Héctor Del Zotto

INGAP-related pentadecapeptide: Its modulatory effect upon insulin secretion

We examined the effects of a pentadecapeptide having the 104-118 aminoacid sequence of islet neogenesis-associated protein (INGAP-PP) on insulin secretion, and the morphological characteristics of adult and neonatal pancreatic rat islets cultured in RPMI and 10 mM glucose for 4 days, with or without different INGAP-PP concentrations (0.1-100 mug/ml). A scrambled 15 aminoacid peptide was used as control for the specificity of INGAP-PP effect. Cultured neonatal and adult islets released insulin in response to glucose (2.8-16.7 mM) in a dose-dependent manner, and to leucine and arginine (10 mM). In all cases, the response was greater in adult islets. INGAP-PP added to the culture medium significantly enhanced glucose- and aminoacid-induced insulin release in both adult and newborn rats; however, no changes were observed with the scrambled peptide. Similar results were obtained incubating freshly isolated adult rat islets with INGAP-PP. Whereas INGAP-PP did not induce significant changes in islet survival rate or proportion/number of islet cells, it increased significantly beta-cell size. This first demonstration of the enhancing effect of INGAP-PP on the beta-cell secretory response of adult and newborn islets opens a new avenue to study its production mechanism and potential use to increase the secretory capacity of endogenous islets in intact animals or of islets preserved for future transplants.

Islet Neogenesis Associated Protein (INGAP) modulates gene expression in cultured neonatal rat islets.

The Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. We currently studied the effects of a pentadecapeptide having the 104-118 amino acid sequence of INGAP (INGAP-PP) on insulin secretion and on transcript profile expression in 4-day-cultured normal pancreatic neonatal rat islets. Islets cultured with INGAP-PP released significantly more insulin in response to 2.8 and 16.7 mM glucose than those cultured without the peptide. The macroarray analysis showed that 210 out of 2352 genes spotted in the nylon membranes were up-regulated while only 4 were down-regulated by INGAP-PP-treatment. The main categories of genes modified by INGAP-PP included several related with islet metabolism, insulin secretion mechanism, beta-cell mass and islet neogenesis. RT-PCR confirmed the macroarray results for ten selected genes involved in growing, maturation, maintenance of pancreatic islet-cells, and exocytosis, i.e., Hepatocyte nuclear factor 3beta (HNF3beta), Upstream stimulatory factor 1 (USF1), K(+)-channel proteins (SUR1 and Kir6.2), PHAS-I protein, Insulin 1 gene, Glucagon gene, Mitogen-activated protein kinase 1 (MAP3K1), Amylin (IAPP), and SNAP-25. INGAP-PP also stimulated PDX-1 expression. The expression of three transcripts (HNF3beta, SUR1, and SNAP-25) was confirmed by Western blotting for the corresponding proteins. In conclusion, our results show that INGAP-PP enhances specifically the secretion of insulin and the transcription of several islet genes, many of them directly or indirectly involved in the control of islet metabolism, beta-cell mass and islet neogenesis. These results, together with other previously reported, strongly indicate an important role of INGAP-PP, and possibly of INGAP, in the regulation of islet function and development.

Molecular mechanisms of tungstate-induced pancreatic plasticity: a transcriptomics approach

BackgroundSodium tungstate is known to be an effective anti-diabetic agent, able to increase beta cell mass in animal models of diabetes, although the molecular mechanisms of this treatment and the genes that control pancreas plasticity are yet to be identified. Using a transcriptomics approach, the aim of the study is to unravel the molecular mechanisms which participate in the recovery of exocrine and endocrine function of streptozotocin (STZ) diabetic rats treated with tungstate, determining the hyperglycemia contribution and the direct effect of tungstate.ResultsStreptozotocin (STZ)-diabetic rats were treated orally with tungstate for five weeks. Treated (STZ)-diabetic rats showed a partial recovery of exocrine and endocrine function, with lower glycemia, increased insulinemia and amylasemia, and increased beta cell mass achieved by reducing beta cell apoptosis and raising beta cell proliferation. The microarray analysis of the pancreases led to the identification of three groups of differentially expressed genes: genes altered due to diabetes, genes restored by the treatment, and genes specifically induced by tungstate in the diabetic animals. The results were corroborated by quantitative PCR. A detailed description of the pathways involved in the pancreatic effects of tungstate is provided in this paper. Hyperglycemia contribution was studied in STZ-diabetic rats treated with phloridzin, and the direct effect of tungstate was determined in INS-1E cells treated with tungstate or serum from untreated or treated STZ-rats, observing that tungstate action in the pancreas takes places via hyperglycemia-independent pathways and via a combination of tungstate direct and indirect (through the serum profile modification) effects. Finally, the MAPK pathway was evaluated, observing that it has a key role in the tungstate-induced increase of beta cell proliferation as tungstate activates the mitogen-activated protein kinase (MAPK) pathway directly by increasing p42/p44 phosphorylation and indirectly by decreasing the expression of raf kinase inhibitor protein (Rkip), a negative modulator of the pathway.ConclusionIn conclusion, tungstate improves pancreatic function through a combination of hyperglycemia-independent pathways and through its own direct and indirect effects, whereas the MAPK pathway has a key role in the tungstate-induced increase of beta cell proliferation.

INGAP-PP up-regulates the expression of genes and proteins related to K-ATP(+) channels and ameliorates Ca2+ handling in cultured adult rat islets

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K(+)(ATP) channel, Ca(2+) handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca(2+) ([Ca(2+)](i)), static and dynamic insulin secretion, and (86)Rb efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 muM tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC(50) of 10.0+/-0.4 vs. 13.7+/-1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 microM tolbutamide and accelerated the velocity of glucose-induced reduction of (86)Rb efflux in perifused islets. These effects were accompanied by a significant increase in [Ca(2+)](i) and the maintenance of a considerable degree of [Ca(2+)](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K(+)(ATP) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca(2+)](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes.

Islet neogenesis-associated protein pentadecapeptide (INGAP-PP): Mechanisms involved in its effect upon β-cell mass and function

The effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) administration to normal male hamsters upon serum glucose and triglyceride levels, beta-cell mass and function was studied. INGAP-PP (500 mug) or saline was injected twice daily during 10 days. Both groups showed comparable body weight, serum glucose and triglyceride levels. INGAP-PP treated animals had significantly higher HOMA-IR and HOMA-beta and their islets released more insulin in response to glucose; they had lower islet DNA content, significantly increased number of islets/unit area, beta-cell replication rate and mass, cells co-expressing Pdx-1/INGAP and islets in contact with ducts, and decreased beta-cell apoptosis rate. The percentage of cells expressing Pdx-1 alone or together with INGAP or insulin increased significantly in ducts. These animals also showed a significantly higher concentration of Pdx-1 and Ngn-3 mRNA and a lower number of INGAP-positive cells. In conclusion, INGAP-PP promoted a controlled and functionally active increase of beta-cell mass; our data demonstrate for the first time the mechanism responsible for such changes; that Ngn-3 would be involved in INGAP-PP-induced neogenesis; and the existence of a negative feedback loop with endogenous INGAP-producing cells. Accordingly, INGAP-PP could be used to induce these effects in people with or at risk of developing diabetes.

Transcription, expression and tissue binding in vivo of INGAP and INGAP-related peptide in normal hamsters

We studied islet neogenesis-associated protein (INGAP) transcription and its immunocytochemical presence in and binding in vivo of (125)I-tyrosylated INGAP pentadecapeptide ((125)I-T-INGAP-PP) to different normal male hamster tissues. (125)I-T-INGAP-PP was injected intraperitoneally with or without unlabeled T-INGAP-PP (0-1 mg/100 g bw), drawing blood samples at different times after injection; radioactivity was measured in serum, brain, skeletal muscle, dorsal root ganglia, liver, kidney, small intestine and pancreas samples, expressing results as organ:serum ratio. INGAP transcription (RT-PCR) and immunopositive cells were investigated in liver, kidney, brain, small intestine and pancreas. Total serum radioactivity increased progressively as a function of time; whereas 71% of this activity was displaced by unlabeled T-INGAP-PP at 5, 10 and 20 min, only 9% was at 60 min. Only liver, pancreas and small intestine specifically bound (125)I-T-INGAP-PP. The pancreas tissue dose-response curve showed a 50% displacement at 3.9x10(4) ng/100 g bw, suggesting a low binding affinity of its receptor. INGAP-mRNA was only identified in pancreatic islets and exocrine tissue. Our results suggest that INGAP transcription/expression is probably restricted to pancreas cells exerting its effect in a paracrine fashion. INGAP would be released and circulate bound to a serum protein from where it is bound and inactivated by the liver. Tissue binding could also explain INGAPs immunocytochemical presence in small intestine, where it could affect epithelial cell turnover.

Selective effect of INGAP-PP upon mouse embryonic stem cell differentiation toward islet cells

We evaluated the effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) upon islet beta- and non-beta cell differentiation from mouse embryonic stem (mES) cells. ES-D3 cell lines were cultured following Lumelskys protocol with or without INGAP-PP (5 microg/ml) at different stages. Gene expression was quantified using qPCR. mES cells were fixed and immunostained using anti insulin-, somatostatin-, glucagon-, Pdx-1-, Ngn-3-, Nkx-6.1 and PGP9.5 specific antibodies. PCNA was used to measure replication rate. Bcl(2) (immunostaining) and caspase-3 (enzyme activity and gene expression) were determined as apoptosis markers. INGAP-PP increased IAPP, Glut-2, Kir-6.2, SUR-1 and insulin gene expression, and the percentage of insulin-immunostained cells. Conversely, INGAP-PP reduced significantly glucagon and somatostatin gene expression and immunopositivity. While nestin gene expression was not affected, there was a significant reduction in the percentage of PGP9.5-immunostained cells. Pdx-1 gene expression increased by 115% in INGAP-PP treated cells, as well as the percentage of Pdx-1, Ngn-3 and Nkx-6.1 immunopositive cells. Neither caspase-3 (expression and activity) nor Bcl(2) positively immunostained cells were affected by INGAP-PP. Accordingly, INGAP-PP would promote stem cell differentiation into a beta-like cell phenotype, simultaneously decreasing its differentiation toward non-beta-cell precursors. Therefore, INGAP-PP would be potentially useful to obtain beta-cells from stem cells for replacement therapy.

Islet Cannabinoid Receptors Cellular Distribution and Biological Function

Objectives This study aimed to determine the cellular distribution of islet cannabinoid receptors (CBs) and their involvement in the development of metabolic and hormonal changes in rats fed a fructose-rich diet (F). Methods In normal rat islets, we determined CBs (immunofluorescence and retrotranscription–polymerase chain reaction) and glucose-stimulated insulin secretion (GSIS) of isolated islets incubated with the CB1 antagonist rimonabant (R) and/or different CBs agonists. In 3-week F-fed rats, we determined the in vivo effect of R on serum glucose, triglyceride, and insulin levels; homeostasis model assessment for insulin resistance, GSIS, and CBs and insulin receptor substrate gene expression levels (real-time polymerase chain reaction). Results Cannabinoid receptors appeared exclusively in islet &agr; cells. Whereas different CB agonists enhanced GSIS in normal rat islets, R did not affect it. F rats had higher serum triglyceride and insulin levels and homeostasis model assessment for insulin resistance than control rats; these alterations were prevented by R coadministration. Although R did not correct the increased GSIS observed in F islets, it modulated CBs and insulin receptor substrate gene expression. Conclusions Islet CBs would exert an important modulatory role in metabolic homeostasis. Administration of R and F affected islet CB expression and prevented the development of F-induced metabolic impairment. Selective islet CB1 blockers could be useful to prevent/treat the alterations induced by the intake of unbalanced/unhealthy diets.

Islet neogenesis-associated protein (INGAP)-positive cell mass, β-cell mass, and insulin secretion: their relationship during the fetal and neonatal periods

Objectives To study the chronological appearance of pancreatic islet neogenesis–associated protein (INGAP)-positive cells and its correlation with the increase in &bgr;-cell mass and function in fetal and neonatal rats. Methods Normal Wistar rat embryos (E) at gestational days 15, 17, and 19 (E15, E17, E19) and 7-day-old postnatal rats (P7) were humanely killed to determine body and pancreas weight; blood glucose; glucose and arginine-induced insulin secretion; real-time polymerase chain reaction of Pdx1 and Ngn3; quantitative immunomorphometric analysis of &bgr;-cell replication and apoptosis rate, cytokeratin and INGAP cell mass, and Pdx-1– and Ngn3-positive cells. Results Body and pancreas weight increased with age (P7 > E19 > E17 > E15; P < 0.05). Neonates had higher blood glucose concentrations than embryos (P < 0.05). We recorded a simultaneous and significant age-dependent trend of increase in the number of &bgr;- and Pdx-1-positive cells, &bgr;- and cytokeratin-positive cell mass and &bgr;-cell capacity to release insulin in response to glucose and arginine, and decreased &bgr;-cell apoptotic rate. These changes closely paralleled the increase in INGAP-positive cell mass. Conclusions These findings suggest that INGAP exerts a positive modulatory effect on &bgr;-cell mass and its secretory function in fetal and neonatal rats, thus becoming a new component in the multifactorial regulation of such processes.