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Background: The liver has been suggested as a suitable target organ for gene therapy of Type 1 diabetes. However, the fundamental issue whether insulin-secreting hepatocytes in vivo will be destroyed by the autoimmune processes that kill pancreatic β cells has not been fully addressed. It is possible that the insulin secreting liver cells will be destroyed by the immune system because hepatocytes express major histocompatibility complex (MHC) class I molecules and exhibit constitutive Fas expression; moreover the liver has antigen presenting activity. Together with previous reports that proinsulin is a possible autoantigen in the development of Type 1 diabetes, the autoimmune destruction of insulin producing liver cells is a distinct possibility. Methods: To address this question, transgenic Non-Obese Diabetic (NOD) mice which express insulin in the liver were made using the Phosphoenolpyruvate Carboxykinase (PEPCK) promoter to drive the mouse insulin I gene (Ins). Results: The liver cells were found to possess preproinsulin mRNA, translate (pro)insulin in vivo and release it when exposed to 100 nmol/l glucagon in vitro. The amount of insulin produced was however significantly lower than that produced by the pancreas. The transgenic PEPCK-Ins NOD mice became diabetic at 20–25 weeks of age, with blood glucose levels of 24.1 ± 1.7 mmol/l. Haematoxylin and eosin staining of liver sections from these transgenic NOD PEPCK-Ins mice revealed the absence of an infiltrate of immune cells, a feature that characterised the pancreatic islets of these mice. Conclusions: These data show that hepatocytes induced to produce (pro)insulin in NOD mice are not destroyed by an ongoing autoimmune response; furthermore the expression of (pro)insulin in hepatocytes is insufficient to prevent development of diabetes in NOD mice. These results support the use of liver cells as a potential therapy for type 1 diabetes. However it is possible that a certain threshold level of (pro)insulin production might have to be reached to trigger the autoimmune response. Page 1 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 would be unsatisfactory to address the issue of insulin Background Genetic alteration of non-pancreatic cells in a diabetic autoantigenicity. person to synthesise, store and secrete insulin in the same manner as a pancreatic β cell is a potential therapy of type The liver of the transgenic PEPCK-Ins NOD mice were 1 diabetes. The hepatocyte has been suggested as a suita- characterised with respect to insulin mRNA transcription, ble target cell for such gene therapy [1-9]. Such cells made (pro)insulin content and (pro)insulin release. The blood in vitro are capable of synthesizing and storing pro(insu- sugar levels of the animals were monitored and the livers lin) and can secrete this peptide in response to a physio- of the animals were analysed for any evidence of immune logical challenge with glucose [1,2]. Moreover, when cell infiltration. transplanted into diabetic mice these cells can lower blood glucose levels to the normal range . Further, Fer- Methods ber et al.  has recently showed that adenovirus-medi- Materials ated in vivo transfer of the PDX-1 transgene to the mouse The PEPCK promoter was generously donated by Dr R. W. liver results in the conversion of a hepatocyte subpopula- Hanson (Case Western Reserve University, Cleveland, tion to the beta cell phenotype. Ferber also showed that Ohio, USA). DNA modifying enzymes and competent this population of trans-differentiated liver cells was bacteria for transformation were purchased from Promega induced to produce the prohormone convertases PC1/3 (Madison, Wisconsin, USA). RPMI 1640, α-MEM and and PC2 leading to complete processing of proinsulin. fetal bovine serum (FBS) were purchased from Trace Bio- The amount of insulin produced was sufficient to amelio- sciences (Castle Hill, Sydney, Australia). Hybond-N+ rate streptozotocin-induced hyperglycaemia in the mice. nylon membrane for the Southern Blot and ribonuclease protection assay (RPA) was from Amersham International For this gene therapy approach to be viable, the insulin (Bucks, UK). Human insulin and rat insulin standards for producing liver cells must not be destroyed by the the in-house radioimmunoassay (RIA) were obtained immune system or else this could lead to liver damage. from Novo Nordisk (Bagsvaerd, Denmark). This fundamental question has not been addressed by previous studies with (pro)insulin producing hepatocytes Transient transfection of HEPG2 cells The transfection of the HEP G2 cells with the PEPCK-Ins as diabetes in these models was induced chemically rather than by autoimmune means [6-9]. The autoimmune transgene was carried out using Lipofectamine (Gibco- destruction of insulin-producing hepatocytes is a possibil- BRL, Madison, Wisconsin, USA). Cells were trypsin har- ity since hepatocytes express major histocompatibility vested and seeded 24 hr prior to transfection at a density complex (MHC) class I molecules , and constitutively of 200,000 cells/well in 24 well tissue culture plates (Bec- express Fas , moreover the liver cells have antigen pre- ton Dickinson, New Jersey, USA) to achieve 60–80% con- senting activity  and are attacked in autoimmune dis- fluence at the time of transfection. 10µl of Lipofectamine eases such as chronic active hepatitis. Furthermore was added to 300µl of serum free media and incubated at (pro)insulin appears to be an autoantigen in the develop- room temperature for 30 minutes. After the incubation, ment of type 1 diabetes [13,14]. Evidence for this comes 300µl of DNA solution (3–4µg) was added and the from the presence insulin and Glutamic Acid Decarboxy- lipid:DNA complex solution incubated at room tempera- lase (GAD) autoantibodies in the sera of people with pre- ture for 15 minutes. The cells were washed once with 1 ml diabetes . It also comes from studies that show that of serum free medium prior to the transfer of 300µl of the diabetes can be adoptively transferred to normoglycaemic lipid:DNA complex solutions to the cells. The cells were mice by the introduction of insulin-specific T cell clones then incubated for 5 h at 37°C in an air incubator. The . These data taken together suggest that hepatocytes DNA/lipid complex media was removed and replaced which produce (pro)insulin might be targeted and with 2 mL of α-MEM 10% FBS which contains Antimy- destroyed by the same autoimmune processes responsible cotic and Antibiotic solution (10µL/mL) (Sigma, St Louis, for the destruction of pancreatic β cells. Missouri, USA). In addition, for the PEPCK-Ins transgene, a final concentration of 100 nM glucagon was added to To address this question in vivo, transgenic Non-Obese the media to upregulate the PEPCK promoter. The cells Diabetic (NOD) mice that express insulin in the liver were were incubated at 37°C in an air incubator for 48–60 hr. created using the Phosphoenolpyruvate Carboxykinase After the incubation, the conditioned culture medium was (PEPCK) promoter  to direct expression of the mouse collected and assayed for mouse (pro)insulin. insulin I gene. This promoter had previously been used to create a transgenic C57BL/6 human insulin secreting Insulin Radioimmunoassay (RIA) mouse line with hepatic insulin expression . However (Pro)insulin was measured by an in-house RIA using rat these mice do not develop autoimmune diabetes and insulin standard (Novo Nordisk Laboratories, Bagsvaerd, Denmark). The lowest value on the rat insulin standard Page 2 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 curve used in the assay was 0.25 ng/ml. The intra and inte- specific for mouse insulin I mRNA. The sequence of prim- rassay coefficients of variance were <5% and 10% ers used were: respectively. (Forward) TAA CCC CCA GCC CTT AGT GAC CAG CTA Generation of transgenic mice TAA To direct the expression of the mouse insulin I gene to the liver of the transgenic mice, the 5' flanking sequences (- (Reverse) AAA GTT TTA TTC ATT GCA GAG GGG TGG 460 bp to +73 bp) of the rat PEPCK gene  were used GGC to drive the expression of the genomic mouse insulin I gene (-25 bp to +557 bp) . The construct was digested The PCR products were run on a 1.2% Tris Acetate EDTA with Xba I and Pst I (Fig 1A) and the PEPCK-Ins transgene agarose gel, photographed and analysed using the Gel was isolated from the agarose gel using the Supelco Gen- Doc 1000 system (Biorad, Hercules, California, USA). Elute Spin Column (Sigma, St Louis, Missouri, USA). Standard procedures were followed to generate transgenic Insulin mRNA in the samples was also visualised using mice . Fertilized mouse eggs were flushed from the the ribonuclease protection assay (RPA) method. The oviducts of superovulated NOD mice 6–8 h after ovula- plasmid used as the template to transcribe the riboprobe tion. Male pronuclei of the fertilized eggs were injected for mouse insulin I was a generous gift from Chirgwin J. with 2pl of the DNA solution (2 ng/µl) and the embryos M. (San Antonio, Texas, USA). The Digoxigenin (DIG) that divide to the 2 cell stage were implanted in the ovi- labelled insulin riboprobe was synthesized by the in vitro ducts of pseudopregnant mice. After birth the animals transcription method using the DIG RNA labeling kit were tested for the presence of the transgene by PCR and (SP6/T7) from Boehringer Mannheim (Mannheim, Ger- Southern blot of the DNA from tail tip samples taken after many), which utilises DIG-dUTP, according to the manu- weaning at 3 weeks of age. facturer's recommendations. Treatment of the animals Hybridisation and RNase treatment were performed using The mice were fed ad libitum with a standard diet and the RPA II kit from Ambion according to the manufac- kept under a light-dark cycle of 12 h in compliance with turer's recommendations. Next the samples were loaded the animal ethics of our institution. The facilities used to and run on a 6% polyacrylamide/8 M urea denaturing breed and maintain the mice were specific pathogen free, mini-gel in Tris borate EDTA buffer. The RNA bands on with air passaged through a HEPA filter. The transgenic the gel were transferred onto a nylon membrane by using PEPCK-Ins NOD mice and the wild type littermates were an electro-gel transfer apparatus and the membrane was monitored for natural development of diabetes. The dia- fixed by using an UV cross linker (Hybaid, UK). betic PEPCK-Ins transgenic NOD mice were maintained by daily insulin injections and sacrificed 4 weeks after For the visualisation of the RNA bands on the nylon mem- they became diabetic. brane, the DIG Wash and Block Buffer Set and the DIG Chemiluminescent Detection Kit, both of which were pur- Blood glucose measurements of mice chased from Boehringer Mannheim, were used. The mem- Blood glucose levels of the wild type as well as the trans- brane was then exposed to a standard Kodak X-ray film for genic NOD mice were performed using the Medisense 30 minutes. The film was subsequently analysed using the Precision QID Blood Glucose Sensor System (Bedford, Gel Doc 1000 system. Massachusetts, USA). The mice were bled by pricking the tail vein and 5µl of the blood placed on to the Precision Organ culture Plus blood glucose electrode. A mouse was classified as Pancreatic and hepatic tissue organ cultures were per- being diabetic if a reading of ≥ 15 mmol/l was obtained formed using organ culture dishes from Becton Dickinson on more than one occasion. (New Jersey, USA) . Briefly, the organs were taken from mice, and an aliquot of 10–30 mg removed from Insulin mRNA analysis each organ. The tissue was then diced into 1 mm explants The total RNA from liver and pancreas was isolated using and spread on a filter paper placed on a sterile wire grid in TRIzol (Life Technologies, Grand Island, New York, USA) the inner well of the organ culture dish such that the tissue . The detection of mouse insulin I mRNA by RT-PCR was exposed to air above and the RPMI 1640 10% FBS was performed using Superscript II RNase H Reverse Tran- medium below. Sterile saline was placed in the outer well scriptase from GibcoBRL (Grand Island, New York, USA) to maintain a humidified environment. The tissue in the to synthesize the first strand cDNA as recommended by organ culture dishes were incubated for 24 hr in a humid- the manufacturer. The cDNA sample was then used as a ified 5% CO and air incubator at 37°C. Culture superna- template in PCR amplification (30 cycles) using primers tant from all media changes of respective wells were Page 3 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 PEPCK promoter (530bp) Mouse insulin 1 + Poly A sequence (600bp) AATAAA Xba I Bgl II Pst I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Genomic mouse Ins I PEPCK-Ins Tg 1 2 3 4 5 6 7 Heterozygous pups Stillborn pups Single Copy Het died before weaning (Homozygous) Tg CTL mouse A-C Figure 1 . The PEPCK-Ins transgene A-C. The PEPCK-Ins transgene. Schematic representation (A). Example of PCR screening of PEPCK-Ins transgenic mice (B). Lanes 1–5 and lanes 10–14 are PCR reactions with genomic DNA extracted from mice that developed from microinjected mouse eggs. Primers specific for the PEPCK-Ins transgene were used for lanes 1–9 (30 cycles) while primers specific for mouse insulin 1 were used for lanes 10–18 (30 cycles). Lanes 6 and 15 are PCR negative controls (no DNA added). Lanes 7 and 16 are PCR reactions to demonstrate the specificity of the primers used (wild type NOD mouse DNA added). Lanes 8, 9, 17 and 18 are half copy spiked and plasmid controls respectively (positive controls). Example of Southern Blotting for the PEPCK-Ins transgene (C). Lane 1 One copy spiked sample (100 ng Balb/c genomic DNA + 102fg PEPCK-Ins plasmid), lane 2 genomic DNA from tail tip of F2 PEPCK-Ins mouse, lanes 3 and 4 are DNA from pups which died before weaning and lanes 5–7 are DNA from still born pups. Each lane was loaded with 15µg of genomic DNA that was digested with Xba I and Pst I to release the transgene. Page 4 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 pooled separately and kept at -20°C until the RIA was per- placed on poly-L-lysine-coated slides. After dewaxing and formed. The results were expressed per mg of tissue. serial alcohol rehydration, the tissue sections were treated O , and then incubated in PBS containing 10% with H 2 2 (Pro)insulin content goat serum or 1% BSA for 20 minutes at room tempera- (Pro)insulin was extracted by homogenising a weighed ture to block nonspecific binding. sample of liver or pancreas in acid-ethanol (solution of 0.18 mol/l HCl in 70% ethanol) and incubating overnight For insulin staining, primary insulin antibody (Dako Cor- at 4°C. The next day, the samples were spun down and the poration, Via Real, Carpinteria, California, USA) at supernatant removed. The (pro)insulin content was deter- 1:1250 was added and the sections were incubated over- mined by RIA and the results expressed per mg tissue. night. The next morning the sections were washed in PBS and incubated with anti-guinea pig IgG (1:400) at room In situ hybridisation of (pro)insulin mRNA temperature for 20 minutes. Thereafter the sections were To detect the transcription expression of (pro)insulin, in washed again with PBS, and biotinylated anti-rabbit / situ hybridisation (ISH) was performed on pancreatic and mouse antibody added to the sections for 15 minutes fol- liver tissues of diabetic transgenic PEPCK-Ins NOD mice lowed by streptavidin-peroxidase conjugate for a further and diabetic wild type NOD mice using a modification of 15 minutes. Finally, the sections were treated with sub- a previously described method (20). Briefly, 4µm thick strate-chromogen AEC. A standard concentration (0.1%) paraffin sections were deparaffinized with xylene, ethanol of haematoxylin was added as a counterstain. The primary and air dried. The sections in 0.01 mol/l citric buffer, pH antibody was omitted for the negative control. The hae- 6.0 were then treated with microwave irradiation for 10 matoxylin and eosin staining of sections was performed min. Following this procedure the sections were treated automatically by the Jung Autostainer XL machine (Leica, with proteinase K (Invitrogen, Carlsbad, CA, USA) 1µg/ml New Jersey, USA). for 15 min at 37°C. They were washed 3 times with phos- phate buffered saline for 5 min and dried with ethanol. Statistical analysis The sections were then hybridized with -S-labelled The log rank-test was used to determine whether the riboprobes (1µg/ml) overnight at 57°C. The -S- occurrence of diabetes in the transgenic mice was signifi- labelled riboprobes were synthesized by in vitro transcrip- cantly different from the wild type NOD mice. The non- tion (SP6/T7) incorporation of -S-dUTP using RNA parametric t-test (Mann-Whitney U test) was used to polymerase (Roche, Mannheim, Germany). The template determine whether the blood glucose levels of the diabetic used was mouse insulin I cDNA that was generously transgenic and wild type NOD mice were significantly donated by Chirgwin JM (San Antonio, Texas, USA). The different. hybridisation buffer consisted of a 25µl hybridisation cocktail (labelled riboprobe 500,000 cpm/section, 50% Results formamide, 0.1% SDS, 0.1% sodium thiosulfate, 0.1 mol/ Transient transfection of HEP G2 with PEPCK-Ins l DTT, 0.3 mol/l NaCl, 20 mmol/l Tris-HCl [pH 7.5], 2 To confirm that the PEPCK-Ins transgene (Figure 1A) was mmol/l EDTA, 20% dextran sulphate, 0.02% sheared functional, 3–4µg of the transgene was transfected into salmon sperm DNA, 0.1% total yeast RNA, 0.02% yeast the human hepatoma cell line HEP G2. After the transfec- tRNA, 1 × Denhardt's solution). After hybridisation, the tion, the cells were incubated in α-MEM supplemented slides were soaked in 2X standard saline citrate (SSC), with 10% FBS. Glucagon was added to upregulate the rinsed with deionised water and then treated with RNase PEPCK promoter expression at a final concentration of A (20µg/ml) for 30 min at room temperature. High strin- 100 nmol/l for 24 hr. At the end of the incubation period, gency washes were then performed on the slides with the conditioned culture medium was positive for rodent 2XSSC followed by 0.2XSSC. The slides were then dried by insulin at a concentration of 0.90 ± 0.04 ng/200,000 HEP ethanol, air dried for 20 min and the results viewed by G2 cells (n = 8). NBT2 emulsion autoradiography (Kodak, Rochester, New York). For controls, sense-probe instead of anti-sense was Generation of transgenic mice used for hybridisation. The photos were taken using the From a series of two microinjections, 43 mice were Olympus IX70 microscope (Melville, New York) with a obtained of which 4 were positive for the PEPCK-Ins dark field condenser. transgene when analysed by PCR (Figure 1B) and South- ern blot (Figure 1C). Two of the 4 transgenic founder mice Histochemistry were found to have established germline transmission of Sections of the liver and pancreas from the transgenic the transgene. From the Southern blot analysis, these mice mice were washed twice in Phosphate Buffered Saline have only a single copy of the transgene (Figure 1C). (PBS), fixed in 10% formalin overnight and embedded into paraffin. Consecutive 5µm sections were cut and Page 5 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 Transcription of insulin I mRNA in the liver and 22 out of 51 females (43%) became diabetic (Figure Of the two lines only one was found to transcribe insulin 4), at 19–28 weeks of age. The mice were followed for a mRNA in the liver, as determined by mouse insulin I RT- total of 15 months but no other transgenic mice became PCR (Figure 2A) and mouse insulin I Ribonuclease Pro- diabetic while 55% of the NOD females became diabetic. tection Assay (RPA) (Figure 2B). No insulin mRNA was The incidence of diabetes in the transgenic PEPCK-Ins detected in the second line despite documented germline NOD females (16% diabetic at 30 weeks) differs signifi- transmission of the transgene. The transgenic mice cantly from the incidence of diabetes in the wild-type appeared healthy and active, and were normoglycaemic. NOD females (43% diabetic at 30 weeks) as determined The blood glucose levels were 6.0 ± 1.1 mmol/l (n = 20) by the log-rank test (chi squared value 16.1, P < 0.001). as compared to 6.5 ± 0.5 (n = 20) for wild type littermates. The overall incidence of diabetes in the transgenic PEPCK- Ins NOD mice (13%) was also significantly lower com- Production of (pro)insulin from liver cells pared to the wild type (28%) NOD mice as determined by The ability of the liver cells from PEPCK-Ins positive trans- the log-rank test (chi squared value 18.7, P < 0.001). The genic mice to produce (pro)insulin was examined on liver blood glucose levels of the diabetic transgenic NOD removed from mice that had been sacrificed. Ideally pro- PEPCK-Ins mice (24.8 ± 1.9 mmol/l) were also signifi- duction of (pro)insulin from the liver would be sought in cantly lower than those of the diabetic wild type NOD vivo. This was not possible since any (pro)insulin released mice (>33 ± 2.1 mmol/l) as determined by the Mann from the liver would be indistinguishable from that Whitney U test, U = 32 and P = 0.004. secreted from pancreatic β cells. Two methods were used to analyse if (pro)insulin was produced from liver tissue The livers of the diabetic transgenic mice were examined removed from the mice. The liver was both extracted for histologically to determine if there was cellular infiltra- its hormonal content, and placed in organ culture with tion and destruction of hepatocytes. This was not so, the analysis of the conditioned culture medium for (pro)insu- liver having a normal architecture with no evidence of lin. In both cases, (pro)insulin was measurable. The necrosis (Figure 5). In contrast, there was a cellular infil- (pro)insulin content was 0.5 ± 1 ng/mg (n = 5), which is trate in the islets of the diabetic (Figure 6A) but not nor- equivalent to 8% of the (pro)insulin content of the pan- moglycaemic transgenic mice (Figure 6B) creas (Table 1) on a weight to weight basis. Organ culture Immunohistochemical staining for (pro)insulin showed a of explants in the presence of 100 nM glucagon resulted in reduced number of β cells (Figure 6C) compared to nor- release of 3.4 ± 1 ng/mg (pro)insulin per day, which is moglycaemic littermates (Figure 6D). 23% of that produced by cultured pancreatic explants. No (pro)insulin could be found either in liver extracts or from Next, the (pro)insulin content of the liver of the diabetic cultured liver explants taken from wild type NOD mice. mice was determined. This was similar to that from the These results show that there was translation of (pro)insu- liver of normoglycaemic transgenic mice (Table 1). lin from preproinsulin mRNA in the liver of transgenic (Pro)insulin release from cultures of liver cells was meas- mice. The amount of (pro)insulin present in the liver cells ured, and again found to be similar to that from the liver was too small for it to be detected immunohistochemi- cells of non-diabetic transgenic mice (Table 1). However, cally (data not shown). as expected, the (pro)insulin content of the pancreas of diabetic transgenic mice was lower than that of normogly- In situ hybridisation of (pro)insulin mRNA caemic transgenic or wild type mice (Table 1). Indeed, the In situ hybridisation confirmed the presence of (pro)insu- level was so low as to be comparable to that seen in the lin mRNA in the pancreatic and liver sections of diabetic liver of either normoglycaemic or diabetic transgenic PEPCK-Ins transgenic mice (Figure 3A and 3C) but not in mice. The amount of (pro)insulin released from pancre- the liver section of the wild type NOD mice (Figure 3E). atic explants from the diabetic transgenic mice was too The (pro)insulin mRNA was localised to the hepatocytes low to be detected. of the diabetic PEPCK-Ins transgenic mice which appear to be evenly distributed throughout the liver section (Fig- Discussion ure 3C). The sense control sections were negative (Figure At the end of the study, 5 heterozygous transgenic PEPCK- 3B,3D and 3E). Ins NOD mice had become diabetic. Nevertheless, pre- proinsulin mRNA was localised to the hepatocytes by in Occurrence of diabetes in transgenic mice situ hybridisation and was detected in total RNA extracts One out of 14 males (7%) and four out of 25 females of the livers of these transgenic mice by both RT-PCR and (16%) of the F1 and F2 progeny of the PEPCK-Ins trans- RPA. (Pro)insulin was detected in acid ethanol extracts of genic NOD mice became diabetic (Figure 4). The age at the livers from the transgenic mice by RIA. Furthermore, which this occurred was 20–25 weeks. Among the wild (pro)insulin was released from explants of transgenic liver type NOD mice in our colony, 5 males out of 45 (11%) Page 6 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 1 2 3 4 5 6 1 2 3 4 5 6 7 A-B Figure 2 . Mouse insulin 1 mRNA A-B. Mouse insulin 1 mRNA. RT-PCR (A). Lanes 1A-6A are RT-PCR reactions using primers specific for mouse insulin 1 mRNA (30 cycles). Lanes 1B-6B are RT-PCR reactions using primers specific for mouse GAPDH mRNA (30 cycles). Lanes 1–4 are total RNA from the liver of progeny from founder T645-18, lane 5 is total RNA from the liver of progeny from founder T647-2 and lane 6 total RNA from the pancreas of progeny from founder T645-18. RPA for mouse insulin 1 mRNA (B). Lane 1 pancreatic total RNA, lane 2 liver total RNA from T647-2 progeny and lanes 3–7 liver total RNA from T645-18 progeny. Page 7 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 Table 1: (Pro)insulin content of and release from pancreatic and hepatic tissue of transgenic and wild type NOD mice. PANCREAS LIVER F2 progeny (Pro)insulin content (ng/ (Pro)insulin secretion (ng/ (Pro)insulin content (ng/ (Pro)insulin release in the mg) mg tissue/ day) mg) presence of 100 nmol/l glucagon (ng/mg tissue/ day) Normoglycaemic 6.3 ± 0.8 15.8 ± 1.6 0.5 ± 0.1 3.4 ± 1.0 transgenic (n = 5) Normoglycaemic wild type 6.3 ± 1.0 15.2 ± 2.3 ND ND (n = 3) Diabetic transgenic (n = 5) 0.5 ± 0.1 (n = 3; 2 ND) ND (n = 3) 0.6 ± 0.2 (n = 5) 3.6 ± 0.3 (n = 3) ND – not detectable (<0.025 ng/mg tissue) Data expressed as X ± SD. Tg Islets Insulitis Tg Liver Wt Liver C E Insulin Antisense BE D Insulin Sense A-F Figure 3 . In-situ hybridisation A-F. In-situ hybridisation. Pancreatic islets from a diabetic transgenic mouse illustrating insulitis, (A) antisense and (B) sense. Transgenic liver from a diabetic transgenic mouse (C) antisense and (D) sense. Wild type liver from a diabetic NOD mouse (E) antisense and (F) sense. Page 8 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 PEPCK Ins NOD females NOD Males NOD Females PEPCK Ins NOD males Time (Weeks) Figure 4 Incidence of diabetes in F1 and F2 PEPCK-Ins transgenic NOD mice versus wild type F1 and F2 NOD mice Incidence of diabetes in F1 and F2 PEPCK-Ins transgenic NOD mice versus wild type F1 and F2 NOD mice. PEPCK-Ins NOD males total n = 14, PEPCK-Ins NOD females n = 25, wild type NOD males n = 45 and wild type NOD females n = 51. placed in organ culture. These results indicate that the diabetes, this reduces the possibility of delayed destruc- liver cells from these transgenic PEPCK-Ins NOD mice did tion of the (pro)insulin expressing hepatocytes. During synthesise and produce (pro)insulin. this time, hepatic insulin expression was at least equal to that found in the pancreas. At the apparently low levels of Histological examination of the livers of the diabetic transgene expression, these results suggest that (pro)insu- transgenic mice showed no infiltration of the liver by lin is not targeted by the immune system in this transgenic immune cells even though insulitis was observed in the PEPCK-Ins mouse model superimposed on the autoim- pancreatic sections. This indicates that hepatic insulin mune diabetes model of the NOD mouse. Likewise, Lipes production did not cause the development of tolerance to et al reported that when pituitary cells in transgenic NOD insulin in these PEPCK-Ins transgenic NOD mice. The mice were made to produce insulin, the cells were not tar- lack of infiltration of immune cells in the livers of the dia- geted by the immune system even though the pancreatic β betic transgenic mice also suggests that the mouse insulin cells were. No cellular infiltrate also was shown when the I mRNA transcribing hepatocytes were not targeted or pituitary cells that produced (pro)insulin were taken out- destroyed by the immune system. In addition, as the side the blood brain barrier and transplanted beneath the transgenic mice were sacrificed 4 weeks after the onset of renal capsule of NOD mice . Our data and those of Page 9 of 12 (page number not for citation purposes) Non-Diabetic Mice (%) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 human (pro)insulin from the livers of the mice (7). Simi- larly, the transgenic mice which Valera et al made diabetic by streptozotocin injections also had lower blood glucose levels compared to the diabetic wild type NOD mice sug- gesting that the constitutive hepatic insulin release low- ered the blood glucose levels of the diabetic mice but not to normal. This lack of therapeutic effect could be due to the fact that the PEPCK promoter is induced by glucagon and cyclic AMP. The high glucose levels in the diabetic transgenic mice might have resulted in the inhibition of endogenous glucagon release and thus shutting down (pro)insulin production in the liver. However it is possible that in some of our transgenic mice, enough (pro)insulin was produced to prevent diabetes. This would explain the significantly lower incidence of diabetes in the transgenic PEPCK-Ins (13%) compared to the wild type (28%) NOD mice (P < 0.001). One possibil- ity is a protective effect of insulin, whether by allowing exhausted beta cells to rest or by altering the makeup of the T cells in the pancreas destroying the β cells there. The first reason was the basis for the large North American trial in pre-diabetic people in the late 90's with small doses of parenteral insulin . The second reason was the basis for the large North American trial in pre-diabetic people in the very late 90's with oral insulin . It is possible that a critical amount of (pro)insulin needs to be produced for an autoimmune effect to be observed. The (pro)insulin content of the transgenic liver cells in normoglycaemic mice was much lower than that of a pan- creatic β cell and was also lower than that in the liver of PEPCK-human insulin C57BL/6 transgenic mice pro- duced by Valera et al . (Pro)insulin was not detected Staining with haematoxylin Figure 5 of the liver of diabet and eosinic transgenic PEPCK-Ins mice immunohistochemically in the liver of our PEPCK-Ins Staining of the liver of diabetic transgenic PEPCK-Ins mice transgenic mice, whereas it was in the liver of Valera's with haematoxylin and eosin. (Black Bar = 20µm). transgenic mice. Attempts by us to increase production of (pro)insulin by producing homozygous mice failed, with all mice being stillborn (Figure 1C). This might be because of transient upregulation of the PEPCK gene at birth , resulting in increased production of (pro)insu- lin that caused hypoglycaemia. Alternatively, it could be Lipes et al suggest that autoimmune destruction of due to a transgene integration effect where the transgene (pro)insulin producing cells in the NOD mouse is specific disrupted a vital gene; with a double copy deletion being to the islets. lethal. The blood glucose levels of the diabetic transgenic NOD Another possible explanation for the lack of an autoim- PEPCK-Ins mice (24.8 ± 1.9 mmol/l) were significantly mune effect is that presentation of insulin peptides by lower (P = 0.004) than those of the diabetic wild type hepatocytes might be limited by the ability of the liver NOD mice (>33 ± 2.1 mmol/l). This is consistent with cells to cleave proinsulin. The enzymes responsible for some (pro)insulin being released from the liver. These this in the β cell, prohormone convertases (PC) 1/3 and results are in agreement with those of Valera et al in which PC2, are absent from normal liver cells, but the the majority of their high copy number PEPCK human endopeptidase furin is present . Cleavage of insulin C57BL/6 mice also became diabetic when injected proinsulin by furin however would require genetic modi- with streptozotocin despite the constitutive release of fication of the peptide . Page 10 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 A C C B D Figure 6 A-D. Haematoxylin and eosin, and insulin staining of pancreatic sections from transgenic PEPCK-Ins NOD mice A-D. Haematoxylin and eosin, and insulin staining of pancreatic sections from transgenic PEPCK-Ins NOD mice. H & E staining of a pancreatic islet from a diabetic transgenic mouse illustrating insulitis (A), and from a normoglycaemic transgenic mouse (B). Insulin staining of a pancreatic islet from a diabetic transgenic mouse showing few remaining β cells (C) and from a normo- glycaemic transgenic mice (D). Black Bar = 20µm for A-C, and 40µm for D. Page 11 of 12 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:3 http://www.jautoimdis.com/content/1/1/3 14. Wegmann DR, Gill RG, Norbury-Glaser M, Schloot N, Daniel D: In summary, we have shown that transgenic NOD mice Analysis of the spontaneous T cell response to insulin in that produce (pro)insulin in their liver do not develop cel- NOD mice. J Autoimmun 1994, 7(6):833-43. lular infiltration of their liver when autoimmune destruc- 15. Wynshaw-Boris A, Lugo TG, Short JM, Fournier RE, Hanson RW: Identification of a cAMP regulatory region in the gene for rat tion of pancreatic β cells occur. Furthermore the cytosolic phosphoenolpyruvate carboxykinase (GTP). 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Diabetes Research Foundation (JDRF) and from an National Health Medical 20. McKenzie KJ, Hind C, Farquaharson MA, McGill M, Foulis AK: Dem- Research Council – JDRF Special Program Grant (GM). We would like to onstration of insulin production and storage in insulinomas thank Dr Alan Baxter for constructive comments on the design of the by in situ hybridization and immunocytochemistry. J Pathol 1997, 181:218-222. experiments and Ms Lindy Williams for her assistance with the insulin 21. Lipes MA, Cooper EM, Skelly R, Rhodes CJ, Boschetti E, Weir GC, immunohistochemical staining. Davalli AM: Insulin-secreting non-islet cells are resistant to autoimmune destruction. Proc Natl Acad Sci USA 1996, References 93:8595-8600. 22. Bowman MA, Campbell L, Darrow BL, Ellis TM, Suresh A, Atkinson 1. 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Scherer MN, Graeb C, Tange S, Dyson C, Jauch KW, Geissler EK: Immunologic considerations for therapeutic strategies uti- lizing allogeneic hepatocytes: hepatocyte-expressed mem- brane-bound major histocompatibility complex class I Publish with Bio Med Central and every antigen sensitizes while soluble antigen suppresses the scientist can read your work free of charge immune response in rats. Hepatology 2000, 32:999-1007. 11. Fujino M, Li XK, Kitazawa Y, Funeshima N, Guo L, Okuyama T, "BioMed Central will be the most significant development for Amano T, Amemiya H, Suzuki S: Selective repopulation of mice disseminating the results of biomedical researc h in our lifetime." liver after Fas-resistant hepatocyte transplantation. Cell Sir Paul Nurse, Cancer Research UK Transplant 2001, 10:353-361. 12. Mehal WZ, Azzaroli F, Crispe IN: Antigen presentation by liver Your research papers will be: cells controls intrahepatic T cell trapping, whereas bone available free of charge to the entire biomedical community marrow-derived cells preferentially promote intrahepatic T cell apoptosis. J Immunol 2001, 167:667-673. peer reviewed and published immediately upon acceptance 13. Gleichmann H, Bottazzo GF, Gries FA: Cytoplasmic islet cell cited in PubMed and archived on PubMed Central autoantibodies: prevalence and pathogenic significance. Adv Exp Med Biol 1988, 246:71-7. yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 12 of 12 (page number not for citation purposes)
Journal of Autoimmune Diseases – Springer Journals
Published: Nov 8, 2004
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