Control of hyperglycemia in male mice by leflunomide: mechanisms of action

in Journal of Endocrinology
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p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKTS473/T308 and S6K1T389 phosphorylation but decreased S6S235/236 and IRS-1S1101 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on ob/ob and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in ob/ob and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKTS473/T308 phosphorylation in the fat and muscle of ob/ob mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity in vitro, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.

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    The mechanisms of leflunomide-mediated anti-hyperglycemic effect. (A) Chemical structure of leflunomide and A77 1726. (B) Mode of action of A77 1726. Overnutrition with high concentrations of fatty acids and amino acids leads to constitutive S6K1 activation, which phosphorylates IRS-1S1101, leading to poor AKT activation. Leflunomide and its active metabolite A77 1726 inhibit S6K1 activity, subsequently leading to the inhibition of IRS-1S1101. Inhibition of IRS-1S1101 phosphorylation leads to insulin receptor sensitization, as revealed by increased insulin receptor tyrosine phosphorylation and increased binding of IRS-1 to the p85 subunit of PI-3 kinase. AKTS473/T308 phosphorylation and activation leads to increased glucose uptake by stimulating GLUT4 membrane translocation, increased glycogen synthesis, and decreased gluconeogenesis. Chronic use of rapamycin leads to inhibition of both mTORC1 and mTORC2, thus exacerbating hyperglycemia. A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0536.

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    Effect of A77 1726 on protein phosphorylation in the PI-3 kinase pathway. C2C12 myotubes were starved in serum-free medium for 4 h and then treated with the indicated concentrations of A77 1726 or rapamycin (50 nM) for 2 h. The cells were left unstimulated or stimulated with insulin (20 nM) for 20 min. Cells were harvested and analyzed for the phosphorylation of AKTS473, AKTT308, and S6K1T389, IRS-1S1101, IRS-1S636, and S6S235/236, followed by reprobing with their specific antibodies for total protein levels. Relative protein phosphorylation was determined by analyzing the density of bands and presented as bar graphs. The results are the mean ± standard deviation (s.d.) from three experiments. A77, A77 1726; *P < 0.05; **P < 0.01, compared to the insulin-stimulated control (no drug treatment).

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    A77 1726 sensitizes insulin receptor in L6 myotubes. This experiment and the remaining ones (Figs 4, 5, 6 and 7) were carried out under the condition of insulin resistance in which the cells were incubated in the presence of high concentrations of amino acids. L6 myotubes were first starved of serum for 4 h and then incubated either in an amino-acid free medium (EBSS) or EBSS medium containing 2× the amino acid concentrations (2×AA) found in MEM in the absence or presence of A77 1726 (200 µM) for 2 h. After stimulation with insulin (20 nM) for 20 min, cells are harvested and analyzed for the phosphorylation of AKTS473/T308 (A), S6K1T389 (B), IRS-1S1101 (C), S6S235/236 (D), and reprobed with their specific antibodies for total proteins. Relative protein phosphorylation was analyzed by using an Image J software. The results are the mean ± s.d. from three experiments. A77, A77 1726; Rapa, rapamycin. *P < 0.05; **P < 0.01, compared to the control (with insulin and 2× amino acids but without drug).

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    A77 1726 sensitizes insulin receptor in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated in Fig. 3 and then incubated either in an amino-acid free medium (EBSS) or EBSS medium containing 2× the amino acid concentrations (2×AA) found in MEM in the absence or presence of A77 1726 for 2 h. Cells were stimulated with insulin (20 nM) for 20 min. The cells were harvested and analyzed for the phosphorylation of AKTS473/T308 (A), S6K1T389 (B), IRS-1S1101 (C), S6S235/236 (D), and reprobed with their specific antibodies for total proteins. The density of bands was analyzed by using an NIH Image-J software and normalized by the arbitrary units of their corresponding total proteins. The results are the mean ± s.d. from three experiments. A77, A77 1726; Rapa, rapamycin. *P < 0.05; **P < 0.01, compared to the control (with insulin and 2× amino acids but without drug).

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    A77 1726 stimulates insulin receptor tyrosine phosphorylation and increases the insulin receptor substrate (IRS-1) binding to the p85α subunit of the PI-3 kinase. (A) 3T3-L1 adipocytes and L6 myotubes were similarly treated as in Fig. 3 and analyzed for the phosphorylation of insulin receptor tyrosine phosphorylation at Y1146. (B) A77 1726 increases the binding of the p85 subunit of the PI-3 kinase to IRS-1. 3T3-L1 adipocytes and L6 myotubes were treated in Fig. 3. Cell lysates were immunoprecipitated with an anti-p85 antibody followed by probing with anti-p85 and anti-IRS-1 antibodies in Western blot. Relative protein phosphorylation was analyzed by using Image J software. The results are the mean ± s.d. from three experiments. A77, A77 1726; *P < 0.05; **P < 0.01.

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    A77 1726 enhances insulin-induced GLUT4 translocation to the plasma membrane. (A) GLUT4 translocation to the plasma membrane. mCherry-GLUT4-myc-transfected L6 cells were starved of serum for 4 h and then incubated in the absence or presence of A77 1726 (200 µM) without or with 2× the amino acid concentrations. Cells were left unstimulated or stimulated with 20 nM insulin for 45 min. After fixation in methanol for 10 min, mCherry-tagged GLUT4 fluorescence was visualized under a Leica LP8 confocal microscope. Arrows denote the mCherry-tagged GLUT4 translocation to the cell membrane. (B) Quantification of the GLUT4 translocation to the plasma membrane. The data represent the mean ± s.d. from one of three experiments with similar results. *P < 0.05; **P < 0.01. A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0536.

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    A77 1726 increases glucose uptake. L6 myotubes (A) and 3T3-L1 adipocytes (B) seeded in 24-well plates were starved in serum-free, low glucose DMEM medium for 4 h. The cells were then incubated in EBSS in the absence or presence of 2×AA and/or A77 1726 (200 µM) or rapamycin (50 nM) for 2 h. The cells were left unstimulated or stimulated with 20 nM insulin for 45 min followed by incubation of [3H]-2-DG for 5 min. The data are the mean ± s.d. of the triplicate in one of three experiments with similar results. *P < 0.05; **P < 0.01, compared to the control (with insulin and 2× amino acids but without drug).

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    Control of hyperglycemia and insulin receptor sensitization by leflunomide. (A) ob/ob mice were treated as described in ‘Materials and methods’ section. The mice were challenged with glucose (1 g/kg) by intraperitoneal injection for GTT. Blood glucose levels were measured at the indicated time. CMC vs leflunomide, uridine or leflunomide + uridine, P < 0.001; leflunomide vs leflunomide + uridine, P = 0.002. Lef, leflunomide; Uri, uridine; The arbitrary values of AUC were calculated and shown in a bar graph (B) **CMC vs leflunomide or leflunomide + uridine, P < 0.001; CMC vs uridine, P = 0.057. (C) Control of hyperglycemia by leflunomide in ITT. ob/ob mice were treated as described in ‘Materials and methods’ section. After fasting for 6 h, insulin (2.5 U/kg) was injected intravenously. Blood glucose levels were measured at the indicated times. CMC vs leflunomide, uridine or leflunomide + uridine, P < 0.001; leflunomide vs leflunomide + uridine, P = 0.006. (D) AUC in the different treatment groups was shown as the bar graph. **CMC vs leflunomide or leflunomide + uridine, P < 0.001; CMC vs uridine, P = 0.115. (E and G) C57BL/6 male mice (5w-weeks-old) fed on normal chow diet (NCD) or high-fat diet (HFD) for 10 weeks were treated daily with CMC or leflunomide (35 mg/kg/day) for 3 days and then evaluated for blood glucose levels in GTT (E) and ITT (G). (F and H) AUC in different treatment group was shown as bar graphs. **CMC vs leflunomide in mice fed with HFD, P < 0.001; CMC vs leflunomide in mice fed with NCD, P > 0.05. A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0536.

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    AKT phosphorylation in the metabolic tissues is enhanced by leflunomide. C57BL/6 (A) and ob/ob (B) mice were treated daily with CMC (1.5%) or leflunomide (35 mg/kg/day) for 3 days. On day 4, the mice were treated with a last dose and then fasted for 6 h. Mice were then injected intravenously with saline or insulin (2.5 unit/kg). Five minutes later, the mice were sacrificed. Gastrocnemius muscle, visceral white adipose and liver tissues were harvested and analyzed for AKTS473/T308 phosphorylation by Western blot with their specific antibodies and reprobed with an antibody against total AKT protein. Immunoblots represent the data from two animals in each group. The experiment was repeated twice. (C and D) The density of the bands in the tissues of normal C57BL/6 mice (C) and ob/ob mice (D) was analyzed by using NIH Image-J software and normalized by the arbitrary units of their corresponding total proteins. The results are the mean ± s.d. from four animals in each group. *P < 0.05; ns, not significant between CMC/insulin-treated and leflunomide/insulin-treated group.

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