FXR activation alleviates tacrolimus-induced post-transplant diabetes mellitus by regulating renal gluconeogenesis and glucose uptake

Abstract

Background: Tacrolimus (FK506)-induced diabetes mellitus is one of the most important factors of post-transplant diabetes mellitus (PTDM). However, the detailed mechanisms underlying PTDM are still unclear. Farnesoid X receptor (FXR) regulates glycolipid metabolism. The objective of this study was to explore whether FXR is involved in the development of tacrolimus-induced diabetes mellitus.

Methods: After C57BL/6J mice were treated with tacrolimus (FK506) for 3 months, the fasting blood glucose levels, body weights, renal morphological alterations, and mRNA expression levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose transporter 2 (GLUT2) among the control group, the FK506 group and the FK506 + GW4064 (a FXR agonist) group (n = 7) were measured. The intracellular location of peroxisome proliferator activated receptor γ coactivator-1α (PGC1α) and forkhead box O1 (FOXO1) was detected by immunofluorescence. Human renal cortex proximal tubule epithelial cells (HK-2) were treated with 15 μM FK506 or 4 μM FXR agonist (GW4064) for 24, 48 and 72 h, and the expression levels of FXR, gluconeogenesis and glucose uptake, representing the enzymes PEPCK and GLUT2, were detected with real-time PCR and western blot analyses. Finally, the mRNA levels of PEPCK and GLUT2 in HK-2 cells were measured after FXR was upregulated.

Results: FK506 significantly inhibited the mRNA and protein levels of FXR at 48 h and 72 h in HK-2 cells (P < 0.05). Meanwhile, FK506 promoted gluconeogenesis and inhibited glucose uptake in HK-2 cells (P < 0.05). However, overexpression of FXR in transfected HK-2 cell lines significantly inhibited gluconeogenesis and promoted glucose uptake (P < 0.05). The FXR agonist GW4064 significantly decreased the fasting blood glucose in mice challenged with FK506 for 3 months (P < 0.05), inhibited gluconeogenesis (P < 0.05) and significantly promoted glucose uptake (P < 0.05). Immunofluorescence staining and western blot analyses further revealed that FXR activation may affect the translocation of PGC1α and FOXO1 from the nucleus to the cytoplasm.

Conclusions: FXR activation may mitigate tacrolimus-induced diabetes mellitus by regulating gluconeogenesis as well as glucose uptake of renal cortex proximal tubule epithelial cells in a PGC1α/FOXO1-dependent manner, which may be a potential therapeutic strategy for the prevention and treatment of PTDM.

Keywords: FXR; Glycometabolism; Kidney; Post-transplant diabetes mellitus; Tacrolimus.

Fig. 1

The FXR agonist GW4064 improved fasting blood glucose and inhibited the translocation of PGC1α and FOXO1 from the nucleus to the cytoplasm in gluconeogenesis with FK506 treatment in mice for 3 months. a After FK506 was administered at a dose of 1 mg/kg/day to the C57BL/6J mice for 3 months, the fasting blood glucose levels obviously increased gradually compared with those of the control group, and the FXR agonist GW4064 downregulated the blood glucose levels. No significant difference was found for b body weights and c morphological alterations among the control group, the FK506 group and the FK506 + GW4064 group. The C-1, C-2 and C-3 groups were the control group, the FK506 group and the FK506 + GW4064 group, respectively. d, f–h, j, k Western blotting and quantitative analysis show the protein subcellular localization of PGC1α and FOXO1. Original magnification, ×3400 in each group. e, i IF staining for PGC1α (e) and FOXO1 (i) in sections of 3 groups. DAPI was used to locate the nuclei of the cells. Data are presented as the mean ± SD (n = 7). *P < 0.05 vs. the control group, #P < 0.05 vs. the FK506 group

Fig. 2

The FXR agonist GW4064 inhibited gluconeogenesis and promoted glucose uptake after FK506 treatment in mice for 3 months. a–c Compared with that of the FK506 group, there was an obvious increase in FXR for the FK506 + GW4064 group at the mRNA and protein levels. d Like FXR, SHP1 was downregulated with FK506 treatment and was promoted with FK506 + GW4064 at the mRNA level. e After FK506 was administered at a dose of 1 mg/kg/day to the C57BL/6J mice for 3 months, the mRNA levels of PEPCK obviously increased at the end of the third month compared with those of the control group, and the FXR agonist GW4064 downregulated PEPCK at the mRNA level. f The uptake protein GLUT2 was significantly downregulated at the mRNA level by GW4064 under FK506 treatment. Data are presented as the mean ± SD (n = 7). *P < 0.05 vs. the control group, #P < 0.05 vs. the FK506 group

Fig. 3

The effects of FK506 on the mRNA and protein expression of FXR and related glycometabolic genes. a, b FK506 (15 µM) inhibited the protein expression of FXR at 24, 48 and 72 h in the HK-2 cell lines. The mRNA levels of FXR (c), SHP1 (d) and GLUT2 (f) were clearly downregulated by FK506 at the same time. e The mRNA level of PEPCK was obviously upregulated by FK506 treatment for 24, 48 and 72 h. Data are presented as the mean ± SD (n = 3 per group). *P < 0.05 vs. the control group

Fig. 4

FXR suppressed gluconeogenesis and enhanced glucose uptake in the HK-2 cell lines. a, b The protein expression of FXR was obviously upregulated by GW4064 at 24, 48 and 72 h in the HK-2 cell lines. GW4064 at 4 µM inhibited the mRNA level of FXR (c) and promoted the mRNA expression of SHP1 (d) and GLUT2 (f) at the same time. e The mRNA level of PEPCK was obviously inhibited by GW4064 treatment for 24, 48 and 72 h. g The mRNA expression of FXR was inhibited by si-FXR at 50 nM. The mRNA expression of SHP-1 (h) and GLUT2 (i) were obviously inhibited when FXR was knocked out and the PEPCK (j) mRNA expression level was promoted at the same time. Data are presented as the mean ± SD (n = 3 per group). *P < 0.05 vs. the control group

Fig. 5

FXR transfected in HK-2 cell lines suppressed gluconeogenesis and enhanced glucose uptake. a–c Western blot and real-time PCR analysis of FXR expression in transfected HK-2 cell lines. GAPDH was used as a loading control for the two analyses. The relative mRNA and protein expression of FXR was calculated by normalizing the FXR optical density against GAPDH. d Immunofluorescence analysis of FXR expression in the experimental cell lines. Strong cytoplasmic staining was observed with cells that were transfected with the pCMV-FXR but not the vector-transfected cells or normal HK-2 cells. The high expression of FXR could upregulate the mRNA expression of SHP1 (e) and GLUT2 (g) in HK-2 cells, but the mRNA levels of PEPCK were clearly downregulated at the same time (f). Data are presented as the mean ± SD (n = 3 per group). *P < 0.05 vs. the control group