(Received for publication, October 17, 1995; and in revised form, October 30, 1995)
From the
Glucose-stimulated insulin secretion is believed to require
metabolism of the sugar via a high Kpathway in
which glucokinase (hexokinase IV) is rate-limiting. In this study, we
have used recombinant adenoviruses to overexpress the liver and islet
isoforms of glucokinase as well as low K
hexokinase I in isolated rat islets of Langerhans. Glucose
phosphorylating activity increased by up to 20-fold in extracts from
islets treated with adenoviruses containing the cDNAs encoding either
tissue isoform of glucokinase, but such cells exhibited no increase in
2- or 5-[
H]glucose usage, lactate production,
glycogen content, or glucose oxidation. Furthermore, glucokinase
overexpression enhanced insulin secretion in response to stimulatory
glucose or glucose plus arginine by only 36-53% relative to
control islets. In contrast to the minimal effects of overexpressed
glucokinases, overexpression of hexokinase I caused a 2.5-4-fold
enhancement in all metabolic parameters except glycogen content when
measured at a basal glucose concentration (3 mM). Based on
measurement of glucose phosphorylation in intact cells, overexpressed
glucokinase is clearly active in a non-islet cell line (CV-1) but not
within islet cells. That this result cannot be ascribed to the levels
of glucokinase regulatory protein in islets is shown by direct
measurement of its activity and mRNA. These data provide evidence for
functional partitioning of glucokinase and hexokinase and suggest that
overexpressed glucokinase must interact with factors found in limiting
concentration in the islet cell in order to become activated and engage
in productive metabolic signaling.
Glucose-stimulated insulin secretion (occurring at
concentrations in excess of 5 mM) is believed to require
metabolism of the sugar via a high K pathway in which glucokinase is
rate-limiting(1, 2) . Consistent with an important
role for glucokinase in the high K
regulatory pathway, it has recently been shown that
mutations in this gene are associated with
-cell dysfunction in a
subtype of non-insulin-dependent diabetes mellitus known as
maturity-onset diabetes of the young(3) . Furthermore,
reduction of glucokinase activity by 70% in
-cells of transgenic
mice expressing a glucokinase-specific ribozyme results in decreased
glucose-stimulated insulin secretion (GSIS)(
)(4) .
Low K
hexokinases are also expressed in
-cells and appear to determine the amount of insulin secreted at
basal glucose concentrations (1, 5, 6, 7) suggesting a functional
segregation from the high K
glucokinase-mediated glucose signaling pathway. Implicit in
such a model is that protection against an inappropriately vigorous
response to a glucose challenge might be achieved by regulating access
of the sugar to the high K
pathway by
requiring that glucokinase couple to factors found in limiting
concentration in the islet cell. Since the impact of glucokinase
overexpression has not been studied, it is not known whether increased
abundance of this enzyme will specifically enhance high K
glucose metabolism and insulin
secretion. In order to address this question, we have used the
recombinant adenovirus system to overexpress two isoforms of
glucokinase and hexokinase I in isolated islets of Langerhans.
A large body of evidence has accumulated in support of the
notion that glucokinase represents the rate-limiting step of glucose
metabolism in pancreatic islet -cells. Based solely on kinetic
considerations, one may have predicted that overexpression of
glucokinase in islets would have a potent enhancing effect on glucose
metabolism and, as a consequence, on GSIS. Indeed glucokinase activity,
glucose usage, and GSIS increase coordinately in proportion to the
glucose concentration of islet culture media(21, 22) ,
and hyperglycemic infusion is reported to increase glucokinase activity
and render islets more sensitive to glucose(23) , leading to
the suggestion that the increase in enzyme activity is the likely cause
of the enhanced glucose response. Interpretation of these experiments
is complicated by the fact that expression of a large number of islet
proteins is increased by culture at high glucose(24) . The
present study was designed to evaluate the effect of glucokinase
overexpression in a more specific manner by introducing the gene
encoding this enzyme into isolated islets via recombinant adenovirus.
Figure 1:
Immunofluorescent
detection of glucokinase and insulin in cultured rat islets. Cultured
islets were treated with AdCMV-GAL (A, C, and E) or AdCMV-GKI (B, D, and F).
Sections were treated with anti-glucokinase antibody
GK-1 (A, B, E, and F) or guinea pig
anti-insulin antibody (C and D). Sections were viewed
at
400 (A, B, C, and D) or
1000 (E and F)
magnification.
Western blot analysis showed that treatment of islets with the AdCMV-GKI virus resulted in large increases in glucokinase protein relative to control islets, as detected with antibody U343 (11) (Fig. 2A), which is specific for the unique N-terminal segment of the islet isoform of glucokinase, or antibody V980 (Fig. 2B), raised against a region of the protein common to both liver and islet glucokinases(11) . As shown in Fig. 2B, treatment of islets with the AdCMV-GKL virus resulted in levels of immunodetectable protein slightly higher than those achieved with the AdCMV-GKI virus. Finally, treatment of islets with the AdCMV-HKI virus resulted in consistent overexpression of hexokinase I protein (Fig. 2C), in agreement with our previous work (5) .
Figure 2: Immunoblot analysis of adenovirus-mediated expression of glucose-phosphorylating enzymes in cultured rat islets. Islets extracts were prepared 3-4 days after treatment with the indicated recombinant adenoviruses (see text for abbreviations) and resolved by SDS-polyacrylamide gel electrophoresis, using 20 µg of total protein/lane. Representative blots are shown. A, immunodetection with antibody U343, which is specific for the islet glucokinase N-terminal sequence(11) . Included in this panel is a control lane (GK STD) containing an extract of bacteria engineered for expression of islet glucokinase. The appearance of two immunoreactive bands in this sample is likely due to partial proteolytic degradation, since other samples that we have analyzed previously contained only a single band(11) . B, immunodetection with antibody V980, which recognizes a C-terminal region common to the liver and islet glucokinase isoforms(11) . C, islets treated with AdCMV-HKI and immunodetection performed with an antibody specific for rat hexokinase I(13) . Samples from three separate islet aliquots treated with AdCMV-HKI are shown.
Glucokinase represents less than half of the total
glucose phosphorylating activity in control islets (4.4 ± 0.7
and 5.6 ± 1.0 units/g measured at 20 mM glucose in the
absence of Glc-6-P and 1.2 ± 0.4 and 2.3 ± 0.5 units/g in
the presence of 10 mM Glc-6-P for untreated and
AdCMV-GAL-treated islets, respectively), consistent with previous
reports(1, 25) . Treatment of islets with AdCMV-GKI or
AdCMV-GKL resulted in 14- and 19.4-fold increases in total glucose
phosphorylation and 37.6- and 53.8-fold increases in glucokinase
activity (measured in the presence of 10 mM Glc-6-P),
respectively (data represent the average of 6-8 groups of islets
per condition). Overexpression of hexokinase I resulted in an 8-fold
increase in total glucose phosphorylation relative to the two control
groups, with nearly all of the observed increase sensitive to Glc-6-P
inhibition.
Isolated islet
preparations are removed from their normal pancreatic environment and
may be deprived of certain nutritional, nervous, and hormonal signals
that might be required to activate overexpressed
glucokinase(26) . To partially address this concern, we
repeated the foregoing experiments with a rich tissue culture medium
(DMEM) containing 5.5 mM glucose in the basal period and 20
mM glucose in the stimulation period. We found that basal
insulin release from all islet groups was increased approximately
5-fold during perifusion with 5.5 mM glucose in DMEM (average
of 190 microunits/ml/1000 islets) compared with insulin release in
Hanks' buffer containing 3 mM glucose (37
microunits/ml/1000 islets). Untreated islets or islets treated with
AdCMV-GAL, AdCMV-GKI, or AdCMV-GKL all responded similarly to 20
mM glucose by increasing insulin release by approximately
2-fold above this new base line (data not shown). Thus, while our data
indicate a minimal secretory impact of overexpressed glucokinases in
isolated islets, it remains possible that future studies of transgenic
animals or in vitro experiments with agents that can
potentiate GSIS such as glucagon-like peptide-1 or acetylcholine
(carbachol) may uncover larger effects.
Figure 3:
Metabolic fate of glucose in
adenovirus-treated islets. Assays were performed on islets incubated at
3 mM (hatched bars) or 20 mM glucose (black bars) (see text for explanation of abbreviations). A, glucose usage in intact islets measured as the amount of H
O release during incubation with
5-[
H]-D-glucose. B, lactate
production from islets. C, glucose usage in intact islets
measured as the amount of
H
O release during
incubation with 2-[
H]-D-glucose. Values
represent the mean ± S.E. for four independent groups of islets
per condition. * indicates those groups in which usage or lactate
production was higher at 20 mM glucose than at 3 mM glucose (p < 0.05). # indicates those groups for which
usage or lactate production was higher than in the respective control
groups (p < 0.05).
Figure 4:
Glucose phosphorylation in whole cells and
cell extracts. Assays were performed with isolated islets or CV-1 cells
treated with the indicated recombinant adenoviruses (see text for
abbreviations). A, accumulation of phosphorylated products in
intact islets or CV-1 cells exposed to
[U-C]glucose for 90 min. B, total
glucose phosphorylating activity (no Glc-6-P added) in islet or CV-1
cell homogenates measured at 3 and 20 mM glucose (hatched and black bars, respectively). In both panels,
data are normalized to the value obtained at 3 mM glucose in
AdCMV-
GAL-treated islets for each cell type. Values represent the
mean ± S.D. of four measurements. * in A indicates that
the accumulation of labeled product in the indicated group was greater
than in all other groups (p < 0.001), and # in B indicates that glucose phosphorylating activity was significantly
increased in AdCMV-GKI-treated islets or CV-1 cells at either 3 or 20
mM glucose relative to the respective AdCMV-
GAL-treated
controls (p < 0.001).
Figure 5: Expression of glucokinase regulatory protein mRNA in pancreatic islets and liver. The levels of glucokinase regulatory protein mRNA were determined using reverse transcriptase-PCR as described under ``Materials and Methods.'' The predicted 276-base pair (bp) amplification product is shown for islet samples from 6-week prediabetic ZDF (fa/fa) (lane 1), 6-week lean control ZDF (fa/- or -/-) (lane 2), 12-week diabetic ZDF (lane 3), and 12-week lean control ZDF (lane 4) and for liver samples from 12-week diabetic ZDF (lane 5) and Wistar (lane 6) rats. Lane 7 contains molecular weight standards.
In summary, our studies show that
overexpression of glucokinase in isolated islets has minimal effects on
glucose metabolism and insulin release. These findings appear to be
specific to the high K glucokinases, since changes
in metabolic parameters and insulin secretion are easily detectable in
islets overexpressing low K
hexokinase I. Our
results indicate that the known glucokinase regulatory protein is
unlikely to be present in islets at levels sufficient to explain our
findings. We therefore favor an alternative model, in which
overexpressed glucokinase must interact with factors found in limiting
concentration in the islet cell in order to become activated and engage
in productive metabolic signaling. This regulatory mechanism may be a
unique feature of islet cells, since overexpressed glucokinase is
clearly active within CV-1 or liver cells, and may explain why a
decrease in
-cell glucokinase activity has demonstrable effects on
glucose metabolism and insulin release, while overexpression of the
enzyme has little impact.