Insulin and insulin-like growth factor-I (IGF-I) binding in fish red muscle: regulation by high insulin levels

Regulatory Peptides 68 (1997) 181–187

Insulin and insulin-like growth factor-I (IGF-I) binding in fish red muscle: regulation by high insulin levels a a ˜ a , *, T.W. Moon b , C. Castejon ´ a , J. Gutierrez ´ N. Banos , I. Navarro a

Departament de Fisiologia, Facultat de Biologia. Universitat de Barcelona. Av. Diagonal 645, 08028 Barcelona, Spain b Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K1 N 6 N5 Received 17 September 1996; revised 1 December 1996; accepted 3 December 1996

Abstract Insulin and IGF-I binding to semi-purified red muscle receptors was characterized in brown trout, Salmo trutta and the common carp, Cyprinus carpio. The yield of glycoprotein obtained after semipurification of receptors with WGA-agarose affinity chromatography in mg g 21 initial tissue was 210.6621 mg g 21 in trout and 108.562.5 mg g 21 in carp. IGF-I specific binding (4.7260.64% / 10 mg glycoprotein) was 4–5-times higher than insulin binding (1.0460.12% / 10 mg glycoprotein) in trout red muscle. This difference in binding was due to a higher number and a greater affinity of the IGF-I (Kd , 0.2160.03 nM) compared with the insulin (Kd , 0.6760.06 nM) receptors in this tissue. Carp red muscle IGF-I binding (9.1460.55% / 10 mg glycoprotein) surpassed insulin binding (2.5960.094% / 10 mg glycoprotein) mainly because of a greater affinity of the IGF-I (Kd , 0.09260.027 nM) compared with the insulin (Kd , 0.151560.0285 nM) receptor. IGF-I and insulin binding in carp red muscle were higher than in trout, as a consequence of a higher affinity of carp red muscle receptors. Arginine injection provoked acute hyperinsulinemia in both trout (23.361.01 ng ml 21 ) and carp (24.361.34 ng ml 21 ). Specific binding of insulin and IGF-I to the red muscle decreased 4 h after injection. In trout, a decrease of insulin and IGF-I binding of 47.0% and 63.3%, respectively was observed compared with controls; in carp, these values were 44.0% and 45.0%. The number of insulin and IGF-I receptors decreased (42–55%) but affinities did not change suggesting that receptor down-regulation is a consequence of high insulin levels.  1997 Elsevier Science B.V. All rights reserved

1. Introduction Insulin and insulin growth factor I (IGF-I) are close structurally related peptides belonging to the insulin superfamily, that exert their biological functions binding to specific cell surface receptors. Insulin binding in liver, white skeletal and heart muscles, brain or gonads has been studied in different fish species [1–8]. Much less is known about IGF-I receptors in non-mammalian species although binding of IGF-I has been reported in avian [9,10] and amphibian cells [11,12]. More recently, IGF-I binding to fish tissues has also been demonstrated [6,7,13]. The principal role of insulin in higher vertebrates is the maintenance of metabolic homeostasis by regulating the fluctuation of major metabolites [14], while the predomi*Corresponding author. 0167-0115 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0167-0115( 96 )02118-0

nate function of IGF-I is to stimulate growth and differentiation [15]. Nevertheless, insulin and IGF-I closely interact in the regulation of metabolism and growth. In mammals, IGF-I affects both uptake and metabolism of glucose [16,17] and protein synthesis in skeletal muscle [18]. The effects of insulin on growth promotion are much less potent than those of IGF-I. In avian muscle satellite cells, IGF-I is a more effective activator of glucose transport and protein synthesis than is insulin [19]. Information regarding IGF-I actions in fish is scarce and incomplete. IGF-I is hypoglycemic in fish [20] and fish insulin showed the same growth potency as IGF-I in the branchial cartilage incubation system [21]. Thus the physiological functions of insulin and IGF-I seem to have become more distinct or separate within vertebrates from fish to mammals. Insulin binding to white muscle of different fish species

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shows clear differences as a function of nutritional status and diet type. Glucose intolerance is more pronounced in carnivorous (such as salmonids) than omnivorous fish species [22] and insulin receptor number in skeletal muscle of omnivorous species are higher and related to the higher level of glucose in their diet [4]. Furthermore, rainbow trout adapted to a high carbohydrate diet, had higher plasma insulin levels and higher number of skeletal muscle insulin receptors [23]. The ability of insulin to stimulate glucose uptake varies as a function of skeletal muscle type in mammals. Fibers with a predominately red muscle composition had higher insulin binding and tyrosine kinase activities than predominately white muscles in rats [24]. In fish, red muscle is anatomically and metabolically clearly differentiated from white muscle. Although it constitutes only 5–10% of the total fish muscle mass, red muscle has a very active metabolism. It operates aerobically and works continuously during locomotory movements of the fish. Glucose consumption of red muscle increases significantly during increased aerobic swimming [25]. In contrast, white muscle is anaerobic and it is used primarily for burst swimming activities. Glucose uptake is lower and more constant in white than red muscle, but it represents a high percentage (70–80%) of the total weight of fish so ultimately its impact upon glucose kinetics may be much higher. Although the metabolism of fish red muscle has been extensively studied [25], little information is available on hormone receptors or function. The aim of this study, therefore, was to analyze insulin and IGF-I binding to semipurified receptors prepared from brown trout and common carp red muscle and to establish their regulation by high insulin levels.

temperature was 98C and experiments were conducted in the fall of the year. Common carp, Cyprinus carpio, (age 3 years, weight 266667.3 g, n 5 8, obtained from the Barcelona Zoo, Spain) were fasted as above, then injected i.p. with either 9 mmol g 21 arginine-free base or saline solution. Sampling was as for trout. Water temperature was 108C and experiments were conducted in the fall of the year.

2.3. Sampling Fish were anaesthetized in 100 ppm 3-aminobenzoic acid ethyl esther (MS222), and blood sampled by caudal puncture. Fish were killed by cranial blow and slices of lateral white and red muscles were rapidly excised and frozen in liquid nitrogen. Tissues were stored in liquid nitrogen until used for receptor purification.

2.4. Semi-purification of receptors Semi-purification of solubilized insulin receptors from red and white muscles was performed at 48C according to ´ Parrizas et al. [4]. Briefly, frozen muscle (8 g) samples were homogenized in HEPES buffer (pH 7.6) containing peptidase inhibitors. Solubilization of homogenate was achieved by adding 2% Triton X-100 (final concentration) and stirring for 1 h. Solubilized homogenates were centrifuged (150 000 g, 90 min at 48C) and the resulting supernatants were applied to affinity columns of wheatgerm agglutinin (WGA) bound to agarose. Glycoproteins were eluted from the WGA column with a HEPES buffer supplemented with 0.3 M N-acetyl-D-glucosamine (pH 7.4).

2. Materials and methods

2.1. Chemicals

2.5. Receptor-binding assays

Wheat-germ agglutinin bound to agarose was purchased from Vector (Burlingame, USA). Porcine insulin was obtained from Lilly (Indianapolis, USA). Human Tyr A14 125 I-monoiodoinsulin and human recombinant 3- 125 I-IGF-I, both with 2000 Ci mmol 21 specific activity, were purchased from Amersham (Arlington Hts., IL, USA). All other chemicals used were purchased from Sigma (St. Louis, MO, USA).

´ Binding assays were performed according to Parrizas et al. [4]. Briefly, 40–50 ml of the WGA eluate (15–30 mg glycoproteins, according to Bradford [26] was incubated for 4–16 h at 48C in HEPES buffer (pH 7.4) containing 0.1% BSA, bacitracin, increasing concentrations of cold hormone (from 0.005 nM to 5 mM) and the radiolabelled ligand as tracer (20 000 cpm). Semi-purified receptors were precipitated by addition of 0.08% bovine t-globulin and 10.4% polyethylene glycol (final concentrations), followed by centrifugation at 14 000 g for 5 min at 48C. Reproducibility of the method was assessed using an inter-assay standard (variations , 10%). Binding data were analyzed by Scatchard plots [27]. Only the high-affinity, low-capacity binding sites calculated from the curvilinear plot are reported. Each binding experiment was performed at least in triplicate, using receptor glycoproteins from different purifications.

2.2. Animals and experimental design Brown trout, Salmo trutta, (age 3 years, weight 374610.5 g, n 5 22, obtained from the fish farm Piscifactoria Baga` near Barcelona) were fasted for 18 h then injected i.p. with either 6.6 mmol g 21 arginine-free base or saline solution (pH 7.6). Fishes were sampled for plasma and tissues at the time of injection and 4 h later. Water

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2.6. Radioimmunoassay Plasma insulin levels were measured by radioimmunoassay using bonito insulin as standard and a rabbit antibonito insulin as antiserum [28].

2.7. Statistical analysis All data are presented as means6S.E.M. Significant differences between groups of fish were tested by analysis of variance (two-way ANOVA). Differences were considered significant at P , 0.05.

3. Results

3.1. Characterization of insulin and IGF-I binding in red muscle 3.1.1. Trout Glycoprotein content in semipurified receptor preparations, calculated per gram of initial tissue sample was 210.6621 mg g 21 . Insulin specific binding (Bsp), as expressed per 10 mg of glycoprotein eluted from red muscle preparations, was 1.0460.12%. The Kd value calculated from Scatchard analysis for insulin high affinity binding was 0.6760.06 nM and the number of receptors obtained was 137.6616.1 fmol mg 21 of glycoprotein eluted (Table 1). IGF-I specific binding was about 4–5-fold higher than insulin binding in this tissue (4.7260.64% / 10 mg of glycoprotein). The Kd for IGF-I binding to red muscle preparations was 0.2160.03 nM and number of receptors was 207.2644.3 fmol mg 21 of glycoprotein. Consequently, specific binding differences between hormones were due to both the presence of a higher number (about 2-fold more) and a higher affinity of the IGF-I than insulin receptors (Table 1). Insulin and IGF binding to red muscle when expressed

Table 1 Characteristics of insulin and IGF-I binding to semipurified receptor preparations from red muscle of trout and carp Trout

Carp

Insulin % Bsp / 10 mg glycoprotein R o (fmol mg 21 glycoprotein) Kd (nM)

1.0460.12 a 137.6616.1 a 0.6760.06 a

2.5960.25 b 93.5620.5 a 0.1560.028 b

IGF-I % Bsp / 10 mg glycoprotein R o (fmol mg 21 glycoprotein) Kd (nM)

4.7260.64 a 207.2644.3 a 0.2160.03 a

9.1460.55 b 160.5649.5 a 0.09260.027 a

Values are means6S.E. Bsp%: percentage of specific hormone binding. R 0 : receptor number. Kd : affinity of the receptors expressed as constant of dissociation. Superscript letters that differ across a row denote values significantly different at P , 0.05.

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as %Bsp / 100 mg of tissue were higher than that found in white muscle (red muscle: insulin, 2.1960.26%, IGF-I, 9.9561.36%; white muscle: insulin, 0.6160.03%, IGF-I, 2.0160.63%).

3.1.2. Carp The yield of glycoprotein obtained after purification of carp red muscle on WGA was 108.562.5 mg g 21 of red muscle sample. Specific binding of insulin to red muscle preparations in carp was higher than that found in trout samples: 2.2160.094% Bsp / 10 mg. Affinity was higher (0.1560.028 nM) in carp muscle, but the number of receptors (93.5620.5 fmol mg 21 ) was not significantly different between the two species (Table 1). IGF-I binding to red muscle was again significantly higher than insulin binding (9.1460.55% Bsp / 10 mg of glycoprotein). These values of IGF-I binding were higher than those of trout (Table 1). IGF-I receptor number and Kd value in carp red muscle (160.5649.5 fmol mg 21 of glycoprotein, and 0.09260.027 nM, respectively) were lower than those of trout. Consequently, the higher IGF-I specific binding found in red muscle of carp was due mainly to the higher affinity rather than a higher number of receptors (Table 1). Insulin and IGF binding to red muscle when expressed as %Bsp / 100 mg of tissue was higher than that found in white muscle (red muscle: insulin, 2.8160.27%, IGF-I, 9.9260.60%; white muscle: insulin, 1.1760.64%, IGF-I, 4.1560.84%). 3.2. Effects of arginine injection on insulin and IGF-I binding 3.2.1. Trout Plasma insulin levels increased significantly above basal values 4 h after arginine injection, increasing from 11.660.97 to 23.361.01 ng ml 21 of plasma (Fig. 1). Glycoprotein yields from red muscle of injected animals (186.5618.1 mg g 21 of tissue), however, were similar to those from control animals. The insulin specific binding to red muscle (0.5560.08% Bsp / 10 mg of glycoprotein) was decreased to 50% of control (Fig. 2A) 4 h after arginine injection. The number of insulin receptors decreased to 72.0610.3 fmol mg 21 of glycoprotein while the affinity did not change (Kd , 0.6660.07 nM) (compare with Table 1). IGF-I binding (1.7360.3%Bsp / 10 mg; Fig. 2B) and number of IGF-I receptors in injected animals (93.3621.8 fmol mg 21 of glycoprotein) decreased significantly compared with controls. Affinity did not differ significantly with respect to control values (0.2760.05 nM) (compare with Table 1). 3.2.2. Carp Plasma insulin levels increased significantly over control

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Fig. 1. Plasma insulin values (ng ml 21 ) of control and arginine injected trout (6.6 mmol g 21 fish) and carp (9 mmol g 21 fish). Different letters indicate values significantly different at P , 0.05.

values 4 h after arginine injection, (16.960.24 ng ml 21 to 24.361.34 ng ml 21 ) (Fig. 1). Glycoprotein yield of injected carp red muscle samples (89.8623.6 mg g 21 ) did not differ from that of control carp red muscle. Insulin specific binding to red muscle of injected carp (1.4560.3% Bsp / 10 mg of glycoprotein) (Fig. 3A) was lower than that of control carp (see Table 1). The affinity of carp red muscle insulin receptors did not vary after arginine injection, so differences in insulin specific binding could be related to changes in the number of receptors. Carp red muscle of injected animals bound 2-times less IGF-I than control animals (5.0860.83% Bsp / 10 mg of glycoprotein) (Fig. 3B) (compared with Table 1), but no changes in affinity were detected (Kd 0.0760.024 nM). Again, differences in specific binding could be explained in terms of changes in receptor number which decreased significantly to 9369 fmol mg 21 of glycoprotein.

4. Discussion This study characterized the binding of insulin and IGF-I to red muscle from brown trout and carp. Receptors were purified using a WGA affinity column, a procedure used routinely in our laboratory for these receptors from other tissues [4,5,13,23]. It also demonstrates that these binding characteristics are modified under conditions when insulin levels are elevated by arginine, in contrast to our previous studies on fish white muscle [23]. The advantage of fish red and white muscles are their anatomical differentiation, a situation not seen in mammals. Using mammalian muscle composed predominately of red fibers, insulin sensitivity, responsiveness and

Fig. 2. Percent of specific hormone binding (%Bsp) expressed per 10 mg of glycoprotein to semi-purified receptor preparations from red muscle of control and arginine injected trout. (A) insulin binding; (B) IGF-I binding. *Indicate values significantly different at P , 0.05.

tyrosine kinase activities are higher than in rat white muscle [24]. This incremented response to insulin affects 2-deoxyglucose uptake and glycogen synthesis [29,30], and amino acid uptake [31]. The specific binding of insulin to red muscle (when expressed as %Bsp / 100 mg of tissue) reported here for both carp and trout was higher than that found in white muscle, in agreement with the mammalian data. Furthermore, IGF-I binding is higher than that of insulin, confirming our previous studies for white muscle [5,8], but adding red muscle reported here for the first time to the list of fish tissues where IGF-I predominates over insulin binding. The affinity of the insulin and IGF-I receptors is similar in both red muscle (Table 1) and white muscle [5], consequently, the high hormone binding in red muscle is due to the higher number of insulin and IGF-I receptors in this

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Fig. 3. Percent of specific hormone binding (%Bsp) expressed per 10 mg of glycoprotein to semi-purified receptor preparations from red muscle of control and arginine injected carp. (A) insulin binding; (B) IGF-I binding. *Indicate values significantly different at P , 0.05.

type of muscle. In contrast, Zorzano et al. [32] observed that IGF-I receptor number and affinity did not differ between red and white muscle fibers in the rat. Differences in hormone binding may be related to the functional roles of the two fish tissues. Red muscle is used continuously and glucose uptake into resting and exercised trout red muscle is higher than in the white muscle which is used primarily during burst exercise [25]. During exercise glucose utilization by red muscle increases between 7 and 20-fold while white muscle rates are relatively constant. A very high binding of insulin and specially IGF-I has also been found in fish heart muscle [6] again a tissue with constant activity and one that needs a continuous input of energy.

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Differences in insulin binding between the two species of fish found in red muscle are in agreement with previous studies in fish white skeletal muscle [4] in which receptors are present with higher affinity and number in omnivorous fish, such as carp, than in carnivorous fish, such as trout. As previously argued [4], this difference reflects a greater tolerance of a high carbohydrate diet by the omnivorous than carnivorous species. In both teleosts, specific binding and affinity of semipurified IGF-I receptors were higher than those of insulin receptors, in agreement with that found in white muscle and other fish tissues [5,8]. IGF-I binding in heart and skeletal muscle predominates over insulin binding, not only in fish but in other ectothermic species such as frog and turtle [5]. This difference in hormone binding has been reported in Xenopus laevis oocytes [11,12] and carp ovaries [8]. Furthermore, IGF-I binding appears to be more specific than insulin in more phylogenetically ancient animals [13]. These findings contrast with data obtained on mammals, where IGF-I binding to muscles is 3–4-times lower than insulin binding [16,32]. This specific, high affinity IGF-I binding in fish muscles suggests some important role for IGF-I in these ectothermal vertebrates, especially considering that the growth promoting and metabolic functions of insulin and IGF-I in fish overlap more in these animals than the endothermic vertebrates [20,21]. The stimulatory effects of arginine on insulin in fish are well known [33–35]. Arginine induced hyperinsulinemia in carp and trout at 3 h (Fig. 1) and levels remained high for up to 12 h [23]. In mammals, high plasma insulin titres decrease the number of insulin receptors (down-regulation) in several tissues, including lymphocytes, hepatocytes and adipocytes [36–38]. Information in this regard within ectothermic vertebrates is scarce. Pharmacological concentrations of insulin provoked down-regulation of insulin receptors of lamprey [39] and salmonid [34] hepatocytes in vitro. Leibush and Lappova [40] described a rapid (10 min) down-regulation of insulin receptors in isolated hepatocytes from lamprey (Lampreta fluviatilis) at ambient temperature and physiological concentrations of insulin. Moon and coworkers [41] recently demonstrated a decrease in the specific binding of insulin and IGF-I to receptors of isolated trout cardiomyocytes maintained in media containing high hormone concentrations. Other pancreatic hormones also induce down-regulation, including that for glucagon binding to hepatocytes of American eel (Anguilla anguilla) and of bullhead exposed to high glucagon concentrations [42]. To our knowledge this is the first report of the regulation of insulin and IGF-I binding to red muscle receptors from any teleost, and in fact, any ectothermic vertebrate red muscle tissue. Hyperinsulinemia provoked a decrease in the specific binding and number of insulin and IGF-I receptors in red muscle, without changing affinity. The decrease in IGF-I binding presumably was produced by an

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increase in plasma IGF-I levels induced by high insulin levels [43]. Down-regulation of the insulin and IGF-I receptors in red muscle contrasts with our data in white muscle of carp and trout were high plasma insulin levels increased the number of insulin receptors [23]. This difference in regulation may reflect the greater number of receptors present in red compared with white muscles. When circulating hormone increases, red muscle decreases receptor numbers to avoid an excessive response to the hormone, since the high number of receptors correlates with high rates of glucose uptake and utilization. This could starve the other glucose-requiring tissues of the body (e.g., nervous system) of essential glucose. Given the different function of white compared with red muscle [25], these hormones may have quite different roles in these two tissues. Certainly fish cardiac muscle which is also a very active aerobic tissue with an abundance of insulin and IGF-I receptors, also demonstrates receptor down-regulation [41]. The present study characterizes the insulin and IGF-I binding to red muscle of trout and carp. The abundance and characteristics of these receptors differ from those of white muscle, but as with white muscle, IGF-I receptors predominate over insulin receptors. We also report the existence of down-regulation of insulin and IGF-I receptors by hyperinsulinemia provoked by arginine, in contrast to these receptors in fish white muscle. The properties of these receptors probably reflect the unique physiology of red muscle in fish species.

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Acknowledgments

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We are indebted to Ciba-Geigy (Basel) for the gift of ´ de human recombinant IGF-I. We thank the Piscifactorıa ` Departament de Medi Natural de la Generalitat de Baga, Catalunya, and especially Sr. Antonino Clemente, as well as L. Colom and V. Prat from Barcelona Zoological Park, for providing the fish. The English version has been corrected by Robin Rycroft of the Language Advisory Service of the University of Barcelona. This study was supported by North Atlantic Treaty Organization grant to J.G. and E.M. Plisetskaya. (5-2-0.5 / RG 921175), grants to J.G. (PB 94-0864, PTR93-0026, GRQ 94-1051 CIRIT), and CIRIT to T.W.M.

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