Yasukiyo Yoshioka

Vialinin A is a ubiquitin-specific peptidase inhibitor.

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin.

Ubiquitin-specific peptidase 5, a target molecule of vialinin A, is a key molecule of TNF-α production in RBL-2H3 cells.

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.

Enzymatically synthesized glycogen inhibits colitis through decreasing oxidative stress

Abstracts Inflammatory bowel diseases are a group of chronic inflammation conditions of the gastrointestinal tract. Disruption of the mucosal immune response causes accumulation of oxidative stress, resulting in the induction of inflammatory bowel disease. In this study, we investigated the effect of enzymatically synthesized glycogen (ESG), which is produced from starch, on dextran sulfate sodium (DSS)‐ and 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced colitis in C57BL/6 mice. Oral administration of ESG suppressed DSS‐ and TNBS‐induced shortening of large intestine in female mice and significant decreased DSS‐induced oxidative stress and TNBS‐induced pro‐inflammatory cytokine expression in the large intestine. ESG increase in the expression levels of heme oxygenase‐1 (HO‐1) and NF‐E2‐related factor‐2 (Nrf2), a transcription factor for HO‐1 expressed in the large intestine. Furthermore, ESG‐induced HO‐1 and Nrf2 were expressed mainly in intestinal macrophages. ESG is considered to be metabolized to resistant glycogen (RG) during digestion with &agr;‐amylase in vivo. In mouse macrophage RAW264.7 cells, RG, but not ESG decreased 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH)‐induced reactive oxygen species (ROS). Knockdown of Nrf2 inhibited RG‐induced HO‐1 expression and negated the decrease in AAPH‐induced ROS brought about by RG. RG up‐regulated the protein stability of Nrf2 to decrease the formation of Nrf2‐Keap1 complexes. RG‐induced phosphorylation of Nrf2 at Ser40 was suppressed by ERK1/2 and JNK inhibitors. Our data indicate that ESG, digested with &agr;‐amylase to RG, suppresses DSS‐ and TNBS‐induced colitis by increasing the expression of HO‐1 in the large intestine of mice. Furthermore, we demonstrate that RG induces HO‐1 expression by promoting phosphorylation of Nrf2 at Ser40 through activation of the ERK1/2 and JNK cascade in macrophages. Graphical abstract Figure. No Caption available. HighlightsESG prevented DSS‐induced colitis in mice by decreasing oxidative stress.ESG administration induced Nrf2 and HO‐1 expression in colon tissues of mice.ESG metabolite RG induced Nrf2 and HO‐1 expression in RAW264.7 macrophages.Knockdown of Nrf2 and HO‐1 in RAW264.7 cells negated RG‐induced antioxidant effect.

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane‐embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis.

Vialinin A and thelephantin G, potent inhibitors of tumor necrosis factor-α production, inhibit sentrin/SUMO-specific protease 1 enzymatic activity

Several p-terphenyl compounds have been isolated from the edible Chinese mushroom Thelephora vialis. Vialinin A, a p-terphenyl compound, strongly inhibits tumor necrosis factor-α production and release. Vialinin A inhibits the enzymatic activity of ubiquitin-specific peptidase 5, one of the target molecules in RBL-2H3 cells. Here we examined the inhibitory effect of p-terphenyl compounds, including vialinin A, against sentrin/SUMO-specific protease 1 (SENP1) enzymatic activity. The half maximal inhibitory concentration values of vialinin A and thelephantin G against full-length SENP1 were 1.64±0.23μM and 2.48±0.02μM, respectively. These findings suggest that p-terphenyl compounds are potent SENP1 inhibitors.

Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1

Theobromine, a methylxanthine derived from cacao beans, reportedly has various health-promoting properties but molecular mechanism by which effects of theobromine on adipocyte differentiation and adipogenesis remains unclear. In this study, we aimed to clarify the molecular mechanisms of the anti-adipogenic effect of theobromine in vitro and in vivo. ICR mice (4week-old) were administered with theobromine (0.1g/kg) for 7days. Theobromine administration attenuated gains in body and epididymal adipose tissue weights in mice and suppressed expression of adipogenic-associated genes in mouse adipose tissue. In 3T3-L1 preadipocytes, theobromine caused degradation of C/EBPβ protein by the ubiquitin-proteasome pathway. Pull down assay showed that theobromine selectively interacts with adenosine receptor A1 (AR1), and AR1 knockdown inhibited theobromine-induced C/EBPβ degradation. Theobromine increased sumoylation of C/EBPβ at Lys133. Expression of the small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) gene, coding for a desumoylation enzyme, was suppressed by theobromine. In vivo knockdown studies showed that AR1 knockdown in mice attenuated the anti-adipogenic effects of theobromine in younger mice. Theobromine suppresses adipocyte differentiation and induced C/EBPβ degradation by increasing its sumoylation. Furthermore, the inhibition of AR1 signaling is important for theobromine-induced C/EBPβ degradation.

Licorice flavonoid oil enhances muscle mass in KK- A y mice

Aims: Muscle mass is regulated by the balance between the synthesis and degradation of muscle proteins. Loss of skeletal muscle mass is associated with an increased risk of developing metabolic diseases such as obesity and type 2 diabetes mellitus. The aim of this study was to clarify the effects of licorice flavonoid oil on muscle mass in KK‐Ay/Ta mice. Main methods: Male genetically type II diabetic KK‐Ay/Ta mice received 0, 1, or 1.5 g/kg BW of licorice flavonoid oil by mouth once daily for 4 weeks. After 4 weeks, the femoral and soleus muscles were collected for western blotting for evaluation of the mTOR/p70 S6K, p38/FoxO3a, and Akt/FoxO3a signaling pathways. Key findings: Ingestion of licorice flavonoid oil significantly enhanced femoral muscle mass without affecting body weight in KK‐Ay/Ta mice. Licorice flavonoid oil also decreased expression of MuRF1 and atrogin‐1, which are both markers of muscle atrophy. The mechanisms by which licorice flavonoid oil enhances muscle mass include activation of mTOR and p70 S6K, and regulation of phosphorylation of FoxO3a. Significance: Ingestion of licorice flavonoids may help to prevent muscle atrophy.

Liquorice flavonoid oil suppresses hyperglycaemia accompanied by skeletal muscle myocellular GLUT4 recruitment to the plasma membrane in KK-Ay mice

Abstract For over 4000 years, liquorice has been one of the most frequently employed botanicals as a traditional herbal medicine. Although previous reports have found that liquorice flavonoids possess various health beneficial effects, the underlying mechanism responsible for the anti-diabetic effect of liquorice flavonoids remains unclear. The present study demonstrates that liquorice flavonoid oil (LFO) improves type 2 diabetes mellitus through GLUT4 translocation to the plasma membrane by activating both the adenosine monophosphate-activated protein kinase (AMPK) pathway and Akt pathway in muscle of KK-Ay mice. Furthermore, LFO lowered postprandial hyperglycaemia in a human study. These results indicate that LFO may exert a therapeutic effect on metabolic disorders, such as diabetes and hyperglycaemia, by modulating glucose metabolism through AMPK- and insulin-dependent pathways in skeletal muscle. Graphical Abstract