Objective-To prospectively characterize acute hyperammonemic episodes in patients with urea cycle... more Objective-To prospectively characterize acute hyperammonemic episodes in patients with urea cycle disorders (UCD) in regards to precipitating factors, treatments and utilization of medical resources. Study design-Prospective, longitudinal observational study of hyperammonemic episodes in patients with UCD enrolled in the NIH sponsored Urea Cycle Disorders Consortium Longitudinal Study. An acute hyperammonemic event was defined as plasma ammonia level > 100 µmol/L. Physician reported data regarding the precipitating event and laboratory and clinical variables were recorded in a central database. Results-A total of 128 patients with UCD experienced 413 hyperammonemia events. Most patients experienced between 1-3 (65%) and 4-6 (23%) hyperammonemia events since the study inception, averaging less than one event/year. The most common identifiable precipitant was Infection (33%), 24% of which were due to upper/lower respiratory tract infections. Indicators of increased morbidity were seen with Infection: increased hospitalization rates (P=0.02), longer hospital stays (+2.0 days, P = 0.003) and increased use of intravenous ammonia scavengers (+45-52%, P = 0.003-0.03). Conclusions-Infection is the most common precipitant of acute hyperammonemia in patients with UCD and is associated with indicators of increased morbidity (ie, hospitalization rate, length of stay, use of IV ammonia scavengers). These findings suggest that the catabolic and immune effects of infection may be a target for clinical intervention in inborn errors of metabolism. The complete urea cycle is present in the liver and plays a critical role in the incorporation of excess nitrogen (ie, ammonia) into urea. Portions of the cycle are present throughout the
We report the identification and characterization of a five-carbon protein posttranslational modi... more We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (K glu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 K glu sites in 191 proteins and showed that K glu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.
Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although ure... more Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high‐protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low‐protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate‐activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5‐aminoimidazole‐4‐carboxamide ribonucleotide (...
Mitochondria are ancient organelles that have co-evolved with their cellular hosts, developing a ... more Mitochondria are ancient organelles that have co-evolved with their cellular hosts, developing a mutually beneficial arrangement. In addition to making energy, mitochondria are multifaceted, being involved in heat production, calcium storage, apoptosis, cell signaling, biosynthesis, and aging. Many of these mitochondrial functions decline with age, and are the basis for many diseases of aging. Despite the vast amount of research dedicated to this subject, the relationship between aging mitochondria and immune function is largely absent from the literature. In this review, three main issues facing the aging immune system are discussed: (1) inflamm-aging; (2) susceptibility to infection and (3) declining T-cell function. These issues are re-evaluated using the lens of mitochondrial dysfunction with aging. With the recent expansion of numerous profiling technologies, there has been a resurgence of interest in the role of metabolism in immunity, with mitochondria taking center stage. Bu...
Despite judicious monitoring and care, patients with fatty acid oxidation disorders may experienc... more Despite judicious monitoring and care, patients with fatty acid oxidation disorders may experience metabolic decompensation due to infection which may result in rhabdomyolysis, cardiomyopathy, hypoglycemia and liver dysfunction and failure. Since clinical studies on metabolic decompensation are dangerous, we employed a preclinical model of metabolic decompensation due to infection. By infecting mice with mouse adapted influenza and using a pair-feeding strategy in a mouse model of long-chain fatty acid oxidation (Acadvl −/−), our goals were to isolate the effects of infection on tissue acylcarnitines and determine how they relate to their plasma counterparts. Applying statistical data reduction techniques (Partial Least Squares-Discriminant Analysis, PLS-DA), we were able to identify critical acylcarnitines that were driving differentiation of our experimental groups for all the tissues studied. While plasma displayed increases in metabolites directly related to mouse VLCAD deficiency (e.g. C16 and C18), organs like the heart, muscle and liver also showed involvement of alternative pathways (e.g. medium chain FAO and ketogenesis), suggesting adaptive measures. Matched correlation analyses showed strong correlations (r > 0.7) between plasma and tissue levels for a small number of metabolites. Overall, our results demonstrate that infection as a stress produces perturbations in metabolism in Acadvl −/− that differ greatly from WT infected and Acadvl −/− pair-fed controls. This model system will be useful for studying the effects of infection on tissue metabolism as well as evaluating interventions aimed at modulating the effects of metabolic decompensation.
Restimulation-induced cell death (RICD) regulates immune responses by restraining effector T cell... more Restimulation-induced cell death (RICD) regulates immune responses by restraining effector T cell expansion and limiting nonspecific damage to the host. RICD is triggered by re-engagement of the TCR on a cycling effector T cell, resulting in apoptosis. It remains unclear how RICD sensitivity is calibrated in T cells derived from different individuals or subsets. In this study we show that aerobic glycolysis strongly correlates with RICD sensitivity in human CD8+ effector T cells. Reducing glycolytic activity or glucose availability rendered effector T cells significantly less sensitive to RICD. We found that active glycolysis specifically facilitates the induction of proapoptotic Fas ligand upon TCR restimulation, accounting for enhanced RICD sensitivity in highly glycolytic T cells. Collectively, these data indicate that RICD susceptibility is linked to metabolic reprogramming, and that switching back to metabolic quiescence may help shield T cells from RICD as they transition into...
Journal of inherited metabolic disease, Jan 11, 2015
Nutritional management of acute metabolic decompensation in amino acid inborn errors of metabolis... more Nutritional management of acute metabolic decompensation in amino acid inborn errors of metabolism (AA IEM) aims to restore nitrogen balance. While nutritional recommendations have been published, they have never been rigorously evaluated. Furthermore, despite these recommendations, there is a wide variation in the nutritional strategies employed amongst providers, particularly regarding the inclusion of parenteral lipids for protein-free caloric support. Since randomized clinical trials during acute metabolic decompensation are difficult and potentially dangerous, mathematical modeling of metabolism can serve as a surrogate for the preclinical evaluation of nutritional interventions aimed at restoring nitrogen balance during acute decompensation in AA IEM. A validated computational model of human macronutrient metabolism was adapted to predict nitrogen balance in response to various nutritional interventions in a simulated patient with a urea cycle disorder (UCD) during acute metab...
ASS1 is a cytosolic enzyme that plays a role in the conversion of citrulline to arginine. In huma... more ASS1 is a cytosolic enzyme that plays a role in the conversion of citrulline to arginine. In human and mouse tissues, ASS1 protein is found in several components of the immune system, including the thymus and T cells. However, the role of ASS1 in these tissues remains to be defined. Considerable attention has been focused recently on the role of metabolism in T cell differentiation and function. Based on the expression of ASS1 in the immune system, we hypothesized that ASS1 deficiency would result in T cell defects. To evaluate this question, we characterized immune function in hypomorphic fold/fold mice. Analysis of splenic T cells by flow cytometry showed a marked reduction in T cell numbers with normal expression of activation surface markers. Gene therapy correction of liver ASS1 to enhance survival resulted in a partial recovery of splenic T cells for characterization. In vitro and in vivo studies demonstrated the persistence of the ASS1 enzyme defect in T cells and abnormal T ...
Free radical formation resulting in oxidative stress is a hallmark of mitochondrial dysfunction. ... more Free radical formation resulting in oxidative stress is a hallmark of mitochondrial dysfunction. Indeed, oxidative stress has been demonstrated to be an underlying pathophysiologic process in various inborn errors of metabolism. Metabolic profiling of oxidative stress may provide a nonspecific measure of disease activity that may further enable physicians to monitor disease. In the present study, we investigated two markers of oxidative damage in urinary samples from IEM subjects and controls: F-2 isoprostanes, a measure of lipid peroxidation and di-tyrosine, a measure of protein oxidation. We also determined urinary antioxidant activity in these samples. Subsets of IEM patients showed significantly higher levels of the damage markers isoprostanes and dityrosine. Of note, patients with cobalamin disorders (i.e., CblB and CblC) consistently had the highest levels of oxidative damage markers. Lower urine antioxidant capacity was seen in all subject categories, particularly cobalamin disorders and propionic acidemia. Longitudinal studies in subjects with MSUD showed good concordance between markers of oxidative damage and acute decompensation. Overall, quantifying oxidative stress offers a unique perspective to IEM. These measures may provide a means of addressing mitochondrial function in IEM and aid in the development of therapeutic targets and clinical monitoring in this diverse set of disorders.
Introduction-Combined methylmalonic aciduria and homocystinuria, cobalamin C (cblC) type, is an i... more Introduction-Combined methylmalonic aciduria and homocystinuria, cobalamin C (cblC) type, is an inherited disorder of vitamin B 12 metabolism caused by mutations in MMACHC. CblC typically presents in the neonatal period with neurological deterioration, failure to thrive, cytopenias, and multisystem pathology including renal and hepatic dysfunction. Rarely, affected individuals present in adulthood with gait ataxia and cognitive decline. Treatment with hydroxycobalamin may ameliorate the clinical features of early-onset disease and prevent clinical late-onset disease. Propionic acidemia (PA), methylmalonic acidemia (MMA), and various disorders of cobalamin metabolism are characterized by elevated propionylcarnitine (C3) on Newborn Screening (NBS). Distinctions can be made between these disorders with secondary analyte testing. Elevated methionine is already routinely used as a NBS marker for cystathionine ß-synthase deficiency. We propose that low methionine may be useful as a secondary analyte for specific detection of cbl disorders among a larger pool of infants with elevated C3 on NBS. Methods-Retrospective analysis of dried blood spot (DBS) data in patients with molecularly confirmed cblC disease. Results-9 out of 10 patients with confirmed cblC born in New York between 2005 and 2008 had methionine below 13.4 μmol/L on NBS. Elevated C3, elevated C3:C2 ratio, and low methionine were incorporated into a simple screening algorithm that can be used to improve the specificity of newborn screening programs and provide a specific and novel method of distinguishing cblC from other disorders of propionate metabolism prior to recall for confirmatory testing. Conclusions-It is anticipated that this algorithm will aid in early and specific detection of cobalamin C, D, and F diseases, with no additional expense to NBS laboratories screening for organic acidemias and classical homocystinuria.
Over 27 cases of liver transplant, kidney transplant and combined liver-kidney transplant have be... more Over 27 cases of liver transplant, kidney transplant and combined liver-kidney transplant have been reported for the treatment of methylmalonic aciduria. We describe a case of a 5-year-old boy who underwent combined liver-kidney transplant (CLKT) for phenotypic mut0 disease. His history was notable for more than 30 hospitalizations for severe acidosis, metabolic strokes, liver disease, pancreatic disease, chronic renal insufficiency with interstitial nephritis, and decreased quality of life. Post-CLKT, there was a marked reduction in serum (80%) and urine MMA levels (90%) as well as a cessation of metabolic decompensations. Neurologic deterioration continued post-CKLT manifested as a cerebellar stroke. The clinical details and therapeutic implications of solid organ transplant for methylmalonic aciduria are discussed.
Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by ... more Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear pa... more Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n=21) and patients (n=12) with either the m.3243A>G mutation or single, large-scale mtDNA deletions, we examined: i) cytokine responses (IL-6, TNF-α, IL-1β) in response to acute lipopolysaccharide (LPS) exposure, and ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC50), relative to controls, leukocytes from patients with mtDNA deletions showed 74 - 79% lower responses for IL-6 and IL-1β (pIL-6=0.031, pIL-1β=0.009). Moreover, IL-6...
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Objective: In individuals with mitochondrial disease, respiratory viral infection can result in m... more Objective: In individuals with mitochondrial disease, respiratory viral infection can result in metabolic decompensation with mitochondrial hepatopathy. Here, we used a mouse model of liver-specific Complex IV deficiency to study hepatic allostasis during respiratory viral infection. Methods: Mice with hepatic cytochrome c oxidase deficiency (LivCox10 À/À) were infected with aerosolized influenza, A/PR/8 (PR8), and euthanized on day five after infection following three days of symptoms. This time course is marked by a peak in inflammatory cytokines and mimics the timing of a common clinical scenario in which caregivers may first attempt to manage the illness at home before seeking medical attention. Metabolic decompensation and mitochondrial hepatopathy in mice were characterized by serum hepatic testing, histology, electron microscopy, biochemistry, metabolomics, and bioenergetic profiling. Results: Following influenza infection, LivCox10 À/À mice displayed marked liver disease including hepatitis, enlarged mitochondria with cristae loss, and hepatic steatosis. This pathophysiology was associated with viremia. Primary hepatocytes from LivCox10 À/À mice cocultured with WT Kupffer cells in the presence of PR8 showed enhanced lipid accumulation. Treatment of hepatocytes with recombinant TNFa implicated Kupffer cell-derived TNFa as a precipitant of steatosis in LivCox10 À/À mice. Eliminating Kupffer cells or blocking TNFa in vivo during influenza infection mitigated the steatosis and mitochondrial morphologic changes. Conclusions: Taken together, our data shift the narrative of metabolic decompensation in mitochondrial hepatopathy beyond the bioenergetic costs of infection to include an underlying susceptibility to immune-mediated damage. Moreover, our work suggests that immune modulation during metabolic decompensation in mitochondrial disease represents a future viable treatment strategy needing further exploration.
Metabolically quiescent T cells circulate throughout the body in search of antigen. Following eng... more Metabolically quiescent T cells circulate throughout the body in search of antigen. Following engagement of their cognate receptors, T cells undergo metabolic reprogramming to support their activation, differentiation, and ultimately function. In the spirit of Sir Archibald Garrod, this metabolic reprogramming actually imparts a chemical individuality which confers advantage, while in others confers vulnerability, depending upon the milieu. Studying T cell immunometabolism in the context of inborn errors of metabolism allows one to define essential pathways of intermediary metabolism as well metabolic vulnerabilities and plasticity. Inborn errors of metabolism, a class of diseases first named by Garrod, have a long history of being informative for common physiologic and pathologic processes. This endeavor may be accomplished through the study of patients, animal models, and in vitro models of inborn errors of metabolism. In this review, the basics of intermediary metabolism and core metabolic pathways will be discussed, along with their relationship to T cell immunometabolism. Due to their pleiotropic nature, the reader will be specifically directed toward various inborn errors of metabolism which may be helpful for answering important questions about the role of metabolism in T cells.
Purpose: Propionic acidemia (PA) is a severe metabolic disorder characterized by multiorgan patho... more Purpose: Propionic acidemia (PA) is a severe metabolic disorder characterized by multiorgan pathology, including renal disease. The prevalence of chronic kidney disease (CKD) in PA patients and factors associated with CKD in PA are not known. Methods: Thirty-one subjects diagnosed with PA underwent laboratory and clinical evaluations through a dedicated natural history study at the National Institutes of Health (ClinicalTrials.gov identifier: NCT02890342). Results: Cross-sectional analysis of the creatinine-based estimated glomerular filtration rate (eGFR) in subjects with native kidneys revealed an age-dependent decline in renal function (P < 0.002). Among adults with PA, 4/8 (50%) had eGFR <60 mL/min/1.73 m 2. There was a significant discrepancy between eGFRs calculated using estimating equations based on serum creatinine compared with serum cystatin C (P < 0.0001). The tubular injury marker, plasma lipocalin-2, and plasma uric acid were strongly associated with CKD (P < 0.0001). The measured 24-hour creatinine excretion was below normal, even after adjusting for age, height, and sex. Conclusion: CKD is common in adults with PA and is associated with age. The poor predictive performance of standard eGFR estimating equations, likely due to reduced creatine synthesis in kidney and liver, could delay the recognition of CKD and management of ensuing complications in this population.
Immunometabolism aims to define the role of intermediary metabolism in immune cell function, with... more Immunometabolism aims to define the role of intermediary metabolism in immune cell function, with bioenergetics and the mitochondria recently taking center stage. To date, the medical literature on mitochondria and immune function extols the virtues of mouse models in exploring this biologic intersection. While the laboratory mouse has become a standard for studying mammalian biology, this model comprises part of a comprehensive approach. Humans, with their broad array of inherited phenotypes, serve as a starting point for studying immunometabolism; specifically, patients with mitochondrial disease. Using this top-down approach, the mouse as a model organism facilitates further exploration of the consequences of mutations involved in mitochondrial maintenance and function. In this review, we will discuss the emerging phenotype of immune dysfunction in mitochondrial disease as a model for understanding the role of the mitochondria in immune function in available mouse models.
T cell subsets including effector (T), regulatory (T), and memory (T) cells are characterized by ... more T cell subsets including effector (T), regulatory (T), and memory (T) cells are characterized by distinct metabolic profiles that influence their differentiation and function. Previous research suggests that engagement of long-chain fatty acid oxidation (LC-FAO) supports Foxp3 T cell and T cell survival. However, evidence for this is mostly based on inhibition of Cpt1a, the rate-limiting enzyme for LC-FAO, with the drug etomoxir. Using genetic models to target Cpt1a specifically in T cells, we dissected the role of LC-FAO in primary, memory, and regulatory T cell responses. Here we show that the ACC2/Cpt1a axis is largely dispensable for T, T, or T cell formation, and that the effects of etomoxir on T cell differentiation and function are independent of Cpt1a expression. Together our data argue that metabolic pathways other than LC-FAO fuel T or T differentiation and suggest alternative mechanisms for the effects of etomoxir that involve mitochondrial respiration.
CD8 T cells are key members of the adaptive immune response against infections and cancer. As we ... more CD8 T cells are key members of the adaptive immune response against infections and cancer. As we discuss in this review, these cells can present diverse metabolic requirements, which have been intensely studied during the past few years. Our current understanding suggests that aerobic glycolysis is a hallmark of activated CD8 T cells, while naive and memory (T ) cells often rely on oxidative phosphorylation, and thus mitochondrial metabolism is a crucial determinant of CD8 T cell development. Moreover, it has been proposed that CD8 T cells have a specific requirement for the oxidation of long-chain fatty acids (LC-FAO), a process modulated in lymphocytes by the enzyme CPT1A. However, this notion relies heavily on the metabolic analysis of in vitro cultures and on chemical inhibition of CPT1A. Therefore, we introduce more recent studies using genetic models to demonstrate that CPT1A-mediated LC-FAO is dispensable for the development of CD8 T cell memory and protective immunity, and q...
Objective-To prospectively characterize acute hyperammonemic episodes in patients with urea cycle... more Objective-To prospectively characterize acute hyperammonemic episodes in patients with urea cycle disorders (UCD) in regards to precipitating factors, treatments and utilization of medical resources. Study design-Prospective, longitudinal observational study of hyperammonemic episodes in patients with UCD enrolled in the NIH sponsored Urea Cycle Disorders Consortium Longitudinal Study. An acute hyperammonemic event was defined as plasma ammonia level > 100 µmol/L. Physician reported data regarding the precipitating event and laboratory and clinical variables were recorded in a central database. Results-A total of 128 patients with UCD experienced 413 hyperammonemia events. Most patients experienced between 1-3 (65%) and 4-6 (23%) hyperammonemia events since the study inception, averaging less than one event/year. The most common identifiable precipitant was Infection (33%), 24% of which were due to upper/lower respiratory tract infections. Indicators of increased morbidity were seen with Infection: increased hospitalization rates (P=0.02), longer hospital stays (+2.0 days, P = 0.003) and increased use of intravenous ammonia scavengers (+45-52%, P = 0.003-0.03). Conclusions-Infection is the most common precipitant of acute hyperammonemia in patients with UCD and is associated with indicators of increased morbidity (ie, hospitalization rate, length of stay, use of IV ammonia scavengers). These findings suggest that the catabolic and immune effects of infection may be a target for clinical intervention in inborn errors of metabolism. The complete urea cycle is present in the liver and plays a critical role in the incorporation of excess nitrogen (ie, ammonia) into urea. Portions of the cycle are present throughout the
We report the identification and characterization of a five-carbon protein posttranslational modi... more We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (K glu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 K glu sites in 191 proteins and showed that K glu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.
Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although ure... more Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high‐protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low‐protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate‐activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5‐aminoimidazole‐4‐carboxamide ribonucleotide (...
Mitochondria are ancient organelles that have co-evolved with their cellular hosts, developing a ... more Mitochondria are ancient organelles that have co-evolved with their cellular hosts, developing a mutually beneficial arrangement. In addition to making energy, mitochondria are multifaceted, being involved in heat production, calcium storage, apoptosis, cell signaling, biosynthesis, and aging. Many of these mitochondrial functions decline with age, and are the basis for many diseases of aging. Despite the vast amount of research dedicated to this subject, the relationship between aging mitochondria and immune function is largely absent from the literature. In this review, three main issues facing the aging immune system are discussed: (1) inflamm-aging; (2) susceptibility to infection and (3) declining T-cell function. These issues are re-evaluated using the lens of mitochondrial dysfunction with aging. With the recent expansion of numerous profiling technologies, there has been a resurgence of interest in the role of metabolism in immunity, with mitochondria taking center stage. Bu...
Despite judicious monitoring and care, patients with fatty acid oxidation disorders may experienc... more Despite judicious monitoring and care, patients with fatty acid oxidation disorders may experience metabolic decompensation due to infection which may result in rhabdomyolysis, cardiomyopathy, hypoglycemia and liver dysfunction and failure. Since clinical studies on metabolic decompensation are dangerous, we employed a preclinical model of metabolic decompensation due to infection. By infecting mice with mouse adapted influenza and using a pair-feeding strategy in a mouse model of long-chain fatty acid oxidation (Acadvl −/−), our goals were to isolate the effects of infection on tissue acylcarnitines and determine how they relate to their plasma counterparts. Applying statistical data reduction techniques (Partial Least Squares-Discriminant Analysis, PLS-DA), we were able to identify critical acylcarnitines that were driving differentiation of our experimental groups for all the tissues studied. While plasma displayed increases in metabolites directly related to mouse VLCAD deficiency (e.g. C16 and C18), organs like the heart, muscle and liver also showed involvement of alternative pathways (e.g. medium chain FAO and ketogenesis), suggesting adaptive measures. Matched correlation analyses showed strong correlations (r > 0.7) between plasma and tissue levels for a small number of metabolites. Overall, our results demonstrate that infection as a stress produces perturbations in metabolism in Acadvl −/− that differ greatly from WT infected and Acadvl −/− pair-fed controls. This model system will be useful for studying the effects of infection on tissue metabolism as well as evaluating interventions aimed at modulating the effects of metabolic decompensation.
Restimulation-induced cell death (RICD) regulates immune responses by restraining effector T cell... more Restimulation-induced cell death (RICD) regulates immune responses by restraining effector T cell expansion and limiting nonspecific damage to the host. RICD is triggered by re-engagement of the TCR on a cycling effector T cell, resulting in apoptosis. It remains unclear how RICD sensitivity is calibrated in T cells derived from different individuals or subsets. In this study we show that aerobic glycolysis strongly correlates with RICD sensitivity in human CD8+ effector T cells. Reducing glycolytic activity or glucose availability rendered effector T cells significantly less sensitive to RICD. We found that active glycolysis specifically facilitates the induction of proapoptotic Fas ligand upon TCR restimulation, accounting for enhanced RICD sensitivity in highly glycolytic T cells. Collectively, these data indicate that RICD susceptibility is linked to metabolic reprogramming, and that switching back to metabolic quiescence may help shield T cells from RICD as they transition into...
Journal of inherited metabolic disease, Jan 11, 2015
Nutritional management of acute metabolic decompensation in amino acid inborn errors of metabolis... more Nutritional management of acute metabolic decompensation in amino acid inborn errors of metabolism (AA IEM) aims to restore nitrogen balance. While nutritional recommendations have been published, they have never been rigorously evaluated. Furthermore, despite these recommendations, there is a wide variation in the nutritional strategies employed amongst providers, particularly regarding the inclusion of parenteral lipids for protein-free caloric support. Since randomized clinical trials during acute metabolic decompensation are difficult and potentially dangerous, mathematical modeling of metabolism can serve as a surrogate for the preclinical evaluation of nutritional interventions aimed at restoring nitrogen balance during acute decompensation in AA IEM. A validated computational model of human macronutrient metabolism was adapted to predict nitrogen balance in response to various nutritional interventions in a simulated patient with a urea cycle disorder (UCD) during acute metab...
ASS1 is a cytosolic enzyme that plays a role in the conversion of citrulline to arginine. In huma... more ASS1 is a cytosolic enzyme that plays a role in the conversion of citrulline to arginine. In human and mouse tissues, ASS1 protein is found in several components of the immune system, including the thymus and T cells. However, the role of ASS1 in these tissues remains to be defined. Considerable attention has been focused recently on the role of metabolism in T cell differentiation and function. Based on the expression of ASS1 in the immune system, we hypothesized that ASS1 deficiency would result in T cell defects. To evaluate this question, we characterized immune function in hypomorphic fold/fold mice. Analysis of splenic T cells by flow cytometry showed a marked reduction in T cell numbers with normal expression of activation surface markers. Gene therapy correction of liver ASS1 to enhance survival resulted in a partial recovery of splenic T cells for characterization. In vitro and in vivo studies demonstrated the persistence of the ASS1 enzyme defect in T cells and abnormal T ...
Free radical formation resulting in oxidative stress is a hallmark of mitochondrial dysfunction. ... more Free radical formation resulting in oxidative stress is a hallmark of mitochondrial dysfunction. Indeed, oxidative stress has been demonstrated to be an underlying pathophysiologic process in various inborn errors of metabolism. Metabolic profiling of oxidative stress may provide a nonspecific measure of disease activity that may further enable physicians to monitor disease. In the present study, we investigated two markers of oxidative damage in urinary samples from IEM subjects and controls: F-2 isoprostanes, a measure of lipid peroxidation and di-tyrosine, a measure of protein oxidation. We also determined urinary antioxidant activity in these samples. Subsets of IEM patients showed significantly higher levels of the damage markers isoprostanes and dityrosine. Of note, patients with cobalamin disorders (i.e., CblB and CblC) consistently had the highest levels of oxidative damage markers. Lower urine antioxidant capacity was seen in all subject categories, particularly cobalamin disorders and propionic acidemia. Longitudinal studies in subjects with MSUD showed good concordance between markers of oxidative damage and acute decompensation. Overall, quantifying oxidative stress offers a unique perspective to IEM. These measures may provide a means of addressing mitochondrial function in IEM and aid in the development of therapeutic targets and clinical monitoring in this diverse set of disorders.
Introduction-Combined methylmalonic aciduria and homocystinuria, cobalamin C (cblC) type, is an i... more Introduction-Combined methylmalonic aciduria and homocystinuria, cobalamin C (cblC) type, is an inherited disorder of vitamin B 12 metabolism caused by mutations in MMACHC. CblC typically presents in the neonatal period with neurological deterioration, failure to thrive, cytopenias, and multisystem pathology including renal and hepatic dysfunction. Rarely, affected individuals present in adulthood with gait ataxia and cognitive decline. Treatment with hydroxycobalamin may ameliorate the clinical features of early-onset disease and prevent clinical late-onset disease. Propionic acidemia (PA), methylmalonic acidemia (MMA), and various disorders of cobalamin metabolism are characterized by elevated propionylcarnitine (C3) on Newborn Screening (NBS). Distinctions can be made between these disorders with secondary analyte testing. Elevated methionine is already routinely used as a NBS marker for cystathionine ß-synthase deficiency. We propose that low methionine may be useful as a secondary analyte for specific detection of cbl disorders among a larger pool of infants with elevated C3 on NBS. Methods-Retrospective analysis of dried blood spot (DBS) data in patients with molecularly confirmed cblC disease. Results-9 out of 10 patients with confirmed cblC born in New York between 2005 and 2008 had methionine below 13.4 μmol/L on NBS. Elevated C3, elevated C3:C2 ratio, and low methionine were incorporated into a simple screening algorithm that can be used to improve the specificity of newborn screening programs and provide a specific and novel method of distinguishing cblC from other disorders of propionate metabolism prior to recall for confirmatory testing. Conclusions-It is anticipated that this algorithm will aid in early and specific detection of cobalamin C, D, and F diseases, with no additional expense to NBS laboratories screening for organic acidemias and classical homocystinuria.
Over 27 cases of liver transplant, kidney transplant and combined liver-kidney transplant have be... more Over 27 cases of liver transplant, kidney transplant and combined liver-kidney transplant have been reported for the treatment of methylmalonic aciduria. We describe a case of a 5-year-old boy who underwent combined liver-kidney transplant (CLKT) for phenotypic mut0 disease. His history was notable for more than 30 hospitalizations for severe acidosis, metabolic strokes, liver disease, pancreatic disease, chronic renal insufficiency with interstitial nephritis, and decreased quality of life. Post-CLKT, there was a marked reduction in serum (80%) and urine MMA levels (90%) as well as a cessation of metabolic decompensations. Neurologic deterioration continued post-CKLT manifested as a cerebellar stroke. The clinical details and therapeutic implications of solid organ transplant for methylmalonic aciduria are discussed.
Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by ... more Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear pa... more Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n=21) and patients (n=12) with either the m.3243A>G mutation or single, large-scale mtDNA deletions, we examined: i) cytokine responses (IL-6, TNF-α, IL-1β) in response to acute lipopolysaccharide (LPS) exposure, and ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC50), relative to controls, leukocytes from patients with mtDNA deletions showed 74 - 79% lower responses for IL-6 and IL-1β (pIL-6=0.031, pIL-1β=0.009). Moreover, IL-6...
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Objective: In individuals with mitochondrial disease, respiratory viral infection can result in m... more Objective: In individuals with mitochondrial disease, respiratory viral infection can result in metabolic decompensation with mitochondrial hepatopathy. Here, we used a mouse model of liver-specific Complex IV deficiency to study hepatic allostasis during respiratory viral infection. Methods: Mice with hepatic cytochrome c oxidase deficiency (LivCox10 À/À) were infected with aerosolized influenza, A/PR/8 (PR8), and euthanized on day five after infection following three days of symptoms. This time course is marked by a peak in inflammatory cytokines and mimics the timing of a common clinical scenario in which caregivers may first attempt to manage the illness at home before seeking medical attention. Metabolic decompensation and mitochondrial hepatopathy in mice were characterized by serum hepatic testing, histology, electron microscopy, biochemistry, metabolomics, and bioenergetic profiling. Results: Following influenza infection, LivCox10 À/À mice displayed marked liver disease including hepatitis, enlarged mitochondria with cristae loss, and hepatic steatosis. This pathophysiology was associated with viremia. Primary hepatocytes from LivCox10 À/À mice cocultured with WT Kupffer cells in the presence of PR8 showed enhanced lipid accumulation. Treatment of hepatocytes with recombinant TNFa implicated Kupffer cell-derived TNFa as a precipitant of steatosis in LivCox10 À/À mice. Eliminating Kupffer cells or blocking TNFa in vivo during influenza infection mitigated the steatosis and mitochondrial morphologic changes. Conclusions: Taken together, our data shift the narrative of metabolic decompensation in mitochondrial hepatopathy beyond the bioenergetic costs of infection to include an underlying susceptibility to immune-mediated damage. Moreover, our work suggests that immune modulation during metabolic decompensation in mitochondrial disease represents a future viable treatment strategy needing further exploration.
Metabolically quiescent T cells circulate throughout the body in search of antigen. Following eng... more Metabolically quiescent T cells circulate throughout the body in search of antigen. Following engagement of their cognate receptors, T cells undergo metabolic reprogramming to support their activation, differentiation, and ultimately function. In the spirit of Sir Archibald Garrod, this metabolic reprogramming actually imparts a chemical individuality which confers advantage, while in others confers vulnerability, depending upon the milieu. Studying T cell immunometabolism in the context of inborn errors of metabolism allows one to define essential pathways of intermediary metabolism as well metabolic vulnerabilities and plasticity. Inborn errors of metabolism, a class of diseases first named by Garrod, have a long history of being informative for common physiologic and pathologic processes. This endeavor may be accomplished through the study of patients, animal models, and in vitro models of inborn errors of metabolism. In this review, the basics of intermediary metabolism and core metabolic pathways will be discussed, along with their relationship to T cell immunometabolism. Due to their pleiotropic nature, the reader will be specifically directed toward various inborn errors of metabolism which may be helpful for answering important questions about the role of metabolism in T cells.
Purpose: Propionic acidemia (PA) is a severe metabolic disorder characterized by multiorgan patho... more Purpose: Propionic acidemia (PA) is a severe metabolic disorder characterized by multiorgan pathology, including renal disease. The prevalence of chronic kidney disease (CKD) in PA patients and factors associated with CKD in PA are not known. Methods: Thirty-one subjects diagnosed with PA underwent laboratory and clinical evaluations through a dedicated natural history study at the National Institutes of Health (ClinicalTrials.gov identifier: NCT02890342). Results: Cross-sectional analysis of the creatinine-based estimated glomerular filtration rate (eGFR) in subjects with native kidneys revealed an age-dependent decline in renal function (P < 0.002). Among adults with PA, 4/8 (50%) had eGFR <60 mL/min/1.73 m 2. There was a significant discrepancy between eGFRs calculated using estimating equations based on serum creatinine compared with serum cystatin C (P < 0.0001). The tubular injury marker, plasma lipocalin-2, and plasma uric acid were strongly associated with CKD (P < 0.0001). The measured 24-hour creatinine excretion was below normal, even after adjusting for age, height, and sex. Conclusion: CKD is common in adults with PA and is associated with age. The poor predictive performance of standard eGFR estimating equations, likely due to reduced creatine synthesis in kidney and liver, could delay the recognition of CKD and management of ensuing complications in this population.
Immunometabolism aims to define the role of intermediary metabolism in immune cell function, with... more Immunometabolism aims to define the role of intermediary metabolism in immune cell function, with bioenergetics and the mitochondria recently taking center stage. To date, the medical literature on mitochondria and immune function extols the virtues of mouse models in exploring this biologic intersection. While the laboratory mouse has become a standard for studying mammalian biology, this model comprises part of a comprehensive approach. Humans, with their broad array of inherited phenotypes, serve as a starting point for studying immunometabolism; specifically, patients with mitochondrial disease. Using this top-down approach, the mouse as a model organism facilitates further exploration of the consequences of mutations involved in mitochondrial maintenance and function. In this review, we will discuss the emerging phenotype of immune dysfunction in mitochondrial disease as a model for understanding the role of the mitochondria in immune function in available mouse models.
T cell subsets including effector (T), regulatory (T), and memory (T) cells are characterized by ... more T cell subsets including effector (T), regulatory (T), and memory (T) cells are characterized by distinct metabolic profiles that influence their differentiation and function. Previous research suggests that engagement of long-chain fatty acid oxidation (LC-FAO) supports Foxp3 T cell and T cell survival. However, evidence for this is mostly based on inhibition of Cpt1a, the rate-limiting enzyme for LC-FAO, with the drug etomoxir. Using genetic models to target Cpt1a specifically in T cells, we dissected the role of LC-FAO in primary, memory, and regulatory T cell responses. Here we show that the ACC2/Cpt1a axis is largely dispensable for T, T, or T cell formation, and that the effects of etomoxir on T cell differentiation and function are independent of Cpt1a expression. Together our data argue that metabolic pathways other than LC-FAO fuel T or T differentiation and suggest alternative mechanisms for the effects of etomoxir that involve mitochondrial respiration.
CD8 T cells are key members of the adaptive immune response against infections and cancer. As we ... more CD8 T cells are key members of the adaptive immune response against infections and cancer. As we discuss in this review, these cells can present diverse metabolic requirements, which have been intensely studied during the past few years. Our current understanding suggests that aerobic glycolysis is a hallmark of activated CD8 T cells, while naive and memory (T ) cells often rely on oxidative phosphorylation, and thus mitochondrial metabolism is a crucial determinant of CD8 T cell development. Moreover, it has been proposed that CD8 T cells have a specific requirement for the oxidation of long-chain fatty acids (LC-FAO), a process modulated in lymphocytes by the enzyme CPT1A. However, this notion relies heavily on the metabolic analysis of in vitro cultures and on chemical inhibition of CPT1A. Therefore, we introduce more recent studies using genetic models to demonstrate that CPT1A-mediated LC-FAO is dispensable for the development of CD8 T cell memory and protective immunity, and q...
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