Jean Simon$#, Robert W. Rosebroughll, John P. McMurtryll, Norman C. Steelell, Jesse Roth$, ...... McMurtry, J. P., Rosebrough, R. W., and Steele, N. C. (1983).
Vol. 261, No. 36, Issue of December 25,pp. 17081-17088,1986 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Fasting andRefeeding Alter the Insulin Receptor Tyrosine Kinase in Chicken Liver but Fail to Affect Brain Insulin Receptors* (Received for publication, July 17, 1986)
Jean Simon$#,Robert W. Rosebroughll, John P. McMurtryll, Norman C. Steelell, Jesse Roth$, Martin AdamoS, and DerekLeRoithSB From the $Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, (Nonruminant Animal NutritionLaboratory, United States Department of Agriculture, Beltsville, Maryland 20705, and the §Stationde Recherches Avicoles, Zmtitut National de la Recherche Agronomique, Nouzilly, 37380 Monnaie, France
Insulin receptors from chicken liver and brain were Insulin receptors are oligomeric glycoproteins composed of studied following alterations in the nutritional state. CY and p subunits (1, 2). The a subunit which binds insulin is Chickens were either fasted for 48 h, fasted for 48 h exclusively extracellular whereas the,B subunit has an extra24 h, or fed a regular diet ad libitum. cellular domain linked by a membrane-spanning region to a and then refed for lZ6I-Porcine insulin binding was significantly elevated larger intracellular domain that contains a tyrosine-specific in liver membranes from the fasted animals andlow- protein kinase. Normally there is coupling of the a and p ered inrefed chickenswhen compared to preparations subunits whereby the binding of insulin stimulates phosphofrom ad libitum fed chickens. These changes in ”‘1- rylation of the /3 subunit as well as stimulation of phosphoinsulin binding were inversely relatedto the levels of rylation of tyrosine-specific substrates (3-8). Although no plasma insulin and since receptor affinities for insulinfunction has as yet been identified for insulin-induced tyrowere similar in each group, they probably represent alterations in receptornumber. Apparent M. of a sub- sine phosphorylation, a number of examples have been deof couplingbetween binding to the a units of the insulin receptors wasunaffected by alter- scribedwherelack subunit and / 3 subunit functionisassociated with insulin ations in the nutritional states. Thepresence of ATPase-like activities thatco-eluted with liver insulin re- resistance. Theseinclude studies demonstrating that tyrosine ceptors from wheat germ agglutinin lectin columns but kinase is markedly reduced in cells from some patients with well as in muscle from not from pea lectin columns necessitated the use of both severe insulin resistance (9, 10) as pea and wheat germ agglutinin for liver insulin recep- diabetic rats (11)and obese mice (12). Chickens normally show some degree of insulin resistance tor purification. The insulin receptors purified from both lectin columns were recognized by anti-insulin as demonstrated by elevated blood glucose and fewer insulin receptors on target tissues despitepresence the of a circulating receptor antiserum and had similar affinities for insulin which were unaltered by the nutritional state. insulinwith a specific biological activitythat is 2-3-fold Insulin-stimulatable autophosphorylationof the B sub- higher than duck insulin and most mammalian insulins(13unit of the insulin receptor was lower in livers from 17). However, in a previous study of chicken insulin receptors fasted chickens and intermediate in refed chickens. in liver and brain, we found normal coupling between insulin Furthermore, basal and insulin-induced phosphoryla- binding to thea subunit and,B subunit phosphorylation (18). tion of the artificial substrate poly(Glu,Tyr)4:1 was Thus, in the present studywe investigated whether the state significantly less in the fasting state and intermediate of nutrition could affect insulin receptor tyrosine kinase acin the refed state compared to the ad libitum fed state. tivity in liverand brain. For this purpose chickens were either Insulin sensitivity (measured as the dose of insulin fasted for 48 h, refed for 24 h after the 48-h fast, or fed ad required for50% maximal stimulation of kinase activpreviously ity) was similarin all three statessuggesting that the libitum. These models were chosen since it has been differences ininsulin-induced phosphorylation are due demonstrated thatprolonged fasting causes insulin resistance to a change in maximal stimulation and not a change and starvation-refeedingcycles may improve insulin sensitivity in rats (19-22) and similarly in chickens (23, 24). in insulin sensitivity. In contrast to the alterations seen with liver receptors, braininsulin receptors were unEXPERIMENTAL PROCEDURES’ affected by these alterationsin nutritional state.These findings suggest that: 1) liver insulin receptors are RESULTS affected by altering the nutritional state; 2) insulin bindingtolivermembranesisinverselyrelatedto Insulin Binding to Liver and Brain Membranes plasma insulin levels; and 3) tyrosine kinase is deBinding of ‘251-porcine insulinto liver membranes was creased both in fasted and refed animals suggesting an uncoupling of the normal interaction between a subunit altered by the different nutritional states.Significantly lower and ~3subunit in liver insulin receptors.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should he addressed Diabetes Branch, NIDDK, Bldg. 10, Room 88243, NIH, Bethesda, MD 20892.
Portions of this paper (including “Experimental Procedures,” Table 1, and Figs. 1-3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-2414, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
17081
Fasting Refeeding and
17082
Kinase
Alter Insulin Receptor Tyrosine
'2sI-insulin binding to receptors in liver membranes was present in refed chickens 0, < 0.01), and slightly though consistently higher binding in liver membranes was found in fasted chickens when compared to the binding from ad libitum fed chickens (Fig. 4). Insulin concentrations required for 50% inhibition of tracer binding were similar (1.92 & 0.05 nM fasted, 1.66 & 0.09 nM ad libitum, and 1.67 & 0.06 nM refed), suggesting that the affinity of liver insulin receptors was not modified by the nutritional states and that the differences observed in insulin binding were the consequence of changes in the number of insulin receptors/mg of protein. Similar conclusions were supported by Scatchard-type analysis (data I I I I 1 not shown). In contrast, in brain the altered nutritional states 0 0.2 0.7 2.3 7 23 70 230 700 23w did not affect insulin binding (Fig. 5 ) ; both the affinity and UNLAEELLED INSULIN (nMl the number of insulin receptors in brain were unchanged by FIG.5 . Competitive inhibition of '251-insulin binding to the altered nutritional states with 50% inhibition of tracer binding of 1.36 & 0.12 nM for fasted, 1.15 f 0.20 nM for ad chicken brain membranes in various nutritional states. Brain membranes were prepared from chickens which wereeither fasted for libitum, and 1.29 f 0.16 nM for refed. These conclusions were 48 h, ad libitum fed, or refed for 24 h after a 48-h fasting period. Y supported by Scatchard-type analysis (data notshown). Porcine insulin (0.03 nM) was incubated with membranes for 4-5 h I
a t room temperaturein
I
I
the absence and presence of increasing
Cross-linking of '251-Insulin to Insulin Receptors concentrations of unlabeled porcine insulin. The results are presented Receptors from chicken liver and brain membranes were as mean rf: S.E. (n = 5 ) . When not presented the S.E. are smaller cross-linked with '"1-insulin, solubilized, immunoprecipitated than thesymbols for the mean. using an anti-insulin antibody, and then subjected to PAGE under reducing conditions. Following autoradiography, a major band representing the a-subunit (18) was seen in both tissues. As shown in chickens and other vertebrates the asubunit migrated faster for brain receptors than for liver receptors (Fig. 6). The mobility of the a-subunit and the 200 difference in the apparent M , between liver and brain receptors (about 10,000) were not modified by the altered nutritional states. 116Phosphorylation of Poly(Glu,Tyr)4:1 by Liver and Brain Receptors Liver Receptors-Two separate preparations of solubilized lectin-purifiedinsulinreceptors were obtained from liver
4
A
66 -
92
Fasted
0 Ad
Libitum Fed
Refed
FIG.6. Cross-linking of lZ6I-porcineinsulin to the a-subunit of the insulin receptors from chicken liver and brain membranes in various nutritional states. Membranes of chicken liver and brain were incubated with '"I-porcine insulin and cross-linked using disuccinimidyl suberate. The cross-linked materials were solubilized (1%Triton X-100) and immunoprecipitated using a guinea pig anti-insulin antiserum and Pansorbin. The immunoprecipitated materials were run on PAGE under reducing conditions. The gel was autoradiographed for 24 h. The migration of molecular weight markers were run in parallel on the same gel as indicated. F, fasted; R, refed; A, ad libitum fed. 0'0.2
0.7
I
I
I
I
I
I
2.3
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23
70
230
700
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UNLAEELLED INSULIN fnM)
FIG.4. Competitive inhibition of '251-porcineinsulin binding to chicken liver membranes in various nutritional states. Liver membranes were prepared from chickens which were either fasted for 48 h, ad libitum fed, or refed for 24 h after a 48-h fasting period. '251-Porcineinsulin (0.03 nM) was incubated with membranes for 4-5 h a t room temperaturein the absence and presence of increasing concentrations of unlabeled porcine insulin. The results are presented as mean k S.E. (n = 5 ) . When not presented the S.E. are smaller than thesymbols for the mean.
membranes: "pea" receptors and "WGA"' receptors (see "Experimental Procedures"). Both lectin-purified preparations had similar binding affinities for insulin (Fig. 3). Since the number of liver insulin receptors/mg of membrane protein was affected by the nutritional state, both pea and WGA receptor preparations from the fasted and the ad libitum fed * T h e abbreviations usedare:
WGA, wheat germ agglutinin;
HEPES,4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
17083
Fasting and Refeeding Alter Insulin Receptor Tyrosine Kinase
states were diluted to achieve similar tracer binding as in the the amount of membrane protein which was solubilized was not the same in each nutritional state, the tracer binding of refed state (Fig. 3). Usingpeareceptorsthephosphorylation of poly(Glu, the solubilized receptors was slightly different. For ease of was adjusted Tyr)4:l was linear (Fig. 2) for at least 60 min. This was seen comparison of kinase activity the tracer binding to 0.08 (bound/free) for each nutritional state prior to the foreach of thenutritionalstates(datanotshown),and, therefore, the kinase activity of these receptors was studied reaction. Since the kinase activity of purified brain insulin at 15 min. In contrast, the kinase activityof WGA receptors receptors is linear fora t least 60 min (18), the phosphorylation was measured at 8 min since phosphorylation was linear for of poly(Glu,Tyr)4:1 substrate was measured at 15 min. Both the basal activity (inset toFig. 9) and the insulin-stimulated about 10 min using these receptors(Fig. 2 and Ref. 18). Basal tyrosine kinase activity in both pea and WGA-puri- activity ( A kinase: stimulated 32Pincorporation minus basal, fied receptors was significantly lower in the fasted state than Fig. 9) were similar in all three nutritional states. The maxiin the adlibitum fed state (p < 0.05, inset toFig. 7, A and B). mal counts were 2.0-2.2 times basal, and half-maximal reBasal activity in receptors from refed animals was interme- sponse was achieved at 1.7 nM insulin. In all 3 nutritional diate between that of fasted and ad libitum. Insulin-dependent states therewas no demonstrable ATPase-like activityin the receptor preparation and only minimal activity in the flow (A kinase, i.e. stimulatedminus tyrosinekinaseactivities basal) was lower in fasted compared to ad libitum (p < 0.05, through of the column (Fig. 8B). Fig. 7, A and B ) . Again, activity in the refed receptors was Phosphorylation of theSubunit bracketed by values in fasted and ad libitum receptors. The insulin concentration required for half-maximal phosphorylLiver Receptors-Autophosphorylation of the p subunit (95ation was unaffected by the altered nutritional states (1.3-2.6 kDa band that immunoprecipitated with the human insulin nM in pea receptors and0.5-0.6 nM in WGA receptors). When anti-receptor antiserum Blo) (Fig. 10, A and B, right lanes) plotting the insulin-dependent phosphorylation as percent was not detectable in the basal state, either on autoradiograincrease above basal, no difference for sensitivity among the phy (Fig. 10,A and B) of the gels or by p scintillation counting three nutritional states was noted (data not shown). Thus, of the excised gel (datanotshown).Insulin-stimulatable the differences in insulin-dependent kinase activity appear tophosphorylation of the p subunit was detectable at lo-' and be closely related to the alterations in basal phosphorylation. M insulin andwas lower in fasted comparedto ad libitum ATPase-like activitywas present in theWGA preparations and intermediate in the refed state. These results were con) (Fig. 8A). No ATPase-like activity was demonstrable in the sistently obvious on the gels at 2 ATP (50 and 5 p ~ concenpea receptors. Since the differences in phosphorylation be- trations. Both pea and WGA preparations demonstrated simtween the various nutritional states (see above) are found in ilar results. However, due to thelow incorporation of 32Pand both pea and WGA preparations, the ATPase of the WGA p scintillation counting of individual bands, quantitative and preparations does not seem to play a role in thesedifferences. statistical analysisof the datawas not possible. Brain Receptors-Basal autophosphorylation of the p subBrain Receptors-Brain insulin receptors do not contain ATPase-like hydrolytic activity when solubilized and purified unit of the insulin receptors in WGA preparations was not M ) stimulated on WGA columns (18).The affinity of the solubilized recep- detectable (Fig. 11). Insulin (IO-' and tors was not changed by the nutritional state (Fig. 1). Since phosphorylation of the specific (Blo immunoprecipitable)95-
h
01
,
1
0.3
1
1
3 INSULIN InM)
10
I 100
t
L 0 1
A
F
I
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I
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INSULIN InMI
FIG. 7. Insulin dose responses for the phosphorylation of the artificialsubstrate poly(Glu,Tyr)l:l in pea ( A )or WGA (B)purified insulin receptors from chicken liver in various nutritional states. Pea and WGA liver insulin receptors were obtained from fasted, ad libitum and refed chickens as described in the legend to Fig. 3. The tracer insulin binding capacities of the partially purified receptors from the different nutritional states were adjusted by dilution in the glucosamine-Triton-HEPESbuffer to thesame level, i.e. bound/free = 0.16 for pea receptors and bound/free = 0.18 for WGA receptors. The initial protein concentrationof the receptors was 6-20 pg/ml for pea receptors and 19-33 pg/ml for WGA receptors. Insulin receptors were first preincubated for 30 min at room temperature in the absence or the presence of porcine insulin at various concentrations. Phosphorylation was then initiated using the combination of M e , Mn2+,CTP, and vanadate and allowed to continue a t room temperature for 15 min using pea receptors or for 8 min using WGA receptors. Data are presented 1) as the amount of 32Pincorporated into the substrate in the absence of insulin (basal activity) inthe upper left insets and 2) as theamount of 32Pincorporated above the basal activity (A kinase) in response to insulin. Data represent the mean S.E. of 5 experiments (pea) and 4 experiments (WGA).
17084
Fasting and RefeedingAlter InsulinReceptor Tyrosine Kinase
0 Ad Llbltum 0 Refed
P
0 Buffer
n WGA
0
6
15
30
TIME, min
0
7
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30
TIME, mln
FIG. 8. Time course of ATP hydrolysis by solubilized lectin-purified insulin receptors from chicken liver (A) and brain (B)in various nutritional states. Pea and WGA liver insulin receptorswere obtained as described in the legend to Fig. 3. Their hydrolytic activitieswere measured using phosphomolybdate precipitation (35, 37) in the presence of M$+, Mn2+,CTP, and vanadate at room temperature and compared to that of buffer only, using the same tracer insulin binding capacity (bound/free = 0.16). The differences in hydrolytic activity among the three nutritional states using WGA-purified liver receptors probably reflectthe differences in protein concentrations in each preparation which were altered to normalize insulin receptor binding ( A ) .The hydrolytic activities of WGA-purified receptors as wellas the flow through from the WGA columns of the different brainsolubilized materials were also measured.The initial protein concentrationof the receptors was 43-50 pg/ml ( B ) . changesininsulinbinding were inversely proportionalto plasma insulin levels, suggesting a n association of circulating insulin with insulin receptor concentration. Thus, chicken liver insulin receptors may be regulated by insulin both in uiuo, as shown in these studies, as well as in vitro (38). In contrast brain insulin receptors were unaffected in the present study by changes in plasma insulin concentration. This lack of change in brain insulin receptors despite altered plasma insulin levels has been previously reported in rats and mice (39, 40). Insulin-dependent tyrosine kinase activity in liver membranes was also affectedby the alteration in nutritional states. Comparison between nutritional states was made using insulin receptors at the same concentration, i.e. similar binding of tracer '251-insulin. Under these conditions, basal and insu01 0.3 1 3 10 1M INSULIN InMl lin-stimulatedphosphorylation of poly(Glu,Tyr)4:1(exFIG. 9. Insulin dose response for the phosphorylation of pressed per unit of receptor) wassignificantlyreduced in artificial substrate poly(Glu,Tyr)4:1 in WGA-purified insulin fasted compared to ad libitum, with refed chickens having receptors from chicken brain in various nutritional states. Solubilized and WGA-purified brain insulin receptorswere prepared intermediate activity. These results may be interpreted as from fasted, ad libitum,and refed chickens.The tracer insulin binding suggesting that although circulating insulin levels probably capacity of the receptors from the different nutritional states was regulate receptor number (41) the insulin-dependent tyrosine adjusted by dilution in the glucosamine-Triton-HEPES buffer to the kinase activity may be affected via some other mechanism(s) same bound/free level(0.08). The initial protein concentrationof the receptors was 43-50 pg/ml. Insulin receptorswere first preincubated in altered nutritional states.Alternatively, the elevated insufor 30 min at room temperature in the absence and the presence of lin levels in the refed state may also be responsible for the a reduction porcine insulin at various concentrations. Phosphorylation was ini- reduction in tyrosine kinase in addition to causing tiated using the combination of M e , Mn2+,CTP, and vanadate and in insulinbinding, as has been proposed in studies of fat cells allowed to continue at room temperature for 15 min. Data are pre- incubated with high insulin levels (42). sented 1) as the amount of 32Pincorporated into the substrate in the In addition to the altered basal tyrosine kinase activity in absence of insulin (basalactivity) in the upper left inset and 2) as the amount of 32Pincorporated above the basal activity (A kinase) in chicken liver membranes, when insulin-induced phosphorylresponse to insulin. Data represent the mean f S.E. of 3 experiments. ation of poly(Glu,Tyr)4:1 was expressed as percent increase above basal, the insulindose response was similar in all three nutritional states. Thus, insulin-induced tyrosine kinase ackDa band. No differences in the insulin-stimulatable phostivity is proportional to the activity in the basal state. Interphorylation were detected between the three nutritional states (Fig. 11).The phosphorylationof other phosphoproteins seen estingly, insulin sensitivity measured as the concentrationof is insulin required to achieve half-maximal phosphorylationwas on the gels was not insulin dependent, and their nature unclear. Quantitative analysisof the radioactivitywas impre- similar in each of the three nutritional states despite the alterations in basal and insulin-dependent tyrosine kinase cise which precluded statistical analysisof these data. activity. Furthermore, similar resultswere found in both pea DISCUSSION and WGA-preparedliver receptors, suggesting that the presWGAIn the present studywe have shown that insulin binding to ence of the contaminating ATPase-like activity in a significant role in these chicken liver membranes was elevated by fasting for 48h and prepared receptors did not play we have identified reduced below normal by fasting and refeeding, compared to studies. In thisregard it is unclear whether ad libitum feeding. The affinityof the receptors for insulin in two subtypes of chicken liver insulin receptors using pea lectin purification; however, similar affinities for insulin and reccrude membranes, as well as in lectin-purified membranes, was similar in all three states. Therefore, changes in binding ognition by human anti-insulin receptor antiserum suggests findings rats reflect changes in receptor concentration. Moreover, these that they may be similar. In contrast to our
v
1
Fasting and Refeeding Alter Insulin Receptor Tyrosine Kinase
17085 WGA Receptors
B
Pea Receptors M,
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lnsulin(M)
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lnsulin(M)
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lo-’ 1 0 - 7 1 0 - 7
N.S. B,o
N.S. B,,
FIG. 10. Autophosphorylation of the@-subunitof pea (A) or WGA ( B )purified insulin receptors from chicken liver in the various nutritional states. Pea and WGA liver insulin receptors were obtained from fasted, ad libitum, and refed chickens. The tracer binding capacity of the receptors from the different nutritional states was adjusted by dilution to thesame bound/free level (0.25) for both pea and WGA receptors (40 pl of these receptors were used in a final volume of 90 pl). Insulin receptors were first preincubated for 30 min a t room temperature in the absence or the presence of porcine insulin at the indicated concentrations. Phosphorylation was initiated using the combination of M e , Mn2+,CTP, andvanadate and allowed to continue a t room temperature for 6 min. The samples were run on sodium dodecyl sulfate-PAGE underreducing conditions after direct application. Samples of insulin-stimulated pea and WGA receptors from refed chickens were also gel electrophoresed after immunoprecipitation with normal human serum (N.S.) or human anti-insulin receptor antiserum (Bloa t 1:60 dilution, extreme right lunes of each panel). The migration of molecular weight markers run in parallel on the same gel is indicated. Following autoradiography the 95-kDa bands were excised from the gels and the 32P-labeled6subunits were extracted and theradioactivity measured by liquid scintillation.
fasted for 72 h or fed high carbohydrate diets showed alterations inbasal tyrosine kinase activityof liver insulin receptors compared to ad libitum fed rats (43), and insulin-induced tyrosine kinase (stimulated minus basal) was similar in all three nutritional statesin that study (43). Insulin-induced autophosphorylation of the /3 subunit from liver insulin receptors was detectable at lo-’ M insulin and M insulin in both pea and WGA further increased at preparations. Qualitative differences were apparent between the various nutritional states. Leastautophosphorylation was noted in fasted animals compared with ad libitum, and the refed group demonstrated intermediateresults. Unfortunately due to low levels of incorporation of 32Pinto the /3 subunit statistical analysis of these data was not possible. The results of our studies on the effect of altered nutritional states on liver insulinreceptortyrosinekinaseshouldbe contrasted with those in the rat.Blackshear et al. (44) found enhanced insulin-stimulatable autophosphorylation of the /3 subunit in livers of rats fasted for 48 h. In contrast to these results 72-h fasted rats and high carbohydrate diet-fed rats had no change in insulin-stimulatable /3 subunit autophosphorylation, despite the alterations in basal kinase activity (43). The reasons for these differences between our results and those in rats may include differences in species, age of animals, and differences in dietary manipulations used. In uiuo studies have demonstrated a state of relative insulin insensitivity in young chickens undergoing prolonged fasting (23). The present study demonstratesdecreased tyrosine ki-
nase activity of the insulin receptors produced by prolonged fasting and probably represents a postbinding defect in the liver of these chickens. These results could explain the insulin insensitivity of earlier experiments (23). On the other hand trained fasted-refed chickens demonstrate increased insulin sensitivity in vivo as shown by improved glucose tolerance after overnight fast (24). Since it is unclear whether the tyrosinekinase of the insulin receptor is involved in the postbinding activity of insulin action we cannot be sure that there is a causal relationship between the altered tyrosine kinase activity of liver receptors in altered nutritional states and the demonstrated insulin resistance in chickens. However, evidence in favor of the possible role of insulin receptor kinaseininsulinaction comes from anumber of studies suggesting that impaired insulin receptor kinase activity is associated with insulin resistance. These include some patients with severe insulin resistance and acanthosis nigricans (9, lo), obese mice (12), streptozotocin-induced diabetic rats (11,45) aswell as cultured rat hepatoma cells (46), melanoma cells (47), IM-9lymphocytes (48), and adipocytes (42,49,50). In contrast to the results in liver receptors, the various nutritional manipulations in thechicken did not affect insulin binding to brain receptors. Further, insulin-stimulated autophosphorylation, as well as basal and insulin-stimulatable phosphorylation of poly(Glu,Tyr(4:1) was similar in all three nutritional states. In conclusion, alterations in nutritional states affect the insulin receptors of chicken livers but fail to influence brain
Fasting Refeeding and Alter Insulin
17086 Fasted
Refed
Receptor Tyrosine Kinase
6. Kasuga, M., Zick, Y., Blith, D. L., Karlsson, F. A., Haring, H. U., and Kahn, C. R. (1982) J. Biol. Chem. 257, 9891-9894 7. Petruzzelli, L. M., Ganguly, S., Smith, C. J., Cobb, M. H., Rubin, C. S., and Rosen, 0.M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,6792-6796 200 8. Van Obberghen, E., and Kowalski, A. (1982) FEBS Lett. 143, 179-182 9. Grunberger, G.,Zick,Y., and Gorden, P. (1984) Science 223, 116932-934 92 10. Grigorescu, F., Flier, J. S., and Kahn, C. R. (1984) J. Biol. Chem. 259,15003-15006 66 11. Burant, C. F., Treutelaar, M. K., and Buse, M. G. (1986) J . Clin. Invest. 77, 260-270 12. Le Marchand-Brustel, Y., Grbmeaux, T., Ballotti, R., and Van Obberghen, E. (1985) Nature 3 1 5 , 676-679 13. Simon, J., Freychet, P., and Rosselin, G. (1977) Diabetologiu 13, 219-228 14. Kemmler, W., Renner, R., Zynamon, A., and Hepp, K. D. (1978) Biochim. Biophys.Acta 543, 349-356 15. Simon, J. (1979) Biochim. Biophys.Acta 585, 563-574 16. Simon, J., Freychet, P., and Rosselin, G. (1974) Endocrinology 95,1439-1449 17. Simon, J., Freychet, P., Rosselin, G., and DeMeyts, P. (1977) Endocrinology 100, 115-121 lnsulin(M) 0 10-310-7 0 1 0 - 9 1 0 - 7 0 10-9 1 0 1- 70 . 7 10-7 18. Simon, J., and LeRoith, D. (1986) Eur. J. Biochem. 158, 125132 E,, N.S. 19. Tzagournis, M., and Skillman, T. G. (1970) Metabolism 19,170FIG. 11. Autophosphorylation of the &subunit of WGA-pu178 rified insulin receptors from chicken brain in the various 20. Olefsky, J. M. (1976) J. Clin. Invest. 58, 1450-1460 nutritional states. Solubilized WGA-purified brain insulin recep- 21. Braun, T., Vrana, A., and Fabry P. (1967) Experientia 23, 468tors were prepared from fasted, ad libitum, and refed chickens. The 470 tracer binding capacity of the receptors from the different nutritional 22. Wiley, J. H., and Leveille, G. A. (1970) J. Nutr. 100, 1073-1080 states was adjusted by dilution to the same bound/free level (0.103), 23. Simon, J., and Rosselin, G. (1978) Horm. Metab. Res. 10, 93-98 and 40 pl of these receptors were used in a final volume of90 p l . 24. Simon, J., and Rosselin, G. (1979) J. Nutr. 109,631-641 Insulin receptors were first preincubated for 30 min a t room temper- 25. McMurtry, J. P., Rosebrough, R.W., and Steele, N. C. (1983) ature in the absence or the presence of porcine insulin at theindicated Poult. Sci. 62, 697-701 concentrations. Phosphorylation was initiated using the combination 26. Havrankova,J.,Roth, J., and Brownstein, M. (1978) Nature of M P , Mn2+, CTP, andvanadate and allowed to continue a t room 272,827-829 temperature for 6 min. The samples were run in PAGE under reducing 27. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. conditions after direct application. Two samples of maximally insulin(1951) J . Bwl. Chem. 193, 265-275 stimulated receptors were also gel electrophoresed after immunopre- 28. Pilch, P. F., and Czech, M. P. (1980) J. Biol. Chem. 255, 1722cipit-tion with human anti-insulin receptor antiserum (Blo a t 1:60 1731 dilw m ) or normal human serum (N.S.) (extreme right lanes). The 29. Kasuga, M., White, M.F., andKahn, C. R. (1985) Methods migration of molecular weight markers run in parallel on the same Enzymol. 109,609-621 gel is indicated. Following autoradiography the 95-kDa bands were 30. Kasuga, M., Van Obberghen, E., Yamada, K. M., and Harrison, excised from the gels, and the 32P-labeled@-subunitswere extracted L. C. (1981) Diabetes 30,354-357 and theradioactivity measured by liquid scintillation. 31. Hedo, J. A., Harrison, L. C., and Roth, J. (1981) Biochemistry 20,3385-3393 insulin receptors. Specifically, the tyrosine kinase of the liver 32. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 insulin receptors canbe modulated by nutrition. Thus, fasting 33. Finn, F. M., Titus, G., Horstman, D., and Hofmann, K. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7328-7332 and refeeding states are associated with a decrease in auto- 34. Cuatrecasas, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1277phosphorylation of the insulin receptor @ subunit aswell as a 1281 similar decrease in basal and insulin-induced tyrosine kinase 35. Zick, Y., Kasuga, M., Kahn, C.R., and Roth, J. (1983) J. Biol. activity. However, the physiological importance of these Chem. 258,75-80 36.Zick, Y., Grunberger, G., Rees-Jones, R.W., and Comi, R. J. changes requires further study. (1985) Eur. J . Biochem. 148, 177-182 Acknowledgments-We wish to thank Simeon I. Taylor for advice 37. Reimann, E. M., and Umfleet, R. A. (1978) Biochim.Biophys. Acta 523,516-521 in planning and executing these studies; J. Hedo, D. Rouiller, and R. Comi for advice regarding lectin columns; Elaine Collier and George 38. Krupp, M., and Lane, M.D. (1981) J. Biol.Chem. 256, 16891694 Delahunty for useful comments in preparationof the manuscript; and 39. 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