Laboratory of Experimental Medicine, Brussels Free University, B-1070 Brussels, Belgium
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ABSTRACT |
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D-mannoheptulose is apparently transported into
cells mainly at the intervention of GLUT-2 and hence was
recently proposed as a tool to label preferentially insulin-producing
-cells in the pancreatic gland. The validity of such a proposal was
investigated in the present study conducted in isolated perfused
pancreatic glands from control and streptozotocin-induced diabetic
rats. After a 30-min equilibration period,
D-[3H]mannoheptulose (0.1 mM) and
[U-14C]sucrose (0.5 mM) were infused for 15 min in the
presence of 30 mM D-glucose. The pancreatic glands were
then perfused for 10 min with a nonradioactive medium during and after
administration of cytochalasin B (0.02 mM). Under these experimental
conditions, the intracellular distribution space of
D-[3H]mannoheptulose averaged 5.42 ± 0.75 nl/mg in control animals, whereas it failed to be significantly
different from zero in the streptozotocin rats. The present procedure
may thus allow the assessment of the relative contribution of islet
-cells to the total mass of the pancreatic gland.
rat pancreas perfusion; D-[3H]mannoheptulose; streptozotocin-induced diabetic rats
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INTRODUCTION |
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IT WAS RECENTLY PROPOSED that D-mannoheptulose is transported across the plasma membrane by GLUT-2 and may be used to preferentially label insulin-producing cells in the pancreatic gland (15). Using a suitable radioactive analog of the heptose may allow one to quantitate the endocrine pancreas by a noninvasive procedure (10, 11).
The major aim of the present experiments, conducted in the isolated
perfused pancreas of control and streptozotocin-induced diabetic rats,
was to investigate whether
D-[3H]mannoheptulose could be used to assess
the relative contribution of islet -cells to the total mass of the
pancreatic gland.
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MATERIALS AND METHODS |
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D-[3H]mannoheptulose and [U-14C]sucrose were purchased from NEN (Boston, MA).
Male Wistar rats (Iffa Credo, L'Arbresle, France) were given free access to food (KM-04-k12; Pavan Service, Oud Turnhout, Belgium) and tap water up to the time of anesthesia with pentobarbital sodium (0.2 µmol/g body wt). Three rats were injected intravenously with streptozotocin (STZ; Sigma, St. Louis, MO) at a dose of 0.25 µmol/g body wt. (6). Three to four days later, they received twice daily a subcutaneous injection of insulin (6 U/rat; Insulatard, Novo Nordisk, Denmark) for 3 days and were anesthetized in the morning of the next day.
The pancreas of each rat was perfused without recirculation through both the celiac and superior mesenteric arteries, as described elsewhere (7, 14). The basal salt-balanced solution (14) contained D-glucose (30 mM), dextran (clinical grade; 40 g/l; Sigma), and bovine serum albumin (RIA grade; 5 g/l; Sigma). D-[3H]mannoheptulose (final concentration: 0.1 mM) and [U-14C]sucrose (0.5 mM) in saline were administered through a sidearm syringe working at a flow rate of 75 µl/min from minutes 31 to 45. Likewise, dimethylsulfoxide (1.0 µl/ml) and, when required (minutes 40-55), cytochalasin B (20 µM; Sigma) in saline were also administered through a sidearm syringe working at the same flow rate.
The methods used to measure plasma insulin (9) and D-glucose (1) concentrations, as well as pancreatic insulin content (9) and release (9), were identical to those described in the cited references.
The effluent radioactivity was measured in samples (10 µl) of the perfusate by liquid scintillation. All results are expressed relative to the reference value found, within the same experiment, from minutes 41 to 45 inclusive. The radioactive content of pieces of pancreas (487 ± 38 mg wet wt; n = 18) was also measured by liquid scintillation in aliquot samples (50 µl) of homogenates prepared by mechanical homogenization and sonification (3 × 10 s) in 1.5 ml of H2O.
The analysis of secretory data was conducted as described elsewhere (4). Briefly, the output of insulin (µU/ml) was expressed in each individual experiment relative to the reference value found from minutes 25 to 33 inclusive. Such normalized values were then converted to absolute values after multiplication by the mean reference value found in the same group of rats (control or STZ rats). The analysis of secretory data takes into account the delay of 2-3 min attributable to the dead space of the perfusion system, as documented by the changes in effluent radioactivity. The statistical significance of changes in insulin output over selected periods of perfusion was assessed by linear regression.
All results, including those already mentioned, are expressed as means ± SE together with the number of determinations (n) or degree of freedom (df). The statistical significance of differences between mean values was assessed by Student's paired or unpaired t-test. Regression and covariance analysis was conducted according to Snedecor (18).
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RESULTS |
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Metabolic and hormonal data.
The control rats weighed 339 ± 18 g (n = 3).
The STZ rats lost 9.1 ± 2.9 g per day (n = 3) over the period of 3-4 days after the administration of STZ.
They then gained 38 ± 3 g (n = 3) while being treated with insulin for 3 days. At the time they were killed, their body weight was not significantly different from that of the
control rats (Table 1). The mean weight
of the pancreatic pieces eventually examined for their radioactive
content and the total weight of the pancreatic gland were also
not significantly different in control and STZ rats. Relative to paired
body weight, the wet weight of the pancreas averaged 0.486 ± 0.011 and 0.531 ± 0.052% in control and STZ rats, respectively
(n = 3 in both cases; P > 0.4).
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Secretory data. The perfusion pressure remained fairly constant throughout the experiments, with a mean value of 22 ± 1 mmHg (n = 6).
In the control rats, the output of insulin between minutes 25 and 33 inclusive averaged 1.17 ± 0.13 mU/ml (n = 27) and increased modestly but significantly (r = 0.4251; df = 25; P < 0.05) over this period (Fig. 1). Such was not the case (r = 0.3548; df = 22; P > 0.05) after introduction of D-mannoheptulose (0.1 mM; minutes 34-41 inclusive). In the presence of the heptose, but after introduction of cytochalasin B, a significant increase in insulin output was again recorded (r = 0.5781; df = 22; P < 0.01) between minutes 41 and 48 inclusive. A further and even steeper increase in secretory rate (r = 0.6970; df = 22; P < 0.001) was observed after removal of D-mannoheptulose but continued administration of cytochalasin B (minutes 48-55 inclusive). Covariance analysis indeed indicated that the slope of the regression line was about two times higher (P < 0.06) from minutes 48 to 55 (64.15 ± 14.07 10
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Radioactive data.
Figure 2 depicts the time course for
changes in 3H and 14C efflux from the pancreas
of either control or STZ rats infused for 15 min with
D-[3H]mannoheptulose (0.1 mM) and
[U-14C]sucrose (0.5 mM), the effluent radioactivity being
expressed relative to the mean reference value found in each experiment between minutes 41 and 45 inclusive. The patterns
observed with each of the two isotopes appeared virtually identical to
one another. Likewise, no obvious difference was found between control
and STZ rats.
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DISCUSSION |
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The STZ rats used in the present study displayed typical features of insulin deficiency, including loss of body weight, hyperglycemia, hypoinsulinemia, and an extremely low pancreatic insulin content. When treated with insulin, they gained weight and their glycemia decreased, albeit remaining higher than that found in control rats.
In the presence of 30 mM D-glucose, the output of insulin was much higher in control animals than in STZ rats. In the former animals, D-mannoheptulose prevented the increase in insulin output otherwise observed before and after administration of the heptose. This indicates that, despite the low concentration of D-mannoheptulose (0.1 mM) and high concentration of D-glucose (30 mM), the heptose antagonized, to a limited extent and in a reversible manner, the insulinotropic action of D-glucose.
As expected (19), the administration of cytochalasin B,
which aimed at avoiding the efflux of
D-[3H]mannoheptulose from the islet -cells
during the washout of extracellular radioactivity (10),
further increased glucose-stimulated insulin release in the pancreas of
control rats. Likewise, in the STZ rats, a biphasic increase in insulin
output was observed in response to the administration of cytochalasin B.
At the onset of D-[3H]mannoheptulose and
[U-14C]sucrose administration, the
3H/14C ratio in the effluent from the pancreas
progressively increased toward its equilibrium value. This is
consistent with the view that the space in the pancreatic gland readily
accessible to the 14C-labeled disaccharide, i.e., the
extracellular space, was smaller than that in which
D-[3H]mannoheptulose gained rapid access.
Moreover, the fact that, at the onset of
D-[3H]mannoheptulose and
[U-14C]sucrose administration, the
3H/14C ratio was lower in the experiments
conducted in pancreases from control rats than in those performed in
the pancreatic glands of STZ rats is consistent with the knowledge that
the major intracellular compartment for
D-[3H]mannoheptulose accumulation, i.e., the
islet -cells, was much larger in control animals than in STZ rats.
Unexpectedly, however, a mirror image was not observed at the end of
D-[3H]mannoheptulose and
[U-14C]sucrose administration. On the contrary, the
3H/14C ratio for effluent radioactivity,
instead of increasing, displayed a modest but significant fall between
minutes 49 and 52. Because this decrease in the
3H/14C effluent ratio took place during
cytochalasin B administration and was of comparable magnitude in
control and STZ rats, it appears unlikely to entail any major change in
the balance between D-[3H]mannoheptulose
influx into and efflux from the islet -cells. It could be
attributable, however, to a limited uptake and catabolism of
[U-14C]sucrose by some pancreatic cells, comparable to
those previously documented in both normal and tumoral islet cells
(3). Such a phenomenon may indeed account for the fall in
the 3H/14C ratio recorded at the end of the
experiments, if D-[3H]mannoheptulose was
rapidly washed out mainly from the extracellular space and some
14C-labeled metabolites from [U-14C]sucrose
were more slowly cleared from an intracellular compartment.
When corrected for extracellular contamination, as judged from the
radioactive data recorded with [U-14C]sucrose, the
intracellular distribution space of
D-[3H]mannoheptulose was not significantly
different from zero in the pancreas of STZ rats. The occasional
occurrence of a positive value for
D-[3H]mannoheptulose intracellular
distribution space in the STZ rats is likely to correspond to a limited
uptake of the tritiated heptose by acinar cells (15)
and/or remaining -cells. It was indeed recently documented that
1) D-[3H]mannoheptulose may be
taken up to a restricted extent by cells not equipped with GLUT-2,
e.g., parotid and pancreatic acinar cells (10, 15), and
that 2) the uptake of the tritiated heptose is not impaired
in islets exposed in vitro to STZ before incubation in the presence of
D-[3H]mannoheptulose (17).
In isolated islets or dispersed islet cells and in hepatocytes, the intracellular distribution space of D-[3H] mannoheptulose is comparable to that of D-[5-3H]glucose and represents about one-half of the intracellular 3HOH space (10). In the pancreas of the control rats, the intracellular distribution space of D-[3H]mannoheptulose averaged 5.42 ± 0.75 nl/mg. The latter value is close to that expected from the relative contribution of the endocrine pancreas to the total pancreatic mass (~1% in male rats with a body weight comparable to that of the control animals used in the present study; see Refs. 2 and 16) and the just-mentioned value for the intracellular distribution space of D-[3H]mannoheptulose in isolated islets relative to their intracellular 3HOH space.
It should be stressed that the value for the intracellular
D-[3H]mannoheptulose distribution space in
the perfused pancreas could conceivably be affected by such factors as
the length of exposure to the tritiated heptose and the concentration
of D-glucose present in the perfusate. We have indeed
previously shown that the uptake of
D-[3H]mannoheptulose by isolated islets and
pieces of pancreas represents a time-related phenomenon
(10). In the islets, it is increased, also in a
time-related manner, by D-glucose (10), which,
on the contrary, decreases
D-[3H]mannoheptulose uptake by erythrocytes
(15) and pieces of pancreas from STZ rats
(12). Such contrasting situations coincide with opposite
effects of D-glucose on the phosphorylation of
D- [3H]mannoheptulose by bovine heart
hexokinase vs. human -cell glucokinase (10). It is
precisely because of these prior observations that the present
experiments were conducted at a high concentration of
D-glucose (30 mM) and included a relatively short period of exposure to D-[3H]mannoheptulose (10 min
before the introduction of cytochalasin B).
The experimental design used in the present experiments could
conceivably be adapted to assess the relative contribution of insulin-producing -cells to the total mass of biological samples other than the perfused pancreas. For instance, we have recently observed that a comparable design can be used to measure the relative contribution of
-cells to the total mass of isolated islets
(13).
In conclusion, therefore, the present approach provides a means for
assessing the contribution of -cells to the total mass of the
pancreatic gland. It opens the way to comparable experiments in other
biological samples and provides further support to the proposal that a
suitably radiolabeled analog of D-mannoheptulose might
eventually represent an adequate tool for imaging and quantification of
the insulin-producing endocrine moiety of the pancreatic gland by a
noninvasive procedure.
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ACKNOWLEDGEMENTS |
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We are grateful to N. Bolaky for technical assistance and C. Demesmaeker for secretarial help.
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FOOTNOTES |
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This study was supported by a grant (3.4513.94) from the Belgian Foundation for Scientific Medical Research.
Address for reprint requests and other correspondence: W. J. Malaisse, Laboratory of Experimental Medicine, Brussels Free Univ., 808 Route de Lennik, B-1070 Brussels, Belgium (E-mail: malaisse{at}med.ulb.ac.be).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 November 2000; accepted in final form 26 March 2001.
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