Insulin resistance in skeletal muscle is an early event in the development of diabetes, with obesity being one of the major contributing factors. In vitro, conditioned medium (CM) from differentiated human adipocytes impairs insulin signaling in human skeletal muscle cells, but it is not known whether insulin resistance is reversible and which mechanisms may underlie this process. CM induced insulin resistance in human myotubes at the level of insulin-stimulated Akt and GSK-3 phosphorylation. In addition, insulin-resistant skeletal muscle cells exhibit enhanced production of reactive oxygen species and ceramide as well as a downregulation of myogenic transcription factors such as myogenin and MyoD. However, insulin resistance was not paralleled by increased apopotosis. Regeneration of myotubes for 24 or 48 h after induction of insulin resistance restored normal insulin signaling. However, the expression level of myogenin could not be reestablished. In addition to decreasing myogenin expression, CM also decreased the release of IL-6 and IL-8 and increased monocyte chemotactic protein-1 (MCP-1) secretion from skeletal muscle cells. Although regeneration of myotubes reestablished normal secretion of IL-6, the release of IL-8 and MCP-1 remained impaired for 48 h after withdrawal of CM. In conclusion, our data show that insulin resistance in skeletal muscle cells is only partially reversible. Although some characteristic features of insulin-resistant myotubes normalize in parallel to insulin signaling after withdrawal of CM, others such as IL-8 and MCP-1 secretion and myogenin expression remain impaired over a longer period. Thus, we propose that the induction of insulin resistance may cause irreversible changes of protein expression and secretion in skeletal muscle cells.
- adipose tissue
- cellular cross-talk
- oxidative stress
obesity is one of the major risk factors contributing to the development of insulin resistance and type 2 diabetes (10). In this context, the negative cross-talk between adipose tissue and skeletal muscle is involved in early metabolic disturbances leading to insulin resistance (31, 33). Adipocytes from obese patients have a different secretion pattern compared with lean donors, with the release of proinflammatory factors and adipokines being increased (28). In fact, these adipose-derived molecules might be key contributors to the development of insulin resistance and other diseases such as endothelial dysfunction and atherosclerosis (36). In vitro, we were able to show that adipocyte-conditioned medium (CM) containing various adipokines induces insulin resistance in skeletal muscle cells (7, 9).
The development of insulin resistance is a reversible process. Reduction of adipose tissue mass by weight loss is a validated approach to reverse insulin resistance (11, 25). In parallel with improved insulin sensitivity, weight reduction also normalizes adipokine blood level, which has been demonstrated for IL-6 (5), high-molecular-weight adiponectin (2), monocyte chemotactic protein-1 (MCP-1) (4), and TNFα (19). It could be shown that insulin resistance disappears in cultured skeletal muscle biopsies from obese patients (3, 22), demonstrating that insulin resistance might be a reversible feature that can be acquired with obesity. However, other studies in muscle biopsies from obese and diabetic patients demonstrated that insulin resistance is retained in culture (3, 13, 39). This study was aimed at analyzing reversibility of adipocyte-induced insulin resistance in skeletal muscle cells and underlying mechanisms.
MATERIALS AND METHODS
BSA (fraction V, fatty acid free) was obtained from Roth (Karlsruhe, Germany). Reagents for SDS-PAGE were supplied by Amersham Pharmacia Biotech (Braunschweig, Germany) and by Sigma (München, Germany). Polyclonal antibodies anti-phospho-GSK-3α/β (Ser21/9), anti-phospho-Akt (Ser473), and anti-glucose transporter 4 (GLUT4) were supplied by Cell Signaling Technology (Frankfurt, Germany) and anti-tubulin by Calbiochem (Darmstadt, Germany). Antibodies for myogenin came from Acris (Hiddenhausen, Germany), for MyoD from Imgenex (San Diego, CA), and the one for myosin heavy chain (MHC) from Upstate (San Diego, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies were purchased from Promega (Mannheim, Germany). Collagenase CLS type 1 was obtained from Worthington (Freehold, NJ), and culture media were obtained from Gibco (Berlin, Germany). Primary human skeletal muscle cells and supplement pack for growth medium were obtained from PromoCell (Heidelberg, Germany). All other chemicals were of the highest analytical grade commercially available and were purchased from Sigma.
Culture of human skeletal muscle cells.
Primary human skeletal muscle cells of four healthy Caucasian donors (2 males, 9 and 47 yr of age; 2 females, 10 and 48 yr of age) were supplied as proliferating myoblasts (5 × 105 cells) and cultured as described previously (9). For an individual experiment, myoblasts were seeded in six-well culture dishes (9.6 cm2/well) at a density of 105 cells/well and were cultured in α-modified Eagle's/Ham's F-12 medium containing Skeletal Muscle Cell Growth Medium Supplement Pack up to near-confluence. The cells were then differentiated and fused by culture in α-modified Eagle's medium for 4 days and used for experiments.
Adipocyte isolation and culture.
Adipose tissue samples were obtained from the mammary fat of normal or moderately overweight women (body mass index 24.5 ± 0.9, aged between 23 and 41 yr) undergoing surgical mammary reduction. The procedure to obtain adipose tissue was approved by the ethics committee of Heinrich-Heine-University, Duesseldorf, Germany. All subjects were healthy, free of medication, and had no evidence of diabetes according to routine laboratory tests. Adipose tissue samples were dissected from other tissues and minced in pieces of ∼10 mg in weight. Preadipocytes were isolated by collagenase digestion as described previously (12). Isolated cell pellets were resuspended in Dulbecco's modified Eagle's/Ham's F-12 medium supplemented with 10% FBS, seeded on membrane inserts (3.5 × 105/4.3 cm2) or in a six-well culture dish, and kept in culture for 16 h. After washing, culture was continued in an adipocyte differentiation medium (DMEM-F-12, 33 μM biotin, 17 μM d-pantothenic acid, 66 nM insulin, 1 nM triiodo-l-thyronin, 100 nM cortisol, 10 μg/ml apotransferrin, 50 μg/μl gentamycin, 15 mM HEPES, 14 mM NaHCO3, pH 7.4). After 15 days, 60–80% of seeded preadipocytes developed to differentiated adipose cells, as defined by cytoplasm filled completely with small or large lipid droplets. These cells were then used for generation of CM, as previously described by us (8). Briefly, after in vitro differentiation, adipocytes were incubated for 48 h in skeletal muscle cell differentiation medium. This CM was then harvested, centrifuged to remove any cell debris, and immediately frozen in aliquots for future use. CM from 350,000 adipocytes was used to stimulate one cavity in a six-well plate of skeletal muscle cells. In control experiments, skeletal muscle cell differentiation medium was incubated for 48 h without adipocytes and tested upon its effect on skeletal muscle. No difference in insulin signaling could be found using this medium compared with fresh skeletal muscle cell differentiation medium (data not shown).
Muscle cells were treated as indicated and lysed in a buffer containing 50 mM HEPES (pH 7.4), 1% (vol/vol) Triton X, 1 mM Na3VO4, and Complete Protease Inhibitor Cocktail from Roche Diagnostics. After incubation for 2 h at 4°C, the suspension was centrifuged at 13,000 g for 15 min. Thereafter, 5 μg of lysates was separated by SDS-PAGE using 10% horizontal gels and transferred to polyvinylidene fluoride filters in a semidry blotting apparatus. For detection, filters were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat dry milk and subsequently incubated overnight with the appropriate antibodies. After extensive washing, filters were incubated with secondary HRP-coupled antibody and processed for enhanced chemiluminescene detection using Uptilight (Interchim, France). Signals were visualized and evaluated on a LUMI Imager workstation using image analysis software (Boehringer Mannheim, Mannheim, Germany).
ELISAs for IL-6, IL-8, and MCP-1 were purchased from Diaclone (Stamford, CT). Undiluted samples from skeletal muscle cell supernatant were measured according to the manufacturer's protocols.
Measurement of reactive oxygen species and nitric oxide production in skeletal muscle cells.
Differentiated skeletal muscle cells were treated with CM overnight to induce insulin resistance. Then, cells were washed in PBS without Ca/Mg and used for the assay. For measurement of reactive oxygen species (ROS), cells were incubated in 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (Molecular Probes, Karlsruhe, Germany) solved in phenol red-free DMEM for 30 min. As a positive control, cells were treated with 0.3% H2O2 for 30 min in parallel to 2′,7′-dichlorodihydrofluorescein diacetate incubation. For measurement of nitric oxide (NO), skeletal muscle cells were incubated with 10 μM 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (Molecular Probes) solved in phenol red-free DMEM for 30 min. As a positive control for NO production, cells were also treated with 500 μM SNAP (Calbiochem, Darmstadt, Germany) for 30 min in parallel with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. Afterward, cells were lysed in the above-mentioned lysis buffer and fluorescence measured using an excitation wavelength of 595 nm on a Fluostar-P (SLT, Salzburg, Austria).
Measurement of SDH activity in skeletal muscle cells.
Differentiated skeletal muscle cells were incubated with CM for the indicated time and lysed in homogenization buffer containing 250 mM glucose, 10 mM Tris·HCl, 0.5 mM EGTA, and 0.5 mM DTT. Succinate dehydrogenase (SDH) activity was measured according to Pennington's method (23). Briefly, ∼200 μg of cell lysate was incubated with 10 mM sodium succinate in 50 mM NaH2PO4 buffer for 20 min at 37°C. Five millimolars p-iodonitrotetrazolium violet solved in 50 mM NaH2PO4 buffer was added to a final concentration of 0.5 mM for an additional 10 min at 37°C. The reaction was stopped by an ethylacetate- ethanol-trichloracid solution (5:5:1, vol/vol/wt). Immediately after 2-min centrifugation at 13,000 g, the supernatant was measured at 490 nm on a spectrophotometer (Beckman, Krefeld, Germany).
Measurement of apoptosis.
Apoptosis was monitored by assessment of caspase-3 activity, and nuclear fragmentation in skeletal muscle cells was treated with CM. The DEVD-cleaving activity of the caspase-3 class of cystein proteases was determined in cell lysates using Ac-DEVD-AMC (BD Biosciences, Heidelberg, Germany) as fluorogenic substrate according to the manufacturer's protocol. The ability of cell lysates to cleave the specific caspase-3 substrate was quantified by spectrofluorometry using an excitation wavelength of 390 nm and an emission wavelength of 460 nm with a microplate reader. For detection of nuclear fragmentation, the cells were double-stained with Hoechst 33342 and propidium iodide. Skeletal muscle cells were washed twice with PBS and stained with 10 μg/ml Hoechst 33342 and 1 μg/ml propidium iodide at 37°C for 15 min. Fluorescence was observed under a Leica DM IRB fluorescence microscope. At least 400 cells were counted for each experiment. Cells with condensed or fragmented nuclei were defined to be apoptotic, and cells with normally shaped nuclei were supposed to be viable.
Quantitative evaluation of ceramide.
Lipids from skeletal muscle cells were extracted in chloroform-methanol-water (2:1:0.1, vol/vol/vol) for 24 h at 48°C. Lipid extracts were applied to thin-layer Silica Gel 60 plates (Merck, Darmstadt, Germany) as described earlier (38). Ceramides were resolved twice using chloroform-methanol-acetic acid (190:9:1, vol/vol/vol) as developing systems. Following development, plates were air-dried, sprayed with 8% (wt/vol) H3PO4 containing 10% (wt/vol) CuSO4, and charred at 180°C for 10 min. Lipids were identified by their Rf value, using authentic lipid samples as references. Individual lipid bands obtained by thin-layer chromatography were evaluated by photodensitometry (Shimadzu, Kyoto, Japan). Assuming constant cholesterol amounts in all samples, densitometric data obtained for ceramide were normalized to cholesterol.
Presentation of data and statistics.
Statistical analysis was performed by ANOVA. All statistical analyses were done using Statview (SAS, Cary, NC), considering a P value of <0.05 as statistically significant. Corresponding significance levels are indicated in the figures.
CM-induced insulin resistance of insulin signaling in skeletal muscle cells is a reversible process.
CM of differentiated human adipocytes impairs insulin signaling at the level of Akt in human skeletal muscle cells (Fig. 1A). Insulin-stimulated GSK-3α/β phosphorylation is only slightly decreased by CM treatment, whereas basal phosphorylation is significantly increased, leading to an insignificant insulin effect (Fig. 1B). Withdrawal of CM for 24 or 48 h reestablishes normal insulin signaling in skeletal muscle cells, with Akt and GSK-3α phosphorylation being similar to control and GSK-3β phosphorylation being even higher than in the control situation.
Insulin resistance is accompanied by reduced expression of myogenic transcription factors in skeletal muscle cells and an irreversible downregulation of myogenin.
During differentiation, skeletal muscle cells display an increased expression of myogenin, MHC, and MyoD, all of which are markers of myogenesis (Fig. 2, A–C). Analysis of myogenic transcription factors revealed that CM-treated skeletal muscle cells have significantly reduced expression of myogenin, MHC, and MyoD (Fig. 3, A–C). Skeletal muscle cells display an increasing GLUT4 level (Fig. 4A, top). However, CM treatment did not affect GLUT4 expression in differentiated myotubes (Fig. 4A, bottom), and the cells exhibited an unaltered morphology compared with control cells (Fig. 4B). Withdrawal of CM for 24 or 48 h reverses the downregulation of MHC and MyoD, whereas the expression of myogenin remains decreased over the whole period compared with control (Fig. 3). Thus, despite reestablished insulin signaling, skeletal muscle cells do not normalize myogenin expression after CM treatment and withdrawal.
CM-treated skeletal muscle cells are characterized by a partially irreversible secretory dysfunction.
Skeletal muscle cells secrete various myokines, including IL-6, IL-8, and MCP-1. Compared with adipocytes that secrete ∼500 pg·ml−1·24 h−1 IL-6, skeletal muscle cells exhibit lower secretion of this cytokine with 23 ± 1 pg·ml−1·24 h−1 (n = 5). Treatment with CM leads to a significantly lower IL-6 secretion during the first 24 h of regeneration of myotubes (Fig. 5A). Forty-eight hours after CM withdrawal, however, IL-6 secretion is comparable with control cells.
IL-8 secretion is also lower in skeletal muscle cells (94 ± 12 pg·ml−1·24 h−1; n = 5) compared with adipocytes (∼500 pg·ml−1·24 h−1). CM-treated skeletal muscle cells display significantly impaired IL-8 secretion over the whole regeneration period of 48 h compared with control. This suggests that IL-8 secretion might be irreversibly disturbed in insulin-resistant myocytes (Fig. 5B).
MCP-1 is a cytokine robustly released from human adipocytes (∼3 ng·ml−1·24 h−1) but also secreted at low levels from myotubes (37 ± 11 pg·ml−1·24 h−1; n = 5). Induction of insulin resistance in skeletal muscle cells significantly stimulates MCP-1 secretion after 24 h of regeneration with an additional increase after 48 h (Fig. 5C).
Insulin-resistant skeletal muscle cells exhibit increased oxidative stress and decreased mitochondrial capacity but no apoptosis.
ROS and NO are both potential players in the induction of insulin resistance. As presented in Fig. 6, a significant increase in both ROS and NO production was observed in skeletal muscle cells treated with CM. SDH activity was measured in whole cell lysates of skeletal muscle cells to assess oxidative capacity. CM treatment slightly but significantly reduced SDH activity in whole cell lysates after 24 h (Fig. 7). Longer incubation with CM over 96 h further reduced the level of SDH activity. The parallel induction of insulin resistance and oxidative stress cannot, however, be assigned to apoptosis in skeletal muscle cells. Measurement of caspase-3 activity revealed no increase in CM-treated cells compared with controls [1.08 ± 0.13 vs. 1.06 ± 0.17 arbitrary units, significantly elevated positive control (campthotecin for 5 h) 1.95 ± 0.03 arbitrary units; n = 3–4]. Furthermore, nuclear fragmentation was not elevated in CM-treated cells compared with controls [2.6 ± 0.1 vs. 2.2 ± 0.2% apoptotic cells, significantly elevated positive control (campthotecin for 5 h) 5.0 ± 1.0% apoptotic cells; n = 3–4].
Insulin-resistant skeletal muscle cells contain higher ceramide levels.
Ceramide constitutes a well-known player in insulin resistance. Fatty acids and ceramide can induce insulin resistance in skeletal muscle cells (26, 37). Analysis of lipid extracts by thin-layer chromatography revealed a nearly threefold increase of ceramide content in insulin-resistant skeletal muscle cells compared with controls (Fig. 8).
Adipose tissue expansion and increased release of adipokines have been shown to play a crucial role in the induction of insulin resistance (14). We could demonstrate in several studies that adipocyte-derived factors can induce insulin resistance in skeletal muscle cells in vitro (7, 9, 32). The data presented here now demonstrate that CM-treated skeletal muscle cells are characterized not only by impaired insulin signaling but also by various other defects. Insulin-resistant skeletal muscle cells downregulate the expression of myogenin and display oxidative stress, lower mitochondrial capacity, and higher ceramide content. Furthermore, insulin-resistant myotubes have disturbed secretion of the myokines IL-6, IL-8, and MCP-1.
In vitro-differentiated skeletal muscle cells are characterized by a high abundance of myogenic transcription factors such as myogenin and MyoD. We demonstrate here for the first time that adipocyte-derived factors lead to a marked downregulation of myogenin in skeletal muscle cells. It is known from the literature that TNFα suppresses the differentiation process in C2C12 myoblasts (34), but nothing is known about its effect on differentiated cells. However, CM contains very low doses of TNFα [<0.02 pmol/l (7)], making it probable that another adipokine with a higher concentration in CM might be the culprit for downregulation of myogenin. The loss of myogenin in insulin-resistant skeletal muscle cells is, however, associated with a conservation of skeletal muscle phenotype, as myotubes display normal morphology and GLUT4 expression. However, it cannot be completely ruled out that the downregulation of multiple markers, including MyoD, MHC, and SDS, points to a dedifferentiation of skeletal muscle cells, and it is impossible so far to speculate on the meaning of this finding for the situation in skeletal muscle in vivo.
IL-6, IL-8, and MCP-1 are known secretory products from skeletal muscle with different roles in myogenesis, exercise, inflammation, and insulin sensitivity. Increased IL-6 levels are associated with insulin resistance in vivo (16), but short-term treatment of skeletal muscle cells with IL-6 can increase insulin sensitivity (40). The reported increase of IL-6 during exercise (21) makes it likely that IL-6 has completely different acute and chronic effects. As for myogenesis, IL-6 is a promyogenic factor (1) explaining the parallel decrease of myogenic markers and IL-6 secretion in the myotubes. Both IL-8 and MCP-1 are proinflammatory chemokines that are increased in serum of obese and diabetic patients (17, 29, 30). MCP-1 is a potent inducer of insulin resistance in skeletal muscle cells (32) and plays a role in myopathies (6). TNFα and IFNγ have been described to induce MCP-1 transcription in myoblasts (6). Although IL-8 secretion is almost completely inhibited in CM-treated skeletal muscle cells, MCP-1 release increases, pointing to an inflammatory effect of CM.
SDH activity is known to be slightly but significantly reduced in skeletal muscle lysates from diabetic patients compared with controls (20). We also observed a reduction in SDH activity in CM-treated skeletal muscle cells, indicating a possible role of decreased oxidative capacity in the initiation of skeletal muscle cell insulin resistance. Notably, in diabetic patients, reduced oxidative capacity in parallel with increased glycolytic activity is due to a significant alteration of skeletal muscle fiber composition.
Oxidative stress is a result of increased ROS or NO production and can lead to oxidation and damage of DNA, protein, and lipids (18). Increasing ROS production as observed in our model could cause damage to mitochondria and so-called mitoptosis and explain the loss of mitochondria observed in states with increased oxidative stress such as insulin resistance and diabetes. Thus, increased ROS or NO levels could also explain decreased SDH activity in insulin-resistant skeletal muscle cells. Other work in L6 muscle cells shows that palmitate-induced insulin resistance is also characterized by higher levels of ROS and NO (27). However, it should be noted that fatty acids are barely detectable in CM when an HPLC approach is used (data not shown). Therefore, we conclude that adipocyte-derived factors produce an increase in ROS and NO similar to that produced by fatty acids.
NO and inducible NO synthase are known to be increased in the diabetic state and are linked to chronic inflammation (15). However, it is not known how NO induces or exacerbates insulin resistance. In C2C12 skeletal muscle cells, the NO donor SNAP inhibits Akt activity, making it possible that an intracellular increase in skeletal muscle cell NO might contribute to insulin resistance (41). Furthermore, diabetic patients are characterized by higher blood levels of nitrates and nitrites as well as higher expression of inducible NO synthase in skeletal muscle (35). In our primary myotubes we also observed an increase in NO production after treatment with CM, which might, together with ROS, contribute to the development of insulin resistance. It should be noted in this context that CM-treated skeletal muscle cells are not apopototic, as shown by unaltered percentage of cells with nuclear fragmentation and similar caspase-3 activity, compared with controls, so NO and ROS elevation cannot be attributed to apoptosis.
The sphingolipid ceramide is described to be a possible link between obesity and diabetes. Fatty acids and resulting higher levels of ceramide can induce insulin resistance in skeletal muscle cells (26, 37). In this study, insulin-resistant skeletal muscle cells are also characterized by increased ceramide levels, which may contribute to adipokine-induced insulin resistance and illustrate disturbed lipid metabolism.
In this study, we were able to show that adipocyte-induced insulin resistance is a reversible process in skeletal muscle cells, at least at the level of insulin signaling. However, some alterations are not fully reversible and may illustrate longer-lasting damage to the myotubes by one-time treatment with CM. Skeletal muscle cells display long-lasting myogenin downregulation and secretory defects of IL-8 and MCP-1. Differentiation of skeletal muscle involves a group of transcription factors, including myogenin and MyoD, that activate muscle-specific gene expression and each have a distinct function during myogenesis (24). In our model, we observe a loss of myogenin expression with preservation of muscle phenotype. At this point, we cannot evaluate the physiological impact of the loss of myogenin. Our data clearly show that the loss of myogenin is unrelated to early steps in insulin signaling, myotube morphology, and GLUT4 expression. Certainly, our model of in vitro-differentiated skeletal muscle cells has limitations as to how our findings on downregulation of myogenic markers underlies obesity-related insulin resistance in vivo. Future work should be aimed to relate our findings to the in vivo situation in diabetic and obese patients in this respect. In summary, we could demonstrate that adipocyte-derived insulin resistance in skeletal muscle cells impacts on various aspects of skeletal muscle cell physiology. The analysis of mechanisms involved in skeletal muscle insulin resistance and its reversibility might lead to a better understanding of this process and a possible discovery of muscular targets for the treatment of type 2 diabetes.
This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium für Gesundheit, the German Diabetes Foundation, and the Commission of the European Communities (Collaborative Project ADAPT, contract no. HEALTH-F2-2008-201100).
We thank Prof. R. Olbrisch and his team, the Department of Plastic Surgery, Florence Nightingale Hospital Düsseldorf, for support in obtaining adipose tissue samples. We also thank Marlis Koenen, Andrea Cramer, Angelika Horrighs, and Daniela Herzfeld de Wiza. The secretarial assistance of Birgit Hurow is gratefully acknowledged.
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- Copyright © 2008 by American Physiological Society