Journal of Molecular Medicine https://doi.org/10.1007/s00109-018-1623-z

ORIGINAL ARTICLE

Chronic hyperinsulinemia induced miR-27b is linked to adipocyte insulin resistance by targeting insulin receptor Ankita Srivastava 1,2 & Kripa Shankar 1 & Muheeb Beg 1 & Sujith Rajan 1,2 & Abhishek Gupta 1 & Salil Varshney 1,2 & Durgesh Kumar 1,2 & Sanchita Gupta 1,2 & Raj Kumar Mishra 3 & Anil Nilkanth Gaikwad 1,2 Received: 17 May 2017 / Revised: 1 February 2018 / Accepted: 5 February 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Defect in insulin signaling leads to the development of insulin resistance followed by type 2 diabetes. Exploiting our previously developed physiological chronic hyperinsulinemia (CI)-mediated insulin resistance (IR) model, we wanted to understand how miRNAs contribute to the development of IR. Amongst the identified and validate miRNAs, the expression of miR-27b was found to be highly upregulated during CI-induced IR in 3T3-L1 adipocytes. We also validated the expression of miR-27b in CIinduced IR in human mesenchymal stem cell (hMSC)-derived adipocytes and in vivo high fat diet (HFD)-induced IR mice model. Bioinformatics target prediction softwares and luciferase reporter assay identified insulin receptor (INSR) as one of a prime target of miR-27b. Lentiviral mediated overexpression of miR-27b impairs insulin signaling by modulating INSR expression that in turn led to decreased glucose uptake in both 3T3-L1 and hMSC-derived adipocytes. Conversely, inhibition of miR27b reversed CI-mediated suppression of target protein INSR and improved phosphorylation of Akt, a nodal protein of insulin signaling that is impaired by CI treatment. Lentiviral mediated overexpression of miR-27b in in vivo C57BL/6 mice impaired whole body glucose tolerance and adipose tissue insulin sensitivity. Furthermore, inhibition of miR-27b in HFD-induced insulin resistance mice model improved glucose tolerance and adipose tissue insulin sensitivity by increasing the expression of its target gene INSR in eWAT. Thus, our results indicate that miR-27b functions as a prime modulator of CI-induced IR via regulating the expression of INSR. Key messages & miR-27b is upregulated in different in vitro and in vivo models of insulin resistance. & miR-27b directly suppresses the expression of INSR by targeting 3’UTR of INSR. & Modulation of miR-27b expression regulates insulin sensitivity by targeting INSR. Keywords Chronic hyperinsulinemia . miR-27b . Insulin resistance . Insulin receptor . High fat diet

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00109-018-1623-z) contains supplementary material, which is available to authorized users. * Anil Nilkanth Gaikwad [email protected] 1

Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031, India

2

Academy of Scientific and Innovative Research, CSIR-CDRI, Lucknow, India

3

SIPS Superspeciality Hospital, 29-Shahmeena Road, Lucknow 226003, India

Introduction In the recent years, the prevalence of obesity has been increasing rapidly worldwide, which is a major risk factor for type 2 diabetes, cardiovascular diseases, and certain types of cancer [1–4]. The critical factor that links obesity to type 2 diabetes is insulin resistance (IR). IR describes the failure of insulin to activate insulin signaling leading to reduced GLUT4 translocation and subsequent extracellular glucose uptake [5]. Mehran et al. revised the old model of hyperinsulinemia as a link between obesity and insulin resistance. The new model states that manifestation of hyperinsulinemia occurs before

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obesity and IR leading to the development of type 2 diabetes [6]. Insulin imparts its biological functions by binding to the insulin receptor (INSR), the prime interactor of insulin signaling. This binding triggers phosphorylation of insulin receptor substrate-1 (IRS-1) at tyrosine residues. Phosphorylated IRS-1 transduces the insulin signals to PI3K/Akt pathway which finally execute GLUT4 translocation to the cell membrane and subsequent uptake of glucose from outside of the cell [7, 8]. miRNAs are small, endogenous, 20–22 nucleotide long non-coding RNAs that bind to the 3’UTR of target mRNA transcript triggering either translational block or mRNA degradation or both [9, 10]. These post-transcriptional regulators affect a variety of processes involved in adipocyte differentiation, insulin secretion, and insulin sensitivity [11–14]. Several miRNAs are dysregulated in obesity, which contributes to secondary complications associated with obesity like cardiovascular diseases and type 2 diabetes [15–18]. Thus, studies on miRNA can provide us opportunities to identify novel pathways and targets as biomarkers associated with obesity and metabolic complications. Previous studies have reported the role of miRNAs in the regulation of lipid and glucose homeostasis that are associated with the pathophysiology of IR and type 2 diabetes [19–23]. miR-143 impairs glucose homeostasis via inhibiting insulin stimulated Akt activation [17]. miR-802 decreases hepatic insulin sensitivity and impair glucose metabolism through targeting Hnf1b [24]. miR-29 suppresses insulin stimulated glucose uptake through indirect inhibition of Akt activation [25]. On the contrary, miR-26a is known to improve insulin sensitivity and glucose and lipid metabolism by decreasing hepatic glucose production and fatty acid synthesis [26]. These earlier findings strongly suggest the role of miRNAs in contributing metabolic disease etiology and provide insights of molecular targets for therapeutic intervention to cure IR and type 2 diabetes. In the present study, following to differential expression profiling of miRNAs in chronic hyperinsulinemia (CI)-induced IR in 3T3-L1 and the control adipocyte, we identified miR-27b as significantly upregulated miRNA in early insulinresistant adipocytes. Using bioinformatics and pathway enrichment analysis, we identified that miR-27b directly targets 3’UTR of INSR. This was further validated in our in vitro and in vivo models of IR and found that it suppresses INSR expression leading to decreased insulin sensitivity in both 3T3L1 and human mesenchymal stem cell (hMSC)-derived IR adipocytes and HFD-induced obesity and IR mice model. Inhibition of miR-27b improved insulin sensitivity impaired by CI treatment. Lentiviral mediated miRNA modulation studies in mice models further validated our in vitro observations. Our studies thus proved that altered miR-27b significantly contributes to adipocyte IR development by functional modulation of the INSR expression.

Materials and methods Cell culture The mouse 3T3-L1 preadipocyte cell line culture and differentiation were described previously [27]. hMSCs were isolated from liposuction samples as approved by the Institutional Committee on Stem Cell Research (ICSCR) of Dr. Ram Manohar Lohia Institute of Medical Science (RMLIMS), Lucknow (IEC no.: 20/14). Cell culture and differentiation of hMSC to mature adipocytes were described elsewhere [28]. Insulin resistance development The development of CIinduced IR was described previously [29]. In brief, the pathophysiological condition of hyperinsulinemia was developed by incubating differentiated adipocytes with 500 pM of insulin (Sigma) for 72 h (CI). The medium was replaced with fresh medium at every 24 h to maintain insulin level. After 72 h, cells were washed extensively using a KRH buffer and proceeded for further experiments. Differential miRNA and mRNA expression profiling Differential expression of miRNA and mRNA were outsourced from iLife Discoveries Pvt. Ltd. (Gurgaon, India). The total RNA was isolated (isolation of total RNA was discussed later in this section) from four samples (two control and two CI-induced insulin-resistant 3T3-L1 adipocytes) and provided for differential miRNA and mRNA expression profiling. The differential expression profiling of miRNA and mRNA was done on Affymetrix and Agilent platform, respectively. Data provided in supplementary files 1 and 2. Lentivirus production and transduction pLenti-miR27b (overexpression of miR-27b, cat. no. MmiR3461-MR03), pLentiEV (control plasmid), pLenti-miR-27b-Inhib (inhibition of miR-27b, cat. no. MmiR-AN0360-AM04), and pLenti-ConInhib (scramble control plasmid, cat. no. CmiR-AN0001AM04) were purchased from GeneCopoeia (Rockville, MD, USA). Packaging plasmids (psPAX2 and pMD2.G) were gifted by Didier Trono (Addgene plasmids: 12260, 12259). Recombinant lentiviral particles for miR-27b overexpression and miR-27b inhibition were produced by the calcium phosphate transfection method and stored at − 80 °C. After 8 days of differentiation, the 3T3-L1 and hMSCderived adipocytes were transduced with lentiviruses. The transduction was carried out with protamine sulfate at 12 μg/ml. [3H] 2-deoxyglucose uptake 2-Deoxyglucose (2-DOG) uptake in mature, lentiviral transduced adipocytes was performed as described previously [28]. Radioactivity retained by the cell lysate was measured by a liquid scintillation counter (Beckman Coulter, LS 6500, Beckman Coulter Inc. USA).

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Animal experiment Six- to eight-week male C57BL/6 mice were used in the study. All the experimental protocols were followed as per CPCSEA guidelines and approved by the Institutional Animal Ethics Committee (IAEC), CSIR-CDRI, Lucknow. Mice were acclimatized for 1 week and maintained at well-ventilated conditions (60–65% RH, 25 ± 2 °C temperature in the institutional animal house) throughout the experiment. Mice were fed with normal chow diet (10% Kcal from fat, Cat No. 12450 K, Research Diets Inc., USA) and high fat diet (60% Kcal from fat, cat. no. D12492, Research Diets Inc., USA) for 6 weeks. Food intake and body weight were monitored weekly. For intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT), mice were fasted for 6 and 5 h, respectively. Two g/kg glucose load and 0.75 U/kg insulin load were administered intraperitoneally (i.p.) for IPGTT and ITT, respectively. Blood glucose was measured at basal, 15, 30, 60, 90, and 120 min using an Accucheck glucometer. At the end of 8 weeks, mice were fasted overnight, pulsed with insulin (0.75 IU/kg, i.p.) for 15 min, perfused with normal saline, and sacrificed. Epididymal white adipose tissue (eWAT) was collected and stored at − 80 °C. Western blotting It is performed as described previously [30]. Monoclonal rabbit antibodies against p-Akt (Ser473) (cat. no. 4060S), Akt (cat. no. 9272S), p-AS160 (Thr642) (cat. no. 8881S), AS160 (cat. no. 2670S), PPARγ (cat. no. 2430S), cMyc (cat. no. 5605S), and mouse INSR β (cat. no. 3020S) were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin (cat. no. sc-47778) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Densitometric quantification of protein bands was performed using the National Institutes of Health (NIH) ImageJ software. RNA isolation and qRT-PCR Total RNA was isolated from in vitro and in vivo samples using a TRIZOL reagent. First strand cDNA synthesis for miRNA and total mRNA was performed using high capacity cDNA reverse transcription kit from isolated RNA. miRNA-specific cDNA and total cDNA were then subsequently used for quantitative real-time PCR analysis on Light Cycler 480 (Roche Diagnostics) using SYBR Green master mix (Kapa Biosystems). The list of primers used is given in Table S1 of the supplementary material. Luciferase assay Luciferase reporter plasmid containing wild type 3’UTR of INSR was purchased from GeneCopoeia (cat. no. MmiT029471b-MT06, Rockville, MD, USA). Mutated 3’UTR of INSR containing three-point mutations within the binding site of miR-27b was synthesized in pUC57 plasmid from GenScript. Mutated 3’UTR of INSR from pUC57 plasmid was then cloned into a luciferase reporter plasmid. The luciferase reporter constructs containing wild-type and mutated 3’UTR of INSR were transfected in Lenti-EV and Lenti-

miR-27b-transduced HEK 293T cells to determine the effects of overexpression of miR-27b on luciferase translation. After 48 h of transfection, luciferase activities were assayed using a luciferase reporter assay system (Promega). Experiments were performed in triplets. Leptin ELISA Leptin was measured in in vitro culture medium and in vivo mice serum sample as per the manufacturer’s protocol using Leptin DuoSet ELISA kit purchased from R&D Systems (cat. no. DY498). Statistical analysis Data were expressed as mean ± SD for in vitro and mean ± SEM for in vivo metabolic parameters. Statistical significance between the two conditions was analyzed by Student’s t test and more than two conditions were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Data were analyzed on Graph Pad Prism (Version3.00). P values less than < 0.05 were considered significant. For differential miRNA and mRNA expression profiling, significance was analyzed by unpaired asymptotic t test method [31, 32]. The significant p value cutoff 0.05 has been taken. Fold change was calculated between the treated and the control sample. miRNAs with P value ≤ 0.05 and fold change ≥ 4 were chosen for further studies.

Results Identification of miR-27b as a regulator of insulin signaling from differential miRNA analysis In order to identify differentially altered miRNAs in our previously developed CI-mediated IR in 3T3-L1 adipocytes [29], the differential miRNA expression profiling was performed using the Affymetrix platform on an RNA isolated from the control and CI-exposed insulin-resistant 3T3-L1 adipocytes. Differential miRNA expression profiling identified 38 miRNAs of which 22 miRNA were significantly upregulated and 16 were downregulated in CI-induced insulin-resistant 3T3-L1 adipocytes (Supplementary file 1). Out of 22 upregulated miRNAs, we chose 10 miRNAs (miR-26a, 23a, 151, 222, let7f, 183, 542, 346, 27b, and 23b) for further validation as they were highly expressed, i.e., more than four folds and have some association with metabolic disorders based on literature evidences [26, 33–39]. Real-time PCR based validation studies showed that out of 10 selected miRNAs, the expression of 8 miRNAs was significantly increased with miR27b to be highly upregulated in the CI-induced insulin-resistant 3T3-L1 adipocyte (Fig. 1a). We utilized open source target prediction softwares (miRWalk, Microt4, miRanda, miRDB, miRNAMAP, PITA, RNAZ2, RNAhybrid, and TargetScan) to identify all possible predicted targets of the above eight upregulated miRNAs. For each miRNA, those

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Fig. 1 Identification of miR-27b in differential miRNA analysis and validation in different models of IR. a Validation of identified miRNAs in CI-induced IR in 3T3-L1 adipocytes. qRT-PCR analysis of 10 identified miRNAs in CI-induced insulin-resistant 3T3-L1 adipocytes. miRNA expression was normalized with U6snRNA as an endogenous reference gene. N = 3, error bars represent SD, *P < 0.05, **P < 0.01, ***P < 0.001 as analyzed by two-way ANOVA followed by

Bonferroni’s post analysis test. b Pathway enrichment of validated targets of miR-27b using the EnrichR software. Arrow denotes insulin signaling pathway in mouse and human. c qRT-PCR analysis of miR-27b expression in the control and CI-induced IR in hMSC-derived adipocytes. d, e qRT-PCR analysis of miR-27b expression in 4-week and 8-week HFD fed mice model of IR, respectively. N = 3, error bars represent SD, **P < 0.01, *P < 0.05 as analyzed by Student’s t test

targets predicted by five or more than five softwares were selected for which downregulation was anticipated. The above anticipated downregulated gene list of individual miRNA was mapped against significantly downregulated differential gene expression profiling data (Agilent platform; Supplementary file 2). This provided us with a list of total validated targets of each miRNA for pathway analysis (Table 1 and

Supplementary file 3–10). Pathway analysis using the EnrichR software showed that out of eight selected miRNAs, only targets of miR-27b were directly involved in insulin signaling pathway, whereas the targets of other miRNAs were involved in MAPK, TGFβ or immune system related pathways (Fig. 1b and Supplementary Fig. 1). So from the pathway analysis, we decided to primarily focus our

J Mol Med Table 1

Total numbers of genes targeted by eight selected miRNAs

miRNA

Total number of validated targets

miR-26a

993

miR-151-5p

278

miR-let-7f miR-183

751 947

miR-542-5p miR-346

251 863

miR-27b

584

miR-23b

627

studies on the possible role of miR-27b in insulin resistance pathophysiology. Further to this, we assessed expression of miR-27b in our later developed model of CI-induced IR in adipocytes differentiated from human mesenchymal stem cells (hMSC) [28]. Similar to 3T3-L1 adipocytes, we found an increased expression of miR-27b in CI-induced IR in hMSC-derived adipocytes (Fig. 1c). We also found significantly increased expression of miR-27b in epididymal white adipose tissue of mice fed with high fat diet for 4 and 8 weeks. It was interesting to note that 8-week HFD fed mice showed higher expression of miR-27b (5-fold increased expression) than 4-week HFD fed mice (Fig. 1d, e). During development of CI-induced insulin resistance model, we did not find insulin resistance like phenotype up to 60 h of chronic insulin exposure. We could observe the IR phenotype only after 72 h of CI treatment [28, 29]. Correlating to this, at 60 h of the CI treatment point, we did not find an increase in miR-27b expression, but its expression was significantly upregulated at 72 h of CI exposure (Supplementary Fig. 2). Chen et al. showed that the transcription factor, c-Myc, suppressed the expression of miR23b and miR-27b via binding to the enhancer sequences (E-box) in the promoter region of miR-23b-27b-24-1 cluster [40]. To explore how the CI treatment upregulates the expression of miR-27b, we assessed the expression of cMyc in CI-induced IR 3T3-L1 adipocytes. Exposure of 3T3-L1 adipocytes to chronic insulin reduced the expression of c-Myc as compared to the control adipocytes. Thus, the decreased expression of c-Myc was negatively correlated with the increased expression of miR-27b in CIinduced IR adipocytes (Supplementary Fig. 3a, b).

Target identification of miR-27b Using the EnrichR software and validated targets of miR-27b, we found that out of total 154 genes from mouse and 160

genes from human genome associated with the insulin signaling pathway, 10 genes were found as targets of miR-27b with possibilities to play role in insulin signaling alteration (Table 2). The inverse correlation study showed insulin receptor (INSR) to be 3.7-fold downregulated whereas its target miR-27b was 5.4-fold upregulated in differential expression data of CI-induced IR adipocytes. Out of above 10 validated targets, INSR is the first and foremost protein of the insulin signaling transduction network and hence found very interesting to focus on. We also found the decrease in INSR expression at mRNA level as well as decreased β subunit of INSR at protein level in CI-induced insulin-resistant 3T3-L1 and hMSC-derived white adipocytes (Fig. 2a–d). To confirm further, whether INSR expression is indeed modulated by miR-27b, we analyzed the binding site of miR-27b within the 3’UTR region of INSR. From bioinformatics analysis, we found a highly conserved binding site of miR-27b in the 3’UTR region of the INSR gene in several species such as mouse, rat, human, rabbit, chimpanzee, and guinea pig (Fig. 2e). The binding site of miR-27b in 3’UTR of INSR was further confirmed by luciferase reporter assay. Herein, luciferase reporter cloned constructs were prepared with wild-type and mutated (three-point mutations in the binding site of miR-27b) 3’UTR of INSR (Fig. 2f). Reporter constructs containing wild-type and mutated 3’UTR of INSR were transfected in Lenti-EV and Lenti-miR-27b transduced HEK 293T cells. Luciferase reporter assays revealed that overexpression of miR-27b reduced the activity of luciferase in reporter construct containing wild-type 3’UTR, but not in the mutated 3’UTR (Fig. 2g, h). These results suggest that INSR might be one of the direct target of miR-27b. The detailed process of pathway analysis and identification of validated targets was given in BSupplementary material^.

Increased miR-27b impairs insulin signaling and reduces glucose uptake in 3T3-L1 and hMSC-derived adipocytes To investigate the putative role of miR-27b in modulating insulin signaling, we overexpressed miR-27b in 3T3-L1 adipocyte using lentivirus particles of miR-27b. Mature adipocytes were transduced with Lenti-EV and Lenti-miR-27b for 72 h (Fig. 3a). An increase in miR-27b expression confirmed the overexpression of miR-27b in 3T3-L1 adipocytes (Fig. 3b). Overexpression of miR-27b suppressed the expression of its target gene INSR which was confirmed at mRNA level and β-chain of INSR at the protein level (Fig. 3c, d). Adipocytes transduced with Lenti-miR-27b impaired insulin signaling and developed an IR-like phenotype. We observed a decrease in phosphorylations of Akt at Ser473, and its downstream substrate AS160 at Thr642 following insulin stimulation (10 nM) in Lenti-miR-27b transduced adipocyte when compared with Lenti-EV transduced adipocytes (Fig. 3e).

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Fig. 2 miR-27b binds to 3’UTR of INSR mRNA. a, c qRT-PCR analysis of INSR in the control and CI-exposed 3T3-L1 and hMSC-derived adipocytes after 72 h of the CI treatment. b, d Western blots of βsubunit of INSR in 3T3-L1 and hMSC-derived adipocytes after 72 h of the CI treatment. Densitometry of representative blots were normalized with β-actin. Data is represented in fold difference. N = 3, error bars represent SD, *P < 0.05, ***P < 0.001 as tested by Student’s t test. e The conserved binding site of miR-27b within the 3’UTRs of different species as identified by the TargetScan software. f The 3’UTR of INSR

contains the binding site for miR-27b as shown by colored sequences. In the mutant construct, the corresponding binding site contains three-point mutations. Luciferase reporter assay was performed to assess the interaction between miR-27b with its target gene INSR. Lenti-EV and Lenti-miR-27b-transduced HEK 293T cells were transfected with a luciferase reporter plasmid containing g wild-type 3’UTR of INSR and h mutated 3’UTR of INSR. Experiments were repeated three times. Data is represented as mean ± SD. ***P < 0.001 as tested by Student’s t test.

Insulin-resistant phenotype was further confirmed by insulin stimulated radiolabeled tritiated 2-4 deoxyglucose uptake assay in Lenti-miR-27b and Lenti-EV-transduced 3T3-L1

adipocytes. We observed a significantly decreased glucose uptake in miR-27b overexpressed adipocytes (Fig. 3f). Similar to 3T3-L1 adipocytes, miR-27b impaired insulin

J Mol Med Table 2 Validated targets of miR27b involved in insulin signaling identified by the EnrichR software

Pathway

Overlap

P value*

Genes

Insulin signaling Mus musculus

10/154

0.003955

Insulin signaling Homo sapiens

10/160

0.005074

MAP3K2, GSK3B, MAP3K1, PRKAA2, INSR, TBC1D4, KIF5B, RAC2, TSC1, SOS1 MAP3K2, GSK3B, MAP3K1, PRKAA2, INSR, TBC1D4, KIF5B, RAC2, TSC1, SOS1

*P value is computed using a standard statistical method: Fisher’s exact test or the hypergeometric test by the EnrichR software. This is a binomial proportion test that assumes a binomial distribution and independence for the probability of any gene belonging to any set

signaling in hMSC-derived adipocytes as confirmed by reduced expression of INSR and phosphorylation of Akt and AS160 proteins. miR-27b-reduced insulin stimulated glucose uptake in hMSC-derived adipocytes also (Fig. 3g–l). We have also found decreased expression of INSR in miR-27b overexpressed HEK 293T cells (Fig. 3m). Following earlier reports, we have also assessed the expression pattern of other targets of miR-27b such as PPARγ and leptin [41–43]. Overexpression of miR-27b significantly decreased the expression of PPARγ at both mRNA and protein level in normal 3T3L1 adipocytes (Fig. 3n, o). However, we did not find decrease in leptin level in culture media of miR-27b overexpressed 3T3L1 adipocytes but leptin level was rather increased as compared to Lenti-EV transduced 3T3-L1 adipocytes (Supplementary Fig. 4a). This might be fact that the earlier report was in chondrocyte cells [43] while all our studies were in 3T3-L1 adipocytes. Thus, we conclude that the increased miR-27b downregulate the expression of INSR as well as PPARγ level concomitant to impaired insulin signaling, decreased glucose uptake, and further lead to the development of an IR-like phenotype.

Inhibition of miR-27b increases INSR expression and restores insulin signaling impaired by CI exposure After validating the expression and role of miR-27b in CIinduced IR, we further wanted to study whether inhibition of miR-27b could reverse the CI-induced IR-like phenotype in 3T3-L1 and hMSC-derived adipocytes. For this, lentiviral construct with inhibitor sequence against miR-27b (LentimiR-27b Inhib) was used to inhibit mature miR-27b expression. Lenti-Con Inhib was used as the negative control. To assess insulin signaling, lentiviral particles of the control inhibitor and miR-27b inhibitor were transduced followed by CI exposure to generate IR in 3T3-L1 adipocytes (Fig. 4a). Reduced expression of miR-27b following CI exposure in 3T3-L1 adipocytes confirm inhibition of miR-27b expression (Fig. 4b). Inhibition of miR-27b during the CI treatment leads to increased mRNA level of INSR as compared to the control condition (Fig. 4c). Furthermore, the expression of β-chain of INSR and insulin-stimulated phosphorylations of Akt at Ser473 and AS160 at Thr642 were also increased after

inhibition of miR-27b in CI-induced insulin-resistant 3T3L1 adipocytes (Fig. 4d). Similarly, the inhibition of miR-27b expression restored insulin signaling in CI-induced insulinresistant hMSC-derived adipocytes also (Fig. 4e–h). Taking clue from earlier studies and our own previous results, we assessed the expression of PPARγ and leptin after the inhibition of miR-27b in CI-induced IR 3T3-L1 adipocytes. We found an increased expression of PPARγ at both mRNA and protein levels, whereas the level of leptin was found to be decreased after the inhibition of miR-27b in CI-induced IR adipocytes (Fig. 4i, j and Supplementary Fig. 4b). Taken together, these results indicate a potential role of miR-27b in CIinduced IR. We also checked the expression of miR-27a to assess whether it has any compensatory role after inhibition of miR-27b in CI-induced IR 3T3-L1 adipocytes. Inhibition of miR-27b in CI-induced IR adipocytes did not alter the miR27a expression level (Supplementary Fig. 5).

miR-27b impairs insulin signaling and generates IR in white adipose tissue of the in vivo mice model As mentioned earlier, we have observed an increased miR-27b expression in the HFD fed insulin-resistant mice model at 4 and 8 weeks. So, we wanted to assess if the overexpression of miR-27b has any role in regulating insulin sensitivity when given in C57BL/6 mice model fed on normal chow diet. The control and miR-27b lentiviral particles were delivered twice at an interval of 7 days by tail vein injection in mice (Fig. 5a). After 2 weeks of treatment, mice receiving Lenti-miR-27b showed impaired glucose tolerance and decreased insulin sensitivity as compared to Lenti-EV control mice (Fig. 5b, c). However, there was no significant gain in body weight and organ weight between mice infected with Lenti-EV and LentimiR-27b (Fig. 5d). Compared to Lenti-EV control mice, Lenti-miR-27b showed a significant increase in miR-27b expression in eWAT which proved overexpression of miR-27b in eWAT (Fig. 5e). The expression of miR-27b was also assessed in brown adipose tissue (BAT), liver, and skeletal muscle. We found a significantly increased level of miR-27b in BAT

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ƒFig. 3

Overexpression of miR-27b impairs insulin signaling in 3T3-L1 and hMSC-derived adipocytes. 3T3-L1 and hMSC-derived adipocytes were transduced with Lenti-EV and Lenti-miR-27b and cultured for the next 72 h. a–f 3T3-L1adipocytes. g–l hMSC-derived adipocytes. a, g Microscopic images of green fluorescent protein (GFP) confirming the efficiency of lentivirus transduction in 3T3-L1 and hMSC-derived adipocytes, respectively. b, h Real time analysis of overexpression of miR-27b in 3T3-L1 and hMSC-derived adipocytes, respectively. U6 snRNA was used as an internal control. c, i Relative expression of INSR in Lenti-EV and Lenti-miR-27b-transduced 3T3-L1 and hMSC-derived adipocytes, respectively. N = 3, error bars represent SD, *P < 0.05, ***P < 0.001 as analyzed by Student’s t test. d, j Western blots of β-subunit of INSR in 3T3-L1 and hMSC-derived adipocytes, respectively, after the transduction with Lenti-EV and Lenti-miR7b. Densitometry of representative blot was normalized with β-actin. Data is represented in fold difference. N = 3, error bars represent SD, *P < 0.05, **P < 0.01, as tested by Student’s t test. e, k Western blot analysis of p-Akt (Ser473) and p-AS160 (Thr642) in Lenti-EV and Lenti-miR-27b-transduced 3T3-L1 and hMSC-derived adipocytes, respectively. Cell lysate was prepared after 10 nM of insulin pulsing. Densitometry of p-Akt and p-AS160 was normalized with Akt and AS160 proteins respectively (eI, eII and kI, kII). Data is represented in fold difference. N = 3, error bars represent SD, *P < 0.05, ***P < 0.001 as tested by one-way ANOVA followed by Bonferroni’s multiple comparison test. f, l Glucose uptake in Lenti-EV and Lenti-miR-27btransduced adipocytes. Experiment was performed with or without insulin pulsing of 10 nM. N = 3, error bars represent SD, ***P < 0.001, **P < 0.01 as tested by one-way ANOVA followed by Bonferroni’s multiple comparison test. m Western blots of β-subunit of INSR in HEK 293T cells after transduction with Lenti-EV and Lenti-miR7b. Densitometry of representative blot was normalized with β-actin. Data is represented in fold difference. N = 3, error bars represent SD, *P < 0.05, as tested by Student’s t test. n Relative expression of PPARγ in Lenti-EV and Lenti-miR-27b-transduced 3T3-L1 adipocytes. N = 3, error bars represent SD, **P < 0.01, as tested by Student’s t test. o Western blot analysis of PPARγ in Lenti-EV and Lenti-miR-27b-transduced 3T3-L1 adipocytes. Densitometry of representative blot was normalized with β-actin. Data is represented in fold difference. N = 3, error bars represent SD, ***P < 0.001, as tested by Student’s t test

and liver, but not in muscle of Lenti-miR-27b injected mice (Fig. 5f–h). Similar to in vitro observations, we found that the INSR mRNA and protein level expression of β chain of INSR was significantly decreased in eWAT of mice overexpressed with miR-27b (Fig. 5i, j). We further examined the phosphorylation of Akt protein in eWAT. Mice injected with Lenti-miR-27b show decreased phosphorylation of Akt in eWAT as compared to the Lenti-EV control mice after insulin pulsing (intraperitoneal injection of 0.75 U/kg of insulin for 15 min) (Fig. 5j). Following earlier reports, we also assessed the expression pattern of PPARγ and leptin, other targets of miR-27b in miR-27b overexpressed mice. Overexpression of miR-27b significantly decreased expression of PPARγ in eWAT of LentimiR-27b injected mice as compared to Lenti-EV at both the mRNA and the protein level (Fig. 5k, l). However, the serum level of leptin was increased in Lenti-miR-27b injected mice (Supplementary Fig. 4c). Earlier data indicate that

miR-27b is involved in the impairment of insulin signaling and the development of IR in adipose tissue of an in vivo mice model too.

Inhibition of miR-27b improves glucose tolerance and insulin sensitivity in the mice model of HFD-induced IR Following abovementioned experiment and our earlier observation of increased miR-27b in the HFD fed mice model of obesity, we decided to undertake studies on inhibition miR27b in HFD fed model of obesity. Four-week HFD fed mice were injected with Lenti-Con-Inhib and Lenti-miR-27b-inhib by tail vein injection method. It was repeated once after 7 days and kept on HFD during the study period (Fig. 6a). After completion of 6 weeks, we observed significantly improved glucose tolerance and insulin tolerance in Lenti-miR-27b injected mice as compared to Lenti-Con-Inhib injected mice (Fig. 6b, c). Lenti-miR-27b-Inhib injected mice significantly reduced body weight and eWAT compared to that of LentiCon-Inhib injected mice (Fig. 6d, e). The decreased expression of miR-27b in eWAT confirmed the inhibition of miR-27b expression in LentimiR-27b-Inhib injected HFD fed mice (Fig. 6f). Inhibition of miR-27b in Lenti-miR-27b Inhib injected mice fed on HFD diet showed a significant reduction in miR-27b expression in BAT and liver as compared to HFD fed LentiCon Inhib mice, but no significant difference was observed in miR-27b expression in muscle of HFD fed Lenti-miR27b Inhib as compared to Lenti-Con Inhib (Fig. 6g–i). Furthermore, Lenti-miR-27b-Inhib infected mice show increased mRNA level of INSR and INSR β chain and phosphorylation of Akt protein (Fig. 6j, k). Suppression of miR27b in HFD fed Lenti-miR-27b Inhib mice found corroborating to increased expression of PPARγ in eWAT at both the mRNA and the protein level and decreased serum leptin level (Fig. 6l, m, Supplementary Fig. 4d). Thus, suppression of miR-27b in eWAT improve impaired insulin signaling caused due to HFD-induced IR.

Discussion Currently, miRNA-mediated regulation of energy homeostasis is increasingly being reported by the scientific community, but how miRNA(s) regulate adipocyte insulin signaling and glucose homeostasis is still a gap area. In the present study exploiting physiological hyperinsulinemic IR model system, we have systematically identified miR-27b as an important microRNA found to be upregulated in insulin-resistant 3T3L1 adipocytes. Our studies demonstrate that miR-27b is

J Mol Med

capable of directly modulating insulin signaling by targeting INSR expression. We further validated the role of miR-27b in

regulating insulin sensitivity and glucose homeostasis exploiting different in vitro and in vivo models of IR.

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ƒFig. 4

Inhibition of miR-27b restores insulin signaling impaired by CI exposure. Lenti-Con-Inhib and Lenti-miR-27b Inhib were transduced in 3T3-L1 and hMSC-derived adipocytes. a–d 3T3-L1 adipocytes. e–h hMSC-derived adipocytes. a, e Microscopic images of red fluorescent protein, confirming the efficiency of lentivirus transduction in 3T3-L1 and hMSC-derived adipocytes, respectively. b, f Real-time analysis of inhibition of miR-27b in CI-exposed Lenti-Con-Inhib and Lenti-miR27b-Inhib-tranduced 3T3-L1 and hMSC-derived adipocytes, respectively. c, g Relative mRNA expression of INSR in Lenti-Con-Inhib and Lenti-miR-27b-Inhib-transduced 3T3-L1 and hMSC-derived adipocytes, respectively. N = 3, error bars represent SD, *P < 0.05, **P < 0.01, ***P < 0.001 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. d, h Lenti-Con-Inhib and LentimiR-27b-Inhib-transduced control and CI-treated 3T3-L1 and hMSCderived adipocytes, respectively, were lysed after 10 nM of insulin pulsing for 20 min and used for immunoblotting to evaluate INSR β-chain and p-Akt and p-AS160 expression. Densitometry of INSR β-chain, pAkt, and p-AS160 was normalized with β-actin Akt and AS160 proteins, respectively. Data is represented in fold difference. N = 3, error bars represent SD, *P < 0.05, **P < 0.01, ***P < 0.001 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. i Relative expression of PPARγ in Lenti-Con-Inhib and Lenti-miR-27b-Inhibtransduced control and CI-treated 3T3-L1 adipocytes, respectively. N = 3, error bars represent SD, ***P < 0.001 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. j Western blot analysis of PPARγ in Lenti-Con-Inhib and Lenti-miR-27b-Inhibtransduced control and CI-treated 3T3-L1 adipocytes. Densitometry of representative blot was normalized with β-actin. Data is represented in fold difference. N = 3, error bars represent SD, ***P < 0.001 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

We have previously reported that adipocytes on exposure to 500 pM insulin for 72 h (CI) develop an IR-like phenotype [28, 29]. In the same model system, we have performed differential mRNA and miRNA expression profiling in the control and CI-induced insulin-resistant 3T3L1 adipocyte. The differential miRNA expression profiling in CI-induced IR in 3T3-L1 adipocytes presented 22 miRNAs to be significantly upregulated. From these 22 upregulated miRNAs, 10 miRNAs (miR-26a, 23a, 151, 222 let7f, 183, 542, 346, 27b, and 23b) were chosen for further confirmation on the basis of their highest level of upregulation and the previously known literature associated with IR and type 2 diabetes. Following systematic in vitro and in vivo approaches, we identified miR-27b to be capable of significantly contributing to IR generation at an early stage by modulating insulin receptor expression and downstream insulin signaling. From the identified miRNAs differentially expressed in CI-induced IR, few were earlier reported to contribute IR pathophysiology. miR-26a is known to regulate hepatic glucose and lipid metabolism and improve insulin sensitivity [26]. Zhu et al. reported that the let-7 family, including the let-7f target insulin-PI3K-mTOR pathway, thereby impaired glucose tolerance and development of IR [33]. miR-183 promotes liver IR by targeting the insulin receptor substrate 1

(IRS1) [34]. miR-27b is very well studied in glucocorticoid-induced body fat accumulation and metabolic dysfunction [37]. Chronic glucocorticoid exposure leads to insulin resistance generation [44, 45]. As these miRNAs were also identified in our studies, it further underlines the importance of our chronic hyperinsulinemiainduced IR adipocyte model system close to physiological condition. Our study provided some additional miRNAs which were not picked up in earlier studies owing to its physiological insulin concentration (500 pM) and exposure time period (72 h) differences. The validated targets of the eight identified miRNAs are mainly involved in MAPK signaling, TGFβ signaling, and signaling associated with the immune modulations. Earlier reports suggest the association of hyperinsulinemia with proinflammatory signaling, which in turn leads to the development of IR and type 2 diabetes [46, 47]. TGFβ/SMAD3 and MAPK signaling pathway play an important role in regulating glucose tolerance and energy homeostasis [48, 49]. A detailed pathway analysis performed for these eight miRNAs identified miR-27b to be the only miRNA capable of targeting insulin signaling. miR-27b is clustered in the miR-23b-27b-24-1 cluster. The miRNA microarray expression profiling did not show any change in expression of miR-24-1 while miR-23b and miR-27b both were significantly altered in CI-induced IR adipocytes. It is well reported that c-Myc, a transcription factor, regulates the expression of miR-23b and the miR27b of miR-23b-27b-24-1 cluster at the transcriptional level via binding to the enhancer sequences (E-box) in the promoter region. Chen et al. showed that the binding of c-Myc suppresses the expression of miR-23b and miR27b [40]. The previous finding is very well corroborated with a decreased c-Myc expression and an increased miR27b level in our CI-induced IR 3T3-L1 adipocytes. Therefore, there might be a strong possibility of inverse correlation between the expression of miR-27b and c-Myc in CI-induced IR adipocytes, and indeed, it was further confirmed by modulating the expression of c-Myc (Supplementary Fig. 3). Further, experimental evidences related to the binding of c-Myc to the promoter region of miR-23b-27b-24-1 are necessary to confirm its role in the development of insulin resistance which is beyond the scope of current manuscript. The validation of eight identified miRNAs showed that miR-27b was highly upregulated in CI-induced IR adipocytes. Sequence homology shows a highly conserved miR-27b sequence between mouse, human, and other species too. Meanwhile, our laboratory has also developed chronic hyperinsulinemia induce IR model using hMSC-derived adipocyte [28]. We, therefore, evaluate the role of miR-27b in this model system also. We found an increased expression of miR27b in CI-induced IR in hMSC-derived adipocytes too. The

J Mol Med

inverse correlation analysis identified increased expression of miR-27b with decreased INSR expression in CI-induced IR

adipocytes. The bioinformatics analysis and luciferase reporter assay confirmed the binding of miR-27b within the 3’UTR

J Mol Med

ƒFig. 5

Overexpression of miR-27b impairs insulin sensitivity and generates IR in eWAT of C57BL/6 mice. a Schematic representation of experimental procedure. C57BL/6 mice were fed on chow diet. Mice were injected with lentivirus injection twice through tail vein at day 1 and day 7. b, c At 14 and 16 days of injection, blood glucose was measured through IPGTT and ITT, respectively. d At day 18, mice were sacrificed and organ weight was measured. N = 5–6 mice per group, error bars represent SEM., *P < 0.05 as analyzed by Student’s t test. e–h qRTPCR analysis of miR-27b in eWAT, BAT, liver, and muscle of Lenti-EV and Lenti-miR-27b injected mice (N = 5–6), respectively. Error bars represent SD, *P < 0.05 as analyzed by Student’s t test. n.s. non-significant. i qRT-PCR analysis of INSR in eWAT of Lenti-EV and Lenti-miR-27b injected mice (N = 5–6). Error bars represent SD, ***P < 0.001 as analyzed by Student’s t test. j Western blot analysis of INSR β and p-Akt (Ser473) in eWAT of Lenti-EV and Lenti-miR-27b injected mice pulsed with insulin (0.75 U/kg, i.p.). Densitometry of INSR β and p-Akt was normalized with β-actin and Akt protein, respectively, N = 5–6 mice per group. Error bars represent SD, *P < 0.05, **P < 0.01 as analyzed by Student’s t test. k qRT-PCR analysis of PPARγ in eWAT of Lenti-EV and Lenti-miR-27b injected mice (N = 5–6). Error bars represent SD, **P < 0.01, as analyzed by Student’s t test. l Western blot analysis of PPARγ in eWAT of Lenti-EV and Lenti-miR-27b injected mice. Densitometry of PPARγ was normalized with β-actin. N = 5–6 mice per group. Error bars represent SD, *P < 0.05, as analyzed by Student’s t test

region of INSR. Given the account, it was imperative to study the interplay between miR-27b and proximal target of insulin signaling, INSR. Friesen et al. reported that the adipocyte specific insulin receptor knockout mice are less insulin sensitive and glucose tolerant as compared to wild-type littermate [50]. Obesity-induced upregulation of miR-15b expression is linked to the pathogenesis of IR in liver through targeting INSR [51]. Our studies also very well corroborate with earlier reports. We also found an increased miR-27b expression in CI-induced IR adipocytes with a decreased INSR expression and a decreased phosphorylation of Akt and AS160 and other parameters of IR. Several studies confirmed the differential expression of miR-27b in different tissues with distinct functions. miR27b post-transcriptionally regulates the expression of TNF-alpha-induced adenosine 2b receptor in the colonic epithelial tissue during colonic inflammation [52]. Rogler et al. showed the role of miR-27b in regulating bone morphogenetic protein signaling and liver stem cell differentiation [53]. miR-27b is involved in the differentiation and development of different types of tissues, such as differentiation of myeloblasts to granulocytes and early phase of ventricular development during cardiogenesis [54, 55]. miR-27b negatively regulates adipogenesis of 3T3-L1 preadipocytes and human multipotent adipose-derived stem cells to adipocytes via targeting PPARγ, the adipogenic key transcription factor [41, 42]. PPARγ is also known to improve insulin sensitivity in type 2 diabetes patients [56]. Our studies positively correlate with the

validation of PPARγ as a target of miR-27b in our overexpression and inhibition of miR-27b experiments. Although Zhou et al. reported leptin as a target of miR27b in osteoarthritic chondrocytes [43], our studies do not exhibit similar pattern. This might be different levels of sensitivities of adipocyte and chondrocytes to insulinmediated leptin secretion. In line with the previous studies of increased plasma leptin level and insulin resistance, we also found an elevated plasma leptin level in miR-27boverexpressed adipocytes. [57, 58]. Kong et al. reported that glucocorticoid upregulates miR-27b expression which is responsible for the inhibition of browning and thermogenic program of WAT via targeting PRDM16 [37]. In our earlier reported study, CI exposure decreases mitochondrial biogenesis and suppresses the expression of brown adipocyte specific markers in hMSC-derived brown adipocytes [28]. The expression of miR-27b was upregulated in the epididymal fat tissue of genetically obese ob/ob mice [41]. The authors also correlate their study of an increased miR-27b expression with the development of hypoxia which inhibits adipogenesis in eWAT of ob/ob mice [41]. The obese fat tissue shows a different tissue microenvironment as compared to that of normal adipose tissue and found to be hypoxic and inflammatory [41]. To summarize, apart from targeting INSR, miR-27b also regulates multiple factors that derive adipose tissue towards IR and obesity. In line with our in vitro studies, we overexpressed miR27b using a lentiviral system in C57BL/6 mice. As anticipated, the overexpression of miR-27b significantly reduced glucose tolerance and insulin sensitivity along with a decreased INSR expression, the target protein, and a decreased phosphorylation of Akt in eWAT. We have also profiled the expression of miR-27b in other organs of miR-27b overexpressed mice such as BAT, liver, and muscle. Overexpression of miR-27b significantly increased the expression of miR-27b in BAT and liver, but we did not find any increase in muscle. The HFD-induced IR model in C57BL/6 mice is an efficient model for IR and obesity with characteristics of hyperglycemia and hyperinsulinemia [59, 60]. Our profiling studies have indicated that miR-27b is increased in the adipose tissue of these animals. Therefore, we decided to use anti-miR27b to inhibit its expression. Inhibition of miR27b using lentiviral system markedly reduced body weight and eWAT weight of mice fed on HFD diet. The expression pattern of miR-27b showed a reduced expression of miR27b in eWAT, BAT, and liver but not in muscle of miR-27binhibited HFD fed mice. In addition, HFD impaired glucose tolerance and insulin sensitivity were improved in mice injected with miR-27b inhibitor. These results indicate that miR-27b significantly contributes to hyperinsulinemiainduced impaired insulin sensitivity in adipose tissue.

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J Mol Med

ƒFig. 6

Inhibition of miR-27b improves glucose tolerance and insulin sensitivity in high fat diet fed mice. a Schematic representation of experimental procedure. C57BL/6J mice were fed 60% high-fat diet (HFD) for 4 weeks. After 4 weeks, HFD fed mice were injected with Lenti-ConInhib and Lenti-miR-27b-Inhib twice through tail vein injection after 7 days of time interval. b, c Blood glucose were measured after 6 weeks for IPGTT and ITT, respectively, in chow fed and HFD with Lenti-ConInhib and Lenti-miR-27b-Inhib injected mice. d Body weight and e Organ weight was measured after sacrifice at the end of experiment. N = 5–6 mice per group, error bars represent SEM., *P < 0.05, **P < 0.01, ***P < 0.001 as tested by one-way and two-way ANOVA followed by Bonferroni’s multiple comparison test. f–i Relative expression of miR-27b in eWAT, BAT, liver, and muscle of chow and HFD fed Lenti-Con-Inhib and Lent-miR-27b-Inhib injected mice (N = 5–6), respectively. Error bars represent SD, *P < 0.05, **P < 0.01 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. n.s. non-significant. j qRT-PCR analysis of INSR in chow and HFD fed Lenti-Con-Inhib and Lent-miR-27b-Inhib injected mice (N = 5–6). Error bars represent SD, *P < 0.05 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. k Western blot analysis of INSR β and p-Akt (Ser473) in eWAT of Lenti-Con-Inhib and LentimiR-27b-Inhib injected mice pulsed with insulin (0.75 U/kg, i.p.). Densitometry of INSR β and p-Akt (N = 5–6) was normalized with βactin and Akt protein, respectively. Error bars represent SD, *P < 0.05, **P < 0.01 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. l qRT-PCR analysis of PPARγ in chow and HFD fed Lenti-Con-Inhib and Lent-miR-27b-Inhib injected mice (N = 5–6). Error bars represent SD, *P < 0.05, ***P < 0.001 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. m Western blot analysis of PPARγ in eWAT of Lenti-Con-Inhib and LentimiR-27b-Inhib injected mice. Densitometry of PPARγ (N = 5–6) was normalized with β-actin. Error bars represent SD, *P < 0.05, **P < 0.01 as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test

Compliance with ethical standards All the experimental protocols were followed as per CPCSEA guidelines and approved by the IAEC Approval: CSIR-CDRI/IAEC/2016/64, Lucknow. Conflict of interest The authors declare that they have no conflicts of interest.

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Thus, our study for the first time implicates the role of miR27b in IR by virtue of its target binding site in the 3’UTR region of INSR. The expression modulation studies, in both in vitro and in vivo models, validate the role of miR-27b in insulin signaling and glucose homeostasis and further strengthen our hypothesis. Thus, our study highlights the importance of miR-27b in the pathophysiology of IR and type 2 diabetes. Acknowledgements We acknowledge Dr. Ram Manohar Lohia Institute of Medical Science (RML-IMS), Lucknow, for providing the ICSCR approval. We also acknowledge the help and support provided by the staff of the Sushrut Institute of Plastic Surgery (SIPS) Hospital for the collection of the liposuction sample. Funding information This work was supported by the CSIR-CDRI Network project: BTowards holistic understanding of complex diseases: Unraveling the threads of complex disease^ (THUNDER) project no.: BSC0102, Department of Biotechnology (DBT) project (GAP0079), and Department of Biotechnology (DBT) project (GAP0179). We are also thankful for technical support from the institutional SAIF division. MB, KS, SR, and AG are supported by the SRF-UGC; AS and DK are supported by the SRF-CSIR, New Delhi. SV is supported by the SRF-ICMR. This Manuscript bears CSIRCDRI communication number: 9638.

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