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. 2008 Jun-Jul;1781(6-7):352-8.
doi: 10.1016/j.bbalip.2008.05.001. Epub 2008 May 15.

Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition

Tal M Lewin  1 , Hendrik de Jong, Nicole J M Schwerbrock, Linda E Hammond, Steven M Watkins, Terry P Combs, Rosalind A Coleman
Affiliations

Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition

Tal M Lewin et al. Biochim Biophys Acta. 2008 Jun-Jul.

Abstract

Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate-limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high-sucrose diet or a 3-month high-fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1(-/-) mice contained 20-80% less TAG than the wildtype controls. In addition, hearts from Gpat1(-/-) mice fed the high-sucrose diet incorporate 60% less [(14)C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long-chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1(-/-)(-/-) mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1(-/-)(-/-) hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high-fat diets and influences the incorporation of 20:4n6 into heart phospholipids.

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Figures

Figure 1
Figure 1. Hearts from Gpat1−/− mice lack Gpat1 mRNA, but retain an NEM-resistant GPAT activity
A: RNA was isolated from backcrossed Bl/6 wildtype (+/+), heterozygous (+/−), and knockout (−/−) Gpat1 mouse hearts (n = 2) and analyzed for the presence or absence of neo and Gpat1 transcripts by reverse transcriptase PCR as described in Materials and Methods. The first lane contains DNA size markers (from top: 1000, 850, and 650 bp). The second lane (C) is a no template control reaction. Heart total membranes (B) and mitochondria (C, D) from wildtype and Gpat1−/− mice were isolated as described and assayed for GPAT activity in the absence or presence of (B, C) 1 mM NEM or (D) 0–10 mM NEM. Data not shown for 5 and 10 mM NEM, as further inhibition was not observed. Data are presented as the mean ± SD (n = 3 – 4).
Figure 1
Figure 1. Hearts from Gpat1−/− mice lack Gpat1 mRNA, but retain an NEM-resistant GPAT activity
A: RNA was isolated from backcrossed Bl/6 wildtype (+/+), heterozygous (+/−), and knockout (−/−) Gpat1 mouse hearts (n = 2) and analyzed for the presence or absence of neo and Gpat1 transcripts by reverse transcriptase PCR as described in Materials and Methods. The first lane contains DNA size markers (from top: 1000, 850, and 650 bp). The second lane (C) is a no template control reaction. Heart total membranes (B) and mitochondria (C, D) from wildtype and Gpat1−/− mice were isolated as described and assayed for GPAT activity in the absence or presence of (B, C) 1 mM NEM or (D) 0–10 mM NEM. Data not shown for 5 and 10 mM NEM, as further inhibition was not observed. Data are presented as the mean ± SD (n = 3 – 4).
Figure 2
Figure 2. Absence of GPAT1 diminishes myocardial triacylglycerol accumulation
Lipids were extracted from hearts of male and female Bl/6 (A, high sucrose diet) or F2 (B, high fat diet) mice (n ≥ 6 for each group) and TAG content was determined as described in Materials and Methods. A: High sucrose (HS) vs control (CT) diet. B: High fat (HF) diet vs baseline. For hearts from mice at baseline or on the control diet, heart TAG did not differ between male and female mice. Data are presented as mean ± SD.
Figure 3
Figure 3. Gpat1−/− mice have lower plasma triacylglycerol than wildtype mice after high sucrose- or high fat-diet
Plasma lipids were extracted from male and female Bl/6 (A, high sucrose diet) or F2 (B, high fat diet) mouse hearts and TAG concentration was determined as described in Materials and Methods. A: High sucrose (HS) vs control (CT) diet. B: High fat (HF) diet vs baseline. Data are presented as the mean ± SD; n = 6 for each group.
Figure 4
Figure 4. Male and female Gpat1−/− mice have lower VLDL secretion rates than wildtype mice after 2 week high sucrose diet
After a 4 h fast, male (A) and female (B) wildtype and Gpat1 null Bl/6 mice were injected with Tyloxapol as described in Materials and Methods and plasma was collected at the times shown. Rates of VLDL secretion were calculated as the concentration of TAG (mg/dl) per unit of liver weight (g) per hour. Data are presented as the mean ± SD; n ≥ 5 for each group.
Figure 5
Figure 5. Absence of GPAT1 decreases [14C]palmitate incorporation into heart triacylglycerol in vivo
Wildtype and Gpat1−/− mice were fed a high-sucrose diet for 2 wks and injected with [14C]palmitate complexed to BSA as described in Materials and Methods. Heart lipids were extracted and separated by TLC and the amount of [14C] incorporated into TAG was quantified by Bioscan analysis. Data are presented as cpm [14C] incorporated into TAG/g protein/cpm [14C] in plasma; n = 3.
Figure 6
Figure 6. Absence of GPAT1 alters heart phospholipid fatty acid composition
Lipids were extracted from hearts and plasma of F2 female wildtype (+/+) and Gpat1−/− (−/−) mice (n = 4/genotype) and fatty acid composition was determined as described in Materials and Methods. A, phosphatidylcholine (PC); B, phosphatidylethanolamine (PE); phosphatidylserine (PS) and phosphatidylinositol (PI); D, cardiolipin (CL) E, plasma TAG. Data are presented as mean ± SD. *P ≤ 0.05 relative to wildtype control.
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