Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy Aims/hypothesis Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells . Exogenous insulin therapy can not achieve precise physiological control of blood glucose concentrations , and debilitating complications develop . Lentiviral vectors are promising tools for liver-directed gene therapy . However , to date , transduction rates in vivo remain low in hepatocytes , without the induction of cell cycling . We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats . Introduction Type 1 diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells -LSB- 1 -RSB- . Treatment requires daily injections of insulin to normalise blood glucose levels . Tight glucose control delays , but does not prevent , the onset of complications , which increase morbidity and mortality . Transplantation therapy is limited by the scarcity of donors and chronic immunosuppressive regimens -LSB- 2 -RSB- . Physiological glucose control could be achieved by genetically engineering surrogate beta cells that are capable of synthesising , storing and secreting insulin in response to metabolic signals . One approach to liver-directed gene therapy of diabetes is the transcriptional control of insulin expression without the induction of storage -LSB- 3 , 4 -RSB- , which is nonresponsive to minute-to-minute fluctuations in blood glucose . Another strategy is the delivery of beta cell transcription factors to the liver to induce pancreatic transdifferentiation . Studies have employed transiently expressing adenoviral vectors to deliver pancreatic and duodenal homeobox gene 1 -LRB- Pdx1 -RRB- and neurogenic differentiation gene 1 -LRB- Neurod1 -RRB- / betacellulin gene -LRB- Btc -RRB- -LSB- 5 -- 8 -RSB- to streptozotocin -LRB- STZ -RRB- - induced diabetic mice and isolated human liver cells , which have resulted in liver-to-pancreas transdifferentiation and reversal of diabetes . However , conversion of significant portions of the liver to ` true ' pancreatic tissue may lead to an increased risk of autoimmune attack and development of hepatitis , due to the exocrine differentiating capacity of PDX1 -LSB- 7 -RSB- . Most studies on the delivery of genes to the liver have employed adenoviral vectors -LSB- 5 -- 8 -RSB- due to their ability to efficiently transduce non-dividing cells . However , the therapeutic gene remains episomal , often with consequent short-term gene expression and detrimental host immune responses -LSB- 9 -RSB- . Short-term euglycaemia has been achieved following delivery of insulin via an adeno-associated vector , with dose-dependent efficacy -LSB- 10 -RSB- . Lentiviral vectors -LRB- LV -RRB- are capable of stably integrating genes into the chromosomes of dividing and non-dividing cells -LSB- 9 , 11 -- 13 -RSB- . However , their ability to transduce quiescent hepatocytes in vivo remains low , as efficient transduction generally requires induction of cell cycling -LSB- 14 -RSB- , which can be achieved by partial hepatectomy -LSB- 15 -RSB- or delivery of hepatotoxins -LSB- 16 , 17 -RSB- . We have developed an improved method for high efficiency transduction of rat hepatocytes in vitro and describe a modified surgical technique for efficient delivery of LV to rat livers resulting in efficient transduction , without the requirement for cell division . We have used this technique to deliver lentivector-encoded furin-cleavable insulin -LRB- INS-FUR -RRB- -LSB- 18 -RSB- , which enables cells to cleave proinsulin to mature insulin , in the livers of diabetic rats . This has resulted in permanent reversal of diabetes , insulin storage and restoration of glucose tolerance . Materials and methods Vector production INS-FUR cDNA -LSB- 18 -RSB- -LRB- proins.IfurIIfur.B10D ; INS-FUR ; gift from Genentech , San Francisco , CA , USA -RRB- was cloned into the multi-cloning site of the LV HIV/MSCV -LRB- HMD -RRB- -LSB- 19 -RSB- -LRB- a gift from J. Choi , Department of Pathology and Laboratory Medicine , University of Pennsylvania , PA , USA -RRB- at the EcoRI site to produce the HMD/INS-FUR construct . This LV vector has an HIV/murine stem cell virus hybrid long terminal repeat as the promoter and an internal ribosomal entry site , allowing bicistronic expression of the genes encoding insulin and enhanced green fluorescent protein -LRB- EGFP -RRB- simultaneously . The vectors were produced by calcium phosphate precipitation cotransfection of three plasmids -LRB- 10 μg HMD/INS-FUR or 10 μg pHR ' CMVGFP -LSB- 20 -RSB- expressing EGFP alone , plus 20 μg pCMVΔR8 .2 and 10 μg of the heterologous VSV-G envelope construct pCVSV-G ; all other vector constructs were gifts from I. Verma , Salk Institute for Biological Sciences , CA , USA -RRB- into 293T cells -LSB- 9 -RSB- . Conditioned medium was collected 48 h after transfection , filtered and pelleted -LRB- 50,000 g -RRB- . Virus titre was determined by transducing 293T cells -LRB- 5 × 105 -RRB- with serially diluted vector stocks and quantifying numbers of EGFP-positive cells by flow cytometry as described -LSB- 21 -RSB- . Viral replication-competency was also assessed . Transduction of hepatocytes in vitro Maintenance and experimental manipulation of male Wistar rats -LRB- 250 -- 300 g ; Gore Hill Research Laboratories , Sydney , NSW , Australia -RRB- were performed in accordance with the NIH principles of laboratory care and regulations of the Australian Research Council and were approved by the University of Technology Sydney . Hepatocytes were isolated using a two-step EDTA/collagenase digestion . The portal vein -LRB- PV -RRB- and the abdominal aorta were cannulated and the liver was flushed in situ with warmed -LRB- 40 °C -RRB- Hanks ' buffered salt solution without Ca2 + and Mg2 + -LRB- Sigma-Aldrich , Castle Hill , NSW , Australia -RRB- , plus 1 mmol/l EDTA for 5 -- 7 min . This was followed by perfusion with 0.05 % collagenase -LRB- C5138 ; Sigma-Aldrich -RRB- in Hanks ' buffered salt solution with Ca2 + and Mg2 + -LRB- Sigma-Aldrich -RRB- for 3 -- 5 min . The hepatocytes were collected , filtered through sterile 250 and 100 μm mesh and washed three times in DMEM plus 10 % FCS . Preparations containing greater than 85 % viable cells were plated onto Matrigel -LRB- gift from C. Liddle , Westmead Hospital , Sydney , NSW , Australia -RRB- and cultured in DMEM supplemented with NaHCO3 -LRB- 44 mmol/l -RRB- , insulin -LRB- 100 mU/l -RRB- , glutamic acid -LRB- 2.5 mmol/l -RRB- , proline -LRB- 1 mmol/l -RRB- , ascorbic acid -LRB- 56 mg/l -RRB- , penicillin -LRB- 100 mg/ml -RRB- , nicotinamide -LRB- 10 mmol/l -RRB- , epidermal growth factor -LRB- 20 ng/ml -RRB- , selenium salts -LRB- 0.1 μg/ml -RRB- and hydrocortisone -LRB- 1 nmol/l -RRB- . After 5 days , 1.5 % DMSO together with CuSO4 -LRB- 2.5 μg/l -RRB- , FeSO4 .7 H2O -LRB- 834 μg/l -RRB- and ZnSO4 -LRB- 1.47 mg/l -RRB- was also added to the medium . Although Matrigel inhibits cell division , hepatocytes were also irradiated -LRB- caesium-137 source ; total doses of 0 , 10 or 20 Gy -RRB- 24 h after seeding and DNA synthesis was assessed by -LSB- 3H -RSB- thymidine incorporation -LSB- 22 -RSB- . The positive control was hepatocytes plated on rat-tail collagen 1 -LRB- Collaborative Biomedical Products , Bedford , MA , USA -RRB- . At 24 h after cell attachment , hepatocytes -LRB- 5 × 105 per well -RRB- were transduced with pHR ' CMVGFP LV -LRB- 2.5 × 107 transduction units -LSB- TDU -RSB- -RRB- for 16 h in the presence of 8 μg/ml polybrene . Transduction efficiency was assessed at 2 , 7 , 14 , 21 and 28 days by fluorescence microscopy and flow cytometry . EGFP-expressing cells were also stained for the liver-specific marker glucose-6-phosphatase . Transduction of hepatocytes in vivo Rats were divided into four groups of 16 animals . For Group 1 , the right adrenal vein was ligated and successively the infrahepatic vena cava -LRB- IVC -RRB- , hepatic artery , PV and suprahepatic vena cava were clamped before pHR ' CMVGFP or HMD LV -LRB- 5 × 108 TDU -RRB- was infused through the PV . During this procedure , blood supply to the liver was stopped for 5 min and then recommenced for 2 min . This procedure of intervallic infusion in full flow occlusion -LRB- FFO -RRB- was repeated three times . Animals in Group 2 were treated similarly ; except 0.9 % saline was infused . A 70 % partial hepatectomy was performed on animals in Group 3 by resecting the left lateral and median liver lobes . The right adrenal vein was ligated and LV was infused 24 h later by the procedure detailed for Group 1 . A fourth group of animals was treated the same as Group 3 , except 0.9 % saline was infused . Livers were removed at 1 , 2 , 4 and 8 weeks . Transduction efficiency was assessed by flow cytometric quantification of EGFP-positive cells.To assess cell proliferation , 5-bromo-2 ′ - deoxyuridine -LRB- 100 mg/kg ; Sigma -RRB- was injected intraperitoneally at 18 , 28 and 48 h after surgery -LRB- partial hepatectomy or sham-operated -RRB- and into untreated animals -LRB- three animals per time-point -RRB- . After 2 h , livers were excised and processed by conventional histological techniques . Incorporation of 5-bromo-2 ′ - deoxyuridine was determined by immunohistochemistry.Diabetes was induced by an intraperitoneal injection of STZ -LRB- 75 mg/kg body weight ; Sapphire Science , Crows Nest , NSW , Australia -RRB- . Animals received HMD/INS-FUR or empty HMD vector administered by FFO without partial hepatectomy . After treatment , animals were monitored for body weight and blood glucose . Animals were killed at 60 or 500 days and body tissues were sampled . To determine the proportion of EGFP-expressing cells that were hepatocytes , liver cells were isolated and stained with anti-cytokeratin 18 -LRB- CK18 -RRB- antibody -LRB- Chemicon , Temecula , CA , USA -RRB- and R-phycoerythrin-conjugated secondary antibody -LRB- Chemicon -RRB- . Numbers of insulin-positive cells were determined using an anti-insulin antibody -LRB- Sigma-Aldrich -RRB- and aminomethylcoumarin-conjugated secondary antibody followed by flow cytometric analysis . Microscopic analyses Livers and pancreases were collected 60 and 500 days after vector infusion . Islet hormones were identified by peroxidase immunohistochemistry using diaminobenzidine as substrate . Primary antibodies against insulin -LRB- BioGenex , San Ramon , CA , USA -RRB- , glucagon -LRB- Santa Cruz Biotechnology , Santa Cruz , CA , USA -RRB- and somatostatin -LRB- Santa Cruz Biotechnology -RRB- were used . All cells in ten random fields were scored . Data were expressed as the number of positive cells per mm2 of tissue.For electron microscopy , tissue was fixed and processed using uranyl acetate block-staining and lead citrate counter-staining of ultra-thin sections -LRB- 80 nm -RRB- . For insulin immunoelectronmicroscopy , a post-embedding immunogold procedure was used . The beta cell line Nit-1 was the positive control . Tissues and cells were embedded in LR white and labelling procedures were performed as previously described -LSB- 23 -RSB- . Functional studies Intravenous glucose tolerance tests were performed on fasted -LRB- 6 -- 8 h -RRB- animals . After infusion of glucose -LRB- 0.5 g/kg body weight -RRB- , blood samples were collected from the tail vein . Insulin content of pancreas and liver was determined after acid/ethanol extraction -LSB- 23 -RSB- . Insulin and proinsulin in acid/ethanol extractions were separated on a Sephadex G50 superfine column -LRB- Sigma-Aldrich -RRB- -LSB- 24 -RSB- . Insulin in serum samples and column fractions was measured using an RIA for human insulin -LRB- Linco Research , St Charles , MO , USA -RRB- , which has less than 1 and 6 % cross-reactivity with rodent insulin and human proinsulin , respectively . Commercial RIAs -LRB- Linco Research -RRB- were used to measure human and rat C-peptide -LSB- 25 -RSB- . To assess liver function , aspartate transaminase -LRB- AST -RRB- and alanine aminotransferase -LRB- ALT -RRB- were measured daily over the first 10 days and then monthly using commercial kits -LRB- Roche Diagnostics , Castle Hill , NSW , Australia -RRB- . PCR analyses For RT-PCR , liver , pancreas , spleen , kidney and lung were collected at 60 and 500 days after vector infusion and snap-frozen in liquid nitrogen . Total RNA was extracted using Trizol -LRB- Invitrogen Australia , Mt Waverly , VIC , Australia -RRB- . Samples were treated with DNase I -LRB- Invitrogen -RRB- . Real-time quantitative PCR was conducted using SYBR green I and a sequence detection system -LRB- ABI 7500 HT ; Applied Biosystems , Scoresby , VIC , Australia -RRB- . Optimal primer and cDNA template concentrations were determined by titration . Amplification efficiencies for each primer set were determined to be similar . Data were represented as differences between threshold cycle values -LRB- ΔCT -RRB- for the transcripts of interest and the internal standard , β-actin . The expression of the genes encoding human insulin and EGFP was examined in normal and HMD/INS-FUR-transduced liver cells using semi-quantitative RT-PCR . Oligonucleotide sequences are available on request . Qualitative western blot analyses Nuclear and cytoplasmic extracts of liver and pancreas were prepared using a nuclear extraction Kit -LRB- Chemicon -RRB- . Samples -LRB- 1 μg protein -RRB- were separated by SDS-PAGE and transferred to nitrocellulose . Primary antibodies against PDX1 and NEUROD1 -LRB- Santa Cruz -RRB- were used . Statistical analyses Differences between groups were determined by Student 's paired t test or , if there were more than two groups , by one-way analysis of variance after log transformation of data . Data are expressed as means ± standard errors -LRB- SEM -RRB- . Results Efficient transduction of hepatocytes Isolation of hepatocytes by dual perfusion of the PV and abdominal aorta resulted in increased cell viability as compared with perfusion of the PV alone -LRB- 85.7 ± 6.2 % vs 67.5 ± 7.8 % , respectively , p = 0.03 -RRB- . The cells were morphologically normal , as assessed by light and electron microscopy , with maintenance of gap junctions between cells and limited vacuolation . Hepatocytes grown on Matrigel in the optimised medium , with or without irradiation , did not undergo cell division , as assessed by -LSB- 3H -RSB- thymidine incorporation . Cells grown on collagen , which enhances cell growth , reached a maximum growth rate at day 2 and this plateaued between days 5 and 6 -LRB- data not shown -RRB- . Expression of EGFP was seen 48 h post-transduction and persisted for over 1 month -LRB- Fig. 1a -RRB- . Up to 87 ± 1.2 % of the irradiated cells expressed EGFP -LRB- Fig. 1b -RRB- . Most EGFP-positive cells -LRB- 80.2 ± 10.7 % -RRB- were also positive for glucose-6-phosphatase , which suggested a hepatocyte lineage -LRB- data not shown -RRB- . Growth of hepatocytes on collagen did not increase transduction efficiency . Delivery of HMD/INS-FUR to hepatocyte cultures resulted in low constitutive insulin expression -LRB- 0.2 pmol per 3 × 105 cells -RRB- and no insulin storage ; therefore transplantation of hepatocytes transduced in vitro was not performed . Fig. 1EGFP expression in hepatocytes in vitro and in vivo . a EGFP expression in primary hepatocytes in vitro at indicated times post transduction with pHR ' CMVGFP . Original magnification : 400 × . The insets show untransduced hepatocytes . b Flow cytometric analysis of irradiated -LRB- 10 Gy -RRB- , non-dividing primary hepatocytes 14 days post-transduction with pHR ' CMVGFP . Negative control cells show background fluorescence only . EGFP-positive cells constituted 87 ± 1.2 % of all cells following transduction . Graphs representative of three independent experiments . c Flow cytometric analysis of liver cells isolated from a normal control rat and a rat transduced with the HMD LV vector 1 month previously . EGFP-positive cells constituted 60 ± 3.2 % of all liver cells . The results are representative of three experiments . d Representative flow cytometry dot-plot showing the proportion of EGFP-expressing cells that were hepatocytes -LRB- CK-18-positive : 58 ± 3.1 % -RRB- from an animal that had been transduced with the HMD/INS-FUR LV 2 months previously and e the proportion of insulin-positive -LRB- 57 ± 2.8 % -RRB- hepatocytes . Graphs are representative of three experimentsWe delivered pHR ' CMVGFP to the livers of diabetic rats by FFO and compared the transduction efficiency in non-hepatectomised and partially-hepatectomised animals . The total wet weights of liver 48 h after partial hepatectomy were similar to those of non-hepatectomised rats -LRB- 14.3 ± 0.4 and 14.7 ± 0.8 g , respectively -RRB- . We also determined whether FFO stimulated cell division . In control rats , 0.1 ± 0.06 % of hepatocytes were proliferating at 18 h and this rate did not significantly alter at any time point . At 18 h in animals that had undergone FFO , 0.3 ± 0.2 % of hepatocytes were proliferating , compared with 0.4 ± 0.3 % at 28 h and 0.1 ± 0.04 % at 48 h . These values were not significantly different to those recorded for control animals . By comparison , in animals that had undergone a partial hepatectomy , 12.8 ± 2.4 % of hepatocytes were proliferating at 18 h . This value increased to 21.4 ± 4.3 % at 28 h and then decreased to 7.4 ± 1.2 % at 48 h.To determine transduction efficiency , liver cells were isolated after transduction with pHR ' CMVGFP and EGFP-positive cells were assessed by flow cytometry . The maximal number of EGFP-expressing cells was detected 2 weeks after transduction -LRB- 57.0 ± 2 % -RRB- and was not significantly different after 2 months -LRB- 58.2 ± 3.5 % -RRB- . In animals that were partially hepatectomised prior to transduction , there was no significant difference in numbers of EGFP-expressing hepatocytes -LRB- 59.2 ± 4 % -RRB- after 2 months compared with non-hepatectomised animals . Use of the HMD vector also resulted in high levels of transduction -LRB- Fig. 1c -RRB- . Cells expressing EGFP/INS-FUR were almost exclusively hepatocytes , as verified by CK18 and insulin labelling -LRB- Fig. 1d , e -RRB- . Transduction with HMD/INS-FUR reversed STZ-diabetes Blood glucose levels of STZ-diabetic rats decreased to subnormal levels immediately after transduction with HMD/INS-FUR vector -LRB- Fig. 2a -RRB- . By day 5 , blood glucose levels of insulin-transduced animals were not different to non-diabetic controls and reversal of diabetes was maintained for 500 days -LRB- experimental endpoint -RRB- . STZ-diabetic rats treated with HMD alone remained hyperglycaemic until day 60 , when they were killed . The body weights of rats treated with HMD/INS-FUR were not significantly different to those of non-diabetic controls until day 125 , lower between days 135 and 275 , becoming not significantly different again after day 300 -LRB- Fig. 2b -RRB- . The body weights of rats treated with the empty vector alone continued to fall from day 50 to 60 . Throughout the experimental period , there was no elevation of liver enzymes in animals treated with HMD/INS-FUR -LRB- AST : 38.4 ± 6 , ALT : 26.9 ± 5 U/l -RRB- compared with control animals -LRB- AST : 44.2 ± 16 , ALT : 19.1 ± 7 -RRB- . Fig. 2Stable expression of INS-FUR ameliorated STZ-induced hyperglycaemia . Blood glucose concentrations -LRB- a -RRB- and body weights -LRB- b -RRB- of non-diabetic control rats -LRB- black circles -RRB- and STZ-diabetic rats treated with HMD/INS-FUR -LRB- white squares -RRB- or HMD alone -LRB- black triangles -RRB- . Values are means ± SEM -LRB- n = 5 -RRB- HMD/INS-FUR transduction stimulated expression of pancreatic hormones We examined pancreatic hormone-producing cells in the livers of rats transduced with LV after 2 months . Figure 3a shows extensive staining for insulin in normal pancreatic islets . By comparison , pancreatic beta cells were rarely observed in STZ-diabetic animals -LRB- 1.5 ± 1.4 cells -RRB- -LRB- Fig. 3b -RRB- . Normal liver was negative for insulin -LRB- Fig. 3c -RRB- , glucagon and somatostatin -LRB- not shown -RRB- . No pancreatic hormones were detected in livers of animals treated with empty vector . Following transduction with HMD/INS-FUR , significant numbers of hepatocytes were converted into insulin-positive cells -LRB- Fig. 3d -RRB- . There was also evidence of some pancreatic transdifferentiation , as glucagon - and somatostatin-positive cells were detected -LRB- Fig. 3e , f -RRB- . Cells positive for pancreatic hormones were distributed throughout the livers of animals transduced with HMD/INS-FUR and not restricted to regions adjacent to the PV . Fig. 3Expression of pancreatic hormones following reversal of STZ-induced hyperglycaemia . Photomicrographs of anti-insulin staining of -LRB- a -RRB- normal pancreas , -LRB- b -RRB- STZ-diabetic HMD/INS-FUR vector-treated pancreas , -LRB- c -RRB- normal liver and -LRB- d -RRB- STZ-diabetic HMD/INS-FUR vector-treated liver at 2 months . Photomicrographs of anti-glucagon -LRB- e -RRB- and anti-somatostatin -LRB- f -RRB- staining in STZ-diabetic HMD/INS-FUR vector-treated liver at 2 months . Positive cells appear brown . Original magnification : 200 × . In a total of 1,200 ± 61 cells the number of insulin-positive cells -LRB- 585.6 ± 73.8 cells/mm2 -RRB- was fivefold higher than the number of glucagon-positive cells -LRB- 109.9 ± 37.1 cells/mm2 -RRB- and 11-fold higher than the number of somatostatin-positive cells -LRB- 52.2 ± 8.1 cells/mm2 -RRB- . The number of EGFP-positive cells was 854 ± 80.9 -LRB- data not shown -RRB- . Therefore , some EGFP-positive cells did not store pancreatic hormones . No pancreatic hormones were detected in liver from animals treated with empty vector or from control animals . g Transmission electron micrograph showing secretory vesicles with dense granules surrounded by a pale halo -LRB- bar : 500 nm -RRB- . Inset , vesicle 320 nm in diameter . h Immuno-electron micrographs showing localisation of insulin in liver 2 months after the transduction of an STZ-diabetic rat with HMD/INS-FUR and -LRB- i -RRB- insulin-secreting granules in Nit-1 beta cells -LRB- positive control -RRB- ; bar : 400 nmElectron microscopy revealed cytoplasmic granules -LRB- 270 -- 330 nm in diameter -RRB- in liver tissue from animals treated with HMD/INS-FUR -LRB- Fig. 3g -RRB- . Immunoelectron microscopy of insulin-producing cells in the liver of STZ-diabetic rats transduced with HMD/INS-FUR was comparable to those observed in Nit-1 beta cells -LRB- Fig. 3h , i -RRB- . Inflammatory cell infiltration and necrosis within the livers of transduced animals were not observed . Insulin-containing granules were absent in rats treated with empty vector and in untreated animals . HMD/INS-FUR transduction induced hepatic insulin production Insulin storage was 4,165 ± 180 pmol per liver in HMD/INS-FUR-treated animals , compared with 10,418 ± 210 pmol in normal rat pancreas -LRB- n = 3 -RRB- . STZ-treated rat pancreas only contained 255 ± 110 pmol insulin . Separation of insulin from proinsulin in liver extracts from animals treated with HMD/INS-FUR yielded two distinct peaks . The first represented proinsulin and partially processed material and the second larger peak corresponded to fully processed insulin -LRB- Fig. 4 -RRB- . Fig. 4Stable expression of INS-FUR resulted in production of mature human insulin . Extracts of rat livers were separated by gel permeation chromatography and assayed for insulin . Arrows indicate mobility of mature human insulin -LRB- I -RRB- -LRB- 5,800 Da -RRB- and proinsulin -LRB- pI -RRB- -LRB- 9,400 Da -RRB- . Results are representative of three independent experiments HMD/INS-FUR transduced animals displayed regulated insulin secretion There was no significant difference between the ability of STZ-diabetic animals transduced with the HMD/INS-FUR vector and untreated animals to normalise blood glucose levels after delivery of a bolus of intravenous glucose -LRB- Fig. 5a -RRB- . Levels of human C-peptide and human insulin in serum samples from HMD/INS-FUR-treated animals -LRB- Fig. 5b , c -RRB- were comparable to levels of rat C-peptide and insulin recorded for non-diabetic animals . Negligible levels of rat C-peptide were detected in serum from HMD/INS-FUR-treated STZ-diabetic animals -LRB- Fig. 5c -RRB- . Fig. 5Plasma glucose -LRB- a -RRB- , insulin -LRB- b -RRB- and C-peptide -LRB- c -RRB- levels after IVGTT . IVGTTs remained normal for animals maintained for 500 days -LRB- data not shown -RRB- . White squares , HMD/INS-FUR-treated animals -LRB- 60 days -RRB- expressing human insulin and human C-peptide ; black diamonds , normal control animals expressing rat insulin and rat C-peptide ; black triangles , rat C-peptide in HMD/INS-FUR-treated animals . Values are means ± SEM -LRB- n = 5 -RRB- HMD/INS-FUR transduction stimulated expression of beta cell transcription factors EGFP expression was only detected in liver tissue of pHR ' CMVGFP - -LRB- Fig. 6a -RRB- and HMD-treated -LRB- data not shown -RRB- rats . Similarly , INS-FUR expression was restricted to the livers of HMD/INS-FUR-treated rats -LRB- Fig. 6a -RRB- . Insulin was not detected in livers from diabetic control animals and diabetic animals injected with HMD alone -LRB- data not shown -RRB- . Mean ratios of insulin : β-actin -LRB- 1.000 ± 0.069 -RRB- and EGFP : β-actin -LRB- 1.088 ± 0.062 -RRB- in HMD/INS-FUR liver , determined by semi-quantitative PCR , were not significantly different . Transduction of STZ-diabetic rats with the HMD/INS-FUR vector induced sustained expression of several pancreatic transcription factors -LRB- Fig. 6b -RRB- . Normal liver and untreated diabetic liver -LRB- not shown -RRB- were negative for all pancreatic transcription factors , while normal pancreas was positive . Fig. 6Beta cell transcription factors and pancreatic hormones expressed in transduced rat livers . a RT-PCR analysis of transcription of the genes encoding EGFP or INS-FUR in animals transduced with pHR ' CMVGFP or HMD/INS-FUR LV , respectively : liver -LRB- lane 1 -RRB- , pancreas -LRB- lane 2 -RRB- , spleen -LRB- lane 3 -RRB- , kidney -LRB- lane 4 -RRB- and brain -LRB- lane 5 -RRB- at 60 days . Results for HMD/INS-FUR transduced animals at 500 days were identical -LRB- not shown -RRB- . b RT-PCR analysis of transcription of the genes for : -LRB- 1 -RRB- beta cell transcription factors -LRB- Pdx1 , Neurod1 , Neurog3 , Nkx2-2 , Nkx6-1 , Pax4 , Pax6 -RRB- ; -LRB- 2 -RRB- the pancreatic endocrine hormones somatostatin -LRB- Sst -RRB- , glucagon -LRB- Gcg -RRB- and pancreatic polypeptide -LRB- Ppy -RRB- ; -LRB- 3 -RRB- GLUT2 -LRB- Slc2a2 -RRB- and glucokinase -LRB- Gck -RRB- ; -LRB- 4 -RRB- rat insulin 1 and 2 -LRB- Ins1 , Ins2 -RRB- ; -LRB- 5 -RRB- insulin proconvertase PC2 -LRB- Pc2 -RRB- ; -LRB- 6 -RRB- the exocrine marker p48 -LRB- P48 -RRB- ; and -LRB- 7 -RRB- β-actin -LRB- Actb -RRB- in normal liver -LRB- lane 1 -RRB- , liver transduced with HMD alone at 60 days -LRB- lane 2 -RRB- , liver transduced with HMD/INS-FUR LV at 60 days -LRB- lane 3 -RRB- and 500 days -LRB- lane 4 -RRB- , and normal pancreas -LRB- lane 5 -RRB- . c Western blot analysis for PDX1 and NEUROD1 in normal pancreas -LRB- lane 1 -RRB- , liver transduced with HMD/INS-FUR LV at 60 days -LRB- lane 2 -RRB- , liver transduced with HMD alone at 60 days -LRB- lane 3 -RRB- and normal liver -LRB- lane 4 -RRB- . PDX1 and NEUROD1 generated bands at 43 and 50 kDa , respectivelyUnexpectedly , both Pdx1 and Neurod1 expression were detected by RT-PCR in empty vector-transduced livers ; however , protein production was not detected -LRB- Fig. 6c -RRB- . HMD/INS-FUR-transduced livers also expressed the genes for neurogenin 3 -LRB- Neurog3 -RRB- and Nkx2-2 -LRB- Nkx2-2 -RRB- . Glucagon and somatostatin were present in liver transduced with HMD/INS-FUR -LRB- confirmed by immunohistochemistry ; Fig. 3e , f -RRB- . Pancreatic polypeptide was not expressed . The transduction of diabetic animals with HMD/INS-FUR did not result in complete pancreatic transdifferentation of liver tissue since expression of the rat insulin genes -LRB- Ins1 , Ins2 -RRB- and the genes encoding the insulin prohormone convertase 2 -LRB- Pc2 , also known as Pcsk2 -RRB- and the exocrine marker p48 -LRB- Ptf1a , also known as P48 -RRB- were not detected -LRB- Fig. 6b -RRB- . Quantitative real time PCR analysis of the expression levels of beta cell transcription factors Pdx1 -LRB- 4.3 ± 0.9 -RRB- , Neurod1 -LRB- 5.7 ± 1.1 -RRB- and Neurog3 -LRB- 7.4 ± 1.8 -RRB- in HMD/INS-FUR-treated livers at 500 days indicated that they were lower -LRB- p < 0.001 -RRB- than levels expressed in normal pancreatic tissue -LRB- Pdx1 : 12.5 ± 2.3 , NeuroD1 : 16.8 ± 1.4 , Neurog3 : 18.5 ± 2.2 -RRB- . Discussion This is the first report of permanent amelioration of STZ-induced hyperglycaemia using LV delivery of INS-FUR . Transduced animals processed , stored and secreted insulin in a glucose-regulated manner . Remission of diabetes , due to the secretion of insulin from residual beta cells , was discounted as insulin-positive pancreatic beta cells were rarely observed and levels of rat C-peptide were negligible . This is also the first report showing that transduction of INS-FUR alone can induce expression of several beta cell transcription factors resulting in hepatocyte differentiation along a pancreatic lineage . All previous reports of liver -- pancreas transdifferentiation have been described in cells that endogenously express beta cell transcription factors -LSB- 23 , 26 -RSB- or have been engineered to express these factors , leading to varying degrees of transdifferentiation -LSB- 5 -- 8 , 27 , 28 -RSB- . Following immediate reduction in blood glucose due to transgene expression , induction of long-term euglycaemia in the present study was probably related to partial hepatic-to-pancreas transdifferentiation triggered by the lentiviral intrahepatic delivery method of INS-FUR or from expression of stably integrated transgene in the host genome . The HMD/INS-FUR transgene alone , which constitutively expresses insulin , could not mimic the glucose-sensitive kinetics demonstrated by IVGTT , nor could it have solely induced insulin storage . Evidently , the hepatic delivery of the LV was a key factor , for expression of HMD alone triggered expression of Pdx1 and Neurod1 , if only at the mRNA level . It is possible that the use of B10-insulin may have been another factor . This insulin is superior to normal insulin in biological activity -LSB- 29 -RSB- and has some mitogenic properties -LSB- 30 -- 32 -RSB- . However , there was no evidence of excessive mitogenic activity or tumour development , while liver function tests remained normal throughout , with no lymphocytic infiltrates . Parallels can be drawn to the partial pancreatic transdifferentiation observed in Huh7ins cells , which endogenously produce NEUROD1 -LSB- 23 -RSB- and PDX1 -LRB- C. Tao , University of Technology Sydney ; unpublished results -RRB- . This cell line was transfected with normal human insulin under the control of a constitutive promoter . After this , secretory granules developed , which stored di-arginyl insulin and secreted it in a glucose-responsive manner , correcting diabetes . However , in Huh7ins cells the expression of fully processed native insulin was absent . Similarly , in the present study , pancreatic transdifferentiation was incomplete , as genes encoding transcription factors such as NKX6-1 and PAX6 , which occur late in the hierarchy ; were not expressed , nor were the rodent insulin genes or the genes for PC2 pancreatic polypeptide or exocrine markers . Therefore , the problems encountered with non-selective pancreatic transdifferentiation , produced after expression of PDX1 alone -LSB- 5 -- 7 , 33 , 34 -RSB- , were not encountered . How the hepatic delivery of INS-FUR induced the expression of Pdx1 and other beta cell transcription factors requires further investigation . Liver and pancreas derive from common progenitor cells . Transdifferentiation between liver and pancreas is controlled by only a few transcription factors -LSB- 35 , 36 -RSB- . Pdx1 is one such master switch gene . In a recent study , liver cells producing pancreatic markers -LRB- insulin and PDX1 -RRB- were induced and localised to areas adjacent to or within injured tissue areas -LSB- 37 -RSB- . Lentiviral transduction may represent a cellular insult , making progenitor cells permissive to a pancreatic developmental shift . The mechanisms that prevented both complete pancreatic transdifferentiation and expression of endogenous rat insulin are yet to be elucidated . Studies on the development of the rat pancreas indicate that chronologically glucagon and somatostatin appear prior to insulin -LSB- 38 -RSB- . The production of these pancreatic hormones may have prevented hypoglycaemia by providing counter-regulatory mechanisms . Complete transdifferentiation into true beta cells may require additional external factors , such as prolonged hyperglycaemia -LSB- 39 , 40 -RSB- . If this is true , then the rapid reversion to euglycaemia following delivery of the insulin gene in our study may have prohibited complete liver-to-pancreas transdifferentiation . Additionally , the expression of human insulin may have exerted a negative feedback mechanism to inhibit endogenous insulin expression . Unlike other studies that have engineered beta cell neogenesis from liver cells -LSB- 5 -- 7 -RSB- , the insulin-containing cells were not isolated to the area surrounding the portal circulation . Insulin storage was 40 % of the insulin content of a rat pancreas , satisfying clinical requirements for insulin independence -LSB- 41 -RSB- . Corroborating previous studies using INS-FUR , granules predominately stored fully processed insulin -LSB- 18 , 42 , 43 -RSB- . EGFP and insulin were produced at equivalent levels in liver tissue transduced with HMD/INS-FUR . The majority of cells that expressed EGFP and insulin were also CK18-positive , indicative of a hepatocyte lineage for transduced cells . Despite the lack of the central polypurine tract , LV pHR ' CMVGFP efficiently transduced non-dividing hepatocytes in vitro , in the absence of cell division . We obtained an improvement in transduction efficiency of up to 87 % in vitro , compared with 68 % obtained by Zahler et al. -LSB- 44 -RSB- . Increased transduction efficiency may be attributable to several factors , including : -LRB- 1 -RRB- the isolation of hepatocytes by perfusion of both the PV and abdominal aorta as opposed to the PV alone , the most widely employed procedure -LSB- 45 -RSB- ; -LRB- 2 -RRB- the optimisation of growth medium ; -LRB- 3 -RRB- the use of LV at high multiplicity of infection -LRB- 50:1 -RRB- ; and/or -LRB- 4 -RRB- the use of the R8 .2 packaging plasmid , which encodes accessory HIV proteins . The use of the attenuated plasmid R8 .91 , used by Zahler et al. , resulted in a 5 % reduction in transduction efficiency -LRB- B. Ren , unpublished observations -RRB- . Similarly , delivery of INS-FUR in the HMD hybrid vector , which lacks the central polypurine tract -LSB- 19 -RSB- , to the portal circulation of STZ-diabetic rats by FFO resulted in up to 60 % transduction of liver cells , without inducing cell division and with transgene expression being confined to the liver . The FFO technique , which employs repeated vector infusion , is probably responsible for the increased transduction efficiency and is more clinically applicable than the widely used perfusion method , where the IVC is cannulated , thereby increasing the risk of IVC narrowing and pelvic limb thrombosis -LSB- 46 , 47 -RSB- . This novel approach offers potential as a beta cell replacement strategy with diabetic patients serving as their own donors . Current studies in our laboratory using nonobese diabetic mice , indicate that this procedure was able to reverse autoimmune diabetes for 5 months with no evidence of autoimmune destruction of insulin-secreting hepatocytes -LRB- B. Ren , unpublished observations -RRB- , giving hope that this procedure will be useful for reversal of autoimmune diabetes . There are distinct advantages to the use of LV for transduction of hepatocytes . LV stably integrates into the genome of non-dividing cells , including hepatocytes , and could provide long-lasting expression of therapeutic genes -LSB- 48 , 49 -RSB- . Whilst LV integration was not specifically examined in this study , the longevity of INS-FUR and EGFP expression provides some evidence for this . Further , human hepatocytes may be more susceptible to LV transduction -LSB- 50 -RSB- than rodent cells . LV are probably superior to adenoviral vectors where problems of short-term expression of genes and immunological responses have been reported -LSB- 9 -RSB- . Adeno-associated vectors are extremely efficient at transducing mouse livers ; however , a recent study using adeno-associated viral 8-mediated delivery of Pdx1 did not result in correction of STZ-diabetes in mice -LSB- 28 -RSB- . The unique results reported in this study point to possible benefits of gene therapy using LV .

O O O O O O O O O O B I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O

beta cell transcription factors
gene therapy
hepatocytes
insulin storage
type 1 diabetes
wistar rats


Epigenetic

regulation

of

in

human

type

diabetic

islets

and

effect

on

insulin

secretion

Aims/hypothesis

Insulin

secretion

in

pancreatic

islets

is

dependent

upon

mitochondrial

function

and

production

of

ATP

The

transcriptional

coactivator

peroxisome

proliferator

activated

receptor

gamma

alpha

-LRB-

protein

gene

-RRB-

is

a

master

regulator

of

mitochondrial

genes

and

its

expression

is

decreased

and

related

to

impaired

oxidative

phosphorylation

in

muscle

from

patients

with

type

diabetes

Whether

it

plays

a

similar

role

in

human

pancreatic

islets

is

not

known

We

therefore

investigated

if

expression

is

altered

in

islets

from

patients

with

type

diabetes

and

whether

this

expression

is

influenced

by

genetic

-LRB-

polymorphism

-RRB-

and

epigenetic

-LRB-

DNA

methylation

-RRB-

factors

We

also

tested

if

experimental

downregulation

of

expression

in

human

islets

influenced

insulin

secretion

Introduction

Type

diabetes

is

characterised

by

chronic

hyperglycaemia

as

a

result

of

impaired

pancreatic

beta

cell

function

and

insulin

resistance

in

peripheral

tissues

i.e.

skeletal

muscle

adipose

tissue

and

liver

Although

each

of

these

pathogenic

defects

could

be

accounted

for

by

specific

mechanisms

impaired

ATP

production

as

a

consequence

of

reduced

oxidative

phosphorylation

might

provide

an

intriguing

common

pathogenic

pathway

for

all

these

defects

The

transcriptional

coactivator

peroxisome

proliferator

activated

receptor

gamma

alpha

-LRB-

protein

gene

-RRB-

is

an

important

factor

regulating

the

expression

of

genes

for

oxidative

phosphorylation

and

ATP

production

in

target

tissues

through

coactivation

of

nuclear

receptors

-LSB-

-RSB-

We

have

previously

shown

that

the

expression

of

and

a

set

of

genes

involved

in

oxidative

phosphorylation

is

reduced

in

skeletal

muscle

from

patients

with

type

diabetes

-LSB-

-RSB-

Furthermore

a

common

polymorphism

in

the

gene

has

been

associated

with

increased

risk

of

type

diabetes

and

an

age-related

reduction

in

muscle

expression

-LSB-

-RSB-

In

addition

genetic

variation

in

the

gene

was

associated

with

indices

of

beta

cell

function

-LSB-

-RSB-

Despite

the

central

role

of

ATP

for

insulin

secretion

the

function

of

in

human

pancreatic

islets

and

beta

cells

is

less

well

established

-LSB-

-RSB-

Obesity

reduced

physical

activity

and

ageing

are

well

known

risk

factors

for

type

diabetes

However

all

individuals

exposed

to

an

affluent

environment

do

not

develop

the

disease

One

likely

reason

is

that

genetic

variation

modifies

individual

susceptibility

to

the

environment

However

the

environment

could

also

modify

genetic

risk

factors

by

influencing

expression

of

a

gene

by

DNA

methylation

or

histone

modifications

Cytosine

residues

occurring

in

CG

dinucleotides

are

targets

for

DNA

methylation

and

gene

expression

is

usually

reduced

when

DNA

methylation

takes

place

at

a

promoter

Whether

DNA

methylation

influences

gene

expression

in

target

tissues

for

type

diabetes

and

thereby

the

pathogenesis

of

the

disease

remains

to

be

demonstrated

The

present

study

investigated

-LRB-

-RRB-

whether

expression

of

is

altered

in

human

islets

from

patients

with

type

diabetes

-LRB-

-RRB-

if

this

expression

is

influenced

by

genetic

-LRB-

the

polymorphism

-RRB-

and

epigenetic

-LRB-

DNA

methylation

-RRB-

factors

and

-LRB-

-RRB-

if

expression

of

influences

insulin

secretion

Methods

Multi-organ

donors

The

characteristics

of

the

non-diabetic

and

type

diabetic

multi-organ

donors

whose

pancreases

were

processed

for

islet

preparation

are

presented

in

Table

Pancreases

were

obtained

and

processed

with

the

approval

of

the

regional

Ethics

Committee

Table

characteristics

of

type

diabetic

and

non-diabetic

donors

Non-diabetic

donorsType

diabetic

donorsp

valuen

-LRB-

male/female

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

Age

-LRB-

years

-RRB-

aBMI

-LRB-

-RRB-

aGly/Gly

-LRB-

-RRB-

Gly/Ser

Ser/Ser

-LRB-

-RRB-

Data

are

expressed

as

mean

SEMaDonors

used

for

DNA

methylation

polymorphism

Human

pancreatic

islets

and

experimental

plan

Isolated

pancreatic

islets

were

prepared

by

collagenase

digestion

and

density

gradient

purification

-LSB-

-RSB-

After

isolation

islets

were

cultured

free

floating

in

culture

medium

-LRB-

Sigma-Aldrich

St

Louis

MO

USA

-RRB-

at

mmol/l

glucose

concentration

and

studied

within

days

from

isolation

Cell

viability

measured

by

Trypan

Blue

exclusion

was

higher

than

in

control

and

diabetic

islets

after

days

in

culture

Insulin

secretion

study

Insulin

secretion

studies

were

performed

as

previously

described

-LSB-

-RSB-

Following

a

min

pre-incubation

period

at

mmol/l

glucose

groups

of

islets

of

comparable

size

were

kept

at

°C

for

min

in

KRB

-LRB-

wt/wt

-RRB-

albumin

pH

containing

mmol/l

glucose

At

the

end

of

this

period

the

medium

was

completely

removed

and

replaced

with

KRB

containing

either

or

mmol/l

glucose

plus

mmol/l

arginine

or

mmol/l

glucose

plus

μmol/l

glibenclamide

After

an

additional

min

incubation

period

the

medium

was

removed

Media

-LRB-

μl

aliquots

from

the

ml

incubation

volume

-RRB-

were

stored

at

°C

until

insulin

concentrations

were

measured

by

immunoradiometric

assay

-LRB-

Pantec

Forniture

Biomediche

Turin

Italy

-RRB-

Animals

Streptozotocin

-LRB-

STZ

-RRB-

nicotinamide

-LRB-

-RRB-

treated

male

Wistar

rats

-LRB-

months

old

-RRB-

were

administered

mg/kg

i.p.

-LRB-

Sigma

St

Louis

MO

USA

-RRB-

dissolved

in

saline

min

before

an

i.v.

injection

of

mg/kg

STZ

-LRB-

Sigma

-RRB-

that

had

been

dissolved

in

citrate

buffer

-LRB-

pH

-RRB-

immediately

before

use

Control

rats

were

injected

with

vehicle

alone

STZ

NA-treated

animals

had

stable

hyperglycaemia

-LRB-

mmol/l

-RRB-

and

they

were

used

for

the

experiments

weeks

after

diabetes

was

induced

Pancreatic

islets

were

isolated

by

the

collagenase

method

using

the

procedure

of

pancreatic

duct

cannulation

and

density

gradient

purification

as

described

elsewhere

-LSB-

-RSB-

GK

rats

were

obtained

from

the

Stockholm

colony

and

bred

as

described

-LSB-

-RSB-

Inbred

normoglycaemic

rats

were

purchased

from

Charles

River

Laboratories

-LRB-

Wilmington

MA

USA

-RRB-

and

maintained

by

sister

brother

mating

Transfer

of

GK

alleles

onto

the

genome

of

rats

by

repeated

backcrossing

-LRB-

ten

generations

-RRB-

established

the

homozygous

congenic

strains

-LRB-

-RRB-

and

-LRB-

-RRB-

carries

of

the

GK

genotype

-LRB-

cM

-RRB-

based

on

genetic

distance

on

a

homozygous

genetic

background

rats

display

hyperglycaemia

accompanied

by

fasting

hyperinsulinaemia

and

increased

epididymal

fat

implicating

insulin

resistance

carries

of

the

GK

genotype

-LRB-

cM

-RRB-

and

display

hyperglycaemia

and

insulin

secretion

defects

-LSB-

-RSB-

Backcrossing

was

designed

to

introduce

mitochondrial

DNA

and

chromosomes

X

plus

Y

from

The

congenic

strains

were

kept

constant

by

sister

brother

mating

for

several

generations

To

avoid

effects

of

the

oestrous

cycle

and

other

sex-specific

influences

only

male

rats

were

included

in

this

study

Rats

were

maintained

at

constant

temperature

and

humidity

in

a

h

light

dark

cycle

with

free

access

to

standard

laboratory

chow

pellets

and

water

All

experiments

were

approved

by

the

local

Ethics

Committees

Isolated

pancreatic

islets

were

prepared

from

rats

at

weeks

of

age

after

a

h

fast

-LRB-

hours

-RRB-

by

injection

of

a

collagenase

solution

via

the

bile

pancreatic

duct

-LSB-

-RSB-

Analysis

of

mRNA

expression

in

pancreatic

islets

Total

RNA

was

extracted

from

human

islets

after

days

in

culture

using

the

RNeasy

Protect

Mini

Kit

-LRB-

Qiagen

Valencia

CA

USA

-RRB-

It

was

quantified

by

absorbance

at

nm

-LRB-

ratio

-RRB-

in

a

Perkin-Elmer

spectrophotometer

-LRB-

Waltham

MA

USA

-RRB-

and

its

integrity

was

assessed

after

electrophoresis

in

-LRB-

wt/wt

-RRB-

agarose

gels

by

ethidium

bromide

staining

Human

and

rat

mRNA

expression

were

quantified

by

RT-PCR

-LSB-

-RSB-

Gene-specific

probes

and

primer

pairs

for

-LRB-

Assays-on-demands

human

and

rat

Applied

Biosystems

Foster

City

CA

USA

-RRB-

were

used

Each

sample

was

run

in

duplicate

and

the

transcript

quantity

was

normalised

to

the

mRNA

level

of

cyclophilin

A

-LRB-

human

and

rat

Applied

Biosystems

-RRB-

For

each

probe/primer

set

a

standard

curve

was

generated

which

was

confirmed

to

increase

linearly

with

increasing

amounts

of

cDNA

Downregulation

of

in

human

islets

using

silencing

RNA

-LRB-

siRNA

-RRB-

In

order

to

test

whether

inhibition

of

expression

could

directly

modulate

insulin

release

isolated

human

islets

were

transfected

with

siRNA

by

using

Arrest-In

Transfection

-LRB-

Open

Biosystem

Celbio

Pero

Italy

-RRB-

This

is

a

polymeric

formulation

developed

and

optimised

for

highly

efficient

delivery

of

siRNA

into

the

nucleus

of

suspension

cells

in

the

presence

of

serum-containing

medium

Pre-designed

Silencer

siRNAs

and

Silencer

non-targeting

siRNAs

-LRB-

negative

control

-RRB-

for

-LRB-

Ambion

Austin

TX

USA

-RRB-

were

used

Transfection

was

performed

according

to

the

manufacturer

's

instructions

Briefly

islets

obtained

from

five

pancreases

were

washed

times

in

KRB

and

exposed

for

min

to

free

and

KRB

to

allow

cell

disaggregation

At

the

end

of

the

incubation

period

islets

per

study

point

were

re-suspended

in

μl

medium

-LRB-

Sigma-Aldrich

-RRB-

added

with

adult

bovine

serum

siRNA

-LRB-

nmol/l

-RRB-

was

diluted

in

μl

while

μg

Arrest-In

solution

-LRB-

Celbio

-RRB-

was

dissolved

in

μl

and

incubated

for

min

after

rapid

mixing

to

allow

formation

of

transfection

complexes

Finally

μl

of

this

solution

were

added

into

wells

containing

islets

and

incubation

was

allowed

for

h

in

a

incubator

at

°C

At

the

end

of

the

incubation

period

well

volume

was

doubled

with

culture

medium

and

samples

were

kept

in

the

incubator

for

another

h

when

islets

function

viability

transfection

efficiency

-LRB-

when

measured

by

Polyfectamine

tied

to

a

fluorescent

probe

-RRB-

and

gene

expression

were

evaluated

Genotyping

Genomic

DNA

was

extracted

from

pancreatic

islets

using

a

Wizard

Genomic

DNA

Purification

kit

-LRB-

Promega

Madison

WI

USA

-RRB-

The

-LRB-

GGT

AGT

-RRB-

polymorphism

of

was

genotyped

using

an

allelic

discrimination

assay

performed

with

an

ABI

system

-LRB-

Applied

Biosystems

-RRB-

using

PCR

primers

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

and

TaqMan

MGB

probes

and

-LSB-

-RSB-

DNA

methylation

A

sequence

starting

bp

upstream

from

the

translation

start

was

used

in

MethPrimer

-LRB-

-RRB-

to

search

for

regions

with

CpG

sites

and

PCR

designs

A

sequence

bases

upstream

from

the

translation

start

including

four

possible

DNA-methylation

sites

and

a

putative

hepatic

nuclear

factor

-LRB-

-RRB-

binding

site

was

selected

and

analysed

for

DNA

methylation

-LRB-

Fig.

-RRB-

Genomic

DNA

isolated

from

pancreatic

islets

of

nine

non-diabetic

and

ten

type

diabetic

multi-organ

donors

was

treated

with

bisulphite

using

the

EZ

DNA

Methylation

Kit

-LRB-

Zymo

Research

Orange

CA

USA

-RRB-

Bisulphite-modified

DNA

was

amplified

by

nested

PCR

with

primers

designed

using

MethPrimer

Primer

pair

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

and

primer

pair

-LRB-

used

for

nested

PCR

-RRB-

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

The

PCR

products

were

then

cloned

into

a

vector

-LRB-

TOPO

TA

Cloning

Kit

for

Sequencing

Invitrogen

Carlsbad

CA

USA

-RRB-

and

ten

colonies

from

each

donor

were

purified

with

a

Miniprep

kit

-LRB-

Qiagen

-RRB-

These

individual

clones

were

sequenced

using

BigDye

Terminator

Cycle

Sequencing

Kit

-LRB-

Applied

Biosystems

-RRB-

The

number

of

methylated

sites

was

determined

and

divided

by

the

total

number

of

methylation

sites

and

then

multiplied

by

to

show

the

percentage

of

methylation

for

each

donor

Fig.

mRNA

expression

in

human

pancreatic

islets

is

influenced

by

type

diabetes

a

polymorphism

and

DNA

methylation

The

influence

of

a

type

diabetes

and

b

the

polymorphism

on

mRNA

expression

in

human

pancreatic

islets

c

The

promoter

sequence

investigated

showing

the

four

DNA

methylation

target

sites

and

and

a

putative

binding-site

for

d

The

influence

of

type

diabetes

on

DNA

methylation

of

the

promoter

The

influence

of

e

type

diabetes

-LRB-

-RRB-

and

f

the

polymorphism

on

absolute

insulin

release

-LRB-

pmol

islet

min

-RRB-

in

response

to

mmol/l

glucose

g

Correlations

between

mRNA

expression

and

absolute

insulin

release

-LRB-

pmol

islet

min

-RRB-

in

response

to

mmol/l

glucose

in

human

pancreatic

islets

-LRB-

r

p

-RRB-

Results

are

expressed

as

mean

SEM

p

Statistical

methods

Differences

in

mRNA

expression

percentage

of

DNA

methylation

and

insulin

secretion

between

the

different

groups

studied

were

analysed

using

Student

's

t

test

or

the

non-parametric

Mann

Whitney

test

where

appropriate

Correlations

were

calculated

using

Pearson

correlation

coefficients

for

normally

distributed

values

and

Spearman

correlation

coefficients

when

normality

was

rejected

Log

values

were

used

in

the

multivariate

regression

analysis

Differences

in

expression

between

GK

and

rats

were

analysed

using

one-way

ANOVA

followed

by

a

Kruskal

Wallis

Z

test

All

p

values

were

two-tailed

and

p

values

less

than

were

considered

significant

Statistical

calculations

were

performed

by

NCSS

software

-LRB-

NCSS

Statistical

Software

Kaysville

UT

USA

-RRB-

Results

The

mRNA

expression

of

was

markedly

reduced

in

pancreatic

islets

from

type

diabetic

compared

with

non-diabetic

donors

-LRB-

vs

p

Fig.

-RRB-

Intriguingly

non-diabetic

carriers

of

a

allele

Gly/Ser

or

Ser/Ser

genotype

carriers

had

markedly

lower

mRNA

expression

compared

with

carriers

of

the

Gly/Gly

genotype

-LSB-

Gly/Ser

Ser/Ser

-LRB-

n

-RRB-

vs

Gly/Gly

-LRB-

n

-RRB-

p

-RSB-

-LRB-

Fig.

-RRB-

However

there

was

no

difference

in

mRNA

expression

between

non-diabetic

carriers

of

the

Gly/Ser

-LRB-

n

-RRB-

or

Ser/Ser

-LRB-

n

-RRB-

genotypes

This

demonstrates

that

the

effect

of

the

genotype

on

gene

expression

is

seen

already

in

the

pre-diabetic

state

The

diabetic

donors

were

significantly

older

and

had

higher

BMI

than

control

donors

-LRB-

Table

-RRB-

We

therefore

tested

whether

these

factors

might

also

relate

to

expression

in

human

islets

using

a

multivariate

regression

analysis

including

age

BMI

and

disease

status

as

covariates

Only

disease

status

was

significantly

associated

with

expression

in

this

analysis

-LRB-

r

p

-RRB-

We

next

evaluated

whether

epigenetic

phenomena

such

as

DNA

methylation

would

influence

expression

in

islets

Cytosine

residues

occurring

in

CG

dinucleotides

are

targets

for

DNA

methylation

and

gene

expression

is

usually

reduced

when

DNA

is

methylated

at

the

promoter

We

used

bisulphite

sequencing

to

assess

DNA

methylation

of

four

methylation

target

sites

and

in

the

promoter

in

pancreatic

islets

from

the

type

diabetic

and

non-diabetic

human

multi-organ

donors

-LRB-

Fig.

-RRB-

Interestingly

there

was

an

approximately

twofold

increase

in

DNA

methylation

of

the

promoter

of

diabetic

compared

with

non-diabetic

human

islets

-LRB-

vs

p

Fig.

-RRB-

Also

in

this

subset

of

donors

the

mRNA

expression

was

significantly

reduced

in

diabetic

compared

with

non-diabetic

islets

-LRB-

p

-RRB-

There

was

a

trend

towards

an

inverse

correlation

between

the

level

of

DNA

methylation

and

mRNA

expression

-LRB-

r

p

after

adjustment

for

disease

status

-RRB-

Insulin

release

-LRB-

pmol

islet

min

-RRB-

in

response

to

mmol/l

glucose

was

reduced

in

type

diabetic

compared

with

non-diabetic

islets

-LRB-

vs

p

-RRB-

as

well

as

in

non-diabetic

islets

carrying

the

allele

-LRB-

Gly/Ser

Ser/Ser

-RRB-

compared

with

carriers

of

the

Gly/Gly

genotype

-LRB-

Gly/Ser

Ser/Ser

vs

Gly/Gly

p

Fig.

f

-RRB-

In

addition

the

mRNA

expression

of

correlated

positively

with

glucose-mediated

insulin

release

-LRB-

r

p

Fig.

-RRB-

Moreover

the

polymorphism

did

not

significantly

affect

basal

insulin

secretion

at

mmol/l

glucose

-LRB-

Gly/Ser

Ser/Ser

vs

Gly/Gly

p

-RRB-

or

insulin

response

to

arginine

-LRB-

Gly/Ser

Ser/Ser

vs

Gly/Gly

p

-RRB-

or

glibenclamide

-LRB-

Gly/Ser

Ser/Ser

vs

Gly/Gly

p

-RRB-

Having

seen

the

relationship

between

expression

and

insulin

secretion

we

set

out

to

determine

whether

modulation

of

expression

would

influence

insulin

secretion

This

was

achieved

by

studying

insulin

secretion

after

experimental

downregulation

of

in

human

islets

transfected

with

siRNA

-LRB-

n

experiments

-RRB-

As

shown

in

Fig.

transfection

with

siRNA

was

associated

with

a

reduction

in

the

mRNA

expression

of

-LRB-

p

-RRB-

reduction

in

insulin

mRNA

-LRB-

p

-RRB-

and

reduction

of

the

insulin

stimulation

index

-LRB-

i.e.

incremental

folds

above

baseline

insulin

release

p

-RRB-

Notably

no

significant

change

was

seen

in

glucagon

mRNA

expression

suggesting

that

the

effect

was

primarily

due

to

a

downregulation

of

in

beta

cells

As

a

negative

control

transfection

with

scrambled

siRNA

was

not

associated

with

any

changes

in

-LRB-

-RRB-

insulin

-LRB-

-RRB-

or

glucagon

-LRB-

-RRB-

mRNA

expression

-LRB-

p

-RRB-

Fig.

of

human

pancreatic

islets

with

siRNA

is

associated

with

reduced

mRNA

levels

of

-LRB-

n

-RRB-

and

b

insulin

-LRB-

n

-RRB-

and

c

concomitant

reduction

of

the

insulin

stimulation

index

-LRB-

ISI

-RRB-

i.e.

incremental

fold

change

above

basal

insulin

release

d

Conversely

inhibition

of

expression

in

human

pancreatic

islets

has

no

effect

on

glucagon

mRNA

expression

-LRB-

n

-RRB-

Results

are

expressed

as

mean

SEM

p

The

finding

of

reduced

expression

in

human

diabetic

islets

was

somewhat

surprising

given

an

earlier

report

of

increased

levels

in

islets

from

diabetic

mice

-LRB-

ob/ob

-RRB-

and

rat

-LRB-

ZDF

and

pancreatectomised

rats

-RRB-

models

-LSB-

-RSB-

To

further

explore

species-specific

differences

we

also

measured

mRNA

expression

in

pancreatic

islets

from

two

diabetic

rat

models

expression

was

reduced

in

islets

from

STZ

diabetic

male

Wistar

rats

compared

with

control

animals

-LRB-

vs

p

Fig.

-RRB-

Moreover

expression

was

reduced

in

islets

from

the

congenic

strain

which

has

insulin

secretion

defects

compared

with

the

insulin-resistant

strain

-LRB-

vs

p

-RRB-

of

the

GK

rat

an

animal

model

of

polygenic

type

diabetes

-LRB-

Fig.

-RRB-

However

no

difference

in

expression

was

found

when

comparing

islets

from

or

GK

rat

strains

-LRB-

Fig.

-RRB-

Fig.

mRNA

expression

in

rodent

pancreatic

islets

a

Male

Wistar

rats

-LRB-

months

old

-RRB-

were

treated

with

STZ

-LRB-

n

-RRB-

or

vehicles

-LRB-

control

n

-RRB-

-LSB-

-RSB-

and

mRNA

expression

together

with

the

internal

standard

cyclophilin

A

was

analysed

in

pancreatic

islets

isolated

from

STZ

NA-treated

animals

showing

a

stable

hyperglycaemia

-LRB-

mmol

glucose/l

-RRB-

and

controls

mRNA

expression

together

with

the

internal

standard

cyclophilin

A

was

analysed

in

pancreatic

islets

prepared

from

rats

at

weeks

of

age

after

a

h

fast

GK

rats

-LRB-

n

-RRB-

were

obtained

from

the

Stockholm

colony

and

bred

as

described

-LSB-

-RSB-

Inbred

normoglycaemic

-LRB-

n

-RRB-

were

purchased

from

Charles

River

Laboratories

Transfer

of

GK

alleles

onto

the

genome

of

rats

by

repeated

backcrossing

-LRB-

ten

generations

-RRB-

established

the

homozygous

congenic

strains

and

rats

-LRB-

n

-RRB-

carry

of

the

GK

genotype

based

on

genetic

distance

on

a

homozygous

genetic

background

-LRB-

cM

-RRB-

and

display

hyperglycaemia

accompanied

by

fasting

hyperinsulinaemia

implicating

insulin

resistance

-LRB-

n

-RRB-

carries

of

the

GK

genotype

-LRB-

cM

-RRB-

and

display

insulin

secretion

defects

-LSB-

-RSB-

Results

are

expressed

as

mean

SEM

p

Discussion

The

key

results

from

the

present

study

were

that

mRNA

expression

and

insulin

secretion

were

reduced

in

pancreatic

islets

of

patients

with

type

diabetes

and

in

non-diabetic

carriers

of

a

allele

a

genotype

previously

associated

with

the

disease

Moreover

we

demonstrate

that

epigenetic

mechanisms

are

likely

to

be

operative

in

the

pathogenesis

of

type

diabetes

since

DNA

methylation

of

the

promoter

was

increased

in

human

diabetic

islets

Experimental

downregulation

of

expression

in

human

islets

by

siRNA

resulted

in

decreased

insulin

secretion

thereby

demonstrating

a

causal

link

between

levels

and

insulin

secretion

Based

on

these

observations

we

propose

a

model

where

combinations

of

genetic

and

epigenetic

factors

can

influence

the

level

of

in

human

islets

and

subsequently

glucose-stimulated

insulin

secretion

The

transcriptional

coactivator

is

an

important

factor

regulating

the

expression

of

genes

for

oxidative

phosphorylation

in

a

number

of

tissues

including

skeletal

muscle

liver

and

adipose

tissue

We

and

others

have

previously

demonstrated

reduced

expression

of

and

a

set

of

genes

involved

in

oxidative

phosphorylation

in

skeletal

muscle

from

patients

with

type

diabetes

-LSB-

-RSB-

However

the

level

and

role

of

in

pancreatic

islets

of

patients

with

type

diabetes

remained

to

be

determined

The

present

results

indicate

that

the

situation

in

human

islets

mirrors

the

situation

in

skeletal

muscle

since

the

expression

level

of

was

reduced

in

human

type

diabetic

islets

thus

providing

evidence

for

a

common

defect

that

may

simultaneously

contribute

to

peripheral

insulin

resistance

and

impairment

of

beta

cell

function

In

contrast

to

the

present

results

in

humans

elevated

levels

have

been

reported

in

islets

from

diabetic

rodent

models

ob/ob

mice

ZDF

rats

and

pancreatectomised

rats

-LSB-

-RSB-

In

these

rodent

models

the

increase

in

was

associated

with

suppressed

beta

cell

metabolism

and

insulin

release

This

could

reflect

true

species

differences

but

also

differences

in

experimental

conditions

To

address

this

issue

we

determined

-LRB-

-RRB-

the

effect

of

inhibiting

production

by

transfecting

human

pancreatic

islets

with

siRNA

and

-LRB-

-RRB-

expression

in

pancreatic

islets

of

different

rodent

models

with

experimental

diabetes

i.e.

STZ-diabetic

Wistar

rats

and

congenic

strains

from

the

type

diabetes-like

GK

rat

Silencing

production

was

associated

with

concomitant

reduction

of

insulin

mRNA

expression

and

insulin

release

in

response

to

glucose

This

effect

appears

to

be

highly

specific

for

insulin

regulation

as

it

had

no

effect

whatsoever

on

glucagon

mRNA

expression

The

congenic

strains

have

been

bred

from

the

GK

rat

based

upon

low

insulin

secretion

or

insulin

resistance

In

line

with

the

results

observed

in

human

islets

expression

was

reduced

in

islets

from

diabetic

animals

and

in

rat

strains

with

suppressed

insulin

secretion

Collectively

these

data

support

the

notion

that

expression

is

reduced

in

animal

models

of

diabetes

and

human

diabetes

and

associated

with

impaired

insulin

secretion

The

risk

of

developing

type

diabetes

increases

with

high-fat/high-energy

diets

reduced

physical

activity

and

age

However

not

all

individuals

respond

to

the

environment

in

the

same

way

This

variation

in

response

to

environmental

factors

has

partially

been

ascribed

to

genetic

factors

The

common

variant

in

the

gene

is

a

plausible

candidate

for

such

a

genetic

factor

-LSB-

-RSB-

We

have

previously

shown

that

this

polymorphism

is

associated

with

an

age-related

decline

in

gene

expression

in

human

skeletal

muscle

-LSB-

-RSB-

The

present

study

shows

that

the

same

risk

allele

is

associated

with

reduced

expression

and

impaired

insulin

secretion

in

human

islets

Thus

our

results

provide

the

ground

for

a

common

genetic

predisposition

towards

type

diabetes

contributing

to

both

impaired

insulin

action

and

secretion

Reduced

expression

may

not

simply

reflect

an

effect

of

the

polymorphism

per

se

but

could

also

occur

through

epigenetic

phenomena

such

as

increased

DNA

methylation

of

the

promoter

Cytosine

residues

occurring

in

CG

dinucleotides

are

targets

for

DNA

methylation

and

gene

expression

is

usually

reduced

when

DNA

methylation

takes

place

at

a

promoter

Epigenetic

changes

are

well

known

to

influence

gene

expression

of

suppressor

genes

and

oncogenes

in

cancer

cells

and

contribute

to

tumour

growth

-LSB-

-RSB-

Increased

DNA

methylation

of

the

promoter

of

the

gene

in

type

diabetic

human

islets

may

contribute

to

beta

cell

dysfunction

by

similar

mechanisms

To

our

knowledge

this

is

the

first

study

to

demonstrate

that

DNA

methylation

may

play

a

role

in

regulating

gene

expression

in

human

diabetic

islets

Our

data

suggest

that

can

modulate

glucose-mediated

insulin

secretion

in

human

islets

most

likely

via

an

effect

on

ATP

production

as

indicated

by

normal

insulin

release

in

response

to

arginine

and

glibenclamide

which

stimulate

insulin

downstream

of

ATP

In

support

of

such

a

role

mRNA

expression

correlated

with

glucose-stimulated

insulin

release

Moreover

inhibition

of

expression

by

siRNA

transfection

in

human

islets

was

associated

with

a

decline

in

insulin

mRNA

and

insulin

release

Similarly

in

human

type

diabetic

islets

reduced

mRNA

levels

were

associated

with

impaired

glucose-mediated

insulin

secretion

The

polymorphism

which

previously

has

been

associated

with

type

diabetes

was

also

associated

with

reduced

expression

and

impaired

insulin

secretion

in

the

human

islets

One

caveat

of

this

study

could

be

that

we

did

not

measure

the

protein

level

of

in

human

islets

However

we

have

previously

demonstrated

a

significant

positive

correlation

between

the

mRNA

and

protein

levels

in

human

skeletal

muscle

-LSB-

-RSB-

and

we

therefore

assume

that

this

might

also

be

the

case

in

human

islets

The

reduction

in

expression

in

non-diabetic

carriers

of

a

allele

was

strong

and

factors

other

than

haploinsufficiency

may

therefore

explain

this

reduction

Moreover

whether

the

genotype

influences

expression

in

diabetic

islets

would

be

of

interest

however

the

present

study

lacks

statistical

power

to

perform

this

analysis

These

relationships

should

be

addressed

in

future

ad

hoc

studies

In

conclusion

we

have

shown

that

mRNA

expression

is

reduced

in

islets

from

patients

with

type

diabetes

and

it

is

influenced

by

both

genetic

-LRB-

-RRB-

and

epigenetic

-LRB-

DNA

methylation

-RRB-

factors

This

reduction

in

expression

correlated

with

impaired

glucose-stimulated

insulin

secretion

which

is

known

to

require

ATP

Thereby

the

present

study

provides

two

novel

insights

into

the

molecular

mechanisms

of

islet

dysfunction

in

type

diabetes

first

that

a

master

transcription

regulator

of

oxidative

phosphorylation

may

be

involved

and

second

that

epigenetic

factors

like

DNA

methylation

should

be

considered

when

searching

for

the

genetic

causes

of

type

diabetes

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O B I I O O B O O O B I O O O O O B I I O O O B O O O B O O O B B I O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I I O B I O O B O O O O O O B I I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O B I O O B O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O B O O O O O O O O O O O O O O O O B I O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O B I I O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O B I I O B I O O B O O O O B O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O B I O O O B I I O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O B O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B B I O B O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O B O O O O O O O O O O O O B O O B I I O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O B I O O B O O O O O O O O O O O B I O O B I O O O O B I O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O O O B I I O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I I O O O O O O B O O O O B O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O B O O B I I O

dna methylation
epigenetic
gene expression
genetic
human
pancreatic islets
ppargc1a
type 2 diabetes