Absolute rates of cholesterol synthesis in rat intestine in vitro and in vivo: a comparison of different substrates in slices and isolated cells

Abstract

In an effort to localize small intestinal sterol synthesis both along the length of the intestine and along the villus-crypt axis, we defined the optimal conditions to measure absolute rates of digitonin-precipitable sterol (DPS) synthesis in whole intestinal slices and in isolated epithelial cells. When [1-(14)C]acetate and [1-(14)C]octanoate were compared in midgut slices, [1-(14)C]octanoate was preferentially metabolized to CO(2) and DPS. Because of its rapid intramitochondrial degradation, [1-(14)C]octanoate effectively swamped-out other sources of acetyl CoA (C(2)) and achieved rates of C(2) flux into DPS virtually identical to the absolute rates determined with [(3)H]water. On the other hand, unlabeled acetate decreased the apparent C(2) flux from [1-(14)C]octanoate into DPS, but not into CO(2), in a dose-dependent fashion up to 8 mM. Under comparable incubation conditions, intestinal epithelial cells isolated by the EDTA chelation technique differed from slices in several respects. First, DPS synthesis from the various substrates was proportional to time only for 30 min compared to 90 min in slices. Second, the preferred (14)C-labeled substrate for DPS and CO(2) synthesis was acetate rather than octanoate. Third, neither of the (14)C-labeled substrates achieved the rates of synthesis found with [(3)H]water. Fourth, sterol synthesis from any of the substrates was essentially zero in the absence of glucose in the incubation medium. When the optimal rates of sterol synthesis in vitro were estimated using [(3)H]water in villus and crypt cell fractions of the jejunum and ileum, all ileal fractions were more active. However, ileal villus cells exceeded the jejunal villus cells by 5.2-fold in their capacity to incorporate [(3)H]water into DPS, whereas the crypt cell fractions differed by only 1.4-fold. The majority of the sterol synthetic capacity resided in the lower villus region in both the proximal and distal intestine and the crypts accounted for about 30% and 14%, respectively, of the total found in the jejunum and ileum. A similar distribution pattern along both the vertical and horizontal axes of the intestine was found after [(3)H]water administration in vivo, although under these conditions the proportion recovered in the crypts increased to 38% and 31%, respectively, of the total [(3)H]DPS found in the jejunum and ileum. These studies demonstrate that [(3)H]water yields optimal rates of sterol synthesis both in intestinal slices and in isolated epithelial cells and is incorporated into DPS mainly in cells of the lower villus and crypt region both in vitro and in vivo.-Stange, E. F., and J. M. Dietschy. Absolute rates of cholesterol synthesis in rat intestine in vitro and in vivo: a comparison of different substrates in slices and isolated cells.