Abstract
The adipose differentiation-related protein (ADRP) was first characterized as a mRNA induced early during adipocyte differentiation (Jiang, H. P., and G. Serrero. 1992. Proc. Natl. Acad. Sci. USA. 89:7856-7860). The present study demonstrates that ADRP mRNA is expressed in a variety of tissues and cultured cell lines. Immunocytochemical examination revealed that ADRP localizes to neutral lipid storage droplets in cultured murine 3T3-L1 adipocytes, murine MA-10 Leydig cells, Chinese hamster ovary (CHO) fibroblasts, and human HepG2 hepatoma cells; the association of ADRP with lipid droplets was confirmed by subcellular fractionation of MA-10 Leydig cells. In addition to ADRP, steroidogenic cells and adipocytes express the perilipins, a family of lipid droplet-associated proteins that share a highly related sequence domain with ADRP. ADRP and perilipins co-localize on lipid droplets in MA-10 Leydig cells. While ADRP was found on small lipid droplets in 3T3-L1 preadipocytes and early differentiated adipocytes, it was absent in maturing adipocytes. In contrast, perilipins were absent early during differentiation, but were found on small and large lipid droplets at later stages. The transition in surface protein composition of adipocyte lipid droplets from ADRP to perilipins occurred 3 days after the initiation of differentiation when cells displayed co-localizatioin of both proteins on the same lipid droplets. The specific localization of adipose differentiation-related protein to lipid droplets in a wide variety of cells suggests that ADRP plays a role in management of neutral lipid stores.
Highlights
adipose differentiation-related protein (ADRP) mRNA is expressed in various tissues and cultured cell lines Based on the finding of the perilipins in steroidogenic cells [13] in addition to adipocytes, and given the structural similarities between the amino termini of ADRP and the perilipins, we reasoned that the expression of ADRP may not be limited to adipocytes
Northern blot analysis of total RNA extracted from cultured cell lines identified ADRP mRNA in murine MA-10 Leydig cells at approximately equal abundance to that in differentiated murine STS-Ll adipocytes (Fig. 1b)
Human HepC2 hepatoma cells (Fig. IC), Chinese hamster ovary (CHO) fibroblasts (Fig. Id), and STS-Ll pre-adipocytes all expressed similar amounts of ADRP mRNA and less than that of MA-10 Leydig cells. Among these cultured cell lines, only differentiated STS-Ll adipocytes and MA-10 Leydig cells express perilipin mRNAs
Summary
Nile Red was obtained from Molecular Probes, Inc. (Eugene, OR). Lissamine rhodamine- or fluoresceinconjugated secondary antibodies raised against rabbit or guinea pig IgGs were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Nile Red was obtained from Molecular Probes, Inc. Lissamine rhodamine- or fluoresceinconjugated secondary antibodies raised against rabbit or guinea pig IgGs were obtained from Jackson ImmunoResearch Laboratories, Inc. TRIzol and powdered Dulbecco’s Modified Eagle’s medium, Waymouth’sMB 752/ 1medium, Ham’sF-10 medium, Ham’s F-12 medium, and Minimum Essential Medium were purchased from Life Technologies, Inc (Grand Island, NY). 3-Isobutyl-l-methylxanthinei,nsulin, and dexamethasone were obtained from Sigma (St. Louis, MO). Horse serum and fetal bovine serum were obtained from Summit Biotechnology (Ft. Collins,CO). Rat R2C Leydig cells, Chinese hamster ovary (CHO) fibroblasts, human HepG2 hepatoma cells, and murine. 3T3-Ll cells were obtained from the American Type Culture Collection (Rockville,MD). MA-10 Leydig cells were generously provided by Dr Mario Ascoli (University of Iowa, Iowa City, IA). RNA from various tissues was purchased from Clontech Laboratories, Inc. RNA from various tissues was purchased from Clontech Laboratories, Inc. (Palo Alto, C4)
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