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 β-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.

Fasting is known to cause physiological changes in the endocrine pancreas, including decreased insulin secretion and increased reactive oxygen species (ROS) production. However, there is no consensus about the long-term effects of intermittent fasting (IF), which can involve up to 24 hours of fasting interspersed with normal feeding days. In the present study, we analyzed the effects of alternate-day IF for 12 weeks in a developing and healthy organism. Female 30-day-old Wistar rats were randomly divided into two groups: control, with free access to standard rodent chow; and IF, subjected to 24-hour fasts intercalated with 24-hours of free access to the same chow. Alternate-day IF decreased weight gain and food intake. Surprisingly, IF also elevated plasma insulin concentrations, both at baseline and after glucose administration collected during oGTT. After 12 weeks of dietary intervention, pancreatic islets displayed increased ROS production and apoptosis. Despite their lower body weight, IF animals had increased fat reserves and decreased muscle mass. Taken together, these findings suggest that alternate-day IF promote β -cell dysfunction, especially in developing animals. More long-term research is necessary to define the best IF protocol to reduce side effects.

Caloric restriction (CR) prevents obesity and increases resilience against pathological stimuli in laboratory rodents. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher Ca²⁺ uptake rates and capacities, avoiding Ca²⁺-induced mitochondrial permeability transition. Dietary restriction has also been shown to increase kidney resistance against damaging stimuli; if these effects are related to similar mitochondrial adaptations has not been uncovered. Here, we characterized changes in mitochondrial function in response to 6 mo of CR in rats and measured bioenergetic parameters, redox balance, and Ca²⁺ homeostasis. CR promoted an increase in succinate-supported mitochondrial oxygen consumption rates. Although CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney, we found that mitochondrial H₂O₂ release was enhanced in a succinate-dependent manner. Surprisingly, and opposite to the effects observed in the brain and liver, mitochondria from CR animals were more prone to Ca²⁺-induced mitochondrial permeability transition, in a manner reversed by the antioxidant dithiothreitol. CR mitochondria also displayed higher Ca²⁺ uptake rates, which were not accompanied by changes in Ca²⁺ efflux rates or related to altered inner mitochondrial membrane potentials or amounts of the mitochondrial Ca²⁺ uniporter. Instead, increased mitochondrial Ca²⁺ uptake rates in CR kidneys correlated with loss of mitochondrial Ca²⁺ uptake protein 2 (MICU2), a mitochondrial Ca²⁺ uniporter modulator. Interestingly, MICU2 is also modulated by CR in the liver, suggesting that it has a broader diet-sensitive regulatory role controlling mitochondrial Ca²⁺ homeostasis. Together, our results highlight the organ-specific bioenergetic, redox, and ionic transport results of CR, with some unexpected deleterious effects in the kidney. NEW & NOTEWORTHY Prevention of obesity through caloric restriction (CR) is well known to protect many tissues but has been poorly studied in kidneys. Here, we determined the effects of long-term CR in rat kidney mitochondria, which are central players in energy metabolism and aging. Surprisingly, we found that the diet increased mitochondrial reactive oxygen production and permeability transition. This suggests that the kidneys respond differently to restricted diets and may be more susceptible under CR.

Nocturnal melatonin secretion is important for preservation of ß-cell mass and function. Knowing that type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by hyperglycemia caused by the elevated resistance of peripheral tissues to insulin, reduction in pineal melatonin and disturbances of insulin secretion by pancreatic ß-cells. In this context, exercise is considered one of the most valuable non-pharmacological approaches for treatment of T2DM. Considering the beneficial role of melatonin on glycemic metabolism in physical exercise, we investigated the effects of moderate-intensity aerobic exercise plus melatonin on glycemic homeostasis, the morphology and architecture of pancreas in spontaneous T2DM animals [Goto-Kakizaki (GK) rats]. The results confirmed that melatonin alone reduced the mass of epididymal white adipose tissue (WAT); however, only the combination of melatonin and physical exercise significantly reduced caloric intake, body weight, WAT and improved glucose tolerance and insulin sensitivity in T2DM rats. This combination also reduced apoptosis of cells in pancreatic islets. We observed either melatonin or the combination was able to reduce insulinemia. However, only the combination improved the morphology of the pancreatic islets. Thus, we conclude that in GK rats, melatonin plays a crucial role in the functionality of the pancreas to improve insulin sensitivity of peripheral tissues and, consequently, to maintain the glucose homeostasis. In addition, the combination is more efficiency to improve glucose tolerance and integrity of pancreatic islets in GK rats than melatonin alone.