Electroacupuncture Mimics Exercise-Induced Changes in Skeletal Muscle Gene Expression in Women With Polycystic Ovary Syndrome.

CONTEXT: Autonomic nervous system activation mediates the increase in whole-body glucose uptake in response to electroacupuncture but the mechanisms are largely unknown. OBJECTIVE: To identify the molecular mechanisms underlying electroacupuncture-induced glucose uptake in skeletal muscle in insulin-resistant overweight/obese women with and without polycystic ovary syndrome (PCOS). DESIGN/PARTICIPANTS: In a case-control study, skeletal muscle biopsies were collected from 15 women with PCOS and 14 controls before and after electroacupuncture. Gene expression and methylation was analyzed using Illumina BeadChips arrays. RESULTS: A single bout of electroacupuncture restores metabolic and transcriptional alterations and induces epigenetic changes in skeletal muscle. Transcriptomic analysis revealed 180 unique genes (q < 0.05) whose expression was changed by electroacupuncture, with 95% of the changes towards a healthier phenotype. We identified DNA methylation changes at 304 unique sites (q < 0.20), and these changes correlated with altered expression of 101 genes (P < 0.05). Among the 50 most upregulated genes in response to electroacupuncture, 38% were also upregulated in response to exercise. We identified a subset of genes that were selectively altered by electroacupuncture in women with PCOS. For example, MSX1 and SRNX1 were decreased in muscle tissue of women with PCOS and were increased by electroacupuncture and exercise. siRNA-mediated silencing of these 2 genes in cultured myotubes decreased glycogen synthesis, supporting a role for these genes in glucose homeostasis. CONCLUSION: Our findings provide evidence that electroacupuncture normalizes gene expression in skeletal muscle in a manner similar to acute exercise. Electroacupuncture might therefore be a useful way of assisting those who have difficulties performing exercise.

Influence of obesity, weight loss, and free fatty acids on skeletal muscle clock gene expression.

The molecular circadian clock plays a role in metabolic homeostasis. We tested the hypothesis obesity and systemic factors associated with insulin resistance affect skeletal muscle clock gene expression. We determined clock gene expression in skeletal muscle of obese women (n = 5) and men (n = 18) before and 6 mo after Roux-en-Y gastric bypass (RYGB) surgery and normal-weight controls (women n = 6, men n = 8). Skeletal muscle clock gene expression was affected by obesity and weight loss. CRY1 mRNA (P = 0.05) was increased and DBP mRNA (P < 0.05) was decreased in obese vs. normal weight women and restored to control levels after RYGB-induced weight loss. CLOCK, CRY1, CRY2, and DBP mRNA (P < 0.05) was decreased in obese men compared with normal weight men. Expression of all other clock genes was unaltered by obesity or weight loss in both cohorts. We correlated clock gene expression with clinical characteristics of the participants. Among the genes studied, DBP and PER3 expression was inversely correlated with plasma lipids in both cohorts. Circadian time-course studies revealed that core clock genes oscillate over time (P < 0.05), with BMAL1, CIART, CRY2, DBP, PER1, and PER3 expression profiles altered by palmitate treatment. In conclusion, skeletal muscle clock gene expression and function is altered by obesity, coincident with changes in plasma lipid levels. Palmitate exposure disrupts clock gene expression in myotubes, indicating that dyslipidemia directly alters the circadian program. Strategies to reduce lipid overload and prevent elevations in nonesterified fatty acid and cholesterol levels may sustain circadian clock signals in skeletal muscle.

IL6 and LIF mRNA expression in skeletal muscle is regulated by AMPK and the transcription factors NFYC, ZBTB14, and SP1.

Adenosine monophosphate-activated protein kinase (AMPK) controls glucose and lipid metabolism and modulates inflammatory responses to maintain metabolic and inflammatory homeostasis during low cellular energy levels. The AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) interferes with inflammatory pathways in skeletal muscle, but the mechanisms are undefined. We hypothesized that AMPK activation reduces cytokine mRNA levels by blocking transcription through one or several transcription factors. Three skeletal muscle models were used to study AMPK effects on cytokine mRNA: human skeletal muscle strips obtained from healthy men incubated in vitro, primary human muscle cells, and rat L6 cells. In all three skeletal muscle systems, AICAR acutely reduced cytokine mRNA levels. In L6 myotubes treated with the transcriptional blocker actinomycin D, AICAR addition did not further reduce Il6 or leukemia inhibitory factor ( Lif) mRNA, suggesting that AICAR modulates cytokine expression through regulating transcription rather than mRNA stability. A cross-species bioinformatic approach identified novel transcription factors that may regulate LIF and IL6 mRNA. The involvement of these transcription factors was studied after targeted gene-silencing by siRNA. siRNA silencing of the transcription factors nuclear transcription factor Y subunit c ( Nfyc), specificity protein 1 ( Sp1), and zinc finger and BTB domain containing 14 ( Zbtb14), or AMPK α1/α2 subunits, increased constitutive levels of Il6 and Lif. Our results identify novel candidates in the regulation of skeletal muscle cytokine expression and identify AMPK, Nfyc, Sp1, and Zbtb14 as novel regulators of immunometabolic signals from skeletal muscle.

Deconstructing metabolic inflammation using cellular systems.

Over the past years, we have embarked in a systematic analysis of the effect of obesity or fatty acids on circulating monocytes, microvascular endothelial cells, macrophages, and skeletal muscle cells. With the use of cell culture strategies, we have deconstructed complex physiological systems and then reconstructed "partial equations" to better understand cell-to-cell communication. Through these approaches, we identified that in high saturated fat environments, cell-autonomous proinflammatory pathways are activated in monocytes and endothelial cells, promoting monocyte adhesion and transmigration. We think of this as a paradigm of the conditions promoting immune cell infiltration into tissues during obesity. In concert, it is possible that muscle and adipose tissue secrete immune cell chemoattractants, and indeed, our tissue culture reconstructions reveal that myotubes treated with the saturated fatty acid palmitate, but not the unsaturated fatty acid palmitoleate, release nucleotides that attract monocytes and other compounds that promote proinflammatory classically activated "(M1)-like" polarization in macrophages. In addition, palmitate directly triggers an M1-like macrophage phenotype, and secretions from these activated macrophages confer insulin resistance to target muscle cells. Together, these studies suggest that in pathophysiological conditions of excess fat, the muscle, endothelial and immune cells engage in a synergistic crosstalk that exacerbates tissue inflammation, leukocyte infiltration, polarization, and consequent insulin resistance.

Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK).

A rise in tissue-embedded macrophages displaying "M1-like" proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues.

Ozone exposure triggers insulin resistance through muscle c-Jun N-terminal kinase activation.

A growing body of evidence suggests that exposure to traffic-related air pollution is a risk factor for type 2 diabetes. Ozone, a major photochemical pollutant in urban areas, is negatively associated with fasting glucose and insulin levels, but most aspects of this association remain to be elucidated. Using an environmentally realistic concentration (0.8 parts per million), we demonstrated that exposure of rats to ozone induced whole-body insulin resistance and oxidative stress, with associated endoplasmic reticulum (ER) stress, c-Jun N-terminal kinase (JNK) activation, and disruption of insulin signaling in skeletal muscle. Bronchoalveolar lavage fluids from ozone-treated rats reproduced this effect in C2C12 myotubes, suggesting that toxic lung mediators were responsible for the phenotype. Pretreatment with the chemical chaperone 4-phenylbutyric acid, the JNK inhibitor SP600125, or the antioxidant N-acetylcysteine alleviated insulin resistance, demonstrating that ozone sequentially triggered oxidative stress, ER stress, and JNK activation to impair insulin signaling in muscle. This study is the first to report that ozone plays a causative role in the development of insulin resistance, suggesting that it could boost the development of diabetes. We therefore provide a potential mechanism linking pollutant exposure and the increased incidence of metabolic diseases.

Nucleotides released from palmitate-challenged muscle cells through pannexin-3 attract monocytes.

Obesity-associated low-grade inflammation in metabolically relevant tissues contributes to insulin resistance. We recently reported monocyte/macrophage infiltration in mouse and human skeletal muscles. However, the molecular triggers of this infiltration are unknown, and the role of muscle cells in this context is poorly understood. Animal studies are not amenable to the specific investigation of this vectorial cellular communication. Using cell cultures, we investigated the crosstalk between myotubes and monocytes exposed to physiological levels of saturated and unsaturated fatty acids. Media from L6 myotubes treated with palmitate-but not palmitoleate-induced THP1 monocyte migration across transwells. Palmitate activated the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway in myotubes and elevated cytokine expression, but the monocyte chemoattracting agent was not a polypeptide. Instead, nucleotide degradation eliminated the chemoattracting properties of the myotube-conditioned media. Moreover, palmitate-induced expression and activity of pannexin-3 channels in myotubes were mediated by TLR4-NF-κB, and TLR4-NF-κB inhibition or pannexin-3 knockdown prevented monocyte chemoattraction. In mice, the expression of pannexin channels increased in adipose tissue and skeletal muscle in response to high-fat feeding. These findings identify pannexins as new targets of saturated fatty acid-induced inflammation in myotubes, and point to nucleotides as possible mediators of immune cell chemoattraction toward muscle in the context of obesity.

Cross-talk between skeletal muscle and immune cells: muscle-derived mediators and metabolic implications.

Skeletal muscles contain resident immune cell populations and their abundance and type is altered in inflammatory myopathies, endotoxemia or different types of muscle injury/insult. Within tissues, monocytes differentiate into macrophages and polarize to acquire pro- or anti-inflammatory phenotypes. Skeletal muscle macrophages play a fundamental role in repair and pathogen clearance. These events require a precisely regulated cross-talk between myofibers and immune cells, involving paracrine/autocrine and contact interactions. Skeletal muscle also undergoes continuous repair as a result of contractile activity that involves participation of myokines and anti-inflammatory input. Finally, skeletal muscle is the major site of dietary glucose disposal; therefore, muscle insulin resistance is essential to the development of whole body insulin resistance. Notably, muscle inflammation is emerging as a potential contributor to insulin resistance. Recent reports show that inflammatory macrophage numbers within muscle are elevated during obesity and that muscle cells in vitro can mount autonomous inflammatory responses under metabolic challenge. Here, we review the nature of skeletal muscle inflammation associated with muscle exercise, damage, and regeneration, endotoxin presence, and myopathies, as well as the new evidence of local inflammation arising with obesity that potentially contributes to insulin resistance.

Chronic treatment with myo-inositol reduces white adipose tissue accretion and improves insulin sensitivity in female mice.

Type 2 diabetes is a complex disease characterized by a state of insulin resistance in peripheral tissues such as skeletal muscle, adipose tissue or liver. Some inositol isomers have been reported to possess insulin-mimetic activity and to be efficient in lowering blood glucose level. The aim of the present study was to assess in mice the metabolic effects of a chronic treatment with myo-inositol, the most common stereoisomer of inositol. Mice given myo-inositol treatment (0.9 or 1.2 mg g(-1) day(-1), 15 days, orally or intraperitoneally) exhibited an improved glucose tolerance due to a greater insulin sensitivity. Mice treated with myo-inositol exhibited a decreased white adipose tissue accretion (-33%, P<.005) compared with controls. The decrease in white adipose tissue deposition was due to a decrease in adipose cell volume (-33%, P<.05), while no change was noticed in total adipocyte number. In skeletal muscle, in vivo as well as ex vivo myo-inositol treatment increased protein kinase B/Akt phosphorylation under baseline and insulin-stimulated conditions, suggesting a synergistic action of myo-inositol treatment and insulin on proteins of the insulin signalling pathway. Myo-inositol could therefore constitute a viable nutritional strategy for the prevention and/or treatment of insulin resistance and type 2 diabetes.

The lipid peroxidation by-product 4-hydroxy-2-nonenal (4-HNE) induces insulin resistance in skeletal muscle through both carbonyl and oxidative stress.

Numerous oxidants are produced as by-products of aerobic cell metabolism, and there is growing evidence that they play key roles in the pathogenesis of insulin resistance. Under conditions of oxidative stress, lipid peroxidation of ω6-polyunsaturated fatty acids leads to the production of 4-hydroxy-2-nonenal (4-HNE). Several lines of evidence suggest that 4-HNE could be involved in the pathophysiology of metabolic diseases; therefore, in this study we assessed the direct effects of 4-HNE on skeletal muscle insulin sensitivity. Gastrocnemius muscle and L6 muscle cells were treated with 4-HNE. Insulin signaling was measured by Western blotting and glucose uptake using 2-deoxy-d-[3H]glucose. Carbonyl stress, glutathione content, and oxidative stress were assessed as potential mechanisms leading to insulin resistance. Protection of cells was induced by pretreatment with 3H-1,2-dithiole-3-thione, N-acetyl-cysteine, aminoguanidine, or S-adenosyl-methionine. 4-HNE induced a time- and dose-dependent decrease in insulin signaling and insulin-induced glucose uptake in muscle. It induced a state of carbonyl stress through adduction of proteins as well as a depletion in reduced glutathione and production of radical oxygen species. A pharmacological increase in glutathione pools was achieved by 3H-1,2-dithiole-3-thione and protected the cells against all deleterious effects of 4-HNE; furthermore, N-acetylcysteine, aminoguanidine, and S-adenosylmethionine prevented 4-HNE noxious effects. 4-HNE can impair insulin action in muscle cells through oxidative stress and oxidative damage to proteins, eventually leading to insulin resistance. These deleterious effects can be prevented by pretreatment with antioxidants, scavengers, or an increase in intracellular glutathione pools. Use of such molecules could represent a novel strategy to combat insulin resistance and other oxidative stress-associated pathologies.