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The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis.

Fryer LG, Jones B, Duncan EJ, Hutchison CE, Ozkan T, Williams PA, Alder O, Nieuwdorp M, Townley AK, Mensenkamp AR, Stephens DJ, Dallinga-Thie GM, Shoulders CC - J. Biol. Chem. (2013)

Bottom Line: The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi.The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation.We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo.

View Article: PubMed Central - PubMed

Affiliation: From the Endocrinology Centre, William Harvey Research Institute, Queen Mary University of London and Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom.

ABSTRACT
Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

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Sar1B:H79G decreases cholesterol biosynthesis gene expression in McArdle-RH7777 cells, independently of reduced Apob and Mttp expression.A, expression values of 701 probe sets were significantly altered (FDR, 0.000215; Δ 6.8) by Sar1B:H79G but not Sar1A (FDR 0.009, Δ1.9) or Sar1B (FDR 0.015, Δ 2.0) overexpression. B, green labeling depicts reduced mRNA levels in stably overexpressing Sar1B:H79G cells. *, expression values are verified by RT-qPCR. Note: in stably overexpressing Sar1A and Sar1B cells, mRNA levels of these genes were not reduced (supplemental Table S5). C and D, relative protein levels of three representative enzymes on the cholesterol biosynthetic pathway (Hmgcs1, Lss, Dhcr7) and a control enzyme, Acsl5, in stably transfected cells overexpressing Sar1B:H79G (C), Sar1A, and Sar1B (D). C and D data (mean ± S.E.) are from at least three independent experiments. ***, p < 0.001 versus cells with empty vector control; **, p < 0.01 versus cells with empty vector control. White line separators indicate that noncontiguous lanes from the same gel are shown.
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Figure 3: Sar1B:H79G decreases cholesterol biosynthesis gene expression in McArdle-RH7777 cells, independently of reduced Apob and Mttp expression.A, expression values of 701 probe sets were significantly altered (FDR, 0.000215; Δ 6.8) by Sar1B:H79G but not Sar1A (FDR 0.009, Δ1.9) or Sar1B (FDR 0.015, Δ 2.0) overexpression. B, green labeling depicts reduced mRNA levels in stably overexpressing Sar1B:H79G cells. *, expression values are verified by RT-qPCR. Note: in stably overexpressing Sar1A and Sar1B cells, mRNA levels of these genes were not reduced (supplemental Table S5). C and D, relative protein levels of three representative enzymes on the cholesterol biosynthetic pathway (Hmgcs1, Lss, Dhcr7) and a control enzyme, Acsl5, in stably transfected cells overexpressing Sar1B:H79G (C), Sar1A, and Sar1B (D). C and D data (mean ± S.E.) are from at least three independent experiments. ***, p < 0.001 versus cells with empty vector control; **, p < 0.01 versus cells with empty vector control. White line separators indicate that noncontiguous lanes from the same gel are shown.

Mentions: We identified 701 probe sets that had altered expression values (FDR <0.000215) in the Sar1B:H79G cell lines only (Fig. 3A). In the control analyses (i.e. native Sar1 overexpression), fewer probe sets were uniquely differentially expressed as follows: three in the Sar1A- and 70 in the Sar1B-overexpressing cell lines (supplemental Fig. S1). The Sar1B:H79G probe set contained an over-representation of genes assigned the Gene Ontology terms “Cholesterol and Lipid Biosynthetic and Metabolic Processes” (Table 1), although neither the Sar1A nor Sar1B datasets did (supplemental Tables S3 and S4). We therefore collated the expression values for genes active in fatty acid, triglyceride, and cholesterol metabolism, and verified the mRNA levels of representative genes in an independent experiment by RT-qPCR (supplemental Table S5).


The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis.

Fryer LG, Jones B, Duncan EJ, Hutchison CE, Ozkan T, Williams PA, Alder O, Nieuwdorp M, Townley AK, Mensenkamp AR, Stephens DJ, Dallinga-Thie GM, Shoulders CC - J. Biol. Chem. (2013)

Sar1B:H79G decreases cholesterol biosynthesis gene expression in McArdle-RH7777 cells, independently of reduced Apob and Mttp expression.A, expression values of 701 probe sets were significantly altered (FDR, 0.000215; Δ 6.8) by Sar1B:H79G but not Sar1A (FDR 0.009, Δ1.9) or Sar1B (FDR 0.015, Δ 2.0) overexpression. B, green labeling depicts reduced mRNA levels in stably overexpressing Sar1B:H79G cells. *, expression values are verified by RT-qPCR. Note: in stably overexpressing Sar1A and Sar1B cells, mRNA levels of these genes were not reduced (supplemental Table S5). C and D, relative protein levels of three representative enzymes on the cholesterol biosynthetic pathway (Hmgcs1, Lss, Dhcr7) and a control enzyme, Acsl5, in stably transfected cells overexpressing Sar1B:H79G (C), Sar1A, and Sar1B (D). C and D data (mean ± S.E.) are from at least three independent experiments. ***, p < 0.001 versus cells with empty vector control; **, p < 0.01 versus cells with empty vector control. White line separators indicate that noncontiguous lanes from the same gel are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3924288&req=5

Figure 3: Sar1B:H79G decreases cholesterol biosynthesis gene expression in McArdle-RH7777 cells, independently of reduced Apob and Mttp expression.A, expression values of 701 probe sets were significantly altered (FDR, 0.000215; Δ 6.8) by Sar1B:H79G but not Sar1A (FDR 0.009, Δ1.9) or Sar1B (FDR 0.015, Δ 2.0) overexpression. B, green labeling depicts reduced mRNA levels in stably overexpressing Sar1B:H79G cells. *, expression values are verified by RT-qPCR. Note: in stably overexpressing Sar1A and Sar1B cells, mRNA levels of these genes were not reduced (supplemental Table S5). C and D, relative protein levels of three representative enzymes on the cholesterol biosynthetic pathway (Hmgcs1, Lss, Dhcr7) and a control enzyme, Acsl5, in stably transfected cells overexpressing Sar1B:H79G (C), Sar1A, and Sar1B (D). C and D data (mean ± S.E.) are from at least three independent experiments. ***, p < 0.001 versus cells with empty vector control; **, p < 0.01 versus cells with empty vector control. White line separators indicate that noncontiguous lanes from the same gel are shown.
Mentions: We identified 701 probe sets that had altered expression values (FDR <0.000215) in the Sar1B:H79G cell lines only (Fig. 3A). In the control analyses (i.e. native Sar1 overexpression), fewer probe sets were uniquely differentially expressed as follows: three in the Sar1A- and 70 in the Sar1B-overexpressing cell lines (supplemental Fig. S1). The Sar1B:H79G probe set contained an over-representation of genes assigned the Gene Ontology terms “Cholesterol and Lipid Biosynthetic and Metabolic Processes” (Table 1), although neither the Sar1A nor Sar1B datasets did (supplemental Tables S3 and S4). We therefore collated the expression values for genes active in fatty acid, triglyceride, and cholesterol metabolism, and verified the mRNA levels of representative genes in an independent experiment by RT-qPCR (supplemental Table S5).

Bottom Line: The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi.The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation.We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo.

View Article: PubMed Central - PubMed

Affiliation: From the Endocrinology Centre, William Harvey Research Institute, Queen Mary University of London and Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom.

ABSTRACT
Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

Show MeSH
Related in: MedlinePlus