Discovery of proqodine A derivatives with antitumor activity targeting NAD(P)H: quinone oxidoreductase 1 and nicotinamide phosphoribosyltransferase.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavin protease highly expressed in various cancer cells. NQO1 catalyzes a futile redox cycle in substrates, leading to substantial reactive oxygen species (ROS) production. This ROS generation results in extensive DNA damage and elevated poly (ADP-ribose) polymerase 1 (PARP1)-mediated consumption of nicotinamide adenine dinucleotide (NAD+), ultimately causing cell death. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage synthesis pathway, emerges as a critical target in cancer therapy. The concurrent inhibition of NQO1 and NAMPT triggers hyperactivation of PARP1 and intensive NAD+ depletion. In this study, we designed, synthesized, and assessed a novel series of proqodine A derivatives targeting both NQO1 and NAMPT. Among these, compound T8 demonstrated potent antitumor properties. Specifically, T8 selectively inhibited the proliferation of MCF-7 cells and induced apoptosis through mechanisms dependent on both NQO1 and NAMPT. This discovery offers a promising new molecular entity for advancing anticancer research.

Dangua Fang regulating tricarboxylic acid cycle and respiratory chain and its mechanism in diabetic rats.

OBJECTIVE: To investigate the influence and possible targets of Dangua Fang on tricarboxylic acid (TCA) cycle and respiratory chain to enrich the prescription's mechanism of effective intervention on glycolipid metabolic diseases such as type 2 diabetes. METHODS: After interventional rats were fed with high glucose and high fat diet ad libitum for 4 weeks, intraperitoneally injected streptozotocin to induce diabetic model. According to blood glucose level,28 diabetic rats were selected and continued to be fed with high glucose and high fat diet, were stratified by body weight, and divided randomly by blood glucose into Model group (was given sterile water by gastric perfusion and injected aquae pro injection intraperitoneally), Dangua group [Dangua liquor 20.5 g·kg-1·d-1 by perfusion and aquae pro injection intraperitoneally], Inhibitor group [sterile water by perfusion and nicotinamide phosphoribosyl transferase (Nampt) specific blocker GEN-617 1.25 mg/kg intraperitoneally], DanInhit group (Dangua liquor and GEN-617 synchronously). Control group were continuously fed with ordinary diet. The intervention was last for 10 weeks. Body weight (BW), liver index (LI), glycosylated hemoglobin (HbA1c), TC, TG, free fatty acids (FFA), creatinine (Cr), and A-ketoglutarate (α-KG), Iso-citric acid (ICA), oxaloacetic acid (OAA) were tested. The cytochrome C oxidase (COX) and Succinate dehydrogenase (SDH) were evaluated by Colorimetry; Nampt protein, Adenosine triphosphate (ATP) synthase (ATPs), Nicotinamide adenine dinucleotide (NAD+)and its reduced (NADH) in liver were measured by enzyme linked immunosorbent assay. The expressions of Nampt and mitochondrialnadhdehydrogenase-1 (mt-ND1) gene in liver was assessed by real-time polymerase chain reaction. Hepatic tissue staining was also completed. RESULTS: The levels of BW, ICA, α-KG and Nampt-mRNA in the Model group are lower than that in the Normal group (P < 0.05), conversely, liver weight, LI, TC, HbA1c, SDH and ATPs, mt-ND1-mRNA, and Nampt protein in the Model group are higher (P < 0.01, P < 0.05). Compared with Model group, the levels of ICA, Nampt-mRNA and Nampt in Dangua group are significantly increased, and FFA obviously raised (P < 0.01 and P < 0.05); liver weight, BW, SDH are obviously lower, and HbA1c decreased significantly (P < 0.01, P < 0.05). TG, FFA and Nampt protein increased in the DanInhit group, TC, TG, BW obviously increased in the Inhibitor group, but SDH is decreased in both the two groups (P < 0.05, P < 0.01). Compared with Dangua group, DanInhib group has the lower levels of ICA, mt-ND1-mRNA, Nampt-mRNA, and the higher level of BW, LI and HbA1c. In the Inhibitor group, ICA and Nampt protein decreased, BW and LI, HbA1c and TG increased (P < 0.01 or P < 0.05). Tissue staining display that, in the model group there is obvious pathologic changes ie: fibrosis, steatosis and inflammatory cell infiltration. Lesions in the Dangua group are mild, and those of Inhibitor group are more obvious than the Model group, and DanInhit group is intermediately affected compared to Dangua group and Inhibitor group. CONCLUSION: Dangua Fang increases the metabolic flux of TCA cycle and optimizes respiratory chain function by up-regulating Nampt expression.

The role of visfatin/ NAMPT in the regulation of feeding in goldfish (Carassius auratus).

The protein NAMPT (nicotinamide phosphoribosyltransferase, encoded by the NAPMT gene) is present in two forms. The intracellular form of NAMPT (iNAMPT) is the rate-limiting enzyme in a major nicotinamide adenine dinucleotide (NAD) biosynthetic pathway and regulates cellular metabolism. NAMPT is also secreted by cells in the extracellular milieu, and referred to as extracellular NAMPT (eNAMPT or visfatin). In mammals, visfatin has been linked to various metabolic disorders. However, the role of visfatin in regulating energy homeostasis in fish is not known. In this study, we assessed the effects of nutritional status on NAMPT mRNA expression and the effects of visfatin peripheral injections on food intake and the expression of appetite regulators in goldfish. Our results show that NAMPT is widely expressed in peripheral tissues and brain. Fasting induced increases in NAMPT expression in liver but had no effect on either brain or intestine NAMPT expression levels. Intraperitoneal injections of visfatin (400 ng/g) induced an increase in food intake and in expression levels of hepatic leptin and sirtuin1. Visfatin injections decreased intestine CCK and PYY, and telencephalon (but not hypothalamic) orexin and NPY expression levels. Visfatin did not affect plasma glucose levels, intestine ghrelin or brain CART, POMC and AgRP expressions. These data suggest that visfatin/NAMPT might be involved in the regulation of feeding and energy homeostasis in goldfish.

NAMPT reduction-induced NAD+ insufficiency contributes to the compromised oocyte quality from obese mice.

Maternal obesity is associated with multiple adverse reproductive outcomes, whereas the underlying molecular mechanisms are still not fully understood. Here, we found the reduced nicotinamide phosphoribosyl transferase (NAMPT) expression and lowered nicotinamide adenine dinucleotide (NAD+ ) content in oocytes from obese mice. Next, by performing morpholino knockdown assay and pharmacological inhibition, we revealed that NAMPT deficiency not only severely disrupts maturational progression and meiotic apparatus, but also induces the metabolic dysfunction in oocytes. Furthermore, overexpression analysis demonstrated that NAMPT insufficiency induced NAD+ loss contributes to the compromised developmental potential of oocytes and early embryos from obese mice. Importantly, in vitro supplement and in vivo administration of nicotinic acid (NA) was able to ameliorate the obesity-associated meiotic defects and oxidative stress in oocytes. Our results indicate a role of NAMPT in modulating oocyte meiosis and metabolism, and uncover the beneficial effects of NA treatment on oocyte quality from obese mice.

NAMPT single-nucleotide polymorphism rs1319501 and visfatin/NAMPT affect nitric oxide formation, sFlt-1 and antihypertensive therapy response in preeclampsia.

Aim: We examined the relationships between visfatin/NAMPT and nitrite concentrations (a marker of nitric oxide [NO] formation) or sFlt-1 levels in 205 patients with preeclampsia (PE) responsive or nonresponsive to antihypertensive therapy, and whether NAMPT SNPs rs1319501 and rs3801266 affect nitrite concentrations in PE and 206 healthy pregnant women. Patients & methods: Circulating visfatin/NAMPT and sFlt-1 levels were measured by ELISA, and nitrite concentrations by using an ozone-based chemiluminescence assay. Results: In nonresponsive PE patients, visfatin/NAMPT levels were inversely related to nitrite concentrations and positively related to sFlt-1 levels. NAMPT SNP rs1319501 affected nitrite concentrations in nonresponsive PE patients and was tightly linked with NAMPT functional SNPs in Europeans. Conclusion:NAMPT SNP rs1319501 and visfatin/NAMPT affect NO formation, sFlt-1 levels and antihypertensive therapy response in PE.

Plasma level and expression of visfatin in the porcine hypothalamus during the estrous cycle and early pregnancy.

Visfatin appears to be an energy sensor involved in the regulation of female fertility, which creates a hormonal link integrating the control of energy homeostasis and reproduction. This study evaluates the expression levels of visfatin gene and protein in selected areas of the porcine hypothalamus responsible for gonadotropin-releasing hormone synthesis: the mediobasal hypothalamus (MBH) and preoptic area (POA), and visfatin concentrations in the blood plasma. The tissue samples were harvested from gilts on days 2-3, 10-12, 14-16, and 17-19 of the estrous cycle, and on days 10-11, 12-13, 15-16, 27-28 of pregnancy. Visfatin was localized in the cytoplasm and nucleus of cells creating both studied hypothalamic structures. The study demonstrated that visfatin gene and protein expression in MBH and POA depends on hormonal status related to the phase of the estrous cycle or early pregnancy. Blood plasma concentrations of visfatin during the estrous cycle were higher on days 2-3 in relation to other studied phases of the cycle, while during early pregnancy, the highest visfatin contents were observed on days 12-13. This study demonstrated visfatin expression in the porcine hypothalamus and its dependence on the hormonal milieu related to the estrous cycle and early pregnancy.

Dingxin recipe alleviates atherosclerosis injury in ApoE-knockout mice via downregulation of visfatin expression and inhibition of the visfatin-induced inflammatory response.

OBJECTIVE: To further elucidate the mechanism underlying the anti-atherosclerotic effect of Dingxin recipe (DXR). METHODS: Fifty 6-week-old male ApoE-/- mice were randomly divided into the following groups: model, simvastatin (5 mg·kg－1·d－1), DXR low-dose (9.30 g·kg－1·d－1), DXR middle-dose (18.59 g·kg－1·d－1) and DXR high-dose (37.18 g·kg－1·d－1) (n = 10). Ten male C57BL/6J mice were used as the control group. All ApoE-/- mice were fed a high-fat diet (HFD) and the control mice received a common diet. After HFD for 12 weeks, the mice were treated with DXR or simvastatin for another 12 weeks. The expression of inflammatory cytokines and visfatin was determined in serum and atherosclerotic lesions by enzyme-linked immunosorbent assay. Visfatin expression was also assessed in aortic atherosclerotic plaques. Cultured vessel endothelial cells (VECs) were pretreated with DXR sera prior to visfatin. The effects of DXR were analyzed to elucidate its protective mechanism against visfatin-induced inflammation in VECs. RESULTS: DXR regulated blood lipids and reduced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and visfatin expression in ApoE-/- mice, particularly at the higher doses. The areas of atherosclerotic lesions in the DXR groups were significantly smaller than those in the model group. DXR alleviated visfatin-induced VEC injury via downregulation of TNF-α, IL-6, ICAM-1 and VCAM-1 through mitogen-activated protein kinase pathways. CONCLUSION: DXR alleviated atherosclerosis injury via downregulation of visfatin expression and inhibition of the visfatin-induced inflammatory response in VECs.

NAMPT and BMAL1 Are Independently Involved in the Palmitate-Mediated Induction of Neuroinflammation in Hypothalamic Neurons.

Obesity is a prominent metabolic disease that predisposes individuals to multiple comorbidities, including type 2 diabetes mellitus, cardiovascular diseases, and cancer. Elevated circulating levels of fatty acids contribute to the development of obesity, in part, by targeting the hypothalamus. Palmitate, the most abundant circulating saturated fatty acid, has been demonstrated to dysregulate NAMPT and circadian clock proteins, as well as induce neuroinflammation. These effects ultimately result in hypothalamic dysregulation of feeding behavior and energy homeostasis. NAMPT is the rate-limiting enzyme of the NAD+ salvage pathway and its expression is under the control of the circadian clock. NAD+ produced from NAMPT can modulate the circadian clock, demonstrating bidirectional interactions between circadian and metabolic pathways. Using NPY/AgRP-expressing mHypoE-46 neurons as well as the novel mHypoA-BMAL1-WT/F and mHypoA-BMAL1-KO/F cell lines, we studied whether there were any interactions between NAMPT and the core circadian clock protein BMAL1 in the palmitate-mediated induction of neuroinflammation. We report that palmitate altered Nampt, Bmal1, Per2 and the inflammatory genes Nf-κb, IκBα, Il-6, and Tlr4. Contrary to studies performed with peripheral tissues, the palmitate-mediated induction in Nampt was independent of BMAL1, and basal Nampt levels did not appear to exhibit rhythmic expression. Palmitate-induced downregulation of Bmal1 and Per2 was independent of NAMPT. However, NAMPT and BMAL1 were both involved in the regulation of Nf-κb, IκBα, Il-6, and Tlr4, as NAMPT inhibition resulted in the repression of basal Nf-κb and IκBα and normalized palmitate-mediated increases in Il-6, and Tlr4. On the other hand, BMAL1 deletion repressed basal Nf-κb, but increased basal Il-6. We conclude that NAMPT and BMAL1 do not interact at the transcriptional level in hypothalamic neurons, but are independently involved in the expression of inflammatory genes.

Bioactive fractions from Securidaca inappendiculata alleviated collagen-induced arthritis in rats by regulating metabolism-related signaling.

Securidaca inappendiculata is a xanthone rich medicinal plant that has been used in the treatment of inflammation and autoimmune diseases like rheumatoid arthritis (RA) for centuries; however, the material base and mechanism of action responsible for its anti-arthritis effect still remains elusive. The objective of this study is to evaluate the therapeutic effects of xanthone-enriched extract of the plant against collagen-induced arthritis (CIA) in rats and explore the underlying mechanisms. The xanthone-deprived fraction (XDF) and xanthone-rich fraction (XRF) were obtained by using a resin adsorption coupled with acid-base treatment method, and their chemical composition difference was characterized by UPLC-MS/MS analysis. Effects of the two on CIA were analyzed using radiographic, histological, and immunohistochemical analyses. The results indicated that XRF alleviated joint structures destructions with the higher efficacy than XDF, and decreased levels of TNF-α, IL-6, and anti-cyclic citrullinated peptide antibody in CIA rats significantly. Furthermore, XRF inhibited nicotinamide phosphoribosyl transferase (NAMPT) mediated fat biosynthesis and utilization indicated by clinical evidences and metabonomics analysis, which thereby disrupted energy-metabolism feedback. In addition, Toll-like Receptor 4 and High Mobility Group Protein 1 expressions were downregulated in XRF-treated CIA rats. Collective evidences suggest NAMPT could be an ideal target for RA treatments and reveal a novel antirheumatic mechanism of S. inappendiculata by regulating NAMPT controlled fat metabolism.

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.

Nicotinamide riboside induces a thermogenic response in lean mice.

AIMS: Nicotinamide Riboside (NR) is a NAD+ booster with wide physiological repercussion including the improvement on glucose and lipid homeostasis, increasing the life expectancy in mammals. However, the effects of NR on metabolism are only partially known. Here, we evaluated the effects of NR on the thermogenic response, highlighting the brown adipose tissue (BAT) in lean mice. MAIN METHODS: Male C57BL/67 mice were supplement with NR (400 mg/Kg/day) during 5 weeks. The Comprehensive Lab Animal Monitoring System (CLAMS) and thermographic images were used to evaluated the physiological effects of NR treatment. The BAT were extracted and analyzed by Western Blotting and qPCR. Also, bioinformatics analyses were performed to establish the connection between the NAD+ synthesis pathway in BAT and thermogenic response in several isogenic strains of BXD mice. KEY FINDINGS: Transcriptomic analysis revealed that genes involved in NAD+ synthesis (Nampt and Nmnat1) in the BAT were negatively correlated with body weight and fat mass. The heat map showed a strong positive correlation between Nampt and Ucp1 mRNA in BAT and body temperature in several strains of BXD lean mice. The experimental approaches demonstrated that oral NR supplementation reduced the abdominal visceral fat depots, with discrete impact on oxygen consumption in C57BL/6J mice. Interestingly, NR significantly increased the body temperature, and this phenomenon was accompanied by high levels of UCP1 protein content and Pgc1α mRNA in BAT. SIGNIFICANCE: This study demonstrated the oral NR supplementation was sufficient to induce the thermogenic response in lean mice changing the BAT metabolism.

Suppressive effects of berberine on atherosclerosis via downregulating visfatin expression and attenuating visfatin-induced endothelial dysfunction.

Berberine (BBR) possesses significant anti-atherosclerosis properties. Visfatin is one of the most promising biomarkers of incoming atherosclerosis. However, research on the effect of BBR on regulating visfatin expression in atherogenesis remains largely unknown. In this study, we investigated the effects of BBR on visfatin expression and atherogenesis in apolipoprotein E knockout (ApoE-/-) mice. The effect of BBR on attenuating visfatin-induced endothelial dysfunction was also evaluated in cultured human umbilical vein endothelial cells (HUVECs). In vivo experiments showed that BBR treatment (5 mg/kg/day) significantly reduced the serum levels of visfatin, lipid, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the protein expression of visfatin, p-p38 MAPK and p-c-Jun N-terminal kinase (JNK) in mice aorta and the distribution of visfatin in the atherosclerotic lesions in ApoE-/- mice fed with a Western diet. In addition, in vitro experiments indicated that visfatin (100 µg/l) significantly increased apoptosis, the contents of IL-6 and TNF-α, the protein levels of p-p38 MAPK, p-JNK and Bax in HUVECs, which were reversed by BBR administration (50 µmol/l). Our findings suggest that BBR significantly ameliorates Western diet-induced atherosclerosis in ApoE-/- mice via downregulating visfatin expression, which is related to the inhibition of p38 MAPK and JNK signaling pathways and subsequent suppression of visfatin-induced endothelial dysfunction.

Nicotinamide Phosphoribosyltransferase Deficiency Potentiates the Antiproliferative Activity of Methotrexate through Enhanced Depletion of Intracellular ATP.

Lower plasma nicotinamide phosphoribosyltransferase (NAMPT) levels are associated with improved response to methotrexate (MTX) in patients with juvenile idiopathic arthritis. Cell-based studies confirmed that reduced cellular NAMPT activity potentiates the pharmacologic activity of MTX; however, the mechanism of this interaction has yet to be defined. Therefore, in this study, we investigate the mechanism of enhanced pharmacologic activity of MTX in NAMPT-deficient A549 cells. Small interfering RNA-based silencing of NAMPT expression resulted in a greater than 3-fold increase in sensitivity to MTX (P < 0.005) that was completely reversed by supplementation with folinic acid. Despite a 68% reduction in cellular NAD levels in NAMPT-deficient cells, no change in expression or activity of dihydrofolate reductase was observed and uptake of MTX was not significantly altered. MTX did not potentiate the depletion of cellular NAD levels, but NAMPT-deficient cells had significant elevations in levels of intermediates of de novo purine biosynthesis and were 4-fold more sensitive to depletion of ATP by MTX (P < 0.005). Supplementation with hypoxanthine and thymidine completely reversed the antiproliferative activity of MTX in NAMPT-deficient cells and corresponded to repletion of the cellular ATP pool without any effect on NAD levels. Together, these findings demonstrate that increased MTX activity with decreased NAMPT expression is dependent on the antifolate activity of MTX and is driven by enhanced sensitivity to the ATP-depleting effects of MTX. For the first time, these findings provide mechanistic details to explain the increase in pharmacological activity of MTX under conditions of reduced NAMPT activity.

Fragment-based discovery of a potent NAMPT inhibitor.

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.

Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy.

BACKGROUND: Myocardial metabolic impairment is a major feature in chronic heart failure. As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress and oxidative stress response, nicotinamide adenine dinucleotide (NAD+) is emerging as a metabolic target in a number of diseases including heart failure. Little is known on the mechanisms regulating homeostasis of NAD+ in the failing heart. METHODS: To explore possible alterations of NAD+ homeostasis in the failing heart, we quantified the expression of NAD+ biosynthetic enzymes in the human failing heart and in the heart of a mouse model of dilated cardiomyopathy (DCM) triggered by Serum Response Factor transcription factor depletion in the heart (SRFHKO) or of cardiac hypertrophy triggered by transverse aorta constriction. We studied the impact of NAD+ precursor supplementation on cardiac function in both mouse models. RESULTS: We observed a 30% loss in levels of NAD+ in the murine failing heart of both DCM and transverse aorta constriction mice that was accompanied by a decrease in expression of the nicotinamide phosphoribosyltransferase enzyme that recycles the nicotinamide precursor, whereas the nicotinamide riboside kinase 2 (NMRK2) that phosphorylates the nicotinamide riboside precursor is increased, to a higher level in the DCM (40-fold) than in transverse aorta constriction (4-fold). This shift was also observed in human failing heart biopsies in comparison with nonfailing controls. We show that the Nmrk2 gene is an AMP-activated protein kinase and peroxisome proliferator-activated receptor α responsive gene that is activated by energy stress and NAD+ depletion in isolated rat cardiomyocytes. Nicotinamide riboside efficiently rescues NAD+ synthesis in response to FK866-mediated inhibition of nicotinamide phosphoribosyltransferase and stimulates glycolysis in cardiomyocytes. Accordingly, we show that nicotinamide riboside supplementation in food attenuates the development of heart failure in mice, more robustly in DCM, and partially after transverse aorta constriction, by stabilizing myocardial NAD+ levels in the failing heart. Nicotinamide riboside treatment also robustly increases the myocardial levels of 3 metabolites, nicotinic acid adenine dinucleotide, methylnicotinamide, and N1-methyl-4-pyridone-5-carboxamide, that can be used as validation biomarkers for the treatment. CONCLUSIONS: The data show that nicotinamide riboside, the most energy-efficient among NAD precursors, could be useful for treatment of heart failure, notably in the context of DCM, a disease with few therapeutic options.

Bioenergetic adaptations of the human liver in the ALPPS procedure - how liver regeneration correlates with mitochondrial energy status.

BACKGROUND: The Associating Liver Partition and Portal Ligation for Staged Hepatectomy (ALPPS) depends on a significant inter-stages kinetic growth rate (KGR). Liver regeneration is highly energy-dependent. The metabolic adaptations in ALPPS are unknown. AIMS: i) Assess bioenergetics in both stages of ALPPS (T1 and T2) and compare them with control patients undergoing minor (miHp) and major hepatectomy (MaHp), respectively; ii) Correlate findings in ALPPS with volumetric data; iii) Investigate expression of genes involved in liver regeneration and energy metabolism. METHODS: Five patients undergoing ALPPS, five controls undergoing miHp and five undergoing MaHp. Assessment of remnant liver bioenergetics in T1, T2 and controls. Analysis of gene expression and protein content in ALPPS. RESULTS: Mitochondrial function was worsened in T1 versus miHp; and in T2 versus MaHp (p < 0.05); but improved from T1 to T2 (p < 0.05). Liver bioenergetics in T1 strongly correlated with KGR (p < 0.01). An increased expression of genes associated with liver regeneration (STAT3, ALR) and energy metabolism (PGC-1α, COX, Nampt) was found in T2 (p < 0.05). CONCLUSION: Metabolic capacity in ALPPS is worse than in controls, improves between stages and correlates with volumetric growth. Bioenergetic adaptations in ALPPS could serve as surrogate markers of liver reserve and as target for energetic conditioning.

NAMPT inhibition synergizes with NQO1-targeting agents in inducing apoptotic cell death in non-small cell lung cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for a number of enzymes and regulatory proteins involved in a variety of cellular processes, including deacetylation enzyme SIRT1 which modulates several tumor suppressors such as p53 and FOXO. Herein we report that NQO1 substrates Tanshione IIA (TSA) and ß-lapachone (ß-lap) induced a rapid depletion of NAD(+) pool but adaptively a significant upregulation of NAMPT. NAMPT inhibition by FK866 at a nontoxic dose significantly enhanced NQO1-targeting agent-induced apoptotic cell death. Compared with TSA or ß-lap treatment alone, co-treatment with FK866 induced a more dramatic depletion of NAD(+), repression of SIRT1 activity, and thereby the increased accumulation of acetylated FOXO1 and the activation of apoptotic pathway. In conclusion, the results from the present study support that NAMPT inhibition can synergize with NQO1 activation to induce apoptotic cell death, thereby providing a new rationale for the development of combinative therapeutic drugs in combating non-small lung cancer.

Preclinical models of nicotinamide phosphoribosyltransferase inhibitor-mediated hematotoxicity and mitigation by co-treatment with nicotinic acid.

Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.

SIRT1-Mediated eNAMPT Secretion from Adipose Tissue Regulates Hypothalamic NAD+ and Function in Mice.

Nicotinamide phosphoribosyltransferase (NAMPT), the key NAD(+) biosynthetic enzyme, has two different forms, intra- and extracellular (iNAMPT and eNAMPT), in mammals. However, the significance of eNAMPT secretion remains unclear. Here we demonstrate that deacetylation of iNAMPT by the mammalian NAD(+)-dependent deacetylase SIRT1 predisposes the protein to secretion in adipocytes. NAMPT mutants reveal that SIRT1 deacetylates lysine 53 (K53) and enhances eNAMPT activity and secretion. Adipose tissue-specific Nampt knockout and knockin (ANKO and ANKI) mice show reciprocal changes in circulating eNAMPT, affecting hypothalamic NAD(+)/SIRT1 signaling and physical activity accordingly. The defect in physical activity observed in ANKO mice is ameliorated by nicotinamide mononucleotide (NMN). Furthermore, administration of a NAMPT-neutralizing antibody decreases hypothalamic NAD(+) production, and treating ex vivo hypothalamic explants with purified eNAMPT enhances NAD(+), SIRT1 activity, and neural activation. Thus, our findings indicate a critical role of adipose tissue as a modulator for the regulation of NAD(+) biosynthesis at a systemic level.

Dietary proanthocyanidins modulate melatonin levels in plasma and the expression pattern of clock genes in the hypothalamus of rats.

SCOPE: Circadian rhythms allow organisms to anticipate and adapt to environmental changes, and food components can adjust internal rhythms. Proanthocyanidins improve cardiovascular risk factors that exhibit circadian oscillations. Therefore, the aim of the current study was to determine whether proanthocyanidins can modulate body rhythms. METHODS AND RESULTS: Male Wistar rats were orally gavaged with 250 mg grape seed proanthocyanidin extract (GSPE)/kg body weight at zeitgeber time (ZT) 0 (light on). Phenotypic biorhythm was evaluated by measuring the concentration of plasma melatonin and metabolites, using MNR-metabolomics, at several ZT. Remarkably, GSPE treatment maintained nocturnal melatonin levels at ZT3 and altered the oscillations of some metabolites in plasma. Quantification of expression of clock-core (Clock, Bmal1, Per2, Rorα, Rev-erbα) and clock-controlled (Nampt) genes in the hypothalamus by RT-PCR showed that this phenotypic alteration was concomitant with the modulation of the expression pattern of Bmal1 and Nampt. However, GSPE did not modulate the nocturnal expression of clock genes when administered at ZT12 (light off). CONCLUSION: PAs could have chronobiological properties, although their activity depends largely on the time of administration.