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The Effects of 4-Methylcatechol in Rat Insulinoma β-cells (INS-1)
Insülinoma is a tumor originating from the beta cells of the Langerhans islets, which form the endocrine part of the pancreas and secretes insulin. In healthy individuals, insulin is secreted from the beta cells in order to decrease the increased blood glucose. When the blood glucose reaches homeostasis, an inhibitory mechanism stops the excessive secretion of insulin. However, insulin secretion could not be regulated by this mechanism in insülinoma and insülinomas continue insulin secrection. 4-methylcatechol, which is also known as 3,4-dihydroxytoluene, is a catecholamine derivative and it is known that it show a cytotoxic effect by generating reactive oxygen species. The aim of this study was to determine the doses of 4-methylcatechol that caused cell death in rat insülinoma β-cells (INS-1), to find out the type of cellular death at these doses, and to investigate the molecular mechanism of cellular death occurred.
In this study, we have used INS-1 cell line. In order to determine the death rate of the cell along with what type of death (early apoptosis, later apoptosis, and necrosis) occurred at the doses causing cellular death, the cells were marked with acridine orange/ethidium bromide and visualized under fluorescent microscope. Under these doses, the apoptosis rate with apoptose kit and necrosis rate with cytotoxicity kit were measured as spectrophotometric method. Reactive oxygen species were shown by using dichlorofluorescein diacetate and mitochondrial membrane potential loss was indicated with 3,3'-dihexyloxycarbocyanine iodide, both spectrophotometric method, as a result of incubation of the cells. High-performance liquid chromatography was used to determine the intracellular ATP and GTP decrease. The amount of insulin in the cell and the secretion were calculated by using insulin ELISA kit. The JNK/ERK cellular pathway was investigated at the cell death-causing doses after exogeneously administered 4-methylcatechol. Both active and total JNK and ERK amounts were shown with the ELISA method. In the MAPK pathway, functional p-RAF1, both active and total Elk1, c-Jun and ATF2 transcription factors, heat shock proteins (Hsp 70 and Hsp 90) were shown in the cell. Betacellulin and inhibin beta-A amounts were shown in both the cell and the secretion with western blot method. Expression levels of the molecules that determined the protein levels were calculated from the Ct values obtained with q-RT-PCR method.
It was shown that cellular death was observed in between 250-450 µM doses of 4-methycatechol in INS-1 cells. We calculated that there was a significant increase of apoptotic cell death at determined doses compared to control group. It was observated more necrotic cells than apoptosis with administration of 350, 400, and 450 µM 4-methylcatechol. Lactate dehydrogenase levels, reactive oxygen species, and mitochondrial potential loss were increased at these doses. Our findings were supported by ATP and GTP losses.
When JNK and ERK cellular pathway were screened, an increase in p-RAF1 activity, a decrease in its expression, a decrease in the total JNK amount, an increase in active JNK amount, a decrease in its expression, an increase in total c-Jun amount were found, but no change in active c-Jun amount and its expression were observed by 4-methylcatechol administration when compared to control group at determinated doses. Total ATF2 and active ATF2 amounts did not lead to any change, but there was a decrease in its expression. Total ERK amount did not change, but active ERK and its expression did decrease. There was no change in total Elk1 and active Elk1 amounts, but it was found a decrease in Elk1 expression. The amounts of Hsp 70 and Hsp 90 from heat shock proteins their expressions were not found a change in between the groups. Betacellulin was decreased in the cell, the secretion and its expression. Inhibin beta A did not lead to a change in the cell and the secretion, while a decrease in its expression was noticed.
Intracellular insulin amount did not change significantly in between the groups, but Ins1 expression showed a decrease. Insulin secrection increased with giving a low dose of glucose in increasing doses of 4-methylcatechol, whereas there was no difference in between the groups with giving a high dose of glucose. In addition, a decrease was observed in GLUT2 expression.
As a result, cellular death occurred when 4-methylcatechol was applied to insülinoma INS-1 cells at 250-450 µM doses. Giving 350, 400, and 450 µM 4-methylcatechol led to more necrotic death of the cells. We concluded that cells perform necrotic death by the following options: i) phosphorylated RAF1 activates the JNK pathway with the activity of transcription factor c-Jun, ii) Hsp 70 and Hsp 90 did not show a change inside the cell, rendering the JNK pathway active.