Chronic rapamycin treatment causes diabetes in male mice

Abstract

Current evidence indicates that the mammalian target of rapamycin inhibitor rapamycin both increases longevity and, seemingly contradictorily, impairs glucose homeostasis. Most studies exploring the dimensions of this paradox have been based on rapamycin treatment in mice for up to 20 wk. We sought to better understand the metabolic effects of oral rapamycin over a substantially longer period of time in HET3 mice. We observed that treatment with rapamycin for 52 wk induced diabetes in male mice, characterized by hyperglycemia, significant urine glucose levels, and severe glucose and pyruvate intolerance. Glucose intolerance occurred in male mice by 4 wk on rapamycin and could be only partially reversed with cessation of rapamycin treatment. Female mice developed moderate glucose intolerance over 1 yr of rapamycin treatment, but not diabetes. The role of sex hormones in the differential development of diabetic symptoms in male and female mice was further explored. HET3 mice treated with rapamycin for 52 wk were gonadectomized and monitored over 10 wk. Castrated male mice remained glucose intolerant, while ovariectomized females developed significant glucose intolerance over the same time period. Subsequent replacement of 17β-estradiol (E₂) in ovariectomized females promoted a recovery of glucose tolerance over a 4-wk period, suggesting the protective role of E₂ against rapamycin-induced diabetes. These results indicate that 1) oral rapamycin treatment causes diabetes in male mice, 2) the diabetes is partially reversible with cessation of treatment, and 3) E₂ plays a protective role against the development of rapamycin-induced diabetes.

mammalian target of rapamycin (mTOR) is a nutrient-sensitive serine-threonine kinase involved in cellular processes ranging from metabolism and autophagy to cytoskeletal regulation and cell survival (22, 32). Rapamycin, commonly used as an immunosuppressant and chemotherapeutic drug (19, 25), has been found to induce longevity in Saccharomyces cerevisae, Drosophila melanogaster, and mice through its inhibitory effects on mTOR (2, 4, 12, 18). Although these are promising beginnings, more recent studies demonstrate significant adverse effects of rapamycin on metabolism, including insulin resistance and glucose intolerance (3, 7, 11, 13, 16, 21, 37, 38).

The seeming ability of rapamycin to extend lifespan, while at the same time compromising metabolic function in a manner expected to reduce longevity is recognized as the “rapamycin paradox” (14). This paradox highlights the gap in our understanding of its effects and the mechanisms by which they occur. Most in vivo rapamycin-based studies to date have used differing modes of administration, length of treatment, and species or strain of study organism, making difficult a direct comparison of variations between their results. Trials in humans indicate development of insulin resistance in patients treated with rapamycin following kidney transplants (16, 37, 38). While long-term studies administering rapamycin to mice report only increased longevity and no noticeable persisting side effects (2, 4, 12, 28), insulin resistance and glucose intolerance have been noted in mice and rats treated with 2–4 mg/kg rapamycin for 2–6 wk in multiple shorter-term studies (3, 7, 11, 13, 21, 28), with decreased pancreatic β-cell mass observed in two cases (11, 13). Most recently, Fang et al. (9) have reported that insulin insensitivity and glucose intolerance develop in mice by 6 wk of rapamycin administration, but independently resolve by the end of a 20-wk period (9). Furthermore, Lamming et al. (20) observed that mice treated for 3 wk with rapamycin have increased HbA1c levels, suggestive of increased blood glucose levels, while those treated for 3 mo do not display the same. Clearly, short-term treatment of mice with rapamycin influences circulating glucose homeostasis.

The picture above is further complicated by differences in the development of diabetes by sex, as noted in humans, mice, and rats. Substantial and growing evidence implicates the sex hormones testosterone (T) and 17β-estradiol (E₂) as key drivers of this difference (17, 27, 7a). Although the sum of evidence regarding T is equivocal (6, 10, 23, 30), multiple studies suggest that E₂ confers a protective effect against diabetes. Laboratory-based studies demonstrate that male mice are more prone to developing chemical- or high-fat diet-induced diabetes (30, 36) and that diabetic symptoms improve after administration of E₂ (30). Female mice and rats have been shown to develop insulin resistance or glucose intolerance following exogenous T administration (23, 30), ovariectomy (35, 36), or knockout/inhibition of E₂ receptors such as ERα (24, 35, 39, 40). In human-based studies, a decrease in E₂ levels in women, or polymorphisms in its receptor ERα in men have been associated with increased risk of diabetes onset (1, 26). In the context of rapamycin-induced metabolic impairment, Lamming et al. (20) showed that only female mice heterozygous for the mTOR complex components mTOR and GβL were long-lived with intact glucose tolerance (21). Additionally, Miller et al. (28) reported greater levels of glucose intolerance in male mice compared with females following 4 wk of oral rapamycin treatment (28). This demonstrates a need to better characterize potential sex-related differences in rapamycin-induced metabolic effects to comprehensively illustrate the consequences of rapamycin administration.

Therefore, we sought to more fully understand the metabolic effects of rapamycin on mice. We hypothesized that long-term oral administration of rapamycin would induce severe glucose intolerance and insulin resistance in mice. Contrary to the suggestions by Fang et al. (9), we found that rapamycin administration for 1 yr resulted in diabetes. We observed that this was true only in male mice, with females only becoming glucose intolerant. Additionally, we more closely characterized the onset of rapamycin-induced diabetes and consequences of rapamycin cessation. We observed progressive and consistent glucose intolerance over an 18-wk period in young mice started on a rapamycin-containing diet after weaning, and, furthermore, found glucose intolerance induced by rapamycin over a 1-yr period to be fully reversible in female mice and only partially reversible in males. Finally, we sought to further characterize the role of sex hormones in rapamycin-induced diabetes. Castration of rapamycin-induced diabetic male mice slowed the progression of glucose intolerance over an 11-wk period. Conversely, ovariectomy of females led to increased glucose intolerance over the same timeframe, which was reversed by E₂ administration over 4 wk.

MATERIALS AND METHODS

Animals and animal diets.

Genetically heterogeneous HET3 mice were used in this study. F2 hybrid mice crossed from Jackson Laboratories no. 10009 CByB6F1J and no. 100004 C3D2F1J were fed modified LabDiets [JL Rat & Mouse Irr/4f (5LG6)] diets: a microencapsulated 3/8″ pellet control or rapamycin diet from Randy Strong's laboratory at the University of Texas Health Science Center at San Antonio (12). The breeding strategy in this study was identical to that previously published (12, 28) and was utilized intentionally to examine the effects of rapamycin on glucose homeostasis in mice independent of substrain. The control diet contained 189 ppm Eudragit, while the rapamycin diet contained 189 ppm (14 mg/kg food) encapsulated rapamycin; mice were started on diets immediately after weaning. Mice were housed four to a cage in standard, clear, polycarbonate mouse cages with maple sani chip bedding (Harlan Tek Labs, no. T.7090M), at 30°C with 12:12-h light-dark phases. Cages had a wire bar lid and a mouse ventilated filter top. The bedding was changed 2 or 3 times weekly. These conditions are not specifically pathogen-free. Two sentinels in the room were euthanized at 3-mo intervals throughout the year for full serology reports (Charles River Animal Research Services), with all reports returning as negative. Food and water were available ad libitum, and food intake and body weight were measured weekly. The sample size for each group was as follows: control-fed group: n = 75 (males), n = 74 (females); rapamycin-fed group: n = 73 (males), n = 70 (females). All experiments were approved by the Williams College Animal Use Committee.

A separate group of 12 mice (6 males and 6 females) were euthanized after 3 mo on either the rapamycin-containing diet or control diet. Blood was drawn directly from the heart in deeply anesthetized mice. The blood was immediately centrifuged at 10,000 g for 2 min, and the plasma was flash frozen in liquid nitrogen. Plasma samples were analyzed for rapamycin using a commercially available kit that has a sensitivity of 1.25 ng/ml (IMx Sirolimus assay; Abbott Laboratories). The heart, kidneys, and triceps muscle were removed, flash frozen in liquid nitrogen, and stored at −80°C until further analysis.

Western analysis for phosphorylation of ribosomal protein S6.

Tissue samples (heart, kidney, and skeletal muscle) were prepared for gel electrophoresis and enzymatic assay by homogenization using a Polytron blade-type blender in homogenization buffer (200 mM Tris·HCl, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 2% Triton X, pH 7.5). Homogenates were centrifuged at 10,000 g for 10 min, and the supernatant was stored at −20°C. Protein concentration of the supernatant was determined using the Bradford method. One microgram of protein was separated by SDS-PAGE gel electrophoresis using prepoured 4–20% minigels (Bio-Rad; cat. no. 456–1093). The proteins were transferred to Amersham Hybond-P PVDF membranes. Membranes were blocked in 5% BSA in TBS-Tween (TBST) for 1 h. Duplicate blots were washed in TBST followed by an overnight incubation with a primary antibody that detects either total ribosomal protein S6 (rpS6) (Cell Signaling Technologies; no. 2217S diluted 1:3,000 in 5% BSA in TBST) or phosphorylated rpS6 (Cell Signaling Technologies; no. 2215S diluted 1:3,000 in 5% BSA in TTBS) at 4°C. Membranes were washed and probed with a secondary antibody (Cell Signaling Technologies; no. 7074S, diluted 1:10,000 in 5% BSA in TTBS) for 1 h at room temperature. After the final washing, membranes were exposed to LumiGLO and peroxide reagents (Cell Signaling Technologies; no.7003), and blots were imaged and developed on blue X-ray film.

Urine collection and analysis.

Urine was collected using a modified version of the polyethylene funnel method originally devised by Perline (31). Mice were placed in urine cages individually 3 or 4 h after the beginning of the light phase, and were given ad libitum access to water through an opening in the cage. To prevent contamination of the urine by glucose originating from food pellets, mice were fasted during the urine collection process. After 7 h of urine collection, mice were replaced in their original cages and once again given ad libitum access to both food and water. The collected urine was stored at −20°C. Urine glucose levels were measured using the glucose LiquiColor assay (Stanbio). The microplate was read at 450 nm on a modulus microplate from Turner Biosystems, and glucose concentrations were calculated from a generated standard curve. The sample sizes used were as follows: control-fed group: n = 7 (males), n = 4 (females) and rapamycin-fed group: n = 9 (males), n = 6 (females).

Fasting blood glucose.

Blood glucose measurements were recorded from a tail bleed after a 6-h fast beginning after the onset of the light phase using TrueResult glucose meter and strips.

Tolerance tests.

Animals were individually housed and fasted for 6 h prior to experimentation, beginning after the onset of the light phase. For glucose tolerance tests, mice were injected intraperitoneally with 1 g glucose/kg body wt prepared in sterile saline solution. For insulin tolerance tests, mice were injected intraperitoneally with insulin at 0.75 U/kg body wt. Insulin solution was prepared by suspension of powdered human insulin (Sigma-Aldrich) in sterile saline. For pyruvate tolerance tests, pyruvate (Sigma-Aldrich) prepared in sterile saline solution was delivered intraperitoneally at 0.7 mg/kg body wt.

Ovariectomies.

Under isofluorane anesthesia, ovaries were removed using diagonal incisions of ∼7 mm in length made directly below the rib cage in the dorsal flanks. The ovary was identified and confirmed beneath adipose layers by locating the uterus attached to it, after which a hemostat was closed at its base. The ovary was removed by cutting along the distal edge of the hemostat, and the wound was closed by cauterization. The body wall was closed using nonabsorbable suture (5–0 Ethilon, Johnson & Johnson). The skin was then closed with 7-mm wound clips (Autoclip), and triple antibiotic ointment (Actavis) was applied to the wound. The above procedure was followed for mice undergoing sham ovariectomies. However, after the incision on the body wall was created and the white adipose was identified, the body wall and skin were immediately closed. Mice were allowed 7 days for recovery before experimentation resumed.

Castrations.

Under isofluorane anesthesia, a vertical 4-mm incision was made in the middle of the scrotal sac. Pressure was applied to one side of the base of the testes, facilitating accessibility of the encased testis on that side. An incision was made in the sac enclosing the testis, and further pressure was gently applied to push the testis out completely. The top of the epididymis was identified and grasped with a hemostat, and both testis and epididymis were removed by cutting along its distal edge. Any remaining tissue was allowed to fall back into the sac, with no need for cauterization. The procedure was repeated on the other side to remove the second testis. The incision was then closed using nonabsorbable suture, and triple antibiotic ointment was applied. Mice undergoing sham surgeries were anesthetized as described and were subsequently replaced in their cage and allowed to recover for 7 days before experimentation resumed.

Implant preparation and procedure.

Implants were composed of 2.5-cm pieces of Silastic tubing (1.37 mm ID, 3.18 mm OD, Dow Corning) containing 17β-estradiol (Steraloids) dissolved in sesame oil (Sigma-Aldrich) at a concentration of 180 μg/ml, or only sesame oil. The tubes were closed on both ends with a 2-mm length of silicone medical adhesive (Type A; Dow Corning). This concentration has been shown to maintain steady, physiological levels of 17β-estradiol in the plasma and, thus, is commonly used (8, 41). Under anesthesia, a vertical, interscapular 7-mm incision was made in the skin. After inserting the Silastic tubing, the wound was closed using a nonabsorbable suture, and triple antibiotic ointment was applied.

Histology analysis of liver and pancreas.

The liver and pancreas were collected from a subset of control-fed (euglycemic) and rapamycin-fed (hyperglycemic) mice and stored in 10% formalin. Samples were sent to Charles River Animal Diagnostic Services, where sections were prepared, stained with hematoxylin and eosin, and analyzed at Charles River.

Calculations and statistical analysis.

Statistical analysis was completed using SPSS software. Single-variable ANOVA tests were performed to determine differences across groups in fasting blood glucose and urine glucose data sets. Repeated-measures ANOVA was performed followed by Tukey's post hoc testing to determine the changes in fasting blood glucose, glucose tolerance, and body weight over time. Statistical analysis did not assume consistent means or normally distributed data. All error bars indicate means ± SE, and significance is defined as P < 0.05.

RESULTS

Chronic (52 wk) treatment with rapamycin.

A pilot group of 12 mice were fed either the rapamycin diet or a normal control diet for 3 mo and euthanized for assessment of rapamycin in the blood. Control-fed mice did not have detectable levels of circulating rapamycin (the lower limit of the assay is 1.25 ng/ml), whereas all mice on the rapamycin diet had detectable levels (4.98 ± 0.69 ng/ml) of rapamycin in the blood. Phosphorylated rpS6, a protein target downstream of mTOR signaling, was assessed by Western blot analysis in the kidney, heart, and skeletal muscle (triceps) of these mice and was found to be significantly blunted in all of the tissues tested from rapamycin-fed mice compared with control-fed mice (Fig. 1).

Fig. 1.

Mammalian target of rapamycin (mTOR) signaling, as assessed by phosphorylation of ribosomal protein S6, in the kidney, heart, and skeletal muscle is decreased with rapamycin feeding. Mice treated with rapamycin show decreased phosphorylation of ribosomal protein S6 relative to total ribosomal protein S6, compared with age-matched control-fed mice in the kidney (A), heart (B), and skeletal muscle (C).

Chronic oral rapamycin treatment caused a decrease in the growth rate of male mice after 5 wk of administration (Fig. 2A) and after 27 wk in female mice (Fig. 2B). After 29 wk of treatment, rapamycin-treated male mice began to eat significantly more food than control-fed mice (Fig. 2C), although this was not seen in female mice (Fig. 2D). Rapamycin treatment adversely affected glucose homeostasis, with male mice having more severe symptoms than female mice (Fig. 3). Hyperglycemia developed in male mice treated for 52 wk with oral rapamycin (Fig. 3A). Additionally, decreased weight in male mice correlated with increased yet variable fasting blood glucose levels (Fig. 3B). Male rapamycin-treated mice also displayed increased levels of urine glucose compared with nontreated controls (Fig. 3C).

Fig. 2.

Body weight and food consumption in mice treated with oral rapamycin show a lack of weight gain and hyperphagia. A: male mice treated with rapamycin have a consistently lower weight than age matched controls, diverging after 5 wk on oral rapamycin treatment (P = 0.008). B: female mice treated with oral rapamycin develop a lower weight than their age-matched controls by 27 wk on rapamycin (P = 0.001). C: food consumption increases after 28 wk on rapamycin treatment in male mice (P = 0.01). This difference persists and becomes more pronounced for the 52-wk duration of the treatment. D: female rapamycin-treated mice do not experience a difference in food consumption compared with age-matched controls. Error bars for all graphs show means ± SE. (*P < 0.05).

Fig. 3.

Chronic rapamycin treatment elevates fasting blood glucose and urine glucose. A: fasting blood glucose levels in mice treated with rapamycin for 52 wk depict hyperglycemia in male mice compared with controls (P < 0.001). Control-fed: n = 18, rapamycin-fed: n = 69. B: an increase in fasting blood glucose is associated with a decrease in body mass in males treated with rapamycin (R² = 0.461) but not females (R² = 0.001). n = 69 in each group. C: analysis of urine during a 7-h fast shows elevated urine glucose in male mice treated with rapamycin (P = 0.02), and negligible glucose in the urine of female mice. Control-fed: n = 7 (males), n = 4 (females), rapamycin-fed: n = 9 (males), n = 6 (females). Error bars represent means ± SE. (*P < 0.05).

Histological analysis was performed on the liver and pancreas from euglycemic (control-fed) and hyperglycemic (rapamycin-fed) males. No morphological abnormalities were noted in liver samples (data not shown). While full quantitative analysis was not performed on the numbers of islets with the pancreas tissues, we noted fewer islets on those samples from rapamycin-fed mice. In addition, select islets were found by the histological analysis to be small and slightly disorganized (Fig. 4).

Fig. 4.

Histological analysis of pancreatic tissue shows minimal abnormalities with chronic rapamycin treatment. The pancreas was removed from 1-yr-old mice, stained with hematoxylin and eosin, and analyzed by Charles River Animal Diagnostic Services. The samples are representative of the pancreas analyzed from control-fed and rapamycin-fed mice.

Tolerance tests were performed with glucose, pyruvate, and insulin (Fig. 5). Mice treated with rapamycin for 52 wk showed glucose intolerance in glucose tolerance tests (GTTs; Fig. 5A). Similarly, pyruvate tolerance tests showed significant pyruvate intolerance in male mice chronically treated with oral rapamycin (Fig. 5B). Finally, rapamycin-treated male mice displayed a normal sensitivity to insulin when examining blood glucose expressed relative to baseline glucose (Fig. 5C, inset). Although female rapamycin-treated mice were glucose-intolerant, they remained sensitive to both pyruvate and insulin (Fig. 5).

Fig. 5.

One year of rapamycin treatment leads to glucose, pyruvate, and insulin intolerance. A: glucose tolerance test (GTT) and area under the curve (AUC) analyses show glucose intolerance in both male and female mice chronically treated with rapamycin compared with controls (P < 0.001 and P = 0.001, respectively). Control-fed: n = 10 (males), n = 9 (females), rapamycin-fed: n = 9 (males), n = 9 (females). B: pyruvate tolerance test (PTT) and AUC analyses indicate that chronic rapamycin treatment leads to pyruvate intolerance in male mice (n = 6 in each group; P = 0.009). C: insulin tolerance test (ITT) shows insulin sensitivity in all groups (n = 6 in each group). Inset depicts the percent of basal glucose after insulin administration. For all tolerance tests, mice were fasted for 6 h, and error bars represent means ± SE. (*P < 0.05).

To better understand the timeline of progression of these diabetic symptoms, a second cohort of male mice was started on oral rapamycin after weaning for 18 wk, while closely monitoring the progression of these glucose homeostasis markers (Fig. 6). As previously demonstrated, hyperglycemia developed in males treated with rapamycin, trending toward consistently increasing fasting blood glucose over time (Fig. 6A). Furthermore, mice on oral rapamycin treatment developed persistent glucose intolerance by 8 wk of treatment, as measured by GTTs (Fig. 6B). These rapamycin-treated mice remained insulin-sensitive relative to nontreated mice (Fig. 6C, inset).

Fig. 6.

Development of glucose intolerance in male mice on 18 wk of oral rapamycin treatment. A: fasting blood glucose levels (after a 6-h fast) are significantly increased over 14 wk of rapamycin treatment by repeated-measures ANOVA (P = 0.043; n = 7 in each group). B: AUC of GTTs shows young male mice develop glucose intolerance after just 4 wk on rapamycin treatment, compared with age-matched controls (n = 7 in each group). C: all groups showed normal insulin sensitivity as measured by ITTs. Inset: percent of basal glucose after insulin administration (n = 7 in each group). Error bars in all graphs represent means ± SE. (*P < 0.05).

Cessation of chronic rapamycin treatment.

A subset of mice (n = 6 of each sex) treated with rapamycin for 52 wk was taken off of rapamycin for a period of 18 wk while monitoring fasting blood glucose (Fig. 7), glucose tolerance (Fig. 7), and insulin tolerance (data not shown). Strikingly, after just 1 wk off of rapamycin treatment, the average fasting blood glucose level in male mice decreased significantly to 135 ± 4 mg/dl, although this remained significantly above that of control mice (96 ± 5 mg/dl; Fig. 7A). Similarly, as monitored by successive GTTs, male mice quickly recovered glucose tolerance after the cessation of rapamycin treatment, observable within 2 days off of rapamycin (Fig. 7, C and E). This became a significant change after 1 wk off of rapamycin treatment, however, these mice never returned to the same level of tolerance as the age-matched controls (Fig. 7E). Insulin sensitivity remained the same over the 18-wk period (data not shown).

Fig. 7.

Cessation of rapamycin treatment leads to a full reversal of glucose intolerance in females and a partial reversal in males treated with rapamycin for 1 year. A: male mice show a partial reversibility of hyperglycemia, displaying significantly lower fasting blood glucose levels than rapamycin-fed controls as early as 1 wk after the cessation of treatment (P = 0.001), yet not reaching the level of control males (P = 0.03). B: female mice taken off rapamycin display fasting blood glucose levels that match the controls, while mice continued on rapamycin treatment have an elevated blood glucose level. C and E: GTTs in male mice show a reduction in glucose intolerance over time after the cessation of rapamycin treatment; however, these mice never reach the glucose tolerance level of the age-matched control. D and F: GTTs and the resultant AUC analysis in female mice show a significant and persistent increase in glucose tolerance 2 wk after the cessation of rapamycin treatment. Prior to cessation, all mice had been treated with rapamycin for 1 yr. n = 6 in all groups. Prior to tolerance tests and fasting blood glucose measurements, mice were fasted for 6 h. Error bars in all graphs represent mean ± SE. (*P < 0.05).

Female mice taken off rapamycin after a period of 52 wk also displayed changes in glucose homeostasis. Fasting blood glucose in female mice, which did not notably change following 52 wk of rapamycin treatment, remained similarly unchanged after cessation of treatment (Fig. 7B). Glucose intolerance, however, was reversed after the cessation of chronic rapamycin treatment in females (Fig. 7D). Female mice taken off of rapamycin became glucose tolerant just 2 wk after the cessation of rapamycin, matching the tolerance of control-fed females (Fig. 7F). Additionally, females remained insulin-sensitive after the cessation of rapamycin treatment (data not shown).

Ovariectomy and replacement of E₂.

Following an initial characterization of diabetes in male mice and glucose intolerance in female mice, gonadectomies were performed on a subset of mice that had been treated with rapamycin for 52 wk in an effort to better understand the role of sex hormones in developing the sex-based differences in the phenotype of chronic rapamycin-treated mice. After ovariectomy, rapamycin-treated females became increasingly glucose intolerant over a period of 10 wk (Fig. 8A). Tolerance to a glucose load did not change in rapamycin-fed, sham-operated females over the same time period (data not shown). Control-fed ovariectomized and nonovariectomized mice monitored over the same period of time did not develop glucose intolerance (data not shown). This suggests a protective role of ovarian-derived hormones in preventing the progression of rapamycin-induced glucose intolerance. To better characterize the role of E₂ specifically in chronically treated females, ovariectomized females were implanted with Silastic tubing containing either E₂ or sesame oil vehicle after 10 wk postovariectomy. Glucose tolerance was monitored in these implanted mice with weekly GTTs, showing an improvement in glucose tolerance over a 4-wk period (Fig. 8B). Vehicle-implanted females did not show a change in glucose tolerance postimplant (data not shown). This evidence suggests a critical protective role of E₂ in preventing the most severe metabolic symptoms in female mice chronically exposed to rapamycin.

Fig. 8.

Ovariectomized females treated with rapamycin display increased glucose intolerance, which can be reversed by E₂ supplement. A: GTTs at week 1, 2, 3, and 10 postovariectomy in chronic rapamycin-treated female mice show a progressive decrease in glucose tolerance (n = 11 in Ovx and n = 5 in sham). B: GTTs at week 0, 1, 2, 3, and 4 post-E₂ replacement shows recovery of glucose tolerance in ovariectomized females that had been chronically treated with rapamycin (n = 6 in E₂ and n = 5 in vehicle). Error bars in all graphs represent means ± SE.

A subset of males that were fed rapamycin for 1 yr was castrated or sham-operated, and monitored for alterations in diabetic status via weekly GTTs (data not shown). Castration did not impact glucose tolerance as assessed by GTTs in rapamycin-treated mice, suggesting that a lack of T does not alleviate glucose intolerance in rapamycin-treated males.

DISCUSSION

Most rapamycin-based studies to date have documented symptoms of prediabetes, such as impaired glucose tolerance and insulin insensitivity in mice in response to varying lengths and modes of rapamycin treatment (3, 7, 9, 11, 13, 20, 21, 28). Our results indicate that long-term (52 wk) oral administration of rapamycin induces diabetes in male mice. Over a 52-wk period, rapamycin-fed male mice had increased food intake and decreased body weight, were hyperglycemic, and had detectable and significantly high levels of urine glucose. High fasting blood glucose in males was associated with low body mass, a characteristic of significant, untreated diabetes in which insulin-mediated glucose uptake and subsequent cell growth is compromised (5). Rapamycin-treated males were further found to be glucose- and pyruvate-intolerant, with intact insulin sensitivity. On the other hand, rapamycin-treated female mice were euglycemic, had negligible glucose in the urine, and no decrease in body mass. Female mice treated with rapamycin were glucose-intolerant, yet pyruvate- and insulin-sensitive.

Diabetes is often associated with altered morphology of liver and pancreatic tissue. Recent studies have shown that islet size is decreased and β-cell mass is reduced in rodents following short-term rapamycin administration (13, 42). In the current study, samples of pancreas and liver from hyperglycemic rapamycin-fed male mice and control-fed mice showed no major morphological abnormalities. Although it was noted that in pancreases of rapamycin-fed mice, islets of Langerhans were slightly smaller, disorganized, and more thinly distributed across samples, these observations were drawn from a limited number of mice. Additionally, this qualitative assessment uses random sampling, which does not account for the plane at which the section was taken. However, these data suggest that chronic rapamycin treatment may have an effect on the integrity and abundance of β-cells, which supports the conclusion that rapamycin-induced diabetes is only partially reversible in males. A more systematic quantification of islet cells should be completed to confirm these observations.

Using a second cohort of outbred male mice to better pinpoint the onset of diabetes, we observed glucose intolerance beginning at 4 wk on oral rapamycin, accompanied by increased fasting blood glucose. Other studies using various modes of administration of rapamycin have also suggested the onset of glucose intolerance and insulin resistance at this approximate time period (9, 11, 13, 28, 42).

Our data support previously reported findings of metabolic impairment following 4–6 wk of rapamycin treatment (11, 13, 20, 21, 28, 42) and further extend the ongoing debate on the metabolic effects of rapamycin. Significant insulin resistance in response to rapamycin treatment has been previously reported in shorter studies (9, 11, 21). It is important to note that rapamycin can cause elevated triglycerides and free fatty acids in rodents (13) and humans (29) and that the dyslipidemia may be partially responsible for the induced insulin resistance. Conversely, Lamming et al. (20) recently reported no insulin resistance in HET3 mice administered with oral rapamycin that were glucose-intolerant (20). This is in agreement with our data in that both male and female rapamycin-treated mice responded normally to insulin.

Data from recent studies also suggest that the rapamycin paradox, the apparent ability of rapamycin to both impair metabolism and induce longevity, might be explained by taking chronology into account. Fang et al. (9) reported an initial worsening, and subsequent independent improvement in metabolic parameters, including glucose intolerance and insulin resistance, by 20 wk on treatment in genetically heterogeneous mice injected daily with rapamycin. Furthermore, Lamming et al. (20) suggested an independent resolution of elevated blood glucose as measured by HbA1c in young (6 mo old) HET3 mice treated with oral rapamycin for 3 mo compared with mice treated for 3 wk. In their study, 21-mo-old mice treated similarly did not develop increased HbA1c levels at all. Recently, Miller et al. (28) reported glucose intolerance and mild insulin resistance after 4 wk on rapamycin treatment in a long-term study involving oral rapamycin administration to HET3 mice until death. However, the mice used in their study were not reported to develop further noticeable diabetic symptoms following a longer period of time on rapamycin, despite having increased longevity. These findings run counter to our observations of consistent worsening of diabetic symptoms in mice chronically treated with rapamycin. Importantly, despite using identical breeding strategies to produce our mice, we began oral rapamycin treatment immediately after weaning, while Lamming et al. (20) initiated treatment at 6 and 21 mo, and Miller et al. (28) at 9 mo of age. Such notable differences between our studies, including the observations described by Fang et al. (9), reinforce the significant role that strain-specific differences, mode of administration, and even the timing of initiation and duration of treatment may play in the metabolic response to rapamycin (9, 20).

Following the cessation of chronic rapamycin treatment after 52 wk, a recovery of glucose tolerance was noted within just a few days in male and female mice. Of note, complete recovery of glucose homeostasis was only observed in female mice taken off rapamycin. Females returned to control levels of glucose tolerance within 2 wk of treatment cessation. Only a partial reversal was noted in male mice taken off rapamycin, with merely reduced hyperglycemia and glucose intolerance. A possible cause of inability to recover glucose tolerance while remaining insulin-sensitive could be reduced function of pancreatic β-cells in rapamycin-treated males. In our case, this might be a result of other direct effects of rapamycin on pancreatic morphology, as has been noted in shorter-term studies (11, 13). This might explain the observed glucose intolerance and slight insulin resistance, as seen in mice following prolonged rapamycin administration (20). Additionally, possible pancreatic β-cell dysfunction may be a result of chronic rapamycin administration causing glucose intolerance, in conjunction with older age in our mice (33, 34).

The dramatic differences in rapamycin-induced glucose intolerance and insulin resistance between the sexes in mice as noted here has previously been alluded to by Lamming et al. (21) and Miller et al. (28). While much has been done to better elucidate the role of E₂ and T in diabetes induced by various means in rats and mice (6, 10, 23, 24, 30, 35, 36, 39, 40), little has been done to explore their role in rapamycin-induced diabetes. We show here that E₂ plays a protective role against rapamycin-induced diabetes, with female mice becoming increasingly glucose-intolerant over a period of 10 wk postovariectomy and recovering glucose tolerance over 4 wk post-E₂ replacement. In our study, control-fed ovariectomized female mice followed during the same 10-wk period did not exhibit increased glucose intolerance, suggesting a specific role of E₂ in the context of rapamycin-induced diabetes. Prior studies on E₂ loss have also reported glucose intolerance and insulin resistance accompanied by weight gain (24, 36), making it difficult to separate the influence of adiposity and direct effects of E₂ deficit on glucose intolerance. In this study, we found no significant increase in body mass or food intake postovariectomy, suggesting that increased glucose intolerance was a result of E₂ loss specifically (data not shown).

In line with the less conclusive literature surrounding T (6, 10, 23, 30), we did not find as pronounced an effect of T deficiency on rapamycin-induced diabetes. Worsening of glucose intolerance was impeded in rapamycin-fed castrated male mice compared with sham-operated males. However, castration did not promote recovery of glucose tolerance in males. Of note, castrated males used in these experiments were over 1 yr of age and severely diabetic. Prolonged untreated diabetes may have resulted in changes in pancreatic β-cell function that prevented males from being able to recover glucose tolerance (33, 34). Given these results, we might infer with caution that T plays a facilitative role in rapamycin-induced diabetes in males. However, further work needs to be done in this regard, including characterization of the effects of T replacement in addition to deficit, with the use of younger mice to explore the potential effect of age on results.

Perspectives and Significance

Our findings illustrate in greater detail the effect of long-term oral rapamycin administration on male and female mice. After 4 wk of daily administration in young mice, rapamycin induces glucose intolerance that is more pronounced in males, and progresses consistently toward diabetes by 52 wk. At this time point, diabetic symptoms are less severe and fully reversible in female mice, and partially reversible in males following cessation of rapamycin treatment. Sex-based differences in the progression of and recovery from diabetic symptoms can be at least partly attributed to the protective effect of circulating E₂ in females, and the possibly facilitative effect of T in males. Thus, our findings suggest that further, more detailed studies are required to better characterize and understand the rapamycin paradox. Future studies to this end should focus on quantifying changes in pancreatic islet number and size following rapamycin treatment, gonadectomy, and hormone replacement to better elucidate other mechanisms by which rapamycin-induced diabetes progresses.

GRANTS

This work was supported by National Institutes of Health Grant R15AG37959-1 to S. J. Swoap.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: C.E.S., U.P., B.K.P., and S.J.S. conception and design of research; C.E.S., U.P., and B.K.P. performed experiments; C.E.S., U.P., and B.K.P. analyzed data; C.E.S., U.P., B.K.P., and S.J.S. interpreted results of experiments; C.E.S. and U.P. prepared figures; C.E.S. and U.P. drafted manuscript; C.E.S., U.P., and S.J.S. edited and revised manuscript; C.E.S., U.P., B.K.P., and S.J.S. approved final version of manuscript.