OBJECTIVE

Ciliary neurotrophic factor (CNTF) reverses muscle insulin resistance by increasing fatty acid oxidation through gp130-LIF receptor signaling to the AMP-activated protein kinase (AMPK). CNTF also increases Akt signaling in neurons and adipocytes. Because both Akt and AMPK regulate glucose uptake, we investigated muscle glucose uptake in response to CNTF signaling in lean and obese mice.

RESEARCH DESIGN AND METHODS

Mice were injected intraperitoneally with saline or CNTF, and blood glucose was monitored. The effects of CNTF on skeletal muscle glucose uptake and AMPK/Akt signaling were investigated in incubated soleus and extensor digitorum longus (EDL) muscles from muscle-specific AMPKα2 kinase-dead, gp130ΔSTAT, and lean and obese ob/ob and high-fat–fed mice. The effect of C2-ceramide on glucose uptake and gp130 signaling was also examined.

RESULTS

CNTF reduced blood glucose and increased glucose uptake in isolated muscles in a time- and dose-dependent manner with maximal effects after 30 min with 100 ng/ml. CNTF increased Akt-S473 phosphorylation in soleus and EDL; however, AMPK-T172 phosphorylation was only increased in soleus. Incubation of muscles from AMPK kinase dead (KD) and wild-type littermates with the PI3-kinase inhibitor LY-294002 demonstrated that PI3-kinase, but not AMPK, was essential for CNTF-stimulated glucose uptake. CNTF-stimulated glucose uptake and Akt phosphorylation were substantially reduced in obesity (high-fat diet and ob/ob) despite normal induction of gp130/AMPK signaling—effects also observed when treating myotubes with C2-ceramide.

CONCLUSIONS

CNTF acutely increases muscle glucose uptake by a mechanism involving the PI3-kinase/Akt pathway that does not require AMPK. CNTF-stimulated glucose uptake is impaired in obesity-induced insulin resistance and by ceramide.

Skeletal muscle glucose uptake is regulated by both intrinsic and circulating factors involving the phosphatidylinositol (PI3)-kinase/Akt and the AMP-activated protein kinase (AMPK) signaling pathways (1). The regulation of muscle glucose uptake by both pathways converges on AS160 (TBC1D4) and TBC1D1 where phosphorylation inhibits the negative regulation of GLUT4 vesicle translocation (2,4) to the plasma membrane (5,6). In insulin-resistant skeletal muscle, reduced insulin receptor substrate (IRS) and Akt activation results in reduced skeletal muscle glucose uptake (7,8). In contrast, activation of AMPK by endogenous circulating factors, including adiponectin (9,10) and interleuken (IL)-6 (11,13), and by pharmacological agents such as 5-aminoimidazole-4-carboxamide riboside (AICAR) (14,15) increases muscle glucose uptake, and this response is maintained in muscle from diabetic rodents and humans (16,,19).

We have recently shown that the ciliary neurotrophic factor (CNTF), a member of the IL-6 family, also activates muscle AMPK and increases lipid oxidation (20). CNTF elicits intracellular signaling pathways by ligand binding to the CNTF receptor α (CNTFRα), which initiates heterodimerization and activation of gp130 and leukemia inhibitory factor (LIF) receptor, the two transmembrane β subunits of the receptor complex (21). This differs from the initial step in IL-6 signaling, which induces homodimerization of two gp130 subunits and does not involve LIF (22). Formation of the CNTFRα-gp130-LIF complex leads to association with isoforms of janus kinases, and these subsequently phosphorylate specific tyrosine residues on the intracellular domains, creating docking sites for Src homology–containing tyrosine phosphatase 2 (SH2)-containing proteins (23,24). Phosphorylation of Y757 of the intracellular domain of the β subunit leads to activation of the PI3-kinase/Akt pathway. Activation of the PI3-kinase by gp130-LIF relies on association of the p85 subunit with the regulatory SH2 domain–containing adapter molecule GAB1 coordinated by SHP2 (25), and the assembling of the complex does not involve IRS-1 (26). Phosphorylation of four more distal tyrosine residues leads to activation of the signal transducer and activator of transcription (STAT) 1 and 3 (23). This distal domain is responsible for AMPK activation by an adenosine nucleotide-dependent mechanism (27). Because both AMPK and PI3 kinase/Akt signaling are enhanced by activation of the gp130-LIF receptor, our first aim was to test whether CNTF increases glucose uptake in skeletal muscle and the signaling pathway(s) invoked.

The etiology of impaired muscle insulin signaling with obesity is multifactorial and appears to be related to at least two major events: chronic low-grade inflammation and intramyocellular lipid accumulation (7,28). Both events activate inhibitory IRS-1 kinases such as IκB kinase-β (29,30), c-jun terminal amino kinase (28), and protein kinase C θ (31), which impair insulin signaling by inhibiting IRS-1 association with PI3-kinase subunits. Although this is believed to be a significant element in muscle insulin resistance, the sphingolipid ceramide also impairs insulin signaling by promoting protein phosphatase 2A–dependent Akt dephosphorylation, thereby inhibiting Akt activation (32,33). Thus, because gp130-LIF signaling to glucose uptake would be expected to be independent of IRS-1, our second aim was to determine whether gp130-LIF signaling toward glucose uptake is maintained in mice with obesity-induced insulin resistance. Analysis of the molecular pathways of CNTF signaling to glucose uptake may provide important insights into the function of gp130-LIF signaling and reveal the potential of a novel therapeutic target for type 2 diabetes.

Animals.

C57Bl6/J, ob/ob, AMPKα2 kinase-dead (KD) and gp130ΔSTAT mice aged 8 to 18 weeks with corresponding littermate controls were used in experiments. Transgenic mice overexpressing a KD form of the AMPKα2 protein under control of the muscle creatine kinase promoter have been described previously (34). Briefly, the KD mutation was generated by Lys45Arg mutagenesis to encode a KD α2 protein that displaces all detectable endogenous α2 and 50–70% of endogenous α1 protein from αβγ heterotrimer complexes (34,35). The knockin mutant gp130ΔSTAT mice express a COOH-terminal truncation mutation of gp130 that prevents activation of STAT1/3 as described previously (36). This mutation has a deletion of the distal intracellular domain responsible for AMPK and STAT activation. All mice were kept at a 12:12 h light:dark cycle at 20–21°C and provided ad libitum access to food and water. Mice were maintained on a standard rodent chow diet/low-fat diet (5% calories fat; Harlan Teklad) except in high-fat diet (HFD) experiments, in which the diet was composed of 45% calories from fat (SF04–027; Specialty Feeds). All procedures were approved by the St. Vincent's Hospital Animal Ethics Committee.

CNTF tolerance test.

C57Bl6/J mice were fasted for 6 h before being injected with either saline or 0.3 mg/kg CNTF, a concentration of CNTF that activates AMPK and induces weight loss without eliciting an inflammatory response (20). Blood glucose was monitored over 150 min.

Muscle incubations.

Soleus (oxidative and glycolytic fibers) and extensor digitorum longus (EDL; primarily glycolytic fibers) muscles were dissected from anesthetized mice (6 mg of pentobarbital per 100 g−1 body weight) and transferred to incubation flasks containing 2 ml of essential buffer (Krebs-Henseleit buffer, pH 7.4, with 2 mmol/l pyruvate, 8 mmol/l mannitol, and 0.1% BSA), gassed with 95% O2 + 5% CO2, and maintained at 30°C as previously described (15). For all experiments, muscles were preincubated for 15 min in this buffer before it was replaced with buffer containing AICAR (2 mmol/l for 40 min; Toronto Research Chemicals Inc, Ontario, Canada), CNTF (at concentrations and durations indicated above, Axokine; Regeneron Pharmaceuticals Inc, New York), or insulin (30 nmol/l [additivity study] or 2.8 nmol/l [HFD study] for 40 min, Actrapid; Novo Nordisk, Bagsvaerd, Denmark). In separate experiments, muscles were incubated in LY-294002 hydrochloride (37) (60 nmol/l for 30 min; Sigma-Aldrich Corp, St. Louis, MO) before treatment with CNTF or insulin as described.

2-deoxy-d-glucose (2DG) uptake was measured over 10 min by replacing existing incubation buffer with the buffer described above but with the addition of 0.5 μCi/ml−12-[2,6-3H]-deoxy-d-glucose, 1 mmol/l 2-deoxy-d-glucose, and 0.2 μCi [1-14C]-mannitol/ml. After preparing muscles as described below, radioactivity was measured in muscle lysates by liquid scintillation counting (Tri-Carb 2000; Packard Instrument Co).

Muscle lysate preparation.

Muscles were homogenized in ice-cold buffer (50 mmol/l Hepes, pH 7.4, 150 mmol/l NaCl, 10 mmol/l NaF, 1 mmol/l sodium pyrophosphate, 0.5 mmol/l EDTA, 250 mmol/l sucrose, 1 mmol/l dithiothreitol, 1% TritonX-100, 1 mmol/l Na3VO4, and one Roche protease inhibitor tablet per 50 ml buffer) using an electrical homogenizer. Lysates were prepared as previously described (38) and stored at −80°C until analysis. Protein content in lysates was measured by the bicinchoninic acid method (Pierce).

Immunoblotting.

Expression or phosphorylation of investigated proteins was determined in muscle lysates by SDS-PAGE and immunoblotting using the following primary antibodies: pan-αAMPK, phospho-AMPK T172, and phospho–acetyl CoA carboxylase (ACC) S222 (as previously described [(39)]), STAT3, phospho-STAT3 Y705, Akt, and phospho-Akt S473 (Cell Signaling Technology Inc). Secondary antibodies were horseradish-conjugated protein G (Bio-Rad Laboratories, Richmond, CA). Bands were visualized using an enhanced chemoluminescence system and quantified using ImageQuant TL 05 software (Amersham Biosciences, U.K.). Values obtained using phospho-specific antibodies are expressed as the ratio to the total content of the protein measured after stripping the membrane and reprobing.

AMPK activity.

AMPKα1 and -α2 activities were measured from 100 μg of muscle lysate protein using rabbit polyclonal AMPK antibodies for immunoprecipitation as previously described (40).

Muscle lipids.

Lipids were extracted from freeze-dried powdered gastrocnemius muscle tissues using chloroform:methanol:PBS (1:2:0.8) and 0.2% SDS. Triacylglycerol were saponified in an ethanol-KOH solution at 60°C, and glycerol content was measured fluorometrically. Diacylglycerols and ceramides were extracted and quantified according to the methods of Preiss et al. (41).

Muscle cell studies.

Muscle cells were differentiated and serum starved overnight. The next morning, 20 nmol/l C2-dihydroceramide (Toronto Research Chemicals Inc, Ontario, Canada) was added for 6 h before cells were treated for 20 min with either CNTF (10 ng/ml) or insulin (10 nmol/l) before collecting protein lysates or measuring 2DG uptake as previously described (20). C2C12 cell were used for signaling experiments and L6 muscle cells for 2DG uptake because of the limited capacity of C2C12 to increase glucose uptake in response to stimuli.

Data are expressed as means ± SE. Statistical evaluations were performed by either Student's t test or two-way ANOVA using the Student-Newman-Keuls method as a post hoc test when appropriate. Differences between groups were considered statistically significant if P < 0.05.

In vivo effects of ciliary neurotrophic factor on blood glucose.

Injection of mice with CNTF was associated with a reduction in blood glucose after 40 min compared with saline-injected controls. Blood glucose remained reduced in the CNTF group throughout the remainder of the test (Fig. 1,A). The AUC was 20% lower in CNTF-treated mice than in saline-injected controls (Fig. 1 B).

Muscle glucose uptake: ciliary neurotrophic factor dose response.

The effects of CNTF to reduce blood glucose in vivo may have been mediated by the suppression of hepatic glucose production or increased glucose uptake into adipose tissue and skeletal muscle. Therefore, to directly test the effects of CNTF on skeletal muscle glucose uptake, we incubated isolated muscles with CNTF at concentrations ranging from 2 to 500 ng/ml. CNTF increased muscle glucose uptake over 30 min in a dose-dependent manner with a significant increase at 2 ng/ml in both soleus and EDL muscles (Fig. 2,A–B). Glucose uptake was elevated with increasing CNTF concentrations up to 100 ng/ml with no further increases observed at 500 ng/ml (Fig. 2,A and B), consistent with dose-dependent effects on fatty acid oxidation observed previously (20). We next investigated if the effect of CNTF on glucose uptake was additive to the effects of insulin and AICAR. In soleus, CNTF, insulin, and AICAR independently increased glucose uptake to a similar degree, and combining AICAR and insulin with CNTF had an additive effect (Fig. 2,C). In EDL, insulin and AICAR increased glucose uptake more strongly than CNTF and no further increase was observed when combining these treatments with CNTF (Fig. 2 D).

Time course of CNTF-stimulated muscle glucose uptake and signaling.

CNTF increased glucose uptake in soleus and EDL after 30 min—an effect that diminished thereafter in both muscle types (Fig. 3,A). CNTF has been reported to increase the activity/phosphorylation of AMPK in muscle (20) and Akt in neurons (42), and because both kinases regulate muscle glucose uptake, we examined their activating phosphorylation. CNTF increased Akt S473-P nearly 200% in soleus and 450% in EDL muscle, and these effects diminished after 90 min (Fig. 3,B). AMPK T172-P was increased 90% in the soleus with CNTF after 30 min, and this was maintained for 60 min (Fig. 3,C). Although CNTF induced an apparent rise in ACCβ S218-P in soleus, this did not achieve statistical significance (P = 0.09) (Fig. 3,D). CNTF had no detectable effect on AMPK- or ACCβ-P in EDL (Fig. 3 C–D), which is consistent with previous observations in glycolytic muscle (20).

Effects of PI3-kinase inhibition and ablated AMPK activity on CNTF actions.

We next examined the relative importance of AMPK and PI3K/Akt signaling for CNTF-mediated glucose uptake. Muscles from wild-type and AMPK KD mice were preincubated with or without the PI3-kinase inhibitor, LY-294002, followed by stimulation with CNTF. Glucose uptake was increased to a similar degree with CNTF in wild-type and AMPK KD muscles, indicating that this response was AMPK independent. CNTF-stimulated glucose uptake was abolished by LY-294002 in both muscle types irrespective of mouse genotype (Fig. 4 A), which is consistent with PI3-kinase/Akt signaling being essential for this response.

We next measured α-isoform–specific AMPK activity to verify that CNTF had activated AMPK signaling in wild-type mice and that the activation was impaired in AMPK KD muscles (Fig. 4,B). As anticipated, overexpression of the AMPK KD construct lowered basal AMPK activity of both α-AMPK isoforms in both muscle types, with the most pronounced effect on α2-AMPK activity (34). In wild-type soleus, CNTF increased α1-AMPK activity by 180% and α2-AMPK activity by 35%, whereas CNTF had no effect on either AMPK isoform in AMPK KD muscles (Fig. 4,B). CNTF did not increase AMPK activity in the EDL (Fig. 4,B), which is consistent with CNTF not increasing AMPK phosphorylation in the EDL muscle (Fig. 3 C).

Lastly, to establish whether inhibition of PI3-kinase or AMPK signaling altered proximal CNTF signaling through the gp130-LIF receptor complex, we examined STAT3 Y705-P in the EDL muscle of wild-type and AMPK KD mice in the absence or presence of LY-294002. Incubation with CNTF increased STAT3 Y705-P by 140% in wild-type muscle, and importantly, the increase was unaffected in AMPK-KD muscles and unaffected by coincubation with LY-294002 (Fig. 4,C). We also verified that increases in Akt-P with CNTF were not affected by overexpression of the AMPK KD construct and that LY 294002 inhibited Akt activation (Fig. 4 D). Similar findings were observed in the soleus muscle (data not shown).

CNTF-stimulated glucose uptake in gp130ΔSTAT muscles.

Signaling from the gp130-LIF receptor complex activates two distinct intracellular signaling pathways. The first depends on phosphorylation of four tyrosine residues (Y765, Y812, Y904, and Y914) on the distal cytoplasmic tail of gp130, which is required and sufficient for activating STAT1/3 (23) and AMPK (20). The second arm requires phosphorylation of the more proximal Y757 residue in gp130, which induces ERK and PI3-kinase/Akt signaling (23). We incubated muscles from gp130ΔSTAT mice, which have a deletion of the cytosolic distal portion of the gp130 receptor required for induction of STAT1/3 and AMPK signaling but retain the Y757 residue that is required for activation of Erk and PI3-kinase/Akt signaling. Incubation with CNTF increased glucose uptake in soleus and EDL of wild-type mice, and this increase was maintained in gp130ΔSTAT knockin mutant mice (Fig. 5,A). CNTF increased Akt S473-P in both genotypes, whereas STAT3 Y705-P was increased in wild-type but not in gp130ΔSTAT muscles as expected (Fig. 5 B). Thus, CNTF increased glucose uptake normally despite impaired gp130 signaling and activation of AMPK.

Effects of obesity on CNTF-stimulated muscle glucose uptake and signaling.

It is well established that obesity is associated with impaired insulin sensitivity in skeletal muscle, and impaired activation of IRS-1 has been attributed a primary role (28,43). Because CNTF stimulates glucose uptake downstream of IRS-1, we tested whether CNTF-stimulated glucose uptake is maintained in muscles from mice fed a HFD for 12 weeks and obese mice deficient in leptin (ob/ob). As dictated by design, the high-fat feeding increased body weight by ∼20% (chow, 28.4 ± 0.5 g; HFD, 34.0 ± 0.7 g), fasting insulin levels (836 ± 76 pg/ml to 1,670 ± 377 pg/ml), and the AUC during a GTT (chow, 539 ± 23; HFD, 870 ± 94). Similarly, ob/ob mice had dramatically increased body mass (wild-type, 29.6 ± 0.7 g; ob/ob, 57.3 ± 2.8 g) and AUC during a GTT (WT, 574 ± 26; ob/ob, 1,347 ± 98). CNTF increased glucose uptake by 80 to 100% in the soleus and EDL in chow-fed mice, whereas CNTF-stimulated glucose uptake was completely blunted in the soleus and reduced by ∼50% in EDL of mice fed a HFD (Fig. 6,A). Similarly, CNTF increased muscle glucose uptake in both muscle types of lean littermates of ob/ob mice; however, this increase was abolished in muscles from ob/ob mice (Fig. 6 B).

We then examined the mechanism by which obesity inhibited CNTF-stimulated glucose uptake. CNTF increased STAT3 Y705-P in skeletal muscle of chow- and HFD-fed animals (Fig. 6,C), indicating no impairment of CNTF action at the receptor level. CNTF-induced Akt S473-P was increased by 4- and 19-fold in soleus and EDL of chow-fed mice, respectively, and this increase was markedly impaired with obesity in both muscle types (Fig. 6,D). In contrast, AMPK T172-P in soleus was also unaffected by diet (Fig. 6 E).

Muscle lipids and CNTF signaling.

High-fat feeding increased muscle contents of triacylglycerol, diacylglycerol, and ceramide by ∼100%, ∼50%, and ∼50%, respectively, compared with chow-fed controls (Fig. 7,A). Because ceramides have been shown to directly inhibit insulin-stimulated Akt phosphorylation (33), we tested whether exposure of cultured myotubes to a short-chain ceramide analog also impaired CNTF-stimulated glucose uptake and Akt phosphorylation. Consistent with our findings in obese skeletal muscle, myotubes treated with C2-ceramide had impaired CNTF-stimulated glucose uptake (Fig. 7,B). Reductions in glucose uptake were not the result of inhibition of the gp130-LIF receptor complex because CNTF increased STAT3-P by ∼350% in both vehicle and C2-ceramide–treated cells (Fig. 7,C). However, C2-ceramide reduced basal and CNTF-stimulated Akt S473-P (Fig. 7,D). AMPK T172-P was increased (P < 0.05) by 60% with CNTF, and this increase was unaffected by C2-ceramide treatment (Fig. 7 E).

CNTF can regulate metabolic and growth signaling pathways in several tissue types. The present study shows that CNTF regulates glucose uptake and delineates the proximal signaling events mediating this response. We show that acute exposure of CNTF reduces blood glucose in vivo and increases glucose uptake in soleus and EDL muscle in vitro through the PI3-kinase/Akt signaling pathway. It was expected that AMPK would also exert a role in this process, but we found that AMPK was not required for CNTF to stimulate glucose uptake. Skeletal muscle is the most important tissue for insulin-stimulated glucose disposal (44), and skeletal muscle insulin resistance is a major defect in most obese phenotypes. Accordingly, we investigated whether CNTF-stimulated glucose uptake was maintained in muscle from obese, insulin-resistant mice. The rationale for these studies was to establish whether gp130-LIF signaling could constitute an alternative pathway to substitute for insufficient insulin signaling to skeletal muscle glucose uptake. However, obesity was associated with impaired CNTF-stimulated glucose uptake, which, at least in part, may have been attributed to increased muscle lipids and impaired PI3-kinase/Akt signaling with CNTF.

CNTF is a peptide hormone of the IL-6 family, which is highly expressed in peripheral nerves and other tissues (24,45,46). CNTF levels are low in serum of healthy individual as a result of the absence of an exocytosis targeting sequence (47,48). Recent reports have shown significant promise for the use of CNTF as an antiobesity therapeutic because it suppresses food intake acutely (49) and induces hypothalamic neurogenesis (50), leading to a new set point in body mass. CNTF also has direct antidiabetic effects in peripheral tissues. Chronic treatment of obese diabetic mice with CNTF increases metabolic rate (51) and reduces liver steatosis by enhancing fat oxidation and reducing synthesis of complex lipids (52). We have recently shown that chronic CNTF treatment of obese diabetic mice reverses obesity-induced insulin resistance by activating AMPK and reducing muscle lipid accumulation (20). In line with this study, CNTF acutely prevents muscle insulin resistance in response to a 2-h lipid infusion by preventing lipid accumulation (27). The clinical efficacy and safety of CNTF as a possible therapeutic for obesity is supported by findings in humans demonstrating weight loss and improved glycemic control (53).

Because we previously showed that CNTF activates AMPK in muscle (20) and AMPK is well known to increase glucose uptake in skeletal muscle (15,54,55), we expected AMPK rather than PI3-kinase/Akt signaling to be required for CNTF-stimulated glucose uptake. However, multiple lines of evidence indicate that CNTF signaling to glucose uptake may be mediated by a PI3-kinase/Akt–dependent signaling pathway: 1) the kinetics in Akt S473 phosphorylation correlated with increases in glucose uptake in both soleus and EDL muscles; 2) incubation with the PI3-kinase inhibitor, LY-294002, completely blocked CNTF-stimulated Akt phosphorylation and glucose uptake; 3) CNTF increased glucose uptake was not impaired in muscles overexpressing a KD AMPKα2; 4) AMPK was not activated by CNTF in the EDL muscle despite a 100% increase in glucose uptake; 5) a knockin mutation of a truncated gp130 receptor, which impairs gp130 signaling toward STAT3 and AMPK, had no effect on CNTF-stimulated glucose uptake; and 6) obesity induced by an HFD or treatment of cells with C2-ceramide impaired CNTF stimulated glucose uptake and was associated with blunted Akt but not AMPK phosphorylation. However, our findings demonstrating that CNTF-stimulated glucose uptake was additive to a maximal dose of insulin in Soleus, but not EDL muscle suggest that under some conditions, AMPK activation may also play a role in the stimulation of glucose uptake in Soleus, albeit to a lesser extent than originally anticipated. A possible explanation for the limited role of AMPK in CNTF-stimulated glucose uptake is that CNTF predominantly activated the α1-AMPK isoform. Studies in AMPKα2-null mice have shown that AMPKα2, but not α1 is required for the regulation of muscle glucose uptake in response to stimuli such as AICAR (15), possibly explaining why CNTF activation of AMPK is not required to increase glucose transport.

Because we found CNTF to increase muscle glucose uptake, it was of interest to test the effects of CNTF in insulin-resistant muscle from obese mice. We found that the efficacy of CNTF to regulate glucose uptake was reduced by ∼50% or more in muscles from obese HFD-fed and ob/ob mice. CNTF resistance with obesity seems not to be the result of impaired activation of the gp130-LIF receptor complex because CNTF-induced phosphorylation of AMPK and STAT3 were normal in obese muscle. In contrast, CNTF-stimulated phosphorylation of Akt was suppressed in obese muscles and was associated with muscle DAG and ceramide accumulation. Ceramides downregulate Akt phosphorylation and membrane translocation by activating protein phosphatase 2A (56). Ceramides also increase the activity of protein kinase C ζ, leading to inhibitory Akt S34 phosphorylation (57). In addition to the finding that ceramides were elevated in muscle from obese mice, our studies in myotubes showed that the C2-ceramide not only reduced basal Akt phosphorylation but also completely prevented CNTF-induced Akt phosphorylation and CNTF-stimulated glucose uptake. These results agree with previous studies in myotubes (33) and isolated rodent (32) and human (58) skeletal muscle demonstrating impaired insulin-stimulated Akt activation and glucose uptake when ceramides are elevated. In the present studies, DAG and a reduction in PI-3 kinase activity may have also contributed to the reduced CNTF activation of Akt in muscles from obese animals. Importantly, the finding that neither the C2-ceramide nor obesity altered CNTF-induced STAT3 and AMPK phosphorylation is consistent with our previous findings that CNTF activation of AMPK and lipid metabolism is maintained in skeletal muscles from obese, insulin-resistant mouse models (20). Thus, although the signaling arm of CNTF to glucose uptake is compromised with obesity, the activation of AMPK and lipid oxidation is preserved.

In summary, our data show that CNTF-stimulated glucose uptake is associated with activation of the PI3-kinase/Akt signaling pathway. Furthermore, CNTF-stimulated Akt S473 phosphorylation and glucose uptake is impaired in muscles from obese insulin-resistant mice despite the maintenance of STAT3 and AMPK signaling. This mechanism may, in part, be attributable to ceramide accumulation with obesity, which in turn impairs CNTF signaling to the PI3-kinase/Akt signaling arm of the gp130 receptor. Because CNTF-induced activation of AMPK was maintained in obesity, CNTF or CNTF analogs provide the bases for a viable therapeutic to prevent muscle lipid accumulation and to restore insulin sensitivity in obesity.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This research was supported by grants from the National Health and Medical Research Council, Australia (G.R.S., M.J.W., and B.E.K.) and the National Institutes of Health (NIH), U.S. (M.J.B.; R01-DK56886). S.B.J. was supported by a Danish Research Council of Health and Diseases postdoctoral fellowship. M.J.W. is an R. Douglas Wright Fellow, and G.R.S. is a National Health and Medical Research Council of Australia Research Fellow and a Canadian Research Chair in Metabolism, Obesity and Type 2 Diabetes. B.E.K. is an Australian Research Council Federation Fellow.

No potential conflicts of interest relevant to this article were reported.

1
Rose
AJ
,
Richter
EA
:
Skeletal muscle glucose uptake during exercise: how is it regulated?
Physiology (Bethesda)
20
:
260
270
,
2005
2
Bruss
MD
,
Arias
EB
,
Lienhard
GE
,
Cartee
GD
:
Increased phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle in response to insulin or contractile activity
.
Diabetes
54
:
41
50
,
2005
3
Treebak
JT
,
Glund
S
,
Deshmukh
A
,
Klein
DK
,
Long
YC
,
Jensen
TE
,
Jorgensen
SB
,
Viollet
B
,
Andersson
L
,
Neumann
D
,
Wallimann
T
,
Richter
EA
,
Chibalin
AV
,
Zierath
JR
,
Wojtaszewski
JF
:
AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits
.
Diabetes
55
:
2051
2058
,
2006
4
Taylor
EB
,
An
D
,
Kramer
HF
,
Yu
H
,
Fujii
NL
,
Roeckl
KS
,
Bowles
N
,
Hirshman
MF
,
Xie
J
,
Feener
EP
,
Goodyear
LJ
:
Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle
.
J Biol Chem
283
:
9787
9796
,
2008
5
Friedman
JE
,
Dudek
RW
,
Whitehead
DS
,
Downes
DL
,
Frisell
WR
,
Caro
JF
,
Dohm
GL
:
Immunolocalization of glucose transporter GLUT4 within human skeletal muscle
.
Diabetes
40
:
150
154
,
1991
6
Sano
H
,
Kane
S
,
Sano
E
,
Miinea
CP
,
Asara
JM
,
Lane
WS
,
Garner
CW
,
Lienhard
GE
:
Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation
.
J Biol Chem
278
:
14599
14602
,
2003
7
Yu
C
,
Chen
Y
,
Cline
GW
,
Zhang
D
,
Zong
H
,
Wang
Y
,
Bergeron
R
,
Kim
JK
,
Cushman
SW
,
Cooney
GJ
,
Atcheson
B
,
White
MF
,
Kraegen
EW
,
Shulman
GI
:
Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle
.
J Biol Chem
277
:
50230
50236
,
2002
8
Cho
H
,
Mu
J
,
Kim
JK
,
Thorvaldsen
JL
,
Chu
Q
,
Crenshaw
EB
 III
,
Kaestner
KH
,
Bartolomei
MS
,
Shulman
GI
,
Birnbaum
MJ
:
Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta)
.
Science
292
:
1728
1731
,
2001
9
Yamauchi
T
,
Kamon
J
,
Minokoshi
Y
,
Ito
Y
,
Waki
H
,
Uchida
S
,
Yamashita
S
,
Noda
M
,
Kita
S
,
Ueki
K
,
Eto
K
,
Akanuma
Y
,
Froguel
P
,
Foufelle
F
,
Ferre
P
,
Carling
D
,
Kimura
S
,
Nagai
R
,
Kahn
BB
,
Kadowaki
T
:
Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase
.
Nat Med
8
:
1288
1295
,
2002
10
Tomas
E
,
Tsao
TS
,
Saha
AK
,
Murrey
HE
,
Zhang Cc
C
,
Itani
SI
,
Lodish
HF
,
Ruderman
NB
:
Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation
.
Proc Natl Acad Sci U S A
99
:
16309
16313
,
2002
11
Carey
AL
,
Steinberg
GR
,
Macaulay
SL
,
Thomas
WG
,
Holmes
AG
,
Ramm
G
,
Prelovsek
O
,
Hohnen-Behrens
C
,
Watt
MJ
,
James
DE
,
Kemp
BE
,
Pedersen
BK
,
Febbraio
MA
:
Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase
.
Diabetes
55
:
2688
2697
,
2006
12
Geiger
PC
,
Hancock
C
,
Wright
DC
,
Han
DH
,
Holloszy
JO
:
IL-6 increases muscle insulin sensitivity only at superphysiological levels
.
Am J Physiol Endocrinol Metab
292
:
E1842
1846
,
2007
13
Glund
S
,
Deshmukh
A
,
Long
YC
,
Moller
T
,
Koistinen
HA
,
Caidahl
K
,
Zierath
JR
,
Krook
A
:
Interleukin-6 directly increases glucose metabolism in resting human skeletal muscle
.
Diabetes
56
:
1630
1637
,
2007
14
Hayashi
T
,
Hirshman
MF
,
Kurth
EJ
,
Winder
WW
,
Goodyear
LJ
:
Evidence for 5′ AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport
.
Diabetes
47
:
1369
1373
,
1998
15
Jorgensen
SB
,
Viollet
B
,
Andreelli
F
,
Frosig
C
,
Birk
JB
,
Schjerling
P
,
Vaulont
S
,
Richter
EA
,
Wojtaszewski
JF
:
Knockout of the alpha2 but not alpha1 5′-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside but not contraction-induced glucose uptake in skeletal muscle
.
J Biol Chem
279
:
1070
1079
,
2004
16
Koistinen
HA
,
Galuska
D
,
Chibalin
AV
,
Yang
J
,
Zierath
JR
,
Holman
GD
,
Wallberg-Henriksson
H
:
5-Amino-imidazole carboxamide riboside increases glucose transport and cell-surface GLUT4 content in skeletal muscle from subjects with type 2 diabetes
.
Diabetes
52
:
1066
1072
,
2003
17
Fiedler
M
,
Zierath
JR
,
Selen
G
,
Wallberg-Henriksson
H
,
Liang
Y
,
Sakariassen
KS
:
5-aminoimidazole-4-carboxy-amide-1-beta-D-ribofuranoside treatment ameliorates hyperglycaemia and hyperinsulinaemia but not dyslipidaemia in KKAy-CETP mice
.
Diabetologia
44
:
2180
2186
,
2001
18
Steinberg
GR
,
Smith
AC
,
Van Denderen
BJ
,
Chen
Z
,
Murthy
S
,
Campbell
DJ
,
Heigenhauser
GJ
,
Dyck
DJ
,
Kemp
BE
:
AMP-activated protein kinase is not down-regulated in human skeletal muscle of obese females
.
J Clin Endocrinol Metab
89
:
4575
4580
,
2004
19
Iglesias
MA
,
Furler
SM
,
Cooney
GJ
,
Kraegen
EW
,
Ye
JM
:
AMP-Activated protein kinase activation by AICAR increases both muscle fatty acid and glucose uptake in white muscle of insulin-resistant rats in vivo
.
Diabetes
53
:
1649
1654
,
2004
20
Watt
MJ
,
Dzamko
N
,
Thomas
WG
,
Rose-John
S
,
Ernst
M
,
Carling
D
,
Kemp
BE
,
Febbraio
MA
,
Steinberg
GR
:
CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK
.
Nat Med
12
:
541
548
,
2006
21
Davis
S
,
Aldrich
TH
,
Stahl
N
,
Pan
L
,
Taga
T
,
Kishimoto
T
,
Ip
NY
,
Yancopoulos
GD
:
LIFR beta and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor
.
Science
260
:
1805
1808
,
1993
22
Murakami
M
,
Hibi
M
,
Nakagawa
N
,
Nakagawa
T
,
Yasukawa
K
,
Yamanishi
K
,
Taga
T
,
Kishimoto
T
:
IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase
.
Science
260
:
1808
1810
,
1993
23
Ernst
M
,
Jenkins
BJ
:
Acquiring signalling specificity from the cytokine receptor gp130
.
Trends Genet
20
:
23
32
,
2004
24
Febbraio
MA
:
gp130 receptor ligands as potential therapeutic targets for obesity
.
J Clin Invest
117
:
841
849
,
2007
25
Takahashi-Tezuka
M
,
Yoshida
Y
,
Fukada
T
,
Ohtani
T
,
Yamanaka
Y
,
Nishida
K
,
Nakajima
K
,
Hibi
M
,
Hirano
T
:
Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase
.
Mol Cell Biol
18
:
4109
4117
,
1998
26
Bertola
A
,
Bonnafous
S
,
Cormont
M
,
Anty
R
,
Tanti
JF
,
Tran
A
,
Le Marchand-Brustel
Y
,
Gual
P
:
Hepatocyte growth factor induces glucose uptake in 3T3–L1 adipocytes through A Gab1/phosphatidylinositol 3-kinase/Glut4 pathway
.
J Biol Chem
282
:
10325
10332
,
2007
27
Watt
MJ
,
Hevener
A
,
Lancaster
GI
,
Febbraio
MA
:
Ciliary neurotrophic factor prevents acute lipid-induced insulin resistance by attenuating ceramide accumulation and phosphorylation of c-Jun N-terminal kinase in peripheral tissues
.
Endocrinology
147
:
2077
2085
,
2006
28
Hotamisligil
GS
,
Peraldi
P
,
Budavari
A
,
Ellis
R
,
White
MF
,
Spiegelman
BM
:
IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance
.
Science
271
:
665
668
,
1996
29
Yuan
M
,
Konstantopoulos
N
,
Lee
J
,
Hansen
L
,
Li
ZW
,
Karin
M
,
Shoelson
SE
:
Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta
.
Science
293
:
1673
1677
,
2001
30
Arkan
MC
,
Hevener
AL
,
Greten
FR
,
Maeda
S
,
Li
ZW
,
Long
JM
,
Wynshaw-Boris
A
,
Poli
G
,
Olefsky
J
,
Karin
M
:
IKK-beta links inflammation to obesity-induced insulin resistance
.
Nat Med
11
:
191
198
,
2005
31
Kim
JK
,
Fillmore
JJ
,
Sunshine
MJ
,
Albrecht
B
,
Higashimori
T
,
Kim
DW
,
Liu
ZX
,
Soos
TJ
,
Cline
GW
,
O'Brien
WR
,
Littman
DR
,
Shulman
GI
:
PKC-theta knockout mice are protected from fat-induced insulin resistance
.
J Clin Invest
114
:
823
827
,
2004
32
Holland
WL
,
Brozinick
JT
,
Wang
LP
,
Hawkins
ED
,
Sargent
KM
,
Liu
Y
,
Narra
K
,
Hoehn
KL
,
Knotts
TA
,
Siesky
A
,
Nelson
DH
,
Karathanasis
SK
,
Fontenot
GK
,
Birnbaum
MJ
,
Summers
SA
:
Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance
.
Cell Metab
5
:
167
179
,
2007
33
Schmitz-Peiffer
C
,
Craig
DL
,
Biden
TJ
:
Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate
.
J Biol Chem
274
:
24202
24210
,
1999
34
Mu
J
,
Brozinick
JT
 Jr
,
Valladares
O
,
Bucan
M
,
Birnbaum
MJ
:
A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport I skeletal muscle
.
Mol Cell
7
:
1085
1094
,
2001
35
Jensen
TE
,
Rose
AJ
,
Hellsten
Y
,
Wojtaszewski
JF
,
Richter
EA
:
Caffeine-induced Ca2+ release increases AMPK-dependent glucose uptake in rodent soleus muscle
.
Am J Physiol Endocrinol Metab
293
:
E286
293
,
2007
36
Ernst
M
,
Inglese
M
,
Waring
P
,
Campbell
IK
,
Bao
S
,
Clay
FJ
,
Alexander
WS
,
Wicks
IP
,
Tarlinton
DM
,
Novak
U
,
Heath
JK
,
Dunn
AR
:
Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation
.
J Exp Med
194
:
189
203
,
2001
37
Wojtaszewski
JF
,
Lynge
J
,
Jakobsen
AB
,
Goodyear
LJ
,
Richter
EA
:
Differential regulation of MAP kinase by contraction and insulin in skeletal muscle: metabolic implications
.
Am J Physiol
277
:
E724
E732
,
1999
38
Jorgensen
SB
,
Nielsen
JN
,
Birk
JB
,
Olsen
GS
,
Viollet
B
,
Andreelli
F
,
Schjerling
P
,
Vaulont
S
,
Hardie
DG
,
Hansen
BF
,
Richter
EA
,
Wojtaszewski
JF
:
The alpha2–5′AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading
.
Diabetes
53
:
3074
3081
,
2004
39
Chen
ZP
,
Mitchelhill
KI
,
Michell
BJ
,
Stapleton
D
,
Rodriguez-Crespo
I
,
Witters
LA
,
Power
DA
,
Ortiz de Montellano
PR
,
Kemp
BE
:
AMP-activated protein kinase phosphorylation of endothelial NO synthase
.
FEBS Lett
443
:
285
289
,
1999
40
Jorgensen
SB
,
Wojtaszewski
JF
,
Viollet
B
,
Andreelli
F
,
Birk
JB
,
Hellsten
Y
,
Schjerling
P
,
Vaulont
S
,
Neufer
PD
,
Richter
EA
,
Pilegaard
H
:
Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle
.
FASEB J
19
:
1146
1148
,
2005
41
Preiss
J
,
Loomis
CR
,
Bishop
WR
,
Stein
R
,
Niedel
JE
,
Bell
RM
:
Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells
.
J Biol Chem
261
:
8597
8600
,
1986
42
Alonzi
T
,
Middleton
G
,
Wyatt
S
,
Buchman
V
,
Betz
UA
,
Muller
W
,
Musiani
P
,
Poli
V
,
Davies
AM
:
Role of STAT3 and PI 3-kinase/Akt in mediating the survival actions of cytokines on sensory neurons
.
Mol Cell Neurosci
18
:
270
282
,
2001
43
Petersen
KF
,
Shulman
GI
:
Etiology of insulin resistance
.
Am J Med
119
:
S10
16
,
2006
44
Baron
AD
,
Brechtel
G
,
Wallace
P
,
Edelman
SV
:
Rates and tissue sites of non-ins
.
Am J Physiol
255
:
E769
E774
,
1988
45
Gupta
SK
,
Altares
M
,
Benoit
R
,
Riopelle
RJ
,
Dunn
RJ
,
Richardson
PM
:
Preparation and biological properties of native and recombinant ciliary neurotrophic factor
.
J Neurobiol
23
:
481
490
,
1992
46
Gearing
DP
,
Comeau
MR
,
Friend
DJ
,
Gimpel
SD
,
Thut
CJ
,
McGourty
J
,
Brasher
KK
,
King
JA
,
Gillis
S
,
Mosley
B
, et al
.:
The IL-6 signal transducer, gp130: an oncostatin M receptor and affinity converter for the LIF receptor
.
Science
255
:
1434
1437
,
1992
47
Wenisch
C
,
Linnau
KF
,
Looaresuwan
S
,
Rumpold
H
:
Plasma levels of the interleukin-6 cytokine family in persons with severe Plasmodium falciparum malaria
.
J Infect Dis
179
:
747
750
,
1999
48
Stockli
KA
,
Lottspeich
F
,
Sendtner
M
,
Masiakowski
P
,
Carroll
P
,
Gotz
R
,
Lindholm
D
,
Thoenen
H
:
Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor
.
Nature
342
:
920
923
,
1989
49
Steinberg
GR
,
Watt
MJ
,
Fam
BC
,
Proietto
J
,
Andrikopoulos
S
,
Allen
AM
,
Febbraio
MA
,
Kemp
BE
:
Ciliary neurotrophic factor suppresses hypothalamic AMP-kinase signaling in leptin-resistant obese mice
.
Endocrinology
147
:
3906
3914
,
2006
50
Kokoeva
MV
,
Yin
H
,
Flier
JS
:
Neurogenesis in the hypothalamus of adult mice: potential role in energy balance
.
Science
310
:
679
683
,
2005
51
Bluher
S
,
Moschos
S
,
Bullen
J
 Jr
,
Kokkotou
E
,
Maratos-Flier
E
,
Wiegand
SJ
,
Sleeman
MW
,
Mantzoros
CS
:
Ciliary neurotrophic factorAx15 alters energy homeostasis, decreases body weight, and improves metabolic control in diet-induced obese and UCP1-DTA mice
.
Diabetes
53
:
2787
2796
,
2004
52
Sleeman
MW
,
Garcia
K
,
Liu
R
,
Murray
JD
,
Malinova
L
,
Moncrieffe
M
,
Yancopoulos
GD
,
Wiegand
SJ
:
Ciliary neurotrophic factor improves diabetic parameters and hepatic steatosis and increases basal metabolic rate in db/db mice
.
Proc Natl Acad Sci U S A
100
:
14297
14302
,
2003
53
Ettinger
MP
,
Littlejohn
TW
,
Schwartz
SL
,
Weiss
SR
,
McIlwain
HH
,
Heymsfield
SB
,
Bray
GA
,
Roberts
WG
,
Heyman
ER
,
Stambler
N
,
Heshka
S
,
Vicary
C
,
Guler
HP
:
Recombinant variant of ciliary neurotrophic factor for weight loss in obese adults: a randomized, dose-ranging study
.
JAMA
289
:
1826
1832
,
2003
54
Merrill
GF
,
Kurth
EJ
,
Hardie
DG
,
Winder
WW
:
AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle
.
Am J Physiol
273
:
E1107
E1112
,
1997
55
Wojtaszewski
JF
,
Jorgensen
SB
,
Hellsten
Y
,
Hardie
DG
,
Richter
EA
:
Glycogen-dependent effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle
.
Diabetes
51
:
284
292
,
2002
56
Stratford
S
,
Hoehn
KL
,
Liu
F
,
Summers
SA
:
Regulation of insulin action by ceramide: dual mechanisms linking ceramide accumulation to the inhibition of Akt/protein kinase B
.
J Biol Chem
279
:
36608
36615
,
2004
57
Fox
TE
,
Houck
KL
,
O'Neill
SM
,
Nagarajan
M
,
Stover
TC
,
Pomianowski
PT
,
Unal
O
,
Yun
JK
,
Naides
SJ
,
Kester
M
:
Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains
.
J Biol Chem
282
:
12450
12457
,
2007
58
Itani
SI
,
Ruderman
NB
,
Schmieder
F
,
Boden
G
:
Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha
.
Diabetes
51
:
2005
2011
,
2002
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