Advertisement

Histochemistry and Cell Biology

, Volume 120, Issue 4, pp 285–292 | Cite as

Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes

  • Seu-Mei WangEmail author
  • Ran-Der Hwang
  • Andrew S. Greenberg
  • Hui-Ling Yeo
Original Paper

Abstract

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination of immunohistochemistry and Nile red staining/Oil red O. We demonstrated that, although most growing lipid droplets appeared to have a lipid core surrounded by a fluorescent rim of ADRP, perilipin, and p200, tiny protein aggregates of ADRP, perilipin, or p200 could also be found to occur in the absence of lipid accumulation. In addition, ADRP associated with nascent lipid droplets prior to that of perilipin or p200. We provide evidence that lipid droplet surface proteins, especially ADRP and perilipin, are important in serving as a nucleation center for the assembly of lipid to form nascent lipid droplets.

Keywords

ADRP Perilipin p200 Lipogenesis 

Notes

Acknowledgements

We thank Dr. Thomas Keenan for providing the rabbit anti-ADRP antibody, and Dr. Thomas Barkas for his critical reading and correction of this paper. The grant sponsor was the National Science Council, Republic of China, grant number NSC 91-2320-B-002-125

References

  1. Atshaves BP, Storey SM, McIntosh AL, Petrescu AD, Lyuksyutova OI, Greenberg AS, Schroeder F (2001) Sterol carrier protein-2 expression modulates protein and lipid composition of lipid droplets. J Biol Chem 276:25324–25335CrossRefPubMedGoogle Scholar
  2. Brasaemle DL, Barber T, Wolins N, Serrero DG, Blanchette-Mackie EJ, Londos C (1997) Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J Lipid Res 38:2249–2263PubMedGoogle Scholar
  3. Brasaemle DL, Rubin B, Harten IA, Gruia-Gray J, Kimmel AR, Londos C (2000) Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis. J Biol Chem 275:38486–38493CrossRefPubMedGoogle Scholar
  4. Clifford GM, Londos C, Kraemer FB, Vernon RG, Yeaman SJ (2000) Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocytes. J Biol Chem 275:5011–5015CrossRefPubMedGoogle Scholar
  5. Coleman R, Bell RM (1976) Triacylglycerol synthesis in isolated fat cells. J Biol Chem 251:4537–4543PubMedGoogle Scholar
  6. Fong TH, Wang SM (1997) Dissection of the signaling mechanism for capsule detachment of lipid droplets in rat adrenocortical cells. J Cell Biochem 65:67–74CrossRefPubMedGoogle Scholar
  7. Fong TH, Yang CC, Greenberg AS, Wang SM (2002) Immunocytochemical studies on lipid droplet-surface proteins in adrenal cells. J Cell Biochem 86:432–439CrossRefPubMedGoogle Scholar
  8. Fritz JD, Swart DR, Greaser ML (1980) Factors affecting polyacrylamide gel electrophoresis and electroblotting of high molecular-weight myofibrillar protein. Anal Biochem 180:205–210Google Scholar
  9. Frolov A, Petrescu A, Atshaves BP, So PTC, Gratton E, Serrero G, Schroeder F (2000) High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach. J Biol Chem 275:12769–12780PubMedGoogle Scholar
  10. Fukumoto S, Fujimoto T (2002) Deformation of lipid droplets in fixed samples. Histochem Cell Biol 118:423–428PubMedGoogle Scholar
  11. Gao J, Serrero G (1999) Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake. J Biol Chem 274:16825–16830CrossRefPubMedGoogle Scholar
  12. Gao J, Ye H, Serrero G (2000) Stimulation of adipose differentiation related protein (ADRP) expression in adipocyte precursors by long chain fatty acids. J Cell Physiol 182:297–302CrossRefPubMedGoogle Scholar
  13. Garcia A, Sekowski A, Subramanian V, Brasaemle (2003) The central domain is required to target and anchor perilipin A to lipid droplets. J Biol Chem 278:625–635CrossRefPubMedGoogle Scholar
  14. Greenberg AS, Egan JJ, Wek SA, Garty NB, Balanchette MEJ, Londos C (1991) Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J Biol Chem 266:11341–11346PubMedGoogle Scholar
  15. Heid HW, Schnolzer M, Keenan TW (1996) Adipocyte differentiation-related protein is secreted into milk as a constitute of milk lipid globule membrane. Biochem J 320:1025–1030PubMedGoogle Scholar
  16. Imamura M, Inoguchi T, Ikuyama S, Taniguchi S, Kobayashi K, Nakashima N, Nawata H (2002) ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts. Am J Endocrinol Metab 283:E775–E783Google Scholar
  17. Jepson CA, Harrison JA, Kraemer FB, Yeaman SJ (1996) Down-regulation of hormone-sensitive lipase in sterol ester-laden J774.2 macrophages. Biochem J 318:173–177PubMedGoogle Scholar
  18. Jiang HP, Serrero G (1992) Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein. Proc Natl Acad Sci U S A 89:7856–7860PubMedGoogle Scholar
  19. Londos C, Brasaemle DL, Gruia-Gray J, Servetnick DA, Schultz CJ, Levin DM, Kimmel AR (1995) Perilipins: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells. Biochem Soc Trans 23:611–615PubMedGoogle Scholar
  20. Londos C, Gruia-Gray J, Brasaemle DL, Rondinone CM, Takeda T, Dwyer NK, Barber T, Kimmel AR, Blanchette-Mackie EJ (1996) Perilipin: possible roles in structure and metabolism of intracellular neutral lipids in adipocytes and steroidogenic cells. Int J Obes Relat Metab Disord 3:S97–S101Google Scholar
  21. Londos C, Brasaemle DL, Schultz CJ, Segrest JP, Kimmel AR (1999) Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells. Semin Cell Dev Biol 10:51–58CrossRefPubMedGoogle Scholar
  22. MacDougald OA, Lane MD (1995) Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64:345–373PubMedGoogle Scholar
  23. Mrotek JJ, Mathew JK, Curtis JC (1981) A method for the isolation of lipid droplet fractions from decapsulated rat adrenals. Steroids 38:229–241CrossRefPubMedGoogle Scholar
  24. Murphy DJ (2001) The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res 40:325–438PubMedGoogle Scholar
  25. Murphy DJ, Vance J (1999) Mechanisms of lipid-body formation. Trends Biochem Sci 24:109–115CrossRefPubMedGoogle Scholar
  26. Reinhart MP, Billheimer JT, Faust JR, Gaylor JL (1987) Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver. J Biol Chem 262:9649–9655PubMedGoogle Scholar
  27. Serrero G, Frolov A, Schroeder F, Tanaka K, Gelhaar L (2000) Adipose differentiation related protein: expression, purification of recombinant protein in Escherichia coli and characterization of its fatty acid binding properties. Biochim Biophys Acta 1488:245–254CrossRefPubMedGoogle Scholar
  28. Servetnick SA, Brasaemle DL, Gruia-Gray J, Kimmel AR, Wolff J, Londos C (1995) Perilipins are associated with cholesterol ester droplets in steroidogenic adrenal cortical and Leydig cells. J Biol Chem 270:16970–16973CrossRefPubMedGoogle Scholar
  29. Souza SC, de Vargas LM, Yamamoto MT, Lien P, Franciosa MD, Moss LG, Greenberg AS (1998) Overexpression of perilipin A and B blocks the ability of tumor necrosis factor alpha to increase lipolysis in 3T3-L1 adipocytes. J Biol Chem 273:24665–24669CrossRefPubMedGoogle Scholar
  30. Tansey JT, Sztalryd C, Gruia-Gray J, Roush DL, Zee JV, Gavrilova O, Reitman ML, Deng C-X, Li C, Kimmel AR, Londos C (2001) Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity. Proc Natl Acad Sci U S A 98:6494–6499CrossRefPubMedGoogle Scholar
  31. Targett-Adams P, Chambers D, Gledhill S, Hope RG, Coy JF, Girod A, MaLauchlan J (2003) Live cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-related protein. J Biol Biochem 278:15998–16007CrossRefGoogle Scholar
  32. Tontonoz P, Hu E, Spiegelman BM (1994) Stimulation of adipogenesis in fibroblasts by PPARγ2, a ligand-activated transcription factor. Cell 79:1147–1156PubMedGoogle Scholar
  33. Wang SM, Fong TH (1995) A lipid droplet-specific capsule is present in rat adrenal cells: evidence from a monoclonal antibody. Biochem Biophys Res Commun 217:81–88CrossRefPubMedGoogle Scholar
  34. Wang SM, Fong TH, Hsu SY, Chien CL, Wu JC (1997) Reorganization of a novel vimentin-associated protein in 3T3-L1 cells during adipose conversion. J Cell Biochem 67:84–91CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Seu-Mei Wang
    • 1
    Email author
  • Ran-Der Hwang
    • 1
  • Andrew S. Greenberg
    • 2
  • Hui-Ling Yeo
    • 1
  1. 1.Department of Anatomy and Cell Biology, College of MedicineNational Taiwan UniversityTaipeiTaiwan
  2. 2.United States Department of AgricultureHuman Nutrition Research Center on Aging at Tufts UniversityBostonUSA

Personalised recommendations