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Originally published In Press as doi:10.1074/jbc.M303509200 on August 18, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42080-42090, October 24, 2003
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Glucose-induced Translational Control of Proinsulin Biosynthesis Is Proportional to Preproinsulin mRNA Levels in Islet {beta}-Cells but Not Regulated via a Positive Feedback of Secreted Insulin*

Barton Wicksteed, Cristina Alarcon, Isabelle Briaud, Melissa K. Lingohr, and Christopher J. Rhodes{ddagger}

From the Pacific Northwest Research Institute and Department of Pharmacology, University of Washington, Seattle, Washington 98122-4302

Received for publication, April 4, 2003 , and in revised form, August 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (<=2h) this is due to increases in the translation of pre-existing mRNA. However, prolonging glucose stimulation (24 h) also increases preproinsulin mRNA levels. It has been proposed that secreted insulin from the pancreatic {beta}-cell regulates its own synthesis through a positive autocrine feedback mechanism. Here the comparative contributions of translation and mRNA levels on the levels of proinsulin biosynthesis were examined in isolated pancreatic islets. Also, the autocrine role of insulin upon four {beta}-cell functions (insulin secretion, proinsulin translation, preproinsulin mRNA levels, and total protein synthesis) was investigated in parallel. The results showed that proinsulin biosynthesis is regulated, in the short term (1 h), solely at the level of translation, through an ~6-fold increase in response to glucose (2.8 mM versus 16.7 mM glucose). In the longer term, when preproinsulin mRNA levels have increased ~2-fold, a corresponding increase was observed in the fold response of proinsulin translation to a stimulatory glucose concentration (>=10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the {beta}-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin plays an important role in the maintenance of mammalian metabolic homeostasis. Secretion of insulin, from the pancreatic {beta}-cell, is the most efficient means whereby the organism can rapidly decrease circulating blood glucose concentrations. The primary function of the {beta}-cell is the production, storage, and regulated secretion of insulin. Under normal circumstances, the {beta}-cell maintains a remarkable condition where there is always a readily available pool of insulin that can be rapidly secreted in response to a stimulus, such as a rise in extracellular glucose concentration (1). Any increase in insulin release is rapidly compensated for a corresponding increase in proinsulin biosynthesis at the translational level (1), so that {beta}-cell insulin stores are constantly upheld. Thus, production of insulin is a highly regulated and dynamic process.

In the short term (<=2 h), proinsulin biosynthesis is predominately regulated at the translational level by many factors including certain nutrients, peptide hormones, and a few pharmacological agents, but of these glucose is the most physiologically relevant (1-4). As for glucose-induced insulin secretion, glucose metabolism is required to stimulate proinsulin biosynthesis in isolated pancreatic islets (5). It is a highly specific process in the pancreatic {beta}-cell that only applies to biosynthesis of proinsulin and a minor subset of proteins, most of which are insulin secretory granule proteins (6). The biosynthesis of the vast majority of {beta}-cell proteins is subject to general translational regulation as found in other eukaryotic cells (7). Stimulatory glucose concentrations (>=4 mM) can increase general protein synthesis in the {beta}-cell up to 2-fold with in 60 min, perhaps via phosphorylation of eukaryotic initiation factor-2B and/or activation of mammalian target of rapamycin (8, 9); however, glucose can induce proinsulin biosynthesis >=15-fold in the same time frame (10, 11). Within this short time period (<=4 h), there is no change in total islet {beta}-cell preproinsulin mRNA levels in response to stimulatory glucose concentrations, despite the marked specific increase in proinsulin biosynthesis (2-4). However, there is a change in distribution of preproinsulin mRNA within the {beta}-cell upon glucose stimulation with a recruitment of preproinsulin mRNA from an inert cytosolic pool to translationally active membrane bound polysomes on the rough endoplasmic reticulum, the site of proinsulin biosynthesis (2, 3). This is consistent with translational control of proinsulin biosynthesis mediated at, or prior to, the initiation phase (1). Recently, we have shown that the specific nature of glucose-induced translational control for proinsulin biosynthesis can be conferred by sequences in the 5'-untranslated region of the preproinsulin mRNA, and that the 3'-untranslated region contains cis-elements involved in glucose-mediated regulation of preproinsulin mRNA stability (4, 12).

For prolonged periods of glucose stimulation (>12 h (13, 14)), situations of refeeding after a period starvation (15), or chronic hyperglycemia (14), proinsulin biosynthesis is also controlled by increases in insulin gene transcription and preproinsulin mRNA stability (16). It should be noted that this longer term regulation, based at the level of increasing preproinsulin mRNA levels, is supplementary to the glucose-induced translational control of proinsulin biosynthesis (1). However, as long as a mRNA is relatively abundant, as is preproinsulin mRNA in the {beta}-cell (12), the extent of the protein synthesis encoded by that mRNA template may not necessarily depend on the mRNA levels if there is specific translational control (7, 17). Nonetheless, this is an unresolved issue in pancreatic {beta}-cells. In some studies, chronic glucose exposure to pancreatic islets appears to indicate that translational control of proinsulin biosynthesis is relative to preproinsulin mRNA levels (14, 18); however, in other studies there is no correlation between preproinsulin mRNA levels and the extent of proinsulin biosynthesis (2, 3, 19). Here we have investigated whether a relationship exists between preproinsulin mRNA levels and the extent of glucose-induced translational control of proinsulin biosynthesis in the same primary {beta}-cells, for both short and long term exposure glucose.

It has been proposed that glucose-induced preproinsulin gene transcription and proinsulin biosynthesis are mediated via a positive feedback of secreted insulin back on the {beta}-cell (20-24). It has been argued that the mild glucose-intolerant phenotype of mice, where the insulin receptor gene has been deleted from {beta}-cells (so-called {beta}IRKO mice) (25), is supportive of this argument (26). However, it is also possible that the {beta}IRKO mouse phenotype might be primarily due to an inadvertent additional knock-out of the insulin receptor in certain hypothalamic neurons that causes insulin resistance (27, 28), which adversely affects {beta}-cell function secondarily (29). Indeed, the idea of insulin having a direct feedback action on {beta}-cell function is controversial. Although insulin can inhibit insulin secretion in vivo (30), it does not in denervated human pancreas (31) or in isolated rat islets (32), suggesting that suppression of insulin secretion by insulin itself is indirect and neurally mediated (33). While insulin has been implicated to increase insulin gene expression in some studies (20-23), in other studies insulin has been shown to decrease insulin gene expression in vivo (34) or have no effect in isolated islet studies (12). Moreover, the mechanism of glucose-induced insulin secretion is distinct from that of glucose-induced proinsulin biosynthesis and insulin gene transcription (1). Somatostatin markedly inhibits glucose-induced insulin secretion but has no effect on glucose-induced proinsulin biosynthesis (35) or insulin gene transcription (36). Although glucose-stimulated insulin secretion and insulin gene transcription are Ca2+-dependent (36, 37), glucose-induced proinsulin biosynthesis is Ca2+-independent (10, 11). Moreover, whereas the KATP-channel sulfonylurea agonists stimulate insulin secretion and diazoxide antagonists inhibit insulin secretion, these reagents have no effect on proinsulin biosynthesis (1, 38, 39). As such, there are circumstances where glucose-induced proinsulin biosynthesis is independent of secreted insulin (1). In this study, we have attempted to address the controversy of whether there are feedback effects of secreted insulin on {beta}-cell function by directly measuring regulated insulin secretion, preproinsulin mRNA levels, and proinsulin biosynthesis in isolated rat islets in parallel experiments (and in some instances in the very same islets). No effect of either secreted or exogenously added insulin could be found on glucose-induced insulin secretion, proinsulin biosynthesis, or preproinsulin mRNA levels in either short-term (1 h) or long term (24 h) exposure to glucose.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—EasyTagTM Expre35S35S protein labeling mix from PerkinElmer Life Sciences, containing 73% of L-[35S]methionine, was used for islet metabolic radiolabeling, referred to as [35S]methionine. IGF-11 was obtained from GroPep Pty. Ltd. (Adelaide, Australia). Unless otherwise stated, all other reagents were from Sigma.

Islet Isolation and Culture—Islets of Langerhans were isolated from 200-250-g male Sprague-Dawley rats (Charles River Laboratories) as described previously (11, 35). Islets used for analysis of short term incubation conditions (1 h) were initially cultured overnight (16-20 h) in RPMI 1640 medium (Invitrogen) containing 5.6 mM glucose, 10% (v/v) fetal bovine serum (Hyclone), 100 units/ml penicillin, and 100 µg/ml streptomycin to allow recovery from the isolation procedure. These islets were washed and then preincubated in Krebs-Ringer HEPES-buffered saline plus 0.1% (w/v) bovine serum albumin at a basal 2.8 mM glucose for 90 min at 37 °C and then transferred to Krebs-Ringer HEPES-buffered saline at a basal 2.8 mM glucose or stimulatory 16.7 mM glucose alone or with the additional presence of 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 or 100 nM insulin, or the absence of extracellular [Ca2+] replaced by 5 mM EGTA as indicated, then incubated for a further 1 h at 37 °C the last 20 min of which was in the additional presence of 100 µCi of [35S]methionine.

Islets used for the analysis of {beta}-cell function over the long term (24 h) were also cultured in RPMI 1640 medium but supplemented with 0.1% (w/v) bovine serum albumin, 100 units/ml penicillin, and 100 µg/ml streptomycin, either at a basal 2.8 mM glucose or stimulatory 16.7 mM glucose. These 24-h incubations were also in the presence or absence of 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 or 100 nM insulin as indicated. These islets were washed and then, in batches of 40-50 islets, continued for a 1-h incubation at 37 °C in Krebs-Ringer HEPES-buffered saline plus 0.1% (w/v) bovine serum albumin under the same conditions at either a basal 2.8 mM glucose or a stimulatory 16.7 mM glucose ± the additional presence of 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, or 10 or 100 nM insulin as indicated, where the last 20 min was in the additional presence of 100 µCi of [35S]methionine.

After the second 1-h islet incubation period, 0.1 ml of medium was collected, centrifuged at 5,000 x g for 2 min, and the supernatant stored at -20 °C pending radioimmunoassay for insulin/C-peptide secretion. The islets were then harvested by centrifugation at 500 x g for 2 min and washed in phosphate-buffered saline containing 2 mM methionine. Those islets to be analyzed for total protein and proinsulin biosynthesis were lysed in 100 µl of 50 mM HEPES (pH 7.6) containing 1.0% (v/v) Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 1 µM E64, 1 µM pepstatin A, 1 µM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 10 µM leupeptin with sonication (25 watts, 10 s, on ice). A 5-µl aliquot was taken from this lysate and stored at -20 °C pending radioimmunoassay for intracellular insulin/C-peptide content. Islets for analysis of mRNA levels were lysed in the lysis buffer supplied with the Direct Lysate kit (Ambion) prior to RNase protection assay.

Protein Synthesis Analysis—Total protein synthesis was determined by trichloroacetic acid precipitation in a 5-µl aliquot of islet lysates as previously described (11, 35). Proinsulin biosynthesis was analyzed by (pro)insulin immunoprecipitation of [35S]methionine-radiolabeled islet lysates as described previously (4, 11, 35). Immunoprecipitated 35S-labeled proinsulin was then subjected to alkaline-urea polyacrylamide gel electrophoresis, fluorography, and quantitative densitometric scanning as described previously (4, 11, 35). Prior to (pro)insulin immunoprecipitation islet lysates were made equivalent for the amount of total protein synthesis as assessed by trichloroacetic acid-precipitation, so as to examine specific glucose-induced proinsulin biosynthesis.

Analysis of Islet mRNA Levels—The mRNA levels in islets were analyzed by the RNase protection assay, using the direct lysis kit (Ambion) as described previously (4). Essentially, RNA was protected using [32P]uridine-labeled antisense RNA fragments corresponding to the coding region of preproinsulin or to part of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding region (Ambion). This yielded protected RNA fragments of 200 bp for the endogenous preproinsulin mRNA and 318 bp of the GAPDH mRNA. Samples were resolved by denaturing 5% acrylamide/TBE (Tris, boric acid, EDTA) gel electrophoresis and analyzed by autoradiography and phosphorimaging (Coulter Beckman).

Radioimmunoassay—Insulin radioimmunoassay was performed using rat insulin standards and antibodies (Linco Research, Inc., St. Charles, MO). Insulin secretion was calculated as a percentage of islet insulin content. C-peptide radioimmunoassay was performed using a rat C-peptide immunoassay kit (Linco Research, Inc.). C-peptide secretion was also calculated as a percentage of islet C-peptide content.

Immunoblot Analysis—Islet cell lysates were first normalized for total protein levels as determined using the BCA protein assay kit (Pierce) and then subjected to immunoblot analysis as described previously (40-42).

Data Analysis—Data are presented as a means ± S.E. Statistical analysis was by were compared by analysis of variance or Student's t test where p <= 0.05 was considered a significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In these parallel studies in isolated islet we have examined whether there is a relationship between glucose-stimulated insulin secretion and the regulation of preproinsulin mRNA levels that in turn may influence the extent of translational control for glucose-induced proinsulin biosynthesis. In many instances these {beta}-cell functional parameters have been measured in the same islets, but the results for insulin secretion, preproinsulin mRNA levels, total proteins synthesis, and specific regulation of proinsulin biosynthesis in short term (1 h) and long term (24 h) periods of glucose stimulation are presented individually.

Insulin/C-peptide Secretion—As anticipated, isolated rat pancreatic islets incubated at a stimulatory 16.7 mM glucose secreted 16-fold more insulin than those incubated at a basal 2.8 mM glucose (Fig. 1A; p <= 0.01) during a 1-h incubation period. After 24 h at a stimulatory 16.7 mM glucose, the rate of insulin secretion was 25-fold greater than that at a basal 2.8 mM glucose (Fig. 2A; p <= 0.01). Glucose-stimulated insulin release in 1-h incubations was inhibited by >90% without extracellular [Ca2+] in the incubation medium (Fig. 1A; p <= 0.01), as observed previously (10, 11). A similar experiment could not be done over a 24-h incubation period, since a prolonged absence of [Ca2+] in the extracellular incubation medium causes pancreatic islets to break up. The added presence of somatostatin inhibited glucose-induced insulin secretion by ~60% over a 1-h incubation period (Fig. 1A; p <= 0.05) and by ~70% during a 24-h period (p <= 0.01; Fig. 2A), as described before (35, 43, 44). The sulfonylurea, glyburide, increased the basal rate of insulin secretion at 2.8 mM glucose 4-5-fold over1h(p <= 0.05; Fig. 1A) and 19-fold over 24 h (p <= 0.01; Fig. 2A), as expected. Likewise, glyburide significantly potentiated 16.7 mM glucose-induced insulin secretion during 1-h (p <= 0.05; Fig. 1A) and 24-h incubation periods (p <= 0.05; Fig. 2A) over that of stimulatory glucose alone. In contrast, the addition of IGF-1 had no significant affect on glucose-induced insulin secretion over 1-h (Fig. 1A) or 24-h (Fig. 2A) incubation periods. To examine whether addition of exogenous insulin can have an effect on glucose-induced insulin secretion, C-peptide secretion (which is normally secreted in equimolar quantities with insulin from pancreatic {beta}-cells) has had to be measured, since the large amount of exogenous insulin added to the incubation medium would interfere with measurement of secreted insulin (32). Stimulatory 16.7 mM glucose increased C-peptide secretion above that at a basal 2.8 mM glucose similarly to that of insulin secretion over 1-h (p <= 0.01; Fig. 1B) and 24-h (p <= 0.01; Fig. 2B) incubation periods. The addition of exogenous insulin at either 10 or 100 nM had no effect on glucose-induced C-peptide secretion over either 1-h or 24-h periods (Figs. 1B and 2B), indicating that insulin did not affect insulin secretion, as observed previously (32).



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  		FIG. 1.
Short term (1 h) glucose-induced insulin/C-peptide secretion in isolated islets. Isolated rat pancreatic islets were preincubated at a basal 2.8 mM glucose for 90 min then at a basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, 100 nM insulin, or the absence of extracellular [Ca2+] replaced by 5 mM EGTA as indicated for 1 h, then the degree of insulin secretion (A) and C-peptide secretion (B) determined as described under "Experimental Procedures." Results are shown as a mean ± S.E. of the percentage of insulin or C-peptide secreted of >=6 individual experiments.

 


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  		FIG. 2.
Long term (24 h) glucose-induced insulin/C-peptide secretion in isolated islets. Isolated rat pancreatic islets were incubated for 24 h at a basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, or 100 nM insulin as indicated and then the degree of insulin secretion (A) and C-peptide secretion (B) determined over a subsequent 1-h incubation under the same conditions as described under "Experimental Procedures." Results are shown as mean ± S.E. of the percentage of insulin or C-peptide secreted in 1 h of >=6 individual experiments.

 

Preproinsulin mRNA Levels—Although this has been recently challenged (20), there is a general majority consensus that total preproinsulin mRNA in islet pancreatic {beta}-cells, when measured directly, are unaltered by stimulatory glucose concentrations over short-term 1-2-h incubation periods (2-4, 45). In general, preproinsulin mRNA levels are only increased by glucose in pancreatic {beta}-cells over longer term periods (>=8 h) (45). In this study, it was found that stimulatory 16.7 mM glucose did not significantly change preproinsulin mRNA levels over a 1-h period in comparison with those at basal 2.8 mM glucose (Fig. 3). In contrast, in isolated islets incubated for 24 h at stimulatory 16.7 mM glucose, preproinsulin mRNA levels were significantly increased between 2- and 3-fold above basal 2.8 mM glucose (p <= 0.05; Fig. 4) as observed previously (61). The removal of extracellular [Ca2+] over a 1-h incubation period, or addition of somatostatin over both 1-h or 24-h periods, where glucose-induced insulin secretion was significantly inhibited (Figs. 1 and 2), did not affect islet preproinsulin mRNA levels (Figs. 3 and 4). Moreover, somatostatin did not significantly alter the glucose-induced increase in preproinsulin mRNA levels in islets over 24 h (Fig. 4), in agreement with previous studies (36). Despite glyburide increasing basal and glucose-stimulated insulin secretion over 1-h and 24-h periods (Figs. 1 and 2), it had no significant effect on preproinsulin mRNA levels at 1 h (Fig. 3) or on glucose-induced increase in preproinsulin mRNA levels over 24 h (Fig. 4). Neither IGF-1 nor insulin affected islet preproinsulin mRNA levels over 1 h (Fig. 3) and the glucose-stimulated increase in preproinsulin mRNA levels over 24 h (Fig. 4).



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  		FIG. 3.
Short term (1 h) regulation of preproinsulin mRNA levels in isolated islets. Isolated rat pancreatic islets were preincubated at a basal 2.8 mM glucose for 90 min then at basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, 100 nM insulin, or the absence of extracellular [Ca2+] replaced by 5 mM EGTA as indicated for 1 h, then preproinsulin mRNA and GAPDH mRNA determined by RNase protection assay as described under "Experimental Procedures." A shows an example autoradiograph of the RNase protection assay. B, results are shown as a mean ± S.E. (n = 4) of the fold increase in islet preproinsulin mRNA levels relative to GAPDH mRNA control levels.

 


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  		FIG. 4.
Long term (24 h) glucose-induced regulation of preproinsulin mRNA levels in isolated islets. Isolated rat pancreatic islets were incubated for 24 h at a basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, or 100 nM insulin as indicated, then preproinsulin mRNA and GAPDH mRNA determined by RNase protection assay as described under "Experimental Procedures." A shows an example autoradiograph of the RNase protection assay. B, results are shown as a mean ± S.E. (n = 4) of the fold increase in islet preproinsulin mRNA levels relative to GAPDH mRNA control levels.

 

Total Protein Synthesis—16.7 mM glucose stimulated total protein synthesis in isolated islets ~2-fold over both 1-h and 24-h incubation periods above basal 2.8 mM glucose (p <= 0.05; Figs. 5 and 6), as found previously (10, 11). Neither somatostatin, glyburide, IGF-1, nor insulin affects glucose-induced total protein synthesis in islets over the short or long term period (Figs. 5 and 6). Removal of extracellular [Ca2+] further increased glucose-induced total protein synthesis in islets incubated over 1 h (Fig. 5), despite the marked inhibition of glucose-stimulated insulin release (Fig. 1).



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  		FIG. 5.
Short term (1 h) glucose-induced regulation of total protein synthesis in isolated islets. Isolated rat pancreatic islets were preincubated at a basal 2.8 mM glucose for 90 min and then at a basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, 100 nM insulin, or absence of extracellular [Ca2+] replaced by 5 mM EGTA as indicated for 1 h, the last 20 min of which was in the additional presence of [35S]methionine, then total protein synthesis determined by trichloroacetic acid precipitation as described under "Experimental Procedures." Results are shown as a mean ± S.E. (n >= 5) of the fold increase in total protein synthesis above basal 2.8 mM glucose base line.

 


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  		FIG. 6.
Long term (24 h) glucose-induced regulation of total protein synthesis in isolated islets. Isolated rat pancreatic islets were incubated for 24 h at a basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, or 100 nM insulin as indicated, then total protein synthesis assessed over a subsequent 1-h incubation, the last 20 min of which was in the additional presence of [35S]methionine, by trichloroacetic acid precipitation as described under "Experimental Procedures." Results are shown as a mean ± S.E. (n >= 5) of the fold increase in total protein synthesis above basal 2.8 mM glucose base line.

 

Proinsulin Biosynthesis—Measurement of glucose-induced proinsulin biosynthesis in isolated islets was corrected for glucose-induced total protein synthesis, so as only the specific effect of glucose on proinsulin biosynthesis was observed (4, 10, 11). In the short term (1 h), 16.7 mM glucose increased proinsulin biosynthesis in isolated islets 4-5-fold (p <= 0.05; Fig. 7), without change in total preproinsulin mRNA levels (Fig. 3) as found previously, indicating translational control (2-4, 10, 11, 35). Removal of extracellular [Ca2+] in the short term 1-h incubation tended to further increase islet glucose-induced proinsulin biosynthesis to ~6-fold (Fig. 7) despite a marked inhibition of glucose-induced insulin secretion in the same islets (Fig. 1). Likewise, although somatostatin significantly inhibited glucose-induced insulin secretion (Fig. 1), in the same islets glucose-induced proinsulin biosynthesis was unaffected (Fig. 7), as described previously (35). Whereas glyburide increased basal and potentiated glucose-stimulated insulin secretion over a 1-h period, there was no significant effect of glyburide on proinsulin biosynthesis in the same islets (Fig. 7), as observed for other sulfonylureas (39). The addition of exogenous IGF-1 or insulin had no effect on short term (1 h) translational control of glucose-induced proinsulin biosynthesis (Fig. 7). In 24-h islet incubations, 16.7 mM glucose increased proinsulin biosynthesis in isolated islets >=10-fold (p <= 0.05; Fig. 8), a greater magnitude than that observed for islets incubate over a short term 1-h period (Fig. 7). However, in this longer term 24-h incubation preproinsulin mRNA levels are increased ~2-fold at stimulatory 16.7 mM glucose compared with basal 2.8 mM glucose (Fig. 4), accounting for the doubling of proinsulin biosynthesis by providing twice the level preproinsulin mRNA template available for translation. As for the short term 1-h incubation (Fig. 7), addition of somatostatin over a 24-h period had no effect on glucose-induced proinsulin biosynthesis (Fig. 8), despite significant inhibition of glucose-induced insulin secretion in the same islets (Fig. 2). Glyburide slightly increased proinsulin biosynthesis at basal 2.8 mM glucose over 24 h (~3-fold; Fig. 8) but had no effect on 16.7 mM glucose-induced proinsulin biosynthesis (Fig. 8). As with the 1-h short term incubation, exogenous IGF-1 or insulin had no effect on long term (24 h) translational control of glucose-induced proinsulin biosynthesis (Fig. 8).



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  		FIG. 7.
Short term (1 h) glucose-induced regulation of proinsulin biosynthesis in isolated islets. Isolated rat pancreatic islets were preincubated at a basal 2.8 mM glucose for 90 min and then at a basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, 100 nM insulin, or absence of extracellular [Ca2+] replaced by 5 mM EGTA as indicated for 1 h, the last 20 min of which was in the additional presence of [35S]methionine, then proinsulin biosynthesis determined as described under "Experimental Procedures." A shows an example fluorograph of the 35S-labeled proinsulin biosynthesis analysis. B, results are shown as mean ± S.E. (n >= 5) of the fold increase in proinsulin biosynthesis above basal 2.8 mM glucose base line.

 


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  		FIG. 8.
Long term (24 h) glucose-induced regulation of proinsulin biosynthesis in isolated islets. Isolated rat pancreatic islets were preincubated at a basal 2.8 mM glucose for 90 min and then at a basal 2.8 mM glucose ({square}) or stimulatory 16.7 mM glucose ({blacksquare}) ± 1 µM somatostatin, 1 µM glyburide, 10 nM IGF-1, 10 nM insulin, or 100 nM insulin as indicated, then proinsulin biosynthesis assessed over a subsequent 1-h incubation, the last 20 min of which was in the additional presence of [35S]methionine, as described under "Experimental Procedures." A shows an example fluorograph of the 35S-labeled proinsulin biosynthesis analysis. B, results are shown as a mean ± S.E. (n >= 5) of the fold increase in proinsulin biosynthesis above basal 2.8 mM glucose base line.

 

Glucose/IGF-1-induced Activation of Erk-1/-2, PKB, and p70S6K in Isolated Rat Islets Is Not Mediated by Secreted Insulin—Neither exogenous insulin nor IGF-1 affected preproinsulin mRNA levels or proinsulin biosynthesis (Figs. 3, 4, 7, and 8), so it was examined whether these were actually capable of activating signal transduction pathways in isolated islets. Isolated rat islets were incubated at basal 2.8 mM or stimulatory 16.7 mM glucose for 15 min ± 5 nM IGF-1 and in the presence or absence of 10 or 100 nM insulin as indicated (Fig. 9A). 16.7 mM glucose increased Erk-1/-2 phosphorylation activation independent of IGF-1 but not that of PKB (Fig. 9A), as observed previously (40, 42). The addition of 5 nM IGF-1 at 16.7 mM glucose further enhanced islet Erk-1/-2 phosphorylation and independently increased PKB phosphorylation activation (Fig. 9), as found previously in pancreatic {beta}-cell lines (40, 42). In contrast to IGF-1, the addition of insulin (10 or 100 nM) had no significant effect on Erk-1/-2 phosphorylation activation (Fig. 9A). Moreover, only at 100 nM insulin was there a slight increase in PKB-Ser473 phosphorylation at 2.8 mM and 16.7 mM glucose, but this was much less than that instigated by a 20-fold less concentration of IGF-1 (Fig. 9A).



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  		FIG. 9.
The effect of glucose, IGF-1, and insulin on activation of Erk-1/-2, PKB, and p70S6K in isolated islets. Isolated rat islets were preincubated for 90 min at basal 2.8 mM glucose, then for 15 min for analysis of Erk-1/2 and PKB phosphorylation or 60 min for analysis of p70S6K phosphorylation at a basal 2.8 mM glucose or stimulatory 16.7 mM glucose ± IGF-1 (5 nM), insulin (10 or 100 nM), or somatostatin (1 µM) as indicated. Phosphorylation of Erk-1/2, PKB, and p70S6K was performed by specific immunoblot analysis in islet cell lysates as described under "Experimental Procedures" (40, 42). A shows example immunoblot analyses of glucose/IGF-1-induced Erk-1/-2 and PKB phosphorylation in the absence or presence of 10 or 100 nM insulin. B shows example immunoblot analyses of glucose/IGF-1-induced Erk-1/-2, PKB, and p70S6K phosphorylation in the absence or presence of 1 µM somatostatin (a concentration that markedly inhibits glucose-induced insulin secretion (Figs. 1 and 2)). The phosphorylation state of p70S6K is indicated by the extent of its electrophoretic retardation giving a "laddering effect" as indicated by the arrows. The immunoblots shown are representative of at least three independent observations.

 

It was also investigated whether glucose induced phosphorylation of Erk-1/-2 and p70S6K in isolated rat pancreatic islets was mediated via secreted insulin. While Erk-1/-2 is phosphorylated after a 5-min glucose stimulus, phosphorylation of p70S6K by glucose is only apparent after 20 min reaching a maximum activation after 40 min (40, 42). Thus, p70S6K phosphorylation in rat islets was assessed over a 60 min incubation period. Isolated rat islets were incubated at basal 2.8 mM or stimulatory 16.7 mM glucose for 15 or 60 min ± 5 nM IGF-1 and in the presence or absence of 1 µM somatostatin that significantly inhibits glucose-induced insulin secretion (Figs. 1 and 2). 16.7 mM glucose significantly increased Erk-1/-2 phosphorylation activation that was further enhanced in the presence of 5 nM IGF-1 (Fig. 9B). This glucose/IGF-1-induced activation of Erk-1/-2 was unaffected in the presence of somatostatin (Fig. 9B), indicating that it was not mediated by secreted insulin. Likewise, somatostatin did not affect IGF-1 induced phosphorylation activation of PKB (Fig. 9B). Immunoblot analysis of p70S6K indicated an increase in p70S6K phosphorylation state in islets incubated for 60 min at a stimulatory 16.7 mM glucose, which was further enhanced by 5 nM IGF-1 (Fig. 9B). Glucose-induced p70S6K phosphorylation activation was unaffected by the presence of somatostatin (Fig. 9B), despite a significant decrease in glucose-induced insulin secretion by somatostatin (Fig. 1). Moreover, addition of 100 nM insulin in the presence of somatostatin did not further enhance glucose-induced p70S6K phosphorylation (data not shown), as was observed for IGF-1 (Fig. 9B). These observations indicate that glucose-induced p70S6K phosphorylation in islet {beta}-cells was not mediated via secreted insulin and complement previous findings where inhibition of glucose-induced insulin secretion by the Ca2+ channel blocker verapamil also had no effect on glucose-induced p70S6K phosphorylation (42).

Finally, it should be noted that total Erk-1/-2 and total PKB levels were equivalent between islet samples in these experiments indicating the specific nature of glucose/IGF-1-induced phosphorylation of these proteins (Fig. 9, A and B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Under normal circumstances glucose-induced proinsulin biosynthesis is predominately controlled at the translational level (14), and this is reiterated in this study. In the short term (1 h), there was a significant increase in glucose-induced proinsulin biosynthesis without change in preproinsulin mRNA levels. This is consistent with the notion that specific translational control of the biosynthesis of a certain protein does not necessarily depend on the abundance of the mRNA template (17, 46). However, this study revealed that this is not always the case for proinsulin biosynthesis. In islets incubated over a longer term 24-h period at a stimulatory glucose there was an increase in preproinsulin mRNA levels, which correlated with a proportional increase in the magnitude of glucose-induced proinsulin biosynthesis at the translational level. The long term (>=12 h) glucose-induced increase in preproinsulin mRNA levels in the {beta}-cell is contributed by both increased transcriptional regulation (45) and a specific stabilization of preproinsulin mRNA half-life (12, 16). However, it should be realized that it is only in relatively unusual circumstances that this long term regulation of preproinsulin mRNA levels becomes physiologically significant for {beta}-cells, since excursions in circulating glucose levels are usually transient and no longer than 2-3 h. Normally, glucose-induced translational control of proinsulin biosynthesis is sufficient to replenish intracellular insulin stores in the {beta}-cell depleted by the concurrent glucose-induced insulin secretion (1). Nonetheless, there are circumstances such as prolonged hyperglycemia (14) or refeeding after a period of starvation (15), where a glucose-induced increase in the preproinsulin mRNA levels can augment the translational control of proinsulin biosynthesis. As such, the pancreatic {beta}-cell has a capacity to supplement translational regulation of proinsulin biosynthesis, at the level of regulating insulin gene transcription and preproinsulin mRNA stability, to further increase proinsulin production under conditions of prolonged glucose-stimulation and compensate for an increased insulin secretory demand. However, it should be noted that such a demand, if it goes on too long as in chronic hyperglycemia and hyperlipidemia found in type-2 diabetes models, can eventually become detrimental to the {beta}-cell so that preproinsulin mRNA levels/proinsulin biosynthesis decrease, and that in turn contributes to {beta}-cell dysfunction and reduced insulin secretory capacity observed in the diabetic state (47-51).

It has been recently proposed that glucose-induced changes in preproinsulin mRNA levels and proinsulin biosynthesis are mediated via a positive feedback of secreted insulin on the {beta}-cell (20-24). However, this is currently a controversial issue. Other studies have found either no effect (12), or a negative feedback effect (34), of insulin on preproinsulin mRNA levels. The majority of studies to date have found no affect of insulin on translational control of proinsulin biosynthesis, which is actually independent from regulation of insulin secretion (1). In this study, insulin secretion, proinsulin biosynthesis, and preproinsulin mRNA levels were directly measured normal in isolated rat islets in parallel and in some instances in exactly the same islets. There was no apparent affect of exogenous insulin or IGF-1 on glucose-induced C-peptide/insulin secretion, proinsulin biosynthesis, or preproinsulin mRNA levels in either short term (1 h) or long term (24 h) incubation periods. This was unlikely due to IGF-1 not having a biological effect on islets since in parallel experiments it was clearly capable of enhancing glucose-induced Erk-1/-2 phosphorylation and independently activating PKB. In contrast to IGF-1, however, insulin was relatively ineffective at activating Erk-1/-2 and PKB, although there was a slight increase in PKB phosphorylation at higher insulin concentrations (>=100 nM). This lack of insulin action on isolated islet cells was most likely due to insulin receptor desensitization and down-regulation caused by locally high insulin concentrations secreted from islets themselves (52-54). It is possible that the modest effect of 100 nM insulin might even be acting via IGF-1 receptors (55). In addition, glucose-induced proinsulin biosynthesis at the translational level and long term stimulation of preproinsulin mRNA levels could not be attributed to a positive feedback of glucose-induced insulin secretion in this study. Inhibition of glucose-induced insulin secretion in the short term, by removal of extracellular [Ca2+], had no adverse effect on translational control of glucose-induced proinsulin biosynthesis as observed previously (10, 11). Indeed, if anything, glucose-induced proinsulin biosynthesis was slightly increased in the absence of extracellular [Ca2+] in line with a similar effect on total protein synthesis. Likewise, somatostatin significantly inhibited glucose-induced insulin secretion with out any negative effect on glucose-induced proinsulin biosynthesis or long term increase in preproinsulin mRNA levels, consistent with previous observations (35, 36). Also, somatostatin did not affect glucose-induced phosphorylation activation of Erk-1/-2 and p70S6K despite significantly inhibiting insulin secretion, thus reaffirming that glucose can activate elements of insulin/IGF-1 signaling pathways independently of secreted insulin (42). In contrast to somatostatin, the sulfonylurea, glyburide, stimulated insulin secretion at both basal and stimulatory glucose concentrations, but had no additional effect on glucose-induced proinsulin biosynthesis or preproinsulin mRNA levels. Glyburide had no effect on augmenting preproinsulin mRNA levels at basal glucose concentrations in the long term, despite a marked glyburide-induced insulin secretion. In the short term (1 h) glyburide had no effect on basal proinsulin biosynthesis; however, in the long term (24 h) basal proinsulin biosynthesis was slightly increased but only in parallel to a similar rise in islet total protein synthesis. All in all, these data suggest that a glucose-induced proinsulin biosynthesis at the translational level and increase in preproinsulin mRNA levels are not mediated by a positive feedback of glucose-induced secreted insulin.

It should be noted that studies showing no effect of insulin on islet preproinsulin mRNA levels (12) have tended to measure preproinsulin mRNA levels directly. Other studies, where an effect of insulin on regulating insulin gene expression in {beta}-cells has been reported, have mostly relied upon either luciferase (23) or enhanced green fluorescent protein (20-22) reporters driven by truncated versions of the rat insulin gene promoter that would be a secondary read-out of preproinsulin mRNA levels. It should be noted that changes in luciferase activity or green fluorescent protein fluorescence could reflect changes in the {beta}-cell intracellular environment, especially in regard to fluctuations in ATP levels (35) and cytosolic pH (56), both of which are influenced by glucose in {beta}-cells (35, 57), in addition to reporting insulin gene promoter activity. Also, it has been reported that glucose-stimulated proinsulin biosynthesis depends on insulin-stimulated insulin gene transcription (24), a finding that is inconsistent with the results presented here. However, this cited study was based on measurement of proinsulin biosynthesis only conducted in 15-min incubation periods (24), a time that is not sufficient to overcome the 15-20-min lag period before significant glucose-induced translational control of proinsulin biosynthesis can be observed (10, 11). These problems in experimental design severely compromise the conclusions of the previous study (24). The results presented in this current study are consistent with the majority of the literature, which indicate that glucose stimulation of proinsulin biosynthesis and preproinsulin mRNA levels are most likely controlled by secondary signals emanating from increased {beta}-cell glucose metabolism (1, 35, 36, 45). Although insulin itself is unlikely to influence regulation of preproinsulin mRNA levels, elements in the insulin signal transduction pathway of {beta}-cells may be involved. In this regard, it should be noted that glucose, independent of insulin or IGF-1, can activate Erk-1/-2, mammalian target of rapamycin/p70S6K, and in the longer term (>2 h) insulin receptor substrate-2 in {beta}-cells (41, 42, 58), which in turn can influence control of insulin gene transcription (59, 60). Notwithstanding, it is emphasized that glucose regulation of preproinsulin mRNA levels is only pertinent to long-term glucose stimulation of {beta}-cells (45), which is supplementary for amplifying glucose-induced translational control of proinsulin biosynthesis. Thus, this study emphasizes the importance of translational control of proinsulin biosynthesis as the predominate means of regulating proinsulin production in the pancreatic {beta}-cell, which is independent of locally secreted insulin.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK47919, DK55269, and DK50610. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Pacific Northwest Research Inst., 720 Broadway, Seattle, WA 98122-4302. Tel.: 206-860-6777; Fax: 206-726-1202; E-mail: cjr{at}pnri.org.

1 The abbreviations used are: IGF-1, insulin-like growth factor 1; GAPDH, glyceradlehyde-3-phosphate dehydrogenase; PKB, protein kinase B (also called Akt); Erk-1/-2, extracellular regulated kinase-1 and -2; p70S6K, 70-kDa S6 protein kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. L. M. Dickson for technical and intellectual contribution to this work.



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