Effects of islet neogenesis associated protein depend on vascular endothelial growth factor gene expression modulated by hypoxia-inducible factor 1-alpha.

BACKGROUND: Pharmacology has provided efficient tools to improve insulin effect/secretion but the decrease in ß-cell mass remains elusive. INGAP-PP could provide a therapeutic alternative to meet that challenge. AIM: To further understand the mechanism that links INGAP-PP effects upon ß-cell mass and function with islet angiogenesis. METHODOLOGY: Normal male Wistar rats were divided into 2 groups and injected with a single dose of 100 mg/Kg suramin or saline. Both groups were divided into 2 subgroups that received daily doses of 2 mg/kg INGAP-PP or saline for ten days. Plasma glucose, triacylglycerol, TBARS, and insulin levels were measured. Pancreas immunomorphometric analyses were also performed. Pancreatic islets were isolated to measure glucose-stimulated insulin secretion (GSIS). Specific islet mRNA levels were studied by qRT-PCR. Statistical analysis was done using ANOVA. RESULTS: No differences were recorded in body weight, food intake, or any other plasma parameter measured in all groups. Islets from INGAP-PP-treated rats significantly increased GSIS, ß-cell mass, and mRNA levels of Bcl-2, Ngn-3, VEGF-A, VEGF-R2, CD31, Ang1 and Ang2, Laminin ß-1, and Integrin ß-1, and decreased mRNA levels of Caspase-8, Bad, and Bax. Islets from suramin-treated rats showed significant opposite effects, but INGAPP-PP administration rescued most of the suramin effects in animals treated with both compounds. CONCLUSION: Our results reinforce the concept that INGAP-PP enhances insulin secretion and ß-cell mass, acting through PI3K/Akt/mTOR pathways and simultaneously activating angiogenesis through HIF-1α-mediated VEGF-A secretion. Therefore, INGAP-PP might be a suitable antidiabetic agent able to overcome two major alterations present in T2D.

Islet adaptive changes to fructose-induced insulin resistance: beta-cell mass, glucokinase, glucose metabolism, and insulin secretion.

Beta-cell mass, hexokinase/glucokinase (HK/GK) activity, glucose metabolism and insulin secretion were studied in the islets of rats with fructose-induced insulin resistance (IR). Normal male Wistar rats were fed a standard commercial diet and water without (control, C) or with 10% fructose-rich diet (FRD) for 3 weeks. Blood glucose (strips), triglyceride (commercial kit), and insulin (RIA) levels were measured at the time of death. Glucose-induced insulin release, glucose metabolism ((14)CO(2) and (3)H(2)O production from D-[U-(14)C]- and D-[5-(3)H]-glucose) and HK/GK activity (G-6-P production), transcription (RT-PCR), protein expression (Western blot), and cellular compartmentalization were measured in isolated islets (collagenase digestion). FRD rats presented normoglycemia but impaired glucose tolerance, hypertriglyceridemia, hyperinsulinemia, and increased HOMA-IR index. In these rats, beta-cell mass decreased significantly by 33%, with a 44% increase in the percentage of apoptotic cells. Glucose-induced insulin release and islet glucose metabolism were higher in FRD rats. While GK activity (total and cytosolic fraction) and protein expression were significantly higher in FRD islets, HK showed no change in any of these parameters. Our results demonstrate that the changes induced by dietary-induced IR upon beta-cell function and mass are strongly conditional on the nutrient model used. In our model (intact animals with impaired glucose tolerance), GK activity increases through mechanisms previously shown only in vitro or under highly hyperglycemic conditions. Such an increase plays a pivotal role in the adaptive increased release of insulin in response to IR, even in the presence of marked beta-cell mass reduction.

Enhanced expression of hexokinase I in pancreatic islets induced by sucrose administration.

Administration of a sucrose-rich diet (SRD) to normal hamsters induces an insulin-resistant state and a significant increase of insulin secretion and beta-cell mass. Islets isolated from these animals had a marked increase in glucose metabolism and glucose-induced insulin secretion, at both low and high glucose concentrations. They also presented increased hexokinase (HK) activity, without measurable changes in glucokinase (GK) activity. In this study we measured HK and GK activity in homogenates of islets isolated from normal control and SRD-fed hamsters, as well as in their particulate and cytosolic fractions. We also measured transcription rate (mRNA by reverse transcriptase PCR) and expression levels (Western blotting) of both enzymes in these islets. We found an increase in HK activity and expression levels, without measurable changes in HK mRNA level in SRD-fed animals. Whereas a similar GK activity was measured in homogenates of islets isolated from both groups, such activity was significantly higher in the cytosolic fraction of SRD islets. On the other hand, GK transcription rate and expression level were similar in both experimental groups. Our results suggest that the increased beta-cell secretory response to low glucose can be partly ascribed to an increased activity of islet HK consecutive to an enhanced expression of the enzyme, while the enhanced response to high glucose could be due to changes in GK compartmentalization.

Islet neogenesis: an apparent key component of long-term pancreas adaptation to increased insulin demand.

This study aimed to determine the relative importance of different functional and morphological pancreatic changes induced by the chronic administration of a sucrose-rich diet (SRD) to maintain normal glucose homeostasis. Male Wistar rats were fed either sucrose (SRD) or starch (CD) for 6 and 12 months. At both periods, serum glucose and triacylglycerol levels were significantly higher (P<0.05; paired and unpaired Student's t-test) in SRD rats. Serum insulin levels were significantly lower in SRD only at 12 months. At 6 months, the insulin secretion dose-response curve in SRD rats showed a shift to the left that was no longer observed at 12 months, when SRD islets decreased their response to 16 mM glucose. At 6 months, SRD rats showed a significant increase in beta-cell volume density (Vvi) and islet cell replication rate, together with a decrease in beta-cell apoptotic rate. Changes were not detected in the percentage of PDX-1- and islet neogenesis associated protein (INGAP)-positive cells. Conversely, at 12 months, there was a significant decrease in beta-cell Vvi and in the percentage of PDX-1-positive cells; the islet cell replication rate was not modified, and the number of apoptotic beta-cells increased significantly. No signs of increased neogenesis or INGAP-positive cells were recorded at any period in SRD rats. Our results show that SRD rats are unable to develop functional and morphological pancreatic reactive changes sufficient to maintain normal glucose and triacylglycerol levels for a long period. Such failure could be ascribed to their inability to increase the rate of neogenesis and of INGAP production.

Expression of islet neogenesis-associated protein in islets of normal hamsters.

The aim of the present study was to test the possible presence and expression of islet neogenesis-associated protein (INGAP) in islet cells of normal adult hamsters. Pancreata from normal male Syrian hamsters were removed to perform the following studies. (i) Western blot analysis using the cytosolic fraction from homogenates of isolated islets, exocrine tIssue and whole pancreas, and rabbit INGAP-specific antibody. (ii) Immunohistochemical identification of INGAP-positive cells in fixed sections of intact pancreata, fresh and 72 h cultured islets (isolated by collagenase digestion), and smears of exocrine pancreatic cells, using the same INGAP-specific antibody and streptavidin-biotin complex. (iii) RT-PCR using total RNA extracted from isolated islets and from exocrine tIssue as template, and a specific pair of primers. (iv) Control of the sequence of the PCR products. INGAP protein was identified by Western blot in the cytosolic fraction of homogenates from fresh isolated islets, exocrine cells and whole fresh pancreas. INGAP-immunopositive cells were observed in duct, exocrine and islet cells in either fixed intact or digested pancreatic tIssue. INGAP mRNA was identified in samples of total RNA from fresh and cultured isolated islets and from exocrine cells. Our data demonstrate that INGAP is present and expressed in islets and in exocrine pancreatic cells of normal hamsters. The ubiquitous localization of INGAP suggests its possible role in the physiological process of islet growth and its protective effect upon streptozotocin-induced diabetes.

Pancreatic duodenal homeobox-1 and islet neogenesis-associated protein: a possible combined marker of activateable pancreatic cell precursors.

The aim of this work was to study the possible relationship between pancreatic duodenal homeobox-1 (Pdx-1) and islet neogenesis-associated protein (INGAP) during induced islet neogenesis. Pregnant hamsters were fed with (S) and without (C) sucrose, and glycemia, insulin secretion in vitro, and pancreas immunomorphometric parameters were measured in their 7-day-old offspring. S offspring had significantly lower glycemic levels than C animals. Insulin release in response to increasing glucose concentrations in the incubation medium (2-16 mM glucose) did not increase in pancreata from either C or S offspring. However, pancreata from S offspring released more insulin than those from C animals. In S offspring, beta-cell mass, beta-cell replication rate and islet neogenesis increased significantly, with a simultaneous decrease in beta-cell apoptotic rate. INGAP- and Pdx-1-positive cell mass also increased in the islets and among acinar and duct cells. We found two subpopulations of Pdx-1 cells: INGAP-positive and INGAP-negative. Pdx-1/INGAP-positive cells did not stain with insulin, glucagon, somatostatin, pancreatic polypeptide, or neurogenin 3 antibodies. The increment of Pdx-1/INGAP-positive cells represented the major contribution to the Pdx-1 cell mass increase. Such increments varied among pancreas subsectors: ductal>insular>extrainsular. Our results suggested that INGAP participates in the regulation of islet neogenesis, and Pdx-1/INGAP-positive cells represent a new stem cell subpopulation at an early stage of development, highly activateable in neogenesis.

Mechanisms involved in the beta-cell mass increase induced by chronic sucrose feeding to normal rats.

The aim of the present study was to clarify the mechanisms by which a sucrose-rich diet (SRD) produces an increase in the pancreatic beta-cell mass in the rat. Normal Wistar rats were fed for 30 weeks either an SRD (SRD rats; 63% wt/wt), or the same diet but with starch instead of sucrose in the same proportion (CD rats). We studied body weight, serum glucose and triacylglycerol levels, endocrine tissue and beta-cell mass, beta-cell replication rate (proliferating cell nuclear antigen; PCNA), islet neogenesis (cytokeratin immunostaining) and beta-cell apoptosis (propidium iodide). Body weight (g) recorded in the SRD rats was significantly (P<0.05) larger than that of the CD group (556.0+/-8.3 vs 470.0+/-13.1). Both serum glucose and triacylglycerol levels (mmol/l) were also significantly higher (P<0.05) in SRD than in CD rats (serum glucose, 8.11+/-0.14 vs 6.62+/-0.17; triacylglycerol, 1.57+/-0.18 vs 0.47+/-0.04). The number of pancreatic islets per unit area increased significantly (P<0.05) in SRD rats (3.29+/-0.1 vs 2.01+/-0.2). A significant increment (2.6 times) in the mass of endocrine tissue was detected in SRD animals, mainly due to an increase in the beta-cell mass (P=0.0025). The islet cell replication rate, measured as the percentage of PCNA-labelled beta cells increased 6.8 times in SRD rats (P<0.03). The number of apoptotic cells in the endocrine pancreas decreased significantly (three times) in the SRD animals (P=0.03). The cytokeratin-positive area did not show significant differences between CD and SRD rats. The increase of beta-cell mass induced by SRD was accomplished by an enhanced replication of beta cells together with a decrease in the rate of beta-cell apoptosis, without any evident participation of islet neogenesis. This pancreatic reaction was unable to maintain serum glucose levels of these rats at the level measured in CD animals.

Expression and cellular distribution pattern of plasma membrane calcium pump isoforms in rat pancreatic islets.

This work is aimed at identifying the presence and cellular distribution pattern of plasma membrane calcium pump (PMCA) isoforms in normal rat pancreatic islet. Microsomal fractions of isolated islets and exocrine tissue were analyzed to detect different PMCA isoforms. The cellular distribution pattern of these PMCAs in the islets was also studied in fixed pancreas sections incubated with antibodies against PMCAs and insulin. Antibody 5F10, which reacts with all PMCA variants, showed multiple bands in the blots in the 127-134 kDa region, indicating the presence of several isoforms. Microsomes also reacted positively with specific antibodies for individual PMCA isoforms, generating a band of the expected size. Antibody 5F10 immunocytochemically labeled the plasma cell membrane of both b- and non-b-cells, but predominantly the former. All islet cells were also labeled with antibodies against isoforms 1 and 4, while the antibody reacting with isoform 3 labeled exclusively b-cells. A few b- and non-b-cells were positively labeled with the antibody reacting with PMCA b variant. Negative results were obtained with the antibody against isoform 2. Further studies, together with previous reports on the modulatory effect of insulin secretagogues and blockers upon PMCA activity, may provide evidence of the importance of this particular PMCA expression for islet function under normal and pathological conditions.

Changes induced by sucrose administration on glucose metabolism in pancreatic islets in normal hamsters.

We correlated the changes in glucose-induced insulin secretion with those observed in glucose metabolism and hexokinase/glucokinase activity in islets from normal sucrose-fed hamsters. Blood glucose and insulin levels were measured in normal male hamsters fed with (S5) or without (C5) 10% sucrose in the drinking water for 5 weeks. Isolated islets (collagenase digestion) from both groups of animals were used to study insulin secretion, (14)CO(2) and (3)H(2)O production from D-[U-(14)C]-glucose and D-[5-(3)H]-glucose respectively, with 3.3 or 16.7 mM glucose in the medium, and hexokinase/glucokinase activity (fluorometric assay) in islet homogenates. Whereas S5 and C5 animals had comparable normal blood glucose levels, S5 showed higher insulin levels than C5 hamsters (2.3+/-0.1 vs 0.6+/-0.03 ng/ml, P<0.001). Islets from S5 hamsters released significantly more insulin than C5 islets in the presence of low and high glucose (3.3 mM glucose: 0.77+/-0.04 vs 0.20+/-0.06 pg/ng DNA/min, P<0.001; 16.7 mM glucose: 2.77+/-0.12 vs 0.85+/-0.06 pg/ng DNA/min, P<0.001) and produced significantly higher amounts of (14)CO(2) and (3)H(2)O at both glucose concentrations ((14)CO(2): 3.3 mM glucose: 0.27+/-0.01 vs 0.18+/-0.01, P<0.001; 16.7 mM glucose: 1.44+/-0.15 vs 0.96+/-0.08, P<0.02; (3)H(2)O: 3.3 mM glucose: 0.31+/-0.02 vs 0.15+/-0.01, P<0.001; 16.7 mM glucose: 1.46+/-0.20 vs 0.76+/-0.05 pmol glucose/ng DNA/min, P<0.005). The hexokinase K(m) and V(max) values from S5 animals were significantly higher than those from C5 ones (K(m): 100.14+/-7.01 vs 59.90+/- 3.95 microM, P<0.001; V(max): 0.010+/-0.0005 vs 0.008+/- 0.0006 pmol glucose/ng DNA/min, P<0.02). Conversely, the glucokinase K(m) value from S5 animals was significantly lower than in C5 animals (K(m): 15.31+/-2.64 vs 35.01+/-1.65 mM, P<0.001), whereas V(max) figures were within a comparable range in both groups (V(max): 0.048+/-0.009 vs 0.094+/-0.035 pmol glucose/ng DNA/min, not significant). The glucose phosphorylation ratio measured at 1 and 100 mM (hexokinase/glucokinase ratio) was significantly higher in S5 (0.26+/-0.02) than in C5 animals (0.11+/-0.01, P<0.005), and it was attributable to an increase in the hexokinase activity in S5 animals. In conclusion, sucrose administration increased the hexokinase/glucokinase activity ratio in the islets, which would condition the increase in glucose metabolism by beta-cells, and in beta-cell sensitivity and responsiveness to glucose. These results support the concept that increased hexokinase rather than glucokinase activity causes the beta-cell hypersensitivity to glucose, hexokinase being metabolically more active than glucokinase to up-regulate beta-cell function.

Mecanismo de producción del aumento de la masa de células B inducido por la administración crónica de sacarosa a ratas normales / Mechanisms producing an increase in B-cell mass induced by the chronic administration of sucrose to normal rats

El objetivo del presente trabajo fue clarificar los mecanismos mediante los cuales una dieta rica en sacarosa produce, en el páncreas de la rata, un aumento de la masa celular B. Se utilizaron ratas Wistar normales, alimentadas durante 30 semanas con una dieta rica en sacarosa (DRS; 63 por ciento o bien con una dieta similar en la que la sacarosa se reemplazó por la misma proporción de almidón (DC). Se estudió el peso corporal, los niveles séricos de glucosa y triacilglicerol, el tejido endocrino, la masa celular B, el grado de replicación de las células B (antígeno de proliferación nuclear celular, PCNA), la neogénesis insular (inmunomarcación de citoqueratina, CK) y la apoptosis de células B (yoduro de propidio). El peso corporal de las ratas DRS fue significativamente mayor (p<0,05) que el registrado en el grupo DC (556,0ñ8,3 vs 470,0ñ13,1). Tanto los niveles séricos de glucosa como los de triacilglicerol (mmol/L) fueron significativamente mayores (p<0,05) en las ratas DRS que en las DC: Glucemia 8,11 ñ 0,14 vs 6,62 ñ 0,17; triacilglicerol 1,57 ñ 0,18 vs 0,47 ñ 0,04. El número de islotes pancreáticos por unidad de área aumentó significativamente (p<0,05) en las ratas DRS (3,29 ñ 0,1 vs 2,01 ñ 0,2). Además, se detectó un incremento significativo (2,6 veces) en la masa de tejido endocrino de los animales DRS, principalmente debido a un aumento en la masa celular B (p=0,0025. El porcentaje de células B insulares en replicación (PCNA positivas) aumentó 6,8 veces en las ratas DRS (p<0,03). El número de células apoptóticas del páncreas endocrino disminuyó significativamente en el grupo DRS (3 veces, p=0,03). No hubo diferencias significativas en el área positiva para CK entre los animales DRS y DC. El aumento de la masa celular B inducido por la DRS se correspondió con un aumento en la replicación de las células B, junto con una disminución en la proporción de dichas células en apoptosis. No hubo evidencias de neogénesisen los islotes. Dichos cambios pancreáticos no pudieron mantener la glucemia de estos animales en valores semejantes a los obtenidos en las ratas DC. Estos resultados muestran que en este modelo de manipulación alimentaria, aunque el aumento de replicación de las células B y la disminución de la apoptosis jugarían un rol importante, la neogénesis insular constituiría el mecanismo compensador decisivo en la adaptación a la mayor demanda de insulina

Mecanismo de producción del aumento de la masa de células B inducido por la administración crónica de sacarosa a ratas normales / Mechanisms producing an increase in B-cell mass induced by the chronic administration of sucrose to normal rats

El objetivo del presente trabajo fue clarificar los mecanismos mediante los cuales una dieta rica en sacarosa produce, en el páncreas de la rata, un aumento de la masa celular B. Se utilizaron ratas Wistar normales, alimentadas durante 30 semanas con una dieta rica en sacarosa (DRS; 63 por ciento o bien con una dieta similar en la que la sacarosa se reemplazó por la misma proporción de almidón (DC). Se estudió el peso corporal, los niveles séricos de glucosa y triacilglicerol, el tejido endocrino, la masa celular B, el grado de replicación de las células B (antígeno de proliferación nuclear celular, PCNA), la neogénesis insular (inmunomarcación de citoqueratina, CK) y la apoptosis de células B (yoduro de propidio). El peso corporal de las ratas DRS fue significativamente mayor (p<0,05) que el registrado en el grupo DC (556,0ñ8,3 vs 470,0ñ13,1). Tanto los niveles séricos de glucosa como los de triacilglicerol (mmol/L) fueron significativamente mayores (p<0,05) en las ratas DRS que en las DC: Glucemia 8,11 ñ 0,14 vs 6,62 ñ 0,17; triacilglicerol 1,57 ñ 0,18 vs 0,47 ñ 0,04. El número de islotes pancreáticos por unidad de área aumentó significativamente (p<0,05) en las ratas DRS (3,29 ñ 0,1 vs 2,01 ñ 0,2). Además, se detectó un incremento significativo (2,6 veces) en la masa de tejido endocrino de los animales DRS, principalmente debido a un aumento en la masa celular B (p=0,0025. El porcentaje de células B insulares en replicación (PCNA positivas) aumentó 6,8 veces en las ratas DRS (p<0,03). El número de células apoptóticas del páncreas endocrino disminuyó significativamente en el grupo DRS (3 veces, p=0,03). No hubo diferencias significativas en el área positiva para CK entre los animales DRS y DC. El aumento de la masa celular B inducido por la DRS se correspondió con un aumento en la replicación de las células B, junto con una disminución en la proporción de dichas células en apoptosis. No hubo evidencias de neogénesisen los islotes. Dichos cambios pancreáticos no pudieron mantener la glucemia de estos animales en valores semejantes a los obtenidos en las ratas DC. Estos resultados muestran que en este modelo de manipulación alimentaria, aunque el aumento de replicación de las células B y la disminución de la apoptosis jugarían un rol importante, la neogénesis insular constituiría el mecanismo compensador decisivo en la adaptación a la mayor demanda de insulina (AU)

Effect of an acute glucose overload on Islet cell morphology and secretory function in the toad.

The aim of this work was to study the effect of induced hyperglycemia on islet cell mass and insulin secretion in normal toads. Immunolabeled beta cell area, replication (bromodeoxyuridine) and apoptosis (propidium iodide) rate, islet neogenesis (cytokeratin), and insulin secretion in vitro were measured in adult male specimens of Bufo arenarum during and after interruption of the injection of either a 50% glucose solution (2 g/100 g) or its vehicle for 4 days. Glucose administration caused hyperglycemia (122.6 +/- 16.7 and 508.3 +/- 115.9 mg/dl vs 23.5 +/- 1.26 and 22.8 +/- 1.8 mg/dl, at days 3 and 5, respectively, P < 0.05) and a significant decrease in the number of islets/mm(2) (day 3: 9.7 +/- 0.9 vs 3.3 +/- 0.4, P < 0.05; day 5: 9.4 +/- 0.8 vs 7.4 +/- 0.6; day 9: 9.6 +/- 0.9 vs 6.2 +/- 0.4, P < 0.05) and in the percentage of immunolabeled beta cell area (day 3: 2.07 +/- 0.2 vs 0.5 +/- 0.1%, P < 0.05; day 5: 1.8 +/- 0.1 vs 0.6 +/- 0.1%; day 9: 1.7 +/- 0.1 vs 0.7 +/- 0.1%, P < 0.05). Glucose-injected animals had a simultaneous significantly higher percentage of BrdU-labeled beta cells (day 3: 0.46 +/- 0.02 vs 0.23 +/- 0.03%; day 5: 0.54 +/- 0.13 vs 0.22 +/- 0.02%; day 9: 0.61 +/- 0.0 vs 0.27 +/- 0.05%, P < 0.05) and cytokeratin-labeled endocrine cells (day 3: 0.21 +/- 0.06 vs 0.01 +/- 0.00%; day 5: 0.17 +/- 0.06 vs 0.01 +/- 0.01%; day 9: 1.25 +/- 0.2 vs 0.01 +/- 0.008%, P < 0.05) and a higher rate of apoptotic beta cells (day 3: 0.14 +/- 0.04 vs 0.05 +/- 0.02%; day 5: 0.4 +/- 0.06 vs 0.05 +/- 0.2, P < 0.05; day 9: 0.47 +/- 0.04 vs 0.06 +/- 0.03, P < 0.05). Comparable amounts of insulin were secreted in vitro by both groups in response to 2 mM glucose, whereas there was a significantly reduced response to 8 mM glucose in treated animals (day 3: 73 +/- 12 vs 165 +/- 20%; day 5: 74 +/- 11 vs 204 +/- 18%, P < 0.05). This decreased response to high glucose reverted to normal after removal of the glucose injection. These results show for the first time that short-term hyperglycemia triggers marked morphological and transient secretory changes in the toad pancreas similar in part to those elicited in the pancreas of several mammals. As with other results previously reported, these results support the usefulness of the toad as an alternative easily handled model to study the growth and secretory function of the endocrine pancreas.

Quantitative ultrastructural changes induced by sucrose administration in the pancreatic B cells of normal hamsters.

We have previously reported that young male Syrian hamsters receiving a sucrose-rich diet presented increased B-cell replication rate and size. The aim of the present study was to analyze, under the same experimental conditions, the ultrastructural changes in B cells. For this purpose, young male Syrian hamsters were fed with a commercial diet and 10% sucrose in their drinking water (S group) while the control group (C) received the same diet and tap water, for 5 weeks. Samples of the pancreas removed after that period were processed for the immunohistochemical identification of B cells as well as for measuring several ultrastructural parameters. S hamsters showed higher serum insulin levels, while similar serum glucose values were obtained in animals from both groups. The B cells from S group exhibited lesser number of dense secretory granules at expenses of an increase of the pale ones, increased number of both exocytosis profiles and fusion-granule images, as well as enlargement of the intercellular space and mitochondrial area. Marked expansions of this space, limited by junctional complexes, were observed between adjacent B cells. These results would indicate that sucrose administration to normal hamsters not only increases the pancreatic B-cell mass but also induces measurable subcellular changes in the individual B-cell characteristic of an enhanced secretory activity. The present model would represent a useful tool for testing strategies in preventing the damage or promoting the recovery of the pancreatic B cells.

Possible relationship between changes in islet neogenesis and islet neogenesis-associated protein-positive cell mass induced by sucrose administration to normal hamsters.

The possible relationship between changes in islet cell mass and in islet neogenesis-associated protein (INGAP)-cell mass induced by sucrose administration to normal hamsters was investigated. Normal hamsters were given sucrose (10% in drinking water) for 5 (S8) or 21 (S24) weeks and compared with control (C) fed hamsters. Serum glucose and insulin levels were measured and quantitative immunocytochemistry of the endocrine pancreas was performed. Serum glucose levels were comparable among the groups, while insulin levels were higher in S hamsters. There was a significant increase in beta-cell mass (P<0.02) and in beta-cell 5-bromo-2'-deoxyuridine index (P<0.01), and a significant decrease in islet volume (P<0.01) only in S8 vs C8 hamsters. Cytokeratin (CK)-labelled cells were detected only in S8 hamsters. INGAP-positive cell mass was significantly larger only in S8 vs C8 hamsters. Endocrine INGAP-positive cells were located at the islet periphery ( approximately 96%), spread within the exocrine pancreas ( approximately 3%), and in ductal cells (<1%) in all groups. INGAP positivity and glucagon co-localization varied according to topographic location and type of treatment. In C8 hamsters, 49.1+/-6. 9% cells were INGAP- and glucagon-positive in the islets, while this percentage decreased by almost half in endocrine extra-insular and ductal cells. In S8 animals, co-expression increased in endocrine extra-insular cells to 36.3+/-9.5%, with similar figures in the islets, decreasing to 19.7+/-6.9% in ductal cells. INGAP-positive cells located at the islet periphery also co-expressed CK. In conclusion, a significant increase of INGAP-positive cell mass was only observed at 8 weeks when neogenesis was present, suggesting that this peptide might participate in the control of islet neogenesis. Thus, INGAP could be a potentially useful tool to treat conditions in which there is a decrease in beta-cell mass.

Quantitative ultrastructural changes induced by sucrose administration in the pancreatic B cells of normal hamsters

We have previously reported that young male Syrian hamsters receiving a sucrose-rich diet presented increased B-cell replication rate and size. The aim of the present study was to analyze, under the same experimental conditions, the ultrastructural changes in B cells. For this purpose, young male Syrian hamsters were fed with a commercial diet and 10 sucrose in their drinking water (S group) while the control group (C) received the same diet and tap water, for 5 weeks. Samples of the pancreas removed after that period were processed for the immunohistochemical identification of B cells as well as for measuring several ultrastructural parameters. S hamsters showed higher serum insulin levels, while similar serum glucose values were obtained in animals from both groups. The B cells from S group exhibited lesser number of dense secretory granules at expenses of an increase of the pale ones, increased number of both exocytosis profiles and fusion-granule images, as well as enlargement of the intercellular space and mitochondrial area. Marked expansions of this space, limited by junctional complexes, were observed between adjacent B cells. These results would indicate that sucrose administration to normal hamsters not only increases the pancreatic B-cell mass but also induces measurable subcellular changes in the individual B-cell characteristic of an enhanced secretory activity. The present model would represent a useful tool for testing strategies in preventing the damage or promoting the recovery of the pancreatic B cells.

Quantitative ultrastructural changes induced by sucrose administration in the pancreatic B cells of normal hamsters

We have previously reported that young male Syrian hamsters receiving a sucrose-rich diet presented increased B-cell replication rate and size. The aim of the present study was to analyze, under the same experimental conditions, the ultrastructural changes in B cells. For this purpose, young male Syrian hamsters were fed with a commercial diet and 10 sucrose in their drinking water (S group) while the control group (C) received the same diet and tap water, for 5 weeks. Samples of the pancreas removed after that period were processed for the immunohistochemical identification of B cells as well as for measuring several ultrastructural parameters. S hamsters showed higher serum insulin levels, while similar serum glucose values were obtained in animals from both groups. The B cells from S group exhibited lesser number of dense secretory granules at expenses of an increase of the pale ones, increased number of both exocytosis profiles and fusion-granule images, as well as enlargement of the intercellular space and mitochondrial area. Marked expansions of this space, limited by junctional complexes, were observed between adjacent B cells. These results would indicate that sucrose administration to normal hamsters not only increases the pancreatic B-cell mass but also induces measurable subcellular changes in the individual B-cell characteristic of an enhanced secretory activity. The present model would represent a useful tool for testing strategies in preventing the damage or promoting the recovery of the pancreatic B cells.(AU)

Quantitative ultrastructural changes induced by sucrose administration in the pancreatic B cells of normal hamsters.

We have previously reported that young male Syrian hamsters receiving a sucrose-rich diet presented increased B-cell replication rate and size. The aim of the present study was to analyze, under the same experimental conditions, the ultrastructural changes in B cells. For this purpose, young male Syrian hamsters were fed with a commercial diet and 10

sucrose in their drinking water (S group) while the control group (C) received the same diet and tap water, for 5 weeks. Samples of the pancreas removed after that period were processed for the immunohistochemical identification of B cells as well as for measuring several ultrastructural parameters. S hamsters showed higher serum insulin levels, while similar serum glucose values were obtained in animals from both groups. The B cells from S group exhibited lesser number of dense secretory granules at expenses of an increase of the pale ones, increased number of both exocytosis profiles and fusion-granule images, as well as enlargement of the intercellular space and mitochondrial area. Marked expansions of this space, limited by junctional complexes, were observed between adjacent B cells. These results would indicate that sucrose administration to normal hamsters not only increases the pancreatic B-cell mass but also induces measurable subcellular changes in the individual B-cell characteristic of an enhanced secretory activity. The present model would represent a useful tool for testing strategies in preventing the damage or promoting the recovery of the pancreatic B cells.

Changes induced by sucrose administration upon the morphology and function of pancreatic islets in the normal hamster.

BACKGROUND: This report documents sequential changes in islet morphology (cell replication and islet neogenesis) and glucose-induced insulin secretion in young normal male Syrian hamsters. METHODS: Three-week-old animals received a control standard commercial diet or this diet supplemented with sucrose--10% (w/v) solution in drinking water, a treatment that stimulated pancreatic growth and function--for 5 (C5/S5) or 21 (C21/S21) weeks. Insulin secretion and content were measured in isolated islets, while several biochemical parameters were assessed in serum. Different morphological features were analysed in the endocrine pancreas by quantitative immunocytochemistry. RESULTS: Serum glucose, triglycerides and total cholesterol levels were comparable among the groups, whereas serum- and pancreatic-insulin levels were higher in the S hamsters. Islets from S21 hamsters released more insulin than those from C21 animals at all glucose concentrations tested. The volume densities of the total endocrine pancreas (1.9 +/- 0.2 vs 1.2 +/- 0.2; p < 0.02) of the beta-cell subpopulation, the islet number per unit area (2.4 +/- 0.1 vs 1.2 +/- 0.1; p < 0.0004) and the beta-cell mass (4.2 +/- 0.5 vs 2.3 +/- 0.5; p < 0.01) were significantly higher in S5 vs C5 animals. Conversely, the islet volume and the number of beta cells/islets were significantly smaller in S5 than in C5 animals. The beta-cell replication rate in S5 hamsters was 10-fold that of C5 animals. All these parameters had comparable values in S21 and C21 animals. We detected cytokeratin-labelled cells located at the islet periphery (in alpha cells) and among the ductular cells, only in the S5 hamsters. CONCLUSIONS: Sucrose administration to young hamsters causes time-dependent pancreatic modifications, with morphological changes (increase in islet- and in beta-cell mass with incremented beta-cell replication rate and evidence of islet neogenesis) occurring at 5 weeks and insulin secretion (increase in insulin sensitivity to glucose) being mainly affected at 21 weeks. This experimental model could prove useful for studying the mechanisms underlying the control of islet-cell population distribution and for developing new strategies in preventing cell damage.

Postnatal sequential changes in islet morphology and insulin secretion of normal hamsters.

We have studied the postnatal development of the endocrine pancreas from normal female Syrian golden hamsters 1, 8, and 24 weeks of age. The observations were made by (a) analysis of insulin secretion in response to glucose using isolated pancreatic islets and (b) identification and quantitation of insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-secreting cells. Glucose-induced insulin secretion showed typical dose-response curves. However, whereas in 24-week-old animals maximal secretion was already present with 8 mM glucose, in younger hamsters such a response was attained only with 20 mM glucose. The volume density of the endocrine pancreas and the number of islets were increased in 1-week-old hamsters compared to the older animals. The islet volume average in 8-week-old hamsters was almost three times higher than that measured in 1-week-old animals. However, the proportion and size of each cell type in the islets did not present significant differences among the groups studied. Our results show that, in hamsters, the endocrine pancreas reaches the adult general characteristics late after birth. Furthermore, the definite morphological pattern is attained far earlier than the secretory response. These observations provide basic information for further studies regarding the mechanisms and factors that control both the growth and the differentiation of endocrine cell populations as well as glucose-induced insulin secretion in a simple experimental model.