Nutr Res Pract.  2014 Oct;8(5):494-500. 10.4162/nrp.2014.8.5.494.

Padina arborescens extract protects high glucose-induced apoptosis in pancreatic beta cells by reducing oxidative stress

Affiliations
  • 1Department of Food and Nutrition, College of Medical and Life Science, Silla University, Busan 617-736, Korea.
  • 2Department of Food Science and Nutrition, Pusan National University, 2, Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 609-735, Korea. hanjs@pusan.ac.kr

Abstract

BACKGROUND/OBJECTIVES
This study investigated whether Padina arborescens extract (PAE) protects INS-1 pancreatic beta cells against glucotoxicity-induced apoptosis.
MATERIALS/METHODS
Assays, including cell viability, lipid peroxidation, generation of intracellular ROS, NO production, antioxidant enzyme activity and insulin secretion, were conducted. The expressions of Bax, Bcl-2, and caspase-3 proteins in INS-1 cells were evaluated by western blot analysis, and apoptosis/necrosis induced by high glucose was determined by analysis of FITC-Annexin V/PI staining.
RESULTS
Treatment with high concentrations of glucose induced INS-1 cell death, but PAE at concentrations of 25, 50 or 100 microg/ml significantly increased cell viability. The treatment with PAE dose dependently reduced the lipid peroxidation and increased the activities of antioxidant enzymes reduced by 30 mM glucose, while intracellular ROS levels increased under conditions of 30 mM glucose. PAE treatment improved the secretory responsiveness following stimulation with glucose. The results also demonstrated that glucotoxicity-induced apoptosis is associated with modulation of the Bax/Bcl-2 ratio. When INS-1 cells were stained with Annexin V/PI, we found that PAE reduced apoptosis by glucotoxicity.
CONCLUSIONS
In conclusion, the present study indicates that PAE protects against high glucose-induced apoptosis in pancreatic beta cells by reducing oxidative stress.

Keyword

Padina arborescens extract; high glucose; apoptosis; oxidative stress; INS-1 pancreatic beta cell

MeSH Terms

Apoptosis*
Blotting, Western
Caspase 3
Cell Death
Cell Survival
Glucose
Insulin
Insulin-Secreting Cells*
Lipid Peroxidation
Oxidative Stress*
Caspase 3
Glucose
Insulin

Figure

  • Fig. 1 Effect of PAE on the viability of high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with 5.5 mM or 30 mM glucose for 48 h, and subsequently incubated for 48 h in the presence or absence of 25, 50, or 100 µg/mL PAE. The use of 5.5 mM glucose was representative of normal glucose conditions and the 30 mM glucose treatments represent high glucose conditions. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

  • Fig. 2 Effect of PAE on intracellular ROS generation in the high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

  • Fig. 3 Effect of PAE on TBRAS generation in the high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

  • Fig. 4 Effect of PAE on NO level in high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-cValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

  • Fig. 5 Effects of PAE on insulin secretion in high glucose-treated INS-1 pancreatic β cells. Cells in wells of 6-well plates (2 × 105 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

  • Fig. 6 Effects of PAE on the expression of Caspase-3, Bax and Bcl-2 (A) and Caspase-3, Bax and Bcl-2 ratio (B) in INS-1 pancreatic β cells. Relative expression was quantified by densitometry using the Multi Gauge V3.1 and calculated according to the reference bands of β-actin (mean, n = 3). a-cValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

  • Fig. 7 Identification of the type of cell death by Annexin V-FITC/PI staining. The status of cell death was determined by counting the cells stained with Annexin V-FITC/PI using glow cytometer. Cells were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. A: 5.5 mM glucose; B: 30 mM glucose; C: 30 mM glucose + PAE 50 µg/mL; D: 30 mM glucose + PAE 100 µg/mL.


Reference

1. Robertson RP. Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem. 2004; 279:42351–42354.
Article
2. Zúrová-Nedelcevová J, Navarová J, Drábiková K, Jancinová V, Petríková M, Bernátová I, Kristová V, Snirc V, Nosál'ová V, Sotníková R. Participation of reactive oxygen species in diabetes-induced endothelial dysfunction. Neuro Endocrinol Lett. 2006; 27:Suppl 2. 168–171.
3. Mandrup-Poulsen T, Helqvist S, Wogensen LD, Mølvig J, Pociot F, Johannesen J, Nerup J. Cytokine and free radicals as effector molecules in the destruction of pancreatic beta cells. Curr Top Microbiol Immunol. 1990; 164:169–193.
4. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. β-cell deficit and increased β-cell apoptosis in humans with type 2 diabetes. Diabetes. 2003; 52:102–110.
Article
5. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes. 2005; 54:1615–1625.
6. Heo SJ, Park EJ, Lee KW, Jeon YJ. Antioxidant activities of enzymatic extracts from brown seaweeds. Bioresour Technol. 2005; 96:1613–1623.
Article
7. Pal Singh I, Bharate SB. Phloroglucinol compounds of natural origin. Nat Prod Rep. 2006; 23:558–591.
Article
8. Park MH, Han JS. Protective effect of Padina arborescens extract against high glucose-induced oxidative damage in human umbilical vein endothelial cells. Prev Nutr Food Sci. 2013; 18:11–17.
Article
9. Park MH, Han JS. Hypoglycemic effect of Padina arborescens extract in streptozotocin-induced diabetic mice. Prev Nutr Food Sci. 2012; 17:239–244.
Article
10. Fautz R, Husein B, Hechenberger C. Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS). Mutat Res. 1991; 253:173–179.
Article
11. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999; 27:612–616.
Article
12. Fraga CG, Leibovitz BE, Tappel AL. Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. Free Radic Biol Med. 1988; 4:155–161.
Article
13. Nath J, Powledge A. Modulation of human neutrophil inflammatory responses by nitric oxide: studies in unprimed and LPS-primed cells. J Leukoc Biol. 1997; 62:805–816.
Article
14. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974; 47:469–474.
Article
15. Aebi H. Catalase in vitro. Methods Enzymol. 1984; 105:121–126.
16. Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun. 1976; 71:952–958.
Article
17. Green CD, Jump DB, Olson LK. Elevated insulin secretion from liver X receptor-activated pancreatic β-cells involves increased de novo lipid synthesis and triacylglyceride turnover. Endocrinology. 2009; 150:2637–2645.
Article
18. Yamabe N, Kang KS, Goto E, Tanaka T, Yokozawa T. Beneficial effect of Corni Fructus, a constituent of Hachimi-jio-gan, on advanced glycation end-product-mediated renal injury in streptozotocin-treated diabetic rats. Biol Pharm Bull. 2007; 30:520–526.
Article
19. Lenzen S. Oxidative stress: the vulnerable beta-cell. Biochem Soc Trans. 2008; 36:343–347.
20. Robertson RP, Harmon J, Tran PO, Poitout V. Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes. Diabetes. 2004; 53:Suppl 1. S119–S124.
21. Tiedge M, Lortz S, Munday R, Lenzen S. Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species. Diabetes. 1998; 47:1578–1585.
Article
22. Evans JL, Goldfine ID, Maddux BA, Grodsky GM. Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev. 2002; 23:599–622.
Article
23. Del Guerra S, Grupillo M, Masini M, Lupi R, Bugliani M, Torri S, Boggi U, Del Chiaro M, Vistoli F, Mosca F, Del Prato S, Marchetti P. Gliclazide protects human islet beta-cells from apoptosis induced by intermittent high glucose. Diabetes Metab Res Rev. 2007; 23:234–238.
Article
24. Robertson R, Zhou H, Zhang T, Harmon JS. Chronic oxidative stress as a mechanism for glucose toxicity of the beta cell in type 2 diabetes. Cell Biochem Biophys. 2007; 48:139–146.
Article
25. Kong Q, Lin CL. Oxidative damage to RNA: mechanisms, consequences, and diseases. Cell Mol Life Sci. 2010; 67:1817–1829.
Article
26. Kappus H. Oxidative stress in chemical toxicity. Arch Toxicol. 1987; 60:144–149.
Article
27. Du X, Stocklauser-Färber K, Rösen P. Generation of reactive oxygen intermediates, activation of NF-kappaB, and induction of apoptosis in human endothelial cells by glucose: role of nitric oxide synthase? Free Radic Biol Med. 1999; 27:752–763.
Article
28. Messmer UK, Reed UK, Brüne B. Bcl-2 protects macrophages from nitric oxide-induced apoptosis. J Biol Chem. 1996; 271:20192–20197.
Article
29. McDaniel ML, Kwon G, Hill JR, Marshall CA, Corbett JA. Cytokines and nitric oxide in islet inflammation and diabetes. Proc Soc Exp Biol Med. 1996; 211:24–32.
Article
30. Kaneto H, Fujii J, Myint T, Miyazawa N, Islam KN, Kawasaki Y, Suzuki K, Nakamura M, Tatsumi H, Yamasaki Y, Taniguchi N. Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction. Biochem J. 1996; 320:855–863.
Article
31. Tajiri Y, Grill V. Aminoguanidine exerts a beta-cell function-preserving effect in high glucose-cultured beta-cells (INS-1). Int J Exp Diabetes Res. 2000; 1:111–119.
Article
32. Grankvist K, Marklund S, Täljedal IB. Superoxide dismutase is a prophylactic against alloxan diabetes. Nature. 1981; 294:158–160.
Article
33. Benhamou PY, Moriscot C, Richard MJ, Beatrix O, Badet L, Pattou F, Kerr-Conte J, Chroboczek J, Lemarchand P, Halimi S. Adenovirus-mediated catalase gene transfer reduces oxidant stress in human, porcine and rat pancreatic islets. Diabetologia. 1998; 41:1093–1100.
Article
34. Krause MS, McClenaghan NH, Flatt PR, de Bittencourt PI, Murphy C, Newsholme P. L-arginine is essential for pancreatic beta-cell functional integrity, metabolism and defense from inflammatory challenge. J Endocrinol. 2011; 211:87–97.
Article
35. Moriscot C, Pattou F, Kerr-Conte J, Richard MJ, Lemarchand P, Benhamou PY. Contribution of adenoviral-mediated superoxide dismutase gene transfer to the reduction in nitric oxide-induced cytotoxicity on human islets and INS-1 insulin-secreting cells. Diabetologia. 2000; 43:625–631.
Article
36. Fariss MW, Chan CB, Patel M, Van Houten B, Orrenius S. Role of mitochondria in toxic oxidative stress. Mol Interv. 2005; 5:94–111.
Article
37. Antonsson B, Martinou JC. The Bcl-2 protein family. Exp Cell Res. 2000; 256:50–57.
Article
38. Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999; 6:99–104.
Article
Full Text Links
  • NRP
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr