Jacques Philippe

Pdx1 level defines pancreatic gene expression pattern and cell lineage differentiation

The absence of Pdx1 and the expression of brain-4 distinguish α-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 INSrαβ and INSrβ to achieve tightly controlled and conditional expression of wild type Pdx1 or its dominant-negative mutant, as well as brain-4. INSrαβ cells express not only insulin but also glucagon and brain-4, while INSrβ cells express only insulin. Overexpression of Pdx1 eliminated glucagon mRNA and protein in INSrαβ cells and promoted the expression of β-cell-specific genes in INSrβ cells. Induction of dominant-negative Pdx1 in INSrαβ cells resulted in differentiation of insulin-producing β-cells into glucagon-containing α-cells without altering brain4 expression. Loss of Pdx1 function alone in INSrβ cells, which do not express endogenous brain-4 and glucagon, was also sufficient to abolish the expression of genes restricted to β-cells and to cause α-cell differentiation. In contrast, induction of brain-4 in INSrβ cells initiated detectable expression of glucagon but did not affect β-cell-specific gene expression. In conclusion, Pdx1 confers the expression of pancreatic β-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 α-cell differentiation.

Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplantations.

BACKGROUND A series of 13 islet autotransplantations and 13 islet allotransplantations performed between 1992 and 1999 at the University Hospital of Geneva are presented. Factors affecting the outcome are analyzed. METHODS Islet autotransplantation has been performed in seven patients with chronic pancreatitis and in six patients with benign tumors undergoing extensive pancreatectomy. Islet allografts were performed in C-peptide-negative patients simultaneously or after a kidney or lung transplantation. Each recipient received islets from one to four donors. Panel-reactive antibodies were monitored by microlymphocytotoxicity test. RESULTS Eleven of 13 patients who underwent autotransplantation maintained insulin independence for 6 months to 5 years. Two years after autologous islet transplantation, five of nine patients were insulin independent with an glycosylated hemoglobin of 5.9%. Three late islet failures occurred in patients with chronic pancreatitis. Islet yield was significantly lower in patients with chronic pancreatitis than in patients with benign tumors (2044 equivalent islet number/gram resected pancreas versus 5184 equivalent islet number/gram; P=0.037). In islet allotransplantation, no early graft loss was found. All 13 patients who underwent allotransplantation had basal C-peptide levels above 0.3 nmol/L for 3 months to 5 years. Mean glycosylated hemoglobin decreased from 9.1% before transplantation to 5.5% at month 3. Insulin independence was achieved in two type I diabetic patients. In four of six patients with graft failure, the graft had induced panel-reactive antibodies. CONCLUSIONS In islet autotransplantation, the reduced number of islets that can be isolated from fibrotic pancreata may be the major limiting factor. In islet allotransplantation, early graft function can now be consistently achieved. Islet allografts seem to be highly immunogenic, and chronic islet failure cannot be prevented consistently by conventional immunosuppression.

Multipotential phenotypic expression of genes encoding peptide hormones in rat insulinoma cell lines.

The developmental origin of the four phenotypically distinct hormone-producing islet cells (insulin, glucagon, somatostatin, pancreatic polypeptide) is unclear. To investigate the potential for phenotypic differentiation of islet cells, we prepared several clonal cell lines from a radiation-induced rat islet tumor and analyzed them for insulin, glucagon, and somatostatin gene expression by cDNA hybridization, immunocytochemistry, and radioimmunoassay. We found expression of all three genes in the tumor and in the parental cell line and mixed variable phenotypes in the clonal lines derived from the parental line. We also observed the ectopic expression of the angiotensinogen gene in the tumor and the cell lines. The relative levels of hormonal gene expression differed among the cell lines but remained fixed during continuous passage. The three islet hormone mRNAs were larger compared to the pancreas owing to longer poly(A) tracts. These observations indicate that neoplastic islet cells retain the potential to differentiate into hormone-specific cellular phenotypes and may mimic developmental pathways of the pancreatic islets.

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.

Transcriptional regulation of genes encoding insulin, glucagon, and angiotensinogen by sodium butyrate in a rat islet cell line.

The state of differentiation of various neoplastic cell lines is inversely correlated with the rate of cellular growth. To delineate the changes in hormone gene expression associated with an induced decrease in the growth rate of rat insulinoma cells, we studied the effects of sodium butyrate on the expression of the genes encoding insulin, glucagon, and angiotensinogen. Sodium butyrate inhibited cellular proliferation and decreased levels of c-myc mRNA. Concomitantly, steady-state levels of mRNAs encoding insulin and glucagon increased by 10- and 8.5-fold, respectively, as a result of a specific increase in the transcription of both genes. Sodium butyrate also inhibited angiotensinogen gene expression, which was ectopic in the insulinoma cells. These observations suggest that sodium butyrate induces a pattern of events leading to the differentiation of the rat insulinoma cells.

Differential Regulation of the Glucagon and Insulin I Gene Promoters by the Basic Helix-Loop-Helix Transcription Factors E47 and BETA2

The insulin and glucagon genes are expressed in the beta and alpha cells of the islets of Langerhans, respectively. The factors controlling their cell- and islet-specific expression are poorly known. Insulin-enhancer factor-1 (IEF1) has previously been shown to interact with the E boxes of the rat insulin I and II genes and was proposed to play a critical role in beta cell-specific expression. BETA2, a recently identified basic helix-loop-helix (bHLH) protein, binds with high affinity and transactivates the rat insulin II gene upon dimerization with the ubiquitous bHLH protein E47. We show here that the heterodimer E47/BETA2 also binds and transactivates the rat insulin I and glucagon genes and exhibits the same characteristics as IEF1. In transfection experiments, the E boxes of the insulin I and glucagon genes confer transcriptional activity in both insulin- and glucagon-producing cells, which is increased by overexpression of E47 and BETA2. However, overexpression of E47 inhibits only E box-mediated glucagon gene expression, whereas it activates insulin gene transcription, indicating that the E boxes of the insulin and glucagon genes display gene-specific characteristics. We conclude that the heterodimer E47/BETA2 represents an islet-specific factor that controls both insulin and glucagon gene transcription and that the E47/BETA2 ratio may be important for regulated gene expression.

The caudal-related Homeodomain Protein Cdx-2/3 Regulates Glucagon Gene Expression in Islet Cells

Glucagon gene transcription in the endocrine pancreas is regulated by at least four cis-acting DNA control elements. We showed previously that G1 is critical for alpha cell-specific expression. G1 contains three AT-rich sequences important for promoter function, which represent candidate binding sites for homeodomain transcription factors. Performing reverse transcription-polymerase chain reaction amplifications with degenerate oligonucleotide primers homologous to the Antennapedia homeobox, cDNA clones corresponding to the caudal-related gene cdx-2/3 were predominantly obtained from glucagon-producing cells and primary non-beta cells. From RNase protection and polymerase chain reaction analyses, cdx-2/3 turned out to be the only caudal-related gene that is expressed at significant levels in cells of the endocrine pancreas. Cdx-2/3 binds with high affinity to an AT-rich motif of G1, which matches the consensus binding site of caudal-related proteins. In the glucagon-producing hamster cell line InR1G9, Cdx-2/3 is a subunit of complex B3 formed on G1. Alternative splicing generates two cdx-2/3 transcripts in islet cells, coding for a full-length protein and an amino-terminally truncated isoform. Although both isoforms bind G1 with similar affinity, only the full-length Cdx-2/3 A protein activates glucagon gene transcription in non-glucagon-producing cells, transcriptional activation being dose-dependent. We therefore conclude that the caudal-related gene cdx-2/3 is implicated in the transcriptional control of glucagon gene expression in the alpha cells of the islets of Langerhans.

Non-alcoholic fatty liver disease and insulin resistance: from bench to bedside.

Non-alcoholic fatty liver disease (NAFLD) is now the most frequent chronic liver disease in the developed countries. There is also growing evidence from basic and clinical research that NAFLD has a strong relationship to insulin resistance, which is a key factor in the development of type 2 diabetes. The aim of this review is to summarize the recent important findings linking NAFLD and insulin resistance. Lipid accumulation, particularly of diacylglycerol, appears to be of major importance in this process. Mitochondrial dysfunction, through decreased mitochondrial biogenesis, increases oxidative stress, and ageing also plays an important role. Finally, endoplasmic reticulum stress and inflammation also probably contribute to the development of insulin resistance via mechanisms that are still not well understood. Clinical aspects of NAFLD, such as its diagnosis and management, are also investigated in this review.

Sequential Kidney/Islet Transplantation: Efficacy and Safety Assessment of a Steroid-Free Immunosuppression Protocol

The aim of this study was to assess the efficiency and safety of the Edmonton immunosuppression protocol in recipients of islet‐after‐kidney (IAK) grafts. Fifteen islet infusions were administered to 8 patients with type 1 diabetes and a functioning kidney graft. Immunosuppression was switched on the day of transplantation to a regimen associating sirolimus‐tacrolimus‐daclizumab. Insulin‐independence was achieved in all patients for at least 3 months, with an actual rate of 71% at 1 year after transplantation (5 of 7 patients). After 24‐month mean follow‐up, five have ongoing insulin independence, 11–34 months after transplantation, with normal HbA1c, fructosamine and mean amplitude of glycemic excursions (MAGE) values. Results of arginine‐stimulation tests improved over time, mostly after the second islet infusion. Severe adverse events included bleeding after percutaneous portal access (n = 2), severe pneumonia attributed to sirolimus toxicity (n = 1), kidney graft loss after immunosuppression discontinuation (n = 1), reversible humoral kidney rejection (n = 1) and fever of unknown origin (n = 1). These data indicate that the Edmonton approach can be successfully applied to the IAK setting. This procedure is associated with significant side effects and only patients with stable function of the kidney graft should be considered. The net harm versus benefit has not yet been established and will require further studies with larger numbers of enrolled subjects.

Cyclic adenosine monophosphate prevents the glucocorticoid-mediated inhibition of insulin gene expression in rodent islet cells.

Dexamethasone negatively regulates insulin gene expression in HIT-15 cells. In vivo, however, an excess of glucocorticoids results in an increase in insulin biosynthesis and peripheral hyperinsulinemia. To resolve this contradiction, we have studied the effects of dexamethasone in primary rat islet cells. We show here that dexamethasone decreases insulin mRNA levels in single islet cells, as in HIT-15 cells, but does not affect these levels in reaggregated islet cells and increases them in intact islets of Langerhans. Because cAMP is an important regulator of insulin gene expression and intracellular cAMP content may be decreased in single beta cells, we investigated whether cAMP could prevent the inhibitory effect of dexamethasone on insulin mRNA levels. In the presence of cAMP analogues, the inhibitory action of dexamethasone was not only prevented, but insulin mRNA increased to levels comparable to those observed when cAMP analogues were used alone. We conclude that the insulin gene is negatively regulated by dexamethasone in single islet cells, but that other factors such as cAMP prevent this effect when the native environment of islet cells is preserved. Our results indicate that insulin gene regulation is influenced by cell to cell contacts within the islet, and that intracellular cAMP levels might be influential in this regulation.