Assessment of islet beta -cell mass in isolated rat pancreases perfused with D-[3H]mannoheptulose

Laurence Ladrière, Viviane Leclercq-Meyer, and Willy J. Malaisse

Laboratory of Experimental Medicine, Brussels Free University, B-1070 Brussels, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -cells to the total mass of the pancreatic gland.

rat pancreas perfusion; D-[3H]mannoheptulose; streptozotocin-induced diabetic rats


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -cells to the total mass of the pancreatic gland.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Metabolic, hormonal and radioactive data

In the STZ rats, the plasma D-glucose averaged, before insulin treatment, 32.16 ± 1.40 mM, as distinct (P < 0.001) from a mean value of 8.77 ± 0.39 mM in the control animals (n = 3 in both cases). The insulin treatment lowered the plasma D-glucose concentration by 17.45 ± 2.34 mM (n = 3; P < 0.02). Nevertheless, the plasma D-glucose concentration remained higher (P < 0.02) in the insulin-treated STZ rats than in the control animals (Table 1).

Before insulin treatment, the plasma insulin concentration was much lower (P < 0.02) in STZ rats than in control animals (Table 1). It increased by 43.6 ± 9.3 µU/ml (n = 3; P < 0.05) after insulin treatment of the STZ rats, reaching a level comparable to that found in control rats. The insulinogenic index (i.e., the paired ratio between plasma insulin and D-glucose concentrations), which averaged 0.35 ± 0.06 U/mol (n = 3) in the STZ rats before insulin treatment, increased (P < 0.02) to 3.85 ± 0.89 U/mol (n = 3) after insulin treatment, the latter value remaining somewhat lower, albeit not significantly so, than that found in control animals (6.37 ± 0.98 U/mol; n = 3).

The insulin content of the pancreas, measured in pancreatic pieces (341 ± 27 mg; n = 6), was much lower in STZ rats than in control animals (Table 1).

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 10-3/min; regression coefficient ± its SD) than from minutes 41 to 48 (31.17 ± 9.38 10-3/min). Such successive changes do not occur in pancreases from control rats perfused in the sole presence of a high concentration of D-glucose, in which case the output of insulin steadily increases over at least the first 80 min of the experiments (8).


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Fig. 1.   Insulin output from the pancreas of control and streptozotocin-induced diabetic (STZ) rats perfused throughout the experiments at 30.0 mM D-glucose. D-mannoheptulose (0.1 mM) and cytochalasin B (20 µM) were administered from minute 31 to minute 50 and from minutes 41 to 55 inclusive, respectively. No correction was introduced for the dead space of the perfusion device. Mean values (±SE) are expressed as µU/ml and refer to 3 individual experiments in each case. In control rats, regression lines from minutes 25 to 33, 34 to 41, 41 to 48, and 48 to 55 are indicated. In the STZ rats, the peak-shaped stimulation of insulin release recorded after introduction of cytochalasin B and the later progressive increase in insulin output are both illustrated.

The output of insulin between minutes 25 and 33 was two orders of magnitude lower (P < 0.001) in STZ rats (26.1 ± 4.3 µU/ml; n = 27) than in the control rats (see Secretory data). It remained fairly stable before and after introduction of D-mannoheptulose. A peak-shaped increase in insulin release was observed, however, at minutes 45-46, shortly after introduction of cytochalasin B. For instance, at minute 45, the output of insulin was 36.4 ± 7.9% (n = 3; P < 0.05) higher than the paired value recorded just before introduction of cytochalasin B (minute 43). A comparable phenomenon was previously observed (5), under comparable experimental conditions and at the same time, in pancreases from either control or STZ rats infused at a lower concentration of D-glucose (8.3 mM). In the STZ rats, as in the control rats, a significant increase in insulin output (r = 0.5287; df = 22; P < 0.01) was also recorded between minutes 48 and 55 inclusive, i.e., in the presence of cytochalasin B and after removal of D-mannoheptulose (Fig. 1).

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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Fig. 2.   3H outflow (top) and 14C outflow (bottom) from pancreases of control (left) and STZ (right) rats perfused for 15 min with both D-[3H]mannoheptulose and [U-14C]sucrose. Mean values (±SE) are expressed relative to the paired reference value recorded from minutes 41 to 45, inclusive, and refer to 3 individual experiments in all cases.

As shown in Fig. 3, the fractional turnover rate of radioactive material in the pancreatic parenchyma, as judged from either the differences between effluent radioactivity from minute 35 to minute 38 and the equilibrium reference value (minutes 41-45 inclusive) or the measurements made from minute 50 to minute 53, yielded comparable values for 3H and 14C, whether in control or STZ rats, with an overall mean paired 3H-to-14C (3H/14C) ratio of 100.4 ± 1.6% (n = 12). The absolute value for such a fractional turnover rate averaged 0.508 ± 0.031 and 0.585 ± 0.041 min-1 in control and STZ rats, respectively (n = 12 in both cases; P > 0.15).


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Fig. 3.   Radioactive data collected at the onset (minutes 35-38; top) and end (minutes 50-53; bottom) of administration of both D-[3H] mannoheptulose and [U-14C]sucrose to the pancreas of either control rats (left) or STZ rats (right). Top: values for 3H () and 14C (open circle ) outflow refer to the difference between experimental readings at each time point and the equilibrium value recorded from minutes 41 to 45 inclusive. Bottom: values for 3H () and 14C (open circle ) outflow refer to experimental readings recorded at each time point. In all cases, data are expressed relative to the above-mentioned equilibrium value, taken as unity. Mean values (±SE) refer to 3 individual experiments.

During the first few minutes of D-[3H]mannoheptulose and [U-14C]sucrose administration, however, the paired 3H/14C ratio for effluent radioactivity increased modestly but significantly (Fig. 4, top). This increase appeared more marked in control animals than in STZ rats. Thus, at minute 33, the 3H/14C ratio was significantly lower than unity (P < 0.05) in control animals, but not so in STZ rats. However, the comparison between the mean values reached at minute 33 in the control and STZ rats yielded a P value only lower than 0.10. 


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Fig. 4.   Paired ratio for outflow of 3H and 14C, both expressed relative to the reference value recorded from minutes 41 to 45 inclusive, from pancreas of control (left) and STZ (right) rats at the onset (top) and end (bottom) of D-[3H]mannoheptulose and [U-14C]sucrose administration. Mean values (±SE) refer to 3 individual experiments in all cases.

Unexpectedly, when the administration of D-[3H] mannoheptulose and [U-14C]sucrose was halted, the changes in the paired ratio between 3H and 14C outflow did not represent the mirror image of those recorded at the onset of the experiments. On the contrary, a modest but significant fall in 3H/14C outflow was observed (Fig. 3, bottom). This phenomenon was comparable in control and STZ rats. Thus the mean values reached between minutes 50 and 52 inclusive were, in control and STZ rats respectively, 3.58 ± 1.09 and 4.71 ± 1.12% lower than unity (n = 9 and P < 0.02 or less in both cases), these two percentages not being significantly different from one another (P > 0.4).

The radioactive content of the pancreatic samples (dpm/mg wet weight) obtained at the end of the perfusion, when divided by the 3H and 14C content (dpm/nl) of the last effluent sample (minute 55), yielded in control rats a higher apparent distribution space (nl/mg) for D-[3H]mannoheptulose than for [U-14C]sucrose, with a mean paired difference of 122.6 ± 12.0 nl/mg (n = 9). Such a difference overestimates, however, the intracellular distribution space of the tritiated heptose, because its uptake by pancreatic cells took place at a time when the extracellular concentration of D-[3H] mannoheptulose was much higher than after the washout of extracellular space during the late phase of the experiments. It was corrected, therefore, by the corresponding paired ratio in 3H outflow at minute 55/minutes 41-45, i.e., 4.33 ± 0.37% (n = 3). It then averaged no more than 5.42 ± 0.75 nl/mg (n = 9). In the STZ rats, the corresponding mean value, i.e., 0.46 ± 0.70 nl/mg (n = 9), was not significantly different from zero (P > 0.5).

The paired ratio between the 14C content of the pancreatic samples (dpm/mg) and that of the last effluent samples (dpm/nl) yielded values not significantly different (P > 0.8) in control and STZ rats (Table 1). The overall mean value for such a variable (842.7 ± 76.7 nl/mg; n = 18) was higher than the extracellular space of the pancreatic gland, as expected from the fact that a sizeable fraction of the perfusion flow (~1.6 ml/min) corresponds to liquid having no access to the pancreatic parenchyma. For instance, in the case of 3HOH, such a fraction represents more than one-half of the perfusion flow (5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -cells to the total mass of isolated islets (13).

In conclusion, therefore, the present approach provides a means for assessing the contribution of beta -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.


    ACKNOWLEDGEMENTS

We are grateful to N. Bolaky for technical assistance and C. Demesmaeker for secretarial help.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bergmeyer, HU, and Berndt E. Glucose determination with glucose oxidase and peroxidase. In: Methods of Enzymatic Analysis. New York: Academic, 1974, p. 1205-1215.

2.   Davis, BJ, and Smith PH. Effects of substantia nigra lesions on the volumes of A, B and D cells and the content of insulin and glucagon in the rat pancreas. Diabetologia 28: 756-762, 1985[ISI][Medline].

3.   Giroix, MH, Sener A, and Malaisse WJ. Artifactual and true uptake of labelled sucrose by rat pancreatic islet cells. Comp Biochem Physiol 85A: 289-296, 1986[ISI].

4.   Jijakli, H, Ulusoy S, and Malaisse WJ. Dissociation between the potency and reversibility of the insulinotropic action of two meglitinide analogues. Pharmacol Res 34: 105-108, 1996[ISI][Medline].

5.  Ladrière L, Leclercq-Meyer V, and Malaisse WJ. D-[1-14C]mannoheptulose uptake by the perfused pancreas of control and streptozotocin-induced diabetic rats. Diabetes Res. In press.

6.   Ladrière, L, Malaisse-Lagae F, and Malaisse WJ. Pancreatic uptake of 65Zn in control and streptozotocin-injected rats. Med Sci Res 28: 43-44, 2000.

7.   Leclercq-Meyer, V, Kadiata MM, and Malaisse WJ. Stimulation by 2-deoxy-D-glucose tetraacetates of hormonal secretion from the perfused rat pancreas. Am J Physiol Endocrinol Metab 276: E689-E696, 1999[Abstract/Free Full Text].

8.   Leclercq-Meyer, V, Marchand J, and Malaisse WJ. The role of calcium in glucagon release. Studies with verapamil. Diabetes 27: 996-1004, 1978[Abstract].

9.   Leclercq-Meyer, V, Marchand J, Woussen-Colle MC, Giroix MH, and Malaisse WJ. Multiple effects of leucine on glucagon, insulin and somatostatin secretion from the perfused rat pancreas. Endocrinology 116: 1168-1174, 1985[Abstract].

10.   Malaisse, WJ. On the track to the beta-cell. Diabetologia 44: 393-406, 2001[ISI][Medline].

11.   Malaisse, WJ, Courtois P, Kadiata MM, and Sener A. GLUT2-mediated transport of D-mannoheptulose: a tool for imaging of the endocrine pancreas? (Abstract) Diabetes 49 Suppl 1: A418, 2000[ISI].

12.  Malaisse WJ, Doherty M, Kadiata MM, Ladrière L, and Malaisse-Lagae F. Pancreatic fate of D-[3H]mannoheptulose. Cell Biochem Funct In press.

13.   Malaisse, WJ, and Ladrière L. Assessment of B-cell mass in isolated islets exposed to D-[3H]mannoheptulose. Int J Mol Med 7: 405-406, 2001[ISI][Medline].

14.   Malaisse, WJ, Leclercq-Meyer V, and Malaisse-Lagae F. Methods for the Measurement of Insulin Secretion. Peptide Hormones: A Practical Approach. Oxford, UK: IRL, 1990, p. 211-231.

15.   Malaisse, WJ, Ramirez R, Courtois P, Rasschaert J, and Sener A. D-[3H]mannoheptulose uptake and phosphorylation in different cell types (Abstract). Eur J Physiol 441: R103, 2001.

16.   McEvoy, RC. Changes in the volumes of the A-, B- and D-cell populations in the pancreatic islets during the postnatal development of the rat. Diabetes 30: 813-817, 1981[Abstract].

17.  Sener A and Malaisse WJ. Unaltered D-[3H]mannoheptulose uptake after prior in vitro exposure of rat pancreatic islets to streptozotocin. Diabetes Res In press.

18.   Snedecor, GW. Statistical Methods. Ames: The Iowa State University Press, 1956.

19.   Van Obberghen, E, Somers G, Devis G, Vaughan GD, Malaisse-Lagae F, Orci L, and Malaisse WJ. Dynamics of insulin release and microtubular-microfilamentous system. I. Effect of cytochalasin B. J Clin Invest 52: 1041-1051, 1973[ISI][Medline].


Am J Physiol Endocrinol Metab 281(2):E298-E303
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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