Background and aims:

Adiponectin (ApN) is an adipokine exhibiting insulin-sensitizing, fat-burning and anti-inflammatory properties on several tissues including muscle. ApN could also modulate oxidative stress. This adipokine is almost exclusively produced by adipose tissue under normal conditions. However, ApN can be induced in the skeletal muscle in response to inflammatory stimuli. The aim of the present work was to examine whether ApN could also be upregulated in muscle in response to metabolic and/or oxidative aggression both in vivo, in a murine model of type 2 diabetes, and in vitro.

Materials and Methods:

Obese, diabetic and leptin-deficient ob/ob mice (O, 13 wk old) were used and compared to lean littermates (L). A group of ob/ob mice was also treated with the antioxidant probucol for three weeks (P). At the end of the experiment, tail vein blood was sampled from fed animals and tibialis anterior muscles were collected for measurement of mRNA levels by RTQ-PCR and of ApN levels by immunohistochemistry. Additional in vitro experiments were performed on C2C12 myotubes for up to 48 h.

Results:

ApN gene expression was paradoxically increased in tibialis anterior muscle of ob/ob mice while it was decreased in their adipose tissue, with a lowering of circulating ApN as a consequence. The abundance of ApN mRNA in the muscle was ~80-fold higher in O mice than in L littermates (P < 0.001) and proved to be positively correlated with systemic oxidative stress. ApN gene upregulation was also shown in laser-microdissected myocytes of O mice. Concomitantly, immunoreactivity for ApN was enhanced in obese muscle fibres together with labelling for two markers of oxidative stress (PRDX3 and PRDX5). Finally, ectopic lipid storage was detected in cryocut sections of tibialis anterior muscle from O but not L mice, using Oil Red O coloration.

We subsequently attempted to identify the mechanisms underlying muscle ApN induction in vitro. We tested on cultured myotubes the effects of glucose/lipid overload, high insulin and ROS to mimic the in vivo diabetic and obese situation where glucotoxicity, lipotoxicity, hyperinsulinemia/insulin resistance and/or oxidative stress prevail. High glucose (25 mM) and insulin (100 nM) did not affect ApN mRNA levels in C2C12 myotubes. By contrast, Structolipid® (a mixture of triglycerides) and ROS (reactive oxygen species) producers (H₂O₂ or the lipoperoxidation product, 4-hydroxy-2-nonenal) were efficient inducers of ApN when added to the C2C12 culture medium (~2.5-, ~2.5- and ~7-fold respectively, P < 0.001 for each). ApN induction was reversed by using an antioxidant. Thus, ApN upregulation was abolished with N-acetylcysteine in vitro and with probucol treatment of obese mice in vivo.

Conclusion:

Adiponectin is upregulated in muscles of ob/ob mice. This induction may result from lipotoxicity and related oxidative stress. This overexpression could be viewed as a local protective mechanism to counteract ectopic fat deposit and oxidative damage in obese and diabetic muscle.