Delta-like Homolog Research Articles

8134 Hypothalamic Dlk1 Expression is Not Essential for Preventing Early Maturation of the Reproductive Axis in Female and Male Mice

Abstract Disclosure: D.B. Macedo: None. H. Kim Kyeol: None. A. Abreu: None. A. Latronico: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Inactivating mutations in Delta-like homolog 1 (DLK1), a maternally imprinted gene on chromosome 14, have been recognized as a genetic cause of central precocious puberty (CPP) in humans. It is well established that the timing of puberty, especially in females, is highly sensitive to energy stores and metabolic cues, with lower body fat negatively correlated with age at puberty and menarche. Mice lacking Dlk1 have pre- and postnatal growth retardation. Despite their considerably lower body weight (BW), we found that Dlk1 deficient females achieved puberty at the same age as controls (‘relative precocious puberty’). Moreover, Dlk1 deficiency led to activation of the reproductive axis despite lower levels of kisspeptin and leptin, an adipose tissue hormone with a permissive/stimulatory effect on metabolic control of reproduction. Dlk1 is a transmembrane protein that plays an essential role in inhibiting adipocyte differentiation and its biologically active form is soluble. Increased hypothalamic expression postnatally suggests a neuroendocrine function, but the precise mechanism by which DLK1 deficiency leads to CPP is yet to be deciphered. In this study, we aimed to investigate the role of hypothalamic Dlk1 expression on pubertal onset without the interference of diminished BW, using a conditional knockout (cKO) mouse model. Methods and Results: We generated a targeted Dlk1 knockout mouse model by crossing mice expressing Cre recombinase under the control of Nestin gene (Nes Cre), expressed primarily in neuro-epithelial cells, with mice harboring the Dlk1 gene flanked by loxP sites (Dlk1fl). Since Dlk1 is imprinted and expressed only by the paternal allele, we bred heterozygous Dlk1fl/+ male mice with Nes Cre females to obtain Dlk1fl/Cre (Dlk1 cKO), Dlk1+/Cre (Nes Cre) and Dlk1+/+ (wild type - WT). We confirmed by RT-qPCR that Dlk1 mRNA was undetectable in the mediobasal hypothalamus of Dlk1 cKO adult male mice, whereas it was present in Nes Cre and WT mice. We noted a lower BW in Nes Cre compared to WT mice, as previously reported. However, no difference in BW was observed between Dlk1 cKO and Nes Cre mice at the timing of puberty (females: Dlk1 cKO 12.6 ± 0.8 g vs. Nes Cre 13.8 ± 0.2 g, P = 0.18; males: Dlk1 cKO 12.4 ± 0.2 g vs. Nes Cre 13.1 ± 0.5 g, P = 0.26). Puberty onset, assessed by age at vaginal opening (females) and at preputial separation (males), was not affected by hypothalamic Dlk1 deletion (Dlk1 cKO: 35.6 ± 2.7 days vs. Nes Cre 34.6 ± 0.7 days, P = 0.73; Dlk1 cKO 29.0 ± 0.6 vs. Nes Cre: 30.3 ± 0.6 days, P = 0.19). Conclusion: In contrast to global Dlk1 deficiency, targeted deletion of Dlk1 from neuronal and glial cells did not affect body weight or timing of pubertal onset. These findings suggest that Dlk1 originates from peripheral tissues, such as adipocytes, to regulate reproduction and metabolism. Presentation: 6/1/2024

Evaluation of the Efficacy of Stem Cells Therapy in the Periodontal Regeneration: A Meta-Analysis and Mendelian Randomization Study.

Stem cell therapy for periodontal defects has shown good promise in preclinical studies. The purpose of this study was to evaluate the impact of stem cell support on the regeneration of both soft and hard tissues in periodontal treatment. PubMed, Cochrane Library, Embase, and Web of Science were searched and patients with periodontal defects who received stem cell therapy were included in this study. The quality of the included articles was assessed using Cochrane's tool for evaluating bias, and heterogeneity was analyzed using the I2 method. An Mendelian randomizationinvestigation was conducted using abstract data from the IEU public databases obtained through GWAS. Nine articles were included for the meta-analysis. Stem cell therapy effectively rebuilds periodontal tissues in patients with periodontal defects, as evidenced by a reduction in probing depth, clinical attachment level andbone defect depth . And delta-like homolog 1 is a protective factor against periodontal defects alternative indicator of tooth loosening. The findings of this research endorse the utilization of stem cell treatment for repairing periodontal defects in individuals suffering from periodontitis. It is recommended that additional extensive clinical investigations be carried out to validate the efficacy of stem cell therapy and encourage its widespread adoption.

A Male Japanese Patient with Temple Syndrome Complicated by Type 2 Diabetes Mellitus.

We herein present the case of a 21-year-old male Japanese diabetic patient with Temple syndrome, caused by maternal uniparental disomy of chromosome 14. The patient was overweight and had type 2 diabetes, dyslipidemia, metabolic dysfunction-associated steatotic liver disease, and microalbuminuria. He had an increased fat mass in the truncal region and a decreased lean mass throughout the body. This may lead to insulin resistance due to the absence of delta-like homolog 1 (DLK1) and retrotransposon gag-like 1 (RTL1). The patient had experienced social withdrawal at home (hikikomori in Japanese), had poorly controlled type 2 diabetes, and was overweight despite receiving diet therapy and oral hypoglycemic agents.

The negative adipogenesis regulator Dlk1 is transcriptionally regulated by Ifrd1 (TIS7) and translationally by its orthologue Ifrd2 (SKMc15).

Delta-like homolog 1 (Dlk1), an inhibitor of adipogenesis, controls the cell fate of adipocyte progenitors. Experimental data presented here identify two independent regulatory mechanisms, transcriptional and translational, by which Ifrd1 (TIS7) and its orthologue Ifrd2 (SKMc15) regulate Dlk1 levels. Mice deficient in both Ifrd1 and Ifrd2 (dKO) had severely reduced adipose tissue and were resistant to high fat diet-induced obesity. Wnt signaling, a negative regulator of adipocyte differentiation was significantly up regulated in dKO mice. Elevated levels of the Wnt/β-catenin target protein Dlk1 inhibited the expression of adipogenesis regulators Pparg and Cebpa, and fatty acid transporter Cd36. Although both, Ifrd1 and Ifrd2, contributed to this phenotype, they utilized two different mechanisms. Ifrd1 acted by controlling Wnt signaling and thereby transcriptional regulation of Dlk1. On the other hand, distinctive experimental evidence showed that Ifrd2 acts as a general translational inhibitor significantly affecting Dlk1 protein levels. Novel mechanisms of Dlk1 regulation in adipocyte differentiation involving Ifrd1 and Ifrd2 are based on experimental data presented here.

Long term in vivo Dlk1 gene transfer protects against myocardial fibrosis-induced cardiac dysfunction

Introduction: Cardiac fibrosis can independently contribute to the impairment of cardiac function and development of heart failure. Early activation of pro-fibrotic genetic pathways has been suggested to occur well before adverse ventricular remodeling occurs in fibrosis as it progresses to heart failure. Delta-like homolog 1 (Dlk1), a paternally imprinted gene that encodes a transmembrane protein that contains multiple epidermal growth factor repeats, has been shown to play a critical role as a regulator of cell differentiation, neuronal differentiation, bone differentiation, and hematopoiesis. We hypothesize that Dlk-1 could regulate myocardial fibrosis via inhibition of fibroblast-myofibroblast conversion and therefore restrain cardiac fibrosis progression. Methods: Wildtype (WT) and Dlk1 knockout (Dlk1-/-) mice were subjected to myocardial infarction (MI) by permanent ligation of the left anterior descending artery to induce heart failure. A recombinant adeno-associated virus 9 (AAV9) was used to deliver Dlk1 into the myocardium in vivo. Cardiac function and hemodynamic changes were measured by echocardiography and left ventricular catheterization. Immunohistochemistry and confocal microscopy were used to determine the expression of pro-fibrotic markers. Total RNA was prepared from left ventricular (LV) tissue or cardiomyocytes and fibroblasts from Dlk1-/- and WT hearts. Masson trichrome staining was used to determine abnormal collagen deposition in the heart. Result: Myocardial fibrosis was demonstrated by increased expression of alpha-smooth muscle actin (αSMA), collagen, fibronectin, connective tissue growth factor, along with a substantial increase in the deposition of collagen as evident from Masson's trichrome staining. We observed downregulated Dlk-1 mRNA and protein expression in ischemic tissues from human patients and infarcted pigs’ hearts. The reduced levels of Dlk1 were associated with increased mRNA expression of pro-fibrotic molecules, collagen1a, LOX and αSMA in the scar area. Echocardiographic analysis revealed substantial LV chamber dilatation and reduced fractional shortening (FS %) in Dlk1 null mice as compared to WT, whereas the mice treated with AAV9-Dlk1 for 8-10 weeks showed a significant improvement in cardiac function. Consistently, Dlk1-deficient mice exhibited impairment in heart function, cardiac fibrosis and myocyte hypertrophy and increased expression of atrial natriuretic peptide, and B-type natriuretic peptide, while AAV9-Dlk1-treated mice significantly reduced cardiac remodeling and myocardial fibrosis. Conclusion: Restoration of Dlk1 in the heart following MI-induced heart failure improved cardiac function and attenuated myocardial fibrosis. The findings from the current study suggest a critical role for Dlk1 in myocardial fibrosis-induced cardiac remodeling, indicating that Dlk1 gene therapy may be a viable therapeutic approach for heart failure. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression patterns and DNA methylation profile of GTL2 gene in goats

Gene trap locus 2 (GTL2), a long non-coding paternal imprinting gene, participates in various biological processes, including cell proliferation, differentiation, and apoptosis, by regulating the transcription of target mRNA, which is tightly related to the growth of the organic and maintenance of function. In this study, DNA methylation patterns of CpG islands (CGI) of GTL2 were explored, and its expression level was quantified in six tissues, rumen epithelium cells, and skeletal muscle cells in goats. GTL2 expression levels were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and the methylation model was confirmed by bisulfite-sequencing PCR (BSP). CGI methylation of GTL2 indicated a moderate methylation (ranging from 81.42 to 86.83%) in the brain, heart, liver, kidney, lung, and longissimus dorsi. GTL2 is most highly expressed in brain tissues, but there is no significant difference in the other five tissues. In addition, in the rumen epithelium cell proliferation, GTL2 expression was highest at 60 h, followed by 72 h, and almost unchanged at 12–48 h. In the skeletal muscle cell differentiation, GTL2 expression was highest at 0 and 24 h, significantly decreasing at 72 and 128 h. Pearson correlation analysis did not indicate a clear relationship between methylation and GTL2 expression levels, suggesting that other regulatory factors may modulate GTL2 expression. This study will provide a better understanding of the expression regulation mechanism of genes in the delta-like homolog 1 gene (DLK1)-GTL2 domain.

Robust protein-based engineering of hepatocyte-like cells from human mesenchymal stem cells.

Cells of interest can be prepared from somatic cells by forced expression of lineage-specific transcription factors, but it is required to establish a vector-free system for their clinical use. Here, we report a protein-based artificial transcription system for engineering hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (MSCs). MSCs were treated for 5 days with 4 artificial transcription factors (4F), which targeted hepatocyte nuclear factor (HNF)1α, HNF3γ, HNF4α, and GATA-binding protein 4 (GATA4). Then, engineered MSCs (4F-Heps) were subjected to epigenetic analysis, biochemical analysis and flow cytometry analysis with antibodies to marker proteins of mature hepatocytes and hepatic progenitors such as delta-like homolog 1 (DLK1) and trophoblast cell surface antigen 2 (TROP2). Functional properties of the cells were also examined by injecting them to mice with lethal hepatic failure. Epigenetic analysis revealed that a 5-day treatment of 4F upregulated the expression of genes involved in hepatic differentiation, and repressed genes related to pluripotency of MSCs. Flow cytometry analysis detected that 4F-Heps were composed of small numbers of mature hepatocytes (at most 1%), bile duct cells (~19%) and hepatic progenitors (~50%). Interestingly, ~20% of 4F-Heps were positive for cytochrome P450 3A4, 80% of which were DLK1-positive. Injection of 4F-Heps significantly increased survival of mice with lethal hepatic failure, and transplanted 4F-Heps expanded to more than 50-fold of human albumin-positive cells in the mouse livers, well consistent with the observation that 4F-Heps contained DLK1-positive and/or TROP2-positive cells. Taken together with observations that 4F-Heps were not tumorigenic in immunocompromised mice for at least 2 years, we propose that this artificial transcription system is a versatile tool for cell therapy for hepatic failures.

Effect of weaning strategy and backgrounding management on growth performance, carcass characteristics, and mRNA expression in the longissimus muscle of beef steers.

We evaluate the effect of the weaning strategy (WS; early, 130 ± 21 d vs. normal, 187 ± 20 d) and backgrounding management (BGM) on growth, carcass characteristics, and relative mRNA expression in the longissimus muscle (LM) of beef steers. One hundred and twenty Angus × SimAngus-crossbred steers (body weight (BW) = 130 ± 11.2 kg) were used in a randomized complete block design. Steers, blocked by age and BW, were randomly assigned to one treatment (2 × 2 factorial). Treatments consisted of early weaned (EW) or normal weaned (NW) steers subsequently backgrounded (BG) on either a forage-based (FB) or concentrates-based (CB) diet. The EW steers (d 0) were ad libitum-fed a grain-based diet for 49 d until nursing calves were NW. Steers were ad libitum-fed either a FB diet for 214 d or a CB diet for 95 d afterward. Steers were finished on a high-grain diet until harvested at an estimated constant 12th-rib fat thickness (1.5 cm). Expression of mRNA in the LM was measured over time. Data were analyzed using PROC MIXED in SAS. The EW steers were heavier (P ˂ 0.01) at the beginning of the backgrounding and finishing period. When the finishing phase began, steers FB were heavier (P ˂ 0.01) than CB steers. There was a tendency for a WS × BGM interaction (P = 0.08) for final BW, where NW-FB steers were heavier than the steers on the other three treatments, which do not differ between them. In the finishing phase, steers BG on a FB diet had greater dry matter intake and average daily gain, but lower gain-to-feed ratio (P ˂ 0.01). There was a WS × BGM interaction (P = 0.03) for days on feed (DOF) in the finishing diet, where backgrounding steers in a FB diet decreased DOF required to reach the harvesting target among EW steers, but not within NW steers. No interactions or treatment effects (P ≥ 0.17) were detected for marbling score (MS). For ZFP423, EW steers showed a greater mRNA expression on d 112 and a lower expression on d 255 than NW steers (P ˂ 0.01). In d 57, steers BG on a CB diet presented a greater delta-like homolog 1 mRNA expression than steers BG on a FB diet, whereas in d 255, this was inverted (P ˂ 0.01). For CCAAT/enhancer binding protein D (C/EBPD) mRNA expression, a tendency for a WS × BGM interaction was observed (P = 0.06), where a greater expression of C/EBPD was observed in steers BG on a FB diet among EW steers, but not within NW steers. In this study, early grain feeding followed by different BGM does not support MS improvements of beef carcasses.

DLK1: A Novel Biomarker of Placental Insufficiency in Stillbirth and Live Birth.

Delta-like homolog 1 (DLK1) is a growth factor that is reduced in maternal sera in pregnancies with small for gestational age neonates. We sought to determine if DLK1 is associated with stillbirth (SB), with and without placental insufficiency. A nested case-control study was performed using maternal sera from a multicenter case-control study of SB and live birth (LB). SB and LB were stratified as placental insufficiency cases (small for gestational age <5% or circulatory lesions on placental histopathology) or normal placenta controls (appropriate for gestational age and no circulatory lesions). Enzyme-linked immunosorbent assay (ELISA) was used to measure DLK1. The mean difference in DLK1 was compared on the log scale in an adjusted linear regression model with pairwise differences, stratified by term/preterm deliveries among DLK1 results in the quantifiable range. In exploratory analysis, geometric means were compared among all data and the proportion of "low DLK1" (less than the median value for gestational age) was compared between groups and modeled using linear and logistic regression, respectively. Overall, 234 SB and 234 LB were analyzed; 246 DLK1 values were quantifiable within the standard curve. Pairwise comparisons of case and control DLK1 geometric means showed no significant differences between groups. In exploratory analysis of all data, adjusted analysis revealed a significant difference for the LB comparison only (SB: 71.9 vs. 99.1 pg/mL, p = 0.097; LB: 37.6 vs. 98.1 pg/mL, p = 0.005). In exploratory analysis of "low DLK1," there was a significant difference between the odds ratio of having "low DLK1" between preterm cases and controls for both SB and LB. There were no significant differences in geometric means nor "low DLK1" between SB and LB. In exploratory analysis, more placental insufficiency cases in preterm SB and LB had "low DLK1." However, low DLK1 levels were not associated with SB. · Maternally circulating DLK1 is correlated with placental insufficiency.. · Maternally circulating DLK1 is not correlated with SB.. · DLK1 is a promising marker for placental insufficiency..

Genetic factors in precocious puberty

Pubertal onset is known to result from reactivation of the hypothalamic-pituitary-gonadal (HPG) axis, which is controlled by complex interactions of genetic and nongenetic factors. Most cases of precocious puberty (PP) are diagnosed as central PP (CPP), defined as premature activation of the HPG axis. The cause of CPP in most girls is not identifiable and, thus, referred to as idiopathic CPP (ICPP), whereas boys are more likely to have an organic lesion in the brain. ICPP has a genetic background, as supported by studies showing that maternal age at menarche is associated with pubertal timing in their offspring. A gain of expression in the kisspeptin gene (KISS1), gain-of-function mutation in the kisspeptin receptor gene (KISS1R), loss-of-function mutation in makorin ring finger protein 3 (MKRN3), and loss-of-function mutations in the delta-like homolog 1 gene (DLK1) have been associated with ICPP. Other genes, such as gamma-aminobutyric acid receptor subunit alpha-1 (GABRA1), lin-28 homolog B (LIN28B), neuropeptide Y (NPYR), tachykinin 3 (TAC3), and tachykinin receptor 3 (TACR3), have been implicated in the progression of ICPP, although their relationships require elucidation. Environmental and socioeconomic factors may also be correlated with ICPP. In the progression of CPP, epigenetic factors such as DNA methylation, histone posttranslational modifications, and noncoding ribonucleic acids may mediate the relationship between genetic and environmental factors. CPP is correlated with short- and long-term adverse health outcomes, which forms the rationale for research focusing on understanding its genetic and nongenetic factors.

Lack of miR-379/miR-544 Cluster Resists High-Fat Diet-Induced Obesity and Prevents Hepatic Triglyceride Accumulation in Mice.

Non-alcoholic fatty liver disease (NAFLD) affects obesity-associated metabolic syndrome, which exhibits hepatic steatosis, insulin insensitivity and glucose intolerance. Emerging evidence suggests that microRNAs (miRNAs) are essential for the metabolic homeostasis of liver tissues. Many hepatic miRNAs located in the miR-379/miR-544 cluster were significantly increased in leptin-receptor-deficient type 2 mice (db/db), a mouse model of diabetes. However, the function of the miR-379/miR-544 cluster in the process of hepatic steatosis remains unclear. Here, we report that the novel function of miR-379/miR-544 cluster in regulating obesity-mediated metabolic dysfunction. Genetical mutation of miR-379/miR-544 cluster in mice displayed resistance to high-fat diet (HFD)-induced obesity with moderate hepatic steatosis and hypertriglyceridemia. In vitro studies revealed that silencing of miR-379 in human hepatocellular carcinoma (HepG2) cells ameliorated palmitic acid-induced elevation of cellular triglycerides, and overexpression of miR-379 had the opposite effect. Moreover, Igf1r (Insulin-like growth factor 1 receptor) and Dlk1 (Delta-like homolog 1) were directly targeted by miR-379 and miR-329, respectively, and elevated in the livers of the miR-379/miR-544 cluster knockout mice fed on HFD. Further transcriptome analyses revealed that the hepatic gene expressions are dysregulated in miR-379/miR-544 knockout mice fed with HFD. Collectively, our findings identify the miR-379/miR-544 cluster as integral components of a regulatory circuit that functions under conditions of metabolic stress to control hepatic steatosis. Thus, this miRNA cluster provides potential targets for pharmacologic intervention in obesity and NAFLD.

Disturbance of the Dlk1-Dio3 imprinted domain may underlie placental Dio3 suppression and extracellular thyroid hormone disturbance in placenta-derived JEG-3 cells following decabromodiphenyl ether (BDE209) exposure

Decabromodiphenyl ether (BDE209) has been widely used as a flame retardant in the past four decades, leading to human health consequences, especially neurological impairments. Our previous in vivo studies have suggested that developmental neurotoxicity in offspring may be the result of BDE209-induced placental type III iodothyronine deiodinase (Dio3) disturbance and consequent thyroid hormone (TH) instability. Dio3 is paternally imprinted gene, and its balanced expression is crucial in directing normal development and growth. In this study, we used placenta-derived cells to investigate how BDE209 affected Dio3 expression through interfering imprinting mechanisms in the delta-like homolog 1 (Dlk1)-Dio3 imprinted region. Gene chip analysis and RT-qPCR identified miR409-3p, miR410-5p, miR494-3p, miR668-3p and miR889-5p as potential candidates involved in Dio3 deregulation. The sodium bisulfite-clonal sequencing revealed the BDE209 affect methylation status of two differentially methylated regions (DMRs), intergenic-DMR (IG-DMR) and maternally expressed gene 3-DMR (MEG3-DMR). Our data indicate that placental Dio3 may be a potential molecular target for future study of BDE209 developmental toxicity. In particular, miRNAs, IG-DMR and MEG3-DMR in the Dlk1-Dio3 imprinted locus may be informative in directing studies in TH disturbance and developmental toxicity induced by in utero exposure to environmental persistent organic pollutants (POPs), and those candidate miRNAs may prove to be convenient and noninvasive biomarkers for future large-scale population studies.

Pubertal Onset Occurs in Female Mice Lacking Paternally Expressed Dlk1 Despite Lower Leptin and Kisspeptin Levels

Abstract The timing of puberty in females is highly sensitive to metabolic cues and energy reserves. Epidemiologic studies indicate a relationship between increased body mass index and earlier puberty in girls. In contrast, a significant delay in puberty and menarche is seen in girls who have diminished body fat. Multiple peripheral hormones are responsible for transmitting metabolic information to hypothalamic kisspeptin and GnRH neurons. Sufficient levels of leptin, an adipose tissue hormone with a permissive/stimulatory effect on the metabolic control of reproduction, are required for puberty onset, reproductive function and fertility. Loss-of-function mutations in the Delta-like homolog 1 (DLK1) gene have been described in girls with central precocious puberty (CPP) and increased body fat, suggesting a link between metabolism and reproduction. DLK1 is a paternally expressed gene located on human chromosome 14q32.2 in a locus associated with Temple syndrome (TS). Dlk1 knockout mice display pre- and postnatal growth retardation, a phenotype that overlaps with TS. We have shown that Dlk1 deficient female mice achieved puberty at the same age as wild type mice, despite a considerably lower body weight (BW) (“relative precocious puberty”). To date, the mechanisms of action of Dlk1 in determining pubertal onset remain unknown. In this study, we used a Dlk1 deficient mouse model to explore the influence of Dlk1 in the regulation of reproductive axis, particularly its effects on leptin and/or kisspeptin, a major excitatory factor of the reproductive axis. By RT-qPCR and Western blot, we confirmed that both Dlk1 mRNA and protein were undetectable in the mediobasal hypothalamus (MBH) of Dlk+/p- (which inherited the mutant allele from their father), but it was present in Dlk+/+ mice. White adipose tissue (WAT) and blood were collected from Dlk+/p- and Dlk+/+ female mice at postnatal day (PND) 26, and MBH tissue was obtained from both groups at PND 15, 26 and 60. Quantification of total WAT showed no significant difference between Dlk1+/p-and Dlk1+/+ mice (p=0.8) at PND26, even after correction for total BW (p=0.29). Hypothalamic mRNA levels of Kiss1 and Socs3, a downstream mediator of leptin signaling, were measured by RT-qPCR. Kiss1 mRNA levels were significantly reduced in the MBH of Dlk1+/p- mice at PND15 and PND60, but no significant difference was found at PND 26. Socs3 expression was significantly lower in Dlk1+/p- mice (p=0.04) as a result of the reduced circulating levels of leptin (ELISA) observed in these mice at PDN26 (p=0.01). Our findings suggest that the absence of Dlk1 may attenuate the metabolic effects of low body weight and low leptin levels on puberty onset and that, as seen in humans, DLK1 is an important link between body weight and pubertal development. Finally, Dlk1 deficiency leads to activation of the reproductive axis despite lower levels of kisspeptin.

DLK1 Expressed in Mouse Orexin Neurons Modulates Anxio-Depressive Behavior but Not Energy Balance.

Lateral hypothalamic area (LHA) neurons expressing the neuropeptide orexin (OX) are implicated in obesity and anxio-depression. However, these neurons release OX as well as a host of other proteins that might contribute to normal physiology and disease states. We hypothesized that delta-like homolog 1 (DLK1), a protein reported to be co-expressed by all OX neurons, contributes to the regulation of energy balance and/or anxio-depression. Consistent with previous reports, we found that all rat OX neurons co-express DLK1. Yet, in mice and humans only a subset of OX neurons co-expressed DLK1. Since human OX-DLK1 distribution is more similar to mice than rats, mice are a comparable model to assess the human physiologic role of DLK1. We therefore used a viral lesion strategy to selectively delete DLK1 within the LHA of adult mice (DLK1Null) to reveal its role in body weight and behavior. Adult-onset DLK1 deletion had no impact on body weight or ingestive behavior. However, DLK1Null mice engaged in more locomotor activity than control mice and had decreased anxiety and depression measured via the elevated plus maze and forced swim tests. These data suggest that DLK1 expression via DLK1-expressing OX neurons primarily contributes to anxio-depression behaviors without impacting body weight.

TRIM28 Regulates Dlk1 Expression in Adipogenesis.

The tripartite motif-containing protein 28 (TRIM28) is a transcription corepressor, interacting with histone deacetylase and methyltransferase complexes. TRIM28 is a crucial regulator in development and differentiation. We would like to investigate its function and regulation in adipogenesis. Knockdown of Trim28 by transducing lentivirus-carrying shRNAs impairs the differentiation of 3T3-L1 preadipocytes, demonstrated by morphological observation and gene expression analysis. To understand the molecular mechanism of Trim28-mediated adipogenesis, the RNA-seq was performed to find out the possible Trim28-regulated genes. Dlk1 (delta-like homolog 1) was increased in Trim28 knockdown 3T3-L1 cells both untreated and induced to differentiation. Dlk1 is an imprinted gene and known as an inhibitor of adipogenesis. Further knockdown of Dlk1 in Trim28 knockdown 3T3-L1 would rescue cell differentiation. The epigenetic analysis showed that DNA methylation of Dlk1 promoter and differentially methylated regions (DMRs) was not altered significantly in Trim28 knockdown cells. However, compared to control cells, the histone methylation on the Dlk1 promoter was increased at H3K4 and decreased at H3K27 in Trim28 knockdown cells. Finally, we found Trim28 might be recruited by transcription factor E2f1 to regulate Dlk1 expression. The results imply Trim28-Dlk1 axis is critical for adipogenesis.

NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

The NOTCH family of receptors and ligands is involved in numerous cell differentiation processes, including adipogenesis. We recently showed that overexpression of each of the four NOTCH receptors in 3T3-L1 preadipocytes enhances adipogenesis and modulates the acquisition of the mature adipocyte phenotype. We also revealed that DLK proteins modulate the adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells in an opposite way, despite their function as non-canonical inhibitory ligands of NOTCH receptors. In this work, we used multipotent C3H10T1/2 cells as an adipogenic model. We used standard adipogenic procedures and analyzed different parameters by using quantitative-polymerase chain reaction (qPCR), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), luciferase, Western blot, and metabolic assays. We revealed that C3H10T1/2 multipotent cells show higher levels of NOTCH receptors expression and activity and lower Dlk gene expression levels than 3T3-L1 preadipocytes. We found that the overexpression of NOTCH receptors enhanced C3H10T1/2 adipogenesis levels, and the overexpression of NOTCH receptors and DLK (DELTA-like homolog) proteins modulated the conversion of cells towards a brown-like adipocyte phenotype. These and our prior results with 3T3-L1 preadipocytes strengthen the idea that, depending on the cellular context, a precise and highly regulated level of global NOTCH signaling is necessary to allow adipogenesis and determine the mature adipocyte phenotype.

Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: tumor suppressor role of lncRNA MEG3.

BackgroundGlioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis.MethodsReal-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3).ResultsLoss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan–Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT).ConclusionIn GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.

SUN-100 Mice Lacking Paternally Expressed DLK1 Reach Puberty at a Lower Body Weight Than Littermate Controls

Body fat content along with a variety of genetic, environmental and psychosocial factors are responsible for the development and maintenance of reproductive function, especially in females. Epidemiologic studies indicate a relationship between increased body mass index and earlier puberty in girls. In contrast, a significant delay in puberty and menarche is seen in girls who are very physically active and have markedly diminished body fat. This link between reproduction and metabolism was reinforced with the recent report of loss-of-function mutations in the Delta-like homolog 1 (DLK1) gene in girls with central precocious puberty (CPP) and increased body fat. DLK1 is a paternally expressed gene located on chromosome 14q32.2 in a locus associated with Temple syndrome (TS), an imprinting disorder caused mainly by maternal parental disomy (mUPD). Dlk1 knockout mice display pre- and postnatal growth retardation, a phenotype that overlaps with human mUPD14. However, precocious puberty, a common finding associated with TS, was not carefully characterized in these mice. We used a Dlk1 deficient mouse model to determine the effects of Dlk1 on pubertal maturation. We confirmed by RT-qPCR that Dlk1 mRNA was undetectable in the mediobasal hypothalamus, where kisspeptin and other regulators of puberty are expressed, of Dlk+/p- mice (which inherited the mutant allele from their father) whereas it was present in Dlk+/+ mice. As reported previously, body weight was significantly lower in juvenile male and female Dlk+/p- mice, compared to wild-type littermate controls. Interestingly, mutant and control female mice achieved vaginal opening, a marker of puberty onset, at a similar age (Dlk+/p-: 29.8 ± 1.5 days, n=11 vs. Dlk+/+: 29.1 ± 0.7 days, n=15, p=0.6) despite a considerably lower body weight in the Dlk1 deficient mice at the time of vaginal opening (Dlk+/p-: 10.1 ± 0.8 g vs. Dlk+/+: 14.3 ± 0.3 g, p<0.0001). Similarly, in the Dlk+/p- males, preputial separation occurred at a lower body weight than in controls (Dlk+/p-: 12.4 ± 0.3 g, n=9 vs. Dlk+/+: 14.1 ± 0.2 g, n=19, p<0.0001). We hypothesize that the lack of Dlk1 at the hypothalamic level may be attenuating the effect of the low body weight on determining pubertal onset. These findings suggest that DLK1 is an important link between body weight and pubertal development in mice, as has been shown in humans.

MON-171 Characterization of the Adrenal Gland and Adrenal Rest Tissues in Congenital Adrenal Hyperplasia

Background: Adrenonodular hyperplasia and tumor formation are common long-term complications of congenital adrenal hyperplasia (CAH) driven by chronic ACTH elevation. Clinical studies indicate that the majority of males with classic CAH have testicular adrenal rest tumors (TART). Ovarian adrenal rest tumors (OART) are less commonly observed. Little is known about the pathophysiology of adrenal rest, however both adrenal cortex and Leydig cell markers have been described in TART, suggesting a pluripotent embryological cell origin. Objective: To characterize adrenals and adrenal rest tissues of patients with CAH in comparison with normal tissues. Materials and Methods: Using immunohistochemistry (IHC) and, real-time qRT-PCR we investigated CAH-affected adrenals (n=5), adrenal rest tissues (n=2; 1 testicular, 1 ovarian), controls [normal adrenal (n=2), testis (n=1), and ovary (n=1)]. Tissue sections prepared from paraffin embedded tissue blocks were immunostained with adrenal [melanocortin 2 receptor (MC2R), delta-like homolog 1 (DLK-1), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR) and other cytochrome P450 genes], inflammatory [interleukin 2 receptor (IL-2R), B-lymphocyte antigen (CD20), cluster of differentiation 3 (CD3), tumor necrosis factor alpha (TNFα), interleukin 6 (IL6)], and gonadal markers [progesterone receptor (PR), androgen receptor (AR), insulin-like 3 (INSL3)]. RNA was isolated and gene expression studies were performed. High-throughput RNA sequencing technology was used to analyze the differential transcriptome profiles between the CAH adrenals, adrenal rest and normal tissues.Results:In contrast to the controls, CAH adrenals and adrenal rest tissues showed the following:(i) IHC studies revealed 95% of tissue positive for adrenal zona-reticularis; (ii) significant nodular lymphocytic infiltration with a predominance of B and T lymphocytes and overexpression of lymphocyte markers IL-2R, CD20, CD3, and inflammatory cytokines TNFα and IL6; (iii) increased expression of adrenocortical specific genes MC2R, DLK1. Conclusion: CAH-affected adrenals and adrenal rest tissue have similar predominance of zona reticularis and demonstrate lymphocytic infiltration. Active inflammation may play a role in the abnormal development of adrenal and adrenal rest tissue in CAH patients.

Loss of TSC complex enhances gluconeogenesis via upregulation of Dlk1-Dio3 locus miRNAs

Loss of the tumor suppressor tuberous sclerosis complex 1 (Tsc1) in the liver promotes gluconeogenesis and glucose intolerance. We asked whether this could be attributed to aberrant expression of small RNAs. We performed small-RNA sequencing on liver of Tsc1-knockout mice, and found that miRNAs of the delta-like homolog 1 (Dlk1)-deiodinase iodothyronine type III (Dio3) locus are up-regulated in an mTORC1-dependent manner. Sustained mTORC1 signaling during development prevented CpG methylation and silencing of the Dlk1-Dio3 locus, thereby increasing miRNA transcription. Deletion of miRNAs encoded by the Dlk1-Dio3 locus reduced gluconeogenesis, glucose intolerance, and fasting blood glucose levels. Thus, miRNAs contribute to the metabolic effects observed upon loss of TSC1 and hyperactivation of mTORC1 in the liver. Furthermore, we show that miRNA is a downstream effector of hyperactive mTORC1 signaling.