Sirtuin 1 suppresses mitochondrial dysfunction of ischemic mouse livers in a mitofusin 2-dependent manner

Abstract

Ischemia/reperfusion (I/R) injury is a major cause of morbidity and mortality after liver surgery. The role of Sirtuin 1 (SIRT1) in hepatic I/R injury remains elusive. Using human and mouse livers, we investigated the effects of I/R on hepatocellular SIRT1. SIRT1 expression was significantly decreased after I/R. Genetic overexpression or pharmacological activation of SIRT1 markedly suppressed defective autophagy, onset of the mitochondrial permeability transition, and hepatocyte death after I/R, whereas SIRT1-null hepatocytes exhibited increased sensitivity to I/R injury. Biochemical approaches revealed that SIRT1 interacts with mitofusin-2 (MFN2). Furthermore, MFN2, but not MFN1, was deacetylated by SIRT1. Moreover, SIRT1 overexpression substantially increased autophagy in wild-type cells, but not in MFN2-deficient cells. Thus, our results demonstrate that the loss of SIRT1 causes a sequential chain of defective autophagy, mitochondrial dysfunction, and hepatocyte death after I/R. Cell Death and Differentiation (2016) 23, 279-290; doi:10.1038/cdd.2015; published online 17 July 2015During hepatic resection and liver transplantation operations, inflow occlusion is employed to temporarily limit blood flow to minimize intraoperative blood loss. Although prolonged ischemia eventually causes tissue injury, severe damage paradoxically does not occur until recovery of blood flow and restitutions of normal physiological pH.1 Ischemia/reperfusion (I/R) injury is a key cause of postoperative liver failure during hemorrhagic shock, hepatectomy, and liver transplantation. Despite continuous efforts, substantial benefits from current strategies have not been realized, mainly because of the multifactorial nature of I/R injury.I/R initiates opening of high-conductance permeability transition pores in the mitochondrial inner membranes, leading to mitochondrial permeability transition (MPT).2 Onset of the MPT uncouples oxidative phosphorylation and depolarizes mitochondrial membrane potential (ΔΨ m ) that in turn causes ATP depletion and cell death.Autophagy is an evolutionarily conserved catabolic process. Among the three forms of autophagy, macroautophagy is of particular importance in the liver, as it not only degrades unneeded intracellular proteins but also digests injured or dysfunctional organelles such as abnormal mitochondria. 3 We have shown that impaired autophagy contributes to liver I/R injury. [4][5][6] Sirtuin1 (SIRT1) deacetylates Lys residues of both histone and nonhistone targets, and is activated in response to fasting and calorie restriction in the liver, a condition inducing autophagy. 7,8 Despite its extramitochondrial localization, SIRT1 appears to affect mitochondrial biogenesis 9 and bioenergetics, 10 but its mechanisms remain elusive. Using isolated hepatocytes, mouse livers, SIRT1-null mice, and human livers, we here demonstrate that I/R depletes livers of SIRT1 and that specific overexpression of SIRT1 mitigates defective autophagy, onset of the MPT, and subsequent hepatocyte death after both in vitro...

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

“…Cells were exposed to 2 hr of simulated ischemia by exposure to anoxic Krebs‐Ringer HEPES (KRH) solution at pH 6.2. Reperfusion was then induced by reoxygenating anoxic cells in normoxic KRH at pH 7.4 for up to 2 hr (Biel et al., 2016; Kim et al., 2008; Wang et al., 2011). Propidium iodide (PI) fluorometry revealed that greater than 50% cell death occurred in old hepatocytes after 2 hr of reperfusion, while young hepatocytes withstood these conditions quite well with minimal cell death, consistent with our previous report (Wang et al., 2011).…”

“…Prolonged I/R depletes young hepatocytes of SIRT1, which in turn causes necrotic cell death after reperfusion (Biel et al., 2016). To examine how short I/R affects SIRT1 in old hepatocytes, we assessed changes in SIRT1 expression before and after ischemia in young and old hepatocytes (Figure 1c).…”

“…Cell death assay, however, showed that SIRT1 overexpression alone did not protect old cells against I/R injury, indicating that other factor(s) are attributable for enhanced cell death in reperfused old hepatocytes. We recently reported that SIRT1 interacts with MFN2, a mitochondrial outer membrane protein and that this interaction is required for SIRT1‐dependent cytoprotection against prolonged ischemia (Biel et al., 2016). To examine how aging influences MFN2 expression following short I/R, changes in MFN2 expression were determined in both young and old hepatocytes (Figure 1d).…”

“…Given the central role of autophagy in supporting hepatocellular viability after I/R, we have previously demonstrated in young hepatocytes and livers that an NAD + ‐dependent deacetylase SIRT1 promotes autophagy and hepatocyte survival after reperfusion (Biel et al., 2016). SIRT1 can activate autophagy through various pathways, including modulation of forkhead box transcription factors (FOX), ATG5, ATG7, LC3, and BECN1 (Huang et al., 2015; Lee et al., 2008; Sun et al., 2015).…”

“…SIRT1 can activate autophagy through various pathways, including modulation of forkhead box transcription factors (FOX), ATG5, ATG7, LC3, and BECN1 (Huang et al., 2015; Lee et al., 2008; Sun et al., 2015). We recently reported that a gradual depletion of this deacetylase with extended ischemia is a causal to mitochondrial dysfunction and cell death (Biel et al., 2016). Furthermore, SIRT1 overexpression reversed pathological events through its interaction with and subsequent deacetylation of MFN2.…”