Exogenous Administration of DLK1 Ameliorates Hepatic Steatosis and Regulates Gluconeogenesis Via Activation of AMPK

ORIGINAL ARTICLE Exogenous administration of DLK1 ameliorates hepatic steatosis and regulates gluconeogenesis via activation of AMPK

Y-h Lee1,5,MRYun1,5, HM Kim2, BH Jeon1, B-C Park3, B-W Lee1, ES Kang1,HCLee1, YW Park3 and B-S Cha1,4

BACKGROUND/OBJECTIVES: Activation of Notch signaling pathologically enhances lipogenesis and gluconeogenesis in the liver causing non-alcoholic fatty liver disease (NAFLD) and diabetes. Delta-like 1 homolog (DLK1), an imprinted gene that can modulate adipogenesis and muscle development in mice, was found as an inhibitory regulator of Notch signaling. Therefore, we investigated the metabolic effect of exogenous DLK1 in vitro and in vivo. SUBJECTS/METHODS: A soluble DLK1 peptide was generated with fusion between a human Fc fragment and extracellular domain of DLK1. Male db/db mice were randomly assigned to two groups: vehicle treated and DLK1-treated group (25 mg kg − 1, intraperitoneal injection, twice a week for 4 weeks). Primary mice hepatocytes and HepG2 cells were used for in vitro experiments. RESULTS: After 4 weeks of DLK1 administration, hepatic triglyceride content and lipid droplets in liver tissues, as well as serum levels of liver enzymes, were markedly decreased in db/db mice. DLK1 treatment induced phosphorylation of AMPK and ACC and suppressed nuclear expression of SREBP-1c in the mouse liver or hepatocytes, indicating regulation of fatty acid oxidation and synthesis pathways. Furthermore, DLK1-treated mice showed significantly lower levels of fasting and random glucose, with improved glucose and insulin tolerance compared with the vehicle-treated group. Macrophage infiltration and proinflammatory cytokine levels in the epididymal fat were decreased in DLK1-treated db/db mice. Moreover, DLK1 suppressed glucose production from hepatocytes, which was blocked after co-administration of an AMPK inhibitor, compound C. DLK1-treated hepatocytes and mouse liver tissues showed lower PEPCK and G6Pase expression. DLK1 triggered AKT phosphorylation followed by cytosolic translocation of FOXO1 from the nucleus in hepatocytes. CONCLUSIONS: The present study demonstrated that exogenous administration of DLK1 reduced hepatic steatosis and hyperglycemia via AMPK activation in the liver. This result suggests that DLK1 may be a novel therapeutic approach for treating NAFLD and diabetes. International Journal of Obesity (2016) 40, 356–365; doi:10.1038/ijo.2015.173

BACKGROUND exhibit upregulated hepatic Notch signaling that correlated Obesity is currently a worldwide epidemic that leads to the positively with the severity of fatty liver and the expression of 7 development of chronic metabolic diseases such as type 2 gluconeogenic genes. Genetic- or chemical-induced suppression diabetes and non-alcoholic fatty liver disease (NALFD).1 Despite of Notch demonstrated an improvement in insulin sensitivity with the development of several classes of therapeutic agents for decreased blood glucose levels5 and protection from diet-induced treating type 2 diabetes, other treatment options that cure the fatty liver and hypertriglyceridemia.6 condition are needed. As of yet, there are no clinically available The canonical Notch signaling pathway is activated by direct drugs to treat NAFLD. interaction between one of four Notch receptors (Notch1–4) and The evolutionarily conserved Notch pathway has an essential transmembrane Notch ligands of the Jagged (−1 and − 2) or Delta- role in many fundamental processes during early development like (−1, − 3, − 4) families on a neighboring cell, resulting in a series and mature tissue homeostasis by regulating cell fate determina- of proteolytic cleavages that induce the transcription of Notch tion, proliferation, differentiation and death.2,3 Aberrant modula- targets.2,8 Delta-like 1 homolog (DLK1) is known as a transmem- tion of Notch signaling pathways has been directly linked to brane protein belonging to the epidermal growth factor-like various diseases such as congenital disorders and cancer.3,4 repeat-containing family, which also includes Notch receptors and Moreover, Notch signaling has been revealed as a crucial their ligands (for example, jagged and delta-like isoforms).9 component involved in the development of NAFLD and Accumulating evidence strongly indicates that DLK1 interacts diabetes.5,6 Notch activation pathologically enhances lipogenesis with Notch1 and functions as an inhibitory regulator of Notch and gluconeogenesis in hepatocytes, thereby resulting in signaling.10–12 Furthermore, the soluble extracellular domain of increased insulin resistance.5,6 type 2 diabetes and NAFLD patients DLK1 produced by a protease of tumor necrosis factor-α

1Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea; 2Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul, Korea; 3Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea and 4Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea. Correspondence: Dr B-S Cha, Department of Internal Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Gu, Seoul 120 752, South Korea. E-mail: [email protected] 5These authors contributed equally to this work. Received 31 March 2015; revised 7 August 2015; accepted 13 August 2015; accepted article preview online 28 August 2015; advance online publication, 22 September 2015 Metabolic effect of DLK1 Y Lee et al 357 converting enzyme suppressed adipogenesis in vitro and in vivo.13 pore mesh. After centrifugation, pellets were resuspended in Dulbecco's Based on these findings, DLK1 may be a promising target for Modified Eagle's medium (DMEM; GE Healthcare Hyclone, Sungnam, modulating metabolic dysfunction found in type 2 diabetes or Korea) supplemented with 2.7 mM D-glucose and 10% fetal bovine serum fi NAFLD by inhibiting Notch signaling. Therefore, we developed for and subsequently incubated at 37 °C in humidi ed air containing 5% CO2. fi After determining cell viability via trypan blue exclusion testing, the rst time an Fc-conjugated DLK1 consisting of the N-terminal 5 extracellular domain of DLK1 fused to an Fc fragment, and hepatocytes were seeded on collagen-coated 6-well plates (5 × 10 per well) and incubated for 24 h before use. HepG2 cells were maintained in investigated the therapeutic effect of the recombinant DLK1 high-glucose DMEM medium supplemented with 10% fetal bovine serum, protein in animal models of fatty liver and diabetes. 100 U penicillin and 100 μg streptomycin.

MATERIALS AND METHODS Protein extraction and immunoblotting Development of soluble DLK1 protein Mouse livers, primary hepatocytes and HepG2 cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA), and the protein To produce a soluble form of DLK1 protein, the extracellular domain of content was measured using the Bradford assay (Bio-Rad, 162-0115, DLK1 (Glu25 to Gly302) was fused to a human Fc fragment. We generated Hercules, CA, USA). Nuclear and cytosolic proteins were extracted from a plasmid, pYK602-sDLK1, which contains a signal sequence for mouse livers, primary hepatocytes and HepG2 cells using the NE-PER kit secretion and the CMV promoter. Expression was performed as described ’ 14 fi (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer s previously. Puri cation was then conducted with using protein instructions. Equal amounts of protein (30 μg) were heat-denatured in 4 × A-Sepharose (GE Healthcare, Uppsala, Sweden). The purities of proteins sample buffer (2% sodium dodecyl sulfate, 62.5 mM Tris (pH 6.8), 0.01% in eluted solutions were assessed by Coomassie staining after sodium bromophenol blue, 1.43 mM β-mercaptoethanol and 0.1% glycerol), dodecyl sulfate-polyacrylamide gel electrophoresis. separated on 10 or 12% sodium dodecyl sulfate-polyacrylamide gels and electrotransferred onto nitrocellulose membrane (Bio-Rad). Membranes Animal procedures were subsequently blotted with antibodies against the following proteins: Seven-week-old db/db male mice and C57BL/6J mice were purchased pAMPK (cat#2535, Cell Signaling Technology), AMPK (cat#2603, Cell from Orient Bio (Sungnam, Korea). Each group contained four animals and Signaling Technology), pACC (cat#3661, Cell Signaling Technology), ACC was housed in an animal facility maintained at a temperature of 23 ± 2 °C (cat#3662, Cell Signaling Technology), pAkt (cat#9271, Cell Signaling and a humidity of 55 ± 5%. The mice were exposed to a 12 h light, 12-h Technology), Akt (cat#4691, Cell Signaling Technology), FOXO1 (cat# sc- β dark cycle and fed a standard unrestricted diet with monitoring for food 67140, Santa Cruz, CA), -actin (cat#sc-47778, Santa Cruz), SREBP1-c fi intake and body weight twice a week. In db/db mice, the DLK1-treated (cat#PA1-46142, Thermo Fisher Scienti c, Rockford, IL), lamin (cat#4777, group (n = 12) received DLK1 intraperitoneally (25 mg kg − 1) twice a week, Cell Signaling Technology) and GAPDH (cat#sc-25778, Santa Cruz). and the vehicle-treated group (n = 12) were given the same volume of PBS − instead of DLK1. C57BL/6J mice were either administered 15 mg kg 1 RNA isolation and real-time PCR DLK1 (n = 6) or vehicle alone (n = 6) intraperitoneally twice a week. Animals Total RNA was extracted using Tirol reagent (Invitrogen, Grand Island, NY, were killed after fasting for 6 h. All animal studies were approved by the USA) according to the manufacturer’s instructions and subjected to reverse Animal Care and Use Committee of the Yonsei University College of transcription with the high capacity complementary DNA transcription kit Medicine (No. 2013-0147-1). (Applied Biosystems, Foster City, CA, USA) followed by quantitative real- time PCR using the ABI 7500 sequence detection system (Applied Glucose and insulin tolerance tests Biosystems). PCR was conducted using the following primers (for SYBR ′ ′ An oral glucose tolerance test was performed in 11-week-old db/db mice Green): PEPCK, forward (5 -CTT CTC TGC CAA GGT CAT CC-3 ) and reverse − ′ ′ ′ after an overnight fast. After 1 g kg 1 of glucose was administered orally, (5 -GTG CCC ATC CCC AAA A-3 ); G6Pase, forward (5 -TCC TGG GAC AGA ′ ′ ′ blood glucose levels were measured at 30, 60, 90, 120, 180 and 240 min CAC ACA AG-3 ) and reverse (5 -CCA ATA GCG TAT ATT AAA GTT G-3 ); ′ using a glucose analyzer (Accu-Check; Roche Diagnostics, Basel, Switzer- acyl-Coenzyme A dehydrogenase, forward (5 -TGA CGG AGC AGC CAA TGA ′ ′ ′ land). The insulin tolerance test was conducted following intraperitoneal -3 ) and reverse (5 -TCG TCA CCC TTC TTC TCT GCT T-3 ); GAPDH, forward − ′ ′ ′ administration of human regular insulin (1.5 U kg 1, Novolin R; Novo (5 -AAC TTT GGC ATT GTG GAA GG-3 ) and reverse (5 -TGT TCC TAC CCC ′ ′ Nordisk, Clayton, NC, USA) after a 6 h fast. Blood samples were collected CAA TGT GT-3 ); CPT-1a, forward (5 -GGG AGG ACA GAG ACT GTA CGC TC ′ ′ ′ before and at 30, 60, 90 and 120 min after insulin injection. -3 ) and reverse (R 5 -TGT AGG AAA CAC CAT AGC CGT CAT-3 ); ACOX, forward (5′-GGG TGG TAT GCT GTC GTA C-3′) and reverse (5′-CAA AGA CCT TAA CGG TCA CGT AGT G-3′); AMPK, forward (5′-TGA CGG AGC AGC CAA Biochemical analyses TGA-3′) and reverse (5′-TCG TCA CCC TTC TTC TCT GCT T-3′). Quantitative Blood samples were obtained with heparinized syringes by puncture into analyses were performed using the ΔΔcycle threshold method and the inferior vena cava and immediately centrifuged at 5000 g for 15 min. StepOne Software version 2.2.2 (Grand Island, NY, USA). Serum levels of aspartate aminotransferase, alanine aminotransferase, and fi non-esteri ed fatty acids were measured by ELISA (BioAssay Systems, Glucose production assay Hayward, CA, USA). Cholesterol and triglycerides were examined using ELISA kits from BioVision (Milipitas, CA, USA), whereas serum insulin levels After a 4 h incubation in serum-free, 25 mM glucose DMEM containing were also measured using an ELISA kit (ALPCO Diagnostics, Salem, NH). 10 nM insulin, primary hepatocytes or HepG2 cells were treated for 6 h with the indicated specific media: 0.5 mM adenosine 3’,5’-cyclic monophosphate (cAMP) plus 1 μM dexamethasone (Dex) to induce gluconeogenesis, − 1 Hepatic triglyceride measurement and Oil Red O staining 150 μgml DLK1 or 100 nM insulin to suppress gluconeogenesis. After homogenization, triglyceride content in liver tissues was measured Compound C (10 μM, Sigma-Aldrich, St Louis, MO, USA) was used to block with the Triglycerides Quantification Kit (K622, Biovision, Milipitas, CA, USA) AMPK activation. After 1–3 h of incubation in gluconeogenic media (20 mM according to the manufacturer’s instructions. Lipid droplets in HepG2 cells sodium lactate and 2 mM sodium pyruvate containing serum-free, glucose- were visualized and subsequently quantified by Oil Red O staining after free, phenol red-free DMEM), glucose production was measured in the treatment with palmitate and DLK1. To measure the quantification of lipid media using a Glucose Assay kit (Abcam, Cambridge, MA, USA). accumulation, Oil Red O was eluted by adding 100% isopropanol and the optical density was measured by spectrophotometry at 520 nm. Immunofluorescence Immunofluorescent staining was conducted to detect the expression of Isolation of primary hepatocytes and cell culture FOXO1 in cultured primary hepatocytes and HepG2 cells as described Primary hepatocytes were harvested according to the two-step perfusion previously.16 Cells were plated at 1 × 104 cells per well on chamber glass method as previously described15 with minor modifications. Livers were slides and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 5 min after perfused with Hank’s Balanced Salt Solution followed by digestion with washing with PBS. Cells were blocked in PBS containing 5% bovine serum buffer containing collagenase type 2 (Gibco, Gaithersburg, MD, USA). Then, albumin for 2 h at room temperature and incubated with the primary the liver was finely chopped in a Petri dish and filtered through a 100-μm FOXO1 antibody (1:200) overnight at 4 °C, followed by incubation with

© 2016 Macmillan Publishers Limited International Journal of Obesity (2016) 356 – 365 Metabolic effect of DLK1 Y Lee et al 358 secondary goat anti-rabbit IgG-FITC (1:400, Invitrogen) for 2 h at room Statistical analyses temperature. Propidium iodide (PI, 1:1000, Invitrogen) was used as a Data were compared using the Student’s t-test or analysis of variance using nuclear counterstain. Images were obtained using a confocal microscope the SPSS 20.0 program (SPSS Institute, Chicago, IL, USA) and graphs were (LSM700; Carl Zeiss Inc., Oberkochen, Germany). plotted using GraphPad Prism (Version 5.0, GraphPad, San Diego, CA, USA). A P-value o0.05 was considered statistically significant. All results are Histological analysis and immunohistochemistry representative of data from three to seven experiments. Variances were fi used depending on the result. The power of our study was estimated as The removed masses were xed using 10% neutral-buffered formalin and α α fi μ 80.3% ( = 0.05) based on Figure 1c, and 97.1% ( = 0.05) based on processed into paraf n blocks. Sections (4 m) were stained with Figure 2a using G*Power (Version 3.1.9.2, Kiel, Germany). hematoxylin and eosin. Immunohistochemical analysis for the expression of F4/80 was performed as described previously.16 After antigen retrieval with citrate buffer (pH 6.0 at 90 °C), specimens were incubated with anti- RESULTS F4/80 antibody (1:400; Abcam), then specific biotinylated secondary antibody (1:100; Vector Laboratories, Burlingame, CA, USA), followed by Soluble DLK1 ameliorates hepatic steatosis in db/db mice streptavidin-peroxidase (DAKO, Kyoto, Japan). diaminobenzidine (Vector To investigate the metabolic effect of DLK1 in the animal model of Laboratories) was used as a chromogen, and counterstaining was fatty liver and diabetes, we generated a soluble DLK1 peptide conducted using hematoxylin. The number of F4/80-positive cells in each consisting of the N-terminal extracellular domain of DLK1 fused to stained section was counted at × 400 magnification. The fraction of F4/80- a human Fc fragment (Figure 1a). This recombinant protein positive macrophages was determined as a percentage of the total contains six epidermal growth factor-like domains and the number of cells in each tissue section. juxtamembrane portion, which corresponds to residues 25–302 Immunohistochemistry of the pancreas was conducted using an anti- of DLK1 (P80370, UniProt Knowledgebase). Blood samples for insulin antibody (sc-9168, Santa Cruz). The percentage of β-cells was pharmacokinetic analysis were collected from mice dosed with calculated as the ratio of the insulin-positive cell area to the total − 1 pancreatic tissue area within the entire section. The β-cell mass was intraperitoneally-injected 15 mg kg until 72 h, demonstrating 17 derived by multiplying the β-cell fraction by the weight of the pancreas. that the mean half-life (T1/2) of DLK1 was 26 h and plasma − 1 Histological images were analyzed using ImageJ software (NIH Image, concentration of DLK1 was maintained approximately 10 μgml Bethesda, MA, USA). (Supplementary Fig. S1).

Figure 1. Exogenous administration of soluble DLK1 ameliorated hepatic steatosis in db/db mice. (a) Schematic of the structure of endogenous DLK1 (upper) and DLK1-human Fc fragment fusion protein (lower). EGF-like repeat domains are colored in purple. Tumor necrosis factor alpha converting enzyme (TACE) cleavage site is marked. S, signal sequence; JM, juxtamembrane domain; TM, transmembrane domain; Cy, cytoplasmic region; hFc, human Fc fragment. (b–f) DLK1 (25 mg kg − 1) was injected intraperitoneally (twice weekly) into db/db mice for 4 weeks we evaluated (b) liver histology (Magniﬁcation, × 200), (c) hepatic TG content, (d) liver weight, and (e) hepatic glycogen content. (f) Serum AST and ALT were measured after 4 weeks of DLK1 treatment. Data are presented as the mean ± s.d. (n = 6). *Po0.05 and **Po0.01. BW, body weight; TG, triglyceride.

Figure 2. Exogenous administration of soluble DLK1 ameliorated hyperglycemia and dyslipidemia in db/db mice. (a) Fasting glucose levels. (b) Random glucose levels. (c, d) Oral glucose tolerance test (c) and insulin tolerance test (d) were performed after a 4-week treatment of DLK1 or vehicle. (e) Serum TG, total cholesterol, and non-esteriﬁed fatty acid (NEFA) were measured after 4 weeks of DLK1 treatment. Data are presented as the mean ± s.d. (n = 6). *Po0.05 and **Po0.01 compared with the vehicle group.

After treatment for 4 weeks with DLK1, db/db mice exhibited proinflammatory cytokine mRNA expression such as interleukin- markedly decreased hepatic triglyceride content and lipid droplets 1β and iNOS in epididymal fat pads (Figures 3f and g). (Figures 1b and c). However, vehicle- and DLK1-treated mice had comparable liver weights (Figure 1d), which may be due to Inhibition of lipid accumulation via AMPK activation by DLK1 increased glycogen content in the liver of DLK1-treated mice NAFLD treatments such as metformin18 and glucagon-like (Figure 1e). Serum levels of aspartate aminotransferase and (ref. 19) fi peptide-1 primarily target AMPK, leading to increased fatty alanine aminotransferase were also signi cantly decreased in acid oxidation and suppression of lipid accumulation in mice administered with DLK1 (Figure 1f). hepatocytes.20 Similarly, we observed that DLK1 treatment in db/db mice significantly enhanced Thr172 phosphorylation on Soluble DLK1 decreased blood glucose levels and macrophage AMPK-α (Figure 4a). Phosphorylation of acetyl–coenzyme A infiltration in adipose tissues of db/db mice carboxylase (ACC), a downstream target of AMPK, was also Furthermore, DLK1-treated mice showed significantly lower levels induced in the liver of db/db mice treated with DLK1. Among the of fasting and random blood glucose levels but not insulin genes involved in fatty acid oxidation, our data demonstrate that compared with the control group (Figures 2a and b and hepatic expression of acyl-Coenzyme A dehydrogenase (ACADM) Supplementary Fig. S2A). This result was supported by an overall was significantly elevated in DLK1-treated mice (Figure 4b). improvement in whole body glucose and insulin tolerance in Phosphorylation of AMPK by DLK1 was also replicated in the liver DLK1-treated mice compared with vehicle-treated mice of normal C57BL/6J mice that received DLK1 (Figure 4c). We also (Figures 2c and d). However, vehicle- and DLK1-treated mice confirmed that the activation of AMPK by DLK1 in primary exhibited comparable body weights and composition in terms of hepatocytes (Figure 4d) and HepG2 cells in vitro (Figure 4e) was skeletal muscle, as well as in subcutaneous and visceral fat depots consistent with in vitro studies. Moreover, DLK1 increased the (Supplementary Fig. S2B–E); they also consumed similar quantities phosphorylation of both AMPK and ACC in a dose-dependent of food (Supplementary Fig. S2F). Plasma triglyceride and non- manner (Figure 4e). However, administration of human Fc esterified fatty acid levels were decreased in DLK1-treated db/db fragment alone showed no effect on AMPK activation mice, whereas total cholesterol levels were not statistically (Figure 4d). These results suggest that DLK1 induces fatty acid different between the two groups (Figure 2e). Pancreatic islet oxidation in hepatocytes by activating AMPK. mass was marginally decreased in control mice compared with To assess whether DLK1 treatment ameliorates lipid accumula- DLK1-treated mice (Figures 3a–c). To assess the role of DLK1 in tion in hepatocytes via AMPK activation in vitro, we stained lipid adipose inflammation, epididymal fat pads were stained with droplets with Oil Red O and quantified them by spectro- F4/80 antibody and then visualized by immunohistochemistry photometry. As shown in Figures 5a and b, DLK1 significantly (Figure 3d). Although adipocyte morphology did not differ reduced intracellular lipid accumulation in the presence of between the two groups, infiltration of F4/80-positive macro- palmitate compared with control. However, lipid accumulation phages was significantly decreased in DLK1-treated db/db was not affected by DLK1 administration after pretreatment with mice (Figure 3e). Furthermore, DLK1 reduced the level of compound C, an inhibitor of AMPK. Furthermore, DLK1 reduced

Figure 3. Effects of DLK1 treatment on pancreatic islets and adipose tissue inflammation in db/db mice. (a–c) Immunohistochemistry of db/db pancreas with anti-insulin antibody (stained in brown; × 40 magnification). Representative images are shown (a). The insulin-positive area of the islet was measured, and the β-cell fraction (b) and mass (c) were calculated. (d) Histology (H&E, upper row; × 100 magnification) and immunohistochemical staining of epididymal fat of db/db mice with anti-F4/80 antibody (stained in brown, lower row; × 200 magnification). (e) The percentage of F4/80-positive cells within epididymal fat pads was significantly reduced in DLK1-Fc-treated compared with vehicle- treated mice (Po0.05). (f,g) Expression of IL-1β (f) and iNOS (g) in epididymal fat pads by real-time PCR. Data are presented as the mean ± s.d. (n = 6). *Po0.05 and **Po0.01 compared with the vehicle group.

the nuclear expression of SREBP-1c in HepG2 cells with either glucose levels in db/db mice (Figure 2a), DLK1-treated normal mice control or palmitate-treated condition (Figure 5c). showed signiﬁcantly decreased plasma levels of fasting glucose but not random glucose compared with vehicle-treated mice (Figures 6a and b). In addition, total body weight and food intake were similar DLK1 suppresses hepatic glucose production by inhibiting G6Pase between the two groups (Supplementary Fig. S3). and PEPCK expression via FOXO1 translocation To elucidate the mechanism underlying the glucose-lowering Next, we investigated the effect of DLK1 treatment on glucose effect of DLK1, we examined the gene expression of essential metabolism in normal C57BL/6J mice, primary hepatocytes and enzymes involved in gluconeogenesis, such as PEPCK and G6Pase, HepG2 cells (Figure 6). Consistent with previous results of low fasting by real-time PCR in normal and db/db mice after DLK1 treatment.

Figure 4. DLK1 stimulated AMPK activation followed by induction of genes related to fatty acid oxidation in db/db mice. (a) db/db mice were treated with DLK1 for 4 weeks and then the levels of phosphorylated and total AMPK, as well as phosphorylated and total ACC, were determined in hepatocytes by immunoblotting. The right graph shows densitometric analysis of the optical density-based data of the phosphorylated AMPK/total AMPK ratio from the immunoblots shown on the left. Data are representative of six specimens. (b) Hepatic mRNA expression of CPT1, ACOX, and ACADM were measured by real-time PCR. (c) Level of phosphorylated and total AMPK, as well as phosphorylated and total ACC, in normal C57BL/6J mice treated with either vehicle or DLK1. Primary hepatocytes (d) and HepG2 cells (e) treated with DLK1 or WY14643 (as a positive control) were analyzed by immunoblotting. The right graph shows densitometric analysis of the optical density-based data of the phosphorylated AMPK/total AMPK ratio from the immunoblots shown on the left. *Po0.05 and **Po0.01.

PEPCK and G6Pase expression in DLK-treated mice were A previous study demonstrated that hepatic gluconeogenesis is decreased by approximately 50% relative to vehicle-treated db/ suppressed by cytosolic translocation of FOXO1 from the nucleus in a db and normal mice, respectively (Figures 6c and d). process mediated by Akt phosphorylation.21 AsshowninFigure6g, We then assessed whether this inhibitory effect of DLK1 on DLK1 treatment induced Akt phosphorylation in HepG2 cells. gluconeogenesis was mediated by AMPK activation. After 6 h of Furthermore, cAMP/Dex-induced nuclear translocation of FOXO1 pretreatment with cAMP and Dex, HepG2 cells and primary was inhibited, whereas cytosolic localization of FOXO1 was increased hepatocytes were treated with DLK1 or compound C and insulin by administration of DLK1 in HepG2 cells (Figure 6h). Immunofluor- for 6 h, and glucose production and gene expression was escent analysis also confirmed this result in HepG2 cells (Figures 6i assessed. As shown in Figure 6e, DLK1 significantly suppressed and j). These results suggest that DLK1 suppresses hepatic glucose production from both HepG2 cells and primary hepato- gluconeogenesis through the Akt and FOXO1 signaling pathway. cytes. However, this inhibitory effect was blocked by pretreatment with compound C. DLK1 also suppressed cAMP/Dex-induced upregulation of PEPCK expression (Figure 6f). These findings DISCUSSION indicate that DLK1 inhibits hepatic glucose production by The present study reports that exogenous DLK1 administration reducing the expression of gluconeogenic genes, such as PEPCK ameliorated hepatic steatosis and alleviated hyperglycemia and and G6Pase via AMPK activation. glucose intolerance in the diabetic animal model. For the first

Figure 5. DLK1 inhibited lipid accumulation in hepatocytes by activation of AMPK and suppression of SREBP-1c. (a, b) Intracellular lipid − 1 accumulation was assessed in HepG2 cells treated with palmitate (0.3 mM) and DLK1 (150 μgml ) by visualization with Oil Red O staining (a), which was quantiﬁed by spectrophotometry (b). Data are presented as the mean ± s.d. (n = 6). (c) Nuclear expression of SREBP-1c in HepG2 cells treated with either vehicle or DLK1. The bottom graph shows densitometric analysis of the optical density-based data. *Po0.05 and **Po0.01.

time, we demonstrate that the metabolic effects of DLK1, namely structure used in the animal experiments because only increased fatty acid oxidation and suppressed gluconeogenesis in membrane-tethered DLK1 inhibited adipose tissue expansion.30 the liver, occurs through the activation of AMPK. By generating a Recent paper demonstrated that DLK1 overexpression amelio- fusion protein containing the extracellular domain of DLK1 and rated lipid accumulation in the liver with weight reduction and the human Fc region, which increases its stability, we show the improvement of insulin sensitivity in obese mice.31 However, our unexpected action of DLK1 on glucose and lipid metabolism in similar findings indicate that DLK1-mediated protection from the liver. hepatic steatosis in the context of excessive energy intake was not DLK1, also referred as preadipocyte factor 1, was first cloned secondary to a decrease in body weight or adipose tissue because from a 3T3-L1 preadipocyte complementary DNA library and has db/db mice in the treated and untreated groups exhibited been linked to the inhibition of adipogenic differentiation.22,23 comparable body composition. This interesting effect of DLK1 DLK1 expression disappears during adipogenesis.24 Thus, it is on hepatosteatosis was attained by activation of AMPK, which considered a preadipocyte marker as is highly and specifically leads to the modulation of genes related to fatty acid oxidation expressed in preadipocytes but not in mature adipocytes. Several and synthesis in the liver. Furthermore, DLK1-treated animals in vivo experiments using DLK1-null mice, as well as transgenic showed lower blood glucose levels via suppression of gluconeo- mice overexpressing DLK1 in adipose tissue, showed that DLK1 genic genes. Together, these DLK1-mediated changes in gene has an essential role as a negative regulator of adipogenesis.25 expression altered whole body and hepatic energy metabolism by DLK1-null mice exhibited accelerated body weight gain with activating the AMPK signaling pathway (Figure 6k). increased mass of adipose tissues.25 Moreover, those fed a high fat AMPK is a major cellular regulator of glucose and lipid diet showed impaired insulin resistance and glucose intolerance metabolism in hepatocytes.32 Its activation induces intracellular compared to wild-type mice.24 Contrary to our results, transgenic metabolic changes that favor the treatment of diabetes, such as DLK1-overexpressing mice under the control of the adipocyte- suppressing biosynthetic pathways (gluconeogenesis and fatty specific FABP4/aP2 promoter demonstrated a marked decrease in acid synthesis) and stimulating catabolic processes (fatty acid adipose tissue content but also a lipodystrophy-like feature with oxidation) in the liver.32 We also confirmed that AMPK activation insulin resistance and hypertriglyceridemia.26,27 Similarly, trans- by exogenous DLK1 further phosphorylated and inactivated ACC, genic mice with liver-specific overexpression of DLK1 also which modulates the proximal and rate-limiting step of lipogen- displayed less white adipose tissue.27 esis. In addition, nuclear SREBP-1c, a key transcription factor for These conflicting findings suggest that proper dosage and fatty acid synthesis, was significantly decreased in hepatocytes timing might be crucial for DLK1 to exert its metabolic effect in following DLK1 treatment as a result of AMPK activation. In terms adipose tissue. This concept is supported by a recent study of gluconeogenesis, we demonstrated that DLK1 effectively showing that tightly regulated dosage control of DLK1 is expelled FOXO1 from the nucleus, leading to the suppression of important for its regulatory function in neurogenesis.28 In PEPCK and G6Pase expression in the liver. Furthermore, DLK1 addition, DLK1 has been identified as a dosage-critical gene effectively reduced adipose tissue inflammation by lowering during development and growth.29 Therefore, it is plausible that macrophage infiltration in db/db mice. We cannot exclude the the effect of exogenous DLK1 administered in adult animal possibility that this effect may be a direct anti-inflammatory models may be different from that of previous genetic animal function of DLK1 or a secondary function that reduces hypergly- models in which DLK1 expression during early life has been cemia. Further study is required to resolve these questions. manipulated. In contrast to previous findings from genetically To date, there is insufficient evidence about the metabolic engineered mice, we observed that exogenous administration of function of DLK1 in the liver (except adipose tissue) because most soluble DLK1 did not affect the development of adipose tissue. research has focused on its role in liver fibrosis and tumor This may be due to differences in the concentration or DLK1 development.33 Huang, et al.34 clearly showed that DLK1 mRNA

Figure 6. DLK1 suppressed hepatic glucose production by inhibiting G6Pase and PEPCK expression via FOXO1 translocation. DLK1 (15 mg kg − 1) was injected intraperitoneally twice weekly into normal C57BL/6J mice for 6 weeks. (a, b) Fasting glucose (a) and random glucose (b) were evaluated during the treatment period. (c) Hepatic expression of G6Pase and PEPCK mRNA in the liver of normal C57BL/6J mice after DLK1 treatment. (d) Hepatic expression of G6Pase and PEPCK mRNA in the liver of db/db mice after DLK1 treatment. (e) Effect of DLK1 on glucose production in HepG2 cells. HepG2 cells were cultured in high-glucose DMEM alone or with cAMP plus dexamethasone (Dex, 1 μM). Cells were treated for 6 h with insulin (10 nM), DLK1 (150 μg/ml), DLK1 with compound C (10 μM) or compound C alone and then cultured in gluconeogenic media. After 3 h, the media was collected and glucose output was measured. *Po0.05 and ***Po0.001 compared to control group (blank box). (f) PEPCK mRNA expression in HepG2 cells was determined by real-time PCR. (g) Immunoblot analysis was performed to determine the level of phosphorylated and total Akt in HepG2 cells treated with DLK1 (150 μgml− 1) or WY14643 (as a positive control) for 6 h. WY, WY14643. (h) Change in FOXO1 nuclear and cytosolic localization by DLK1 in HepG2 cells pre-treated with cAMP plus Dex. (i) Immunofluorescent assay using anti-FOXO1 antibody (stained green) and propidium iodide (red nuclear stain) was performed on HepG2 cells cultured in high-glucose DMEM alone or with cAMP plus dexamethasone (Dex, 1 μM). Cells were treated with insulin (10 nM) or DLK1 (150 μgml− 1) for 6 h. Magnification, × 200. (j) Quantification of a representative experiment is shown (at least 200 cells were examined, right). All results are representative of three independent experiments. Data are presented as the mean ± s.d. (n = 6). *Po0.05 and ***Po0.001. (k) A schematic illustration showing the putative mechanism by which DLK1 improves hepatic steatosis and hyperglycemia.

© 2016 Macmillan Publishers Limited International Journal of Obesity (2016) 356 – 365 Metabolic effect of DLK1 Y Lee et al 364 was present only in hepatocytes from human liver. In the 3 Bolos V, Grego-Bessa J, de la Pompa JL. Notch signaling in development experimental model of partial hepatectomy, DLK1 was found to and cancer. Endocr Rev 2007; 28: 339–363. be highly upregulated in hepatocytes, leading to proliferation and 4 Ntziachristos P, Lim JS, Sage J, Aifantis I. From fly wings to targeted cancer regeneration of the liver.35 Although hepatic expression of DLK1 therapies: a centennial for notch signaling. Cancer Cell 2014; 25: 318–334. was not evaluated, RNA-mediated gene regulation at the Dlk1- 5 Pajvani UB, Shawber CJ, Samuel VT, Birkenfeld AL, Shulman GI, Kitajewski J et al. Dio3 domain perturbed hepatic glycogenolysis, gluconeogenesis, Inhibition of Notch signaling ameliorates insulin resistance in a FoxO1- dependent manner. 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