Insulin and IGF-I response to a glucose load in European sea bass (Dicentrarchus labrax) juveniles

Aquaculture 315 (2011) 321–326

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Insulin and IGF-I response to a glucose load in European sea bass (Dicentrarchus labrax) juveniles P. Enes a,⁎, H. Peres a, J. Sanchez-Gurmaches c, I. Navarro c, J. Gutiérrez c, A. Oliva-Teles a,b a b c

CIMAR/CIIMAR — Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, Edifício FC4, 4169-007 Porto, Portugal Departamento de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain

a r t i c l e

i n f o

Article history: Received 16 June 2010 Received in revised form 19 October 2010 Accepted 24 February 2011 Available online 5 March 2011 Keywords: European sea bass Glucose IGF-I Insulin Tolerance test

a b s t r a c t A glucose tolerance test was performed in European sea bass juveniles to evaluate the effect of a glucose load on plasma insulin and insulin-like growth factor-I (IGF-I) levels. Interaction between these hormones, plasma triacylglycerides and liver glycogen was also determined. After being fasted for 48 h, fish were intraperitoneally injected with either 1 g of glucose per kg body weight or a saline solution. Plasma glucose levels peaked at 23–25 mmol l− 1, 2–6 h after the glucose injection and fish exhibited hyperglycemia until 12 h, when plasma glucose recovered to basal levels. Plasma insulin levels did not change significantly between sampling points, but insulin level was higher in the glucose group than in the control 4–6 h and 24 h after injection. Though plasma IGF-I levels remained constant for a long time (except at time 12 h) in the glucose injected fish, at times 6–9 h, 24 h and 48 h IGF-I levels were higher in the glucose group than in the control. Glucose administration led to lower plasma triacylglyceride levels than saline solution at times 9–12 h. Although liver glycogen content was not affected by the glucose injection (except at times 4 h and 48 h), comparative to saline solution injection, there was a significant increase of liver glycogen at 6 h and 9 h. Results of this study indicate that under these experimental conditions glucose is probably not the most important stimulator of insulin release. Insulin may have contributed to the increase of IGF-I levels and to the enhancement of glucose uptake by the liver in glucose injected fish. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Fish, and particularly carnivorous species, appear to have limited capacity to use glucose for energy purposes (Hemre et al., 2002; Enes et al., 2009). In fact, although fish have the whole enzymatic machinery required for carbohydrate utilization (Cowey and Walton, 1989; Dabrowski and Guderley, 2002; Enes et al., 2009) most species have an impaired glucose tolerance and prolonged hyperglycemia is commonly observed after an acute glucose load (Moon, 2001). Glucose tolerance is however species dependent, with herbivorous and omnivorous fish showing significantly shorter periods of hyperglycemia than carnivorous species (Furuichi and Yone, 1981; Lin et al., 1995; Peres et al., 1999; Legate et al., 2001). Nevertheless, compared to mammals even the most glucose tolerant fish require longer time for clearing a glucose load, as expected, as fish are poikilothermic and their metabolic rate is normally lower than that of mammals (Moon, 2001). There are a number of possible explanations for the lower glucose tolerance in fish, including those related with hormonal response to

⁎ Corresponding author. Tel.: +351 22 040 2736; fax: +351 22 040 2709. E-mail address: [email protected] (P. Enes). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.02.042

glucose loads. It is well known that glucose can stimulate insulin secretion in fish and that, as in mammals, insulin stimulates glucose uptake by peripheral tissues such as liver and skeletal muscle, and promotes glyconeogenesis and lipogenesis in an attempt to maintain glucose homeostasis (Mommsen and Plisetskaya, 1991). Depending on nutritional condition and blood collection point the concentrations of plasma insulin in systemic circulation of fish range from 1 to 30 ng ml− 1 (Mommsen and Plisetskaya, 1991; Navarro and Gutiérrez, 1995). According to Gutiérrez et al. (1984, 1987) insulin levels in European sea bass ranged between 9 and 12 ng ml− 1, being among the highest to be found in teleosts. In common carp (Cyprinus carpio), red sea bream (Chrysophrys major), yellowtail (Seriola quinqueradiata), Chinook salmon (Oncorhynchus tshawytscha) and hybrid tilapia (Oreochromis niloticus × O. aureus), oral administration of a high glucose dose (1.7 g kg body weight (BW)− 1) results in an increase in plasma insulin levels, with maximal values being attained approximately 1 h after glucose administration in tilapia and at 2 h for the other species (Furuichi and Yone, 1981; Mazur et al., 1992; Lin et al., 1995). Some authors have argued that this delayed and lower insulin secretion in comparison to mammals after a glucose load could be related to the higher sensitivity to glucose of pancreatic somatostatin-secreting cells than the insulin-secreting cells; indeed, somatostatin is a hormone known to decrease insulin secretion in fish

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(Sheridan et al., 1987; Harmon et al., 1991; Eilertson and Sheridan, 1993). Moreover, although insulin release is stimulated by glucose, amino acids are considered to be a more potent secretagogue of insulin in fish (Mommsen and Plisetskaya, 1991). As far as we know insulin response to a glucose load has not been analyzed in European sea bass, a species of high interest in aquaculture that has the peculiarity of presenting high plasma insulin levels (Gutiérrez et al., 1984, 1987). Insulin-like growth factor-I (IGF-I) is structurally and functionally related to insulin and its biological action in fish includes growth regulation, stimulation of tissue differentiation, reproduction and osmoregulation (Duan, 1998). Furthermore, IGF-I was more effective than insulin in stimulating glucose and amino acid uptake in rainbow trout (Oncorhynchus mykiss) muscle cells suggesting that this hormone is also involved in the regulation of carbohydrate metabolism and could even have more relevance than insulin itself (Castillo et al., 2004). However, available data suggest that dietary carbohydrate has no effect on plasma IGF-I levels in coho salmon (Oncorhynchus kisutch) and barramundi (Lates calcarifer) (Nankervis et al., 2000; Higgs et al., 2009). Though similar to mammals, piscine circulating IGF-I levels in brown trout (Salmo trutta) seem to be regulated by mechanisms in which insulin is partially involved (Baños et al., 1999). To our knowledge the effect of a glucose load on plasma IGF-I levels was not yet studied in fish. Insulin and IGF-I-specific receptors have been detected in several fish species and in different tissues, including liver, skeletal muscle and adipose tissue (Gutiérrez et al., 1991a; Plisetskaya et al., 1993; Párrizas et al., 1995; Baños et al., 1997; Planas et al., 2000). Opposite to mammals, it was observed in all fish tissues examined to date that IGF-I receptors were more abundant than insulin receptors. Moreover, IGF-I receptors have also higher affinity for IGF-I than insulin receptors for insulin suggesting that IGF-I may have an important role in the metabolism of lower vertebrates (Párrizas et al., 1995; Baños et al., 1997; Planas et al., 2000). The number of insulin and IGF-I receptors is regulated by the nutritional state and diet composition. Thus, in rainbow trout and European sea bass it was observed that high carbohydrate diets showed an up-regulation of insulin and IGF-I receptor number and binding in the skeletal muscle compared to fish fed with low carbohydrate diets (Gutiérrez et al., 1991a; Baños et al., 1998). A glucose tolerance test (GTT) is a rapid method widely used to study the ability of fish to utilize carbohydrates (Palmer and Ryman, 1972; Furuichi and Yone, 1981; Wilson and Poe, 1987; Harmon et al., 1991; Lin et al., 1995; Garcia-Riera and Hemre, 1996; Peres et al., 1999; Legate et al., 2001; Booth et al., 2006) as glucose is the main monosaccharide resulting from the digestion and absorption of dietary carbohydrate. Data from a previous GTT performed in European sea bass by Peres et al. (1999) pointed to a need of hormonal response studies for a better understanding of glucose metabolism in this species. Therefore, the present study aims at evaluating the effect of a glucose load on plasma insulin and IGF-I levels in order to gain further knowledge on the contribution of each hormone to glucose regulation in European sea bass, a species of great interest in aquaculture. The knowledge on the hormonal control of carbohydrate metabolism in this species would help to improve the formulation and efficiency of new diets for the production of this species. 2. Materials and methods 2.1. Experimental animals and procedures A glucose tolerance test was performed in European sea bass (Dicentrarchus labrax) juveniles at the Marine Zoology Station, Porto University (Portugal). Fish were obtained from a commercial fish farm (Viveiros Vila Nova, Vila Nova de Mil Fontes, Portugal) and after

transportation to the experimental facilities animals were submitted to a quarantine period of 2 weeks during which the fish were fed on a commercial diet. The experiment was performed in a thermoregulated water recirculation system equipped with a battery of fiberglass cylindrical tanks of 100 l capacity. During the trial, tanks were supplied with a continuous flow of filtered seawater (6.0 l min− 1), temperature averaged 22 ± 0.5 °C, salinity averaged 34 ± 1.0 g l− 1 and dissolved oxygen was kept near saturation (7.0 mg l− 1). Fifteen tanks were each one stocked with 10 European sea bass weighing 60–80 g. Before the trial, fish were allowed to adapt to the experimental conditions for 4 weeks, and during that period were fed by hand, once daily to apparent visual satiety with a commercial diet (crude protein of 53%, crude lipid of 16%). Then, fish were fasted for 48 h, slightly anaesthetized with ethylene glycol monophenyl ether (0.3 ml l− 1), immediately weighed and injected intraperitoneally with 1 g of glucose per kg BW− 1. A glucose solution of 100 mg ml− 1 concentration was used for that purpose and control fish were injected with a 0.9% saline solution. Equal volumes (10 ml kg BW− 1) were injected in test and control fish. In order to minimize stress due to sampling, two different tanks were used for each sampling time, one for glucose injection and the other for saline solution injection and only one tank was used for time 0 h. Blood and liver samples were collected just before injections (time 0 h) and then at 2, 4, 6, 9, 12, 24 and 48 h after the glucose or saline solution injections. Blood was collected from the caudal vein with a heparinized syringe and immediately centrifuged. Plasma aliquots were established for glucose, triacylglycerides, insulin and IGF-I analysis and frozen at −80 °C. After collection, livers were weighed for determination of hepatosomatic indices and frozen at −80 °C until glycogen content analysis. 2.2. Biochemical analysis 2.2.1. Plasma glucose, plasma triacylglycerides and liver glycogen Plasma glucose and triacylglycerides were determined using an enzymatic-colorimetric method (Spinreact, Girona, Spain; glucose Kit, cod. 1001191 and triacylglycerides Kit, cod. 1001312). Hepatic glycogen content was determined as described by Plummer (1987). 2.3. Radioimmunoassays 2.3.1. Plasma insulin Plasma insulin was measured by radioimmunoassay (RIA) using bonito insulin as the standard and rabbit anti-bonito insulin as antiserum, according to the method described by Gutiérrez et al. (1984). 2.3.2. Plasma IGF-I For quantification IGF-I plasma was previously submitted to an acid–ethanol extraction to release IGFs from binding proteins. Thus, 40 μl plasma was treated with 160 μl acid–ethanol (12.5% 2 N HCl, 87.5% ethanol) and incubated at room temperature for 30 min. Thereafter the acid–ethanol mixture was neutralized with 80 μl 0.855 M Tris base and centrifuged at 10,000 g for 10 min at 4 °C. Supernatant was collected and stored at −80 °C until analysis. Plasma IGF-I levels were measured using a generic fish IGF-I RIA Kit (GroPep, Adelaide, Australia). The assay was based on the use of recombinant red sea bream (Pagrus auratus) IGF-I as tracer and standard and anti-barramundi IGF-I serum (final assay dilution 1:25,000) as the primary antibody. A goat anti-rabbit IgG (1:20) (Millipore, Massachusetts, USA) was used as the precipitating antibody. The effectiveness of this assay for measurement of IGF-I in European sea bass was confirmed by the parallelism of a dilution/ displacement curve of European sea bass plasma to that of the recombinant red sea bream IGF-I standard provided with the kit (Fig. 1). Sensitivity and midrange of the assay were 0.05 and 0.7– 0.8 ng ml− 1, respectively.

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100

%B/Bo

80 60 40 20 0 0

50

100

150

200

250

300

IGF-I (ng ml-1) Fig. 1. Validation of the radioimmunoassay method for European sea bass plasma IGF-I samples. The parallelism between the standard curve (white squares) and plasma serial dilutions (black squares) confirms the effectiveness of the method for this species.

2.4. Statistical analysis Statistical analysis of data was done by two-way ANOVA using the SPSS 17.0 for Windows software package. The probability level of 0.05 was used for rejection of the null hypothesis. When the effect of sampling time and/or type of injection was significant, the two factors were analyzed separately by one-way ANOVA. Significant differences among groups were determined by the Tukey multiple range test. 3. Results The glucose injection resulted in a significantly elevated plasma glucose concentration with maximum levels being attained between 2 and 6 h after injection (Fig. 2A). Thereafter, plasma glucose levels decreased to the basal value, which was attained within 12 h after the

Glucose (mmol l-1)

A

GLU 30

c*

c*

c*

25

SS b*

20 15 10

a

a

a

a

5

glucose load. No differences in plasma glucose levels between sampling times were noticed at 12, 24 or 48 h after the glucose load. The saline solution injection did not affect plasma glucose levels over the course of the sampling period. Plasma glucose levels were identical between the glucose and the saline solution injected fish at times 12, 24 and 48 h. No differences in plasma insulin levels during the sampling period were observed within the glucose or saline solution injected groups (Fig. 2B; Table 1). However, significant differences were noticed at times 4, 6 and 24 h between fish injected with the glucose and the saline solution, with higher values being attained in the glucose injected fish. Though statistically different (Table 1) plasma IGF-I levels were constant for a long time, except at time 12 h, which presented an unexpected low value in the glucose injected group (Fig. 2C). Significantly higher plasma IGF-I values were however noticed at times 6, 9, 24 and 48 h in the glucose injected fish comparative to the control fish. In the glucose injected fish, plasma triacylglyceride levels were constant and only at time 48 h the levels presented a lower value than at times 0 and 4 h (Fig. 2D). The saline solution injection did not affect plasma triacylglyceride levels during the sampling period. Overall, glucose injected fish presented lower plasma triacylglyceride levels than the saline solution injected fish, with values being statistically significant at times 9 and 12 h. Hepatic somatic index (HSI) was not affected by the injection treatment (Table 1); however differences among sampling times were noticed and overall reflected the variation of glycogen values (Fig. 3A). Liver glycogen content after the glucose injection was lower than the basal level at times 4 and 48 h and after a saline solution injection at times 2 h to 9 h and 48 h (Fig. 3B). The average liver glycogen content was also affected by the injection treatment, being significantly higher in glucose injected fish than in saline solution injected fish (Table 1). Significantly higher glycogen values were noticed at times 6 and 9 h in the glucose injected fish comparative to the control fish (Fig. 3B). A significant (p b 0.05) and negative

B Insulin (ng ml-1)

120

323

0

20

*

*

4

6

16 14 12 10

0

2

4

6

9

12

24

48

0

2

Hours

9

12

24

48

Hours

C 20

ab

ab

ab

b*

b* ab*

15

ab*

a

10 5 0 0

2

4

6

9

Hours

12

24

48

Tryacylglycerides (mmol l-1)

D 25

IGF-I (ng ml-1)

*

18

*

*

ab

ab

ab

ab

6

9

12

24

3,5 3 2,5 b

2

ab

b

a

1,5 1 0

2

4

48

Hours

Fig. 2. Plasma glucose (A), insulin (B), IGF-I (C) and triacylglyceride (D) levels in European sea bass after glucose (GLU) or saline solution (SS) injections. Values represent means ± SD (n = 10). Significant differences (p b 0.05) among sampling times within treatment are indicated by different letters.*Indicates a significant difference (p b 0.05) between treatments at each sampling time.

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Table 1 Two-way ANOVA analysis of variance of plasma and liver results in European sea bass after a glucose or a saline solution injection. Variation source

Hour

Type of injection

Interaction

Plasma Glucose Insulin IGF-I Triacylglycerides

*** ns ** ***

*** ** ** **

*** * ns ns

Liver HSI Glycogen

*** ***

ns *

ns ns

*p b 0.05; **p b 0.01; ***p b 0.001; ns — non significant.

correlation (r = − 0.239) was found between the variation pattern with time of liver glycogen content and plasma insulin level. 4. Discussion Plasma glucose response to a glucose load showed a similar pattern to that previously observed in this species (Peres et al., 1999). However, peak values attained in the present study (23– 25 mmol l− 1) were considerably higher than the ones observed in the mentioned work (14–16 mmol l− 1). Since the route and dose of glucose administrated were similar in both studies, differences in fish size may explain such results, as bigger fish (133 g vs. 60–80 g) were used by Peres et al. (1999). In fact, it has been reported that carbohydrate utilization is influenced by fish weight with bigger fish showing better glucose tolerance than smaller ones (Tung and Shiau, 1993). In addition, differences in the duration of the fasting period before the glucose injection (48 h vs. 24 h) may also have

GLU

A

SS

2.0 c

1.8

b

1.6

HSI

1.4

bc

ab

ab

bc bc a

c

1.2 1.0

ab

ab

abc

abc

bc abc a

0.8 0.6 0.4 0.2 0.0 0

2

4

6

9

12

24

48

Hours

Glycogen (g 100g liver-1)

B 14 c

12

bc

c*

c

c

cd

cd

c*

10 d

8

ab

6

bc

bc

4

ab

ab

2 0

a

a 0

2

4

6

9

12

24

48

Hours Fig. 3. Hepatosomatic index (HSI) (A) and liver glycogen (B) content in European sea bass after glucose (GLU) or saline solution (SS) injections. Values represent means ± SD (n = 8). Significant differences (p b 0.05) among sampling times within treatment are indicated by different letters. *Indicates a significant difference (p b 0.05) between treatments at each sampling time.

contributed to the higher values observed in the present study. Indeed, fasted fish generally have an extended period of hyperglycemia when compared with routinely fed fish (Moon, 2001). Handling stress is also known to raise plasma glucose level in fish (Hemre et al., 1991; Garcia-Riera and Hemre, 1996). However, plasma glucose concentration in the saline-injected fish allows a correction for this effect and the constant values observed suggest that the effect of handling was minor in our study as already observed in GTTs performed in other species (Harmon et al., 1991; Mazur et al., 1992; Stone et al., 2003; Booth et al., 2006). In the present study a glucose peak was attained 2 h after the glucose load, and then a plateau was maintained up to 6 h after the glucose load. Previously, Peres et al. (1999) reported a glucose peak 3 h after injection. However time intervals in the two studies were not coincident; Peres et al. (1999) evaluated plasma glucose 1 and 3 h after the glucose load while in the present study time intervals were 2 and 4 h after the glucose load. The short time to reach a plasma glucose peak observed in the present study is similar to that observed in red sea bream after an oral administration of 1.7 g glucose kg BW− 1 (Furuichi and Yone, 1981), whereas 1 g glucose kg BW− 1 intraperitoneally administrated to snapper (P. auratus), turbot (Scophthalmus maximus) and Atlantic salmon (Salmo salar) or orally administrated to white sturgeon (Acipenser transmontanus) led to plasma glucose peaks at 3 h (Hemre et al., 1995; Garcia-Riera and Hemre, 1996; Gisbert et al., 2003; Booth et al., 2006). Earlier peaks, 1 h after glucose administering, were observed in gilthead sea bream (Sparus aurata) and silver perch (Bydianus bydianus) intraperitoneally injected with the same glucose dose used in our study (Peres et al., 1999; Stone et al., 2003) and in Nile tilapia (Oreochromis nilotica) injected with 0.2, 0.4 or 2 g glucose kg BW− 1 (Wright et al., 1998). A glucose load of 1.7 g glucose kg BW− 1 orally administrated to common carp, also led to maximum plasma glucose concentration within 1 h (Furuichi and Yone, 1981). Therefore, European sea bass presented a medium timing of glucose peak in comparison with other fish species. These differences in timing of glucose peak cannot be attributed exclusively to the species, since glucose absorption can be affected by the route of glucose administration, size of fish and experimental conditions (Moon, 2001). Taking these considerations into account, the duration of hyperglycemia is related to the degree of glucose tolerance of the species. In this line, present data showed that European sea bass juveniles require circa 12 h for re-establishing basal values of circulating levels of glucose. Such a prolonged hyperglycemia is commonly observed in carnivorous teleosts (Harmon et al., 1991; Garcia-Riera and Hemre, 1996; Peres et al., 1999; Booth et al., 2006), while omnivorous and herbivorous species usually show significantly shorter hyperglycemia periods of 4–6 h (Furuichi and Yone, 1981; Wilson and Poe, 1987; Lin et al., 1995) even though when maintained at similar rearing temperatures. Hence, it does not seem that hyperglycemia duration is related to temperature (which influences normal metabolic rate) but it should be somehow related with other mechanisms that help maintain glucose homeostasis, such as enzymatic activity or expression, or hormonal regulation of glucose metabolism. In general, plasma glucose peak correlates with that of insulin secretion, with maximal insulin levels in teleosts being observed 1–3 h after a glucose load (Furuichi and Yone, 1981; Lin et al., 1995; Navarro et al., 2002). Insulin levels in the control group were in the range and even higher than the plasma levels previously described in this species (Gutiérrez et al., 1984, 1987). Our data showed that plasma insulin increased moderately at 4–6 h after the glucose load when compared to the saline solution injected fish. Accordingly, other species showed slight increases in insulin after glucose administration. For instance, in rainbow trout and American eel (Anguilla rostrata) insulin levels increased after glucose injection (0.25 g glucose kg BW− 1) from 2 to 7 ng ml− 1 and from 6 to 11 ng ml− 1, respectively (Legate et al., 2001). A glucose load of 1.7 g glucose

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kg BW− 1 orally administrated to Chinook salmon increased plasma insulin levels from 4 to 7 ng ml− 1 (Mazur et al., 1992). Furthermore, the authors also reported that glucose tolerance curves were poorly correlated with plasma glucose concentrations of insulin. In contrast, in common carp and hybrid tilapia a considerable increase in plasma insulin levels (from 20 to 60 μU ml− 1 and from 7.5 to 40 μU ml− 1, respectively) was observed after oral administration of 1.7 g glucose kg BW− 1 (Furuichi and Yone, 1981; Lin et al., 1995). Overall, these results suggested that insulin release is stimulated by glucose, although glucose is not probably the more important insulin secretagogue. Indeed, amino acids and especially arginine and lysine are more potent insulin secretagogues than glucose in fish (Mommsen et al., 2001). Furthermore, although not confirmed in European sea bass, somatostatin-producing pancreatic cells seem to be more sensitive than insulin-producing pancreatic cells to glucose in fish. Since somatostatin inhibits insulin secretion this may explain the reduced insulin secretion during the initial period after glucose administration (Ronner and Scarpa, 1984; Harmon et al., 1991). Therefore, European sea bass although having relatively high basal levels of insulin (9–12 ng ml− 1) in comparison to other species (Gutiérrez et al., 1984, 1987) showed a moderate insulin response to glucose that fits well with its carnivorous nature. In mammals, one of the roles attributed to insulin is promoting glycogen synthesis in tissues such as liver and muscle. However, an increased glucose uptake or glycogen deposition in liver and muscle due to insulin does not seem to occur in all fish species (Mommsen and Plisetskaya, 1991). In our study, liver glycogen and insulin levels showed similar variation pattern with time. Slightly lower liver glycogen content was in general observed in the saline-injected fish, which could be due to the longer period of deprivation of glucose in this group as compared to the glucose group. Indeed, in this species one day fasting proved to be enough to significantly drop hepatic glycogen levels (Perez-Jimenez et al., 2007). These differences were specifically observed between 6 h and 9 h in the glucose injected fish, just after being exposed to high levels of plasma glucose. Although muscle glycogen was not analyzed in our study, Peres et al. (1999) showed that in this species no effect on muscle glycogen was observed in fish submitted to a similar glucose load. IGF-I has an important role in the regulation of fish carbohydrate metabolism, and was more effective than insulin in stimulating glucose uptake in rainbow trout muscle cells (Castillo et al., 2004). Furthermore, and in contrast to mammals, a predominance of IGF-I receptors over insulin receptors was also reported in fish skeletal muscle (Párrizas et al., 1995). To our knowledge this is the first study evaluating IGF-I response to a glucose load in fish. Our data showed that plasma IGF-I peaked at 6–9 h after the glucose load when compared to the saline solution injected fish. This represents a 2 h delay relative to the insulin peak, and may be related to it. Indeed, it was reported both in fish and in mammals, that circulating IGF-I levels may be regulated by mechanisms in which insulin is partially involved (Plisetskaya and Duan, 1994; Baños et al., 1998, 1999). Accordingly, a hyperinsulinemia induced either by arginine or insulin injection increased IGF-I levels in brown trout, whereas hypoinsulinemia induced by a streptozotocin injection had the opposite effect (Baños et al., 1999). With the exception of time 12 h which presented an unexpected value, lower plasma IGF-I levels were observed between times 6 and 48 h in the saline injected fish compared to the glucose injected fish. In fact, a decrease in plasma insulin and IGF-I levels with food deprivation was reported in several species (Gutiérrez et al., 1991b; Navarro and Gutiérrez, 1995; Baños et al., 1999; Planas et al., 2000; Montserrat et al., 2007a,b). Furthermore, in European sea bass it was previously shown that fasting also decreased the expression of hepatic and muscle IGF-I mRNA levels (Terova et al., 2007). As the biological activity of insulin and IGF-I depends on binding to their specific cell surface receptors, studies concerning regulation at the receptor level would clarify the contribution of each hormone to the

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restoration of plasma glucose levels. Nevertheless, it is possible to consider that the observed IGF-I increase may in part be responsible for plasma glucose clearance in European sea bass. In general, saline solution injected fish showed higher plasma triacylglyceride levels than glucose injected fish, which is in line with previous results in European sea bass and in other species (Harmon et al., 1991; Garcia-Riera and Hemre, 1996; Hemre and Hansen, 1998; Peres et al., 1999). In fact, Harmon et al. (1991) observed in rainbow trout that a glucose load led to higher hepatic triacylglycerol lipase activity and thus to higher plasma fatty acid levels than a saline solution load. Furthermore, the activity of the lipogenic enzyme glucose-6-phosphate dehydrogenase was lowered by the glucose injection. This catabolic pattern was related with a glucose induced release of somatostatin which may have enhanced lipolysis thus inhibiting triacylglyceride synthesis (Harmon et al., 1991). Accordingly, administration of somatostatin-25 to salmon was followed by hyperlipidemia caused by hepatic lipolysis, whereas an acute insufficiency of this hormone decreased the lipolytic rate (Sheridan et al., 1987; Plisetskaya et al., 1989). Overall, it is possible that a similar mechanism exists in European sea bass. Moreover, the lipogenic role of insulin may have been masked by the somatostatin release which may have contributed to the absence of an evident insulin peak as discussed above. In conclusion, our data showed that an intraperitoneal injection of 1 g glucose kg BW− 1 resulted in a hyperglycemic peak 2 h after injection that was not corrected until 12 h post-glucose administration. Overall, our results indicate that under these experimental conditions glucose is probably not the most important stimulator of insulin release whereas insulin may have contributed to the increase of plasma IGF-I levels, which in turn may help insulin to enhance the glucose uptake by the liver in glucose injected fish. Further studies, namely at the receptor level, would help to clarify the contribution of each hormone in restoring basal plasma glucose level. Acknowledgments The first author was supported by a grant (BPD/39688/2007) from Fundação para a Ciência e a Tecnologia, Portugal. We would like to express our thanks to Mr. P. Correia for the technical assistance during the trial. References Baños, N., Moon, T.W., Castejón, C., Gutiérrez, J., Navarro, I., 1997. Insulin and insulinlike growth factor-I (IGF-I) binding in fish red muscle: regulation by high insulin levels. Regul. Pept. 68, 181–187. Baños, N., Baró, J., Castejón, C., Navarro, I., Gutiérrez, J., 1998. Influence of highcarbohydrate enriched diets on plasma insulin levels and insulin and IGF-I receptors in trout. Regul. Pept. 77, 55–62. Baños, N., Planas, J.V., Gutiérrez, J., Navarro, I., 1999. Regulation of plasma insulin-like growth factor-I levels in brown trout (Salmo trutta). Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 124, 33–40. Booth, M.A., Anderson, A.J., Allan, G.L., 2006. Investigation of the nutritional requirements of Australian snapper Pagrus auratus (Bloch & Schneider 1801): digestibility of gelatinized wheat starch and clearance of an intra-peritoneal injection of D-glucose. Aquac. Res. 37, 975–985. Castillo, J., Codina, M., Martínez, M.L., Navarro, I., Gutiérrez, J., 2004. Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R935–R941. Cowey, C.B., Walton, M.J., 1989. Intermediary metabolism, In: Halver, J.E. (Ed.), Fish Nutrition, 2nd edn. Academic Press, San Diego, California, pp. 260–329. Dabrowski, K., Guderley, H., 2002. Intermediary metabolism, In: Halver, J.E., Hardy, R.W. (Eds.), Fish Nutrition, 3rd Edition. Academic Press, San Diego, California, pp. 309–365. Duan, C., 1998. Nutritional and developmental regulation of insulin-like growth factors in fish. J. Nutr. 128, 306S–314S. Eilertson, C.D., Sheridan, M.A., 1993. Differential effects of somatostatin-14 and somatostatin-25 on carbohydrate and lipid metabolism in rainbow trout Oncorhynchus mykiss. Gen. Comp. Endocrinol. 92, 62–70. Enes, P., Panserat, S., Kaushik, S., Oliva-Teles, A., 2009. Nutritional regulation of hepatic glucose metabolism. Fish Physiol. Biochem. 35, 519–539. Furuichi, M., Yone, Y., 1981. Change of blood sugar and plasma insulin levels of fishes in glucose tolerance test. Bull. Jpn Soc. Sci. Fish. 47, 761–764.

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