Despite tremendous research advancements in nonalcoholic fatty liver disease (NAFLD), our understanding of sex differences in NAFLD remains insufficient. This review summarizes the current knowledge on sex differences in NAFLD, identifies gaps, and discusses important considerations for future research. The prevalence and severity of NAFLD are higher in men than in women during the reproductive age. However, after menopause, NAFLD occurs at a higher rate in women, suggesting that estrogen is protective. Sex differences also exist for the major risk factors of NAFLD. In general, animal models of NAFLD recapitulate the sex differences observed in patients, with more severe steatosis and steatohepatitis, more proinflammatory/profibrotic cytokines, and a higher incidence of hepatic tumors in male than female subjects. Based on computer modeling, female and male livers are metabolically distinct with unique regulators modulating sex-specific metabolic outcomes. Analysis of the literature reveals that most published clinical and epidemiological studies fail to examine sex differences appropriately. Considering the paucity of data on sex differences and the knowledge that regulators of pathways relevant to current therapeutic targets for NAFLD differ by sex, clinical trials should be designed to test drug efficacy and safety according to sex, age, reproductive stage (i.e., menopause), and synthetic hormone use. Conclusion: Sex differences do exist in the prevalence, risk factors, fibrosis, and clinical outcomes of NAFLD, suggesting that, while not yet incorporated, sex will probably be considered in future practice guidelines; adequate consideration of sex differences, sex hormones/menopausal status, age, and other reproductive information in clinical investigation and gene association studies of NAFLD are needed to fill current gaps and implement precision medicine for patients with NAFLD.

Sustained c-Jun N-terminal kinase (JNK) activation has been implicated in many models of cell death and tissue injury. Phosphorylated JNK (p-JNK) interacts with the mitochondrial outer membrane SH3 homology associated BTK binding protein (Sab, or SH3BP5). Using knockdown or liver-specific deletion of Sab, we aimed to elucidate the consequences of this interaction on mitochondrial function in isolated mitochondria and liver injury models in vivo. Respiration in isolated mitochondria was directly inhibited by p-JNK 1 adenosine triphosphate. Knockdown or liver-specific knockout of Sab abrogated this effect and markedly inhibited sustained JNK activation and liver injury from acetaminophen or tumor necrosis factor/galactosamine. We then elucidated an intramitochondrial pathway in which interaction of JNK and Sab on the outside of the mitochondria released protein tyrosine phosphatase, nonreceptor type 6 (SHP1, or PTPN6) from Sab in the inside of the mitochondrial outer membrane, leading to its activation and transfer to the inner membrane, where it dephosphorylates P-Y419Src (active), which required a platform protein, docking protein 4 (DOK4), on the inner membrane. Knockdown of mitochondrial DOK4 or SHP1 inhibited the inactivation of mitochondrial p-Src and the effect of p-JNK on mitochondria. Conclusions: The binding to and phosphorylation of Sab by p-JNK on the outer mitochondrial membrane leads to SHP1-dependent and DOK4-dependent inactivation of p-Src on the inner membrane; inactivation of mitochondrial Src inhibits electron transport and increases reactive oxygen species release, which sustains JNK activation and promotes cell death and organ injury.

Although necrosis in the acetaminophen (APAP) model is known to be regulated by c-Jun NH2- terminal kinase, (JNK) through interaction with mitochondria, the role of necroptosis through receptor interacting proteins 1 and 3 (RIPK1 and RIPK3) has also been suggested. Our aim was to determine the relationship between these two mechanisms of cell death. To verify the participation of RIPK1, we used antisense knockdown and confirmed protection comparable to the RIPK1 inhibitor, necrostatin in vivo and in vitro. However, we found no evidence that RIPK3 is expressed in primary mouse hepatocytes under basal conditions or after APAP and RIPK3−/− mice were not protected. RIPK3 was exclusively expressed in nonparenchymal cells. RIPK1 knockdown protected RIPK3−/− mice to the same extent as wild type mice, underscoring the independent role of RIPK1. We confirmed necroptosis is not involved in APAP toxicity by using mixed lineage kinase domain-like protein (MLKL) −/− mice, which were not protected from APAP. Next we addressed if there is interplay between RIPK1 and JNK. RIPK1 knockdown decreased the level of JNK activation and translocation to mitochondria and abrogated subsequent translocation of dynamin-related protein-1 (Drp1). Interestingly, APAP induced the translocation of RIPK1 to mitochondria which was unaffected by the knockdown of the mitochondrial JNK docking protein, Sh3 homology3 binding protein5 (Sab). Conclusion RIPK1 participates in APAP induced necrosis upstream of JNK activation while RIPK3 and MLKL are dispensable, indicating that necroptosis does not contribute to APAP-induced necrosis and RIPK1 has a unique, independent role.

Sustained JNK activation plays a critical role in hepatotoxicity by acetaminophen or GalN/TNF-␣. To address the importance of JNK translocation to mitochondria that accompanies sustained activation in these models, we assessed the importance of the expression of a potential initial target of JNK in the outer membrane of mitochondria, namely Sab (SH3 domain-binding protein that preferentially associates with Btk), also known as Sh3bp5 (SH3 domain-binding protein 5). Silencing the expression of Sab in the liver using adenoviral shRNA inhibited sustained JNK activation and mitochondrial targeting of JNK and the upstream MKK4 (MAPK kinase 4), accompanied by striking protection against liver injury in vivo and in cultured hepatocytes in both toxicity models. We conclude that mitochondrial Sab may serve as a platform for the MAPK pathway enzymes and that the interaction of stress-activated JNK with Sab is required for sustained JNK activation and toxicity.

Our aim was to better understand the mechanism and importance of sustained c-Jun N-terminal kinase (JNK) activation in endoplasmic reticulum (ER) stress and effects of ER stress on mitochondria by determining the role of mitochondrial JNK binding protein, Sab. Tunicamycin or brefeldin A induced a rapid and marked decline in basal mitochondrial respiration and reserve-capacity followed by delayed mitochondrial-mediated apoptosis. Knockdown of mitochondrial Sab prevented ER stress-induced sustained JNK activation, impaired respiration, and apoptosis, but did not alter the magnitude or time course of activation of ER stress pathways. P-JNK plus adenosine 5′-triphosphate (ATP) added to isolated liver mitochondria promoted superoxide production, which was amplified by addition of calcium and inhibited by a blocking peptide corresponding to the JNK binding site on Sab (KIM1). This peptide also blocked tunicamycin-induced inhibition of cellular respiration. In conclusion, ER stress triggers an interaction of JNK with mitochondrial Sab, which leads to impaired respiration and increased mitochondrial reactive oxygen species, sustaining JNK activation culminating in apoptosis.