Cell Biochemistry and Biophysics

, Volume 67, Issue 3, pp 1157–1169 | Cite as

Non-Esterified Fatty Acids Activate the AMP-Activated Protein Kinase Signaling Pathway to Regulate Lipid Metabolism in Bovine Hepatocytes

  • Xinwei Li
  • Xiaobing Li
  • Hui Chen
  • Liancheng Lei
  • Juxiong Liu
  • Yuan Guan
  • Zhaoxi Liu
  • Liang Zhang
  • Wentao Yang
  • Chenxu Zhao
  • Shixin Fu
  • Peng Li
  • Guowen Liu
  • Zhe Wang
Original Paper


Non-esterified fatty acids (NEFAs) act as signaling molecules involved in regulating genes expression to modulate lipid metabolism. However, the regulation mechanism of NEFAs on lipid metabolism in dairy cows is unclear. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of NEFAs and AMPKα inhibitors (BML-275). NEFAs increased AMPKα phosphorylation through up-regulating the protein levels of liver kinase B1. Activated AMPKα increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α (PPARα). NEFAs also directly activate the PPARα independent of AMPKα. Activated PPARα increased the lipolytic genes expression to increase lipid oxidation. Furthermore, activated AMPKα inhibited the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid synthesis. Activated AMPKα phosphorylated and inhibited acetyl-CoA carboxylase and increased carnitine palmitoyltransferase-1 activity, which increased lipid oxidation. Consequently, the triglyceride content in the NEFAs-treated hepatocytes was significantly decreased. These results indicate that NEFAs activate the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in hepatocytes, which in turn, generates more ATP to relieve the negative energy balance in transition dairy cows.


Non-esterified fatty acids AMP-activated protein kinase signaling pathway Lipid oxidation Lipid synthesis 



Negative energy balance


Non-esterified fatty acids


AMP-activated protein kinase


Liver kinase B1


Peroxisome proliferator-activated receptor α


Sterol regulatory element-binding protein 1c


Carbohydrate responsive element-binding protein




Acyl-CoA oxidase


Carnitine palmitoyltransferase 1


Carnitine palmitoyltransferase 2


Liver fatty acid-binding protein


Acyl-CoA synthetase long-chain


Acetyl-CoA carboxylase 1




Fatty acid synthase


Stearoyl-CoA desaturase-1




Electrophoretic mobility shift assay


Polyunsaturated fatty acids



This study was supported by the National Key Basic Research Program of China (No. 2011CB100800).


  1. 1.
    Dann, H. M., & Drackley, J. K. (2006). Carnitine palmitoyltransferase I in liver of periparturient dairy cows: effects of prepartum intake, postpartum induction of ketosis, and periparturient disorders. Journal of Dairy Science, 88, 3851–3859.CrossRefGoogle Scholar
  2. 2.
    Loor, J. J., Everts, R. E., Bionaz, M. H., Dann, M., Morin, D. E., Oliveira, R., et al. (2007). Nutrition-induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy cows. Physiological Genomics, 32, 105–116.PubMedCrossRefGoogle Scholar
  3. 3.
    Wathes, D. C., Cheng, Z., Fenwick, M. A., Fitzpatrick, R., & Patton, J. (2011). Influence of energy balance on the somatotrophic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow. Reproduction, 141, 269–281.PubMedCrossRefGoogle Scholar
  4. 4.
    Xu, C., Wang, Z., Liu, G. W., Li, X. B., Xie, G. H., Xia, C., et al. (2008). Metabolic characteristic of the liver of dairy cows during ketosis based on comparative proteomics. Asian-Australian Journal of Animal Sciences, 21, 1003–1010.Google Scholar
  5. 5.
    Jorritsma, R., Jorritsma, H., Schukken, Y. H., Bartlettd, P. C., Wensinga, T., & Wentink, G. H. (2001). Prevalence and indicators of post partum fatty infiltration of the liver in nine commercial dairy herds in The Netherlands. Livestock Production Science, 68, 53–60.CrossRefGoogle Scholar
  6. 6.
    Zhang, Z., Li, X., Liu, G., Gao, L., Guo, C., Kong, T., et al. (2011). High insulin concentrations repress insulin receptor gene expression in calf hepatocytes cultured in vitro. Cellular Physiology and Biochemistry, 27, 637–640.PubMedCrossRefGoogle Scholar
  7. 7.
    Duplus, E., Glorian, M., & Forest, C. (2000). Fatty acid regulation of gene transcription. Journal of Biological Chemistry, 275, 30749–30752.PubMedCrossRefGoogle Scholar
  8. 8.
    Jump, D. B., Botolin, D., Wang, Y., Xu, J., Christian, B., & Demeure, O. (2005). Fatty acid regulation of hepatic gene transcription. Journal of Nutrition, 135, 2503–2506.PubMedGoogle Scholar
  9. 9.
    Kahn, B. B., Alquier, T., Carling, D., & Hardie, D. G. (2005). AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabolism, 1, 15–25.PubMedCrossRefGoogle Scholar
  10. 10.
    Hardie, D. G. (2003). The AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology, 144, 5179–5183.PubMedCrossRefGoogle Scholar
  11. 11.
    Viollet, B., Foretz, M., Guigas, B., Horman, S., Dentin, R., Bertrand, L., et al. (2006). Activation of AMP-activated protein kinase in the liver: A new strategy for the management of metabolic hepatic disorders. Journal of Physiology, 574(Pt 1), 41–53.PubMedCrossRefGoogle Scholar
  12. 12.
    Long, Y. C., & Zierath, J. R. (2006). AMP-activated protein kinase signaling in metabolic regulation. The Journal of Clinical Investigation, 116, 1776–1783.PubMedCrossRefGoogle Scholar
  13. 13.
    Martínez, N., White, V., Kurtz, M., Higa, R., Capobianco, E., & Jawerbaum, A. (2011). Activation of the nuclear receptor PPARα regulates lipid metabolism in foetal liver from diabetic rats: Implications in diabetes-induced foetal overgrowth. Diabetes/Metabolism Research and Reviews, 27, 35–46.PubMedCrossRefGoogle Scholar
  14. 14.
    Uyeda, K., & Repa, J. J. (2006). Carbohydrate response element binding protein, ChREBP, a transcription factor coupling hepatic glucose utilization and lipid synthesis. Cell Metabolism, 4, 107–110.PubMedCrossRefGoogle Scholar
  15. 15.
    Kawaguchi, T., Osatomi, K., Yamashita, H., Kabashima, T., & Uyeda, K. (2002). Mechanism for fatty acid “sparing” effect on glucose-induced transcription: Regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase. Journal of Biological Chemistry, 277, 3829–3835.PubMedCrossRefGoogle Scholar
  16. 16.
    Li, Y., Xu, S., Mihaylova, M. M., Zheng, B., Hou, X., Jiang, B., et al. (2011). AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice. Cell Metabolism, 13, 376–388.PubMedCrossRefGoogle Scholar
  17. 17.
    Shaw, R. J., Lamia, K. A., Vasquez, D., Koo, S. H., Bardeesy, N., Depinho, R. A., et al. (2005). The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science, 310, 1642–1646.PubMedCrossRefGoogle Scholar
  18. 18.
    Xu, C., Wang, Z., Zhang, R. H., Zhang, H. Y., Fu, S. X., & Xia, C. (2011). Effect of NEFA and glucose levels on CPT-I mRNA expression and translation in cultured bovine hepatocytes. Journal of Veterinary Medical Science, 73, 97–101.PubMedCrossRefGoogle Scholar
  19. 19.
    Zhang, Z. G., Li, X. B., Gao, L., Liu, G. W., Kong, T., Li, Y. F., et al. (2012). An updated method for the isolation and culture of primary calf hepatocytes. Veterinary Journal, 191, 323–326.CrossRefGoogle Scholar
  20. 20.
    Li, X., Li, X., Bai, G., Chen, H., Deng, Q., Liu, Z., et al. (2012). Effects of non-esterified fatty acids on the gluconeogenesis in bovine hepatocytes. Molecular and Cellular Biochemistry, 359, 385–388.PubMedCrossRefGoogle Scholar
  21. 21.
    Ramnanan, C. J., McMullen, D. C., Groom, A. G., & Storey, K. B. (2010). The regulation of AMPK signaling in a natural state of profound metabolic rate depression. Molecular and Cellular Biochemistry, 335, 91–105.PubMedCrossRefGoogle Scholar
  22. 22.
    Yu, P. B., Hong, C. C., Sachidanandan, C., Babitt, J. L., Deng, D. Y., Hoyng, S. A., et al. (2008). Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nature Chemical Biology, 4, 33–41.PubMedCrossRefGoogle Scholar
  23. 23.
    Cameron, R. E., Dyk, P. B., Herdt, T. H., Kaneene, J. B., Miller, R., Bucholtz, H. F., et al. (1998). Dry cow diet, management, and energy balance as risk factors for displaced abomasums in high producing dairy herds. Journal of Dairy Science, 81, 132–139.PubMedCrossRefGoogle Scholar
  24. 24.
    Li, P., Li, X. B., Fu, S. X., Wu, C. C., Wang, X. X., Yu, G. J., et al. (2012). Alterations of fatty acid β-oxidation capability in the liver of ketotic cows. Journal of Dairy Science, 95, 1759–1766.PubMedCrossRefGoogle Scholar
  25. 25.
    Andreelli, F., Foretz, M., Knauf, C., Cani, P. D., Perrin, C., Iglesias, M. A., et al. (2006). Liver adenosine monophosphate-activated kinase-alpha 2 catalytic subunit is a key target for the control of hepatic glucose production by adiponectin and leptin but not insulin. Endocrinology, 147, 2432–2441.PubMedCrossRefGoogle Scholar
  26. 26.
    Foretz, M., Ancellin, N., Andreelli, F., Saintillan, Y., Grondin, P., Kahn, A., et al. (2005). Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver. Diabetes, 54, 1331–1339.PubMedCrossRefGoogle Scholar
  27. 27.
    Suchankova, G., Tekle, M., Saha, A., Ruderman, N. B., Clarke, S. D., & Gettys, T. W. (2005). Dietary polyunsaturated fatty acids enhance hepatic AMP-activated protein kinase activity in rats. Biochemical and Biophysical Research Communications, 326, 851–858.PubMedCrossRefGoogle Scholar
  28. 28.
    Clark, H., Carling, D., & Saggerson, D. (2004). Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids. European Journal of Biochemistry, 271, 2215–2224.PubMedCrossRefGoogle Scholar
  29. 29.
    Fediuc, S., Gaidhu, M. P., & Ceddia, R. B. (2006). Regulation of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation by palmitate in skeletal muscle cells. Journal of Lipid Research, 47, 412–420.PubMedCrossRefGoogle Scholar
  30. 30.
    Watt, M. J., Steinberg, G. R., Chen, Z. P., Kemp, B. E., & Febbraio, M. A. (2006). Fatty acids stimulate AMP activated protein kinase and enhance fatty acid oxidation in L6 myotubes. The Journal of Physiology, 574, 139–147.PubMedCrossRefGoogle Scholar
  31. 31.
    Zhou, G., Myers, R., Li, Y., Chen, Y., Shen, X., Fenyk-Melody, J., et al. (2001). Role of AMP-activated protein kinase in mechanism of metformin action. The Journal of Clinical Investigation, 108, 1167–1174.PubMedGoogle Scholar
  32. 32.
    Qiu, L., Wu, X., Chau, J. F., Szeto, I. Y., Tam, W. Y., Guo, Z., et al. (2008). Aldose reductase regulates hepatic peroxisome proliferator-activated receptor alpha phosphorylation and activity to impact lipid homeostasis. Journal of Biological Chemistry, 283, 17175–17183.PubMedCrossRefGoogle Scholar
  33. 33.
    Sugden, M. C., Bulmer, K., Gibbons, G. F., Knight, B. L., & Holness, M. J. (2002). Peroxisome proliferator-activated receptor-a (PPARa) deficiency leads to dysregulation of hepatic lipid and carbohydrate metabolism by fatty acids and insulin. Biochemical Journal, 364, 361–368.PubMedCrossRefGoogle Scholar
  34. 34.
    Everett, L., Galli, A., & Crabb, D. (2000). The role of hepatic peroxisome proliferator-activated receptors (PPARs) in health and disease. Liver, 20, 191–199.PubMedCrossRefGoogle Scholar
  35. 35.
    Pawar, A., & Jump, D. B. (2003). Unsaturated fatty acid regulation of PPARα activity in primary rat hepatocytes. Journal of Biological Chemistry, 278, 35931–35939.PubMedCrossRefGoogle Scholar
  36. 36.
    Steven, D. C. (2001). Polyunsaturated fatty acid regulation of gene transcription: A molecular mechanism to improve. American Society for Nutritional Sciences, 60, 1129–1132.Google Scholar
  37. 37.
    Shimomura, I., Bashmakov, Y., & Horton, J. D. (1999). Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. Journal of Biological Chemistry, 274, 30028–30032.PubMedCrossRefGoogle Scholar
  38. 38.
    Iizuka, K., Bruick, R. K., Liang, G., Horton, J. D., & Uyeda, K. (2001). Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis. Proceedings of the National Academy of Sciences of the United States of America, 101, 7281–7286.CrossRefGoogle Scholar
  39. 39.
    Dobrzyn, A., Dobrzyn, P., Miyazaki, M., & Ntambi, J. M. (2005). Polyunsaturated fatty acids do not activate AMP-activated protein kinase in mouse tissues. Biochemical and Biophysical Research Communications, 332, 892–896.PubMedCrossRefGoogle Scholar
  40. 40.
    Dentin, R., Benhamed, F., Pégorier, J. P., Foufelle, F., Viollet, B., Vaulont, S., et al. (2005). Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation. The Journal of Clinical Investigation, 115, 2843–2854.PubMedCrossRefGoogle Scholar
  41. 41.
    Hardie, D. G. (2004). The AMP-activated protein kinase pathway-new players upstream and downstream. Journal of Cell Science, 117, 5479–5487.PubMedCrossRefGoogle Scholar
  42. 42.
    Ruderman, N. B., Saha, A. K., Vavvas, D., & Witters, L. A. (1999). Malonyl-CoA fuel sensing, and insulin resistance. American Journal of Physiology, Endocrinology and Metabolism, 276, 1–18.Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Xinwei Li
    • 1
  • Xiaobing Li
    • 1
  • Hui Chen
    • 1
  • Liancheng Lei
    • 1
  • Juxiong Liu
    • 1
  • Yuan Guan
    • 1
  • Zhaoxi Liu
    • 1
  • Liang Zhang
    • 1
  • Wentao Yang
    • 1
  • Chenxu Zhao
    • 1
  • Shixin Fu
    • 2
  • Peng Li
    • 3
  • Guowen Liu
    • 1
  • Zhe Wang
    • 1
  1. 1.Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary MedicineJilin UniversityChangchunChina
  2. 2.Institute of Animal Science and TechnologyHeilongjiang Bayi Agriculture UniversityDaqingChina
  3. 3.College of Animal Science and Veterinary MedicineShenyang Agricultural UniversityShenyangChina

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