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Prolonged Exposure of Human ß-Cells to High Glucose Increases Their Release of Proinsulin during Acute Stimulation with Glucose or Arginine¹

Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium

Address all correspondence and requests for reprints to: Dr. D. Pipeleers, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: dpip{at}mebo.vub.ac.be

	Abstract

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The disproportionate hyperproinsulinemia in type 2 diabetes has been attributed to either a primary ß-cell defect or a secondary dysregulation of ß cells under sustained hyperglycemia. This study examines the effect of a 10- to 13-day exposure to 20 mmol/L glucose on subsequent proinsulin and insulin release by human islets isolated from nondiabetic donors. Compared to control preparations kept at 6 mmol/L glucose, the high glucose cultured ß-cells released more proinsulin and less insulin during perifusion at 5, 10, or 20 mmol/L glucose. The lower amounts of secreted insulin resulted from a marked reduction in cellular insulin content (5-fold lower than in controls). The higher amount of secreted proinsulin is attributed to the sustained state of cellular activation that is known to occur after prolonged exposure to high glucose levels. This activated state of the ß-cell population is also held responsible for its higher secretory responsiveness to 5 mmol/L arginine at a submaximal (5 mmol/L) glucose concentration (8-fold higher proinsulin levels than in the control population). It results, together with the reduction in cellular insulin content, in 7- to 10-fold higher proinsulin over insulin ratios in the medium; at 5 mmol/L glucose, this extracellular ratio is similar to that in the cells. These data add direct support to the view that a disproportionate hyperproinsulinemia can result from a sustained activation of human ß-cells after prolonged exposure to elevated glucose levels.

	Introduction

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PROINSULIN levels are increased in patients with type 2 diabetes (1, 2, 3, 4) as well as in subjects at risk for this disease (5, 6, 7, 8). This may result from a delay in proinsulin processing and/or from an accelerated release of immature granules, which are known to contain higher proportions of proinsulin (9, 10, 11). The disproportionate elevation of circulating proinsulin has been related to the degree of impairment in the ß-cell secretory capacity (12), thus supporting the view that it reflects a primary ß-cell defect (13, 14). There is also evidence that this alteration is the consequence of an increased secretory demand, like that occurring in persistent hyperglycemia (9). In rat models of hyperglycemia, both islet tissue and plasma were indeed characterized by increased proportions of proinsulin over insulin (15, 16, 17, 18). In hemipancreatectomized patients, hyperproinsulinemia was also found to develop as a result of an increased ß-cell demand (19). Chronically elevated glucose levels do not necessarily impair ß-cell functions in the sense of reducing their activity. Prolonged exposure of isolated rat or human ß-cells to high glucose concentrations was shown to induce a hyperactivated state, which is maintained after subsequent incubations at lower glucose levels (20, 21). In the present work, we examine whether this condition leads to a disproportionately increased release of proinsulin by human ß-cells.

	Materials and Methods

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Preparation of human islet cells

Pancreata were obtained from organ donors at European hospitals affiliated with ß Cell Transplant (Brussels, Belgium) and Eurotransplant (Leiden, The Netherlands) (22). After collagenase digestion, the tissue suspensions were gently dispersed and submitted to gradient centrifugation (21). The fractions enriched in islet cell clumps were isolated and cultured in serum-free medium as described previously (21). Preparations for this study were precultured for 2–3 days before distribution over two dishes containing Ham’s F-10 medium with 1% BSA, 2 mmol/L glutamine, and either 6 or 20 mmol/L glucose. They were then further cultured for 10–13 days, with medium replacements every other day. At the end of this culture period, the two preparations were collected from the dishes and washed before samples were taken for immunocytochemistry and for DNA (21), proinsulin, and insulin assays; the rest of the material was used for perifusion. The cellular composition of the test fractions and their total number of ß-cells were determined as previously described (21). At the time of perifusion, the preparations contained 76 ± 2% endocrine cells (57 ± 2% ß-cells and 21 ± 2% -cells); dead cells represented less than 6%. They are called islet cell preparations instead of islets because the isolation and culture procedures resulted in a progressive dispersing of the initial islet structures, a step that we consider useful for enrichment of living endocrine cells.

Perifusion of human islet cells

A multiple microchamber module (Endotronics, Inc., MN) with build-in pump and thermostat was used for perifusion of the human islet cells (23). Cultured islet preparations were loaded on preformed Bio-Gel P2 columns (Bio-Rad Laboratories, Inc., Richmond, CA) and perifused with Ham’s F-10 medium supplemented with 0.5% (wt/vol) BSA (fraction V, RIA grade, Sigma Chemical Co., St. Louis, MO), 2 mmol/L glutamine, and 2 mmol/L CaCl₂ (final concentration) and equilibrated with 95% O₂-5% CO₂ (23). During the first 20 min, the medium contained 2.5 mmol/L glucose. The cells were then exposed to 10-min pulses of increasing glucose concentration in the presence or absence of 5 mmol/L arginine, each pulse alternating with a 10-min phase at 2.5 mmol/L glucose (Fig. 1). The flow rate was 1 mL/min; samples were collected over 1 min and assayed for immunoreactive insulin and proinsulin.

View larger version (30K): [in this window] [in a new window]   Figure 1. Effect of prolonged culture at high glucose (20 mmol/L) on insulin (upper panel) and proinsulin (lower panel) release from human ß-cells. Human islets cells were cultured at 20 () or 6 () mmol/L glucose before perifusion at different glucose concentrations, in the absence or presence of arginine (5 mmol/L). The rate of hormone release is expressed as femtomoles of insulin or proinsulin per min/103 ß-cells. Data represent the mean ± SEM of six independent experiments.

The human insulin RIA was carried out as described previously (23). Human proinsulin displays a 25% cross-reactivity in this assay. As the amount of proinsulin measured in the samples represents maximally 12% of the corresponding insulin levels, it can be concluded that this cross-reactivity causes maximally a 3% error in the quantification of insulin and a 1% error in the ratio of the measured proinsulin over insulin values. The proinsulin RIA is based on a two-step nonequilibrium procedure with negligible cross-reactivity with human insulin and C peptide (24). We used polyclonal goat antihuman proinsulin from Linco Research, Inc. (St. Charles, MO) and human proinsulin standard donated by F. Sodoyez-Goffaux (University of Liege, Liege, Belgium). Assay samples were incubated with the antibody for 18 h at 20 C before [¹²⁵I]human proinsulin (also provided by Dr. F. Sodoyez-Goffaux) was added, and the incubation was continued for 24 h. Bound and free proinsulin were separated by centrifugation after incubation (20 min, 20 C) with horse antisheep-coated Sepharose (Pharmacia Decanting Suspension 2, Pharmacia Biotech, Uppsala, Sweden), and pellets were washed with phosphate-buffered saline containing 0.25% BSA and counted. Standard curves were calculated on-line by RIA-Calc software Pharmacia, Wallac, Finland), and values were accepted if they showed two linear dilutions. The sensitivity of the assay was 5 pmol/L (blank - 3 SD), and the interassay coefficients of variation were, respectively, 11%, 6%, and 5% for 20, 60, and 100 pg proinsulin/tube. The intraassay coefficients of variation were, respectively, 3%, 4%, and 8% for 70, 35, and 20 pg/tube. Statistical analysis was performed with StatView SE⁺ graphics for Macintosh (Abacus Concepts, Berkeley, CA). Results are expressed as the mean ± SEM. Statistical significance of differences was calculated by Wilcoxon rank sum test.

	Results

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Effect on cellular insulin and proinsulin content

Culture at 20 mmol/L glucose markedly reduced the cellular insulin content in each of the six preparations, but had little effect on the cellular proinsulin content. The mean value for insulin was 5-fold lower (0.9 ± 0.2 pmol/10³ ß-cells; P < 0.05) than that in the control condition with 6 mmol/L (4.3 ± 1.3 pmol/10³ ß-cells), whereas that of proinsulin was only 25% lower (0.08 ± 0.02 pmol/10³ ß-cells vs. 0.11 ± 0.03 in controls; P < 0.05). Consequently, the molar ratio of cellular proinsulin over insulin was 4-fold higher in the 20 mmol/L glucose-cultured cells (Table 1).

At low glucose concentration (2.5 mmol/L), both preparations released comparable amounts of insulin (0.21 ± 0.03 and 0.24 ± 0.03 fmol/10³ ß-cells after culture at, respectively, 6 and 20 mmol/L; P > 0.05) and proinsulin (0.010 ± 0.003 and 0.015 ± 0.004 fmol/10³ ß-cells after culture at, respectively, 6 and 20 mmol/L; P > 0.05).

Rapid insulin secretory responses were measured after a rise in glucose to 5, 10, or 20 mmol/L (Fig. 1). The insulin release rate during these stimulations was 2- to 3-fold lower (P < 0.05) after culture at 20 mmol/L glucose than in control preparations (Table 2). This difference disappeared when insulin release was expressed as a function of the corresponding insulin content (Table 3); it even reversed for the 5 mmol/L glucose stimulus, which caused, in relative terms, a 2-fold higher insulin release from 20 mmol/L glucose cultured cells (P < 0.05; Table 3). Addition of arginine (5 mmol/L) to the glucose stimuli had little effect in control preparations; only a small (20%) stimulation was seen with the 10 mmol/L glucose pulse (Tables 2 and 3); on the other hand, it induced 60% and 300% higher responses in ß-cells cultured in 20 mmol/L glucose that were stimulated by, respectively, 10 and 5 mmol/L glucose (Tables 2 and 3). During stimulation with 5 mmol/L glucose plus 5 mmol/L arginine, the fractional release rate was 8-fold higher in 20 mmol/L glucose-cultured cells than in the control preparation (Fig. 2 and Table 3).

View larger version (34K): [in this window] [in a new window]   Figure 2. Effect of prolonged culture at high glucose (20 mmol/L) on insulin (upper panel) and proinsulin (lower panel) release from human ß-cells. Human islets cells were cultured at 20 () or 6 () mmol/L glucose before perifusion at different glucose concentrations in the absence or presence of arginine (5 mmol/L). The rate of insulin or proinsulin release is expressed as a percentage of the cellular insulin or proinsulin content measured at start of the perifusion. Data represent the mean ± SEM of six independent experiments.

Ratio of proinsulin over insulin in effluent

The molar proinsulin over insulin ratio was markedly higher in the effluent of ß-cell preparations that were exposed to chronically elevated glucose levels. During perifusion at 5 or 20 mmol/L glucose, these ratios were, respectively, 7- and 10-fold higher in 20 mmol/L glucose-cultured cells than in control preparations (Table 1). It was noticed that the ratio in the 5 mmol/L glucose effluent was comparable to that measured in the cells, whether they were cultured at control or high glucose levels (Table 1).

	Discussion

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Chronic hyperglycemia can cause abnormalities in insulin and proinsulin release (reviewed in Ref. 9). It is therefore considered a pathogenic factor in the ß-cell dysfunction of type 2 diabetes (9, 25). The degree of fasting hyperglycemia has been related to the disproportionate increase in proinsulin levels and, hence, to the rise in the proinsulin over insulin ratio (12). It remains questionable, however, to what extent this relationship expresses an effect of chronically elevated glucose levels on the ß-cells (10). The possibility cannot be excluded that an increasing severity of the ß-cell defect leads to both a higher degree of hyperglycemia and a further dissociation between proinsulin and insulin levels (10). The present study demonstrates that prolonged exposure of normal human ß-cells to high glucose concentrations (20 mmol/L) brings them to a state in which they release more proinsulin and less insulin upon glucose stimulation. This leads to a 7- to 10-fold higher medium proinsulin over insulin ratio than that for control cells that were cultured at 6 mmol/L glucose. These extracellular changes are comparable to those described in type 2 diabetes (1, 2, 3, 4). They reflect in the first place the changes in cellular proinsulin and insulin contents. The cellular insulin content was markedly decreased by culture at high glucose, probably as a result of an excessive release that is not compensated by synthesis; this leads to lower amounts of released insulin (20, 21). When insulin release was expressed as a function of the cellular insulin content, the ß-cell secretory activity was not decreased, confirming the view that exposure to high glucose does not reduce ß-cell functions, at least not over 2-week periods (20, 21). Previous work has indicated that this condition activates the majority of cells, increasing their mean sensitivity to glucose (20, 21), a feature that has also been noticed in type 2 diabetic patients.

The present data suggest that proinsulin release might represent a better marker for the functional state of ß-cells with a history of sustained secretory demand, such as persistently high glucose levels. At all tested glucose concentrations, more proinsulin is released during stimulation than from control cells. This increased release of the precursor hormone is not caused by a delay in conversion (26). We believe, rather, that it results from the sustained state of cellular activation. That proinsulin can be used as marker for this condition is in part attributable to the fact that high glucose exposure only marginally decreases the cellular proinsulin content at least under the present conditions. We do not know whether this will still be the case after the longer exposure periods that characterize the in vivo situation.

Prior exposure to high glucose increased not only the ß-cell secretory responsiveness to a subsequent glucose stimulus, but also that to the nonglucose secretagogue, arginine, when supplemented at a submaximal glucose concentration. Priming for a subsequent arginine stimulus was detected in terms of both insulin and proinsulin release rates. As for glucose stimuli, proinsulin again appeared to be a better marker, as its extracellular concentration increased with priming. Thus, when arginine was administered at 5 mmol/L glucose, proinsulin levels were 8-fold higher for high glucose-cultured ß-cells than for control cells.

In conclusion, after prolonged exposure to elevated glucose concentrations, human ß-cells release more proinsulin and less insulin when stimulated by glucose or arginine. The markedly increased proinsulin over insulin ratio in the medium reflects the changes in cellular hormone content that result from a persistent state of cellular activation. The high glucose-exposed ß-cells exhibit a higher responsiveness to glucose and arginine. The proinsulin levels in the medium appear to be a valid marker for measuring the functional state of ß-cells, in particular during or after conditions of increased secretory demand.

	Acknowledgments

 
The authors thank the personnel of the central unit of ß-Cell Transplant for preparing the human islet cells, and Lutgart Heylen and Gabriel Schoonjans for technical assistance in the present work. They are grateful to Dr. F. Sodoyez-Goffaux, P. Houssa, and M. Deberg (University of Liege, Liege, Belgium) for their help in setting up the proinsulin assay.

	Footnotes

 
¹ This work was supported by grants from the Juvenile Diabetes Foundation (JDF-DIRP 995004), the Belgian Fonds voor Wetenschappelijk Onderzoek (FWO G.0376.97), and the services of the Prime Minister (Interuniversity Attraction Pole P4/21).

² Research Assistant of the Belgian Fonds voor Wetenschappelijk Onderzoek.

Received August 18, 1998.

Revised January 11, 1999.

Accepted January 12, 1999.

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