Lipotoxicity in pancreatic beta cells may contribute to type 2 diabetes pathogenesis [2, 36]. Cholesterol is a functional component of cell membranes, which maintains beta cell secretory granules and plasma membrane function and the fluidity of beta cells [37]. However, excessive cholesterol may directly impair beta cell function. Cholesterol can be oxidated into several oxysterols in vivo, and previous studies have shown that oxysterols were higher in the type 2 diabetes subjects [38, 39]. As the predominant and most toxic oxidation product [13, 15], 7-ketocholesterol can have various side effects and is associated with several diseases (atherosclerosis and cardiovascular diseases, alzheimer disease, age-related macular degeneration) [17–22]. A previous study [25] demonstrated that diabetic serum contained higher levels of 7-ketocholesterol. Our GSIS results confirmed that 7-ketocholesterol could lead to impaired insulin secretion. Pancreatic beta cell insulin secretion is a biphasic, where it consists of a rapid and transient first phase, followed by a slowly developing and sustained second phase to respond glucose stimulation [40]. In type 2 diabetes, the first phase may be completely absent and the second phase reduced. And Our study demonstrated that the first phase and second phase insulin secretion were decreased obviously after 7-ketocholesterol treatment. The results further confirmed the impact of oxysterols on insulin secretion.
Insulin secretion is derived by glucose entry into the pancreatic beta cell by Glut2 and metabolism into ATP that subsequently shuts off the ATP-sensitive K+-channels, leading to membrane depolarization, voltage-dependent Ca2+ channel opening, Ca2+ influx, and insulin granule exocytosis [29, 30, 41]. Xu et al. used a TIRFM image of time-lapse visualization performed to observe the fusion processes in exocytosis. Excess cholesterol was found to reduce the number of glucose-stimulated fusion events, modulate the proportion of full fusion, and kiss-and-run fusion events [8]. However, there is currently no evidence to show the effect of 7-ketocholesterol on exocytosis. Our data shows that the full fusion events were reduced, and the proportion of kiss and run events was increased following 7-ketocholesterol treatment. These changes might lead to a reduction in insulin secretion. During the course of insulin release, the t-SNARE proteins, syntaxin 1 (STX1) and SNAP25 are localized in the plasma membrane, whereas the v-SNARE protein, VAMP2, is associated with insulin secretory granules [41, 42]. Our data show the SNAP25 was down-regulated, whereas STX1 and VAMP2 were unchanged following 7-ketocholesterol treatment. Therefore, 7-ketocholesterol was inhibited insulin granule binding with binding partner, SNAP25 to decrease insulin granule fusion on the plasma membrane. Furthermore, the calcium acts as a stimulus for insulin secretion and also a signal to increase insulin synthesis[43]. Ca2+ stimulates insulin secretion by regulating docking and initiating fusion of secretory granules with the plasma membrane, a process mediated by SNARE proteins. Ca2+ entry is actually directed to the sites of exocytosis via the binding of the L-type Ca2 + channels to SNARE proteins[44]. Calcium imaging in our study showed that calcium influx was reduced under high glucose stimulation after 7-ketocholesterol treatment, the result is consisting with the downregulation SNAP25 and exocytosis. Glut2 was associated with glucose sensing and is necessary for transporting glucose into the cell during the initiation phase of GSIS[45]. FFAs lipotoxicity was damaged islet β-cells insulin secretion and inhibited the Glut2 expression[46]. The level of GLUT2 expression were reduced to further demonstrate that insulin secretion was dysfunctional after 7-ketocholesterol treatment.
Characterized by increased ROS levels, oxidative stress is a notable factor in the pathogenesis of beta cell dysfunction in type 2 diabetes [47]. Excessive accumulation of saturated fatty acids can cause the generation of reactive oxygen species, resulting in oxidative stress, mitochondrial dysfunction, loss of mitochondrial membrane potential, impaired ATP production, and fracture and fragmentation of mitochondria, which ultimately leads to cell injuries [48]. Pancreatic beta cells in both rodents and humans are reportedly rich in mitochondria [49] and have low levels of classical antioxidant enzymes compared to other cell types, leading to vulnerable to mitochondria dysfunction and increasing oxidative damage[50]. Following pretreatment with 7-ketocholesterol, there was increased cellular ROS, decreased MMP and ATP levels, which indicated that 7-ketocholesterol enhanced cellular oxidative stress and damaged mitochondrial function. The evidence showed the NAC could reduced oxidative stress in many disease [51]. Our data showed the NAC was obviously eliminate cellular ROS and strength mitochondrial function; and the insulin secretion function recovered obviously. Through the regulation of cytoprotective gene expression, the KEAP1-NRF2 stress response pathway is the principal inducible defense against oxidative and electrophilic stresses. In response to stress, an intricate molecular mechanism facilitated by sensor cysteines within KEAP1 allows NRF2 to escape ubiquitination, accumulate within the cell, and translocate to the nucleus, where it can promote its antioxidant transcription program[52]. Our further study showed that antioxidant NRF2 was translocated from the cytoplasm into the nucleus after 7-ketocholesterol treatment, which then triggered the up-regulation of antioxidant genes to decrease oxidative stress. Maintaining redox homeostasis is important for cell function, while the antioxidation genes were up-regulated to alleviate injury, the antioxidant level could not prevent intracellular injury by 7-ketocholesterol, which accelerated beta cell damage in INS1 cells.
Pancreatic beta cells undergo dynamic compensation and decompensation processes during the development of type 2 diabetes, in which metabolic stresses such as oxidative stress, endoplasmic reticulum stress and inflammatory signals are key regulators of beta cell dynamics [47]. The present study only focused on an oxidative stress pathway in insulin secretion and exocytosis, antioxidants can partly restore the insulin secretion. There must be other mechanisms that 7-ketocholesterol can affect insulin secretion. Thus, future studies may employ such other mechanisms of affecting insulin secretion to induce 7-ketocholesterol injury.
Oxysterols are derived from cholesterol and provide a feedback mechanism of cholesterol biosynthesis to maintain cholesterol homeostasis. 7-ketocholesterol is one of the most important oxysterols and can be obtained either from food intake or from free-radical oxidation or the enzymatic oxidation of cholesterol in vivo [16, 53–55]. Additionally, 7-ketocholesterol can be metabolized to 27-hydroxylated 7-ketocholesterol and aqueous products by cholesterol 27-hydroxylase (CYP27A1), reduced to 7β-hydroxycholesterol by hydroxysteroid dehydrogenase (HSD11B1) and/or esterified by sterol O-acyltransferase (SOAT). Following the inhibition of CYP27A1, increased flux was diverted to reduction and esterification. When esterification was inhibited, further reduction and increased metabolism followed [16]. 7-ketocholesterol was observed to undergo greater hepatic metabolism and excretion, and no increased accumulation was observed in the tissues in the Cyp27−/− animals compared with the wild-type control mice [54]. Although 7-ketocholesterol has been shown to accumulate in human macrophage-foam cells and atherosclerotic lesions [55], dietary 7-ketocholesterol intake did not increase the levels of 7-ketocholesterol in the artery wall in mice. Moreover, the excessive dietary intake of 7-ketocholesterol does not affect cholesterol and glucose metabolism, which might be related to 7-ketocholesterol rebalance in vivo. The complexity of the generation and destination of 7-ketocholesterol makes it difficult to mimic a higher concentration of 7-ketocholesterol in vivo using a gene editing model or diet intervention model to accurately observe the adverse effects of 7-ketocholesterol in various organs.