Pharmacological inhibition of S6K1 increases glucose metabolism and Akt signalling in vitro and in diet-induced obese mice

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1 Diabetologia (6) 9:9 6 DOI.7/s ARTICLE Pharmaologial inhibition of K inreases gluose metabolism and Akt signalling in vitro and in diet-indued obese mie Mihael Shum & Kerstin Bellmann & Philippe St-Pierre & André Marette Reeived: 8 June /Aepted: November /Published online: January 6 # Springer-Verlag Berlin Heidelberg 6 Abstrat Aims/hypothesis The mammalian target of rapamyin omplex (mtorc)/p7 ribosomal kinase (K) pathway is overativated in obesity, leading to inhibition of phosphoinositide -kinase (PIK)/Akt signalling and insulin resistane. However, hroni mtorc inhibition by rapamyin impairs gluose homeostasis beause of robust indution of liver gluoneogenesis. Here, we ompared the effet of rapamyin with that of the seletive K inhibitor, PF-7867, on gluose metabolism in vitro and in vivo. Methods We used L6 myoytes and FAO hepatoytes to explore the effet of PF-7867 on the regulation of gluose uptake, gluose prodution and insulin signalling. We also treated high-fat (HF)-fed obese mie for 7 days with PF-7867 in omparison with rapamyin to assess gluose tolerane, insulin resistane and insulin signalling in vivo. Results Chroni rapamyin treatment indued insulin resistane and impaired gluose metabolism in hepati and musle ells. Conversely, hroni K inhibition with PF-7867 redued gluose prodution in hepatoytes and enhaned gluose uptake in myoytes. Whereas rapamyin treatment inhibited Akt phosphorylation, PF-7867 inreased Akt phosphorylation in both ell lines. These opposite effets of the mtorc and K Eletroni supplementary material The online version of this artile (doi:.7/s--89-6) ontains peer-reviewed but unedited supplementary material, whih is available to authorised users. André Marette andre.marette@riupq.ulaval.a Department of Mediine, Quebe Heart and Lung Institute, Hôpital Laval, Pavillon Marguerite d Youville, Room Y8, 7 Chemin Ste-Foy, Québe, Canada GV G inhibitors were also observed in vivo. Indeed, while rapamyin treatment indued gluose intolerane and failed to improve Akt phosphorylation in liver and musle of HF-fed mie, PF-7867 treatment improved gluose tolerane and inreased Akt phosphorylation in metaboli tissues of these obese mie. Conlusions/interpretation Chroni K inhibition by PF-7867 improves gluose homeostasis in obese mie through enhaned Akt ativation in liver and musle. Our results suggest that speifi K blokade is a valid pharmaologial approah to improve gluose disposal in obese diabeti individuals. Keywords Akt. Gluose tolerane. Gluose uptake. Hepati gluose prodution. resistane. mtorc. PF Rapamyin. K. Type diabetes Abbreviations AMPK AMP kinase CREB AMP response element-binding protein FOXO Forkhead box O G6Pase Gluose-6-phosphatase GTT Gluose tolerane test HF High fat HFD High-fat diet HNFα Hepatoyte nulear fator α KO Knokout MSK Mitogen and stress ativated kinase mtorc/ Mammalian target of rapamyin omplex / PGCα Peroxisome proliferator-ativated reeptor γ oativator α PIK Phosphoinositide -kinase PTT Pyruvate tolerane test RSK p9 ribosomal kinase

2 Diabetologia (6) 9:9 6 9 K TG Introdution p7 ribosomal kinase Triaylglyerol is an essential hormone that regulates many physiologial pathways suh as ellular growth, proliferation, gluose and lipid homeostasis. Impaired insulin ation has been shown to play major roles in metaboli diseases, suh as obesity and type diabetes. Several Ser/Thr kinases have been found to phosphorylate multiple Ser residues in IRS- in response to growth fators, nutrient exess or inflammatory stimuli, whih inhibit insulin signalling and ontribute to insulin resistane. These inlude the mammalian target of rapamyin (mtor) and p7 ribosomal kinase (K) as well as -Jun NH -terminal kinase (JNK) and inhibitor of nulear fator-κb (IKK)[, ]. Both ribosomal kinases p7 ribosomal kinase (K) and p9 ribosomal kinase (RSK) are well reognised key insulin signalling proteins [, ]. The two K isoforms, K and K, were originally thought to be redundant proteins due to their high homology. However, reent studies suggest distint roles for eah isoform (reviewedin[]). Indeed, K partiipates in insulinmediated ell growth but like mtorc also operates negative feedbak loops at various of regulation. For example, several studies have shown that both mtorc and/or K phosphorylate speifi serine residues on IRS proteins [6, 7]. Moreover, some of these IRS- sites, notably Ser and Ser 66, were differentially regulated by mtor and K [8, 9]. We have further reported that Ser is an important target of K and that this site is hyperphosphorylated in animal models of obesity and upon nutrient exess in humans []. Reently, Liu et al [] reported that K an also disrupt mtorc by phosphorylating Sin on both T86 and T98 residues. All these negative feedbak ontrol mehanisms reveal the importane of K in the regulation of insulin s pleiotropi ations. The metaboli funtions of K were also demonstrated using geneti models. Indeed, K knokout (KO) mie are resistant to high-fat (HF)-diet-indued obesity, and showed improvement of gluose tolerane and insulin signalling []. However, K KO mie on standard low-fat diet are also hypoinsulinaemi, gluose intolerant and have redued beta ell mass due to a lesion in gluose sensing or insulin prodution []. Younis et al [] further observed an improvement in gluose tolerane using antisense oligonuleotides against K in mie for weeks; however, this was assoiated with a derease in body weight and food intake. Interestingly, adeno-assoiated virus (AAV)-mediated hepati K/ depletion was reently shown to improve systemi insulin resistane, hepati gluoneogenesis and hepati steatosis []. These studies suggest that K inhibition may be an interesting therapeuti option to alleviate obesitylinked insulin resistane and type diabetes. Aute pharmaologial inhibition of the mtorc/k pathway with rapamyin inreases insulin signalling and gluose metabolism in vitro and in animal models of insulin resistane [, 6 8]. However, hroni rapamyin treatment auses diabetes-like symptoms due to alterations in hepati gluoneogeni genes and lipid storage in fat [9, 9, ]. However, no studies have yet evaluated the effet of speifially inhibiting K on ellular insulin signalling, gluose metabolism and whole-body gluose homeostasis. Reently, the seletive K inhibitor PF-7867 has been haraterised [], allowing us to test the effet of pharmaologial inhibition of K on gluose metabolism for the first time. Here, we observed that PF-7867 redued hepati gluose prodution and stimulated musle gluose uptake in vitro, in assoiation with inreased Akt phosphorylation in both hepatoytes and myoytes. Moreover, PF-7867 improved gluose tolerane in HF-fed obese mie by restoring Akt S7 phosphorylation in metaboli tissues, suggesting that K inhibition may represent a valid approah to improve gluose ontrol in obese individuals with type diabetes. Methods Reagents and antibodies Reagents and antibodies are inluded in Eletroni Supplementary Material (ESM) Methods. Cell ulture L6 myoblasts were grown in α-mem (Invitrogen, Burlington, ON, Canada) supplemented with % (vol./vol.) FBS and differentiated into myotubes in α-mem with % (vol./vol.) FBS as previously desribed []. L6 ells were serum-deprived for h prior to the experiments, and nmol/l of insulin was used to stimulate the ells during the last min of deprivation. FAO hepatoytes were maintained in RPMI media (Invitrogen), serum-deprived overnight and stimulated with the indiated onentration of insulin. Vehile (dimethyl sulfoxide, DMSO.% [vol./vol.]) or rapamyin ( nmol/l) or PF-7867 ( μmol/l) were used sine p7 kinase is fully inhibited at this dose []. Only myoplasma free ells were used. Western analyses Western blots were performed as desribed [9]. Briefly, equal amounts of proteins were separated by SDS-PAGE (7.% [wt/vol.]) and transferred onto nitroellulose membrane. Membranes were bloked in % (wt/vol.) fish gelatin diluted in Tris ph 7. +.% (vol./vol.) Tween (TBS-T) and inubated overnight at C with the respetive antibodies diluted in % (wt/vol.) fish gelatin in TBS-T.

3 9 Diabetologia (6) 9:9 6 Gluose uptake Gluose (-DG) uptake was performed as previously desribed [] (seeesmmethods). Gluose prodution Gluose prodution was evaluated in FAO hepatoytes as desribed previously [] (seeesm Methods). Animal studies Animal handling and treatment were approved by the Animal Care and Handling Committee of Laval University. Male C7Bl/6 mie (6 weeks old) were purhased from Charles River Laboratories (St Constant, QC, Canada) and housed individually in ages in a room kept at ± C with a h light/ h dark yle. Mie were randomly distributed among groups but mathed for body weight at the beginning of the studies. The gluose and pyruvate tolerane tests were done blinded. Male C7Bl/6 mie (6 weeks old) were fed high-fat diet (HFD, 6% energy from fat, Researh Diets, D9 (New Brunswik, NJ, USA)) for weeks before being randomly assigned to three groups: () ontrol (HF) reeiving vehile (8% EtOH [vol./vol.],.% [wt/vol.] arboxymethylellulose sterile); () treated with PF-7867 ( mg kg day, i.p.); or () treated with rapamyin ( mg kg day, i.p.) for 7 days while being kept on the same HFD (ESM Methods). i.p. gluose and pyruvate tolerane tests After weeks of HFD feeding, mie were fasted for 6 h and injeted i.p. with gluose ( g/kg) diluted in saline,.9% (wt/vol.) (NaCl mmol/l). For the pyruvate tolerane test (PTT), mie were fasted for 6 h and injeted i.p. with pyruvate ( g/kg; Sigma-Aldrih, Oakville, ON, Canada) diluted in saline,. 9% (wt/vol.). Plasma/tissue determinations Blood gluose were measured by a One Touh Mini Ultra Gluometer (LifeSan, CA, USA). Plasma insulin was determined by radioimmunoassay (Lino Researh, St Charles, MO, USA). Triaylglyerol (TG) in plasma and liver were measured by enzymati methods aording to the manufaturers instrutions (Randox Lab kit, WV, USA). Total lipid was isolated from. g frozen liver using hloroform methanol extration []. Hepati TG ontent was enzymatially determined from reonstituted lipid extrat. Liver glyogen was determined using the phenolsulphuri aid reation []. Nulear extrats Liver nulear extrats were prepared as previously desribed [9] and subjeted to immunoblotting as desribed above. RNA extration and quantitative PCR analysis RNA extration and quantitative PCR analysis were performed as desribed previously [9] (seeesmmethods). Quantifiation and statistial analyses One- and two-way ANOVA with Newman Keuls post ho was performed with Sigma Plot version (Systat Software, San Jose, CA, USA); p values were onsidered signifiant if they were <.. SEMs are represented in the graphs. The riterion for exlusion of animals was a >% deviation from the mean weight of the other animals of the same group. The riterion for the exlusion of any data point was a distane > ±. SD from the mean of the other data from the same group. Results K inhibition dereases phosphorylation and inreases Akt phosphorylation in hepatoytes As the main target of K, ribosomal protein phosphorylation was first measured after treatment with PF-7867 in hepatoytes. Both basal and insulin-stimulated phosphorylation on S/6 were rapidly inhibited by PF-7867, and the inhibitory effet was maintained for up to h in insulintreated ells (Fig. ). Moreover, K Thr89 phosphorylation inreased after PF-7867 treatment, a finding that is in line with previous reports, and that was suggested to involve some feedbak pathways by whih K might regulate its own phosphorylation by mtorc [, 6]. As previously shown using geneti tools in ells [], pharmaologial K inhibition by PF-7867 redued insulin-stimulated IRS S phosphorylation. PF-7867 treatment inreased insulin-indued Akt phosphorylation from h and this effet was sustained for up to h (Fig. b, ). We also ruled out that PF-7867 might inhibit mitogen and stress ativated kinase (MSK) sine this inhibitor did not inhibit Thr8 MSK and Ser AMP response element-binding protein (CREB) indued by phorbol myristate aetate (PMA) in hepatoytes (ESM Fig. ), in line with previous work by Peare et al []. K inhibition dereases gluose prodution in hepati ells To evaluate the role of K in liver gluose metabolism, we measured basal and insulin-suppressed gluose prodution in FAO hepati ells. PF-7867 treatment for h dereased basal gluose prodution, whih was further redued with inreasing onentrations of insulin (Fig. a). We previously reported that hroni rapamyin treatment inreased hepati gluose prodution in rats and FAO ells [9]. We thus ompared the effet of hroni PF-7867 treatment with that of rapamyin by treating FAO ells for 7 h with either drug before measuring gluose prodution. As illustrated in Fig. b, hroni K inhibition redued basal gluose prodution while hroni rapamyin treatment inreased it. When orreted for differenes in basal rates of gluose prodution, the results revealed that rapamyin treatment redued the

4 Diabetologia (6) 9:9 6 9 Fig. Time-dependent K inhibition by PF signalling analysis after timedependant K inhibition by PF-7867 (a, b). FAO ells were treated with μmol/l PF for the indiated times followed by min insulin treatment ( nmol/l). These blots are representative of at least three separate experiments. () Quantifiation of basal (white bars) and insulin-stimulated (blak bars) phospho-akt are shown; n =.p <., vehile vs PF-7867 treated ells a PF h h h 8h h - h h h 8h h p /6 pthr89 K K ps IRS IRS b PF h h h 8h h - h h h 8h h ps7 Akt ps7 Akt/Akt (fold inrease vs basal) pt8 Akt/Akt (fold inrease vs basal) 7 Veh h h h 8h h Veh h h h 8h h pt8 Akt Akt suppressing effet of insulin while insulin ation on gluose prodution remained unaffeted by K inhibition (Fig., d). Nulear extrations of proteins involved in gluoneogeni enzyme expression revealed that hepatoyte nulear fator α (HNFα), forkhead box O (FOXO) and CREB were dereased after 7 h inhibition of K while hroni rapamyin treatment inreased the of these proteins ompared with vehile (Fig. e). However, peroxisome proliferator-ativated reeptor γ, oativator α (PGCα) expression in the nuleus was not affeted by the treatments. In line with these results, FAO ells treated with PF-7867 showed redued mrna expression of the gluoneogeni enzymes PEPCK (also known as Pk)andG6Pase (also known as G6p)(Fig.f). Chroni rapamyin treatment inreased G6Pase protein ontent but PF-7867 failed to affet gluose-6-phosphate (G6Pase) and PEPCK protein (Fig. g). As expeted, both aute and hroni PF-7867 treatment redued insulin-mediated phosphorylation (Fig. h). On the other hand, hroni rapamyin treatment ompletely redued both basal and insulin-stimulated phosphorylation, even below the level of vehile-treated ontrol ells. Interestingly, aute and hroni inhibition of K inreased insulin-indued Akt phosphorylation while this was fully abrogated in ells hronially exposed to rapamyin (Fig. h). Whereas rapamyin treatment inreased basal Akt T8 phosphorylation, insulin-indued Akt S7 phosphorylation was disrupted by the inhibitor. K inhibition inreases gluose uptake in L6 myoytes We next determined whether K inhibition improves gluose uptake using L6 myoytes. PF-7867 markedly inreased basal gluose uptake after h and this effet persisted up to 8 h ompared with vehile-treated ells (Fig. a). did not further inrease the effet of the K inhibitor on gluose uptake. Conversely, 8 h rapamyin treatment signifiantly redued basal and insulin-mediated gluose uptake in these musle ells. The stimulatory effet of PF-7867 on gluose uptake was fully abolished by Akt inhibition using the Akt inhibitor, Viii (Fig. b). As in hepatoytes, PF-7867 redued insulin-indued phosphorylation while hroni rapamyin treatment fully abrogated this response in L6 musle ells (Fig. ). After 8 h treatment, K inhibition inreased Akt phosphorylation. However, rapamyin was found to derease Akt S7 while inreasing both basal and insulinmediated Akt T8 phosphorylation (Fig. ). Reently, PF-7867 was reported to enhane AMP kinase (AMPK) phosphorylation in mouse embryoni fibroblasts laking K [7]. However, AMPK Thr7 phosphorylation was not inreased after and 7 h treatment of L6 myoytes with the K inhibitor (Fig. d). Colletively, these results demonstrate that gluose metabolism is inreased after PF-7867 exposure in both musle and hepati ells. In vivo treatment with PF-7867 improves gluose homeostasis in obese mie To evaluate the therapeuti potential of PF-7867 in vivo, we treated HFD-fed obese mie with PF-7867 for week and ompared its effet with that of rapamyin. As expeted, the HFD indued body weight gain, adiposity, hyperglyaemia and hyperinsulinemia as ompared with mie fed the how diet (Table ). While PF-7867 did not affet body weight or adiposity, rapamyin treatment was found to signifiantly redue body weight, whih was partly related to dereased inguinal adipose tissue weight. Interestingly, only week of PF-7867 treatment was found to improve fasting gluose whereas rapamyin further inreased fasting hyperglyaemia in obese mie. In addition to fasting gluose, rapamyin treatment was also found to inrease fasting insulinaemia as previously reported in various rodent models [9,, 8, 9].

5 96 Diabetologia (6) 9:9 6 a b e Guose prodution (fold over basal from vehile) Gluose prodution (% vs basal) g Protein gluoneogeni enzyme expression (fold inrease over basal)... x - x - Rapa h - PF - h 7h G6Pase PEPCK x - x - x - x - x -9 x -8 x -7 (mol/l) (mol/l) x - x - x -9 x -8 x -7 Veh PF h PF 7h R 7h I Guose prodution (fold over basal from vehile) Gluose prodution (% vs basal).... Veh PF h PF 7h R 7h p /6 ps7 Akt pt8 Akt Akt Fig. K inhibition dereased gluose prodution and inreased Akt phosphorylation in hepatoytes. Gluose prodution was evaluated in FAO ells as desribed in Methods. Cells were treated with vehile (blak irles) or with μmol/l PF-7867 (blak squares) for h (a, ) or 7 h (b, d) and ompared with ells treated with nmol/l rapamyin (blak triangles) for 7 h; n = ; p <. for rapamyin- vs vehileand PF-7867-treated ells; p <., p <. for PF-7867 vs vehile. (e) Representative blots and quantifiation of CREB (white bars), HNFα (grey bars), PGCα (blak bars), FOXO (hathed bars) and histone H of nulear extrats from FAO ells treated with μmol/l PF-7867 or nmol/l rapamyin. p <. vs vehile from the same treatment group. (f) mrna of G6pase (white bars) and Pepk (blak bars) were assessed by semi-quantitative RT-PCR in FAO ells d h x - x - x - x - x -9 x -8 x -7 (mol/l) x - x - x - x - x -9 x -8 x -7 (mol/l) CREB HNFα PGCα FOXO Histone H f Fold inrease over vehile mrna gluoneogeni enzyme expression (fold inrease over basal) ps7 Akt/Akt (fold inrease vs basal) pt8 Akt/Akt (fold inrease vs basal)... Veh PF h PF 7h R 7h Veh PF h PF 7h R 7h Veh PF h PF 7h I Veh PF h PF 7h R 7h Veh PF h PF 7h R 7h treated with PF-7867 or insulin ( nmol/l, h). The graphs depit mrna expression in FAO ells of target genes orreted for the expression of 6B (also known as Rplp) as a ontrol gene, n =,p <.vs vehile. (g) Protein of G6Pase (white bars) and PEPCK (blak bars) were measured in FAO ells treated with PF-7867, rapamyin or insulin ( nmol/l, h), n =,p <. vs vehile. (h) Phospho- /6 and Akt phosphorylation were measured in FAO ells treated with μmol/l PF-7867 or nmol/l rapamyin and basal (white bars) or min insulin-stimulated ells (blak bars) ( nmol/l) were analysed. p <. vs insulin- and PF-insulin-treated groups, p <., p <.vs vehile treated ells, as indiated. I, insulin; PF, PF-7867; R, rapamyin; Veh, vehile To determine the impat of K inhibition on gluose homeostasis, we next performed a gluose tolerane test (GTT). The HFD indued gluose intolerane, whih was partially reversed by K inhibition but further exaerbated by rapamyin treatment (Fig. a, b). The improvement of gluose tolerane by K inhibition was independent of

6 Diabetologia (6) 9: a -DG uptake (fold inrease over basal) Veh PF h PF 8hR 8h p /6 ps7 Akt pt8 Akt Akt ps7 Akt/Akt (fold inrease vs basal) b -DG uptake (fold inrease over basal) Veh PF 8h Aktin PF h PF 8h R 8h pt8 Akt/Akt (fold inrease vs basal) Veh PF h PF 8h R 8h d pthr7 AMPK Veh PF h PF 8h AMPK p /6 Fig. K inhibition inreased gluose uptake and inreased Akt phosphorylation in L6 myotubes. (a) Gluose (-DG) uptake was evaluated in L6 ells treated with vehile (white bars) or μmol/l PF-7867 ( h, grey bars; 8 h, blak bars) or nmol/l rapamyin for 8 h (hathed bars) as desribed in the Methods. Results are presented as fold over basal onditions and are the means ± SEM of three independent experiments. p <. for insulin vs basal onditions (white bars). (b) Cells were used as in (a) with or without Akt inhibitor-viii (Akt i; μmol/l) for the last hour. () Phospho- /6 and Akt phosphorylation were measured in L6 ells treated with μmol/l PF-7867 or nmol/l rapamyin and basal (white bars) or min insulin-stimulated ells (blak bars) ( nmol/l) were analysed, n =. p <. vs insulin- and PF-insulin-treated groups (blak bars), p <. vs vehile treated ells. (d) Phospho-Thr7 AMPK and phospho- /6 were measured in L6 ells pre-treated with μmol/l PF-7867 or vehile (DMSO.%) and basal (white bars) or min insulin-stimulated ells (blak bars) ( nmol/l) were analysed, n =.p <.,p <., p <. in all figure parts, as indiated. AMPK, AMP kinase; PF, PF-7867; R/Rapa, rapamyin; Veh, vehile hanges in plasma insulin while rapamyin inreased the insulin response during the GTT (Fig. ). HF feeding also inreased liver TG but dereased liver glyogen ontent (Fig. d, e). PF-7867 tended to derease slightly liver TG and glyogen ontent ompared with the HF group while rapamyin tended to inrease them leading to a statistially signifiant differene between the drugs. Chroni PF-7867 and rapamyin effet on gluoneogenesis in vivo The rapamyin-indued deterioration of gluose tolerane was also assoiated with inreased gluoneogenesis as measured by a PTT, whih was not affeted by PF treatment (Fig. a). Aordingly, PF treated mie expressed a similar level of nulear PGCα, HNFα andcrebomparedwithhf-fed ontrol mie (Fig. b). As expeted from the inreased hepati gluoneogenesis in the rapamyin-treated mie, we found higher nulear expression of CREB and HNFα in the liver of these animals. HF-fed mie treated with the K inhibitor expressed similar mrna of gluoneogeni enzymes ompared with vehile-treated HF-fed ontrols while G6Pase mrna were inreased in liver of rapamyin-treated mie (Fig. ). Chroni PF-7867 improved Akt S7 phosphorylation in HF-fed mie tissues To further explore the mehanisms underlying the metaboli phenotype of PF-7867-treated mie, we next analysed Akt phosphorylation in liver, musle and adipose tissues. In liver, the HFD indued a derease in insulin-stimulated Akt S7 phosphorylation in all tissues, whih was fully restored by PF-7867 in liver and musle, but only partially improved in adipose tissue. In ontrast,

7 98 Diabetologia (6) 9:9 6 Table Effet of 7-day PF-7867 and rapamyin treatment on weight, food intake and metaboli variables of treated mie Metaboli variable Standard diet HFD Vehile Vehile PF Rapa Body weight (g) 8.78 ±. a 9. ±. b 8.7 ±.7 b,. ±.9 Energy intake (kj/day).6 ±.8.9 ±..9 ±.96. ±. Fasting gluose (mmol/l) 8.8 ±.6 a.6 ±. b 9.8 ±.6 a.8 ±. Fasting insulin (pmol/l).6 ±. a 68.9 ±. b. ±. b 69.6 ± 8. TG (mmol/l). ±.6 a.6 ±. a, b. ±. a, b.6 ±. b Liver (g). ±..8 ±.8. ±.. ±. Panreas (g). ±..9 ±.. ±..9 ±. Gastronemius (g). ±.. ±.. ±.. ±. Heart (g). ±. a. ±. a, b. ±. a, b. ±. b Inguinal WAT (g). ±. a. ±. b,.7 ±. b.99 ±. Ret WAT (g). ±. a.7 ±. b.68 ±. b.6 ±. b Epididymal WAT (g). ±. a. ±. b. ±.9 b.9 ±.9 b BAT (g).9 ±.. ±.. ±.. ±. Fat mass (%). ±.6 a.99 ±.7 b. ±.78 b.8 ±.9 b Lean mass (g).6 ±.. ±.7.8 ±..6 ±. Data are means ± SE (n =7 ontrol and HFD group; n =8 for PF-7867 and rapamyin group) Means not sharing a ommon supersript are signifiantly different from eah other, p <. BAT, brown adipose tissue; PF, PF-7867; Rapa, rapamyin; Ret, retroperitoneal; WAT, white adipose tissue rapamyin treatment only partially restored Akt S7 phosphorylation in liver but not in musle and adipose tissues (Fig. 6a ). The effet of the drug on Akt Thr8 phosphorylation was less lear. Whereas both PF-7867 and rapamyin treatments tended to improve insulin-indued Thr8 phosphorylation in liver and musle (Fig. 6a, b), only rapamyin was found to improve the phosphorylation of this site in adipose tissue of obese mie (Fig. 6). Both PF-7867 and rapamyin were found to blunt the HFD-indued inreased S phosphorylation of IRS- a Gluose (mmol/l) d TG (mg/g liver),,, Time (min) HFD Time (min) HFD GTT (AUC) (mmol/l min) Glyogen (mg/g liver) Fig. K inhibition improved gluose tolerane while rapamyin exaerbated gluose intolerane in HF-fed mie. Mie were treated with PF-7867 and rapamyin as desribed in the Methods and fasted for 6 h before i.p. GTTs. (a)plasmagluose,(b) AUC of plasma gluose and () insulin were measured during a GTT. -fed mie (white irles and white bars), and HF-fed mie treated with vehile (blak b e HFD (pmol/l) 8 6 squares and grey bars), PF-7867 (white squares and blak bars) and rapamyin (white triangles and hathed bars), n =6 8 for eah group. (d) Liver TG and (e) liver glyogen ontent were measured as desribed in the Methods. n =6 8 for eah group. p <., p <.. PF, PF-7867; Rapa, rapamyin; Veh, vehile

8 Diabetologia (6) 9: a Gluose (mmol/l) 6 9 Time (min) PGCα HNFα CREB SP inliverandadiposetissue(esmfig.). IRS- phosphorylation on S ould not be deteted in musle (data not shown). To determine whether the tissue-speifi effets of PF-7867 on Akt phosphorylation may be related to differential inhibition of K in liver, musle and adipose tissue, we further evaluated the phosphorylation state of in those tissues. Unexpetedly, PF-7867 did not inhibit phosphorylation, whih was, however, ompletely inhibited by rapamyin under both basal and insulinstimulated onditions (Fig. 7a ). On the other hand, treatment with PF-7867, but not rapamyin, aused a marked inrease in K phosphorylation, at least in liver and adipose tissue (Fig. 7a ). This is onsistent with the inhibition of K by PF-7867, as seen in vitro (Fig. ) and previous reports [, 6], suggesting that other kinases might phosphorylate ribosomal proteins in those tissues in vivo. Disussion HF diet HF diet HF diet The mtorc/k pathway regulates many essential proesses suh as growth, proliferation, protein synthesis and lipogenesis. In addition, several studies have emphasised the b mrna gluoneogeni enzyme expression (fold over vehile from how) Transriptional fator expression (arbitrary units) Fig. PF-7867 did not reprodue the deleterious effet of hroni rapamyin on hepati gluoneogenesis. (a) Plasma gluose were measured during an i.p. PTT. n =6 8 for eah group. -fed mie (white irles), HF-fed mie (blak squares), HF-fed mie treated with PF-7867 (white squares) and HF-fed mie treated with rapamyin (white triangles). (b) mrna of G6pase (white bars) and Pepk (blak bars) were assessed by semi-quantitative RT-PCR; n =6 8. () Representative blots and densitometri analysis of normalisation of total protein/sp protein of PGCα (blak bars), HNFα (grey bars), CREB (white bars) and SP proteins in nulear extrats prepared from liver samples. n = for eah group. p <., p <., HF group treated with vehile vs PF-7867 and rapamyin-treated ells. PF, PF-7867; Rapa, rapamyin; Veh, vehile.... role of mtorc/k in metabolism sine this pathway senses nutrients and energy (ATP) but also regulates insulin signalling at various. Aute treatment (< h) with the mtorc inhibitor, rapamyin, improves the metaboli ations of insulin through disrupting the negative feedbak loop towards phosphoinositide -kinase (PIK)/Akt signalling in both ellular and animal models [, 6 8]. However, when administered hronially (> h) rapamyin also has important adverse metaboli effets in rodent models and in humans, ausing hyperlipidaemia, hyperglyaemia and hyperinsulinaemia [9, 9,, ]. In the present study, we show that hroni K inhibition does not reprodue the adverse effets observed after hroni rapamyin treatment. In vitro studies using hepati and musle ells revealed that K inhibition with PF-7867 redued hepati gluose prodution and inreased musle gluose uptake, and these effets were seen even in the absene of insulin stimulation. In both ell types we found that, in ontrast to rapamyin treatment, PF-7867 inreased insulin-indued Akt phosphorylation. The therapeuti relevane of these ellular findings was further demonstrated in HF-fed mie, in whih hroni treatment with PF-7867 for 7 days was found to improve gluose tolerane, while hroni rapamyin exaerbated gluose intolerane in these obese animals. This improvement in gluose homeostasis after K inhibition was assoiated with a restoration of Akt phosphorylation in liver, musle and adipose tissues. Although PF-7867 did not inhibit MSK (ESM Fig. ) orrsk[] inmusleand hepati ells, we annot ompletely exlude that part of its benefiial effets on gluose homeostasis in vivo is explained by partial inhibition of these enzymes. However, our finding that pharmaologial inhibition of K improves gluose metabolism is in line with previous studies using geneti models of K defiieny. Um et al [] first showed that K / mie are proteted against diet-indued obesity and insulin resistane, and this was assoiated with inreased Akt S7 phosphorylation in insulin target tissues. More reent studies have also shown that antisense oligonuleotides against K in rats or liver-speifi downregulation of K/ using a lentiviral approah in the liver of HF-fed mie improved gluose tolerane and insulin sensitivity, whih was assoiated with inreased Akt S7 phosphorylation in the liver and adipose tissue in the latter study [, ]. Our results show that K inhibition represents a more suitable target to improve gluose homeostasis as ompared with abrogating the mtorc/k pathway with rapamyin, whih has been reported to impair gluose and lipid metabolism after hroni in vivo exposure [9, 9, 9]. The adverse effets of hroni rapamyin treatment are partly due to off-target effets on mtorc [9,, ], whih impairs Akt S7 phosphorylation and ativity [, ], but these side effets were not observed with seletive K inhibition in the present study. In ontrast, we found that

9 6 Diabetologia (6) 9:9 6 a HF diet ps7 Akt pt8 Akt Akt Relative ps7 Akt/Akt 8 6 Relative pt8 Akt/Akt b HF diet ps7 Akt pt8 Akt Akt eef Relative ps7 Akt/Akt..... Relative pt8 Akt/Akt ps7 Akt pt8 Akt Akt HF diet Relative ps7 Akt/Akt Fig. 6 K inhibition inreased Akt phosphorylation. -fed mie (white bars), HF-fed mie treated with vehile (grey bars), HF-fed mie treated with PF-7867 (blak bars) and HF-fed mie treated with rapamyin (hathed bars) were fasted for 6 h before saline or insulin (.8 U/kg audal, min) vein injetion and tissues were analysed for Akt phosphorylation. Liver (a), gastronemius musle (b) and... Relative pt8 Akt/Akt epididymal white adipose tissue () were lysed and equal amounts of proteins were separated on SDS-PAGE and proessed for western blot analyses using the indiated antibodies. n = 8 for eah group.p <., p <., p <.. PF, PF-7867; Rapa, rapamyin; Veh, vehile PF-7867 inreased Akt S7 phosphorylation, whih was assoiated with blunted inhibitory S phosphorylation on IRS in vitro and in vivo. However, K is also involved in a negative feedbak loop towards mtorc/akt signalling, whih has been reently linked to inhibitory phosphorylation of the mtorc subunit Sin on Thr86 and Thr98 by K [,, 6]. Indeed, Sin phosphorylation by K inhibits the ability of mtorc to phosphorylate Akt at S7. This may explain the preferential effet of PF-7867 on Akt S7 and not Akt T8 as revealed by immunoblot analysis of Akt phosphorylation sites in FAO ells and insulin target tissues of HF-fed obese mie. In line with these results, PF has been also shown to be ardioprotetive against myoardial infartion in mie by inreasing Akt phosphorylation [7]. Thus, inreased Akt ativity likely plays a major role in the benefiial metaboli outomes of K inhibition by PF Our studies in hepati and musle ells further suggest that PF-7867 inreases gluose metabolism independently from insulin ation. Indeed, we observed that gluose prodution by hepatoytes is redued after aute and hroni treatment with PF-7867, whereas insulin-mediated suppression was not further enhaned by K inhibition. Similarly, PF-7867 robustly inreased basal gluose uptake in L6 myoytes and insulin did not further inrease the stimulatory effet of PF-7867 on this proess. This insulinindependent effet of the K inhibitor is onsistent with the finding that K-mediated Sin phosphorylation negatively regulates mtorc independently from insulin signalling []. K inhibition in hepati ells was assoiated with inreased Akt S7 phosphorylation and lower mrna expression of PEPCK and G6Pase gluoneogeni enzymes as well as a redution in CREB, FOXO and HFNα nulear. However, PF-7867 failed to signifiantly redue PEPCK or G6Pase protein expression despite lower mrna expression of these enzymes. It is possible that G6Pase and PEPCK proteins are only redued in speifi ellular ompartments [8 ]. Indeed, we have used a PEPCK-speifi antibody that reognises the ytosoli isoform, whereas PEPCK is predominantly a mitohondrial form. Moreover, other important enzymes for gluoneogeni flux suh as pyruvate arboxylase and frutose,6-bisphosphatase, whih are regulated by CREB, HNFα and FOXO, might also be affeted by PF In addition, previous studies have showed that hepati gluoneogenesis an be regulated

10 Diabetologia (6) 9:9 6 6 a HF diet ps/6 pthr89 K K Relative ps/6 / Relative pthr89 K/K b eef HF diet ps/6 pthr89 K K Relative ps/6 / Relative pthr89 K/K ps/6 pthr89 K K HF diet Relative ps/6 / Relative pthr89 K/K Fig. 7 K inhibition did not derease phosphorylation in HF-fed obese mie. -fed mie (white bars), and HF-fed mie treated with vehile (grey bars), PF-7867 (blak bars) and rapamyin (hathed bars) were fasted for 6 h before saline or insulin (.8 U/kg audal, min) vein injetion and tissues were analysed for and K phosphorylation. Liver (a), gastronemius musle (b) and epididymal white adipose tissue () were lysed and equal amounts of proteins were separated on SDS-PAGE and proessed for western blot analyses using the indiated antibodies. n = 8 for eah group.p <., p <., p <.. PF, PF-7867; Rapa, rapamyin; Veh, vehile independently from the expression of these enzymes. Indeed, hanges in gluoneogeni flux have been previously desribed in humans without hanges in PEPCK/ or G6Pase protein/mrna expression []. Moreover, Foretz et al [] showed that metformin dereases hepati gluose prodution via a transription-independent proess, through hanges in hepati energy state, whih were also independent from AMPK ativation. The detailed moleular mehanisms underlying the suppressive effet of PF-7867 on hepati gluose prodution still need to be fully eluidated. Whereas PF-7867 treatment was found to improve gluose tolerane in HF-fed obese mie, the drug failed to improve gluoneogenesis, as measured during the PTT and by determination of the gluoneogeni programme. Nevertheless, other observations suggest that liver insulin sensitivity was improved in PF-7867-treated obese mie, espeially when ompared with mie hronially exposed to rapamyin. Indeed, liver Akt S7 phosphorylation was normalised in these mie and liver glyogen ontent was improved, while hepati TG were dereased in these animals when ompared with rapamyin-treated obese mie. Although gluoneogenesis is unaffeted, one potential mehanism underlying this hepati phenotype of PF treated obese mie may involve a ompensatory inrease in K ativity. Previous studies have shown that K an ompensate for the loss of K (also known as Rps6kb)inK KO mie and that K is the major kinase phosphorylating ribosomal proteins [, ]. In mie with partial geneti deletion of both K and K (also known as Rps6kb) in the liver, suppression of hepati gluose prodution by insulin was inreased [], suggesting that K might also play a role in gluoneogenesis. Indeed, K- defiient mie are more insulin sensitive than wild-type mie exposed to HFD [] espeially given that K also interats with Yin Yang [6], a transriptional fator that promotes hepati gluoneogenesis [7]. In addition, based on the study of Kim et al [8], K is more ativated in liver of ob/ob mie suggesting that K might also be more ative in our HF-diet-indued obesity model. The potential role of ompensatory mehanisms after hroni inhibition of K by PF-7867 warrants further investigation in future studies. Current pharmaologial treatments fail to ahieve optimal glyaemi ontrol and it is therefore ritial to develop new therapeuti options for patients with type diabetes. In this

11 6 Diabetologia (6) 9:9 6 study, we provide evidene that the speifi K inhibitor PF-7867 dereased hepati gluose prodution and musle gluose uptake in vitro, and improved gluose tolerane in vivo, whih may be of linial relevane for type diabetes treatment. Indeed, unlike mtorc/k inhibition by rapamyin whih has adverse metaboli effets when hronially given in vivo seletive K inhibition learly improved gluose tolerane and insulin signalling in metaboli tissues, as revealed by improvement of Akt phosphorylation in HF-fed obese mie. Our results thus suggest that pharmaologial inhibition of K is a valid therapeuti approah for treatment of obesity-linked type diabetes. Aknowledgements The authors aknowledge V. Dumais, C. Dallaire and C. Dion for their tehnial help at Cardiology Axis of the Institut Universitaire de Cardiologie et de Pneumologie de Québe (CRIUCPQ), Hôpital Laval, Québe, Canada. Funding This work was funded by a grant from Canadian Institutes of Health Researh (CIHR) to AM (FRN- 7878); MS was supported by a PhD studentship from Fond de reherhe du Québe en santé (FRQS). AM holds a CIHR/Pfizer researh Chair on the pathogenesis of insulin resistane and ardiovasular diseases. Duality of interest The authors delare that there is no duality of interest assoiated with this manusript. Contribution statement All authors were responsible for the oneption and design of the study, for the analysis and interpretation of data, and for writing and editing the manusript, and thus all ontributed to its intelletual ontent. All authors gave final approval of the version to be published. AM is the guarantor of this work and, as suh, had full aess to all the data in the study and takes responsibility for the integrity of the data and the auray of the data analysis. Referenes. 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