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INTRODUCTION |
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
cells and
hepatocytes (4, 6-10), although the relative contributions of each
cell type have not been determined. Both
cells and hepatocytes are
key sites of GK expression, and both play central roles in glucose
homeostasis. In pancreatic
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
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
cell GK cause
MODY-2.
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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
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 [
-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-
-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
actin promoter-driven
Cre-activable
-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
-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
-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.
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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.
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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.
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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.
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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.
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Use of an Insulin-cre Transgene to Eliminate GK Expression only in
Cells--
To evaluate independently the effects of diminished
cell GK on glucose homeostasis, we next sought to generate mice that lacked only
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
cells (82% of 1770
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
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
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 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 -galactosidase staining
in several pancreatic cells. lacZ staining was
restricted to cells in these animals. d,
lacZ-stained liver from lacZ+Alb-cre
mice showing cytoplasmic -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.
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To generate
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,
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
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
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.
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."
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To determine the effect of a loss of a single copy of the GK gene only
in
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.
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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
-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.
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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.
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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
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
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
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).
Cell-specific Knock-out of GK--
Mice that lack GK only in
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
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
cell glucose sensing. Also, the fact that the phenotype of the
gklox/lox+Rip-cre mice, which lack GK
only in
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.
cell, brain, and gut),
indicates that the phenotype of the islet GK knock-out mice is due
principally to the loss of
cell GK. Thus, the results of both of
these studies are complementary with each pointing to an indispensible
role for pancreatic
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
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
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
cell function are well documented but
poorly understood (50, 51). Sustained hyperglycemia has been shown to
cause
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
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
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
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
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
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
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
cell GK as models of either hepatic insulin -resistance
or impaired
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
cell GK in adult animals or in tissues removed from animals
bearing conditional gk alleles.