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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.

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.

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.

Aims/hypothesis: Changes in cardiac metabolism and ion homeostasis precede and drive cardiac remodeling and heart failure development. We previously demonstrated that sodium/glucose cotransporter 2 inhibitors (SGLT2i's) have direct cardiac effects on ion homeostasis, possibly through inhibition of the cardiac sodium/hydrogen exchanger (NHE-1). Here, we hypothesize that Empagliflozin (EMPA) also possesses direct and acute cardiac effects on glucose and fatty acid metabolism of isolated type II diabetes mellitus (db/db) mouse hearts. In addition, we explore whether direct effects on glucose metabolism are nullified in the presence of an NHE-1 inhibitor.Methods: Langendorff-perfused type II diabetic db/db mouse hearts were examined in three different series: 1: 13C glucose perfusions (n = 32); 2: 13C palmitate perfusions (n = 13); and 3: 13C glucose + 10 μM Cariporide (specific NHE-1 inhibitor) perfusions (n = 17). Within each series, EMPA treated hearts (1 μM EMPA) were compared with vehicle-perfused hearts (0.02% DMSO). Afterwards, hearts were snap frozen and lysed for stable isotope analysis and metabolomics using LC-MS techniques. Hearts from series 1 were also analyzed for phosphorylation status of AKT, STAT3, AMPK, ERK, and eNOS (n = 8 per group).Results: Cardiac mechanical performance, oxygen consumption and protein phosphorylation were not altered by 35 min EMPA treatment. EMPA was without an overall acute and direct effect on glucose or fatty acid metabolism. However, EMPA did specifically decrease cardiac lactate labeling in the 13C glucose perfusions (13C labeling of lactate: 58 ± 2% vs. 50 ± 3%, for vehicle and EMPA, respectively; P = 0.02), without changes in other glucose metabolic pathways. In contrast, EMPA increased cardiac labeling in α-ketoglutarate derived from 13C palmitate perfusions (13C labeling of α-KG: 79 ± 1% vs. 86 ± 1% for vehicle and EMPA, respectively; P = 0.01). Inhibition of the NHE by Cariporide abolished EMPA effects on lactate labeling from 13C glucose.Conclusions: The present study shows for the first time that the SGLT2 inhibitor Empagliflozin has acute specific metabolic effects in isolated diabetic hearts, i.e., decreased lactate generation from labeled glucose and increased α-ketoglutarate synthesis from labeled palmitate. The decreased lactate generation by EMPA seems to be mediated through NHE-1 inhibition.

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

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.

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

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.

Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid β-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4. ispartof: FASEB JOURNAL vol:33 issue:3 pages:4355-4364 ispartof: location:United States status: published

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.

Aberrant mitochondrial fission plays a pivotal role in the pathogenesis of skeletal muscle insulin resistance. However, fusion-fission dynamics are physiologically regulated by inherent tissue-specific and nutrient-sensitive processes that may have distinct or even opposing effects with respect to insulin sensitivity. Based on a combination of mouse population genetics and functional in vitro assays, we describe here a regulatory circuit in which peroxisome proliferator–activated receptor γ (PPARγ), the adipocyte master regulator and receptor for the thiazolidinedione class of antidiabetic drugs, controls mitochondrial network fragmentation through transcriptional induction of Bnip3. Short hairpin RNA–mediated knockdown of Bnip3 in cultured adipocytes shifts the balance toward mitochondrial elongation, leading to compromised respiratory capacity, heightened fatty acid β-oxidation-associated mitochondrial reactive oxygen species generation, insulin resistance, and reduced triacylglycerol storage. Notably, the selective fission/Drp1 inhibitor Mdivi-1 mimics the effects of Bnip3 knockdown on adipose mitochondrial bioenergetics and glucose disposal. We further show that Bnip3 is reciprocally regulated in white and brown fat depots of diet-induced obesity and leptin-deficient ob/ob mouse models. Finally, Bnip3−/− mice trade reduced adiposity for increased liver steatosis and develop aggravated systemic insulin resistance in response to high-fat feeding. Together, our data outline Bnip3 as a key effector of PPARγ-mediated adipose mitochondrial network fragmentation, improving insulin sensitivity and limiting oxidative stress.

Mitochondrial fatty acid β-oxidation (FAO) is the major pathway for the degradation of fatty acids and is essential for maintaining energy homeostasis in the human body. Fatty acids are a crucial energy source in the postabsorptive and fasted states when glucose supply is limiting. But even when glucose is abundantly available, FAO is a main energy source for the heart, skeletal muscle, and kidney. A series of enzymes, transporters, and other facilitating proteins are involved in FAO. Recessively inherited defects are known for most of the genes encoding these proteins. The clinical presentation of these disorders may include hypoketotic hypoglycemia, (cardio)myopathy, arrhythmia, and rhabdomyolysis and illustrates the importance of FAO during fasting and in hepatic and (cardio)muscular function. In this review, we present the current state of knowledge on the biochemistry and physiological functions of FAO and discuss the pathophysiological processes associated with FAO disorders.

Aims Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism. Methods and results We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis. Conclusions Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.

Zellweger spectrum disorders (ZSDs) are autosomal recessive diseases caused by defective peroxisome assembly. They constitute a clinical continuum from severe early lethal to relatively milder presentations in adulthood. Liver disease is a prevalent symptom in ZSD patients. The underlying pathogenesis for the liver disease, however, is not fully understood. We report a hypomorphic ZSD mouse model, which is homozygous for Pex1-c.2531G>A (p.G844D), the equivalent of the most common pathogenic variant found in ZSD, and which predominantly presents with liver disease. After introducing the Pex1-G844D allele by knock-in, we characterized homozygous Pex1-G844D mice for survival, biochemical parameters, including peroxisomal and mitochondrial functions, organ histology, and developmental parameters. The first 20 post-natal days (P20) were critical for survival of homozygous Pex1-G844D mice (~20% survival rate). Lethality was likely due to a combination of cholestatic liver problems, liver dysfunction and caloric deficit, probably as a consequence of defective bile acid biosynthesis. Survival beyond P20 was nearly 100%, but surviving mice showed a marked delay in growth. Surviving mice showed similar hepatic problems as described for mild ZSD patients, including hepatomegaly, bile duct proliferation, liver fibrosis and mitochondrial alterations. Biochemical analyses of various tissues showed the absence of functional peroxisomes accompanied with aberrant levels of peroxisomal metabolites predominantly in the liver, while other tissues were relatively spared. ur findings show that homozygous Pex1-G844D mice have a predominant liver disease phenotype, mimicking the hepatic pathology of ZSD patients, and thus constitute a good model to study pathogenesis and treatment of liver disease in ZSD patients. ispartof: BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE vol:1865 issue:10 pages:2774-2787 ispartof: location:Netherlands status: published

Aim: Acylcarnitines are fatty acid oxidation (FAO) intermediates, which have been implicated in diet-induced insulin resistance. Elevated acylcarnitine levels are found in obese, insulin resistant humans and rodents, and coincide with lower free carnitine. We hypothesized that increasing free carnitine levels by administration of the carnitine precursor gamma-butyrobetaine (gamma BB) could facilitate FAO, thereby improving insulin sensitivity. Methods: C57BL/6N mice were fed with a high fat or chow diet with or without gamma BB supplementation (n = 10 per group). After 8 weeks of diet, indirect calorimetry, glucose tolerance and insulin sensitivity tests were performed. AC profiles and carnitine biosynthesis intermediates were analyzed in plasma and tissues by tandem mass spectrometry (MS) and liquid chromatography tandem MS. Results: gamma BB supplementation did not facilitate FAO, was unable to curb bodyweight and did not prevent impaired glucose homeostasis in the HFD fed mice in spite of marked alterations in the acylcarnitine profiles in plasma and liver. Remarkably, gamma BB did not affect the acylcarnitine profile in other tissues, most notably muscle. Administration of a bolus acetylcarnitine also caused significant changes in plasma and liver, but not in muscle acylcarnitine profiles, again without effect on glucose tolerance. Conclusion: Altogether, increasing carnitine availability affects acylcarnitine profiles in plasma and liver but does not modulate glucose tolerance or insulin sensitivity. This may be due to the lack of an effect on muscle acylcarnitine profiles, as muscle tissue is an important contributor to whole body insulin sensitivity. These results warrant caution on making associations between plasma acylcarnitine levels and insulin resistance. (C) 2016 Elsevier B.V. All rights reserved

Background & Aims: Severe malnutrition in young children is associated with signs of hepatic dysfunction such as steatosis and hypoalbuminemia, but its etiology is unknown. Peroxisomes and mitochondria play key roles in various hepatic metabolic functions including lipid metabolism and energy production. To investigate the involvement of these organelles in the mechanisms underlying malnutrition-induced hepatic dysfunction we developed a rat model of malnutrition.Methods: Weanling rats were placed on a low protein or control diet (5% or 20% of calories from protein, respectively) for four weeks. Peroxisomal and mitochondrial structural features were characterized using immunofluorescence and electron microscopy. Mitochondrial function was assessed using high resolution respirometry. A novel targeted quantitative proteomics method was applied to analyze 47 mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle and fatty acid beta-oxidation pathways.Results: Low protein diet-fed rats developed hypoalbuminemia and hepatic steatosis, consistent with the human phenotype. Hepatic peroxisome content was decreased and metabolomic analysis indicated peroxisomal dysfunction. This was followed by changes in mitochondrial ultrastructure and increased mitochondrial content. Mitochondrial function was impaired due to multiple defects affecting respiratory chain complex I and IV, pyruvate uptake and several 13-oxidation enzymes, leading to strongly reduced hepatic ATP levels. Fenofibrate supplementation restored hepatic peroxisome abundance and increased mitochondrial beta-oxidation capacity, resulting in reduced steatosis and normalization of ATP and plasma albumin levels.Conclusions: Malnutrition leads to severe impairments in hepatic peroxisomal and mitochondrial function, and hepatic metabolic dysfunction. We discuss the potential future implications of our findings for the clinical management of malnourished children.Lay summary: Severe malnutrition in children is associated with metabolic disturbances that are poorly understood. In order to study this further, we developed a malnutrition animal model and found that severe malnutrition leads to an impaired function of liver mitochondria which are essential for energy production and a loss of peroxisomes, which are important for normal liver metabolic function. (C) 2016 European Association for the Study of the Liver. Published by Elsevier B.V.

(31)P MRS provides a unique non-invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of (31)P MRS in the in vivo mouse heart has been limited. The small-sized, fast-beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three-dimensional image-selected in vivo spectroscopy (3D ISIS) for localized (31)P MRS of the in vivo mouse heart at 9.4 T. Cardiac (31)P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory-gated, cardiac-triggered 3D ISIS. Localization and potential signal contamination were assessed with (31)P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5'-triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one-dimensional (1D) ISIS resulted in two-fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo (31)P MRS were within the normal physiological range. Our results show that respiratory-gated, cardiac-triggered 3D ISIS allows for non-invasive assessments of in vivo mouse myocardial energy homeostasis with (31)P MRS under physiological conditions.

Alpha-aminoadipic and alpha-ketoadipic aciduria is an autosomal recessive inborn error of lysine, hydroxylysine, and tryptophan degradation. To date, DHTKD1 mutations have been reported in two alpha-aminoadipic and alpha-ketoadipic aciduria patients. We have now sequenced DHTKD1 in nine patients diagnosed with alpha-aminoadipic and alpha-ketoadipic aciduria as well as one patient with isolated alpha-aminoadipic aciduria, and identified causal mutations in eight. We report nine novel mutations, including three missense mutations, two nonsense mutations, two splice donor mutations, one duplication, and one deletion and insertion. Two missense mutations, one of which was reported before, were observed in the majority of cases. The clinical presentation of this group of patients was inhomogeneous. Our results confirm that alpha-aminoadipic and alpha-ketoadipic aciduria is caused by mutations in DHTKD1, and further establish that DHTKD1 encodes the E1 subunit of the alpha-ketoadipic acid dehydrogenase complex.

High-fat diets (HFDs) have been shown to interfere with skeletal muscle energy metabolism and cause peripheral insulin resistance. However, understanding of HFD impact on skeletal muscle primary function, i.e., contractile performance, is limited. Male C57BL/6J mice were fed HFD containing lard (HFL) or palm oil (HFP), or low-fat diet (LED) for 5 weeks. Fast-twitch (FT) extensor digitorum Iongus (EDL) and slow-twitch (ST) soleus muscles were characterized with respect to contractile function and selected biochemical features. In EDL muscle, a 30%-50% increase in fatty acid (FA) content and doubling of long-chain acylcarnitine (C14-C18) content in response to HFL and HFP feeding were accompanied by increase in protein levels of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, mitochondrial oxidative phosphorylation complexes and acyl-CoA dehydrogenases involved in mitochondrial FA beta-oxidation. Peak force of FT EDL twitch and tetanic contractions was unaltered, but the relaxation time (RT) of twitch contractions was 30% slower compared to LFD controls. The latter was caused by accumulation of lipid intermediates rather than changes in the expression levels of proteins involved in calcium handling. In ST soleus muscle, no evidence for lipid overload was found in any HFD group. However, particularly in HFP group, the peak force of twitch and tetanic contractions was reduced, but RT was faster than LFD controls. The latter was associated with a fast-to-slow shift in troponin T isoform expression. Taken together, these data highlight fiber-type-specific sensitivities and phenotypic adaptations to dietary lipid overload that differentially impact fast- versus slow-twitch skeletal muscle contractile function. (C) 2015 Elsevier Inc. All rights reserved.

Purpose: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid beta-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may-present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is -unpredictable. We investigated predictive markers that may determine clinical severity. Methods: We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading. Results: The strongest correlation (r = 0.93; P

Aim To determine the effect of pioglitazone treatment on in vivo and ex vivo muscle mitochondrial function in a rat model of diabetes. Methods Both the lean, healthy rats and the obese, diabetic rats are Zucker Diabetic Fatty (ZDF) rats. The homozygous fa/fa ZDF rats are obese and diabetic. The heterozygous fa/+ ZDF rats are lean and healthy. Diabetic Zucker Diabetic Fatty rats were treated with either pioglitazone (30 mg/kg/day) or water as a control (n = 6 per group), for 2 weeks. In vivo 1H and 31P magnetic resonance spectroscopy was performed on skeletal muscle to assess intramyocellular lipid (IMCL) content and muscle oxidative capacity, respectively. Ex vivo muscle mitochondrial respiratory capacity was evaluated using high-resolution respirometry. In addition, several markers of mitochondrial content were determined. Results IMCL content was 14-fold higher and in vivo muscle oxidative capacity was 26% lower in diabetic rats compared with lean rats, which was, however, not caused by impairments of ex vivo mitochondrial respiratory capacity or a lower mitochondrial content. Pioglitazone treatment restored in vivo muscle oxidative capacity in diabetic rats to the level of lean controls. This amelioration was not accompanied by an increase in mitochondrial content or ex vivo mitochondrial respiratory capacity, but rather was paralleled by an improvement in lipid homeostasis, that is lowering of plasma triglycerides and muscle lipid and long-chain acylcarnitine content. Conclusion Diminished in vivo muscle oxidative capacity in diabetic rats results from mitochondrial lipid overload and can be alleviated by redirecting the lipids from the muscle into adipose tissue using pioglitazone treatment.

Saccharopine, a nonproteinogenic amino acid originally isolated from the yeast Saccharomyces cerevisiae, is an intermediate in lysine metabolism. In this issue, Zhou et al. (2019. J. Cell Biol. https://doi.org./10.1083/jcb.201807204) show that abnormal accumulation of saccharopine results in defective mitochondrial dynamics and function in worm and mouse models.

The hallmark of nonalcoholic fatty liver disease is steatosis of unknown etiology. To test how dietary protein decreases steatosis, we fed female C57BL/6 J mice low-fat (8 en%) or high-fat (42 en%) combined with low-protein (11 en%), high-protein (HP; 35 en%) or extra-high-protein (HPX; 58 en%) diets for 3 weeks. The 35 en% protein diets reduced hepatic triglyceride, free fatty acid, cholesterol and phospholipid contents to ~50% of that in 11 en% protein diets. Every additional 10 en% protein reduced hepatic fat content ~1.5 g%. HP diets had no effect on lipogenic or fatty acid-oxidizing genes except Ppargc1alpha (+30%), increased hepatic PCK1 content 3- to 5-fold, left plasma glucose and hepatic glycogen concentration unchanged, and decreased inflammation and cell stress (decreased Fgf21 and increased Gsta expression). The HP-mediated decrease in steatosis correlated inversely with plasma branched-chain amino-acid (BCAA) concentrations and hepatic content of BCAA-derived monomethyl branched-chain fatty acids (mmBCFAs) 14-methylpentadecanoic (14-MPDA; valine-derived) and, to a lesser extent, 14-methylhexadecanoic acid (isoleucine-derived). Liver lipid content was 1.6- to 1.8-fold higher in females than in males, but the anti-steatotic effect of HP diets was equally strong. The strong up-regulation of PCK1 and literature data showing an increase in phosphoenolpyruvate and a decline in tricarboxylic acid cycle intermediates in liver reveal that an increased efflux of these intermediates from mitochondria represents an important effect of an HP diet. The HP diet-induced increase in 14-MPDA and the dietary response in gene expression were more pronounced in females than males. Our findings are compatible with a facilitating role of valine-derived mmBCFAs in the antisteatotic effect of HP diets.

Purpose Proton magnetic resonance spectroscopy (1H-MRS) for quantitative in vivo assessment of mouse myocardial metabolism requires accurate acquisition timing to minimize motion artifacts and corrections for T1-dependent partial saturation effects. In this study, mouse myocardial water and metabolite T1 relaxation time constants were quantified. Methods Cardiac-triggered and respiratory-gated PRESS-localized 1H-MRS was employed at 9.4 T to acquire signal from a 4-µL voxel in the septum of healthy mice (n = 10) while maintaining a steady state of magnetization using dummy scans during respiratory gates. Signal stability was assessed via standard deviations (SD) of zero-order phases and amplitudes of water spectra. Saturation-recovery experiments were performed to determine T1 values. Results Phase SD did not vary for different repetition times (TR), and was 13.1° ± 4.5°. Maximal amplitude SD was 14.2% ± 5.1% at TR = 500 ms. Myocardial T1 values (mean ± SD) were quantified for water (1.71 ± 0.25 s), taurine (2.18 ± 0.62 s), trimethylamine from choline-containing compounds and carnitine (1.67 ± 0.25 s), creatine-methyl (1.34 ± 0.19 s), triglyceride-methylene (0.60 ± 0.15 s), and triglyceride-methyl (0.90 ± 0.17 s) protons. Conclusion This work provides in vivo quantifications of proton T1 values for mouse myocardial water and metabolites at 9.4 T. Magn Reson Med 73:2069–2074, 2015. © 2014 Wiley Periodicals, Inc.

The role of anesthetics in animal research models is crucial, yet often ignored, and is almost never the primary focus of examination. Here, we investigated the impact of anesthetic regimens on different parameters of hemodynamics (blood pressure (BP) and heart rate (HR)), metabolism (glucose, insulin, and free fatty acids (FFA)), and inflammation (IL-6 and TNF-α) in two frequently used mouse strains (C57BL/6 and FVB). All animals were at a similar surgical plane of anesthesia, mechanically ventilated, and monitored for 60 min. The following anesthetic regimens were studied: (1) fentanyl–ketamine–midazolam (FKM), (2) fentanyl–midazolam–haldol (FMH), (3) pentobarbital (P), (4) fentanyl–fluanisone–midazolam (FFM), (5) fentanyl–midazolam–acepromazine (FMA), (6) ketamine–medetomidine–atropine (KMA), (7) isoflurane (ISO), and (8) propofol–fentanyl–midazolam (PFM). Metabolic and inflammatory parameters were compared with those obtained from non-anesthetized animals. Hemodynamics: BP >80 mm Hg were only obtained with KMA, whereas hypotension (BP 500 beats/min was observed with ISO and PFM, whereas HR

Contains fulltext : 202680.pdf (Publisher’s version ) (Closed access) Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Acylcarnitines have been linked to obesity-induced insulin resistance. However the majority of these studies have focused on acylcarnitines in plasma. It is currently unclear to what extent plasma levels of acylcarnitines reflect tissue acylcarnitine metabolism. We investigated the correlation of plasma acylcarnitine levels with selected tissue acylcarnitines as measured with tandem mass spectrometry, in both fed and fasted BALB/cJ (BALB) and C57BL/6N (Bl6) mice. Fasting affected acylcarnitine levels in all tissues. These changes varied substantially between the different tissue compartments. No significant correlations were found between plasma acylcarnitine species and their tissue counterparts in both mouse strains, with the exception of plasma C4OH-carnitine in BALB mice. We suggest that this lack of correlation is due to differences in acylcarnitine turnover rates between plasma and tissue compartments and the fact that the plasma acylcarnitine profile is a composition of acylcarnitines derived from different compartments. Therefore, plasma acylcarnitine levels do not reflect tissue levels and should be interpreted with caution. A focus on tissue acylcarnitine levels is warranted in metabolic studies.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inherited disorder of mitochondrial long-chain fatty acid β-oxidation (FAO). Patients with VLCAD deficiency may present with hypoglycemia, hepatomegaly, cardiomyopathy, and myopathy. Although several mouse models have been developed to aid in the study of the pathogenesis of long-chain FAO defects, the muscular phenotype is underexposed. To address the muscular phenotype, we used a newly developed mouse model on a mixed genetic background with a more severe defect in FAO (LCAD(-/-); VLCAD(+/-)) in addition to a validated mouse model (LCAD(-/-); VLCAD(+/+)) and compared them with wild-type (WT) mice. We found that both mouse models show a 20% reduction in energy expenditure (EE) and a 3-fold decrease in locomotor activity in the unfed state. In addition, we found a 1.7°C drop in body temperature in unfed LCAD(-/-); VLCAD(+/+) mice compared with WT body temperature. We conclude that food withdrawal-induced inactivity, hypothermia, and reduction in EE are novel phenotypes associated with FAO deficiency in mice. Unexpectedly, inactivity was not explained by rhabdomyolysis, but rather reflected the overall reduced capacity of these mice to generate heat. We suggest that mice are partly protected against the negative consequence of an FAO defect.-Diekman, E. F., van Weeghel, M., Wanders, R. J. A., Visser, G., Houten, S. M. Food withdrawal lowers energy expenditure and induces inactivity in long-chain fatty acid oxidation-deficient mouse models.

Dienoyl-CoA reductase (DECR) deficiency with hyperlysinemia is a rare disorder affecting the metabolism of polyunsaturated fatty acids and lysine. The molecular basis of this condition is currently unknown. We describe a new case with failure to thrive, developmental delay, lactic acidosis and severe encephalopathy suggestive of a mitochondrial disorder. Exome sequencing revealed a causal mutation in NADK2. NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts. DECR and also the first step in lysine degradation are performed by NADP-dependent oxidoreductases explaining their in vivo deficiency. DECR activity was also deficient in lysates of patient fibroblasts and could only be rescued by transfecting patient cells with functional NADK2. Thus NADPH is not only crucial as a cosubstrate, but can also act as a molecular chaperone that activates and stabilizes enzymes. In addition to polyunsaturated fatty acid oxidation and lysine degradation, NADPH also plays a role in various other mitochondrial processes. We found decreased oxygen consumption and increased extracellular acidification in patient fibroblasts, which may explain why the disease course is consistent with clinical criteria for a mitochondrial disorder. We conclude that DECR deficiency with hyperlysinemia is caused by mitochondrial NADP(H) deficiency due to a mutation in NADK2.

Objective Cardiac hexokinase II (HKII) can translocate between cytosol and mitochondria and change its cellular expression with pathologies such as ischemia–reperfusion, diabetes and heart failure. The cardiac metabolic consequences of these changes are unknown. Here we measured energy substrate utilization in cytosol and mitochondria using stabile isotopes and oxygen consumption of the intact perfused heart for 1) an acute decrease in mitochondrial HKII (mtHKII), and 2) a chronic decrease in total cellular HKII. Methods/results We first examined effects of 200 nM TAT (Trans-Activator of Transcription)-HKII peptide treatment, which was previously shown to acutely decrease mtHKII by ~ 30%. In Langendorff-perfused hearts TAT-HKII resulted in a modest, but significant, increased oxygen consumption, while cardiac performance was unchanged. At the metabolic level, there was a nonsignificant (p = 0.076) ~ 40% decrease in glucose contribution to pyruvate and lactate formation through glycolysis and to mitochondrial citrate synthase flux (6.6 ± 1.1 vs. 11.2 ± 2.2%), and an 35% increase in tissue pyruvate (27 ± 2 vs. 20 ± 2 pmol/mg; p = 0.033). Secondly, we compared WT and HKII+/− hearts (50% chronic decrease in total HKII). RNA sequencing revealed no differential gene expression between WT and HKII+/− hearts indicating an absence of metabolic reprogramming at the transcriptional level. Langendorff-perfused hearts showed no significant differences in glycolysis (0.34 ± 0.03 μmol/min), glucose contribution to citrate synthase flux (35 ± 2.3%), palmitate contribution to citrate synthase flux (20 ± 1.1%), oxygen consumption or mechanical performance between WT and HKII+/− hearts. Conclusions These results indicate that acute albeit not chronic changes in mitochondrial HKII modestly affect cardiac oxygen consumption and energy substrate metabolism., final draft, peerReviewed

The hallmark of NAFLD is steatosis of unknown etiology. We tested the effect of a high-protein (HP)22AA: arachidonic acid; Acac: acetyl-CoA carboxylase; Acox: acyl-coenzyme A oxidase; Akt: thymoma viral proto-oncogene 1 (a.k.a. PKB-protein kinase B); Alas1: aminolevulinic acid synthase 1; Arntl (a.k.a. Bmal1): aryl hydrocarbon receptor nuclear translocator-like protein; BCAA: branched-chain amino acids; BHB: β-hydroxybutyrate; ITGAM: Integrin-αM; Chop/Ddit3: C/EBP-homologous protein/DNA damage-inducible transcript 3 protein; Chrebp: carbohydrate-responsive element-binding protein; Cpt1: carnitine palmitoyltransferase 1; DHA: docosahexaenoic acid; eIF2α: eukaryotic translation–initiation factor 2α; Elovl: elongation of very long-chain fatty acids; en%: energy percent; EPA: eicosapentaenoic acid; Fasn: fatty acid synthase; Fgf21: fibroblast growth factor 21; FFA: free fatty acids; G6Pase: glucose-6-phosphatase; GCN2: general control nonrepressed 2; HF: high fat; HF/LPres: high fat, low protein restricted; HP: high protein; LF: low fat; LP: low protein; Mcp1: monocyte chemotactic protein 1; Mlxipl (a.k.a. Chrebp): Mlx-interacting protein-like; mAco: mitochondrial aconitase; mmBCFA: monomethyl branched-chain fatty acid; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; Nfil3: nuclear factor interleukin-3-regulated protein; NFκB: nuclear factor κB; Nr1d1 (a.k.a. rev-erbα): nuclear receptor subfamily 1, group D, member 1; PCK1: phosphoenolpyruvate carboxykinase 1; PERK: protein kinase RNA-like endoplasmic reticulum kinase; Pgc1α: pparγ-coactivator1-α, PL: choline-containing phospholipids; Ppar: peroxisome proliferator-activated receptor; rs: Spearman correlation coefficient; Scd1: stearoyl-CoA desaturase 1; Srebf: sterol regulatory element-binding transcription factor; TC: total cholesterol; TG: triglycerides. diet on diet-induced steatosis in male C57BL/6 mice with and without pre-existing fatty liver. Mice were fed all combinations of semisynthetic low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for 3weeks. To control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP group was included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets decreased hepatic lipids to ~40% of corresponding LP-containing diets, were more efficient in this respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP-containing diets showed increased mitochondrial oxidative capacity (elevated Pgc1α, mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C0]/[C16+C18] carnitine ratio); increased gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma–glucose concentration without increased G6pase mRNA); reduced fatty-acid desaturation (decreased Scd1 expression and [C16:1n−7]/[C16:0] ratio) and increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced phosphorylated eIF2α, and Fgf21 and Chop expression); and a trend toward less inflammation (lower Mcp1 and Cd11b expression and less phosphorylated NFκB). Conclusion: HP diets prevent and reverse steatosis independently of fat and carbohydrate intake more efficiently than a 20% reduction in energy intake. The effect appears to result from fuel-generated, highly distributed small, synergistic increases in lipid and BCAA catabolism, and a decrease in cell stress.

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.

Background & Aims The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice. Methods We generated MNADK knockout (KO) mice on a C57BL/6NTac background; mice with a wild-type Mnadk gene were used as controls. Some mice were placed on an atherogenic high-fat diet (16% fat, 41% carbohydrate, and 1.25% cholesterol supplemented with 0.5% sodium cholate) or given methotrexate intraperitoneally. We measured rates of fatty acid oxidation in primary hepatocytes using radiolabeled palmitate and in mice using indirect calorimetry. We measured levels of reactive oxygen species in mouse livers and primary hepatocytes. Metabolomic analyses were used to quantify serum metabolites, such as amino acids and acylcarnitines. Results The KO mice had metabolic features of MNADK-deficient patients, such as increased serum concentrations of lysine and C10:2 carnitine. When placed on the atherogenic high-fat diet, the KO mice developed features of nonalcoholic fatty liver disease and had increased levels of reactive oxygen species in livers and primary hepatocytes, compared with control mice. During fasting, the KO mice had a defect in fatty acid oxidation. MNADK deficiency reduced the activation of cAMP-responsive element binding protein-hepatocyte specific and peroxisome proliferator–activated receptor alpha, which are transcriptional activators that mediate the fasting response. The activity of mitochondrial sirtuins was reduced in livers of the KO mice. Methotrexate inhibited the catalytic activity of MNADK in hepatocytes and in livers in mice with methotrexate injection. In mice given injections of methotrexate, supplementation of a diet with nicotinamide riboside, an NAD precursor, replenished hepatic NADP and protected the mice from hepatotoxicity, based on markers such as increased level of serum alanine aminotransferase. Conclusion MNADK facilitates fatty acid oxidation, counteracts oxidative damage, maintains mitochondrial sirtuin activity, and prevents metabolic stress-induced non-alcoholic fatty liver disease in mice.

A heterozygous nonsense variant was identified in dapper, antagonist of beta-catenin, 1 (DACT1) via whole-exome sequencing in family members with imperforate anus, structural renal abnormalities, genitourinary anomalies, and/or ear anomalies. The DACT1 c.1256G>A;p.Trp419* variant segregated appropriately in the family consistent with an autosomal dominant mode of inheritance. DACT1 is a member of the Wnt-signaling pathway, and mice homozygous for null alleles display multiple congenital anomalies including absent anus with blind-ending colon and genitourinary malformations. To investigate the DACT1 c.1256G>A variant, HEK293 cells were transfected with mutant DACT1 cDNA plasmid, and immunoblotting revealed stability of the DACT1 p.Trp419* protein. Overexpression of DACT1 c.1256G>A mRNA in Xenopus embryos revealed a specific gastrointestinal phenotype of enlargement of the proctodeum. Together, these findings suggest that the DACT1 c.1256G>A nonsense variant is causative of a specific genetic syndrome with features overlapping Townes-Brocks syndrome.

Acylcarnitines, fatty acid oxidation (FAO) intermediates, have been implicated in diet-induced insulin resistance and type 2 diabetes mellitus, as increased levels are found in obese insulin resistant humans. Moreover plasma acylcarnitines have been associated with clinical parameters related to glucose metabolism, such as fasting glucose levels and HbA1c. We hypothesized that plasma acylcarnitines would correlate with energy expenditure, insulin sensitivity and other clinical parameters before and during a weight loss intervention. We measured plasma acylcarnitines in 60 obese subjects before and after a 12 week weight loss intervention. These samples originated from three different interventions (diet alone (n = 20); diet and exercise (n = 21); diet and drug treatment (n = 19)). Acylcarnitine profiles were analysed in relation to clinical parameters of glucose metabolism, insulin sensitivity and energy expenditure. Conclusions were drawn from all 60 subjects together. Despite amelioration of HOMA-IR, plasma acylcarnitines levels increased during weight loss. HOMA-IR, energy expenditure and respiratory exchange ratio were not related to plasma acylcarnitines. However non-esterified fatty acids correlated strongly with several acylcarnitines at baseline and during the weight loss intervention (p < 0.001). Acylcarnitines did not correlate with clinical parameters of glucose metabolism during weight loss, questioning their role in insulin resistance and type 2 diabetes mellitus.

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.

The incidence of obesity and insulin resistance is growing, and the increase in type 2 diabetes mellitus (DM2) constitutes one of the biggest challenges for our healthcare systems. Many theories are proposed for the induction of insulin resistance in glucose and lipid metabolism and its metabolic sequelae. One of these mechanisms is lipotoxicity (1–4): excess lipid supply and subsequent lipid accumulation in insulin-sensitive tissues such as skeletal muscle interfere with insulin-responsive metabolic pathways. Various lipid intermediates, like ceramides, gangliosides, diacylglycerol, and other metabolites, have been held responsible for insulin resistance (2,3,5–10). These intermediates can exert such effects because they are signaling molecules and building blocks of cellular membranes, which harbor the insulin receptor. In addition, lipids play an important role in energy homeostasis. Fatty acids (FA) can be metabolized via mitochondrial FA oxidation (FAO), which yields energy (11). As such, FAO competes with glucose oxidation in a process known as the glucose-FA, or Randle, cycle (12). Muoio and colleagues (1,13,14) proposed an alternative mechanism in which FAO rate outpaces the tricarboxylic acid cycle (TCA), thereby leading to the accumulation of intermediary metabolites such as acylcarnitines that may interfere with insulin sensitivity. This accumulation of acylcarnitines corroborates with some human studies showing that acylcarnitines are associated with insulin resistance (15–17). In addition, acylcarnitines have a long history in the diagnosis and neonatal screening of FAO defects and other inborn errors of metabolism (18). This knowledge may aid to understand the interaction between FAO and insulin resistance and fuel future research. In this review, we discuss the role of acylcarnitines in FAO and insulin resistance as emerging from animal and human studies. ### Carnitine biosynthesis and regulation of tissue carnitine content. To guarantee continuous energy supply, the human body oxidizes considerable amounts of fat besides glucose. …

Hydroxybutyrylcarnitine (HB-carnitine) is a metabolite that has been associated with insulin resistance and type 2 diabetes mellitus. It is currently unknown whether HB-carnitine can be produced from D-3-hydroxybutyrate (D-3HB), a ketone body; but its formation from L-3-HB-CoA, a fatty acid beta-oxidation intermediate, is well established. We aimed to assess which stereoisomers of 3-HB-carnitine are present in vivo. Ketosis and increased fatty acid oxidation were induced in 12 lean healthy men by a 38-hour fasting period. The D-3HB kinetics (stable isotope technique) and stereoisomers of muscle 3-HB-carnitine (high-performance liquid chromatography/ultra-performance liquid chromatography tandem mass spectrometry) were measured. Muscle D-3HB-carnitine content was much higher compared with L-3HB-carnitine. In addition, muscle D-3HB-carnitine correlated significantly with D-3-HB production. Following the finding that a ketone body can be converted into a carnitine ester in vivo, we show in vitro that D-3-HB can be converted into HB-carnitine (ketocarnitine) via an acyl-CoA synthetase reaction in several tissues including human muscle. During fasting, HB-carnitine in muscle is derived mainly from the ketone body D-3HB. The role of D-3HB-carnitine synthesis in metabolism remains to be elucidated. (c) 2012 Elsevier Inc. All rights reserved