Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 mM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing b-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.-

Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by endolysosomal cholesterol accumulation. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that non-enzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1−/− mouse model and found several cholesterol oxidation products that were elevated in Npc1−/− mice, were detectable prior to the onset of symptoms, and were associated with disease progression. Non-enzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1 I1061T , is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1 I1061T mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1 I1061T mutation. Despite similar NPC1 mRNA levels between wild type and NPC1I1061T fibroblasts, NPC1 protein levels are decreased by 85% in NPC1 I1061T cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1I1061T protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t1 ⁄ 2 6.5 h) versus wild type Endo H-resistant species (t1 ⁄ 2 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1I1061T protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1 I1061T in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1 I1061T protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1 I1061T mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1 I1061T mutation.

Niemann-Pick type C1 (NPC1) disease is a fatal neurodegenerative disease characterized by neuronal lipid storage and progressive Purkinje cell loss in the cerebellum. We investigated whether therapeutic approaches to bypass the cholesterol trafficking defect in NPC1 disease might delay disease progression in the npc1 ؊/؊ mouse model. We show that the neurosteroid allopregnanolone (ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset of neurological symptoms and prolong the lifespan of npc1 ؊/؊ mice. ALLO and T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of microglial-associated genes and inflammatory mediators, and reduced infiltration of activated microglia in the cerebellar tissue. To establish whether the mechanism of neuroprotection in npc1 ؊/؊ mice involves GABAA receptor activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer that has identical physical properties of natural ALLO but is not a GABA A receptor agonist. ent-ALLO provided identical functional and survival benefits as natural ALLO in npc1 ؊/؊ mice, strongly supporting a GABAA receptor-independent mechanism for ALLO action. On the other hand, the efficacy of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these compounds to activate pregnane X receptor-dependent pathways in vivo. These findings suggest that treatment with pregnane X receptor ligands may be useful clinically in delaying the progressive neurodegeneration in human NPC disease.cholesterol ͉ neurosteroid ͉ allopregnanolone ͉ neurodegeneration N iemann-Pick type C (NPC) disease is an autosomal recessive neurodegenerative disorder characterized by accumulation of cholesterol and other lipids in the viscera and central nervous system and patterned Purkinje cell death in the cerebellum (1). Mutations in the NPC1 gene are responsible for Ϸ95% of human NPC disease. NPC1 loss-of-function mutants exhibit marked impairment of low-density lipoprotein (LDL) cholesterol esterification and mobilization of newly hydrolyzed LDL cholesterol to the plasma membrane (2-4), resulting in lysosomal sequestration of LDL cholesterol, delayed down-regulation of the LDL receptor and de novo cholesterol biosynthesis, and impaired ABCA1-mediated cholesterol efflux (5-7). Despite recent progress in characterizing the biochemical and genetic defects in NPC disease, the mechanisms underlying the neurodegenerative phenotype are not well understood. Moreover, at present there are no effective therapies that delay progression of human NPC disease.Many of the prominent neuropathological features of human NPC disease [e.g., neuronal lipid storage and progressive loss of Purkinje neurons (1)] are recapitulated in the BALB͞c NPC nih (npc1 Ϫ/Ϫ ) mouse, a naturally occurring murine model that harbors a retroposon insertion in the Npc1 gene (8, 9). In NPC1 mice, accumulation of unesterified cholesterol and gangliosides occurs in morphologically normal neurons as early as postnatal day 9 (P9) and precedes neuronal injury and c...

Mutations in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene have been identified in individuals affected with congenital generalized lipodystrophy (CGL). AGPAT2 catalyzes acylation of lysophosphatidic acid to phosphatidic acid, a precursor for both triacylglycerol (TAG) and phospholipid synthesis. Recent studies suggest that reduced AGPAT2 enzymatic activity may underlie the CGL clinical phenotype. To gain insight into how altered AGPAT2 activity causes lipodystrophy, we examined the effect of knockdown of AGPAT2 expression in preadipocytes on TAG synthesis and storage, and on adipocyte differentiation. We show that AGPAT2 mRNA expression is induced 30-fold during adipocyte differentiation and that AGPAT2 enzymatic activity is required for TAG mass accumulation in mature adipocytes. We demonstrate that small interference RNA-mediated knockdown of AGPAT2 expression prevents appropriate early induction of C/EBP␤ and PPAR␥, key transcriptional activators of the adipogenic program, and delays expression of multiple adipocyte-related genes. The unexpected finding, that levels of several phospholipid species, including phosphatidic acid (PA), are elevated in TAG-depleted adipocytes with AGPAT2 knockdown, suggests that impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synthesis. These findings underscore the importance of an AGPAT2-mediated metabolic pathway in adipocyte differentiation. Congenital generalized lipodystrophy (CGL),3 also referred to as Berardinelli-Seip syndrome, is an autosomal recessive disorder characterized by paucity of adipose tissue, severe insulin resistance, hypertriglyceridemia, and hepatic steatosis in childhood (1). Positional cloning strategies have identified mutations in the Berardinelli-Seip congential lipodistrophy 2 gene, which encodes the seipin protein of unknown function, and in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene in individuals affected with CGL (2, 3). AGPAT2 (also referred to as lysophosphatidic acid acyltransferase or LPAAT-␤) encodes an enzyme that catalyzes the acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA), which serves as a precursor for triacylglycerol (TAG) and phospholipid synthesis (4, 5).AGPAT2 is one of five murine lysophosphatidic acid acyltransferase isoforms that have been identified (AGPAT1-5), each of which contains a highly conserved catalytic (NHX 4 D) and substrate binding motif (EGTR) (6). Although there are differences in the tissue distribution of the AGPAT isoforms, the physiological significance of this diversity is not understood. AGPAT1 and AGPAT3 are ubiquitously expressed, whereas AGPAT2, AGPAT4, and AGPAT5 are expressed in a tissue-specific manner (6). In human tissues AGPAT2 is highly expressed in liver, pancreas, skeletal muscle, and small intestine (7) and is the most highly expressed AGPAT isoform in adipose tissue (3).Structure-function studies of AGPAT2 mutations identified in CGL patients dem...