Relevant Mechanism Of Adiponectin Activating The AMPK Signaling Pathway To Regulate Lipid Metabolism In Bovine Hepatocytes

Adiponectin (ADPN) is an adipokine secreted by adipose tissue with a highplasma level, and it plays a crucial role in anti-insulin resistance and beneficiallyregulating metabolic syndrome, especially in hepatic lipid metabolism. However, theregulation mechanism of hepatic lipid metabolism by ADPN in dairy cows is unclear.Therefore, the objective of this study was to investigate the mechanism of ADPN inthe regulation of lipid metabolism in bovine hepatocytes cultured in vitro in order tofurther understand ADPN signaling pathways of mediating lipid metabolism in bovinehepatocytes.First，for time-response experiments, hepatocytes from a newborn calf wereincubated with64ng/mL ADPN for0h,2h,4h,8h,12h and24h. The resultshowed that the protein levels of AdipoR1and AdipoR2and p-AMPKα/AMPKαratio were both higher after ADPN-treatment compared with the control (0h), peakingat4h. That indicates the numbers of AdipoR1and AdipoR2bound by ADPN werelargest after4h with ADPN-treatment and maybe phosphorylation of AMPKα couldbe activated instantaneously.Hepatocytes cultured in vitro were treated with different concentrations ofADPN and BML-275(an AMPKα inhibitor) for both4h and8h, including12groups,control group (0ng/mL ADPN, GC), low-dose group (16ng/mL ADPN, GL),medial-dose group (64ng/mL ADPN, GM), high-dose group (128ng/mL ADPN,GH), BML group (10μmol/L BML-275, BML), and BML+ADPN group (10μmol/LBML-275+64ng/mL ADPN, BML+ADPN). The mRNA, total protein and nuclearprotein were extracted and then were detected by various methods. The resultsshowed that ADPN significantly increased the mRNA and protein expression levels oftwo adiponectin receptors after4h treatment, while there was no statisticalsignificance between the BML and GC groups. However, the expression of AdipoR2 after ADPN-treatment was significantly higher than that of AdipoR1, indicating thatADPN could promote the expression of AdipoR1and AdipoR2, while BML did notaffect their levels. And ADPN exerts its biological function by binding to two specificreceptors, AdipoR1and AdipoR2, especially AdipoR2. Furthermore, thephosphorylation and activity of AMPKα was also up-regulated by ADPN, and whenBML was added, the p-AMPKα levels and activity were significantly decreased.These results indicated that the inhibitory effect of BML on phosphorylation ofAMPKα was obvious, but the effect of ADPN on AMPKα could compensate thisinhibition. Moreover, the expression levels and transcriptional activity of peroxisomeproliferator activated receptor-α (PPARα), as well as the mRNA levels of its targetgenes involved in lipid oxidation, ACO, CPT-1, CPT-2, L-FABP and ACSL, showed acorresponding up-regulation trend as the p-AMPKα levels and activity. By contrast,with the administration of ADPN, the expression levels and transcriptional activity ofsterol regulatory element binding protein1c (SREBP-1c) and carbohydrate-responsiveelement-binding protein (ChREBP) and their target genes involved in fatty acidsynthesis and lipid transport, such as ACCα, SCD-1, FAS, ApoB100, ApoE and MTP,decreased in a similar manner. When BML was added, the expression and activity ofPPARα and its target genes were significantly decreased, while the expression ofSREBP-1c, ChREBP and their target genes showed an increased trend. Furthermore,the triglyceride (TG) and very low-density lipoprotein (VLDL) contents weresignificantly decreased in the ADPN-treated groups. These results indicate that ADPNcould mediate hepatic lipid metabolism by activating these three transcription factors,PPARα, SREBP-1c and ChREBP, and then up-regulating or down-regulating mRNAlevels of their target genes, and they are all the downstream elements of AMPKα.After8h of incubation with ADPN, the up-regulation or down-regulation trend of thethree transcription factors and their target genes remained and even become enhanced.In conclusion, ADPN activates the AMPKα signaling pathway, subsequentlyresults in an enhancement of lipid oxidation by promoting expression level andactivity of PPARα, and a decreasing of lipid synthesis and lipid transport by suppressing those of SREBP1c and ChREBP, and decreasing the contents of TG andVLDL, and finally reducing lipid accumulation in bovine hepatocytes cultured invitro.