(Received for publication, May 26, 1995; and in revised form, August 23, 1995)
From the
Pancreatic beta cells from mice that overexpress the
Ca-binding protein calmodulin have a unique secretory
defect that leads to chronic hyperglycemia. To further understand the
molecular basis underlying this defect, we have studied signaling
pathways in these beta cells. Measurements of cytosolic free
Ca
concentration
([Ca
]
) using fura-2 or
indo-1 revealed a markedly reduced response when glucose was the
stimulant. However, eliciting membrane depolarization with 50 mM K
or the addition of the ATP-sensitive
K
(K
) channel
antagonist tolbutamide restored
[Ca
]
transients to
near normal levels. Electrophysiological analysis of the beta cell ion
channels revealed that Ca
currents, delayed rectifier
K
currents, and K
channel currents were similar in transgenic and nontransgenic
cells, suggesting that these ion channels were able to function
normally. However, whereas K
channel
currents in control cells were reduced by 50% by the presence of high
glucose, those in transgenic cells were unaltered. Addition of
tolbutamide inhibited this channel and enhanced the secretion of
insulin in response to glucose for both control and transgenic cells.
As these observations implicated a metabolic defect, glucose
utilization, which is an indicator of glucose metabolism and ATP
production in beta cells, was measured and found to be reduced by 40%
in the transgenic cells. These data support the contention that
excessive levels of calmodulin may compromise the ability of the beta
cell to metabolize glucose and to modulate the state of the
K
channel, resulting in an inadequate
control of the membrane potential, which collectively impair
[Ca
]
and thus insulin
secretion in response to glucose.
Targeted overexpression of the ubiquitous
Ca-binding protein calmodulin to mouse pancreatic
beta cells results in the generation of an early onset nonimmune
hyperglycemic condition(1) . This condition is manifested by
the first postnatal day and progressively worsens with
age(1, 2) . Further analysis of these transgenic mice
revealed that the hyperglycemic condition is the result of a reduced
first phase and virtually absent second phase insulin secretory
response when glucose is the secretogogue(2) . These secretory
defects are apparent even when pancreatic insulin reserves are at
levels that should be sufficient to prevent the hyperglycemic
condition(1, 3) . However, insulin secretion in young
animals can be restored to normal if agents that release Ca
from internal stores, such as muscarinic agonists or phorbol
esters, are used together with glucose(2, 3) . This
led us to consider the presence of a
Ca
/CaM(
)-dependent pathway that might
converge with the glucose-dependent stimulus-secretion coupling pathway
in the transgenic cells.
It is well known that glucose-mediated
stimulus-secretion coupling in pancreatic beta cells is the result of a
complex intracellular signal transduction pathway that is designed to
efficiently release insulin in a biphasic
manner(4, 5, 6) . During both phases of
insulin secretion, the uptake and metabolism of glucose lead to signals
that result in closure of a K channel located on the
membrane that is regulated by ATP(7) . The closure of these
K
channels leads to depolarization of
the membrane and the opening of voltage-gated Ca
channels(8) . The ensuing transient rise in
[Ca
]
leads to insulin
secretion(9) , but it is not clear exactly how the
Ca
rise regulates the complex series of steps that
culminate in hormone release. Evidence exists that implicates
Ca
-binding proteins as being important primary and
secondary mediators of the stimulus-secretion response in beta cells
following the rise in [Ca
]
(10, 11) .
Since the primary response in beta
cells to elevated levels of glucose is an increase in
[Ca]
prior to the
release of insulin and our transgenic mice did not respond to glucose,
we have investigated the possibility that a defect in Ca
signaling is responsible. This was accomplished by systematically
examining several key components of the glucose-induced secretion
coupling pathway of beta cells. These components included
glucose-induced changes in [Ca
]
as well as beta cell ion channel properties and glucose
metabolism. Our results suggest that the overexpression of CaM results
in a metabolic defect that is distal to the membrane and apparently
involves reduced metabolism of glucose, resulting in the inability to
regulate the membrane potential via closure of the
K
channels and subsequent opening of the
voltage-dependent Ca
channels.
Fura-2 measurements were made on a Zeiss standard microscope
utilizing the instrumentation described by Neher(14) . In
brief, a spinning wheel was used to alternately excite the dye at 360
and 390 nm, while a photomultiplier was used to measure resulting dye
emission at wavelengths >510 nm.
[Ca]
was calculated from the
ratio of the light emitted at the two excitation wavelengths (13, 14) using acquisition software written in
AxoBasic language by Hui Qiu. Indo-1 fluorescence was measured on a
Nikon Optiphot microscope coupled to an Odyssey real-time confocal
microscope (Noran Instruments Inc., Middleton, WI). This system used a
water-cooled argon ion laser to excite the dye at 350-360 nm, and
fluorescence emission was measured at two emission wavelengths
(400-450 and 450-500 nm). Images were acquired and analyzed
utilizing Image-1 software (Universal Imaging Corp., Philadelphia, PA).
Ca currents were measured in isolation by
perifusion of cells with an external solution composed of 130 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl
, 5.4
mM CaCl
, 24 mM NaHCO
, 20
mM HEPES (pH 7.4), and 2.8 mM glucose and pregassed
with 95% O
, 5% CO
to maintain a pH of 7.2. The
internal pipette solution consisted of 145 mM CsCl, 4
mM MgCl
, 3 mM Na
ATP, 10
mM HEPES, 10 mM Cs
EGTA, and O.3 mM GTP and was adjusted to pH 7.35 with CsOH.
K currents were measured during perifusion with an external
solution that contained 133 mM NaCl, 5.4 mM KCl, 2.4
mM CaCl, 1.2 mM MgCl
, 1.1 mM
KH
PO
, 24 mM NaHCO
, 20
mM HEPES (pH 7.4), and 2.8 mM glucose. For the
perforated patch recordings, the perifusion medium contained 16.8
mM glucose in order to measure the closure of the
K
channels. The internal pipette
solution used for measuring delayed rectifier K
currents consisted of 140 mM KCl, 10 mM HEPES,
2 mM Na
ATP, 2 mM MgCl
, and 1
mM Na
EGTA and was adjusted to pH 7.35 with KOH.
For whole-cell K
channel currents, the
internal pipette solution was modified by decreasing the
Na
ATP concentration from 2 to 0.3 mM. The internal
pipette solution for perforated patch experiments on the
K
channel consisted of 140 mM KCl, 10 mM HEPES, 2 mM MgCl
, and 500
µg/ml nystatin and was adjusted to pH 7.35.
Transgenic mice overexpressing CaM in their pancreatic beta
cells have a reduced secretory response to glucose at a very early
age(2) . This reduced secretion is evident when the
extracellular Ca concentration is within the normal
physiological range (2.5 mM). Neither increasing the
extracellular Ca
concentration by 3-fold in the
presence of 16.8 mM glucose nor the addition of Ca
channel agonists (
)induced secretion in beta cells
from CaM mice. Since Ca
is necessary for insulin
secretion(9) , this suggests that CaM overexpression produces a
defect in some aspect of Ca
regulation of insulin
secretion. We have systematically examined the possible sources of this
beta cell defect in isolated islets and single cells from normal and
transgenic mice.
Figure 1:
Representative recordings
of [Ca]
in islets from
control and CaM mice. The bars indicate both the perifusion
solutions that were used during the time of the recording and the time
at which the solutions were switched at the reservoir. The solutions
reached the islets within 50 s afterwards. The values above the bars
are all millimolar glucose, except K
, which is 50
mM.
Figure 2:
A, resting
[Ca]
in control and
CaM islets; B, net rise in
[Ca
]
produced by
stimulating islets from control and CaM mice with 16.8 mM glucose (*, p < 0.05); C, net rise in
[Ca
]
obtained when the
same islets were exposed to 16.8 mM glucose plus 50
mM K
.
Because it has been suggested that the
glucose-induced [Ca]
response
of isolated beta cells differs from that of islets(16) , we
also measured [Ca
]
in single
dissociated beta cells loaded with Indo-I. As was found for isolated
islets, individual beta cells from either control or CaM animals that
were treated with low glucose (2.8 mM) solution showed similar
resting [Ca
]
values, while
exposure to solutions containing 16.8 mM glucose resulted in
robust rises in [Ca
]
in control
cells, but much smaller responses in CaM cells. The peak rise in
[Ca
]
produced by 16.8 mM glucose was 428 ± 85 nM in 29 control cells and 50
± 7 nM in 31 CaM cells (p < 0.05). Because
this defect in [Ca
]
signaling
could be responsible for the attenuation of glucose-sensitive insulin
secretion in the CaM mice, we performed additional experiments to
identify the source of the defect in
[Ca
]
signaling and the role of
this defect in the secretory phenotype of the mice.
A more direct
measure of Ca channel activity can be obtained from
electrical measurements of beta cell Ca
currents(17, 18) . We therefore used whole-cell
patch clamp recording methods (19) to examine the currents
resulting from Ca
channel gating. Cesium ions were
added to the intracellular medium to block currents flowing through
K
channels and thereby allow measurement of
Ca
channel currents in isolation. Families of
Ca
currents recorded from both control and CaM beta
cells are shown in Fig. 3A. These Ca
currents do not obviously differ between the two types of cells.
In both cases, stepping the membrane potential from a holding level of
-70 mV to potentials of -50 mV or more positive resulted in
activation of an inward Ca
current. There were no
consistent differences in the voltage dependence of these currents, as
can been seen in the current-voltage curves shown in Fig. 3B. In both cases, peak current was attained at
0 mV, and the current reversed its polarity at approximately
+50 mV. Peak current density, determined after dividing by the
membrane capacitance to normalize for cell-to-cell variations in cell
area, was not statistically different when comparing 23 control (18.1
± 1.3 pA/pF) and 21 CaM (15.2 ± 1.43 pA/pF) beta cells (p > 0.10). Furthermore, there were no consistent
differences in the kinetics of the Ca
currents
between beta cells from normal and CaM mice (Fig. 3A).
These data indicate that the deficit in glucose-induced
[Ca
]
signals in the CaM beta
cells is not due to a change in the density or gating properties of
Ca
channels.
Figure 3:
Whole-cell Ca currents
in pancreatic beta cells. A, representative family of
Ca
currents recorded from control and CaM beta cells.
The membrane potential was held at -70 mV and stepped to the
indicated voltages for 200 ms at 30-s intervals. B, average
current-voltage relationships for Ca
currents from
control (n = 23) and CaM (n = 21) beta
cells. The currents were divided by the membrane capacitance to take
into account any differences in cell membrane
area.
It is possible that the CaM mutation
affects Ca entry indirectly by altering the
K
conductances responsible for repolarization of the
membrane potential of the beta cell. We began our consideration of this
possibility by characterizing delayed rectifier K
currents. These currents were examined in isolation by including,
in the intracellular patch pipette solution, EGTA (a Ca
buffer that blocks elevation of
[Ca
]
that would activate
Ca
-dependent K
currents) and ATP to
block ATP-sensitive K
currents(20) . The
currents measured under these conditions were blocked by extracellular
application of tetraethylammonium ions (data not shown), a well known
blocker of delayed rectifier K
current in beta
cells(20) , providing pharmacological validation of the
isolation procedure. Fig. 4A illustrates families of delayed
rectifier K
currents activated by depolarization
under these conditions. The time course and magnitude of these outward
currents were very similar in beta cells taken from the control and CaM
lines. Membrane current density was calculated by dividing the current
amplitude by the membrane capacitance, to take into account differences
in membrane area. The voltage dependence of this parameter was very
similar for delayed rectifier K
currents from both
control (n = 25) and CaM (n = 15) beta
cells (Fig. 4B). In both cases, delayed rectifier
currents appeared at membrane potentials of -60 mV and increased
in magnitude at more positive potentials. The peak delayed rectifier
conductance measured at +30 mV was calculated to be 1.21 ±
0.24 nS/pF in control cells and 0.90 ± 0.14 nS/pF in CaM cells,
a difference that is not statistically significant (p >
0.20). These data indicate that the delayed rectifier K
channels are functionally equivalent between control and CaM beta
cells.
Figure 4:
Delayed rectifier K currents in control and CaM beta cells. A, families of
K
current recordings from both cell types. The
membrane potential was held at -70 mV and stepped to the
indicated voltages for 200 ms at 30-s intervals. B, voltage
dependence of delayed rectifier K
currents in both
control (n = 25) and CaM (n = 15) beta
cells.
Because the K channel plays a
key role in the membrane depolarization produced by
glucose(21) , we next examined whether the defect in CaM cells
was caused by changes in K
channels. We
first examined the actions of the sulfonylurea drug tolbutamide. This
drug acts by blocking ATP-sensitive K
channels; the
resulting depolarization of the beta cell membrane potential normally
results in an increase in [Ca
]
and triggering of insulin secretion(8) . We measured
[Ca
]
in islets loaded with
fura-2 to determine whether tolbutamide causes an elevation of
[Ca
]
in CaM islets. Addition of
tolbutamide (1 mM) to the perifusate of resting islets
resulted in sustained and reversible increases in
[Ca
]
in both CaM and control
mice (Fig. 5A). These increases in
[Ca
]
presumably are due to
tolbutamide depolarizing the membrane by blocking ATP-sensitive
K
channels. The peak rises in
[Ca
]
produced by tolbutamide
did not differ significantly (p > 0.05) in control and CaM
islets (Fig. 5B). Likewise, tolbutamide restored the
ability of CaM islets to secrete insulin. At resting conditions (2.8
mM glucose), both the CaM and control islets secreted insulin
at about the same rate (Fig. 6). Addition of 1 mM tolbutamide increased the sensitivity of the islets to 2.8 mM glucose in both the control and CaM islets, with secretion levels
attaining a 4-fold increase over basal rates. Further addition of 16.8
mM glucose potentiated the tolbutamide response in both lines
by
15-fold over low glucose alone and 3.5-fold over low glucose
plus tolbutamide. These data indicate that the tolbutamide block of
K
channels bypasses the defects in
[Ca
]
signaling and insulin
secretion characteristic of CaM islets.
Figure 5:
Tolbutamide induced rises in
[Ca]
. A,
[Ca
]
was measured in
islets from control and CaM mice. Perifusion of the islets was done in
2.8 mM glucose for 45 min to establish a base line. Addition
of 1 mM tolbutamide to the solution (bar) increased
[Ca
]
in both types of
islets. B, peak changes in
[Ca
]
for eight control
and eight CaM islets (p >
0.05).
Figure 6: Insulin secretion induced by tolbutamide in control and CaM islets. After perifusing the islets with Krebs-Ringer bicarbonate containing 2.8 mM glucose, the islets were perifused with Krebs-Ringer bicarbonate containing 2.8 mM glucose and 1 mM tolbutamide (bar) for 20 min. 16.8 mM glucose was then added (bar), and the response was monitored for another 50 min. The data are the means ± S.E. of three independent experiments.
To further understand the
role of K channels in the CaM phenotype,
we next measured currents flowing through these channels. This was done
by using the patch pipette to lower intracellular ATP concentrations
while measuring the resultant changes in K
conductance(21) . The procedure used in our experiments
on beta cells from both control and CaM mice is illustrated in the
current recordings shown in Fig. 7A. Establishment of
diffusional contact between the patch pipette and the inside of the
cell, evident as a large increase in membrane capacitance (19) , occurred at the times indicated by arrows.
Because the patch pipette contained only a minimal concentration of ATP
(0.3 mM), the K
conductance of the membrane
grew larger as ATP was dialyzed out of the cytosol. During this time,
the membrane potential was repeatedly alternated between three values,
-60, -70, and -80 mV. Because the equilibrium
potential for K
ions is approximately -77 mV
under our experimental conditions, the increase in ATP-sensitive
K
conductance was evident as a gradual increase in
outward current when the potential was at -60 mV and as an
increase in inward current when the potential was at -80 mV.
These currents reached peak levels within 10 min of starting
intracellular dialysis and declined at later times because of run-down
(evident at the end of each of the two traces). Addition of tolbutamide
(0.1 mM) to the external solution blocked the currents
measured in both the CaM and control beta cells, showing that these
currents were carried by the K
channel (Fig. 7A). These K
channel currents appeared similar in magnitude in both types of cells.
To quantify the conductance of the K
channels of these two cell types, peak currents were measured at
all three membrane potentials, and the conductance was calculated as
the slope of the current-voltage relationship. These conductances, when
normalized for variations in membrane area, were very similar for the
two types of cells (Fig. 7B). These data indicate that
the density, ATP sensitivity, and pharmacological properties of
K
channels are similar in both types of
cells. When combined with the observations that tolbutamide is able to
activate [Ca
]
signaling and
insulin secretion in the beta cells from CaM mice, these data further
indicate that the defect in glucose-induced insulin secretion in CaM
mice is neither due to changes in the intrinsic properties of
K
channels nor at steps that follow
closure of these channels. Instead, the defect must be at, or proximal
to, the glucose-induced rise in intracellular ATP concentration that
normally leads to blocking of the K
channels.
Figure 7:
ATP-sensitive K conductance in beta cells from control and CaM mice. A,
currents recorded while the membrane potential was alternated between
-60 -70, and -80 mV in 2-s intervals. The patch
pipette contained 0.3 mM ATP to dialyze out cytosolic ATP.
Removal of intracellular ATP beginning at the time indicated (Breakthrough) caused a progressive increase in K
conductance, evident as increased outward current at -60 mV
and increased inward current at -80 mV. Addition of 0.1 mM tolbutamide (bar) caused inhibition of these currents,
confirming that they result from K
channels. Also note the variable amplitude and speed between the
traces due to variations in the series resistance of the pipette and
normal cell to cell differences. B, normalized membrane
conductance for control (0.53 ± 0.12 nS/pF) and CaM (0.68
± 0.15 nS/pF) cells. Data are from 27 control and 24 CaM cells (p > 0.20).
Figure 8: Glucose utilization in islets from control and CaM mice. Measurements were made as described under ``Materials and Methods'' from a minimum of 23 determinations for either 2.8 or 16.8 mM glucose. The data have been normalized by expressing the rates per microgram of total protein to compensate for islet size differences (*, p < 0.05).
If the CaM mutation produces a defect in
production of ATP from glucose, then glucose should be less capable of
closing K channels even though these
channels appear normal in CaM cells (see above). This prediction was
tested by measuring the ability of high glucose to close these channels
in single beta cells. While whole-cell patch clamp methods are the
ideal way to measure such currents, dialysis of the intracellular
medium during such recordings would necessarily perturb intracellular
ATP generation during glucose treatment. To avoid this complication, we
turned to perforated patch recordings as a means of measuring the
K
channel current while keeping
intracellular metabolism intact(15, 21) . Fig. 9A shows an example of the recording of the
K
channel currents activated while
treating a control beta cell with an extracellular solution containing
16.8 mM glucose. In control cells, this treatment consistently
produced a large decrease in the conductance associated with the
K
channels; this conductance decrease is
reflected in a progressive decrease in current during glucose exposure.
The resting conductance of cells from CaM islets (0.24 ± 0.05
nS/pF), measured in low (2.8 mM) glucose conditions, was
similar to that of control cells (0.24 ± 0.04 nS/pF) presumably
because, as in control cells, intracellular ATP levels were low and the
K
channels were open. However, elevation
of glucose concentration to 16.8 mM produced much smaller
decreases in conductance in cells from CaM islets (Fig. 9A). This difference in conductance sensitivity
to glucose was most evident after the K
channel conductance was calculated, as described above, using
measurements of currents recorded at three potentials and normalized
for variations in cell area (Fig. 9B). In control
cells, glucose treatment produced a large decrease in conductance (to
0.13 ± 0.03 nS/pF), while there was no consistent decrease in
conductance (0.24 ± 0.05 nS/pF) in beta cells from CaM mice (p < 0.05). These data are consistent with the hypothesis
that the metabolism of glucose is impaired in CaM cells, so that
glucose metabolism does not result in the usual elevation of ATP
concentration and subsequent blocking of K
channels in these cells.
Figure 9:
ATP-sensitive K currents
measured in beta cells under physiological conditions. A,
current recordings during treatment with 16.8 mM glucose (bar); B, normalized currents averaged from eight
control and eight CaM cells at 2.8 and 16.8 mM glucose (*, p < 0.05).
The early stages of the CaM phenotype are characterized by a
reduction in the first phase of glucose-induced insulin secretion and
the abolition of the second phase of insulin
secretion(2, 3) . In this report, we have considered
the molecular mechanisms responsible for the defects in insulin
secretion resulting from targeted overexpression of CaM in beta cells.
Our most striking finding is that these cells have a greatly attenuated
ability to elevate [Ca]
in
response to glucose stimulation. Because the ionic currents of CaM beta
cells were indistinguishable from those of normal beta cells, it
appears that the reduction in [Ca
]
is due to a defect that is distal to the beta cell membrane. This
defect, however, appears to be directly linked to the modulation of the
ion channels that control membrane potentials, ion fluxes, and insulin
secretion. The fact that glucose utilization and glucose-induced
closure of ATP-sensitive K
channels are decreased in
CaM cells suggests that the defect apparently is due to an impaired
ability of these beta cells to metabolize glucose into intracellular
ATP. Thus, we conclude that beta cells from CaM mice probably produce
less ATP when exposed to glucose, and this defect prevents the cells
from depolarizing their membrane potential, opening voltage-gated
Ca
channels, or elevating
[Ca
]
sufficiently to produce
the phasic secretion of insulin responses characteristic of normal beta
cells(22) .
Elevation of
[Ca]
is necessary for producing
glucose-induced insulin secretion(23) . Therefore, it is likely
that the impaired ability of glucose to elevate
[Ca
]
(Fig. 1) underlies
this defective insulin secretion in CaM beta cells. Direct support for
this contention comes from experiments demonstrating that conditions
that elevate [Ca
]
by
depolarizing the membrane with agents such as tolbutamide (Fig. 6) or by increasing the external K
concentration (2, 3) are able to restore insulin
secretion from CaM islets. In addition, agents that release internal
stores of Ca
, such as phorbol esters
(12-O-tetradecanoylphorbol-13-acetate) or muscarinic agonists
(carbachol), also restore insulin secretion in the transgenic beta
cells(2, 3) . Thus, the phenotype exhibited by the CaM
mice appears to result secondarily from impaired Ca
signaling within the beta cell.
We have also demonstrated that there
is an absence of any changes in the intrinsic properties of
voltage-gated or ATP-sensitive ion currents in CaM beta cells (Fig. 3, Fig. 4, and Fig. 7). It is also clear
that the K channel currents of these
cells can be inhibited by compounds that interact directly with this
channel (Fig. 7). This defect in
[Ca
]
signaling also means that
Ca
-activated K
channels, which
normally would aid in the resetting of the membrane potential (8) , should not be activated by glucose in the CaM mice. It is
possible that excessive CaM alters the properties of the
Ca
-activated K
channels, but because
the secretory defect can be rescued by providing internal
Ca
, this is not likely to be a significant part of
the CaM phenotype. Taken together, these findings demonstrate that the
usual complement of ion channels are present in the CaM beta cell and
are functional. While there is some evidence that CaM-regulated
pathways may modulate ion channels in beta
cells(24, 25) , the 5-fold overexpression of CaM in
these beta cells (1) appears to have no direct effects on the
ion channels.
Increasing the intracellular concentration of anything
that will bind Ca will slow and attenuate transient
rises in [Ca
]
(26) .
Given that CaM binds 4 Ca
ions and that there is a
much higher concentration of CaM in beta cells from CaM
mice(1) , it is possible that Ca
signaling is
defective in CaM cells simply because the extra CaM acts as a
Ca
buffer. Previous calculations alluded to the
possibility that up to 1 mM Ca
might be
bound during stimulatory Ca
transients in these cells (1) . To test this possibility, we reported that overexpression
in the beta cells from mice of an inactive CaM that only binds
Ca
also impairs insulin secretion(3) .
However, more recent studies indicate that this secretory defect is the
result of a different molecular mechanism than the secretory defect
seen in the CaM mice, (
)suggesting that Ca
buffering is not the cause of the phenotype in the CaM mice. Our
observation that depolarization of the membrane with tolbutamide,
elevated concentrations of K
, or agonists of internal
Ca
release restore
[Ca
]
signaling and insulin
secretion in CaM islets is a more direct indication that Ca
buffering is not altered in the CaM beta cells. It is therefore
unlikely that overexpression of CaM impairs Ca
signaling in the beta cell simply by buffering Ca
ions.
Our results instead indicate that CaM overexpression
causes a metabolic defect in beta cells. The clearest indication of
this is that K channels were not closed
during exposure of CaM cells to high extracellular glucose
concentrations. This could result from attenuation of ATP production
from glucose metabolism or from changes in the ability of the
K
channel to bind ATP. The former possibility is more
likely for two reasons. First, tolbutamide appears to bind and close
the K
channel similarly in both control
and CaM cells, suggesting that the channel is conformationally correct
and can bind ATP. Second, our measurements of glucose metabolism (Fig. 8) indicate low rates of glucose utilization in the beta
cells from CaM mice.
Decreased glucose utilization in the CaM beta cells points to a defect somewhere in the glycolytic pathway. It is unlikely that this defect occurs at the most proximal steps of glycolysis, such as glucose transport, because down-regulation of the primary glucose transporter (GLUT2) does not result in hyperglycemia(27) . Furthermore, the glucose-sensing mechanism of the beta cell is still functional in the CaM mice because elevated glucose still increases utilization rates (12, 28) . Thus, it is more likely that the defect of the CaM beta cells lies in the distal steps of glycolysis.
It is not yet clear which distal
step in glucose metabolism is responsible for the impaired ATP
production in the CaM mice. In neonatal streptozotocin-induced diabetic
rats, impaired insulin secretion is linked to reduced ATP production
resulting from defective glucose metabolism in the mitochondria of beta
cells(29, 30) . Furthermore, up to 40% of the total
glucose utilized by beta cells during the stimulus-secretion response
can be shunted through this mitochondrial pathway to produce
ATP(31) . The CaM mice are markedly similar to these neonatal
streptozotocin-induced diabetic rats because they have an 40%
lower glucose utilization rate (Fig. 8) than control mice. In
addition, ketoisocaproic acid, which can be readily metabolized
directly in the mitochondria and induce secretion via direct effects on
the K
channel(32) , failed to do
so in the CaM mice(2) . This indicates a defect in
mitochondrial metabolism in the CaM beta cells.
Dukes et al.(33) have suggested that in beta cells, ATP is produced by
two interrelated metabolic pathways in the mitochondria. While one of
these pathways does not appear to have any CaM-regulated reactions, the
second pathway uses an FAD-linked glycerol-3-phosphate dehydrogenase (33) that possesses an EF hand motif in its carboxyl terminus
and thus could be regulated by Ca, CaM, or some
CaM-dependent event(34) . Thus, overexpression of CaM might
somehow prevent the glycerol-3-phosphate dehydrogenase from generating
the reduced intermediates required for the generation of ATP in
mitochondria. In addition, any inhibition of this FAD-linked shuttle
would also result in lower yields of NAD
, a metabolite
needed for augmentation of glycolysis(33) .
In summary, the phenotype seen in the CaM mice apparently is associated with a defect within the glycolytic pathway. Other transgenic mice overexpressing proteins in the beta cell do not have similar metabolic abnormalities(35) , demonstrating that the reduction in glucose utilization and ATP production is a specific consequence of overexpressing CaM. These data also suggest that this defect is associated with mitochondrial production of ATP, although it is not yet clear why overexpression of CaM leads to this diminished metabolic response.