MATERIALS AND METHODS

In vivo experiment. Fed, male, 9-to 10-week old, Swiss-Webster mice (Taconic Farms, Germantown, NY) weighing between 30-35 g were used. On the morning of the experiment, each mouse was mildly restrained by placing it inside a plastic tube. Animals were allowed to rest quietly for 30 min before hrIL-6 (2.5 mg/10 g; Upstate Biotechnology Inc., Lake Placid, NY) or an equal volume (200 ml) of vehicle (0.1% BSA) was injected via percutaneous puncture of the tail vein. Mice were returned to individual cages and sacrificed by decapitation 4 h later. Blood was collected in heparinized tubes and plasma stored at 070 C. This time point was chosen because previous studies have indicated that IGFBP-1 is increased at this time in animals injected with endotoxin or IL-1b (3, 6). Cell culture. HepG2 cells were grown in 24-well plates (Falcon; BD, Lincoln Parks, NJ) containing 500 ml of MEM (Sigma; St. Louis, MO; 1.5 mM calcium) supplemented with 5% FCS, penicillin (100 U/ml), streptomycin (100 mg/ml) and amphotericin B (25 mg/ml) for 5-7 days after subculture, at which time cells were near confluence. HepG2 cells were used because they are cytokine-responsive (16), and have a constitutive secretion of IGFBP-1 that is under hormonal regulation (17). On the day of the experiment, the medium was replaced with serum-free MEM containing either IL-6 (0-100 ng/ml), mrTNFa (0-100 ng/ml; gift of Genetech, South San Francisco, CA), hrIL-1b (0-100 ng/ml; gift of Biological Response Modifiers Program, Division of Cancer Treatment/NCI), dexamethasone (3 mM), endotoxin (Escherichia coli 026: B6; 1 mg/ml; Difco, Detroit, MI), insulin (0.1-10 nM), dantrolene or diltiazem (25-200 mM). The latter three compounds were obtained from Sigma. Cells were incubated for various periods of time, and the medium collected and stored at 070 C.

IGFBP-1 determination. The IGFBPs in plasma were determined by ligand blot analysis as previously described (3). Protein content in the supernatant was assayed to ensure that each sample had an equal concentration. Western blot analysis was used to determine the relative concentration of IGFBP-1 in medium from HepG2 cells (7, 8). Samples were separated on a 12.5% SDS-PAGE gel under nonreducing conditions. Proteins were electroblotted overnight onto nitrocellulose, and blocked with TRIS-buffered saline containing 1% nonfat dry milk. Membranes were then incubated with a 1: 2000 dilution of antiserum against human IGFBP-1 (UBI, Lake Placid, NY). Antigen-antibody complexes were identified with goat anti-rabbit IgG tagged with horseradish peroxidase (Sigma) and exposed to the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). Band intensities were determined using a laser densitometer (Hoeffer, San Francisco, CA).