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Mitochondrial β-oxidation is the most prominent pathway for fatty acid oxidation but alternative oxidative metabolism exists. Fatty acid ω-oxidation is one of these pathways and forms dicarboxylic acids as products. These dicarboxylic acids are metabolized through peroxisomal β-oxidation representing an alternative pathway, which could potentially limit the toxic effects of fatty acid accumulation. Although dicarboxylic acid metabolism is highly active in liver and kidney, its role in physiology has not been explored in depth. In this review, we summarize the biochemical mechanism of the formation and degradation of dicarboxylic acids through ω- and β-oxidation, respectively. We will discuss the role of dicarboxylic acids in different (patho)physiological states with a particular focus on the role of the intermediates and products generated through peroxisomal β-oxidation. This review is expected to increase the understanding of dicarboxylic acid metabolism and spark future research.

Aims Cardiomyopathy and arrhythmias can be severe presentations in patients with inherited defects of mitochondrial long-chain fatty acid β-oxidation (FAO). The pathophysiological mechanisms that underlie these cardiac abnormalities remain largely unknown. We investigated the molecular adaptations to a FAO deficiency in the heart using the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse model. Methods and results We observed enrichment of amino acid metabolic pathways and of ATF4 target genes among the upregulated genes in the LCAD KO heart transcriptome. We also found a prominent activation of the eIF2α/ATF4 axis at the protein level that was independent of the feeding status, in addition to a reduction of cardiac protein synthesis during a short period of food withdrawal. These findings are consistent with an activation of the integrated stress response (ISR) in the LCAD KO mouse heart. Notably, charging of several transfer RNAs (tRNAs), such as tRNAGln was decreased in LCAD KO hearts, reflecting a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the ISR in the hearts of mice with muscle-specific deletion of carnitine palmitoyltransferase 2. Conclusions Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signalling, in particular the ISR. These results may serve as a foundation for investigating the role of the ISR in the cardiac pathology associated with long-chain FAO defects. Translational Perspective: The heart relies mainly on mitochondrial fatty acid β-oxidation (FAO) for its high energy requirements. The heart disease observed in patients with a genetic defect in this pathway highlights the importance of FAO for cardiac health. We show that the consequences of a FAO defect extend beyond cardiac energy homeostasis and include amino acid metabolism and associated signalling pathways such as the integrated stress response.

Malonyl-CoA-acyl carrier protein transacylase (MCAT) is an enzyme involved in mitochondrial fatty acid synthesis (mtFAS) and catalyzes the transfer of the malonyl moiety of malonyl-CoA to the mitochondrial acyl carrier protein (ACP). Previously, we showed that loss-of-function of mtFAS genes, includingMcat, is associated with severe loss of electron transport chain (ETC) complexes in mouse immortalized skeletal myoblasts (Nowinski et al., 2020). Here, we report a proband presenting with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Using whole exome sequencing, we identified biallelic variants inMCAT. Protein levels for NDUFB8 and COXII, subunits of complex I and IV respectively, were markedly reduced in lymphoblasts and fibroblasts, as well as SDHB for complex II in fibroblasts. ETC enzyme activities were decreased in parallel. Reexpression of wild-typeMCATrescued the phenotype in patient fibroblasts. This is the first report of a patient withMCATpathogenic variants and combined oxidative phosphorylation deficiency.

Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short-chain enoyl-CoA hydratase (ECHS1 or crotonase) deficiency, 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA) and methylmalonic aciduria (MMA). Isobutyryl-CoA dehydrogenase (ACAD8) and short/branched-chain acyl-CoA dehydrogenase (SBCAD,ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl-CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2-methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase, but not ACAD8. MCPA treatment of wild-type and PA HEK-293 cells caused a pronounced decrease in C3-carnitine. Furthermore, deletion ofACADSBin HEK-293 cells led to an equally strong decrease in C3-carnitine when compared to wild-type cells. Deletion ofECHS1in HEK-293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued byACAD8deletion. MCPA was able to rescue lipoylation inECHS1KO cells, but only in cells with priorACAD8deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK-293 cells for the isobutyryl-CoA substrate. Substrate promiscuity appeared less prominent for 2-methylbutyryl-CoA at least in HEK-293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further.

Background Proximal tubular (PT) cells are enriched in mitochondria and peroxisomes. Whereas mitochondrial fatty acid oxidation (FAO) plays an important role in kidney function by supporting the high-energy requirements of PT cells, the role of peroxisomal metabolism remains largely unknown. EHHADH, also known as L-bifunctional protein, catalyzes the second and third step of peroxisomal FAO. Methods We studied kidneys of WT and Ehhadh KO mice on a C57BL/6N background using histology, immunohistochemistry, immunofluorescence, immunoblot, RNA-sequencing, and metabolomics. To assess the role of androgens in the kidney phenotype of Ehhadh KO mice, mice underwent orchiectomy. Results We observed male-specific kidney hypertrophy and glomerular filtration rate reduction in adult Ehhadh KO mice. Transcriptome analysis unveiled a gene expression signature similar to PT injury in acute kidney injury mouse models. This was further illustrated by the presence of KIM-1 (kidney injury molecule-1), SOX-9, and Ki67-positive cells in the PT of male Ehhadh KO kidneys. Male Ehhadh KO kidneys had metabolite changes consistent with peroxisomal dysfunction as well as an elevation in glycosphingolipid levels. Orchiectomy of Ehhadh KO mice decreased the number of KIM-1 positive cells to WT levels. We revealed a pronounced sexual dimorphism in the expression of peroxisomal FAO proteins in mouse kidney, underlining a role of androgens in the kidney phenotype of Ehhadh KO mice. Conclusions Our data highlight the importance of EHHADH and peroxisomal metabolism in male kidney physiology and reveal peroxisomal FAO as a sexual dimorphic metabolic pathway in mouse kidneys.

The human 2-oxoadipate dehydrogenase complex (OADHc) in L-lysine catabolism is involved in the oxidative decarboxylation of 2-oxoadipate (OA) to glutaryl-CoA and NADH (+H+). Genetic findings have linked the DHTKD1 encoding 2-oxoadipate dehydrogenase (E1a), the first component of the OADHc, to pathogenesis of AMOXAD, eosinophilic esophagitis (EoE), and several neurodegenerative diseases. A multipronged approach, including circular dichroism spectroscopy, Fourier Transform Mass Spectrometry, and computational approaches, was applied to provide novel insight into the mechanism and functional versatility of the OADHc. The results demonstrate that E1a oxidizes a non-cognate substrate 2-oxopimelate (OP) as well as OA through the decarboxylation step, but the OADHc was 100-times less effective in reactions producing adipoyl-CoA and NADH from the dihydrolipoamide succinyltransferase (E2o) and dihydrolipoamide dehydrogenase (E3). The results revealed that the E2o is capable of producing succinyl-CoA, glutaryl-CoA, and adipoyl-CoA. The important conclusions are the identification of: (i) the functional promiscuity of E1a and (ii) the ability of the E2o to form acyl-CoA products derived from homologous 2-oxo acids with five, six, and even seven carbon atoms. The findings add to our understanding of both the OADHc function in the L-lysine degradative pathway and of the molecular mechanisms leading to the pathogenesis associated with DHTKD1 variants.

Lysine degradation via formation of saccharopine is a pathway confined to the mitochondria. The second pathway for lysine degradation, the pipecolic acid pathway, is not yet fully elucidated and known enzymes are localized in the mitochondria, cytosol and peroxisome. The tissue-specific roles of these two pathways are still under investigation. The lysine degradation pathway is clinically relevant due to the occurrence of two severe neurometabolic disorders, pyridoxine-dependent epilepsy (PDE) and glutaric aciduria type 1 (GA1). The existence of three other disorders affecting lysine degradation without apparent clinical consequences opens up the possibility to find alternative therapeutic strategies for PDE and GA1 through pathway modulation. A better understanding of the mechanisms, compartmentalization and interplay between the different enzymes and metabolites involved in lysine degradation is of utmost importance.

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by acute encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. We investigated the efficacy of substrate reduction through inhibition of 2-aminoadipic semialdehyde synthase (AASS), an enzyme upstream of the defective glutaryl-CoA dehydrogenase (GCDH), in a cell line and mouse model of GA1. We show that loss of AASS function in GCDH-deficient HEK-293 cells leads to an approximately fivefold reduction in the established GA1 clinical biomarker glutarylcarnitine. In the GA1 mouse model, deletion of Aass leads to a 4.3-, 3.8-, and 3.2-fold decrease in the glutaric acid levels in urine, brain, and liver, respectively. Parallel decreases were observed in urine and brain 3-hydroxyglutaric acid levels, and plasma, urine, and brain glutarylcarnitine levels. These in vivo data demonstrate that the saccharopine pathway is the main source of glutaric acid production in the brain and periphery of a mouse model for GA1, and support the notion that pharmacological inhibition of AASS may represent an attractive strategy to treat GA1.

2-Oxoadipate dehydrogenase (E1a, also known as DHTKD1, dehydrogenase E1, and transketolase domain-containing protein 1) is a thiamin diphosphate-dependent enzyme and part of the 2-oxoadipate dehydrogenase complex (OADHc) in l-lysine catabolism. Genetic findings have linked mutations in the DHTKD1 gene to several metabolic disorders. These include α-aminoadipic and α-ketoadipic aciduria (AMOXAD), a rare disorder of l-lysine, l-hydroxylysine, and l-tryptophan catabolism, associated with clinical presentations such as developmental delay, mild-to-severe intellectual disability, ataxia, epilepsy, and behavioral disorders that cannot currently be managed by available treatments. A heterozygous missense mutation, c.2185G→A (p.G729R), in DHTKD1 has been identified in most AMOXAD cases. Here, we report that the G729R E1a variant when assembled into OADHc in vitro displays a 50-fold decrease in catalytic efficiency for NADH production and a significantly reduced rate of glutaryl-CoA production by dihydrolipoamide succinyl-transferase (E2o). However, the G729R E1a substitution did not affect any of the three side-reactions associated solely with G729R E1a, prompting us to determine the structure–function effects of this mutation. A multipronged systematic analysis of the reaction rates in the OADHc pathway, supplemented with results from chemical cross-linking and hydrogen–deuterium exchange MS, revealed that the c.2185G→A DHTKD1 mutation affects E1a–E2o assembly, leading to impaired channeling of OADHc intermediates. Cross-linking between the C-terminal region of both E1a and G729R E1a with the E2o lipoyl and core domains suggested that correct positioning of the C-terminal E1a region is essential for the intermediate channeling. These findings may inform the development of interventions to counter the effects of pathogenic DHTKD1 mutations.

Secondary carnitine deficiency is commonly observed in inherited metabolic diseases characterized by the accumulation of acylcarnitines such as mitochondrial fatty acid oxidation (FAO) disorders. It is currently unclear if carnitine deficiency and/or acylcarnitine accumulation play a role in the pathophysiology of FAO disorders. The long-chain acyl-CoA dehydrogenase (LCAD) KO mouse is a model for long-chain FAO disorders and is characterized by decreased levels of tissue and plasma free carnitine. Tissue levels of carnitine are controlled by the SLC22A5, the plasmalemmal carnitine transporter. Here, we have further decreased carnitine availability in the LCAD KO mouse through a genetic intervention by introducing one defective Slc22a5 allele (jvs). Slc22a5 haploinsufficiency decreased free carnitine levels in liver, kidney and heart of LCAD KO animals. The resulting decrease in the tissue long-chain acylcarnitines levels had a similar magnitude as the decrease in free carnitine. Levels of cardiac deoxycarnitine, a carnitine biosynthesis intermediate, were elevated due to Slc22a5 haploinsufficiency in LCAD KO mice. A similar increase in heart and muscle deoxycarnitine was observed in an independent experiment using Slc22a5(jvs/jvs) mice. Cardiac hypertrophy, fasting-induced hypoglycemia and increased liver weight, the major phenotypes of the LCAD KO mouse, were not affected by Slc22a5 haploinsufficiency. This may suggest that secondary carnitine deficiency does not play a major role in the pathophysiology of these phenotypes. Similarly, our data do not support a major role for toxicity of long-chain acylcarnitines in the phenotype of the LCAD KO mouse.

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid.

Coronary atherosclerosis results from the delicate interplay of genetic and exogenous risk factors, principally taking place in metabolic organs and the arterial wall. Here we show that 224 gene-regulatory coexpression networks (GRNs) identified by integrating genetic and clinical data from patients with (n = 600) and without (n = 250) coronary artery disease (CAD) with RNA-seq data from seven disease-relevant tissues in the Stockholm–Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study largely capture this delicate interplay, explaining >54% of CAD heritability. Within 89 cross-tissue GRNs associated with clinical severity of CAD, 374 endocrine factors facilitated inter-organ interactions, primarily along an axis from adipose tissue to the liver (n = 152). This axis was independently replicated in genetically diverse mouse strains and by injection of recombinant forms of adipose endocrine factors (EPDR1, FCN2, FSTL3 and LBP) that markedly altered blood lipid and glucose levels in mice. Altogether, the STARNET database and the associated GRN browser (http://starnet.mssm.edu) provide a multiorgan framework for exploration of the molecular interplay between cardiometabolic disorders and CAD.

Peroxisomes metabolize a specific subset of fatty acids, which include dicarboxylic fatty acids (DCAs) generated by ω-oxidation. Data obtained in vitro suggest that the peroxisomal transporter ABCD3 (also known as PMP70) mediates the transport of DCAs into the peroxisome, but in vivo evidence to support this role is lacking. In this work, we studied an Abcd3 KO mouse model generated by CRISPR-Cas9 technology using targeted and untargeted metabolomics, histology, immunoblotting, and stable isotope tracing technology. We show that ABCD3 functions in hepatic DCA metabolism and uncover a novel role for this peroxisomal transporter in lipid homeostasis. The Abcd3 KO mouse presents with increased hepatic long-chain DCAs, increased urine medium-chain DCAs, lipodystrophy, enhanced hepatic cholesterol synthesis and decreased hepatic de novo lipogenesis. Moreover, our study suggests that DCAs are metabolized by mitochondrial fatty acid β-oxidation when ABCD3 is not functional, reflecting the importance of the metabolic compartmentalization and communication between peroxisomes and mitochondria. In summary, this study provides data on the role of the peroxisomal transporter ABCD3 in hepatic lipid homeostasis and DCA metabolism, and the consequences of peroxisomal dysfunction for the liver.

A key goal of geroscience research is to identify effective interventions to extend human healthspan, the years of healthy life. Currently, majority of the geroprotectors are found by screening compounds in model organisms; whether they will be effective in humans is largely unknown. Here we present a new strategy called ANDRU (aging network based drug discovery) to help the discovery of human geroprotectors. It first identifies human aging subnetworks that putatively function at the interface between aging and age-related diseases; it then screens for pharmacological interventions that may “reverse” the age-associated transcriptional changes occurred in these subnetworks. We applied ANDRU to human adipose gene expression data from the Genotype Tissue Expression (GTEx) project. For the top 31 identified compounds, 19 of them showed at least some evidence supporting their function in improving metabolic traits or lifespan, which include type 2 diabetes drugs such as pioglitazone. As the query aging genes were refined to the ones with more intimate links to diseases, ANDRU identified more meaningful drug hits than the general approach without considering the underlying network structures. In summary, ANDRU represents a promising human data-driven strategy that may speed up the discovery of interventions to extend human healthspan. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-019-00106-x) contains supplementary material, which is available to authorized users.

Peroxisomes metabolize a specific subset of fatty acids, which include dicarboxylic fatty acids (DCAs) generated by ω-oxidation. Data obtained in vitro suggest that the peroxisomal transporter ABCD3 (also known as PMP70) mediates the transport of DCAs into the peroxisome, but in vivo evidence to support this role is lacking. In this study, we studied an Abcd3 KO mouse model generated by CRISPR-Cas9 technology using targeted and untargeted metabolomics, histology, immunoblotting, and stable isotope tracing technology. We show that ABCD3 functions in DCA metabolism and uncover a novel role for this peroxisomal transporter in lipid metabolic homeostasis. The Abcd3 KO mouse presents with lipodystrophy, increased circulating free fatty acids, decreased ketone bodies, enhanced hepatic cholesterol synthesis and decreased hepatic de novo lipogenesis. Moreover, our study suggests that DCAs are metabolized by mitochondrial β-oxidation when ABCD3 is not functional, reflecting the importance of the metabolic compartmentalization and communication between peroxisomes and mitochondria. In summary, this study provides data on the role of the peroxisomal transporter ABCD3 in hepatic lipid homeostasis and DCA metabolism, and the consequences of peroxisomal dysfunction for the liver.

SUMMARYPluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARD activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs and a role for PPARD signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new and easily implemented opportunities to generate mature hPSC-CMs for disease modeling and regenerative therapy.

Peroxisomes play an essential role in the β-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA β-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA β-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA β-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA β-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA β-oxidation is a regulator of hepatic cholesterol biosynthesis.

Patients with a disorder of mitochondrial long-chain fatty acid β-oxidation (FAO) have reduced fasting tolerance and may present with hypoketotic hypoglycemia, hepatomegaly, (cardio)myopathy and rhabdomyolysis. Patients should avoid a catabolic state because it increases reliance on FAO as energy source. It is currently unclear whether weight loss through a reduction of caloric intake is safe in patients with a FAO disorder. We used the long-chain acyl-CoA dehydrogenase knockout (LCAD KO) mouse model to study the impact of dietary restriction (DR) on the plasma metabolite profile and cardiac function. For this, LCAD KO and wild type (WT) mice were subjected to DR (70% of ad libitum chow intake) for 4 weeks and compared to ad libitum chow fed mice. We found that DR had a relatively small impact on the plasma metabolite profile of WT and LCAD KO mice. Echocardiography revealed a small decrease in left ventricular systolic function of LCAD KO mice, which was most noticeable after DR, but there was no evidence of DR-induced cardiac remodeling. Our results suggest that weight loss through DR does not have acute and detrimental consequences in a mouse model for FAO disorders.

The heart relies mainly on mitochondrial fatty acid β-oxidation (FAO) for its high energy requirements. Cardiomyopathy and arrhythmias can be severe complications in patients with inherited defects in mitochondrial long-chain FAO, reinforcing the importance of FAO for cardiac health. However, the pathophysiological mechanisms that underlie the cardiac abnormalities in long-chain FAO disorders remain largely unknown. Here, we investigated the cardiac transcriptional adaptations to the FAO defect in the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. We found a prominent activation of the integrated stress response (ISR) mediated by the eIF2α/ATF4 axis in both fed and fasted states, accompanied by a reduction in cardiac protein synthesis during a short period of food withdrawal. Notably, we found an accumulation of uncharged tRNAs in LCAD KO hearts, consistent with a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the cardiac ISR in hearts of mice with a muscle-specific deletion of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signaling, in particular the ISR, and may play a role in the associated cardiac pathology.

Broad-scale adoption of genomic data in health systems offers opportunities for extending methods for the discovery of variation linked to underlying genomic disease risk. We applied a population-scale linkage mapping approach in a large multi-ethnic biobank to a spectrum of disease outcomes derived from Electronic Health Records (EHRs) and uncovered a risk locus for liver disease. We used genome sequencing and in silico approaches to fine-map the signal to a non-coding variant (c.2784-12T>C) in the gene ABCB4. In vitro analysis confirmed the variant disrupted splicing of the ABCB4 pre-mRNA. Four of five homozygotes had evidence of advanced liver disease, and there was a significant association with liver disease among heterozygotes, suggesting the variant is linked to increased risk of liver disease in an allele dose-dependent manner. Population-level screening revealed the variant to be at a carrier rate of 1.95% in Puerto Rican individuals, likely as the result of a Puerto Rican founder effect. This work demonstrates that integrating EHR and genomic data at a population-scale can facilitate novel strategies for understanding the continuum of genomic risk for common diseases, particularly in populations underrepresented in genomic medicine.

BackgroundProximal tubular (PT) cells are enriched in mitochondria and peroxisomes. Whereas mitochondrial fatty acid oxidation (FAO) plays an important role in kidney function by supporting the high-energy requirements of PT cells, the role of peroxisomal metabolism remains largely unknown. EHHADH, also known as L-bifunctional protein, catalyzes the second and third step of peroxisomal FAO.MethodsWe studied kidneys of WT andEhhadhKO mice using histology, immunohistochemistry, immunofluorescence, immunoblot, RNA-sequencing, metabolomics and orchiectomy.ResultsWe observed male-specific kidney hypertrophy and glomerular filtration rate reduction in adultEhhadhKO mice. Transcriptome analysis unveiled a gene expression signature similar to PT injury in acute kidney injury mouse models. This was further illustrated by the presence of KIM-1 (kidney injury molecule-1), SOX-9, and Ki67-positive cells in the PT of maleEhhadhKO kidneys. MaleEhhadhKO kidneys had metabolite changes consistent with peroxisomal dysfunction as well as an elevation in glycosphingolipids levels. Orchiectomy ofEhhadhKO mice reversed kidney enlargement and decreased the number of KIM-1 positive cells. We reveal a pronounced sexual dimorphism in the expression of peroxisomal FAO proteins in mouse kidney, underlining a role of androgens in the kidney phenotype ofEhhadhKO mice.ConclusionsOur data highlight the importance of EHHADH and peroxisomal metabolism in male kidney physiology and reveal peroxisomal FAO as a sexual dimorphic metabolic pathway in mouse kidneys.

Elevated plasma cholesterol and type 2 diabetes (T2D) are associated with coronary artery disease (CAD). Individuals treated with cholesterol-lowering statins have increased T2D risk, while individuals with hypercholesterolemia have reduced T2D risk. We explore the relationship between lipid and glucose control by constructing network models from the STARNET study with sequencing data from seven cardiometabolic tissues obtained from CAD patients during coronary artery by-pass grafting surgery. By integrating gene expression, genotype, metabolomic, and clinical data, we identify a glucose and lipid determining (GLD) regulatory network showing inverse relationships with lipid and glucose traits. Master regulators of the GLD network also impact lipid and glucose levels in inverse directions. Experimental inhibition of one of the GLD network master regulators, lanosterol synthase (LSS), in mice confirms the inverse relationships to glucose and lipid levels as predicted by our model and provides mechanistic insights., Some cholesterol-lowering drugs can increase the risk of type 2 diabetes, but the mechanism behind this is not fully understood. Here the authors show that there is a single genetic regulatory module that influences both cholesterol levels and glucose levels, providing a link between cholesterol levels and diabetes.

BackgroundNOD-like receptors (NLR) are intracellular sensors of the innate immune system, with the NLRP3 being a pro-inflammatory member that modulates cardiac ischemia-reperfusion injury (IRI) and metabolism. No information is available on a possible role of anti-inflammatory NLRs on IRI and metabolism in the intact heart. Here we hypothesize that the constitutively expressed, anti-inflammatory mitochondrial NLRX1, affects IRI and metabolism of the isolated mouse heart.MethodsIsolated C57Bl/6J and NLRX1 knock-out (KO) mouse hearts were perfused with a physiological mixture of the essential substrates (lactate, glucose, pyruvate, fatty acid, glutamine) and insulin. For the IRI studies, hearts were subjected to either mild (20 min) or severe (35 min) ischemia and IRI was determined at 60 min reperfusion. Inflammatory mediators (IL-6, TNFα) and survival pathways (mito-HKII, p-Akt, p-AMPK, p-STAT3) were analyzed at 5 min of reperfusion. For the metabolism studies, hearts were perfused for 35 min with either 5.5 mM 13C-glucose or 0.4 mM 13C-palmitate under normoxic conditions, followed by LC-MS analysis and integrated, stepwise, mass-isotopomeric flux analysis (MIMOSA).ResultsNLRX1 KO significantly increased IRI (infarct size from 63% to 73%, end-diastolic pressure from 59 mmHg to 75 mmHg, and rate-pressure-product recovery from 15% to 6%), following severe, but not mild, ischemia. The increased IRI in NLRX1 KO hearts was associated with depressed Akt signaling at early reperfusion; other survival pathways or inflammatory parameters were not affected. Metabolically, NLRX1 KO hearts displayed increased lactate production and glucose oxidation relative to fatty acid oxidation, associated with increased pyruvate dehydrogenase flux and 10% higher cardiac oxygen consumption.ConclusionDeletion of the mitochondrially-located NOD-like sensor NLRX1 exacerbates severe cardiac IR injury, possibly through impaired Akt signaling, and increases cardiac glucose metabolism.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

The glutaric acidurias are a group of inborn errors of metabolism with different etiologies. Glutaric aciduria type 3 (GA3) is a biochemical phenotype with uncertain clinical relevance caused by a deficiency of succinyl-CoA:glutarate-CoA transferase (SUGCT). SUGCT catalyzes the succinyl-CoA-dependent conversion of glutaric acid into glutaryl-CoA preventing urinary loss of the organic acid. Here, we describe the presence of a GA3 trait in mice of 129 substrains due to SUGCT deficiency, which was identified by screening of urine organic acid profiles obtained from different inbred mouse strains including 129S2/SvPasCrl. Molecular and biochemical analyses in an F2 population of the parental C57BL/6J and 129S2/SvPasCrl strains (B6129F2) confirmed that the GA3 trait occurred in Sugct129/129 animals. We evaluated the impact of SUGCT deficiency on metabolite accumulation in the glutaric aciduria type 1 (GA1) mouse model. We found that GA1 mice with SUGCT deficiency have decreased excretion of urine 3-hydroxyglutaric acid and decreased levels glutarylcarnitine in urine, plasma and kidney. Our work demonstrates that SUGCT contributes to the production of glutaryl-CoA under conditions of low and pathologically high glutaric acid levels. Our work also highlights the notion that unexpected biochemical phenotypes can occur in widely used inbred animal lines.Take home messageGlutaric aciduria type 3 is a naturally occurring trait in mice of the 129 substrains

DHTKD1 is the E1 component of the 2-oxoadipic acid dehydrogenase complex (OADHc), which functions in the L-lysine degradation pathway. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q (CMT2Q) and eosinophilic esophagitis (EoE). A crystal structure and inhibitors of DHTKD1 could improve the understanding of these clinically distinct disorders, but are currently not available. Here we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.1 Å. The protein assembles as a dimer with residues from both monomers contributing to cofactor binding. We also report the impact of ten DHTKD1 missense mutations on the encoded proteins by enzyme kinetics, thermal stability and structural modeling. Some DHTKD1 variants displayed impaired folding (S777P and S862I), whereas other substitutions rendered the enzyme inactive (L234G, R715C and R455Q) or affected the thermal stability and catalytic efficiency (V360A and P773L). Three variants (R163Q, Q305H and G729R) surprisingly showed wild type like properties. Our work provides a structural basis for further understanding of the function of DHTKD1 and a starting point for selective small molecule inhibitors of the enzyme, which could help tease apart the role of this enzyme in several human pathologies.

Background and Aims The presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within enterocytes. Methods Here, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation, and IBD treatment. Results A striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and nonbiologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. In addition, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD. Conclusions These data generate a novel appreciation of the confluence of COVID-19– and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19. Preprint doi: https://doi.org/10.1101/2020.05.21.109124

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.

Polygenic and environmental factors are underlying causes of inflammatory bowel disease (IBD). We hypothesized that integration of the genetic loci controlling a metabolite's abundance, with known IBD genetic susceptibility loci, may help resolve metabolic drivers of IBD.We measured the levels of 1300 metabolites in the serum of 484 patients with ulcerative colitis (UC) and 464 patients with Crohn's disease (CD) and 365 controls. Differential metabolite abundance was determined for disease status, subtype, clinical and endoscopic disease activity, as well as IBD phenotype including disease behavior, location, and extent. To inform on the genetic basis underlying metabolic diversity, we integrated metabolite and genomic data. Genetic colocalization and Mendelian randomization analyses were performed using known IBD risk loci to explore whether any metabolite was causally associated with IBD.We found 173 genetically controlled metabolites (metabolite quantitative trait loci, 9 novel) within 63 non-overlapping loci (7 novel). Furthermore, several metabolites significantly associated with IBD disease status and activity as defined using clinical and endoscopic indexes. This constitutes a resource for biomarker discovery and IBD biology insights. Using this resource, we show that a novel metabolite quantitative trait locus for serum butyrate levels containing ACADS was not supported as causal for IBD; replicate the association of serum omega-6 containing lipids with the fatty acid desaturase 1/2 locus and identify these metabolites as causal for CD through Mendelian randomization; and validate a novel association of serum plasmalogen and TMEM229B, which was predicted as causal for CD.An exploratory analysis combining genetics and unbiased serum metabolome surveys can reveal novel biomarkers of disease activity and potential mediators of pathology in IBD.

Objective The transcription factor Kruppel-like factor 14 (KLF14) has been associated with type 2 diabetes and high-density lipoprotein-cholesterol (HDL-C) through genome-wide association studies. The mechanistic underpinnings of KLF14's control of metabolic processes remain largely unknown. We studied the physiological roles of KLF14 in a knockout (KO) mouse model. Methods Male whole body Klf14 KO mice were fed a chow or high fat diet (HFD) and diet induced phenotypes were analyzed. Additionally, tissue-specific expression of Klf14 was determined using RT-PCR, RNA sequencing, immunoblotting and whole mount lacZ staining. Finally, the consequences of KLF14 loss-of-function were studied using RNA sequencing in tissues with relatively high Klf14 expression levels. Results KLF14 loss-of-function did not affect HFD-induced weight gain or insulin resistance. Fasting plasma concentrations of glucose, insulin, cholesterol, HDL-C and ApoA-I were also comparable between Klf14 +/+ and Klf14 −/− mice on chow and HFD. We found that in mice expression of Klf14 was the highest in the anterior pituitary (adenohypophysis), lower but detectable in white adipose tissue and undetectable in liver. Loss of KLF14 function impacted on the pituitary transcriptome with extracellular matrix organization as the primary affected pathway and a predicted link to glucocorticoid receptor signaling. Conclusions Whole body loss of KLF14 function in male mice does not result in metabolic abnormalities as assessed under chow and HFD conditions. Mostly likely there is redundancy for the role of KLF14 in the mouse and a diverging function in humans.

Background Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. Results In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the “sequence varying mRNAs” transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. Conclusions Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well. Electronic supplementary material The online version of this article (10.1186/s12864-017-4359-1) contains supplementary material, which is available to authorized users.

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322−10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

BACKGROUND: Hypoglycemia is an increasingly recognized complication of bariatric surgery. Mechanisms contributing to glucose lowering remain incompletely understood. We aimed to identify differentially abundant plasma proteins in patients with post-bariatric hypoglycemia (PBH) after roux-en-Y gastric bypass (RYGB), compared to asymptomatic post-RYGB. METHODS: Proteomic analysis of blood samples collected after overnight fast and mixed meal challenge in individuals with PBH, asymptomatic RYGB, severe obesity, or overweight recruited from outpatient hypoglycemia or bariatric clinics. RESULTS: The top-ranking differentially abundant protein at 120 minutes after mixed meal was fibroblast growth factor 19 (FGF-19), an intestinally-derived hormone regulated by bile acid/FXR signaling; levels were 2.4-fold higher in PBH vs. asymptomatic post-RYGB (mean ± SEM: 1094±141 vs. 428±45, P