Characterization of amylin binding sites in a human hepatoblastoma cell line.

PEPTIDES 13(6) 1193-1199, 1992.--Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ~25I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (Kd = 0.11 + 0.04 nM) and low (Kd = 1.3 _+ 0.4 #M) affinity binding sites for t25I-amylin in HepG2 cells. The dissociation experiments also showed that n~I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ~251-amylin bound to HepG2 cells. Nonhomologous peptides did not displace J2~l-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ~251[Tyr°]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of J25I- amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8-37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells, and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction. Amylin Human hepatoblastoma cell line Calcitonin gene-related peptide AMYLIN is a 37-residue peptide originally isolated from the amyloid-rich pancreas of non-insulin-dependent diabetic (NIDD) (10) and insulinoma (29) patients. Subsequently, amylin has also been isolated from the normal pancreas of rats (1). Amylin is now regarded as a normal component of the pancreas, being costored and cosecreted with insulin from the islet secretory granules (16). Recent investigations suggest that amylin inhibits insulin-stimulated glucose uptake and glycogen synthesis by so- leus muscles (19). The peripheral effects of amylin have also been demonstrated in dogs (24) and rats (17,20) using euglycemic clamp technique. These in vivo investigations revealed that amylin also causes hepatic insulin resistance and that the liver is more sensitive to amylin than other peripheral tissues (17). Adipocyte glucose metabolism, however, was not altered by amylin (10). These observations have led to the speculation that amylin may play a role in non-insulin--dependent diabetes mel- litus (NIDDM). Calcitonin gene-related peptide (CGRP), which exhibits >40% homology with amylin, has also been shown to cause peripheral and hepatic insulin resistance (19,20). Furthermore, hCGRP(8-37), an antagonist of CGRP, inhibited the stimulatory adenylate cyclase activity of CGRP as well as that of amylin (21,30). These observations and the failure to demonstrate spe- cific binding ofamylin on various mammalian membrane prep- arations (15,30) have led investigators to speculate that amylin actions on liver and muscles are mediated by CGRP receptors. However, recent findings that amylin causes peripheral insulin resistance through a cAMP-independent pathway ( 1 1) and that N-a-Ac-amylin(8-37) is a more selective antagonist to amylin than CGRP (12) suggest that specific receptors of amylin may mediate its actions. This investigation was therefore undertaken to develop a model system to study amylin receptors and amylin- mediated signal transductions. The human liver carcinoma cell line (HepG2) was chosen for this study because amylin has pre- viously been shown to increase gluconeogenesis and glycoge- nolysis in HepG2 (9). This investigation shows that distinct re- ceptors of amylin and CGRP are present in HepG2. METHOD