Reports of research work funded by grants prior to 2013
University of Otago Wellington, School of Medicine and Health Sciences
Changes in Gene Expression in Hepg2 Cells Exposed to Portal Serum of Diabetics Before and After Loss of Diabetes
RS Stubbs and MH Hayes
Wakefield Biomedical Research Unit
Department of Pathology and Molecular Medicine
Gastric bypass surgery has been observed by us, to bring about remission or possibly resolution of Type 2 diabetes (T2DM) in the majority of diabetic subjects undergoing this form of surgery for severe obesity. This observation was first reported by Professor Walter Pories in the USA in 1995, and has been repeatedly substantiated by others from around the World. We have hypothesized that this dramatic change is brought about by alteration in hormones being released from the gut and acting on the liver, to improve hepatic glucose uptake and to reduce endogenous hepatic glucose production, which is known to be a key feature of T2DM.
In an effort to prove this we undertook a series of experiments in which we looked at alterations in gene expression within cultured liver cells (HepG2 cells) brought about by exposure to blood coming from the gut (portal venous blood), obtained from three diabetic individuals (a) before gastric bypass and (b) again six days after gastric bypass (when diabetes had gone).
The human HepG2 cells were seeded at a density of 2 x 106 cells per well in 6-well plates and cultured to near confluence in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) at 37 ºC in a 5% CO2 air atmosphere (protocol provided by Arun Kanakkanthara, School of Biological Sciences, VUW). The cells were serum-starved for 12 hours and exposed to 20% portal venous serum, taken (a) before and (b) six days following gastric bypass surgery from three diabetic individuals for either 4 hours or 24 hours. Each serum treatment was performed in triplicate. The cells were harvested, washed twice with phosphate-buffered saline (PBS), and the cell pellets were stored at -80 ºC and sent to the National Children’s Research Centre in Washington where RNA was extracted, QC tested and run on cRNA arrays (Illumina HumanHT-12 v4 Expression BeadChip). The resultant data was analysed with BRB array Tools (Class comparison) and Partek (ANOVA).
A number of significant changes in gene expression were noted between the two time points (before and after surgery). These were noted in eight genes after only 4 hours of exposure to post-operative portal venous serum (two were upregulated and six were downregulated) and in 15 genes after 24 hours of incubation (eleven were upregulated and 4 downregulated). Of particular note the gene CTNNB1 was upregulated after 4 hours and G6PC was downregulated after 24 hours. The upregulation of the former would have the effect of increasing glycogen formation within the liver and downreglation of G6PC would have the effect of substantially reducing endogenous glucose production (gluconeogenesis) and also inhibiting the breakdown of glycogen by the liver. All three actions would have a major normalising effect on blood glucose levels, which is what we observe after gastric bypass surgery.
These results support our hypothesis that gastric bypass surgery alters the hormones being released from the gut and passing to the liver (in the portal venous blood) which in turn has a major impact on blood glucose control. These findings have important implications for the direction of further research aimed at finding a pharmacologic agent capable of bringing about remission of T2DM, through the same or similar mechanism to that achieved by gastric bypass surgery.
In order to complete this project funded by WMRF, confirmatory qRTPCR will be conducted on the genes noted to change in the experiment described above, using an applied Biosystems Quantstudio 12k flex real time PCR system and qRTPCRssay on demand plates (3000 assays per plate). mRNA samples will be sent back from Washington and converted to cDNA at John Curtin School of Medical Research describing the results and our previous in vitro assays will be prepared for publication.