Insights into energy balance dysregulation from a mouse model of methylmalonic aciduria

Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria, present unique challenges to energetic homeostasis by disrupting energy-producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut)–type methylmalonic aciduria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared with littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator–activated receptor and Fgf21-controlled pathways. Together, these shed light on the mechanisms and adaptations behind energy imbalance in methylmalonic aciduria and provide insight into metabolic responses to chronic energy shortage, which may have important implications for disease understanding and patient management

Insights into energy balance dysregulation from a mouse model of methylmalonic aciduria

Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria (MMAuria), present unique challenges to energetic homeostasis by disrupting energy producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut) type MMAuria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared to littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator-activated receptor and Fgf21-controlled pathways. Together, these indicate hypometabolism, energetic inflexibility and increased stores at the expense of active tissue as energy shortage consequences

Ethylmalonyl-CoA decarboxylase (ECHDC1) prevents the synthesis of methyl- and ethyl-branched fatty acids

The purpose of the present thesis was to evaluate the function of ECHDC1, a cytosolic enzyme that decarboxylates ethylmalonyl-CoA (em-CoA) and to a lesser extent, methylmalonyl-CoA (mm-CoA). As reviewed in the Introduction, fatty acid synthase (FASN), a cytosolic enzyme, usually elongates acetyl-CoA by successive additions of malonyl-CoA to synthesize straight chain fatty acids (FAs). Less frequently, methyl-branched FAs can be synthesized by starting with CoA esters derived from branched-chain amino acids (replacing acetyl-CoA and forming iso and anteiso FAs), or by using mm-CoA instead of malonyl-CoA during the elongation. Cytosolic mmCoA is formed by a side-reaction of acetyl-CoA carboxylase (ACC) on propionyl-CoA. ACC also acts on butyryl-CoA, thereby forming em-CoA. Incorporation of em-CoA by FASN should lead to the formation of ethyl-branched FAs, but this formation has never been demonstrated. Our working hypothesis on the function of ECHDC1 was that by destroying the two side products of ACC, it would prevent the formation of branched-FAs. We first showed in the Results section that mammalian FASN is not only able to synthesize straight or methyl-branched FAs, but also ethyl-branched FAs. Yet, the catalytic activity observed with emCoA is very weak compared to those observed with malonyl-CoA and mm-CoA. Metabolomic analysis of ECHDC1 KO adipocytes revealed a several-fold increase in the amount of methyl-branched FAs, derived from propionate as shown by isotopic labeling. Feeding the KO cells with labeled ethylmalonate led to the formation of ethyl-branched FAs. Analysis of ECHDC1 KO mice confirmed an increase in the formation of methyl-branched FAs. Yet, the most striking observation was that this deficiency led to the appearance of ethyl-branched FAs in tissues. We also discovered a remarkable accumulation of medium chain ethyl-branched FAs. These species were found in liver mitochondria as CoA esters and were excreted in urine mainly as glycine and taurine conjugates in various states of oxidation. Stable isotope labeling experiments indicated that they were probably ethyl-branched. These findings highlight that, contrary to methyl-branched FAs, mammals are not able to metabolize ethyl-branched FAs. Metabolism owns an elegant double control to prevent their formation, (1) the FASN activity towards em-CoA is very weak and (2) ECHDC1 decarboxylates em-CoA, preventing its incorporation by FASN.(MED - Sciences médicales) -- UCL, 201

Disorders of Purine and Pyrimidine Metabolism

Purine nucleotides are essential cellular constituents that are involved in energy transfer, metabolic regulation, and synthesis of DNA and RNA. Purine metabolism can be divided into three pathways (. Fig. 32.1). 1. De novo purine synthesis (DNPS) comprises a series of 10 enzymatic reactions that are critical to purine formation. This pathway starts with the formation of phosphoribosyl pyrophosphate (PRPP) and leads to the synthesis of inosine monophosphate (IMP). Purine nucleotide synthesis is completed by the interconversion of IMP to adenosine and guanosine nucleotides. Adenosine monophosphate (AMP) synthesis from IMP requires adenylosuccinate synthase (ADSS) and adenylosuccinate lyase (ADSL), while the synthesis of guanosine monophosphate (GMP) occurs via IMP dehydrogenase (IMPDH) and GMP synthetase. Mononucleotides can further be phosphorylated into di- and trinucleotides and reduced into deoxyribonucleotides. Nucleoside triphosphates (NTPs) and their deoxy counterparts (dNTPs) are incorporated into RNA and DNA, involved in cell signaling or used as energy transfer in cells. 2. The catabolic pathway starts from GMP, IMP, and AMP. Its final product is uric acid, a poorly soluble metabolite excreted in urine, that tends to crystallize once its plasma concentration exceeds 6.5–7.0 mg/ dL (0.38–0.47 mmol/L). 3. The salvage pathway allows the recovery of purine bases and nucleosides. Guanine, hypoxanthine, and adenine, which are provided by food intake or catabolic pathways, are reconverted into GMP, IMP, and AMP, respectively. Salvage of the nucleosides adenosine and guanosine, and their deoxy counterparts, is catalyzed by several specific kinases. The salvage pathway additionally converts several pharmacological anticancer agents and antiviral nucleoside analogs into their active forms. Similar to that of purine nucleotides, the metabolism of pyrimidine nucleotides can be divided into three pathways (. Fig. 32.2). 1. The biosynthetic, de novo pathway starts with the formation of carbamoyl phosphate by cytosolic carbamoyl phosphate synthetase II (CPS II), which differs from mitochondrial CPS I, the latter catalyzing the first step of ureagenesis (7 Chap. 19). This is followed by the synthesis of uridine monophosphate (UMP) and, hence, of (deoxy)nucleoside triphosphates ((d)NTPs) used for RNA and DNA synthesis. 2. The catabolic pathway starts from CMP, UMP, and dTMP and yields β-alanine and β-aminoisobutyrate, which are converted into citric acid cycle intermediates. 3. The salvage pathway, which is composed of several kinases, converts pyrimidine nucleosides cytidine, uridine, and thymidine into their corresponding nucleotides CMP, UMP, and dTMP, respectively. This pathway also converts several pharmacological anticancer and antiviral nucleoside analogs into their active forms

Neurological presentations of inborn errors of purine and pyrimidine metabolism

Purines and pyrimidines are essential components as they are the building blocks of vital molecules, such as nucleic acids, coenzymes, signalling molecules, as well as energy transfer molecules. Purine and pyrimidine metabolism defects are characterised by abnormal concentrations of purines, pyrimidines and/or their metabolites in cells or body fluids. This phenomenon is due to a decreased or an increased activity of enzymes involved in this metabolism and has been reported in humans for over 60 years. This review provides an overview of neurological presentations of inborn errors of purine and pyrimidine metabolism. These conditions can lead to psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscular symptoms. Clinical signs are often nonspecific and thus overlooked, but some diseases are treatable and early diagnosis may improve the child’s future. Although these metabolic hereditary diseases are rare, they are most probably under-diagnosed. When confronted with suggestive clinical or laboratory signs, clinicians should prescribe genetic testing in association with a biochemical screening including thorough purine and pyrimidine metabolites analysis and/or specific enzyme evaluation. This is most likely going to increase the number of confirmed patients

Disorders of purine biosynthesis metabolism.

Purines are essential molecules that are components of vital biomolecules, such as nucleic acids, coenzymes, signaling molecules, as well as energy transfer molecules. The de novo biosynthesis pathway starts from phosphoribosylpyrophosphate (PRPP) and eventually leads to the synthesis of inosine monophosphate (IMP) by means of 10 sequential steps catalyzed by six different enzymes, three of which are bi-or tri-functional in nature. IMP is then converted into guanosine monophosphate (GMP) or adenosine monophosphate (AMP), which are further phosphorylated into nucleoside di- or tri-phosphates, such as GDP, GTP, ADP and ATP. This review provides an overview of inborn errors of metabolism pertaining to purine synthesis in humans, including either phosphoribosylpyrophosphate synthetase (PRS) overactivity or deficiency, as well as adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), and adenylosuccinate synthetase (ADSS) deficiencies. ITPase deficiency is being described as well. The clinical spectrum of these disorders is broad, including neurological impairment, such as psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscle presentations or consequences of hyperuricemia, such as gouty arthritis or kidney stones. Clinical signs are often nonspecific and, thus, overlooked. It is to be hoped that this is likely to be gradually overcome by using sensitive biochemical investigations and next-generation sequencing technologies

DBS are suitable for 1,5-anhydroglucitol monitoring in GSD1b and G6PC3-deficient patients taking SGLT2 inhibitors to treat neutropenia

Glycogen storage disease type Ib (GSD1b) and G6PC3-deficiency are rare autosomal recessive diseases caused by inactivating mutations in SLC37A4 (coding for G6PT) and G6PC3, respectively. Both diseases are characterized by neutropenia and neutrophil dysfunction due to the intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases. We recently showed that the use of SGLT2 inhibitor therapy to reduce tubular reabsorption of its precursor, 1,5-anhydroglucitol (1,5-AG), a glucose analog present in blood, successfully restored the neutropenia and neutrophil function in G6PC3-deficient and GSD1b patients. The intra-individual variability of response to the treatment and the need to adjust the dose during treatment, especially in pediatric populations, can only be efficiently optimized if the concentration of 1,5-AG in blood is monitored during treatment, together with the patients’ clinical signs and symptoms. Monitoring the 1,5-AG levels would be greatly simplified if it could be performed on dry blood spots (DBS) which are easy to collect, store and transport. The challenge is to know if a suitable method can be developed to perform accurate and reproducible assays for 1,5-AG using DBS. Here, we describe and validate an assay that quantifies 1,5-AG in DBS using isotopic dilution quantitation by LC-MS/MS that should greatly facilitate patients’ follow-up. 1,5-AG levels measured in plasma and DBS give comparable values. This assay was used to monitor the levels of 1,5-AG in DBS from 3 G6PC3-deficient and 6 GSD1b patients during treatment with SGLT2 inhibitors. We recommend this approach to verify the adequate therapeutical response and compliance to the treatment in G6PC3-deficient and GSD1b patients treated with SGLT2 inhibitors

DBS are suitable for 1,5-anhydroglucitol monitoring in GSD1b and G6PC3-deficient patients taking SGLT2 inhibitors to treat neutropenia

Glycogen storage disease type Ib (GSD1b) and G6PC3-deficiency are rare autosomal recessive diseases caused by inactivating mutations in SLC37A4 (coding for G6PT) and G6PC3, respectively. Both diseases are characterized by neutropenia and neutrophil dysfunction due to the intracellular accumulation of 1,5-anhydroglucitol-6-phos- phate (1,5-AG6P), a potent inhibitor of hexokinases. We recently showed that the use of SGLT2 inhibitor therapy to reduce tubular reabsorption of its precursor, 1,5-anhydroglucitol (1,5-AG), a glucose analog present in blood, successfully restored the neutropenia and neutrophil function in G6PC3-deficient and GSD1b patients. The intra- individual variability of response to the treatment and the need to adjust the dose during treatment, especially in pediatric populations, can only be efficiently optimized if the concentration of 1,5-AG in blood is monitored during treatment, together with the patients' clinical signs and symptoms. Monitoring the 1,5-AG levels would be greatly simplified if it could be performed on dry blood spots (DBS) which are easy to collect, store and transport. The challenge is to know if a suitable method can be developed to perform accurate and reproducible assays for 1,5-AG using DBS. Here, we describe and validate an assay that quantifies 1,5-AG in DBS using isotopic dilution quantitation by LC-MS/MS that should greatly facilitate patients' follow-up. 1,5-AG levels measured in plasma and DBS give comparable values. This assay was used to monitor the levels of 1,5-AG in DBS from 3 G6PC3- deficient and 6 GSD1b patients during treatment with SGLT2 inhibitors. We recommend this approach to verify the adequate therapeutical response and compliance to the treatment in G6PC3-deficient and GSD1b patients treated with SGLT2 inhibitors