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Objective To explore the effect of vitamin D (VD) supplementation during pregnancy and lactation on the endochondral osteogenesis process and mechanistic target of rapamycin (mTOR) pathway in offspring rats. Methods After twenty-four female Wistar rats and twelve male rats were housed together, female rats were randomly divided into Control group, ow- dose VD group (VD-Low group), high-dose VD group (VD-High group), and mTOR inhibitor rapamycin group (RAPA group), with six rats in each group. VD (10,20 ng/kg) and rapamycin (1 mg/kg) were administered during the pregnancy and lactation period from day 0 of pregnancy (GD 0) to day 21 after birth (PND 21)]. After feeding the offspring for 4 weeks, The weight and length of offspring mice were measured; The tibia of the offspring mice were separated and subjected to micro CT scanning and reconstruction; Whole body and local skeletal staining were used to observe the skeletal development of offspring mice; HE staining was used to observe the morphology of chondrocytes in tibial growth plate; Immunohistochemical staining was used to detect proliferation related protein [Ki67, proliferating cell nuclear antigen (PCNA)], differentiation related protein [collagen II a1 chain (Col2A1), SRY-box transcription factor 9 (SOX9)], hypertrophy related protein [collagen X al chain (Col10A1), matrix metalloproteinase 13 (MMP-13), osteopontin (OPN)] and osteogenic related protein [collagen I (Col I), osteocalcin (OCN), alkaline phosphatase (ALP) levels in chondrocyte of tibial tissue; RT-qPCR was used to detect mTOR, ribosomal protein S6 kinase (p70S6K) and elF4E-binding protein (4E-BP1) mRNA levels in tibial tissue; Western blot was used to detect microtubule- associated protein 1 light chain 3 (LC3), Beclin1, p62, mTOR, p-mTOR, p70S6K, p-p70S6K, 4E-BP1 and p-4E-BP1 levels in tibial tissue. Results Compared with Control group, body weight and length of offspring mice in VD Low group, VD High group and RAPA group were increased, trabecular thickness (Tb. Th) in tibial tissue were increased, The arrangement of chondrocytes in tibial tissue growth plate was neat, the thickness of the growth plate, proliferative zone and hypertrophic zone were increased, Ki67, PCNA, Col2A1, SOX9, Col10A1, MMP-13, OPN, Col I, OCN, ALP, LC3 II/I, Beclin1 protein levels were increased, mTOR, p70S6K, 4E-BP1 mRNA levels, p62, p-mTOR/mTOR, p-p70S6K/p70S6K and p-4E-BP1/4E-BP1 protein levels were decreased (all P<0.05). Conclusion VD supplementation during pregnancy and lactation could promote endochondral osteogenesis in offspring mice, which might play a role by inhibiting mTOR signaling pathway and activating autophagy. [ABSTRACT FROM AUTHOR]

Simple Summary: Nutritional cues during embryonic development significantly impact growth, although the mechanism behind this influence remains unclear. Amino acids such as leucine can affect the nutrient-sensing pathway that regulates growth. We injected 2.5 mg of leucine or saline into Japanese quail eggs on the tenth day of incubation. Treatment groups showed no significant difference in hatching success, body mass, gastrointestinal length, and morphological traits (wing, tarsal, and head lengths). However, from day 3 to day 7 post-hatch, chicks hatching from leucine-treated eggs showed increased wing length, body mass, tarsal length, and head and intestinal lengths, which lasted up to 21 days. Similarly, the growth-related genes in the liver were upregulated in leucine-treated quail chicks. However, protein degradation genes remained unchanged. These results suggest that the slight increase in embryonic leucine can promote growth, highlighting the potential for improvement in poultry growth performance. Nutritional cues during embryonic development can alter developmental trajectories and affect postnatal growth. However, the specific mechanisms by which nutrients influence avian growth remain largely unknown. Amino acids can directly interact with the nutrient-sensing pathways, such as the insulin-like growth factor 1 (IGF-1)/mechanistic target of rapamycin (mTOR) pathways, which are known to regulate growth. We examined the effects of embryonic leucine on gene expression and phenotypic growth in Japanese quails by injecting 2.5 mg leucine or saline (control) into Japanese quail eggs on the tenth day of incubation and incubating them under standard conditions. The treatment groups had similar hatching success and size at hatching. However, between 3 and 7 days post-hatching, quails treated with embryonic leucine showed increased growth in body mass and wing, tarsus, head, and intestinal lengths, lasting up to 21 days. The hepatic expression of IGF1, IGF1R, mTOR, and RPS6K1 was upregulated in leucine-treated quails, while the expression of FOXO1 remained unaffected. In conclusion, a subtle increase in embryonic leucine may induce developmental programming effects in Japanese quail by interacting with the IGF-1/mTOR nutrient-sensing pathway to promote growth. This study highlights the role of embryonic amino acids as crucial nutrients for enhancing growth. It provides valuable insight into nutrient intervention strategies during embryonic development to potentially improve poultry growth performance. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 (GLP-1) is a gut-derived hormone secreted by intestinal L cells and vital for postprandial glycemic control. As open-type enteroendocrine cells, whether L cells can sense mechanical stimuli caused by chyme and thus regulate GLP-1 synthesis and secretion is unexplored. Molecular biology techniques revealed the expression of Piezo1 in intestinal L cells. Its level varied in different energy status and correlates with blood glucose and GLP-1 levels. Mice with L cell-specific loss of Piezo1 (Piezo1 IntL-CKO) exhibited impaired glucose tolerance, increased body weight, reduced GLP-1 production and decreased CaMKKβ/CaMKIV-mTORC1 signaling pathway under normal chow diet or high-fat diet. Activation of the intestinal Piezo1 by its agonist Yoda1 or intestinal bead implantation increased the synthesis and secretion of GLP-1, thus alleviated glucose intolerance in diet-induced-diabetic mice. Overexpression of Piezo1, Yoda1 treatment or stretching stimulated GLP-1 production and CaMKKβ/CaMKIV-mTORC1 signaling pathway, which could be abolished by knockdown or blockage of Piezo1 in primary cultured mouse L cells and STC-1 cells. These experimental results suggest a previously unknown regulatory mechanism for GLP-1 production in L cells, which could offer new insights into diabetes treatments.

ABSTRACT: Balanced ratios of branched-chain amino acids (BCAAs) can enhance chicken growth, immunity, and muscle synthesis. However, these ratios can be affected by changes in crude protein (CP) levels or the substitution of protein sources, leading to BCAA antagonism. This, in turn, can have a negative impact on chicken growth. In Experiment 1, a total of 960 0-d-old male Cobb 500 broilers were divided into 6 treatments with 8 replicates. Three different BCAA ratios were used in High or Low CP diets as follows: 1) Low Leu group (Low level of leucine with increased valine and isoleucine levels), 2) Med Leu group, and 3) High Leu group (High level of leucine with reduced valine and isoleucine levels) for a total of 6 diets. In Experiment 2, a total of 640 0-d-old male Cobb 500 broilers were divided into 4 treatments with 8 replicates. The four diets had either High or Low CP and one of two protein sources with the same medium levels of BCAAs: 1) the soybean meal (SBM) group, which had SBM as the main protein source (protein bound AA), and 2) the wheat middlings with non-bound AAs (WM+AA) group (non-bound AA), which had additional non-bound AAs to replace SBM. The High Leu diet had a negative effect on overall growth performance, carcass weight, breast muscle weight, and body mineral composition compared to the Low Leu and Med Leu groups, particularly in the High CP diet (P < 0.05). The SBM group showed increased growth performance, breast muscle weight, expression levels of genes promoting muscle growth, and improved bone mineral composition compared to the WM+AA group, and the High CP group intensified the negative effect of the WM+AA diet (P < 0.05). In summary, balanced BCAA ratios and SBM-based diets have positive effects on chicken growth and muscle accretion, whereas excessive leucine and non-bound AA levels in the diets may negatively affect growth performance and meat yield in chickens.

Tumors in dogs and humans share many similar molecular and genetic features, incentivizing a better understanding of canine neoplasms not only for the purpose of treating companion animals, but also to facilitate research of spontaneously developing tumors with similar biologic behavior and treatment approaches in an immunologically competent animal model. Multiple tumor types of both species have similar dysregulation of signal transduction through phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB; AKT), and mechanistic target of rapamycin (mTOR), collectively known as the PI3K-AKT-mTOR pathway. This review aims to delineate the pertinent aspects of the PI3K-AKT-mTOR signaling pathway in health and in tumor development. It will then present a synopsis of current understanding of PI3K-AKT-mTOR signaling in important canine cancers and advancements in targeted inhibitors of this pathway. [ABSTRACT FROM AUTHOR]

Bottom-of-sulcus dysplasia (BOSD) is increasingly recognized as a cause of drug-resistant, surgically-remediable, focal epilepsy, often in seemingly MRI-negative patients. We describe the clinical manifestations, morphological features, localization patterns and genetics of BOSD, with the aims of improving management and understanding pathogenesis. We studied 85 patients with BOSD diagnosed between 2005–2022. Presenting seizure and EEG characteristics, clinical course, genetic findings and treatment response were obtained from medical records. MRI (3 T) and 18F-FDG-PET scans were reviewed systematically for BOSD morphology and metabolism. Histopathological analysis and tissue genetic testing were performed in 64 operated patients. BOSD locations were transposed to common imaging space to study anatomical location, functional network localization and relationship to normal MTOR gene expression. All patients presented with stereotyped focal seizures with rapidly escalating frequency, prompting hospitalization in 48%. Despite 42% patients having seizure remissions, usually with sodium channel blocking medications, most eventually became drug-resistant and underwent surgery (86% seizure-free). Prior developmental delay was uncommon but intellectual, language and executive dysfunction were present in 24%, 48% and 29% when assessed preoperatively, low intellect being associated with greater epilepsy duration. BOSDs were missed on initial MRI in 68%, being ultimately recognized following repeat MRI, 18F-FDG-PET or image postprocessing. MRI features were grey-white junction blurring (100%), cortical thickening (91%), transmantle band (62%), increased cortical T1 signal (46%) and increased subcortical FLAIR signal (26%). BOSD hypometabolism was present on 18F-FDG-PET in 99%. Additional areas of cortical malformation or grey matter heterotopia were present in eight patients. BOSDs predominated in frontal and pericentral cortex and related functional networks, mostly sparing temporal and occipital cortex, and limbic and visual networks. Genetic testing yielded pathogenic mTOR pathway variants in 63% patients, including somatic MTOR variants in 47% operated patients and germline DEPDC5 or NPRL3 variants in 73% patients with familial focal epilepsy. BOSDs tended to occur in regions where the healthy brain normally shows lower MTOR expression, suggesting these regions may be more vulnerable to upregulation of MTOR activity. Consistent with the existing literature, these results highlight (i) clinical features raising suspicion of BOSD; (ii) the role of somatic and germline mTOR pathway variants in patients with sporadic and familial focal epilepsy associated with BOSD; and (iii) the role of 18F-FDG-PET alongside high-field MRI in detecting subtle BOSD. The anatomical and functional distribution of BOSDs likely explain their seizure, EEG and cognitive manifestations and may relate to relative MTOR expression. [ABSTRACT FROM AUTHOR]

Protein turnover defines the balance between two continuous and complex processes of protein metabolism, synthesis and degradation, which determine their deposition in tissues. Although the liver and intestine have been studied extensively for their important roles in protein digestion, absorption and metabolism, the study of protein metabolism has focused mainly on skeletal muscle tissue to understand the basis for its growth. Due to the high adaptability of skeletal muscle, its protein turnover is greatly affected by different internal and external factors, contributing to carcass lean‐yield and animal growth. Amino acid (AA) labelling and tracking using isotope tracer methodology, together with the study of myofiber type profiling, signal transduction pathways and gene expression, has allowed the analysis of these mechanisms from different perspectives. Positive stimuli such as increased nutrient availability in the diet (e.g., AA), physical activity, the presence of certain hormones (e.g., testosterone) or a more oxidative myofiber profile in certain muscles or pig genotypes promote increased upregulation of translation and transcription‐related genes, activation of mTORC1 signalling mechanisms and increased abundance of satellite cells, allowing for more efficient protein synthesis. However, fasting, animal aging, inactivity and stress, inflammation or sepsis produce the opposite effect. Deepening the understanding of modifying factors and their possible interaction may contribute to the design of optimal strategies to better control tissue growth and nutrient use (i.e., protein and AA), and thus advance the precision feeding strategy. [ABSTRACT FROM AUTHOR]

Lingling Dong,1,&ast; Yong Wang,1,&ast; Haipin Chen,1 Zhouyang Li,1 Xuchen Xu,1 Jiesen Zhou,1 Huahao Shen,1,2 Zhihua Chen1 1Key Laboratory of Respiratory Disease of Zhejiang Province, Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 2State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhihua Chen, Department of Respiratory and Critical Care Medicine, Key Laboratory of Respiratory Disease of Zhejiang Province, Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, People’s Republic of China, Tel +86-571-8898-1913, Fax +86-571-8778-3729, Email zhihuachen@zju.edu.cnBackground: Macrophage-derived matrix metalloproteinase 12 (MMP12) can cause destruction of lung tissue structure and plays a significant role in the development and progression of chronic obstructive pulmonary disease (COPD). MTOR is a serine/threonine kinase that plays a crucial role in cell growth and metabolism. The activity of MTOR in the lung tissues of COPD patients also shows significant changes. However, it is unclear whether MTOR can regulate the development and progression of COPD by controlling MMP12. This study primarily investigates whether MTOR in macrophages can affect the expression of MMP12 and participate in the progression of COPD.Methods: We tested the changes in MTOR activity in macrophages exposed to cigarette smoke (CS) both in vivo and in vitro. Additionally, we observed the effect of MTOR on the expression of MMP12 in macrophages and on lung tissue inflammation and structural damage in mice, both in vivo and in vitro, using MTOR inhibitors or gene knockout mice. Finally, we combined inhibitor treatment with gene knockout to demonstrate that MTOR primarily mediates the expression of MMP12 through the NF-κB signaling pathway.Results: Exposure to CS can enhance MTOR activity in mouse alveolar macrophages. Inhibiting the activity of MTOR or suppressing its expression leads to increased expression of MMP12. Myeloid-specific knockout of MTOR expression can promote the occurrence of CS-induced pulmonary inflammation and emphysema in mice. Inhibiting the activity of NF-κB can eliminate the effect of MTOR on MMP12.Conclusion: Macrophage MTOR can reduce the expression of MMP12 by inhibiting NF-κB, thereby inhibiting the occurrence of COPD inflammation and destruction of lung tissue structure. Activating the activity of macrophage MTOR may be beneficial for the treatment of COPD.Keywords: matrix metalloproteinase 12, MMP12, pulmonary emphysema, cigarette smoke, CS, mechanistic target of rapamycin, MTOR

Abstract Background There is limited published information regarding the expression of mechanistic target of rapamycin (mTOR) in vessels that encapsulate tumor cluster (VETC)‐positive hepatocellular carcinoma (HCC). The mTOR inhibitor, everolimus, has been approved as an immunosuppressant for use in HCC patients after living donor liver transplantation (LDLT). Methods Using a database of 214 patients who underwent LDLT for HCC, we examined the mTOR protein and angiopoietin‐2 (Ang‐2) in VETC‐positive HCC by immunohistochemical staining. The presence of VETC and mTOR expression were evaluated in both primary and recurrent HCC lesions. Results Forty‐three of the 214 patients (20.1%) were VETC‐positive, and 29 of these 43 patients (67.4%) expressed mTOR. Relative Ang‐2 expression was significantly higher in the mTOR‐positive than in the mTOR‐negative group (p = 0.037). Thirty‐four of the 214 patients experienced HCC recurrence after LDLT; 20 of these were operable. The primary lesions of six of these 20 patients were VETC‐positive; five of these six patients also had VETC‐positive recurrent lesions (p

Abstract Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA‐stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1‐acylglycerol‐3‐phosphate‐O‐acyltransferase‐4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus.

Enhancing hepatic gluconeogenesis is one of the main modes of meeting the glucose requirement of dairy cows. This study attempted to determine whether the gluconeogenesis precursor propionate had an effect on the expression of the main genes involved in gluconeogenesis in calf hepatocytes and elucidate the associated mechanisms. Calf hepatocytes were obtained from 5 healthy calves (1 d old; 30 to 40 kg) and exposed to 0-, 1-, 2.5-, or 5-mM sodium propionate (NaP), which is known to promote the expression of genes involved in the gluconeogenesis pathway, including fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase. With regard to the underlying mechanism, propionate promoted the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, hepatocyte nuclear factor 4, and forkhead box O1 (transcription factors that regulate the expression of hepatic gluconeogenic genes) by promoting mammalian target of rapamycin complex 1 (mTORC1), but inhibiting mTORC2 activity (P

Nutritional cues during embryonic development can alter developmental trajectories and affect postnatal growth. However, the specific mechanisms by which nutrients influence avian growth remain largely unknown. Amino acids can directly interact with the nutrient-sensing pathways, such as the insulin-like growth factor 1 (IGF-1)/mechanistic target of rapamycin (mTOR) pathways, which are known to regulate growth. We examined the effects of embryonic leucine on gene expression and phenotypic growth in Japanese quails by injecting 2.5 mg leucine or saline (control) into Japanese quail eggs on the tenth day of incubation and incubating them under standard conditions. The treatment groups had similar hatching success and size at hatching. However, between 3 and 7 days post-hatching, quails treated with embryonic leucine showed increased growth in body mass and wing, tarsus, head, and intestinal lengths, lasting up to 21 days. The hepatic expression of IGF1, IGF1R, mTOR, and RPS6K1 was upregulated in leucine-treated quails, while the expression of FOXO1 remained unaffected. In conclusion, a subtle increase in embryonic leucine may induce developmental programming effects in Japanese quail by interacting with the IGF-1/mTOR nutrient-sensing pathway to promote growth. This study highlights the role of embryonic amino acids as crucial nutrients for enhancing growth. It provides valuable insight into nutrient intervention strategies during embryonic development to potentially improve poultry growth performance.

Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA‐stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1‐acylglycerol‐3‐phosphate‐O‐acyltransferase‐4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus. [ABSTRACT FROM AUTHOR]

Background: There is limited published information regarding the expression of mechanistic target of rapamycin (mTOR) in vessels that encapsulate tumor cluster (VETC)‐positive hepatocellular carcinoma (HCC). The mTOR inhibitor, everolimus, has been approved as an immunosuppressant for use in HCC patients after living donor liver transplantation (LDLT). Methods: Using a database of 214 patients who underwent LDLT for HCC, we examined the mTOR protein and angiopoietin‐2 (Ang‐2) in VETC‐positive HCC by immunohistochemical staining. The presence of VETC and mTOR expression were evaluated in both primary and recurrent HCC lesions. Results: Forty‐three of the 214 patients (20.1%) were VETC‐positive, and 29 of these 43 patients (67.4%) expressed mTOR. Relative Ang‐2 expression was significantly higher in the mTOR‐positive than in the mTOR‐negative group (p = 0.037). Thirty‐four of the 214 patients experienced HCC recurrence after LDLT; 20 of these were operable. The primary lesions of six of these 20 patients were VETC‐positive; five of these six patients also had VETC‐positive recurrent lesions (p < 0.001). The expression of mTOR was significantly higher in the VETC‐positive lesions (p = 0.0018). Conclusions: We showed that mTOR expression was higher in the VETC‐positive primary and recurrent lesions than in the VETC‐negative ones. [ABSTRACT FROM AUTHOR]

Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco’s Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium.

Introduction: The mechanistic target of rapamycin (mTOR) regulates bone homeostasis, a crucial factor in osteoporosis (OP) development. However, most research is based on observational studies, and the causality remains uncertain. Therefore, we analyzed two samples of mendelian randomization (MR) to determine whether there is a causal relationship between mTOR-dependent circulating proteins and OP. Methods: Mendelian weighting (weighted median [WM], inverse variance weighting [IVW], and MR-Egger regression) were applied to analyze the causality between bone phenotypes (bone mineral density [BMD] in forearm, femoral neck, lumbar spine, and heel) and mTOR-dependent circulating proteins (RP-S6K, 4EBP, EIF-4E, EIF-4A, and EIF-4G). Horizontal pleiotropy and heterogeneities were detected using Cochran's Q test, MR-Pleiotropy RE-Sidual Sum and Outlier (MR-PRESSO), and "leave-one-out" analysis. The proteomics-GWAS INTERVAL study was used to select the instrumental variables (IVs) for mTOR proteins. Results: As phenotypes for OP, estimations of BMD were taken in four different sites: forearm (FA) (n = 8143), femoral neck (FN) (n = 32,735), lumbar spine (LS) (n = 28,498), and heel (eBMD) (n = 426,824). Based on IVW analysis, EIF4E is causally related to FA-BMD (OR = 0.938, 95% CI 0.887, 0.991, p = 0.024) but not to BMD elsewhere. Conclusion: MR analysis revealed a causal relationship between EIF-4E and FA-BMD, which may provide new insights into the underlying pathogenesis of OP and a new therapeutic target for OP. [ABSTRACT FROM AUTHOR]

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

Introduction: We investigated whether the infiltration of tumor-infiltrating lymphocytes (TILs) in gastric cancer (GC), as evaluated by hematoxylin and eosin (H&E) staining, could be a prognostic marker. We also explored on the relationship between TILs and mechanistic target of rapamycin (mTOR) and how it regulates immune effector responses in GC. Methods: A total of 183 patients with available data on TIL were included. TIL infiltration was evaluated using H&E staining. We also conducted immunohistochemistry to determine mTOR expression. Results: Positive TIL infiltration was defined as TILs ≥20%. There were 72 (39.3%) and 111 (60.7%) positive and negative cases, respectively. TILs positivity significantly correlated with both absence of lymph node metastasis (p = 0.037) and negative p-mTOR expression (p = 0.040). TIL infiltration correlated with a significantly better overall (p = 0.046) and disease-free (p = 0.020) survival. Conclusion: mTOR possibly suppresses TIL infiltration in GC. H&E staining is an effective tool for evaluating the immune status of GC patients. H&E staining may be used in clinical practice to monitor treatment response in GC. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy-related Cdc42-binding kinase alpha (MRCKα) is an integral component of signaling pathways controlling vital cellular processes, including cytoskeletal reorganization, cell proliferation and cell survival. In this study, we investigated the physiological role of MRCKα in milk protein and fat production in dairy cows, which requires a dynamic and strict organization of the cytoskeletal network in bovine mammary epithelial cells (BMEC). Within a selection of 9 Holstein cows, we found that both mRNA and protein expression of MRCKα in the mammary gland were upregulated during lactation and correlated positively (r > 0.89) with the mRNA and protein levels of β-casein. Similar positive correlations (r > 0.79) were found in a primary culture of BMEC stimulated with prolactin for 24 h. In these cells, silencing of MRCKα decreased basal β-casein, sterol-regulatory element binding protein (SREBP)-1 and cyclin D1 protein level, phosphorylation of mTOR, triglyceride secretion, cell number and viability—while overexpression of MRCKα displayed the reversed effect. Notably, silencing of MRCKα completely prevented the stimulatory action of prolactin on the same parameters. These data demonstrate that MRCKα is a critical mediator of prolactin-induced lactogenesis via stimulation of the mTOR/SREBP1/cyclin D1 signaling pathway.

Abstract Background Facial angiofibroma is the most predominant cutaneous manifestation of tuberous sclerosis complex (TSC), a rare autosomal dominant genetic disorder impacting the mechanistic target of rapamycin (mTOR). Facial angiofibroma can bleed spontaneously, impair eyesight, and cause aesthetic disfiguration causing psychological and social stress. To date, there is little or no evidence on the demographics, and other TSC features associated with facial angiofibroma or the use of mTOR inhibitor for the management of facial angiofibroma. This is a retrospective study of TSC Alliance’s Natural History Database aimed to characterize facial angiofibroma and to evaluate features associated with a higher risk of facial angiofibroma or the use of topical mTOR inhibitors for the management of facial angiofibroma. Data in the NHD was obtained from 18 clinical sites in the US since 2006. Results Of the 2240 patients, 2088 patients were enrolled in the US and data from 2057 patients were included in this analysis. The mean (median) age of overall TSC patients was 22.4 (19.0) years. A total of 69 patients were ≤ 5 years of age. Facial angiofibroma was noted in 1329 (64.6%) patients with TSC. Patients with facial angiofibroma were older on average (Mean: 25.9 [median, 23.0] vs. 16.0 [12.4 years] years, p

Cognitive impairment caused by chronic cerebral hypoperfusion (CCH) is associated with white matter injury (WMI), possibly through the alteration of autophagy. Here, the autophagy–lysosomal pathway (ALP) dysfunction in white matter (WM) and its relationship with cognitive impairment were investigated in rats subjected to two vessel occlusion (2VO). The results showed that cognitive impairment occurred by the 28th day after 2VO. Injury and autophagy activation of mature oligodendrocytes and neuronal axons sequentially occurred in WM by the 3rd day. By the 14th day, abnormal accumulation of autophagy substrate, lysosomal dysfunction, and the activation of mechanistic target of rapamycin (MTOR) pathway were observed in WM, paralleled with mature oligodendrocyte death. This indicates autophagy activation was followed by ALP dysfunction caused by autophagy inhibition or lysosomal dysfunction. To target the ALP dysfunction, enhanced autophagy by systemic rapamycin treatment or overexpression of Beclin1 (BECN1) in oligodendrocytes reduced mature oligodendrocyte death, and subsequently alleviated the WMI and cognitive impairment after CCH. These results reveal that early autophagy activation was followed by ALP dysfunction in WM after 2VO, which was associated with the aggravation of WMI and cognitive impairment. This study highlights that alleviating ALP dysfunction by enhancing oligodendrocyte autophagy has benefits for cognitive recovery after CCH. Improving autophagy–lysosomal pathway dysfunction caused by autophagy inhibition and lysosomal dysfunction in white matter after chronic cerebral hypoperfusion (CCH) by enhancing oligodendrocyte autophagy may reduce oligodendrocyte death, thus alleviating CCH-induced white matter injury and cognitive impairment in rats. [Display omitted] [ABSTRACT FROM AUTHOR]

Despite advances in the nutritional support of low birth weight and early-weaned piglets, most experience reduced extrauterine growth performance. To further optimize nutritional support and develop targeted intervention strategies, the mechanisms that regulate the anabolic response to nutrition must be fully understood. Knowledge gained in these studies represents a valuable intersection of agriculture and biomedical research, as low birth weight and early-weaned piglets face many of the same morbidities as preterm and low birth weight infants, including extrauterine growth faltering and reduced lean growth. While the reasons for poor growth performance are multifaceted, recent studies have increased our understanding of the role of nutrition in the regulation of skeletal muscle growth in the piglet. The purpose of this review is to summarize the published literature surrounding advances in the current understanding of the anabolic signaling that occurs after a meal and how this response is developmentally regulated in the neonatal pig. It will focus on the regulation of protein synthesis, and especially the upstream and downstream effectors surrounding the master protein kinase, mechanistic target of rapamycin complex 1 (mTORC1) that controls translation initiation. It also will examine the regulatory pathways associated with the postprandial anabolic agents, insulin and specific amino acids, that are upstream of mTORC1 and lead to its activation. Lastly, the integration of upstream signaling cascades by mTORC1 leading to the activation of translation initiation factors that regulate protein synthesis will be discussed. This review concludes that anabolic signaling cascades are stimulated by both insulin and amino acids, especially leucine, through separate pathways upstream of mTORC1, and that these stimulatory pathways result in mTORC1 activation and subsequent activation of downstream effectors that regulate translation initiation Additionally, it is concluded that this anabolic response is unique to the skeletal muscle of the neonate, resulting from increased sensitivity to the rise in both insulin and amino acid after a meal. However, this response is dampened in skeletal muscle of the low birth weight pig, indicative of anabolic resistance. Elucidation of the pathways and regulatory mechanisms surrounding protein synthesis and lean growth allow for the development of potential targeted therapeutics and intervention strategies both in livestock production and neonatal care.

BackgroundIn Alzheimer's disease, there are striking changes in CSF composition that relate to altered choroid plexus (CP) function. Studying CP tissue gene expression at the blood-cerebrospinal fluid barrier could provide further insight into the epithelial and stromal responses to neurodegenerative disease states.MethodsTranscriptome-wide Affymetrix microarrays were used to determine disease-related changes in gene expression in human CP. RNA from post-mortem samples of the entire lateral ventricular choroid plexus was extracted from 6 healthy controls (Ctrl), 7 patients with advanced (Braak and Braak stage III-VI) Alzheimer's disease (AD), 4 with frontotemporal dementia (FTD) and 3 with Huntington's disease (HuD). Statistics and agglomerative clustering were accomplished with MathWorks, MatLab; and gene set annotations by comparing input sets to GeneGo ( http://www.genego.com ) and Ingenuity ( http://www.ingenuity.com ) pathway sets. Bonferroni-corrected hypergeometric p-values of

Several amino acids can stimulate milk synthesis in mammary epithelial cells (MEC); however, the regulatory role of isoleucine (Ile) and underlying molecular mechanism remain poorly understood. In this study, we aimed to evaluate the regulatory effects of Ile on milk protein and fat synthesis in MEC and reveal the mediation mechanism of Brahma-related gene 1 (BRG1) on this regulation. Ile dose dependently affected milk protein and fat synthesis, mechanistic target of rapamycin (mTOR) phosphorylation, sterol regulatory element binding protein 1c (SREBP-1c) expression and maturation, and BRG1 protein expression in bovine MEC. Phosphatidylinositol 3 kinase (PI3K) inhibition by LY294002 treatment blocked the stimulation of Ile on BRG1 expression. BRG1 knockdown and gene activation experiments showed that it mediated the stimulation of Ile on milk protein and fat synthesis, mTOR phosphorylation, and SREBP-1c expression and maturation in MEC. ChIP-PCR analysis detected that BRG1 bound to the promoters of mTOR and SREBP-1c , and ChIP-qPCR further detected that these bindings were increased by Ile stimulation. In addition, BRG1 positively regulated the binding of H3K27ac to these two promoters, while it negatively affected the binding of H3K27me3 to these promoters. BRG1 knockdown blocked the stimulation of Ile on these two gene expressions. The expression of BRG1 was higher in mouse mammary gland in the lactating period, compared with that in the puberty or dry period. Taken together, these experimental data reveal that Ile stimulates milk protein and fat synthesis in MEC via the PI3K-BRG1-mTOR/SREBP-1c pathway. [ABSTRACT FROM AUTHOR]

Stearoyl-CoA desaturase-1 (SCD1) catalyzes the rate-liming step of monounsaturated fatty acid biosynthesis and is a key regulator of systemic glucose metabolism. Mice harboring either a global (GKO) or liver-specific deletion (LKO) of Scd1 display enhanced insulin signaling and whole-body glucose uptake. Additionally, GKO and LKO mice are protected from high-carbohydrate diet-induced obesity. Given that high-carbohydrate diets can lead to chronic metabolic diseases such as obesity, diabetes, and hepatic steatosis, it is critical to understand how Scd1 deficiency confers metabolically beneficial phenotypes. Here we show that insulin-like growth factor-binding protein 1 (IGFBP1), a hepatokine that has been reported to enhance insulin signaling, is significantly elevated in the liver and plasma of GKO and LKO mice fed a low-fat high-carbohydrate diet. We also observed that the expression of hepatic Igfbp1 is regulated by oleic acid (18:1n9), a product of SCD1, through the mTORC1-FGF21 axis both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects an individual’s reciprocal social interaction and communication ability. Numerous genetic and environmental conditions are associated with ASD, including tuberous sclerosis complex, phosphatase and tensin homolog hamartoma tumor syndrome, fragile X syndrome, and neurofibromatosis 1. The pathogenic molecular mechanisms of these diseases are integrated into the hyperactivation of mTORC1 (mechanistic target of rapamycin complex 1). Rodent models of these diseases have shown high mTORC1 activity in the brain and ASD-related behavioral deficits, which were reversed by the mTORC1 inhibitor rapamycin. Environmental stress can also affect this signaling pathway. In utero exposure to valproate caused ASD in offspring and enhanced mTORC1 activity in the brain, which was sensitive to mTORC1 inhibition. mTORC1 is a signaling hub for diverse cellular functions, including protein synthesis, through the phosphorylation of its targets, such as ribosomal protein S6 kinases. Metabotropic glutamate receptor 5–mediated synaptic function is also affected by the dysregulation of mTORC1 activity, such as in fragile X syndrome and tuberous sclerosis complex. Reversing these downstream changes that are associated with mTORC1 activation normalizes behavioral defects in rodents. Despite abundant preclinical evidence, few clinical studies have investigated the treatment of ASD and cognitive deficits. Therapeutics other than mTORC1 inhibitors failed to show efficacy in fragile X syndrome and neurofibromatosis 1. mTORC1 inhibitors have been tested mainly in tuberous sclerosis complex, and their effects on ASD and neuropsychological deficits are promising. mTORC1 is a promising target for the pharmacological treatment of ASD associated with mTORC1 activation.

Sensory nerves promote osteogenesis through the release of neuropeptides. However, the potential application and mechanism in which sensory nerves promote healing of bone defects in the presence of biomaterials remain elusive. The present study identified that new bone formation was more abundantly produced after implantation of silicified collagen scaffolds into defects created in the distal femur of rats. The wound sites were accompanied by extensive nerve innervation and angiogenesis. Sensory nerve dysfunction by capsaicin injection resulted in significant inhibition of silicon-induced osteogenesis in the aforementioned rodent model. Application of extracellular silicon in vitro induced axon outgrowth and increased expression of semaphorin 3 A (Sema3A) and semaphorin 4D (Sema4D) in the dorsal root ganglion (DRG), as detected by the upregulation of signaling molecules. Culture medium derived from silicon-stimulated DRG cells promoted proliferation and differentiation of bone marrow mesenchymal stem cells and endothelial progenitor cells. These effects were inhibited by the use of Sema3A neutralizing antibodies but not by Sema4D neutralizing antibodies. Knockdown of Sema3A in DRG blocked silicon-induced osteogenesis and angiogenesis almost completely in a femoral defect rat model, whereas overexpression of Sema3A promoted the silicon-induced phenomena. Activation of “mechanistic target of rapamycin” (mTOR) pathway and increase of Sema3A production were identified in the DRG of rats that were implanted with silicified collagen scaffolds. These findings support the role of silicon in inducing Sema3A production by sensory nerves, which, in turn, stimulates osteogenesis and angiogenesis. Taken together, silicon has therapeutic potential in orthopedic rehabilitation.

Abstract Background The mTOR-PI3K-Akt pathway influences cell metabolism and (malignant) cell growth. We generated sex-specific polygenic risk scores capturing natural variation in 7 out of 10 top-ranked genes in this pathway. We studied the scores directly and in interaction with energy balance-related factors (body mass index (BMI), trouser/skirt size, height, physical activity, and early life energy restriction) in relation to colorectal cancer (CRC) risk in the Netherlands Cohort Study (NLCS) (n=120,852). The NLCS has a case-cohort design and 20.3 years of follow-up. Participants completed a baseline questionnaire on diet and cancer in 1986 when 55–69 years old. ~75% of the cohort returned toenail clippings used for DNA isolation and genotyping (n subcohort=3,793, n cases=3,464). To generate the scores, the dataset was split in two and risk alleles were defined and weighted based on sex-specific associations with CRC risk in the other dataset half, because there were no SNPs in the top-ranked genes associated with CRC risk in previous genome-wide association studies at a significance level p

Controlled ovarian hyperstimulation (COH) is essential for the success of in vitro fertilization (IVF). Evidence showing the comparison of different COH protocols remains predominantly of low certainty and derives from unspecified infertile and highly heterogeneous populations. Thus, personalized approaches to examine the response of patients to the various COH protocols need to be investigated. Data from in vitro and animal studies have identified the mechanistic target of rapamycin (mTOR) and Hippo signaling pathways play a key role in follicular homeostasis and oocyte quality. To be specific, current data indicate the controlled activation of mTOR and the controlled inhibition of the Hippo pathway within the ovarian granulosa cells (GC). Both are reported to lead to a nurturing follicular microenvironment, increase oocyte quality, and potentially improve reproductive outcomes. As intracellular markers, phosphorylated/unphosphorylated levels of the pathways' main downstream mediators could be included among the candidate "personalized" predictors of patients' response to COH protocols and final IVF outcomes. Based on these hypotheses, we make a preliminary attempt to investigate their validity: We propose a prospective cohort study to compare the levels of certain phosphorylated/unphosphorylated components of the investigated pathways (mTOR, ribosomal protein S6 kinase beta-1 (p70S6K-1), yes-associated protein-1 (YAP-1), and transcriptional coactivator with PDZ-binding motif (TAZ)) within the follicular fluid-isolated GC between women undergoing gonadotropin-releasing hormone (GnRH) antagonist/"short" protocols and those receiving GnRH agonist/"long 21" protocols. A case-control design comparing these levels between women achieving pregnancy and those who did not is further planned. Additional analyses addressing the population's expected heterogeneity are planned after the completion of the pilot phase, during which 100 participants undergoing IVF are intended to be recruited. At this stage, these hypotheses are solely based on in vitro/animal data, and thus, similar studies on humans in this respect are necessary for the investigation of their potential validity. [ABSTRACT FROM AUTHOR]

Background: Facial angiofibroma is the most predominant cutaneous manifestation of tuberous sclerosis complex (TSC), a rare autosomal dominant genetic disorder impacting the mechanistic target of rapamycin (mTOR). Facial angiofibroma can bleed spontaneously, impair eyesight, and cause aesthetic disfiguration causing psychological and social stress. To date, there is little or no evidence on the demographics, and other TSC features associated with facial angiofibroma or the use of mTOR inhibitor for the management of facial angiofibroma. This is a retrospective study of TSC Alliance's Natural History Database aimed to characterize facial angiofibroma and to evaluate features associated with a higher risk of facial angiofibroma or the use of topical mTOR inhibitors for the management of facial angiofibroma. Data in the NHD was obtained from 18 clinical sites in the US since 2006.Results: Of the 2240 patients, 2088 patients were enrolled in the US and data from 2057 patients were included in this analysis. The mean (median) age of overall TSC patients was 22.4 (19.0) years. A total of 69 patients were ≤ 5 years of age. Facial angiofibroma was noted in 1329 (64.6%) patients with TSC. Patients with facial angiofibroma were older on average (Mean: 25.9 [median, 23.0] vs. 16.0 [12.4 years] years, p < 0.0001). In patients with vs. without facial angiofibroma, TSC2 mutation (38.9% vs. 34.8%) was more common than TSC1 mutation (12.3% vs. 18.1%), and the incidence rate of most of the other TSC-related manifestations was significantly higher in patients with facial angiofibroma. Majority of patients had focal seizures (72.8% vs. 60.7%), followed by angiomyolipoma (63.7% vs. 21.8%) and renal cysts (59.4% vs. 33.5%). The age groups, 11-17 (odds ratio [OR], 2.53) and 18-45 years (5.98), TSC2 mutation (1.31), focal seizures (1.50), ADHD (1.47) angiomyolipoma (2.79), and renal cysts (2.63) were significantly associated with a higher risk of facial angiofibroma based on multivariate logistic regression. Abrasive or laser therapy was used by 17.1% and 2.6% patients, respectively. Topical mTOR inhibitor use was noted for 329 (24.8%) patients with facial angiofibroma. Overall systemic mTOR inhibitor use was observed in 399 (30.0%) patients for management of one or more TSC manifestations. Use of systemic mTOR inhibitor for facial angiofibroma was noted for 163 (12.3%) patients, among whom only 9 (0.7%) patients used exclusively for the management of facial angiofibroma. Of the patients with facial angiofibroma, 44.6% did not receive any treatment. Significantly higher use of topical mTOR inhibitor was associated with the 11-17 years age group (OR, 1.67), anxiety (1.57), angiomyolipoma (1.51), and renal cysts (1.33).Conclusions: The presence of TSC2 mutations and most other TSC-related manifestations was significantly higher in patients with facial angiofibroma. About one-fourth of patients with facial angiofibroma used a topical mTOR inhibitor and use of systemic mTOR inhibitor for the management of facial angiofibroma or for the other manifestations was noted for 30.0%. About 44.6% of patients did not receive any treatment for the management of facial angiofibroma. [ABSTRACT FROM AUTHOR]

Metabolic homeostasis and the ability to link energy supply to demand are essential requirements for all living cells to grow and proliferate. Key to metabolic homeostasis in all eukaryotes are AMPK and mTORC1, two kinases that sense nutrient levels and function as counteracting regulators of catabolism (AMPK) and anabolism (mTORC1) to control cell survival, growth and proliferation. Discoveries beginning in the early 2000s revealed that AMPK and mTORC1 communicate, or cross-talk, through direct and indirect phosphorylation events to regulate the activities of each other and their shared protein substrate ULK1, the master initiator of autophagy, thereby allowing cellular metabolism to rapidly adapt to energy and nutritional state. More recent reports describe divergent mechanisms of AMPK/mTORC1 cross-talk and the elaborate means by which AMPK and mTORC1 are activated at the lysosome. Here, we provide a comprehensive overview of current understanding in this exciting area and comment on new evidence showing mTORC1 feedback extends to the level of the AMPK isoform, which is particularly pertinent for some cancers where specific AMPK isoforms are implicated in disease pathogenesis., (© 2024 The Author(s).)

Inherited or sporadic loss of the TSC2 gene can lead to pulmonary lymphangioleiomyomatosis (LAM), a rare cystic lung disease caused by protease-secreting interstitial tumor nodules. The nodules arise by metastasis of cells that exhibit features of neural crest and smooth muscle lineage ('LAM cells'). Their aberrant growth is attributed to increased activity of 'mechanistic target of rapamycin complex 1' (mTORC1), an anabolic protein kinase that is normally suppressed by the TSC1-TSC2 protein complex. The mTORC1 inhibitor rapamycin slows the progression of LAM, but fails to eradicate disease, indicating a role for mTORC1-independent mechanisms in LAM pathogenesis. Our previous studies revealed G-protein coupled urotensin-II receptor (UT) signaling as a candidate mechanism, but how it promotes oncogenic signaling in TSC2 -deficient cells remained unknown. Using a human pluripotent stem cell-derived in vitro model of LAM, we now show hyperactivation of UT, which was required for their enhanced migration and pro-neoplastic signaling in a rapamycin-insensitive mechanism that required heterotrimeric Gαq/11 (Gαq). Bioluminescence resonance energy transfer assays in HEK 293T cells lacking TSC2 demonstrated selective and enhanced activation of Gαq and its RhoA-associated effectors compared to wild-type control cells. By immunoprecipitation, recombinant UT was physically associated with Gαq and TSC2. The augmented Gαq signaling in TSC2 -deleted cells was independent of mTOR activity, and associated with increased endosomal targeting of p63RhoGEF, a known RhoA-activating effector of Gαq. These studies identify potential mTORC1-independent pro-neoplastic mechanisms that can be targeted for prevention or eradication of pulmonary and extrapulmonary LAM tumors.

Glucagon-like peptide 1 (GLP-1) is a gut-derived hormone secreted by intestinal L cells and vital for postprandial glycemic control. As open-type enteroendocrine cells, whether L cells can sense mechanical stimuli caused by chyme and thus regulate GLP-1 synthesis and secretion is unexplored. Molecular biology techniques revealed the expression of Piezo1 in intestinal L cells. Its level varied in different energy status and correlates with blood glucose and GLP-1 levels. Mice with L cell-specific loss of Piezo1 ( Piezo1 IntL-CKO) exhibited impaired glucose tolerance, increased body weight, reduced GLP-1 production and decreased CaMKKβ/CaMKIV-mTORC1 signaling pathway under normal chow diet or high-fat diet. Activation of the intestinal Piezo1 by its agonist Yoda1 or intestinal bead implantation increased the synthesis and secretion of GLP-1, thus alleviated glucose intolerance in diet-induced-diabetic mice. Overexpression of Piezo1, Yoda1 treatment or stretching stimulated GLP-1 production and CaMKKβ/CaMKIV-mTORC1 signaling pathway, which could be abolished by knockdown or blockage of Piezo1 in primary cultured mouse L cells and STC-1 cells. These experimental results suggest a previously unknown regulatory mechanism for GLP-1 production in L cells, which could offer new insights into diabetes treatments., Competing Interests: YH, HM, JY, LG, TT, QS, WG, YZ, JL, QW, JG, HZ, LZ, HC, GX No competing interests declared, (© 2024, Huang, Mo, Yang et al.)

Balanced ratios of branched-chain amino acids (BCAAs) can enhance chicken growth, immunity, and muscle synthesis. However, these ratios can be affected by changes in crude protein (CP) levels or the substitution of protein sources, leading to BCAA antagonism. This, in turn, can have a negative impact on chicken growth. In Experiment 1, a total of 960 0-d-old male Cobb 500 broilers were divided into 6 treatments with 8 replicates. Three different BCAA ratios were used in High or Low CP diets as follows: 1) Low Leu group (Low level of leucine with increased valine and isoleucine levels), 2) Med Leu group, and 3) High Leu group (High level of leucine with reduced valine and isoleucine levels) for a total of 6 diets. In Experiment 2, a total of 640 0-d-old male Cobb 500 broilers were divided into 4 treatments with 8 replicates. The four diets had either High or Low CP and one of two protein sources with the same medium levels of BCAAs: 1) the soybean meal (SBM) group, which had SBM as the main protein source (protein bound AA), and 2) the wheat middlings with non-bound AAs (WM+AA) group (non-bound AA), which had additional non-bound AAs to replace SBM. The High Leu diet had a negative effect on overall growth performance, carcass weight, breast muscle weight, and body mineral composition compared to the Low Leu and Med Leu groups, particularly in the High CP diet (P < 0.05). The SBM group showed increased growth performance, breast muscle weight, expression levels of genes promoting muscle growth, and improved bone mineral composition compared to the WM+AA group, and the High CP group intensified the negative effect of the WM+AA diet (P < 0.05). In summary, balanced BCAA ratios and SBM-based diets have positive effects on chicken growth and muscle accretion, whereas excessive leucine and non-bound AA levels in the diets may negatively affect growth performance and meat yield in chickens., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Necroptosis is a lytic and pro-inflammatory form of programmed cell death executed by the terminal effector, the MLKL (mixed lineage kinase domain-like) pseudokinase. Downstream of death and Toll-like receptor stimulation, MLKL is trafficked to the plasma membrane via the Golgi-, actin- and microtubule-machinery, where activated MLKL accumulates until a critical lytic threshold is exceeded and cell death ensues. Mechanistically, MLKL's lytic function relies on disengagement of the N-terminal membrane-permeabilising four-helix bundle domain from the central autoinhibitory brace helix: a process that can be experimentally mimicked by introducing the R30E MLKL mutation to induce stimulus-independent cell death. Here, we screened a library of 429 kinase inhibitors for their capacity to block R30E MLKL-mediated cell death, to identify co-effectors in the terminal steps of necroptotic signalling. We identified 13 compounds - ABT-578, AR-A014418, AZD1480, AZD5363, Idelalisib, Ipatasertib, LJI308, PHA-793887, Rapamycin, Ridaforolimus, SMI-4a, Temsirolimus and Tideglusib - each of which inhibits mammalian target of rapamycin (mTOR) signalling or regulators thereof, and blocked constitutive cell death executed by R30E MLKL. Our study implicates mTOR signalling as an auxiliary factor in promoting the transport of activated MLKL oligomers to the plasma membrane, where they accumulate into hotspots that permeabilise the lipid bilayer to cause cell death., (© 2024 The Author(s).)

Hydrogen sulfide (H 2 S) as a gaseous molecule prevents gastrointestinal (GI)-tract against various injuries. This study aimed to evaluate for the first time the detailed molecular mechanism of mitochondria-targeting H 2 S-prodrugs, AP39 and RT01 in gastroprotection against ischemia/reperfusion (I/R)-induced lesions. Wistar rats exposed to I/R were pretreated i.g. with vehicle, AP39 (0.004–2 mg/kg), RT01 (0.1 mg/kg), or with AP219 (0.1 mg/kg) as structural control without ability to release H 2 S. AP39 was also administered with mTOR1 inhibitor, rapamycin (1 mg/kg i.g.). Gastric damage area was assessed micro−/macroscopically, gastric blood flow (GBF) by laser flowmetry, mRNA level of HIF-1α, GPx, SOD1, SOD2, annexin-A1, SOCS3, IL-1RA, IL-1β, IL-1R1, IL-1R2, TNFR2, iNOS by real-time PCR. Gastric mucosal and/or serum content of IL-1β, IL-4, IL-5, IL-10, G-CSF, M-CSF, VEGFA, GRO, RANTES, MIP-1α, MCP1, TNF-α, TIMP1, FABP3, GST-α, STAT3/5 and phosphorylation of mTOR, NF-κB, ERK, Akt was evaluated by microbeads-fluorescent assay. Mitochondrial complexes activities were measured biochemically. RNA damage was assessed as 8-OHG by ELISA. AP39 and RT01 reduced micro−/macroscopic gastric I/R-injury increasing GBF. AP39-gastroprotection was accompanied by maintained activity of mitochondrial complexes, prevented RNA oxidation and enhanced mRNA/protein expression of SOCS3, IL-1RA, annexin-A1, GST-α, HIF-1α. Rapamycin reversed AP-39-gastroprotection. AP39-gastroprotection was followed by decreased NF-κB, ERK, IL-1β and enhanced Akt and mTOR proteins phosphorylation. AP39-prevented gastric mucosal damage caused by I/R-injury, partly by mitochondrial complex activity maintenance. AP39-mediated attenuation of gastric mucosal oxidation, hypoxia and inflammation involved mTOR1 and Akt pathways activity and modulation of HIF-1α, GST-α, SOCS3, IL1RA and TIMP1 molecular interplay. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background The mechanistic target of rapamycin (mTOR) pathway promoted by positive energy imbalance and insulin-like growth factors can be a mechanism by which obesity influences breast cancer risk. We evaluated the associations of body fatness with the risk of breast cancer varied with phosphorylated (p)-mTOR protein expression, an indication of the pathway activation. Methods Women with newly diagnosed breast cancer (n = 715; 574 [80%] Black and 141 [20%] White) and non-cancer controls (n = 1983; 1280 [64%] Black and 713 [36%] White) were selected from the Women’s Circle of Health Study. Surgical tumor samples among the cases were immunostained for p-mTOR (Ser2448) and classified as p-mTOR-overexpressed, if the expression level ≥ 75th percentile, or p-mTOR-negative/low otherwise. Anthropometrics were measured by trained staff, and body composition was determined by bioelectrical impedance analysis. Odds ratios (ORs) of p-mTOR-overexpressed tumors and p-mTOR-negative/low tumors compared to controls were estimated using polytomous logistic regression. The differences in the associations by the p-mTOR expression status were assessed by tests for heterogeneity. Results Cases with p-mTOR-overexpressed tumors, but not cases with p-mTOR-negative/low tumors, compared to controls were more likely to have higher body mass index (BMI), percent body fat, and fat mass index (P-heterogeneity

B cells contribute to the pathogenesis of polycystic ovary syndrome (PCOS). Clinically, metformin is used to treat PCOS, but it is unclear whether metformin exerts its therapeutic effect by regulating B cells. Here, we showed that the expression level of tumor necrosis factor-alpha (TNF-α) in peripheral blood B cells from PCOS patients was increased. Metformin used in vitro and in vivo was able to reduce the production of TNF-α in B cells from PCOS patients. Administration of metformin improved mouse PCOS phenotypes induced by dehydroepiandrosterone (DHEA) and also inhibited TNF-α expression in splenic B cells. Furthermore, metformin induced metabolic reprogramming of B cells in PCOS patients, including the alteration in mitochondrial morphology, the decrease in mitochondrial membrane potential, Reactive Oxygen Species (ROS) production and glucose uptake. In DHEA-induced mouse PCOS model, metformin altered metabolic intermediates in splenic B cells. Moreover, the inhibition of TNF-α expression and metabolic reprogramming in B cells of PCOS patients and mouse model by metformin were associated with decreased mTOR phosphorylation. Together, TNF-α-producing B cells are involved in the pathogenesis of PCOS, and metformin inhibits mTOR phosphorylation and affects metabolic reprogramming, thereby inhibiting TNF-α expression in B cells, which may be a new mechanism of metformin in the treatment of PCOS.

Simple Summary: The accumulation of alpha-synuclein (α-syn) is considered a pathological hallmark of the neurodegenerative disorders known as synucleinopathies. The clearance of α-syn depends on autophagy activity, which is inhibited by the mechanistic target of rapamycin (mTOR). Thus, it is likely that α-syn accumulation may occur whenever mTOR is overactive and autophagy is suppressed. In fact, the lack of effective autophagy increases the amount of α-syn and may produce protein aggregation. Therefore, in the present study, we questioned whether cells from glioblastoma multiforme (GBM), a lethal brain neoplasm, wherein mTOR is upregulated and autophagy is suppressed, may overexpress α-syn. In fact, a large quantity of α-syn is measured in GBM cells compared with astrocytes, which includes proteinase K-resistant α-syn. Rapamycin, while inhibiting mTOR activity, significantly reduces the amount of α-syn and allocates α-syn within autophagy-like vacuoles. Alpha-synuclein (α-syn) is a protein considered to be detrimental in a number of degenerative disorders (synucleinopathies) of which α-syn aggregates are considered a pathological hallmark. The clearance of α-syn strongly depends on autophagy, which can be stimulated by inhibiting the mechanistic target of rapamycin (mTOR). Thus, the overexpression of mTOR and severe autophagy suppression may produce α-syn accumulation, including the proteinase K-resistant protein isoform. Glioblastoma multiforme (GBM) is a lethal brain tumor that features mTOR overexpression and severe autophagy inhibition. Cell pathology in GBM is reminiscent of a fast, progressive degenerative disorder. Therefore, the present work questions whether, as is analogous to neurons during degenerative disorders, an overexpression of α-syn occurs within GBM cells. A high amount of α-syn was documented in GBM cells via real-time PCR (RT-PCR), Western blotting, immunohistochemistry, immuno-fluorescence, and ultrastructural stoichiometry, compared with the amount of β- and γ-synucleins and compared with the amount of α-syn counted within astrocytes. The present study indicates that (i) α-syn is overexpressed in GBM cells, (ii) α-syn expression includes a proteinase-K resistant isoform, (iii) α-syn is dispersed from autophagy-like vacuoles to the cytosol, (iv) α-syn overexpression and cytosol dispersion are mitigated by rapamycin, and (v) the α-syn-related GBM-like phenotype is mitigated by silencing the SNCA gene. [ABSTRACT FROM AUTHOR]

The global increase in lifespan noted not only in developed nations, but also in large developing countries parallels an observed increase in a significant number of non-communicable diseases, most notable neurodegenerative disorders. Neurodegenerative disorders present a number of challenges for treatment options that do not resolve disease progression. Furthermore, it is believed by the year 2030, the services required to treat cognitive disorders in the United States alone will exceed $2 trillion annually. Mammalian forkhead transcription factors, silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae), the mechanistic target of rapamycin, and the pathways of autophagy and apoptosis offer exciting avenues to address these challenges by focusing upon core cellular mechanisms that may significantly impact nervous system disease. These pathways are intimately linked such as through cell signaling pathways involving protein kinase B and can foster, sometimes in conjunction with trophic factors, enhanced neuronal survival, reduction in toxic intracellular accumulations, and mitochondrial stability. Feedback mechanisms among these pathways also exist that can oversee reparative processes in the nervous system. However, mammalian forkhead transcription factors, silent mating type information regulation 2 homolog 1, mechanistic target of rapamycin, and autophagy can lead to cellular demise under some scenarios that may be dependent upon the precise cellular environment, warranting future studies to effectively translate these core pathways into successful clinical treatment strategies for neurodegenerative disorders.

Background: The mTOR-PI3K-Akt pathway influences cell metabolism and (malignant) cell growth. We generated sex-specific polygenic risk scores capturing natural variation in 7 out of 10 top-ranked genes in this pathway. We studied the scores directly and in interaction with energy balance-related factors (body mass index (BMI), trouser/skirt size, height, physical activity, and early life energy restriction) in relation to colorectal cancer (CRC) risk in the Netherlands Cohort Study (NLCS) (n=120,852). The NLCS has a case-cohort design and 20.3 years of follow-up. Participants completed a baseline questionnaire on diet and cancer in 1986 when 55–69 years old. ~75% of the cohort returned toenail clippings used for DNA isolation and genotyping (n subcohort=3,793, n cases=3,464). To generate the scores, the dataset was split in two and risk alleles were defined and weighted based on sex-specific associations with CRC risk in the other dataset half, because there were no SNPs in the top-ranked genes associated with CRC risk in previous genome-wide association studies at a significance level p<1*10−5. Results: Cox regression analyses showed positive associations between the sex-specific polygenic risk scores and colon but not rectal cancer risk in men and women, with hazard ratios for continuously modeled scores close to 1.10. There was no modifying effect observed of the scores on associations between the energy balance-related factors and CRC risk. However, BMI (in men), non-occupational physical activity (in women), and height (in men and women) were associated with the risk of CRC, in particular (proximal and distal) colon cancer, in the direction as expected in the lower tertiles of the sex-specific polygenic risk scores. Conclusions: Current data suggest that the mTOR-PI3K-Akt pathway may be involved in colon cancer development. This study thereby sheds more light on colon cancer etiology through use of genetic variation in the mTOR-PI3K-Akt pathway. [ABSTRACT FROM AUTHOR]

Insulin stimulates protein synthesis in skeletal muscles. Protein synthesis is controlled by the mechanistic target of rapamycin (mTOR) signaling in skeletal muscles. This study was conducted to investigate the effect of insulin on protein synthesis and mTOR signaling in chick myotube cultures. Chick myotubes were incubated with insulin (1 µg/ml) for 1 h. Protein synthesis, measured using the surface sensing of translation method, was significantly increased by insulin. The phosphorylation of AKT (Thr308 and Ser473), p70 ribosomal S6 kinase 1 (S6K1, Thr389), S6 ribosomal protein (Ser235/236), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46) was also significantly increased by insulin. These results suggest that insulin stimulates protein synthesis via mTOR signaling (phosphorylation of AKT, S6K1, S6 ribosomal protein, and 4E-BP1) in chick myotube cultures.

Autophagy in the skeletal muscle increases under catabolic conditions resulting in muscle atrophy. This study investigated the effect of inhibition of mechanistic target of rapamycin (mTOR) on autophagy in chick skeletal muscle. We examined the effects of Torin1, an mTOR inhibitor, on autophagy. Chick myotubes were incubated with Torin1 (100 nM) for 3 h. It was observed that Torin1 inhibited the phosphorylation of AKT (Ser473), p70 ribosomal S6 kinase 1 (S6K1, Thr389), S6 ribosomal protein (Ser235/236), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46), which are used for measurement of mTOR activity. Torin1 significantly (P< 0.01) increased the LC3-II/LC3-I ratio, an index for autophagosome formation, while it did not influence the expression of autophagy-related genes (LC3B, GABARAPL1, and ATG12). In addition, Torin1 increased atrogin-1/MAFbx (a muscle-specific ubiquitin ligase) mRNA expression. Fasting for 24 h inhibited the phosphorylation of AKT (Ser473), S6K1 (Ther389), S6 ribosomal protein (Ser235/236), and 4E-BP1 (Thr37/46) in chick skeletal muscle and significantly (P

Environmental enrichment is known to be beneficial for cognitive improvement. In many animal models of neurological disorders and brain injury, EE has also demonstrated neuroprotective benefits in neurodegenerative diseases and in improving recovery after stroke or traumatic brain injury. The exact underlying mechanism for these phenomena has been unclear. Recent findings have now indicated that neuronal activity elicited by environmental enrichment induces Ca2+ influx in dorsal root ganglion neurons results in lasting enhancement of CREB-binding protein-mediated histone acetylation. This, in turn, increases the expression of pro-regeneration genes and promotes axonal regeneration. This mechanism associated with neuronal activity elicited by environmental enrichment-mediated pathway is one of several epigenetic mechanisms which modulate axon regeneration upon injury that has recently come to light. The other prominent mechanisms, albeit not yet directly associated with environmental enrichment, include DNA methylation/demethylation and N6-methyladenosine modification of transcripts. In this brief review, I highlight recent work that has shed light on the epigenetic basis of environmental enrichment-based axon regeneration, and discuss the mechanism and pathways involved. I further speculate on the implications of the findings, in conjunction with the other epigenetic mechanisms, that could be harness to promote axon regeneration upon injury.

Placental System L amino acid transporter activity is decreased in pregnancies complicated by intrauterine growth restriction (IUGR) and increased in fetal overgrowth. However, it is unknown if changes in the expression/activity of placental Large Neutral Amino Acid Transporter Small Subunit 1 (Slc7a5/LAT1) are mechanistically linked to placental function and fetal growth. We hypothesized that trophoblast-specific Slc7a5 overexpression increases placental transport of essential amino acids, activates the placental mechanistic target of rapamycin (mTOR) signaling, and promotes fetal growth in mice. Using lentiviral transduction of blastocysts with a Slc7a5 transgene, we achieved trophoblast-specific overexpression of Slc7a5 (Slc7a5 OX) with increased fetal (+27%) and placental weights (+10%). Trophoblast-specific Slc7a5 overexpression increased trophoblast plasma membrane (TPM) LAT1 protein abundance and TPM System L transporter (+53%) and System A transporter activity (+ 21%). Slc7a5 overexpression also increased transplacental transport of leucine (+ 85%) but not of the System A tracer, 14C-methylamino isobutyric acid, in vivo. Trophoblast-specific overexpression of Slc7a5 activated placental mTORC1, as assessed by increased (+44%) phosphorylation of S6 ribosomal protein (Ser 235/236), and mTORC2 as indicated by phosphorylation of PKCα-Tyr-657 (+47%) and Akt-Ser 473 (+96%). This is the first demonstration that placental transport of essential amino acids is mechanistically linked to fetal growth. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter in some cases of fetal overgrowth may directly contribute to the development of these pregnancy complications., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Aims: Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA‐34a (miR‐34a) among the most upregulated miRs in tubers. Here, we characterised miR‐34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR‐34a. Methods: We analysed the expression of miR‐34a in resected cortical tubers (n = 37) compared with autopsy‐derived control tissue (n = 27). The effect of miR‐34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from three patients with TSC and in SH‐SY5Y cells following miR‐34a transfection. Results: The peak of miR‐34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. miR‐34a was also strongly expressed in foetal TSC cortex. Overexpression of miR‐34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR‐34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1. Conclusions: MicroRNA‐34a is most highly overexpressed in tubers during foetal and early postnatal brain development. miR‐34a can negatively regulate mTORC1; however, it may also contribute to abnormal corticogenesis in TSC. [ABSTRACT FROM AUTHOR]

The kinase called mechanistic Target of Rapamycin (mTOR) belongs to a complex network pathway responsible for many physiological mechanisms, responses to multiple factors and usually is deregulated in various types of diseases. In vitro studies support the idea that the binding of mammalian lethal with SEC13 protein 8 (mLst8) subunit on mTOR is necessary to initiate the kinase phosphotransference process. Thus, the mLst8 subunit is important for the regulation of mTOR complex, but its function, especially on catalytic site, needs to be defined. In addition, the contribution of mLst8 to the development and control of pathologies, particularly those in which the mTOR pathways seems deregulated, remains unsolved or with few available data and could be an important step to develop new strategies avoiding the superexpression of this pathway. In our study, we employed coarse-grained molecular dynamics simulation to analyse the behaviour of the mTOR catalytic site when the subunit mLst8 is associated or not, providing insights about the importance of mTOR-mLst8 interaction for the kinase mechanism. [ABSTRACT FROM AUTHOR]