SIRT3 deficiency is resistant to erastin-induced autophagy-dependent ferroptosis by inhibiting the AMPK/mTOR pathway and promoting glutathione peroxidase 4 levels CURRENT STATUS: POSTED

Background: Ferroptosis, an autophagy-dependent cell death, is characterized by lipid peroxidation and iron accumulation, closely associated with pathogenesis of gestational diabetes mellitus (GDM). Sirtuin 3 (SIRT3) has positive regulation on phosphorylation of AMPK, related to maintainance of cellular redox homeostasis. However, whether SIRT3 can confer autophagy by activating the AMPK-mTOR pathway and consequently promote induction of ferroptosis is unknown. Methods: We used human trophoblastic cell line HTR-8/SVneo and porcine trophoblastic cell line pTr2 to deterimine the mechanism of SIRT3 on autophagy and ferroptosis. Results: The expression of SIRT3 protein was significantly elevated in trophoblastic cells exposed to high concentrations of glucose and ferroptosis-inducing compounds. Increased SIRT3 expression contributed to classical ferroptotic events and autophagy activation, whereas SIRT3 silencing led to resistance against both ferroptosis and autophagy. In addition, autophagy inhibition impaired SIRT3-enhanced ferroptosis. On the contrary, autophagy induction had a synergistic effect with SIRT3. Based on mechanistic investigations, SIRT3 depletion inhibited activation of the AMPK-mTOR pathway and enhanced glutathione peroxidase 4 (GPX4) level, thereby suppressing autophagy and ferroptosis. Furthermore, depletion of AMPK blocked induction of ferroptosis in trophoblasts. Conclusions: We concluded that upregulated SIRT3 enhanced autophagy activation by promoting AMPK-mTOR pathway and decreasing GPX4 level to induce ferroptosis in trophoblastic cells. SIRT3 deficiency was resistant to high glucose- and erastin-induced autophagy-dependent ferroptosis and is therefore a potential therapeutic approach for treating GDM.


Background
Gestational diabetes mellitus (GDM), one of the most common metabolic complications of pregnancy, is characterized by aberrant placental functions, resulting in a significantly increased risk of developing type 2 diabetes mellitus (T2DM) in both mother and child [1][2][3]. There are indications that induction of anti-angiogenic factors and a proinflammatory milieu, including impaired trophoblast migration and invasion, are closely associated with abnormal placental function in development of GDM [4,5]. Moreover, appropriate proliferation, migration and differentiation of trophoblasts dominate progression of steroids, hormones and cytokines secretion, vascularizationa and the immunological interface required for normal pregnancy [6][7][8]. Multiple studies verified that diabetic placenta was usually accompanied by abnormal trophoblast proliferation, autophagy and cell cycle control; thereby, development and progression of GDM was dramatically induced [4,9,10]. In addition, overproduction of reactive oxygen species (ROS) occurs in trophoblastic cells after stimulation with high glucose concentrations and superabundant accumulation of ROS can result; eventually, this continuous oxidative stress initiates induction and pathogenesis of GDM [11,12]. Elucidating preventive mechanisms against oxidative stress-triggered lipid metabolism disorder is expected to facilitate development of evidence-based treatment strategies for GDM.
Autophagy is programmed cell death, with an important role in a degradative pathway for eliminating excessive misfolded protein and damaged organelle aggregates [13].
Accumulation of ROS promotes autophagy-dependent cell death of trophoblasts by increasing lipid peroxidation and reducing activities of antioxidant enzymes [14,15]. As a homeostatic dysfunction of autophagy is believed to be a cause of GDM, it is critical to explore how trophoblastic autophagy is physiologically regulated. Ferroptosis is a regulated cell death-exhibiting production of ROS, from iron-dependent formation of lipid peroxidation, rupture of mitochondrial outer membrane and collapse of cellular redox homeostasis [16,17]. However, crosstalk between autophagy and ferroptosis, as well as underlying mechanisms responsible for these two types of cell death, have not been fully elucidated.
Sirtuin 3 (SIRT3), a prototypical NAD + -dependent mitochondrial protein deacetylase, is responsible for bulk mitochondrial protein deacetylation that regulates metabolism and oxidative stress [18]. SIRT3 is involved in maintenance of mitochondrial function, including ATP generation, nutrient oxidation, ROS production, and even cell death [18,19] A novel role of SIRT3 in regulating phosphorylation of activated protein kinase (AMPK) to promote autophagy was recently reported [20]. However, plausible crosstalk between AMPK and SIRT3 during oxidative stress-mediated cell death has apparently not been studied. Based on capability of AMPK to promote autophagy via mTOR and SIRT3 to inhibit oxidative stress-induced ferroptosis [20,21], we speculated that protective effects of SIRT3 on autophagy and ferroptosis would be associated with AMPK. Therefore, the objective was to investigate underlying mechanisms in SIRT3-mediated protection of trophoblasts against autophagy and ferroptosis, with a special focus on the AMPK-mTOR pathway.

Materials and Methods
Reagents and antibodies DME/F12 medium, Opti-MEM medium and fetal bovine serum (FBS) were obtained from  Abcam, Cambridge, MA, USA). Briefly, cells were lysed on ice, and then supernatants were collected after centrifugation at 13000 x g for 10 min. Supernatants were co-incubated with 5 µL Iron Reducer solution at 37℃ for 30 min. Subsequently, 100 µL Iron Probe were added and incubated for 1 h at 37℃ in dark; thereafter, absorbance was detected at 593 nm using a microplate reader.
Subsequently, 20 µL substrate working solution was added and incubated for 10 min at Millipore, MA, USA). After blocking with 5% skimmed milk, membranes were probed overnight at 4 o C with specific primary antibodies, prior to incubation with corresponding secondary antibodies and protein was detected with an enhanced chemiluminescence method (ECL, Solarbio, Beijing, China). 8

Statistical analyses
Results were expressed as mean ± standard deviation (SD). Statistical significance was assessed by Student's t-test or one-way analysis of variance (ANOVA), followed by post hoc Tukey's test. For all statistical analyses, P < 0.05 was considered significant.

SIRT3 is increased during trophoblast ferroptosis
High glucose-induced trophoblast cell death contributes to pathogenesis of GDM [12]. High As SIRT3 has critical roles in regulating oxidative stress and mitochondrial protein acetylation [18], we explored effects of various ferroptosis-inducing compounds on SIRT3 protein expression to determine whether SIRT3 has a pivotal regulatory role during ferroptosis. Cultivated trophoblasts exposed to erastin, sorafenib and BSO in the presence of high glucose had upregulation of SIRT3 protein in both human (HTR8/SVeno) and porcine (pTr2) trophoblast cells (Fig. 1e).
Increased SIRT3 expression contributes to trophoblast ferroptosis 9 To determine if increased SIRT3 is associated with induction of ferroptosis, siRNA of SIRT3 was introduced to HTR8/SVeno and pTr2 cells stimulated with erastin in high glucose condition. SIRT3 siRNA significantly decreased SIRT3 protein level in both HTR8/SVeno and pTr2 cells (Supporting Fig. S2). As expected, SIRT3 knockdown abolished high glucose and erastin-induced growth inhibition (Fig. 2a), redox-active iron overload (Fig. 2b)

Increased SIRT3 expression promotes autophagy activation
To test whether high glucose exerts any influence on autophagy in cultivated trophoblastic cells, Western blot analysis was employed to determine conversion of LC3B-I to LC3B-II and protein level of beclin1. When trophoblastic cells were exposed to high glucose alone, there were significant increases in LC3B-II accumulation and beclin1 protein; furthermore, chloroquine (a lysosomal inhibitor that prevents autophagic degradation in lysosomes) and bafilomycin (a lysosomal inhibitor that prevents autophagosomes from fusion with lysosomes to form autophagosomes) dramatically elevated this increase. Conversely, rapamycin promoted high glucose-induced autophagy activation, evidenced by enhanced LC3B-II conversion and increased beclin1 protein expression (Supporting Fig. S4). To further clarify influence of autophagy activation on ferroptosis, markers of autophagy were examined by western blot analysis in both control siRNA and SIRT3 siRNA-treated cells. As expected, SIRT3 sliencing significantly blocked erastin-induced accumulation of 10 LC3B-II (Fig. 3a) and remarkably decreased expression of beclin1 (Fig. 3b).
Enhanced ferroptosis by SIRT3 is associated with autophagy activation Several lines of evidence reinforced the notion that ferroptosis is an autophagic cell death process. 22− 25 To evaluate whether autophagy is involved in SIRT3-mediated ferroptosis, rapamycin and chloroquine were exploited to activate or inhibit autophagy, respectively.

SIRT3 upregulates autophagy by promoting AMPK
AMPK is a positive regulator of autophagy via suppressing mammalian target of rapamycin C1 (mTORC1) [26]. To study the role of AMPK in SIRT3-mediated autophagy activation, we initially detected AMPK activation after SIRT3 depletion using western blot. SIRT3 knockdown was associated with suppressed AMPK activation, evidenced by decreased protein phosphorylation of AMPK (Fig. 5a), as well as increased mTORC1 activation (Fig. 5b). The AMPK inhibitor, compound C, was used to further determine if AMPK is mechanistically implicated in effect of SIRT3 on autophagy. Protein expression of p-AMPK was decreased to < 30% in compound C-treated cells (Supporting Fig. S5).Furthermore, compound C abolished LC3-II conversion (Fig. 5c) and decreased expression of beclin1 ( Fig. 5d) resulting from SIRT3 activation.

Suppression of AMPK impairs SIRT3-enhanced ferroptpsis
To explore whether AMPK activation triggers SIRT3-enhanced ferroptosis, compound C was used to inhibit phosphorylation of AMPK. Compound C not only inhibited phosphorylation of AMPK, it also reduced erastin-induced growth inhibition in human trophoblastic HTR8/SVeno cells (Fig. 6a). Similarly, pretreatment with compound C significantly abolished high glucose and erastin-induced redox-active iron accumulation (Fig. 6b), GSH depletion (Fig. 6c), and MDA production (Fig. 6d), in the absence or presence of SIRT3 siRNA.
There is increasing evidence iron-related oxidative stress may contribute to lipid oxidation and consequently produce distinct types of cell death, e.g. apoptosis, necrosis, ferroptosis and autophagy [16,27,28]. Mounting evidence has confirmed that ferroptosis, an ironand lipotoxicity-dependent regulated cell death, is driven by an iron-catalyzed lipid peroxidation accumulation initiated via a Fenton reactions-triggered production of hydroxyl radicals and enzymatic lipoxygenase mechanisms [22,29]. Lipophagy, intracellular lipid droplets degradation by autophagy, promotes ferroptotic cell death through induction of lipid peroxidation in hepatocytes, both in vivo and in vitro [30].
However, potential mechanistic consequences of how uncontrolled lipid peroxidation involving in autophagy-dependent ferroptosis are still under debate. In the present study, exposure to high glucose dramatically reduced cell viability and increased production of oxidants in trophoblasts, resulting in ferroptosis in both human and porcine trophoblast cells. We also reported that erastin, sorafenib and BSO can trigger accumulation of ferroptotic trophoblast in vitro.
Overexpression of SIRT3 modulated protein acetylation and bolstered cellular resistance to mitochondrial stress and ferroptosis [21,31,34]. Therefore, effects and underlying mechanisms of SIRT3 on trophoblastic ferroptosis were explored in the current study.
SIRT3 protein level was remarkedly upregulated after cultivated trophoblasts were exposed to ferroptosis-inducing agents in high glucose condition. Moreover, depletion of SIRT3 blocked oxidative stress and ferroptotic effects of high glucose and erastin. Therefore, we inferred that SIRT3 was involved in ferroptosis of trophoblastic cells.
Interestingly, there is a complex crosstalk between ferroptosis and autophagy through complex feedback loops [23,35]. Ferroptosis induction is coupled to an increase in turnover of MAP1LC3 and autophagosome formation, consistent with the notion that lipid peroxidation as well as oxidized lipids can promote autophagy activation [16,36]. Recent study indicates that clockophagy, a novel mode of selective autophagic degradation, facilitates ferroptosis through induction of oxidative injury [37]. In the present study, SIRT3 silencing significantly decreased LC3B-II accumulation and beclin1 protein expression, implicating SIRT3 as having a critical role as a positive regulator of autophagy. These observations were in direct contrast with a previous report that reduced 13 SIRT3 expression enhanced autophagy flux [33]. Evidence supported the notion that excessive activation of autophagy promoted ferroptosis activator-induced induction of ferroptotic cell death [16,23,24,36]. Furthermore, a pharmacological approach or genetic inhibition of the autophagic pathway blocked ferroptotic cell death, at least in some instances. However, functional contributions of SIRT3 to ferroptosis have not been fully understood. We inferred that SIRT3-mediated autophagy, identified as an upstream mechanism, is required to regulate cellular iron homeostasis and cellular oxidative stress during ferroptosis, which was also apparent based on the aggravated effect of rapamycin and reversed effects of chloroquine, respectively.
It has been reported that SIRT3 can confer protection by regulating phosphorylation of AMPK, thereby inhibiting mTOR activity, which was paralleled by autophagy induction [38].

AMPK, with a critical role in lipophagy by initiation of chaperone-mediated autophagy and
in governing cellular defenses against hypoxia, ischemia and oxidative stress [25,[39][40][41], serves as a regulator of cell survival or death. Given the well-documented positive role of AMPK activity in regulation of autophagy, we hypothesized that SIRT3 activates that AMPK-mTOR pathway and consequently induces autophagy. As expected, the present study clarified that increased SIRT3 expression upregulated the phosphorylation level of AMPK, whereas inhibited mTOR activity. AMPK is mechanistically implicated in the effect of SIRT3 on autophagy and the same effect was also confirmed in this study [20,33,38].
Compound C was partially blocked the protein level of p-AMPK in SIRT3-induced circumstance, which was accompanied by decreases in LC3-II conversion as well as beclin1 protein level. Thus, it indicates that SIRT3 promotes induction of autophagy via activating the AMPK-mTOR pathway. Autophagy is activated, leading to lipid peroxidation and iron accumulation, in response to induction of ferroptosis [17,22,23]. Importantly, lysosomal dysfunction and uncontrolled or inappropriate autophagic flux are involved in iron dysbolism in ferroptosis [16,23]. The function of AMPK in mediation of ferroptosis is required for beclin1 phosphorylation, with inhibition of system X c − activity being reported [25]. Perhaps AMPK represents a mechanistic link between ferroptosis and autophagy; this was supported by our data that pharmacological inhibition of AMPK partially abolished SIRT3-induced ferroptotic events in our in vitro model.
Emerging studies have revealed that downregulation of GPX4 could promote induction of ferroptosis, it depletion in cells and mice results in the accumulation of lipid peroxides and lipid ROS [42][43][44]. Our data suggest that inhibition of GPX4 induces ferroptosis by increasing lipid ROS level in trophoblastic cells. Moreover, we also found that depletion of SIRT3 blocked RSL3-induced ferroptosis, indicating that decreased SIRT3 prevented ferroptosis by increasing GPX4 level. In the present study, we elucidated a novel role of SIRT3 in autophagy and ferroptosis via regulating AMPK-mTOR pathway and GPX4 level.
Taken together, our findings demonstrated a scenario where ferroptotic-inducing agents not only induced ferroptotic events, but also promoted autophagy activation, paralleled by increased SIRT3 expression. Thus, depletion of SIRT3 decreased the phosphorylation level of AMPK, whereas increased mTOR activity, eventually inhibiting autophagy activation as well as blocking induction of ferroptosis. Therefore, we inferred that mediation of SIRT3 on the p-AMPK-mTOR pathway partially contributed to regulated cell death, including autophagy and ferroptosis. In paraller, depletion of SIRT3 inhibited ferroptosis by increasing GPX4 level (Fig. 8). However, further investigations are needed to confirm whether the same mechanisms are responsible in vivo. Additionally, whether SIRT3 can regulate AMPK-mediated beclin1 phosphorylation to promote ferroptosis by directly blocking system X c − activity, and the crosstalk between AMPK and GPX4, also requires further study.

Conclusions
High glucose exposure of trophoblasts lead to upregulation of SIRT3 protein level, which in turn exacerbated ferroptotic-inducing compounds-triggered ferroptosis by positively regulating autophagy induction. We inferred that increased SIRT3 expression contributed to autophagy activation by activating AMPK-mTOR pathway, promoting iron accumulation and lipid peroxidation, and in turn, inducing ferroptosis. Whereas, decreased SIRT3 inhibits ferroptosis through regulating GPX4 level. Finally, SIRT3 deficiency was resistant to erastin-induced autophagy-dependent ferroptosis by inhibiting the AMPK/mTOR pathway and increasing GPX4 level in trophoblastic cells.

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The data that support the finding of this study are available from the corresponding author upon reasonable request.

Competing interests
There is no conflict of interest regarding the publication of this manuscript.     Enhanced ferroptosis by SIRT3 is associated with autophagy activation.
HTR8/SVeno and pTr2 cells were transfected with control siRNA or SIRT3 siRNA.  Enhanced ferroptosis by SIRT3 is associated with autophagy activation.
HTR8/SVeno and pTr2 cells were transfected with control siRNA or SIRT3 siRNA.