Fig 1: Adiponectin depletion abrogated DPP4 inhibition‐related vascular benefits in stressed mice. A and B, During the stress protocol, anagliptin (30 mg/kg per d)‐loaded stress mice were injected subcutaneously with control IgG (N‐APN[−]) or neutralizing antibody against APN (N‐APN [+]) every 5 d. Representative blood perfusion images and/or quantitative data show the blood flow (A) and capillary density (B) (n=5). C, Quantification of circulating CD31+/c‐Kit+ in both experimental groups (n=5). D and E, APN−/− mice pretreated with vehicle or anagliptin (30 mg/kg per d) for 3 d underwent ischemic surgery and were subjected to the LSBFI and immunoblotting analyses. Representative images and quantitative data show blood flow recovery and the levels of PPAR‐γ and PGC‐1α proteins in muscles of APN−/− mice (n=3–5). Data are mean±SE. *P<0.01 vs corresponding controls; NS, not significant by Student unpaired t test or ANOVA and Tukey's post hoc tests. APN indicates adiponectin; DPP4, dipeptidyl peptidase‐4; LSBFI, laser speckle blood flow imaging; PGC‐1α, PPAR‐γ co‐activator 1α; PPAR‐γ, peroxisome proliferator‐activated receptor‐γ.
Fig 2: Overexpression of miR-204-5p reversed the effect of HCG11 on hAdMSCs. (A) The expression of adipogenic marker protein and inflammatory factor was detected by Western blot. Expression of (B) C/EBPα, (C) PPARγ2, (D) AdipoQ, (E) FABP4, (F) LPL, (G) IL-6, and (H) TNF-α in hAdMSCs transfected with miR-204-5p mimic, pcDNA-HCG11, and their control was quantified using Image J software. The levels of lipogenesis enzymes such as (I) ACC and (J) FAS were detected by spectrophotometry. (K) Cell proliferation ability was tested by CCK-8. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 versus control. ACC: acetyl coenzyme A carboxylase; AdipoQ: adiponectin; C/EBPα: CCAAT-enhancer-binding protein α; CCK: cell counting kit; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; hAdMSCs: human adipose-derived mesenchymal stem cells; HCG11: human leukocyte antigen complex group 11; IL-6: interleukin-6; LPL: lipoprotein lipase; PPARγ: peroxisome proliferator-activated receptor gamma; TNF-α: tumor necrosis factor alpha.
Fig 3: Cytokines and APN system key factors altered in response to RAP and metformin treatment. A, B, Representative DHE staining and quantitative analysis to detect ROS in the LA and in the EAT in the sham‐operated group, RAP group and RAP + MET group, 6 weeks after pacemaker implantation (n = 6 animals/group, 400 × magnification, scale bar = 50 μm). C‐E, Representative immunoblots and quantitative analysis of the relative changes in NF‐κB and pNF‐κB expression in the LA and the EAT. F‐H, TGF‐β1, IL‐6, TNF‐α, and APN concentrations in the LA and EAT in the sham‐operated group, RAP group and RAP + MET group, 6 weeks after surgery (n = 6 animals/group). I‐L, Representative immunoblots and quantitative analysis of the relative changes in PPARγ and AdipoR1 in the LA and EAT. * P < .05 compared with the sham‐operated group; # P < .05 compared with the RAP group. DHE, dihydroethidium; LA, left atrium; EAT, epicardial adipose tissue; TGF‐β1, transforming growth factor‐β1; IL‐6, interleukin‐6; TNF‐α, tumour necrosis factor‐α; APN, adiponectin
Fig 4: Ablation of Arfrp1 in adipocytes selectively decreases adiponectin and adipsin secretion. (A) Circulating levels of indicated adipokines detected in plasma of 7-week-old Arfrp1iAT−/− (white bars) and control (Arfrpflox/flox, black bars) mice (n = 5–19 mice per genotype). (B) Adiponectin isoforms measured in plasma of 7-week-old animals. HMW: high molecular weight adiponectin. (C) Adiponectin transcript levels (Adipoq) determined in gonadal and subcutaneous white adipose tissue (gonWAT, scWAT) and in brown adipose tissue (BAT) of 7-week-old mice (n = 3–6 mice per genotype). (D, E) Released levels of adiponectin (D) and leptin (E) from gonWAT and scWAT explants of Arfrp1iAT−/− and control mice (n = 4–6 mice per genotype). All data are presented as mean ± SEM, **P ≤ 0.01, ***P ≤ 0.001 by unpaired Student's t-test.
Fig 5: Inhibition of DPP4 reverses GLP‐1 and adiponectin levels in plasma and/or adipose of stressed mice. A through D, Mice were given by oral gavage vehicle (distilled water, Stress), a low dose (30 mg/kg per day, S‐DL) or a high dose of the DPP4 inhibitor anagliptin (60 mg/kg per d, S‐DH) every day from 3 d before undergoing the surgery. At d 4 after surgery, harvested blood and adipose tissues (subcutaneous fat) were analyzed by ELISA or quantitative real‐time PCR, respectively, for the levels of DPP4 activity (A), GLP‐1 protein (B) and adiponectin protein (C), or adiponectin gene (D). Data are mean±SE (n=5–9). *P<0.01 by ANOVA and Tukey's post hoc tests. E, Blood flow was measured by representative laser speckle perfusion imaging, and capillary density was measured by fluorescent staining with tomato lectin. Representative images and quantitative data for blood flow recovery (expressed as the ischemia‐to‐nonischemia LSBFI ratio) (E) and capillary density (expressed as capillary‐to‐myofiber ratio) (F) are shown. Data are mean±SE (n=5–7). *P<0.01 by 1‐way ANOVA and Tukey's post hoc tests. Scale bar: 50 μm. APN indicates adiponectin; DPP4, dipeptidyl peptidase‐4; GAPDH, glyceraldehyde‐3‐phosphate‐dehydrogenase; GLP‐1, glucagon‐like peptide‐1; LSBFI, laser speckle blood flow imaging; PCR, polymerase chain reaction.
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