Tacrolimus and sirolimus have distinct effects on insulin signaling in male and female rats

Abstract

Although the contribution of the immunosuppressants tacrolimus (TAC) and sirolimus (SIR) to the development of posttransplant diabetes mellitus (PTDM) are being increasingly recognized, the mechanisms of immunosuppressant-induced hyperglycemia are unclear. SIR induces insulin resistance predominantly, but is associated with β-cell dysfunction in rodents. TAC affects islet function but is associated with worsening insulin sensitivity in a few, and improvement in some, clinical studies. We sought to clarify the contributions of TAC and SIR to insulin resistance and islet function. Four groups of male and female Sprague-Dawley rats received TAC, SIR, TAC and SIR, or control for 2 weeks. All rats were administered an oral glucose challenge at the end of treatment. Half the groups were sacrificed 10 minutes after administration of regular insulin whereas the other half did not receive insulin before sacrifice. Liver, pancreas, fat, and muscle were harvested subsequently. Quantification of Western blots revealed that SIR and TAC plus SIR suppressed the phospho-Akt (pAkt)-to-Akt ratios in liver, muscle, and fat compared with control, regardless of sex. TAC alone did not impair the pAkt-to-Akt ratios in any of the tissues in male and female rats. β-Cell mass was reduced significantly after TAC treatment in male rats. SIR did not affect β-cell mass, regardless of sex. Our study demonstrated very clearly that SIR impairs insulin signaling, without any effect on β-cell mass, and TAC does not impair insulin signaling but reduces β-cell mass. Our efforts are key to understanding the mechanisms of immunosuppressant-induced hyperglycemia and to tailoring treatments for PTDM.

Fig 1.

Random blood glucose over time. (A) Daily glucose concentration for the groups treated for 2 weeks are shown. Tacrolimus (TAC), sirolimus (SIR), and TAC and SIR increased random blood glucose levels over time in male rats. (B) TAC plus SIR increased random blood glucose levels over time in female rats. Data are presented as mean ± standard error of the mean, with n = 12 per group. *Significantly different from CTRL, P < 0.05. ^α Significantly different from CTRL, P < 0.01. ^β Significantly different from CTRL, P < 0.001. ^μ Significantly different from CTRL, P < 0.001.

Fig 2.

The pAkt-to-Akt ratios in livers of male rats. Western blots of pAkt, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in livers of male rats before and after insulin stimulation are shown. The graphs depict the ratios of pAkt to Akt after quantification of respective bands and normalization to the control (CTRL; insulin negative). White bars, pAkt-to-Akt ratios before insulin stimulation (Insulin negative); black bars, pAkt-to-Akt ratios after insulin stimulation (Insulin positive). Data are presented as mean ± standard error of the mean, with n = 6 per group. ^aCTRL− vs CTRL+, P < 0.05. ^bCTRL+ vs SIR+, P < 0.01. ^cCTRL + vs TAC− and SIR+, P < 0.01. ^dTAC− vs TAC+, P < 0.01. SIR, sirolimus; TAC, tacrolimus.

Fig 3.

pAkt-to-Akt ratios in livers of female rats. Western blots of pAkt, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in livers of female rats before and after insulin stimulation are shown. The graphs depict the ratios of pAkt to Akt after quantification of respective bands and normalization to the control (CTRL; Insulin negative). White bars, pAkt-to-Akt ratios before insulin stimulation (Insulin negative); black bars, pAkt-to-Akt ratios after insulin stimulation (Insulin positive). Data are presented as mean ± standard error of the mean, with n = 6 per group. ^aCTRL− vs CTRL+, P < 0.001. ^bCTRL+ vs SIR+, P < 0.01. ^cCTRL+ vs TAC+, P < 0.05. ^dCTRL+ vs TAC− and SIR+, P < 0.001. ^eTAC− vs TAC+, P < 0.05. SIR, sirolimus; TAC, tacrolimus.

Fig 4.

pAkt-to-Akt ratios in fat of male rats. Western blots of pAkt, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in fat of male rats before and after insulin stimulation are shown. The graphs depict the ratios of pAkt to Akt after quantification of respective bands and normalization to control (CTRL; Insulin negative). White bars, pAkt-to-Akt ratios before insulin stimulation (Insulin negative); black bars, pAkt-to-Akt ratios after insulin stimulation (Insulin positive). Data are presented as mean ± standard error of the mean, with n = 6 per group. ^aCTRL− vs CTRL+, P < 0.01. ^bCTRL+ vs SIR+, P < 0.01. ^cCTRL+ vs TAC− and SIR+, P < 0.01. ^dTAC− vs TAC+, P < 0.01. SIR, sirolimus; TAC, tacrolimus.

Fig 5.

pAkt-to-Akt ratios in fat of female rats. Western blots of pAkt, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in fat of female rats before and after insulin stimulation are shown. The graphs depict the ratios of pAkt to Akt after quantification of respective bands and normalization to control (CTRL; Insulin negative). White bars, pAkt-to-Akt ratios before insulin stimulation (Insulin negative); black bars, pAkt-to-Akt ratios after insulin stimulation (Insulin positive). Data are presented as mean ± standard error of the mean, with n = 6 per group. ^aCTRL− vs CTRL+, P < 0.05. ^bTAC− vs TAC+, P < 0.01. SIR, sirolimus; TAC, tacrolimus.

Fig 6.

pAkt-to-Akt ratios in muscle of male rats. Western blots of pAkt, Akt, and in muscle of male rats before and after insulin stimulation are shown. The graphs depict the ratios of pAkt to Akt after quantification of respective bands and normalization to control (CTRL; Insulin negative). White bars, pAkt-to-Akt ratios before insulin stimulation (Insulin negative); black bars, pAkt-to-Akt ratios after insulin stimulation (Insulin positive). Data are presented as mean ± standard error of the mean, with n = 6 per group. ^aCTRL− vs CTRL+, P < 0.001. ^bCTRL+ vs SIR+, P < 0.001. ^cCTRL+ vs TAC+, P < 0.05. ^dCTRL+ vs TACSIR+, P<0.001. ^eTAC− vs TAC+, P < 0.05. SIR, sirolimus; TAC, tacrolimus.

Fig 7.

pAkt-to-Akt ratios in muscle of female rats. Western blots of pAkt, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in muscle of female rats before and after insulin stimulation are shown. The graphs depict the ratios of pAkt to Akt after quantification of respective bands and normalization to control (CTRL; Insulin negative). White bars, pAkt-to-Akt ratios before insulin stimulation (Insulin negative); black bars, pAkt-to-Akt ratios after insulin stimulation (Insulin positive). Data are presented as mean ± standard error of the mean, with n = 6 per group. ^aCTRL− vs CTRL+, P < 0.01. ^bCTRL+ vs SIR+, P < 0.05. ^cCTRL+ vs TACSIR+, P < 0.01. SIR, sirolimus; TAC, tacrolimus.

Fig 8.

(A, B) β-Cell (A) and α-cell (B) mass in male rats. Tacrolimus (TAC) and TAC and sirolimus (SIR) reduced mass significantly. α-Cell mass was not affected by any of the treatments. Data are presented as mean ± standard error of the mean, with n = 12 per group. *P < 0.05 vs control (CTRL).

Fig 9.

Representative pictures of insulin staining in male rats of various groups. SIR, sirolimus; TAC, tacrolimus.

Fig 10.

Representative pictures of glucagon staining in male rats of various groups. SIR, sirolimus; TAC, tacrolimus.

Fig 11.

(A, B) β-Cell (A) and α-cell (B) mass in female rats. Tacrolimus (TAC) plus sirolimus (SIR) reduced mass significantly. SIR increased the α-cell mass and was not affected by TAC or TAC plus SIR. Data are presented as mean ± standard error of the mean, with n = 12 per group. *P < 0.05 vs control (CTRL).

Fig 12.

Representative pictures of insulin and glucagon staining in female rats. Brown staining represents insulin and red staining represents glucagon. SIR, sirolimus; TAC, tacrolimus.

Fig 13.

Liver glycogen content in male and female rats. Glycogen content was not different among any of the treatment groups in male (A) or female (B) rats. Data are presented as mean ± standard error of the mean, with n = 6 per group.