Chronic Rapamycin Treatment Causes Glucose Intolerance and Hyperlipidemia by Upregulating Hepatic Gluconeogenesis and Impairing Lipid Deposition in Adipose Tissue
Article Figures & Tables
Figures
- 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.
Tables
- TABLE 1
Effect of 15-day rapamycin treatment on weight, food intake, and metabolic parameters of treated rats
Control Rapamycin Body weight (g) 303.6 ± 3.68 226.3 ± 4.34*** Weight gain (g) 91.4 ± 3.57 12.8 ± 3.87*** Food intake (g) 256.4 ± 6.53 217.7 ± 8.53** Food efficiency (body weight gain/food intake) 0.117 ± 0.004 0.019 ± 0.006*** Glucose (mmol/l) 8.5 ± 0.33 13.3 ± 2.53* Insulin (pmol/l) 351.5 ± 58.7 1,265.1 ± 395.1* Glucagon (ng/l) 127.25 ± 10.65 130.47 ± 14.33 Triglycerides (mmol/l) 0.644 ± 0.050 0.848 ± 0.060* NEFAs (mmol/l) 0.115 ± 0.020 0.190 ± 0.040* Glycerol (mg/dl) 1.025 ± 0.084 1.177 ± 0.107 Lactate (mmol/l) 0.904 ± 0.050 0.852 ± 0.039 Adiponectin (μg/ml) 11.89 ± 1.53 12.11 ± 0.83 Leptin (ng/ml) 9.48 ± 1.86 4.94 ± 1.51* -
Data are average ± SE (n = 12 control, n = 10 for rapamycin). Refed plasma data are average ± SE (n = 6–12 for control, n = 6–10 for rapamycin).
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*P ≤ 0.05,
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**P ≤ 0.01,
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***P ≤ 0.001 vs. control. NEFAs, nonesterified fatty acids.
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- TABLE 2
Effect of 15-day rapamycin treatment on muscle and liver triglycerides
Control Rapamycin Muscle triglycerides (mmol/g tissue) 9.52 ± 0.92 10.40 ± 0.93 Liver triglycerides (mmol/g tissue) 18.69 ± 4.02 12.94 ± 0.95 -
Data are average ± SE (n = 6 for each group).
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