Cytotoxicity and cytoprotective effects of citrus flavonoids on insulin-secreting cells BRIN-BD11: beneficial synergic effects.

Flavonoids, in general, have potent antioxidant activity and they can be used in treating chronic diseases involving oxidative stress, such as diabetes mellitus. The purpose of this study was to evaluate the cytotoxicity and cytoprotective effects of citrus flavonoids on the functionality of BRIN-BD11 cells. The assessment of cytotoxic and cytoprotective flavonoid tested was performed using the MTT reduction assay. The flavonoids did not show cytotoxic effects in any of the tested concentrations (5-20 µM) and also negative insulinotropic effects were not observed. To cytoprotective assay, the IC50 of H2O2 in treatment of 2 h (acute oxidative stress) was measured (350 µM). Moreover, under acute oxidative stress, the isolated flavonoids (10 µM) had no cytoprotective effects. Besides an antioxidant role of the flavonoids was only observed when using in association. Thus future experiments are needed, varying the experimental condition, to better evaluate the possible mechanisms of action of these flavonoids.

Changes in food intake, metabolic parameters and insulin resistance are induced by an isoenergetic, medium-chain fatty acid diet and are associated with modifications in insulin signalling in isolated rat pancreatic islets.

Long-chain fatty acids are capable of inducing alterations in the homoeostasis of glucose-stimulated insulin secretion (GSIS), but the effect of medium-chain fatty acids (MCFA) is poorly elucidated. In the present study, we fed a normoenergetic MCFA diet to male rats from the age of 1 month to the age of 4 months in order to analyse the effect of MCFA on body growth, insulin sensitivity and GSIS. The 45% MCFA substitution of whole fatty acids in the normoenergetic diet impaired whole body growth and resulted in increased body adiposity and hyperinsulinaemia, and reduced insulin-mediated glucose uptake in skeletal muscle. In addition, the isolated pancreatic islets from the MCFA-fed rats showed impaired GSIS and reduced protein kinase Ba (AKT1) protein expression and extracellular signal-related kinase isoforms 1 and 2 (ERK(1/2)) phosphorylation, which were accompanied by increased cellular death. Furthermore, there was a mildly increased cholinergic sensitivity to GSIS. We discuss these findings in further detail, and advocate that they might have a role in the mechanistic pathway leading to the compensatory hyperinsulinaemic status found in this animal model.

Angiotensin II-induced JNK activation is mediated by NAD(P)H oxidase in isolated rat pancreatic islets.

Angiotensin II (AII), the active component of the renin angiotensin system (RAS), plays a vital role in the regulation of physiological processes of the cardiovascular system, but also has autocrine and paracrine actions in various tissues and organs. Many studies have shown the existence of RAS in the pancreas of humans and rodents. The aim of this study was to evaluate potential signaling pathways mediated by AII in isolated pancreatic islets of rats. Phosphorylation of MAPKs (ERK1/2, JNK and p38MAPK), and the interaction between proteins JAK/STAT were evaluated. AII increased JAK2/STAT1 (42%) and JAK2/STAT3 (100%) interaction without altering the total content of JAK2. Analyzing the activation of MAPKs (ERK1/2, JNK and p38MAPK) in isolated pancreatic islets from rats we observed that AII rapidly (3 min) promoted a significant increase in the phosphorylation degree of these proteins after incubation with the hormone. Curiously JNK protein phosphorylation was inhibited by DPI, suggesting the involvement of NAD(P)H oxidase in the activation of protein.

Association of NAD(P)H oxidase with glucose-induced insulin secretion by pancreatic beta-cells.

We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H]oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C]glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown.

Angiotensin II induces superoxide generation via NAD(P)H oxidase activation in isolated rat pancreatic islets.

Angiotensin II (Ang II) controls blood pressure, electrolyte balance, cell growth and vascular remodeling. Ang II activates NAD(P)H oxidase in several tissues with important function in the control of insulin secretion. Considering the concomitant occurrence of hypertension, insulin resistance and pancreatic B cell secretion impairment in the development of type II diabetes the aim of the present study was to evaluate the effect of ANG II on NAD(P)H oxidase activation in isolated pancreatic islets. We found that ANGII-induced superoxide generation via NAD(P)H oxidase activation and increased protein and mRNA levels of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)).

Activation of insulin and IGF-1 signaling pathways by melatonin through MT1 receptor in isolated rat pancreatic islets.

Melatonin diminishes insulin release through the activation of MT1 receptors and a reduction in cAMP production in isolated pancreatic islets of neonate and adult rats and in INS-1 cells (an insulin-secreting cell line). The pancreas of pinealectomized rats exhibits degenerative pathological changes with low islet density, indicating that melatonin plays a role to ensure the functioning of pancreatic beta cells. By using immunoprecipitation and immunoblotting analysis we demonstrated, in isolated rat pancreatic islets, that melatonin induces insulin growth factor receptor (IGF-R) and insulin receptor (IR) tyrosine phosphorylation and mediates the activities of the PI3K/AKT and MEK/ERKs pathways, which are involved in cell survival and growth, respectively. Thus, the effects of melatonin on pancreatic islets do not involve a reduction in cAMP levels only. This indoleamine may regulate growth and differentiation of pancreatic islets by activating IGF-I and insulin receptor signaling pathways.

Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity.

It is now widely accepted, given the current weight of experimental evidence, that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage caused by glucolipotoxicity in diabetes. The source of ROS in the insulin secreting pancreatic beta-cells and in the cells which are targets for insulin action has been considered to be the mitochondrial electron transport chain. While this source is undoubtably important, we provide additional information and evidence for NADPH oxidase-dependent generation of ROS both in pancreatic beta-cells and in insulin sensitive cells. While mitochondrial ROS generation may be important for regulation of mitochondrial uncoupling protein (UCP) activity and thus disruption of cellular energy metabolism, the NADPH oxidase associated ROS may alter parameters of signal transduction, insulin secretion, insulin action and cell proliferation or cell death. Thus NADPH oxidase may be a useful target for intervention strategies based on reversing the negative impact of glucolipotoxicity in diabetes.

Glucose, palmitate and pro-inflammatory cytokines modulate production and activity of a phagocyte-like NADPH oxidase in rat pancreatic islets and a clonal beta cell line.

AIMS/HYPOTHESIS: Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47(phox) component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). METHODS: Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47(phox) production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays. RESULTS: Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47(phox) protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47(phox) was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1beta-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47(phox). Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation. CONCLUSIONS/INTERPRETATION: NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise.

Changes of fatty acid composition in incubated rat pancreatic islets.

OBJECTIVE: The hypothesis that changes in fatty acId composition of pancreatic islets occur during incubation was investigated. METHODS: The content and composition of fatty acIds (FA) from rat pancreatic islets and culture medium after incubation for 1 and 3 hours in the absence or in the presence of 5.6, 8.3, or 16.7 mM glucose were determined by HPLC analysis. RESULTS: The FA content of pancreatic islets was reduced after 1 hour incubation in the absence of glucose. However, the total FA content was restored by incubating in the presence of 5.6 mM glucose and exceeded by incubating in the presence of 8.3 mM or 16.7 mM glucose. Saturated FA contributed a substantially greater proportion of the total FA increase in comparison to unsaturated FA, being palmitic and stearic acIds the most important. The total lipId content of pancreatic islets was not increased if the period of incubation in the presence of glucose was extended to 3 hours. A substantial amount of FA was found in the medium after 1 hour incubation in the absence of glucose: 141 ng per 80 islets for saturated and 75 ng per 80 islets for unsaturated. The release of FA from islets is increased in the presence of glucose. CONCLUSION: The release of FA from islets is a novel finding and may be related to modulation of B-cell function.

Pleiotropic effects of fatty acids on pancreatic beta-cells.

Hyperlipidemia is frequently associated with insulin resistance states as found in type 2 diabetes and obesity. Effects of free fatty acids (FFA) on pancreatic beta-cells have long been recognized. Acute exposure of the pancreatic beta-cell to FFA results in an increase of insulin release, whereas a chronic exposure results in desensitization and suppression of secretion. We recently showed that palmitate augments insulin release in the presence of non-stimulatory concentrations of glucose. Reduction of plasma FFA levels in fasted rats or humans severely impairs glucose-induced insulin release. These results imply that physiological plasma levels of FFA are important for beta-cell function. Although, it has been accepted that fatty acid oxidation is necessary for its stimulation of insulin secretion, the possible mechanisms by which fatty acids (FA) affect insulin secretion are discussed in this review. Long-chain acyl-CoA (LC-CoA) controls several aspects of the beta-cell function including activation of certain types of protein kinase C (PKC), modulation of ion channels, protein acylation, ceramide- and/or nitric oxide (NO)-mediated apoptosis, and binding to nuclear transcriptional factors. The present review also describes the possible effects of FA on insulin signaling. We showed for the first time that acute exposure of islets to palmitate upregulates the intracellular insulin-signaling pathway in pancreatic islets. Another aspect considered in this review is the source of FA for pancreatic islets. In addition to be exported to the medium, lipids can be transferred from leukocytes (macrophages) to pancreatic islets in co-culture. This process consists an additional source of FA that may plays a significant role to regulate insulin secretion.

Insulin secretion induced by palmitate--a process fully dependent on glucose concentration.

The effect of 0.1 mM palmitate on insulin secretion by 1 hr incubated pancreatic islets was examined in the presence of different glucose concentrations (5.6 and 16.7 mM). The oxidation of both glucose and palmitate and the incorporation of [U-14C]-palmitate into lipid fractions and phospholipid species were determined. In the presence of 5.6 mM glucose, palmitate reduced insulin release by 80%. In contrast, in the presence of 16.7 mM glucose, palmitate raised the amount of insulin released by 49%. Palmitate (0.1 mM) caused a significant reduction (52%) of [U-14C]-glucose decarboxylation at 5.6 mM but it did not have any effect at 16.7 mM glucose. The decarboxylation of [U-14C]-palmitate was markedly lower (94%) in the presence of 16.7 mM, as compared to 5.6 mM glucose. [U-14C]-Palmitate was significantly incorporated into total lipid fractions in the presence of both glucose concentrations. The increase in glucose concentration from 5.6 to 16.7 mM raised by 138% the incorporation of [U-14C]-palmitate into phospholipids: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI). PC and PA at 0.1 mM raised by three and four-fold, respectively, insulin release by incubated pancreatic islets. We postulated that palmitate (at 0.1 mM) promotes a deviation of glycerol-phosphate to lipid synthesis, decreasing glucose oxidation (at 5.6 mM) and possibly ATP/ADP ratio in the cytosol, leading to a reduction in insulin secretion. At 16.7 mM glucose concentration, the high glycolytic flux is now enough to provide glycerol-phosphate for lipid synthesis and carbons for the Krebs cycle. So, under this condition, ATP production might be not reduced. The increase in the production of PA and PC may explain the increase in insulin secretion observed at 16.7 mM glucose.

Macrophages transfer [14C]-labelled fatty acids to pancreatic islets in culture.

Macrophages are able to produce, export, and transfer fatty acids to lymphocytes in culture. The purpose of this study was to examine if labelled fatty acids could be transferred from macrophages to pancreatic islets in co-culture. We found that after 3 h of co-culture the transfer of fatty acids to pancreatic islets was: arachidonic >> oleic > linoleic = palmitic. Substantial amounts of the transferred fatty acids were found in the phospholipid fraction; 87.6% for arachidonic, 59.9% for oleic, 53.1% for palmitic, and 36.9% for linoleic acids. The remaining radioactivity was distributed among the other lipid fractions analysed (namely polar lipids, cholesterol, fatty acids, triacylglycerol and cholesterol ester), varying with the fatty acid used. For linoleic acid, a significant proportion (63.1%) was almost equally distributed in these lipid fractions. Also, it was observed that transfer of fatty acids from macrophages to pancreatic islets is time-dependent up to 24 h, being constant and linear with time for palmitic acid and remaining constant after 12 h for oleic acid. These results lead us to postulate that in addition to the serum, circulating monocytes may also be a source of fatty acids to pancreatic islets, mainly arachidonic acid.

Tolerable dietary lipid content that does not alter insulin secretion.

Lipids, either as membrane components or as fuel, are important nutrients that can affect insulin secretion. The aim of this study was to establish the maximum tolerable amount of fat present in the diet, which does not induce significant alteration in the process of insulin secretion. For that, just-weaned male albino rats (70-90 g body weight) were fed during 6 weeks with diets for growing rodents containing 7% fat (A Group) as recommended by the American Institute of Nutrition-AIN. Two other groups in which the fat content of the diet was increased to reach 10% (B Group) or 13% (C Group) were also included. Insulin release, 86Rb+ and 45Ca2+ Fractional Outflow Rate (FOR) during the process of glucose-induced insulin secretion was determined in perfused islets isolated from these animals. No statistical differences in these parameters were detected between A and B rats. However, in the C group, a lower 86Rb+ FOR was found during the whole experiment and a poor insulin secretory response to glucose stimulus was observed. These results led us to postulate that the maximal limiting amount of total lipids present in the diet that does not impair the process of glucose-induced insulin secretion is 10%. These findings authorize future studies on the interference of different dietary lipid sources, in a content 43% more elevated than that recommended (10% against 7%), on the mechanisms of insulin secretion.

Modulation of insulin secretion by leptin.

The present study examines the acute effect of leptin (50 nM) on insulin secretion and on the fractional outflow rates of 45Ca2+ and 86Rb+ from pancreatic islets isolated from male lean albino rats. Under a constant physiological glucose concentration (5.6 mM), the addition of leptin to the perifusion medium led to an increment in 45Ca2+ fractional outflow rate followed by a significant (p < 0.05) increase (26%) in the insulin release. At low glucose concentration (2.8 mM), leptin also elicited a significant (p < 0.05; 50-60%) increase in insulin secretion. However, under supraphysiological (16.7 mM) glucose concentration, the rapid first-phase insulin secretion response was abolished. At low glucose levels, islets perifused in the presence of leptin presented a lower 86Rb+ fractional outflow rate compared with perifused controls. In contrast, when glucose was switched to 16.7 mM, compared with controls, a slight increase in the 86Rb+ fractional outflow rate was observed instead. These in vitro data provide evidence that, by changing K+ fluxes, leptin might modulate insulin secretion from pancreatic islets.

Glucose induces an acute increase of superoxide dismutase activity in incubated rat pancreatic islets.

The activity of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSP) in isolated rat pancreatic islets exposed to high glucose concentration for a short period of time (60 min) was determined. High glucose concentration (16.7 mM) did not significantly alter catalase activity. GSP activity was increased by glucose at 5.6 mM, remaining elevated at higher concentrations up to 16.7 mM. However, the activity of SOD increased with glucose concentration, and this increment was closely correlated with the rate of insulin secretion (r = 0.96). High potassium (30 mM) did not increase SOD activity, suggesting that the increase in intracellular ionic calcium concentration does not stimulate this enzyme activity. alpha-Ketoisocaproic acid and pyruvate, which are metabolized through the TCA cycle, did not increase SOD activity, indicating that the stimulation of SOD activity might be triggered by a factor produced through glycolysis or the pentose phosphate pathway.

Soybean- and olive-oils-enriched diets increase insulin secretion to glucose stimulus in isolated pancreatic rat islets.

Islets isolated from rats fed a lipid-enriched diet have shown an impairment of insulin secretion, but there is no available data comparing the effect of diet containing different dietary fat. This may be important in preventing or facilitating the establishment of diabetes. In this study, the effect of diets enriched (10%) with different fatty acids on insulin secretion by isolated pancreatic islets was investigated. The sources of the fatty acids tested were: saturated long chain from animal fat (AF), polyunsaturated from soybean oil (SO), and monounsaturated from olive oil (OL). The results were compared with those from rats receiving a diet enriched (10%) with a balanced mixture of fatty acids (the same proportion of AF, SO, and OL). The effect of fat-rich diets on insulin release was tested in vivo by giving a glucose load (glucose tolerance test-GTT) and in vitro in perfused islets. The mechanism involved was also examined by measuring 45Ca2+ and 86Rb+ fluxes, GLUT-2 content, and glucose oxidation in isolated islets. A significant increase of insulin secretion and glucose oxidation without any alteration of the ionic movements were detected in islets from SO and OL rats. GLUT-2 content was increased in islets of the OL group but diminished in AF rats. The results led us to postulate that soybean and olive oils may increase the response of insulin secretion to glucose stimulus in pancreatic islets.

Pivotal role of leptin in insulin effects.

The OB protein, also known as leptin, is secreted by adipose tissue, circulates in the blood, probably bound to a family of binding proteins, and acts on central neural networks regulating ingestive behavior and energy balance. The two forms of leptin receptors (long and short forms) have been identified in various peripheral tissues, a fact that makes them possible target sites for a direct action of leptin. It has been shown that the OB protein interferes with insulin secretion from pancreatic islets, reduces insulin-stimulated glucose transport in adipocytes, and increases glucose transport, glycogen synthesis and fatty acid oxidation in skeletal muscle. Under normoglycemic and normoinsulinemic conditions, leptin seems to shift the flux of metabolites from adipose tissue to skeletal muscle. This may function as a peripheral mechanism that helps control body weight and prevents obesity. Data that substantiate this hypothesis are presented in this review.

Pivotal role of leptin in insulin effects

The OB protein, also known as leptin, is secreted by adipose tissue, circulates in the blood, probably bound to a family of binding proteins, and acts on central neural networks regulating ingestive behavior and energy balance. The two forms of leptin receptors (long and short forms) have been identified in various peripheral tissues, a fact that makes them possible target sites for a direct action of leptin. It has been shown that the OB protein interferes with insulin secretion from pancreatic islets, reduces insulin-stimulated glucose transport in adipocytes, and increases glucose transport, glycogen synthesis and fatty acid oxidation in skeletal muscle. Under normoglycemic and normoinsulinemic conditions, leptin seems to shift the flux of metabolites from adipose tissue to skeletal muscle. This may function as a peripheral mechanism that helps control body weight and prevents obesity. Data that substantiate this hypothesis are presented in this review

Possible modulatory role of non-activated lymphocytes on insulin secretion.

In order to study the probable physiological role of non-activated lymphocytes on islet B-cells, we incubated and perfused rat pancreatic islets in the presence of low (2.8 mM) and high (16.7 mM) glucose concentrations after pre-exposure for 60 min to rat lymphocytes or to substances secreted by lymphocytes. Insulin secretion and 86Rb+, 45Ca2+ and [3H]-phosphoinositide metabolite fluxes were lower compared to controls when islets were pre-exposed to lymphocytes but were not different when islets were pre-exposed to substances secreted by lymphocytes. These alterations in isotope flux suggest that, when lymphocytes and islets are in contact, closure of potassium channels and a paradoxical effect of glucose load on insulin release occur in the presence of low glucose concentrations. The alterations observed are probably due to a swift and direct action of lymphocyte secretion perhaps induced by a direct of islet cells.

Possible modulatory role of non-activated lymphocytes on insulin secretion

In order to study the probable physiological role of non-activated lymphocytes on islet B-cells, we incubated and perfused rat pancreatic islets in the presence of low (2.8 mM) and high (l6.7 mM) glucose concentrations after pre-exposure for 60 min to rat lymphocytes or to substances secreted by lymphocytes. Insulin secretion and 86Rb+, 45Ca2+ and [3H]-phosphoinositide metabolite fluxes were lower compared to controls when islets were pre-exposed to lymphocytes but were not different when islets were pre-exposed to substances secreted by lymphocytes. These alterations in isotope flux suggest that, when lymphocytes and islets are in contact, closure of potassium channels and a paradoxical effect of glucose load on insulin release occur in the presence of low glucose concentrations. The alterations observed are probably due to a swift and direct action of lymphocyte secretion perhaps induced by a direct contact of islet cells.