Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue

Abstract

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-gamma coactivator-1alpha (PGC-1alpha) 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.

FIG. 1.

Chronic rapamycin treatment decreases adiposity. Sprague-Dawley rats were treated with vehicle or rapamycin (2 mg/kg/day) for 15 days. A: Relative retroperitoneal fat weight. Total DNA tissue content and adipocyte diameter (μm) (B) and representative images of retroperitoneal fat (C) from control and rapamycin-treated rats (magnification ×10) D: Representative Western blots of adipose tissue lysates are shown for phosphorylated S6 (Ser240/244), Akt (Ser473 and Thr308), GSK-3α/β (Ser21/9), and total proteins (two representative animals of six). The graphs depict densitometric analysis of normalization of phospho-Akt/Akt protein. E: Adipose tissue proteins (500 μg) were immunoprecipitated with total Akt antibody. Immunoprecipitates were analyzed for Akt activity. The graphs depict densitometric analysis of total Akt activity. n = 6 for each group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. CTRL, control; RAP, rapamycin.

FIG. 2.

Chronic rapamycin treatment coordinately downregulates genes required for triglyceride hydrolysis, fatty acid transport, and esterification in adipose tissue. Rats were treated with rapamycin as described in the legend to Fig. 1, and adipose tissue was sampled and processed as described in the research design and methods section for determinations of LPL, FATP1, FAT/CD36, Lipin1, PEPCK, MGL, HSL, ATGL, PPARγ1, and PPARγ2 mRNA expression. The graphs depict mRNA expression in the adipose tissue of target genes corrected for the expression of 36B4 as a control gene. n = 12 for each group. *P ≤ 0.05, **P ≤ 0.01.

FIG. 3.

Chronic rapamycin treatment induces glucose and insulin intolerance in rats. Rats were treated with rapamycin as described in the legend to Fig. 1 and fasted for 6 h before intraperitoneal tolerance tests. Plasma glucose (A) and insulin levels (B) were measured during a glucose tolerance test. C: Plasma glucose levels were measured during an insulin tolerance test. n = 12 for each group. Black squares: CTRL; white squares: RAP. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIG. 4.

Chronic rapamycin treatment impairs β-cell mass and insulin clearance in rats. Rats were treated with rapamycin as described in the legend to Fig. 1. Pancreatic sections were stained with an antibody against insulin for the determination of islet size and β-cell mass as described in the research design and methods section. A: Size distribution of islets expressed as percentage of total islets. n = 10 for each group. B: Pancreatic β-cell mass. n = 6 for each group. C: Plasma C-peptide levels. n = 12. D: Ratio of insulin over C-peptide, a measure of insulin clearance. n = 12 for each group. E: Insulin secretion was determined in MIN6 cells treated with rapamycin (25 nmol/l; white squares) or vehicle (black squares) for 24 h followed by glucose stimulation (10 mmol/l) for various time points. n = 3 independent experiments. *P ≤ 0.05, ***P ≤ 0.001.

FIG. 5.

Chronic rapamycin treatment induces glucose intolerance by upregulating gluconeogenesis in rats. Rats were treated with rapamycin as described in the legend to Fig. 1. A: G6Pase, PEPCK, and PGC-1α mRNA expression. The graphs depict mRNA expression in the liver of target genes corrected for the expression of 36B4 as a control gene. B: Representative Western blots of PGC-1, FoxO1, CRTC2, and CREB proteins in nuclear extracts prepared from liver samples (two representative animals of six are shown). The graphs depict densitometric analysis of normalization of total protein/Histone H1 protein. n = 6 for each group. C: Plasma glucose levels measured during a pyruvate tolerance test on rats fasted for 12 h followed by 3 h of refeeding. n = 6 for each group. Black squares: CTRL; white squares: RAP. *P ≤ 0.05, **P ≤ 0.01.

FIG. 6.

Chronic rapamycin treatment improves insulin signaling in liver and muscle. Rats were treated with rapamycin as described in the legend to Fig. 1. A: Representative Western blots of liver lysates are shown for phosphorylated S6 (Ser240/244) and phospho-tyrosine on IRS-1 or IRS-2 immunoprecipitates (1 mg) (two representative animals of six). The graphs depict densitometric analysis of normalization of phospho-tyrosine/IRS protein. B: Representative Western blots of phosphorylated IRS-1 (Ser1101 and Ser636/639) and total proteins (two representative animals of six are shown) in liver lysates. The graphs depict densitometric analysis of normalization of phospho-IRS-1/IRS-1 protein. C: Representative Western blots of total IRS-1 and IRS-2 protein in liver lysates. The graphs depict densitometric analysis of normalization of total IRS/Actin. D: Equal amounts of liver protein (1 mg) were immunoprecipitated with IRS-1 and IRS-2 antibodies. Immunoprecipitates were analyzed for PI 3-kinase activity. The graphs depict densitometric analysis of IRS-1– and IRS-2–associated PI 3-kinase activity. E: Representative Western blots of liver lysates are shown for phosphorylated Akt (Ser473 and Thr308) and total proteins (two representative animals of six). The graphs depict densitometric analysis of normalization of phospho-Akt/Akt protein. F: Liver proteins (500 μg) were immunoprecipitated with total Akt antibody. Immunoprecipitates were analyzed for Akt activity. The graphs depict densitometric analysis of total Akt activity. G: Representative Western blots of muscle lysates are shown for phospho-tyrosine on IRS-1 immunoprecipitates (1 mg). The graphs depict densitometric analysis of IRS-1 phospho-tyrosine corrected for IRS-1 protein content. n = 6 animals. H: Muscle proteins (500 μg) were immunoprecipitated with total Akt antibody. Immunoprecipitates were analyzed for Akt activity and the graph depicts the densitometric analysis of several independent determinations. n = 6 for each group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.