Exendin-4 Improves Diabetic Kidney Disease in C57BL/6 Mice Independent of Brown Adipose Tissue Activation

Figures

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  Figure 1. Exendin-4 treatment improved metabolism in diabetic mice induced by high-fat diet (HFD) and STZ. The body weight (a), random blood glucose (b, d), and food intake (c) were recorded during the period of building the model of diabetic mice. The serum levels of TG (e), LDL (f), and HDL (g) were measured by ELISA. BAT-specific genes (h) were analyzed. n = 6‐8 for each group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. control group; #P < 0.05 and ###P < 0.001 vs. DM+Exc group; &P < 0.05 and &&&P < 0.001 vs. DM-Con group; +P < 0.05 vs. DM+E4 group.

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  Figure 2. Exendin-4 ameliorated the progress of diabetic nephropathy and activated the renal AMPK pathway. The 24-hour urinary albumin (a) and 24-hour urinary 8-OH-dG (b) were measured by ELISA. (c) Periodic acid-Schiff (PAS) and Masson staining of kidney. Magnification ×400. (d–f) The renal mRNA expression of TGF-β1, Col-1, and FN, respectively (n = 6 per group). Western blot analysis of renal TGF-β1 (g, h) and AMPK (i, j) protein and the quantitative analysis results (n = 5 per group). n = 6~8 per group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. DM+Exc group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 vs. DM-Con group.

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  Figure 3. Effects of exendin-4 on adipogenesis-related genes during differentiation of 3T3-L1 and brown preadipocytes (WT-1). The mRNA expression of PPAR-γ, CEBPα, CEBPβ, UCP-1, PGC-1α, CIDEA, CD36, COX2, Cytoc1, and ATGL in mature 3T3-L1 cells (a) and brown preadipocytes (b) with exendin-4 (10 nM) stimulation for 24 hours and days 3 and 8 of differentiation in 3T3-L1 cells (c, d) and brown preadipocytes (e, f), stimulated by exendin-4 (10 nM) (n = 3 per group). ∗P < 0.05 vs. control.

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  Figure 4. Effect of FFA-BSA complexes on myofibroblast-like phenotype transition and lipid accumulation in cultured mesangial cells. Quiescent mesangial cells were treated with media (control, Con), 1% fatty acid-free BSA (BSA) alone, 200 μM oleate- or palmitate-1% fatty acid-free BSA (OA-BSA and PA-BSA, respectively), or exendin-4 of 0 (vehicle control), 0.1, 1, 10, 20, and 100 nM for 48 hours. (a) Proliferation of MCs by MTT assay. (b) Expressions of TGF-β1, FN, α-SMA, and collagen I by quantitative RT-PCR. (c) Lipid accumulation in MCs determined by Oil Red O assay. Inset presents ×10 magnification to illustrate size and location of lipid droplets in cytoplasm. (d) Western bolt analysis of TGF-β1, FN, α-SMA, and collagen I. Quantitative analyses of the results are also shown: TGF-β1 (e), FN (f), α-SMA (g), and Col-1 (h). Values are the mean ± SD of 3 independent experiments. ∗P < 0.05 and ∗∗P < 0.01 vs. BSA group, &P < 0.05 and &&P < 0.01 vs. OA-BSA with 0 nM exendin-4 group. (i) Dose response of MC viability in response to increasing concentration of exendin-4. ^P < 0.05 vs. other groups.

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  Figure 5. Exendin-4 regulates the expressions of TGF-β1, FN, α-SMA, and Col-1 in MCs treated by oleate via AMPK activation. EX: exendin-4; CC: compound C; AI: AICAR. MCs were cultured in DMEM and 1% BSA or 200 μM oleate-BSA for 48 hours. Exendin-4 (20 nM), compound C (10 μM), or AICAR (1 mM) were used to assess the effect of AMPK on myofibroblast marker protein expression. Supernatants were assayed by ELISA after treatment with a different reagent. Western bolt of p-AMPK (a), p-ACC (b), TGF-β1, FN, α-SMA, and Col-1 (c) and quantitative analyses of the results are also shown (d–g). (h) Secreted TGF-β1 in the cell culture supernatants. Values are the mean ± SD of 3 independent experiments. ∗∗P < 0.01 vs. BSA group; &&P < 0.01 vs. OA group; #P < 0.05 and ##P < 0.01 vs. OA+EX group.

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  Figure 6. Effects of exendin-4 on lipid accumulation and mRNA expression of SREBP-1, FAS, PPAR-α, and CPT-1 mediated by AMPK activation. Exendin-4 (20 nM), compound C (10 μM), or AICAR (1 mM) were used to assess the effect of AMPK in MCs cultured with 1% BSA or 200 μM oleate-BSA. Deposition of neutral lipid was characterized by Oil Red O (a), and triglyceride (TG) contents of the cell were measured by glyceridase assay (b). The mRNA expressions of SREBP-1, FAS, PPAR-α, and CPT-1 were measured by quantitative RT-PCR (c–f). ∗P < 0.05 vs. BSA group; &P < 0.05 and &&P < 0.01 vs. OA group; #P < 0.05 and ##P < 0.01 vs. OA+EX group, n = 3.