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Glucose-Regulated Proinsulin Gene Expression Is Required for Adequate Insulin Production during Chronic Glucose Exposure

Endocrine and Metabolism Service, Division of Internal Medicine, and Hadassah Diabetes Center, Hebrew University-Hadassah Medical Center, 91120 Jerusalem, Israel

Address all correspondence and requests for reprints to: Gil Leibowitz, M.D., Endocrine and Metabolism Service, Hadassah University Hospital, P.O. Box 12000, 91120 Jerusalem, Israel. E-mail: gleib{at}hadassah.org.il.

	Abstract

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Psammomys obesus, an animal model of type 2 diabetes, shows rapid and marked depletion of pancreatic insulin content as hyperglycemia develops when fed a high-calorie diet. P. obesus islets do not increase proinsulin gene expression when exposed to high glucose, which may be related to absence of the conserved form of the transcription factor insulin promoter factor 1/pancreatic-duodenal homeobox 1. The present study assesses the importance of regulation of proinsulin gene expression by glucose for insulin production. Islets of diabetes-prone P. obesus and diabetes-resistant Wistar rats, cultured at various glucose concentrations for up to 24 h, were analyzed for proinsulin mRNA by quantitative RT-PCR, proinsulin biosynthesis by leucine incorporation into proinsulin, and insulin content and secretion by RIA. No increase in proinsulin mRNA was observed in P. obesus islets during 24-h exposure to increasing concentrations of glucose. In contrast, rat islets exposed to high glucose responded with a 2- to 3-fold stimulation of proinsulin mRNA. The failure of P. obesus islets to increase proinsulin mRNA was accompanied by a reduced proinsulin biosynthetic response: after 24 h, maximal proinsulin biosynthesis was blunted, associated with depletion of islet insulin content. Inhibition of glucose-stimulated proinsulin gene transcription in rat islets by actinomycin D did not affect the early proinsulin biosynthetic response, which, however, was reduced to the level of P. obesus islets after 24 h in culture.

We conclude that stimulation of proinsulin gene transcription by glucose is necessary for maintaining proinsulin biosynthesis and hence conserving pancreatic insulin stores, under conditions of sustained secretory drive, but not for short-term regulation of proinsulin biosynthesis Our findings support the hypothesis that inadequate regulation of proinsulin gene expression by glucose contributes to the failure of P. obesus to cope with the increased demand for insulin associated with caloric excess, leading to depletion of insulin stores and diabetes.

	Introduction

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UNDER PHYSIOLOGICAL CONDITIONS, the secretion and biosynthesis of insulin are coordinated. Indeed, stimulation of insulin release is associated with a rapid and sustained increase of proinsulin biosynthesis thus preventing the depletion of pancreatic insulin stores (1). Glucose is the main physiological regulator of insulin secretion and production in pancreatic ß-cells (2, 3). It is believed that over short periods (2 h or less), glucose regulates proinsulin biosynthesis mainly by increasing the translation of proinsulin mRNA (4). Under resting conditions, the ß-cell contains a large pool of inert proinsulin mRNA, which is recruited to membrane-bound polyribosomes in the endoplasmic reticulum (ER) in response to elevation of the glucose concentration. Activation of translation results from phosphorylation of translation initiation factors (5) and release of the signal recognition peptide from the ribosome/nascent polypeptide complex, allowing its transport into the ER lumen (6). Recently, it was shown that 5' and 3' untranslated regions (UTR) of proinsulin mRNA act cooperatively to increase glucose-induced proinsulin biosynthesis (7). Elements within the 5' UTR stimulate proinsulin translation and others within the 3' UTR stabilize the mRNA. This translational response, which is independent of changes in proinsulin mRNA concentration, leads to a 30- to 50-fold increase in the rate of proinsulin biosynthesis (8). Over longer time intervals, glucose also increases proinsulin mRNA content by both stimulating proinsulin gene transcription and stabilizing the mRNA (9, 10). In light of the efficient increase in proinsulin mRNA translation in response to glucose, the contribution of the relatively modest and late increase in proinsulin mRNA content to insulin biosynthesis has been questioned (11).

In the present study, we show that regulation of proinsulin gene expression by glucose is important for maintenance of sustained insulin production but not for the early proinsulin biosynthetic response to glucose. This slower adaptive response is required to replenish the pancreatic insulin stores under conditions of prolonged demand for insulin. To demonstrate this, we have exploited the Psammomys obesus model of nutrition-dependent diabetes, in which hyperglycemia is associated with rapid depletion of pancreatic insulin content (12, 13, 14), and evaluated the response of islets derived from normoglycemic diabetes-prone P. obesus and diabetes-resistant Wistar rats to elevated glucose levels under controlled in vitro conditions.

	Materials and Methods

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Experimental animals
P. obesus (Hebrew University colony) and Wistar rats were obtained from Harlan (Jerusalem, Israel). After weaning at 3 wk, P. obesus were maintained on low energy (LE) diet (2.38 kcal/g, Koffolk, Petach-Tikva, Israel), and rats on standard laboratory chow, until studied. Animals were anesthetized with ketamine hydrochloride (Ketalar, Park-Davis), and exsanguinated by cardiac puncture. The animal studies were approved by the Institutional Animal Care and Use Committee of the Hebrew University and Hadassah Medical Organization.

Islet isolation and culture
Islets were prepared by collagenase digestion (Collagenase P, Roche Diagnostics, Mannheim, Germany) as described (12). When large quantities were required, islets were hand-picked once under the stereomicroscope, followed by purification on Histopaque 1083 density gradient (Sigma, St. Louis, MO), 10 min at 480 x g for rat islets and 15 min at 660 x g for P. obesus islets. Islets were used after repeated washes with Hanks’ balanced salt solution. Batches of 200–300 islets of similar size were collected and maintained in suspension in 5 ml Roswell Park Memorial Institute (RPMI) 1640 medium (Biological Industries, Beit-Hemeek, Israel) containing the required glucose concentrations and 10% fetal bovine serum (Biological Industries). Islets were cultured for up to 24 h at 37 C in a 5% CO₂ atmosphere. In studies in which actinomycin D was used to inhibit gene transcription, islets were first incubated with 5 µg/ml actinomycin D (Sigma) in RPMI 1640 for 1 h at 37 C, washed once in fresh RPMI 1640, then transferred to incubation media as indicated by the various experimental protocols. Islets were exposed to actinomycin D for only one hour to reduce potential toxicity to the islets, as this was sufficient to inhibit glucose-stimulated proinsulin mRNA levels for at least 24 h, without affecting basal proinsulin mRNA.

Insulin release and content during culture
At the end of the incubation period, the culture medium was collected, centrifuged, and frozen at -20 C pending insulin analysis. Islets were collected and counted for reference. Islet insulin content was determined by RIA in extracts of batches of 50 islets subjected to repeated freeze-thaw cycles in 1.5 ml microfuge tubes containing 450 µl 0.1% BSA in 0.1 N HCl, followed by centrifugation.

Proinsulin biosynthesis
Groups of 50 islets were collected following culture and preincubated for 1 h at 37 C in Modified Krebs-Ringer bicarbonate buffer containing 20 mM HEPES and 0.25% BSA (KRBH-BSA), supplemented with glucose at the concentration used during culture. After preincubation, the islets were centrifuged and labeled in 50 µl fresh KRBH-BSA buffer containing glucose as above, and 50 µCi L-[4,5-³H]leucine (150 Ci/mmol; Amersham Pharmacia Biotech, Aylesbury, UK). Leucine incorporation was terminated after 15-min incubation at 37 C by the addition of 1-ml ice-cold glucose-free KRBH-BSA buffer and rapid centrifugation. The islet pellet was suspended in 450 µl of 0.1% BSA in 0.1 N HCl, extracted by repeated freeze-thaw cycles, and the high-speed microfuge supernatant collected and stored at -20 C for further processing by HPLC, as described (15). Aliquots were used for determination of total insulin content by RIA, as well as for measurement of total protein biosynthesis by trichloroacetic acid precipitation (16).

Insulin determination
Insulin was determined by RIA using antiinsulin antibody-coated tubes (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and ¹²⁵I-insulin from Linco Research, Inc. (St. Charles, MO). The routine intraassay coefficient variation was 4–6%, interassay coefficient variation 6–10%. Human and rat insulin standards, both from Novo-Nordisk (Bagsvaerd, Denmark), were used for determination of P. obesus and rat insulin-like immunoreactivity, respectively (15). P. obesus produces varying amounts of insulin, proinsulin, and proinsulin conversion intermediates depending on the diet (12), all cross-reacting with our assay antiserum; hence, the term insulin is used for summed quantification of all insulin-like immunoreactive products in this study.

Quantification of proinsulin mRNA by RT-PCR
Total islet RNA was extracted from 200–300 islets using RNAzol B (Tel-Test, Friendswood, TX). For PCR analysis, total RNA was reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI). The resulting cDNAs were amplified by PCR using oligonucleotides complementary to sequences in the proinsulin gene: 5'-TTGTCAACCAGCACCTTTGT-3' and 5'-GTTGCAGTAGTTCTCCAGCT-3' for P. obesus proinsulin, and 5'-CCTGCCCAGGCTTTTGTCA-3' and 5'-GGTGCAGCACTGATCCACAATG-3' for rat proinsulin I. Primers were designed to cross an intron and amplified fragments of 257 bp and 208 bp of the coding sequence of the P. obesus and rat proinsulin genes. 18S rRNA (QuantumRNA kit, Ambion, Inc., Austin, TX) was used as an internal control. Polymerization reaction was performed in a 25-µl reaction volume containing 2.5 µl cDNA (25ng RNA equivalents), 80 µM cold 2'-deoxynucleoside 5'-triphosphates, 2.5 µCi (-³²P)deoxy-CTP, 100 pM of appropriate oligonucleotide primers and 1.5 U of Taq polymerase (MBI Fermentas, Amherst, NY). PCR amplification conditions were as follows: 5 min at 94 C followed by 14 cycles of 94 C, 60 C, and 72 C, 30 sec each step. The amplimers were separated on a 6% polyacrylamide gel in Tris-borate EDTA buffer, the gel was dried and the incorporated (-³²P)deoxy-CTP measured by PhosphorImager. The number of cycles and the final reaction conditions were adjusted to the exponential range phase of the amplification curve for each product (17). For quantitation of proinsulin mRNA in islets of P. obesus and Wistar rats, the ratio of proinsulin/18S rRNA band intensity was determined for each reaction. Because actinomycin D decreased the 18S rRNA in rat islets, proinsulin mRNA/18S rRNA ratio in islets incubated at 16.7 mM glucose with actinomycin D was compared with similarly treated islets at 3.3 mM glucose.

Data presentation and statistical analysis
Data are expressed as mean ± SE for the indicated number of individual experiments, each done on a batch of islets pooled from at least 6 animals. Groups were compared using ANOVA followed by Newman-Keuls test using the InStat statistical program from GraphPad Software, Inc. (San Diego, CA). A paired sample t test was used when the difference between a reference (taken as 100%) and a test was analyzed. P < 0.05 was considered significant.

	Results

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Insulin production is reduced in P. obesus islets
Exposure of P. obesus and rat islets to high glucose for 24 h resulted in 70% and 30% depletion of islet insulin content, respectively (Fig. 1). Insulin depletion occurred earlier and at a lower glucose concentration in P. obesus than in rat islets. Maximal depletion was observed at 8.3 mM and 16.7 mM glucose in P. obesus and rat islets, respectively, with a significant depletion occurring at 6 h of culture in P. obesus vs. 24 h in rat islets. There was a glucose-dependent increase in insulin secretion into the incubation medium in both P. obesus and rat islets (Fig. 2). In accordance with our previous report (14), the dose-response curve for insulin secretion was shifted to the left in P. obesus; however, 24-h cumulative insulin secretion at 16.7 mM glucose was lower in P. obesus than in rat islets. Accelerated depletion of islet insulin content despite lower insulin secretion in P. obesus suggests that insulin production is decreased in this species. We investigated whether deficient insulin production resulted from reduced proinsulin gene expression, or decreased proinsulin mRNA translation.

View larger version (42K): [in this window] [in a new window]   Figure 1. Insulin content in islets exposed to different glucose concentrations. P. obesus and rat islets were incubated for 1 (Fresh), 3, 6, and 24 h in RPMI 1640 medium containing different glucose concentrations. White bars, 1.67 mM glucose; lined bars, 3.3 mM glucose; dotted bars, 8.3 mM glucose; black bars, 16.7 mM glucose. Intracellular insulin content was determined in islet extracts by RIA. Results are mean ± SE of 5–12 individual experiments, each using islets pooled from 6–12 animals. *, P < 0.01 relative to rat islets at 3.3 mM glucose; , P < 0.05 relative to P. obesus islets at 1.67 or 3.3 mM glucose; #, P < 0.01 relative to P. obesus islets at 1.67 or 3.3 mM glucose.

View larger version (14K): [in this window] [in a new window]   Figure 2. Dose-response of insulin secretion from islets exposed for 24 h to different glucose concentrations. P. obesus (circles) and rat (squares) islets were incubated for 24 h in RPMI 1640 medium containing different glucose concentrations. Cumulative insulin secretion into the medium was determined by RIA. Results are mean ± SE of three to six individual experiments, each done on islets pooled from at least six animals. *, P < 0.01 for the difference between rat and P. obesus islets in the same glucose concentration.

View larger version (58K): [in this window] [in a new window]   Figure 3. Proinsulin mRNA in P. obesus islets exposed to high glucose in vitro. Islets were cultured at various glucose concentrations for 3, 6, and 24 h. Lined bars, 3.3 mM glucose; dotted bars, 8.3 mM glucose; black bars, 16.7 mM glucose. Relative quantitative PCR for proinsulin mRNA and 18S rRNA was performed on cDNA prepared from total RNA extracted from 200–300 islets from each group. Proinsulin gene expression was first normalized to 18S rRNA (internal control) and then expressed as percent of values of islets at 3.3 mM glucose. The upper panel shows a representative experiment; the lower, quantification of the results relative to islets at 3.3 mM glucose. Results are mean ± SE. The number of individual experiments, each performed on islets pooled from 6–12 animals, is indicated above each bar.

View larger version (55K): [in this window] [in a new window]   Figure 4. Proinsulin mRNA in rat islets exposed to high glucose in vitro. Islets were cultured and processed as described in Fig. 3 legend. Lined bars, 3.3 mM glucose; black bars, 16.7 mM glucose. Proinsulin gene expression was quantified using primers complementary to the 5' and 3' ends of rat proinsulin I gene. The upper panel shows a representative experiment; the lower, quantification of the results relative to islets at 3.3 mM glucose. Results are mean ± SE. The number of individual experiments, each done on islets pooled from at least 12 animals, is indicated above each bar. *, P < 0.001, and #, P < 0.01 relative to islets cultured for the same time at 3.3 mM glucose.

View larger version (18K): [in this window] [in a new window]   Figure 5. Glucose effect on proinsulin biosynthesis. Dose-response curves of proinsulin biosynthesis in islets from normoglycemic P. obesus (circles) and rats (squares). A, Islets after 1-h culture at different glucose concentrations; B, islets after 6-h culture; C, islets after 24-h culture. Fifty islets were pulsed for 15 min with 50 µCi of 3H-leucine, at the same glucose concentration at which they were cultured. Proinsulin-related peptides were resolved by HPLC and their radioactivity determined by liquid scintillation counting. *, P < 0.001, and , P < 0.05 for the difference between rat and P. obesus islets in the same glucose concentration.

View larger version (37K): [in this window] [in a new window]   Figure 6. Actinomycin D effect on glucose-regulated proinsulin gene expression. Rat islets were cultured at different glucose concentrations for 24 h with and without a 1-h actinomycin D incubation. Relative quantitative PCR was performed on cDNA as described in Fig. 3 legend. The upper panel shows a representative experiment. The lower panel shows results quantified relative to control islets at 3.3 mM glucose. Results are mean ± SE of 4 individual experiments, each done on islets pooled from 6–12 animals.

View larger version (27K): [in this window] [in a new window]   Figure 7. Effect of actinomycin D on proinsulin and total protein biosynthesis in rat islets. Rat islets were cultured for 24 h in RPMI 1640 medium containing 3.3 and 16.7 mM glucose with and without actinomycin D as in Fig. 6. At the end of the culture, 50 islets from each group were pulsed with 50 µCi of 3H-leucine for 15 min. Incorporation into proinsulin-related peptides was determined by liquid scintillation counting of the appropriate fractions resolved by HPLC. Incorporation into total protein was assayed by trichloroacetic acid precipitation. Results are mean ± SE of 6 individual experiments, each using islets pooled from 8–12 rats. *, P < 0.01 relative to islets at 3.3 mM glucose without actinomycin D; the rest of the P values are indicated on the figure itself for clarity.

	Discussion

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Previously we have shown that restoration of the proinsulin mRNA response to glucose in P. obesus islets by adenoviral expression of PDX1 resulted in increased basal insulin content and prevented the depletion of islet insulin following exposure to high glucose (17). In rat islets, the 24-h glucose-dependent increase in proinsulin mRNA levels (Fig. 6) was associated with a similar increase in proinsulin biosynthesis (Fig. 7), suggesting that for a given number of mRNA molecules the long-term biosynthetic response is unchanged. Taken together, these data suggest that glucose-stimulated proinsulin gene expression is a sine qua non condition for long-term maintenance of proinsulin biosynthesis and consequently ß-cell insulin stores.

Interestingly, in P. obesus islets, decreased biosynthetic response to glucose was observed only from the 6-h exposure period onwards, supporting the idea that glucose-stimulated proinsulin mRNA production is not necessary for short-term stimulation of insulin biosynthesis Similarly, in rat islets treated with actinomycin D, the biosynthetic response to glucose was decreased after 24 h. Leibiger and colleagues (19, 20) reported that increased proinsulin biosynthesis paralleled by severalfold increased proinsulin mRNA levels occurred within less than a 1-h exposure of rat islets to high glucose. Fifty percent of this early increase in proinsulin biosynthesis was attributed to the rapid regulation of proinsulin gene transcription by glucose. We did not observe glucose- dependent increase in proinsulin mRNA levels following a 1-h exposure to high glucose, and actinomycin D did not inhibit the early increase in proinsulin biosynthesis in rat islets (Leibowitz, G., and N. Kaiser, unpublished data). Furthermore, in P. obesus islets that lack glucose-stimulation of proinsulin gene expression, proinsulin biosynthesis was similar to that of rat islets at 1 and 6 h. These results are thus difficult to reconcile with the findings of Leibiger and colleagues (19, 20) but are in line with those of several other groups (1, 8, 11, 16).

Development of diabetes in P. obesus is accompanied by a profound depletion of pancreatic insulin stores (12, 13, 15). The depletion of islet insulin content can be reproduced in vitro by exposure of islets derived from normoglycemic, diabetes-prone P. obesus to elevated glucose levels (15, 17). Analysis of the biosynthetic response to glucose in islets derived from nonfasted P. obesus and rats reveals a lower response (evaluated as fold of basal) in P. obesus islets, progressively deteriorating during a 24-h time course. This difference is initially due to an increased basal biosynthetic rate in P. obesus, later accompanied by marked diminution of the maximal response. It should be noted, however, that the in vivo environment of the islets may be different in nonfasted P. obesus and rat. Whereas random nonfasted morning blood glucose levels in Wistar rats were in the range of 4.8–6.2 mM, those of P. obesus were 3.6–5.5 mM. This may explain, in part, the shift to the left of both the curves for insulin release (14) and for proinsulin biosynthesis in P. obesus compared with rat islets (EC₅₀ 2.5–3 vs. 5–6 mM glucose). The increased basal biosynthetic activity enables the ß-cells of P. obesus to function adequately in the animal’s natural habitat, feeding on the low calorie salt-bush, or in the laboratory environment when fed the LE diet. Moreover, it may account for the remarkable ability in vivo to quickly refill the insulin stores when demand is markedly reduced, e.g. by fasting (12, 14). However, this is not sufficient to replenish the insulin lost during periods of increased demand (e.g. high energy diet) because a compensatory increase in maximal biosynthetic capacity is lacking and the maximal response to high glucose levels is markedly reduced.

Are these findings in P. obesus of relevance for human type 2 diabetes? IPF1/PDX1 mediates the glucose effect on proinsulin gene transcription (21, 22). Chronic exposure of the ß-cell to high glucose concentrations in vivo and in vitro was shown to induce defects in proinsulin gene transcription resulting from down-regulation of key ß-cell transcription factors, mainly IPF1/PDX1 (23, 24), including in human islets (25). Therefore, it is possible that hyperglycemia-induced loss of glucose-stimulated proinsulin gene transcription participates in the reduction of insulin production in type 2 diabetic patients.

In summary, our data suggest that impairment of glucose-regulated proinsulin gene expression decreases the maximal proinsulin biosynthetic capacity. The demonstration that glucose-regulated proinsulin gene expression is required for efficient proinsulin production during chronic glucose exposure may have important implications for understanding the interactions between transcriptional and translational regulation of insulin production, and for identifying pathophysiological mechanisms of nutrition-related type 2 diabetes.

	Acknowledgments

 
The authors are grateful to Yaffa Ariav for dedicated technical assistance.

	Footnotes

 
This work was supported in part by grants from the Yael Fund (to G.L.), the Juvenile Diabetes Foundation International (to N.K.), the Israel Science Foundation (to N.K. and D.J.G.), The Israel Ministry of Health (to G.L. and N.K.), and from the European Community (BMH4-CT98-3448, to E.C.).

Abbreviations: ER, Endoplasmic reticulum; IPF1, insulin promoter factor 1; KRBH, Krebs-Ringer bicarbonate buffer containing 20 mM HEPES; LE, low energy; PDX1, pancreatic-duodenal homeobox 1; RPMI, Roswell Park Memorial Institute; UTR, untranslated region.

Received February 12, 2002.

Accepted for publication May 3, 2002.

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