Reports of research work funded by grants prior to 2013
University of Otago Wellington, School of Medicine and Health Sciences
Confirmation of Changes in the Expression Profile of miRNA and mRNA in Abdominal and Omental Fat Samples from Morbidly Obese Patients at the Time of Gastric Bypass Surgery and after Weight Loss and Remission of Diabetes and Insulin Resistance
MT Hayes, D Macartney-Coxson and RS Stubbs
Wakefield Biomedical Research Unit and Water Group, ESR
Department of Pathology and Molecular Medicine
This is a supplementary report on this application to indicate what further work has been done in the last 12 months.
The overall purpose of this proposal was to investigate which genes and miRNA species change significantly with major weight loss or loss of diabetic or insulin resistant status. By so doing we hoped to identify mechanisms by which weight or diabetic status might ultimately be manipulated through the development of new drug targets.
The original application which was funded by WMRF in 2011 had two aims;
- To confirm changes in gene expression profile in liver, abdominal and omental fat in samples taken at the time of gastric bypass surgery and at the time of a second surgery after significant weight loss and resolution of diabetes and insulin resistance using Quantitative RTPCR.
- To confirm changes in miRNA expression profile in abdominal and omental fat in samples taken at the time of gastric bypass surgery and at the time of a second surgery after significant weight loss and resolution of diabetes and insulin resistance using Quantitative RTPCR.
The second of these aims has been completed and was reported on last year, by Dr Macarney-Coxson. In essence one miRNA species (mir-223) was identified as having potential importance in the change in diabetes status following gastric bypass surgery and a further two species were thought to also have potential importance in major weight loss. These findings are being followed up on and were the subject of a further application to WMRF in 2012 which has now been reported on this year (A further investigation of candidate miRNAs involved in obesity and type 2 diabetes mellitus in adipose tissue and plasma, AM Jones, RS Stubbs, D Macartney-Coxon).
The first aim was only partially completed last year, due to delays in the arrays being performed in Washington, DC. Furthermore, because the initial experiment contained only 3 diabetic subjects, it was felt important to expand the samples being tested to include more numbers. For this reason an additional 31 Illumina Human HT12 V4 arrays were run in Washington, on total RNA extracted from abdominal and omental fat samples from a further eight obese patients taken (a) at the time of the initial gastric bypass surgery and (b) at a second operation approximately one year later. These data have recently become available and combined with that from the original 60 arrays mean we now have data for analysis on eight diabetic, seven insulin resistant patients and eight insulin sensitive patients.
Initial analysis indicates that gene expression in fat derived from insulin resistant and diabetic patients does not differ significantly and therefore these two groups can be combined to compare insulin resistant with insulin sensitive states. Predictive analysis of microarrays on genes from omental fat obtained at gastric bypass from insulin resistant vs insulin sensitive individuals allows classification of resistant status with 71% accuracy on the basis of expression of GPR183 – a G protein coupled receptor which is upregulated in response to Epstein Barr infection.
Further analysis will be conducted to identify a number of candidate genes for confirmatory qRTPCR. Washington has been requested to return mRNA samples to John Curtin School of Medical Research for conversion to cDNA for use in qRTPCR experiments. Confirmatory qRTPCR will be conducted on the genes selected using an applied Biosystems Quantstudio 12k flex real time PCR system and qRTPCR assay on demand plates (3000 assays per plate). This work will be completed before the end of 2013.