GLUCOSE-STIMULATED INSULIN SECRETION FROM AN ENGINEERED HUMAN LIVER CELL LINE IS REGULATED BY ATP-SENSITIVE POTASSIUM CHANNELS (KATP) AND L-TYPE CALCIUM CHANNELS

Abstract

P669 Aims: An alternative approach to the treatment of type 1 diabetes is the creation of an ‘artificial beta cell’. It has been previously shown that the stable transfection of the full length insulin cDNA into the human liver cell line HuH7 resulted in synthesis, storage and regulated release of insulin to the physiological stimulus glucose (Huh7ins cells). The aim of the present study was to determine if Huh7ins cells respond to glucose via the blockage of KATP and the activation of voltage-gated calcium channels, as is the case with pancreatic beta cells. Methods: To reveal the mechanisms underlying glucose-stimulated insulin secretion, HuH7 cells were stably transfected with insulin fused to enhanced green fluorescent protein (EGFP): HuH7-EGFPins. As a positive control the glucose responsive pancreatic beta cell line Min6 was also transfected with EGFP: Min6-EGFPins cells. The effect of 20 mM glucose and the KATP activator diazoxide (150 μM) and the blocker glibenclamide (20 μM) on acute insulin secretion from the cells lines was assessed by confocal microscopy and biochemically by static stimulation experiments. Insulin secretion was measured by radioimmunoassay. Specific primers were designed to the KATP subunits Kir 6.2 and SUR 2A together with the L-type calcium channel (D subunit) of pancreatic beta cells. RT-PCRs were perfomed on cDNA prepared from Huh7, Huh7ins, Huh7ins-EGFPins and human pancreatic beta cells (positive control). Results: Confocal microscopy revealed that Huh7ins-EGFPins and Min 6-EGFPins cells responded to a glucose stimulus via KATP. The glucose and glibenclamide responses were seen as a time-dependent loss of fluorescence as insulin granules were released by exocytosis. Cells exposed to diazoxide, accumulated brighter fluorescence. Exposure to glucose for 1 hr resulted in an increase in insulin secretion from 22.8 ± 1.5 to 52.5 ± 3.8 and glibenclamide from 19.4 ± 0.7 to 59.5 ± 3.6 μU insulin/ 106 HuH7ins cells (n=3). Diazoxide completely inhibited glucose-stimulated insulin release. Very similar results were recorded for the Huh7ins-EGFP cells. PCR analysis revealed the presence of the KATP channel subunits Kir 6.2 and SUR 2A together with the L-type calcium channel in all liver cell lines. Conclusions: These results indicate that that Huh7ins cells respond to glucose by the same mechanism seen in β cells. This provides useful information with regards to the future engineering of insulin-secreting liver cells and gives hope that the transplantation of such cells will provide a cure for Type I diabetes.