H19 Lncrna Promotes Skeletal Muscle Insulin Sensitivity in Part by Targeting AMPK

Diabetes Volume 67, November 2018 2183

H19 lncRNA Promotes Skeletal Muscle Insulin Sensitivity in Part by Targeting AMPK

Tingting Geng,1,2 Ya Liu,1,3 Yetao Xu,1,4 Ying Jiang,5 Na Zhang,6 Zhangsheng Wang,1,7 Gordon G. Carmichael,6 Hugh S. Taylor,1 Da Li,1,8 and Yingqun Huang1

Diabetes 2018;67:2183–2198 | https://doi.org/10.2337/db18-0370

Skeletal muscle plays a pivotal role in regulating sys- homeostasis. Muscle insulin resistance contributes criti- temic glucose homeostasis in part through the con- cally to the pathogenesis of major metabolic disorders, served cellular energy sensor AMPK. AMPK activation including type 2 diabetes (T2D) and cardiovascular dis- increases glucose uptake, lipid oxidation, and mitochon- eases. The evolutionarily conserved AMPK is a key sensor drial biogenesis, leading to enhanced muscle insulin of the cellular energy balance that exerts a fundamental sensitivity and whole-body energy metabolism. Here role in metabolic regulation. Expressed in major metabolic we show that the muscle-enriched H19 long noncoding organs such as muscle, liver, fat, and hypothalamus, AMPK RNA (lncRNA) acts to enhance muscle insulin sensitivity, is activated by energy deprivation, exercise, and hormones at least in part, by activating AMPK. We identify the that affect cellular metabolism. In muscle, AMPK activa- METABOLISM atypical dual-speciﬁcity phosphatase DUSP27/DUPD1 tion enhances glucose uptake, fatty acid oxidation, and as a potentially important downstream effector of H19. mitochondrial biogenesis, leading to increased muscle in- We show that DUSP27, which is highly expressed in sulin sensitivity and whole-body energy metabolism. Thus, muscle with previously unknown physiological function, interacts with and activates AMPK in muscle cells. Con- AMPK has emerged as a promising therapeutic target for sistent with decreased H19 expression in the muscle of metabolic diseases (1). insulin-resistant human subjects and rodents, mice with AMPK is a heterotrimeric Ser/Thr kinase consisting of genetic H19 ablation exhibit muscle insulin resistance. one catalytic a subunit and two regulatory subunits, b and Furthermore, a high-fat diet downregulates muscle H19 g. AMPK is activated by upstream kinases that phosphor- via both posttranscriptional and epigenetic mechanisms. ylate a conserved threonine (Thr172) in the a subunit. The Our results uncover an evolutionarily conserved, highly primary kinases that phosphorylate Thr172 are LKB1 expressed lncRNA as an important regulator of muscle and CaMKKb. Binding of AMP to the g subunit leads to insulin sensitivity. allosteric activation of AMPK and protection of Thr172 from dephosphorylation. AMPK can also be inactivated by protein phosphatases and by inhibitory phosphor- Skeletal muscle, the largest insulin-sensitive organ, ac- ylation (1–3). AMPK modulates many downstream targets, counting for up to 60% of whole-body glucose disposal, including acetyl-CoA carboxylase (ACC) and peroxisome plays an essential role in maintaining systemic glucose proliferator–activated receptor-g coactivator 1a (PGC-1a).

1Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School Shengjing Hospital, China Medical University, Shenyang, Liaoning, People’s Re- of Medicine, New Haven, CT public of China 2 fi ’ Department of Endocrinology, First Af liated Hospital of Xi an Jiaotong University Corresponding author: Yingqun Huang, [email protected], or Da Li, ’ ’ School of Medicine, Xi an, Shaanxi, People s Republic of China [email protected]. 3Department of Veterinary Medicine, College of Animal Science and Technology, Received 2 April 2018 and accepted 24 August 2018. Anhui Agricultural University, Hefei, Anhui, People’s Republic of China 4Department of Obstetrics and Gynecology, First Affiliated Hospital of Nanjing This article contains Supplementary Data online at http://diabetes Medical University, Nanjing, Jiangsu, People’s Republic of China .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0370/-/DC1. 5Department of Obstetrics, Women’s Hospital, Zhejiang University School of T.G., Y.L., and Y.X. contributed equally to this work. Medicine, Hangzhou, Zhejiang, People’s Republic of China © 2018 by the American Diabetes Association. Readers may use this article as 6Department of Genetics and Genome Sciences, University of Connecticut Health long as the work is properly cited, the use is educational and not for profit, and the Center, Farmington, CT work is not altered. More information is available at http://www.diabetesjournals 7Department of Cardiology, Fifth People’s Hospital of Shanghai, Fudan University, .org/content/license. Shanghai, People’s Republic of China 8Center of Reproductive Medicine, Department of Obstetrics and Gynecology, 2184 H19 lncRNA, Insulin Sensitivity, and AMPK Diabetes Volume 67, November 2018

ACC is a rate-limiting enzyme for the synthesis of Hyperinsulinemic-Euglycemic Clamp and 14 malonyl-CoA, an essential substrate for fatty acid bio- Measurement of Muscle 2-[ C]deoxy-D-glucose synthesis and a potent inhibitor of lipid oxidation. Phos- Uptake phorylation by AMPK inactivates ACC, resulting in The experiments were performed on 11-week-old WT and fi inhibition of lipogenesis while increasing lipid oxidation KO mice as previously described with minor modi cation (1). PGC-1a is a master regulator of mitochondrial bio- (8). In brief, mice were anesthetized and cannulated. After genesis and lipid oxidation (4,5). AMPK activates PGC-1a recovery for 7 days, mice were fasted overnight (14 h) 3 through multiple mechanisms, including direct phos- followed by infusion of D-[3- H]glucose to assess the basal phorylation. As a transcriptional coactivator, PGC-1a rate of whole-body glucose turnover. After the basal pe- activates different transcription factors to drive tran- riod, a 2-h hyperinsulinemic-euglycemic clamp was con- fi $ scription of vast gene networks involved in all aspects ducted with a xed amount of insulin (4 mU/[kg min]) of energy homeostasis, including glucose utilization and and a variable amount of 20% dextrose to maintain eugly- 14 fatty acid oxidation, as well as mitochondrial biogenesis cemia. A 10-mCi bolus of 2-[ C]deoxy-D-glucose was in- fi (including mitochondrial DNA replication) and function. jected into mice at 90 min to determine tissue-speci c fi Among the numerous genes activated by PGC-1a are Pgc- glucose uptake. After collection of the nal blood sample, 1a (autoregulation), carnitine palmitoyltransferase 1b themicewereanesthetizedandtissueswereharvested, 2 (Cpt1b), pyruvate dehydrogenase kinase 4 (Pdk4), and frozen in liquid nitrogen, and stored at 80°C until later use. mitofusin-2 (Mfn2). As PGC-1a positively regulates its Plasmid Construction own transcription, activation by AMPK can also increase Plasmids pAV-PGK-DUSP27 (wt-DUSP27) and pAV-PGK- PGC-1a protein levels. DUSP27C146S (mt-DUSP27), which express WT and mu- The evolutionarily conserved H19 long noncoding RNA tant dual-specificity protein phosphatase 27 (DUSP27), (lncRNA) is abundantly expressed in skeletal muscle, but respectively, were constructed by cloning the open reading its expression is significantly decreased in patients with frames of WT and mutant mouse DUSP27 (NM_001013826.2) T2D and insulin-resistant rodents (6). Whether H19 is into the pAV-PGK expression vector (Vigene Biosciences involved in regulation of muscle insulin sensitivity and Inc., Rockville, MD). The expression of both proteins was how H19 is downregulated in the diabetic muscle are not driven by a phosphoglycerate kinase 1 (PGK-1) promoter. clear. The accuracy of the clones was confirmed by sequencing.

RESEARCH DESIGN AND METHODS RNA Extraction and Real-time Quantitative PCR Animals Total RNAs were extracted from frozen tissue samples or All animal work was approved by the Yale University In- from cultured myotubes using the PureLink RNA Mini Kit stitutional Animal Care and Use Committee. All mice used (12183018A; Ambion). cDNA was synthesized using the in this report were male. The wild-type (WT) and knockout PrimeScript RT Reagent Kit (TAKARA, RR037A; Invitro- (KO) mice on a background of C57BL/6J were gifts from gen, Grand Island, NY) in a 20-mL reaction containing – Dr. Luisa Dandolo (Institut Cochin, Paris, France). Mice 0.2 0.5 mg of total RNA. Real-time quantitative PCR were housed at 22–24°C with a 12-h light/12-h dark cycle (RT-qPCR) was performed in a 15-mL reaction containing – with regular chow (RC) (Teklad no. 2018, 18% calories 0.5 1 mL of cDNA using iQSYBRGreen (Bio-Rad) in a from fat; Harlan) or high-fat diets (HFDs) (D12451, 45% Bio-Rad iCycler. PCR was performed by initial denatur- calories from fat; Research Diets) and water provided ad ation at 95°C for 5 min, followed by 40 cycles of 30 s at fi fi libitum. For HFD studies, 7-week-old WT mice were ex- 95°C, 30 s at 60°C, and 30 s at 72°C. Speci city was veri ed posed to HFD or RC for 11 days or 13 weeks, followed by by melting curve analysis and agarose gel electrophoresis. the indicated experiments. The threshold cycle (Ct) values of each sample were used in the post-PCR data analysis. Gene expression levels were Body Composition and Glucose and Insulin Tolerance normalized against the following housekeeping genes: Tests b-tubulin for muscle tissues, b-actin for myotubes, and Body composition was measured in vivo by MRI (EcoMRI; U6 for let-7. Real-time PCR primers are listed in Supple- Echo Medical Systems). Glucose tolerance tests (GTTs) mentary Table 2. were performed in 16 h–fasted mice as previously described (7). Each animal received an i.p. injection of 2 g/kg Western Blot Analysis body weight glucose (DeltaSelect) in sterile saline. Blood Myotubes were detached from plates by trypsin digestion glucose levels were measured at 0, 15, 30, 45, 60, 90, and and collected in culture medium. After gentle centrifuga- 120 min after the injection. Insulin tolerance tests (ITTs) tion, the cell pellet was quickly lysed in 23 SDS sample were performed in ad libitum fed mice (7). Each animal buffer (100 mL per well of a 24-well plate) by heating at received an i.p. injection of insulin, 1 unit/kg (Humulin R; 100°C for 5 min with occasional vortexing. For muscle Eli Lilly and Company). Blood glucose levels were moni- tissue samples, 5 mg of frozen tissue was quickly homog- tored at 0, 15, 30, 45, 60, 90, and 120 min after the enized on ice in 200 mLof23 SDS sample buffer in the injection. presence of phosphatase inhibitors (Cocktail 2 [P5726] diabetes.diabetesjournals.org Geng and Associates 2185 and Cocktail 3 [P0044]; Sigma-Aldrich) at a final concen- 48 h, followed by protein extraction and Western blot tration of 13. Samples were then heated at 100°C for analysis. 5 min with occasional vortexing. Additional phosphatase Glucose Uptake Assay inhibitors were added at a final concentration of 13 before These were performed in a 96-well plate scale using the loading the samples onto a 10% SDS-PAGE gel, followed by Glucose Uptake Cell-Based Assay Kit (600470; Cayman Western blot analysis. Detailed information on antibodies Chemical) according to the manufacturer’s instructions and conditions is listed in Supplementary Table 3. Western with minor modifications. To prepare the siRNA mix blot gels were quantified using ImageJ (https://imagej.nih per well of myotubes, 25 pmol of siRNA (control siRNA .gov/ij). b-Actin was used for normalization. For DUSP27 [siCon] or siH19) was mixed with 30 mL of OPTI-MEM by Western blot analysis, mouse IgG k binding protein (m-IgGk gentle pipetting. In a separate tube, 2.5 mL of Lipofect- BP, sc-516102; Santa Cruz Biotechnology) conjugated to horse- amine 3000 was mixed with 30 mL of OPTI-MEM. After radish peroxidase was used instead of anti-mouse IgM second- 5 min incubation at room temperature, the two were aryantibodytoincreasespecificity and minimize background. mixed and incubated at room temperature for 10 min. Myotube Culture and Transfection To prepare the plasmid DNA mix per well of myotubes, Undifferentiated mouse C2C12 myoblasts (91031101-iVL; 0.1 mg of Vec or wt-DUSP27 was mixed with 0.3 mLof Sigma-Aldrich) were maintained in growth medium (GM) P3000 and 10 mL of OPTI-MEM by gentle pipetting. In (DMEM, 11965-092, supplemented with 10% FBS, heat a separate tube, 0.2 mL of Lipofectamine 3000 was mixed inactivated, 1% penicillin/streptomycin, 1% L-glutamine, with 10 mL of OPTI-MEM. After 5 min incubation at room and 1 mmol/L sodium pyruvate; Gibco). To prepare for temperature, the two were mixed and incubated at room differentiation, cells were seeded at a density of 1.6 3 105 temperature for 10 min. The resulting 60 mL of siRNA mix cells/well or 2.0 3 104 cells/well in GM in 6-well or 24-well and 20 mL of plasmid DNA mix were combined to make fi plates, respectively. Differentiation was initiated 2 days a nal transfection cocktail of 80 mL, which was gently later when cells became confluent by replacing GM with added to one well of myotubes. After 6 h of incubation in differentiation medium (DM) containing 2% horse serum a tissue culture incubator, 200 mL of DM was added and in place of 10% FBS. The medium was changed every other incubation was continued for an additional 48 h. On the day until transfection, which was performed on day 4–5 day of the glucose uptake assay, DM was replaced with after initiation of differentiation. 200 mL of glucose-free DMEM (11966-025; Gibco) and For small interfering RNA (siRNA) or let-7 transfection, incubation was carried out for 2 h. Then, the medium was culture medium was gently removed, followed by the replaced with 100 mL of new glucose-free DMEM in the – addition of transfection cocktails to cover the myotubes. presence or absence of 100 nmol/L of insulin for 15 To prepare transfection cocktails for each well of 24-well 20 min. Subsequently, 100 mL of new glucose-free DMEM fl fi plates, 125 pmol of siRNA (with a stock solution of containing uorescent 2-NBDG at a nal concentration of 5 mmol/L) was mixed with 150 mL of OPTI-MEM by gentle 150 mg/mL was added. Incubation was performed in the pipetting. For iLet7 rescue experiments, 125 pmol of H19- dark for an additional 15 min in the tissue culture in- specific siRNA (siH19) and 75 pmol of iLet7 were mixed cubator. The medium was then removed, and the myo- together with 150 mL of OPTI-MEM. In parallel, 6.25 mLof tubeswerewashedoncewith200mLofice-coldPBS. Lipofectamine 2000 was diluted in 150 mLofOPTI-MEMby After adding 100 mL of new ice-cold PBS to the myotubes, fl gentle pipetting. After 5 min incubation at room tempera- uorescent intensity was immediately determined using fl ture, the two were combined by gentle pipetting. After the uorescent plate reader (FilterMax F3 and F5 Multi- incubation at room temperature for 25 min, the resulting Mode Microplate Reader; Molecular Devices). Results are cocktail (300 mL) was gently added to the myotubes. After 8– presented with myotubes without insulin stimulation ar- 10 h incubation in a tissue culture incubator, the cocktail was bitrarily set as 1. gently replaced with fresh DM. Forty-eight hours after the RNA-Seq and Data Analysis transfection, RNA and protein were extracted for analysis. RNAs were extracted from WT and KO soleus muscle tissue Plasmid DNA (Vec; wt-DUSP27 or mt-DUSP27) trans- samples using the Purelink RNA Mini Kit (12183018A). fections were performed in a 24-well plate scale. To pre- Genome-wide transcriptome analysis (RNA-Seq) libraries pare plasmid DNA mix per well of myotubes, 0.4 mgof were prepared using the Illumina TruSeq Stranded Total DNA was mixed with 1 mL of P3000 (L3000-008; Invi- RNA LT Kit with Ribo-Zero Human/Mouse/Rat, set A (rs- trogen) and 40 mL of OPTI-MEM by gentle pipetting. In 122-2201) according to the sample preparation protocol. a separate tube, 0.75 mL of Lipofectamine 3000 was mixed In brief, 1 mg of total RNA was subjected to Ribo-Zero with 40 mL of OPTI-MEM. After 5 min incubation at room depletion to remove rRNAs. The remaining RNA was pu- temperature, the two were mixed and incubated at room rified, fragmented, and primed with random hexamers temperature for 10 min. The resulting 80 mL of trans- for cDNA synthesis. After first and second cDNA synthe- fection cocktail was gently added to myotubes. After 6 h of sis, cDNA fragments were adenylated and then ligated incubation in a tissue culture incubator, 800 mLofDMwas to indexing adapters. The cDNA fragments were enriched added and incubation was continued for an additional by PCR, purified, and then sequenced on an Illumina 2186 H19 lncRNA, Insulin Sensitivity, and AMPK Diabetes Volume 67, November 2018

NextSeq500 using paired-end chemistry and 76–base pair were washed briefly with TBS (20 mmol/L Tris-HCl, pH cycles. Sequences are available from the GEO with acces- 7.4, 225 mmol/L NaCl), fixed with 3% paraformaldehyde/ sion number GSE103202. TBS for 20 min, permeabilized with 1% SDS/TBS for Illumina BaseSpace (https://basespace.illumina.com/) 5 min, and blocked with 10% BSA/0.1% goat serum/TBS embedding tools were used to analyze the RNA-Seq data. for 1 h. Cells were then incubated with anti-DUSP27 and RNA-Seq Alignment version 1.0.0 was used to map sequenc- anti-AMPKa antibody at a dilution of 1:50 at 4°C over- ing reads to mm10 genome and quantify reads of genes. night, washed with TBS, and then incubated with Alexa DESeq2 version 1.0.0 was applied to calculate differential Fluor 555 (for DUSP27) and Alexa Fluor 488 (for AMPKa) expression of genes. conjugated secondary antibodies (1:500 dilution; Invitro- gen) for 1 h. After washing with TBS, cells were mounted Immunoprecipitation with mounting medium containing DAPI (Vector Labora- To prepare antibodies, 8 mLofpackedbeads(protein tories, Burlingame, CA). Images were obtained under L agarose beads [sc-2336; Santa Cruz Biotechnology] confocal microscopy (Leica SP5) at a magnification of 603. for anti-DUSP27 and rabbit preimmune IgM; protein A sepharose beads for anti-AMPKa [2032; Cell Signaling In Vitro Pulldown Assays Technology] and rabbit preimmune IgG) was incubated For glutathione S-transferase (GST) pulldown, GST- with 16 mg of anti-DUSP27, anti-AMPKa, preimmune AMPKa (4 mg, H00005563-P01; R&D Systems) or GST IgM, or preimmune IgG in 250 mL immunoprecipitation (202039; GenScript) was incubated with 8 mL of glutathi- (IP) buffer (1% Triton X-100, 300 mmol/L NaCl, one beads (16100; ThermoFisher) in 200 mL of binding 10 mmol/L Tris-HCl at pH 7.5, and 10 mmol/L EDTA) buffer (BB; 25 mmol/L HEPES [pH 7.3], 100 mmol/L NaCl, at 4°C overnight. The next day, the beads were washed 10% glycerol, 1% Triton X-100, 1 mmol/L dithiothreitol, three times with IP buffer and kept on ice until used. 0.25 mmol/L phenylmethylsulfonyl fluoride, and 13 pro- To prepare tissue lysates, 35 mg of frozen muscle tissue tease inhibitor cocktail [Calbiochem]) in the presence of was homogenized on ice in 100 mL of freshly prepared 0.1% BSA at 4°C for 6 h. The beads were then washed with gentle lysis buffer (GLB; 1% Triton X-100, 10 mmol/L cold BB once, followed by incubation with 2 mgof NaCl, 10 mmol/L Tris-HCl at pH 7.5, 10 mmol/L EDTA, FL-DUSP27 (TP314361; OriGene) in 200 mL of BB (with- 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L out BSA) at 4°C overnight. The next day, beads were dithiothreitol, and 13 protease inhibitor cocktail [Calbio- subjected to quick wash with BB once, slow wash (by chem]), followed by the addition of 900 mL of GLB and 10 s rotating the tube at 4°C for 3 min) with medium-salt BB centrifugation in a benchtop microcentrifuge. After trans- (final NaCl concentration to 200 mmol/L), and then high- ferring the supernatant (;900 mL) to a new tube on ice, salt BB (final NaCl concentration to 300 mmol/L) once each. the remaining tissue pellet was homogenized again in the Bound (pellet) and unbound (Sup) fractions were resolved residual ;100 mL of GLB. Then, the supernatant was on SDS-PAGE, followed by Western blot analysis. added back to the tube containing the tissue pellet and For FLAG-DUSP27 pulldown, FL-DUSP27 (2.5 mg) or the tube was incubated on ice for 10 min. After removing FLAG peptide (F3290-4MG; Sigma-Aldrich) was incubated insoluble materials by centrifugation at 12,000 rpm at 4°C with 8 mL of anti-FLAG magnetic agarose beads (M8823; for 15 min, 4 mol/L NaCl was added to the lysate at a final Sigma-Aldrich) in 200 mL of BB in the presence of 0.1% concentration of 300 mmol/L. The lysate was then split BSA at 4°C for 6 h. The beads were then washed with cold into two tubes (;500 mL per tube) and incubated with BB once, followed by incubation with 1 mg of GST-AMPKa 10 mL of packed protein L beads (for anti-DUSP27 IP) or in 200 mL of BB (without BSA) at 4°C overnight. The next protein A beads (for anti-AMPKa IP) at 4°C for 1 h to day, beads were subjected to quick wash with BB once and minimize nonspecific binding. The precleared lysates were slow wash with medium-salt BB and then high-salt BB once then transferred to tubes containing the respective anti- each. Bound (pellet) and unbound (Sup) fractions were body or preimmune IgM/IgG-coated beads (each tube con- resolved on SDS-PAGE, followed by Western blot analysis. ; m tained 250 L of precleared lysate), and IP was carried Quantitative Methylation-Specific PCR out by rotating the tubes at 4°C for 3 h. After IP, the beads Genomic DNA was extracted from soleus muscle tissues were quickly washed with 1 mL of cold IP buffer twice from WT mice after exposure to RC or HFD using Quick- and then washed an additional three times by rotating gDNA MicroPrep (D3021; Zymo Research). Fifteen micro- the tube at 4°C for 3 min each time. After the final wash, fl liters of elution buffer was used to elute DNA from each residual liquid was completely removed using a long at column. For bisulfite treatment, 200 ng of DNA was used pipette tip. Beads were eluted with 20 mLof23 SDS for each column using the EZ DNA Methylation-Gold Kit at 100°C for 3 min. Ten microliters of eluant was loaded (D5006; Zymo Research). Elution buffer (100 mL) was used onto each well of 12% SDS-PAGE gels. to elute DNA from each column. RT-qPCR was performed Immunofluorescence in a 15-mL reaction containing 5 mL of the eluant using For DUSP27 and AMPKa double immunostaining, myo- iQSYBRGreen (Bio-Rad) in a Bio-Rad iCycler. The PCR tubes grown in a 1-m Slide eight-well ibiTreat microscopy primers (Supplementary Table 2) for methylated NCTC1 chamber (80826; Research Products International Corp.) were used at a final concentration of 0.6 mmol/L in each diabetes.diabetesjournals.org Geng and Associates 2187

PCR reaction. PCR was performed by initial denaturation phosphorylation and activation of AKT, which mediates at 95°C for 5 min, followed by 40 cycles of 30 s at 95°C, most of insulin’s metabolic effects, including increasing 30 s at 60°C, and 30 s at 72°C. Specificity was verified glucose uptake in muscle (reviewed in Boucher et al. [12]). by melting curve analysis and agarose gel electrophoresis. Thus, INSR expression and AKT phosphorylation at Ser473 The threshold cycle (Ct) values of each sample were used in (phosphorylation at this site is required for full activation the post-PCR data analysis. Albumin DNA was used as of AKT [12]) were examined in WT and KO muscles. We loading controls for all quantitative methylation-specific observed decreased INSR expression at both mRNA (Fig. 2A) PCR (QMSP) normalizations. and protein levels (Fig. 2B, top blot in left panel [compare Statistical Analysis lane 2 to lane 1], and middle panel) in KO as compared with WT muscles. This is consistent with the previously reported Statistical analyses and figure construction were per- mechanism of H19-dependent regulation of INSR expression formed using GraphPad Prism version 7.01 for Mac in both human and rodent muscles (6). Unexpectedly, we (www.graphpad.com; GraphPad Software, La Jolla, CA). observed an increase in AKT phosphorylation in KO muscle A value of P , 0.05 was considered statistically significant. (Fig. 2B, middle two blots in left panel [compare lane 2 to Statistical significance was determined by unpaired, two- lane1],andrightpanel).Giventhatanincreaseinthebasal tailed Student t test between two groups. One-way ANOVA, level of AKT-dependent insulin signaling was similarly followed by Tukey multiple comparisons test, was used for comparisons between three groups. Nonparametric Spear- reported in HFD-induced insulin-resistant mouse muscle (13), we speculate that the basal AKT phosphorylation in- man correlations were performed for gene correlation anal- crease in insulin-resistant muscle might serve as a compen- yses. One asterisk represents P , 0.05, and two asterisks satory mechanism that warrants future investigation. represent P , 0.01. Error bars are shown as mean 6 SEM. Next, we tested whether AMPK activity is altered in KO fi RESULTS muscle, as chronic muscle-speci cAMPKinactivation exacerbates the development of diet-induced muscle Genetic Ablation of H19 Leads to Muscle Insulin insulin resistance (14) and constitutive muscle-specific Resistance AMPK activation enhances insulin sensitivity (15). Thus, H19 is highly expressed in skeletal muscle (Fig. 1A), we performed Western blot analysis to assess phosphor- confirming previous reports (9). We also observed that its ylation of AMPKa at Thr172 in WT and KO muscles. The expression is the highest in the oxidative fiber-enriched phosphorylation was significantly decreased in KO as soleus muscle (Fig. 1B). As siRNA-mediated H19 knock- compared with WT muscle (Fig. 2C, top two blots in left down impaired insulin-stimulated glucose uptake in cul- panel [compare lane 2 to lane 1], and middle panel). As tured mouse myotubes (6), we tested whether H19 expected, phosphorylation of ACC at Ser79 was also de- regulates muscle insulin sensitivity in vivo. To this end, creased (Fig. 2C, left and right panels). The apparent de- we used a whole-body H19 KO mouse model that carries crease in ACC total protein level (Fig. 2C, second blot from a targeted deletion of the entire H19 transcription unit bottom in left panel, compare lane 2 to lane 1) was not (10). The KO mice exhibited an overgrowth phenotype surprising because an increase in both ACC protein abun- (Fig. 1C), consistent with previous observations (10,11). dance and Ser79 phosphorylation was reported in mice with Overgrowth could be attributed to an increase in the lean chronic muscle-specific AMPK activation (15). As decreased mass (Fig. 1D and E), which was not surprising because the (or increased) ACC phosphorylation would inhibit (or en- KO mice exhibit skeletal muscle hyperplasia and hyper- hance) lipid oxidation, the total ACC protein level decrease trophy (11). Yet, ITTs performed in ad libitum fed mice (or increase) may reflect a failed compensatory mechanism. unexpectedly revealed decreased whole-body insulin sen- It was previously shown that mice with phosphodefec- sitivity in the KO mice (Fig. 1F). To assess sites where tive ACC1 and/or ACC2 (the mutated enzymes hence were insulin action was affected, hyperinsulinemic-euglycemic not sensitive to AMPK-dependent phosphorylation) had clamp studies after overnight fasting were carried out. decreased fatty acid oxidation, as indicated by increased Results showed that insulin-stimulated glucose uptake in levels of triacylglycerol (TAG) and diacylglycerol in muscle muscles was significantly lower in the KO compared with the (16,17). To assess whether altered ACC phosphorylation/ WT mice (Fig. 1G and Supplementary Fig. 1). Taken together expression in H19 KO muscle affects lipid accumulation, with our previous observation that H19 knockdown by muscle TAG contents were measured, which showed no siRNA in myotubes impaired insulin-stimulated glucose significant difference between the WT and KO animals uptake (6), we suggest that H19 is important for appropri- (Supplementary Fig. 2). Our results highlight the complex- ate physiological regulation of insulin action in muscle. ity of an ACC-mediated mechanism of lipid metabolism AMPK Activity Is Decreased in KO Muscle regulation. Indeed, one study reported that global ACC2 To begin to elucidate the molecular mechanism underlying KO mice had increased fatty acid oxidation and muscle the insulin resistance observed in the H19 KO muscle, insulin sensitivity in addition to reduced fat mass (18), we first tested whether the insulin signaling pathway is whereas another study showed no change in fatty acid affected. Binding of insulin to insulin receptor (INSR) oxidationandlittleeffectonbodyweightandfatmassin initiates a cascade of phosphorylation events, leading to muscle-specificACC2KOmice(19).Further,thelatest 2188 H19 lncRNA, Insulin Sensitivity, and AMPK Diabetes Volume 67, November 2018

Figure 1—H19 deletion impairs muscle insulin sensitivity. A: Results of RT-qPCR analysis of H19 expression in WT mice (n = 4). The H19 level in gastrocnemius muscle is arbitrarily set as 100%. BAT, brown adipose tissue; Pan, pancreas, WAT, white adipose tissue. B: H19 expression in various muscle types from WT mice (n = 4). EDL, estensor digitorum; Gas, gastrocnemius; Qua, quadriceps; Sol, soleus; TA, tibialis anterior. C: Growth curve of WT and KO mice. Each growth point represents n =15–20 mice. D and E: Body composition as assessed by EcoMRI. Each point represents n =15–20 mice. F: ITT in ad libitum fed WT (n = 8) and KO (n = 8) mice, with quantiﬁcation on the right. AUC, area under curve. G: Insulin-stimulated glucose uptake in gastrocnemius and soleus muscles during hyperinsulinemic-euglycemic clamp studies. n =5–8 animals in each genotype. All data represent mean 6 SEM. C–G:*P , 0.05 and **P , 0.01, compared with WT mice.

research ﬁndings have demonstrated that not all muscle in- 1a expression via the PGC-1a autoregulatory pathway (4,5). sulin resistance shows increases in total intramuscular lipids Further, a decrease in expression of PGC-1a targets, Cpt1b, and/or lipid intermediates (20–22). Future studies are cer- Mfn2, and Pdk4 (Fig. 2E), as well as in mitochondrial DNA tainly needed to determine how decreased AMPK activity in contents (Fig. 2F), was evident in the KO muscle, consistent H19KOmuscleaffectslipidmetabolism–mediated ACC. with PGC-1a’s role in stimulating mitochondrial biogenesis As an additional readout for AMPK activity, Western blot and fat oxidation. Based on the ﬁnding that H19 KO muscle analysis was performed on PGC-1a and showed decreased exhibits both decreased ACC phosphorylation and decreased protein level in KO muscle (Fig. 2D). This is in agreement expression of PGC-1a, we conclude that H19 positively with the well-established role of AMPK in promoting PGC- affects AMPK activity in muscle. diabetes.diabetesjournals.org Geng and Associates 2189

Figure 2—H19 deletion leads to decreased AMPK activity and DUSP27 expression in muscle. A: RT-qPCR results of INSR mRNA from WT and KO muscle. n =4–5 animals in each genotype. B: Representative Western blots (left) and densitometry (middle and right) results for INSR, phosphorylation of AKT at Ser473 (pAKT), total AKT (AKT), and b-actin (ACTB, as a loading control) in muscle samples from WT and KO mice fed ad libitum. Muscle extracts from ﬁve mice from each genotype were loaded. C: Representative Western blots (left) and densitometry (middle and right) results for phosphorylation of AMPKa at Thr172 (pAMPK), total AMPKa (AMPK), phosphorylation of ACC at Ser79 (pACC), total ACC (ACC), and ACTB in muscle samples from WT and KO mice fed ad libitum. Muscle extracts from three mice from each genotype were loaded. D: Representative Western blots (left) and densitometry (right) results of PGC-1a protein. Muscle extracts from three mice from each genotype were loaded. E: RT-qPCR results of indicated mRNAs from WT and KO muscle. n =4–5 animals in each genotype. F: Relative mitochondrial DNA (mtDNA) copy numbers from WT and KO muscle. n = 7 animals in each genotype. G: RT-qPCR results of DUSP27 mRNA from WT and KO muscle. n = 5 animals in each genotype. H: Representative Western blots (left) and densitometry (middle and right) results of DUSP27 and DUSP1 proteins. All data are shown as mean 6 SEM. *P , 0.05 and **P , 0.01, compared with WT mice.

DUSP27 Expression Is Downregulated in KO Muscle a recent report that in human cardiac fibroblast cells, To delineate the molecular mechanism linking H19 to H19activatesERKbyregulatingexpressionofDUSP5 AMPK activation, we analyzed gene expression profiles (although the mechanism by which H19 regulates DUSP5 of muscles from WT and KO mice by RNA-Seq. We noted was not defined) (23). This was intriguing because ERK 2190 H19 lncRNA, Insulin Sensitivity, and AMPK Diabetes Volume 67, November 2018

Figure 3—H19 regulates DUSP27 expression via the H19/let-7 axis. A: Bioinformatics predicted three let-7 binding sites at the 39-UTR of mouse DUSP27 mRNA. Sequences of three let-7 subtypes (top strands) and partial sequences of DUSP27 mRNA (bottom strands) are shown. Numbers are in nucleotides relative to the transcriptional start site of DUSP27. B: RT-qPCR results of DUSP27 and H19 expression in myotubes 48 h after transfection of siCon, siH19, or DUSP27-speciﬁc siRNA (siDusp27). C: Western blots (left) and densitometry results (right) of DUSP27 and ACTB proteins from experiments shown in B. Lanes were loaded in increasing amounts of cell lysates. D:RT-qPCRresultsofH19andDUSP27 expression in myotubes 48 h after transfection of siCon, siH19, and siH19 plus iLet-7. E: Western blots (left) and densitometry (right) results of DUSP27 and ACTB protein levels from experiments shown in D. F: RT-qPCR results of DUSP27 mRNA levels in myotubes 24 h after transfection of miCon (negative control miRNA) or let-7. Lanes were loaded in duplicate. G: Western blots (left) and densitometry (right) results of DUSP27 and ACTB protein levels from experiments shown in F. All data are shown as mean 6 SEM (n =3).*P , 0.05; **P , 0.01. ns, no statistical difference.

inactivates AMPK by phosphorylating AMPKa at Ser487/ DUSP27 is evolutionarily conserved; the human DUSP27 491 (3). Therefore, we first examined expression and phos- protein shares 87%, 86%, and 81% identity with mouse, phorylation of ERK in WT and KO muscle but did not find rat, and dog, respectively (24). DUSP27 is a member of a any significant difference between the two groups (data heterogeneous group of protein phosphatases capable of notshown),suggestingthatERKislikelynotinvolvedin dephosphorylating both phosphotyrosine and phosphoser- AMPK inactivation in H19 KO muscle. Next, we turned our ine/phosphothreonine residues within the same substrate attention to DUSP27 (also called DUPD1) (24), as our RNA- (25). DUSPs are divided into six subgroups that include Seq analysis revealed a significant decrease in its expression mitogen-activated protein kinase phosphatases (MKPs) and in KO versus WT muscles, whereas no significant difference atypical DUSPs (25). DUSP27 falls in the atypical category was detected in other DUSPs, including DUSP1 (Supple- (24) and has been shown to act as a phosphatase to de- mentary Table 1 and GEO accession no. GSE103202). The activate ERK and p38 MAPKs in ovarian cells (26). Notably, DUSP27 decrease was confirmed by RT-qPCR (Fig. 2G)and in mice, DUSP27 is expressed highly in skeletal muscle, fat, Western blotting (Fig. 2H). and liver (24), whereas in humans, its mRNA is detected diabetes.diabetesjournals.org Geng and Associates 2191

Figure 4—DUSP27 interacts with AMPKa. A: Mouse gastrocnemius muscle extract was immunoprecipitated with rabbit IgM DUSP27 antibody (preimmune rabbit IgM as a negative control). Representative Western blot results are shown; 3% of input was loaded. B: Mouse soleus muscle extract was immunoprecipitated with rabbit AMPKa antibody (preimmune rabbit IgG as a negative control). Representative Western blot results are shown. HC, IgG heavy chain. C: Representative immunofluorescence image of a myotube with DUSP27 stained red, AMPKa green, and nuclei blue. Arrowheads indicate colocalization of DUSP27 and AMPKa. Original magnification 360. Scale bar, 10 mm. D: Purified recombinant protein FL-DUSP27 (or FLAG peptide as a negative control) prebound on anti-FLAG beads was incubated with purified recombinant protein GST-AMPKa. Bound (pellet) and unbound (Sup) fractions were resolved on SDS-PAGE, followed by Western blot analysis using the anti-AMPKa antibody. E: Purified GST-AMPKa (or GST as a negative control) prebound on glutathione beads was incubated with FL-DUSP27. Bound and unbound fractions were resolved on SDS-PAGE, followed by Western blot analysis using the anti- DUSP27 antibody.

only in skeletal muscle (http://www.proteinatlas.org/ amounts of let-7 but rather the relative abundance be- ENSG00000188716-DUPD1/tissue). Although the physio- tween H19 and let-7 that determines the expression level logical significance of this striking tissue-specific expres- of genes regulated by let-7. Let-7 inhibits target gene sion is unclear, its decreased expression in H19 KO muscle expression by binding to complementary sequences in suggests a regulation of DUSP27 by H19. the mRNA, leading to translational repression and/or mRNA degradation. Therefore, we performed bioinfor- H19 Regulates DUSP27 Expression at the matics analysis and predicted three let-7–binding sites Posttranscriptional Level in the 39-UTR of DUSP27 mRNA (Fig. 3A). To determine We have previously reported the H19/let-7 axis where H19 whether H19 regulates DUSP27 via the H19/let-7 axis, we functions to reduce the bioavailability of microRNA let-7 first tested the effect of H19 siRNA knockdown on by acting as a molecular sponge. H19 contains six let-7– DUSP27 expression in mouse C2C12 myotubes (herein binding sites that sequester let-7 and prevent it from called myotubes). While knocking down DUSP27 (Fig. 3B, binding to target mRNAs (27). Thus, it is not the absolute left column, compare white bar to black bar) expectedly 2192 H19 lncRNA, Insulin Sensitivity, and AMPK Diabetes Volume 67, November 2018

Figure 5—DUSP27 contributes to H19-dependent modulation of AMPK activity. A: RT-qPCR results of DUSP27 expression in myotubes 48 h after transfection of siCon, siH19, or DUSP27-speciﬁc siRNA (siDusp27). B: Western blots and densitometry results of phosphorylation of AMPK (pAMPK), AMPK, pACC, ACC, and ACTB proteins from experiments shown in A. Lanes were loaded in increasing amounts of proteins. C: RT-qPCR results of DUSP27 expression in myotubes 24 h after transfection of miCon (negative control miRNA) or let-7. D: Western blots and densitometry results of pAMPK, AMPK, pACC, ACC, and ACTB proteins from experiments shown in C. E and F: Myotubes were transfected with empty vector (Vec), wt-DUSP27, or mt-DUSP27, followed by Western blot analysis 48 h later. Western blots and densitometry results of DUSP27, pAMPK, AMPK, pACC, ACC, and ACTB proteins are shown. G: Myotubes were transfected with siCon, siH19, or siH19 plus wt-DUSP27, followed by Western blot analysis 48 h later. Western blots and densitometry results of DUSP27, pAMPK, AMPK, and GAPDH proteins are shown. H: Glucose uptake of myotubes from experiments shown in G. Results are presented as relative glucose uptake, with values in absence of insulin stimulation set as 1. I: A proposed model of H19-dependent regulation of AMPK activity. All data are shown as mean 6 SEM (n = 3). *P , 0.05; **P , 0.01. ns, no statistical difference.

reduced its protein level (Fig. 3C, top blots in left panel, (Fig. 3C, top blots in left panel, compare lane 2 to lane 1; compare lane 3 to lane 1; right panel, compare blue dots right panel, compare red dots to black dots), suggesting to black dots), downregulation of H19 (Fig. 3B,middle that H19 positively regulates DUSP27 expression. Next, column, compare gray bar to black bar) also decreased we tested whether this regulation is mediated by let-7. We DUSP27 expression at both the mRNA (Fig. 3B,right have previously documented in other cell types that H19 column, compare gray bar to black bar) and protein levels knockdown increases the bioavailability of let-7, but in the diabetes.diabetesjournals.org Geng and Associates 2193

Figure 6—HFD-induced downregulation of H19, DUSP27, and AMPK signaling in muscle. A: Results of GTTs of WT mice after 13-week exposure to RC or HFD. B: RT-qPCR results of H19 and DUSP27 expression from RC and HFD muscles. C: Representative Western blots of DUSP27, phosphorylation of AMPK (pAMPK), AMPK, pACC, ACC, and ACTB proteins from experiments shown in B. Extracts from three mice were loaded in each group. D: Quantiﬁcation results of Western blots. All data are shown as mean 6 SEM of four to ﬁve animals per group. *P , 0.05 and **P , 0.01, compared with the RC group. AUC, area under the curve.

presence of iLet7 (a let-7–specific inhibitor), the effects of lane 3). Next, immunofluorescence was performed on H19 downregulation (i.e., let-7 release and target gene myotubes using the same anti-AMPKa antibody whose inhibition) were attenuated (6,28–30). As expected, in specificity had been previously validated for both IP (31) myotutes, H19 knockdown (Fig. 3D, left column, compare and immunofluorescence (32). Results showed that a small gray bar to black bar) led to a decrease in DUSP27 fraction of DUSP27 colocalizes with AMPKa in the cyto- expression at the levels of both mRNA (Fig. 3D, right plasm (Fig. 4C, left panel, yellow dots indicated by white column, compare gray bar to black bar) and protein (Fig. arrowheads). The observation that not all DUSP27 and 3E, top blots in left panel, compare lane 2 to lane 1; right AMPKa colocalize with each other is consistent with panel, compare red dots to black dots). However, when AMPKa being a multifunctional protein known to have H19 was knocked down in the presence of iLet7 (Fig. 3D, many interacting partners. Our results indicate that an left column, compare white bar to black bar), there was no appreciable fraction of DUSP27 is in complex with longer a decrease in DUSP27 mRNA (Fig. 3D, right column, AMPKa. To determine whether a direct protein-protein compare white bar to black bar) or protein (Fig. 3E). Further, interaction is involved, in vitro pulldown experiments when let-7 was transfected, it suppressed DUSP27 expres- using purified recombinant proteins were conducted. As sion at both the mRNA (Fig. 3F) and protein levels (Fig. 3G). seen in Fig. 4D, FLAG-tagged DUSP27 (FL-DUSP27) was Taken together, these results suggest that in muscle cells, able to pull down GST-AMPKa (lane 4), whereas FLAG H19 posttranscriptionally regulates DUSP27 expression by peptide alone did not (lane 3). On the other hand, when reducing the bioavailability of let-7. GST-AMPKa (but not GST alone) was used as a bait, it DUSP27 Interacts With AMPKa pulled down FL-DUSP27 (Fig. 4E, compare lane 4 to lane 3). Collectively, our results identify DUSP27 as a new To determine whether DUSP27 mediates, at least in part, interacting partner of AMPKa. the H19-dependent regulation of AMPK, coimmunopre- cipitation (co-IP) experiments were performed using DUSP27 Contributes to H19-Dependent AMPK extracts from WT muscle. The DUSP27 antibody (anti- Activation DUSP27) was able to pull down DUSP27 (Fig. 4A, top blot, To determine whether DUSP27 affects AMPK activity, lane 3), together with AMPKa (bottom blot, lane 3), DUSP27 was knocked down in myotubes, followed by whereas the preimmune IgM control antibody did not Western blot analysis. Downregulation of DUSP27 (Fig. (lane 2). In reciprocal experiments, co-IP using an 5A, compare white bar to black bar) decreased AMPK AMPKa-specific antibody (31) pulled down AMPKa (Fig. phosphorylation (Fig. 5B, top blots in left panel, compare 4B, top blot, lane 3), together with DUSP27 (bottom blot, lane 3 to lane 1; middle panel, compare blue dots to black 2194 H19 lncRNA, Insulin Sensitivity, and AMPK Diabetes Volume 67, November 2018

Figure 7—Mechanisms of H19 downregulation in diabetic muscle. A: A proposed model. Short-term (B–F) and long-term (G–K) effects of HFD on gene expression and NCTC1 methylation. B and G: RT-qPCR results of let-7, H19, and DUSP27 RNA from RC and HFD muscles. C and H: QMSP results of NCTC1 gene body methylation of RC and HFD muscles. D–F and I–K: Spearman correlations with P values and total animal numbers (RC plus HFD) marked. B, C, G, and H: Data are shown as mean 6 SEM of ﬁve to eight animals per group. *P , 0.05 and **P , 0.01, compared with the RC group. ns, no statistical signiﬁcance.

dots), as well as phosphorylation of ACC (Fig. 5B, left and DUSP27, in which the catalytically critical Cys-146 was right panels), suggesting that DUSP27 positively affects replaced by Ser) were transfected into myotubes. Western AMPK activity. Next, when DUSP27 expression was re- blot analysis showed that increasing either wt-DUSP27 (Fig. duced as a result of H19 downregulation (Fig. 5A, compare 5F, top blot in left panel, compare lane 2 to lane 1; right gray bar to black bar), decreased AMPK phosphorylation panel, compare red dots to black dots) or mt-DUSP27 (Fig. (Fig. 5B, left and middle panels) accompanied by decreased 5E, top blot in left panel, compare lane 3 to lane 1; right phosphorylation of ACC (Fig. 5B, left and right panels) was panel, compare blue dots to black dots) was able to increase observed. Further, when DUSP27 expression was inhibited phosphorylation of both AMPK and ACC (Fig. 5F). These by let-7 (Fig. 5C), there was a decrease in phosphorylation results suggest that DUSP27 likely acts as a cofactor for of both AMPK (Fig. 5D, left and middle panels) and ACC AMPK and that the catalytic activity of DUSP27 does not (Fig. 5D, left and right panels). To determine whether appear to be required for this regulation. To determine increasing DUSP27 affects AMPK activity, plasmids express- whether DUSP27 contributes to H19-mediated AMPK ac- ing WT DUSP27 (wt-DUSP27) or mutant DUSP27 (mt- tivation, H19 knockdown in combination with exogeneous diabetes.diabetesjournals.org Geng and Associates 2195

DUSP27 expression experiments were conducted. As in skeletal muscle; let-7 binds to H19 and induces H19 expected, H19 knockdown decreased DUSP27 expression degradation, thereby temporally reducing H19 levels (6) (Fig. 5G, left panel, top blot, compare lane 2 to lane 1; middle (Fig. 7A). The system then corrects itself to restore H19 to panel, compare red dots to black dots) and AMPK phos- normal levels at later time points after acute hyperinsu- phorylation (left and right panels). Exogeneous expression linemia. This regulation thus appears to serve as a pro- of DUSP27 with H19 knockdown (left panel, top blot, tective mechanism in individuals without diabetes to compare lane 3 to lanes 2 and 1, and middle panel) partially prevent muscle from overusing circulating glucose, which restored AMPK phosphorylation to the control level (left otherwise would be toxic to the muscle (6). Importantly, and right panels). Glucose uptake assays showed that H19 the high-dose insulin–induced let-7 increase and H19 knockdown impaired insulin-stimulated glucose uptake (Fig. decrease are dependent on intact insulin signaling, as 5H, compare middle column to left column), whereas exoge- such effects were no longer observed in HFD mus- neous DUSP27 expression partially restored insulin-stimu- cle (6). We also showed that H19 functions to modulate lated glucose uptake to the control level (right column). The DNA methylation by inhibiting S-adenosylhomocysteine lack of full restoration of AMPK phosphorylation and hydrolase (SAHH) (the H19/SAHH axis) (33,34). Specifi- glucose uptake to control levels likely reflected the lack of cally, in mouse myotubes, H19 siRNA knockdown activates full restoration of DUSP27 expression, as the balance be- SAHH (H19 normally binds to SAHH and inhibits its tween H19 siRNA knockdown efficiency and DUSP27 over- enzymatic activity), leading to increases in gene body expression without compromising cell viability had limited methylation and subsequently increased transcription of the amount of DUSP27 to be transfected. Taken together, NCTC1, a lncRNA-encoding gene located 20 kb down- these results support the notion that the H19/DUSP27/ stream of H19 (33) (Fig. 7A). This NCTC1 transcription AMPK axis regulates muscle insulin sensitivity in a cell- increase leads to inhibition of H19 expression via a mech- autonomous manner (Fig. 5I). anism involving promoter competition (the NCTC1 promoter recruits RNA polymerase II for its own transcription DUSP27 Is Downregulated in Muscle of HFD-Induced at the expense of H19 transcription) (33,35). Taken to- Glucose-Intolerant Mice gether, these previous findings led us to hypothesize that Our previous studies showed that H19 is downregulated in the pathway illustrated in Fig. 7A may contribute to the muscle of insulin-resistant patients and rodents (6). As underlying mechanism of H19 repression in diabetic DUSP27 is regulated by H19 (Fig. 3) and as its expression is muscles. decreased in KO muscle (Fig. 2G and H), we asked whether To further explore this model, we examined the expres- DUSP27 expression is altered in the muscle of HFD- sion of let-7, H19, and DUSP27 as well as NCTC1 gene induced glucose-intolerant mice (Fig. 6A). The expression body methylation in muscles of mice after 11-day (pre- of H19 was expectedly decreased in the HFD as compared diabetes) and 13-week (chronic diabetes) exposure to RC or with RC muscle (Fig. 6B, left column), and the expression HFD, respectively. These time points were chosen based on of DUSP27 was also reduced at levels of both mRNA (Fig. a previous study showing that HFD-fed mice started to 6B, right column) and protein (Fig. 6C, top blot, compare exhibit glucose intolerance (as determined by GTTs) at day lane 2 to lane 1, and Fig. 6D, left panel). This was 3, developed hepatic insulin resistance (as determined by accompanied by a decrease in phosphorylation of both hyperinsulinemic-euglycemic clamp studies) at day 7, and AMPK (Fig. 6C and middle panel in Fig. 6D) and ACC (Fig. developed muscle insulin resistance at day 21 (36). After 6C and right panel in Fig. 6D). The decreased ACC total 11 days of HFD feeding, the mice started to show increased protein level in the HFD muscle is consistent with our let-7 expression (Fig. 7B, left column), likely as a result of observation in H19 KO muscle (Fig. 2C), possibly reflecting hyperinsulinemia-induced let-7 upregulation. Importantly, a failed compensatory mechanism. Collectively, our results there was a statistically significant negative correlation further support that the H19/DUSP27/AMPK signaling between let-7 and H19 levels (Fig. 7D). A significantly pathway contributes to muscle insulin sensitivity regulation. positive correlation between H19 and DUSP27 was also H19 Downregulation Involves Both Posttranscriptional observed (Fig. 7E), underscoring an in vivo regulation of and Epigenetic Mechanisms DUSP27 by H19. The apparent heterogeneity in the levels So far, our results strongly suggest that H19 plays an of let-7, H19, and DUSP27 among the individual mice important role in muscle insulin sensitivity regulation, at highlights their differential responses to HFD exposure least in part, by activating AMPK via the putative cofac- during early stages of diabetic development. The lack of tor DUSP27. An important question remains: what in- difference in the average levels of H19 (Fig. 7B, middle duces and maintains H19 downregulation in the muscle of column) and DUSP27 (Fig. 7B, right column) between the humans and rodents with diabetes? Addressing this ques- two groups likely reflected self-correction after hyperinsu- tion has an important clinical implication as it will help to linemia assaults at early stages of diabetes (6). When identify potential molecular targets for the prevention and NCTC1 methylation was assessed using QMSP (33), no treatment of insulin resistance. Our previous work showed significant differences were detected between the two that in nondiabetic mice, acute hyperinsulinemia upregu- groups (Fig. 7C), nor was there a statistically signifi- lates let-7 via activation of the insulin-PI3K-AKT pathway cant correlation between NCTC1 methylation and H19 2196 H19 lncRNA, Insulin Sensitivity, and AMPK Diabetes Volume 67, November 2018 expression (Fig. 7F). Together, these results suggest a mech- being a multifunctional lncRNA, we are certain that anism of let-7–mediated downregulation of H19 in the DUSP27 is not the only downstream effector of H19. early-stage diabetic muscle. Nonetheless, DUSP27 likely plays an important part in When muscles were analyzed after 13 weeks of RC or the H19-mediated regulation of AMPK activity for the HFD exposure, there was no longer a difference in let-7 following reasons. First, both H19 and DUSP27 are evo- levels between the two groups (Fig. 7G, left column), lutionarily conserved and both are highly (and almost although the levels of both H19 (middle column) and exclusively) expressed in human and mouse skeletal mus- DUSP27 (right column) remained significantly lower in cle. Second, there is a strong in vivo positive correlation of the HFD group than in the RC group. The lack of let-7 expression between H19 and DUSP27 in the context of KO difference between the two groups was consistent with muscle (Fig. 2G and H) and prediabetic and chronic di- impaired insulin signaling in the HFD group, as intact abetic muscle (Fig. 7E and J). Third, H19 siRNA knock- insulin signaling is required for let-7 upregulation (6). down in vitro leads to decreased DUSP27 expression via Moreover, whereas a negative correlation between let-7 the H19/let-7 axis (Fig. 3B–G). Fourth, siRNA knockdown and H19 was lost (Fig. 7I), a positive correlation between of either H19 or DUSP27 alone is sufficient to decrease H19 and DUSP27 persisted (Fig. 7J), again consistent with AMPK signaling (Fig. 5A and B). Finally, exogenous ex- an in vivo regulation of DUSP27 by H19. Remarkably, an pression of DUSP27 under the condition of H19 knock- increase in NCTC1 methylation (Fig. 7H), as well as a neg- down led to partial restoration of AMPK phosphorylation ative correlation between NCTC1 methylation and H19 and insulin-stimulated glucose uptake to control levels expression (Fig. 7K), became evident, supporting the hy- (Fig. 5G and H). Collectively, our study supports a role pothesis that chronic hyperinsulinemia (a hallmark of of DUSP27 in linking H19 to AMPK activation and glucose T2D) leads to a stable increase in muscle NCTC1 gene uptake. Future studies using KO and transgenic DUSP27 body methylation and transcription, which in turn leads to mouse models will be necessary to firmly establish the decreased H19 transcription via promoter competition. physiological role of DUSP27 in H19-mediated muscle insulin sensitivity regulation. DISCUSSION DUSPs have been implicated as major modulators of This work identifies H19 lncRNA as a novel modulator of critical signaling pathways that are dysregulated in various AMPK activity, with DUSP27 being an important down- diseases (25). Unlike many DUSPs that have been exten- stream effector of H19 in muscle insulin sensitivity reg- sively studied, little is known about the regulation of ex- ulation. We show that DUSP27 is regulated by H19 at the pression, substrate specificity, mode of action, and especially posttranscriptional level and contributes, at least in part, the biological and physiological functions of DUSP27 (24), to the ability of H19 to enhance AMPK activity. The except for one study showing that DUSP27 can dephos- demonstration that both H19 and DUSP27 expression phorylate and deactivate ERK and p38 MAPKs in ovarian are responsive to dietary cues and can influence signal cells (26). In the current study, we identify DUSP27 as transduction pathways related to T2D highlights their po- a target of the H19/let-7–mediated posttranscriptional tential physiological importance in the regulation of systemic regulation and AMPK as a potentially physiologically rele- glucose metabolism. Further, our exploration of underlying vant target of DUSP27. Although we show evidence that mechanisms leading to H19 and DUSP27 reduction in di- DUSP27 and AMPK interact, the exact mode of action of abetic muscles holds the promise of discovering new ther- DUSP27 in AMPK regulation is unclear, which warrants apeutic targets for T2D and other metabolic diseases. future investigation. Although we cannot exclude the possibility that tissues Despite the identification of numerous lncRNAs in the other than muscle may contribute to the observed in vivo human genome, the physiological role of the vast majority effects (Fig. 1) due to the use of whole-body KO mice, our has remained elusive. In the mouse, a number of lncRNAs results from both cell and animal models together strongly have been shown to play important roles in metabolism point to the H19/DUSP27/AMPK pathway as a novel con- (41–47). For example, the liver-specific triglyceride regu- tributing mechanism of muscle insulin sensitivity regulation. lator (lncLSTR) regulates systemic lipid homeostasis by Notably, we observed a modest increase in Igf2 expres- inhibiting triglyceride uptake into adipose tissues and sion in the H19 KO muscle (data not shown), consistent skeletal muscle (42). The fasting-induced liver glucokinase with previous reports (10,11). However, it is very unlikely (GCK) repressor (LncLGR) inhibits hepatic glycogen syn- that this Igf2 increase contributes to the muscle insulin thesis and lipogenesis by downregulating transcription of resistance seen in the H19 KO mice because it should exert GCK (43). The brown fat lncRNA1 (Blnc1) forms ribonu- effects opposite to those observed. Indeed, it has been well cleoprotein complexes with transcription factor EBF2 to documented that Igf2 binds to INSR and activates the stimulate thermogenic gene expression, thereby promot- insulin-PI3K-mTOR signaling pathway in muscle to increase ing thermogenic adipocyte differentiation (44). Finally, the glucose uptake both in mice (37) and humans (38–40). dietary-inducible liver LeXis regulates cholesterol biosyn- Based on our genome-wide transcriptome studies re- thetic gene expression through interaction with the het- vealing extensive gene expression changes in KO versus erogeneous ribonucleoprotein Raly (47). Notably, all these WT muscle (GEO accession no. GSE103202) and on H19 lncRNAs (lncLSTR, LncLGR, Blnc1, and LeXis) are nuclear diabetes.diabetesjournals.org Geng and Associates 2197 and function by forming ribonucleoprotein complexes to resistance in conditional X-box-binding protein-1 (XBP1) knock-out mice. J Biol affect gene expression at the transcriptional level, albeit Chem 2012;287:2558–2567 with distinct modes of action. Although no human coun- 9. Gabory A, Jammes H, Dandolo L. The H19 locus: role of an imprinted non- – terparts have yet been found, the identification and char- coding RNA in growth and development. BioEssays 2010;32:473 480 acterization of these mouse lncRNAs have shed critical 10. Ripoche MA, Kress C, Poirier F, Dandolo L. Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element. Genes Dev light on our understanding of mechanisms of how lncRNAs 1997;11:1596–1604 impact metabolism. Our findings are of particular impor- fi 11. Martinet C, Monnier P, Louault Y, Benard M, Gabory A, Dandolo L. H19 tance to the eld for the following reasons. First, they controls reactivation of the imprinted gene network during muscle regeneration. fi represent the rst example of an evolutionarily conserved, Development 2016;143:962–971 abundantly expressed lncRNA in muscle metabolic regu- 12. Boucher J, Kleinridders A, Kahn CR. Insulin receptor signaling in normal and lation. Second, the regulation of AMPK, an evolutionarily insulin-resistant states. Cold Spring Harb Perspect Biol 2014;6:a009191 conserved key metabolic fuel gauge, by an lncRNA has not 13. Liu HY, Hong T, Wen GB, et al. Increased basal level of Akt-dependent insulin been previously documented. Finally, the way by which signaling may be responsible for the development of insulin resistance. Am J H19 regulates AMPK is unique in that it involves a little- Physiol Endocrinol Metab 2009;297:E898–E906 studied dual-specificity protein phosphatase DUSP27, which 14. Fujii N, Ho RC, Manabe Y, et al. Ablation of AMP-activated protein kinase is highly expressed in skeletal muscle with previously un- alpha2 activity exacerbates insulin resistance induced by high-fat feeding of known function. mice. Diabetes 2008;57:2958–2966 15. Schönke M, Myers MG Jr., Zierath JR, Björnholm M. Skeletal muscle AMP- activated protein kinase g1(H151R) overexpression enhances whole body energy Acknowledgments. The authors thank Dr. Luisa Dandolo for providing homeostasis and insulin sensitivity. Am J Physiol Endocrinol Metab 2015;309: the H19 KO mice and the Yale Mouse Metabolic Phenotyping Center In Vivo E679–E690 Metabolism Core for performing hyperinsulinemic-euglycemic clamp studies 16. Fullerton MD, Galic S, Marcinko K, et al. Single phosphorylation sites in Acc1 and measurements of muscle TAG contents. and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of met- Funding. This work was supported by the American Diabetes Association formin. Nat Med 2013;19:1649–1654 (1-15-BS-084), the National Institutes of Health (R01-HD-072418 to G.G.C.), 17. O’Neill HM, Lally JS, Galic S, et al. AMPK phosphorylation of ACC2 is required the National Natural Science Foundation of China (81402130 to D.L.), and the for skeletal muscle fatty acid oxidation and insulin sensitivity in mice. Diabetologia Albert Mckern Foundation (GE001347 to Y.H.). 2014;57:1693–1702 Duality of Interest. No potential conflicts of interest relevant to this article 18. Choi CS, Savage DB, Abu-Elheiga L, et al. Continuous fat oxidation in acetyl- were reported. CoA carboxylase 2 knockout mice increases total energy expenditure, reduces Author Contributions. T.G. designed and performed the experiments, fat mass, and improves insulin sensitivity. Proc Natl Acad Sci U S A 2007;104: collected and analyzed the data, and wrote the manuscript. Y.L., Y.X., Y.J., and 16480–16485 N.Z. performed the experiments and collected and analyzed the data. Z.W. 19. 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