Insulin Decreases Myocardial Adiponectin Receptor 1 Expression Via PI3K/Akt and Foxo1 Pathway

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Cardiovascular Research (2012) 93,69–78 doi:10.1093/cvr/cvr273

Insulin decreases myocardial adiponectin receptor 1 expression via PI3K/Akt and FoxO1 pathway

Xiao-Bing Cui1,2, Cheng Wang1,2,LiLi1,2, Dong Fan1,2, Yun Zhou1,2, Dan Wu1,2, Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021 Qing-Hua Cui3, Feng-Ying Fu1,2, and Li-Ling Wu1,2,4*

1Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100191, China; 2Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100191, China; 3Department of Biomedical Informatics, Peking University Health Science Center, Beijing 100191, China; and 4Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health Peking University, Beijing 100191, China

Received 4 May 2011; revised 8 October 2011; accepted 12 October 2011; online publish-ahead-of-print 19 October 2011

Time for primary review: 26 days

Aims Adiponectin is considered an important adipokine protecting against diabetes, atherosclerosis, and cardiovascular disease. Because adiponectin receptors (AdipoRs) are critical components in the adiponectin signalling cascade, we investigated the effect of insulin on the expression of myocardial AdipoRs and explored the possible molecular mechanism...... Methods The hyperinsulinaemia rat model was induced by infusion of insulin (1 U/day) for 28 days: serum and myocardial adi- and results ponectin levels were increased, and skeletal muscle and myocardial AdipoR1 expression and AMP-activated protein kinase (AMPK) phosphorylation were decreased. In primary cultured neonatal rat ventricular myocytes (NRVMs), insulin decreased AdipoR1 but not AdipoR2 expression and AMPK phosphorylation; high glucose had no affect on AdipoRs expression. Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was increased in insulin-treated hearts and in NRVMs. P13K inhibitor LY294002 and Akt1/2 kinase inhibitor but not the ERK1/2 kinase (MEK) inhibitors PD98059 and U0126 blocked the insulin-induced reduction in AdipoR1 expression and AMPK phosphorylation. Insulin induced forkhead/winged helix box gene group O-1 (FoxO1) phosphorylation and translocation from the nucleus to the cytosol, and this was blocked by LY294002. FoxO1 small interfering RNA reduced AdipoR1 expression and AMPK phosphorylation. In electrophoretic mobility shift assay and chromatin immunoprecipitation, FoxO1 bound to the putative site from 2167 to 2157 bp of the AdipoR1 promoter both in vitro and in living cells; insulin suppressed this binding, which was blocked by LY294002...... Conclusion Insulin inhibits myocardial AdipoR1 expression via PI3K/Akt and FoxO1 pathways, and FoxO1 mediates AdipoR1 transcription by binding to its promoter directly. ------Keywords Adiponectin receptor † Cardiomyocyte † Insulin † FoxO1

1. Introduction important endogenous adipokine protecting against diabetes, atherosclerosis, and cardiovascular disease. Adiponectin is an adipocyte-specific adipokine that plays an important Two types of adiponectin receptors (AdipoRs) mediate the activa- role in energy metabolism, regulation, and cardiovascular protection. tion of AMP-activated protein kinase (AMPK) and peroxisome The circulating adiponectin level is decreased in obesity, type 2 dia- proliferator-activated receptor-a, as well as fatty acid oxidation and betes, and coronary artery disease.1,2 Adiponectin-deficient mice glucose uptake by adiponectin.9,10 AdipoR1 is ubiquitously expressed, exhibit insulin resistance, diabetes, and concentric cardiac hyper- with a relatively high level in skeletal muscle, whereas AdipoR2 is pre- trophy.3,4 Administration of adiponectin reduces blood glucose dominantly expressed in liver.11 AdipoRs expression is significantly level, ameliorates insulin resistance, and protects against cardiac decreased in adipose tissue, liver, and muscle of obese diabetic remodeling in adiponectin-knockout and db/db mice.5,6 Adiponectin animal and individuals and in infarcted mouse hearts.12,13 AdipoRs also protects against myocardial ischaemia-reperfusion injury and are critical components in the adiponectin signalling cascade, with improves systolic dysfunction.7,8 Therefore, adiponectin is an the decrease in plasma adiponectin level and down-regulation of

* Corresponding author. Tel: +86 10 8280 2403; fax: +86 10 8280 2403, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2011. For permissions please email: [email protected]. 70 X.-B. Cui et al.

AdipoR expression being involved in insulin resistance, type 2 dia- 2.4 Measurement of serum and heart betes, and coronary artery disease. However, AdipoR mRNA levels adiponectin in human skeletal muscle have been found to be positively associated Frozen heart tissues were homogenized in lysis buffer and centrifuged as 14 with obesity and insulin resistance and to be up-regulated in skeletal described.17 The supernatants were collected and the protein content was mea- 15 muscle from obese mice induced by a high-fat diet. Therefore, the sured by the Bradford method. Serum and heart adiponectin levels were mea- relationship between the expression of AdipoRs and insulin resistance sured by use of a commercially available ELISA kit (Adipo Biotech, Beijing). and type 2 diabetes still needs further investigation. Both AdipoR1 and AdipoR2 are expressed in cardiomyocytes.11 Re- 2.5 Primary culture of neonatal rat ventricular cently, myocardial AdipoR1 expression was found to be decreased in myocytes rats with high-fat and high-sugar diets, possibly associated with Neonatal rat ventricular myocytes (NRVMs) were prepared as described decreased heart function.16 However, the regulatory mechanisms previously.19 Briefly, ventricles of 1- to 3-day-old Sprague-Dawley rats involved in myocardial AdipoR1 expression remain unclear. Also, myo- were minced and digested in phosphate-buffered saline containing 0.1% cardial AdipoR1 expression was found to be increased in streptozoto- trypsin and 0.05% type I collagenase for 8–10 cycles, then cells were cen- Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021 cin-induced diabetic rats,17 which suggests that the myocardial AdipoR1 trifuged and suspended in Dulbecco’s modified Eagle’s medium (Invitro- gen, Carlsbad, CA, USA) containing 15% foetal bovine serum (FBS) and level may be negatively correlated with the serum insulin level. antibiotics (1% penicillin and streptomycin). Myocytes that did not Because hyperinsulinaemia is usually associated with obesity, type 2 attach to culture dishes were plated at 1 × 106 cells/cm2 in the same diabetes, and cardiovascular diseases, and insulin enhances while adi- medium and supplemented with 0.1 mM bromodeoxyuridine to inhibit 18,19 ponectin inhibits angiotensin-induced cardiac hypertrophy, we the proliferation of fibroblasts and obtain higher purity of cardiomyocytes. hypothesized that insulin might interfere with the myocardial adipo- Cells were placed in a serum-free medium for 24 h before experiments. nectin signalling cascade by regulating the expression of AdipoRs. The identity of NRVMs was confirmed by morphological examination We examined the effect of insulin on AdipoR expression in a rat and by staining with anti-sarcomeric a-actin antibody; most (.95%) of model of continuous insulin infusion and in cultured cardiomyocytes. the cells were identified as NRVMs. Moreover, we investigated the molecular mechanism by which insulin regulates AdipoR expression. 2.6 RNA isolation and RT–PCR Total RNA of myocardium and NRVMs was isolated by use of Trizol reagent (Invitrogen). cDNA was generated from total RNA by use of 2. Methods the RevertAid First Strand cDNA Synthesis kit (Fermentas, Burlington, ON, Canada) and amplified by PCR with the gene-specific primers for All experimental procedures were approved by the Ethics Committee of AdipoR1, which were not within the mRNA coding sequence, but able Animal Research, Peking University Health Science Center, and the inves- to evaluate the AdipoR1 mRNA levels, forward, 5′-AGGCAA tigation conformed to the Guide for the Care and Use of Laboratory Animals CTGCTGGTCCTTCAC-3′, reverse, 5′-TGCCAAGCGGTCTGTACTT published by the US National Institutes of Health (NIH Publication No. TC-3′; AdipoR2, forward, 5′-AACCCACAACCTTGCTTCATC-3′, 85-23, revised 1996). reverse, 5′-TCACAGCGCATCCTCTTCAGT-3′; FoxO1 forward, 5′-GG ′ ′ 2.1 Materials TGAACACCATGCCTCACAC-3 , reverse, 5 -GTCGTTCCGAATGATG GACTC-3′; and b-actin forward, 5′-TCCTCCCTGGAGAAGAGCTA-3′, Antibodies for phospho- and total AMPK, Akt, p38 mitogen-activated ′ ′ reverse, 5 -TCAGGAGGAGCAATGATCTTG-3 as a housekeeping protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK), and fork- control. The PCR products were electrophoresed on a 1.5% agarose head/winged helix box gene group O-1 (FoxO1) were from Cell Signaling gel and stained with ethidium bromide. The bands were visualized by Gen- Technology (Beverly, MA, USA). Antibodies for AdipoR1, AdipoR2, eGenius Gel Imaging System (Syngene, Synoptics, Inc., Frederick, MD, b-tubulin, and phospho- and total extracellular signal-regulated kinase USA) and processed by use of Genetools 3.06 (Syngene). 1/2 (ERK1/2) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Insulin, LY294002, Akt1/2 kinase inhibitor, PD98059, and U0126 2.7 Western blot analysis were from Sigma-Aldrich Co. (St Louis, MO, USA). Myocardial tissues or NRVMs were lysed in a buffer containing 50 mM Tris– 2.2 In vivo rat model of continuous insulin HCl, pH 7.2, 0.1% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 40 mM NaF, 2.175 mM NaVO4, 0.1% SDS, infusion 0.1% aprotinin, and 1 mM PMSF. The lysates were centrifuged and the super- Male Sprague-Dawley rats weighing 250–280 g were randomly divided natants were collected. Equal amounts of protein (40 mg) underwent SDS– into control and insulin groups. An osmotic mini-pump (model 2004, PAGE and were transferred to polyvinylidene difluoride membranes. Mem- Durect Corp., Cupertino, CA, USA) was subcutaneously embedded in branes were incubated with primary antibodies and probed with horserad- rats under anaesthesia with sodium pentobarbital (50 mg/kg, i.p.). Insulin ish peroxidase-conjugated secondary antibodies. Bands were detected with (1 U/day) or normal saline was infused constantly for 28 days, then an enhanced chemiluminescence kit (Amersham Biosciences Inc., Piscat- blood samples were collected from abdominal aortas, and hearts were away, NJ, USA). The densities of bands were quantified by use of the excised under anaesthesia as above. No neuromuscular blocking or para- LEICA550IW image analysis system (Leica, Mannheim, Germany). lytic agents were used. The adequacy of anaesthesia was determined by monitoring blood pressure and heart rate by electrocardiography. 2.8 Immunofluorescence NRVMs were cultured on glass coverslips and fixed with 4% paraformal- 2.3 Measurement of blood glucose and serum dehyde, permeabilized with 0.2% Triton X-100 and incubated with 1% insulin bovine serum albumin for 30 min. Then NRVMs were incubated with anti- The blood glucose level was measured by use of Accu-Chek Active Blood FoxO1 antibody (1:200) followed by FITC-labelled secondary antibody Glucose Test Strips (Roche Diagnostics GmbH, Mannheim, Germany). (1:400). Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Serum insulin content was measured by use of an insulin radioimmuno- Sigma-Aldrich). Fluorescence images were captured by the Leica TCS assay kit (Linco Research, St. Charles, MO, USA). SP5 confocal system (Leica, Wetzlar, Germany). Insulin decreases myocardial AdipoR1 expression 71

2.9 Small interfering RNA (siRNA) glucose, ratio of left ventricle to body weight, or cardiac myocyte siRNA targeting FoxO1 and negative control siRNA were from Invitrogen. cross-sectional area (Figure 1A–E). Insulin infusion increased serum The sequence for FoxO1 was 5′-GGAGAAGAGCUGCAUCCAUTT-3′ and cardiac adiponectin levels (Figure 1F and G) but reduced myocar- and for non-specific control 5′-UUCUCCGAACGUGUCACGUTT-3′. dial AdipoR1 protein by 40% (P , 0.01) with no change in AdipoR2 NRVMs were transfected with the siRNA (100 nM for 48 h) by (Figure 1H and I). Phosphorylation of AMPK-a at Thr-172, a down- use of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s stream kinase of AdipoRs, was significantly decreased in the instructions. insulin-infused heart (Figure 1J). Moreover, the mRNA and protein expression of AdipoR1 in skeletal muscle was decreased by insulin infu- 2.10 Electrophoretic mobility shift assay sion, but the AdipoR2 mRNA level was not affected (see 20 Nuclear protein fractions were separated as described. Briefly, NRVMs Supplementary material online, Figure S1A–C). were suspended in hypotonic buffer containing 10 mM HEPES, 10 mM To further explore the effect of insulin on AdipoR expression, KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 1 mM DTT, 1 mM NRVMs were incubated with 1–200 nmol/L insulin for 24 h. NaVO4, and 10 mg/mL aprotinin for 15 min. After adding NP-40 (10%), Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021 the mixture was centrifuged twice and supernatants were collected. Insulin dose-dependently (Figure 2A and B) and time-dependently The DNA sequence of AdipoR1 was obtained from the UCSC (Figure 2C and D) reduced the mRNA expression of AdipoR1 but Genome Browser Database (http://www.genome.ucsc.edu). Putative not AdipoR2. We further verified that insulin decreased the binding sites for FoxO1 (142 to 152 bp and 2167 to 2157 bp) were protein level of AdipoR1 in a time- and dose-dependent manner identified by use of PROMO 3.0 with the database TRANSFAC 8.3. (Figure 2E and F). H2O2, reported to inhibit AdipoR expression in Electrophoretic mobility shift assay (EMSA) involved use of the NRVMs,13 was used as a positive control. Insulin also decreased TM LightShift Chemilumiescent EMSA kit (Pierce Biotechnology, Rockford, AMPK phosphorylation in NRVMs (Figure 2G). These results IL, USA). Briefly, nuclear protein (8 mg) was incubated with biotin-labelled suggest that insulin inhibited AdipoR1 expression and function in DNA probes containing two putative binding sites (underlined sequence) NRVMs. for FoxO1, 5′-biotin-CACACTTCGGTATCTTGTTTAGATTCTAG Because hyperinsulinaemia and hyperglycaemia usually coexist in CTGA-3′ (sense, 131 to 163 bp) and 5′-biotin-GGGAGGGGCC ′ obesity and type 2 diabetes, and high glucose decreases AdipoR1 GGAAATGTTTACGGTGGACCTTT-3 (sense, 2178 to 2146 bp). 22 After reaction, the DNA–protein complexes underwent 6% native expression in L6 myoblasts, we further tested the effect of high PAGE and were transferred to a nitrocellulose membrane, then cross- glucose on AdipoR expression. NRVMs were incubated with constant linked to the membrane at 120 mJ/cm2 and detected by use of horseradish high glucose (25.5 mmol/L) or intermittent high glucose (changing 5.5 peroxidase-conjugated streptavidin. and 25.5 mmol/L glucose medium every 12 h) for 24–96 h. The mRNA expression of AdipoRs was not affected (see Supplementary 2.11 Chromatin immunoprecipitation assay material online, Figure S2A–C). Furthermore, when NRVMs were incu- Chromatin immunoprecipitation (ChIP) assay was performed as bated with both 100 nmol/L insulin and 25.5 mmol/L glucose, the inhibi- 21 described. Briefly, NRVMs were cross-linked with 1% formaldehyde and tory effect on AdipoR1 protein expression did not differ from that with collected into lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH insulin incubation alone. 8.1, 1× protease inhibitor cocktail). After sonication, the lysates underwent immunoclearing and were immunoprecipitated with anti-FoxO1 antibody 3.2 Insulin decreases AdipoR1 expression in or normal rabbit IgG as a negative control. After incubation with protein A- Sepharose and salmon sperm DNA and a sequential washing, precipitates NRVMs via the PI3K/Akt pathway were heated at 658C for 6 h to reverse the formaldehyde cross-linking. Phosphatidylinositol 3-kinase (PI3K) and Akt are the key molecules DNA fragments were purified by use of the TIANquick Midi Purification mediating the insulin signal pathway.23 We found the phosphoryl- kit (Tiangen Biotech, Beijing). PCR analysis involved the following primer ation of Akt at Ser-473 significantly increased in insulin-treated sequences for putative FoxO1 binding sites: site 1 forward, rat hearts and NRVMs (Figure 3A and E). ERK1/2, p38MAPK, and ′ ′ 5 -TTGCGAGTTGGGTACGGG-3 (218 to 21 bp) and reverse, JNK are major members of the MAPK family. We found that 5′-TCGTCCAGCGTCTTCTTCA-3′ (292 to 310 bp); site 2 forward, ′ ′ insulin significantly increased the phosphorylation of ERK1/2 but 5 -CAGCCAGGCCGTAAGTGTC-3 (2291 to 2273 bp) and reverse, not JNK or p38 MAPK in vivo (Figure 3B–D) and in vitro 5′-TGTTCCCGTACCCAACTCG-3′ (215 to 4 bp). (Figure 3F–H ). To further ascertain the roles of Akt and ERK1/2 2.12 Statistical analysis in insulin-reduced AdipoR1 expression, NRVMs were preincubated with the PI3K inhibitor LY294002 (10 mmol/L), Akt1/2 kinase inhibi- Data are presented as means + SD. Differences between groups were evaluated by Student’s t-test or one-way ANOVA followed by Tukey’s tor (1 mmol/L), or PD98059 (10 mmol/L) and U0126 (10 mmol/L), multiple comparison post hoc tests by use of GraphPad Prism 5.0 software. inhibitors of ERK1/2 kinase (MEK), before insulin treatment. P , 0.05 was considered statistically significant. LY294002 and Akt1/2 kinase inhibitor but not PD98059 or U0126 blocked insulin-induced AdipoR1 decrease in expression (Figure 3I–L). The effects of these inhibitors on phosphorylation 3. Results of Akt and ERK1/2 were confirmed in NRVMs (see Supplementary material online, Figure S3A–D), and each of them alone had no 3.1 Insulin decreases AdipoR1 expression effect on AdipoR1 expression. These results suggested that and AMPK phosphorylation in vivo and in insulin decreased AdipoR1 expression via a PI3K/Akt pathway. Fur- vitro thermore, when NRVMs were preincubated with LY294002 and Compared with controls, insulin-infused rats showed significantly Akt1/2 kinase inhibitor, the insulin-induced decrease in p-AMPK increased serum insulin level but no change in body weight, blood was reversed (Figure 3M and N). 72 X.-B. Cui et al. Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021

Figure 1 The chronic hyperinsulinaemia rat model (n ¼ 8) was induced by insulin infusion (1 U/day) for 28 days; normal saline was used as control (n ¼ 8). (A) Serum insulin. (B) Body weight. (C) Blood glucose. (D) Ratio of left ventricle to body weight. (E) Representative HE staining and cross- sectional area of cardiomyocytes. (F) Serum adiponectin. (G) Heart adiponectin. Western blot analysis of myocardial AdipoR1 (H ) and AdipoR2 (I) protein levels. b-Tubulin was an endogenous control. (J ) Western blot analysis of myocardial AMPK-a phosphorylation (p-AMPK). Total AMPK (t-AMPK) was a control. *P , 0.05, **P , 0.01 vs. Con.

3.3 Insulin induces FoxO1 phosphorylation translocation. These data suggest that insulin induced FoxO1 phosphor- and exclusion from the nucleus ylation and exclusion from the nucleus via a PI3K/Akt pathway. FoxO1 is a critical nuclear transcription factor regulated by insulin- dependent phosphorylation.24 Phosphorylation of FoxO1 at Ser-256 3.4 FoxO1 siRNA reduces FoxO1 levels was signiﬁcantly increased in insulin-infused hearts and insulin-treated and decreases AdipoR1 expression in NRVMs (Figure 4A and B). Under the basal condition, FoxO1 localized NRVMs mainly in the nucleus (Figure 4C). After insulin treatment (100 nmol/L, To further verify the importance of FoxO1 in inhibiting AdipoR1 gene 1 h), FoxO1 translocated from the nucleus to the cytosol. Preincubation expression, NRVMs were transfected with control or FoxO1 siRNA. with the PI3K inhibitor LY294002 blocked the insulin-induced FoxO1 The mRNA and protein expression of FoxO1 was signiﬁcantly Insulin decreases myocardial AdipoR1 expression 73 Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021

Figure 2 Expression of AdipoRs and AMPK phosphorylation in NRVMs. RT–PCR analysis of AdipoR1 (A and C) and AdipoR2 (B and D) mRNA in NRVMs treated with insulin for 24 h at indicated concentrations or with 100 nmol/L insulin for the indicated times. b-Actin was an endogenous control. Western blot analysis of the AdipoR1 protein level in NRVMs treated with insulin for 24 h at the indicated concentrations (E) or with

100 nmol/L insulin for the indicated times (F). b-Tubulin was an endogenous control. H2O2 (10 mmol/L, 24 h) was a positive control. (G) Western blot analysis of p-AMPK and t-AMPK in NRVMs treated with insulin (100 nmol/L, 24 h). Data are mean + SD of three independent experiments. *P , 0.05, **P , 0.01 vs. Con. 74 X.-B. Cui et al. Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021

Figure 3 Signalling molecules mediating insulin-mediated AdipoR1 expression. Western blot analysis of Akt (A and E), ERK1/2 (B and F), JNK (C and G) and p38MAPK (D and H ) phosphorylation in rat hearts and NRVMs, respectively. The phosphorylation levels were normalized to their total protein levels. Western blot analysis of AdipoR1 protein in NRVMs treated with insulin (100 nmol/L, 24 h) with or without preincubation with LY294002 (I), Akt1/2 kinase inhibitor (J ), PD98059 (K), or U0126 (L). b-Tubulin was an endogenous control. Western blot analysis of AMPK phosphorylation in NRVMs treated with insulin (100 nmol/L, 24 h) with or without preincubation with LY294002 (M) or Akt1/2 kinase inhibitor (N). The phosphorylation level was normalized to t-AMPK. Data are mean + SD of three independent experiments. *P , 0.05, **P , 0.01 vs. Con. #P , 0.05 vs. insulin. Insulin decreases myocardial AdipoR1 expression 75 Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021

Figure 4 Effect of insulin on FoxO1 phosphorylation and translocation. Western blot analysis of FoxO1 phosphorylation in rat hearts (A) and NRVMs (B). The phosphorylation level was normalized to total FoxO1. (C) Immunoﬂuorescence analysis of subcellular localization of FoxO1 (green) in NRVMs preincubated with or without LY294002 before insulin (100 nmol/L, 1 h) treatment. FBS (10%, 1 h) was a positive control. Nuclei (blue) were stained with DAPI. Data are mean + SD of three independent experiments. *P , 0.05 vs. Con.

decreased (Figure 5A and B). Transfection with FoxO1 siRNA caused a corresponding to site 2 under the same experimental conditions. 54% (P , 0.01) and 33% (P , 0.01) decrease in AdipoR1 mRNA and Insulin suppressed the binding of FoxO1 to site 2, and LY294002 protein levels, respectively, with no change in AdipoR2 mRNA ex- blocked the inhibitory effect of insulin. These data suggested that pression (Figure 5A and B). Moreover, FoxO1 siRNA inhibited FoxO1 regulated AdipoR1 transcription by binding to its promoter AMPK phosphorylation by 40% (P , 0.01). These results suggested region directly, and insulin inhibited FoxO1 binding via a PI3K/Akt that AdipoR1 was a downstream target gene of FoxO1 in NRVMs. pathway.

3.5 FoxO1 is required for insulin-reduced 4. Discussion AdipoR1 expression In the present study, we demonstrated that insulin decreased the To further reveal the mechanism of FoxO1-mediated AdipoR1 cardiac expression of AdipoR1 but not AdipoR2 in rats with continu- expression, we screened the rat AdipoR1 promoter sequence. We ous insulin infusion and in cultured NRVMs. Furthermore, FoxO1 found two putative FoxO1 binding sites located from 22000 to mediated AdipoR1 transcription by binding to the AdipoR1 promoter 200 bp of the promoter sequence: site 1 between 142 and 152 bp region directly. The activation of PI3K/Akt and translocation of FoxO1 and site 2 between 2167 and 2157 bp (Figure 6A). EMSA results were involved in the inhibitory effect of insulin on AdipoR1 showed the formation of a protein–DNA complex when nuclear expression. extracts were incubated with only site 2 oligonucleotides, which Insulin plays a crucial role in regulating glucose and lipid metabol- could be competed out with 100-fold unlabelled oligonucleotides ism, protein synthesis, growth, and contractility in cardiomyocytes, and suppressed by insulin incubation (Figure 6B). To determine and hyperinsulinaemia is usually associated with obesity, type 2 dia- whether FoxO1 bound to either of the two putative binding sites in betes, and cardiovascular disease.23 Previous studies showed that intact NRVMs, ChIP assay was performed (Figure 6C). PCR analysis insulin decreases AdipoR1 but not AdipoR2 expression in C2C12 of input samples revealed a product for both sites, whereas analysis myoblasts and in mouse skeletal muscle.25 Insulin deﬁciency induced of FoxO1 antibody-immunoprecipitated samples revealed a product by streptozotocin increases and insulin replacement reduces 76 X.-B. Cui et al.

improves myocardial function.30 AdipoRs play an important role in mediating the insulin-sensitizing effect of adiponectin.11 Our results suggest that insulin-reduced myocardial AdipoR1 expression may blunt myocardial insulin sensitivity and contribute to the insulin-induced cardiac dysfunction. Although circulating adiponectin levels are usually decreased in animals and subjects with obesity, insulin resistance, and cardiovascular disease,1,2,31 a recent study reported increased plasma adiponectin level and decreased cardiac AdipoR1 expression in the early stage of type 1 diabetes and adiponectin level was gradually decreased during the progression of diabetes.32 In type 2 diabetic rats, cardiac hypertrophy and decreased heart function are associated with decreased

plasma and myocardial adiponectin levels, myocardial AdipoR1 Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021 expression, and AMPK phosphorylation.16 These results suggest that adiponectin levels may vary by stage of disease and disease type. Samuelsson et al. reported that insulin infusion (2 U/day) for 7 weeks induced cardiac hypertrophy and reduced cardiac output.29 However, we observed no signiﬁcant cardiac hypertrophy in our hyperinsulinaemia model (insulin 1 U/day, for 28 days). The increased serum and heart adiponectin levels might be explained at least in part by a compensatory change during the early stage of hyperinsulinaemia. Adiponectin exerts its metabolic modulation and cardioprotective effects by the AMPK signalling pathway.7,10 Targeted disruption of AdipoR1 results in the abrogation of adiponectin-induced AMPK activation in mouse liver.33 We found myocardial AMPK phosphorylation decreased in insulin-treated hearts and NRVMs. Despite an increase in serum and myocardial adiponectin levels, the decreased AdipoR1 expression and AMPK phosphorylation suggested that adiponectin resistance might occur in the early stage of hyperinsulinaemia. These observations further support the possibility of impaired AdipoR1-mediated biological effects in insulin-treated cardiomyocytes, which may contribute in part to the occurrence and progres- Figure 5 Effect of FoxO1 siRNA on AdipoR expression and AMPK phosphorylation. FoxO1 or negative control (NC) siRNA sion of insulin-induced cardiac dysfunction. was transfected into NRVMs for 48 h. (A) RT–PCR analysis of PI3K/Akt and MAPK pathways play crucial roles in the signal trans- FoxO1, AdipoR1, and AdipoR2 mRNA expression. b-Actin was an duction of insulin. PI3K/Akt is considered the main player of the meta- endogenous control. (B) Western blot analysis of FoxO1, AdipoR1 bolic action of insulin, whereas the MAPK pathway is principally protein expression and AMPK phosphorylation. b-Tubulin was an involved in cell growth and differentiation.23 In the present study, endogenous control. The AMPK phosphorylation level was normal- insulin induced Akt phosphorylation in vivo and in vitro, and ized to t-AMPK protein. Data are mean + SD of three independent LY294002 and Akt1/2 kinase inhibitor blocked the effect of insulin experiments. **P , 0.01 vs. Con. on AdipoR1 expression, which indicates that PI3K/Akt is an important signalling pathway mediating the insulin-inhibited AdipoR1 expression in cardiomyocytes. Insulin increased the phosphorylation of ERK1/2, AdipoR expression in skeletal muscle and liver.26 However, insulin but pretreatment with ERK1/2 kinase (MEK) inhibitor did not affect does not alter the expression of AdipoRs in adipocytes,27 and insulin-reduced AdipoR1 expression, which suggests that the MAPK AdipoR expression is reduced in liver and elevated in muscles in family might not be involved in the signal transduction of hyperinsulinaemic mice.15 Therefore, the exact role of insulin on insulin-inhibited AdipoR1 expression. The results agree with those AdipoR expression may depend on the model and tissue investigated of previous studies of C2C12 cells.26,34 A recent study showed that and the cause of insulin resistance. insulin activates a feed-forward inhibitory pathway via PI3K/Akt to Here, we provide more evidence that insulin inhibited not only cause insulin resistance in 3T3-L1 adipocytes.35 High-fat diet-induced skeletal muscle but also myocardial AdipoR1 expression in chronic insulin resistance/hyperinsulinaemia is associated with increased Akt hyperinsulinaemia rats. Furthermore, insulin down-regulated phosphorylation, and blockade of the elevated Akt phosphorylation AdipoR1 but not AdipoR2 levels in NRVMs. Our results also show by LY294002 reversed the insulin sensitivity.36 Thus, insulin-induced similar plasma glucose levels in rats with insulin infusion and controls, PI3K/Akt activation may contribute to the development of insulin as reported previously.28,29 Constant and intermittent high glucose resistance. had no effect on AdipoR expression in NRVMs. These data indicated FoxO1 is an important nuclear transcription factor that that insulin selectively inhibited myocardial AdipoR1 but not AdipoR2 represses or activates target gene transcription after insulin- expression independent of change in the glucose level in vivo and in dependent phosphorylation. Akt-mediated FoxO1 phosphorylation vitro. Myocardial insulin resistance is associated with myocardial dys- at Ser-256 is crucial for subsequent nuclear exclusion.24 The function, and the restoration of insulin sensitivity in the failing heart AdipoR1 mRNA level is associated with FoxO1 mRNA expression Insulin decreases myocardial AdipoR1 expression 77 Downloaded from https://academic.oup.com/cardiovascres/article/93/1/69/412091 by guest on 01 October 2021

Figure 6 Effect of FoxO1 on insulin-mediated AdipoR1 expression. (A) Two putative FoxO1 binding sites between 142 and 152 bp (site 1) and 2167 and 2157 bp (site 2) of the AdipoR1 promoter sequence. (B) Nuclear extracts of NRVMs treated with or without insulin (100 nmol/L, 6 h) were incubated with biotin-labelled probes corresponding to site 1 or site 2 before being applied to a non-denaturing gel. Com- petition were performed by preincubation with 100-fold of the corresponding unlabelled probe (cold). (C) NRVMs were preincubated with or without LY294002, then treated with insulin (100 nmol/L, 6 h) and underwent ChIP assay. PCR analysis of the input-, FoxO1-, and normal rabbit IgG-immunoprecipitated (negative) samples involved the primer sets corresponding to sites 1 and 2. All results are representative of three independent experiments.

in obese insulin-resistant subjects, and FoxO1 enhances transcrip- AdipoR expression is mediated by various physiological and patho- tion of the AdipoR1 promoter in HepG2 cells.37 We showed that logical factors. However, the precise transcription mechanism insulin induced FoxO1 phosphorylation and translocation from the involved in AdipoR expression has not been fully evaluated. Recently, nucleus to the cytosol. The levels of AdipoR1 mRNA and protein C2C12 cells were found to contain a novel insulin-responsive region were decreased by FoxO1 silencing, which suggests that FoxO1 in the AdipoR1 promoter with a strong repressor element, termed acted positively on the expression of AdipoR1. Furthermore, nuclear inhibitory protein, involved in the negative regulation of LY294002 and Akt1/2 kinase inhibitor blocked the inhibitory AdipoR1 promoter by insulin.34 In the present study, we found that effects of insulin on AMPK phosphorylation, and FoxO1 siRNA the AdipoR1 promoter sequence from 2167 to 2157 bp contains inhibited AMPK phosphorylation. These data further support that a critical regulatory site for FoxO1. EMSA and ChIP assay provided insulin decreased cardiac AdipoR1 expression and function the first evidence that FoxO1 bound directly to the AdipoR1 pro- through a PI3K/Akt and FoxO1 pathway. moter both in vitro and in living cells. Moreover, we demonstrated 78 X.-B. Cui et al. that insulin suppressed FoxO1 binding to the sequence via a PI3K/Akt TNF-alpha-treated cardiomyocytes in association with improved glucose uptake. pathway. Am J Physiol Heart Circ Physiol 2007;293:H3490–H3497. 14. Bluher M, Bullen JW Jr, Lee JH, Kralisch S, Fasshauer M, Kloting N et al. Circulating In summary, we demonstrate that insulin decreased myocardial adiponectin and expression of adiponectin receptors in human skeletal muscle: asso- AdipoR1 but not AdipoR2 expression in vivo and in NRVMs. Our ciations with metabolic parameters and insulin resistance and regulation by physical results also indicate that insulin decreases AdipoR1 expression via training. J Clin Endocrinol Metab 2006;91:2310–2316. 15. Barnea M, Shamay A, Stark AH, Madar Z. A high-fat diet has a tissue-specific effect on the PI3K/Akt pathway and that FoxO1 plays an important role in adiponectin and related enzyme expression. Obesity (Silver Spring) 2006;14: insulin-mediated AdipoR1 transcription in cardiomyocytes by 2145–2153. binding directly to the AdipoR1 promoter. These findings may 16. Guo Z, Zheng C, Qin Z, Wei P. Effect of telmisartan on the expression of cardiac adiponectin and its receptor 1 in type 2 diabetic rats. J Pharm Pharmacol 2010;63: improve our understanding of the molecular mechanism involved in 87–94. insulin-associated myocardial dysfunction and provide new insights 17. Guo Z, Xia Z, Yuen VG, McNeill JH. Cardiac expression of adiponectin and its recep- for future therapeutic targets. tors in streptozotocin-induced diabetic rats. Metabolism 2007;56:1363–1371. 18. Yu W, Chen C, Fu Y, Wang X, Wang W. Insulin signaling: a possible pathogenesis of cardiac hypertrophy. Cardiovasc Ther 2010;28:101–105.

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