Role of L-glutamine in the regulation of rat Sertoli cell proliferation by FSH.

In brief: FSH leads to glutamine dependence, which is required for mTORC1 activation and in consequence Sertoli cell proliferation. Abstract: The spermatogenic capacity of adult individuals depends on, among other factors, the number of Sertoli cells (SCs) that result from the proliferative waves during development. FSH upregulates SC proliferation at least partly, through the activation of the PI3K/Akt/mTORC1 pathway, among other mechanisms. It is widely known that mTORC1 is a sensor of amino acids. Among amino acids, glutamine acquires relevance since it might contribute to cell cycle progression through the modulation of mTORC1 activity. It has not been studied yet whether glutamine intervenes in FSH-mediated regulation of SC proliferation and cell cycle progression, or if FSH has any effect on glutamine metabolism. Eight-day-old rat SCs were incubated in culture media without glutamine or with glutamine in the absence or presence of a glutamine transporter inhibitor or a glutaminase activity inhibitor under basal conditions or stimulated with FSH. The results obtained show that FSH does not promote SC proliferation and mTORC1 activation in the absence of glutamine. Also, FSH modulates glutamine metabolism increasing glutaminase isoform 2 and reducing glutamine synthetaseexpression. FSH did not promote SC proliferation and mTORC1 activation when glutaminase activity was inhibited. The results suggest that glutamine or its metabolites might cooperate with FSH in the upregulation of SC proliferation through mTORC1. In addition, as FSH modulates glutamine metabolism through the induction of glutaminase isoform 2, the hormonal control of glutamine metabolism might be part of the intricate signaling network triggered by FSH, which is crucial to establish the population of mature SCs that supports the reproductive function.

Metformin induces ZFP36 by mTORC1 inhibition in cervical cancer-derived cell lines.

BACKGROUND: Metformin, a widely prescribed antidiabetic drug, has shown several promising effects for cancer treatment. These effects have been shown to be mediated by dual modulation of the AMPK-mTORC1 axis, where AMPK acts upstream of mTORC1 to decrease its activity. Nevertheless, alternative pathways have been recently discovered suggesting that metformin can act through of different targets regulation. METHODS: We performed a transcriptome screening analysis using HeLa xenograft tumors generated in NOD-SCID mice treated with or without metformin to examine genes regulated by metformin. Western Blot analysis, Immunohistochemical staining, and RT-qPCR were used to confirm alterations in gene expression. The TNMplot and GEPIA2 platform were used for in silico analysis of genes found up-regulated by metformin, in cervical cancer patients. We performed an AMPK knock-down using AMPK-targeted siRNAs and mTOR inhibition with rapamycin to investigate the molecular mechanisms underlying the effect of metformin in cervical cancer cell lines. RESULTS: We shown that metformin decreases tumor growth and increased the expression of a group of antitumoral genes involved in DNA-binding transcription activator activity, hormonal response, and Dcp1-Dcp2 mRNA-decapping complex. We demonstrated that ZFP36 could act as a new molecular target increased by metformin. mTORC1 inhibition using rapamycin induces ZFP36 expression, which could suggest that metformin increases ZFP36 expression and requires mTORC1 inhibition for such effect. Surprisingly, in HeLa cells AMPK inhibition did not affect ZFP36 expression, suggesting that additional signal transducers related to suppressing mTORC1 activity, could be involved. CONCLUSIONS: These results highlight the importance of ZFP36 activation in response to metformin treatment involving mTORC1 inhibition.

Glutaminolysis regulates endometrial fibrosis in intrauterine adhesion via modulating mitochondrial function.

BACKGROUND: Endometrial fibrosis, a significant characteristic of intrauterine adhesion (IUA), is caused by the excessive differentiation and activation of endometrial stromal cells (ESCs). Glutaminolysis is the metabolic process of glutamine (Gln), which has been implicated in multiple types of organ fibrosis. So far, little is known about whether glutaminolysis plays a role in endometrial fibrosis. METHODS: The activation model of ESCs was constructed by TGF-ß1, followed by RNA-sequencing analysis. Changes in glutaminase1 (GLS1) expression at RNA and protein levels in activated ESCs were verified experimentally. Human IUA samples were collected to verify GLS1 expression in endometrial fibrosis. GLS1 inhibitor and glutamine deprivation were applied to ESCs models to investigate the biological functions and mechanisms of glutaminolysis in ESCs activation. The IUA mice model was established to explore the effect of glutaminolysis inhibition on endometrial fibrosis. RESULTS: We found that GLS1 expression was significantly increased in activated ESCs models and fibrotic endometrium. Glutaminolysis inhibition by GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES or glutamine deprivation treatment suppressed the expression of two fibrotic markers, α-SMA and collagen I, as well as the mitochondrial function and mTORC1 signaling in ESCs. Furthermore, inhibition of the mTORC1 signaling pathway by rapamycin suppressed ESCs activation. In IUA mice models, BPTES treatment significantly ameliorated endometrial fibrosis and improved pregnancy outcomes. CONCLUSION: Glutaminolysis and glutaminolysis-associated mTOR signaling play a role in the activation of ESCs and the pathogenesis of endometrial fibrosis through regulating mitochondrial function. Glutaminolysis inhibition suppresses the activation of ESCs, which might be a novel therapeutic strategy for IUA.

The exception to the rule? TORC1 triggers growth under low nutrient environments.

Eukaryotic cells' proliferation and growth are controlled by the target of rapamycin kinase (TOR). TOR usually activates in favorable energy and nutritional circumstances. This is challenged by recent research, suggesting that plant cells optimized for nutrient absorption in low nutritional conditions may activate the TOR pathway in a polarized manner.

mTORC1 Signaling in AgRP Neurons Is Not Required to Induce Major Neuroendocrine Adaptations to Food Restriction.

Hypothalamic mTORC1 signaling is involved in nutrient sensing. Neurons that express the agouti-related protein (AgRP) are activated by food restriction and integrate interoceptive and exteroceptive signals to control food intake, energy expenditure, and other metabolic responses. To determine whether mTORC1 signaling in AgRP neurons is necessary for regulating energy and glucose homeostasis, especially in situations of negative energy balance, mice carrying ablation of the Raptor gene exclusively in AgRP-expressing cells were generated. AgRPΔRaptor mice showed no differences in body weight, fat mass, food intake, or energy expenditure; however, a slight improvement in glucose homeostasis was observed compared to the control group. When subjected to 5 days of food restriction (40% basal intake), AgRPΔRaptor female mice lost less lean body mass and showed a blunted reduction in energy expenditure, whereas AgRPΔRaptor male mice maintained a higher energy expenditure compared to control mice during the food restriction and 5 days of refeeding period. AgRPΔRaptor female mice did not exhibit the food restriction-induced increase in serum corticosterone levels. Finally, although hypothalamic fasting- or refeeding-induced Fos expression showed no differences between the groups, AgRPΔRaptor mice displayed increased hyperphagia during refeeding. Thus, some metabolic and neuroendocrine responses to food restriction are disturbed in AgRPΔRaptor mice.

Glycolysis as key regulatory step in FSH-induced rat Sertoli cell proliferation: Role of the mTORC1 pathway.

The definitive number of Sertoli cells (SCs), achieved during the proliferative periods, defines the spermatogenic capacity in adulthood. It is recognized that FSH is the main mitogen targeting SC and that it exerts its action, at least partly, through the activation of the PI3K/Akt/mTORC1 pathway. mTORC1 controls a large number of cellular functions, including glycolysis and cell proliferation. Interestingly, recent evidence revealed that the glycolytic flux might modulate mTORC1 activity and, consequently, cell cycle progression. Although mature SC metabolism has been thoroughly studied, several aspects of metabolism regulation in proliferating SC are still to be elucidated. The objective of this study was to explore whether aerobic glycolysis is regulated by FSH through mTORC1 pathway in proliferating SC, and to assess the involvement of glycolysis in the regulation of SC proliferation. The present study was carried out utilizing 8-day-old rat SC cultures. The results obtained show that FSH enhances glycolytic flux through the induction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and lactate dehydrogenase A (LDHA) in an mTORC1 dependent manner. In addition, PFKFB3 and LDH inhibitors prevent FSH from activating mTORC1 and stimulating SC proliferation and glycolysis, presumably through mTORC1 pathway inhibition. In summary, FSH simultaneously regulates SC proliferation and glycolysis in an mTORC1 dependent manner, and glycolysis seems to cooperate with FSH in the stimulation of both cellular functions through the modulation of the same signalling pathway. Therefore, a positive feedback between the mTORC1 pathway and glycolysis triggered by FSH is hypothesized.

AMPK/mTOR pathway significance in healthy liver and non-alcoholic fatty liver disease and its progression.

Obesity is related to several organs, but the liver is particularly affected. Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor and regulator of liver lipid dysfunction and glucose metabolism. The mechanistic target of rapamycin (mTOR) is a protein kinase regulating cell growth, survival, metabolism, and immunity. Together, these pathways are involved in obesity, insulin resistance, non-alcoholic fatty liver disease (NAFLD) and its progression, and autophagy. During energy demand, liver kinase B (LKB) phosphorylation helps activate the AMPK/mTOR pathways. Likewise, the protein forkhead box O family (FOXO) negatively regulates adipogenesis by binding to the promoter sites of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha, initiating adipogenesis. In addition, acetyl-CoA carboxylase, which regulates de novo lipogenesis, is linked to LKB and FOXO in developing NAFLD. The kinase complex, consisting of Unc-51-like autophagy-activating kinase 1 or 2 (ULK1, ULK2) by stimulating autophagy, and eliminating fat droplets in NAFLD, is regulated by mTORC1 and negatively regulated by AMPK that suppresses liver lipogenesis and increases fatty acid oxidation. Also, ULK1 is essential for initiating phagophore formation, establishing macrophagy, and generating autophagosomes. The selective breakdown of lipid droplets through macroautophagy, or macrolipophagy, occurs on a cellular energy level using free fatty acids. In addition, mTORC1 promotes lipogenesis by activating sterol regulatory element-binding protein. Finding new components and novel regulatory modes in signaling is significant for a better understanding of the AMPK/mTOR pathways, potentially facilitating the development of future diagnostic and therapeutic strategies for NAFLD and its progression to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma.

Palbociclib-Induced Cellular Senescence Is Modulated by the mTOR Complex 1 and Autophagy.

Despite not dividing, senescent cells acquire the ability to synthesize and secrete a plethora of bioactive molecules, a feature known as the senescence-associated secretory phenotype (SASP). In addition, senescent cells often upregulate autophagy, a catalytic process that improves cell viability in stress-challenged cells. Notably, this "senescence-related autophagy" can provide free amino acids for the activation of mTORC1 and the synthesis of SASP components. However, little is known about the functional status of mTORC1 in models of senescence induced by CDK4/6 inhibitors (e.g., Palbociclib), or the effects that the inhibition of mTORC1 or the combined inhibition of mTORC1 and autophagy have on senescence and the SASP. Herein, we examined the effects of mTORC1 inhibition, with or without concomitant autophagy inhibition, on Palbociclib-driven senescent AGS and MCF-7 cells. We also assessed the pro-tumorigenic effects of conditioned media from Palbociclib-driven senescent cells with the inhibition of mTORC1, or with the combined inhibition of mTORC1 and autophagy. We found that Palbociclib-driven senescent cells display a partially reduced activity of mTORC1 accompanied by increased levels of autophagy. Interestingly, further mTORC1 inhibition exacerbated the senescent phenotype, a phenomenon that was reversed upon autophagy inhibition. Finally, the SASP varied upon inhibiting mTORC1, or upon the combined inhibition of mTORC1 and autophagy, generating diverse responses in cell proliferation, invasion, and migration of non-senescent tumorigenic cells. Overall, variations in the SASP of Palbociclib-driven senescent cells with the concomitant inhibition of mTORC1 seem to depend on autophagy.

What Do the Transcriptome and Proteome of Menstrual Blood-Derived Mesenchymal Stem Cells Tell Us about Endometriosis?

Given the importance of menstrual blood in the pathogenesis of endometriosis and the multifunctional roles of menstrual mesenchymal stem cells (MenSCs) in regenerative medicine, this issue has gained prominence in the scientific community. Moreover, recent reviews highlight how robust the integrated assessment of omics data are for endometriosis. To our knowledge, no study has applied the multi-omics approaches to endometriosis MenSCs. This is a case-control study at a university-affiliated hospital. MenSCs transcriptome and proteome data were obtained by RNA-seq and UHPLC-MS/MS detection. Among the differentially expressed proteins and genes, we emphasize ATF3, ID1, ID3, FOSB, SNAI1, NR4A1, EGR1, LAMC3, and ZFP36 genes and MT2A, TYMP, COL1A1, COL6A2, and NID2 proteins that were already reported in the endometriosis. Our functional enrichment analysis reveals integrated modulating signaling pathways such as epithelial-mesenchymal transition (↑) and PI3K signaling via AKT to mTORC1 (↓ in proteome), mTORC1 signaling, TGF beta signaling, TNFA signaling via NFkB, IL6 STAT3 signaling, and response to hypoxia via HIF1A targets (↑ in transcriptome). Our findings highlight primary changes in the endometriosis MenSCs, suggesting that the chronic inflammatory endometrial microenvironment can modulate these cells, providing opportunities for endometriosis etiopathogenesis. Moreover, they identify challenges for future research leveraging knowledge for regenerative and precision medicine in endometriosis.

Angiotensin II Regulates Mitochondrial mTOR Pathway Activity Dependent on Acyl-CoA Synthetase 4 in Adrenocortical Cells.

Two well-known protein complexes in mammalian cells, mTOR type 1 and type 2 (mTORC1/2) are involved in several cellular processes such as protein synthesis, cell proliferation, and commonly dysregulated in cancer. An acyl-CoA synthetase type 4 (ACSL4) is one of the most recently mTORC1/2 regulators described, in breast cancer cells. The expression of ACSL4 is hormone-regulated in adrenocortical cells and required for steroid biosynthesis. mTORC1/2 have been reported to be crucial in the proliferation of human adrenocortical tumor cells H295R and interestingly reported at several subcellular locations, which has brought cell biology to the vanguard of the mTOR signaling field. In the present work, we study the regulation of mTORC1/2 activation by angiotensin II (Ang II)-the trophic hormone for adrenocortical cells-the subcellular localization of mTORC1/2 signaling proteins and the role of ACSL4 in the regulation of this pathway, in H295R cells. Ang II promotes activation by phosphorylation of mTORC1/2 pathway proteins in a time-dependent manner. Mitochondrial pools of ribosomal protein S6, protein kinase B (Akt) in threonine 308, and serine 473 and Rictor are phosphorylated and activated. Glycogen synthase kinase type 3 (GSK3) is phosphorylated and inactivated in mitochondria, favoring mTORC1 activation. Epidermal growth factor, a classic mTORC1/2 activator, promoted unique activation kinetics of mTORC1/2 pathway, except for Akt phosphorylation. Here, we demonstrate that ACSL4 is necessary for mTORC1/2 effectors phosphorylation and H295R proliferation, triggered by Ang II. Ang II promotes activation of mitochondrial mTORC1/2 signaling proteins, through ACSL4, with a direct effect on adrenocortical cellular proliferation.

Ginseng total saponins and Fuzi total alkaloids exert antidepressant-like effects in ovariectomized mice through BDNF-mTORC1, autophagy and peripheral metabolic pathways.

BACKGROUND: Shenfu decoction (SFD) is a classic Chinese medicine prescription that has a strong cardiotonic effect. The combination of ginseng (the dried root of Panax ginseng C. A. Meyer) and Fuzi (processed product of sub-root of Aconitum carmichaeli Debx), the main constituents of SFD, has been reported to improve the pharmacological effect of each other. Moreover, research has shown that the main active components of SFD, ginseng total saponins (GTS) and Fuzi total alkaloids (FTA), have antidepressant activity. However, the effects of these ingredients on depressive-like behavior induced by ovariectomy, a model of menopausal depression, have not been studied. PURPOSE: Our research aims to elucidate the antidepressant-like effects of GTS and FTA compatibility (GF) in ovariectomized mice and the potential mechanisms. METHODS: To elucidate the antidepressant-like effects of GF in mice in ovariectomy condition, behavioral tests were performed after 7 days of intragastric administration of different doses of GF. Underlying molecular mechanisms of CREB-BDNF, BDNF-mTORC1 and autophagy signaling were detected by western blotting, serum metabolites were examined by UPLC-QE plus-MS and dendritic spine density was determined by Golgi-Cox staining. RESULTS: GF remarkably decreased the immobility time in the forced swim test. GF also increased levels of pCREB/CREB, BDNF, Akt, mTORC1 and p62 in the prefrontal cortex and hippocampus, as well as decreased LC3-II/LC3-I in the prefrontal cortex and hippocampus of ovariectomized mice. Furthermore, 15 serum differential metabolites (9 of which are lipids and lipid molecules) were identified by metabonomics. Next, the antidepressant-like effects of GF was blocked by rapamycin, an inhibitor of mTORC1. The antidepressant actions of GF on levels of pCREB, mTORC1, LC3-Ⅱ/LC3-Ⅰ and p62 in the prefrontal cortex and the levels of BDNF, Akt, mTORC1 and p62 in the hippocampus were inhibited by rapamycin, and the dendritic spines density was also regulated. CONCLUSION: GF has antidepressant effects in ovariectomized mice, and like other antidepressants, these effects involve activation of BDNF-mTORC1, autophagy regulation and consequent effects on hippocampal synaptic plasticity. Moreover, metabolomic results suggest that GF also has effects on peripheral lipid profiles that may provide potential biomarkers for these antidepressant-like effects. These results indicate that GF is worthy of further exploration as a promising pharmaceutical treatment for depression. This study provides a new direction for the development of new indications for traditional Chinese medicine compounds.

Rare CACNA1H and RELN variants interact through mTORC1 pathway in oligogenic autism spectrum disorder.

Oligogenic inheritance of autism spectrum disorder (ASD) has been supported by several studies. However, little is known about how the risk variants interact and converge on causative neurobiological pathways. We identified in an ASD proband deleterious compound heterozygous missense variants in the Reelin (RELN) gene, and a de novo splicing variant in the Cav3.2 calcium channel (CACNA1H) gene. Here, by using iPSC-derived neural progenitor cells (NPCs) and a heterologous expression system, we show that the variant in Cav3.2 leads to increased calcium influx into cells, which overactivates mTORC1 pathway and, consequently, further exacerbates the impairment of Reelin signaling. Also, we show that Cav3.2/mTORC1 overactivation induces proliferation of NPCs and that both mutant Cav3.2 and Reelin cause abnormal migration of these cells. Finally, analysis of the sequencing data from two ASD cohorts-a Brazilian cohort of 861 samples, 291 with ASD; the MSSNG cohort of 11,181 samples, 5,102 with ASD-revealed that the co-occurrence of risk variants in both alleles of Reelin pathway genes and in one allele of calcium channel genes confer significant liability for ASD. Our results support the notion that genes with co-occurring deleterious variants tend to have interconnected pathways underlying oligogenic forms of ASD.

Metabolic changes induced by DNA damage in Ramos cells: exploring the role of mTORC1 complex.

DNA damage induces the activation of many different signals associated with repair or cell death, but it is also connected with physiological events, such as adult neurogenesis and B-cell differentiation. DNA damage induces different signaling pathways, some of them linked to important metabolic changes. The mTORC1 pathway has a central role in the regulation of growth processes and cell division in response to environmental changes and also controls protein synthesis, lipid biogenesis, nucleotide synthesis, and expression of glycolytic genes. Here, we report that double-strand breaks induced with etoposide affect the expression of genes encoding different enzymes associated with specific metabolic pathways in Ramos cells. We also analyzed the role of mTOR signaling, demonstrating that double-strand breaks induce downregulation of mTOR signaling. Specific inhibition of mTORC1 using rapamycin also induced changes in the expression of metabolic genes. Finally, we demonstrated that DNA damage and rapamycin can regulate glucose uptake. In summary, our findings show that etoposide and rapamycin affect the expression of metabolic genes as well as apoptotic and proliferation markers in Ramos cells, increasing our understanding of cancer metabolism.

Chronic rapamycin treatment decreases hepatic IL-6 protein, but increases autophagy markers as a protective effect against the overtraining-induced tissue damage.

Regular endurance exercise is a non-pharmacological strategy to protect the liver against diseases. Conversely, exercise may be harmful when excessive, the so-called overtraining. As expected, mice who underwent an overtraining protocol presented higher levels of proinflammatory cytokines in the serum and liver. Based on the relationship among overtraining, inflammation and mammalian target of rapamycin complex 1 (mTORC1) upregulation, the present study verified if animals submitted to an overtraining protocol, but with inhibition of the mTOR pathway via rapamycin injections could mitigate the liver and serum inflammation. Once autophagy can be linked to the improvement of hepatic dysfunction, we also investigated if the inhibition of mTORC1 by rapamycin can improve hepatic autophagy. The animals were randomized into four groups: control (CT; sedentary mice), overtraining by downhill running (OT; mice submitted to the downhill running-based overtraining protocol), overtraining by downhill running with chronic administration of rapamycin (OT/Rapa; mice submitted to the downhill running-based overtraining protocol with intraperitoneal injections of rapamycin) and aerobic (AER; submitted to aerobic training protocol). The serum and liver of the animals were used for biochemical analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunoblotting. The main results are (a) OT and OT/Rapa protocols decreased the performance; (b) the protein levels of interleukin 6 (IL-6) were higher for the OT group; the OT/Rapa group reduced the autophagic genes, increased the microtubule-associated protein light chain 3 II/I (LC3II/LC3I) protein ratio and decreased the sequestosome 1 (SQSTM1) protein. In conclusion, rapamycin appears efficiently to increase the autophagy proteins and decrease IL-6 protein in the liver of overtraining mice.

The mTORC1/AMPK pathway plays a role in the beneficial effects of semaglutide (GLP-1 receptor agonist) on the liver of obese mice.

PURPOSE: The liver regulates lipid metabolism. Decreasing mTOR (mechanistic target of rapamycin complex 1) and enhancing AMPK (AMP-activated protein kinase) help degrade hepatic diet-induced accumulated lipids. Therefore, the glucagon-like peptide type 1 receptor agonist (GLP-1) is indicated to treat obesity-related liver metabolic alterations. Then, we investigated the effects of semaglutide (recent GLP-1) by analyzing the liver mTORC1/AMPK pathway genes in obese mice. BASIC PROCEDURES: C57BL/6 male mice were separated into two groups and submitted for 16 weeks of obesity induction. Then they were treated for an additional four weeks with semaglutide (subcutaneous, 40 µg/kg once every three days). The groups formed were: C, control group; CS, control group plus semaglutide; HF, high-fat group; HFS, high-fat group plus semaglutide. Next, the livers were dissected, and rapidly fragments of all lobes were kept and frozen at -80° C for analysis (RT-qPCR). MAIN FINDINGS: Liver markers for the mTOR pathway associated with anabolism and lipogenesis de novo were increased in the HF group compared to the C group but comparatively attenuated by semaglutide. Also, liver markers for the AMPK pathway, which regulates chemical pathways involving the cell's primary energy source, were impaired in the HF group than in the C group but partly restored by semaglutide. CONCLUSION: the mTOR pathway was attenuated, and the insulin signaling and the AMPK pathway were enhanced by semaglutide, ameliorating the liver gene expressions related to the metabolism of obese mice. These findings are promising in delaying the progression of nonalcoholic fatty liver disease.

The Role of IL-6 Released During Exercise to Insulin Sensitivity and Muscle Hypertrophy.

Interleukin-6 (IL-6) influences both inflammatory response and anti-inflammatory processes. This cytokine can be released by exercising skeletal muscle, which characterizes it as a myokine. Unlike what is observed in inflammation, IL-6 produced by skeletal muscle is not preceded by the release of other pro-inflammatory cytokines, but it seems to be dependent on the lactate produced during exercise, thus causing different effects from those seen in inflammatory state. After binding to its receptor, myokine IL-6 activates the PI3K-Akt pathway. One consequence of this upregulation is the potentiation of insulin signaling, which enhances insulin sensitivity. IL-6 increases GLUT-4 vesicle mobilization to the muscle cell periphery, increasing the glucose transport into the cell, and also glycogen synthesis. Muscle glycogen provides energy for ATP resynthesis, and regulates Ca2+ release by the sarcoplasmic reticulum, influencing muscle contraction, and, hence, muscle function by multiple pathways. Another implication for the upregulation of the PI3K-Akt pathway is the activation of mTORC1, which regulates mRNA translational efficiency by regulating translation machinery, and translational capacity by inducing ribosomal biogenesis. Thus, IL-6 may contribute to skeletal muscle hypertrophy and function by increasing contractile protein synthesis.

Guanosine boosts the fast, but not sustained, antidepressant-like and pro-synaptogenic effects of ketamine by stimulating mTORC1-driven signaling pathway.

The mTORC1-dependent dendritic spines formation represents a key mechanism for fast and long-lasting antidepressant responses, but it remains to be determined whether this mechanism may account for the ability of guanosine in potentiating ketamine's actions. Here, we investigated the ability of ketamine plus guanosine to elicit fast and sustained antidepressant-like and pro-synaptogenic effects in mice and the role of mTORC1 signaling in these responses. The combined administration of subthreshold doses of ketamine (0.1 mg/kg, i.p.) and guanosine (0.01 mg/kg, p.o.) caused a fast (1 h - 24 h), but not long-lasting (7 days) reduction in the immobility time in the tail suspension test. This behavioral effect was paralleled by a rapid (started in 1 h) and transient (back to baseline in 24 h) increase on BDNF, p-Akt (Ser473), p-GSK-3ß (Ser9), p-mTORC1 (Ser2448), p-p70S6K (Thr389) immunocontent in the hippocampus, but not in the prefrontal cortex. Conversely, ketamine plus guanosine increased PSD-95 and GluA1 immunocontent in the prefrontal cortex, but not the hippocampus after 1 h, whereas increased levels of these proteins in both brain structures were observed after 24 h, but these effects did not persist after 7 days. The combined administration of ketamine plus guanosine raised the dendritic spines density in the ventral hippocampal DG and prefrontal cortex after 24 h Rapamycin (0.2 nmol/site, i.c.v.) abrogated the antidepressant-like effect and pro-synaptogenic responses triggered by ketamine plus guanosine. These results indicate that guanosine may boost the antidepressant-like effect of ketamine for up to 24 h by a mTORC1-dependent mechanism.

mTOR Activity and Autophagy in Senescent Cells, a Complex Partnership.

Cellular senescence is a form of proliferative arrest triggered in response to a wide variety of stimuli and characterized by unique changes in cell morphology and function. Although unable to divide, senescent cells remain metabolically active and acquire the ability to produce and secrete bioactive molecules, some of which have recognized pro-inflammatory and/or pro-tumorigenic actions. As expected, this "senescence-associated secretory phenotype (SASP)" accounts for most of the non-cell-autonomous effects of senescent cells, which can be beneficial or detrimental for tissue homeostasis, depending on the context. It is now evident that many features linked to cellular senescence, including the SASP, reflect complex changes in the activities of mTOR and other metabolic pathways. Indeed, the available evidence indicates that mTOR-dependent signaling is required for the maintenance or implementation of different aspects of cellular senescence. Thus, depending on the cell type and biological context, inhibiting mTOR in cells undergoing senescence can reverse senescence, induce quiescence or cell death, or exacerbate some features of senescent cells while inhibiting others. Interestingly, autophagy-a highly regulated catabolic process-is also commonly upregulated in senescent cells. As mTOR activation leads to repression of autophagy in non-senescent cells (mTOR as an upstream regulator of autophagy), the upregulation of autophagy observed in senescent cells must take place in an mTOR-independent manner. Notably, there is evidence that autophagy provides free amino acids that feed the mTOR complex 1 (mTORC1), which in turn is required to initiate the synthesis of SASP components. Therefore, mTOR activation can follow the induction of autophagy in senescent cells (mTOR as a downstream effector of autophagy). These functional connections suggest the existence of autophagy regulatory pathways in senescent cells that differ from those activated in non-senescence contexts. We envision that untangling these functional connections will be key for the generation of combinatorial anti-cancer therapies involving pro-senescence drugs, mTOR inhibitors, and/or autophagy inhibitors.

A low-dose combination of ketamine and guanosine counteracts corticosterone-induced depressive-like behavior and hippocampal synaptic impairments via mTORC1 signaling.

Ketamine exhibits rapid and sustained antidepressant responses, but its repeated use may cause adverse effects. Augmentation strategies have been postulated to be useful for the management/reduction of ketamine's dose and its adverse effects. Based on the studies that have suggested that ketamine and guanosine may share overlapping mechanisms of action, the present study investigated the antidepressant-like effect of subthreshold doses of ketamine and guanosine in mice subjected to repeated administration of corticosterone (CORT) and the role of mTORC1 signaling for this effect. The ability of the treatment with ketamine (0.1 mg/kg, i.p.) plus guanosine (0.01 mg/kg, p.o.) to counteract the depressive-like behavior induced by CORT (20 mg/kg, p.o., for 21 days) in mice, was paralleled with the prevention of the CORT-induced reduction on BDNF levels, Akt (Ser473) and GSK-3ß (Ser9) phosphorylation, and PSD-95, GluA1, and synapsin immunocontent in the hippocampus. No changes on mTORC1 and p70S6K immunocontent were found in the hippocampus and prefrontal cortex of any experimental group. No alterations on BDNF, Akt/GSK-3ß, mTORC1/p70S6K, and synaptic proteins were observed in the prefrontal cortex of mice. The antidepressant-like and pro-synaptogenic effects elicited by ketamine plus guanosine were abolished by the pretreatment with rapamycin (0.2 nmol/site, i.c.v., a selective mTORC1 inhibitor). Our results showed that the combined administration of ketamine and guanosine at low doses counteracted CORT-induced depressive-like behavior and synaptogenic disturbances by activating mTORC1 signaling. This study supports the notion that the combined administration of guanosine and ketamine may be a useful therapeutic strategy for the management of MDD.