Colony-enhancing Factor Research Articles

Visfatin Serum Levels Predict Mortality in Critically Ill Patients.

The adipokine visfatin, also termed pre-B-cell colony-enhancing factor (PBEF), is mainly derived from adipose tissue but has been implicated in the regulation of innate immune responses. We hypothesized that visfatin could be a potential circulating biomarker in critical illness and sepsis. We therefore measured serum levels of visfatin in a cohort of 229 critically ill medical patients upon admission to the intensive care unit (ICU). In comparison to 53 healthy controls, visfatin levels were significantly elevated in medical ICU patients, especially in patients with sepsis. Visfatin serum concentrations were strongly associated with disease severity and organ failure but did not differ between patients with or without obesity or type 2 diabetes. Visfatin levels correlated with biomarkers of renal failure, liver dysfunction, and other adipokines (e.g., resistin, leptin, and adiponectin) in critically ill patients. High visfatin levels at ICU admission indicated an increased mortality, both at the ICU and during long-term follow-up of approximately two years. Our data therefore demonstrate that circulating visfatin is a valuable biomarker for risk and prognosis assessment in critically ill patients. Furthermore, visfatin seems to be involved in the pathogenesis of excessive systemic inflammation, supporting further research on visfatin as a therapeutic target.

Clinical Significance of Serum Visfatin in Renal Transplant Recipients

Chronic antibody-mediated rejection is the most common cause of late graft loss in renal transplant recipients. Visfatin is a pre-B cell colony-enhancing factor secreted by activated lymphocytes. We hypothesize that visfatin may play a role in the augmentation of B cell colonies and facilitate antibody-mediated rejection. Renal transplant recipients were randomly selected for the study. Fasting blood samples were obtained for the assay of visfatin. The participants were prospectively followed up for 3 years. A total of 146 patients were recruited for the study and were divided into 3 groups according to tertile of serum visfatin level. At the end of follow-up, 6 patients had graft loss, including 1 graft loss in tertile 1, 3 in tertile 2, and 2 in tertile 3 (P = .60). Fourteen patients experienced at least 1 episode of acute rejection, while 21 patients were diagnosed as having chronic rejection. The distribution of acute rejection was 10.2% in tertile 1, 10.2% in tertile 2, and 8.3% in tertile 3 (P = .94); chronic rejection occurred in 10.2%, 16.3%, and 16.7%, respectively (P = .59). We conclude that serum visfatin level was not correlated with either graft failure or patient mortality in a 3-year observation period.

The role of pre-B-cell colony-enhancing factor in Egyptian children with hemophagocytic lymphohistiocytosis

Background Hemophagocytic lymphohistiocytosis (HLH) is caused by a highly active but ineffective immune response, including impaired or absent function of natural killer cells and cytotoxic T cells, and the release of proinflammatory cytokines. Pre-B-cell colony-enhancing factor (PBEF) is an inflammatory cytokine involved in several inflammatory diseases, and it has been identified to react with several cytokines involved in HLH. Objective We aimed to study the clinicoepidemiological characteristics of Egyptian children with HLH and to evaluate the role of PBEF as a diagnostic and prognostic marker in Egyptian children with HLH. Patients and methods This is a cross-sectional study that recruited fifteen children with HLH from the Pediatric Hematology/Oncology unit. There were 11 male and four female, and their median age (interquartile range) was 13 months. Patients underwent thorough clinical assessment, laying stress on disease manifestation, classification, treatment, and prognosis. Plasma concentration of PBEF was determined using an enzyme-linked immunosorbent assay. Results Four patients were classified as having primary HLH, seven patients were as having secondary HLH and four patients had unknown classification owing to waiting for genotyping. The risk factors before presentation were as follow: four had viral infection, three patients had malignancy, and two patients had immune deficiency before presentation. Seven patients of the study group died and eight patients were still alive. PBEF was significantly increased in the patient group than the control group. PBEF level showed a significant positive correlation with serum ferritin and triglycerides level and a negative correlation with fibrinogen level. Conclusion The elevated PBEF level and its correlation with the widely available biochemical markers for diagnosis of pediatric HLH indicates that it may be involved in its inflammatory process.

Polydatin Attenuates Atherosclerosis in ApoE Mice through PBEF Mediated Reduction of Cholesterol Deposition.

Cholesterol metabolism becomes imbalanced during the formation of macrophage-derived foam cells. Pre-B-cell colony-enhancing factor (PBEF) has recently been found to affect lipid deposition and inflammation in atherosclerosis. Here, we aimed to study the effects and molecular mechanism of Polydatin on atherosclerosis in ApoE-knockout (ApoE ) mice. Thirty ApoE mice were fed a high-fat diet (HFD) for 12 weeks, and then treated with Polydatin for another 12 weeks. Whole aortas and cryosections were stained with oil red O. Blood lipid, PBEF and cytokine levels were measured by ELISA. The mRNAs of cholesterol metabolism-related genes were determined by qRT-PCR and protein levels by Western blotting. Cell cholesterol content and viability were determined in macrophages and RAW 264.7 cells. PBEF siRNA was used to study the effect of Polydatin on cholesterol metabolism in macrophages incubated with ox-LDL. Polydatin lowered blood lipids and decreased atherosclerotic lesions in ApoE mice. The expression of cytokines and the mRNA of cholesterol metabolism-related genes were markedly regulated by Polydatin. Meanwhile, PBEF mRNA and protein were both greatly down-regulated by Polydatin. In vitro, Polydatin protected RAW 264.7 cells treated by ox-LDL and inhibited cholesterol uptake by macrophages. The PBEF siRNA result indicates that Polydatin can modulate cholesterol metabolism in macrophages, partly through down-regulation of PBEF. In conclusion, Polydatin relieves atherosclerosis injury in ApoE mice, mainly through down-regulation of PBEF and inhibition of PBEF-inducing cholesterol deposits in macrophages.

Emodin alleviates severe acute pancreatitis-associated acute lung injury by decreasing pre-B-cell colony-enhancing factor expression and promoting polymorphonuclear neutrophil apoptosis.

The present study aimed to evaluate the protective effects of emodin on severe acute pancreatitis (SAP)-associated acute lung injury (ALI), and investigated the possible mechanism involved. SAP was induced in Sprague-Dawley rats by retrograde infusion of 5% sodium taurocholate (1 ml/kg), after which, rats were divided into various groups and were administered emodin, FK866 [a competitive inhibitor of pre-B-cell colony-enhancing factor (PBEF)] or dexamethasone (DEX). DEX was used as a positive control. Subsequently, PBEF expression was detected in polymorphonuclear neutrophils (PMNs) isolated from rat peripheral blood by reverse transcription-quantitative polymerase chain reaction and western blotting. In addition, histological alterations, apoptosis in lung/pancreatic tissues, apoptosis of peripheral blood PMNs and alterations in the expression of apoptosis-associated proteins were examined by hematoxylin and eosin staining, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay, Annexin V/propidium iodide (PI) assay and western blotting, respectively. Serum amylase activity and wet/dry (W/D) weight ratios were also measured. An in vitro study was also conducted, in which PMNs were obtained from normal Sprague-Dawley rats and were incubated with emodin, FK866 or DEX in the presence of lipopolysaccharide (LPS). Apoptosis of PMNs and the expression levels of apoptosis-associated proteins were examined in cultured PMNs in vitro by Annexin V/PI assay and western blotting, respectively. The results demonstrated that emodin, FK866 and DEX significantly downregulated PBEF expression in peripheral blood PMNs. In addition, emodin, FK866 and DEX reduced serum amylase activity, decreased lung and pancreas W/D weight ratios, alleviated lung and pancreatic injuries, and promoted PMN apoptosis by regulating the expression of apoptosis-associated proteins: Fas, Fas ligand, B-cell lymphoma (Bcl)-2-associated X protein, cleaved caspase-3 and Bcl-extra-large. In addition, the in vitro study demonstrated that emodin, FK866 and DEX significantly reversed the LPS-induced decrease of apoptosis in PMNs by regulating the expression of apoptosis-associated proteins. In conclusion, the present study demonstrated that emodin may protect against SAP-associated ALI by decreasing PBEF expression, and promoting PMN apoptosis via the mitochondrial and death receptor apoptotic pathways.

Possible interaction between visfatin, periodontal infection, and other systemic diseases: A brief review of literature.

Gingivitis and periodontitis are common bacterial infections caused by a variety of microorganisms. Despite the microorganisms’ roles as etiologic agents, inflammation-induced substances also have crucial parts in the loss of connective tissue and the supporting alveolar bone. Visfatin is a pleiotropic mediator, which acts as growth factor, cytokine, and pre-B-cell colony-enhancing factor. A positive correlation was detected between the serum/plasma levels of visfatin and inflammatory disorders such as diabetes mellitus and cardiovascular disease. In addition, the visfatin level was higher in saliva and the gingival crevicular fluid (GCF) of subjects with periodontal disease. This review defined current, predictable patterns of possible interaction of visfatin with periodontal infection and other systemic diseases, using PubMed and Medline databases searching for articles written in English. Peer-reviewed articles were targeted using the following keywords: “visfatin,” “periodontal disease,” “inflammatory mediator,” and “biomarker.” Available full-text articles were read, and related articles were also scrutinized, while a hand search was also performed. Search was confined to human studies, and articles written in English and published between 1985 and 2016 were selected. It was concluded that periodontal infection and other systemic diseases could be related to the levels of visfatin in GCF, saliva, and serum as a biomarker of these diseases.

Inhibition of pre-B cell colony-enhancing factor attenuates inflammation induced by hyperoxia in EA.hy926 cells.

The aim of this study was to investigate the role of pre-B cell colony-enhancing factor(PBEF) in the pathogenesis of bronchopulmonary dysplasia(BPD) using an established cell model of BPD. For this purpose, EA.hy926 cell cultures were divided into 4groups as follows: the air group as the blank control, the hyperoxia group, the hyperoxia plus PBEF siRNA group and the hyperoxia plus scramble siRNA group. Cell viability and the generation of reactive oxygen species(ROS) were determined using respective kits. Moreover, the protein and mRNA expression levels of PBEF, interleukin-8(IL-8) and tumor necrosis factor-α(TNF-α) were also detected by corresponding methods. Compared with the hyperoxia group, the ROS levels in the hyperoxia plus PBEFsiRNA group were significantly reduced(P<0.01). The silencing of PBEF increased cell viability compared with the hyperoxia group. The protein and mRNA expression levels of PBEF, IL-8 and TNF-α were all decreased in the hyperoxia plus PBEFsiRNA group compared with the hyperoxia group(P<0.01). Our study thus demonstrates that the inhibition of PBEF attenuates oxidative stress and inflammation induced by hyperoxia in EA.hy926 cells, suggesting that PBEF may be a potential diagnostic and therapeutic target, which may be used for the development of novel treatment strategies for BPD.

Study of Visfatin Level in Type 1 Diabetic Children and Adolescents.

BACKGROUND:Visfatin is an intracellular enzyme, known as nicotinamide phosphoribosyltransferase (Nampt) and pre-B-cell colony-enhancing factor (PBEF-1). It has insulin-mimetic effects and lowers plasma glucose levels.AIM:The aim of the work was to assess serum concentration of Visfatin in type 1 diabetic children and adolescents and study its relationships with duration of diabetes, body mass index (BMI), glycemic control, insulin dosage, lipid profile and microvascular complications.MATERIAL AND METHODS:Fifty children and adolescents with type 1 diabetes mellitus were recruited with 30 ages and gender-matched healthy subjects. They were subjected to history taking; anthropometric measurements and chronic diabetic complications were recorded if present. Laboratory analysis included urinary microalbumin, serum triglycerides, HDL, LDL, cholesterol, fasting blood glucose, glycosylated Hb (HbA1c) and serum visfatin which was measured with enzyme-linked immunosorbent assay.RESULTS:Diabetic patients showed highly significant decrease in the level of visfatin compared to the control group (P = 0.0001). There was significant further decrease in visfatin level in diabetics with microalbuminuria (n = 13) compared to normoalbuminuric patients (n = 37) (P = 0.015). There was highly significant inverse correlation between visfatin level with age (r = -0.379, p = 0.007), BMI (r = -0.418, p = 0.003), waist circumference (r = -0.430, p = 0.002), hip circumference (r = -0.389, p = 0.005) and microalbuminuria (r = -0.323, p = 0.022).CONCLUSIONS:Type 1 diabetic children and adolescents had a significantly lower visfatin level compared to controls. A marked decrease in the level of visfatin was shown in patients with microalbuminuria with an inverse correlation with BMI suggesting an important role of visfatin in the pathogenesis of type 1 diabetics and type 1 diabetic nephropathy.

Regulation and Function of Extracellular Nicotinamide Phosphoribosyltransferase/Visfatin.

Nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine-enzyme, which was described as to play bioactivities both in the intracellular and in the extracellular environment. However, while the functions of intracellular NAMPT (iNAMPT) are well known, much less is known on extracellular NAMPT (eNAMPT), also called visfatin or pre-B cell colony-enhancing factor. iNAMPT catalyzes the rate-limiting step in the NAD+ biosynthesis pathway from nicotinamide. Its inhibition severely reduces intracellular NAD+ levels, achieving anti-inflammatory and anti-cancer effects. eNAMPT can be detected in the human circulation and in many extracellular environments. Studies show that eNAMPT can act as a growth factor, as an enzyme, and as a cytokine, but its true mechanism of secretion and its physiological functions are still debated. Increased levels of eNAMPT have been associated with different metabolic disorders and cancers. eNAMPT was demonstrated to modulate the pathways involved in the pathophysiology of obesity, diabetes, atherosclerosis, and cardiovascular events by regulating the oxidative stress response, apoptosis, and inflammation. In cancer, eNAMPT was shown to play a pivotal role in modulating cancer cell metabolism, in promoting epithelial-to-mesenchymal transition and in shaping the tumor microenvironment. In line with these functions, circulating eNAMPT levels are frequently increased in cancer patients. Given these pleiotropic roles of eNAMPT in human disease, this protein has attracted attention as a therapeutic target. In this narrative review, we will discuss recent evidence on eNAMPT-driven signalling, highlighting the emerging pathophysiological roles of this protein in different disorders and the potential therapeutic opportunities linked to its targeting. © 2017 American Physiological Society. Compr Physiol 7:603-621, 2017.

Self-Association and Conformational Stability of NAMPT Protein

Nicotinamide phosphoribosyltransferase (NAMPT), also known as Visfatin and Pre B-cell colony enhancing factor (PBEF), is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide (NAD) biosynthesis. It is a highly conserved 52-kDa protein, found in living species from bacteria to humans [1]. The protein serves as a cytokine and is involved in cellular regulation influencing cancer, ischemia, obesity, and type-II diabetes [2, 3]. Despite being involved in many regulatory pathways, there is a paucity of information concerning the function of NAMPT, due to the limitations of in vivo assays, and lack of expression systems for the protein. Here, we successfully expressed the NAMPT protein using the pET-SUMO expression vector in E.coli strain SHuffle containing a hexa-His tag for protein purification. Activity assays demonstrated functionality of the protein. Moreover, initial biophysical characterization of the protein using circular dichroism revealed secondary structural elements consistent with crystallographic data. Dynamic light scattering showed the protein exists as large oligomeric units potentially involved in the NAMPT signal amplification pathway. Hydropathy analysis indicated possible hydrophobic patches on the protein surface that explains the native oligomeric state. Most striking, we discovered that NAMPT can be solubilized in n-dodecyl-β-D-maltopyranoside detergent in monomeric form. These findings open opportunities for further structural and functional investigations. Presently we are optimizing conditions for NMR experiments on NAMPT protein. These methods [4] are complementary to X-ray crystallography, and provide valuable information on the structure and dynamics, offering an important tool for understanding biological functioning.[1] T. Wang et al. (2006) Nat. Struct. Mol. Biol 13, 661.[2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135.[3] A. Fukuhara et al. (2005) Science 307, 426.[4] T. R. Molugu et al. (2016) Chem. Rev. (in press).

Nicotinamide phosphoribosyltransferase inhibits receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation invitro.

The adipokine nicotinamide phosphoribosyltransferase (Nampt), also known as pre-B-cell colony-enhancing factor or the insulin-mimetic hormone visfatin, has a crucial role in the conversion of nicotinamide to nicotinamide mononucleotide during biosynthesis of the coenzyme nicotinamide adenine dinucleotide. Previous reports have demonstrated the inhibitory effects of Nampt on osteoclast formation from human peripheral blood mononuclear cells and CD14+ monocytes. However, the effects of Nampt on bone marrow macrophage (BMM)-derived osteoclastogenesis and its precise role in the process remain unclear. The present in vitro study used recombinant Nampt and BMMs as osteoclast precursors demonstrated that Nampt suppresses receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis by decreasing the phosphorylation of various early signal transducers, including c-Jun N-terminal kinase, Akt, glycogen synthase kinase-3 β, Bruton's tyrosine kinase and phospholipase C γ-2. In addition, western blotting and reverse transcription-quantitative polymerase chain reaction analysis indicated that Nampt downregulates the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1, leading to a decrease in the expression of osteoclast-specific genes including tartrate-resistant acid phosphatase, osteoclast-associated receptor and cathepsin K. However, the bone-resorbing activity of mature osteoclasts treated with Nampt was similar to untreated control osteoclasts. This finding indicates that Nampt exerts its anti-osteoclastogenic activity by targeting osteoclast precursor cells rather than mature osteoclasts. Consequently, the present study demonstrated that Nampt acts as a negative regulator of RANKL-mediated differentiation of BMMs into osteoclasts, suggesting the potential therapeutic targets to treat bone-related disorders such as osteoporosis.

Pre-B-cell colony enhancing factor expression and its clinical significance in colorectal cancer

Objective To explore the expression of pre-B-cell colony enhancing factor (PBEF) in colorectal cancer, and analyze the relationship between PBEF expression and the clinical pathological characte-ristics and its diagnostic and prognostic value. Methods Sixty-eight human colorectal tumor and normal colo-rectal tissues were collected, and the expression of PBEF was detected by immunohistochemical method. The relationship between the expression level of PBEF and the characteristics of tumors and prognosis was analyzed. Results The positive expression rate of PBEF in colorectal cancer tissues was 80.9%, higher than that in normal colorectal tissues, which was 29.4% (χ2=36.41, P 0.05), sex (χ2=0.39, P>0.05), histology type (χ2=0.54, P>0.05) or penetrating serosa (χ2=1.55, P>0.05), but significantly associated with the degree of differentiation (χ2=6.29, P 0.05). Conclusion The abnormal high expression of PBEF may be closely related to the occurrence and development of colorectal cancer. Key words: Colorectal neoplasms; Immunohistochemistry; Pre-B-cell colony enhancing factor

Induction of Apoptosis and Autophagy By Nampt Inhibition in Adult T-Cell Leukemia/Lymphoma and Leukemic Cell Lines

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus type I (HTLV-1). Despite the recent advances in chemotherapy, allogeneic hematopoietic stem cell transplantation, and supportive care, the prognosis for patients with acute, lymphoma, or unfavorable chronic subtypes is one of the poorest among hematological malignancies. The identification of new molecular targets for ATL prevention and treatment is desired. SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, plays crucial roles in various physiological processes, including aging and apoptosis. We previously reported that ATL patients had significantly higher SIRT1 protein levels and novel small-molecule SIRT1 inhibitors are highly effective against ATL cells.1,2 Nicotinamide phosphoribosyltransferase (Nampt) also known as pre-B-cell colony-enhancing factor 1 or visfatin is a rate-limiting enzyme in NAD+ biosynthesis, and it regulates intracellular ATP levels in mammalian cells. Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma) is sensitive to low concentrations of FK866, Nampt inhibitor, as measured in cytotoxicity and clonogenic assays.3Here, we assessed how Nampt is regulated in ATL cells and leukemic cell lines.Results: We observed that ATL patients had significantly higher SIRT1 and Nampt protein levels than healthy controls. FK866 induced significant growth inhibition and apoptosis (Annexin V+ cells and TUNEL) in leukemia/lymphoma cell lines (HTLV-1-related cell lines: S1T, MT-2; Jurkat and HL60). FK866 showed potent activities with GI50values of 0.63, 3.7, 1.0, and 3,4 nM for S1T, MT-2, Jurkat, and HL60 cells, respectively. FK866 also activated caspase activity (caspase-3, 8, and 9) with DNA fragmentation. However, a caspase inhibitor did not inhibit this caspase-dependent cell death. Interestingly, FK866 increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Autophagy detection was also performed using the CytoID Autophagy detection kit. Autophagy levels are increased in the presence of STF-62247 pre-treated with bafilomycin A1, a specific inhibitor of vacuolar proton ATPase, whose inhibition is known to block the fusion of autophagosomes with lysosomes for 2 h. Thus, FK866 simultaneously caused apoptosis and autophagy.Conclusion:These results suggest that Nampt inhibitor is highly effective against ATL cells in caspase-dependent or -independent manners with autophagy, and that its clinical application might improve the prognosis of patients with this fatal disease.1. Kozako T, Aikawa A, Shoji T, et al. High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells. Int J Cancer. 2012;131:2044-2055.2. Kozako T, Suzuki T, Yoshimitsu M, et al. Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells. Sci Rep. 2015;5:11345.3. Nahimana A, Attinger A, Aubry D, et al. The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies. Blood. 2009;113:3276-3286. DisclosuresYoshimitsu:HUYA Bioscience International: Research Funding.

Aberrant Expression of TIMP-2 and PBEF Genes in the Placentae of Cloned Mice Due to Epigenetic Reprogramming Error.

Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2), which is involved in extracellular matrix degradation and tissue remodeling, and downregulation of pre-B cell colony enhancing factor 1 (PBEF), which inhibits apoptosis and induces spontaneous labor. Here, we used Western blotting to further analyze the protein expression levels of TIMP-2 and PBEF in cloned placentae derived from cumulus cells, TSA-treated cumulus cells, intracytoplasmic sperm injection (ICSI), and natural mating (NM control). Cloned and TSA-treated cloned placentae had higher expression levels of TIMP-2 compared with NM control and ICSI-derived placentae, and there was a positive association between TIMP-2 expression and the placental weight of cloned mouse concepti. Conversely, PBEF protein expression was significantly lower in cloned and ICSI placentae compared to NM controls. To examine whether the observed differences were due to abnormal gene expression caused by faulty epigenetic reprogramming in clones, we investigated DNA methylation and histone modification in the promoter regions of the genes encoding TIMP-2 and PBEF. Sodium bisulfite sequencing did not reveal any difference in DNA methylation between cloned and NM control placentae. However, ChIP assays revealed that the level of H3-K9/K14 acetylation at the TIMP-2 locus was higher in cloned placentae than in NM controls, whereas acetylation of the PBEF promoter was lower in cloned and ICSI placenta versus NM controls. These results suggest that cloned placentae appear to suffer from failure of histone modification-based reprogramming in these (and potentially other) developmentally important genes, leading to aberrant expression of their protein products. These changes are likely to be involved in generating the abnormalities seen in cloned mouse placentae, including enlargement and/or a lack of proper placental function.

Abstract 12475: Mechanisms of Akt1-mediated Hypothermia Protection After Cardiac Arrest: Metabolism, Inflammation and Contractile Function

Introduction: Induction of intra-arrest cooling (IC) improves cardiac, neurological and survival outcomes in preclinical models of cardiac arrest. Previous studies demonstrate that IC protection is mediated by Akt1 activation through initiating survival signaling. Hypothesis: We hypothesize that IC-induced Akt1 activation modulates protective calcium cycling proteins, metabolic, and inflammatory pathways. Methods: Adult female Akt1 +/- underwent 8 min of KCl-induced asystolic arrest and were randomized to receive IC (30 ± 0.5°C) or normothermia (NT) before CPR (n = 10 per group). 72 h survival and mean blood pressure (MAP) were measured. Indicators of glucose utilization, inflammatory responses and contractile function in heart and brain of Akt1 +/+ and Akt1 +/- mice were assessed (n=5 per group). Results: No difference on survival was seen for IC and NT treated Akt1 +/- mice. IC induced transient improvement in MAP at 30 min following resuscitation (53.6 ± 2.2 vs 71.2 ± 2.6 mmHg p&lt;0.05) and increased phospholamban phosphorylation in the heart of Akt1 +/+ mice which were not seen in Akt1 +/- mice. Compared to IC-treated Akt1 +/+ , pyruvate dehydrogenase phosphorylation and sorbitol accumulation were significantly increased, and NAD and ATP contents were markedly reduced in the hearts and brains of Akt1 +/- mice. IC significantly inhibited IκBα degradation and NF-κB nuclear translocation, and reduced concentrations of blood lactate and Pre-B cell Colony Enhancing Factor in Akt1 +/+ mice, but not Akt1 +/- mice. Conclusion: Akt1 is a critical mediator for TH protection during early post resuscitation in heart and brain through regulating calcium regulatory proteins, enhancing glucose utilization and downregulating inflammatory responses.

Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia.

We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD+ synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy.

Thermal Denaturation and Domain Stability of NAMPT Protein

Nicotinamide phosphoribosyltransferase (NAMPT) is also known as Pre-B cell colony enhancing factor (PBEF) or visceral fat protein (Visfatin). It is a highly conserved 52-kDa protein, found in living species from bacteria to humans [1], and acts as a rate-limiting step in the salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. NAMPT is involved in several inflammatory deceases notably, sepsis, acute lung injury, and rheumatoid arthritis [2]. It is also known to activate insulin receptor causing type II diabetes [3]. Because of such medicinal relevance, NAMPT has been extensively investigated by X-ray crystallography, aiming at developing inhibitors [1]. However, physiologically relevant biochemical characterization including nuclear magnetic resonance (NMR) spectroscopy for NAMPT is scarce. NMR methods are complementary to X-ray crystallography, and provide valuable information on the structure and functional dynamics simultaneously. They offer an important tool for understanding biological functioning [4]. Bacterial expression, biochemical characterization, and thermal stability studies on NAMPT have been undertaken with the objective of optimizing conditions for NMR experiments. Secondary structure estimates using circular dichroism (CD) spectroscopy are consistent with crystallographic data. Thermal unfolding studies indicated an unusually high degree of thermal stability of NAMPT showing a melting temperature of about 90 °C. Results for dynamic light scattering (DLS) experiments showed a salt-dependent multimeric state. Presently, we are optimizing solvent conditions for obtaining the stable oligomeric state relevant to NMR experiments. Calorimetric studies are also under progress. We successfully expressed NAMPT in E.coli and are developing strategies for better yields and isotope labelling. [1] T. Wang et al. (2006) Nat. Struct. Mol. Biol. 13, 661. [2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135. [3] A. Fukuhara et al. (2005) Science 307, 426. [4] T. R. Molugu et al. (2015) Concepts Magn. Reson. (in press).

Strong association of the polymorphisms in PBEF1 and knee OA risk: a two-stage population-based study in China.

The association of Pre-B cell colony enhancing factor 1 (PBEF1) with obesity, together with its pro-inflammatory properties suggests that PBEF1 might be another crucial mediator that links inflammation with obesity and primary osteoarthritis (OA). We hypothesized that polymorphisms in PBEF1 may modify the risk of developing OA. Thus we systematically screened 4 tagging polymorphisms (rs4730153, rs2058540, rs3801267 and rs16872158) in PBEF1 and evaluated the association between the genetic variants and OA risk in a two-stage case-control study including 196 cases and 442 controls in the first stage and 143 cases and 238 controls in the second stage. In the first stage, two SNPs (rs4730153 and rs16872158) were found to be potentially associated with OA risk (P < 0.05), which were further confirmed in the second stage with similar effects. After combining the two stages, we found that rs4730153 was significantly associated with decreased risk of OA in an additive genetic model (P < 0.05), while rs16872158 showed increased risk of developing OA (P < 0.05). Combined analysis of these 2 SNPs showed a significant allele-dosage association between the number of risk alleles and OA risk (Ptrend = 5.25 × 10−5). These findings indicate that genetic variants in PBEF1 gene may modify individual susceptibility to OA in the Chinese population.

Pre-B-cell colony-enhancing factor as a target for protecting against apoptotic neuronal death and mitochondrial damage in ischemia.

Pre-B-cell colony-enhancing factor as a target for protecting against apoptotic neuronal death and mitochondrial damage in ischemia.

PBEF promotes the apoptosis of pulmonary microvascular endothelial cells and regulates the expression of inflammatory factors and AQP1 through the MAPK pathways.

Pre-B cell colony-enhancing factor (PBEF) has been shown to have a variety of biological functions. Studies have proven that PBEF plays a functional role in acute lung injury (ALI). Therefore, in this study, we aimed to confirm the importance of PBEF in ALI. The effects of PBEF overexpression on the apoptosis of human pulmonary microvascular endothelial cells (HPMECs) were analyzed by flow cytometry, and the results indicated that PBEF promoted the apoptosis of HPMECs, which aggravated the development of ALI. Comparative experiments involving increasing and decreasing PBEF expression demonstrated that PBEF promoted the expression of inflammatory factors, such as interleukin (IL)‑1β, IL‑6 and IL‑8 in the HPMECs , thus intensifying the inflammatory response. PBEF also inhibited the expression of aquaporin 1 (AQP1), which caused a dysfunction and imbalance in water transport. Moreover, we also found that tumor necrosis factor (TNF)‑α promoted the expression of PBEF in the HPMECs. After blocking the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, we found that PBEF regulated the expression of inflammatory factors and AQP1, mainly through the MAPK pathways. Taken together, these results demonstrate that the increase in intracellular PBEF expression promoted the apoptosis of HPMECs and the expression of inflammatory factors and thus enhanced the inflammatory response and inhibited the expression of AQP1, which resulted in abnormal water transport, diminishing the regulatory effects of AQP1 on water transport.