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The skeletal muscle is a metabolically heterogeneous tissue that plays a key role in maintaining whole-body glucose homeostasis. It is well known that muscle insulin resistance (IR) precedes the development of type 2 diabetes. There is a consensus that the accumulation of specific lipid species in the tissue can drive IR. However, the role of the mitochondrial fatty-acid β-oxidation in IR and, consequently, in the control of glucose uptake remains paradoxical: interventions that either inhibit or activate fatty-acid β-oxidation have been shown to prevent IR. We here discuss the current theories and evidence for the interplay between β-oxidation and glucose uptake in IR. To address the underlying intricacies, we (1) dive into the control of glucose uptake fluxes into muscle tissues using the framework of Metabolic Control Analysis, and (2) disentangle concepts of flux and catalytic capacities taking into account skeletal muscle heterogeneity. Finally, we speculate about hitherto unexplored mechanisms that could bring contrasting evidence together. Elucidating how β-oxidation is connected to muscle IR and the underlying role of muscle heterogeneity enhances disease understanding and paves the way for new treatments for type 2 diabetes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Defects in mitochondrial fatty acid β-oxidation (FAO) impair metabolic flexibility, which is an essential process for energy homeostasis. Very-long-chain acyl-CoA dehydrogenase (VLCADD; OMIM 609575) deficiency is the most common long-chain mitochondrial FAO disorder presenting with hypoglycemia as a common clinical manifestation. To prevent hypoglycemia, triheptanoin-a triglyceride composed of three heptanoates (C7) esterified with a glycerol backbone-can be used as a dietary treatment, since it is metabolized into precursors for gluconeogenesis. However, studies investigating the effect of triheptanoin on glucose homeostasis are limited. To understand the role of gluconeogenesis in the pathophysiology of long-chain mitochondrial FAO defects, we injected VLCAD-deficient (VLCAD -/- ) mice with 13 C 3 -glycerol in the presence and absence of heptanoate (C7). The incorporation of 13 C 3 -glycerol into blood glucose was higher in VLCAD -/- mice than in WT mice, whereas the difference disappeared in the presence of C7. The result correlates with 13 C enrichment of liver metabolites in VLCAD -/- mice. In contrast, the C7 bolus significantly decreased the 13 C enrichment. These data suggest that the increased contribution of gluconeogenesis to the overall glucose production in VLCAD -/- mice increases the need for gluconeogenesis substrate, thereby avoiding hypoglycemia. Heptanoate is a suitable substrate to induce glucose production in mitochondrial FAO defect.

Background: Monogenetic inborn errors of metabolism cause a wide phenotypic heterogeneity that may even differ between family members carrying the same genetic variant. Computational modelling of metabolic networks may identify putative sources of this inter-patient heterogeneity. Here, we mainly focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common inborn error of the mitochondrial fatty acid oxidation (mFAO). It is an enigma why some MCADD patients-if untreated-are at risk to develop severe metabolic decompensations, whereas others remain asymptomatic throughout life. We hypothesised that an ability to maintain an increased free mitochondrial CoA (CoASH) and pathway flux might distinguish asymptomatic from symptomatic patients., Results: We built and experimentally validated, for the first time, a kinetic model of the human liver mFAO. Metabolites were partitioned according to their water solubility between the bulk aqueous matrix and the inner membrane. Enzymes are also either membrane-bound or in the matrix. This metabolite partitioning is a novel model attribute and improved predictions. MCADD substantially reduced pathway flux and CoASH, the latter due to the sequestration of CoA as medium-chain acyl-CoA esters. Analysis of urine from MCADD patients obtained during a metabolic decompensation showed an accumulation of medium- and short-chain acylcarnitines, just like the acyl-CoA pool in the MCADD model. The model suggested some rescues that increased flux and CoASH, notably increasing short-chain acyl-CoA dehydrogenase (SCAD) levels. Proteome analysis of MCADD patient-derived fibroblasts indeed revealed elevated levels of SCAD in a patient with a clinically asymptomatic state. This is a rescue for MCADD that has not been explored before. Personalised models based on these proteomics data confirmed an increased pathway flux and CoASH in the model of an asymptomatic patient compared to those of symptomatic MCADD patients., Conclusions: We present a detailed, validated kinetic model of mFAO in human liver, with solubility-dependent metabolite partitioning. Personalised modelling of individual patients provides a novel explanation for phenotypic heterogeneity among MCADD patients. Further development of personalised metabolic models is a promising direction to improve individualised risk assessment, management and monitoring for inborn errors of metabolism., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Diet modulates the development of insulin resistance during aging. This includes tissue-specific alterations in insulin signaling and mitochondrial function, which ultimately affect glucose homeostasis. Exercise stimulates glucose clearance and mitochondrial lipid oxidation and also enhances insulin sensitivity (IS). It is not well known how exercise interacts with age and diet in the development of insulin resistance. To investigate this, oral glucose tolerance tests with tracers were conducted in mice ranging from 4 to 21 months of age, fed a low-fat diet (LFD) or high-fat diet (HFD) with or without life-long voluntary access to a running wheel (RW). We developed a computational model to derive glucose fluxes, which were commensurate with independent values from steady-state tracer infusions. Values for an IS index derived for peripheral tissues (IS-P) and one for the liver (IS-L) were steeply decreased by aging and an HFD. This preceded the age-dependent decline in the mitochondrial capacity to oxidize lipids. In young animals fed an LFD, RW access enhanced the IS-P concomitantly with the muscle β-oxidation capacity. Surprisingly, RW access completely prevented the age-dependent IS-L decrease; however this only occurred in animals fed an LFD. Therefore, this study indicates that endurance exercise can improve the age-dependent decline in organ-specific IS if paired with a healthy diet., Article Highlights: Exercise is a known strategy to improve insulin sensitivity (IS), whereas aging and a lipid-rich diet decrease IS. Using a tracer-based oral glucose tolerance test, we investigated how exercise, age, and diet interact in the development of tissue-specific insulin resistance. Exercise (voluntary access to a running wheel) mainly improved IS in animals fed a low-fat diet. In these animals, exercise improved peripheral IS only at young age but fully prevented the age-dependent decline of hepatic IS. The prevention of age-dependent decline in IS by exercise is tissue-specific and blunted by a lipid-rich diet., (© 2023 by the American Diabetes Association.)

Hyperglycaemia in pregnancy (HIP) is a pregnancy complication characterized by mild to moderate hyperglycaemia that negatively impacts short- and long-term health of mother and child. However, relationships between severity and timing of pregnancy hyperglycaemia and postpartum outcomes have not been systemically investigated. We investigated the impact of hyperglycaemia developing during pregnancy (gestational diabetes mellitus, GDM) or already present pre-mating (pre-gestational diabetes mellitus, PDM) on maternal health and pregnancy outcomes. GDM and PDM were induced in C57BL/6NTac mice by combined 60% high fat diet (HF) and low dose streptozotocin (STZ). Animals were screened for PDM prior to mating, and all underwent an oral glucose tolerance test on gestational day (GD)15. Tissues were collected at GD18 or at postnatal day (PN)15. Among HFSTZ-treated dams, 34% developed PDM and 66% developed GDM, characterized by impaired glucose-induced insulin release and inadequate suppression of endogenous glucose production. No increased adiposity or overt insulin resistance was observed. Furthermore, markers of non-alcoholic fatty liver disease (NAFLD) were significantly increased in PDM at GD18 and were positively correlated with basal glucose levels at GD18 in GDM dams. By PN15, NAFLD markers were also increased in GDM dams. Only PDM affected pregnancy outcomes such as litter size. Our findings indicate that GDM and PDM, resulting in disturbances of maternal glucose homeostasis, increase the risk of postpartum NAFLD development, related to the onset and severity of pregnancy hyperglycaemia. These findings signal a need for earlier monitoring of maternal glycaemia and more rigorous follow-up of maternal health after GDM and PDM pregnancy in humans. KEY POINTS: We studied the impact of high-fat diet/streptozotocin induced hyperglycaemia in pregnancy in mice and found that this impaired glucose tolerance and insulin release. Litter size and embryo survival were compromised by pre-gestational, but not by gestational, diabetes. Despite postpartum recovery from hyperglycaemia in a majority of dams, liver disease markers were further elevated by postnatal day 15. Maternal liver disease markers were associated with the severity of hyperglycaemia at gestational day 18. The association between hyperglycaemic exposure and non-alcoholic fatty liver disease signals a need for more rigorous monitoring and follow-up of maternal glycaemia and health in diabetic pregnancy in humans., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Diet modulates the development of insulin resistance during aging. This includes tissue-specific alterations in insulin signaling and mitochondrial function, which ultimately affect glucose homeostasis. Exercise stimulates glucose clearance, mitochondrial lipid oxidation and enhances insulin sensitivity. It is not well known how exercise interacts with age and diet in the development of insulin resistance. To investigate this, oral glucose tolerance tests (OGTT) with a tracer were conducted in mice ranging from 4 to 21 months of age, fed a low- (LFD) or high-fat diet (HFD), with or without life-long voluntary access to a running wheel (RW). We developed a computational model to derive glucose fluxes, which were commensurate with independent values from steady-state tracer infusions. Both insulin sensitivity indices derived for peripheral tissues and liver (IS-P and IS-L, respectively) were steeply decreased by aging and a HFD. This preceded the age-dependent decline in the mitochondrial capacity to oxidize lipids. In LFD young animals, RW access enhanced the IS-P concomitantly with the muscle β- oxidation capacity. Surprisingly, RW access completely prevented the age-dependent IS-L decrease, but only in LFD animals. This study indicates, therefore, that endurance exercise can improve the age-dependent decline in organ-specific IS mostly in the context of a healthy diet., (© 2023 by the American Diabetes Association.)

Skeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin resistance, butyrate, a short-chain fatty acid (SCFA) produced from dietary fibre fermentation, prevents it. This preventive role of butyrate has been attributed to histone deacetylase (HDAC)-mediated transcription regulation and activation of mitochondrial fatty-acid oxidation. Here we address the interplay between butyrate and the long-chain fatty acid palmitate and investigate how transcription, signalling and metabolism are integrated to result in the butyrate-induced skeletal muscle metabolism remodelling. Butyrate enhanced insulin sensitivity in palmitate-treated, insulin-resistant C2C12 cells, as shown by elevated insulin receptor 1 (IRS1) and pAKT protein levels and Slc2a4 (GLUT4) mRNA, which led to a higher glycolytic capacity. Long-chain fatty-acid oxidation capacity and other functional respiration parameters were not affected. Butyrate did upregulate mitochondrial proteins involved in its own oxidation, as well as concentrations of butyrylcarnitine and hydroyxybutyrylcarnitine. By knocking down the gene encoding medium-chain 3-ketoacyl-CoA thiolase (MCKAT, Acaa2), butyrate oxidation was inhibited, which amplified the effects of the SCFA on insulin sensitivity and glycolysis. This response was associated with enhanced HDAC inhibition, based on histone 3 acetylation levels. Butyrate enhances insulin sensitivity and induces glycolysis, without the requirement of upregulated long-chain fatty acid oxidation. Butyrate catabolism functions as an escape valve that attenuates HDAC inhibition. Thus, inhibition of butyrate oxidation indirectly prevents insulin resistance and stimulates glycolytic flux in myotubes treated with butyrate, most likely via an HDAC-dependent mechanism., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire essential pathway paves the way for establishment of species-, tissue-, and disease-specific metazoan models., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this study we demonstrated through analytic considerations and numerical studies that the mitochondrial fatty-acid β-oxidation can exhibit bistable-hysteresis behavior. In an experimentally validated computational model we identified a specific region in the parameter space in which two distinct stable and one unstable steady state could be attained with different fluxes. The two stable states were referred to as low-flux (disease) and high-flux (healthy) state. By a modular kinetic approach we traced the origin and causes of the bistability back to the distributive kinetics and the conservation of CoA, in particular in the last rounds of the β-oxidation. We then extended the model to investigate various interventions that may confer health benefits by activating the pathway, including (i) activation of the last enzyme MCKAT via its endogenous regulator p46-SHC protein, (ii) addition of a thioesterase (an acyl-CoA hydrolysing enzyme) as a safety valve, and (iii) concomitant activation of a number of upstream and downstream enzymes by short-chain fatty-acids (SCFA), metabolites that are produced from nutritional fibers in the gut. A high concentration of SCFAs, thioesterase activity, and inhibition of the p46Shc protein led to a disappearance of the bistability, leaving only the high-flux state. A better understanding of the switch behavior of the mitochondrial fatty-acid oxidation process between a low- and a high-flux state may lead to dietary and pharmacological intervention in the treatment or prevention of obesity and or non-alcoholic fatty-liver disease., Competing Interests: The authors have declared that no competing interests exist.

Background: The skeletal muscle plays a central role in glucose homeostasis through the uptake of glucose from the extracellular medium in response to insulin. A number of factors are known to disrupt the normal response to insulin leading to the emergence of insulin resistance (IR). Advanced age and a high-fat diet are factors that increase the susceptibility to IR, with lipid accumulation in the skeletal muscle being a key driver of this phenomenon. It is debated, however, whether lipid accumulation arises due to dietary lipid overload or from a decline of mitochondrial function. To gain insights into the interplay of diet and age in the flexibility of muscle lipid and glucose handling, we combined lipidomics, proteomics, mitochondrial function analysis and computational modelling to investigate young and aged mice on a low- or high-fat diet (HFD)., Results: As expected, aged mice were more susceptible to IR when given a HFD than young mice. The HFD induced intramuscular lipid accumulation specifically in aged mice, including C18:0-containing ceramides and diacylglycerols. This was reflected by the mitochondrial β-oxidation capacity, which was upregulated by the HFD in young, but not in old mice. Conspicuously, most β-oxidation proteins were upregulated by the HFD in both groups, but carnitine palmitoyltransferase 1B (CPT1B) declined in aged animals. Computational modelling traced the flux control mostly to CPT1B, suggesting a CPT1B-driven loss of flexibility to the HFD with age. Finally, in old animals, glycolytic protein levels were reduced and less flexible to the diet., Conclusion: We conclude that intramuscular lipid accumulation and decreased insulin sensitivity are not due to age-related mitochondrial dysfunction or nutritional overload alone, but rather to their combined effects. Moreover, we identify CPT1B as a potential target to counteract age-dependent intramuscular lipid accumulation and thereby IR., (© 2021. The Author(s).)

Dietary protein restriction has been demonstrated to improve metabolic health under various conditions. However, the relevance of ageing and age-related decline in metabolic flexibility on the effects of dietary protein restriction has not been addressed. Therefore, we investigated the effect of short-term dietary protein restriction on metabolic health in young and aged mice. Young adult (3 months old) and aged (18 months old) C57Bl/6J mice were subjected to a 3-month dietary protein restriction. Outcome parameters included fibroblast growth factor 21 (FGF21) levels, muscle strength, glucose tolerance, energy expenditure (EE) and transcriptomics of brown and white adipose tissue (WAT). Here, we report that a low-protein diet had beneficial effects in aged mice by reducing some aspects of age-related metabolic decline. These effects were characterized by increased plasma levels of FGF21, browning of subcutaneous WAT, increased body temperature and EE, while no changes were observed in glucose homeostasis and insulin sensitivity. Moreover, the low-protein diet used in this study was well-tolerated in aged mice indicated by the absence of adverse effects on body weight, locomotor activity and muscle performance. In conclusion, our study demonstrates that a short-term reduction in dietary protein intake can impact age-related metabolic health alongside increased FGF21 signalling, without negatively affecting muscle function. These findings highlight the potential of protein restriction as a strategy to induce EE and browning of WAT in aged individuals., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

PKAN, CoPAN, MePAN, and PDH-E2 deficiency share key phenotypic features but harbor defects in distinct metabolic processes. Selective damage to the globus pallidus occurs in these genetic neurodegenerative diseases, which arise from defects in CoA biosynthesis (PKAN, CoPAN), protein lipoylation (MePAN), and pyruvate dehydrogenase activity (PDH-E2 deficiency). Overlap of their clinical features suggests a common molecular etiology, the identification of which is required to understand their pathophysiology and design treatment strategies. We provide evidence that CoA-dependent activation of mitochondrial acyl carrier protein (mtACP) is a possible process linking these diseases through its effect on PDH activity. CoA is the source for the 4'-phosphopantetheine moiety required for the posttranslational 4'-phosphopantetheinylation needed to activate specific proteins. We show that impaired CoA homeostasis leads to decreased 4'-phosphopantetheinylation of mtACP. This results in a decrease of the active form of mtACP, and in turn a decrease in lipoylation with reduced activity of lipoylated proteins, including PDH. Defects in the steps of a linked CoA-mtACP-PDH pathway cause similar phenotypic abnormalities. By chemically and genetically re-activating PDH, these phenotypes can be rescued, suggesting possible treatment strategies for these diseases., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

During fasting, mitochondrial fatty-acid β-oxidation (mFAO) is essential for the generation of glucose by the liver. Children with a loss-of-function deficiency in the mFAO enzyme medium-chain acyl-Coenzyme A dehydrogenase (MCAD) are at serious risk of life-threatening low blood glucose levels during fasting in combination with intercurrent disease. However, a subset of these children remains asymptomatic throughout life. In MCAD-deficient (MCAD-KO) mice, glucose levels are similar to those of wild-type (WT) mice, even during fasting. We investigated if metabolic adaptations in the liver may underlie the robustness of this KO mouse. WT and KO mice were given a high- or low-fat diet and subsequently fasted. We analyzed histology, mitochondrial function, targeted mitochondrial proteomics, and transcriptome in liver tissue. Loss of MCAD led to a decreased capacity to oxidize octanoyl-CoA. This was not compensated for by altered protein levels of the short- and long-chain isoenzymes SCAD and LCAD. In the transcriptome, we identified subtle adaptations in the expression of genes encoding enzymes catalyzing CoA- and NAD(P)(H)-involving reactions and of genes involved in detoxification mechanisms. We discuss how these processes may contribute to robustness in MCAD-KO mice and potentially also in asymptomatic human subjects with a complete loss of MCAD activity.

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanistic target of rapamycin complex 1 (mTORC1) loss of function reduces adiposity whereas partial mTORC1 inhibition enhances fat deposition. Herein we evaluated how constitutive mTORC1 activation in adipocytes modulates adiposity in vivo. Mice with constitutive mTORC1 activation in adipocytes induced by tuberous sclerosis complex (Tsc)1 deletion and littermate controls were evaluated for body mass, energy expenditure, glucose and fatty acid metabolism, mitochondrial function, mRNA and protein contents. Adipocyte-specific Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, as well as adipocyte number and diameter, phenotypes that were associated with increased lipolysis, UCP-1 content (browning) and mRNA levels of pro-browning transcriptional factors C/EBPβ and ERRα. Adipocyte Tsc1 deletion enhanced mitochondrial oxidative activity, fatty acid oxidation and the expression of PGC-1α and PPARα in both visceral and subcutaneous fat. In brown adipocytes, however, Tsc1 deletion did not affect UCP-1 content and basal respiration. Adipocyte Tsc1 deletion also reduced visceral adiposity and enhanced glucose tolerance, liver and muscle insulin signaling and adiponectin secretion in mice fed with purified low- or high-fat diet. In conclusion, adipocyte-specific Tsc1 deletion enhances mitochondrial activity, induces browning and reduces visceral adiposity in mice., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Intermittent fasting (IF) is a dietary intervention often used as an alternative to caloric restriction (CR) and characterized by 24 hour cycles alternating ad libitum feeding and fasting. Although the consequences of CR are well studied, the effects of IF on redox status are not. Here, we address the effects of IF on redox state markers in different tissues in order to uncover how changes in feeding frequency alter redox balance in rats. IF rats displayed lower body mass due to decreased energy conversion efficiency. Livers in IF rats presented increased mitochondrial respiratory capacity and enhanced levels of protein carbonyls. Surprisingly, IF animals also presented an increase in oxidative damage in the brain that was not related to changes in mitochondrial bioenergetics. Conversely, IF promoted a substantial protection against oxidative damage in the heart. No difference in mitochondrial bioenergetics or redox homeostasis was observed in skeletal muscles of IF animals. Overall, IF affects redox balance in a tissue-specific manner, leading to redox imbalance in the liver and brain and protection against oxidative damage in the heart.