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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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Quantification of western blots.Immunoblot results of apoB-100 and apoB-48 proteins in (A) Figure 2E and (B) Figure 2D were quantified by densitometry.DOI:http://dx.doi.org/10.7554/eLife.06557.007
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fig2s2: Quantification of western blots.Immunoblot results of apoB-100 and apoB-48 proteins in (A) Figure 2E and (B) Figure 2D were quantified by densitometry.DOI:http://dx.doi.org/10.7554/eLife.06557.007

Mentions: (A) Strategy for generating tissue-specific Lpcat knockout mice. Lpcat3−/+ mice carrying the conditional-ready knockout allele were mated with Flpe transgenic mice to generate the Lpcat3fl/fl mice. Lpcat3fl/fl mice were bred with tissue-specific Cre transgenic mice to generate tissue-specific Lpcat3 knockout mice. (B) Expression of Lpcat family members in liver of Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice fed a chow diet. Gene expression was quantified by real-time PCR (n ≥ 6/group) (left panel). Primary hepatocytes from Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice were treated overnight with 1 μM LXR agonist GW3965 (GW). Lpcat3 expression was quantified by real-time PCR (right panel). Ct values in Lpcat3fl/fl liver samples were shown. Values are means ± SEM. (C) Plasma lipid levels in chow diet-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice under fed (ad libitum) or overnight (o/n) fasting (n ≥ 8/group). Plasma levels of cholesterol and NEFA were measured after an overnight fast. Values are means ± SEM. (D) Plasma was harvested from chow diet-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice after an overnight fast. ApoB protein was analyzed by western blotting and quantified (Figure 2—figure supplement 2B). (E) Plasma from Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-Lpcat3-KO) mice fasted overnight was pooled (n = 5). Lipoprotein profiles were analyzed by fast protein liquid chromatography (FPLC) (upper panel). ApoB protein in each corresponding fraction was analyzed by western blot (lower panel) and quantified (Figure 2—figure supplement 2A). (F) Lipid contents in livers of chow diet-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice fasted overnight (n ≥ 8/group). Values are means ± SEM. (G) Hematoxylin and eosin staining of liver sections from chow-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice after an overnight fast. (H) Liver expression of Lpcat3 (left panel) and plasma TG levels (right panel) in Lpcat3fl/fl mice 3 weeks after being injected with an adenovirus encoding GFP or Cre. Mice were sacrificed under fed conditions (ad libitum) or after overnight fasting (o/n) (n ≥ 6/group). Gene expression was measured by real-time. Values are means ± SEM. Statistical analysis was performed with a Student's t-test (B, C, F, H). *p < 0.05; **p < 0.01.


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)

Quantification of western blots.Immunoblot results of apoB-100 and apoB-48 proteins in (A) Figure 2E and (B) Figure 2D were quantified by densitometry.DOI:http://dx.doi.org/10.7554/eLife.06557.007
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fig2s2: Quantification of western blots.Immunoblot results of apoB-100 and apoB-48 proteins in (A) Figure 2E and (B) Figure 2D were quantified by densitometry.DOI:http://dx.doi.org/10.7554/eLife.06557.007
Mentions: (A) Strategy for generating tissue-specific Lpcat knockout mice. Lpcat3−/+ mice carrying the conditional-ready knockout allele were mated with Flpe transgenic mice to generate the Lpcat3fl/fl mice. Lpcat3fl/fl mice were bred with tissue-specific Cre transgenic mice to generate tissue-specific Lpcat3 knockout mice. (B) Expression of Lpcat family members in liver of Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice fed a chow diet. Gene expression was quantified by real-time PCR (n ≥ 6/group) (left panel). Primary hepatocytes from Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice were treated overnight with 1 μM LXR agonist GW3965 (GW). Lpcat3 expression was quantified by real-time PCR (right panel). Ct values in Lpcat3fl/fl liver samples were shown. Values are means ± SEM. (C) Plasma lipid levels in chow diet-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice under fed (ad libitum) or overnight (o/n) fasting (n ≥ 8/group). Plasma levels of cholesterol and NEFA were measured after an overnight fast. Values are means ± SEM. (D) Plasma was harvested from chow diet-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice after an overnight fast. ApoB protein was analyzed by western blotting and quantified (Figure 2—figure supplement 2B). (E) Plasma from Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-Lpcat3-KO) mice fasted overnight was pooled (n = 5). Lipoprotein profiles were analyzed by fast protein liquid chromatography (FPLC) (upper panel). ApoB protein in each corresponding fraction was analyzed by western blot (lower panel) and quantified (Figure 2—figure supplement 2A). (F) Lipid contents in livers of chow diet-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice fasted overnight (n ≥ 8/group). Values are means ± SEM. (G) Hematoxylin and eosin staining of liver sections from chow-fed Lpcat3fl/fl (F/F) and Lpcat3fl/flAlbumin-Cre (L-KO) mice after an overnight fast. (H) Liver expression of Lpcat3 (left panel) and plasma TG levels (right panel) in Lpcat3fl/fl mice 3 weeks after being injected with an adenovirus encoding GFP or Cre. Mice were sacrificed under fed conditions (ad libitum) or after overnight fasting (o/n) (n ≥ 6/group). Gene expression was measured by real-time. Values are means ± SEM. Statistical analysis was performed with a Student's t-test (B, C, F, H). *p < 0.05; **p < 0.01.

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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