Virus-delivered GCGR Predator system impairs hepatic glucagon sensitivity ex vivo
As a major optimization comparing to the previously reported Trim-away system, we proposed that our GCGR predator system can be genetically encoded and easily packed into virus vectors for possible in vivo usage. To demonstrate that, we packed Adenovirus carrying CMV-GCGR Predator cassette and a GFP reporter. Using primary mouse hepatocytes as the target cells, fluorescent microscopy showed that the GCGR-Predator system did not affect the packaging and infection of the adenovirus (Figure 1a). To determine if the adenovirus delivered GCGR predator functions in primary cells as we expected, we preformed western blotting analysis to examine the protein abundance of GCGR in primary hepatocytes infected by GFP encoding CTR virus or GCGR Predator carrying viruses. As shown in figure 1b, GCGR Predator transfected hepatocytes showed ~60% lower GCGR abundance comparing to control group (Figure 1b, n>3), indicating that GCGR predator system is capable of degrading GCGR in primary cells. To further explore if such degradation would cause similar metabolic program changes as we observed in HepG2 cell lines, we preformed glucose production assay to evaluate the glucagon sensitivity of the GCGR Predator transfected primary cells. Results suggested that the overall glucose production under glucagon stimulation in GCGR Predator transfected group is significantly lower than CTR virus infected group (Figure 1c). Quantitative PCR analysis and glycogen staining assay also indicated significantly impaired gluconeogenesis and glycogenolysis activity in GCGR Predator transfected groups (Figure 1d and e).
Figure 1. Virus-delivered GCGR Predator system impairs hepatic glucagon sensitivity ex vivo. (a). Representative fluorescent microscopy of the adeno-GCGR Predator infected primary hepatocytes; (b). Adenovirus delivered GCGR Predator mediates GCGR degradation in primary mouse hepatocytes. Left panel: representative blotting of GCGR degradation; right panel: statistical gray scale analysis of blotting assays; (c). Glucose production analysis of primary hepatocytes transfected with GCGR predator; (d). Quantitative PCR assay of G6Pase and PEPCK in primary hepatocytes transfected with GCGR predator; (e). Representative glycogen staining microscopy of GCGR predator transfected primary hepatocytes. “GCGR Pr” stands for GCGR Predator. Relative gene expression was calculated using the 2-ΔΔCT method, with initial normalization of genes against Hprt1 mRNA within each group. The expression levels of each gene in the control groups were arbitrarily set to 1.0. Relative protein expression levels were calculated by using β-actin expression level as initial normalization and then set the protein expression level in the control groups arbitrarily as 1.0. Error bar represents SD of at least 3 biological replicates, ** p<0.01.
Generally, these results indicated that GCGR Predator worked as we expected not only in cultured cell lines, but also in primary cells under virus-based delivery system. They also implied that our system would highly likely to work in the in vivo conditions.
Novel glucose sensor integrating endogenous metabolic network and exogenous transgene expression
With the advice obtained by our HP group, we designed a novel glucose sensing device that is capable of rewiring endogens glucose sensing signal to the expression of exogenous genes (Figure 2a). To demonstrate that such design works as we expected, Glucose sensing promoters (GSPs) were cloned into pGL3 vector to control the expression of firefly luciferase. Corresponding plasmids were co-transfected into HepG2 cell line with SV40-rluc internal control plasmid. As is shown in Figure 2b, comparing to mini-Promoter, 3x GSP, 6x GSP and 9x GSP all showed significant increase of luciferase signal. The transcriptional strength showed a significant or margnially significant improvement of 9×GSP comparing to that of 3×GSP or 6×GSP counterparts. To validate the glucose responsiveness, we challenged the 9x GSP-luciferase carrying cells by culturing cells in 5mM or 20mM glucose after overnight starvation. Results showed a ~1.5-fold increase of luciferase signal in 20 mM treatment group comparing to the 5 mM group (Figure 2a).
To further characterize the glucose sensing module, we generated a 9x GSP-GFP reporter plasmid to increase the detection throughput. By using CMV-mcherry to normalize the effect of glucose on general exogenous gene expression level, we demonstrated that the 9x GSP is capable of activating gene expression in a glucose dose dependent manner (Figure 2d).
Figure 2. Glucose sensing by CHREBP binding hybrid promoter. (a) Schematic representation of the promoter design; (b). Transcriptional strength analysis of different GSPs; (c) Glucose response of 9x GSP-luc cassette; (d) Glucose response profiling of 9x GSP-GFP cassette. Fluorescent intensity of GFP or RFP was calculated by imaging gray scale analysis. Relative luminance was calculated by normalizing to Renilla luciferase signal, relative Fluorescent Intensity was calculated normalizing RFP signal. To calculate fold changes, levels of each control groups (minip or low glucose groups) were arbitrarily set to 1.0. Error bar represents SD of at least 3 biological replicates, * p<0.05, ** p<0.01.
Glucose sensor-GCGR Predator chimera restores glucose homeostasis of T2D patients in silico
To further demonstrate the in vivo performance of our circuit, we developed a set of mathematical models on molecular, cellular and whole-body level (Figure 3a). By integrating these models, we obtained glycemia simulation results under different conditions. Healthy object showed stabled glycemia level at around 96 mg/dL. By decreasing insulin sensitivity by 50%, object showed significant hyperglycemia at around 133 mg/dL. When GCGR-Predator is introduced into the model with a strong constitutive promoter, object showed mild hypoglycemia (~53 mg/mL), which is consistent with the suggestions provided by HP expert interview as well as previous literatures1-3. However, when GCGR Predator system is controlled by glucose sensing device, which provides feed-back control over the degradation of GCGR, simulation showed that the glycemia level would be stabled at around 86.7 mg/dL, indicating that the close-loop controlling system may provide a more robust control over the glycemia level (Figure 3b).
Figure 3. Glucose sensor-GCGR Predator chimera restores glucose homeostasis of T2D patients in silico. (a). Schematic representation of the modeling structure; (b). Glycemia simulation under different conditions.
Reference
1 Christine, L. et al. Liver-specific disruption of the murine glucagon receptor produces α-cell hyperplasia: evidence for a circulating α-cell growth factor. Diabetes 62, 1196-1205 (2013).
2 Lok, S., . et al. The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization. Gene 140, 203-209 (1994).
3 Rivero-Gutierrez, B. et al. Deletion of the glucagon receptor gene before and after experimental diabetes reveals differential protection from hyperglycemia. Molecular metabolism 17, 28-38, doi:10.1016/j.molmet.2018.07.012 (2018).