Comparison of cellular and medium insulin and GABA content as markers for living
-cells
Chen Wang,
Zhidong Ling, and
Daniel Pipeleers
Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium
Submitted 26 May 2004
; accepted in final form 17 September 2004
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ABSTRACT
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Experimental and therapeutic use of islet cell preparations could benefit from assays that measure variations in the mass of living
-cells. Because processes of cell death can be followed by depletion and/or discharge of cell-specific substances, we examined whether in vitro conditions of
-cell death resulted in changes in tissue and medium content of insulin and of
-aminobutyric acid (GABA), two
-cell-specific compounds with different cellular localization and turnover. Exposure of rat purified
-cells to streptozotocin (5 mM, 120 min) or to the nitric oxide donor GEA-3162 (GEA; 50 µM, 120 min) caused 80% necrosis within 24 h; at the end of this period, cellular insulin content was not significantly decreased, but cellular GABA content was reduced by 70%; when cultured at basal glucose (6 mM), the toxin-exposed cells did not discharge less insulin but released 80% less GABA in the period 824 h. As in rat
-cell purification, GABA comigrated with insulin during human islet cell isolation. Twenty-four hours after GEA (500 µM, 120 min), human islet cell preparations exhibited 90% dead cells and a 45 and 90% reduction, respectively, in tissue insulin and GABA content; in the period 924 h, insulin discharge in the medium was not reduced, but GABA release was decreased by 90%. When rat
-cells were cultured for 24 h with nontoxic interleukin (IL)-1
concentrations that suppressed glucose-induced insulin release, cellular GABA content was not decreased and GABA release increased by 90% in the period 824 h. These data indicate that a reduction in cellular and medium GABA levels is more sensitive than insulin as a marker for the presence of dead
-cells in isolated preparations. Pancreatic GABA content also rapidly decreased after streptozotocin injection and remained unaffected by 12 h of hyperglycemia. At further variance with insulin, GABA release from living
-cells depends little on its cellular content but increases with IL-1
-induced alterations in
-cell phenotype.
islet; diabetes;
-cell death;
-aminobutyric acid
PROCESSES OF CELL DEATH can be associated with depletion and/or discharge of cell-specific substances. Cell losses can then be monitored through assays of these compounds in tissue and/or blood. Such method is not yet available to detect destruction of pancreatic
-cells during development of diabetes, but release of insulin-mRNA might qualify as a marker for a massive destruction of a
-cell graft (22). There is not an established method for quantifying the mass of living
-cells in isolated tissue fractions. This lack makes it difficult to assess the therapeutic potential of islet cell preparations and to compose grafts accordingly. Tissue insulin content and secretory activity have been used as quality control tests in islet transplantation (9, 10, 13, 21), but their significance as indexes for the living
-cell mass needs to be further validated. Living
-cells can indeed markedly vary in their insulin content and can variably degranulate during isolation and culture procedures; furthermore, dead
-cells may still contain significant amounts of insulin, in particular in isolated preparations where cellular debris is more slowly cleared than in vivo. In view of the relatively slow turnover of the cellular insulin pool, and the slow insulin diffusion of the crystalline packages, it appears conceivable that the tissue insulin content is not a sensitive index for the living
-cell mass. The present study examined this question in vitro, using previously described conditions of
-cell death and dysfunction (3, 12, 18). It reports how these conditions induce changes in cellular and extracellular insulin and compares these effects with those on (extra)cellular GABA, another
-cell-specific compound (5, 14, 17, 23, 29) that is characterized by a relatively small storage compartment and a higher turnover rate (26, 30).
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MATERIALS AND METHODS
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Preparation and culture of rat pancreatic cells.
Adult male Wistar rats were bred according to Belgian regulations of animal welfare. Rat islet cells were purified as previously described (19). Briefly, after collagenase (Roche Diagnostics) digestion and elutriation of the pancreatic digest, the particles <100 µm in diameter were used as exocrine fraction, and the fraction with larger particles served for handpicking of islets under the dissection microscope. Isolated islets were dissociated in calcium-free medium containing trypsin (Boehringer) and DNase (Boehringer) and then separated into single
-cells (90% pure) and islet non-
-cells (at least 70%
-cells) by autofluorescence-activated cell sorting using cellular light-scatter and FAD(H) autofluorescence as discriminating parameters (19). Purified
-cells were reaggregated for 2 h in a rotary shaking incubator at 37°C (12) and then statically cultured for 16 h in Hams F-10 medium (GIBCO) supplemented with 2 mM glutamine (Cambrex), 6.1 mM glucose, 0.5% (wt/vol) charcoal-extracted BSA (type V, RIA grade; Boehringer Mannheim), 0.075 g/l penicillin, and 0.1 g/l streptomycin. After this overnight culture period,
-cell aggregates were exposed for 2 h to streptozotocin (STZ, 50 mM; Sigma) or nitric oxide (NO) donor GEA-3162 (GEA, 50 µM; Alexis), washed, and further cultured for an additional 24 h without toxins. In the second series of experiments, overnight-cultured
-cell aggregates were cultured for 24 h in the presence or absence of 30 U/ml recombinant human interleukin-1
(IL-1
, sp act: 19 U/ng), a gift kindly provided by McKesson HBOC C & B Services, with or without 1 mM NG-methlthyl-L-arginine (L-NMA; Sigma). The IL-1
concentration of 30 U/ml was selected from previous studies (12). In all experiments, culture medium was collected for measuring its
-aminobutyric acid (GABA) and insulin content and nitrite production (IL-1
experiments). Different tissue fractions and isolated cells were also analyzed for their GABA and insulin content, and
-cell aggregates were examined for the degree of cell death.
Preparation and culture of human pancreatic cells.
Human pancreata were obtained from organ donors (age 46 ± 6 yr) at European hospitals affiliated with Eurotransplant Foundation (Leiden) or with the Surgical Hospital at Helsinki University (Dr. K. Salmela). They were processed by the
-cell Bank of the Juvenile Diabetes Research Foundation Center for
-Cell Therapy in Brussels, with the purpose of preparing islet cell grafts for a clinical trial in diabetic patients. During this procedure, the endocrine-enriched fraction is separated from exocrine-enriched fractions (11). When isolated islet cell fractions did not fulfill the quality control criteria for transplantation, they could be made available to approved research projects when fulfilling the guidelines of Eurotransplant and of the ethics committee of the Vrije Universiteit Brussel (VUB); these guidelines also applied to use of exocrine fractions.
The exocrine cell preparation (composed of 5565% acinar cells, 3040% duct cells, and <3% endocrine cells) was lysed for GABA and insulin measurement. Islet cell preparations were precultured for 12 days in Hams F-10 medium containing 0.075 g/l penicillin, 0.1 g/l streptomycin, 6.1 mM glucose, 2 mM glutamine, 0.5% BSA, 2% human serum, and 2 mM nicotinamide (Biowhittaker) and then for 411 days in the above medium but without serum and nicotinamide (11). After this preculture period, islet cell preparations (4070%
-cells, 1025%
-cells) were exposed for 2 h to the NO donor GEA (500 µM), washed, and cultured for an additional 24 h without GEA. Medium and cell samples were collected at different time points and analyzed for their GABA and insulin content. Cells were also used for assays of viability (7) and DNA content (4).
STZ treatment in vivo.
Male Wistar rats (250300 g) were injected intravenously with STZ [60 mg/kg in 0.1 M citrate buffer (pH 4.5)] and killed 4, 12, or 24 h later. Pancreata were extracted for insulin, GABA, and DNA assays.
GABA, insulin, and nitrite assays.
GABA was measured by HPLC (27) in filtered media and pancreatic tissue/cell extracts after sonication in PBS with 0.1% BSA and centrifugation. Insulin was assayed in culture media and in acetic acid extracts of tissue and cells (19). NO production was derived from the nitrite content of culture media (6).
Viability assays.
Living and dead cells were visualized by fluorescence microscopy after staining with Hoechst 33324 (HO324; Sigma) and propidium iodide (PI; Sigma) (6). Living cells were identified by their intact nuclei with blue fluorescence (HO324) and dead cells by pink fluorescent nuclei (HO324 + PI). We also used an MTT viability assay (MTT is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma; see Refs. 2 and 15), in which cells were incubated with MTT (0.5 mg/ml) for 2 h, washed with PBS, and then lysed with DMSO before measuring absorbance at 595 nm in a Benchmark Microplate Reader (Bio-Rad). A count of the percent living (HO-pos) and dead (HO + PI-pos) cells in cell aggregates is not as accurate as in single cell preparations (6), but it allows detection of the presence of massive cell death, as was intended in the conditions with the toxins. This was then confirmed with the MTT assay, which has the advantage of measuring changes in viability of the entire cell population but which is known to be influenced by alterations in mitochondrial activity in living cells, a variable that should not be neglected in
-cells.
Data expression and statistical analysis.
Insulin and GABA were expressed as nanomoles per microgram DNA or picomoles per 103 cells at the start of experiments. Data represent means ± SE of n no. of independent experiments. Statistical significance of differences was determined using either the Students t-test or ANOVA.
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RESULTS
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GABA and insulin content in pancreatic fractions.
Isolation of islets from the rat and human pancreas resulted in >30-fold enrichment in tissue insulin content, as shown by the increase in the insulin-over-DNA ratio (Table 1). The discarded exocrine fraction still contained insulin, but the hormone-over-DNA ratio was 200-fold lower than that in the islet fraction and 5-fold lower than in the intact pancreas. Purification of
-cells from the rat islet fraction further increased this ratio (Table 1), whereas the ratio on the separated islet non-
-cell fraction was only threefold lower than in the islets, reflecting its contamination with 1520%
-cells.
Similar changes were seen when tissue GABA content was measured during isolation and purification of pancreatic fractions (Table 1). That GABA comigrates with insulin and thus with
-cells is illustrated by the constant GABA-over-insulin ratio during purification of rat islets into
- and non-
-cell fractions. This ratio also remained constant during islet isolation from the human pancreas. However, it decreased twofold during islet isolation from the rat pancreas, which may result from the presence of GABA-containing non-
-cells, such as neurons, in the intact tissue or from preferential discharge of GABA vs. insulin from
-cells during the isolation procedure.
Comparison of GABA and insulin content during
-cell death.
A 2-h exposure of rat
-cells to STZ (5 mM) or to the NO donor GEA (50 µM) did not immediately induce cell death (<20% dead cells in the PI-HO324 viability assay at 2 h; Fig. 1) but resulted in >80% dead cells after 24 h, as determined by fluorescence staining with PI and HO324 (Fig. 1) and by the MTT assay (MTT reduction was decreased by >90% in STZ-treated cells). At the latter time point, cellular insulin content was not decreased in GEA cells, and only marginally decreased in STZ cells (30%, P > 0.05; Table 2), with more insulin discharged in the 24-h medium (Table 2). These data indicate that an in vitro process of dying
-cells cannot be detected through a reduction in cellular or medium insulin content, at least not within 24 h; they also question the significance of these measurements as indexes of living
-cells.
On the other hand, cellular GABA content was significantly decreased 24 h after the 2-h treatment with STZ (68% reduction) or GEA (74% reduction; Table 2); this was associated with a marked decrease of GABA discharge in the medium (Table 2). Cellular and medium GABA thus rapidly decrease during and after
-cell death and can therefore be considered as indexes for living
-cells. When measured within 4 h after exposure to the toxins, no reductions were noticed: cellular GABA was 10.6 ± 3.7 pmol/103 STZ cells vs. 7.6 ± 2.2 pmol/103 control cells, and medium GABA was even increased (Table 3). However, in the period of massive cell death (824 h after the 2-h drug treatment), GABA release in the medium fell to 1020% of control levels (Table 3). In contrast, no reduction in insulin discharge was detected during this period (Table 3).
Similar observations were made with human
-cell preparations that had been exposed to GEA. At 500 µM, this toxin caused 30% necrotic cells after 2 h and 90% after a subsequent 24-h culture in its absence (Fig. 2 and >90% decrease in MTT reduction assay). At the end of this culture period, cellular GABA content was only 7% of control values and 24-h GABA discharge 30% of control (Table 2). On the other hand, cellular insulin content was still 55% of control values, and 24-h insulin discharge was not decreased. When 24-h GABA discharge was examined over shorter time periods, it was increased during GEA exposure, but then rapidly fell to 10% of control levels in the 8- to 24-h period (Table 4); in none of the time periods was a decrease seen in medium insulin levels.

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Fig. 2. Fluorescence micrographs after double staining with PI and HO342 of human islet cells. Human islet cells were first exposed to 500 µM GEA for 2 h and then cultured for 24 h without GEA. Although the majority of control cells was viable (blue-stained nuclei), GEA-treated preparations consisted of predominantly dead cells (pink-stained nuclei).
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Pancreatic insulin and GABA content after
-cell destruction in vivo.
After STZ injection (4 h), pancreatic insulin and GABA content were comparable to control values (Table 5). After 12 h, GABA content had decreased to 22% of control and insulin content to 43%. After 24 h, GABA content was comparable to the 12-h value (15% of control, P > 0.05, vs. 12 h), but insulin content was now only 2% of control. It is to be noticed that 24 h post-STZ, animals had been hyperglycemic for at least 12 h (Table 5).
Comparison of (extra)cellular GABA and insulin content during cytokine-induced
-cell dysfunction.
Exposure of rat
-cells to IL-1
for 24 h did not cause cell death during this period (Fig. 3) but suppressed their insulin release; cellular insulin content was not affected (Tables 6 and 7). This effect was NO dependent, since it was associated with an increased NO production and did not occur in the presence of the inducible NO synthase blocker, L-NMA (Table 7).
IL-1
-exposed
-cells exhibited a higher GABA content (15 ± 1 pmol/103 cells vs. 11 ± 2 pmol/103 control cells), but the difference was not statistically significant. They released more GABA in the medium, both in the 4- to 9-h and the 9- to 24-h periods during which NO production was also elevated (Table 7). This increased GABA release was NO dependent, since it was not detected in the presence of L-NMA (Table 7). At the end of the 24-h exposure, total GABA recovery (cells + medium) was 50% higher (107 ± 5 pmol/103 cells) than in control cells (72 ± 9 pmol/103 cells, P < 0.01).
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DISCUSSION
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Insulin qualifies as a
-cell marker in view of its cell specificity, its high cellular content, and its easy detection in extracts and sections. However, our data indicate that it is an insensitive index for the mass of living
-cells. When isolated rat or human
-cells were exposed to toxins that cause massive necrosis within 24 h, their insulin content was not significantly decreased at the end of this period. Measurement of tissue insulin is thus unlikely to distinguish between living and dead
-cells. It has therefore limited value for assessing the functional
-cell mass in fractions prepared for transplantation. The period between brain death of the pancreas donor and implantation of an isolated islet cell preparation extends over 24 h and longer and exposes the tissue to variable potentially cytotoxic conditions. It will thus be associated with variable degrees of
-cell death in the isolated fractions, and we therefore need ways to monitor the number of living
-cells. Likewise, experiments on isolated islets also occur after and during conditions that affect the proportion of living and dead cells; islet insulin content is therefore not a reliable standard for expressing and comparing
-cell activities. Furthermore, the insulin content of living cells can markedly and rapidly vary with environmental conditions, adding another limitation to tissue insulin content as a measure of the living
-cell mass.
There is so far not yet a simple straightforward way for quantifying the number of living
-cells in isolated tissue or islet fractions. We have developed a method using tissue DNA content, mean DNA content per cell, and percent insulin-positive cells in the tissue (8). It is used to quantify
-cell numbers in grafts for our transplantation trial (9) and was also adopted in the Edmonton series (25). By determining the percent dead cells in electron micrographs, we can estimate the number of living
-cells, but this technique is not sensitive and is difficult to apply on large series.
The present study indicates that cellular and medium GABA content are sensitive and cell-specific parameters of living
-cells in pancreatic cell fractions. Although this compound is also detected in pancreatic nerves (24), it is quantitatively most predominant in the
-cell population, as indicated by its comigration with insulin during islet and
-cell purification and by the marked decline in pancreatic GABA content shortly after injection of the
-cell toxin STZ in rats. When expressed in molar units, the
-cell content in GABA is similar to that in insulin for freshly isolated preparations and becomes higher for cultured cells as a result of their degranulation during culture. Compared with insulin, the marker function of cellular GABA is much less influenced by conditions of degranulation, which is consistent with earlier observations that GABA is not coreleased with insulin (16, 20, 26). On the other hand, it is much more dependent on the living state of the cells, as shown by the rapid and marked decline in cellular GABA content during necrosis of the cells, whereas cellular insulin remained little affected. This diversity is explained by differences in storage of these
-cell compounds. First, the hourly rate of insulin synthesis represents only a small proportion of the cellular insulin content (<2% at 7.5 mM glucose; see Ref. 28), whereas that of GABA is estimated at 2025% of its cellular content (26, 30, and the present study); consequently, a block in synthetic activities, as will occur in damaged cells, will more rapidly deplete the cellular GABA pool. Second, insulin is stored as crystalline complex in secretory vesicles from which it only slowly diffuses after membrane damage, whereas the major part of the smaller GABA molecule resides in an extravesicular space (1) from which its discharge in the medium can be expected to be much more rapid.
The in vitro observed changes in cellular GABA and insulin content after STZ-induced
-cell death are only partly reproduced in vivo. After injection of the toxin (12 h), pancreatic GABA content was reduced by 80%, which is consistent with massive
-cell destruction and the concomitant state of hyperglycemia; no further reduction was measured 12 h later, which is compatible with STZs short-lived action, also indicating that 12 h of hyperglycemia did not affect pancreatic GABA content. Whereas tissue insulin content was only moderately (30%) decreased 24 h after in vitro exposure, it was reduced by >95 percent at 24 h after injection. A decline was noted after 12 h but became more pronounced during the following 12 h, suggesting that the hyperglycemic state depleted insulin stores of surviving
-cells. Further studies will now investigate whether GABA can serve as a marker for these surviving
-cells.
Medium GABA levels also rapidly decline after
-cell death, as can be expected from their high dependency on the rate of GABA production (26) and from the rapid depletion of cellular GABA. This was not the case for medium insulin levels. In fact, medium insulin was increased during the first 8 h after STZ exposure, probably as a result of diffusion of the hormone from damaged cells. However, medium insulin measurements after GEA exposure indicated that this is not a consistent finding; furthermore, medium insulin levels depend on the size of the cellular hormone store and can also be increased after stimulation of living
-cells. GABA release in the medium can thus be considered as a more reliable index for living
-cells than insulin release. It can, however, also reflect regulatory influences on living cells (26, 30), just as the rate of insulin release, but it will be less dependent on the size of its cellular store.
Because stored GABA exhibits a >20-fold higher fractional release than stored insulin, medium GABA levels can also serve as a marker for changes in
-cell function. This is illustrated in rat
-cells that were exposed for 24 h to IL-1
. As previously reported (12), this condition did not induce
-cell death but suppressed glucose-stimulated insulin synthesis and release. It was now found to increase medium GABA levels by 50%, which is consistent with a lack of cytotoxicity and which might express the change in phenotype that we described earlier in this condition (12). Like other features of this phenotypic alteration (12), the IL-1
-induced increase in GABA release was completely dependent on NO production.
In conclusion, extracellular and cellular GABA levels can serve as markers for living
-cells in isolated pancreatic and islet cell fractions. Cellular and medium GABA rapidly and markedly decline after
-cell death. GABA release from living
-cells depends little on cellular GABA content but can vary with the phenotype of the cells. Insulin measurements in medium and cells were found less adequate for these purposes. Further studies are needed to identify the relevance of GABA measurements in in vivo models.
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GRANTS
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This study was supported by grants from the Belgian Science Policy (Interuniversity Attraction Pole P5/17), Juvenile Diabetes Research Fund (Grant no. 4-2001-434), and the Belgian Fund Scientific Research-FWO (Grant no. G0375.00).
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ACKNOWLEDGMENTS
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We thank Drs. Y. Michotte, S. Sarre, and I. Smolders (Farmaceutisch Instituut, Brussels Free University-VUB) for help in HPLC determinations and R. De Proft for technical assistance.
Part of this work was presented at the 18th International Diabetes Federation Congress, Paris, Aug. 2429, 2003.
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FOOTNOTES
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Address for reprint requests and other correspondence: D. Pipeleers, Diabetes Research Center, Brussels Free Univ.-VUB, Laarbeeklaan 103, B-1090 Brussels, Belgium (E-mail: Daniel.Pipeleers{at}vub.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.
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