Chronic Rapamycin Treatment Causes Glucose Intolerance and Hyperlipidemia by Upregulating Hepatic Gluconeogenesis and Impairing Lipid Deposition in Adipose Tissue
OBJECTIVE The mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) pathway is a critical signaling component in the development of obesity-linked insulin resistance and operates a nutrient-sensing negative feedback loop toward the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Whereas acute treatment of insulin target cells with the mTOR complex 1 (mTORC1) inhibitor rapamycin prevents nutrient-induced insulin resistance, the chronic effect of rapamycin on insulin sensitivity and glucose metabolism in vivo remains elusive.
RESEARCH DESIGN AND METHODS To assess the metabolic effects of chronic inhibition of the mTORC1/S6K1 pathway, rats were treated with rapamycin (2 mg/kg/day) or vehicle for 15 days before metabolic phenotyping.
RESULTS Chronic rapamycin treatment reduced adiposity and fat cell number, which was associated with a coordinated downregulation of genes involved in both lipid uptake and output. Rapamycin treatment also promoted insulin resistance, severe glucose intolerance, and increased gluconeogenesis. The latter was associated with elevated expression of hepatic gluconeogenic master genes, PEPCK and G6Pase, and increased expression of the transcriptional coactivator peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) as well as enhanced nuclear recruitment of FoxO1, CRTC2, and CREB. These changes were observed despite normal activation of the insulin receptor substrate/PI 3-kinase/Akt axis in liver of rapamycin-treated rats, as expected from the blockade of the mTORC1/S6K1 negative feedback loop.
CONCLUSIONS These findings unravel a novel mechanism by which mTORC1/S6K1 controls gluconeogenesis through modulation of several key transcriptional factors. The robust induction of the gluconeogenic program in liver of rapamycin-treated rats underlies the development of severe glucose intolerance even in the face of preserved hepatic insulin signaling to Akt and despite a modest reduction in adiposity.
The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that belongs to the phosphatidylinositol (PI) kinase-related protein kinase family (1). mTOR integrates signals from growth factors, hormones, nutrients, and cellular energy level to regulate protein translation and cell growth, proliferation, and survival (1). mTOR exists in two complexes. The mTOR complex 1 (mTORC1) is composed of mTOR, GβL, PRAS40, deptor, and raptor and regulates cell growth, whereas the complex 2 (mTORC2) consists of mTOR, GβL, protor-1, mSIN1, deptor, and rictor and controls cytoskeleton regulation and cell survival (1,2). mTORC1 activates cellular processes by phosphorylating two downstream effectors, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (3). Both mTORC1 and S6K1 are activated by insulin through the insulin receptor/insulin receptor substrate (IRS)/phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway (4). We and others have shown that chronic activation of the mTORC1/S6K1 pathway by nutrients or prolonged insulin treatment promotes insulin resistance through increased IRS-1 serine phosphorylation, leading to a reduction in IRS-1 function and impaired activation of the PI 3-kinase/Akt pathway, thereby creating a negative feedback loop on insulin action. Accordingly, acute inhibition of mTORC1 by rapamycin was found to restore insulin action on the PI 3-kinase/Akt pathway and to prevent the insulin-resistant effects of excess nutrients on insulin-mediated glucose transport in muscle and adipose cells (3,5–7).
Because rapamycin protects from insulin resistance caused by excess nutrients in vitro, it was proposed that it may hold promise as a drug for treatment of insulin resistance in obesity. In fact, rapamycin is already used in the clinic as an effective immunosuppressant in human transplant recipients to prevent graft rejection (8). However, somewhat paradoxically it appears that a significant side effect of chronic inhibition of mTORC1/S6K1 signaling by rapamycin treatment is deranged lipid and glucose metabolism (9). Indeed chronic rapamycin administration was found to cause hyperlipidemia and to reduce fat mass, as well as to promote glucose intolerance and a diabetes-like syndrome (8,10,11). However, the underlying mechanisms behind these metabolic derangements remain unknown and particularly intriguing when considering that rapamycin exerts positive metabolic actions when used in vitro in insulin target cells.
In the present study, we have evaluated the effects of chronic rapamycin treatment in rats and explored potential mechanisms by which this mTORC1 inhibitor impacts on lipid and glucose metabolism. We show that rapamycin treatment reduces fat deposition in white adipose tissue through altered expression of key enzymes required for fatty acid uptake and triglyceride synthesis. Despite such loss in fat mass, rapamycin treatment also causes insulin resistance and glucose intolerance, in part through inducing hepatic gluconeogenesis by enhancement of the expression of gluconeogenic enzymes and nuclear recruitment of important gluconeogenic transcriptional regulators. This hepatic phenotype was observed despite preservation of insulin signaling through the IRS/PI 3-kinase/Akt pathway as expected from the ability of rapamycin to block the mTORC1/S6K1-dependent phosphorylation of IRS-1 on serine residues. Our data thus provide a mechanism to explain the higher prevalence of glucose intolerance and a diabetes-like syndrome in rapamycin-treated patients who underwent a transplant and further highlight the importance of the mTORC1/S6K1 pathway in modulating glucose homeostasis.
RESEARCH DESIGN AND METHODS
Animal handling and treatment were approved by the Animal Care and Handling Committee of Laval University. Male Sprague-Dawley rats (200 g) were purchased from Charles River Laboratories (St. Constant, QC, Canada) and housed individually in cages in a room kept at 23 ± 1°C with a 12-h light/12-h dark cycle. After 4-day adaptation, rats were matched by weight and divided into control and rapamycin-treated groups. Vehicle (0.1% Me2SO, 0.2% carboxymethylcellulose) or rapamycin (2 mg/kg/day; Biomol, Plymouth-Meeting, PA) was injected intraperitoneally once daily. Because the mTORC1 complex acts as a cellular nutrient sensor, we performed experiments (except where indicated) on rats that had been fasted for 12 h followed by 3 h of refeeding to achieve physiological activation of insulin signaling. After 15 days of treatment, control and rapamycin-treated rats were killed by decapitation. Tissues and blood were rapidly harvested and frozen in liquid nitrogen.
Intraperitoneal glucose, insulin, and pyruvate tolerance tests.
Rats were fasted for 6 h and injected intraperitoneally with glucose (2 g/kg) or insulin (2.5 units/kg; human insulin; Eli Lilly, Toronto, ON, Canada) diluted in saline 0.9%. For the pyruvate tolerance test, rats were fasted for 12 h and refed for 3 h before being injected with pyruvate (2 g/kg; Sigma-Aldrich, Oakville, ON, Canada) diluted in saline 0.9%. Blood was collected from the tail tip prior to and at various times after injection.
Blood glucose levels were measured by a Precision PCx glucometer (MediSense; Abbott Laboratories, Mississauga, ON, Canada). Plasma insulin, C-peptide, and glucagon were determined by radioimmunoassay (Linco Research, St. Charles, MO). Plasma adiponectin and leptin were measured by ELISA following the suppliers' recommendations (ALPCO Diagnostics, Salem, NH; Millipore, Nepean, ON, Canada). Triglycerides in plasma and in muscle/liver lipid extracts (Roche Diagnostics, Montreal, QC, Canada) and nonesterified fatty acids (WakoChemicals, Richmond, VA), glycerol (Sigma-Aldrich), and lactate (Eton Bioscience, Hayward, CA) levels were measured by enzymatic methods according to the manufacturers' instructions.
Immunoblotting and immunoprecipitation.
Tissue samples were homogenized in buffer, subjected to SDS-PAGE, and transferred to nitrocellulose membranes as previously described (3). For immunoprecipitation of IRS-1 and IRS-2, liver samples were homogenized in buffer (20 mmol/l Tris, pH 7.5, 140 mmol/l NaCl, 1% Nonidet P-40, 2 mmol/l Na4P2O7, 10 mmol/l NaF, 2 mmol/l Na3VO4, and protease inhibitors). Equal amounts of protein (1 mg) were immunoprecipitated with protein A–Sepharose and subjected to SDS-PAGE as described above. Antibodies used for immunoblotting are listed in supplemental Table 1, available in an online appendix at http://diabetes.diabetesjournals.org/content/early/2010/03/10/db09-1324/suppl/DC1. Densitometric analysis was performed with ImageQuant TL software (GE Healthcare, Little Chalfont, U.K.).
Liver nuclear extracts were prepared as previously described (12) and submitted to immunoblotting as described above.
PI 3-kinase activity was measured in IRS-1 or IRS-2 immunoprecipitates, and Akt kinase activity was measured in Akt immunoprecipitates as previously described using a commercial peptide (crosstide) (3).
RNA extraction and quantitative PCR analysis.
RNA extraction and quantitative PCR analysis were performed as described previously (13). The primers used are listed in supplemental Table 2. Data are expressed as the ratio between the expression of the target gene and the housekeeping gene 36B4 (also known as ARBP), the expression of which was not significantly affected by treatment.
Adipose tissue DNA content and adipocyte diameter.
Adipocyte diameter was measured as described previously (14). Tissue DNA content was determined using the DNeasy tissue kit (QIAGEN, Mississauga, ON, Canada) following the manufacturer's instructions.
Morphometric analysis of islets.
Islet size and β-cell mass were measured as previously described (15).
MIN6 cells at 70% confluence were seeded in 24-well plates 1 day before treatments. On the day of the experiment, cells were incubated for 2 h in Krebs-Ringer bicarbonate HEPES buffer containing 2.8 mmol/l glucose before incubation for 2.5–30 min at 10 mmol/l glucose. Culture medium was collected and assayed for insulin content by ELISA (ALPCO Diagnostics).
ANOVA was performed using the general linear model procedure of Statistical Analysis Software (SAS, Version 9.2; SAS Institute, Cary, NC). When appropriate, initial body weight was included in the statistical model as a covariate.
Chronic rapamycin treatment reduces adiposity and causes hyperlipidemia.
Rapamycin treatment significantly reduced (86%) body weight gain, which could be partly attributed to reduced (15%) food intake (Table 1). Rapamycin also decreased food efficiency by 84% and plasma leptin levels by 52%, suggesting that the weight-reducing effect of the drug is explained mostly by increased energy expenditure (Table 1). This is consistent with the recent finding that disruption of adipose mTORC1 signaling through raptor knockout increased energy expenditure (16). Accordingly, we found that rapamycin did not affect the amount of feces calories per gram of food ingested, indicating that intestinal lipid absorption was not affected by the drug (data not shown). Whereas the absolute mass of all tissues sampled was significantly decreased in rapamycin-treated rats (supplementary Table 3) on a relative mass basis (tissue mass/body weight), only white adipose tissue was significantly reduced compared with control rats, suggesting that rapamycin treatment preferentially impaired adipogenesis beyond its effect on body growth (supplementary Table 3 and Fig. 1A). This is supported by both the strong tendency toward smaller adipocyte diameter (P = 0.057, Fig. 1B and C) and significantly fewer adipocytes (lower DNA content per depot, Fig. 1B and C) in the rapamycin-treated group relative to control. In line with these observations, chronic rapamycin treatment increased plasma triglycerides (32%) and nonesterified fatty acids (65%) (Table 1), suggesting that adipose triglyceride deposition was impaired.
Because clinical studies have reported that rapamycin treatment causes hyperlipidemia and reduces body fat mass, it was deemed important to explore the potential underlying mechanisms by which the mTORC1 inhibitor may affect adipose tissue growth and metabolism. mTORC1 and, more recently, raptor have been shown to control adipogenesis (17) through Akt-mediated phosphorylation of tuberous sclerosis complex 2 (18). However, although rapamycin is rather selective for mTORC1, prolonged treatment with the drug can also inhibit mTORC2 and Akt (19). We thus explored whether chronic rapamycin treatment decreased adipogenesis indirectly through inhibition of mTORC2 and Akt. As expected, the phosphorylation of S6 (Ser240/244), a downstream substrate of the mTORC1/S6K1 pathway, was blunted in adipose tissue by rapamycin (Fig. 1D). Furthermore, Akt phosphorylation on Ser473 was severely inhibited in rapamycin-treated rats (Fig. 1D) indicating that mTORC2 may also have been inhibited by chronic drug exposure. In contrast, however, Akt Thr308 phosphorylation was not decreased in adipose tissue of rapamycin-treated rats (Fig. 1D) and Akt kinase activity was actually increased (Fig. 1E), suggesting that phosphorylation on Thr308 more than compensates for the reduction in Ser473 phosphorylation. This is supported by the fact that phosphorylation of the Akt substrate glycogen synthase kinase-3 (GSK-3) was not reduced in adipose tissue of rapamycin-treated rats (Fig. 1D). Thus, chronic rapamycin administration likely affected adiposity via mTORC1 but not mTORC2 inhibition.
Chronic rapamycin treatment coordinately downregulates genes required for lipid uptake and storage in adipose tissue.
To better understand the mechanism by which chronic rapamycin treatment impairs adipogenesis and causes hyperlipidemia, we next measured the mRNA levels of several genes involved in the clearance of lipids from the circulation and their deposition in adipose tissue as triglycerides. Retroperitoneal adipose tissue expression of lipoprotein lipase (LPL), which catalyzes the hydrolysis of lipoprotein-bound triglycerides supplying fatty acids and glycerol for adipose depot uptake (20), along with the fatty acid transporters (FATP1 and FAT/CD36) were all downregulated by rapamycin treatment (Fig. 2). In addition, phosphoenolpyruvate carboxykinase (PEPCK), which generates the glycerol 3-phosphate backbone for fatty acid esterification (21), and lipin 1, an important enzyme involved in triglyceride synthesis (22), were also significantly reduced by rapamycin treatment. We also evaluated the mRNA levels of the enzymes adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacyglycerol lipase (MGL), which catalyze the sequential hydrolysis of triglycerides to fatty acids and glycerol (23). Chronic rapamycin treatment markedly reduced the expression of ATGL and MGL without affecting those of HSL, excluding possible participation of increased lipolysis to the adipose tissue phenotype and hyperlipidemia. Interestingly, the reduced expression of genes involved in lipid uptake and storage and lipolysis was associated with a significant decrease in the mRNA levels of peroxisome proliferator-activated receptor-γ2 (PPARγ2), a master regulator of adipogenesis, lipogenesis, and lipolysis (Fig. 2) (13,20,24).
Chronic rapamycin treatment impairs glucose tolerance, β-cell mass, insulin clearance, and insulin sensitivity and results in higher expression of crucial elements of the gluconeogenic pathway in the liver.
Treatment with rapamycin markedly affected glucose and insulin homeostasis as reflected by hyperglycemia and hyperinsulinemia (Table 1). Furthermore, rapamycin led to an exaggerated response to glucose administration during a glucose tolerance test, indicative of impaired glucose tolerance (Fig. 3A). The insulin levels during the glucose tolerance test were very high in the rapamycin-treated group and did not return to basal levels as they did in the control group (Fig. 3B). The glycemic response to insulin was also reduced in rapamycin-treated rats (Fig. 3C). However, plasma adiponectin, glucagon, glycerol, and lactate were not altered by the inhibitor (Table 1). The insulin response during the glucose tolerance test suggested a possible defect in islet function. Indeed, as shown in Fig. 4A, islet histology showed that rapamycin-treated rats had more small islets and fewer large islets compared with control rats as well as reduced β-cell mass (Fig. 4B). Nevertheless, plasma C-peptide levels were unchanged 3 h after refeeding, although the elevated ratio of insulin/C-peptide suggests that a marked impairment of hepatic insulin clearance underlies the hyperinsulinemic state of rapamycin-treated rats (Fig. 4C and D). To test whether rapamycin directly affected β-cell function, we also tested the effect of the drug on glucose-induced insulin secretion in MIN6 cells but found no alterations (Fig. 4E).
Given the severity of glucose intolerance, we first focused on the contribution of the liver to the diabetes-like phenotype of rapamycin-treated rats. We studied the mRNA and protein expression of transcriptional regulators of hepatic gluconeogenesis and key enzymes under their control. Notably, chronic rapamycin treatment resulted in greatly elevated liver expression of PPARγ coactivator-1α (PGC-1α) mRNA as well as higher expression of the two key gluconeogenic enzymes, PEPCK and glucose-6-phosphatase (G6Pase) (Fig. 5A). Nuclear localization of forkhead box O1 (FoxO1), PGC-1, cAMP response element-binding protein (CREB)–regulated transcription coactivator 2 (CRTC2; also known as TORC2), and CREB was also significantly enhanced after chronic rapamycin treatment (Fig. 5B). We also performed a pyruvate tolerance test to provide functional evidence that the increased gluconeogenic gene expression was associated with elevated gluconeogenesis. As shown in Fig. 5C, the blood glucose level of rapamycin-treated rats was different from control rats for the last 90 min of the test, suggesting increased hepatic gluconeogenesis. Neither liver glycogen (data not shown) nor triglyceride (Table 2) content was altered by rapamycin treatment. To confirm that rapamycin could directly alter hepatic glucose metabolism, we treated FAO hepatoma cells with rapamycin for 24 h and observed a marked augmentation of glucose production in the basal state as well as in the presence of insulin (supplementary Fig. 1A and B).
Chronic rapamycin treatment improves insulin signaling despite enhancing hepatic gluconeogenesis.
Insulin inhibits hepatic glucose production at least in part through its ability to bind and activate the insulin receptor, leading to the phosphorylation of IRS proteins and subsequent activation of PI 3-kinase and Akt. Because Akt negatively modulates hepatic gluconeogenesis through phosphorylation of FoxO1 (25), it was of major interest to test whether insulin signaling to Akt was impaired in the liver of rapamycin-treated rats. We found, however, that rapamycin-mediated inhibition of the mTORC1/S6K1 pathway, as revealed by complete loss of S6 Ser240/244 phosphorylation, improved insulin signaling in the liver, as shown by increased tyrosine phosphorylation of both IRS-1 and IRS-2 (Fig. 6A) and, conversely, diminished inhibitory phosphorylation of IRS-1 on serine residues (Ser1101 and Ser636/639) (Fig. 6B). IRS-1 and IRS-2 protein expression was also increased in the liver of rapamycin-treated rats (Fig. 6C). Further downstream in the insulin signaling cascade, we found that hepatic IRS-1 (P < 0.01)– and IRS-2 (P = 0.06)–associated PI 3-kinase activity was increased by rapamycin (Fig. 6D), whereas Akt phosphorylation on Ser473 and Thr308 as well as Akt kinase activity were not affected (Fig. 6E and F). We also found that tyrosine phosphorylation of IRS-1 was increased (Fig. 6G), whereas Akt kinase activity was not affected (Fig. 6H) in skeletal muscle of rapamycin-treated rats. Moreover, rapamycin treatment did not affect muscle triglyceride content (Table 2).
The mTORC1/S6K1 pathway has recently emerged as a critical signaling component in the development of obesity-linked insulin resistance by operating a negative feedback loop toward PI 3-kinase/Akt through increasing inhibitory serine phosphorylation of IRS-1 (4–6,26). Accordingly, we and others have demonstrated that acute treatment with rapamycin in vitro prevents nutrient-induced insulin resistance by interfering with this negative feedback loop and limiting phosphorylation of IRS-1 on multiple serine residues (3,5,7,27). Moreover, we recently reported that S6K1 plays a key role in hepatic insulin action through phosphorylation of both Ser1101 and Ser636/639 in hepatic cells and liver of obese insulin-resistant high-fat–fed mice (6). Other studies have shown that long-term activation of the mTORC1/S6K1 pathway also promotes insulin resistance through the proteasomal degradation of IRS-1 (28) and IRS-2 (29). In the present study, we found that chronic treatment with rapamycin also improves insulin signaling to IRS/PI 3-kinase and maintains activation of Akt in the liver, in agreement with our previous findings in hepatocytes acutely treated with the drug (4). Accordingly, we found that chronic inhibition of the mTORC1/S6K1 pathway in rats increases IRS-1 and IRS-2 protein expression, enhances their tyrosine phosphorylation while reducing IRS-1 inhibitory phosphorylation on Ser1101 and Ser636/639. This led to upregulation of IRS-dependent PI 3-kinase activation and normal Akt activity in liver, despite the development of severe whole-body glucose intolerance and insulin resistance. This apparent uncoupling between hepatic insulin signaling and glucose homeostasis is in large part explained by augmented gluconeogenesis, unraveling a novel mechanism of regulation of hepatic glucose production by the mTORC1/S6K1 pathway.
Liver gluconeogenesis is driven by the availability of gluconeogenic substrates and the activity of two important gluconeogenic enzymes, PEPCK and G6Pase (30). In the refed state, insulin suppresses gluconeogenesis by transcriptional downregulation of PEPCK and G6Pase (31). Our data indicate that rapamycin upregulates gluconeogenesis in the refed state by increasing the expression of PEPCK and G6Pase. This was observed despite hyperinsulinemia and without any change in the concentrations of circulating glucagon or major gluconeogenic substrates. We also found that rapamycin markedly increases PGC-1α mRNA expression, which likely contributes to the upregulation of the gluconeogenic enzymes, as observed in type 2 diabetes (32). Because rapamycin blocks a major nutrient-sensing pathway, it has been suggested that it mimics a starvation-like signal even in the presence of nutrients (33) and as such could explain the increase in PGC-1α expression. This is the first report of PGC-1α regulation by mTORC1/S6K1 in liver but recent studies have linked this nutrient-sensing pathway to PGC-1α and the regulation of skeletal muscle metabolism (34–36).
PGC-1α increases gluconeogenic gene expression at least in part by coactivating transcription by FoxO1 (37), possibly through its ability to target O-GlcNAc transferase for GlcNAcylation of FoxO1 (38). Conversely, insulin suppresses gluconeogenesis through Akt-dependent phosphorylation of FoxO1, leading to its nuclear exclusion and degradation (39). We found that chronic rapamycin treatment increases FoxO1 nuclear content where it activates the transcription of PEPCK and G6Pase (40). Thus, FoxO1 nuclear exclusion was not observed, despite the presence of hyperinsulinemia and the maintenance of normal hepatic insulin signaling to Akt in rapamycin-treated rats. Further studies will be needed to test whether rapamycin increases gluconeogenic gene transcription via a PGC1α-dependent GlcNAcylation of FoxO1, thus bypassing insulin-induced Akt activation and phosphorylation of FoxO1.
Interestingly, rapamycin treatment increases nuclear recruitment of both CREB and CRTC2. In response to glucagon and other cAMP-elevating hormones, CRTC2 binds to CREB and stimulates the gluconeogenic program by triggering the dephosphorylation and nuclear translocation of CRTC2, whereas insulin blunts this process through activation of salt-inducible kinase 2, which phosphorylates and induces the cytoplasmic translocation and proteasomal degradation of CRTC2 (41,42). CREB may also promote gluconeogenesis through increased PGC-1α expression (43). The observation that both nuclear CRTC2 and FoxO1 remained elevated 3 h after refeeding in the liver of rapamycin-treated rats likely explains the robust activation of gluconeogenesis in these animals. A recent study by Liu et al. (44) has demonstrated the existence of a temporal switch from CRTC2 to FoxO1 as a critical inducer of gluconeogenesis during a prolonged fast. This switch is operated by a complex series of acetylation and deacetylation events that affect stability and activity of both CRTC2 and FoxO1. The fact that both of these redundant transcriptional factors are activated even after refeeding of rapamycin-treated rats, and in the presence of a fully operative hepatic insulin signaling pathway, suggests that mTORC1/S6K1 exerts a previously unsuspected powerful control over the gluconeogenic program.
We also found that rats treated chronically with rapamycin have a decreased β-cell mass. This is consistent with recent reports that chronic activation of mTOR leads to β-cell mass expansion (15,45). Despite the reduced β-cell mass, insulin secretion as revealed by C-peptide levels after refeeding was not altered in rapamycin-treated rats, suggesting that their hyperinsulinemic state is explained rather by impaired hepatic insulin clearance. Although the reduced β-cell mass could explain the lack of further insulin release during the glucose tolerance test, two observations suggest that the liver defects precede the development of β-cell dysfunction in this model. First, we found that liver gluconeogenesis was affected already after only 2 days of rapamycin treatment (supplementary Fig. 2) even before hyperglycemia develops. Second, insulin secretion by MIN6 cells is not altered by rapamycin treatment for up to 48 h. Nevertheless, the reduction in β-cell mass suggests that chronic exposure to rapamycin may eventually cause major perturbations in β-cell function and further contribute to the diabetes-like syndrome induced by the mTOR inhibitor.
Reduced accretion of body fat and hyperlipidemia constitute another important phenotypic feature of rapamycin-treated rats. The inhibitor appears to affect adiposity mainly through a reduction in cell number, with a small contribution from reduced adipocyte size. This is consistent with studies in cell lines in which chronic rapamycin treatment or small interfering RNA–mediated downregulation of either mTOR, raptor, or S6K1 can have direct effects on adipogenesis and lipid content (17,46,47). The gene expression data suggest a robust reduction in lipid flux in and out of adipose tissue. Hence, the coordinated downregulation of the expression of enzymes required for intravascular hydrolysis (LPL), cellular uptake (FAT/CD36 and FATP1), and storage of lipid (PEPCK and lipin), all known to impact adiposity (48), appeared to be counterbalanced by the downregulation of lipolytic (ATGL, MGL) enzyme expression, consistent with the near maintenance of cell size in rapamycin-treated rats. Reduced lipid uptake and fat cell number impairs the capacity of adipose tissue for plasma lipid clearance, which likely contributes to hyperlipidemia.
Several lines of evidence suggest that the rapamycin-induced decrease in the expression of lipid metabolism genes in adipose tissue might be the result of a reduced transcriptional activity of PPARγ. Indeed, all such genes are known to be positively modulated by PPARγ (13,20,24). Furthermore, rapamycin inhibits PPARγ transcriptional activity in adipocytes (46), and adipose-specific disruption of raptor in mice reduces adiposity (16). Conversely, activation of mTORC1 increases PPARγ expression in adipocytes (18). In the present study, rapamycin reduced the expression of both PPARγ2 and lipin 1, a key lipogenic enzyme and PPARγ coactivator (22). Collectively, these congruent findings strongly suggest a molecular link between mTORC1 and PPARγ transcriptional activity.
We also considered the possibility that the effects of rapamycin on PPARγ expression and adipogenesis might be linked to secondary inhibition of mTORC2, leading to a reduction of Akt activation. However, the reduced Akt Ser473 phosphorylation was possibly compensated by normal phosphorylation of Thr308 because Akt kinase activity and GSK-3 phosphorylation were not reduced in adipose tissue of rapamycin-treated rats. GSK-3 phosphorylation was recently found to be preserved despite complete loss of Akt Ser473 phosphorylation in mouse embryo fibroblasts from rictor knockout animals (49). Our conclusion that mTORC2 inhibition cannot account for the reduction of adiposity is also supported by recent studies in mice with adipose tissue–specific disruption of rictor and mTORC2 signaling, which failed to reduce adipogenesis or fat mass in chow-fed animals (50).
In conclusion, chronic inhibition of the mTORC1/S6K1 pathway by rapamycin causes glucose intolerance mainly through inducing transcriptional activation of gluconeogenic genes via the coordinated activation of PGC-1α, CRTC2, CREB, and FoxO1. The robust activation of the gluconeogenic program was observed even under hyperinsulinemia in the refed state, and despite the maintenance of normal activation of hepatic insulin signaling to Akt owing to the blockade of the mTORC1/S6K1 inhibitory feedback loop by rapamycin. The mTORC1 inhibitor has also marked effects on lipid deposition in adipose tissues that likely contribute to elevated concentrations of plasma triglycerides and nonesterified fatty acids. Our findings therefore further emphasize the critical importance of the mTORC1/S6K1 pathway in the physiological regulation of glucose and lipid metabolism and unravel a new role for this nutrient-sensing pathway in the control of hepatic gluconeogenesis through the modulation of several key transcription factors of the gluconeogenic program. This unrestrained activation of hepatic gluconeogenesis likely underlies the occurrence of a diabetes-like syndrome in patients treated with rapamycin.
This work was supported by a grant (144336) from the Canadian Institutes of Health Research (CIHR). A.M. holds a CIHR/Pfizer Research Chair in the pathogenesis of insulin resistance and cardiovascular diseases. V.P.H. was supported by a PhD studentship from the Fonds de la Recherche en Santé du Québec (FRSQ). S.B. and W.T.F. were supported by a postdoctoral fellowship from the CIHR-funded Obesity Training Program of the Quebec Heart and Lung Institute Research Centre. P.-G.B. was the recipient of a PhD studentship from CIHR.
No potential conflicts of interest relevant to this article were reported.
We thank Philip White and Drs. Geneviève Pilon and Mathieu Laplante for critical reading of the article. We also thank Simon Pelletier, Sébastien Morasse, Marc Majaron, and Véronique Turcotte for their help with animal care, pyruvate tolerance test, quantitative PCR, and statistical analysis.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received September 4, 2009.
- Accepted February 23, 2010.
- © 2010 by the American Diabetes Association.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.