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Effects of Somatostatin on Lipid Metabolism of Larvae and Metamorphosing Landlocked Sea Lamprey,Petromyzon marinus
General and Comparative Endocrinology 111, 177–185 (1998) Article No. GC987107
Effects of Somatostatin on Lipid Metabolism of Larvae and Metamorphosing Landlocked Sea Lamprey, Petromyzon marinus Yung-hsi Kao,* John H. Youson,† John A. Holmes,† and Mark A. Sheridan* *Department of Zoology and Regulatory Bioscience Center, North Dakota State University, Fargo, North Dakota 58105–5517; and †Department of Zoology and Division of Life Sciences, University of Toronto at Scarborough, Scarborough, Ontario M1C 1A4, Canada Accepted April 22, 1998
This study was designed to examine the role of somatostatin in regulating changes in lipid metabolism of larvae and metamorphosing landlocked sea lamprey, Petromyzon marinus. Larvae and animals in late metamorphosis (stage 6 on a 7-stage scale) were injected intraperitoneally once per day for 2 days with either saline (0.6%) or somatostatin-14 (SS-14; 500 ng/g body wt). Injection of SS-14 into larval and stage 6 metamorphosing animals resulted in elevated plasma fatty acids levels. In larvae, SS-14-induced hyperlipidemia was supported by enhanced lipolysis, as indicated by increased triacylglycerol lipase (TGL) activity in the liver and kidney. Mobilization of larval renal lipid was accompanied by reduced TG synthesis, as indicated by decreased diacylglycerol acyltransferase (DGAT) activity. In stage 6 metamorphosing lamprey, SS-14 did not significantly affect TGL activity; however, SS-14 significantly reduced fatty acid synthesis, as measured by acetyl-CoA carboxylase activity, in kidney, liver, and muscle, as well as muscular TG synthesis. SS-14-stimulated lipid depletion is reminiscent of the pattern of lipid metabolism displayed by P. marinus during their spontaneous metamorphosis—an observation which suggests that somatostatin may play a role in metamorphosis-associated changes in lipid metabolism in this species. r 1998 Academic Press Key Words: somatostatin; lipolysis; lipogenesis; lamprey; Petromyzon marinus; metamorphosis.
0016-6480/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
Somatostatins (SSs) are a peptide hormone family that display considerable structural and functional diversity among vertebrates. The first described SS was a 14-amino-acid peptide originally isolated from ovine hypothalamus and found to inhibit growth hormone secretion from the pituitary gland (Brazeau et al., 1973). Subsequent research revealed that SSs exist in a variety of molecular forms and that they are important in coordinating various aspects of animal growth, development, and metabolism (Patel, 1992; Sheridan, 1994). Some of the molecular heterogeneity is explained by differential processing of a single precursor to yield peptides of different lengths; in some tissues the precursor is processed to SS-14, while in others it may be processed to longer molecules that contain SS-14 at their C-terminus (e.g., mammalian somatostatin-28, which contains SS-14 at its C-terminus; the precursor that contains SS-14 at its C-terminus is known as preprosomatostatin-1, PPSS-1; Conlon et al., 1997). The distribution of SSs among nonmammalian vertebrates suggests that there has been strong selection to conserve the tetradecapeptide, SS-14. Peptides identical in sequence to that originally isolated from sheep brain have been isolated from birds, reptiles, amphibians, teleost fish, and elasmobranchs, as well as from both extant groups of cyclostomes, lamprey and hagfish (reviewed by Conlon et al., 1997). The molecular heterogeneity of the SS family also is
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explained by the existence of alternate SS genes that give rise to precursors other than PPSS-1. Lamprey, numerous teleost fish (Oyama et al., 1980; Plisetskaya et al., 1986; Conlon et al., 1987, 1988; Cutfield et al., 1987), and frogs (Vaudry et al., 1992) appear to express PPSS-1 in addition to a second preprosomatostatin. In sea lamprey, Petromyzon marinus, for example, SS-14 was localized from the brain of adults (Sower et al., 1994) while four alternate forms of SS were isolated from the pancreas: a 14-amino-acid form that contains a Ser for Thr substitution at position 12 and three N-terminally extended peptides with 31, 34, or 37 amino acids all with [Ser12]-SS-14 at their C-termini (all presumably derived from the same precursor by differential processing; Andrews et al., 1988). Conlon and co-workers isolated an SS-35 from the pancreas of brook lamprey, Lampetra fluviatilis (1995a), and an SS-33 from the pancreas of the Southern Hemisphere lamprey, Geotria australis (Conlon et al., 1995b), both containing [Ser12]SS-14 at their C-terminus. Immunocytochemical evidence suggests that only SS-14 is present in larval and metamorphosing animals; SS-14-immunoreactive cells were detected in brain, gut, and pancreas (Elliot and Youson, 1987; Youson et al., 1988; Cheung et al., 1990, 1991; Cheung and Youson, 1991; Youson and Potter, 1993). Changes in intestinal-pancreas levels of SS as measured by a heterologous radioimmunoassay were recently measured during the various phases of the life cycle of sea lamprey, P. marinus, and were shown to increase during their spontaneous metamorphosis (Elliot and Youson, 1991). Increased tissue SS concentration from stage 4 to stage 7 of lamprey metamorphosis, most likely explained by increased size of the endocrine pancreas (Youson and Cheung, 1990), is, interestingly, consistent with (1) decreased lipid content both from the whole body and from lipid depot organs (e.g., liver, kidney, and muscle), and with (2) enhanced lipolysis and reduced lipogenesis from the lipid depots (reviewed by Sheridan and Kao, 1997). The first report of a role of SS in coordinating lipid metabolism of lamprey was cited by Plisetskaya (1980), who pointed out that injection of mammalian SS-14 into adult lamprey, L. fluviatilis, elevated plasma fatty acid levels; however, this response was dependent on the dose of mammalian SS-14 administration and the season when animals were used. There has been no study of the role
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Kao et al.
of SS in regulating lipid metabolism in larval and metamorphosing lampreys. In this study, the landlocked form of sea lamprey, P. marinus, was used to evaluate the role of SS-14 in regulating lipid metabolism in larvae and metamorphosing lamprey. Our hypothesis was that SS-14 stimulates lipid depletion from storage sites and results in elevated plasma fatty acid levels.
MATERIALS AND METHODS Experimental Animals Sea lamprey, P. marinus, larvae were first collected from Oshawa Creek, a tributary of Lake Ontario, Ontario, Canada, on May 31 and June 1, 1996, and transported to the University of Toronto at Scarborough Campus, Ontario, Canada, where they were fed baker’s yeast once a week (18 g/25 larvae). Animals were maintained in 21-L glass aquaria with a sandy substrate and 12 L of dechlorinated City of Toronto water at an ambient temperature of 15–18°C under a photoperiod of 15 h light and 9 h dark.
In Vivo SS-14 Injection Larvae and animals in late metamorphosis (stage 6 on a 7-stage scale; cf. Youson and Potter, 1979) were segregated and placed into separate aquaria (10 larvae/ tank; 5 stage 6 metamorphosing animals/tank) 1 week before experimentation. Feeding was suspended 2 days prior to experimentation. Lampreys were anesthetized by immersion in buffered 0.05% (w/v) tricaine methanesulfonate (MS-222) and injected intraperitoneally (10 µl/g body wt) with either 0.6% saline (control) or mammalian SS-14 (Sigma, St. Louis, MO) at a dose of 500 ng SS-14 (dissolved in 0.6% NaCl)/g body wt once per day for a 2-day period. During the period of experimentation from September 18, 1996, to September 20, 1996, water and sand substrate in the aquaria were not changed. Twelve to 14 h after the last injection, animals were anesthetized individually with buffered MS-222 as described above and were weighed and measured. Blood was collected into heparinized capillary tubes from the severed caudal vasculature,
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allowed to clot overnight at 4°C, and then centrifuged (6000g for 5 min). Plasma was collected and stored at 270°C for later determination of fatty acids. Tissues (liver, kidney, muscle, and intestine) were removed, frozen in liquid N2, and then stored at 270°C for later determination of total lipid and enzyme activities. This protocol was developed based upon preliminary experiments that showed that SS-14 doses similar to those used previously by Plisetskaya (1980) resulted in a somatostatin-induced biological response (i.e., elevated plasma fatty acids; cf. Sheridan, 1994).
Biochemical Analysis Plasma fatty acids were measured by the micromethod of Noma et al. (1973). Total lipids and enzyme activities in selected tissues (liver, kidney, muscle, and intestine) were analyzed essentially as described by Kao et al. (1997a,b). Protein content of the enzyme preparations was determined by dye binding (Bradford, 1976) using a Bio-Rad (Richmond, CA) microplate reader and bovine serum albumin (Sigma) as a standard. The rate of lipid breakdown was evaluated by assessing triacylglycerol lipase (TGL; EC 3.1.1.2) activity. Units of enzyme activity are expressed as picomoles of [3H]oleic acid released per hour per milligram of protein from [3H]triolein (tri-[9,10(n)3H]oleate glycerol). The rate of lipid synthesis was evaluated by assessing separatively de novo fatty acid synthesis, as determined by acetyl-CoA carboxylase (ACC; EC 6.4.1.2) activity, and triacylglycerol synthesis, as determined by diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity. Units of ACC activity and DGAT activity are expressed as femtomoles of [14C]malonyl-CoA formed per hour per milligram of protein and picomoles of [14C]TG formed per minute per milligram of protein, respectively.
Statistical Analysis Data are expressed as means 6 SEM. The Student t test was used to examine differences between the control and the SS-14-treated group. A probability level of 0.05 was used to indicate significance. All statistics were performed using SigmaStat (Jandel Scientific, Palo Alto, CA).
RESULTS Body Characteristics While most body characteristics, including body weight, body length, and condition factor, of larval and metamorphic lampreys were not affected by SS-14 treatment over the course of the 2-day experiment (Table 1), the total mass of liver and the hepatosomatic index were significantly lower in larvae after SS-14 administration compared to controls.
Plasma Fatty Acid Concentration There was a development-dependent difference (p , 0.05) in plasma FA levels. The FA levels in control animals were 0.27 6 0.03 µmol/ml in larvae and 0.49 6 0.02 µmol/ml in stage 6 metamorphosing animals, respectively. Treatment with SS-14 significantly elevated FA levels in larvae and in stage 6 transformers (Fig. 1).
Tissue Lipid Content Somatostatin-14 injection into larvae and stage 6 metamorphosing animals did not affect total lipid content of kidney, muscle, and intestine. However, hepatic total lipid content of larvae treated with SS-14 was significantly decreased by 28% (Fig. 2). The hepatic total lipid content of stage 6 metamorphosing animals was not affected by SS-14 administration.
The Rate of Lipolysis The rate of lipolysis was assessed by changes in TGL activity (Fig. 3). Following SS-14 injection, generally enhanced rates of lipid breakdown were observed in larvae and stage 6 metamorphosing animals. In larval kidney and liver, TGL activity was significantly increased when animals were treated with the SS-14. In stage 6 animals, TGL activity increased, although not significantly so. Triacylglycerol lipase activity in muscle and intestine was not affected by SS-14.
The Rate of de Novo Fatty Acid Synthesis The rate of de novo fatty acid synthesis was assessed by changes in ACC activity (Fig. 4). In larvae, injection
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TABLE 1 In Vivo Effects of Mammalian Somatostatin-14 (SS-14) on Body Characteristics of Larvae and Stage 6 Metamorphosing Lamprey, Petromyzon marinusa Larvae
Stage 6
Parameter
Control
SS-14
Control
SS-14
Body weight (g) Body length (cm) Condition factorb Wet organ weight (mg) Liver Kidney Intestine Muscle Organosomatic Index [(wet organ, g/body wt, g) 3 100%] HSIc KSIc ISIc MSIc
2.78 6 0.07 123.1 6 1.2 1.49 6 0.03
2.71 6 0.09 120.9 6 0.9 1.53 6 0.02
3.04 6 0.23 127.8 6 2.9 1.45 6 0.02
3.39 6 0.29 131.0 6 5.0 1.50 6 0.04
23.1 6 1.5 79.9 6 8.0 38.8 6 3.4 170.8 6 7.7
18.1 6 1.4* 88.8 6 11.0 43.9 6 2.5 161.8 6 6.9
14.4 6 0.9 114.7 6 15.4 76.8 6 6.3 170.0 6 16.8
15.6 6 1.4 132.5 6 32.9 81.9 6 9.7 171.1 6 16.1
0.47 6 0.01 3.81 6 0.56 2.55 6 0.20 5.56 6 0.21
0.46 6 0.01 3.71 6 0.65 2.41 6 0.17 5.07 6 0.34
0.84 6 0.07 2.85 6 0.26 1.39 6 0.11 6.14 6 0.21
0.66 6 0.04* 3.22 6 0.35 1.64 6 0.11 5.99 6 0.24
a SS-14 was injected into each animal at a dose of 500 ng per gram of body weight; control animals received 0.6% saline. Data are presented as means 6 SEM, where n 5 10 for larvae and n 5 5 for stage 6 animals. Statistical differences between the control and SS-14 treatment groups were assessed by the Student t test (*P , 0.05). b Calculated as body weight (g)/(body length, mm)3 3 106. c Abbreviations: HSI, hepatosomatic index; KSI, kidney-somatic index; ISI, intestinal-somatic index; MSI, muscular-somatic index.
of SS-14 tended to reduce ACC activity in kidney, liver, and intestine. In stage 6 metamorphosing animals, ACC activity was significantly decreased in the kidney, liver, and muscle after SS-14 administration.
The Rate of Triacylglycerol Synthesis The rate of triacylglycerol synthesis was assessed by changes in DGAT activity (Fig. 5). In larval lamprey, treatment with SS-14 significantly decreased DGAT activity in the kidney and tended to decrease the activity of this enzyme in liver and muscle. In stage 6 metamorphosing animals, SS-14 did not significantly affect DGAT activity in kidney and liver, but hormone treatment did significantly decrease DGAT activity in muscle.
FIG. 1. Effects of somatostatin-14 (SS-14) on plasma fatty acid levels in larvae and stage 6 metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM (n 5 10 for larvae and n 5 5 for stage 6 metamorphosing animals at each treatment). An asterisk indicates a significant difference (P , 0.05) between the control group (saline-treated) and SS-14-treated animals.
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DISCUSSION The results of this study indicate that SS-14 modulates lipid metabolism when administered in vivo to larval and metamorphosing lampreys. The specific
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effects of SS-14 were tissue- and development-dependent. Somatostatin-14-stimulated lipid depletion proceeded from the hydrolysis of triacylglycerol in storage sites and resulted in elevated plasma fatty acid levels. Somatostatin-14 also inhibited fatty acid and triacylglycerol synthesis. It should be noted that we do not yet know the circulating levels of SS-14 in the plasma of lamprey and that the other forms of somatostatin (e.g., forms with [Ser12]-SS-14 at their C-terminus) also may be important in modulating aspects of lamprey growth, development, and metabolism. This is the first time that SS-14 induction of hyperlip-
FIG. 3. Effects of somatostatin-14 (SS-14) on the rate of lipolysis, as indicated by in triacylglycerol lipase (TGL) activity, in the (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (saline-treated) and SS-14-treated animals.
FIG. 2. Effects of somatostatin-14 (SS-14) on total lipid content of (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (salinetreated) and SS-14-treated animals.
idemia in larval and metamorphosing lampreys has been shown. This observation is consistent with that reported by Plisetskaya (1980) for adult lamprey injected with mammalian SS-14 in January (no effect of SS-14 was observed in animals injected in November). Hyperlipidemic effects of SS-14 also have been observed in salmonids (Sheridan, 1994) and mammals (Peracchi et al., 1974; Christensen et al., 1974; Gerich et al., 1975). In contrast, SS-14 is hypolipidemic in birds (Strosser et al., 1983). The plasma fatty acid levels in larval and metamorphosing P. marinus observed in this
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study were somewhat lower than those reported in spawning adult P. marinus (John et al., 1977) and adult L. fluviatilis (Plisetskaya and Mazina, 1969). These differences in plasma fatty acid levels of lamprey may depend on environmental and physiological factors (e.g., season, age, sex, intrinsic activity, other hormones) (Plisetskaya, 1980). The pronounced rise in plasma fatty acids after SS-14 administration observed in this study results from mobilization of depot reserves. This conclusion is supported by the observation that SS-14-induced in-
FIG. 5. Effects of somatostatin-14 (SS-14) on the rate of triacyglycerol synthesis, as indicated by diacylglycerol acyltransferase (DGAT) activity, in the (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (saline-treated) and SS-14-treated animals.
FIG. 4. Effects of somatostatin-14 (SS-14) on the rate of de novo fatty acid synthesis, as indicated by acetyl-CoA carboxylase (ACC) activity, in the (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (salinetreated) and SS-14-treated animals.
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creases in plasma fatty acid levels in larval lamprey were attended by enhanced renal and hepatic rates of lipolysis. Because of the short duration of the experiment, it was not surprising that significant lipid depletion as measured by tissue parameters (e.g., tissue mass, lipid concentration; except hepatic total lipid content) was not observed. Somatostatin-14-stimulated lipid depletion in larval lamprey kidney also was supported by reduced TG synthesis and, to a lesser extent, a lower rate of fatty acid synthesis. In SS-14treated stage 6 metamorphosing animals, elevated plasma fatty acid levels were attended by a reduced
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rate of fatty acid synthesis in the kidney, liver, and muscle and by a reduced rate of TG synthesis in the muscle. The difference in SS-14 responsiveness between larval lamprey and stage 6 metamorphosing animals (the later displaying refractoriness to SS-14 with regard to TGL) is not known but may be related to development-associated differences in SS receptor characteristics. Lipid depletion also has been observed in salmon (Eilertson and Sheridan, 1993) and mammals (Simon and Calle, 1987) treated with SS-14 in vivo. In salmonids, SS-14-stimulated lipolysis was accompanied by reduced lipogenesis, as indicated by decreased glucose-6-phosphate dehydrogenase activity (Eilertson and Sheridan, 1993). The mechanism by which SS-14 modulates lipid depletion in lamprey is not known. It appears likely, however, that SS-14 acts indirectly by interacting with thyroid hormones (THs) and insulin (INS). This conclusion is supported by several lines of evidence. First, acute immunoneutralization of lamprey SS-14 resulted in increased plasma TH levels and in increased plasma INS (Youson et al., 1992; unpublished data). Second, treatment with THs, which blocked potassium perchlorate (KClO4, a goitrogen)-induced lamprey metamorphosis, completely reversed the KClO4-induced lipid depletion pattern (Kao et al., 1996). Third, INS injection resulted in decreased plasma fatty acid levels of larval and metamorphosing lamprey as well as reduced lipolysis and enhanced lipogenesis (Kao et al., 1997c). Finally, increased intestinal-pancreatic SS concentration (Elliott and Youson, 1991) during spontaneous metamorphosis of sea lamprey, P. marinus, was observed to coincide with decreased thyroid hormone concentrations (Youson et al., 1994), increased plasma INS levels (Youson et al., 1994), and depleted total lipids from depot tissues (reviewed by Sheridan and Kao, 1997). The possibility also remains that SS-14 acts directly on lamprey target organs as reported for salmonid liver (Sheridan and Bern, 1986) and adipose tissues (Eilertson and Sheridan, 1994). In summary, we have shown that SS-14 modulates lipid metabolism of larval and metamorphosing lamprey. Somatostatin-14-stimulated lipid depletion is reminiscent of the pattern of lipid metabolism (phase II; characterized by enhanced lipid catabolism) displayed by lamprey during their spontaneous metamorphosis (Kao et al., 1997a,b) and suggests that SS-14 may
play a role, possibly in concert with other factors, in coordinating metamorphosis-associated changes in lipid metabolism.
ACKNOWLEDGMENTS We gratefully acknowledge R. G. Manzon and J. A. Heinig for their technical assistance. This work was supported by grants from the National Science Foundation, U.S.A. (IBN 9723058 and OSR 9452892) to M.A.S., from the Great Lakes Fishery Commission to J.H.Y. and J.A.H., and from the Natural Sciences and Engineering Council of Canada (A5945) to J.H.Y.
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184 L. (1988). Somatostatin-related and glucagon-related peptides with unusual structural features from the European eel (Anguilla anguilla). Gen. Comp. Endocrinol. 72, 181–189. Conlon, J. M., Bondareva, V., Rusakov, Y., Plisetskaya, E. M., Mynarcik, D. C., and Whittaker, J. (1995a). Characterization of insulin, glucagon, and somatostatin from the river lamprey, Lampetra fluviatilis. Gen. Comp. Endocrinol. 100, 96–105. Conlon, J. M., Nielsen, P. F., Youson, J. H., and Potter, I. C. (1995b). Proinsulin and somatostatin from the islet organ of the southernhemisphere lamprey Geotria australis. Gen. Comp. Endocrinol. 100, 413–422. Conlon, J. M., Tostivint, H., and Vaudry, H. (1997). Somatostatin- and urotensin II-related peptides: Molecular diversity and evolutionary perspectives. Regul. Peptides 69, 95–103 Cutfield, S. M., Carne, A., and Cutfield, J. F. (1987). The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY. FEBS Lett. 214, 57–61. Eilertson, C. D., and 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. Eilertson, C. D., and Sheridan, M. A. (1994). Effects of somatostatin-25 on lipid mobilization from rainbow trout Oncorhynchus mykiss, liver and adipose tissue incubated in vitro: Comparison with somatostatin-14. J. Comp. Physiol. B 164, 256–260. Elliott, W. M., and Youson, J. H. (1987). Immunohistochemical observations of the endocrine pancreas during metamorphosis of the sea lamprey, Petromyzon marinus L. Cell Tissue Res. 247, 351–357. Elliott, W. M., and Youson, J. H. (1991). Somatostatin concentrations in the pancreatic-intestinal tissues of the sea lamprey, Petromyzon marinus L., at various periods of its life cycle. Comp. Biochem. Physiol. A 99, 357–360. Gerich, J. E., Lorenzi, M., Hane, S., Gustafson, G., Guillemin, R., and Forsham, P. H. (1975). Evidence for a physiological role of pancreatic glucagon in human glucose homeostasis: studies with somatostatin. Metabolism 24, 175–182. John, T. M., Tomas, E., George, J. C., and Beamish, F. W. H. (1977). Effect of vasotocin on plasma free fatty acid level in the migrating anadromous sea lamprey. Arch. Int. Physiol. Biochim. 85, 865–870. Kao, Y.-H., Youson, J. H., Manzon, R. G., and Sheridan, M. A. (1996). Role of thyroid hormones on lipid metabolism of lamprey (Petromyzon marinus) associated with metamorphosis. Am. Zool. 36, 97A. Kao, Y.-H., Youson, J. H., and Sheridan, M. A. (1997a). Differences in the total lipid and lipid class composition of larvae and metamorphosing sea lamprey, Petromyzon marinus. Fish Physiol. Biochem. 16, 281–290. Kao, Y.-H., Youson, J. H., Holmes, J. A., and Sheridan, M. A. (1997b). Changes in lipolysis and lipogenesis in selected tissues of the landlocked lamprey, Petromyzon marinus, during metamorphosis. J. Exp. Zool. 277, 301–312. Kao, Y.-H., Youson, J. H., Holmes, J. A., and Sheridan, M. A. (1997c). ‘‘Role of Pancreatic Hormones on Lipid Metabolism of Lamprey, Petromyzon marinus, Associated with Metamorphosis.’’ XIII International Congress of Comparative Endocrinology, Yokohama, Japan. [Abstract]
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Kao et al. Noma, A., Okabe, H., and Kita, M. (1973). A new colorimetric micro-determination of free fatty acids in serum. Clin. Chim. Acta 43, 317–320. Oyama, H., Bradshaw, R. A., Bates, O. J., and Permutt, A. (1980). Amino acid sequence of catfish pancreatic somatostatin I. J. Biol. Chem. 255, 2251–2254. Patel, Y. C. (1992). General aspects of the biology and function of somatostatin. In ‘‘Basic and Clinical Aspects of Neuroscience’’ (C. Weil, E. E. Muller, and M. D. Thorner, Eds.), Vol. 4, pp. 1–16. Springer-Verlag, New York. Peracchi, M., Reschini, E., Cantalamessa, L., Giustina, G., Cavagnini, F., Pinto, M., and Bulgheroni, P. (1974). Effect of somatostatin on blood glucose, plasma growth hormone, insulin, and free fatty acids in normal subjects and acromegalic patients. Metabolism 23, 1009–1015. Plisetskaya, E. M. (1980). Fatty acid levels in blood of cyclostomes and fish. Environ. Biol. Fish. 5, 273–290. Plisetskaya, E. M., and Mazina, T. I. (1969). The influence of hormones on nonesterified fatty acids in the blood of the Baltic lamprey, Lampetra fluviatilis. J. Evol. Biochem. Physiol. 5, 374–378. Plisetskaya, E. M., Pollock, H. G., Rouse, J. B., Hamilton, J. W., Kimmel, J. R., Andrews, P. C., and Gorbman, A. (1986). Characterization of coho salmon (Oncorhynchus kisutch) islet somatostatins. Gen. Comp. Endocrinol. 63, 242–263. Sheridan, M. A. (1994). Regulation of lipid metabolism in poikilothermic vertebrates. Comp. Biochem. Physiol. B 107, 495–508. Sheridan, M. A., and Bern, H. A. (1986). Both somatostatin and the caudal neuropeptide, urotensin II, stimulate lipid mobilization from coho salmon liver incubated in vitro. Regul. Peptides 14, 333–344. Sheridan, M. A., and Kao, Y.-Y. (1997). Regulation of metamorphosisassociated changes in the lipid metabolism of selected vertebrates. Am. Zool. 38, 350–368. Simon, M. A., and Calle, C. (1987). Lipolytic effect of somatostatin in rat adipose tissue: an in vivo and in vitro study. In ‘‘Gut Regulatory Peptides—Their Role in Health and Disease: Frontiers in Hormone Research’’ (E. Blazquez, Ed.), Vol. 16, pp. 111–120. Karger, Basel. Sower, S. A., Chiang, Y.-C., and Conlon, J. M. (1994). Polygenic expression of somatostatin in lamprey. Peptides 15, 151–154. Strosser, M. T., Foltzer, C., Cohen, L., and Mialhe, P. (1983). Evidence for an indirect effect of somatostatin on glucagon secretion via inhibition of free fatty acid release in the duck. Horm. Metab. Res. 15, 279–283. Vaudry, H., Chartrel, N., and Conlon, J. M. (1992). Isolation of [Pro2, Met13]-somatostatin-14 and somatostatin-14 from the frog brain reveals the existence of a somatostatin gene family in a tetrapod. Biochem. Biophys. Res. Commun. 188, 477–482. Youson, J. H., and Cheung, R. (1990). Morphogenesis of somatostatin- and insulin-secreting cells in the lamprey endocrine pancreas. Fish Physiol. Biochem. 8, 389–397. Youson, J. H., Elliott, W. M., Beamish, R. J., and Wang, D. W. (1988). A comparison of endocrine pancreatic tissue in adults of four species of lampreys in British Columbia: A morphological and immunohistochemical study. Gen. Comp. Endocrinol. 70, 247–261.
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Youson, J. H., Leatherland, J. F., Plisetskaya, E. M., and Sheridan, M. A. (1992). ‘‘Metabolic and Hormonal Changes in Larval Lampreys (Petromyzon marinus) Following Acute Neutralization with Anti-insulin and Anti-Somatostatin.’’ 2nd International Symposium on Fish Endocrinology, Saint-Malo, France. Youson, J. H., and Potter, I. C. (1979). A description of the stages in the metamorphosis of the anadromous sea lamprey, Petromyzon marinus L. Can. J. Zool. 57, 1808–1817. Youson, J. H., and Potter, I. C. (1993). An immunohistochemical
185 study of enteropancreatic endocrine cells in larvae and juveniles of the Southern-Hemisphere lampreys Geotria australis and Mordacia mordax. Gen. Comp. Endocrinol. 92, 151–167. Youson, J. H., Plisetskaya, E. M., and Leatherland, J. F. (1994). Concentrations of insulin and thyroid hormones in the serum of landlocked sea lampreys (Petromyzon marinus) of three larval year classes, in larva exposed to two temperature regimes, and in individuals during and after metamorphosis. Gen. Comp. Endocrinol. 94, 294–304.
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Effects of Somatostatin on Lipid Metabolism of Larvae and Metamorphosing Landlocked Sea Lamprey, Petromyzon marinus Yung-hsi Kao,* John H. Youson,† John A. Holmes,† and Mark A. Sheridan* *Department of Zoology and Regulatory Bioscience Center, North Dakota State University, Fargo, North Dakota 58105–5517; and †Department of Zoology and Division of Life Sciences, University of Toronto at Scarborough, Scarborough, Ontario M1C 1A4, Canada Accepted April 22, 1998
This study was designed to examine the role of somatostatin in regulating changes in lipid metabolism of larvae and metamorphosing landlocked sea lamprey, Petromyzon marinus. Larvae and animals in late metamorphosis (stage 6 on a 7-stage scale) were injected intraperitoneally once per day for 2 days with either saline (0.6%) or somatostatin-14 (SS-14; 500 ng/g body wt). Injection of SS-14 into larval and stage 6 metamorphosing animals resulted in elevated plasma fatty acids levels. In larvae, SS-14-induced hyperlipidemia was supported by enhanced lipolysis, as indicated by increased triacylglycerol lipase (TGL) activity in the liver and kidney. Mobilization of larval renal lipid was accompanied by reduced TG synthesis, as indicated by decreased diacylglycerol acyltransferase (DGAT) activity. In stage 6 metamorphosing lamprey, SS-14 did not significantly affect TGL activity; however, SS-14 significantly reduced fatty acid synthesis, as measured by acetyl-CoA carboxylase activity, in kidney, liver, and muscle, as well as muscular TG synthesis. SS-14-stimulated lipid depletion is reminiscent of the pattern of lipid metabolism displayed by P. marinus during their spontaneous metamorphosis—an observation which suggests that somatostatin may play a role in metamorphosis-associated changes in lipid metabolism in this species. r 1998 Academic Press Key Words: somatostatin; lipolysis; lipogenesis; lamprey; Petromyzon marinus; metamorphosis.
0016-6480/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
Somatostatins (SSs) are a peptide hormone family that display considerable structural and functional diversity among vertebrates. The first described SS was a 14-amino-acid peptide originally isolated from ovine hypothalamus and found to inhibit growth hormone secretion from the pituitary gland (Brazeau et al., 1973). Subsequent research revealed that SSs exist in a variety of molecular forms and that they are important in coordinating various aspects of animal growth, development, and metabolism (Patel, 1992; Sheridan, 1994). Some of the molecular heterogeneity is explained by differential processing of a single precursor to yield peptides of different lengths; in some tissues the precursor is processed to SS-14, while in others it may be processed to longer molecules that contain SS-14 at their C-terminus (e.g., mammalian somatostatin-28, which contains SS-14 at its C-terminus; the precursor that contains SS-14 at its C-terminus is known as preprosomatostatin-1, PPSS-1; Conlon et al., 1997). The distribution of SSs among nonmammalian vertebrates suggests that there has been strong selection to conserve the tetradecapeptide, SS-14. Peptides identical in sequence to that originally isolated from sheep brain have been isolated from birds, reptiles, amphibians, teleost fish, and elasmobranchs, as well as from both extant groups of cyclostomes, lamprey and hagfish (reviewed by Conlon et al., 1997). The molecular heterogeneity of the SS family also is
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explained by the existence of alternate SS genes that give rise to precursors other than PPSS-1. Lamprey, numerous teleost fish (Oyama et al., 1980; Plisetskaya et al., 1986; Conlon et al., 1987, 1988; Cutfield et al., 1987), and frogs (Vaudry et al., 1992) appear to express PPSS-1 in addition to a second preprosomatostatin. In sea lamprey, Petromyzon marinus, for example, SS-14 was localized from the brain of adults (Sower et al., 1994) while four alternate forms of SS were isolated from the pancreas: a 14-amino-acid form that contains a Ser for Thr substitution at position 12 and three N-terminally extended peptides with 31, 34, or 37 amino acids all with [Ser12]-SS-14 at their C-termini (all presumably derived from the same precursor by differential processing; Andrews et al., 1988). Conlon and co-workers isolated an SS-35 from the pancreas of brook lamprey, Lampetra fluviatilis (1995a), and an SS-33 from the pancreas of the Southern Hemisphere lamprey, Geotria australis (Conlon et al., 1995b), both containing [Ser12]SS-14 at their C-terminus. Immunocytochemical evidence suggests that only SS-14 is present in larval and metamorphosing animals; SS-14-immunoreactive cells were detected in brain, gut, and pancreas (Elliot and Youson, 1987; Youson et al., 1988; Cheung et al., 1990, 1991; Cheung and Youson, 1991; Youson and Potter, 1993). Changes in intestinal-pancreas levels of SS as measured by a heterologous radioimmunoassay were recently measured during the various phases of the life cycle of sea lamprey, P. marinus, and were shown to increase during their spontaneous metamorphosis (Elliot and Youson, 1991). Increased tissue SS concentration from stage 4 to stage 7 of lamprey metamorphosis, most likely explained by increased size of the endocrine pancreas (Youson and Cheung, 1990), is, interestingly, consistent with (1) decreased lipid content both from the whole body and from lipid depot organs (e.g., liver, kidney, and muscle), and with (2) enhanced lipolysis and reduced lipogenesis from the lipid depots (reviewed by Sheridan and Kao, 1997). The first report of a role of SS in coordinating lipid metabolism of lamprey was cited by Plisetskaya (1980), who pointed out that injection of mammalian SS-14 into adult lamprey, L. fluviatilis, elevated plasma fatty acid levels; however, this response was dependent on the dose of mammalian SS-14 administration and the season when animals were used. There has been no study of the role
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Kao et al.
of SS in regulating lipid metabolism in larval and metamorphosing lampreys. In this study, the landlocked form of sea lamprey, P. marinus, was used to evaluate the role of SS-14 in regulating lipid metabolism in larvae and metamorphosing lamprey. Our hypothesis was that SS-14 stimulates lipid depletion from storage sites and results in elevated plasma fatty acid levels.
MATERIALS AND METHODS Experimental Animals Sea lamprey, P. marinus, larvae were first collected from Oshawa Creek, a tributary of Lake Ontario, Ontario, Canada, on May 31 and June 1, 1996, and transported to the University of Toronto at Scarborough Campus, Ontario, Canada, where they were fed baker’s yeast once a week (18 g/25 larvae). Animals were maintained in 21-L glass aquaria with a sandy substrate and 12 L of dechlorinated City of Toronto water at an ambient temperature of 15–18°C under a photoperiod of 15 h light and 9 h dark.
In Vivo SS-14 Injection Larvae and animals in late metamorphosis (stage 6 on a 7-stage scale; cf. Youson and Potter, 1979) were segregated and placed into separate aquaria (10 larvae/ tank; 5 stage 6 metamorphosing animals/tank) 1 week before experimentation. Feeding was suspended 2 days prior to experimentation. Lampreys were anesthetized by immersion in buffered 0.05% (w/v) tricaine methanesulfonate (MS-222) and injected intraperitoneally (10 µl/g body wt) with either 0.6% saline (control) or mammalian SS-14 (Sigma, St. Louis, MO) at a dose of 500 ng SS-14 (dissolved in 0.6% NaCl)/g body wt once per day for a 2-day period. During the period of experimentation from September 18, 1996, to September 20, 1996, water and sand substrate in the aquaria were not changed. Twelve to 14 h after the last injection, animals were anesthetized individually with buffered MS-222 as described above and were weighed and measured. Blood was collected into heparinized capillary tubes from the severed caudal vasculature,
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allowed to clot overnight at 4°C, and then centrifuged (6000g for 5 min). Plasma was collected and stored at 270°C for later determination of fatty acids. Tissues (liver, kidney, muscle, and intestine) were removed, frozen in liquid N2, and then stored at 270°C for later determination of total lipid and enzyme activities. This protocol was developed based upon preliminary experiments that showed that SS-14 doses similar to those used previously by Plisetskaya (1980) resulted in a somatostatin-induced biological response (i.e., elevated plasma fatty acids; cf. Sheridan, 1994).
Biochemical Analysis Plasma fatty acids were measured by the micromethod of Noma et al. (1973). Total lipids and enzyme activities in selected tissues (liver, kidney, muscle, and intestine) were analyzed essentially as described by Kao et al. (1997a,b). Protein content of the enzyme preparations was determined by dye binding (Bradford, 1976) using a Bio-Rad (Richmond, CA) microplate reader and bovine serum albumin (Sigma) as a standard. The rate of lipid breakdown was evaluated by assessing triacylglycerol lipase (TGL; EC 3.1.1.2) activity. Units of enzyme activity are expressed as picomoles of [3H]oleic acid released per hour per milligram of protein from [3H]triolein (tri-[9,10(n)3H]oleate glycerol). The rate of lipid synthesis was evaluated by assessing separatively de novo fatty acid synthesis, as determined by acetyl-CoA carboxylase (ACC; EC 6.4.1.2) activity, and triacylglycerol synthesis, as determined by diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity. Units of ACC activity and DGAT activity are expressed as femtomoles of [14C]malonyl-CoA formed per hour per milligram of protein and picomoles of [14C]TG formed per minute per milligram of protein, respectively.
Statistical Analysis Data are expressed as means 6 SEM. The Student t test was used to examine differences between the control and the SS-14-treated group. A probability level of 0.05 was used to indicate significance. All statistics were performed using SigmaStat (Jandel Scientific, Palo Alto, CA).
RESULTS Body Characteristics While most body characteristics, including body weight, body length, and condition factor, of larval and metamorphic lampreys were not affected by SS-14 treatment over the course of the 2-day experiment (Table 1), the total mass of liver and the hepatosomatic index were significantly lower in larvae after SS-14 administration compared to controls.
Plasma Fatty Acid Concentration There was a development-dependent difference (p , 0.05) in plasma FA levels. The FA levels in control animals were 0.27 6 0.03 µmol/ml in larvae and 0.49 6 0.02 µmol/ml in stage 6 metamorphosing animals, respectively. Treatment with SS-14 significantly elevated FA levels in larvae and in stage 6 transformers (Fig. 1).
Tissue Lipid Content Somatostatin-14 injection into larvae and stage 6 metamorphosing animals did not affect total lipid content of kidney, muscle, and intestine. However, hepatic total lipid content of larvae treated with SS-14 was significantly decreased by 28% (Fig. 2). The hepatic total lipid content of stage 6 metamorphosing animals was not affected by SS-14 administration.
The Rate of Lipolysis The rate of lipolysis was assessed by changes in TGL activity (Fig. 3). Following SS-14 injection, generally enhanced rates of lipid breakdown were observed in larvae and stage 6 metamorphosing animals. In larval kidney and liver, TGL activity was significantly increased when animals were treated with the SS-14. In stage 6 animals, TGL activity increased, although not significantly so. Triacylglycerol lipase activity in muscle and intestine was not affected by SS-14.
The Rate of de Novo Fatty Acid Synthesis The rate of de novo fatty acid synthesis was assessed by changes in ACC activity (Fig. 4). In larvae, injection
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TABLE 1 In Vivo Effects of Mammalian Somatostatin-14 (SS-14) on Body Characteristics of Larvae and Stage 6 Metamorphosing Lamprey, Petromyzon marinusa Larvae
Stage 6
Parameter
Control
SS-14
Control
SS-14
Body weight (g) Body length (cm) Condition factorb Wet organ weight (mg) Liver Kidney Intestine Muscle Organosomatic Index [(wet organ, g/body wt, g) 3 100%] HSIc KSIc ISIc MSIc
2.78 6 0.07 123.1 6 1.2 1.49 6 0.03
2.71 6 0.09 120.9 6 0.9 1.53 6 0.02
3.04 6 0.23 127.8 6 2.9 1.45 6 0.02
3.39 6 0.29 131.0 6 5.0 1.50 6 0.04
23.1 6 1.5 79.9 6 8.0 38.8 6 3.4 170.8 6 7.7
18.1 6 1.4* 88.8 6 11.0 43.9 6 2.5 161.8 6 6.9
14.4 6 0.9 114.7 6 15.4 76.8 6 6.3 170.0 6 16.8
15.6 6 1.4 132.5 6 32.9 81.9 6 9.7 171.1 6 16.1
0.47 6 0.01 3.81 6 0.56 2.55 6 0.20 5.56 6 0.21
0.46 6 0.01 3.71 6 0.65 2.41 6 0.17 5.07 6 0.34
0.84 6 0.07 2.85 6 0.26 1.39 6 0.11 6.14 6 0.21
0.66 6 0.04* 3.22 6 0.35 1.64 6 0.11 5.99 6 0.24
a SS-14 was injected into each animal at a dose of 500 ng per gram of body weight; control animals received 0.6% saline. Data are presented as means 6 SEM, where n 5 10 for larvae and n 5 5 for stage 6 animals. Statistical differences between the control and SS-14 treatment groups were assessed by the Student t test (*P , 0.05). b Calculated as body weight (g)/(body length, mm)3 3 106. c Abbreviations: HSI, hepatosomatic index; KSI, kidney-somatic index; ISI, intestinal-somatic index; MSI, muscular-somatic index.
of SS-14 tended to reduce ACC activity in kidney, liver, and intestine. In stage 6 metamorphosing animals, ACC activity was significantly decreased in the kidney, liver, and muscle after SS-14 administration.
The Rate of Triacylglycerol Synthesis The rate of triacylglycerol synthesis was assessed by changes in DGAT activity (Fig. 5). In larval lamprey, treatment with SS-14 significantly decreased DGAT activity in the kidney and tended to decrease the activity of this enzyme in liver and muscle. In stage 6 metamorphosing animals, SS-14 did not significantly affect DGAT activity in kidney and liver, but hormone treatment did significantly decrease DGAT activity in muscle.
FIG. 1. Effects of somatostatin-14 (SS-14) on plasma fatty acid levels in larvae and stage 6 metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM (n 5 10 for larvae and n 5 5 for stage 6 metamorphosing animals at each treatment). An asterisk indicates a significant difference (P , 0.05) between the control group (saline-treated) and SS-14-treated animals.
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DISCUSSION The results of this study indicate that SS-14 modulates lipid metabolism when administered in vivo to larval and metamorphosing lampreys. The specific
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effects of SS-14 were tissue- and development-dependent. Somatostatin-14-stimulated lipid depletion proceeded from the hydrolysis of triacylglycerol in storage sites and resulted in elevated plasma fatty acid levels. Somatostatin-14 also inhibited fatty acid and triacylglycerol synthesis. It should be noted that we do not yet know the circulating levels of SS-14 in the plasma of lamprey and that the other forms of somatostatin (e.g., forms with [Ser12]-SS-14 at their C-terminus) also may be important in modulating aspects of lamprey growth, development, and metabolism. This is the first time that SS-14 induction of hyperlip-
FIG. 3. Effects of somatostatin-14 (SS-14) on the rate of lipolysis, as indicated by in triacylglycerol lipase (TGL) activity, in the (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (saline-treated) and SS-14-treated animals.
FIG. 2. Effects of somatostatin-14 (SS-14) on total lipid content of (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (salinetreated) and SS-14-treated animals.
idemia in larval and metamorphosing lampreys has been shown. This observation is consistent with that reported by Plisetskaya (1980) for adult lamprey injected with mammalian SS-14 in January (no effect of SS-14 was observed in animals injected in November). Hyperlipidemic effects of SS-14 also have been observed in salmonids (Sheridan, 1994) and mammals (Peracchi et al., 1974; Christensen et al., 1974; Gerich et al., 1975). In contrast, SS-14 is hypolipidemic in birds (Strosser et al., 1983). The plasma fatty acid levels in larval and metamorphosing P. marinus observed in this
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study were somewhat lower than those reported in spawning adult P. marinus (John et al., 1977) and adult L. fluviatilis (Plisetskaya and Mazina, 1969). These differences in plasma fatty acid levels of lamprey may depend on environmental and physiological factors (e.g., season, age, sex, intrinsic activity, other hormones) (Plisetskaya, 1980). The pronounced rise in plasma fatty acids after SS-14 administration observed in this study results from mobilization of depot reserves. This conclusion is supported by the observation that SS-14-induced in-
FIG. 5. Effects of somatostatin-14 (SS-14) on the rate of triacyglycerol synthesis, as indicated by diacylglycerol acyltransferase (DGAT) activity, in the (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (saline-treated) and SS-14-treated animals.
FIG. 4. Effects of somatostatin-14 (SS-14) on the rate of de novo fatty acid synthesis, as indicated by acetyl-CoA carboxylase (ACC) activity, in the (A) kidney, (B) liver, (C) muscle, and (D) intestine of larvae (n 5 10) and stage 6 (n 5 5) metamorphosing lamprey, Petromyzon marinus. Data are expressed as means 6 SEM. An asterisk indicates a significant difference (P , 0.05) between the control group (salinetreated) and SS-14-treated animals.
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creases in plasma fatty acid levels in larval lamprey were attended by enhanced renal and hepatic rates of lipolysis. Because of the short duration of the experiment, it was not surprising that significant lipid depletion as measured by tissue parameters (e.g., tissue mass, lipid concentration; except hepatic total lipid content) was not observed. Somatostatin-14-stimulated lipid depletion in larval lamprey kidney also was supported by reduced TG synthesis and, to a lesser extent, a lower rate of fatty acid synthesis. In SS-14treated stage 6 metamorphosing animals, elevated plasma fatty acid levels were attended by a reduced
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rate of fatty acid synthesis in the kidney, liver, and muscle and by a reduced rate of TG synthesis in the muscle. The difference in SS-14 responsiveness between larval lamprey and stage 6 metamorphosing animals (the later displaying refractoriness to SS-14 with regard to TGL) is not known but may be related to development-associated differences in SS receptor characteristics. Lipid depletion also has been observed in salmon (Eilertson and Sheridan, 1993) and mammals (Simon and Calle, 1987) treated with SS-14 in vivo. In salmonids, SS-14-stimulated lipolysis was accompanied by reduced lipogenesis, as indicated by decreased glucose-6-phosphate dehydrogenase activity (Eilertson and Sheridan, 1993). The mechanism by which SS-14 modulates lipid depletion in lamprey is not known. It appears likely, however, that SS-14 acts indirectly by interacting with thyroid hormones (THs) and insulin (INS). This conclusion is supported by several lines of evidence. First, acute immunoneutralization of lamprey SS-14 resulted in increased plasma TH levels and in increased plasma INS (Youson et al., 1992; unpublished data). Second, treatment with THs, which blocked potassium perchlorate (KClO4, a goitrogen)-induced lamprey metamorphosis, completely reversed the KClO4-induced lipid depletion pattern (Kao et al., 1996). Third, INS injection resulted in decreased plasma fatty acid levels of larval and metamorphosing lamprey as well as reduced lipolysis and enhanced lipogenesis (Kao et al., 1997c). Finally, increased intestinal-pancreatic SS concentration (Elliott and Youson, 1991) during spontaneous metamorphosis of sea lamprey, P. marinus, was observed to coincide with decreased thyroid hormone concentrations (Youson et al., 1994), increased plasma INS levels (Youson et al., 1994), and depleted total lipids from depot tissues (reviewed by Sheridan and Kao, 1997). The possibility also remains that SS-14 acts directly on lamprey target organs as reported for salmonid liver (Sheridan and Bern, 1986) and adipose tissues (Eilertson and Sheridan, 1994). In summary, we have shown that SS-14 modulates lipid metabolism of larval and metamorphosing lamprey. Somatostatin-14-stimulated lipid depletion is reminiscent of the pattern of lipid metabolism (phase II; characterized by enhanced lipid catabolism) displayed by lamprey during their spontaneous metamorphosis (Kao et al., 1997a,b) and suggests that SS-14 may
play a role, possibly in concert with other factors, in coordinating metamorphosis-associated changes in lipid metabolism.
ACKNOWLEDGMENTS We gratefully acknowledge R. G. Manzon and J. A. Heinig for their technical assistance. This work was supported by grants from the National Science Foundation, U.S.A. (IBN 9723058 and OSR 9452892) to M.A.S., from the Great Lakes Fishery Commission to J.H.Y. and J.A.H., and from the Natural Sciences and Engineering Council of Canada (A5945) to J.H.Y.
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