Down-regulation of microRNAs in type 2 diabetes implicates non-coding RNA in insulin action

1Robin

A McGregor, Keller, Gallagher, R Nielsen, 2Christian P Fischer, 2Bente K Pedersen and 1James A Timmons
of Life Sciences, Heriot-Watt University, Edinburgh, UK 2Centre of Inflammation & Metabolism, University of Copenhagen, Denmark
1School

1,2Pernille

1Iain

2Anders

BACKGROUND
Skeletal muscle insulin resistance is a hallmark of type 2 diabetes and metabolic syndrome. Microarray profiling has consistently failed to identify differentially expressed gene networks1,2 and this includes our recent analysis (n=124, U133+2 Affymetrix arrays, unpublished observation). The lack of a modified global transcriptome in human type 2 diabetes patients suggests post-transcriptional regulation must play a role in the disease process. MicroRNAs are regulators of mRNA translation. miR-1, miR-133a and miR-206 are muscle specific and regulate muscle development3,4. It is plausible that microRNA modulation could help explain the lack of transcriptional changes in human skeletal muscle insulin resistance. Thus, we proposed that impairment in glucose tolerance is regulated at the post-transcriptional level by alterations in muscle miRNA expression.

METHODS
Human skeletal muscle biopsies (n=30) were taken from 3 groups defined by glucose tolerance: type 2 diabetes (DM2), impaired glucose tolerance (IGT) or normal glucose tolerance (Control). Participants were matched for age, BMI and VO2max. Real-time PCR for the detection of microRNA expression Taqman MicroRNA assay (Applied Biosystems) which detects mature miRNA was used to measure the expression of miR-1, miR-133a, and miR-206. The miRNA expression levels were normalized to RNU48. All reactions were run singleplex and quantified using the !Ct method. Data are expressed relative to healthy control subject values and analyzed using ANOVA to compare differences in !Ct values between the three groups followed by a post-hoc t-test where appropriate to identify specific group differences. Expression of the miRNAs were plotted against metabolic parameters to identify possible correlations. For all analyses P<0.05 was considered significant. In the figures significant differences are indicated by *** P<0.001, ** P<0.01, *P<0.05. Bioinformatics MicroRNA targets were obtained from PicTar (http://pictar.bio.nyu.edu/) and TargetScan ( http://www.targetscan.org/). We used EASE (http://david.abcc.ncifcrf.gov/home.jsp) to examine the miRNA target lists for gene ontology enrichment using an FDR <0.05.

RESULTS
Table 1. Subject Characteristics Subject Characteristics Age BMI VO2max Fasting glucose 2-h glucose HbA1c Type 2 diabetes Impaired Glucose Tolerance 60.4 14 60.2 7.1 26.6 1.9 26.3 1.7 28.8 8.6 29.4 6.9 11.3 2.9*** 5.9 0.5** 21.1 5.1*** 7.5 1.8** 8.3 1.3*** 5.8 0.2* Healthy Control 60.3 7.5 25.9 1.9 28.4 6.0 5.0 0.4 5.1 1.6 5.6 0.3

Figure 3. Fasting plasma glucose concentration associated with miR-133a expression

Figure 4. Longer-term insulin resistance indicator associated with miR-133a expression

Figure 2. Expression of miR-133a and miR-206 downregulated in type 2 diabetes individuals

Figure 5. Main gene ontologies targeted by miR-133a miR-133a

GO CELLULAR COMPONENTS Intracellular organelle Organelle Envelope

e.g. BNIP3L; COX6A1; MCL1; NUP153; RANBP2; TIMM17A; TIMM8A
.

GO BIOLOGICAL PROCESSES Intracellular protein transport Protein Dephosphorylation

e.g. CLTA; CLTC; GABARAPL1; RANBP2; SNX1; TIMM17A; TIMM8A; TRAM1; TRAM2

GO MOLECULAR FUNCTION Protein Binding Phosphoprotein phosphatase activity

e.g.SOCS5; PTPN12; PTPRD; CDC42

CONCLUSION
We provide the first evidence for altered miRNA expression in human insulin resistance. There is a robust down-regulation of miR-133a (and miR-206) in skeletal muscle and this was strongly associated with clinical status. Gene ontology analysis of the 186 predicted targets of miR-133a (expressed in skeletal muscle) revealed plausible interactions with genes involved with the insulin signalling pathway and thus the disease process.

REFERENCES
1.! Nguyen et al. (2006) Insulin resistance does not influence gene expression in skeletal muscle. Journal of Biochemistry and Molecular Biology, 39: 457. 2.! Mootha et al. (2003) PGC-1" responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature Genetics,
3.! Chen et al. (2006) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nature Genetics, 38: 228. 4.! Rao et al. (2006) Myogenic factors that regulate expression of muscle-specific microRNAs. PNAS, 103: 8721. 34: 267.

This study was funded by The Chief Scientists Office, Scotland (JT), P. Keller was funded by the Lundbeck Foundation. R.McGregor was funded by Heriot-Watt University and The Physiological Society UK. We thank John Fox for excellent technical support.