Overexpression of Dominant-Negative Mutant Hepatocyte ...ton, KY) or anti–E-cadherin antibody (kindly provided by Dr. R. Kemler, Max-Planck-Institute, Freiburg, Germany) (11) overnight

Overexpression of Dominant-Negative MutantHepatocyte Nuclear Factor-1� in Pancreatic �-CellsCauses Abnormal Islet Architecture With DecreasedExpression of E-Cadherin, Reduced �-cell Proliferation,and DiabetesKazuya Yamagata,

1Takao Nammo,

1Makoto Moriwaki,

1Arisa Ihara,

1Katsumi Iizuka,

1Qin Yang,

1

Tomomi Satoh,1

Ming Li,1

Rikako Uenaka,1

Kohei Okita,1

Hiromi Iwahashi,1

Qian Zhu,1

Yang Cao,1

Akihisa Imagawa,1

Yoshihiro Tochino,1

Toshiaki Hanafusa,2

Jun-ichiro Miyagawa,1

and Yuji Matsuzawa1

One subtype of maturity-onset diabetes of the young(MODY)-3 results from mutations in the gene encodinghepatocyte nuclear factor (HNF)-1�. We generatedtransgenic mice expressing a naturally occurring domi-nant-negative form of human HNF-1� (P291fsinsC) inpancreatic �-cells. A progressive hyperglycemia withage was seen in these transgenic mice, and the micedeveloped diabetes with impaired glucose-stimulatedinsulin secretion. The pancreatic islets exhibited abnor-mal architecture with reduced expression of glucosetransporter (GLUT2) and E-cadherin. Blockade of E-cadherin–mediated cell adhesion in pancreatic isletsabolished the glucose-stimulated increases in intracel-lular Ca2� levels and insulin secretion, suggesting thatloss of E-cadherin in �-cells is associated with impairedinsulin secretion. There was also a reduction in �-cellnumber (50%), proliferation rate (15%), and pancreaticinsulin content (45%) in 2-day-old transgenic mice anda further reduction in 4-week-old animals. Our findingssuggest various roles for HNF-1� in normal glucosemetabolism, including the regulation of glucose trans-port, �-cell growth, and �-cell–to–�-cell communication.Diabetes 51:114–123, 2002

Maturity-onset diabetes of the young (MODY) isa group of disorders characterized by earlyonset diabetes (usually before 25 years ofage), pancreatic �-cell dysfunction, and auto-

somal dominant inheritance (1). MODY3 results from

heterozygous mutations in the homeodomain-containingtranscription factor hepatocyte nuclear factor (HNF)-1�(2). HNF-1� is known to be expressed in liver, kidney,intestine, and pancreas (3). Most MODY3 subjects underthe age of 10 years have a normal fasting blood glucoseand a normal glucose tolerance. However, they developmarked hyperglycemia with a progressive impairment ininsulin secretion (4–6). The molecular mechanisms bywhich mutations in the HNF-1� gene lead to �-cell dys-function and diabetes are unclear. Mice lacking HNF-1�exhibit a defect in glycolytic signaling of insulin secretion,suggesting low expression of proteins in this signalingpathway (7,8). However, unlike MODY3 patients, HNF-1�knockout mice exhibit severe liver and kidney dysfunc-tion.

We have recently shown that the most common muta-tion found in HNF-1�, P291fsinsC, acts in a dominant-negative manner (9). To gain a better understanding of themolecular basis of HNF-1� diabetes, we generated trans-genic mice expressing a hybrid rat insulin promoter (RIP)-P291fsinsC–HNF-1� transgene. The transgenic micedeveloped progressive hyperglycemia. Our data suggestthat HNF-1� plays an important role in maintenance ofvarious functions of normal �-cells, including glucosetransport, �-cell growth, and �-cell communication.

RESEARCH DESIGN AND METHODS

Generation of transgenic mice. Human P291fsinsC–HNF-1� cDNA (9) wasligated to the rat insulin II promoter containing expression vector (kindlyprovided by Dr. R. Palmiter, University of Washington, Seattle, WA). A 3.4-kbHindIII expression unit was microinjected into the fertilized eggs of (C57BL/6 � SJL)F2 mice. Transgenic mice were selected by Southern blot analysis andpolymerase chain reaction (PCR) and were backcrossed to C57BL/6 mice (�4generations) for experiments. RIP wild type (WT)–HNF-1� transgene (9) wasalso constructed, and transgenic mice expressing WT–HNF-1� in pancreatic�-cells were generated. Age- and sex-matched transgene-negative littermateswere used as control mice throughout the study. The experimental protocolwas approved by the Ethics Review Committee for Animal Experimentation ofOsaka University.Western blot analysis. Pancreatic islets were isolated from mice by thecollagenase digestion method (10). Cells were lysed in extraction buffer [100mmol/l NaCl, 50 mmol/l Tris-HCl (pH 8.0), 20 mmol/l EDTA, and 1% SDS], andWestern blot was performed as described (9). The membrane was incubatedwith monoclonal anti–HNF-1� antibody (Transduction Laboratories, Lexing-

From the 1Department of Internal Medicine and Molecular Science, GraduateSchool of Medicine, Osaka University, Osaka, Japan; and the 2First Depart-ment of Internal Medicine, Osaka Medical College, Osaka, Japan.

Address correspondence and reprint requests to Kazuya Yamagata, MD,Department of Internal Medicine and Molecular Science, Graduate School ofMedicine, B5, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871,Japan. E-mail: [email protected].

Received for publication 12 July 2001 and accepted in revised form 17October 2001.

K.Y. and T.N. contributed equally to this study.BrdU, bromo-2�-deoxyuridine; HKRB, HEPES-balanced Krebs-Ringer bicar-

bonate; HNF, hepatocyte nuclear factor; MODY, maturity-onset diabetes of theyoung; PCR, polymerase chain reaction; PP, pancreatic polypeptide; RIP, ratinsulin promoter; RT, reverse transcription; WT, wild type.

114 DIABETES, VOL. 51, JANUARY 2002

ton, KY) or anti–E-cadherin antibody (kindly provided by Dr. R. Kemler,Max-Planck-Institute, Freiburg, Germany) (11) overnight at 4°C and furtherincubated with horseradish peroxidase–conjugated anti-IgG antibody (Pro-mega, Osaka, Japan). The binding antibody was visualized using enhancedchemiluminescence Western Blotting detection reagents (Amersham LifeSciences, Little Chalfont, U.K.).Reverse transcription-PCR. Total RNA was isolated from pancreas, liver,kidney, and brain using TRIzol reagent (Life Technologies, Rockville, Mary-land). Contaminating genomic DNA was removed by treatment with DNase I.cDNA synthesis was performed with 1 �g of total RNA using Moloney murineleukemia virus reverse transcriptase (Life Technologies) with dNTPs andoligo dT primers. The following primers were used for the specific PCRamplification of human HNF-1�: 5�-AGGACCTGAGCCTGCCGAGCAAC-3� and5�-AGGGCTCTCCATAGGCCCAGGCT-3� (annealing temperature 60°C andproduct size 281 bp) and mouse GAPDH: 5�-TGACAACTCACTCAAGATTG-3�and 5�-CACGTCAGATCCACGACGGA-3� (annealing temperature 60°C andproduct size 321 bp).Measurements of blood glucose and insulin level. Glucose tolerancetesting was performed in female mice (8 weeks of age) because male miceexhibited severe hyperglycemia. Mice were fasted for 16 h and then loadedwith 1 g/kg glucose through the tail vein. Serum insulin levels were determinedby Glazyme insulin enzyme immunoassay kit (Wako, Osaka, Japan). Insulincontent was measured after extraction by the acid-ethanol method (12).Immunohistochemical analysis. Male mice were used in all histologicalstudies unless otherwise mentioned. Immunohistochemistry was performedas described (13). The following primary antibodies were used: guinea piganti-insulin (Dako, Tokyo, Japan), rabbit anti-glucagon (Linco, St. Charles,MO), rabbit anti-somatostatin (Dako), rabbit anti–pancreatic polypeptide (PP)(Dako), rabbit anti–PDX-1 (13), goat anti-glucokinase (Santa Cruz, Santa Cruz,CA), rabbit anti-GLUT2 (kindly provided by Dr. B. Thorens, Institute ofPharmacology and Toxicology, Lausanne, Switzerland), rat anti–N-cadherin(kindly provided by Dr. M. Takeichi, Kyoto University, Kyoto, Japan), and ratanti–E-cadherin (11). The anti-Pax6 antiserum was prepared after immunizinga rabbit with synthetic peptides (QVPGSEPDMSQYWPRLQ, amino acid resi-dues 396 to 422) (14). Immunofluorescence was viewed using a laser scanconfocal microscope (LSM510; Carl Zeiss, Jena, Germany) or a light micro-scope.Morphometry. Male Tg-1 and control mice (2 days old and 4 weeks old) wereused for morphometric analysis. Morphometric analysis was performed aspreviously described (13). Three sets of 10 serial sections were obtained fromeach pancreas. The first section in each set was immunostained for insulin,and the second one was stained for glucagon, somatostatin, and PP. The totalarea of the pancreas was measured under a television monitor. �-Cell andnon–�-cell counts were determined in six sections in each pancreas. Therelative numbers of �-cells and non–�-cells were expressed as the number perone square millimeter of the pancreatic area.Cell proliferation rate. �-cell and non–�-cell proliferation rates weredetermined by 5 bromo-2�-deoxyuridine (BrdU) incorporation (13). Male Tg-1and control mice were injected intraperitoneally with 100 mg/kg of BrdU (CellProliferation Kit; Amersham Pharmacia Biotech) and killed 6 h later. Four setsof five serial sections were cut out from a paraffin block. The first section wasimmunostained for insulin and BrdU, and the second section was stained fornon–� cell hormones (glucagon, somatostatin, and PP) and BrdU. Data areshown as the sum of the four separate pancreas sections from four differentmice in each group.Batch incubation experiments. Ten islets isolated from 6- to 7-week-oldfemale Tg-1 and control mice were preincubated at 37°C for 60 min inHEPES-balanced Krebs-Ringer bicarbonate (HKRB) buffer (15). The pancre-atic islets were then incubated for 20 min in a buffer containing 3.3 or 20mmol/l glucose.Measurements of intracellular calcium concentrations. Isolated isletsfrom Tg-1 mice were plated on microwell dishes (MatTek, Ashland, MA). Cellswere loaded with 5 �mol/l fura-2 acetoxymethyl ester (Dojin, Kumamoto,Japan) for 30 min (15). Changes in [Ca2�]i in response to 15 mmol/l glucoseand 20 mmol/l KCl were expressed as the ratio of the emitted light intensity(detected at 510 nm) after excitation at 340 and 380 nm (ratio 340/380) (7).Islet cells of control mice were incubated in the presence of rat anti–E-cadherin IgG1 or rat nonimmune IgG1 (200 ng/ml) for 48 h, and changes in[Ca2�]i were also measured.Perifusion experiments. Islet cells were plated on 24-well dishes andcultured in the presence of rat anti–E-cadherin IgG1 or rat nonimmune IgG1(200 ng/ml) for another 48 h. Islet cells were perifused at 1 ml/min with HKRBbuffer. After preincubation for 60 min, 26-mmol/l glucose stimulation wasapplied.

Statistical analysis. Results are expressed as means � SD. Differences wereanalyzed using a two-tailed unpaired Student’s t test. P 0.05 was consideredstatistically significant.

RESULTS

P291fsinsC–HNF-1� transgenic mice have impaired

�-cell function. We generated transgenic mice express-ing human dominant-negative mutant (P291fsinsC–HNF-1�) (Fig. 1A) in �-cells. Two founder animals wereobtained and bred with C57BL/6 mice to generate trans-genic lines. They had different transgene copy numbers (5Tg-1 and 20 Tg-2 copies). Western blot analysis wasperformed using proteins prepared from isolated islets.More than 100 islets were generally found after prepara-tion by the collagenase digestion method from a 6- to8-week-old control mouse, whereas only 10–20 isletscould be observed from a transgenic mouse. The levels ofexpression of mutant proteins in Tg-1 and Tg-2 mice wereestimated to be 6- and 24-fold greater than that of endog-enous HNF-1�, respectively (Fig. 1B). We also generatedtransgenic mice with WT–HNF-1� for comparison. Thelevel of expression of the transgene was fivefold greatercompared with that of endogenous HNF-1�. Reverse tran-scription (RT)-PCR analysis was performed to examinethe extra-pancreas expression of P291fsinsC–HNF-1� gene(Fig. 1C). Expression of the transgene was not detected inliver and heart, but leaky expression was found in brain, aspreviously reported (16). P291fsinsC–HNF-1� mice weregrossly indistinguishable from their control littermates atbirth. Nonfasting blood glucose concentrations and bodyweights were similar between HNF-1� mutant mice andcontrol littermates at 2 days of age (Fig. 1D). Nonfastingblood glucose levels at 4 weeks were higher in maletransgenic mice than in control mice and increased furtherto 406 � 154 mg/dl (Tg-1, n 18, P 0.001) and 485 � 147mg/dl (Tg-2, n 16, P 0.001) at 8 weeks of age (Fig. 1E).Nonfasting blood glucose levels of female Tg-1 mice at 4weeks were comparable with those of control mice,whereas blood glucose concentration of most female Tg-2mice (13/16) were higher than the average glucose levels(136 mg/dl) in control mice. Nonfasting blood glucoseconcentrations in female transgenic mice were lower at 8weeks of age than in male mutant mice, but the concen-tration was still significantly higher than in control mice(Fig. 1E). Sexual dimorphism of sensitivity to diabetes hasbeen reported in rodent models of diabetes (17).

Insulin levels at 5 min after intravenous glucose loadwere significantly low both in Tg-1 and Tg-2 mice (Fig. 2A).Pancreatic islets were isolated from 6- to 7-week-oldfemale Tg-1 mice and their littermates, and insulin secre-tion was examined. Glucose at a concentration of 20mmol/l stimulated insulin secretion in the islets of controlmice by 9.7-fold. Significant impairment of insulin secre-tion (2.1-fold, P 0.01) in response to the same dose ofglucose, however, was observed in Tg-1 mice (Fig. 2B).Changes in [Ca2�]i after glucose stimulation in �-cells of 6-to 8-week-old female Tg-1 mice were measured to examinewhether the reduction in glucose-stimulated insulin secre-tion is associated with reduced [Ca2�]i changes (Fig. 2C).Changes in [Ca2�]i in islets of transgenic mice were reducedcompared with islets in the control mice. However, theresponses in [Ca2�]i to 20 mmol/l KCl were similar be-

K. YAMAGATA AND ASSOCIATES

DIABETES, VOL. 51, JANUARY 2002 115

FIG. 1. Phenotypes of P291fsinsC–HNF-1� Tg mice. A: Schematic representation of P291fsinsC–HNF-1� protein. B: Western blot analysis ofexpression levels of WT and P291fsinsC–HNF-1� in pancreatic islets of control and mutant mice. C: Extra-pancreas expression of theP291fsinsC–HNF-1� transgene. Total RNA was prepared from several tissues of control (lanes 1-4), Tg-1 (lanes 5-8) and Tg-2 (lanes 9-12) miceand used for RT-PCR. Lanes 1, 5, and 9: pancreas; lanes 2, 6, and 10: liver; lanes 3, 7, and 11: heart; and lanes 4, 8, and 12: brain. Leaky expressionwas detected in brain. D: Nonfasting blood glucose level and body weight in 2-day-old P291fsinsC–HNF-1� mice. E: Nonfasting glucoseconcentrations of 4-week-old male mice (�) and 8-week-old male mice (f). F: Nonfasting blood glucose concentrations of 4-week-old female mice(�) and 8-week-old female mice (f). Data shown in D– F are means � SE of the indicated number of mice. N.S., not significant. *P < 0.05, **P <0.01, ***P < 0.001.

DOMINANT-NEGATIVE HNF-1� TRANSGENIC MICE


tween control and mutant mice. A defective response toglucose has been reported in HNF-1� knockout mice (7).

We also measured pancreatic insulin content in malemice (Fig. 2D). Insulin content of transgenic mice wasalready significantly reduced (Tg-1 45% of control P 0.001 and Tg-2 31% of control, P 0.001) at day 2. Insulincontent in the pancreas of mutant mice was furtherreduced to 4% (Tg-1 P 0.001) and 3.8% (Tg-2 P 0.001)of that in the control mice at 8 weeks of age.P291fsinsC–HNF-1� transgenic mice have abnormal

islet architecture. Staining for insulin in the pancreas of8-week-old Tg-1 mice revealed that islets were small in size

and contained fewer insulin-positive cells (Fig. 3A and B).We also analyzed the islets of Tg-1 mice at 2 days and at 4weeks of age (Fig. 3C– N). Mature islet structure with acore of �-cells surrounded by a mantle of glucagon cellswas observed 2 days postnatally in control mice (Fig. 3C

and D). In contrast, the endocrine cells did not formwell-organized islets in the pancreas of 2-day-old Tg-1 mice(Fig. 3E and F), indicating that the abnormality of isletstructure is evident even before the onset of diabetes.Islets of transgenic mice were smaller than those ofcontrol mice and morphologically irregular in shape at 4weeks of age. �-Cells intermingled with non–�-cells within

FIG. 2. Impaired glucose-stimulated insulin secretion and reduced insulin content in P291fsinsC–HNF-1� transgenic mice. A: intravenous glucosetolerance tests were performed in 8-week-old female P291fsinsC–HNF-1� and control mice. Insulin levels before and 5 min after glucose injectionare shown. Horizontal bars represent the mean insulin levels of each group. B: Insulin secretion from isolated islets from 6- to 7-week-old femaleTg-1 and control mice in response to 3.3 mmol/l (�) and 20 mmol/l (f) glucose stimulation. C: Changes of [Ca2�]i in islets in response to 26 mmol/lglucose and 20 mmol/l KCl in 6- to 8-week-old female mice. Representative data of seven experiments are shown. D: Reduced insulin content inthe pancreas of P291fsinsC–HNF-1� transgenic mice. Pancreata were removed from 2-day-old (Day 2), 1-week-old (Day 7), 4-week-old (4W), and8-week-old (8W)male mice, and insulin was extracted by the acid-ethanol method and measured. �, control mice; t, Tg-1 mice; f, Tg-2 mice. Datashown in B and D are means � SE of the indicated number of animals. N.S., not significant. *P < 0.05, **P < 0.01, ***P < 0.001.



the islet core (Fig. 3K– N). There were no apparentdifferences in the histological features between Tg-1 andTg-2 mice or between male and female mutants.

Morphometric analysis showed no significant differencein non–�-cell number between control and Tg-1 mice.However, �-cell numbers in transgenic mice were signifi-cantly decreased by 50% (P 0.05) and 58% (P 0.05)compared with the respective control at 2 days and at 4weeks of age (Fig. 4A and B). Pancreatic weight was

similar between control and Tg mice [2 days: Tg-1 7.1 � 0.6mg (n 5), Tg-2 7.9 � 1.4 (n 10), and control 7.7 � 1.4mg (n 7); 4 weeks: Tg-1 182 � 44 mg (n 5), Tg-2 169 �18 mg (n 5), and control 175 � 35 mg (n 10)]. Thus,reduction of �-cell number was still significant, even afteradjustment for pancreas weight.Reduced �-cell proliferation in islets of P291fsinsC–

HNF-1� transgenic mice. We determined the cell prolif-eration rate in 2-day- and 4-week-old mice by BrdU

FIG. 3. Immunohistochemistry of pancreatic hormones in male control (A, C, D, and G– J) and Tg-1 mice (B, E, F, and K– N). Paraffin-embeddedsections of pancreata from 8-week-old (A and B), 2-day-old (C–F), and 4-week-old (G–N) mice were immunostained with antibodies directedagainst insulin (A, B, C, E, G, and K), glucagon (D, F, H, and L), somatostatin (I and M) and pancreatic polypeptide (J and N). Scale bar � 470�m (A and B) and 25 �m (C–H).



incorporation. There was no significant difference in non–�-cell proliferation rate between control mice [2 days: 49of 3,682 (1.3%), 4 weeks: 57 of 9,854 (0.58%)] and male Tg-1mutant mice [2 days: 64 of 5,361 (1.2%), 4 weeks: 27 of4,730 (0.57%)]. However, �-cell proliferation rate in Tg-1mice was significantly reduced to 14.6% at 2 days, relativeto the control mice [control: 92 of 6,543 (1.4%), Tg-1: 13 of6,116 (0.21%); P 0.01]. The proliferation rate in Tg-1 micewas also significantly reduced at 4 weeks of age [control:988 of 18,704 (5.2%), Tg-1: 91 of 10,360 (0.87%); P 0.01], suggesting that �-cell proliferation is reduced inP291fsinsC–HNF-1� transgenic mice. Terminated deoxy-nucleotidyl transferase ( (TDT)-mediated dUTP nick andlabeling (TUNEL)-positive cells were not detected among�-cells of the 4-week-old male control and Tg-2 mice.These data suggest a lack of accelerated apoptosis in�-cells of mutant mice.Reduced expression of GLUT2 in islets of P291fsinsC–

HNF-1� transgenic mice. We also analyzed the expres-sion of several markers associated with mature functional�-cells. Expression of PDX-1, Pax6, GK (Fig. 5), andNkx2.2 (data not shown) in transgenic mice was compa-rable with that of control mice, suggesting that RIP-drivenexpression of P291fsinsC–HNF-1� does not result in anonspecific loss of �-cell proteins. GLUT2 facilitates glu-cose transport in �-cells, and the transcription of GLUT2 isregulated by HNF-1� in vitro (18). The expression ofGLUT2 was reduced on the islets of 4-week-old malemutant mice (Fig. 5O). GLUT2 expression was also re-duced in �-cells of 4-week-old female Tg-1 and Tg-2 micewith normal glucose levels (data not shown). In sharpcontrast, the expression of GLUT2 was not reduced in�-cells of 4-week-old male transgenic mice overexpressing

WT–HNF-1� (Fig. 5Q). These results suggest that GLUT2 isa target of HNF-1� in vivo.Reduced expression of E-cadherin in islets of

P291fsinsC–HNF-1� transgenic mice. Cell adhesionmolecules have functional roles in the aggregation andorganization of islets (19,20). Cadherins constitute a su-perfamily of transmembrane glycoproteins that mediateCa2�-dependent homophilic interactions between cells atthe level of adherens junctions (21). E- and N-cadherinsare both expressed in pancreatic islets and are thought tomediate cell adhesion in islet cells (19,20). Immunostain-ing with E-cadherin–specific antibody revealed a uniformexpression of E-cadherin on the surface of islet cells aswell as exocrine cells in control mice. In contrast, theexpression of E-cadherin was reduced in cell-to-cell con-tacts between most �-cells in Tg-1 transgenic mice at 2days of age (Fig. 6A– D). �-Cells of the transgenic micewere scattered in the islets, and compacted �-cell–to–�-cell contact was abolished. Reduced expression of E-cadherin in �-cells was also observed in 4-week-oldtransgenic mice (Fig. 6F and H). Tg-2 mice and femalemutant mice also showed the abnormality (data notshown). The reduced expression of E-cadherin was con-firmed by Western blot analysis using isolated islets (Fig.6L). In contrast, E-cadherin expression in WT–HNF-1� Tgmice was not reduced (Fig. 6J and K). Expression ofN-cadherin, another cadherin in pancreatic islets, ap-peared normal in transgenic mice (Fig. 6L– O).Inhibition of E-cadherin–mediated �-cell contacts

impairs glucose-stimulated insulin secretion. It hasbeen suggested that �-cell–to–�-cell contact is required fornormal insulin secretion (22–24). Mouse islets were cul-tured in the presence of rat anti–E-cadherin, blocking IgG1

FIG. 4. Reduced number of �-cell in male Tg-1 mice. �-cells and non–�-cells were counted in three separate pancreas sections from mice (A,2-day-old mice; B, 4-week-old mice). Relative numbers of �-cells and non–�-cells are shown as the number per one square millimeter of thepancreatic area. Data are means � SE of the indicated number of animals. N.S., not significant. *P < 0.05.



antibody (DECMA-1) or control IgG1, and changes inglucose-stimulated [Ca2�]i and insulin secretion were mea-sured. This antibody can block E-cadherin–mediated ad-herens junction formation in various types of cells (11).Incubation with DECMA-1 antibody (200 ng/ml) inhibitedthe formation of the structure (Fig. 7A). In contrast,control rat IgG1 had no effect on the structure. Culture ofislet cells with DECMA-1 antibody for 48 h abolished theresponse of [Ca2�]i to glucose stimulation, whereas 20mmol/l KCl stimulation increased [Ca2�]i to levels compa-rable with islet cells incubated with control IgG1 (Fig. 7B).Glucose-stimulated insulin secretion was impaired in isletstreated with DECMA-1 antibody (Fig. 7C). These datasuggest that reduced expression of E-cadherin might affectinsulin secretion as well as islet organogenesis.

DISCUSSION

P291fsinsC–HNF-1� transgenic mice developed diabeteswith impaired glucose-stimulated insulin secretion, whichis a characteristic feature of human MODY3 (4–6). How-

ever, HNF-1� knockout mouse (25) exhibited severe liverand kidney dysfunction, and another kind of HNF-1� nullmice (26) exhibited Laron-type dwarfism, infertility, andliver dysfunction. In contrast, human MODY3 patients donot show such signs. Leaky expression of the transgenewas detected in the brain of P291fsinsC–HNF-1� trans-genic mice, but there were no apparent abnormalities ingeneral appearance or behavior in the mice. Histologicalstudies revealed that P291fsinsC–HNF-1� transgenic micedisplayed no abnormalities in liver and kidney (data notshown). Thus, the P291fsinsC–HNF-1� transgenic mouseis a novel animal model of human MODY3. Diabetes in theP291fsinsC–HNF-1� transgenic mouse is more severe thanthat in the HNF-1� knockout mouse. The mutant HNF-1�protein may sequester other �-cell proteins, and this couldaccount at least in part for the more severe phenotype.HNF-1� forms a heterodimer with structurally relatedtranscription factor HNF-1�, which is also expressed inpancreatic islets. The heterodimer formation between invitro translated P291fsinsC–HNF-1� and HNF-1� was con-

FIG. 5. Reduced expression of GLUT2 in �-cells of Tg-1 mice. Pancreatic sections derived from male control (A– D, I– K, and L), Tg-1 (E– H, M–P), and WT–HNF-1� Tg mice (Q and R) were double-stained for insulin [fluorescein isothiocyanate label, green (B, D, F, and H) and rhodaminlabel, red (J, L, N, P, and R)], PDX-1 (3,3�-diaminobenzidine tetrahydrochloride label [DAB] brown) (A and E), Pax6 (DAB label, brown) (C andG), GK (fluorescein isothiocyante label, green) (I and M), and GLUT2 (fluorescein isothiocyante label, green) (K, O, and Q). Scale bar � 20 �m.



firmed by electrophoretic mobility shift assay (data notshown). HNF-1� might be a victim of P291fsinsC–HNF-1�in �-cells.

Expression levels of PDX-1, Pax6, Nkx2.2, GK, andN-cadherin appeared to be comparable in the Tg mice.However, the expression levels of GLUT2 and E-cadherinwere reduced in the mutant mice. Because the downregu-lation of the molecules was found before the onset ofdiabetes, it is not a secondary effect of hyperglycemia.Reduced levels of expressions of GLUT2 and E-cadherinwere not found in WT–HNF-1� Tg mice. These datasuggest that these molecules are targets of HNF-1� in vivo.Abnormal islet structure with loosely connected and scat-

tered �-cells was observed in the P291fsinsC–HNF-1�transgenic mice. The alteration of the islet structure is nota general occurrence with RIP-driven transgenic mice(16,27,28). Because E-cadherin is important for proper celladhesion, its reduced expression in Tg mice could berelated to the abnormal structure. Similar abnormalitieshave been reported in the islets of transgenic mice ex-pressing mutant E-cadherin with a dominant-negative ef-fect (19). Defect in pancreatic islet morphology has beenreported in other transcription factor knockout mice(29,30), but there is no information about the expressionlevels of cadherins in the mice.

Intercellular communication among �-cells appears to

FIG. 6. Reduced expression of E-cadherin on �-cells in male Tg-1 mice. Pancreata of 2-day-old mice (A–D) and 4-week-old mice (E– H, J, and K)were immunostained with an antibody against E-cadherin (Alexa 488 label, green) and an antibody against insulin (rhodamine label, red).Expression of E-cadherin on the surface of �-cells was detected in control mice (C and G). Note the marked decrease in the green fluorescenceon �-cells in Tg-1 mice (D and H). I: Western blot analysis using isolated islets. Expression of E-cadherin in 6- to 7-week-old female Tg-1 micewas reduced. Expression of E-cadherin in WT–HNF-1� Tg mice was normal (G and K). Pancreata of 4-week-old control mice (L and M) and Tgmice (N and O) were immunostained with an antibody against N-cadherin (Cy3 label, red) and an antibody against insulin (fluoresceinisothiocyanate label, green). Scale bar � 10 �m.



be necessary for normal insulin secretion (22–24). Treat-ment of islet cells with the antibody resulted in inhibitionof the structure and marked suppression of glucose-stimulated insulin secretion. Thus, the reduced expressionof E-cadherin in Tg mice might account at least in part forthe impaired glucose-stimulated insulin secretion in themice. The molecular mechanism of reduced E-cadherinexpression in the P291fsinsC–HNF-1� is not currentlyclear. Chicken E-cadherin (L-CAM) gene has a binding sitefor HNF-1� in the intron, and HNF-1� acts as an enhancerthrough the binding site (31). We found a putative HNF-1�binding site in intron 2 of human E-cadherin gene (acces-sion number AC009082) on the human genome sequencedatabase search. Further studies are necessary to clarifythe regulation of E-cadherin by HNF-1�.

Our results showed reduced GLUT2 expression in�-cells in transgenic mice before the onset of diabetes.Homozygous mice deficient in GLUT2 develop diabeteswith impaired glucose-stimulated insulin secretion (32).Then, loss of GLUT2 on �-cells might also be associatedwith impaired insulin secretion in P291fsinsC–HNF-1�transgenic mice.

Reduction of �-cell mass and insulin content have beenreported in HNF-1� knockout (�/�) mice (7) and was alsoobserved in P291fsinsC–HNF-1� Tg mice. However, theknockout mice demonstrate hepatic dysfunction, phenyl-ketonuria, and renal Fanconi syndrome and show severegrowth retardation (25,26). Thus, �-cell mass adjusted forbody weight was not changed in the knockout mice (7). Incontrast, P291fsinsC–HNF-1� Tg mice did not show suchsevere growth retardation, and the differences in �-cellmass were still significant, even after adjustment for bodyweight, suggesting that loss of HNF-1� affects �-cell massand insulin content. It might be worth noting that theGLUT2 knockout mouse displays a decrease in �-cell mass(32). Because a 90% partial pancreatectomy leads toimpaired glucose-induced insulin secretion in rats (33), thedecreased insulin content in Tg mice (3.2–4% of thecontrol in 8-week-old mice) could be related to the onsetof diabetes.

Earlier studies have suggested that loss of HNF-1�affects glycolysis and mitochondrial oxidation (8,9,18).Our findings suggest that HNF-1� plays a variety of roles indetermining normal �-cell function by regulating GLUT2and E-cadherin expression and �-cell mass. Further stud-ies are necessary to determine whether the same abnor-malities are implicated in human HNF-1� diabetes.Detailed studies of Tg mice may lead to a better under-standing of the target genes of HNF-1� in �-cells and themolecular basis of MODY3.

ACKNOWLEDGMENTS

This work was supported by grants from the JapaneseMinistry of Education, Culture, Sports, Science and Tech-nology, Japan Diabetes Foundation, Yamanouchi Founda-tion for Research on Metabolic Disorders, Japan InsulinStudy Group, and Research for the Future Program of theJapan Society for the Promotion of Science (97L00801).

We thank R. Palmiter (RIP vector), B. Thorens (anti-GLUT2), Y. Kajimoto (anti–PDX-1), R. Kemler (anti–E-cad-herin), and M. Takeichi (anti–N-cadherin) for generouslyproviding the respective reagents. We also thank G.I. Bell(University of Chicago, IL) and C.B. Wollheim (UniversityMedical Center, Switzerland) for their continuous encour-agement of our work.

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Overexpression of Dominant-Negative Mutant Hepatocyte ...ton, KY) or anti–E-cadherin antibody (kindly provided by Dr. R. Kemler, Max-Planck-Institute, Freiburg, Germany) (11) overnight