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Endocrinology Vol. 145, No. 8 3941-3949
Copyright © 2004 by The Endocrine Society

Selective Deletion of the Hnf1ß (MODY5) Gene in ß-Cells Leads to Altered Gene Expression and Defective Insulin Release

Li Wang, Catherine Coffinier, Melissa K. Thomas, Lionel Gresh, Ganiu Eddu, Tal Manor, Lynne L. Levitsky, Moshe Yaniv and David B. Rhoads

Pediatric Endocrine Unit (L.W., G.E., T.M., L.L.L., D.B.R.), MassGeneral Hospital for Children, and Laboratory of Molecular Endocrinology and Diabetes Unit (M.K.T.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; and Unité Expression Génétique et Maladies (C.C., L.G., M.Y.), Unité de Recherche Associée, Centre National de la Recherche Scientifique 1644, Institut Pasteur, 75724 Paris, France

Address all correspondence and requests for reprints to: David B. Rhoads, Ph.D., Pediatric Endocrine Unit, MassGeneral Hospital for Children, 55 Fruit Street BHX410, Boston, Massachusetts 02114-2696. E-mail: drhoads{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hepatocyte nuclear factor 1{alpha} (HNF1{alpha}) and HNF1ß (or vHNF1) are closely related transcription factors expressed in liver, kidney, gut, and pancreatic ß-cells. Many HNF1 target genes are involved in carbohydrate metabolism. Human mutations in HNF1{alpha} or HNF1ß lead to maturity-onset diabetes of the young (MODY3 and MODY5, respectively), and patients present with impaired glucose-stimulated insulin secretion. The underlying defect in MODY5 is not known. Analysis of HNF1ß deficiency in mice has not been possible because HNF1ß null mice die in utero. To examine the role of HNF1ß in glucose homeostasis, viable mice deleted for HNF1ß selectively in ß-cells (ß/H1ß-KO mice) were generated using a Cre-LoxP strategy. ß/H1ß-KO mice had normal growth, fertility, fed or fasted plasma glucose and insulin levels, pancreatic insulin content, and insulin sensitivity. However, ß/H1ß-KO mice exhibited impaired glucose tolerance with reduced insulin secretion compared with wild-type mice but preserved a normal insulin secretory response to arginine. Moreover, ß/H1ß-KO islets had increased HNF1{alpha} and Pdx-1, decreased HNF4 mRNA levels, and reduced glucose-stimulated insulin release. These results indicate that HNF1ß is involved in regulating the ß-cell transcription factor network and is necessary for glucose sensing or glycolytic signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MATURITY-ONSET DIABETES OF the young (MODY) is an autosomal dominant, monogenic form of diabetes that usually develops before age 25 with underlying genetic heterogeneity (1). To date, mutations leading to MODY have been identified in six genes: HNF4{alpha}/MODY1 (2), glucokinase/MODY2 (3), HNF1{alpha}/MODY3 (4), PDX1/MODY4 (5), HNF1ß/MODY5 (6), and BETA2/NeuroD1/MODY6 (7). Notably, five of these genes encode transcription factors. Because patients present with a defect in glucose-stimulated insulin secretion, MODY appears to result from the inability of the ß-cell to sense and/or to respond to secretagogue signals.

MODY5 mutations also lead to varying degrees of nephropathy and/or genitourinary defects (6, 8, 9, 10) and are associated with familial hypoplastic glomerulocystic kidney disease (11). This pleiotropic pattern of presentation corresponds to the presence of hepatocyte nuclear factor 1ß (HNF1ß) in pancreas, renal tubules, and the genitourinary tract (12, 13). HNF1ß is also expressed in liver, the tissue from which it was first cloned (14, 15, 16), as well as the gastrointestinal tract (17). HNF1ß along with its homologous heterodimerization partner HNF1{alpha} (15, 16) supports the expression of many genes in target tissues (18). Several HNF1 targets, including the insulin gene itself (19, 20), are involved in carbohydrate metabolism (21). In zebrafish, the Hnf1ß homologue vhnf1 regulates the development of the liver, kidney, and pancreas (22). Work in our laboratory (23) and in others (24, 25) has focused on the role of HNF1 in regulating genes involved in glucose disposal. These considerations led us to postulate a similar role for genes controlling glycolysis in the ß-cell.

In pancreas development, HNF1ß expression precedes that of neurogenin 3 (Ngn3) in visceral endoderm cells destined to become endocrine cells (26). Both HNF1ß immunostaining and DNA binding activity were detected in fetal islets with lower levels postnatally (26). HNF1{alpha} is abundant in pancreatic islets (27) and is also found in cultured ß-cell lines (19). Mice lacking HNF1{alpha} develop diabetes (28) with a defect in glucose-stimulated insulin secretion (29), but their islets are responsive to some secretagogues not metabolized via glycolysis (29). These results suggest that HNF1{alpha} contributes to glucose homeostasis by regulating glucose metabolism in ß-cells. A similar analysis of HNF1ß function has not been possible because mice lacking HNF1ß die soon after implantation (30, 31).

To circumvent the developmental requirement for HNF1ß, we used a Cre-LoxP strategy (32) to generate mice selectively lacking the Hnf1ß gene in pancreatic ß-cells (ß/H1ß-KO mice). ß/H1ß-KO mice at 2 months of age exhibited impaired glucose tolerance with impaired insulin secretion that became progressively worse with age. In contrast, insulin secretory responses to arginine were similar between ß/H1ß-KO and control mice. Pancreatic islets appeared histologically normal and preserved insulin expression. However, ß/H1ß-KO islets exhibited altered expression of MODY transcription factors and reduced glucose-stimulated insulin secretion. These results suggest that loss of HNF1ß disrupts the regulatory pathways necessary for glucose signaling in ß-cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and genotyping
Mice were housed in a specific-pathogen-free facility maintained at 24 C with a 12-h light, 12-h dark cycle and provided water and standard rodent chow ad libitum. Study protocols were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. Transgenic mice expressing Cre recombinase under control of the rat insulin 2 promoter (RIP-Cre) were previously described (33). RIP-Cre mice were hybrids derived from 129sv, C57BL/6, and DBA-2 strains. Transgenic mice in which exon 1 of the Hnf1ß gene was flanked with LoxP sites (floxed HNF1ß) were derived from 129sv and C57BL/6 strains (34). Mice from these two lines were interbred to generate mice homozygous for the floxed Hnf1ß gene and either positive (ß/H1ß-KO) or negative (controls) for the RIP-Cre transgene. Age- and gender-matched littermates were used for each experiment.

Genotyping was performed by PCR on tail DNA of 2- to 3-wk-old mice. The primers to detect the RIP-Cre transgene were 5'-ATGTCCAATTTACTGACCG (forward) and 5'-CGCCGCATAACCAGTGAAAC (reverse). The primers to distinguish the Hnf1ß alleles were 5'-AGCTCCACTTTCGCTACTC (VX4; forward), 5'-ACCCTACAACAAACTTTCCAC (VX5; deleted/reverse), and 5'-CTTTAGGCGCGGTTACAACG (VX8; intact/reverse); these amplified a unique product from each allele. Hnf1ß gene recombination in specific tissues was detected with either PCR or Southern blotting (35). The genotyping strategies are shown in Fig. 1AGo.



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  		FIG. 1. Hnf1ß gene recombination. A, Schematic of Hnf1ß alleles (not to scale). Exon 1 of the Hnf1ß gene is on a 3.0-kb HindIII genomic fragment (HNF1ßWT) that increases to 3.6 kb when targeted with LoxP sites (closed arrowheads; HNF1ßflox). Cre-mediated recombination generates a 1.6-kb remnant (HNF1ßdel). Southern blotting using an intronic probe (PROBE) or PCR using a set of three primers (open arrows) distinguishes the three alleles. VX4 and VX8 generate the indicated PCR products HNF1ßWT and HNF1ßflox, but the products predicted from VX5 are more than 1 kb and not observed under our conditions. Deletion of exon 1 removes the VX8 priming site and reduces the VX4-VX5 distance sufficiently to generate the product shown. B, Southern blot analysis: Lanes: 1, tail DNA from wild-type homozygote; 2, tail DNA from floxed HNF1ß homozygote; 3, islet DNA from control mouse; 4, islet DNA from ß/H1ß-KO mouse. C, PCR analysis of ß/H1ß-KO tissue DNA (12 wk of age): Left, islets; right, liver; CTL, control DNA preparation containing all three Hnf1ß alleles.

 
Tolerance tests
Mice were fasted overnight for 14 h, weighed and injected ip with 2 mg/g D-glucose, 2 mg/g L-arginine, or 0.75 mU/g human insulin (Lilly, Indianapolis, IN). Venous blood was obtained from the tail before (0 min) and at several times after the injection. Blood glucose levels were measured with an AccuChek Advantage glucose meter (Roche Diagnostics, Indianapolis, IN). Additional blood was collected at selected times in heparinized capillary tubes for subsequent measurement of plasma insulin using the rat insulin ELISA kit and the rat insulin standard (Crystal Chem, Inc., Chicago, IL).

Pancreatic insulin content
Mice were killed by CO2 inhalation, and pancreata were quickly removed, weighed, and rapidly frozen in liquid nitrogen. Insulin was extracted with acid ethanol (36) and measured by RIA (rat insulin RIA kit, Linco Research, Inc., St. Charles, MO).

Islet isolation
Islets were isolated using a modification of a published method (37). Mice were anesthetized with sodium pentobarbital (3 mg/g ip; Sigma Chemical Co., St. Louis, MO). Pancreata were inflated via the pancreatic duct with cold 0.8 mg/ml Liberase (Roche) in Hanks’ balanced saline solution, excised, and incubated in a stationary water bath at 37 C. Released islets were enriched by handpicking under a stereomicroscope.

Immunohistochemistry
Fluorescent immunocytochemistry was performed as previously described (38). A Nikon epifluorescence microscope equipped with a SPOT RT Slider camera (Diagnostic Instruments, Inc., Sterling Heights, MI) was used to capture images to a Power Mac G4 computer. Images were processed with the accompanying SPOT camera software and Adobe PhotoShop (Adobe Systems, San Jose, CA).

RT-PCR analysis
RNA was extracted from liver or isolated islets using TRIzol (Invitrogen Corp., Carlsbad, CA). For RT, 2 µg total RNA was mixed with 0.5 µg oligo-dT (Promega, Madison, WI) and incubated for 2 min at 72 C. The cDNA was synthesized with SuperScript II (Invitrogen) for 60 min at 42 C and diluted 5-fold with H2O, and 1 µl was used for PCR. Amplifications used published primer sequences for HNF1{alpha} and HNF1ß (39), GLUT2 (40), glucokinase (41), Pdx-1, HNF4{alpha}, and HNF4{gamma} (42) using a RapidCycler (Idaho Technology, Inc., Salt Lake City, UT). HNF1 PCR products were resolved on 6% polyacrylamide gels (39) and other gene products on 2% agarose gels. Images from ethidium bromide-stained gels were quantified using an Eagle Eye II gel documentation system (Stratagene, La Jolla, CA). Specific conditions are available upon request.

Islet insulin release
Islets isolated as described above were incubated at 37 C for 30 min in islet buffer [119 mM NaCl, 20 mM Na-HEPES (pH 7.4), 4.6 mM KCl, 1 mM CaCl2, 2 mM MgSO4, 0.1 mg/ml RIA-grade BSA, 1.55 mM Na2HPO4, 0.4 mM KH2PO4, 5 mM NaHCO3] containing low glucose (2.8 mM). The buffer was aspirated and replaced with fresh islet buffer containing low glucose (2.8 mM), high glucose (16.7 mM), or K+ (2.8 mM glucose + 20 mM KCl). After incubation at 37 C for 30 min, buffer aliquots were removed for insulin RIA as above.

Statistical methods
All values are expressed as mean ± SD. Statistical analyses were carried out using Student’s unpaired t test with P < 0.05 accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of ß-cell-specific HNF1ß knockout (ß/H1ß-KO) mice
We generated ß/H1ß-KO mice by conditional gene targeting (32). Mice harboring the floxed Hnf1ß allele, in which exon 1 was flanked with LoxP sites (34), were interbred with RIP-Cre transgenic mice, which express Cre under control of the rat insulin promoter to target it to pancreatic ß-cells (33). A schematic of the recombination event and the detection strategies are shown in Fig. 1AGo. The targeted Exon 1 of the Hnf1ß gene contains the first 115 codons encoding the dimerization domain and part of the POU-like domain (34). Loss of exon 1 would be expected to eliminate HNF1ß synthesis by removing the translational start site. Moreover, should translation occur from a downstream AUG or cryptic ribosome entry site, loss of the dimerization domain (15, 16) would prevent truncated proteins from forming dimers necessary for DNA binding. Offspring homozygous for the floxed Hnf1ß allele and hemizygous for the RIP-Cre transgene should have exon 1 deleted and HNF1ß function lost only in the insulin-producing ß-cells of pancreatic islets. Offspring were genotyped by PCR analysis of tail DNA to detect the presence of the RIP-Cre transgene (not shown) and Southern blotting to confirm the LoxP insertion in the Hnf1ß allele (Fig. 1BGo, lanes 1 and 2). Cre-mediated excision of HNF1ß exon 1 in islets isolated from ß/H1ß-KO mice was demonstrated by the appearance of the 1.6-kb genomic deletion fragment (Fig. 1BGo, lanes 3 and 4). RIP-Cre-mediated recombination was specific (Fig. 1CGo). Islets isolated from two ß/H1ß-KO mice contained the deleted allele and the inherited targeted allele (Fig. 1CGo, left). The first lane is a control sample containing all three alleles. Liver DNA contained only the targeted allele (Fig. 1CGo, right). Some mice exhibited less recombination in the islets by this assay. To assure that the ß/H1ß-KO mice examined had ß-cell-selective loss of the Hnf1ß gene, only those with ≥40% islet deletion were used for further analysis.

ß/H1ß-KO mice were born with typical Mendelian frequency and were indistinguishable from controls in appearance or behavior. ß/H1ß-KO mice were similar to control mice over the first 2 months of life in growth rate (Fig. 2Go) and in adult weight (not shown). No deficiency in fertility was noted for either gender.



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  		FIG. 2. Growth rates. A, Male mice ({blacksquare}, ß/H1ß-KO, n = 5; {square}, controls, n = 8); B, female mice ({bullet}, ß/H1ß-KO, n = 11; {circ}, controls, n = 5). No significant differences were detected between ß/H1ß-KO and control mice of either gender.

 
Glucose and insulin levels in ß/H1ß-KO mice
To assess the effect of HNF1ß gene deletion in ß-cells on glucose homeostasis, glucose and insulin levels were measured in ß/H1ß-KO mice. All samples were obtained between 0900 and 1100 h to minimize potentially confounding diurnal effects. Both glucose and insulin levels were within normal ranges in either fed or fasted ß/H1ß-KO mice and were indistinguishable from control mice (Table 1Go). No significant differences were detected when data were segregated by gender or age (not shown).


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  		TABLE 1. Plasma glucose and insulin levels

 
Glucose tolerance
To examine the impact of the ß-cell HNF1ß deletion on responses to an acute glucose challenge, glucose disposal was assessed in fasted mice by ip glucose tolerance tests. At all ages examined (2–8 months), fasted glucose levels were similar between ß/H1ß-KO and control mice. Glucose responses to ip glucose injections in the two groups were compared in mice aged 2–3 months (Fig. 3AGo). Most ß/H1ß-KO mice (23 of 25) showed a significantly greater excursion in plasma glucose than control mice regardless of gender. The average plasma glucose concentration rose to approximately 400 mg/dl by 30 min and remained at that level at 60 min. In contrast, glucose levels in control mice reached a peak of 262 ± 70 mg/dl at 15 min and then declined. ß/H1ß-KO mice had statistically higher plasma glucose at all times tested up to 90 min. The impairment worsened as ß/H1ß-KO mice aged, with excursions at 60 min exceeding 600 mg/dl in some mice at 5–6 months (not shown). The two ß/H1ß-KO mice with normal glucose tolerance at 3 months became impaired when tested at 5 months.



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  		FIG. 3. Glucose and insulin tolerance tests. Mice aged 2–3 months were fasted 14 h, injected with glucose (2 mg/g ip; n = 22–25), or insulin (0.75 mU/g ip; n = 11–12) at t = 0, and tail vein blood was collected ({bullet}, ß/H1ß-KO; {circ}, control). Glucose tolerance: A, plasma glucose levels; B, plasma insulin levels. Differences between the two groups were not statistically significant at any time examined (P > 0.05). C, Insulin/glucose ratios. Insulin tolerance: D, blood glucose levels. Differences between the two groups were not statistically significant at any time examined (P > 0.05). *, P < 0.05; **, P < 0.01; {dagger}, P < 0.001; {ddagger}, P < 0.0001.

 
Plasma insulin levels rose in all mice after glucose injection (Fig. 3BGo). Although ß/H1ß-KO mice had lower plasma insulin than controls at all times, this difference did not reach statistical significance. However, when the insulin/glucose ratios were compared as a measure of ß-cell responsiveness, a significant divergence occurred at 30 min (P < 0.05), the first time examined (Fig. 3CGo). Thus, despite the apparent modest difference in the insulin secretory profile, ß/H1ß-KO mice were unable to mount an adequate insulin response.

Insulin sensitivity
Glucose intolerance could arise from either insufficient insulin secretion or reduced glucose disposal from peripheral insulin resistance. To distinguish between these possibilities, we examined insulin sensitivity directly by measuring plasma glucose after an insulin challenge (Fig. 3DGo). At 30 min, plasma glucose dropped to approximately 75% of initial levels in either control or ß/H1ß-KO mice. From there, ß/H1ß-KO glucose levels declined to approximately 50% of the initial level, whereas control levels stabilized, although the difference was not statistically significant (P = 0.075; 60 min). Thus, ß/H1ß-KO mice are at least as sensitive to insulin as controls. We conclude that ß/H1ß-KO mice are impaired in glucose-stimulated insulin secretion.

Arginine stimulation test
To examine whether the observed defect in ß/H1ß-KO insulin secretion was specific for glucose, an arginine stimulation test was performed. Arginine leads to insulin release by mechanisms independent of those used by glucose, although the final pathway of secretion is common (43). After an arginine challenge (2 mg/g ip), both ß/H1ß-KO and control mice showed an identically robust release of insulin (Fig. 4AGo) and similar changes in plasma glucose (Fig. 4BGo). By 30 min, plasma insulin had increased approximately 3-fold. As anticipated, and in contrast to the glucose tolerance test, the insulin/glucose ratios after arginine stimulation were essentially identical between the two groups (Fig. 4CGo). The ability of ß/H1ß-KO mice to release normal amounts of insulin in response to arginine suggests that their defect is in glucose sensing or metabolism.



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  		FIG. 4. A–C, Arginine stimulation test for ß/H1ß-KO mice. Mice aged 2–3 months were fasted 14 h, injected with arginine (2 mg/g ip; n = 12 each genotype), and tail vein blood was collected ({bullet}, ß/H1ß-KO; {circ}, control). A, Plasma insulin levels; B, plasma glucose levels; C, insulin/glucose ratios. No significant differences were detected. D, Arginine stimulation test for HNF1{alpha}-null mice: plasma insulin levels in 8-wk-old HNF1{alpha} null mice ({bullet}) vs. HNF1{alpha}+/– heterozygote littermates ({circ}). Differences were statistically significant for each time examined (n = 3 each genotype; P < 0.01).

 
In contrast to the intact arginine response observed in ß/H1ß-KO mice, islets isolated from HNF1{alpha} null mice do not secrete insulin in response to arginine (29). To compare these genetic models, an arginine stimulation test was performed on HNF1{alpha} null mice and matched normal heterozygous littermates. The heterozygous mice showed an excellent insulin response after arginine stimulation that was essentially absent in HNF1{alpha} null mice (Fig. 4DGo).

Pancreatic histology and islet insulin content
To evaluate whether the glucose intolerance observed after selective deletion of HNF1ß in ß-cells was associated with changes in islet development, histology and insulin expression were assessed. ß/H1ß-KO mice did not exhibit any gross alterations in pancreas morphology. Pancreata from ß/H1ß-KO mice exhibited histology indistinguishable from control mice (Fig. 5Go). Although not systematically quantified, there was no obvious difference in the number, distribution, or islet sizes between the two genotypes. ß/H1ß-KO mice exhibited similar immunostaining for both insulin and glucagon (Fig. 6Go). In mice of both genotypes, islets had a normal core of insulin-staining ß-cells surrounded by glucagon-staining {alpha}-cells. The ratio and distribution of ß-cells vs. {alpha}-cells in ß/H1ß-KO islets were similar to controls. Pancreatic insulin content at 5 months of age was also not statistically different between the two genotypes: ß/H1ß-KO = 62 ± 20 ng/mg (n = 9); control = 58 ± 14 ng/mg (n = 5). Moreover, pancreatic weights were indistinguishable (not shown).



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  		FIG. 5. Pancreas histology. Pancreata from mice 8–13 wk old were examined by hematoxylin and eosin staining of paraffin-embedded sections. Representative low-power views (40x; A, B, E, and F) and high-power views (200x; C, D, G, and H) from ß/H1ß-KO (A–D) and control mice (E–H) are shown.

 


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  		FIG. 6. Islet insulin immunostaining. Paraffin-embedded pancreata from 10-wk-old mice were stained for insulin with Cy2-conjugated secondary antibody (green) and for glucagon with Cy3-conjugated secondary antibody (red). Typical control (left) and ß/H1ß-KO (right) islets are shown.

 
Gene expression changes in ß/H1ß-KO islets
To assess changes in gene expression in HNF1ß-deficient islets, RT-PCR was performed on RNA extracted from isolated islets (Fig. 7Go; Table 2Go). The primers used for the two Hnf1 genes are directed to conserved regions and amplify three electrophoretically distinguishable products derived, respectively, from the long HNF1ß transcript, the single HNF1{alpha} transcript, and the short HNF1ß transcript (39). Both HNF1ß transcripts were reduced 4-fold in ß/H1ß-KO mice. In contrast, islet HNF1{alpha} expression increased 3-fold in these mice. This increase suggested that HNF1{alpha} expression is negatively regulated by HNF1ß (either directly or indirectly). In addition, the expression of several other genes was altered in ß/H1ß-KO islets. Pdx-1 expression was also increased approximately 3-fold in ß/H1ß-KO islets, whereas both HNF4 transcripts were reduced at least 3-fold. Neither of the glucose-sensing genes, GLUT2 or glucokinase, appeared to be significantly altered in ß/H1ß-KO islets.



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  		FIG. 7. Islet gene expression. Semiquantitative RT-PCR analysis of islet RNA from mice aged 10–14 wk. See Materials and Methods for details.

 

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  		TABLE 2. Relative mRNA levels in isolated islets

 
Insulin secretion in ß/H1ß-KO islets
To assess ß-cell function, insulin release was examined in isolated islets (Table 3Go). Control islets exhibited normal release to glucose or to KCl. However, islets from ß/H1ß-KO mice did not increase insulin release in high glucose but did respond to KCl. The similar insulin responses after KCl stimulation indicate that depolarization-induced insulin secretion is normal in ß/H1ß-KO islets. Thus, the loss of HNF1ß in ß-cells leads to impaired glucose tolerance due to the inability of ß-cells to respond adequately to glucose stimulation.


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  		TABLE 3. Insulin release by isolated islets

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using a Cre-LoxP strategy, we generated mice with selective deletion of the Hnf1ß gene in pancreatic ß-cells as a model to examine the islet defects in human MODY5. ß/H1ß-KO mice exhibited a loss of glucose-induced insulin secretion that resulted in impaired glucose tolerance. Glucose intolerance was observed as early as 2 months of age with glycemic excursions in excess of 400 mg/dl for ß/H1ß-KO mice vs. approximately 200 mg/dl for control mice. Both normal insulin sensitivity and reduced glucose-induced islet insulin release pointed to defective ß-cell function in ß/H1ß-KO mice rather than impaired glucose disposal. Thus, the Rip-Cre-mediated deletion of the Hnf1ß gene and associated loss of HNF1ß function in pancreatic ß-cells was sufficient to cause glucose intolerance.

HNF1ß mRNA is first detected in the primitive endoderm at d 4.5 post coitus (30) and later in the yolk sac and the developing gut, including the pancreas primordium (12, 44). HNF1{alpha} expression generally follows HNF1ß (44, 45). HNF1{alpha} null mice are viable despite an early-onset type 2-like form of diabetes and severe renal tubular defects (28, 29, 46). In contrast, Hnf1ß gene deletion leads to embryonic lethality shortly after implantation due to a defect in the visceral endoderm (30, 31). This finding suggests that HNF1ß is essential particularly during embryogenesis with a more supportive role for HNF1{alpha} in the activation of late target genes. Furthermore, lack of HNF1{alpha} expression in HNF1ß-null embryoid bodies (31) suggests that HNF1ß expression is necessary for HNF1{alpha} expression in this tissue, either directly or indirectly. Alternatively, the recent study showing that overexpression of HNF1{alpha} in HNF1ß-null embryonic stem cells can restore a normal endodermal-like differentiation program suggests that HNF1ß is necessary to induce HNF1{alpha} but not the immediate downstream targets examined (47). In another study, it was shown that the Ngn3-positive cells that are thought to be precursors of pancreatic endocrine cells arise from HNF1ß-positive ductal cells during embryogenesis (26). These investigators found that the majority of HNF1ß immunostaining in endocrine cells and DNA binding activity in pancreas occurred before embryonic d 14.5. Activation of the rat insulin promoter in the pancreatic bud at embryonic d 10 (48) would initiate Cre-mediated Hnf1ß gene recombination in insulin-expressing islet cell precursors of the visceral endoderm as well as in adult ß-cells. Thus, the phenotype of ß/H1ß-KO mice may result from reduced HNF1ß function in both developmental and postnatal stages. However, because the RIP promoter is not very active in early embryonic pancreas development, the ß/H1ß-KO mouse model primarily addresses the function of HNF1ß in differentiated pancreatic ß-cells. Resolution of the functions of HNF1ß in early embryonic pancreas development may require the generation of additional genetically modified mouse models.

No physical abnormalities other than impaired glucose tolerance were observed in ß/H1ß-KO mice. They were normoglycemic and normoinsulinemic in both the fed and fasted states. Moreover, deletion of the Hnf1ß gene in ß-cells did not disrupt pancreatic architecture, alter islet histology, or reduce insulin content. Most notably, arginine-induced insulin secretion did not appear to be affected in ß/H1ß-KO mice. This finding indicates that ß/H1ß-KO islets are still capable of insulin secretion but not in response to glucose (or similarly metabolized secretagogues). Our data showing that isolated ß/H1ß-KO islets did not have a normal increase in insulin release in response to glucose supports this conclusion.

Hnf1ß gene recombination led to altered expression of several transcription factors in adult islets. In particular, mRNA levels of both HNF1{alpha} and Pdx-1 were increased, whereas those of both HNF4 transcripts were decreased. The greater Pdx-1 expression was possibly due to increased HNF1{alpha}, reduced HNF1ß, or a combination. This result is consistent with studies of the human IPF1 gene, which contains an enhancer element that is activated by HNF1{alpha} but not by HNF1ß (49). Although Pdx-1 expression is dependent on HNF1ß during zebrafish development (22), this pathway has not been examined in mice. Possibly, Pdx-1 expression is initiated by HNF1ß during mouse development but maintained by HNF1{alpha} in adults. In contrast, both HNF4 transcripts were reduced, similar to that observed in HNF1{alpha} null mouse islets (42, 50). HNF1{alpha} has been shown to control ß-cell HNF4{alpha} expression via an alternative upstream promoter in the Hnf4{alpha} gene (42, 51). Our result suggests that HNF1ß is also necessary for HNF4{alpha} expression in ß-cells. GLUT2 and glucokinase did not appear to be significantly altered in ß/H1ß-KO islets, perhaps due to increased Pdx-1 and/or HNF1{alpha} levels (40, 52). These results suggest that the initial steps of glucose metabolism, i.e. uptake and phosphorylation, are not impaired in ß/H1ß-KO ß-cells. Although direct assay will be needed to verify this conclusion, these data point to downstream steps as responsible for impaired glucose sensing. Nevertheless, the dysregulation of several important ß-cell transcription factors in opposite directions raises the possibility that compensatory mechanisms minimized the impact of reduced HNF1ß expression on ß-cell function.

The present study of ß-cell HNF1ß expression can be placed in context with other studies. Our previous study using a ß-galactosidase transgene knocked into the Hnf1ß locus indicated weak, but positive, Hnf1ß gene expression in adult ß-cells (12). More recently, HNF1ß protein and DNA-binding activity were detected in adult islets, but immunodetection required an additional amplification step and the DNA binding activity was significantly weaker than in fetal islets (26). Our semiquantitative RT-PCR data (Fig. 7Go) clearly demonstrate HNF1ß mRNA in control islets with significantly less in ß/H1ß-KO islets. Thus, despite its relatively low expression levels, the phenotype that we observed in ß/H1ß-KO mice indicates that HNF1ß is a potent transcription factor that is important for ß-cell function.

We recognize that the Hnf1ß gene may be only partially recombined in ß/H1ß-KO mice. Nevertheless, the extent of deletion obtained was sufficient to impair glucose tolerance. Similarly, partial loss of Pdx-1 expression in the Cre-LoxP MODY4/Pdx-1 model was sufficient to impair ß-cell metabolic function (53). In many cases, small changes in transcription factor mRNA levels (<2-fold) can have a large impact. Indeed, many MODY mutations result in a loss of transcription factor function with the predicted 50% reduction in mRNA levels that presumably leads to diabetes. Also, the 50% reduction of Pdx-1 expression in heterozygous knockout mice resulted in reduced ß-cell mass, target gene expression, and insulin secretion (52, 54). Furthermore, HNF1{alpha} heterozygous null mice have reduced Pdx-1 expression and, consequently, diminished insulin gene expression (50). The observed ß/H1ß-KO phenotype suggests that the HNF1ß level, however low, is rate limiting for normal ß-cell function. Although a more severe phenotype may have ensued with a total loss of HNF1ß, our results show that even a partial reduction results in metabolic dysfunction.

It is instructive to compare the phenotypes of HNF1{alpha} null mice and ß/H1ß-KO mice. HNF1{alpha} null mice have disorganized islets, which are smaller with a reduced ß-cell fraction (29). The preservation of islet morphology in the ß/H1ß-KO mice suggests that HNF1ß, at the time it is inactivated, is not necessary for islet development or adult maintenance. HNF1{alpha} null ß-cells exhibit impaired glucose- and arginine-stimulated insulin secretion (29) due to reduced ATP synthesis (55) and defective glucose sensing due to lower GLUT2 and glucokinase expression (50). The selective loss of glucose-stimulated insulin secretion in ß/H1ß-KO mice suggests that different pathway defects are responsible for impaired glucose signaling. In addition, insulin gene expression is reduced in HNF1{alpha} null mice (50) but apparently unaffected in ß/H1ß-KO mice. The insulin gene, a target for both HNF1{alpha} and HNF1ß (20), is activated both by HNF1{alpha} directly and by HNF1{alpha}-dependent Pdx-1 expression (50). The increased levels of both HNF1{alpha} and Pdx-1 in ß/H1ß-KO mice may be sufficient to maintain insulin expression. Also in this context, overexpression of a dominant-negative Hnf1{alpha} allele impaired mitochondrial metabolism (56). Disruption of different metabolic pathways in ß/H1ß-KO mice could explain the disparate responses to glucose and arginine. Thus, within the limitations of these two mouse models, loss of HNF1ß vs. HNF1{alpha} leads to different changes in islet function.

Our ß/H1ß-KO model differs from human MODY5 in that the mice acquire a reduced dosage of the Hnf1ß gene selectively in ß-cells (insulin-expressing cells) during development rather than receiving from conception a single global mutant allele, potentially encoding a dominant-negative protein (57). Glucose homeostasis in ß/H1ß-KO mice differs from human MODY5 in at least two ways. First, ß/H1ß-KO mice are glucose intolerant only during an ip glucose tolerance test, whereas MODY5 patients have fasting plasma hyperglycemia as well as other clinical manifestations (6). Mutant HNF1ß protein in nonpancreatic tissues in MODY5 may contribute to the MODY5 phenotype, for instance by deregulating hepatic glucose output. Second, most mice were glucose intolerant by 2 months of age, whereas the age of MODY5 onset varies. This difference may suggest distinct functions for HNF1ß in the two species. Alternatively, the severity of the ß/H1ß-KO phenotype may depend on comodifier genes that vary with genetic background.

In summary, selective deletion of HNF1ß in ß-cells leads to impaired glucose tolerance, dysregulated islet gene expression, and reduced glucose-stimulated insulin secretion. The relevance of this finding is emphasized by studies showing that impaired glucose tolerance is one of the best predictors of the development of diabetes (58). However, as is the case for HNF1{alpha} null mice, the secretory defect in ß/H1ß-KO mice is specific to the secretagogue. The normal insulin response to arginine in the latter mice suggests that losing either HNF1{alpha} or HNF1ß results in different aberrations of the glucose-signaling and insulin-secretion pathways. A more comprehensive assessment of gene expression changes and insulin secretory responses will help to define these differences. Finally, generation of viable ß/H1ß-KO mice will be useful to study the islet defects in MODY5 diabetes.


   Acknowledgments
 
We extend thanks to Dr. Mark Magnuson (Vanderbilt University, Nashville, TN) and Rohit Kulkarni (Joslin Diabetes Clinic, Boston, MA) for providing the RIP-Cre mice, Frank J. Gonzalez (National Cancer Institute, Bethesda, MD) for providing the HNF1{alpha} null mice, Heather Hermann and Jamie Volinic for the immunostaining, and Lihua Zhang and Dayana Duguerre for expert technical assistance.


   Footnotes
 
This work was supported by U.S. Public Health Service Grants HD31215 (L.L.L.) and DK54399 (D.B.R.), Boston Area Diabetes and Endocrinology Research Center (BADERC) Award DK57521 (L.L.L.), and March of Dimes Grant 1-FY99-221 (D.B.R.). We gratefully acknowledge summer research fellowship awards from the American Pediatric Society & Society for Pediatric Research (T.M.) and the Lawson Wilkins Pediatric Endocrine Society (G.E.).

L.W. and C.C. contributed equally to the work.

Present address for L.W.: Eli Lilly Asia, Inc., 16th Floor Harbor Ring Plaza, 18 XiZhang Middle Road, Shanghai 200001, People’s Republic of China.

Present address for C.C.: Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, California 90095.

Abbreviations: ß/H1ß-KO, Selectively lacking the HNF1ß gene in pancreatic ß-cells; HNF1ß, hepatocyte nuclear factor 1ß; MODY, maturity-onset diabetes of the young; RIP, rat insulin 2 promoter.

Received March 4, 2004.

Accepted for publication May 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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