TNF-α stimulates glucose uptake in L6 myoblasts

ELSEVIER

Diabetes Research and Clinical Practice 32 (1996) 1I 18

TNF-a stimulates glucose uptake in L6 myoblasts Hironori Yamasaki”,“, Yoshihiko Yamaguchi”, Hirofumi Takino”, Hiroshi Matsuo”, Kazunari Matsumoto”, Shigeo Uotani”, Shoichi Akazawa”, Shunichi Yamashitab, Shigenobu Nagataki” “The First Department os Internal Medicine, Nagasaki University School of Medicine, l-7-J Sakamoto, ,Vagasaki 852, Japan bDepartment of Cell PhysiologJj, Atomic Disease Institute, Nagasaki Unicersit~~ School of Medicine, Nagasaki 852. Japan

Received 3 July 1995: revised 16 February 1996: accepted 20 February 1996

Abstract

The mechanism of TNF-a to regulate glucose metabolism remains unclear. To further delineate the TNF-c( signal transduction pathway mediating glucose metabolism, we utilized L6 rat myoblasts which contain the receptors for the insulin-like growth factor-I (IGF-I) and TNF-a, and the ability of both ligands to stimulate glucose uptake was compared. IGF-I (6.5 nM) maximally stimulated glucose uptake 7-fold after 24 h incubation, while 23 nM TNF-x maximally stimulated glucose uptake 3-fold only after 48 h incubation. IGF-I receptor a-subunit, insulin receptor substrate-l (IRS-l), and mitogen-activated protein (MAP) kinase were all phosphorylated in response to 6.5 nM IGF-I after 10 min incubation. In contrast, the treatment with 23 nM TNF-c( failed to phosphorylate either IGF-I receptor ,/?-subunit or IRS-1 but did phosphorylate MAP kinase as much as IGF-I did. Despite a similar extent to which TNF-r induced MAP kinase phosphorylation as IGF-I did, TNF-r stimulated glucose uptake less compared to IGF-I. The results indicate that MAP kinase phosphorylation is not sufficient for glucose uptake in L6 myoblasts. TNF-a-elicited signal transduction to glucose uptake may utilize a different pathway from that seen with IGF-I. Keywords:

IGF-I;

TNF-a;

MAP kinase; L6 myoblasts

1. Introduction TNF-a secretory spectrum

is an endotoxin-induced macrophage protein which evokes an extremely wide of biological actions at a target cell [1,2].

TNF-a acts as a cytotoxic factor for many tumor cells [3] as well as a metabolic regulator for lipid and glucose metabolism [4]. Several lines of evi* Corresponding author, Tel.: + 81 958 497264: fax: +81 958 497270.

dence point to the role of TNF-a physiological glucose metabolism stimulated

glucose

uptake

in mediating [S]. TNF-c(

by increasing

GLUT-1

mRNA gene expression in 3T3-Ll fibroblasts [6]. In contrast, an inhibitory action on glucose uptake by TNF-x has also been demonstrated by a decrease in levels of GLUT-4 mRNA in adipose tissue [7]. Thus, biological actions of TNF-CY on glucose metabolism appear to be complex. TNF-c( receptors widely expressed on several cell types, have been identified and cloned [8,9]. Binding of

0021-9150/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SOl68-8227(96)01221-l

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H. Yamasaki et al. / Diabetes Research and Clinical Practice 32 (1996) II

TNF-cr to its receptor leads to the activation of multiple signal transduction pathways [lo]. However, intracellular signal transduction to glucose metabolism induced by TNF-c( remains to be established. Insulin/IGF-I, which are the most critical ligands for glucose metabolism, induced a cascade of intracellular protein phosphorylation [ll- 131. MAP kinase, a serine/threonine kinase with 41-43 kDa MW, is thought to play an important role in the phosphorylation cascade initiated by insulin/IGF-I and other growth factors [14-171. To clarify TNF-a signal transduction pathway on glucose metabolism, we utilized L6 rat myoblast cells, where the IGF-I receptor rather than the insulin receptor have been shown to be dominantly expressed [l&19]. The ability of TNF-c( to regulate glucose uptake and to interfere with IGF-I-induced substrate phosphorylation of the intracellular second messenger proteins were therefore compared in L6 myoblasts. 2. Materials

and methods

2.1. Materials [‘251]IGF-I, [‘251]insulin, [1251]TNF-a and [3sS]methionine were purchased from Amersham (Arlington Heights, IL), [3H]2-deoxyglucose from ICN (Irvin, CA), Protein A-Sepharose from Pharmacia (Piscataway, NJ), monoclonal anti-phosphotyrosine antibody (PY-20) from Oncogene (Uniondale, NY), polyclonal anti-MAP kinase (Erk-CT) from UBI (Lake Placid, NY) and the other reagents from Sigma (St. Louis, MO). Recombinant human IGF-I was kindly provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). Recombinant TNF-cr was kindly provided by Dainippon Pharmaceutical Co. (Osaka, Japan). 2.2. Tissue culture L6 myoblasts were kindly provided by Dr. I. Morimoto (University of Occupational and Environmental Health, Fukuoka, Japan). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum in 5% CO, at 37°C to prevent L6 myoblasts from further differentiation [20].

18

2.3. Ligand binding study Binding of radiolabeled ligand was performed directly on the cell monolayers. A sample of 3 x lo5 cells were plated on 9-cm2 multiwell plates and allowed to grow for 16 h. Cells were washed in PBS (pH 7.3) 3 times and then incubated with indicated radioligand (50 000 counts/min, specific activity 2000 Ci/mmol for insulin or IGF-I, and 70 000 counts/min, specific activity 800 Ci/mmol for TNF-a) and increasing concentrations of ligand, in a final volume of 1 ml 50 mM HEPES buffer (pH 7.8) with 1% BSA, 120 mM NaCl, 1.2 mM MgSO,, and 5.5 mM glucose for 16 h at 4°C. Non-specific binding for [‘251]IGF-I, [“‘I]insulin and [12’I]TNF-a were defined as the binding observed in the presence of 100 nM unlabeled IGFI, 1 ,vM insulin, and 170 nM TNF-c(, respectively. At the end of the incubation period, cells were washed 3 times with PBS at 4°C and solubilized in 1 ml 1 N NaOH for determination of cell-associated radioactivity. To determine cell number, parallel control plates were tested as described above, and the cells were counted after the respective incubation. Receptor number and affinity were determined by Scatchard analysis [21]. 2.4. 2-deoxyglucose (2DOG) uptake A sample of 1 x lo5 cells were plated on 2-cm2 multiwell plates and allowed to grow for 16 h. The medium was then changed and replenished with 1 ml DMEM containing 0.3% BSA with or without the indicated concentrations of IGF-I, insulin, or TNF-X. After the indicated incubation period, cells were washed 3 times with PBS and incubated with 300 ~1 2DOG incorporation buffer (20 mM HEPES buffer containing 140 mM NaCl, 1 mM CaCl,, 5 mM KCl, 2.5 mM MgSO, and 1% BSA) with [3H]2DOG (1 ~Cci, specific activity 30 Ci/mmol) and 100 PM 2DOG for 20 min at 37°C. Non-specific incorporation was defined as the incorporation observed in the presence of 25 PM cytochalasin B. At the end of the incubation period cells were washed 3 times with PBS at 4°C and solubilized with 300 ~1 1 N NaOH to determine the incorporated radioactivity [22].

13

H. Yamasaki et al. ))/Diabetes Research and Clinical Practice 32 (1996) I l-18

2.5. Tyrosinr phosphorylution of the IGF-I receptor, IRS-I, und MAP kinasr Cells labeled for 16 h with [“Slmethionine (250 PCi, specific activity 1174 Ci/mmol) were incubated with 13 nM IGF-I and/or 23 nM TNF-x for IO min at 37°C. Cells were then lysed with lysis buffer (1% Triton X-100, 30 mM sodium pyrophosphate, 10 mM Tris (pH 7.6), 5 mM EDTA (pH X), 50 mM NaCl, 0.1% BSA, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride) and centrifuged for 15 min at 3000 x g at 4°C. Immunoprecipitation was performed using a monoclonal anti-phosphotyrosine antibody (PY-20) or polyclonal anti-MAP kinase (Erk-CT) at 4°C for 16 h. Protein-A-Sepharose was incubated with the antibodies-cell lysate complex for 3 h at room temperature. Immunoprecipitates were washed 3 times in lysis buffer, resuspended in sample buffer, boiled for 5 min and electrophoresed on 7.5% sodium dodecyl sulfate-polyacrylamide gel [23]. 2.6. Data analysis Data are shown as mean f S.E.M., and differences were assessed by non-paired t-testing.

incubation. The values are expressed as percent of control. Parallel experiments revealed no significant differences in cell number between the IGF-I treated group and the untreated control cells (data not shown). IGF-I stimulated 2DOG uptake in a dose-dependent manner up to 7-fold as compared to control levels. Fifty percent of maximal stimulation (ED,,) was achieved with 2.0 nM IGF-I. This concentration was relatively similar to that observed for the IC,, in the IGF-I displacement curve. In contrast, insulin failed to stimulate 2DOG uptake even at the high concentrations that produced the maximal IGF-I effect. Taken together, L6 myoblasts were characterized by insulin insensitivity, and glucose metabolism in L6 myoblasts was regulated dominantly by IGF-I. Fig. 4 shows [“‘I]TNF-x displacement by TNFSI. The IC,, was achieved with 1.5 nM TNF-x. Scatchard analysis for TNF-cc binding showed a Kd of 0.5 nM and total binding capacity of 5800 sites/cell. To evaluate the TNF-x effect on glucose uptake, L6 myoblasts treated with 5.7 nM TNF-cc for the indicated incubation period were subjected to 2DOG uptake (Fig. 5). Maximal stimulation of 2DOG uptake (2.6-fold) was, however, achieved only after 48 h treatment. Treatment for 24 h showed just 1.4-fold induction which was much

3. Results To characterize receptor binding in L6 myoblasts, IGF-I and insulin tracer binding were performed (Fig. 1). [1251]IGF-I specific binding was 14”/0 bound/O.6 x lo6 cells. In contrast, 0.7% [‘251]insulin specific binding is indicating negligible availability of insulin binding sites in L6 myoblasts. Fig. 2 shows [‘251]IGF-I displacement by IGF-I and insulin in L6 myoblasts. Fifty percent displacement of maximal binding (IC,,) was achieved with 0.65 nM IGF-I. The association constant (KJ and the number of IGF-I binding sites were obtained from Scatchard analysis shown in the inset. Kd and total binding capacity for IGF-I were found to be 0.5 nM and 83 x lo3 sites/cell, respectively. Fig. 3 shows the dose-response curve for the effects of IGF-I on 2DOG uptake after 24 h

IGF-I

Insulin

Fig. 1. [‘2iI]IGF-I and [““I]insulin tracer binding to L6 tnyoblasts. Cells were incubated with radiolabeled l&and at 4°C for 16 h with or without excess amount of unlabeled ligand. The values were represented as the mean of 2 independent experiments.

14

H. Yamasaki et al. 1 Diabetes Research and Clinical Practice 32 (1996) ll-

18

Kd:

0

zal bound

Binding

sites:

400

ooo

05IlM 83 x lo3

/cell

IGF-I @g/ml)

Fig. 2. Displacement of [“‘I]IGF-I binding by increasing concentrations of unlabeled IGF-I and insulin. IGF-I binding was performed as described in Materials and methods. The binding data were subjected to Scatchard analysis shown in the inset. Total binding sites and the affinity for IGF-I were found to be 83000 sites/cell and 0.5 nM, respectively.

lower than 6.5 nM IGF-I (7.0-fold). The dose response of TNF-cr effect was tested after 48 h of treatment (Fig. 6). Fifty percent of the maximum effect was achieved with 1.0 nM TNF-a which was found to be similar to the Kd observed in the Scatchard analysis obtained from TNF-a binding. Maximal stimulation achieved with 23 nM TNF-a was found to be no more than 3.0-fold induction. To clarify IGF-I and TNF-a signal transduction, cells metabolically labeled with [“5S]methionine were treated with 13 nM IGF-I and/or 23 nM TNF-c( for 10 min. Fig. 7A shows that, after IGF-I stimulation, the 97-kDa phosphorylated IGF-I receptor P-subunit was immunoprecipitated as well as a larger phosphorylated protein corresponding to the predicted size of IRS-l. As expected, the 135-kDa IGF-I receptor cc-subunit was also immunoprecip-

itated, indicating the integrity of the covalent bonding of the receptor subunits prior to resolution. In contrast, TNF-a treatment failed to phosphorylate either IGF-I receptor p-subunit or IRS-l. Fig. 7B shows that IGF-I treatment induced 43 kDa phosphorylated protein corresponding to the predicted size of MAP kinase. To confirm that 43 kDa protein represents MAP kinase in L6 myoblasts, anti-MAP kinase (ErkCT) was employed for a parallel immunoprecipitation study. The 43 kDa protein immunoprecipitated by Erk-CT was observed to be identical in size to the protein immunoprecipitated by PY-20 (Fig. 7B). In contrast to the inability of TNF-cr to phosphorylate IGF-I receptor p-subunit and IRS-l, TNF-a did induce the phosphorylation of MAP kinase to a similar extent to which IGF-I did.

H. Yarnasaki et al. / Diabetes Research and Clinical Practice 32 (1996) I l-18

To examine the interaction of TNF-a to IGF-I signalling, L6 cells were treated with both 13 nM IGF-I and 23 nM TNF-x followed by substrate phosphorylation analysis (Fig. 7A and B). Combination treatment with the two ligands (IGF-I and TNF-c() showed no additive effects on the phosphorylation of MAP kinase as compared to the level of that treated with IGF-I alone.

4. Discussion The present study demonstrates that TNF-r stimulated both glucose uptake and MAP kinase phosphorylation without inducing IRS-l phosphorylation in L6 myoblasts. MAP kinase is a cytoplasmic enzyme which phosphorylates serine/threonine residues of specific substrates, including myelin basic protein and microtubule-associated protein-2 [ 141. IGF-I treatment induced protooncogene ~21’“” activation and subsequent Raf-l-MAP kinase kinaseMAP kinase activation [24&26]. Therefore the MAP kinase phosphorylation cascade is a critical nmsoSOuaamzal-

insulin

IGF-I or insulin (nM) Fig. 3. Dose-response curve of IGF-I and insulin on 2DOG uptake to L6 myoblasts. Cells were treated with increasing concentrations of IGF-I for 24 h at 37°C. Then, 1 PCi [‘H]2DOG and 100 ,uM unlabeled 2DOG were added and cells were incubated for 20 min at 37°C to determine the incorporation. Results are mean & S.E.M., representative of 2 independent experiments. Each treated group of cells was compared to the untreated control cells. 6.5 nM IGF-I significantly stimulated 2DOG incorporation 7-fold as compared to control, while insulin failed to stimulate it even at 14 nM insulin.

15

pathway for IGF-I signalling [16]. As expected, in this study, MAP kinase was rapidly phosphorylated by IGF-I treatment of L6 cells. Similar to IGF-I, TNF-x exposure to L6 cells induces the phosphorylation of MAP kinase. These results in this study are supported by the recent demonstration that MAP kinase activation was induced by TNF-a treatment of NIH3T3 fibroblasts [27]. TNF-x receptor signal transduction leading to MAP kinase phosphorylation, however, remains unclear. In our study, TNF-ainduced MAP kinase phosphorylation is not accompanied by IRS-l phosphorylation. It is not surprising that TNF-c( receptor failed to induce IRS-l phosphorylation, since IRS-l has been shown to be a common signalling component for IGF-I and insulin receptor [28], and IRS-l phosphorylation requires the association of IRS-l molecule with the tyrosine at the position of 950 in the IGF-I receptor or 960 in the insulin receptor NPXY motif present in the submembrane region of these receptors, but not present in TNFSI receptor [9,23,29]. These results suggest that unlike IGF-I, TNF-c( may in part employ a distinct pathway leading to interacting with MAP kinase activation. Sphingomyelin and its metabolite, ceramide, have been recently shown to mediate TNF-x activation to downstream signalling including MAP kinase stimulation [30]. Treatment with sphingomyelinase that catalyze sphingomyelin to ceramide mimicked TNF-c( action, inducing tyrosine phosphorylation of MAP kinase [31]. These findings in our study and others suggest that MAP kinase cascade appears to play an important role in TNF-a signal transduction. Interestingly, the even small number of TNF-x receptors compared to that for IGF-I receptors appeared to be sufficient to phosphorylate MAP kinase as much as IGF-I. Both IGF-I and TNF-(x stimulated glucose uptake in a dose-response manner in L6 cells which express abundant amount of GLUTl, but less GLUT4 [20,32]. IGF-I induced an increase in the level of GLUT1 mRNA and protein content (Imamura H et al., unpublished results). Furthermore, for TNF-a, several lines of evidence indicated that this cytokine stimulated glucose uptake in several cell types [6,33]. As similar

16

H. Yamasaki et al. 1 Diabetes Research and Clinical Practice 32 (1996) 11-18

0.007 -

Binding sites: 5900 / cell Kd: 0.5 nM 0.0010.003 0.002 0.001

0

20

10

30

Bound TNF-a

TNF-a

40

60

60

(pg/ml)

(nM)

Fig. 4. Displacement of [“‘I]TNF-ol by increasing concentrations of unlabeled TNF-GI. TNF-G( binding was performed as described in Materials and methods. The binding data were subjected to Scatchard analysis shown in the inset. Total binding sites and the affinity for TNF-a were found to be 5300 sites/cell and 0.5 nM, respectively.

to the case of IGF-I, TNF-cr increased GLUT1 mRNA level in preadipocytes [6]. The mechanism would be explained by the stabilization of GLUT1 mRNA where the 3’-untranslated region of the mRNA is interacted with adenosine-uridine binding factor which is activated by TNF-c( [34]. However, how MAP kinase activation is participating in regulating TNF-a associated GLUT1 mRNA alteration remains to be determined. Overexpression of MAP kinase gene will allow us to know the importance of MAP kinase activity for GLUT1 gene expression. In this study, the phosphorylation level of MAP kinase was similarly stimulated by both IGF-I and TNF-cr treatment, nevertheless, TNF-cr-induced glucose uptake seen in the dose-response curve showed smaller responsiveness as compared to IGF-I. The data suggest that MAP kinase phosphorylation is not a major signalling component for TNF-a-mediated glucose uptake. Alternatively, in addition to stimulatory effect to glucose uptake, unknown inhibitory signalling

/,

I,

I,

I.

20

40

60

60

time

I

100

(hrs)

Fig. 5. Time course of TNF-5c on 2DOG uptake in L6 myoblasts. Cells were treated with 5.7 nM TNF-a for the indicated times. 2DOG incorporation was performed as in Fig. 3. Results are the mean * S.E.M. representative of 2 independent experiments. Each treated group of cells was compared to the untreated control group. 5.7 nM TNF-or stimulated 2DOG uptake in a time-dependent manner with maximal stimulation after 48 h.

17

H. Yamasaki et al. / Diabetes Research and Clinical Practice 32 (1996) 11-18 ’ a

IGF-I(nM) TNF-cr

(nU)

-

,3

-

,3

-

-

23

23

200 c-IRS-1

97 -

TNF (nM)

Fig. 6. Dose-response of TNF-a on 2DOG incorporation in L6 myoblasts. Cells were treated with increasing concentration of TNF-n for 48 h. Then 2DOG incorporation was performed as in Fig. 3. TNF-a maximally stimulated 2DOG uptake 3-fold as compared to the untreated control group of cells. Results are mean k S.E.M. of 3 independent experiments.

elicited by TNF-a may be cross-talking downstream to MAP kinase cascade. In conclusion, the present study demonstrates that TNF-cr and IGF-I similarly phosphorylate MAP kinase in L6 myoblasts with a different magnitude for glucose uptake. Acknowledgements This work was supported by the Japanese Diabetes Foundation. The authors thank Miss. Mami Ikenaga for her excellent technical assistance.

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.I-

c-

-

IGF-I A

MAP llinase

Fig. 7. Phosphorylation of cellular protein in response to IGF-I and TNF-a. Cells metabolically labeled with [?S]methionine were treated with 13 nM IGF-I or 23 nM TNF-a at 37°C for 10 min. Phosphotyrosine-containing protein was immunoprecipitated with anti-phosphotyrosine (PY-20) in (A) and in the four lanes from the left in (B). In order to determine non-specific immunoprecipitation, an immunoprecipitation with or without adding anti-MAP kinase antibody was shown in the two lanes from the right in (B).

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