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. 2014 May;55(5):905-18.
doi: 10.1194/jlr.M047761. Epub 2014 Feb 25.

Selective Evaluation of High Density Lipoprotein From Mouse Small Intestine by an in Situ Perfusion Technique

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Free PMC article

Selective Evaluation of High Density Lipoprotein From Mouse Small Intestine by an in Situ Perfusion Technique

Satoshi Yamaguchi et al. J Lipid Res. .
Free PMC article

Abstract

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.

Keywords: atherosclerosis; in situ perfusion; intestine; lipoprotein; regulation.

Figures

Fig. 1.
Fig. 1.
Establishment of an in situ perfusion model of mouse SI and analyses of lipoproteins in the SI lymph perfusates. A: A schematic representation of our in situ perfusion system for assessing HDL production in mouse SI. The abdominal aorta and its branches were ligated as shown in red bars, and the aorta was punctured and cannulated to serve as the inlet (supplementary Fig. I). The portal vein and intestinal lymph duct were punctured and cannulated to serve as outlets. Flow of the perfusion buffer was shown by the arrows. The mouse body and perfusion buffer were warmed to 35°C before perfusion was started. B: Non-SDS-PAGE analysis of HDL-apo AI (upper panel) and SDS-PAGE analysis of apo AI (lower panel) in SI lymph perfusates from WT (left lane) and ABCA1 KO (right lane) mice. C: Non-SDS-PAGE analysis of HDL-apo AI (upper panel) and SDS-PAGE analysis of apo B48 and apo B100 (lower panel) in perfusates from the SI lymph duct and portal vein of ABCA1 KO mice perfused with buffer containing serum from WT mice.
Fig. 2.
Fig. 2.
Comparison of plasma HDL, intestinal HDL, and hepatic HDL in WT mice by peptide mapping using LC/MS. A–D: LC/MS total ion chromatograms of peptides, in which peptide-ion intensity is shown as a function of the peptide retention time, for HDL separated by ultracentrifugation from plasma (P-HDL) (A), SI lymph perfusates (SI-HDL) obtained using a novel in situ perfusion model (B), mesenteric lymph (C-HDL) obtained by conventional intestinal lymph cannulation experiments (C), and liver perfusates (L-HDL) (D) of WT mice. The numerical value of each peak shows m/z of representative peptides included in the peak. Arrows indicate peptides with m/z 542 and 524 detected specifically in SI-HDL.
Fig. 3.
Fig. 3.
HPLC analyses of lipids and preparative ultracentrifugation followed by Western blotting for apos in SI lymph perfusates. A: HPLC analyses of SI lymph perfusates from WT mice. Two hundred microliters of SI lymph perfusates was run on HPLC as described in the Methods. TC, FC, TG, and PL were measured enzymatically. Arrows denote the average elution time of indicated plasma lipoproteins in WT mice. B: Apo distribution among lipoproteins in SI lymph perfusates from WT mice separated by preparative ultracentrifugation. Lipoproteins in plasma and SI lymph perfusates were subjected to small-scale preparative ultracentrifugation to concentrate samples, and the concentrated samples were then run on SDS-PAGE followed by Western blot analysis using antibodies against the indicated apos. CM, VLDL, LDL, and HDL denote the density range of the indicated plasma lipoprotein fractions.
Fig. 4.
Fig. 4.
Lipid and apo composition of HDL in SI lymph perfusates from WT mice. A: Lipid and protein composition of HDL in plasma (P-HDL), SI lymph perfusates (SI-HDL), and liver perfusates (L-HDL) separated by serial preparative ultracentrifugation. HDL was further separated by HPLC to determine its lipid composition. B: Comparison of apo compositions of SI-HDL and L-HDL. SI-HDL and L-HDL were obtained by in situ perfusion followed by serial preparative ultracentrifugation and subjected to SDS-PAGE followed by Western blot analysis using antibodies against the indicated apos.
Fig. 5.
Fig. 5.
Electron micrographs of negatively stained HDL from SI lymph perfusates. A: Representative negative-stain EM of HDL separated by serial ultracentrifugation from SI lymph perfusates (SI-HDL, lower panel) and plasma (P-HDL, upper panel). Magnification: 200,000× scale bar: 100 nm. B: Size distribution of SI-HDL and P-HDL particles from negative-stain electron micrographs. The frequency distributions of the size of SI-HDL (pink bars; n = 913) and P-HDL (gray bars; n = 1,412) were plotted together, and the red bars represent the overlaid parts. Two measurements were made for the diameter of each HDL particle, and the mean diameter was used to calculate the size frequency. C: Box-and-whisker plots showing the mean (■), median (middle bar in the rectangle), and 10th (bottom bar), 25th (bottom of rectangle), 75th (top of rectangle), and 90th (top bar) percentiles of the sizes of SI-HDL (black) and P-HDL (red) particles. The individual data are shown on the left of the boxes. * P < 0.001, SI-HDL versus P-HDL, assessed by the Wilcoxon rank sum test.
Fig. 6.
Fig. 6.
Effects of glyburide and DTNB on the assembly of SI-HDL. A: Possible mechanism for the assembly of SI-HDL. Glyburide and DTNB are known inhibitors of ABCA1 and LCAT, respectively. B: Effect of glyburide on immunoblot patterns of HDL in SI lymph perfusates from WT mice. WT mice were subjected to in situ SI perfusion in the presence (right panel) and absence (left panel) of glyburide in the perfusion buffer. SI-HDL was run on non-SDS-PAGE followed by the detection of apo AI. Arrowhead represents free apo AI. C: Effect of DTNB on the formation of SI-HDL. SI lymph perfusates were obtained from WT mice that were perfused using a perfusion buffer in the presence (right panel) and absence (left panel) of DTNB in the perfusion buffer.
Fig. 7.
Fig. 7.
Nutritional and genetic regulation of SI-HDL. The HDL-C concentration (mg/dl) in different perfusates was measured by HPLC as described in the Methods and is shown under each column. A: Effect of fasting on SI-HDL in WT mice. Mice were subjected to in situ perfusion at ad libitum and after 24 h of fasting. SI lymph perfusates were analyzed by using non-SDS-PAGE followed by Western blot analysis for apo AI. B: Effect of a high-fat diet on SI-HDL in WT mice. Mice were subjected to in situ SI perfusion after being fed a high-fat diet for 4 weeks. C: Comparison of SI-HDL production in 14-week-old WT and apo E KO mice.

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