Melatonin regulates maternal pancreatic remodeling and B-cell function during pregnancy and lactation.

The endocrine pancreas of pregnant rats shows evident plasticity, which allows the morphological structures to return to the nonpregnant state right after delivery. Furthermore, it is well-known the role of melatonin in the maintenance of the endocrine pancreas and its tropism. Studies indicate increasing nocturnal serum concentrations of maternal melatonin during pregnancy in both humans and rodents. The present study investigated the role of melatonin on energy metabolism and in pancreatic function and remodeling during pregnancy and early lactation in rats. The results confirm that the absence of melatonin during pregnancy impairs glucose metabolism. In addition, there is a dysregulation in insulin secretion at various stages of the development of pregnancy and an apparent failure in the glucose-stimulated insulin secretion during the lactation period, evidencing the role of melatonin on the regulation of insulin secretion. This mechanism seems not to be dependent on the antioxidant effect of melatonin and probably dependent on MT2 receptors. We also observed changes in the mechanisms of death and cell proliferation at the end of pregnancy and beginning of lactation, crucial periods for pancreatic remodeling. The present observations strongly suggest that both functionality and remodeling of the endocrine pancreas are impaired in the absence of melatonin and its adequate replacement, mimicking the physiological increase seen during pregnancy, is able to reverse some of the damage observed. Thus, we conclude that pineal melatonin is important to metabolic adaptation to pregnancy and both the functionality of the beta cells and the remodeling of the pancreas during pregnancy and early lactation, ensuring the return to nonpregnancy conditions.

Effects of metformin on insulin resistance and metabolic disorders in tumor-bearing rats with advanced cachexia.

Metformin (MET) is widely used in the correction of insulin (INS) resistance and metabolic abnormalities in type 2 diabetes. However, its effect on INS resistance and metabolic disorders associated with cancer cachexia is not established. We investigated the MET effects, isolated or associated with INS, on INS resistance and metabolic changes induced by Walker-256 tumor in rats with advanced cachexia. MET (500 mg·kg-1, oral) and MET + INS (1.0 IU·kg-1, s.c.) were administered for 12 days, starting on the day of tumor cell inoculation. Tumor-bearing rats showed adipose and muscle mass wasting, body mass loss, anorexia, decreased Akt phosphorylation in retroperitoneal and mesenteric adipose tissue, peripheral INS resistance, hypoinsulinemia, reduced INS content and secretion from pancreatic islets, and also inhibition of glycolysis, gluconeogenesis, and glycogenolysis in liver. MET and MET + INS treatments did not prevent these changes. It can be concluded that treatments with MET and MET + INS did not prevent the adipose and muscle mass wasting and body mass loss of tumor-bearing rats possibly by not improving INS resistance. Therefore, MET, used for the treatment of INS resistance in type 2 diabetes, is not effective in improving INS resistance in the advanced stage of cancer cachexia, evidencing that the drug does not have the same beneficial effect in these 2 diseases.

Dual effect of advanced glycation end products in pancreatic islet apoptosis.

BACKGROUND: Loss of ß-cell function hastens deterioration of metabolic control in type 2 diabetes patients. Besides amyloid deposit and glucolipotoxicity, advanced glycation end products (AGEs) acting through their receptors (RAGE) seem to contribute to this process by promoting islet apoptosis. In order to investigate the role of AGEs in ß-cell deterioration, we evaluated the temporal and dose effects of AGE compounds on apoptosis rate, reactive oxygen species generation and expression of pro-apoptotic and anti-apoptotic genes in cultured islets. METHODS: Rat pancreatic islets were exposed or not for 24, 48, 72 and 96 h to albumin modified by glycoaldehyde. Apoptosis, reactive oxygen species and superoxide content and NADPH oxidase activity were evaluated as well as RNA expression of the genes Ager (codes for RAGE), Bax, Bcl2 and Nfkb1. RESULTS: In 24 and 48 h, glycoaldehyde elicited a decrease in apoptosis rate in comparison with the control condition concomitantly with a reduction in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In contrast, after 72 and 96 h, glycoaldehyde promoted an increase in apoptosis rate concomitantly with an increase in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In 24 h, glycoaldehyde elicited a decrease in the islet content of reactive oxygen species, whereas after 48 and 72 h, it promoted an opposite effect, increasing superoxide generation. The NADPH oxidase inhibitor VAS2870 attenuated superoxide production, implicating NADPH oxidase as an important source of reactive oxygen species in islets exposed to AGEs. CONCLUSIONS: Albumin modified by glycoaldehyde exerted a dual effect in cultured pancreatic islets, being protective against apoptosis after short exposure but pro-apoptotic after prolonged exposure.

Melatonin improves insulin sensitivity independently of weight loss in old obese rats.

In aged rats, insulin signaling pathway (ISP) is impaired in tissues that play a pivotal role in glucose homeostasis, such as liver, skeletal muscle, and adipose tissue. Moreover, the aging process is also associated with obesity and reduction in melatonin synthesis from the pineal gland and other organs. The aim of the present work was to evaluate, in male old obese Wistar rats, the effect of melatonin supplementation in the ISP, analyzing the total protein amount and the phosphorylated status (immunoprecipitation and immunoblotting) of the insulin cascade components in the rat hypothalamus, liver, skeletal muscle, and periepididymal adipose tissue. Melatonin was administered in the drinking water for 8- and 12 wk during the night period. Food and water intake and fasting blood glucose remained unchanged. The insulin sensitivity presented a 2.1-fold increase both after 8- and 12 wk of melatonin supplementation. Animals supplemented with melatonin for 12 wk also presented a reduction in body mass. The acute insulin-induced phosphorylation of the analyzed ISP proteins increased 1.3- and 2.3-fold after 8- and 12 wk of melatonin supplementation. The total protein content of the insulin receptor (IR) and the IR substrates (IRS-1, 2) remained unchanged in all investigated tissues, except for the 2-fold increase in the total amount of IRS-1 in the periepididymal adipose tissue. Therefore, the known age-related melatonin synthesis reduction may also be involved in the development of insulin resistance and the adequate supplementation could be an important alternative for the prevention of insulin signaling impairment in aged organisms.

Maternal moderate physical training during pregnancy attenuates the effects of a low-protein diet on the impaired secretion of insulin in rats: potential role for compensation of insulin resistance and preventing gestational diabetes mellitus.

The effects of pregestational and gestational low-to-moderate physical training on insulin secretion in undernourished mothers were evaluated. Virgin female Wistar rats were divided into four groups as follows: control (C, n = 5); trained (T, n = 5); low-protein diet (LP, n = 5); trained with a low-protein diet (T + LP, n = 5). Trained rats ran on a treadmill over a period of 4 weeks before mate (5 days week⁻¹ and 60 min day⁻¹, at 65% of VO(2max)). At pregnancy, the intensity and duration of the exercise were reduced. Low-protein groups were provided with an 8% casein diet, and controls were provided with a 17% casein diet. At third day after delivery, mothers and pups were killed and islets were isolated by collagenase digestion of pancreas and incubated for a further 1 h with medium containing 5.6 or 16.7 mM glucose. T mothers showed increased insulin secretion by isolated islets incubated with 16.7 mM glucose, whereas LP group showed reduced secretion of insulin by isolated islets when compared with both C and LP + T groups. Physical training before and during pregnancy attenuated the effects of a low-protein diet on the secretion of insulin, suggesting a potential role for compensation of insulin resistance and preventing gestational diabetes mellitus.

BMAL1 modulates ROS generation and insulin secretion in pancreatic ß-cells: An effect possibly mediated via NOX2.

The pancreatic ß cells circadian clock plays a relevant role in glucose metabolism. NADPH oxidase (NOX) family is responsible for producing reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide, using NADPH as an electron donor. In pancreatic ß-cells, NOX-derived ROS inhibits basal and glucose-stimulated insulin secretion. Thus, we hypothesized that the absence of BMAL1, a core circadian clock component, could trigger an increase of NOX2-derived ROS in pancreatic ß cells, inhibiting insulin secretion under basal and stimulated glucose conditions. To test such hypothesis, Bmal1 knockdown (KD) was performed in cultured clonal ß-cell line (INS-1E) and knocked out in isolated pancreatic islets, using a tissue-specific ß-cells Bmal1 knockout (KO) mice. The insulin secretion was assessed in the presence of NOX inhibitors. The Bmal1 KD within INS-1E cells elicited a rise of intracellular ROS content under both glucose stimuli (2.8 mM and 16.7 mM), associated with an increase in Nox2 expression. Additionally, alterations of glutathione levels, CuZnSOD and catalase activities, reduction of ATP/ADP ratio, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and aconitase activities, followed by glucokinase and Slc2a2 (Glut2) expression were also observed in INS-1E ß-cells, reflecting in a diminished insulin secretion pattern. The isolated islets from ß-cell Bmal1-/- mice have shown a similar cellular response, where an increased NOX2-derived ROS content and a reduced basal- and glucose-stimulated insulin secretion were observed. Therefore, together with NOX inhibition (Apocynin), polyethene-glycol linked to superoxide dismutase (PEG-SOD), phorbol myristate acetate (PMA), and diethyldithiocarbamate (DDC) data, our findings suggest a possible BMAL1-mediated NOX2-derived ROS generation in pancreatic ß cells, leading to the modulation of both basal- and glucose-stimulated insulin secretion.

Lipotoxicity and ß-Cell Failure in Type 2 Diabetes: Oxidative Stress Linked to NADPH Oxidase and ER Stress.

A high caloric intake, rich in saturated fats, greatly contributes to the development of obesity, which is the leading risk factor for type 2 diabetes (T2D). A persistent caloric surplus increases plasma levels of fatty acids (FAs), especially saturated ones, which were shown to negatively impact pancreatic ß-cell function and survival in a process called lipotoxicity. Lipotoxicity in ß-cells activates different stress pathways, culminating in ß-cells dysfunction and death. Among all stresses, endoplasmic reticulum (ER) stress and oxidative stress have been shown to be strongly correlated. One main source of oxidative stress in pancreatic ß-cells appears to be the reactive oxygen species producer NADPH oxidase (NOX) enzyme, which has a role in the glucose-stimulated insulin secretion and in the ß-cell demise during both T1 and T2D. In this review, we focus on the acute and chronic effects of FAs and the lipotoxicity-induced ß-cell failure during T2D development, with special emphasis on the oxidative stress induced by NOX, the ER stress, and the crosstalk between NOX and ER stress.

Transient NADPH oxidase 2-dependent H2O2 production drives early palmitate-induced lipotoxicity in pancreatic islets.

Modern lifestyles, including lack of physical activity and poor nutritional habits, are driving the rapidly increasing prevalence of obesity and type 2 diabetes. Increased levels of free fatty acids (FFAs), particularly saturated FFAs, in obese individuals have been linked to pancreatic ß-cell failure. This process, termed lipotoxicity, involves activation of several stress responses, including ER stress and oxidative stress. However, the molecular underpinnings and causal relationships between the disparate stress responses remain unclear. Here we employed transgenic mice, expressing a genetically-encoded cytosolic H2O2 sensor, roGFP2-Orp1, to monitor dynamic changes in H2O2 levels in pancreatic islets in response to chronic palmitate exposure. We identified a transient increase in H2O2 levels from 4 to 8 h after palmitate addition, which was mirrored by a concomitant decrease in cellular NAD(P)H levels. Intriguingly, islets isolated from NOX2 knock-out mice displayed no H2O2 transient upon chronic palmitate treatment. Furthermore, NOX2 knockout rescued palmitate-dependent impairment of insulin secretion, calcium homeostasis and viability. Chemical inhibition of NOX activity protected islets from palmitate-induced impairment in insulin secretion, however had no detectable impact upon the induction of ER stress. In summary, our results reveal that transient NOX2-dependent H2O2 production is a likely cause of early palmitate-dependent lipotoxic effects.

Evidence for NADPH oxidase activation by GPR40 in pancreatic ß-cells.

Objective: Investigate the involvement of the fatty acids receptor GPR40 in the assembly and activation of NADPH oxidase and the implications on pancreatic ß-cell function.Methods: BRIN-BD11 ß-cells were exposed to GPR40 agonist (GW9508) or linoleic acid in different glucose concentrations. Superoxide and H2O2 were analyzed, respectively, by DHE fluorescence and by fluorescence of the H2O2 sensor, roGFP2-Orp1. Protein contents of p47phox in plasma membrane and cytosol were analyzed by western blot. NADPH oxidase role was evaluated by p22phox siRNA or by pharmacological inhibition with VAS2870. NOX2 KO islets were used to measure total cytosolic calcium and insulin secretion.Results: GW9508 and linoleic acid increased superoxide and H2O2 contents at 5.6 and 8.3 mM of glucose. In addition, in 5.6 mM, but not at 16.7 mM of glucose, activation of GPR40 led to the translocation of p47phox to the plasma membrane. Knockdown of p22phox abolished the increase in superoxide after GW9508 and linoleic acid. No differences in insulin secretion were found between wild type and NOX2 KO islets treated with GW9508 or linoleic acid.Discussion: We report for the first time that acute activation of GPR40 leads to NADPH oxidase activation in pancreatic ß-cells, without impact on insulin secretion.

Chronic activation of GPR40 does not negatively impact upon BRIN-BD11 pancreatic ß-cell physiology and function.

BACKGROUND: Free fatty acids (FFAs) are known for their dual effects on insulin secretion and pancreatic ß-cell survival. Short-term exposure to FFAs, such as palmitate, increases insulin secretion. On the contrary, long-term exposure to saturated FFAs results in decreased insulin secretion, as well as triggering oxidative stress and endoplasmic reticulum (ER) stress, culminating in cell death. The effects of FFAs can be mediated either via their intracellular oxidation and consequent effects on cellular metabolism or via activation of the membrane receptor GPR40. Both pathways are likely to be activated upon both short- and long-term exposure to FFAs. However, the precise role of GPR40 in ß-cell physiology, especially upon chronic exposure to FFAs, remains unclear. METHODS: We used the GPR40 agonist (GW9508) and antagonist (GW1100) to investigate the impact of chronically modulating GPR40 activity on BRIN-BD11 pancreatic ß-cells physiology and function. RESULTS: We observed that chronic activation of GPR40 did not lead to increased apoptosis, and both proliferation and glucose-induced calcium entry were unchanged compared to control conditions. We also observed no increase in H2O2 or superoxide levels and no increase in the ER stress markers p-eIF2α, CHOP and BIP. As expected, palmitate led to increased H2O2 levels, decreased cell viability and proliferation, as well as decreased metabolism and calcium entry. These changes were not counteracted by the co-treatment of palmitate-exposed cells with the GPR40 antagonist GW1100. CONCLUSIONS: Chronic activation of GPR40 using GW9508 does not negatively impact upon BRIN-BD11 pancreatic ß-cells physiology and function. The GPR40 antagonist GW1100 does not protect against the deleterious effects of chronic palmitate exposure. We conclude that GPR40 is probably not involved in mediating the toxicity associated with chronic palmitate exposure.

The insulin resistance is reversed by exogenous 3,5,3'triiodothyronine in type 2 diabetic Goto-Kakizaki rats by an inflammatory-independent pathway.

PURPOSE: Diabetes mellitus (DM) has a multifactorial etiology that imparts a particular challenge to effective pharmacotherapy. Thyroid hormone actions have demonstrated beneficial effects in diabetic as well as obese rats. In both conditions, inflammation status plays a crucial role in the development of insulin resistance. Taking this into consideration, the present study aimed to demonstrate another possible pathway of thyroid hormone action on insulin sensitivity in a spontaneous type 2 diabetic rat model: the Goto-Kakizaki (GK) rats. GK animals present all typical hallmarks of type 2 DM (T2DM), except the usual peripheric inflammatory condition, observed in the other T2DM animal models. METHODS: GK rats were treated or not with 3,5,3'triiodothyronine (T3). Insulin sensitivity, glucose tolerance, and proteins related to glucose uptake and utilization were evaluated in the skeletal muscle, white adipose tissue, and liver. RESULTS: GK rats T3-treated presented enhanced insulin sensitivity, increased GLUT-4 content in the white adipose tissue and skeletal muscle, and increased hexokinase and citrate synthase content in skeletal muscle. Both non-treated and T3-treated GK rats did not present alterations in cytokine content in white adipose tissue, skeletal muscle, liver, and serum. CONCLUSIONS: These results indicate that T3 improves insulin sensitivity in diabetic rats by a novel inflammatory-independent mechanism.

Chronic treatment with dexamethasone alters clock gene expression and melatonin synthesis in rat pineal gland at night.

BACKGROUND: Melatonin is a neuroendocrine hormone that regulates many functions involving energy metabolism and behavior in mammals throughout the light/dark cycle. It is considered an output signal of the central circadian clock, located in the suprachiasmatic nucleus of the hypothalamus. Melatonin synthesis can be influenced by other hormones, such as insulin and glucocorticoids in pathological conditions or during stress. Furthermore, glucocorticoids appear to modulate circadian clock genes in peripheral tissues and are associated with the onset of metabolic diseases. In the pineal gland, the modulation of melatonin synthesis by clock genes has already been demonstrated. However, few studies have shown the effects of glucocorticoids on clock genes expression in the pineal gland. RESULTS: We verified that rats treated with dexamethasone (2 mg/kg body weight, intraperitoneal) for 10 consecutive days, showed hyperglycemia and pronounced hyperinsulinemia during the dark phase. Insulin sensitivity, glucose tolerance, melatonin synthesis, and enzymatic activity of arylalkylamine N-acetyltransferase, the key enzyme of melatonin synthesis, were reduced. Furthermore, we observed an increase in the expression of Bmal1, Per1, Per2, Cry1, and Cry2 in pineal glands of rats treated with dexamethasone. CONCLUSION: These results show that chronic treatment with dexamethasone can modulate both melatonin synthesis and circadian clock expression during the dark phase.

Zinc Supplementation Improves Glucose Homeostasis in High Fat-Fed Mice by Enhancing Pancreatic ß-Cell Function.

Zinc is an essential component of the insulin granule and it possibly modulates insulin secretion and signaling. Since insulin resistance is a hallmark in the development of type 2 diabetes mellitus, this study aimed at investigating if zinc supplementation is able to improve glucose tolerance and ß-cell function in a model of insulin resistance. Male C57BL/6 mice were distributed in four groups according to the diet: normal fat (NF); normal fat supplemented with ZnCl2 (NFZ); high-fat (HF); and, high-fat chow supplemented with ZnCl2 (HFZ). Intraperitoneal glucose (ipGTT) and insulin (ipITT) tolerance, glycemia, insulinemia, HOMA-IR, and HOMA-ß were determined after 15 weeks in each diet. Glucose-stimulated insulin secretion (GSIS) was investigated in isolated islets. The insulin effect on glucose uptake, metabolism, and signaling was investigated in soleus muscle. ZnCl2 did not affect body mass or insulin sensitivity as assessed by ipITT, HOMA-IR, muscle glucose metabolism, and Akt and GSK3-ß phosphorylation. However, glucose tolerance, HOMA-ß, and GSIS were significantly improved by ZnCl2 supplementation. Therefore, ZnCl2 supplementation improves glucose homeostasis in high fat-fed mice by a mechanism that enhances ß-cell function, rather than whole-body or muscle insulin sensitivity.

Insulin, not glutamine dipeptide, reduces lipases expression and prevents fat wasting and weight loss in Walker 256 tumor-bearing rats.

Cachexia is the main cause of mortality in advanced cancer patients. We investigated the effects of insulin (INS) and glutamine dipeptide (GDP), isolated or associated, on cachexia and metabolic changes induced by Walker 256 tumor in rats. INS (NPH, 40 UI/kg, sc) or GDP (1.5g/kg, oral gavage) was once-daily administered during 11 days after tumor cell inoculation. GDP, INS or INS+GDP treatments did not influence the tumor growth. However, INS and INS+GDP prevented retroperitoneal fat wasting and body weight loss of tumor-bearing rats. In consistency, INS and INS+GDP prevented the increased expression of triacylglycerol lipase (ATGL) and hormone sensitive lipase (HSL), without changing the expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in the retroperitoneal adipose tissue of tumor-bearing rats. INS and INS+GDP also prevented anorexia and hyperlactatemia of tumor-bearing rats. However, INS and INS+GDP accentuated the loss of muscle mass (gastrocnemius, soleus and long digital extensor) without affecting the myostatin expression in the gastrocnemius muscle and blood corticosterone. GDP treatment did not promote beneficial effects. It can be concluded that treatment with INS (INS or INS+GDP), not with GDP, prevented fat wasting and weight loss in tumor-bearing rats without reducing tumor growth. These effects might be attributed to the reduction of lipases expression (ATGL and LHS) and increased food intake. The results show the physiological function of INS in the suppression of lipolysis induced by cachexia mediators in tumor-bearing rats.

Pioglitazone improves insulin sensitivity and reduces weight loss in Walker-256 tumor-bearing rats.

AIM: The lipogenic effect of pioglitazone (PGZ), an insulin (INS) sensitizer, is well established. However, few studies have evaluated PGZ effects in preventing weight loss in cancer. We investigated PGZ effects, alone or associated with INS, on INS resistance, cachexia and metabolic abnormalities induced by Walker-256 tumor in rats. MAIN METHODS: PGZ (5.0mg·kg-1, oral) or PGZ+INS (NPH, 1.0UI·kg-1, sc), were once-daily administered during 12days, starting on the day inoculation of Walker-256 tumor cells. Rats were separated in small (about 17g) and big (about 30g) tumor-bearing. KEY FINDINGS: Big tumor-bearing rats showed greater cachexia, blood triacylglycerol and free fatty acids and INS resistance. PGZ and PGZ+INS treatments did not change tumor growth and food intake, but reduced several abnormalities such as INS resistance, increased blood free fatty acids, retroperitoneal fat wasting and body weight loss in small tumor-bearing rats. The prevention of retroperitoneal fat wasting did not involve reduction of tumor necrosis factor-α expression increased. In big tumor-bearing rats, PGZ and PGZ+INS treatments reversed the high blood triacylglycerol and free fatty acids levels, but had no effect on other parameters. SIGNIFICANCE: PGZ and PGZ+INS improved INS peripheral sensitivity, possibly by decreasing blood free fatty acids, and reduced fat tissue wasting and body weight loss in small tumor-bearing rats. The results suggest clinical benefits of PGZ in preventing INS resistance, adipose tissue wasting and weight loss when the tumor is small, i.e., in less severe cachexia.

Control of Insulin Secretion by Production of Reactive Oxygen Species: Study Performed in Pancreatic Islets from Fed and 48-Hour Fasted Wistar Rats.

Mitochondria and NADPH oxidase are important sources of reactive oxygen species in particular the superoxide radical (ROS) in pancreatic islets. These molecules derived from molecular oxygen are involved in pancreatic ß-cells signaling and control of insulin secretion. We examined the involvement of ROS produced through NADPH oxidase in the leucine- and/or glucose-induced insulin secretion by pancreatic islets from fed or 48-hour fasted rats. Glucose-stimulated insulin secretion (GSIS) in isolated islets was evaluated at low (2.8 mM) or high (16.7 mM) glucose concentrations in the presence or absence of leucine (20 mM) and/or NADPH oxidase inhibitors (VAS2870-20 µM or diphenylene iodonium-DPI-5 µM). ROS production was determined in islets treated with dihydroethidium (DHE) or MitoSOX Red reagent for 20 min and dispersed for fluorescence measurement by flow cytometry. NADPH content variation was examined in INS-1E cells (an insulin secreting cell line) after incubation in the presence of glucose (2.8 or 16.7 mM) and leucine (20 mM). At 2.8 mM glucose, VAS2870 and DPI reduced net ROS production (by 30%) and increased GSIS (by 70%) in a negative correlation manner (r = -0.93). At 16.7 mM glucose or 20 mM leucine, both NADPH oxidase inhibitors did not alter insulin secretion neither net ROS production. Pentose phosphate pathway inhibition by treatment with DHEA (75 µM) at low glucose led to an increase in net ROS production in pancreatic islets from fed rats (by 40%) and induced a marked increase (by 144%) in islets from 48-hour fasted rats. The NADPH/NADP+ ratio was increased when INS-1E cells were exposed to high glucose (by 4.3-fold) or leucine (by 3-fold). In conclusion, increased ROS production through NADPH oxidase prevents the occurrence of hypoglycemia in fasting conditions, however, in the presence of high glucose or high leucine levels, the increased production of NADPH and the consequent enhancement of the activity of the antioxidant defenses mitigate the excess of ROS production and allow the secretory process of insulin to take place.

Melatonin modifies basal and stimulated insulin secretion via NADPH oxidase.

Melatonin is a hormone synthesized in the pineal gland, which modulates several functions within the organism, including the synchronization of glucose metabolism and glucose-stimulated insulin secretion (GSIS). Melatonin can mediate different signaling pathways in pancreatic islets through two membrane receptors and via antioxidant or pro-oxidant enzymes modulation. NADPH oxidase (NOX) is a pro-oxidant enzyme responsible for the production of the reactive oxygen specie (ROS) superoxide, generated from molecular oxygen. In pancreatic islets, NOX-derived ROS can modulate glucose metabolism and regulate insulin secretion. Considering the roles of both melatonin and NOX in islets, the aim of this study was to evaluate the association of NOX and ROS production on glucose metabolism, basal and GSIS in pinealectomized rats (PINX) and in melatonin-treated isolated pancreatic islets. Our results showed that ROS content derived from NOX activity was increased in PINX at baseline (2.8 mM glucose), which was followed by a reduction in glucose metabolism and basal insulin secretion in this group. Under 16.7 mM glucose, an increase in both glucose metabolism and GSIS was observed in PINX islets, without changes in ROS content. In isolated pancreatic islets from control animals incubated with 2.8 mM glucose, melatonin treatment reduced ROS content, whereas in 16.7 mM glucose, melatonin reduced ROS and GSIS. In conclusion, our results demonstrate that both basal and stimulated insulin secretion can be regulated by melatonin through the maintenance of ROS homeostasis in pancreatic islets.

L-Arginine supplementation improves insulin sensitivity and beta cell function in the offspring of diabetic rats through AKT and PDX-1 activation.

Maternal hyperglycemia can result in defects in glucose metabolism and pancreatic ß-cell function in offspring. The purpose of this study was to evaluate the impact of maternal diabetes mellitus on pancreatic islets, muscle and adipose tissue of the offspring, with or without oral l-Arginine supplementation. The induction of diabetes was performed using streptozotocin (60mg/kg). Animals were studied at 3 months of age and treatment (sucrose or l-Arginine) was administered from weaning. We observed that l-Arg improved insulin sensitivity in the offspring of diabetic mothers (DA), reflected by higher insulin-induced phosphorylation of Akt in muscle and adipose tissue. Insulin resistance is associated with increased oxidative stress and the NADPH oxidase enzyme plays an important role. Our results showed that the augmented interaction of p47PHOX with gp91PHOX subunits of the enzyme in skeletal muscle tissue in the offspring of diabetic rats (DV) was abolished after l-Arg treatment in DA rats. Maternal diabetes caused alterations in the islet functionality of the offspring leading to increased insulin secretion at both low (2.8mM) and high (16.7mM) concentrations of glucose. l-Arg reverses this effect, suggesting that it may be an important modulator in the insulin secretory process. In addition it is possible that l-Arg exerts its effects directly onto essential molecules for the maintenance and survival of pancreatic islets, decreasing protein expression of p47PHOX while increasing Akt phosphorylation and PDX-1 expression. The mechanism by which l-Arg exerts its beneficial effects may involve nitric oxide bioavailability since treatment restored NO levels in the pancreas.

DHEA supplementation in ovariectomized rats reduces impaired glucose-stimulated insulin secretion induced by a high-fat diet.

Dehydroepiandrosterone (DHEA) and the dehydroepiandrosterone sulfate (DHEA-S) are steroids produced mainly by the adrenal cortex. There is evidence from both human and animal models suggesting beneficial effects of these steroids for obesity, diabetes mellitus, hypertension, and osteoporosis, conditions associated with the post-menopausal period. Accordingly, we hypothesized that DHEA supplementation in ovariectomized (OVX) female rats fed a high-fat diet would maintain glucose-induced insulin secretion (GSIS) and pancreatic islet function. OVX resulted in a 30% enlargement of the pancreatic islets area compared to the control rats, which was accompanied by a 50% reduction in the phosphorylation of AKT protein in the pancreatic islets. However, a short-term high-fat diet induced insulin resistance, accompanied by impaired GSIS in isolated pancreatic islets. These effects were reversed by DHEA treatment, with improved insulin sensitivity to levels similar to the control group, and with increased serine phosphorylation of the AKT protein. These data confirm the protective effect of DHEA on the endocrine pancreas in a situation of diet-induced overweight and low estrogen concentrations, a phenotype similar to that of the post-menopausal period.

Evidence for the involvement of GPR40 and NADPH oxidase in palmitic acid-induced superoxide production and insulin secretion.

G protein coupled receptor 40 (GPR40) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex have been shown to be involved in the fatty acid amplification of glucose-stimulated insulin secretion (GSIS). The effect of palmitic acid on superoxide production and insulin secretion by INS-1E cells and the possible involvement of GPR40 and NADPH oxidase in these processes were examined in this study. Cells were incubated during 1 h with palmitic acid in low and high glucose concentrations, a GPR40 agonist (GW9508) and inhibitors of NADPH oxidase (diphenyleneiodonium, DPI) and PKC (calphostin C). GW9508 induced superoxide production at 2.8 and 5.6 mM glucose concentrations and stimulated insulin secretion at 16.7 mM glucose concentration involving both PKC and NADPH oxidase activation. Palmitic acid induced superoxide production through NADPH oxidase and GPR40-dependent pathways and the stimulation of insulin secretion in the presence of a high glucose concentration was reduced by knockdown of GPR40 using siRNA. Our results suggest that palmitic acid induces superoxide production and potentiates GSIS through NADPH oxidase and GPR40 pathways in pancreatic ? cells.