(Received for publication, June 28, 1995; and in revised form, July 21, 1995)
From the
Glucokinase catalyzes a rate-limiting step in glucose metabolism
in hepatocytes and pancreatic cells and is considered the
``glucose sensor'' for regulation of insulin secretion.
Patients with maturity-onset diabetes of the young (MODY) have
heterozygous point mutations in the glucokinase gene that result in
reduced enzymatic activity and decreased insulin secretion. However, it
remains unclear whether abnormal liver glucose metabolism contributes
to the MODY disease. Here we show that disruption of the glucokinase
gene results in a phenotype similar to MODY in heterozygous mice.
Reduced islet glucokinase activity causes mildly elevated fasting blood
glucose levels. Hyperglycemic clamp studies reveal decreased glucose
tolerance and abnormal liver glucose metabolism. These findings
demonstrate a key role for glucokinase in glucose homeostasis and
implicate both islets and liver in the MODY disease.
Pancreatic cells and hepatocytes are the two major cell
types responsible for maintaining glucose homeostasis.
cells
respond to changes in plasma glucose levels by regulating their insulin
release, whereas hepatocytes adjust their glucose uptake and glucose
production to changes in plasma insulin. Both cell types express a
specialized high K
member of the
hexokinase family of enzymes, glucokinase (GK), (
)which
catalyzes a rate-limiting step in glucose metabolism, the
phosphorylation of glucose to glucose 6-phosphate. In
cells
glucose metabolism generates signals for insulin secretion, and GK is
considered the major ``glucose sensor'' that couples the
extracellular glucose levels to insulin secretion(1) . Recent
DNA polymorphism studies of patients with maturity-onset diabetes of
the young (MODY), a form of non-insulin-dependent (type II) diabetes
mellitus, have established that heterozygous point mutations in the GK
gene are associated with the development of
diabetes(2, 3) . The mutations result in reduced
enzymatic activity(4, 5) , which causes abnormal
glucose sensing and decreased insulin
secretion(6, 7, 8) . In
cells the GK
promoter is constitutively active, and GK activity is regulated by
glucose at post-transcriptional levels(9) . The dominance of
the GK mutations in MODY has therefore been explained as a gene dosage
effect. In contrast, hepatocytes utilize a different GK promoter, which
is regulated by insulin(10, 11) . This promoter has
the potential to up-regulate the expression of the normal allele in
hepatocytes of MODY patients to compensate for the reduced GK activity
caused by the mutant allele. However, this transcriptional regulation
depends on plasma insulin levels, which in MODY patients are reduced
due to the islet abnormality. Hence, it remains unclear whether
abnormal liver glucose metabolism contributes to the MODY disease.
Previously we have attenuated GK function specifically in
cells
in transgenic mice using an antisense approach(12) . These mice
manifested a decreased insulin secretory response to glucose; however,
they did not show changes in fasting plasma glucose levels or glucose
tolerance. Here we used homologous recombination in mouse embryonic
stem cells to assess the effects of disrupted GK function in both
cells and hepatocytes and generate an animal model for MODY.
A targeting vector was constructed in which a fragment of the mouse GK gene spanning exon 2 was replaced with a neomycin resistance gene, resulting in a deletion and frameshift in the transcript (Fig. 1A). This vector was used to disrupt the GK gene in embryonic stem (ES) cells and generate mice heterozygous for the mutation (Fig. 1B).
Figure 1: Panel A, targeting and screening strategy for disruption of the mouse GK gene. Topline, GK targeting construct. The pgk-Neo gene is flanked by 5 kb of GK gene sequences at the 5` end and 0.9 kb at the 3` end. The herpes TK gene under control of the pgk promoter is attached at the 3` end. Middleline, a region of the mouse GK gene spanning exons 1 and 2. Bottomline, the targeted locus followinghomologous recombination. The SacI-SmaI fragment including exon 2 is replaced by the pgk-Neo gene. The indicated oligomers were used in PCR screening of ES cell clones and mouse tail DNA. Restriction sites: B, BamHI; Bg, BglII; H, HindIII; R, EcoRI; S, SacI; Sm, SmaI; X, XhoI (the restriction map includes minor corrections from sequence data kindly provided by M. Magnuson). Panel B, Southern blot analysis of tail DNA from GK +/+ and +/- mice and the E14 ES cell line, digested with BglII. The endogenous locus and the targeted locus generate different sizes of BglII fragments (9 and 2.5 kb, respectively), which are detected by the indicated HindIII fragment probe.
Analysis of glucose phosphorylation activity in islet homogenates of GK +/- mice revealed a 37% reduction compared with GK +/+ littermates (Table 1). This value is less than the expected 50% reduction in activity and may represent compensation by increased transcription of the wild-type allele, increased translation or stability of the wild-type GK mRNA, or by post-translational mechanisms. Liver GK activity in GK +/- mice was reduced by 28% compared with GK +/+ controls (Table 1). Based on the documented inducibility of the liver GK promoter by insulin(10, 11) , the wild-type allele could be expected to fully compensate for the reduced GK activity caused by the mutant allele. However, the liver GK activity observed in the GK +/- mice represents only partial compensation. There were no statistically significant differences in activity of the ubiquitous hexokinase in both islets and liver of GK +/- compared with control mice (Table 1).
Plasma glucose levels in fed GK +/- mice were normal; however, overnight fasting levels were 25% higher than those of GK +/+ littermates (Table 2). Thus, the reduced GK activity induces mildly elevated fasting glucose levels in the GK +/- mice, as is the case in MODY patients(6, 7, 8) . Plasma insulin levels were not significantly different in the GK +/- mice, compared with GK +/+ controls (Table 2).
To
determine the -cell and hepatic response to elevated blood glucose
levels, hyperglycemic clamp studies were performed in chronically
catheterized conscious mice. The plasma glucose concentration was
raised to an average of 15-18 mM during a 90-min
infusion period, and the rate of glucose infusion (GIR) required to
maintain the plasma glucose at this level provided an index of glucose
tolerance. The average GIR during the last 50 min of the clamp was
decreased by 40% in GK +/- as compared to GK +/+
mice (417 ± 50 versus 700 ± 33
µmol/kg
min) (Fig. 2C). The rate of glucose
uptake (R
) was decreased by 44% in the GK
+/- mice (Fig. 2B). These values reflect a
decreased tolerance to glucose, which is similar to that observed in
MODY patients(6, 7, 8) . The reduced GIR
could be due to decreased glucose-induced insulin secretion and/or
altered glucose metabolism. Glucose-induced insulin secretion during
the hyperglycemic clamp was 37% lower in GK +/- compared
with GK +/+ mice (6.9 ± 2.6 versus 11.0
± 3.1 ng of insulin/µmol of plasma glucose; n = 6-8). However, this difference did not achieve
statistical significance, due to data variability.
Figure 2:
Glucose disposal in response to
hyperglycemia. A, plasma glucose concentrations during the
basal period and during the hyperglycemic clamp. B, rate of
whole body glucose uptake (R) during the basal
period and during the hyperglycemic clamp. C, rate of glucose
infusion (GIR) required to raise and maintain the plasma glucose
concentrations at 15-18 mM during the hyperglycemic
clamp. Values are mean ± S.E. (n = 6 for GK
+/+ and n = 8 for GK +/- mice). *. p < 0.05 GK +/- versus GK +/+
by t test.
Hyperglycemia
inhibits hepatic glucose production (HGP) and stimulates hepatic
glucose uptake and glycogen synthesis. This process requires enhanced
phosphorylation of plasma glucose in hepatocytes, resulting in
increased flux through GK. An impairment in hepatic glucose
phosphorylation capacity may cause impaired hepatic adaptation to
changes in plasma glucose concentrations. These parameters were
assessed by infusing [3-H]glucose during the
hyperglycemic clamp studies and determining HGP and hepatic UDPG formed
from plasma glucose. Hyperglycemia and hyperinsulinemia reduced HGP by
72% in GK +/+ mice and by only 47% in GK +/- mice (Table 3). The relative contribution of plasma glucose to liver
glycogen repletion was evaluated by calculating the ratio of the
specific activities of hepatic [
H]UDPG and portal
vein plasma glucose, which provides a measure of the contribution of
plasma glucose to the hepatic UDPG pool (``direct'' pathway),
as opposed to UDPG generated by gluconeogenesis. As shown in Table 3, the contribution of the direct pathway to hepatic UDPG
was markedly decreased in GK +/- mice compared with GK
+/+ mice. A significant correlation (r =
0.47; p < 0.05) was demonstrated between the GIR during the
hyperglycemic clamp studies and the fraction of hepatic UDPG derived
from the direct pathway in the two groups combined. Thus a modest
decrease in liver GK activity in the GK +/- mice is
sufficient to affect hepatic glucose metabolism. These findings suggest
that abnormalities in liver glucose metabolism contribute to the MODY
disease.
Breeding of heterozygous mutants failed to generate GK
-/- mice. Approximately one third of the embryos in timed
pregnancies were found to be resorbed starting at E9.5, indicating that
the absence of GK is lethal to embryos. At this stage, only primordial
buds of liver and exocrine pancreas without islets are observed.
Nevertheless, RT-PCR analysis of total RNA from wild-type E9.5 embryos
revealed significant GK expression (Fig. 3), and E9.5 embryo
extracts contained GK enzymatic activity (data not shown). Breeding of
GK mutant mice with a transgenic mouse line expressing a GK transgene
rescued the homozygous mutant, ()demonstrating that the
embryonic lethality results from the absence of GK activity. These
findings suggest an important function for GK during development;
however, the cell types in which its activity is required remain to be
identified.
Figure 3:
RT-PCR analysis of wild-type E9.5 mouse
embryos for GK expression. Total embryo RNA, as well as RNA from adult
mouse kidney, which does not express GK, and from the rat insulinoma
cell line TC6, was reverse transcribed and amplified with GK
primers. The amplified DNA fragments were blotted onto filters and
hybridized with a GK cDNA probe to confirm the identity of the band
(not shown). Size markers are in base
pairs.
In conclusion, disruption of one allele of the GK gene
is associated with decreased tolerance to glucose and moderate
impairments in both hepatic and -cell glucose sensing. Thus a
partial reduction in mouse islet and liver GK activity causes a disease
very similar to human MODY, underscoring the key role of GK in
maintaining glucose homeostasis. In contrast, a
-cell specific
reduction of GK activity in transgenic mice, which results in a
decreased insulin secretory response to glucose, is not sufficient to
cause detectable changes in fasting plasma glucose levels or glucose
tolerance(12) . These findings indicate that the MODY disease
results from abnormal glucose metabolism in both islets and liver. The
GK +/- mice will allow studies on genetic and environmental
factors that may interact with the impaired GK activity to cause overt
diabetes, as well as testing of therapeutic approaches to restore
normal glucose homeostasis.