Roderick Nakayama

Effect of dietary cholesterol on apolipoprotein E synthesis in the rabbit

Female New Zealand White rabbits were fed either rabbit chow or rabbit chow plus 1% (w/w) cholesterol for 14 days. The chow-fed rabbits had normal plasma lipoprotein profiles on agarose gel electrophoresis, 59 +/- 5 mg of cholesterol and 5.5 +/- 0.4 mg of apolipoprotein E (apoE) per dl of serum. The cholesterol-fed rabbits had significant amounts of beta-VLDL in their serum, 1870 +/- 140 mg of cholesterol and 96 +/- 12 mg of apoE per dl of serum. Relative rates of apoE synthesis were determined by incubating hepatocytes in culture medium containing [3H]leucine for 15 min at 37 degrees C and expressing the radioactivity incorporated into immunoprecipitable apoE as a percentage of the radioactivity incorporated into total protein. Hepatocytes from cholesterol-fed rabbits had twice the relative rate of apoE synthesis (1.05 +/- 0.18%) of hepatocytes from chow-fed rabbits (0.55 +/- 0.07%). This increase in synthesis could be a major contributor to the 17-fold increase in serum apoE levels in the cholesterol-fed rabbit.

Regulation of glucose-6-P dehydrogenase activity in primary rat hepatocyte cultures

Abstract The glucose-6-P dehydrogenase specific activity in rat hepatocytes increases approximately 10-fold when the cells are placed into culture for three days. The induction requires insulin with maximum enzyme levels occurring at 10 −7 M. Pulse-labeling experiments revealed a 10-fold increase in the enzymes relative rate of synthesis after only 8 hours in culture.

Regulation of glucose-6-P dehydrogenase synthesis in rat epididymal fat pads☆

Abstract The relative rate of synthesis of glucose-6-P dehydrogenase increases up to 8-fold when fasted rats are fed a 60% carbohydrate, fat-free diet for 3 days but the specific activity of the enzyme only increases 2 to 3 fold. This suggests that the high carbohydrate diet also causes a 2 to 3 fold increase in the rate of glucose-6-P dehydrogenase degradation. The nutritional induction of this enzyme in adipose tissue is primarily due to a large increase in the rate of its synthesis.

Solution hybridization quantitation of G6PD mRNA in rat epididymal fat pads

A solution hybridization assay is systematically characterized and used to quantitate glucose-6-phosphate dehydrogenase (G6PD) mRNA from epididymal fat pads in fasted and glucose-induced rats. G6PD mRNA and specific activity increase 9-fold and 2-fold, respectively. The 9-fold increase in G6PD synthesis reported previously (Wolfe et al. (1979) Biochem. Biophys. Res. Commun. 89, 108-115) can, therefore, be accounted for by the increase in G6PD mRNA. This solution hybridization assay is sensitive enough to quantitative levels of G6PD mRNA in total liver RNA from a fasted rat, one of the least abundant sources of this mRNA. It can, therefore, be used to answer several questions about the regulation of G6PD synthesis in rat tissues. Preliminary results suggest that the dietary regulation of G6PD mRNA in rat liver is much larger than previously reported.

Quantitation of glucose-6-phosphate dehydrogenase mRNA by solution hybridization: correlation with rates of synthesis.

Rat liver glucose-6-phosphate dehydrogenase (G6PD) is one of several proteins involved in lipid metabolism whose synthesis is regulated by diet. In experiments reported here, rats were fasted or fed diets until a new steady state level of G6PD was produced. Livers were used to measure G6PD activity, synthesis and mRNA simultaneously. Since accurate quantitation of G6PD mRNA by Northern blots was found to be difficult in noninduced animals a new solution hybridization assay was also used. Noninduced rats have approx. One molecule of G6PD mRNA per liver cell. Changes in G6PD mRNA are larger than previously reported and, at the steady state, can completely account for the 33-fold change in G6PD activity and synthesis when fasted rats are refed a high carbohydrate diet. In contrast, a high fat carbohydrate-free diet does not increase G6PD mRNA and dibutyryl cAMP lowers G6PD mRNA. Since changes in G6PD synthesis and activity are closely correlated, degradation of G6PD is not significantly regulated.

Regulation of liver glucose-6-P dehydrogenase levels in female rats

1. Gender differences in the dietary regulation of rat liver glucose-6-P dehydrogenase activity, synthesis and mRNA levels were examined. 2. As expected, in normal rats fed a standard chow diet, females have higher G6PD activity than males because they have more G6PD mRNA and therefore a higher rate of G6PD synthesis. 3. In contrast, the decreased dietary induction in female rats is due to a more rapid rate of G6PD degradation rather than a decrease in G6PD mRNA or synthesis.