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.

The number of solid-organ transplants being performed is increasing as a result of longer graft and host survival, and improved immunosuppression strategies. However, an important side effect of 2 commonly used immunosuppressants—tacrolimus (TAC) and sirolimus (SIR)—is new-onset diabetes, also known as posttransplant diabetes mellitus (PTDM)^1-7 PTDM has varying incidence rates based on the population studied, criteria for diagnosis of PTDM, organ transplanted, and the prevailing immunosuppression strategy of the time.^8-14 Despite the abundance of data on the incidence and consequence of PTDM, the pathophysiology and appropriate treatment options have not been well studied. Currently, it is thought that insulin deficiency and insulin resistance play significant roles in the development of PTDM.¹⁵ The majority of available data suggests that TAC, a calcineurin inhibitor, affects islet function predominantly.^6,16-19 However, different clinical studies have suggested variably that TAC increases, decreases, or has no effect on insulin sensitivity.^20-25 SIR, an mTOR inhibitor, has been shown to impair insulin signaling, and this is likely a predominant mechanism contributing to hyperglycemia,^2,26,27 but rodent studies have also suggested that SIR impacts β-cell mass and function.^16,28 Therefore, there is a lack of consensus on the underlying mechanisms for PTDM caused by TAC and SIR. Understanding the mechanisms of immunosuppressant-induced hyperglycemia will be key to formulating appropriate treatments for and/or prevention of PTDM.

We have demonstrated in previous studies that treatment of normal male and female rats for 2 weeks with TAC and SIR, individually and in combination, induces hyperglycemia.^6,19 SIR also induced hyperinsulinemia, suggestive of insulin resistance.^6,19 However, because TAC, and TAC and SIR resulted in impaired islet function, it is unclear whether TAC also contributed to insulin resistance. In light of the contradictory evidence in the literature, we sought to define the contributions of TAC and SIR to liver, muscle, and adipose insulin resistance and islet function. There is no current literature to support that there is a difference in incidence between males and females in the incidence of PTDM. Because there was no data available, we wanted to explore any possible sex differences in immunosuppressant-induced hyperglycemia. We hereby demonstrate that SIR impairs insulin signaling, which contributes to both hyperglycemia and hyperinsulinemia, whereas TAC contributes to hyperglycemia by impairing islet function. Our results also show that β-cell mass is reduced by TAC and not SIR. These results clarify the understanding of the mechanisms of immunosuppressant-induced hyperglycemia and are imperative to modify treatments for PTDM based on their immunosuppressant regimen.

METHODS

Animal care.

Adult (age, 42–45 days), male Sprague-Dawley rats were kept under 12-hour light and dark conditions and were fed standard lab chow. Food, but not water, was removed for 12 hours to conduct the oral glucose challenge test, as described later. This study was carried out in strict accordance with the recommendations for care and use of laboratory animals by the National Institutes of Health. The protocol was approved by Institutional Animal Care and Use Committee of the VA-Nebraska-Western Iowa Health Care System. All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.²⁹

Experimental design.

Experiments on male and female rats were done separately. A total of 96 rats were studied: males, n = 48 rats, 12 rats per group × groups; females, n = 48 rats, 12 rats per group × 4 groups. Animals (n = 12 rats per group) were given 1 of 4 daily treatment regimens that consisted of 2 subcutaneous injections. One group received a daily injection of TAC (2 mg/kg/d; LC Laboratories, Woburn, Mass) in diluent (10% ethanol in sunflower oil) subcutaneously for 2 weeks. A second group received daily injections of SIR (0.8 mg/kg/d; LC Laboratories) in diluent subcutaneously for 2 weeks. The third group received a daily subcutaneous injection of TAC (2 mg/kg/d) and SIR (0.8 mg/kg/d) for 2 weeks. Last, the fourth group received 2 injections of diluent daily and served as the control group. These drug doses were chosen based on our previously published studies.¹⁹ Weights were obtained daily, and nonfasting blood glucose concentrations were measured by glucometer (FreeStyle Freedom Lite; Abbot, Alameda, Calif) every other day.

After 2 weeks of treatment, an oral glucose challenge was performed on each group (1.5 g/kg by gavage) before sacrifice. To obtain fasting samples, food was removed from the animals 12 hours before the oral glucose challenge. Baseline glucose and insulin were measured from blood taken from the tail vein before administration of the glucose load. Blood was removed from the tail vein 15, 30, 60, and 120 minutes after glucose challenge to measure glucose and insulin concentrations. The next day, groups of rats were sacrificed under anesthesia. Just before sacrifice, half of the rats in each group were injected with regular insulin (0.5 U/kg; Humulin R; Eli Lilly, Indianapolis, Ind) intraperitoneally 10 minutes before tissues were harvested. Cardiac blood, pancreas, liver, soleus muscle, and epididymal fat were harvested.

Insulin concentrations.

Plasma insulin concentration was measured using a sensitive radioimmunoassay (Millipore Research, St. Charles, Mo; limit of sensitivity, 0.02 ng/mL)(http://www.millipore.com/catalogue/item/ezrmi-13k). TAC and SIR levels were measured by liquid chromatography-tandem mass spectrometry on blood samples taken 24 hours after the last subcutaneous injections at the Clinical Laboratory of The Nebraska Medical Center.³⁰ Intra-assay variations were 3.5% and 3.9%, and interassay variations were 2.7% and 5.0%, for TAC and SIR, respectively.

Pancreas pathology.

The pancreata were fixed in 10% formalin and set in paraffin blocks. Sections were cut at the Tissue Sciences Facility at the University of Nebraska Medical Center and were stained for cell apoptosis using the terminal deoxynucleotidyl transferase-mediated 2′-Deoxyuridine, 5′-Triphosphate dUTP-biotin nick end labeling of DNA fragmentation (TUNEL) method (DeadEnd Colorimetric TUNEL System; Promega, Madison, Wisc). Each pancreatic section was photographed, and islets were analyzed at ×20 magnification. Islets were evaluated for apoptosis; mean apoptotic cells per islet were compared among groups. Exocrine cell apoptosis was quantified by counting TUNEL-+ cells in 10 representative, randomized fields under ×10 magnification. Mean number of apoptotic cells per field were compared among groups.

Light microscopy was used to study TUNEL-stained sections for the following measurements: the number of islets of Langerhans, mean islet area, and islet cell and exocrine cell apoptosis. Image J software was used to quantify mean islet area. These data are represented as the integrated density.³¹ The analysts were blinded to the treatment groups before evaluation. Area values were calculated in square pixels and converted to square micrometers.

Immunohistochemistry.

Additional slides were stained for insulin and glucagon by immunohistochemistry by the Tissue Sciences Facility at the University of Nebraska Medical Center using guinea pig anti-insulin antibody (Dako Cytomation, Carpentaria, Calif) and rabbit antiglucagon antibody (Cell Signaling Technology, Danvers, Mass). Stained slides were visualized using light microscopy and were photographed at ×10 magnification. Image J software was used to quantify insulin and glucagon content, and represented the integrated density.³¹ Average integrated density values were calculated in square pixels and converted to square micrometers. β-Cell mass was expressed as the percentage of insulin-positive cells contained in the total surveyed area, and α-cell mass as the percentage of glucagon-positive cells contained in the total surveyed area.³²

Western blot analysis.

Liver, muscle, and fat cell lysates were analyzed using the standard Western blotting procedure³³ for the relative quantities of Akt and phospho-Akt. Western blot cell lysates were fractionated by sodium dodecylsulfate polyacrylamide gel electrophoresis, and membranes were probed at 4°C overnight with the antibodies mouse anti-Akt (pan, 40D4, 1:1000 dilution, Cell Signaling Technology) and rabbit anti-pAkt (Ser473, 1:2000 dilution, Cell Signaling Technology). Membranes were also probed with antiglyceraldehyde-3-phosphate dehydrogenase (1:5000 dilution, Millipore Research), which served as a lane loading control. Blots were visualized on an Odyssey infrared imaging system. Protein levels were then quantified by densitometry using Odyssey imaging software.

Glycogen assay.

Whole tissues were not available for measurement of glycogen content from the original set of experiments. We performed glycogen analysis in tissues from another experiment with identical conditions pertaining to TAC and SIR treatment, but with the sole difference of insulin stimulation 10 minutes before sacrifice. The hepatic glycogen content was determined using the method of Roehrig and Allred.³⁴ Briefly, glycogen in liver homogenates was converted to glucose using amyloglucosidase, followed by measurement of glucose using a glucose oxidase assay (Sigma Chemical, St. Louis, Mo). Protein content in the homogenates was determined using the bicinchoninic acid assay (Thermo Scientific, Rockford, Ill).

Statistical analysis.

The change in weight among groups over time was compared by 2-way analysis of variance (ANOVA), with Bonferroni posttests used for analysis. Mean random blood glucose concentrations were compared by 2-way ANOVA with Bonferroni test post hoc differences. Glucose and insulin concentration responses to oral glucose load were calculated as area under the curve and were compared by 1-way ANOVA with Tukey’s multiple comparison tests for post hoc differences. One-way ANOVA was used to compare islet cell apoptosis, islet area, liver glycogen content, and insulin and glucagon integrated density among treatment groups. The differences in insulin-stimulated pAkt-to-Akt ratios among groups were compared using 2-way ANOVA with Bonferroni posttests. Similar testing was used to analyze data from male and female rats, separately. Parametric tests were done after verification of normality. When the normality assumptions were not met, Kruskal-Wallis tests with pair-wise Mann Whitney U tests and a manual Bonferroni correction were applied. All data are represented as mean ± standard error of the mean unless otherwise specified. P values less than 0.05 were considered significant. GraphPad Prism software was used for analyses.

RESULTS

Treatment with TAC plus SIR increased random glucose levels in male and female rats compared with controls (Fig 1; P < 0.05). Treatment with TAC and SIR individually increased random glucose levels in male rats (Fig 1, A; P < 0.05) but not in female rats (Fig 1, B). Weight gains at the end of the treatment period compared with the baseline in various groups are as follows: control, 53 ± 1.5%; SIR, 23.1 ± 1.3%; TAC, 26.6 ± 1.3%; and TAC plus SIR, −7.4 ± 1.7%. TAC, SIR, and TAC and SIR treatment reduced weight gain compared with controls, with the most profound effect seen in the TAC plus SIR-treated group (P < 0.05). The following results are applicable to male and female rats, are consistent with our previously published data, and thus not shown.^6,19 Blood glucose levels were also elevated in the TAC-, SIR-, and TAC and SIR-treated groups after glucose challenge compared with control (P < 0.05). Treatment with SIR elevated the insulin concentration significantly in response to the glucose challenge compared with controls (P < 0.05). The TAC- and TAC and SIR-treated groups appeared to have decreased insulin concentrations compared with controls after glucose challenge, but this did not reach statistical significance.

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.

Insulin stimulation increased pAkt-to-Akt ratios significantly in liver, muscle, and fat of control rats and TAC rats (male and female) compared with the respective tissues from control and TAC rats (male and female) without insulin stimulation (P < 0.05; Figs 2-7). Insulin stimulation did not increase pAkt-to-Akt ratios significantly in the liver, muscle, and fat of SIR rats (male and female) compared with their noninsulin-stimulated counterparts (Figs 2-7). In addition, the insulin-stimulated pAkt-to-Akt ratios in liver, fat, and muscle of SIR-treated rats were significantly less than the control (insulin positive) group (P < 0.05; Figs 2-7). Insulin-stimulated pAkt-to-Akt ratios in the liver, muscle, and fat of the combination group (TAC and SIR) were not different from their noninsulin-stimulated counterparts, regardless of sex (Figs 2-7).

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 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.

In the male rats, the TAC, and TAC and SIR treatment decreased the β-cell mass compared with controls in male rats (P < 0.05; Fig 8, A). α-Cell mass was not decreased significantly in any of the groups (Fig 8, B). Representative pictures from each group are presented in Fig 9 (insulin staining) and Fig 10 (glucagon staining). Treatment with TAC and SIR combined also increased significantly the number of apoptotic endocrine and exocrine cells in the pancreas, consistent with our previously published data.⁶ The mean numbers of apoptotic cells per islet (±standard error of the mean) across the groups were as follows: control, 1.9 ± 0.8; SIR, 21.8 ± 2.5; TAC, 21.9 ± 3.1; and TAC and SIR, 46.7 ± 5.7. TAC, SIR, and TAC and SIR combined had significantly more apoptotic cells per islet compared with the control group (P < 0.05).

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.

In the female rats, the TAC and TAC and SIR combined treatment decreased the β-cell mass compared with the control, but only TAC and SIR combined reached statistical significance (P < 0.05; Fig 11, A). α-Cell mass was increased significantly in the SIR group compared with the control (P < 0.05; Fig 11, B). Representative pictures from each group are presented in Fig 12. Treatment with TAC and SIR combined also increased significantly the number of apoptotic endocrine and exocrine cells in the pancreas, consistent with our previously published data.⁶ The mean numbers of apoptotic cells per islet (±standard error of the mean) across the groups were as follows: control, 1.4 ± 1.1; SIR, 1.2 ± 0.7; TAC, 0.7 ± 0.5; and TAC and SIR combined, 8.7 ± 3. TAC and SIR combined had significantly more apoptotic cells per islet compared with the control (P < 0.05). The glycogen content in liver from TAC-treated male rats appeared to be lower than controls; however, none of the groups were significantly different from the control in male or female rats (Fig 13).

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.

DISCUSSION

TAC contributes to hyperglycemia after solid-organ transplantation.^1-7 Literature from in vivo and in vitro studies suggests that the predominant effect of TAC on hyperglycemia is its affect on insulin production and secretion.^6,16-19 After transplantation of syngeneic islets in the livers of C57BL/6 diabetic mice, 4 weeks of TAC treatment induced hyperglycemia and reduced the β-cell area and proliferation.¹⁶ We have shown that 2-week treatment of normal Sprague-Dawley rats with TAC induced hyperglycemia without inducing hyperinsulinemia in response to oral glucose challenge, and reduced islet area compared with control rats.^6,19 TAC increased β-cell apoptosis in isolated human islets and reduced β-cell proliferation in rodents.¹⁷ TAC impairs insulin secretion and transcriptional regulation in INS-1E β cells.²¹

TAC has also been associated variably with insulin resistance. One study linked TAC to insulin resistance via enhanced lipolysis, inhibition of lipid storage, and expression of lipogenic genes in human adipose tissue.²⁰ Another study demonstrated that 8–10 days of treatment with TAC impairs insulin sensitivity in patients undergoing hemodialysis.²¹ Crucial limitations of that study included the studied population had endstage renal disease and not kidney transplant, no control group was available, and a very small number of patients was studied.²¹ Another clinical study suggested that tissue insulin sensitivity estimated by the euglycemic hyperinsulinemic clamp technique did not change significantly after conversion from cyclosporine to TAC therapy in patients after kidney transplantation.²² A separate clinical study of type 1 diabetes mellitus kidney/pancreas transplant recipients using an intravenous glucose tolerance test showed that TAC had no effect on glucose metabolism during the late posttransplant period (more than 8 years after transplant).²³ Interestingly, a clinical study of healthy volunteers showed that acute intravenous infusions of TAC increased insulin sensitivity.²⁴ A retrospective analysis of kidney transplant recipients in Spain demonstrated a high incidence of PTDM and metabolic syndrome among those treated with TAC as the main immunosuppressive agent.²⁵ Insulin resistance is thought to be the main driving force of metabolic syndrome in the nontransplant population.¹⁸ In summary, although there is strong evidence that TAC affects β-cell mass and function, the available evidence had been unclear about the effects of TAC on insulin resistance, given the conflicting results and the mixed population.

We sought to clarify the role of TAC in insulin resistance and islet function. TAC individually and in combination with SIR induced hyperglycemia in both male and female rats. TAC individually and in combination with SIR reduced β-cell mass. In response to an oral glucose challenge, insulin secretion did not increase in the TAC-treated rats, confirming impairment of islet function. Although there was a significant difference between control positive and TAC+ in the liver of female rats (Fig 3) and the muscle of male rats (Fig 6), the insulin-stimulated pAkt-to-Akt ratios in these groups were significantly higher than the noninsulin-stimulated pAkt-to-Akt ratios in the respective groups. Hence, TAC does not impair insulin-induced Akt phosphorylation in any of the tissues studied. Glycogen content in liver tissue was not different among various treatment groups in male or female rats, likely because of the short-term nature of the treatment in our model as well as the insulin stimulation 10 minutes before sacrifice. In summary, short-term treatment with TAC in normal rats induced hyperglycemia. The predominant cause of TAC-induced hyperglycemia appeared to be a result of its effect on β cells and not on insulin sensitivity.

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 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.

SIR has been thought to be an independent risk factor for PTDM,⁵ and has been associated with hypertriglyceridemia in clinical studies, which is one of the predominant features of insulin resistance.^18,35,36 We have shown that SIR induces hyperglycemia and hyperinsulinemia in normal rats, suggestive of insulin resistance.^6,19 Recent studies have elucidated further the mechanisms of SIR-induced insulin resistance. Ye et al² demonstrated that mTORC2 disruption, rather than inhibition of mitochondria (mTORC1 disruption), causes insulin resistance in SIR-treated myotubes. They demonstrated that SIR has a biphasic effect on insulin sensitivity in C2C12 myotubes as a result of sequential disruption of mTORC1 and mTORC2.² Blattler²⁶ et al showed that chronic SIR treatment in mice led to insulin resistance with suppression of insulin/IGF signaling and genes associated within this pathway, such as IGF12, IRS1-2, and Akt1-3. In addition, skeletal muscle-specific YY1 knockout mice were protected from SIR-induced diabeticlike symptoms.²⁶ Deblon et al²⁷ demonstrated that SIR-treated rats were glucose intolerant, hyperinsulinemic, and hyperglycemic, but not hyperlipidemic. Euglycemic hyperinsulinemic clamp measurements showed skeletal muscle to be a major site of SIR-induced insulin resistance.²⁷ Long-term SIR administration attenuated glucose uptake and metabolism in skeletal muscle by preventing full insulin-induced Akt activation, and altering the expression and translocation of glucose transporters to the plasma membrane.²⁷ Rodent studies have also suggested that SIR impacts β-cell mass and function.^16,28 Four weeks of SIR treatment induced hyperglycemia and reduced the β-cell area and proliferation after transplantation of syngeneic islets in livers of C57BL/6 diabetic mice.¹⁶ SIR treatment of MIN6 cells and isolated rat and human islets resulted in a loss of cell function and viability.²⁸

Our data demonstrated that SIR suppressed insulin-stimulated Akt phosphorylation in liver, fat, and muscle. In the rats treated with TAC and SIR combined, the insulin signaling pathway appeared to be impaired in all the insulin-sensitive tissues of male and female rats, because insulin-stimulated Akt phosphorylation was impaired. SIR did not affect liver glycogen content in male or female rats.

The limitations of our study include the short-term nature of the treatment, and that our model is not a transplant model. Nonetheless, our model allows us to study very clearly the mechanisms of TAC- and SIR-induced hyperglycemia.

In conclusion, TAC and SIR induce hyperglycemia in normal rats (male and female) after short-term treatment. The predominant mechanism of TAC-induced hyperglycemia is by reducing β-cell mass and thus insulin production. Contrary to some studies, TAC does not appear to affect insulin sensitivity. SIR impaired insulin signaling in liver, fat, and muscle, consistent with previous studies. Mechanisms of TAC- and SIR-induced hyperglycemia do not appear to be different between male and female rats. The combination of TAC and SIR, a regimen frequently used in solid-organ and islet transplantation, affected insulin signaling and islet mass, and induced pronounced endocrine and exocrine apoptosis.

Speculations.

These data give us better insight for tailoring treatment strategies for PTDM based on their immunosuppression regimen (ie, targeting insulin resistance or insulin deficiency or both in a given patient). Antihyperglycemic therapies aimed at β-cell preservation could be used in patients with a TAC-based immunosuppression regimen, whereas those that minimize insulin resistance could be of importance in SIR-based immunosuppression. Our published data using metformin suggested that SIR-induced hyperglycemia may be minimized using metformin.⁶ Pioglitazone did not appear to impact any of the glycemic parameters in our model, although the lack of effect may have been primarily a result of the short-term nature of treatment with pioglitazone (2 weeks; unpubl. obs.). Linagliptin appears to improve hyperglycemia induced by TAC in our model (unpubl. obs.). Longer treatment periods are necessary to tease out these issues.

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.

AT A GLANCE COMMENTARY.

Shivaswamy V, et al.

Background

We have shown previously that commonly used immunosuppressants—tacrolimus and sirolimus—induce hyperglycemia in normal rats. Clinical studies have shown that tacrolimus and sirolimus contribute to new onset of diabetes after solid-organ transplantation, which reduces graft and host survival. The mechanisms of tacrolimus- and Sirolimus-induced hyperglycemia are unclear.

Translational Significance

We demonstrate clearly that tacrolimus reduces β-cell mass and sirolimus impairs insulin signaling in male and female rats. These results have a significant impact on treatment strategies for new onset of diabetes after transplant, which could potentially improve graft and host survival.

Abbreviations:

PTDM

posttransplant diabetes mellitus

SIR

sirolimus

TAC

tacrolimus

TUNEL

terminal deoxynucleotidyl transferase-mediated 2′-Deoxyuridine, 5′-Triphosphate-biotin nick end labeling of DNA fragmentation