Insulin

Binding

and Receptor Tyrosine Kinase Activity Muscle: Effect of Starvation

in Rat Liver and Skeletal

M. Balage, J. Grizard, C. Sornet,

and M. Manin

J. Simon,

D. Dardevet,

Insulin binding and insulin receptor kinase activity were measured in solubilized and partially purified receptor preparations from liver and skeletal muscles of rats that were either fed a standard diet or subjected to a 72-hour fasting period. Insulin binding capacity was increased in both tissues from fasted rats as determined by Scatchard analysis. The affinity of the receptors was not modified by fasting. Affinity labeling of the a-subunit of insulin receptors also suggested an increase in the number of insulin receptors in both tissues. The ability of insulin to stimulate the autophosphorylation of the p-subunit as well as the phosphorylation of the artificial substrate Glu”-Ty? was significantly impaired in liver from fasted rats and by contrast unchanged in skeletal muscles. These findings indicate that in rats, fasting produces changes in insulin receptor kinase activity in liver but not in muscle. The physiological significance of this tissue-specific regulation of receptor kinase activity in relation to insulin action during fasting remains to be established. @ 1990

by W.S.

Saunders

Company.

I

NSULIN BINDING to specific cell surface receptors is the initial event in insulin action on target tissues. Insulin receptors are oligomeric glycoproteins consisting of (Ysubunits (130 to 135 Kd), which bind insulin, and P-subunits (90 to 95 Kd), which possess an intrinsic tyrosine kinase activity. Insulin binding to the a-subunits stimulates autophosphorylation of the P-subunits, as well as phosphorylation of exogenous substrates. It has been postulated that the activation of the protein kinase activity of the insulin receptor is an important step in transmembrane signaling by insulin. There are several examples where alterations in receptor kinase activity could explain changes in insulin action’-5; however, this was not always the case.6.9 In addition, the mechanism by which insulin resistance occurs appears to differ from tissue to tissue.5.7.8.‘0 Prolonged fasting in rats is characterized by insulin deficiency and insulin resistance. In vivo, both peripheral and hepatic insulin resistance has been observed on glucose metabolism”.‘* despite an increase in insulin receptor number.‘3-‘” To understand this paradox. the insulin receptor kinase activity has been studied in liver from fasted rats; however, conflicting results were obtained.“.” Surprisingly, this topic has never been considered in skeletal muscle despite

the fact that

weight

and

stimulated

glucose kinase

for disposal

this, we investigated tyrosine

this tissue

accounts

the

about

proportion

in the whole

both insulin activity

represents major

binding

in liver and

40% of body

body.”

In view of receptor

muscle

of

72-hour fasted rats. MATERIALS AND METHODS

Chemicals Purified monocomponent porcine insulin was obtained from Novo, Bagsvaerd, Denmark. [‘251-TyrA’4] human monoiodoinsulin (IM 166) and [r-“PI adenosine triphosphate (ATP) (3,000 Ci/mmol) were purchased from Amersham International, Amersham, England and New England Nuclear, Boston, MA, respectively. The wheatFrom the Laboratoire d’Eiude du M+tabolisme Azotb. Centre de Clermont-Theix. Ceyrat, France: and the Station de Recherches Avicoles. Nouzilly. Monnaie. France. Address reprint requests to M. Balage, Laboratoire d’Etude du Metabolisme Azote, INRA-Theix, 63122 Ceyrat. France. B 1990 by W.B. Saunders Company. 0026-0495/90/3904-0006$3.00/O 366

Animals Male Wistar rats (150 g) were used. They were housed in controlled environmental conditions at 22OC, 60% relative humidity, with a 12-hour dark period starting at 8 PM. The animals were either fed ad libitum a standard rat chow or fasted for 72 hours. They were killed by decapitation under nembutal anesthesia at approximately 10 AM. Livers and skeletal muscles from hind legs (ie, a mixture of red muscles containing various proportions of fiber type) were quickly excised, frozen, and stored at - 80°C until analyzed. Insulin

Receptor

Preparations

of insulin-

and insulin

skeletal

germ-agglutinin agarose (WGA; Glycaminosylex) and the bovine y-globulins (Pentex, Fraction II) were obtained from Miles Scientific, Napperville, IL. Artificial substrate, G1u*“-Tyr’o; N-2-hydroxyethylpiperarine-N’-2-ethanesulfonic acid (HEPES), phenylmethylsulfonylfluoride (PMSF); N-acetyl-D glucosamine and bovine serum albumin (BSA, fraction V) were from Sigma Chemical, St Louis, MO. All reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad (Richmond, CA). Serum from patient B,, with autoantibodies to insulin receptors was a gift of Drs J. Roth and P. Gorden (National Institutes of Health, Bethesda, MD). Protein A-bearing Staphylococcus aureus (Pansorbin) was from Calbiochem-Behring, Los Angeles. CA. All other chemicals were reagent grade.

Liver and skeletal muscle membranes and insulin receptors were prepared by the method of Havrankova et ali9 and Hedo et al.” Briefly, muscle (5 g) was homogenized in 40 mL of I mmol/L NaHCO, at 4°C with a Polytron (setting 5,0.5 minutes). Total liver was homogenized in 1 mmol/L NaHCO, (5 mL per 1 g liver weight) using an all glass Dounce homogenizer. We verified that homogenization using either Dounce or Polytron yields the same final insulin binding in liver. Thereafter, liver and muscle homogenates were centrifuged at 4°C for 30 minutes at 600 xg. The pellets were discarded, whereas the supernatants were centrifuged at 4°C for 30 minutes at 20 000 xg. The resulting pellets were washed once with 1 mmol/L NaHCO,. The final pellet was solubilized in 50 mmol/L HEPES buffer (pH 7.6) containing 1% Triton X-100, 2 mmol/L PMSF at 4°C for 30 min. In pilot experiments, we showed that addition of proteolytic inhibitors (pepstatin, leupeptin, and aprotinin) during solubilization did not produce any improvement in tyrosine kinase activity both in liver and muscle insulin receptor preparations. Insoluble material was discarded by centrifugation at 100,000 xg for 30 minutes at 4°C. The supernatant solution diluted to the same protein amount was applied five times to a column containing WGA (1 mL packed vol). After extensive washing with 50 mmol/L HEPES buffer (pH 7.6) containing 150 mmol/L NaCl Metabolism, Vol39, No 4 (April), 1990: pp 366-373

INSULIN RECEPTOR KINASE ACTIVITY

IN FASTED RATS

and 0.1% Triton X-100, the bound glycoproteins were eluted with buffer supplemented with 0.3 mol/L N-acetyl-o-glucosamine. This method provides a 30-fold purification for the insulin binding capacity per milligram protein in both liver and muscle. In both fed and fasted rats, 85% and 95% of the applied binding activity was recovered in the eluate for liver and muscle receptors, respectively.

Insulin Binding Assays Aliquot samples (50 rL) of partially purified receptor preparations diluted to the same protein concentration (8 pg/mL for liver, 5 gg/mI. for muscle) in WGA-elution buffer were incubated with a tracer amount of [‘25I-A’4] insulin (-0.01 nmol/L) overnight at 4OC in the absence or the presence of increasing concentrations of unlabeled insulin (0 to 100 nmol/L) in a total volume of 200 rL 50 mmol/L HEPES buffer (pH 7.6) containing 150 mmol/L NaCl, 0.025% Triton X-100, 0.075 mol/L N-acetyl-glucosamine, 0.3% BSA. The receptor-bound insulin was precipitated for IO minutes at 4°C by the addition of 100 @L of 0.3% y-globulin and 300 pL of 25% polyethylene glycol and collected by centrifugation in a Beckman Microfuge B. The resulting pellets were washed with 300 rL of 12.5% polyethylene glycol and recentrifuged. The tips of the tubes containing the final pellets were sliced off and counted for radioactivity in a gamma counter. The nonspecific “‘I-insulin binding, which was less than 5% of total binding, was determined at 5 x 10m6 mol/L insulin and subsequently subtracted from each value. Scatchard analysis was used for determination of total insulin binding capacity of each receptor preparation.

Ajinity Labeling of Insulin Receptors With ‘2SI-Insulin Following “‘I-insulin (2 x lo-” mol/L) binding in the presence or the absence of unlabeled insulin (3 x 10m6 mol/L) using equal amounts of solubilized proteins, the “51-insulin-receptor complex was covalently cross-linked with I mmol/L disuccinimidyl suberate for 15 minutes at O°C by the method of Pilch and Czech.2’ The reaction was stopped by adding 50 pL of 200 mmol/L HEPES buffer (pH 7.6) containing 10% SDS, 25% 2-mercaptoethanol, 50% glycerol, 0.05% bromophenol blue and boiled immediately for 7 minutes. Analysis of samples were performed by one-dimensional SDSPAGE according to the method of Laemmli** in a 7.5% gel. The gels were then fixed, stained with Coomassie blue, dried, and autoradiographed at - 70°C with Amersham Hypcrfilm-MP using lighteningplus screen.

Tyrosine Kinase Activity:Phosphorylation of Exogenous Substrate Partially purified insulin receptors (20 rL) were preincubated in a total volume of 70 FL containing 0.2 mg Glu*‘-Tyr*’ in the absence or presence of different insulin concentrations at room temperature for 30 minutes. For this, insulin receptor preparations from fasted and fed rats were diluted in order to equalize their binding activity. Phosphorylation was initiated by the addition of 20 rL of a mixture containing 20 mmol/L MgCl,, I mmol/L cytidine 5’-triphosphate (CTP). and 50 pmol/L [y”P] ATP (1 pCi/nmol) as a final concentration. In these conditions, the phosphorylation reaction was linear for at least 1 hour. After a 20-minute incubation at room temperature, the reaction was stopped by applying samples (70 pL) to filter paper squares (3 x 3 cm, Whatmann 3 MM) and soaking three times in 10% trichloracetic acid containing IO mmol/L sodium pyrophosphate. Papers were then washed extensively in 90% ethanol and dried. “P incorporation was quantified by liquid scintillation. Incorporation of j2P into trichloracetic acid (TCA) precipitable material observed in the absence of exogenous substrate was subtracted to yield actual substrate phosphorylation. Reactions were also performed in the absence of insulin receptors (using WGA-

367

elution buffer) to correct for background. Results are expressed as fentomoles of ‘*P phosphate incorporated per milligram of GlugOTyr”’ per picomole binding capacity per minute.

Insulin Receptor Autophosphorylation Partially purified receptor preparations (20 pL) (diluted to equalize binding activity in fasted and fed rats) were preincubated as previously described in the absence or presence of insulin (100 nmol/L). Autophosphorylation was initiated by adding [r-“PI ATP (I2 rmol/L), Mn acetate (3 mmol/L), CTP (I mmol/L). After 10 minutes at room temperature, aliquots (70 pL) of the reaction mixture were combined with 50 rL of a solution containing 0.2% Triton X-100, 10 mmol/L EDTA, 100 mmol/L NaF, 20 mmol/L Na pyrophosphate, 20 mmol/L NaH,PO,, 20 mmol/L ATP, 5% SDS, 12.5% 2-mercaptoethanol, 25% glycerol, 0.025% bromophenol blue, and 60 mmol/L HEPES (pH 7.6). After heating at 97OC for 7 minutes, the samples were analized by SDS-PAGE as described. In some experiments, phosphorylated insulin receptors were immunoprecipitated. In this case, the autophosphorylation reaction was stopped by adding 30 /IL of a stopping buffer: 0.4% Triton X-100, 20 mmol/L EDTA, 200 mmol/L NaF, 40 mmol/L Na pyrophosphate, 40 mmol/L NaH,PO,, 40 mmol/L ATP, 40 mmol/L HEPES pH 7.6; then l/100 final dilution of polyclonal human insulin receptor antibody BlO was added. After overnight incubation at 4°C 25 PL of Pansorbin was added and incubation was carried out for an additional 1 hour in an ice bath. After centrifugation, the immunoprecipitates were washed three times with 50 mmol/L HEPES buffer containing 150 mmol/L NaCl and 0.1% Triton X-100. Finally, the pellets were suspended in 40 mmol/L HEPES buffer pH 7.6 containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue, boiled for 7 minutes, and subjected to SDSPAGE. The gels were then processed as described previously. The ATPase activity of the partially purified insulin receptors was tested by measuring hydrolysis of ATP (determination of inorganic phosphate release) according to the molybdate technique used by Simon et al.’

Other Analysis Plasma insulin was determined by a standard as previously described.‘l Protein determination the Bradford dye method24 using the Bio-Rad used as the standard.

radioimmunoassay was performed by reagent. BSA was

Statistical Analysis Experiments so comparisons i test.

were always performed in parallel with both groups, between fed and fasted rats were made using a paired

RESULTS

Animal Characteristics Final body weights were 112.8 r 2.1 and 166.7 2 2.2 g (mean k SE, n = 15) in fasted and fed rats, respectively. Fasting also resulted in a dramatic decrease of liver weight (3.4 + 0.2 v 8.9 - 0.3 g). As previously reported,“,25 fasted rats had low plasma insulin levels when compared with fed rats (21 k 3 v91 + 13 &/mL, n = 10).

Insulin Binding to Partially Purified Solubilized Insulin Receptors As shown in Fig 1, insulin binding to partially purified insulin receptors from liver and muscle was higher in fasted than in fed rats (P < .025 at all insulin concentrations in both

BALAGE ET AL

368

LIVER

MUSCLE

-“~O,_o

‘ ‘..., 0.1

0.2

.a3

Bound (nhl)

INSULIN

CONCENTRATION

(r&I)

tissues). The concentration of unlabeled insulin needed to produce 50% decrease in insulin binding (ID 50) was unaffected by nutritional state (about 1 and 0.5 nmol/L in liver and muscle, respectively). According to Scatchard analysis, this increase in binding was mainly related to an increase in binding capacity rather than to an increase in binding affinity (Fig 1, insets) in both tissues (the maximal binding capacities were 89 i 7 v 46 k 2 pmol/mg protein in liver WGA eluates and 61 i 7 v 36 + 6 in muscle WGA eluates in fasting and fed states, respectively). When affinity-labeled insulin receptors from liver and muscle of fasted and fed rats were subjected to SDS-PAGE under reducing conditions, only one protein band was labeled with an approximate molecular weight of 135 Kd, which corresponds to the molecular weight of the a-subunit of the

Fig 1. Insulin binding to solubilized, WGA-purified insulin receptors from liver end muscle in 72-hour fasted (0) and fed (0) rats. WGA-purified insulin receptors (50 pL) from liver (about 1.6 ug protein) and muscle (about 1 fig protein) were incubated with a tracer amount of A14’261-insulin (approximately 0.01 nmol/L) end increasing concentrations of unlabeled insulin in a final volume of 200 uL buffer (pli 7.6) under conditions previously described (see Materiels and Methods). The receptor-bound insulin was precipitated with polyethylene glycol using bovine y-globulin as carrier protein. The results are presented as mean + SE of 12 paired individual experiments. The insets represent the Scatchard plot of the data.

insulin receptor (Fig 2). In addition, the radioactivity of this band was totally displaced by an excess of unlabeled insulin, which supports its specificity as the insulin binding site and substantiates the integrity of the insulin receptor cr-subunit in partially purified receptor preparations. In both liver and muscle receptors, the bands were clearly darker for preparations from fasted rats (almost twofold using densitometry measurements). Since the same amounts of WGA-purified proteins were used for cross-linking experiments, this result also suggests an increase in insulin receptor number in both tissues following prolonged fasting. Exogenous

Substrate

Kinase Activity

The ability of insulin to stimulate the phosphorylation of the artificial substrate Glu*‘-Tyr” was used to assess the

INSULIN RECEPTOR KINASE ACTIVITY IN FASTED RATS

LIVER fasted

Insulin

-

+

-

+

369

MUSCLE fed fasted

-

+

-

ma1 rates of phosphorylation (stimulated minus basal) (Table 1) was lower in fasted than fed rats. Thus, the ability of insulin to stimulate the insulin receptor kinase activity was greatly depressed in liver from fasted rats as compared with fed rats. In contrast, the insulin concentration required for half-maximal insulin effect did not appeared affected by starvation (about 1 nmol/L in each group). Skeletal muscle was different from liver regarding tyrosine kinase activity, ie. both the basal and insulin-stimulated tyrosine kinase activity of muscle insulin receptors were similar in fasted and fed rats (Fig 3, Table 1). Thus, the ability of insulin to stimulate the insulin receptor kinase from skeletal muscle seemed to be unaffected by starvation. In fed rats, tyrosine kinase activity towards the exogenous substrate was approximately threefold higher in muscle-derived receptors than in liver-derived receptors; this is in agreement with the data of Burant et al.‘” It could be argued that the lower levels of 32P incorporation into exogenous substrate found in liver insulin receptors, especially with preparations from fasted rats, might be related to enhanced ATP hydrolysis. It was not the case since less than 2% of “P-ATP was degraded during phosphorylation assays for receptors from both tissues (data not shown). In addition, it does not seem likely that insulin receptor kinase activity might be impaired in liver when compared with muscle, since the efficiency of insulin to maximally stimulate the insulin receptor kinase was very high in liver (nine- to 12-fold increase above basal level).

+

(3.1o-%l) Fig 2. Affinity labeling of insulin receptors from liver and muscle in fed or fasted rats. Partially purified insulin receptors (equalized to same protein content in fed and fasted groups) were incubated as described in Fig 1 with ‘Z61-insulin (2 x lo-” mol/L) in the presence or absence of an excess of unlabeled insulin (3 x 10 ’ mol/L). The ‘*‘l-insulin receptor complexes were crosslinked with 1 mmol/L disuccinimidyl suberate for 16 minutes at 0°C. The samples were subjected to SDS electrophoresis in 7.6% acrylamide gel under reducing conditions. The gels were then stained, dried, and eutoradiographed. Autoradiographs were tested by densitometry (the ratio between fasted and fed group was 1.9 + 0.2 and 1.8 t 0.3 in liver and muscle, respectively: mean * SE, n = 4). The molecular weight markers: myosin (Mr = 205,DOO). B-galactosidase (Mr = 118.OOD). phosphorylsse b (Mr = 97.400). bovine albumin (Mr = 88,000). and ovalbumin (46,000) were run in parallel.

Phosphorylation of the /3-Subunit The autophosphorylation of the @-subunit of the insulin receptor and the insulin receptor kinase activity toward exogenous substrate do not always exhibit parallel changes6.“,” So we next explored the autophosphorylation of the insulin receptor in both liver and skeletal muscle from fasted and fed rats. In the basal state, autophosphorylation of insulin receptor was not detectable either in liver or skeletal muscle (Fig 4). Insulin increased the incorporation of ‘*P from [y3’P] ATP into a protein of a molecular weight between 95 and 100 Kd, which corresponds to the @-subunit of the insulin receptor. In liver, insulin-stimulated phosphorylation of the @-subunit was lower in fasted than in fed rats (Fig 4). Like for exogenous substrate phosphorylation, skeletal muscle receptors differed from liver receptors regarding the autophosphorylation, ie, the insulin-stimulated phosphorylation of the P-subunit of muscle receptors was similar in fasted and fed rats (Fig 4). In skeletal muscle as in the liver (data not shown), the labeled protein obtained after phosphorylation of

protein kinase activity of the insulin receptors. Measurements were performed using receptors from fasted and fed animals at the same binding capacity. In liver, basal tyrosine kinase activity was not significantly different in the two groups (Table 1). Figure 3 clearly shows that, whereas insulin stimulated ‘*P incorporation into Glu*“Tyr*” in the two groups, it had a blunted effect in the liver insulin receptors from fasted rats at all insulin concentrations (P < ,025). incremental increase between basal and maxiTable 1. Insulin Receptor

Tyrosine

Kinase Activity in Liver and Skeletal

Muscle From Fasted and Fed Rats

Liver Fed Rasal

activity

Maximal insulin-stimulated activity

MUSCltl Fasted

Fed

Fasted

1.6 ? 0.3

20 + 4

25 k 6

33.6

+ 5.7

14.8 +_ 2.8’

113 i- 19

102 + 13

33.3

+ 6.4

13.2 + 2.8”

93 k 16

77 t 12

2.7 r 0.4

incremental increase (stimulated minus basal)

NOTE. Values are means + SE of five individual experiments. They are derived from data shown in Fig 3 and expressed as fentomoles 32P incorporated per milligram substrate per minute per picomole binding capacity. lP < .05

BALAGE ET AL

LIVER

1 /--___--fed

/’

,

/’

,/’

I

MUSCLE fed

1

1

Fig 3. Dose-response curves of insulin-stimulated phosphorylation of Glu=-Tyr” by liver and skeletal muscle receptor preperetions from 7Zhour fasted (0) and fed (0) rats. Aliquotr of WGA-purified inrulin receptors diluted to the rnme binding ectivity in fed end fasted groups were first preincubeted In the absence or the presence of insulin at verious concentretlons at room temperaturs for 30 minutes. Phosphorylrtlon wes then oerriod out In the prrsonoe of [Y-~P] ATP in condltlons dosoribed Isee Materlslr and Methods) st room tempereture for 20 minutes. The deta are expressed OS the amount of “P inoorporetsd per mllllgrem substrate per minute per plcomole binding capacity in each group. The results are presented as the mean f SE of five paired individual experiments.

insulin receptors was immunoprecipitable with anti-insulin receptor antiserum B,,, which confirms its identity as the P-subunit of the insulin receptor (Fig 4, extreme right lanes). DISCUSSION

In the present study, rats increased insulin receptor preparations These changes were

capacity per milligram glycoprotein without any change in binding affinity. Affinity labeling of the insulin receptor confirmed the increase in insulin binding sites in both tissues. Increase in insulin binding in liver from fasted rats was not surprising, as similar data have been widely described in plasma membranes,“’ isolated hepatocytes,‘3x’5 or partially purified insulin receptors.” Contrarily to liver, increased insulin binding to partially purified muscle insulin receptors from fasted rats was a new information using such a receptor preparation. Previous studies using isolated soleus muscles from fasted rats28,29 or mice, ” also showed an increase in insulin binding; however, this was related to an improvement of binding affinity rather than binding capacity. Using partially purified insulin receptors, we found an increase in insulin binding sites by either Scatchard analysis or affinity labeling. These discrepancies may reflect the differences in interpreting changes in insulin binding kinetics using solubilized insulin receptor preparations instead of intact tissues. Moreover, insulin binding in intact muscle reflects cell surface receptor binding, whereas the present data represent extractable the binding to a “total pool” of detergent membrane insulin receptors. These improvements in insulin binding sites in both liver and skeletal muscle from fasted rats probably resulted from an “up-regulation” process that was induced by the low circulating insulin levels. However, they cannot explain the state of insulin unresponsiveness demonstrated by others in both liver and peripheral tissues of such animals using the euglycemic clamp technic.” Thus, we studied the insulin receptor kinase activity. which possibly represents the first post binding event involved in insulin action. Indeed, several observations suggested that the tyrosine kinase activity intrinsic to the insulin receptor /?subunit was important for its transmembrane signaling. Inhibition of the receptor kinase activity by either selective mutations in specific regions of the p-subunit or introduction into cells of antibodies directed against the receptor psubunit significantly reduces or eliminates many of the cellular insulin actions (reviewed in reference 31). In view of this, insulin receptor kinase activity has been widely investigated in various conditions of insulin resistance.‘-9.27.32,33

we have shown that 72-hour fasting in binding in partially purified insulin from both liver and skeletal muscles. mainly related to increased binding

In the present study, we demonstrated that in rat, following a 72-hour fasting period, insulin receptor kinase activity was dramatically depressed in liver and in contrast unchanged in muscle. In liver we found that both the insulininduced autophosphorylation of the receptor P-subunit and the insulin-stimulated phosphorylation of the exogenous substrate were greatly reduced by fasting, whereas the basal activity (at least for the phosphorylation of exogenous substrate) was unaltered. Previous studies looking at the effect of fasting on liver insulin receptor tyrosine kinase activity have led to qualitatively similar results in chickens’ and to various results in rats.‘6.‘7 Blackshear et a116found enhanced insulin receptor autophosphorylation in liver of 4%hour fasted rats. In contrast, Freidenberg et al” observed no change in insulin stimulated P-subunit phosphorylation in liver of 72-hour fasted rats despite a decrease in insulin-stimulated artificial substrate phosphorylation, which in that situation was accounted for by a decrease in the basal tyrosine kinase activity of the insulin receptor. There are several experimental

INSULIN RECEPTOR KINASE ACTIVITY

IN FASTED RATS

371

LIVER fasted

Mrx,0_3

Fig 4. Autophosphorylation of the B-subunit of purified insulin receptors liver and muscle in fed or fasted rats. Partially purified receptors diluted to the same binding activity in fed and fasted group5 were preincubated in the absence or the presence of insulin (1 X lo-’ mol/Lj at room temperature for 30 minutes. Phosphorylation was then initiated by adding (Y-~*P) ATP in the presence of Mn++ and CTP. After 10 minutes at room temperature, the reaction was stopped in conditions previously described (see Material and Methods). The samples were then subjected to SDS-PAGE under reducing conditions as described in Fig 2. In some experiments, phosphorylated insulin receptors were immunoprecipitated with insulin receptor antibody B,, (1:lOO dilution) before electrophoresis (extreme right lanes). After migration, the gels were stained, dried, and autoradiographed.

MUSCLE fed

fasted

fed

fasted fed

11697-c

66-

45-c

Insulin

(lo-‘M)

-+

and/or methodological differences between these studies. First, Freidenberg et al and Blackshear et al used Sprague Dawley rats whereas we used Wistar rats; it has been shown that different strains of the same animal species may differ in their biological responses34 and, therefore, the changes induced by fasting and the extent of those changes may differ from one strain to another. Second, Blackshear et alI6 used a crude detergent extract of microsomes, whereas we used partially purified receptors. However, Friedenberg et al” also used partially purified liver receptors; their data differ from ours with regard to the efficiency of insulin to stimulate the tyrosine kinase activity (twofold stimulation at 4OC v loto 12-fold at room temperature in our experiment) and the ATP hydrolysis during the phosphorylation reactions (10% v 2% in the present experiment). The origin of these discrepancies is currently unknown. Similarly, the mechanisms leading to an alteration of the insulin receptor kinase in the liver but not in skeletal muscles following prolonged fasting await further elucidation. Thus, fasting in rats is another example where data generated in one tissue may not be extrapolated to another tissue. Such phenomenon has also been obtained when insulin receptor kinase was compared among different tissues (liver, brain, muscles, white or brown adipocyte) in various nutritional or pathophysiological conditions in patients or several animal species, ie, non-insulin-dependent diabetic patientqds6 chronic uremic rats,’ young obese Zucker rats,3s gold-thioglucose obese mice,‘,” and fasted chickens.5*7 If we hypothetize that the insulin-stimulated tyrosine kinase activity plays a role in the expression of insulin action, it is tempting to speculate that the decrease in the ability of insulin to stimulate the insulin receptor kinase activity we observed in the liver of fasted rats can explain, at least in part, the impairment of liver insulin responsiveness recorded either in vivo (with respect to hepatic glucose production),” or in vitro (with respect to cu-aminoisobutyrate uptake in

+-

+Bl,+

hepatocytes36) following a prolonged fasting period. In the present study, both insulin-stimulated exogenous substrate and P-subunit phosphorylation were not altered in skeletal muscle from fasted rats, whereas fasting-induced insulin resistance in peripheral tissues has been documented in viva.” Thus, the mechanisms by which insulin resistance occurs can differ from tissue to tissue and very likely involve various regulating mechanisms. So, our data in skeletal muscle from fasted rats is a new result that increases the list of examples where in vivo insulin resistant states were not related to abnormal insulin receptor tyrosine kinase activity.6-9 Some hypotheses could explain these discrepancies. First, the tyrosine kinase activity of the insulin receptor may not be involved in all of insulin’s action. For example, inhibition of receptor kinase activity by indomethacine did not suppress all of the insulin effects in cell (reviewed in reference 31). Moreover, it has been demonstrated that monoclonal antibodies directed to the o-subunit of the insulin receptor could stimulate glucose transport in isolated human adipocytes without any stimulation of receptor kinase activity. ” Second, the insulin resistant state could be related to a defect distal to the receptor kinase activity. Third, receptor-associated kinase activity was measured in vitro, whereas insulin resistance state was generally recorded in vivo. In that direction, insulin resistance in vitro has not been demonstrated in muscle of fasted rodents28.30;however, this may be inherent to the difficulties encountered in getting an insulin response in vitro in muscle. Four, insulin has multiple effects on muscle metabolism, some of them being unaffected by fasting. For instance, it has been shown that insulinstimulated glucose utilization was greatly impaired in fasted humans or rats,“,‘* whereas insulin inhibition of proteolysis was preserved in humans38 or increased in rats.3g Unfortunately, the involvement of insulin receptor tyrosine kinase activity in the various insulin effects has not been elucidated.

BALAGE ET AL

372

In conclusion, the present data bear new informations on the physiological regulation of the insulin receptor kinase activity in rats. Fasting resulted in an improved insulin binding capacity in both liver and skeletal muscle. In contrast, insulin-induced receptor tyrosine kinase activity was greatly depressed in liver, whereas it was unchanged in muscle. Thus, fasting is another example of the growing list where insulin-resistant states are not always related to

abnormal tyrosine kinase activity of the insulin receptor. The biological significance of these findings remains to be established.

ACKNOWLEDGMENTS

We would like to thank A. Genest for the preparation of this manuscript.

REFERENCES

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