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. 2019 Jan 24;10(1):421.
doi: 10.1038/s41467-019-08287-x.

TAZ couples Hippo/Wnt signalling and insulin sensitivity through Irs1 expression

Jun-Ha Hwang  1 , A Rum Kim  1 , Kyung Min Kim  1 , Jung Il Park  1 , Ho Taek Oh  1 , Sung A Moon  1 , Mi Ran Byun  1 , Hana Jeong  2 , Hyo Kyung Kim  2 , Michael B Yaffe  3 , Eun Sook Hwang  4 , Jeong-Ho Hong  5
Affiliations

TAZ couples Hippo/Wnt signalling and insulin sensitivity through Irs1 expression

Jun-Ha Hwang et al. Nat Commun. .

Abstract

Insulin regulates blood glucose levels by binding its receptor and stimulating downstream proteins through the insulin receptor substrate (IRS). Impaired insulin signalling leads to metabolic syndrome, but the regulation of this process is not well understood. Here, we describe a novel insulin signalling regulatory pathway involving TAZ. TAZ upregulates IRS1 and stimulates Akt- and Glut4-mediated glucose uptake in muscle cells. Muscle-specific TAZ-knockout mice shows significantly decreased Irs1 expression and insulin sensitivity. Furthermore, TAZ is required for Wnt signalling-induced Irs1 expression, as observed by decreased Irs1 expression and insulin sensitivity in muscle-specific APC- and TAZ-double-knockout mice. TAZ physically interacts with c-Jun and Tead4 to induce Irs1 transcription. Finally, statin administration decreases TAZ, IRS1 level and insulin sensitivity. However, in myoblasts, the statin-mediated decrease in insulin sensitivity is counteracted by the expression of a constitutively active TAZ mutant. These results suggest that TAZ is a novel insulin signalling activator that increases insulin sensitivity and couples Hippo/Wnt signalling and insulin sensitivity.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
TAZ stimulates Irs1 expression and insulin signalling. a Insulin was intraperitoneally injected into wild-type (WT) and muscle-specific TAZ-knockout (mKO) mice. After 15 min, muscle lysates were analysed by immunoblotting. Eight- to ten-week-old mice were used for experiment. b The three independent experiments shown in a were assessed, and the ratio of phosphorylated to total AKT was analysed, as well as the protein levels of ribosomal protein S6 kinase and AS160. c Serum-starved WT and TAZ-knockout (KO) mouse embryonic fibroblasts (MEFs) were treated with 1 nM insulin. Cell lysates were analysed by immunoblotting at the indicated time points. β-Actin was used as a loading control. d Serum-starved control (Con) and TAZ-knockdown (Ti) C2C12 myoblasts were treated with 1 nM insulin. Cell lysates prepared at the indicated time points were analysed by immunoblot analysis. β-Actin was used as a loading control. e Irs1 transcription was analysed by quantitative reverse transcription (qRT)-PCR in skeletal muscle from WT and mKO mice (n = 7, left), WT and TAZ-KO MEFs (n = 3, middle), Con and Ti C2C12 cells (n = 3, right). For skeletal muscle data, 8–10-week-old mice were used. Data are presented as mean ± SD for panels b and e (middle and right), and mean ± SEM for panel e (left). Statistical analysis was performed using a Student’s t-test. *p < 0.05; ***p < 0.005
Fig. 2
Fig. 2
TAZ stimulates glucose uptake and increases insulin sensitivity. a Glucose uptake was analysed in control (Con) and TAZ-knockdown (Ti) C2C12 myotubes (n = 3). b Con and Ti C2C12 myotubes were treated with 100 nM insulin. After 30 min, the plasma membrane (PM) and cytosol (Cyt) were fractionated and analysed by immunoblotting using Glut4 antibodies. The insulin receptor β-subunit and α-tubulin were used as PM and Cyt loading controls, respectively. c For the glucose tolerance test, WT and mKO mice were fasted for 16 h, and d-glucose was injected intraperitoneally. Blood glucose was measured at the indicated time points (n = 6, left). d For the insulin tolerance test, WT and mKO mice were fasted for 4 h, and insulin was administered intraperitoneally. Blood glucose levels were measured at the indicated time points (n = 7, right). e WT and mKO mice were fasted for 16 h or fed ad libitum, and serum insulin concentrations were analysed (n = 6). For c–e, 8–10-week-old mice were used. Data are presented as mean ± SD for panel a, and mean ± SEM for panels c–e. Statistical analysis was performed using a Student’s t-test. *p < 0.05; **p < 0.01
Fig. 3
Fig. 3
TAZ stimulates Irs1 transcription with c-Jun and Tead4. a Chromatin immunoprecipitation (ChIP) sequencing analysis was assessed in FLAG-tagged TAZ (F-TAZ)-overexpressing or control C2C12 myoblasts. Sites of F-TAZ binding were visualised in the mouse genome. The primary target site for TAZ-binding (TAZ-binding element 1, TBE1) is marked with an asterisk. The histone modification status (H3K27ac, H3K4 me3, and H3K4 me1) was obtained from raw ChIP sequencing read data downloaded from the NCBI Sequence Read Archive (SRP009088). b Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) was performed to validate the TAZ-binding element 1 (TBE1) site. ChIP was performed in control and F-TAZ-overexpressing C2C12 cells. ChIPed DNA was analysed by qPCR with primer sets spanning the TBE1 site (n = 3). c Scrambled or two different siRNAs for both c-Jun and Tead4 were co-transfected into C2C12 cells. Transfected cells were analysed by qRT-PCR for Irs1 transcripts 48 h after transfection (n = 3). d Depleting c-Jun and Tead4 decreased IRS1 level. C2C12 cells were treated with two different siRNAs for c-Jun and Tead4 and lysates were analysed by immunoblotting. e TAZ and c-Jun stimulate a TBE1-containing luciferase reporter gene. Approximately 500 bp of intronic elements surrounding TBE1 were cloned into the pGL3-basic luciferase vector (TBE1-luc), and the completed plasmid was transfected into 293T cells together with c-Jun-, Tead4-, and/or TAZ-expressing plasmids. Luciferase activity was analysed 24 h after transfection. The pGL3-basic vector was used as a control (n = 3). f TAZ stimulated TBE1-driven gene transcription. Control and TAZ-knockdown 293T cells (Ti) were transfected with pGL3-basic or TBE1-luc, and a luciferase assay was performed 24 h after transfection (n = 3). Data are presented as mean ± SD. Statistical analysis was performed using a Student’s t-test. ***p < 0.005
Fig. 4
Fig. 4
TAZ interacts with c-Jun for Irs1 transcription. a Mouse gastrocnemius muscle genomic DNA was sheared by sonication, and chromatin fragments were immunoprecipitated with c-Jun antibodies. ChIPed DNA was analysed by qRT-PCR with primer sets flanking TBE1 (n = 3). Eight- to ten-week-old mice were used. b TAZ interacted with c-Jun. 293T cells were co-transfected with F-TAZ- and c-Jun-expressing plasmids. After immunoprecipitating with anti-FLAG antibodies, the eluted samples were analysed by immunoblotting with TAZ and c-Jun antibodies. c Endogenous c-Jun interacted with TAZ. C2C12 cell lysates were immunoprecipitated with IgG or c-Jun antibodies, and immune complexes were detected using TAZ and c-Jun antibodies. d Mutating c-Jun-binding sites in TBE1-luc decreased TAZ- and c-Jun-mediated reporter activity. The mutant c-Jun-binding site was generated by site-directed mutagenesis. Luciferase reporter plasmids harbouring wild-type (TBE-luc) or mutant c-Jun-binding sites (TBE1m-luc) were transfected into 293T cells together with c-Jun- and TAZ-expressing plasmids. Luciferase reporter gene activity was analysed 24 h after transfection (n = 3). e Wild-type (amino acids 1–395), N-terminus-deleted TAZ (amino acids 124–395 and 164–395) and WW domain-truncated TAZ expression plasmids (ΔWW) were transfected into 293T cells together with a c-Jun expression plasmid. Cell lysates were immunoprecipitated 24 h after transfection with FLAG antibodies, and the eluted samples were analysed by immunoblotting using TAZ and c-Jun antibodies. f The transcriptional activity of the TAZ deletion mutant was assessed using a TBE1-luc reporter plasmid. 293T cells were co-transfected with the TAZ deletion mutant together with a c-Jun expression plasmid as described in e. A Renilla luciferase plasmid was used for transfection normalisation. Cells were lysed, and luciferase activity was measured 24 h after transfection (n = 3). Data are presented as mean ± SD. Statistical analysis was performed using Student’s t-test. ns not significant; ***p < 0.005
Fig. 5
Fig. 5
TAZ stimulates Wnt signalling-induced Irs1 transcription. a Wild-type (WT) and TAZ-knockout (KO) mouse embryonic fibroblasts (MEF) were treated with Wnt3a-conditioned or control medium from L cells, for 24 h. Cell lysates were analysed by immunoblotting. b Irs1 expression of cells described in a was analysed by qRT-PCR (n = 3). c Control (Con) and TAZ-knockdown (Ti) C2C12 myotubes were treated with control or Wnt3a-conditioned medium for 24 h. Level of IRS1 and TAZ was then analysed by immunoblotting. d Total RNAs from cells described in c were prepared, and Irs1 expression was analysed by qRT-PCR (n = 3). e Gastrocnemius muscles from WT, muscle-specific TAZ-knockout (TAZmKO), muscle-specific adenomatous polyposis coli (APC)-KO (APCmKO), and muscle-specific APC and TAZ double-KO (DbmKO) mice were analysed by immunoblotting, using the indicated antibodies. For β-catenin data, non-phospho (active) β-catenin antibody was used. f Irs1 transcription was analysed in the gastrocnemius muscles of WT, TAZmKO, APCmKO, and DbmKO mice by qRT-PCR (n = 8). g Glucose tolerance and h insulin tolerance were assessed in WT, TAZ-mKO, APC-mKO, and DbmKO mice (n = 6). For e–h, 8–10-week-old mice were used. Data are presented as mean ± SD for panels b and d, and as mean ± SEM for panels f–h. Statistical analysis was performed using a Student’s t-test. *p < 0.05; ***p < 0.005
Fig. 6
Fig. 6
Statins decrease IRS1 and TAZ level, and insulin sensitivity. a Six-week-old mice were administered simvastatin or vehicle for 3 weeks. Mice were sacrificed, the gastrocnemius muscles were isolated, and IRS1 and TAZ level was analysed by immunoblotting. α-Tubulin was used as a loading control. b RNA was isolated from gastrocnemius muscles in panel a. Irs1 transcription was analysed by qRT-PCR and normalised to Gapdh expression (n = 8). c Six-week-old mice were administered simvastatin or vehicle for 3 weeks. Mice were fasted for 16 h, and d-glucose was intraperitoneally injected at 2 g per kg body weight. Serum glucose levels were measured using a glucometer at the indicated time points. The area under the curve for each mouse was calculated and presented as a fold-change relative to that for the vehicle condition (n = 8). d Six-week-old mice were administered simvastatin or vehicle for 3 weeks. Mice were fasted for 4 h, then administered insulin at 1.25 U per kg body weight. Serum glucose levels were analysed at the indicated time points. The area under the curve for each mouse was calculated and presented a fold-change relative to that for the vehicle condition (n = 8). e C2C12 myotubes were treated with DMSO or simvastatin at the indicated concentration for 48 h, and cell lysates were analysed by immunoblotting. f Cells described in e were harvested, and Irs1 expression was analysed by qRT-PCR and normalised to Gapdh expression (n = 3). g The same experiment in panel e was performed with cerivastatin instead of simvastatin. h The same experiment in panel f was assessed with cerivastatin instead of simvastatin. Data are presented as the relative fold induction (n = 3). Data are presented as mean ± SD for panels f and h, and as mean ± SEM for panels b–d. Statistical analysis was performed using a Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.005
Fig. 7
Fig. 7
Statin treatment inhibits insulin signalling via TAZ degradation. a Insulin was intraperitoneally injected into vehicle- or simvastatin-administered mice. After 15 min, muscle lysates were analysed by immunoblotting. α-Tubulin was used as a loading control. Simvastatin or vehicle was administered to 6-week-old mice for 3 weeks. b Three independent experiments performed as described in a were assessed, and the ratios of IRS1 and TAZ to α-tubulin were analysed; in addition, the ratios of phosphorylated to total AKT, AS160, and ribosomal protein S6 kinase were determined (n = 3). c C2C12 cells were transduced with control, wild-type TAZ, or proteolytic degradation-resistant TAZ (TAZ4SA; TAZ S58, 62, 306, 309A) retroviral vectors to establish stable cell lines. Then, 10 μM simvastatin or DMSO was added to cells for 48 h, and IRS1 and TAZ level in harvested cells was analysed by immunoblotting. d Irs1 expression in the cells described in c was analysed by qRT-PCR and normalised to Gapdh expression (n = 3). e Glucose uptake was analysed in control, wild-type TAZ, and TAZ4SA-overexpressing C2C12 myotubes (n = 3). Data are presented as mean ± SD. Statistical analysis was performed using a Student’s t-test. ns not significant; *p < 0.05; **p < 0.01; ***p < 0.005
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