Triglyceride accumulation protects against fatty acid-induced lipotoxicity

Laura Listenberger

Xianlin Han

Sarah Lewis

Sylvaine Cases

Robert Farese

Daniel Ory

Jean Schaffer

^{Center for Cardiovascular Research and}^{Division of Bioorganic Chemistry, Departments of}^{Internal Medicine, of Molecular Biology and Pharmacology, and of}^{Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110-1010; and}^{Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, CA 94141-9100}

^{To whom correspondence should be addressed at: Center for Cardiovascular Research, Washington University School of Medicine, 660 South Euclid Avenue, Box 8086, St. Louis, MO 63110-1010. E-mail:}ude.ltsuw.mi@ffahcsj.

Communicated by David M. Kipnis, Washington University School of Medicine, St. Louis, MO

Received 2002 Dec 20; Accepted 2003 Jan 30.

Abstract

Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic β-cell apoptosis and heart failure. Here, we demonstrate in cultured cells that the relative toxicity of two common dietary long chain fatty acids is related to channeling of these lipids to distinct cellular metabolic fates. Oleic acid supplementation leads to triglyceride accumulation and is well tolerated, whereas excess palmitic acid is poorly incorporated into triglyceride and causes apoptosis. Unsaturated fatty acids rescue palmitate-induced apoptosis by channeling palmitate into triglyceride pools and away from pathways leading to apoptosis. Moreover, in the setting of impaired triglyceride synthesis, oleate induces lipotoxicity. Our findings support a model of cellular lipid metabolism in which unsaturated fatty acids serve a protective function against lipotoxicity though promotion of triglyceride accumulation.

Abstract

The striking prevalence of obesity world-wide is a significant health problem due to serious medical complications that include hypertension, insulin resistance, diabetes, coronary artery disease, and heart failure (1). Evidence is emerging that elevated serum free fatty acid (FA) levels contribute to the pathogenesis of the metabolic syndrome and heart disease. Whereas adipocytes have a unique capacity to store excess FAs in the form of triglyceride in lipid droplets, non-adipose tissues such as cardiac myocytes and pancreatic β-cells have a limited capacity for storage of lipids. In hyperlipidemic states, accumulation of excess lipid in non-adipose tissues leads to cell dysfunction and/or cell death, a phenomenon known as lipotoxicity. Excess lipid accumulation in skeletal muscle is associated with the development of insulin resistance (2). Lipid overload in pancreatic β-cells leads to dysregulated insulin secretion (3, 4) and apoptotic cell death (5), both of which may contribute to the genesis of diabetic states. Lipoapoptosis is also observed in the heart, in which it leads to the development of heart failure (6, 7).

In a variety of experimental systems, saturated and unsaturated FAs differ significantly in their contributions to lipotoxicity. Previous studies in Chinese hamster ovary (CHO) cells (8), cardiac myocytes (9), pancreatic β-cells (10, 11), breast cancer cell lines (12), and hematopoietic precursor cell lines (13) all suggest that lipotoxicity from accumulation of long chain FAs is specific for saturated FAs. This selectivity has been attributed to the generation of specific proapoptotic lipid species or signaling molecules in response to saturated but not unsaturated FAs. The nature of such signals may differ across cell types, but includes reactive oxygen species generation (8), de novo ceramide synthesis (14), nitric oxide generation (15), decreases in phosphatidylinositol-3-kinase (12), and primary effects on mitochondrial structure or function (16). Long chain FAs may also suppress anti-apoptotic factors such as BclII (17). Cosupplementation with unsaturated FAs (9, 10, 12) has been shown to rescue saturated FA-induced lipoapoptosis by an unknown mechanism.

The present study was designed to characterize the fundamental cellular mechanisms though which the common saturated and unsaturated dietary lipids, palmitic and oleic acids, exert their differential effects on survival of cultured cells. We provide evidence that the differential toxicity of these FAs is directly related to their ability to promote triglyceride accumulation. We show that exogenous or endogenously generated unsaturated FAs rescue palmitate-induced apoptosis by promoting palmitate incorporation into triglyceride. Moreover, oleic acid, as well as palmitic acid, is toxic in cells with impaired triglyceride synthetic capacity. In vivo, triglyceride accumulation in non-adipose tissues occurs in the setting of mismatch between cellular lipid influx and lipid utilization. Our study suggests that triglyceride accumulation in non-adipose cells in response to acute lipid overload represents an initial cellular defense against lipotoxicity.

CHO, 25RA, and SCD-overexpressing CHO cells (SCD) were incubated in media supplemented where indicated with 500 μM palmitate (palm) or 500 μM palmitate plus 200 μM oleate (palm/oleate) for 6 h. The amount of palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18:1), and linoleate (18:2) present in triglyceride pool is reported and represents the mean of three independent experiments ± SE.

CHO and SCD-overexpressing cells (SCD) were incubated in media supplemented with 500 μM deuterated palmitate for 6 h. Incorporation of deuterated palmitate (d16:0), deuterated palmitoleate (d16:1), deuterated stearate (d18:0), and deuterated oleate (d18:1) in the triglyceride fraction is reported and represents the mean of three independent experiments ± SE.

Acknowledgments

We thank R. O. L. Wong for providing access to the confocal microscopy resources in the Bakewell NeuroImaging Laboratory, and the Washington University Mass Spectrometer Facility Center for allowing use of the electrospray ionization mass spectrometer. This work was supported by grants from the American Heart Association (EIA 0040040N), the National Institutes of Health (T32 HL07275, P01HL57278), and the Washington University Pharmacia Biomedical Research Program.

Acknowledgments

Abbreviations

CHO	Chinese hamster ovary
SCD	stearoyl-CoA desaturase
DGAT1	acyl CoA:diacylglycerol transferase 1
FA	fatty acid
PI	propidium iodide

Abbreviations

References

1. Kopelman P G. Nature. 2000;404:635–643.[PubMed]
2. Shulman G I. J Clin Invest. 2000;106:171–176.
3. Zhou Y P, Grill V. J Clin Endocrinol Metab. 1995;80:1584–1590.[PubMed]
4. Prentki M, Vischer S, Glennon M C, Regazzi R, Deeney J T, Corkey B E. J Biol Chem. 1992;267:5802–5810.[PubMed]
5. Shimabukuro M, Zhou Y T, Levi M, Unger R H. Proc Natl Acad Sci USA. 1998;95:2498–2502.
6. Chiu H C, Kovacs A, Ford D A, Hsu F F, Garcia R, Herrero P, Saffitz J E, Schaffer J E. J Clin Invest. 2000;107:813–822.
7. Zhou Y-T, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens D, Orci L, Unger R H. Proc Natl Acad Sci USA. 2000;97:1784–1789.
8. Listenberger L L, Ory D S, Schaffer J E. J Biol Chem. 2001;276:14890–14895.[PubMed]
9. deVries J E, Vork M M, Roemen T H M, deJong Y F, Cleutjens J P M, van der Vusse G J, van Bilsen M. J Lipid Res. 1997;38:1384–1394.[PubMed]
10. Maedler K, Spinas G A, Dyntar D, Moritz W, Kaiser N, Donath M Y. Diabetes. 2001;50:69–76.[PubMed]
11. Cnop M, Hannaert J C, Hoorens A, Eizirik D L, Pipeleers D G. Diabetes. 2001;50:1771–1777.[PubMed]
12. Hardy S, Langelier Y, Prentki M. Cancer Res. 2000;60:6353–6358.[PubMed]
13. Paumen M B, Ishida Y, Muramatsu M, Yamamoto M, Honjo T. J Biol Chem. 1997;272:3324–3329.[PubMed]
14. Shimabukuro M, Higa M, Zhou Y T, Wang M Y, Newgard C B, Unger R H. J Biol Chem. 1998;273:32487–32490.[PubMed]
15. Shimabukuro M, Ohneda M, Lee Y, Unger R H. J Clin Invest. 1997;100:290–295.
16. Ostrander D B, Sparagna G C, Amoscato A A, McMillin J B, Dowhan W. J Biol Chem. 2001;276:38061–38067.[PubMed]
17. Shimabukuro M, Wang M Y, Zhou Y T, Newgard C B, Unger R H. Proc Natl Acad Sci USA. 1998;95:9558–9561.
18. Smith S J, Cases S, Jensen D R, Chen H C, Sande E, Tow B, Sanan D A, Raber J, Eckel R H, Farese Jr R V. Nat Genet. 2000;25:87–90.[PubMed]
19. Millard E E, Srivastava K, Traub L M, Schaffer J E, Ory D S. J Biol Chem. 2000;275:38445–38451.[PubMed]
20. Bligh E G, Dyer W J. Can J Biochem Physiol. 1959;37:911–917.[PubMed]
21. Han X, Gross R W. Anal Biochem. 2001;295:88–100.[PubMed]
22. Han X. Anal Biochem. 2002;302:199–212.[PubMed]
23. Ory D S, Neugeborn B A, Mulligan R C. Proc Natl Acad Sci USA. 1996;93:11400–11406.
24. Obukowics M G, Welsch D J, Salsgiver W J, Martin-Berger C L, Chinn K S, Duffin K L, Raz A, Needleman P. J Pharmacol Exp Ther. 1998;287:157–166.[PubMed]
25. Kurien V A, Oliver M F. Lancet. 1966;2:122–127.[PubMed]
26. Kleinfeld A M, Protho D, Brown D L, Davis R C, Richieri G V, DeMaria A. Am J Cardiol. 1996;78:1350–1354.[PubMed]
27. Miyazaki M, Kim Y C, Gray-Keller M P, Attie A D, Ntambi J M. J Biol Chem. 2000;275:30132–30138.[PubMed]
28. Miyazaki M, Kim Y C, Ntambi J M. J Lipid Res. 2001;42:1018–1024.[PubMed]
29. Hua X, Nohturfft A, Goldstein J L, Brown M S. Cell. 1996;87:415–426.[PubMed]
30. Shimano H, Horton J D, Hammer R E, Shimomura I, Brown M S, Goldstein J L. J Clin Invest. 1996;98:1575–1584.
31. Enoch H G, Catala A, Strittmatter P. J Biol Chem. 1976;251:5095–5103.[PubMed]
32. Chen H C, Smith S J, Ladha Z, Jensen D R, Ferreira L D, Pulawa L K, McGuire J G, Pitas R E, Eckel R H, Farese R V. J Clin Invest. 2002;109:1049–1055.
33. Rosenthal M D. Lipids. 1981;16:173–182.[PubMed]
34. Cohen P, Miyazaki M, Socci N D, Hagge-Greenberg A, Liedtke W, Soukas A A, Sharma R, Hudgins L C, Ntambi J M, Friedman J M. Science. 2002;297:240–243.[PubMed]
35. Kliewer S A, Sundseth S S, Jones S A, Brown P J, Wisely G B, Koble C S, Devchand P, Wahli W, Wilson T M, Lenhard J M, Lehmann J M. Proc Natl Acad Sci USA. 1997;94:4318–4323.
36. Bell R M, Coleman R A. Annu Rev Biochem. 1980;1980:459–487.[PubMed]