NAD + biosynthesis and metabolic fluxes of tryptophan in hepatocytes isolated from rats fed a clofibrate-containing diet

Abstract

Hepatocytes were isolated from rats fed a diet with or without 0.25% clofibrate, and NAD + synthesis by the hepatocytes was determined using either [ carboxyl- 14C]nicotinic acid or [5- 3H]tryptophan. NAD + and total pyridine nucleotides synthesized from [ 14C]nicotinic acid by the clofibrate-treated cells were not significantly different from those synthesized by the control cells when expressed on the basis of nanomoles per hour per milligram of DNA. On the contrary, NAD + synthesized from [ 3H]tryptophan was significantly higher in the clofibrate-treated cells (158% of the control cells) on the basis of nanomoles per hour per milligram of DNA. Clofibrate was inhibitory to tryptophan metabolism as a whole, affecting the glutarate pathway more (decreased to 37% of control) than the kynureninase flux (decreased to 64% of control). As a result, the quinolinate-NAD flux, estimated as the difference in the amounts of tryptophan metabolized by the two metabolic pathways, increased in the clofibrate-treated hepatocytes. The increase in quinolinate during the incubation was 8 times more in the clofibrate-treated cells than in the control cells, which confirmed alteration in the metabolic fluxes of tryptophan in the clofibrate-treated cells. Hepatic quinolinate phosphoribosyltransferase (EC 2.4.2.19) activity increased with dietary clofibrate and returned to the control level 1 week after removing clofibrate from the diet. Nicotinate phosphoribosyltransferase (EC 2.4.2.11) and NAD + glycohydrolase (EC 3.2.2.5) activities remained unchanged with dietary clofibrate.