Wnt9a deficiency discloses a repressive role of Tcf7l2 on endocrine differentiation in the embryonic pancreas.

Transcriptional and signaling networks establish complex cross-regulatory interactions that drive cellular differentiation during development. Using microarrays we identified the gene encoding the ligand Wnt9a as a candidate target of Neurogenin3, a basic helix-loop-helix transcription factor that functions as a master regulator of pancreatic endocrine differentiation. Here we show that Wnt9a is expressed in the embryonic pancreas and that its deficiency enhances activation of the endocrine transcriptional program and increases the number of endocrine cells at birth. We identify the gene encoding the endocrine transcription factor Nkx2-2 as one of the most upregulated genes in Wnt9a-ablated pancreases and associate its activation to reduced expression of the Wnt effector Tcf7l2. Accordingly, in vitro studies confirm that Tcf7l2 represses activation of Nkx2-2 by Neurogenin3 and inhibits Nkx2-2 expression in differentiated ß-cells. Further, we report that Tcf7l2 protein levels decline upon initiation of endocrine differentiation in vivo, disclosing the downregulation of this factor in the developing endocrine compartment. These findings highlight the notion that modulation of signalling cues by lineage-promoting factors is pivotal for controlling differentiation programs.

The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice.

AIMS/HYPOTHESIS: Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function. METHODS: Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment. RESULTS: Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice. CONCLUSIONS/INTERPRETATION: These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.

Sodium tungstate regulates food intake and body weight through activation of the hypothalamic leptin pathway.

AIMS: Sodium tungstate is an anti-obesity drug targeting peripheral tissues. In vivo, sodium tungstate reduces body weight gain and food intake through increasing energy expenditure and lipid oxidation, but it also modulates hypothalamic gene expression when orally administered, raising the possibility of a direct effect of sodium tungstate on the central nervous system. METHODS: Sodium tungstate was administered intraperitoneally (ip) to Wistar rats, and its levels were measured in cerebrospinal fluid through mass spectrometry. Body weight gain and food intake were monitored for 24 h after its administration in the third ventricle. Hypothalamic protein was obtained and subjected to western blot. In vitro, hypothalamic N29/4 cells were treated with 100 µM sodium tungstate or 1 nM leptin, and protein and neural gene expression were analysed. RESULTS: Sodium tungstate crossed the blood-brain barrier, reaching a concentration of 1.31 ± 0.07 mg/l in cerebrospinal fluid 30 min after ip injection. When centrally administered, sodium tungstate decreased body weight gain and food intake and increased the phosphorylation state of the main kinases and proteins involved in leptin signalling. In vitro, sodium tungstate increased the phosphorylation of janus kinase-2 (JAK2) and extracellular signal-regulated kinase-1/2 (ERK1/2), but the activation of each kinase did not depend on each other. It regulated c-myc gene expression through the JAK2/STAT system and c-fos and AgRP (agouti-related peptide) gene expression through the ERK1/2 pathway simultaneously and independently. CONCLUSIONS: Sodium tungstate increased the activity of several kinases involved in the leptin signalling system in an independent way, making it a suitable and promising candidate as a leptin-mimetic compound in order to manage obesity.

The role of transmembrane protein 27 (TMEM27) in islet physiology and its potential use as a beta cell mass biomarker.

AIMS/HYPOTHESIS: Transmembrane protein 27 (TMEM27) is a membrane protein cleaved and shed by pancreatic beta cells that has been proposed as a beta cell mass biomarker. Despite reports of its possible role in insulin exocytosis and cell proliferation, its function in beta cells remains controversial. We aimed to characterise the function of TMEM27 in islets and its potential use as a beta cell mass biomarker. METHODS: To determine TMEM27 function, we studied TMEM27 gene expression and localisation in human healthy and diabetic islets, the correlation of its expression with cell cycle and insulin secretion genes in human islets, its expression in tungstate-treated rats, and the effects of its overproduction on insulin secretion and proliferation in a beta cell line and islets. To elucidate its utility as a beta cell mass biomarker, we studied TMEM27 cleavage in a beta cell line, islets and primary proximal tubular cells. RESULTS: TMEM27 mRNA levels in islets are lower in diabetic donors than in controls. Its gene expression correlates with that of insulin and SNAPIN in human islets. TMEM27 expression is downregulated in islets of tungstate-treated rats, which exhibit decreased insulin secretion and increased proliferation. TMEM27 overproduction in a beta cell line and islets significantly enhanced glucose-induced insulin secretion, with modest or no effects on proliferation. Finally, TMEM27 is cleaved and shed by renal proximal tubular cells and pancreatic islets. CONCLUSIONS/INTERPRETATION: Our data support a role for TMEM27 in glucose-induced insulin secretion but not in cell proliferation. The finding that its cleavage is not specific to beta cells challenges the current support for its use as a potential beta cell mass biomarker.

Factores de transcripción y desarrollo del páncreas endocrino / Transcription factors and endocrine pancreas development

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Inflammatory mechanisms in diabetes: lessons from the beta-cell.

Inflammation plays an important role in the destruction of pancreatic islet beta-cells that leads to type I diabetes. This involves infiltration of T-cells and macrophages into the islets and local production of inflammatory cytokines such as interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. Our laboratory has developed several strategies for protecting beta-cells against oxidative stress and cytokine-induced cytotoxicity. These include a cytokine selection strategy that results in cell lines that are resistant to the combined effects of IL-1 beta+IFN-gamma. More recently, we have combined the cytokine selection procedure with overexpression of the antiapoptotic gene bcl-2, resulting in cell lines with greater resistance to oxidative stress and cytokine-induced damage than achieved with either procedure alone. This article summarizes this work and the remarkably divergent mechanisms by which protection is achieved in the different model systems. We also discuss the potential relevance of insights gained from these approaches for enhancing islet cell survival and function in both major forms of diabetes.

High glucose concentration favors the selective secretion of islet amyloid polypeptide through a constitutive secretory pathway in human pancreatic islets.

We studied the contribution of the constitutive and the regulated pathways to the total secretion of islet amyloid polypeptide (IAPP) in human pancreatic islets after prolonged culture at either 5.5 or 24.4 mM glucose. In islets cultured in low concentrations of glucose, the secretion of IAPP in response to glucose was unaffected by brefeldin A (BFA) and completely blocked by ethyleneglycoltetraacetic acid. In islets cultured in high glucose concentrations, it was strongly inhibited by both agents. BFA had no effect on the glucose-induced insulin secretion. The determination of the islet peptide contents and the mRNA levels revealed a several-fold increase in the IAPP/insulin molar ratio of islets cultured in high glucose concentrations. Thus, prolonged exposure of human islets to high concentrations of glucose results in an increase in the synthesis of IAPP with respect to insulin. As a result, the release of IAPP through a mechanism sensitive to BFA is favored. These data support the hypothesis that IAPP and insulin are regulated in a noncoordinated way in human pancreatic islets.

Distinctive regulatory and metabolic properties of glycogen-targeting subunits of protein phosphatase-1 (PTG, GL, GM/RGl) expressed in hepatocytes.

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.

Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity.

Engineered insulinoma cell lines may represent an alternative to isolated islets for transplantation therapy of type 1 diabetes. Success of this approach may require development of cell lines that can withstand cytokine-mediated damage. To this end, we have cultured INS-1 insulinoma cells in increasing concentrations of interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium+ ++ bromide (MTT) viability assay, the selected cells, termed INS-1res, were 100% viable after 5 days of treatment with 10 ng/ml of IL-1beta. These cells were also 78 +/- 1.2% viable after 5 days of exposure to the combination of 10 ng/ml IL-1beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1res cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of cytokines. Stable transfection of INS-1res cells with a plasmid containing the human insulin cDNA and expansion of the transfected colonies in the absence of cytokines produced cell lines that were on average more resistant to IL-1beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1res-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range. With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60% reduction in expression of the IL-1 receptor type I (IL-1RI) in INS-1res cells compared with parental INS-1 cells. IL-1beta signaling through p38 MAP kinase was found to be normal in INS-1res cells, suggesting that their expression of IL-1RI is sufficient to maintain cytokine action. However, normal IL-1beta-mediated translocation of NF-kappaB and induction of inducible nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1res cell lines, suggesting a mechanism for the IL-1beta resistance. In sum, this study defines a strategy for isolation of cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.

The role of glucose and its metabolism in the regulation of glucokinase expression in isolated human pancreatic islets.

Previous reports concerning the regulation of glucokinase expression in beta cells have been done using cell models from rodent origin. Evidence is lacking so far to implicate the same regulatory mechanisms in human cells. In this study, we investigate the effects of glucose on the expression of glucokinase using isolated human pancreatic islets. High glucose (16.7 mM), in a time-dependent manner, increases the amount of immunoreactive glucokinase (+150% after 7 days culture, P < 0.01) without apparent changes in glucokinase gene expression, suggesting that glucose exerts its effect at a posttranscriptional level. Mannose, but not the nonmetabolized hexoses, 3-O-methylglucose or 2-deoxyglucose, increases glucokinase protein content. Even though these findings are compatible with an involvement of signals derived from glucose metabolism, additional data argue against this hypothesis: (i) a glucokinase inhibitor (mannoheptulose) does not block glucose-induced increase in glucokinase content and (ii) other metabolic fuels (amino acids) are ineffective. We suggest that the glucose molecule, by mechanisms yet to be defined, but probably not involving its metabolism, regulates human glucokinase expression.

Overexpression of G11alpha and isoforms of phospholipase C in islet beta-cells reveals a lack of correlation between inositol phosphate accumulation and insulin secretion.

It has been suggested that insulin secretion from pancreatic islets may be mediated in part by activation of phospholipases C (PLCs) and phosphoinositide hydrolysis. The purpose of this study was to determine whether the relatively modest fuel-stimulated insulin secretion responses of rodent beta-cell lines might be explained by inadequate expression or activation of PLC isoforms. We have found that two insulinoma cell lines, INS-1 and betaG 40/110, completely lack PLC-delta1 expression but have levels of expression of PLC-beta1, -beta2, -beta3, -delta2, and -gamma1 that are similar to or slightly reduced from those found in fresh rat islets. Adenovirus-mediated overexpression of PLC-delta1, -beta1, or -beta3 in INS-1 or betaG 40/110 cells results in little or no enhancement in inositol phosphate (IP) accumulation and no improvement in insulin secretion when the cells are stimulated with glucose or carbachol, despite the fact that the overexpressed proteins are fully active in cell extracts. Overexpression of PLC-beta1 or -beta3 in normal rat islets elicits a larger increase in IP accumulation but, again, has no effect on insulin secretion. Because the effect of carbachol on insulin secretion is thought to be mediated through muscarinic receptors that link to the Gq/11 class of heterotrimeric G proteins, we also overexpressed G11alpha in INS-1 cells, either alone or in concert with overexpression of PLC-beta1 or -beta3. Overexpression of G11alpha enhances IP accumulation, an effect slightly potentiated by co-overexpression of PLC-beta1 or -beta3, but these maneuvers do not affect glucose or carbachol-stimulated insulin secretion. In sum, our studies show a lack of correlation between IP accumulation and insulin secretion in INS-1 cells, betaG 40/110 cells, or cultured rat islets. We conclude that overexpression of PLC isoforms and/or G11alpha is not an effective means of enhancing fuel responsiveness in the insulinoma cell lines studied.

Effect of high glucose concentration on proinsulin biosynthesis and conversion by human islets.

In the present study we investigate whether glucose concentration could have an effect on proinsulin biosynthesis and processing. We cultured control human islets under chronic high and low glucose concentrations. After the culture period, islets were pulse-labeled and chased for different periods of time. Proteins from islets were collected, insulin immunoprecipitated, and analyzed by alkaline-urea gel electrophoresis. We have found an accelerated rate of proinsulin conversion by those islets exposed to high glucose concentration (at 24.4 mM of glucose), but not by those islets cultured at low glucose concentration (at 5.5 mM of glucose). However, we do not observe any decrease or increase on newly proinsulin synthesis in any of these conditions.

Glucose regulation of islet amyloid polypeptide gene expression in rat pancreatic islets.

Intracellular pathways by which glucose regulates islet amyloid polypeptide (IAPP) gene expression in pancreatic islets were studied. IAPP mRNA levels were threefold higher in islets cultured with 16.7 mM glucose compared with control (5.5 mM glucose). Mannose and amino acids but not 2-deoxyglucose or 6-deoxyglucose mimicked the effect of glucose. Mannoheptulose (a glycolysis inhibitor) and verapamil and diazoxide (which affect calcium signaling pathway) abolished the difference in islet IAPP mRNA content between high and low glucose. At low glucose, IAPP mRNA levels were increased 1.9-fold in islets treated with forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) but not with 12-O-tetradecanoylphorbol 13-acetate. Insulin mRNA levels were 1.6-fold higher in islets cultured at high glucose than controls; glucose metabolism was required, whereas no effects of cAMP or diazoxide were observed. IAPP and insulin were cosecreted into the media. We conclude that glucose regulation of IAPP mRNA abundance requires intracellular metabolism of the hexose and that calcium may serve as a mediator of this effect; cAMP but not protein kinase C possibly participates in this regulation.

Signals related to glucose metabolism regulate islet amyloid polypeptide (IAPP) gene expression in human pancreatic islets.

Intracellular pathways involved in glucose stimulation of IAPP gene expression were studied in human pancreatic islets. Glucose (16.7 mM), but not mannose, caused a 2.3-fold increase in IAPP mRNA levels; this effect was inhibited by actinomycin D. In the presence of the non-metabolizable 6-deoxyglucose (16.7 mM) IAPP mRNA levels were markedly depleted. Both mannoheptulose and verapamil blocked glucose-induced stimulation of the IAPP gene. The magnitude of the insulin gene response to glucose was smaller (1.3-fold); none of the above-mentioned agents had significant effects on insulin mRNA content. Tunicamycin elicited a 2.4- and 2.7-fold increase in IAPP mRNA levels in the low and high glucose media, respectively; however, it did not change insulin mRNA. It had no effect on rat IAPP or insulin mRNAs, either. We conclude that IAPP gene expression is regulated by signals derived from glucose metabolism and that intracellular calcium may be involved in this response. IAPP and insulin genes are not co-regulated in cultured human pancreatic islets.

Autoantibodies against mitochondrial glycerophosphate dehydrogenase in patients with IDDM.

The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (mGDH) plays a key role in the recognition of D-glucose as a stimulus for insulin release from the pancreatic islet B-cell. This study reveals that autoantibodies against this enzyme are not uncommonly found in patients with insulin-dependent diabetes mellitus (IDDM) examined at the onset of the disease. Antibodies reacting with a recombinant mGDH fragment product were observed in the serum of four out of 15 type-1 diabetics, but in none of 15 control subjects. The serum of patients positive for the recombinant mGDH fragment also recognized native mGDH in a rat testis extract, provided that the enzymatic protein was first exposed to an anti-mGDH rabbit serum. Antibodies against mGDH were also found in four out 12 patients with autoimmune thyroiditis. These findings reveal that a mitochondrial enzyme, that represents an essential component of the islet B-cell glucose-sensing device, may act as an antigenic determinant in patients with IDDM or other autoimmune diseases.

Apparent starvation-induced repression of pancreatic islet glucokinase.

Conflicting data were previously reported concerning the effect of starvation upon the glucokinase protein and glucokinase mRNA content of pancreatic islets. In the present study, conducted in fed rats and animals starved for 48 h, the catalytic activity of glucokinase in islet homogenates and the content of this enzyme in islets were both decreased by starvation to the same relative extent. These findings support the view that the altered metabolic and secretory response to D-glucose found in islets from starved rats may be attributable, in part at least, to an apparent repression of glucokinase.