Sirt1 activator induces proangiogenic genes in preadipocytes to rescue insulin resistance in diet-induced obese mice

Figure 1. SRT1720 treatment promotes angiogenesis. (A) Relative mRNA expression of SRT1720 (1 μM)-treated 3T3-L1 preadipocytes, compared with control (n = 6 independent experiment). (B) Relative mRNA expression of siRNA Sirt1-treated 3T3-L1, compared with non-target control. (C) Relative mRNA expression of SRT1720 (1 μM)-treated ex vivo preadipocytes, compared with control. (D) Relative mRNA expression of eWAT from high-fat diet (HFD)-fed myeloid cell-specific Sirt1 knockout mice (Mye-Sirt1 KO, Sirt1flox/flox; LysM-CreKI/+), compared with HFD-fed Sirt1flox/flox control mice. The data are shown as the means ± SEM. *P < 0.05, **P < 0.01.

Figure 2. SRT1720 improves glucose metabolism in DIO mice. (A,B) Glucose and insulin concentrations for an intraperitoneal glucose tolerance test (IP-GTT) and an intraperitoneal insulin tolerance test (IP-ITT) in SRT1720-treated DIO mice and control DIO mice (n = 5–6 per group). The data are shown as the means ± SEM. *P < 0.05, **P < 0.01. (C) Relative mRNA expression of mitochondrial genes in eWAT (n = 4–5 per group). Each data point was normalized to the 18 S mRNA level. The data are shown as the means ± SEM. *P < 0.05, **P < 0.01. (D) Hematoxylin and eosin (H&E) staining of eWAT, showing crown-like structures (scale bar, 200 μm). (E) Relative mRNA expression of M1/M2 macrophage markers in eWAT (n = 5–6 mice per group). The data are shown as the means ± SEM. *P < 0.05, **P < 0.01.

Figure 3. SRT1720 treatment enhances vasculature in eWAT. (A) mRNA expression of genes related to angiogenesis in eWAT. (n = 6–7). The results are shown as the mean ± SEM. *P < 0.05, **P < 0.01. (B) Immunostaining of eWAT with anti-CD31 and CD34 antibody. (C) Immunofluorescence labeling of eWAT with anti-PDGFRb. (D) Immunofluorescence labeling of eWAT with anti-CD31 (green) and anti-CD13 (red) antibodies. Scale bar, 100 μm. (E) Relative mRNA expression of hypoxia-related genes in eWAT from DIO mice treated with or without SRT1720 (n = 4–5 per group). The results are shown as the mean ± SEM. *P < 0.05, **P < 0.01.

Figure 4. SRT1720 treatment upregulates the expression of angiogenic factors in PDGFRα+ preadipocytes. (A) Representative flow cytometry analysis of eWAT. Cells in the SVF of eWAT from DIO WT mice were analyzed using flow cytometry. Cells were isolated from enzymatically digested mouse eWAT. After the exclusion of doublets and debris, dead cells were excluded using 7AAD staining. Live cells were further stained with anti-CD31 and anti-PDGFRb for the detection of endothelial cells and pericytes, respectively. (B) Representative flow cytometry images of lineage-negative PDGFRα + cells (PDGFRα + preadipocytes) in eWAT. (C) mRNA expression of angiogenic factors in the PDGFRα + preadipocyte fraction (n = 3). The results are shown as the mean ± SEM. *P < 0.05, **P < 0.01. AU, arbitrary units.

Figure 5. SRT1720 treatment reduces fibrosis of eWAT. (A) Representative images of Azan-stained sections of eWAT from NC, HFD, and HFD + SRT1720-treated mice (Scale bars, 100 μm). The fibrotic area (blue) was analyzed using ImageJ software. (B) Immunostaining of eWAT stained with anti-collagen type I antibody. The data are shown as the means ± SEM (n = 6 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 (Student t-test).