Impact of biological sex and sex hormones on molecular signatures of skeletal muscle at rest and in response to distinct exercise training modes.

Substantial divergence in cardio-metabolic risk, muscle size, and performance exists between men and women. Considering the pivotal role of skeletal muscle in human physiology, we investigated and found, based on RNA sequencing (RNA-seq), that differences in the muscle transcriptome between men and women are largely related to testosterone and estradiol and much less related to genes located on the Y chromosome. We demonstrate inherent unique, sex-dependent differences in muscle transcriptional responses to aerobic, resistance, and combined exercise training in young and older cohorts. The hormonal changes with age likely explain age-related differential expression of transcripts. Furthermore, in primary human myotubes we demonstrate the profound but distinct effects of testosterone and estradiol on amino acid incorporation to multiple individual proteins with specific functions. These results clearly highlight the potential of designing exercise programs tailored specifically to men and women and have implications for people who change gender by altering their hormone profile.

Characterization of cellular senescence in aging skeletal muscle.

Senescence is a cell fate that contributes to multiple aging-related pathologies. Despite profound age-associated changes in skeletal muscle (SkM), whether its constituent cells are prone to senesce has not been methodically examined. Herein, using single cell and bulk RNA-sequencing and complementary imaging methods on SkM of young and old mice, we demonstrate that a subpopulation of old fibroadipogenic progenitors highly expresses p16 Ink4a together with multiple senescence-related genes and, concomitantly, exhibits DNA damage and chromatin reorganization. Through analysis of isolated myofibers, we also detail a senescence phenotype within a subset of old cells, governed instead by p2 Cip1 . Administration of a senotherapeutic intervention to old mice countered age-related molecular and morphological changes and improved SkM strength. Finally, we found that the senescence phenotype is conserved in SkM from older humans. Collectively, our data provide compelling evidence for cellular senescence as a hallmark and potentially tractable mediator of SkM aging.

Impact of obesity on the molecular response to a single bout of exercise in a preliminary human cohort.

OBJECTIVE: The health benefits of exercise are well documented, but several exercise-response parameters are attenuated in individuals with obesity. The goal of this pilot study was to identify molecular mechanisms that may influence exercise response with obesity. METHODS: A multi-omics comparison of the transcriptome, proteome, and phosphoproteome in muscle from a preliminary cohort of lean individuals (n = 4) and individuals with obesity (n = 4) was performed, before and after a single bout of 30 minutes of unilateral cycling at 70% maximal oxygen uptake (VO2 peak). Mass spectrometry and RNA sequencing were used to interrogate the proteome, phosphoproteome, and transcriptome from muscle biopsy tissue. RESULTS: The main findings are that individuals with obesity exhibited transcriptional and proteomic signatures consistent with reduced mitochondrial function, protein synthesis, and glycogen synthesis. Furthermore, individuals with obesity demonstrated markedly different transcriptional, proteomic, and phosphoproteomic responses to exercise, particularly biosynthetic pathways of glycogen synthesis and protein synthesis. Casein kinase II subunit alpha and glycogen synthase kinase-3ß signaling was identified as exercise-response pathways that were notably altered by obesity. CONCLUSIONS: Opportunities to enhance exercise responsiveness by targeting specific molecular pathways that are disrupted in skeletal muscle from individuals with obesity await a better understanding of the precise molecular mechanisms that may limit exercise-response pathways in obesity.

Skeletal muscle mitochondrial dysfunction and muscle and whole body functional deficits in cancer patients with weight loss.

Reductions in skeletal muscle mass and function are often reported in patients with cancer-associated weight loss and are associated with reduced quality of life, impaired treatment tolerance, and increased mortality. Although cellular changes, including altered mitochondrial function, have been reported in animals, such changes have been incompletely characterized in humans with cancer. Whole body and skeletal muscle physical function, skeletal muscle mitochondrial function, and whole body protein turnover were assessed in eight patients with cancer-associated weight loss (10.1 ± 4.2% body weight over 6-12 mo) and 19 age-, sex-, and body mass index (BMI)-matched healthy controls to characterize skeletal muscle changes at the whole body, muscle, and cellular level. Potential pathways involved in cancer-induced alterations in metabolism and mitochondrial function were explored by interrogating skeletal muscle and plasma metabolomes. Despite similar lean mass compared with control participants, patients with cancer exhibited reduced habitual physical activity (57% fewer daily steps), cardiorespiratory fitness [22% lower V̇o2peak (mL/kg/min)] and leg strength (35% lower isokinetic knee extensor strength), and greater leg neuromuscular fatigue (36% greater decline in knee extensor torque). Concomitant with these functional declines, patients with cancer had lower mitochondrial oxidative capacity [25% lower State 3 O2 flux (pmol/s/mg tissue)] and ATP production [23% lower State 3 ATP production (pmol/s/mg tissue)] and alterations in phospholipid metabolite profiles indicative of mitochondrial abnormalities. Whole body protein turnover was unchanged. These findings demonstrate mitochondrial abnormalities concomitant with whole body and skeletal muscle functional derangements associated with human cancer, supporting future work studying the role of mitochondria in the muscle deficits associated with cancer.NEW & NOTEWORTHY To our knowledge, this is the first study to suggest that skeletal muscle mitochondrial deficits are associated with cancer-associated weight loss in humans. Mitochondrial deficits were concurrent with reductions in whole body and skeletal muscle functional capacity. Whether mitochondrial deficits are causal or secondary to cancer-associated weight loss and functional deficits remains to be determined, but this study supports further exploration of mitochondria as a driver of cancer-associated losses in muscle mass and function.

Impaired Muscle Mitochondrial Function in Familial Partial Lipodystrophy.

CONTEXT: Familial partial lipodystrophy (FPL), Dunnigan variety is characterized by skeletal muscle hypertrophy and insulin resistance besides fat loss from the extremities. The cause for the muscle hypertrophy and its functional consequences is not known. OBJECTIVE: To compare muscle strength and endurance, besides muscle protein synthesis rate between subjects with FPL and matched controls (n = 6 in each group). In addition, we studied skeletal muscle mitochondrial function and gene expression pattern to help understand the mechanisms for the observed differences. METHODS: Body composition by dual-energy X-ray absorptiometry, insulin sensitivity by minimal modelling, assessment of peak muscle strength and fatigue, skeletal muscle biopsy and calculation of muscle protein synthesis rate, mitochondrial respirometry, skeletal muscle transcriptome, proteome, and gene set enrichment analysis. RESULTS: Despite increased muscularity, FPL subjects did not demonstrate increased muscle strength but had earlier fatigue on chest press exercise. Decreased mitochondrial state 3 respiration in the presence of fatty acid substrate was noted, concurrent to elevated muscle lactate and decreased long-chain acylcarnitine. Based on gene transcriptome, there was significant downregulation of many critical metabolic pathways involved in mitochondrial biogenesis and function. Moreover, the overall pattern of gene expression was indicative of accelerated aging in FPL subjects. A lower muscle protein synthesis and downregulation of gene transcripts involved in muscle protein catabolism was observed. CONCLUSION: Increased muscularity in FPL is not due to increased muscle protein synthesis and is likely due to reduced muscle protein degradation. Impaired mitochondrial function and altered gene expression likely explain the metabolic abnormalities and skeletal muscle dysfunction in FPL subjects.

Adipose tissue macrophage populations and inflammation are associated with systemic inflammation and insulin resistance in obesity.

Obesity is accompanied by numerous systemic and tissue-specific derangements, including systemic inflammation, insulin resistance, and mitochondrial abnormalities in skeletal muscle. Despite growing recognition that adipose tissue dysfunction plays a role in obesity-related disorders, the relationship between adipose tissue inflammation and other pathological features of obesity is not well-understood. We assessed macrophage populations and measured the expression of inflammatory cytokines in abdominal adipose tissue biopsies in 39 nondiabetic adults across a range of body mass indexes (BMI 20.5-45.8 kg/m2). Skeletal muscle biopsies were used to evaluate mitochondrial respiratory capacity, ATP production capacity, coupling, and reactive oxygen species production. Insulin sensitivity (SI) and ß cell responsivity were determined from test meal postprandial glucose, insulin, c-peptide, and triglyceride kinetics. We examined the relationships between adipose tissue inflammatory markers, systemic inflammatory markers, SI, and skeletal muscle mitochondrial physiology. BMI was associated with increased adipose tissue and systemic inflammation, reduced SI, and reduced skeletal muscle mitochondrial oxidative capacity. Adipose-resident macrophage numbers were positively associated with circulating inflammatory markers, including tumor necrosis factor-α (TNFα) and C-reactive protein (CRP). Local adipose tissue inflammation and circulating concentrations of TNFα and CRP were negatively associated with SI, and circulating concentrations of TNFα and CRP were also negatively associated with skeletal muscle oxidative capacity. These results demonstrate that obese humans exhibit increased adipose tissue inflammation concurrently with increased systemic inflammation, reduced insulin sensitivity, and reduced muscle oxidative capacity and suggest that adipose tissue and systemic inflammation may drive obesity-associated metabolic derangements.NEW AND NOTEWORTHY Adipose inflammation is proposed to be at the nexus of the systemic inflammation and metabolic derangements associated with obesity. The present study provides evidence to support adipose inflammation as a central feature of the pathophysiology of obesity. Adipose inflammation is associated with systemic and peripheral metabolic derangements, including increased systemic inflammation, reduced insulin sensitivity, and reduced skeletal muscle mitochondrial respiration.

Cerebellar and multi-system metabolic reprogramming associated with trauma exposure and post-traumatic stress disorder (PTSD)-like behavior in mice.

Mitochondrial metabolism is increasingly implicated in psychopathologies and mood disorders, including post-traumatic stress disorder (PTSD). We recently reported that mice exposed to a novel paradigm for the induction of PTSD-like behavior displayed reduced mitochondrial electron transport chain (mtETC) complex activity as well as decreased multi-system fatty acid oxidation (FAO) flux. Based on these results, we hypothesized that stressed and PTSD-like animals would display evidence of metabolic reprogramming in both cerebellum and plasma consistent with increased energetic demand, mitochondrial metabolic reprogramming, and increased oxidative stress. We performed targeted metabolomics in both cerebellar tissue and plasma, as well as untargeted nuclear magnetic resonance (NMR) spectroscopy in the cerebellum of 6 PTSD-like and 7 resilient male mice as well as 7 trauma-naïve controls. We identified numerous differences in amino acids and tricarboxylic acid (TCA) cycle metabolite concentrations in the cerebellum and plasma consistent with altered mitochondrial energy metabolism in trauma exposed and PTSD-like animals. Pathway analysis identified metabolic pathways with significant metabolic pathway shifts associated with trauma exposure, including the tricarboxylic acid cycle, pyruvate, and branched-chain amino acid metabolism in both cerebellar tissue and plasma. Altered glutamine and glutamate metabolism, and arginine biosynthesis was evident uniquely in cerebellar tissue, while ketone body levels were modified in plasma. Importantly, we also identified several cerebellar metabolites (e.g. choline, adenosine diphosphate, beta-alanine, taurine, and myo-inositol) that were sufficient to discriminate PTSD-like from resilient animals. This multilevel analysis provides a comprehensive understanding of local and systemic metabolite fingerprints associated with PTSD-like behavior, and subsequently altered brain bioenergetics. Notably, several transformed metabolic pathways observed in the cerebellum were also reflected in plasma, connecting central and peripheral biosignatures of PTSD-like behavior. These preliminary findings could direct further mechanistic studies and offer insights into potential metabolic interventions, either pharmacological or dietary, to improve PTSD resilience.

The secretome of senescent preadipocytes influences the phenotype and function of cells of the vascular wall.

Senescent cells accumulate in numerous tissues in several chronic conditions such as aging, obesity, and diabetes. These cells are in a state of irreversible cell-cycle arrest and secrete inflammatory cytokines, chemokines and other immune modulators that have paracrine effects on nearby tissues. Adipose tissue, in particular, harbors senescent cells, which have been linked with numerous chronic conditions and age-related comorbidities. Here we performed a series of in vitro experiments to determine the influence of senescent preadipocytes on key cell types found in vessel walls, including vascular smooth muscle cells (VSMCs), endothelial cells (ECs), macrophages (MQs), and adipose-derived stromal/stem cells (ASCs). Primary human preadipocytes were irradiated to trigger a senescence-like phenotype. VSMCs, ECs, MQs, and ASCs were exposed to conditioned media collected from irradiated preadipocytes or control preadipocytes. Additional experiments were performed where VSMCs, ECs, MQs, and ASCs were co-cultured with irradiated or control preadipocytes. The secretome of irradiated cells induced an inflammatory phenotype, decreased cell viability, disrupted proliferation and migration, and impaired metabolic function of these cell types in vitro. These maladaptive changes in response to senescent cell exposure provide early evidence in support of a hypothesis that senescent preadipocytes trigger phenotypic and functional changes in key cellular components of blood vessels that may contribute to vascular disease.

Extramyocellular interleukin-6 influences skeletal muscle mitochondrial physiology through canonical JAK/STAT signaling pathways.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has been shown to be produced acutely by skeletal muscle in response to exercise, yet chronically elevated with obesity and aging. The mechanisms by which IL-6 influences skeletal muscle mitochondria acutely and chronically are unclear. To better understand the influence of extramyocellular IL-6 on skeletal muscle mitochondrial physiology, we treated differentiated myotubes with exogenous IL-6 to evaluate the dose- and duration-dependent effects of IL-6 on salient aspects of mitochondrial biology and the role of canonical IL-6 signaling in muscle cells. Acute exposure of myotubes to IL-6 increased the mitochondrial reactive oxygen species (mtROS) production and oxygen consumption rates (JO2 ) in a manner that was dependent on activation of the JAK/STAT pathway. Furthermore, STAT3 activation by IL-6 was partly attenuated by MitoQ, a mitochondrial-targeted antioxidant, suggesting that mtROS potentiates STAT3 signaling in skeletal muscle in response to IL-6 exposure. In concert with effects on mitochondrial physiology, acute IL-6 exposure induced several mitochondrial adaptations, consistent with the stress-induced mitochondrial hyperfusion. Exposure of myotubes to chronically elevated IL-6 further increased mtROS with eventual loss of respiratory capacity. These data provide new evidence supporting the interplay between cytokine signaling and mitochondrial physiology in skeletal muscle.

Metabo- and mechanoreceptor expression in human heart failure: Relationships with the locomotor muscle afferent influence on exercise responses.

NEW FINDINGS: What is the central question of this study? How do locomotor muscle metabo- and mechanoreceptor expression compare in heart failure patients and controls? Do relationships exist between the protein expression and cardiopulmonary responses during exercise with locomotor muscle neural afferent feedback inhibition? What is the main finding and its importance? Heart failure patients exhibited greater protein expression of transient receptor potential vanilloid type 1 and cyclooxygenase-2 than controls. These findings are important as they identify receptors that may underlie the augmented locomotor muscle neural afferent feedback in heart failure. ABSTRACT: Heart failure patients with reduced ejection fraction (HFrEF) exhibit abnormal locomotor group III/IV afferent feedback during exercise; however, the underlying mechanisms are unclear. Therefore, the purpose of this study was to determine (1) metabo- and mechanoreceptor expression in HFrEF and controls and (2) relationships between receptor expression and changes in cardiopulmonary responses with afferent inhibition. Ten controls and six HFrEF performed 5 min of cycling exercise at 65% peak workload with lumbar intrathecal fentanyl (FENT) or placebo (PLA). Arterial blood pressure and catecholamines were measured via radial artery catheter. A vastus lateralis muscle biopsy was performed to quantify cyclooxygenase-2 (COX-2), purinergic 2X3 (P2X3 ), transient receptor potential vanilloid type 1 (TRPV 1), acid-sensing ion channel 3 (ASIC3 ), Piezo 1 and Piezo 2 protein expression. TRPV 1 and COX-2 protein expression was greater in HFrEF than controls (both P < 0.04), while P2X3 , ASIC3 , and Piezo 1 and 2 were not different between groups (all P > 0.16). In all participants, COX-2 protein expression was related to the percentage change in ventilation (r = -0.66) and mean arterial pressure (MAP) (r = -0.82) (both P < 0.01) with FENT (relative to PLA) during exercise. In controls, TRPV 1 protein expression was related to the percentage change in systolic blood pressure (r = -0.77, P = 0.02) and MAP (r = -0.72, P = 0.03) with FENT (relative to PLA) during exercise. TRPV 1 and COX-2 protein levels are elevated in HFrEF compared to controls. These findings suggest that the elevated TRPV 1 and COX-2 expression may contribute to the exaggerated locomotor muscle afferent feedback during cycling exercise in HFrEF.

Methylarginine metabolites are associated with attenuated muscle protein synthesis in cancer-associated muscle wasting.

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week-old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Impaired cardiac performance, protein synthesis, and mitochondrial function in tumor-bearing mice.

BACKGROUND: To understand the underlying mechanisms of cardiac dysfunction in cancer, we examined cardiac function, protein synthesis, mitochondrial function and gene expression in a model of heart failure in mice injected with Lewis lung carcinoma (LLC1) cells. EXPERIMENTAL DESIGN: Seven week-old C57BL/J6 male and female mice were injected with LLC1 cells or vehicle. Cardiac ejection fraction, ventricular wall and septal thickness were reduced in male, but not female, tumor-bearing mice compared to vehicle-injected control mice. Cardiac protein synthesis was reduced in tumor-bearing male mice compared to control mice (p = 0.025). Aspect ratio and form factor of cardiac mitochondria from the tumor-bearing mice were increased compared control mice (p = 0.042 and p = 0.0032, respectively) indicating a more fused mitochondrial network in the hearts of tumor-bearing mice. In cultured cardiomyocytes maximal oxygen consumption and mitochondrial reserve capacity were reduced in cells exposed to tumor cell-conditioned medium compared to non-conditioned medium (p = 0.0059, p = 0.0010). Whole transcriptome sequencing of cardiac ventricular muscle from tumor-bearing vs. control mice showed altered expression of 1648 RNA transcripts with a false discovery rate of less than 0.05. Of these, 54 RNA transcripts were reduced ≤ 0.5 fold, and 3 RNA transcripts were increased by ≥1.5-fold in tumor-bearing mouse heart compared to control. Notably, the expression of mRNAs for apelin (Apln), the apelin receptor (Aplnr), the N-myc proto-oncogene, early growth protein (Egr1), and the transcription factor Sox9 were reduced by >50%, whereas the mRNA for growth arrest and DNA-damage-inducible, beta (Gadd45b) is increased >2-fold, in ventricular tissue from tumor-bearing mice compared to control mice. CONCLUSIONS: Lung tumor cells induce heart failure in male mice in association with reduced protein synthesis, mitochondrial function, and the expression of the mRNAs for inotropic and growth factors. These data provide new mechanistic insights into cancer-associated heart failure that may help unlock treatment options for this condition.

Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Despite the strong association between diabetes and dementia, it remains to be fully elucidated how insulin deficiency adversely affects brain functions. We show that insulin deficiency in streptozotocin-induced diabetic mice decreased mitochondrial ATP production and/or citrate synthase and cytochrome oxidase activities in the cerebrum, hypothalamus, and hippocampus. Concomitant decrease in mitochondrial fusion proteins and increased fission proteins in these brain regions likely contributed to altered mitochondrial function. Although insulin deficiency did not cause any detectable increase in reactive oxygen species (ROS) emission, inhibition of monocarboxylate transporters increased ROS emission and further reduced ATP production, indicating the causative roles of elevated ketones and lactate in counteracting oxidative stress and as a fuel source for ATP production during insulin deficiency. Moreover, in healthy mice, intranasal insulin administration increased mitochondrial ATP production, demonstrating a direct regulatory role of insulin on brain mitochondrial function. Proteomics analysis of the cerebrum showed that although insulin deficiency led to oxidative post-translational modification of several proteins that cause tau phosphorylation and neurofibrillary degeneration, insulin administration enhanced neuronal development and neurotransmission pathways. Together these results render support for the critical role of insulin to maintain brain mitochondrial homeostasis and provide mechanistic insight into the potential therapeutic benefits of intranasal insulin.-Ruegsegger, G. N., Manjunatha, S., Summer, P., Gopala, S., Zabeilski, P., Dasari, S., Vanderboom, P. M., Lanza, I. R., Klaus, K. A., Nair, K. S. Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Urinary Metabolites Associated with Blood Pressure on a Low- or High-Sodium Diet.

Dietary salt intake has significant effects on arterial blood pressure and the development of hypertension. Mechanisms underlying salt-dependent changes in blood pressure remain poorly understood, and it is difficult to assess blood pressure salt-sensitivity clinically. Methods: We examined urinary levels of metabolites in 103 participants of the Dietary Approaches to Stop Hypertension (DASH)-Sodium trial after nearly 30 days on a defined diet containing high sodium (targeting 150 mmol sodium intake per day) or low sodium (50 mmol per day). Targeted chromatography/mass spectrometry analysis was performed in 24 h urine samples for 47 amino metabolites and 10 metabolites related to the tricarboxylic acid cycle. The effect of an identified metabolite on blood pressure was examined in Dahl salt-sensitive rats. Results: Urinary metabolite levels improved the prediction of classification of blood pressure salt-sensitivity based on race, age and sex. Random forest and generalized linear mixed model analyses identified significant (false discovery rate <0.05) associations of 24 h excretions of ß-aminoisobutyric acid, cystine, citrulline, homocysteine and lysine with systolic blood pressure and cystine with diastolic blood pressure. The differences in homocysteine levels between low- and high-sodium intakes were significantly associated with the differences in diastolic blood pressure. These associations were significant with or without considering demographic factors. Treatment with ß-aminoisobutyric acid significantly attenuated high-salt-induced hypertension in Dahl salt-sensitive rats. Conclusion: These findings support the presence of new mechanisms of blood pressure regulation involving metabolic intermediaries, which could be developed as markers or therapeutic targets for salt-sensitive hypertension.

1α,25-dihydroxyvitamin D3 mitigates cancer cell mediated mitochondrial dysfunction in human skeletal muscle cells.

Cancer cachexia is associated with muscle weakness and atrophy. We investigated whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), which has previously been shown to increase skeletal myoblast oxygen consumption rate, could reverse the deleterious effects of tumor cell conditioned medium on myoblast function. Conditioned medium from Lewis lung carcinoma (LLC1) cells inhibits oxygen consumption, increases mitochondrial fragmentation, inhibits pyruvate dehydrogenase activity, and enhances proteasomal activity in human skeletal muscle myoblasts. 1α,25(OH)2D3 reverses the tumor cell-mediated changes in mitochondrial oxygen consumption and proteasomal activity, without changing pyruvate dehydrogenase activity. 1α,25(OH)2D3 might be useful in treatment of weakness seen in association with CC.

Very-long-chain ω-3 fatty acid supplements and adipose tissue functions: a randomized controlled trial.

Background: Increased omega-3 (n-3) fatty acid consumption is reported to benefit patients with metabolic syndrome, possibly due to improved adipose tissue function.Objective: We tested the effects of high-dose, very-long-chain ω-3 fatty acids on adipose tissue inflammation and insulin regulation of lipolysis.Design: A double-blind, placebo-controlled study compared 6 mo of 3.9 g eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)/d (4.2 g total ω-3/d; n = 12) with a placebo (4.2 g oleate/d; n = 9) in insulin-resistant adults. Before and after treatment, the volunteers underwent adipose tissue biopsies to measure the total (CD68+), pro- (CD14+ = M1), and anti- (CD206+ = M2) inflammatory macrophages, crown-like structures, and senescent cells, as well as a 2-step pancreatic clamping with a [U-13C]palmitate infusion to determine the insulin concentration needed to suppress palmitate flux by 50% (IC50(palmitate)f).Results: In the ω-3 group, the EPA and DHA contributions to plasma free fatty acids increased (P = 0.0003 and P = 0.003, respectively), as did the EPA and DHA content in adipose tissue (P < 0.0001 and P < 0.0001, respectively). Despite increases in adipose and plasma EPA and DHA in the ω-3 group, there were no significant changes in the IC50(palmitate)f (19 ± 2 compared with 24 ± 3 µIU/mL), adipose macrophages (total: 31 ± 2/100 adipocytes compared with 33 ± 2/100 adipocytes; CD14+: 13 ± 2/100 adipocytes compared with 14 ± 2/100 adipocytes; CD206+: 28 ± 2/100 adipocytes compared with 29 ± 3/100 adipocytes), crown-like structures (1 ± 0/10 images compared with 1 ± 0/10 images), or senescent cells (4% ± 1% compared with 4% ± 1%). There were no changes in these outcomes in the placebo group.Conclusions: Six months of high-dose ω-3 supplementation raised plasma and adipose ω-3 fatty acid concentrations but had no beneficial effects on adipose tissue lipolysis or inflammation in insulin-resistant adults. This trial was registered at clinicaltrials.gov as NCT01686568.

Noninvasive Monitoring of the Mitochondrial Function in Mesenchymal Stromal Cells.

PURPOSE: Mitochondria are a gatekeeper of cell survival and mitochondrial function can be used to monitor cell stress. Here we validate a pathway-specific reporter gene to noninvasively image the mitochondrial function of stem cells. PROCEDURES: We constructed a mitochondrial sensor with the firefly luciferase (Fluc) reporter gene driven by the NQO1 enzyme promoter. The sensor was introduced in stem cells and validated in vitro and in vivo, in a mouse model of myocardial ischemia/reperfusion (IR). RESULTS: The sensor activity showed an inverse relationship with mitochondrial function (R (2) = -0.975, p = 0.025) and showed specificity and sensitivity for mitochondrial dysfunction. In vivo, NQO1-Fluc activity was significantly higher in IR animals vs. controls, indicative of mitochondrial dysfunction, and was corroborated by ex vivo luminometry. CONCLUSIONS: Reporter gene imaging allows assessment of the biology of transplanted mesenchymal stromal cells (MSCs), providing important information that can be used to improve the phenotype and survival of transplanted stem cells.

Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice.

Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and ß-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of ß-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a ß-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.