Sex differences in skeletal muscle revealed through fiber type, capillarity, and transcriptomics profiling in mice.

Skeletal muscle anatomy and physiology are sexually dimorphic but molecular underpinnings and muscle-specificity are not well-established. Variances in metabolic health, fitness level, sedentary behavior, genetics, and age make it difficult to discern inherent sex effects in humans. Therefore, mice under well-controlled conditions were used to determine female and male (n = 19/sex) skeletal muscle fiber type/size and capillarity in superficial and deep gastrocnemius (GA-s, GA-d), soleus (SOL), extensor digitorum longus (EDL), and plantaris (PLT), and transcriptome patterns were also determined (GA, SOL). Summed muscle weight strongly correlated with lean body mass (r2  = 0.67, p < 0.0001, both sexes). Other phenotypes were muscle-specific: e.g., capillarity (higher density, male GA-s), myofiber size (higher, male EDL), and fiber type (higher, lower type I and type II prevalences, respectively, in female SOL). There were broad differences in transcriptomics, with >6000 (GA) and >4000 (SOL) mRNAs differentially-expressed by sex; only a minority of these were shared across GA and SOL. Pathway analyses revealed differences in ribosome biology, transcription, and RNA processing. Curation of sexually dimorphic muscle transcripts shared in GA and SOL, and literature datasets from mice and humans, identified 11 genes that we propose are canonical to innate sex differences in muscle: Xist, Kdm6a, Grb10, Oas2, Rps4x (higher, females) and Ddx3y, Kdm5d, Irx3, Wwp1, Aldh1a1, Cd24a (higher, males). These genes and those with the highest "sex-biased" expression in our study do not contain estrogen-response elements (exception, Greb1), but a subset are proposed to be regulated through androgen response elements. We hypothesize that innate muscle sexual dimorphism in mice and humans is triggered and then maintained by classic X inactivation (Xist, females) and Y activation (Ddx3y, males), with coincident engagement of X encoded (Kdm6a) and Y encoded (Kdm5d) demethylase epigenetic regulators that are complemented by modulation at some regions of the genome that respond to androgen.

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Myoglobin (Mb) is a regulator of O2 bioavailability in type I muscle and heart, at least when tissue O2 levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb-/-) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males (n = 10/genotype) and females (n = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb-/- adiposity was ∼42%-48% higher versus WT females (P = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb-/-, specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb-/- mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency.NEW & NOTEWORTHY The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.

On the potential role of globins in brown adipose tissue: a novel conceptual model and studies in myoglobin knockout mice.

Myoglobin (Mb) regulates O2 bioavailability in muscle and heart as the partial pressure of O2 (Po2) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher Po2 and converting NO2- to NO as Po2 falls. Myoglobin binding of fatty acids may also signal a role in fat metabolism. Interestingly, Mb is expressed in brown adipose tissue (BAT), but its function is unknown. Herein, we present a new conceptual model that proposes links between BAT thermogenic activation, concurrently reduced Po2, and NO pools regulated by deoxy/oxy-globin toggling and xanthine oxidoreductase (XOR). We describe the effect of Mb knockout (Mb-/-) on BAT phenotype [lipid droplets, mitochondrial markers uncoupling protein 1 (UCP1) and cytochrome C oxidase 4 (Cox4), transcriptomics] in male and female mice fed a high-fat diet (HFD, 45% of energy, ∼13 wk), and examine Mb expression during brown adipocyte differentiation. Interscapular BAT weights did not differ by genotype, but there was a higher prevalence of mid-large sized droplets in Mb-/-. COX4 protein expression was significantly reduced in Mb-/- BAT, and a suite of metabolic/NO/stress/hypoxia transcripts were lower. All of these Mb-/--associated differences were most apparent in females. The new conceptual model, and results derived from Mb-/- mice, suggest a role for Mb in BAT metabolic regulation, in part through sexually dimorphic systems and NO signaling. This possibility requires further validation in light of significant mouse-to-mouse variability of BAT Mb mRNA and protein abundances in wild-type mice and lower expression relative to muscle and heart.NEW & NOTEWORTHY Myoglobin confers the distinct red color to muscle and heart, serving as an oxygen-binding protein in oxidative fibers. Less attention has been paid to brown fat, a thermogenic tissue that also expresses myoglobin. In a mouse knockout model lacking myoglobin, brown fat had larger fat droplets and lower markers of mitochondrial oxidative metabolism, especially in females. Gene expression patterns suggest a role for myoglobin as an oxygen/nitric oxide-sensor that regulates cellular metabolic and signaling pathways.

Exercise training and diet-induced weight loss increase markers of hepatic bile acid (BA) synthesis and reduce serum total BA concentrations in obese women.

Regular exercise has profound metabolic influence on the liver, but effects on bile acid (BA) metabolism are less well known. BAs are synthesized exclusively in the liver from cholesterol via the rate-limiting enzyme cholesterol 7 alpha-hydroxylase (CYP7A1). BAs contribute to the solubilization and absorption of lipids and serve as important signaling molecules, capable of systemic endocrine function. Circulating BAs increase with obesity and insulin resistance, but effects following exercise and diet-induced weight loss are unknown. To test if improvements in fitness and weight loss as a result of exercise training enhance BA metabolism, we measured serum concentrations of total BAs (conjugated and unconjugated primary and secondary BAs) in sedentary, obese, insulin-resistant women (N = 11) before (PRE) and after (POST) a ∼14-wk exercise and diet-induced weight loss intervention. BAs were measured in serum collected after an overnight fast and during an oral glucose tolerance test (OGTT). Serum fibroblast growth factor 19 (FGF19; a regulator of BA synthesis) and 7-alpha-hydroxy-cholesten-3-one (C4, a marker of CYP7A1 enzymatic activity) also were measured. Using linear mixed-model analyses and the change in V̇O2peak (mL/min/kg) as a covariate, we observed that exercise and weight loss intervention decreased total fasting serum BA by ∼30% (P = 0.001) and increased fasting serum C4 concentrations by 55% (P = 0.004). C4 was significantly correlated with serum total BAs only in the POST condition, whereas serum FGF19 was unchanged. These data indicate that a fitness and weight loss intervention modifies BA metabolism in obese women and suggest that improved metabolic health associates with higher postabsorptive (fasting) BA synthesis. Furthermore, pre- vs. postintervention patterns of serum C4 following an OGTT support the hypothesis that responsiveness of BA synthesis to postprandial inhibition is improved after exercise and weight loss.NEW & NOTEWORTHY Exercise and weight loss in previously sedentary, insulin-resistant women facilitates a significant improvement in insulin sensitivity and fitness that may be linked to changes in bile acid metabolism. Diet-induced weight loss plus exercise-induced increases in fitness promote greater postabsorptive bile acid synthesis while also sensitizing the bile acid metabolic system to feedback inhibition during a glucose challenge when glucose and insulin are elevated.

Two SARS-CoV-2 Genome Sequences of Isolates from Rural U.S. Patients Harboring the D614G Mutation, Obtained Using Nanopore Sequencing.

Two coding-complete sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained from samples from two patients in Arkansas, in the southeastern corner of the United States. The viral genome was obtained using the ARTIC Network protocol and Oxford Nanopore Technologies sequencing.

Coupling of energy intake and energy expenditure across a temperature spectrum: impact of diet-induced obesity in mice.

Obesity and its metabolic sequelae are implicated in dysfunction of the somatosensory, sympathetic, and hypothalamic systems. Because these systems contribute to integrative regulation of energy expenditure (EE) and energy intake (EI) in response to ambient temperature (Ta) changes, we hypothesized that diet-induced obesity (DIO) disrupts Ta-associated EE-EI coupling. C57BL/6N male mice were fed a high-fat diet (HFD; 45% kcal) or low-fat diet (LFD; 10% kcal) for ∼9.5 wk; HFD mice were then split into body weight (BWT) quartiles (n = 8 each) to study DIO-low gainers (Q1) versus -high gainers (Q4). EI and indirect calorimetry (IC) were measured over 3 days each at 10°C, 20°C, and 30°C. Responses did not differ between LFD, Q1, and Q4; EI and BWT-adjusted EE increased rapidly when transitioning toward 20°C and 10°C. In all groups, EI at 30°C was not reduced despite lower EE, resulting in positive energy balance and respiratory exchange ratios consistent with increased de novo lipogenesis, energy storage, and relative hyperphagia. We conclude that 1) systems controlling Ta-dependent acute EI/EE coupling remained intact in obese mice and 2) rapid coupling of EI/EE at cooler temperatures is an important adaptation to maintain energy stores and defend body temperature, but less critical at thermoneutrality. A post hoc analysis using digestible EI plus IC-calculated EE suggests that standard IC assumptions for EE calculation require further validation in the setting of DIO. The experimental paradigm provides a platform to query the hypothalamic, somatosensory, and sympathetic mechanisms that drive Ta-associated EI/EE coupling.

Carnitine palmitoyltransferase 2 knockout potentiates palmitate-induced insulin resistance in C2C12 myotubes.

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.

Neonatal diet impacts liver mitochondrial bioenergetics in piglets fed formula or human milk.

BACKGROUND: Neonatal diet impacts many physiological systems and can modify risk for developing metabolic disease and obesity later in life. Less well studied is the effect of postnatal diet (e.g., comparing human milk (HM) or milk formula (MF) feeding) on mitochondrial bioenergetics. Such effects may be most profound in splanchnic tissues that would have early exposure to diet-associated or gut microbe-derived factors. METHODS: To address this question, we measured ileal and liver mitochondrial bioenergetics phenotypes in male piglets fed with HM or MF from day 2 to day 21 age. Ileal and liver tissue were processed for mitochondrial respiration (substrate only [pyruvate, malate, glutamate], substrate + ADP, and proton "leak" post-oligomycin; measured by Oroboros methods), mitochondrial DNA (mtDNA) and metabolically-relevant gene expression analyses. RESULTS: No differences between the diet groups were observed in mitochondrial bioenergetics indices in ileal tissue. In contrast, ADP-dependent liver Complex I-linked OXPHOS capacity and Complex I + II-linked OXPHOS capacity were significantly higher in MF animals relative to HM fed piglets. Interestingly, p53, Trap1, and Pparß transcript abundances were higher in MF-fed relative to HM-fed piglets in the liver. Mitochondrial DNA copy numbers (normalized to nuclear DNA) were similar within-tissue regardless of postnatal diet, and were ~ 2-3 times higher in liver vs. ileal tissue. CONCLUSION: While mechanisms remain to be identified, the data indicate that neonatal diet can significantly impact liver mitochondrial bioenergetics phenotypes, even in the absence of a change in mtDNA abundance. Since permeabilized liver mitochondrial respiration was increased in MF piglets only in the presence of ADP, it suggests that formula feeding led to a higher ATP turnover. Specific mechanisms and signals involved with neonatal diet-associated differences in liver bioenergetics remain to be elucidated.

Blood cytokine patterns suggest a modest inflammation phenotype in subjects with long-chain fatty acid oxidation disorders.

Excessive cellular accumulation or exposure to lipids such as long-chain acylcarnitines (LCACs), ceramides, and others is implicated in cell stress and inflammation. Such a situation might manifest when there is a significant mismatch between long-chain fatty acid (LCFA) availability versus storage and oxidative utilization; for example, in cardiac ischemia, increased LCACs may contribute to tissue cell stress and infarct damage. Perturbed LCFAß-oxidation is also seen in fatty acid oxidation disorders (FAODs). FAODs typically manifest with fasting- or stress-induced symptoms, and patients can manage many symptoms through control of diet and physical activity. However, episodic clinical events involving cardiac and skeletal muscle myopathies are common and can present without an obvious molecular trigger. We have speculated that systemic or tissue-specific lipotoxicity and activation of inflammation pathways contribute to long-chain FAOD pathophysiology. With this in mind, we characterized inflammatory phenotype (14 blood plasma cytokines) in resting, overnight-fasted (~10 h), or exercise-challenged subjects with clinically well-controlled long-chain FAODs (n = 12; 10 long-chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD]; 2 carnitine palmitoyltransferase 2 [CPT2]) compared to healthy controls (n = 12). Across experimental conditions, concentrations of three cytokines were modestly but significantly increased in FAOD (IFNγ, IL-8, and MDC), and plasma levels of IL-10 (considered an inflammation-dampening cytokine) were significantly decreased. These novel results indicate that while asymptomatic FAOD patients do not display gross body-wide inflammation even after moderate exercise, ß-oxidation deficiencies might be associated with chronic and subtle activation of "sterile inflammation." Further studies are warranted to determine if inflammation is more apparent in poorly controlled long-chain FAOD or when long-chain FAOD-associated symptoms are present.

Is palmitate truly proinflammatory? Experimental confounders and context-specificity.

Based primarily on cell culture results, saturated fatty acids (SFAs) are proposed to promote inflammation and contribute to metabolic dysfunction through Toll-like receptor activation. Studies are often complicated by a requirement for carriers (e.g., BSA) or solvents (e.g., ethanol) to increase SFA solubility. To ascertain whether these factors influence interpretations of SFA-associated inflammation activity, we measured responses of RAW264.7 monocyte/macrophages and C2C12 myotubes to various BSA, ethanol, and cyclodextrin (alternative FA carrier) conditions. Fatty acid-free, low-endotoxin BSA preparations (0.33% to 2% wt/vol) activated whereas 0.5-1.0% ethanol inhibited RAW264.7 TNFα release. Ethanol modestly increased IL-6 secretion in C2C12 myotubes. Cyclodextrins (0.3-6.0 mM) were tested as alternative carriers of palmitate, but their usefulness was limited due to toxicity and solubility issues. Using a lower-inflammation BSA source and no ethanol, ∼24-h sodium palmitate treatment (≤600 µM) failed to trigger RAW264.7 TNFα release and, in fact, significantly dampened BSA-induced inflammation by >50%. In C2C12 myotubes, only high palmitate concentrations (500-600 µM) elicited IL-6 secretion (>2.5-fold increase). Acute palmitate (200 or 500 µM) treatment did not activate MAP kinase pathways above that of fresh BSA-containing media alone in either cell type. These results highlight the importance of experimental conditions in studies exploring SFA inflammation effects. The limited (or even anti-inflammatory) effects of palmitate that we observed indicate that immunomodulatory effects of SFAs are context-specific. Thus, caution is needed when interpreting the literature related to putative proinflammatory effects of SFA.

Transgenic mice with ectopic expression of constitutively active TLR4 in adipose tissues do not show impaired insulin sensitivity.

INTRODUCTION: Chronic low-grade inflammation is associated with obesity and diabetes. However, what causes and mediates chronic inflammation in metabolic disorders is not well understood. Toll-like receptor 4 (TLR4) mediates both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns and endogenous molecules, respectively. Saturated fatty acids can activate TLR4, and TLR4-deficient mice were protected from high fat diet (HFD)-induced obesity and insulin resistance, suggesting that TLR4-mediated inflammation may cause metabolic dysfunction, such as obesity and insulin resistance. METHODS: We generated two transgenic (TG) mouse lines expressing a constitutively active TLR4 in adipose tissue and determined whether these TG mice would show increased insulin resistance. RESULTS: TG mice fed a high fat or a normal chow diet did not exhibit increased insulin resistance compared to their wild-type controls despite increased localized inflammation in white adipose tissue. Furthermore, females of one TG line fed a normal chow diet had improved insulin sensitivity with reduction in both adiposity and body weight when compared with wild-type littermates. There were significant differences between female and male mice in metabolic biomarkers and mRNA expression in proinflammatory genes and negative regulators of TLR4 signaling, regardless of genotype and diet. CONCLUSIONS: Together, these results suggest that constitutively active TLR4-induced inflammation in white adipose tissue is not sufficient to induce systemic insulin resistance, and that high fat diet-induced insulin resistance may require other signals in addition to TLR4-mediated inflammation.

Application of an In Vivo Hepatic Triacylglycerol Production Method in the Setting of a High-Fat Diet in Mice.

High-fat (HF) diets typically promote diet-induced obesity (DIO) and metabolic dysfunction (i.e., insulin resistance, hypertriglyceridemia, and hepatic steatosis). Dysfunction of triacylglycerol (TAG) metabolism may contribute to the development of hepatic steatosis, via increased de novo lipogenesis or repackaging of circulating nonesterified fatty acids (NEFAs). Hepatic TAG production (HTP) rate can be assessed through injecting mice with nonionic detergents that inhibit tissue lipoprotein lipase. Potential confounding effects of detergent-based HTP tests (HTPTs) used in longitudinal studies-including the impact on food intake, energy balance, and weight gain-have not been reported. To examine this, male C57BL/6J mice were fed a 10% or 60% kcal diet. After 4 weeks, the mice underwent an HTPT via poloxamer 407 intraperitoneal injections (1000 mg/kg). Weight gain, energy intake, and postabsorptive TAG levels normalized 7-10 days post-HTPT. The post-HTPT recovery of body weight and energy intake suggest that, in metabolic phenotyping studies, any additional sample collection should occur at least 7-10 days after the HTPT to reduce confounding effects. Diet-specific effects on HTP were also observed: HF-fed mice had reduced HTP, plasma TAG, and NEFA levels compared to controls. In conclusion, the current study highlights the procedural and physiological complexities associated with studying lipid metabolism using a HTPT in the DIO mouse model.

Postprandial Inflammatory Responses and Free Fatty Acids in Plasma of Adults Who Consumed a Moderately High-Fat Breakfast with and without Blueberry Powder in a Randomized Placebo-Controlled Trial.

BACKGROUND: Saturated fatty acids (FAs) released from triglyceride-rich lipoproteins (TGRLs) activate Toll-like receptor 2 (TLR-2) and induce the expression of proinflammatory cytokines in monocytes. Certain plant polyphenols inhibit TLR-mediated signaling pathways. OBJECTIVE: We determined whether plasma free FAs (FFAs) after a moderately high-fat (MHF, 40% kcal from fat) breakfast modulate the inflammatory status of postprandial blood, and whether blueberry intake suppresses FFA-induced inflammatory responses in healthy humans. METHODS: Twenty-three volunteers with a mean ± SEM age and body mass index (in kg/m(2)) of 30 ± 3 y and 21.9 ± 0.4, respectively, consumed an MHF breakfast with either a placebo powder or 2 or 4 servings of blueberry powder in a randomized crossover design. The placebo powder was provided on the first test day and the blueberry powder doses were randomized with a 2-wk washout period. Plasma concentrations of lipids, glucose, and cytokines were determined. To determine whether FFAs derived from TGRL stimulate monocyte activation, and whether this is inhibited by blueberry intake, whole blood was treated with lipoprotein lipase (LPL). RESULTS: The median concentrations of FFAs and cytokines [tumor necrosis factor-α, interleukin (IL)-6 and IL-8] in postprandial plasma (3.5 h) decreased compared with fasting plasma regardless of the blueberry intake (P < 0.001 for FFAs and P < 0.05 for cytokines). However, concentrations of FFAs and cytokines including IL-1ß increased in LPL-treated whole blood compared with untreated blood samples from participants who consumed the placebo powder. Blueberry intake suppressed IL-1ß and IL-6 production in LPL-treated postprandial blood compared with the placebo control when fasting changes were used as a covariate. CONCLUSIONS: The plasma FFA concentration may be an important determinant affecting inflammatory cytokine production in blood. Supplementation with blueberry powder did not affect plasma FFA and cytokine concentrations; however, it attenuated the cytokine production induced by ex vivo treatment of whole blood with LPL. This trial was registered at clinicaltrials.gov as NCT01594008.

Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and -independent effects.

Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with l-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. l-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations ≥25 µM, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 µM. Additionally, l-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase between 2.5- and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. l-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 µM, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted l-C16 carnitine-mediated IL-6 production by >65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the l-C16 carnitine-associated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiological-to-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology.

Acylcarnitines: potential implications for skeletal muscle insulin resistance.

Insulin resistance may be linked to incomplete fatty acid ß-oxidation and the subsequent increase in acylcarnitine species in different tissues including skeletal muscle. It is not known if acylcarnitines participate in muscle insulin resistance or simply reflect dysregulated metabolism. The aims of this study were to determine whether acylcarnitines can elicit muscle insulin resistance and to better understand the link between incomplete muscle fatty acid ß-oxidation, oxidative stress, inflammation, and insulin-resistance development. Differentiated C2C12, primary mouse, and human myotubes were treated with acylcarnitines (C4:0, C14:0, C16:0) or with palmitate with or without carnitine acyltransferase inhibition by mildronate. Treatment with C4:0, C14:0, and C16:0 acylcarnitines resulted in 20-30% decrease in insulin response at the level of Akt phosphorylation and/or glucose uptake. Mildronate reversed palmitate-induced insulin resistance concomitant with an ∼25% decrease in short-chain acylcarnitine and acetylcarnitine secretion. Although proinflammatory cytokines were not affected under these conditions, oxidative stress was increased by 2-3 times by short- or long-chain acylcarnitines. Acylcarnitine-induced oxidative stress and insulin resistance were reversed by treatment with antioxidants. Results are consistent with the conclusion that incomplete muscle fatty acid ß-oxidation causes acylcarnitine accumulation and associated oxidative stress, raising the possibility that these metabolites play a role in muscle insulin resistance.

Acylcarnitines activate proinflammatory signaling pathways.

Incomplete ß-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete ß-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 µM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.

Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-κB α, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-κB subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88(-/-) macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations.