Inhibition of mitochondrial complex 1 by the S6K1 inhibitor PF-4708671 partly contributes to its glucose metabolic effects in muscle and liver cells

Abstract

mTOR complex 1 (mTORC1) and p70 S6 kinase (S6K1) are both involved in the development of obesity-linked insulin resistance. Recently, we showed that the S6K1 inhibitor PF-4708671 (PF) increases insulin sensitivity. However, we also reported that PF can increase glucose metabolism even in the absence of insulin in muscle and hepatic cells. Here we further explored the potential mechanisms by which PF increases glucose metabolism in muscle and liver cells independent of insulin. Time course experiments revealed that PF induces AMP-activated protein kinase (AMPK) activation before inhibiting S6K1. However, PF-induced glucose uptake was not prevented in primary muscle cells from AMPK α1/2 double KO (dKO) mice. Moreover, PF-mediated suppression of hepatic glucose production was maintained in hepatocytes derived from AMPK α1/2-dKO mice. Remarkably, PF could still reduce glucose production and activate AMPK in hepatocytes from S6K1/2 dKO mice. Mechanistically, bioenergetics experiments revealed that PF reduces mitochondrial complex I activity in both muscle and hepatic cells. The stimulatory effect of PF on glucose uptake was partially reduced by expression of the Saccharomyces cerevisiae NADH:ubiquinone oxidoreductase in L6 cells. These results indicate that PF-mediated S6K1 inhibition is not required for its effect on insulin-independent glucose metabolism and AMPK activation. We conclude that, although PF rapidly activates AMPK, its ability to acutely increase glucose uptake and suppress glucose production does not require AMPK activation. Unexpectedly, PF rapidly inhibits mitochondrial complex I activity, a mechanism that partially underlies PF's effect on glucose metabolism.

Keywords: AMPK-activated protein kinase (AMPK); PF-4708671; diabetes; gluconeogenesis; glucose homeostasis; glucose metabolism; mechanistic target of rapamycin (mTOR); mitochondrial complex I; obesity; p70 S6 kinase (S6K1).

Figure 1.

PF-4708671 rapidly increased AMPK phosphorylation before inhibiting S6K1. A and B, L6 differentiated myocytes (A) and FAO hepatoma cells (B) were treated with the indicated concentrations of PF-4708671 for 2 h in the presence of FBS (10%). Cells were then harvested and processed for SDS-PAGE and Western blotting using the indicated antibodies. C and D, L6 differentiated myocytes (C) and FAO hepatoma cells (D) were treated for the indicated times with 10 μm PF in the presence of FBS (10%). Actin levels are presented as a loading control. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle-treated cells as indicated. Shown are representative blots and mean ± S.D. from three to five independent experiments.

Figure 2.

PF-4708671 did not increase glucose uptake or decrease hepatic glucose production by activating AMPK. A, WT and AMPK α1/2 KO primary muscle cells in the absence or presence of insulin (100 nm, 45 min) and/or PF-4708671 (10 μm, 5 or 48 h) were lysed and immunoblotted using the indicated antibodies. B, glucose uptake was assessed under similar conditions. a, p < 0.05 versus vehicle-treated WT cells; b, p < 0.05 versus vehicle-treated AMPK α1/2 KO cells; c, p < 0.05 versus vehicle-treated WT cells with insulin; d, p < 0.05 versus vehicle-treated AMPK α1/2 KO cells with insulin. White columns, AMPK α1/2 WT; gray columns, AMPK α1/2 KO. C and D, hepatocytes were isolated from WT and AMPK α1/2 KO mice. After 2 days, the cells were treated with PF-4708671 (10 μm), metformin (0.5 mm), and/or glucagon (25 nm) for 8 h in hepatic glucose production medium, followed by cell lysis for immunoblotting using the indicated antibodies (C), and hepatic glucose production was assessed (D). Met, metformin. White columns, AMPK α1/2 WT; gray columns, AMPK α1/2 KO. Glucose values were corrected for protein content. Shown are the mean ± S.D. of three to four independent experiments and a representative blot from three independent experiments. a, p < 0.05 versus vehicle-treated WT cells without glucagon; b, p < 0.05 versus vehicle-treated AMPK α1/2 KO cells without glucagon; c, p < 0.05 versus vehicle-treated WT cells with glucagon; d, p < 0.05 versus vehicle-treated AMPK α1/2 KO cells with glucagon.

Figure 3.

PF-4708671 decreased basal hepatic glucose production in S6K1/2-deficient hepatocytes. A, hepatic glucose production was assessed in primary hepatocytes culture isolated from WT and S6K1 1/2 KO mice. After 2 days, the cells were treated with PF-4708671 (10 μm), metformin (Met, 0.5 mm), and/or glucagon (Gluc, 25 nm) for 8 h in HGP medium, followed by cell lysis and immunoblotting using the indicated antibodies. B, glucose produced by hepatocytes was measured, and values were corrected for protein content. White columns, S6K1/2 WT; gray columns, S6K1/2 KO. Shown are the mean ± S.D. of at least four independent experiments and a representative blot from three independent experiments. a, p < 0.05 versus vehicle-treated WT cells without glucagon; b, p < 0.05 versus vehicle-treated S6K 1/2 KO cells without glucagon; c, p < 0.05 versus vehicle-treated WT cells with glucagon; d, p < 0.05 versus vehicle-treated S6K 1/2 KO cells with glucagon.

Figure 4.

PF-4708671 decreased mitochondrial respiration and mitochondrial spare respiratory capacity in muscle cells and hepatocytes. L6 cells were seeded at 20,000 cells/well in 24-well XF plates and incubated for 24 h with 10% bovine growth serum (BGS) in MEM at 37 °C/5% CO₂. For differentiation, growth medium was replaced with differentiation medium (2% BGS in MEM). Well-differentiated myocytes were used on day 5 for all assays. A, mitochondrial parameters determined from the oxygen consumption trace following PF, oligomycin (Oligo), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (Rot/AA) injection. B and C, L6 cells were acutely treated with 1–20 μm PF-4708671 by using a port from the Seahorse cartridge followed by a Mitostress protocol. D and E, FAO cells were seeded at 20,000 cells/well in 24-well XF plates and incubated for 3 days with 10% BGS in MEM at 37 °C/5% CO₂. Mitochondrial parameters were assessed with the Mitostress protocol, and PF acute is the decrease in basal respiration induced by PF after 400 min. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle-treated cells as indicated. Shown is the mean ± S.D. of four to five independent experiments.

Figure 5.

PF-4708671 reduced mitochondrial complex 1 activity. On the day of the experiment, cells were washed twice with mannitol/sucrose buffer containing 4 mm ADP and treated with the indicated PF-4708671 concentrations before cell permeabilization with the XF plasma membrane permeabilizer (1 nm) from Seahorse Bioscience. Then the plate was inserted into the XF 24-well apparatus. A and C, mitochondrial complex I and II activity was assessed by successively adding Pyr/Mal (10 mm/1 mm), rotenone (Rot, 1 μm), succinate (10 mm), and antimycin A (AA, 1 μm) in differentiated L6 cells (A) and FAO hepatocytes (C). B–D, mitochondrial complex I activity was calculated from OCR differences between pyruvate/malate and rotenone. Mitochondrial complex II activity was calculated from OCR differences between succinate and antimycin A. *, p < 0.05; **, p < 0.01 versus vehicle-treated cells as indicated. Shown is the mean ± S.D. of four to five independent experiments.

Figure 6.

PF-induced glucose uptake is partially blocked by NDI1 expression in L6 myotubes. Stable L6-pMXS– and L6-NDI1 (NADH:ubiquinone oxidoreductase)–transfected myoblasts were differentiated as described under “Experimental procedures.” NDI1 overexpression was verified by purifying total RNA followed by RNA reverse transcription. A, real-time PCR was then performed, and NDI1 expression was corrected to ARBP gene expression. N.D., not detected. A.U., arbritrary units. B, glucose uptake was measured in L6 cell transfected with either the control vector pMXS or NDI1 and then differentiated. Cells were treated with 10 μm PF for 5 h and/or with insulin (Ins, 100 nm, 45 min). White columns, pMXS; gray columns, NDI1. 2-DG, 2-deoxy-glucose. C, changes in glucose uptake were corrected for the basal glucose uptake level under the respective conditions and are reported as percent. White columns, pMXS; gray columns, NDI1. ***, p < 0.001 versus respective PF-treated pMXS cells; a and b, p < 0.001 versus the respective basal level. Shown is the mean ± S.D. of four independent experiments.