Preproinsulin mrna in the Rat Eye

Preproinsulin mrna in the Rat Eye G. Colin Budd,* Ben Pansky,\ and Lou Glatzer\ Purpose. The goal of this study was to extend the results of previous immunoassay, immunocytochemistry, and in situ hybridization studies showing the presence of insulin-related peptide in the rat retina by confirming the expression of insulin genes in the rat eye. Methods. Total and poly(a) + RNA were isolated from whole rat eyes, and separately from the retina, choroid, iris, lens, and vitreous. The poly (A) + RNA was used for preparation of insulinspecific cdna according to a coupled reverse transcription polymerase chain reaction (RT- PCR) protocol under high stringency conditions. Southern transfers, restriction fragment analyses, and nucleotide sequencing were used to characterize and identify the amplified cdna products. Results. Amplified cdna fragments of 329 ± 6 base pairs (bp) were derived from whole rat eye and rat retina poly(a) + RNA, but not from other regions of the eye. Southern blots probed with preproinsulin-specific primer demonstrated homology with similar-sized cdna from rat pancreas. Restriction digests with 10 restriction enzymes and direct nucleotide sequencing confirmed that the 329-bp cdna was identical to the previously known coding sequence for rat pancreatic preproinsulin, DNA. Conclusions. The identification of retinal preproinsulin, mrna was confirmed. This correlates with previous studies showing insulin immunoreactivity in rat eyes and in cultured retina, and verifies in situ hybridization evidence for the presence of insulin-related mrna in retinal glial cells. Invest Ophthalmol Vis Sci 1993; 34:463-469..Although other peptide products of the pancreatic islets are found in the brain and regions of the gastrointestinal tract, insulin traditionally has been considered to be a unique product of the vertebrate pancreatic islets of Langerhans. In previous studies using immunoassay, 1 immunocytochemistry, 2 ' 3 and in situ hybridization, 3 ' 4 we have demonstrated insulin immunoreactivity in the brain, pituitary, retina, and retinoblastoma cells, and the presence of cell-specific ex- From the Departments of * Physiology and Biophysics, and \ Anatomy, Medical College of Ohio, and the %Biology Department, University of Toledo, Toledo, Ohio. Supported by grants from the National Eye Institute, NIH. (ROI-EY06SS6) and from, the Ohio Lions Eye Research Foundation. Submitted for publication: April 29, 1992; accepted June 24, 1992. Proprietary interest category: N. Reprint requests: G. Colin Build, Department of Physiology and Biophysics, Medical. College of Ohio, 3000 Arlington Avenue, Toledo, OH 43699. pression of cytoplasmic insulin-related mrna in cultured retinal glial cells, anterior pituitary cells, and in Y-79 human retinoblastoma cells. In the current studies, total RNA from whole rat eyes and poly(a) + RNA from specific parts of the rat eye were investigated for the presence of preproinsulin mrna using the combined techniques of reverse transcription and polymerase chain reaction amplification (RT-PCR) of specific cdna using preproinsulin-specific oligonucleotide primers under conditions of high stringency. A 329- base pair (bp) DNA was amplified from whole eye total RNA and retinal mrna that comigrated on electrophoresis with a cdna amplified by the same RT-PCR methods from rat pancreas total RNA. Restriction enzyme digestion and nucleotide sequencing demonstrated that the DNA fragments derived from whole eyes and retina corresponded precisely to the codingsequence of rat pancreatic preproinsulin, DNA. Invesiigaiivc Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2 Copyright Association for Research in Vision and Ophthalmology 463

464 Investigative Ophthalmology 8c Visual Science, February 1993, Vol. 34, No. 2 MATERIALS AND METHODS Total RNA was isolated, free of DNA and protein, from 16 pairs of 2-year-old Sprague-Dawley rat eyes and separately from rat pancreas by the quanidinium isothiocyanate-cscl method. 5 All animals were handled in accordance with the ARVO Statement for the Use of Animals in Opthalmic and Vision Research. Poly(A) + RNA was then purified directly from the total RNA on oligo (dt) cellulose according to the Polyattract and Ribosep mrna isolation procedures. 6 " 8 Alternatively, poly(a) + RNA was purified individually from whole eyes, retina, choroid, iris, lens, and vitreous using a micro method (Micro Fastrack, Invitrogen, Inc., San Diego, CA). Aliquots (1-10 ^g) of the poly(a) + RNA were reverse-transcribed and then insulin-specific cdna was generated according to the coupled RT-PCR procedure of Rappolee et al. 9 The oligonucleotide primers (3'-GTT GAC CTC TTG ATG ACG TTG) and (5'-ATG GCC CTG TGG ATG CGC TTC) corresponding to 21 bp at each end of the coding region equivalent to bases 4182 to 4202 and 4491 to 4511 in the rat preproinsulin, sequence (RATINSI, Genebank database tape release version 73.1; NCBI, Bethesda, MD), were specific for the known, highly conserved, rat preproinsulin, DNA sequence. PCR amplification was carried out with Ampli-Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT) for 25 to 40 cycles 10 in an Eppendorf programmable temperature cycler under conditions of high stringency to eliminate nonspecific annealing of primer to template (denaturation temperature: 94 C; annealing temperatures: 55 C or 65 C; and extension temperature: 72 C). The amplified DNA fragments were evaluated by electrophoresis in 4% agarose (Nusieve GTG/agarose-3:l; FMC Bioproducts, Rockland, ME) stained with ethidium bromide. Southern transfers were performed and probed with 32 P-nick-translated rat preproinsulin, cdna (3 X 10 6 cpm for 18 hr in 50% formamide hybridization buffer) and washed at 55 C (0.2 XSSC + 0.1%SDS). DNA fragments amplified during the PCR procedure were excised from agarose gels, purified according to the Prep-A-Gene procedure (Biorad, Inc., Richmond, CA) and reamplified with the PCR procedure to obtain 1 to 2 jug DN A/100 ix\ amplification reaction. In some cases, the reamplified DNA was concentrated and washed in Centricon-30 tubes (Amicon Corp., Danvers, MA) to remove excess dntps and primers and also Prep-a-gene purified before sequencing. The concentrated, purified DNA was evaluated for size by electrophoresis in 4% agarose in comparison with size control fragments (0X174 DNA digested with Hae III, a 123-bp DNA ladder [BRL, Inc., Gaithersburg, MD] or Biomarker Low [Bioventures, Inc., Murfreesboro, TN ). Aliquots of the purified DNA were directly sequenced according to a modification" 12 of the Sanger method 13 using 32 P-end-labeled primer and Sequenase-II T7 DNA polymerase (U.S. Biochemical, Inc., Cleveland, OH). Both of the 3'-oligonucleotide and 5'-oligonucleotide primers used for PCR amplification were end-labeled and used as primers for doublestranded sequencing in both directions. Electrophoresis was carried out in 8% polyacrylamide/8 mol/1 urea at approximately 2000 V, 60 W. After drying under vacuum at 65 C, the sequencing gels were autoradiographed with XARfilm (Kodak, Rochester, NY) for 24 to 72 hr. Sequences were read, by hand or with an IBI gel scanner (IBI, Inc., New Haven, CT), and compared with sequences derived from cdna prepared from rat pancreatic tissue and sequences recorded in the Genbank database, National Center for Biotechnology Information, Bethesda, Maryland. To confirm that the amplified products were derived from RNA and not from genomic DNA, it was verified that no amplification occurred when aliquots of total RNA were subjected to 30 cycles of PCR without prior reverse transcription or when reverse transcribed products were PCR'd in the absence of Ampli- Taq DNA polymerase. Restriction analysis was performed using standard protocols, the appropriate unique buffer (Promega, Inc., Madison, WI) or with React buffers (BRL, Inc.) for 2 hr at 37 C. Enzymes used were: BamHI, ECoRI, Hindi, Hindlll, PstI, Xhol, Hinfl, Hpall, Haelll, and Alul. Four percent analytical agarose gels (3% NuSieve/1% agarose; FMC, Inc.) were used and typically run for 4400 voltminutes in a minigel apparatus. They were prestained with ethidium bromide at 0.5 jug/ml agarose or poststained at 1 Mg/ml in water for 20 to 30 min. Estimations of fragment sizes were carried out with a linear regression program and distances were measured relative to the size standards. Estimates were made with three points above and below the experimental point and no standards were used with a regression coefficient less than 0.995. In all procedures, special care was taken to eliminate the possibility of cross-contamination between preparations, including isolation of tissues and poly(a) + RNA in separate areas, sterile gloved handling of all samples, and meticulous care in the use of instruments and reagents. 14 RESULTS The application of RT-PCR to poly(a) + RNA derived separately from whole rat eyes and from rat retina produced amplified cdna fragments with an estimated size of 329 ± 6 bp (Fig. 1A). No comparable fragments were obtained from our sampling of other regions of the eye (ie, choroid, iris, lens, and vitreous). Negative control studies, eliminating either the template or the Ampli-Taq DNA polymerase, showed no

465 Preproinsulin mrna in the Rat Eye 1 FIGURE l. (A) Composite aga- rose gel of PCR products from rat pancreas and rat eye total RNA after combined RT and PCR with preproinsulin-specific oligonucleotide primers. Lane 1 (rat pancreas) demonstrates a 329-bp cdna. Lane 2 W>XI74/HaeIlI standards). Lane 3 (rat eyes) shows a 329bp cdna similar to that in lane 1. (B) Southern transfer showing homology of 329-bp cdna fragments in lanes 1 and 3 with 3J ' P-labeled rat insulin, cdna probe (3 X 106cpm). B 329 ± 6 bp bands after PCR amplification. Amplified cdna fragments with an estimated size of 329 ± 6 bp also were visualized when the pancreas was used as the source of poly(a)+ RNA (Fig. 1A). Southern blots probed with 32P-end-labeled 3'-primer are shown in Figure IB. Both pancreas and eye products demonstrate rat preproinsulin ^homologous bands at the same position, just above the 310-bp marker. These results indicate that the products obtained from the eye are similar, if not identical to the pancreas-derived rat preproinsulin, cdna. The identities of the rat eye and rat pancreas PCR products were investigated further by comparing the restriction patterns of the cdnas amplified from both tissues. Restriction enzymes were used that should cut rat preproinsulin cdna once, twice, or not at all (Fig. 2). Although a total of 10 restriction enzymes was used, the results of restriction endonuclease cleavage with 6 of the enzymes are shown in Figure 2. Lanes 1-3 and 8-10 are digests of the rat eye RT-PCR product. Lanes 5-7 and 12-14 are digests of the rat pancreas RT-PCR product. Lane 4 is a 1:1 mixture of uncut (no enzyme), purified 329-bp PCR product derived from both rat eye and rat pancreas RNA extracts used as a marker. Lane 11 is the 0X174/Hae III standard marker. Lanes 1-7 represent the uncut products with nonrestricting enzymes (excluding lane 4, which has no enzymes present). Lanes 8-10 and 12 14 are cleaving restriction endonucleases. It should be noted that two of the enzymes, Hinfl and Alul, are able to distinguish between preproinsulin, cdna and preproinsulin2 cdna. The close correspondence of the observed fragment sizes to the expected values for rat preproinsulin, cdna underlines the identity of the product. As expected for rat preproinsulin, (or preproinsulin2) cdna, the noncutting enzymes BamHI, EcoRI, Hindi, Hindlll, PstI, Xhol, and Hinfl did not cleave the 329-bp linear band from either the pancreatic or rat eye products. Haelll was predicted to generate four fragments, two of which (113 and 178 bp) would be expected to be visible in our gels. Lanes 9 and 13 (Fig. 2) show the observed fragments, estimated to be 115 and 182 bp. Hpall also produced four fragments whose visible bands should be 78 and 213 bp, respectively. The observed fragment (lanes 8,14) was 218 bp. Alul was predicted to cleave rat preproinsulin, cdna into two fragments of 130 and 200 bp, The actual

466 Investigative Ophthalmology 8c Visual Science, February 1993, Vol. 34, No. 2 FIGURE 2. Agarose gel demonstrating the results of restriction enzyme digestions of rat eye and rat pancreas PCR products. Lanes 1-3 and 8-10 demonstrate digests of rat eye amplified product. Lanes 5-7 and 12-14 show digests of rat pancreas amplified product. Noncutting enzymes are shown in lanes 1 and 7 (EcoRl), lanes 2 and 6 (Xhol), and lanes 3 and 5 (Hinfl). The enzymes that cut are shown in lanes 8 and 14 (Hpall, 218-bp fragment), lanes 9 and 13 (Haelll, 182- and 1 15-bp fragments) and lanes 10 and J2 (Alul, 200- and 130-bp fragments). Lane 4 contains a 1:1 mixture of undigested pancreas and eye amplified products. Lane 11 contains phix174/haeili standards. observed fragments (lanes 10,12) were 130 and 200 bp. If the material were preproinsulin 2 cdna, three fragments of 89, 110, and 130 bp would have been expected with AluJ.. The differentiation between preproinsulin, cdna and preproinsulin 2 cdna is further confirmed by the failure of Hinfl to cut the eye and pancreas products. Hinfl normally cuts preproinsulin2 cdna into two fragments of 140 and 190 bp. A Southern transfer from a gel containing the RTPCR-amplified products from both rat eye and rat pancreas was probed with 32P-end-labeled 5r-primer and a strong annealed signal was seen at the 329-bp position of both the rat eye and rat pancreas products (Figs. 1A, B). The nucleotide sequences of purified cdna obtained separately from whole rat eyes and rat retina were determined. The sequences were found to be identical to the previously known coding sequence of rat pancreatic preproinsuiin, DNA15 and to the sequence of the 329-bp amplified DNA that we generated after application of RT-PCR to poly(a)+ RNA isolated from rat pancreatic tissue. The sequences were identical in all respects, including the sequence encoding the insulin! signal peptide, B-chain, C-peptide, and A-chain (Fig. 3). DISCUSSION The purified 329-bp cdnas separately obtained from the whole eye and the rat retina were found by gel electrophoi esis, Southern blotting, and restriction enzyme digestion to resemble the rat preproinsuiin, coding sequence obtained from rat pancreas RT-PCR products. The identification of retinal preproinsulin, mrna was confirmed by the direct sequencing of rat preproinsuiinj cdna in the reverse transcription products from total rat eye RNA and rat retina poly(a)+ RNA. This appears to be the first evidence for the expression of an insulin mrna in normal mammalian eye tissue. When nonreverse transcribed total RNA was subjected to PCR amplification with the preproinsulin-specific primers, no detectable contamination with genomic preproinsuiin DNA was observed. The sequenced rat eye cdna product was identical to the preproinsuiin! coding sequence obtained by RT-PCR of rat pancreas poly(a)+ RNA and also identical to the published sequence.15 These findings correlate with our previous studies demonstrating immunoreactivity to insulin antisera in

Preproinsulin mrna in the Rat Eye 467 RAT RETINA PREPROINSULIN-I CODING SEQUENCE (NUMBERING CORRESPONDS TO RAT PANCREAS PREPROINSULIN I SEQUENCE IN GENBANK DATABASE) (5'oligonucleotide primer) 419O 42OO 421O 4220 423O 4240 425O ATG GCC CTG TGG ATG CGC TTC CTG CCC CTG CTG GCC CTG CTC GTC CTC TGG GAG CCC AAG CCT GCC CAG GCT TAC CGG GAC ACC TAC GCG AAG GAC GGG GAC GAC CGG GAC GAG CAG GAG ACC CTC GGG TTC GGA CGG GTC CGA Het Ala Leu Trp Met Arg Phe Leu Pro Leu Leu Ala Leu Leu Val Leu Trp Glu Pro Lys Pro Ala Gin Ala a a a a a a a a a a PREPROINSULIN I _a a a a a a a a a b b b b, b b b b INSULIN I SIGNAL PEPTIDE b b b b b b b b e e e e e e e e e e INS-1 MRNA e e e e e e e e e e 4260 4270 4280 429O 43OO 431O 4320 TTT GTC AAA CAG CAC CTT TGT GGT CCT CAC CTG GTG GAG GCT CTG TAC CTG GTG TGT GGG GAA CGT GGT TTC AAA CAG TTT GTC GTG GAA ACA CCA GGA GTG GAC CAC CTC CGA GAC ATG GAC CAC ACA CCC CTT GCA CCA AAG Phe Val Lys Gin His Leu Cys Gly Pro His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe a a a a a a a a a a PREPROINSULIN I _a a a a a a a a a c c c c c c c c c c _B CHAIN c c c c c c c c c c e e e e e e e e e e INS-1 MRNA e e e e e e e a e 433O 434O 435O 436O 4370 4380 4390 TTC TAC ACA CCC AAG TCC CGT CGT GAA GTG GAG GAC CCG CAA GTG CCA CAA CTG GAG CTG GGT GGA GGC CCG AAG ATG TGT GGG TTC AGG GCA GCA CTT CAC CTC CTG GGC GTT CAC GGT GTT GAC CTC GAC CCA CCT CCG GGC Phe Tyr Thr Pro Lys Ser Arg Arg Glu Val Glu Asp Pro Gin Val Pro Gin Leu Glu Leu Gly Gly Gly Pro a a a a a a a a a a PREPROINSULIN I a a a a a a a a a C B CHAIN C > f f f f f C-PEPTIDE f f f f f f f f f e e e e a e e a e e INS-1 MRNA a «e e e e a a a a 44OO 441O 442O 443O 444O 4450 446O 447O GAG GCC GGG GAT CTT CAG ACC TTG GCA CTG GAG GTT GCC CGG CAG AAG CGT GGC ATT GTG GAT CAG TGC TGC CTC CGG CCC CTA GAA GTC TGG AAC CGT GAC CTC CAA CGG GCC GTC TTC GCA CCG TAA CAC CTA GTC ACG ACG Glu Ala Gly Asp Leu Gin Thr Leu Ala Leu Glu Val Ala Arg Gin Lys Arg Gly lie Val Asp Gin Cys Cys a a a a a a a a a a PREPROINSULIN I a a a a a a a a a t f f t f f" f f f C-PEPTIDE f f t > d d A CHAIN d d e e e e e e e e e e INS-1 MRNA e e e e e e e e e e 448O ( 3'oligonucleotlde primer) 449O 45OO 451O ACC AGC ATC TGC TCC CTC TAC CAA CTG GAG AAC TAC TGC AAC TGA TGG TCG TAG ACG AGG GAG ATG GTT GAC CTC TTG ATG ACG TTG ACT Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Asn End a a a a a a a a a a a a a a d d d d d d d d d d d d d > e e e d e e e e e e e e e e > FIGURE 3. Nucleotide sequence of the 329-bp amplified DNA produced by combined reverse transcription and PCR from poly(a) + RNA purified from whole rat eye total RNA. tissue slices of rat eye and retina and immunoreactivity to insulin, and the glial cell markers S-100 protein and glial fibrillary acidic protein, in retinal Miiller cells in primary culture. 2-316 Correlations can also be made with our previous in situ hybridization experiments using labeled rat preproinsulin cdna probes to locate insulin-related mrna in the cytoplasm of retinal glial cells cultured in serum- and insulin-free media. 3 ' 4 The evidence that cells in the eye can synthesize preproinsulin j mrna raises the possibility that insulin may have a local physiologic role in extrapancreatic tissues, being involved in intracellular (autocrine) and intercellular (paracrine) signaling. Also, locally produced insulin could serve as a growth factor, a neurotrophic factor, in organelle organization, in protein storage, in modulation of specific metabolic functions, including monoamine metabolism, or as a neuromodulator. It is known that insulin stimulates the synaptosomal uptake of neurotransmitter amino acids in the adult rat brain and thus may be important as a neuromodulator 17 or neurotransmitter, 18 and that insulin has growth factor-like properties in the brain that are mediated through phosphorylation. 19 The importance of insulin in glucose or amino acid metabolism in neural tissue, however, remains uncertain. 20 " 22 The known neurotrophic effects of insulin (as well as nerve growth factor and insulin-like growth factor-i) 23-24 suggests the possibility that insulin may interact with the cholinergic system and other systems in the brain and other tissues. Insulin has been shown to affect brain monoamine metabolism, 25 dopamine release, 26 and cerebroside synthesis, 27 and can regulate membrane transport and influence enzyme activities as well as DNA and RNA synthesis. 28 Treatment of neurons in culture with a depolarizing solution has been shown to induce a threefold stimulation of insulin release into the culture medium. 29 Thus, locally produced insulin may be important for the growth and maintenance of neural tissue 1830 or in the control of neural and periph-

468 Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2 eral carbohydrate 3132 and catecholamine metabolism. 33 Because Muller cells contain glycolytic enzymes and can synthesize and store glycogen, 34 it has been suggested that locally produced insulin could play a role in glucose or amino acid metabolism in the retina. Furthermore, the appearance of insulin in the developing retina and other areas of the developing eye before the initiation of pancreatic insulin synthesis 3536 strongly suggests a significant role for insulin as a growth or trophic factor in the developing eye. These findings are strengthened by evidence showing that insulin-related molecules arose early in evolution and that such molecules can stimulate sugar uptake, lipid mobilization, and growth stimulation in nervous tissue and other tissues of invertebrates and vertebrates. 37 Overall, much remains to be known about the control and importance of insulin synthesis occurring in the eye and other nonpancreatic tissues. Further work is in progress to determine the quantity of mrna being produced in the eye, the identification of the specific localization and specific cells that are involved in its production, and the developmental time course leading to the appearance of insulin mrna and insulin immunoreactivity in the fetal eye. Key Words cdna, mrna, polymerase chain reaction, preproinsulin synthesis, retina. Acknowledgments The authors thank Angela Lineen and Donald Zhou for expert technical assistance in the preparation of RNA samples and the amplification of some of the specific cdna fragments. References 1. Das A, Pansky B, Budd GC, Kollarits CR. Immunocytochemistry of mouse and human retina with antisera to insulin and S-100 protein. Curr Eye Res. 1984; 3:1397. 2. Das A, Pansky B, Budd GC, Kollarits CR. Binding of antisera to insulin and S-100 protein in mammalian retina and optic nerve. The Physiologist. 1984; 27(4):254. 3. Das A, Budd GC, Pansky B, Cordell B, Kollarits CR. Insulin-specific mrna in rat retinal glial cells. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1985; 26(suppl):337. 4. Das A, Pansky B, Budd GC. Demonstration of insulinspecific mrna in cultured rat retinal glial cells. Invest Ophthalmol Vis Sci. 1987; 28:1800. 5. Chirgwin JJ, Przbyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. 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