Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells.

As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of ß-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1ß, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known ß-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial ß-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D.

Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy.

BACKGROUND: Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing ß-cells of the pancreas. The present study aimed to reverse T1D by gene therapy. METHODS: We used a novel surgical technique, which involves isolating the liver from the circulation before the delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR) or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared with the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests were performed. Expression of insulin and pancreatic transcription factors was determined by the reverse transcriptase-polymerase chain reaction and immunohistochemistry and immunoelectron microscopy was used to localise insulin. RESULTS: Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed the expression of ß-cell transcription factors, murine insulin, glucagon and somatostatin, and hepatic storage of insulin in granules. The expression of hepatic markers, C/EBP-ß, G6PC, AAT and GLUI was down-regulated in INS-FUR-treated livers. Liver function tests remained normal, with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, and no pancreatic transdifferentiation or normal glucose tolerance was observed. CONCLUSIONS: This gene therapy protocol has, for the first time, permanently reversed T1D with normal glucose tolerance in NOD mice and, as such, represents a novel therapeutic strategy for the treatment of T1D.

Growth-promoting effect of Rh(D) antibody on human pancreatic islet cells.

CONTEXT/OBJECTIVE: Hyperinsulinism with islet cell hyperplasia is a frequent complication, of unknown cause, in hemolytic disease of the newborn, occurring in Rh(D)-positive infants of Rh-isoimmunized Rh(D)-negative mothers, but not in infants with other hemolytic disorders. We investigated the possibility that trans-placentally acquired anti-D Ig is the cause of both conditions. DESIGN: Monolayer cultures of human islet cells were exposed to sera from Rh-isoimmunized mothers and newborns, where jaundice, hyperinsulinism, and hypoglycemia in the infant had ensued. Parallel cultures with anti-D, specific anti-D monoclonal antibodies, normal human Ig (15 microg/ml), and serum controls were also undertaken. Islet cell proliferation was determined by [3H]thymidine incorporation. Insulin storage and chronic and acute insulin secretion to glucose were analyzed by RIA. Rh(D) surface antigen expression was determined on islet cells by flow cytometric analysis. RESULTS: Islet cell proliferation and insulin secretion were significantly greater in coculture with test sera (P < 0.01; n = 8) and with anti-D (P < 0.001; n = 8), compared with either controls or Ig. After 8 d of growth, the static incubation experiment showed a 3.5-fold response to glucose stimulus in all sera. Rh(D) antigen expression was detected on the islet cell surface by flow cytometry, and islet cell morphology was normal. Colocalization of the proliferation marker Ki67 with insulin by immunofluorescent staining further indicated that Rh(D) antibody promoted islet growth. CONCLUSIONS: The anti-Rh(D) islet cell proliferative effect generates neonatal hyperinsulinism in Rh isoimmunization. Anti-Rh(D) may have application for islet cell proliferation in diabetes mellitus treatment for Rh(D)-positive subjects. Further analysis is required.

ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT 2 transporter regulate glucose-stimulated insulin secretion.

As part of our research into the liver-directed gene therapy of Type I diabetes, we have engineered a human hepatoma cell line (HEPG2ins/g cells) to store and secrete insulin to a glucose stimulus. The aim of the present study was to determine whether HEPG2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels (KATP). Using patch-clamp electrophysiology with symmetrical KCl solutions, the single-channel conductance of KATP was 61pS. KATP was inhibited by ATP (1 mM) or cAMP (50 microM) applied to the cytosolic side of the membrane. Single KATP channels and macroscopic whole-cell currents were inhibited by glucose (20 mM) and glibenclamide (20 microM) and were activated by diazoxide (150 microM). Immunoprecipitation and Western blot analysis confirmed the presence of Kir6.2 KATP channel subunit protein in HEPG2ins/g and HEPG2ins cells. Using radioimmunoassay techniques, we report that exposure of the cells to tolbutamide (100 microM) resulted in an increase in insulin secretion from 0.3 +/- 0.05 to 1.8 +/- 0.2 pmol insulin/10(6) cells and glibenclamide (20 microM) from 0.4 +/- 0.06 to 2.1 +/- 0.3 (n=4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide (150 microM) completely inhibited glucose-stimulated insulin release. Glucose 20 mM and glibenclamide 100 microM increased intracellular Ca2+ level in the HEPG2ins/g cells. However, glucose 20 mM did not stimulate a rise in intracellular Ca2+ in the un-transfected parent cell-line HEPG2. We used confocal microscopy to confirm that glucose (20 mM) stimulated the release of insulin from the fluorescently labeled secretion granules in the cells. Furthermore, glibenclamide (20 microM) also stimulated the release of insulin from fluorescently labeled secretion granules, and diazoxide (150 microM) blocked that stimulated release of insulin. Our results suggest that HEPG2ins/g cells respond to glucose via signaling pathways that depend on KATP, similar to a normal pancreatic beta cell.

Function of a genetically modified human liver cell line that stores, processes and secretes insulin.

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic beta cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for beta cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic beta cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic beta cells and become a therapy for the treatment of type I diabetes.