Daniel S. Ory

Triglyceride accumulation protects against fatty acid-induced lipotoxicity

Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic β-cell apoptosis and heart failure. Here, we demonstrate in cultured cells that the relative toxicity of two common dietary long chain fatty acids is related to channeling of these lipids to distinct cellular metabolic fates. Oleic acid supplementation leads to triglyceride accumulation and is well tolerated, whereas excess palmitic acid is poorly incorporated into triglyceride and causes apoptosis. Unsaturated fatty acids rescue palmitate-induced apoptosis by channeling palmitate into triglyceride pools and away from pathways leading to apoptosis. Moreover, in the setting of impaired triglyceride synthesis, oleate induces lipotoxicity. Our findings support a model of cellular lipid metabolism in which unsaturated fatty acids serve a protective function against lipotoxicity though promotion of triglyceride accumulation.

Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.

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 μM 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 β-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.

miR-33 links SREBP-2 induction to repression of sterol transporters

The sterol regulatory element binding protein 2 (SREBP-2) and the liver X receptor (LXR) control antagonistic transcriptional programs that stimulate cellular cholesterol uptake and synthesis, and cholesterol efflux, respectively. The clinical importance of SREBP-2 is revealed in patients with hypercholesterolemia treated with statins, which reduce low-density lipoprotein (LDL) cholesterol levels by increasing hepatic expression of SREBP-2 and its target, the LDL receptor. Here we show that miR-33 is encoded within SREBP-2 and that both mRNAs are coexpressed. We also identify sequences in the 3′ UTR of ABCA1 and ABCG1, sterol transporter genes both previously shown to be regulated by LXR, as targets for miR-33–mediated silencing. Our data show that LXR-dependent cholesterol efflux to both ApoAI and serum is ameliorated by miR-33 overexpression and, conversely, stimulated by miR-33 silencing. Finally, we show that ABCA1 mRNA and protein and plasma HDL levels decline after hepatic overexpression of miR-33, whereas they increase after hepatic miR-33 silencing. These results suggest novel ways to manage hypercholesterolemic patients.

Small molecule inhibitors reveal Niemann–Pick C1 is essential for Ebola virus infection

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann–Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.

Transgenic Expression of Fatty Acid Transport Protein 1 in the Heart Causes Lipotoxic Cardiomyopathy

Evidence is emerging that systemic metabolic disturbances contribute to cardiac myocyte dysfunction and clinically apparent heart failure, independent of associated coronary artery disease. To test the hypothesis that perturbation of lipid homeostasis in cardiomyocytes contributes to cardiac dysfunction, we engineered transgenic mice with cardiac-specific overexpression of fatty acid transport protein 1 (FATP1) using the &agr;-myosin heavy chain gene promoter. Two independent transgenic lines demonstrate 4-fold increased myocardial free fatty acid (FFA) uptake that is consistent with the known function of FATP1. Increased FFA uptake in this model likely contributes to early cardiomyocyte FFA accumulation (2-fold increased) and subsequent increased cardiac FFA metabolism (2-fold). By 3 months of age, transgenic mice have echocardiographic evidence of impaired left ventricular filling and biatrial enlargement, but preserved systolic function. Doppler tissue imaging and hemodynamic studies confirm that these mice have predominantly diastolic dysfunction. Furthermore, ambulatory ECG monitoring reveals prolonged QTc intervals, reflecting reductions in the densities of repolarizing, voltage-gated K+ currents in ventricular myocytes. Our results show that in the absence of systemic metabolic disturbances, such as diabetes or hyperlipidemia, perturbation of cardiomyocyte lipid homeostasis leads to cardiac dysfunction with pathophysiological findings similar to those in diabetic cardiomyopathy. Moreover, the MHC-FATP model supports a role for FATPs in FFA import into the heart in vivo.

Erk is essential for growth, differentiation, integrin expression,and cell function in human osteoblastic cells

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of αβ1, αvβ3, and αvβ5 integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.

Chronic Cyclodextrin Treatment of Murine Niemann-Pick C Disease Ameliorates Neuronal Cholesterol and Glycosphingolipid Storage and Disease Progression

Background Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused most commonly by a defect in the NPC1 protein and characterized by widespread intracellular accumulation of unesterified cholesterol and glycosphingolipids (GSLs). While current treatment therapies are limited, a few drugs tested in Npc1−/− mice have shown partial benefit. During a combination treatment trial using two such compounds, N-butyldeoxynojirimycin (NB-DNJ) and allopregnanolone, we noted increased lifespan for Npc1−/− mice receiving only 2-hydroxypropyl-β-cyclodextrin (CD), the vehicle for allopregnanolone. This finding suggested that administration of CD alone, but with greater frequency, might provide additional benefit. Methodology/Principal Findings Administration of CD to Npc1−/− mice beginning at either P7 or P21 and continuing every other day delayed clinical onset, reduced intraneuronal cholesterol and GSL storage as well as free sphingosine accumulation, reduced markers of neurodegeneration, and led to longer survival than any previous treatment regime. We reasoned that other lysosomal diseases characterized by cholesterol and GSL accumulation, including NPC disease due to NPC2 deficiency, GM1 gangliosidosis and mucopolysaccharidosis (MPS) type IIIA, might likewise benefit from CD treatment. Treated Npc2−/− mice showed benefits similar to NPC1 disease, however, mice with GM1 gangliosidosis or MPS IIIA failed to show reduction in storage. Conclusions/Significance Treatment with CD delayed clinical disease onset, reduced intraneuronal storage and secondary markers of neurodegeneration, and significantly increased lifespan of both Npc1−/− and Npc2−/− mice. In contrast, CD failed to ameliorate cholesterol or glycosphingolipid storage in GM1 gangliosidosis and MPS IIIA disease. Understanding the mechanism(s) by which CD leads to reduced neuronal storage may provide important new opportunities for treatment of NPC and related neurodegenerative diseases characterized by cholesterol dyshomeostasis.

Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease

Oxysterols are biomarkers for diagnosis and drug treatment in Niemann-Pick C1 disease. Turning the Tables on Cholesterol A big push in disease research is to identify biochemical markers (biomarkers) in the blood that are early indicators of a disease that is already silently under way. By detecting the disease in its earliest stages, drugs and other therapeutic interventions have the best chance of halting or reversing the course of the disease before major tissue damage has been done. In a new study, Porter and colleagues set out to identify blood biomarkers for Niemann-Pick C1, a childhood neurological disease that is usually fatal. Niemann-Pick C1 disease is caused by mutations in the NPC1 or NPC2 proteins that result in mishandling of cholesterol and lipids in the endolysosomal system of cells. This leads to aberrant deposition of free cholesterol in the central nervous system, the death of neurons, and increasing motor and intellectual impairment, usually resulting in death during adolescence. The early symptoms of the disease are often difficult to distinguish from other childhood diseases, and thus, intervention in the form of a drug such as miglustat often comes too late. This prompted Porter and coworkers to search for possible molecules in the blood that could be used for early diagnosis of the disease and also to monitor the effectiveness of new drugs. On the basis of reports that aberrantly deposited free cholesterol is associated with increased oxidative stress, these investigators reasoned that cholesterol oxidation products (oxysterols) might be the long-sought biomarkers for Niemann-Pick C1 disease. Working in mice lacking the Npc1 gene, the researchers quickly identified two oxysterols that were markedly elevated in the plasma and tissues of the sick mice but not their healthy counterparts. Furthermore, the concentrations of these two oxysterols increased as the disease progressed. Moving into cats carrying an NPC1 mutation, which exhibit similar disease symptoms and progression as human patients, Porter and coworkers were able to decrease elevated concentrations of the two oxysterols and ameliorate disease symptoms by treating the animals with the experimental drug cyclodextrin. But could oxysterols be used as biomarkers in the human disease? The investigators demonstrated that the blood concentrations of two related oxysterol molecules were almost 10 times higher in Niemann-Pick C1 patients than in age-matched healthy controls or those with other diseases such as atherosclerosis or diabetes. Together, these compelling results suggest that the two oxysterol molecules are accurate diagnostic markers of early clinical disease and can be used not only to monitor disease progression but also to demonstrate drug efficacy. Free cholesterol may be at the root of Niemann-Pick C1 disease, but now, there is a way to turn the tables on cholesterol by using its oxidation products to diagnose and treat the disease in its earliest stages. Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic 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 before the onset of symptoms, and were associated with disease progression. Nonenzymatically 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 the 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.

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

A Glanzmann’s mutation in β3 integrin specifically impairs osteoclast function

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the αvβ3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the β3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of β3–/– osteoclast function following expression of a full-length β3 integrin. In contrast, truncated β3, lacking a cytoplasmic domain (hβ3Δc), is completely ineffective in restoring function to β3–/– osteoclasts. To identify the components of the β3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate β integrin signaling. Of the six, only the S752P substitution, which also characterizes a form of the human bleeding disorder Glanzmann’s thrombasthenia, fails to rescue β3–/– osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y747F/Y759F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the β3 integrin regulates platelets and osteoclasts.