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Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased ~10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4-6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released lAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins. Caco-2 cells; lipid absorption; small intestine; enzyme-labeled fluorescence-97

Intestinal alkaline phosphatase (IAP) is involved in the process of fat absorption, a conclusion confirmed by an altered lipid transport and a faster body weight gain from 10 to 30 wk in both male and female mice with a homozygous null mutation of the IAP coding gene (Akp[3.sup.-/-] mice). This study was aimed to delineate morphologically and quantitatively the accelerated lipid absorption in male Akp[3.sup.-/-] mice. Feeding a corn oil bolus produced an earlier peak of triacylglycerol in serum (2 vs. 4 h for Akp[3.sup.-/-] and wild-type mice, respectively) and an approximately twofold increase in serum triacylglycerol concentration in Akp[3.sup.-/-] mice injected with a lipolysis inhibitor, Triton WR-1339. A corn oil load induced the threefold enlargement of the Golgi vacuoles in male wild-type mice but not in Akp[3.sup.-/-] mice, indicating that absorbed lipids rarely reached the Golgi complex and that the transcytosis of lipid droplets does not follow the normal pathway in male Akp[3.sup.-/-] mice. Force feeding an exaggerated fat intake by a 30% fat chow for 10 wk induced obesity in both male Akp[3.sup.-/-] and wild-type mice, and therefore no phenotypic difference was observed between the two. On the other hand, the forced high-fat chow induced an 18% greater body weight gain, hepatic steatosis, and visceral fat accumulation in female Akp[3.sup.-/-] mice but not in female wild-type controls. These results provide further evidence that IAP is involved in the regulation of the lipid absorption process and that its absence leads to progressive metabolic abnormalities in certain fat-forced conditions. fat absorption; metabolic abnormality; obesity; small intestine doi:10.1152/ajpgi.00331.2006.

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43[degrees]C induced the expression of IAP-I and HSP72 mRNAs time dependently ( isozyme; heat shock protein; sodium arsenite; glutathione

Peroxynitrite has been implicated in the oxidative modification of low-density lipopro-tein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipoproteins modified by peroxynitrite lead to the onset of atherosclerotic vascular disease. We therefore prepared in vitro lipoproteins oxidatively modified by peroxynitrite (NO2-lipoprotein) and investigated the effect of NO2-lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular complexes of apolipoproteins in HDL were detected on immunoblotting with monoclonal antibodies against apolipoprotein AI and AH, suggesting that nitration of HDL by peroxynitrite causes intermolecular cross-linking of the apolipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prepared NO2-HDL, as quantitated by HPLC, and the amount in NO2-lipoprotein depended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whether or not NO2-lipoproteins affect the function of endothelial cells, we first examined the viability of cultured human aortic endothelial cells (HAECs) exposed to NOs-lipopro-teins. Incubation with either NO2-HDL or NO2-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO2-HDL markedly suppressed at 48 h not only the expressed levels of mRNA and protein but also the activity of catalase in HAECs. In contrast, NO2-LDL significantly reduced the expression and activity of Cu2+,Zn2+-superoxide dismutase (CuZn-SOD) in the cells. Neither NO2-HDL nor NO2-LDL interfered with nitric oxide production or expression of cycloox-ygenases and NADPH oxidase in HAECs. Increased radical production in NOs-lipopro-tein-treated HAECs implied that reactive oxygen species such as superoxide anions and hydroxyl radicals may contribute to the mechanism of the toxic effect induced in endothelial cells by NO2-lipoprotein, Overall, NO2-lipoprotein may lead to deterioration of the vascular function through these endothelial cell responses. [ABSTRACT FROM AUTHOR]