THE JOURNAL OF BIOLOGICAL CHEMWCRY Vol. 265, No. 2, Issue of January 15, PP. 1124-112’3,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

The Insulin-like Effects of Selenate in Rat Adipocytes”

(Received for publication, April 28, 1989)

Osamu Ezaki From the Division of Nutrition and Diseases, National Institute of Nutrition, Toyama, Shinjuku-ku, Tokyo 162, Japan

Selenate was found to have several insulin-like ef- fects in rat adipocytes: stimulation of glucose transport activity by translocation of two types of glucose trans- porters from intracellular sites to the plasma mem- brane, stimulation of CAMP phosphodiesterase activ- ity, and stimulation of ribosomal S6 protein phos- phorylation. Furthermore, in intact cells addition of 1 IIIM selenate stimulated tyrosyl phosphorylation of 210-, 1’70-, 120-, 95-, 70-, and 60-kDa proteins but failed to stimulate insulin receptor kinase activity, suggesting that selenate stimulated other tyrosine ki- nase. In the presence of insulin, selenate enhances in- sulin receptor kinase activity and phosphorylations of insulin-stimulated tyrosyl phosphoproteins. These re- sults may provide clues for the elucidation of the role of selenium in animals and the mechanism of insulin action.

The essential role of selenium has been clarified by animal experiments. Chicks or rats fed a low selenium diet showed poor growth and high mortality, and this state could not be reversed by adding vitamin E (1, 2). In humans, an endemic cardiomyopathy known as Keshan disease appeared in a certain area of China and is assumed to be caused by selenium deficiency (3). During intravenous nutrition, some patients showed selenium deficiency and developed skeletal myopathy (4). On the other hand, in developing serum-free medium for cell cultures, selenium as selenite was used as one of the nutrient factors for promoting cell growth (5-7). As the well established biochemical role of selenium is its function in glutathione peroxidase (8), the selenium effect in cell culture was considered to be due to protection of cells from toxic effects of peroxides (9). It was first observed in this study that selenate is a potent insulin-like agent.

EXPERIMENTAL PROCEDURES

Muterials--32Pi was purchased from Amersham (Buckinghamshire, United Kingdom), and sodium selenate and sodium selenite from Wako (Tokyo, Japan). The sources of the other materials have been described previously (10, 11).

Polvclonal anti-human ervthrocvtes glucose transporter (GTl)’ antibddies whose characteristics hake be& described previously (11, 12) were kindlv provided bv Dr. M. Kasahara (Teikvo University, Japan). Polycl&& antibodies to the C-terminal region ITyr-Leu-Gl;- Pro-Asp-Glu-Asn-Asp) of glucose transporter (GT2) in insulin-sen- sitive tissue (13) were raised by injection to rabbits as a conjugate

* This work was supported by a lump sum budget for the Environ- mental Institute of the Environment Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

1 The abbreviations used are: GTl, erythrocyte glucose transporter; GT2, glucose transporter mainly expressed in insulin-sensitive tissue; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate.

form with keyhole limpet hemocyanin using glutaraldehyde. Poly- clonal antibodies to phosphotyrosine were raised as described by Pang et al. (14) and were purified by affinity chromatography coupled with phosphotyrosine and by protein A affinity chromatography.

Preparation of Isolated Rat Adipocytes and Subcellular Fractions- Adipocytes were isolated from epididymal adipose tissues of Sprague- Dawley rats (150-200 g each) by the method reported heretofore (15, 16). Following isolation, the cells were washed four times with a solution containing 119 mM NaCl, 4.7 mM KCl, 2.6 mM Ca& 1.2 mM KH,POI, 1.2 mM MgSOa, 32.3 mM HEPES, pH 7.4, 20 mg/ml bovine serum albumin, and 2 mM D-ghCOSe (buffer A), suspended in 10 ml of buffer A, and incubated for 30 min at 37 “C. Subcellular fractionation of rat adipocytes was made using the method described by Kono et al. (16). Each aliquot of cells was first incubated with buffer alone or with 1 nM insulin or with 1 mM sodium selenate in 10 ml of buffer A for 10 min at 37 “C in a polycarbonate Erlenmeyer flask. Homogenization was performed in a buffer containing 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.5 (buffer B). Each aliquot of cells was washed twice with 10 ml of buffer B, suspended in 9 ml of buffer B at 13 “C, and homogenized. The homogenate thus obtained was centrifuged at 2,200 x g for 2 min. The aqueous solution under the fat layer was withdrawn and centrifuged at 100,000 X g for 60 min. The pellet was designated as crude membrane fraction. This was suspended in buffer B and applied to the top of a 12-ml linear sucrose aadient (15-32.5% (w/w)) containing buffer C (1 mM EDTA , ,, and 10 mM Tris-HCl, pH 7.5). After noneqiilibrium centrifugation for 40 min at 150,000 X g, the fractions containing 24-31% sucrose were recovered as plasma membrane fraction and those containing 14-19% sucrose as low density microsome fraction. Vesicles in each fraction were washed with buffer C at 175,000 x g for 90 min, suspended in 100 ~1 of buffer C, and stored at -70°C.

In experiments where inhibition of kinase and phosphatase activity was intended (Figs. 4-6) buffer supplemented with 5 mM EDTA, 25 mM sodium fluoride, 20 mM sodium pyrophosphate, 2 mM sodium vanadate, 2.5 mM phenylmethylsulfonyl fluoride, and 400 trypsin inhibitor units/ml was used.

Electrophoresis-The pellet of the fractions was solubilized by boiling for 3 min in a solution containing 2.5% sodium dodecyl sulfate, 75 mM dithiothreitol, 12.5% glycerol, 0.025% bromphenol blue, and 12.5 mM sodium phosphate, pH 7.0. Sodium dodecyl sulfate-polyacryl- amide gel electrophoresis was performed according to Laemmli (17) with 4% stacking gel and 7% resolving gel for detection of phospho- rylated proteins or with 9.5% resolving gel for glucose transporter. Immunoblotting of electrophoresis gels was performed as described previously (10, 11). To quantify the glucose transporters and phos- phorylated proteins, pieces of sheet containing aimed proteins were cut out, and radioactivity was counted in a y-counter. The background was estimated by counting a region with no labeled band and then subtracted. Radioactivity determined in this manner was proportional to the amount of the transporter or the phosphorylated proteins applied over the range used in this study.

Measurement of Ribosomal S6 Protein Phosphorylution-Aliquots of adipocytes were incubated with 32Pi at 100 &i/ml for 120 min at 37 “C in phosphate-free buffer A. Then, cells (5 X lo6 cells) were incubated with the agents. The cells were washed once with 20 ml of buffer B containing kinase and phosphatase inhibitors (see above) and homogenized with a glass homogenizer. The homogenate was centrifuged at 3,000 x g for 2 min. The aqueous solution below the fat laver was withdrawn and centrifuged at 17,000 x g for 30 min. The sipernatant was then centrifuged-& 100,000 x g for 60 min, and one-third of the resultant pellet was solubilized by boiling for 3 min in Laemmli’s sample buffer (17). Sodium dodecyl sulfate-polyacryl- amide gel electrophoresis was performed with a 5-20% gradient gel.

1124

by guest on October 16, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Insulin-like Effects of Selenate

The phosphorylated bands were visualized by autoradiography. Measurement of Kinase Activity of Insulin Receptors Activated in

Intact Cells-The method of measuring the kinase activity of insulin receptors activated in intact cells as described by Klein et al. (18, 19) was used with modifications (11). Briefly, the solubilized crude mem- brane fractions in the presence of kinase and phosphatase inhibitors (see above) were applied to wheat germ-agarose. Phosphorylation assays using wheat germ-agarose eluate having similar insulin binding activities were initiated by incubation with 8 mM MnClZ, 12 mM MgCl*, 1 mg/ml histone 2B, and 50 1M [32P]ATP (10 &i). After 5 min at 20 “C, reactions were terminated by adding 10 mM ATP (final concentration) and heating to 100 “C for 5 min in Laemmli’s sampling buffer. Phosphorylated proteins were analyzed by 12% sodium dode- cyl sulfate-polyacrylamide gel electrophoresis followed by autoradi- ography. Areas on the gels containing phosphorylated proteins were cut, and radioactivity was determined in a @counter.

Measurement of Phosphotyrosine Phosphatase Activity in Cell-free System-The isolated adipo&tes (2.5 x i0” cells) were -incubated in buffer A with 10 nM insulin for 10 min at 37 “C. Then, the cells were quickly washed twice with 10 mM buffer D (150 mM’ NaCl, 25 mM HEPES, pH 7.4), solubilized by 1% Triton X-100, and then divided into several aliquots. One aliquot was immediately treated with Laem- mli’s sampling buffer and boiled as control of phosphorylated sample. Another aliquot was incubated with buffer D alone or with each agent as indicated at 37 “C. The reaction was terminated by adding Laem- mli’s sampling buffer and boiling. Tyrosyl phosphorylation of these lvsates was analvzed bv 7% uolvacrvlamide-SDS gel electrophoresis and immunoblo&ing u-sing anti-phosphotyrosine antibody followed by autoradiography.

Other Assays-The glucose transport activity of adipocytes was assessed by measuring the transport of 1 mM 3-O-[“Clmethyl-D- glucose for 3 s using an oil flotation method (11, 20). Adipocyte cell number was measured using a cell-counting plate (Fuchs Rosenthal, Nitirin, Tokyo, 0.2 mm in depth). Protein was assayed by Bradford’s method (21), sucrose concentration by refractometry, phosphodies- terase activity as described by Kono et al. (22), 5’-nucleotidase by the method of Avruch and Wallach (23), and UDP-galactose N-acetylglu- cosamine galactosyltransferase by the method of Fleisher (24).

RESULTS

As shown in Fig. lA, selenate (SeOi-) stimulated glucose transport activity in a dose-dependent manner. The stimula- tive effect was observed at a concentration of 1 PM, and its half-maximal and maximal transport activity was with 100 pM and 1 mM, respectively. Its maximal transport activity was almost equipotent to that with 1 nM insulin. Addition of 1 mM selenite (SeO?) also stimulated glucose transport activ- ity, and its effect was about 50% of the maximal insulin- stimulated transport activity (data not shown). Stimulation of transport activity by 1 mM selenate was observed 2 min after addition of selenate, and the activity reached a steady state within 10 min (Fig. 1B). Since the insulin-stimulated glucose transport activity is largely due to the translocation mechanism of glucose transporters (25, 26) the effects of selenate on the subcellular distribution of transporters were studied by immunoblotting using antibody to human eryth- rocyte glucose transporter (ll), termed GTl, or to C-terminal region of another transporter, termed GTZ, expressed in adi- poeytes (13). The addition of 1 mM selenate as well as 1 nM insulin translocated 71 f 4 and 69 f 3% (mean + S.E., n = 3), respectively, of GTl and 34 + 2 and 32 k 3% of GT2 which had been located in the intracellular site to the plasma membrane (Fig. 2). These data suggest that each of the insulin-sensitive transporters is translocated by selenate. As the relative enrichment of marker enzymes, 5’-nucleotidase activity for the plasma membrane, and galactosyltransferase activity for Golgi apparatus from 1 mM selenate-treated cells were not associated with significant changes, the change of distribution patterns by selenate may not be due to different sedimentation characteristics of the vesicles (data not shown).

Examination was next made whether selenate also stimu- lates another effector pathway of insulin signaling. For this

,

O %+ 10-6

I I I 1

1 o-5 10-a 10-S 10-Z

CONCENTRATION, M

I

”

0 10 20 30 INCUBATION TIME, min

FIG. 1. Selenate-stimulated glucose transport activity. In panel A, isolated rat adipocytes in buffer A were exposed to the indicated concentrations of selenate for 30 min at 37 “C (0). In panel B, cells were incubated with 1 mM selenate for the indicated periods at 37 ‘C (0). At the end of the incubations, 3-O-[“Clmethyl-D-glucose uptake was measured. As control, cells were incubated with 1 nM insulin for 10 min (0). The data show mean values -t S.E. (n = 3).

purpose, insulin-stimulated CAMP phosphodiesterase activity was examined. Selenate also stimulated phosphodiesterase activity, but its dose dependence curve was biphasic. Its effect was maximum with 1 mM but declined with 10 mM (Fig. 3). The dose dependence of insulin-stimulated activity was also biphasic (22), suggesting a similar activation mechanism might be involved in both agents.

Insulin is also known to stimulate phosphorylation of ri- bosomal S6 protein in rat adipocytes, and its role is considered to be associated with initiation of protein synthesis (27). In our previous study, insulin stimulated phosphorylation of 35- kDa protein in the crude microsome fraction, and this protein was identified as ribosomal S6 protein by reactivity to anti- S6 antibody (28). As shown in Fig. 4, addition of selenate stimulated S6 phosphorylation as effectively as 1 nM insulin, suggesting its role in protein synthesis stimulation. Also, addition of either insulin or selenate stimulated phosphoryla- tions of 200-, 117-, 93-, and 66-kDa proteins.

As reported in previous studies (11, 29), when adipocytes were incubated with insulin, tyrosyl phosphorylation of the 95-kDa subunit of insulin receptor was increased, and two endogenous proteins of 170 and 60 kDa were also phospho- rylated at tyrosine residue(s) using an immunoblot technique with an anti-phosphotyrosine antibody. As shown in Fig. 5, in cell lysate addition of 1 mM selenate stimulated tyrosyl phosphorylation of 210-, 170-, 120-, 95-, and 60-kDa proteins. The insulin-induced phosphorylation of 170-, 95-, and 60-kDa

by guest on October 16, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Insulin-like Effects of Selenate 1126

FIG. 2. A representative autoradiogram of two types of glu- cose transporter (GTl and GT2) distributions in plasma mem- brane (PM) and low density microsome (L&Z) from cells treated with buffer alone, insulin, or selenate. Aliquots of pooled adipocytes in buffer A were incubated with buffer alone, 1 nM insulin for 10 min. or 1 mM sodium selenate for 10 min at 37 “C. Then, they were homogenized and fractionated as described under “Experimental Procedures.” The glucose transporters in each fraction were detected by immunoblotting using antibody to GTl (panel A) or to the C-terminal peptide of GT2 (panel B). The cell number used in each line of plasma membrane or low density microsome fraction was 1.0 X 10” cells.

proteins was further enhanced by mixed incubation of selen- ate and insulin. As most of the insulin receptor and 30% of 60-kDa protein were located in membrane fractions (ll), tyrosyl phosphorylation of crude membrane fractions was also examined. Addition of 1 mM selenate also stimulated mem- brane-bound tyrosyl phosphorylation of 210-, 170-, 120-, 95-, 70-, and BO-kDa proteins. The increased phosphorylation of 170-, 95-, 70-, and 60-kDa proteins was also further enhanced by mixed incubation of selenate and insulin. Insulin binding to cells was unaffected significantly by adding selenate (data not shown).

As the foregoing data suggest that 1 mM selenate stimulates insulin receptor kinase or other phosphotyrosine kinase or inhibits phosphotyrosine phosphatase, the kinase activity of insulin receptor was next examined. When the insulin recep-

5: 0 10-e 10-s lO-4 10-Z 10-Z

CONCENTRATION, M

FIG. 3. Selenate-stimulated CAMP phosphodiesterase activ- ity. Aliquots of cells in buffer A were exposed to the indicated concentrations of selenate (0). As control, cells were incubated with 1 nM insulin (0) for 10 min. At the end of incubation, they were homogenized. Phosphodiesterase activity was measured as described previously (22). The data show mean values t S.E. (n = 3).

! Mr

-200 K

S6-

-1

; , - 21 K

FIG. 4. Selenate-stimulated ribosomal S6 protein phos- phorylation. Aliquots of cells were incubated with ‘*P8 at 100 &i/ ml for 120 min at 37 “C in phosphate-free buffer A. Then, cells (5 X lo6 cells) were incubated with buffer alone or with 1 nM insulin for 10 min or with 1 mM selenate for 10 min. The cells were homogenized and fractionated as described under “Experimental Procedures.” The phosphorylated bands were visualized by autoradiography.

tors are isolated in the presence of phosphatase and kinase inhibitors, the activation state of the kinase, resulting from exposure of the intact cells to insulin, can be preserved (18, 19). By this procedure, the kinase activity of the receptor was measured in a cell-free system in an insulin-free buffer using histone 2B as substrate. To determine receptor kinase activ-

by guest on October 16, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Insulin-like Effects of Selenate 1127

CELL LYSATE MEMBRANE -A I- ( - ) SELENATE ( - ) SELENATE

INSULIN - ‘-+‘ ‘- ‘- Mr

170K-

95K-

60K-

,.‘x -2()@(

-116K

- 93K

FIG. 5. Effects of selenate in intact cells on tyrosyl phos- phorylation in whole cell lysate and membrane fraction. Ali- quots of cells were incubated with buffer alone or with 1 mM selenate for 30 min, mixed with or without 1 nM insulin, and incubated for an additional 10 min at 37 “C. In experiments of cell lysate, the cells were quickly washed with buffer B containing phosphatase and kinase inhibitors, solubilized by Laemmli’s sampling buffer, and boiled. In experiments of membrane fraction, the cells were homogenized in buffer B containing phosphatase and kinase inhibitors, and crude membrane fraction was obtained as described under “Experimental Procedures.” Tyrosyl phosphoprotein of these lysate and crude mem- brane proteins were analyzed by 7% polyacrylamide-SDS gel electro- phoresis and immunoblotting using anti-phosphotyrosine antibody followed by autoradiography. A typical autoradiogram of several experiments is shown. The cell number used in each line of Cell lysate and Membrane was 2.5 x lo” and 1.0 X lo6 cells, respectively.

ity, the receptor preparations were adjusted to contain similar binding activities. Fig. 6 shows a representative autoradi- ogram where adipocytes were incubated with 1 mM selenate in the absence and presence of 1 nM insulin. However, in this experiment, as the insulin-induced autophosphorylation of the 95-kDa subunit of insulin receptor was not apparent though insulin-induced phosphorylation of histone 2B was markedly observed, only the kinase activity to histone 2B was examined. Addition of 1 mM selenate did not significantly stimulate insulin receptor kinase, but enhanced insulin stim- ulated its receptor kinase about P-fold.

Examination was next made to determine whether selenate inhibits phosphotyrosine phosphatase activity in a cell-free system. As shown in Fig. 7, when the cell lysate-obtained insulin-treated cells were incubated with buffer D (see “Ex- perimental Procedures”) at 37 “C for 10 min, the phosphoty- rosy1 proteins were dephosphorylated (see second line of Fig. 7). Addition of 1 mM selenate failed to prevent dephosphoryl- ation of the phosphotyrosyl proteins. Addition of 1 mM van-

adate or lib group metal ions (1 mM Zn’+, 1 mM Cd”, 0.1 mM Hg’+) but not 1 mM Mn”, 1 mM Mg2+ could prevent dephosphorylation of these proteins. These data suggested that in this cell-free system, 1 mM selenate does not stimulate tyrosine kinase activity and does not act as an inhibitor of phosphotyrosine phosphatase.

DISCUSSION

In this study, selenate was found to have several physiolog- ical insulin-like effects in rat adipocytes: stimulation of glu- cose transport activity, CAMP phosphodiesterase activity, and ribosomal S6 protein phosphorylation. Selenate similar to

& & (4 9i --Yc-\

INSULIN - + - +

95K-

FIG. 6. Effects of selenate in intact cells with or without insulin on insulin receptor kinase activity. Aliquots of cells in buffer A were incubated with buffer alone or with 1 mM selenate for 30 min, mixed with or without 1 nM insulin, and incubated for additional 10 min. Incubations were terminated by homogenization in buffer B containing phosphatase and kinase inhibitors, and the crude membrane fraction obtained by centrifugation was solubilized by 1% Triton X-100. Insulin receptors were partially purified, and an equal number of receptors was used for receptor kinase activity to histone 2B as described under “Experimental Procedures.” A typical autoradiogram from several experiments is shown.

insulin translocated two different types of glucose transporter from the intracellular site to the plasma membrane. About 70% of GTl and about 30% of GT2, which had been located in the intracellular site, were translocated to the plasma membrane by either insulin or selenate, suggesting that a large portion of intracellular GTl and a small portion of GT2 are located in the translocatable glucose transporter-contain- ing vesicles.

Addition of 1 mM selenate stimulated the tyrosyl phos- phorylation of several proteins including 170-, 95-, and 60- kDa proteins, which were also phosphorylated by insulin. However, selenium failed to stimulate insulin receptor kinase and to inhibit phosphotyrosine phosphatase, suggesting that selenium may stimulate other tyrosine kinase. Thus, selenate stimulated tyrosyl phosphorylation of the 95-kDa subunit of insulin receptor but failed to stimulate its kinase activity, suggesting that selenate stimulates phosphorylation at the site(s) of insulin receptor, which does not induce stimulation of receptor kinase activity. In this regard, the discrepancy between autophosphorylation of the 95-kDa subunit of insulin receptor and its kinase activity has been reported (30). HOW- ever, as the study for inhibition of phosphotyrosine phospha- tase was made in the cell-free system, it is conceivable that selenate inhibited phosphotyrosine phosphatase in intact cells. The insulin-like effects of vanadate (31, 32) and IIb group metal ions (ll), which act as inhibitors of phosphoty-

by guest on October 16, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Insulin-like Effects of Selenate

Mr

- 200K

- 116K

95K - I . ..*

60K - 66K

FIG. 7. Effects of agents in cell lysate on inhibition of phos- photyrosine phosphatase activity. The cell lysates obtained by solubilized 1% Triton X-100 from insulin-treated cells were divided into 9 aliquots. One aliquot was immediately treated with Laemmli’s sampling buffer and boiled as control of phosphorylated sample (first left line). Another aliquot was incubated with buffer D alone or 1 mM selenate, 1 mM vanadate, 1 mM &SO,, 1 mM CdCb 0.1 mM HgC&, 1 mM MnCI?, or 1 mM MgCl, for 10 min at 37 “C. The reaction was terminated by the addition of Laemmli’s sampling buffer and boiling. Tyrosyl phosphorylation of these lysates was analyzed by 7% poly- acrylamide-SDS gel electrophoresis and immunoblotting using anti- phosphotyrosine antibody followed by autoradiography. A typical autoradiogram of several experiments is shown. The cell number used in each line was 2 x lo” cells.

rosine phosphatase in the cell-free system, were not correlated with insulin receptor kinase activity. Selenate similar to these metal ions may act as an insulin-like agent by a post-insulin receptor kinase mechanism. It is unclear, at present, whether these tyrosyl-phosphorylatedproteins are mediators of several insulin- or selenate-induced insulin-like effects. The enhance- ment of insulin-stimulated tyrosyl phosphorylation of 170-, 95, and 60-kDa proteins by the addition of selenate may be due to the enhancement of insulin-stimulated receptor kinase.

Selenate and selenite are also known as catalysts of the oxidation of SH groups, and oxidation of 4 mol of SH/mol of selenite is required (33). It is conceivable that the oxidation of SH groups of the receptor causes aggregation of the receptor or conformational changes of the receptor, and this leads to mediate insulin-like effects. Recently, Debant et al. (34) have reported that receptor cross-linking restores the insulin met- abolic effect altered by mutation of the kinase domain of the receptor, suggesting the importance of receptor aggregation in signal transduction.

These insulin-like effects of selenate may well explain its growth-promoting effects observed in various organisms. Sou- ness et al. (35) reported that adipocytes from a selenium- deficient rat showed a decrease of maximal insulin-stimulated glucose oxidation activity. Adipocytes from insulin-depleted rats induced by injection of streptozotocin also showed a similar decrease in insulin-stimulated glucose transport activ- ity (36). The actual concentration of selenium in rat blood is about 1 FM (37), and selenium might express in uiuo a rela- tively weak insulin-like effect. It is, however, conceivable that the biological effects of selenium are different among its compound forms. Further studies are necessary to confirm in uiuo the insulin-like effects of selenium.

1. 2.

3.

4.

5.

6.

7.

8.

9.

10.

11. 12.

13.

14.

15. 16.

17. 18.

19.

20.

21. 22.

23.

24. 25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

Acknou~ledgments--I am grateful to Dr. Michihiro Kasahara and Dr. Hiroshige Itakura for their continued interest in this study and for their useful suggestions.

REFERENCES

Thompson, J. N., and Scott, M. L. (1969) J. Nutr. 97, 335-342 McCoy, K. E. M., and Weswig, P. H. (1969) J. Nutr. 98, 383-

389 Chen. X., Yang, G., Chen, J., Chen, X., Wen, Z., and Ge, X.

(1980) Biol. %ace Element Res. 2,91-107 Van Rii. A. M.. Thomson. C. D.. McKenzie, J. M., and Robinson,

M. Fy’(1979)‘Am. J. C&z. Nutr. 32, 20762085 Eccleston, P. A., and Silberberg, D. H. (1984) Deo. Brain Res.

16, l-9 Nakabayashi, H., Takeda, K.,Yamane, T., Miyazaki, M., Miyano,

K., and Sato, J. (1984) Gann 75, 151-158 Nakanishi. Y.. Cuttitta. F.. Kasprzvk. P. G., Avis, I., Steinberg,

S. M., Gazd&, A. F., &d’Mulshine,‘J. L. (i988) Exp. Cell Bioj. 56, 74-85

Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G., and Hoekstra, W. G. (1973) Science 179,588- 590

McKeehan, W. L., Hamilton, W. G., and Ham, R. G. (1976) Proc. Natl. Acad. Sci. U. S. A. ‘73, 2023-2027

Ezaki, O., Kasuga, M., Akanuma, Y., Takata, K., Hirano, H., Fujita-Yamaguchi, Y., and Kasahara, M. (1986) J. Biol. Chem. 261.3295-3305

Ezaki, 0. (1989) J. Biol. Chem. 264, 16118-16122 Ezaki. 0.. Bono, N.. Itakura. H.. and Kasahara, M. (1989)

&o&em. Biophys. ies. Commun. 159, 1368-1374 James, D. E., Strube, M., and Mueckler, M. (1989) Nature 338,

83-87 Pang, D. T., Sharma, B. R., and Shafer, J. A. (1985) Arch.

B&hem. Biophys. 242, 176-186 Rodbell. M. (1964) J. Biol. Chem. 239, 375-380 Kono, $., Robinson, F. W., Blevins, T. L., and Ezaki, 0. (1982)

J. Biol. Chem. 257, 10942-10947 Laemmli, U. K. (1970) Nature 227,680-685 Klein, H. H., Freidenberg, G. R., Kladde, M., and Olefsky, J. M.

(1986) J. Biol. Chem. 261,4691-4697 Klein, H. H., Freidenberg, G. R., Matthaei, S., and Olefsky, J. M.

(1987) J. Biol. Chem. 262, 10557-10564 Gliemann, J., Bsterlind, K., Vinten, J., and Gammeltoft, S. (1972)

Biochim. Biophys. Acta 286, l-9 Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 Kono, T., Robinson, F. W., and Sarver, J. A. (1975) J. Biol. Chem.

250,7826-7835 Avruch, J., and Wallach, D. F. H. (1971) Biochim. Biophys. Acta

233,334-347 Fleisher, B. (1974) Methods Enzymol. 31, 180-191 Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255,

4758-4762 Suzuki, K., and Kono, T. (1980) Proc. Natl. Acad. Sci. U. S. A.

77, 2542-2545 Hansson, A., and Ingelman-Sundberg, M. (1985) Eur. J. Biochem.

151,97-100 Ezaki, O., Itakura, H., Kasuga, M., Anraku, Y., and Kasahara,

M. (1986) in Contemporary Themes in Biochemistry, ICSU Short Report (Kon, 0: L., id) Vol. 6, pp. 440-441, Cambridge University Press, Cambridge

Momomura, K., Tobe, K., Seyama, Y., Takaku, F., and Kasuga, M. (1988) Biochem. Biophys. Res. Commun. 155, 1181-1186

Morrison. B. D.. and Pessin. J. E. (1987) J. Eiol. Chem. 262, 2861-2868

Mooney, R. A., Bordwell, K. L., Luhowskyj, S., and Casnellie, J. E. (1988) Endocrinology 124, 422-429

Strout, H. V., Vicaro, P. P., Saperstein, R., and Slater, E. E. (1989) Endocrinolopy 124. 1918-1924

Tsen, C. C., and Tappel, A. L. (1958) J. Biol. Chem. 233, 1230- 1232

Debant, A., Ponzio, G., Clauser, E., Contreres, J. O., and Rossi, B. (1989) Biochemistry 28, 14-17

Souness, J. E., Stouffer, J. E., and Chagoya De Sanchez, V. (1983) Biochem. J. 214,471-477

Karnieli, E., Hissin, P. J., Simpsom, I. A., Salans, L. B., and Cushman. S. W. (1981) J. Clin. Invest. 68.811-814

Abdel Rahim, A. G., Arther, J. R., and Mills, C. F. (1986) J. Nutr. 116,403-411

by guest on October 16, 2020

http://ww

w.jbc.org/

Dow

nloaded from

O EzakiThe insulin-like effects of selenate in rat adipocytes.

1990, 265:1124-1128.J. Biol. Chem. 

  http://www.jbc.org/content/265/2/1124Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/2/1124.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on October 16, 2020

http://ww

w.jbc.org/

Dow

nloaded from