Obesity-induced overexpression of miRNA-143 inhibits insulin-stimulated AKT activation and impairs glucose metabolism

Journal name:
Nature Cell Biology
Volume:
13,
Pages:
434–446
Year published:
DOI:
doi:10.1038/ncb2211
Received
Accepted
Published online

Abstract

The contribution of altered post-transcriptional gene silencing to the development of insulin resistance and type 2 diabetes mellitus so far remains elusive. Here, we demonstrate that expression of microRNA (miR)-143 and 145 is upregulated in the liver of genetic and dietary mouse models of obesity. Induced transgenic overexpression of miR-143, but not miR-145, impairs insulin-stimulated AKT activation and glucose homeostasis. Conversely, mice deficient for the miR-143–145 cluster are protected from the development of obesity-associated insulin resistance. Quantitative-mass-spectrometry-based analysis of hepatic protein expression in miR-143-overexpressing mice revealed miR-143-dependent downregulation of oxysterol-binding-protein-related protein (ORP) 8. Reduced ORP8 expression in cultured liver cells impairs the ability of insulin to induce AKT activation, revealing an ORP8-dependent mechanism of AKT regulation. Our experiments provide direct evidence that dysregulated post-transcriptional gene silencing contributes to the development of obesity-induced insulin resistance, and characterize the miR-143–ORP8 pathway as a potential target for the treatment of obesity-associated diabetes.

At a glance

Figures

  1. Dysregulated expression of the miR-143-145 cluster in insulin target tissues of obese and diabetic mice.
    Figure 1: Dysregulated expression of the miR-143–145 cluster in insulin target tissues of obese and diabetic mice.

    (a) Northern-blot analysis of hepatic miR-143 expression in db/db mice (n=5), compared with wild-type controls (n=5) . 5S ribosomal RNA was used as a loading control. (b) Northern-blot analysis of hepatic miR-143 expression in mice fed high-fat diet (HFD; n=5) or normal chow diet (NCD) (n=5). 5S rRNA was used as a loading control. (c) Real-time PCR analysis of hepatic miR-143 expression in db/db mice (n=10), compared with wild-type controls (n=10). (d) Real-time PCR analysis of hepatic miR-143 expression in mice fed high-fat diet (HFD; n=8) or normal chow diet (NCD; n=5 ). (e) Real-time PCR analysis of hepatic miR-145 expression in db/db mice (n=10), compared with wild-type controls (n=10). (f) Real-time PCR analysis of hepatic miR-145 expression in mice fed high-fat diet (HFD; n=8) or normal chow diet (NCD; n=5). (g) Real-time PCR analysis of miR-143 expression in the indicated tissues of db/db mice (skeletal muscle (SM), n=8; heart, n=7; white adipose tissue (WAT), n=7; brown adipose tissue (BAT), n=8 ), compared with wild-type controls (SM, n=7; heart, n=9; WAT, n=9; BAT, n=9 ). (h) Real-time PCR analysis of pancreatic miR-143 expression in db/db mice (n=6), compared with wild-type controls (n=5). (i) Real-time PCR analysis of miR-145 expression in the indicated tissues of db/db mice (SM, n=7; heart, n=6; WAT, n=7; BAT, n=8 ), compared with wild-type controls (SM, n=8; heart, n=7; WAT, n=8; BAT, n=6 ). (j) Real-time PCR analysis of pancreatic miR-145 expression in db/db mice (n=6 , 20 weeks old), compared with wild-type controls (n=5 , 20 weeks old). Expression of miRNAs was normalized to that of control RNAs (northern blot, 5S rRNA; real-time PCR, sno234) and set to unity in wild-type controls. All error bars indicate s.e.m. *P≤0.05,**P≤0.01. Uncropped images of blots are shown in Supplementary Fig. S9.

  2. Conditional overexpression of miR-143 in mice.
    Figure 2: Conditional overexpression of miR-143 in mice.

    (a) Scheme of a single vector configuration for inducible miRNA expression. Recombinase-mediated cassette exchange (RMCE) through Flpe-mediated recombination using the exchange vector generates the Rosa26 (RMCE-exchanged) allele. The exchange vector carries the miR-143 coding region under the control of the H1-tetO promoter, the codon-optimized itetR gene under the control of the CAGGS promoter (chicken β-actin promoter) and a truncated neoR gene for positive selection of clones on successful RMCE. (b) Southern-blot analysis of genomic DNA from embryonic stem cells. In clones 1–4 successful RMCE had occurred. The positions of probe and restriction sites (H=HindIII) are indicated in a. Clone 2 was used for generation of transgenic mice. (c) Schematic representation of transgene for inducible miR-143 overexpression. Expression of transgenic miR-143 relies on the RNA polymerase III (polIII)-dependent H1 promoter, containing the operator sequences (tetO) of the Escherichia coli tetracycline-resistance operon. Binding of the tetracycline-resistance-operon repressor (itetR) to tetO prevents transcription. Doxycycline sequesters itetR and enables the binding of polIII to the H1 promoter, resulting in transcription of the extra miR-143 allele. (d) Northern-blot analysis of mature miR-143 and 5S rRNA (loading control) in liver, skeletal muscle (SM) and white (WAT) and brown adipose tissue (BAT) of miR-143DOX mice and wild-type littermate controls. (e) Quantification of northern-blot analysis shown in d (miR-143DOX mice, n=3; wild-type littermate controls, n=3). Expression of miR-143 in the indicated tissues was normalized to that of 5S rRNA and set to unity in the respective wild-type tissue. All error bars indicate s.e.m. *P≤0.05. (f) Northern-blot analysis of mature miR-143 and 5S rRNA (loading control) in hepatocytes isolated from miR-143DOX mice and wild-type littermate controls. (g) Northern-blot analysis of mature miR-143 and 5S rRNA (loading control) in liver and skeletal muscle (SM) of miR-143DOX mice (T) and wild-type littermate controls (W) without and after 1, 2, 4 and 8 days of doxycycline administration. Uncropped images of blots are shown in Supplementary Fig. S9.

  3. Impaired glucose metabolism in miR-143-overexpressing mice.
    Figure 3: Impaired glucose metabolism in miR-143-overexpressing mice.

    (a) Blood glucose concentrations of miR-143DOX mice (−Doxycycline (Dox), n=22; +Dox, n=11) and wild-type littermate controls (−Dox, n=22, +Dox, n=10 ) before and after doxycycline administration. Concentrations were measured in overnight-fasted mice. (b) Serum insulin levels of miR-143DOX mice (−Dox, n=7; +Dox, n=13) and wild-type littermate controls (−Dox, n=9; +Dox, n=13 ) before and after doxycycline administration. Concentrations were measured in overnight-fasted mice. (c) Glucose-tolerance test of miR-143DOX mice (−Dox, n=15; +Dox, n=13) and wild-type littermate controls (−Dox, n=16; +Dox, n=13) before and after doxycycline administration. (d) Insulin-tolerance test of miR-143DOX mice (−Dox, n=14; +Dox, n=22) and wild-type littermate controls (−Dox, n=15; +Dox, n=19) before and after doxycycline administration. Blood glucose concentrations of miR-143DOX mice and wild-type controls before i.p. administration of insulin were set to 100%. (e) HOMA of miR-143DOX mice (−Dox, n=16; +Dox, n=13) and wild-type littermate controls (−Dox, n=16; +Dox, n=13) before and after doxycycline administration. (f) Plasma insulin concentrations after glucose bolus injection in miR-143DOX mice (n=8) and wild-type littermate controls (n=9). (g) Haematoxylin and eosin (H&E), insulin and glucagon stainings of pancreatic islets in miR-143DOX mice and wild-type littermate controls. Scale bars 100μm. (h) Percentage of β -cell mass in miR-143DOX mice (n=5) and wild-type littermate controls (n=5). All error bars indicate s.e.m. *P≤0.05, **P≤0.01.

  4. Conditional overexpression of LacZ[thinsp]shRNA or miR-145 does not impair glucose homeostasis.
    Figure 4: Conditional overexpression of LacZshRNA or miR-145 does not impair glucose homeostasis.

    (a) Schematic representation of transgene for inducible LacZshRNA expression. (b) Body weight of LacZshRNADOX mice (n=10) and wild-type littermate controls (n=10). (c) Serum insulin levels of LacZshRNADOX mice (n=10) and wild-type littermate controls (n=10). Concentrations were measured in overnight-fasted mice. (d) Glucose-tolerance test of LacZshRNADOX mice (n=10) and wild-type littermate controls (n=10). (e) Insulin-tolerance test of LacZshRNADOX mice (n=10) and wild-type littermate controls (n=10). Blood glucose concentrations of LacZshRNADOX mice and wild-type controls before i.p. administration of insulin were set to 100%. (f) HOMA of LacZshRNADOX mice (n=10) and wild-type littermate controls (n=10). (g) Schematic representation of transgene for inducible miR-145 overexpression. (h) Body weight of miR-145DOX mice (n=6) and wild-type littermate controls (n=7). (i) Serum insulin levels of miR-145DOX mice (n=8) and wild-type littermate controls (n=8). Concentrations were measured in overnight-fasted mice. (j) Glucose-tolerance test of miR-145DOX mice (n=6) and wild-type littermate controls (n=7). (k) Insulin-tolerance test of miR-145DOX mice (n=6) and wild-type littermate controls (n=7). Blood glucose concentrations of miR-145DOX mice and wild-type controls before i.p. administration of insulin were set to 100%. (l) Homeostatic model assessment of miR-145DOX mice (n=8) and wild-type littermate controls (n=8). All error bars indicate s.e.m.

  5. Conditional miR-143 overexpression impairs insulin-stimulated AKT activation in liver.
    Figure 5: Conditional miR-143 overexpression impairs insulin-stimulated AKT activation in liver.

    (a) Representative western-blot analysis and quantification of expression and insulin-stimulated phosphorylation of IR and AKT in the liver of miR-143DOX mice (pIR, n=15; pAKTSer473, n=14; pAKTThr308, n=15 ) and wild-type littermate controls (pIR, n=16; pAKTSer473, n=17, pAKTThr308, n=17). β -actin was used as a loading control. (b) Representative western-blot analysis and quantification of expression and insulin-stimulated phosphorylation of IR and AKT in skeletal muscle of miR-143DOX mice (n=5) and wild-type littermate controls (n=5). α -tubulin was used as a loading control. Mice were injected with either saline (−) or insulin (+). Immunoreactive phospho-proteins were normalized to the total expression of the respective protein and the quotient of wild-type controls was set to unity. All error bars indicate s.e.m. **P≤0.01. Uncropped images of blots are shown in Supplementary Fig. S9.

  6. miR-143-145-deficient mice are protected from diet-induced insulin resistance and hepatic AKT inhibition.
    Figure 6: miR-143–145-deficient mice are protected from diet-induced insulin resistance and hepatic AKT inhibition.

    (a) Glucose-tolerance test of miR-143–145 knockout mice (n=12) and wild-type littermate controls (n=12), on a high-fat diet. (b) Insulin-tolerance test of miR-143–145 knockout mice (n=12) and wild-type littermate controls (n=12) on a high-fat diet. Blood glucose concentrations of miR-143–145 knockout mice and wild-type controls before i.p. administration of insulin were set to 100%. (c) Representative western-blot analysis and quantification of expression and insulin-stimulated phosphorylation of indicated proteins in the liver of miR-143–145 knockout mice (pIR, n=7; pAKT, n=7) and wild-type controls (pIR, n=8; pAKT, n=8 ) on a high-fat diet. Mice were injected with either saline (−) or insulin (+). Immunoreactive phospho-proteins were normalized to the total expression of the respective protein and the quotient of wild-type controls was set to unity. β -actin was used as a loading control. (d) Haematoxylin and eosin (H&E) staining and adipocyte size distribution of epigonadal white-adipose-tissue sections from miR-143–145 knockout mice (n=3) and wild-type controls (n=3) on a high-fat diet. Scale bar 100μm. (e) Mac-2 staining and quantification of white adipose tissue sections from miR-143–145 knockout mice (n=3) and wild-type controls (n=3) on a high-fat diet. Scale bar 100μm. Red arrows indicate cells surrounded by a Mac-2-positive area. (f) Real-time PCR analysis of F4/80, Il-6 and Tnf- α mRNA expression in white adipose tissue of miR-143–145 knockout mice (n=5), compared with wild-type controls (n=5 ), on a high-fat diet. Expression of mRNAs was normalized to Gusb and Hprt mRNA and set to unity in wild-type controls. (g) Representative western-blot analysis of in vitro phosphorylation of c-Jun (p-c-Jun) in liver and skeletal muscle (SM) lysates from miR-143–145 knockout mice (n=4) and wild-type controls (n=4) on a high-fat diet. Immunoreactive phospho-c-Jun was normalized to total JNK input and the quotient of wild-type controls was set to unity. All error bars indicate s.e.m. *P≤0.05, **P≤0.01, ***P≤0.001. Uncropped images of blots are shown in Supplementary Fig. S9.

  7. In vivo SILAC identifies ORP8 as an miR-143 target.
    Figure 7: In vivo SILAC identifies ORP8 as an miR-143 target.

    (a) General scheme of the in vivo SILAC approach. Protein expression in the liver of 13C6-lysine-labelled (SILAC) mice was analysed and compared with doxycycline-treated unlabelled wild-type and miR-143DOX mice, respectively. Differences in protein expression between SILAC and miR-143DOX mice were normalized for the ratio SILAC/unlabelled wild type, thus allowing for indirect comparison of protein expression between miR143DOX mice and wild-type littermate controls. (b) Relative luciferase activity of the indicated reporter constructs. Firefly luciferase activity was normalized to the activity of co-expressed Renilla luciferase. Luciferase activity of the reporter construct, containing the wild-type miR-143 binding sites to ORP8 (open bar), was set to unity. Results represent five independent experiments. All error bars indicate s.e.m. *P≤0.05. (c) Representative western-blot analysis of ORP8 expression in the liver of miR-143DOX mice (n=12) and wild-type littermate controls (n=12). β -actin was used as a loading control. (d) Real-time PCR analysis of Orp8 mRNA expression in the liver of miR-143DOX mice (n=11), compared with wild-type controls (n=14). Expression of mRNAs was normalized to Gusb and Hprt mRNA. (e) Representative western-blot analysis of ORP8 expression in the liver of miR-143–145 knockout mice (n=9) and wild-type littermate controls (n=9). miR-143–145 knockout mice but not miR-143DOX mice were on a high-fat diet. β -actin was used as a loading control. (f) Real-time PCR analysis of Orp8 mRNA expression in the liver of miR-143–145 knockout mice (n=6), compared with wild-type controls (n=6 ), on a high-fat diet. Expression of mRNAs was normalized to Gusb and Hprt mRNA. For western-blot and real-time PCR analysis ORP8 expression in wild-type controls was set to unity. All error bars indicate s.e.m. *P≤0.05, ***P≤0.001. Uncropped images of blots are shown in Supplementary Fig. S9.

  8. Downregulation of ORP8 in cultured liver cells impairs insulin-stimulated AKT activation.
    Figure 8: Downregulation of ORP8 in cultured liver cells impairs insulin-stimulated AKT activation.

    (a) Western-blot analysis of ORP8 expression in the indicated tissues of wild-type C57BL/6 mice. AKT was used as a loading control. (b) Western-blot analysis of ORP8 expression in HepG2 cells transfected with the indicated siRNA oligonucleotides. β -actin was used as a loading control. (c) Western-blot analysis of insulin-stimulated phosphorylation of AKT and GSK3 in HepG2 cells transfected with the indicated siRNA oligonucleotides. β -actin was used as a loading control. Serum-depleted cells were stimulated with either saline (−) or increasing concentrations of insulin (0.1–100nM). (d) Western-blot analysis of ORP8 expression in retroviral-transformed Hepa1–6 cell clones stably expressing the indicated shRNA. β -actin was used as a loading control. (e) Representative western-blot analysis of insulin-stimulated phosphorylation of AKT, GSK3 and FOXO levels in Hepa1–6 cell clones stably expressing the indicated shRNA. β -actin was used as a loading control. Serum-depleted cells were stimulated with either saline (−) or increasing concentrations of insulin (0.1–100nM). (f) Quantification of dose-dependent insulin-stimulated AKT phosphorylation. Protein expression was quantified in six independent control-shRNA- and Orp8-shRNA-expressing Hepa1–6 cell clones. Relative values represent the average of three independent experiments for each clone. (g) Quantification of dose-dependent insulin-stimulated FOXO phosphorylation. Protein expression was quantified in six independent control-shRNA- and Orp8-shRNA-expressing Hepa1–6 cell clones. Relative values represent the average of three independent experiments for each clone. All error bars indicate s.e.m. *P≤0.05,**P≤0.01,***P≤0.001. Uncropped images of blots are shown in Supplementary Fig. S9.

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Author information

Affiliations

  1. Department of Mouse Genetics and Metabolism, Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC) University of Cologne, and Center for Endocrinology, Diabetes and Preventive Medicine (CEDP), University Hospital Cologne, Max Planck Institute for Neurological Research, Zülpicher Straße 47, D-50674 Cologne, Germany

    • Sabine D. Jordan,
    • Diana M. Willmes,
    • Nora Redemann,
    • F. Thomas Wunderlich &
    • Jens C. Brüning
  2. Max Planck Institute for Heart and Lung Research, D-61231 Bad Nauheim, Germany

    • Markus Krüger,
    • Thomas Böttger &
    • Thomas Braun
  3. Phenotyping Facility of the Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), D-50674 Cologne, Germany

    • Hella S. Brönneke
  4. Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, D-50674 Cologne, Germany

    • Carsten Merkwirth
  5. Institute for Medical Microbiology, Immunology and Hygiene, Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, D-50674 Cologne, Germany

    • Hamid Kashkar
  6. Minerva Foundation Institute for Medical Research, Biomedicum, FI-00290 Helsinki, Finland

    • Vesa M. Olkkonen
  7. TaconicArtemis Pharmaceuticals GmbH, D-51063 Cologne, Germany

    • Jost Seibler

Contributions

S.D.J. and J.C.B. designed the research; S.D.J. carried out most of the experiments; M.K. carried out in vivo SILAC analyses; D.M.W. and N.R. provided extra technical assistance; F.T.W. helped to design cloning strategies; H.S.B. analysed energy expenditure in miR-143DOX mice; C.M. carried out luciferase assays; H.K. helped with lentivirus experiments; V.M.O. provided ORP8 antibody and shRNA ORP8 lentiviruses. T. Böttger and T. Braun provided miR143–145 knockout mice; J.S. in part generated miR-143DOX and miR-145DOX mice and provided LacZshRNADOX mice. S.D.J. and J.C.B. wrote the manuscript. All authors participated in the interpretation of the data and production of the final manuscript.

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The authors declare no competing financial interests.

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  1. Supplementary Table 1 (100K)

    Supplementary Information

  2. Supplementary Table 3 (100K)

    Supplementary Information

Additional data