO1) significantly increased, whereas FoxO1 in the nucleus decreased and phosphorylated FoxO1 in the cytoplasm also significantly raised. In addition, the expression of Sirt1 significantly declined, and Ac-FoxO1 in the nucleus also significantly increased. These data indicated that GDL may promote the phosphorylation of FoxO1 and promote its nucleation by activating the PI3K/AKT/FoxO1 signaling pathway and inhibit Ac-FoxO1 hydrolysis in the nucleus through the Sirt1/FoxO1 signaling pathway to suppress the transcriptional activity of FoxO1. Furthermore, it inhibited the expression of autophagy genes Atg12 and Gabarapl1. In summary, our work provides new insights into the potential mechanisms of GDL repairing WD neuronal damage through autophagy pathways.

Acne Transcriptomics: Fundamentals of Acne Pathogenesis and Isotretinoin Treatment.

This review on acne transcriptomics allows for deeper insights into the pathogenesis of acne and isotretinoin's mode of action. Puberty-induced insulin-like growth factor 1 (IGF-1), insulin and androgen signaling activate the kinase AKT and mechanistic target of rapamycin complex 1 (mTORC1). A Western diet (hyperglycemic carbohydrates and milk/dairy products) also co-stimulates AKT/mTORC1 signaling. The AKT-mediated phosphorylation of nuclear FoxO1 and FoxO3 results in their extrusion into the cytoplasm, a critical switch which enhances the transactivation of lipogenic and proinflammatory transcription factors, including androgen receptor (AR), sterol regulatory element-binding transcription factor 1 (SREBF1), peroxisome proliferator-activated receptor γ (PPARγ) and signal transducer and activator of transcription 3 (STAT3), but reduces the FoxO1-dependent expression of GATA binding protein 6 (GATA6), the key transcription factor for infundibular keratinocyte homeostasis. The AKT-mediated phosphorylation of the p53-binding protein MDM2 promotes the degradation of p53. In contrast, isotretinoin enhances the expression of p53, FoxO1 and FoxO3 in the sebaceous glands of acne patients. The overexpression of these proapoptotic transcription factors explains isotretinoin's desirable sebum-suppressive effect via the induction of sebocyte apoptosis and the depletion of BLIMP1(+) sebocyte progenitor cells; it also explains its adverse effects, including teratogenicity (neural crest cell apoptosis), a reduced ovarian reserve (granulosa cell apoptosis), the risk of depression (the apoptosis of hypothalamic neurons), VLDL hyperlipidemia, intracranial hypertension and dry skin.

Importance of smooth muscle p110alpha/AKT/FOXO1 signaling for the development of abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs) and extracellular matrix (ECM) degradation. The catalytic PI 3-kinase isoform p110α signals downstream of growth factor receptors. Mice harboring an SMC-specific p110α deficiency (SM-p110α-/-) are characterized by impaired SMC proliferation and survival. Aim We hypothesized that p110α deficiency in SMCs exacerbates the development and progression of AAA as it promotes SMC loss and thus weakens the aortic wall in healthy and diseased animals. Therefore, we investigated how p110α deficiency in SMCs affects AAA formation, vascular integrity as well as downstream signaling in SMCs. Methods and results Medial thickness of the abdominal aorta in SM-p110α-/- mice was significantly decreased compared to wild-type (WT) littermate controls (29.0±3.1µm vs. 42.5±4.1µm). Morphological characterization of the aortic wall by transmission electron microscopy revealed impaired elastic fibers, detached SMCs and apoptotic cells in SM-p110α-/- mice. SMC differentiation markers including calponin and myosin heavy chain were downregulated in the aortic wall of SM-p110α-/- mice. Western blots demonstrated that p110α deficiency impaired the expression of elastin. Consequently, elastin fibers were characterized by reduced fiber width and length. In addition, growth factor stimulated proliferation of p110α-/- SMCs was significantly impaired compared to WT controls. AAA development in SM-p110α-/- mice and WT littermates were studied using the porcine pancreatic elastase (PPE) model. PPE was infused into the infrarenal aorta. Ultrasound examination of the aorta revealed an increased aortic diameter in all PPE-treated mice. However, the increase in aortic diameter was significantly elevated (p&amp;lt;0.05) in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10). Furthermore, PPE treated SM-p110-/- mice were characterized by increased ECM degradation, decreased vascular remodelling, and less proliferating cells. Mechanistically, growth factor-dependent phosphorylation and inactivation of crucial phenotypic modulators of SMCs - FOXO-1, -3 and -4 - were shown to be significantly decreased in p110α-/- SMCs compared to WT cells. Specific inhibition of FOXO1 rescued the impaired proliferation of p110α-/- SMCs and enhanced the expression of elastin and calponin. Analysis of FOXO expression in humans revealed that FOXO1 was significantly upregulated in AAA patients compared to healthy individuals. Conclusion p110α deficiency in SMCs promotes the development and progression of AAA, as p110α controls crucial SMC functions which are important for structural processes in the aortic wall. Thereby, SMC proliferation is controlled by p110α dependent phosphorylation and inactivation of FOXO1. Since FOXO1 is upregulated in AAA patients, the p110α/AKT/FOXO1 signaling cascade may provide a key target for modulating aortic wall stability in AAA.

Abstract 18545: GSK-3β Prevents Lipotoxicity in Obesity Cardiomyopathy Through Upregulation of ATGL

Introduction: Obesity induces accumulation of lipids in cardiomyocytes, leading to cardiac dysfunction, termed lipotoxic cardiomyopathy. We have shown previously that GSK-3α promotes fatty acid uptake in the heart. In contrast, GSK-3β may mediate lipid catabolism. In this study, we aimed to elucidate the role of GSK-3β in lipolysis, primarily focusing on the post-translational regulation of FoxO1 and its association with ATGL, which is the first lipase to hydrolyze lipids. Methods: Wild-type (WT) and homozygous cardiac-specific GSK-3β knock-out (GSK-3β-cKO) mice were fed a normal diet (ND) or a high fat diet (HFD) for 3 months. Results: HFD consumption in WT mice activated GSK-3β in the heart, as indicated by decreases in Ser9-phosphorylated GSK-3β/total GSK-3β (0.5-fold, p&lt;0.05 compared to ND consumption), and upregulated ATGL (2.4-fold, p&lt;0.05) in the heart. Echocardiographic analyses showed preserved systolic and diastolic function in WT mice at this time point (LVEF: 56.3 ± 1.5 %, E/A: 2.06 ± 0.22, n = 13). However, ATGL was not increased (1.6-fold) and both systolic and diastolic dysfunction were observed (LVEF: 45.8 ± 1.5 %, p&lt;0.01 compared to WT, E/A: 1.43 ± 0.20, p&lt;0.05, n =12) in GSK-3β-cKO mice fed a HFD. GSK-3β overexpression in neonatal rat ventricular cardiomyocytes increased ATGL in a dose-dependent manner (p&lt;0.05). FoxO1 knock-down on this background inhibited upregulation of ATGL, suggesting that GSK-3β-induced upregulation of ATGL is mediated through a FoxO1-dependent mechanism. Immunoprecipitation showed that GSK-3β interacts with FoxO1, while mass spectrometry analyses revealed that GSK-3β phosphorylates FoxO1 at 8 Serine/Threonine residues. A FoxO1 mutant in which Serine/Threonine residues at the GSK-3β phosphorylation sites are mutated to Alanine was excluded from the nucleus, and its overexpression prevented upregulation of ATGL in the presence of GSK-3β overexpression. Conclusions: GSK-3β is activated in the presence of HFD consumption in the heart, which may, in turn, prevent lipotoxic cardiomyopathy through phosphorylation of FoxO1 and upregulation of ATGL.

The liver-derived exosomes stimulate insulin gene expression in pancreatic beta cells under condition of insulin resistance.

An insufficient functional beta cell mass is a core pathological hallmark of type 2 diabetes (T2D). Despite the availability of several effective pharmaceuticals for diabetes management, there is an urgent need for novel medications to protect pancreatic beta cells under diabetic conditions. Integrative organ cross-communication controls the energy balance and glucose homeostasis. The liver and pancreatic islets have dynamic cross-communications where the liver can trigger a compensatory beta cell mass expansion and enhanced hormonal secretion in insulin-resistant conditions. However, the indispensable element(s) that foster beta cell proliferation and insulin secretion have yet to be completely identified. Exosomes are important extracellular vehicles (EVs) released by most cell types that transfer biological signal(s), including metabolic messengers such as miRNA and peptides, between cells and organs. We investigated whether beta cells can take up liver-derived exosomes and examined their impact on beta cell functional genes and insulin expression. Exosomes isolated from human liver HepG2 cells were characterized using various methods, including Transmission Electron Microscopy (TEM), dynamic light scattering (DLS), and Western blot analysis of exosomal markers. Exosome labeling and cell uptake were assessed using CM-Dil dye. The effect of liver cell-derived exosomes on Min6 beta cells was determined through gene expression analyses of beta cell markers and insulin using qPCR, as well as Akt signaling using Western blotting. Treatment of Min6 beta cells with exosomes isolated from human liver HepG2 cells treated with insulin receptor antagonist S961 significantly increased the expression of beta cell markers Pdx1, NeuroD1, and Ins1 compared to the exosomes isolated from untreated cells. In line with this, the activity of AKT kinase, an integral component of the insulin receptor pathway, is elevated in pancreatic beta cells, as represented by an increase in AKT's downstream substrate, FoxO1 phosphorylation. This study suggests that liver-derived exosomes may carry a specific molecular cargo that can affect insulin expression in pancreatic beta cells, ultimately affecting glucose homeostasis.

Diosmetin induces apoptosis and protective autophagy in human gastric cancer HGC-27 cells via the PI3K/Akt/FoxO1 and MAPK/JNK pathways.

Gastric cancer represents a significant global health concern, necessitating the exploration of novel therapeutic options. Diosmetin, a natural flavonoid derived from citrus and vegetables, has demonstrated promising anti-tumor activity against various tumor cells. However, the potential anticancer effect of diosmetin in gastric cancer and its underlying mechanism have yet to be elucidated. In this study, we aimed to investigate the impact of diosmetin on cell proliferation, migration, cell cycle progression and apoptosis in human gastric cancer HGC-27 cells. Our findings revealed that diosmetin effectively suppressed cell proliferation, induced G2/M phase cell cycle arrest, and triggered cell apoptosis. Mechanistically, diosmetin downregulated the expression of antiapoptotic proteins Bcl-2 and Bcl-xL, while upregulated the level of proapoptotic proteins such as Bax, cleaved PARP and cleaved caspase-3. Additionally, diosmetin inhibited Akt and FoxO1 phosphorylation, while activated the MAPK signaling pathway. Notably, pretreatment of IGF-1, an Akt activator, attenuated the diosmetin-induced apoptosis. Furthermore, pretreatment with SP600125, a JNK inhibitor, significantly reduced the protein level of LC3B, while promoted the expression of cleaved caspase-3 and cleaved PARP. Collectively, our results suggest that diosmetin holds promise as an effective therapeutic agent against gastric cancer by inducing apoptosis through inhibition of the Akt/FoxO1 pathway and promoting protective autophagy via the MAPK/JNK signaling pathway.

SAT038 Dietary Trans-10,cis-12 Conjugated Linoleic Acid Regulates Glucose Levels In Mice With Severe Insulin Resistance: Potential Effects On The Liver Via HNF4α And FOX01

Abstract Disclosure: S.M. Lee: None. J.T. Muratalla: None. C.W. Liew: None. J. Cordoba-Chacon: None. Plasma trans-10 cis-12-conjugated linoleic acid CLA (t10c12-CLA) is inversely associated with increased risk of diabetes in humans. In mice, it is well-known that t10c12-CLA induces whole-body insulin resistance due to severe lipodystrophy (loss of whole-body adipose tissue). However, t10c12-CLA-fed mice show normal glucose levels and, t10c12-CLA improved glucose clearance in models with preexisting insulin resistance: Zucker fatty/diabetic rats, obese and insulin resistant Ldlr-/- mice, and diet-induced obese and insulin resistant male mice. In this study, we assessed whether supplementation of t10c12-CLA in a low-fat diet reduces glucose levels in adipocyte-specific insulin receptor knockout (IRFKO) mice, which is a mouse model of congenital lipodystrophy. In addition to lipodystrophy, IRFKO mice show liver steatosis and increased blood glucose levels that are consequences of whole-body insulin resistance and increased hepatic gluconeogenesis. Briefly, 5-6-week-old male and female control and IRFKO mice were fed with a low-fat diet supplemented with 0.8% t10c12-CLA for 5 weeks. In IRFKO mice, dietary t10c12-CLA did not reduce adipose tissue mass. However, dietary t10c12-CLA increased liver steatosis and plasma insulin levels. Surprisingly, dietary t10c12-CLA reduced glucose levels in both male and female IRFKO mice. Also, dietary t10c12-CLA reduced food intake and polydipsia, which were increased in IRFKO mice due to lipodystrophy and hyperglycemia, respectively. A transcriptomic analysis (RNA-seq) of livers of control and IRFKO male mice fed with a t10c12-CLA diet showed that t10c12-CLA reduced the expression of the key gluconeogenic and mitochondrial fatty oxidation genes. Previous studies have suggested that t10c12-CLA reduces the expression of Forkhead box 01 (Fox01) and Hepatocyte nuclear factor 4 alpha (HNF4α). Of note, the livers of t10c12-CLA-fed control and IRFKO male mice showed a downregulation of positive targets of FoxO1 and HNF4α. In addition, livers of t10c12-CLA-fed mice showed reduced total FOXO1 protein levels, and increased phosphorylation of FOXO1 in t10c12-CLA-fed IRFKO mice. These findings suggest that t10c12-CLA could be directly acting on the liver to alter HNF4α and FOXO1-dependent regulation of hepatic fatty acid oxidation and glucose production. These actions may explain why t10c12-CLA-fed mice do not show increased glucose levels and is potentially linked to the inverse association between t10c12-CLA levels and diabetes risk. Presentation: Saturday, June 17, 2023

TRIM11 attenuates Treg cell differentiation by p62-selective autophagic degradation of AIM2

Ubiquitination is an important protein modification that regulates diverse biological processes, including CD4+ Tcell differentiation and functions. However, the function of most E3 ubiquitin ligases in CD4+ Tcell differentiation and CD4+ Tcell-mediated pathological diseases remains unclear. In this study, we find that tripartite motif-containing motif 11 (TRIM11) specifically negatively regulates regulatory T (Treg) cell differentiation in CD4+ Tcells and promotes autoimmune disease development in an AIM2-dependent manner. Mechanistically, TRIM11 interacts with absent in melanoma 2 (AIM2) and promotes the selective autophagic degradation of AIM2 by inducing AIM2 ubiquitination and binding to p62 in CD4+ Tcells. AIM2 attenuates AKT and FOXO1 phosphorylation, MYC signaling, and glycolysis, thereby promoting the stability of Treg cells during experimental autoimmune encephalomyelitis (EAE). Our findings suggest that TRIM11 serves as a potential target for immunotherapeutic intervention for dysregulated immune responses that lead to autoimmunity and cancers.

Noncanonical CDK4 signaling rescues diabetes in a mouse model by promoting β cell differentiation.

Expanding β cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain β cell number. β cell failure in the IRS2-deletion mouse type 2 diabetes model is, in part, due to loss of CDK4 regulator cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of β cell mass in IRS2-deficient mice. Surprisingly, not only β cell mass but also β cell dedifferentiation was effectively rescued, despite no improvement in whole body insulin sensitivity. Ex vivo studies in primary islet cells revealed a mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical β cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation and reduced FOXO1 abundance. Taken together, these results demonstrate a differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that β cell mass can be expanded without compromising function.

Klotho increases antioxidant defenses in astrocytes and ubiquitin–proteasome activity in neurons

Klotho is an antiaging protein, and its levels decline with age and chronic stress. The exogenous administration of Klotho can enhance cognitive performance in mice and negatively modulate the Insulin/IGF1/PI3K/AKT pathway in terms of metabolism. In humans, insulin sensitivity is a hallmark of healthy longevity. Therefore, this study aimed to determine if exogenous Klotho, when added to neuronal and astrocytic cell cultures, could reduce the phosphorylation levels of certain insulin signaling effectors and enhance antioxidant strategies in these cells. Primary cell cultures of cortical astrocytes and neurons from mice were exposed to 1 nM Klotho for 24 h, with or without glucose. Klotho decreased pAKT and mTOR levels. However, in astrocytes, Klotho increased FOXO-3a activity and catalase levels, shielding them from intermediate oxidative stress. In neurons, Klotho did not alter FOXO-3 phosphorylation levels but increased proteasome activity, maintaining lower levels of PFKFB3. This study offers new insights into the roles of Klotho in regulating energy metabolism and the redox state in the brain.

The roles of stress-induced premature senescence and Akt/FoxO1 signaling in periapical lesions.

There is little knowledge about oxidative stress-induced senescence involvement in apical periodontitis. Here, we explored its molecular mechanism in periapical lesions. Ten cases of radicular cysts and five cases of periapical granulomas were randomly selected. Immunohistochemical analysis was performed to detect the expression and correlation between Senescence-associated factor polymerase I and transcript release factor (PTRF) and Akt/FoxO1 signaling. Human periodontal ligament cells (hPDLCs) pretreated with LY294002 were exposed to H2O2-induced oxidative stress conditions and then cell proliferation, senescence, apoptosis, and associated signaling were evaluated by EdU labeling, β-galactosidase assay, RT-qPCR, and western blot analysis, respectively. Polymerase I and transcript release factor and Akt/FoxO1 signaling were more frequently expressed in the radicular cyst than in periapical granulomas. Notably, cells in radicular cysts showed Akt activation, FoxO1 phosphorylation, and cytoplasmic translocation. In vitro, prominent H2O2-induced senescence was observed in hPDLCs. LY294002, a PI3K inhibitor, attenuated the expression levels of senescence (Klotho, P16INK4), apoptosis (Bad, Fas), phosphorylated Akt, and phosphorylated FoxO1; however, did not affect cell proliferation. Our data indicated that senescence is present in clinical periapical lesions, and Akt/FoxO1 signaling is involved in the H2O2-induced cellular senescence, which could serve as a potential therapeutic target.

Protective Effect and Mechanism of Autophagy in Endothelial Cell Injury Induced by Hyperoxia.

Bronchopulmonary dysplasia is a chronic lung disease in premature infants with alveolar simplification and pulmonary vascular development disorder as the main pathological feature and hyperoxia as the main etiology. Autophagy is a highly conserved cytological behavior of self-degrading cellular components and is accompanied by oxidative stress. Studies have reported that autophagy is regulated by FOXO1 posttranslational modification. However, whether autophagy can be involved in the regulation of endothelial cell injury induced by hyperoxia and its mechanism are still unclear. We have activated and inhibited autophagy in human umbilical vein endothelial cells under hyperoxia and verified the role of autophagy in endothelial cell-related functions from both positive and negative aspects. Our research showed that the expression level of autophagy-related proteins decreased, accompanied by decreased cell migration ability and tube formation ability and increased cell reactive oxygen species level and cell permeability under hyperoxia conditions. Using an autophagy agonist alleviated hyperoxia-induced changes and played a protective role. However, inhibition of autophagy aggravated the cell damage induced by hyperoxia. Moreover, the decrease in autophagy proteins was accompanied by the upregulation of FOXO1 phosphorylation and acetylation. We concluded that autophagy was a protective mechanism against endothelial cell injury caused by hyperoxia. Autophagy might participate in this process by coregulating posttranslational modifications of FOXO1. · Hyperoxia induces vascular endothelial cell injury.. · Autophagy may has a protective role under hyperoxia conditions.. · FOXO1 posttranslational modification may be involved in the regulation of autophagy..