Abstract
Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.
Highlights
The ability of an organism to efficiently extract energy-rich nutrients from its diet is critical for survival
GPAT3 Was Abundantly Expressed in the Absorptive Epithelial Cells of the Small Intestine—Previous studies have documented a role for GPAT3 in the synthesis of triacylglycerol in adipocytes [17, 18, 28, 29]
Our study demonstrates that the GPAT3 protein is abundantly expressed in the enterocytes of the jejunum where it is strongly up-regulated during fasting
Summary
Animal Studies—GPAT3 knock-out (Gpat3Ϫ/Ϫ) mice were generated as described previously [18]. FFAs were extracted using methanol: water (80:20 v/v) and heptadecanoic acid (200 nM) as an internal standard and analyzed by ultra performance liquid chromatography (UPLC)-MS using a Waters Acquity UPLC coupled to a Thermo LTQ Orbitrap Velos mass spectrometer. Tissues were homogenized as described above, and long chain acylcarnitines were extracted with dichloromethane:isopropyl alcohol:methanol (25:10:65, v/v/v) containing palmitoyl-L-carnitine (N-methyld3) hydrochloride (200 nM) as the internal standard. Primary enterocytes were isolated and incubated with 0.5 Ci/ml of [3H]oleic acid or [14C]cholesterol for 1 h, and cell pellets were collected and washed as described above after incubation. High performance Liquid Chromatography (HPLC), Tissue Lipid and Bile Acid Analysis—Liver or intestinal tissues were weighed, and lipids were extracted using a modified Bligh and Dyer method. Statistical Analysis—Statistical tests were performed (GraphPad Prism software, version 6.0, GraphPad, La Jolla, CA)
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