Evidence for the link between defective autophagy and inflammation in peripheral blood mononuclear cells of type 2 diabetic patients

  • Samira Alizadeh
  • Hossein Mazloom
  • Asie Sadeghi
  • Solaleh Emamgholipour
  • Abolfazl Golestani
  • Farshid Noorbakhsh
  • Mohsen Khoshniatnikoo
  • Reza Meshkani
Original Article

Abstract

Autophagy was shown to modulate inflammation in immune cells. This study was designed to evaluate the association between autophagy and inflammation in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic (T2D) and non-diabetic (ND) subjects. The autophagy markers were measured by real-time PCR and western blot. The gene expression of pro- and anti-inflammatory cytokines was assessed by real-time PCR. Reduced transcription of BECN1 and LAMP2 and unchanged expression of MAP1LC3B and ATG5 were observed in PBMCs of T2D patients. Decreased LC3B-II and increased p62/SQSTM1 levels were found in PBMCs of diabetic patients. The p-mTOR level was higher in PBMCs of diabetic patients. An increase in both IL-1β and TNF-α gene expression, along with a decrease in the expression of IL-10, was observed in PBMCs of T2D patients. TNF-α mRNA expression was inversely correlated with the mRNA expression of BECN1 and LAMP2. TNF-α and IL-1β expression were negatively correlated with the protein levels of LC3B-II. TNF-α and IL-1β expression had also a positive correlation with protein level of p62. IL-10 mRNA expression was positively correlated with the mRNA expression of BECN1 and LAMP2 and protein levels of LC3B-II and negatively correlated with protein level of p62. In addition, p-mTOR level was positively correlated with IL-1β and TNF-α mRNA expression. The results revealed a reduced autophagy in PBMCs of T2D patients that is liked with an enhanced inflammation. The suppression of autophagy in PBMCs of diabetic patients may be associated with the activation of the mTOR signaling.

Keywords

Autophagy Type 2 diabetes Peripheral blood mononuclear cells Inflammation mTOR Cytokine 

Abbreviations

AMPK

AMP-activated protein kinase

DBP

Diastolic blood pressure

IL-6

Interleukin 6

IL-1β

Interleukin 1β

LC3B

Light chain 3B

LAMP-2

Lysosome-associated membrane protein 2

MAP1LC3B

Microtubule-associated protein 1 light chain 3 beta

mTOR

Mammalian target of rapamycin

PBMC

Peripheral blood mononuclear cell

SBP

Systolic blood pressure

TNF-α

Tumor necrosis factor alpha

T2D

Type 2 diabetes

Notes

Acknowledgements

We greatly appreciate the assistance provided by the staff of the Endocrinology and Metabolism Research Institute of Tehran University of Medical Sciences. We also thank all volunteers for their participation in the study. This work was financially supported by grants from the Deputy of Research, Tehran University of Medical Sciences (grant 91-03-30-19266).

Authors’ contributions

SA: Conducted research, analyzed data or performed statistical analysis, wrote paper. HM: Conducted research. AS: Conducted research. SE: Wrote paper. AG: Edited paper. FN: Analyzed data or performed statistical analysis. MKN: Helped in patients’ recruitment. RM: Designed research, analyzed data, or performed statistical analysis, had primary responsibility for final content.

Compliance with ethical standards

Conflict of interest

Nothing to declare

References

  1. 1.
    Alberti KG, Zimmet PZ (1998) Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabetic medicine: a journal of the British Diabetic Association 15:539–553.  https://doi.org/10.1002/(sici)1096-9136(199807)15:7<539::aid-dia668>3.0.co;2-s CrossRefGoogle Scholar
  2. 2.
    Alexandraki KI, Piperi C, Ziakas PD, Apostolopoulos NV, Makrilakis K, Syriou V, Diamanti-Kandarakis E, Kaltsas G, Kalofoutis A (2008) Cytokine secretion in long-standing diabetes mellitus type 1 and 2: associations with low-grade systemic inflammation. J Clin Immunol 28:314–321.  https://doi.org/10.1007/s10875-007-9164-1 CrossRefPubMedGoogle Scholar
  3. 3.
    Barlow AD, Thomas DC (2015) Autophagy in diabetes: beta-cell dysfunction, insulin resistance, and complications. DNA Cell Biol 34:252–260.  https://doi.org/10.1089/dna.2014.2755 CrossRefPubMedGoogle Scholar
  4. 4.
    Bohensky J, Leshinsky S, Srinivas V, Shapiro IM (2010) Chondrocyte autophagy is stimulated by HIF-1 dependent AMPK activation and mTOR suppression. Pediatr Nephrol 25:633–642.  https://doi.org/10.1007/s00467-009-1310-y CrossRefPubMedGoogle Scholar
  5. 5.
    Boland B, Kumar A, Lee S, Platt FM, Wegiel J, Yu WH, Nixon RA (2008) Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease. The Journal of neuroscience: the official journal of the Society for Neuroscience 28:6926–6937.  https://doi.org/10.1523/jneurosci.0800-08.2008 CrossRefGoogle Scholar
  6. 6.
    Cai D, Liu T (2012) Inflammatory cause of metabolic syndrome via brain stress and NF-kappaB. Aging 4:98–115.  https://doi.org/10.18632/aging.100431 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Crisan TO, Plantinga TS, van de Veerdonk FL, Farcas MF, Stoffels M, Kullberg BJ, van der Meer JW, Joosten LA, Netea MG (2011) Inflammasome-independent modulation of cytokine response by autophagy in human cells. PLoS One 6:e18666.  https://doi.org/10.1371/journal.pone.0018666 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Deng H-P, Chai J-K, Shen C-A, Zhang X-B, Ma L, Sun T-J, Hu Q-G, Chi Y-F, Dong N (2015) Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes. Biosci Rep 35:e00242.  https://doi.org/10.1042/BSR20150017 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dunlop EA, Tee AR (2014) mTOR and autophagy: a dynamic relationship governed by nutrients and energy. Semin Cell Dev Biol 36:121–129.  https://doi.org/10.1016/j.semcdb.2014.08.006 CrossRefPubMedGoogle Scholar
  10. 10.
    Eskelinen EL, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 1793:664–673.  https://doi.org/10.1016/j.bbamcr.2008.07.014 CrossRefPubMedGoogle Scholar
  11. 11.
    Gacka M, Dobosz T, Szymaniec S, Bednarska-Chabowska D, Adamiec R, Sadakierska-Chudy A (2010) Proinflammatory and atherogenic activity of monocytes in type 2 diabetes. J Diabetes Complicat 24:1–8.  https://doi.org/10.1016/j.jdiacomp.2008.07.001 CrossRefPubMedGoogle Scholar
  12. 12.
    Harris J (2011) Autophagy and cytokines. Cytokine 56:140–144.  https://doi.org/10.1016/j.cyto.2011.08.022 CrossRefPubMedGoogle Scholar
  13. 13.
    Harris J, Hartman M, Roche C, Zeng SG, O'Shea A, Sharp FA, Lambe EM, Creagh EM, Golenbock DT, Tschopp J, Kornfeld H, Fitzgerald KA, Lavelle EC (2011) Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation. J Biol Chem 286:9587–9597.  https://doi.org/10.1074/jbc.M110.202911 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kobayashi S, Xu X, Chen K, Liang Q (2012) Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury. Autophagy 8:577–592.  https://doi.org/10.4161/auto.18980 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kruse R, Vind BF, Petersson SJ, Kristensen JM, Hojlund K (2015) Markers of autophagy are adapted to hyperglycaemia in skeletal muscle in type 2 diabetes. Diabetologia 58:2087–2095.  https://doi.org/10.1007/s00125-015-3654-0 CrossRefPubMedGoogle Scholar
  16. 16.
    Lee DF, Kuo HP, Chen CT, Hsu JM, Chou CK, Wei Y, Sun HL, Li LY, Ping B, Huang WC, He X, Hung JY, Lai CC, Ding Q, Su JL, Yang JY, Sahin AA, Hortobagyi GN, Tsai FJ, Tsai CH, Hung MC (2007) IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell 130:440–455.  https://doi.org/10.1016/j.cell.2007.05.058 CrossRefPubMedGoogle Scholar
  17. 17.
    Lee HM, Shin DM, Yuk JM, Shi G, Choi DK, Lee SH, Huang SM, Kim JM, Kim CD, Lee JH, Jo EK (2011) Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1. J Immunol (Baltimore, Md: 1950) 186:1248–1258.  https://doi.org/10.4049/jimmunol.1001954 CrossRefGoogle Scholar
  18. 18.
    Liu HY, Han J, Cao SY, Hong T, Zhuo D, Shi J, Liu Z, Cao W (2009) Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia: inhibition of FoxO1-dependent expression of key autophagy genes by insulin. J Biol Chem 284:31484–31492.  https://doi.org/10.1074/jbc.M109.033936 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods (San Diego, Calif) 25:402–408.  https://doi.org/10.1006/meth.2001.1262 CrossRefGoogle Scholar
  20. 20.
    Masini M, Bugliani M, Lupi R, del Guerra S, Boggi U, Filipponi F, Marselli L, Masiello P, Marchetti P (2009) Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 52:1083–1086.  https://doi.org/10.1007/s00125-009-1347-2 CrossRefPubMedGoogle Scholar
  21. 21.
    McKnight NC, Zhenyu Y (2013) Beclin 1, an essential component and master regulator of PI3K-III in health and disease. Current pathobiology reports 1:231–238.  https://doi.org/10.1007/s40139-013-0028-5 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Meijer AJ, Codogno P (2006) Signalling and autophagy regulation in health, aging and disease. Mol Asp Med 27:411–425.  https://doi.org/10.1016/j.mam.2006.08.002 CrossRefGoogle Scholar
  23. 23.
    Meng Q, Cai D (2011) Defective hypothalamic autophagy directs the central pathogenesis of obesity via the IkappaB kinase beta (IKKbeta)/NF-kappaB pathway. J Biol Chem 286:32324–32332.  https://doi.org/10.1074/jbc.M111.254417 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Meshkani R, Adeli K (2009) Hepatic insulin resistance, metabolic syndrome and cardiovascular disease. Clin Biochem 42:1331–1346.  https://doi.org/10.1016/j.clinbiochem.2009.05.018 CrossRefPubMedGoogle Scholar
  25. 25.
    Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3:542–545CrossRefPubMedGoogle Scholar
  26. 26.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326.  https://doi.org/10.1016/j.cell.2010.01.028 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Moruno F, Perez-Jimenez E, Knecht E (2012) Regulation of autophagy by glucose in mammalian cells. Cell 1:372–395.  https://doi.org/10.3390/cells1030372 CrossRefGoogle Scholar
  28. 28.
    Navarro JF, Mora C (2006) Diabetes, inflammation, proinflammatory cytokines, and diabetic nephropathy. TheScientificWorldJOURNAL 6:908–917.  https://doi.org/10.1100/tsw.2006.179 CrossRefPubMedGoogle Scholar
  29. 29.
    Navarro JF, Mora C, Gomez M, Muros M, Lopez-Aguilar C, Garcia J (2008) Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor-alpha and interleukin-6 in type 2 diabetic patients. Nephrol Dial Transplant 23:919–926.  https://doi.org/10.1093/ndt/gfm674 CrossRefPubMedGoogle Scholar
  30. 30.
    Ost A, Svensson K, Ruishalme I, Brannmark C, Franck N, Krook H, Sandstrom P, Kjolhede P, Stralfors P (2010) Attenuated mTOR signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes. Mol Med (Cambridge, Mass) 16:235–246.  https://doi.org/10.2119/molmed.2010.00023 Google Scholar
  31. 31.
    Plantinga TS, Joosten LA, van der Meer JW, Netea MG (2012) Modulation of inflammation by autophagy: consequences for Crohn's disease. Curr Opin Pharmacol 12:497–502.  https://doi.org/10.1016/j.coph.2012.01.017 CrossRefPubMedGoogle Scholar
  32. 32.
    Qian M, Fang X, Wang X (2017) Autophagy and inflammation. Clin Transl Med 6:24.  https://doi.org/10.1186/s40169-017-0154-5 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Saftig P, Beertsen W, Eskelinen EL (2008) LAMP-2: a control step for phagosome and autophagosome maturation. Autophagy 4:510–512CrossRefPubMedGoogle Scholar
  34. 34.
    Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, Omori H, Noda T, Yamamoto N, Komatsu M, Tanaka K, Kawai T, Tsujimura T, Takeuchi O, Yoshimori T, Akira S (2008) Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 456:264–268.  https://doi.org/10.1038/nature07383 CrossRefPubMedGoogle Scholar
  35. 35.
    Strisciuglio C, Duijvestein M, Verhaar AP, Vos AC, van den Brink GR, Hommes DW, Wildenberg ME (2013) Impaired autophagy leads to abnormal dendritic cell-epithelial cell interactions. Journal of Crohn's & colitis 7:534–541.  https://doi.org/10.1016/j.crohns.2012.08.009 CrossRefGoogle Scholar
  36. 36.
    Todde V, Veenhuis M, van der Klei IJ (2009) Autophagy: principles and significance in health and disease. Biochim Biophys Acta 1792:3–13.  https://doi.org/10.1016/j.bbadis.2008.10.016 CrossRefPubMedGoogle Scholar
  37. 37.
    Tsiotra PC, Tsigos C, Yfanti E, Anastasiou E, Vikentiou M, Psarra K, Papasteriades C, Raptis SA (2007) Visfatin, TNF-alpha and IL-6 mRNA expression is increased in mononuclear cells from type 2 diabetic women. Horm Metab Res 39:758–763.  https://doi.org/10.1055/s-2007-990288 CrossRefPubMedGoogle Scholar
  38. 38.
    Xie S, Chen M, Yan B, He X, Chen X, Li D (2014) Identification of a role for the PI3K/AKT/mTOR signaling pathway in innate immune cells. PLoS One 9:e94496CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Yang Z, Klionsky DJ (2010) Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol 22:124–131.  https://doi.org/10.1016/j.ceb.2009.11.014 CrossRefPubMedGoogle Scholar
  40. 40.
    Yang H, Wang X, Zhang Y, Liu H, Liao J, Shao K, Chu Y, Liu G (2014) Modulation of TSC-mTOR signaling on immune cells in immunity and autoimmunity. J Cell Physiol 229:17–26.  https://doi.org/10.1002/jcp.24426 CrossRefPubMedGoogle Scholar
  41. 41.
    Yoshizaki T, Kusunoki C, Kondo M, Yasuda M, Kume S, Morino K, Sekine O, Ugi S, Uzu T, Nishio Y, Kashiwagi A, Maegawa H (2012) Autophagy regulates inflammation in adipocytes. Biochem Biophys Res Commun 417:352–357.  https://doi.org/10.1016/j.bbrc.2011.11.114 CrossRefPubMedGoogle Scholar
  42. 42.
    Zimmet PZ (1999) Diabetes epidemiology as a tool to trigger diabetes research and care. Diabetologia 42:499–518.  https://doi.org/10.1007/s001250051188 CrossRefPubMedGoogle Scholar

Copyright information

© University of Navarra 2018

Authors and Affiliations

  1. 1.Department of Clinical Biochemistry, Faculty of MedicineTehran University of Medical SciencesTehranIslamic Republic of Iran
  2. 2.Department of Biochemistry, Afzalipour School of MedicineKerman University of Medical SciencesKermanIslamic Republic of Iran
  3. 3.Department of Immunology, Faculty of MedicineTehran University of Medical SciencesTehranIslamic Republic of Iran
  4. 4.Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences InstituteTehran University of Medical SciencesTehranIslamic Republic of Iran

Personalised recommendations