2.1. Cell Cultures

Rat mesangial cell lines (HBZY-1, MCs) were cultured in complete DMEM culture medium (HyClone, MA, USA), 10% fetal bovine serum, penicillin, and streptomycin. The cells were incubated at 37°C in a humidified 95% air, 5% CO₂ atmosphere-designated incubator. Original oleic acid or palmitic acid (both from Sigma-Aldrich, Santa Clara, CA, USA) were added to mimic the FFA-albumin complexes in obesity. MCs were cultured in DMEM medium alone for 12 hours for synchronization and then were exposed to FFA-BSA complexes (200 μmol/L), with or without the additional application of exendin-4 (0.1, 1, 10, 20, or 100 nM), compound C (10 μM), or AICAR (1 mM). The MTT (Cell Signaling Technology, Danvers, MA, USA) method was used to examine the cell proliferation.

Mouse 3T3-L1 preadipocytes were obtained from the Chinese Academy of Sciences, and the brown preadipocytes were established as previously described [14]. First, we isolated the interscapular BAT of C57BL/6, then we use collagenase to digest the BAT. Differentiation of preadipocytes was carried out according to the protocol which has been described in detail in the previous study [15, 16]. Cells were incubated with or without exendin-4 (10 nmol/L) from day 0 to day 8. Furthermore, the differentiated cells were cultured with serum-free DMEM after differentiation, and 12 hours before treatment, the cells were then treated with exendin-4 (10 nmol/L) or vehicle.

2.2. Animal Experiments

Male C57BL/6J mice (six weeks old) were purchased from the Guangdong Medical Laboratory Animal Center. All mice were nurtured under controlled conditions (temperature 22°C, 12-hour light-dark cycle) in the specific pathogen-free (SPF) barrier facility at the Southern Medical University.

2.3. Mouse Models of T2DM

The male C57BL/6J mice were randomized to the chow diet (CD, n = 8) or high-fat diet (HFD, 60% of total calorie from fat, n = 39) groups. After 4 weeks, the HFD group was made diabetic by STZ (120 μg/g body weight, pH 4.5; Sigma S0130) diluted in 10 mmol/L sodium citrate buffer; the control mice were infused with the same amount of citrate buffer. The mice were judged to be diabetic when the blood glucose exceeded 250 mg/dL. Then, the diabetic mice were divided into 4 groups (n = 8-10 per group): diabetic mice (DM-Con), BAT-excision diabetic mice (DM+Exc), exendin-4-treated diabetic mice (DM+E4), and exendin-4-treated diabetic mice with BAT excision (DM+Exc+E4). The BAT was removed from the intrascapular region of the diabetic mice in the DM+Exc group and the DM+Exc+E4 group. Exendin-4 (sigma E7144) was administered to DM+E4 mice and DM+Exc+E4 mice by intraperitoneal injection for 8 weeks, and the DM-Con mice received the injection of saline. Mice were euthanized after 12 weeks, and the kidneys were obtained for further analyses.

2.4. Biochemical Measurements

Blood glucose levels were measured using an Accu-Chek glucose monitor (Bayer Corp.) every week. After mice were euthanized, collected blood and the serum was stored at -80°C. The concentrations of triglycerides (TG), HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) were measured by commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Twenty-four-hour urine collections for measurements were obtained from mice placed in metabolic cages. Urinary microalbumin concentrations and the levels of 8-hydroxy-2 ^′-deoxyguanosine (8-OH-dG) were measured using enzyme-linked immunosorbent assay (ELISA) kits from Bethyl Laboratories, Inc., Montgomery, TX, USA, and CUSABIO, USA, respectively.

2.5. Quantitative Real-Time PCR and Western Blot Analysis

Total RNA was extracted with TRIzol (Takara, Dalian, China), and the RNA quality was detected by the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). The cDNA was synthesized by M-MLV Kit (Invitrogen, Carlsbad, CA, USA). The SYBR Green qPCR kit (Takara, Dalian, China) was used to perform real-time quantitative PCR (RT-qPCR) in the Roche LightCycler 480 II system (Roche, Basle, Switzerland). The expression of the transcript is normalized by the expression level of β-actin.

RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract the total protein of the sample, and the protein concentration was measured by a BCA assay (Takara, Dalian, China). The protein was separated by 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to the PVDF membrane (Merck Millipore, MA, USA). Blocking with 5% skim milk in TBST for 1-2 hours, the PVDF membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies were as follows: phospho-adenosine 5 ^′-monophosphate- (AMP-) activated protein kinase (AMPK) α (Thr172) and AMPKα, phospho-acetyl-CoA carboxylase (ACC) (Ser79) and ACC, transforming growth factor- (TGF-) β1 (all from Cell Signaling Technology, Danvers, MA, USA), collagen I (Col-1) and fibronectin (FN) (both from Abcam, Cambridge, MA, USA), and α-smooth muscle actin (SMA) and β-actin (both from Boster Bio Co., Wuhan, China).

2.6. Histological Analysis

Oil Red O (Sigma-Aldrich) staining was used to determine TG accumulation as previously described [17]. The total TG of MCs was extracted and measured by a TG Assay Kit (Applygen Technologies Inc., Beijing, China). After being fixed for 48 h by 4% paraformaldehyde, the renal tissue was dehydrated and then embedded in paraffin, cut into 4 μm thick slices, and stained with periodic acid-Schiff (PAS) and Masson’s trichrome. All sections were detected by Olympus B ×40 upright light microscope (Olympus, Tokyo, Japan).

2.7. Statistical Analysis

All data were represented as the mean ± SD. The differences between two independent groups were examined by two-tailed Student’s t-test, and the differences among three independent groups were examined by one-way ANOVA. Statistical analysis was performed by using SPSS 20. There was a significant difference when P < 0.05.

3.1. Exendin-4 Improved Glucose and Lipid Metabolism in Diabetic Mice

After 8 weeks, the body weight and blood glucose of diabetic mice increased significantly compared with those of the control group (Figures 1(a) and 1(b)). However, there was no significant difference between the two groups with regard to food intake (Figure 1(c)). The blood glucose level of the DM+Exc group was significantly higher than that of the other groups. Exendin-4 treatment could restore this effect to some extent with no significance (Figure 1(d)). To explore the effect of exendin-4 on lipid metabolism, we measured the contents of TG, LDL, and HDL. DM-Con mice and DM+Exc mice showed great higher levels of TG and LDL, which could be reversed by exendin-4 treatment (Figures 1(d)–1(g)). But there was no significant effect on the HDL level.

The expressions of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupled protein-1 (UCP-1), cluster of differentiation 36 (CD36), and adipose triglyceride lipase (ATGL) in BAT of DM mice were all downregulated. Exendin-4 treatment could upregulate ATGL and CD36 significantly and nonsignificantly increased the expression of UCP-1 and PGC-1α (Figure 1(h)).

3.2. Exendin-4 Ameliorated the Progress of Diabetic Nephropathy and Activated Renal AMPK Pathway

Exendin-4 decreased urinary albumin excretion and the urinary 8-OH-dG level (Figures 2(a)–2(b)). Diabetic mice also showed obvious glomerular mesangial expansion; the PAS and Masson staining indicated that the increase in DM-Con mice and DM+Exc mice was suppressed by exendin-4 (Figure 2(c)). We also detected the mRNA abundance of renal TGF-β1, Col-1, and FN to explore the renal fibrosis in mice, a major pathological change in DKD. The mRNA of TGF-β1, Col-1, and FN (Figures 2(d)–2(f)) and the protein of TGF-β1 (Figures 2(g) and 2(h)) were significantly increased in DM-Con mice and DM+Exc mice. And exendin-4 completely reduced these increases. These results demonstrated that BAT excision could aggravate the damage of renal function, and exendin-4 treatment could improve this damage.

AMPK regulates a series of physiological events, including mitochondrial function and cellular growth [18, 19]. Our previous study found that exendin-4 could alleviate rat mesangial cell proliferation induced by high glucose through decrease of the phosphorylation of extracellular regulated protein kinases (ERK) and the expression of rapamycin (mTOR) via AMPK activation [20]. In this study, we found that renal AMPK phosphorylation was significantly decreased in diabetic mice, which could be improved by exendin-4 treatment (Figures 2(i) and 2(j)).

3.3. Exendin-4 Has No Significant Effect on Adipocytes In Vitro

We further analyzed the effect of exendin-4 on white and brown adipogenesis in vitro. The expressions of UCP-1, PGC-1α, Cell Death-Inducing DFFA-Like Effector A (CIDEA), peroxisome proliferator-activated receptor- (PPAR-) γ, CCAAT Enhancer Binding Protein alpha (CEBPα), CEBPβ, CD36, cyclooxygenase (COX) 2, cytochrome (Cytoc) 1, and ATGL were determined during the differentiation process of 3T3-L1 and brown preadipocytes. The results showed that only PPAR-γ was significantly downregulated by exendin-4 in mature 3T3-L1 cells (Figure 3(a)) and that there was no effect in brown mature adipocytes (Figure 3(b)). During the differentiation, only CD36 was upregulated by exendin-4 at day 3 in WT-1 cells (Figures 3(c) and 3(d)), and there was no significant effect on the differentiation of 3T3-L1 (Figures 3(e) and 3(f)). Thus, exendin-4 did not exert obvious effect on brown adipogenesis and activity in vitro.

3.4. Exendin-4 Improved the Myofibroblast-Like Phenotype Transition in MCs Induced by Oleate

The cell activity measured by MTT metabolism was decreased by 13.8 ± 0.8% in the oleate group at 48 hours compared to the BSA group (Figure 4(a)). Oil Red O staining revealed that exposure to oleate greatly increased the size and quantity of neutral lipid droplets in the cytosol, while the body of cells stimulated with palmitate shrank and became rounded with pyknotic nuclei compared to that of the control (Figure 4(c)). Both mRNA (Figure 4(b)) and protein (Figure 4(d)–4(h)) expressions of TGF-β1, FN, α-SMA, and Col-1 were significantly increased in the oleate group compared with the BSA control, while this increase is suppressed by treatment with 10 nM and 20 nM of exendin-4. There were no significant changes observed in the cell viability between groups (Figure 4(i)).

3.5. AMPK Pathway Involved in the Effects of Exendin-4 on Myofibroblast-Like Phenotype Transition and Lipid Accumulation in MCs

The phosphorylation of AMPK and ACC was inhibited in MCs cultured with oleate, while 20 nM of exendin-4 significantly restored their phosphorylation (Figures 5(a) and 5(b)). The effect of exendin-4 was reversed by compound C. On the contrary, AICAR had a similar effect as exendin-4. AICAR and exendin-4 significantly inhibited oleate-induced protein expression of fibrogenic markers (Figures 5(c)–5(g)), which can be reversed by compound C. Moreover, exendin-4, as well as AICAR, significantly decreased the TGF-β1 secretion induced by oleate in cell culture supernatants, which was also reversed by compound C (Figure 5(h)).

Oleate greatly increased the lipid accumulation in MCs which can be decreased by exendin-4 (Figure 6(a)). A glyceridase assay was used to accurately reflect TG changes in MCs. The result showed that exendin-4 inhibited oleate-induced TG accumulation significantly (Figure 6(b)), which can be attenuated by compound C. The expressions of SREBP-1 and FAS were inhibited by exendin-4 and AICAR (Figures 6(c) and 6(d)), which were reversed by compound C. Oleate downregulated the expressions of PPAR-α and CPT-1, which were reversed by exendin-4 (Figures 6(e) and 6(f)).