Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria.

Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes.

Mitochondrial dynamics, Leydig cell function, and age-related testosterone deficiency.

The mitochondrial translocator protein (18 kDa; TSPO) is a high-affinity cholesterol-binding protein that is an integral component of the cholesterol trafficking scaffold responsible for determining the rate of cholesterol import into the mitochondria for steroid biosynthesis. Previous studies have shown that TSPO declines in aging Leydig cells (LCs) and that its decline is associated with depressed circulating testosterone levels in aging rats. However, TSPO's role in the mechanistic decline in LC function is not fully understood. To address the role of TSPO depletion in LC function, we first examined mitochondrial quality in Tspo knockout mouse tumor MA-10 nG1 LCs compared to wild-type MA-10 cells. Tspo deletion caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1) overexpression resulted in the restoration of mitochondrial function and mitochondrial morphology as well as in steroid formation in TSPO-depleted nG1 LCs. LCs isolated from aged rats form less testosterone than LCs isolated from young rats. Treatment of aging LCs with M1 improved mitochondrial function and increased androgen formation, suggesting that aging LC dysfunction may stem from compromised mitochondrial dynamics caused by the age-dependent LC TSPO decline. These results, taken together, suggest that maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain or restore testosterone levels with aging.

In vitro and in vivo studies on the effect of a mitochondrial fusion promoter on Leydig cell integrity and function.

Background: The interstitial testicular Leydig cells are responsible for the production of testosterone, which functionally deteriorate with normal aging. Decreased expression of mitochondrial steroidogenic interactome proteins and diminished mitochondrial function in aging Leydig cells suggest that mitochondrial dynamics play a role in maintaining adequate levels of testosterone. Optic atrophy 1 (OPA1) protein regulates mitochondrial dynamics and cristae formation in many cell types. Previous studies showed that increasing OPA1 expression in dysfunctional Leydig cells restored mitochondrial function and recovered androgen production to levels found in healthy Leydig cells. These findings suggested that mitochondrial dynamics may be a promising target to ameliorate diminished testosterone levels in aging males. Methods: We used twelve-month-old rats to explore the relationship between mitochondrial dynamics and Leydig cell function. Isolated Leydig cells from aged rats were treated ex vivo with the cell-permeable mitochondrial fusion promoter 4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl)hydrazono)ethyl) phenol (mitochondrial fusion promoter M1), which enhances mitochondrial tubular network formation. In parallel, rats were treated with 2 mg/kg/day M1 for 6 weeks before Leydig cells were isolated. Results: Ex vivo M1-treated cells showed enhanced mitochondrial tubular network formation by transmission electron microscopy, enhanced Leydig cell mitochondrial integrity, improved mitochondrial function, and higher testosterone biosynthesis compared to controls. However, in vivo treatment of aged rats with M1 not only failed to re-establish testosterone levels to that of young rats, it also led to further reduction of testosterone levels and increased apoptosis, suggesting M1 toxicity in the testis. The in vivo M1 toxicity seemed to be tissue-specific, however. Conclusion: Promoting mitochondrial fusion may be one approach to enhancing cell health and wellbeing with aging, but more investigations are warranted. Our findings suggest that fusion promoters could potentially enhance the productivity of aged Leydig cells when carefully regulated.

Testosterone recovery therapy targeting dysfunctional Leydig cells.

Reduced serum testosterone affects millions of men across the world and has been linked to several comorbidities, metabolic dysfunctions, and quality of life changes. The standard treatment for testosterone deficiency remains testosterone replacement therapy. However, limitations on its use and the risk of significant adverse effects make alternative therapeutics desirable. Studies on the mechanisms regulating and synthesizing testosterone formation in testicular Leydig cells demonstrate numerous endogenous targets that could increase testosterone biosynthesis, which could alleviate reduced testosterone effects. Testosterone biosynthesis is facilitated by a conglomerate of cytosolic and mitochondrial proteins that facilitate cholesterol translocation into the mitochondria, the rate-limiting step in steroidogenesis. An effective therapeutic approach would be required to increase endogenous testosterone formation by enhancing steroidogenesis in Leydig cells. Numerous ligands for steroidogenic proteins have been developed, which increase steroid hormone formation. However, off-target effects on neurosteroid and adrenal steroid formation may limit their clinical use. First-in-class biologics, such as voltage-dependent anion channel peptides and transplantation of induced human Leydig-like cells offer advances in the development of specific strategies that could be used to enhance endogenous steroid formation in hormone deficient patients.

Impact of endocrine-disrupting chemicals on steroidogenesis and consequences on testicular function.

Testicular steroidogenesis is a tightly regulated process that produces the androgens important for the development, maintenance and function of the male reproductive system. These androgens are also essential for overall health, and well-being. Disruptions in the ability of the testis to form steroids can result in developmental abnormalities, dysfunction, and infertility. Endocrine-disrupting chemicals (EDCs) can interfere with the intricate signaling and metabolizing networks that produce androgens and promote their dysfunction. These chemicals are found ubiquitously in our environment, as they are integral components of products that are used every day. The effects of EDCs, such as bisphenols, phthalates, and alkyl chemicals, have been studied independently, revealing deleterious effects; but the combined influence of these structures on steroidogenesis has yet to be completely elucidated. This manuscript presents an updated review on EDC mixtures and their impact on testicular function and fertility, highlighting new findings that illustrate the anti-androgenic capabilities of EDC mixtures.

Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease.

Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Synchronous Fibrolamellar Hepatocellular Carcinoma and Auricular Myxoma.

Synchronic occurrence of benign and malignant tumors is extremely rare. Fibrolamellar hepatocellular carcinoma represents 1% to 2% of all hepatocarcinomas, while myxomas represent about half of all the cases of primary tumors of the heart. We present the case of a 53-year-old woman with a left atrial myxoma that was surgically removed. Several weeks later, the patient returned to the hospital with abdominal pain. CT scan showed a mass in the left lobe of the liver that was resected and diagnosed as fibrolamellar hepatocellular carcinoma. As of this writing, the patient is healthy.