Chinese Medical Journal 2013;126(5):937-941
Liraglutide prevents high glucose level induced insulinoma cells apoptosis by targeting autophagy

Correspondence to:LI Yan-bo,Department of Endocrinology, First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China (Tel: 86-451-85555637. Fax:. E-mail:liyanbo65@163.com)
Keywords
autophagy; pancreatic beta-cell; type 2 diabetes; liraglutide; apoptosis
Abstract
Background  The pathophysiology of type 2 diabetes is progressive pancreatic beta cell failure with consequential reduced insulin secretion. Glucotoxicity results in the reduction of beta cell mass in type 2 diabetes by inducing apoptosis. Autophagy is essential for the maintenance of normal islet architecture and plays a crucial role in maintaining the intracellular insulin content by accelerating the insulin degradation rate in beta cells. Recently more attention has been paid to the effect of autophagy in type 2 diabetes. The regulatory pathway of autophagy in controlling pancreatic beta cells is still not clear. The aim of our study was to evaluate whether liraglutide can inhibit apoptosis and modulate autophagy in vitro in insulinoma cells (INS-1 cells).
Methods  INS-1 cells were incubated for 24 hours in the presence or absence of high levels of glucose, liraglutide (a long-acting human glucagon-like peptide-1 analogue), or 3-methyadenine (3-MA). Cell viability was measured using the Cell Counting Kit-8 (CCK8) viability assay. Autophagy of INS-1 cells was tested by monodansylcadaverine (MDC) staining, an autophagy fluorescent compound used for the labeling of autophagic vacuoles, and by Western blotting of microtubule-associated protein I light chain 3 (LC3), a biochemical markers of autophagic initiation.
Results  The viability of INS-1 cells was reduced after treatment with high levels of glucose. The viability of INS-1 cells was reduced and apoptosis was increased when autophagy was inhibited. The viability of INS-1 cells was significantly increased by adding liraglutide to supplement high glucose level medium compared with the cells treated with high glucose levels alone. 
Conclusions  Apoptosis and autophagy were increased in rat INS-1 cells when treated with high level of glucose, and the viability of INS-1 cells was significantly reduced by inhibiting autophagy. Liraglutide protected INS-1 cells from high glucose level-induced apoptosis that is accompanied by a significant increase of autophagy, suggesting that liraglutide plays a role in beta cell apoptosis by targeting autophagy. Thus, autophagy may be a new target for the prevention or treatment of diabetes.
The pathophysiology of type 2 diabetes is progressive pancreatic beta cell failure with consequential reduced insulin secretion, and insulin resistance in peripheral tissues. The beta cell mass and acquired beta cell dysfunction can not secrete adequate insulin to overcome peripheral insulin resistance, which contributes to development of diabetes. Glucotoxicity results in the reduction of beta cell mass in type 2 diabetes by inducing apoptosis. In addition, autophagy is also thought to be responsible for the loss of beta cell mass.1,2
 
Autophagy is a biological process in which double-membrane cytosolic vesicles called autophagosomes sequester cytoplasm, subsequently delivering it to lysosomes where the engulfed cellular components are degraded.3,4 Autophagy is markedly upregulated during conditions of nutrient or growth factor deficiency to maintain the normal functioning of cellular structures and provide energy for cell survival.3,5 In recent articles, signs of altered autophagy were observed in the beta cells of the Zucker diabetic fatty rat and in a beta cell line following prolonged exposure to high glucose.6 Autophagy is essential for the maintenance of normal islet architecture and plays a crucial role in maintaining the intracellular insulin content by accelerating the insulin degradation rate in beta cells of the Rab3A–/– null mouse.7,8 Further demonstration of the importance of normal autophagy for the preservation of beta cells has been recently provided; impaired glucose tolerance and lower insulin levels in beta cell-specific Atg7 knockout mice have been reported.9 These phenotypes are accompanied by vacuolar changes, mitochondrial swelling, and endoplasmic reticulum (ER) distension in beta cells. Nonetheless, autophagy may also represent a form of programmed cell death called autophagic cell death or type 2 programmed cell death, and altered autophagy is associated with loss of beta cell mass in diabetes.1
 
Recently more attention has been paid on the effect of autophagy in type 2 diabetes,10 although the regulatory pathway of autophagy for controlling pancreatic beta cells is still not clear. We hypothesized that autophagy can be precisely modulated in the development of diabetes. Glucagon-like peptide-1 (GLP-1) is a 37-amino acid peptide secreted from the L-cells of the intestinal epithelium in response to food. GLP-1 has been shown to increase glucose-dependent insulin secretion, expand islet cell mass, and inhibit β-cell apoptosis in animal models of diabetes. Liraglutide is a human incretins-GLP-1 analogue with high homology to the native hormone. It shares 97% sequence identity with native human GLP-1.11 A recent study has demonstrated that liraglutide increases islet cell proliferation and total β-cell mass in Zucker diabetic rats. It has also been proven that liraglutide inhibits in a dose-dependent manner the cytokine-induced apoptosis in primary rat islet cells, and reduces free fatty acid induced apoptosis by approximately 50%.12 In this study, we determined the role of autophagy in pancreatic beta cells exposed to high glucose levels and investigated the capability of GLP-1 to preserve high glucose level induced INS-1 cell apoptosis by targeting autophagy.
 
METHODS
 
Cell culture
INS-1 rat insulinoma cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, China) supplemented with 15% fetal bovine serum (FBS, Sigma, USA), and antibiotics. Cells were maintained at 37°C in an atmosphere of 5% CO2 and 100% humidity. Purified INS-1 cells were divided into six groups and cultured for 24 hour using RPMI-1640 medium in the presence of 5 mmol/L glucose (control group), 5 mmol/L glucose + 5 mmol/L 3-methyadenine (3-MA, Sigma), 5 mmol/L glucose + 100 nmol/L liraglutide (Novo Nordisk, Denmark), 25 mmol/L glucose, 25 mmol/L glucose + 5 mmol/L 3-MA, and 25 mmol/L glucose + 100 nmol/L liraglutide.13
 
Cell counting kit-8 (CCK8) viability assay
INS-1 cells were treated with CCK8 (Japan-Dojindo Laboratories, Japan) at 37°C for 1 hour. Absorbance was measured at 450 nm using a microplate reader (Tecan (Shanghai) Trading Company, Switzerland).
 
MDC staining
INS-1 cells were stained with 50 µm MDC (Sigma) in phosphate buffer saline (PBS) at 37°C for 1 hour. After washing with PBS, cells were immediately analyzed under a fluorescence microscopy at 355 nm excitation and 460 nm emission (Becton, Dickinson Company, USA).
 
Annexin V-FITC/PI staining
INS-1 cells were stained with Annexin V-FITC and propidium iodide (Annexin V-FITC/PI) (Baosai Corporation, China) at room temperature for 5 to 15 minutes, then immediately analyzed by flow cytometry.
 
Western blotting analyses
Equal amounts of protein (50 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, and immunoblotted with antibodies described above. After incubation with secondary antibody conjugated to horseradish peroxidase, the bands were detected with the enhanced chemiluminescence (ECL) system (GE healthcare, UK).
 
Statistical analysis
Data were presented as mean±standard deviation (SD) of at least three independent experiments. Statistical differences between the various groups were determined using the Student’s t test by SPSS 20.0 (SPSS Inc., USA). P values less than 0.05 were considered statistically significant.
 
RESULTS
 
Chronic exposure to high glucose increases INS-1 cell death
To test the effect of high glucose on cell viability, INS-1 cells were incubated in 25 mmol/L glucose for 24 hours and cell viability was measured using the CCK8 assay. The results show that the viability of INS-1 cells treated with 25 mmol/L glucose was reduced by 20% when compared with the control (Figure 1). The apoptotic cells accounted for 19% of the dead cells compared with 5% in the control group (Figure 2). Autophagy of INS-1 cells was test by monodansylcadaverine (MDC) stain, an autofluorescent compound used for the in vivo labeling of autophagic vacuoles, and by Western blotting of LC3. Results showed dot-like structures appeared in cytoplasm and in perinuclear regions in the high glucose treated INS-1 cells after MDC staining, and the proportion of cells with MDC stained dots and the level of LC3 were dramatically increased in INS-1 cells induced by high glucose. This demonstrated that autophagy was enhanced in INS-1 cells incubated in 25 mmol/L glucose (Figures 3 and 4).
 

view in a new window
Figure 1. The viability of INS-1 cell with the treatment of high glucose, 3-MA and liraglutide. The cell viability was reduced with a high glucose level (25 mmol/L glucose) for 24 hours (P <0.01). When autophagy was inhibited (treated with 5 mmol/L 3-MA), the viability of INS-1 cells in high glucose was significantly reduced (P <0.01) and the viability of INS-1 cells was also reduced in normal glucose (P <0.01).
 

view in a new window
Figure 2. The apoptosis of INS-1 cells with high glucose treatment, 3-MA and liraglutide. The apoptosis of INS-1 cells was increased by high glucose after 24 hours (P <0.01). When autophagy was inhibited, the apoptosis of INS-1 cells was significantly increased compared with treatment with high glucose alone (P <0.01) and the apoptosis of INS-1 cells with high glucose alone was also increased in normal glucose (P <0.01). The apoptosis of INS-1 cells was significantly increased by adding liraglutide to high glucose medium (P <0.01).
 
Role of autophagy in INS-1 cells
To test the effect of autophagy in high glucose level induced INS-1 cell death, INS-1 cells were treated with 3-MA, an autophagy inhibitor. Pre-treated with 3-MA, the viability of INS-1 cells incubated in high glucose was reduced by 60% (Figure 1), and apoptosis was significantly increased to 23% (Figure 2). It demonstrated that autophagy plays a protective role in high glucose level induced INS-1 cell apoptosis. The viability of INS-1 cells in the control group was also reduced by 40% (Figure 1), and apoptosis was increased to 10% after treated with 3-MA (Figure 2), suggesting that autophagy is indispensable in the survival of cells in a normal environment.
 
Role of liraglutide in INS-1 cells
In our study, the viability of INS-1 cells in high glucose was significantly improved (increased by 10%) by liraglutide (Figure 1). The apoptosis of INS-1 cell was reduced to 7% compared with cells treated with high glucose alone (Figure 2). The MDC fluorescence absorbency and the LC3 levels were also increased compared with the high glucose group (Figures 3 and 4).
 

view in a new window
Figure 3. The autophagy of INS-1 cells after treatment with high glucose, 3-MA and liraglutide. MDC is an autofluorescent compound used for the in vivo labeling of autophagic vacuoles. The proportion of INS-1 cells with MDC stained dots was dramatically increased after treatment with high glucose (P <0.01). When autophagy was inhibited with 3-MA, the proportion of INS-1 cells with MDC stained dots was remarkably reduced (P <0.01). The proportion of INS-1 cells with MDC stained dots was significantly increased after treatment of liraglutide.
 

view in a new window
Figure 4. The expression of LC3 in INS-1 cells. A: 5 mmol/L glucose (control group); B: 5 mmol/L glucose + 5 mmol/L 3-MA; C: 5 mmol/L glucose + 100 nmol/L liraglutide; D: 25 mmol/L glucose; E: 25 mmol/L glucose + 5 mmol/L 3-MA; F: 25 mmol/L glucose + 100 nmol/L liraglutide. The expression of LC3 was enhanced with the treatment of liraglutide and significantly decreased with 3-MA.
 
DISCUSSION
 
The main functions of pancreatic beta cells are insulin synthesis and secretion, both of which play an important role in glucose homeostasis. Type 2 diabetes is a progressive disease, characterized of insulin resistance and the beta cell disfunction, which lead to impaired glucose-induced insulin secretion. In our study, the viability of beta cells was remarkably reduced and the apoptosis was significantly increased when treated with high glucose levels, while the autophagy of beta cell was enhanced. After inhibiting autophagy with 3-MA, the apoptosis of beta cells was significantly increased. These results indicate that autophagy plays a protective role in high-glucose induced INS-1 beta cell apoptosis. Autophagy is regarded as an adaptive respond to ER stress and oxidative stress that eventually results in degradation of intracellular proteins and organelles.
 
Glucotoxic and dyslipidemic states are part of the metabolic syndrome associated with type 2 diabetes. Glucotoxicity and lipotoxicity contribute to the reduction of beta cell mass in type 2 diabetes. Glucose is a well-known stimulus of proinsulin biosynthesis. Insulin biosynthesis approaches 50% of total protein synthesis in stimulated beta cells, and the ER is responsible for the synthesis of almost all secreted proteins. These proteins are correctly folded and assembled by chaperones in the ER lumen. When the glucose concentration increases, the insulin demand also is markedly increased. As a result, the chaperones become overloaded and the ER fails to fold and export newly synthesized proteins, leading to ER stress.14 ER stress can result in the accumulation of misfolded proteins, and is necessary for lipid-induced apoptosis in beta cells.15,16 In addition to ER stress, pancreatic beta cells are prone to oxidative stress, due to the fact that antioxidants such as superoxide dismutase (SOD), glutathione peroxidase and catalase are present at low levels in beta cells. Oxidative stress can also lead to the accumulation of misfolded proteins. Lenzen et al17 studied gene expression of SOD, catalase, and glutathione peroxidase in pancreatic islets and for comparison in various other mouse tissues using a sensitive Northern blot hybridization technique. Gene expression of the antioxidant enzymes is usually in the range of ±50% of that in the liver. They found that in pancreatic islets gene expression is substantially lower. The levels of the cytoplasmic Cu/Zn SOD and the mitochondrial Mn SOD gene expression are in the range of 30%–40% of the liver. Glutathione peroxidase gene expression is 15%, and catalase gene expression is not detectable in pancreatic islets.18
 
Protein degradation is mediated by two major systems; the ubiquitin proteasome system and autophagy. The ubiquitin proteasome system is a major degradation system for short-lived proteins, whereas autophagy is mainly responsible for the degradation of long-lived proteins and other cellular contents. Autophagy is activated when the ubiquitin proteasome system is inhibited, which suggests the two systems are functionally linked.19 Ubiquitinated-protein aggregates increase in INS-1 cells in response to high glucose. The formation and clearance of ubiquitinated-protein aggregates are regulated by autophagy. Inhibition of the proteasome does not affect the clearance of ubiquitinated proteins in INS-1 cells. In contrast, an increase in ubiquitinated-protein aggregates is observed in cells treated with 3-MA, an autophagy inhibitor; the level of aggregates is higher than that seen in cells grown in high glucose alone.20 Similarly, deletion of Atg7 also leads to accumulation of ubiquitinated-protein aggregates.21 These results demonstrate that autophagy plays a critical role in the degradation of cellular components including ubiquitinated proteins that are associated with diabetes.
 
In our study, we also found that the viability of the control group was reduced, and INS-1 cell apoptosis was increased after treated with 3-MA. This indicates that a minimum basal level of autophagy is required for INS-1 cell survival. This is consistent with a recent study1. Beclin 1 and Atg5 are key mediators of autophagosome formation. Beclin 1 is the mammalian orthologue of the yeast Atg6/Vps30 protein and a regulator of the class III phosphatidylinositol 3-kinase complex involved in autophagosome formation. Fujimoto et al21 reported that reducing beclin 1 expression in Pdx1 KD MIN6 cells (mouse insulinoma cells) by 30% and 50% decreases MIN6 cell death. However, MIN6 cell death increases when beclin 1 expression is reduced by 90%.16
 
Overt type 2 diabetes melitus occurs only when β-cells fail and can no longer compensate for the increased insulin secretion required to maintain normoglycemia. Agents that can prevent deterioration of β-cell function are much needed for the management of type 2 diabetes. In our study, the viability of INS-1 cells was significantly increased and apoptosis decreased compared with the cells treated with high glucose alone by adding liraglutide to high glucose level medium. Liraglutide is a human incretins GLP-1 analogue with high homology to the native hormone. GLP-1 is a key factor in maintaining the normal balance between insulin and glucagon levels. Intestinal absorption of glucose stimulates secretion of these hormones, which act to increase insulin and decrease glucagon secretion. GLP-1 is associated with enhanced satiety, reduced food intake, and weight loss. GLP-1 preserves beta cell morphology and function and reduces cellular apoptosis.7 The antiapoptotic action of GLP-1 is mediated by a cyclic adenosine monophosphate and phosphatidylinositol 3-kinase-dependent signaling pathway, and by an increased expression of the antiapoptotic proteins bcl-2 and bcl-xL.22 In our study, we also found that liraglutide inhibits INS-1 cell apoptosis, accompanied by a significant increase of autophagy. This indicates liraglutide may also prevent high glucose level induced INS-1 cell apoptosis by mediating autophagy. Thus, autophagy may be a new target for the prevention or treatment of diabetes.
 
REFERENCES
 
1.  Masini M, Bugliani M, Lupi R, del Guerra S, Boggi U, Filipponi F, et al. Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 2009; 52: 1083-1086.
2.  Chen ZF, Li YB, Han JY, Wang J, Yin JJ, Li J, et al. The double-edged effect of autophagy in pancreatic beta cells and diabetes. Autophagy 2011; 7: 12-16.
3.  Mizushima N. Autophagy: process and function. Genes Dev 2007; 21: 2861-2873.
4.  Mora R, Régnier-Vigouroux A. Autophagy-driven cell fate decision maker: activated microglia induce specific death of glioma cells by a blockade of basal autophagic flux and secondary apoptosis/necrosis. Autophagy 2009; 5: 419-421.
5.  Kadowaki M, Karim MR, Carpi A, Miotto G. Nutrient control of macroautophagy in mammalian cells. Mol Aspects Med 2006; 27: 426-443.
6.  Kaniuk NA, Kiraly M, Bates H, Vranic M, Volchuk A, Brumell JH. Ubiquitinated-protein aggregates form in pancreatic beta-cells during diabetes-induced oxidative stress and are regulated by autophagy. Diabetes 2007; 56: 930-939.
7.  Marsh BJ, Soden C, Alarcón C, Wicksteed BL, Yaekura K, Costin AJ, et al. Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine cells. Mol Endocrinol 2007; 21: 2255-2269.
8.  Gonzalez CD, Lee MS, Marchetti P, Pietropaolo M, Towns R, Vaccaro MI, et al. The emerging role of autophagy in the pathophysiology of diabetes mellitus. Autophagy 2011; 7: 2-11.
9.  Jung HS, Chung KW, Won Kim J, Kim J, Komatsu M, Tanaka K, et al. Loss of autophagy diminishes pancreatic β cell mass and function with resultant hyperglycemia. Cell Metab 2008; 8: 318-324.
10.  Jing K, Lim K. Why is autophagy important in human diseases? Exp Mol Med 2012; 44: 69-72.
11.  Rossi MC, Nicolucci A. Liraglutide in type 2 diabetes: from pharmacological development to clinical practice. Acta Biomed 2009; 80: 93-101.
12.  Wajcberg E, Amarah A. Liraglutide in the management of type 2 diabetes. Drug Des Devel Ther 2010; 4: 279-290.
13.  Wang Q, Li L, Xu E, Wong V, Rhodes C, Brubaker PL. Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells. Diabetologia 2004; 47: 478-487.
14.  Huang CJ, Lin CY, Haataja L, Gurlo T, Butler AE, Rizza RA, et al. High expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress-mediated β-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes. Diabetes 2007; 56: 2016-2027.
15.  Wang MR, Crager M, Pugazhenthi S. Modulation of apoptosis pathways by oxidative stress and autophagy in β cells. Exp Diabetes Res 2012; 2012: 647914.
16.  Laybutt DR, Preston AM, Akerfeldt MC, Kench JG, Busch AK, Biankin A, et al. Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia 2007; 50: 752-763.
17.  Lenzen S, Drinkgern J, Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic Biol Med 1996; 20: 463-466.
18.  Ding WX, Ni HM, Gao W, Yoshimori T, Stolz DB, Ron D, et al. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am J Pathol 2007; 171: 513-524.
19.  Fujitani Y, Kawamori R, Watada H. The role of autophagy in pancreatic β cell and diabetes. Autophagy 2009; 5: 280-282.
20.  Marchetti P, Masini M. Autophagy and the pancreatic beta-cell in human type 2 diabetes. Autophagy 2009; 5: 1055-1056.
21.  Fujimoto K, Hanson PT, Tran H, Ford EL, Han Z, Johnson JD, et al. Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation. J Biol Chem 2009; 284: 27664-27673.
22.  Farilla L, Bulotta A, Hirshberg B, Calzi SL, Khoury N, Noushmehr H. Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology 2003; 144: 5149-5158.
  1. the grants from the Natural Science Foundation of Heilongjiang Province (No. 200940), the Science Foundation of the Education Department of Heilongjiang Province (No. 11591157), and the Science Foundation of the Health Department of Heilongjiang Province (No. 2012-540).,;