Hepatocytes from Sprague-Dawley rats were isolated aseptically by collagenase perfusion as described by Berry et al.7. The initial viability of isolated hepatocytes was more than 85% as determined by 0.2% (w/v) Trypan blue exclusion. The freshly isolated hepatocytes were resuspended in Williams E (WE) medium to give a final cell concentration of 7.5 x 105 cells per ml. The basal WE medium contained 1 ^g/ml insulin, 50 ng/ml EGF, 50 nM dexamethasone, 3 nM Na2SeO3, 100 units/ml penicillin G, 100 ^g/ml streptomycin sulfate, 0.25 ^g/ml amphotericin B. Five ml of the diluted cell suspension (0.18 ml per cm2) were plated on each 60 mm diameter collagen-coated dish14. Cells were allowed to attach in basal medium over for a 4-h period. At 4 h, the basal medium was replaced with either fresh basal medium or the designated treatment medium. Cells were cultured with either standard WE with or without 2 mM methionine plus 0.05 mM bathocuproine disulfonate (BCS) for a total culture time of 48 h.
Table 4. Effects of experimental diets on food intake and weight gain of rats and on hepatic protein concentrations for Feeding Study # 1
Average daily dietconsump-tion, g
Average daily 4.1 ± 0.6a 6.1 ± 0.3b 6.0 ± 0.4b 5.0 ± 0.3ab 5.4 ± 0.2ab weight gain, g
Body weight at 281 ± 9.1a 321 ± 4.3b 321 ± 5.8b 313±5.1b 312 ± 2.7b end offeeding period, g
Liver protein, 210.2 ± 3.9 215.2 ± 4.2 223.2 ± 1.9 204.7 ±4.2 200.5 ±5.1 mg/g liver_
Values are means ± SE. Within a row, values with different superscripts are significantly different (p < 0.05) by ANOVA and Tukey-Kramer's ra-procedure.
For measurement of CDO activity and CDO concentration (western blot analysis), cultured hepatocytes were disrupted by sonication for three 15-s intervals using a High Intensity Ultrasonic Processor (Sonics and Materials Inc., Dansbury, CT) in 50 mM Mes [2-(N-morpholino)ethanesulfonic acid], pH 6.0. The supernatant fraction of cell homogenates was obtained by cen-
trifugation at 20,000 x g for 30 min at 4°C. Cells were stored in denaturation buffer (described above) for further analysis of mRNA via northern blot analyses. Each experiment was conducted in triplicate.
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