The Adipose-Derived Insulin Sensitizer Adiponectin Can Activate Intracellular Signaling Independent of the Well Known AdipoR1 or AdipoR2 Receptors

Adiponectin (ADN) is a crucial mediator of insulin sensitivity and vascular health. The discoveries of the adiponectin receptors AdipoR1 and AdipoR2 (R1 and R2) prompted efforts to identify ADN-mimetics. R1 and R2 are especially difficult drug targets due to the complex higher-order structures of ADN as well as challenges in creating receptor knock-out cell lines. We approached R1 and R2 as possible targets beginning with a rigorous validation of the ligand and receptors. ADN preparations from multiple internal and external sources were characterized in physicochemical, cell binding and in vitro assays (pAMPK/pACC and ceramidase). It is important to note that ADN from only one source passed this evaluation. This ADN triggered in vitro activities associated with this protein, including stimulation of phosphorylation of ACC (EC50 = 9.3 ug/ml), calcium influx (EC50 = 2.2 ug/ml), glucose uptake, fatty acid oxidation and ceramidase activity. With a biologically active ADN preparation identified, we strove to understand the interactions of ADN with R1 and R2. However, both R1 and R2 are ubiquitously expressed, complicating the in vitro confirmation of receptor-dependent functional activity. We selected HAP1 cells, an ADN-responsive haploid CML derived line well-suited for gene deletion via CRISPR-Cas9 technology, to generate R1, R2 and R1/R2 deficient cells. Surprisingly, the stimulatory activity of adiponectin in pACC and ceramidase assays in HAP1 cells deficient in R1, R2 or both R1 and R2 were indistinguishable from those in wild type HAP1 cells. In addition, transient overexpression of R1 in HAP1 cells or pretreatment with antibodies to block R1 did not alter their pACC responses to ADN. These data indicate R1 and R2 are not responsible for all the in vitro biological activity attributed to ADN and suggest that there must be other yet unidentified receptors for ADN. Disclosure J.W. Gunnet: Employee; Self; Janssen Research & Development. W. Li: None. Y. Wang: None. J.A. Chavez: None. M. Husovsky: None. R. Meng: None. W. Lang: None. J.R. Jeyaseelan: Employee; Self; Janssen Pharmaceuticals, Inc.. G.W. Caldwell: None. S. Edavettal: Employee; Self; Janssen Research & Development.