Effect of treatment with conditioned media derived from C2C12 myotube on adipogenesis and lipolysis in 3T3-L1 adipocytes

Abstract

Regular exercise is an effective strategy that is used to prevent and treat obesity as well as type 2 diabetes. Exercise-induced myokine secretion is considered a mechanism that coordinates communication between muscles and other organs. In order to examine the possibility of novel communications from muscle to adipose tissue mediated by myokines, we treated 3T3-L1 adipocytes with C2C12 myotube electrical pulse stimulation-conditioned media (EPS-CM), using a C2C12 myotube contraction system stimulated by an electrical pulse. Continuous treatment with myotube EPS-CM promoted adipogenesis of 3T3-L1 pre-adipocytes via the upregulation of the peroxisome proliferator-activated receptor-gamma (PPARγ) 2 and PPARγ-regulated gene expression. Furthermore, our results revealed that myotube EPS-CM induces lipolysis and secretion of adiponectin in mature adipocytes. EPS-CM obtained from a C2C12 myoblast culture did not induce such changes in these genes, suggesting that contraction-induced myokine(s) secretion occurs particularly in differentiated myotubes. Thus, contraction-induced secretion of myokine(s) promotes adipogenesis and lipid metabolism in 3T3-L1 adipocytes. These findings suggest the possibility that skeletal muscle communicates to adipose tissues during exercise, probably by the intermediary of unidentified myokines.

Fig 1. Cell contraction and preparation of C2C12 myotube electrical pulse stimulation-conditioned media (EPS-CM).

(A) AMPK phosphorylation (Thr172) level in C2C12 myotubes by 3h EPS. (B) Lactate dehydrogenase activity in the conditioned media. (C, D) Lactic acid and glucose concentration in the conditioned media. The initial concentration of glucose in the medium was 5.5 mM. (E, F) Immunoblotting of IL-6 and IL-15 in the conditioned media. Data are expressed as mean ± SEM (n = 8 (A-E), n = 4 (F)). **; p < 0.01. N.S., not significant.

Fig 2. Effects of myotube EPS-CM treatment on differentiation and lipid accumulation of 3T3-L1 adipocytes.

Differentiating 3T3-L1 adipocytes were continuously treated with 50% myotube EPS-CM. (A) Representative images of adipocytes stained with Oil Red O on days 4, 6, and 10 post-differentiation, (B) followed by the measuring of total lipid amount. (C-E) The expression levels of PPARγ2, C/EBPα, and SREBP-1 were evaluated via qRT-PCR on days 4, 6, and 10. Data are expressed as mean ± SEM (n = 3–4). *; p < 0.05. N.S., not significant. Scale bar, 200 μm.

Fig 3. Effects of myotube EPS-CM treatment on lipolysis of 3T3-L1 adipocytes.

(A) Following the final 48 h treatment with myotube EPS-CM (days 8–10), glycerol levels in the culture media of 3T3-L1 adipocytes were determined. (B) Representative images of stained mature adipocytes (day 10) containing lipid droplets are shown for the Control-CM and EPS-CM states. (C) The distribution of lipid droplets with respect to their sizes (μm²) is shown for the Control-CM and EPS-CM states. (D) Perilipin-1 protein levels in 3T3-L1 adipocytes (day 10) were quantified using immunoblotting. (E, F) The expression of HSL and ATGL was evaluated via qRT-PCR on days 6 and 10. Data are expressed as mean ± SEM (n = 4 (A, C), n = 10 (D), n = 3–4 (E, F)). *; p < 0.05. **; p<0.01. Scale bar, 50 μm.

Fig 4. Effects of myotube EPS-CM treatment on adiponectin secretion of 3T3-L1 adipocytes.

(A) 3T3-L1 adipocytes were continuously treated with 50% myotube EPS-CM, and on day 10 post-differentiation, cell lysates were subjected to immunoblotting for adiponectin. (B) Following the final 48 h treatment with myotube EPS-CM (day 8–10), the levels of adiponectin in the culture media of 3T3-L1 adipocytes were determined using ELISA. Data are expressed as mean ± SEM (n = 12 (A), n = 8 (B)). *; p < 0.05. **; p<0.01.

Fig 5. Effects of myotube EPS-CM treatment on mRNA expression of 3T3-L1 adipocytes.

The mRNA expression level of 3T3-L1 adipocytes subjected to continuous treatment with C2C12 myotube/myoblast derived CM with and without EPS (EPS-CM and Control-CM) were measured on day 10 post-differentiation. (A-H) The expression levels of PPARγ2, FAS, LPL, aP2, adiponectin, leptin, TNF-α, and UCP-1 were evaluated by qRT-PCR. Data are expressed as mean ± SEM (n = 4–6). Different letters represent significant differences (p < 0.05).