Targeted metabolomics connects thioredoxin-interacting protein (TXNIP) to mitochondrial fuel selection and regulation of specific oxidoreductase enzymes in skeletal muscle.

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of ß-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.

Obesity and lipid stress inhibit carnitine acetyltransferase activity.

Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control.

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 diminution 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 beta-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.

Characterization of a primary brown adipocyte culture system derived from human fetal interscapular fat.

Brown fat has gained widespread attention as a potential therapeutic target to treat obesity and associated metabolic disorders. Indeed, the anti-obesity potential of multiple targets to stimulate both brown adipocyte differentiation and recruitment have been verified in rodent models. However, their therapeutic potential in humans is unknown due to the lack of a human primary brown adipocyte cell culture system. Likewise, the lack of a well-characterized human model has limited the discovery of novel targets for the activation of human brown fat. To address this current need, we aimed to identify and describe the first primary brown adipocyte cell culture system from human fetal interscapular brown adipose tissue. Pre-adipocytes isolated from non-viable human fetal interscapular tissue were expanded and cryopreserved. Cells were then thawed and plated alongside adult human subcutaneous and omental pre-adipocytes for subsequent differentiation and phenotypic characterization. Interscapular pre-adipocytes in cell culture differentiated into mature adipocytes that were morphologically indistinguishable from the adult white depots. Throughout differentiation, cultured human fetal interscapular adipocytes demonstrated increased expression of classical brown fat markers compared to subcutaneous and omental cells. Further, functional analysis revealed an elevation in fatty acid oxidation as well as maximal and uncoupled oxygen consumption in interscapular brown adipocytes compared to white control cells. These data collectively identify the brown phenotype of these cells. Thus, our primary cell culture system derived from non-viable human fetal interscapular brown adipose tissue provides a valuable tool for the study of human brown adipocyte biology and for the development of anti-obesity therapeutics.

Muscle-Specific Overexpression of PGC-1α Does Not Augment Metabolic Improvements in Response to Exercise and Caloric Restriction.

This study used mice with muscle-specific overexpression of PGC-1α, a transcriptional coactivator that promotes mitochondrial biogenesis, to determine whether increased oxidative potential facilitates metabolic improvements in response to lifestyle modification. MCK-PGC1α mice and nontransgenic (NT) littermates were fed a high-fat diet (HFD) for 10 weeks, followed by stepwise exposures to voluntary wheel running (HFD+Ex) and then 25% caloric restriction with exercise (Ex/CR), each for an additional 10 weeks with continued HFD. Running and CR improved weight and glucose control similarly in MCK-PGC1α and NT mice. Sedentary MCK-PGC1α mice were more susceptible to diet-induced glucose intolerance, and insulin action measured in isolated skeletal muscles remained lower in the transgenic compared with the NT group, even after Ex/CR. Comprehensive profiling of >200 metabolites and lipid intermediates revealed dramatic group-specific responses to the intervention but did not produce a lead candidate that tracked with changes in glucose tolerance irrespective of genotype. Instead, principal components analysis identified a chemically diverse metabolite cluster that correlated with multiple measures of insulin responsiveness. These findings challenge the notion that increased oxidative capacity defends whole-body energy homeostasis and suggest that the interplay between mitochondrial performance, lipotoxicity, and insulin action is more complex than previously proposed.

Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue during Exercise.

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.

Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility.

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.