Novel acyl-coenzyme A:monoacylglycerol acyltransferase plays an important role in hepatic triacylglycerol secretion

Abstract

Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays a predominant role in the resynthesis of triacylglycerol in the small intestine, but its contribution to triacylglycerol synthesis in other tissues, such as the liver, is not clear. In this study, we identified a novel MGAT gene, which is identical with lysophosphatidylglycerol acyltransferase1 (LPGAT1). Mouse LPGAT1 is expressed in a number of tissues and most highly expressed in the liver. Hepatic LPGAT1 expression in diabetic db/db mice is higher than that in the control db/m mouse, which is consistent with increased hepatic MGAT activity in db/db mouse. To elucidate the role of LPGAT1 gene in lipid metabolism in db/db mice, we constructed an adenovirus of short hairpin RNA (shRNA) targeting LPGAT1 to selectively knockdown LPGAT1 gene expression in the liver. Hepatic MGAT activity and LPGAT1 expression in db/db mice infected with LPGAT1 shRNA adenovirus were significantly lower than those in mice infected with the control virus. Notably, treatment with LPGAT1 shRNA adenovirus caused a marked reduction in serum triacylglycerol and cholesterol levels and a significant increase in hepatic cholesterol level. These findings indicate that LPGAT1, a newly identified MGAT enzyme, plays a significant role in hepatic triacylglycerol synthesis and secretion in db/db mice.

Fig. 1.

Tissue distribution of mouse LPGAT1 mRNA . LPGAT1 expression in mouse tissues was determined by Northern blot analysis. Lane 1, brain; lane 2, heart; lane 3, kidney; lane 4, liver; lane 5, lung; lane 6, skeletal muscle; lane 7, skin; lane 8, small intestine; lane 9, spleen; lane 10, stomach; lane 11, testis; lane 11, thymus.

Fig. 2.

In vitro MGAT activity and substrate specificity of LPGAT1 enzyme. A: CHO cells were transfected with empty vector or mouse LPGAT1, and cell lysates were analyzed for acyltransferase activity. Activity was measured by incorporating [¹⁴C]palmitoyl CoA into lysophosphatidic acid, DAG, cholesterol, and monoacylglycerol, resulting in phosphatidic acid (AGPAT), triacylglycerol (DGAT), cholesterol ester (ACAT), and diacylglycerol (MGAT), respectively. Values are given mean ± SEM (n = 3). Significant differences from the control group were determined using Student's t-test. **P < 0.01. B: Preference of LPGAT1 for rac-1-monoacylglycerol and sn-2-monoacylglycerol as acyl acceptor. MGAT activity was determined with 30 μM [¹⁴C]palmitoyl CoA and 0.1 mM rac-1-monoacylglycerol or sn-2-monoacylglycerol.

Fig. 3.

MGAT activity and LPGAT1 expression in db/m and db/db mice. A: Microsomes were prepared from liver of db/m or db/db mice, and hepatic MGAT activity was measured using 30 μM [¹⁴C]palmitoyl CoA and 0.1 mM sn-2-monoacylglycerol. B: Total RNA was prepared from liver of db/m or db/db mice and used for real-time quantitative PCR to evaluate the expression of LPGAT1 gene. Values are given as mean ± SEM (n = 5). Significant differences from the control group were determined using Student's t-test. *P < 0.05.

Fig. 4.

LPGAT1 shRNA adenovirus-mediated knockdown of LPGAT1 expression in CHO cells. CHO cells stably expressing FLAG-tagged mouse LPGAT1 gene were infected with increasing doses (MOI) mLPGAT1 shRNA adenovirus or control virus, which contained mouse lamin shRNA sequence. Membrane fractions were then prepared, and expression of FLAG-tagged protein was detected by immunoblotting of membrane samples with M2 anti-FLAG antibody.

Fig. 5.

LPGAT1 shRNA adenovirus-mediated knockdown of LPGAT1 expression in liver samples of db/db mice. Male db/db mice were injected intravenously either with the control virus or LPGAT1 shRNA adenovirus. Animals were euthanized 5 days (A) or 16 days (B) after infection, and gene expression levels in liver samples were analyzed by quantitative RT-PCR. Values are given as mean ± SEM (n ≥ 4). Significant differences from the control group were determined using Student's t-test. *P < 0.05.

Fig. 6.

Hepatic MGAT and LPGAT activity in db/db mice treated with LPGAT1 shRNA adenovirus. Hepatic MGAT activity (A) and LPGAT activity (B) in mice infected either with the control virus or LPGAT1 shRNA adenovirus were analyzed. Values are given mean ± SEM (n = 5). Significant differences from the control group were determined using Student's t-test. *P < 0.05.

Fig. 7.

Food consumption and body weight of db/db mice treated with the control virus or LPGAT1 shRNA adenovirus. Animals infected either with the control virus or LPGAT1 shRNA adenovirus were fed ad libitum with standard chow. Food consumption (A) and body weight (B) were recorded for 16 days. Values are given as mean ± SEM (n = 5). Significant differences from the control group were determined using Student's t-test. *P < 0.05.

Fig. 8.

Serum lipid concentrations in db/db mice treated with LPGAT1 shRNA adenovirus. Serum TAG (A), total cholesterol (B), FFA (C), and HDL-cholesterol (D) levels of control- or LPGAT1 shRNA-treated db/db mice were determined. Values are given as mean ± SEM (n = 5). Significant differences from the control group were determined using Student's t-test. *P < 0.05; **P < 0.01.

Fig. 9.

Hepatic lipid contents in db/db mice treated with the control virus or LPGAT1 shRNA adenovirus. Hepatic TAG (A) and total cholesterol (B) levels in control- or LPGAT1 shRNA-treated db/db mice were determined. Values are given as mean ± SEM (n = 5). Signifi cant differences from the control group were determined using Student's t-test. *P < 0.05.