The daily insulin secretion pattern correlates with the circadian rhythm including melatonin in pancreatic islets and beta cells [26, 27]. Melatonin suppresses insulin secretion in both rats and mice [28–31]. Moreover, melatonin can have direct or indirect effects under physiological conditions. Such differences could be attributed to the different time courses for melatonin administration and the requirements for daily rhythmicity. In this condition, melatonin has direct effects on insulin secretion in pancreatic islets and beta cells, effects that can be dependent on melatonin receptors. The non-hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) and the melatonin antagonist luzindole block the effects of melatonin on insulin secretion in neonatal rat islets . INS-1 cells [33, 34], rat islets [35, 36], and human islets  express MTNR1A mRNA. Also, MTNR1B mRNA has been detected in rat [35, 36] and human  islets. MIN-6 cells express both forms of the melatonin receptor . The present study showed that 10–100 nM melatonin concentrations can increase the insulin level in cells and decrease the insulin level in media in a melatonin dose-dependent manner and with dependence on melatonin receptors.
Physiological melatonin concentrations lower than nanomolar concentrations are involved with the regulation of many biological and physiological processes in the human body and are dependent on melatonin receptor functions within the gastrointestinal system, heart, brain, and immune system [37, 38]. In vitro, nanomolar melatonin can directly suppress human breast cancer cell survival through the estrogen-response pathway, as well as a decrease in the activity and expression of aromatase, sulfatase, and 17 beta-hydroxysteroid dehydrogenase, and an increase in the activity and expression of estrogen sulfotransferase. These observations support the suggestion that melatonin is mainly associated with antiestrogenic actions and interactions in tumor cells’ estrogen-signaling pathway . Arese et al.  demonstrated that a 1 nM concentration of melatonin in HaCaT cells incubated for 3–6 h induces a 4-fold increase in neuronal NOS mRNA expression above the basal level. HaCaT cells exposed to melatonin concentrations in the 1 nM range for 5 h showed the entry of melatonin into cells and heterogeneous distribution of melatonin levels among organs and tissues, resulting in a cell melatonin concentration of 52.86 × 10− 3 ± 0.0063 pM, which is similar to the melatonin concentration flowing in the human blood at night (approximately 100–200 pg/mL), the peak melatonin concentration-time [40, 41]. Melatonin treatment at 1 pM to 1 nM showed a nitrergic inhibition in hamster retina , suggesting that melatonin probably modulates the nitrergic activity through a receptor-mediated mechanism in the retina. This effect of melatonin on the nitrergic activity was shown to be dependent on the physiological concentration in rat hypothalamus and cerebellum [42, 43]. Melatonin, a highly lipophilic molecule, can enter cells and cross all physiological barriers. However, this effect of melatonin can be restricted to cell types that express both melatonin receptors and nitrergic activity . The present study demonstrated that nanomolar melatonin concentrations can influence insulin synthesis in rat insulinoma INS-1E cells and insulin secretion in media, which actions are dependent on melatonin receptors.
This study demonstrated the insulin secretion and vesicle trafficking-related proteins include Rab5, GOPC, caveolin-1, EEA1, clathrin, APPL1, and syntaxin-6. In an early endosome fusion, GOPC interacts with syntaxin- 6 , and syntaxin-6 is a member of the syntaxin family of SNAREs that are Rab5 effectors. This family comprises at least 17 members, all of which localize to membrane compartments along the endocytic and exocytic pathways [46, 47]. Syntaxin-6 can interact with the EEA1 protein, a Rab5 effector , suggesting that syntaxin-6 may promote the tethering of post-Golgi vesicles to early endosomes. Cytosolic syntaxin-6 has predominantly negative effects on the endosomal system of pancreatic β-cells and can provide indirect interference with insulin secretory pathways . The APPL1 protein leads to the phosphatidylinositol 3-kinase (PI3K)-dependent formation of EEA1-positive early endosomes  which activate the Rab5 effector [50, 51]. APPL1 protein regulates insulin secretion in pancreatic β cells by regulating the expression of SNARE proteins through the PI3K/Akt-dependent pathway, suggesting that APPL1 protein is a master coordinator and controller of insulin secretion . Clathrin, a cargo protein of transport vesicles, is not required in the formation of β-cell secretory granules or to process or regulate exocytosis of proinsulin/insulin .
The key target of Akt/PKB and insulin in a multitude of cellular events including glucose uptake, glycogen synthesis, gluconeogenesis, and lipid storage is phosphoinositide 3-kinase (PI3K) to specific sites on IRS1/IRS2 that are tyrosine-phosphorylated by the insulin receptor [21, 52–55]. A major down-regulator of insulin action is mTOR, which belongs to the PI3K-related kinase protein family. mTOR functions in a mitogenic pathway downstream of PI3K and is activated by insulin and growth factors in the presence of sufficient nutrients including amino acids and glucose [56, 57]. mTOR Complex 1 (mTORC1) is composed of rapamycin-sensitive adaptor protein of mTOR (Raptor), G-protein β-subunit-like protein (GβL), and the Akt/ PKB substrate 40 kDa (PRAS40) [58, 59]. The mTORC1 is activated by insulin, growth factors, serum, phosphatidic acid, amino acids (particularly leucine), and oxidative stress [58, 60]. mTOR Complex 2 (mTORC2) is composed of mTOR, rapamycin-insensitive companion of mTOR (Rictor), GβL, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1) [61, 62]. mTORC2 phosphorylates the Akt/PKB at serine 473 residue. Phosphorylation of serine stimulates Akt phosphorylation at threonine 308 residue by PDK1 and leads to full Akt activation [63, 64]. It has been reported that mTORC2 is regulated by insulin, growth factors, serum, and nutrient levels . The present study shows that insulin biosynthesis and secretion through the Akt/mTORC1 pathway.
The Bcl-2 family proteins including Bcl-2, Bcl-xL, and Bax have been studied as a pro- and antioxidant [62–64]. The localization of Bcl-2 to the mitochondria indicates it has a role in generating a slight pro- and antioxidant state via mitochondrial machinery [65, 66]. Chen and Pervaiz  proposed that mitochondrial respiration, as a novel mechanism, can control survival even in the presence of ROS, indicating that this protective mechanism, including antioxidant defense systems such as SOD, can prevent ROS production. Mn-SOD, localized to the mitochondrial matrix, predominantly quenches mitochondrial O2− and increases activity through Bcl-2 overexpression. In this study, the expression levels of Bcl-2, Bcl-xL, and Bax proteins involved in the regulation of insulin synthesis or its secretion from β-cells.