Glucose regulates proinsulin and prosomatostatin but not proglucagon messenger ribonucleic acid levels in rat pancreatic islets.

Insulin and glucagon are the major hormones involved in the control of fuel metabolism and particularly of glucose homeostasis; in turn, nutrients tightly regulate insulin and glucagon secretion from the islets of Langerhans. Nutrients have clearly been shown to affect insulin secretion, as well as insulin biosynthesis and proinsulin gene expression; by contrast, the effects of nutrients on proglucagon gene expression have not been studied. We have investigated the effect of glucose, arginine, and palmitate on glucagon release, glucagon cell content, and proglucagon messenger RNA (mRNA) levels from isolated rat islets in 24-h incubations. We report here that concentrations of glucose that clearly regulate insulin and somatostatin release as well as proinsulin and prosomatostatin mRNA levels, do not significantly affect glucagon release, glucagon cell content or proglucagon mRNA levels. In addition, though both 10 mM arginine and 1 mM palmitate strongly stimulated glucagon release, they did not affect proglucagon mRNA levels. We conclude that, in contrast to insulin and somatostatin, glucose does not affect glucagon release and proglucagon mRNA levels, and arginine and palmitate do not coordinately regulate glucagon release and proglucagon mRNA levels.

Chronic exposure to high glucose concentrations increases proglucagon messenger ribonucleic acid levels and glucagon release from InR1G9 cells.

Alpha cell function is impaired in diabetes. In diabetics, plasma levels of glucagon are high despite persistently elevated glucose levels and may even rise paradoxically in response to a glucose load; high plasma glucagon levels are accompanied by increased proglucagon gene expression. We have investigated the effects of high glucose concentrations on InR1G9 cells, a glucagon-producing cell line. We show here that chronically elevated glucose concentrations increase glucagon release by 2.5- to 4-fold, glucagon cell content by 2.5- to 3-fold, and proglucagon messenger RNA levels by 4- to 8-fold, whereas changes for 24 h have no effect on proglucagon messenger RNA levels. Persistently elevated glucose affects proglucagon gene expression at the level of transcription and insulin is capable of preventing this effect. We conclude that chronically elevated glucose may be an important factor in the alpha cell dysfunction that occurs in diabetes and thus that glucose may not only affect the beta cell but also the alpha cell.

Glucose-induced preproinsulin gene expression is inhibited by the free fatty acid palmitate.

Prolonged exposure to elevated FFA levels has been shown to induce peripheral insulin resistance and to alter the beta-cell secretory response to glucose. To investigate the effects of FFAs on preproinsulin gene expression, we measured insulin release, cell content, and messenger RNA (mRNA) levels in rat islets after a 24-h exposure to 1 mM palmitate. Insulin release increased at all glucose concentrations studied; in contrast, preproinsulin mRNA levels were specifically reduced by palmitate at high glucose with a decrease in insulin stores, suggesting that palmitate inhibits the glucose-stimulated increase in preproinsulin gene expression. The mechanisms by which palmitate affects preproinsulin gene expression implicate both preproinsulin mRNA stability and transcription, as suggested by an actinomycin D decay assay, quantification of primary preproinsulin transcripts, and transient transfection experiments in Min6 cells. Metabolism of palmitate is not required to obtain these effects, inasmuch as they can be reproduced by 2-bromopalmitate. However, oleate and linoleate did not significantly influence preproinsulin mRNA levels. We conclude that insulin release and preproinsulin gene expression are not coordinately regulated by palmitate and that chronically elevated FFA levels may interfere with beta-cell function and be implicated in the development of noninsulin-dependent diabetes.

Insulin, but not glucose lowering corrects the hyperglucagonemia and increased proglucagon messenger ribonucleic acid levels observed in insulinopenic diabetes.

The factors that regulate glucagon biosynthesis and proglucagon gene expression are poorly defined. We previously reported that insulin inhibits proglucagon gene expression in vitro. In vivo, however, the effects of insulin on the regulation of the proglucagon gene have been controversial. Furthermore, whether glucose plays any role alone or in conjunction with insulin on proglucagon gene expression is unknown. We investigated the consequences of insulinopenic diabetes on glucagon gene expression in the endocrine pancreas and intestine and whether insulin and/or glucose could correct the observed abnormalities. We show here that in the first 3 days after induction of hyperglycemia by streptozotocin, rats have levels of plasma glucagon and proglucagon messenger RNA comparable to those of normoglycemic controls despite hyperglycemia. With more prolonged diabetes, plasma glucagon and proglucagon messenger RNA levels increase; this increase is corrected by insulin treatment, but not by phloridzin despite normalization of the glycemia by both treatments. Proglucagon gene expression exhibits the same regulatory response to glucose and insulin in both pancreas and ileum. We conclude that insulin tonically inhibits proglucagon gene expression in the pancreas and ileum and that glucose plays a minor, if any, role in this regulation.

Detection of insulin mRNA in the peripheral blood after human islet transplantion predicts deterioration of metabolic control.

Recent updates of the Edmonton trial have shown that insulin independence is progressively lost in approximately 90% of islet transplant recipients over the first 5 years. Early prediction of islet graft injury could prompt the implementation of strategies attempting to salvage the transplanted islets. We hypothesize that islet damage is associated with the release and detection of insulin mRNA in the circulating blood. Whole blood samples were prospectively taken from 19 patients with type 1 diabetes receiving 31 islet transplants, immediately prior to transplantation and at regular time-points thereafter. After RNA extraction, levels of insulin mRNA were determined by quantitative reverse tran-scriptase-polymerase chain reaction. All patients exhibited a primary peak of insulin mRNA immediately after transplantation, without correlation of duration and amplitude with graft size or outcome. Twenty-five subsequent peaks were observed during the follow-up of 17 transplantations. Fourteen secondary peaks (56%) were closely followed by events related to islet graft function. Duration and amplitude of peaks were higher when they heralded occurrence of an adverse event. Peaks of insulin mRNA can be detected and are often associated with alterations of islet graft function. These data suggest that insulin mRNA detection in the peripheral blood is a promising method for the prediction of islet graft damage.

The paired homeodomain transcription factor Pax-2 is expressed in the endocrine pancreas and transactivates the glucagon gene promoter.

Glucagon gene expression is controlled by at least four DNA elements within the promoter; G2, G3, and G4 confer islet-specific expression, while G1 restricts glucagon transcription to alpha cells. Two islet-specific complexes are formed on G3, the insulin-responsive element of the glucagon gene; one of these corresponds to the paired homeodomain protein Pax-6, a major glucagon gene transactivator that plays a crucial role in alpha cell development. We describe here the identification of the second complex as Pax-2, another member of the paired box family. Pax-2 is known to be crucial for the development of the urogenital tract and of the central nervous system, but its presence in the endocrine pancreas has not been reported. We detected Pax-2 gene expression by RT-PCR; in islets, Pax-2 is present as two alternative splicing isoforms, Pax-2A and Pax-2B, whereas in the glucagon- and insulin-producing cell lines alphaTC1 and Min6, a distinct isoform, Pax-2D2, is found in addition to Pax-2B. Both islet-specific isoforms bind to the enhancer element G3 and to the alpha-specific promoter element G1 that also interacts with Pax-6. Pax-2A and Pax-2B dose-dependently activate transcription from the G3 and the G1 elements both in heterologous and in glucagon-producing cells. Our data indicate that Pax-2 is the third paired domain protein present in the endocrine pancreas and that one of its roles may be the regulation of glucagon gene expression.

Pax-6 and Cdx-2/3 interact to activate glucagon gene expression on the G1 control element.

The promoter element G1, critical for alpha-cell-specific expression of the glucagon gene, contains two AT-rich sequences important for transcriptional activity. Pax-6, a paired homeodomain protein previously shown to be required for normal alpha-cell development and to interact with the enhancer element G3 of the glucagon gene, binds as a monomer to the distal AT-rich site of G1. However, although the paired domain of Pax-6 is sufficient for interaction with the G3 element, the paired domain and the homeodomain are required for high affinity binding to G1. In addition to monomer formation, Pax-6 interacts with Cdx-2/3, a caudal-related homeodomain protein binding to the proximal AT-rich site, to form a heterodimer on G1. Both proteins are capable of directly interacting in the absence of DNA. In BHK-21 cells, Pax-6 activates glucagon gene transcription both through G3 and G1, and heterodimerization with Cdx-2/3 on G1 leads to more than additive transcriptional activation. In glucagon-producing cells, both G1 and G3 are critical for basal transcription, and the Pax-6 and Cdx-2/3 binding sites are required for activation. We conclude that Pax-6 is not only critical for alpha-cell development but also for glucagon gene transcription by its independent interaction with the two DNA control elements, G1 and G3.

Pdx1 level defines pancreatic gene expression pattern and cell lineage differentiation.

The absence of Pdx1 and the expression of brain-4 distinguish alpha-cells from other pancreatic endocrine cell lineages. To define the transcription factor responsible for pancreatic cell differentiation, we employed the reverse tetracycline-dependent transactivator system in INS-I cell-derived subclones INSralphabeta and INSrbeta to achieve tightly controlled and conditional expression of wild type Pdx1 or its dominant-negative mutant, as well as brain-4. INSralphabeta cells express not only insulin but also glucagon and brain-4, while INSrbeta cells express only insulin. Overexpression of Pdx1 eliminated glucagon mRNA and protein in INSralphabeta cells and promoted the expression of beta-cell-specific genes in INSrbeta cells. Induction of dominant-negative Pdx1 in INSralphabeta cells resulted in differentiation of insulin-producing beta-cells into glucagon-containing alpha-cells without altering brain4 expression. Loss of Pdx1 function alone in INSrbeta cells, which do not express endogenous brain-4 and glucagon, was also sufficient to abolish the expression of genes restricted to beta-cells and to cause alpha-cell differentiation. In contrast, induction of brain-4 in INSrbeta cells initiated detectable expression of glucagon but did not affect beta-cell-specific gene expression. In conclusion, Pdx1 confers the expression of pancreatic beta-cell-specific genes, such as genes encoding insulin, islet amyloid polypeptide, Glut2, and Nkx6.1. Pdx1 defines pancreatic cell lineage differentiation. Loss of Pdx1 function rather than expression of brain4 is a prerequisite for alpha-cell differentiation.

Ectopic expression of the beta-cell specific transcription factor Pdx1 inhibits glucagon gene transcription.

AIMS/HYPOTHESIS: The transcription factor Pdx1 is required for the development and differentiation of all pancreatic cells. Beta-cell specific inactivation of Pdx1 in developing or adult mice leads to an increase in glucagon-expressing cells, suggesting that absence of Pdx1could favour glucagon gene expression by a default mechanism. METHOD: We investigated the inhibitory role of Pdx1 on glucagon gene expression in vitro. The glucagonoma cell line InR1G9 was transduced with a Pdx1-encoding lentiviral vector and insulin and glucagon mRNA levels were analysed by northern blot and real-time PCR. To understand the mechanism by which Pdx1 inhibits glucagon gene expression, we studied its effect on glucagon promoter activity in non-islet cells using transient transfections and gel-shift analysis. RESULTS: In glucagonoma cells transduced with a Pdx1-encoding lentiviral vector, insulin gene expression was induced while glucagon mRNA levels were reduced by 50 to 60%. In the heterologous cell line BHK-21, Pdx1 inhibited by 60 to 80% the activation of the alpha-cell specific element G1 conferred by Pax-6 and/or Cdx-2/3. Although Pdx1 could bind three AT-rich motifs within G1, two of which are binding sites for Pax-6 and Cdx-2/3, the affinity of Pdx1 for G1 was much lower as compared to Pax-6. In addition, Pdx1 inhibited Pax-6 mediated activation through G3, to which Pdx1 was unable to bind. Moreover, a mutation impairing DNA binding of Pdx1 had no effect on its inhibition on Cdx-2/3. Since Pdx1 interacts directly with Pax-6 and Cdx-2/3 forming heterodimers, we suggest that Pdx1 inhibits glucagon gene transcription through protein to protein interactions with Pax-6 and Cdx-2/3. CONCLUSION/INTERPRETATION: Cell-specific expression of the glucagon gene can only occur when Pdx1 expression extinguishes from the early alpha cell precursor.