The objective of the research was to identify the presence of adiponectin receptors and to study adiponectin action on glucose uptake and growth in mouse mammary epithelial cells. These cells expressed adiponectin receptors, AdipoR1 and AdipoR2. Insulin (10 ng/ml) or insulin like growth factor-I (IGF-I, 10 ng/ml) alone did not alter the degree of AdipoR1 and AdipoR2 genes expression from 0 to 4 h incubation. Prolactin (10 ng/ml) or epidermal growth factor (EGF, 10 ng/ml) alone also did not induce the two genes’ mRNA in the incubation time. Adiponectin (1 μg/ml) alone or pre-incubation of insulin alone (100 ng/ml) for 2 h prior to adiponectin stimulation did not increase 2-deoxy-D-glucose,[1,2-3H] uptake but adiponectin+pre-incubation of insulin significantly increased glucose uptake compare to control (p<0.05). In a similar way, insulin alone or pre-incubation of adiponectin alone (2 h) did not increase glucose uptake but insulin+pre-incubation of adiponectin increased glucose uptake compare to control (p<0.05). Insulin sensitization for 2 h prior to adiponectin stimulation tended to increase glucose uptake response by the following adiponectin stimulation showing small interaction effect between insulin and adiponectin (p<0.1). However, adiponectin sensitization for 2 hours prior to insulin stimulation did not shown interaction effect between adiponectin and insulin (p>0.1). The glucose uptake by both of hormones seems to be not interactive but additive (p<0.05). Adiponectin in the presence of 2% FBS decreased DNA synthesis of mammary epithelia (p<0.05). AICAR (100 or 200 μM), AMPK activator, decreased mammary epithelial cell growth in the presence of 2% FBS. These results indicate that adiponectin pathway has inhibitory effect on mammary epithelial cell growth.

This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (～35 kDa), dimer (～70 kDa), trimer (～105 kDa) and hexamer (～210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (～300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher’s study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (β- MeOH), anionic detergent (SDS) and heat (95℃) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.