Palmitate-induced Down-regulation of Sortilin and Impaired GLUT4 Trafficking in C2C12 Myotubes*

Elevated saturated FFAs including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKCθ and TNF-α-converting enzyme, but not p38, JNK, or mitochondrial reactive oxygen species generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKCθ activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the peroxisome proliferator-activated receptor γ agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKCθ states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKCθ-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of peroxisome proliferator-activated receptor γ agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.

Skeletal muscle cells as well as adipocytes exhibit the highest levels of insulin-stimulated glucose uptake, which is achieved by translocation of the insulin-responsive glucose transporter (GLUT4) from intracellular storage compartment(s) to the plasma membrane (1). It has become increasingly apparent that sortilin, a type I transmembrane protein originally identified as a major component of GLUT4-containing vesicles from rat adipocytes (2,3), plays crucial roles in the development of the insulin-responsive GLUT4 translocation system not only in adipocytes (4,5) but also in skeletal muscle cells (6). Evidence available thus far indicates sortilin to be directly involved in the biogenesis of insulin-responsive GLUT4 storage vesicles by regulating sorting events of GLUT4 protein between the trans-Golgi network and endosomes (4,5,7), and experimental suppression of sortilin in adipocytes has been shown to inhibit insulin-responsive GLUT4 translocation (4).
Intriguingly, a recent report demonstrated sortilin expression to be significantly reduced in skeletal muscle and adipose tissues of obese and diabetic db/db mice and human patients, appearing to be inversely correlated with the expression levels of pro-inflammatory TNF-␣ in adipose tissue (8). In addition, injecting TNF-␣, which can induce insulin resistance, into mice resulted in late onset down-regulation of sortilin mRNA and protein levels in skeletal muscle and adipose tissues (8), suggesting possible involvement of sortilin reduction in the pathogenesis of chronic insulin resistance induced by TNF-␣, especially in terms of insulin-responsive GLUT4 translocation.
Insulin resistance is defined as the pathophysiological condition in which the ability of insulin to regulate glucose homeostasis in target cells is reduced, a state commonly associated with obesity (9). Indeed, high fat feeding and increased levels of circulating FFAs progressively led to the induction of peripheral insulin resistance characterized by impaired insulin-responsive GLUT4 translocation in skeletal muscle (10,11). The deleterious effects of saturated FFAs such as palmitate (C16:0) in skeletal muscle have been attributed to abnormal accumulation of palmitoyl-CoA, diacylglycerol, and/or ceramide, which subsequently leads to aberrant activation of various serine/ threonine kinases such as PKC (12)(13)(14).
PKC is a novel PKC isoform abundantly expressed in skeletal muscle, which reportedly may be relevant to FFA-induced muscle insulin resistance (15). One of the established pathogenic effects of PKC activity is detrimental phosphorylation of serine residues on insulin receptor substrate (IRS) 3 proteins, which in turn reduces the ability of the IRS proteins to activate phosphatidylinositol 3-kinase cascades (13,16,17). In addition, PKC has the unique ability to activate transcriptional factor NF-B, and the PKC-NF-B signaling cascade has been directly implicated in the expression of various pro-inflammatory cytokines including TNF-␣ (14). TNF-␣ has also been shown to trigger phosphorylation of the critical serine residues of IRS proteins (18,19). Thus, it is generally accepted that, as a consequence of the impaired insulin signaling competency resulting from at least these two distinct mechanisms involving PKC, insulin-responsive glucose uptake/disposal in skeletal muscle is diminished.
In the present study, we treated C2C12 myotubes with various saturated and unsaturated FFAs to study the molecular mechanisms underlying the FFA-induced insulin resistance in skeletal muscle cells as assessed by insulin-responsive GLUT4 recycling. We demonstrated saturated FFAs, especially palmitate (C16:0), but not unsaturated FFAs, to induce down-regulation of sortilin via a mechanism involving PKC, leading to impaired GLUT4 trafficking in differentiated C2C12 myotubes. In addition, we demonstrated a crucial role for sortilin in maintaining proper insulin-responsive GLUT4 trafficking even in palmitate-treated cells, because a PPAR␥ agonist restored sortilin abundance and GLUT4 recycling without improving palmitate-induced impairments of signaling cascades. These findings provide novel insights into the beneficial actions of PPAR␥ agonists and the pathogenesis of PKC-dependent insulin resistance elicited by palmitate, which could occur via a modulation of sortilin, a sorting receptor involved in GLUT4 sorting/trafficking in skeletal muscle cells.
Cell Culture-Mouse skeletal muscle cell lines, C2C12 myoblasts, were maintained in DMEM supplemented with 10% FBS, 30 g/ml penicillin, and 100 g/ml streptomycin (growth medium) at 37°C under a 5% CO 2 atmosphere. For biochemical study, the cells were grown on six-well plates (BD Biosciences) at a density of 3 ϫ 10 4 cells/well in 3 ml of growth medium. Three days after plating, the cells had reached ϳ80 -90% confluence (day 0). Differentiation was then induced by replacing the growth medium with DMEM (4.5 g/liter glucose) supplemented with 2% calf serum, 1 nM insulin, 30 g/ml penicillin, and 100 g/ml streptomycin (differentiation medium) (20). The differentiation medium was changed every 24 h, and the differentiated cells (at days 4 and 5) were used for subsequent experiments. FFA-containing media were prepared by preincubation of FFA with DMEM supplemented with 2% FFA-free BSA as described previously (20). Briefly, FFAs were dissolved in ethanol and diluted 1:150 in DMEM containing 2% (w/v) FFA-free BSA and 2% calf serum, and the lipid-containing media were incubated for 1 h at 37°C before administration to the culture. Under these conditions, the molar ratio of FFA (0 -1.0 mM) to 2% BSA (calculated molecular weight of 665,000) would be ϳ0 -3.3. The inhibitors were prepared in dimethyl sulfoxide and then added to the media at 0.1% (v/v).
Quantitative Real Time PCR-Total RNA was prepared using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Quantitative real time PCR analysis was performed using the Light Cycler instrument and SYBR Green detection kit according to the manufacturer's instructions (Roche Applied Science). PCR primers for sortilin were: 5Ј-CCT CTG TGA CTT TGG CTA CTT C-3Ј and 5Ј-ATC CCA CCT TGG CAT TTG T-3Ј. PCR primers for ␤-actin as a control were: 5Ј-CGT TGA CAT CCG TAA AGA CCT C-3Ј and 5Ј-AGC CAC CGA TCC ACA CAG A-3Ј.
Western Blot Analysis-Cell lysates were prepared using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 g/ml pepstatin, 5 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 6500 IU/ml aprotinin, phosphatase inhibitor mixture-1; Sigma), and the protein concentrations of cell lysates were then measured using the BCA protein assay kit (Pierce). Proteins (20 g) were subjected to 10% SDS-PAGE and then transferred to a PVDF membrane (Immobilon-P; Millipore), and the membranes were then blocked for 2 h at room temperature with 5% BSA in TBS containing 0.1% Tween 20. The membranes were next immunoblotted with primary antibodies at dilutions of 1:500 to 1:1000. Specific total or phospho-proteins were visualized after subsequent incubation with a 1:10000 dilution of anti-mouse or rabbit IgG conjugated to horseradish peroxidase and the SuperSignal Chemiluminescence detection procedure (Pierce). Three independent experiments were performed for each condition.
Detection of Secreted Fragment of Sortilin in Conditioned Media-Conditioned media were collected and concentrated using Amicon Ultra-15/30K (Millipore Corp., Billerica, MA), followed by immunoprecipitation using anti-sortilin antibody. Immunoprecipitated materials were then subjected to Western blot analysis.
ELISA for TNF-␣-TNF-␣ levels in media were evaluated using the mouse TNF-␣ assay kit (R & D Systems).
Caspase-3 Assay-Cellular caspase-3 activity was evaluated using the EnzChek Caspase-3 assay kit (Invitrogen) according to the manufacturer's instructions. Briefly, the cell lysates were incubated with a caspase-3 substrate, Z-DEVD-AMC, which upon cleavaged by active caspase-3, generates fluorescent products. Fluorescent intensity was measured using a SpectraMax M5 (excitation, 342 nm; emission 441 nm) and subtracted from that of the cell lysate-free sample, followed by normalization using the corresponding protein amount levels.
Anti-c-Myc Antibody Uptake Assay-GLUT4 recycling was analyzed as described previously (21). Briefly, C2C12 myotubes expressing Myc-GLUT4-ECFP were serum-starved, washed three times with Krebs-Ringer/phosphate/HEPES (KRPH) buffer, and then placed in a CO 2 incubator with 2 ml of KRPH buffer. After 10 min of incubation, 4 g/ml of the anti-c-Myc antibody were added to the buffer, and the cells were stimulated with or without 100 nM insulin for 30 min. After incubation for 30 min with the anti-Myc antibody, the cells were placed on ice to stop the reaction, and washed five times with PBS. The cells were harvested using 1ϫ Laemmli buffer and subjected to Western blot analysis using anti-mouse IgG and anti-c-Myc antibodies. In the same experiments, we also measured amounts of Alexa 555-conjugated anti-c-Myc antibody bound to cell surface-exposed Myc-GLUT4-ECFP at 4°C using a laser-induced fluorescence scanner (PharosFX Plus; Bio-Rad) following SDS-PAGE of the whole cell lysates.  Palmitate-induced sortilin reduction is not mediated through autocrine secreted TNF-␣. A, C2C12 myotubes were treated with 0.25-1 mM palmitate for 16 h, and the conditioned medium was then subjected to ELISA for TNF-␣ concentration measurement. B, C2C12 myotubes were treated with 1-100 ng/ml of TNF-␣, and the cell lysates were subjected to Western blot analysis. C, C2C12 myotubes were treated with the indicated concentrations of palmitate for 16 h in the absence or presence of anti-TNF-␣ neutralizing antibody, and the cell lysates were then subjected to Western blot analysis. Three independent experiments were performed, and representative results were obtained.

Live Cell Imaging of Intracellular Trafficking and Data
Analysis-C2C12 myoblasts were maintained and differentiated into myotubes on poly-L-lysine-coated glass-bottomed recording chamber (thickness, 0.15-0.18 mm; Matsunami Glass, Osaka, Japan). The differentiated myotubes treated with fatty acids were stained with 50 nM LysoTracker Red DND-99 (Invitrogen) according to the manufacturer's instructions. Then the cells were washed with imaging medium (150 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES-NaOH, pH 7.4) containing 5.5 mM D-glucose. The images were acquired every 1 s for 100 s with an inverted microscope (IX81; Olympus, Tokyo, Japan) equipped with a laser scanner (FV1000; Olympus) and an oil-immersion objective lens (Pla-nApo 60ϫ, numerical aperture of 1.40). The fluorescence was excited at 543 nm and detected at Ͼ560 nm. The movement of stained acidic compartments was tracked with G-Track software with a centroid fitting mode (G-angstrom; Sendai). We tracked each object that was successfully fitted with 6 ϫ 6-pixel regions-of-interest for at least 30 consecutive frames. We showed the movement speed in two ways: individual and effective speed (22). The former was calculated based on displace-ment between two consecutive frames, and the latter was determined by dividing the distance between the origin and the end of the trace by the trace duration. The movement speed calculated as above inevitably contains a factor resulting from instrumental noise. Therefore, we corrected the "apparent" speeds by subtracting the value obtained from cells fixed with 1% paraformaldehyde after staining with LysoTracker.
Statistical Analysis-The data are expressed as the means Ϯ S.E. Statistical significance was determined by analysis of variance followed by the Williams' test unless otherwise indicated. The differences were considered significant when the p values were less than 0.05.
It should be noted that high concentrations of saturated FFAs and TNF-␣ (100 ng/ml), but not unsaturated FFAs, induced significant increases in caspase-3 activity (data not shown), indicating that myotubes were undergoing apoptotic responses in response to either saturated FFAs or TNF-␣ under these culture conditions, as reported previously (23,24). PKC and TACE/ADAM17, but Neither p38 MAPK nor NF-B Are Involved in Palmitate-induced Reduction of Sortilin in C2C12 Myotubes-Because treatment with palmitate resulted in aberrant activations of a wide variety of intracellular signaling cascades including p38 MAPK, JNK, and PKC in C2C12 myotubes as we previously reported (20), we next attempted to define intracellular signaling cascades involved in the palmitate-induced reduction of sortilin expression. Pharmacological inhibition of PKC by rottlerin abolished the effect of palmitate, and the sortilin amount was restored when the cells were treated with palmitate in the presence of 30 M rottlerin (Fig. 3, A and B). Involvement of PKC in the palmitateinduced sortilin reduction was also confirmed by shRNA-mediated knockdown experiments, and the effect of palmitate was attenuated in C2C12 myotubes treated with PKC-specific, but not scramble control, shRNA lentiviral particles (Fig. 3C).
To further investigate the underlying mechanism of the PKC-dependent sortilin suppression elicited by palmitate treatment, we examined the effect of TAPI-1, a selective inhibitor for TACE (also known as ADAM17) because sortilin reportedly undergoes shedding of its luminal domain with TACE (25), which could be induced by phorbol 12-myristate 13-acetate, a PKC activator (26). Palmitate-induced sortilin reduction was significantly blunted by TAPI-1 in a dose-dependent manner, although 30 M of TAPI-1 could not reverse the sortilin amount to the control levels (Fig. 4). Consistent with these findings, the secreted fragment of sortilin was detected in the conditioned media after palmitate treatment (16 h) but then disappeared with the addition of TAPI-1 in a dose-dependent manner (Fig. 4).
We also examined the possible involvement of mitochondria-derived reactive oxygen species by using pharmacological inhibitors for enzymes related to fatty acid oxidation. Etomoxir (carnitine palmitoyltransferase-1 inhibitor), thenoyltrifluoroacetone (electron transport complex II inhibitor), and carbonyl cyanide m-chlorophenylhydrazone (an uncoupler of oxidative phosphorylation) all failed to exert any inhibitory effect on palmitate-induced sortilin reduction (Fig. 5D), whereas these inhibitors completely abolished reactive oxygen species production (data not shown). We also found that administration of ceramide, a possible mediator of the actions of palmitate (27,28), did not mimic palmitate-induced sortilin down-regulation at least with 16 h of C2-ceramide (1-100 M) treatment (data not shown).
PPAR␥, but Neither PPAR␣ nor PPAR␤/␦, Is Involved in the Protective Action of Unsaturated FFAs against Palmitate-induced Sortilin Reduction-We also examined the possible involvement of PPARs in the protective actions of unsaturated FFAs on palmitate-induced events, because unsaturated FFAs have been shown to serve as potent ligands for these PPARs (31,32). The palmitate-induced sortilin reduction was not reversed by either Wy-14643 (PPAR␣ agonist) or GW501516 (PPAR␤/␦ agonist) (33,34). However, troglitazone (PPAR␥ agonist) (35) displayed a remarkable dose-dependent inhibitory effect on palmitate-induced sortilin reduction with no obvious alterations in palmitate-induced PKC phosphorylation (Fig. 7). Although relatively higher concentrations of troglitazone were required for its protective effect against palmitate-induced sortilin down-regulation to be exerted, this is perhaps due to sup-  NOVEMBER 5, 2010 • VOLUME 285 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 34375 plementation with BSA, which reportedly binds troglitazone and affects its potency (36,37).

Involvement of Sortilin Reduction in Palmitate-induced Impairment of Insulin-responsive GLUT4 Recycling in C2C12
Myotubes-Finally, we examined the involvement of sortilin reduction in the generation of insulin resistance in C2C12 myotubes, because sortilin plays important roles in establishing the insulin-responsive glucose transport system in skeletal muscle cells (6). To address this possibility, we employed a Myc-GLUT4 recycling assay using anti-Myc antibody as we reported previously (21). Consistent with a previous report (20), palmitate treatment impaired insulin-responsive GLUT4 recycling, concomitantly with the sortilin reduction, with impaired insulin responsiveness being partially attributable to the increased GLUT4 recycling even under basal conditions (Fig. 8). The palmitate-induced impairment of insulin-responsive GLUT4 recycling was reversed by adding concentrations of palmitoleate (C16:1), oleate (C18:1), or troglitazone sufficient to restore sortilin expression levels (Fig. 8). The impairment of insulin-induced Akt phosphorylation was reversed by unsaturated FFAs, but not by troglitazone. Insulin-induced GLUT4 translocation was significantly inhibited by shRNA-mediated sortilin knockdown in C2C12 myotubes without influencing Akt phosphorylation (Fig. 8C).
Palmitate Impairs Lysosome Motility-To examine whether palmitate affects intracellular trafficking events other than GLUT4 translocation, we analyzed the movement of acidic compartments including lysosomes in C2C12 myotubes by staining with LysoTracker Red (Fig.  9A). The dye-positive structures showed highly dynamic movement in the cells (Fig. 9B), as in other types of cells (38,39). We quantitatively analyzed the movement by calculating the movement speed based on displacement between two consecutive frames or between the origin and the end of the trace (Fig.  9, C-E; see "Experimental Procedures"). Interestingly, treatment with palmitate for 16 h significantly reduced the movement of intracellular acidic compartments (Fig. 9, C-E). These effects of palmitate were dose-and time-dependent (Fig. 9, F and G). Importantly, oleate significantly inhibited the deleterious effects of palmitate and completely restored the movement of acidic compartments to levels comparable with those under intact conditions (Fig. 9, D and E). These observations indicate that palmitate affects not only GLUT4 trafficking but also intracellular trafficking more widely and that oleate has a restorative effect on palmitate-induced derangements of intracellular trafficking in C2C12 myotubes.

DISCUSSION
The role of PKC activity elicited by the palmitate-induced accumulation of DAG and acyl-CoA has been well established in generating pathogenic insulin resistance (15), which is generally explained by impaired insulin signaling cascades resulting from detrimental PKC-induced phosphorylation of serine residues on IRS proteins (13,17,40) and PDK1 (41). In the present study, we demonstrated that palmitate-induced PKC activation is also attributable to the down-regulation of sortilin (Fig. 3), a sorting receptor protein implicated in generating insulin-responsive GLUT4 vesicles. Importantly, unsaturated FFAs including palmitoleate (C16:1) and oleate (C18:1) effectively reversed palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin protein abundance by preventing aberrant PKC phosphorylation (Fig. 6). On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes slightly but significantly inhibited insulin-induced GLUT4 recycling without dampen- ing Akt phosphorylation (Fig. 8). In addition, palmitate treatment appeared to significantly impair lysosome motility, which was again effectively restored by oleate administration (Fig. 9). Although the degree to which detrimental PKC actions on the generation of insulin signaling defects or the down-regulation of sortilin expression contributes to the development of impaired GLUT4 recycling remains to be determined, our findings shed novel mechanistic insights into the pathogenic roles of PKC in generating insulin resistance in skeletal muscle cells (Fig. 10).
Involvement of PKC and TACE/ADAM17 in Palmitate-induced Sortilin Down-regulation-It was recently reported that exogenous administration of TNF-␣, an established inducer of insulin resistance (42), resulted in decreased sortilin expression in both adipose and skeletal muscle tissues in vivo and that TNF-␣-induced sortilin suppression was also observed in cultured adipocytes (8). However, we demonstrated the contribution of autocrine TNF-␣ to the palmitate-induced sortilin reduction in C2C12 myotubes to be minimal at least under the culture conditions of the present study, because the anti-TNF-␣ neutralizing antibody had no impact (Fig. 2), with palmitate instead directly inducing sortilin down-regulation through a mechanism involving PKC as an intracellular signaling intermediate (Fig. 3). In agreement with this observation, pharmacological inhibition of NF-B cascades by pyrrolidine dithiocarbamate, which completely abolished palmitate-induced TNF-␣ secretion (data not shown), failed to interfere with the sortilin reduction (Fig. 5A). Because high concentra-tions of TNF-␣ suppressed sortilin expression in C2C12 myotubes (Fig. 2), similar to observations in cultured adipocytes (8), sortilin expression in skeletal muscle is perhaps regulated by various different and/or combined insults such as saturated FFAs and circulating TNF-␣ triggering insulin resistance in vivo.
We provide compelling evidence that PKC activity is predominantly involved in palmitate-induced sortilin down-regulation by showing that inhibition of PKC activity and phosphorylation by either the pharmacological inhibitor rottlerin (Fig. 3) or unsaturated FFAs (Fig. 6) completely prevent this palmitate-induced event. However, shRNA-mediated reduction of PKC only partially prevented the decrease in sortilin abundance by palmitate treatment (Fig. 3D). We also found that palmitate-induced suppression of sortilin mRNA expression was obvious at concentrations higher than 0.75 mM, whereas sortilin protein was significantly reduced at 0.5 mM palmitate (Fig. 1). Taken together, these results suggest that palmitateinduced sortilin down-regulation is complex, being induced at both the transcriptional and the post-transcriptional level, possibly involving multiple harmful intermediates evoked by palmitate in addition to PKC activity.
In this regard, we found that palmitate-induced sortilin reduction is significantly inhibited by TAPI-1, the selective TACE/ADAM17 inhibitor (Fig. 4), indicating PKC-dependent  A, the cell lysates were subjected to Western blot analysis. Three independent experiments were performed, and representative results were obtained. B, specific bands in the Western blots were quantified using a densitometer. The data are expressed as the means Ϯ S.E. of three independent experiments. *, p Ͻ 0.05 versus C16:0 alone. NOVEMBER 5, 2010 • VOLUME 285 • NUMBER 45 sortilin reduction to be mediated at least partially through the post-translational modification (shedding) involving TACE activity (25), which could be activated by a PKC activator, TPA (26). Intriguingly, several recent studies have demonstrated that TACE activity is increased in the skeletal muscles of obese type 2 diabetes patients and in lipid-induced insulin resistance (43,44). In addition, increased TACE activity has been directly implicated in the pathogenesis of insulin resistance, which is generally explained by the augmented secretion of TNF-␣ (43,45,46). Thus, our findings provide an important connection between TACE activity and PKC-dependent sortilin reduction in skeletal muscle cells under palmitate-induced insulinresistant conditions, which appeared to be directly involved in the pathogenesis of impaired GLUT4 trafficking as discussed below.

Sortilin Reduction and Insulin Resistance in C2C12 Myotubes
At present, we have not yet defined the molecular mechanism underlying the palmitate-induced sortilin mRNA downregulation. PKC has been shown to modulate the activities of many transcription factors including nuclear factor of activated T cell c2, NF-B, and activator protein-1 (47), and several lines of evidence indicate PKC to also be involved in transcriptional repression by modulating nuclear receptor-corepressor and/or -coactivator interaction in T lymphoid cells (48) (49). Because significant increases in caspase-3 activity were detected in C2C12 myotubes exposed to saturated FFAs but not unsaturated FFAs, the suppression of sortilin mRNA expression observed herein might be a consequence of apoptotic responses to some degree. Further studies are needed to clarify this important issue.
Palmitate-induced Sortilin Down-regulation and Impaired GLUT4 Trafficking-In our previous study designed to elucidate the role of sortilin in the development of insulin-responsive GLUT4 translocation in C2C12 myocytes, we observed that sortilin serves as an important factor in generating the insulin-responsive glucose transport system (6), essentially as seen in the 3T3L1 adipogenic cell line (4). In addition, we reported sortilin to also be directly involved in the early phase of myogenic differentiation processes by cooperatively functioning with p75 neurotrophin receptor and pro-form of NGF of nerve growth factor in C2C12 myocytes (6). Because siRNA-mediated sortilin knockdown in C2C12 myoblasts severely inhibited their myogenic differentiation (data not shown) (6), we utilized shRNA lentiviral technology to knock down sortilin expression in differentiated C2C12 myotubes in the present study (Fig. 8). Similar to observations in the 3T3L1 adipogenic cell line (4), our data clearly demonstrated that experimental suppression of sortilin abundance in differentiated C2C12 myotubes resulted in impaired insulin-responsive GLUT4 recycling, which is partially attributable to increased GLUT4 recycling even in the basal state, without affecting insulin-induced Akt phosphorylation (Fig. 8).
Supporting an essential role for sortilin in the formation of insulin-responsive GLUT4 vesicles (4 -7), our data (shown in To evaluate Akt phosphorylation status, the serum-starved cells were treated with 100 nM insulin for 5 min. A, the cell lysates were subjected to Western blot analysis using anti-phospho Akt (Ser 473 ) and anti-Akt antibodies. B, the results from A, uptake of anti-Myc antibody in response to insulin, were subjected to densitometric analysis for quantification. C, differentiated C2C12 myotubes expressing Myc-GLUT4-ECFP (day 3) were treated with or without lentiviral particles containing PKC-specific shRNA or scramble control shRNA and then further cultured for 72 h. The Myc-GLUT4 recycling assay and Western blot analysis were performed as described above for A and B. The data are expressed as the means Ϯ S.E. of four independent experiments. *, p Ͻ 0.05 versus each basal conditions. Representative immunoblots obtained from two to four independent experiments are shown. Fig. 8) strongly suggest that sortilin down-regulation induced by palmitate treatment is involved in the pathogenesis of impaired GLUT4 trafficking and, therefore, that restoration of sortilin abundance either by unsaturated FFAs or troglitazone is crucial for maintaining GLUT4 trafficking in response to insulin stimulation in C2C12 myotubes. These findings are consistent with previous studies showing that unsaturated FFAs such as palmiteoleate (C16:1) and oleate (C18:1) have protective effects on palmitate-induced insulin resistance at the level of glucose uptake in L6 myotubes (50,51). At present we have not yet defined in detail the mechanism(s) underlying the impacts of sortilin reduction on impaired GLUT4 trafficking. However, because our data indicate the palmitate-induced sortilin reduction to be detectable after 16 h of treatment with relatively high levels of palmitate ( Fig. 1), this palmitate-induced late onset event perhaps contributes to generating the chronic, but not the acute, phase of insulin resistance (52-54), possibly resulting from impairments in the formation of insulin-responsive GLUT4 storage vesicles. The most plausible explanation for the impaired GLUT4 trafficking elicited by prolonged palmitate treatment is the derangement in sorting/targeting events of GLUT4 governed by sortilin, possibly contributing to the higher basal GLUT4 localization at the plasma membrane with a subsequent reduction in insulin-responsive recycling as we observed herein (Fig. 8). Given that the formation of insulin-responsive GLUT4 storage vesicles is a prerequisite for skeletal muscle cells to properly respond to the intracellular signals evoked by insulin stimulation (6), future studies are warranted to clarify the mechanism(s) underlying the impacts of sortilin reduction on impaired GLUT4 trafficking, especially on GLUT4 storage vesicle formation.
An interesting observation made in the present study was that troglitazone, a PPAR␥ agonist (35), but neither ␣ nor ␤/␦  Palmitate-induced activation of PKC, but not mitochondrial reactive oxygen species, p38, JNK, or NF-B, contributes to sortilin down-regulation resulting from actions at both transcriptional (mRNA expression) and post-transcriptional (TACE-mediated shedding) levels. Unsaturated FFA such as oleate effectively prevented aberrant PKC activation elicited by palmitate and thereby restored sortilin contents as well as insulin-responsive GLUT4 translocation. Thus, palmitate-induced deleterious PKC activation contributes to not only dampening of insulin receptor signals (signaling defects), but also the induction of sortilin down-regulation, possibly resulting in derangements in GLUT4 sorting/targeting (trafficking defects). In addition to these PKC-dependent events, palmitate also induces significant impairments in lysosome motility, indicating that various intracellular trafficking events are severely deranged under the insulin-resistant conditions elicited by palmitate.
agonist, prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling impairment (Fig. 8) without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKC states (Fig. 7) nor impaired insulin-responsive Akt phosphorylation was affected (Fig. 7). These data strongly suggest that sortilin restoration, rather than signaling competency involving Akt phosphorylation, is crucial for maintaining the insulin-responsive GLUT4 recycling in skeletal muscle cells at least under the present culture conditions. In excellent agreement with this idea, it has become increasingly apparent that the most deleterious defects of skeletal muscle insulin resistance including those evoked by palmitate/high fat occur independently of the IRS signaling cascades (53,(55)(56)(57). Moreover, several lines of evidence have also demonstrated that despite its therapeutic benefits, rosiglitazone, a PPAR␥ agonist, failed to ameliorate the IRS signaling cascade defects in the skeletal muscles of FFA-induced insulin resistant rodents (58) as well as that in human patients with type 2 diabetes (59). On the other hand, as mentioned above, Kaddai et al. (8) recently reported that sortilin expression was significantly down-regulated in adipose and muscle tissues in obese mice and humans. Importantly, they also found that rosiglitazone prevented the sortilin reduction triggered by TNF-␣ treatment in cultured adipocytes (8). Taken together with the results of our present study, these compelling lines of evidence have now set the stage for the hypothesis that insulin resistance, especially as regards insulinresponsive GLUT4 translocation, could occur not only through insulin signaling defects but also via malfunction/reduction of protein(s) such as sortilin that directly controls trafficking and/or sorting of GLUT4. In addition, our data provide novel insights into the beneficial actions of PPAR␥ agonists, which are highly likely to exert their insulin-sensitizing actions in skeletal muscle cells via effects on multiple cellular functions (60), including sortilin restoration, perhaps in concert with the improvements in insulin signaling cascades in skeletal muscles under conditions of insulin resistance (61)(62)(63).
Finally, our findings that palmitate treatment resulted in a significant impairment in lysosomal motility strongly suggest multiple intracellular trafficking events in addition to GLUT4 trafficking to also be deranged under insulin-resistant conditions. Although the underlying mechanisms and pathogenic relevance to development of the impaired insulin-responsive GLUT4 translocation remain to be clarified, our novel findings raise the possibility that insulin-resistant states could also be characterized as intracellular trafficking defects (65) in addition to the pathophysiological conditions characterized as insulin receptor signaling defects (Fig. 10).
Given that muscle insulin resistance has been implicated as one of the earliest defects in type 2 diabetes development, characterized by decreased glucose transport and metabolism (66,67), it is clear that further understanding of the events underlying palmitate-induced events, both sortilin down-regulation and trafficking defects, could have major implications not only for our knowledge and understanding of glucose homeostasis but also for the development of new treatment strategies and therapies for type 2 diabetes.