165 research outputs found
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Characterization of an Acute Muscle Contraction Model Using Cultured C2C12 Myotubes
A cultured C2C12 myotube contraction system was examined for application as a model for acute contraction-induced phenotypes of skeletal muscle. C2C12 myotubes seeded into 4-well rectangular plates were placed in a contraction system equipped with a carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. Approximately 80% of the myotubes were observed to contract microscopically, and the contractions lasted for at least 3 h with electrical stimulation. Calcium ion transient evoked by the electric pulses was detected fluorescently with Fluo-8. Phosphorylation of protein kinase B/Akt (Akt), 5′ AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38), and c-Jun NH2-terminal kinase (JNK)1/2, which are intracellular signaling proteins typically activated in exercised/contracted skeletal muscle, was observed in the electrically stimulated C2C12 myotubes. The contractions induced by the electric pulses increased glucose uptake and depleted glycogen in the C2C12 myotubes. C2C12 myotubes that differentiated after exogenous gene transfection by a lipofection or an electroporation method retained their normal contractile ability by electrical stimulation. These findings show that our C2C12 cell contraction system reproduces the muscle phenotypes that arise in vivo (exercise), in situ (hindlimb muscles in an anesthetized animal), and in vitro (dissected muscle tissues in incubation buffer) by acute muscle contraction, demonstrating that the system is applicable for the analysis of intracellular events evoked by acute muscle contraction
Contraction and AICAR Stimulate IL-6 Vesicle Depletion From Skeletal Muscle Fibers In Vivo
Recent studies suggest that interleukin 6 (IL-6) is released from contracting skeletal muscles; however, the cellular origin, secretion kinetics, and signaling mechanisms regulating IL-6 secretion are unknown. To address these questions, we developed imaging methodology to study IL-6 in fixed mouse muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP–containing vesicles and protein by 62% (P < 0.05), occurring rapidly and progressively over 25 min of contraction. However, contraction-mediated IL-6-EGFP reduction was normal in muscle-specific AMP-activated protein kinase (AMPK) α2-inactive transgenic mice. In contrast, the AMPK activator AICAR decreased IL-6-EGFP vesicles, an effect that was inhibited in the transgenic mice. In conclusion, resting skeletal muscles contain IL-6–positive vesicles that are expressed throughout myofibers. Contractions stimulate the rapid reduction of IL-6 in myofibers, occurring through an AMPKα2-independent mechanism. This novel imaging methodology clearly establishes IL-6 as a contraction-stimulated myokine and can be used to characterize the secretion kinetics of other putative myokines
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Ablation of AMP-Activated Protein Kinase 2 Activity Exacerbates Insulin Resistance Induced by High-Fat Feeding of Mice
Objective: We determined whether muscle AMP-activated protein kinase (AMPK) has a role in the development of insulin resistance. Research Design and Methods: Muscle-specific transgenic mice expressing an inactive form of the AMPK 2 catalytic subunit (2i TG) and their wild-type littermates were fed either a high-fat (60% kcal fat) or a control (10% kcal fat) diet for 30 weeks. Results: Compared with wild-type mice, glucose tolerance in 2i TG mice was slightly impaired on the control diet and significantly impaired on the high-fat diet. To determine whether the whole-body glucose intolerance was associated with impaired insulin sensitivity in skeletal muscle, glucose transport in response to submaximal insulin (450 U/ml) was measured in isolated soleus muscles. On the control diet, insulin-stimulated glucose transport was reduced by 50% in 2i TG mice compared with wild-type mice. High-fat feeding partially decreased insulin-stimulated glucose transport in wild-type mice, while high-fat feeding resulted in a full blunting of insulin-stimulated glucose transport in the 2i TG mice. High-fat feeding in 2i TG mice was accompanied by decreased expression of insulin signaling proteins in gastrocnemius muscle. Conclusions: The lack of skeletal muscle AMPK 2 activity exacerbates the development of glucose intolerance and insulin resistance caused by high-fat feeding and supports the thesis that AMPK 2 is an important target for the prevention/amelioration of skeletal muscle insulin resistance through lifestyle (exercise) and pharmacologic (e.g., metformin) treatments
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Exercise Alleviates Lipid-Induced Insulin Resistance in Human Skeletal Muscle–Signaling Interaction at the Level of TBC1 Domain Family Member 4
Excess lipid availability causes insulin resistance. We examined the effect of acute exercise on lipid-induced insulin resistance and TBC1 domain family member 1/4 (TBCD1/4)-related signaling in skeletal muscle. In eight healthy young male subjects, 1 h of one-legged knee-extensor exercise was followed by 7 h of saline or intralipid infusion. During the last 2 h, a hyperinsulinemic-euglycemic clamp was performed. Femoral catheterization and analysis of biopsy specimens enabled measurements of leg substrate balance and muscle signaling. Each subject underwent two experimental trials, differing only by saline or intralipid infusion. Glucose infusion rate and leg glucose uptake was decreased by intralipid. Insulin-stimulated glucose uptake was higher in the prior exercised leg in the saline and the lipid trials. In the lipid trial, prior exercise normalized insulin-stimulated glucose uptake to the level observed in the resting control leg in the saline trial. Insulin increased phosphorylation of TBC1D1/4. Whereas prior exercise enhanced TBC1D4 phosphorylation on all investigated sites compared with the rested leg, intralipid impaired TBC1D4 S341 phosphorylation compared with the control trial. Intralipid enhanced pyruvate dehydrogenase (PDH) phosphorylation and lactate release. Prior exercise led to higher PDH phosphorylation and activation of glycogen synthase compared with resting control. In conclusion, lipid-induced insulin resistance in skeletal muscle was associated with impaired TBC1D4 S341 and elevated PDH phosphorylation. The prophylactic effect of exercise on lipid-induced insulin resistance may involve augmented TBC1D4 signaling and glycogen synthase activation
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Tribbles 3 Mediates Endoplasmic Reticulum Stress-Induced Insulin Resistance in Skeletal Muscle
Endoplasmic Reticulum (ER) stress has been linked to insulin resistance in multiple tissues but the role of ER stress in skeletal muscle has not been explored. ER stress has also been reported to increase tribbles 3 (TRB3) expression in multiple cell lines. Here, we report that high fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases TRB3 and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signaling. Incubation of C2C12 cells and mouse skeletal muscle with ER stressors thapsigargin and tunicamycin increases TRB3 and impairs insulin signaling and glucose uptake, effects reversed in cells overexpressing RNAi for TRB3 and in muscles from TRB3 knockout mice. Furthermore, TRB3 knockout mice are protected from high fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that TRB3 mediates ER stress-induced insulin resistance in skeletal muscle
Exercise training induces depot-specific adaptations to white and brown adipose tissue
Exercise affects whole-body metabolism through adaptations to various tissues, including adipose tissue (AT). Recent studies investigated exercise-induced adaptations to AT, focusing on inguinal white adipose tissue (WAT), perigonadal WAT, and interscapular brown adipose tissue (iBAT). Although these AT depots play important roles in metabolism, they account for only ∼50% of the AT mass in a mouse. Here, we investigated the effects of 3 weeks of exercise training on all 14 AT depots. Exercise induced depot-specific effects in genes involved in mitochondrial activity, glucose metabolism, and fatty acid uptake and oxidation in each adipose tissue (AT) depot. These data demonstrate that exercise training results in unique responses in each AT depot; identifying the depot-specific adaptations to AT in response to exercise is essential to determine how AT contributes to the overall beneficial effect of exercise11425439This work was supported by National Institutes of Health grants R01-HL138738 and K01-DK105109 (to K.I.S.), R01-DK099511 (to L.J.G.), and 5P30 DK36836 (Joslin Diabetes Center DRC). The authors thank Nathan Makarewicz for editorial contribution
Exercise intensity regulates cytokine and klotho responses in men
Background Short-term exercise training programs that consist of moderate intensity endurance training or high intensity interval training have become popular choices for healthy lifestyle modifications, with as little as two weeks of training being shown to improve cardiorespiratory fitness and whole-body glucose metabolism. An emerging concept in exercise biology is that exercise stimulates the release of cytokines and other factors into the blood that contribute to the beneficial effects of exercise on metabolism, but whether these factors behave similarly in response to moderate and high intensity short term training is not known. Here, we determined the effects of two short-term exercise training programs on the concentrations of select secreted cytokines and Klotho, a protein involved in anti-aging. Methods Healthy, sedentary men (n = 22) were randomized to moderate intensity training (MIT) or sprint intensity training (SIT) treatment groups. SIT consisted of 6 sessions over 2 weeks of 6 x 30 s all out cycle ergometer sprints with 4 min of recovery between sprints. MIT consisted of 6 sessions over 2 weeks of cycle ergometer exercise at 60% VO2peak, gradually increasing in duration from 40 to 60 min. Blood was taken before the intervention and 48 h after the last training session, and glucose uptake was measured using [18F]FDG-PET/CT scanning. Cytokines were measured by multiplex and Klotho concentrations by ELISA. Results Both training protocols similarly increased VO2peak and decreased fat percentage and visceral fat (P Conclusion Short-term exercise training at markedly different intensities similarly improves cardiovascular fitness but results in intensity-specific changes in cytokine responses to exercise.</div
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