Luis Emilio Flores

Islet NADPH oxidase activity modulates β-cell mass and endocrine function in rats with fructose-induced oxidative stress.

BACKGROUND Islet NADPH oxidase activity is modulated by glucose and other insulin secretagogues and it might be part of the regulatory mechanism of insulin secretion. We studied its modulatory role of islet NADPH oxidase upon β-cell function in rats with fructose-induced oxidative stress. METHODS Normal rats were fed for 3weeks with a standard diet, a fructose-rich diet or both diets plus apocynin. We measured plasma glucose, insulin, triacylglycerol and lipid peroxidation levels and the homeostasis model assessment-insulin resistance (HOMA-IR) and HOMA-β indexes, and performed an oral glucose tolerance test. β-cell volume density and the number of islets per mm(2) were determined by immunomorphometric analysis of the pancreas. Insulin secretion, glucose metabolism, glucokinase and NADPH oxidase activities were studied in islets isolated from each experimental group. RESULTS Fructose-fed rats had increased plasma triacylglycerol, insulin and lipid peroxidation levels associated with an insulin resistance state; the reactive higher secretion was unable to cope with the increased demand of insulin, leading to an impaired glucose tolerance. They also have a lower number of islets per area unit with a decreased β-cell volume density. All these alterations were prevented by blocking NADPH oxidase activity with apocynin. CONCLUSION Fructose-induced changes are partly mediated by modulation of NADPH oxidase activity. GENERAL SIGNIFICANCE The metabolic dysfunctions and enhanced oxidative stress measured in fructose-fed rats resemble those recorded in human prediabetes; thus, successful strategies employed in this model could be later used to prevent the progression of this state towards type 2 diabetes in human beings.

Changes in islet plasma membrane calcium-ATPase activity and isoform expression induced by insulin resistance.

We studied the effect of insulin resistance (IR) induced by administration of a fructose-rich diet (FRD) to normal Wistar rats for 21days, upon islet plasma membrane calcium ATPases (PMCAs) and insulin secretion. FRD rats showed significantly higher triglyceride and insulin levels, insulin:glucose ratio and HOMA-IR index than controls. FRD islets released significantly more insulin in response to glucose and showed (a) marked changes in PMCA isoform protein content (decreased PMCA 2 and increased PMCA 3), (b) a decrease in total PMCAs activity, and (c) higher levels of cytosolic calcium [Ca(2+)](i). The lower PMCAs activity with the resultant increase in [Ca(2+)](i) would favor the compensatory greater release of insulin necessary to cope with the IR state present in FRD rats and to maintain normal glucose homeostasis. Thus, changes in PMCAs activity and isoform expression play a modulatory role upon insulin secretion during long-term adaptation to an increased hormone demand.

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.

Modulatory effect of hormones on insulin secretion in vitro in the toad

The aim of this work was to demonstrate the possible direct effect of several hormones upon glucose-induced insulin secretion in amphibians. Hence, pancreas pieces of Bufo arenarum were incubated for 60 min at 25 degrees with 2 and 8 mM glucose plus the addition of hormones known to affect insulin secretion in mammals, measuring the release of insulin by radioimmunoassay. Glucagon (1 microM), ACTH (2.5 microM), human and bovine growth hormone (4.6 and 2.1 microM), prolactin (0.27 microM), corticosterone (0.4 microM), androstanolone (10(-2) microM), estradiol and estrone (10 microM), triiodothyronine and thyroxine (1 microM) enhanced significantly the glucose-induced insulin secretion. Androstanolone, human and bovine growth hormone, triiodothyronine and thyroxine only exerted such effect in the presence of 8 mM glucose. Conversely, somatostatin (1 microM), adrenalin (1 microM), clonidine (2 microM), dexamethasone (0.4 microM), and 2-hydroxyestradiol (5 microM) decreased significantly the glucose-induced insulin release. However, the effect of somatostatin was only apparent in the presence of high glucose. The direct effect of all these hormones--tested for the first time in the amphibian pancreas--was similar to that described in the mammalian pancreas, thus suggesting that such hormones might participate, at least in vitro, in the fine-tuning of insulin secretion in amphibians.

Dual effect of 2‐deoxy‐D‐glucose tetraacetate upon glucose‐induced insulin release

The effect of 2‐deoxy‐D‐glucose tetraacetate upon glucose‐stimulated insulin release was explored in pancreatic islets from either normal or hereditary diabetic rats. At a high concentration (10 mM), it decreased the secretory response to D‐glucose, such an inhibitory effect being more marked in the case of the α‐ than β‐anomer of the ester. At lower concentrations (0.19 to 1.7 mM), however, 2‐deoxy‐D‐glucose tetraacetate augmented insulin secretion evoked by 8.3 mM D‐glucose, with again a preference for the α‐anomer of the ester. In relative terms, such an enhancing action was more marked in Goto‐Kakizaki than normal rats. Hence, it is proposed that selected esters of non‐nutrient carbohydrates could be used as insulinotropic tools in the treatment of non‐insulin‐dependent diabetes mellitus.

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.

Failure of streptozotocin tetraacetate to undergo extensive hydrolysis and to inhibit D-glucose metabolism and insulinotropic action in rat pancreatic islets

The esterification of several monosaccharides, such as D‐glucose, D‐mannoheptulose and 2‐deoxy‐D‐glucose was recently reported to increase their biological efficiency as either nutrient or antimetabolic agent. In the present study, however, the tetraacetate ester of streptozotocin was unexpectedly found to be less potent than unesterified streptozotocin in inhibiting D‐glucose metabolism and insulinotropic action in isolated rat pancreatic islets. This coincided with a much lower rate for the hydrolysis of streptozotocin tetraacetate than D‐glucose pentaacetate in islet homogenates. These findings document that the esterification of single sugars is not always a successful procedure to enhance their biological potency, for instance because of too low a rate for the intracellular hydrolysis of the ester. To the extent that the activity of the concerned esterase(s) may differ in distinct cell types, as suggested by a prior observation, advantage could be taken of such a situation to target selected esters towards specific, e.g. tumoural cells. Copyright

Liver carbohydrates metabolism: A new islet-neogenesis associated protein peptide (INGAP-PP) target

HighlightsINGAP‐PP significantly increases liver glucose metabolism.INGAP‐PP effects were possibly mediated by P‐Akt signaling pathway.INGAP‐PP might become an effective pharmacological tool to treat people with T2D. &NA; Islet‐Neogenesis Associated Protein‐Pentadecapeptide (INGAP‐PP) increases &bgr;‐cell mass and enhances glucose and amino acids‐induced insulin secretion. Our aim was to demonstrate its effect on liver metabolism. For that purpose, adult male Wistar rats were injected twice‐daily (10 days) with saline solution or INGAP‐PP (250 &mgr;g). Thereafter, serum glucose, triglyceride and insulin levels were measured and homeostasis model assessment (HOMA‐IR) and hepatic insulin sensitivity (HIS) were determined. Liver glucokinase and glucose‐6‐phosphatase (G‐6‐Pase) expression and activity, phosphoenolpyruvate carboxykinase (PEPCK) expression, phosphofructokinase‐2 (PFK‐2) protein content, P‐Akt/Akt and glycogen synthase kinase‐3&bgr; (P‐GSK3/GSK3) protein ratios and glycogen deposit were also determined. Additionally, glucokinase activity and G‐6‐Pase and PEPCK gene expression were also determined in isolated hepatocytes from normal rats incubated with INGAP‐PP (5 &mgr;g/ml). INGAP‐PP administration did not modify any of the serum parameters tested but significantly increased activity of liver glucokinase and the protein level of its cytosolic activator, PFK‐2. Conversely, INGAP‐PP treated rats decreased gene expression and enzyme activity of gluconeogenic enzymes, G‐6‐Pase and PEPCK. They also showed a higher glycogen deposit and P‐GSK3/GSK3 and P‐Akt/Akt ratio. In isolated hepatocytes, INGAP‐PP increased GK activity and decreased G‐6‐Pase and PEPCK expression. These results demonstrate a direct effect of INGAP‐PP on the liver acting through P‐Akt signaling pathway. INGAP‐PP enhances liver glucose metabolism and deposit and reduces its production/output, thereby contributing to maintain normal glucose homeostasis. These results reinforce the concept that INGAP‐PP might become a useful tool to treat people with impaired islet/liver glucose metabolism as it occurs in T2D.

Impaired endocrine-metabolic homeostasis: underlying mechanism of its induction by unbalanced diet

To characterize the intrinsic mechanism by which sucrose induces β-cell dysfunction. Normal rats received for 3 weeks a standard diet supplemented with 10% sucrose in the drinking water (high sucrose (HS)) with/out an antioxidant agent (R/S α-lipoic acid). We measured plasma glucose, insulin, triglyceride, leptin, and lipid peroxidation levels; homeostasis model assessment (HOMA)-insulin resistance (HOMA-IR) and HOMA for β-cell function (HOMA-β) indexes were also determined. Insulin secretion, β-cell apoptosis, intracellular insulin and leptin mediators, and oxidative stress (OS) markers were also measured in islets isolated from each experimental group. HS rats had increased plasma triglyceride, insulin, leptin, and lipid peroxidation (OS marker) levels associated with an insulin-resistant state. Their islets developed an initial compensatory increase in glucose-induced insulin secretion and mRNA and protein levels of β-cell apoptotic markers. They also showed a significant decrease in mRNA and protein levels of insulin and leptin signaling pathway mediators. Uncoupling protein 2 (UCP2), peroxisome proliferator-activated receptor (PPAR)-α and -δ mRNA and protein levels were increased whereas mRNA levels of Sirtuin-1 (Sirt-1), glutathione peroxidase, and catalase were significantly lower in these animals. Development of all these endocrine-metabolic abnormalities was prevented by co-administration of R/S α-lipoic acid together with sucrose. OS may be actively involved in the mechanism by which unbalanced/unhealthy diet induces β-cell dysfunction. Since metabolic-endocrine dysfunctions recorded in HS rats resembled those measured in human pre-diabetes, knowledge of its molecular mechanism could help to develop appropriate strategies to prevent the progression of this metabolic state toward type 2 diabetes (T2D).