Evidence for a Hyperglycemic Effect of Methionine– Enkephalin in the Prawns Penaeus indicus and Metapenaeus monocerus

General and Comparative Endocrinology 123, 90 –99 (2001) doi:10.1006/gcen.2001.7655, available online at http://www.idealibrary.com on

Evidence for a Hyperglycemic Effect of Methionine– Enkephalin in the Prawns Penaeus indicus and Metapenaeus monocerus B. Kishori, B. Premasheela, R. Ramamurthi, and P. Sreenivasula Reddy 1 Department of Biotechnology, Sri Venkateswara University, Tirupati 517502, India Accepted March 5, 2001

Panulirus interruptus, and red swamp crayfish, Procambarus clarkii. Through immunocytochemistry, they found leucine– enkephalin-like immunoreactivity in retinular cells of the ommatidia and in axons that run from the medulla interna to the medulla terminalis in the eyestalks. Later, Fingerman et al. (1983, 1985) reported the occurrence of methionine– enkephalin-like and leucine– enkephalin-like material in the neurosecretory cells of the eyestalks of fiddler crabs, Uca pugilator. Luschen et al. (1991) isolated methionine– enkephalin and leucine– enkephalin from the thoracic ganglia of the shore crab, Carcinus maenas. Although opioid peptides have been described in several crustaceans (see Nagabhushanam et al., 1995), the physiological functions of these peptides are not fully established. Methionine– enkephalin appears to stimulate release of the black pigment-dispersing and red pigment-concentrating hormones (Quackenbush and Fingerman, 1984) and the distal retinal pigment dark-adapting hormone (Kulkarni and Fingerman, 1987) from the neurosecretory cells of U. pugilator. Martinez et al. (1988) reported increased locomotor activity in the land crab Gecarcinus lateralis after injection of FK 33824, a stable methionine– enkephalin analogue. Rothe et al. (1991) provided evidence that leucine– enkephalin injection decreased hemolymph glucose concentration in C. maenas. Sarojini et al. (1995) observed hypoglycemia in P. clarkii after leucine– enkephalin injection. Injection of methionine– enkepha-

The influence of methionine– enkephalin on carbohydrate metabolism of the prawns Penaeus indicus and Metapenaeus monocerus was studied. Injection of the opioid methionine– enkephalin into intact prawns induced significant hyperglycemia in a dose-dependent manner. Total tissue (midgut gland and muscle) carbohydrate and glycogen levels decreased following methionine– enkephalin injection, with a significant activation of phosphorylase in intact prawns, indicating glycogenolysis leading to hyperglycemia. In contrast, injection of methionine– enkephalin into eyestalk-ablated crabs did not affect the levels of hemolymph glucose, total tissue carbohydrates and glycogen, and activity of phosphorylase. These results support an earlier hypothesis for crabs which proposed that methionine– enkephalin acts as a neurotransmitter in crustaceans and stimulates the release of hyperglycemic hormone in inducing hyperglycemia. © 2001 Academic Press Key Words: methionine– enkephalin; hyperglycemia; midgut gland and muscle; phosphorylase; hyperglycemic hormone; glycogen and total carbohydrates.

INTRODUCTION Mancillas et al. (1981) were the first to demonstrate the presence of an opioid peptide in spiny lobster, 1

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Opioid-Induced Hyperglycemia in Prawns

lin slowed ovarian maturation significantly in U. pugilator (Sarojini et al., 1995) and P. clarkii (Sarojini et al., 1996). Evidence for a neurotransmitter role for methionine– enkephalin in regulating hemolymph sugar level has been presented in the fresh water crab Oziotelphusa senex senex (Reddy, 1999) and an antagonistic action of opioid peptides in the regulation of ovarian maturation in the crab has been shown (Kishori and Reddy, 2000). Evidence for endocrine control of carbohydrate metabolism was first obtained by Abramowitz et al. (1944). They found that sinus gland extracts of U. pugilator and Callinectes sapidus increased the reducing sugar concentrations of the blood in these crabs. The hormone(s) responsible for hyperglycemic activity has been identified in the past 10 years and is commonly referred to as the crustacean hyperglycemic hormone(s) (CHH). This hormone is synthesized by the medulla terminalis ganglionic X-organ in the eyestalks of decapod crustaceans and is transported to the sinus gland, where it is stored and released. Considerable work has since been carried out on the chemical nature, mode, and site of action of CHH (see Keller et al., 1985; Sedlmeir, 1985). The amino acid sequences of several CHHs (Kegel et al., 1989, 1991; Chang et al., 1990; Martin et al., 1993; Huberman et al., 1993; Yang et al., 1995) and the nucleotide sequences of cDNA cloning (Weideman et al., 1989; Tensen et al., 1991; De Kleijn et al., 1995; Ohira et al., 1997) have been determined. It is also known that CHH(s) is present in several isoforms (Tensen et al., 1989; Chang et al., 1990; Yasuda et al., 1994; Yang et al., 1997). Recently, the differential expression of the hyperglycemic hormone gene during different molt stages of the American lobster, Homarus americanus, has been reported (Reddy et al., 1997). There are several pertinent observations that prompted the present study: (a) methionine– enkephalin-like immunoreactivity was observed in the neurosecretory cells of eyestalks of crustaceans (Fingerman et al., 1983, 1985), (b) the neurosecretory cells are a site of hyperglycemic hormone secretion (Keller, 1992; Reddy and Ramamurthi, 1999), and (c) methionine– enkephalin has a neurotransmitter role (Reddy, 1999). The present study was undertaken to examine the possibility that methionine– enkephalin has a role in regulating hemolymph sugar level in Penaeus indicus

and Metapenaeus monocerus and, if so, to determine whether it acts as a hormone and/or as a neurotransmitter.

MATERIALS AND METHODS Prawns, M. monocerus (Fabricius) and P. indicus (H. Milne Edwards), were collected from the Buckingham canal, near the Kavali sea coast, Andhra Pradesh, India (14°50⬘E and 80°5⬘N). Only intermolt prawns (80 ⫾ 5 mm length and 4.0 ⫾ 0.3 g body weight) were selected and acclimatized to laboratory conditions for a period of 1 week under constant salinity (15 ⫾ 1 ppt), pH (7.1 ⫾ 0.1), and temperature (23 ⫾ 1°). During acclimatization they were fed ad libitum with oil cake powder. The medium in which they were placed was changed every day and continuous aeration was provided. Only intermolt (stage C 4) intact prawns were used for experimentation. Methionine– enkephalin was obtained from Sigma Chemical Co. and dissolved in crustacean saline (Van Harreveld, 1936). The methionine– enkephalin concentration used was 10 ⫺10 to 10 ⫺8 M/prawn. The prawns were injected with either crustacean saline or methionine– enkephalin in 10-␮l portions. Hemolymph was collected from control and experimental prawns and analyzed for hemolymph glucose concentration. Midgut gland and muscle tissues were isolated from control and injected prawns, and levels of total carbohydrates and glycogen and activity levels of phosphorylase were determined. The data were analyzed with Student’s t test.

Estimation of Hemolymph Glucose Level One hundred microliters of hemolymph were collected through the arthrodial membrane and mixed with 300 ␮l of 95% ethanol. After deproteinization (4°, 14,000g, 10 min), the sample was mixed with a mixture of glucose enzyme reagent (glucose-6-phosphate dehydrogenase and NADP) and color reagents (phenazine methosulfate and iodonitrotetrazolium chloride) (kit from Sigma). After 30 min, the intensity of the color was measured at 490 nm and quantified with standards.

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Estimation of Tissue Glycogen and Total Carbohydrate Levels The tissue (midgut gland and muscle) total carbohydrates level was estimated in the 10% trichloroacetic acid supernatants (3% w/v). Glycogen was measured in the ethanolic precipitate of the trichloroacetic acid supernatants, according to the method of Carroll et al. (1956).

Assay of Glycogen Phosphorylase Activity The phosphorylase activity was assayed in midgut gland and muscle by colorimetric determination of inorganic phosphate released from glucose-1-phosphate (Cori et al., 1955); 0.4 ml of the enzyme was incubated with 2.0 mg of glycogen for 30 min at 35°. The reaction was started by the addition of 0.2 ml of 0.016 M glucose-1-phosphate to one tube (phosphorylase a) and by the addition of a mixture of 0.2 ml of glucose-1-phosphate and 0.004 M adenosine-5-monophosphate to another tube (phosphorylase ab). The reaction mixture was incubated for 15 min for phosphorylase ab (total) and for 30 min for phosphorylase a (active). The reaction was arrested by the addition of 5 ml of 5 N sulfuric acid, and the amount of inorganic phosphate released was estimated.

Estimation of Protein Content The protein content in the enzyme source was estimated (Lowry et al., 1951) with bovine serum albumin as standard.

RESULTS Effect of Methionine–Enkephalin on Hemolymph Glucose Level To determine whether there is any effect of methionine– enkephalin on hemolymph glucose level, and, if so, whether it is dose dependent, 50 prawns were divided into five groups of 10 each. The first group, which served as normal, did not receive any treatment. The second group served as control and re-

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Kishori et al.

ceived 10 ␮l of physiological saline (Van Harreveld, 1936). The prawns in the third through fifth groups were injected with methionine– enkephalin at a dose of 10 ⫺8, 10 ⫺9, or 10 ⫺10 M/prawn, respectively, in a 10-␮l volume. Hemolymph was collected 2 h after injection. Hemolymph glucose level increased significantly (P ⬍ 0.001) in a dose-dependent fashion in prawns that received methionine– enkephalin compared to normal prawns (Fig. 1), whereas injection of physiological saline did not produce any significant change in hemolymph glucose level in M. monocerus (Fig. 1A) and P. indicus (Fig. 1B).

Effect of Methionine–Enkephalin on the Levels of Total Tissue Carbohydrates and Glycogen and on the Activity Level of Phosphorylase in Intact Prawns Administration of methionine– enkephalin (10 ⫺8 M/prawn) to P. indicus significantly decreased glycogen and total carbohydrate levels in the midgut gland (Table 1). The former corresponds to a ⫺36.63% decrease and the latter to a ⫺25.49% decrease from the levels of the control. Muscle glycogen (⫺29.21%) and total carbohydrate (⫺32.25%) levels also decreased significantly. There was a parallel increase in phosphorylase activity levels in both midgut gland and muscle after the injection of methionine– enkephalin (Table 1). The ratio of active (a) to total (ab) phosphorylase was also elevated, indicating interconversion of inactive to active phosphorylase (Table 1). Injection of methionine– enkephalin into M. monocerus also significantly decreased tissue glycogen (midgut gland, ⫺32.6%; muscle, ⫺28.04%) and total carbohydrate (midgut gland, ⫺17.44%; muscle, ⫺43.73%) levels with an increase in tissue phosphorylase activity (Table 2).

Effect of Eyestalk Ablation and Injection of Eyestalk Extract and Methionine–Enkephalin into Eyestalkless Prawns on the Levels of Hemolymph Glucose, Total Tissue Carbohydrates and Glycogen, and Activity of Phosphorylase Eyestalk ablation is a classical operation in crustacean endocrinology; it removes the X-organ and the sinus gland, a neurohormonal organ containing the

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changes in hemolymph glucose level, tissue carbohydrate level, and phosphorylase activity. Eyestalk ablation significantly decreased hemolymph glucose level in P. indicus (⫺28.87%) and M. monocerus (⫺18.45%) with significant increases in total tissue carbohydrate and glycogen levels and a decrease in phosphorylase activity (Tables 3 and 4). Injection of eyestalk extract into eyestalkless prawns reversed these changes. This meets the classical criteria of endocrine control and demonstrates that the hemolymph sugar level of the prawns is regulated by a hormonal agent originating in the eyestalks. Similar results were reported earlier for several crustaceans (Keller et al., 1985; Keller, 1992). However, injection of methionine– enkephalin (10 ⫺8 M/prawn) into eyestalkless crabs had no effect on the levels of hemolymph glucose, total tissue carbohydrates and glycogen, and phosphorylase activity (Tables 3 and 4).

Hyperglycemic Effect of Eyestalk Extract of the Prawns P. indicus and M. monocerus Injected with Methionine–Enkephalin

FIG. 1. Effect of different doses of methionine– enkephalin (met– enkephalin) on the hemolymph glucose levels of Metapenaeus monocerus (A) and Penaeus indicus (B). 1, Normal; 2, saline-injected control; 3, met– enkephalin, 10 ⫺10 M/prawn; 4, met– enkephalin, 10 ⫺9 M/prawn; 5, met– enkephalin, 10 ⫺8 M/prawn. Numbers in parentheses indicate the percentage change from the normal values. Error bars are SD; N ⫽ 10. Statistically significant increases in hemolymph glucose levels, compared with normal, are marked with an asterisk.

neuron ending of the neurosecretory system, which is the secretory and release site for an array of hormones, including the hyperglycemic hormone. Eyestalks were removed from the prawns by the cutting off of the organs at the base without prior ligation but with cautery of the wound after operation. After 24 h of eyestalk ablation, the prawns were analyzed for

Eyestalks were collected from the prawns injected with 10 ⫺8, 10 ⫺9, and 10 ⫺10 M/prawn after 2 h of injection. Medulla terminalis ganglionic X-organ–sinus gland complexes were removed from the eyestalks under a stereo microscope. Aqueous extracts of the glands were prepared for bioassay as described earlier (Reddy, 1992). Hyperglycemic activity of the extract was measured by the injection of eyestalk extract (two eyestalk equivalents) into prawns. Concentrations of hemolymph glucose levels were determined before the injection and 120 min thereafter as described earlier. The hyperglycemic activity of eyestalk extracts of methionine– enkephalin-injected prawns was less than that produced by eyestalks of normal prawns (Table 5). The different amounts of hyperglycemic activity in the eyestalks of the prawns injected with methionine– enkephalin relative to the eyestalks of the control prawns suggest that methionine– enkephalin triggers hyperglycemic hormone release in the penaeid prawns. It has been speculated that methionine– enkephalin induces hyperglycemia in O. senex senex by triggering release of the hyperglycemic hormone from eyestalks (Reddy, 1999).

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TABLE 1 Effect of Methionine–Enkephalin (Met-enk) (10 ⫺8 M/Prawn) Injection on the Levels of Total Carbohydrate and Glycogen and Activity Levels of Phosphorylase in the Midgut Gland and Muscle of Penaeus indicus Parameter Total carbohydrate (mg glucose/g tissue) Glycogen (mg glucose/g tissue) Phosphorylase a (iP released/mg protein/h) Phosphorylase ab (iP released/mg protein/h) Phosphorylase a/ab

Tissue Midgut Muscle Midgut Muscle Midgut Muscle Midgut Muscle Midgut Muscle

gland gland gland gland gland

Normal

Met-enk injected

10.24 ⫾ 1.29 8.03 ⫾ 0.97 1.01 ⫾ 0.11 0.89 ⫾ 0.09 1.66 ⫾ 0.09 1.73 ⫾ 0.12 2.32 ⫾ 0.42 2.44 ⫾ 0.66 0.71 0.70

7.63* ⫾ 0.92 (⫺25.49) 5.44* ⫾ 0.43 (⫺32.25) 0.64* ⫾ 0.09 (⫺36.63) 0.53* ⫾ 0.07 (⫺29.21) 2.31* ⫾ 0.21 (39.15) 2.55* ⫾ 0.28 (47.39) 2.96* ⫾ 0.51 (27.59) 3.39* ⫾ 0.54 (38.93) 0.78 0.75

Note. Values are mean ⫾ SD of 10 individuals. Values in parentheses are percentage change from normal. * Values are significant at P ⬍ 0.001.

DISCUSSION

M. monocerus in a dose-dependent manner (Fig. 1). Methionine– enkephalin-induced hyperglycemia has also been demonstrated in the crab O. senex senex (Reddy, 1999). The observation that methionine– enkephalin was ineffective in increasing glucose levels in eyestalkless P. indicus and M. monocerus (Tables 3 and 4) is consistent with observations in O. senex senex (Reddy, 1999) and the mud crab Scylla serrata (B. Kishori and P. S. Reddy, unpublished observations) and suggests that the hyperglycemic effect of methionine– enkephalin results from an enhanced release of eyestalk hyperglycemic factor(s), presumably CHH(s) from eyestalks (Keller, 1992; Soyez, 1997). This hy-

A neurotransmitter role for methionine– enkephalin has been reported in the fiddler crab U. pugilator (Sarojini et al., 1995) and the red swamp crayfish P. clarkii (Sarojini et al., 1997). Rothe et al. (1991) reported that leucine– enkephalin inhibits hyperglycemic hormone release in C. maenas and lowers the hemolymph glucose concentration when injected into intact U. pugilator, but not eyestalkless U. pugilator. Similar results were also observed in P. clarkii (Sarojini et al., 1995). The present study shows that methionine– enkephalin elicited a hyperglycemic response in P. indicus and

TABLE 2 Effect of Methionine–Enkephalin (Met-enk) (10 ⫺8 M/prawn) Injection on the Levels of Total Carbohydrate and Glycogen and Activity Levels of Phosphorylase in the Midgut Gland and Muscle of Metapenaeus monocerus Parameter Total carbohydrate (mg glucose/g tissue) Glycogen (mg glucose/g tissue) Phosphorylase a (iP released/mg protein/h) Phosphorylase ab (iP released/mg protein/h) Phosphorylase a/ab

Tissue Midgut Muscle Midgut Muscle Midgut Muscle Midgut Muscle Midgut Muscle

gland gland gland gland gland

Normal

Met-enk injected

9.23 ⫾ 1.33 7.66 ⫾ 0.89 0.92 ⫾ 0.16 0.82 ⫾ 0.08 1.93 ⫾ 0.11 2.33 ⫾ 0.31 3.63 ⫾ 0.65 4.09 ⫾ 0.59 0.53 0.56

7.62* ⫾ 0.88 (⫺17.44) 4.31* ⫾ 0.52 (⫺43.73) 0.62* ⫾ 0.08 (⫺32.60) 0.59* ⫾ 0.07 (⫺28.04) 2.43* ⫾ 0.31 (25.38) 2.98* ⫾ 0.29 (27.89) 3.91 NS ⫾ 0.68 (7.71) 4.62 NS ⫾ 0.44 (12.95) 0.61 0.64

Note. Values are mean ⫾ SD of 10 individuals. Values in parentheses are percentage change from normal. NS Not significant. * Values are significant at P ⬍ 0.001.

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Opioid-Induced Hyperglycemia in Prawns

TABLE 3 Effect of Eyestalk Ablation and Injection of Eyestalk Extract and Methionine–Enkephalin (10 ⫺8 M/prawn) into Eyestalkless Penaeus indicus on the Levels of Hemolymph Glucose, Tissue Glycogen, and Total Carbohydrates and Activity Levels of Phosphorylase

Parameter Hemolymph glucose Midgut gland Total carbohydrates Glycogen Phosphorylase a Phosphorylase ab Phosphorylase a/ab Muscle Total carbohydrates Glycogen Phosphorylase a Phosphorylase ab Phosphorylase a/ab

Normal prawns

Eyestalk-ablated prawns (ESX)

ESX injected with eyestalk extract

4.26 ⫾ 0.35

3.03* ⫾ 0.41 (⫺28.87)

10.24 ⫾ 1.29 1.01 ⫾ 0.11 1.66 ⫾ 0.09 2.69 ⫾ 0.42 0.61

13.66* ⫾ 1.55 (33.39) 1.91* ⫾ 0.18 (89.11) 1.17* ⫾ 0.05 (⫺29.52) 1.96* ⫾ 0.14 (⫺27.13) 0.59

9.57* ⫾ 1.94 (⫺29.94) 1.21* ⫾ 0.11 (⫺36.64) 1.72* ⫾ 0.31 (47.01) 2.56* ⫾ 0.44 (30.61) 0.67

13.44 NS ⫾ 1.44 (⫺1.61) 1.92 NS ⫾ 0.10 (0.52) 1.19 NS ⫾ 0.08 (1.71) 1.91 NS ⫾ 0.11 (⫺2.55) 0.62

8.03 ⫾ 0.97 0.82 ⫾ 0.09 1.73 ⫾ 0.12 2.44 ⫾ 0.66 0.71

10.14* ⫾ 0.91 (26.28) 1.01* ⫾ 0.09 (23.17) 1.29* ⫾ 0.09 (⫺25.43) 1.81* ⫾ 0.12 (⫺35.82) 0.71

7.62* ⫾ 0.71 (⫺24.85) 0.84* ⫾ 0.06 (⫺16.83) 1.91* ⫾ 0.08 (48.06) 2.32* ⫾ 0.34 (28.18) 0.82

10.29 NS ⫾ 0.65 (1.48) 0.99 NS ⫾ 0.09 (⫺1.98) 1.30 NS ⫾ 0.11 (0.78) 1.86 NS ⫾ 0.22 (2.76) 0.70

5.29* ⫾ 0.54 (74.59)

ESX injected with methionine–enkephalin 3.04 NS ⫾ 0.51 (4.03)

Note. Values are mean ⫾ SD of 10 individual prawns. For calculation of percentage change and evaluation of P for ESX prawns, normals served as control; for ESX-injected prawns, ESX prawns served as control. NS Not significant. * P ⬍ 0.001.

pothesis is further supported by the finding that in O. senex senex, the methionine– enkephalin antagonist naloxone blocked the release of CHH from the eyestalk (P. S. Reddy and Riyaz Basha, unpublished observations).

The effect of eyestalk hormones on the tissue carbohydrate levels and phosphorylase activity has been extensively studied in several crustaceans (Keller, 1965; Ramamurthi et al., 1968; Sagardia, 1969). Eyestalk removal inactivates the phosphorylase system

TABLE 4 Effect of Eyestalk Ablation and Injection of Eyestalk Extract and Methionine–Enkephalin (10 ⫺8 M/prawn) into Eyestalkless Metapenaeus monocerus on the Levels of Hemolymph Glucose, Tissue Glycogen, and Total Carbohydrates and Activity Levels of Phosphorylase

Parameter Hemolymph glucose Midgut gland Total carbohydrates Glycogen Phosphorylase a Phosphorylase ab Phosphorylase a/ab Muscle Total carbohydrates Glycogen Phosphorylase a Phosphorylase ab Phosphorylase a/ab

Normal prawns

Eyestalk-ablated prawns (ESX)

ESX injected with eyestalk extract

5.04 ⫾ 0.36

4.11* ⫾ 0.52 (⫺18.45)

9.23 ⫾ 1.33 0.92 ⫾ 0.16 1.93 ⫾ 0.11 3.63 ⫾ 0.65 0.53

12.09* ⫾ 0.98 (30.98) 1.31* ⫾ 0.26 (42.39) 1.21* ⫾ 0.08 (⫺37.30) 2.78* ⫾ 0.08 (⫺23.41) 0.43

9.78* ⫾ 0.99 (⫺19.10) 0.98* ⫾ 0.09 (⫺25.19) 2.09* ⫾ 0.36 (72.72) 3.64* ⫾ 0.99 (30.93) 0.57

12.14 NS ⫾ 0.96 (0.41) 1.36 NS ⫾ 0.17 (3.81) 1.22 NS ⫾ 0.09 (0.82) 2.72 NS ⫾ 0.09 (⫺2.15) 0.44

7.66 ⫾ 0.89 0.82 ⫾ 0.08 2.33 ⫾ 0.31 4.09 ⫾ 0.59 0.56

10.43* ⫾ 0.98 (36.16) 1.12* ⫾ 0.08 (36.58) 1.67* ⫾ 0.08 (⫺28.32) 3.23* ⫾ 0.39 (⫺21.02) 0.51

7.01* ⫾ 0.92 (⫺32.79) 0.76* ⫾ 0.07 (⫺32.14) 2.68* ⫾ 0.41 (60.47) 4.12* ⫾ 0.61 (27.55) 0.65

10.51 NS ⫾ 1.16 (0.76) 1.11 NS ⫾ 0.09 (⫺0.89) 1.72 NS ⫾ 0.09 (2.99) 3.22 NS ⫾ 0.41 (⫺0.30) 0.53

6.58* ⫾ 0.51 (60.09)

ESX injected with methionine–enkephalin 4.21 NS ⫾ 0.82 (2.43)

Note. Values are mean ⫾ SD of 10 individual prawns. For calculation of percentage change and evaluation of P for ESX prawns, normals served as control; for ESX-injected prawns, ESX prawns served as control. NS Not significant. * P ⬍ 0.001.

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TABLE 5 Effect of Injection of Eyestalk Extract of Prawns that Received Different Doses of Methionine–Enkephalin on the Hemolymph Glucose Level of Penaeus imdicus and Metapenaeus monocerus Hemolymph glucose level

Group Penaeus indicus Control prawns Control prawn eyestalk extract injected into prawns 10 ⫺8 M/prawn 10 ⫺9 M/prawn 10 ⫺10 M/prawn Metapenaeus monocerus Control prawns Control prawn eyestalk extract injected into prawns 10 ⫺8 M/prawn 10 ⫺9 M/prawn 10 ⫺10 M/prawn

0 min before injection

120 min after injection

4.24 ⫾ 0.82

4.29

⫾ 0.74 (1.18)

4.42 ⫾ 0.41 4.40 ⫾ 0.44 4.33 ⫾ 0.51 4.39 ⫾ 0.43

7.82* ⫾ 0.86 (61.54) 4.98 NS ⫾ 0.56 (13.18) 5.26* ⫾ 0.46 (21.48) 6.08* ⫾ 0.45 (38.50)

5.12 ⫾ 0.44

5.18

5.14 ⫾ 0.38 5.15 ⫾ 0.42 5.10 ⫾ 0.31 5.14 ⫾ 0.42

8.04* ⫾ 0.32 (56.42) 5.82 NS ⫾ 0.51 (13.01) 6.11* ⫾ 0.44 (19.80) 6.91* ⫾ 0.47 (34.44)

⫾ 0.55 (1.17)

Note. Prawns were injected with the different doses of methionine– enkephalin. At 24 h postinjection, eyestalks were excised from the injected animals. Eyestalk extract was prepared and two eyestalk equivalents of extract in 10 ␮l volume was injected into prawns. Hemolymph was collected from the prawns before and 120 min after injection, and glucose level was determined. Values are mean (mg glucose/100 ml) ⫾ SD of 10 individual prawns. Values in parentheses are percentage change from 0 min samples. NS Not significant. * P ⬍ 0.001.

and activates uridine– diphosphate– glucose glycogen transglucosylase (glycogen synthetase) (Keller, 1965; Ramamurthi et al., 1968). Ramamurthi et al. (1968) also observed stimulation of uptake and incorporation of glucose 14C into the glycogen fraction of muscle tissue after eyestalk removal with a decrease in hemolymph sugar level, whereas injection of eyestalk extract reversed these changes. It has been reported that the hyperglycemic hormone of crab (O. senex senex) and prawn (Penaeus monodon) eyestalks enhances the activity of the phosphorylase system (Reddy, 1992; Reddy et al., 1982, 1984). Decreased hemolymph sugar levels, increased total carbohydrate and glycogen levels of midgut gland and muscle observed in destalked (24 h) prawns, and the total/partial reversal of these changes after eye-

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stalk extract injection indicate that sugar molecules are mobilized between hemolymph and storage organ carbohydrate pools and that this process is under the control of hyperglycemic hormone present in the eyestalks. Hohnke and Scheer (1970) suggested that the primary role of the so-called hyperglycemic hormone is not to elevate hemolymph sugar level but to elevate intracellular glucose through the degradation of glycogen by activating the enzyme phosphorylase. These glucose molecules leak into hemolymph, causing hyperglycemia. This view has been supported by Keller and Andrew (1973) and Telford (1975). The decreased phosphorylase activity in the tissues of eyestalk-ablated prawns and an increased phosphorylase activity after eyestalk extract injection (Tables 3 and 4) is in good agreement with the earlier findings. An increased tissue phosphorylase activity and a decrease in glycogen and total carbohydrate levels of prawns, followed by hyperglycemia after the injection of methionine– enkephalin into intact prawns, indicate glycogenolysis and mobilization of sugar molecules into hemolymph. The present study suggests that methionine– enkephalin triggers release of hyperglycemic hormone from the sinus glands of eyestalks, which is responsible for hyperglycemia. The hyperglycemic hormone acts directly by activating the phosphorylase system in muscle and the midgut gland, leading to degradation of glycogen and mobilization of free sugars from tissues to hemolymph, ultimately resulting in hyperglycemia, a typical crustacean hyperglycemic hormone action (Hohnke and Scheer, 1970). The experiments on the effect of methionine– enkephalin on the carbohydrate metabolism of the prawns P. indicus and M. monocerus provide evidence that the opioid system is indeed involved in the regulation of hemolymph glucose level. Previous studies on the freshwater field crab O. senex senex (Reddy, 1999) have shown that methionine– enkephalin triggers the release of hyperglycemic hormone, which is responsible for the hyperglycemia. Since methionine– enkephalin induces hyperglycemia and since methionine– enkephalin does not affect hemolymph glucose levels in eyestalkless crabs, the following hypotheses can be put forth. Methionine– enkephalin in penaeid prawns stimulates the release of hyperglycemic hormone, which in turn, stimulates the phosphorylase system, resulting in glycogenolysis and hyperglyce-

97

Opioid-Induced Hyperglycemia in Prawns

mia. The decreased hyperglycemic activity of eyestalks of prawns injected with methionine– enkephalin supports the hypotheses. In summary, methionine– enkephalin is a potent hyperglycemic regulator in intact P. indicus and M. monocerus, but not in eyestalk-ablated prawns; the most likely sites of action of methionine– enkephalin are the eyestalks where the X-organ–sinus glands are present. Presumably, the action of methionine– enkephalin is effected by stimulation of the release of the CHH(s) from the sinus gland of eyestalk. It is suggested that the source of methionine– enkephalin-induced hyperglycemia is tissue carbohydrates. Significantly decreased tissue carbohydrate levels with an elevated phosphorylase activity are responsible for hyperglycemia following methionine– enkephalin injection. That the hyperglycemic activity of eyestalk extract of methionine– enkephalin-injected prawns was less than that produced by eyestalk extract of control prawns supports this hypothesis. Similar stimulatory action was also observed for the release of red pigment-concentrating hormone and black pigmentdispersing hormone (Quackenbush and Fingerman, 1984) and gonad-inhibiting hormone (Sarojini et al., 1995) from eyestalks of other crustacean species by methionine– enkephalin. Recently, a neurotransmitter role of methionine– enkephalin in O. senex senex in stimulating the release of CHH(s) from the eyestalks has been noted (Reddy, 1999). A similar neurotransmitter role reported earlier for methionine– enkephalin in the release of LH and oxytocin in the mammalian system (Grossman et al., 1981; Bicknel et al., 1985) shows an interesting parallel with that in the crustacean system.

ACKNOWLEDGMENTS Special thanks go to Dr. M. Srinivasulu Reddy, Assistant Professor, S.V.U.P.G. Centre, Kavali for providing laboratory facilities for maintenance of prawns. We thank Dr. K. V. S. Sarma, Associate Professor, Sri Venkateswara University for statistical analysis of data. We also thank the anonymous reviewers who improved our manuscript. This work was generously supported by Department of Science and Technology grant (SP/SO/CO4/96) to Dr. P. Sreeniva-

sula Reddy. S. Umasankar and G. Suneetha provided valuable technical assistance.

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