Dual Roles for Glucokinase in Glucose Homeostasis as Determined by Liver and Pancreatic beta  Cell-specific Gene Knock-outs Using Cre Recombinase*

Catherine PosticDagger , Masakazu ShiotaDagger , Kevin D. NiswenderDagger §, Thomas L. JettonDagger , Yeujin ChenDagger , J. Michael Moates, Kathy D. SheltonDagger , Jill LindnerDagger , Alan D. CherringtonDagger , and Mark A. MagnusonDagger parallel

From the Departments of Dagger  Molecular Physiology and Biophysics and of  Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxP technology in combination with gene targeting to perform global, beta  cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. beta  cell- and hepatocyte-specific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a beta  cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in beta  cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the beta  cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both beta  cell and hepatic GK contribute to the hyperglycemia of MODY-2.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Glucokinase (GK)1 plays an essential role in blood glucose homeostasis. In humans, GK gene mutations cause maturity onset diabetes of the young, type 2 (MODY-2) (1-4), a disease that is characterized by early onset and persistent hyperglycemia (5). The pathophysiological basis of GK-deficient diabetes is generally viewed as involving both pancreatic beta  cells and hepatocytes (4, 6-10), although the relative contributions of each cell type have not been determined. Both beta  cells and hepatocytes are key sites of GK expression, and both play central roles in glucose homeostasis. In pancreatic beta  cells GK determines glucose utilization and thus is necessary for glucose-stimulated insulin secretion. In liver, GK is thought to determine rates of both glucose uptake and glycogen synthesis and is also viewed as being essential for the regulation of various glucose-responsive genes (11). Although GK is also expressed in certain hypothalamic nuclei of the brain, where it might contribute to feeding behavior and counter regulatory responses, and in the gut, where it might contribute to the secretion of enteroincretins such as GLP-1 (12), the functional importance of the enzyme in both of these sites is undefined.

Both GK gene knock-outs (13-15) and GK transgenic mice (16-18) have been generated and used as model systems to determine the effects of either increased or diminished GK gene expression on blood glucose homeostasis. These studies have revealed a reciprocal relationship between GK gene copy number and the blood glucose concentration (19). GK gene knock-out mice have provided useful animal models for MODY-2, but analysis of these mice has been limited by several issues. First, mice that totally lack both islet and hepatic isoforms of GK, or even just the islet isoform, do not live for more than a few days, thereby preventing detailed physiological studies in adult animals. Second, the phenotype of mice that globally lack GK remains unsettled. Two different lines of global GK null mice differ markedly in their age of death; one is lethal at mid-gestation (13), whereas the other dies shortly after birth (14). Third, the role of the liver in the pathogenesis of hyperglycemia that characterizes MODY-2 has not been clearly resolved.

Characterization of the cell-specific roles of GK in glucose homeostasis requires an animal model in which the enzyme can be selectively eliminated in selected sites without affecting expression in other sites. To achieve this, we made use of the cre-loxP gene targeting strategy since it enables mice containing cell-specific gene deletions to be created (20-24). We describe here the generation of mice bearing two variant conditional gk alleles. We converted one of these conditional, loxed gk alleles to a null allele, and we also generated mice that express either insulin-cre (Rip-cre) or albumin-cre (Alb-cre) transgenes. By intercrossing two cell-specific Cre transgenes with the loxed gk allele, we generated mice with either hepatic or beta  cell-specific defects in GK expression. Analysis of these different global and tissue-specific GK gene knock-outs has allowed us to more clearly define how deficiencies in both hepatic and beta  cell GK cause MODY-2.

    EXPERIMENTAL PROCEDURES

Targeting Vector-- The key features of the targeting vector used, shown in Fig. 1a, are a phosphoglycerol kinase-neomycin resistance gene cassette (neoR), a phosphoglycerol kinase-herpes simplex virus type I thymidine kinase gene cassette, and three 34-bp loxP sequences (25). Two of the loxP sites flank neoR, and the third is located between exons 8 and 9 of the GK gene. The vector was assembled in pNTK(A) (a gift from Dr. Richard Mortensen, Harvard Medical School) using loxP sites isolated from pBS246 (25) and mouse GK gene fragments isolated from clone lambda 21 which was obtained from a 129/Ola P1 clone (26). A detailed description of the 11 DNA manipulations required to assemble the targeting vector are available on request. Correct assembly of this vector was confirmed by DNA sequencing. The sequences altered in the gklox allele were deposited in GenBankTM (accession numbers AF047362 and AF47830).

Gene Targeting and Blastocyst Injections-- 50 µg of the targeting vector was linearized with NotI and then electroporated into 5 × 107 TL-1 ES cells, a line derived from 129/SvEvTacBR mice (27). Analysis of several clones resistant to both G418 and ganciclovir revealed one clone, BG7, that had undergone the desired recombination event. NeoR was removed from this clone by partial Cre-mediated recombination. Briefly, 5 × 107 cells were electroporated with 50 µg of uncut pBS185, a CMV-cre expression vector (25). The transfected cells were plated at low dilution without G418 or ganciclovir selection, and ~200 clones were picked and characterized. From these, a single clone (BE8) was identified that had undergone partial recombination, thereby removing neoR and one loxP site but leaving both exons 9 and 10 and its flanking loxP sites intact. Both the BG7 and BE8 ES cell clones were microinjected into C57Bl/6 blastocysts and implanted into pseudopregnant ICR female recipients (28). Male chimeras were mated with C57Bl/6 females, and germline transmission was identified by both Southern blot and PCR analysis of tail DNA (29). Except for the gklox/lox mice, which were maintained as both inbred (129/SvEvTac) and hybrid lines, all other combinations of the conditional gk alleles and cell-specific Cre transgenes were studied as hybrid lines derived from 129/SvEvTac, C57Bl/6, and DBA-2 strains.

Animals-- All mice were housed in specific pathogen-free barrier facilities, maintained on a 12-h light/dark cycle, and fed a standard rodent chow (Purina Mills, Inc., St. Louis, MO). Pups from both sexes were used for analyses at day 2. For the rest of the studies only males were used. Animals were killed during the post-absorptive state (between 8:00 and 10:00 a.m.) except as specified.

Conversion of the gklox+neo Allele to a gkdel Allele-- Cre-mediated recombination of the gklox+neo allele into a gkdel allele was performed by pronuclear microinjection of supercoiled pBS185 DNA (25) into single cell mouse embryos derived from mating gklox+neo males with superovulated B6D2 F1 hybrid females. Conversion of the gklox+neo allele into a gkdel allele was detected by both Southern blot and PCR analysis of tail DNA from all resulting pups. The removal of exons 9 and 10 eliminates 125 of the 465 amino acids in both isoforms of GK.

Cre Transgenic Mice-- An insulin-cre transgene (Rip-cre) was assembled by removing nls-cre coding sequences from pmL78 (kindly provided by Mark Lewandowsky, University of California, San Francisco) by digestion with SalI and EcoRI and cloning them into the corresponding sites of pBSIIKS. A 668-bp fragment of the rat insulin 2 promoter (Rip) was ligated into the XhoI and KpnI sites of this vector, and a 2.1-kilobase pair fragment of the human growth hormone (hGH) gene was inserted into the SpeI site. Finally, the plasmid was digested with SalI and religated to remove a small intervening DNA fragment that had been created. The albumin-cre transgene (Alb-cre) was made by removing the nls-cre and hGH fusion fragment from the Rip-cre-hGH vector by digestion with XbaI. This fragment was blunt-ended by Klenow fill-in and then ligated into an EcoRV site of a plasmid containing the 2335-bp rat albumin enhancer/promoter fragment (kindly provided by Richard Palmiter, University of Washington) (30). The Rip-cre-hGH and Alb-cre-hGH plasmids were digested with KpnI and NotI, and the transgenes were isolated by gel electrophoresis and agarase digestion. These DNA fragments were microinjected into the pronuclei of B6D2 F2 hybrid mice by the Vanderbilt Transgenic/ES Cell Shared Resource. Tail DNA from potential founder mice was screened by Southern blot analysis for DNA integration. Rip-cre founder 25 and Alb-cre founder 21, the two Cre transgenic mice selected for use in this study, were estimated to contain 9 and 7 transgene copies, respectively.

Genotype Analysis-- Three different gk alleles (gkw, gklox, and gkdel) were routinely distinguished by PCR analysis. The gkw allele was detected using primers 1 (5'-TGTCTCAATTTGCTGTGTCCTCCA) and 2 (5'-TCTGTTAATGCAAATGCTCGTGTT), which amplify a 605-bp fragment. The gklox allele was detected as a 710-bp fragment after amplification with primers 1 and 2. The gkdel allele was detected as a 435-bp DNA fragment using primers 2 and 3 (5'-TTGAGACCCGTTTTGTGTCG). Both the Alb-cre and Rip-cre transgenes were detected using the primers 5'-ACCTGAAGATGTTCGCGATTATCT and 5'-ACCGTCAGTACGTGAGATATCTT, which amplify a 370-bp fragment. The lacZ transgenes were detected using the primers 5'-TGCTGATGAAGCAGAACAACTT and 5'-TATTTAATCAGCGACTGTCC, which amplify a 602-bp fragment.

Reverse Transcriptase-PCR Assay of Cre-hGH mRNA-- RNA was isolated from mouse tissues according to Chomczynski and Sacchi (31). First strand cDNA synthesis was carried out by first diluting 1 µg of RNA in 20 µl of diethylpyrocarbonate-treated water, denaturing at 85 °C for 3 min, and chilling on ice. In a 20-µl reaction containing 750 ng of the denatured RNA, 2 µl of 10× reaction buffer, 3 µM MgCl2 (Perkin-Elmer), 1 mM dNTPs (U. S. Biochemical Corp.), 10 units of reverse transcriptase, 20 units of RNasin, and 5 µg of oligo(dT) primer, first strand cDNA synthesis was carried out by incubating the reaction at 25 °C for 10 min, 37 °C for 30 min, 42 °C for 30 min, 95 °C for 5 min, and 5 °C for 5 min. After cDNA synthesis, PCR was carried out in 50-µl reactions containing 5 µl of 10× PCR mix, 1.5 mM MgCl2, 40 µM dATP, dTTP, dGTP, 20 µM dCTP, 2.5 µCi of [alpha -32P]dCTP, 2.5 units of Taq DNA polymerase (Promega), and 10 pmol of the primers 5'-GAAGCCTATATCCCAAAGGAA and 5'-GACCTTCAACGGTGAGGTCA, which amplify a fragment of 548 bp. Thermal cycling consisted of a 5-min denaturation at 95 °C followed by 26 cycles of 1 min denaturation at 95 °C, 1 min annealing at 59 °C, and 1 min extension at 72 °C. After a final extension at 72 °C for 7 min, 5 µl of the final reaction was mixed with 5 µl of sequencing gel loading buffer and denatured, and 4 µl was separated in a 6% denaturing polyacrylamide gel prior to autoradiography.

lacZ Reporter Mice-- A Cre-inducible nls-lacZ reporter gene (CMV-RAGE-beta -Gal) was provided by Dr. Alan Naftalin (Division of Cardiology, Vanderbilt University), which contains, in sequential order, a cytomegalovirus promoter, loxP, yeast His3 (stop) sequences, loxP, and nls-lacZ. The transgene was separated from vector DNA by digestion with SphI and NotI and gel electrophoresis. CATZ transgenic mice (32), which contain a beta  actin promoter-driven Cre-activable beta -galactosidase fusion gene, were kindly provided by M. Schneider (Baylor College of Medicine). Both lacZ reporter mice were estimated to have ~35 copies of the transgenes in their genome by quantitative Southern analysis.

Assessment of Cre Recombination-- Tissues were rapidly frozen in liquid nitrogen after removal from the animal, and genomic DNA was prepared according to Hogan et al. (29). The efficiency of Cre recombination was usually assessed by Southern blot analysis. DNA was digested with either BglII or XmnI, and the blots were probed with either an ~900-bp DNA fragment containing GK exons 9 and 10 or a ~600-bp BamHI-EcoRI fragment located at the 3' end of the GK gene locus (Fig. 1a).

Analytical Procedures-- Blood glucose concentrations were determined with a Hemocue blood glucose analyzer (Hemocue, Mission Viejo, CA). Plasma glucose concentrations were determined using a micro-assay procedure (33). The values obtained from the Hemocue were ~20% lower than the values determined by plasma glucose assay. Plasma insulin concentrations were determined by radioimmunoassay (RIA) using a rat insulin RIA kit from Linco Research (St. Louis, MO). Plasma non-esterified fatty acids were measured with a NEFA C kit from Wako Pure Chemical Industries. Hepatic glycogen content was analyzed as described by Keppler et al. (34). Plasma triglycerides and beta -hydroxybutyrate concentrations were measured using a colorimetric kit from Sigma.

Western Blot Analysis and GK Activity Measurements-- Western blot analysis and GK activities in crude liver extracts were determined as described previously (35).

Histological Analyses-- Organs were quickly removed after cervical dislocation and fixed in 4.0% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.6, for 2-4 h, and washed in PBS overnight. Tissues were either infiltrated in 30% sucrose/PBS for cryostat sectioning or dehydrated in ethanol for paraffin embedding. lacZ expression was detected by incubating tissues in stain solution (8.4 mM KCl; 84 mM phosphate buffer, pH 7.5; 2 mM MgCl2; 5 mM K4Fe(CN)6; 5 mM K4FeCN6; 0.01% sodium deoxycholate; 0.02% Nonidet P-40; 1% 5-bromo-4-chloro-3-indolyl beta -D-galactosidase) for 12 h at room temperature. Samples processed for histological examination were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. For detection of glycogen, cryostat sections of liver were subjected to the periodic acid-Schiff technique. Sections were oxidized 10 min in 0.5% periodic acid, washed, and stained with Schiff's reagent for 5 min. For the detection of neutral lipid, liver cryosections were stained with the Oil Red O technique using 0.23% dye dissolved in 65% isopropyl alcohol for 10 min. Following washing, sections were counterstained with hematoxylin.

Immunohistochemical Analysis-- Tissues were processed for immunocytochemical analysis as described previously (36). For Cre immunostaining, whole pancreas was fixed and equilibrated in 30% sucrose in PBS overnight at 4 °C. Tissues were then frozen in OCT in a cryostat chamber at -20 to -35 °C, sectioned, mounted onto electrostatically charged slides, and allowed to dry at room temperature for at least 30 min. Cre was then detected using a rabbit polyclonal antisera to Cre (kindly provided by Barbara Morris, Novagen).

In Vivo Glucose Kinetics-- Hyperglycemic clamp studies were performed using chronically cannulated, conscious mice as described previously (18). All groups of mice were fasted 6-8 h prior to the experimentation. Body weight, hematocrit, and general appearance were used as indices of health. A variable infusion of 50% glucose was used to raise blood glucose levels to ~300 mg/dl. A 4 µCi bolus of tracer ([3-3H]glucose, NEN Life Science Products) was given at -100 min, followed by a constant 0.04 µCi/min for the duration of the study. The glucose turnover rate (mg/kg/min) was calculated as the rate of tracer infusion (dpm/min) divided by the blood glucose specific activity (dpm/mg) corrected to the body weight of the mouse. Glycogen synthesis via the direct pathway was determined by measuring incorporation of [3H] into glycogen (dpm/g liver)/[3H] specific activity in plasma glucose (dpm/mg).

Statistical Analysis-- All results are presented as the mean ± S.E. of the mean. Statistical significance was determined by Student's t test.

    RESULTS

Conditional gk Alleles-- A conditional GK gene allele (gklox+neo) that contains three loxP sites and a neomycin resistance (neoR) cassette was created by gene targeting in ES cells (Fig. 1a). This allele contains two loxP sites flanking exons 9 and 10 and a third loxP site downstream of neoR. By partial Cre-mediated recombination in ES cells, the gklox+neo allele was converted to a second conditional allele (gklox) that lacks neoR and contains only two loxP sites (Fig. 1a). Correct gene targeting and gene conversion in ES cells was confirmed by Southern blot (Fig. 1, b and c) and PCR analysis (not shown). ES cells containing either a gklox+neo or gklox allele were then used to generate mice with these two conditional alleles, and each were bred to homozygosity. Both lines of mice obtained in this manner were viable, but each exhibited subtle perturbations in their blood glucose concentration. Eight-week-old gklox+neo/lox+neo mice had a blood glucose concentration of 242 ± 9 mg/dl compared with 194 ± 3 mg/dl for the gklox/lox mice and 175 ± 8 mg/dl for mice with two high activity (gka) wild type alleles (hereafter designated as gkw) (37). The mice were otherwise normal after at least six generations of inbreeding. Since the mixing of different inbred strains of mice may have contributed to the minor differences observed, we also compared the basal blood glucose concentrations of mice with two wild type alleles (gkw/w) with the gklox/lox mice in an inbred 129SvEvTac background (Table I). A persistent difference was also observed, suggesting that the insertion of a loxP site and some flanking sequences between exons 8 and 9 caused a slight attenuation in GK gene expression. Indeed, measurements of hepatic GK activity indicated it was reduced by 28% in gklox/lox mice compared with gkw/w mice (15 ± 1 milliunits/mg protein versus 21 ± 3 milliunits/mg protein, p < 0.05).


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Fig. 1.   Gene targeting and Cre deletion events. a, top, a partial map of the wild type gkw allele. Exons are indicated as solid rectangles. The location of the DNA fragment used as the hybridization probe in b and c is shown. Middle, a map of the GK gene targeting vector is shown. The vector contains a phosphoglycerol kinase-neomycin resistance gene cassette (neoR), a phosphoglycerol kinase-herpes simplex virus type I thymidine kinase gene cassette, and three loxP sequences (represented as triangles). Two of the loxP sites flank neoR, and the third is located between exons 8 and 9 in the GK gene. The gklox+neo allele was created by homologous recombination (HR) in ES cells. Bottom, the gklox allele was derived from the gklox+neo allele through partial Cre recombination. Exons 9 and 10 and neoR were excised by Cre DNA microinjection or cell-specific Cre expression in transgenic mice. b and c, Southern blot analysis. Tail DNA was digested either with XmnI (b) or BglII (c) and then probed with a PCR DNA fragment corresponding to mouse GK exons 9/10 (~900 bp). The location of the probe is indicated in a. Expected alleles and sizes are shown on the right and the left sides, respectively. The genotypes shown (left to right) are gkw,w, gklox+neo/w, gklox+neo, lox+neo, gklox/w, and gklox/lox. kb, kilobase pairs.

                              
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Table I
Comparison of post-absorptive (basal) plasma glucose concentrations (mg/dl) in different lines of mice
Blood samples were collected, quickly centrifuged, and plasma removed and frozen immediately at -80 °C. Statistical analysis was performed with a two-tailed unpaired Student's t test. Values are represented as means ± S.E. Comparisons were made among mice of similar age and genetic background. Significance was accepted at p < 0.05. ND, not determined.

Characterization of Mice with a Global Knock-out of GK-- To determine the effect of a total deletion of GK, we converted the gklox+neo allele into a deleted allele (gkdel) by microinjecting different concentrations of a CMV-cre expression plasmid (pBS185) into single cell gklox+neo/w mouse embryos (38). A total of 56 pups were born from 398 microinjected embryos. Tail DNAs from these animals were analyzed by both Southern blot (Fig. 2a) and PCR analysis (Fig. 2b), and each recombination event was categorized as either complete or partial (results not shown). At concentrations of 0.1 ng/µl or greater, Cre efficiently excised both loxP-flanked DNA fragments, thereby creating a total of 10 mice that were heterozygous for the gkdel allele (e.g. gkdel/w). Mice that were homozygous null for GK (e.g. gkdel/del) were then obtained by intercrossing gkdel/w mice. Genotype analysis of 115 newborn pups revealed frequencies of 32 gkw/w, 59 gkdel/w, and 24 gkdel/del mice, consistent with Mendelian inheritance. gkdel/del mice appeared normal at birth, but died within 4 days of birth from severe diabetes, as observed previously by Grupe et al. (14). gkdel/w mice had an ~50% reduction in hepatic GK activity (9.7 ± 1.8 compared with 21 ± 3 milliunits/mg protein in gkw/w mice, p < 0.05). GK immunoreactivity was not detected in either the liver or in islets of gkdel/del mice (not shown).


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Fig. 2.   Conversion of the gklox+neo allele to a gkdel allele. a, Southern blot analysis. Tail DNA was digested with XmnI and then probed with the 3' end probe (location is shown in Fig. 1a). Lane 1, genomic DNA from a gklox+neo/w mouse; lane 2, DNA from a gkdel/w mouse, showing modification in size of the gene as a consequence of Cre-mediated recombination. Expected alleles and sizes are shown on the right and the left of the panel, respectively. b, example of PCR genotype analysis. 605- and a 435-bp DNA fragments corresponding to the gkw and gkdel alleles, respectively, were amplified using specific primers as described under "Experimental Procedures." kb, kilobase pairs.

To determine the effects of diminished GK activity plasma glucose, insulin, and free fatty acid concentrations were determined at postnatal day 2 (P2). Two-day old gkdel/del mice were severely diabetic with plasma glucose concentrations of 566 ± 64 mg/dl (Table I). Plasma glucose concentrations in gkdel/w mice differed from that of gkw/w mice as early as P2 and became more pronounced with age (Table I). Plasma insulin concentrations were reduced by ~70% in gkdel/del mice compared with gkw/w mice at P2 (0.11 ± 0.01 versus 0.33 ± 0.08 ng/ml, p < 0.001), but no difference in insulin concentrations were detected between gkdel/w and gkw/w mice, even though gkdel/w had elevated plasma glucose concentrations (Table I). Free fatty acid levels were ~1.8-fold higher in gkdel/del pups (0.55 ± 0.07 versus 0.3 ± 0.04 mEq/l in gkw/w mice, p < 0.05), consistent with the observed hypoinsulinemia. As previously reported by Grupe et al. (14), the livers of the gkdel/del mice were markedly steatotic (Fig. 3c). Oil red O staining of liver sections from gkdel/del mice showed large fat droplets within hepatocytes (Fig. 3f). Hepatic glycogen was also diminished, as was evident by periodic acid-Schiff staining (Fig. 3i). Livers of gkdel/w mice showed intermediate amounts of steatosis and glycogen content compared with livers of both gkw/w and gkdel/del pups (Fig. 3, b, e, and h).


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Fig. 3.   Gross and histochemical analyses of livers from perinatal normal and GK knock-out mice. a-c, fatty liver phenotype. Necropsies were performed on gkw/w, gkdel/w, and gkdel/del mice at P2 to demonstrate differences in the appearances of the livers due to steatosis. Dissecting microscope view showing the progressive loss of the normal red color with diminished GK gene copy number. d-f, oil red O histochemistry for neutral lipid in liver sections. Only small lipid droplets (red) are observed in livers of normal gkW/W mice, whereas more numerous and larger lipid droplets are observed in livers of both gkdel/w and gkdel/del mice. The sections were counterstained with hematoxylin. g-i, periodic acid-Schiff histochemistry for glycogen in liver sections. High levels of glycogen are observed in liver of gkw/w mice, which is diminished most notably in gkdel/del mice. Scale bars represent 50 µm. n = 3-6 in each group.

In Vivo Analysis of Glucose Metabolism in Adult gkdel/w Mice-- To elucidate the physiological mechanisms of the hyperglycemic phenotype basal and hyperglycemic clamp studies were performed using adult gkw/w and gkdel/w mice. Under basal conditions, mice that are heterozygous null for GK are hyperglycemic compared with mice with two wild type alleles (Table II). gkw/w and gkdel/w mice did not show any difference in their fasting insulin concentrations (Table II). In hyperglycemic clamp studies, during which blood glucose levels were raised to ~300 mg/dl, gkdel/w mice demonstrated marked glucose intolerance as reflected by an ~70% reduction in glucose infusion rate compared with controls (Table II). Bali et al. (13) have previously reported a similar amount of glucose intolerance in adult mice with only one functional GK gene copy. The peak of insulin secretion in response to the hyperglycemic clamp was decreased by 60% in gkdel/w mice (Table II). Using [3-3H]glucose as a metabolic tracer, we measured glucose turnover and clearance rates, as well as hepatic glycogen synthesis, under hyperglycemic conditions (Table II). A striking difference in newly synthesized hepatic glycogen was observed at the end of the hyperglycemic clamp study with synthesis being decreased by >90% in gkdel/w mice compared with gkw/w mice (Table II).

                              
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Table II
Summary of metabolic parameters from basal and hyperglycemic clamp experiments of gkw/w and gkdel/w mice
Several metabolic parameters were experimentally determined, and indices of glucose metabolism were calculated. The gkdel/w/gkw/w ratio for each parameter is shown. For the hyperglycemic clamp studies, a variable infusion of 50% glucose was used to raise blood glucose levels to ~300 mg/dl. [3-3H]Glucose was used as a metabolic tracer to measure glucose turnover and glucose clearance rates. Glycogen synthesis from direct pathway was also calculated as described under "Experimental Procedures." n = 3-6 for each group.

Use of an Insulin-cre Transgene to Eliminate GK Expression only in beta  Cells-- To evaluate independently the effects of diminished beta  cell GK on glucose homeostasis, we next sought to generate mice that lacked only beta  cell GK. To accomplish this, Rip-cre transgenic mice were made that expressed Cre under the control of a 668-bp fragment of the rat insulin II promoter. One of the five founder lines produced showed a high level of expression nearly exclusively in the pancreas (Fig. 4a). Analysis of Cre expression by immunocytochemistry (Fig. 4b) identified recombinase protein in most beta  cells (82% of 1770 beta  cells examined) of this line. No Cre immunoreactivity was detected in acinar cells or in other endocrine cell types (Fig. 4b). To verify that the Rip-cre transgene itself was not affecting beta  cell function and glucose homeostasis, the blood glucose concentrations of Rip-cre transgenic mice were determined and found not to be significantly different from that of the non-transgenic controls (179 ± 7 mg/dl in gkw/w mice compared with 159 ± 7 mg/dl in gkw/w+Rip-cre mice). The ability of the Rip-cre transgene to promote recombination only in beta  cells was assessed by crossing Rip-cre mice with CATZ mice, a line bearing a Cre-inducible lacZ reporter gene (32). lacZ-expressing cells were only observed within islets (Fig. 4c) with no staining seen in any other tissue examined (not shown); however, the number of blue cells observed was fewer than expected, especially in light of the results shown below. When the Rip-cre transgenic mice were also crossed with another line of mice containing a different Cre-inducible lacZ transgene a similar result was obtained (not shown).


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Fig. 4.   Rip-cre and Alb-cre transgenic mice. a, amplification by reverse transcriptase-PCR of hGH mRNA in pancreas (Pan) of Rip-cre mouse (founder 25) and in liver (Liv) of Alb-cre mouse (founder 21). b, confocal image of a frozen section from a representative islet from Rip-cre line immunostained for insulin (green) and Cre (red-orange). Cre is expressed in most beta  cells but not other cell types in the adult animal. Scale bar represents 50 µm. c, lacZ-stained islet from CATZ +Rip-cre mice showing cytoplasmic beta -galactosidase staining in several pancreatic beta  cells. lacZ staining was restricted to beta  cells in these animals. d, lacZ-stained liver from lacZ+Alb-cre mice showing cytoplasmic beta -galactosidase activity in most hepatocytes. lacZ staining was specific to the liver in this line of mice. Spl, spleen; Kid, kidney; Hrt, heart; SkM, skeletal muscle; Brn, brain. Scale bar for c and d represents 50 µm.

To generate beta  cell-specific GK knock-out mice (e.g. gklox/lox +Rip-cre), animals heterozygous for the loxed GK gene and that carried the Rip-cre transgene were mated with gklox/lox mice. Genotype analysis of 127 pups alive at 3 weeks of age identified only a few surviving gklox/lox+Rip-cre pups (40 gklox/w, 40 gklox/lox, 38 gklox/w+Rip-cre, and 9 gklox/lox+Rip-cre, chi 2 = 21.81, p < 0.001). The blood glucose concentrations of these few surviving mice were highly variable (range 175-327 mg/dl) but generally greater than that of the gklox/lox mice. Several of the viable gklox/lox+Rip-cre mice were then mated to gklox/lox mice, and 30 of their offspring were killed at P2 for analysis. gklox/lox+Rip-cre P2 pups had blood glucose concentrations of 335 ± 35 mg/dl (range 219-511) compared with 118 ± 8 (range 41-191) in gklox/lox mice (Table I). Analysis of plasma insulin concentrations revealed that they were decreased by ~70% in gklox/lox+Rip-cre P2 pups compared with gklox/lox mice (0.14 ± 0.02 ng/ml versus 0.43 ± 0.07 ng/ml, p < 0.001). Blood glucose concentrations in gklox/w+Rip-cre mice were also increased by ~50% compared with gklox/w controls (Table I).

Southern blot analysis of a variety of tissue DNAs from gklox/lox+Rip-cre mice did not reveal recombination in any tissue (Fig. 5a), although PCR analysis of genomic revealed recombination within the pancreas (Fig. 5c). GK immunostaining was also nearly absent in beta  cells of gklox/lox+Rip-cre compared with gklox/lox mice (not shown). Together, these results indicate that co-expression of the Rip-cre transgene with the gklox/lox allele causes >80% neonatal mortality as a result of severe hyperglycemia. The absence of 100% mortality may be due to minor variegation in the expression of the Rip-cre transgene, as suggested by the Cre immunocytochemistry (Fig. 4b). The same metabolic and biochemical alterations that were observed in livers of gkdel/del neonates, namely steatosis and glycogen depletion, were also observed in livers from gklox/lox+Rip-cre mice (not shown). gklox/w+Rip-cre mice, which bear only one functional gk allele in the beta  cells, were moderately hyperglycemic and survived (Table I). At 6-10 weeks of age these animals showed ~30% increase in basal (post-absorptive) blood glucose, without any difference in their insulin concentrations (0.30 ± 0.07 ng/ml in gklox/w+Rip-cre mice compared with 0.36 ± 0.15 ng/ml in gklox/w mice).


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Fig. 5.   beta cell or liver-restricted recombination in tissues of gklox/lox+Rip-cre or gklox/lox+Alb-cre mice. a, Southern blot analysis of DNA from tissues prepared from a gklox/lox+Rip-cre mouse. b, Southern blot analysis of DNA from tissues from a gklox/lox+Alb-cre mouse. Blots were then probed with the 3' probe (Fig. 1a) to detect either the gklox (top) or gkdel (bottom) alleles. c, PCR analysis showing amplification of the gkdel allele in pancreas from gklox/lox+Rip-cre mice. The primers used are described under "Experimental Procedures."

To determine the effect of a loss of a single copy of the GK gene only in beta  cells on insulin secretion and on hepatic fluxes, a 2-h hyperglycemic clamp study was performed in adult gklox/w +Rip-cre mice and gklox/w controls (Table III). Under fasting conditions, gklox/w+Rip-cre mice had a 25% increase in the blood glucose concentration, without a detectable difference in basal insulin concentrations (Table III). However, during the hyperglycemic clamp insulin secretion was 70% lower than that of gklox/w+Rip-cre mice (Table III). Glucose turnover and glucose infusion rates during the hyperglycemic clamp were also reduced by ~60 and 70% respectively, in gklox/w+Rip-cre mice. Net glycogen synthesis in liver, measured using [3-3H]glucose as a metabolic tracer, was reduced by ~50% in liver of gklox/w+Rip-cre (Table III), presumably as a consequence of diminished insulin secretion.

                              
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Table III
Summary of metabolic parameters from basal and hyperglycemic clamp experiments of gklox/w and gklox/w + Rip-cre mice
Several metabolic parameters were experimentally determined, and indices of glucose metabolism were calculated as described under "Experimental Procedures." n = 3-6 for each group.

Use of an Albumin-cre Transgene to Eliminate Hepatic GK-- To determine the independent effects of diminished hepatic GK on glucose homeostasis, we next generated mice with a liver-specific knock-out of GK. A transgene containing the rat albumin promoter/enhancer (Alb-cre) was used to produce four gklox/w+Alb-cre founder mice. By reverse transcriptase-PCR analysis, one founder expressed the Alb-cre transgene specifically in the liver (Fig. 4a). A line was established from this animal and used in all subsequent analysis. The ability of the Alb-cre transgene to specifically cause recombination in the liver was assessed by intercrossing it with a Cre-inducible lacZ reporter gene, as above. This experiment revealed recombination in liver (Fig. 4d) but not in any other tissue examined (not shown).

Hepatic GK knock-out mice (e.g. gklox/lox+Alb-cre) were obtained by intercrossing gklox/w+Alb-cre transgenic founders with gklox/w mice. In contrast to the lethality caused by intercrossing the Rip-cre transgene, the Alb-cre transgene did not adversely affect animal survival. Like the Rip-cre transgene, the Alb-cre transgene did not affect basal blood glucose in the presence of two wild type gk alleles (not shown). Southern blot analysis of tissue DNAs from gklox/lox+Alb-cre mice showed over 80% conversion of the gklox to gkdel allele in liver, with no sign of recombination in other tissues (Fig. 5b). Total recombination was not expected to occur in liver since this tissue also contains several other cell types besides hepatocytes (39). To assess further the efficiency of recombination within hepatocytes, both Western blot analysis and glucose phosphorylation assays were performed. As shown in Fig. 6, a and b, gklox/lox+Alb-cre mice lacked detectable GK immunoreactivity and had at least a 95% reduction in GK activity in liver. Immunocytochemical analysis of livers from gklox/lox+Alb-cre mice also showed no evidence of nuclear GK immunoreactivity, which was readily observed for gklox/lox mice (Fig. 6c). Interestingly, despite the virtually complete elimination of hepatic GK, adult gklox/lox+Alb-cre mice showed only a ~10% increase in their fed blood glucose concentrations compared with gklox/lox controls (Table I). However, insulin levels in fed hepatic GK knock-out mice were twice as high as the appropriate control (1.2 ± 0.2 ng/ml for gklox/lox+Alb-cre mice versus 0.6 ± 0.08 ng/ml for gklox/lox mice, p < 0.05). Measurement of plasma free fatty acid, triglyceride, and beta -hydroxybutyrate concentrations in gklox/lox+Alb-cre mice showed no differences in these plasma metabolites (Table III). The hepatic GK knock-out mice also had normal levels of both hepatic and muscle glycogen (Table IV).


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Fig. 6.   Liver-specific inactivation of GK using Alb-cre transgenic mice. a, a representative Western blot analysis of liver homogenates demonstrating lack of expression of GK in liver of gklox/lox+Alb-cre mice is shown (n = 5 per group; p < 0.001 compared with controls). b, hepatic GK phosphorylating activity in liver of gklox/lox and gklox/lox+Alb-cre mice (n = 5 per group; p < 0.001 compared with controls). c, immunoperoxidase staining for GK (light brown) in liver. Nuclear GK immunoreactivity was only detected in hepatocytes from gklox/lox but not from gklox/lox+Alb-cre mice. Scale bar represents 50 µm.

                              
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Table IV
Plasma circulating intermediates and glycogen content in gklox/lox and gklox/lox + Alb-cre mice
Blood samples were taken from the tail vein, and metabolite concentrations were measured as described under "Experimental Procedures." Hepatic and muscle glycogen content was measured according to Kepler et al. (34). The gklox/lox + Alb-cre/gklox/lox ratio for each parameter is shown.

Finally, to explore to what extent the absence of hepatic GK had on both insulin secretion and hepatic fluxes, we performed basal and hyperglycemic clamp studies in both gklox/lox+Alb-cre and gklox/lox mice (Table V). In these animals, which had been fasted for 8 h prior to the study, blood glucose concentrations in gklox/lox+Alb-cre mice were ~40% higher than the controls, with no differences being detected in either plasma insulin concentration or glucose turnover rate (Table V). During the hyperglycemic clamp phase of the study, glucose turnover and glucose infusion rates were markedly reduced in the gklox/lox+Alb-cre mice (Table V). Net hepatic glycogen synthesis, measured at the end of the 2-h hyperglycemic clamp, was reduced by ~90% in liver of gklox/lox+Alb-cre mice compared with their controls (Table V). Surprisingly, gklox/lox+Alb-cre mice secreted 70% less insulin in response to the glucose stimulus compared with controls (Table V).

                              
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Table V
Summary of metabolic parameters from basal and hyperglycemic clamp experiments of gklox/lox and gklox/lox + Alb-cre mice
n = 3-6 for each group.


    DISCUSSION

More than 40 different GK gene mutations have been found to cause MODY-2 (1-4). Since these mutations are located in exons shared by both the islet and the hepatic GK isoforms, diminished GK activity is predicted to occur in all sites where the gene is expressed. Although prior studies have suggested that diminished beta  cell GK is primarily responsible for the hyperglycemia in MODY-2, the design of these studies did not allow the contributions of individual tissues, particularly the liver, to be accurately assessed. Thus, we have used the cre-loxP strategy to selectively eliminate GK expression in both the liver and pancreatic beta  cell, thereby enabling us to independently determine the contributions of these two cell types to the pathogenesis of hyperglycemia in MODY-2.

Creation of Mice with Conditional gk Alleles-- Mice that were homozygous for either of the two conditional GK alleles (e.g. gklox+neo/lox+neo or gklox/lox) had blood glucose concentrations that were slightly increased compared with mice with two high activity (gkw) wild type alleles (37). The blood glucose concentration was greatest in mice with two gklox+neo alleles, which is readily understandable given the well described potential for adverse effects of placing a functional transcription unit close to other genes (40). Although mice with two gklox alleles had blood glucose concentrations ~10% greater than wild type, and hepatic GK activity 28% lower than wild type, these variations were small and did not adversely affect our studies. However, it is noteworthy that even small differences in GK gene expression, such as that apparently caused by inserting a loxP site into the transcribed region of the gene, manifest themselves as an alteration in the plasma glucose concentration. Whereas this finding provides even further evidence of the high control strength of this enzyme (41), it also suggests that the insertion of loxP sequences within introns will not always be benign, and thus must be done cautiously.

Effect of a Global GK Knock-out-- Mice that totally lack GK (e.g. gkdel/del) are severely hyperglycemic at P2 and die within the first few days of life. This phenotype matches that previously reported by Grupe et al. (14) but differs from that of Bali et al. (13) who observed embryonic lethality beginning at about embryonic day 9.5. This difference may be due to the random insertion of a second copy of the targeting vector in the genome of the mice made by Bali et al. (13), as indicated by the presence of an unexpected band after hybridization of DNA from these mice with a probe containing neoR sequences (35). This may have led to the inadvertent knock-out of a gene that is essential during early embryogenesis. Whereas the lethal embryonic phenotype seems to have been rescued by an 83-kilobase pair transgene containing the entire GK gene locus (35), which argues against this possibility, the experiment did not take into account the possibility of allele segregation.

The metabolic alterations observed in gkdel/del pups, particularly the hepatic steatosis, are all consistent with insufficient insulin secretion due the inability of beta  cells in the GK null mice to respond to an elevated plasma glucose concentration. Mice that have induced mutations of both insulin genes have nearly an identical phenotype (42). Pups that totally lack insulin also have marked hepatic steatosis and die of severe hyperglycemia within 48 h of birth (42). Hepatic glycogen content is also markedly reduced in gkdel/del pups, consistent with a rapid depletion of hepatic glycogen content after birth as a consequence of insulin deficiency (43).

beta Cell-specific Knock-out of GK-- Mice that lack GK only in beta  cells are phenotypically similar to animals with a global GK knock-out since most gklox/lox+Rip-cre pups die shortly after birth of severe diabetes. The lack of total lethality in the gklox/lox+Rip-cre animals is probably due to minor variegation in the expression of the Rip-cre transgene, as was demonstrated by immunohistochemistry. The finding of severe diabetes in mice with a nearly complete knock-out of GK in the beta  cell, but which still have the islet isoform of GK in rare glucose-sensitive cells in both the brain and gut (12), indicates that extrapancreatic glucose-sensing mechanisms are incapable of overriding a major defect in beta  cell glucose sensing. Also, the fact that the phenotype of the gklox/lox+Rip-cre mice, which lack GK only in beta  cells, is very similar to that of the mutant mice of Terauchi et al. (15), which lack expression of the islet isoform of GK in all sites (e.g. beta  cell, brain, and gut), indicates that the phenotype of the islet GK knock-out mice is due principally to the loss of beta  cell GK. Thus, the results of both of these studies are complementary with each pointing to an indispensible role for pancreatic beta  cell GK in glucose homeostasis. The extent to which GK in brain and gut enteroendocrine cells contributes to glucose homeostasis, if at all, remains to be determined.

It is also interesting to note that the hyperglycemic phenotype of the gklox/w+Rip-cre mice is greater than that previously observed in animals that express an insulin-GK ribozyme transgene (44). Whereas the results of these two different studies may not be directly comparable, it is likely that the Cre strategy, compared with use of a GK ribozyme transgene strategy, is more efficient in reducing the amount of islet GK.

Liver-specific Knock-out of GK-- In contrast to the perinatal lethality associated with the lack of beta  cell GK, the total absence of hepatic GK did not cause the mice to die. However, the in vivo analysis performed clearly indicates that hepatic GK, through both direct and indirect effects, plays an important role in glucose homeostasis. Although hepatic GK knock-out mice show only a ~10% increase in their glucose concentrations, they have post-absorptive (fed) plasma insulin concentrations twice that of controls. The latter finding suggests that diminished hepatic GK leads to a state of mild insulin resistance. It has long been known that a major action of insulin in the liver is to stimulate hepatic GK gene expression, which in turn has been thought to enhance hepatic glucose disposal. Thus, the absence of hepatic GK appears to severely attenuate at least one major effect of insulin in the liver, namely to enhance glucose utilization. However, since hyperglycemia and hyperinsulinemia are also known to cause diminished glucose utilization in the muscle, the loss of hepatic GK may cause additional metabolic disturbances that interfere not only with hepatic glucose disposal but also attenuate insulin action and glucose utilization elsewhere.

Impaired glycogen synthesis and a decreased contribution of the direct pathway to glycogen synthesis has previously been reported in patients with MODY-2 (45). We have found that liver-specific GK knock-out mice have normal glycogen content post-feeding but show a marked impairment in their ability to synthesize glycogen during a hyperglycemic clamp. These results indicate that while hepatic GK is essential for the synthesis of glycogen from the direct pathway during hyperglycemia, normal glycogen levels can be maintained as a consequence of the indirect pathway. Interestingly, the gklox/lox+Alb-cre mice are able to synthesize small amounts of glycogen in liver (0.5 ± 0.1 versus 4.0 ± 0.8 mg/g in controls) even in the virtual or total absence of hepatic GK. Previous studies have shown that glucose 6-phosphate produced by GK, and not by hexokinase I, promotes the activation of glycogen synthase and glycogen accumulation in hepatocytes (46, 47). Partial inhibition of hepatic GK in the rat, achieved by the infusion of glucosamine, has been shown to impair the ability of hyperglycemia to suppress endogenous glucose production (48). Thus, the gklox/lox+Alb-cre mice, which have virtually a total defect in hepatic GK, must be able to maintain normal levels of glycogen either by using glucose 6-phosphate derived from hexokinase I or substrate flux originating via the indirect pathway. The consequences of the lack of hepatic GK on various hepatic substrate fluxes remain to be explored further.

An unexpected finding of these studies is that the loss of hepatic GK leads to impaired insulin secretion. This defect appears to be caused by the mild but sustained hyperglycemia in these mice. Whereas chronic hyperglycemia might eventually be expected to lead to an increase in beta  cell mass sufficient to match the need for increased insulin production (49), this does not seem to be the case. The effects of chronic hyperglycemia on beta  cell function are well documented but poorly understood (50, 51). Sustained hyperglycemia has been shown to cause beta  cells to become unresponsive to glucose, thereby leading to an impairment in glucose-induced insulin secretion. The degree of dysfunction depends both on the glucose level and the duration of the exposure, with even mild hyperglycemia having been shown to cause beta  cell dysfunction in diabetic rat models (52-54). The highly defined nature of the metabolic defect in the gklox/lox+Alb-cre may enable these animals to be used as a model system to determine the mechanisms by which hyperglycemia impairs beta  cell function.

Role of the Liver in the Hyperglycemic Phenotype of MODY-2-- These studies have allowed us to determine directly the role of hepatic GK in glucose homeostasis. By using the cre-loxP strategy in mice, information has been obtained that helps us to understand better why GK gene defects in humans cause hyperglycemia. First, the gklox/w+Alb-cre mice, in which only one of the GK genes is deleted only in the liver, have blood glucose concentrations as adult animals that are ~13% greater than their gklox/w controls. Second, indirect support of a role for hepatic GK is suggested by comparing the blood glucose concentrations of the gkdel/w and gklox/w+Rip-cre mice, since this presumably reflects the net effect of GK in all sites of expression besides the beta  cell. Adult gkdel/w mice have an ~74% increase in basal blood glucose compared with gkw/w mice (Table I), whereas the blood glucose concentration of the gklox/w+Rip-cre mice is only elevated by ~34%, compared with gklox/w controls. Thus, the ~40% difference between these two different groups of mice is probably due largely to hepatic GK, although in principle some of this difference could also indicate a role for brain or gut GK in glucose homeostasis. Finally, a role for the liver in determining the hyperglycemia of MODY-2 is also suggested by the lack of differences in insulin levels in gkdel/w, gklox/w+Rip-cre, and gklox/lox+Alb-cre mice after an 8-h fast (in each case compared with the appropriate controls), even though all of these mice remained hyperglycemic.

A critical role for GK in hepatic glucose metabolism has previously been demonstrated by overexpressing the enzyme. Increased GK, both in primary hepatocytes and in transgenic mice, has been shown to enhance both glycolysis and glycogen synthesis (16-18, 46, 47). Whereas the lack of hepatic GK has less of an effect on the plasma glucose concentration than might have been expected, these data, as well as those obtained previously from GK gene locus transgenic mice (18), clearly indicate that variations in the amount of hepatic GK can profoundly affect basal glucose concentrations. The finding that hepatic GK, in addition to that of beta  cell GK, plays a role in causing the hyperglycemia of MODY-2 is consistent with studies showing impairments in liver glucose metabolism in patients with this disease (45, 55).

Issues Pertaining to the Use of Cre-loxP Strategy in Physiologic Studies-- These studies clearly indicate the feasibility of using cre-loxP technology to study the tissue-specific contributions of certain genes in glucose homeostasis despite the fact that certain experimental aspects of this methodology remain suboptimal. For instance, it remains problematic to determine the precise efficiency of recombination in rare cell types, such as pancreatic beta  cells. Our use of two different lines of Cre-inducible lacZ reporter mice, similar to those described by other investigators (32, 38), failed to provide this information. Although such reporter mice have been advocated as reliable indicators of recombination (20), both lines of mice we used appeared to under-report the actual amount of recombination that was achieved at a loxed gk allele. The Alb-cre transgene, which caused a total reduction in hepatic GK expression, induced expression of lacZ in only about half of hepatocytes when assessed by histochemical analysis. A similar situation was observed for the Rip-cre transgene, which was shown to express Cre in at least 82% of beta  cells by immunocytochemistry and which was lethal in combination with two loxed gk alleles but did not cause lacZ expression in nearly as many cells. The reason why the lacZ reporter mice were less useful than expected is not known but could be due either to multiple tandem copies of the transgenes, which might cause unexpected recombination events, or that these fusion genes become hypermethylated and thus might be silent in some cells. Despite these issues, the lacZ reporter mice were useful in demonstrating sites of recombination.

Conclusion-- This study has demonstrated the feasibility of using the cre-loxP methodology to explore a complex metabolic and physiologic question, namely the cell-specific roles of GK in glucose homeostasis. Based on the knowledge gained by this analysis, it is now feasible to use mice with specific defects in hepatic or beta  cell GK as models of either hepatic insulin -resistance or impaired beta  cell function. We expect that by using either inducible Cre transgenes (22-24, 56) or recombinant adenovirus that express Cre (57), it should soon be feasible to induce defects in either hepatic and/or beta  cell GK in adult animals or in tissues removed from animals bearing conditional gk alleles.

    ACKNOWLEDGEMENTS

ES cell and DNA microinjections were performed by the Vanderbilt Transgenic/ES Cell Shared Resource; confocal micrographs were acquired using the Cell Imaging Shared Resource, and radioimmunoassays were performed by RIA core laboratory in the Vanderbilt Diabetes Center (these facilities). We thank Catherine Pan for technical assistance; Chris Wright for helpful comments; Mark Lewandowsky for nls-cre DNA; Barbara Norris for the anti-Cre antisera; and M. Schneider for the LacZ reporter mice.

    FOOTNOTES

* These studies were supported in part by National Institutes of Health Grants DK42612 and DK 42502. Vanderbilt Transgenic/ES Cell Shared Resource, Cell Imaging Shared Resource, and RIA core laboratory in the Vanderbilt Diabetes Center are supported by National Institutes of Health Grants CA68485 and DK20593.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Vanderbilt Medical Scientist Trainee supported by National Institutes of Health Grant 5T3Z C-M07347.

parallel To whom correspondence should be addressed: 723 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0615. Tel.: 615-322-7006; Fax: 615-322-7236; E-mail: mark.magnuson{at}mcmail.vanderbilt.edu.

    ABBREVIATIONS

The abbreviations used are: GK, glucokinase; MODY-2, maturity onset diabetes of the young, type 2; PCR, polymerase chain reaction; bp, base pair(s); PBS, phosphate-buffered saline; hGH, human growth hormone; P2, postnatal day 2..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Froguel, P., Vaxillaire, M., Sun, F., Velho, G., Zouali, H., Butel, M. O., Lesage, S., Vionnet, N., Clément, K., Fougerousse, F., Tanizawa, Y., Weissenbach, J., Beckman, S. J., Lathrop, J. M., Passa, P., Permutt, M. A., and Cohen, D. (1992) Nature 356, 162-164[CrossRef][Medline] [Order article via Infotrieve]
  2. Velho, G., Froguel, P., Clement, K., Pueyo, M. E., Rakotoambinina, B., Zouali, H., Passa, P., Cohen, D., and Robert, J. J. (1992) Lancet 340, 1162-1163[Medline] [Order article via Infotrieve]
  3. Velho, G., Blanche, H., Vaxillaire, M., Bellanne-Chantelot, C., Pardini, V. C., Timsit, J., Passa, P., Deschamps, I., Robert, J.-J., Weber, I. T., Marotta, D., Pilkis, S. J., Lipkind, G. M., Bell, G. I., and Froguel, P. (1997) Diabetologia 40, 217-224[CrossRef][Medline] [Order article via Infotrieve]
  4. Vionnet, N., Stoffel, M., Takeda, J., Yasuda, K., Bell, G. I., Zouali, H., Sesage, S., Lesage, S., Velho, G., Iris, F., Passa, P., Froguel, P., and Cohen, D. (1992) Nature 356, 721-722[CrossRef][Medline] [Order article via Infotrieve]
  5. Velho, G., and Froguel, P. (1997) Diabetes Metab. Rev. 23, 34-37
  6. Froguel, P., Zouali, H., Vionnet, N., Velho, G., Vaxillaire, M., Sun, F., Lesage, S., Stoffel, M., Takeda, J., Passa, P., Permutt, M. A., Beckman, J. S., Bell, G. I., and Cohen, D. (1993) N. Engl. J. Med. 328, 697-702[Abstract/Free Full Text]
  7. Hager, J., Blanche, H., Sun, F., Vionnet, N., Vaxillaire, M., Poller, W., Cohen, D., Czernichow, P., Velho, G., Robert, J.-J., Cohen, N., and Froguel, P. (1994) Diabetes 43, 730-733[Abstract]
  8. Stoffel, M., Froguel, P., Takeda, J., Zouali, H., Vionnet, N., Nishi, S., Weber, I. T., Harrison, R. W., Pilkis, S. J., Lesage, S., Vaxillaire, M., Velho, G., Sun, F., Iris, F., Passa, P., Cohen, D., and Bell, G. I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7698-7702[Abstract]
  9. Sun, F., Knebelmann, B., Pueyo, M. E., Zouali, H., Lesage, S., Vaxillaire, M., Passa, P., Cohen, D., Velho, G., Antignac, C., and Froguel, P. (1993) J. Clin. Invest. 92, 1174-1180[Medline] [Order article via Infotrieve]
  10. Zouali, H., Vaxillaire, M., Lesage, S., Sun, F., Velho, G., Vionnet, N., Chiu, K., Passa, P., Permutt, A., Demenais, F., Cohen, D., Beckman, J. S., and Froguel, P. (1993) Diabetes 42, 1238-1245[Abstract]
  11. Girard, J. (1997) Annu. Rev. Nutr. 17, 325-352[CrossRef][Medline] [Order article via Infotrieve]
  12. Jetton, T. L., Liang, Y., Petterpher, C. C., Zimmerman, E. C., Cox, F. G., Horvath, K., Matchinsky, F. M., and Magnuson, M. A. (1994) J. Biol. Chem. 269, 3641-3654[Abstract/Free Full Text]
  13. Bali, D., Svetlanov, A., Lee, H.-W., Fusco-DeMane, D., Leiser, M., Li, B., Barzilai, N., Surana, M., Hou, H., Fleischer, N., DePinho, R., Rossetti, L., and Efrat, S. (1995) J. Biol. Chem. 270, 21464-21467[Abstract/Free Full Text]
  14. Grupe, A., Hultgren, B., Ryan, S., Ma, Y. H., Bauer, M., and Stewart, T. A. (1995) Cell 83, 69-78[Medline] [Order article via Infotrieve]
  15. Terauchi, Y., Sakura, H., Yasuda, K., Iwamoto, K., Takahashi, N., Ito, K., Kasai, H., Suzuki, H., Ueda, O., Kamada, N., Jishage, K., Komeda, K., Noda, M., Kanazawa, Y., Taniguchi, S., Miwa, I., Akanuma, Y., Kodama, T., Yazaki, Y., and Kadowaki, T. (1995) J. Biol. Chem. 270, 30253-30256[Abstract/Free Full Text]
  16. Ferre, T., Riu, E., Bosch, F., and Valera, A. (1996) FASEB J. 10, 1213-1218[Abstract/Free Full Text]
  17. Hariharan, N., Farrelly, D., Hagan, D., Hillyer, D., Arbeeny, C., Sabrah, T., Treloar, A., Brown, K., Kalinowski, S., and Mookhtiar, K. (1997) Diabetes 46, 11-16[Abstract]
  18. Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22570-22575[Abstract/Free Full Text]
  19. Niswender, K. D., Postic, C., Shiota, M., Jetton, T. L., and Magnuson, M. A. (1997) Biochem. Soc. Trans. 25, 113-117[Medline] [Order article via Infotrieve]
  20. Tsien, J. Z., Chen, D. F., Gerber, D., Tom, C., Mercer, E. H., Anderson, D. J., Mayford, M., Kandel, E. R., and Tonegawa, S. (1996) Cell 87, 1317-1326[Medline] [Order article via Infotrieve]
  21. Kuhn, R., Schenk, F., Aguet, M., and Rajewsky, K. (1995) Nature 269, 1427-1429
  22. Metzger, D., Clifford, J., Chiba, H., and Chambon, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6991-6995[Abstract]
  23. Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10887-10890[Abstract/Free Full Text]
  24. Kellendonk, C., Tronche, F., Monaghan, A.-P., Angrand, P.-O., Stewart, F., and Schutz, G. (1996) Nucleic Acids Res. 24, 1404-1411[Abstract/Free Full Text]
  25. Sauer, B. (1993) Methods Enzymol. 225, 890-900[Medline] [Order article via Infotrieve]
  26. Postic, C., Niswender, K. D., Decaux, J.-F., Parsa, R., Shelton, K. D., Gouhot, B., Pettepher, C. C., Granner, D. K., Girard, J., and Magnuson, M. A. (1995) Genomics 29, 740-750[CrossRef][Medline] [Order article via Infotrieve]
  27. Labosky, P. A., Barlow, D. P., and Hogan, B. L. M. (1994) Development 120, 3197-3204[Abstract/Free Full Text]
  28. Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (Robertson, E. D., ed), pp. 71-112, IRL Press at Oxford University Press, Oxford
  29. Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating the Mouse Embryo: A Laboratory Manual, pp. 293-295, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  30. Pinkert, C. A., Ornitz, D. M., Brinster, R. L., and Palmiter, R. D. (1987) Genes Dev. 3, 268-276
  31. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  32. Agah, R., Frenkel, P. A., French, B. A., Michael, L. H., Overbeek, P. A., and Schneider, M. D. (1997) J. Clin. Invest. 100, 169-179[Abstract/Free Full Text]
  33. Bergmeyer, H. U. (1984) in Methods in Enzymatic Analysis (Bergmeyer, J., and Grabl, M., eds), pp. 163-172, Verlag Chemie, Weinheim, Germany
  34. Keppler, D., and Decker, K. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed), pp. 11-18, Verlag Chemie, Weinheim, Germany
  35. Niswender, K. D., Postic, C., Jetton, T. L., Bennet, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22564-22569[Abstract/Free Full Text]
  36. Jetton, T. L., and Magnuson, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2619-2623[Abstract]
  37. Moates, J. M., Postic, C., Decaux, J.-F., Girard, J., and Magnuson, M. A. (1997) Genomics 45, 185-193[CrossRef][Medline] [Order article via Infotrieve]
  38. Araki, K., Araki, M., Miyazaki, J., and Vassalli, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 160-164[Abstract]
  39. Bouwens, L., De Bleser, P., Vanderkerken, K., Geerts, B., and Wisse, E. (1992) Enzyme 46, 155-168[Medline] [Order article via Infotrieve]
  40. Olson, E. N., Arnold, H.-H., Rigby, P. W. J., and Wold, B. J. (1996) Cell 85, 1-4[Medline] [Order article via Infotrieve]
  41. Matschinsky, F. M. (1990) Diabetes 39, 647-652[Abstract]
  42. Duvillie, B., Cordonnier, N., Deltour, L., Dandoy-Dron, F., Itier, J.-M., Monthioux, E., Jami, J., Joshi, R. L., and Bucchini, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5137-5140[Abstract/Free Full Text]
  43. Girard, J., Ferre, P., Pegorier, J.-P., and Duee, P.-H. (1992) Physiol. Rev. 72, 507-561[Free Full Text]
  44. Efrat, S., Leiser, M., Wu, Y. J., Fusco-DeMane, D., Emran, O. A., Surana, M., Jetton, T., Magnuson, M. A., Weir, G., and Fleischer, N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2051-2055[Abstract]
  45. Velho, G., Petersen, K. F., Perseghin, G., Hwang, J.-H., Rothman, D. L., Pueyo, M. E., Cline, G. W., Froguel, P., and Shulman, G. I. (1996) J. Clin. Invest. 98, 1755-1761[Abstract/Free Full Text]
  46. Seoane, J., Gomez-Foix, A. M., O'Doherty, R. M., Gomez-Ara, C., Newgard, C. B., and Guinovart, J. J. (1996) J. Biol. Chem. 271, 23756-23760[Abstract/Free Full Text]
  47. O'Doherty, R. M., Lehman, D. L., Seoane, J., Gomez-Foix, A. M., Guinovart, J. J., and Newgard, C. B. (1996) J. Biol. Chem. 271, 20524-20530[Abstract/Free Full Text]
  48. Barzilai, N., Hawkins, M., Angelov, I., Hu, M., and Rossetti, L. (1996) Diabetes 45, 1329-1335[Abstract]
  49. Bonner-Weir, S., Deery, D., Leahy, J. L., and Weir, G. C. (1989) Diabetes 38, 49-53[Abstract]
  50. Leahy, J. L., Bonner-Weir, S., and Weir, G. C. (1992) Diabetes Care 15, 442-455[Abstract]
  51. Rossetti, L., Giaccari, A., and Defronzo, R. A. (1990) Diabetes Care 13, 610-630[Abstract]
  52. Leahy, J. L., Bonner-Weir, S., and Weir, G. C. (1988) J. Clin. Invest. 81, 1407-1414[Medline] [Order article via Infotrieve]
  53. Leahy, J. L., Cooper, H. E., and Weir, G. C. (1987) Diabetes 36, 459-464[Abstract]
  54. Trent, D. F., Fletcher, D. J., May, J. M., Bonner-Weir, S., and Weir, G. C. (1984) Diabetes 33, 170-175[Abstract]
  55. Tappy, L., Dussoix, P., Iynedjian, P., Henry, S., Schneiter, P., Zahnd, G., Jequier, E., and Philippe, J. (1997) Diabetes 46, 204-208[Abstract]
  56. Wang, H., DeMayo, F. J., Tsai, Y. S., and O'Malley, B. W. (1997) Nature Biotech. 15, 239-243[Medline] [Order article via Infotrieve]
  57. Anton, M., and Graham, F. L. (1995) J. Virol. 69, 4600-4606[Abstract]


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