Inhibition Of mTORC1 Activity Research Articles

Mitochondrial localization of SESN2.

SESN2 is a member of the evolutionarily conserved sestrin protein family found in most of the Metazoa species. The SESN2 gene is transcriptionally activated by many stress factors, including metabolic derangements, reactive oxygen species (ROS), and DNA-damage. As a result, SESN2 controls ROS accumulation, metabolism, and cell viability. The best-known function of SESN2 is the inhibition of the mechanistic target of rapamycin complex 1 kinase (mTORC1) that plays a central role in support of cell growth and suppression of autophagy. SESN2 inhibits mTORC1 activity through interaction with the GATOR2 protein complex preventing an inhibitory effect of GATOR2 on the GATOR1 protein complex. GATOR1 stimulates GTPase activity of the RagA/B small GTPase, the component of RagA/B:RagC/D complex, preventing mTORC1 translocation to the lysosomes and its activation by the small GTPase Rheb. Despite the well-established role of SESN2 in mTORC1 inhibition, other SESN2 activities are not well-characterized. We recently showed that SESN2 could control mitochondrial function and cell death via mTORC1-independent mechanisms, and these activities might be explained by direct effects of SESN2 on mitochondria. In this work, we examined mitochondrial localization of SESN2 and demonstrated that SESN2 is located on mitochondria and can be directly involved in the regulation of mitochondrial functions.

Exercise Improves the Fasting Regulation of Skeletal Muscle Protein Synthesis in Tumor Bearing Mice

IntroductionCancer is a debilitating disease that is often accompanied by decreased physical activity and chronic energetic stress that disrupts muscle proteostasis. Skeletal muscle protein turnover is highly sensitive to changes in feeding and activity. Raptor (Ser 792) and TSC1/2 (Ser 1387) phosphorylation can inhibit mTORC1 activation and suppress anabolic signaling, which has been implicated in cancer‐induced skeletal muscle wasting.PurposeTo examine the effect of the cancer environment on the fasting regulation of Raptor and TSC1/2 in male ApcMin/+ mice and determine if increased physical activity could alter this response.MethodsMale C57BL/6 (B6, N=24) and ApcMin/+ (MIN, N=31) mice were either sacrificed under ad libitum conditions (B6‐fed, M‐fed), fasted for 12hrs (B6‐fast, M‐fast), or fasted for 12hrs following 4wks of voluntary wheel running (B6+W, M+W). TSC1/2 serine 1387 and Raptor serine 792 were measured in the gastrocnemius muscle as phosphorylation to total ratio by western blot. Protein synthesis was measured by puromycin incorporation.ResultsAll MIN mice exhibited body weight loss (p<0.001) and reduced gastrocnemius mass (p<0.001) when compared to all B6 mice. Raptor phosphorylation (pRaptor) was induced in M‐fast compared to M‐fed (p=0.019), which was not changed by fasting in B6 mice (p=0.414). TSC1/2 phosphorylation was induced in M‐fast compared to M‐fed (p=0.001) and in B6‐fast to B6‐fed (p=0.039). Puromycin was trending to be reduced in M‐fast compared to MIN‐fed (p=0.070), but there was no change in B6‐fast to B6‐fed (p=0.323). Raptor phosphorylation was not different in M‐fast compared to M+W (p=0.302), however pRaptor was reduced in B6+W compared B6‐fast (p=0.028). TSC1/2 phosphorylation (p=0.001) and puromycin (p=0.016) were induced in MIN+W compared to M‐fast, with no changes in the B6‐fast to B6+W (p=0.360, p=0.196; respectively).ConclusionsOur results provide evidence that the cancer environment disrupts fasting regulation of mTORC1 in skeletal muscle. Interestingly, TSC1/2 phosphorylation was sensitive to fasting independent of the tumor environment, while wheel activity induced protein synthesis independent of Raptor phosphorylation. Therefore, further study is warranted to determine physical activities regulation of fasting skeletal muscle protein synthesis in tumor bearing mice.Support or Funding InformationAcknowledgementsNCI R01‐CA121249

Mitochondrial Localization and Function of SESN2

The goal of this study was to understand the potential mechanisms responsible for the regulation of metabolism and mitochondrial functions by SESTRIN2 (SESN2) and other members of the SESTRIN protein family. Our studies are based on cell fractionation and microscopy analyses to determine the localization of SESN2 and on theC. elegans model to define the role of SESTRINs in mitochondrial respiration.SESN2 is a member of evolutionarily conserved SESTRIN protein family found in the majority of theMetazoa species. SESN2 is transcriptionally activated by many stress factors including metabolic derangements, oxidants, and DNA‐damage. As a result, SESN2 suppresses oxidative stress, tunes up metabolic pathways, and regulates cell viability. Many of the activities of SESN2 are linked to regulation of mechanistic target of rapamycin complex 1 kinase (mTORC1) that plays the central role in the regulation of cell growth, metabolism, and autophagy. SESN2 inhibits mTORC1 activity through the interaction with the GATOR2 protein complex that prevents the inhibitory effect of GATOR2 on the GATOR1 protein complex and relieves an inhibitory effect of GATOR1 towards RagA/B GTPases, the major activators of mTORC1. Despite the well‐established role of SESN2 in the mTORC1 inhibition, some other SESN2 activities are not well characterized. As established by us and other groups, SESN2 can control mitochondrial respiration and cell death via mTORC1‐independent mechanisms. We hypothesized that these processes can be explained by the direct effects of SESN2 on mitochondria. To study the potential localization of SESN2 on mitochondria we performed mitochondrial fractionation from different human cancer cell lines and observed that considerable amounts of SESN2 protein were co‐purified with mitochondria. According to the results of treatment with proteases, SESN2 was located on the outer mitochondrial membrane. While we observed no association of SESN2 with the AKAP1 protein, as was previously reported, we found that the mitochondrial localization of SESN2 depends on GATOR2. We determine that mitochondrial SESN2 might be responsible for the regulation of the accumulation of reactive oxygen species and respiration in mammalian cells andC. elegans.We have shown that SESN2 is associated with mitochondria and can be directly involved in direct regulation of mitochondria functions.Support or Funding InformationThis work was supported by Russian Science Foundation grant 17‐14‐01420.

Ibudilast enhances the clearance of SOD1 and TDP-43 aggregates through TFEB-mediated autophagy and lysosomal biogenesis: The new molecular mechanism of ibudilast and its implication for neuroprotective therapy

A key feature of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders including Alzheimer disease (AD), Parkinson disease (PD) and Huntington’s disease (HD) is abnormal aggregation and deposition of misfolded proteins. Previous studies have shown that autophagy plays an important role in the clearance of disease-linked protein aggregates. In the current study, we report that ibudilast, which is a non-selective inhibitor of phosphodiesterases (PDEs) and an anti-inflammation drug, can induce autophagy and lysosomal biogenesis through mammalian target of rapamycin complex 1 - transcription factor EB (mTORC1-TFEB) signaling. We have found that ibudilast significantly enhances the clearance of disease-linked TAR DNA binding protein (TDP-43) and superoxide dismutase 1 (SOD1) protein aggregates in transfected cellular models carrying corresponding gene mutations. The mechanistic study revealed that ibudilast could markedly enhance TFEB nuclear translocation and increase the autolysosomes by inhibiting mTORC1 activity. We have also demonstrated that ibudilast could protect TDP-43-induced cytotoxicity in motor neuron-like NSC-34 cells. Collectively, our study identifies ibudilast as an autophagy enhancer and provides insights into the molecular basis of ibudilast for the potential treatment of several neurodegenerative disorders.

ESC-sEVs Rejuvenate Senescent Hippocampal NSCs by Activating Lysosomes to Improve Cognitive Dysfunction in Vascular Dementia.

Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism is unclear and there is still lack of effective medications. In this study, the VD rats exhibit a progressive cognitive impairment, as well as a time‐related increasing in hippocampal neural stem cells (H‐NSCs) senescence, lost and neurogenesis decline. Then, embryonic stem cell‐derived small extracellular vesicles (ESC‐sEVs) are intravenously injected into VD rats. ESC‐sEVs treatment significantly alleviates H‐NSCs senescence, recovers compromised proliferation and neuron differentiation capacity, and reverses cognitive impairment. By microarray analysis and RT‐qPCR it is identified that several miRNAs including miR‐17‐5p, miR‐18a‐5p, miR‐21‐5p, miR‐29a‐3p, and let‐7a‐5p, that can inhibit mTORC1 activation, exist in ESC‐sEVs. ESC‐sEVs rejuvenate H‐NSCs senescence partly by transferring these miRNAs to inhibit mTORC1 activation, promote transcription factor EB (TFEB) nuclear translocation and lysosome resumption. Taken together, these data indicate that H‐NSCs senescence cause cell depletion, neurogenesis reduction, and cognitive impairment in VD. ESC‐sEVs treatment ameliorates H‐NSCs senescence by inhibiting mTORC1 activation, and promoting TFEB nuclear translocation and lysosome resumption, thereby reversing senescence‐related neurogenesis dysfunction and cognitive impairment in VD. The application of ESC‐sEVs may be a novel cell‐free therapeutic tool for patients with VD, as well as other aging‐related diseases.

Podocyte-specific deletion of tubular sclerosis complex 2 promotes focal segmental glomerulosclerosis and progressive renal failure.

Obesity can initiate and accelerate the progression of kidney diseases. However, it remains unclear how obesity affects renal dysfunction. Here, we show that a newly generated podocyte-specific tubular sclerosis complex 2 (Tsc2) knockout mouse model (Tsc2Δpodocyte) develops proteinuria and dies due to end-stage renal dysfunction by 10 weeks of age. Tsc2Δpodocyte mice exhibit an increased glomerular size and focal segmental glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis and proteinaceous casts. Podocytes isolated from Tsc2Δpodocyte mice show nuclear factor, erythroid derived 2, like 2-mediated increased oxidative stress response on microarray analysis and their autophagic activity is lowered through the mammalian target of rapamycin (mTOR)—unc-51-like kinase 1 pathway. Rapamycin attenuated podocyte dysfunction and extends survival in Tsc2Δpodocyte mice. Additionally, mTOR complex 1 (mTORC1) activity is increased in podocytes of renal biopsy specimens obtained from obese patients with chronic kidney disease. Our work shows that mTORC1 hyperactivation in podocytes leads to severe renal dysfunction and that inhibition of mTORC1 activity in podocytes could be a key therapeutic target for obesity-related kidney diseases.

MTORC1 signaling pathway regulates macrophages in choroidal neovascularization

Macrophages are involved in choroidal neovascularization (CNV). The mechanistic target of rapamycin complex 1 (mTORC1) is a central cell regulator, but mTORC1 function in macrophages in CNV is not fully understood. We explored the effect of mTORC1 pathway regulation on macrophages in CNV. A laser-induced murine CNV model was performed. Expression of phospho-S6 and F4/80 in CNV lesions was analyzed by immunofluorescence. Macrophages in CNV lesions were found at 1 day after laser treatment, reached a peak at 5 days, and decreased at 7 and 14 days. mTORC1 activity of cells in CNV lesions was increased from 3 to 7 days, and deceased at 14 days. Most infiltrating macrophages in CNV lesions had strong mTORC1 activity at 3 and 5 days that subsequently decreased. In vitro, THP-1 macrophages were polarized to M1 or M2 with rapamycin or siRNA treatment. The human retinal pigment epithelium (RPE) cell line ARPE-19 was co-cultured with macrophages. Cytokine expression of macrophages and ARPE-19 cells was detected by quantitative PCR. Inhibiting mTORC1 activity of macrophages reduced M1 and strengthened M2, which was reversed by mTORC1 hyperactivation. Both M1 and M2 macrophages induced RPE cells to express less PEDF and more MMP9, IL-1β and MCP-1. Inhibiting or enhancing mTORC1 activity of macrophages changed cytokine expression of RPE cells. Together, we demonstrated that macrophage functions in CNV were regulated partly by the mTORC1 pathway, and mTORC1 activity of macrophages influenced the expression of cytokines that are associated with CNV development in RPE cells. This study provides more understanding about the regulatory mechanism of macrophages in CNV.

CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy

Mitochondria house anabolic and catabolic processes that must be balanced and adjusted to meet cellular demands. The RNA‐binding protein CLUH (clustered mitochondria homolog) binds mRNAs of nuclear‐encoded mitochondrial proteins and is highly expressed in the liver, where it regulates metabolic plasticity. Here, we show that in primary hepatocytes, CLUH coalesces in specific ribonucleoprotein particles that define the translational fate of target mRNAs, such as Pcx, Hadha, and Hmgcs2, to match nutrient availability. Moreover, CLUH granules play signaling roles, by recruiting mTOR kinase and the RNA‐binding proteins G3BP1 and G3BP2. Upon starvation, CLUH regulates translation of Hmgcs2, involved in ketogenesis, inhibits mTORC1 activation and mitochondrial anabolic pathways, and promotes mitochondrial turnover, thus allowing efficient reprograming of metabolic function. In the absence of CLUH, a mitophagy block causes mitochondrial clustering that is rescued by rapamycin treatment or depletion of G3BP1 and G3BP2. Our data demonstrate that metabolic adaptation of liver mitochondria to nutrient availability depends on a compartmentalized CLUH‐dependent post‐transcriptional mechanism that controls both mTORC1 and G3BP signaling and ensures survival.

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0–0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.

Dstyk mutation leads to congenital scoliosis-like vertebral malformations in zebrafish via dysregulated mTORC1/TFEB pathway

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.

Scutellarin protects against myocardial ischemia-reperfusion injury by suppressing NLRP3 inflammasome activation

BackgroundActivation of NLRP3 inflammasome plays a key role in cardiac dysfunction for acute myocardial ischemia-reperfusion injury. Scutellarin (Scu) is a flavonoid purified from Erigeron breviscapus. Whether Scu has any influence on the activation of NLRP3 inflammasome in cardiomyocytes remains unknown. PurposeWe aimed to examine the therapeutic effect of Scu on cardiomyocyte ischemia-reperfusion (I/R) injury and its effect on NLRP3 inflammasome in rats with acute myocardial I/R injury and anoxia/reoxygenation (A/R)-induced H9c2 injuries. MethodsHeart injuries were induced through 30 min of ischemia followed by 24 h of reperfusion. Scu was intraperitoneally administered 15 min before vascular ligation. Effects of Scu on cardiac injury were detected by echocardiograms, TTC staining, and histological and immunohistochemical analyses. The effects of Scu on biochemical parameters were analyzed. H9c2 cells were pretreated with different concentrations of Scu for 6 h before A/R exposure. Afterward, cell viability, LDH release, and Hoechst 33342 and peromide iodine double staining were determined. Western blot analyses of proteins, including those involved in autophagy, NLRP3, mTOR complex 1 (mTORC1), and Akt signaling, were conducted. ResultsIn vivo study revealed that Scu improved diastolic dysfunction, ameliorated myocardium structure abnormality, inhibited myocyte apoptosis and inflammatory response, and promoted autophagy. Scu reduced NLRP3 inflammasome activation, inhibited mTORC1 activity, and increased Akt phosphorylation. In vitro investigation showed the same results. The Scu-mediated NLRP3 inflammasome and mTORC1 inhibition and cardioprotection were abolished through the genetic silencing of Akt by siRNA. ConclusionsThe cardioprotective effect of Scu was achieved through its anti-inflammatory effect. It suppressed the activation of NLRP3 inflammasome. In addition, inflammasome restriction by Scu was dependent on Akt activation and mTORC1 inhibition.

MTORC1 in the orbitofrontal cortex promotes habitual alcohol seeking.

The mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in dendritic translation and in learning and memory. We previously showed that heavy alcohol use activates mTORC1 in the orbitofrontal cortex (OFC) of rodents (Laguesse et al., 2017a). Here, we set out to determine the consequences of alcohol-dependent mTORC1 activation in the OFC. We found that inhibition of mTORC1 activity in the OFC attenuates alcohol seeking and restores sensitivity to outcome devaluation in rats that habitually seek alcohol. In contrast, habitual responding for sucrose was unaltered by mTORC1 inhibition, suggesting that mTORC1's role in habitual behavior is specific to alcohol. We further show that inhibition of GluN2B in the OFC attenuates alcohol-dependent mTORC1 activation, alcohol seeking and habitual responding for alcohol. Together, these data suggest that the GluN2B/mTORC1 axis in the OFC drives alcohol seeking and habit.

Abstract B108: 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors

Abstract Aberrantly activated PI3K/mTOR signaling is frequently implicated as an oncogenic driver in human cancer. In response to nutrient and growth factor stimuli, mTOR complex 1 (mTORC1) regulates cell growth and cap-dependent protein translation through phosphorylation and activation of S6K, and through phosphorylation and inactivation of the translational repressor 4EBP1. While suppression of mTORC1 activity causes rapid dephosphorylation of its substrates S6K and 4EBP1, it has also been reported that the translational repressor 4EBP3 is transcriptionally induced during prolonged mTORC1 inhibition.1 We have designed a series of complete and selective bi-steric inhibitors of mTORC1 by covalently linking rapamycin, a selective yet incomplete mTORC1 inhibitor, with mTOR kinase active-site inhibitors. These compounds potently and durably suppress phosphorylation of S6K and 4EBP1, induce growth suppression and apoptosis in multiple cancer cell lines, and cause tumor growth inhibition in xenograft models. In the present study we evaluated the utility of 4EBP3 mRNA expression as a pharmacodynamic (PD) biomarker of mTORC1 inhibition by our bi-steric inhibitors. We further interrogated whether tumor mTORC1 inhibition and anti-tumor efficacy could be predicted by monitoring this PD marker in surrogate tissue. First, we demonstrated that 4EBP3 mRNA and protein levels increased in response to mTORC1 inhibition in a concentration- and time-dependent manner in MDA-MB-468, MCF-7, and HCC1954 breast carcinoma cells. Kinetic studies in which we compared the response of bi-steric inhibitors to that of short duration-of-action active-site inhibitors revealed that 4EBP3 induction requires sustained mTORC1 inhibition, as loss of mTORC1 inhibition correlated with a rapid return to baseline 4EBP3 expression. Treatment with inhibitors of varying potency against mTORC1 also demonstrated a strong correlation between magnitude of cell growth inhibition and 4EBP3 mRNA induction. Additionally, suppression of tumor 4EBP1 phosphorylation and induction of 4EBP3 mRNA was observed in MCF-7 and HCC9154 tumor xenografts following in vivo administration of bi-steric inhibitors with a concomitant induction of murine 4ebp3 in peripheral blood mononuclear cells (PBMCs). Initial studies indicate that ex vivo exposure of human PBMCs to a mTORC1 inhibitor also promotes 4EBP3 mRNA expression. Together, these data suggest that 4EBP3 could provide a sensitive mRNA-based in vivo biomarker of prolonged mTORC1 inhibition, as is the case following administration of a single dose of a bi-steric mTORC1 inhibitor. The concordance between the responses in tumor and PBMC suggests it may be possible to monitor tumor mTORC1 inhibition by monitoring 4EBP3 in surrogate tissue. In summary, 4EBP3 mRNA represents a potential PD biomarker of mTORC1 inhibitor response as an alternative to measurement of the phosphorylation status of mTORC1 substrates. 1. Tsukumo, T., et al. Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3. Nat Commun 7:11776. doi: 10.1038/ncomms11776 (2016). Citation Format: Bianca J. Lee, Nuntana Dinglasan, Tram Nguyen, Ed Lorenzana, Stacy Wilson, G. Leslie Burnett, James B. Aggen, Robert J. Nichols, Mallika Singh, David Wildes, Jaqueline A.M. Smith. 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B108. doi:10.1158/1535-7163.TARG-19-B108

The tumor suppressor NDRG2 cooperates with an mTORC1 inhibitor to suppress the Warburg effect in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most common malignancies worldwide, and metabolic reprogramming has a profound effect on RCC tumorigenesis. mTORC1 inhibitors are widely used in RCC treatment, yet some types of RCC cells are resistant to these compounds. Thus, clarification of the metabolic mechanism of mTORC1 inhibitors and exploration of new therapeutic approaches are urgently needed. In this study, we found that the mTORC1 pathway was hyperactive in RCC. Immunohistochemistry and western blot analysis showed that phosphorylation of the mTORC1 substrate 4EBP1 at threonine 37/46 increased in RCC tissues compared with that in normal renal tissues. It was also found that mTORC1 inhibitor everolimus suppressed glucose consumption, lactate production, and multiple catalytic enzymes involved in glycolysis in 786-O and ACHN cells, but the accumulation of HIF1α induced by CoCl2 blocked the inhibitory effect of everolimus on aerobic glycolysis. Interestingly, western blot and metabolite analysis showed that the tumor suppressor NDRG2 (N-Myc downstream regulated gene 2) was able to inhibit mTORC1 activity and cooperate with an mTOR inhibitor to decrease aerobic glycolysis in 786-O and ACHN cells. These results demonstrate that NDRG2 may potentially synergize with mTORC1 inhibitors to suppress malignant phenotype of RCC. Taken together, these data provided preclinical evidence that the combination of NDRG2 and mTORC1 inhibitors might be a promising strategy for RCC therapy.

MTORC1 to AMPK switching underlies β-cell metabolic plasticity during maturation and diabetes.

Pancreatic beta cells (β-cells) differentiate during fetal life, but only postnatally acquire the capacity for glucose-stimulated insulin secretion (GSIS). How this happens is not clear. In exploring what molecular mechanisms drive the maturation of β-cell function, we found that the control of cellular signaling in β-cells fundamentally switched from the nutrient sensor target of rapamycin (mTORC1) to the energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK), and that this was critical for functional maturation. Moreover, AMPK was activated by the dietary transition taking place during weaning, and this in turn inhibited mTORC1 activity to drive the adult β-cell phenotype. While forcing constitutive mTORC1 signaling in adult β-cells relegated them to a functionally immature phenotype with characteristic transcriptional and metabolic profiles, engineering the switch from mTORC1 to AMPK signaling was sufficient to promote β-cell mitochondrial biogenesis, a shift to oxidative metabolism, and functional maturation. We also found that type 2 diabetes, a condition marked by both mitochondrial degeneration and dysregulated GSIS, was associated with a remarkable reversion of the normal AMPK-dependent adult β-cell signature to a more neonatal one characterized by mTORC1 activation. Manipulating the way in which cellular nutrient signaling pathways regulate β-cell metabolism may thus offer new targets to improve β-cell function in diabetes.

Pentapeptide Protects INS-1 Cells From hIAPP-Mediated Apoptosis by Enhancing Autophagy Through mTOR Pathway.

The human islet amyloid polypeptide (hIAPP), the major component of islet amyloid deposition, is one of the amyloidogenic peptides and has been associated with β cell loss and dysfunction in type 2 diabetes (T2D). Autophagy plays a central role in the clearance of hIAPP aggregates, thereby diminishing the hIAPP-induced cytotoxicity. Conversely, hIAPP has been reported to have interfering effects on the autophagy. The pentapeptide FLPNF developed in our previous study has been shown to have effects on the level of the downstream proteins of mTOR and autophagy–lysosome pathway. In the present study, the peptide FLPNF-mediated increase in autophagy flux and its underlying mechanisms, as well as its protecting effect on INS-1 cells, were investigated. Autophagy flux in INS-1 cells overexpressing hIAPP (hIAPP-INS-1 cells) markedly increased after exposure to peptide FLPNF for 24 h and peaked at a concentration of 200 µM. Peptide FLPNF enhanced the autophagy by inhibiting the mTORC1 activity. Flow cytometry results showed the peptide FLPNF bind to mammalian target of rapamycin (mTOR), and further molecular docking analysis revealed a direct interaction between peptide FLPNF and the FRB domain of mTOR. Meanwhile, both peptide FLPNF and rapamycin significantly decreased the hIAPP-induced apoptosis, whereas 3-MA increased the apoptosis. Furthermore, peptide FLPNF reduced the hIAPP oligomer and improved the hIAPP-INS-1 cells insulin release function at high glucose concentration. Taken together, the peptide FLPNF decreased the hIAPP oligomer via upregulating autophagy by inhibiting mTORC1 activity, thus protecting the INS-1 cells from hIAPP-induced apoptosis and improving the insulin release function of INS-1 cells.

Regulation of YAP by Mammalian Target of Rapamycin Complex 1 in Endothelial Cells Controls Blood Pressure Through COX-2/mPGES-1/PGE2 Cascade.

Endothelial cells regulate vascular tone by producing both relaxing and contracting factors to control the local blood flow. Hypertension is a common side effect of mTORC1 (mammalian target of rapamycin complex 1) inhibitors. However, the role of endothelial mTORC1 in hypertension remains elusive. The present study aimed to determine the role of endothelial mTORC1 in Ang II (angiotensin II)-induced hypertension and the underlying mechanism. Endothelial mTORC1 activity was increased by Ang II both in vitro and in vivo. Blood pressure was higher in Tie-2-Cre-mediated regulatory associated protein of mTOR (mammalian target of rapamycin; Raptor) heterozygous-deficient (Tie2Cre-RaptorKD) mice than control mice both before and after Ang II infusion. Acetylcholine-evoked endothelium-dependent relaxation of mesenteric arteries was impaired in Tie2Cre-RaptorKD mice. Treatment with indomethacin or a specific COX (cyclooxygenase)-2 inhibitor, NS-398, but not L-NG-nitroarginine methyl ester reduced endothelium-dependent relaxation in Raptorflox/- mice to a similar extent as in Tie2Cre-RaptorKD mice. Metabolomic profiling revealed that the plasma content of prostaglandin E2 was reduced in Tie2Cre-RaptorKD mice with or without Ang II infusion. In endothelial cells, reduction of the protein level of YAP (yes-associated protein) with siRNA-mediated RPTOR deficiency was autophagy dependent and transcriptionally regulated the expression of COX-2 and mPGES-1 (microsomal prostaglandin E synthase-1). Hence, overexpression of YAP in endothelial cells enhanced the mRNA and protein levels of COX-2 and mPGES-1 and reversed the endothelial dysfunction and hypertension in Tie2Cre-RaptorKD mice. The present results demonstrate that suppression of mTORC1 activity in endothelial cells reduces prostaglandin E2 production and causes hypertension by reducing YAP-mediated COX-2/mPGES-1 expression.

GPCR signaling inhibits mTORC1 via PKA phosphorylation of Raptor.

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth, metabolism, and autophagy. Extensive research has focused on pathways that activate mTORC1 like growth factors and amino acids; however, much less is known about signaling cues that directly inhibit mTORC1 activity. Here, we report that G-protein coupled receptors (GPCRs) paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA) and inhibit mTORC1. Mechanistically, PKA phosphorylates the mTORC1 component Raptor on Ser 791, leading to decreased mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, mTORC1 activity is partially rescued even after PKA activation. Gαs-coupled GPCRs stimulation leads to inhibition of mTORC1 in multiple cell lines and mouse tissues. Our results uncover a signaling pathway that directly inhibits mTORC1, and suggest that GPCRs paired to Gαs proteins may be potential therapeutic targets for human diseases with hyperactivated mTORC1.

Vitamin D increases glucocorticoid efficacy via inhibition of mTORC1 in experimental models of multiple sclerosis

The limited efficacy of glucocorticoids (GCs) during therapy of acute relapses in multiple sclerosis (MS) leads to long-term disability. We investigated the potential of vitamin D (VD) to enhance GC efficacy and the mechanisms underlying this VD/GC interaction. In vitro, GC receptor (GR) expression levels were quantified by ELISA and induction of T cell apoptosis served as a functional readout to assess synergistic 1,25(OH)2D3 (1,25D)/GC effects. Experimental autoimmune encephalomyelitis (MOG35–55 EAE) was induced in mice with T cell-specific GR or mTORc1 deficiency. 25(OH)D (25D) levels were determined in two independent cohorts of MS patients with stable disease or relapses either responsive or resistant to GC treatment (initial cohort: n = 110; validation cohort: n = 85). Gene expression of human CD8+ T cells was analyzed by microarray (n = 112) and correlated with 25D serum levels. In vitro, 1,25D upregulated GR protein levels, leading to increased GC-induced T cell apoptosis. 1,25D/GC combination therapy ameliorated clinical EAE course more efficiently than respective monotherapies, which was dependent on GR expression in T cells. In MS patients from two independent cohorts, 25D deficiency was associated with GC-resistant relapses. Mechanistic studies revealed that synergistic 1,25D/GC effects on apoptosis induction were mediated by the mTOR but not JNK pathway. In line, 1,25D inhibited mTORc1 activity in murine T cells, and low 25D levels in humans were associated with a reduced expression of mTORc1 inhibiting tuberous sclerosis complex 1 in CD8+ T cells. GR upregulation by 1,25D and 1,25D/GC synergism in vitro and therapeutic efficacy in vivo were abolished in animals with a T cell-specific mTORc1 deficiency. Specific inhibition of mTORc1 by everolimus increased the efficacy of GC in EAE. 1,25D augments GC-mediated effects in vitro and in vivo in a T cell-specific, GR-dependent manner via mTORc1 inhibition. These data may have implications for improvement of anti-inflammatory GC therapy.

Activation of the mTORC1/PGC-1 axis promotes mitochondrial biogenesis and induces cellular senescence in the lung epithelium.

Cellular senescence is a biological process by which cells lose their capacity to proliferate yet remain metabolically active. Although originally considered a protective mechanism to limit the formation of cancer, it is now appreciated that cellular senescence also contributes to the development of disease, including common respiratory ailments such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. While many factors have been linked to the development of cellular senescence, mitochondrial dysfunction has emerged as an important causative factor. In this study, we uncovered that the mitochondrial biogenesis pathway driven by the mammalian target of rapamycin/peroxisome proliferator-activated receptor-γ complex 1α/β (mTOR/PGC-1α/β) axis is markedly upregulated in senescent lung epithelial cells. Using two different models, we show that activation of this pathway is associated with other features characteristic of enhanced mitochondrial biogenesis, including elevated number of mitochondrion per cell, increased oxidative phosphorylation, and augmented mitochondrial reactive oxygen species (ROS) production. Furthermore, we found that pharmacological inhibition of the mTORC1 complex with rapamycin not only restored mitochondrial homeostasis but also reduced cellular senescence to bleomycin in lung epithelial cells. Likewise, mitochondrial-specific antioxidant therapy also effectively inhibited mTORC1 activation in these cells while concomitantly reducing mitochondrial biogenesis and cellular senescence. In summary, this study provides a mechanistic link between mitochondrial biogenesis and cellular senescence in lung epithelium and suggests that strategies aimed at blocking the mTORC1/PGC-1α/β axis or reducing ROS-induced molecular damage could be effective in the treatment of senescence-associated lung diseases.