Objectives RNASET2 is a lysosomal RNase whose enzymatic function is required for early events in lipotoxicity. However, the endogenous RNA substrates of RNASET2 that modulate lipid-induced cell death are not known. The purpose of this study was to identify RNASET2 substrates that impact lipotoxic stress. Methods RNA sequencing was used to identify RNAs that increase in abundance in human cells upon RNASET2 knockdown, and actinomycin D assays were used to show that RNASET2 impacted decay rates of these RNAs. We tested for the presence of these RNAs in immunoisolated lysosomes and determined the contribution of the lysosomal membrane transporter SIDT2 in delivery of these RNAs to the lysosome. A role for these RNAs in lipotoxic cell death was directly tested in loss- and gain of function analysis. Results RNASET2 knockdown increased steady-state abundance of UCHL3, PFN2 and PRDX3 mRNAs and prolonged their decay rate, leading to increased protein expression. These mRNAs were delivered to the lysosomal lumen by the lysosomal membrane transporter SIDT2 that mediates RNautophagy. While UCHL3 and PFN2 have not previously been implicated in lipotoxic responses, expression of these proteins protected against lipid-induced cell death. Conclusions Our study identified specific mRNA substrates of RNASET2 and uncovered a previously unexplored function for lysosomes and RNautophagy in regulation of the response to metabolic stress. Moreover, we demonstrated that RNautophagy selectively regulates turnover of specific endogenous RNAs and thereby impacts regulation of gene expression.

Pancreatic β-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair β-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on β-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also of mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our findings uncover a translational regulatory circuit during β-cell glucose toxicity that impairs expression of proteins with critical roles in β-cell function.

Ceramides and cardiorespiratory (CR) fitness are both related to cardiovascular diseases. The associations of three blood plasma ceramides (C16:0, C22:0, and C24:0) with CR fitness in the population-based Study of Health in Pomerania (SHIP-START-1; n = 1,102; mean age 50.3 years, 51.5% women) are investigated. In addition, subgroup analysis according to age (</≥54 years) and sex (female/male) is performed. Ceramides are quantified by liquid chromatography/mass spectrometry (LC/MS). CR fitness is assessed by a cardiopulmonary exercise test. Sex and age independent associations are found for higher levels of C24:0 and C24:0/C16:0 ratio with higher maximal oxygen consumption (VO2peak) kg-1 and oxygen consumption at the anaerobic threshold (VO2@AT1) as well as for the relation of C24:0/C16:0 with maximum workload (Wattmax kg-1). In contrast, age/sex subgroup specific inverse associations with Wattmax kg-1 are found in women <54 years for C22:0, while a positive association in men ≥54 years. Higher levels of C24:0 are associated with higher Wattmax kg-1, except for women <54 years, where no significant association can be found. The findings suggest that the use of single ceramides as cardiovascular biomarkers may be inferior, compared to ceramide ratio C24:0/C16:0. Therefore C24:0/C16:0 ratio may be a more suitable and robust cardiovascular biomarker and should be preferred over single ceramides.

The pathway used by mammalian cells to make triglyceride lipids when supplies of fat molecules are high has long been known. A route that works when fat supplies are low has now been discovered.

Background GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in & beta;-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy.Methods The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/ MS methods.Findings We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with & beta;-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient.Interpretations These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the ef-ficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients.Funding This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.Copyright & COPY; 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction: Ceramides are promising new predictive biomarkers for cardiovascular diseases (CVD), but their main mode of action is still unknown. As low cardiorespiratory fitness (CRF) is also linked with CVD, we investigated the association of three of the most abundant blood plasma ceramides and their respective ratios with CRF in a large population-based study. Hypothesis: Plasma long-chain ceramide levels are positively associated with higher CRF in the general population, while higher short-chain ceramide levels are associated with lower CRF. Methods: Levels of C16:0, C22:0, and C24:0 ceramide were quantified by a liquid chromatography-mass spectrometry and CRF was measured by a cardiopulmonary exercise test in the first follow-up of the Study of Health in Pomerania (SHIP-START-1; n = 1,105; mean age 50.3 years). Multivariable linear regression models were used to assess the association between ceramides and CRF. Results: Higher C24:0 levels as well as C24:0/C16:0 ratio were significantly associated with higher CRF, while higher levels of C22:0 or C22:0/C16:0 ratio were associated with lower CRF. Furthermore, we found that body mass index (BMI) interacts with these associations, whereas neither, sex, beta blockers nor lipid-lowering medication had any effect modifying influence. Conclusions: This study showed that ceramides are associated with CRF, one of the most important risk factors of CVD. Ceramide C24:0 as well as C24:0/C16:0 ratio might be beneficial regarding CRF, whereas C22:0 and C22:0/C16:0 ratio might be detrimental. Our study results were independent of many cardiovascular risk factors, which highlights ceramides as potential predictive biomarkers. Future studies with larger numbers of subjects are needed to verify these results, and to evaluate whether the observed interaction with BMI could limit the use to specific BMI subgroups.

Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3 ) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.

Nutrient excess is linked to the pathogenesis of complications of obesity and type 2 diabetes. Fatty acid accumulation in non‐adipose tissues is initially well tolerated and leverages the nearly universal ability of cells to store fatty acids as triglycerides in lipid droplets. However, lipid overload eventually precipitates cell dysfunction through generation of toxic metabolites, activation of signaling cascades, endoplasmic reticulum stress, and oxidative stress. These pathways contribute to cell death, organ dysfunction and disease pathogenesis. Mechanisms that regulate the transition from adaptive lipid storage to initiation of lipotoxicity are not well understood. New insights have come from genetic screens in model cell culture systems. By leveraging the ability of retroviral promotor trap mutagenesis to disrupt genetic elements regardless of coding potential and using selection for resistance to palmitate‐induced cell death, our group discovered that disruption of specific small nucleolar RNAs (snoRNAs) protects against lipotoxic and oxidative stress in cultured cells. By extending our findings to in vivo models, we have demonstrated that knockdown or knockout of these snoRNAs rewires metabolism in ways that increase lipid metabolism, enhance oxidative stress tolerance, and confer resistance to lipotoxicity. Our studies highlight novel roles for snoRNAs as metabolic modulators and suggest that expression and function of this class of intronic non‐coding RNAs may have relevance to human health and disease.Support or Funding InformationNIH R01 DK064989NIH R01 DK108357

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

Cardiac dysfunction in T2D is associated with excessive FA uptake, oxidation, and generation of toxic lipid species by the heart. It is not known whether decreasing lipid delivery to the heart can effect improvement in cardiac function in humans with T2D. Thus, our objective was to test the hypothesis that lowering lipid delivery to the heart would result in evidence of decreased "lipotoxicity," improved cardiac function, and salutary effects on plasma biomarkers of cardiovascular risk. Thus, we performed a double-blind randomized placebo-controlled parallel design study of the effects of 12 weeks of fenofibrate-induced lipid lowering on cardiac function, inflammation, and oxidation biomarkers, and on the ratio of two plasma ceramides, Cer d18:1 (4E) (1OH, 3OH)/24:0 and Cer d18:1 (4E) (1OH, 3OH)/16:0 (i.e., "C24:0/C16:0"), which is associated with decreased risk of cardiac dysfunction and heart failure. Fenofibrate lowered plasma TG and cholesterol but did not improve heart systolic or diastolic function. Fenofibrate treatment lowered the plasma C24:0/C16:0 ceramide ratio and minimally altered oxidative stress markers but did not alter measures of inflammation. Overall, plasma TG lowering correlated with improvement of cardiac relaxation (diastolic function) as measured by tissue Doppler-derived parameter e '. Moreover, lowering the plasma C24:0/C16:0 ceramide ratio was correlated with worse diastolic function. These findings indicate that fenofibrate treatment per se is not sufficient to effect changes in cardiac function; however, decreases in plasma TG may be linked to improved diastolic function. In contrast, decreases in plasma C24:0/C16:0 are linked with worsening cardiac function.

Introduction: NT-proBNP is a predictor of mortality in patients with or without HF. Ceramides are bioactive lipids involved in cell signaling and membrane structure. Our group showed that the ratio of specific plasma ceramides (C24:0/C16:0) is inversely related to death. However, NT-proBNP levels were not available for multivariate modeling of mortality in that cohort. Hypothesis: We hypothesized that plasma C24:0/C16:0 would add to a predictive model that includes NT-proBNP and classical cardiovascular risk factors (CVRs) of mortality. We also hypothesized that body mass index (BMI) modulates the predictive value of plasma C24:0/C16:0. Methods: We performed multivariable modeling on data from 2,760 Caucasian subjects in the SHIP (Study of Health in Pomerania) cohort from northern Europe. Plasma C24:0/C16:0 was measured using targeted liquid chromatograph/tandem mass spectrometry. CVRs in the modeling included: age, sex, systolic blood pressure, current smoking, diabetes, and total/high-density cholesterol. Model performance was evaluated using Akaike’s information criterion (AIC) and Harrel’s c-statistic. Results: There were 529 deaths over a median of 14.6 y. Model selection via AIC revealed that the best model with the least number of variables to predict mortality included plasma C24:0/16:0 and BMI (+ their interaction), CVRs, and NT-proBNP, P < 0.001. The evidence ratio showed that this best model is 540 times more likely to be the ideal model than the model that only includes CVRs. The best model was also much more informative than a model that added only NT-proBNP to the CVRs (ΔAIC = 19.865, Akaike weight < 0.001). With increasing BMI, the protective effect of plasma C24:0/16:0 on survival probability diminished. Post hoc analysis showed the best model predicted CVD mortality but not non-CVD mortality. Conclusions: A mortality prediction model that includes plasma C24:0/C16:0, NT-proBNP, and CVRs is superior to CVRs alone or CVRs + NT-proBNP and helps further stratify patients’ risk. BMI modifies the predictive value of plasma C24:0/C16:0. Plasma C24:0/C16:0 may be a novel target for modifying CVD mortality risk.

Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.

Excess fatty acid accumulation in nonadipose tissues leads to cell dysfunction and cell death that is linked to the pathogenesis of inherited and acquired human diseases. Study of this process, known as lipotoxicity, has provided new insights into the regulation of lipid homeostasis and has revealed new molecular pathways involved in lipid-induced cellular stress. The discovery that disruption of specific small nucleolar RNAs protects against fatty acid–induced cell death and remodels metabolism in vivo opens new opportunities for understanding how nutrient signals influence cellular and systemic metabolic homeostasis through RNA biology. Excess fatty acid accumulation in nonadipose tissues leads to cell dysfunction and cell death that is linked to the pathogenesis of inherited and acquired human diseases. Study of this process, known as lipotoxicity, has provided new insights into the regulation of lipid homeostasis and has revealed new molecular pathways involved in lipid-induced cellular stress. The discovery that disruption of specific small nucleolar RNAs protects against fatty acid–induced cell death and remodels metabolism in vivo opens new opportunities for understanding how nutrient signals influence cellular and systemic metabolic homeostasis through RNA biology. Fatty acids perform many critical cellular functions. These amphipathic molecules are building blocks of membrane lipids that demarcate the boundaries of cells and organize biochemical reactions within intracellular organelles. Fatty acids also serve as precursors for the biosynthesis of signaling lipids, such as ceramides and hydroxylated fatty acids, which regulate physiological processes. Moreover, fatty acids are nutrients that can be oxidized to produce ATP or efficiently stored as triglycerides. They are major nutrients for tissues with high energy demands, such as muscle, and a critical source of potential energy for organisms during periods of fasting. Under physiological conditions, cellular fatty acid supplies derive from de novo synthesis, recycling of other lipid classes, and import of exogenous lipids, distinct processes that must be coordinated with cells' needs for these lipids for diverse functions. Failure to balance acquisition and use of fatty acids underlies the pathophysiology of human diseases, ranging from rare monogenic disorders of metabolism to common complications of metabolic syndrome and type 2 diabetes mellitus (T2DM). In the 1970s and 1980s, clinicians discovered that disorders presenting as hypoglycemia in the setting of viral illness or fasting could be linked to mutations in genes encoding enzymes in fatty acid oxidation (1Stanley C.A. Hale D.E. Coates P.M. Hall C.L. Corkey B.E. Yang W. Kelley R.I. Gonzales E.L. Williamson J.R. Baker L. Medium-chain acyl-CoA dehydrogenase deficiency in children with non-ketotic hypoglycemia and low carnitine levels.Pediatr. Res. 1983; 17 (6646897): 877-88410.1203/00006450-198311000-00008Crossref PubMed Scopus (161) Google Scholar). Evidence of tissue dysfunction—failure of the liver to produce critical ketone bodies during starvation or frank damage to skeletal and cardiac muscle—coupled with observations of triglyceride buildup in these tissues suggested that excess lipid was toxic (2DiMauro S. DiMauro P.M. Muscle carnitine palmityltransferase deficiency and myoglobinuria.Science. 1973; 182 (4745596): 929-93110.1126/science.182.4115.929Crossref PubMed Scopus (395) Google Scholar). Similarly, associations between triglyceride accumulation and myopathy in the tissues of children and young adults with neutral lipid storage diseases suggested that excess lipid impairs muscle function (3Chanarin I. Patel A. Slavin G. Wills E.J. Andrews T.M. Stewart G. Neutral-lipid storage disease: a new disorder of lipid metabolism.Br. Med. J. 1975; 1 (1139147): 553-55510.1136/bmj.1.5957.553Crossref PubMed Scopus (225) Google Scholar). More recently, as the prevalence of obesity, metabolic syndrome, and T2DM has increased globally, there is increasing recognition that nonalcoholic fatty liver disease and diabetic cardiomyopathy are diseases in which tissue level accumulation of excess lipids, related to hyperlipidemia and/or insulin resistance, is associated with significant impairment of organ function (4James O. Day C. Non-alcoholic steatohepatitis: another disease of affluence.Lancet. 1999; 353 (10335777): 1634-163610.1016/S0140-6736(99)00163-4Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 5Sharma S. Adrogue J.V. Golfman L. Uray I. Lemm J. Youker K. Noon G.P. Frazier O.H. Taegtmeyer H. Intramyocardial lipid accumulation in the failing human heart resembles the lipotoxic rat heart.FASEB J. 2004; 18 (15522914): 1692-170010.1096/fj.04-2263comCrossref PubMed Scopus (588) Google Scholar). These clinical presentations inspired animal studies in the laboratory to address key questions regarding the pathogenesis of these diseases. Knockout mouse models of monogenic disorders of fatty acid oxidation and dietary and genetic models of metabolic syndrome and T2DM phenocopy both tissue level lipid accumulation and organ dysfunction. Fatty acid oxidation disorders are characterized by impaired utilization in the setting of a normal supply of fatty acids, whereas models of metabolic syndrome and T2DM are examples of supply exceeding the capacity to metabolize fatty acids. These models recapitulate the lipid-induced activation of endoplasmic reticulum (ER) and oxidative stress observed in tissues of affected patients (6Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Gorgun C. Glimcher L.H. Hotamisligil G.S. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.Science. 2004; 306 (15486293): 457-46110.1126/science.1103160Crossref PubMed Scopus (2978) Google Scholar, 7Boudina S. Sena S. Theobald H. Sheng X. Wright J.J. Hu X.X. Aziz S. Johnson J.I. Bugger H. Zaha V.G. Abel E.D. ED Mitochondrial energetics in the heart in obesity-related diabetes: direct evidence for increased uncoupled respiration and activation of uncoupling proteins.Diabetes. 2007; 56 (17623815): 2457-246610.2337/db07-0481Crossref PubMed Scopus (464) Google Scholar, 8Ljubkovic M. Gressette M. Bulat C. Cavar M. Bakovic D. Fabijanic D. Grkovic I. Lemaire C. Marinovic J. Disturbed fatty acid oxidation, endoplasmic reticulum stress, and apoptosis in left ventricle of patients with type 2 diabetes.Diabetes. 2019; 68 (31391173): 1924-193310.2337/db19-0423Crossref PubMed Scopus (38) Google Scholar, 9Anderson E.J. Kypson A.P. Rodriguez E. Anderson C.A. Lehr E.J. Neufer P.D. Substrate-specific derangements in mitochondrial metabolism and redox balance in the atrium of the type 2 diabetic human heart.J. Am. Coll. Cardiol. 2009; 54 (19892241): 1891-189810.1016/j.jacc.2009.07.031Crossref PubMed Scopus (295) Google Scholar). Other genetically modified mouse models with tissue-specific overexpression of proteins involved in fatty acid transport have provided compelling evidence that tissue lipid overload precipitates similar toxicity, even in the absence of insulin resistance, hyperglycemia, hyperlipidemia, or other systemic metabolic perturbation (10Chiu H.C. Kovacs A. Ford D.A. Hsu F.F. Garcia R. Herrero P. Saffitz J.E. Schaffer J.E. A novel mouse model of lipotoxic cardiomyopathy.J. Clin. Invest. 2001; 107 (11285300): 813-82210.1172/JCI10947Crossref PubMed Scopus (617) Google Scholar, 11Yagyu H. Chen G. Yokoyama M. Hirata K. Augustus A. Kako Y. Seo T. Hu Y. Lutz E.P. Merkel M. Bensadoun A. Homma S. Goldberg I.J. Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy.J. Clin. Invest. 2003; 111 (12569168): 419-42610.1172/JCI16751Crossref PubMed Scopus (298) Google Scholar, 12Chiu H.C. Kovacs A. Blanton R.M. Han X. Courtois M. Weinheimer C.J. Yamada K.A. Brunet S. Xu H. Nerbonne J.M. Welch M.J. Fettig N.M. Sharp T.L. Sambandam N. Olson K.M. et al.Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy.Circ. Res. 2005; 96 (15618539): 225-23310.1161/01.RES.0000154079.20681.B9Crossref PubMed Scopus (349) Google Scholar). Clinically, accumulation of lipid droplets in nonadipose tissue is the sine qua non of lipid overload states when supply exceeds demand. Fatty acids are delivered to tissues in the form of circulating lipoprotein particles from which fatty acids are released by lipoprotein lipase–mediated hydrolysis at the capillary endothelium, by endocytosed lipoproteins that are hydrolyzed intracellularly, and through uptake of free fatty acids liberated by adipose tissue lipolysis. In cells and in tissues that do not need the fatty acids for membrane biogenesis, synthesis of signaling molecules, or oxidation, fatty acids are re-esterified to glycerol, and resulting triglycerides are stored in the neutral lipid core of intracellular lipid droplets. Our understanding of the regulation of dynamic lipid trafficking into and out of these organelles has been advanced as the identities and function of proteins associated with the limiting monolayer of the lipid droplet have been illuminated (13Wilfling F. Wang H. Haas J.T. Krahmer N. Gould T.J. Uchida A. Cheng J.X. Graham M. Christiano R. Fröhlich F. Liu X. Buhman K.K. Coleman R.A. Bewersdorf J. Farese Jr., R.V. et al.Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.Dev. Cell. 2013; 24 (23415954): 384-39910.1016/j.devcel.2013.01.013Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). Several physical features of lipid droplets have the potential to contribute to cellular and organ level dysfunction. Lipid droplet membrane contact sites with the ER, mitochondria, peroxisomes, lysosomes, and Golgi provide potential platforms through which particularly abundant or large lipid droplets may interfere with the functions of other organelles (14Valm A.M. Cohen S. Legant W.R. Melunis J. Hershberg U. Wait E. Cohen A.R. Davidson M.W. Betzig E. Lippincott-Schwartz J. Applying systems-level spectral imaging and analysis to reveal the organelle interactome.Nature. 2017; 546 (28538724): 162-16710.1038/nature22369Crossref PubMed Scopus (549) Google Scholar). The physical imposition of large lipid droplets also has the potential to alter cytoskeletal structures and perturb the biochemical reactions or trafficking events they regulate (15Kim J.I. Park J. Ji Y. Jo K. Han S.M. Sohn J.H. Shin K.C. Han J.S. Jeon Y.G. Nahmgoong H. Han K.H. Kim J. Kim S. Choe S.S. Kim J.B. During adipocyte remodeling, lipid droplet configurations regulate insulin sensitivity through F-actin and G-actin Reorganization.Mol. Cell Biol. 2019; 39 (31308132): e00210-e0021910.1128/MCB.00210-19Crossref PubMed Scopus (25) Google Scholar). Moreover, the ability of lipid droplets to recruit proteins to their surface raises the possibility that sequestration of proteins at these sites could be maladaptive (16Welte M.A. Proteins under new management: lipid droplets deliver.Trends Cell Biol. 2007; 17 (17766117): 363-36910.1016/j.tcb.2007.06.004Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The precise contributions of such mechanisms to the pathogenesis of lipotoxicity remains to be determined. On the other hand, evidence in cell culture models of fatty acid overload suggests that triglyceride molecules per se are inert with respect to lipotoxicity. Excess saturated long-chain fatty acids (e.g. palmitate) are substantially more toxic than unsaturated long-chain fatty acids (e.g. oleate), and toxicity is inversely associated with cells' capacity to incorporate the excess lipid into triglycerides (17Cnop M. Hannaert J.C. Hoorens A. Eizirik D.L. Pipeleers D.G. Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation.Diabetes. 2001; 50 (11473037): 1771-177710.2337/diabetes.50.8.1771Crossref PubMed Scopus (455) Google Scholar). The ability to synthesize triglyceride from palmitate can be increased by up-regulation of stearoyl-CoA desaturase overexpression, which converts saturated palmitate into monounsaturated palmitoleate, or by exposure to mixtures of both saturated and unsaturated fatty acids (18Listenberger L.L. Han X. Lewis S.E. Cases S. Farese Jr., R.V. Ory D.S. Schaffer J.E. Triglyceride accumulation protects against fatty acid-induced lipotoxicity.Proc. Natl. Acad. Sci. U.S.A. 2003; 100 (12629214): 3077-308210.1073/pnas.0630588100Crossref PubMed Scopus (1402) Google Scholar). Importantly, these strategies that increase incorporation of excess fatty acids into triglyceride stores are associated with enhanced cell tolerance of the lipid load, whereas genetic disruption of triglyceride synthesis pathways renders cells similarly susceptible to saturated and monounsaturated fatty acid–induced cell death. Collectively, these data suggest that the toxicity of excess fatty acids is precipitated either by lipid that is not effectively incorporated into triglyceride stores in lipid droplets or by fatty acids released from these dynamic structures through ongoing cycles of lipolysis. Studies in mice show that diverse genetic manipulations alter lipid droplet homeostasis and impact lipotoxicity in different ways. In the heart, only some of these data support the hypothesis that triglyceride accumulation is protective. In models in which lipotoxicity results from increased fatty acid import into cardiomyocytes, systolic heart failure is observed both when incorporation of lipid into triglyceride droplets is increased (10Chiu H.C. Kovacs A. Ford D.A. Hsu F.F. Garcia R. Herrero P. Saffitz J.E. Schaffer J.E. A novel mouse model of lipotoxic cardiomyopathy.J. Clin. Invest. 2001; 107 (11285300): 813-82210.1172/JCI10947Crossref PubMed Scopus (617) Google Scholar) and when excess lipid is diverted to alternate metabolic fates, resulting in little triglyceride accumulation (11Yagyu H. Chen G. Yokoyama M. Hirata K. Augustus A. Kako Y. Seo T. Hu Y. Lutz E.P. Merkel M. Bensadoun A. Homma S. Goldberg I.J. Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy.J. Clin. Invest. 2003; 111 (12569168): 419-42610.1172/JCI16751Crossref PubMed Scopus (298) Google Scholar). Models with knockout or overexpression of enzymes in triglyceride homeostasis, such as adipose tissue triglyceride lipase and diacylglycerol acyltransferase show seemingly divergent consequences of cardiomyocyte triglyceride accumulation on cardiac function (19Kienesberger P.C. Pulinilkunnil T. Nagendran J. Young M.E. Bogner-Strauss J.G. Hackl H. Khadour R. Heydari E. Haemmerle G. Zechner R. Kershaw E.E. Dyck J.R. 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Excess fatty acid accumulation in nonadipose tissues leads to cell dysfunction and cell death that is linked to the pathogenesis of inherited and acquired human diseases. Study of this process, known as lipotoxicity, has provided new insights into the regulation of lipid homeostasis and has revealed new molecular pathways involved in lipid-induced cellular stress. The discovery that disruption of specific small nucleolar RNAs protects against fatty acid?induced cell death and remodels metabolismin vivoopens new opportunities for understanding how nutrient signals influence cellular and systemic metabolic homeostasis through RNA biology.

Objective: Total ceramide concentrations are linked with increased insulin resistance and cardiac dysfunction.However, recent studies have demonstrated that plasma concentrations of specific very-long-chain fatty ceramides (C24:0 and C22:0) are associated with a reduced incidence of coronary heart disease and all-cause mortality.We hypothesized that specific genetic loci are associated with plasma C22:0 and C24:0 concentrations.Methods: Heritability and genome-wide association studies of plasma C24:0 and C22:0 ceramide concentrations were performed among 2,217 participants in the Framingham Heart Study Offspring Cohort, adjusting for cardiovascular risk factor covariates and cardiovascular drug treatment.Results: The multivariable-adjusted heritability for C22:0 and C24:0 ceramides was 0.42 (standard error [SE], 0.07; p=1.8E-9) and 0.25 (SE, 0.08; p=0.00025), respectively.Nineteen single nucleotide polymorphisms (SNPs), all on chromosome 20, significantly associated with C22:0 concentrations; the closest gene to these variants was SPTLC3.The lead SNP (rs4814175) significantly associated with 3% lower plasma C22:0 concentrations (p=2.83E-11).Nine SNPs, all on chromosome 20 and close to SPTLC3, were significantly associated with C24:0 ceramide concentrations.All 9 were also significantly related to plasma C22:0 levels.The lead SNP (rs168622) was significantly associated with 10% lower plasma C24:0 ceramide concentrations (p=9.94E-09).Conclusion: SNPs near the SPTLC3 gene, which encodes serine palmitoyltransferase long chain base subunit 3 (SPTLC3; part of the enzyme that catalyzes the rate-limiting step of de novo sphingolipid synthesis) were associated with plasma C22:0 and C24:0 ceramide concentrations.These results are biologically plausible and suggest that SPTLC3 may be a potential therapeutic target for C24:0 and C22:0 ceramide modulation.

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disorder caused by mutations in either the NPC1 or the NPC2 gene. A new class of lipids, N-acyl-O-phosphocholineserines were recently identified as NPC biomarkers. The most abundant species in this class of lipid, N-palmitoyl-O-phosphocholineserine (PPCS), was evaluated for diagnosis of NPC disease and treatment efficacy assessment with 2-hydroxypropyl-β-cyclodextrin (HPβCD) in NPC. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated to measure PPCS in human plasma and cerebrospinal fluid (CSF). A cutoff of 248 ng/mL in plasma provided a sensitivity of 100.0% and specificity of 96.6% in identifying NPC1 patients from control and NPC1 carrier subjects. PPCS was significantly elevated in CSF from NPC1 patients, and CSF PPCS levels were significantly correlated with NPC neurological disease severity scores. Plasma and CSF PPCS did not change significantly in response to intrathetical (IT) HPβCD treatment. In an intravenous (IV) HPβCD trial, plasma PPCS in all patients was significantly reduced. These results demonstrate that plasma PPCS was able to diagnose NPC1 patients with high sensitivity and specificity, and to evaluate the peripheral treatment efficacy of IV HPβCD treatment.

Niemann-Pick disease type C (NPC) is a neurodegenerative disease in which mutation of NPC1 or NPC2 gene leads to lysosomal accumulation of unesterified cholesterol and sphingolipids. Diagnosis of NPC disease is challenging due to non-specific early symptoms. Biomarker and genetic tests are used as first-line diagnostic tests for NPC. In this study, we developed a plasma test based on N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine (TCG) that was markedly increased in the plasma of human NPC1 subjects. The test showed sensitivity of 0.9945 and specificity of 0.9982 to differentiate individuals with NPC1 from NPC1 carriers and controls. Compared to other commonly used biomarkers, cholestane-3β,5α,6β-triol (C-triol) and N-palmitoyl-O-phosphocholine (PPCS, also referred to as lysoSM-509), TCG was equally sensitive for identifying NPC1 but more specific. Unlike C-triol and PPCS, TCG showed excellent stability and no spurious generation of marker in the sample preparation or aging of samples. TCG was also elevated in lysosomal acid lipase deficiency (LALD) and acid sphingomyelinase deficiency (ASMD). Plasma TCG was significantly reduced after intravenous (IV) 2-hydroxypropyl-β-cyclodextrin (HPβCD) treatment. These results demonstrate that plasma TCG was superior to C-triol and PPCS as NPC1 diagnostic biomarker and was able to evaluate the peripheral treatment efficacy of IV HPβCD treatment.

Abstract Background We determined the association between ratios of plasma ceramide species of differing fatty‐acyl chain lengths and incident dementia and Alzheimer’s disease (AD) dementia in a large, community‐based sample. Methods We measured plasma ceramide levels in 1892 [54% women, mean age 70.1 (SD 6.9) yr.] dementia‐free Framingham Offspring Study cohort participants between 2005 and 2008. We related ratios of very long‐chain (C24:0, C22:0) to long‐chain (C16:0) ceramides to subsequent risk of incident dementia and AD dementia. Structural MRI brain measures were included as secondary outcomes. Results During a median 6.5 year follow‐up, 81 participants developed dementia, of whom 60 were diagnosed with AD dementia. In multivariable Cox‐proportional hazards analyses, each standard deviation (SD) increment in the ratio of ceramides C24:0/C16:0 was associated with a 27% reduction in the risk of dementia (HR 0.73, 95% CI 0.56–0.96) and AD dementia (HR 0.73, 95% CI 0.53–1.00). The ratio of ceramides C22:0/C16:0 was also inversely associated with incident dementia (HR per SD 0.75, 95% CI 0.57–0.98), and approached statistical significance for AD (HR 0.73, 95% CI 0.53–1.01, P = 0.056). Higher ratios of ceramides C24:0/C16:0 and C22:0/C16:0 were also cross‐sectionally associated with lower white matter hyperintensity burden on MRI (−0.05 ± 0.02, P = 0.02; −0.06 ± 0.02, P = 0.003; respectively per SD increase), but not with other MRI brain measures. Conclusions Higher plasma ratios of very long‐chain to long‐chain ceramides are associated with a reduced risk of incident dementia and AD dementia in our community‐based sample. Circulating ceramide ratios may serve as potential biomarkers for predicting dementia risk in cognitively healthy adults.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPβCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.