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Biochemical Society Transactions (2007) Volume 35, part 5
Glucokinase activators: molecular tools for
studying the physiology of insulin-secreting cells
D. Johnson*, R.M. Shepherd*, D. Gill†, T. Gorman†, D.M. Smith† and M.J. Dunne*1
*Faculty of Life Sciences, University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, U.K., and †AstraZeneca,
Diabetes and Obesity Drug Discovery, Mereside, Alderley Park, Cheshire SK10 4TG, U.K.
Abstract
GK (glucokinase) catalyses the phosphorylation of glucose to glucose 6-phosphate in glucosensitive cells.
In pancreatic β-cells, this reaction is the rate-limiting step of insulin release. Recent work has led to the
discovery of synthetic small-molecule activators of GK that stimulate β-cell physiology and subsequently
enhance the glucose-dependent release of insulin. It is currently recognized that these compounds may
represent a significant advance in the development of new agents in the treatment of diabetes. In addition,
GKAs (GK activators) are emerging as reagents that are useful tools with which to probe the function of
pancreatic β-cells and other glucosensitive cells. This includes providing insights into the physiology of the
β-cell by helping to elucidate the kinetic cycle of GK, confirming the central role of glucose metabolism to
the β-cell and highlighting subtle species-dependent differences in insulin secretion between rodent and
human islets of Langerhans.
GK (glucokinase) and the development
of GKAs (GK activators)
GK (EC 2.7.1.1; hexokinase IV or D) catalyses the phosphorylation of glucose to glucose 6-phosphate in glucosensitive
cells, including pancreatic β-cells [1]. GK has an affinity for
glucose that is within the physiological plasma glucose range
[S0.5 (half-saturation constant) for glucose of ∼7 mM] [2].
The release of insulin from β-cells tightly correlates with the
activity of GK and, as a result, GK is often referred to as
the ‘glucose sensor’ [1]. The glucose sensor concept is reinforced by the fact that mutations of the GK gene can cause
hyperglycaemia as a result of ‘loss-of-function’ mutations
or hypoglycaemia through ‘gain-of-function’ mutations [3].
GK is therefore a pivotal determinant of regulated insulin
release.
There has been intense interest in identifying and then
developing novel GKA compounds, and several have been
recently described in the literature [4–14]. Although each
GKA has subtly different pharmacokinetics, their general
mode of action is broadly similar. GKAs specifically target
GK over the other hexokinases, and allosterically activate
the enzyme, resulting in an increase in the affinity of GK for
glucose as demonstrated by a reduced S0.5 value for glucose
[4–6,8,13]. In addition, it has also been shown that some
GKAs also increase the V max for glucose. In islets, we have
recently shown in detail that the activation of GK results
in marked increases in the cytosolic Ca2+ concentration and
that this is correlated with subsequent glucose-dependent
enhancement of insulin release [12] (Table 1). However,
Key words: calcium signalling, glucokinase activator, insulin release, islet cell, pancreatic β-cell.
Abbreviations used: [Ca2+ ]i , intracellular Ca2+ concentration; GFP, green fluorescent protein; GK,
glucokinase; GKA, GK activator; MIP, mouse insulin promoter; S0.5 , half-saturation constant.
1
To whom correspondence should be addressed (email [email protected]).
C The
C 2007 Biochemical Society
Authors Journal compilation islets contain several different cell types and as there has
been some discussion about the existence of GK in non
β-cell populations [15–18], we have furthered our initial
studies using MIP (mouse insulin promoter)–GFP (green
fluorescent protein) mice. In this mouse, GFP is coupled
with the insulin promoter [19], and islet β-cells can therefore
be reliably identified. Figure 1 shows that GKA50-induced
Ca2+ signals in MIP β-cells are directly comparable with
those obtained from intact islets. GKA50-enhanced insulin
release is characterized by a leftward shift in the glucose
response curve but no significant effect on the maximal rate
of secretion at high glucose concentrations [12]. It therefore
appears that these classes of agents act on β-cells as Ca2+ mediated and glucose-dependent insulin secretagogues. (For
specific details on the discovery and action of GKAs, the
reader is directed to the reference list of the present article.)
GKAs as a molecular tool for probing β-cell
physiology
Regardless of the therapeutic benefit of these compounds,
GKAs have great potential as molecular tools that can be
used to probe the function of β-cells and other glucosensitive
cells. The body of published work describing the function
of several different GKAs has revealed insights into the
physiology of the β-cell, and some of these ideas and
concepts will be discussed in the present study.
The structure and subsequent elucidation of the
kinetic cycles of GK
The main benefit of GKAs is their high specificity for the GK
enzyme over the other hexokinases, allowing the investigator
to specifically manipulate glucosensitive cells. GK has an
Metabolism
Table 1 Effects of GKA50 on islets of Langerhans and MIN6 β-cell physiological parameters
Results are adapted from Johnson et al. [12] and show the shift or fold increase (EC50 or glucose-stimulated insulin secretion respectively) and
the change in [Ca2+ ]i relative to ‘baseline’ results induced by GKA50 compared with untreated vehicle controls; * denotes a significant difference
(P 0.05), NS denotes no significant change and ND denotes value not determined.
Fold increase in glucosestimulated insulin secretion
EC50 glucose (mM)
2 mM
3 mM
5 mM
Average [Ca2+ ]i at 2 mM glucose
Rat islets
3.4*
NS
1.6*
2.5*
15 ± 3 nM decrease (n = 10)
Mouse islets
Human islets
MIN6 β-cells
ND
ND
10.7*
3.4*
ND
2*
5.5*
NS
3.5*
8.3*
4*
4*
23 ± 2 nM decrease and 76 ± 12 nM rise (n = 10)
ND
ND
Figure 1 GKA50-induced calcium signals in mouse β-cells
Islet cells from MIP–GFP mice were used to definitively identify β-cells,
which were then exposed to 1 µM GKA50 in the presence of 2 mM glucose. Note that the rises in [Ca2+ ]i (increase in fura 2 fluorescence ratio)
in response to GKA50 is preceded by a decrease in [Ca2+ ]i . These results
mirror the reported actions of GKA50 in mouse islets [12]. Typical
results from two regions of GFP fluorescence are illustrated (n = 12
experiments).
allosteric domain that is not present in the other members
of the hexokinase family, and GKAs are thought to bind to
this site [6,8]. The specificity of GKAs for GK was utilized
when the crystal structure of GK was solved [6]. These studies
revealed that GK adopts three structurally and kinetically distinct conformations that are termed closed, open and superopen. Both the closed and open states are kinetically active,
whereas the super-open state is inactive [6]. The identification
of these states has allowed for a convincing hypothesis to be
put forward to explain how GK can, uniquely for a monomeric enzyme, display a positive co-operativity for its substrate. The hypothesis allows GK to enter two distinct glucose
concentration-dependent catalytic cycles that are termed
‘fast’ and ‘slow’ respectively. At low glucose concentrations,
most of the GKs will enter the ‘slow’ cycle that involves the
inactive super-open conformation. As glucose levels increase,
the enzyme will preferentially enter the ‘fast’ catalytic cycle
that involves the two active conformations of GK. GKAs are
postulated to shift the equilibrium between the two cycles in
favour of the ‘fast’ cycle by stabilizing GK in a kinetically
active conformation [20]. The use of GKAs has therefore enhanced the understanding of GK-mediated phosphorylation
and helped to solve a long-standing kinetic question.
Species-dependent differences in calcium
handling in insulin-secreting cells
GKA-dependent modulation of insulin release is broadly
similar in rodent and human insulin-secreting cells (see
above). However, the use of GKAs has revealed subtle
species-dependent differences in β-cell nutrient sensing
and subsequent insulin release. For example, GKA50
significantly stimulates insulin release from mouse islets of
Langerhans (and MIN6 cells) at a concentration of 2 mmol/l
glucose (Table 1) [12]. However, it is without effect on insulin
release from rat or human islets at this concentration of
glucose [12]. Interestingly, GKA50 has a similar affinity for
both recombinant mouse and rat GKs, suggesting that the
differences in sensitivity may lie downstream of the enzyme,
and (at lease in part) at the level of cytosolic calcium handling
[[Ca2+ ]i (intracellular Ca2+ concentration)]. Thus, in mouse
islets and mouse β-cells (Figure 1), GKA50 causes a biphasic
change in [Ca2+ ]i at 2 mM glucose that is characterized by
an initial decrease in cytosolic Ca2+ (caused by sequestration
of calcium from the cytoplasm) and is followed by a marked
rise in [Ca2+ ]i . Conversely, rat islets show only the initial
decrease in [Ca2+ ]i without the subsequent rise at 2 mM
glucose [12]. These results were unexpected and resolution
of these differences will provide valuable insights into the
function of calcium-handling proteins in insulin-releasing
cells and their subsequent regulation of insulin release.
Long-term nutrient sensing by the β-cell
The physiology of insulin-releasing cells is acutely dependent
on the availability of glucose; for example, Glauser et al.
[21] have recently reported how glucose and cAMP alter the
transcriptome of the β-cell in the long term. The changes in
gene expression signify a complex molecular mechanism by
which the β-cell senses and adapts to long-term changes in
nutrient availability. Most of the observed up-regulated genes
were those encoding proteins involved in the metabolism
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Biochemical Society Transactions (2007) Volume 35, part 5
of glucose and secretion of insulin [21]. At this time, the
use of GKAs to modulate gene expression has not been
described. However, it would not be surprising to learn that
chronic treatment of insulin-releasing cells with GKAs leads
to adaptive changes in gene expression very similar to those
recently reported by Glauser et al. [21]. Not only would such
a report signify the long-term protective effects of GKAs but
GKAs would be useful tools to manipulate gene expression
in glucose-sensitive cells in general and β-cells in particular.
Summary and conclusions
The development of GKAs represents a possible significant
advance in diabetes therapy. As glucose-sensitive cells
are, by their very nature, acutely dependent on glucose
availability, GKAs enhance the responses of these cells to
glucose. The work characterizing the mode of action of
this class of compounds on insulin release has revealed
novel observations in β-cell function and physiology, some
examples of which are described here. It is expected that
experiments studying the long-term chronic effects of GKAs
will reveal more unexpected insights into β-cell physiology.
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Received 9 July 2007
doi:10.1042/BST0351208