title: Lipocalin-2 inhibits autophagy and induces insulin resistance in H9c2 cells.
abstract: Lipocalin-2 (Lcn2; also known as neutrophil gelatinase associated lipocalin, NGAL) levels are increased in obesity and diabetes and associate with insulin resistance. Correlations exist between Lcn2 levels and various forms or stages of heart failure. Insulin resistance and autophagy both play well-established roles in cardiomyopathy. However, little is known about the impact of Lcn2 on insulin signaling in cardiomyocytes. In this study, we treated H9c2 cells with recombinant Lcn2 for 1 h followed by dose- and time-dependent insulin treatment and found that Lcn2 attenuated insulin signaling assessed via phosphorylation of Akt and p70S6K. We used multiple assays to demonstrate that Lcn2 reduced autophagic flux. First, Lcn2 reduced pULK1 S555, increased pULK1 S757 and reduced LC3-II levels determined by Western blotting. We validated the use of DQ-BSA to assess autolysosomal protein degradation and this together with MagicRed cathepsin B assay indicated that Lcn2 reduced lysosomal degradative activity. Furthermore, we generated H9c2 cells stably expressing tandem fluorescent RFP/GFP-LC3 and this approach verified that Lcn2 decreased autophagic flux. We also created an autophagy-deficient H9c2 cell model by overexpressing a dominant-negative Atg5 mutant and found that reduced autophagy levels also induced insulin resistance. Adding rapamycin after Lcn2 could stimulate autophagy and recover insulin sensitivity. In conclusion, our study indicated that acute Lcn2 treatment caused insulin resistance and use of gain and loss of function approaches elucidated a causative link between autophagy inhibition and regulation of insulin sensitivity by Lcn2.
authors.affiliation: Department of Biology, York University, Toronto, ON, M3J 1P3, Canada.
authors.forename: Yee Kwan
authors.initials: YK
authors.lastname: Chan
authors.affiliation: Department of Biology, York University, Toronto, ON, M3J 1P3, Canada.
authors.forename: Hye Kyoung
authors.initials: HK
authors.lastname: Sung
authors.affiliation: Department of Biology, York University, Toronto, ON, M3J 1P3, Canada.
authors.forename: James Won Suk
authors.initials: JW
authors.lastname: Jahng
authors.affiliation: Department of Biology, York University, Toronto, ON, M3J 1P3, Canada.
authors.forename: Grace Ha Eun
authors.initials: GH
authors.lastname: Kim
authors.affiliation: Department of Biology, York University, Toronto, ON, M3J 1P3, Canada.
authors.forename: Meng
authors.initials: M
authors.lastname: Han
authors.affiliation: Department of Biology, York University, Toronto, ON, M3J 1P3, Canada. Electronic address: gsweeney@yorku.ca.
authors.forename: Gary
authors.initials: G
authors.lastname: Sweeney
mesh_terms: D000818:Animals; D000071182:Autophagosomes / Q000378:metabolism / Q000648:ultrastructure; D001343:Autophagy / Q000187:drug effects*; D002401:Cathepsin B / Q000378:metabolism; D002460:Cell Line; D007328:Insulin / Q000378:metabolism; D007333:Insulin Resistance; D000071068:Lipocalin-2 / Q000494:pharmacology*; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000648:ultrastructure; D059748:Proteolysis; D051381:Rats; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D007328:Insulin; D000071068:Lipocalin-2; D012710:Serum Albumin, Bovine; D002401:Cathepsin B
keywords: Adipokine; Autophagy; Cardiomyopathy; Heart failure; Insulin resistance; Lipocalin-2 (Lcn2); Neutrophil gelatinase associated lipocalin (NGAL)