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The Expression of Preprosomatostatin II mRNAs in the Brockmann Bodies of Rainbow Trout, Oncorhynchus mykiss, Is Regulated by Glucose
General and Comparative Endocrinology 118, 150–160 (2000) doi:10.1006/gcen.1999.7452, available online at http://www.idealibrary.com on
The Expression of Preprosomatostatin II mRNAs in the Brockmann Bodies of Rainbow Trout, Oncorhynchus mykiss, Is Regulated by Glucose Melissa M. Ehrman, Gregory T. Melroe, Jeffrey D. Kittilson, and Mark A. Sheridan Department of Zoology and Regulatory Biosciences Center, North Dakota State University, Fargo, North Dakota 58105 Accepted December 15, 1999
We previously characterized two cDNAs that encode for distinct preprosomatostatin molecules containing [Tyr7, Gly10]-somatostatin-14 at their C-termini (PPSS II8 and PPSS II9) and found that these cDNAs were differentially expressed in the endocrine pancreas (Brockmann body) of rainbow trout, Oncorhynchus mykiss. In this study, we examined the control of PPSSII8 mRNA and PPSS II9 mRNA expression by glucose. Fish injected with glucose displayed elevated plasma levels of glucose in association with nearly three-fold higher levels of PPSS II mRNAs compared to saline-injected control animals. Glucose directly stimulated the expression of both PPSS II mRNAs in vitro in a dose-dependent manner; however, glucose was a more potent stimulator of PPSS II9 expression than of PPSS II8 expression. The hexoses, mannose, galactose, and fructose, as well as glucose, all induced the expression of PPSS II mRNAs, whereas, sucrose and the glucose analogs, 3-o-methylglucose and 2-deoxyglucose, were without effect. In addition, the expression of PPSS II mRNAs was stimulated by dihydroxyacetone, pyruvate, lactate, acetate, and citrate. Furthermore, the expression of PPSS II mRNAs was inhibited by iodoacetate, an inhibitor of glycolysis, but was stimulated by dichloroacetate, a stimulator of Krebs cycle flux via pyruvate dehydrogenase activation. Finally, glucose-stimulated PPSS II expression was inhibited by actinomycin. These results indicate that the expression of PPSS II mRNAs in the Brockmann body of trout is regulated by nutrients such as glucose and suggest that
glucose-stimulated expression of PPSS II mRNAs requires the uptake and subsequent metabolism of the sugar and is transcription sensitive. r 2000 Academic Press Key Words: preprosomatostatin expression; somatostatin; glucose; rainbow trout
INTRODUCTION Somatostatins (SSs) are a multifunctional family of peptide hormones (Sheridan et al., 1999). Several groups of vertebrates, including lamprey, teleost fish, and frogs, produce two or more SSs (Conlon et al., 1997). Salmonid fish, for example, possess somatostatin-14 (SS-14), a peptide identical in sequence to that of mammals, as well as a second, more abundant peptide, salmonid SS-25, which contains [Tyr7, Gly10]-SS-14 at its C-terminus (Plisetskaya et al., 1986). We recently showed that rainbow trout express two distinct mRNAs that encode for separate preprosomatostatins (PPSSs) containing [Tyr7, Gly10]-SS-14 at their C-terminus, designated PPSS II8 and PPSS II9 (Moore et al., 1995, 1999; Sheridan et al., 1997), and that these mRNAs are differentially expressed (Moore et al., 1999). Based on the presence of putative processing sites, PPSS II8 could yield [Tyr7, Gly10]-SS-14, as well as an N-terminally extended 28-amino acid peptide, whereas, PPSS II9 could yield [Tyr7, Gly10]-SS-14 in addition to salmonid SS-25 (Moore et al., 1999). 150
0016-6480/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.
151
Glucose Regulates Somatostatin Expression in Trout
In this study, we examined for the first time the effects of glucose on the expression of PPSS mRNAs. The rationale for this work stemmed from previous studies in rainbow trout which revealed that fasting, a condition that is accompanied by short-term hyperglycemia (Sheridan and Mommsen, 1991), altered pancreatic levels of PPSS mRNAs (Ehrman et al., 1999). The results of this study contribute to an overall understanding of how the production and secretion of SS is controlled.
MATERIALS AND METHODS Animals Juvenile rainbow trout (Oncorhynchus mykiss) of both sexes were obtained from Dakota Trout Ranch near Carrington, North Dakota and transported to North Dakota State University where they were maintained in 800-L circular tanks supplied with recirculated (10% make-up volume per day) dechlorinated municipal water at 14°C under a 12:12 h, light–dark photoperiod. Fish were fed to satiety twice daily with Supersweet Feeds (Glenco, MN) trout grower, except for 24–36 h before in vivo or in vitro manipulations. Animals were acclimated to laboratory conditions for at least 4 weeks prior to experiments.
Experimental Conditions For in vivo experiments, animals were transferred to 50-L aquaria (10 fish/aquarium; one aquarium for each treatment of four treatment groups) and their feeding was suspended. Thirty-six hours later, the fish were anesthetized with 0.05% (v/v) 2-phenoxyethanol (Sigma, St. Louis, MO), weighed, injected (10 µL/g body weight) with either 0.75% (w/v) NaCl (control) or 3000 mg/dL glucose (treated) as described previously (Harmon et al., 1991), and then replaced into their aquarium. At various times after injection (3 and 12 h), the animals were reanesthetized and blood and Brockmann bodies were removed. Serum and tissues were immediately frozen on dry ice and stored at 290°C until further analysis, usually within 2 weeks. Hematocrits between the saline- and glucose-injected animals did not differ significantly.
For in vitro experiments, the fish were anesthetized and their Brockmann bodies removed and prepared for culture as described previously (Eilertson and Sheridan, 1995). Isolated hemi-islets were placed in 24-well culture plates (ca. 2–3 hemi-islets per well) and preincubated (14°C, 100% O2, shaken at 100 rpm with a gyratory shaker) for 2 h in 1 mL of basal medium [in mM: 137 NaCl, 5.4 KCl, 4 NaHCO3, 1.7 CaCl2, 0.8 MgSO4, 0.5 KH2PO4, 0.3 Na2HPO4, 10 Hepes, 4 glucose with 0.24% (w/v) bovine serum albumin, pH 7.6]. Following preincubation, the medium was removed, the islets were washed gently with 1 mL of fresh basal medium, and 1 mL of test medium was added to each well. Test solutions were prepared by adjusting the NaCl concentration so that all solutions were isoosmotic. All of the test agents, including hexoses, analogs, and inhibitors, were obtained from Sigma. Incubation proceeded under the same conditions as preincubation for up to 24 h, after which time the medium was removed and the islets were immediately frozen on dry ice. Islets were stored at 290°C until RNA extraction and quantitation.
Analyses Total RNA was extracted from Brockmann bodies using a modification of the RNAzol method (Moore et al., 1995). Preprosomatostatin II8 and PPSS II9 mRNA was measured via a quantitative slot-blot technique (Moore et al., 1999) in which in vitro-synthesized cRNA standards were blotted onto a nylon membrane along with sample RNA. The membranes were hybridized with specific, 32P-labeled oligonucleotide probes (Moore et al., 1999) and quantified with the Packard Cyclone Imaging System (Fig. 1). Sample blots were then stripped and rehybridized with a 32P-labeled fulllength g-actin probe (human fibroblast; Gunning et al., 1983). Sample RNA abundance was calculated after correction for background and differences in probe specific activity and after g-actin normalization, as described previously (Moore et al., 1999). Plasma glucose was determined by the o-toluidine colorimetric assay of Hu¨varinen and Nikkila (1962).
Statistics Statistical differences were estimated by analysis of variance; multiple comparisons among means were
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FIG. 1. Quantitative slot-blot analysis for preprosomatostatin (PPSS) II8 and PPSS II9 mRNAs using gene-specific oligonucleotide probes. Serial dilutions of in vitro-synthesized cRNA standards for PPSS II8 and PPSS II9 mRNA (A) and total RNA extracted from rainbow trout Brockmann bodies (B) were subjected to slot-blot analysis using 32P-labeled oligonucleotide probes. Blots were exposed on a Packard Cyclone imaging system and a standard curve (C) was developed after correction for background.
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made with the Student–Newman–Keul’s test. Differences were considered significant at P , 0.05. For ease of comparison, data were expressed as percentage change (final level 2 initial level/initial level 3 100); statistics were calculated on untransformed data.
mRNA 12 h after injection. Whether or not the effects of glucose on the expression of PPSS II mRNAs was direct was evaluated by in vitro incubation of Brockmann bodies.
In Vitro Experiments
RESULTS In Vivo Experiment The effects of glucose on pancreatic expression of PPSS II mRNAs were first studied in vivo. Injection of fish with glucose (3000 mg/dL) resulted in hyperglycemia that lasted for up to 12 h (Fig. 2), a pattern that was similar to that of our previous observations (Harmon et al., 1991). Steady-state levels of PPSS II8 mRNA and PPSS II9 mRNA in the Brockmann bodies of trout injected with glucose were significantly higher than levels of these mRNAs in control fish 3 h after injection (Fig. 3). Glucose-injected trout continued to display elevated levels of both PPSS II8 mRNA and PPSS II9
Glucose directly stimulated the expression of PPSS II8 mRNA and PPSS II9 mRNA in Brockmann bodies incubated in vitro. Steady-state levels of both PPSS II mRNAs increased in a dose-dependent manner (Fig. 4); the maximum response to glucose was at a concentration of 10 mM. In addition, glucose stimulated the expression of PPSS II9 mRNA to a greater extent than that of PPSS II8 mRNA. The time course of glucose-stimulated (10 mM) PPSS II mRNA expression is shown in Fig. 5. Steady-state levels of both PPSS II8 mRNA and of PPSS II9 mRNA rose quickly in response to glucose, reaching maximum expression after 6 h, and then declined. The mechanism(s) by which glucose stimulated the expression of PPSS II mRNAs was(were) studied in several experiments. The hexoses, mannose, galactose, and fructose, as well as glucose, all induced the
FIG. 2. Effects of glucose injection (3000 mg/dL at 10 µL/g body weight) on plasma glucose concentration. Data are presented as percentage change (mean 6 SEM; n 5 10) from the saline-injected control group. Control levels of glucose were 88.4 6 8.6 and 92.7 6 9.2 mg/dl, respectively, 3 and 12 h after injection. *Significantly different from control (P , 0.05).
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FIG. 3. Effects of glucose injection (3000 mg/dL at 10 µL/g body weight) on the expression of preprosomatostation II mRNAs in the Brockmann bodies of rainbow trout. Data are presented as percentage change (mean 6 SEM; n 5 10) from the saline-injected control group. Control values at 3 and 12 h, respectively, for PPSS II8 were 1.1 6 0.2 molecules of RNA 3 1028/µg total RNA and 0.9 6 0.1 units and for PPSS II9 were 2.4 6 0.3 units and 2.2 6 0.2 units. *Significantly different from control (P , 0.05).
FIG. 4. Effects of varying concentrations of glucose on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 1 mM glucose group (1.4 6 0.3 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 1.9 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another.
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FIG. 5. Time course of glucose (10 mM)-stimulated expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro. Data are presented as percentage change (mean 6 SEM; n 5 6) from the group sampled at time 0 (0.8 6 0.1 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 1.9 6 0.2 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species.
expression of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 6). Sucrose, a disaccharide which does not enter cells readily, did not affect the expression of PPSS II mRNAs. In addition, neither 3-o-methyl glucose, a compound that enters cells but is not phosphorylated, nor 2-deoxyglucose, a compound that enters cells and is then phosphorylated but not metabolized further, altered the steady-state levels of PPSS II mRNAs (Fig. 7). On the other hand, metabolites such as dihydroxyacetone, pyruvate, and lactate significantly stimulated the levels of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 8). An inhibitor of glycolysis, iodoacetate, significantly reduced the levels of PPSS II mRNAs (Fig. 8). Compounds that enter or are intermediates of the Krebs cycle (e.g., acetate, citrate) also elevated PPSS II mRNA levels (Fig. 9). Dichloroacetate, a compound which increases flux through the Krebs cycle by stimulating mitochondrial pyruvate dehydrogenase activity, had a pronounced effect on the levels of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 9). Actinomycin D, an inhibitor of RNA transcription, inhibited glucose-stimulated expression of PPSS II mRNAs (Fig. 10).
DISCUSSION The results of this study indicate that glucose modulates the expression of the two distinct mRNAs in the Brockmann bodies of rainbow trout that encode for PPSSs containing [Tyr7, Gly10]-SS at their C-termini (PPSS II8 and PPSS II9). The effects of glucose on the expression of PPSS II mRNAs are direct and rapid; however, glucose is a more potent stimulator of PPSS II9 mRNA expression than of PPSS II8 mRNA expression. These findings, the first reporting the effects of metabolites on the levels of PPSS mRNAs, suggest that glucose stimulates the biosynthesis of SSs, peptides important in the regulation of growth, development, and metabolism of vertebrates. Glucose-stimulated expression of PPSS II mRNAs requires the uptake and subsequent metabolism of the sugar. This conclusion is supported by several observations. First, a number of hexoses in addition to glucose which enter cells and undergo phosphorylation, isomerization, and metabolism to varying degrees were all
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FIG. 6. Effects of various hexoses on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (1.8 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 3.4 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species; all groups are different from control.
FIG. 7. Effects of sucrose and various glucose analogs on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (1.6 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 3.7 6 0.5 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control.
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FIG. 8. Effects of intermediates and an inhibitor of glycolysis on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (2.1 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 4.0 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control.
FIG. 9. Effects of intermediates and a stimulator of the Krebs cycle on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (1.3 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 2.7 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control. Dichloroacetate increases flux through the Krebs cycle by stimulating mitochondrial pyruvate dehydrogenase activity.
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FIG. 10. Effects of actinomycin D on glucose-stimulated expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (0.8 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 1.8 6 0.3 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control.
capable of stimulating PPSS II mRNA expression. Second, the lack of an effect of sucrose, a disaccharide which does not enter cells readily, excludes the possibility that hexose-stimulated PPSS II mRNA expression was an osmotic effect. Third, that hexose-stimulated PPSS II mRNA expression requires phosphorylation was supported by the failure of the glucose analog 3-o-methyl glucose to alter steady-state levels of PPSS II mRNAs. Phosphorylation alone, however, does not appear to be sufficient for hexose-stimulated PPSS II mRNA expression. This is indicated by the inability of the glucose analog 2-deoxyglucose to affect steadystate levels of PPSS II mRNAs. Last, the requirement for the subsequent metabolism of glucose through glycolysis was indicated by the ability of metabolites such as dihydroxyacetone, pyruvate, and lactate to stimulate the levels of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 8). Moreover, inhibition of glycolysis with iodoacetate significantly reduced the levels of PPSS II mRNAs. The further dependence of glucose-stimulated PPSS II mRNA expression on metabolism of
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substrates through the Krebs cycle was indicated by several observations, including the effects of acetate, citrate, and the pyruvate dehydrogenase stimulator dichloroacetate. Together, the present findings suggest that the proximate mediator of glucose-stimulated PPSS II expression is generated after the aldolase step of glycolysis. The present results with actinomycin D suggest that glucose-stimulated increases in the steady state levels of PPSS II mRNAs arise from altered rates of transcription. Whether the proximate mediators produced during the metabolism of glucose activate specific transacting factors that, in turn, modulate gene expression via carbohydrate-responsive elements in promoter regions of the SS genes is not known. It also should be noted that an influence of glucose metabolism on the stability of PPSS II mRNAs cannot be ruled out. The finding that glucose stimulates the expression of PPSS II mRNAs extends our knowledge of the effects of glucose on SS production. Previously, we have shown that glucose administration in vivo to rainbow
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trout (Harmon et al., 1991) elevated plasma SS levels. In addition, glucose stimulated the secretion of SS-14 from perfused pancreata of mammals (Ipp et al., 1977), eel (Ince and So, 1983), and channel catfish (Ronner and Scarpa, 1987). Glucose also stimulated the release of SS-14 from islets isolated from anglerfish (Milgram et al., 1991), as well as the release of both SS-14 and salmonid SS-25 (a PPSS II9 product containing [Tyr7, Gly10]-SS-14) from islets isolated from rainbow trout (Eilertson and Sheridan, 1995). The regulation of SS biosynthesis and secretion by glucose may have important implications for the nutritional and metabolic physiology of vertebrates. Glucose-modulated SS production provides an important feedback control on the release of other metabolically important hormones, such as insulin and glucagon, in so far as SSs have been shown to inhibit the release of these factors in both mammals (Patel, 1992) and fish (Eilertson and Sheridan, 1993). Particularly noteworthy in the present study was the preferential effects of glucose (in vitro) on the expression of PPSS II9. Based on the sequence of trout PPSS IIs (Moore et al., 1999) and our understanding of precursor processing (Conlon, 1989), such preferential expression could lead to the synthesis of a preponderance of one SS peptide form, salmonid SS-25, over another, salmonid SS-28, suggesting distinctive roles for the various SS isoforms. Growing evidence suggests that there are important structure–function relationships among the SS family of peptides (Sheridan et al., 1999). For example, salmonid SS-25 was more potent in inhibition of glucagon release than SS-14 (Eilertson and Sheridan, 1993). In summary, we have demonstrated that glucose regulates the expression of PPSS II8 mRNA and of PPSS II9 mRNA in the Brockmann body of rainbow trout. Glucose-stimulated alterations in the steadystate levels of PPSS II mRNAs depends upon the phosphorylation and subsequent metabolism of the hexose and is actinomycin sensitive.
ACKNOWLEDGMENTS We thank Leslie Alexander, Darlene Knutson, Marty Pesek, and Bart Slagter for their assistance with these experiments. This research was supported by grants from the U.S. National Science
Foundation (IBN 9723058) and the U.S. Department of Agriculture (98-35206-6410) to M.A.S.
REFERENCES Conlon, J. M. (1989). Biosynthesis of regulatory peptides—Evolutionary perspectives. In ‘‘The Comparative Physiology of Regulatory Peptides’’ (S. Holmgren, Ed.), pp. 174–202. Chapman & Hall, New York. Conlon, J. M., Tostivint, H., and Vaudry, H. (1997). Somatostatin- and urotensin II-related peptides: Molecular diversity and evolutionary perspectives. Regul. Pept. 69, 95–103. Ehrman, M. M., Moore, C. A., Kittilson, J. D., Eilertson, C. D., and Sheridan, M. A. (1999). Nutritional regulation of somatostatin expression in rainbow trout, Oncorhynchus mykiss. J. Endocrinol., submitted. 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. (1995). Pancreatic somatostatin-14 and somatostatin-25 release in rainbow trout is stimulated by glucose and arginine. Am. J. Physiol. 269, R1017–R1023. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H., and Kedes, L. (1983). Isolation and characterization of full-length cDNA clones for a-, b-, g-actin mRNAs: Skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell. Biol. 3, 787–795. Harmon, J. S., Eilertson, C. D., Sheridan, M. A., and Plisetskaya, E. M. (1991). Insulin suppression is associated with hypersomatostatinemia and hyperglucagonemia in glucose-injected rainbow trout. Am. J. Physiol. 261, R609–R613. Hu¨varinen, A., and Nikkila, E. A. (1962). Specific determination of blood glucose with o-toluidine. Clin. Chim. Acta 7, 140–143. Ince, B. W., and So, S. T. (1983). Differential secretion of glucagon-like and somatostatin-like immunoreactivity from the perfused eel pancreas in response to D-glucose. Gen. Comp. Endocrinol. 53, 389–397. Ipp, E., Dobbs, R. E., Arimura, A., Vale, W., Harris, V., and Unger, R. A. (1977). Release of immunoreactive somatostatin from the pancreas in response to glucose, amino acids, pancreaozymincholecystokinin, and tolbutamide. J. Clin. Invest. 60, 760–765. Milgram, S. L., McDonald, J. K., and Noe, B. D. (1991). Neuronal influence on hormone release from anglerfish islet cells. Am. J. Physiol. 261, E444–E456. Moore, C. A., Kittilson, J. D., Dahl, S. K., and Sheridan, M. A. (1995). Isolation and characterization of a cDNA encoding for preprosomatostatin containing [Tyr7, Gly10]-somatostatin-14 from the endocrine pancreas of rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 98, 253–261. Moore, C. A., Kittilson, J. D., Ehrman, M. M., and Sheridan, M. A. (1999). Rainbow trout (Oncorhynchus mykiss) possess two somatostatin mRNAs that are differentially expressed. Am. J. Physiol. 277, R1553–R1561.
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160 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. O. Thorner, Eds.), Vol. 4, pp. 1–16. Springer-Verlag, Berlin. 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. Ronner, P., and Scarpa, A. (1987). Secretagogues for pancreatic hormone release in the channel catfish (Ictalurus punctatus). Gen. Comp. Endocrinol. 65, 354–362.
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Ehrman et al. Sheridan, M. A., and Mommsen, T. P. (1991). Effects of nutritional state on in vivo lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 81, 473–483. Sheridan, M. A., Kittilson, J. D., Ehrman, M. M., and Moore, C. A. (1997). Polygenic expression of somatostatin in rainbow trout. In ‘‘Advances in Comparative Endocrinology’’ (S. Kawashima and S. Kikuyama, Eds.), pp. 291–294. Monduzzi Editore, Bologna. Sheridan, M. A., Kittilson, J. D., and Slagter, B. A. (2000). Structure– function relationships of the signaling system for the somatostatin peptide hormone family. Am. Zool., in press.
The Expression of Preprosomatostatin II mRNAs in the Brockmann Bodies of Rainbow Trout, Oncorhynchus mykiss, Is Regulated by Glucose Melissa M. Ehrman, Gregory T. Melroe, Jeffrey D. Kittilson, and Mark A. Sheridan Department of Zoology and Regulatory Biosciences Center, North Dakota State University, Fargo, North Dakota 58105 Accepted December 15, 1999
We previously characterized two cDNAs that encode for distinct preprosomatostatin molecules containing [Tyr7, Gly10]-somatostatin-14 at their C-termini (PPSS II8 and PPSS II9) and found that these cDNAs were differentially expressed in the endocrine pancreas (Brockmann body) of rainbow trout, Oncorhynchus mykiss. In this study, we examined the control of PPSSII8 mRNA and PPSS II9 mRNA expression by glucose. Fish injected with glucose displayed elevated plasma levels of glucose in association with nearly three-fold higher levels of PPSS II mRNAs compared to saline-injected control animals. Glucose directly stimulated the expression of both PPSS II mRNAs in vitro in a dose-dependent manner; however, glucose was a more potent stimulator of PPSS II9 expression than of PPSS II8 expression. The hexoses, mannose, galactose, and fructose, as well as glucose, all induced the expression of PPSS II mRNAs, whereas, sucrose and the glucose analogs, 3-o-methylglucose and 2-deoxyglucose, were without effect. In addition, the expression of PPSS II mRNAs was stimulated by dihydroxyacetone, pyruvate, lactate, acetate, and citrate. Furthermore, the expression of PPSS II mRNAs was inhibited by iodoacetate, an inhibitor of glycolysis, but was stimulated by dichloroacetate, a stimulator of Krebs cycle flux via pyruvate dehydrogenase activation. Finally, glucose-stimulated PPSS II expression was inhibited by actinomycin. These results indicate that the expression of PPSS II mRNAs in the Brockmann body of trout is regulated by nutrients such as glucose and suggest that
glucose-stimulated expression of PPSS II mRNAs requires the uptake and subsequent metabolism of the sugar and is transcription sensitive. r 2000 Academic Press Key Words: preprosomatostatin expression; somatostatin; glucose; rainbow trout
INTRODUCTION Somatostatins (SSs) are a multifunctional family of peptide hormones (Sheridan et al., 1999). Several groups of vertebrates, including lamprey, teleost fish, and frogs, produce two or more SSs (Conlon et al., 1997). Salmonid fish, for example, possess somatostatin-14 (SS-14), a peptide identical in sequence to that of mammals, as well as a second, more abundant peptide, salmonid SS-25, which contains [Tyr7, Gly10]-SS-14 at its C-terminus (Plisetskaya et al., 1986). We recently showed that rainbow trout express two distinct mRNAs that encode for separate preprosomatostatins (PPSSs) containing [Tyr7, Gly10]-SS-14 at their C-terminus, designated PPSS II8 and PPSS II9 (Moore et al., 1995, 1999; Sheridan et al., 1997), and that these mRNAs are differentially expressed (Moore et al., 1999). Based on the presence of putative processing sites, PPSS II8 could yield [Tyr7, Gly10]-SS-14, as well as an N-terminally extended 28-amino acid peptide, whereas, PPSS II9 could yield [Tyr7, Gly10]-SS-14 in addition to salmonid SS-25 (Moore et al., 1999). 150
0016-6480/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.
151
Glucose Regulates Somatostatin Expression in Trout
In this study, we examined for the first time the effects of glucose on the expression of PPSS mRNAs. The rationale for this work stemmed from previous studies in rainbow trout which revealed that fasting, a condition that is accompanied by short-term hyperglycemia (Sheridan and Mommsen, 1991), altered pancreatic levels of PPSS mRNAs (Ehrman et al., 1999). The results of this study contribute to an overall understanding of how the production and secretion of SS is controlled.
MATERIALS AND METHODS Animals Juvenile rainbow trout (Oncorhynchus mykiss) of both sexes were obtained from Dakota Trout Ranch near Carrington, North Dakota and transported to North Dakota State University where they were maintained in 800-L circular tanks supplied with recirculated (10% make-up volume per day) dechlorinated municipal water at 14°C under a 12:12 h, light–dark photoperiod. Fish were fed to satiety twice daily with Supersweet Feeds (Glenco, MN) trout grower, except for 24–36 h before in vivo or in vitro manipulations. Animals were acclimated to laboratory conditions for at least 4 weeks prior to experiments.
Experimental Conditions For in vivo experiments, animals were transferred to 50-L aquaria (10 fish/aquarium; one aquarium for each treatment of four treatment groups) and their feeding was suspended. Thirty-six hours later, the fish were anesthetized with 0.05% (v/v) 2-phenoxyethanol (Sigma, St. Louis, MO), weighed, injected (10 µL/g body weight) with either 0.75% (w/v) NaCl (control) or 3000 mg/dL glucose (treated) as described previously (Harmon et al., 1991), and then replaced into their aquarium. At various times after injection (3 and 12 h), the animals were reanesthetized and blood and Brockmann bodies were removed. Serum and tissues were immediately frozen on dry ice and stored at 290°C until further analysis, usually within 2 weeks. Hematocrits between the saline- and glucose-injected animals did not differ significantly.
For in vitro experiments, the fish were anesthetized and their Brockmann bodies removed and prepared for culture as described previously (Eilertson and Sheridan, 1995). Isolated hemi-islets were placed in 24-well culture plates (ca. 2–3 hemi-islets per well) and preincubated (14°C, 100% O2, shaken at 100 rpm with a gyratory shaker) for 2 h in 1 mL of basal medium [in mM: 137 NaCl, 5.4 KCl, 4 NaHCO3, 1.7 CaCl2, 0.8 MgSO4, 0.5 KH2PO4, 0.3 Na2HPO4, 10 Hepes, 4 glucose with 0.24% (w/v) bovine serum albumin, pH 7.6]. Following preincubation, the medium was removed, the islets were washed gently with 1 mL of fresh basal medium, and 1 mL of test medium was added to each well. Test solutions were prepared by adjusting the NaCl concentration so that all solutions were isoosmotic. All of the test agents, including hexoses, analogs, and inhibitors, were obtained from Sigma. Incubation proceeded under the same conditions as preincubation for up to 24 h, after which time the medium was removed and the islets were immediately frozen on dry ice. Islets were stored at 290°C until RNA extraction and quantitation.
Analyses Total RNA was extracted from Brockmann bodies using a modification of the RNAzol method (Moore et al., 1995). Preprosomatostatin II8 and PPSS II9 mRNA was measured via a quantitative slot-blot technique (Moore et al., 1999) in which in vitro-synthesized cRNA standards were blotted onto a nylon membrane along with sample RNA. The membranes were hybridized with specific, 32P-labeled oligonucleotide probes (Moore et al., 1999) and quantified with the Packard Cyclone Imaging System (Fig. 1). Sample blots were then stripped and rehybridized with a 32P-labeled fulllength g-actin probe (human fibroblast; Gunning et al., 1983). Sample RNA abundance was calculated after correction for background and differences in probe specific activity and after g-actin normalization, as described previously (Moore et al., 1999). Plasma glucose was determined by the o-toluidine colorimetric assay of Hu¨varinen and Nikkila (1962).
Statistics Statistical differences were estimated by analysis of variance; multiple comparisons among means were
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FIG. 1. Quantitative slot-blot analysis for preprosomatostatin (PPSS) II8 and PPSS II9 mRNAs using gene-specific oligonucleotide probes. Serial dilutions of in vitro-synthesized cRNA standards for PPSS II8 and PPSS II9 mRNA (A) and total RNA extracted from rainbow trout Brockmann bodies (B) were subjected to slot-blot analysis using 32P-labeled oligonucleotide probes. Blots were exposed on a Packard Cyclone imaging system and a standard curve (C) was developed after correction for background.
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made with the Student–Newman–Keul’s test. Differences were considered significant at P , 0.05. For ease of comparison, data were expressed as percentage change (final level 2 initial level/initial level 3 100); statistics were calculated on untransformed data.
mRNA 12 h after injection. Whether or not the effects of glucose on the expression of PPSS II mRNAs was direct was evaluated by in vitro incubation of Brockmann bodies.
In Vitro Experiments
RESULTS In Vivo Experiment The effects of glucose on pancreatic expression of PPSS II mRNAs were first studied in vivo. Injection of fish with glucose (3000 mg/dL) resulted in hyperglycemia that lasted for up to 12 h (Fig. 2), a pattern that was similar to that of our previous observations (Harmon et al., 1991). Steady-state levels of PPSS II8 mRNA and PPSS II9 mRNA in the Brockmann bodies of trout injected with glucose were significantly higher than levels of these mRNAs in control fish 3 h after injection (Fig. 3). Glucose-injected trout continued to display elevated levels of both PPSS II8 mRNA and PPSS II9
Glucose directly stimulated the expression of PPSS II8 mRNA and PPSS II9 mRNA in Brockmann bodies incubated in vitro. Steady-state levels of both PPSS II mRNAs increased in a dose-dependent manner (Fig. 4); the maximum response to glucose was at a concentration of 10 mM. In addition, glucose stimulated the expression of PPSS II9 mRNA to a greater extent than that of PPSS II8 mRNA. The time course of glucose-stimulated (10 mM) PPSS II mRNA expression is shown in Fig. 5. Steady-state levels of both PPSS II8 mRNA and of PPSS II9 mRNA rose quickly in response to glucose, reaching maximum expression after 6 h, and then declined. The mechanism(s) by which glucose stimulated the expression of PPSS II mRNAs was(were) studied in several experiments. The hexoses, mannose, galactose, and fructose, as well as glucose, all induced the
FIG. 2. Effects of glucose injection (3000 mg/dL at 10 µL/g body weight) on plasma glucose concentration. Data are presented as percentage change (mean 6 SEM; n 5 10) from the saline-injected control group. Control levels of glucose were 88.4 6 8.6 and 92.7 6 9.2 mg/dl, respectively, 3 and 12 h after injection. *Significantly different from control (P , 0.05).
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FIG. 3. Effects of glucose injection (3000 mg/dL at 10 µL/g body weight) on the expression of preprosomatostation II mRNAs in the Brockmann bodies of rainbow trout. Data are presented as percentage change (mean 6 SEM; n 5 10) from the saline-injected control group. Control values at 3 and 12 h, respectively, for PPSS II8 were 1.1 6 0.2 molecules of RNA 3 1028/µg total RNA and 0.9 6 0.1 units and for PPSS II9 were 2.4 6 0.3 units and 2.2 6 0.2 units. *Significantly different from control (P , 0.05).
FIG. 4. Effects of varying concentrations of glucose on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 1 mM glucose group (1.4 6 0.3 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 1.9 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another.
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FIG. 5. Time course of glucose (10 mM)-stimulated expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro. Data are presented as percentage change (mean 6 SEM; n 5 6) from the group sampled at time 0 (0.8 6 0.1 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 1.9 6 0.2 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species.
expression of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 6). Sucrose, a disaccharide which does not enter cells readily, did not affect the expression of PPSS II mRNAs. In addition, neither 3-o-methyl glucose, a compound that enters cells but is not phosphorylated, nor 2-deoxyglucose, a compound that enters cells and is then phosphorylated but not metabolized further, altered the steady-state levels of PPSS II mRNAs (Fig. 7). On the other hand, metabolites such as dihydroxyacetone, pyruvate, and lactate significantly stimulated the levels of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 8). An inhibitor of glycolysis, iodoacetate, significantly reduced the levels of PPSS II mRNAs (Fig. 8). Compounds that enter or are intermediates of the Krebs cycle (e.g., acetate, citrate) also elevated PPSS II mRNA levels (Fig. 9). Dichloroacetate, a compound which increases flux through the Krebs cycle by stimulating mitochondrial pyruvate dehydrogenase activity, had a pronounced effect on the levels of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 9). Actinomycin D, an inhibitor of RNA transcription, inhibited glucose-stimulated expression of PPSS II mRNAs (Fig. 10).
DISCUSSION The results of this study indicate that glucose modulates the expression of the two distinct mRNAs in the Brockmann bodies of rainbow trout that encode for PPSSs containing [Tyr7, Gly10]-SS at their C-termini (PPSS II8 and PPSS II9). The effects of glucose on the expression of PPSS II mRNAs are direct and rapid; however, glucose is a more potent stimulator of PPSS II9 mRNA expression than of PPSS II8 mRNA expression. These findings, the first reporting the effects of metabolites on the levels of PPSS mRNAs, suggest that glucose stimulates the biosynthesis of SSs, peptides important in the regulation of growth, development, and metabolism of vertebrates. Glucose-stimulated expression of PPSS II mRNAs requires the uptake and subsequent metabolism of the sugar. This conclusion is supported by several observations. First, a number of hexoses in addition to glucose which enter cells and undergo phosphorylation, isomerization, and metabolism to varying degrees were all
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FIG. 6. Effects of various hexoses on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (1.8 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 3.4 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species; all groups are different from control.
FIG. 7. Effects of sucrose and various glucose analogs on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (1.6 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 3.7 6 0.5 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control.
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FIG. 8. Effects of intermediates and an inhibitor of glycolysis on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (2.1 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 4.0 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control.
FIG. 9. Effects of intermediates and a stimulator of the Krebs cycle on the expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (1.3 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 2.7 6 0.4 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control. Dichloroacetate increases flux through the Krebs cycle by stimulating mitochondrial pyruvate dehydrogenase activity.
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FIG. 10. Effects of actinomycin D on glucose-stimulated expression of preprosomatostatin (PPSS) II mRNAs in rainbow trout Brockmann bodies incubated in vitro for 6 h. Data are presented as percentage change (mean 6 SEM; n 5 6) from the 4 mM glucose control group (0.8 6 0.2 molecules of RNA 3 1028/µg total RNA for PPSS II8 and 1.8 6 0.3 units for PPSS II9). Groups with different letters are significantly (P , 0.05) different from one another for a given mRNA species. *Significantly different from control.
capable of stimulating PPSS II mRNA expression. Second, the lack of an effect of sucrose, a disaccharide which does not enter cells readily, excludes the possibility that hexose-stimulated PPSS II mRNA expression was an osmotic effect. Third, that hexose-stimulated PPSS II mRNA expression requires phosphorylation was supported by the failure of the glucose analog 3-o-methyl glucose to alter steady-state levels of PPSS II mRNAs. Phosphorylation alone, however, does not appear to be sufficient for hexose-stimulated PPSS II mRNA expression. This is indicated by the inability of the glucose analog 2-deoxyglucose to affect steadystate levels of PPSS II mRNAs. Last, the requirement for the subsequent metabolism of glucose through glycolysis was indicated by the ability of metabolites such as dihydroxyacetone, pyruvate, and lactate to stimulate the levels of PPSS II8 mRNA and PPSS II9 mRNA (Fig. 8). Moreover, inhibition of glycolysis with iodoacetate significantly reduced the levels of PPSS II mRNAs. The further dependence of glucose-stimulated PPSS II mRNA expression on metabolism of
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substrates through the Krebs cycle was indicated by several observations, including the effects of acetate, citrate, and the pyruvate dehydrogenase stimulator dichloroacetate. Together, the present findings suggest that the proximate mediator of glucose-stimulated PPSS II expression is generated after the aldolase step of glycolysis. The present results with actinomycin D suggest that glucose-stimulated increases in the steady state levels of PPSS II mRNAs arise from altered rates of transcription. Whether the proximate mediators produced during the metabolism of glucose activate specific transacting factors that, in turn, modulate gene expression via carbohydrate-responsive elements in promoter regions of the SS genes is not known. It also should be noted that an influence of glucose metabolism on the stability of PPSS II mRNAs cannot be ruled out. The finding that glucose stimulates the expression of PPSS II mRNAs extends our knowledge of the effects of glucose on SS production. Previously, we have shown that glucose administration in vivo to rainbow
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trout (Harmon et al., 1991) elevated plasma SS levels. In addition, glucose stimulated the secretion of SS-14 from perfused pancreata of mammals (Ipp et al., 1977), eel (Ince and So, 1983), and channel catfish (Ronner and Scarpa, 1987). Glucose also stimulated the release of SS-14 from islets isolated from anglerfish (Milgram et al., 1991), as well as the release of both SS-14 and salmonid SS-25 (a PPSS II9 product containing [Tyr7, Gly10]-SS-14) from islets isolated from rainbow trout (Eilertson and Sheridan, 1995). The regulation of SS biosynthesis and secretion by glucose may have important implications for the nutritional and metabolic physiology of vertebrates. Glucose-modulated SS production provides an important feedback control on the release of other metabolically important hormones, such as insulin and glucagon, in so far as SSs have been shown to inhibit the release of these factors in both mammals (Patel, 1992) and fish (Eilertson and Sheridan, 1993). Particularly noteworthy in the present study was the preferential effects of glucose (in vitro) on the expression of PPSS II9. Based on the sequence of trout PPSS IIs (Moore et al., 1999) and our understanding of precursor processing (Conlon, 1989), such preferential expression could lead to the synthesis of a preponderance of one SS peptide form, salmonid SS-25, over another, salmonid SS-28, suggesting distinctive roles for the various SS isoforms. Growing evidence suggests that there are important structure–function relationships among the SS family of peptides (Sheridan et al., 1999). For example, salmonid SS-25 was more potent in inhibition of glucagon release than SS-14 (Eilertson and Sheridan, 1993). In summary, we have demonstrated that glucose regulates the expression of PPSS II8 mRNA and of PPSS II9 mRNA in the Brockmann body of rainbow trout. Glucose-stimulated alterations in the steadystate levels of PPSS II mRNAs depends upon the phosphorylation and subsequent metabolism of the hexose and is actinomycin sensitive.
ACKNOWLEDGMENTS We thank Leslie Alexander, Darlene Knutson, Marty Pesek, and Bart Slagter for their assistance with these experiments. This research was supported by grants from the U.S. National Science
Foundation (IBN 9723058) and the U.S. Department of Agriculture (98-35206-6410) to M.A.S.
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160 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. O. Thorner, Eds.), Vol. 4, pp. 1–16. Springer-Verlag, Berlin. 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. Ronner, P., and Scarpa, A. (1987). Secretagogues for pancreatic hormone release in the channel catfish (Ictalurus punctatus). Gen. Comp. Endocrinol. 65, 354–362.
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Ehrman et al. Sheridan, M. A., and Mommsen, T. P. (1991). Effects of nutritional state on in vivo lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 81, 473–483. Sheridan, M. A., Kittilson, J. D., Ehrman, M. M., and Moore, C. A. (1997). Polygenic expression of somatostatin in rainbow trout. In ‘‘Advances in Comparative Endocrinology’’ (S. Kawashima and S. Kikuyama, Eds.), pp. 291–294. Monduzzi Editore, Bologna. Sheridan, M. A., Kittilson, J. D., and Slagter, B. A. (2000). Structure– function relationships of the signaling system for the somatostatin peptide hormone family. Am. Zool., in press.