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.

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.