Modulated skeletal muscle microRNA processing within the invariant transcriptional landscape of type 2 diabetes

1Robin McGregor, 1,2Pernille Keller, 1Iain J Gallagher, 2Anders R Nielsen, 2Camilla Scheele, 3Judit Remenyi 4Claus-Dieter Mayer, 3Gyorgy Hutvagner, 2Christian P Fischer, 5Claes Wahlestedt, 1James A Timmons, and 2Bente K Pedersen

1Heriot-Watt University Edinburgh; 2Centre of Inflammation and Metabolism, University of Copenhagen; 3Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee; 4Biomathematics & Statistics Scotland; 5Scripps Research Institute Florida

BACKGROUND Global transcript abundance profiling is a powerful systems biology tool for mapping alterations in phenotype only when careful consideration of the physiological context is maintained. Detection of regulated mRNA allows for reconstruction of modulated gene networks, including identification of key protein regulators (by inference), and hence provides detailed insight into to the development of a disease phenotype. Skeletal muscle insulin resistance is a hallmark of metabolic syndrome and OXPHOS gene expression has been claimed to be down-regulated in patients with diabetes1.

Herein we present the first robust global transcriptome analysis of human skeletal muscle (vastus lateralis) in type 2 diabetes from 118 subjects (DM2, type 2 diabetes (n=45), impaired glucose tolerance (IGT, n=26) and normal glucose tolerance (NGT, n=47)). MicroRNAs are regulators of mRNA translation. miR-1, miR-133a and miR-206 are muscle specific and regulate muscle development2,3 and so we also considered that altered muscle phenotype is regulated at the post-transcriptional level, through alterations in muscle miRNA expression.

RESULTS

.

CONCLUSION The skeletal muscle transcriptome does not differ from control subjects in type II diabetes subjects, and this includes OXPHOS gene expression. We then provide the first evidence for altered miRNA expression in human skeletal muscle insulin resistance. There is a robust down-regulation of miR-133a (and miR-206) in skeletal muscle and this was associated with clinical status. Numerous protein targets involved in phospho-signalling and glucose transport have both miR-133a and miR-206 seed regions in their 3` UTR, suggesting plausible interactions between these microRNAs and genes involved with insulin action.

METHODS The study and analysis was approved by the appropriate ethics committees and performed according to the Declaration of Helsinki. Patients were free from diabetic treatment for 1 week prior to assessment. RNA was isolated using TriZol and profiled on the Affymetrix™ platform covering >47,000 mRNA sequences. We also utilized the TaqMan microRNA (miRNA) real-time qPCR method, to determine the expression of the muscle specific microRNAs (miR-1, miR-133a and miR-206). The miRNA expression levels were normalized to RNU48 and 18s. All reactions were run singleplex and quantified using the ΔCt method. Data are expressed relative to healthy control subject values and analyzed using ANOVA to compare differences in ΔCt 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.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

mir-1 mir-133a mir-206

Fold

chan

ge fr

om co

ntro

ls

DM2 IGT Control

*

**

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. We thank John Fox for excellent technical support.

Subject Characteristics

Type 2 diabetes

Impaired Glucose Tolerance

Healthy Control

Age 60.4 ±14 60.2 ±7.1 60.3 ±7.5 BMI 26.6 ±1.9 26.3 ±1.7 25.9 ±1.9 VO2max 28.8 ±8.6 29.4 ±6.9 28.4 ±6.0 Fasting glucose 11.3 ±2.9*** 5.9 ±0.5** 5.0 ±0.4 2-h glucose 21.1 ±5.1*** 7.5 ±1.8** 5.1 ±1.6 HbA1c 8.3 ±1.3*** 5.8 ±0.2* 5.6 ±0.3

REFERENCES 1.  Mootha et al. (2003) PGC-1α responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature Genetics, 2.  Chen et al. (2006) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nature Genetics, 38: 228. 3.  Rao et al. (2006) Myogenic factors that regulate expression of muscle-specific microRNAs. PNAS, 103: 8721. 34: 267.

Expression of mir-133a and mir-206 down-regulated in type 2 diabetes individuals

Subject characteristics (diabetes only) for miRNA profiling

-10.00

-9.00

-8.00

-7.00

-6.00

-5.00

-4.00

-3.00

-2.004 6 8 10 12 14 16 18

Fasting glucose (mmol/L)

miR

-133

a ex

pres

sion

(ΔC

T)

r = 0.64

Global Clustering All Subjects (n=118)

Affym

etrix Probes

Med

ian

log(

2) in

tens

ity fo

r T2D

sub

ject

s The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

Median log(2) intensity for NGT subjects

4 6 8 10 12 14

4

6

8

10

12

14

NGT T2D

12 10 8 6 4 2 0

NGT IGT DM2

T2D NGT Age 54.8 ± 10.2 51.3 ± 10.7 BMI 31.4 ± 6.2 31.1 ± 7.2 VO2max 26.9 ± 8.4 29.5 ± 10.5 Fasting glucose 9.8 ± 4.4* 5.0 ± 0.4

OXPHOS genes expression in NGT vs DM2

20

21

22

23

24

25

26

27

28

29

30

pri-mir-133a-1

pri-mir-1-2

pri-mir-1-1

pri-mir-133a-2

pri-mir 133a-1

pri-mir 1-2

pri-mir 1-1

pri-mir 133a-2

Cyc

le T

hres

hold

Chrom-18 Chrom-20

Chromosome 20 - C20orf166

Pre-miR 1-1 Pre-miR-133a-2

A1 A2

Chromosome 18 - EST

Pre-miR 133a-1 Pre-miR 1-2

A3 A4

Enr

ichm

ent s

core

(ES

) R

anke

d lis

t met

ric (S

igna

l to

Noi

se)

T2D (positively correlated)

GSEA Mir-1 and mir133a precursors (pri-transcripts) appear distinctly expressed but are not altered by disease

Expression of mir-133a correlates with fasting glucose