[Effect of Erchen Decoction on liver mitochondrial function by inhibiting mTORC1/SREBP1/CAV1 pathway in mice with high-fat diet].

This study aims to investigate the effect of Erchen Decoction(ECD) on liver mitochondrial function in mice with a high-fat diet and its possible mechanism. A total of sixty C57BL/6J mice were randomly divided into a normal group, high-fat group, ECD group, mTORC1 activator(MHY) group, ECD+MHY group, and polyene phosphatidyl choline(PPC) group, with 10 rats in each group. The normal group was given a normal diet, and the other groups were fed a high-fat diet for 20 weeks. At the 17th week, the ECD group and ECD+MHY group were given ECD(8.7 g·kg~(-1)) daily, and the PPC group was given PPC(0.18 g·kg~(-1)) daily, while the remaining groups were given normal saline(0.01 mL·g~(-1)) daily for four weeks. In the 19th week, the MHY group and ECD+MHY group were injected intraperitoneally with MHY(5 mg·kg~(-1)) every other day for two weeks. During the experiment, the general conditions of the mice were observed. The contents of triglyceride(TG) and total cholesterol(TC) in serum were measured. Morphological changes in liver tissue were examined through HE and oil red O staining. The content of adenosine triphosphate(ATP) was determined using chemiluminescence, and mitochondrial membrane potential was assessed using a fluorescence probe(JC-1). Western blot was performed to detect the expression of rapamycin target protein complex 1(mTOR1), ribosomal protein S6 kinase B1(S6K), sterol regulatory element binding protein 1(SREBP1), and caveolin 1(CAV1). RESULTS:: revealed that compared with the normal group, the mice in the high-fat group exhibited significant increases in body weight and abdominal circumference(P<0.01). Additionally, there were significant increases in TG and TC levels(P<0.01). HE and oil red O staining showed that the boundaries of hepatic lobules were unclear; hepatocytes were enlarged, round, and irregularly arranged, with obvious lipid droplet deposition and inflammatory cell infiltration. The liver ATP content and mitochondrial membrane potential decreased significantly(P<0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 increased significantly(P<0.01), while the expression of CAV1 decreased significantly(P<0.01). Compared with the high-fat group, the body weight and TG content of mice in the ECD group and PPC group decreased significantly(P<0.05). Improvements were observed in hepatocyte morphology, lipid deposition, and inflammatory cell infiltration. Furthermore, there were significant increases in ATP content and mitochondrial membrane potential(P<0.05 or P<0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly in the ECD group(P<0.01), while CAV1 expression increased significantly(P<0.01). However, the indices mentioned above did not show improvement in the MHY group. When the ECD+MHY group was compared with the MHY group, there were significant reductions in body weight and TG contents(P<0.05). The morphological changes of hepatocytes, lipid deposition, and inflammatory cell infiltration were recovered. Moreover, there were significant increases in liver ATP content and mitochondrial membrane potential(P<0.05 or P<0.05). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly(P<0.01), while CAV1 expression increased significantly(P<0.01). In conclusion, ECD can improve mitochondrial function by regulating the mTORC1/SREBP1/CAV1 pathway. This mechanism may be involved in the resolution of phlegm syndrome and the regulation of lipid metabolism.

Fraxetin pretreatment alleviates cisplatin-induced kidney injury by antagonizing autophagy and apoptosis via mTORC1 activation.

Cisplatin-induced kidney injury (CKI) is a common complication of chemotherapy. Fraxetin, derived from Fraxinus bungeana A. DC. bark, has antioxidant, anti-inflammatory, and anti-fibrotic effects. This study aims to investigate fraxetin's effects on CKI and its underlying mechanism in vivo and in vitro. Tubular epithelial cells (TECs) and mice were exposed to cisplatin with and without fraxetin preconditioning assess fraxetin's role in CKI. TECs autophagy was observed using transmission electron microscopy. Apoptosis levels in animal tissues were measured using TUNEL staining. The protective mechanism of fraxetin was explored through pharmacological and genetic regulation of mTORC1. Molecular docking was used to identify potential binding sites between fraxetin and mTORC1. The results indicated that fraxetin pretreatment reduced cisplatin-induced kidney injury in a time- and concentration-dependent way. Fraxetin also decreased autophagy in TECs, as observed through electron microscopy. Tissue staining confirmed that fraxetin pretreatment significantly reduced cisplatin-induced apoptosis. Inhibition of mTORC1 using rapamycin or siRNA reversed the protective effects of fraxetin on apoptosis and autophagy in cisplatin-treated TECs, while activation of mTORC1 enhanced fraxetin's protective effect. Molecular docking analysis revealed that fraxetin can bind to HEAT-repeats binding site on mTORC1 protein. In  summary, fraxetin pretreatment alleviates CKI by antagonizing autophagy and apoptosis via mTORC1 activation. This provides evidence for the potential therapeutic application of fraxetin in CKI.

MTMR7 suppresses the phenotypic switching of vascular smooth muscle cell and vascular intimal hyperplasia after injury via regulating p62/mTORC1-mediated glucose metabolism.

BACKGROUND AND AIMS: Myotubularin-related protein 7 (MTMR7) suppresses proliferation in various cell types and is associated with cardiovascular and cerebrovascular diseases. However, whether MTMR7 regulates vascular smooth muscle cell (VSMC) and vascular intimal hyperplasia remains unclear. We explored the role of MTMR7 in phenotypic switching of VSMC and vascular intimal hyperplasia after injury. METHODS AND RESULTS: MTMR7 expression was significantly downregulated in injured arteries. Compared to wild type (WT) mice, Mtmr7-transgenic (Mtmr7-Tg) mice showed reduced intima/media ratio, decreased percentage of Ki-67-positive cells within neointima, and increased Calponin expression in injured artery. In vitro, upregulating MTMR7 by Len-Mtmr7 transfection inhibited platelet derived growth factor (PDGF)-BB-induced proliferation, migration of VSMC and reversed PDGF-BB-induced decrease in expression of Calponin and SM-MHC. Microarray, single cell sequence, and other bioinformatics analysis revealed that MTMR7 is highly related to glucose metabolism and mammalian target of rapamycin complex 1 (mTORC1). Further experiments confirmed that MTMR7 markedly repressed glycolysis and mTORC1 activity in PDGF-BB-challenged VSMC in vitro. Restoring mTORC1 activity abolished MTMR7-mediated suppression of glycolysis, phenotypic shift in VSMC in vitro and protection against vascular intimal hyperplasia in vivo. Furthermore, upregulating MTMR7 in vitro led to dephosphorylation and dissociation of p62 from mTORC1 in VSMC. External expression of p62 in vitro also abrogated the inhibitory effects of MTMR7 on glycolysis and phenotypic switching in PDGF-BB-stimulated VSMC. CONCLUSIONS: Our study demonstrates that MTMR7 inhibits injury-induced vascular intimal hyperplasia and phenotypic switching of VSMC. Mechanistically, the beneficial effects of MTMR7 are conducted via suppressing p62/mTORC1-mediated glycolysis.

A novel therapeutic target for kidney diseases: Lessons learned from starvation response.

The prevalence of chronic kidney disease (CKD) is increasing worldwide, making the disease an urgent clinical challenge. Caloric restriction has various anti-aging and organ-protective effects, and unraveling its molecular mechanisms may provide insight into the pathophysiology of CKD. In response to changes in nutritional status, intracellular nutrient signaling pathways show adaptive changes. When nutrients are abundant, signals such as mechanistic target of rapamycin complex 1 (mTORC1) are activated, driving cell proliferation and other processes. Conversely, others, such as sirtuins and AMP-activated protein kinase, are activated during energy scarcity, in an attempt to compensate. Autophagy, a cellular self-maintenance mechanism that is regulated by such signals, has also been reported to contribute to the progression of various kidney diseases. Furthermore, in recent years, ketone bodies, which have long been considered to be detrimental, have been reported to play a role as starvation signals, and thereby to have renoprotective effects, via the inhibition of mTORC1. Therefore, in this review, we discuss the role of mTORC1, which is one of the most extensively studied nutrient-related signals associated with kidney diseases, autophagy, and ketone body metabolism; and kidney energy metabolism as a novel therapeutic target for CKD.

Role of mammalian target of rapamycin in the formation and progression of retinopathy of prematurity-like vascular abnormalities in neonatal rats.

Retinopathy of prematurity (ROP), a retinal disease that can occur in premature infants, can lead to severe visual impairment. In this study, we examined the preventive and therapeutic effects of mammalian target of rapamycin complex 1 (mTORC1) inhibition on abnormal retinal blood vessels in a rat model of ROP. To induce ROP-like vascular abnormalities, rats were subcutaneously treated with KRN633, an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, on postnatal day 7 (P7) and P8. KRN633-treated (ROP) rats were treated subcutaneously with the mTORC1 inhibitor rapamycin according to preventive and therapeutic protocols, i.e., from P11 to P13 (P11-P13) and from P14 to P20 (P14-P20), respectively. To compare with the effects of VEGF inhibition, KRN633 was administered according to similar protocols. Changes in retinal vasculature, phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, and the proliferative status of vascular cells were evaluated at P14 and P21 using immunohistochemistry. Rapamycin treatment from P11 to P13 prevented increases in arteriolar tortuosity, capillary density, and the number of proliferating vascular cells, and eliminated pS6 immunoreactivity in ROP rats. KRN633 treatment at P11 and P12 (P11/P12) also prevented the appearance of ROP-like retinal blood vessels. Rapamycin treatment from P14 to P20 failed to attenuate arteriolar tortuosity but prevented increases in capillary density and proliferating vascular cell number at the vascular front, but not at the central zone. KRN633 treatment from P14 to P20 significantly reduced abnormalities in the retinal vasculature; however, the effects were inferior to those of KRN633 treatment on P11/P12. These results suggest that activation of the mTORC1 pathway in proliferating endothelial cells contributes to the appearance and progression of ROP-like retinal blood vessels. Therefore, inhibition of mTORC1 may be a promising approach for selectively targeting abnormal retinal blood vessels in ROP.

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway.

Rosmarinic acid (RA) has gained attraction in bioprocessing as a media supplement to improve cellular proliferation and protein production. Here, we observe up to a two-fold increase in antibody production with RA-supplementation, and a concentration-dependent effect of RA on cell proliferation for fed-batch Chinese hamster ovary (CHO) cell cultures. Contrary to previously reported antioxidant activity, RA increased the reactive oxygen species (ROS) levels, stimulated endoplasmic reticulum (ER) stress, activated the unfolded protein response (UPR), and elicited DNA damage. Despite such stressful events, RA appeared to maintained cell health via mammalian target of rapamycin (mTOR) pathway activation; both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) were stimulated in RA-supplemented cultures. By reversing such mTOR pathway activity through either chemical inhibitor addition or siRNA knockdown of genes regulating the mTORC1 and mTORC2 complexes, antibody production, UPR signaling, and stress-induced DNA damage were reduced. Further, the proliferative effect of RA appeared to be regulated selectively by mTORC2 activation and have reproduced this observation by using the mTORC2 stimulator SC-79. Analogously, knockdown of mTORC2 strongly reduced X-box binding protein 1 (XBP1) splicing, which would be expected to reduce antibody folding and secretion, sugging that reduced mTORC2 would correlate with reduced antibody levels. The crosstalk between mTOR activation and UPR upregulation may thus be related directly to the enhanced productivity. Our results show the importance of the mTOR and UPR pathways in increasing antibody productivity, and suggest that RA supplementation may obviate the need for labor-intensive genetic engineering by directly activating pathways favorable to cell culture performance.

RGS10 inhibits proliferation and migration of pulmonary arterial smooth muscle cell in pulmonary hypertension via AKT/mTORC1 signaling.

Objective: Excessive proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is a core event of pulmonary hypertension (PH). Regulators of G protein signaling 10 (RGS10) can regulate cellular proliferation and cardiopulmonary diseases. We demonstrate whether RGS10 also serves as a regulator of PH.Methods: PASMC was challenged by hypoxia to induce proliferation and migration. Adenovirus carrying Rgs10 gene (Ad-Rgs10) was used for external expression of Rgs10. Hypoxia/SU5416 or MCT was used to induce PH. Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were used to validate the establishment of PH model.Results: RGS10 was downregulated in hypoxia-challenged PASMC. Ad-Rgs10 significantly suppressed proliferation and migration of PASMC after hypoxia stimulus, while silencing RGS10 showed contrary effect. Mechanistically, we observed that phosphorylation of S6 and 4E-Binding Protein 1 (4EBP1), the main downstream effectors of mammalian target of rapamycin complex 1 (mTORC1) as well as phosphorylation of AKT, the canonical upstream of mTORC1 in hypoxia-induced PASMC were negatively modulated by RGS10. Both recovering mTORC1 activity and restoring AKT activity abolished these effects of RGS10 on PASMC. More importantly, AKT activation also abolished the inhibitory role of RGS10 in mTORC1 activity in hypoxia-challenged PASMC. Finally, we also observed that overexpression of RGS10 in vivo ameliorated pulmonary vascular wall thickening and reducing RVSP and RVHI in mouse PH model.Conclusion: Our findings reveal the modulatory role of RGS10 in PASMC and PH via AKT/mTORC1 axis. Therefore, targeting RGS10 may serve as a novel potent method for the prevention against PH."

A focus on leucine in the nutritional regulation of human skeletal muscle metabolism in ageing, exercise and unloading states.

Muscle protein synthesis (MPS) and muscle protein breakdown (MPB) are influenced through dietary protein intake and physical (in)activity, which it follows, regulate skeletal muscle (SKM) mass across the lifespan. Following consumption of dietary protein, the bio-availability of essential amino acids (EAA), and primarily leucine (LEU), drive a transient increase in MPS with an ensuing refractory period before the next MPS stimulation is possible (due to the "muscle full" state). At the same time, MPB is periodically constrained via reflex insulin actions. Layering exercise on top of protein intake increases the sensitivity of SKM to EAA, therefore extending the muscle full set-point (∼48 h), to permit long-term remodelling (e.g., hypertrophy). In contrast, ageing and physical inactivity are associated with a premature muscle full set-point in response to dietary protein/EAA and contractile activity. Of all the EAA, LEU is the most potent stimulator of the mechanistic target of rapamycin complex 1 (mTORC1)-signalling pathway, with the phosphorylation of mTORC1 substrates increasing ∼3-fold more than with all other EAA. Furthermore, maximal MPS stimulation is also achieved following low doses of LEU-enriched protein/EAA, negating the need for larger protein doses. As a result, LEU supplementation has been of long term interest to maximise muscle anabolism and subsequent net protein accretion, especially when in tandem with resistance exercise. This review highlights current knowledge vis-à-vis the anabolic effects of LEU supplementation in isolation, and in enriched protein/EAA sources (i.e., EAA and/or protein sources with added LEU), in the context of ageing, exercise and unloading states.

Targeting TLK2 inhibits the progression of gastric cancer by reprogramming amino acid metabolism through the mTOR/ASNS axis.

Several recent studies have suggested that TLKs are related to tumor progression. However, the function and mechanism of action of TLK2 in gastric cancer (GC) remain elusive. In this study, TLK2 was found to be significantly upregulated in patients with GC and was identified as an independent prognostic factor for GC. Consistently, TLK2 knockdown markedly reduced the aggressiveness of GC, whereas its overexpression had the opposite effect. IP-MS revealed that the effects of TLK2 on GC were mainly associated with metabolism reprogramming. TLK2 knockdown suppressed amino acid synthesis by downregulating the mTORC1 pathway and ASNS expression in GC cells. Mechanistically, mTORC1 directly interacts with the ASNS protein and inhibits its degradation. Further experiments validated that the ASNS protein was degraded via ubiquitination instead of autophagy. Inhibiting and activating the mTORC1 pathway can upregulate and downregulate ASNS ubiquitination, respectively, and the mTORC1 pathway can reverse the regulatory effects of TLK2 on ASNS. Furthermore, TLK2 was found to regulate the mRNA expression of ASNS. TLK2 directly interacted with ATF4, a transcription factor of ASNS, and promoted its expression. The kinase inhibitor fostamatinib significantly inhibited the proliferative, invasive, and migratory capabilities of GC cells by inhibiting TLK2 activity. Altogether, this study reveals a novel functional relationship between TLK2 and the mTORC1/ASNS axis in GC. Therefore, TLK2 may serve as a potential therapeutic target for GC.

miR-146a-5p regulates autophagy and NLRP3 inflammasome activation in epithelial barrier damage in the in vitro cell model of ulcerative colitis through the RNF8/Notch1/mTORC1 pathway.

Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon that can be influenced by microRNAs (miRNAs). This study aims to investigate the impact of miR-146a-5p on lipopolysaccharide (LPS)-induced Caco-2/HT-29 cell autophagy and NLRP3 inflammasome activation and the underlying mechanism, with the aim of identifying potential therapeutic targets. We used LPS to establish Caco-2/HT-29 cell models and measured cell viability by CCK-8. The levels of miR-146a-5p, RNF8, markers of NLRP3 inflammasome activation and autophagy, proteins involved in the Notch1/mTORC1 pathway, and inflammatory factors were assessed by RT-qPCR, Western blot, and ELISA. Intestinal epithelial barrier function was evaluated by measuring transepithelial electrical resistance. Autophagic flux was measured using tandem fluorescent-labeled LC3. miR-146a-5p was highly-expressed in LPS-induced Caco-2/HT-29 cells, and autophagy flux was blocked at the autolysosomal stage after LPS induction. Inhibition of miR-146a-5p suppressed NLRP3 inflammasome activation, reduced intestinal epithelial barrier damage, and facilitated autophagy inhibition in LPS-induced Caco-2/HT-29 cells. The autophagy inhibitor NH4Cl partially nullified the inhibitory effects of miR-146a-5p inhibition on NLRP3 inflammation activation. miR-146a-5p targeted RNF8, and silencing RNF8 partly abrogated the action of miR-146a-5p inhibition on promoting autophagy and inhibiting NLRP3 inflammasome activation. miR-146a-5p inhibition suppressed the Notch1/mTORC1 pathway activation by upregulating RNF8. Inhibition of the Notch1/mTORC1 pathway partially nullified the function of silencing RNF8 on inhibiting autophagy and bolstering NLRP3 inflammasome activation. In conclusion, miR-146a-5p inhibition may be a potential therapeutic approach for UC, as it facilitates autophagy of LPS-stimulated Caco-2/HT-29 cells, inhibits NLRP3 inflammasome activation, and reduces intestinal epithelial barrier damage by upregulating RNF8 and suppressing the Notch1/mTORC1 pathway.

Core Target and Key Signal Pathway of Xiaoluo in the Treatment of Thyroid Cancer.

Context: At present, hormone therapy and surgery are the main treatments for thyroid cancer, and they have a quick effect but a high recurrence rate. Also, the side effects are significant. it's extremely urgent to explore treatments that can take into account both therapeutic benefits and side effects. Objective: The study intended to explore whether Xiaoluo has an inhibitory effect on the proliferation of thyroid-cancer cells in vitro and to examine the core target and key signaling pathway of Xiaoluo in the treatment of thyroid cancer, using the thyroid-cancer cell line SW579. Design: The research team performed an in-vitro study. Setting: The study took place at the College of Pharmacy at Harbin University of Commerce in Harbin, China. Outcome Measures: The research team used a Western blot analysis to detect the expression of apoptosis proteins-B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3-and the activity related to the signaling pathways phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin 1 (mTORC1). The team measured optical densities and inhibition rates for the 1, 2, 5, 10, and 15 mg/mL Xiaokuo groups and for a negative control group. The research team measured apoptosis, expression of Bcl-2, Bax, and Caspase-3, and expression of P13K, AKT, and mTor for the 10 µmol/L LY294002, 10 mg/mL Xiaoluo, 100 ng/mL IGF-1, and 100 ng/mL IGF-1+10 mg/mL Xiaoluo groups and for the blank control group. Results: The inhibition of SW579 cell proliferation increased with each increase in the Xiaoluo concentration from 1-15 mg/mL, and the inhibition rate reached 49.63% when the concentration was 15 mg/ml. The Xiaoluo group's late and total apoptosis rates were significantly higher (both P < .01) than those of the blank control group. The Xiaoluo group's expression of the Bcl-2 protein was significantly lower (P < .05), and its expressions of Bax and Caspase-3 were significantly higher (both P < .01) than those of the blank control group. The Xiaoluo group's expressions of P-PI3K, P-Akt, and P-MTOR were significantly lower than those of the blank group (all P < .01). These findings were comparable to those that occurred with use of the PI3K/AKT/mTORC1 signaling pathway inhibitor LY294002. Conclusions: Xiaoluo exerts its antithyroid-cancer effects through the induction of apoptosis in thyroid cancer cells through the inhibition of the PI3K/AKT/mTORC1 signaling pathway. Xiaoluo may serve as a potential therapeutic agent for the treatment of thyroid cancer.

Mesenchymal stromal cells regulate THP-1-differentiated macrophage cytokine production by activating Akt/mammalian target of rapamycin complex 1 pathway.

BACKGROUND AIMS: The Akt/mammalian target of rapamycin (mTOR) pathway in macrophages converges inflammatory and metabolic signals from multiple receptors to regulate a cell's survival, metabolism and activation. Although mesenchymal stromal cells (MSCs) are well known to modulate macrophage activation, the effects of MSCs on the Akt/mTOR pathway in macrophages have not been elucidated. METHODS: We herein investigated whether MSCs affect the Akt/mTOR complex 1 (mTORC1) pathway to regulate macrophage polarization. RESULTS: Results showed that human bone marrow-derived MSCs induced activation of Akt and its downstream mTORC1 signaling in THP-1-differentiated macrophages in a p62/sequestosome 1-independent manner. Inhibition of Akt or mTORC1 attenuated the effects of MSCs on the suppression of tumor necrosis factor-α and interleukin-12 production and the promotion of interleukin-10 and tumor growth factor-ß1 in macrophages stimulated by lipopolysaccharide/ATP. Conversely, activation of Akt or mTORC1 reproduced and potentiated MSC effects on macrophage cytokine production. MSCs with cyclooxygenase-2 knockdown, however, failed to activate the Akt/mTORC1 signaling in macrophages and were less effective in the modulation of macrophage cytokine production than control MSCs. CONCLUSIONS: These data demonstrate that MSCs control THP-1-differentiated macrophage activation at least partly through upregulation of the Akt/mTORC1 signaling in a cyclooxygenase-2-dependent manner.

Combination of the LARS1 Inhibitor, BC-LI-0186 with a MEK1/2 Inhibitor Enhances the Anti-Tumor Effect in Non-Small Cell Lung Cancer.

PURPOSE: The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth and proliferation by growth factor coordination and amino acid availability. Leucyl-tRNA synthetase 1 (LARS1) senses the intracellular leucine concentration and mediates amino acid-induced activation of mTORC1. Thus, LARS1 inhibition could be useful in cancer treatment. However, the fact that mTORC1 can be stimulated by various growth factors and amino acids suggests that LARS1 inhibition alone has limitations in inhibiting cell growth and proliferation. We investigated the combined effects of BC-LI-0186, a LARS1 inhibitor, and trametinib, an MEK inhibitor, on non-small cell lung cancer (NSCLC). Materials and Methods: Protein expression and phosphorylation were observed by immunoblotting, and genes differentially expressed between BC-LI-0186-sensitive and -resistant cells were identified by RNA sequencing. The combined effect of the two drugs was inferred from the combination index values and a xenograft model. RESULTS: LARS1 expression was positively correlated with mTORC1 in NSCLC cell lines. BC-LI-0186 treatment of A549 and H460 cells maintained in media supplemented with fetal bovine serum revealed paradoxical phosphorylation of S6 and activation of mitogen- activated protein kinase (MAPK) signaling. Compared with BC-LI-0186-sensitive cells, -resistant cells showed enrichment of the MAPK gene set. The combination of trametinib and BC-LI-0186 inhibited the phosphorylation of S6, MEK, and extracellular signal-regulated kinase and their synergistic effects were confirmed in a mouse xenograft model. CONCLUSION: The combination of BC-LI-0186 and trametinib inhibited the non-canonical mTORC1-activating function of LARS1. Our study demonstrated a new therapeutic approach for NSCLC without targetable driver mutations.

mTORC1 signaling pathway regulates tooth repair.

Tooth germ injury can lead to abnormal tooth development and even tooth loss, affecting various aspects of the stomatognathic system including form, function, and appearance. However, the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited. Therefore, it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model. Here, we constructed a Tg(dlx2b:Dendra2-NTR) transgenic line that labeled tooth germ specifically. Taking advantage of the NTR/Mtz system, the dlx2b+ tooth germ cells were depleted by Mtz effectively. The process of tooth germ repair was evaluated by antibody staining, in situ hybridization, EdU staining and alizarin red staining. The severely injured tooth germ was repaired in several days after Mtz treatment was stopped. In the early stage of tooth germ repair, the expression of phosphorylated 4E-BP1 was increased, indicating that mTORC1 is activated. Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ. Normally, mouse incisors were repaired after damage, but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress. Overall, we are the first to construct a stable and repeatable repair model of severe tooth germ injury, and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair, providing a potential target for clinical treatment of tooth germ injury.

Glycolysis and de novo fatty acid synthesis cooperatively regulate pathological vascular smooth muscle cell phenotypic switching and neointimal hyperplasia.

Switching of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a dedifferentiated (proliferative) phenotype contributes to neointima formation, which has been demonstrated to possess a tumor-like nature. Dysregulated glucose and lipid metabolism is recognized as a hallmark of tumors but has not thoroughly been elucidated in neointima formation. Here, we investigated the cooperative role of glycolysis and fatty acid synthesis in vascular injury-induced VSMC dedifferentiation and neointima formation. We found that the expression of hypoxia-inducible factor-1α (HIF-1α) and its target 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), a critical glycolytic enzyme, were induced in the neointimal VSMCs of human stenotic carotid arteries and wire-injured mouse carotid arteries. HIF-1α overexpression led to elevated glycolysis and resulted in a decreased contractile phenotype while promoting VSMC proliferation and activation of the mechanistic target of rapamycin complex 1 (mTORC1). Conversely, silencing Pfkfb3 had the opposite effects. Mechanistic studies demonstrated that glycolysis generates acetyl coenzyme A to fuel de novo fatty acid synthesis and mTORC1 activation. Whole-transcriptome sequencing analysis confirmed the increased expression of PFKFB3 and fatty acid synthetase (FASN) in dedifferentiated VSMCs. More importantly, FASN upregulation was observed in neointimal VSMCs of human stenotic carotid arteries. Finally, interfering with PFKFB3 or FASN suppressed vascular injury-induced mTORC1 activation, VSMC dedifferentiation, and neointima formation. Together, this study demonstrated that PFKFB3-mediated glycolytic reprogramming and FASN-mediated lipid metabolic reprogramming are distinctive features of VSMC phenotypic switching and could be potential therapeutic targets for treating vascular diseases with neointima formation. © 2023 The Pathological Society of Great Britain and Ireland.

Intranasal Administration of Resolvin E1 Produces Antidepressant-Like Effects via BDNF/VEGF-mTORC1 Signaling in the Medial Prefrontal Cortex.

Intracerebroventricular infusion of resolvin E1 (RvE1), a bioactive metabolite derived from eicosapentaenoic acid, exerts antidepressant-like effects in a mouse model of lipopolysaccharide (LPS)-induced depression; these effects are blocked by systemic injection of rapamycin, a mechanistic target of rapamycin complex 1 (mTORC1) inhibitor. Additionally, local infusion of RvE1 into the medial prefrontal cortex (mPFC) or dorsal hippocampal dentate gyrus (DG) produces antidepressant-like effects. To evaluate the potential of RvE1 for clinical use, the present study examined whether treatment with RvE1 via intranasal (i.n.) route, a non-invasive route for effective drug delivery to the brain, produces antidepressant-like effects in LPS-challenged mice using tail suspension and forced swim tests. Intranasal administration of RvE1 significantly attenuated LPS-induced immobility, and these antidepressant-like effects were completely blocked by an AMPA receptor antagonist or L-type voltage-dependent Ca2+ channel blocker. The antidepressant-like effects of both i.n. and intra-mPFC administrations of RvE1 were blocked by intra-mPFC infusion of a neutralizing antibody (nAb) for brain-derived neurotrophic factor (BDNF) or vascular endothelial growth factor (VEGF). Intra-mPFC infusion of rapamycin completely blocked the antidepressant-like effects of both i.n. and intra-mPFC administrations of RvE1 as well as those of intra-mPFC infusion of BDNF and VEGF. Moreover, i.n. RvE1 produced antidepressant-like effects via mTORC1 activation in the mPFC of a mouse model of repeated prednisolone-induced depression. Intra-dorsal DG infusion of BDNF and VEGF nAbs, but not rapamycin, blocked the antidepressant-like effects of i.n. RvE1. These findings suggest that i.n. administration of RvE1 produces antidepressant-like effects through activity-dependent BDNF/VEGF release in the mPFC and dorsal DG, and mTORC1 activation in the mPFC, but not in the dorsal DG. Thus, RvE1 can be a promising candidate for a novel rapid-acting antidepressant.

Combination of mTOR inhibitor PP242 and AMPK activator metformin exerts enhanced inhibitory effects on colorectal carcinoma cells in vitro by blocking multiple kinase pathways.

The second-generation mammalian target of rapamycin (mTOR) inhibitor PP242 has demonstrated limited success in some rapamycin-insensitive tumours. We examined the therapeutic potential of combining PP242 with adenosine 50- monophosphate-activated protein kinase (AMPK) activator metformin, using a panel of colorectal carcinoma (CRC) cell lines. We found that the PP242 and metformin combination enhanced the suppression of CRC cell proliferation, colony formation, and cancer cell apoptosis induction. The effect of this combination was observed on AMPK phosphorylation. Western blotting showed that PP242 inhibited mTORC1 activation, as indicated by the reduced expression of its major substrate p-S6K1 and the partially reduced phosphorylation of eIF4E-binding protein 1 (4E-BP1). The inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT Ser473) was transient and occurred in the first few hours of PP242 treatment; metformin exposure decreased the PP242 activity, counteracting AKT activation. We further demonstrated that this was related to direct AMPK-mediated phosphorylation of IRS-1 at Ser789. Thus, the combination of PP242 and metformin completely blocked the activity of both mTORC1 and mTORC2 kinase. This study suggests that this combination could be a more effective strategy for the treatment of CRC.

mTORC1 signaling pathway regulates tooth repair / 国际口腔科学杂志·英文版

Tooth germ injury can lead to abnormal tooth development and even tooth loss, affecting various aspects of the stomatognathic system including form, function, and appearance. However, the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited. Therefore, it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model. Here, we constructed a Tg(dlx2b:Dendra2-NTR) transgenic line that labeled tooth germ specifically. Taking advantage of the NTR/Mtz system, the dlx2b+ tooth germ cells were depleted by Mtz effectively. The process of tooth germ repair was evaluated by antibody staining, in situ hybridization, EdU staining and alizarin red staining. The severely injured tooth germ was repaired in several days after Mtz treatment was stopped. In the early stage of tooth germ repair, the expression of phosphorylated 4E-BP1 was increased, indicating that mTORC1 is activated. Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ. Normally, mouse incisors were repaired after damage, but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress. Overall, we are the first to construct a stable and repeatable repair model of severe tooth germ injury, and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair, providing a potential target for clinical treatment of tooth germ injury.

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy.

BACKGROUND & AIMS: Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation. METHODS: Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas. CONCLUSIONS: These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy. LAY SUMMARY: Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.