Mitochondrial Cholesterol Accumulation Research Articles

PCSK9 Regulates Cardiac Mitochondrial Cholesterol by Promoting TSPO Degradation.

Cholesterol is critical for mitochondrial membrane structure and function. Given the emergence of mitochondria as a key factor in the pathogenesis of heart failure, mitochondrial cholesterol homeostasis may be crucial for maintaining mitochondrial properties and thus cardiac function. We previously showed that CM-Pcsk9-/- mice (mice with cardiomyocyte-specific deletion of the gene encoding PCSK9 [proprotein convertase subtilisin-kexin type 9]) have impaired cardiomyocyte mitochondrial bioenergetics and heart function, paralleled by cardiomyocyte mitochondrial cholesterol accumulation and an increased number of mitochondria-endoplasmic reticulum contacts. However, the mechanisms linking PCSK9 to mitochondrial cholesterol homeostasis remain unclear. We hypothesized that PCSK9 acts on proteins involved in mitochondrial cholesterol trafficking in the heart to maintain cardiac mitochondrial function. By performing RNA sequencing and immunoblot on hearts from CM-Pcsk9-/- and CM-Pcsk9+/+ (without cardiomyocyte-specific deletion of Pcsk9) mice, we showed that TSPO (translocator protein) was increased by Pcsk9 deficiency. To investigate the relationship between TSPO levels and heart function in humans, we compared the transcriptome of human left ventricles with high versus low TSPO levels. We used H9c2 (a rat cardiomyoblast cell line) cardiomyocytes to explore the mechanism linking PCSK9/TSPO to mitochondrial cholesterol content and function. The impact of reduced TSPO levels on cardiac function and mitochondrial oxidation in CM-Pcsk9-/- mice was tested using adeno-associated virus serotype 9 short hairpin TSPO. Both gene and protein levels of TSPO, a mitochondrial protein involved in cholesterol transport, were increased in CM-Pcsk9-/- mouse hearts. Transcriptome analysis showed that high TSPO expression in human left ventricles was associated with impaired mitochondrial and cardiac function. We showed that PCSK9 induced TSPO degradation through a proteasomal mechanism that occurs in cardiomyocytes but not hepatocytes and contributes to maintaining normal mitochondrial cholesterol composition and function. At the molecular level, endoplasmic reticulum-resident PCSK9 interacted with GRP78 (glucose regulatory protein 78) , reducing GRP78-TSPO interactions and leading to TSPO misfolding and degradation by the ubiquitin-proteasome pathway. Importantly, gene therapy-induced downregulation of TSPO in CM-Pcsk9-/- mice prevented mitochondrial cholesterol accumulation and improved cardiac function. These findings indicate that PCSK9 regulates mitochondrial cholesterol levels by modulating the TSPO degradation in the heart. Modulation of mitochondrial cholesterol by targeting TSPO may be a promising therapeutic approach for heart failure.

Increased Steroidogenic Acute Regulatory Protein Contributes to Cholesterol-induced β-Cell Dysfunction.

Hypercholesterolemia is often observed in individuals with type 2 diabetes. Cholesterol accumulation in subcellular compartments within islet β-cells can result in insulin secretory dysfunction, which is a key pathological feature of diabetes. Previously, we demonstrated that expression of the mitochondrial cholesterol transport protein, steroidogenic acute regulatory protein (StAR), is induced in islets under conditions of β-cell dysfunction. However, whether it contributes to mitochondrial cholesterol accumulation in β-cells and cholesterol-induced β-cell dysfunction has not been determined. Thus, we sought to examine the role of StAR in isolated mouse islets under conditions of excess exogenous cholesterol. Cholesterol treatment of islets upregulated StAR expression, which was associated with cholesterol accumulation in mitochondria, decreased mitochondrial membrane potential and impaired mitochondrial oxidative phosphorylation. Impaired insulin secretion and reduced islet insulin content were also observed in cholesterol-laden islets. To determine the impact of StAR overexpression in β-cells per se, a lentivirus was used to increase StAR expression in INS-1 cells. Under these conditions, StAR overexpression was sufficient to increase mitochondrial cholesterol content, impair mitochondrial oxidative phosphorylation, and reduce insulin secretion. These findings suggest that elevated cholesterol in diabetes may contribute to β-cell dysfunction via increases in StAR-mediated mitochondrial cholesterol transport and accumulation.

Instationary metabolic flux analysis reveals that NPC1 inhibition increases glycolysis and decreases mitochondrial metabolism in brain microvascular endothelial cells.

Instationary metabolic flux analysis reveals that NPC1 inhibition increases glycolysis and decreases mitochondrial metabolism in brain microvascular endothelial cells.

Stard1 promotes cholestatic liver injury and disease progression by sensitizing to bile acid hepatotoxicity.

Cholestatic liver diseases (CLD) are often accompanied by hepatocellular injury, fibrosis, and cirrhosis due to the intracellular accumulation of solutes that cannot be excreted into bile, including bile acids (BAs). These are synthesized in hepatocytes from cholesterol mainly via the classic pathway and in a lower proportion through the mitochondrial acidic pathway. The latter requires STARD1-dependent cholesterol transport to the mitochondrial inner membrane for metabolism, whose contribution to BA-induced hepatotoxicity and CLD is unknown. Here we show that patients with primary biliary cholangitis exhibit increased expression of STARD1 compared to control subjects. Mice with hepatocyte-specific Stard1 deletion (Stard1Δhep) were more resistant to experimental models of complete (bile duct ligation, BDL) and chemical obstructive cholestasis-induced liver injury, inflammation and fibrosis than Stard1f/f mice. Stard1Δhep mice exhibited reduced hepatic BAs and mitochondrial cholesterol accumulation but increased mitochondrial GSH (mGSH) levels following BDL compared to Stard1f/f mice. Pharmacological mGSH depletion sensitized primary mouse hepatocytes to a mix of BAs mimicking the profile seen in Stard1f/f mice after BDL leading to increased inflammatory response and cytotoxicity. These findings highlight a role for STARD1 in cholestatic liver injury and suggest that its targeting may be of relevance for CLD.

M1-derived extracellular vesicles polarize recipient macrophages into M2-like macrophages and alter skeletal muscle homeostasis in a hyper-glucose environment

BackgroundMacrophages release not only cytokines but also extracellular vesicles (EVs). which are small membrane-derived nanovesicles with virus-like properties transferring cellular material between cells. Until now, the consequences of macrophage plasticity on the release and the composition of EVs have been poorly explored. In this study, we determined the impact of high-glucose (HG) concentrations on macrophage metabolism, and characterized their derived-EV subpopulations. Finally, we determined whether HG-treated macrophage-derived EVs participate in immune responses and in metabolic alterations of skeletal muscle cells.MethodsTHP1-macrophages were treated with 15mM (MG15) or 30mM (MG30) glucose. Then, M1/M2 canonical markers, pro- and anti-inflammatory cytokines, activities of proteins involved in glycolysis or oxidative phosphorylation were evaluated. Macrophage-derived EVs were characterized by TEM, NTA, MRSP, and 1H-Nuclear magnetic resonance spectroscopy for lipid composition. Macrophages or C2C12 muscle cells were used as recipients of MG15 and MG30-derived EVs. The lipid profiles of recipient cells were determined, as well as proteins and mRNA levels of relevant genes for macrophage polarization or muscle metabolism.ResultsUntreated macrophages released small and large EVs (sEVs, lEVs) with different lipid distributions. Proportionally to the glucose concentration, glycolysis was induced in macrophages, associated to mitochondrial dysfunction, triacylglycerol and cholesterol accumulation. In addition, MG15 and MG30 macrophages had increased level of CD86 and increase release of pro-inflammatory cytokines. HG also affected macrophage sphingolipid and phospholipid compositions. The differences in the lipid profiles between sEVs and lEVs were abolished and reflected the lipid alterations in MG15 and MG30 macrophages. Interestingly, MG15 and MG30 macrophages EVs induced the expression of CD163, Il-10 and increased the contents of triacylglycerol and cholesterol in recipient macrophages. MG15 lEVs and sEVs induced insulin-induced AKT hyper-phosphorylation and accumulation of triacylglycerol in myotubes, a state observed in pre-diabetes. Conversely, MG30 lEVs and sEVs induced insulin-resistance in myotubes.ConclusionsAs inflammation involves first M1 macrophages, then the activation of M2 macrophages to resolve inflammation, this study demonstrates that the dialog between macrophages through the EV route is an intrinsic part of the inflammatory response. In a hyperglycemic context, EV macrophages could participate in the development of muscle insulin-resistance and chronic inflammation.

Identification of a mechanism promoting mitochondrial sterol accumulation during myocardial ischemia-reperfusion: role of TSPO and STAR.

Hypercholesterolemia is a major risk factor for coronary artery diseases and cardiac ischemic events. Cholesterol per se could also have negative effects on the myocardium, independently from hypercholesterolemia. Previously, we reported that myocardial ischemia-reperfusion induces a deleterious build-up of mitochondrial cholesterol and oxysterols, which is potentiated by hypercholesterolemia and prevented by translocator protein (TSPO) ligands. Here, we studied the mechanism by which sterols accumulate in cardiac mitochondria and promote mitochondrial dysfunction. We performed myocardial ischemia-reperfusion in rats to evaluate mitochondrial function, TSPO, and steroidogenic acute regulatory protein (STAR) levels and the related mitochondrial concentrations of sterols. Rats were treated with the cholesterol synthesis inhibitor pravastatin or the TSPO ligand 4'-chlorodiazepam. We used Tspo deleted rats, which were phenotypically characterized. Inhibition of cholesterol synthesis reduced mitochondrial sterol accumulation and protected mitochondria during myocardial ischemia-reperfusion. We found that cardiac mitochondrial sterol accumulation is the consequence of enhanced influx of cholesterol and not of the inhibition of its mitochondrial metabolism during ischemia-reperfusion. Mitochondrial cholesterol accumulation at reperfusion was related to an increase in mitochondrial STAR but not to changes in TSPO levels. 4'-Chlorodiazepam inhibited this mechanism and prevented mitochondrial sterol accumulation and mitochondrial ischemia-reperfusion injury, underlying the close cooperation between STAR and TSPO. Conversely, Tspo deletion, which did not alter cardiac phenotype, abolished the effects of 4'-chlorodiazepam. This study reveals a novel mitochondrial interaction between TSPO and STAR to promote cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion. This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury.

Helicobacter pylori promotes gastric cancer through CagA-mediated mitochondrial cholesterol accumulation by targeting CYP11A1 redistribution.

Cholesterol and Helicobacter pylori (H. pylori) are both risk factors for gastric cancer (GC). However, the relationship between cholesterol and H. pylori and their function in the progression of GC are controversial. In this study, we addressed that H. pylori could induce mitochondrial cholesterol accumulation and promote GC proliferation and protect GC cells against apoptosis via cholesterol. Metabolomic and transcriptomic sequencing were used to identify CYP11A1 responsible for H. pylori-induced cholesterol accumulation. In vitro and in vivo function experiments revealed that cholesterol could promote the proliferation of GC and inhibit apoptosis. Mechanically, the interaction of Cytotoxin-associated gene A (CagA) and CYP11A1 redistributed mitochondrial CYP11A1 outside the mitochondria and subsequently caused mitochondrial cholesterol accumulation. The CYP11A1-knockdown upregulated cholesterol accumulation and reproduced the effect of cholesterol on GC in a cholesterol-dependent manner. Moreover, CYP11A1-knockdown or H. pylori infection inhibited mitophagy and maintained the mitochondria homeostasis. H. pylori could contribute to the progression of GC through the CagA/CYP11A1-mitoCHO axis. This study demonstrates that H. pylori can contribute to the progression of GC via cholesterol, and eradicating H. pylori is still prognostically beneficial to GC patients.

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Mitochondrial cholesterol: Metabolism and impact on redox biology and disease

Mitochondrial cholesterol: Metabolism and impact on redox biology and disease

Abstract P016: Endomembranes accumulate unesterified cholesterol in Apc mutant models and humans with CRC as assessed by RNAseq and confocal microscopy

Abstract Background: Increased rates of cholesterol synthesis have been recognized as an important aspect of the metabolism of transformed cells. However, precisely how cholesterol dysmetabolism affects membrane homeostasis is not yet fully understood. Since free cholesterol can be transported from the plasma membrane to other organelles in a dynamic and bidirectional fashion, there is an urgent need to determine to what extent endomembranes are affected by driver oncogene-mediated distortions in cholesterol homeostasis. Aim: Determine if cholesterol is increased in endomembranes, relative to the plasma membrane in Apc mutant cell lines and primary Apc null mouse colonocytes amd CRC samples. Methods: Three mouse epithelial isogenic wild type and mutant Apc colonic cell lines (YAMC (Apc +/+), IMCE (Apc +/-), IMCE βcat (Apc +/- + ΔN89 βcat) were used. In addition, primary colonocytes from Apc wildtype and null mice were examined. mRNA was sequenced using a TruSeq Illumina Stranded mRNA kit. Data analysis was conducted as follows: EdgeR was used to identify differentially expressed genes. Human CRC RNAseq data was obtained from a public reservoir and analyzed. A gene target list (~ 290 genes) related to organelle cholesterol transport was subsequently queried. Membranes from the endoplasmic reticulum (ER), mitochondria and lysosomes were assessed using fluorescent probes: ERTracker, MitoTracker and LysoTracker, respectively. Cells were fixed and stained with Filipin III and imaged using confocal microscopy. Fluorescence colocalization was calculated and statistical analysis was performed using a one-way ANOVA. Results: Apc mutant colonocyte cell lines and Apc null mouse models and human CRC samples exhibited differential expression of cholesterol trafficking genes including Acaa1b, Acaa2, Pcsk9, Stard5. Genes related to mitochondrial cholesterol metabolism, transport, and accumulation, e.g., Tspo, Ppargc1a and Cyp27a, were also found differentially expressed in the three models. Imaging experiments revealed that mitochondrial cholesterol was 17% higher in IMCE and IMCE βcat cell lines. Lysosomal cholesterol was 21% higher in IMCE cells. ER cholesterol levels were unaffected. Conclusions: Our preliminary findings indicate dysregulation of mitochondrial and lysosomal unesterified cholesterol levels in colonocytes expressing mutant APC. Citation Format: Monica Munoz-Vega, Alfredo Erazo-Oliveras, Michael L. Salinas, Xiaoli Wang, Jennifer S. S Goldsby, Robert S. Chapkin. Endomembranes accumulate unesterified cholesterol in Apc mutant models and humans with CRC as assessed by RNAseq and confocal microscopy. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P016.

Hypoxia signaling and cholesterol/steroidogenic acute regulatory protein 1 axis: interplay and role in alcohol and non-alcohol-related liver diseases

Metabolic zonation in the liver carries out the maintenance of organ and body homeostasis. Hypoxia is an inherent physiological feature of the liver and contributes to the zonal properties of the hepatic parenchyma. As a master regulator of hypoxia, the transcription factor hypoxia-inducing factor (HIF) is stabilized primarily by oxygen availability, and it is thought to contribute to steatohepatitis due to alcohol-related (ASH) and non-alcohol-related liver disease (NASH). Cholesterol has emerged as an important player in both diseases, and hypoxia increases hepatic cholesterol levels. Steroidogenic acute regulatory protein 1 (STARD1) is a mitochondrial outer membrane protein that transfers cholesterol to mitochondrial inner membrane for metabolic processing and acts as the rate-limiting step in the alternative pathway of bile acid synthesis in hepatocytes. STARD1 expression increases in ASH and NASH and determines the accumulation of cholesterol in mitochondria, which impacts the physico-chemical mitochondrial membranes properties and as a consequence impairs the activity of specific mitochondrial solute carriers, such as the 2-oxoglutarate carrier (2-OGC), limiting the exchange between cytosolic glutathione and mitochondrial 2-oxoglutarate (2-OG). Although HIF-1 is stabilized in hypoxia largely due to the requirement of prolylhydroxylases (PHDs) for oxygen to signal HIF degradation, PHDs are also dependent on 2-OG, and therefore it is conceivable that impairment of 2-OGC by STARD1-mediated cholesterol accumulation may contribute to HIF-1 stabilization due in part to decreased availability of cytosolic 2-OG. In this perspective, this review explores the interplay between HIF-1 stabilization and STARD1 induction and the potential contribution of this functional relationship to ASH and NASH.

Cholesterol trapping by SOAT1 induces mitochondrial cholesterol accumulation and decrease oxidative metabolism

Cholesterol trapping by SOAT1 induces mitochondrial cholesterol accumulation and decrease oxidative metabolism

1457-P: Cholesterol Accumulation in Islets Increases Steroidogenic Acute Regulatory (StAR) Protein Expression and Decreases Islet Cell Viability and ß-Cell Function

Diabetes is associated with elevated plasma cholesterol (Chol) , with its intracellular accumulation resulting in β-cell dysfunction. In steroidogenic tissues, StAR facilitates Chol transport from the outer to inner mitochondrial membrane for subsequent metabolism. We have shown that StAR is expressed in β cells and its upregulation increases mitochondrial Chol content and decreases mitochondrial function. To determine whether excess Chol per se increases StAR expression and reduces islet viability, we cultured mouse islets for 24 hours in Chol and quantified mRNA levels of Star and genes involved in Chol synthesis (Hmgcr) , uptake (Ldlr) and efflux (Abca1) , cell viability and glucose stimulated insulin secretion (GSIS) . Islet Chol content increased with increasing Chol concentrations (27.1±1.7, 55.3±0.5 and 72.7±5.2 µg/mg protein for 0, 0.25 and 0.5 mM respectively; n=3, p<0.0by ANOVA) , with mitochondrial Chol increasing 2.4-fold in 0.5 mM (9.1±0.2 to 22.4±1.9 µg/mg protein; n=3, p<0.005) . This was associated with increased Star mRNA expression (1.0±0.1, 3.0±0.9 and 7.0±1.5; n=3, p=0.014) . Further, mRNA expression of Hmgcr tended to decrease (1.0±0.2, 0.8±0.3 and 0.3±0.0; n=3, p=0.088) , whereas expression of Ldlr and Abca1 did not change (Ldlr: 1.7±0.8, 1.1±0.7 and 0.5±0.3; n=3, p=0.508; Abca1: 1.1±0.3, 1.9±0.7 and 1.1±0.1; n=3, p=0.413) . These changes were associated with reductions in cell viability (100±15.7%, 68.0±12.6% and 57.3±9.6%; n=2, p=0.025) and GSIS in response to 20 mM glucose (fold increase over 2.8 mM: 0 mM Chol 28.5±0.6, 0.25 mM Chol 8.9±0.5, 0.5 mM Chol 10.8±0.9; n=2, p<0.0001) . In summary, islet cholesterol accumulation is associated with increased StAR expression, decreased cell viability and reduced β-cell function. These data suggest that elevated cholesterol in diabetes may contribute to β-cell dysfunction by increasing StAR expression and mitochondrial cholesterol accumulation. Disclosure R.Akter: None. M.F.Hogan: Employee; NanoString. N.Esser: None. J.J.Castillo: None. R.L.Hull-meichle: Research Support; Casma Therapeutics. S.Zraika: None. S.E.Kahn: Advisory Panel; Bayer AG, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Inc., Merck & Co., Inc., Novo Nordisk, Pfizer Inc. Funding VA (I01BX001060) ,VA (IBX004063) ,NIDDK (P30 DK017047) ,NIDDK (T32 DK007247)

Acid ceramidase improves mitochondrial function and oxidative stress in Niemann-Pick type C disease by repressing STARD1 expression and mitochondrial cholesterol accumulation

Acid ceramidase improves mitochondrial function and oxidative stress in Niemann-Pick type C disease by repressing STARD1 expression and mitochondrial cholesterol accumulation

Mitochondrial Cholesterol in Alzheimer's Disease and Niemann-Pick Type C Disease.

Mitochondrial dysfunction has been recognized as a key player in neurodegenerative diseases, including Alzheimer's disease (AD) and Niemann–Pick type C (NPC) disease. While the pathogenesis of both diseases is different, disruption of intracellular cholesterol trafficking has emerged as a common feature of both AD and NPC disease. Nutritional or genetic mitochondrial cholesterol accumulation sensitizes neurons to Aβ-mediated neurotoxicity in vitro and promotes cognitive decline in AD models. In addition to the primary accumulation of cholesterol and sphingolipids in lysosomes, NPC disease is also characterized by an increase in mitochondrial cholesterol levels in affected organs, predominantly in brain and liver. In both diseases, mitochondrial cholesterol accumulation disrupts membrane physical properties and restricts the transport of glutathione into mitochondrial matrix, thus impairing the mitochondrial antioxidant defense strategy. The underlying mechanisms leading to mitochondrial cholesterol accumulation in AD and NPC diseases are not fully understood. In the present manuscript, we discuss evidence for the potential role of StARD1 in promoting the trafficking of cholesterol to mitochondria in AD and NPC, whose upregulation involves an endoplasmic reticulum stress and a decrease in acid ceramidase expression, respectively. These findings imply that targeting StARD1 or boosting the mitochondrial antioxidant defense may emerge as a promising approach for both AD and NPC disease.

NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress

Transport of dietary cholesterol from endocytic organelles to the endoplasmic reticulum (ER) is essential for cholesterol homoeostasis, but the mechanism and regulation of this transport remains poorly defined. Membrane contact sites (MCS), microdomains of close membrane apposition, are gaining attention as important platforms for non-vesicular, inter-organellar communication. Here we investigate the impact of ER-endocytic organelle MCS on cholesterol transport. We report a role for Niemann-Pick type C protein 1 (NPC1) in tethering ER-endocytic organelle MCS where it interacts with the ER-localised sterol transport protein Gramd1b to regulate cholesterol egress. We show that artificially tethering MCS rescues the cholesterol accumulation that characterises NPC1-deficient cells, consistent with direct lysosome to ER cholesterol transport across MCS. Finally, we identify an expanded population of lysosome-mitochondria MCS in cells depleted of NPC1 or Gramd1b that is dependent on the late endosomal sterol-binding protein STARD3, likely underlying the mitochondrial cholesterol accumulation in NPC1-deficient cells.

Cholesterol enrichment in liver mitochondria impairs oxidative phosphorylation and disrupts the assembly of respiratory supercomplexes

Cholesterol enrichment in liver mitochondria impairs oxidative phosphorylation and disrupts the assembly of respiratory supercomplexes

The 2-oxoglutarate carrier promotes liver cancer by sustaining mitochondrial GSH despite cholesterol loading

The 2-oxoglutarate carrier promotes liver cancer by sustaining mitochondrial GSH despite cholesterol loading

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease

Regular treadmill exercise inhibits mitochondrial accumulation of cholesterol and oxysterols during myocardial ischemia-reperfusion in wild-type and ob/ob mice

Regular treadmill exercise inhibits mitochondrial accumulation of cholesterol and oxysterols during myocardial ischemia-reperfusion in wild-type and ob/ob mice