Adiponectin receptor 1 resists the decline of serum osteocalcin and GPRC6A expression in ovariectomized mice

<div><p>Hormonal changes that cause metabolic complications are a common problem in postmenopausal women. Adiponectin and osteocalcin are cytokines associated with glucose regulatory and insulin sensitized function in postmenopausal stages. The current study investigated the role of adiponectin signaling and osteocalcin mediated function in glucose metabolism in ovariectomized mice. In a mouse menopausal-related metabolic disorder model, overexpression of adiponectin receptor 1 improved glucose tolerance and caused resistance to body weight increase and decline of serum osteocalcin. Furthermore, adiponectin receptor 1 transgenic ovariectomized mice had higher GPRC6A (the putative osteocalcin receptor) expression in muscle tissue. Immunofluorescence indicated that GPRC6A and adiponectin receptor 1 were co-localized in mouse muscle tissues. The present finding suggested adiponectin receptor 1 can mediate the improvement of glucose metabolism by osteocalcin in ovariectomized mice. Our findings imply the possibility to ameliorate menopause-induced metabolic disorder by GPRC6A and adiponectin signaling.</p></div

Immunofluorescence stain of GPRC6A and AdipoR1 in mouse muscle tissue.

<p>Cell nulei were stained with DAPI (blue). Muscle tissue sections were stained with GPRC6A (red) and AdipoR1 (green) antibody and viewed at X400 magnification with scale bars indicating 20 mm. Arrow refers to the co-localization of GPRC6A and AdipoR1.</p

Additional file 2: of Adiponectin and adiponectin receptor 1 overexpression enhance inflammatory bowel disease

Figure S1. Sequence alignment of human, pig and mouse AdipoR1. The sequence of Homo sapiens, Sus scrofa, and Mus musculus AdipoR1 were aligned using the software BioEdit. The sequences and their GenBank accession numbers are: Homo sapiens (NP_057083.2), Sus scrofa (NP_001007194) and Mus musculus (NP_082596.2). Boxes show the zinc-binding site (residues 187–212, 333–347) and C-terminal extracellular region/CTR (residues 365–375) of AdipoR1 which are conserved between pig and mouse. (PDF 216 kb

Additional file 1: of Adiponectin and adiponectin receptor 1 overexpression enhance inflammatory bowel disease

Table S1. Primer sets for quantitative real-time PCR. Primers were designed using the Primer-BLAST tool of National Center for Biotechnology Information (NCBI). Each primer sequence was confirmed by aligning its reference sequence in the NCBI database. (PDF 202 kb

Additional file 4: of Adiponectin and adiponectin receptor 1 overexpression enhance inflammatory bowel disease

Figure S3. Mouse ADN bound to porcine AdipoR1 in pAdipoR1 mice. (A) Mouse ADN (mADN) bound with mouse AdipoR1 in wild type (WT) mice, and mADN bound with mouse and flag-conjugated porcine AdipoR1 in pAdipoR1 mice. (B) Co-immunoprecipitation (co-IP) between m-ADN and flag-pAdipoR1. To confirm the mADN binds with pAdipoR1, IP was performed using anti-ADN antibody followed by immunoblotting using anti-flag antibody and anti-ADN antibody in the colon of WT and AdipoR1 mice. Flag-pAdpoR1 was detectable in the protein of AdipoR1 mice. ADN was detectable both in the WT and AdipoR1 mice. mADN: mouse adiponectin; mAdipoR1: mouse adiponectin receptor 1; f-pAdipoR1: porcine adiponectin receptor 1 conjugated with flag; IB: immunoblotting. (PDF 187 kb

Sequence similarity of Soat1 among various animal species.

<p>Zebrafish Soat1 amino acid sequences are aligned with the SOAT1 orthologs from human (ENSP00000356591), rat (ENSRNOP00000005677), mouse (ENSMUSP00000058344), chicken (ENSGALP00000006691) and <i>Xenopus</i> (ENSXETP00000050224). The amino acid sequence of zebrafish Soat1 was predicted according to the cloned sequence. The MBOAT domain is predicted between 168 to 517 residue (arrow under sequences). The acyl-CoA binding domain (FYXDWWN, orange box above the sequences) and the postulated catalytical residue for esterification (His457, red asterike above the sequences) are conservative through species. The identical amino acids are shown in the black box, while the less conservative regions are enclosed by white box.</p

A schematic illustration of the molecular mechanism for yolk cholesterol trafficking in zebrafish embryo.

<p>Before the formation of YSL, free cholesterol diffuses from yolk to blastomeres directly. After YSL is formed at about 4 hpf, free cholesterol are transported from yolk to embryo through Npc1l1. The blood circulation in zebrafish embryos begins at about 24 hpf, and the importance of lipoproteins in yolk lipids absorption could be observed at 48 hpf when Mttp or Apo C-II was defective. The mRNA of <i>soat2</i> is expressed after 12 hpf, but its enzyme does not evidently contribute to the yolk cholesterol trafficking until the assembly of lipoproteins become prominent after 48 hpf. The level of CEs surged at 72 hpf and the loss of Soat2 activity resulted in delayed yolk consumption most prominently at 72 hpf indicated that the activity of Soat2 peaked at 72 hpf.</p

Expression patterns of zebrafish <i>soat1</i> during embryogenesis.

<p>Whole-mount <i>in situ</i> hybridization shows that <i>soat1</i> can be detected in all blastomeres at 1 cell stage (A), 3 hpf (B), and 6 hpf (C). The expression of <i>soat1</i> was prominent at yolk sac at 12 hpf (D) and 24 hpf (E). By 48 hpf (F, G), <i>soat1</i> was detected in the brain, retina, pectoral fin, hatching gland and pericardium. By 72 hpf (H, I), <i>soat1</i> was also observed prominently in the liver primordia and intestine.</p

Increased intracellular accumulation of neutral lipid and CEs as a result of esterification of cholesterol to fatty acyl-CoA.

<p>(A) Oil Red O (ORO) staining of eGFP stable expressing (Ctrl), zebrafish <i>saot1</i> stable expressing (Soat1), and zebrafish <i>soat2</i> stable expressing (Soat2) HEK293 cells after incubation with low (15 μM oleic acids and 1 μg/mL cholesterol) or high (150 μM oleic acids and 10 μg/mL cholesterol) level of substrate at room temperature for 6 hrs. The expression of zebrafish Soat2 significantly increased the intracellular accumulation of neutral lipid indicated its enzymatic activity of cholesterol esterification, and this accumulation of neutral lipid is significantly increased with the increased levels of substrates. Note the significantly increased neutral lipid in WT-high compared to WT-low reflected the activity of endogenous human SOAT1 expressed by HEK293 cells. The letters above each column indicate the statistical groups, and the data sharing the same letters indicates no significant difference. (B) The cell lysate of HEK293 cells with zebrafish <i>soat2</i> overexpression contained significantly higher CE content than either control group or <i>soat1</i> overexpression group. Consistent with the ORO staining, the CE content in <i>soat1</i> group was comparable to the control group. (C) RT-PCR confirmed the expression of both zebrafish <i>soat1</i> and eGFP in Soat1-overexpressing HEK293 cells, zebrafish <i>soat2</i> and eGFP in Soat2-overexpressing HEK293 cells and only eGFP in control group in which the HEK293 cells were transfect with empty vectors. (D) The HEK293 cells overexpressing zebrafish Soat2 (Soat2-DsRed) contained more and larger intracellular lipid droplets containing NBD-cholesterol as compared to the HEK293 cells overexpressing DsRed only (DsRed). (E) Avasimibe (AVA) significantly reduced the intracellular accumulation of neutral lipid, but the cells with zebrafish <i>soat2</i> overexpression still showed a significantly more ORO staining than other AVA-treated cells. (F) Pyripyropene A (PPPA) could significantly reduce the intracellular accumulation of neutral lipid by inhibiting the enzymatic activity of both endogenous human SOAT1 and zebrafish Soat2 when compared to the vehicle control (DMSO). The letters above each column indicate the statistical groups, and the data sharing the same letters indicates no significant difference.</p

Sequences of the oligos used in this study.

<p>Sequences of the oligos used in this study.</p