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

Abstract

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 b 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 b 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 b cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic b cells and become a therapy for the treatment of type I diabetes.

Gene expression profiling of HUH7-ins: Lack of a granulogenic function for Chromogranin A.

“…The 140-fold lower insulin content of both CGA and NR cells, as compared to GR cells (Fig. 8) 10 and is consistent with this conclusion. The comparatively low proinsulin levels in medium bathing CGA and NR cells (proinsulin:insulin ratio 0.54 and 0.73% respectively) are consistent with the effect of the converting enzymes PC1 and PC2 found in the cytoplasm.…”

“…According to current literature the combination of CgB and CgA in a cell should lead to the formation of granulelike structures in the cytoplasm. 1,2 The absence of such structures in HUH7 cells, 10 but their presence in HUH7-ins cells, cannot be explained by previous knowledge of SGB.…”

“…21 Cells were cultured for a maximum of 10 passages before being used in experiments, and their insulin secretion (HUH7-ins cells) confirmed by RIA prior to conducting experiments. The generation of HUH7-ins cells has been described previously, 10 gene expression. According to current literature the combination of CgB and CgA in a cell should lead to the formation of granulelike structures in the cytoplasm.…”

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“…Also as long as the cells remain in the capsule, agents such as viruses might not be released (although there are reports that at least retroviruses can be released from encapsulated cells [6]. Other sources of cells include human stem cells that can be differentiated into insulin producing cells such as mesenchymal stem cells [7] as well as cells that are genetically modified to produce insulin [8].There has been some concern that certain earlier clinical • Ability to reproducibly source, produce and characterize the starting material.…”

Hepatic Insulin Gene Therapy in Insulin-Dependent Diabetes Mellitus

“…An emerging approach that holds promise is the transdifferentiation of hepatocytes into insulin-secreting cells that are capable of storing and secreting insulin in a very similar way like b-cells. Gene transfer of the transcription factors pancreatic and duodenal homeobox gene 1 (PDX1) and NeuroD into hepatocytes or a modified neoplastic human liver cell line (HUH7) has been demonstrated to ameliorate STZ-induced hyperglycemia in mice (79,106). Recently, Kojima et al showed that hepatocytes transfected with NeuroD and betacellulin, a b-cell growth factor, is able to drive the formation of insulin-producing cells and normalizes blood glucose levels in STZ-induced diabetic mice (107).…”