Endocrine Abstracts

Insulin resistance (IR), characterized by an impairment of target tissues to insulin responsiveness, is the core defect preceding diabetes. Preclinical and clinical data have suggested a role for the dopaminergic system in glucose homeostasis control. The aim of the current study was to investigate the role of the dopamine agonist cabergoline (CAB), displaying high affinity for dopamine type 2 (DR2) receptor and low affinity for serotonin receptors (5-HTRs), in the regulation of muscle IR. An in vitro model of mouse skeletal muscle (C2C12) has been used in baseline and IR palmitate (1 mM for 16 h)-induced conditions. DRs and 5-HTRs messenger and protein levels have been evaluated by RT-qPCR and WB, respectively. DR2 protein expression has been analyzed also through liquid chromatography tandem-mass spectrometry (LC-MS/MS). The effect of escalating doses of CAB (10⁻¹⁰ M, 10^-8 M, 10⁻⁶ M) alone or combined with 2*10⁻⁸M insulin on glucose uptake (GU) was evaluated by a colorimetric assay, while intracellular signaling has been investigated by WB. To evaluate GLUTs translocation, membrane proteins have been isolated from cytoplasmic proteins and analyzed by WB. As revealed by RT-qPCR and WB, C2C12 did not express DRs but expressed messenger and protein levels of 5-HT2RA and B. LC-MS/MS confirmed the complete absence of D2 protein. In C2C12 at baseline condition, the percentage (%) of GU was significantly stimulated by 10⁻¹⁰ M and 10⁻⁸ M CAB, either alone or combined with insulin. A significant increase of IRS-1 Tyr608 and Akt Ser473 phosphorylation levels as well as membrane GLUT4 translocation were shown after the 10⁻⁸ M CAB combined with insulin exposure, suggesting that CAB might stimulate GU by activating insulin receptor (INS-R) signaling. In condition of IR, C2C12 exposed to 10⁻⁶ M CAB with insulin showed a significant increase of % GU as well as GLUT1 membrane translocation after 10⁻⁶ M CAB alone. Moreover, 10⁻⁶ M CAB either alone or combined with insulin triggered a significant increase of pAMPK Thr172 and a significant decrease of pERK Thr202/Tyr204, suggesting that in IR state, CAB might stimulate glucose internalization through AMPK pathway independently from INS-R signaling activation. Pre-treatment with 5-HT2RA (altanserin) and 5-HT2RB (Ly272015) antagonists inhibited CAB-regulated intracellular signaling and GU suggesting that CAB acts through 5-HTRs. In conclusion, these data show that CAB induces muscle GU both at baseline and IR conditions by improving insulin and AMPK pathways respectively, through the serotoninergic pathway.