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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.

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.

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.

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

SUMMARYGlutaric 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 ofDHTKD1in GCDH-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine, and demonstrate that OGDH is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, DLST and DLD to form a hybrid α-ketoglutaric and α-ketoadipic acid dehydrogenase complex. In summary, α-ketoadipic acid is an in vivo substrate for DHTKD1, but also OGDH. The classic α-ketoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards α-ketoadipic acid.

A key goal of geroscience research is to discover effective interventions to extend human healthspan, the years of healthy life. Currently, majority of the geroprotectors are found by testing compounds in model organisms; whether these compounds will be effective in humans is largely unknown. Here we present a novel strategy called ANDRU (aging network based drug discovery) to help the discovery of human geroprotectors. Instead of relying on model organisms, this approach is driven by human genomic and pharmacogenomic data. It first identifies human aging subnetworks that putatively function at the interface between aging and age-related diseases; it then screens for pharmacological or genetic interventions that may “reverse” the age-associated transcriptional changes seen in these subnetworks. We applied ANDRU to human adipose and artery tissues. In adipose tissue, PTPN1, a target for diabetes treatment and APOE, a known genetic factor for human longevity and diseases like Alzheimer’s disease, were ranked at the top. For small molecules, conjugated linoleic acid and metformin, a drug commonly used to treat type 2 diabetes, were ranked among the top compounds. In artery tissue, N-methyl-D-aspartate antagonists and curcumin were ranked at the top. In summary, ANDRU represents a promising human data-driven strategy that may speed up the discovery of interventions to extend human healthspan.

The small heterodimer partner (SHP) is an atypical nuclear receptor known mainly for its role in bile acid homeostasis in the enterohepatic tract. We explore here the role of SHP in the testis. SHP is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. SHP there inhibits the expression of steroidogenic genes, on the one hand by inhibiting the expression of the nuclear receptors steroidogenic factor-1 and liver receptor homolog-1 (lrh-1), and on the other hand by directly repressing the transcriptional activity of LRH-1. Consequently, in SHP knockout mice, testicular testosterone synthesis is increased independently of the hypothalamus–pituitary axis. Independent of its action on androgen synthesis, SHP also determines the timing of germ cell differentiation by controlling testicular retinoic acid metabolism. Through the inhibition of the transcriptional activity of retinoic acid receptors, SHP controls the expression of stimulated by retinoic acid gene 8 (stra8)—a gene that is indispensable for germ cell meiosis and differentiation. Together, our data demonstrate new roles for SHP in testicular androgen and retinoic acid metabolism, making SHP a testicular gatekeeper of the timing of male sexual maturation.