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Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material
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European Journal of Cell Biology 79, 27 ± 34 (2000, January) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
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Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material Lucien F. Harthoorn1), Jacques H. B. Diederen, Rob C. H. M. Oudejans, Monique M. A. Verstegen, Henk G. B. Vullings, Dick J. Van der Horst Biochemical Physiology Research Group and Institute of Biomembranes, Utrecht University, Utrecht/The Netherlands Received December 16, 1998 Received in revised version August 20, 1999 Accepted October 10, 1999
Intracisternal granules ± ergastoplasmic granules ± neuroendocrine cells ± adipokinetic cells ± Locusta migratoria (Insecta) The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material. Abbreviations. AcPase Acid phosphatase. ± AKH Adipokinetic hormone. ± CC Corpus cardiacum. ± HPLC High performance liquid chromatography. ± ICG Intracisternal granule. ± TGN trans-Golgi network. ± (WGA-)HRP (Wheat-germ agglutinin-conjugated) horseradish peroxidase. Dr. L. F. Harthoorn, Biochemical Physiology Research Group and Institute of Biomembranes, Utrecht University, Padualaan 8, NL-3584 CH Utrecht/The Netherlands, e-mail: [email protected], Fax: 31 30 253 28 37.
1)
Introduction Intracisternal granules (ICGs) are present in both exocrine (Tooze et al., 1989; Tooze et al., 1990) and endocrine cells (Hopkins, 1972; Noda and Farquhar, 1992; Bassetti et al., 1995) and originate from premature condensation of hormones within cisternae of the rough endoplasmic reticulum. It is proposed that hormones, presumably in their unprocessed form, condense into ICGs when the rate of biosynthesis of hormones is too high for storage in and release from ordinary secretory granules (Nunez and Gershon, 1976; Wood and Keough, 1987; Noda and Farquhar, 1992). Alternatively, ICGs might contain improperly folded or non-correctly assembled or in some other way defective, transport-incompetent secretory proteins (Hurtley and Helenius, 1989). ICGs have been reported to be degraded by autophagy (Tooze et al., 1990) or by a non-autophagic, crinophagy-like process (Noda and Farquhar, 1992; Bassetti et al., 1995). The neuroendocrine adipokinetic cells of the migratory locust, Locusta migratoria, constitute an adequate and accessible model system for studying functional significance and fate of ICGs. The ICGs in these cells, which originally have been called ergastoplasmic granules (Lafon-Cazal, 1975), may attain diameters of 5 mm and more (Figs. 1a, b). The adipokinetic cells within the glandular lobes of the locust corpus cardiacum produce the adipokinetic hormones (AKHs) I, II and III (Bogerd et al., 1995), which control the release of carbohydrate and lipid from the insect fat body during flight (Beenakkers et al., 1985). The synthesis of the adipokinetic prohormones, their packaging into secretory granules, and their processing to the bioactive hormones is completed after approximately 75 min (Oudejans et al., 1990). After their formation in the adipokinetic cell bodies, the secretory granules are transported to the cell processes for storage or release into the hemolymph (Jansen et al., 1989; Diederen et al., 1992; Diederen et al., 1993; Diederen and Vullings, 1995). Flight activity is the only known natural stimulus for triggering the release of AKHs.
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Fig. 1. a: Semithin section of the corpus cardiacum of a locust (43 days after imaginal ecdysis) containing several intracisternal granules (arrows) of various diameters. b: Ultrathin section of the corpus
cardiacum with an intracisternal granule enveloped by a ribosomestudded membrane. ICG intracisternal granule; SG secretory granule. Bar in a 10 mm and in b 1 mm.
Both the secretory granules and the ICGs react with antisera specific for AKHs (Diederen et al., 1987). AKHs appear to be synthesized continuously, and with the age of the insect the amount of hormones within the adipokinetic cells increases (Oudejans et al., 1993), as do the numbers of secretory granules (Diederen et al., 1992) and ICGs (Lafon-Cazal and Michel, 1977; Michel and Lafon-Cazal, 1978). The number and volume of ICGs decrease when locusts fly for a short period of time and the ICGs disappear even completely during sustained flight (Michel and Lafon-Cazal, 1978). Furthermore, locusts reared under solitary conditions have more and larger ICGs than locusts in the gregarious phase induced by rearing under crowded conditions, which is most probably due to a lower locomotor activity of solitary locusts requiring less fuel mobilisation and consequently a limited release of AKHs (Diederen et al., 1999). These data suggest that prohormones stored within the ICGs can be mobilized and released after proper processing if required. The ICGs thus may be considered to represent a spatially economic way for the cell to store extra adipokinetic prohormones. Alternatively, similar to ICGs in other secretory cell types, ICGs in adipokinetic cells might also contain products that are in some way defective and not transport-competent and thus presumably destined for degradation. The present study deals with the question whether the ICGs in the adipokinetic cells are destined for degradation or, alternatively, whether they represent a supplementary storage compartment of (pro)hormones. The first issue to investigate was whether the ICGs are degraded by autophagy. After administration of the endocytic tracers horseradish peroxidase (HRP) and wheat-germ agglutinin-conjugated HRP (WGAHRP) to adipokinetic cells, these tracers can be visualized cytochemically in endosomal, autophagic and lysosomal organelles (Jansen et al., 1989; Diederen and Vullings, 1995) and
thus, eventually, in autophagosomes together with ICGs (Tooze et al., 1990; Seglen and Bohley, 1992; Dunn, 1994). An alternative way for degradation of ICGs may result from a crinophagy-like process including the direct fusion of ICGs with lysosomes, resulting in the appearance of lysosomal enzymes within the ICGs (Noda and Farquhar, 1992; Seglen and Bohley, 1992; Dunn, 1994; Bassetti et al., 1995). The latter was investigated by enzyme-cytochemical methods for the demonstration of the lysosomal enzyme acid phosphatase (AcPase). For establishing the significance of ICGs in case they are not degraded, the rate of formation of ICGs was assessed by studying the biosynthetic incorporation of radioactive amino acids into the content of ICGs using autoradiography. In addition, biochemical analysis of the content of ICGs in comparison to the content of regular secretory granules was performed to disclose whether ICGs may function as stores of intact secretory material.
Materials and methods
Male locusts (Locusta migratoria) were used, 43 days after the imaginal ecdysis. Animals were taken from the laboratory s normal breeding stock reared in cages under crowded conditions at 30 8C, at a relative humidity of 40%, under a daily photoperiod of 16 h, and on a diet of reed grass supplemented with rolled oats. For each experiment, the animals were decapitated without previous anaesthesia.
Endocytic tracer experiments
HRP was administered to locusts by injecting 10 ml physiological saline (7.50 g/l NaCl and 0.375 g/l KCl in distilled water) containing 14% HRP (type II, No P-8250, Sigma, St.Louis, MO, USA) into the abdomen. The injection was repeated after 6 and 15 h. The animals were sacrificed 3 h after the third injection. WGA-HRP was administered by injecting 15 ml physiological saline containing 1% WGA-HRP (type IV, No L-3892, Sigma, St.Louis, MO,
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USA) into the abdomen. The injection was repeated after 9 and 14 h. The animals were sacrificed 7.5 h after the third injection. After the (WGA-)HRP uptake experiments, the corpora cardiaca were quickly dissected from the animals and fixed for 1 h in an ice-cooled, freshly prepared mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). After fixation, the glands were rinsed (3 5 min) and stored overnight in the same buffer, at 4 8C. (WGA-)HRP activity was visualized and the corpora cardiaca were prepared for transmission electron microscopy as previously described (Diederen et al., 1993). Control reactions for endogenous peroxidase activity were performed on corpora cardiaca from locusts injected with saline only.
in semi-thin (1 mm) sections, post-stained for 2 min in a 1% methyleneblue solution in 1% borax : azure II (1 : 1) in distilled water.
Acid phosphatase cytochemistry
Endocytic tracer experiments
Corpora cardiaca were quickly dissected and fixed for 1 h in an icecooled, freshly prepared mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.3). After fixation, the glands were rinsed (2 5 min) in ice-cooled 0.1 M cacodylate buffer (pH 7.3), embedded in 7.5% agar and stored overnight in the same buffer, at 4 8C. AcPase was visualized in 60 mm Vibratome sections and the tissue prepared for transmission electron microscopy as previously reported (Jansen et al., 1989).
Radiolabelling and autoradiography
Corpora cardiaca were dissected and collected in insect saline buffer (ISB) (pH 7.0) (10 mM HEPES, 150 mM NaCl, 10 mM KCl, 4 mM CaCl2, 2 mM MgCl2 in distilled water). The glands were then incubated in 200 ml ISB containing 0.74 MBq of a 3H-amino acid mixture (TRK 440; Amersham International, Buckinghamshire, England), for 1 h, at 30 8C, under gentle shaking. Next, the corpora cardiaca were rinsed with ISB (3 10 min) at 30 8C. This allowed the adipokinetic cells to incorporate radioactive amino acids into the products of their biosynthesis for maximally 1.5 h. The glands were then fixed and embedded in an epoxy resin, and the radioactive labelling was visualized in ultrathin sections as previously reported (Sharp-Baker et al., 1995). The autoradiographic exposure time was 7 weeks. Controls for negative and positive chemography were included.
Biochemical analysis
To isolate and separate ICGs and secretory granules, firstly corpora cardiaca were quickly dissected from locusts and collected in 200 ml ISB. After rinsing three times in ISB, the glands were homogenized in 500 ml ice-cold sucrose (0.44 M) with a Potter-Elvehjem teflon homogenizer (five strokes at 1500 rpm). The homogenate was centrifuged at 100g and 4 8C, for 10 min in a Sorvall RC-5B centrifuge using a SS-24 rotor. The supernatant was collected and filtered over a 0.65 mm Ultrafree-MC Durapore filter (Millipore, Bedford, MA, USA). After pelleting the filtrate, both the 100g pellet and the filtrate pellet were washed two times with ISB and dissolved. Each of the pellet solutions were divided into two parts, and pelleted again. One part of both fractions was used for biochemical analysis, while the other part was used for microscopy to check the homogeneity of the fractions. For biochemical analysis, the pellets were sonified and extracted according to the procedure reported previously (Oudejans et al., 1991). The extracts were dissolved in 2.5 M acetic acid and subjected to high performance liquid chromatography (HPLC). A wide-pore reversedphase HPLC column (Adsorbosphere XL-C8, 4.6 250 mm, particle size 5 mm, pore size 300 ; Alltech, Breda, The Netherlands) was used, the elution conditions were as described previously (Oudejans et al., 1991), and fluorescence was measured (Shimadzu RF10A Fluorospectrometer, Shimadzu Corporation, Kyoto, Japan) at an excitation wavelenght of 276 nm and an emission wavelenght of 360 nm. An extract of whole corpora cardiaca was also subjected to HPLC, as described previously (Oudejans et al., 1991). For microscopy, the pellets were fixed for 1 h with 2% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.3), and 1 h with osmium tetroxide in 0.1 M Na-cacodylate buffer (pH 7.3), all at 4 8C. After fixation, the pellets were enclosed in 2% agar and dehydrated and embedded as previously reported (Diederen et al., 1993). Fractions were visualized
Results Morphologically, ICGs are clearly distinguishable from regular secretory granules in locust adipokinetic cells (Figs. 1a, b). Based on their size, ICGs can easily be observed and followed in (electron) microscopy and be separated from secretory granules to determine their content biochemically. The endocytic tracers HRP and WGA-HRP were found in all categories of endosomal, autophagic and lysosomal organelles in the adipokinetic cells. Unlike unconjugated HRP, WGAHRP occasionally was found also in trans-Golgi networks (TGNs), secretory granules and in transport vesicles with a diameter of about 100 nm, some of which may have originated from the TGN and may be delivering lysosomal enzymes and other lysosomal proteins to lysosomes. The ICGs, however, could not be found to contain (WGA-)HRP reaction deposits, nor could ICGs be found within tracer-containing lysosomal or autophagic structures (Figs. 2a, b, c). There was also no morphological evidence that fusion may occur between ICGs and any organelle of the endocytic and lysosomal systems.
Acid phosphatase cytochemistry
Enzyme-cytochemical detection of AcPase was used to visualize organelles belonging to the lysosomal and autophagic systems which participate in crinophagy-like processes. AcPase was found in all categories of organelles belonging to these lysosomal and autophagic systems. AcPase was never seen within ICGs (Figs. 3a, b), nor could ICGs be found within any AcPase-positive organelle.
Radiolabelling experiments
Biosynthetic incorporation of a mixture of tritiated amino acids for maximally 1.5 h followed by autoradiography allowed us to get an indication of the rate of formation of ICGs. The intensity of labelling of the ICGs varied greatly. After autoradiography some ICGs were covered by many silver grains, which were more or less evenly spread over these ICGs (Fig. 4a). Other ICGs were covered by fewer silver grains (Fig. 4b), which sometimes appeared to be confined to the peripheral regions of the ICG (Fig. 4c). Still other ICGs were not labelled by silver grains at all (Fig. 4d).
Biochemical analysis
Using previously described biochemical procedures and reversed-phase HPLC conditions (Oudejans et al., 1991), peptide hormones or related compounds could be identified and quantified. A selective detection of AKHs and their prohormones could be achieved by application of fluorospectrometry, using excitation and emission wavelengths specific for tryptophan-containing compounds (Teale and Weber, 1957). Due to the absence of tryptophan in the flanking peptides of prohormones of AKHs, cleaved rest products did not show any signal and therefore did not interfere in fluorometric detection. The chromatographic profile of whole corpora cardiaca extracts showed peaks expected from our previous study (Oudejans et al., 1991) (Fig. 5a). The three AKHs (peak I, II
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Fig. 2. Parts of adipokinetic cells after endocytosis of HRP (a) or WGA-HRP (b, c). Both tracers are amply present in endosomal, autophagic and lysosomal structures. WGA-HRP also appears in secretory granules. Some of the small tracer-containing vesicles with a diameter of about 100 nm (arrowheads in b, c) may be TGN-derived
transport vesicles. No tracer is present in the intracisternal granules. AS autophagic structure; ES endosomal structure; ICG intracisternal granule; IS intercellular space; LS lysosomal structure; TGN transGolgi network; WS WGA-HRP-labelled secretory granule. Bars 1 mm.
Fig. 3. Parts of adipokinetic cells with several AcPase-positive autophagic and lysosomal structures. No AcPase activity is present in
the intracisternal granules. AS autophagic structure; ICG intracisternal granule; LS lysosomal structure. Bars 1 mm.
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Fig. 4. Autoradiographs of intracisternal granules after incorporation of radioactive amino acids. The radioactive labelling varies from
intense (a) to intermediate (b, c) to zero (d). ICG intracisternal granule. Bars 1 mm.
and III) could be measured and are relatively less hydrophobic than prohormones which showed two peaks (peak IV and V) with retention times of 1.705 and 1.716, respectively, relative to that of AKH III. However, due to the extremely small difference in retention time of peak IV and V, and the typical appearance of one prohormone peak (peak V) from the ICG fraction (Fig. 5b) and the complete other one (peak VI) from the secretory granule fraction (Fig. 5c), it is most likely that
one type of prohormone is present in two structurally different forms in cells of the corpus cardiacum of 43-days-old locusts. In this case, this only prominent prohormone can be that of AKH I due to its relatively highest amount. Remarkable is the presence of significant amounts of AKH I, II and III in the ICG fraction, whereas in the secretory granule fraction relatively more unidentified minor peaks were present compared to the ICG fraction. Figures 5b and 5c show also the
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Fig. 5. Reversed-phase HPLC analysis and fluorescence detection (excitation at 276 nm, emission at 360 nm) of peptides isolated from extracts of corpora cardiaca from 43-days-old locusts (Locusta migratoria). a: HPLC profile of whole corpora cardiaca extracts showing peaks of AKH I (I), AKH II (II), AKH III (III) and prohormones (IV and V). b: HPLC profile of a fraction containing ICGs showing peaks for all three
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AKHs (I ± III) and a prohormone form of AKH I characterized as peak V, together with a semithin section of the pelleted fraction containing ICGs (large arrows) and nuclei (small arrows). c: HPLC profile of the secretory granule fraction showing all three AKH peaks and a prohormone form of AKH I characterized as peak IV, together with a semithin section of the pelleted secretory granule fraction. Bars 1 mm.
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microscopic photographs (insets) of the fractions containing ICGs and secretory granules, respectively. ICGs appear to be structures with a large density, because they sediment at a very low centrifugal gravitation together with nuclei. The relative amounts of prohormone of AKH I in whole corpora cardiaca extracts, expressed as the prohormone : AKH III ratio, is 0.66 for peak V and 0.27 for peak IV (Table I). The fraction containing the ICGs showed a relative amount of prohormone of AKH I (peak V) of 0.52, whereas the secretory granule fraction had a relative amount of prohormone of AKH I (peak IV) of 0.27 (Table I).
Discussion The results obtained with the endocytic tracers HRP and WGA-HRP offer insight into the relation between the ICGs and the endocytic and autophagic pathways. (WGA-)HRP injected into locusts was found within adipokinetic cells in early and late endosomes, in autophagic vacuoles, and in lysosomes (see also Jansen et al., 1989; Diederen et al., 1993). If ICGs were to be degraded by autophagy, they would have been taken up in autophagic vacuoles, which would also have received (WGA-)HRP by fusing with endosomes or lysosomes that contained these tracers. However, no ICGs could be found in any (WGA-)HRP-containing autophagic or lysosomal structure. Apparently, ICGs in locust adipokinetic cells are not degraded by means of autophagy. If (WGA-)HRP-labelled lysosomes were to fuse with ICGs directly, as has to be expected if ICGs were to be degraded by a crinophagy-like process, then ICGs should be found which also were labelled with (WGA)-HRP. However, no WGA-HRP-labelled ICGs could be detected. Unlike unconjugated HRP, WGA-HRP sticks to the plasma membrane by means of its lectin component WGA and is then endocytosed. Endocytosed fragments of plasma membrane with bound WGA-HRP are transported to the TGNs and are used in the production of new secretory granules (Diederen and Vullings, 1995) and vesicles that transport lysosomal proteins from the TGN to their lysosomal destinations. If ICGs were among these destinations, as might be expected in case of crinophagy-like degradation of ICGs, then again ICGs should receive WGA-HRP, which obviously does not occur. The absence of crinophagy-like degradation of ICGs is confirmed by the results of the AcPasecytochemistry. No AcPase activity could be found in any ICG, which renders it unlikely that lysosomes or TGN-derived transport vesicles with lysosomal enzymes would have fused with ICGs. No ICGs could be found fusing or in close association with AcPase-containing (autophago)lysosomal structures either, which also makes crinophagy-like and autophagic degradation of ICGs unlikely. The presence of heavily labelled ICGs in autoradiographs of adipokinetic cells which had been allowed to incorporate
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radioactive amino acids for 1.5 h, indicates that these ICGs can be formed largely within this 1.5-h period. Synthesis of the non-labelled ICGs apparently was already completed before the incubation with radioactive amino acids started, whereas weakly labelled ICGs can be considered to be nearly completed ICGs to which some radioactive synthetic products were added during the incubation period. The silver grains over the latter ICGs tended to be located over the peripheral region of the ICGs, which suggests the deposition of new material to occur peripherally by addition to the older material within the ICGs. Biochemical analysis of fractions containing either ICGs or secretory granules, reveals that among the presence of the three AKHs only one type of prohormone, most likely the prohormone of AKH I due to its relatively high amount, could be measured. Remarkable is the presence of two structurally different forms of prohormone of AKH I in cells of the corpus cardiacum of 43-days-old locusts (Fig. 5a). This occurrence most presumably might be caused by dimerization, a wellknown phenomenon in the biosynthesis and processing of AKHs (O Shea and Rayne, 1992). The presence of one form of AKH I prohormone in ICGs and another form in secretory granules points to that way of structure shift. Since ICGs are due to their ribosome-studded membranes (Fig. 1b) pre-Golgi structures, they can contain unprocessed secretory material, i. e. in a non-dimerized form, whereas ordinary secretory granules contain also prohormone of AKH I, but most likely in the dimerized form. The presence of prohormone in secretory granules indicates that prohormone processing is a relatively late event in the secretory pathway. Concerning the relative quantities of prohormones (Table I), the content of (dimerized) prohormone in corpora cardiaca is totally present in secretory granules. The ICGs contain approximately 80% of the total amount of (non-dimerized) prohormone present in corpora cardiaca. The ICGs contain, relatively to the amount of AKHs, approximately two times more prohormone than secretory granules do. Rather surprising is the presence of all three AKHs in ICGs. A possible contamination of secretory granules content in the ICG fraction can be excluded, because no dimerized prohormone (peak IV, Fig. 5b) can be traced. This suggests that processed and releasable AKHs, which are principally stored in secretory granules, can apparently be taken up as supplementary material to these pre-Golgi granules, however, via an as yet unknown process. In this way, ICGs seem to represent a spatially economic way for the cell to store not only prohormones, but AKHs as well. Further studies remain to be done on transport routes of secretory material among compartmentalized granular structures in neuroendocrine cells. In conclusion, the data presented in this study indicate that, unlike the ICGs in other secretory cell types, the ICGs in the neuroendocrine adipokinetic cells of locusts are not degraded. Since the number of ICGs decreases after flight activity of the locusts, which implies a higher AKH release (Michel and
Tab. I. Prohormone detection: relative quantities (prohormone: AKH III ratios) and retention times (relative to that of AKH III) from whole corpora cardiaca extracts and extracts of fractions containing intracisternal granules or secretory granules. organ/organelle
prohormone peak
relative retention time
prohormone:AKH III ratio
corpus cardiacum
IV V V IV
1.705 1.716 1.716 1.705
0.27 0.66 0.52 0.27
intracisternal granules secretory granules
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Lafon-Cazal, 1978), whereas the number and size of ICGs increases when locusts are reared under solitary conditions, which implies a lower AKH release (Diederen et al., 1999), apparently the ICGs can be considered to be supplementary stores of secretory material, which can be mobilized and released if called upon.
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Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material Lucien F. Harthoorn1), Jacques H. B. Diederen, Rob C. H. M. Oudejans, Monique M. A. Verstegen, Henk G. B. Vullings, Dick J. Van der Horst Biochemical Physiology Research Group and Institute of Biomembranes, Utrecht University, Utrecht/The Netherlands Received December 16, 1998 Received in revised version August 20, 1999 Accepted October 10, 1999
Intracisternal granules ± ergastoplasmic granules ± neuroendocrine cells ± adipokinetic cells ± Locusta migratoria (Insecta) The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material. Abbreviations. AcPase Acid phosphatase. ± AKH Adipokinetic hormone. ± CC Corpus cardiacum. ± HPLC High performance liquid chromatography. ± ICG Intracisternal granule. ± TGN trans-Golgi network. ± (WGA-)HRP (Wheat-germ agglutinin-conjugated) horseradish peroxidase. Dr. L. F. Harthoorn, Biochemical Physiology Research Group and Institute of Biomembranes, Utrecht University, Padualaan 8, NL-3584 CH Utrecht/The Netherlands, e-mail: [email protected], Fax: 31 30 253 28 37.
1)
Introduction Intracisternal granules (ICGs) are present in both exocrine (Tooze et al., 1989; Tooze et al., 1990) and endocrine cells (Hopkins, 1972; Noda and Farquhar, 1992; Bassetti et al., 1995) and originate from premature condensation of hormones within cisternae of the rough endoplasmic reticulum. It is proposed that hormones, presumably in their unprocessed form, condense into ICGs when the rate of biosynthesis of hormones is too high for storage in and release from ordinary secretory granules (Nunez and Gershon, 1976; Wood and Keough, 1987; Noda and Farquhar, 1992). Alternatively, ICGs might contain improperly folded or non-correctly assembled or in some other way defective, transport-incompetent secretory proteins (Hurtley and Helenius, 1989). ICGs have been reported to be degraded by autophagy (Tooze et al., 1990) or by a non-autophagic, crinophagy-like process (Noda and Farquhar, 1992; Bassetti et al., 1995). The neuroendocrine adipokinetic cells of the migratory locust, Locusta migratoria, constitute an adequate and accessible model system for studying functional significance and fate of ICGs. The ICGs in these cells, which originally have been called ergastoplasmic granules (Lafon-Cazal, 1975), may attain diameters of 5 mm and more (Figs. 1a, b). The adipokinetic cells within the glandular lobes of the locust corpus cardiacum produce the adipokinetic hormones (AKHs) I, II and III (Bogerd et al., 1995), which control the release of carbohydrate and lipid from the insect fat body during flight (Beenakkers et al., 1985). The synthesis of the adipokinetic prohormones, their packaging into secretory granules, and their processing to the bioactive hormones is completed after approximately 75 min (Oudejans et al., 1990). After their formation in the adipokinetic cell bodies, the secretory granules are transported to the cell processes for storage or release into the hemolymph (Jansen et al., 1989; Diederen et al., 1992; Diederen et al., 1993; Diederen and Vullings, 1995). Flight activity is the only known natural stimulus for triggering the release of AKHs.
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Fig. 1. a: Semithin section of the corpus cardiacum of a locust (43 days after imaginal ecdysis) containing several intracisternal granules (arrows) of various diameters. b: Ultrathin section of the corpus
cardiacum with an intracisternal granule enveloped by a ribosomestudded membrane. ICG intracisternal granule; SG secretory granule. Bar in a 10 mm and in b 1 mm.
Both the secretory granules and the ICGs react with antisera specific for AKHs (Diederen et al., 1987). AKHs appear to be synthesized continuously, and with the age of the insect the amount of hormones within the adipokinetic cells increases (Oudejans et al., 1993), as do the numbers of secretory granules (Diederen et al., 1992) and ICGs (Lafon-Cazal and Michel, 1977; Michel and Lafon-Cazal, 1978). The number and volume of ICGs decrease when locusts fly for a short period of time and the ICGs disappear even completely during sustained flight (Michel and Lafon-Cazal, 1978). Furthermore, locusts reared under solitary conditions have more and larger ICGs than locusts in the gregarious phase induced by rearing under crowded conditions, which is most probably due to a lower locomotor activity of solitary locusts requiring less fuel mobilisation and consequently a limited release of AKHs (Diederen et al., 1999). These data suggest that prohormones stored within the ICGs can be mobilized and released after proper processing if required. The ICGs thus may be considered to represent a spatially economic way for the cell to store extra adipokinetic prohormones. Alternatively, similar to ICGs in other secretory cell types, ICGs in adipokinetic cells might also contain products that are in some way defective and not transport-competent and thus presumably destined for degradation. The present study deals with the question whether the ICGs in the adipokinetic cells are destined for degradation or, alternatively, whether they represent a supplementary storage compartment of (pro)hormones. The first issue to investigate was whether the ICGs are degraded by autophagy. After administration of the endocytic tracers horseradish peroxidase (HRP) and wheat-germ agglutinin-conjugated HRP (WGAHRP) to adipokinetic cells, these tracers can be visualized cytochemically in endosomal, autophagic and lysosomal organelles (Jansen et al., 1989; Diederen and Vullings, 1995) and
thus, eventually, in autophagosomes together with ICGs (Tooze et al., 1990; Seglen and Bohley, 1992; Dunn, 1994). An alternative way for degradation of ICGs may result from a crinophagy-like process including the direct fusion of ICGs with lysosomes, resulting in the appearance of lysosomal enzymes within the ICGs (Noda and Farquhar, 1992; Seglen and Bohley, 1992; Dunn, 1994; Bassetti et al., 1995). The latter was investigated by enzyme-cytochemical methods for the demonstration of the lysosomal enzyme acid phosphatase (AcPase). For establishing the significance of ICGs in case they are not degraded, the rate of formation of ICGs was assessed by studying the biosynthetic incorporation of radioactive amino acids into the content of ICGs using autoradiography. In addition, biochemical analysis of the content of ICGs in comparison to the content of regular secretory granules was performed to disclose whether ICGs may function as stores of intact secretory material.
Materials and methods
Male locusts (Locusta migratoria) were used, 43 days after the imaginal ecdysis. Animals were taken from the laboratory s normal breeding stock reared in cages under crowded conditions at 30 8C, at a relative humidity of 40%, under a daily photoperiod of 16 h, and on a diet of reed grass supplemented with rolled oats. For each experiment, the animals were decapitated without previous anaesthesia.
Endocytic tracer experiments
HRP was administered to locusts by injecting 10 ml physiological saline (7.50 g/l NaCl and 0.375 g/l KCl in distilled water) containing 14% HRP (type II, No P-8250, Sigma, St.Louis, MO, USA) into the abdomen. The injection was repeated after 6 and 15 h. The animals were sacrificed 3 h after the third injection. WGA-HRP was administered by injecting 15 ml physiological saline containing 1% WGA-HRP (type IV, No L-3892, Sigma, St.Louis, MO,
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USA) into the abdomen. The injection was repeated after 9 and 14 h. The animals were sacrificed 7.5 h after the third injection. After the (WGA-)HRP uptake experiments, the corpora cardiaca were quickly dissected from the animals and fixed for 1 h in an ice-cooled, freshly prepared mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). After fixation, the glands were rinsed (3 5 min) and stored overnight in the same buffer, at 4 8C. (WGA-)HRP activity was visualized and the corpora cardiaca were prepared for transmission electron microscopy as previously described (Diederen et al., 1993). Control reactions for endogenous peroxidase activity were performed on corpora cardiaca from locusts injected with saline only.
in semi-thin (1 mm) sections, post-stained for 2 min in a 1% methyleneblue solution in 1% borax : azure II (1 : 1) in distilled water.
Acid phosphatase cytochemistry
Endocytic tracer experiments
Corpora cardiaca were quickly dissected and fixed for 1 h in an icecooled, freshly prepared mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.3). After fixation, the glands were rinsed (2 5 min) in ice-cooled 0.1 M cacodylate buffer (pH 7.3), embedded in 7.5% agar and stored overnight in the same buffer, at 4 8C. AcPase was visualized in 60 mm Vibratome sections and the tissue prepared for transmission electron microscopy as previously reported (Jansen et al., 1989).
Radiolabelling and autoradiography
Corpora cardiaca were dissected and collected in insect saline buffer (ISB) (pH 7.0) (10 mM HEPES, 150 mM NaCl, 10 mM KCl, 4 mM CaCl2, 2 mM MgCl2 in distilled water). The glands were then incubated in 200 ml ISB containing 0.74 MBq of a 3H-amino acid mixture (TRK 440; Amersham International, Buckinghamshire, England), for 1 h, at 30 8C, under gentle shaking. Next, the corpora cardiaca were rinsed with ISB (3 10 min) at 30 8C. This allowed the adipokinetic cells to incorporate radioactive amino acids into the products of their biosynthesis for maximally 1.5 h. The glands were then fixed and embedded in an epoxy resin, and the radioactive labelling was visualized in ultrathin sections as previously reported (Sharp-Baker et al., 1995). The autoradiographic exposure time was 7 weeks. Controls for negative and positive chemography were included.
Biochemical analysis
To isolate and separate ICGs and secretory granules, firstly corpora cardiaca were quickly dissected from locusts and collected in 200 ml ISB. After rinsing three times in ISB, the glands were homogenized in 500 ml ice-cold sucrose (0.44 M) with a Potter-Elvehjem teflon homogenizer (five strokes at 1500 rpm). The homogenate was centrifuged at 100g and 4 8C, for 10 min in a Sorvall RC-5B centrifuge using a SS-24 rotor. The supernatant was collected and filtered over a 0.65 mm Ultrafree-MC Durapore filter (Millipore, Bedford, MA, USA). After pelleting the filtrate, both the 100g pellet and the filtrate pellet were washed two times with ISB and dissolved. Each of the pellet solutions were divided into two parts, and pelleted again. One part of both fractions was used for biochemical analysis, while the other part was used for microscopy to check the homogeneity of the fractions. For biochemical analysis, the pellets were sonified and extracted according to the procedure reported previously (Oudejans et al., 1991). The extracts were dissolved in 2.5 M acetic acid and subjected to high performance liquid chromatography (HPLC). A wide-pore reversedphase HPLC column (Adsorbosphere XL-C8, 4.6 250 mm, particle size 5 mm, pore size 300 ; Alltech, Breda, The Netherlands) was used, the elution conditions were as described previously (Oudejans et al., 1991), and fluorescence was measured (Shimadzu RF10A Fluorospectrometer, Shimadzu Corporation, Kyoto, Japan) at an excitation wavelenght of 276 nm and an emission wavelenght of 360 nm. An extract of whole corpora cardiaca was also subjected to HPLC, as described previously (Oudejans et al., 1991). For microscopy, the pellets were fixed for 1 h with 2% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.3), and 1 h with osmium tetroxide in 0.1 M Na-cacodylate buffer (pH 7.3), all at 4 8C. After fixation, the pellets were enclosed in 2% agar and dehydrated and embedded as previously reported (Diederen et al., 1993). Fractions were visualized
Results Morphologically, ICGs are clearly distinguishable from regular secretory granules in locust adipokinetic cells (Figs. 1a, b). Based on their size, ICGs can easily be observed and followed in (electron) microscopy and be separated from secretory granules to determine their content biochemically. The endocytic tracers HRP and WGA-HRP were found in all categories of endosomal, autophagic and lysosomal organelles in the adipokinetic cells. Unlike unconjugated HRP, WGAHRP occasionally was found also in trans-Golgi networks (TGNs), secretory granules and in transport vesicles with a diameter of about 100 nm, some of which may have originated from the TGN and may be delivering lysosomal enzymes and other lysosomal proteins to lysosomes. The ICGs, however, could not be found to contain (WGA-)HRP reaction deposits, nor could ICGs be found within tracer-containing lysosomal or autophagic structures (Figs. 2a, b, c). There was also no morphological evidence that fusion may occur between ICGs and any organelle of the endocytic and lysosomal systems.
Acid phosphatase cytochemistry
Enzyme-cytochemical detection of AcPase was used to visualize organelles belonging to the lysosomal and autophagic systems which participate in crinophagy-like processes. AcPase was found in all categories of organelles belonging to these lysosomal and autophagic systems. AcPase was never seen within ICGs (Figs. 3a, b), nor could ICGs be found within any AcPase-positive organelle.
Radiolabelling experiments
Biosynthetic incorporation of a mixture of tritiated amino acids for maximally 1.5 h followed by autoradiography allowed us to get an indication of the rate of formation of ICGs. The intensity of labelling of the ICGs varied greatly. After autoradiography some ICGs were covered by many silver grains, which were more or less evenly spread over these ICGs (Fig. 4a). Other ICGs were covered by fewer silver grains (Fig. 4b), which sometimes appeared to be confined to the peripheral regions of the ICG (Fig. 4c). Still other ICGs were not labelled by silver grains at all (Fig. 4d).
Biochemical analysis
Using previously described biochemical procedures and reversed-phase HPLC conditions (Oudejans et al., 1991), peptide hormones or related compounds could be identified and quantified. A selective detection of AKHs and their prohormones could be achieved by application of fluorospectrometry, using excitation and emission wavelengths specific for tryptophan-containing compounds (Teale and Weber, 1957). Due to the absence of tryptophan in the flanking peptides of prohormones of AKHs, cleaved rest products did not show any signal and therefore did not interfere in fluorometric detection. The chromatographic profile of whole corpora cardiaca extracts showed peaks expected from our previous study (Oudejans et al., 1991) (Fig. 5a). The three AKHs (peak I, II
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Fig. 2. Parts of adipokinetic cells after endocytosis of HRP (a) or WGA-HRP (b, c). Both tracers are amply present in endosomal, autophagic and lysosomal structures. WGA-HRP also appears in secretory granules. Some of the small tracer-containing vesicles with a diameter of about 100 nm (arrowheads in b, c) may be TGN-derived
transport vesicles. No tracer is present in the intracisternal granules. AS autophagic structure; ES endosomal structure; ICG intracisternal granule; IS intercellular space; LS lysosomal structure; TGN transGolgi network; WS WGA-HRP-labelled secretory granule. Bars 1 mm.
Fig. 3. Parts of adipokinetic cells with several AcPase-positive autophagic and lysosomal structures. No AcPase activity is present in
the intracisternal granules. AS autophagic structure; ICG intracisternal granule; LS lysosomal structure. Bars 1 mm.
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Fig. 4. Autoradiographs of intracisternal granules after incorporation of radioactive amino acids. The radioactive labelling varies from
intense (a) to intermediate (b, c) to zero (d). ICG intracisternal granule. Bars 1 mm.
and III) could be measured and are relatively less hydrophobic than prohormones which showed two peaks (peak IV and V) with retention times of 1.705 and 1.716, respectively, relative to that of AKH III. However, due to the extremely small difference in retention time of peak IV and V, and the typical appearance of one prohormone peak (peak V) from the ICG fraction (Fig. 5b) and the complete other one (peak VI) from the secretory granule fraction (Fig. 5c), it is most likely that
one type of prohormone is present in two structurally different forms in cells of the corpus cardiacum of 43-days-old locusts. In this case, this only prominent prohormone can be that of AKH I due to its relatively highest amount. Remarkable is the presence of significant amounts of AKH I, II and III in the ICG fraction, whereas in the secretory granule fraction relatively more unidentified minor peaks were present compared to the ICG fraction. Figures 5b and 5c show also the
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Fig. 5. Reversed-phase HPLC analysis and fluorescence detection (excitation at 276 nm, emission at 360 nm) of peptides isolated from extracts of corpora cardiaca from 43-days-old locusts (Locusta migratoria). a: HPLC profile of whole corpora cardiaca extracts showing peaks of AKH I (I), AKH II (II), AKH III (III) and prohormones (IV and V). b: HPLC profile of a fraction containing ICGs showing peaks for all three
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AKHs (I ± III) and a prohormone form of AKH I characterized as peak V, together with a semithin section of the pelleted fraction containing ICGs (large arrows) and nuclei (small arrows). c: HPLC profile of the secretory granule fraction showing all three AKH peaks and a prohormone form of AKH I characterized as peak IV, together with a semithin section of the pelleted secretory granule fraction. Bars 1 mm.
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microscopic photographs (insets) of the fractions containing ICGs and secretory granules, respectively. ICGs appear to be structures with a large density, because they sediment at a very low centrifugal gravitation together with nuclei. The relative amounts of prohormone of AKH I in whole corpora cardiaca extracts, expressed as the prohormone : AKH III ratio, is 0.66 for peak V and 0.27 for peak IV (Table I). The fraction containing the ICGs showed a relative amount of prohormone of AKH I (peak V) of 0.52, whereas the secretory granule fraction had a relative amount of prohormone of AKH I (peak IV) of 0.27 (Table I).
Discussion The results obtained with the endocytic tracers HRP and WGA-HRP offer insight into the relation between the ICGs and the endocytic and autophagic pathways. (WGA-)HRP injected into locusts was found within adipokinetic cells in early and late endosomes, in autophagic vacuoles, and in lysosomes (see also Jansen et al., 1989; Diederen et al., 1993). If ICGs were to be degraded by autophagy, they would have been taken up in autophagic vacuoles, which would also have received (WGA-)HRP by fusing with endosomes or lysosomes that contained these tracers. However, no ICGs could be found in any (WGA-)HRP-containing autophagic or lysosomal structure. Apparently, ICGs in locust adipokinetic cells are not degraded by means of autophagy. If (WGA-)HRP-labelled lysosomes were to fuse with ICGs directly, as has to be expected if ICGs were to be degraded by a crinophagy-like process, then ICGs should be found which also were labelled with (WGA)-HRP. However, no WGA-HRP-labelled ICGs could be detected. Unlike unconjugated HRP, WGA-HRP sticks to the plasma membrane by means of its lectin component WGA and is then endocytosed. Endocytosed fragments of plasma membrane with bound WGA-HRP are transported to the TGNs and are used in the production of new secretory granules (Diederen and Vullings, 1995) and vesicles that transport lysosomal proteins from the TGN to their lysosomal destinations. If ICGs were among these destinations, as might be expected in case of crinophagy-like degradation of ICGs, then again ICGs should receive WGA-HRP, which obviously does not occur. The absence of crinophagy-like degradation of ICGs is confirmed by the results of the AcPasecytochemistry. No AcPase activity could be found in any ICG, which renders it unlikely that lysosomes or TGN-derived transport vesicles with lysosomal enzymes would have fused with ICGs. No ICGs could be found fusing or in close association with AcPase-containing (autophago)lysosomal structures either, which also makes crinophagy-like and autophagic degradation of ICGs unlikely. The presence of heavily labelled ICGs in autoradiographs of adipokinetic cells which had been allowed to incorporate
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radioactive amino acids for 1.5 h, indicates that these ICGs can be formed largely within this 1.5-h period. Synthesis of the non-labelled ICGs apparently was already completed before the incubation with radioactive amino acids started, whereas weakly labelled ICGs can be considered to be nearly completed ICGs to which some radioactive synthetic products were added during the incubation period. The silver grains over the latter ICGs tended to be located over the peripheral region of the ICGs, which suggests the deposition of new material to occur peripherally by addition to the older material within the ICGs. Biochemical analysis of fractions containing either ICGs or secretory granules, reveals that among the presence of the three AKHs only one type of prohormone, most likely the prohormone of AKH I due to its relatively high amount, could be measured. Remarkable is the presence of two structurally different forms of prohormone of AKH I in cells of the corpus cardiacum of 43-days-old locusts (Fig. 5a). This occurrence most presumably might be caused by dimerization, a wellknown phenomenon in the biosynthesis and processing of AKHs (O Shea and Rayne, 1992). The presence of one form of AKH I prohormone in ICGs and another form in secretory granules points to that way of structure shift. Since ICGs are due to their ribosome-studded membranes (Fig. 1b) pre-Golgi structures, they can contain unprocessed secretory material, i. e. in a non-dimerized form, whereas ordinary secretory granules contain also prohormone of AKH I, but most likely in the dimerized form. The presence of prohormone in secretory granules indicates that prohormone processing is a relatively late event in the secretory pathway. Concerning the relative quantities of prohormones (Table I), the content of (dimerized) prohormone in corpora cardiaca is totally present in secretory granules. The ICGs contain approximately 80% of the total amount of (non-dimerized) prohormone present in corpora cardiaca. The ICGs contain, relatively to the amount of AKHs, approximately two times more prohormone than secretory granules do. Rather surprising is the presence of all three AKHs in ICGs. A possible contamination of secretory granules content in the ICG fraction can be excluded, because no dimerized prohormone (peak IV, Fig. 5b) can be traced. This suggests that processed and releasable AKHs, which are principally stored in secretory granules, can apparently be taken up as supplementary material to these pre-Golgi granules, however, via an as yet unknown process. In this way, ICGs seem to represent a spatially economic way for the cell to store not only prohormones, but AKHs as well. Further studies remain to be done on transport routes of secretory material among compartmentalized granular structures in neuroendocrine cells. In conclusion, the data presented in this study indicate that, unlike the ICGs in other secretory cell types, the ICGs in the neuroendocrine adipokinetic cells of locusts are not degraded. Since the number of ICGs decreases after flight activity of the locusts, which implies a higher AKH release (Michel and
Tab. I. Prohormone detection: relative quantities (prohormone: AKH III ratios) and retention times (relative to that of AKH III) from whole corpora cardiaca extracts and extracts of fractions containing intracisternal granules or secretory granules. organ/organelle
prohormone peak
relative retention time
prohormone:AKH III ratio
corpus cardiacum
IV V V IV
1.705 1.716 1.716 1.705
0.27 0.66 0.52 0.27
intracisternal granules secretory granules
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Lafon-Cazal, 1978), whereas the number and size of ICGs increases when locusts are reared under solitary conditions, which implies a lower AKH release (Diederen et al., 1999), apparently the ICGs can be considered to be supplementary stores of secretory material, which can be mobilized and released if called upon.
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