Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we ...identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.
•SIRT4 represses fatty acid oxidation and stimulates lipogenesis•SIRT4 deacetylates and represses MCD, regulating malonyl coA levels in vivo•SIRT4 loss increases exercise capacity and protects against diet-induced obesity
The concept of “metabolic inflexibility” was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to ...nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.
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► Comprehensive metabolic profiling links CrAT to whole body glucose homeostasis ► Muscle-specific ablation of Crat disrupts systemic glucose tolerance in mice ► CrAT deficiency disrupts nutrient control of PDH activity and substrate switching ► L-carnitine supplements improve glucose control in insulin-resistant humans
In vitro models of contractile human skeletal muscle hold promise for use in disease modeling and drug development, but exhibit immature properties compared to native adult muscle. To address this ...limitation, 3D tissue-engineered human muscles (myobundles) were electrically stimulated using intermittent stimulation regimes at 1 Hz and 10 Hz. Dystrophin in myotubes exhibited mature membrane localization suggesting a relatively advanced starting developmental maturation. One-week stimulation significantly increased myobundle size, sarcomeric protein abundance, calcium transient amplitude (∼2-fold), and tetanic force (∼3-fold) resulting in the highest specific force generation (19.3mN/mm2) reported for engineered human muscles to date. Compared to 1 Hz electrical stimulation, the 10 Hz stimulation protocol resulted in greater myotube hypertrophy and upregulated mTORC1 and ERK1/2 activity. Electrically stimulated myobundles also showed a decrease in fatigue resistance compared to control myobundles without changes in glycolytic or mitochondrial protein levels. Greater glucose consumption and decreased abundance of acetylcarnitine in stimulated myobundles indicated increased glycolytic and fatty acid metabolic flux. Moreover, electrical stimulation of myobundles resulted in a metabolic shift towards longer-chain fatty acid oxidation as evident from increased abundances of medium- and long-chain acylcarnitines. Taken together, our study provides an advanced in vitro model of human skeletal muscle with improved structure, function, maturation, and metabolic flux.
Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, ...including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.
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•CrAT is a mitochondrial enzyme that interconverts acetyl-CoA and acetylcarnitine•Acetylcarnitine in blood and tissues has emerged as a biomarker of energy surplus•Muscle contraction stimulates acetylcarnitine uptake, recycling, and oxidation•CrAT-mediated acetyl group buffering is essential for optimal exercise performance
Seiler et al. investigate exercise fatigue due to metabolic inertia and show that the nutritionally regulated enzyme carnitine acetyltransferase (CrAT), which interconverts acetyl-CoA and acetylcarnitine, plays a key role in modulating muscle energy economy. CrAT modulation could prove a useful strategy in delaying muscle fatigue and combatting exercise intolerance.
Chronic metabolic diseases have been linked to molecular signatures of mitochondrial dysfunction. Nonetheless, molecular remodeling of the transcriptome, proteome, and/or metabolome does not ...necessarily translate to functional consequences that confer physiologic phenotypes. The work here aims to bridge the gap between molecular and functional phenomics by developing and validating a multiplexed assay platform for comprehensive assessment of mitochondrial energy transduction. The diagnostic power of the platform stems from a modified version of the creatine kinase energetic clamp technique, performed in parallel with multiplexed analyses of dehydrogenase activities and ATP synthesis rates. Together, these assays provide diagnostic coverage of the mitochondrial network at a level approaching that gained by molecular “-omics” technologies. Application of the platform to a comparison of skeletal muscle versus heart mitochondria reveals mechanistic insights into tissue-specific distinctions in energy transfer efficiency. This platform opens exciting opportunities to unravel the connection between mitochondrial bioenergetics and human disease.
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•Bridging the gap between molecular and functional mitochondrial phenomics•An assay platform for diagnosing perturbations in mitochondrial energy transduction•Use of the creatine kinase energetic clamp technique to model in vivo bioenergetics•Insights into tissue-specific distinctions in energy transfer efficiency
Fisher-Wellman et al. develop and validate a multiplexed assay platform that permits deep and comprehensive phenotyping of mitochondrial bioenergetics under conditions that model in vivo fluctuations in energy supply and demand. The report provides a blueprint for building this platform and a workflow for executing and interpreting the assays.
Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary ...to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive β-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial β-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus,
mcd
−/− mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.
It has been proposed that skeletal muscle insulin resistance arises from the accumulation of intramyocellular lipid metabolites that impede insulin signaling, including diacylglycerol and ceramide. ...We determined the role of de novo ceramide synthesis in mediating muscle insulin resistance.
Mice were subjected to 12 weeks of diet-induced obesity (DIO), and then treated for 4 weeks with myriocin, an inhibitor of serine palmitoyl transferase-1 (SPT1), the rate-limiting enzyme of de novo ceramide synthesis.
After 12 weeks of DIO, C57BL/6 mice demonstrated a doubling in gastrocnemius ceramide content, which was completely reversed (141.5 ± 15.8 vs. 94.6 ± 10.2 nmol/g dry wt) via treatment with myriocin, whereas hepatic ceramide content was unaffected by DIO. Interestingly, myriocin treatment did not alter the DIO-associated increase in gastrocnemius diacyglycerol content, and the only correlation observed between lipid metabolite accumulation and glucose intolerance occurred with ceramide (R = 0.61). DIO mice treated with myriocin showed a complete reversal of glucose intolerance and insulin resistance which was associated with enhanced insulin-stimulated Akt and glycogen synthase kinase 3β phosphorylation. Furthermore, myriocin treatment also decreased intramyocellular ceramide content and prevented insulin resistance development in db/db mice. Finally, myriocin-treated DIO mice displayed enhanced oxygen consumption rates (3,041 ± 124 vs. 2,407 ± 124 ml/kg/h) versus their control counterparts.
Our results demonstrate that the intramyocellular accumulation of ceramide correlates strongly with the development of insulin resistance, and suggests that inhibition of SPT1 is a potentially promising target for the treatment of insulin resistance.
This study sought to examine the functional significance of mitochondrial protein acetylation using a double knockout (DKO) mouse model harboring muscle-specific deficits in acetyl-CoA buffering and ...lysine deacetylation, due to genetic ablation of carnitine acetyltransferase and Sirtuin 3, respectively. DKO mice are highly susceptible to extreme hyperacetylation of the mitochondrial proteome and develop a more severe form of diet-induced insulin resistance than either single KO mouse line. However, the functional phenotype of hyperacetylated DKO mitochondria is largely normal. Of the >120 measures of respiratory function assayed, the most consistently observed traits of a markedly heightened acetyl-lysine landscape are enhanced oxygen flux in the context of fatty acid fuel and elevated rates of electron leak. In sum, the findings challenge the notion that lysine acetylation causes broad-ranging damage to mitochondrial quality and performance and raise the possibility that acetyl-lysine turnover, rather than acetyl-lysine stoichiometry, modulates redox balance and carbon flux.
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•Mitochondria lacking CrAT and Sirt3 are susceptible to extreme protein acetylation•Hyperacetylation is accompanied by disturbances in redox balance and insulin action•Hyperacetylation does not affect mitochondrial respiration and enhances fat oxidation•Sirt3 flux and acetyl-lysine turnover promote a fuel switch from fat to glucose
Williams et al. show that double knockout (DKO) mice harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation are susceptible to extreme mitochondrial hyperacetylation and insulin resistance. However, DKO mitochondria have normal respiratory function and increased fat oxidation. The findings suggest that acetyl-lysine turnover, not stoichiometry, regulates mitochondrial fuel use.
In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export ...of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine dimunition was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete β-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.
The impaired capacity of skeletal muscle to switch between the oxidation of fatty acid (FA) and glucose is linked to disordered metabolic homeostasis. To understand how muscle FA oxidation affects ...systemic glucose, we studied mice with a skeletal muscle-specific deficiency of long-chain acyl-CoA synthetase (ACSL)1. ACSL1 deficiency caused a 91% loss of ACSL-specific activity and a 60-85% decrease in muscle FA oxidation. Acsl1(M-/-) mice were more insulin sensitive, and, during an overnight fast, their respiratory exchange ratio was higher, indicating greater glucose use. During endurance exercise, Acsl1(M-/-) mice ran only 48% as far as controls. At the time that Acsl1(M-/-) mice were exhausted but control mice continued to run, liver and muscle glycogen and triacylglycerol stores were similar in both genotypes; however, plasma glucose concentrations in Acsl1(M-/-) mice were ∼40 mg/dL, whereas glucose concentrations in controls were ∼90 mg/dL. Excess use of glucose and the likely use of amino acids for fuel within muscle depleted glucose reserves and diminished substrate availability for hepatic gluconeogenesis. Surprisingly, the content of muscle acyl-CoA at exhaustion was markedly elevated, indicating that acyl-CoAs synthesized by other ACSL isoforms were not available for β-oxidation. This compartmentalization of acyl-CoAs resulted in both an excessive glucose requirement and severely compromised systemic glucose homeostasis.
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