The Activities of Lysyl Hydroxylase 3 (LH3) Regulate the Amount and Oligomerization Status of Adiponectin

<div><p>Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDL-cholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.</p> </div

The distribution of adiponectin oligomers is altered in the serum of LH mutant mice.

<p>(A) The ratio of HMW/total adiponectin was significantly increased and MMW/total decreased in serum of 2 months old male mice, even though the total amount of adiponectin was lowered as seen in the elution profile of adiponectin (B). The oligomeric forms were separated with gel filtration chromatography and quantified either from adiponectin immunoblots (A) or by ELISA (B). The level of oligomeric forms (A) was calculated as a proportion of total adiponectin. The values represent the average ± SD of the experiments. P values were calculated using unpaired homoscedastic student t-tests with two-tailed distribution.* p<0.05, ** p<0.01, *** p<0.001.</p

The expression of genes involved in β-oxidation, fatty acid synthesis and gluconeogenesis in LH mutant mice.

<p>Quantitative RT-PCR was used to determine changes in the gene expressions in LH mutant mice after 4 months of high fat diet. The columns represent the relative expression levels of muscular genes involved in the mitochondrial β-oxidation; CPT-1 and VLCAD and hepatic genes involved in the mitochondrial β-oxidation, fatty acid synthesis and gluconeogenesis; VLCAD, ACACA and PEPCK-C, respectively. For relative quantification of gene expression, the results were normalized using GADPH and β-actin as endogenous controls, and the expression of the wild type samples were set to 1. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050045#s3" target="_blank">Results</a> represent means with 95% confidence interval of 10 independently analyzed mice for each genotype. P values were calculated using unpaired heteroscedastic student t-test with one-tailed distribution. * p<0.05, ** p<0.01, *** p<0.001. Abbreviations: CPT-1 = carnitine palmitoyltransferase, VLCAD = very long chain acyl-CoA dehydrogenase, Acaca = acetyl-CoA carboxylase, PEPCK-C = phosphoenoylpyruvate carboxykinase.</p

Distribution of recombinant adiponectin in MEF cell lysate and cell culture media.

A<p>Average of three experiments in which equal number of cells were used.</p>B<p>The results are calculated as a percentage of adiponectin from the total adiponectin on a cell culture plate (ng in lysate+ng in media measured by ELISA) and presented as average values ± S.D. from 3 different transfections.</p

Immunoblot and mass analyses of adiponectin produced in LH3 manipulated cell lines.

<p>(A) Recombinant adiponectin was produced in MEF cells and the medium containing the secreted adiponectin was collected for the analysis. The immunoblot analysis of recombinant adiponectin monomers from the concentrated medium of wild type, LH3^−/− knockout and LH mutant MEF cells indicates a clear size difference. (B) Schematic picture of the collagenous domain of mouse adiponectin shows the positions of posttranslational modifications of lysine and proline residues reported earlier. (C) Mass spectrometry identification of peptides and modifications from tryptic digests of mouse adiponectin produced in wild type, LH3^−/− knockout and LH mutant MEFs. Abbreviations: WT = wild type; KO = LH3^−/− knockout; MUT = LH mutant; Hyl = hydroxylysine; Gal = galactosyl; Glc = glucosyl.</p

Total adiponectin levels in LH mutant mice and heterozygous LH3 knockout mice.

<p>(A) Total adiponectin levels were significantly reduced in the serum of LH mutant mice (MUT), but not in the heterozygous LH3 knockout (LH3^+/−) mice. Adiponectin level analyses were done from the serum of the 5 months old male (n = 6 wt, 5 ko^+/−) and female (n = 6 wt, 6 ko^+/−) LH3^+/− mice, and 6 months (n = 6 wt, 5 mut) and 10 months (n = 7 wt, 5 mut) old male, and 2 months (n = 7 wt, 5 mut) and 10 months (n = 9 wt, 7 mut) old female LH mutant mice with ELISA. (B) Total leptin levels were comparable in LH mutant and wild type mice. Leptin analyses were done from the serum of the 5 months (n = 6 wt, 4 mut) and 10 months (n = 4 wt, 3 mut) old male, and 8 months (n = 5 wt, 5 mut) old female LH mutant mice with ELISA. (C) Adiponectin monomers from LH mutant and wild type serum exhibited differences in electrophoretic mobility. In order to evaluate the electrophoretic mobility of adiponectin, different volumes, 5 µl and 10 µl, of diluted (1/300) serum of wild type and LH mutant mice were separated near the end of a 15% SDS-PAGE. (D) The immunoblot analysis of adipose tissue homogenate (41 µg of protein) of wild type and LH mutant male mice did not show differences in the levels of adiponectin monomers, but the electrophoretic mobility of LH mutant adiponectin was clearly increased compared to that of the wild type. (E) In the adipose tissue of LH3^+/− mice (50 µg of protein loaded on the gel) no shift in the mobility or in the amount of adiponectin monomers was detected. In immunoblots C, D and E two representative wild type, LH mutant and LH3^+/− samples are shown. The values represent the average ± SD of the experiments. P values were calculated using unpaired homoscedastic student t-test with two-tailed distribution. * p<0.05, ** p<0.01, *** p<0.001.</p

Oligomer distribution of recombinant adiponectin is altered in LH3^−/− knockout MEF cells.

<p>The oligomeric forms of recombinant adiponectin were separated with gel filtration chromatography and quantified by ELISA. Comparison of typical gel filtration elution profiles of recombinant adiponectin produced in (A) wild type and (B) LH3^−/− knockout MEFs indicates that HMW and MMW oligomers are not secreted from knockout cells. (C) Double transfection with adiponectin and LH3 constructs normalized the elution profile in LH3^−/− knockout MEFs. (D) Recombinant adiponectin produced in LH mutant MEFs formed similar oligomers as the wild type. The adiponectin oligomers corresponding to the peaks seen with gel filtration chromatography were also detected on immunoblot (E) separated under non-reducing and non-heat-denaturing conditions. Equal volumes of concentrated cell culture media were used in all analysis. Representative elution profiles are shown. Abbreviations: WT = wild type; KO = LH3^−/− knockout; LH3 = lysyl hydroxylase isoform 3; MUT = LH mutant.</p