Mariko Shin

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

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.

Effect of Thioridazine or Chlorpromazine on Increased Hepatic NAD+ Level in Rats Fed Clofibrate, a Hypolipidaemic Drug

The effect of the phenothiazines, thioridazine and chlorpromazine, on the increased hepatic NAD+ level of rats fed clofibrate, a hypolipidaemic drug, has been investigated.

Nad Levels In The Rat Primary Cultured Hepatocytes Affected By Peroxisome- Proliferators

The effect of peroxisome-proliferators (PPs) on the NAD level in primary cultured rat hepatocytes was investigated and compared with that in the liver of rat administered with PPs. Various PPs, including fibrates, phthalate esters and steroid, increased NAD level in the cultured hepatocytes as in whole animal with a little exception. The NAD level decreased after once reaching the peak by the addition of most PPs. The gradual decrease of NAD observed in primary cultured hepatocytes, was partially inhibited by the addition of poly(ADP-ribose) polymerase and/or NAD glycohydrolase inhibitors. In the presence of these inhibitors, the increase of NAD by PPs still remained. The mechanism of increasing NAD level by PPs will be due to the stimulation of tryptophan (Trp)-NAD biosynthesis, with the possibility of the transcriptional regulation of genes related to Trp-NAD pathway by PPs. This in vitro system, therefore, can be used to clarify detailed mechanism of the stimulation of hepatic NAD biosynthesis in rats administered PPs.

Effects of peroxisome-proliferators on the TRP-NAD pathway.

The mechanism of the elevation of hepatic NAD level in the rats administered clofibrate and other peroxisome-proliferators were investigated. In the hepatocytes from the clofibrate-fed rats, NAD biosynthesis from Trp, but not from nicotinic acid, was specifically stimulated. The activities of peroxisomal marker enzymes, the peroxisome-proliferator activated receptors (PPAR)-dependent enzymes and key enzymes in the Trp-NAD pathway changed in parallel with the hepatic NAD increase; the activity of quinolinate phosphori-bosyltransferase (QPRT) was increased whereas that of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) was drastically reduced. The results strongly suggest that the hepatic NAD increase might be caused by transcription of genes coding the key enzymes of the Trp-NAD pathway via PPAR.

Effect of leucine on intestinal absorption of tryptophan in rats

1. To elucidate the causal relation between leucine and the lowering of hepatic NAD content of rats fed on a leucine-excessive diet (Yamada et al. 1979), the effect of leucine on intestinal absorption of tryptophan was investigated. 2. Co-administration of [3H]tryptophan and leucine, with leucine at ten times the level of tryptophan, delayed absorption of L-[side chain 2,3-3H]tryptophan from the digestive tract and incorporation of [3H]tryptophan into portal blood, the liver and a protein fraction of the liver. After 120 min, more than 95% of tryptophan was absorbed whether [3H]tryptophan was administered with or without leucine. 3. Co-administration of a mixture of ten essential amino acids, in proportions simulating casein, with [3H]tryptophan markedly delayed absorption of tryptophan from the digestive tract. The addition of supplementary leucine to the amino acid mixture, however, caused no further delay. 4. In rats prefed a leucine-excessive diet for 1 week [3H]tryptophan was absorbed at the same rate as in rats fed on a control diet. 5. The results indicate that competition between tryptophan and leucine for intestinal absorption did not cause lowering of hepatic NAD.