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Chronic hyperglycemia impairs insulin secretion by affecting insulin receptor expression, splicing, and signaling in RIN ß cell line and human islets of Langerhans¹

MARTA L. HRIBAL², LUCIA PEREGO^*,,2, SARAH LOVARI, FRANCESCO ANDREOZZI, ROSSELLA MENGHINI, CARLA PEREGO, GIOVANNA FINZI^**, LUCIANA USELLINI^**, CLAUDIA PLACIDI^**, CARLO CAPELLA^**, VALERIA GUZZI^*, DAVIDE LAURO, FEDERICO BERTUZZI, ALBERTO DAVALLI^*, GUIDO POZZA^||, ANTONIO PONTIROLI, MASSIMO FEDERICI, RENATO LAURO, ANTONIO BRUNETTI, FRANCO FOLLI^*,3 and GIORGIO SESTI^,3

Laboratory of Molecular Medicine, Department of Internal Medicine, University of Rome-Tor Vergata, Rome;
^* Department of Medicine, Unit of Endocrinology and Metabolic Disease, IRCCS HS Raffaele, Milan;
Department of Internal Medicine, University of Milan,
Department of Experimental and Clinical Medicine, University of Catanzaro-Magna Græcia, Catanzaro;
CNR, Centro per lo Studio della Farmacologia Cellulare e Molecolare, Milan;
^** Department of Surgical Pathology, Università dell’Insubria, Varese;
Department of Surgery, HS Raffaele; and
^|| Università Vita-Salute San Raffaele, Milan, Italy

³Correspondence: G.S., Dipartimento di Medicina Sperimentale e Clinica, Università di Catanzaro-Magna Græcia, Via Tommaso Campanella, 115; 88100 Catanzaro-Italy. E-mail: sesti{at}unicz.it or F.F., Department of Medicine, HS Raffaele, Via Olgettina, 60; 20132 Milan, Italy. E-mail: folli.franco{at}hsr.it

SPECIFIC AIMS

Recent evidence suggests that insulin stimulates insulin gene transcription via the Ex11- rather than the Ex11+ isoform. Since chronic hyperglycemia negatively affects insulin receptor (IR) function and regulates alternative splicing of IR, we inquired whether chronic exposure of pancreatic ß cells to high glucose results in alterations in insulin signaling due to changes in IR expression and relative abundance of its spliced isoforms.

PRINCIPAL FINDINGS

1. Localization of IR in human pancreas and effect of high glucose on human pancreatic islets and RIN ß cells
Immunofluorescence analysis in human pancreatic sections using the MA-10 monoclonal anti-IR antibody, which recognizes the subunit of both isoforms or an anti-insulin antibody showed that IR was prevalently expressed in the ß cells, where it colocalized with insulin. Immunoelectron microscopy analysis showed that IR was prevalently localized in the secretory granules of ß cells and, to a lesser extent, cells and at the plasma membrane (Fig. 1 d). Intracellular insulin content was decreased in human islets exposed to high glucose concentrations (16.7 mM) compared with islets exposed to normal glucose concentrations (5.5 mM) (P<0.01). Basal insulin secretion was reduced by 25% in human islets exposed to high glucose concentrations (P<0.05). Insulin release stimulated by 30 mM glucose was reduced by 74% in human islets exposed to high glucose concentrations compared with islets exposed to normal glucose concentrations (P<0.01). Steady-state insulin mRNA levels were not increased in human islets exposed to high glucose levels compared with controls (Fig. 1a ). These results indicate human islets exposed to high glucose failed to compensate by increasing insulin transcription for sustained secretory demand secondary to elevations in ambient glucose concentrations. We took advantage of a well-characterized pancreatic ß cell model of glucose toxicity, rodent ß cell lines continuously cultured under high glucose concentrations. We continuously cultured RIN ß cells in a medium containing 11.1 mM glucose and used them at passage 16-25 (early passage=RIN-E) or at passage 45-55 (late passage=RIN-L). Basal and glucose-stimulated fractional insulin secretion was significantly reduced in RIN-L compared with RIN-E cells. Insulin content was decreased by 25% in RIN-L compared with RIN-E cells (P<0.002). Insulin mRNA levels were reduced by 50% in RIN-L compared with RIN-E cells (P<0.0009). PDX-1 mRNA levels were reduced by 50% in RIN-L compared with RIN-E cells (P<0.002), whereas mRNA levels of glucose transporter GLUT2, glucokinase, and Foxa2 (formerly HNF-3ß) were not affected by exposure to high glucose.

View larger version (102K): [in this window] [in a new window]   Figure 1. Effects of high glucose on insulin mRNA levels, and IR expression in human islets. a) Northern blot of insulin mRNA levels in human islets exposed to normal (NG islets) or high glucose (HG islets). Data are the means ± SE of 7 independent experiments. b) RT-PCR analysis of IR in NG or HG islets. Data (expressed as arbitrary units) are the mean ± SE of 5 independent experiments. c) RT-PCR analysis of IR isoforms expression in NG or HG islets. Data are the means ± SE of 4 independent experiments. d) Immunoelectron microscopy of NG islets (original magnification13,800x; 34,500x for inset). e) Immunoelectron microscopy of HG islets (original magnification 13,000).

2. Effect of high glucose on IR expression in human islets and RIN ß cells
Analysis of IR expression by semiquantitative RT-PCR showed a 35% decrease in human islets exposed to high glucose compared with controls (P<0.002) (Fig. 1b ). Immunoelectron microscopic analysis of human islets exposed to high glucose concentrations showed a marked reduction in insulin-containing granules and IR (Fig. 1d, e ). Slot blot and immunoblotting analysis showed that IR expression was significantly reduced in RIN-L compared with RIN-E cells at protein and mRNA levels (P<0.001).

3. Effect of high glucose on the high-mobility group (HMG) protein HMGI(Y) in the RIN ß cells
HMGI(Y) is required for the proper transcription of IR gene and its expression correlates with IR protein levels. DNA binding activity assessed by EMSA in nuclear extracts from RIN-L was significantly reduced compared with RIN-E cells. Immunoblotting analysis showed that HMGI(Y) nuclear protein expression was significantly lower in RIN-L than RIN-E cells (P<0.002).

4. Effect of high glucose on relative abundance of the two IR isoforms in human islets and RIN ß cells
We inquired whether changes observed in human islets and RIN ß cells exposed to high glucose concentrations were accompanied by alterations in abundance of the two spliced IR isoforms. RT-PCR assay revealed that human islets exposed to normal glucose concentrations express IR-Ex11- and IR-Ex11+ receptor isoforms accounting for 40 and 60%, respectively, of total IR mRNA molecules (Fig. 1c ). The percentage of Ex11⁺ isoform was increased up to 80% of total IR mRNA molecules in human islets exposed to high glucose (P<0.01) (Fig. 1c ). RIN-E cells express predominantly IR-Ex11- rather than IR-Ex11+ (80% and 20%, respectively). However, after exposure to high glucose, the percentage of Ex11⁺ isoform increased up to 40% of total IR mRNA molecules in RIN-L cells (P<0.001). Likewise, protein level of IR-Ex11⁺ isoform, determined by immunoblotting of the immunoprecipitated isoform, was increased by twofold in RIN-L compared with RIN-E cells (P<0.02). When RIN-L cells were transiently transfected with expression vector containing the IR-Ex11- cDNA, insulin mRNA level was restored to 85% of that in RIN-E cells; transfection with expression vector containing the IR-Ex11+ cDNA failed to restore insulin expression.

5. Effect of high glucose on IR, IRS-1, and IRS-2 phosphorylation and PI-3-kinase activation in RIN ß cells
Next we inquired whether changes in IR expression induced by high glucose were associated with alterations in IR signaling. Insulin-stimulated tyrosine phosphorylation of IR was decreased by 29% in RIN-L compared with RIN-E cells (P<0.001). Insulin-stimulated association of p85 subunit of PI 3-kinase with IR was reduced by 25% (P<0.001) under basal condition and by 29% (P<0.002) upon insulin stimulation in RIN-L compared with RIN-E cells. Accordingly, insulin-stimulated PI 3-kinase activity associated with IR was reduced by 30% in RIN-L vs. RIN-E cells (P<0.001). Total PI 3-kinase activity precipitated with anti-p85 antibody was reduced by 35% in RIN-L compared with RIN-E cells (P<0.005). By contrast, the extent of basal and insulin-stimulated tyrosine phosphorylation of IRS-1 or IRS-2 was similar in RIN-E and RIN-L cells.

6. Effect of high glucose on Akt, PHAS-I and p70S6 kinase activation and on insulin biosynthesis in RIN ß cells
The Ser/Thr kinase Akt is a critical downstream effector of PI 3-kinase. Expression of Akt did not differ between RIN-E and RIN-L cells. Basal Ser473 Akt phosphorylation was decreased by 18% in RIN-L compared with RIN-E cells (P<0.01) whereas insulin-induced Ser473 Akt phosphorylation was reduced by 33% (P<0.001). No difference was observed between RIN-E and RIN-L cells in Thr308 Akt phosphorylation. Akt promotes protein synthesis by modulating downstream proteins involved in the control of the translation process including the kinase mTOR (mammalian target of rapamycin) and its downstream targets p70S6 kinase and PHAS-I (phosphorylated heat and acid stable). Expression and insulin-stimulated phosphorylation at Thr389 of p70S6 kinase did not differ between RIN-E and RIN-L cells. Exposure to insulin promotes the phosphorylation of PHAS-I and its dissociation from the eukaryotic translation initiation factor 4F eIF4F, allowing eIF4F to form a productive translation initiation complex. Compared with RIN-E cells, phosphorylation of PHAS-I ß and forms was markedly reduced in the basal state and upon insulin stimulation in RIN-L. Specific insulin biosynthesis was reduced by 50% upon glucose stimulation (P<0.001) and by 80% upon insulin stimulation (P<0.001) vs. RIN-E cells.

CONCLUSIONS AND SIGNIFICANCE

In this study, we demonstrate that IR is expressed in human pancreatic ß cells where it is localized in insulin-containing granules. Chronic exposure of human islets and rat ß cells to high glucose concentrations reduced insulin content and glucose-stimulated insulin secretion. We demonstrate that these defects are associated with a marked reduction in insulin-containing granules, a significant decrease in IR expression, and an increase in abundance of the IR-Ex11⁺ isoform. The reduction in IR expression in these cells could be accounted for by a decrease in protein expression of HMGI(Y), which is required for the proper transcription of IR gene. That no changes were observed in expression of molecules involved in glucose sensing such as GLUT2 and glucokinase indicates that defects in insulin secretion caused by elevation in ambient glucose levels are not the result of altered expression of key proteins involved in glucose sensing, but of alterations in intracellular mechanisms of glucose-stimulated insulin release. We also found that depletion of insulin stores in human islets exposed to high glucose as a result of increased secretory demand was not compensated for by an increase in insulin mRNA synthesis. This defect in compensatory insulin mRNA synthesis was more evident in RIN ß cells cultured longer at high glucose concentrations, which showed a reduction in insulin mRNA levels. Reexpression of IR-Ex11-, but not IR-EX11+, in RIN-L cells restored insulin mRNA expression. It is tempting to suggest that the changes in expression of the two IR isoforms, acting in concert with the reduction in IR, may contribute to the observed alterations in insulin secretion and synthesis induced by elevated glucose concentrations (Fig. 2 ). We found a significant impairment in the insulin signaling involving the IR-associated PI 3-kinase/Akt pathway after exposure of rat pancreatic ß cells to high glucose. Under these conditions, we also observed a reduction of insulin protein synthesis upon glucose and insulin stimulation. Analysis of the signaling pathway downstream of PI 3-kinase/Akt, which is implicated in the control of insulin protein translation, revealed that elevated glucose concentration affected activation of PHAS-I rather than p70S6 kinase. Because we closely reproduced diabetic condition in vitro by exposing human islets to 16.7 mM glucose and pancreatic ß cell lines to 11.1 mM, the results of the present study may be clinically relevant. In conclusion, the present data suggest that changes in early steps of IR signaling may play a role in determining or aggravating ß cell dysfunction caused by chronic hyperglycemia in patients with type 2 diabetes.

View larger version (57K): [in this window] [in a new window]   Figure 2. Schematic diagram of functional consequences of changes in IR expression and splicing in pancreatic ß cells exposed to high glucose concentrations. In ß cells exposed to normal glucose, released insulin may stimulate insulin gene transcription and biosynthesis via IR-Ex11- rather than IR-Ex11+ isoform. In ß cells exposed to elevated glucose, increased abundance of Ex11+ receptor isoform may contribute to reduction in insulin secretion, expression, and biosynthesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0685fje; doi: 10.1096/fj.02-0685fje

² These authors contributed equally to this manuscript.

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