Ian A. Semple

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 chronic increase of cytosolic calcium concentration in hepatocytes upon 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 thirty years, our results suggest they may be a safe therapeutic option for restoring autophagic flux and treating metabolic pathologies in obese patients.

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/EBPβ). 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 liver of 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 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.

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.

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

Significance Antioxidant therapy was once considered useful for treating metabolic syndrome because excessive accumulation of reactive oxygen species (ROS) was identified as an inducer of diverse metabolic pathologies. However, the effectiveness of dietary antioxidants in treating obesity-associated diseases had been largely controversial in numerous animal and human clinical studies, some of which actually show adverse effects upon antioxidant consumption. Here, we show that Sestrin2 and other antioxidants can interfere with uncoupling protein 1 (Ucp1) expression through suppression of ROS-mediated p38 MAPK activation. Ucp1, a protein responsible for heat generation and energy dissipation, is known to suppress diverse metabolic pathologies associated with obesity and aging. Thus, our results explain why some antioxidant therapies were not successful in treating obesity-associated diseases and extending health and lifespan in mammals. 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.

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. DOI: http://dx.doi.org/10.7554/eLife.12204.001

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, 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.

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).