Comparative Biochemistry and Physiology, Part A 151 (2008) 180–184
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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p a
Drosophila ia2 modulates secretion of insulin-like peptide Jihyun Kim, Hyojoo Bang, Syungkyun Ko, Inhee Jung, Haehee Hong, Jeongsil Kim-Ha ⁎ Department of Molecular Biology, Sejong University, 98 Kunja-dong, Kwangjin ku 143-747, Seoul, South Korea
A R T I C L E
I N F O
Article history: Received 17 March 2008 Received in revised form 19 June 2008 Accepted 19 June 2008 Available online 25 June 2008 Keywords: Autoantigen Diabetes Drosophila Hexokinase IA-2 ia2 Gut Pancreas
A B S T R A C T Islet antigen-2 (IA-2) is a major autoantigen in type I diabetes. To throw light on the function of IA-2 we examined the role of ia2, a Drosophila homologue, during Drosophila development. In situ hybridization showed that ia2 was expressed in the central nervous system and midgut region. The neuronal expression pattern of ia2 was very similar to that of IA-2 in mammals. Disruption of gut-specific ia2 expression by double stranded RNA interference (dsRNAi) resulted in defects in gut development, and this phenotype was rescued by overexpression of hexokinase. Until now the roles of IA-2 and hexokinase in insulin signaling have been described separately but we found that ia2 modulated the expression of both insulin and hexokinase. Moreover this modulation seems to be important for gut development during metamorphosis. As the pancreas develops from the gut during vertebrate development, our results suggest a possible role of IA-2 in insulin and hexokinase regulation. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Approximately 70% of newly diagnosed Type I diabetic patients have autoantibodies to IA-2 (Lan et al., 1996; Leslie et al., 1999). IA-2 is a member of the protein tyrosine phosphatase family (Lan et al., 1994) but it is not clear whether IA-2 truly functions as a phosphatase because it failed to show any phosphatase activity. Endogenous IA-2 is present in the secretory vesicles of neuroendocrine cells throughout the body, including the alpha and beta cells of pancreatic islets (Solimena et al., 1996). Disruption of IA-2 in mice does not lead to a dramatic increase of blood glucose level but it reduces glucose-dependent insulin secretion (Saeki et al., 2002; Henquin et al., 2008). Conversely, overexpression of IA-2 results in increased glucose-induced insulin secretion (Harashima et al., 2005). These observations point to a role of IA-2 in regulating insulin secretion and thus in the development of diabetes. However, knockout of IA-2 in the non-obese diabetic mouse, the most widely studied animal model of type I diabetes, did not lead to the development of diabetes (Kubosaki et al., 2004). Therefore, the physiological role of IA2 in the pathogenesis of Type I diabetes is still not clear. Drosophila melanogaster contains probable orthologs of human disease genes that match 75% of the 1378 human disease loci examined (Lasko, 2002). Recent research on the Drosophila insulin-signaling pathway has raised the possibility that certain features of diabetes mellitus are shared by both Drosophila and humans. In the Drosophila genome, seven insulin-like peptides (ilps) are found that are expressed
⁎ Corresponding author. Tel.: +82 2 3408 3644; fax: +82 2 312 8834. E-mail address:
[email protected] (J. Kim-Ha). 1095-6433/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2008.06.020
in brain and other tissues, such as imaginal discs and gut. The most studied region that produces the ilps is an area of seven neuroendocrine cells in each brain hemisphere. The expression of ilp-2, -3, and -5 was detected in these insulin-producing cells (IPCs); additionally, IPC ablation was shown to cause an elevation of carbohydrate levels in hemolymph (Rulifson et al., 2002). In addition to carbohydrate regulation, the roles of the ilps were demonstrated in growth and reproduction (Claeys et al., 2002). However, the specificity of each ilp, as well as additional roles of the ilps, has not been investigated seriously. A homolog of the human IA-2 gene, ia2, was found in Drosophila. As the possible use of Drosophila in the study of diabetes has been suggested from the ilp results, we expected to find another regulatory mechanism from the study of ia2 in Drosophila. Therefore, we examined the tissue-specific expression of ia2 and analyzed its function in vivo. 2. Materials and methods 2.1. Tissue preparation and in situ hybridization Flies were embedded in OCTcompound and frozen in liquid nitrogen. Serial 15 µm cryosections were placed on polylysine-coated slides, fixed with 4% paraformaldehyde in 1× phosphate-buffered saline (PBS) for 1 h, and washed in PBST (0.1% Triton X-100 in PBS). After incubation in acetylation buffer (0.25% acetic anhydride in 0.1M triethanolamine), they were washed in PBST, and prehybridized in hybridization buffer (5X SSC, 50% formamide, 100 µg/mL salmon sperm DNA, 50 µg/mL heparin, and 0.1% Tween-20) at room temperature for 10 min. Hybridizations were performed at 55 °C overnight. After washing with PBST, the slides were incubated with alkaline phosphatase-conjugated anti-DIG
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antibody (Roche, Mannheim, Germany), and hybridization signals were visualized by incubation with BCIP and NBT. Incubation was continued until the hybridization signals were sufficiently strong, and the reactions were stopped by washing in 1× PBS. 2.2. Transgenic flies harboring ia2 dsRNA and a hexokinaseoverexpressing construct To generate a construct expressing a stem-loop structured ia2 dsRNA, ia2 cDNA fragments corresponding to nucleotides 3728–4257 and 3728–4397 (nucleotide numbers according to GenBank accession number NM134718) were incorporated into the pCaSpeR-UAS vector in opposite orientation. A full length 1493 nucleotide Hexokinase (Hex) cDNA (GenBank accession number AF237469) was cloned into pCaSpeR-UAS in the sense orientation. Transgenic flies were generated by injecting embryos with these constructs together with a helper plasmid, delta 2-3. UAS-InR and UAS-Akt-expressing flies were obtained from Dr. JK Jeong at KAIST. 2.3. Hexokinase antibody production and western analysis Polyclonal antibodies against Hex protein corresponding to the C terminal half (nucleotides 599 to 1339 of GenBank AF237469) were raised in rats. Affinity purified Hex antibody was tested for specificity by transfecting Schneider cells with full length Hex cDNA cloned into pCaSpeR-hs vector (Thummel et al., 1988) and examining extracts of the transfected cells for reactivity with the Hex antibodies. Elevated amounts of Hex protein were detected in transfected and 2 h heatinduced cells (data not shown). For Western analysis, larval extracts were incubated with anti-Hex and anti-GFP (Santa Cruz, USA) antibodies at 1: 4000 and 1:1000 dilutions, respectively.
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ia2-PB, from a single genomic region (CG31795) were found with a high degree of similarity to human IA-2. ia2-PA and ia2-PB are 1144 and 1288 amino acids in length, respectively. ia2-PB (GenBank number Q59E11) differs only slightly from ia2-PA: it contains a 163 amino acid N-terminal extension, and its final C-terminal 12 amino acid sequence is different from that of ia2-PA (GenBank number Q9VPV8). Most of the extracellular N-terminal region of human IA-2 is not conserved in the Drosophila ia2 proteins (Grumbling and Strelets, 2006). The greatest similarity is found in the intracellular protein tyrosine phosphatase domain, which shares 65% identity and 77% homology to IA-2. In what follows the two homologues will be treated as one protein and referred to as Drosophila ia2. 3.2. Expression of Drosophila ia2 In humans and the mouse, IA-2 is exclusively expressed in brain and pancreas. In Drosophila, small clusters of neuroendocrine cells in the brain called insulin producing cells (IPCs) have been shown to contribute to pancreas function (Rulifson et al., 2002). No other tissues that function as pancreas equivalents have been identified in Drosophila. To compare the expression pattern of Drosophila ia2 with that of human and mouse, we examined ia2 expression in various developmental stages. Drosophila ia2 transcripts were detected from the larval stage and persisted throughout development to the adult (Fig. 1). To
2.4. RT-PCR RNA was extracted from 3rd instar larvae of the wild type and the Kr-GAL4 N UAS-ia2RNAi strain. From 5 µg of total RNA, single strand cDNA was synthesized with oligo dT primer and the final volume was adjusted with D.W to 200 µL at the end of the reaction. Five µL of the cDNA was used for PCR which consisted of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, repeated twenty eight times (Master cycler gradient 5331, Eppendorf Inc. Germany) for all the insulin-likepeptide (Ilp) genes except for Ilp4; 35 cycles were performed for the latter. Primers used were: Ilp1 (5′ CGGAGCAGGAGGTGCAGGAT 3′/ 5′ CTATTTCGGTAGACAGTAGA 3′), Ilp2 (5′ ACCTAAGCAGTAAACCCATA 3′/ 5′ TCCAGATCGCTGTCGGCACC 3′), Ilp3 (5′ ATGGGCATCGAGATGAGGTG 3′/ 5′ GGAACGGTCTTCGAAGCCAT 3′), Ilp4 (5′ ATGAGCCTGATTAGACTGGG 3′/ 5′ GGTCTCGCACTCTAGCATCC 3′), Ilp5 (5′ ATGTTCCGCTCCGTGATCCC 3′/ 5′ GGAGCTATCCAAATCCGCCA 3′), Ilp6 ( 5′ ATGGTTCTCAAAGTGCCGAC 3′/ 5′ CCTGCGCTTCCCGAAACTGT 3′). Ilp7 (5′ TCGGACTGGGAGAACGTGTG 3′/ 5′ GGAGTGTTTCCATCCGATCG 3′) and rp49 (5′ CAGTCGGATCGATATGCTAAGCTGT 3′/ 5′ TAACCGATGTTGGGCATCAGATACT 3′). For real-time PCR analysis, 5 µL of the synthesized cDNA was used per reaction. SYBR green dye (Invitrogen, USA) and EF-Taq polymerase (Solgent, Korea) was used. PCR consisted of 50 °C for 2 min, 95 °C for 1 min followed by 95 °C for 10 s, 55 °C for 25 s and 72 °C for 30 s, repeated forty times. The amounts of Ilps relative to the rp49 control gene product were measured using the ddCt relative quantitation study program in the 7300 Real Time PCR (Applied Biosystems, USA) system SDS software. 3. Results 3.1. Drosophila homologs of the human IA-2 gene The 979 amino acid sequence of human IA-2 was used to search a Drosophila protein database. Two hypothetical proteins, ia2-PA and
Fig. 1. Expression of the ia2 gene during development. A. Developmental RT-PCR analysis of ia2 gene expression. L1, L2, L3 indicates 1st, 2nd and 3rd larvae, respectively, and P1, P2, P3 are one day white, two day yellow and three day-old pupae, respectively. The ribosomal protein gene rp49 was used as a loading control. B. In situ hybridization of sectioned larvae and adult flies with a Drosophila ia2 riboprobe. The riboprobe was made from a DNA template corresponding to nucleotides 3728 to 4257 in GenBank accession number NM134718. ia2 expression was detected in the brain and midgut (arrows) of first and third instar larvae (top and middle), and expression was conserved in adult flies (bottom).
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examine the tissue specific expression of ia2 we sectioned wild type larvae and adult flies and carried out in situ hybridization with an ia2 probe. In larval and adult stages ia2 was exclusively expressed in the central nervous system (CNS), which consists of brain and thoracic ganglion (Fig. 1B). The neuronal expression pattern of ia2 is very similar to that of IA-2 in the mouse, where transcripts are found in brain, pituitary and pancreas (Shimizu et al., 2001). ia2 was also expressed in midgut cells (Fig. 1B). The Drosophila larval midgut consists of two types of cells: polytene enterocytes and diploid gut imaginal gland cells (Yee and Hynes, 1993). During metamorphosis the former cells die and the latter expand to form the adult gut. ia2 was expressed in both the polytene and diploid cells (data not shown). 3.3. Disruption of ia2 expression in the abdominal region causes a defect in abdomen formation during metamorphosis Since we could not locate any existing ia2 mutant flies we inhibited ia2 function by double stranded RNA interference (dsRNAi). We made a construct consisting of a 529 nucleotide inverted repeat sequence from the C- terminus of the ia2 coding region, with a UAS sequence in the promoter, and generated transgenic flies harboring this construct. The dsRNA was expressed specifically in neurons and gut cells, using tissue specific GAL4 drivers. When an elav-Gal4 driver was used to suppress ia2 expression in the CNS region we did not detect any reduction of ia2 mRNA expression (data not shown), and the flies did not show any defects. On the other hand, Kr-Gal4 driven lines (we will refer to these as Kr-Gal4 N UAS-ia2RNAi lines) showed a tissue-specific reduction of ia2 expression: CNS expression was normal, but gut expression was significantly reduced (Fig 2A, B). Since ia2 expression
Fig. 3. Hexokinase is reduced when ia2 expression is suppressed. Expression of Hex was examined using Hex antibody. A dramatic decrease of Hex protein was observed in KrGAL4 N UAS-ia2RNAi 3rd instar larvae compared to Kr-GAL4 strains. As the Kr-GAL4 chromosome also contains UAS-GFP, GFP protein was used as a loading control.
in the CNS remained normal, we were only able to confirm the efficiency of RNAi in the gut region by in situ hybridization. 80% of the flies did not eclose (Fig. 2C-E); they had eyes and wings but did not complete development. To examine the cause of this lethality we removed the bodies from the pupal cases. Although most of the body parts were complete, the abdominal region was covered by a very thin film-like layer instead of hard cuticle (Fig. 2D). When this thin film was removed almost no abdominal structures could be seen. We conclude that the internal organs of the abdominal region did not develop properly in these flies at metamorphosis. 3.4. Overexpression of hexokinase rescues the abdominal defect caused by dsRNA interference of ia2 Since IA-2 is reported to be involved in the secretion of insulin (Saeki et al., 2002), we tested whether the lethal RNAi phenotype could be rescued by overexpressing genes encoding proteins of the insulin signaling pathway. We overexpressed InR, Akt and Hex in midgut cells using the same Gal4 driver that was used to drive ia2 dsRNA expression. Overexpression of InR or Akt did not rescue the lethal phenotype of ia2 dsRNA flies (data not shown) but overexpression of Hex did (Fig. 2 E and F). We therefore asked whether Hex expression is lower in the dsRNA interference strains. We examined Hex protein levels by Western blot analysis and found a marked reduction in the Hex band when ia2 was knocked down (Fig. 3). 3.5. Insulin-like-peptide expression is reduced by knockdown of ia2 In Drosophila, there are seven insulin-like-peptides (ilps). We examined the expression of all seven ilps in wild type and Kr-GAL4 N UAS-ia2RNAi larvae. A reduction of mRNA expression was detected only in ilp6 (Fig. 4). While no change of expression of any of the other ilps was detected by reverse transcription-polymerase chain reaction (RT-PCR), a reduction of about 40% of the ilp6 PCR product was observed (Fig. 4A). The same result was obtained by real time PCR (Fig. 4B). 4. Discussion
Fig. 2. Tissue specific disruption of ia2. In wild type adult flies ia2 is expressed in midgut cells (A). In flies expressing ia2 dsRNA under the Krüppel-GAL4 (Kr-GAL4) driver expression of ia2 was reduced only in tissues where Kr-GAL4 is expressed: expression in the midgut (arrows) was dramatically reduced (B). Larvae harboring both Kr-GAL4 and UAS-dsRNA (ia2) also had a reduced rate of eclosion (E). Eighty percent of the flies of the expected genotype arrested at the 3-day pupa stage and died without eclosion (C, E). Flies harboring both Kr-GAL4 and UAS-dsRNA (ia2) had darker eye pigment because of the white gene inserted in the P element vector (bottom in C and D) whereas flies with either one of the transposons had lighter eye pigment (top in C and D). Overexpression of Hex together with the ia2 dsRNAi construct reversed the pupal stage lethal phenotype, as can be seen by the empty pupal case (F).
Human IA-2 and IA-2 beta share 74% identity and are major autoantigens in type I diabetes. The IA-2 gene is conserved in many species such as macaco, rat, mouse, C. elegans, zebrafish and Drosophila (Cai et al., 2001). The function of IA-2 has only been tested in mice and an attempt to determine its function in C. elegans using dsRNA was unsuccessful (Cai et al., 2001). Drosophila has a homologous ia2 gene located at 21E3 on the second chromosome. The corresponding two transcripts from this single genomic region generate two protein isoforms (Grumbling and
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Fig. 4. Expression of insulin-like peptides in ia2 RNAi strains. RNA was extracted from Kr-GAL4 N UAS-ia2RNAi and control Kr-Gal4 third instar larvae and examined for all the seven insulin-like peptides. A. Reduction of expression was only observed for the ilp6 transcript. The amount of PCR product was quantitated using the GelQuant program provided by DNR Bio-imaging Systems Ltd (Jerusalem, Israel). B. To confirm the data, expression was re-examined by real-time PCR. The relative quantification method was used and a reduction of about 40% of the Ilp6 transcript was observed.
Strelets, 2006). Our findings and those of other workers show that expression of the IA-2 family has similar tissue specificity: in human and mouse there is a high level of IA-2 expression in brain and moderate levels in the pancreas (Shimizu et al., 2001). We also found a high level of Drosophila ia2 expression in brain and the thoracic ganglion region. Although a test of pancreas-specific expression is not possible since there is apparently no pancreas in Drosophila, we observed expression of ia2 in the gut region, which is the organ from which the pancreas develops in vertebrates. Therefore, not only their sequence conservation but also the conservation of their tissue expression pattern makes it worthwhile to compare the function of IA-2 and its homologues in various organisms. Insulin stimulates the expression of genes that encode glycolytic enzymes, including glucokinase/hexokinase, while inhibiting the expression of those genes that encode gluconeogenic enzymes (Saltiel and Kaha, 2001). The selective stimulation of hexokinase mRNA and protein synthesis by insulin has been reported (Osawa et al., 1995), and reduced hexokinase activity is observed in diabetes (Vestergaard, 1999). We also observed a dramatic decrease in Hex expression within ia2-knockdown Drosophila strains. In addition, a reduction in iIp-6 expression was observed. Since ia2 knockdown was shown to result in the down-regulation of both ilp6 and Hex, we next examined the blood sugar levels in these flies. Since the major sugar in Drosophila hemolymph is trehalose, we measured hemolymph carbohydrate levels as the sum of both trehalose and glucose according to the method of Rulifson et al. (2002). Unexpectedly, we observed a decrease in carbohydrate levels (data not shown). During metamorphosis, larval gut cells die and diploid gut imaginal gland cells expand to form the adult gut structure. The absence of ia2 does not appear to interfere with the hydrolysis of the larval cells but does prevent the expansion of new adult-specific gut cells. Certain roles in growth and reproduction have been demonstrated for certain ilps but the role of ilp6 has not yet been defined. Various ilp-expression patterns were examined by Brogiolo et al. (Brogiolo et al., 2001) who found that an ilp6 signal could be detected in the gut regions of Drosophila larvae. One of the best-documented effects of insect ilp genes in Manduca sexta and Bombyx mori was the stimulation of ecdysteroidogenesis (Hayes et al., 1995; Nagata et al., 1992). Ecdysone is a key regulatory molecule in metamorphosis. The severe defective phenotype that we observed in abdominal development might be caused by an ecdysone deficiency. We observed normal metamorphosis in both the head and thorax but metamorphosis in the abdomen appeared to be defective. To determine whether
ia2 functions in the gut imaginal gland to either regulate hormonal signaling or promote cell survival for adult gut tissue formation requires further analysis. Evidence for the inter-regulation of IA-2/ia2, insulin, and hexokinase is increasing. An association of glucokinase/hexokinase with insulin secretory vesicles as a means to regulate glucokinase/hexokinase stability by preventing its degradation has been suggested (Saltiel and Kaha, 2001). Therefore, the destabilization of insulin secretory vesicles by ia2 knockdown could lead to decreases in both insulin and hexokinase levels. Although the mechanism for the rescue of the ia2 deficient, abdominal morphogenesis-defective phenotype by Hex is not clear at this point, it is clear that ia2 and Hex function in the same pathway during gut development. It would be interesting to determine whether increased hexokinase activity can also compensate for a reduction of IA-2 in the mammalian pancreas. We show that Drosophila ia2 shares many regulatory features with mammalian IA-2. A further extension of our study would lead to a better understanding of the regulatory components in the IA-2 pathway. Acknowledgements This work was supported by grant 02-PJ1-PG3-21002-0001 from the Korean Ministry of Health and Welfare, and R01-2006-000-10783 from the Korea Science and Engineering Foundation to J. K-H. References Brogiolo, W., Stocker, H., Ikeya, T., Rintelen, F., Fernandez, R., Hafen, E., 2001. An evolutionarily conserved function of the Drosophila insulin receptor and insulinlike peptides in growth control. Curr. Biol. 11, 213–221. Cai, T., Krause, M.W., Odenwald, W.F., Toyama, R., Notkins, A.L., 2001. The IA-2 family: homolog in Caenorhabditis elegans, Drosophila and zebrafish. Daibetologia 44, 81–88. Claeys, I., Simonet, G., Poels, J., Loy, T.V., Vercammen, L., De Loof, A., Broeck, J.V., 2002. Insulin-related peptides and their conserved signal transduction pathway. Peptides 23, 807–816. Grumbling, G., Strelets, V., 2006. The FlyBase consortium, FlyBase: anatomical data, images and queries. Nucleic Acids Res. 34, D484–D488. Harashima, S., Clark, A., Christie, M.R., Notkins, A.L., 2005. The dense core transmembrane vesicle protein IA-2 is a regulator of vesicle number and insulin secretion. Proc. Natl. Acad. Sci. U. S. A. 102, 8704–8909. Hayes, G.C., Muehleisen, D.P., Bollenbacher, W.E., Watson, R.D., 1995. Stimulation of ecdysteroidogenesis by small prothoracicotropic hormone: role of calcium. Mol. Cell. Endocrinol. 115, 105–112. Henquin, J.-C., Nenquin, M., Szollosi, A., Kubosaki, A., Notkins, A.L., 2008. Insulin secretion in islets from mice with a double knockout for the dense core vesicle proteins islet antigen-2 (IA-2) and IA-2β. J. Endocrinol. 196, 573–581.
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