We have investigated the in vitro effects of increased levels of glucose and free fatty acids on autophagy activation in pancreatic beta cells. INS-1E cells and isolated rat and human pancreatic islets were incubated for various times (from 2 to 24 h) at different concentrations of glucose and/or palmitic acid. Then, cell survival was evaluated and autophagy activation was explored by using various biochemical and morphological techniques. In INS-1E cells as well as in rat and human islets, 0.5 and 1.0 mM palmitate markedly increased autophagic vacuole formation, whereas high glucose was ineffective alone and caused little additional change when combined with palmitate. Furthermore, LC3-II immunofluorescence co-localized with that of cathepsin D, a lysosomal marker, showing that the autophagic flux was not hampered in PA-treated cells. These effects were maintained up to 18–24 h incubation and were associated with a significant decline of cell survival correlated with both palmitate concentration and incubation time. Ultrastructural analysis showed that autophagy activation, as evidenced by the occurrence of many autophagic vacuoles in the cytoplasm of beta cells, was associated with a diffuse and remarkable swelling of the endoplasmic reticulum. Our results indicate that among the metabolic alterations typically associated with type 2 diabetes, high free fatty acids levels could play a role in the activation of autophagy in beta cells, through a mechanism that might involve the induction of endoplasmic reticulum stress.
References
[1]
Todde V, Veenhuis M, van der Klei IJ (2009) Autophagy: principles and significance in health and disease. Biochim Biophys Acta 1792: 3–13.
Tsukada M, Ohsumi Y (1993) Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett 333: 169–174.
[4]
Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, et al. (2004) The role of autophagy during the early neonatal starvation period. Nature 432: 1032–1036.
[5]
Kadowaki M, Karim MR, Carpi A, Miotto G (2006) Nutrient control of macroautophagy in mammalian cells. Mol Aspects Med 27: 426–443.
[6]
Mizushima N, Hara T (2006) Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration? Autophagy 2: 302–304.
[7]
Bergamini E (2006) Autophagy: a cell repair mechanism that retards ageing and age-associated diseases and can be intensified pharmacologically. Mol Aspects Med 27: 403–410.
[8]
Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6: 463–477.
[9]
Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451: 1069–1075.
[10]
Tsujimoto Y, Shimizu S (2005) Another way to die: autophagic programmed cell death. Cell Death Differ 12: Suppl 21528–1534.
[11]
Kroemer G, Levine B (2008) Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 9: 1004–1010.
[12]
Levine B, Yuan J (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115: 2679–2688.
[13]
Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132: 27–42.
[14]
Rhodes CJ (2005) Type 2 diabetes-a matter of beta-cell life and death? Science 307: 380–384.
[15]
Prentki M, Nolan CJ (2006) Islet beta cell failure in type 2 diabetes. J Clin Invest 116: 1802–1812.
[16]
Kaniuk NA, Kiraly M, Bates H, Vranic M, Volchuk A, et al. (2007) Ubiquitinated-protein aggregates form in pancreatic beta-cells during diabetes-induced oxidative stress and are regulated by autophagy. Diabetes 56: 930–939.
[17]
Li N, Frigerio F, Maechler P (2008) The sensitivity of pancreatic beta-cells to mitochondrial injuries triggered by lipotoxicity and oxidative stress. Biochem Soc Trans 36: 930–934.
[18]
Hartley T, Brumell J, Volchuk A (2009) Emerging roles for the ubiquitin-proteasome system and autophagy in pancreatic beta-cells. Am J Physiol Endocrinol Metab 296: E1–10.
[19]
H？yer-Hansen M, J？？ttel？ M (2007) Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell Death Differ 14: 1576–1582.
[20]
Chen H, Chan DC (2009) Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases. Hum Mol Genet 18: R169–176.
[21]
Ebato C, Uchida T, Arakawa M, Komatsu M, Ueno T, et al. (2008) Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metab 8: 325–332.
[22]
Jung HS, Chung KW, Won Kim J, Kim J, Komatsu M, et al. (2008) Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia. Cell Metab 8: 318–324.
[23]
Choi SE, Lee SM, Lee YJ, Li LJ, Lee SJ, et al. (2008) Protective role of autophagy in palmitate-induced INS-1 beta-cell death. Endocrinology 150: 126–134.
[24]
Masini M, Bugliani M, Lupi R, del Guerra S, Boggi U, et al. (2009) Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 52: 1083–1086.
[25]
Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, et al. (2004) Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 145: 667–678.
[26]
Novelli M, Fabregat ME, Fernandez-Alvarez J, Gomis R, Masiello P (2001) Metabolic and functional studies on isolated islets in a new rat model of type 2 diabetes. Mol Cell Endocrinol 175: 57–66.
[27]
Marchetti P, Bugliani M, Lupi R, Marselli L, Masini M, et al. (2007) The endoplasmic reticulum in pancreatic beta cells of type 2 diabetes patients. Diabetologia 50: 2486–2494.
[28]
Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, et al. (2002) Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 51: 1437–1442.
[29]
Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M, et al. (2006) Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis. Endocrinology 147: 3398–3407.
[30]
Galluzzi L, Maiuri MC, Vitale I, Zischka H, Castedo M, et al. (2007) Cell death modalities: classification and pathophysiological implications. Cell Death Differ 14: 1237–1243.
[31]
Eskelinen EL, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 1793: 664–673.
[32]
Weibel ER (1979) Stereological Methods vol. 1 : Pratical methods for Biological Morphometry. Acad Press, New York.
[33]
Poitout V, Olson LK, Robertson RP (1996) Insulin-secreting cell lines: classification, characteristics and potential applications. Diabetes Metab 22: 7–14.
[34]
Marchetti P, Dotta F, Lauro D, Purrello F (2008) An overview of pancreatic beta-cell defects in human type 2 diabetes: implications for treatment. Regul Pept 146: 4–11.
[35]
El-Assaad W, Buteau J, Peyot ML, Nolan C, Roduit R, et al. (2003) Saturated fatty acids synergize with elevated glucose to cause pancreatic beta-cell death. Endocrinology 144: 4154–4163.
[36]
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, et al. (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19: 5720–5728.
[37]
Las G, Serada SB, Wikstrom JD, Twig G, Shirihai OS (2011) Fatty Acids Suppress Autophagic Turnover in β-Cells. J Biol Chem 286: 42534–42544.
[38]
Shvets E, Fass E, Elazar Z (2008) Utilizing flow cytometry to monitor autophagy in living mammalian cells. Autophagy 4: 621–628.
[39]
Misler S (2010) The isolated pancreatic islet as a micro-organ and its transplantation to cure diabetes: celebrating the legacy of Paul Lacy. Islets 2: 210–224.
[40]
Weir GC, Marselli L, Marchetti P, Katsuta H, Jung MH, et al. (2009) Towards better understanding of the contributions of overwork and glucotoxicity to the beta-cell inadequacy of type 2 diabetes. Diabetes Obes Metab 11: Suppl 482–90.
[41]
Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M, et al. (2006) Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis. Endocrinology 147: 3398–3407.
[42]
Kaushik S, Singh R, Cuervo AM (2010) Autophagic pathways and metabolic stress. Diabetes Obes Metab 12: Suppl 24–14.
[43]
Kovsan J, Bashan N, Greenberg AS, Rudich A (2010) Potential role of autophagy in modulation of lipid metabolism. Am J Physiol Endocrinol Metab 298: E1–E7.
[44]
Rabinowitz JD, White E (2010) Autophagy and metabolism. Science 330: 1344–1348.
[45]
Singh R, Cuervo AM (2011) Autophagy in the cellular energetic balance. Cell Metab 13: 495–504.
[46]
Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, et al. (2009) Autophagy regulates lipid metabolism. Nature 458: 1131–1135.
[47]
Czaja MJ, Cuervo AM (2009) Lipases in lysosomes, what for? Autophagy 5: 866–867.
[48]
Koga H, Kaushik S, Cuervo AM (2010) Altered lipid content inhibits autophagic vesicular fusion. FASEB J 24: 3052–3065.
[49]
Kharroubi I, Ladrière L, Cardozo AK, Dogusan Z, Cnop M, et al. (2004) Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress. Endocrinology 145: 5087–5096.
[50]
Cunha DA, Hekerman P, Ladrière L, Bazarra-Castro A, Ortis F, et al. (2008) Initiation and execution of lipotoxic ER stress in pancreatic beta-cells. J Cell Sci 121: 2308–2318.
[51]
H？yer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, et al. (2007) Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell 25: 193–205.
[52]
Ding WX, Ni HM, Gao W, Hou YF, Melan MA, et al. (2007) Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol Chem 282: 4702–4710.
[53]
Ladrière L, Igoillo-Esteve M, Cunha DA, Brion JP, Bugliani M, et al. (2010) Enhanced signaling downstream of ribonucleic Acid-activated protein kinase-like endoplasmic reticulum kinase potentiates lipotoxic endoplasmic reticulum stress in human islets. J Clin Endocrinol Metab 95: 1442–1449.
[54]
Meidute Abaraviciene S, Lundquist I, Galvanovskis J, Flodgren E, Olde B, et al. (2008) Palmitate-induced beta-cell dysfunction is associated with excessive NO production and is reversed by thiazolidinedione-mediated inhibition of GPR40 transduction mechanisms. PLoS One 3: e2182.
[55]
Las G, Serada SB, Wikstrom JD, Twig G, Shirihai OS (2011) Fatty acids suppress autophagic turnover in β-cells. J Biol Chem 286: 42534–42544.
[56]
Zhou YP, Grill VE (1994) Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest 93: 870–876.
[57]
Elks ML (1993) Chronic perifusion of rat islets with palmitate suppresses glucose-stimulated insulin release. Endocrinology 133: 208–214.
[58]
Shimabukuro M, Zhou YT, Levi M, Unger RH (1998) Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci U S A 95: 2498–2502.
[59]
Wu JJ, Quijano C, Chen E, Liu H, Cao L, et al. (2009) Mitochondrial dysfunction and oxidative stress mediate the physiological impairment induced by the disruption of autophagy. Aging (Albany NY) 1: 425–437.
Bernales S, Schuck S, Walter P (2007) ER-phagy: selective autophagy of the endoplasmic reticulum. Autophagy 3: 285–287.
[62]
Ogata M, Hino S, Saito A, Morikawa K, Kondo S, et al. (2006) Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 26: 9220–9231.
[63]
Eizirik DL, Cardozo AK, Cnop M (2008) The role for endoplasmic reticulum stress in diabetes mellitus. Endocr Rev 29: 42–61.
[64]
Cnop M, Ladrière L, Igoillo-Esteve M, Moura RF, Cunha DA (2010) Causes and cures for endoplasmic reticulum stress in lipotoxic β-cell dysfunction. Diabetes Obes Metab 12: Suppl 276–82.
[65]
Cunha DA, Hekerman P, Ladrière L, Bazarra-Castro A, Ortis F, et al. (2008) Initiation and execution of lipotoxic ER stress in pancreatic beta-cells. J Cell Sci 121: 2308–2318.
[66]
Gwiazda KS, Yang TL, Lin Y, Johnson JD (2009) Effects of palmitate on ER and cytosolic Ca2+ homeostasis in beta-cells. Am J Physiol Endocrinol Metab 296: E690–701.
[67]
Komiya K, Uchida T, Ueno T, Koike M, Abe H, et al. (2010) Free fatty acids stimulate autophagy in pancreatic β-cells via JNK pathway. Biochem Biophys Res Commun 401: 561–567.
[68]
Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A (2009) Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 16: 966–975.
[69]
Gump JM, Thorburn A (2011) Autophagy and apoptosis: what is the connection? Trends Cell Biol 21: 387–392.
[70]
Meijer AJ, Codogno P (2008) Autophagy: a sweet process in diabetes. Cell Metab 8: 275–276.
[71]
Czaja MJ (2011) Functions of autophagy in hepatic and pancreatic physiology and disease. Gastroenterology 140: 1895–1908.
[72]
Ito S, Koshikawa N, Mochizuki S, Takenaga K (2007) 3-Methyladenine suppresses cell migration and invasion of HT1080 fibrosarcoma cells through inhibiting phosphoinositide 3-kinases independently of autophagy inhibition. Int J Oncol 31: 261–268.
[73]
Hwang SL, Li X, Lee JY, Chang HW (2012) Improved insulin sensitivity by rapamycin is associated with reduction of mTOR and S6K1 activities in L6 myotubes. Biochem Biophys Res Commun 418: 402–407.
[74]
Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, et al. (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4: 151–175.
