Sestrins are evolutionarily conserved mediators of exercise benefits.

Exercise is among the most effective interventions for age-associated mobility decline and metabolic dysregulation. Although long-term endurance exercise promotes insulin sensitivity and expands respiratory capacity, genetic components and pathways mediating the metabolic benefits of exercise have remained elusive. Here, we show that Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits. In both fly and mouse models, genetic ablation of Sestrins prevents organisms from acquiring metabolic benefits of exercise and improving their endurance through training. Conversely, Sestrin upregulation mimics both molecular and physiological effects of exercise, suggesting that it could be a major effector of exercise metabolism. Among the various targets modulated by Sestrin in response to exercise, AKT and PGC1α are critical for the Sestrin effects in extending endurance. These results indicate that Sestrin is a key integrating factor that drives the benefits of chronic exercise to metabolism and physical endurance.

Concurrent activation of growth factor and nutrient arms of mTORC1 induces oxidative liver injury.

mTORC1 is a protein kinase important for metabolism and is regulated by growth factor and nutrient signaling pathways, mediated by the Rheb and Rag GTPases, respectively. Here we provide the first animal model in which both pathways were upregulated through concurrent mutations in their GTPase-activating proteins, Tsc1 and Depdc5. Unlike former models that induced limited mTORC1 upregulation, hepatic deletion of both Tsc1 and Depdc5 (DKO) produced strong, synergistic activation of the mTORC1 pathway and provoked pronounced and widespread hepatocyte damage, leading to externally visible liver failure phenotypes, such as jaundice and systemic growth defects. The transcriptome profile of DKO was different from single knockout mutants but similar to those of diseased human livers with severe hepatitis and mouse livers challenged with oxidative stress-inducing chemicals. In addition, DKO liver cells exhibited prominent molecular pathologies associated with excessive endoplasmic reticulum (ER) stress, oxidative stress, DNA damage and inflammation. Although DKO liver pathologies were ameliorated by mTORC1 inhibition, ER stress suppression unexpectedly aggravated them, suggesting that ER stress signaling is not the major conduit of how hyperactive mTORC1 produces liver damage. Interestingly, superoxide scavengers N-acetylcysteine (NAC) and Tempol, chemicals that reduce oxidative stress, were able to recover liver phenotypes, indicating that mTORC1 hyperactivation induced liver damage mainly through oxidative stress pathways. Our study provides a new model of unregulated mTORC1 activation through concomitant upregulation of growth factor and nutrient signaling axes and shows that mTORC1 hyperactivation alone can provoke oxidative tissue injury.

Cardioprotective roles of sestrin 1 and sestrin 2 against doxorubicin cardiotoxicity.

Doxorubicin is a chemotherapy medication widely used to treat a variety of cancers. Even though it offers one of the most effective anti-cancer treatments, its clinical use is limited because of its strong cardiotoxicity that can lead to fatal conditions. Here, we show that sestrin 1 and sestrin 2, members of the sestrin family of proteins that are stress-inducible regulators of metabolism, are critical for suppressing doxorubicin cardiotoxicity and coordinating the AMPK-mammalian target of rapamycin complex 1 (mTORC1) autophagy signaling network for cardioprotection. Expression of both sestrin 1 and sestrin 2 was highly increased in the mouse heart after doxorubicin injection. Genetic ablation of sestrin 1 and sestrin 2 rendered mice more vulnerable to doxorubicin and exacerbated doxorubicin-induced cardiac pathologies including cardiomyocyte apoptosis and cardiac dysfunction. These pathologies were associated with strong dysregulation of the cardiac signaling network, including suppression of the AMPK pathway and activation of the mTORC1 pathway. Consistent with AMPK downregulation and mTORC1 upregulation, autophagic activity of heart tissue was diminished, leading to prominent accumulation of autophagy substrate, p62/SQSTM1. Taken together, our results indicate that sestrin 1 and sestrin 2 are important cardioprotective proteins that coordinate metabolic signaling pathways and autophagy to minimize cardiac damage in response to doxorubicin insult. Augmenting this protective mechanism could provide a novel therapeutic rationale for prevention and treatment of doxorubicin cardiotoxicity. NEW & NOTEWORTHY Doxorubicin is a highly efficient chemotherapeutic medicine; however, its use is limited because of its strong cardiotoxicity. Here, we show that sestrin 1 and sestrin 2 are critical protectors of cardiomyocytes from doxorubicin damage. By upregulating AMPK and autophagic activities and suppressing mammalian target of rapamycin complex 1 and oxidative stress, sestrins counteract detrimental effects of doxorubicin on cardiomyocytes. Correspondingly, loss of sestrin 1 and sestrin 2 produced remarkable dysregulation of these pathways, leading to prominent cardiac cell death and deterioration of heart function.

Autophagy Dysregulation and Obesity-Associated Pathologies.

Autophagy is one of the major degradative mechanisms that can eliminate excessive nutrients, toxic protein aggregates, damaged organelles and invading microorganisms. In response to obesity and obesity-associated lipotoxic, proteotoxic and oxidative stresses, autophagy plays an essential role in maintaining physiological homeostasis. However, obesity and its associated stress insults can often interfere with the autophagic process through various mechanisms, which result in further aggravation of obesity-related metabolic pathologies in multiple metabolic organs. Paradoxically, inhibition of autophagy, within specific contexts, indirectly produces beneficial effects that can alleviate several detrimental consequences of obesity. In this minireview, we will provide a brief discussion about our current understanding of the impact of obesity on autophagy and the role of autophagy dysregulation in modulating obesity-associated pathological outcomes.

Lipotoxicity induces hepatic protein inclusions through TANK binding kinase 1-mediated p62/sequestosome 1 phosphorylation.

Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause nonalcoholic steatohepatitis (NASH). NASH, in turn, is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). Formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here, we show that, upon saturated fatty acid-induced lipotoxicity, TANK binding kinase 1 (TBK1) is activated and phosphorylates p62. TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of ubiquitin-p62 aggregates not only in cultured hepatocytes, but also in mouse models of obesity and NASH. CONCLUSION: These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. (Hepatology 2018).

Tumor suppressive role of sestrin2 during colitis and colon carcinogenesis.

The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery of epithelial cells after inflammatory injury. During colitis-promoted tumorigenesis, Sestrin2 was shown to be an important mediator of p53's control over mTORC1 signaling and tumor cell growth. These results highlight Sestrin2 as a novel tumor suppressor, whose downregulation can accelerate both colitis and colon carcinogenesis.

Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay.

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.

Sestrin2, a Regulator of Thermogenesis and Mitohormesis in Brown Adipose Tissue.

Sestrin2 is a stress-inducible protein that functions as an antioxidant and inhibitor of mTOR complex 1. In a recent study, we found that Sestrin2 overexpression in brown adipocytes interfered with normal metabolism by reducing mitochondrial respiration through the suppression of uncoupling protein 1 (UCP1) expression. The metabolic effects of Sestrin2 in brown adipocytes were dependent on its antioxidant activity, and chemical antioxidants produced similar effects in inhibiting UCP1-dependent thermogenesis. These observations suggest that low levels of reactive oxygen species (ROS) in brown adipocytes can actually be beneficial and necessary for proper metabolic homeostasis. In addition, considering that Sestrins are ROS inducible and perform ROS detoxifying as well as other metabolism-controlling functions, they are potential regulators of mitohormesis. This is a concept in which overall beneficial effects result from low-level oxidative stress stimuli, such as the ones induced by caloric restriction or physical exercise. In this perspective, we incorporate our recent insight obtained from the Sestrin2 study toward a better understanding of the relationship between ROS, Sestrin2, and mitochondrial metabolism in the context of brown adipocyte physiology.

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis.

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, we identified CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.

Sestrin2 inhibits mTORC1 through modulation of GATOR complexes.

Sestrins are stress-inducible metabolic regulators that suppress a wide range of age- and obesity-associated pathologies, many of which are due to mTORC1 overactivation. Upon various stresses, the Sestrins inhibit mTORC1 activity through an indirect mechanism that is still unclear. GATORs are recently identified protein complexes that regulate the activity of RagB, a small GTPase essential for mTORC1 activation. GATOR1 is a GTPase activating protein (GAP) for RagB whereas GATOR2 functions as an inhibitor of GATOR1. However, how the GATORs are physiologically regulated is unknown. Here we show that Sestrin2 binds to GATOR2, and liberates GATOR1 from GATOR2-mediated inhibition. Released GATOR1 subsequently binds to and inactivates RagB, ultimately resulting in mTORC1 suppression. Consistent with this biochemical mechanism, genetic ablation of GATOR1 nullifies the mTORC1-inhibiting effect of Sestrin2 in both cell culture and Drosophila models. Collectively, we elucidate a new signaling cascade composed of Sestrin2-GATOR2-GATOR1-RagB that mediates stress-dependent suppression of mTORC1 activity.

Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers.

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that a chronic increase of the cytosolic calcium concentration in hepatocytes during obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome-lysosome fusion in liver, preventing accumulation of protein inclusions and lipid droplets and suppressing inflammation and insulin resistance. As calcium channel blockers have been safely used in clinics for the treatment of hypertension for more than 30 years, our results suggest they may be a safe therapeutic option for restoring autophagic flux and treating metabolic pathologies in obese patients.

Sestrin2 promotes Unc-51-like kinase 1 mediated phosphorylation of p62/sequestosome-1.

Autophagy is a homeostatic process that is important for degrading protein aggregates, nutrient deposits, dysfunctional organelles and several signaling molecules. p62/sequestosome-1 is a protein that binds to several autophagy substrates, such as ubiquitinated proteins, damaged mitochondria and signaling molecules such as an Nrf2 inhibitor Keap1, promoting their autophagic degradation. Sestrin2, a stress-inducible protein, has recently been shown to bind to p62 and promote autophagic degradation of such p62 targets. Because Sestrin2 is a metabolic regulator that suppresses diverse age- and obesity-associated pathologies, the autophagy-controlling function of Sestrin2 may be important for its other physiological functions. However, the molecular mechanism of how Sestrin2 can promote clearance of p62-associated proteins has been unclear. Here we show that Sestrin2 physically associates with Unc-51-like protein kinase 1 (ULK1) and p62 to form a complex in which both Sestrin2 and p62 become phosphorylated by ULK1 at multiple sites. Ser403 of p62, whose phosphorylation is known to promote autophagic degradation of p62 and its targets, is among the sites phosphorylated by ULK1. ULK1-mediated p62 phosphorylation was facilitated by Sestrin2 in cells as well as in in vitro kinase assays. Consistent with this finding, oligomycin-induced energy deprivation, which strongly activates ULK1, provoked a robust Ser403 phosphorylation of p62 in wild-type mouse embryonic fibroblasts. However, in ULK1/2- and Sestrin2-deficient mouse embryonic fibroblasts, oligomycin-induced p62 phosphorylation was dramatically attenuated, suggesting that endogenous Sestrin2-ULK1/2 mainly mediates p62 phosphorylation in response to energetic stress. Taken together, this study identifies ULK1 as a new p62 Ser403 kinase and establishes Sestrin2 as a promoter of ULK1-mediated p62 phosphorylation.

Hepatoprotective role of Sestrin2 against chronic ER stress.

Upon prolonged endoplasmic reticulum (ER) stress, cells attenuate protein translation to prevent accumulation of unfolded proteins. Here we show that Sestrin2 is critical for this process. Sestrin2 expression is induced by an ER stress-activated transcription factor CCAAT-enhancer-binding protein beta (c/EBPß). Once induced, Sestrin2 halts protein synthesis by inhibiting mammalian target of rapamycin complex 1 (mTORC1). As Sestrin2-deficient cells continue to translate a large amount of proteins during ER stress, they are highly susceptible to ER stress-associated cell death. Accordingly, dietary or genetically induced obesity, which does not lead to any pathological indication other than simple fat accumulation in the liver of wild-type (WT) mice, can provoke Sestrin2-deficient mice to develop severe ER stress-associated liver pathologies such as extensive liver damage, steatohepatitis and fibrosis. These pathologies are suppressed by liver-specific Sestrin2 reconstitution, mTORC1 inhibition or chemical chaperone administration. The Sestrin2-mediated unfolded protein response (UPR) may be a general protective mechanism against ER stress-associated diseases.

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.