Structural and Functional Characterization of the Fat-Derived Anti-Diabetic Hormone Adiponectin
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. l. The fat-derived hormone, adiponectin, has recently been shown to be a key factor that links obesity with its related metabolic syndromes, such as Type 2 diabetes and atherosclerosis. Functional studies by other groups demonstrated that this novel hormone has direct anti-diabetic, anti-atherogenic and anti-inflammatory functions. This study describes the use of a modern proteomics-based approach to identify and characterize multiple posttranslationally modified isoforms of adiponectin secreted from adipocytes. 2. Two-dimensional gel electrophoresis and NH2-terminal amino acid sequencing revealed that adiponectin secreted from 3T3-L1 adipocytes existed as eight isoforms with different molecular weights and pI values. Carbohydrate detection demonstrated that six of the adiponectin isoforms are glycosylated, whereas bacterially produced adiponectin has no such modifications. 3. The glycosylation sites were mapped to several conserved lysines (residues 68, 71, 80 and 104) located within the collagenous domain of adiponectin, each having a surrounding motif of GXKGE(D). Analysis using mass spectrometry, reversed phase HPLC and amino acid analyzer showed that these four lysines were hydroxylated and subsequently glycosylated possibly by a glucosylgalactosyl group. Mutational studies revealed that replacement of these four lysines by arginines largely abolished glycosylation of adiponectin, suggesting that glycosylation mainly occurs on these four lysine residues. 4. Ex vivo functional studies demonstrated that full-length adiponectin produced by mammalian cells potently enhanced the ability of subphysiological concentrations of insulin to inhibit glucose production in primary rat hepatocytes. This insulinsensitizing activity was dramatically attenuated when the four-glycosylated hydroxylysines were substituted by arginines. Bacterially generated full-length adiponectin that lacks posttranslational modifications has little activity with respect to inhibition of hepatic glucose production. 5. Endogenous circulating adiponectin purified from fetal bovine serum also existed as multiple posttranslationally modified isoforms. Preliminary analysis using Q-STAR® MS/MS analysis confirmed that hydroxylation and glycosylation also occurred on this protein. Studies in C57BL/6J mice demonstrated that endogenous bovine adiponectin with proper posttranslational modifications, is much more potent than bacterially generated adiponectin in decreasing blood glucose levels and in enhancing lipid clearance after a high fat meal. 6. These results collectively suggest that full-length adiponectin with proper posttranslational modification is a functionally active insulin-sensitizer, and that hydroxylation and glycosylation of the four lysine residues within its collagenous domain contribute to this activity.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/2003