mTOR-related Proteins Research Articles

Rumen-protected methionine supplementation improves lactation performance and alleviates inflammation during a subclinical mastitis challenge in lactating dairy cows

This study aimed to evaluate the effects of rumen-protected Met on lactation performance, inflammation, and immune response, and liver glutathione of lactating dairy cows during a subclinical mastitis challenge (SMC). Thirty-two Holstein cows (145 ± 51 DIM) were enrolled in a randomized complete block design. At -21 d relative to the SMC, cows were assigned to dietary treatments, and data were collected before and during the SMC. Cows were blocked according to parity, DIM, and milk yield and received a basal diet (17.4% CP; Lys 7.01% MP and Met 2.14% MP) plus 100 g/d of ground corn (CON; n = 16) or a basal diet plus 100 g/d of ground corn and rumen-protected Met (SM, Smartamine M at 0.09% of dietary DM; n = 16), fed as a top-dress. At 0 d, the mammary gland's rear right quarter was infused with 100,000 cfu of Streptococcus uberis (O140J). Milk yield was recorded twice daily from 0 until 3 d relative to SMC. Milk samples were collected during each milking from 0 to 3 d relative to SMC, blood samples were collected at 0, 6, 12, 24, 48, and 72 h relative to SMC. The mTOR pathway activation was assessed in immune cells in blood and milk samples by measuring quantity and phosphorylation status of mTOR-related proteins, including AKT, S6RP, and 4EBP1. For the ratio of phosphorylated to total AKT, S6RP, and 4EBP1, blood samples were collected at 0, 12, and 24 h, and milk samples at 24 h relative to SMC. Liver biopsies were performed at -10 d and 24 h relative to SMC for measurement of glutathione. Linear mixed models with repeated measures were used to analyze the results. There was a trend for greater milk yield per milking (+ 0.8 kg) and per day (+1.7 kg) after SMC in SM cows compared with CON. The DMI was not affected by dietary treatments. Reactive oxygen metabolites were lower in SM cows than in CON. Milk somatic cell linear score was not affected by dietary treatments, and a score >4 at 24 h confirmed subclinical mastitis. The SM cows had greater milk fat percentage at 24 and 36 h post SMC, resulting in overall greater milk fat. Milk protein tended to be greater in SM cows than in CON. We observed greater liver glutathione in SM cows than in CON. Among inflammation biomarkers, ceruloplasmin was lower for SM cows compared with CON. In milk, greater phosphorylated (p) AKT:AKT and pS6RP:S6RP ratios were observed in immune cell populations from SM cows compared with CON. Blood neutrophils had a greater p4EBP1:4EBP1 ratio in SM cows compared with CON. Overall, our results show that Met supplementation during an SMC positively affected milk performance, lowered the risk of oxidative stress, and attenuated inflammation partially by increasing liver glutathione and immune cells' protein synthesis via mTOR signaling.

The casual association between mTOR-related proteins and rheumatoid arthritis: Mendelian randomization in European populations

The casual association between mTOR-related proteins and rheumatoid arthritis: Mendelian randomization in European populations

IL-32γ Induced Autophagy Through Suppression of MET and mTOR Pathways in Liver Tumor Growth Inhibition.

Interleukin-32γ (IL-32γ) has diverse functions in various malignancies. In this study, we investigated the role of IL-32γ in autophagy induction in liver cancer cells and delineated the underlying mechanisms. We found that the increased IL-32γ expression inhibited the growth, cell cycle progression, and migration of HepG2 and Hep3B cell lines; it also decreased the expression of related proteins. Furthermore, the IL-32γ overexpression induced autophagy, as indicated by the number of puncta, the expression of LC3, and the expression of autophagy-related markers. The expression levels of LAMP1, a protein essential for autophagosome formation, and colocalization with LC3 also increased. Big data analysis revealed that the expression of MET, a well-known target of autophagy, and the expression of mTOR and mTOR-related proteins were decreased by the IL-32γ overexpression. The combination treatment of MET inhibitor, cabozantinib (2 µM), and IL-32γ overexpression further increased the number of puncta, the colocalization of LC3 and LAMP1, and the expression of autophagy-related proteins. In vivo, liver tumor growth was suppressed in the IL-32γ-overexpressing mouse model, and autophagy induction was confirmed by the increased expression of LC3 and LAMP1 and the decreased expression of autophagy pathway markers (MET and mTOR). Autophagy was also decreased in the liver tumor sample of human patients. ROC curve and spearman analysis revealed that the expression levels of LC3 and IL-32γ were significantly correlated in human tumor serum and tissues. Therefore, IL-32γ overexpression induced autophagy in liver tumors through the suppression of MET and mTOR pathways critical for tumor growth inhibition.

Rumen-protected methionine supplementation during the transition period under artificially induced heat stress: Effects on cow-calf performance

This study aimed to evaluate the effects of rumen-protected Met on lactation performance, inflammation and immune response, and liver glutathione of lactating dairy cows during a subclinical mastitis challenge (SMC). Thirty-two Holstein cows (145 ± 51 DIM) were enrolled in a randomized complete block design. At -21 d relative to the SMC, cows were assigned to dietary treatments, and data were collected before and during the SMC. Cows were blocked according to parity, DIM, and milk yield and received a basal diet (17.4% CP; Lys 7.01% MP and Met 2.14% MP) plus 100 g/d of ground corn (CON; n = 16) or a basal diet plus 100 g/d of ground corn and rumen-protected Met (SM, Smartamine M at 0.09% of dietary DM; n = 16), fed as a top-dress. At 0 d, the mammary gland's rear right quarter was infused with 100,000 cfu of Streptococcus uberis (O140J). Milk yield was recorded twice daily from 0 until 3 d relative to SMC. Milk samples were collected during each milking from 0 to 3 d relative to SMC, blood samples were collected at 0, 6, 12, 24, 48, and 72 h relative to SMC. The mTOR pathway activation was assessed in immune cells in blood and milk samples by measuring quantity and phosphorylation status of mTOR-related proteins, including AKT, S6RP, and 4EBP1. For the ratio of phosphorylated to total AKT, S6RP, and 4EBP1, blood samples were collected at 0, 12, and 24 h, and milk samples at 24 h relative to SMC. Liver biopsies were performed at -10 d and 24 h relative to SMC for measurement of glutathione. Linear mixed models with repeated measures were used to analyze the results. There was a trend for greater milk yield per milking (+ 0.8 kg) and per day (+1.7 kg) after SMC in SM cows compared with CON. The DMI was not affected by dietary treatments. Reactive oxygen metabolites (ROM) were lower in SM cows than in CON. Milk somatic cell linear score was not affected by dietary treatments, and a score >4 at 24 h confirmed subclinical mastitis. The SM cows had greater milk fat percentage at 24 and 36 h post SMC, resulting in overall greater milk fat. Milk protein tended to be greater in SM cows than in CON. We observed greater liver glutathione in SM cows than in CON. Among inflammation biomarkers, ceruloplasmin was lower for SM cows compared with CON. In milk, greater pAKT:AKT and pS6RP:S6RP ratios were observed in immune cell populations from SM cows compared with CON. Blood neutrophils had a greater p4EBP1:4EBP1 ratio in SM cows compared with CON. Overall, our results show that Met supplementation during an SMC positively affected milk performance, lowered the risk of oxidative stress, and attenuated inflammation partially by increasing liver glutathione and immune cells' protein synthesis via mTOR signaling.

Tumorigenic role of tacrolimus through mTORC1/C2 activation in post-transplant renal cell carcinomas.

Kidney transplant recipients (KTRs) face an increased risk of renal cell carcinoma (RCC), in which the immunosuppressive regimen plays an important role. This study aimed to identify intracellular signalling alterations associated with post-transplant (post-tx) tumour formation. Expression of mTOR-related proteins were analysed in kidneys obtained from end-stage renal disease (ESRD) patients and RCCs developed in KTRs or non-transplant patients. The effects of tacrolimus (TAC) and rapamycin (RAPA) on mTOR activity, proliferation, and tumour growth were investigated through different in vitro and in vivo experiments. Elevated mTORC1/C2 activity was observed in post-tx RCCs and in kidneys of TAC-treated ESRD patients. In vitro experiments demonstrated that TAC increases mTOR activity in a normal tubular epithelial cell line and in the investigated RCC cell lines, moreover, promotes the proliferation of some RCC cell line. In vivo, TAC elevated mTORC1/C2 activity in ischaemic kidneys of mice and enhanced tumour growth in xenograft model. We observed significantly increased mTOR activity in ischaemic kidneys and post-tx RCCs, which highlights involvement of mTOR pathway both in the healing or fibrotic processes of kidney and in tumorigenesis. TAC-treatment further augmented the already elevated mTOR activity of injured kidney, potentially contributing to tumorigenesis during immunosuppression.

Protein interaction network analysis of mTOR signaling reveals modular organization

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (phosphoinositide 3-kinases), protein kinases (AKT), and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex coimmunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum-deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after 1h, despite phosphorylation changes observed after only 5min. Using small molecule inhibitors of phosphoinositide 3-kinase, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed "modules", that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to the activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.

KLF5‑mediated expression of CARD11 promotes the progression of gastric cancer.

Caspase recruitment domain-containing protein 11 (CARD11) has been reported as functioning in multiple types of cancers. In the present study, the role and mechanism of CARD11 in gastric cancer was investigated. First, CARD11 expression in gastric cancer tissues and the association of CARD11 with overall survival were analyzed by the encyclopedia of RNA interactomes database. CARD11 expression in gastric cancer cells was detected by western blotting and reverse transcription-quantitative PCR analyses. After CARD11 silencing, cell proliferation was evaluated by Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining and flow cytometry analysis. Wound healing and Transwell assays were used to measure the capacities of cell migration and invasion. Additionally, the expression levels of epithelial-mesenchymal transition (EMT)-related proteins and mTOR-related proteins were detected by western blot analysis. HumanTFDB predicted the binding of the transcription factor Krüppel-like factor 5 (KLF5) to the CARD11 promoter, which was confirmed by dual luciferase reporter and chromatin immunoprecipitation assays. To explore the regulatory effects between KLF5 and CARD11, KLF5 was overexpressed to perform the rescue experiments in gastric cancer cells with CARD11 silencing. Results revealed that CARD11 was highly expressed in gastric cancer and was associated with poor prognosis. CARD11 interference inhibited the proliferation of gastric cancer cells and induced cell cycle arrest. Additionally, CARD11 silencing suppressed the migration, invasion and EMT of gastric cancer cells, accompanied by upregulated E-cadherin expression and downregulated N-cadherin and vimentin expression. Moreover, the transcription factor KLF5 positively regulated the transcription of CARD11 in gastric cancer. KLF5 overexpression reversed the effects of interference of CARD11 expression in gastric cancer cells to promote their proliferation, migration, invasion and EMT. KLF5 overexpression also led to a reduction in cell cycle arrest. Finally, interference of CARD11 expression suppressed the mTOR pathway, which was activated by KLF5. In conclusion, KLF5-mediated CARD11 promoted the proliferation, migration and invasion of gastric cancer cells.

ID: 204592 Differential Target Multiplexed and Low-Rate SCS regulate mTOR Signaling in a Model of Neuropathic Pain

Neuropathic pain is mediated by neuroinflammation. We have demonstrated that SCS using differential target multiplexed programming (DTMP) provides significant relief of pain-like behavior in an animal model of neuropathic pain. Transcriptomics and proteomics of the stimulated spinal cord indicates that DTMP regulates biological process involved in neuroinflammation via balancing of perturbed neuron-glial interactions. The mammalian target of rapamycin (mTOR) signaling pathway (directed by protein complexes mTORC1 and mTORC2) has a direct effect on inflammatory pathways. It contains various regulating proteins that can activate or inhibit a pain state. The effect of DTMP and Low-Rate (LR) SCS on mTOR-related proteins will be presented.

Tuberous sclerosis complex-1 (TSC1) contributes to selective neuronal vulnerability in Alzheimer's disease.

Selective neuronal vulnerability of hippocampal Cornu Ammonis (CA)-1 neurons is a pathological hallmark of Alzheimer's disease (AD) with an unknown underlying mechanism. We interrogated the expression of tuberous sclerosis complex-1 (TSC1; hamartin) and mTOR-related proteins in hippocampal CA1 and CA3 subfields. A human post-mortem cohort of mild (n= 7) and severe (n= 10) AD and non-neurological controls (n= 9) was used for quantitative and semi-quantitative analyses. We also developed an in vitro TSC1 knockdown model in rat hippocampal neurons, and transcriptomic analyses of TSC1 knockdown neuronal cultures were performed. We found a selective increase of TSC1 cytoplasmic inclusions in human AD CA1 neurons with hyperactivation of one of TSC1's downstream targets, the mammalian target of rapamycin complex-1 (mTORC1), suggesting that TSC1 is no longer active in AD. TSC1 knockdown experiments showed accelerated cell death independent of amyloid-beta toxicity. Transcriptomic analyses of TSC1 knockdown neuronal cultures revealed signatures that were significantly enriched for AD-related pathways. Our combined data point to TSC1 dysregulation as a key driver of selective neuronal vulnerability in the AD hippocampus. Future work aimed at identifying targets amenable to therapeutic manipulation is urgently needed to halt selective neurodegeneration, and by extension, debilitating cognitive impairment characteristic of AD.

MiR-926-3p influences myocardial injury in septic mice through regulation of mTOR signaling pathway by targeting TSC1.

The purpose of this study is to investigate the influence of miR-926-3p on myocardial injury and its mechanisms. An animal model of sepsis was constructed by CLP, and animals were randomly divided into 4 groups: C group, miR-926-3p inhibitor group, CLP + NC group, and CLP + miR-926-3p inhibitor group. Compared with those in C group, echocardiographic parameters remarkably declined in CLP + NC group. Compared with CLP + NC group, miR-926-3p inhibitor group indicated elevated echocardiographic parameters in mice, pathological improvement tendency of myocardial tissues and distinct reduction in cardiomyocyte apoptosis. It could be observed by electron microscopy that the number of lysosomes in miR-926-3p inhibitor group was greatly increased relative to CLP + NC group. Immunofluorescence exhibited that the number of green fluorescent puncta was significantly higher in miR-926-3p inhibitor group as compared to that in CLP + NC group. The autophagic flow was verified by observing the relative expression of LC3II at different times. The results of Western blotting manifested that miR-926-3p inhibitor up-regulated mTOR-related protein expressions and down-regulated the protein expression of p-mTOR. LPS was adopted to induce cardiomyocyte injury in vitro, and the results confirmed that, identical to in vivo experiments, miR-926-3p inhibitor was able to up-regulate the protein expressions of mTOR-related protein and down-regulate p-mTOR protein expression in cardiomyocytes. After addition of MHY1485, The expression of mTOR-related proteins changes in each group. Inhibition of miR-926-3p enhances autophagy through regulation of the mTOR signaling pathway, thus ameliorating myocardial injury in septic mice.

Activation of Neuroinflammation via mTOR Pathway is Disparately Regulated by Differential Target Multiplexed and Traditional Low-Rate Spinal Cord Stimulation in a Neuropathic Pain Model.

IntroductionSpinal cord stimulation (SCS) has been used for decades to treat neuropathic pain conditions with limited understanding of its mechanisms of action. The mTOR pathway is a well-known co-factor in chronic pain and has not been previously linked to SCS therapy. Proteomic and phosphorylation analyses allow capturing a broad view of tissue response to an injury model and subsequent therapies such as SCS. Here, we evaluated the effect of differential target multiplexed SCS programming (DTMP) and traditional low-rate spinal cord stimulation (LR-SCS) on the mTOR pathway using proteomic and phosphoproteomic analyses.MethodsThe spared nerve injury (SNI) model of neuropathic pain in animals was established followed by continuous treatment with either DTMP or LR-SCS for 48 hours. Control groups included sham-stimulated (No-SCS) and uninjured animals (No-SNI). Proteins were extracted from spinal cord tissue removed post-stimulation and subjected to liquid chromatography/tandem mass spectrometry to assess changes in protein expression and states of phosphorylation. Bioinformatics tools and literature were used to identify mTOR-related proteins in the various groups.ResultsOver 7000 proteins were identified and filtered to find 1451 and 705 proteins significantly affected by DTMP and LR-SCS (p < 0.05), respectively, relative to No-SCS. Literature and bioinformatic tools yielded 192 mTOR-related proteins that were cross-referenced to the list of DTMP and LR-SCS affected proteins. Of these proteins, 49 were found in the proteomic dataset. Eight of these proteins showed a significant response to the pain model, 25 were significantly modulated by DTMP, and 8 by LR-SCS. Phosphoproteomic analyses yielded 119 mTOR-related phosphoproteins affected by the injury model with a 66% reversal following DTMP versus a 58% reversal by LR-SCS.ConclusionProteomic and phosphoproteomic analyses support the hypothesis that DTMP, and to a lesser extent LR-SCS, reverse injury induced changes of the mTOR pathway while treating neuropathic pain.

Jiedu Tongluo Baoshen formula enhances podocyte autophagy and reduces proteinuria in diabetic kidney disease by inhibiting PI3K/Akt/mTOR signaling pathway

Ethnopharmacological relevanceTraditional Chinese medicine (TCM) has been applied to diabetic kidney disease (DKD). A large number of animal trials each year focus on TCM for DKD, but the evidence for these preclinical studies is not clear. Aim of the studyThe aim of this study was to study the therapeutic effect of Jiedu Tongluo Baoshen formula (JTBF) on DKD proteinuria and renal protection. At the same time, it is verified that JTBF can reduce podocyte injury by enhancing autophagy function, and then achieve the effect of proteinuria. Materials and methodsWe use high performance liquid chromatography to detect and analyze the fingerprint of JTBF to find the chemical composition. Subsequently, we constructed a DKD rat model induced by high-fat diet and streptozocin (HFD + STZ). Urine and blood biochemical automatic analyzer were used to detect 24-h urine protein quantification (24 h-UP) and renal function. The renal pathological changes were observed by H&E and transmission electron microscopy (TEM), and the levels of autophagy-related proteins and mRNA in podocytes were detected by immunohistochemistry, RT-qPCR and Western Blot. The chemical composition of JTBF was screened from traditional Chinese medicine systems pharmacol (TCMSP) and PubChem databases, and the potential targets and associated pathways of JTBF were predicted using kyoto encyclopedia of genes and genomes (KEGG) and protein-protein interaction (PPI) network analysis in network pharmacology, and confirmed in animal experiments and histopathological methods. ResultsWe discovered 77 active ingredients of JTBF. Through animal experiments, it was found that JTBF reduced 24 h-UP and promoted the expression of podocin, nephrin, and WT-1 in podocytes, thereby reducing podocyte damage. At the same time, JTBF activates the expression of podocyte autophagy-related proteins (beclin-1, LC3 and P62). Subsequently, through network pharmacology predictions, 208 compounds were obtained from JTBF, and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) was a potential signal pathway. JTBF was obtained in DKD rat kidney tissue to inhibit the expression of PI3K, Akt and mTOR related proteins. ConclusionsJTBF enhance podocyte autophagy to reduce podocyte damage, thereby effectively treating DKD proteinuria and protecting kidney function.

ASPP2 Coordinates ERS-Mediated Autophagy and Apoptosis Through mTORC1 Pathway in Hepatocyte Injury Induced by TNF-α.

Background: Though ASPP2 plays an important role in regulating cell apoptosis and autophagy in case of liver injury, there remains a lack of clarity on the molecular mechanism of ASPP2 regulating autophagy and apoptosis. Methods: A hepatocyte injury model was constructed using HL7702 cell line and TNF-α. The cells were treated by ASPP2 overexpression adenovirus or short hairpin RNA lentivirus and endoplasmic reticulum stress (ERS) or the mammalian target of rapamycin (mTOR) inhibitor or agonist, respectively. The autophagy was detected by means of western blot and Green fluorescent protein-labeled- Microtubule-associated protein light chain 3 (GFP-LC3) plasmid transfection, while the apoptosis was detected through western blot, flow cytometry and TUNEL assay. Besides, the proteins related to ERS and mTOR were detected by western blot. Results: The low level of ASPP2 expression was accompanied by high-level autophagy and low-level apoptosis and vice versa in case of hepatocyte injury induce by TNF-α. By upregulating the proteins related to mTORC1 and ERS, ASPP2 induced apoptosis but inhibited autophagy. However, the effect of ASPP2 on autophagy and apoptosis can be reversed by the use of mTORC1 and ERS interfering agent, which indicates that ASPP2 regulated autophagy and apoptosis through mTORC1and ERS pathway. ERS treatment made no difference to the expression of ASPP2 and mTOR-related proteins, which suggests the possibility that the regulation of ERS on apoptosis and autophagy could occur in the downstream of ASPP2 and mTOR. Conclusion: ASPP2 could inhibit autophagy and induce apoptosis through mTORC1-ERS pathway in case of the hepatocyte injury induce by TNF-α. The role of ASPP2-mTORC1-ERS axis was verified in hepatocyte injury, which suggests the possibility that ASPP2 is an important regulatory molecule for the survival and death of hepatocyte.

A Profound Basic Characterization of eIFs in Gliomas: Identifying eIF3I and 4H as Potential Novel Target Candidates in Glioma Therapy.

Simple SummaryGliomas are brain tumors with currently limited therapy options. Glioma growth and proliferation is regulated by the mTOR pathway together with eukaryotic initiation factors (eIFs). In this work we show a profound basic characterization of eIFs in human gliomas and demonstrate increased mRNA and protein expressions of several eIFs in gliomas compared to healthy control brain tissue. Moreover, increased eIF3I and eIF4H levels seem to have a negative influence on the survival of patients. Our work suggests eIF3I and eIF4H as potential targets for future glioma therapy.Glioblastoma (GBM) is an utterly devastating cerebral neoplasm and current therapies only marginally improve patients’ overall survival (OS). The PI3K/AKT/mTOR pathway participates in gliomagenesis through regulation of cell growth and proliferation. Since it is an upstream regulator of the rate-limiting translation initiation step of protein synthesis, controlled by eukaryotic initiation factors (eIFs), we aimed for a profound basic characterization of 17 eIFs to identify potential novel therapeutic targets for gliomas. Therefore, we retrospectively analyzed expressions of mTOR-related proteins and eIFs in human astrocytoma samples (WHO grades I–IV) and compared them to non-neoplastic cortical control brain tissue (CCBT) using immunoblot analyses and immunohistochemistry. We examined mRNA expression using qRT-PCR and additionally performed in silico analyses to observe the influence of eIFs on patients’ survival. Protein and mRNA expressions of eIF3B, eIF3I, eIF4A1, eIF4H, eIF5 and eIF6 were significantly increased in high grade gliomas compared to CCBT and partially in low grade gliomas. However, short OS was only associated with high eIF3I gene expression in low grade gliomas, but not in GBM. In GBM, high eIF4H gene expression significantly correlated with shorter patient survival. In conclusion, we identified eIF3I and eIF4H as the most promising targets for future therapy for glioma patients.

Suppression of Allograft Fibrosis by Regulation of Mammalian Target of Rapamycin-Related Protein Expression in Kidney-Transplanted Recipients Treated with Everolimus and Reduced Tacrolimus.

BackgroundAlthough renoprotective effects of everolimus (EVR) in kidney transplantation (KTx) have been widely reported, its pathophysiological mechanism remains unclear.Material/MethodsWe compared changes in eGFR (ΔGFR, ml/min/1.73 m2) and the ratio of the fibrotic area in biopsy specimens (ΔFI,%) from 3 months to 3 years after KTx between the EVR+ group (EVR addition and Tac reduction early after KTx, n=32), and the EVR− group (normal Tac without EVR, n=28). We also immunohistochemically evaluated mTOR-related protein expression.ResultsΔGFR and ΔFI in the EVR+ vs. EVR− groups were −0.27±6.8 vs. −9.8±12.8 (p<0.001) and 2.4±4.9 vs. 9.5±10.5 (p<0.001), respectively. Phosphorylated mTOR and phosphorylated 4EBP1 expression at 3 years in the EVR+ group was significantly lower than that in the EVR− group. Moreover, in the subgroup analysis comparing ΔGFR and ΔFI among groups stratified by immunosuppressive regimen and mTOR signal enhancement, the ΔFI in patients with EVR+ with decreased mTOR signal enhancement was significantly milder than that in other patients. In addition, in the multivariate analysis, EVR addition was the only independent predictor for allograft fibrosis, whereas the Tac C0 concentration at neither 1 nor 3 years proved to be a risk factor.ConclusionsThese results suggested that EVR addition and Tac reduction may attenuate kidney allograft fibrosis, and that the suppression of mTOR signaling process may be involved in the anti-fibrotic effect of this immunosuppressive regimen. These results provide suggestions of how to utilize EVR for patients with KTx and improve graft function.

In Situ Metabolic Characterisation of Breast Cancer and Its Potential Impact on Therapy

Simple SummaryMetabolic rewiring, as an important hallmark of cancer supports the bioenergetic needs of growing tumours at the primary site or in metastases. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to metabolic plasticity and progression in cancers. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Using a set of immunoreactions, we observed that the characterisation of metabolic pathways has a prognostic potential in human cases. High in situ metabolic plasticity was considered with high mTOR activity and high expression of at least two other studied metabolic enzymes (lactate dehydrogenase A, glutaminase, fatty acid synthase or carnitine palmitoyltransferase). According to our metabolic analyses and immunohistochemistry results, breast cancer is characterised by considerable metabolic diversity in individual cases. Consequently, we suggest selecting patients who may benefit from more accurate follow-up and specific therapies (including the combination of anti-metabolic treatments and recent therapies).In spite of tremendous developments in breast cancer treatment, the relatively high incidence of relapsing cases indicates a great need to find new therapeutic strategies in recurrent, metastatic and advanced cases. The bioenergetic needs of growing tumours at the primary site or in metastases—accumulating genomic alterations and further heterogeneity—are supported by metabolic rewiring, an important hallmark of cancer. Adaptation mechanisms as well as altered anabolic and catabolic processes balance according to available nutrients, energy, oxygen demand and overgrowth or therapeutic resistance. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to this metabolic plasticity and progression in breast carcinomas. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Cellular metabolism and mTOR-related protein expression were characterised in ten cell lines, along with their sensitivity to specific mTOR and other metabolic inhibitors. Selected immunohistochemical reactions were performed on ~100 surgically removed breast cancer specimens. The obtained protein expression scores were correlated with survival and other clinicopathological data. Metabolic and mTOR inhibitor mono-treatments had moderate antiproliferative effects in the studied cell lines in a subtype-independent manner, revealing their high adaptive capacity and survival/growth potential. Immunohistochemical analysis of p-S6, Rictor, lactate dehydrogenase A, glutaminase, fatty acid synthase and carnitine palmitoyltransferase 1A in human samples identified high mTOR activity and potential metabolic plasticity as negative prognostic factors for breast cancer patients, even in subtypes generally considered as low-risk. According to our results, breast cancer is characterised by considerable metabolic diversity, which can be targeted by combining antimetabolic treatments and recent therapies. Alterations in these pathways may provide novel targets for future drug development in breast cancer. We also propose a set of immunostainings for scoring metabolic heterogeneity in individual cases in order to select patients who may benefit from more accurate follow-up and specific therapies.

Effects of Endurance Exercise Bouts in Hypoxia, Hyperoxia, and Normoxia on mTOR-Related Protein Signaling in Human Skeletal Muscle.

Przyklenk, A, Aussieker, T, Gutmann, B, Schiffer, T, Brinkmann, C, Strüder, HK, Bloch, W, Mierau, A, and Gehlert, S. Effects of endurance exercise bouts in hypoxia, hyperoxia, and normoxia on mTOR-related protein signaling in human skeletal muscle. J Strength Cond Res 34(8): 2276-2284, 2020-This study investigated the effects of short-term hypoxia (HY), hyperoxia (PER), and normoxia on anabolic signaling proteins in response to an acute bout of moderate endurance exercise (EEX) before and after an endurance exercise training intervention. Eleven healthy male subjects conducted one-legged cycling endurance exercise (3 × 30 min·wk for 4 weeks). One leg was trained under hypoxic (12% O2) or hyperoxic conditions (in a randomized cross-over design), and the other leg was trained in normoxia (20.9% O2) at the same relative workload. Musculus vastus lateralis biopsies were taken at baseline (T0) as well as immediately after the first (T1) and last (T2) training session to analyze anabolic signaling proteins and the myofiber cross-sectional area (FCSA). No significant differences were detected for FCSA between T0 and T2 under all oxygen conditions (p > 0.05). No significant differences (p > 0.05) were observed for BNIP3, phosphorylated RSK1, ERK1/2, FoxO3a, mTOR, and S6K1 between all conditions and time points. Phosphorylated Akt/PKB decreased significantly (p < 0.05) at T1 in PER and at T2 in HY and PER. Phosphorylated rpS6 decreased significantly (p < 0.05) at T1 only in PER, whereas nonsignificant increases were shown in HY at T2 (p = 0.10). Despite no significant regulations, considerable reductions in eEF2 phosphorylation were detected in HY at T1 and T2 (p = 0.11 and p = 0.12, respectively). Short-term hypoxia in combination with moderate EEX induces favorable acute anabolic signaling responses in human skeletal muscle.

Increased mTOR and suppressed autophagic flux in the heart of a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease

Cardiac hypertrophy is common in autosomal dominant polycystic kidney disease (ADPKD) patients. We found increased heart weight in Pkd1RC/RC and Pkd2WS25/+ mouse models of ADPKD. As there is a link between increased heart weight and mammalian target of rapamycin (mTOR), the aim of the study was to determine mTOR complex 1 and 2 signaling proteins in the heart in the Pkd1RC/RC mouse model of PKD. In 70 day old Pkd1RC/RC hearts, on immunoblot analysis, there was a large increase in p-AMPKThr172, a known autophagy inducer, and an increase in p-AktSer473 and p-AktThr308, but no increase in other mTORC1/2 proteins (p-S6Ser240/244, p-mTORSer2448). In 150 day old Pkd1RC/RC hearts, there was an increase in mTORC1 (p-S6Ser240/244) and mTOR-related proteins (p-AktThr308, p-GSK3βSer9, p-AMPKThr172). As the mTOR pathway is the master regulator of autophagy, autophagy proteins were measured. There was an increase in p-Beclin-1 (BECN1), an autophagy regulator and activating molecule in Beclin-1-regulated autophagy (AMBRA1), a regulator of Beclin that play a role in autophagosome formation, an early stage of autophagy. There was a defect in the later stage of autophagy, the fusion of the autophagosome with the lysosome, known as autophagic flux, as evidenced by the lack of an increase in LC3-II, a marker of autophagosomes, with the lysosomal inhibitor bafilomycin, in both 70 day old and 150 day old hearts. To determine the role of autophagy in causing increased heart weight, Pkd1RC/RC were treated with 2-deoxyglucose (2-DG) or Tat-Beclin1 peptide, agents known to induce autophagy. 2-DG treatment from 150 to 350 days of age, a time period when increased heart weight developed, did not reduce the increased heart weight. Unexpectedly, Tat-Beclin 1 peptide treatment from 70 to 120 days of age resulted in increased heart weight. In summary, there is suppressed autophagic flux in the heart at an early age in Pkd1RC/RC mice. Increased mTOR signaling in older mice is associated suppressed autophagic flux. There was a large increase in p-AMPKThr172, a known autophagy inducer, in both young and old mice. 2-DG treatment did not impact increased heart weight and Tat-Beclin1 peptide increased heart weight.

Differential accumulation of storage bodies with aging defines discrete subsets of microglia in the healthy brain.

To date, microglia subsets in the healthy CNS have not been identified. Utilizing autofluorescence (AF) as a discriminating parameter, we identified two novel microglia subsets in both mice and non-human primates, termed autofluorescence-positive (AF+) and negative (AF-). While their proportion remained constant throughout most adult life, the AF signal linearly and specifically increased in AF+ microglia with age and correlated with a commensurate increase in size and complexity of lysosomal storage bodies, as detected by transmission electron microscopy and LAMP1 levels. Post-depletion repopulation kinetics revealed AF- cells as likely precursors of AF+ microglia. At the molecular level, the proteome of AF+ microglia showed overrepresentation of endolysosomal, autophagic, catabolic, and mTOR-related proteins. Mimicking the effect of advanced aging, genetic disruption of lysosomal function accelerated the accumulation of storage bodies in AF+ cells and led to impaired microglia physiology and cell death, suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

1894-P: Interruption of Glucagon Signaling Increases Pancreas Mass

Interruption of glucagon signaling by glucagon receptor gene inactivation or treatment with a glucagon receptor blocking monoclonal antibody (GCGR-Ab) stimulates alpha cell proliferation and expansion. Surprisingly, pancreas weight is also increased in mice with GCG gene inactivation. To investigate mechanisms linking glucagon signaling and pancreas size, we treated C57BL/6J mice with GCGR-Ab or a control IgG. Eight weeks of treatment (N=4-15 mice/treatment) increased absolute pancreas weight (0.42±0.013 vs. 0.32±0.008; p&amp;lt;0.0001), pancreas weight normalized to body weight (1.39±0.03% vs. 1.11±0.02%; p&amp;lt;0.0001), and serum levels of 15 amino acids (AA), including glutamine (1.7-4.67 fold, p&amp;lt;0.0001). GCGR-Ab treatment promoted acinar cell proliferation (Ki67+, 0.65±0.14% vs. 0.20±0.03%; p=0.0058), increased acinar cell size (peri-islet region: 438.4±12.77 vs. 341.7±22.05μm2; p=0.0045; peripheral region: 431.1±9.34 vs. 334.2±25μm2, p=0.0055; central region: 437.0±10.45 vs. 338.7±13.6 μm2, p=0.0006), and increased pancreas protein content (248.3±9.84 vs. 194±8.67μg/mg tissue; p=0.0003). By electron microscopy, GCGR-Ab treatment increased acinar cell zymogen granule number (129±9 vs. 49±6; p&amp;lt;0.0001; n=28-37 cells/treatment) and led to acinar cell ER dilatation, but did not induce pancreatitis or alter acinar cell apoptosis or pancreatic macrophage number. Serum cholecystokinin and total glucagon-like peptide-1, trophic hormones linked to exocrine growth, were not changed by GCGR-Ab treatment. GCGR-Ab treatment upregulated mRNA and protein levels of the glutamine transporter, Slc38a3, and increased mTOR-related ribosomal protein S6 phosphorylation in acinar cells. Together, these data indicate that interruption of glucagon signaling increases pancreas mass by inducing acinar cell hyperplasia and hypertrophy and activates protein synthesis and mTOR signaling; whether this is mediated by elevated glutamine or AA levels is not known. Disclosure C. Dai: None. A. Bradley: None. K.C. Coate: None. G. Poffenberger: None. E. Spears: None. J.J. Wright: None. S. Shrestha: None. D. Dean: None. M. Brissova: None. A.C. Powers: None. Funding National Institutes of Health (DK117147)