Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact.

Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles. This association was visualized by fluorescence microscopy and computer-aided three-dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells. A mitochondria-associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re-associated with mitochondria in vitro. In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine. Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine. Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine. In contrast with previous observations with permeabilized yeast cells [Achleitner, G., Zweytick, D., Trotter, P., Voelker, D. & Daum, G. (1995) J. Biol. Chem. 270, 29836-29842], export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system.     

Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondrion-associated membranes of human cells

The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent antiapoptotic protein, which prevents cytochrome c release from mitochondria. The UL37x1 NH₂-terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase 1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using subcellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria, and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37_NH2 and gpUL37_COOH were both detected in the ER and MAM fraction, even though only pUL37_NH2 is preferentially imported into mitochondria but gpUL37_COOH is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH₂-terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation.     

Insights into the requirement of phosphatidylcholine synthesis for liver function in mice

Phosphatidylcholine (PC) is made in the liver by the CDP-choline pathway and via phosphatidylethanolamine N-methyltransferase (PEMT), which catalyzes the conversion of phosphatidylethanolamine to PC. Unexpectedly, hepatic apolipoprotein B-100 secretion is inhibited in male, but not female, Pemt-/- mice (Noga, A. A., Y. Zhao, and D. E. Vance. 2002. J. Biol. Chem. 277: 42358-42365; Noga, A. A., and D. E. Vance. 2003. J. Biol. Chem. 278: 21851-21859). To gain further insight into this process, we compared PC metabolism in male and female mice fed chow or a high-fat/high-cholesterol (HF/HC) diet. Immunoblot analyses demonstrated that twice as much PEMT2 was present in livers from female compared with male mice. In contrast, assays of CTP:phosphocholine cytidylyltransferase from livers of Pemt+/+ mice demonstrated more active cytidylyltransferase in male than in female mice. Secretion of PEMT-derived PC into lipoproteins was examined in vivo by injection of mice with [methyl-3H]methionine in the presence of Triton WR1339. The PEMT-derived PC shifts to smaller-sized particles in response to a HF/HC diet, but only in male mice. Secretion of PEMT-derived PC into bile was enhanced in mice fed a HF/HC diet. These results demonstrate that the synthesis and targeting of PC produced by the PEMT pathway in the livers of mice differs in a gender- and diet-specific manner.     

CNTF overproduction hastens onset of symptoms in motor neuron degeneration (mnd) mice

Ciliary neurotrophic factor (CNTF) promotes the survival of motor neurons, in vitro and in vivo. Moreover, CNTF can block the degeneration of injured or diseased motor neurons in young rodents. Motor neuron degeneration (mnd) mutant mice display adult onset symptoms reflecting progressive motor debilitation and provide a model in which to test the hypothesis that CNTF can prevent the loss of these motor functions. We generated mnd mice that harbor a genomically integrated transgene, resulting in overexpression of the encoded CNTF protein in these mice. In contrast to the beneficial effects of CNTF in preventing motor neuron degeneration in other experimental paradigms, we report that overproduction of CNTF increased the rate of onset of motor disease symptoms in mnd mice and the presence of the transgene correlated with low adult body weight in mnd and wild-type genetic backgrounds. © 1996 John Wiley & Sons, Inc.     

Respiratory State and Phosphatidylserine Import in Brain Mitochondria In Vitro

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with ¹⁴C-PS containing liposomes, ¹⁴C-PS became a substrate of PS decarboxylase, as monitored by the formation of ¹⁴C-phosphatidylethanolamine (PE), indicating translocation of ¹⁴C-PS to the inner membrane. The kinetics of ¹⁴C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, ¹⁴C-PE formation decreased in the presence of NaN₃. Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P₂) containing MAM, synaptosomes and myelin was prelabeled with ¹⁴C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of ¹⁴C-PS in P₂ membranes were also studied by incubating P₂ with ¹⁴C-serine. In the resting state ¹⁴C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of ¹⁴C-PS and its conversion to ¹⁴C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane. Received: 7 April 1999/Revised: 21 September 1999     

MAM (mitochondria-associated membranes) in mammalian cells: Lipids and beyond

One mechanism by which communication between the endoplasmic reticulum (ER) and mitochondria is achieved is by close juxtaposition between these organelles via mitochondria-associated membranes (MAM). The MAM consist of a region of the ER that is enriched in several lipid biosynthetic enzyme activities and becomes reversibly tethered to mitochondria. Specific proteins are localized, sometimes transiently, in the MAM. Several of these proteins have been implicated in tethering the MAM to mitochondria. In mammalian cells, formation of these contact sites between MAM and mitochondria appears to be required for key cellular events including the transport of calcium from the ER to mitochondria, the import of phosphatidylserine into mitochondria from the ER for decarboxylation to phosphatidylethanolamine, the formation of autophagosomes, regulation of the morphology, dynamics and functions of mitochondria, and cell survival. This review focuses on the functions proposed for MAM in mediating these events in mammalian cells. In light of the apparent involvement of MAM in multiple fundamental cellular processes, recent studies indicate that impaired contact between MAM and mitochondria might underlie the pathology of several human neurodegenerative diseases, including Alzheimer's disease. Moreover, MAM has been implicated in modulating glucose homeostasis and insulin resistance, as well as in some viral infections.     

Bilayer-penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria.

In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV-DHFR, which exhibit mitochondrial import pathways with different characteristics. In-vitro-synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein [Rietveld, A. & de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6707; Dumont, M. E. & Richards, F. M. (1984) J. Biol. Chem. 259, 4147-4156]. Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in-vitro-synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria. In contrast, the purified and in-vitro-synthesized precursor fusion protein pCOX IV-DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer. The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins. The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.     

Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

We previously demonstrated that pharmacological induction of autophagy protected against acetaminophen (APAP)-induced liver injury in mice by clearing damaged mitochondria. However, the mechanism for removal of mitochondria by autophagy is unknown. Parkin, an E3 ubiquitin ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkin-mediated mitophagy in protection against APAP-induced liver injury. We found that Parkin translocated to mitochondria in mouse livers after APAP treatment followed by mitochondrial protein ubiquitination and mitophagy induction. To our surprise, we found that mitophagy still occurred in Parkin knock-out (KO) mice after APAP treatment based on electron microscopy analysis and Western blot analysis for some mitochondrial proteins, and Parkin KO mice were protected against APAP-induced liver injury compared with wild type mice. Mechanistically, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK), increased induction of myeloid leukemia cell differentiation protein (Mcl-1) expression, and increased hepatocyte proliferation after APAP treatment in their livers compared with WT mice. In contrast to chronic deletion of Parkin, acute knockdown of Parkin in mouse livers using adenovirus shRNA reduced mitophagy and Mcl-1 expression but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury. Therefore, chronic deletion (KO) and acute knockdown of Parkin have differential responses to APAP-induced mitophagy and liver injury in mice.     

Lipids of mitochondria

A unique organelle for studying membrane biochemistry is the mitochondrion whose functionality depends on a coordinated supply of proteins and lipids. Mitochondria are capable of synthesizing several lipids autonomously such as phosphatidylglycerol, cardiolipin and in part phosphatidylethanolamine, phosphatidic acid and CDP-diacylglycerol. Other mitochondrial membrane lipids such as phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sterols and sphingolipids have to be imported. The mitochondrial lipid composition, the biosynthesis and the import of mitochondrial lipids as well as the regulation of these processes will be main issues of this review article. Furthermore, interactions of lipids and mitochondrial proteins which are highly important for various mitochondrial processes will be discussed. Malfunction or loss of enzymes involved in mitochondrial phospholipid biosynthesis lead to dysfunction of cell respiration, affect the assembly and stability of the mitochondrial protein import machinery and cause abnormal mitochondrial morphology or even lethality. Molecular aspects of these processes as well as diseases related to defects in the formation of mitochondrial membranes will be described.     

Deficient mitochondrial Ca(2+) buffering in the Cln8(mnd) mouse model of neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses (NCLs) are a group of genetic childhood-onset progressive brain diseases characterized by a decline in mental and motor capacities, epilepsy, visual loss and premature death. Using patch clamp, fluorescence imaging and caged Ca²⁺ photolysis, we evaluated the mechanisms of neuronal Ca²⁺ clearance in Cln8^mnd mice, a model of the human NCL caused by mutations in the CLN8 gene. In Cln8^mnd hippocampal slices, Ca²⁺ clearance efficiency in interneurons and, to some extent, principal neurons declined with age. In cultured Cln8^mnd hippocampal neurons, clearance of large Ca²⁺ loads was inefficient due to impaired mitochondrial Ca²⁺ uptake. In contrast, neither Ca²⁺ uptake by sarco/endoplasmic reticulum Ca²⁺ ATPase, nor Ca²⁺ extrusion through plasma membrane was affected by the Cln8 mutation. Excitotoxic glutamate challenge caused Ca²⁺ deregulation more readily in Cln8^mnd than in wt neurons. We propose that neurodegeneration in human CLN8 disorders is primarily caused by reduced mitochondrial Ca²⁺ buffering capacity.     

Fatty acid composition of membrane lipids and energy metabolism in mitochondria of pea seedlings under water deficit

The impacts of water deficit and melamine salt of bis(oximethyl)phosphonic acid (melaphen) on the fatty acid (FA) composition of membrane lipids and energy metabolism in mitochondria of 5-day-old pea (Pisum sativum L., cv. Flora-2) seedlings were studied. Insufficient watering resulted in the accumulation of saturated and a decrease in the content of unsaturated FAs with 18 and 20 carbon atoms. Seed treatment with 3 × 10^?10 M melaphen prevented these changes in the FA composition in the mitochondrial membrane lipids. Changes in the FA compositions of membrane lipids were correlated with changes in energy metabolism in mitochondria: the efficiency of oxidative phosphorylation and the rate of NAD-dependent substrate oxidation in the presence of ADP and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) were reduced. A close correlation was observed between changes in the highest rates of NAD-dependent substrate oxidation and the relative content of FAs with 18 (r = 0.76489) and 20 (r = 0.9637) carbon atoms. The regulatory role of C₁₈ and C₂₀ unsaturated FAs in the mitochondrial energy metabolism of pea seedlings is discussed.     

Redox proteomic identification of HNE-bound mitochondrial proteins in cardiac tissues reveals a systemic effect on energy metabolism after doxorubicin treatment

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria after DOX treatment. A redox proteomics method involving two-dimensional electrophoresis followed by mass spectrometry and investigation of protein databases identified several HNE-modified mitochondrial proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle and electron transport chain. The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE-adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate was also significantly reduced after DOX treatment. Treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE–protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury, suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.     

Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart

Mitochondrial proteins have been shown to be common targets of S-nitrosylation (SNO), but the existence of a mitochondrial source of nitric oxide remains controversial. SNO is a nitric oxide-dependent thiol modification that can regulate protein function. Interestingly, trans-S-nitrosylation represents a potential pathway for the import of SNO into the mitochondria. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown to act as a nuclear trans-S-nitrosylase, has also been shown to enter mitochondria. However, the function of GAPDH in the mitochondria remains unknown. Therefore, we propose the hypothesis that S-nitrosylated GAPDH (SNO-GAPDH) interacts with mitochondrial proteins as a trans-S-nitrosylase. In accordance with this hypothesis, SNO-GAPDH should be detected in mitochondrial fractions, interact with mitochondrial proteins, and increase mitochondrial SNO levels. Our results demonstrate a four-fold increase in GAPDH levels in the mitochondrial fraction of mouse hearts subjected to ischemic preconditioning, which increases SNO-GAPDH levels. Co-immunoprecipitation studies performed in mouse hearts perfused with the S-nitrosylating agent S-nitrosoglutathione (GSNO), suggest that SNO promotes the interaction of GAPDH with mitochondrial protein targets. The addition of purified SNO-GAPDH to isolated mouse heart mitochondria demonstrated the ability of SNO-GAPDH to enter the mitochondrial matrix, and to increase SNO for many mitochondrial proteins. Further, the overexpression of GAPDH in HepG2 cells increased SNO for a number of different mitochondrial proteins, including heat shock protein 60, voltage-dependent anion channel 1, and acetyl-CoA acetyltransferase, thus supporting the role of GAPDH as a potential mitochondrial trans-S-nitrosylase. In further support of this hypothesis, many of the mitochondrial SNO proteins identified with GAPDH overexpression were no longer detected with GAPDH knock-down or mutation. Therefore, our results suggest that SNO-GAPDH can act as a mitochondrial trans-S-nitrosylase, thereby conferring the transfer of SNO from the cytosol to the mitochondria.     

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233–1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.     

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt^−/− mice than in Pemt^+/+ mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt^−/− mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.     

Import of phosphatidylethanolamine for the assembly of rat brain mitochondrial membranes

Mitochondria can synthesize phosphatidyl-ethanolamine (PE) through phosphatidylserine decarboxylase (PS decarboxylase) activity or can import this lipid from the endoplasmic reticulum. In this work, we studied the factors influencing the import of PE in brain mitochondria and its utilization for the assembly of mitochondrial membranes. Incubation of rat brain homogenate with [1-³H]ethanolamine resulted in the synthesis and distribution of ³H-PE to subcellular fractions. T-wenty-one percent of labeled PE was recovered in purified mitochondria. The import of PE in mitochondria was studied in a reconstituted system made of microsomes (donor particles) and purified mitochondria (acceptor particles). Ca⁺² and nonspecific lipid transfer protein purified from liver tissue (nsL-TP) enhanced the translocation process. ³H-PE synthesized in membrane associated to mitochondria (MAM) could also translocate to mitochondria in the reconstituted system. Exposure of mitochondria to trinitrobenzensulfonic acid (TNBS) resulted in the reaction of more than 60% of ³H-PE imported from endoplasmic reticulum and of about 25% of ¹⁴C-PE produced in mitochondria by decarboxylation of ¹⁴C-PS. Moreover, the removal of the outer mitochondrial membrane by digitonin treatment, resulted in the loss of ³H-PE, but not ¹⁴C-PE. These results indicate that labeled PE imported in mitochondria is mainly localized in the outer mitochondrial membrane, whereas PE produced by PS decarboxylase activity is confined to the inner mitochondrial membrane. Phospholipase C hydrolyzed 25–30% of both PE radioactivity and mass of the outer mitochondrial membrane indicating an asymmetrical distribution of this lipid across the membrane.Mr. Carlo Ricci is thanked for his skillful technical assistance. This work has been supported by a grant from the Ministry of Education, Rome, Italy.     

Ups1p and Ups2p antagonistically regulate cardiolipin metabolism in mitochondria

Cardiolipin, a unique phospholipid composed of four fatty acid chains, is located mainly in the mitochondrial inner membrane (IM). Cardiolipin is required for the integrity of several protein complexes in the IM, including the TIM23 translocase, a dynamic complex which mediates protein import into the mitochondria through interactions with the import motor presequence translocase–associated motor (PAM). In this study, we report that two homologous intermembrane space proteins, Ups1p and Ups2p, control cardiolipin metabolism and affect the assembly state of TIM23 and its association with PAM in an opposing manner. In ups1Δ mitochondria, cardiolipin levels were decreased, and the TIM23 translocase showed altered conformation and decreased association with PAM, leading to defects in mitochondrial protein import. Strikingly, loss of Ups2p restored normal cardiolipin levels and rescued TIM23 defects in ups1Δ mitochondria. Furthermore, we observed synthetic growth defects in ups mutants in combination with loss of Pam17p, which controls the integrity of PAM. Our findings provide a novel molecular mechanism for the regulation of cardiolipin metabolism.     

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1^−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1^−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1^−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1^−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.