Identification of glyoxylate cycle enzymes in chick liver--the effect of vitamin D3: cytochemistry and biochemistry.

Information regarding the presence of the glyoxylate cycle in chick liver was sought. This metabolic pathway has long been thought to be absent from vertebrate tissues. Previous studies in other tissues have shown that, when present, this pathway is sensitive to vitamin-D. Thus, the effect of long-term vitamin-D deficiency and subsequent vitamin-D replacement on liver structure was studied by light microscopy. In addition, specific biochemical assays for the presence of glyoxylate cycle enzymes were performed. Light microscopy of lipid extracted tissues, light microscopic histochemistry, and quantitative histochemistry showed that the hepatocytes from vitamin-D-deficient animals contained primarily lipid. Hepatocytes from normal and vitamin-D-replete livers contained primarily carbohydrate as judged by their staining with periodic acid-Schiff (PAS). Also, malate synthase positive peroxisomes were seen in hepatocytes from normal and vitamin-D-treated chicks. Structures positive for this glyoxylate cycle enzyme were rarely seen in the hepatocytes from vitamin-D-deficient animals. Biochemical analyses showed the presence of the two unique glyoxylate cycle enzymes, isocitrate lyase and malate synthase, in chick hepatocytes. The activity of these enzymes was markedly increased in the vitamin-D-replete livers. In addition, chick hepatocytes demonstrated the capacity to oxidize fatty acid in the presence of cyanide. This activity, which is characteristic of peroxisomal B-oxidation rather than mitochondrial, was stimulated by vitamin-D treatment. Lastly, incubation of chick liver in the presence of a fatty acid substrate (palmitate) led to higher tissue glycogen content. The latter was further increased in liver from vitamin-D-replete animals. These data show the presence of glyoxylate cycle enzymes in a higher vertebrate and indicate that this tissue is endowed with the capacity to convert lipid to carbohydrate.