Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes

Cartwright, Ian J.; Higgins, Joan A.; Wilkinson, Jane; Bellavía, S. L.; Kendrick, John; Graham, John M.

doi:10.1016/s0022-2275(20)37261-8

Cited by 31 publications

(11 citation statements)

References 55 publications

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“…However, to minimize experimental manipulations, during which apoB may be lost or degraded, despite addition of a cocktail of protease inhibitors, we investigated the possibility of determination of the incorporation of [ 35 S]methionine into apoB after separation of the total cell proteins on 3-20% gradient gels. We have successfully used this method previously in studies of rabbit or rat hepatocytes (13)(14)(15)19). Essentially the same results were obtained using either immunoprecipitation or counting of apoB bands on SDS-PAGE gels ( Fig.…”

Section: Determination Of the Incorporation Of [ 35 S]methionine Into Cellular And Secreted Apobmentioning

confidence: 58%

“…The incubation flasks were gassed with 95% oxygen/5% carbon dioxide for a few seconds at approximately 15-min intervals throughout the incubation. In some experiments the hepatocytes were incubated for a series of times; in other experiments, the cells were incubated with radiolabeled substrate for 30 min, isolated by centrifugation, and reincubated with unlabeled substrate (45 m m ) for up to 120 min to follow the fate of the radiolabeled apoB (13)(14)(15)19). At each time interval the cells were pelleted by centrifugation at 1000 g for 5 min.…”

Section: Preparation and Incubation Of Hepatocytesmentioning

confidence: 99%

“…A large fraction of this pool of apoB is degraded in the RER lumen (13,15) presumably because it has not acquired the correct complement of lipids, is not properly folded, or is prevented in some way from moving on through the secretory pathway. Most of the VLDL lipids are transferred to the lumen of the smooth endoplasmic reticulum (SER) and are assembled with the apoB-containing VLDL precursors between the SER and the cis -Golgi (14,19). The initial events in the intracellular transit of apoB and the assembly of precursor particles in the lumen of the RER are similar in HepG2 cells and hepatocytes (20,21).…”

mentioning

confidence: 99%

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Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes

Kendrick

Higgins

1999

Journal of Lipid Research

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Dietary fish oils inhibited secretion and stimulated intracellular degradation of apolipoprotein (apo)B in hamster hepatocytes, while dietary sunflower oils stimulated secretion and had no effect on degradation of apoB. To investigate the intracellular site at which fish oils act, we have made use of our previous observations that inhibition of degradation by N-acetyl-leucyl-leucyl-norleucinal (ALLN) results in accumulation of apoB in the trans -Golgi membrane and does not stimulate secretion, while inhibition of degradation by o -phenanthroline results in accumulation of apoB in the rough endoplasmic reticulum membrane and stimulates secretion. Thus, ALLN protects apoB which has been diverted from secretion and o -phenanthroline protects apoB which is targetted for secretion. Addition of o -phenantholine to the incubation medium of hepatocytes from fish oil-fed hamsters inhibited degradation of apoB and stimulated its secretion in particles of the density of VLDL, while addition of ALLN had no effect. These observations suggest that dietary fish oils reversibly inhibit early steps in the assembly of very low density lipoprotein precursors and target apoB for degradation in the rough endoplasmic reticulum. -Kendrick, J. S., and J. A. Higgins. Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes.

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Section: Determination Of the Incorporation Of [ 35 S]methionine Into Cellular And Secreted Apobmentioning

confidence: 58%

Section: Preparation and Incubation Of Hepatocytesmentioning

confidence: 99%

mentioning

confidence: 99%

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Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes

Kendrick

Higgins

1999

Journal of Lipid Research

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“…Cellular and secreted lipids were extracted and separated by HPTLC, as described previously (15,19). Quantitation of 3 H radioactivity in TAG was performed by scintillation counting.…”

Section: Analysis Of Triacylglycerolmentioning

confidence: 99%

“…Quantitation of 3 H radioactivity in TAG was performed by scintillation counting. TAG mass was determined by laser densitometry as described previously (15,19).…”

Section: Analysis Of Triacylglycerolmentioning

confidence: 99%

Isolated rabbit enterocytes as a model cell system for investigations of chylomicron assembly and secretion

Cartwright

Higgins

1999

Journal of Lipid Research

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A method is described for the isolation of viable enterocytes from rabbit small intestine. The procedure can also be used to isolate populations of epithelial cells from the crypt/villus gradient. The isolated enterocytes synthesized and secreted apoB-48 and triacylglycerol in particles of the density of chylomicrons. Secretion was stimulated by addition of bile salt/lipid micelles. Pulse-chase experiments demonstrated that newly synthesized apoB-48 is degraded intracellularly and that degradation is inhibited by provision of lipid micelles, suggesting that regulation of chylomicron assembly and secretion is broadly similar to that of very low density lipoprotein assembly in hepatocytes. This procedure for preparation of isolated enterocytes will provide a useful model system for investigation of the molecular details of chylomicron assembly. -Cartwright, I. J., and J. A. Higgins. Isolated rabbit enterocytes as a model cell system for investigations of chylomicron assembly and secretion.

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Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation

Graham

2001

CP Cell Biology

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This unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran‐treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self‐generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self‐generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.

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Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes

Cited by 31 publications

References 55 publications

Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes

Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes

Isolated rabbit enterocytes as a model cell system for investigations of chylomicron assembly and secretion

Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation

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