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Obesity is a critical global health issue and a major risk factor for type 2 diabetes mellitus (T2DM). However, molecular mechanism by which obesity leads to T2DM has not been fully elucidated. Here, we investigated the role of adipocyte nardilysin (NRDC) in insulin sensitivity using adipocyte-specific NRDC-knockout mice (Adipo-KO). Despite no difference in body weight, Adipo-KO demonstrated enhanced glucose tolerance and insulin sensitivity exclusively under high fat diet (HFD) conditions. Epididymal white adipose tissue (eWAT) in Adipo-KO showed the decreased crown-like structures and F4/80-positive cells. Consistently, insulin-stimulated Akt phosphorylation was elevated in the eWAT of HFD-fed Adipo-KO and 3T3-L1 adipocytes with NRDC knockdown. In addition, NRDC knockdown led to a decrease in cellular oxygen consumption rate and hypoxia-induced HIF1α expression in 3T3-L1 adipocytes. The augmented Akt phosphorylation by NRDC knockdown was reversed by daprodustat, which stabilizes HIF1α, suggesting that the effect of NRDC on insulin sensitivity is mediated by HIF1α. Upstream analysis of the differentially expressed genes (DEGs) in NRDC knockdown 3T3-L1 adipocytes revealed that PPARγ is enriched in the promoters of the DEGs. We also confirmed the complex formation of NRDC and PPARγ. In conclusion, NRDC in adipocyte regulates insulin sensitivity through HIF1α and PPARγ.

Yoshiya Kawaguchi and colleagues show that a Sox9-positive progenitor pool located in the epithelia of the biliary and pancreatic ducts of adult mice serves as a continuous source of intestinal cells, hepatocytes and pancreatic acinar cells, identifying a shared pathway underlying the cellular renewal of these adult endodermal organs. The liver and exocrine pancreas share a common structure, with functioning units (hepatic plates and pancreatic acini) connected to the ductal tree. Here we show that Sox9 is expressed throughout the biliary and pancreatic ductal epithelia, which are connected to the intestinal stem-cell zone. Cre-based lineage tracing showed that adult intestinal cells, hepatocytes and pancreatic acinar cells are supplied physiologically from Sox9-expressing progenitors. Combination of lineage analysis and hepatic injury experiments showed involvement of Sox9-positive precursors in liver regeneration. Embryonic pancreatic Sox9-expressing cells differentiate into all types of mature cells, but their capacity for endocrine differentiation diminishes shortly after birth, when endocrine cells detach from the epithelial lining of the ducts and form the islets of Langerhans. We observed a developmental switch in the hepatic progenitor cell type from Sox9-negative to Sox9-positive progenitors as the biliary tree develops. These results suggest interdependence between the structure and homeostasis of endodermal organs, with Sox9 expression being linked to progenitor status.

Brown adipose tissue (BAT) dissipates chemical energy as heat through uncoupling protein 1 (UCP1). The induction of mitochondrial reactive oxygen species (ROS) in BAT was recently identified as a mechanism that supports UCP1-dependent thermogenesis. We previously demonstrated that nardilysin (NRDC) plays critical roles in body temperature homeostasis. Global NRDC-deficient ( Nrdc –/– ) mice show hypothermia due to a lower set point for body temperature, whereas BAT thermogenesis at room temperature (RT) is enhanced mainly to compensate for poor thermal insulation. To examine the primary role of NRDC in BAT thermogenesis, we generated adipocyte-specific NRDC-deficient (Adipo-KO) mice by mating Nrdc floxed ( Nrdc flox/flox ) mice with adiponectin-Cre mice. Adipo-KO mice showed hyperthermia at both RT and thermoneutrality. They were also more cold-tolerant than Nrdc flox/flox mice. However, UCP1 mRNA levels were significantly lower in Adipo-KO BAT at RT, thermoneutrality, and 4 °C, whereas no significant differences were observed in UCP1 protein levels at RT and 4 °C. We examined the protein stability of UCP1 using the cycloheximide chase assay and found that NRDC negatively regulated its stability via the ubiquitin–proteasome pathway. NRDC may be also involved in ROS-mediated in vivo thermogenesis because the inhibitory effects of N-acetyl cysteine, an ROS scavenger, on β3 agonist-induced thermogenesis were stronger in Adipo-KO mice. Collectively, the present results demonstrate that NRDC in BAT controls adaptive thermogenesis and body temperature homeostasis possibly via the regulation of UCP1 protein stability and ROS levels.

MYOD-induced microRNA-494-3p expression inhibits fast oxidative myotube formation by downregulating myosin heavy chain 2 (MYH2) in human induced pluripotent stem cells (hiPSCs) during skeletal myogenesis. However, the molecular mechanisms regulating MYH2 expression via miR-494-3p remain unknown. Here, using bioinformatic analyses, we show that miR-494-3p potentially targets the transcript of the E1A-binding protein p300 at its 3′-untranslated region (UTR). Myogenesis in hiPSCs with the Tet/ON-myogenic differentiation 1 ( MYOD1 ) gene (MyoD-hiPSCs) was induced by culturing them in doxycycline-supplemented differentiation medium for 7 days. p300 protein expression decreased after transient induction of miR-494-3p during myogenesis. miR-494-3p mimics decreased the levels of p300 and its downstream targets MYOD and MYH2 and myotube formation efficiency. p300 knockdown decreased myotube formation efficiency, MYH2 expression, and basal oxygen consumption rate. The binding of miR-494-3p to the wild type p300 3′-UTR, but not the mutated site, was confirmed using luciferase assay. Overexpression of p300 rescued the miR-494-3p mimic-induced phenotype in MyoD-hiPSCs. Moreover, miR-494-3p mimic reduced the levels of p300, MYOD, and MYH2 in skeletal muscles in mice. Thus, miR-494-3p might modulate MYH2 expression and fast oxidative myotube formation by directly regulating p300 levels during skeletal myogenesis in MyoD-hiPSCs and murine skeletal muscle tissues.

Inflammation is closely associated with cerebrovascular diseases, cardiovascular diseases, diabetes, and cancers, and it is accompanied by the development of autoantibodies in the early stage of inflammation-related diseases. Hence, it is meaningful to discover novel antibody biomarkers targeting inflammation-related diseases. In this study, Jumonji C-domain-containing 6 (JMJD6) was identified by the serological identification of antigens through recombinant cDNA expression cloning. In particular, JMJD6 is an antigen recognized in serum IgG from patients with unstable angina pectoris (a cardiovascular disease). Then, the serum antibody levels were examined using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay and a purified recombinant JMJD6 protein as an antigen. We observed elevated levels of serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with inflammation-related diseases such as ischemic stroke, acute myocardial infarction (AMI), diabetes mellitus (DM), and cancers (including esophageal cancer, EC; gastric cancer; lung cancer; and mammary cancer), compared with the levels in healthy donors. The s-JMJD6-Ab levels were closely associated with some inflammation indicators, such as C-reactive protein and intima–media thickness (an atherosclerosis index). A better postoperative survival status of patients with EC was observed in the JMJD6-Ab-positive group than in the negative group. An immunohistochemical analysis showed that JMJD6 was highly expressed in the inflamed mucosa of esophageal tissues, esophageal carcinoma tissues, and atherosclerotic plaques. Hence, JMJD6 autoantibodies may reflect inflammation, thereby serving as a potential biomarker for diagnosing specific inflammation-related diseases, including stroke, AMI, DM, and cancers, and for prediction of the prognosis in patients with EC.

Some cancers are related to atherosclerotic diseases; therefore, these two types of disease may share some antibody biomarkers in common. To investigate this, a first screening of sera was performed from patients with esophageal squamous cell carcinoma (ESCC) or acute ischemic stroke (AIS) for serological identification of antigens using recombinant cDNA expression cloning (SEREX). The amplified luminescent proximity homogeneous assay‐linked immunosorbent assay (AlphaLISA) method, which incorporates glutathione donor beads and anti‐human IgG acceptor beads, was used to evaluate serum antibody levels. SEREX screening identified low‐density lipoprotein receptor–related protein–associated protein 1 (LRPAP1) as a target antigen of serum IgG antibodies in the sera of patients with ESCC or AIS. Antigens, including recombinant glutathione S‐transferase–fused LRPAP1 protein, were prepared to examine serum antibody levels. AlphaLISA revealed significantly higher antibody levels against the LRPAP1 protein in patients with solid cancers such as ESCC and colorectal carcinoma and some atherosclerosis‐related diseases such as AIS and diabetes mellitus compared with healthy donors. Correlation analysis revealed that the elevated serum antibody levels against LRPAP1 were associated with smoking, a well‐known risk factor for both cancer and atherosclerosis. Serum LRPAP1 antibody is therefore a common marker for the early diagnosis of some cancers and atherosclerotic diseases and may reflect diseases caused by habitual smoking. Some cancers and arteriosclerosis may share some antibody biomarkers in common. To investigate this, a first screening of sera was performed from patients with esophageal squamous cell carcinoma for SEREX. Serum LRPAP1 antibody is a useful marker for the early diagnosis of some cancers and atherosclerotic diseases.

It has been reported that nardilysin (NRDC), a metalloendopeptidase which regulates various growth factors and cytokines, is associated with malignancies in a conflicting manner, in which it promoted gastric, hepatocellular, and colorectal cancers and suppressed pancreatic ductal adenocarcinoma. However, it has not been investigated how NRDC is associated with cutaneous malignancies for now. Immunohistochemical staining has revealed that NRDC expression is observed in all extramammary Paget's disease (EMPD) cases. Notably, other cutaneous malignancies including basal cell carcinoma, squamous cell carcinoma, and eccrine porocarcinoma, did not show increased NRDC expression in immunohistochemistry. EMPD typically presents several types of lesions including nodules, and positive staining of NRDC on EMPD was observed regardless of the type of lesions. Examination using samples taken from nodular lesions showed that some cases showed heterogenous NRDC expression within each lesion. We also found that NRDC staining was weaker in the marginal parts of EMPD lesion than in the central parts in several cases, and tumor cells tend to be distributed beyond the macroscopic skin lesions in these cases. It was speculated that decreased NRDC expression in the marginal zones of the skin lesions may be associated with the ability of tumor cells to produce the cutaneous manifestation of EMPD. This study suggests that NRDC may be associated with EMPD like other malignancies reported previously.

ObjectiveNardilysin (NRDC), a zinc peptidase, exhibits multiple localisation-dependent functions including as an enhancer of ectodomain shedding in the extracellular space and a transcriptional coregulator in the nucleus. In this study, we investigated its functional role in exocrine pancreatic development, homeostasis and the formation of pancreatic ductal adenocarcinoma (PDA).DesignWe analysed Ptf1a-Cre; Nrdcflox/flox mice to investigate the impact of Nrdc deletion. Pancreatic acinar cells were isolated from Nrdcflox/flox mice and infected with adenovirus expressing Cre recombinase to examine the impact of Nrdc inactivation. Global gene expression in Nrdc-cKO pancreas was analysed compared with wild-type pancreas by microarray analysis. We also analysed Ptf1a-Cre; KrasG12D; Nrdcflox/flox mice to investigate the impact of Nrdc deletion in the context of oncogenic Kras. A total of 51 human samples of pancreatic intraepithelial lesions (PanIN) and PDA were examined by immunohistochemistry for NRDC.ResultsWe found that pancreatic deletion of Nrdc leads to spontaneous chronic pancreatitis concomitant with acinar-to-ductal conversion, increased apoptosis and atrophic pancreas in mice. Acinar-to-ductal conversion was observed mainly through a non-cell autonomous mechanism, and the expression of several chemokines was significantly increased in Nrdc-null pancreatic acinar cells. Furthermore, pancreatic deletion of Nrdc dramatically accelerated KrasG12D -driven PanIN and subsequent PDA formation in mice. These data demonstrate a previously unappreciated anti-inflammatory and tumour suppressive functions of Nrdc in the pancreas in mice. Finally, absence of NRDC expression was observed in a subset of human PanIN and PDA.ConclusionNrdc inhibits pancreatitis and suppresses PDA initiation in mice.

Post-translational histone modifications, such as acetylation and methylation, are prerequisites for transcriptional regulation. The metalloendopeptidase nardilysin (Nrdc) is a H3K4me2-binding protein that controls thermoregulation and β-cell functions through its transcriptional coregulator function. We herein combined high-throughput ChIP-seq and RNA-seq to achieve the first genome-wide identification of Nrdc target genes. A ChIP-seq analysis of immortalized mouse embryo fibroblasts (iMEF) identified 4053 Nrdc-binding sites, most of which were located in proximal promoter sites (2587 Nrdc-binding genes). Global H3K4me2 levels at Nrdc-binding promoters slightly increased, while H3K9ac levels decreased in the absence of Nrdc. Among Nrdc-binding genes, a comparative RNA-seq analysis identified 448 candidates for Nrdc target genes, among which cell cycle-related genes were significantly enriched. We confirmed decreased mRNA and H3K9ac levels at the promoters of individual genes in Nrdc-deficient iMEF, which were restored by the ectopic introduction of Nrdc. Reduced mRNA levels, but not H3K9ac levels were fully restored by the reintroduction of the peptidase-dead mutant of Nrdc. Furthermore, Nrdc promoted cell cycle progression at multiple stages, which enhanced cell proliferation in vivo . Collectively, our integrative studies emphasize the importance of Nrdc for maintaining a proper epigenetic status and cell growth.

Nardilysin (NRDC) is a metalloendopeptidase of the M16 family. We previously showed that NRDC activates inflammatory cytokine signaling, including interleukin‐6‐signal transducer and activator of transcription 3 (STAT3) signaling. NRDC has been implicated in the promotion of breast, gastric and esophageal cancer, as well as the development of liver fibrosis. In this study, we investigated the role of NRDC in the promotion of hepatocellular carcinoma (HCC), both clinically and experimentally. We found that NRDC expression was upregulated threefold in HCC tissue compared to the adjacent non‐tumor liver tissue, which was confirmed by immunohistochemistry and western blotting. We also found that high serum NRDC was associated with large tumor size (>3 cm, P = 0.016) and poor prognosis after hepatectomy (median survival time 32.0 vs 73.9 months, P = 0.003) in patients with hepatitis C (n = 120). Diethylnitrosamine‐induced hepatocarcinogenesis was suppressed in heterozygous NRDC‐deficient mice compared to their wild‐type littermates. Gene silencing of NRDC with miRNA diminished the growth of Huh‐7 and Hep3B spheroids in vitro. Notably, phosphorylation of STAT3 was decreased in NRDC‐depleted Huh‐7 spheroids compared to control spheroids. The effect of a STAT3 inhibitor (S3I‐201) on the growth of Huh‐7 spheroids was reduced in NRDC‐depleted cells relative to controls. Our results show that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC promotes tumor growth through activation of STAT3. We investigated the role of nardilysin (NRDC) in the promotion of HCC, and showed that (i) NRDC is up‐regulated in HCC tissue; (ii) high serum NRDC is associated with increased tumor size and poor prognosis among patients with hepatitis C; (iii) diethylnitrosamine‐induced hepatocarcinogenesis is suppressed in NRDC‐deficient mice; and (iv) NRDC promotes the growth of HCC spheroids via activation of STAT3. These results indicate that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC could be a therapeutic target for HCC.