The Ciliary Neutrofic Factor stimulates muscle glucose uptake through SHP2 and PI3-kinase but ...

22 Pages·2009·0.34 MB·English

by  Sebastian Beck Jorgensen

Diabetes Publish Ahead of Print, published online January 9, 2009
CNTF Stimulates Muscle Glucose Uptake by a PI3-Kinase Dependent
Pathway that is Impaired with Obesity

Gregory R. Steinberg1, Matthew J. Watt1,4, Matthias Ernst3, Morris J. Birnbaum6, Bruce E.
Kemp1,2 and Sebastian Beck Jørgensen1,5

1St. Vincent’s Institute of Medical Research and Department of Medicine, University of
Melbourne, Fitzroy, 3065, 2CSIRO Molecular Health Technologies, Parkville, 3Ludwig Inst. for
Cancer Research, Parkville 3052, 4Department of Physiology, Monash University, Clayton 3800,
Victoria, Australia. 5Section of Human Physiology, Copenhagen Muscle Research Centre, Dep.
of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark. 6Institute of
Diabetes, Obesity and Metabolism, University of Pennsylvania, Philadelphia, USA

Correspondence:
Gregory R. Steinberg, Ph. D.
St. Vincent’s Institute, 41 Victoria Parade, Fitzroy, Victoria, 3065, Australia
Email: [email protected]
or
Sebastian B. Jørgensen, Ph.D.
13 Universitetsparken, 2100 Copenhagen, Denmark
Email: [email protected]

Submitted 20 May 2008 and accepted 6 January 2009.

This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association,
publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it
by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at
http://diabetes.diabetesjournals.org.
Copyright American Diabetes Association, Inc., 2009
ABSTRACT

Objective. Ciliary neurotrophic factor (CNTF) reverses muscle insulin resistance by increasing
fatty acid oxidation via gp130/LIF receptor signaling to the AMP-activated protein kinase
(AMPK). CNTF also increases Akt signaling in neurons and adipocytes. As 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 monitored. The effects of CNTF on skeletal muscle glucose uptake and
AMPK/Akt signaling were investigated in incubated soleus and EDL muscles from muscle
specific AMPKα2 kinase-dead (KD), 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-1. CNTF
increased Akt-S473 phosphorylation in soleus and EDL however; AMPK-T172 phosphorylation
was only increased in soleus. Incubation of muscles from AMPK-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 (HFD 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.
2
S
keletal muscle glucose uptake is PI3-kinase by gp130/LIF relies on association
regulated by both intrinsic and of the p85 subunit with the regulatory SH2
circulating factors involving the PI3- domain-containing adapter molecule GAB1
kinase/Akt and the AMP-activated protein coordinated by SHP2 (25) and the assembling
kinase (AMPK) signaling pathways (1). The of the complex does not involve IRS-1 (26).
regulation of muscle glucose uptake by both Phosphorylation of four more distal tyrosine
pathways converges on AS160 (TBC1D4) residues leads to activation of the “signal
and TBC1D1 where phosphorylation inhibits transducer and activator of transcription”
the negative regulation of GLUT4 vesicle (STAT) 1 and 3 (23). This distal domain is
translocation (2-4) to the plasma membrane responsible for AMPK activation by an
(5; 6). In insulin resistant skeletal muscle, adenosine nucleotide-dependent mechanism
reduced insulin receptor substrate (IRS) and (27). As both AMPK- and PI3 kinase/Akt
Akt activation results in reduced skeletal signaling is enhanced by activation of the
muscle glucose uptake (7; 8). In contrast, gp130/LIF receptor, our first aim was to test
activation of AMPK by endogenous whether CNTF increases glucose uptake in
circulating factors including adiponectin (9; skeletal muscle and the signaling pathway/s
10) and interleukin (IL)-6 (11-13), and by invoked.
pharmacological agents such as 5- The etiology of impaired muscle insulin
Aminoimidazole-4-carboxamide riboside signaling with obesity is multi-factorial and
(AICAR) (14; 15), increases muscle glucose appears to be related to at least two major
uptake and this response is maintained in events: chronic low-grade inflammation and
muscle from diabetic rodents and humans intra-myocellular lipid accumulation (7; 28).
(16-19). Both events activate inhibitory IRS-1 kinases
We have recently shown that the ciliary such as IκB kinase-β (IKK-β) (29; 30), c-jun
neurotrophic factor (CNTF), a member of the terminal amino kinase (JNK) (28) and protein
IL-6 family, also activates muscle AMPK and kinase C theta (PKC θ) (31), which impair
increases lipid oxidation (20). CNTF elicits insulin signaling by inhibiting IRS-1
intracellular signaling pathways by ligand association with PI3-kinase subunits. While
binding to the CNTF receptor α (CNTFRα) this is believed to be a significant element in
which initiates heterodimerization and muscle insulin resistance, the sphingolipid
activation of gp130 and leukemia inhibitory ceramide, also impairs insulin signaling by
factor (LIF) receptor, the two transmembrane promoting PP2A dependent Akt
β-subunits of the receptor complex (21). This dephosphorylation thereby inhibiting Akt
differs from the initial step in IL-6 signaling activation (32; 33). Thus, as gp130-LIF
which induces homo-dimerization of two signaling to glucose uptake would be
gp130 subunits and does not involve LIF (22). expected to be independent of IRS-1, our
Formation of the CNTFRα-gp130-LIF second aim was to determine whether gp130-
complex leads to association with isoforms of LIF signaling towards glucose uptake is
janus kinases (JAKs) and these subsequently maintained in mice with obesity-induced
phosphorylate specific tyrosine residues on insulin-resistance. Analysis of the molecular
the intracellular domains, creating docking pathways of CNTF signaling to glucose
sites for SH2 containing proteins (23; 24). uptake may provide important insights into
Phosphorylation of Y757 of the intracellular the function of gp130-LIF signaling, and
domain of the β-subunit leads to activation of reveal the potential of a novel therapeutic
the PI3-kinase/Akt pathway. Activation of the target for type 2 diabetes.
3
8 mM mannitol and 0.1% BSA), gassed with
METHODS 95% O + 5% CO and maintained at 30°C as
2 2
Animals. C57Bl6/J, ob/ob, AMPKα2 kinase- described (15). For all experiments, muscles
dead (KD) and gp130ΔSTAT mice age 8-18 were pre-incubated for 15 min in this buffer
weeks with corresponding littermate controls before it was replaced with buffer containing
were used in experiments. Transgenic mice AICAR (2 mM for 40 min, Toronto Research
over-expressing a KD form of the AMPK α2 Chemicals Inc., ON, Canada), CNTF (at
protein under control of the muscle creatine concentrations and durations as indicated,
kinase promoter have been described Axokine®, Regeneron Pharmaceuticals Inc,
previously (34). Briefly, the KD mutation NY, USA) or insulin (30 nM (additivity
was generated by Lys45Arg mutagenesis to study) or 2.8 nM (HFD study) for 40 min,
encode a kinase-dead α2 protein that displaces Actrapid®, Novo Nordisk A/S, Denmark). In
all detectable endogenous α2 and 50-70% of separate experiments muscles were incubated
endogenous α1 protein from αβγ heterotrimer in LY-294002 hydrochloride (37) (60 nM for
complexes (34; 35). The knockin mutant 30 min, Sigma-Aldrich Corp. MO, USA)
gp130ΔSTAT mice expresses a COOH- before treatment with CNTF or insulin as
terminal truncation mutation of gp130 that described above.
prevents activation of STAT1/3 as described 2-deoxy-D-glucose (2DG) uptake was
previously (36). This mutation has a deletion measured over 10 min by replacing existing
of the distal intracellular domain responsible incubation buffer with the same buffer
for AMPK and STAT activation. All mice described above but with the addition of 0.5
were kept at a 12:12 hr light-dark cycle at 20- μCi ml-12-[2,6-3H]-deoxy-D-glucose, 1 mM
21°C and provided ad libitum access to food 2-deoxy-D-glucose and 0.2 μCi [1-14C]-
and water. Mice were maintained on a mannitol ml-1. After preparing muscles as
standard rodent chow diet/low fat diet (5% described below, radioactivity was measured
calories fat, Harlan Teklad) except in high-fat in muscle lysates by liquid scintillation
diet (HFD) experiments, where the diet was counting (Tri-Carb 2000, Packard Instrument
composed of 45% calories from fat (SF04- Co.).
027, Specialty Feeds, WA, Australia). All Muscle lysate preparation. Muscles were
procedures were approved by the St. homogenized in ice-cold buffer (50 mM
Vincent’s Hospital Animal Ethics Committee. Hepes, pH 7.4, 150 mM NaCl, 10 mM NaF, 1
CNTF Tolerance Test. C57Bl6/J mice were mM sodium pyrophosphate, 0.5 mM EDTA,
fasted for 6 hours before being injected with 250 mM Sucrose, 1 mM dithiothreitol, 1%
either saline or 0.3 mg/kg CNTF, a TritonX-100, 1 mM Na VO and 1 Roche
3 4
concentration of CNTF which activates protease inhibitor tablet per 50 ml buffer)
AMPK and induces weight loss without using an electrical homogenizer. Lysates were
eliciting an inflammatory response (20). prepared as previously described (38) and
Blood glucose was monitored over 150 min. stored at -80°C until analysis. Protein content
Muscle incubations. Soleus (oxidative and in lysates was measured by the bicinchoninic
glycolytic fibers) and extensor digitorum acid method (Pierce).
longus (EDL, primarily glycolytic fibers) Immunoblotting. Expression or
muscles were dissected from anesthetized phosphorylation of investigated proteins was
mice (6 mg of pentobarbital 100 g-1 body determined in muscle lysates by SDS-PAGE
weight) and transferred to incubation flasks and immunoblotting using the following
containing 2 ml of essential buffer (Krebs- primary antibodies: pan-αAMPK, phospho-
Henseleit buffer, pH 7.4, with 2 mM pyruvate, AMPK T172 and phospho-ACC S222 (as
4
described (39)), STAT3, phospho-STAT3 between groups were considered statistically
Y705, Akt and phospho-Akt S473 (Cell significant if p<0.05.
Signaling Technology Inc., MA, USA).
Secondary antibodies were horseradish RESULTS
conjugated protein G (Bio-Rad Laboratories, In vivo effects of CNTF on blood glucose.
CA USA). Bands were visualized using an Injection of mice with CNTF was associated
enhanced chemoluminescence system and with a reduction in blood glucose after 40
quantified using ImageQuant TL 05 software minutes compared with saline injected
(Amersham Biosciences, UK). Values controls. Blood glucose remained reduced in
obtained using phospho-specific antibodies the CNTF group throughout the remainder of
are expressed as the ratio to the total content the test (Fig 1A). The area under the curve
of the protein measured after stripping the (AUC) was 20% lower in CNTF treated mice
membrane and re-probing. than saline injected controls (Fig 1B).
AMPK activity. AMPK α1 and α2 activities Muscle glucose uptake; CNTF dose
were measured from 100 μg of muscle lysate response. The effects of CNTF to reduce
protein using rabbit polyclonal AMPK blood glucose in vivo may have been
antibodies for immunoprecipitation as mediated by the suppression of hepatic
previously described (40). glucose production or increased glucose
Muscle lipids. Lipids were extracted from uptake into adipose tissue and skeletal muscle.
freeze-dried powdered gastrocnemius muscle Therefore, to directly test the effects of CNTF
tissues using chloroform: methanol: PBS on skeletal muscle glucose uptake we
(1:2:0.8) and 0.2% SDS. Triacylglycerol were incubated isolated muscles with CNTF at
saponified in an ethanol-KOH solution at concentrations ranging from 2-500 ng·ml-1.
60°C and glycerol content was measured CNTF increased muscle glucose uptake over
fluorometrically. Diacylglycerols and 30 min in a dose dependent manner with a
ceramides were extracted and quantified significant increase at 2 ng·ml-1 in both soleus
according to the methods of Preiss et al. (41). and EDL muscles (Fig 2A&B). Glucose
Muscle cell studies. Muscle cells were uptake was elevated with increasing CNTF
differentiated and serum starved overnight. concentrations up to 100 ng·ml-1 with no
The following morning 20 nM C2- further increases observed at 500 ng·ml-1 (Fig
dihydroceramide (Toronto Research 2A&B), consistent with dose dependent
Chemicals Inc., ON, Canada) was added for 6 effects on fatty acid oxidation observed
hr before cells were treated for 20 min with previously (20). We next investigated if the
either CNTF (10 ng·ml-1) or insulin (10 nM) effect of CNTF on glucose uptake was
before collecting protein lysates or measuring additive to the effects of insulin and AICAR.
2DG uptake as described (20). C2C12 cell In soleus, CNTF, insulin and AICAR
were used for signaling experiments and L6 independently increased glucose uptake to a
muscle cells for 2DG uptake because of the similar degree, and combining AICAR and
limited capacity of C2C12 to increase glucose insulin with CNTF had an additive effect (Fig
uptake in response to stimuli. 2C). In EDL, insulin and AICAR increased
Statistics. Data are expressed as means ± SE. glucose uptake more strongly than CNTF and
Statistical evaluations were performed by no further increase was observed when
either Student’s t-test or two-way ANOVA combining these treatments with CNTF (Fig
using the Student-Newman-Keuls method as a 2D).
post hoc test when appropriate. Differences Time-course of CNTF stimulated muscle
glucose uptake and signaling. CNTF
5
increased glucose uptake in soleus and EDL activity (34). In WT soleus, CNTF increased
after 30 min, an effect which thereafter α1-AMPK activity by 180% and α2-AMPK
diminished in both muscle types (Fig 3A). activity by 35% whereas CNTF had no effect
CNTF has been reported to increase the on either AMPK isoform in AMPK-KD
activity/phosphorylation of AMPK in muscle muscles (Fig 4B). CNTF did not increase
(20) and Akt in neurons (42), and since both AMPK activity in the EDL (Fig 4B)
kinases regulate muscle glucose uptake, we consistent with CNTF not increasing AMPK
examined their activating phosphorylation. phosphorylation in the EDL muscle (Fig 3C).
CNTF increased Akt S473-P nearly 200% in Lastly, to establish whether inhibition of
soleus and 450% in EDL muscle and these PI3-kinase or AMPK signaling altered
effects diminished after 90 min (Fig 3B). proximal CNTF signaling through the gp130-
AMPK T172-P was increased 90% in the LIF receptor complex, we examined STAT3
soleus with CNTF after 30 min and this was Y705-P in the EDL muscle of WT and
maintained for 60 min (Fig 3C). Although AMPK KD mice in the absence or presence
CNTF-induced an apparent rise in ACCβ of LY-294002. Incubation with CNTF
S218-P in soleus this did not achieve increased STAT3 Y705-P by 140% in WT
statistical significance (p=0.09) (Fig 3D). muscle, and importantly, the increase was
CNTF had no detectable effect on AMPK- or unaffected in AMPK-KD muscles and
ACCβ-P in EDL (Fig 3C and 3D) consistent unaffected by co-incubation with LY-294002
with previous observations in glycolytic (Fig 4C). We also verified that increases in
muscle (20). Akt-P with CNTF was not affected by over-
Effects of PI3-kinase inhibition and ablated expression of the AMPK KD construct and
AMPK activity on CNTF actions. We next that LY 294002 inhibited Akt activation (Fig
examined the relative importance of AMPK, 4D). Similar findings were observed in the
and PI3K/Akt signaling for CNTF-mediated soleus muscle (data not shown).
glucose uptake. Muscles from WT and CNTF-stimulated glucose uptake in
AMPK KD mice were pre-incubated with or gp130ΔSTAT muscles. Signaling from the
without the PI3-kinase inhibitor, LY-294002, gp130-LIF receptor complex activates two
followed by stimulation with CNTF. Glucose distinct intracellular signaling pathways. The
uptake was increased to a similar degree with first depends on phosphorylation of four
CNTF in WT and AMPK KD muscles tyrosine residues (Y765, Y812, Y904, Y914)
indicating this response was AMPK on the distal cytoplasmic tail of gp130 which
independent. CNTF-stimulated glucose is required and sufficient for activating
uptake was abolished by LY-294002 in both STAT1/3 (23) and AMPK (20). The second
muscle types, irrespective of mouse genotype arm requires phosphorylation of the more
(Fig 4A), consistent with PI3-kinase/Akt proximal Y757 residue in gp130 which
signaling being essential for this response. induces ERK and PI3-kinase/Akt signaling
We next measured α-isoform specific (23). We incubated muscles from
AMPK activity to verify that CNTF had gp130ΔSTAT mice, which have a deletion of
activated AMPK signaling in WT mice, and the cytosolic distal portion of the gp130
that the activation was impaired in AMPK- receptor required for induction of STAT1/3
KD muscles (Fig 4B). As anticipated, over- and AMPK signaling, but retain the Y757
expression of the AMPK KD construct residue that is required for activation of Erk
lowered basal AMPK activity of both α- and PI3-kinase/Akt signaling. Incubation with
AMPK isoforms in both muscle types with CNTF increased glucose uptake in soleus and
the most pronounced effect on α2-AMPK EDL of WT mice and this increase was
6
maintained in gp130ΔSTAT knockin mutant EDL of chow fed mice, respectively, and this
mice (Fig 5A). CNTF increased Akt S473-P increase was markedly impaired with obesity
in both genotypes whereas STAT3 Y705-P in both muscle types (Fig 6D). In contrast,
was increased in WT, but not in AMPK T172-P in soleus was also unaffected
gp130ΔSTAT muscles as expected (Fig 5B). by diet (Fig 6E).
Thus, CNTF increased glucose uptake Muscle lipids and CNTF signaling. High-fat
normally despite impaired gp130 signaling feeding increased muscle contents of TAG,
and activation of AMPK. DAG and ceramide by ~100%, ~50% and
Effects of obesity on CNTF-stimulated ~50%, respectively, compared to chow fed
muscle glucose uptake and signaling. It is controls (Fig 7A). Since ceramides have been
well established that obesity is associated shown to directly inhibit insulin stimulated
with impaired insulin sensitivity in skeletal Akt phosphorylation (33) we tested whether
muscle and impaired activation of IRS-1 have exposure of cultured myotubes to a short-
been attributed a primary role (28; 43). Since chain ceramide analog also impaired CNTF
CNTF stimulates glucose uptake downstream stimulated glucose uptake and Akt
of IRS1 we tested whether CNTF-stimulated phosphorylation. Consistent with our findings
glucose uptake is maintained in muscles from in obese skeletal muscle, myotubes treated
mice fed a high-fat diet (HFD) for 12 weeks with C2-ceramide had impaired CNTF-
and obese mice deficient in leptin (ob/ob). As stimulated glucose uptake (Fig 7B).
dictated by design, the high-fat feeding Reductions in glucose uptake were not due to
increased body weight by ~20% (chow; inhibition of the gp130-LIF receptor complex
28.4±0.5g, HFD; 34.0±0.7g ), fasting insulin as CNTF increased STAT3-P by ~350% in
levels (836±76 pg/ml to 1670±377 pg/ml) and both vehicle and C2-ceramide treated cells
the AUC during a GTT (Chow; 539±23, (Fig 7C), However, C2-ceramide reduced
HFD; 870±94). Similarly, ob/ob mice had basal and CNTF stimulated Akt S473-P (Fig
dramatically increased body mass (WT; 7D). AMPK T172-P was increased (p<0.05)
29.6±0.7g, ob/ob:57.3±2.8g) and AUC during by 60% with CNTF and this increase was
a GTT (WT; 574±26, ob/ob;1347±98). unaffected by C2-ceramide treatment (Fig 7E).
CNTF increased glucose uptake by 80-100%
in the soleus and EDL in chow-fed mice DISCUSSION
whereas CNTF-stimulated glucose uptake CNTF can regulate metabolic and growth
was completely blunted in the soleus and signaling pathways in several tissue types.
reduced by ~50% in EDL of mice fed a HFD The present study shows that CNTF regulates
(Fig 6A). Similarly, CNTF increased muscle glucose uptake, and delineates the proximal
glucose uptake in both muscle types of lean signaling events mediating this response. We
littermates of ob/ob mice, however, this show that acute exposure of CNTF reduces
increase was abolished in muscles from ob/ob blood glucose in vivo and increases glucose
mice (Fig 6B). uptake in soleus and EDL muscle in vitro via
We then examined the mechanism by the PI3-kinase/Akt signaling pathway. It was
which obesity inhibited CNTF-stimulated expected that AMPK would also exert a role
glucose uptake. CNTF increased STAT3 in this process, but we found that AMPK was
Y705-P in skeletal muscle of chow and high- not required for CNTF to stimulate glucose
fat fed animals (Fig 6C), indicating no uptake. Skeletal muscle is the most important
impairment of CNTF action at the receptor tissue for insulin-stimulated glucose disposal
level. CNTF-induced Akt S473-P was (44) and skeletal muscle insulin resistance is a
increased by 4- and 19-fold in soleus and major defect in most obese phenotypes.
7
Accordingly, we investigated whether CNTF- skeletal muscle (15; 54; 55), we expected
stimulated glucose uptake was maintained in AMPK rather than PI3-kinase/Akt signaling
muscle from obese, insulin resistant mice. to be required for CNTF stimulated glucose
The rationale for these studies was to uptake. However, multiple lines of evidence
establish whether gp130-LIF signaling could indicate that CNTF signaling to glucose
constitute an alternative pathway to substitute uptake may be mediated by a PI3-kinase/Akt
for insufficient insulin signaling to skeletal dependent signaling pathway: 1) The kinetics
muscle glucose uptake. However, obesity was in Akt S473 phosphorylation correlated with
associated with impaired CNTF-stimulated increases in glucose uptake in both soleus and
glucose uptake, which, at least in part, may EDL muscles. 2) Incubation with the PI3-
have been attributed to increased muscle kinase inhibitor, LY-294002, completely
lipids and impaired PI3-kinase/Akt signaling blocked CNTF-stimulated Akt
with CNTF. phosphorylation and glucose uptake. 3) CNTF
CNTF is a peptide hormone of the increased glucose uptake was not impaired in
interleukin-6 family which is highly muscles over-expressing a kinase-dead
expressed in peripheral nerves and other AMPKα2. 4) AMPK was not activated by
tissues (24; 45; 46). CNTF levels are low in CNTF in the EDL muscle despite a 100%
serum of healthy individual due to the increase in glucose uptake. 5) A knockin
absence of an exocytosis targeting sequence mutation of a truncated gp130 receptor, which
(47; 48). Recent reports have shown impairs gp130 signaling towards STAT3 and
significant promise for the use of CNTF as an AMPK, had no effect on CNTF-stimulated
anti-obesity therapeutic since it suppresses glucose uptake. 6) Obesity induced by a HFD
food intake acutely (49) and induces or treatment of cells with C2 ceramide
hypothalamic neurogenesis (50), leading to a impaired CNTF stimulated glucose uptake
new set-point in body-mass. CNTF also has and was associated with blunted Akt but not
direct anti-diabetic effects in peripheral AMPK phosphorylation. However, our
tissues. Chronic treatment of obese diabetic findings demonstrating that CNTF-stimulated
mice with CNTF increases metabolic rate (51) glucose uptake was additive to a maximal
and reduces liver steatosis by enhancing fat dose of insulin in SOLEUS but not EDL
oxidation and reducing synthesis of complex muscle suggest that under some conditions
lipids (52). We have recently shown that AMPK activation may also play a role in the
chronic CNTF treatment of obese diabetic stimulation of glucose uptake in SOLEUS,
mice reverses obesity-induced insulin albeit to a lesser extent than originally
resistance by activating AMPK and reducing anticipated. A possible explanation for the
muscle lipid accumulation (20). In line with limited role of AMPK in CNTF stimulated
this study, CNTF acutely prevents muscle glucose uptake is that CNTF predominantly
insulin-resistance in response to a 2 hr lipid activated the α1-AMPK isoform. Studies in
infusion by preventing lipid accumulation AMPK α2 null mice have shown that AMPK
(27). The clinical efficacy and safety of α2, but not α1, is required for the regulation
CNTF as a possible therapeutic for obesity is of muscle glucose uptake in response to
supported by findings in humans stimuli such as AICAR (15), possibly
demonstrating weight loss and improved explaining why CNTF activation of AMPK is
glycaemic control (53). not required to increase glucose transport.
As we previously showed that CNTF Since we found CNTF to increase muscle
activates AMPK in muscle (20) and AMPK is glucose uptake, it was of interest to test the
well known to increase glucose uptake in effects of CNTF in insulin resistant muscle
8
from obese mice. We found that the efficacy AMPK and lipid metabolism is maintained in
of CNTF to regulate glucose uptake was skeletal muscles from obese, insulin resistant
reduced by ~50% or more in muscles from mouse models (20). Thus, while the signaling
obese HFD fed and ob/ob mice. CNTF arm of CNTF to glucose uptake is
resistance with obesity seems not to be due to compromised with obesity, the activation of
impaired activation of the gp130/LIF receptor AMPK and lipid oxidation is preserved.
complex because CNTF-induced In summary, our data show that CNTF
phosphorylation of AMPK and STAT3 were stimulated glucose uptake is associated with
normal in obese muscle. In contrast, CNTF activation of the PI3-kinase/Akt signaling
stimulated phosphorylation of Akt was pathway. Furthermore, CNTF-stimulated Akt
suppressed in obese muscles and was S473 phosphorylation and glucose uptake is
associated with muscle DAG and ceramide impaired in muscles from obese insulin
accumulation. Ceramides down-regulate Akt resistant mice despite the maintenance of
phosphorylation and membrane translocation STAT3 and AMPK signaling. This
by activating protein phosphatase 2A (PP2A) mechanism may, in part, be attributable to
(56). Ceramides also increase the activity of ceramide accumulation with obesity which in
PKC ζ leading to inhibitory Akt S34 turn impairs CNTF signaling to the PI3-
phosphorylation (57). In addition to the kinase/Akt signaling arm of the gp-130
finding that ceramides were elevated in receptor. Since CNTF-induced activation of
muscle from obese mice, our studies in AMPK was maintained in obesity, CNTF or
myotubes showed that the C2 ceramide not CNTF analogues provide the bases for a
only reduced basal Akt phosphorylation but viable therapeutic to prevent muscle lipid
also completely prevented CNTF-induced Akt accumulation and the restore insulin
phosphorylation and CNTF-stimulated sensitivity in obesity.
glucose uptake. These results agree with
previous studies in myotubes (33) isolated ACKNOWLEDGEMENTS
rodent (32) and human (58) skeletal muscle These studies were supported by grants
demonstrating impaired insulin-stimulated from the National Health and Medical
Akt activation and glucose uptake when Research Council, AU, (GRS, MJW, BEK)
ceramides are elevated. In the present studies, and the National Institute of Health, USA,
DAG and a reduction in PI-3 kinase activity (MJB, R01-DK56886). SBJ was supported
may have also contributed to the reduced by Danish Research Council of Health and
CNTF activation of Akt in muscles from Diseases postdoctoral fellowship. MJW is a R.
obese animals. Importantly, the finding that Douglas Wright Fellow and GRS a National
neither the C2 ceramide or obesity altered Health and Medical Research Council of
CNTF-induced STAT3 and AMPK Australia Research Fellow. BEK is an
phosphorylation is consistent with our Australian Research Council Federation
previous findings that CNTF activation of Fellow.

9
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