The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 1 R A D S J . B i o l . R e s . A p p l . S c i . 1 Op e n Ac c e s s F u l l L e n g t h A r t i c l e The Action of Cytomegalovirus (CMV) Promoter on Expression of Genetically Engineered Insulin in Rat Hepatocytes Gehad Elsayed Mahmoud Department of Genetics, Faculty of Science, Alexandria, Egypt A B S T R A C T Background: Background: Insulin is hormone production in the pancreas that stimulates glucose uptake from blood to enter the body's cells, where it is converted into energy needed by muscles and tissues to function. The pancreatic cells are responsible for the secretion of insulin. In diabetes, the body cannot produce enough insulin or cannot use insulin effectively. Objectives: In this study, we attempted to activate the hepatic cells to secrete insulin instead of the pancreatic cells. Methodology: This was done by gene therapy; an excellent strategy to treat diabetes by supplying the correct wild type copy of a furin cleavable sites preproinsulin. The preproinsulin was extracted from the rat DNA, cloned and mutated to generate the two furin cleavable sites responsible for the removal of C-peptide to form the two chains (A and B) for mature insulin production. This mutated insulin was derived by Cytomegalovirus (CMV) promoter to express insulin by its transfection inside the primary rat hepatocytes using a non-viral vehicle to keep the hepatic cells healthier against the transfection. In vitro, the rat hepatocytes could not divide well as in vivo, but special hormones like insulin and dexamethasone lived longer and kept their function. Results: The CMV promoter is strong and lead to overexpression of mature insulin inside rat hepatocytes leading to deterioration, the toxicity of hepatocytes and finally cell death. Hepatocytes in vitro were more fragile and needed some modification to adapt to the secretion of insulin. Conclusion: Glucokinase or glucose transporter promoters were much more perfect than CMV. They can activate the hepatocytes to modulate the glucose level and so limiting the amount of insulin secreted. These promoters are weaker than CMV but much more perfect for hepatocytes. Keywords Cloning, CMV promoter, Gene therapy, Hepatocytes, Mutation, Non-viral Vector, Preproinsulin, Transfection. *Address of Correspondence gehadelsayedkg@gmail.com Article info. Received: September 15, 2020 Accepted: August 16, 2020 Cite this article: Mahmoud GE. The Action of Cytomegalovirus (CMV) Promoter on Expression of Genetically Engineered Insulin in Rat Hepatocytes. RADS J Biol Res Appl Sci. 2020; 11(1):1-8. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding Source: Nil Conflict of Interest: Nil I N T R O D U C T I O N The intra-cellular conversion for all known mammalian proinsulin to insulin involves cleavage at two paired basic sites present at either end of the C-peptide1. In both rat and human proinsulins, these are Arg31-Arg32 at the B- chain/C-peptide junction and Lys64-Arg65 at the C- peptide/A-chain junction. The cleavage is carried out by the endopeptidases PC2 and PC3/12-4. Most cell engineering approaches to target the non- neuroendocrine cells that lack the specific endopeptidases (PC2 and PC3/1) required to process proinsulin into active mature insulin. To overcome this O R I G I N A L A R T I C L E The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 2 R A D S J . B i o l . R e s . A p p l . S c i . 2 problem, many researchers used site-directed mutations to engineer proinsulin to be a substrate for furin5-7. The enzyme Paired Basic Amino Acid cleaving enzyme also known as (PACE) is a Golgi-associated propeptide endoprotease that is present in the constitutive secretory pathway of virtually all cells8. The most chosen target cells for insulin gene therapy are hepatocytes. Although hepatocytes do not have the machinery to store insulin within secretory vesicles and secrete it in a regulated fashion, hepatocytes are attractive targets for insulin expression because they are closely related to the pancreatic β cells developmentally, play a very important role in glucose homeostasis, and are relatively easy to target9-11. M A T E R I A L S A N D M E T H O D S Amplification of the Preproinsulin Gene from the Rat Spleen We extracted the rat genomic DNA from the rat spleen by genomic extraction kit. Amplification of the preproinsulin gene by PCR was performed using the designed primers: F: 5’CATGGCCCTGTGGATGCGCTTCCTGCCCCTG3’ R: 5’GAGTTGCAGTAGTTCTCCAGTTGGTAGAGGA3’ The PCR cycle step was done as: initial denaturation (94°C, 3min, and 1cycle), denaturation (94°C, 30sec), annealing (55°C, 30sec), extension (72°C, 1min, 35 cycles), final extension (72°C, 5min, 1cycle). The amplified gene was desalted and purified, then cloned into a cloning NEB® PCR cloning vector (Cat. No. E1202). Further, the gene was mutated by substitution using site directed mutagenesis (Cat. No. E0554S) to substitute lysine instead of glutamic acid in A chain, arginine instead of valine in the connected C peptide, and arginine instead of glutamine in the B chain. Translated protein MALWMRFLPLLALLVLWEPKPAQAFVKQHLCGPHLVEA LYLVCGERGFFYTPKSRREVEDPQVPQLELGGGPEAG DLQTLALEVARQKRGIVDQCCTSICSLYQLENYCNStop C-peptide Wild type Insulin R EVEDPQVPQLELGGGPEAGDLQTLALEVARQ K Mutant type R KREDPQVPQLELGGGPEAGDLQTLALEVARR K C- peptide with Tetrabasic furin cleavage site According to the NEBase Changer, the primers are at the B-chain/C-peptide: F:5'CAAGTCCCGTCGTAAACGGGAGGACCCGCAAG3' R: 5'GGTGTGTAGAAGAAACCACGTTCCC3' At the C-peptide/A-chain: F:5’GAGGTTGCCCGGCGGAAGCGTGGCATTG3' R:5’CAGTGCCAAGGTCTGAAG A TCCC3' The vector used for mutation now contains the preproinsulin with the new three amino acids. After mutation, transformation into bacteria and isolation of the pure vector has proceeded. Then digested the vector by one restriction enzyme (NruI), amplify the gene of interest by: F:TTGGATCCACCATGGCCCTGTGGATGCGCTTCCTG CCCCTG, R:GTGAATTCGTTGCAGTAGTTCTCCAGTTGGTAGAG GGA Insertion of Preproinsulin Gene into the Expressing Plasmid The expressing plasmid (#13031) with CMV promoter was received from the Addgene, isolated, purified, and digested by the two restriction enzymes (ECORI & BamHI), then purified and desalted by using purification kit (extraction kit Cat. No. 20021), then by the same restriction enzymes we digested the amplified preproinsulin gene, the ligation step was later proceeded for the digested plasmid and digested gene. Digested expressing vector (0.3µg/ul) 1.5µl Digested purified preproinsulin gene (1µg/ul) 3µl Ligase buffer 2µl Distilled H2O 13µl (Mix well) Ligase 1µl Culture of Hepatocytes The culture of hepatocytes was performed according to the protocol by Ling et al., with some modifications12. Rat hepatic cells were purchased from cell biologics, shipped in suspension (Cat. No. RA-6224F), centrifuged, then The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 3 R A D S J . B i o l . R e s . A p p l . S c i . 3 suspended in 30ml warm William's complete medium (Add the following to Williams' Medium E: L-glutamine 2mM, fetal calf serum (FCS) to 5%, dexamethasone to 100nM, cadmium chloride to 9µg/l, Dimethyl sulfoxide (DMSO) to 0.5%, penicillin to 100IU/ml and streptomycin to 100mg/ml), and then centrifuged and resuspended again in 20ml warm William's complete medium. Cells were counted within the cell suspension using a hemocytometer and cell viability was determined by trypan blue staining. Cells were plated in collagen coated plates, cultured at 37°C in 95% air and 5% CO2. After 4hrs of culture, the cells were replaced with another medium that is serum free to maintain the morphology of the cells. After 24hrs of culture images, every 2hrs on the first day of culture and every 2 days was captured by Olympus inverted microscope. After optimization the cell culture condition, transfection takes place using 1.5µl TurboFect transfection reagent (Cat. No. R0531) in 1ml culture medium after 24hrs of culture. Examination of the Released Green Fluorescent Protein The expressed green fluorescent protein was detected under the inverted green fluorescent microscope. Measurement of the Amount of Insulin Released The insulin release was measured according to the concentration of glucose added by adding different concentrations of glucose from 5 to 25mM glucose to the media of the transfected cells. We measured the amount of insulin released according to the concentration of glucose added by the Cohesion rat insulin ELISA kit (Cat. No. CEK1622). Sequencing The designing plasmid containing the genetically engineered insulin was forward and reverse sequenced using the ABI prism Big DyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems, Germany). R E S U L T S To get the rat preproinsulin gene, we extracted the genomic DNA from the rat spleen. Then we amplified it by PCR amplification technique using the specific primer designed for the rat preproinsulin gene. The amplified preproinsulin gene was inserted into the cloning vector, transformed to get high yield and isolated by midiprep kit. The mutation proceeded in the two chains A & B. Then gene of interest was amplified by digesting the vector and inserted into the expressing plasmid. So, we obtained a vector containing the preproinsulin gene with sites of cleavage derived by the CMV promoter. This vector was measured for its concentration to detect its purity (Table 1) and sequenced for both forward and reverse sequence to be sure from the location of mutated preproinsulin gene (Fig. 1 & 2). Table 1. Measurement of the Concentration and Purity of the Extracted Plasmid using UV Spectrophotometer. (The dilution factor was X300). S. No. Plasmid A260 A280 Concentration (µg/ml) Ratio 01 Designing CMV expressin g plasmid 0.270 0.140 4050 1.92 Fig 1. The forward sequence for designing CMV expressing plasmid showing the area between CMV promoter and the Enhanced green fluorescent protein (EGFP). Fig 2. The reverse sequence for designing CMV expressing plasmid showing the area between EGFP and CMV promoter. The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 4 R A D S J . B i o l . R e s . A p p l . S c i . 4 The expressing plasmid was now ready to transfect into the primary rat hepatic cells. Primary rat hepatocytes were washed, diluted, counted (Table 2), then cultured in 6 well coated collagen plates with complete Williams’ E medium in a humified atmosphere incubator for 2hrs and after that demonstrate the morphology of the cells (Fig. 3). Fig 3. The rat hepatocytes immediately after plating on 6 well collagen coated plate with Williams’ E complete medium under an inverted microscope. Shows the cells X200. Table 2. The Count of Live and Dead Cells on Haemocytometer to Detect the Viability and Cell Density of the Rat Hepatic Cells. S. No. No. of squares Live cells Dead cells Total count 01 1 71 8 79 02 2 52 0 52 03 3 85 4 89 04 4 68 6 74 05 Average 69 5 74 Viable cells = 69 X 104 x 2 = 138 x 104 cells/ml. Total count = 74 X104 x 2 = 148 x104 cells/ml. Viability % = viable/total =138/148 = 93% Cell density = Total count The rat hepatic cells were investigated after plating under the inverted microscope every 48hrs to detect the cell viability, division, and morphology (Fig. 4). The cells show high viability already after plating, the viable cells count decreases day after day to reach about 32% at day 19 after plating. After optimization of the cell culture condition, transfection takes place using 1.5µl TurboFect transfection reagent in 1ml culture medium after 24hrs of culture, in a 12 well collagen coated culture plate. Then transgenic expression was analyzed 24-48hrs later. We will transfect the cells with the prepared expressing plasmid then examine the cell morphology and viability after transfection (Fig. 5). The rat hepatocytes after transfection with the construct containing CMV promoter showed a high change in the morphology and viability, hepatocytes cannot stand more than 5 or 6 days, the viability decreases fast and day after day to reach about 5% on day 5 from transfection. The morphology high changed; the cells appeared very tired. The CMV promoter is strong which resulted overexpression for the gene controlled by it, the rat hepatocytes are fragile and cannot divide well in vitro to stand with this over expression. Under the inverted fluorescent microscope, we examined the fluorescent cells which released the fluorescent fused insulin-EGFP (Fig. 6). The media around the hepatocytes showed no fluorescent even under addition different concentrations of glucose, but the cells show high fluorescent from the first day but, this fluorescence decreases every day till reach day 6 in which almost no fluorescence found. The over expression of the fused protein trapped inside the cells lead to the failure of hepatocytes to stand. Fig 4. X100 rat hepatic cells after plating. (A) After 24hrs of plating. (B) After 5 days of plating. (C) After 7 days of plating. (D) After 11 days of plating. (E) After 17 days of plating. (F) After 19 days of plating. The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 5 R A D S J . B i o l . R e s . A p p l . S c i . 5 Fig 5. The rat hepatic cells after the transfection with the designing plasmid with CMV promoter X100. (A) The rat hepatocytes after 7hrs from transfection. (B) The rat hepatocytes after 24hrs from transfection. (C) The rat hepatocytes after 2 days from transfection. (D) The rat hepatocytes after 3 days from transfection. (E) The rat hepatocytes after 4 days from transfection. (F) The rat hepatocytes after 5 days from transfection. CMV promoter has not the ability to make balance out and in the cells, has not the ability to transport glucose from media to cells and vice versa. So, the insulin still trapped inside. Glucose was measured in cell lysates and cell media of transfected culture cells 3 times by colorimetric glucose assay (Table 3), after the addition of different concentrations of glucose (0-25mM) to the media and obtained the average. Fig 6. The transfected rat hepatocytes with the designing plasmid with CMV promoter under the green fluorescent microscope X200. (A) The media after stimulation with 25mM glucose. (B) The media of free glucose. (C) The transfected hepatocytes after 24hrs from transfection. (D) The transfected hepatocytes after 2 days from transfection. (E) The transfected hepatocytes after 3 days from transfection. (F) The transfected hepatocytes after 5 days from transfection. (G) The transfected hepatocytes after 6 days from transfection. The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 6 R A D S J . B i o l . R e s . A p p l . S c i . 6 Table 3. The Colorimetric Method for the Detection of the Amount of Glucose. S. No. Parameters Calorimetric glucose assay in mg/dl after 24hrs from the addition of different doses of glucose in the transfected primary hepatocyte culture medium 01 Different doses of glucose added in mM Free glucose 5mM 10mM 15mM 25mM 02 The designing plasmid with CMV promoter In media ND 81 210 240 301 In Cell lysate ND ND ND ND ND Note: Present in both cell media & lysates after the addition of different amount concentration of glucose to the media of transfected cells with (the designing plasmid with cmv promoter) no detected amount of glucose can be found inside the cells, as the cmv promoter has not the ability to transport glucose in and out the cells. Due to the amount of mature insulin released from the designing plasmid with CMV promoter, we measured it 3 times by cohesion rat insulin kit Cat. No. CEK1622). and get the standard curve for rat insulin (Fig. 7). From this standard curve, we can obtain the different amounts of insulin released from the construct in both cell lysate and media in pg/ml from its relative O.D. And so make a relation between the concentration of glucose in mM and the concentration of insulin in pg/ml. Fig 7. Standard curve for rat insulin. For the designing of a plasmid with CMV promoter, this construct releases overdose for insulin only inside the cells not outside the medium, as this promoter has not the ability to transport the glucose in and out the cell and the insulin remain trapped inside the cells and not out to the media. So, the relation between the glucose and insulin inside the cell be constant and overexpression of insulin found in case of low dose or high dose glucose, and outside the cells (in media), no insulin found to make a relation (Fig. 8). Fig. 8. The relation between the concentrations of insulin released corresponding to different doses of glucose in cell lysate using the designing plasmid with CMV promoter. No response to glucose appeared even in high expression of insulin. D I S C U S S I O N Many alternative approaches to treat Type 1 Diabetes mellitus (T1DM) using the correct type of gene or genetically engineered gene, without using pancreas transplantation, have been attempted. Examples of such attempts include insulin production from various native cells, such as liver cells13-15. In our study, we engineered insulin to be correctly secreted from the liver cells. The mammalian CMV promoter is strong, constitutive transgene expression and would drive consistent, high- level expression of insulin even during using low concentration of glucose. Thus, a weaker promoter must be used to maintain low levels of insulin production to gain a successful insulin gene therapy in treating T1DM16. The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 7 R A D S J . B i o l . R e s . A p p l . S c i . 7 C O N C L U S I O N Our current study aimed to express insulin in vitro in rat hepatocytes, a strategy to try to adapt hepatic cells to secrete insulin instead of pancreatic cells, using a construct containing the furin cleavable sites preproinsulin derived by a CMV promoter. The mature insulin secreted successfully but the usage of this promoter was a bad choice, as the CMV promoter lead the insulin trapped inside the cells and no passage for the glucose in and out the media. The rat hepatocytes in vitro are fragile and not divide to stand with this insulin overexpression. The usage of the weaker promoter will work better like glucose transporter 2 promoter (GLuT2) or glucokinase promoter which can transport glucose in and out the cells. A C K N O W L E D G E M E N T S I am gratefully acknowledged Mr Karim Morsi, Eng. Mohammed Abdalla, Dr. Necolas and The Research and Medical Technology Institute, Smouha, Alexandria, Egypt and the research lab of Faculty of Science, Alexandria, Egypt. L I S T O F A B B R E V I A T I O N S CMV Cytomegalovirus DMSO Dimethyl sulfoxide FCS Fetal Calf Serum GLuT2 Glucose Transporter 2 PACE Paired basic Amino acid Cleaving Enzyme R E F E R E N C E S 1. Steiner DF. The proprotein convertases. Curr Opin Chem Bio. 1998: 2(1);31-9. 2. Seidah NG, Gaspar L, Mion P, Marcinkiewicz M, Mbikay M, Chretien M. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA Cell Bio. 1990; 9(6):415-24. 3. Smeekens SP, Steiner DF. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J Biol Chem. 1990; 265(6):2997-3000. 4. Smeekens SP, Avruch AS, LaMendola J, Chan SJ, Steiner DF. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proceedings of the Nat Acad Sci. 1991; 88(2):340-4. 5. Yanagita M, Nakayama K, Takeuchi T. Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non‐endocrine cell line, COS‐7. FEBS Lett. 1992; 311(1):55-9. 6. Groskreutz DJ, Sliwkowski MX & Gorman CM. Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin. J Bio Chem. 1994. 269; 6241-5. 7. Muzzin P, Eisensmith RC, Copeland KC, Woo SL. Hepatic insulin gene expression as treatment for type 1 diabetes mellitus in rats. Mol Endocrinol. 1997; 11(6):833-7. 8. Van de Ven WJ, Voorberg J, Fontijn R, Pannekoek H, van den Ouweland AM, van Duijnhoven HL, Roebroek AJ, Siezen RJ. Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes. Mol Bio Rep. 1990; 14(4):265-75. 9. Hovorka R. Closed-loop insulin delivery: From bench to clinical practice. Nature Reviews Endocrinol. 2011; 7(7):385-95. 10. Vehik K, Ajami NJ, Hadley D, Petrosino JF, Burkhardt BR. The changing landscape of type 1 diabetes: recent developments and future frontiers. Curr Diab Rep. 2013; 13(5):642-50. 11. Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. New England J Med. 1993; 329(14):977-86. 12. Shen L, Hillebrand A, Wang DQ, Liu M. Isolation, and primary culture of rat hepatic cells. JoVE. 2012; (64):e3917. 13. Auricchio A, Gao GP, Yu QC, Raper S, Rivera VM, Clackson T, Wilson JM. Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer. Gene Ther. 2002; 9(14):963- 71. 14. Dong H, Altomonte J, Morral N, Meseck M, Thung SN, Woo SL. Basal insulin gene expression significantly improves conventional insulin therapy in type 1 diabetic rats. Diab. 2002; 51(1):130-8. The Action of CMV Promoter on Expression of Insulin in Rat Hepatocytes Vol. 11 (1), June 2020 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 8 R A D S J . B i o l . R e s . A p p l . S c i . 8 15. Burkhardt BR, Parker MJ, Zhang YC, Song S, Wasserfall CH, Atkinson MA. Glucose transporter-2 (GLUT2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice. FEBS Lett. 2005; 579(25):5759-64. 16. Roep BO, Peakman M. Antigen targets of type 1 diabetes autoimmunity. Cold Spring Harbor perspectives in medicine. 2012; 2(4):a007781.