(Received for publication, September 11, 1995; and in revised form, October 27, 1995)
From the
Mice carrying a null mutation in the glucokinase (GK) gene in
pancreatic -cells, but not in the liver, were generated by
disrupting the
-cell-specific exon. Heterozygous mutant mice
showed early-onset mild diabetes due to impaired insulin-secretory
response to glucose. Homozygotes showed severe diabetes shortly after
birth and died within a week. GK-deficient islets isolated from
homozygotes showed defective insulin secretion in response to glucose,
while they responded to other secretagogues: almost normally to
arginine and to some extent to sulfonylureas. These data provide the
first direct proof that GK serves as a glucose sensor molecule for
insulin secretion and plays a pivotal role in glucose homeostasis.
GK-deficient mice serve as an animal model of the insulin-secretory
defect in human non-insulin-dependent diabetes mellitus.
Glucokinase (GK), ()mainly expressed in pancreatic
-cells and the liver, is thought to constitute a rate-limiting
step in glucose metabolism in these tissues (1, 2, 3, 4) . Since insulin
secretion parallels glucose metabolism and the high K
of GK (5-8 mM) ensures
that it can change its enzymatic activity within the physiological
range of glucose concentrations, GK has been proposed to act as a
glucose sensor in the pancreatic
-cell(1, 5) .
Recently, mutations of the GK gene have been identified in patients
with maturity-onset diabetes of the young, a subtype of early-onset
non-insulin-dependent diabetes mellitus
(NIDDM)(6, 7, 8) . However, since all the
mutations in humans so far occur in the region of the gene that is
common to pancreatic
-cells and hepatocytes(9) , and are
heterozygous, it may not have been possible to fully reveal
physiological roles of pancreatic
-cell GK either in vivo or in vitro. To this end, mice carrying a null mutation
in the GK gene in pancreatic
-cells, but not in the liver, were
generated by homologous recombination. Heterozygous mutant mice showed
early-onset mild diabetes resembling the phenotype for human
maturity-onset diabetes of the young. Homozygotes showed severe
diabetes shortly after birth and died within a week. GK-deficient
islets showed defective insulin secretion in response to glucose, while
they responded to other secretagogues: almost normally to arginine and
to some extent to sulfonylureas. These data provide the first direct
proof that GK serves as a glucose sensor molecule for insulin secretion
and plays a pivotal role in glucose homeostasis.
Figure 1:
Targeted
disruption of glucokinase gene in pancreatic -cells. A,
schematic representation of the mouse GK gene (top), the
targeting vector (middle), and the targeted gene (bottom). Top, a BamHI site was introduced
in exon 1
. Middle, a neomycin resistance gene (neo
) was substituted for the XbaI-BamHI fragment in exon 1
. A diphtheria
toxin A fragment gene (DTA) was ligated on the 3` terminus
across the vector backbone. Bottom, expected structure of the
GK gene following successful gene targeting. B, Southern blot
analysis of ES cell clones and the siblings generated by a cross
between heterozygous GK mutants (F2 mice). The genomic DNA was digested
with BamHI and SmaI, and hybridized with probe A
under high stringency. The 8.8-kilobase band corresponds to the
wild-type gene (+/+), and 7.1-kilobase band to the targeted
gene (-/-). C, glucose phosphorylating activities
of islets by HK and GK (V
). Solid bar denotes the wild-type, and open bar the homozygotes.
Values are expressed in mol kg DNA
h
, as mean and standard error of the mean
(mean ± S.E.) (n =
4).
Figure 2:
Mild
diabetes in heterozygous GK knock-out mice. Results of a glucose
tolerance test are shown. Wild-type (open circles) and
heterozygous mice (open triangles) were loaded with 1.5 mg
g (body weight) glucose, and blood glucose (upper panel) and serum insulin (lower panel) levels
were determined at the indicated time points. The data points indicate
mean ± S.E. (n = 15).**, p < 0.01;
*, p < 0.05.
Figure 3: Characterization of homozygous GK knock-out mice. A, changes in body weight of neonates. The genotype of the neonates was determined by Southern blot analysis (Fig. 1B). The body weight of the wild-type (open circles), the heterozygotes (open triangles), the homozygotes (crosses) is plotted against the number of days after birth.**, p < 0.01; *, p < 0.05 compared with the wild-type. B, relationship between the blood glucose levels and the serum insulin levels at 3-4 days of age. Mice of each genotype were fed freely, and blood samples were collected by decapitation.
9 out of 10 homozygous mutant pups
subcutaneously injected with 10-20 milliunits of human insulin
(Novolin R, Novo) twice a day survived beyond 7 days of age, and 4 out
of 6 homozygous mutant pups orally administered with 20 µmol of
glibenclamide (kindly provided by Yamanouchi Pharmaceutical Co.) per
day survived beyond 10 days of age, while all untreated pups (n = 50) died before 7 days of age. Both of these treatments
significantly lowered blood glucose levels (by 20-40%) and caused
a gain in body weight (to approximately 80% of the wild-type
littermate). Since GK is also expressed in rare neuroendocrine cells in
the brain and gut(22) , it is possible that the absence of
glucose sensing in the brain or gut by GK may have modulated the
phenotype of homozygous mutant pups. Nevertheless, it seems likely that
hyperglycemia due to a lack of -cell GK is the major cause of
severe metabolic failure and early death in several days, since insulin
or sulfonylurea improved these features. Immunostaining for glucagon,
insulin, and somatostatin revealed that differentiation into pancreatic
,
, and
cells also appeared to be unaffected (Fig. 4). Although there may be subtle changes in the
architecture of
,
, and
cells in the islets, its gross
appearance was normal. The insulin contents of the wild-type,
heterozygous, and homozygous GK-deficient islets were 1.7 ± 0.3 (n = 5), 2.2 ± 0.7 (n = 6), and
2.5 ± 0.2 (n = 10) ng of insulin/islet,
respectively. These data indicate that
-cell GK is not essential
for normal development, differentiation of endocrine pancreas, or
insulin biosynthesis.
Figure 4: Immunohistochemistry of GK-deficient islets. Islets were stained for insulin (a and d), glucagon (b and e), or somatostatin (c and f). a-c are the same section of an islet from a wild-type mouse, while d-f are the same section of an islet from a homozygous mutant mouse. Scale bar, 100 µm.
Figure 5:
Characterization of GK-deficient islets. A, increase in intracellular calcium concentration of islets
elicited by 20 mM glucose, 10 µM glibenclamide,
or 20 mM arginine. Basal calcium levels in the wild-type,
heterozygous, and homozygous islets are 78 ± 15, 116 ±
30, and 110 ± 34 nM, respectively. Values are expressed
in nM, as mean ± S.E. (n = 4-7). B, insulin secretion in response to the indicated
secretagogues. Values are expressed in nanograms of insulin 10
islets h
, as mean ± S.E. (n = 6-15). Solid bar denotes the
wild-type (Wild), hatched bar the heterozygotes (Hetero), and open bar the homozygotes (Null). Insulin secretion in response to 3 or 10 mM glucose from null islets was not determined. **, p <
0.01; *, p < 0.05 compared with the
wild-type.
We next examined insulin secretion using the batch incubation method (Fig. 5B). Insulin secretion from heterozygous GK-deficient islets in response to 0.1 mM or 3 mM glucose was normal, but that in response to 10 mM glucose was significantly impaired compared with wild-type islets. Impairment in insulin secretion in response to 20 mM glucose was less evident. On the other hand, insulin secretion in response to glibenclamide or arginine was unaffected. In homozygous GK-deficient islets, although there was some basal insulin secretion at 0.1 mM glucose, presumably due to the activity of HK, increase in insulin secretion in response to 20 mM glucose was completely abolished. In contrast, insulin secretion in response to arginine was essentially preserved. Regarding insulin secretion in response to glibenclamide, it was decreased by about 50-80% depending on the method of estimation (Fig. 5B). Nevertheless, since the islets used here were supposed to be a mixture of those with normal and those with higher basal calcium levels (expected to be 20% and 80% of the population, respectively), which were responsive and unresponsive to glibenclamide in calcium study, we interpreted these results as suggesting that islets with normal basal calcium levels may have responded to sulfonylurea in insulin secretion.
The insulin secretory response to a physiological increment in glucose concentration was impaired in heterozygous GK-deficient islets and completely defective in homozygous GK-deficient islets despite the presence of HK, supporting the concept that GK serves as a glucose sensor molecule for insulin secretion. This is consistent with the smaller increments in serum insulin levels after a glucose load in heterozygous mice (Fig. 2), lack of increments in insulin levels in homozygous mice in spite of hyperglycemia (Fig. 3B), and the secretory abnormalities in human subjects with GK mutations(23) . GK-deficient islets responded to non-glucose secretagogues in insulin secretion (almost normally to arginine and to some extent to sulfonylureas), indicating that GK is not absolutely required for insulin secretion in response to these secretagogues. It should also be noted that insulin secretion in response to glibenclamide was impaired, suggesting that GK may play an important role in insulin secretion in response to some of non-glucose secretagogues such as glibenclamide. This possibility would be examined in future. The heterozygous or insulin-treated homozygous mutant mice described here provide the first animal model of diabetes with a defined genetic defect in insulin secretion, and should give important insights into the pathogenesis and development of human NIDDM.