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Glycerolipid Biosynthesis in Porcine Adipose Tissue In Vitro. I. Assay Conditions for Homogenates

U.S. Department of Agriculture^,3,4,5,, Clay Center, NE 68933 and Texas A&M University², College Station 77843

Abstract

Previous studies on glycerolipid biosynthesis in swine adipose tissue in vitro resulted in synthesis of primarily phospholipid, whereas triacylglycerol represents the vast majority of adipose tissue lipids. The objectives of this research were to maximize synthesis of triacylglycerol in vitro using the 700 x g infranatant fraction of a swine adipose tissue homogenate as the enzyme source. The capacity for total lipid synthesis was increased by > 50%, and the proportion of lipids synthesized as triacylglycerols was increased by increasing the length of incubation time from 20 to 60 min and the concentration of enzyme in the incubation from that obtained from 33 to that obtained from 120 mg adipose tissue. It is recommended that glycerolipid biosynthesis be assessed using two assays. An assay of up to 10 min was linear with incubation time and measured the initial incorporation of glycerol-3-phosphate into the pathway (GPAT); this incorporation was mostly into phospholipids. An assay of about 60 min was not linear with incubation time, but incorporation into total lipids (LSC) was predominantly into the triacylglycerol fraction. Although the LSC assay was not linear with time, it represents steady-state conditions that more closely typify conditions in situ. Oleate at .6 mM was inhibitory with enzyme extracted from 33 or 75 mg adipose tissue, whereas palmitate was not. Palmitoyl-CoA was not a suitable substrate because it produced low LSC and little triacylglycerol. Fluoride increased LSC but inhibited conversion of phospholipids into triacylglycerols, so its presence is not recommended. Use of this assay to compare animals with divergent adipocyte size seems inappropriate because animal differences probably will result in divergent enzyme concentrations in the incubation.

¹ Present address: Dept. of Anim. Sci., Univ. of Wyoming, Laramie 82701.

² Dept. of Anim. Sci., association of D. C. Rule and location of S. B. Smith. Tech. Article No. 23029, Texas Agric. Exp. Sta.

³ Roman L. Hruska U. S. Meat Anim. Res. Center, ARS, USDA, Clay Center, NE. To whom all correspondence should be sent.

⁴ We appreciate the assistance of T. W. Acton and associated, especially J. A. Dague, for adipose tissue biopsy of pigs and M. M. Bierman for secretarial assistance.

⁵ Mention of trade names, proprietary products or specific equipment does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that also may be suitable.