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Comparative Study
. 2012 Apr;109(4):1074-82.
doi: 10.1002/bit.24373. Epub 2011 Nov 21.

Combinatorial Insulin Secretion Dynamics of Recombinant Hepatic and Enteroendocrine Cells

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Free PMC article
Comparative Study

Combinatorial Insulin Secretion Dynamics of Recombinant Hepatic and Enteroendocrine Cells

Kiranmai Durvasula et al. Biotechnol Bioeng. .
Free PMC article

Abstract

One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies.

Figures

Figure 1
Figure 1
Adenoviruses used for transduction: a) AdG3-2xfur, b) AdG3-InsTail and c) AdG3-Track. Each adenovirus contained the furin-compatible human proinsulin gene (PPI2xfur) under the transcriptional regulation of the glucose-responsive promoter G3.
Figure 2
Figure 2
Insulin accumulation profile of GLUTag-INS under basal and stimulation conditions. The vertical lines indicate medium changes. The inset details the time course of insulin secreted right after the cells are stimulated with either 2% MH only or cocktail medium (2% MH and 20 mM glucose). #Significant difference between cocktail and basal groups at these times, p<.05. *Significant difference between MH and basal groups at these times, p<0.05.
Figure 3
Figure 3
Bright field and fluorescent images of AdG3-Track transduction of a) HepG2 cells at MOI = 1, b) H4IIE cells at MOI = 1, c) H4IIE cells at MOI = 4 and d) H4IIE cells at MOI = 10.
Figure 4
Figure 4
Insulin accumulation profile of HepG2 cells transduced with a) AdG3-Track, b) AdG3-2xfur and c) AdG3-InsTail. The vertical lines indicate medium changes.*p<0.05.
Figure 5
Figure 5
Combinatorial secretion profile with stimulation in cocktail medium: a) HepG2 transduced with AdG3-2xfur (H) with GLUTag-INS (G); b) HepG2 transduced with AdG3-InsTail (H) with GLUTag-INS (G).

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