The ANK1 rs508419-C T2D-risk allele increases the expression of sAnk1.5 and miR486 but their overexpression in transgenic mice does not significantly alter glucose tolerance

Genome wide association studies (GWAS) identified the ANK1 gene as a common type 2 diabetes mellitus (T2D) susceptibility locus. More recently, GWAS studies identified a novel SNP associated to T2D susceptibility, namely rs508419, in the internal promoter of ANK1 gene, which drives the expression of small ankyrin 1.5 (sAnk1.5), a striated muscle-specific protein localized in the sarcoplasmic reticulum (SR) membrane. sAnk1.5 interacts with the giant sarcomeric protein obscurin, allowing the correct localization of SR around the myofibrils. The rs508419 SNP is characterized by the substitution of a thymine with a cytosine, which determines an increased transcriptional activity of the ANK1 internal promoter, resulting in high protein levels of sAnk1.5 in skeletal muscle biopsies of individuals carrying the C/C variant of the SNP, with respect to individuals carrying either T/T or C/T genotype. Interestingly, the sequence of microRNA-486 (miR-486), a small non-coding RNA, is positioned in the intron between exon 41 and exon 42 of the coding sequence of sAnk1.5. Therefore, miR-486 is expressed under the transcriptional control of both the principal and of the internal promoters of ANK1. miR-486 plays a role in the regulation of the PI3K/AKT signalling pathway, which regulates several cellular processes, such as growth, cellular proliferation and survival, protein synthesis and degradation, lipid and glucose metabolism. In our laboratory we analysed human skeletal muscle biopsies to evaluate whether miR-486 expression levels were increased in individuals carrying the SNP rs508419. These results showed a significant 3-fold increase of miR-486 expression levels in the skeletal muscle of individuals carrying the C/C variant of the SNP compared to those carrying the T/T variant of the SNP. Given that skeletal muscle is one of the main tissues involved in regulating glucose disposal, the aim of this thesis was to verify whether sAnk1.5 and miR-486 overexpression in mouse skeletal muscle might associate with T2D susceptibility. To this goal, we generated a double transgenic mouse model, (TgsAnk1.5/+//TgmiR486/+), hereafter referred to as double Tg (D-Tg), where the sAnk1.5 coding sequence and the miR-486 sequence are under the transcriptional control of the skeletal muscle-specific rat myosin light chain (MLC) promoter, and of the mouse muscle-specific creatine kinase (CKM) promoter, respectively. Accordingly, the D-Tg mouse was expected to present a skeletal muscle-specific increase of sAnk1.5 and miR-486 expression. Indeed, RT-PCR experiments indicated that the expression of the transgene was restricted to striated muscles and sAnk1.5 and miR-486 mRNAs levels were both robustly increased in transgenic mice compared to WT. Accordingly, western blot analysis of protein extracts from gastrocnemius, 4 extensor digitorum longus (EDL) and soleus muscles revealed an increase in sAnk1.5 protein levels of 45%, 60% and 35%, respectively, with respect to control mice. Notably, in the D-Tg mouse model we observed a significant discrepancy between the levels of sAnk1.5 protein expression and the levels of the corresponding mRNA. Indeed, in the gastrocnemius muscle, sAnk1.5 mRNA was about 12 times more expressed in transgenic mice, compared to only 1.5-fold increase in protein expression, suggesting a post-translational regulation of sAnk1.5 expression. Evidence of this was confirmed by muscular administration of the proteasome inhibitor MG132 in transgenic mice. Results showed sAnk1.5 protein expression levels significantly increased of about 35% compared to those observed in the contralateral untreated gastrocnemius muscles. To evaluate the potential association between sAnk1.5 and miR-486 overexpression with T2D, glucose and insulin tolerance were monitored during a period of 12 months in male transgenic mice. Blood glucose levels after overnight fasting were significantly higher in 2 and 6 months old transgenic mice compared to WT controls. However, overall glucose and insulin tolerance was not altered between the two experimental groups. Finally, considering the miR-486 role in regulating PI3K/AKT signalling pathway, which in skeletal muscle fiber is activated following insulin stimulation thus being a key pathway in maintaining glucose homeostasis, we characterized the expression pattern of PTEN, p85α, FOXO1a, pAKTser473, and GLUT4 in the skeletal muscles of D-Tg mice. Currently, we are performing additional experiments where D-Tg mice are fed with a high-fat diet to better evaluate whether sAnk1.5 and miR-486 overexpression might predispose to T2D susceptibility.