miR-876-3p regulates glucose homeostasis and insulin sensitivity by targeting adiponectin

in Journal of Endocrinology
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miRNA has been known to regulate diverse cellular and molecular functions. In the earlier study, we have reported that adipocytes differentiated from human mesenchymal stem cells (hMSC) on 72-h chronic insulin (CI) treatment exhibit insulin resistance (IR). Present study has further explored above model to investigate the role of early expressed miRNAs within human adipocytes to modulate differential adipokine expression as observed during IR. Our results highlight that miR-876-3p regulate glucose homeostasis and its dysregulation leads to IR. We found that miR-876-3p level is a critical determinant of adiponectin expression by virtue of its target within adiponectin 3′UTR. Regulatory effect of miR-876-3p impacts crosstalk between adiponectin and insulin signaling. Rosiglitazone treatment in CI-induced IR adipocytes drastically reduced miR-876-3p expression and increased adiponectin level. In line with this, lentiviral-mediated inhibition of miR-876-3p expression ameliorated CI and high-fat diet (HFD)-induced IR in adipocytes differentiated from hMSC and C57BL/6 mice, respectively. Our findings thus suggest that modulating miR-876-3p expression could provide novel opportunities for therapeutic intervention of obesity-associated metabolic syndrome.

Downloadable materials

  • Supplementary information
  • Supplementary Table 1: 72 hours RNA-seq read data statistics. Each control and CI condition samples of 72 hours having three biological replicates. All replicates read data were quality assessment and control statistics given below in table.
  • Supplementary Table 2: 72 hours small RNA-seq read data statistics. Quality assessment and filtering reads statistic result of 72 hours small RNA-Seq data of adipose tissue having biological replicates.
  • File 1: Adipokine array coordinates and corresponding proteins.
  • File 2: Gene enrichment of validated miRNA target genes having significant Gene ontology tested by hyper-geometric test at 5% and bonferroni p-value adjustment. It included biological process and molecular function.
  • File 3: miRNA and mRNA primer list.
  • File 4: Target genes of miR-876-3p
  • Supplementary Figure 5
  • Supplementary Material 1
  • Supplementary Material 2
  • Supplementary Material 3
  • Supplementary Material 4

 

      Society for Endocrinology

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    Transcriptome analysis of control and CI-induced IR adipocytes. Western blot analysis of proteins involved in insulin signaling in control and CI-treated adipocytes, n = 3 (A). Schematic diagram representing experimental design of differential miRNA and mRNA profiling (B). Adipocytes differentiated from hMSC (five different liposuction samples BMI <30) were given CI treatment for 72 h and protein was isolated with/without insulin stimulation. Proteins were isolated and subjected to Western blot analysis for pAKT (Ser473), pAS160 (Thr642), AKT and AS160 protein, n = 3. (C). MA plots and Heat map of differentially expressed mRNA and miRNA in control and CI-induced IR adipocytes (D and E). A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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    Identification of reciprocal relationships between miRNAs and targets genes. qPCR validation of ten differentially expressed miRNAs from control and CI condition (A). Expression heatmap of ten validated miRNAs whose anti-correlation with target gene is significant (PCC <−0.6) (B). Significant pathway bar chart of miRNA target genes (C). LogFC value difference association between miRNAs and target genes. Red colored bar for target gene and blue colored bar for miRNAs (D). A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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    CI-induced IR adipocytes show differential adipokine profiling: miR-876-3p targets adiponectin. Supernatant of control and CI-induced IR adipocytes were collected and different adipokines were analyzed using adipokine array kit. The dot blot of different adipokines in supernatant of control and CI-induced IR adipocytes, n = 2 (A). Densitometry analysis of dot blots of fig A, error bars represent s.d., *P < 0.05, **P < 0.01, ***P < 0.001 as tested by Student t-test (B). Quantitative gene expression analysis of PGC1α, CPT1A in control and CI-induced IR adipocytes. n = 3, error bars represent s.d., **P < 0.01 and ***P < 0.001 as tested by one-way ANOVA and Bonferroni post-test analysis (C). A case of miR-876-3p binding model to ADIPOQ in 3′UTR (D). HEK293T cells were transduced with Lenti-miR-876-3p and incubated overnight. miR-876-3p overexpressing cells were further transduced with reporter plasmid contain ADIPOQ 3′ UTR with either miR-876-3p target binding site or without target binding site. After 2 days of incubation luciferase activity was measured in luminometer. Renilla luciferase activity was used for normalization and readings are presented in fold difference. n = 3, error bars represent s.d., **P > 0.01 as tested by Student t-test (E). A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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    A network view of miR-876-3p target genes. Out of 300 target genes of miR-876-3p, more than 50% were found interacting with each other. Majority of which belonged to a single cluster centered around three hub genes: MAP2K2, CAD and EP300. Most of these genes, including the hub genes, have been reported to be associated with adipocytes, muscles, insulin response, energy and glucose metabolism related process. A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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    Overexpression of miR-876-3p decreased adiponectin expression and caused insulin resistance in human adipocytes. Adipocytes transduced with either Lenti-EV or Lenti-miR876-3p. Images were taken after 48 h of transduction at 10× magnification with Leica DFC 450C (A). qPCR of miR-876-3p in adipocytes transduced with either Lenti-EV or Lenti-876-3p. n = 3, error bars represent s.d., ***P < 0.001 as tested by Student t-test (B). qPCR analysis of ADIPOQ in adipocytes transduced with either Lenti-EV or Lenti-miR876-3p. RNA was isolated after 48 h of transduction. n = 3, error bars represents s.d., ***P < 0.001 as tested by Student’s t-test (C). qPCR analysis of genes involved in inflammation and beta-oxidation. n = 3, error bars represent s.d., *P < 0.05, ***P < 0.001 as tested by one-way ANOVA and Bonferroni post-test analysis (D). Adiponectin was measured in the supernatant of Lenti-EV or Lenti-miR-876-3p transduced adipocytes at 24 h interval using ELISA. The concentration of adiponectin is measured in pg/mL of supernatant. n = 3, error bars represent s.d. **P < 0.01 as tested by Student t-test (E). Protein isolated from adipocytes transduced with either Lenti-EV or Lenti-miR-876-3p was subjected to western blot analysis. Following antibodies were probed pACC (ser79), pAMPK (Thr172), pATF2 and pP38 (Thr180/Tyr182). The densitometry of the blots normalized with either respective protein or actin is given adjacent to the blots. n = 3, error bars represent s.d., **P < 0.01 and ***P < 0.001 as tested by Student t-test (F). Fully mature adipocytes transduced with either Lenti-EV or Lenti-miR-876-3p. Protein was isolated after stimulation with insulin (10 nm) for 20 min. Isolated protein was subjected to western blot analysis and probed against pAKT (Ser473), pAKT (Thr308), pAS160 (Thr642) and their respective proteins. Densitometry analysis is shown adjacent to the blots normalized with respective proteins. n = 3, error bars represent s.d., *P < 0.05 and **P < 0.01 as tested by Student’s t-test (G). Adipocytes transduced with either Lenti-EV or Lenti-miR-876-3p were stimulated with insulin (10 nm) for 20 min and glucose uptake was measured. Data is represented as fold difference. Glucose uptake readings were normalized with respective protein concentration. n = 3, error bar represents s.d., *P < 0.05 and **P < 0.01 as compared by unpaired Student’s t-test (H). A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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    Inhibition of miR-876-3p in CI-induced IR adipocytes increased adiponectin level and restored insulin signaling. CI-treated adipocytes were either transduced with Lenti-con inhibitor or Lenti-miR876-3p inhibitor and the images were taken after 48 h of transduction at 10× magnification with Leica DFC 450 (A). Quantitative gene expression analysis of miR-876-3p in control and CI adipocytes transduced with either Lenti-Con inhibitor or Lenti-876-3p inhibitor. n = 3, error bars represent s.d., ***P < 0.001 as tested by one-way ANOVA followed by Bonfferoni post-test analysis (B). Quantitative gene expression analysis of ADIPOQ gene in control and CI-treated adipocytes transduced with either Lenti-Con inhibitor or Lenti-miR-876-3p inhibitor. n = 3, error bars represents s.d., ***P < 0.001 as tested by Student’s t-test (C). qPCR analysis of genes involved in inflammation and beta-oxidation in control and CI-treated adipocytes transduced with either Lenti-con inhibitor or Lenti-miR-876-3p inhibitor. n = 3, error bars represent s.d., *P < 0.05, ***P < 0.001 as tested by one-way ANOVA and Bonferroni post-test analysis (D). Control and CI-treated adipocytes transduced with either Lenti-Con inhibitor or Lenti-miR-876-3p inhibitor were grown in six-well plate. Supernatant was collected at 24 h interval and adiponectin level was measured using ELISA. The concentration of adiponectin is measured in pg/mL of supernatant. n = 3, error bars represent s.d. **P < 0.01 as tested by Student’s t-test (E). Protein isolated from control and CI-treated adipocytes transduced with either Lenti-Con inhibitor or Lenti-miR-876-3p inhibitor was subjected to western blot analysis. Following antibodies were probed pACC (ser79), pAMPK (Thr172), pATF2 and pP38 (Thr180/Tyr182). The densitometry of the blots normalized with either respective protein or actin is given adjacent to the blots. n = 3, error bars represent s.d., **P < 0.01 and ***P < 0.001 as tested by Student’s t-test (F). Control and CI-treated adipocytes were transduced with either Lenti-Con inhibitor or Lenti-miR876-3p inhibitor. Protein was isolated after stimulation with insulin (10 nm). Isolated protein was subjected to Western blot analysis and probed against pAKT (Ser473), pAKT (Thr308), pAS160 (Thr642) and their respective proteins. Densitometry analysis is shown adjacent to the blots normalized with respective proteins. n = 3, error bars represent s.d., *P < 0.05 and **P < 0.01 as tested by Student’s t-test (G). Glucose uptake of control and CI-treated adipocytes transduced with either Lent-Con inhibitor or Lenti-miR-876-3p inhibitor. Insulin (10 nM) stimulation was given. Data are represented as fold difference. Glucose uptake readings were normalized with respective protein concentration. n = 3, error bar represent s.d., ***P < 0.01 as compared by one-way ANOVA and Bonferroni post-test analysis (H). A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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    miR-876-3p inhibition reverts insulin resistance by regulating the action of adiponectin. siRNA-mediated adiponectin knockdown CI-induced IR adipocytes were transfected with either Lenti-Con inhibitor or Lenti-miR-876-3p inhibitor and 10 nM insulin pulsing was given for 20 min. Isolated protein was subjected to Western blot analysis and probed against adiponectin, pAKT (Ser473), pAS160 (Thr642) and their respective proteins. Densitometry analysis is shown adjacent to the blots normalized with respective proteins or actin. n = 3, error bars represent s.d., *P < 0.05 and **P < 0.01 as compared by one-way ANOVA and Bonferroni post-test analysis (A). Mitochondrial respiration study of adipocyte transduced with either Lenti-EV or Lenti-miR-876-3p. OCR at basal level and in the presence of ATP synthase inhibitor (1 µM oligomycin), proton uncoupler (1 µM FCCP) and electron transport chain inhibitors (0.5 µM Rotenone/Antimycin mix) was measured with Seahorse Bioscience XFp Extracellular Flux analyzer. n = 3, s.d. represent standard deviation, **P < 0.01 and ***P < 0.001 as tested by Student t-test (B). Mitochondrial respiration study of CI-induced IR adipocytes transduced with either Lenti-Con inhibitor or Lenti-miR-876-3p inhibitor. OCR was measured similar to above experiment. n = 3, s.d. represent standard deviation, **P < 0.01 and ***P < 0.001 as tested by Student t-test (C). Adiponectin level was measured in supernatant of control adipocytes transduced with either Lenti-EV or Lenti-miR-876-3p with or without 24 h rosiglitazone treatment. n = 3, error bars represent s.d., ***P < 0.001 as tested by one-way ANOVA Bonferroni post-test analysis (D). Adiponectin level was measured in supernatant of CI-induced IR adipocytes transduced with either Lenti-Con inhibitor or Lenti-miR-876-3p inhibitor. Rosiglitazone treatment was given as shown in figure for 24 h. n = 3, error bars represent s.d., **P < 0.01, ***P < 0.001 as tested by one-way ANOVA Bonferroni post-test analysis (E). Quantitative gene expression analysis of miR-876-3p in con and CI-treated adipocytes after 24 h rosiglitazone (1 μM) as shown in figure. n = 3, error bars represent s.d., ***P < 0.001 as tested by one-way ANOVA and Bonferroni post-test analysis (F). A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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    Inhibition of miR-876-3p lead to improved insulin sensitivity in 4-week HFD-fed mice. Quantitative gene expression analysis of miR-876-3p in eWAT of chow and HFD mice, n = 6, error bars represent s.d., ***P < 0.001 as tested by Student’s t-test (A). qPCR analysis of ADIPOQ in eWAT of chow and HFD mice. n = 6, error bars represent s.d., ***P < 0.001 as tested by Student’s t-test (B). Serum adiponectin level of chow and HFD mice represented in ng/mL. n = 6, error bars represent s.e.m., ***P < 0.001 as tested by Student’s t-test (C). Schematic diagram of representing Lenti-miR-876 inhibition experiment in 4-week HFD-fed mice (D). Quantitative gene expression analysis of miR-876-3p in eWAT of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lenti-miR-876-3p inhibitor. n = 5, error bars represent s.d., **P < 0.01 as tested by one-way ANOVA and Bonferroni post-test analysis (E). Quantitative gene expression analysis of adipoQ in eWAT of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lenti-miR-876-3p inhibitor, n = 5, error bars represent s.d. **P < 0.01 and ***P < 0.001 as tested by one-way ANOVA followed by Bonferroni post-test analysis (F). Serum adiponectin level of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lenti-miR-876-3p inhibitor. n = 5, error bars represent s.e.m., **P < 0.01 as tested by one-way ANOVA followed by Bonferroni post-test analysis (G). Body weight in percentage of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lent-miR-876-3p inhibitor. n = 5 (H). Weight of eWAT, iBAT, liver, kidney of chow and HFD-fed mice injected with Lenti-con inhibitor or Lenti-miR-876-3p inhibitor (I). Fasting blood glucose level of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lenti-miR-876-3p inhibitor, n = 5, error bars represent s.e.m., *P < 0.05 and ***P < 0.001 as tested by one-way ANOVA followed by Bonferroni post-test analysis (J). IPGTT and its area under curve (AUC) of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lenti-876-3p inhibitor. n = 5, error bars represent s.e.m., **P < 0.01 and ***P < 0.001 as tested by one-way ANOVA and Bonferroni post-test analysis (K and L). ITT and it AUC of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lenti-miR-876-3p inhibitor. n = 5, error bars represent s.e.m., *P < 0.01 and **P < 0.001 as tested by one-way ANOVA and followed by Bonferroni post-test analysis (M and N). Protein isolated after insulin pulsing from eWAT of chow and HFD-fed mice injected with either Lenti-con inhibitor or Lenti-miR-876-3p inhibitor were subjected to western blot analysis. Following proteins were probed to check insulin and adiponectin signaling as shown in figure, n = 3 (O). A full colour version of this figure is available at https://doi.org/10.1530/JOE-17-0387.

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