Oral administration of insulin in winter-acclimatized carp (Cyprinus carpio) induces hepatic ultrastructural changes

0300-9629/93 $6.00 + 0.00 0 1993 Pergamon Press Ltd

Camp. Bioehem. Physioi. Vol. 106A. No. 4, pp. 477-682, 1993 Printed in Great Britain

ORAL ADMINISTRATrON OF INSULIN IN WINTER-ACCLIMATIZED CARP (CYPR1lVUS CARPlo) INDUCES HEPATIC ULTRASTRUCTURAL CHANGES MARIA IN&SVERA,FRANCIXO ROMERO,JAM FIGUEROA, RODOLFOAMTIUUJZR, GLORIALEON, JULIETAVILLAN~VA and MANUELKRAUSKOPF* Instituto de Bioquimica, Fact&ad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. (Fax 56-63-219410) (Received 2 Feb 1993; accepted 4 March 1993)

Abstract-l.

The intestinal absorption of insulin in carps was assessed examining the tran~pithelial passage of ingested gold-ladle hormone by electron microscopy. Insulin transfer occurred mainly through the intercellular spaces between the enterocytes. 2. When reaching the lamina propria, the gold-labeled hormone gathered predominantly around the granules of the granular cells, and therefore can enter the circulatory system via the blood capillaries which are found in close contact with these cells. 3. Winter-acclimatized carp were also capable of internalizing the hormone when fed with insulin. 4. Furthermore, the absorbed hormone revealed full activity in regard to the observed changes in the ultrastructnre of the liver cells of the treated cold-adapted fish. 5. The fish ingesting the hormone underwent the same type of hepatic ultrastructure reprogr~ing observed when winter-acclimatized carps are injected intraperitoneally with insulin, i.e. conversion to a phenotype corresponding to hepatocytes from summer-adapted carp. 6. The oral absorption of insulin by winter-acclimatized fish and its effect in reversing the cold-adaptive state might be useful for the fish culturing industry.

INTRODUCTION Seasonal acclimatization of eurythermal fish demands compensatory responses involving the gene expression process at both the transcriptional and translational level (Plant et al., 1977; Saez et al., 1982; Krauskopf et al., 1981, 1988; Inostroza et al., 1990; Gerlach et al.,1990).In the carp hepatocyte, the required adaptive adjustment involves many cellular and molecular changes. As a consequence of this, the liver cells of summer- and winter-acclimatized carp exhibit profound ultrastruct~al ~pro~~in~ affecting the nuclei and cytoplasm (Saez et at., 1984a). One of the most prominent seasonal changes in the cytoarchitecture of the carp hepatocytes is found in the nucleolus where winter acclimatization leads to a dramatic segregation of the nucleolar components, revealing that ribosomal RNA ~an~~ption is strongly affected (Saez et uf., 1984a; Krauskopf et al., 1988). Another dramatic shift is observed in the cytoplasm with regard to its glycogen content (Saez et al., 1984a). The cyclic seasonal reprogramming of the cellular arrangement can be altered when winter carps receive in~~~ton~l injections of porcine insulin (Saez et al., 1984b). The treatment decreases the glycogen *Author to whom correspondence should be addressed at Instituto de Bioquimica, Universidad Austral de Chile, Valdivia, Chile. 677

content, restores the merging of the nucleolar components, and increases the total RNA level in liver cells of winter-acclimatized carp (Saez et al., 1984b). Because in the carp, as in other fish, high density lipoproteins (HDL) comprise the major serum protein fraction (Chapman, 1980; Krauskopf et al., 1988; Babin and Vernier, 1989) one of its apolipoprotein, apo A-I has been well characterized (Amthauer et al., 1989; Hare1 et al., 1989) and its synthesis examined upon acclimatization of the fish (Inostroza et al., 1990). As with a11the distinctive features of winteracclimatized carp liver cells so far examined in our laboratory (Saez et al., 1984b Krauskopf et al., 1988), insulin leads to hepatocytes resembling those of the summer-acclimatized state by also reversing the low apolipoprotein A-I content found in the liver of the cold-adapted carp (Inostroza et al., 1990). We have shown recently that carp apo A-I is absorbed by the intestine and that the undegraded protein is transferred to the fish circulatory system. The internalization process is also affected by seasonal acclimatization (Vera et al., 1992). Absorption of proteins by the intestinal tract of fish has been shown by using horseradish peroxidase (Rombout et al., 1985; McLean and Ash, 1986, 1987), and bovine growth hormone (Le Bail er al., 1989), among others. Thus, we have postulated that if insulin could be also internalized by the carp intestine, it could be possible to reverse the features which

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characterize winter-adapted fish by feeding them with the hormone. Herein we present evidence that indeed insulin is absorbed by the intestinal tract and that its transfer in winter-acclimatized carp induces ultrastructural changes in the liver cells leading to features which characterize the summer phenotype. While this article was being written Hertz et al. (1992) reported that as other proteins, bovine insulin was absorbed by the intestine of carp acclimated to 21-24°C. Our findings suggest that it might be possible to approach the management of the growth capabilities of fish undergoing seasonal acclimatization by nourishing them with insulin. Such a process could be very profitable to surmount the limited development of fish during the cold season in the fish culturing industry.

MATERIALS AND METHODS

Carp (Cyprinus carpio) of both sexes, weighing NO-15OOg were caugth in the Calle-Calle river during s~mer and winter and maintained in an outdoor tank with running river water (Saez et a!., 1984a). The temperature of the river and outdoor tank water in summer and winter was 18-20°C and 9-10°C respectively. Electron microscopy was performed in samples of summer- and inter-a~limatiz~ carp (the controls and those treated with insulin) liver tissue prepared as described earlier (Saez et al., l984a). The samples of proximal intestine tissue were taken 3-5 cm caudal from the first imprinted folding in the stretching of

the anterior segment which follows the opening of the bile duct and processed as reported (Vera et al., 1992).

Carp were injected in the coelomic cavity with 2.5 I.U. of porcine insulin (Novo Industri A/S) per 100 g of fish body weight, three times at 24 hr intervals as described elsewhere (Inostroza et al., 1990). Oral administration of insulin or insulin-colloidal gold in phosphate buffered saline was carried out by intubation with a ~lyethylene catheter attached to a syringe. The tube end reached the portion anterior to the opening of the bile duct. To examine the biological activity of the hormone reaching the circulatory system after its ingestion, winter-acclimatized carp were fed by intubation with 7.5 IU of porcine insulin~l~g fish body weight, three times at 24 hr intervals. Colloidal gold (12-15 nm) was obtained after Frens (1973) and porcine insulin-colloidal gold following Roth (1983).

RESULTS AND DISCU~ION

To visualize the uptake of insulin, one dose of 7 mg of gold-labeled insulin was administered to an orally intubated winter-acclimatized carp. The proximal intestine was removed 45 min later and processed for ultrastructural analyses. As shown in Fig. I the transepithelial passage of the labeled hormone occurs mainly through the intercellular spaces. This can be observed at the luminal border of the enterocytes (Fig. 1A). Towards the basal membrane it is possible

Fig. 1. (A) Upon ingestion of colloidal gold-lab&d insulin by a winter-acclimatized carp, most of the labeled-hormone is detected mainly within the intercellular spaces between the enterocytes ( x 15,200). (B) In the basal border of the epithclial cells, the gold-labeled hormone can be observed proximal to the basal membrane ( x 11,300). (C) In the lamina propria, the gold-labeled hormone surrounds the granules of the granular cells ( x 8400).

Oral administration of insulin to carp to detect gold-labeled insulin approaching the lamina propria (Fig. 1B). Beneath the epithelial cells, in the laminal proptia, there are blood capillaries in close contact with the granular cells (Vera et al., 1992). Figure lc shows that the gold-labeled hormone gathers predominantly around the granules of the granular cells. The pattern is the same as that occurring during the intestinal transfer of carp apolipoprotein A-I into the systemic circulation of the fish (Vera et al., 1992). The passage of bovine growth hormone (bGH) through the intercellular spaces of the enterocytes in the intestine of Salmo gairdnerii follows a similar order, except that the granule cells of the lamina propria behave as immune cells and trap a high number of the bGH molecules (Le Bail et al., 1989). Nevertheless, most bGH molecules are absorbed by the enterocytes and degraded. Thus, molecules which escape degradation are found in the intercellular space between the enterocytes, the interstitial space of the lamina propria and capillaries resembling mammalian lymphatic capillaries, thus reaching the circulatory system. Studying the kinetic of appearance of bGH in the rainbow trout plasma, Le Bail et al. (1989) found that the peak was situated between 30 and 180 min. The oral absorption of bovine insulin in carp was recently determined by Hertz et al. (1992). Although the peak of absorption appeared always 3&45 min after intubation, the extent of absorption was extremely variable. To study the biological activity of the orally absorbed hormone, a group of winter-acclimatized carp were fed by intubation with insulin, and another group received three injections of insulin as detailed in Materials and Methods. One day after the last insulin administration, the fish were killed and the livers were processed for electron microscope examination. Liver tissue from summer- and winter-acclimatized carp was also examined. Figure 2A and B depicts the distinctive ultrastructural features of hepatocytes from summer- and winter-acclimatized fish. Clearly the amount and distribution of glycogen in the cytoplasm of liver cells from cold acclimatized fish differs from that found in summer. In winter, glycogen particles occupy most of the carp hepatocyte cytoplasm, confining the Golgi apparatus, the rough endoplasmic reticulum, and also the mitochondria, to a perinuclear location (Saez et al., 1984a). In contrast, during summer the rough endoplasmic reticulum is distributed throughout the cytoplasm, as are the rest of the organelles. In summer, glycogen decreases dramatically and it is found scattered throughout the cytoplasm. In this season, the components of the nucleolus appear highly intermingled, while a strong nucleolar segregation characterizes the liver cell of cold-acclimatized carp. The differences in the carp hepatocyte cytoarchitecture between both seasons have been associated with the reprogramming of gene expression conforming part of the compensatory responses

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that acclimatization demands (Saez et al., 1984a; Krauskopf et al., 1988; Inostroza et al., 1990). Environment-induced morphological changes in the liver cells of teleosts have been studied with regard to nutrition (Gas and Serfaty, 1972; Segner and Mailer, 1984), temperature (Berlin and Dean, 1967: Braunbeck et al., 1987) and seasonal acclimatization (Saez et al., 1984a; Segner and Braunbeck, 1990; Robertson and Bradley, 1991). There is increasing evidence that during environmental acclimatization the cellular and molecular reprogramming differs from that attained when the fish is subjected to laboratory acclimation (Segner and Braunbeck, 1990). Winter acclimatization also affects the hepatocytes of frogs, inducing ultrastructrual changes comparable to those featured in the cold-acclimatized carp (Bami and Bernocchi, 1991). We have previously shown that the cyclic seasonal ultrastructrural conversion of the carp liver cells is sex independent (Saez et al., 1984a). We documented also, that insulin injections of winter-acclimatized carp caused a morphological change in the liver cell which shows striking similarities with the ultrastructure found in hepatocytes of the summer-adapted fish (Saez et al., 1984b). Figure 2C depicts the features of a carp hepatocyte from a winter-acclimatized fish injected for 3 days with porcine insulin. Glycogen, which in control fish appears concentrated in large masses, becomes arranged in small clusters distributed along the cytoplasm, intermixed with cell organelles. The rough endoplasmic reticulum and mitochondria undergoes the same kind of redistribution losing their perinuclear localization (Saez et al., 1984b). Figure 2C also shows the remarkable change of the nucleolar body of a winter-adapted carp liver cell treated with insulin. It is also noteworthy that the three nucleolar components become highly intermingled, thus resembling the nucleolar organization of hepatoctyes from summer adapted carps. Figure 2D depicts a liver cell from a winteracclimatized carp fed with insulin by oral intubation. Clearly, the morphological changes are the same as those attained when the winter-adapted fish are injected with the hormone. The results presented in this communication, demonstrate that the intestinal absorption of insulin in carp fish follows a similar pattern as that observed for bGH (Le Bail et al., 1989) in rainbow trout and for apo A-I in carp (Vera et al., 1992). Because the intestinal transfer of proteins can be profoundly affected in cold-adapted fish (Vera et al., 1992) the oral absorption of insulin in winter-acclimatized fish is particularly interesting. The recent studies in that Hertz et al., (1992) showed by radioimmuno assay (RIA) the oral absorption of active bovine insulin were performed in carps acclimated to 21-24°C. Our results indicate that winter-acclimatized carps internalize ingested insulin, and that the biological activity of the absorbed hormone causes the same hepatic

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Fig. 2. (A) Hepatocyte of a summer-acclimatized carp. The lipid droplets, glycogen, rough endoplasmic reticulum and mitochondria, appear evenly distributed in the cytoplasm. The nucleolar components are highly intermingled. ( x 9100). (B) Hepatocyte from a winter-acclimatized carp. Large masses of glycogen occupy most of the cytoplasm, whereas the organelles are confined to the perinucleus. The nucleolar components are clearly segregated. ( x 9100). (C) Hepatocyte from a winter-acclimatized carp which was injected insulin intraperitoneally. The effect on the glycogen content and distribution of rough endoplasmic reticulum and mitochondria is shown. Also, the nucleolar body exhibiting its intermingled components ( x 9100). (D) Hepatocyte from a winter-acclimatized carp which was fed insulin. The observed ultrastructural features are the same of those depicted in (C). ( x 9100).

ultrastructure reprogramming as the one achieved upon injection of the hormone. It is well known that carp liver glycogen content reaches values of up to 15-20 mg/lOO mg wet tissue and that insulin injections can reduce sharply its level (Murat and Serfaty 1975; Murat, 1976a; Saez et al., 1984b) and increases the activity of the liver y-

amylase (Murat, 1976b). Apart from this particular action of insulin in the carp fish, it has been shown that the hormone promotes protein synthesis in isolated hepatocytes of coho salmon (Plisetskaya et al., 1984), and that it might be involved in the parr-smolt transformation of coho (Plisetskaya et al., 1988) and Atlantic salmon (Robertson and Bradley, 1991).

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Oral administration of insulin to carp

The feeding strategies in commercial fish culture are attracting increasing interest. As shown by Storebakken et al. (1991) the body weight gains and feed efficiencies in the rainbow trout (Oncorhynchus mykiss) are significantly affected by feeding rates. Increasing the feeding rate results in gained weight which correlates also with increased concentration of insulin (Storebakken et al., 1991). The growth promoting effects of parenterally administered growth hormone has been known for some years (Donaldson et al., 1979). Therefore the intestinal transfer of this hormone which has been recently shown to occur in Salmo gairdnerii (Le Bail et al., 1989) and in Cyprinus carpio (Hertz et al., 1991), has been thought to offer a practical alternative to its use in aquaculture. Another explored option has been the construction of transgenic fish (Du et al., 1992). However, the seasonal environmental conditions imposing metabolic restrictions which affect the weight gain of fish during winter, require growth promoting factors capable of reversing the coldadapted physiological state which impedes significant growth. As we have observed previously, insulin appears to have the required properties to restore summer metabolic features in winter-acclimatized carp (Saez et al., 1984b; Krauskopf et al., 1988; Inostroza et al., 1990; Vera et al., 1992). Thus, its oral absorption with retention of biological activity confirmed in this communication, provides a new means of approaching feeding in commercial aquaculture in areas where winter temperature affects the growth of the fish Acknowledgements-We thank Professor Haraldo Peria for critical reading of the manuscript. This work was supported

by grants 905191 and 007/90 from FONDECYT S-90- 15 from DID-UACH.

and

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