Effects of metformin on hyperglycemia in an experimental model of tacrolimus- and sirolimus-induced diabetic rats

Abstract

Background/aims: Metformin (MET) is a first-line drug for type 2 diabetes mellitus (DM); its effect on new-onset diabetes after transplantation caused by immunosuppressant therapy is unclear. We compared the effects of MET on DM caused by tacrolimus (TAC) or sirolimus (SRL).

Methods: DM was induced by injection of TAC (1.5 mg/kg) or SRL (0.3 mg/kg) for 2 weeks in rats, and MET (200 mg/kg) was injected for 2 more weeks. The effects of MET on DM caused by TAC or SRL were evaluated using an intraperitoneal glucose tolerance test (IPGTT) and by measuring plasma insulin concentration, islet size, and glucose-stimulated insulin secretion (GSIS). The effects of MET on the expression of adenosine monophosphate-activated protein kinase (AMPK), a pharmacological target of MET, were compared between TAC- and SRL-treated islets.

Results: IPGTT showed that both TAC and SRL induced hyperglycemia and reduced plasma insulin concentration compared with vehicle. These changes were reversed by addition of MET to SRL but not to TAC. Pancreatic islet cell size was decreased by TAC but not by SRL, but addition of MET did not affect pancreatic islet cell size in either group. MET significantly increased GSIS in SRL- but not in TAC-treated rats. AMPK expression was not affected by TAC but was significantly decreased in SRL-treated islets. Addition of MET restored AMPK expression in SRL-treated islets but not in TAC-treated islets.

Conclusions: MET has different effects on hyperglycemia caused by TAC and SRL. The discrepancy between these drugs is related to their different mechanisms causing DM.

Keywords: Metformin; New onset diabetes after transplantation; Sirolimus; Tacrolimus.

Figure 1.

Experimental design for this study. After a low-salt diet for 1 week, animals were divided into five groups of nine rats each and were treated with vehicle group (VH), tacrolimus (TAC), sirolimus (SRL), or TAC or SRL plus metformin (MET). S.C., subcutaneous; P.O., oral gavage.

Figure 2.

Schematic workflow for the isolation of islets from rats and the glucose-stimulated insulin secretion test. Rat pancreatic islets were isolated from rats by digesting the pancreatic duct with collagenase P. After digestion, the islets were separated on Histopaque 1077 (Sigma). Islets were picked up with a glass loop under a dissecting microscope. Islets were preincubated in conditioning media for 1 day. On the next day, islets were initially exposed to tacrolimus (TAC; 30 ng/mL) or sirolimus (SRL; 90 ng/mL) for 12 hours, followed by addition of metformin (165 ng/mL) for a further 12 hours. At the end of the incubation period, islets were incubated for 30 minutes in 25 mM glucose, and the supernatant fluid was sampled and insulin concentration measured.

Figure 3.

Effects of metformin (MET) on tacrolimus (TAC)- or sirolimus (SRL)-induced pancreatic islet dysfunction. (A) Intraperitoneal glucose tolerance test. (B) Area under the curve for glucose (AUCg). (C) Plasma insulin concentration. Note that the glucose and insulin concentrations were restored in the SRL + MET group compared with the SRL group but not in the TAC and TAC + MET groups. The results are expressed as the mean ± SE of nine individual animals. VH, vehicle group. ^ap < 0.05 vs. VH, ^bp < 0.05 vs. SRL.

Figure 4.

Effects of metformin (MET) on pancreatic islet size in tacrolimus (TAC)- or sirolimus (SRL)-induced pancreatic islet injury. (A) Immunostaining for insulin showing pancreatic islet morphology and size in the experimental group. Note that irregular islet boundaries and vacuolization (arrowheads) in the TAC and SRL groups. (B) Quantitative analysis of insulin-positive areas in islets. Note the significantly reduced islet size in the TAC and TAC + MET groups but not in the SRL and SRL + MET groups. The results are expressed as the mean ± SE of nine individual animals. VH, vehicle group. ^ap < 0.05 vs. VH.

Figure 5.

Direct effects of metformin (MET) on insulin secretion by tacrolimus (TAC) or sirolimus (SRL) assessed by the glucose-stimulated insulin secretion (GSIS) test. Insulin release in response to glucose was significantly lower in the TAC and SRL groups compared with the vehicle group. Note that the GSIS was significantly increased by MET combined with SRL but not by TAC compared with the corresponding non-MET-treated group. The results are expressed as the mean ± SE of at least three independent experiments. ^ap < 0.05 vs. VH, ^bp < 0.05 vs. SRL.

Figure 6.

Effects of metformin (MET) on the expression of adenosine monophosphate-activated protein kinase (AMPK) during treatment with tacrolimus (TAC) or sirolimus (SRL). Representative immunoblot of phosphorylated (p-AMPK) and total AMPK (t-AMPK) in primary cultured rat islets treated with TAC or SRL with or without MET. The results of quantitative analysis are expressed as a ratio of p-AMPK to t-AMPK. The density in the vehicle group (VH) was assigned a relative value of 100%. The results are expressed as the mean ± SE of at least three independent experiments. ^ap < 0.05 vs. VH, TAC, and TAC + MET, ^bp < 0.05 vs. SRL.