Cardiolipin deficiency disrupts CoQ redox state and induces steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disorder marked by lipid accumulation, leading to steatohepatitis (MASH). A key feature of the transition to MASH involves oxidative stress resulting from defects in mitochondrial oxidative phosphorylation (OXPHOS). Here, we show that pathological alterations in the lipid composition of the inner mitochondrial membrane (IMM) directly instigate electron transfer inefficiency to promote oxidative stress. Specifically, cardiolipin (CL) was downregulated across four mouse models of MASLD. Hepatocyte-specific CL synthase knockout (CLS-LKO) led to spontaneous MASH with elevated mitochondrial electron leak. Loss of CL interfered with the ability of coenzyme Q (CoQ) to transfer electrons, promoting leak primarily at sites IIF and IIIQ0. Data from human liver biopsies revealed a highly robust correlation between mitochondrial CL and CoQ, co-downregulated with MASH. Thus, reduction in mitochondrial CL promotes oxidative stress and contributes to pathogenesis of MASH.

Ketone flux through BDH1 supports metabolic remodeling of skeletal and cardiac muscles in response to intermittent time-restricted feeding.

Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart.

Weight loss increases skeletal muscle mitochondrial energy efficiency in obese mice.

Weight loss from an overweight state is associated with a disproportionate decrease in whole-body energy expenditure that may contribute to the heightened risk for weight regain. Evidence suggests that this energetic mismatch originates from lean tissue. Although this phenomenon is well documented, the mechanisms have remained elusive. We hypothesized that increased mitochondrial energy efficiency in skeletal muscle is associated with reduced expenditure under weight loss. Wildtype (WT) male C57BL6/N mice were fed with high fat diet for 10 weeks, followed by a subset of mice that were maintained on the obesogenic diet (OB) or switched to standard chow to promote weight loss (WL) for additional 6 weeks. Mitochondrial energy efficiency was evaluated using high-resolution respirometry and fluorometry. Mass spectrometric analyses were employed to describe the mitochondrial proteome and lipidome. Weight loss promoted ~50% increase in the efficiency of oxidative phosphorylation (ATP produced per O2 consumed, or P/O) in skeletal muscle. However, weight loss did not appear to induce significant changes in mitochondrial proteome, nor any changes in respiratory supercomplex formation. Instead, it accelerated the remodeling of mitochondrial cardiolipin (CL) acyl-chains to increase tetralinoleoyl CL (TLCL) content, a species of lipids thought to be functionally critical for the respiratory enzymes. We further show that lowering TLCL by deleting the CL transacylase tafazzin was sufficient to reduce skeletal muscle P/O and protect mice from diet-induced weight gain. These findings implicate skeletal muscle mitochondrial efficiency as a novel mechanism by which weight loss reduces energy expenditure in obesity.

Assessment of Bioenergetics in BAT Mitochondria.

High-resolution respirometry is commonly used to quantify mitochondrial respiratory rates. In the respirometry chamber, a change in oxygen concentration is measured by a polarographic electrode to derive the rate of oxygen consumption (JO2). Here, we describe our adapted protocol to bioenergetically phenotype mitochondria from mouse brown adipose tissue (BAT). Given the presence of uncoupling protein 1 (UCP1), mitochondria from BAT provide unique challenges and opportunities in applying high-resolution respirometry to understand energy transduction through oxidative phosphorylation (OXPHOS).

Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles.

Even-chain acylcarnitine (AC) metabolites, most of which are generated as byproducts of incomplete fatty acid oxidation (FAO), are viewed as biomarkers of mitochondrial lipid stress attributable to one or more metabolic bottlenecks in the ß-oxidation pathway. The origins and functional implications of FAO bottlenecks remain poorly understood. Here, we combined a sophisticated mitochondrial phenotyping platform with state-of-the-art molecular profiling tools and multiple two-state mouse models of respiratory function to uncover a mechanism that connects AC accumulation to lipid intolerance, metabolic inflexibility, and respiratory inefficiency in skeletal muscle mitochondria. These studies also identified a short-chain carbon circuit at the C4 node of FAO wherein reverse flux of glucose-derived acetyl CoA through medium-chain ketothiolase enhances lipid tolerance and redox stability in heart mitochondria by regenerating free CoA and NAD+. The findings help to explain why diminished FAO capacity, AC accumulation, and metabolic inflexibility are tightly linked to poor health outcomes.

Lipid hydroperoxides promote sarcopenia through carbonyl stress.

Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here, we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacological neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacological suppression.

Mitochondrial phosphatidylethanolamine modulates UCP1 to promote brown adipose thermogenesis.

Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of adenosine triphosphate (ATP) synthase. Here, we provide evidence that phosphatidylethanolamine (PE) modulates UCP1-dependent proton conductance across the IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to ß3 adrenergic receptor agonist-induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PE-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.

Blocking RAGE improves wound healing in diabetic pigs.

Receptor for Advanced Glycated End-products (RAGE) is highly expressed in diabetes and impairs wound healing. We proposed that administering an antibody that blocks RAGE will hasten the healing of dorsal wounds in diabetic pigs compared with a non-immune IgG. Two purpose-bred diabetic (D) Yucatan minipigs (Sinclair, Auxvasse MO) each underwent 12 2 × 2 cm full thickness dorsal wounds: four wounds received decellularized porcine skin patches (Xylyx Bio, Bklyn NY): four anti-RAGE Ab (CR-3) infused patches, four saline infused patches and four wounds were left open. One pig received anti-RAGE Ab (CR-3) 1 mg/kg IM q 10 days and other received non-immune IgG. Wounds were measured at 2 and 4 weeks followed by euthanasia and wound harvesting. At 2 weeks few of the patches appeared to be incorporated into the wound. By 4 weeks all patches in pigs treated systemically with CR-3 were detached and the wounds almost healed. For all 24 wounds for both pigs regardless of presence of patch or type of patch, the average IgG treated pig wound size at 4 weeks was 69.2 ± 14.6% of initial size and the average CR-3 treated pig wound size was 40.9 ± 11.3% of initial size (P = 0.0002). Quantitative immunohistology showed greater staining for collagen in the CR-3 treated wounds compared with IgG treated. Staining was positive for RAGE, Mac, and IL-6 in the IgG treated wounds and negative in the CR-3 treated wounds. From these pilot experiments, we conclude that a RAGE blocking antibody given parenterally improved wound healing in a diabetic pig while patches were not effective.

Lipid peroxidation does not mediate muscle atrophy induced by PSD deficiency.

Mechanisms by which disuse promotes skeletal muscle atrophy is not well understood. We previously demonstrated that disuse reduces the abundance of mitochondrial phosphatidylethanolamine (PE) in skeletal muscle. Deletion of phosphatidylserine decarboxylase (PSD), an enzyme that generates mitochondrial PE, was sufficient to promote muscle atrophy. In this study, we tested the hypothesis that muscle atrophy induced by PSD deletion is driven by an accumulation of lipid hydroperoxides (LOOH). Mice with muscle-specific knockout of PSD (PSD-MKO) were crossed with glutathione peroxidase 4 (GPx4) transgenic mice (GPx4Tg) to suppress the accumulation of LOOH. However, PSD-MKO × GPx4Tg mice and PSD-MKO mice demonstrated equally robust loss of muscle mass. These results suggest that muscle atrophy induced by PSD deficiency is not driven by the accumulation of LOOH.

Short-term exposure to a clinical dose of metformin increases skeletal muscle mitochondrial H2O2 emission and production in healthy, older adults: A randomized controlled trial.

BACKGROUND AND AIMS: Metformin is the most commonly prescribed medication to treat diabetes. Emerging evidence suggests that metformin could have off target effects that might help promote healthy muscle aging, but these effects have not been thoroughly studied in glucose tolerant older individuals. The purpose of this study was to investigate the short-term effects of metformin consumption on skeletal muscle mitochondrial bioenergetics in healthy older adults. METHODS: We obtained muscle biopsy samples from 16 healthy older adults previously naïve to metformin and treated with metformin (METF; 3F, 5M), or placebo (CON; 3F, 5M), for two weeks using a randomized and blinded study design. Samples were analyzed using high-resolution respirometry, immunofluorescence, and immunoblotting to assess muscle mitochondrial bioenergetics, satellite cell (SC) content, and associated protein markers. RESULTS: We found that metformin treatment did not alter maximal mitochondrial respiration rates in muscle compared to CON. In contrast, mitochondrial H2O2 emission and production were elevated in muscle samples from METF versus CON (METF emission: 2.59 ± 0.72 SE Fold, P = 0.04; METF production: 2.29 ± 0.53 SE Fold, P = 0.02). Furthermore, the change in H2O2 emission was positively correlated with the change in type 1 myofiber SC content and this was biased in METF participants (Pooled: R2 = 0.5816, P = 0.0006; METF: R2 = 0.674, P = 0.0125). CONCLUSIONS: These findings suggest that acute exposure to metformin does not impact mitochondrial respiration in aged, glucose-tolerant muscle, but rather, influences mitochondrial-free radical and SC dynamics. CLINICAL TRIAL REGISTRATION: NCT03107884, clinicaltrials.gov.

Low lysophosphatidylcholine induces skeletal muscle myopathy that is aggravated by high-fat diet feeding.

Obesity alters skeletal muscle lipidome and promotes myopathy, but it is unknown whether aberrant muscle lipidome contributes to the reduction in skeletal muscle contractile force-generating capacity. Comprehensive lipidomic analyses of mouse skeletal muscle revealed a very strong positive correlation between the abundance of lysophosphatidylcholine (lyso-PC), a class of lipids that is known to be downregulated with obesity, with maximal tetanic force production. The level of lyso-PC is regulated primarily by lyso-PC acyltransferase 3 (LPCAT3), which acylates lyso-PC to form phosphatidylcholine. Tamoxifen-inducible skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) was sufficient to reduce muscle lyso-PC content in both standard chow diet- and high-fat diet (HFD)-fed conditions. Strikingly, the assessment of skeletal muscle force-generating capacity ex vivo revealed that muscles from LPCAT3-MKI mice were weaker regardless of diet. Defects in force production were more apparent in HFD-fed condition, where tetanic force production was 40% lower in muscles from LPCAT3-MKI compared to that of control mice. These observations were partly explained by reductions in the cross-sectional area in type IIa and IIx fibers, and signs of muscle edema in the absence of fibrosis. Future studies will pursue the mechanism by which LPCAT3 may alter protein turnover to promote myopathy.

Lysophospholipid acylation modulates plasma membrane lipid organization and insulin sensitivity in skeletal muscle.

Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analysis of primary myocytes from individuals who were insulin-sensitive and lean (LN) or insulin-resistant with obesity (OB) revealed several species of lysophospholipids (lyso-PLs) that were differentially abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle-specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lysophosphatidylcholine/phosphatidylcholine, concomitant with improved skeletal muscle insulin sensitivity. Conversely, skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) promoted glucose intolerance. The absence of LPCAT3 reduced phospholipid packing of cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.

Neutralizing mitochondrial ROS does not rescue muscle atrophy induced by hindlimb unloading in female mice.

Excess reactive oxygen species (ROS) induced by physical inactivity is associated with muscle atrophy and muscle weakness. However, the role of mitochondrial ROS on disuse-induced muscle atrophy is not fully understood. The purpose of this study was to utilize a genetic strategy to examine the effect of neutralizing mitochondrial ROS on disuse-induced skeletal muscle atrophy. This was accomplished by placing wild-type (WT) and mitochondrial-targeted catalase-expressing (MCAT) littermate mice on 7 days of hindlimb unloading. After assessment of body weight and composition, muscles were analyzed for individual muscle mass, force-generating capacity, fiber type, cross-sectional area, and mitochondrial function, including H2O2 production. Despite a successful attenuation of mitochondrial ROS, MCAT mice were not protected from muscle atrophy. No differences were observed in body composition, lean mass, individual muscle masses, force-generating capacity, or muscle fiber cross-sectional area. These data suggest that neutralizing mitochondrial ROS is insufficient to suppress disuse-induced loss of skeletal muscle mass and contractile function.NEW & NOTEWORTHY The premise of this study was to examine the efficacy of genetic suppression of mitochondrial reactive oxygen species (ROS) to attenuate disuse-induced muscle atrophy and muscle weakness. Neutralization of mitochondrial ROS by MCAT expression was insufficient to rescue muscle atrophy and muscle weakness.

Alternative splicing of UCP1 by non-cell-autonomous action of PEMT.

OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Mitochondrial PE potentiates respiratory enzymes to amplify skeletal muscle aerobic capacity.

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. Here, 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. Unexpectedly, skeletal muscle-specific inhibition of mitochondria-autonomous synthesis of PE caused respiratory failure because of 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.

Peroxisome-derived lipids regulate adipose thermogenesis by mediating cold-induced mitochondrial fission.

Peroxisomes perform essential functions in lipid metabolism, including fatty acid oxidation and plasmalogen synthesis. Here, we describe a role for peroxisomal lipid metabolism in mitochondrial dynamics in brown and beige adipocytes. Adipose tissue peroxisomal biogenesis was induced in response to cold exposure through activation of the thermogenic coregulator PRDM16. Adipose-specific knockout of the peroxisomal biogenesis factor Pex16 (Pex16-AKO) in mice impaired cold tolerance, decreased energy expenditure, and increased diet-induced obesity. Pex16 deficiency blocked cold-induced mitochondrial fission, decreased mitochondrial copy number, and caused mitochondrial dysfunction. Adipose-specific knockout of the peroxisomal ß-oxidation enzyme acyl-CoA oxidase 1 (Acox1-AKO) was not sufficient to affect adiposity, thermogenesis, or mitochondrial copy number, but knockdown of the plasmalogen synthetic enzyme glyceronephosphate O-acyltransferase (GNPAT) recapitulated the effects of Pex16 inactivation on mitochondrial morphology and function. Plasmalogens are present in mitochondria and decreased with Pex16 inactivation. Dietary supplementation with plasmalogens increased mitochondrial copy number, improved mitochondrial function, and rescued thermogenesis in Pex16-AKO mice. These findings support a surprising interaction between peroxisomes and mitochondria regulating mitochondrial dynamics and thermogenesis.

Phospholipid methylation regulates muscle metabolic rate through Ca2+ transport efficiency.

The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins.1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca2+-ATPase (SERCA) to affect cellular metabolism.3-5 Recent evidence suggests that transport efficiency (Ca2+ uptake / ATP hydrolysis) of skeletal muscle SERCA can be uncoupled to increase energy expenditure and protect mice from diet-induced obesity.6,7 In isolated SR vesicles, membrane phospholipid composition is known to modulate SERCA efficiency.8-11 Here we show that skeletal muscle SR phospholipids can be altered to decrease SERCA efficiency and increase whole-body metabolic rate. The absence of skeletal muscle phosphatidylethanolamine (PE) methyltransferase (PEMT) promotes an increase in skeletal muscle and whole-body metabolic rate to protect mice from diet-induced obesity. The elevation in metabolic rate is caused by a decrease in SERCA Ca2+-transport efficiency, whereas mitochondrial uncoupling is unaffected. Our findings support the hypothesis that skeletal muscle energy efficiency can be reduced to promote protection from obesity.

Targeted overexpression of catalase to mitochondria does not prevent cardioskeletal myopathy in Barth syndrome.

Barth Syndrome (BTHS) is an X-linked recessive disorder characterized by cardiomyopathy and muscle weakness. The underlying cause of BTHS is a mutation in the tafazzin (TAZ) gene, a key enzyme of cardiolipin biosynthesis. The lack of CL arising from loss of TAZ function results in destabilization of the electron transport system, promoting oxidative stress that is thought to contribute to development of cardioskeletal myopathy. Indeed, in vitro studies demonstrate that mitochondria-targeted antioxidants improve contractile capacity in TAZ-deficient cardiomyocytes. The purpose of the present study was to determine if resolving mitochondrial oxidative stress would be sufficient to prevent cardiomyopathy and skeletal myopathy in vivo using a mouse model of BTHS. To this end we crossed mice that overexpress catalase in the mitochondria (MCAT mice) with TAZ-deficient mice (TAZKD) to produce TAZKD mice that selectively overexpress catalase in the mitochondria (TAZKD+MCAT mice). TAZKD+MCAT mice exhibited decreased mitochondrial H2O2 emission and lipid peroxidation compared to TAZKD littermates, indicating decreased oxidative stress. Despite the improvements in oxidative stress, TAZKD+MCAT mice developed cardiomyopathy and mild muscle weakness similar to TAZKD littermates. These findings indicate that resolving oxidative stress is not sufficient to suppress cardioskeletal myopathy associated with BTHS.