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Originally published In Press as doi:10.1074/jbc.M507496200 on December 23, 2005

J. Biol. Chem., Vol. 281, Issue 8, 5246-5257, February 24, 2006
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Hepatocyte Nuclear Factor-4{alpha} Is Essential for Glucose-stimulated Insulin Secretion by Pancreatic beta-Cells*Formula

Atsuko Miura{ddagger}, Kazuya Yamagata{ddagger}1, Masafumi Kakei§, Hiroyasu Hatakeyama, Noriko Takahashi||, Kenji Fukui{ddagger}, Takao Nammo, Kazue Yoneda{ddagger}, Yusuke Inoue**, Frances M. Sladek{ddagger}{ddagger}, Mark A. Magnuson§§, Haruo Kasai, Junichiro Miyagawa{ddagger}, Frank J. Gonzalez**, and Iichiro Shimomura{ddagger}

From the {ddagger}Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan, the §Department of Internal Medicine, Division of Endocrinology, Metabolism, and Geriatric Medicine, Akita University School of Medicine, Hondo, Akita 010-8543, Japan, the Department of Cell Physiology, National Institute for Physiological Sciences and Graduate University of Advanced Studies, Okazaki 444-8787, Japan, the ||Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan, the {ddagger}{ddagger}Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521-0314, the §§Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and the **Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 11, 2005 , and in revised form, December 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the hepatocyte nuclear factor (HNF)-4{alpha} gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of glucose-stimulated insulin secretion by pancreatic beta-cells. HNF-4{alpha}, a transcription factor belonging to the nuclear receptor superfamily, is expressed in pancreatic islets as well as in the liver, kidney, and intestine. However, the role of HNF-4{alpha} in pancreatic beta-cell is unclear. To clarify the role of HNF-4{alpha} in beta-cells, we generated beta-cell-specific HNF-4{alpha} knock-out (betaHNF-4{alpha}KO) mice using the Cre-LoxP system. The betaHNF-4{alpha}KO mice exhibited impairment of glucose-stimulated insulin secretion, which is a characteristic of MODY1. Pancreatic islet morphology, beta-cell mass, and insulin content were normal in the HNF-4{alpha} mutant mice. Insulin secretion by betaHNF-4{alpha}KO islets and the intracellular calcium response were impaired after stimulation by glucose or sulfonylurea but were normal after stimulation with KCl or arginine. Both NAD(P)H generation and ATP content at high glucose concentrations were normal in the betaHNF-4{alpha}KO mice. Expression levels of Kir6.2 and SUR1 proteins in the betaHNF-4{alpha}KO mice were unchanged as compared with control mice. Patch clamp experiments revealed that the current density was significantly increased in betaHNF-4{alpha}KO mice compared with control mice. These results are suggestive of the dysfunction of KATP channel activity in the pancreatic beta-cells of HNF-4{alpha}-deficient mice. Because the KATP channel is important for proper insulin secretion in beta-cells, altered KATP channel activity could be related to the impaired insulin secretion in the betaHNF-4{alpha}KO mice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte nuclear factor (HNF)2-4{alpha}, a transcription factor belonging to the nuclear hormone receptor superfamily (NR2A1), is expressed in the liver, kidney, intestine, and pancreas (1). Similar to other nuclear receptors, HNF-4{alpha} has several functional domains, including the N-terminal transactivation domain (AF-1), a DNA-binding domain, a functionally complex C-terminal region that forms a ligand-binding domain, and a dimerization interface and a transactivation domain (AF-2). In the liver, HNF-4{alpha} plays an important role in regulating various genes involved in glucose, fatty acid, amino acid, and cholesterol metabolism, as well as blood coagulation and hepatic development and differentiation (2, 3).

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic disorder that accounts for 2-5% of type 2 diabetes. It is characterized by autosomal dominant inheritance and an early age of onset (usually at <25 years old) (4). We have shown previously that heterozygous mutations of the HNF-4{alpha} gene can cause a particular form of MODY (MODY1) (5). Functional studies of the mutations in MODY1 patients have shown that the diabetes is caused by loss-of-function mutations (6, 7). Clinical studies have also shown that the primary cause of MODY1 is an impairment of glucose-stimulated insulin secretion by pancreatic beta-cells rather than liver dysfunction (8, 9), indicating that loss of HNF-4{alpha} leads to abnormal insulin secretion from the beta-cells. In addition, recent genetic studies have shown that single nucleotide polymorphisms in the P2 (pancreatic beta-cell type) promoter of the HNF-4{alpha} gene are associated with type 2 diabetes in some populations (10-12). Thus, not only do HNF-4{alpha} mutations cause MODY, but variations of the HNF-4{alpha} gene are associated with a genetic predisposition to common type 2 diabetes. These findings suggest that HNF-4{alpha} in pancreatic beta-cells has an important role in maintaining normal glucose metabolism. Previous in vitro studies have revealed that HNF-4{alpha} regulates the expression of pancreatic beta-cell genes involved in glucose metabolism (e.g. insulin and glucose transporter-2 (GLUT2)) as well as HNF-1{alpha} (13). HNF-1{alpha} is a target gene of HNF-4{alpha} in the liver (14), and mutations of the HNF-1{alpha} gene cause type 3 of MODY (MODY3) (15). However, the role of HNF-4{alpha} in the regulation of pancreatic beta-cell gene expression and its correlation with metabolism-secretion coupling are still unclear. Targeted disruption of the hnf4{alpha} gene in mice results in early embryonic death because of dysfunction of the visceral endoderm (2), and this embryonic lethality prevents further analysis of the role of HNF-4{alpha} in pancreatic beta-cells. Furthermore, in contrast to humans, heterozygous hnf4{alpha} (+/-) mice exhibit normal glucose tolerance (6). To examine the role of HNF-4{alpha} in pancreatic beta-cells, we specifically disrupted HNF-4{alpha} in beta-cells by employing the Cre-loxP system. Pancreatic beta-cell-specific HNF-4{alpha} knock-out mice (betaHNF-4{alpha}KO mice) were viable and exhibited impairment of glucose-stimulated insulin secretion, which is a characteristic of human MODY1. The secretory response of insulin and the intracellular Ca2+ response to glucose or tolbutamide were reduced in isolated HNF-4{alpha} mutant islets. Insulin secretion and the intracellular Ca2+ response to glucose or tolbutamide were decreased in isolated mutant HNF-4{alpha} islets. In addition, the responsiveness of the KATP channel current density to high glucose was decreased in betaHNF-4{alpha}KO mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Genotyping—HNF-4{alpha}flox/flox mice with loxP sites flanking exons 4 and 5 of the HNF-4{alpha} gene were generated as described previously (16). Mice expressing Cre recombinase under control of the rat insulin 2 gene promoter (RIP-Cre) have been described elsewhere (17). RIP-Cre mice were backcrossed onto the C57BL/6 genetic background more than seven generations and maintained. Heterozygous (flox/wt) mice carrying one copy of the RIP-Cre transgene were interbred with flox/wt littermates lacking Cre (more than three generations) to generate pancreatic beta-cell-specific HNF-4{alpha} knock-out mice (betaHNF-4{alpha}KO) and littermate control mice (flox/flox (FLOX), RIP-Cre (CRE), and wt/wt (WT)). Age- and sex-matched littermates were used as the controls. The mice were genetic hybrids of C57BL/6 and 129/SvJ mice. Genotyping of HNF-4{alpha} was performed by PCR, as described previously (16). PCR to detect the Cre transgene was achieved with 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s using the following specific primers: RIP-CreF (5'-TTACCGGTCGATGCAACGAGTGATG-3') and RIP-CreR (5'-TTCCATGAGTGAACGAACCTGGTCG-3'). This study was performed according to the guidelines of the Animal Ethics Committee of Osaka University.

Western Blot Analysis—Pancreatic islets were isolated from mice by collagenase digestion, and the cells were lysed in extraction buffer (100 mmol/liter NaCl, 50 mmol/liter Tris-HCl (pH 8.0), 20 mmol/liter EDTA, and 1% SDS). Total protein was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with an antibody for HNF-4{alpha} (1, 18) and HNF-1{alpha} (BD Transduction Laboratories), as well as antibodies targeting actin (A5060, Sigma), Kir6.2 (P4621, Sigma; AB5495, Chemicon), and SUR1 (sc-25683, Santa Cruz Biotechnology). Bound antibodies were detected with a horseradish peroxidase-conjugated secondary antibody (Promega), and immune complexes were visualized using ECL Western blotting detection reagents (Amersham Biosciences). The images were scanned and quantified using ScanningImager and ImageQuant software (Amersham Biosciences).

Measurement of Glucose and Insulin Levels—Blood glucose levels were determined with a glucometer (Glutest Ace, Sanwa Kagaku, Japan). Plasma insulin concentrations were determined with a Glazyme insulin enzyme immunoassay kit (Wako, Japan). The pancreatic insulin content or islet insulin content was measured after extraction by the acid-ethanol method (19). A glucose tolerance test was performed at 10 and 24 weeks of age. Mice were fasted for 14 h, and blood was collected before and at 15, 30, 60, and 120 min after the intraperitoneal injection of glucose (1 g/kg or 2 g/kg). For the insulin tolerance test, mice were injected with 1.0 units/kg of regular human insulin after a 4-h fast.

Immunohistochemical Analysis—Under general anesthesia, 24-week-old mice were perfused with 4% paraformaldehyde in 0.1 mmol/liter phosphate buffer. Then the pancreas was harvested and fixed in the same solution for 12 h. Immunohistochemical analysis was performed using 6-µm sections of paraffin-embedded pancreatic tissues and the following primary antibodies: guinea pig anti-swine insulin (1:5,000) (Dako, Carpinteria, CA); rabbit anti-human glucagon (1:1,000) (Linco Research Inc., St. Charles, MO); rabbit anti-human somatostatin (1:200) (Dako); rabbit anti-human pancreatic polypeptide (1:1,000) (Dako); and mouse anti-human HNF-4{alpha} (1:200) (H1415, Perseus, Japan). The fluorescent-labeled secondary antibodies were biotinylated goat anti-guinea pig IgG (for insulin) (1:200) (Vector Laboratories), biotinylated goat anti-rabbit IgG (for glucagon, somatostatin, and pancreatic polypeptide) (1:200) (Vector Laboratories), and biotin-SP-conjugated AffiniPure donkey anti-mouse IgG (Jackson Immuno Research) (1:200). For amplification of the HNF-4{alpha} signal, a TSA kit (Molecular Probes) was used according to the manufacturer's instructions. Immunofluorescence was detected under a light microscope (Olympus, Tokyo) or a laser scan confocal microscope (Carl Zeiss, Jena). For quantitation of beta-cell mass, three mice of each genotype were analyzed at 24 weeks of age. Sections of paraffin-embedded pancreatic tissue were cut at 300-µm intervals and immunostained. The beta-cell area was calculated as a percentage of the total pancreatic area, as described previously (19).

Quantitation of mRNA by Real Time RT-PCR—Total RNA was isolated from the pancreatic islets of betaHNF-4{alpha}KO mice and flox/flox (FLOX) mice in Trizol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized by using a High capacity cDNA archive kit (Applied Biosystems), and real time PCR was performed with an ABI PRISM 7900 (Applied Biosystems, Foster City, CA) using specific primers and fluorescent probes. The primers and probes for GLUT2, insulin 1, insulin 2, glucokinase, glucose-6-phosphatase, aldolase B, SNAP-25, syntaxin 1, VDCC, Kir6.2, PDX-1, NeuroD, Nkx6.1, Pax6, Foxo1, PGC-1{alpha}, HNF-1beta, SHP, PPAR{alpha}, and TATA box-binding protein were all purchased from Applied Biosystems.

Perifusion Experiments—Pancreatic islets were isolated from betaHNF4-{alpha}KO mice and control mice at the age of 20-24 weeks by collagenase digestion. Islets were cultured overnight prior to the following experiments. Ten islets were perifused at 37 °C with Hepes-Krebs buffer (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.2 mM MgSO4, 20 mM NaHCO3, 2.8 mM glucose, and 10 mM Hepes) containing 0.5% (w/v) bovine serum albumin (BSA) (equilibrated with 5% CO2, 95% air (pH 7.4)) at a flow rate of 1 ml/min. After perifusion with 2.8 mM glucose for 40 min, the solutions were altered as indicated in Fig. 5.

Batch Incubation Experiments—Batch incubation was performed as described previously (19). Briefly, pancreatic islets (10 per tube) were preincubated at 37 °C for 60 min in Hepes-Krebs buffer supplemented with 0.5% (w/v) BSA. Then the islets were incubated for 30 min in 350 µl of the same buffer containing 500 µM tolbutamide or the buffer containing 16.7 mM glucose in the presence or absence of 1 nM GLP-1 (Peptide Institution, Osaka, Japan).

Intracellular Ca2+ Concentration of Pancreatic Islets—The cytosolic Ca2+ concentration was measured with fura-2 acetoxymethyl ester using two-photon excitation imaging as described previously (20, 21). In brief, isolated islets were incubated at 37 °C for 30 min in serum-free Dulbecco's modified Eagle's medium culture medium supplemented with bovine serum albumin (1 mg/ml), 0.1% Pluronic F-127, and 20 µM fura-2 acetoxymethyl ester (Molecular Probes), followed by washing with Sol A (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes-NaOH (pH 7.3), and 2.8 mM glucose). The laser power at the specimen was set at 10 milliwatts, and the excitation wavelength was 830 nm. Images were acquired every 2.5 s. The cytosolic Ca2+ concentrations in response to 20 mM glucose, 1 µM glibenclamide, and 30 mM KCl were calculated as (F0 - F)/F0, where F0 and F represent the resting and post-stimulation fluorescence, respectively.

Measurement of NAD(P)H Fluorescence—Autofluorescence of NAD(P)H (400-510 nm) in the islets was imaged using two-photon excitation microscopy at an excitation wavelength of 720 nm (22).

Measurement of the Islet ATP Content—The ATP content of pancreatic islets was measured by a quantitative bioluminescence assay using a modification of the protocol described previously (23). Experiments were performed on islets from betaHNF-4{alpha}KO mice and control mice after incubation for 48 h in RPMI 1640 medium containing 11 mM glucose. Groups of 10 islets (diameter 0.15-0.2 mm) were selected manually and preincubated in KRB containing 3 mM glucose for 60 min. After preincubation, the islets were incubated for 30 min in buffer containing 3 or 20 mM glucose. Then ATP was extracted from the islets and assayed using an ATP bioluminescence assay kit (CLS II, Roche Applied Science) according to the manufacturer's instructions.


Figure 1
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  		FIGURE 1.
Generation of beta-cell-specific HNF-4{alpha} knock-out mice. A, schematic representation of the floxed and null alleles. The null allele lacks exons 4 and 5 of the HNF-4{alpha} gene as a result of recombination between LoxP sites. B, Western blot analysis of HNF-4{alpha} and HNF-1{alpha} in the islets of control (WT, FLOX, CRE) and betaHNF-4{alpha}KO (KO) mice (10- and 24-week-old female mice). Expression of HNF-4{alpha} in betaHNF-4{alpha}KO islets was reduced by about 80% compared with that in control mice. HNF-1{alpha} expression was not affected in the betaHNF-4{alpha}KO islets. C, double immunofluorescent staining for the HNF-4{alpha} (green) and insulin (red) or somatostatin (SM) (red). The intensity of HNF-4{alpha} staining in beta-cells (arrows) was reduced in betaHNF-4{alpha}KO mice. Arrowheads indicate the expression of HNF-4{alpha} in non-beta-cells. Magnification, x40. D, Western blotting of HNF-4{alpha} in the liver and kidney of control and betaHNF-4{alpha}KO mice. The expression of HNF-4{alpha} was similar between control and KO mice.

 
Immunofluorescent Staining of Kir6.2—Islets were dispersed by trituration (200 passes through a pipette with a 200-µl tip) in Ca2+- and Mg2+-free Hanks' solution. The cells were then plated on Lab-TekII chamber slides (Nalge Nunc) and cultured for 2-3 days. For surface staining (24), the cells were incubated with anti-Kir6.2 antibody (Santa Cruz Biotechnology, sc-11228) at 4 °C. For total cellular staining of Kir6.2 or SUR1, the cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline and then incubated with anti-Kir6.2 antibody (sc-11228) or anti-SUR1 antibody (Santa Cruz Biotechnology, sc-25683).

Patch Clamp Recording—A patch clamp technique (25, 26) was used to record whole-cell currents from perforated cells with the pipette solution containing nystatin (150 µg/ml) dissolved in 0.1% Me2SO. Membrane currents were recorded by using an amplifier (200B Axopatch, Molecular Devices, Foster City, CA), and data were stored on-line in a computer with pCLAMP8 software. Voltage clamp in the perforated mode was defined as adequate when series resistance was less than 20 megohms. The patch pipettes were pulled from glass tubes (Narishige Co. Ltd., Tokyo, Japan). The resistance of each pipette was between 4 and 7 megohms when filled with the following pipette solution (content in mM): K2SO4 40, KCl 50, MgCl2 5, EGTA 0.5, and Hepes 10 (pH 7.2) with KOH. The perforated whole-cell clamp mode was established within 5-10 min after formation of a gigaohm seal between the patch pipette and the membrane of an isolated beta-cell. These cells were superfused with BSA-free Hepes-Krebs buffer containing 2.8 mM glucose for at least 30 min before the experiments. The capacitance of cells from control mice and betaHNF-4{alpha}KO mice was 7.4 ± 2.1 picofarads (n = 20) and 7.6 ± 2.7 picofarads (n = 16), respectively. The beta-cells were voltage-clamped at the holding potential of -70 mV, stepped to -100 mV for voltage ramping from -100 to -50 mV at a rate of 5 mV/100 ms, and then stepped back to -70 mV every 10 s. These experiments were performed at room temperature (22-25 °C).


Figure 2
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  		FIGURE 2.
Effect of beta-cell-specific ablation of HNF-4{alpha} on glucose metabolism. A and B, body weight (A) and nonfasting blood glucose level (B)of betaHNF-4{alpha}KO mice (n = 15, each genotype). C-F, intraperitoneal glucose tolerance tests performed in control and betaHNF-4{alpha}KO mice (C, 10-week-old males; D, 10-week-old females; E, 24-week-old males; F, 24-week-old females). Glucose (1 g/kg body weight (C, D, and F) and 2 g/kg body weight (E)) was injected intraperitoneally after a 14-h fast, and blood samples were collected at the indicated time intervals. G, blood glucose and plasma insulin concentrations of 24-week-old female mice in the fed state. Mice (n = 5, each genotype) were fed for 1 h after 14 h of fasting. H, blood glucose concentrations during insulin tolerance test in control and betaHNF-4{alpha}KO mice. Values are mean ± S.E. of the indicated number of mice. *, p < 0.05; **, p < 0.01.

 


Figure 3
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  		FIGURE 3.
A, immunohistochemistry of pancreatic hormones in 24-week-old female mice. Pancreatic sections were immunostained with insulin, glucagons, somatostatin, and pancreatic polypeptide (PP). Magnification, x40. B-D, quantitation of islet mass, islet number, and beta-cell area in FLOX and betaHNF-4{alpha}KO mice. Data are mean ± S.E. of values from three mice of each genotype. N.S., not significant. E and F, insulin content of FLOX and betaHNF-4{alpha}KO mice. Pancreata were removed from 24-week-old female mice (n = 4, each genotype), and insulin was extracted by the acid-ethanol method (E). For measurement of islet insulin content, islets were solubilized in acid-ethanol (F). Data are mean ± S.E.

 
Statistical Analysis—Results were expressed as the mean ± S.E. Statistical analysis was performed with the unpaired t test, and differences were considered to be significant at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of beta-Cell-specific HNF-4{alpha} Knock-out Mice—Pancreatic beta-cell-specific HNF-4{alpha} knock-out mice (betaHNF-4{alpha}KO) were generated by breeding heterozygous HNF-4{alpha}flox/wt mice carrying one copy of the RIP-Cre transgene with HNF-4{alpha}flox/wt mice lacking the Cre gene (Fig. 1A). Because exons 4 and 5 of the HNF-4{alpha} gene are flanked by loxP sites, recombination with Cre leads to production of a truncated HNF-4{alpha} protein lacking part of the DNA-binding domain and the C-terminal ligand-binding and transactivation domain (16). We examined the expression of HNF-4{alpha} in these mice by immunoblot analysis of pancreatic islets, liver, and kidney with HNF-4{alpha} antibody that recognizes the very C terminus of HNF-4{alpha} (1, 18). The expression of HNF-4{alpha} in the islets of betaHNF-4{alpha}KO mice was found to be reduced by 81-84% (Fig. 1B). As expected, HNF-4{alpha} is expressed in pancreatic beta-cells of wild-type mice (Fig. 1C, arrows). Immunostaining of pancreatic sections confirmed the efficient ablation of HNF-4{alpha} in beta-cells of betaHNF-4{alpha}KO mice (Fig. 1C). HNF-4{alpha} staining was detected in pancreatic non-beta-cells of betaHNF-4{alpha}KO mice (Fig. 1C, arrowheads). In contrast to the pancreatic beta-cells, expression of HNF-4{alpha} was not decreased in the liver or kidney (Fig. 1D), indicating that there was a beta-cell-selective loss of HNF-4{alpha} expression in these mice. Previous studies have shown that HNF-4{alpha} is a major regulator of HNF-1{alpha} gene expression in the liver, whereas HNF-1{alpha} regulates HNF-4{alpha} expression by pancreatic beta-cells (27). These findings suggest the existence of a positive feedback regulatory loop between HNF-4{alpha} and HNF-1{alpha} in the pancreatic beta-cells. However, the expression of HNF-1{alpha} was not decreased in betaHNF-4{alpha}KO islets (Fig. 1B), indicating that HNF-4{alpha} is not essential for HNF-1{alpha} expression by adult mouse beta-cells.


Figure 4
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  		FIGURE 4.
Gene expression in the islets ofbetaHNF-4{alpha}KO mice. Total RNA was extracted from islets of female betaHNF-4{alpha}KO (n = 4-5) and FLOX (n = 4-7) mice and subjected to real time RT-PCR. Data were normalized to the value for the control gene TATA box-binding protein (TBP) to yield the relative abundance. Data are mean ± S.E. *, p < 0.05.

 
Impaired Glucose Tolerance in betaHNF-4{alpha} Knock-out Mice—betaHNF-4{alpha}KO mice were born in a Mendelian fashion (data not shown). On inspection, betaHNF-4{alpha}KO mice were indistinguishable from their control littermates (wt/wt (WT), flox/flox (FLOX), and RIP-Cre (CRE)). Body weight (Fig. 2A) and the nonfasting blood glucose concentration (Fig. 2B) were similar in betaHNF4{alpha}KO mice and control mice at both 10 and 24 weeks. Because random blood glucose levels were similar in the betaHNF-4{alpha}KO mice and control littermates, the response to intraperitoneal administration of glucose was measured because this is a more sensitive determinant of glucose tolerance. Glucose tolerance was found to be normal in the male betaHNF-4{alpha}KO mice at 10 weeks (Fig. 2C). However, glucose levels at 15, 30, 60, and 120 min after administration of a glucose load were significantly higher in female KO mice at 10 weeks (Fig. 2D). Female mutant mice also showed a poor initial response of insulin secretion after intraperitoneal injection of glucose. At 24 weeks, both male and female betaHNF-4{alpha}KO mice exhibited glucose intolerance and an impaired insulin response to glucose loading (Fig. 2E). Most interestingly, the HNF-4{alpha}-deficient mice generated in this study showed no evidence of a decrease in blood glucose or elevation of plasma insulin levels in the fasting state (Fig. 2, C--F), as was reported in another strain of beta-cell-specific HNF-4{alpha} knock-out mice (28). Their knock-out mice also exhibited hyperinsulinemic hypoglycemia in the fed state (28), but our mutant animals also did not show hyperinsulinemic hypoglycemia in the fed state (Fig. 2G). The insulin tolerance test did not reveal any significant difference in insulin sensitivity between betaHNF-4{alpha}KO mice and control mice (Fig. 2H), excluding the possibility that the mutant mice were insulin-resistant.

Pancreatic Histology and Insulin Content in betaHNF-4{alpha}KO Mice—MODY3 model mice have an abnormal islet structure (19, 23). To determine whether any abnormalities of islet morphology were present, pancreatic sections from 24-week-old mice with different genotypes were examined by immunohistochemistry (Fig. 3A). It was found that the islet architecture was normal in betaHNF-4{alpha}KO mice, featuring a core of beta-cells surrounded by a layer of non-beta-cells. Also, the islet levels of insulin, glucagon, somatostatin, and pancreatic polypeptide expression were similar in KO and control animals. Quantitative analysis revealed no significant difference in the islet mass, islet number, islet size distribution, and beta-cell size between FLOX and KO mice (Fig. 3, B-D and supplemental Fig. 1). Furthermore, the pancreatic insulin content and insulin content of islets of betaHNF-4{alpha}KO mice were similar to that of control mice (Fig. 3, E and F). These results suggested that loss of HNF-4{alpha} from pancreatic beta-cells led to the impairment of beta-cell function rather than causing abnormalities of islet differentiation or insulin biosynthesis.

Pancreatic Islet Gene Expression—The effect of HNF-4{alpha} deficiency on gene expression was evaluated in the islets of 24-week-old betaHNF-4{alpha} mice and FLOX mice by real time RT-PCR (Fig. 4). Previous in vitrostudies have suggested that insulin, HNF-1{alpha}, GLUT2, and aldolase B are target genes for HNF-4{alpha} in beta-cells (13). However, expression of the insulin 1, insulin 2, and HNF-1{alpha} genes was unchanged in the present study, in agreement with the results mentioned above (Figs. 1B and 3E). Expression of the genes encoding GLUT2, aldolase B, glucokinase, glucose-6-phosphatase, VDCC, syntaxin-1, and SNAP-25, which are involved in insulin secretion by beta-cells, was also similar in betaHNF-4{alpha} islets and control islets. Furthermore, we found that the expression of several transcription factors that are abundant in the islets, including PDX-1, NeuroD, Nkx6.1, Pax6, Foxo1, PGC-1{alpha}, and HNF-1beta, did not differ between betaHNF-4{alpha}KO mouse islets and control mouse islets. It has been reported that HNF-4{alpha} may directly regulate the expression of the small heterodimer partner (SHP), an orphan nuclear receptor that forms heterodimers with various nuclear receptors, including HNF-4{alpha} (29). However, SHP expression was unchanged in betaHNF-4{alpha}KO mice. PPAR{alpha} is a transcription factor belonging to the nuclear receptor family that regulates the expression of genes involved in the beta-oxidation of fatty acids (30). Expression of PPAR{alpha} was reduced in liver-specific HNF-4{alpha}KO mice (16). Consistent with this result, PPAR{alpha} gene expression was reduced in betaHNF-4{alpha}KO islets relative to the level seen in control animals, suggesting that PPAR{alpha} was a target of HNF-4{alpha} in pancreatic beta-cells.


Figure 5
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  		FIGURE 5.
A and B, insulin secretory responses to glucose (A), KCl (A), and arginine (B) in perifused islets of control and betaHNF-4{alpha}KO mice. The peak insulin secretory response to 20 mM glucose was significantly reduced in the betaHNF-4{alpha}KO islets. Data represent the mean ± S.E. of four experiments in each genotype. *, p < 0.05. C, insulin secretion in batch-incubated islets of betaHNF-4{alpha}KO and FLOX mice. Insulin release by KO mice in response to 1 nM GLP-1 was similar to that by control islets. Data represent the mean ± S.E. of 10-16 experiments in each genotype. *, p < 0.05.

 
Insulin Secretion Profile of Perifused Islets and Changes of Intracellular Ca2+—To define the defect of insulin secretion in betaHNF-4{alpha}KO mice, isolated islets were stimulated with several different secretagogues. Glucose stimulates insulin secretion by inducing the closure of KATP channels via generation of ATP. Glycolysis generates ATP both directly and indirectly via the production of NADH. An increase of the ATP/ADP ratio leads to membrane depolarization and to an increase of cytosolic [Ca2+]i, via the VDCC, and eventually results in the exocytosis of insulin-containing secretory granules (31). First, we tested insulin secretion from perifused pancreatic islets after stimulation with glucose. There was no significant difference in basal insulin secretion between betaHNF-4{alpha}KO islets and control islets in the presence of 2.8 mM glucose. However, insulin secretion in response to a high glucose concentration (20 mM) was significantly reduced in betaHNF-4{alpha}KO islets compared with control islets (peak insulin response, WT; 102.2 pg/min/islet, FLOX; 141.2 pg/min/islet, CRE; 141.1 pg/min/islet, betaHNF-4{alpha}KO; 51.1 pg/min/islet, p < 0.05) (Fig. 5A and data not shown). In contrast, insulin secretion in response to depolarization induced by 25 mM KCl (Fig. 5A) or 20 mM arginine (Fig. 5B) was normal in betaHNF-4{alpha}KO mice, indicating that there was no defect of the later steps of insulin secretion after elevation of [Ca2+]i in betaHNF-4{alpha}KO islets. GLP-1 (glucagon-like peptide-1) potentiates insulin secretion from pancreatic beta-cells by increasing intracellular cAMP concentration (32). The insulinotropic action of GLP-1 was also preserved in betaHNF-4{alpha}KO islets (FLOX, 2.2-fold; KO, 2.5-fold) (Fig. 5C). The glycerol-3-phosphate dehydrogenase reaction forms NADH + H+, which can be oxidized by the respiratory chain in mitochondrion to produce ATP. To evaluate further the reasons for defective glucose-stimulated insulin secretion by betaHNF-4{alpha}KO islets, we measured NAD(P)H generation by two-photon excitation microscopy (Fig. 6A). Changes in the fluorescence intensity in response to a glucose load were similar for betaHNF-4{alpha}KO and control islets, suggesting that there was no defect in NADH formation. The ATP content was also measured in islets from betaHNF-4{alpha}KO mice and control mice after incubation with 3 and 20 mM glucose using a quantitative bioluminescence assay (Fig. 6B). It was found that the ATP levels in betaHNF-4{alpha}KO islets were similar to those in control islets, suggesting that glucose metabolism was normal in the mutant islets.

Changes of Islet Intracellular Ca2+—Next, we examined whether the impairment of glucose-stimulated insulin secretion was associated with a reduced intracellular Ca2+ level. Changes of [Ca2+]i in betaHNF-4{alpha}KO islets after a glucose load were significantly smaller compared with those in control islets (Fig. 7A). In contrast, both the amplitude and time course of the KCl-induced increase of [Ca2+]i did not differ between the two groups (Fig. 7B). Sulfonylurea-like glibenclamide stimulates insulin secretion by blocking KATP channels. As was the case for glucose, the [Ca2+]i response to glibenclamide stimulation was significantly decreased (Fig. 7C). The rise of [Ca2+]i in response to tolbutamide stimulation was also decreased (Fig. 7D). In agreement with the above results, stimulation of betaHNF-4{alpha}KO islets using tolbutamide only produced 37% of the insulin secretion seen with control islets (Fig. 7E). Taken together, these results indicate that KATP channel function is defective in betaHNF-4{alpha}KO mice.

Expression of Kir6.2 and SUR1 Proteins by betaHNF-4{alpha}KO Islets—The KATP channels in pancreatic beta-cells are composed of Kir6.2 and SUR1 (33). Because lack of Kir6.2 or SUR1 expression leads to defective insulin secretion in response to glucose or sulfonylurea stimulation (34, 35), we evaluated the level of Kir6.2 and SUR1 expression in betaHNF-4{alpha}KO islets by Western blotting. Expression of Kir6.2 was significantly reduced in the previously reported beta-cell-specific HNF-4{alpha}(-/-) mice (28). In marked contrast, expression levels of Kir6.2 protein were not reduced in the islets of our betaHNF-4{alpha}KO mice (Fig. 8, A and B, and supplemental Fig. 2). Expression of Kir6.2 mRNA was also unchanged in betaHNF-4{alpha}KO mice (Fig. 8C). Adequate surface expression of KATP channels is necessary for normal insulin secretion (24, 36). To examine the expression of Kir6.2 on the cell surface, we performed immunofluorescent staining using Kir6.2 antibody (sc-11228), which recognizes the extracellular domain of Kir6.2 (33). As shown in Fig. 8D, the level of Kir6.2 expression was similar in FLOX and KO islets. In addition, SUR1 protein was also expressed at similar levels in both betaHNF-4{alpha}KO mice and control mice (Fig. 8, D and E). These results indicated that the KATP channel defect in betaHNF-4{alpha}KO mice was not caused by reduced expression of Kir6.2 or SUR1.


Figure 6
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  		FIGURE 6.
A, NAD(P)H response to glucose in betaHNF-4{alpha}KO (n = 4) and FLOX (n = 4) islets. Averaged autofluorescence intensity is shown. Responses to 20 mM glucose were similar between control and betaHNF-4{alpha}KO islets. B, ATP content in betaHNF-4{alpha}KO and control islets. ATP content was measured in islets from betaHNF-4{alpha}KO (closed box) and control (open box) mice following incubation at 3 and 20 mM glucose, using quantitative bioluminescence assay. ATP production by betaHNF-4{alpha}KO islets was similar to that by control islets. Data are mean ± S.E. **, p < 0.01 (3 mM versus 20 mM). N.S., not significant.

 
Changes of KATP Channel Current Density in betaHNF-4{alpha}KO Islets—Fig. 9A displays the KATP channel currents recorded during voltage ramping from -100 to -50 mV. Because the KATP channel has voltage-independent kinetics and is a major determinant of the resting potential of beta-cells, the currents elicited by a voltage ramp run through these channels. In control cells exposed to 2.8 mM glucose, the current direction was reversed around -70 mV, and the current was substantially inhibited from 3 to 10 min after increasing the glucose concentration to 8.3 mM (Fig. 9A, left panel). The inhibited current did not reverse its direction during the ramp procedure, indicating that the resting membrane potential was higher than -50 mV at this glucose concentration. On the other hand, the beta-cells from betaHNF-4{alpha}KO mice yielded a current that crossed 0 even at 8.3 mM, with a subtle shift of the reversal potential despite marked reduction of the current amplitude (right panel). Thus, in beta-cells from betaHNF-4{alpha}KO mice, the KATP channel current showed a decrease of amplitude in response to 8.3 mM glucose, but the reduction was insufficient to allow membrane depolarization to reach the threshold potential of VDCC and produce an action potential. Among the 10 betaHNF-4{alpha}KO beta-cells tested, three cells were responsive to glucose stimulation and showed a concurrent reduction of the KATP channel current without any action potential (Fig. 9A). Three cells responded to 8.3 mM glucose with an action potential, but the other four cells were unresponsive to glucose stimulation regarding the current inhibition. In contrast to these cells from betaHNF-4{alpha}KO mice, we observed good glucose responsiveness in all four control beta-cells, and action potentials occurred after an increase of the glucose concentration to 8.3 mM. The current density recorded from cells of betaHNF-4{alpha}KO mice at 2.8 mM glucose was significantly increased compared with that from control cells at the same glucose concentration (Fig. 9B). There was a nonsignificant decrease of the current density recorded from betaHNF-4{alpha}KO beta-cells as the glucose concentration increased to 8.3 mM, whereas the current density obtained from control cells was significantly inhibited by 8.3 mM glucose. Thus, the responsiveness of KATP channel current density to 8.3 mM glucose was impaired in betaHNF-4{alpha}KO mice. Enhanced activity of the KATP channels in betaHNF-4{alpha}KO mice was not attributable to reduced ATP sensitivity of the channels, because the 50% inhibitory concentration for ATP was 1.5 µM when tested on inside-out membrane patches excised from the beta-cells of betaHNF-4{alpha}KO mice (data not shown). This value was rather lower than previously reported for mouse beta-cells (37). Next, the tolbutamide sensitivity of these beta-cells was tested (Fig. 9C). The 50% inhibitory concentration was 6.2 and 3.6 µM for cells from control and betaHNF-4{alpha}KO mice, respectively. Therefore, the channels had a similar sensitivity to tolbutamide in both types of mice, but the inhibitory effect of 10-100 µM tolbutamide may be insufficient to produce insulin secretion in betaHNF-4{alpha}KO mice (Fig. 9C).


Figure 7
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  		FIGURE 7.
A-C, the response of [Ca2+]i to glucose, KCl, and glibenclamide in betaHNF-4{alpha}KO islets. Changes in [Ca2+]i in response to 20 mM glucose (A), 30 mM KCl (B), 1 µM glibenclamide (C), and 500 µM tolbutamide (D) were measured in islets with fura-2 using two-photon excitation imaging in pancreatic beta-cells of either FLOX or betaHNF-4{alpha}KO mice and was represented by (F0 - F)/F0, where F0 and F indicate resting fluorescence and fluorescence after stimulation, respectively. (F0 - F)/F0 (10 s after the rise) in response to each secretagogue was calculated, and the mean ± S.E. is presented. N.S., not significant. **, p < 0.01. E, insulin secretion in batch-incubated islets of betaHNF-4{alpha}KO and control mice. Insulin release in response to 500 µM tolbutamide was significantly impaired in betaHNF-4{alpha}KO islets. Data are mean ± S.E. **, p < 0.01.

 


Figure 8
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  		FIGURE 8.
Expression of Kir6.2 and SUR1 proteins in betaHNF-4{alpha}KO islets. Islets isolated from 24-week-old female control and betaHNF-4{alpha}KO mice were subjected to immunoblot analysis with antibodies against Kir6.2 (A) and SUR1 (E). B, Kir6.2 expression levels in isolated islets were normalized to actin protein levels. Data are mean ± S.E. N.S., not significant. C, levels of Kir6.2 mRNA in control (n = 7) and betaHNF-4{alpha}KO (n = 5) islets. D, control and KO islets showed similar levels of surface staining of Kir6.2 and SUR1. The scale bar represents 5 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mutations in the gene encoding HNF-4{alpha} cause MODY associated with impaired insulin secretion (5). In addition, variation of the beta-cell-specific promoter in the HNF-4{alpha} gene is associated with common type 2 diabetes (10-12), so investigation of the role of HNF-4{alpha} in pancreatic beta-cells could lead to a better understanding of type 2 diabetes as well as MODY. In the present study, we generated beta-cell-specific HNF-4{alpha} knock-out mice using the Cre-LoxP system with a rat insulin promoter-driven Cre transgene. Expression of HNF-4{alpha} protein was reduced by about 80% in betaHNF-4{alpha}KO islets compared with control islets, and the betaHNF-4{alpha}KO mice exhibited impaired glucose tolerance with defective insulin secretion, which is characteristic of human MODY1. However, the mice did not develop diabetes at 6 months of age. Because a dominant negative effect cannot be a major cause of human MODY1 (6, 7, 38), the reason why these mice did not develop diabetes remains to be solved. Because the insulin response to GLP-1 was normal in betaHNF-4{alpha}KO islets, the response to incretins may be one of the potential mechanisms compensating for the impaired response to glucose.

HNF-4{alpha} plays an essential role in hepatic development and organogenesis, and it is also expressed by the developing pancreas in mice (39).3 Although the insulin promoter-driven Cre expression occurs in neonates (40), the possibility exists that excision of the floxed allele may occur too late to prevent HNF-4{alpha} from acting in developing beta-cells. This possibility could have been tested by using another promoter system (e.g. PDX-1-Cre). Although there is a possibility that perturbation of HNF-4{alpha} activity in another organ such as liver may affect the MODY1 phenotype, our results obtained with isolated islets clearly indicated that HNF-4{alpha} was necessary for normal insulin secretion by beta-cells.

Gupta et al. (28) recently reported another line of beta-cell-specific HNF-4{alpha} knock-out mice, but there were several important differences between our mutant mice and theirs. First, their mice showed a significant decrease of blood glucose levels in both the fed and fasting states. In contrast, there were no significant differences of blood glucose levels between our betaHNF-4{alpha}KO and control animals. Second, they reported that plasma insulin levels were significantly elevated in their mutant mice, but the insulin levels in our KO mice were indistinguishable from that of control mice. Third, Kir6.2 expression was significantly reduced in the islets of their HNF-4{alpha} mutant mice but not in the islets of our animals. Although expression of HNF-4{alpha} was significantly decreased (<10%) in the islets of HNF-1{alpha} knock-out mice, expression of Kir6.2 was also unchanged in the islets (29). At this point, we have no adequate explanation for these differences. Although the same RIP-Cre transgene (17) was used to generate both HNF-4{alpha}beta-cell-specific null mouse lines, different background strains of mice were used in the two studies (129 Svj x C57BL/6 mice in our work versus CD1 mice). This may contribute in part to the different phenotypes obtained in the two studies. Because hyperinsulinemic hypoglycemia has not been reported clinically in MODY1 patients (8, 9, 41), we believe that our new beta-cell-specific HNF-4{alpha} knock-out mice is more reflective of human MODY1 and thus may provide a useful animal model of this disease.


Figure 9
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  		FIGURE 9.
Response of the KATP channel current to glucose and tolbutamide in control and betaHNF-4{alpha}KO mice. A, KATP channel currents recorded during voltage ramping from -100 to -50 mV at glucose concentrations of 2.8 and 8.3 mM. Voltage-clamped beta-cells in the perforated mode from a control mouse (left panel) and a betaHNF-4{alpha}KO mouse (right panel) are shown. Dotted lines indicate the zero current level. Note that calibration bars indicating the current magnitude are different in the two panels. B, comparison of current density response to changes of the glucose concentration in control mice and betaHNF-4{alpha}KO mice. KATP channel conductance was measured from the slope of the current trace during voltage ramping, and the current density was obtained as the conductance divided by the cell capacitance. *, p < 0.05 versus control cells with 2.8 mM glucose. C, dose-response relation between the KATP channel current density and the tolbutamide concentration. The glucose concentration in the external solution was 2.8 mM throughout these experiments. Curves were fitted by a Michaelis-Menten type equation. Data are shown as the mean ± S.E. *, p < 0.05; **, p < 0.02 versus control.

 
Our patch clamp experiments revealed that KATP channel activity was up-regulated in betaHNF-4{alpha}KO mice. It has been reported that decreased responsiveness of the KATP channel to ATP leads to impaired insulin secretion and diabetes in both mice and humans (42, 43). However, our data did not support this possibility in betaHNF-4{alpha}KO mice because their ATP sensitivity was not reduced. The molecular mechanism by which HNF-4{alpha} regulates the KATP channel is still unclear, but a defect of this channel could be related to the impaired insulin secretion observed in betaHNF-4{alpha}KO mice. KATP channel activity is regulated by various factors, e.g. Kir6.2 is phosphorylated by protein kinase A and its phosphorylation increases KATP channel activity (44). GTP-binding proteins have also been shown to modulate KATP channel activity (44). Expression of PPAR{alpha} was reduced in betaHNF-4{alpha}KO islets (Fig. 4), but its effect on KATP channel activity is unknown. Further studies will be necessary to define the mechanism of KATP channel regulation by HNF-4{alpha}.

Recently, Odom et al. (45) reported that HNF-4{alpha} showed binding to the promoters of 11% of islet genes. In our mice, however, expression of the many genes involved in insulin secretion was unchanged. Detailed studies of our betaHNF-4{alpha}KO mice may lead to a better understanding of the target genes of HNF-4{alpha} in pancreatic beta-cells, as well as the molecular basis of both MODY and common type 2 diabetes.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. Back

1 To whom correspondence should be addressed: Dept. of Metabolic Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3732; Fax: 81-6-6879-3739; E-mail: kazu{at}imed2.med.osaka-u.ac.jp.

2 The abbreviations used are: HNF, hepatocyte nuclear factor; RT, reverse transcription; BSA, bovine serum albumin; WT, wild type; KO, knock out; MODY1, maturity-onset diabetes of the young; PPAR, peroxisome proliferator-activated receptor; VDCC, voltage-dependent calcium channel; SHP, small heterodimer partner. Back

3 T. Nammo and K. Yamagata, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank T. Miki for helpful discussions and T. Tanaka, and T. Kodama for providing HNF-4{alpha} antibody. We also thank A. Ihara, F. Katsube, Y. Tochino, and M. Onishi for technical assistance.



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