Intestinal GATA4 deficiency induces proximal fibroblast growth factor 15 expression and represses hepatic gluconeogenesis
Authors: Jay V. Patankar, Vinay Sachdev, Sascha Obrowsky, Sanja Levak-Frank, Dagmar Kratky
Journal: Journal of Hepatology (2013)

We have recently reported that conditional deletion of intestinal GATA4 leads to an increased protection from obesity-associated hepatic steatosis [[1]Patankar J.V. Obrowsky S. Doddapattar P. Hoefler G. Battle M. Levak-Frank S. et al.Intestinal GATA4 deficiency protects from diet-induced hepatic steatosis.J Hepatol. 2012; 57: 1061-1068Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. Using two independent murine models of diet-induced hepatic steatosis, we demonstrated that intestine specific GATA4 knockout (GATA4iKO) mice are protected from high fat diet-induced hepatic steatosis. Our findings showed that intestinal GATA4 deficiency was protective only in case where hepatic de novo lipogenesis was involved, secondary to insulin resistance. GATA4iKO mice were not protected upon induction of steatosis via dietary deficiency of methionine and choline. Methionine and choline-deficient diet leads to hepatic steatosis by suppressing VLDL release and beta-oxidation and does not significantly impact de novo lipogenesis. The underlying mechanism for the observed benefit is the ectopic upregulation of GLP-1 and higher number of GLP-1 producing L-cells in the proximal intestine of GATA4iKO mice.Previous studies have shown that the levels of the important enterokine fibroblast growth factor 15 (FGF-15) are also upregulated several fold in the proximal intestine of GATA4iKO mice [2Battle M.A. Bondow B.J. Iverson M.A. Adams S.J. Jandacek R.J. Tso P. et al.GATA4 is essential for jejunal function in mice.Gastroenterology. 2008; 135: e1671Abstract Full Text Full Text PDF Scopus (67) Google Scholar, 3Beuling E. Kerkhof I.M. Nicksa G.A. Giuffrida M.J. Haywood J. aan de Kerk D.J. et al.Conditional Gata4 deletion in mice induces bile acid absorption in the proximal small intestine.Gut. 2010; 59: 888-895Crossref PubMed Scopus (34) Google Scholar]. Under physiological conditions, transcript levels of intestinal FGF-15 are restricted to the mucosa of the ileum and are sensitive to bile acid availability and consequent activation of farnesoid X receptor (FXR). However, it is not clear whether the ectopic FGF-15 produced in the proximal small intestine of GATA4iKO mice is subjective to bile acid-mediated regulation. FGF-15 is a crucial determinant of hepatic bile acid synthesis and recent reports indicate an important role of this enterokine in the regulation of postprandial hepatic gluconeogenesis, secondary to insulin [[4]Potthoff M.J. Boney-Montoya J. Choi M. He T. Sunny N.E. Satapati S. et al.FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway.Cell Metab. 2011; 13: 729-738Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Unlike insulin, which peaks at 15 min post-feeding, ileal FGF-15 transcripts peak 1 h postprandially, when bile acid concentrations increase in the small intestine [[4]Potthoff M.J. Boney-Montoya J. Choi M. He T. Sunny N.E. Satapati S. et al.FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway.Cell Metab. 2011; 13: 729-738Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Hepatic effects of ectopic FGF-15 expressed from the proximal intestine and its regulation have not been studied.Battle et al. have shown that transcript levels of hepatic cholesterol 7-alpha hydroxylase (Cyp7a1) in GATA4iKO mice are repressed [[2]Battle M.A. Bondow B.J. Iverson M.A. Adams S.J. Jandacek R.J. Tso P. et al.GATA4 is essential for jejunal function in mice.Gastroenterology. 2008; 135: e1671Abstract Full Text Full Text PDF Scopus (67) Google Scholar]. This finding indicates that proximally produced FGF-15 in GATA4iKO mice is physiologically active and can repress hepatic bile acid synthesis. We have previously shown that GATA4iKO mice are resistant to diet-induced obesity and insulin resistance and maintain lower plasma glucose levels [[5]Patankar J.V. Chandak P.G. Obrowsky S. Pfeifer T. Diwoky C. Uellen A. et al.Loss of intestinal GATA4 prevents diet-induced obesity and promotes insulin sensitivity in mice.Am J Physiol Endocrinol Metab. 2010; 300: E478-E488Crossref PubMed Scopus (12) Google Scholar]. However, important questions as to whether its expression is regulated by bile acids and whether it can in turn regulate hepatic gluconeogenic gene expression have not been addressed. To answer these questions, we performed quantitative real-time PCR analysis of intestinal and hepatic transcripts in fasted and 1 h post-gavage control and GATA4iKO mice. We observed that the expression of proximal FGF-15 in GATA4iKO mice is constitutively upregulated even in the fasted state where bile acid availability is negligible (Table 1). However, in line with the observations of Beuling et al. [[3]Beuling E. Kerkhof I.M. Nicksa G.A. Giuffrida M.J. Haywood J. aan de Kerk D.J. et al.Conditional Gata4 deletion in mice induces bile acid absorption in the proximal small intestine.Gut. 2010; 59: 888-895Crossref PubMed Scopus (34) Google Scholar] we also found ileal FGF-15 transcripts in GATA4iKO mice to be severely repressed in both fasted condition as well as 1 h post-gavage states. Additionally, transcript levels of proximal FGF-15 after 1 h post-gavage of cholic acid (25 mmol/kg in sodium bicarbonate buffer) were induced 88 fold in GATA4iKO vs. control mice (Table 1). In addition, we noticed that the mRNA expression of important gluconeogenic genes in the liver of fasted GATA4iKO mice was downregulated (Table 1). As reported previously [[2]Battle M.A. Bondow B.J. Iverson M.A. Adams S.J. Jandacek R.J. Tso P. et al.GATA4 is essential for jejunal function in mice.Gastroenterology. 2008; 135: e1671Abstract Full Text Full Text PDF Scopus (67) Google Scholar], we also found repression of hepatic Cyp7a1 in fasted livers of GATA4iKO mice (Table 1). GATA4iKO mice had lower body weight in fasted (33.4 ± 1.3 vs. 27.1 ± 1.4) condition and reduced fasting glucose levels compared with controls (Table 1).Table 1mRNA fold change and p value for genes involved in entero-hepatic bile acid and gluconeogenic gene regulation in fasted and 1 h post re-feeding of a liquid diet (Ensure Plus™, Abbott GmbH, Austria) or 25 mmol cholic acid per kg body weight (Sigma, GmbH, Austria).n.a., not available.Data represent fold change and p value or mean ± SEM (n = 6 per group for plasma glucose; n = 3 per group for gene expression analysis).∗∗p <0.01. Open table in a new tab These findings clearly indicate that the ectopic FGF-15 expression in the proximal intestine of GATA4iKO mice remains physiologically active and impacts hepatic glucose metabolism. Unlike ileal FGF-15, ectopic proximal expression of FGF-15 in GATA4iKO mice persists even in fasted conditions. Our finding of suppressed hepatic gluconeogenic gene expression could explain the previous observation of reduced fasting plasma glucose levels in high fat diet-fed GATA4iKO mice [[5]Patankar J.V. Chandak P.G. Obrowsky S. Pfeifer T. Diwoky C. Uellen A. et al.Loss of intestinal GATA4 prevents diet-induced obesity and promotes insulin sensitivity in mice.Am J Physiol Endocrinol Metab. 2010; 300: E478-E488Crossref PubMed Scopus (12) Google Scholar]. Further studies aimed at understanding bile acid mediated effects in proximal intestinal enterocytes in GATA4iKO mice are warranted to identify possible interaction between intestinal FXR and GATA4.Financial supportThis work was supported by the PhD program “Molecular Medicine” of the Medical University of Graz, the Austrian Science Fund grants ( P22832 , DK-MCD W1226 , SFB LIPOTOX F30 ), and the Austrian National Bank ( 12929 ).Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. We have recently reported that conditional deletion of intestinal GATA4 leads to an increased protection from obesity-associated hepatic steatosis [[1]Patankar J.V. Obrowsky S. Doddapattar P. Hoefler G. Battle M. Levak-Frank S. et al.Intestinal GATA4 deficiency protects from diet-induced hepatic steatosis.J Hepatol. 2012; 57: 1061-1068Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. Using two independent murine models of diet-induced hepatic steatosis, we demonstrated that intestine specific GATA4 knockout (GATA4iKO) mice are protected from high fat diet-induced hepatic steatosis. Our findings showed that intestinal GATA4 deficiency was protective only in case where hepatic de novo lipogenesis was involved, secondary to insulin resistance. GATA4iKO mice were not protected upon induction of steatosis via dietary deficiency of methionine and choline. Methionine and choline-deficient diet leads to hepatic steatosis by suppressing VLDL release and beta-oxidation and does not significantly impact de novo lipogenesis. The underlying mechanism for the observed benefit is the ectopic upregulation of GLP-1 and higher number of GLP-1 producing L-cells in the proximal intestine of GATA4iKO mice. Previous studies have shown that the levels of the important enterokine fibroblast growth factor 15 (FGF-15) are also upregulated several fold in the proximal intestine of GATA4iKO mice [2Battle M.A. Bondow B.J. Iverson M.A. Adams S.J. Jandacek R.J. Tso P. et al.GATA4 is essential for jejunal function in mice.Gastroenterology. 2008; 135: e1671Abstract Full Text Full Text PDF Scopus (67) Google Scholar, 3Beuling E. Kerkhof I.M. Nicksa G.A. Giuffrida M.J. Haywood J. aan de Kerk D.J. et al.Conditional Gata4 deletion in mice induces bile acid absorption in the proximal small intestine.Gut. 2010; 59: 888-895Crossref PubMed Scopus (34) Google Scholar]. Under physiological conditions, transcript levels of intestinal FGF-15 are restricted to the mucosa of the ileum and are sensitive to bile acid availability and consequent activation of farnesoid X receptor (FXR). However, it is not clear whether the ectopic FGF-15 produced in the proximal small intestine of GATA4iKO mice is subjective to bile acid-mediated regulation. FGF-15 is a crucial determinant of hepatic bile acid synthesis and recent reports indicate an important role of this enterokine in the regulation of postprandial hepatic gluconeogenesis, secondary to insulin [[4]Potthoff M.J. Boney-Montoya J. Choi M. He T. Sunny N.E. Satapati S. et al.FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway.Cell Metab. 2011; 13: 729-738Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Unlike insulin, which peaks at 15 min post-feeding, ileal FGF-15 transcripts peak 1 h postprandially, when bile acid concentrations increase in the small intestine [[4]Potthoff M.J. Boney-Montoya J. Choi M. He T. Sunny N.E. Satapati S. et al.FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway.Cell Metab. 2011; 13: 729-738Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Hepatic effects of ectopic FGF-15 expressed from the proximal intestine and its regulation have not been studied. Battle et al. have shown that transcript levels of hepatic cholesterol 7-alpha hydroxylase (Cyp7a1) in GATA4iKO mice are repressed [[2]Battle M.A. Bondow B.J. Iverson M.A. Adams S.J. Jandacek R.J. Tso P. et al.GATA4 is essential for jejunal function in mice.Gastroenterology. 2008; 135: e1671Abstract Full Text Full Text PDF Scopus (67) Google Scholar]. This finding indicates that proximally produced FGF-15 in GATA4iKO mice is physiologically active and can repress hepatic bile acid synthesis. We have previously shown that GATA4iKO mice are resistant to diet-induced obesity and insulin resistance and maintain lower plasma glucose levels [[5]Patankar J.V. Chandak P.G. Obrowsky S. Pfeifer T. Diwoky C. Uellen A. et al.Loss of intestinal GATA4 prevents diet-induced obesity and promotes insulin sensitivity in mice.Am J Physiol Endocrinol Metab. 2010; 300: E478-E488Crossref PubMed Scopus (12) Google Scholar]. However, important questions as to whether its expression is regulated by bile acids and whether it can in turn regulate hepatic gluconeogenic gene expression have not been addressed. To answer these questions, we performed quantitative real-time PCR analysis of intestinal and hepatic transcripts in fasted and 1 h post-gavage control and GATA4iKO mice. We observed that the expression of proximal FGF-15 in GATA4iKO mice is constitutively upregulated even in the fasted state where bile acid availability is negligible (Table 1). However, in line with the observations of Beuling et al. [[3]Beuling E. Kerkhof I.M. Nicksa G.A. Giuffrida M.J. Haywood J. aan de Kerk D.J. et al.Conditional Gata4 deletion in mice induces bile acid absorption in the proximal small intestine.Gut. 2010; 59: 888-895Crossref PubMed Scopus (34) Google Scholar] we also found ileal FGF-15 transcripts in GATA4iKO mice to be severely repressed in both fasted condition as well as 1 h post-gavage states. Additionally, transcript levels of proximal FGF-15 after 1 h post-gavage of cholic acid (25 mmol/kg in sodium bicarbonate buffer) were induced 88 fold in GATA4iKO vs. control mice (Table 1). In addition, we noticed that the mRNA expression of important gluconeogenic genes in the liver of fasted GATA4iKO mice was downregulated (Table 1). As reported previously [[2]Battle M.A. Bondow B.J. Iverson M.A. Adams S.J. Jandacek R.J. Tso P. et al.GATA4 is essential for jejunal function in mice.Gastroenterology. 2008; 135: e1671Abstract Full Text Full Text PDF Scopus (67) Google Scholar], we also found repression of hepatic Cyp7a1 in fasted livers of GATA4iKO mice (Table 1). GATA4iKO mice had lower body weight in fasted (33.4 ± 1.3 vs. 27.1 ± 1.4) condition and reduced fasting glucose levels compared with controls (Table 1). n.a., not available. Data represent fold change and p value or mean ± SEM (n = 6 per group for plasma glucose; n = 3 per group for gene expression analysis). ∗∗p <0.01. These findings clearly indicate that the ectopic FGF-15 expression in the proximal intestine of GATA4iKO mice remains physiologically active and impacts hepatic glucose metabolism. Unlike ileal FGF-15, ectopic proximal expression of FGF-15 in GATA4iKO mice persists even in fasted conditions. Our finding of suppressed hepatic gluconeogenic gene expression could explain the previous observation of reduced fasting plasma glucose levels in high fat diet-fed GATA4iKO mice [[5]Patankar J.V. Chandak P.G. Obrowsky S. Pfeifer T. Diwoky C. Uellen A. et al.Loss of intestinal GATA4 prevents diet-induced obesity and promotes insulin sensitivity in mice.Am J Physiol Endocrinol Metab. 2010; 300: E478-E488Crossref PubMed Scopus (12) Google Scholar]. Further studies aimed at understanding bile acid mediated effects in proximal intestinal enterocytes in GATA4iKO mice are warranted to identify possible interaction between intestinal FXR and GATA4. Financial supportThis work was supported by the PhD program “Molecular Medicine” of the Medical University of Graz, the Austrian Science Fund grants ( P22832 , DK-MCD W1226 , SFB LIPOTOX F30 ), and the Austrian National Bank ( 12929 ). This work was supported by the PhD program “Molecular Medicine” of the Medical University of Graz, the Austrian Science Fund grants ( P22832 , DK-MCD W1226 , SFB LIPOTOX F30 ), and the Austrian National Bank ( 12929 ). Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors thank Dr. M. Battle and Dr. S. Duncan for providing intestine-specific GATA4 knockout mice and Mr. A. Ibovnik, Ms. S. Povoden and Ms. I. Hindler for their technical support and animal care.