Insulin binding and receptor tyrosine kinase activity in skeletal muscle ...

Insulin binding and receptor tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish MARCELINA PARRIZAS, JO& PLANAS, E. M. PLISETSKAYA, AND JOAQUIM GUTIfiRREZ Departament de Fisiologia Animal, Facultat de Biologia, Universitat de Barcelona, Barcelona 08028, Spain; and School of Fisheries, University of Washington, Seattle, Washington 98195 Phrrizas, Marcelina, Jo& Planas, E. M. Plisetskaya, and Joaquim Gutibrrez. Insulin binding and receptor tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish. Am. J. Physiol. 266 (Regulatory Integrative Comp. Physiol. 35): R1944-R1950, 1994.-We characterized the insulin receptors in skeletal muscle from several fish species with different nutritional preferences: brown trout (Salmo trutta fario), gilthead sea bream (Sparus aurata), tilapia (Tilapia mossambica >, and carp (Cyprinus carpio), semipurified by affinity chromatography (wheat germ agglutinin-agarose). Total specific binding and number of receptors per unit weight of piscine white skeletal muscle were lower than those values found in mammalian skeletal muscle. The same parameters in carp muscle receptor preparations were severalfold higher than in trout muscle (binding capacity 440 2 47 fmol/mg glycoprotein in carp and 82 t 23 fmol/mg glycoprotein in trout). Piscine insulin receptors phosphorylated exogenous substrate poly(Glu,Tyr) but less so than mammalian receptors. Tyrosine kinase activity of receptors, calculated as percent of 32P incorporated into substrate in the presence of insulin compared with basal incorporation, was also highest in carp (2 10 -+ 4%) and lowest in trout (150 t 2%). In both trout and carp deprived of food for 15 days, specific binding of insulin decreased. Nevertheless, differences between the two species were retained. Our results demonstrate that particular properties of insulin receptors in fish skeletal muscle may be related to nutritional preferences. This finding coincides with the phenomenon of differential glucose tolerance in fish: carnivorous fish, such as trout, are less tolerant, whereas omnivorous fish, such as carp, readily utilize a carbohydrate-rich diet. fish; skeletal muscle; insulin feeding preferences; fast

receptor;

tyrosine

kinase activity;

OF SOME FISH species to diets high in content of carbohydrates has been well known for a long time (8). Fish species with different preferences in natural food show different degrees of glucose tolerance when adapted to mixed diets used in aquaculture (12, 33). Omnivorous species, such as Cyprinidae, accept more carbohydrates in the diet than do carnivorous species, such as Salmonidae. Recent studies, using homologous radioimmunoassays (RIA) for insulin, have demonstrated that, in fish intolerant to carbohydrates, neither systemic levels of insulin nor insulin secretion in response to glucose seem to be impaired (26). This implies that glucose intolerance in such fish as salmonids may be related to factors at the tissue level. Because of their mass, skeletal muscles have the major role in glucose uptake and are the most important targets for insulin action in vertebrate animals (5). The first step in the biological action of insulin is binding of the hormone to its receptor on the plasma INTOLERANCE

R1944

0363-6119/94

$3.00

Copyright

membrane. The insulin receptor in all vertebrate species studied so far, including a primitive Agnathan hagfish, is a heterotetramer containing two extracellular a-subunits and two transmembrane p-subunits (9). When insulin binds to the a-subunits, a conformational change takes place, which activates tyrosine kinase activity of the cytoplasmatic domain of the P-subunits, initiating a phosphorylation cascade that leads eventually to signal transduction (19). The process can be modulated by changes in insulin receptor capacity and affinity as well and in tyrosine kinase activity. Decline of one or more of these parameters has been found in insulin-resistant (obese) mammals (7, 25) and in humans with non-insulin-dependent diabetes mellitus (NIDDM) (7, 11). In contrast, either fasting or high-carbohydrate diet caused in mammals an upregulation of both binding capacity and tyrosine kinase activity of insulin receptors (3,lO). Insulin-receptor binding in liver, skeletal and heart muscle, brain, red blood cells, and gonads has been studied in variety of fish species (13, 16, 17, 22, 27, 28, 30). Autophosphorylation of the insulin receptor of the stingray Dasyatis americana liver and lamprey Lampetra fZuviatilis liver have been reported (32, 24), and the phosphorylation of exogenous substrates by insulin receptor of carp ovaries was demonstrated (16). Although Leibush (22) has compared different characteristics of insulin-receptor binding in liver, muscles, brain, and red blood cells of scorpion fish (Scorpaena porcus), pink salmon (Oncorhynchus gorbusha) and Baltic lamprey, L. fluviatilis (all carnivorous species, collected from their natural habitats), similar comparisons between carnivorous and omnivorous fish specieshave not been reported. Thus the aim of the present study is to characterize and compare certain features of insulin-receptor binding in skeletal muscle of several fish species with different dietary preferences: carnivorous brown trout (Salmo trutta fario) and gilthead sea bream (Sparus aurata), semicarnivorous tilapia (Tilapia mossambica), and omnivorous carp (Cyprinus carpio). We attempt to determine whether the variations in tolerance to carbohydrates, reported in fish, correlate with any pattern of insulin binding to the skeletal muscle. MATERIALS Animals

AND METHODS

and Experimental

Design

First experiment. To obtain some general characteristics of insulin receptors in skeletal muscles of different fish species we collected muscle tissue from five immature specimens of brown trout S. trutta fario, weighing 123 t 10 g, raised at the fish farm Piscifactoria Baga in Barcelona area, Spain; five immature carp C. carpio, weighing 158 t 11 g; four mature

o 1994 the American

Physiological

Society

INSULIN

RECEPTORS

IN FISH

tilapia T. mossambica weighing 82 * 5 g (both species from Zoological Park of Barcelona); and four immature gilthead sea bream S. aurata weighing 130 2 5 g (from fish-holding facilities of Department of Zoology, University of Barcelona). Fish were kept at the above-mentioned locations for at least several months before sampling and fed daily diets specially designed for each species: trout (Sterling Silver Cup: 13% lipids, 44% protein, and 21% carbohydrates), gilthead sea bream (Ewos: 12% lipids, 49% protein, and 10% starch), and carp and tilapia (half-natural and half-commercial diet, Tetrapond: 1.5% lipids, 25% protein, and 2% fiber). Muscle tissue samples were collected from fish 18 h after last feeding. Second experiment. To compare insulin receptors of carp and trout while avoiding differences caused by external factors, we transferred carp to the Piscifactoria Baga where they were maintained under the same environmental conditions (daylight and water temperature) and fed the same commercial diet (Sterling Silver Cup, Lerida, Spain) as trout raised at this fish farm. Carps were adapted to the holding facilities for at least 30 days before the beginning of sampling. After 1 wk, carps actively fed on the new diet. Six fish of similar weight (trout 222 t 41 g, carp 243 t 23 g) were selected from both species, and muscle samples were collected 18 h after last feeding. Third experiment. In a separate experiment, carried out at Piscifactoria Baga, 30 carp and 30 trout were deprived of food for 15 days before tissue sampling. Sampling Fish were killed by cranial blow, and strips of white lateral muscle were rapidly excised and frozen in liquid nitrogen. Muscles were stored in liquid nitrogen until receptor purification. Semipurification

of Receptors

Processing of the muscle tissue and semipurification of solubilized insulin receptors were performed at 4°C according to Gutierrez et al. (13). Frozen muscle samples (8-9 g) were macerated in a cooled mortar and then homogenized with Ultraturrax in a buffer containing 25 mM N-2-hydroxyethylpiperazine-2\r’-2-ethanesulfonic acid (HEPES), 4 mM EDTA, 4 mM ethylene glycol-bis( P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2 mM phenylmethylsulfonyl fluoride (PMSF), and peptidase inhibitors: 1 unit/ml of aprotinin, 1 mM bacitracin, 1 mM leupeptin, 1 mM pepstatin, and 25 mM benzamidine, pH 7.6. Solubilization of homogenate was achieved by adding Triton X-100, followed by stirring for 1 h at 4°C. Several concentrations of Triton X-100, such as 1, 2, 3, and 6%, were tested. Solubilized homogenate was ultracentrifugated at 150,000 g for 90 min, and the ultracentrifugation pellet was tested for presence of receptor glycoproteins. Best results (no receptor glycoprotein could be recovered from the pellet) were obtained when a final concentration of 2% Triton X-100 in the homogenate was used. Insignificant increases in receptor yields were gained by using 3 or 6% Triton X-100. Supernatants after ultracentrifugation were recycled three times through a disposable polypreparative column packed with 1 ml of wheat germ agglutinin (WGA) bound to agarose (Vector Lab oratories). The column was washed with 100 ml of a buffer containing 25 mM HEPES and 0.1% Triton X-100, pH 7.4. Glycoproteins were eluted from the WGA column with the same buffer supplemented with 0.3 M N-acetyl-D-glucosamine. Receptor-Binding

Assays

Binding assays were performed according to the method of James et al. (20) adapted by us for fish. Briefly, 50 ~1 of the

SKELETAL

MUSCLE

R1945

WGA eluate, corresponding to 15-25 pg glycoproteins as measured according to Bradford (6) were incubated in 30 mM HEPES buffer containing 0.1% bovine serum albumin (BSA), 100 units/ml of bacitracin (pH 7.4), increasing concentrations of unlabeled porcine insulin (from 0.005 nM to 5 FM) and the radiolabeled insulin as tracer (20,000 counts/min of human TyrA14-1251-monoiodoinsulin, 2,000 Ci/mmol specific activity, Amersham Life Science). Specificity of the assay was determined by displacing labeled insulin with increasing concentrations of insulin-like growth factor I (IGF-I). Incubation time course and temperature were established in separate assays, and subsequent experiments were performed under optimal conditions (4-16 h of incubation at 4°C) (data not shown). Semipurified receptors were precipitated by addition of 0.5 ml of bovine T-globulin (0.2% in 50 mM phosphate buffer, pH 7.4) and 0.5 ml of polyethylene glycol (25% in 10 mM phosphate buffer, pH 7.4) and centrifugation at 14,000 g for 5 min in Eppendorf microfuge. Nonspecific binding was estimated as radioactivity bound in the presence of a maximal concentration of unlabeled insulin (5 ~.LM) and subtracted from the total counts. Nonspecific binding for preparations of carp and trout semipurified receptors constituted 2 t 0.08% of total counts when incubation was carried out for 1 h at 22OC, and 1.25 I~I 0.07% after incubation for 16 h at 4°C. Reproducibility of the method was assessed using an interassay standard (interassay variations were < 10%). Binding data were analyzed in Scatchard coordinates (29). Only the high-affinity, low-capacity binding sites calculated from the curvilinear plot were taken into account. Each experiment was performed at least in triplicate using receptor glycoproteins isolated from skeletal muscles of different fish. Tyrosine

Kinase Activity

Tyrosine kinase activity was determined according to James et al. (20) with some modifications. Receptor glycoproteins (lo-20 ~1) were preincubated for 4-16 h at 4°C with increasing insulin concentrations (from 0.003 nM to 3 PM) in a buffer containing 30 mM HEPES and 100 mM MgC12, pH 7.4 (final volume 70 ~1). Then receptors were incubated with 50 PM ATP for 10 min to allow autophosphorylation. Synthetic substrate poly(Glu,Tyr)4:1 was added in a final concentration 0.25 mglml and, after a further incubation for 30 min, the reaction was stopped by transferring samples to filter paper squares (3 x 3 cm, Whatman 3M) and soaking them in 10% trichloroacetic acid (TCA) containing 10 mM sodium pyrophosphate. Papers were washed at least five times over a 2-3 h period, air dried, and counted in a scintillation counter (Packard, 1500 Tri-Carb). Results of these experiments were presented either as picomoles of 32P transferred to the substrate per femtomole of receptor protein, or as radioactivity incorporated into the substrate as percent of the basal values (phosphorylation of the receptor without insulin added); the highest percentage obtained is called percent maximal stimulation. At least three receptor preparations from different fish were used for each experiment. Each point of the assay was done in triplicate. Presence of ATPase activity in the samples was tested by the method described in Ref. 31 to measure free 32P in the incubation mixture. Radioimmunoassay Plasma insulin was measured in fish (bonito) RIA system, according to Gutierrez et al. (14). Plasma dilution curves of the species of fish used in this study were parallel to the standard curve (15).

R1946

INSULIN

Statistical

RECEPTORS

IN FISH

SKELETAL

Analysis

All data are presented as means 2 SE. Statistical between groups of fish were tested by analysis (two-way ANOVA). Differences were considered significant at P < 0.05.

A 6007 .

differences of variance statistically

sootc ..

RESULTS

Insulin Binding to the Muscle Receptors of Different Fish Species Glycoprotein content in semipurified insulin-receptor preparations calculated per gram of initial tissue sample ranged from 64.0 -+ 10.0 pg/g in carp muscle to 185.0 t 15 kg/g in sea bream muscle. Values for trout and tilapia muscle were 77.0 t 6.0 and 137 t 37 pg/g, respectively (nine specimens for each species). Insulin bound to the muscle receptors of all fish species studied. This process is time and temperature dependent, with optimal binding reaching equilibrium conditions at 4 h and remaining equilibrated for up to 16 h of incubation at 4°C. Displacement of labeled insulin by mammalian IGF-I was observed only at very high concentrations of this peptide [in carp, Michaelis constant (&) 1.19 t 0.02 nM for IGF-I and 0.29 t 0.02 nM for insulin], which implied that the binding of insulin was specific. Receptor number (binding capacity, RO) and affinity in skeletal muscle varied markedly among the fish species investigated. It was especially noticeable that there were higher number of receptors, higher affinity, and higher specific binding of insulin to carp muscle receptors, the latter parameter being almost twofold higher than in other three species of fish (Table 1, Fig. LA). The concentration of cold insulin that displaced 50% of the tracer (EC& was 0.35 t 0.1 nM for trout, 0.89 t 0.13 nM for sea bream, 0.32 t 0.07 nM for tilapia, and 0.29 t 0.03 nM for carp. The most striking difference was observed between carp and trout (Fig. 2). These results were again confirmed in a second experiment on carp and trout maintained under identical conditions and fed the same diet (Fig. 3). Although higher than in trout, binding capacity of carp muscle receptors is much lower than that of mammalian muscle tissue. When a rat muscle sample was processed exactly like the piscine sample, we found the binding capacity of semipurified insulin receptor to Table 1. Characteristics of insulin binding sites in receptor preparations from muscle of several fish species Trout

Ro, fmol/mg Kd,

nM

%B,,/lO

82 + 23* 0.38 2 0.02* J-Lg 1.15 + 0.03*

Values are means + experiments done with no. (binding capacity) wheat germ agglutinin Bsp, % specific binding/l0 differ across rows denote at P < 0.05.

MUSCLE

Sea Bream

Tilapia

carp

175+33*t 0.95 + 0.12t 1.30 + O.lO*

214 2 20t 0.38 + 0.12” 3.15 f 0.60”

440 f 47$ 0.28 + 0.02* 7.22 + 0.90-F

SE. Results are representative of at least 3 different receptor purifications. Ro, receptor fmol/mg glycoprotein eluted from agarosecolumn; Kd, affinity (dissociation constant); kg glycoprotein. Superscript symbols that values significantly different from each other

s 4 E =

................... ................... ................... ...................

400'

................... ................... ................... ................... ................... ................... ................... ................... ................... ...................

. T

300E

SB

T

................... ................... ................... ................... .................. ...................

C

0

K

a

b

h

I

.................. ................... ................... ................... ................... ................... ................... ................... ................... ...................

200:

100' . .

B

Trout Seabream Tilapia

Carp

2501

b

Q 3 .IE

a

z

a 1

.................. ................. .................. ................. .................. ................. .................. ................. 1 ..................

1

a

iF 150-

100

Trout

Seabream Tilapia

Carp

Fig. 1. A: specific binding of insulin (Bsp, inset); binding capacity of receptors, Ro; B: maximal stimulation of tyrosine kinase activity presented as %basal activity in 4 species of fish. Inset: trout (T) seabream (SB), tilapia (T), and carp (C). Here and in Figs. 2-5 results are means 2 SE of 3 experiments, each done in duplicate. Lower case letters over columns (a-c) denote values significantly different from each other at P < 0.05.

be twofold higher in rat than in carp muscle (980 t 65 fmol/mg glycoprotein compared with 410 t 42 fmol/mg, respectively, five specimens in each group). Tyrosine Kinase in Muscle Insulin Receptors of Different Fish Species Insulin receptors semipurified from skeletal muscle of all four fish phosphorylated artificial exogenous substrate poly( Glu,Tyr)4: 1. Tyrosine kinase activity was stimulated by insulin in a dose-dependent manner (Fig. 4). Receptors incubated in the absence of insulin disslaved similar basal rates of nhosnhotransferase activitv

INSULIN

l

20

IN FISH

r

SKELETAL

R1947

MUSCLE

r

h

Fig. and sent was ratio.

RECEPTORS

0 Trout l Carp

0 Carp

2. Insulin binding to trout (0) presented in mean from 7 different done in duplicate. B,

semipurified muscle receptors of carp (0) Scatchard coordinates. Each point repreexperiments in which each measurement insulin bound; B/F, bound-to-free insulin

(3.4 t 0.4 pm01 32P transferred/fmol receptor protein for trout; 2.1 t 0.7 pmol/fmol for gilthead sea bream; 5.1 t 1.2 pmol/fmol for tilapia, and 3.5 t 0.7 pmol/fmol for carp, n = 4). Receptors incubated with increasing concentrations of insulin reached a higher level of

........... .......... ........... .......... .......................... ......................... ........... .......................... ......................... l---l iI

800:

21 0

3

7ooj

b

.......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... .......................... ..........................

C ........... .......... ........... .......... ........... .......... ........... .......... :.:.:.:.:.:;.:.:.:.: .......... ........... .......... ...........

.......... a ........... ........... .......... ........... a .......... ........... .......... ........... .......... LL T C T C Control I Fasted

C

.......................... ......................... ............ .............. ......................... .............................................................................. t.......................... II ........................ .............. ... ... ..................*............

I

Trout Carp Control Fig. 3. Specific binding must zle insulin receptors

' 1 ' rllln' 10.0

' ' ' ,,,uJ 100.00

Insulin (M/l)

010

600:

' ' ' r11aa' ' ' "1111' 0.1 1.0

Trout Carp Fasted

of insulin (in set) and binding capacity (Ro) of starved and fed trout and carp.

of

Fig. 4. Insulin effect on tyrosine kinase activity in trout and carp muscle receptors. Tyrosine kinase activity is presented either as %ba.sal activity found in abscence of insulin (A) or as pmol P transfered/fmol receptor protein (B).

phosphotransferase activity and, consequently, higher percentage of stimulation compared with basal activity (Fig. 4A). Apparent differences were found between the specieswhen receptors were incubated with a concentration of insulin (60 nM) sufficient for maximal stimulation of 32Ptransfer (Fig. 1B). Although the stimulation profiles were similar, the major differences in activity were found again between carp and trout. Carp and trout receptor preparations were tested for the presence of free phosphorus to assess whether differences in tyrosine kinase activity were due to adenosinetriphosphatase (ATPase) activity. Little ATPase activity was observed. Values of free phosphorus in carp and trout samples did not differ significantly from those in blank tubes. Values were 1.00 t 0.16% of total counts for blank; 1.53 t 0.15% for carp muscle, and 1.58 t 0.17%

R1948

INSULIN

RECEPTORS

IN FISH

for trout muscle (72 = 3). Rat samples presented even higher ATPase activity (2.50 t 0.20%). Trout receptors reached a maximal phosphotransferase activity of 4.9 t 0.2 pmol/fmol of binding capacity, whereas activated carp receptors reached 7.0 t 0.9 pmol/fmol. The maximal percentages of phosphorylation obtained in fish species ranged from 150 (trout) to 210% (carp), which is much lower than maximal stimulation of 513 t 36% found by us in the rat muscle gastrocnemius. Effect of Food Deprivation From Carp and Trout

on Muscle

Insulin

Receptors

Fifteen days of starvation significantly decreased plasma insulin concentration in trout (from 9.6 t 0.7 to 5 5 + 0.5 rig/ml) but not in carp (9.1 t 0.5 and 9.1 t 0:45&g/ml in fed and starved groups, respectively; 6 fish/group). Notwithstanding systemic levels of insulin, receptorbinding capacity and specific binding in food-deprived fish of both species decreased significantly to 50% of the respective control values (Fig. 3). Even so, parameters of insulin binding in carp muscles remained higher than those in trout. In both carp and trout starved for 15 days, insulin caused a dose-dependent stimulation of tyrosine kinase in the muscle receptors (data not shown). Although average values of maximal stimulation were higher in food-deprived groups of fish, a significant difference was found only in trout receptors (increase from 150 t 8 to 175 t 8%) (Fig. 5). DISCUSSION

Some fish, including salmonids, have a low ability to regulate the hyperglycemia that develops as a consequence of either glucose injection, oral glucose load, or 250-

b 200.I5 z Y E z * O 150-

ioo-

a I

b I , .................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... b.................... *

Trout Carp Control

l..................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... I.. ................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... ...................... ..................... , ..................... ..................... ..................... ..................... , ..................... ..................... ...................... .....................

Trout Carp Fasted

kinase in muscle receptors of Fig. 5. Maximal stimulation of tyrosine starved and fed trout and carp. Data are presented as %basal activity.

SKELETAL

MUSCLE

feeding high-carbohydrate diets (see Ref. 8 for review). Such glucose intolerance may be related to the nutritional preferences of the particular fish species, because Furuichi and Yone (12) have demonstrated that glucose intolerance is more pronounced in carnivorous species, whose natural food contains lower levels of carbohydrates than the food of omnivorous species. There are only three reports in the literature focused on either comparison of fish insulin receptors in agnathan with receptors in two carnivorous teleosts (22) or on comparison of binding affinity and capacity of insulin receptors in salmonid fish fed different diets (l), or deprived of food for different periods of time (18). The present experiments are the first to compare binding patterns and tyrosine kinase activity of piscine insulin receptor in skeletal muscle of several teleost fishes with different feeding preferences (carnivorous vs. omnivorous). In our study on fish, we employed a mammalian tracer and unlabeled insulin, whereas our RIA system included only homologous piscine components. Justification for doing this has been given by us and others in earlier publications (17,22,26,27). The muscle preparations from all fish species tested bound insulin. However, receptor characteristics, such as the quantity of insulin bound specific and the binding capacity, were apparently lower than those described in the mammalian literature (20). These results are in agreement with our earlier data on rainbow trout skeletal muscle (13) and with the data on skeletal and heart muscles of rat, chicken, pink salmon, and lamprey (22). Binding capacity (receptor number) per milligram of glycoprotein elu ted from rat gastrocnemius muscle Wi3 .s twofold higher than that obtained for carp skeletal muscle. James et al. (20) also have reported 300% maximal stimulation of tyrosine kinase activity in muscle insulin receptors of rat ‘, wherea s Balage et al. (3) found 500-600% and Azhar et al. (2) up to l,OOO%. Using our experimental procedure, we confirm these data, having obtained 450 -550% stimulation in rat compared with 150-200% in fish insulin receptor. When we adapted a technique of receptor purification (20) to stud ies on fish, we found that solubilization of homogenate with 2% instead of 1% of Triton X-100 increased the yield of receptor protein without affecting either specific binding of insulin or tyrosine kinase activity. The same holds true for rat skeletal muscle. Among teleost fish under investigation, omnivorous species (carp) appeared to have more numerous insulin receptors, with higher tyrosine kinase activity, than do carnivorous species (trout, sea bream). Species such as T. mossambica, which fed mostly, but not exclusively, on plants, resembled carp more closely than trout with respect to insulin receptor characteristics. Maximal differences were found between strictly omnivorous carp vs. carnivorous trout. Interestingly, this difference was retained even when fish of both species were fed an identical commercial diet. Thus characteristics of insu. lin receptors in the skeletal muscle of omnivorou s carp and carnivorous trout favor utilization of higher level of carbohydrates by carp more than by trout.

INSULIN

RECEPTORS

IN FISH

Ablett et al. (1) reported an increase in insulinreceptor concentration in the liver, but not in skeletal muscles, of trout fed a high-carbohydrate diet. In our earlier study, an increase in the binding capacity of muscle receptors was found 3 h after feeding trout with a high-carbohydrate diet (13). Thus we may speculate that some adaptation in insulin-receptor numbers can be achieved depending on nature of the diet. After a 72-h fast, an increase in the binding capacity of skeletal muscle insulin receptors occurred in the rat (3). This agrees with a finding that insulin receptor mRNA level in rat muscle tissue increases after a 40-h fast (21). In chickens, the increase in binding capacity after a 48-h fast affects only the liver but not brain receptors (31). Carp and trout in the present study responded to 15 days of food deprivation by a twofold decrease in specific binding and number of muscle insulin receptors. Similar data concerning insulin receptors in the heart muscle of upstream migrating, naturally fasting lamprey were reported by Leibush and Bondareva (23). Thus different animals appear to use different strategies for regulation of insulin binding during fasting periods. Specific binding of insulin in mammalian tissues may depend on a variety of conditions other than dietary composition or food deprivation. Decrease in insulin binding to the receptors in skeletal muscle is a wellinvestigated phenomenon in obese animals and humans (7) and in pat ients with NIDDM (l&25). Insulin receptors of mammals are downregulated by the systemic levels of insulin (3). Relationships between plasma insulin levels and receptor number in fish are more complicated, because specific binding of insulin in fish tissues may be regulated by plasma insulin either up or down, or remain unchanged (13, 18, 23, 28). In the present study, we have found that, while in starving trout a decrease in insulin specific binding and binding capacity are coincident with hypoinsulinemia, in carp the same phenomenon was not accompanied by any changes in plasma insulin concentration. Similar absence of hypoinsulinemia after 16 days of starvation was reported previously (4). Thus the decrease in number of insulin receptors in carp muscle may be triggered by modulators other than the circulating levels of the hormone. Stuart (32) was the first to analyze the autophosphorylation capacity of piscine (skate) insulin receptor in liver. Recently, Leibush et al. (24) studied autophosphorylation of semipurified insulin receptors from the lamprey liver. Both groups of researchers found that phosphorylation dynamics of piscine insulin receptors are similar to those of mammals (rat). As far as we know, the only study on phosphorylation of exogenous substrate by insulin receptor in fish has been done by us on carp ovaries (16). Maximal stimulation of tyrosine kinase (-200%) (16) was identical to that described in the present study in carp muscle. In both fasted carp and trout, tyrosine kinase activity was increased, although only in trout was this increase significant. In contrast, a decrease in tyrosine kinase activity has been found in insulin receptors isolated from liver but not muscle from the fasted rat. It was

SKELETAL

R1949

MUSCLE

assumed that the increase in receptor numbers found in both liver and muscle might compensate for a decrease in hepatic tyrosine kinase activity (3). Results obtained in this study imply that insulin receptors semipurified from skeletal muscles of omnivorous fish have higher affinity, higher tyrosine kinase activity, and are more numerous than in carnivorous fish. This general feature, which resembles insulinreceptor properties of endothermic vertebrates, leads to an improved tolerance to higher quantities of carbohydrates in the diet. We thank the Piscifactoria de Baga, Departament de Medi Natural de la Generalitat de Catalunya, and especially Antonino Clemente, as well as Barcelona Zoological Park, for providing the trout, carp, and tilapia; and Department of Zoology, IJniversity of Barcelona, for donating the sea bream. Critical reading of the manuscript by Dr. Antonio Zorzano and Dr. Aubrey Gorbman is highly appreciated. The English version has been corrected by Robin Rycroft of the Language Advisory Service of the University of Barcelona. We acknowledge the gift of human recombinant IGF-I by Ciba-Geigy (Basel, Switzerland). This work was supported by the grants from Direction General de Ciencia y Tecnologia (PB 91-0471) to J. Gutierrez; the US National Science Foundation (DCB 8915935) to E. M. Plisetskaya; the North Atlantic Treaty Organization (52-0.5/RG 921175) to J. Gutierrez and E. M. Pisetskaya; and by grants from Formation de Personal Investigador, Ministerio de Education y Ciencia and Comissio Interdepartamental de Recerca i Tecnologia, Generalitat de Catalunya to M. Parrizas. This study was reported in part at 13th Intl. Congr. of Comparative Endocrinology, Toronto, Canada, 1993 (Parrizas et al., 1993). Address for reprint requests: J. Gutierrez, Departament de Fisiologia Animal, Facultat de Biologia, Universitat de Barcelona, Barcelona 08028, Spain. Received

9 September

1993; accepted

in final

form

7 December

1993.

REFERENCES 1. Ablett, R. F., M. J. Taylor, and D. P. Selivonchick. The effect of high-protein and high-carbohydrate diets on [ 1251]iodoinsulin binding in skeletal muscle plasma membranes and isolated hepatocytes of rainbow trout S&no gairdneri. Br. J. Nutr. 50: 129-139, 1983. 2. Azhar, S., J. C. Butte, R. F. Santos, C. E. Mondon, andG. M. Reaven. Characterization of insulin receptor kinase activity and autophosphorylation in different skeletal muscle types. Am. J. PhysioZ. 260 (EndocrinoZ. Metab. 23): El-E7, 1991. M., J. Grizard, C. Sornet, J. Simon, D. Dardevet, 3. Balage, and M. Manin. Insulin binding and receptor tyrosine kinase activity in rat liver and skeletal muscle: effect of starvation. MetaboZism 39: 366-373, 1990. 4. Blasco, J., J. Fernandez, and J. Gutierrez. Fasting and refeeding in carp Cyprinus carpio: the mobilization of reserves, plasma metabolites and hormone variations. J. Comp. PhysioZ. B Biochem. Syst. Environ. Physiol. 162: 539-546, 1992. 5. Bogardus, C. Does insulin resistance primarily affect skeletal muscle? Diabetes Metab. Rev. 5: 527-528, 1989. M. M. A rapid and sensitive method for the quantita6. Bradford, tion of microgram quantities of protein utilizing the principle of protein-dye binding. AnaZ. Biochem. 72: 248-254,1976. 7. Caro, J. F., M. K. Sinha, S. M. Raju, 0. Ittoop, W. J. Pories, E. G. Flickinger, D. Meelheim, and G. L. Dohm. Insulin receptor kinase in human skeletal muscle from obese subjects with and without noninsulin dependent diabetes. J. CZin. Invest. 79: 1330-1337,1987. 8. Cowey, C. B., and M. J. Walton. Intermediary metabolism. In: Fish Nutrition, edited by J. E. Halver. New York: Academic, 1989, p. 260-329. 9. Czech, M. P., and J. Massague. Subunit structure and dynamics of the insulin receptor. Federation Proc. 41: 2719-2723, 1985. 10. Freidenberg, G. R., H. H. Klein, R. Cordera, and J. M. Olefsky. Insulin receptor kinase activity in rat liver: regulation

R1950

11.

12

13

14

15

16

17

18.

19.

20.

21.

INSULIN

RECEPTORS

IN FISH

by fasting and high-carbohydrate feeding. J. Biol. Chem. 260: 12444-12453,1985. Freidenberg, G. R., R. R. Henry, H. H. Klein, D. R. Reichart, and J. M. Olefsky. Decreased kinase activity of insulin receptors fromadipocytes of non-insulin-dependent diabetic subjects. J. CZin. Invest. 79: 240-250, 1987. Furuichi, M., and Y. Yone. Changes of blood sugar and plasma insulin levels of fishes in glucose tolerance test. B&Z. Jpn. Sot. Sci. Fish. 47: 761-764, 1981. Gutierrez, J., T. Asgard, E. Fabbri, and E. M. Plisetskaya. Insulin-receptor binding in skeletal muscle of trout. Fish Physiol. Biochem. 9: 351-360,199l. Gutierrez, J., M. Carrillo, S. Zanuy, and J. Planas. Daily rhytms of insulin and glucose levels in the plasma of sea bass Dicentrarchus Zabrax after experimental feeding. Gen. Comp. EndocrinoZ. 55: 393-397,1984. Gutierrez, J., J. Fernandez, J. Blasco, J. M. Gesse, and J. Planas. Plasma glucagon levels in different species of fish. Gen. Comp. EndocrinoZ. 63: 328-333,1986. Gutierrez, J., M. Parrizas, N. M. Carneiro, J. L. Maestro, M. A. Maestro, and J. Planas. Insulin and IGF-I receptors and tyrosine kinase activity in carp ovaries: changes with reproductive cycle. Fish Physiol. Biochem. 11: 247-25, 1993. Gutierrez, J., and E. M. Plisetskaya. Insulin binding to liver plasma membranes of coho salmon during smoltification. Gen. Comp. EndocrinoZ. 82: 446-475,199l. Gutierrez, J., and E. M. Plisetskaya. Insulin binding to liver plasma membranes in salmonids with modified plasma insulin levels. Can. J. ZooZ. 69: 2745-2750, 1991. Haring, H. U. The insulin receptor: signalling mechanism and contribution to the pathogenesis of insulin resistance. DiabetoZogia 34: 848-861, 1992. James, D. E., A. Zorzano, M. Boni-Schnetzler, R. A. Nemenoff, A. Powers, P. F. Pilch, and N. B. Ruderman. Intrinsic differences of insulin receptor kinase activity in red and white muscle. J. BioZ. Chem. 261: 14939-14944, 1986. Knott, R. M., P. Trayhurn, and J. E. Hesketh. Changes in insulin receptor mRNA levels in skeletal muscle and brown adipose tissue of weaning rats during fasting and refeeding. Br. J. Nutr. 68: 583-592,1992.

SKELETAL

22.

23.

24.

25.

26.

27.

28

.

29. 3. . 31 .

32. 88.

MUSCLE

Leibush, B. N. Insulin-Receptors Interactions in EvoZution of Vertebrates (PhD thesis). Leningrad, USSR: Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences, 1989 (In Russian). Leibush, B. N., and V. M. Bondareva. Insulin receptors in the river lamprey (Lampetra fluviatilis) during prepawning natural fasting. Zh. EvoZ. Biokhim. FizioZ. 23: 193-198, 1987. Leibush, B. N., Y. Shchepkina, and M. Pertseva. The insulin receptor down regulation and autophosphorylation in isolated hepatocytes of lamprey (Abstract). Conf Eur. Comparative Endocrinologists 16th, Padova, Italy, 1992, p. 139. Le Marchand-Brustel, Y., T. Gremeaux, R. Ballotti, and E. van Obberghen. Insulin receptor tyrosine kinase is defective in skeletal muscle of insulin-resistant obese mice. Nature Lond. 3 15: 676-679,1985. Mommsen, T. P., and E. M. Plisetskaya. Insulin in fishes and agnathans: history, structure, and metabolic regulation. Rev. Aquat. Sci. 4: 225-259,199l. Muggeo, M., B. M. Ginsberg, J. Roth, D. M. Neville, Jr., P. De Meyts and C. R. Kahn. The insulin receptor in vertebrates is functionally more conserved during evolution than insulin itself. Endocrinology 104: 1393-1402,1979. Plisetskaya, E. M., E. Fabbri, T. W. Moon, J. Gutierrez, and C. Ottolenghi. Insulin binding to isolated hepatocytes of Atlantic salmon and rainbow trout. Fish Physiol. Biochem. 11: 401-109, 1993. Scatchard, G. The attractions of proteins for small molecules and ions. Ann. NYAcad. Sci. 51: 660-672,1949. Segner, H., R. Bohm, and W. Kloas. Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes. Fish Physiol. Biochem. 11: 411-420, 1993. Simon, J., R. W. Rosebrough, J. P. McMurtry, N. C. Steele, J. Roth, M. Adamo, and D. Leroith. Fasting and refeeding alter the insulin receptor tyrosine kinase in chicken liver but fail to affect brain insulin receptors. J. BioZ. Chem. 261: 1708117088,1986. Stuart, C. A. Characterization of a novel insulin receptor from stingray liver. J. BioZ. Chem. 263: 7881-7886, 1988. Yone, Y. The utilization of carbohydrates by fish. Proc. 7th Japan-Soviet Symp. on Aquaculture, Tokyo, Japan, 1979, p. 39-48.