oa Characterization of Circulating miRNAs in Insulin Sensitive and Insulin Resistant Arab Individuals in Qatar

Summary

Insulin resistance manifest in skeletal muscle, liver and adipocytes. Insulin stimulates muscle glucose uptake, suppresses the rate of hepatic glucose production (HGP) and restrains lipolysis. In insulin resistant individuals, insulin-stimulated glucose uptake is markedly reduced in skeletal muscle, impaired insulin-mediated suppression of hepatic glucose production (HGP) and inhibition of lipolysis. It is evident from human and animal studies that diabetes alters miRNAs expression in metabolically important organs (adipose, liver and skeletal muscle).micRNA are small non coding RNA molecules. However, they play critical role in the regulation of gene expression via post-transcriptional mechanisms. They are small in size (21?23 nucleotides) and recent studies have demonstrated that they play important role in many human complex diseases including diabetes. The ability of miRNA that simultaneously regulate many target genes makes them attractive candidates for regulating insulin production/secretion and action. mRNA are secreted to the circulation and are taken up from the circulation by several tissues, making them an attractive mechanism for inter-tissue signaling molecules. Thus, if micro RNA play role in the pathogenesis of insulin resistance or in communicating between insulin resistant tissues and the beta cell to regulate the level of beta cell function in concert with the prevailing level of insulin resistance, we anticipate a change in the circulatory profile of micro RNA in insulin resistant individuals compared to insulin sensitive individuals. Such a distinct circulatory profile of micro RNA in insulin sensitive versus insulin resistant individuals could provide a signature for the identification of insulin resistant individuals. Moreover, differentially expressed micro RNA molecules in insulin resistant individuals could serve signaling molecules which convey messages between insulin resistant tissues, i.e. liver, skeletal muscle and adipocytes and the beta cell. Insulin sensitivity is commonly assessed to predict the risk and evaluate the management of type 2 diabetes in clinical and research settings. Insulin sensitivity can be measured using a variety of techniques that are commonly employed in diabetes research and care. Among them, euglycemic hyperinsulemic clamp is the gold-standard method to quantify the sensitivity to insulin.

The aim of the present study is to determine the profile of miRNA in the plasma in insulin sensitive Arab individuals and compare the result to that in insulin resistant Arab individuals.

specific aims are;

1. To discover the circulatory profile of microRNA in insulin sensitive and insulin resistant Arab individuals by using next generation sequencing technology.

2. To identify novel microRNA/targets which are differentially expressed in insulin resistant individuals by real time Taq-Man PCR.

3. To explore the effects changes of these miRNAs in insulin resistance diabetes β-Cell Biology. The experiments range from relevant tissue and cell cultures systems to human physiology. Design and methods: The circulating miRNA profile was assessed in a pilot study of 20 healthy volunteers whom glucose tolerance status was determined by the OGTT, and their insulin sensitivity was quantitated by the gold standard method the euglycemic hyperinsulemic clamp. Based on Glucose Infusion rate (GIR: mg/kg/min), before and after a 6-h hyperinsulinemeic euglycemic clamp, we selected two groups: 10 with high levels of GIR (insulin-sensitive group) and 10 with Very low levels of GIR (insulin resistant group).The serum samples was collected from the two groups for miRNA extraction and subsequent sequencing using the True-seq Illumina protocol. A small RNA library was generated from samples using the Illumina Truseq™ Small. The purified cDNA library was used for cluster generation on Illumina's Cluster Station and then sequenced on Illumina GAIIx following vendor's instruction for running the instrument. Raw sequencing reads (40 nts) were obtained using Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70). Result: Sequencing of the small circulating RNA isolated from insulin-resistant and insulin-sensitive healthy samples produced 69,559,764 raw reads. A total of 41,697,300 mappable reads was obtained after sequence filtering. Using a 2-fold expression difference as a cutoff, 33 miRNAs showed significally differential expression between insulin sensitive and insulin-resistant group. Among which, 10 were up-regulated and 22 were down-regulated. This RNA-seq discovery study shows that the most changes in miRNAs expression is in accordance with previous studies performed to explore circulating miRNAs in human insulin resistance or T2DM. Indeed, among the 54 selected miRNAs, we have identified the same changes in mir-126 expression that has described by Zampetaki and colleagues. This group reported that mir-126 is a unique plasma miRNA signature in 800 patients with T2D and observed that the reduced levels of mir-126 showed the strongest association with T2D. From the 33 miRNAs, a relative number of them are much documented to be associated with insulin resistance and type 2 diabetes. Nevertheless, some identified miRNAs are potential novel candidates and need to be deeply exploring during the event of the pathogenesis of T2D. Conclusion: This study provides a comparative profiling of circulating miRNAs in insulin-resistant versus insulin–sensitive subjects and the identification of specific miRNAs in circulation that changes are associated with insulin action. To our knowledge, this is the first study to investigate insulin resistance in Arab population using euglycemic hyperinsulemic clamp the gold-standard method to assess insulin sensitivity and aim to identify changes in circulating miRNAs expression associated to insulin action and signaling. The study also provides evidence that Insulin Resistance in human is related to changes in multiples microRNAs. Functional Validation studies of the differentially expressed miRNAs and relevant target and signaling pathways are ongoing.