miR-146a regulates insulin sensitivity via NPR3

The pathogenesis of obesity-related metabolic diseases has been linked to the inflammation of white adipose tissue (WAT), but the molecular interconnections are still not fully understood. MiR-146a controls inflammatory processes by suppressing pro-inflammatory signaling pathways. The aim of this study was to characterize the role of miR-146a in obesity and insulin resistance. MiR-146a−/− mice were subjected to a high-fat diet followed by metabolic tests and WAT transcriptomics. Gain- and loss-of-function studies were performed using human Simpson–Golabi–Behmel syndrome (SGBS) adipocytes. Compared to controls, miR-146a−/− mice gained significantly more body weight on a high-fat diet with increased fat mass and adipocyte hypertrophy. This was accompanied by exacerbated liver steatosis, insulin resistance, and glucose intolerance. Likewise, adipocytes transfected with an inhibitor of miR-146a displayed a decrease in insulin-stimulated glucose uptake, while transfecting miR-146a mimics caused the opposite effect. Natriuretic peptide receptor 3 (NPR3) was identified as a direct target gene of miR-146a in adipocytes and CRISPR/Cas9-mediated knockout of NPR3 increased insulin-stimulated glucose uptake and enhanced de novo lipogenesis. In summary, miR-146a regulates systemic and adipocyte insulin sensitivity via downregulation of NPR3. Electronic supplementary material The online version of this article (10.1007/s00018-020-03699-1) contains supplementary material, which is available to authorized users.


Figure S1 |
Figure S1 | Basal metabolic characteristics of miR-146a -/-mice.Female miR-146a -/-(KO) and respective control mice (WT) were studied at an age of 10 weeks.(A) Body weight, (B) fasted blood glucose, (C) fasted plasma insulin concentrations, and (D) HOMA-IR.(E) To assess insulin sensitivity, mice were intraperitoneally injected with 0.75 IU insulin per kg body weight and blood glucose was monitored for 120 min (ITT).Area under the curve (AUC) of ITT.(F) To assess glucose tolerance, mice were gavaged with 2.5 g glucose per kg body weight and blood glucose was monitored for 120 min (OGTT).AUC of OGTT.Data are displayed as mean and SEM of 5 animals per group.Statistics: (A-D, E and F AUCs) unpaired t-test, (E and F curves) two-way ANOVA with Bonferroni correction.* p<0.05, **** p<0.0001.

Figure S2 |
Figure S2 | Body weight development.Female miR-146a -/-(KO) and respective control mice (WT) at an age of 10 weeks were fed a high fat diet (HFD) or normal diet (ND).Body weight was measured twice a week.Data are displayed as mean and SEM of 10 animals per group.Statistics: two-way ANOVA with Bonferroni correction.* p<0.05, * p<0.01, * p<0.001, * p<0.0001.

Figure S3 |
Figure S3 | Inguinal fat pad histology.After 10 weeks of high fat diet (HFD) or respective normal diet (ND), inguinal white adipose tissue (iWAT) of miR-146a -/-(KO) or respective control mice (WT) was collected and processed for histological analysis.Shown are representative microphotographs of H&E stained iWAT sections.

Figure S4 |
Figure S4 | Metabolic characterisation of miR-146a -/-mice after 5 weeks on a high fat diet.Female miR-146a -/-(KO) and respective control mice (WT) were metabolically characterized at the age of 15 weeks, after 5 weeks high fat (HFD) or respective normal diet (ND).(A and B) Insulin tolerance test (ITT) and oral glucose tolerance test (OGTT) in mice fed a ND.(C and D) ITT and OGTT in mice fed an HFD.(E) Area under the curve (AUC) of ITT.(F) AUC of OGTT.Data are displayed as mean and SEM of 10 animals per group.Statistics: (A-D) two-way ANOVA with Bonferroni correction.(E and F) one-way ANOVA with Tukey correction.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure S8 |
Figure S8 | IRAK1 is regulated by miR-146a in SGBS adipocytes but does not regulate insulin stimulated glucose uptake (A) Representative IRAK1 and TRAF6 Western blot of SGBS adipocytes overexpressing miR-146a with densitometric analysis of 3 independent performed experiments displayed as mean and SEM.SGBS adipocytes were transfected with 20 nM control (Ctrl) or IRAK1 siRNA and (B) IRAK1 expression was controlled by qPCR and (C) glucose uptake experiments were performed with 0 nM and 1 nM insulin.Data are displayed as mean and SEM fold increase to Ctrl siRNA 0 nM insulin.Statistics: (A,B) paired t-test, * p<0.05; (C) one-way ANOVA with Tukey correction, ns = not significant, * p<0.05, ** p<0.01.

Figure
Figure S11 | miR-146a and NPR3 mRNA levels of NPR3 KO cells after NT and control (NT) and miR-146a mimic transfection.Control (EV) or NPR3 KO cells were transfected with 20 nM non-targeting (NT) or miR-146a mimic and (A) miR-146a expression and (B) NPR3 mRNA expression was quantified by qPCR in mature adipocytes.Data are displayed as mean and SEM of 3 independent experiments with sno68 or HPRT as reference gene.Statistics: one-way ANOVA with Tukey correction, ns= not significant, * p<0.05, ** p<0.01.