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. 2020 Jul 16;12(4):87–98. doi: 10.1080/19382014.2020.1783162

Melatonin protects INS-1 pancreatic β-cells from apoptosis and senescence induced by glucotoxicity and glucolipotoxicity

Yu Hee Lee a, Hye Sook Jung b, Min Jeong Kwon a, Jung Eun Jang a, Tae Nyun Kim a, Soon Hee Lee a, Mi-Kyung Kim a,b, Jeong Hyun Park a,b,✉
PMCID: PMC7527021  PMID: 32673151

ABSTRACT

Introduction

Melatonin is a hormone known as having very strong anti-oxidant property. Senescence is a biological state characterized by the loss of cell replication and the changes consisting of a pro-inflammatory phenotype, leading to Senescence Associated Secretory Phenotype (SASP) which is now regarded as one of the fundamental processes of many degenerative diseases. Increased cell division count induces cell senescence via DNA damage in response to elevated Reactive Oxygen Species (ROS). We wanted to test whether melatonin could reduce apoptosis and stress induced premature pancreatic β-cell senescence induced by glucotoxicity and glucolipotoxicity.

Materials and method

Cultured rodent pancreatic β-cell line (INS-1 cell) was used. Glucotoxicity (HG: hyperglycemia) and glucolipotoxicity (HGP: hyperglycemia with palmitate) were induced by hyperglycemia and the addition of palmitate. The degrees of the senescence were measured by SA-β-Gal and P16lnk4A staining along with the changes of cell viabilities, cell cycle-related protein and gene expressions, endogenous anti-oxidant defense enzymes, and Glucose Stimulated Insulin Secretion (GSIS), before and after melatonin treatment.

Results

Cultured INS-1 cells in HG and HGP conditions revealed accelerated senescence, increased apoptosis, cell cycle arrest, compromised endogenous anti-oxidant defense, and impaired glucose-stimulated insulin secretion. Melatonin decreased apoptosis and expressions of proteins related to senescence, increase the endogenous anti-oxidant defense, and improved glucose-stimulated insulin secretion.

Conclusion

Melatonin protected pancreatic β-cell from apoptosis, decreased expressions of the markers related to the accelerated senescence, and improved the biological deteriorations induced by glucotoxicity and glucolipotoxicity.

KEYWORDS: Melatonin, pancreatic β-cell, Senescence, glucotoxicity, glucolipotoxicity

Introduction

Diabetes mellitus is a chronic metabolic disease represented by the persistently high blood sugar levels over a prolonged time and is related to many metabolic diseases. To prevent the development of complications, adequate metabolic control is necessary. But, usually, the degree of metabolic control of type 2 diabetes mellitus deteriorates over time. Aging process itself with decreased insulin sensitivity of the various tissues, along with the time-dependent deterioration of pancreatic β-cell function may be involved in this phenomenon.

Glucotoxicity and glucolipotoxicity denote the toxic effects of high blood glucose with or without hyperlipidemia frequently seen in uncontrolled diabetes patients. It usually increases the oxidative stresses, leading to increased insulin resistance of the muscle and adipose tissues, along with the functional decline of pancreatic β-cells.1

Senescence of the cells is a course that normal cells cease to divide. Cells can be induced to the state of senescence (SIPS: stress-induced premature senescence) via DNA damage in response to the elevated oxidative stress.2 Senescent cells remain metabolically active, and commonly transforms into SASP (Senescence Associated Secretory Phenotype) secreting inflammatory cytokines, growth factors, and proteases.3 SASP is associated with many chronic metabolic diseases, including type 2 diabetes and atherosclerosis.2

The inter-connections between senescence and diabetes mellitus are very sophisticated. The contributions of cellular senescence on the pathophysiologies of type 2 diabetes mellitus are through a direct impact on pancreatic β-cell function, SASP-mediated tissue and organ damage, and the effects on adipose tissue dysfunction. High circulating blood glucose levels and altered lipid metabolism can accelerate the degree of senescence of the cells.4 The diabetic microenvironment enhances cellular senescence by high blood glucose and increases the synthesis of ceramide, which in turn, causes so-called lipotoxicity.4 Diabetes mellitus and resulting oxidative stress are also associated with telomere shortening in various kinds of cell types.5,6

Melatonin is primarily secreted from the pineal gland and regulates the circadian rhythm like sleep–wake timing, blood pressure, etc.7 Many of its effects are through activation of melatonin receptors, while others are due to its role as an antioxidant.8-10 Melatonin promotes the expression of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase.11 In plants, it functions to defend against oxidative stress.12 From before there are some studies that melatonin receptors are expressed in pancreas β-cells and melatonin regulates insulin secretion through these receptors.13,14

We wanted to study that melatonin can prevent apoptosis and the several changes associated with stress-induced accelerated senescence by glucotoxicity and glucolipotoxicity in INS-1 rodent pancreatic β-cells.

Method

Cell culture

Rat insulinoma cell line, INS-1 cells, was a kind gift from Professor Won at Yeungnam University in South Korea.15 INS-1 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 11 mM Glucose, and 50 μM 2-mercaptoethanol. All the cell cultures were incubated at 37°C with 5% CO2, and studied between 30th and 40th passages.

Cell viability measurement (MTT assay)

INS-1 cells were seeded in 48-well plates at 2 × 104 cells per well and incubated for 48 hours. To make glucotoxic or glucolipotoxic environment, we used high glucose concentration or high glucose with palmitate in the culture medium. To assess the protective effect of melatonin (Sigma, MO, USA), the normal culture mediums were aspirated, and the new mediums containing 33 mM glucose or 33 mM glucose with 300 μM palmitate were applied, along with the various concentrations of melatonin inside, and further incubated for 96 hours. After 96 hours, the culture mediums were removed, the plates were washed with PBS, and the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reagents were applied and incubated for 2 more hours. The optical densities were measured at 570 nm by automatic plate reader.

Apoptosis assessment by Annexin V staining

INS-1 cells were plated in 6-well plates at 1 × 105 cells per well and incubated in the appropriate condition. The cells were treated with trypsin-EDTA and centrifuged in 1500 rpm for 5 minutes at 4°C. After the aspiration of supernatants, the cells were washed with 1 ml of Annexin V binding solution (140 mM NaCl, 10 mM HEPES pH 7.4, 2.5 mM CaCl2), and centrifuged in 1200 rpm for 5 more minutes at 4°C. The supernatants were removed, and 3 μl of Annexin V-FITC and 10 μl of propidium iodide were added. After incubation for 15 more minutes in the darkness, 300 μl of FACS buffer (1% FBS, 0.1% NaN3) was added and analyzed by FACSort (BECTON DICKINSON, BD bioscience).

Assessment of cell senescence by β-galactosidase staining

INS-1 cells were grown on a cover-slide in 6-well plates at 1 × 105 cells per well. The culture mediums were removed from the wells of the plates, and the plates were gently washed with PBS. Then, the cells were fixed and were gently washed with PBS again. All subsequent experiments were performed with Senescence β-Galactosidase Staining Kit according to the protocol provided by the manufacturer (Cell Signaling Company).

Assessment of the expression of p16 protein

INS-1 cells were plated onto cover-slides. After the appropriate incubation condition, cells were fixed and permeabilized with Cytoperm/Cytofix (BD, CA, USA) for 15 minutes and gently washed with PBS. Cells were blocked with blocking solution (5% normal goat serum, 0.3% Triton X-100 in PBS) for 1 hour at room temperature, and were added with anti-p16 for 1 hour at room temperature. After washing three times with PBS for 5 minutes, anti-Rabbit-alexa 555-conjugated secondary antibody and DAPI (1 μg/ml) were applied, and incubated for 1 hour at room temperature and gently washed with PBS. FISH analysis was performed using a green fluorescence microscope, Olympus BX-51 (Olympus, Tokyo, Japan)

Western blot analysis

After the incubation in the appropriate condition, INS-1 cells were gently washed with PBS and lysed in mammalian tissue lysis/extraction reagent including protease inhibitor (Roche, CA, USA). We performed the protein quantification by BCA protein assay kit (Pierce, CA, USA), added with 1× SDS sample buffer (50 mM Tris pH6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.01% bromophenol blue). Proteins were separated in 12% SDS-PAGE, transferred onto PVDF membrane, and immunoblotted with anti-p38 MAPK (1:1000, Cell signaling, MA, USA), anti-phospho p38 MAP kinase (Thr180/Tyr182) (1:1000, Cell signaling, MA, USA), Sirt1 (1:1000, Abcam, MA, USA), p16 (1:1000, Abcam, MA, USA), anti-p53 (1:1000, Millipore, MA, USA), anti-MnSOD (1:1000, Cell signaling, MA, USA), anti-Catalase (1:1000, Abcam, MA, USA), anti-CDK4 (1:1000, Abcam, MA, USA), and anti-alpha tubulin (1:1000, Sigma, MA, USA) at 4°C overnight. Secondary antibodies (goat anti-rabbit conjugated alkaline phosphatase and goat anti-mouse conjugated alkaline phosphatase, Cell Signaling, MA, USA) were applied for 1 hour at room temperature, and the membranes were developed via AP-conjugated development kit (Bio-rad, CA, USA). Developed protein bands were quantified by Multi Gauge V2.2 program (Tokyo, Japan).

Cell cycle analysis

Cultured INS-1 cells were treated with 1 μM of nocodazole for 24 hours, and reacted with high glucose or high glucose and palmitate with melatonin for additional 24 hours, and the cell cycle analysis was performed. The cells were harvested and for fixation, 70% ethanol was added dropwise and reacted at 4°C for 30 minutes. RNase (50 μg/ml) and Propidium Iodide (50 μg/ml) were added in each tube and reacted in the darkness for additional 30 minutes. And the cell cycle was analyzed by FACSort (BECTON DICKINSON, BD bioscience).

Insulin secretion (GSIS)

After the culture of INS-1 cells in the appropriate condition, the cells were gently washed with PBS. To quantify the degree of glucose-stimulated insulin secretion (GSIS), the INS-1 cells were starved for 1 hour in the RPMI medium containing 3 mM glucose and 2% FBS. Then, the medium was changed into KRBB solution (4.74 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgCl2∙6H2O, 35 mM NaHCO3, 10 mM HEPES) containing 3 mM glucose or 15 mM glucose, and the cells were incubated for the additional 1 hour. The intracellular insulin was isolated with the mammalian tissue lysis/extraction reagent including protease inhibitor (Roche, CA, USA). Secreted insulin was measured using Rat/Mouse Insulin ELISA kit (Linco Research, MO, USA). The amount of the secreted insulin was analyzed by calculating medium/intracellular insulin.

Statistical analysis

All experiments were repeated three times. Values are expressed as the means ± S.E.M. The differences between the two groups were analyzed using Student’s t-test (2-tailed), and multiple comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. P values less than.05 were considered as statistically significant.

Results

The effects of melatonin on HG and HGP induced apoptosis of INS-1 cells

The viabilities of the cultured INS-1 cells were measured in glucotoxic (HG: High Glucose) and glucolipotoxic (HGP: High Glucose with Palmitate) conditions. Both in HG and HGP conditions, the INS-1 cells showed significantly decreased cell viability as compared with the control group. The HGP condition did not significantly affect the cell viability compared with the only HG condition. In both HG and HGP culture conditions, melatonin supplementation significantly increased the cell viabilities in a dose-dependent manner (Figure 1A). HGP condition showed a more sensitive response to the administered melatonin for improving viability than HG condition. The effects of melatonin for the INS-1 cell apoptosis were tested. Compared with the control group, the degrees of cell apoptosis were markedly increased in both HG and HGP culture. But the addition of the melatonin completely abolished the harmful effects of HG and HGP culture conditions (Figure 1B).

Figure 1.

Figure 1.

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Effect of melatonin on cell viability and cell apoptosis.

The cultured INS-1 cells were maintained in 33 mM glucose or 33 mM glucose with palmitate 300 μM, and these cells were treated with the melatonin 500 nM, 1000 nM. The degree of viability (A) and apoptosis (B) of the cultured INS-1 cells were measured. Each bar represents mean ± standard deviation. Statistically significant differences are indicated by * (p < .05).

Assessment of the effects of melatonin on the SA-β-Gal expressions in INS-1 cells in HG and HGP culture conditions

Senescence-Associated β-Galactosidase (SA-β-Gal) is a hydrolase enzyme, and exerts its enzymatic activity only in the senescent cells. With p16Ink4A, this enzyme is routinely used as a marker for the cellular senescence.16 Decreased expression of SA-β-Gal can be interpreted as the rejuvenation of the cells. We performed SA-β-Gal staining (Figure 2A) and immunostaining for the p16Ink4A protein (Figure 2B) for the verification of the changes of the degree of INS-1 cell senescence. The SA-β-Gal expressions were markedly increased in HG and HGP culture conditions. However, this phenomenon was dramatically decreased when melatonin was added.

Figure 2.

Figure 2.

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The protective effects of melatonin on the senescence of INS-1 cells assessed by the expressions of senescence-associated β-galactosidase (SA β-Gal) and p16Ink4A.

INS-1 cells were cultured in the hyperglycemic (HG) or hyperglycemic and hyperlipidemic (HGP) conditions for 96 hours. After that, melatonin 1000 nM was added to the culture media, and the staining for senescence-associated β-galactosidase (SA β-Gal) was carried out (A) and immune-staining of p16Ink4A protein was also performed (B).

The effects of the melatonin on the cell cycle

The cells in the sub-G1 phase of the cell cycle are in the state of the progression of the apoptosis.17 The percentage of the INS-1 cells in sub-G1 phase increased by more than 30% in the HGP culture condition as compared with the control group. Melatonin treatment significantly reduced the percentage of the INS-1 cells in the sub-G1 phase in the HGP culture condition. However, in the HG culture condition, the administration of melatonin did not show significant effects on the cell cycle of INS-1 cells, and this might be the one reason that the addition of melatonin showed less powerful effects for the viability of INS-1 cells in the HG culture condition than HGP (Figure 3).

Figure 3.

Figure 3.

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The effect of the melatonin on the cell cycle.

The INS-1 cells in each cell cycle were assessed by the flow cytometry analysis (A). The percentages of each cell cycle were expressed by bar graphs (B). Statistically significant differences were indicated by * (p < .05).

The effects of melatonin on the expressions of proteins related with the cell cycle

One distinct feature of the cellular senescence is the arrest of the cell cycle, and this phenomenon is associated with the increased expression of p53 protein. The p53 protein expression is usually related to the induction of cell senescence and the increased cell apoptosis.12 We also examined the expressions of the other proteins related to the cell cycle and the cellular senescence. The p53, p16 and phosphorylated p38, which were related to the cellular senescence and the arrest of the cell cycle, were measured. All these proteins showed the increased expressions in the HG and HGP culture conditions, and the treatment with melatonin significantly decreased the expressions (Figure 4).

Figure 4.

Figure 4.

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The effects of the melatonin on the expressions of proteins related with the cell cycle genes.

Western blot analyses were performed for examining the changes of the protein expressions related to the cell cycle (A). Bar graphs represent the changes of the expressions of these proteins (B,C,D). Each bar represents mean ± standard deviation. Statistically significant differences are indicated by * (p < .05).

The effects of melatonin on the anti-oxidant enzymes

Reactive oxygen species (ROS) are continuously formed inside the body during the routine metabolic processes, especially in the mitochondria. Increased reactive oxygen species (ROS) by HG and HGP conditions overwhelmed the endogenous anti-oxidant defense capacities, eventually leading to cellular dysfunctions and increased apoptosis. The melatonin has been known as a very strong anti-oxidant, we checked the changes of the proteins related to anti-oxidant defense mechanisms. We measured the changes of the protein levels of Mn-SOD and catalase. In the HG culture condition, increased levels of Mn-SOD and catalase were observed, and these increases were normalized after the addition of the melatonin. In the HGP culture condition, the initial levels of Mn-SOD and catalase were not increased, and the addition of the melatonin significantly increased the levels of both anti-oxidant enzymes. We speculate that the increased levels of Mn-SOD and catalase by the addition of the melatonin can partially explain the findings that the melatonin exerts superior protective effects in HGP culture condition rather than HG (Figure 5).

Figure 5.

Figure 5.

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The effects of the melatonin for the anti-oxidant enzymes.

The changes of the levels of anti-oxidant proteins were evaluated in HG and HGP culture conditions by Western blot analysis, before and after melatonin treatment (A). Bar graphs represent the changes of the expressions of these proteins (B,C). Each bar represents mean ± standard deviation. Statistically significant differences were indicated by * (p < .05).

The effects of melatonin on GSIS (Glucose-induced insulin secretion)

The INS-1 cell used in our experiments is the rodent pancreatic β-cell line which can secrete insulin according to the ambient glucose in a dose-dependent manner. In HG and HGP conditions, just like advanced type 2 diabetes mellitus, the INS-1 cells show decreased capacity for GSIS (Glucose Induced Insulin Secretion). After using melatonin, partial, but significant improvements in GSIS capacity were only observed in HGP culture conditions (Figure 6).

Figure 6.

Figure 6.

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Improved GSIS of INS-1 cells by melatonin treatment.

The amounts of insulin secretion from the INS-1 cells in hyperglycemia (GSIS) were measured by ELISA. The amount of the secreted insulin was analyzed by calculating medium/intracellular insulin. Each bar represents mean ± standard deviation. Statistically significant differences are indicated by * (p < .05).

Discussion

To prevent chronic diabetic complications, tight or intensive blood glucose control is needed.18 But, the previous long-term clinical trials showed that the absolute value of blood glucose levels in type 2 diabetes progressively deteriorated over time despite the best clinical efforts.19 One of the main reasons for the time-dependent aggravation of metabolic control is the decline of pancreatic β-cell function.20 Uncontrolled hyperglycemia and hyperlipidemia in type 2 diabetes induce glucotoxicity and lipotoxicity.1 Multiple factors are believed to be responsible for the progressive decline of the residual β-cell function. Over-load of pancreatic β-cells for the excessive insulin requirement by pathologic insulin resistance, increased circulating free fatty acid, pro-inflammatory cytokines, excessive oxidative stress, and hyperglycemia per se might be related to this phenomenon.21 Through the upregulation of proteins involved in the various pathways, pancreatic β-cells in gluco-lipotoxic states ultimately undergo apoptosis.1

Senescence of the cell is characterized by which normal cells cease to divide. It is now known as “replicative senescence”, or the Hayflick limit. Replicative senescence is mechanistically caused by the shortening of the telomeres at the end of each chromosomal DNA during cellular division. Cells can also be aged in response to the excessive oxidative stress caused by the elevated reactive oxygen species (ROS).2 As cells age, they lose cloning ability, change into a pro-inflammatory phenotype and stain positive for senescence-associated β-galactosidase activity (SA – β Gal).22 P16INK4A is a tumor suppressor protein and a cyclin kinase inhibitor, and increased expression is a primary driver of cellular senescence, an irreversible form of cell cycle arrest.23 SA- β Gal and p16Ink4A are useful biomarkers of cellular senescence. Also, proteins p53, p21, p16ink4a and Bmi-1 involve in senescence signaling, some of them can serve as markers.24 A Senescence-Associated Secretory Phenotype (SASP) consisting of inflammatory cytokines, growth factors, and proteases is another characteristic feature of the senescent cells.3 SASP is associated with many chronic metabolic diseases like type 2 diabetes and atherosclerosis.25 This provided researchers with the opportunity to make anti-aging drugs as a new chronic metabolic disease control agent.25 Removing or targeted elimination of senescent pancreatic β-cells in diabetes mellitus has shown the possibility of improving the function of residual β-cells.26-28

The pathophysiologic mechanisms for the age-dependent deterioration of the pancreas β-cell function have been extensively studied, yet is still incompletely understood. While senescence prevents β-cell replication and regeneration, it also acts to increase the capacity of insulin secretion from the β-cells.29 The senescent β-cells secrete more insulin under basal conditions. It might have a negative impact on the glucose homeostasis and induce increased insulin resistance. Another important phenotype of senescent β-cells is the reduced potential for proliferation. It obviously poses a severe limitation on the flexibility of the system for controlling the intermediary metabolism. When β-cell enters the state of senescence, there will be no resources to increase β-cell function in response to the age-dependent deterioration of systemic insulin sensitivity.30

Melatonin is a product of the pineal gland and it regulates circadian rhythm.31 In humans, melatonin is a full agonist of melatonin receptor 1 and melatonin receptor 2, both of which belong to the class of G-protein coupled receptors (GPCRs).11 Melatonin occurs at high concentrations within the mitochondrial fluid which greatly exceeds the plasma concentration of melatonin.24,32,33 Due to its capacity for free radical scavenging, indirect effects on the expression of antioxidant enzymes, and its significant concentrations within mitochondria, a number of authors have indicated that melatonin has an important physiological function as a mitochondrial antioxidant.34

The role of melatonin in control of whole-body metabolism and insulin release has been controversial.35 Several previous studies have suggested a negative effect on insulin release,36 some studies showed stimulatory effects.37,38 Both improved and worsened glucose tolerance have been shown following melatonin treatment.39,40 The use of different animal species might be a reason, and the most human studies did not standardize the time of the experiments, because endogenous secretion of melatonin varies so much according to time. A decreased blood insulin level in night time, when melatonin levels are high but metabolic requirements are low, may be a physiological defense against dangerous nocturnal hypoglycemia.41

We showed in our experiments that hyperglycemia with or without hyperlipidemic condition increased the expressions of p16 protein and SA-β-Gal stain positivity in INS-1 pancreatic β-cells, which would be the characteristic features seen in senescence state. But our cell cycle analysis did not show the typical senescent pattern of cell cycle arrest. Instead, markedly increased subG1 cell population indicative of increased apoptosis in HGP culture condition was observed. It might be due to the overwhelming toxicity exerted by glucolipotoxicity, which drove the cells into the massive apoptosis before typical senescence induced cell cycle arrest. It should be studied in detail in the near future.

Melatonin in our experiments showed different effects on INS-1 pancreatic β-cells cultured in HG and HGP conditions. Although the overall effects of the protection of apoptosis were nearly the same, the degree of anti-oxidant enzyme expressions was quite different. The underlying mechanisms for the harmful effects exerted by glucotoxicity and glucolipotoxicity would be different. We speculate that the main action of melatonin in HG condition might be the direct suppression of ROS in mitochondria which resulted in a lower level of oxidative stress, and decreased the expressions of antioxidant enzymes. While in HGP condition, the toxic effects would greater that HG condition, which stimulated the expressions of strong anti-oxidant enzymes. This hypothesis should be verified in the near future.

Melatonin is a strong endogenous anti-oxidant. In our experiments, we could see that the melatonin not only possesses anti-oxidant property but also strong anti-senescent activity. Some previous reports showed that the administration of melatonin would cause decreased insulin secretion via binding with endogenous melatonin receptors. Usually, the amounts of in-vivo melatonin secretion are higher at night time, the suppression of endogenous insulin secretion at the time of food deprivation would be beneficial for avoiding un-necessary hypoglycemia. The actual net effects of melatonin supplementation for the type 2 diabetes patients should be determined in the near future through delicate and precise clinical trial designs with sufficient duration.

Several points should be expressed as the limitations of our study. Firstly, we used only one immortalized β-cell line for the entire experiments. The results of our experiments might be different when we use other cells, especially primary β-cells harvested from the actual animal models which would be very vulnerable to senescence than the cell lines. But the separation of insulin-secreting pure β-cells from the harvested animal islets is technically very difficult. And using actual whole animal islets to these experiments usually poses extreme difficulty in interpreting the results because of the contaminated responses from other islet cells. Secondly, we used supra-physiologic dose of melatonin in our experiments. But like many other in-vitro experiments, using high doses of testing substances would be the common procedure for the so-called “proof of concept” experiment. Surely, the results of our experiments could not be extrapolated to the physiologic conditions, and this should be regarded as an important limitation of this study. Thirdly, we used SA-β-Gal staining and p16 expression as senescence markers. Although they are regarded as representative senescence markers, they also could be elevated in inflammation and other situations. And especially in experiments for SIPS (stress-induced premature senescence), clear discriminations between senescence and stress-induced inflammation would be difficult. The increase in both SA-β-Gal and p16 levels in our experiment clearly suggests the possibility of the involvement of the senescence process, more delicate and detailed molecular procedures would be necessary in the future experiments. Lastly, we could not clearly discriminate the anti-oxidant and anti-senescence effects of melatonin on the stressed β-cells. We believe that the strong anti-oxidant property of melatonin partly contributes to its anti-senescence effects. Our lab will perform further experiments using other anti-oxidants with nearly the same efficacy as an active control to differentiate anti-senescence from anti-oxidant efficacies of melatonin.

In conclusion, we showed that the administration of melatonin to the stressed INS-1 pancreatic β-cells by hyperglycemia and/or hyperlipidemia significantly reduced apoptosis and the expressions of important senescence-related molecular markers. Further studies are needed to clarify this issue, especially in the view-point of long-term stability or durability of metabolic control of type 2 diabetes patients.

Funding Statement

This work was supported by grant from Inje University Busan Paik Hospital, 2017.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

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