S6K1 inhibition decreases S6 phosphorylation and increases Akt phosphorylation in hepatocytes
As the main target of S6K1, ribosomal S6 protein phosphorylation was first measured after treatment with PF-4708671 in hepatocytes. Both basal and insulin-stimulated S6 phosphorylation on S235/6 were rapidly inhibited by PF-4708671, and the inhibitory effect was maintained for up to 24 h in insulin-treated cells (Fig. 1). Moreover, S6K1 Thr389 phosphorylation increased after PF-4708671 treatment, a finding that is in line with previous reports, and that was suggested to involve some feedback pathways by which S6K1 might regulate its own phosphorylation by mTORC1 [21, 26]. As previously shown using genetic tools in cells , pharmacological S6K1 inhibition by PF-4708671 reduced insulin-stimulated IRS1 S1101 phosphorylation. PF-4708671 treatment increased insulin-induced Akt phosphorylation from 5 h and this effect was sustained for up to 24 h (Fig. 1b, c). We also ruled out that PF-4708671 might inhibit mitogen and stress activated kinase 1 (MSK1) since this inhibitor did not inhibit Thr581 MSK1 and Ser133 cAMP response element-binding protein (CREB) induced by phorbol myristate acetate (PMA) in hepatocytes (ESM Fig. 1), in line with previous work by Pearce et al .
S6K1 inhibition decreases glucose production in hepatic cells
To evaluate the role of S6K1 in liver glucose metabolism, we measured basal and insulin-suppressed glucose production in FAO hepatic cells. PF-4708671 treatment for 5 h decreased basal glucose production, which was further reduced with increasing concentrations of insulin (Fig. 2a). We previously reported that chronic rapamycin treatment increased hepatic glucose production in rats and FAO cells . We thus compared the effect of chronic PF-4708671 treatment with that of rapamycin by treating FAO cells for 72 h with either drug before measuring glucose production. As illustrated in Fig. 2b, chronic S6K1 inhibition reduced basal glucose production while chronic rapamycin treatment increased it. When corrected for differences in basal rates of glucose production, the results revealed that rapamycin treatment reduced the suppressing effect of insulin while insulin action on glucose production remained unaffected by S6K1 inhibition (Fig. 2c, d). Nuclear extractions of proteins involved in gluconeogenic enzyme expression revealed that hepatocyte nuclear factor 4α (HNF4α), forkhead box O (FOXO) and CREB were decreased after 72 h inhibition of S6K1 while chronic rapamycin treatment increased the levels of these proteins compared with vehicle (Fig. 2e). However, peroxisome proliferator-activated receptor γ, coactivator 1α (PGC1α) expression in the nucleus was not affected by the treatments. In line with these results, FAO cells treated with PF-4708671 showed reduced mRNA expression of the gluconeogenic enzymes PEPCK (also known as Pck1) and G6Pase (also known as G6pc) (Fig. 2f). Chronic rapamycin treatment increased G6Pase protein content but PF-4708671 failed to affect glucose-6-phosphate (G6Pase) and PEPCK protein levels (Fig. 2g). As expected, both acute and chronic PF-4708671 treatment reduced insulin-mediated S6 phosphorylation (Fig. 2h). On the other hand, chronic rapamycin treatment completely reduced both basal and insulin-stimulated S6 phosphorylation, even below the level of vehicle-treated control cells. Interestingly, acute and chronic inhibition of S6K1 increased insulin-induced Akt phosphorylation while this was fully abrogated in cells chronically exposed to rapamycin (Fig. 2h). Whereas rapamycin treatment increased basal Akt T308 phosphorylation, insulin-induced Akt S473 phosphorylation was disrupted by the inhibitor.
S6K1 inhibition increases glucose uptake in L6 myocytes
We next determined whether S6K1 inhibition improves glucose uptake using L6 myocytes. PF-4708671 markedly increased basal glucose uptake after 5 h and this effect persisted up to 48 h compared with vehicle-treated cells (Fig. 3a). Insulin did not further increase the effect of the S6K1 inhibitor on glucose uptake. Conversely, 48 h rapamycin treatment significantly reduced basal and insulin-mediated glucose uptake in these muscle cells. The stimulatory effect of PF-4708671 on glucose uptake was fully abolished by Akt inhibition using the Akt inhibitor, Viii (Fig. 3b). As in hepatocytes, PF-4708671 reduced insulin-induced S6 phosphorylation while chronic rapamycin treatment fully abrogated this response in L6 muscle cells (Fig. 3c). After 48 h treatment, S6K1 inhibition increased Akt phosphorylation. However, rapamycin was found to decrease Akt S473 while increasing both basal and insulin-mediated Akt T308 phosphorylation (Fig. 3c). Recently, PF-4708671 was reported to enhance AMP kinase (AMPK) phosphorylation in mouse embryonic fibroblasts lacking S6K . However, AMPK Thr172 phosphorylation was not increased after 5 and 72 h treatment of L6 myocytes with the S6K1 inhibitor (Fig. 3d). Collectively, these results demonstrate that glucose metabolism is increased after PF-4708671 exposure in both muscle and hepatic cells.
In vivo treatment with PF-4708671 improves glucose homeostasis in obese mice
To evaluate the therapeutic potential of PF-4708671 in vivo, we treated HFD-fed obese mice with PF-4708671 for 1 week and compared its effect with that of rapamycin. As expected, the HFD induced body weight gain, adiposity, hyperglycaemia and hyperinsulinemia as compared with mice fed the chow diet (Table 1). While PF-4708671 did not affect body weight or adiposity, rapamycin treatment was found to significantly reduce body weight, which was partly related to decreased inguinal adipose tissue weight. Interestingly, only 1 week of PF-4708671 treatment was found to improve fasting glucose whereas rapamycin further increased fasting hyperglycaemia in obese mice. In addition to fasting glucose, rapamycin treatment was also found to increase fasting insulinaemia as previously reported in various rodent models [19, 20, 28, 29].
To determine the impact of S6K1 inhibition on glucose homeostasis, we next performed a glucose tolerance test (GTT). The HFD induced glucose intolerance, which was partially reversed by S6K1 inhibition but further exacerbated by rapamycin treatment (Fig. 4a, b). The improvement of glucose tolerance by S6K1 inhibition was independent of changes in plasma insulin levels while rapamycin increased the insulin response during the GTT (Fig. 4c). HF feeding also increased liver TG but decreased liver glycogen content (Fig. 4d, e). PF-4708671 tended to decrease slightly liver TG and glycogen content compared with the HF group while rapamycin tended to increase them leading to a statistically significant difference between the drugs.
Chronic PF-4708671 and rapamycin effect on gluconeogenesis in vivo
The rapamycin-induced deterioration of glucose tolerance was also associated with increased gluconeogenesis as measured by a PTT, which was not affected by PF-4798671 treatment (Fig. 5a). Accordingly, PF-4798671-treated mice expressed a similar level of nuclear PGC1α, HNF4α and CREB compared with HF-fed control mice (Fig. 5b). As expected from the increased hepatic gluconeogenesis in the rapamycin-treated mice, we found higher nuclear expression of CREB and HNF4α in the liver of these animals. HF-fed mice treated with the S6K1 inhibitor expressed similar mRNA levels of gluconeogenic enzymes compared with vehicle-treated HF-fed controls while G6Pase mRNA levels were increased in liver of rapamycin-treated mice (Fig. 5c).
Chronic PF-4708671 improved Akt S473 phosphorylation in HF-fed mice tissues
To further explore the mechanisms underlying the metabolic phenotype of PF-4708671-treated mice, we next analysed Akt phosphorylation in liver, muscle and adipose tissues. In liver, the HFD induced a decrease in insulin-stimulated Akt S473 phosphorylation in all tissues, which was fully restored by PF-4708671 in liver and muscle, but only partially improved in adipose tissue. In contrast, rapamycin treatment only partially restored Akt S473 phosphorylation in liver but not in muscle and adipose tissues (Fig. 6a–c). The effect of the drug on Akt Thr308 phosphorylation was less clear. Whereas both PF-4708671 and rapamycin treatments tended to improve insulin-induced Thr308 phosphorylation in liver and muscle (Fig. 6a, b), only rapamycin was found to improve the phosphorylation of this site in adipose tissue of obese mice (Fig. 6c). Both PF-4708671 and rapamycin were found to blunt the HFD-induced increased S1101 phosphorylation of IRS-1 in liver and adipose tissue (ESM Fig. 2). IRS-1 phosphorylation on S1101 could not be detected in muscle (data not shown).
To determine whether the tissue-specific effects of PF-4708671 on Akt phosphorylation may be related to differential inhibition of S6K1 in liver, muscle and adipose tissue, we further evaluated the phosphorylation state of S6 in those tissues. Unexpectedly, PF-4708671 did not inhibit S6 phosphorylation, which was, however, completely inhibited by rapamycin under both basal and insulin-stimulated conditions (Fig. 7a–c). On the other hand, treatment with PF-4708671, but not rapamycin, caused a marked increase in S6K1 phosphorylation, at least in liver and adipose tissue (Fig. 7a–c). This is consistent with the inhibition of S6K1 by PF-4708671, as seen in vitro (Fig. 1) and previous reports [21, 26], suggesting that other kinases might phosphorylate ribosomal S6 proteins in those tissues in vivo.