Hepatic Mitochondrial Dysfunction Research Articles

Environmentally Relevant Concentrations of S-6PPD-Quinone Caused More Serious Hepatotoxicity Than R-Enantiomer and Racemate in Oncorhynchus mykiss.

6PPD-quinone (6PPD-Q) is frequently detected in various environmental media, and the environmentally relevant concentrations can be fatal to Oncorhynchus mykiss. Notably, 6PPD-Q has two enantiomers (S-6PPD-Q and R-6PPD-Q). In this study, O. mykiss was separately exposed to each enantiomer and racemate of 6PPD-Q for 96 h at environmentally relevant concentrations, and livers were collected. Effects on the biochemical, pathological, and ultrastructural changes were assessed, and metabolomics was conducted to elucidate the potential hepatotoxicity mechanism. Compared with the control treatment, the levels of catalase (CAT, all treatments except for 0.1 μg/L rac-6PPD-Q), and glutathione-S-transferase (GST, all treatments) significantly declined. Hepatocyte space became smaller, nuclear morphology changed, and nucleolysis occurred. Mitochondrial malformation and vesicle-like structure dilation of the endoplasmic reticulum (ER) were observed in the hepatocytes, which was most serious after S-6PPD-Q exposure. Some amino acid metabolism, folate biosynthesis, taurine and hypotaurine metabolism and purine metabolism were disturbed, consistent with mitochondrial dysfunction and ER stress. The differential metabolites were in the order of S-6PPD-Q (216) > rac-6PPD-Q (88) > R-6PPD-Q (56). Thus, 6PPD-Q-induced hepatic mitochondrial dysfunction and ER stress, causing metabolic disturbance and oxidative stress might be the toxic mechanism of 6PPD-Q in O. mykiss liver, and S-6PPD-Q effects were the most serious.

GTPBP8 mitigates nonalcoholic steatohepatitis (NASH) by depressing hepatic oxidative stress and mitochondrial dysfunction via PGC-1α signaling

Nonalcoholic steatohepatitis (NASH) is emerging as a major cause of liver transplantation and hepatocellular carcinoma (HCC). Regrettably, its pathological mechanisms are still not fully comprehended. GTP-binding protein 8 (GTPBP8), belonging to the GTP-binding protein superfamily, assumes a crucial role in RNA metabolism, cell proliferation, differentiation, and signal transduction. Its aberrant expression is associated with oxidative stress and mitochondrial dysfunctions. Nevertheless, its specific functions and mechanisms of action, particularly in NASH, remain elusive. In our current study, we initially discovered that human hepatocytes L02 displayed evident mitochondrial respiratory anomaly, mitochondrial damage, and dysfunction upon treatment with palmitic acids and oleic acids (PO), accompanied by significantly reduced GTPBP8 expression levels through RNA-Seq, RT-qPCR, western blotting, and immunofluorescence assays. We then demonstrated that GTPBP8 overexpression mediated by adenovirus vector (Ad-GTPBP8) markedly attenuate lipid accumulation, inflammatory response, and mitochondrial impair and dysfunction in hepatocytes stimulated by PO. Conversely, adenovirus vector-mediated GTPBP8 knockdown (Ad-shGTPBP8) significantly accelerated lipid deposition, inflammation and mitochondrial damage in PO-treated hepatocytes in vitro. Furthermore, we constructed an in vivo NASH murine model by giving a 16-week high fat high cholesterol diet (HFHC) diet to hepatocyte specific GTPBP8-knockout (GTPBP8HKO) mice. We firstly found that HFHC feeding led to metabolic disorder in mice, including high body weight, blood glucose and insulin levels, and liver dysfunctions, which were accelerated in these NASH mice with GTPBP8 deficiency in hepatocytes. Consistently, GTPBP8HKO remarkably exacerbated the progression of NASH phenotypes induced by HFHC, as proved by the anabatic lipid accumulation, inflammation, fibrosis and reactive oxygen species (ROS) production in liver tissues, which could be largely attributed to the severe mitochondrial damage and dysfunction. Mechanistically, we further identified that GTPBP8 interacted with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in hepatocytes. Importantly, the hepaprotective effects of GTPBP8 against mitochondrial dysfunction, oxidative stress and inflammation was largely dependent on PGC-1α expression. Collectively, GTPBP8 may exert a protective role in the progression of NASH, and targeting the GTPBP8/PGC-1α axis may represent a potential strategy for NASH treatment by improving mitochondrial functions.

Mettl3-m6A-YTHDF1 axis promotion of mitochondrial dysfunction in metabolic dysfunction–associated steatotic liver disease

BackgroundN6-methyladenosine (m6A) mRNA modification and mitochondrial function hold paramount importance in the advancement of metabolic dysfunction-associated steatotic liver disease (MASLD). AimThe aim of this study was to elucidate the impact of m6A on hepatic mitochondrial dysfunction and provide a novel perspective for a more comprehensive understanding of the pathogenesis of MASLD. MethodsHigh-throughput screening methods were used to identify the underlying transcriptome and proteome changes in MASLD model mice. Western blotting, blue native gel electrophoresis (BNGE), dot blot, and Seahorse analyses were conducted to identify and validate the underlying regulatory mechanisms of m6A on mitochondria. ResultsIn vivo, abnormal m6A modification in MASLD was attributed to the upregulation of methyltransferase like 3 (Mettl3) and the downregulation of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) induced by high-fat foods. In vitro, knockdown of Mettl3 inhibited hepatic oxidative phosphorylation (OXPHOS) and the mitochondrial respiratory chain (MRC), while overexpression of Mettl3 promoted these processes. However, knockout of the reader protein YTHDF1, which plays a crucial role in the m6A modification process, counteracted the effect of Mettl3 and suppressed mitochondrial OXPHOS. ConclusionsIn MASLD, damage to the MRC may be regulated by the Mettl3-m6A-YTHDF1 axis, particularly by the role of YTHDF1. Modulation of the Mettl3-m6A-YTHDF1 axis has the potential to improve mitochondrial function, alleviate MASLD symptoms, and decrease the likelihood of disease progression.

Patchouli alcohol alleviates metabolic dysfunction-associated steatohepatitis via inhibiting mitochondria-associated endoplasmic reticulum membrane disruption-induced hepatic steatosis and inflammation in rats

Metabolic dysfunction-associated steatohepatitis (MASH) is a severe metabolic dysfunction-associated steatotic liver disease (MASLD) characterized by abnormal hepatic steatosis and inflammation. Previous studies have shown that Patchouli alcohol (PA), the primary component of Pogostemonis Herba, can alleviate digestive system diseases. However, its protection against MASH remains unclear. This study explored the protective effects and underlying mechanism of PA against high-fat diet-induced MASH in rats. Results showed that PA considerably reduced body weight, epididymal fat, and liver index and attenuated liver histological injury in MASH rats. PA alleviated hepatic injury by inhibiting steatosis and inflammation. These effects are associated with the improvement of SREBP-1c- and PPARα-mediated lipid metabolism and inhibition of the STING-signaling pathway-mediated inflammatory response. Moreover, PA-inhibited hepatic endoplasmic reticulum (ER) stress and mitochondrial dysfunction, reducing SREBP-1c and STING expressions and enhance PPARα expression. PA treatment had the strongest effect on the regulation of mitogen fusion protein 2 (Mfn2) in inhibiting mitochondrial dysfunction. Mfn2 is an important structural protein for binding ERs and mitochondria to form mitochondria-associated ER membranes (MAMs). MASH-mediated disruption of MAMs was inhibited after PA treatment-induced Mfn2 activation. Therefore, the pharmacological effect of PA on MASH is mainly attributed to the inhibition of MAM disruption-induced hepatic steatosis and inflammation. The findings of this study may have implications for MASH treatment that do not neglect the role of Mfn2-mediated MAMs.

α-Ketoglutarate prevents hyperlipidemia-induced fatty liver mitochondrial dysfunction and oxidative stress by activating the AMPK-pgc-1α/Nrf2 pathway

α-Ketoglutarate (AKG), a crucial intermediate in the tricarboxylic acid cycle, has been demonstrated to mitigate hyperlipidemia-induced dyslipidemia and endothelial damage. While hyperlipidemia stands as a major trigger for non-alcoholic fatty liver disease, the protection of AKG on hyperlipidemia-induced hepatic metabolic disorders remains underexplored. This study aims to investigate the potential protective effects and mechanisms of AKG against hepatic lipid metabolic disorders caused by acute hyperlipidemia. Our observations indicate that AKG effectively alleviates hepatic lipid accumulation, mitochondrial dysfunction, and loss of redox homeostasis in P407-induced hyperlipidemia mice, as well as in palmitate-injured HepG2 cells and primary hepatocytes. Mechanistic insights reveal that the preventive effects are mediated by activating the AMPK-PGC-1α/Nrf2 pathway. In conclusion, our findings shed light on the role and mechanism of AKG in ameliorating abnormal lipid metabolic disorders in hyperlipidemia-induced fatty liver, suggesting that AKG, an endogenous mitochondrial nutrient, holds promising potential for addressing hyperlipidemia-induced fatty liver conditions.

Genistein alleviates chronic heat stress-induced lipid metabolism disorder and mitochondrial energetic dysfunction by activating the GPR30-AMPK-PGC-1α signaling pathways in the livers of broiler chickens

The objective of this study was to investigate the preventive effects and mechanisms of genistein (GEN) on production performance and metabolic disorders in broilers under chronic heat stress (HS). A total of 120 male three-week-old Ross broilers were randomly assigned to 5 groups: a thermoneutral zone (TN) group maintained at normal temperature (21 ± 1°C daily), an HS group subjected to cyclic high temperature (32 ± 1°C for 8 h daily), and three groups exposed to HS with varying doses of GEN (50, 100, or 150 mg/kg diet). The experimental period lasted for three weeks. Here, HS led to a decline in growth performance parameters and hormone secretion disorders (P < 0.05), which were improved by 100 and 150 mg/kg GEN treatment (P < 0.05). Moreover, the HS-induced increases in the liver index (P < 0.01) and abdominal fat rate (P < 0.05) were attenuated by 150 mg/kg GEN (P < 0.05). The HS-induced excessive lipid accumulation in the liver and serum (P < 0.01) was ameliorated after 100 and 150 mg/kg GEN treatment (P < 0.05). Furthermore, the HS-induced decreases in lipolysis-related mRNA levels and increases in lipid synthesis-related mRNA levels in the liver (P < 0.01) were effectively blunted after 100 and 150 mg/kg GEN treatment (P < 0.05). Importantly, the HS-stimulated hepatic mitochondrial energetic dysfunction and decreases in the mRNA or protein levels of peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial transcription factor A in the liver were ameliorated by 150 mg/kg GEN (P < 0.05). Moreover, 50-150 mg/kg GEN treatment resulted in a significant increase in the mRNA or protein levels of G protein-coupled estrogen receptor (GPR30), AMP-activated protein kinase (AMPK) α1, phosphorylated AMPKα, and phosphorylated acetyl-CoA carboxylase α. Collectively, GEN alleviated metabolic disorders and hepatic mitochondrial energetic dysfunction under HS, possibly through the activation of GPR30-AMPM-PGC-1α pathways. These data provide a sufficient basis for GEN as an additive to alleviate HS in broilers.

The PNPLA3 I148M variant increases ketogenesis and decreases hepatic de novo lipogenesis and mitochondrial function in humans

The PNPLA3 I148M variant is the major genetic risk factor for all stages of fatty liver disease, but the underlying pathophysiology remains unclear. We studied the effect of this variant on hepatic metabolism in homozygous carriers and non-carriers under multiple physiological conditions with state-of-the-art stable isotope techniques. After an overnight fast, carriers had higher plasma β-hydroxybutyrate concentrations and lower hepatic de novo lipogenesis (DNL) compared to non-carriers. After a mixed meal, fatty acids were channeled toward ketogenesis in carriers, which was associated with an increase in hepatic mitochondrial redox state. During a ketogenic diet, carriers manifested increased rates of intrahepatic lipolysis, increased plasma β-hydroxybutyrate concentrations, and decreased rates of hepatic mitochondrial citrate synthase flux. These studies demonstrate that homozygous PNPLA3 I148M carriers have hepatic mitochondrial dysfunction leading to reduced DNL and channeling of carbons to ketogenesis. These findings have implications for understanding why the PNPLA3 variant predisposes to progressive liver disease.

Chronic Intermittent Hypoxia-Induced Dysmetabolism Is Associated with Hepatic Oxidative Stress, Mitochondrial Dysfunction and Inflammation.

The association between obstructive sleep apnea (OSA) and metabolic disorders is well-established; however, the underlying mechanisms that elucidate this relationship remain incompletely understood. Since the liver is a major organ in the maintenance of metabolic homeostasis, we hypothesize that liver dysfunction plays a crucial role in the pathogenesis of metabolic dysfunction associated with obstructive sleep apnea (OSA). Herein, we explored the underlying mechanisms of this association within the liver. Experiments were performed in male Wistar rats fed with a control or high fat (HF) diet (60% lipid-rich) for 12 weeks. Half of the groups were exposed to chronic intermittent hypoxia (CIH) (30 hypoxic (5% O2) cycles, 8 h/day) that mimics OSA, in the last 15 days. Insulin sensitivity and glucose tolerance were assessed. Liver samples were collected for evaluation of lipid deposition, insulin signaling, glucose homeostasis, hypoxia, oxidative stress, antioxidant defenses, mitochondrial biogenesis and inflammation. Both the CIH and HF diet induced dysmetabolism, a state not aggravated in animals submitted to HF plus CIH. CIH aggravates hepatic lipid deposition in obese animals. Hypoxia-inducible factors levels were altered by these stimuli. CIH decreased the levels of oxidative phosphorylation complexes in both groups and the levels of SOD-1. The HF diet reduced mitochondrial density and hepatic antioxidant capacity. The CIH and HF diet produced alterations in cysteine-related thiols and pro-inflammatory markers. The results obtained suggest that hepatic mitochondrial dysfunction and oxidative stress, leading to inflammation, may be significant factors contributing to the development of dysmetabolism associated with OSA.

FRI029 Chronic Free Fatty Acid Elevation Is Associated With An Overcompensatory Increase In Hepatic Mitochondrial Function In Patients With Overweight Or Obesity

Abstract Disclosure: F. Bril: None. S. Kalavalapalli: None. K. Cusi: None. The obesity epidemic has resulted in a significant increase in the prevalence of nonalcoholic fatty liver disease (NAFLD). Some patients with NAFLD progress to more severe forms of the disease, such as nonalcoholic steatohepatitis (NASH) and cirrhosis. Our central hypothesis is that the inability of hepatic mitochondria to enhance nutrient oxidation in the setting of nutrient oversupply plays a key role in the progression of NAFLD. Specifically, the aim of this study was to assess if free fatty acid (FFA) excess contributes to hepatic mitochondrial dysfunction in patients with overweight or obesity. Patients with BMI ≥ 25kg/m2, without diabetes were included in the study. Patients underwent a 2-hour oral glucose tolerance test (OGTT) and a liver proton magnetic resonance spectroscopy (1H-MRS) to measure liver fat. In vivo hepatic mitochondrial ATP levels were measured by phosphorus (31P)-MRS every 30 minutes during a 2-hour oral fructose challenge (OFC). Due to unregulated phosphorylation of fructose upon entering hepatocytes, the OFC provides a dynamic measurement of hepatic ATP consumption and re-synthesis. Hepatic ATP levels were estimated as β-ATP/total phosphorus. A total of 37 patients were recruited for this study (age: 54 ± 11 years; sex: 43% male/57% female; BMI: 34.4 ± 6.3 kg/m2; A1c: 5.7 ± 0.4%; NAFLD: 51%). No differences were found in hepatic ATP levels in patients with or without NAFLD (0.019 ± 0.015 vs. 0.016 ± 0.012, p=0.44). Patients were then divided in three groups (tertiles) based on fasting FFA levels. We did not observe any significant differences among these three groups in key clinical variables, such as age, sex, BMI, A1c or prevalence of NAFLD. At baseline (pre OFC), we did not observe any difference in hepatic ATP levels (i.e., β-ATP/total phosphorus) among FFA tertiles (0.020 ± 0.015, 0.020 ± 0.012, and 0.012 ± 0.012, p=0.25). During the OFC, we estimated changes over time in hepatic ATP levels as the decremental area under the curve (AUROC) of β-ATP/total phosphorus. We observed that the decremental AUROC of hepatic ATP levels during OFC was significantly different among groups (0.57 ± 0.86, 0.14 ± 0.59, -0.40 ± 0.54, p=0.006). Patients in the highest tertile of FFAs showed no consumption of ATP after the OFC, but rather an overall increase in ATP AUROC over time. Plasma FFA levels correlated well with hepatic ATP AUROC during the OFC (-0.47, p =0.007). Conclusion: Chronic elevations in FFA levels are associated with changes in hepatic mitochondrial ATP synthesis, which were unmasked after an oral fructose challenge. While long-term, FFA elevations may impair mitochondrial function, in our study we observed an overcompensatory response in patients with high plasma FFA, characterized by an increase in hepatic ATP levels over time after fructose consumption. At what point this compensation is overwhelmed leading to inflammation and cellular lipotoxicity remains to be determined. Presentation: Friday, June 16, 2023

Uroguanylin prevents hepatic steatosis, mitochondrial dysfunction and fibrosis in obesity-associated NAFLD.

The biological mediators supporting the resolution of liver steatosis, inflammation and fibrosis after bariatric surgery in patients with obesity and NAFLD remain unclear. We sought to analyze whether uroguanylin and guanylin, two gut hormones involved in the regulation of satiety, food preference and adiposity, are involved in the amelioration of obesity-associated NAFLD after bariatric surgery. Proguanylin (GUCA2A) and prouroguanylin (GUCA2B) were measured in 214 participants undergoing bariatric surgery with biopsy-proven NAFLD diagnosis. Pathways involved in lipid metabolism, mitochondrial network and fibrogenesis were evaluated in liver biopsies (n=137). The effect of guanylin and uroguanylin on these metabolic functions was assessed in HepG2 hepatocytes and LX-2 hepatic stellate cells (HSC) under lipotoxic and profibrogenic conditions. Plasma and hepatic expression of GUCA2B were decreased in obesity-associated NAFLD. Both GUCA2A and GUCA2B levels were increased after sleeve gastrectomy and Roux-en-Y gastric bypass in parallel to the improved liver function. The liver of patients with type 2 diabetes showed impaired mitochondrial β-oxidation, biogenesis, dynamics as well as increased fibrosis. Uroguanylin diminished the lipotoxicity in palmitate-treated HepG2 hepatocytes, evidenced by decresased steatosis and lipogenic factors, as well as increased mitochondrial network expression, AMPK-induced β-oxidation and oxygen consumption rate. Additionally, uroguanylin, but not guanylin, reversed HSC myofibroblast transdifferentiation as well as fibrogenesis after TGF-β1 stimulation. Uroguanylin constitutes a protective factor against lipotoxicity, mitochondrial dysfunction and fibrosis. Increased GUCA2B levels might contribute to improve liver injury in patients with obesity-associated NAFLD after bariatric surgery.

Maternal hepatic adaptations during obese pregnancy encompass lobe-specific mitochondrial alterations and oxidative stress.

Maternal obesity(MO) is rising worldwide, affecting half of all gestations, constituting a risk-factor for pregnancy-associated liver diseases (PALD) and hepatic diseases. PALD occur in approximately 3% of pregnancies and are characterized by maternal hepatic oxidative stress(OS) and mitochondrial dysfunction. Understanding the role of MO on liver function and pathophysiology could be crucial for understanding the altered pathways leading to PALD and liver disease. We investigated specific hepatic metabolic alterations in mitochondria and oxidative stress during MO at late-gestation. Maternal hepatic tissue was collected at 90% gestation in Control and MO ewes. Maternal hepatic redox state, mitochondrial Respiratory Chain(MRC) and OS markers were investigated. MO decreased MRC complex-II activity and SDHA and SDHB protein, increased complex-I and complex-IV activities despite reduced mtCO1 protein, and increased ATP5a protein. Hepatic MO-metabolic remodeling was characterized by decreased ANT-1/2 and VDAC protein expression and PKA activity, and augmented NAD+/NADH ratio due to reduced NADH levels. MO showed an altered redox state with increased OS, increased lipid peroxidation, decreased GSH/GSSG ratio, increased SOD and decreased catalase antioxidant enzymatic activities, lower catalase, glutathione peroxidase (Gpx)-4 and glutathione reductase protein expression, and increased Gpx-1 abundance. MO-related hepatic changes were more evident in the right lobe, corroborated by the integrative data analysis. Hepatic tissue from obese pregnant ewes showed alterations in the redox state, consistent with OS and MRC and metabolism remodeling. These are hallmarks of PALD and hepatic disease, supporting MO as a risk-factor and highlighting OS and mitochondrial dysfunction as mechanisms responsible for liver disease predisposition.

Unveiling the Link: Exploring Mitochondrial Dysfunction as a Probable Mechanism of Hepatic Damage in Post-Traumatic Stress Syndrome.

PTSD is associated with disturbed hepatic morphology and metabolism. Neuronal mitochondrial dysfunction is considered a subcellular determinant of PTSD, but a link between hepatic mitochondrial dysfunction and hepatic damage in PTSD has not been demonstrated. Thus, the effects of experimental PTSD on the livers of high anxiety (HA) and low anxiety (LA) rats were compared, and mitochondrial determinants underlying the difference in their hepatic damage were investigated. Rats were exposed to predator stress for 10 days. Then, 14 days post-stress, the rats were evaluated with an elevated plus maze and assigned to HA and LA groups according to their anxiety index. Experimental PTSD caused dystrophic changes in hepatocytes of HA rats and hepatocellular damage evident by increased plasma ALT and AST activities. Mitochondrial dysfunction was evident as a predominance of small-size mitochondria in HA rats, which was positively correlated with anxiety index, activities of plasma transaminases, hepatic lipids, and negatively correlated with hepatic glycogen. In contrast, LA rats had a predominance of medium-sized mitochondria. Thus, we show links between mitochondrial dysfunction, hepatic damage, and heightened anxiety in PTSD rats. These results will provide a foundation for future research on the role of hepatic dysfunction in PTSD pathogenesis.

Phosphatidylserine decarboxylase downregulation in uric acid‑induced hepatic mitochondrial dysfunction and apoptosis.

The molecular mechanisms underlying uric acid (UA)-induced mitochondrial dysfunction and apoptosis have not yet been elucidated. Herein, we investigated underlying mechanisms of UA in the development of mitochondrial dysfunction and apoptosis. We analyzed blood samples of individuals with normal UA levels and patients with hyperuricemia. Results showed that patients with hyperuricemia had significantly elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, which may indicate liver or mitochondrial damage in patients with hyperuricemia. Subsequently, lipidomic analysis of mouse liver tissue mitochondria and human liver L02 cell mitochondria was performed. Compared with control group levels, high UA increased mitochondrial phosphatidylserine (PS) and decreased mitochondrial phosphatidylethanolamine (PE) levels, whereas the expression of mitochondrial phosphatidylserine decarboxylase (PISD) that mediates PS and PE conversion was downregulated. High UA levels also inhibited signal transducer and activator of transcription 3 （STAT3） phosphorylation as well as mitochondrial respiration, while inducing apoptosis both in vivo and in vitro. Treatment with allopurinol, overexpression of PISD, and lyso-PE (LPE) administration significantly attenuated the three above-described effects in vitro. In conclusion, UA may induce mitochondrial dysfunction and apoptosis through mitochondrial PISD downregulation. This study provides a new perspective on liver damage caused by hyperuricemia.

1597-P: KCNH6 Regulates Hepatic Mitochondrial Function and Glucose Metabolism via NF-κB Pathway

KCN, a human ether-a-go-go-related gene, encodes KCN ion channel in the Kv11 family, which ranks 11th in abundance among different ion channel families. KCNH6, a subfamily of KCN potassium channel family, has been proven to affect glucose metabolism and insulin secretion both in humans and mice. However, the precise function of KCNH6 in the liver remains unknown. In this study, we showed that knockout (KO) of Kcnh6 impairs mitochondrial function and insulin tolerance as well as promotes high-fat-diet-induced insulin resistance. In line, overexpression of Kcnh6 in hepatocytes ameliorates fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes ameliorating insulin resistance. Similarly, induction of Kcnh6 by tail vein injection into KO mice decreased insulin tolerance. Mechanistically, Kcnh6 improved mitochondrial function and insulin secretion via the the NFκB-IκB kinase (IKK) pathway both in vitro and in vivo. Therefore, we suggest Kcnh6 as a potential novel therapeutic target to treat hepatic energy metabolism and mitochondrial dysfunction and insulin resistance. Disclosure R.Zhao: None. J.Lu: None. F.Xiong: None.

Albumosomes formed by cytoplasmic pre-folding albumin maintain mitochondrial homeostasis and inhibit nonalcoholic fatty liver disease

Hepatic mitochondrial dysfunction contributes to the progression of nonalcoholic fatty liver disease (NAFLD). However, the factors that maintain mitochondrial homeostasis, especially in hepatocytes, are largely unknown. Hepatocytes synthesize various high-level plasma proteins, among which albumin is most abundant. In this study, we found that pre-folding albumin in the cytoplasm is completely different from folded albumin in the serum. Mechanistically, endogenous pre-folding albumin undergoes phase transition in the cytoplasm to form a shell-like spherical structure, which we call the “albumosome”. Albumosomes interact with and trap pre-folding carnitine palmitoyltransferase 2 (CPT2) in the cytoplasm. Albumosomes control the excessive sorting of CPT2 to the mitochondria under high-fat-diet-induced stress conditions; in this way, albumosomes maintain mitochondrial homeostasis from exhaustion. Physiologically, albumosomes accumulate in hepatocytes during murine aging and protect the livers of aged mice from mitochondrial damage and fat deposition. Morphologically, mature albumosomes have a mean diameter of 4μm and are surrounded by heat shock protein Hsp90 and Hsp70 family proteins, forming a larger shell. The Hsp90 inhibitor 17-AAG promotes hepatic albumosomal accumulation in vitro and in vivo, through which suppressing the progression of NAFLD in mice.

Mitochondrial DNA methylation in metabolic associated fatty liver disease.

Hepatic lipid accumulation and mitochondrial dysfunction are hallmarks of metabolic associated fatty liver disease (MAFLD), yet molecular parameters underlying MAFLD progression are not well understood. Differential methylation within the mitochondrial DNA (mtDNA) has been suggested to be associated with dysfunctional mitochondria, also during progression to Metabolic Steatohepatitis (MeSH). This study further investigates whether mtDNA methylation is associated with hepatic lipid accumulation and MAFLD. HepG2 cells were constructed to stably express mitochondria-targeted viral and prokaryotic cytosine DNA methyltransferases (mtM.CviPI or mtM.SssI for GpC or CpG methylation, respectively). A catalytically inactive variant (mtM.CviPI-Mut) was constructed as a control. Mouse and human patients' samples were also investigated. mtDNA methylation was assessed by pyro- or nanopore sequencing. Differentially induced mtDNA hypermethylation impaired mitochondrial gene expression and metabolic activity in HepG2-mtM.CviPI and HepG2-mtM.SssI cells and was associated with increased lipid accumulation, when compared to the controls. To test whether lipid accumulation causes mtDNA methylation, HepG2 cells were subjected to 1 or 2 weeks of fatty acid treatment, but no clear differences in mtDNA methylation were detected. In contrast, hepatic Nd6 mitochondrial gene body cytosine methylation and Nd6 gene expression were increased in mice fed a high-fat high cholesterol diet (HFC for 6 or 20 weeks), when compared to controls, while mtDNA content was unchanged. For patients with simple steatosis, a higher ND6 methylation was confirmed using Methylation Specific PCR, but no additional distinctive cytosines could be identified using pyrosequencing. This study warrants further investigation into a role for mtDNA methylation in promoting mitochondrial dysfunction and impaired lipid metabolism in MAFLD.

Verbenalin attenuates hepatic damage and mitochondrial dysfunction in alcohol-associated steatohepatitis by regulating MDMX/PPARα-mediated ferroptosis.

Verbenalin is a major compound in Verbena officinalis L. Verbena officinalis L was first recorded in the 'Supplementary Records of Famous Physicians.' Verbenalin (VE) is its active constituent and has been found to have many biological effects, including anti-obesity, anti-inflammatory, and antioxidant activities, removing jaundice, and treating malaria. It could treat lump accumulation, dysmenorrhea, throat obstruction, edema, jaundice, and malaria. Palmitic acid (PA), oleic acid (OA), ethanol, and acetaminophen liver injuries have been proven to benefit from verbenalin. To study the effects of verbenalin on the prevention of alcoholic steatohepatitis (ASH) through the regulation of oxidative stress and mitochondrial dysfunction by regulating MDMX (Murine double minute X)/PPARα (Peroxisome proliferator-activated receptor alpha)-mediated ferroptosis. C57BL/6 mice treated with alcohol followed by the Gao-Binge protocol were administered verbenalin by gavage simultaneously. The mitochondrial mass and morphology were visualized using TEM. AML-12cells were stimulated with ethanol to mimic ASH in vitro. Western blotting, co-immunoprecipitation, and kit determination were simultaneously performed. The target protein of verbenalin was identified by molecular docking, and cellular thermal shift assay (CETSA) further confirmed its interactions. Verbenalin alleviates oxidative stress and ferroptosis in alcohol-associated steatohepatitis. To elucidate the molecular mechanism by which verbenalin inhibits abnormal mitochondrial dysfunction, molecular docking was performed, and MDMX was identified as the target protein of verbenalin. CETSA assays revealed a specific interaction between MDMX and verbenalin. Co-immunoprecipitation demonstrated that PPARα played a critical role in promoting the ability of MDMX to affect ferroptosis. Verbenalin regulates MDMX/PPARα-mediated ferroptosis in AML-12cells. Verbenalin regulates ferroptosis and highlights the therapeutic potential of verbenalin and ferroptosis inhibition in reducing alcoholic steatohepatitis.

Chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) leaf extracts regulate mitochondrial bioenergetics and fatty acid oxidation in C2C12 myotubes and primary hepatocytes

Ethnopharmacological relevanceChaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) is an important component of the regular diet and traditional medicine of indigenous communities in Mexico. Customarily, Chaya is consumed as a beverage made of macerated leaf, cooked, or prepared in teas or infusions to empirically treat obesity, diabetes, gastrointestinal disorders, and kidney stones. The beneficial effects of Chaya can be attributed to the presence of protein, dietary fiber, vitamins, and especially polyphenols, which regulate mitochondrial function. Therefore, polyphenols present in Chaya extracts could be used to develop novel strategies to prevent and treat metabolic alterations related to mitochondrial dysfunction in the muscle and liver of subjects with obesity, type 2 diabetes, and cardiovascular diseases. However, limited information is available concerning the effect of Chaya extracts on mitochondrial activity in those tissues. Aim of the studyThe aim of this study was to evaluate the antioxidant capacity of an aqueous extract (AE) or mixed (methanol/acetone/water) extract (ME) of Chaya leaf and their effect on C2C12 myotubes and primary hepatocyte mitochondrial bioenergetics and fatty acid oxidation (FAO). Materials and methodsTotal polyphenol content and antioxidant activity were determined using the Folin–Ciocalteu method and the oxygen radical absorbance capacity assay, respectively. The effect of AE and ME from Chaya leaf on mitochondrial activity and FAO of C2C12 myotubes and primary hepatocytes was evaluated using an extracellular flux analyzer. ResultsThe AE and ME from Chaya leaf exhibited antioxidant activity and a polyphenol content similar to nopal, another plant used in Mexican traditional medicine. AE significantly (p < 0.05) decreased the maximal respiration and spare respiratory capacity (SRC) of C2C12 cells, whereas ME had little effect on C2C12 mitochondrial function. Conversely, ME significantly (p < 0.05) decreased SRC in primary hepatocytes, whereas AE increased maximal respiration and SRC at low doses (5 and 10 μM). Moreover, low doses of Chaya AE significantly (p < 0.05) increased AMPK phosphorylation, acyl-coenzyme A oxidase protein abundance, and palmitate oxidation in primary hepatocytes. ConclusionThe AE of Chaya leaf increases mitochondrial function and FAO of primary hepatocytes, indicating its potential to treat hepatic mitochondrial dysfunction underlying metabolic diseases.

From Non-Alcoholic Fatty Liver to Hepatocellular Carcinoma: A Story of (Mal)Adapted Mitochondria.

Non-alcoholic fatty liver disease (NAFLD) is a global pandemic affecting 25% of the world's population and is a serious health and economic concern worldwide. NAFLD is mainly the result of unhealthy dietary habits combined with sedentary lifestyle, although some genetic contributions to NAFLD have been documented. NAFLD is characterized by the excessive accumulation of triglycerides (TGs) in hepatocytes and encompasses a spectrum of chronic liver abnormalities, ranging from simple steatosis (NAFL) to steatohepatitis (NASH), significant liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although the molecular mechanisms that cause the progression of steatosis to severe liver damage are not fully understood, metabolic-dysfunction-associated fatty liver disease is strong evidence that mitochondrial dysfunction plays a significant role in the development and progression of NAFLD. Mitochondria are highly dynamic organelles that undergo functional and structural adaptations to meet the metabolic requirements of the cell. Alterations in nutrient availability or cellular energy needs can modify mitochondria formation through biogenesis or the opposite processes of fission and fusion and fragmentation. In NAFL, simple steatosis can be seen as an adaptive response to storing lipotoxic free fatty acids (FFAs) as inert TGs due to chronic perturbation in lipid metabolism and lipotoxic insults. However, when liver hepatocytes' adaptive mechanisms are overburdened, lipotoxicity occurs, contributing to reactive oxygen species (ROS) formation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Impaired mitochondrial fatty acid oxidation, reduction in mitochondrial quality, and disrupted mitochondrial function are associated with a decrease in the energy levels and impaired redox balance and negatively affect mitochondria hepatocyte tolerance towards damaging hits. However, the sequence of events underlying mitochondrial failure from steatosis to hepatocarcinoma is still yet to be fully clarified. This review provides an overview of our understanding of mitochondrial adaptation in initial NAFLD stages and highlights how hepatic mitochondrial dysfunction and heterogeneity contribute to disease pathophysiology progression, from steatosis to hepatocellular carcinoma. Improving our understanding of different aspects of hepatocytes' mitochondrial physiology in the context of disease development and progression is crucial to improving diagnosis, management, and therapy of NAFLD/NASH.

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered 13C3, 15N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.