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The Journal of Clinical Endocrinology & Metabolism 91(9):3592–3597 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2006-0638

The Suppressor of Cytokine Signaling 3 Inhibits Leptin Activation of AMP-Kinase in Cultured Skeletal Muscle of Obese Humans Gregory R. Steinberg, Andrew J. McAinch, Michael B. Chen, Paul E. O’Brien, John B. Dixon, David Cameron-Smith, and Bruce E. Kemp St. Vincent’s Institute and Department of Medicine (G.R.S., M.B.C., B.E.K.), University of Melbourne, Fitzroy, Victoria 3065, Australia; Commonwealth Scientific and Industrial Research Organization (B.E.K.), Molecular and Health Technologies, Parkville, Victoria 3052, Australia; School of Exercise and Nutrition Sciences (A.J.M., D.C.-S.), Deakin University, Burwood, Victoria 3125, Australia; and Centre of Obesity Research and Education (P.E.O., J.B.D.), Monash University, Alfred Hospital, Melbourne, Victoria 3181, Australia Context: Leptin is thought to regulate whole-body adiposity and insulin sensitivity, at least in part, by stimulating fatty acid metabolism via activation of AMP-kinase (AMPK) in skeletal muscle. Human obesity is associated with leptin resistance, and recent studies have demonstrated that hypothalamic expression of the suppressors of cytokine signaling 3 (SOCS3) regulates leptin sensitivity in rodents. Objective: The objective of the study was to investigate the effects of leptin on fatty acid oxidation and AMPK signaling in primary myotubes derived from lean and obese skeletal muscle and evaluate the contribution of SOCS3 to leptin resistance and AMPK signaling in obese humans. Results: We demonstrate that leptin stimulates AMPK activity and increases AMPK Thr172 and acetyl-CoA carboxylase-␤ Ser222 phosphorylation and fatty acid oxidation in lean myotubes but that in

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UPPRESSED RATES OF skeletal muscle fatty acid oxidation (1) combined with increased rates of fatty acid uptake (2) and esterification (3) contribute to the accumulation of im lipid and facilitate the development of skeletal muscle insulin resistance in obese humans (4). In rodents leptin acutely (5) and chronically (6) stimulates skeletal muscle fatty acid oxidation reducing im triacylglycerol storage (7). These acute (8) and chronic (9) effects are attributed to the activation of AMP-activated protein kinase (AMPK), which stimulates fatty acid oxidation by phosphorylation and inhibition of acetyl-CoA carboxylase (ACC)-␤ leading to reduced malonyl-CoA and increased carnitine palmitoyltransferase-1 activity and mitochondrial fatty acid flux (10). In skeletal muscle the activity of AMPK shows an absolute dependence on phosphorylation of Thr172 in the activation loop of the ␣-subunit by the upstream kinase LKB1 (11). AMP acts allosterically via the ␥-subunit to enhance phosphoryFirst Published Online July 5, 2006 Abbreviations: ACC, Acetyl-CoA carboxylase; AMPK, AMP kinase; BMI, body mass index; F, forward; LH, high concentration of leptin; LL, low concentration of leptin; R, reverse; SOCS, suppressors of cytokine signaling. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

obese subjects leptin-dependent AMPK signaling and fatty acid oxidation are suppressed. Reduced activation of AMPK was associated with elevated expression of IL-6 (⬃3.5-fold) and SOCS3 mRNA (⬃2.5fold) in myotubes of obese subjects. Overexpression of SOCS3 via adenovirus-mediated infection in lean myotubes to a similar degree as observed in obese myotubes prevented leptin but not AICAR (5amino-imidazole-4-carboxamide-1-␤-D-ribofuranoside) activation of AMPK signaling. Conclusions: These data demonstrate that SOCS3 inhibits leptin activation of AMPK. These data suggest that this impairment of leptin signaling in skeletal muscle may contribute to the aberrant regulation of fatty acid metabolism observed in obesity and that pharmacological activation of AMPK may be an effective therapy to bypass SOCS3-mediated skeletal muscle leptin resistance for the treatment of obesity-related disorders. (J Clin Endocrinol Metab 91: 3592–3597, 2006)

lation of AMPK Thr172 by LKB1 kinase (12, 13) as well as further activating the phosphorylated holoenzyme. Although leptin has pronounced effects in rodents fed a high-carbohydrate chow diet (6) and rodents with lipodystrophy (14), we and others (15–17) have demonstrated the presence of leptin resistance after high-fat feeding. Human obesity is characterized by elevated levels of circulating leptin (18), and a blunting of leptin’s effects on skeletal muscle fatty acid oxidation suggests the presence of skeletal muscle leptin resistance (19). A potential mediator of leptin resistance is the suppressors of cytokine signaling (SOCS)-3. SOCS3 is a member of a family of proteins (SOCS1-SOCS7, and cytokine-induced SH2-containing protein) that bind with their central SH2 domains to phosphotyrosine residues in cytokine receptors (20). Initial studies by Bjorbaek et al. (21) demonstrated the inhibition of leptin signaling via the signal transducer and activator of transcription-3 in hypothalamic nuclei was blocked by SOCS3 binding of pTyr985 of the leptin receptor (22). Recent studies in vivo have supported these findings as mutation of the leptin receptor at Tyr985 to Leu prevents SOCS3 binding resulting in enhanced activation of hypothalamic signal transducer and activator of transcription-3, reductions in food intake, and reduced adiposity in mice harboring the Y985L mutation, demonstrating a critical role of this residue in mediating hypothalamic leptin

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resistance (23). In addition, both SOCS3 hypothalamicspecific null mice (24) and SOCS3 mice with haploinsufficiency (25) have recently been found to have enhanced leptin sensitivity and are resistant to diet-induced obesity. In liver, SOCS3 expression is elevated in insulin-resistant rodents, and inhibition of SOCS3 with antisense oligonucleotides rescues impaired insulin sensitivity and improves hepatic steatosis in vivo (26). In skeletal muscle, SOCS3 is up-regulated after high-fat feeding, an effect that was associated with skeletal muscle leptin resistance (17, 27). In this study we used cultured primary myotubes derived from skeletal muscle of lean and obese subjects to directly examine the role of leptin on skeletal muscle fatty acid oxidation and AMPK signaling. The use of primary myotubes represents an excellent model to study the effects of leptin on AMPK signaling and fatty acid oxidation because they express protein characteristics of differentiated muscle cells (28) and maintain the metabolic phenotypes of suppressed fatty acid oxidation under basal conditions (29) and in response to alterations in substrates (30) and hormones (31) analogous to these conditions in vivo. Therefore, the purpose of this study was to investigate the effects of leptin on fatty acid oxidation and AMPK signaling in primary myotubes derived from lean and obese skeletal muscle and evaluate the contribution of SOCS3 to leptin resistance and AMPK signaling in obese humans.

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Aldrich Co., St. Louis, MO), 0.1% fetal bovine serum, 1% penicillin, streptomycin, and amphotericin B (PSA) ␣MEM at 37 C for 1 h. Cells were then treated with the same media but with the addition of 1 ␮Ci/ml palmitate (Amersham Biosciences, Buckinghamshire, UK) with vehicle (PBS), AICAR (2 mm), or low and high concentrations of recombinant human leptin for 2 h. Total palmitate oxidation was calculated by the addition of aqueous phase counts representing acid soluble metabolites to 14CO2 counts captured within the benzethonium hydroxide. The quantity of palmitate oxidized was calculated from the specific activity of labeled palmitate in the incubation medium (15).

SOCS3 mRNA Total cellular RNA and real-time RT-PCR was performed as previously described (31). Briefly, forward and reverse primers and cDNA (12 ng), were run for 40 cycles of PCR in a total volume of 20 ␮l. To compensate for variations in input RNA amounts, and efficiency of reverse transcription, cyclophilin (GenBank cyclophilin X52851) mRNA was quantified, and all results were normalized to these values. Cyclophilin expression did not differ between groups. Fluorescent emission data were analyzed for the critical threshold (CT) values, with the expression of the gene of interest normalized to cyclophilin and expressed as 2⫺⌬CT (33). The sequences of the forward (F) and reverse (R) primers are listed from 5⬘-3⬘ and are as follows: SOCS3 (NM_003955) (F) GACCAGCGCCACTTCTTCA, (R) CTGGATGCGCAGGTTCTTG; IL-6 (NM_000600) (F) GTG ACA TCC TCG ACG GCA TCT, (R) GTG CCT CTT TGC TGC TTT CAC; and cyclophilin (X52851) (F) CATCTGCACTGCCAAGACTGA, (R) TTCATGCCTTCTTTCACTTTGC. Leptin receptor expression (NM_001003679) was determined using an Assay-onDemand gene expression kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions.

SOCS3 adenovirus infection Subjects and Methods Subjects The participants were seven (five males and two females in each group) lean [body mass index (BMI) ⬍ 25 kg/m2] and obese (BMI ⬎ 35 kg/m2) subjects undergoing general abdominal surgery or lap-band surgery, respectively. Written informed consent was obtained from all participants following approval by the Human Ethics Research Committee of Deakin University, the Avenue Hospital, and Cabrini Hospital, Melbourne. After a fast (12–18 h), general anesthesia was induced with a short-acting propofol and maintained by a fentanyl and rocuronium volatile anesthetic mixture, and a muscle biopsy of rectus abdominus muscle was removed.

Cell culture Primary skeletal muscle cell culture was performed as recently described (31). All experiments were conducted on cells that were on passage four.

AMPK activity For the determination of AMPK-related activity, cells were cultured in 10-cm plates and treated with vehicle (PBS), 2 mm 5-amino-imidazole4-carboxamide-1-␤-d-ribofuranoside (AICAR; Toronto Research Chemicals, Toronto, Canada), or low (LL; 0.l ␮g/ml) and high (LH, 0.5 ␮g/ml) concentrations of recombinant human leptin (R&D Systems, Minneapolis, MN). The leptin concentration and time point used in this study were the same as previously demonstrated to activate AMPK in rodent skeletal muscle (8). AICAR concentrations were selected based on previous studies in cell culture demonstrating activation of AMPK in primary human myotubes (31, 32). AMPK expression, activity, and Thr172 phosphorylation as well as ACC expression and phosphorylation were determined as previously described (31).

Fatty acid oxidation Fatty acid oxidation was measured in myotubes cultured on 10-cm plates as previously described (31). Briefly, cells were preincubated with 3 ml of 4% fatty acid-free bovine albumin (ICN), 1 mm palmitate (Sigma-

Primary human myotubes from a lean subject were differentiated as described above. After 4 d of differentiation, cells were infected with a SOCS3 adenovirus (Ad-SOCS3, 100 PFU/plate) or control vector (AdNull, 100 PFU/plate). Two days after infection, cells were serum starved overnight and treated the following morning with 0.5 ␮g/ml leptin or 2 mm AICAR for 30 min, lysed, snap frozen in liquid nitrogen, and analyzed for AMPK activity and ACC␤ phosphorylation as described above and LKB1 activity as previously described (31). Briefly, for LKB1 activity, sheep anti-LKB1 antibody (Upstate Biotechnology, Lake Placid, NY) was prebound to protein A beads and incubated with the muscle lysates for 2 h at 4 C. LKB1 assays were performed on the beads using a two-step reaction with full-length, bacterially expressed, human AMPK (␣1␤1␥1) as substrate. The AMPK (␣1␤1␥1) activity was then determined using the AMPK SAMS peptide assay. SOCS3 expression was measured via immunoblot using anti-SOCS3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) from 100 ␮g of protein resolved on a 12% acrylamide gel. In separate experiments, cells treated with vehicle or leptin for 30 min were lysed in 0.5 m perchloric acid (1 mm EDTA) and neutralized with 2.2 m KHCO3. This extract was used for the determination of AMP (ATP), phosphocreatine, creatine, and lactate by spectrophotometric assays as described (34).

Calculations and statistical analysis Data are reported as mean ⫾ se for figures and mean ⫾ sd for tables. AMPK and ACC phosphorylation were corrected for AMPK ␣-pan (␣1 and ␣2) and ACC␤ expression, respectively. One experimental replicate per subject (n ⫽ 7) was used to determine differences between lean and obese subjects. Adenovirus infection of SOCS3 was conducted in one leptin-responsive lean cell line cultured to passage four in two separate experiments with three repeats per condition (n ⫽ 6). Results were analyzed using ANOVA procedures, and a Tukey’s post hoc test was used to test for significant differences revealed by the ANOVA. A Student’s t test for unpaired samples was used where appropriate. Significance was accepted at P ⱕ 0.05.

Results

Clinical characteristics for lean and obese subjects are listed in Table 1. Obese subjects had greater body mass and

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TABLE 1. Clinical characteristics

Age (yr) BMI (kg/m2) Weight (kg) Fasting glucose (mmol/liter) Plasma insulin (␮IU/ml) Cholesterol (mmol/liter) Fasting tryglycerides (mmol/liter)

Lean (n ⫽ 7)

Obese (n ⫽ 7)

44.1 ⫾ 10.1 21.7 ⫾ 1.85 62.8 ⫾ 9.7 5.18 ⫾ 0.41 4.21 ⫾ 3.81 4.81 ⫾ 1.14 1.04 ⫾ 0.46

36.2 ⫾ 10.9 47.3 ⫾ 12.0a 136.5 ⫾ 48.0a 5.00 ⫾ 0.27 15.71 ⫾ 6.98a 5.66 ⫾ 1.36 1.31 ⫾ 0.63

Data are means ⫾ SD. a Significantly different from lean, P ⬍ 0.05.

BMI than lean subjects and had elevated fasting plasma insulin levels relative to lean controls, suggesting the presence of peripheral insulin resistance. Two of the seven obese subjects had a family history of diabetes, but because responses to leptin and basal AMPK expression/activity and fatty acid oxidation were similar between these individuals and the rest of the group, their data were included in the analysis. Neither lean nor obese subjects were taking any medications known to alter carbohydrate or fatty acid metabolism. AICAR stimulated AMPK␣1 and AMPK␣2 activities (Fig. 1, A and B) in myotubes from lean and obese subjects. In skeletal muscle myotubes from lean subjects, a LH increased

FIG. 1. AMPK␣1 (A) and AMPK␣2 (B) activity, AMPK Thr172 phosphorylation (C), and ACC␤ Ser222 phosphorylation (D) in cultured myotubes of lean and obese subjects after 30 min of treatment with vehicle (PBS), AICAR (2 mM), or LL (0.1 ␮g/ ml) and LH (0.5 ␮g/ml). Data are shown as means ⫾ SEM, n ⫽ 7. a, Significantly different from vehicle treatment; b, same condition from lean cells.

Steinberg et al. • SOCS3 and Skeletal Muscle Leptin Resistance

AMPK␣2 activity relative to vehicle-treated cells (P ⬍ 0.001) or a low dose of leptin (Fig. 1B). A LH also increased AMPK␣1 activity (Fig. 1A). Unlike skeletal muscle from lean subjects, in obese cultured skeletal muscle, LH did not increase AMPK␣1 or AMPK␣2 activities (Fig. 1, A and B). In agreement with previous findings (31), we found that AMPK␣1 was approximately 10-fold more active than AMPK␣2 in primary human myotubes. In human skeletal muscle, AMPK␣1 and AMPK␣2 display similar activities (19, 35), and AMPK␣2 is more sensitive to activation by AICAR (19). The reason for this shift in isoform activity in primary myotubes is unknown. Consistent with increases in AMPK activity, AICAR increased AMPK Thr172 phosphorylation (Fig. 1C) and ACC␤ Ser222 phosphorylation (Fig. 1D) in both lean and obese subjects. AMPK␣ and ACC␤ protein expression was unaltered between lean and obese subjects (data not shown). In lean subjects, AMPK Thr172 phosphorylation was elevated after treatment with a LH but not a LL (Fig. 1C), but both LL and LH elevated ACC␤ phosphorylation (Fig. 1D). Consistent with a lack of AMPK activation in obese skeletal muscle, AMPK Thr172 phosphorylation (Fig. 1C) and ACC␤ Ser222 phosphorylation (Fig. 1D) were unaltered after leptin treatment.

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2). In contrast, whereas leptin treatment in Ad-Null increased AMPK␣1, AMPK␣2, ACC␤ phosphorylation, and fatty acid oxidation, this effect was suppressed in Ad-SOCS3 (Table 2). To examine mechanisms mediating the suppression of AMPK signaling by SOCS3, we measured ATP, AMP, phosphocreatine, creatine, and lactate after vehicle or leptin treatment in Ad-Null- and Ad-SOCS3-infected cells. Leptin increased cellular AMP and reduced ATP in Ad-Null, but this effect was abolished in Ad-SOCS3 (Table 3). Phosphocreatine, creatine, or lactate was unaltered by either leptin or SOCS3. LKB1 activity was not affected by either leptin or SOCS3 (Table 3). FIG. 2. Palmitate oxidation in cultured myotubes from lean and obese subjects treated with vehicle (PBS), AICAR (2 mM), or LL (0.1 ␮g/ml) and LH (0.5 ␮g/ml) of leptin for 120 min. Data are shown as means ⫾ SEM, n ⫽ 7. a, Significantly different from vehicle treatment; b, same condition from lean cells.

AICAR treatment increased palmitate oxidation in both lean and obese myotubes (Fig. 2). Consistent with changes in ACC␤ Ser222 phosphorylation, a LH increased palmitate oxidation in lean myotubes but did not alter palmitate oxidation in myotubes from lean subjects. To investigate potential mechanisms contributing to leptin resistance observed in obese myotubes, we examined the expression of SOCS3. Skeletal muscle myotubes from obese subjects had greater SOCS3 mRNA expression relative to myotubes derived from lean subjects (lean, 3.69 ⫾ 1.56; obese, 7.71 ⫾ 2.02 arbitrary units, P ⫽ 0.034). SOCS3 expression was related to BMI (R ⫽ 0.524, P ⫽ 0.030). Similarly, IL-6 expression was also elevated in obese subjects (lean, 1.0 ⫾ 0.22; obese, 3.76 ⫾ 1.2 arbitrary units, P ⫽ 0.019) and was also positively correlated with BMI (r ⫽ 0.501, P ⫽ 0.034). Leptin receptor expression was not significantly altered between lean and obese subjects (lean, 2.70 ⫾ 1.66; obese, 4.24 ⫾ 2.43 arbitrary units). To determine whether there was a causal link between elevated SOCS3 mRNA expression and reduced leptin sensitivity in myotubes from obese subjects, we infected primary human myotubes from a lean subject with a SOCS3 adenovirus (Ad-SOCS3) or green fluorescent protein control virus (Ad-Null). Seventy-two hours after infection, SOCS3 was overexpressed in skeletal muscle myotubes from lean subjects in Ad-SOCS3-infected (2.64 ⫾ 0.94 arbitrary density units) but not Ad-Null-infected (1.00 ⫾ 0.94 arbitrary density units) cells. AMPK␣-pan (␣1 and ␣2) expression was not different between Ad-Null- and Ad-SOCS3-infected cells. AICAR increased AMPK␣1 and -␣2 activities and increased ACC␤ phosphorylation and fatty acid oxidation to a similar degree in both Ad-Null- and Ad-SOCS3-infected cells (Table

Discussion

More than a decade ago, leptin was found to rapidly restore euglycemia independent of weight loss in ob/ob mice, suggesting a role of leptin in regulating insulin sensitivity in peripheral tissues (36). However, it was later found that rodents with diet-induced obesity (37) and obese humans (18) have elevated levels of leptin, suggesting the development of leptin resistance in human obesity. Indeed clinical trials subsequently demonstrated that leptin was largely ineffective in treating most cases of human obesity (38). Leptin signaling is mediated by short and long leptin receptor isoforms, both of which are expressed in skeletal muscle (39). Skeletal muscle is an important target tissue for leptin action because it is the primary tissue contributing to basal metabolic rate and whole-body fatty acid metabolism, and the aberrant regulation of skeletal muscle fatty acid metabolism contributes to the development of insulin resistance (4). In the present study, we provide the first evidence that leptin also stimulates AMPK activity, ACC␤ phosphorylation, and fatty acid oxidation in cultured skeletal muscle of lean subjects. The most significant finding of this study is that leptin activation of AMPK signaling is blunted with obesity. We (19) and others (40) recently demonstrated that the mRNA and protein expression of AMPK isoforms is not altered in skeletal muscle of obese or type 2 diabetic subjects and that pharmacological activation of AMPK by AICAR is maintained (19, 31, 41). AMPK activation by AICAR is also maintained in myotubes in culture, indicating that the suppressed activation of AMPK by leptin in obesity is not due to defects in AMPK signaling. These findings are consistent with leptin resistance in myotubes cultured from obese individuals being due to defects upstream of AMPK. A potential mediator of leptin resistance is SOCS3. In the present study, we illustrate that SOCS3 is up-regulated in skel-

TABLE 2. AMPK ␣1 and ␣2 activities, ACC phosphorylation and palmitate oxidation from a lean subject differentiated for 4 d and infected with a control (Ad-Null) or SOCS3 (Ad-SOCS3) adenovirus for 72 h followed by treatment with vehicle, AICAR, or leptin for 30 min (AMPK and ACC) or 120 min (palmitate oxidation) Ad-Null

AMPK␣1 (pmol/min䡠mg protein) AMPK␣2 (pmol/min䡠mg protein) pS222/ACC (arbitrary units) Palmitate oxidation (nmol/h)

Ad-SOCS3

Vehicle

AICAR

Leptin

Vehicle

AICAR

Leptin

18.3 ⫾ 1.7 3.8 ⫾ 0.4 2.3 ⫾ 0.9 3.0 ⫾ 1.2

38.5 ⫾ 7.9a 7.4 ⫾ 1.3a 9.7 ⫾ 0.2a 6.9 ⫾ 2.5a

27.9 ⫾ 6a 6.9 ⫾ 2.3a 6.5 ⫾ 3.5a 5.2 ⫾ 1.1a

13.5 ⫾ 1.8 5.5 ⫾ 0.2 2.1 ⫾ 0.7 2.8 ⫾ 0.8

46.5 ⫾ 11a 6.7 ⫾ 0.2a 8.4 ⫾ 4.7a 5.1 ⫾ 0.5a

18.5 ⫾ 8.7 4.2 ⫾ 1.4b 2.7 ⫾ 0.3b 2.9 ⫾ 1.3

Data shown as means ⫾ SD, n ⫽ 6 of two independent experiments. Significantly different from avehicle and bsame condition in Ad-Null infected cells.

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TABLE 3. Adenine nucleotides, muscle metabolites, and LKB1 activity in primary myotubes from a lean subject differentiated for 4 d and infected with a control (Ad-Null) or SOCS3 (Ad-SOCS3) adenovirus for 72 h followed by treatment with vehicle or leptin for 30 min Ad-Null

ATP (nmol/mg protein) AMP (pmol/mg protein) Phosphocreatine (nmol/mg protein) Creatine (nmol/mg protein) Lactate (nmol/mg protein) LKB1 activity (pmol/min䡠mg protein)

Ad-SOCS3

Vehicle

Leptin

Vehicle

Leptin

6.3 ⫾ 2.0 0.81 ⫾ 0.20 16.3 ⫾ 3.9 48.8 ⫾ 6.3 10 ⫾ 2.7 10.7 ⫾ 1.8

4.3 ⫾ 1.0a 2.28 ⫾ 1.2a 15.6 ⫾ 1.7 36.9 ⫾ 8.5 12.9 ⫾ 5.1 11.5 ⫾ 0.8

7.5 ⫾ 0.8 1.24 ⫾ 0.80 18.1 ⫾ 4.6 42.7 ⫾ 5.4 11.5 ⫾ 7.0 11.9 ⫾ 2.5

7.1 ⫾ 1.1 1.84 ⫾ 1.1 18.4 ⫾ 4.0 36.0 ⫾ 8.7 15.3 ⫾ 4.8 8.9 ⫾ 1.4

Data are means ⫾ SD, n ⫽ 6 of two separate experiments. a Significantly different from vehicle for Ad-Null, P ⬍ 0.05.

etal muscle myotubes of obese humans. The SOCS family of proteins were first identified by their rapid up-regulation in response to IL-6 acting in a classical negative feedback loop to regulate cytokine signal transduction (42). Human obesity is characterized by elevated levels of circulating IL-6 (43). Recent studies demonstrated that IL-6 is highly expressed in skeletal muscle (44); therefore, we hypothesized that elevated levels of SOCS3 in obese myotubes may be due to increased IL-6 expression. We found that IL-6 expression was positively associated with BMI and was elevated in obese myotubes. Interestingly, whereas Rieusset et al. (45) demonstrated the upregulation of SOCS3 by IL-6 in primary myotubes and elevated SOCS3 in type 2 diabetic skeletal muscle, SOCS3 was not elevated in skeletal muscle of obese subjects (BMI 32.9 kg/m2). One potential reason for the disparity between our data and Riessuet et al. (45) may be due to the morbidly obese subjects (BMI 47.3 kg/m2) used in the present study. In support of this possibility, we observed a positive correlation between both IL-6 and SOCS3 expression and BMI. These data suggest that elevated IL-6 levels in obese myotubes may be of genetic origin because myotubes were passaged four times before experiments, thus most likely eliminating any contribution from their in vivo environment. The mechanisms contributing to elevated IL-6 in obese myotubes may involve the C-174G promoter polymorphism of the IL-6 gene, which is associated with elevated circulating IL-6, reduced energy expenditure and insulin sensitivity, and an increased predisposition for obesity (46). To examine whether there was a relationship between the elevation of SOCS3 in myotubes of obese subjects and reduced AMPK activation by leptin, SOCS3 was overexpressed in leptin-sensitive myotubes of a lean subject via adenovirus infection to a similar degree as observed in obese myotubes. The up-regulation of SOCS3 inhibited leptin activation of AMPK but importantly, not activation of AMPK by AICAR, demonstrating a direct effect of SOCS3 on leptin signaling upstream of AMPK. Reduced activation of AMPK was accompanied by reduced ACC␤ phosphorylation and fatty acid oxidation after leptin treatment, indicating that increased SOCS3 expression with obesity may contribute to the suppressed rates of skeletal muscle fatty acid oxidation observed in vivo and may therefore contribute to the accumulation of im lipid and subsequent insulin resistance common in obesity (1). Leptin directly activates AMPK, stimulating fatty acid oxidation acutely in skeletal muscle by altering the AMP to ATP ratio (8). We provide evidence that a similar mechanism exists for activating AMPK in human myotubes and that

SOCS3 inhibits leptin-induced increases in cellular AMP, which in turn suppresses leptin activation of AMPK signaling. The mechanism by which leptin and other hormones such as adiponectin (47) and ciliary neurotrophic factor (17) increase cellular AMP is currently unknown but may involve mitochondrial uncoupling or futile cycling. In the present study, we illustrate that LKB1 is not activated by leptin. These findings are in agreement with previous studies in a variety of cell systems (12, 13) that indicate that LKB1 is constitutively active despite large alterations in AMPK Thr172 phosphorylation and activity, suggesting that increases in nucleotide levels and localization of the LKB1 accessory proteins MO25 and STRAD may be more critical determinants of AMPK phosphorylation/activity. In conclusion, the principal findings of this study are that leptin stimulates AMPK activity, ACC␤ phosphorylation, and fatty acid oxidation in skeletal muscle myotubes from lean subjects. Of significance in myotubes of morbidly obese subjects, increased SOCS3 expression was associated with blunted leptin-dependent activation of AMPK signaling, an effect that was replicated in lean skeletal muscle myotubes overexpressing SOCS3. These data indicate that SOCS3 expression in skeletal muscle may be an important factor contributing to the development of skeletal muscle leptin resistance and the suppressed rates of skeletal muscle fatty acid oxidation observed in obesity. Acknowledgments We thank AMRAD Operations Pty. Ltd. (Richmond, Victoria, Australia) for providing the SOCS3 adenovirus construct and Dr. Walter Thomas (Baker Heart Research Institute, Melbourne, Australia) for assistance with purification. We gratefully acknowledge the invaluable contributions of the surgeons, Dr. Simon Woods, F.R.A.C.S. (Monash University Academic Surgery Unit, Cabrini Hospital) and Dr. Mark Lawrence (Network Director, Obstetrics and Gynaecology, Bayside Health). We thank Ms. Janelle Mollica for assisting with the preparation of the human muscle cell culture. Received March 23, 2006. Accepted June 26, 2006. Address all correspondence and requests for reprints to: Gregory R. Steinberg, Ph.D., St. Vincent’s Institute, 9 Princes Street, Fitzroy, Victoria 3065, Australia. E-mail: [email protected]. This work was supported by the National Health and Medical Research Council (to B.E.K., G.R.S.), Diabetes Australia Research Trust (to G.R.S.), and the National Heart Foundation (to B.E.K.). G.R.S. is supported by a Target Obesity Research Fellowship from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada, and the Canadian Diabetes Association. B.E.K. is an Australian Research Council Federation Fellow. Disclosure statement: The authors have nothing to declare.

Steinberg et al. • SOCS3 and Skeletal Muscle Leptin Resistance

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