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.
The objective of this study was to test the hypothesis that exercise-stimulated muscle glucose uptake (MGU) is augmented by increasing mitochondrial reactive oxygen species (mtROS) scavenging capacity. This hypothesis was tested in genetically altered mice fed chow or a high-fat (HF) diet that accelerates mtROS formation. Mice overexpressing SOD2 ( sod2 Tg ), mitochondria-targeted catalase ( mcat Tg ), and combined SOD2 and mCAT (mtAO) were used to increase mtROS scavenging. mtROS was assessed by the H 2 O 2 emitting potential ( JH 2 O 2 ) in muscle fibers. sod2 Tg did not decrease JH 2 O 2 in chow-fed mice, but decreased JH 2 O 2 in HF-fed mice. mcat Tg and mtAO decreased JH 2 O 2 in both chow- and HF-fed mice. In parallel, the ratio of reduced to oxidized glutathione (GSH/GSSG) was unaltered in sod2 Tg in chow-fed mice, but was increased in HF-fed sod2 Tg and both chow- and HF-fed mcat Tg and mtAO. Nitrotyrosine, a marker of NO-dependent, reactive nitrogen species (RNS)-induced nitrative stress, was decreased in both chow- and HF-fed sod2 Tg , mcat Tg , and mtAO mice. This effect was not changed with exercise. Kg, an index of MGU was assessed using 2-[ 14 C]-deoxyglucose during exercise. In chow-fed mice, sod2 Tg , mcat Tg , and mtAO increased exercise Kg compared with wild types. Exercise Kg was also augmented in HF-fed sod2 Tg and mcat Tg mice but unchanged in HF-fed mtAO mice. In conclusion, mtROS scavenging is a key regulator of exercise-mediated MGU and this regulation depends on nutritional state.
Phosphatidylethanolamine methyltransferase (PEMT) generates phosphatidylcholine (PC), the most abundant phospholipid in the mitochondria and an important acyl chain donor for cardiolipin (CL) biosynthesis. Mice lacking PEMT (PEMTKO) are cold-intolerant when fed a high-fat diet (HFD) due to unclear mechanisms. The purpose of this study was to determine whether PEMT-derived phospholipids are important for the function of uncoupling protein 1 (UCP1) and thus for maintenance of core temperature.To test whether PEMT-derived phospholipids are important for UCP1 function, we examined cold-tolerance and brown adipose (BAT) mitochondria from PEMTKO mice with or without HFD feeding. We complemented these studies with experiments on mice lacking functional CL due to tafazzin knockdown (TAZKD). We generated several conditional mouse models to study the tissue-specific roles of PEMT, including mice with BAT-specific knockout of PEMT (PEMT-BKO).Chow- and HFD-fed PEMTKO mice completely lacked UCP1 protein in BAT, despite a lack of difference in mRNA levels, and the mice were accordingly cold-intolerant. While HFD-fed PEMTKO mice exhibited reduced mitochondrial CL content, this was not observed in chow-fed PEMTKO mice or TAZKD mice, indicating that the lack of UCP1 was not attributable to CL deficiency. Surprisingly, the PEMT-BKO mice exhibited normal UCP1 protein levels. Knockout of PEMT in the adipose tissue (PEMT-AKO), liver (PEMT-LKO), or skeletal muscle (PEMT-MKO) also did not affect UCP1 protein levels, suggesting that lack of PEMT in other non-UCP1-expressing cells communicates to BAT to suppress UCP1. Instead, we identified an untranslated UCP1 splice variant that was triggered during the perinatal period in the PEMTKO mice.PEMT is required for UCP1 splicing that yields functional protein. This effect is derived by PEMT in nonadipocytes that communicates to BAT during embryonic development. Future research will focus on identifying the non-cell-autonomous PEMT-dependent mechanism of UCP1 splicing.
Although nicotinamide nucleotide transhydrogenase (NNT)–deficient C57BL/6J (6J) mice are known to be highly susceptible to diet-induced metabolic disease, this notion stems primarily from comparisons of 6J mice to other inbred strains. To date, very few studies have directly compared metabolic disease susceptibility between NNT-deficient 6J mice and NNT-competent C57BL/6 substrains. In this study, comprehensive profiling of the metabolic response to a high-fat/high-sucrose diet (HFD) were compared across time in 6J and C57BL/6NJ (6N) mice. Given that increased peroxide exposure drives insulin resistance, coupled with the fact that NNT regulates peroxide detoxification, it was hypothesized that 6J mice would experience greater derangements in redox homeostasis/metabolic disease upon HFD exposure. Contrary to this, both lines were found to be highly susceptible to diet-induced metabolic disease, as evidenced by impairments in glucose tolerance as early as 24 h into the HFD. Moreover, various markers of the metabolic syndrome, as well as peroxide stress, were actually blunted, rather than exacerbated, in the 6J mice, likely reflecting compensatory increases in alterative redox-buffering pathways. Together, these data provide evidence that the susceptibility to HFD-induced metabolic disease is similar in the 6J and 6N substrains. Given the numerous genetic variances in the 6J stain, including loss of NNT function, these findings suggest that the 6N substrain is the more logical and representative genetic background model for metabolic studies.
Mitochondria generate and maintain a redox or “electrical” charge that is distributed throughout cells. The current work identifies a redox mechanism by which energy balance is continuously sensed and coupled to compensatory changes in energy expenditure.
We recently found that treatment with the creatine analogue β-guanidine propionic acid (βGPA) is sufficient to prevent increased mitochondrial H2O2 emission (mEH2O2) potential and to maintain insulin sensitivity in high fat (HF) fed SD rats. βGPA treatment lowers cellular energy charge raising the question as to whether AMPK or mEH2O2 is the primary factor linking βGPA to the prevention of insulin resistance. PURPOSE: To determine if βGPA induced improvements in mitochondrial function and insulin sensitivity during a HF diet are mediated by AMPK. METHODS: AMPK·2 subunit dominant negative mice and their wild-type littermates were fed (10 wks) standard chow (SC), 60% HF or HF plus βGPA (HF βGPA, 0.4 mg/g/d by gavage). RESULTS: Regardless of genotype, βGPA prevented the HF-induced body weight gain while both food intake and whole body oxygen consumption were significantly (P<0.05) increased. Respiratory quotient was lower with HF and further lowered by βGPA but unaffected by genotype. Locomotor activity was similar in all groups. HF decreased both whole body and muscle specific glucose uptake whereas both were preserved in HF- βGPA regardless of genotype. In permeabilized red gastrocnemius myofibers, even though mitochondrial maximal respiration was similar between all groups, mEH2O2 potential was ∼60% higher in HF vs SC. βGPA treatment prevented the HF effect and, remarkably, reduced mEH2O2 potential to levels ∼50% lower than SC, regardless of genotype. CONCLUSION: These data indicate that increased energy expenditure induced by βGPA, independent of AMPK, prevents the HF-induced increase in mEH2O2 potential and the development of insulin resistance, providing further evidence that the governance of mEH2O2 is a primary factor regulating insulin sensitivity in skeletal muscle. Supported by NIH DK073488TABLE
Abstract Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are regulated. Herein, we show that exercise training or muscle disuse alters mitochondrial membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, whereas removal of PE diminished, mitochondrial respiratory capacity. Surprisingly, skeletal muscle-specific inhibition of mitochondrial-autonomous synthesis of PE caused a respiratory failure due to metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. These findings highlight the previously underappreciated role of mitochondrial membrane phospholipids in dynamically controlling skeletal muscle energetics and function.
Biochem. J. (2019) 476 1521–1537, DOI: 10.1042/BCJ20190182The authors would like to include an additional acknowledgement for their article. The authors acknowledge the assistance of Dr Deborah M. Muoio from Duke University who helped develop the biochemical assay platform used in this article and whose intellectual discussions contributed to the initial idea for this study. The authors apologize for the omission of Dr Muoio from the article.
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