Accumulation of 1,2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats.

Choline deficiency, via deprivation of labile methyl groups, is associated with a greatly increased incidence of hepatocarcinoma in experimental animals. This dietary deficiency also causes fatty liver, because choline is needed for hepatic secretion of lipoproteins. We hypothesized that fatty liver might be associated with the accumulation of 1,2-sn-diradylglycerol and subsequent activation of protein kinase C. Several lines of evidence indicate that cancers might develop secondary to abnormalities in protein kinase C-mediated signal transduction. We observed that rats fed a choline-deficient diet for 1, 6, or 27 weeks had increased hepatic concentrations of 1,2-diradylglycerol. At 1 and 6 weeks, hepatic plasma membrane from choline-deficient rats had increased concentrations of 1,2-sn-diacylglycerol and 1-alkyl, 2-acylglycerol, with the latter accounting for 20-26% of membrane 1,2-sn-diradylglycerol (as compared with only 2-5% in controls). Protein kinase C activity was increased in hepatic plasma membrane at 1 week of choline deficiency. By Western blotting there was an increase in the amount of protein kinase C zeta and a decrease in the amount of protein kinase C delta in liver at 1 week. By 6 weeks of choline deficiency, hepatic plasma membrane and cytosolic protein kinase C (PKC) activities were increased significantly, with increased amounts of hepatic plasma membrane protein kinase C alpha, and delta detected by Western blotting. Glycogen synthase activity in liver was diminished after 1 week of choline deficiency; this enzyme is inhibited by PKC-mediated phosphorylation. We suggest that choline deficiency perturbed PKC-mediated transmembrane signaling within liver and that this contributed to the development of hepatic cancer in these animals.

Positional and temporal regulation of lipogenic gene expression in mouse liver.

We have examined the dynamics of positional gene expression in mouse liver using the carbohydrate induction of lipogenic genes as a model. Using a protocol of fasting and refeeding a high-carbohydrate, no-fat diet to obtain maximal induction, we investigated the temporal expression and localization of malic enzyme (ME) and fatty acid synthase (FAS). In situ hybridization showed that both ME and FAS were expressed at low basal levels in all hepatocytes in livers of mice fed a control diet. Furthermore, dietary induction of ME and FAS mRNA occurred in periportal cells within 6 hours. After 12 hours, the portal cells were maximal; and after 24-36 hours, all cells showed high levels of message. This was coincident with expression of ME and FAS mRNAs, which appeared to be maximal between 24 and 36 hours. Both steady-state mRNA levels and pericentral localization then declined, until only periportal hepatocytes showed strong expression of ME and FAS. Nuclear transcription rates measured by run-on assay demonstrated that maximal transcription rates preceded maximum mRNA levels by peaking at 12 hours. Furthermore, run-on assays showed that the periportal induction by carbohydrates is primarily a transcriptional response for FAS, and both transcriptional and posttranscriptional for ME. These results indicate that lipogenic gene expression is a temporal response induced by carbohydrate feeding and is regulated by both positional and transcriptional mechanisms.

Effect of vitamin B-6 deficiency on fasting plasma homocysteine concentrations.

The catabolism of homocysteine through cystathionine synthesis requires pyridoxal-5'-phosphate, thus the effect of vitamin B-6 deficiency on plasma homocysteine concentrations was evaluated. Total fasting plasma homocysteine concentrations were measured in 11 elderly subjects aged 64.4 +/- 1.7 y (mean +/- SE) who consumed a vitamin B-6-deficient diet for less than or equal to 20 d. Only 1 of the 11 subjects was found to have elevated homocysteine concentrations even though all subjects exhibited high urinary xanthurenic acid concentrations after a tryptophan load, a measure indicative of vitamin B-6 deficiency. In a supporting study, fasting plasma homocysteine concentrations were measured in 3- and 23-mo-old rats fed vitamin B-6-deficient diets and were compared with those of vitamin B-6-replete, pair-fed controls. There was no difference in homocysteine concentrations between deficient and pair-fed animals after 6 wk of the dietary regimen for either age group; after 9 wk a modest elevation was observed in the 3-mo-old deficient rats whereas no difference was observed for the 23-mo-old rats. It is concluded that fasting plasma homocysteine concentrations are not initially elevated in vitamin B-6 deficiency and therefore fasting plasma homocysteine concentrations are not a good indicator of vitamin B-6 status.

Expression of plasmolipin in oligodendrocytes.

Plasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602-610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligodendrocyte cultures. Double-label immunofluorescence showed that plasmolipin was expressed by galactocerebroside (GC)-positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC-positive cells. During the following 3 weeks in culture plasmolipin staining of oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmolipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenates from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by oligodendrocytes.

Presence of the plasma membrane proteolipid (plasmolipin) in myelin.

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.

Subtle abnormalities of gait detected early in vitamin B6 deficiency in aged and weanling rats with hind leg gait analysis.

Motor abnormalities have been observed in every species made vitamin B6 deficient, and have been detected and quantified early in vitamin B6 deficiency in young adult female Long-Evans rats with hind leg gait analysis. Our objective was to determine if hind leg gait analysis could be used to detect vitamin B6 deficiency in weanling (3 weeks) and aged (23 months) Fischer 344 male rats. Rats (n = 10 per group) were fed: the control diet ad libitum (AL-CON); the control diet devoid of added pyridoxine hydrochloride (DEF); or the control diet pair-fed to DEF (PF-CON). At 10 weeks, plasma pyridoxal phosphate concentration confirmed deficiency in both age groups. Gait abnormalities were detected in the absence of gross motor disturbances in both aged and weanling DEF rats at 2-3 weeks. Width of step was significantly reduced (16%, p less than 0.003) in DEF aged rats compared to AL- and PF-CON. This pattern of response was similar to that reported previously in young adult rats. In weanling rats, pair feeding alone reduced mean width of step (+/- SEM) by 25% compared to ad libitum feeding (2.7 +/- 0.1 vs 3.6 +/- 0.1 cm for PF- vs AL-CON, respectively, p less than 0.05). In DEF weanling rats, width (3.0 +/- 0.1 cm) was increased compared to PF-CON (11%, p less than 0.05) but decreased compared to AL-CON (16%, p less than 0.05). Width of step was significantly altered early in B6 deficiency in rats of different ages and strains and in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging and vitamin B-6 depletion: effects on plasma pyridoxal-5'-phosphate and erythrocyte aspartate aminotransferase activity coefficients in rats.

Plasma pyridoxal-5'-phosphate (PLP) and erythrocyte aspartate aminotransferase activity coefficients were longitudinally determined in rats. Blood was obtained at weeks 0, 1, 2, 4, 6, 9, and 11 from rats initially aged 3 wk, 3 mo, 12 mo, and 23 mo (weeks 0, 1, 4, 6, and 9 only). The diet groups were ad libitum control (ALC), deficient (DEF), and pair-fed control (PF). Plasma PLP concentrations of controls were highest at 3 mo, intermediate at 3 wk and 12 mo, and lowest at 23 mo. When an additional group of 22-mo-old rats was fed a high-vitamin B-6 diet for 4 wk, their low baseline plasma PLP concentrations did not increase significantly. Plasma PLP decreased significantly within 1 wk and activity coefficients increased significantly by week 4 in all DEF rats. Depletion was most rapid and severe in the youngest DEF rats and least in 12-mo-old DEF rats. Mechanisms for the low plasma PLP control values and resistance to depletion in aged rats remain to be determined.