The effect of dietary n-3 and n-6 polyunsaturated fatty acids on the expression of cyclooxygenase 1 and 2 and levels of p21ras in rat mammary glands.

Dietary n-6 polyunsaturated fatty acids (PUFAs) promote rat mammary cancer while n-3 PUFAs are inhibitory. The purpose of this study was to determine whether the fats exert their effects by altering the expression of genes that affect cancer development. Therefore, we have examined the effect of PUFAs on the expression of the cyclooxygenase (COX) 1 and 2 genes that are involved in prostaglandin biosynthesis. We also investigated the effect of dietary PUFAs on the expression of the p21ras protein and Ha-ras mRNA. Rats were fed either low- (7%; LF) or high- (21%; HF) fat diets that were rich in either n-6 PUFAs (safflower oil, S) or n-3 PUFAs (menhaden oil, M) for 3 weeks. COX-1 mRNA levels were approximately the same in groups fed diets containing either level of menhaden oil, but were increased by approximately 30% in the LFS and HFS groups (P < 0.05). Transcripts of the inducible COX-2 gene were not detectable in the menhaden oil groups, but this gene was expressed in animals fed either level of safflower oil and in the HFS group was associated with increased levels of COX enzymatic activity and production of PGE2. Animals fed safflower oil had elevated levels of p21ras protein compared to animals fed menhaden oil. Ha-ras mRNA was increased by approximately 35% in animals fed HFS compared to the group fed HFM (P < 0.05). These results demonstrate that dietary n-6 PUFAs upregulate COX-2 and, to some extent, COX-1 expression. There was a concomitant increase in COX enzyme activity and PG synthesis in the mammary glands of rats fed high levels of n-6 PUFAs. Together with associated changes in p21ras expression, these results may explain, at least in part, the promoting effects of dietary n-6 PUFAs on mammary carcinogenesis.

Very low protein diets induce a rapid decrease in hepatic cAMP-dependent protein kinase followed by a lower increase in adenylyl cyclase activity in rats.

Recent evidence indicates that cAMP-mediated responses are desensitized in liver during malnutrition. While receptor-stimulated production of cAMP is increased in hepatocytes from rats fed very low protein diets for 14 d, activity of cAMP-dependent protein kinase (PKA) is decreased in liver cytosol. The present study investigated the time course for this desensitization. Weanling rats were fed either a 0.5 (malnourished) or 15% protein (control) diet for 1, 3, 7 or 14 d. Total PKA activity decreased after only 3 d of feeding the low protein diet. This decrease was confined to the cytosolic compartment and was associated with a lower quantity of immunoreactive RI regulatory subunit of PKA, with no difference in the quantity of immunoreactive RII regulatory subunit. In contrast, basal-, MnCl2- and guanine nucleotide regulatory protein-stimulated adenylyl cyclase activities were not greater in liver membranes of malnourished rats than in those of the control rats until the 2nd wk of feeding. Greater activity was paralleled by an increase in the quantity of the stimulatory guanine nucleotide regulatory protein at d 14. The inhibitory guanine nucleotide regulatory protein quantity did not differ between dietary groups. Greater cAMP production was not mediated by changes in PKA phosphorylation of adenylyl cyclase because preincubation of membranes with purified PKA catalytic subunit decreased MnCl2-stimulated cAMP production equally in liver membranes of both control and malnourished rats. Similarly, treatment with alkaline phosphatase decreased adenylyl cyclase activity but did not eliminate the difference in adenylyl cyclase activity between control and malnourished rats. These data demonstrate that loss of PKA activity is an early response to a low protein diet and that, subsequently, a number of molecular adaptations occur which increase cAMP production. These changes may be adaptive responses to malnutrition that maintain essential cAMP-dependent functions.

Activity of cAMP-dependent protein kinase is reduced in protein-energy malnourished rats.

Glucagon decreases glutathione synthesis in hepatocytes from well-nourished rats. However, in hepatocytes from malnourished rats, glucagon does not inhibit glutathione synthesis, suggesting a desensitization of cAMP-mediated signal transduction. We investigated the mechanism for this desensitization of cAMP-mediated responsiveness in malnourished rats by comparing the signal transduction pathways in rats fed very low protein diets (0.5 g protein/100 g diet) with those of rats fed diets adequate in protein (15 g protein/100 g diet) for 2 wk. Glucagon receptor and forskolin-stimulated cAMP production were greater in hepatocytes from malnourished rats. Stimulation of adenylyl cyclase with forskolin, guanine nucleotides or manganese in hepatic membranes was also enhanced after malnutrition. Moreover, quantity of the stimulatory guanine nucleotide regulatory protein was 70-80% greater in hepatocytes from malnourished rats but the inhibitory guanine nucleotide regulatory protein was not different. These results suggested that desensitization of cAMP-mediated signal transduction after malnutrition occurred at a site distal to cAMP production. Maximal activity of cAMP-dependent protein kinase was 60% lower in liver homogenates from malnourished rats compared with controls. This difference in activity was confined to the cytosolic compartment, with no difference in activity observed in the particulate fraction. Lower activity of cAMP-dependent protein kinase in the cytosol of malnourished rats was associated with a 43% reduction in the quantity of regulatory subunit type I, with no difference in the regulatory subunit type II. These data indicate that desensitization of cAMP signal transduction in rat liver after malnutrition is due to a decrease in the quantity and activity of cAMP-dependent protein kinase.