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Lpcat3-dependent production of arachidonoyl phospholipids is a key determinant of triglyceride secretion.

Rong X, Wang B, Dunham MM, Hedde PN, Wong JS, Gratton E, Young SG, Ford DA, Tontonoz P - Elife (2015)

Bottom Line: Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma TGs.Mice lacking Lpcat3 in the liver show reduced plasma TGs, hepatosteatosis, and secrete lipid-poor very low-density lipoprotein (VLDL) lacking arachidonoyl PLs.Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, United States.

ABSTRACT
The role of specific phospholipids (PLs) in lipid transport has been difficult to assess due to an inability to selectively manipulate membrane composition in vivo. Here we show that the phospholipid remodeling enzyme lysophosphatidylcholine acyltransferase 3 (Lpcat3) is a critical determinant of triglyceride (TG) secretion due to its unique ability to catalyze the incorporation of arachidonate into membranes. Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma TGs. Mice lacking Lpcat3 in the liver show reduced plasma TGs, hepatosteatosis, and secrete lipid-poor very low-density lipoprotein (VLDL) lacking arachidonoyl PLs. Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles. These data identify Lpcat3 as a key factor in lipoprotein production and illustrate how manipulation of membrane composition can be used as a regulatory mechanism to control metabolic pathways.

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Related in: MedlinePlus

Generation and analysis of global Lpcat3 knockout mice.(A) Strategy for generating global Lpcat3 knockout mice. A ‘knock-out first/conditional-ready’ gene-targeting vector was used to generate targeted cells. A gene-trap cassette is located between the two FRT sites. LacZ, β-galactosidase; neo, neomycin phosphotransferase II. (B) Genotyping of Lpcat3+/+ (WT), Lpcat3+/− (Het), and Lpcat3−/− (KO) mice. Genomic DNA was prepared from tail biopsies, and PCR products were separated on a 1% agarose gel. (C) Expression of Lpcat3 in liver and small intestine of newborn Lpcat3−/− and Lpcat3+/+ pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. (D) The body weight and blood glucose of Lpcat3+/+ (WT), Lpcat3−/+ (Het), and Lpcat3−/− (KO) newborn pups (n ≥ 8/group). Values are means ± SEM. (E) Kaplan–Meier survival curve of Lpcat3+/+ (WT), Lpcat3−/+ (Het) and Lpcat3−/− (KO) pups after birth (n ≥ 20 mice/group). The neonatal lethality can be delayed by injection of 50 μl 10% glucose solution once per day after born. 5 Lpcat3+/+ (WT), 9 Lpcat3−/+ (Het) and 6 Lpcat3−/− (KO) mice were used in the rescue experiment. (F) Representative photograph of Lpcat3+/+ (WT) and Lpcat3−/− (KO) pups after 5 days of glucose injections. (G–H) Gene expression in livers (G) and small intestines (H) of Lpcat3+/+ (WT) and Lpcat3−/− (Lpcat3 KO) newborn pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. Statistical analysis was performed using Student's t-test (C, G and H) and one-way ANOVA with Bonferroni post-hoc tests (D). *p < 0.05; **p < 0.01.DOI:http://dx.doi.org/10.7554/eLife.06557.003
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fig1: Generation and analysis of global Lpcat3 knockout mice.(A) Strategy for generating global Lpcat3 knockout mice. A ‘knock-out first/conditional-ready’ gene-targeting vector was used to generate targeted cells. A gene-trap cassette is located between the two FRT sites. LacZ, β-galactosidase; neo, neomycin phosphotransferase II. (B) Genotyping of Lpcat3+/+ (WT), Lpcat3+/− (Het), and Lpcat3−/− (KO) mice. Genomic DNA was prepared from tail biopsies, and PCR products were separated on a 1% agarose gel. (C) Expression of Lpcat3 in liver and small intestine of newborn Lpcat3−/− and Lpcat3+/+ pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. (D) The body weight and blood glucose of Lpcat3+/+ (WT), Lpcat3−/+ (Het), and Lpcat3−/− (KO) newborn pups (n ≥ 8/group). Values are means ± SEM. (E) Kaplan–Meier survival curve of Lpcat3+/+ (WT), Lpcat3−/+ (Het) and Lpcat3−/− (KO) pups after birth (n ≥ 20 mice/group). The neonatal lethality can be delayed by injection of 50 μl 10% glucose solution once per day after born. 5 Lpcat3+/+ (WT), 9 Lpcat3−/+ (Het) and 6 Lpcat3−/− (KO) mice were used in the rescue experiment. (F) Representative photograph of Lpcat3+/+ (WT) and Lpcat3−/− (KO) pups after 5 days of glucose injections. (G–H) Gene expression in livers (G) and small intestines (H) of Lpcat3+/+ (WT) and Lpcat3−/− (Lpcat3 KO) newborn pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. Statistical analysis was performed using Student's t-test (C, G and H) and one-way ANOVA with Bonferroni post-hoc tests (D). *p < 0.05; **p < 0.01.DOI:http://dx.doi.org/10.7554/eLife.06557.003

Mentions: To examine the consequence of Lpcat3 deficiency in vivo, we generated Lpcat3-deficient mice from targeted ES cells (Figure 1A). The targeted allele was ‘conditional-ready’, making it possible to create both global and tissue-specific knockout mice. The global knockout mice (i.e., homozygous for the targeted allele) showed markedly reduced levels of Lpcat3 transcripts in liver and intestine (Figure 1B,C). Global Lpcat3−/− mice on a C57BL/6 background were born at the expected Mendelian frequency, and their weights were indistinguishable from WT mice at birth (Figure 1D, Table 1). However, the blood glucose levels of Lpcat3−/− mice were very low at birth, and none survived beyond day 2 (Figure 1D,E). Lpcat3−/− pups survived for up to 6 days when given subcutaneous glucose injections, but the pups did not thrive and invariably died (Figure 1E,F). Analysis of gene expression in liver and small intestines of the pups revealed changes in a number of genes linked to lipid metabolism, some of which were common in both tissues (Figure 1G,H). Although these changes appeared consistent with a role for Lpcat3 in lipid metabolism, it was impossible to exclude the possibility that these gene-expression alterations were simply due to the extremely poor health of the mice. We therefore turned our attention to tissue-selective knockout mice, with the hope that we could obtain viable mice and decipher the function of Lpcat3.10.7554/eLife.06557.003Figure 1.Generation and analysis of global Lpcat3 knockout mice.


Lpcat3-dependent production of arachidonoyl phospholipids is a key determinant of triglyceride secretion.

Rong X, Wang B, Dunham MM, Hedde PN, Wong JS, Gratton E, Young SG, Ford DA, Tontonoz P - Elife (2015)

Generation and analysis of global Lpcat3 knockout mice.(A) Strategy for generating global Lpcat3 knockout mice. A ‘knock-out first/conditional-ready’ gene-targeting vector was used to generate targeted cells. A gene-trap cassette is located between the two FRT sites. LacZ, β-galactosidase; neo, neomycin phosphotransferase II. (B) Genotyping of Lpcat3+/+ (WT), Lpcat3+/− (Het), and Lpcat3−/− (KO) mice. Genomic DNA was prepared from tail biopsies, and PCR products were separated on a 1% agarose gel. (C) Expression of Lpcat3 in liver and small intestine of newborn Lpcat3−/− and Lpcat3+/+ pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. (D) The body weight and blood glucose of Lpcat3+/+ (WT), Lpcat3−/+ (Het), and Lpcat3−/− (KO) newborn pups (n ≥ 8/group). Values are means ± SEM. (E) Kaplan–Meier survival curve of Lpcat3+/+ (WT), Lpcat3−/+ (Het) and Lpcat3−/− (KO) pups after birth (n ≥ 20 mice/group). The neonatal lethality can be delayed by injection of 50 μl 10% glucose solution once per day after born. 5 Lpcat3+/+ (WT), 9 Lpcat3−/+ (Het) and 6 Lpcat3−/− (KO) mice were used in the rescue experiment. (F) Representative photograph of Lpcat3+/+ (WT) and Lpcat3−/− (KO) pups after 5 days of glucose injections. (G–H) Gene expression in livers (G) and small intestines (H) of Lpcat3+/+ (WT) and Lpcat3−/− (Lpcat3 KO) newborn pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. Statistical analysis was performed using Student's t-test (C, G and H) and one-way ANOVA with Bonferroni post-hoc tests (D). *p < 0.05; **p < 0.01.DOI:http://dx.doi.org/10.7554/eLife.06557.003
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fig1: Generation and analysis of global Lpcat3 knockout mice.(A) Strategy for generating global Lpcat3 knockout mice. A ‘knock-out first/conditional-ready’ gene-targeting vector was used to generate targeted cells. A gene-trap cassette is located between the two FRT sites. LacZ, β-galactosidase; neo, neomycin phosphotransferase II. (B) Genotyping of Lpcat3+/+ (WT), Lpcat3+/− (Het), and Lpcat3−/− (KO) mice. Genomic DNA was prepared from tail biopsies, and PCR products were separated on a 1% agarose gel. (C) Expression of Lpcat3 in liver and small intestine of newborn Lpcat3−/− and Lpcat3+/+ pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. (D) The body weight and blood glucose of Lpcat3+/+ (WT), Lpcat3−/+ (Het), and Lpcat3−/− (KO) newborn pups (n ≥ 8/group). Values are means ± SEM. (E) Kaplan–Meier survival curve of Lpcat3+/+ (WT), Lpcat3−/+ (Het) and Lpcat3−/− (KO) pups after birth (n ≥ 20 mice/group). The neonatal lethality can be delayed by injection of 50 μl 10% glucose solution once per day after born. 5 Lpcat3+/+ (WT), 9 Lpcat3−/+ (Het) and 6 Lpcat3−/− (KO) mice were used in the rescue experiment. (F) Representative photograph of Lpcat3+/+ (WT) and Lpcat3−/− (KO) pups after 5 days of glucose injections. (G–H) Gene expression in livers (G) and small intestines (H) of Lpcat3+/+ (WT) and Lpcat3−/− (Lpcat3 KO) newborn pups. Gene expression was quantified by real-time PCR (n ≥ 5/group). Values are means ± SEM. Statistical analysis was performed using Student's t-test (C, G and H) and one-way ANOVA with Bonferroni post-hoc tests (D). *p < 0.05; **p < 0.01.DOI:http://dx.doi.org/10.7554/eLife.06557.003
Mentions: To examine the consequence of Lpcat3 deficiency in vivo, we generated Lpcat3-deficient mice from targeted ES cells (Figure 1A). The targeted allele was ‘conditional-ready’, making it possible to create both global and tissue-specific knockout mice. The global knockout mice (i.e., homozygous for the targeted allele) showed markedly reduced levels of Lpcat3 transcripts in liver and intestine (Figure 1B,C). Global Lpcat3−/− mice on a C57BL/6 background were born at the expected Mendelian frequency, and their weights were indistinguishable from WT mice at birth (Figure 1D, Table 1). However, the blood glucose levels of Lpcat3−/− mice were very low at birth, and none survived beyond day 2 (Figure 1D,E). Lpcat3−/− pups survived for up to 6 days when given subcutaneous glucose injections, but the pups did not thrive and invariably died (Figure 1E,F). Analysis of gene expression in liver and small intestines of the pups revealed changes in a number of genes linked to lipid metabolism, some of which were common in both tissues (Figure 1G,H). Although these changes appeared consistent with a role for Lpcat3 in lipid metabolism, it was impossible to exclude the possibility that these gene-expression alterations were simply due to the extremely poor health of the mice. We therefore turned our attention to tissue-selective knockout mice, with the hope that we could obtain viable mice and decipher the function of Lpcat3.10.7554/eLife.06557.003Figure 1.Generation and analysis of global Lpcat3 knockout mice.

Bottom Line: Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma TGs.Mice lacking Lpcat3 in the liver show reduced plasma TGs, hepatosteatosis, and secrete lipid-poor very low-density lipoprotein (VLDL) lacking arachidonoyl PLs.Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, United States.

ABSTRACT
The role of specific phospholipids (PLs) in lipid transport has been difficult to assess due to an inability to selectively manipulate membrane composition in vivo. Here we show that the phospholipid remodeling enzyme lysophosphatidylcholine acyltransferase 3 (Lpcat3) is a critical determinant of triglyceride (TG) secretion due to its unique ability to catalyze the incorporation of arachidonate into membranes. Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma TGs. Mice lacking Lpcat3 in the liver show reduced plasma TGs, hepatosteatosis, and secrete lipid-poor very low-density lipoprotein (VLDL) lacking arachidonoyl PLs. Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles. These data identify Lpcat3 as a key factor in lipoprotein production and illustrate how manipulation of membrane composition can be used as a regulatory mechanism to control metabolic pathways.

Show MeSH
Related in: MedlinePlus