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.

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.

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.

S6K1 Controls DNA Damage Signaling Modulated by the MRN Complex to Induce Radioresistance in Lung Cancer.

Radiation is a mainstay of lung cancer treatment; however, resistance frequently develops. Identifying novel therapeutic targets to increase radiation sensitivity is crucial. S6K1 is a serine/threonine kinase known to regulate protein translation which is associated with radioresistance, but the mechanisms involved are unknown. We proposed to determine whether S6K1 promotes radioresistance by regulating DNA repair in lung cancer. Colony formation, protein expression and proliferation were assessed. S6K1 was modulated pharmacologically by either PF-4708671 or genetically by Crispr-Cas9. Higher radioresistance levels in lung cancer cells were associated with lower phosphoactivation of MRN complex members, a key activator of radiation-induced DNA repair signaling. We also found lower levels of p-ATM, a target of the MRN complex, in more radioresistant cells, which was associated with a lower expression of γ-H2AX cafter radiation. Further, genetic and pharmacological S6K1 targeting sensitized lung cancer cells to low doses of radiation (p ≤ 0.01). Additionally, S6K1-/- deletion increased the phosphoactivation of MRN complex members, indicating that S6K1 itself can shut down DNA damage regulated by MRN signaling. This is the first report showing that S6K1 inhibition radiosensitizes lung cancer cells by decreasing MRN complex-regulated DNA repair signaling. Future studies should evaluate the role of S6K1 as a target to overcome radioresistance.

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.

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.

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.

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.

Hyperbaric oxygen treatment increases intestinal stem cell proliferation through the mTORC1/S6K1 signaling pathway in Mus musculus.

BACKGROUND: Hyperbaric oxygen treatment (HBOT) has been reported to modulate the proliferation of neural and mesenchymal stem cell populations, but the molecular mechanisms underlying these effects are not completely understood. In this study, we aimed to assess HBOT somatic stem cell modulation by evaluating the role of the mTOR complex 1 (mTORC1), a key regulator of cell metabolism whose activity is modified depending on oxygen levels, as a potential mediator of HBOT in murine intestinal stem cells (ISCs). RESULTS: We discovered that acute HBOT synchronously increases the proliferation of ISCs without affecting the animal's oxidative metabolism through activation of the mTORC1/S6K1 axis. mTORC1 inhibition by rapamycin administration for 20 days also increases ISCs proliferation, generating a paradoxical response in mice intestines, and has been proposed to mimic a partial starvation state. Interestingly, the combination of HBOT and rapamycin does not have a synergic effect, possibly due to their differential impact on the mTORC1/S6K1 axis. CONCLUSIONS: HBOT can induce an increase in ISCs proliferation along with other cell populations within the crypt through mTORC1/S6K1 modulation without altering the oxidative metabolism of the animal's small intestine. These results shed light on the molecular mechanisms underlying HBOT therapeutic action, laying the groundwork for future studies.

The independent effects of local heat application on muscle growth program associated mRNA and protein phosphorylation.

The development and maintenance of skeletal muscle is crucial for the support of daily function. Recent evidence suggests that genes coded for proteins associated with the human muscle growth program (myogenic and proteolytic genes) are sensitive to local heat application. Therefore, the purpose of this investigation was to determine the effect of 4 h of local heat application to the vastus lateralis at rest on acute phosphorylation (mTORSer2448, p70-S6K1Thr389, and 4E-BP1Thr47/36) and gene expression changes for proteins associated with the muscle growth program. Intramuscular temperature of the HOT limb was 1.2 ± 0.2 °C higher than CON limb after 4 h of local heating. However, this local heat stimulus did not influence transcription of genes associated with myogenesis (MSTN, p = 0.321; MYF5, p = 0.445; MYF6, p = 0.895; MEF2a, p = 0.809; MYO-G, p = 0.766; MYO-D1, p = 0.118; RPS3, p = 0.321; and RPL-3L, p = 0.577), proteolysis (Atrogin-1, p = 0.573; FOXO3a, p = 0.452; MURF-1, p = 0.284), nor protein phosphorylation (mTORSer2448, p = 0.981; P70-S6K1Thr389, p = 0.583; 4E-BP1Thr37/46, p = 0.238) associated with the muscle growth program. These findings suggest little to no association between the local application of heat, at rest, and the activation of the observed muscle growth program-related markers.

Amino acid transporters revisited: New views in health and disease.

Amino acid transporters (AATs) are membrane-bound transport proteins that mediate transfer of amino acids into and out of cells or cellular organelles. AATs have diverse functional roles ranging from neurotransmission to acid-base balance, intracellular energy metabolism, and anabolic and catabolic reactions. In cancer cells and diabetes, dysregulation of AATs leads to metabolic reprogramming, which changes intracellular amino acid levels, contributing to the pathogenesis of cancer, obesity and diabetes. Indeed, the neutral amino acid transporters (NATs) SLC7A5/LAT1 and SLC1A5/ASCT2 are likely involved in several human malignancies. However, a clinical therapy that directly targets AATs has not yet been developed. The purpose of this review is to highlight the structural and functional diversity of AATs, their diverse physiological roles in different tissues and organs, their wide-ranging implications in human diseases and the emerging strategies and tools that will be necessary to target AATs therapeutically.

S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer.

BACKGROUND: CDK4/6 inhibitors combined with endocrine therapy has become the preferred treatment approach for patients with estrogen receptor-positive metastatic breast cancer. However, the predictive biomarkers and mechanisms of innate resistance to CDK4/6 inhibitors remain largely unknown. We sought to elucidate the molecular hallmarks and therapeutically actionable features of patients with resistance to CDK4/6 inhibitors. METHODS: A total of 36 patients received palbociclib and endocrine therapy were included in this study as the discovery cohort. Next-generation sequencing of circulating tumour DNA in these patients was performed to evaluate somatic alterations associated with innate resistance to palbociclib. Then the candidate biomarker was validated in another independent cohort of 104 patients and publicly available datasets. The resistance was verified in parental MCF-7 and T47D cells, as well as their derivatives with small interfering RNA transfection and lentivirus infection. The relevant mechanism was examined by RNA sequencing, chromatin immunoprecipitation and luciferase assay. Patient-derived organoid and patient-derived xenografts studies were utilized to evaluated the antitumor activity of rational combinations. RESULTS: In the discovery cohort, S6K1 amplification (3/35, 9%) was identified as an important reason for innate resistance to CDK4/6 inhibitors. In the independent cohort, S6K1 was overexpressed in 15/104 (14%) patients. In those who had received palbociclib treatment, patients with high-expressed S6K1 had significantly worse progression free survival than those with low S6K1 expression (hazard ratio = 3.0, P = 0.0072). Meta-analysis of public data revealed that patients with S6K1 amplification accounted for 12% of breast cancers. Breast cancer patients with high S6K1 expression had significantly worse relapse-free survival (hazard ratio = 1.31, P < 0.0001). In breast cancer cells, S6K1 overexpression, caused by gene amplification, was sufficient to promote resistance to palbociclib. Mechanistically, S6K1 overexpression increased the expression levels of G1/S transition-related proteins and the phosphorylation of Rb, mainly through the activation of c-Myc pathway. Notably, this resistance could be abrogated by the addition of mTOR inhibitor, which blocked the upstream of S6K1, in vitro and in vivo. CONCLUSIONS: S6K1 amplification is an important mechanism of innate resistance to palbociclib in breast cancers. Breast cancers with S6K1 amplification could be considered for combinations of CDK4/6 and S6K1 antagonists.

Nuclear S6K1 regulates cAMP-responsive element-dependent gene transcription through activation of mTOR signal pathway.

S6K1 serves as an important signaling regulator of cell proliferation and growth in the mTOR signaling pathway. Excessive activation of the mTOR/S6K1 signaling pathway promotes abnormal cell growth and survival, thereby resulting in tumorigenesis. The roles of S6K1 in protein synthesis and metabolism are well known, but an additional role of S6K1 as a gene transcription regulator has not been much understood. Here, we demonstrated that S6K1 is dynamically distributed in the cytoplasm and nuclei of human cervical cancer cells. S6K1 nuclear localization was serum dependent and serum deprivation or rapamycin treatment inhibited S6K1 Thr389 phosphorylation and, thereby, S6K1 was retained in the cytoplasm. Furthermore, we found that endogenous S6K1 interacted with CREB in the cervical cancer cells. Additionally, S6K1 upregulated the CRE-driven promoter luciferase activity. The proto-oncogene c-JUN, which has several CREs, was attenuated in the S6K1 knockdown cervical cancer cells. The binding of CREB/S6K1 to the c-JUN promoter, altered by serum restimulation, was associated with active epigenetic markers. In HeLa cell, 891 promoter regions, to which S6K1 directly binds, were detected. Our findings suggested that active S6K1, which is dynamically translocated into the nucleus, directly binds to chromatin and could play a role in epigenetic mechanisms or transcription factor recruitment.

Sexual dimorphism in the molecular mechanisms of insulin resistance during a critical developmental window in Wistar rats.

BACKGROUND: Insulin resistance (IR) is a condition in which the response of organs to insulin is impaired. IR is an early marker of metabolic dysfunction. However, IR also appears in physiological contexts during critical developmental windows. The molecular mechanisms of physiological IR are largely unknown in both sexes. Sexual dimorphism in insulin sensitivity is observed since early stages of development. We propose that during periods of accelerated growth, such as around weaning, at postnatal day 20 (p20) in rats, the kinase S6K1 is overactivated and induces impairment of insulin signaling in its target organs. This work aimed to characterize IR at p20, determine its underlying mechanisms, and identify whether sexual dimorphism in physiological IR occurs during this stage. METHODS: We determined systemic insulin sensitivity through insulin tolerance tests, glucose tolerance tests, and blood glucose and insulin levels under fasting and fed conditions at p20 and adult male and female Wistar rats. Furthermore, we quantified levels of S6K1 phosphorylated at threonine 389 (T389) (active form) and its target IRS1 phosphorylated at serine 1101 (S1101) (inhibited form). In addition, we assessed insulin signal transduction by measuring levels of Akt phosphorylated at serine 473 (S473) (active form) in white adipose tissue and skeletal muscle through western blot. Finally, we determined the presence and function of GLUT4 in the plasma membrane by measuring the glucose uptake of adipocytes. Results were compared using two-way ANOVA (With age and sex as factors) and one-way ANOVA with post hoc Tukey's tests or t-student test in each corresponding case. Statistical significance was considered for P values < 0.05. RESULTS: We found that both male and female p20 rats have elevated levels of glucose and insulin, low systemic insulin sensitivity, and glucose intolerance. We identified sex- and tissue-related differences in the activation of insulin signaling proteins in p20 rats compared to adult rats. CONCLUSIONS: Male and female p20 rats present physiological insulin resistance with differences in the protein activation of insulin signaling. This suggests that S6K1 overactivation and the resulting IRS1 inhibition by phosphorylation at S1101 may modulate to insulin sensitivity in a sex- and tissue-specific manner. Video Abstract.

Insulin regulates the synthesis of carbohydrates, lipids and proteins differently between males, and females. One of its primary functions is maintaining adequate blood glucose levels favoring glucose entry in muscle and adipose tissue after food consumption. Insulin resistance (IR) is a condition in which the response of organs to insulin is impaired. IR is frequently associated with metabolic dysfunction such as inflammation, obesity, or type 2 diabetes. However, physiological IR develops in healthy individuals during periods of rapid growth, pregnancy, or aging by mechanisms not fully understood. We studied the postnatal development, specifically around weaning at postnatal day 20 (p20) of Wistar rats. In previous works, we identified insulin resistance during this period in male rats. This work aimed to characterize IR at p20, determine its underlying mechanisms, and identify whether sexual dimorphism in physiological IR occurs during this stage. We found that p20 rats of both sexes have elevated blood glucose and insulin levels, low systemic insulin sensitivity, and glucose intolerance. We identified differences in insulin-regulated protein activation (S6K1, IRS1, Akt, and GLUT4) between sexes in different tissues and adipose tissue depots. Studying these mechanisms and their differences between males and females is essential to understanding insulin actions and their relationship with the possible development of metabolic diseases in both sexes.

Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/ß-Catenin Transcriptional Complex Formation.

Ribosomal protein S6 kinase 1 (S6K1), a key downstream effector of the mammalian target of rapamycin (mTOR), regulates diverse functions, such as cell proliferation, cell growth, and protein synthesis. Because S6K1 was previously known to be localized in the cytoplasm, its function has been mainly studied in the cytoplasm. However, the nuclear localization and function of S6K1 have recently been elucidated and other nuclear functions are expected to exist but remain elusive. Here, we show a novel nuclear role of S6K1 in regulating the expression of the Wnt target genes. Upon activation of the Wnt signaling, S6K1 translocated from the cytosol into the nucleus and subsequently bound to ß-catenin and the cofactors of the Wnt/ß-catenin transcriptional complex, leading to the upregulation of the Wnt target genes. The depletion or repression of S6K1 downregulated the Wnt target gene expression by inhibiting the formation of the Wnt/ß-catenin transcriptional complex. The S6K1-depleted colon cancer cell lines showed lower transcription levels of the Wnt/ß-catenin target genes and a decrease in the cell proliferation and invasion compared to the control cell lines. Taken together, these results indicate that nuclear S6K1 positively regulates the expression of the Wnt target genes by inducing the reciprocal interaction of the subunits of the transcriptional complex.

Bempedoic Acid Restores Liver H2S Production in a Female Sprague-Dawley Rat Dietary Model of Non-Alcoholic Fatty Liver.

We previously demonstrated that treatment with BemA (bempedoic acid), an inhibitor of ATP citrate lyase, significantly reduces fatty liver in a model of liver steatosis (HFHFr-female Sprague-Dawley rat fed a high-fat high-fructose diet). Since the hepatic production of the gasotransmitter H2S is impaired in liver disorders, we were interested in determining if the production of H2S was altered in our HFHFr model and whether the administration of BemA reversed these changes. We used stored liver samples from a previous study to determine the total and enzymatic H2S production, as well as the expression of CBS (cystathionine ß-synthase), CSE (cystathionine γ-lyase), and 3MST (3-mercaptopiruvate sulfurtransferase), and the expression/activity of FXR (farnesoid X receptor), a transcription factor involved in regulating CSE expression. Our data show that the HFHFr diet reduces the total and enzymatic production of liver H2S, mainly by decreasing the expression of CBS and CSE. Furthermore, BemA treatment restored H2S production, increasing the expression of CBS and CSE, providing evidence for the involvement of FXR transcriptional activity and the mTORC1 (mammalian target of rapamycin1)/S6K1 (ribosomal protein S6 kinase beta-1)/PGC1α (peroxisome proliferator receptor gamma coactivator1α) pathway.

GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin.

Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR ROP2 physically interacts with and, when GTP-bound, activates TOR in vitro TOR activation in response to auxin is abolished in ROP-deficient rop2 rop6 ROP4 RNAi plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin.

MicroRNA-506 modulates insulin resistance in human adipocytes by targeting S6K1 and altering the IRS1/PI3K/AKT insulin signaling pathway.

The incidence of obesity has increased rapidly, becoming a worldwide public health issue that involves insulin resistance. A growing number of recent studies have demonstrated that microRNAs play a significant role in controlling the insulin signaling network. For example, miR-506-3p expression has been demonstrated to correlate with insulin sensitivity; however, the underlying mechanism remains unknown. In this study, we found that miR-506-3p enhanced glucose uptake by 2-deoxy-D-glucose uptake assays and regulated the protein expression of key genes involved in the PI3K/AKT insulin signaling pathway including IRS1, PI3K, AKT, and GlUT4. We next predicted ribosomal protein S6 kinase B1 (S6K1) to be a candidate target of miR-506-3p by bioinformatics analysis and confirmed using dual-luciferase assays that miR-506-3p regulated S6K1 expression by binding to its 3'-UTR. Moreover, modulating S6K1 expression counteracted the effects of miR-506-3p on glucose uptake and PI3K/AKT pathway activation. In conclusion, miR-506-3p altered IR in adipocytes by regulating S6K1-mediated PI3K/AKT pathway activation. Taken together, these findings provide novel insights and potential targets for IR therapy.