Stange E F, Dietschy J M
J Lipid Res. 1983 Jan;24(1):72-82.
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.
为了确定小肠中胆固醇合成在肠管长度方向以及绒毛 - 隐窝轴上的定位，我们确定了在全肠切片和分离的上皮细胞中测量洋地黄皂苷可沉淀胆固醇（DPS）合成绝对速率的最佳条件。当在中肠切片中比较[1 - （14）C]乙酸盐和[1 - （14）C]辛酸酯时，[1 - （14）C]辛酸酯优先代谢为CO₂和DPS。由于其在线粒体内快速降解，[1 - （14）C]辛酸酯有效地排除了其他乙酰辅酶A（C₂）来源，并使C₂流入DPS的速率几乎与用[（3）H]水测定的绝对速率相同。另一方面，未标记的乙酸盐以剂量依赖方式降低了从[1 - （14）C]辛酸酯到DPS的表观C₂通量，但不影响到CO₂的通量，最高可达8 mM。在可比的孵育条件下，通过EDTA螯合技术分离的肠上皮细胞在几个方面与切片不同。首先，与切片中90分钟相比，各种底物的DPS合成仅在30分钟内与时间成正比。其次，用于DPS和CO₂合成的首选（14）C标记底物是乙酸盐而非辛酸酯。第三，两种（14）C标记底物均未达到用[（3）H]水发现的合成速率。第四，在孵育培养基中不存在葡萄糖时，任何底物的胆固醇合成基本为零。当使用[（3）H]水估计空肠和回肠绒毛和隐窝细胞部分的体外最佳胆固醇合成速率时，所有回肠部分都更活跃。然而，回肠绒毛细胞将[（3）H]水掺入DPS的能力比空肠绒毛细胞高5.2倍，而隐窝细胞部分仅相差1.4倍。大部分胆固醇合成能力存在于近端和远端肠道的绒毛下部区域，隐窝分别占空肠和回肠中发现的总量的约30％和14％。在体内给予[（3）H]水后，在肠道的垂直和水平轴上发现了类似的分布模式，尽管在这些条件下，在隐窝中回收的比例分别增加到空肠和回肠中发现的总[（3）H]DPS的38％和31％。这些研究表明，[（3）H]水在肠切片和分离的上皮细胞中均产生最佳的胆固醇合成速率，并且在体外和体内主要掺入绒毛下部和隐窝区域的细胞中的DPS中。 - 施坦格，E.F.，和J.M.迪茨基。大鼠小肠体外和体内胆固醇合成的绝对速率：切片和分离细胞中不同底物的比较。
