The AlfpCre mouse revisited: Evidence for liver steatosis related to growth hormone deficiency

Authors

  • Vincent P.E.G. Pruniau M.Sc.,

  • Els Louagie M.Sc., Ph.D.,

  • Bas Brouwers M.Sc.,

  • Jeroen Declercq M.Sc., Ph.D.,

  • John W.M. Creemers Ph.D.


  • Potential conflict of interest: Nothing to report.

  • This work was supported, in part, by g ants from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) and Geconcerteerde Onderzoeksacties (GOA; GOA/12/016). V.P. and B.B. are aspirant fellows of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

To the Editor:

With great interest we read the article by Mueller et al. on the development of steatosis and hepatocellular carcinoma in mice by disrupting hepatic growth hormone (GH) and glucocorticoid receptor signaling.[1] They demonstrate that abrogation of hepatic signal transducer and activator of transcription 5 (Stat5) signaling caused liver steatosis by up-regulation of genes involved in lipid uptake and synthesis. However, we have concerns regarding the use of AlfpCre+ transgenic mice to inactivate Stat5 in the liver. While generating the AlfpCre transgene, the entire human GH (hGH) gene was inserted for its introns and polyadenylation signal.[2] As a consequence, a polycistronic messenger RNA (mRNA) transcript encoding Cre Recombinase and hGH was transcribed in the liver, hypothalamus, and pituitary (data not shown). However, hGH was only translated in the hypothalamus and pituitary (Fig. 1A). It has previously been described that targeted expression of hGH in the hypothalamus reduces expression of hypothalamic GH releasing hormone (GHRH), leading to GH deficiency (GHD).[3] This is also the case in AlfpCre+ mice in a C57Bl6/J background, as shown by quantitative real-time polymerase chain reaction (qRT-PCR). In the hypothalamus, expression levels of GHRH were reduced by 70% (P < 0.01) and mouse GH messenger RNA (mRNA) in the adenopituitary by 61% (P < 0.05). AlfpCre+ mice also showed hypoplasia of the pituitary (Fig. 1B). Hepatic insulin-like growth factor 1 (Igf1) mRNA and Igf1 plasma protein levels were decreased by 71% (P < 0.001) and 60% (P < 0.001), respectively, and reduced phosphorylation of Stat5 in AlfpCre+ livers was shown by western blotting (Fig. 1C), confirming GHD in AlfpCre+ mice. Consequently, AlfpCre+ mice exhibit postnatal growth retardation (Fig. 1D), a finding that was also observed in AlfpCre+/−c-JunF/F mice.[4] Downstream of the impaired GH signaling, AlfpCre+ livers showed increased levels of triglycerides (TGs), free fatty acids (FFAs), and cholesterol, indicating liver steatosis (Fig. 1E). Finally, genes involved in lipid uptake and synthesis, including CD36, very low-density lipoprotein receptor, and peroxisome proliferator-activated receptor gamma, were significantly up-regulated by 2.52- (P < 0.01), 5.44- (P < 0.01), and 2.09-fold (P < 0.001), respectively, an expression profile indicating liver steatosis (Fig. 1F). Altogether, AlfpCre+ mice show liver steatosis related to GHD, and therefore care should be taken to use the right controls (AlfpCre+ unfloxed mice instead of AlfpCre floxed mice) while conditionally inactivating or overexpressing genes in livers of mice.

Figure 1.

Liver steatosis and growth retardation related to GHD in AlfpCre+ mice. (A) hGH enzyme-linked immunosorbent assay (Invitrogen, Carlsbad, CA) analysis on pituitary, hypothalamus, and liver lysates. (B) Microscopic analysis of the pituitary. Pituitary is indicated with arrow. (C) Western blotting of Stat5 and phospho-Stat5 (Cell Signaling Technology, Danvers, MA). (D) Growth curve of AlfpCre+ and AlfpCre mice. (E) Biochemical liver lipid content analysis. TG, cholesterol (Chol), and FFA concentrations were measured (Wako Diagnostics, Richmond, VA) and corrected for tissue weight. (F) mRNA expression analysis of genes involved in lipid uptake and synthesis by qRT-PCR. Data are the mean ± standard error of the mean of at least three samples. Statistical analysis was performed using the Student t test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Vincent P.E.G. Pruniau, M.Sc.

  • Els Louagie, M.Sc., Ph.D.

  • Bas Brouwers, M.Sc.

  • Jeroen Declercq, M.Sc., Ph.D.

  • John W.M. Creemers, Ph.D.

  • Laboratory for Biochemical Neuroendocrinology

  • Department for Human Genetics

  • KU Leuven

  • Leuven, Belgium

  • Els Louagie is currently affiliated with the Laboratory for Molecular Immunology and Inflammation

  • Department of Rheumatology

  • Ghent University Hospital

  • Ghent, Belgium

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