(Received for publication, September 20, 1995; and in revised form, November 9, 1995)
From the
Studies of dispersed cells have been used to infer their
behavior in the intact pancreatic islet. When dispersed,
cells
exhibit multiple metabolic glucose-response populations with different
insulin secretion properties. This has led to a model for
glucose-dependent insulin secretion from the islet based on a step-wise
recruitment of individual
cells. However, previously reported
synchronous and uniform Ca
activity and electrical
responses indicate that
cell behavior within intact islets is
more uniform. Therefore, uncertainty remains whether
cell
metabolic heterogeneity is functionally important in intact islets. We
have used two-photon excitation microscopy to measure and compare the
glucose-induced NAD(P)H autofluorescence response in dispersed
cells and within intact islets. Over 90% of
cells in intact
islets responded to glucose with significantly elevated NAD(P)H levels,
compared with less than 70% of dispersed
cells. In addition, all
responding
cells within intact islets exhibited a sigmoidal
glucose dose response behavior with inflection points of
8 mM glucose. These results suggest that
cell heterogeneity may
be functionally less important in the intact islet than has been
predicted from studies of dispersed
cells and support the role of
glucokinase as the rate-limiting enzyme in the
cell glucose
response.
Insulin secretion from pancreatic cells is tightly coupled
to glucose metabolism(1, 2, 3, 4) .
When dispersed, marked variability in the metabolic responses of
cells to glucose has been observed(5, 6) using
NAD(P)H autofluorescence as an index of the cellular redox state (7) . Based on this, it has been suggested that metabolic
heterogeneity plays a fundamental role in whole islet insulin secretion
by a mechanism of variable activation thresholds of individual
cells (8) . The observation that glucokinase, which has been
postulated as the rate-limiting step in glucose transduction by
cells(3) , exhibits heterogeneous immunoreactivity among
cell also supports this model (9) . However, other indicators
of
cell function within intact islets, such as synchronous
intracellular [Ca
]
(10) and electrical (11) responses, indicate that
intraislet
cells constitute a functionally more homogeneous
population. Direct measurement of the metabolic behavior of cells
within the intact islet, however, has not been performed. Here we
report the first measurement of glucose-induced NAD(P)H
autofluorescence changes within the intact islet at subcellular
resolution using TPEM (
)(12, 13) . Using
the same instrument, we also measured the glucose-induced metabolic
behavior of isolated cells, thereby allowing a quantitative comparison
of the metabolic response of
cells under both conditions.
A new alternative to confocal microscopy, TPEM allows collection of optical sections within thick samples, such as pancreatic islets, with greatly reduced photobleaching and photodamage. Previously, we have described a laser scanning microscope that is optimized for TPEM of UV excitable fluorophores, such as NAD(P)H(14) . This instrument allows extended dynamic studies of many cells simultaneously, thereby permitting observation of the temporal and spatial organization of metabolic activity within the intact islet.
For the dynamic NAD(P)H images, the perifusion was turned off, image acquisition was started, and 8 s later a small volume of test agent was added at the far edge of the sample chamber, which initially contained 3 ml of basal buffer (DME base (Sigma) with 2 mM glutamine and 1 mM glucose). In these experiments, it was more convenient to use a glass-bottomed 35-mm dish (PG35G-0-14-gm, Matek Corporation, Ashland, MA) than the coverslip chamber, but temperature was still maintained by heating both the stage and the objective lens. Pre-heated test solutions were added as follows: 100 µl of 1 M glucose, 60 µl of 1 M mannoheptulose, or 100 µl 100 mM NaCN to give the approximate final concentrations of 30, 20, and 3 mM, respectively. TPEM images were acquired continuously for 136 s at 8 s/scan.
For the steady-state glucose
response measurements, islets were perifused at 1.2 ml/min and 37
°C with Krebs-Ringer bicarbonate buffer (120 mM NaCl, 4.8
mM KCl, 2.5 mM CaCl, 1.2 mM MgCl
, 5 mM NaHCO
, and 10 mM HEPES, pH 7.4) containing 3, 5, 7.5, 12, or 23 mM glucose, and NAD(P)H autofluorescence was measured after 10 min of
equilibration with each different concentration of glucose. In a
parallel experiment using 12 mM glucose, images acquired at
5-min intervals after the addition of glucose, showed that 10 min was
sufficient to reach the response plateau.
NAD(P)H measurements of dispersed cells were performed by the same techniques as the glucose response, except that perifusion rate was lowered to 0.5 ml/min and glucose levels were varied between 1 mM and 30 mM to compare directly with the dynamic NAD(P)H measurements in whole islets.
We have measured NAD(P)H autofluorescence at the subcellular
level throughout intact pancreatic islets (Fig. 1) by combining
TPEM (14) with established quantitative laser scanning
microscopy methods(18) . Subcellular structures, such as the
nuclei (which appear dark in the NAD(P)H image) are clearly visible.
The spatial discrimination and collection efficiency are sufficient to
quantitatively assess NAD(P)H levels in individual cells, with
temporal resolutions of 4-8 s/image. A series of control
experiments were performed to demonstrate that the fluorescence changes
observed were due to changes in [NAD(P)H] and to establish
the limits of islet viability during TPEM (Fig. 2). Inhibition
of respiration using cyanide produced about a 2-fold elevation in
autofluorescence, whereas inhibition of glucose phosphorylation with
mannoheptulose led to a
50% reduction. The effect of these
metabolic perturbations on NAD(P)H levels agrees with similar
measurements using UV excitation(7, 19) . Laser
irradiation of
3 mW (average power at the sample) generated
signals sufficient for imaging without evidence of cellular damage (e.g. autofluorescence increase, detectable photobleaching, or
degradation of glucose response). However, extended laser irradiation
>5 mW resulted in increased autofluorescence (Fig. 2) and the
loss of any glucose-induced autofluorescence response (data not shown).
To determine if the laser-induced autofluorescence increase was caused
by two-photon excitation or the incident red light, islets were exposed
to intense laser illumination focused into the coverslip. This exposed
the entire islet to unfocused red light without generating any two
photon excitation within cells. In this situation, even 15 mW of
unfocused red light did not affect autofluorescence, and the subsequent
glucose-induced NAD(P)H response was unaffected. Thus, islet cells are
sensitive only to the amount of two-photon excitation and not
single-photon interactions from the red light.
Figure 1:
NAD(P)H autofluorescence in an optical
section of an isolated rat islet. Individual cells and their
nuclei are visible 40 µm into an islet mounted on a coverslip. The
reduced light scattering and absorption associated with TPEM (red light
instead of UV light is used to excite NAD(P)H) affords unprecedented
clarity in optical sections throughout the islet. The scale bar is 10 µm.
Figure 2:
Cellular autofluorescence detected in
islets by TPEM is due to NAD(P)H. Digital image analysis was used to
measure autofluorescence in single cells after image acquisition.
NaCN (3 mM), which inhibits cellular respiration, caused a
>2.5-fold rise in cellular autofluorescence (7) (
, n = 5 islets). Mannoheptulose (20 mM), which
inhibits glucose utilization by competitive inhibition of glucokinase,
resulted in a
50% decrease in autofluorescence (
, typical
cell, n = 3 islets). Glucose stimulation (30
mM) increased autofluorescence >2-fold (
, typical
cell, n = 8 islets). An average laser power of 3
mW at the sample produced NAD(P)H autofluorescence signals adequate for
imaging without detectable photobleaching (
). Continuous
exposure to excessive doses of two-photon irradiation (>5 mW average
power) caused a 1.5-fold increase in autofluorescence after 136 s
(
). This cellular photodamage was caused by two-photon
interactions because islets pre-exposed to 15 mW of defocused red light
showed no change in NAD(P)H levels (data not shown). The scale bar is 10 µm.
To allow quantitative
comparison of cells in the intact islet and after dispersion, we
measured NAD(P)H responses of cells in both situations. In the case of
intact islets, dynamic behavior was also observed over several
successive images. The temporal metabolic response to glucose of a
single optical section of cells in an intact islet is shown in Fig. 3(A-H). To attribute responses to specific
islet cell types after TPEM, and
cells were identified by
retrospective immunofluorescent staining and conventional laser
scanning confocal microscopy (Fig. 3I, green for insulin and red for glucagon).
Figure 3:
Serial images showing the temporal
metabolic responses of cells visible in an optical section of an intact
islet. Two-photon excitation of cellular autofluorescence is shown in A-H. After measuring dynamic changes, and
cells (I, green for insulin and red for
glucagon) were identified by immunofluorescent staining and confocal
microscopy. The NAD(P)H images shown (time course generated as in Fig. 2) begin one scan before any increase was detected and
continue at 8-s intervals thereafter. NAD(P)H autofluorescence began to
increase in peripheral cells 32 s after glucose addition (B)
and then spread inward toward the center of the islet (C-H). Peak NAD(P)H autofluorescence in central
cells lagged that of the periphery by about 40 s, consistent with
glucose diffusion into the islet.
Increased NAD(P)H
autofluorescence was observed 32 s after switching from low to
high glucose. The increase was observed first at the islet periphery
with a gradual spread of the autofluorescence response to the central
cells about 40 s later, consistent with glucose diffusion into
the islet. Except for this time delay, the NAD(P)H autofluorescence
increase of all
cells was strikingly uniform. Some cells began
and ended the experiment with relatively low autofluorescence, thus
giving the appearance of heterogeneity in single images. However, these
cells showed a proportionate increase in NAD(P)H, which could be
readily detected by animation (rapid playback of time series images,
animation is available by World Wide Web at http://160.129.157.26) and
could be quantitated by digital image analysis of single cells (see
below). In
cells, the NAD(P)H signal did not increase in response
to increased glucose; these cells were excluded from further analysis.
To quantitatively assess the metabolic response of the intraislet
cell population, we determined the ratio of peak NAD(P)H
signal/initial NAD(P)H signal for every
cell present in single
optical sections from five islets (46-133
cells/islet
section). The average
cell response ratio in a single islet
section ranged from 1.74 to 2.45 (for the five islets studied), each
with a standard deviation <25% of the mean. To compare these results
to isolated
cells, nonresponding
cells were defined as
those that showed responses less than two standard deviations below the
mean. By this measure, nonresponding
cells in five islets
constituted 14, 6, 13, 5, and 6% (mean = 9%) of insulin
immunopositive cells. A similar result was obtained when nonresponding
cells were identified and counted by animation. By either method, all
of the nonresponding
cells were located at the periphery of the
islet.
Measurements of NAD(P)H levels at various steady-state
glucose concentrations were performed to further examine responses at
the single-cell level in intact islets (Fig. 4). The response
curves generated from eight typical cells within two intact
islets, which are shown in Fig. 4, are sigmoidal with inflection
points at
8 mM glucose. This dose response behavior is
consistent with the rate-limiting role of glucokinase, the high K
hexokinase in the
cell(3, 20, 21) .
Figure 4:
Metabolic response measurement within
intact islets at steady-state glucose concentrations. These sigmoidally
shaped glucose concentration-dependent response curves were obtained.
The inflection point at 8 mM is consistent with a
rate-limiting role for glucokinase in cell glucose
usage(3) . These response curves were taken from eight
representative cells across two separate islet
preparations.
To validate the results
obtained from intact islets, we also examined the NAD(P)H response of
isolated cells. Fig. 5shows a typical field of
dissociated islet cells in the presence of 1 mM (Fig. 5A) or 30 mM glucose (Fig. 5B). As with intact islets, retrospective
immunostaining was performed to confirm that all analyzed cells were
cells. Parallel control experiments on the same cell preparations
showed that >95% of the cells were viable as determined by either
trypan blue exclusion (negative control for membrane integrity) or
Fura-2/AM uptake (positive control for esterase activity). As seen in
all fields of dispersed
cells, Fig. 5shows much greater
variability in the NAD(P)H response to glucose than the intact islets
(equivalent images are shown in Fig. 3, A and H). The mean NAD(P)H autofluorescence increase for dispersed
cells was
1.9 (similar to that seen in whole islets), but
the standard deviation of the increase was
35% of the mean
(greater variation than the 25% observed in intact islets). The
percentage of nonresponding isolated
cells was also quantified
using the same definitions as in the intact islets. This analysis
showed that 17 of 53 (
32%) dispersed
cells, obtained from
two separate islet preparations, were found to be nonresponsive.
Figure 5:
NAD(P)H autofluorescence measured by TPEM
in isolated cells. The responses of these dissociated cells as
glucose is increased from 1 (A) to 30 mM (B)
are much more heterogeneous than those of
cells in intact islets.
The scale bar is 12 µm.
We have used two-photon excitation microscopy to measure and
compare the metabolic responses to glucose of individual cells within
intact pancreatic islets. We confirmed that the observed
autofluorescence was largely due to NAD(P)H because its responses to
cyanide and mannoheptulose exposure were consistent with those from
established techniques on isolated
cells(7, 22) . Measurement of NAD(P)H autofluorescence
in an intact pancreatic islet requires a sophisticated technique, such
as TPEM, because of the tissue thickness and high levels of
fluorescence background. Whole islet experiments were possible because
the lack of out-of-focus fluorescence generation inherent in two-photon
imaging allowed acquisition of optical sections deep within the islets (Fig. 1). Although NAD(P)H is a very poor fluorophore, having
both a low absorption cross-section and quantum yield, TPEM enabled
extended time course measurements without measurable photobleaching and
photodamage. This allowed continuous NAD(P)H imaging of a single islet
for several minutes without killing the cells (Fig. 2). Even
using TPEM, however, there is always a trade-off between the signal to
noise ratio and temporal resolution (13) , and in order to
obtain sufficient signal for these NAD(P)H autofluorescence
measurements, we were usually limited to 8 s/image.
The
glucose-induced metabolic responses from the cells in intact
islets were strikingly uniform and contrast significantly with the
heterogeneous behavior of isolated
cells ( Fig. 3and Fig. 5). In our experiments, greater than 90% of
cells in
intact islets were metabolically active, whereas less than 70% of
dispersed
cells responded to the same glucose concentrations.
These isolated cell measurements are in excellent agreement with
previous reports where the metabolically responsive population of
isolated
cells in the presence of 20 mM glucose was
determined to be 70%(5, 8) . In addition, as shown in Fig. 5, the amplitude of response varied more greatly in the
dispersed cells.
Two explanations for the differences observed
between isolated cells and those in the intact islet seem possible, and
each has different implications. The first explanation is that
proteolytic damage during islet and single-cell isolation procedures
may degrade the NAD(P)H response of certain cells without compromising
their viability. In intact islets, all nonresponsive cells were at the
periphery, and cells on the islet exterior would obviously be more
prone to this kind of damage. A similar damage mechanism may also be
responsible for the increased fraction of nonresponsive dispersed
cells, because islet dispersion requires even more aggressive
proteolytic treatment. If this is the case, then models developed from
studies of isolated
cells are unlikely to accurately describe
islet function.
A second explanation for the differences between
isolated cells and those in intact islets could be that
intercellular communication might act to overcome the heterogeneities
of individual
cells. Cell-cell contact may allow less responsive
cells to be entrained by the more responsive cells, but this was not
observed in the dissociated cells (Fig. 5) where cells in
contact were not found to respond more uniformly than completely
isolated ones. In intact islets, individual
cells never displayed
earlier responses than neighboring cells (Fig. 3), so any
entrainment mechanism would need to act faster than a few seconds. Such
coupling cannot be electrical because electrophysiological and
[Ca
]
measurements show a time
lag between the NAD(P)H increase and membrane depolarization of >15
s(23) . Such a mechanism would likely involve a small, easily
diffusible messenger. This messenger could also diffuse rapidly toward
the islet core and generate an activation signal ahead of the primary
glucose-induced autofluorescence wave, but no such signal was observed (Fig. 3). Although it may be possible to construct a model with
a messenger that can equilibrate between adjacent cells within a few
seconds and not trigger cells toward the islet center sooner than the
measured 40 s delay, we conclude that such an entrainment mechanism is
unlikely.
In the absence of a defined entrainment mechanism, the
uniform metabolic response results in the whole islet appear to be
inconsistent with significant metabolic heterogeneity among
cells, but the images do show autofluorescence heterogeneity at the
initial glucose concentration of 1 mM (Fig. 3A), as do the isolated
cells (Fig. 5A). This basal autofluorescence heterogeneity
suggests that a metabolic thresholding based on the absolute ATP/ADP
ratio (related to the absolute NAD(P)H/NAD(P)
ratio)
could generate heterogeneous
cell function even in the presence
of a uniform NAD(P)H response. However, such basal heterogeneities in
whole islets are difficult to interpret, because they could possibly
arise due to variable optical density or the subcellular distribution
of NAD(P)H. First, cells in the middle of the islet generally have more
tissue between them and the objective lens than do cells on the
periphery, and more fluorescence signal is indeed detected in the
peripheral cells (Fig. 3). Secondly, because the 1-µm-thick
optical section passes through each cell differently, the amount of
NAD(P)H detected in each cell may differ as well.
The metabolic
heterogeneity found in dispersed cells has been shown to
correlate with heterogeneity in insulin secretion and has thus led to a
model for islet function based on variable activation thresholds (8) . However, our measurements of NAD(P)H changes as a
function of glucose concentration indicated a sigmoidal dose response
with an inflection point at
8 mM glucose (Fig. 4).
This coincides with the K
of glucokinase, the
cell glucose sensor(3, 21) , and contradicts a
model where the sigmoidal insulin secretory response from the whole
islet is generated by a stepwise recruitment of individual
cells
with variable activation thresholds(8) . None of the
cells examined here exhibited different inflection points, as would be
predicted by the stepwise recruitment model. We conclude that a
stepwise recruitment of
cells does not describe insulin secretion
from whole islets. However, in the absence of direct insulin secretion
measurements from individual cells within the islet, models of
functional heterogeneity cannot be ruled out. The results may also be
inconsistent with previous descriptions of pronounced
cell
glucokinase heterogeneity as detected by immunohistochemical
methods(9) . Further studies of the differences between intact
islet and isolated
cells using TPEM should lead to a resolution
of these controversies and a greater understanding of intraislet
metabolic response dynamics.