NAMPT-dependent NAD+ biosynthesis controls circadian metabolism in a tissue-specific manner.

Molecular clocks in the periphery coordinate tissue-specific daily biorhythms by integrating input from the hypothalamic master clock and intracellular metabolic signals. One such key metabolic signal is the cellular concentration of NAD+, which oscillates along with its biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT). NAD+ levels feed back into the clock to influence rhythmicity of biological functions, yet whether this metabolic fine-tuning occurs ubiquitously across cell types and is a core clock feature is unknown. Here, we show that NAMPT-dependent control over the molecular clock varies substantially between tissues. Brown adipose tissue (BAT) requires NAMPT to sustain the amplitude of the core clock, whereas rhythmicity in white adipose tissue (WAT) is only moderately dependent on NAD+ biosynthesis, and the skeletal muscle clock is completely refractory to loss of NAMPT. In BAT and WAT, NAMPT differentially orchestrates oscillation of clock-controlled gene networks and the diurnality of metabolite levels. NAMPT coordinates the rhythmicity of TCA cycle intermediates in BAT, but not in WAT, and loss of NAD+ abolishes these oscillations similarly to high-fat diet-induced circadian disruption. Moreover, adipose NAMPT depletion improved the ability of animals to defend body temperature during cold stress but in a time-of-day-independent manner. Thus, our findings reveal that peripheral molecular clocks and metabolic biorhythms are shaped in a highly tissue-specific manner by NAMPT-dependent NAD+ synthesis.

MetaboLink: A web application for Streamlined Processing and Analysis of Large-Scale Untargeted Metabolomics Data.

MOTIVATION: The post-processing and analysis of large-scale untargeted metabolomics data face significant challenges due to the intricate nature of correction, filtration, imputation, and normalization steps. Manual execution across various applications often leads to inefficiencies, human-induced errors, and inconsistencies within the workflow. RESULTS: Addressing these issues, we introduce MetaboLink, a novel web application designed to process LC-MS metabolomics datasets combining established methodologies and offering flexibility and ease of implementation. It offers visualization options for data interpretation, an interface for statistical testing, and integration with PolySTest for further tests and with VSClust for clustering analysis. AVAILABILITY: Fully functional tool is publicly available at https://computproteomics.bmb.sdu.dk/Metabolomics/. The source code is available at https://github.com/anitamnd/MetaboLink and a detailed description of the app can be found at https://github.com/anitamnd/MetaboLink/wiki. A tutorial video can be found at https://youtu.be/ZM6j10S6Z8Q. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Loss of Sucrase-Isomaltase Function Increases Acetate Levels and Improves Metabolic Health in Greenlandic Cohorts.

BACKGROUND & AIMS: The sucrase-isomaltase (SI) c.273_274delAG loss-of-function variant is common in Arctic populations and causes congenital sucrase-isomaltase deficiency, which is an inability to break down and absorb sucrose and isomaltose. Children with this condition experience gastrointestinal symptoms when dietary sucrose is introduced. We aimed to describe the health of adults with sucrase-isomaltase deficiency. METHODS: The association between c.273_274delAG and phenotypes related to metabolic health was assessed in 2 cohorts of Greenlandic adults (n = 4922 and n = 1629). A sucrase-isomaltase knockout (Sis-KO) mouse model was used to further elucidate the findings. RESULTS: Homozygous carriers of the variant had a markedly healthier metabolic profile than the remaining population, including lower body mass index (ß [standard error], -2.0 [0.5] kg/m2; P = 3.1 × 10-5), body weight (-4.8 [1.4] kg; P = 5.1 × 10-4), fat percentage (-3.3% [1.0%]; P = 3.7 × 10-4), fasting triglyceride (-0.27 [0.07] mmol/L; P = 2.3 × 10-6), and remnant cholesterol (-0.11 [0.03] mmol/L; P = 4.2 × 10-5). Further analyses suggested that this was likely mediated partly by higher circulating levels of acetate observed in homozygous carriers (ß [standard error], 0.056 [0.002] mmol/L; P = 2.1 × 10-26), and partly by reduced sucrose uptake, but not lower caloric intake. These findings were verified in Sis-KO mice, which, compared with wild-type mice, were leaner on a sucrose-containing diet, despite similar caloric intake, had significantly higher plasma acetate levels in response to a sucrose gavage, and had lower plasma glucose level in response to a sucrose-tolerance test. CONCLUSIONS: These results suggest that sucrase-isomaltase constitutes a promising drug target for improvement of metabolic health, and that the health benefits are mediated by reduced dietary sucrose uptake and possibly also by higher levels of circulating acetate.

Distinct maternal metabolites are associated with obesity and glucose-insulin axis in the first trimester of pregnancy.

BACKGROUND/OBJECTIVES: Obesity in pregnancy associates with changes in the glucose-insulin axis. We hypothesized that these changes affect the maternal metabolome already in the first trimester of human pregnancy and, thus, aimed to identify these metabolites. PATIENTS/METHODS: We performed untargeted metabolomics (HPLC-MS/MS) on maternal serum (n = 181, gestational weeks 4+0-11+6). For further analysis, we included only non-smoking women as assessed by serum cotinine levels (ELISA) (n = 111). In addition to body mass index (BMI) and leptin as measures of obesity and adiposity, we metabolically phenotyped women by their fasting glucose, C-peptide and insulin sensitivity (ISHOMA index). To identify metabolites (outcome) associated with BMI, leptin, glucose, C-peptide and/or ISHOMA (exposures), we used a combination of univariable and multivariable regression analyses with multiple confounders and machine learning methods (Partial Least Squares Discriminant Analysis, Random Forest and Support Vector Machine). Additional statistical tests confirmed robustness of results. Furthermore, we performed network analyses (MoDentify package) to identify sets of correlating metabolites that are coordinately regulated by the exposures. RESULTS: We detected 2449 serum features of which 277 were annotated. After stringent analysis, 15 metabolites associated with at least one exposure (BMI, leptin, glucose, C-peptide, ISHOMA). Among these, palmitoleoyl ethanolamine (POEA), an endocannabinoid-like lipid endogenously synthesized from palmitoleic acid, and N-acetyl-L-alanine were consistently associated with C-peptide in all the analyses (95% CI: 0.10-0.34; effect size: 21%; p < 0.001; 95% CI: 0.04-0.10; effect size: 7%; p < 0.001). In network analysis, most features correlating with palmitoleoyl ethanolamide and N-acetyl-L-alanine and associated with C-peptide, were amino acids or dipeptides (n = 9, 35%), followed by lipids (n = 7, 27%). CONCLUSIONS: We conclude that the metabolome of pregnant women with overweight/obesity is already altered early in pregnancy because of associated changes of C-peptide. Changes of palmitoleoyl ethanolamide concentration in pregnant women with obesity-associated hyperinsulinemia may reflect dysfunctional endocannabinoid-like signalling.

Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains.

The type III CRISPR-Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR-Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA5 and cA6. cA6 acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm6 domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cAns to switch signaling off. CARF ring nuclease converts cA6 to linear A6>p and to the final A3>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA6 or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Csm effector and the cA6-activated Csm6 ribonuclease.

Free Fatty Acids Interfere with the DNA Binding Activity of the Virulence Regulator PrfA of Listeria monocytogenes.

Naturally occurring free fatty acids (FFAs) are recognized as potent antimicrobial agents that also affect the production of virulence factors in bacterial pathogens. In the foodborne pathogen Listeria monocytogenes, some medium- and long-chain FFAs act as antimicrobial agents as well as signaling compounds, causing a repression of transcription of virulence genes. We previously observed that the master virulence regulator PrfA is involved in both the antimicrobial and virulence-inhibitory response of L. monocytogenes to selected FFAs, but the underlying mechanisms are presently unknown. Here, we present a systematic analysis of the antimicrobial and PrfA-inhibitory activities of medium- and long-chain FFAs of various carbon chain lengths and degrees of saturation. We observed that exposure to specific antimicrobial and nonantimicrobial FFAs prevented PrfA-dependent activation of virulence gene transcription and reduced the levels of PrfA-regulated virulence factors. Thus, an antimicrobial activity was not compulsory for the PrfA-inhibitory ability of an FFA. In vitro binding experiments revealed that PrfA-inhibitory FFAs were also able to prevent the constitutively active variant PrfA* from binding to the PrfA box in the promoter region of the virulence gene hly, whereas noninhibitory FFAs did not affect its ability to bind DNA. Notably, the unsaturated FFAs inhibited the DNA binding activity of PrfA* most efficiently. Altogether, our findings support a model in which specific FFAs orchestrate a generalized reduction of the virulence potential of L. monocytogenes by directly targeting the key virulence regulator PrfA.IMPORTANCEListeria monocytogenes is a Gram-positive pathogen able to cause foodborne infections in humans and animals. Key virulence genes in L. monocytogenes are activated by the transcription regulator PrfA, a DNA binding protein belonging to the CRP/FNR family. Various signals from the environment are known to affect the activity of PrfA, either positively or negatively. Recently, we found that specific medium- and long-chain free fatty acids act as antimicrobial agents as well as signaling compounds in L. monocytogenes Here, we show that both antimicrobial and nonantimicrobial free fatty acids inhibit PrfA-dependent activation of virulence gene transcription by interfering with the DNA binding activity of PrfA. Our findings suggest that free fatty acids could be candidates for alternative therapies against L. monocytogenes.

Regulation of very-long acyl chain ceramide synthesis by acyl-CoA-binding protein.

Ceramide and more complex sphingolipids constitute a diverse group of lipids that serve important roles as structural entities of biological membranes and as regulators of cellular growth, differentiation, and development. Thus, ceramides are vital players in numerous diseases including metabolic and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very-long-chain acyl-CoA esters, which is required for its ability to stimulate CerS activity. We also show that high-speed liver cytosol from wild-type mice activates CerS3 activity, whereas cytosol from ACBP knock-out mice does not. Consistently, CerS2 and CerS3 activities are significantly reduced in the testes of ACBP-/- mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial importance in understanding the regulation of ceramide metabolism in pathogenesis.

Changes in kynurenine pathway metabolism in Parkinson patients with L-DOPA-induced dyskinesia.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective drug in the symptomatic treatment of Parkinson's disease, but chronic use is associated with L-DOPA-induced dyskinesia in more than half the patients after 10 years of treatment. L-DOPA treatment may affect tryptophan metabolism via the kynurenine pathway. Altered levels of kynurenine metabolites can affect glutamatergic transmission and may play a role in the development of L-DOPA-induced dyskinesia. In this study, we assessed kynurenine metabolites in plasma and cerebrospinal fluid of Parkinson's disease patients and controls. Parkinson patients (n = 26) were clinically assessed for severity of motor symptoms (UPDRS) and L-DOPA-induced dyskinesia (UDysRS). Plasma and cerebrospinal fluid samples were collected after overnight fasting and 1-2 h after intake of L-DOPA or other anti-Parkinson medication. Metabolites were analyzed in plasma and cerebrospinal fluid of controls (n = 14), Parkinson patients receiving no L-DOPA (n = 8), patients treated with L-DOPA without dyskinesia (n = 8), and patients with L-DOPA-induced dyskinesia (n = 10) using liquid chromatography-mass spectrometry. We observed approximately fourfold increase in the 3-hydroxykynurenine/kynurenic acid ratio in plasma of Parkinson's patients with L-DOPA-induced dyskinesia. Anthranilic acid levels were decreased in plasma and cerebrospinal fluid of this patient group. 5-Hydroxytryptophan levels were twofold increased in all L-DOPA-treated Parkinson's patients. We conclude that a higher 3-hydroxykynurenine/kynurenic acid ratio in plasma may serve as a biomarker for L-DOPA-induced dyskinesia. Longitudinal studies including larger patients cohorts are needed to verify whether the changes observed here may serve as a prognostic marker for L-DOPA-induced dyskinesia.

Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice.

The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic cholesteryl esters, and that lipids featuring an 18:1 fatty acid moiety are increased in Acbp depleted mice across all tissues investigated. Our results also show that the perturbation of systemic lipid metabolism in Acbp knockout mice is transient and becomes normalized and similar to that of wild type as mice grow older. These findings demonstrate that ACBP serves crucial functions in maintaining lipid metabolic homeostasis in mice during weaning.

Elimination of the last reactions in ergosterol biosynthesis alters the resistance of Saccharomyces cerevisiae to multiple stresses.

The sterol composition of membranes is known to influence many phenotypes of yeast. However, a systematic study of the relationship between sterol composition and stress resistances has not been conducted. Here, we therefore constructed single or double gene deletion mutants of the last four enzymes in ergosterol biosynthesis in a prototrophic genetic background of Saccharomyces cerevisiae. Identification of the sterol composition of these mutants revealed a high flexibility of the sterol-processing steps instead of the previously proposed sequential conversion. Compared with the wild type, the mutants showed altered resistances to different exogenous stresses regarding the specific growth rate and duration of lag phase. The erg5 deletion mutant whose sterol has a saturated side chain exhibited overall robust growth under the tested stress conditions. The thermotolerant phenotype of erg5 deletion mutant was reproduced in filamentous fungus Penicillium oxalicum. These results highlight the important role of sterols in the response of yeast cells to environmental stresses, and suggest the possibility of improving the robustness of industrial yeast strains by engineering their sterol composition.

Compromised epidermal barrier stimulates Harderian gland activity and hypertrophy in ACBP-/- mice.

Acyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed adaptation to weaning, including the induction of the hepatic lipogenic and cholesterogenic gene programs. Here we show that ACBP is highly expressed in the Harderian gland, a gland that is located behind the eyeball of rodents and involved in the production of fur lipids and lipids used for lubrication of the eye lid. We show that disruption of the Acbp gene leads to a significant enlargement of this gland with hypertrophy of the acinar cells and increased de novo synthesis of monoalkyl diacylglycerol, the main lipid species produced by the gland. Mice with conditional targeting of the Acbp gene in the epidermis recapitulate this phenotype, whereas generation of an artificial epidermal barrier during gland development reverses the phenotype. Our findings indicate that the Harderian gland is activated by the compromised epidermal barrier as an adaptive and protective mechanism to overcome the barrier defect.

Acyl-CoA binding protein and epidermal barrier function.

The acyl-CoA binding protein (ACBP) is a 10kDa intracellular protein expressed in all eukaryotic species and mammalian tissues investigated. It binds acyl-CoA esters with high specificity and affinity and is thought to act as an intracellular transporter of acyl-CoA esters between different enzymatic systems; however, the precise function remains unknown. ACBP is expressed at relatively high levels in the epidermis, particularly in the suprabasal layers, which are highly active in lipid synthesis. Targeted disruption of the ACBP gene in mice leads to a pronounced skin and fur phenotype, which includes tousled and greasy fur, development of alopecia and scaling of the skin with age. Furthermore, epidermal barrier function is compromised causing a ~50% increase in transepidermal water loss relative to that of wild type mice. Lipidomic analyses indicate that this is due to significantly reduced levels of non-esterified very long chain fatty acids in the stratum corneum of ACBP(-/-) mice. Here we review the current knowledge of ACBP with special focus on the function of ACBP in the epidermal barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

A novel role for central ACBP/DBI as a regulator of long-chain fatty acid metabolism in astrocytes.

Acyl-CoA-binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl-CoA esters. Several studies have suggested that ACBP acts as an acyl-CoA pool former and regulates long-chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam-Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABAA receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism-related gene expression using ACBP-deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA-CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes. Acyl-CoA-binding protein (ACBP) or diazepam-binding inhibitor is a secreted peptide acting centrally as a GABAA allosteric modulator. Using brain slices, cortical, and hypothalamic astrocyte cultures from ACBP KO mice, we demonstrate that ACBP mainly localizes in astrocytes and regulates unsaturated but not saturated long-chain fatty acids (LCFA) metabolism. In addition, ACBP deficiency alters FA metabolism-related genes and results in intracellular FA accumulation while affecting their release. Our results support a novel role for ACBP in brain lipid metabolism. FA, fatty acids; KO, knockout; PL, phospholipids; TAG, triacylglycerol.

Autophagy in the light of sphingolipid metabolism.

Maintenance of cellular homeostasis requires tight and coordinated control of numerous metabolic pathways, which are governed by interconnected networks of signaling pathways and energy-sensing regulators. Autophagy, a lysosomal degradation pathway by which the cell self-digests its own components, has over the past decade been recognized as an essential part of metabolism. Autophagy not only rids the cell of excessive or damaged organelles, misfolded proteins, and invading microorganisms, it also provides nutrients to maintain crucial cellular functions. Besides serving as essential structural moieties of biomembranes, lipids including sphingolipids are increasingly being recognized as central regulators of a number of important cellular processes, including autophagy. In the present review we describe how sphingolipids, with special emphasis on ceramides and sphingosine-1-phosphate, can act as physiological regulators of autophagy in relation to cellular and organismal growth, survival, and aging.

Quantitative proteomics by amino acid labeling in C. elegans.

We demonstrate labeling of Caenorhabditis elegans with heavy isotope-labeled lysine by feeding them with heavy isotope-labeled Escherichia coli. Using heavy isotope-labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi-mediated knockdown of the nuclear hormone receptor 49 in C. elegans. The combined use of quantitative proteomics and selective gene knockdown is a powerful tool for C. elegans biology.

Glucose- and nitrogen sensing and regulatory mechanisms in Saccharomyces cerevisiae.

Pro- and eukaryotic cells are constantly challenged by varying concentrations of nutrients in their environment. Perceiving and adapting to such changes are therefore crucial for cellular viability. Thus, numerous specialized cellular receptors continuously sense and react to the availability of nutrients such as glucose and nitrogen. When stimulated, these receptors initiate various cellular signaling pathways, which in concert constitute a complex regulatory network. To ensure a highly specific response, these pathways and networks cross-communicate with each other and are regulated at several steps and by numerous different regulators. As numerous of these regulating proteins, biochemical mechanisms, and cellular pathways are evolutionary conserved, complex biochemical information relevant to humans can be obtained by studying simple organisms. Thus, the yeast Saccharomyces cerevisiae has been recognized as a powerful model system to study fundamental biochemical processes. In the present review, we highlight central signaling pathways and molecular circuits conferring nitrogen- and glucose sensing in S. cerevisiae.

Assessment of the Inflammatory Effects of Gut Microbiota from Human Twins Discordant for Ulcerative Colitis on Germ-free Mice.

Disturbances in gut microbiota are prevalent in inflammatory bowel disease (IBD), which includes ulcerative colitis (UC). However, whether these disturbances contribute to development of the disease or are a result of the disease is unclear. In pairs of human twins discordant for IBD, the healthy twin has a higher risk of developing IBD and a gut microbiota that is more similar to that of IBD patients as compared with healthy individuals. Furthermore, appropriate medical treatment may mitigate these disturbances. To study the correlation between microbiota and IBD, we transferred stool samples from a discordant human twin pair: one twin being healthy and the other receiving treatment for UC. The stool samples were transferred from the disease-discordant twins to germ-free pregnant dams. Colitis was induced in the offspring using dextran sodium sulfate. As compared with offspring born to mice dams inoculated with stool from the healthy cotwin, offspring born to dams inoculated with stool from the UC-afflicted twin had a lower disease activity index, less gut inflammation, and a microbiota characterized by higher α diversity and a more antiinflammatory profile that included the presence and higher abundance of antiinflammatory species such as Akkermansia spp., Bacteroides spp., and Parabacteroides spp. These findings suggest that the microbiota from the healthy twin may have had greater inflammatory properties than did that of the twin undergoing UC treatment.

Pulmonary maternal immune activation does not cross the placenta but leads to fetal metabolic adaptation.

The fetal development of organs and functions is vulnerable to perturbation by maternal inflammation which may increase susceptibility to disorders after birth. Because it is not well understood how the placenta and fetus respond to acute lung- inflammation, we characterize the response to maternal pulmonary lipopolysaccharide exposure across 24 h in maternal and fetal organs using multi-omics, imaging and integrative analyses. Unlike maternal organs, which mount strong inflammatory immune responses, the placenta upregulates immuno-modulatory genes, in particular the IL-6 signaling suppressor Socs3. Similarly, we observe no immune response in the fetal liver, which instead displays metabolic changes, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. The maternal liver and plasma display similar metabolic alterations, potentially increasing bioavailability of docosahexaenoic acid for the mother and fetus. Thus, our integrated temporal analysis shows that systemic inflammation in the mother leads to a metabolic perturbation in the fetus.

Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia.

Pseudoexons are nonfunctional intronic sequences that can be activated by deep-intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The PCCA c.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder propionic acidemia by deficiency of the propionyl-CoA carboxylase enzyme encoded by PCCA and PCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. The PCCA c.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. Interestingly, the PCCA pseudoexon offers an unexploited potential to upregulate gene expression because healthy tissues show relatively high inclusion levels. By blocking inclusion of the nonactivated wild-type pseudoexon, we can increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels in not only patient fibroblasts harboring PCCA missense variants but also those harboring PCCB missense variants. This is a potential treatment strategy for propionic acidemia.

Regulation of lipid droplet size and phospholipid composition by stearoyl-CoA desaturase.

Fatty acid desaturation regulates membrane function and fat storage in animals. To determine the contribution of stearoyl-CoA desaturase (SCD) activity on fat storage and development in the nematode Caenorhabditis elegans, we analyzed the lipid composition and lipid droplet size in the fat-6;fat-7 desaturase mutants independently and in combination with mutants disrupted in conserved lipid metabolic pathways. C. elegans with impaired SCD activity displayed both reduced fat stores and decreased lipid droplet size. Mutants in the daf-2 (insulin-like growth factor receptor), rsks-1 (homolog of p70S6 kinase, an effector of the target of rapamycin signaling pathway), and daf-7 (transforming growth factor ß) displayed high fat stores, the opposite of the low fat observed in the fat-6;fat-7 desaturase mutants. The metabolic mutants in combination with fat-6;fat-7 displayed low fat stores, with the exception of the daf-2;fat-6;fat-7 triple mutants, which had increased de novo fatty acid synthesis and wild-type levels of fat stores. Notably, SCD activity is required for the formation of large-sized lipid droplets in all mutant backgrounds, as well as for normal ratios of phosphatidylcholine (PC) to phosphatidylethanolamine (PE). These studies reveal previously uncharacterized roles for SCD in the regulation of lipid droplet size and membrane phospholipid composition.