Glucose tolerance is associated with differential expression of microRNAs in skeletal muscle: results from studies of twins with and without type 2 diabetes

Aims/hypothesis We aimed to identify microRNAs (miRNAs) associated with type 2 diabetes and risk of developing the disease in skeletal muscle biopsies from phenotypically well-characterised twins. Methods We measured muscle miRNA levels in monozygotic (MZ) twins discordant for type 2 diabetes using arrays. Further investigations of selected miRNAs included target prediction, pathway analysis, silencing in cells and association analyses in a separate cohort of 164 non-diabetic MZ and dizygotic twins. The effects of elevated glucose and insulin levels on miRNA expression were examined, and the effect of low birthweight (LBW) was studied in rats. Results We identified 20 miRNAs that were downregulated in MZ twins with diabetes compared with their non-diabetic co-twins. Differences for members of the miR-15 family (miR-15b and miR-16) were the most statistically significant, and these miRNAs were predicted to influence insulin signalling. Indeed, miR-15b and miR-16 levels were associated with levels of key insulin signalling proteins, miR-15b was associated with the insulin receptor in non-diabetic twins and knockdown of miR-15b/miR-16 in myocytes changed the levels of insulin signalling proteins. LBW in twins and undernutrition during pregnancy in rats were, in contrast to overt type 2 diabetes, associated with increased expression of miR-15b and/or miR-16. Elevated glucose and insulin suppressed miR-16 expression in vitro. Conclusions Type 2 diabetes is associated with non-genetic downregulation of several miRNAs in skeletal muscle including miR-15b and miR-16, potentially targeting insulin signalling. The paradoxical findings in twins with overt diabetes and twins at increased risk of the disease underscore the complexity of the regulation of muscle insulin signalling in glucose homeostasis. Electronic supplementary material The online version of this article (doi:10.1007/s00125-014-3434-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.


AntagomiR experiment
For antagomiR transfection targeting miR-15b and miR-16, satellite cells were isolated from vastus lateralis muscle biopsies from five healthy donors (three males and two females) aged 59±7 as previously described (1,11). Proliferating myoblasts were seeded into 6-well plates. At day 3 with differentiation media (DMEM, high glucose, 2% horse serum) (Invitrogen), cells were transfected using Lipofectamine 2000 (Invitrogen) with 10 nmol/l antagomiRs (Exiqon, Vedbaek, Denmark) targeting miR-15b/miR-16 or with a non-targeting antagomiR (Exiqon). The simultaneous knockdown of miR-15b and miR-16 was performed to hinder a possible rescue effect that could occur when inhibiting them separately. An untreated sample and a Lipofectamine 2000 only sample were included as additional controls. Cells were incubated with the transfection reagents for 48 hours. Cells were harvested after 5-10 minutes incubation with 0, 1, 10, or 100 nmol/l insulin (Actrapid, Novo Nordisk, Bagsvaerd, Denmark) in Trizol (Invitrogen) for RNA isolation or in lysis buffer (Cell signaling technology, Beverly, MA, USA) for protein isolation.

Rat protocol
Female Wistar rats were bred locally at a designated animal unit of the University of Cambridge (Cambridge, UK). Adult females weighing between 235 and 250 grams were fed ad libitum either a control diet (20% protein) n=11 or an isocaloric low protein (LP) (8% protein) diet n=11 (Arie Blok, Woerden, Netherlands) during gestation and lactation as described previously (28). Three days after birth, litter sizes were randomly standardized to four males and four females. At 21 days of age, male offspring were weaned onto a standard rat diet (LAD1; Special Diet Services, Witham, UK). At three months of age, 22 males, one from each of the eleven control litters and one from each of the eleven LP litters was killed by a rising concentration of carbon dioxide. Vastus lateralis muscle biopsies were excised and snap frozen in liquid nitrogen before storing at -80 o C for later use.

RNA isolation
Total RNA from human muscle biopsies were extracted with TRI reagent (Sigma-Aldrich, St. Louis,MO), from human cell cultures with Trizol (Invitrogen) and from rat muscle biopsies and L6 cells with mirVana total RNA isolation kit (Ambion, Applied Biosystems, Warrington, UK), all according to the manufacturers protocols.

LNA Arrays
For each of the discordant twins (n=22), 1 µg total RNA was labeled with Hy3 using the miRCURY labeling kit according to the manufacturer's instructions and hybridized to a miRCURY LNA miRNA array (Exiqon, Vedbaek, Denmark) representing version 9.2 of the Sanger miRBase using the HS-400-Pro microarray hybridization station (Tecan, Grödig, Austria). The 22 arrays were scanned using the ArrayWoRx white-light CCD-based scanner (Applied Precision, Issaquah, WA) and ImaGene 8.0 (BioDiscovery, El Segundo, CA) was used for image analysis including flagging of bad quality spots. The resulting text-files were normalized using normexp (20) and quantile normalization (21) in R (version 2.9.1). Updated annotation files representing miRBase version 14 were used and the 571 human miRNAs on the array were used in the downstream analysis.

Gene expression
ABI-PRISM 7900 Sequence Detection system (Applied Biosystems, Foster City, CA, USA (ABI)) was used for both miRNA and mRNA detection.
Human muscle biopsies -miRNA quantification; 1 µg of total RNA was reverse transcribed using Megaplex™ primer pools A v2.1. The product was loaded in duplicates and quantitative real-time PCR (qRT-PCR) was performed with TaqMan® miRNA assays for miR-15b, miR-16, miR-185, miR-494 and RNU48. The geometric mean of the three small non-coding reference RNAs (miR-185, miR-494 and RNU48) was used to normalize the expression of miR-15b and miR-16. miR-185 and miR-494 was chosen based on stability calculations (39). The SLqPCR package in R was used to rank miRNAs from population 1 (n=22), and 16 additional skeletal muscle miRNA expression profiles from our laboratory, based on their stability. RNU48 is recommended as an internal control by ABI.
Cell culture -mRNA quantification; 0.5 µg of total RNA was reverse-transcribed using cDNA high capacity kit (ABI) according to the manufacturer's protocol. cDNA samples were loaded in triplicates and qRT-PCR was performed with primers designed using Roche Universal probe library (Roche), INSR forward primer: 5΄-gctggattattgcctcaaagg, INSR reverse primer: 5΄tgagaatcttcagactcgaatgg, PI3KR1 forward primer: 5΄-ggaatgttcaactctatacagaacaca, PI3KR1 reverse primer: 5΄agtccacggagggtgtatca. 18S rRNA was used as endogenous control (ABI). The comparative delta CT method was used to calculate relative abundance of miRNA or mRNA levels between samples.
Rat muscle biopsies and L6 cells; TaqMan® individual miRNA assays for miR-15b, miR-16 and miR-185 were used as well as assays for Peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1 alpha (PGC-1a) and Cytochrome c, somatic (CYTC). The expression of mir-15b and mir-16 were then normalized to the geometric mean of the housekeeping genes miR-185, PGC-1a and CYTS (none of which were influenced by maternal diet, insulin or glucose).

Muscle cell cultures -Meso-Scale discovery
Cell samples were harvested in protein lysis buffer (Cell signaling technology, Danvers, MA, USA). Phosphatase inhibitor cocktail 1 and 2 (Sigma Aldrich) and protease inhibitor complete mini (Roche) was added fresh to the buffer. Protein lysates were centrifuged at maximum speed for 1 h at 4°C, and the pellet was discarded. Protein concentration was measured using a colorimetric protein assay (Bio-Rad, Laboratories, Richmond, CA, USA.). INSR levels were measured according to the