Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

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

Highlights of the 2012 Research Workshop: Using Nutrigenomics and Metabolomics in Clinical Nutrition Research

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Glycerol-3-phosphate Acyltransferase (GPAT)-1, but Not GPAT4, Incorporates Newly Synthesized Fatty Acids into Triacylglycerol and Diminishes Fatty Acid Oxidation

Four glycerol-3-phosphate acyltransferase (GPAT) isoforms, each encoded by a separate gene, catalyze the initial step in glycerolipid synthesis; in liver, the major isoforms are GPAT1 and GPAT4. To determine whether each of these hepatic isoforms performs a unique function in the metabolism of fatty acid, we measured the incorporation of de novo synthesized fatty acid or exogenous fatty acid into complex lipids in primary mouse hepatocytes from control, Gpat1−/−, and Gpat4−/− mice. Although hepatocytes from each genotype incorporated a similar amount of exogenous fatty acid into triacylglycerol (TAG), only control and Gpat4−/− hepatocytes were able to incorporate de novo synthesized fatty acid into TAG. When compared with controls, Gpat1−/− hepatocytes oxidized twice as much exogenous fatty acid. To confirm these findings and to assess hepatic β-oxidation metabolites, we measured acylcarnitines in liver from mice after a 24-h fast and after a 24-h fast followed by 48 h of refeeding with a high sucrose diet to promote lipogenesis. Confirming the in vitro findings, the hepatic content of long-chain acylcarnitine in fasted Gpat1−/− mice was 3-fold higher than in controls. When compared with control and Gpat4−/− mice, after the fasting-refeeding protocol, Gpat1−/− hepatic TAG was depleted, and long-chain acylcarnitine content was 3.5-fold higher. Taken together, these data demonstrate that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acids into TAG and to divert them away from oxidation

Mitochondrial Overload and Incomplete Fatty Acid Oxidation Contribute to Skeletal Muscle Insulin Resistance

SummaryPrevious 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

Myocardial Lipin 1 knockout in mice approximates cardiac effects of human LPIN1 mutations

Lipin 1 is a bifunctional protein that is a transcriptional regulator and has phosphatidic acid (PA) phosphohydrolase activity, which dephosphorylates PA to generate diacylglycerol. Human lipin 1 mutations lead to episodic rhabdomyolysis, and some affected patients exhibit cardiac abnormalities, including exercise-induced cardiac dysfunction and cardiac triglyceride accumulation. Furthermore, lipin 1 expression is deactivated in failing heart, but the effects of lipin 1 deactivation in myocardium are incompletely understood. We generated mice with cardiac-specific lipin 1 KO (cs-Lpin1-/-) to examine the intrinsic effects of lipin 1 in the myocardium. Cs-Lpin1-/- mice had normal systolic cardiac function but mild cardiac hypertrophy. Compared with littermate control mice, PA content was higher in cs-Lpin1-/- hearts, which also had an unexpected increase in diacylglycerol and triglyceride content. Cs-Lpin1-/- mice exhibited diminished cardiac cardiolipin content and impaired mitochondrial respiration rates when provided with pyruvate or succinate as metabolic substrates. After transverse aortic constriction-induced pressure overload, loss of lipin 1 did not exacerbate cardiac hypertrophy or dysfunction. However, loss of lipin 1 dampened the cardiac ionotropic response to dobutamine and exercise endurance in association with reduced protein kinase A signaling. These data suggest that loss of lipin 1 impairs cardiac functional reserve, likely due to effects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling

Inhibition of De Novo Ceramide Synthesis Reverses Diet-Induced Insulin Resistance and Enhances Whole-Body Oxygen Consumption

OBJECTIVE - 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. RESEARCH DESIGN AND METHODS - 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. RESULTS - 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. CONCLUSIONS - 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. © 2010 by the American Diabetes Association.link_to_subscribed_fulltex

Mitochondrial Glycerol-3-phosphate Acyltransferase-1 Is Essential in Liver for the Metabolism of Excess Acyl-CoAs

In vitro studies suggest that the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform catalyzes the initial and rate-controlling step in glycerolipid synthesis and aids in partitioning acyl-CoAs toward triacylglycerol synthesis and away from degradative pathways. To determine whether the absence of mtGPAT1 would increase oxidation of acyl-CoAs and restrict the development of hepatic steatosis, we fed wild type and mtGPAT1-/- mice a diet high in fat and sucrose (HH) for 4 months to induce the development of obesity and a fatty liver. Control mice were fed a diet low in fat and sucrose (LL). With the HH diet, absence of mtGPAT1 resulted in increased partitioning of acyl-CoAs toward oxidative pathways, demonstrated by 60% lower hepatic triacylglycerol content and 2-fold increases in plasma beta-hydroxybutyrate, acylcarnitines, and hepatic mRNA expression of mitochondrial HMG-CoA synthase. Despite the increase in fatty acid oxidation, liver acyl-CoA levels were 3-fold higher in the mtGPAT1-/- mice fed both diets. A lack of difference in CPT1 and FAS mRNA expression between genotypes suggested that the increased acyl-CoA content was not because of increased de novo synthesis, but instead, to an impaired ability to use long-chain acyl-CoAs derived from the diet, even when the dietary fat content was low. Hyperinsulinemia and reduced glucose tolerance on the HH diet was greater in the mtGPAT1-/- mice, which did not suppress the expression of the gluconeogenic genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This study demonstrates that mtGPAT1 is essential for normal acyl-CoA metabolism, and that the absence of hepatic mtGPAT1 results in the partitioning of fatty acids away from triacylglycerol synthesis and toward oxidation and ketogenesis

Compartmentalized Acyl-CoA Metabolism in Skeletal Muscle Regulates Systemic Glucose Homeostasis

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. Acsl1M−/− mice were more insulin sensitive, and, during an overnight fast, their respiratory exchange ratio was higher, indicating greater glucose use. During endurance exercise, Acsl1M−/− mice ran only 48% as far as controls. At the time that Acsl1M−/− 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 Acsl1M−/− 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

Metabolomic analysis of insulin resistance across different mouse strains and diets

Insulin resistance is a major risk factor for many diseases. However, its underlying mechanism remains unclear in part because it is triggered by a complex relationship between multiple factors, including genes and the environment. Here, we used metabolomics combined with computational methods to identify factors that classified insulin resistance across individual mice derived from three different mouse strains fed two different diets. Three inbred ILSXISS strains were fed high-fat or chow diets and subjected to metabolic phenotyping and metabolomics analysis of skeletal muscle. There was significant metabolic heterogeneity between strains, diets, and individual animals. Distinct metabolites were changed with insulin resistance, diet, and between strains. Computational analysis revealed 113 metabolites that were correlated with metabolic phenotypes. Using these 113 metabolites, combined with machine learning to segregate mice based on insulin sensitivity, we identified C22:1-CoA, C2-carnitine, and C16-ceramide as the best classifiers. Strikingly, when these three metabolites were combined into one signature, they classified mice based on insulin sensitivity more accurately than each metabolite on its own or other published metabolic signatures. Furthermore, C22:1-CoA was 2.3-fold higher in insulin-resistant mice and correlated significantly with insulin resistance. We have identified a metabolomic signature composed of three functionally unrelated metabolites that accurately predicts whole-body insulin sensitivity across three mouse strains. These data indicate the power of simultaneous analysis of individual, genetic, and environmental variance in mice for identifying novel factors that accurately predict metabolic phenotypes like whole-body insulin sensitivity