pp 1–14 | Cite as

Liver-derived FGF21 is essential for full adaptation to ketogenic diet but does not regulate glucose homeostasis

  • Mikiko Watanabe
  • Garima Singhal
  • Ffolliott M. Fisher
  • Thomas C. Beck
  • Donald A. Morgan
  • Fabio Socciarelli
  • Marie L. Mather
  • Renata Risi
  • Jared Bourke
  • Kamal Rahmouni
  • Owen P. McGuinness
  • Jeffrey S. Flier
  • Eleftheria Maratos-FlierEmail author
Original Article



Fibroblast growth factor 21 (FGF21) is expressed in several metabolically active tissues, including liver, fat, and acinar pancreas, and has pleiotropic effects on metabolic homeostasis. The dominant source of FGF21 in the circulation is the liver.

Objective and methods

To analyze the physiological functions of hepatic FGF21, we generated a hepatocyte-specific knockout model (LKO) by mating albumin-Cre mice with FGF21 flox/flox (fl/fl) mice and challenged it with different nutritional models.


Mice fed a ketogenic diet typically show increased energy expenditure; this effect was attenuated in LKO mice. LKO on KD also developed hepatic pathology and altered hepatic lipid homeostasis. When evaluated using hyperinsulinemic-euglycemic clamps, glucose infusion rates, hepatic glucose production, and glucose uptake were similar between fl/fl and LKO DIO mice.


We conclude that liver-derived FGF21 is important for complete adaptation to ketosis but has a more limited role in the regulation of glycemic homeostasis.


Fibroblast Growth Factor 21 Ketogenic diet Nonalcoholic fatty liver disease Cholesterol Energy metabolism Adipose tissue 



Fibroblast growth factor 21




FGF21 liver-specific knockout


Ketogenic diet


High fat diet


Peroxisome proliferator-activated receptor α


Fibroblast growth factor 21 knockout


Wild type






Glucose metabolic Index


Standard error of the mean


Comprehensive lab animal monitoring system oxymax


Quantitative nuclear magnetic resonance


Alanine aminotransferase


PhosphoGlycerate Kinase


Nonalcoholic fatty liver disease


Brown adipose tissue


Uncoupling protein 1


Sympathetic nerve activity


Analysis of variance




Epididymal white adipose tissue


Inguinal white adipose tissue


Intraperitoneal glucose tolerance test


Insulin tolerance test


Respiratory exchange ratio


Methionine choline deficient


Sympathetic nervous system


Diet induced obesity


Fatty acids synthase


Stearoyl-CoA desaturase


Cluster of differentiation 36


Long chain acyl-CoA dehydrogenase


Very long chain acyl-CoA dehydrogenase


Peroxisomal acyl-coenzyme A oxidase 1


Carnitine palmitoyltransferase I α


Peroxisome proliferator-activated receptor

PGC1 α

Peroxisome proliferator-activated receptor (PPAR) γ Coactivator 1α

PGC1 β

Peroxisome proliferator-activated receptor (PPAR) γ Coactivator 1β


ATP-binding cassette sub-family G member 5


ATP-binding cassette sub-family G member 8


Liver X receptor


Cytochrome P450 7A1,


Small heterodimer partner


3-hydroxy-3-methylglutaryl-CoA synthase


3-hydroxy-3-methylglutaryl-CoA reductase


Sterol regulatory element-binding protein 2


Proprotein convertase subtilisin/kexin type 9


LDL receptor

TGF1 β

Transforming growth factor 1 β


Monocyte chemoattractant protein-1


Matrix metalloproteinase-2


Smooth muscle actin

IL1 β

Interleukin 1 β


Metallopeptidase inhibitor 1



We thank Dr Pavlos Pissios for scientific discussions and Dr Thomas Webb for technical assistance.

Author contributions

G.S., F.M.F., T.C.B., O.P.M., J.S.F. and E.M.F. conceived and designed the experiments. M.W., G.S., J.B., M.M., T.C.B., D.A.M. and R.R. and Vanderbilt MMPC performed the experiments. M.W., G.S., F.M.F., T.C.B., F.S., O.P.M. analyzed the data. E.M.F., J.S.F., F.M.F., K.R. and O.P.M. contributed with reagents/materials/analysis tools/critical revisions. M.W., G.S. and E.M.F. drafted the manuscript.


This work was supported by NIH Grant DK028082 (to E.M.F. and J.S.F.). M.W. was supported by funds from The Rotary Club of Rome. We are grateful for the help from the HDDC Core supported by NIH Grant NIDDK P30 DK034854. Generation of genetically altered floxed mice was made under the auspices of BADERC 5P30DK057521 and the BNORC 5P30DK046200. K.R was supported by funds from the NIH (HL084207), the American Heart Association (14EIA18860041) and the University of Iowa Fraternal Order of Eagles Diabetes Research Center. O.P.M. was supported by funds from the NIH (DK059637). The Vanderbilt Mouse Metabolic Phenotyping Center (DK059637) and the Hormone Assay and Analytical Services Core (DK059637 and DK020593) provided surgical and analytical support for clamp studies.

Supplementary material

12020_2019_2124_MOESM1_ESM.eps (156 kb)
Supplementary Information
12020_2019_2124_MOESM2_ESM.eps (204 kb)
Supplementary Information
12020_2019_2124_MOESM3_ESM.eps (305 kb)
Supplementary Information
12020_2019_2124_MOESM4_ESM.docx (14 kb)
Supplementary Information


  1. 1.
    T. Inagaki, M. Choi, A. Moschetta, L. Peng, C.L. Cummins, J.G. McDonald et al. Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis. Cell Metab. 2, 217–225 (2005)PubMedCrossRefGoogle Scholar
  2. 2.
    T. Shimada, S. Mizutani, T. Muto, T. Yoneya, R. Hino, S. Takeda et al. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc. Natl Acad. Sci. USA 98, 6500–6505 (2001)PubMedCrossRefGoogle Scholar
  3. 3.
    T. Shimada, Y. Yamazaki, M. Takahashi, H. Hasegawa, I. Urakawa, T. Oshima et al. Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism. Am. J. Physiol. Ren. Physiol. 289, F1088–F1095 (2005)CrossRefGoogle Scholar
  4. 4.
    A. Kharitonenkov, T.L. Shiyanova, A. Koester, A.M. Ford, R. Micanovic, E.J. Galbreath et al. FGF-21 as a novel metabolic regulator. J. Clin. Investig. 115, 1627–1635 (2005)PubMedCrossRefGoogle Scholar
  5. 5.
    T. Coskun, H.A. Bina, M.A. Schneider, J.D. Dunbar, C.C. Hu, Y. Chen et al. Fibroblast growth factor 21 corrects obesity in mice. Endocrinology 149, 6018–6027 (2008)PubMedCrossRefGoogle Scholar
  6. 6.
    G. Gaich, J.Y. Chien, H. Fu, L.C. Glass, M.A. Deeg, W.L. Holland et al. The effects of LY2405319, an FGF21 analog, in obese human subjects with type 2 diabetes. Cell Metab. 18, 333–340 (2013)PubMedCrossRefGoogle Scholar
  7. 7.
    S. Talukdar, Y. Zhou, D. Li, M. Rossulek, J. Dong, V. Somayaji et al. A long-acting FGF21 molecule, PF-05231023, decreases body weight and improves lipid profile in non-human primates and type 2 diabetic subjects. Cell Metab. 23, 427–440 (2016)PubMedCrossRefGoogle Scholar
  8. 8.
    J. Xu, S. Stanislaus, N. Chinookoswong, Y.Y. Lau, T. Hager, J. Patel et al. Acute glucose-lowering and insulin-sensitizing action of FGF21 in insulin-resistant mouse models-association with liver and adipose tissue effects. Am. J. Physiol. Endocrinol. Metab. 297, E1105–E1114 (2009)PubMedCrossRefGoogle Scholar
  9. 9.
    M.K. Badman, P. Pissios, A.R. Kennedy, G. Koukos, J.S. Flier, E. Maratos-Flier, Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab. 5, 426–437 (2007)PubMedCrossRefGoogle Scholar
  10. 10.
    T. Inagaki, P. Dutchak, G. Zhao, X. Ding, L. Gautron, V. Parameswara et al. Endocrine regulation of the fasting response by PPARalpha-mediated induction of fibroblast growth factor 21. Cell Metab. 5, 415–425 (2007)PubMedCrossRefGoogle Scholar
  11. 11.
    K.J.P. Schwenger, S.E. Fischer, T.D. Jackson, A. Okrainec, J.P. Allard, Non-alcoholic fatty liver disease in morbidly obese individuals undergoing bariatric surgery: prevalence and effect of the pre-bariatric very low calorie diet. Obes. Surg. 28, 1109–1116 (2018)PubMedCrossRefGoogle Scholar
  12. 12.
    F.M. Fisher, P.C. Chui, I.A. Nasser, Y. Popov, J.C. Cunniff, T. Lundasen et al. Fibroblast growth factor 21 limits lipotoxicity by promoting hepatic fatty acid activation in mice on methionine and choline-deficient diets. Gastroenterology 147(1073–1083), e1076 (2014)Google Scholar
  13. 13.
    M.J. Potthoff, T. Inagaki, S. Satapati, X. Ding, T. He, R. Goetz et al. FGF21 induces PGC-1alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response. Proc. Natl Acad. Sci. USA 106, 10853–10858 (2009)PubMedCrossRefGoogle Scholar
  14. 14.
    B.N. Desai, G. Singhal, M. Watanabe, D. Stevanovic, T. Lundasen, F.M. Fisher et al. Fibroblast growth factor 21 (FGF21) is robustly induced by ethanol and has a protective role in ethanol associated liver injury. Mol. Metab. 6, 1395–1406 (2017)PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    J.R. Dushay, E. Toschi, E.K. Mitten, F.M. Fisher, M.A. Herman, E. Maratos-Flier, Fructose ingestion acutely stimulates circulating FGF21 levels in humans. Mol. Metab. 4, 51–57 (2015)PubMedCrossRefGoogle Scholar
  16. 16.
    F.M. Fisher, M. Kim, L. Doridot, J.C. Cunniff, T.S. Parker, D.M. Levine et al. A critical role for ChREBP-mediated FGF21 secretion in hepatic fructose metabolism. Mol. Metab. 6, 14–21 (2017)PubMedCrossRefGoogle Scholar
  17. 17.
    M.K. Badman, A. Koester, J.S. Flier, A. Kharitonenkov, E. Maratos-Flier, Fibroblast growth factor 21-deficient mice demonstrate impaired adaptation to ketosis. Endocrinology 150, 4931–4940 (2009)PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    G. Singhal, G. Kumar, S. Chan, F.M. Fisher, Y. Ma, H.G. Vardeh et al. Deficiency of fibroblast growth factor 21 (FGF21) promotes hepatocellular carcinoma (HCC) in mice on a long term obesogenic diet. Mol Metab. 13, 56–66 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    S. Huang, J. He, X. Zhang, Y. Bian, L. Yang, G. Xie et al. Activation of the hedgehog pathway in human hepatocellular carcinomas. Carcinogenesis 27, 1334–1340 (2006)PubMedCrossRefGoogle Scholar
  20. 20.
    D. Ye, Y. Wang, H. Li, W. Jia, K. Man, C.M. Lo et al. Fibroblast growth factor 21 protects against acetaminophen-induced hepatotoxicity by potentiating peroxisome proliferator-activated receptor coactivator protein-1alpha-mediated antioxidant capacity in mice. Hepatology 60, 977–989 (2014)PubMedCrossRefGoogle Scholar
  21. 21.
    G. Singhal, F.M. Fisher, M.J. Chee, T.G. Tan, A. El Ouaamari, A.C. Adams et al. Fibroblast growth factor 21 (FGF21) protects against high fat diet induced inflammation and islet hyperplasia in pancreas. PLoS ONE 11, e0148252 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    M.K. Badman, A.R. Kennedy, A.C. Adams, P. Pissios, E. Maratos-Flier, A very low carbohydrate ketogenic diet improves glucose tolerance in ob/ob mice independently of weight loss. Am. J. Physiol. Endocrinol. Metab. 297, E1197–E1204 (2009)PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    N. Douris, T. Melman, J.M. Pecherer, P. Pissios, J.S. Flier, L.C. Cantley et al. Adaptive changes in amino acid metabolism permit normal longevity in mice consuming a low-carbohydrate ketogenic diet. Biochim Biophys. Acta 1852, 2056–2065 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    A.R. Kennedy, P. Pissios, H. Otu, R. Roberson, B. Xue, K. Asakura et al. A high-fat, ketogenic diet induces a unique metabolic state in mice. Am. J. Physiol. Endocrinol. Metab. 292, E1724–E1739 (2007)PubMedCrossRefGoogle Scholar
  25. 25.
    E.D. Berglund, C.Y. Li, H.A. Bina, S.E. Lynes, M.D. Michael, A.B. Shanafelt et al. Fibroblast growth factor 21 controls glycemia via regulation of hepatic glucose flux and insulin sensitivity. Endocrinology 150, 4084–4093 (2009)PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    J.P. Camporez, F.R. Jornayvaz, M.C. Petersen, D. Pesta, B.A. Guigni, J. Serr et al. Cellular mechanisms by which FGF21 improves insulin sensitivity in male mice. Endocrinology 154, 3099–3109 (2013)PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    F.M. Fisher, P.C. Chui, P.J. Antonellis, H.A. Bina, A. Kharitonenkov, J.S. Flier et al. Obesity is a fibroblast growth factor 21 (FGF21)-resistant state. Diabetes 59, 2781–2789 (2010)PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    H. Li, G. Wu, Q. Fang, M. Zhang, X. Hui, B. Sheng et al. Fibroblast growth factor 21 increases insulin sensitivity through specific expansion of subcutaneous fat. Nat. Commun. 9, 272 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
  30. 30.
    J.E. Ayala, D.P. Bracy, C. Malabanan, F.D. James, T. Ansari, P.T. Fueger et al. Hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice. J Vis Exp. (2011)Google Scholar
  31. 31.
    J.E. Ayala, D.P. Bracy, O.P. McGuinness, D.H. Wasserman, Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes 55, 390–397 (2006)PubMedCrossRefGoogle Scholar
  32. 32.
    K.X. Mulligan, R.T. Morris, Y.F. Otero, D.H. Wasserman, O.P. McGuinness, Disassociation of muscle insulin signaling and insulin-stimulated glucose uptake during endotoxemia. PLoS ONE 7, e30160 (2012)PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    E.W. Kraegen, D.E. James, A.B. Jenkins, D.J. Chisholm, Dose-response curves for in vivo insulin sensitivity in individual tissues in rats. Am. J. Physiol. 248, E353–E362 (1985)PubMedGoogle Scholar
  34. 34.
    G. Singhal, N. Douris, A.J. Fish, X. Zhang, A.C. Adams, J.S. Flier et al. Fibroblast growth factor 21 has no direct role in regulating fertility in female mice. Mol. Metab. 5, 690–698 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    E.M. Brunt, D.E. Kleiner, L.A. Wilson, P. Belt, B.A. Neuschwander-Tetri, N.C.R. Network, Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings. Hepatology 53, 810–820 (2011)PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    S.M. Martinez, G. Crespo, M. Navasa, X. Forns, Noninvasive assessment of liver fibrosis. Hepatology 53, 325–335 (2011)PubMedCrossRefGoogle Scholar
  37. 37.
    J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957)PubMedGoogle Scholar
  38. 38.
    P. Seoane-Collazo, J. Roa, E. Rial-Pensado, L. Linares-Pose, D. Beiroa, F. Ruiz-Pino et al. SF1-specific AMPKalpha1 deletion protects against diet-induced obesity. Diabetes 67, 2213–2226 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    A. Guilherme, D.J. Pedersen, F. Henriques, A.H. Bedard, E. Henchey, M. Kelly et al. Neuronal modulation of brown adipose activity through perturbation of white adipocyte lipogenesis. Mol. Metab. 16, 116–125 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    K. Shinohara, P. Nakagawa, J. Gomez, D.A. Morgan, N.K. Littlejohn, M.D. Folchert et al. Selective deletion of renin-b in the brain alters drinking and metabolism. Hypertension 70, 990–997 (2017)PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    A.I. Mina, R.A. LeClair, K.B. LeClair, D.E. Cohen, L. Lantier, A.S. Banks, CalR: a web-based analysis tool for indirect calorimetry experiments. Cell Metab. 28(656–666), e651 (2018)Google Scholar
  42. 42.
    F.M. Fisher, E. Maratos-Flier, Understanding the physiology of FGF21. Annu Rev. Physiol. 78, 223–241 (2016)PubMedCrossRefGoogle Scholar
  43. 43.
    A. Kharitonenkov, R. DiMarchi, FGF21 revolutions: recent advances illuminating FGF21 biology and medicinal properties. Trends Endocrinol. Metab. 26, 608–617 (2015)PubMedCrossRefGoogle Scholar
  44. 44.
    N. Douris, D.M. Stevanovic, F.M. Fisher, T.I. Cisu, M.J. Chee, N.L. Nguyen et al. Central fibroblast growth factor 21 browns white fat via sympathetic action in male mice. Endocrinology 156, 2470–2481 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    F.M. Fisher, S. Kleiner, N. Douris, E.C. Fox, R.J. Mepani, F. Verdeguer et al. FGF21 regulates PGC-1alpha and browning of white adipose tissues in adaptive thermogenesis. Genes Dev. 26, 271–281 (2012)PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    N.Y. Kalaany, D.J. Mangelsdorf, LXRS and FXR: the yin and yang of cholesterol and fat metabolism. Annu Rev. Physiol. 68, 159–191 (2006)PubMedCrossRefGoogle Scholar
  47. 47.
    W. Luu, L.J. Sharpe, I.C. Gelissen, A.J. Brown, The role of signalling in cellular cholesterol homeostasis. IUBMB Life 65, 675–684 (2013)PubMedCrossRefGoogle Scholar
  48. 48.
    J. Ye, R.A. DeBose-Boyd, Regulation of cholesterol and fatty acid synthesis. Cold Spring Harb. Perspect. Biol 3, 1–14 (2011).CrossRefGoogle Scholar
  49. 49.
    M.M. Chen, C. Hale, S. Stanislaus, J. Xu, M.M. Veniant, FGF21 acts as a negative regulator of bile acid synthesis. J. Endocrinol. 237, 139–152 (2018)PubMedCrossRefGoogle Scholar
  50. 50.
    J. Zhang, J. Gupte, Y. Gong, J. Weiszmann, Y. Zhang, K.J. Lee et al. Chronic over-expression of fibroblast growth factor 21 increases bile acid biosynthesis by opposing FGF15/19 action. EBioMedicine 15, 173–183 (2017)PubMedCrossRefGoogle Scholar
  51. 51.
    I. Tabas, Consequences of cellular cholesterol accumulation: basic concepts and physiological implications. J. Clin. Invest. 110, 905–911 (2002)PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    K.R. Markan, M.C. Naber, M.K. Ameka, M.D. Anderegg, D.J. Mangelsdorf, S.A. Kliewer et al. Circulating FGF21 is liver derived and enhances glucose uptake during refeeding and overfeeding. Diabetes 63, 4057–4063 (2014)PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    S. Vernia, J. Cavanagh-Kyros, T. Barrett, C. Tournier, R.J. Davis, Fibroblast growth factor 21 mediates glycemic regulation by hepatic JNK. Cell Rep. 14, 2273–2280 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    J.S. Flier, Irreproducibility of published bioscience research: diagnosis, pathogenesis and therapy. Mol. Metab. 6, 2–9 (2017)PubMedCrossRefGoogle Scholar
  55. 55.
    D.J. Drucker, Never waste a good crisis: confronting reproducibility in translational research. Cell Metab. 24, 348–360 (2016)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Mikiko Watanabe
    • 1
    • 2
  • Garima Singhal
    • 1
  • Ffolliott M. Fisher
    • 1
  • Thomas C. Beck
    • 3
  • Donald A. Morgan
    • 4
  • Fabio Socciarelli
    • 5
  • Marie L. Mather
    • 1
  • Renata Risi
    • 2
  • Jared Bourke
    • 1
  • Kamal Rahmouni
    • 4
  • Owen P. McGuinness
    • 3
  • Jeffrey S. Flier
    • 1
    • 6
  • Eleftheria Maratos-Flier
    • 1
    Email author
  1. 1.Department of Medicine, Beth Israel Deaconess Medical CenterHarvard Medical SchoolBostonUSA
  2. 2.Department of Experimental Medicine, Section of Medical Pathophysiology, Food Science and EndocrinologySapienza University of RomeRomeItaly
  3. 3.Department of Molecular Physiology and BiophysicsVanderbilt University School of MedicineNashvilleUSA
  4. 4.Department of PharmacologyUniversity of Iowa Carver College of MedicineIowa CityUSA
  5. 5.Department of Oncology-PathologyKarolinska InstitutetStockholmSweden
  6. 6.Department of NeurobiologyHarvard Medical SchoolBostonUSA

Personalised recommendations