Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Expansion of adult ␤-cell mass in response to increased metabolic demand is dependent on HNF-4␣ Rana K. Gupta,1,3 Nan Gao,1,3 Regina K. Gorski,1,2 Peter White,1 Olga T. Hardy,1 Kiran Rafiq,1 John E. Brestelli,1 Guang Chen,2 Christian J. Stoeckert Jr.,1,2 and Klaus H. Kaestner1,4 1Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA; 2Center for Bioinformatics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA The failure to expand functional pancreatic ␤-cell mass in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing ␤-cells is the principle mechanism for ␤-cell expansion in adult mice. Here we demonstrate that the proliferative response of ␤-cells is dependent on the orphan nuclear receptor hepatocyte nuclear factor-4␣ (HNF-4␣), the gene that is mutated in Maturity-Onset Diabetes of the Young 1 (MODY1). Computational analysis of microarray expression profiles from isolated islets of mice lacking HNF-4␣ in pancreatic ␤-cells reveals that HNF-4␣ regulates selected genes in the ␤-cell, many of which are involved in proliferation. Using a physiological model of ␤-cell expansion, we show that HNF-4␣ is required for ␤-cell replication and the activation of the Ras/ERK signaling cascade in islets. This phenotype correlates with the down-regulation of suppression of tumorigenicity 5 (ST5) in HNF-4␣ mutants, which we identify as a novel regulator of ERK phosphorylation in ␤-cells and a direct transcriptional target of HNF-4␣ in vivo. Together, these results indicate that HNF-4␣ is essential for the physiological expansion of adult ␤-cell mass in response to increased metabolic demand. [Keywords: Ras; extracellular regulated kinase; mitogen activated protein kinase; ␤-cells; HNF-4␣; type 2 diabetes; gestational diabetes] Supplemental material is available at http://www.genesdev.org. Received January 26, 2007; revised version accepted February 9, 2007. Diabetes mellitus is a metabolic disorder that currently and pancreatic polypeptide-producing PP cells. The im- affects >150 million people worldwide. This number is portance of gene transcription in the regulation of ␤-cell predicted to grow to as many as 300 million people by growth and function is highlighted by the genetic and the year 2025 (Zimmet et al. 2001). The disease can be molecular analysis of Maturity-Onset Diabetes of the characterized by either absolute insulin deficiency due Young (MODY). MODY, an autosomal dominantly in- to the autoimmune destruction of pancreatic insulin- herited form of type 2 diabetes, results from mutations producing ␤-cells (type 1 diabetes mellitus), or relative in at least six different genes. One of these encodes the insulin deficiency due to defective insulin secretion and/ glycolytic enzyme glucokinase (MODY2), which is an or insulin sensitivity (type 2 diabetes mellitus). The re- important glucose sensor, while all others encode tran- sulting hyperglycemia can ultimately result in organ scription factors: Hepatocyte nuclear factor (HNF)-4␣ failure and death. (MODY1); HNF-1␣ (MODY3); insulin promoter factor-1 Insulin production is limited to the endocrine pan- (IPF1/Pdx-1; MODY4); HNF-1␤ (MODY5); and neuro- creas, a micro-organ within the exocrine pancreas con- genic differentiation factor 1 (NeuroD1; MODY6) (Shih sisting of the islets of Langerhans. The pancreatic islets and Stoffel 2002). MODY transcription factors represent comprise <5% of the total pancreatic volume, and them- crucial links in a transcriptional hierarchy that controls selves are composed of five endocrine cell types: gluca- both the specification of endocrine cell types during gon-producing ␣-cells, insulin-producing ␤-cells, so- early pancreatic development as well as the maintenance matostatin-producing ␦-cells, ghrelin-producing ␧-cells, of the differentiated state in the adult (Jonsson et al. 1994; Ahlgren et al. 1998; Wang et al. 1998, 2000, 2004; Hagenfeldt-Johansson et al. 2001; Fukui et al. 2005; 3These authors contributed equally to this work. Gupta et al. 2005; Miura et al. 2006). Complementing 4Corresponding author. E-MAIL [email protected]; FAX (215) 573-5892. the molecular identification of the MODY transcription Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1535507. factors by human genetics, developmental biologists 756 GENES & DEVELOPMENT 21:756–769 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press HNF-4␣ and ␤-cell expansion have elucidated regulatory pathways by targeted disrup- tion of the genes encoding pancreatic-enriched transcrip- tion factors, and by the analysis of the expression pat- terns of these genes (Wilson et al. 2003). A detailed un- derstanding of how these transcription factors and their downstream targets govern ␤-cell differentiation in vivo is essential in order to effectively manipulate these pro- cesses as a therapeutic strategy for diabetes. Among the transcription factors required for the main- tenance of the adult ␤-cell is the orphan nuclear receptor hepatocyte nuclear factor-4␣ (HNF-4␣). Targeted disrup- tion of HNF-4␣ in ␤-cells results in impaired glucose tolerance due to attenuated glucose-stimulated insulin secretion (Gupta et al. 2005; Miura et al. 2006). This can be explained, in part, by a defect in the function of the ATP-sensitive potassium channel (KATP), a central com- ponent of the glucose-sensing machinery. Both studies reported that ␤-cells devoid of HNF-4␣ exhibit only few changes in the expression of genes involved in regulating insulin secretion (Gupta et al. 2005; Miura et al. 2006). However, the effects of HNF-4␣ deficiency on global Figure 1. Gene expression profiling of HNF-4␣loxP/loxP;Ins.Cre gene expression in the ␤-cell have not been explored. mice. Total RNA was prepared from isolated islets of 4- to Therefore, in order to determine the molecular net- 5-mo-old control and mutant mice. The RNA was amplified, ␣ works controlled by HNF-4 , we sought to identify all of fluorescently labeled, and hybridized to the PancChip 5.0 its targets, whether regulated directly or indirectly, by cDNA microarray. (A) A scatterplot of average intensity values comparing gene expression profiles of HNF-4␣loxP/loxP; for all expressed genes illustrates that the vast majority of Ins.Cre mice to those of littermate controls. Gene ex- genes were not differentially expressed. Approximately 2% pression analysis revealed that numerous genes and of expressed genes (128 genes significantly up-regulated and pathways involved in the regulation of ␤-cell pro- 57 significantly down-regulated) were differentially expressed loxP/loxP liferation are dependent on HNF-4␣. As a result, in HNF-4␣ mutant islets (n =5 HNF-4␣ , n =3 ␣loxP/loxP HNF-4␣loxP/loxP;Ins.Cre mice fail to expand ␤-cell mass HNF-4 ;Ins.Cre). (B) Quantitative real-time PCR confir- mation of differentially expressed genes. Total RNA was ex- in response to increased metabolic demands. Moreover, tracted from isolated islets of a second cohort of animals, and our analysis indicates that Ras/ERK signaling in the cDNA derived from unamplified RNA was used for PCR. Thir- ␤ ␣ -cell is dependent on HNF-4 . This defect can be ex- teen of 14 (92%) genes were confirmed as differentially ex- plained in part by the down-regulation of suppression of pressed, consistent with the 10% FDR setting chosen for mi- tumorigenicity 5 (ST5) in islets of HNF-4␣loxP/loxP; croarray analysis (n = 4–6 for PCR). (*) p-value from Student’s Ins.Cre mice, which we identify as a novel regulator of t-test <0.05. ERK activation in ␤-cells and a direct transcriptional tar- get of HNF-4␣ in vivo. genes in HNF-4␣loxP/loxP;Ins.Cre mice (Table 1). Consis- tent with the known functions of HNF-4␣ in the liver, Results the largest percentage of HNF-4␣-regulated genes are in- HNF-4␣ regulates proliferative pathways in the ␤-cell volved in metabolism (Hayhurst et al. 2001; Parviz et al. 2003). However, an almost equal number of differen- In order to identify targets of HNF-4␣ in the ␤-cell, we tially expressed genes function in signal transduction performed large-scale expression profiling on pancreatic and cell proliferation. These results were surprising loxP/loxP islets from 4-mo-old HNF-4␣ ;Ins.Cre mice in since ␤-cell mass was unchanged in 4-mo-old HNF- which HNF-4␣ is deleted in ∼85% of ␤-cells (Gupta et al. 4␣loxP/loxP;Ins.Cre (Gupta et al. 2005; Miura et al. 2006), 2005). This microarray expression analysis identified 128 and the frequency of proliferative and apoptotic ␤-cells significantly up-regulated and 57 significantly down- in adult mutants was indistinguishable from controls regulated genes in HNF-4␣ mutants (Fig. 1A; Supple- (data not shown). In addition, thus far a direct role for mentary Table 1). Using quantitative real-time PCR, we HNF-4␣ in the regulation of ␤-cell proliferation has not confirmed 13 of 14 (92%) randomly chosen differentially been reported. Nevertheless, our gene expression analy- expressed genes as HNF-4␣-dependent, as expected by sis suggested that HNF-4␣ mutants might display im- the 10% false discovery rate chosen in the initial analysis paired ␤-cell growth in response to proliferative stimuli. (Fig. 1B). However, the expression levels of the vast major- ity (98%) of the genes expressed in pancreatic islets were HNF-4␣ is required for the expansion of maternal not significantly modulated by the absence of HNF-4␣. ␤-cell mass during pregnancy To elucidate the physiological processes in the ␤-cell that are dependent on HNF-4␣, we first determined the Assessing the rate of ␤-cell proliferation and cell death in Gene Ontology functions for the differentially expressed the pancreatic islet is challenging given the low rate of GENES & DEVELOPMENT 757 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Gupta et al.