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

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (/=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.     

A cis-element in the 5' untranslated region of the preproinsulin mRNA (ppIGE) is required for glucose regulation of proinsulin translation

Insulin production in pancreatic beta cells is predominantly regulated through glucose control of proinsulin translation. Previously, this was shown to require sequences within the untranslated regions (UTRs) of the preproinsulin (ppI) mRNA. Here, those sequences were found to be sufficient for specific glucose-regulated proinsulin translation. Furthermore, an element 40-48 bp from the 5' end of the ppI mRNA specifically bound a factor present in islets of Langerhans. Glucose-responsive factor binding to this cis-element exhibited temporal and glucose-concentration-dependent patterns that paralleled proinsulin biosynthesis. Mutating this cis-element abolished the ability of ppI mRNA UTRs to confer glucose regulation upon translation. Like the rat 5'UTR, the human ppI 5'UTR conferred glucose regulation of translation. However alternative splicing of the human 5'UTR that disrupts the cis-element abolished glucose-regulated translation. These data indicate that glucose regulation of cis-element/trans-acting factor interaction is a key component of the mechanism by which glucose regulates insulin production.     

RNA-binding protein HuD controls insulin translation

Although expression of the mammalian RNA-binding protein HuD was considered to be restricted to neurons, we report that HuD is present in pancreatic β cells, where its levels are controlled by the insulin receptor pathway. We found that HuD associated with a 22-nucleotide segment of the 5' untranslated region (UTR) of preproinsulin (Ins2) mRNA. Modulating HuD abundance did not alter Ins2 mRNA levels, but HuD overexpression decreased Ins2 mRNA translation and insulin production, and conversely, HuD silencing enhanced Ins2 mRNA translation and insulin production. Following treatment with glucose, HuD rapidly dissociated from Ins2 mRNA and enabled insulin biosynthesis. Importantly, HuD-knockout mice displayed higher insulin levels in pancreatic islets, while HuD-overexpressing mice exhibited lower insulin levels in islets and in plasma. In sum, our results identify HuD as a pivotal regulator of insulin translation in pancreatic β cells.     

Acute inhibition of proinsulin biosynthesis at the translational level by palmitic acid

The effects of fatty acids on pancreatic beta cell are still controversial. Here, in order to determine whether free fatty acids acutely affect beta cell functions, we studied the effect of palmitic acid (PA) on proinsulin biosynthesis and insulin secretion using rat islets in vitro. Exposure of islets to PA for 1 h reduced glucose-stimulated proinsulin biosynthesis in a dose-dependent manner; in contrast, no change in insulin secretion was observed after 1 h incubation with PA. Furthermore, PA treatment did not cause any change of preproinsulin mRNA level during 1-h incubation period. Thus, our data indicate that PA primarily suppresses glucose-induced proinsulin biosynthesis within 1 h at the translational level.     

Direct effect of glucose on the preproinsulin mRNA level in isolated pancreatic islets

The effects of glucose on the preproinsulin mRNA level and the rate of (pro)insulin biosynthesis were examined in isolated mouse pancreatic islets. Relative concentrations of preproinsulin mRNA were quantitated by a RNA-dot hybridization procedure. The level of preproinsulin mRNA in islets incubated for up to 7 days at 20 mM glucose remained constant. In islets incubated at 3.3 mM glucose the preproinsulin mRNA level decreased and was after 24 h reduced to one tenth of the level at 20 mM glucose. Subsequent incubation at 20 mM glucose completely restored the preproinsulin mRNA level but only after 3 days of culture, while the insulin release was restored within 24 h. The insulin-biosynthetic activity of the islets was correlated to the variation in the level of the preproinsulin mRNA. These results suggest that glucose does have a direct influence on the level of preproinsulin mRNA and that the rate of (pro)insulin biosynthesis is limited by the level of the preproinsulin mRNA.     

p38 mitogen-activated protein kinase/Hog1p regulates translation of the AU-rich-element-bearing MFA2 transcript

AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3' untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3'-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3'-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3'-UTR in a regulated manner to control MFA2 3'-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3'-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3'-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3'-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.     

Cell-free processing and segregation of insulin precursors

The biosynthesis, segregation, and processing of preproinsulin (116 amino acids) was investigated to determine the mechanism(s) by which it is translocated across the endoplasmic reticulum membrane. Islet mRNA was translated in the wheat germ cell-free system, and at various times during preproinsulin synthesis, puromycin was added, followed by addition of microsomal membranes. Neither processing of preproinsulin nor translocation of proinsulin into microsomal membranes occurred in the presence of puromycin. Synchronization of preproinsulin translation by addition of 7-methylguanosine 5'-phosphate enabled the timing of preproinsulin synthesis and proinsulin (91 amino acids) segregation into microsomal membranes to be determined. Membrane binding occurs when about 60 amino acids have been polymerized, i.e. prior to the completion of the polypeptide chain. The binding of signal recognition particle to the nascent signal is demonstrated to be an absolute requirement for translocation and processing of preproinsulin. The results indicate that segregation and processing of preproinsulin are co-translational events; no evidence for a post-translational mechanism was found. Furthermore, this work, together with similar studies, suggests that presecretory polypeptides must be synthesized as part of a precursor with a minimum size of 60-80 amino acids in order to effect membrane binding and translocation of the polypeptide chain within the intracisternal space of the endoplasmic reticulum.     

Glucose-stimulated translation regulation of insulin by the 5' UTR-binding proteins

Insulin is the key regulator of glucose homeostasis in mammals, and glucose-stimulated insulin biosynthesis is essential for maintaining glucose levels in a narrow range in mammals. Glucose specifically promotes the translation of insulin in pancreatic β-islet, and the untranslated regions of insulin mRNA play a role in such regulation. Specific factors in the β-islets bind to the insulin 5' UTR and regulate its translation. In the present study we identify protein-disulfide isomerase (PDI) as a key regulator of glucose-stimulated insulin biosynthesis. We show that both in vitro and in vivo PDI can specifically associate with the 5' UTR of insulin mRNA. Immunodepletion of PDI from the islet extract results in loss of glucose-stimulated translation indicating a critical role for PDI in insulin biosynthesis. Similarly, transient overexpression of PDI resulted in specific translation activation by glucose. We show that the RNA binding activity of PDI is mediated through PABP. PDI catalyzes the reduction of the PABP disulfide bond resulting in specific binding of PABP to the insulin 5' UTR. We also show that glucose stimulation of the islets results in activation of a specific kinase that can phosphorylate PDI. These findings identify PDI and PABP as important players in glucose homeostasis.     

Isolation and partial purification of catfish pancreatic islet messenger RNA.

Poly(a)-rich mRNA has been isolated from catfish pancreatic islet total nucleic acid. Cell-free translation of the mRNA by wheat germ extracts yielded a protein of 11 000-12 000 molecular weight, estimated by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. This peptide is larger than catfish proinsulin, but contains tryptic peptides of proinsulin. Its synthesis comprises up to 23% of the cell-free product, depending on the conditions of cell-free synthesis. Synthesis is inhibited by 7-methylguanosine 5'-monophosphate suggesting the presence of a 7-methylguanosine cap on the 5' end of catfish proinsulin mRNA. Sucrose gradient centrifugation of the islet poly(A)-rich mRNA yielded 8S and 12S peaks. These fractions were translated with wheat germ extracts and it was determined that over 60% of the islet mRNA-dependent protein from the 8S fraction was preproinsulin. The 8S mRNA fraction was electrophoresed on 3% agarose-6 M urea gels and demonstrated to be several bands, ranging from 100 000-200 000 molecular weight.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5′-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5′-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5′-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40–100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic β-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5′-UTR of the human insulin mRNA.     

Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'-UTR, or sandwiched between the 5'- and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.     

Jagn1 Is Induced in Response to ER Stress and Regulates Proinsulin Biosynthesis

The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic β-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.