Loss of mitochondria long-chain fatty acid oxidation impairs skeletal muscle contractility by disrupting myofibril structure and calcium homeostasis.

OBJECTIVE: Abnormal lipid metabolism in mammalian tissues can be highly deleterious, leading to organ failure. Carnitine Palmitoyltransferase 2 (CPT2) deficiency is an inherited metabolic disorder affecting the liver, heart, and skeletal muscle due to impaired mitochondrial oxidation of long-chain fatty acids (mLCFAO) for energy production. METHODS: However, the basis of tissue damage in mLCFAO disorders is not fully understood. Mice lacking CPT2 in skeletal muscle (Cpt2Sk-/-) were generated to investigate the nexus between mFAO deficiency and myopathy. RESULTS: Compared to controls, ex-vivo contractile force was reduced by 70% in Cpt2Sk-/- oxidative soleus muscle despite the preserved capacity to couple ATP synthesis to mitochondrial respiration on alternative substrates to long-chain fatty acids. Increased mitochondrial biogenesis, lipid accumulation, and the downregulation of 80% of dystrophin-related and contraction-related proteins severely compromised the structure and function of Cpt2Sk-/- soleus. CPT2 deficiency affected oxidative muscles more than glycolytic ones. Exposing isolated sarcoplasmic reticulum to long-chain acylcarnitines (LCACs) inhibited calcium uptake. In agreement, Cpt2Sk-/- soleus had decreased calcium uptake and significant accumulation of palmitoyl-carnitine, suggesting that LCACs and calcium dyshomeostasis are linked in skeletal muscle. CONCLUSIONS: Our data demonstrate that loss of CPT2 and mLCFAO compromise muscle structure and function due to excessive mitochondrial biogenesis, downregulation of the contractile proteome, and disruption of calcium homeostasis.

High-dose atorvastatin therapy progressively decreases skeletal muscle mitochondrial respiratory capacity in humans.

BACKGROUNDWhile the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODSEight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTSMaximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSIONThese findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDINGNIH R01 AR071263.

High Fat Diet-Induced Obesity Dysregulates Splenic B Cell Mitochondrial Activity.

Diet-induced obesity impairs mitochondrial respiratory responses in tissues that are highly metabolically active, such as the heart. However, less is known about the impact of obesity on the respiratory activity of specific cell types, such as splenic B cells. B cells are of relevance, as they play functional roles in obesity-induced insulin resistance, inflammation, and responses to infection. Here, we tested the hypothesis that high-fat-diet (HFD)-induced obesity could impair the mitochondrial respiration of intact and permeabilized splenic CD19+ B cells isolated from C57BL/6J mice and activated ex vivo with lipopolysaccharide (LPS). High-resolution respirometry was used with intact and permeabilized cells. To reveal potential mechanistic targets by which HFD-induced obesity dysregulates B cell mitochondria, we conducted proteomic analyses and 3D serial block face scanning electron microscopy (SBFEM). High-resolution respirometry revealed that intact LPS-stimulated B cells of obese mice, relative to controls, displayed lower ATP-linked, as well as maximal uncoupled, respiration. To directly investigate mitochondrial function, we used permeabilized LPS-stimulated B cells, which displayed increased H2O2 emission and production with obesity. We also examined oxidative phosphorylation efficiency simultaneously, which revealed that oxygen consumption and ATP production were decreased in LPS-stimulated B cells with obesity relative to controls. Despite minimal changes in total respiratory complex abundance, in LPS-stimulated B cells of obese mice, three of the top ten most downregulated proteins were all accessory subunits of respiratory complex I. SBFEM showed that B cells of obese mice, compared to controls, underwent no change in mitochondrial cristae integrity but displayed increased mitochondrial volume that was linked to bioenergetic function. Collectively, these results establish a proof of concept that HFD-induced obesity dysregulates the mitochondrial bioenergetic metabolism of activated splenic B cells.

Salvage surgeries for splanchnic artery aneurysms after failed endovascular therapy: case series.

INTRODUCTION: Splanchnic arterial aneurysms are a rare but potentially lethal disease with a mortality rate of more than 10% after rupture. Endovascular therapy is the first-line treatment for splanchnic aneurysms. However, appropriate management for splanchnic aneurysms after failed endovascular therapy remained inconclusive. MATERIALS AND METHODS: A retrospective review was performed for consecutive patients (from 2019 to 2022) who underwent salvage surgeries for splanchnic artery aneurysms following failed endovascular therapy. The authors defined failed endovascular therapy as the technical infeasibility to apply endovascular therapy, the incomplete exclusion of the aneurysm, or the incomplete resolution of preoperative aneurysm-associated complications. Salvage operations included aneurysmectomy with vascular reconstruction and partial aneurysmectomy with directly closing of bleeders from the intraluminal space of the aneurysms. RESULTS: Seventy-three patients received endovascular therapies for splanchnic aneurysms, and 13 failed endovascular trials. The authors performed salvage surgeries for five patients and enrolled them in this study, including four false aneurysms of the celiac or superior mesenteric arteries and a true aneurysm of the common hepatic artery. The causes of failed endovascular therapy included coil migration, insufficient space for safely deploying the covered stent, a persistent mass effect from the postembolized aneurysm, or infeasibility for catheter cannulation. The mean hospital stay was nine days (mean±SD, 8.8±1.6 days), with no one suffering 90-day surgical morbidity and mortality, and all patients getting symptoms improvement. During the follow-up period (mean±SD, 24±10 months), one patient suffered a small residual asymptomatic celiac artery aneurysm (8 mm in diameter) and was treated conservatively due to underlying liver cirrhosis. CONCLUSION: Surgical management is a feasible, effective, and safe alternative for splanchnic aneurysms after failed endovascular therapy.

TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling.

Cholinergic and sympathetic counter-regulatory networks control numerous physiological functions, including learning/memory/cognition, stress responsiveness, blood pressure, heart rate, and energy balance. As neurons primarily utilize glucose as their primary metabolic energy source, we generated mice with increased glycolysis in cholinergic neurons by specific deletion of the fructose-2,6-phosphatase protein TIGAR. Steady-state and stable isotope flux analyses demonstrated increased rates of glycolysis, acetyl-CoA production, acetylcholine levels, and density of neuromuscular synaptic junction clusters with enhanced acetylcholine release. The increase in cholinergic signaling reduced blood pressure and heart rate with a remarkable resistance to cold-induced hypothermia. These data directly demonstrate that increased cholinergic signaling through the modulation of glycolysis has several metabolic benefits particularly to increase energy expenditure and heat production upon cold exposure.

Skeletal muscle undergoes fiber type metabolic switch without myosin heavy chain switch in response to defective fatty acid oxidation.

OBJECTIVE: Skeletal muscle is a heterogeneous and dynamic tissue that adapts to functional demands and substrate availability by modulating muscle fiber size and type. The concept of muscle fiber type relates to its contractile (slow or fast) and metabolic (glycolytic or oxidative) properties. Here, we tested whether disruptions in muscle oxidative catabolism are sufficient to prompt parallel adaptations in energetics and contractile protein composition. METHODS: Mice with defective mitochondrial long-chain fatty acid oxidation (mLCFAO) in the skeletal muscle due to loss of carnitine palmitoyltransferase 2 (Cpt2Sk-/-) were used to model a shift in muscle macronutrient catabolism. Glycolytic and oxidative muscles of Cpt2Sk-/- mice and control littermates were compared for the expression of energy metabolism-related proteins, mitochondrial respiratory capacity, and myosin heavy chain isoform composition. RESULTS: Differences in bioenergetics and macronutrient utilization in response to energy demands between control muscles were intrinsic to the mitochondria, allowing for a clear distinction of muscle types. Loss of CPT2 ablated mLCFAO and resulted in mitochondrial biogenesis occurring most predominantly in oxidative muscle fibers. The metabolism-related proteomic signature of Cpt2Sk-/- oxidative muscle more closely resembled that of glycolytic muscle than of control oxidative muscle. Respectively, intrinsic substrate-supported mitochondrial respiration of CPT2 deficient oxidative muscles shifted to closely match that of glycolytic muscles. Despite this shift in mitochondrial metabolism, CPT2 deletion did not result in contractile-based fiber type switching according to myosin heavy chain composition analysis. CONCLUSION: The loss of mitochondrial long-chain fatty acid oxidation elicits an adaptive response involving conversion of oxidative muscle toward a metabolic profile that resembles a glycolytic muscle, but this is not accompanied by changes in myosin heavy chain isoforms. These data suggest that shifts in muscle catabolism are not sufficient to drive shifts in the contractile apparatus but are sufficient to drive adaptive changes in metabolic properties.

Genetically increasing flux through ß-oxidation in skeletal muscle increases mitochondrial reductive stress and glucose intolerance.

Elevated mitochondrial hydrogen peroxide (H2O2) emission and an oxidative shift in cytosolic redox environment have been linked to high-fat-diet-induced insulin resistance in skeletal muscle. To test specifically whether increased flux through mitochondrial fatty acid oxidation, in the absence of elevated energy demand, directly alters mitochondrial function and redox state in muscle, two genetic models characterized by increased muscle ß-oxidation flux were studied. In mice overexpressing peroxisome proliferator-activated receptor-α in muscle (MCK-PPARα), lipid-supported mitochondrial respiration, membrane potential (ΔΨm), and H2O2 production rate (JH2O2) were increased, which coincided with a more oxidized cytosolic redox environment, reduced muscle glucose uptake, and whole body glucose intolerance despite an increased rate of energy expenditure. Similar results were observed in lipin-1-deficient, fatty-liver dystrophic mice, another model characterized by increased ß-oxidation flux and glucose intolerance. Crossing MCAT (mitochondria-targeted catalase) with MCK-PPARα mice normalized JH2O2 production, redox environment, and glucose tolerance, but surprisingly, both basal and absolute insulin-stimulated rates of glucose uptake in muscle remained depressed. Also surprising, when placed on a high-fat diet, MCK-PPARα mice were characterized by much lower whole body, fat, and lean mass as well as improved glucose tolerance relative to wild-type mice, providing additional evidence that overexpression of PPARα in muscle imposes more extensive metabolic stress than experienced by wild-type mice on a high-fat diet. Overall, the findings suggest that driving an increase in skeletal muscle fatty acid oxidation in the absence of metabolic demand imposes mitochondrial reductive stress and elicits multiple counterbalance metabolic responses in an attempt to restore bioenergetic homeostasis.NEW & NOTEWORTHY Prior work has suggested that mitochondrial dysfunction is an underlying cause of insulin resistance in muscle because it limits fatty acid oxidation and therefore leads to the accumulation of cytotoxic lipid intermediates. The implication has been that therapeutic strategies to accelerate ß-oxidation will be protective. The current study provides evidence that genetically increasing flux through ß-oxidation in muscle imposes reductive stress that is not beneficial but rather detrimental to metabolic regulation.

Preference and detrimental effects of high fat, sugar, and salt diet in wild-caught Drosophila simulans are reversed by flight exercise.

High saturated fat, sugar, and salt contents are a staple of a Western diet (WD), contributing to obesity, metabolic syndrome, and a plethora of other health risks. However, the combinatorial effects of these ingredients have not been fully evaluated. Here, using the wild-caught Drosophila simulans, we show that a diet enriched with saturated fat, sugar, and salt is more detrimental than each ingredient separately, resulting in a significantly decreased lifespan, locomotor activity, sleep, reproductive function, and mitochondrial function. These detrimental effects were more pronounced in female than in male flies. Adding regular flight exercise to flies on the WD markedly negated the adverse effects of a WD. At the molecular level, the WD significantly increased levels of triglycerides and caused mitochondrial dysfunction, while exercise counterbalanced these effects. Interestingly, fruit flies developed a preference for the WD after pre-exposure, which was averted by flight exercise. The results demonstrate that regular aerobic exercise can mitigate adverse dietary effects on fly mitochondrial function, physiology, and feeding behavior. Our data establish Drosophila simulans as a novel model of diet-exercise interaction that bears a strong similarity to the pathophysiology of obesity and eating disorders in humans.

Aglycemic growth enhances carbohydrate metabolism and induces sensitivity to menadione in cultured tumor-derived cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent form of liver malignancy and carries poor prognoses due to late presentation of symptoms. Treatment of late-stage HCC relies heavily on chemotherapeutics, many of which target cellular energy metabolism. A key platform for testing candidate chemotherapeutic compounds is the intrahepatic orthotopic xenograft (IOX) model in rodents. Translational efficacy from the IOX model to clinical use is limited (in part) by variation in the metabolic phenotypes of the tumor-derived cells that can be induced by selective adaptation to subculture conditions. METHODS: In this study, a detailed multilevel systems approach combining microscopy, respirometry, potentiometry, and extracellular flux analysis (EFA) was utilized to examine metabolic adaptations that occur under aglycemic growth media conditions in HCC-derived (HEPG2) cells. We hypothesized that aglycemic growth would result in adaptive "aerobic poise" characterized by enhanced capacity for oxidative phosphorylation over a range of physiological energetic demand states. RESULTS: Aglycemic growth did not invoke adaptive changes in mitochondrial content, network complexity, or intrinsic functional capacity/efficiency. In intact cells, aglycemic growth markedly enhanced fermentative glycolytic substrate-level phosphorylation during glucose refeeding and enhanced responsiveness of both fermentation and oxidative phosphorylation to stimulated energy demand. Additionally, aglycemic growth induced sensitivity of HEPG2 cells to the provitamin menadione at a 25-fold lower dose compared to control cells. CONCLUSIONS: These findings indicate that growth media conditions have substantial effects on the energy metabolism of subcultured tumor-derived cells, which may have significant implications for chemotherapeutic sensitivity during incorporation in IOX testing panels. Additionally, the metabolic phenotyping approach used in this study provides a practical workflow that can be incorporated with IOX screening practices to aid in deciphering the metabolic underpinnings of chemotherapeutic drug sensitivity.

Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure.

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H2O2 (JH2O2) production. H2O2 is reduced to H2O by electrons supplied by NADPH. NADP+ is reduced back to NADPH by activation of mitochondrial membrane potential-dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH2O2 production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through ß-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP- and low-ADP-stimulated respiration that accelerating flux through ß-oxidation generates a corresponding increase in mitochondrial JH2O2 production, that the majority (∼70-80%) of H2O2 produced is reduced to H2O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within ß-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH2O2 production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.

Estrogen receptor-α in female skeletal muscle is not required for regulation of muscle insulin sensitivity and mitochondrial regulation.

OBJECTIVE: Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development. METHODS: A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women. RESULTS: Twelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity. CONCLUSIONS: Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete.

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.

Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform.

Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

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.

Differential Dopamine D1 and D3 Receptor Modulation and Expression in the Spinal Cord of Two Mouse Models of Restless Legs Syndrome.

Restless Legs Syndrome (RLS) is often and successfully treated with dopamine receptor agonists that target the inhibitory D3 receptor subtype, however there is no clinical evidence of a D3 receptor dysfunction in RLS patients. In contrast, genome-wide association studies in RLS patients have established that a mutation of the MEIS1 gene is associated with an increased risk in developing RLS, but the effect of MEIS1 dysfunction on sensorimotor function remain unknown. Mouse models for a dysfunctional D3 receptor (D3KO) and Meis1 (Meis1KO) were developed independently, and each animal expresses some features associated with RLS in the clinic, but they have not been compared in their responsiveness to treatment options used in the clinic. We here confirm that D3KO and Meis1KO animals show increased locomotor activities, but that only D3KO show an increased sensory excitability to thermal stimuli. Next we compared the effects of dopaminergics and opioids in both animal models, and we assessed D1 and D3 dopamine receptor expression in the spinal cord, the gateway for sensorimotor processing. We found that Meis1KO share most of the tested behavioral properties with their wild type (WT) controls, including the modulation of the thermal pain withdrawal reflex by morphine, L-DOPA and D3 receptor (D3R) agonists and antagonists. However, Meis1KO and D3KO were behaviorally more similar to each other than to WT when tested with D1 receptor (D1R) agonists and antagonists. Subsequent Western blot analyses of D1R and D3R protein expression in the spinal cord revealed a significant increase in D1R but not D3R expression in Meis1KO and D3KO over WT controls. As the D3R is mostly present in the dorsal spinal cord where it has been shown to modulate sensory pathways, while activation of the D1Rs can activate motoneurons in the ventral spinal cord, we speculate that D3KO and Meis1KO represent two complementary animal models for RLS, in which the mechanisms of sensory (D3R-mediated) and motor (D1R-mediated) dysfunctions can be differentially explored.

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.

Impact of 17ß-estradiol on complex I kinetics and H2O2 production in liver and skeletal muscle mitochondria.

Naturally or surgically induced postmenopausal women are widely prescribed estrogen therapies to alleviate symptoms associated with estrogen loss and to lower the subsequent risk of developing metabolic diseases, including diabetes and nonalcoholic fatty liver disease. However, the molecular mechanisms by which estrogens modulate metabolism across tissues remain ill-defined. We have previously reported that 17ß-estradiol (E2) exerts antidiabetogenic effects in ovariectomized (OVX) mice by protecting mitochondrial and cellular redox function in skeletal muscle. The liver is another key tissue for glucose homeostasis and a target of E2 therapy. Thus, in the present study we determined the effects of acute loss of ovarian E2 and E2 administration on liver mitochondria. In contrast to skeletal muscle mitochondria, E2 depletion via OVX did not alter liver mitochondrial respiratory function or complex I (CI) specific activities (NADH oxidation, quinone reduction, and H2O2 production). Surprisingly, in vivo E2 replacement therapy and in vitro E2 exposure induced tissue-specific effects on both CI activity and on the rate and topology of CI H2O2 production. Overall, E2 therapy protected and restored the OVX-induced reduction in CI activity in skeletal muscle, whereas in liver mitochondria E2 increased CI H2O2 production and decreased ADP-stimulated respiratory capacity. These results offer novel insights into the tissue-specific effects of E2 on mitochondrial function.

High Incomplete Skeletal Muscle Fatty Acid Oxidation Explains Low Muscle Insulin Sensitivity in Poorly Controlled T2D.

Context: Almost 50% of type 2 diabetic (T2D) patients are poorly controlled [glycated hemoglobin (HbA1c) ≥ 7%]; however, the mechanisms responsible for progressively worsening glycemic control are poorly understood. Lower skeletal muscle mitochondrial respiratory capacity is associated with low insulin sensitivity and the development of T2D. Objective: We investigated if skeletal muscle insulin sensitivity (SI) was different between well-controlled T2D (WCD) and poorly controlled T2D (PCD) and if the difference was associated with differences resulting from mitochondrial respiratory function. Design: Vastus lateralis muscle mitochondrial respiration, mitochondrial content, mitochondrial enzyme activity, and fatty acid oxidation (FAO) were measured. SI and the acute response to glucose (AIRg) were calculated by MINMOD analysis from glucose and insulin obtained during a modified, frequently sampled, intravenous glucose tolerance test. Results: SI and AIRg were lower in PCD than WCD. Muscle incomplete FAO was greater in PCD than WCD and greater incomplete FAO was associated with lower SI and higher HbA1c. Hydroxyacyl-coenzyme A dehydrogenase expression and activity were greater in PCD than WCD. There was no difference in maximal mitochondrial respiration or content between WCD and PCD. Conclusion: The current results suggest that greater skeletal muscle incomplete FAO in poorly controlled T2D is due to elevated ß oxidation and is associated with worsening muscle SI.