Nucleobindin-2 Protein Research Articles

Role of Nucleobindin-2 in the Clinical Pathogenesis and Treatment Resistance of Glioblastoma.

Glioblastoma (GBM) stands as the most prevalent primary malignant brain tumor, typically resulting in a median survival period of approximately thirteen to fifteen months after undergoing surgery, chemotherapy, and radiotherapy. Nucleobindin-2 (NUCB2) is a protein involved in appetite regulation and energy homeostasis. In this study, we assessed the impact of NUCB2 expression on tumor progression and prognosis of GBM. We further evaluated the relationship between NUCB2 expression and the sensitivity to chemotherapy and radiotherapy in GBM cells. Additionally, we compared the survival of mice intracranially implanted with GBM cells. High NUCB2 expression was associated with poor prognosis in patients with GBM. Knockdown of NUCB2 reduced cell viability, migration ability, and invasion ability of GBM cells. Overexpression of NUCB2 resulted in reduced apoptosis following temozolomide treatment and increased levels of DNA damage repair proteins after radiotherapy. Furthermore, mice intracranially implanted with NUCB2 knockdown GBM cells exhibited longer survival compared to the control group. NUCB2 may serve as a prognostic biomarker for poor outcomes in patients with GBM. Additionally, NUCB2 not only contributes to tumor progression but also influences the sensitivity of GBM cells to chemotherapy and radiotherapy. Therefore, targeting NUCB2 protein expression may represent a novel therapeutic approach for the treatment of GBM.

Circ_0000274 contributes to renal cell carcinoma progression by regulating miR-338-3p/NUCB2 axis and JAK1/STAT3 pathway

BackgroundKidney transplant recipients (KTRs) are at increased risk of developing renal cell carcinoma (RCC). Accumulating evidence has demonstrated that circular RNAs (circRNAs) are essential players in tumor advancement. However, the functions of circ_0000274 in renal cell carcinoma (RCC) are barely explored. MethodsThe primary RCC cell lines 786-O and A498 were used in this study. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was employed for the RNA levels of circ_0000274, microRNA-338-3p (miR-338-3p) and nucleobindin 2 (NUCB2). RNase R assay was conducted to analyze the feature of circ_0000274.Cell Counting Kit-8 (CCK-8) assay, colony formation assay, transwell assay, tube formation assay and flow cytometry analysis were conducted for cell viability, colony formation, metastasis, angiogenesis and apoptosis, respectively. Western blot assay was utilized for protein levels. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were adopted to analyze the associations of circ_0000274 RNA, miR-338-3p RNA and NUCB2 protein. Murine xenograft model was established to explore the function of circ_0000274 RNA in vivo. Immunohistochemistry (IHC) assay was used to analyze NUCB2 protein level in xenograft tumors. ResultsCompared to normal tissues and cells, circ_0000274 RNA level was elevated in RCC tissues and cells. Knockdown of circ_0000274 RNA suppressed cell viability, colony formation, metastasis and tube formation and promoted apoptosis in RCC cells in vitro. Circ_0000274 RNA sponged miR-338-3p RNA to positively regulate NUCB2 protein in RCC cells. Inhibition of miR-338-3p reversed the impacts of circ_0000274 knockdown on RCC cell malignant behaviors. MiR-338-3p RNA overexpression repressed the malignant phenotypes of RCC cells, while NUCB2 protein elevation could abrogate the effect. Moreover, circ_0000274 RNA knockdown blocked tumorigenesis in vivo. Besides, circ_0000274 RNA knockdown inactivated the JAK1/STAT3 protein signaling pathway. ConclusionCirc_0000274 RNA functioned as an oncogene in RCC development by regulating miR-338-3p RNA/NUCB2 protein axis and activating the JAK1/STAT3 protein signaling pathway.

Abstract 4884: To Investigate the mechanism and function of nucleobindin-2 in human hepatoma cells

Abstract Hepatocellular carcinoma is a common malignancy affecting approximately one million people worldwide annually. Recent studies have shown that appetite-associated nucleobindin-2 (NUCB-2) is over-expressed in clinical tumor samples such as gastric cancer, prostate cancer, renal cell carcinoma, endometrial carcinoma cells, colon cancer and breast cancer. The survival rate of high-level expression of NUCB-2 in cancer patients was lower than cancer patients with low-level expression of NUCB-2. In addition, overexpression of NUCB-2 was observed in liver cancer by using bioinformatics analysis. The liver cancer patients with NUCB-2 overexpression are associated with poor prognosis. However, the effect of NUCB-2 overexpression on tumor cells is still unclear. In this project, we evaluated the novel role and mechanisms of NUCB-2 overexpression in hepatoma cells. Our results indicated NUCB-2 mRNA expression level was induced by ER stress as determined with RT-PCR and real-time PCR, and induction of NUCB-2 protein level was significantly increased by ER stress. Increased ER stress by pre-S2Δ protein was significantly enhanced NUCB-2 expression. We also observed that the overexpression of NUCB-2 was corrected with ER stress in human hepatocellular carcinoma tumor samples. Consequently, this mechanistic research may provide a molecular basis to develop a biomarker and novel cancer chemotherapeutic agent for high level expression of NUCB-2 in cancer patients. keywords:Endoplasmic reticulum stress, Nucleobindin-2, real-time PCR, RT-PCR, HBV large surface mutant protein pre-S2Δ, Hepatocellular carcinoma, Huh-7, HepG2, MCF-7, Tunicamycin, Brefeldin A. Citation Format: Jui-Hsiang Hung, Hsiu-Ping Tsao, Ren-Hao Li, Chih-Han Li, Hut-Min Chang. To Investigate the mechanism and function of nucleobindin-2 in human hepatoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4884.

Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma.

Nucleobindin 2 (NUCB-2) is a multifunctional protein that contains several functional domains and is associated with a wide variety of biological processes, such as food intake and energy homeostasis. NUCB-2 has been demonstrated to be associated with worse malignant outcomes and cell migration in breast and prostate cancer. However, to the best of our knowledge, its clinical and biological significance in renal cell carcinoma remains unknown. In the present study, tissue specimens from 68 patients with renal cell carcinoma and 10 normal controls were collected for NUCB-2 mRNA and protein assays. The NUCB-2 level in the patients with renal cell cancer was significantly increased compared with the normal control patients. NUCB-2-knockout in the renal cancer cell line SK-RC-52 inhibited migration and invasion. In addition, the expression levels of molecules associated with epithelial-mesenchymal transition (EMT), including E-cadherin, β-catenin, Slug and Twist, were affected by NUCB-2 suppression and the zinc finger E-box binding to homeobox 1 (ZEB1)-dependent pathway. The AMP-dependent protein kinase (AMPK)/target of rapamycin complex (mTORC) 1 signaling pathway participates in the regulation of NUCB-2-mediated metastasis and EMT. Suppression of NUCB-2 also inhibited tumor nodule formation in a murine renal cell carcinoma tumor model. In summary, NUCB-2 increased migration, invasion and EMT in renal cell carcinoma cells through the AMPK/TORC1/ZEB1 pathway in vitro and in vivo.

High expression of Nucleobindin‐2 is associated with poor prognosis in gastric cancer

Nucleobindin‐2 (NUCB2) is a 396‐amino acid protein, cleaved into the N‐terminal nesfatin‐11–82, nesfatin‐285–163 and the C‐terminal nesfatin‐3166–396. NUCB2 contains a signal peptide, a leucine zipper structure, two Ca2+ binding EF‐hand domains, and has a wide variety of basic cellular functions. NUCB2 is also a precursor protein of nesfatin‐1, which was originally identified in hypothalamic nuclei, and which is a regulatory factor involved in the central control of food intake and energy balance.There are several reports indicating that NUCB2 is also expressed in various human peripheral tissues. Moreover, recent studies have reported that high levels of NUCB2 mRNA and protein are a potent prognostic factor for prostate cancer, endometrial carcinoma, and breast cancer. However, the clinicopathological significance of NUCB2 expression in gastric cancer has still not been elucidated. Therefore, we examined NUCB2 expression in a large number of gastric cancer patients, using immunohistochemistry, to explore its clinicopathological significance. To explore this, we aimed to investigate the NUCB2 expression in gastric cancer tissues and adjacent non‐tumor tissues and its potential relevance to clinicopathological factors and prognosis using immunohistochemistry analysis. In our study, NUCB2 level in gastric cancer tissues was higher than in non‐tumor tissues. A high expression of NUCB2 is significantly associated with tumor depth, lymph node metastasis, lymphatic invasion, venous invasion and clinical stage. Furthermore, the expression level of NUCB2 protein was independent predictor of progression‐free survival.In summary, NUCB2 might play a crucial role in gastric cancer development and could serve as an independent predictor of prognosis of gastric cancer patients.Progression‐free survival and overall survival curves of 328 gastric cancer patients divided into two groups according to NUCB2 expression levels. The significant differences were given as p = 0.0214 for progression‐free survival and p=0.0211 for overall survival.Figure 1

NUCB2 polymorphisms are associated with an increased risk for type 2 diabetes in the Chinese population.

BackgroundThe nucleobindin 2 (NUCB2) gene encodes the NUCB2 protein, which plays a critical role in glucose metabolism and diabetes. This study explored the correlation between NUCB2 genetic variants and type 2 diabetes mellitus (T2DM). The study further examined the different NUCB2 variants that confer risk to T2DM in Chinese Han populations.MethodsThis study evaluated the anthropometric and glycemic profiles of 578 T2DM patients and 1,609 healthy controls. Subsequently, we genotyped five single nucleotide polymorphisms (SNPs) (rs10832756, rs1330, rs10766383, rs10832757, and rs11024251) in all the study participants using a Sequenom Mass ARRAY SNP genotyping platform.ResultsThe distribution of polymorphisms was significantly different between the T2DM patients and healthy controls. Our logistic regression analysis results showed that the five NUCB2 SNPs are significantly correlated with the risk for T2DM, especially rs11024251(P=2.97×10−6). Interestingly, analysis of male and female sub-populations separately showed that only two of the SNPs (rs10832757 and rs11024251) have significant correlation to T2DM in males [P=0.0244, odds ratio (OR) 1.28 and P=0.0062, OR 1.35, respectively). In females however, we identified four significant SNPs (rs1330, rs10766383, rs10832757, and rs11024251; P<0.05, OR 1.31–1.42). Furthermore, we found that rs1330 is associated with body mass index of female subpopulation only (P=0.0174, β =0.0060).ConclusionsNUCB2 polymorphisms could have a pivotal role in the presence of T2DM. Sex-specific SNPs of NUCB2 could account for the differences in clinical features of T2DM between male and female subpopulations. Nevertheless, our results should be replicated using larger sample sizes, and experimental investigations are needed to elucidate the molecular mechanisms of the associations observed in this study.

MON-313 High Expression of Nucleobindin-2 Is Associated with Poor Prognosis in Gastric Cancer

Nucleobindin-2 (NUCB2) is a 396-amino acid protein, cleaved into the N-terminal nesfatin-11-82, nesfatin-285-163and the C-terminal nesfatin-3166-396. NUCB2 contains a signal peptide, a leucine zipper structure, two Ca2+binding EF-hand domains, and has a wide variety of basic cellular functions. NUCB2 is also a precursor protein of nesfatin-1, which was originally identified in hypothalamic nuclei, and which is a regulatory factor involved in the central control of food intake and energy balance. There are several reports indicating that NUCB2 is also expressed in various human peripheral tissues. Moreover, recent studies have reported that high levels of NUCB2 mRNA and protein are a potent prognostic factor for prostate cancer, endometrial carcinoma, and breast cancer. Suzuki et al.described high NUCB2 immunoreactivity in patients with breast cancer, and NUCB2 protein expression associated significantly with lymph node metastasis and an increased risk of recurrence. They found that NUCB2 significantly increased tumor cell proliferation, migration, and invasion, suggesting that NUCB2 plays a crucial role in the metastatic process of breast cancer. Several researchers have reported that the expression level of NUCB2 was significantly higher in prostate cancer tissues than in non-cancerous tissues, and was associated with lymph node metastasis and shorter survival. However,theclinicopathologicalsignificance of NUCB2 expression in gastric cancer has still not beenelucidated. Therefore, we examined NUCB2 expression in a large number of gastric cancer patients, using immunohistochemistry, to explore its clinicopathologicalsignificance. To explore this, we aimed to investigate the NUCB2 expression in gastric cancer tissues and adjacent non-tumor tissues and its potential relevance to clinicopathological factors and prognosis using immunohistochemistry analysis. In our study, NUCB2 level in gastric cancertissues was higher than in non-tumor tissues. A high expression of NUCB2 is significantly associated with tumor depth, lymph node metastasis, lymphatic invasion, venous invasion and clinical stage. Furthermore, the expression level of NUCB2 protein was independent predictor of progression-free survival. In summary, NUCB2 might play a crucial role in gastric cancerdevelopment and could serve as an independent predictor of prognosis of gastric cancerpatients.

High NUCB2 expression level is associated with metastasis and may promote tumor progression in colorectal cancer.

Nucleobindin 2 (NUCB2) is mainly expressed in the hypothalamic nuclei and has a proven role in energy homeostasis. It has also been recently reported to have a key role in tumor progression. However, the clinical significance of NUCB2 in colorectal cancer (CRC) remains unknown. In the present study, the level of NUCB2 mRNA was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in 34 paired fresh tissues from patients with CRC. RT-qPCR was followed by immunohistochemical (IHC) staining of NUCB2 protein in tissue microarrays of 251 samples to evaluate the clinical significance of NUCB2 in CRC. The RT-qPCR indicated an upregulation of NUCB2 mRNA in CRC tissues compared with normal tissues (P=0.027). IHC staining indicated a positive association between elevated NUCB2 expression and lymph node metastasis or tumor-node-metastasis (TNM) stage. Patients with CRC and lymph node metastasis demonstrated a higher expression of NUCB2 (49.5%, 50/101) compared with those without lymph node metastasis (36.7%, 55/150; P=0.043). Furthermore, NUCB2 expression was also higher in patients with CRC and TNM stage III–IV compared with those with TNM stage I–II (50.9% vs. 35.0%; P=0.011). However, Kaplan-Meier analysis indicated no significant association between NUCB2 expression and disease-free survival of patients. Additionally, multivariate analysis did not identify the upregulation of NUCB2 as an independent prognostic predictor in patients with CRC (P=0.755). In conclusion, the present study demonstrated that upregulation of NUCB2 is significantly associated with CRC metastasis, indicating that NUCB2 may be a cancer-associated oncogene associated with the aggressive progression of CRC.

Erratum to: Gonadotropin-releasing hormone, kisspeptin, and gonadal steroids directly modulate nucleobindin-2/nesfatin-1 in murine hypothalamic gonadotropin-releasing hormone neurons and gonadotropes.

Neuroendocrine regulation of metabolism and reproduction are tightly interlinked. Nesfatin-1 is an 82 amino acid metabolic peptide derived from nucleobindin-2 (NUCB2). NUCB2 mRNA and protein significantly increase in the hypothalamus of rats during puberty-to-adult transition. Administration of nesfatin-1 modulates circulating LH and testosterone in male rats. However, whether nesfatin-1 acts directly on neurons and gonadotropes remain unknown. In addition, whether reproductive hormones of the hypothalamo-pituitary gonadal axis modulate NUCB2/nesfatin-1 is unclear. To address these, we employed murine hypothalamic (GT1-7) and pituitary (LβT2) cells in vitro. Nucb2 expression, and NUCB2/nesfatin-1 immunoreactivity were observed in both GT1-7 and LβT2 cells, and in the hypothalamus of mice. Nesfatin-1 co-localized GnRH in GT1-7 cells, and in the hypothalamic perikarya of mice. Cells were treated with kisspeptin, GnRH, and estradiol and testosterone, as well as nesfatin-1 for 2, 6 or 24 hours. Synthetic nesfatin-1 increased Kiss1r and Gnrh expression in GT1-7 cells and Lhβ in LβT2. Nesfatin-1 increased GnRH and LHβ protein expression in GT1-7 and LβT2 at 6-hour post incubation respectively. Both NUCB2 mRNA and protein were increased in GT1-7 cells treated with kisspeptin. Testosterone increased NUCB2 mRNA and protein expression in GT1-7 and LβT2. 17β-estradiol increased NUCB2 mRNA and protein expression in LβT2. Nesfatin-1 acts directly on hypothalamic neurons and gonadotropes to elicit a generally positive influence on the endocrine milieu regulating reproduction in mice. Reproductive hormones, in turn, modulate brain and pituitary NUCB2/nesfatin-1. In conclusion, we provide additional information to designate nesfatin-1 as a novel, additional factor that helps reproductive success.

Gonadotropin-releasing hormone, kisspeptin, and gonadal steroids directly modulate nucleobindin-2/nesfatin-1 in murine hypothalamic gonadotropin-releasing hormone neurons and gonadotropes.

Neuroendocrine regulation of metabolism and reproduction are tightly interlinked. Nesfatin-1 is an 82 amino acid metabolic peptide derived from nucleobindin-2 (NUCB2). NUCB2 mRNA and protein significantly increase in the hypothalamus of rats during puberty-to-adult transition. Administration of nesfatin-1 modulates circulating LH and testosterone in male rats. However, whether nesfatin-1 acts directly on neurons and gonadotropes remain unknown. In addition, whether reproductive hormones of the hypothalamo-pituitary gonadal axis modulate NUCB2/nesfatin-1 is unclear. To address these, we employed murine hypothalamic (GT1-7) and pituitary (LβT2) cells in vitro. Nucb2 expression, and NUCB2/nesfatin-1 immunoreactivity were observed in both GT1-7 and LβT2 cells, and in the hypothalamus of mice. Nesfatin-1 co-localized GnRH in GT1-7 cells, and in the hypothalamic perikarya of mice. Cells were treated with kisspeptin, GnRH, and estradiol and testosterone, as well as nesfatin-1 for 2, 6 or 24 hours. Synthetic nesfatin-1 increased Kiss1r and Gnrh expression in GT1-7 cells and Lhβ in LβT2. Nesfatin-1 increased GnRH and LHβ protein expression in GT1-7 and LβT2 at 6-hour post incubation respectively. Both NUCB2 mRNA and protein were increased in GT1-7 cells treated with kisspeptin. Testosterone increased NUCB2 mRNA and protein expression in GT1-7 and LβT2. 17β-estradiol increased NUCB2 mRNA and protein expression in LβT2. Nesfatin-1 acts directly on hypothalamic neurons and gonadotropes to elicit a generally positive influence on the endocrine milieu regulating reproduction in mice. Reproductive hormones, in turn, modulate brain and pituitary NUCB2/nesfatin-1. In conclusion, we provide additional information to designate nesfatin-1 as a novel, additional factor that helps reproductive success.

Nucleobindin 2 (NUCB2) in human endometrial carcinoma: a potent prognostic factor associated with cell proliferation and migration.

Nucleobindin 2 (NUCB2) is a multifunctional protein containing several functional domains, and associated with wide variety of biological process such as food intake and energy homeostasis. Recently, NUCB2 has been implicated in not only normal human tissues but also some kinds of human malignancies. However, its clinical and/or biological significance has largely remained unknown in endometrial carcinomas. We therefore immunolocalized NUCB2 protein in 87 endometrial carcinoma tissues and examined its clinical significance. NUCB2 immunoreactivity was detected in 19 out of 87 (22%) of endometrial carcinoma cases examined, and positively correlated with Ki67 labeling index, while there was no significant correlation between NUCB2 and stage, histological grade, and progesterone receptor status. Furthermore, NUCB2 immunoreactivity was significantly correlated with increased risk of recurrence and worse clinical outcome regardless of stage or histological grade. Subsequent multivariate analyses did reveal that NUCB2 immunoreactivity was an independent prognostic factor for both disease-free survival and endometrial cancer specific survival. In vitro experiments demonstrated that knockdown of NUCB2 using specific siRNA for NUCB2 significantly impaired cell proliferation and migration of the endometrial carcinoma cell lines, Ishikawa and Sawano cells, and that nesfatin-1 treatment significantly promoted cell proliferation and migration in Ishikawa cells. These findings possibly suggested that NUCB2 and/or nesfatin-1 had pivotal roles in the progression of endometrial carcinomas. Immunohistochemical NUCB2 status may therefore serve as a potent biomarker for endometrial carcinomas.

Nucleobindin 2 and its role in tumorigenesis and progression

Nucleobindin2protein（NUCB2）isanewlydiscoveredneuropeptideprecursorprotein, which has a comprehensive cytology function and is expressed in the hypothalamus nucleus and many peripheral tissues.There aren′t many studies about its signaling pathway,where neuroendocrine regulation,cell survival growth,tumor suppressor,cytokine secretion were found to involve in it.Besides,it has also been confirmed that breast cancer,lung cancer,ovarian cancer and prostate cancer are closely related to NUCB2.Therefore, several downstream pathways of NUCB2 may be related to the formation and progression of tumor.Further stud-ies are still needed to clarify the signal pathways of NUCB2 to provide a reliable basis for clinical cancer preven-tion. Key words: Histones; Neoplasms

Increased nucleobindin-2 (NUCB2) transcriptional activity links the regulation of insulin sensitivity in Type 2 diabetes mellitus.

The protein nucleobindin-2 (NUCB2) has been recently identified as a novel satiety regulator. However, its pathophysiological role in humans remains unknown. The aims of the present study are to explore whether plasma NUCB2-1 and NUCB2 transcription activity are increased in newly diagnosed Type 2 diabetes mellitus (nT2DM) and, if so, whether changing NUCB2-1 level is a physiologic response or a compensatory mechanism for impaired insulin action. The nT2DM, impaired glucose tolerance (IGT), and healthy people (NGT, normal glucose tolerance) groups were enrolled in this study. The peripheral and hepatic insulin actions in rats with intracerebroventricular (ICV) NUCB2-1 administration were examined by euglycemic-hyperinsulinemic clamps. Plasma NUCB2-1 levels were elevated in subjects with both nT2DM and IGT compared with normal controls. NUCB2 mRNA and protein contents of muscle and adipose tissues in T2DM patients were also significantly increased compared to controls. ICV NUCB2-1 infusion in rats inhibited hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity, and this was sufficient to induce insulin sensitivity in the liver and peripheral tissues during euglycemic-hyperinsulinemic clamps. In T2DM patients, there were increases in plasma NUCB2-1 levels and increases in NUCB2 mRNA and protein contents in muscle and adipose tissues. These increases are presumably a compensatory response to defective insulin action.

MTOR-dependent Modulation of Gastric Nesfatin-1/NUCB2

Background: Nesfatin-1, an 82 amino acid peptide derived from the prohormone nucleobindin-2 (NUCB2), is a novel satiety hormone acting through a leptin-independent mechanism in the hypothalamus. The mechanisms by which production of nesfatin-1/NUCB2 is regulated remain unknown. Methods: Nesfatin-1/NUCB2 mRNA and immunoreactivity were examined in gastric tissue and Min-6 cells by RT-PCR and immunofluorescent staining or Western blotting. Results: Nesfatin-1/NUCB2 is co-localized with pS6K1, the downstream target of mammalian target of rapamycin (mTOR), in gastric X/A like cells. A parallel relationship between gastric mTOR signaling and nesfatin-1/NUCB2 was observed during changes in energy status. Both mTOR activity and gastric nesfatin-1/NUCB2 were down-regulated by fasting, and returned to basal levels with re-feeding. In high fat diet induced obese mice, gastric mTOR signaling and nesfatin-1/NUCB2 were increased. Inhibition of the gastric mTOR signaling by rapamycin attenuated the expression of gastric nesfatin-1/NUCB2 mRNA and protein in both lean and obese mice. Attenuation of mTOR activity by rapamycin or over-expression of TSC1 or TSC2 reduced the expression of nesfatin-1/NUCB2 in Min-6 cells, suggesting a direct effect of mTOR signaling. Conclusion: Gastric mTOR is a gastric energy sensor whose activity is linked to the regulation of gastric nesfatin-1/NUCB2.

Lipopolysaccharide increases gastric and circulating NUCB2/nesfatin-1 concentrations in rats

Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to Gram-negative bacteria mimicking symptoms of infection including reduction of food intake. LPS decreases acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. We also recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration, ad libitum fed rats were equipped with an intravenous (iv) catheter. LPS was injected intraperitoneally (ip, 100μg/kg) and blood was withdrawn before and at 2, 5, 7 and 24h post injection and processed for NUCB2/nesfatin-1 radioimmunoassay. Gastric corpus was collected to measure NUCB2 mRNA expression by RT-qPCR and NUCB2/nesfatin-1 protein concentration by Western blot. Injection of LPS increased plasma NUCB2/nesfatin-1 concentrations by 43%, 78% and 62% compared to vehicle at 2h, 5h and 7h post injection respectively (p<0.05) and returned to baseline at 24h. The plasma NUCB2/nesfatin-1 increase at 2h was associated with increased corpus NUCB2 mRNA expression (p<0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (p<0.01). These data show that gastric NUCB2 production and release are increased in response to LPS. These changes are opposite to those of ghrelin in response to LPS supporting a differential gastric regulation of NUCB2/nesfatin-1 and ghrelin expression derived from the same cell by immune challenge.

Hypoxia Induces Intestinal Isocitrate Dehydrogenase Expression and Enhances 13C-Octanoate Metabolism in Healthy Mountaineers After Rapid Ascent to 4559 M

Background: Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to gram-negative bacteria mimicking symptoms of infection including reduction of food intake. We previously reported that LPS decreased acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. In addition, we recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. Aim: To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration. Methods: Ad libitum fed male Sprague-Dawley rats were equipped with an intravenous (IV) catheter placed in the right jugular vein and allowed to recover for four days. LPS was injected intraperitoneally (IP, 100μg/kg) and blood was withdrawn before and at 2, 5, 7 and 24h post injection and processed for NUCB2/nesfatin-1 measurement by radioimmunoassay (n= 5/group). Gastric corpus was collected at different time points to measure NUCB2 mRNA expression by quantitative RT-PCR and NUCB2/nesfatin-1 protein concentration by Western blot. Results: Injection of LPS significantly increased plasma NUCB2/nesfatin-1 concentrations by 55% compared to vehicle at 2h post injection (248.5 ± 45.5 vs. 161.2 ± 14.3 pg/ ml, P<0.05), whereas at all other time points (5, 7, 24h) no significant alterations were observed. The plasma NUCB2/nesfatin-1 increase at 2h was associated with an increased corpus NUCB2 mRNA expression compared to vehicle (1.22 ± 0.11 vs. 0.58 ± 0.17 pg/ml, P<0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (concentration normalized to housekeeping protein β-actin: 1.17 ± 0.06 vs. 0.72 ± 0.05, P<0.01). Conclusions: These data show that NUCB2 production and release are increased under conditions of inflammation in response to LPS. The stomach is likely to represent the source of NUCB2/nesfatin-1 as gastric mRNA and protein content was increased at the same time indicating stimulated production, whereas NUCB2 mRNA was not detectable in circulating white blood cells. These changes are in opposition to those observed for ghrelin and support the assumption of a differential regulation of these two food intake regulatory peptides derived from the same cell. The decrease of ghrelin and the increase of nesfatin-1 may act in concert to decrease food intake giving rise to the potential benefits of stimulating ghrelin and reducing nesfatin-1 signaling under conditions of inflammation. Acknowledgement: VA Merit Award (NWGL), Center grant DK-41301 (Animal Core, YT)

Distribution of nesfatin-1/NUCB2 in the digestive system of humans and rodents

Objective To investigate the regional distribution and morphological features of nesfatin-1/NUCB2 in digestive system of the humans, Sprague-Dawley (SD) rats and the institute of cancer research (ICR) mice, so as to lay the foundation for further study of its functions in the digestive system. Methods The specimens were obtained from SD rats and ICR mice as well as 20 patients with digestive disease, who were admitted to the First hospital affiliated to Nanjing Medical University and receired surgercal treatment. The specimens from patients with malignant tumors were obtained 5 cm apart from cancerous tissues and from patients with benign tumors were obtained near the focus. The resected tissues included pancreas, stomach, duodenum, esophagus, liver, small intestine or colon. The distribution of nesfatin-1/NUCB2 was examined with immunohistochemical (IHC) staining and its protein level in each organ was measured using Western blotting. Results The immuinohistocemical study revealed the similar distribution pattern of nesfatin-1/NUCB2 in the digestive system of the patients, SD rats and ICR mice. Nesfatin-1/NUCB2 was found to localize in the center of the pancreatic islets, the lower 1/3 to 1/2 of the gastric mucosal glands, as well as the submucosa of the duodenum. Western blotting examination showed the expression of NUCB2 in all tissues from patients, SD rats and ICR mice, whereas the protein level of the nesfatin-1/NUCB2 was higher in pancreas (0.84±0.03, 0. 84±0.05 and 0. 84±0.04, respectively), stomach (0.86±0.06,0.81±0.02 and 0. 78±0.02, respectively) or duodenum (0.79±0.09,0. 79±0.04 and 0.78±0.05)than that in esophagus (0.43±0.04,0.44 ± 0.02 and 0.47 ± 0.06, respectively), liver (0.42±0.01,0.44±0.04 and 0.43 ± 0.01, objectively), small intestine (0.32±0.04,0. 32 ± 0. 04 and 0.34 ±0.04, respectively) or colon (0. 29±0.01,0.32±0.03 and 0. 28±0.03, respectively)(all P values=0. 000). Conclusion Nesfatin-1/NUCB2 is widely expressed in the pancreatic islets, gastric mucosal glands and duodenum of the patients, SD rats and ICR mice, which indicates that nesfatin-1/NUCB2 may be involved in the regulation of food intake, carbohydrate metabolism and gastrointestinal motility. Key words: Nesfatin-1 protein; Nucleobindin2 protein; Pancreas; Stomach; Duodenum; Brunner glands; Immunohistochemistry

Expression of nesfatin-1/NUCB2 in rodent digestive system

To observe the regional distributions and morphological features of nesfatin-1/nucleobindin-2 (NUCB2) immunoreactive (IR) cells in the rodent digestive system. Paraffin-embedded sections of seven organs (pancreas, stomach, duodenum, esophagus, liver, small intestine and colon) dissected from sprague-dawley (SD) rats and imprinting-control-region (ICR) mice were prepared. The regional distributions of nesfatin-1/NUCB2 IR cells were observed by immunohistochemical staining. The morphological features of the nesfatin-1/NUCB2 IR cells were evaluated by hematoxylin and eosin (HE) staining. Fresh tissues of the seven organs were prepared for Western blotting to analyze the relative protein levels of NUCB2 in each organ. Immunohistochemical staining showed that the nesfatin-1/NUCB2 IR cells were localized in the central part of the pancreatic islets, the lower third and middle portion of the gastric mucosal gland, and the submucous layer of the duodenum in SD rats and ICR mice. HE staining revealed that the morphological features of nesfatin-1/NUCB2 IR cells were mainly islet cells in the pancreas, endocrine cells in the stomach, and Brunner's glands in the duodenum. Western blotting revealed that NUCB2 protein expression was higher in the pancreas, stomach and duodenum than in the esophagus, liver, small intestine and colon (P = 0.000). Nesfatin-1/NUCB2 IR cells are expressed in the pancreas, stomach and duodenum in rodents. These cells may play an important role in the physiological regulation of carbohydrate metabolism, gastrointestinal function and nutrient absorption.

Identification and Characterization of Nesfatin-1 Immunoreactivity in Endocrine Cell Types of the Rat Gastric Oxyntic Mucosa

Hypothalamic nesfatin-1, derived from the nucleobindin2 (NUCB2) precursor, inhibits nocturnal food intake and body weight gain in rats. Nesfatin-1 is able to cross the blood-brain barrier, suggesting a peripheral source of nesfatin-1. Many centrally acting food intake regulatory neuropeptides are also produced in the periphery, especially in the gastrointestinal tract. Therefore, we investigated the gene expression of NUCB2 and distribution of nesfatin-1-immunoreactive cells in the stomach. Microarray mRNA expression profiles in purified small endocrine cells of the gastric mucosa substantiated by quantitative RT-PCR showed significantly higher NUCB2 mRNA expression compared with brain and heart. Western blot confirmed the expression of NUCB2 protein and its transport into a secretory soluble fraction of gastric mucosal endocrine cell homogenates. Immunohistochemical colabeling for nesfatin-1 and ghrelin, histidine decarboxylase, or somatostatin revealed two subtypes of nesfatin-1-positive endocrine cells. Cells in the midportion of the glands coexpressed nesfatin-1 and ghrelin, whereas few cells in the glandular base coexpressed nesfatin-1 and somatostatin or histidine decarboxylase. High-resolution three-dimensional volume imaging revealed two separate populations of intracytoplasmic vesicles in these cells, one containing nesfatin-1 and the other ghrelin immunoreactivity. Microarray rat genome expression data of NUCB2 in small gastric endocrine cells confirmed by quantitative RT-PCR showed significant down-regulation of NUCB2 after 24 h fasting. In summary, NUCB2 mRNA expression as well as protein content is present in a specific subset of gastric endocrine cells, most of which coexpress ghrelin. NUCB2 gene expression is significantly regulated by nutritional status, suggesting a regulatory role of peripheral nesfatin-1 in energy homeostasis.

CAMP-dependent Protein Kinase A (PKA) Signaling Induces TNFR1 Exosome-like Vesicle Release via Anchoring of PKA Regulatory Subunit RIIβ to BIG2

The 55-kDa TNFR1 (type I tumor necrosis factor receptor) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-bromo-cyclic AMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a protein kinase A (PKA)-dependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIbeta modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIbeta to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIbeta to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide exchange protein that activates class I ADP-ribosylation factors and as an AKAP for RIIbeta that localizes PKA signaling within cellular TNFR1 trafficking pathways.