title: Long-term elevation of free fatty acids leads to delayed processing of proinsulin and prohormone convertases 2 and 3 in the pancreatic beta-cell line MIN6.
abstract: To explore the role of chronically elevated free fatty acids (FFAs) in the pathogenesis of the hyperproinsulinemia of type 2 diabetes, we have investigated the effect of FFAs on proinsulin processing and prohormone convertases PC2 and PC1/PC3 in MIN6 cells cultured in Dulbecco's modified Eagle's medium with or without 0.5 mmol/l FFA mixture (palmitic acid:oleic acid = 1:2). After 7 days of culture, the percent of proinsulin in FFA-exposed cells was increased (25.9 +/-0.3% intracellular and 75.4 +/- 1.2% in medium vs. 13.5 +/-0.2 and 56.2 +/- 4.1%, respectively, in control cells). The biosynthesis and secretion of proinsulin and insulin were analyzed by comparing the incorporation of [3H]Leu and [35S]Met. In pulse-chase studies, proinsulin-to-insulin conversion was inhibited, and proinsulin in the medium was increased by 50% after 3 h of chase, while insulin secretion was decreased by 50% after FFA exposure. Levels of cellular PC2 and PC3 analyzed by Western blotting were decreased by 23 and 15%, respectively. However, PC2, PC3, proinsulin, and 7B2 mRNA levels were not altered by FFA exposure. To test for an effect on the biosynthesis of PC2, PC3, proinsulin, and 7B2, a protein required for PC2 activation, MIN6 cells were labeled with [35S]Met for 10-15 min, followed by a prolonged chase. Most proPC2 was converted after 6 h of chase in control cells, but conversion was incomplete even after 6 h of chase in FFA-exposed MIN6 cells. Media from chase incubations showed that FFA-exposed cells secreted more proPC2 than controls. Similar inhibitory effects were noted on the processing of proPC3, proinsulin, and 7B2. In conclusion, prolonged exposure of beta-cells to FFAs may affect the biosynthesis and posttranslational processing of proinsulin, PC2, PC3, and 7B2, and thereby contribute to the hyperproinsulinemia of type 2 diabetes. The mechanism of inhibition of secretory granule processing by FFAs may be through changes in Ca2+ concentration, the pH in the secretory granules, and/or other factors that may influence the activation and function of the convertases.
authors.affiliation: Department of Biochemistry, University of Chicago, Illinois 60637, USA.
authors.forename: H
authors.initials: H
authors.lastname: Furukawa
authors.forename: R J
authors.initials: RJ
authors.lastname: Carroll
authors.forename: H H
authors.initials: HH
authors.lastname: Swift
authors.forename: D F
authors.initials: DF
authors.lastname: Steiner
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D002460:Cell Line; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D011384:Proinsulin / Q000378:metabolism*; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics*; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D005230:Fatty Acids, Nonesterified; D012333:RNA, Messenger; D011384:Proinsulin; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases