Journal: Diabetologia

Article Title: Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α

doi: 10.1007/s00125-013-2950-9

Figure Lengend Snippet: miR-221 targets the 3′ UTRs of ADIPOR1 and ETS1 and decreases their protein levels. ( a,b ) TargetScan conserved predicted miR-221 binding site on the 3′ UTRs of ADIPOR1 and ETS1 . ( c,d ) Quantification of dual-luciferase assay in HEK 293 cells co-transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars) and ADIPOR1/2 ( c ) or ETS1 ( d ) 3′ UTR reporter plasmids. ( d ) Two overlapping fragments of the ETS1 3′-UTR were subcloned into separate vectors, indicated (A) and (B). Firefly/renilla luciferase ratio (luc ratio) was normalised to no-UTR controls. Error bars indicate SD ( n = 5). * p < 0.05 (two-tailed Mann–Whitney U test). ( e ) Quantification of QRT-PCR for ETS1 mRNA in human pre-adipocytes transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars). Error bars indicate SD ( n = 3). ( f ) Immunoblots for ADIPOR1, ETS1 and β-actin in human pre-adipocytes transfected with miR-221 mimic (+) or control oligonucleotide. ( g ) Quantification of the immunoblots shown in ( f ). Background-subtracted mean signal ( n = 2) for ADIPOR1 and ETS1, normalised to the loading control (β-actin). Black bars, miR-221 mimic; white bars, control oligonucleotide

Article Snippet: For 3′ untranslated (UTR) reporter assays, JetPrime (Polyplus-transfection SA, Illkirch, France) was used to co-transfect HEK 293 cells (ATCC) with miRNA 3′ UTR target expression vectors for human ETS1 , ADIPOR1 , ADIPOR2 or control (Genecopoeia, Germantown, MD, USA), and human miR-221 mimic (Dharmacon, Lafayette, CO, USA) or scrambled control oligonucleotide, in 24-well plates.

Techniques: Binding Assay, Luciferase, Transfection, Control, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

doi: 10.1371/journal.pone.0049915

Figure Lengend Snippet: (A) Plasma adiponectin levels in the control, AngII, and losartan groups were detected by ELISA. (B) Levels of AdipoR1 mRNA were analyzed by qRT-PCR, and β-actin was used as an internal control. (C) Myocardial extracts were immunoblotted with antibody specific for AdipoR1. Blots were reprobed with actin to confirm equal loading. (D) Representative immunostaining images and averaged bar graphs of AdipoR1 density. AdipoR1 is shown in brown and cell nuclei in blue. Scale bar represents 10 µm (6 fields in each sample were scanned and averaged). (E) Myocardial extracts were immunoblotted with antibody specific for p-AMPKα at Thr-172. Blots were reprobed with antibody for AMPK to confirm equal loading. (F) Myocardial extracts were immunoblotted with antibody specific for p-ACC at Ser-79. Blots were reprobed with antibody for ACC to confirm equal loading. Data represent mean ± SE. n = 6 in each group. ** P <0.01 vs. control. # P <0.05, ## P <0.01 vs. AngII.

Article Snippet: Antibodies for AdipoR1, AdipoR2, phospho- and total extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor (NF)-κB, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Clinical Proteomics, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Immunostaining

Journal: PLoS ONE

Article Title: Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

doi: 10.1371/journal.pone.0049915

Figure Lengend Snippet: (A, C) Levels of AdipoR1 mRNA were analyzed by qRT-PCR. (B, D) Expression of AdipoR1 protein was determined by Western blot analysis. (E) Representative immunofluorescence images and averaged bar graphs of AdipoR1 density in unstimulated control NRVMs and cells treated with 0.1 µmol/L AngII for 48 h. Green fluorescence signals represent AdipoR1 protein. Scale bar represents 20 µm (6 fields in each sample were scanned and averaged). (F) NRVMs were pretreated with 0.1 µmol/L AngII for 48 h and then stimulated with 1 µg/ml globular adiponectin for 10 min. AMPK phosphorylation in cell lysates was determined by Western blot analysis. Data represent mean ± SE of three independent experiments. ** P <0.01 vs. control. ## P <0.01 vs. AngII.

Article Snippet: Antibodies for AdipoR1, AdipoR2, phospho- and total extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor (NF)-κB, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Control, Fluorescence, Phospho-proteomics

Journal: PLoS ONE

Article Title: Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

doi: 10.1371/journal.pone.0049915

Figure Lengend Snippet: NRVMs were pretreated with losartan (10 µmol/L) or PD123319 (1 µmol/L) for 1 h and then stimulated with 0.1 µmol/L AngII for 48 h, or incubated with CGP42112A (1 µmol/L) for 48 h. (A) Levels of AdipoR1 mRNA were analyzed by qRT-PCR, and β-actin was used as an internal control. (B) Western blot was performed to detect levels of AdipoR1 and actin protein expression. Data represent mean ± SE of three independent experiments. * P <0.05, ** P <0.01 vs. control. # P <0.05 vs. AngII.

Article Snippet: Antibodies for AdipoR1, AdipoR2, phospho- and total extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor (NF)-κB, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Incubation, Quantitative RT-PCR, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

doi: 10.1371/journal.pone.0049915

Figure Lengend Snippet: (A, B) NRVMs were pretreated with SB202190 (5 µmol/L), PD98059 (10 µmol/L), or SP600125 (10 µmol/L) for 1 h, then stimulated with 0.1 µmol/L AngII for 48 h. Expression of AdipoR1 mRNA and protein was analyzed by qRT-PCR and Western blot respectively. (C) Representative fluorescence images and histogram of relative fluorescence density of ROS. NRVMs were pretreated with NAC (10 mmol/L), apocynin (100 µmol/L), rotenone (10 µmol/L), or allopurinol (100 µmol/L) for 1 h, followed by DCF-DA incubation for 30 min, and then stimulated with 0.1 µmol/L AngII for 30 min. DCF fluorescence was visualized using confocal microscopy. The green color represents ROS and the fluorescence density was normalized to cell count. Scale bar represents 20 µm (6 fields in each sample were scanned and averaged). (D, E) NRVMs were pretreated with NAC (10 mmol/L), apocynin (100 µmol/L), rotenone (10 µmol/L), or allopurinol (100 µmol/L) for 1 h and then stimulated with 0.1 µmol/L AngII for 30 min (D) or 48 h (E). Western blot was performed to detect levels of p-ERK1/2 (D) or AdipoR1 (E). Blots were reprobed with antibody for ERK1/2 (D) or actin (E) to confirm equal loading. (F, G) NRVMs were pretreated with 10 µmol/L gp91 ds-tat or scrambled gp91 ds-tat for 1 h and then stimulated with 0.1 µmol/L AngII for 30 min (F) or 48 h (G). Western blot was performed to detect levels of p-ERK1/2 (F) or AdipoR1 (G). Data represent mean ± SE of three independent experiments. * P <0.05, ** P <0.01 vs. control. # P <0.05, ## P <0.01 vs. AngII.

Article Snippet: Antibodies for AdipoR1, AdipoR2, phospho- and total extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor (NF)-κB, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Fluorescence, Incubation, Confocal Microscopy, Cell Counting, Control

Journal: PLoS ONE

Article Title: Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

doi: 10.1371/journal.pone.0049915

Figure Lengend Snippet: Putative binding sites of c-Myc, STAT5, and NF-κB in the promoter region of AdipoR1.

Article Snippet: Antibodies for AdipoR1, AdipoR2, phospho- and total extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor (NF)-κB, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

doi: 10.1371/journal.pone.0049915

Figure Lengend Snippet: (A) NRVMs were transfected with c-Myc or control siRNA (100 nmol/L) for 48 h. Western blot was performed to detect levels of c-Myc and actin. (B) NRVMs were transfected with c-Myc or control siRNA (100 nmol/L) for 48 h and serum-deprived for 24 h, followed by incubation with 0.1 µmol/L AngII for 48 h. The level of AdipoR1 protein was analyzed by Western blot analysis with actin as an internal control. Data represent mean ± SE of three independent experiments. * P <0.05, ** P <0.01 vs. control. ## P <0.01 vs. AngII.

Article Snippet: Antibodies for AdipoR1, AdipoR2, phospho- and total extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor (NF)-κB, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Control, Western Blot, Incubation

Journal: Cancer Medicine

Article Title: Exploring the Relationship Between Adipocytokines and Endometrial Cancer: Identifying Correlations With Clinico‐Pathological Prognostic Factors

doi: 10.1002/cam4.71007

Figure Lengend Snippet: (A, B) Bar‐graph demonstrating the expression of (A) biomarkers and (B) their receptors in endometrial cancer tissue ( n = 39). X axis‐ biomarkers (A), receptors (B). Y axis–expressions of biomarkers as fold change over benign endometrial calibrator sample (assigned value = 1, for comparison with the cancer samples). The error bars represent standard errors of mean. 1 (C) Heat map demonstrating the expression of all 6 biomarkers and their receptors in endometrial cancer tissues ( n = 39). AdipoQ, adiponectin; ADIPOR1/R2, adiponectin receptors; OBR, leptin receptor; TNF1A, TNFRSF1A; TNF1B, TNFRSF1B (TNF receptors). Gene expressions are plotted on the Y ‐axis against the individual patients on the X ‐axis. Gene expressions are plotted as fold changes over a single pooled calibrator sample value. The color scale ranges from black (lowest value) to white (median) to yellow (highest value).

Article Snippet: The antibodies used were: ADIPOR1 (Proteintech, Manchester, UK, #66619‐1‐Ig; anti‐mouse antibody) and ADIPOR2 (Abcam, Cambridge, UK, #ab223752; anti‐rabbit antibody).

Techniques: Expressing, Comparison

Journal: Cancer Medicine

Article Title: Exploring the Relationship Between Adipocytokines and Endometrial Cancer: Identifying Correlations With Clinico‐Pathological Prognostic Factors

doi: 10.1002/cam4.71007

Figure Lengend Snippet: Heat map comparing the expression of (A) adiponectin, leptin and their receptors in endometrial cancer tissue and parametrial adipose tissue ( n = 39) (B) all 6 biomarkers and their receptors in endometrial cancer tissue and lymph nodes ( n = 12). AdipoQ, adiponectin; AdipoR1/R2, adiponectin receptors; ObR, leptin receptor; TNF1A, TNFRSF1A; TNF1B, TNFRSF1B (TNF receptors). Gene expressions are plotted on the Y ‐axis against the individual patients on the X ‐axis. Gene expression plotted as fold changes over a single pooled calibrator sample. The color scale ranges from black (lowest value) to white (median) to yellow (highest value).

Article Snippet: The antibodies used were: ADIPOR1 (Proteintech, Manchester, UK, #66619‐1‐Ig; anti‐mouse antibody) and ADIPOR2 (Abcam, Cambridge, UK, #ab223752; anti‐rabbit antibody).

Techniques: Expressing, Gene Expression

Journal: Cancer Medicine

Article Title: Exploring the Relationship Between Adipocytokines and Endometrial Cancer: Identifying Correlations With Clinico‐Pathological Prognostic Factors

doi: 10.1002/cam4.71007

Figure Lengend Snippet: Illustration of chromogenic 3,3′‐Diaminobenzidine (DAB) staining for ADIPOR1 (A) and ADIPOR2 (B) receptor in endometrial cancer tissue and benign tissues. Image taken at 10× magnification, Scalebar = 100 μm.

Article Snippet: The antibodies used were: ADIPOR1 (Proteintech, Manchester, UK, #66619‐1‐Ig; anti‐mouse antibody) and ADIPOR2 (Abcam, Cambridge, UK, #ab223752; anti‐rabbit antibody).

Techniques: Staining

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Sequences of real-time PCR primers used for quantification of mRNA levels

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Real-time Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Expression of monocyte AdipoRs, AdipoR1 and AdipoR2, is altered in IR (GINF < 4.0) subjects compared with IS (GINF > 7.5) subjects. IS is determined by euglycemic clamp and expressed as the GINF. Data are mean ± SD, n = 5/group; *P < 0.01 vs. IS.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Expressing

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: APN up-regulates AdipoR1 and AdipoR2 expression in human monocytes. A) APN-induced phosphorylation of AMPK and p38 MAPK. Cells were incubated with 5 µg/ml APN for 30 min, and cell lysates were separated by SDS-PAGE and immunoblotted with antibodies to phospho-AMPK and phospho-p38 MAPK. B) AdipoR1 and AdipoR2 mRNA levels in human THP-1 monocytes treated with 10 µg/ml APN or 10 ng/ml TNF-α. Data are mean ± sd, n = 5/group; *P < 0.01 vs. control; **P < 0.01 vs. TNF-α. C) Western blot analysis of AdipoR1 and AdipoR2 protein expression in human THP-1 monocytes treated with APN or TNF-α. APN signaling in human THP-1 monocytes.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Expressing, Phospho-proteomics, Incubation, SDS Page, Control, Western Blot

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Effect of APN on AdipoR1 and AdipoR2 expression in mouse peritoneal macrophages. A) mRNA levels of AdipoR1 and AdipoR2 in mouse peritoneal macrophages treated with 10 µg/ml APN or 10 ng/ml TNF-α for 24 h. Data are mean ± sd, n = 5/group; *P < 0.01 vs. control; **P < 0.01 vs. TNF-α. B) Western blot analysis of AdipoR1 and AdipoR2 protein levels in mouse peritoneal macrophages treated 10 µg/ml APN or 10 ng/ml TNF-α for 24 h. C) mRNA levels of LXRα and LXRβ in mouse macrophages treated with 10 µg/ml APN or 10 ng/ml TNF-α for 24 h. Data are mean ± sd, n = 5/group; *P < 0.01 vs. control and TNF-α.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Expressing, Control, Western Blot

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Macrophage-polarized activation regulates the expression of AdipoR1 and AdipoR2. Bone marrow-derived macrophages were differentially activated into M1 (IFN-γ/LPS) or M2 (IL-4 or IL-10) for 24 h, and mRNA levels of macrophage polarization markers, IL-12 and MR, as well as AdipoR1 and AdipoR2, were measured by qRT-PCR. Data are mean ± sd, n = 5; *P < 0.05 vs. control; **P < 0.05 vs. M1. Representative of 3 experiments. ND, Not detected.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Activation Assay, Expressing, Derivative Assay, Quantitative RT-PCR, Control

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Regulation of AdipoR expression in differentially polarized mouse peritoneal macrophages, which were activated into M1 (IFN-γ/LPS) or M2 (IL-4 or IL-10) for 24 h, and mRNA levels of IL-12, MR, AdipoR1, and AdipoR2 were measured by qRT-PCR. Data are mean ± sd, n = 5; *P < 0.001 vs. Control; **P < 0.001 vs. M1. B) Western blot analysis of AdipoR1 and AdipoR2 protein levels in mouse peritoneal macrophages treated with 10 µg/ml APN or 10 ng/ml TNF-α for 24 h. Data are representative of 3 experiments.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Exogenous M1/M2 cytokine environment alters macrophage inflammatory status and AdipoR expression. Mouse peritoneal macrophages were activated into M1 (IFN-γ/LPS) and M2 (IL-10) for 24 h, after which, the inducer media were removed, and the cells were incubated further for an additional 24 h in the presence or absence of 10 µg/ml APN without the M1/M2 inducer cytokines. IL-12, MR, AdipoR1, and AdipoR2 mRNA levels were measured by qRT-PCR. Data are mean ± sd, n = 6/group; *P < 0.01 vs. control.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Macrophage polarization differentially regulates AdipoR1 and AdipoR2 expression and inflammatory cytokine expression in response to APN. Bone marrow macrophages differentially activated into the M1 (IFN-γ/LPS)- or M2 (IL-4 or IL-10)-polarized phenotype were incubated in the presence or absence of 10 µg/ml APN for 48 h. A) IL-12, MR, TNF-α, IL-6, IL-10, AdipoR1, and AdipoR2. B) LXRβ, LXRα, and PPARγ. C) p38 MAPK mRNA levels were measured by qRT-PCR. Data are mean ± sd, n = 6/group; *P < 0.05 control vs. APN-treated macrophages.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control

Journal: The FASEB Journal

Article Title: Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

doi: 10.1096/fj.14-253831

Figure Lengend Snippet: Macrophage polarization regulates AdipoR expression and an APN-mediated inflammatory response. This study provides important evidence that differential polarization of macrophages into M1 or M2 phenotype profoundly alters their AdipoR (AdipoR1 and AdipoR2) expression, leading to divergent inflammatory responses to APN. Profound suppression of LXRs and PPARγ, accompanied by activation of p38 MAPK in M1 macrophages, contributes to low AdipoR levels. Relatively high and sustained LXRs and PPARγ levels and IL-10 accompanied by reduced p38 MAPK levels contribute to preservation of AdipoR expression in M2 macrophages. APN-mediated up-regulation of AdipoR expression in M1 macrophages is accompanied by marked induction of proinflammatory cytokines, whereas in M2 macrophages, APN increased the anti-inflammatory response without affecting AdipoR expression.

Article Snippet: Mouse AdipoR1 , , Mm01291331_m1 (ABI).

Techniques: Expressing, Activation Assay, Preserving

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Activation of Adiponectin Receptor Regulates Proprotein Convertase Subtilisin/Kexin Type 9 Expression and Inhibits Lesions in ApoE-Deficient Mice

doi: 10.1161/atvbaha.117.309630

Figure Lengend Snippet: Figure 4. Induction of proprotein convertase subtilisin kexin type 9 (PCSK9) expression by ADP355 (ADP) and AdipoRon (AR) is related to activation of AMP-activated protein kinase α (AMPKα). A, Expression of AdipoR1 and AdipoR2 in the stable AdipoR1 and AdipoR2 knockdown HepG2 (shAR1i and shAR2i) cells was determined by real-time RT-PCR. **P<0.01 vs control (shNSi) cells (n=3). B, shNSi, shAR1i, or shAR2i cells received indicated treatment overnight. Expression of PCSK9, LDLR (low-density lipoprotein receptor), AMP- activated protein kinase α (AMPKα), and π-AMPKα was determined by Western blot. C and D, HepG2 cells were treated with ADP355 and AR at the indicated concentrations overnight. Expression of AMPKα and π-AMPKα was determined by Western blot. AICAR was used as a positive control. E, HepG2 cells were treated with AICAR or metformin (Met) at the indicated concentrations overnight followed by determination of PCSK9, AMPKα, π-AMPKα and PPARγ protein expression. F, CRISPR-Ctrl (clustered regulatory interspaced short palindromic repeat-associated 9) and CRISPR-AMPKα1 HepG2 cells were treated with ADP at the indicated concentrations overnight followed by determination of AMPKα1, PPARγ, and PCSK9 protein expression. G and H, Primary hepatocytes isolated from PPARγfl/fl and HepPPARγ KO mice were treated with AICAR or Met at the indicated concentrations overnight. Expression of PCSK9 protein (G) and mRNA (H) was determined by Western blot and real-time RT-PCR, respectively. *P<0.05 vs control (n=3).

Article Snippet: 4 Inhibition of PPAR AdipoR1 and AdipoR2 expression by siRNA The siRNA was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Activation Assay, Knockdown, Quantitative RT-PCR, Control, Western Blot, Positive Control, CRISPR, Isolation