Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

mTORC1 promotes cell growth via m6A-dependent mRNA degradation.

Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.

Multisite Phosphorylation of S6K1 Directs a Kinase Phospho-code that Determines Substrate Selection.

Multisite phosphorylation of kinases can induce on-off or graded regulation of catalytic activity; however, its influence on substrate specificity remains unclear. Here, we show that multisite phosphorylation of ribosomal protein S6 kinase 1 (S6K1) alters target selection. Agonist-inducible phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) by S6K1 in monocytes and adipocytes requires not only canonical phosphorylation at Thr389 by mTORC1 but also phosphorylation at Ser424 and Ser429 in the C terminus by cyclin-dependent kinase 5 (Cdk5). S6K1 phosphorylation at these additional sites induces a conformational switch and is essential for high-affinity binding and phosphorylation of EPRS, but not canonical S6K1 targets, e.g., ribosomal protein S6. Unbiased proteomic analysis identified additional targets phosphorylated by multisite phosphorylated S6K1 in insulin-stimulated adipocytes-namely, coenzyme A synthase, lipocalin 2, and cortactin. Thus, embedded within S6K1 is a target-selective kinase phospho-code that integrates signals from mTORC1 and Cdk5 to direct an insulin-stimulated, post-translational metabolon determining adipocyte lipid metabolism.

Selective Inhibition of mTORC1 Signaling Supports the Development and Maintenance of Pluripotency.

Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency.

S6K1 deficiency in tumor stroma impairs lung metastasis of melanoma in mice.

Accumulating data suggest that ribosomal protein S6 kinase 1 (S6K1), an effector in the mammalian target of rapamycin (mTOR) pathway, plays pleiotropic roles in tumor progression. However, to date, while the tumorigenic function of S6K1 in tumor cells has been well elucidated, its role in the tumor stroma remains poorly understood. We recently showed that S6K1 mediates vascular endothelial growth factor A (VEGF-A) production in macrophages, thereby supporting tumor angiogenesis and growth. As macrophage-derived VEGF-A is crucial for both tumor cell intravasation and extravasation across the vascular endothelium, our previous findings suggest that stromal S6K1 signaling is required for tumor metastatic spread. Therefore, we aimed to determine the impact of host S6K1 depletion on tumor metastasis using a murine model of pulmonary metastasis (S6k1-/- mice implanted with B16F10 melanoma). The ablation of S6K1 in the host microenvironment significantly reduced the metastasized B16F10 melanoma cells on the lung surface in both spontaneous and intravenous lung metastasis mouse models without affecting the incidence of metastasis to distant lymph nodes. In addition, stromal S6K1 loss decreased the number of tumor cells circulating in the peripheral blood of mice bearing B16F10 xenografts without affecting the vascular leakage induced by VEGF-A in vivo. These observations demonstrate that S6K1 signaling in host cells other than endothelial cells is required to modulate the host microenvironment to facilitate the metastatic spread of tumors via blood circulation, thus revealing its novel role in the tumor stroma during tumor progression.

Curcumin inhibits propofol-induced autophagy of MN9D cells via Akt/mTOR/p70S6K signaling pathway.

The rapid rise in propofol dependency and abuse has highlighted limited resources for addressing substance abuse-related cognitive impairment, prompting the development of novel therapies. Dysregulated autophagy flow accelerates neuronal cell death, and interventions countering this dysregulation offer an appealing strategy for neuronal protection. Curcumin, a potent natural polyphenol derived from turmeric rhizomes, is renowned for its robust antineurotoxic properties and enhanced cognitive function. Utilizing CCK-8 and Ki67 fluorescent staining, our study revealed that curcumin treatment increased cell viability and proliferative potential in MN9D cells exposed to propofol-induced neurotoxicity. Furthermore, enzyme-linked immunosorbent assay and western blot analysis demonstrated the partial restoration of dopamine synthesis, secretion levels, and TH expression in damaged MN9D cells treated with curcumin. Scanning electrode microscope images displayed reduced autolysosomes and phagosomes in curcumin-treated cells compared to the propofol group. Immunoblotting revealed that curcumin mitigated the degradation of LC3I to LC3II and p62 induced by propofol stimulation, with green fluorescence expression of LC3 postcurcumin treatment resembling that following autophagy inhibitor HCQ treatment, indicating that modulating autophagy flow can alleviate propofol's toxic effects. Moreover, curcumin treatment upregulated the Akt/mTOR/p70S6K signaling pathway, suggesting that curcumin potentially curtails autophagy dysregulation in nerve cells by activating Akt/mTOR/p70S6K. In conclusion, our findings suggest that curcumin can ameliorate propofol abuse-induced neurotoxicity, partially through autophagy regulation and Akt/mTOR/p70S6K signaling activation.

The involvement of Akt, mTOR, and S6K in the in vivo effect of IGF-1 on the regulation of rat cardiac Na+/K+-ATPase.

BACKGROUND: We previously demonstrated that insulin-like growth factor-1 (IGF-1) regulates sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) in vascular smooth muscle cells (VSMC) via phosphatidylinositol-3 kinase (PI3K). Taking into account that others' work show that IGF-1 activates the PI3K/protein kinase B (Akt) signaling pathway in many different cells, we here further questioned if the Akt/mammalian target of rapamycin (mTOR)/ribosomal protein p70 S6 kinase (S6K) pathway stimulates Na+/K+-ATPase, an essential protein for maintaining normal heart function. METHODS AND RESULTS: There were 14 adult male Wistar rats, half of whom received bolus injections of IGF-1 (50 µg/kg) for 24 h. We evaluated cardiac Na+/K+-ATPase expression, activity, and serum IGF-1 levels. Additionally, we examined the phosphorylated forms of the following proteins: insulin receptor substrate (IRS), phosphoinositide-dependent kinase-1 (PDK-1), Akt, mTOR, S6K, and α subunit of Na+/K+-ATPase. Additionally, the mRNA expression of the Na+/K+-ATPase α1 subunit was evaluated. Treatment with IGF-1 increases levels of serum IGF-1 and stimulates Na+/K+-ATPase activity, phosphorylation of α subunit of Na+/K+-ATPase on Ser23, and protein expression of α2 subunit. Furthermore, IGF-1 treatment increased phosphorylation of IRS-1 on Tyr1222, Akt on Ser473, PDK-1 on Ser241, mTOR on Ser2481 and Ser2448, and S6K on Thr421/Ser424. The concentration of IGF-1 in serum positively correlates with Na+/K+-ATPase activity and the phosphorylated form of mTOR (Ser2448), while Na+/K+-ATPase activity positively correlates with the phosphorylated form of IRS-1 (Tyr1222) and mTOR (Ser2448). CONCLUSION: These results indicate that the Akt/mTOR/S6K signalling pathway may be involved in the IGF-1 regulating cardiac Na+/K+-ATPase expression and activity.

Inhibition Effects and Mechanisms of Marine Compound Mycophenolic Acid Methyl Ester against Influenza A Virus.

Influenza A virus (IAV) can cause infection and illness in a wide range of animals, including humans, poultry, and swine, and cause annual epidemics, resulting in thousands of deaths and millions of hospitalizations all over the world. Thus, there is an urgent need to develop novel anti-IAV drugs with high efficiency and low toxicity. In this study, the anti-IAV activity of a marine-derived compound mycophenolic acid methyl ester (MAE) was intensively investigated both in vitro and in vivo. The results showed that MAE inhibited the replication of different influenza A virus strains in vitro with low cytotoxicity. MAE can mainly block some steps of IAV infection post adsorption. MAE may also inhibit viral replication through activating the cellular Akt-mTOR-S6K pathway. Importantly, oral treatment of MAE can significantly ameliorate pneumonia symptoms and reduce pulmonary viral titers, as well as improving the survival rate of mice, and this was superior to the effect of oseltamivir. In summary, the marine compound MAE possesses anti-IAV effects both in vitro and in vivo, which merits further studies for its development into a novel anti-IAV drug in the future.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

Investigating the Regulation of Ribosomal Protein S6 Kinase 1 by CoAlation.

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.

Triple targeting of RSK, AKT, and S6K as pivotal downstream effectors of PDPK1 by TAS0612 in B-cell lymphomas.

B-cell lymphomas (BCLs) are the most common disease entity among hematological malignancies and have various genetically and molecularly distinct subtypes. In this study, we revealed that the blockade of phosphoinositide-dependent kinase-1 (PDPK1), the master kinase of AGC kinases, induces a growth inhibition via cell cycle arrest and the induction of apoptosis in all eight BCL-derived cell lines examined, including those from activated B-cell-like diffuse large B-cell lymphoma (DLBCL), double expressor DLBCL, Burkitt lymphoma, and follicular lymphoma. We also demonstrated that, in these cell lines, RSK2, AKT, and S6K, but not PLK1, SGK, or PKC, are the major downstream therapeutic target molecules of PDPK1 and that RSK2 plays a central role and AKT and S6K play subsidiary functional roles as the downstream effectors of PDPK1 in cell survival and proliferation. Following these results, we confirmed the antilymphoma efficacy of TAS0612, a triple inhibitor for total RSK, including RSK2, AKT, and S6K, not only in these cell lines, regardless of disease subtypes, but also in all 25 patient-derived B lymphoma cells of various disease subtypes. At the molecular level, TAS0612 caused significant downregulation of MYC and mTOR target genes while inducing the tumor suppressor TP53INP1 protein in these cell lines. These results prove that the simultaneous blockade of RSK2, AKT, and S6K, which are the pivotal downstream substrates of PDPK1, is a novel therapeutic target for the various disease subtypes of BCLs and line up TAS0612 as an attractive candidate agent for BCLs for future clinical development.

AMPK/mTOR/p70S6K axis prevents apoptosis of Porphyromonas gingivalis-infected gingival epithelial cells via BadSer136 phosphorylation.

Epithelial disruption is the initiation of most infectious disease. Regulation of epithelium apoptosis may play a key role in balance the survival competition between resident bacteria and host cells. The role of the mTOR/p70S6K pathway in preventing apoptosis of human gingival epithelial cells (hGECs) infected with Porphyromonas gingivalis (Pg) was investigated in order to further understand the survival strategy of the epithelial cells in during Pg infecting. hGECs was challenged with Pg for 4, 12, and 24 h. Additionally, hGECs was pretreated with LY294002 (PI3K signaling inhibitor) or Compound C (AMPK inhibitor) for 12 h and exposed them to Pg for 24 h. Subsequently, apoptosis was detected using flow cytometry, and expression and activity of Bcl-2, Bad, Bax, PI3K, AKT, AMPK, mTOR, and p70S6K proteins were analyzed using western blotting. Pg-infecting did not increase apoptosis of hGECs; but the expression ratio of Bad to Bcl-2 was increased after infecting. In contrast, BadSer136 phosphorylation was promoted, accompanied by a significant reduction of mTOR/p70S6K and PI3K/AKT signaling, along with the upregulation of AMPKThr172 signaling. Morrover, the PI3K inhibitor LY294002 promoted Pg-mediated reduction of mTOR/p70S6K expression, and the increase of AMPK signaling and BadSer136 phosphorylation rate, eventually decreasing apoptosis. While Compound C inhibited Pg-mediated activation of AMPK and downregulation of mTOR/p70S6K signaling, significantly reduced the BadSer136 phosphorylation rate, thereby increasing apoptosis. Thus, hGECs prevent apoptosis via an inherent cellular-homeostasis, pro-survival mechanism during Pg infection, the AMPK/mTOR/p70S6K pathway helps prevent apoptosis in hGECs infected with Pg by regulating BadSer136 phosphorylation.

The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). METHODS: Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein-protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. RESULTS: We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. CONCLUSIONS: Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract.

Tricin promoted ATG-7 dependent autophagic degradation of α-synuclein and dopamine release for improving cognitive and motor deficits in Parkinson's disease.

Tricin, a natural nontoxic flavonoid distributed in grasses and euphorbia plants, has been reported to scavenge free radicals, possess anti-inflammatory and antioxidative effects. However, its autophagic effect on Parkinson's disease (PD) has not been elucidated. By adopting cellular and C. elegans models of PD, the autophagic effect of tricin was identified based on the level of autophagy markers (LC3-II and p62). Besides, the pharmacological effects on neurotransmitters (dopamine), inflammatory cytokines (IFN γ, TNFα, MCP-1, IL-10, IL-6 and IL-17A), histology (hematoxylin & eosin and Nissl staining) and behavioural pathology (open-field test, hindlimb clasping, Y-maze, Morris water-maze and nest building test) were also confirmed in the A53T-α-synuclein transgenic PD mouse model. Further experiments demonstrated that tricin induced autophagic flux and lowered the level of α-synuclein through AMPK-p70s6K- and ATG7-dependent mechanism. Compared to the existing clinical PD drugs, tricin mitigated pathogenesis and symptoms of PD with no observable side effects. In summary, tricin is proposed as a potential adjuvant remedy or nutraceutical for the prevention and treatment of PD.

Correlation and influencing factors analysis of colorectal polyps with Helicobacter pylori Infection and p-S6K1 expression.

OBJECTIVE: To investigate the correlation between colorectal polyps (CRP) and Helicobacter pylori (H. pylori) infection, and the correlation between CRP and the expression of phosphorylated ribosomal protein S6 kinase (p-S6K1). Besides, its related influencing factors were determined in the present study. METHODS: A total of 191 subjects who underwent colonoscopy in our hospital from January 2020 to February 2022 were selected for this study. Among them, 141 patients were diagnosed with CRP, and the other 50 subjects were no significant colorectal abnormalities. 141 CRP patients were divided into H. pylori-positive group (n = 89) and H. pylori-negative group (n = 52) according to the results of the H. pylori test. The expression of p-S6K1 in CRP tissue was detected. The relationship between the p-S6K1 expression and the clinicopathological characteristics of CRP patients was analyzed. The logistic analysis of factors influencing the occurrence of CRP was performed. RESULTS: There were significant differences in pathological type, site of disease, the number and size of polyps between the H. pylori negative group and the H. pylori positive group (P < 0.001, P = 0.037, P = 0.042 and P = 0.039). The percentage of the p-S6K1 positive expression in polyp tissues was higher than that in normal tissue and parapolyp tissues (P < 0.001). The p-S6K1 negative group showed significant difference in the number and pathological type of polyps and the presence or absence of a pedicle as compared with the p-S6K1 positive group (P = 0.006, P < 0.001 and P = 0.012). Logistic multifactor analysis showed that BMI, H. pylori infection, smoking history, ApoB, Lp(a) and the p-S6K1 positive expression were all risk factors for the development of CRP (P = 0.025, P = 0.020, P = 0.010, P = 0.005, P = 0.043 and P < 0.001). CONCLUSION: H. pylori infection was closely related to the pathological type, location, and the number and size of CRP. p-S6K1 was highly expressed in CRP, and was positively related to the number, the pathological type and pedicle of polyps. H. pylori infection and the positive p-S6K1 expression were independent risk factors for CRP. By exploring the association between H. pylori infection as well as p-S6K1 and CRP, it is hoped that it will help to formulate a more rigorous colorectal cancer screening program for H. pylori-positive individuals, and at the same time find a new direction for the prevention of CRP and colorectal cancer, and provide some help for future research.

Canonical insulin signaling is not significantly impaired in early stages of depression.

Patients with major depression (MD) are at high-risk for insulin resistance (IR), type-2 diabetes, metabolic syndrome, cardiovascular morbidity and mortality. However, our recent study published in this journal [Eur Arch Psychiatry Clin Neurosci. 2019 Jun;269(4):373-377], found no evidence of IR in acutely-ill drug-naive first-episode MD (FEMD) using the homeostatic model assessment of insulin resistance (HOMA-IR). We concluded, that MD may be related to impaired glucose/insulin homeostasis in the long-term but not in early disease stages. Now, we performed a complementary analysis of the canonical insulin signalling pathway containing the set of control and FEMD samples from the study mentioned above. The first node (pS312-IRS-1, pY-IRS-1) and downstream pathway which affects glucose and lipid homeostasis (phosphorylated proteins: pS473-AKT, pS9-GSK3ß, pS2448-mTOR, pT389-p70S6K; total proteins AKT, GSK3ß, mTOR, p70S6K) were analyzed by electrochemiluminescence (ECL) in neuronal extracellular vesicles (nEVs) enriched for L1 neural cell adhesion molecule (L1CAM) expression. No significant diagnosis-related differences were observed for the pS312-IRS-1 / pYIRS-1 ratio (P = 0.093), but the mean ratio was reduced by ~ 70% in FEMD versus controls. Moreover, omnibus analysis of downstream phosphorylated / total signaling protein ratios and respective post-hoc analyses revealed no significant changes in FEMD patients versus controls (P = 0.734). HAMD-21 scores were not correlated with pS312-IRS-1 / pY-IRS-1 or downstream phosphorylated/total signaling protein ratios. In summary, we did not find evidence for altered neuronal insulin signaling in early disease stages of MD. This is in contrast to schizophrenia, where we and other researchers have seen evidence of IR in first-episode patients.

Coronarin A modulated hepatic glycogen synthesis and gluconeogenesis via inhibiting mTORC1/S6K1 signaling and ameliorated glucose homeostasis of diabetic mice.

Promotion of hepatic glycogen synthesis and inhibition of hepatic glucose production are effective strategies for controlling hyperglycemia in type 2 diabetes mellitus (T2DM), but agents with both properties were limited. Herein we report coronarin A, a natural compound isolated from rhizomes of Hedychium gardnerianum, which simultaneously stimulates glycogen synthesis and suppresses gluconeogenesis in rat primary hepatocytes. We showed that coronarin A (3, 10 µM) dose-dependently stimulated glycogen synthesis accompanied by increased Akt and GSK3ß phosphorylation in rat primary hepatocytes. Pretreatment with Akt inhibitor MK-2206 (2 µM) or PI3K inhibitor LY294002 (10 µM) blocked coronarin A-induced glycogen synthesis. Meanwhile, coronarin A (10 µM) significantly suppressed gluconeogenesis accompanied by increased phosphorylation of MEK, ERK1/2, ß-catenin and increased the gene expression of TCF7L2 in rat primary hepatocytes. Pretreatment with ß-catenin inhibitor IWR-1-endo (10 µM) or ERK inhibitor SCH772984 (1 µM) abolished the coronarin A-suppressed gluconeogenesis. More importantly, we revealed that coronarin A activated PI3K/Akt/GSK3ß and ERK/Wnt/ß-catenin signaling via regulation of a key upstream molecule IRS1. Coronarin A (10, 30 µM) decreased the phosphorylation of mTOR and S6K1, the downstream target of mTORC1, which further inhibited the serine phosphorylation of IRS1, and subsequently increased the tyrosine phosphorylation of IRS1. In type 2 diabetic ob/ob mice, chronic administration of coronarin A significantly reduced the non-fasting and fasting blood glucose levels and improved glucose tolerance, accompanied by the inhibited hepatic mTOR/S6K1 signaling and activated IRS1 along with enhanced PI3K/Akt/GSK3ß and ERK/Wnt/ß-catenin pathways. These results demonstrate the anti-hyperglycemic effect of coronarin A with a novel mechanism by inhibiting mTORC1/S6K1 to increase IRS1 activity, and highlighted coronarin A as a valuable lead compound for the treatment of T2DM.

Glutamine stimulates the S6K/4E-BP branch of insulin signalling pathway to mitigate human poly(Q) disorders in Drosophila disease models.

OBJECTIVE AND METHODS: Since, the S6K/4E-BP sub-pathway can be stimulated by various amino acids; we extended our investigation to examine if oral feeding of amino acids delivers rescue against human poly(Q) toxicity in Drosophila. We utilised Drosophila models of two different poly(Q) disorders to test our hypothesis. Glutamine was fed to the test flies orally mixed in the food. Control and treated flies were then tested for different parameters, such as formation of poly(Q) aggregates and neurodegeneration, to evaluate glutamine's proficiency in mitigating poly(Q) neurotoxicity. RESULTS: Our study, for the first time, reports that glutamine feeding stimulates the growth promoting S6K/4E-BP branch of insulin signalling pathway and restricts pathogenesis of poly(Q) disorders in Drosophila disease models. We noted that glutamine treatment restricts the formation of neurotoxic poly(Q) aggregates and minimises neuronal deaths. Further, glutamine treatment re-establishes the chromatin architecture by improving the histone acetylation which is otherwise compromised in poly(Q) expressing neuronal cells. DISCUSSION: Since, the insulin signalling pathway as well as mechanism of action of glutamine are fairly conserved between human and Drosophila, our finding strongly suggests that glutamine holds immense potential to be developed as an intervention therapy against the incurable human poly(Q) disorders.

B3galt5 deficiency attenuates hepatocellular carcinoma by suppressing mTOR/p70s6k-mediated glycolysis.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with high morbidity and mortality. Beta-1,3-galactosyltransferase 5 (b3galt5) plays crucial roles in protein glycosylation, but its function in HCC remains unclear. Here, we investigated the role and underlying mechanism of b3galt5 in HCC. We found that b3galt5 is highly expressed and associated with a poor prognosis in HCC patients. In vitro studies showed that b3galt5 promoted the proliferation and survival of HCC cells. We also demonstrated that b3galt5 deficiency suppressed hepatocarcinogenesis in DEN/TCPOBOP-induced HCC. Further investigation confirmed that b3galt5 promoted aerobic glycolysis in HCC. Mechanistically, b3galt5 promoted glycolysis by activating the mTOR/p70s6k pathway through O-linked glycosylation modification on mTOR. Moreover, p70s6k inhibition reduced the expression of key glycolytic enzymes and the glycolysis rate in b3galt5-overexpressing cells. Our study uncovers a novel mechanism by which b3galt5 mediates glycolysis in HCC and highlights the b3galt5-mTOR/p70s6k axis as a potential target for HCC therapy.

The isolated effects of local cold application on proteolytic and myogenic signaling.

Post-exercise cooling studies reveal inhibitory effects on markers of skeletal muscle growth. However, the isolated effect of local cold application has not been adequately addressed. It is unclear if the local cold or the combination of local cold and exercise is driving negatively altered skeletal muscle gene expression. The purpose was to determine the effects of a 4 h local cold application to the vastus lateralis on the myogenic and proteolytic response. Participants (n = 12, 27 ± 6 years, 179 ± 9 cm, 82.8 ± 13.0 kg, 18.4 ± 7.1 %BF) rested with a thermal wrap placed on each leg with either circulating cold fluid (10 °C, COLD) or no fluid circulation (room temperature, RT). Muscle samples were collected to quantify mRNA (RT-qPCR) and proteins (Western Blot) associated with myogenesis and proteolysis. Temperatures in COLD were lower than RT at the skin (13.2 ± 1.0 °C vs. 34.8 ± 0.9 °C; p < 0.001) and intramuscularly (20.5 ± 1.3 °C vs. 35.6 ± 0.8 °C, p < 0.001). Myogenic-related mRNA, MYO-G and MYO-D1, were lower in COLD (p = 0.001, p < 0.001, respectively) whereas myogenic-mRNA, MYF6, was greater in COLD (p = 0.002). No other myogenic associated genes were different between COLD and RT (MSTN, p = 0.643; MEF2a, p = 0.424; MYF5, p = 0.523; RPS3, p = 0.589; RPL3-L, p = 0.688). Proteolytic-related mRNA was higher in COLD (FOXO3a, p < 0.001; Atrogin-1, p = 0.049; MURF-1, p < 0.001). The phosphorylation:total protein ratio for the translational repressor of muscle mass, 4E-BP1Thr37/46, was lower in COLD (p = 0.043), with no differences in mTORser2448 (p = 0.509) or p70S6K1Thr389 (p = 0.579). Isolated local cooling over 4 h exhibits inhibited myogenic and higher proteolytic skeletal muscle molecular response.