Angelo R. Carpinelli

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 β‐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 β‐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.

Pleiotropic effects of fatty acids on pancreatic β‐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 β‐cells have long been recognized. Acute exposure of the pancreatic β‐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 β‐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 β‐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.

Insights into the critical role of NADPH oxidase(s) in the normal and dysregulated pancreatic beta cell

It is now widely accepted that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage in diabetes. The source of ROS in the insulin secreting pancreatic beta cells has traditionally been considered to be the mitochondrial electron transport chain. While this source is undoubtedly important, we fully describe in this article recent information and evidence of NADPH oxidase-dependent generation of ROS in pancreatic beta cells and identify the various isoforms that contribute to O2•− and H2O2 production in various conditions. While glucose-stimulated ROS generation may be important for acute regulation of insulin secretion, at higher levels ROS may disrupt mitochondrial energy metabolism. However, ROS may alter other cellular processes such as signal transduction, ion fluxes and/or cell proliferation/death. The various beta cell isoforms of NADPH oxidase (described in this review) may, via differences in the kinetics and species of ROS generated, positively and negatively regulate insulin secretion and cell survival.

Association of NAD(P)H Oxidase with Glucose-Induced Insulin Secretion by Pancreatic β-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.

The stimulus-secretion coupling of glucose-induced insulin release XLVI. Physiological role of l-glutamine as a fuel for pancreatic islets

Exogenous L-glutamine is actively metabolized in rat pancreatic islets. The rate of L-glutamine deamidation largely exceeds the rate of glutamate conversion to gamma-aminobutyrate and alpha-ketoglutarate. The latter conversion occurs in part by oxidative deamination, and in part by transamination reactions coupled with the conversion of 2-keto acids (pyruvate, oxaloacetate), themselves derived from the metabolism of glutamine, to their corresponding amino acids (alanine, aspartate). An important fraction of malate formed from alpha-ketoglutarate leaves the Krebs cycle and is converted to pyruvate, the process being apparently associated with the induction of a more reduced state in cytosolic redox couples. L-Glutamine abolishes the oxidation of endogenous nutrients is documented by the fact that the glutamine-induced increase in O2 consumption is much lower than expected from the rate of 14CO2 output from islets exposed to L-[U-14C]glutamine, L-Glutamine, although decreasing K+ conductance, fails to stimulate insulin release both in the absence and presence of D-glucose. It is proposed that L-glutamine represents a major fuel for pancreatic islets under physiological conditions.

Melatonin inhibits insulin secretion and decreases PKA levels without interfering with glucose metabolism in rat pancreatic islets

Abstract: The effect of melatonin (0.1 μM) on freshly isolated islets from adult rats was investigated. Melatonin caused a marked decrease of insulin secretion by islets in response to glucose. The mechanism involved was then examined. Melatonin did not interfere with glucose metabolism as indicated by the measurement of glucose oxidation. However, the content of the protein kinase A (PKA) catalytic α‐subunit was significantly decreased in islets exposed to melatonin for 1 hr in the presence of 8.3 mM glucose, whereas that of the protein kinase C (PKC) α‐subunit remained unchanged. Melatonin also inhibited forskolin‐induced insulin secretion, a well known activator of adenylate cyclase (AC) activity. This may explain the low content of insulin found in islets incubated in the presence of melatonin for 3 hr. In fact, 3′,5′ ‐cyclic adenosine monophosphate (cAMP), a product of AC activity, stimulates insulin synthesis. These findings led us to postulate that a down‐regulation of the PKA signaling pathway may be the mechanism involved in the melatonin inhibition of the process of glucose‐induced insulin secretion.

The Stimulus-secretion Coupling of Amino Acid-induced Insulin Release: Metabolism and Cationic Effects of Leucine

When isolated rat pancreatic islets are exposed to L-leucine (20 mM), the rate of NH4 production is close to the summed rates of L-[1-14 C] leucine decarboxylation and α-ketoisocaproate production, whereas the rates of acetoacetate production and L-[U-14 C]-leucine oxidation are compatible with the conversion of each mole of the amino acid to one mole of acetoacetate and three moles of CO2. ATP content, ATP/ADP ratio, and adenylate charge are maintained at normal values by L-leucine, whereas the NADH/NAD+ ratio (but not the NADPH/NADP+ ratio) is significantly increased. The release of insulin evoked by L-leucine is potentiated by 2-ketoisovalerate, unaffected by L-valine, and inhibited by menadione. L-leucine mimicks the effect of D-glucose on 86Rb+ and 45Ca2+ handling by the islets. However, relative to its rate of oxidation, the insulinotropic effect of L-leucine is less marked than that of D-glucose. This may be due, in part at least, to a decrease in the oxidation of endogenous nutrients. It is concluded that the metabolic, cationic, and secretory effects of L-leucine in isolated islets are not incompatible with the fuel hypothesis for insulin release.

Daily rhythm of glucose‐induced insulin secretion by isolated islets from intact and pinealectomized rat

Abstract: It is well known that pinealectomy induces in rats a diminished glucose tolerance, insulin resistance, a reduction in GLUT4 content in adipose and muscular tissues, a decrease in hepatic and muscular glycogenesis, impairment of glucagon action and an increase in blood pyruvate concentration. In addition, it has been shown that melatonin suppresses insulin secretion in several experimental conditions. The objective of the present study was to investigate the daily rhythm of glucose‐induced insulin secretion and glucose oxidation by isolated pancreatic islets and to investigate the effect of chronic absence of melatonin (30 days of pinealectomy) on this rhythmic process. The data obtained confirmed the presence of a strong 24‐hr rhythm of insulin secretion by isolated pancreatic islets. In addition, it was demonstrated that the glucose‐metabolizing ability of the B‐cell follows a daily rhythm phase locked to insulin secretion rhythm. Most interesting, however, was the demonstration that the daily rhythmic processes of insulin secretion and B‐cell –[U‐14C]‐glucose oxidation by isolated pancreatic islets is completely modified by the chronic absence of the pineal gland. Thus, pinealectomy induced in all groups an increase in 24‐hr mean glucose‐stimulated insulin secretion and [U‐14C]‐glucose oxidation, in addition to some alterations in the rhythmic amplitude and a remarkable phase‐advancing of the daily curves for 8.3 mm glucose (a condition similar to that observed in fed animals and where the B‐cells are supposedly more active). These observations strongly suggest that the presence of the pineal gland may be necessary for the proper synchronization of these metabolic rhythms with other circadian rhythms like activity–rest and feeding.

New Insights into Fatty Acid Modulation of Pancreatic β‐Cell Function

Insulin resistance states as found in type 2 diabetes and obesity are frequently associated with hyperlipidemia. Both stimulatory and detrimental effects of free fatty acids (FFA) on pancreatic β cells have long been recognized. Acute exposure of the pancreatic β cell to both high glucose concentrations and saturated FFA results in a substantial increase of insulin release, whereas a chronic exposure results in desensitization and suppression of secretion. Reduction of plasma FFA levels in fasted rats or humans severely impairs glucose‐induced insulin release but palmitate can augment insulin release in the presence of nonstimulatory concentrations of glucose. These results imply that changes in physiological plasma levels of FFA are important for regulation of β‐cell function. Although it is widely accepted that fatty acid (FA) metabolism (notably FA synthesis and/or formation of LC‐acyl‐CoA) is necessary for stimulation of insulin secretion, the key regulatory molecular mechanisms controlling the interplay between glucose and fatty acid metabolism and thus insulin secretion are not well understood but are now described in detail in this review. Indeed the correct control of switching between FA synthesis or oxidation may have critical implications for β‐cell function and integrity both in vivo and in vitro . LC‐acyl‐CoA (formed from either endogenously synthesized or exogenous FA) controls several aspects of β‐cell function including activation of certain types of PKC, modulation of ion channels, protein acylation, ceramide‐ and/or NO‐mediated apoptosis, and binding to and activating nuclear transcriptional factors. The present review also describes the possible effects of FAs on insulin signaling. We have previously reported that acute exposure of islets to palmitate up‐regulates some key components of the intracellular insulin signaling pathway in pancreatic islets. Another aspect considered in this review is the potential source of fatty acids for pancreatic islets in addition to supply in the blood. Lipids can be transferred from leukocytes (macrophages) to pancreatic islets in coculture. This latter process may provide an additional source of FAs that may play a significant role in the regulation of insulin secretion.

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

Abstract:  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.