MiR-155 Enhances Insulin Sensitivity by Coordinated Regulation of Multiple Genes in Mice

<div><p>miR-155 plays critical roles in numerous physiological and pathological processes, however, its function in the regulation of blood glucose homeostasis and insulin sensitivity and underlying mechanisms remain unknown. Here, we reveal that miR-155 levels are downregulated in serum from type 2 diabetes (T2D) patients, suggesting that miR-155 might be involved in blood glucose control and diabetes. Gain-of-function and loss-of-function studies in mice demonstrate that miR-155 has no effects on the pancreatic β-cell proliferation and function. Global transgenic overexpression of miR-155 in mice leads to hypoglycaemia, improved glucose tolerance and insulin sensitivity. Conversely, miR-155 deficiency in mice causes hyperglycemia, impaired glucose tolerance and insulin resistance. In addition, consistent with a positive regulatory role of miR-155 in glucose metabolism, miR-155 positively modulates glucose uptake in all cell types examined, while mice overexpressing miR-155 transgene show enhanced glycolysis, and insulin-stimulated AKT and IRS-1 phosphorylation in liver, adipose tissue or skeletal muscle. Furthermore, we reveal these aforementioned phenomena occur, at least partially, through miR-155-mediated repression of important negative regulators (i.e. C/EBPβ, HDAC4 and SOCS1) of insulin signaling. Taken together, these findings demonstrate, for the first time, that miR-155 is a positive regulator of insulin sensitivity with potential applications for diabetes treatment.</p></div

A proposed model on the positive roles of miR-155 in glucose metabolism by coordinated regulation of multiple genes.

<p>A proposed model on the positive roles of miR-155 in glucose metabolism by coordinated regulation of multiple genes.</p

Regulation of gene expression and insulin signalling by miR-155.

<p><b>(A)</b> qRT-PCR analysis of the indicated gene expression in liver, WAT, BAT and SM of RL-m155 mice (4m). n = 5–10 mice per indicated genes. Values are statistically significant at *<i>P</i><0.05; **<i>P</i><0.01 and ^#<i>P</i><0.001. <b>(B)</b> Western blot analysis of the expression of the indicated proteins in liver, adipose tissue and SM of RL-m155 mice (4m; n = 4). <b>(C)</b> Western blot analysis for SOCS1, SOCS3 and HDAC4 expression in miR-155-expressing 7402 cells, 7402 cells transfected with miR-155 inhibitor, and hepa1-6 cells transfected with miR-155 mimics or miR-155 inhibitor. <b>(D)</b> Representative Western-blot analysis of insulin-stimulated AKT and IRS-1 (Insulin receptor substrate 1) phosphorylation in liver, adipose tissue or SM of control and RL-m155 mice (5m). <b>(E)</b> Representative immunoblot analysis of insulin-stimulated AKT phosphorylation versus total AKT protein levels in hepa1-6 cells transfected with miR-155 mimics or miR-155 inhibitor. <b>(F)</b> In vitro evaluation of cellular ¹⁸F-FDG uptake in miR-155-expressing 7402 cells, and hepa1-6 and C2C12 cells transiently transfected with miR-155 mimics by MicroPET/CT. The data on the fold changes in ¹⁸F-FDG uptake between miR-155-expressing indicated cells and the corresponding control cells were shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006308#pgen.1006308.s009" target="_blank">S9 Fig</a>. UC: untransfected cells.</p

Improved glucose tolerance and insulin sensitivity in RL-m155 mice.

<p><b>(A-C)</b> Blood glucose concentrations in fed-state (A), 12-hour–fasted (B) and 24-hour–fasted (C) mice. Control mice: n = 9 (3m) and n = 8 (5m); RL-m155 mice: n = 7 (3m) and n = 9 (5m). <b>(D)</b> Serum insulin concentrations in fed-state and 12-hour–fasted mice (5m). <b>(E-F)</b> Glucose tolerance test (GTT) in 12-hour–fasted mice (E) and area under the curve (AUC) (F) for this GTT (E). <b>(G)</b> Serum insulin measurements performed in 12-hour–fasted mice (3m) during a GTT (E). <b>(H)</b> GTT in 12-hour–fasted control (Con) and RL-m155 mice. <b>(I)</b> AUC analysis for this GTT (H). <b>(J)</b> Insulin tolerance test (ITT) of 12-hour–fasted control and RL-m155 mice. <b>(K)</b> AUC analysis for this ITT (J). <b>(L)</b> ITT performed on 12-hour–fasted 5-month-old control and RL-m155 mice. <b>(M)</b> AUC calculated from mice in (L). Values are statistically significant at *<i>P</i><0.05; **<i>P</i><0.01 and ^#<i>P</i><0.001.</p

RL-m155 mice and miR-155 knockout (KO) mice displayed the unaltered β-cell proliferation and hormone profiles in pancreas.

<p><b>(A)</b> Morphology of the entire pancreas from RL-m155 mice and miR-155 KO mice. <b>(B)</b> Haematoxylin and eosin (H&E), insulin and glucagon stainings of pancreatic islets in RL-m155 mice and miR-155 KO mice. <b>(C)</b> qRT-PCR analysis of Ins1 and Ins2 mRNA expression in pancreas tissue of RL-m155 mice and miR-155 KO mice. <b>(D)</b> Percentage of β-cell mass in RL-m155 mice and miR-155 KO mice. <b>(E)</b> BrdU and Ki67 stainings of pancreatic islets in RL-m155 mice and miR-155 KO mice. <b>(F)</b> Percentage of Ki67-positive cells in pancreatic islets in RL-m155 mice and miR-155 KO mice. In this study, the non-transgenic littermates/wild-type littermates [i.e., control (con) mice] were used as controls of RL-m155 mice, while wild-type C57BL/6J mice (i.e., WT) of the same age and sex were used as controls of miR-155^–/–C57BL/6J mice.</p

Downregulation of C/EBPβ, HDAC4 and PDK4 in cultured liver cells enhanced insulin-stimulated AKT activation and cellular ¹⁸F-FDG uptake.

<p><b>(A)</b> Sequence alignment of 3’-UTR of human (Hsa), mouse (Mmu), rhesus (Mml) and rat (Rno) C/EBPβ highlighting miR-155 binding site. <b>(B)</b> Both C/EBPβ and HDAC4 are target genes of miR-155. The luciferase reporter assay was performed using Hepa1-6 cells as described in the Materials and methods section. Values are statistically significant at ^#<i>P</i><0.001. <b>(C)</b> Immunoblot analysis of C/EBPβ, HDAC4 and PDK4 expression in livers of RL-m155 mice and miR-155 ^-/- mice. <b>(D)</b> Western blot analysis for C/EBPβ, HDAC4 and PDK4 expression in miR-155-expressing 7402 cells, 7402 cells transfected with miR-155 inhibitor, and hepa1-6 cells transfected with miR-155 mimics or miR-155 inhibitor. <b>(E)</b> Cell extracts from hepa1-6 cells transfected with the indicated siRNA oligonucleotides were analyzed by immunoblotting with antibodies against the indicated proteins. <b>(F)</b> Western blot analysis of insulin-stimulated phosphorylation of AKT in hepa1-6 cells transfected with the indicated siRNA oligonucleotides. <b>(G)</b> In vitro evaluation of cellular ¹⁸F-FDG uptake in hepa1-6 cells transfected with the indicated siRNA oligonucleotides by MicroPET/CT. The data on the fold changes in ¹⁸F-FDG uptake between siRNA-transfected hepa1-6 cells and the corresponding control cells were shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006308#pgen.1006308.s010" target="_blank">S10 Fig</a>. UC: untransfected cells</p

Dysregulated miR-155 levels in serum from T2D patients.

<p><b>(A-B)</b> Basal levels of miR-107(A), miR-155 (B) and miR-146a (B) in healthy subjects (HS) (n = 30) and T2D patients (n = 30) detected by qRT-PCR. Values are statistically significant at **<i>P</i><0.01. <b>(C-D)</b> miR155 levels and homeostasis model indicators (HOMA-IR and HOMA-β). HOMA-IR: homeostasis model assessment of insulin resistance; HOMA-β: homeostasis model assessment of β-cell function.</p