Tipepidine activates AMPK and improves adipose tissue fibrosis and glucose intolerance in high-fat diet-induced obese mice.

Tipepidine (3-[di-2-thienylmethylene]-1-methylpiperidine) (TP) is a non-narcotic antitussive used in Japan. Recently, the potential application of TP in the treatment of neuropsychiatric disorders, such as depression and attention deficit hyperactivity disorder, has been suggested; however, its functions in energy metabolism are unknown. Here, we demonstrate that TP exhibits a metabolism-improving action. The administration of TP reduced high-fat diet-induced body weight gain in mice and lipid accumulation in the liver and increased the weight of epididymal white adipose tissue (eWAT) in diet-induced obese (DIO) mice. Furthermore, TP inhibited obesity-induced fibrosis in the eWAT. We also found that TP induced AMP-activated protein kinase (AMPK) activation in the eWAT of DIO mice and 3T3-L1 cells. TP-induced AMPK activation was abrogated by the transfection of liver kinase B1 siRNA in 3T3-L1 cells. The metabolic effects of TP were almost equivalent to those of metformin, an AMPK activator that is used as a first-line antidiabetic drug. In summary, TP is a potent AMPK activator, suggesting its novel role as an antidiabetic drug owing to its antifibrotic effect on adipose tissues.

Sulconazole induces pyroptosis promoted by interferon-γ in monocyte/macrophage lineage cells.

Imidazole derivatives are commonly used as antifungal agents. Here, we aimed to investigate the functions of imidazole derivatives on macrophage lineage cells. We assessed the expression levels of inflammatory cytokines in mouse monocyte/macrophage lineage (RAW264.7) cells. All six imidazole derivatives examined, namely ketoconazole, sulconazole, isoconazole, luliconazole, clotrimazole, and bifonazole, reduced the expression levels of inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor-α, after induction by lipopolysaccharide (LPS) in RAW264.7 cells. These imidazole derivatives also induced cell death in RAW264.7 cells, regardless of the presence or absence of LPS. Since the cell death was characteristic in morphology, we investigated the mode of the cell death. An imidazole derivative, sulconazole, induced gasdermin D degradation together with caspase-11 activation, namely, pyroptosis in RAW264.7 cells and peritoneal macrophages. Furthermore, priming with interferon-γ promoted sulconazole-induced pyroptosis in RAW264.7 cells and macrophages and reduced the secretion of the inflammatory cytokine, IL-1ß, from sulconazole-treated macrophages. Our results suggest that imidazole derivatives suppress inflammation by inducing macrophage pyroptosis, highlighting their modulatory potential for inflammatory diseases.

Photoexcited Carrier Transfer in CuInS2 Nanocrystal Assembly by Suppressing Resonant-Energy Transfer.

High-density assemblies or superlattice structures composed of colloidal semiconductor nanocrystals have attracted attention as key materials for next-generation photoelectric conversion devices such as quantum-dot solar cells. In these nanocrystal solids, unique transport and optical phenomena occur due to quantum coupling of localized energy states, charge-carrier hopping, and electromagnetic interactions among closely arranged nanocrystals. In particular, the photoexcited carrier dynamics in nanocrystal solids is important because it significantly affects various device parameters. In this study, we report the photoexcited carrier dynamics in a solid film of CuInS2 nanocrystals, which is one of the potential nontoxic substitutes with Cd- and Pb-free compositions. Meanwhile, these subjects have been extensively studied in nanocrystal solids formed by CdSe and PbS systems. A carrier-hopping mechanism was confirmed using temperature-dependent photoluminescence spectroscopy, which yielded a typical value of the photoexcited carrier-transfer rate of (2.2±0.6)×107  s-1 by suppressing the influence of the excitation-energy transfer.

Effects of Citrus kawachiensis Peel in Frailty-like Model Mice Induced by Low Protein Nutrition Disorders.

"Frailty" caused by a decline in physiological reserve capacity, chronic inflammation, and oxidative stress in the elderly has recently become a major social issue. The present study examined the effects of the peel of Citrus kawachiensis (CK), which exhibits anti-inflammatory, antioxidant, and pro-neurogenesis activities in frailty-like model mice. Male C57BL/6 mice (15 weeks old) were fed an 18% protein diet (CON), a 2.5% protein diet (PM), and PM mixed with 1% dried CK peel powder for approximately 1 month. Mice were euthanized 2 or 8 days after a single intraperitoneal administration of lipopolysaccharide (LPS) and tissues were dissected. Among peripheral tissues, muscle weight, liver weight, and blood glucose levels were significantly higher in the PM-LPS-CK group than in the PM-LPS group. In the behavioral analysis, locomotive activity was significantly lower in the PM-LPS group than in the PM group. The reduction in locomotive activity in the PM-LPS-CK group was significantly smaller than that in the PM-LPS group. The quantification of microglia in the hippocampal stratum lacunosum-moleculare revealed that increases in the PM-LPS group were significantly suppressed by the dried CK peel powder. Furthermore, the quantification of synaptic vesicle membrane proteins in the hippocampal CA3 region showed down-regulated expression in the PM-LPS group, which was significantly ameliorated by the administration of the dried CK peel powder. Collectively, these results suggest that CK inhibits inflammation and oxidative stress induced by PM and LPS in the central nervous system and peripheral tissue. Therefore, C. kawachiensis is highly effective against "frailty".

N-Caffeoyltryptophan enhances adipogenic differentiation in preadipocytes and improves glucose tolerance in mice.

Coffee consumption has been shown to reduce the risk of developing type 2 diabetes mellitus (T2DM) in humans; however, the exact mechanism is not completely understood. Here, we demonstrate that N-caffeoyltryptophan (CTP), an ingredient of coffee, enhances adipogenic differentiation and promotes glucose uptake into adipocytes. CTP increased lipid accumulation and adipogenic markers (PPARγ, C/EBPα, and FABP4) expression in mouse 3T3-L1 preadipocyte cell lines and primary preadipocytes. In addition, CTP promoted glucose uptake in 3T3-L1 cells. In the oral glucose tolerance test, daily administration of CTP (30 mg/kg/day, i.p.) for a week reduced blood glucose levels in mice. In 3T3-L1 cells, adipogenic differentiation and increased adipogenic markers expression induced by CTP were inhibited by U0126, a selective MEK1/2 inhibitor. Furthermore, mRNA induction of Pparg by CTP was abrogated in SIRT1 siRNA-transfected 3T3-L1 cells. These results suggest the involvement of the MEK/ERK signaling and SIRT1 in the mechanism of adipogenic function of CTP. Taken together, CTP might contribute to the reduction in postprandial glycemia and a subsequent reduction in onset risk for T2DM.

Ibudilast Reduces IL-6 Levels and Ameliorates Symptoms in Lipopolysaccharide-Induced Sepsis Mice.

In Japan, ibudilast (IBD) is a therapeutic agent used to treat asthma, allergic conjunctivitis, and dizziness caused by cerebrovascular disease. Previously, we have reported that IBD could reduce the secretion of proinflammatory cytokines, including interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in lipopolysaccharide (LPS)-treated RAW264.7 monocyte-linage cells in vitro. In the present study, we examined the anti-inflammatory effects of IBD in vivo. As IL-6 is a biomarker for sepsis and has been suggested to exacerbate symptoms, we determined whether IBD reduces IL-6 levels in vivo and improves sepsis symptoms in animal models. We observed that IBD treatment reduced IL-6 levels in the lungs of LPS-treated mice and improved LPS-induced hypothermia, one of the symptoms of sepsis. In addition, IBD reduced IL-6 and attenuated plasminogen activator inhibitor-1 (PAI-1) and alanine aminotransferase (ALT) levels in the serum of LPS-treated mice. Elevated PAI-1 levels exacerbate sepsis-induced disseminated intravascular coagulation (DIC), and ALT is a biomarker for liver dysfunction. IBD improved the survival of mice administered a lethal dose of LPS. IBD administration ameliorated kidney pathology of model mice. Overall, these results suggest that IBD exerts anti-inflammatory functions in vivo and could be a drug candidate for treating endotoxemia, including sepsis.

Cynandione A causes a dynamic change in SIRT1 nuclear trafficking via PKA signaling and beige adipocyte differentiation in 3T3-L1 cells.

Inducible brown-like adipocytes, also known as beige adipocytes, dissipate energy through thermogenesis. Although recent reports suggest that silent information regulator 2 homolog 1 (SIRT1) promotes beige adipocyte differentiation (beiging), the activation mechanism of SIRT1 remains unknown. Here, we report that cynandione A (CA), a major component of Cynanchum wilfordii, causes dynamic changes in SIRT1 nuclear trafficking via protein kinase cAMP-dependent (PKA) signaling and induces the beiging process in adipocyte lineage cells. SIRT1 is located in both the cytoplasm and the nucleus of 3T3-L1 cells. Using cell fractionation and RNA interference experiments, we found that the translocation of SIRT1 from the cytoplasm to the nucleus was enhanced after CA treatment and was followed by upregulation of beige adipocyte-related gene expression. Moreover, we found that CA-induced SIRT1 nuclear trafficking is dependent on the PKA signaling pathway. These results suggest a novel mechanism of CA by which PKA signaling promotes SIRT1 nuclear trafficking, which permits the docking of SIRT1 to its nuclear substrates, leading to beiging in 3T3-L1 cells.

Oenothein B, a Bioactive Ellagitannin, Activates the Extracellular Signal-Regulated Kinase 2 Signaling Pathway in the Mouse Brain.

(1) Background: Oenothein B, a cyclic dimeric ellagitannin present in various medicinal plants, has been reported to exert diverse effects that are beneficial for the treatment and prevention of diseases, including cancer and infections. We recently showed that oenothein B also functions in the brain because its oral administration to systemic inflammatory model mice reduced inflammatory responses in the brain and suppressed abnormal behavior. (2) Results: The present in vivo results demonstrated that oenothein B activated extracellular signal-regulated kinase 2 and cAMP response element-binding protein in the brain, both of which play important roles in synaptic transmission and learning/memory in the central nervous system (CNS). (3) Conclusions: These results suggest that oenothein B exerts neuroprotective effects on the CNS by not only its anti-inflammatory activity but also by enhancing neuronal signaling pathways.

[The Search for Citrus Components with Neuroprotective and Anti-dementia Effects in the Mouse Brain].

Citrus kawachiensis (Kawachi Bankan), is a citrus species grown in Ehime, Japan, and its peel is abundant in 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF). We have recently revealed that HMF, one of the citrus flavonoids, has anti-inflammatory activity and neuroprotective abilities in the brain against global cerebral ischemia. HMF rescued neuronal cell death in the hippocampus and suppressed the activation of microglia, whose activation have been shown to significantly aggravate neurological deficit scores for ischemic mice. In the Y-maze test, HMF showed protection against ischemia-induced short-term memory dysfunction; in addition, HMF enhanced the expression of brain-derived neurotrophic factor and accelerated neurogenesis in the hippocampus. Similarly, the powder of the peel of C. kawachiensis showed anti-inflammatory activity and neuroprotective abilities in the ischemic brain. To further examine the effect of the peel of C. kawachiensis, we administered it to senescence-accelerated-mouse prone 8 (SAMP8) mice, which show memory impairment and brain inflammation, tau hyperphosphorylation, and neuronal dysfunction. The C. kawachiensis treatment inhibited microglial activation and the hyperphosphorylation of tau protein in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in SAMP8. These results suggest that HMF and the peel of C. kawachiensis have potential effects as neuroprotective and anti-dementia agents.

[Mapping the Metallo-maze to Memory Loss: Does Neuronal Metal Ion Deficiency Contribute to Dementia?]

Dementia has no cure and is an international health crisis. In addition to the immeasurable loss of QOL caused by dementia, the global economic cost is predicted to reach $2 trillion (USD) by 2030. Although much remains unknown about the biochemical pathways driving cognitive decline and memory loss during dementia, metals have been implicated in neurodegenerative disease. For example, total levels of Fe and Cu increase, which has been proposed to drive oxidative stress; and Fe, Cu, and Zn can bind amyloid-ß, catalysing aggregation and formation of amyloid plaques. Unfortunately, despite these known facets through which metal ions may induce pathology, studies in greater detail have been hampered by a lack of microscopy methods to directly visualise metal ions, and their chemical form, within brain cells. Herein we report the use of synchrotron X-ray fluorescence microscopy to simultaneously image Fe, Cu, and Zn within neurons in ex vivo brain tissue sections. Using animal models of dementia, we now demonstrate for the first time that despite global increases in brain metal content and metal ion accumulation within amyloid plaques, key brain regions may also become metal ion deficient. Such deficiency could contribute to cognitive decline because of the essential roles metal ions play in neurotransmitter synthesis and energy metabolism. These recent findings are discussed in the context of memory loss, and the impact that metal ion dis-homeostasis may have on diagnostic and therapeutic development.

T-Cell Activation-Inhibitory Assay to Screen Caloric Restriction Mimetics Drugs for Drug Repositioning.

We previously reported a screening method for caloric restriction mimetics (CRM), a group of plant-derived compounds capable of inducing good health and longevity. In the present study, we explored the possibility of using this method to screen CRM drugs for drug repositioning. The method, T-cell activation-inhibitory assay, is based on inductive logic. Most of CRM such as resveratrol have been reported to suppress T-cell activation and have anti-inflammatory functions. Here, we assessed the activity of 12 antiallergic drugs through T-cell activation-inhibitory assay and selected four that showed the lowest IC50 values-ibudilast (IC50 0.97 µM), azelastine (IC50 7.2 µM), epinastine (IC50 16 µM), and amlexanox (IC50 33 µM)-for further investigation. Because azelastine showed high cytotoxicity, we selected only the remaining three drugs to study their biological functions. We found that all the three drugs suppressed the expression of interleukin (IL)-6, an inflammatory cytokine, in lipopolysaccharide-treated macrophage cells, with ibudilast being the strongest suppressor. Ibudilast also suppressed the secretion of another inflammatory cytokine, tumor necrosis factor (TNF)-α, and the expression of an inflammatory enzyme, cyclooxygenase-2, in the cells. These results suggest that T-cell activation-inhibitory assay can be used to screen potential CRM drugs having anti-inflammatory functions for the purpose of drug repositioning.

Isolation and Characterization of Neuroprotective Components from Citrus Peel and Their Application as Functional Food.

The elderly experience numerous physiological alterations. In the brain, aging causes degeneration or loss of distinct populations of neurons, resulting in declining cognitive function, locomotor capability, etc. The pathogenic factors of such neurodegeneration are oxidative stress, mitochondrial dysfunction, inflammation, reduced energy homeostatis, decreased levels of neurotrophic factor, etc. On the other hand, numerous studies have investigated various biologically active substances in fruit and vegetables. We focused on the peel of citrus fruit to search for neuroprotective components and found that: 1) 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) and auraptene (AUR) in the peel of Kawachi Bankan (Citrus kawachiensis) exert neuroprotective effects; 2) both HMF and AUR can pass through the blood-brain barrier, suggesting that they act directly in the brain; 3) the content of AUR in the peel of K. Bankan was exceptionally high, and consequently the oral administration of the dried peel powder of K. Bankan exerts neuroprotective effects; and 4) intake of K. Bankan juice, which was enriched in AUR by adding peel paste to the raw juice, contributed to the prevention of cognitive dysfunction in aged healthy volunteers. This review summarizes our studies in terms of the isolation/characterization of HMF and AUR in K. Bankan peel, analysis of their actions in the brain, mechanisms of their actions, and trials to develop food that retains their functions.

[Neuroprotective effects of the peel of Citrus kawachiensis (Kawachi Bankan) and auraptene in the hippocampus of hyperglycemia mice and global cerebral ischemia/reperfusion injury mice].

The peel of Citrus kawachiensis (Kawachi Bankan), a citrus species grown in Ehime, Japan, is abundant in auraptene. Auraptene, a coumarin compound, have been shown to exert anti-inflammatory effects in peripheral tissues, but it was still unclear of the effect in the brain. Hyperglycemia and brain ischemia induce inflammation and oxidative stress and cause massive damage in the brain; therefore, we examined the anti-inflammatory and other effects of the dried peel powder of C. kawachiensis and auraptene in a hyperglycemia and global cerebral ischemia models. The C. kawachiensis treatment inhibited astroglial activation in the hippocampus and the hyperphosphorylation of tau protein in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in the type 2 diabetic db/db mice. The C. kawachiensis treatment inhibited microglial and astroglial activation, and neuronal cell death in the hippocampus of transient global cerebral ischemia mice. It was suggested that the dried peel powder of C. kawachiensis exerts anti-inflammatory and neuroprotective effects in the brain. We attempted to demonstrate the effect of auraptene in the brains in streptozotocin-induced hyperglycemic mice and transient global cerebral ischemia mice. Auraptene administration showed the similar effects as the peel of C. kawachiensis in the hippocampus of these mice models. These results suggested that auraptene have potential effects as a neuroprotective agent in the peel of C. kawachiensis.

Citrus Auraptene Induces Expression of Brain-Derived Neurotrophic Factor in Neuro2a Cells.

(1) Background: Our published data have indicated that 1) auraptene (AUR), a citrus ingredient, has neuroprotective effects on the mouse brain, owing to its ability to suppress inflammation, such as causing a reduction in hyperactivation of microglia and astrocytes; 2) AUR has the ability to trigger phosphorylation (activation) of extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in neuronal cells; 3) AUR has the ability to induce glial cell line-derived neurotrophic factor (GDNF) synthesis/secretion in rat C6 glioma cells. The well-established fact that the ERK-CREB pathway plays an important role in the production of neurotrophic factors, including GDNF and brain-derived neurotrophic factor (BDNF), prompted us to investigate whether AUR would also have the ability to induce BDNF expression in neuronal cells. (2) Methods: Mouse neuroblastoma neuro2a cells were cultured and the effects of AUR on BDNF mRNA expression and protein content were evaluated by RT-PCR and ELISA, respectively. (3) Results: The levels of BDNF mRNA and secreted BDNF were significantly increased by AUR in a dose- and time-dependent manner in neuro2a cells. (4) Conclusion: The induction of BDNF in neuronal cells might be, in part, one of the mechanisms accounting for the neuroprotective effects of AUR.

Identification of the characteristic components in walnut and anti-inflammatory effect of glansreginin A as an indicator for quality evaluation.

Walnut is a nutritious food material, but only a few studies have been conducted on the mechanisms of its functions and the technique for quality evaluation. Therefore, we analyzed the components in aqueous methanol extract of walnut, and characterized 30 components, including three new compounds, glansreginin C, ellagic acid 4-O-(3'-O-galloyl)-ß-D-xyloside, and platycaryanin A methyl ester. We analyzed the extracts of other nuts using HPLC and clarified that a characteristic peak corresponding to glansreginin A was mainly observed in walnut. These results suggested that glansreginin A might be an indicator component of the quality of walnut. We then examined whether glansreginin A has neuroprotective effect, using lipopolysaccharide (LPS)-induced inflammatory model mice. The results revealed that oral administration of glansreginin A prevented LPS-induced abnormal behavior and LPS-induced hyper-activation of microglia in the hippocampus. These results suggested that glansreginin A has the ability to exert neuroprotective effect via anti-inflammation in the brain.

T-cell activation-inhibitory assay: a proposed novel method for screening caloric restriction mimetics.

Caloric restriction (CR) is a major contributor to good health and longevity. CR mimetics (CRMs) are a group of plant-derived compounds capable of inducing the benefits of CR. Since a longevity gene, SIRT1, inhibits T-cell activation and SIRT1 loss results in increased T-cell activation, we hypothesized that compounds capable of activating SIRT1 signaling can inhibit T-cell activation and function as CRMs. Thus we propose, in the present study, the application of a T-cell activation-inhibitory assay to screen candidate CRMs. Well-known CRMs, such as resveratrol, butein, and fisetin, suppressed the anti-CD3/CD28 antibody-induced activation of mouse spleen T-cells. We next randomly assessed 68 plant-derived compounds for screening novel candidate CRMs using this bioassay and found that all four compounds showing IC50 values <5 µM, such as curcumin, α-mangostin, nobiletin, and heptamethoxyflavone, have beneficial functions for health such as anti-inflammatory effect. These results suggest that the T-cell activation-inhibitory assay can be used to screen candidate CRMs.

Heptamethoxyflavone inhibits adipogenesis via enhancing PKA signaling.

3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), a naturally occurring polymethoxyflavone found in citrus peel, is known to have neuroprotective, anti-inflammatory, and immunomodulatory effects. However, the effects of HMF on adipogenesis remain unclear. Here, we demonstrate that HMF inhibits the early stage of adipogenesis and maturation in 3T3-L1 adipocytes. HMF treatment during preadipocyte differentiation for 8 days reduced lipid accumulation in a dose-dependent manner, and the expression levels of key adipogenic transcription factors (peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα)) and the lipogenic transcription factor, sterol regulatory element-binding protein (SREBP1), were lower after the initial 4 days of the differentiation. Moreover, PPARγ expression level was lower even after the initial 2 days, but C/EBPα and SREBP1 expression was not. HMF upregulated the phosphorylation of protein kinase A catalytic subunit α (PKACα), AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in 3T3-L1 cells. The phosphorylation of ACC leads to the inhibition of adipogenesis. Furthermore, the induction of phosphorylation of AMPK and ACC by HMF was abolished by RNA interference targeting PKACα. Taken together, our results suggest that HMF might inhibit the early stage of adipogenesis via the activation of PKA signaling in 3T3-L1 cells.

Citrus flavonoid 3,5,6,7,8,3',4'-heptamethoxyflavone induces BDNF via cAMP/ERK/CREB signaling and reduces phosphodiesterase activity in C6 cells.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is associated with onset of several central nervous system disorders, e.g., Parkinson's disease, Alzheimer's disease, depression, epilepsy, and chronic pain. In our previous in vivo studies using ischemic and depression mouse models, we revealed that citrus flavonoid 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) exerts neuroprotective effects by enhancing the expression of BDNF in astrocytes within the hippocampus. Therefore, in the present study, we examined the mechanism of BDNF induction by HMF in vitro using rat C6 glioma cells. METHODS: C6 glioma cells were treated with HMF (10 µM) or HMF + U0126 (10 µM), HMF + H89 (1 µM), or HMF + K252a (200 nM) for 48 h. The protein level of mature BDNF (m-BDNF), phosphorylated-ERK (p-ERK) and phosphorylated-cAMP-response element binding protein (p-CREB) were measured using western blot analysis. To clarify the mechanism of HMF for increasing m-BDNF, the inhibitory effect of phosphodiesterase 4B (PDE4B) and PDE4D, and intracellular cAMP levels were examined by ELISA. RESULTS: Our findings revealed that the m-BDNF-inducing activity of HMF was abolished by U0126 but not by H89 or K252a. HMF was found to phosphorylate (activate) ERK and cAMP-response element binding protein (CREB), a BDNF transcription factor. HMF inhibited PDE4B and PDE4D activity. Moreover, 10 µM HMF elevated intracellular cAMP levels in C6 cells. CONCLUSIONS: These findings suggest that HMF might exert its neuroprotective effects by inducing m-BDNF expression in C6 cells, model cell line of astrocytes, via the activation of cAMP/ERK/CREB signaling and inhibiting PDE4B or PDE4D.