ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload.
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ABSTRACT:
SUBMITTER: Egnatchik RA
PROVIDER: S-EPMC4099508 | biostudies-literature | 2014 Aug
REPOSITORIES: biostudies-literature
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Project description:The association between obesity and osteoarthritis (OA) in joints not subjected to mechanical overload, together with the relationship between OA and metabolic syndrome, suggests that there are systemic factors related to metabolic disorders that are involved in the metabolic phenotype of OA. The aim of this work is study the effects of palmitate and oleate on cellular metabolism in an "in vitro" model of human chondrocytes. The TC28a2 chondrocyte cell line was used to analyze the effect of palmitate and oleate on mitochondrial and glycolytic function, Adenosine triphosphate (ATP) production and lipid droplets accumulation. Palmitate, but not oleate, produces mitochondrial dysfunction observed with a lower coupling efficiency, maximal respiration and spare respiratory capacity. Glycolytic function showed lower rates both glycolytic capacity and glycolytic reserve when cells were incubated with fatty acids (FAs). The production rate of total and mitochondrial ATP showed lower values in chondrocytes incubated with palmitic acid (PA). The formation of lipid droplets increased in FA conditions, being significantly higher when the cells were incubated with oleic acid (OL). These results may help explain, at least in part, the close relationship of metabolic pathologies with OA, as well as help to elucidate some of the factors that can define a metabolic phenotype in OA.
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Project description:Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca(2+) increase due to depletion of luminal ER Ca(2+). Palmitate-induced ER Ca(2+) depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca(2+) pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca(2+) depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca(2+) loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca(2+) depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to mitochondrial dysfunction and ER Ca(2+) depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury.
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Project description:Saturated fatty acids contribute to β-cell dysfunction in the onset of type 2 diabetes mellitus. Cellular responses to lipotoxicity include oxidative stress, endoplasmic reticulum (ER) stress, and blockage of autophagy. Palmitate induces ER Ca2+ depletion followed by notable store-operated Ca2+ entry. Subsequent elevation of cytosolic Ca2+ can activate undesirable signaling pathways culminating in cell death. Mitochondrial Ca2+ uniporter (MCU) is the major route for Ca2+ uptake into the matrix and couples metabolism with insulin secretion. However, it has been unclear whether mitochondrial Ca2+ uptake plays a protective role or contributes to lipotoxicity. Here, we observed palmitate upregulated MCU protein expression in a mouse clonal β-cell, MIN6, under normal glucose, but not high glucose medium. Palmitate elevated baseline cytosolic Ca2+ concentration ([Ca2+]i) and reduced depolarization-triggered Ca2+ influx likely due to the inactivation of voltage-gated Ca2+ channels (VGCCs). Targeted reduction of MCU expression using RNA interference abolished mitochondrial superoxide production but exacerbated palmitate-induced [Ca2+]i overload. Consequently, MCU knockdown aggravated blockage of autophagic degradation. In contrast, co-treatment with verapamil, a VGCC inhibitor, prevented palmitate-induced basal [Ca2+]i elevation and defective [Ca2+]i transients. Extracellular Ca2+ chelation as well as VGCC inhibitors effectively rescued autophagy defects and cytotoxicity. These observations suggest enhanced mitochondrial Ca2+ uptake via MCU upregulation is a mechanism by which pancreatic β-cells are able to alleviate cytosolic Ca2+ overload and its detrimental consequences.
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Project description:Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.
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Project description:DPP4 has been shown to induce diabetes-associated mitochondrial dysfunction and cognitive impairment through its non-canonical function. Here, we report that enhanced DPP4 expression in diabetes contributes to IP3R2-mediated mitochondria-associated ER membrane (MAM) formation, mitochondria calcium overload, and cognitive impairment, and its knockdown showed opposite effects. Mechanistically, DPP4 binds to PAR2 in hippocampal neurons and activates ERK1/2/CEBPB signaling, which upregulates ERp29 expression and promotes its binding to IP3R2, thereby inhibiting IP3R2 degradation and promoting MAM formation, mitochondria calcium overload, and cognitive impairment. Meanwhile, targeting DPP4-mediated PAR2/ERK1/2/CEBPB/ERp29 signaling achieved satisfactory therapeutic effects on MAM formation, mitochondria calcium overload, and cognitive impairment. Notably, DPP4 activates this pathway in an enzymatic activity-independent manner, suggesting the non-canonical role of DPP4 in the pathogenesis of mitochondria calcium overload and cognitive impairment in diabetes. Together, these results identify DPP4-mediated PAR2/ERK1/2/CEBPB/ERp29 signaling as a promising therapeutic target for the treatment of cognitive impairment in type 2 diabetes.
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Project description:Diabetic cardiomyopathy is a secondary complication of diabetes with an unclear etiology. Based on a functional genomic evaluation of obesity-associated cardiac gene expression, we previously identified and cloned the gene encoding apolipoprotein O (APOO), which is overexpressed in hearts from diabetic patients. Here, we generated APOO-Tg mice, transgenic mouse lines that expresses physiological levels of human APOO in heart tissue. APOO-Tg mice fed a high-fat diet exhibited depressed ventricular function with reduced fractional shortening and ejection fraction, and myocardial sections from APOO-Tg mice revealed mitochondrial degenerative changes. In vivo fluorescent labeling and subcellular fractionation revealed that APOO localizes with mitochondria. Furthermore, APOO enhanced mitochondrial uncoupling and respiration, both of which were reduced by deletion of the N-terminus and by targeted knockdown of APOO. Consequently, fatty acid metabolism and ROS production were enhanced, leading to increased AMPK phosphorylation and Ppara and Pgc1a expression. Finally, we demonstrated that the APOO-induced cascade of events generates a mitochondrial metabolic sink whereby accumulation of lipotoxic byproducts leads to lipoapoptosis, loss of cardiac cells, and cardiomyopathy, mimicking the diabetic heart-associated metabolic phenotypes. Our data suggest that APOO represents a link between impaired mitochondrial function and cardiomyopathy onset, and targeting APOO-dependent metabolic remodeling has potential as a strategy to adjust heart metabolism and protect the myocardium from impaired contractility.
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Project description:Hepatocyte lipotoxicity is characterized by aberrant mitochondrial metabolism, which predisposes cells to oxidative stress and apoptosis. Previously, we reported that translocation of calcium from the endoplasmic reticulum to mitochondria of palmitate-treated hepatocytes activates anaplerotic flux from glutamine to α-ketoglutarate (αKG), which subsequently enters the citric acid cycle (CAC) for oxidation. We hypothesized that increased glutamine anaplerosis fuels elevations in CAC flux and oxidative stress following palmitate treatment. To test this hypothesis, primary rat hepatocytes or immortalized H4IIEC3 rat hepatoma cells were treated with lipotoxic levels of palmitate while modulating anaplerotic pathways leading to αKG. We found that culture media supplemented with glutamine, glutamate, or dimethyl-αKG increased palmitate lipotoxicity compared with media that lacked these anaplerotic substrates. Knockdown of glutamate-oxaloacetate transaminase activity significantly reduced the lipotoxic effects of palmitate, whereas knockdown of glutamate dehydrogenase (Glud1) had no effect on palmitate lipotoxicity. 13C flux analysis of H4IIEC3 cells co-treated with palmitate and the pan-transaminase inhibitor aminooxyacetic acid confirmed that reductions in lipotoxic markers were associated with decreases in anaplerosis, CAC flux, and oxygen consumption. Taken together, these results demonstrate that lipotoxic palmitate treatments enhance anaplerosis in cultured rat hepatocytes, causing a shift to aberrant transaminase metabolism that fuels CAC dysregulation and oxidative stress.
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