Adenovirus vector-mediated macrophage erythroblast attacher (MAEA) overexpression in primary mouse hepatocytes attenuates hepatic gluconeogenesis.

Japanese patients with type 2 diabetes mellitus present a different responsiveness in terms of insulin secretion to glucose and body mass index (BMI) from other populations. The genetic background that predisposes Japanese individuals to type 2 diabetes mellitus is under study. Recent genetic studies demonstrated that the locus mapped in macrophage erythroblast attacher (MAEA) increases the susceptibility to type 2 diabetes mellitus in East Asians, including Japanese individuals. MAEA encodes a protein that plays a role in erythroblast enucleation and in the normal differentiation of erythroid cells and macrophages. However, the contribution of MAEA to type 2 diabetes mellitus remains unknown. In this study, to overexpress MAEA in the mouse liver and primary mouse hepatocytes, we generated a MAEA-expressing adenovirus (Ad) vector using a novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-MAEA). Blood glucose and insulin levels in Ad-MAEA-treated mice were comparable to those in control Ad-treated mice. Primary mouse hepatocytes transduced with Ad-MAEA showed lower levels of expression of gluconeogenesis genes than those transduced with the control Ad vector. Hepatocyte nuclear factor-4α (HNF-4α) mRNA expression in primary mouse hepatocytes was also suppressed by MAEA overexpression. These results suggest that MAEA overexpression attenuates hepatic gluconeogenesis, which could potentially lead to improvement of type 2 diabetes mellitus.

Cilostazol and enzymatically modified isoquercitrin attenuate experimental colitis and colon cancer in mice by inhibiting cell proliferation and inflammation.

We previously reported the anti-inflammatory effects of cilostazol, a selective inhibitor of phosphodiesterase 3, and two antioxidants, enzymatically modified isoquercitrin and α-lipoic acid in a dextran sodium sulphate-induced colitis mouse model. We further examined the chemopreventive effects of these substances in a murine azoxymethane/dextran sodium sulphate -induced colorectal carcinoma model and compared the effects with those of the well-known anticancer natural plant pigment, anthocyanin. In addition, the effects on cell proliferation activity were evaluated in colon cancer cell lines and mucosal epithelial cells in a model of acute dextran sodium sulphate-induced colitis. Cilostazol and enzymatically modified isoquercitrin improved the outcome of azoxymethane/dextran sodium sulphate-induced colorectal cancer along with anthocyanin though inhibiting inflammation and cell proliferation, but the effect of α-lipoic acid was minimal. Inhibition of cell proliferation by cilostazol was confirmed in vitro. In the acute dextran sodium sulphate-induced colitis model, cilostazol and enzymatically modified isoquercitrin prevented the decrease in epithelial proliferative cells. These results indicate that cilostazol and enzymatically modified isoquercitrin first exhibited an anti-dextran sodium sulphate effect at the initial stage of colitis and then showed antitumour effects throughout subsequent inflammation-related cancer developmental stages.

Use of capillary electrophoresis with dual-opposite end injection for simultaneous analysis of small ions in saliva samples from wrestlers undergoing a weight training program.

Capillary electrophoresis-capacitively coupled contactless conductivity detection (CE-C4D), conducted using an in-house-developed polyvinyl alcohol (PVA)-coated capillary system, was applied for the simultaneous analysis of small anions and cations in saliva samples from wrestlers undergoing a weight training program. Use of the PVA capillary for CE provided good reproducible ion separation with minimization of the electroosmotic flow and suppression of protein adsorption onto the capillary wall. Four cations and eight anions were separated in 12min, using a background electrolyte of 20mM MES/20mM histidine and 18-crown-6 ether (pH 6) at 20kV. The relative standard deviations (n=5) of the migration times and peak areas were <1% and <8%, respectively. The detection limit at a signal-to-noise ratio of 3 ranged from 1.6 to 10µM. Using the optimized CE-C4D system, we investigated the correlations between the concentrations of salivary ions and cortisol, which is commonly used as a stress marker. Analysis of saliva samples from ten wrestlers, who were attempting rapid weight loss before a competition, showed the following trends: (1) all ion concentrations, except for Ca(2+), Na(+), and Cl(-), increased between the first and last days of weight loss; (2) Mg(2+) increased to 166% (from 0.50mM to 1.4mM) between the first and last days of weight loss, being the highest increase of all the ions; and (3) K(+), Mg(2+), NO3(-), and SCN(-) levels were strongly correlated (P<0.05) with cortisol. The CE-C4D rapidly produced useful data on saliva ion contents, with good ion recovery as determined by the standard addition method (89-110%).

Involvement of hepatic IL-1 in the strain-dependent sex differences in serum total cholesterol levels in rats.

The strain and sex differences in serum total cholesterol (TC) levels were examined in F344 and Sprague-Dawley (SD) rats. A sex difference (male

Protein kinase C (PKC) increases TACE/ADAM17 enzyme activity in porcine ovarian somatic cells, which is essential for granulosa cell luteinization and oocyte maturation.

During in vitro maturation of porcine cumulus cell-oocyte complexes and in vitro luteinization of porcine granulosa cells, FSH induces the expression of the protease TNFα-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) and the epidermal growth factor (EGF)-like factors, which activate the EGF receptor (EGFR)-MAPK3/1 pathway in both cumulus and granulosa cells. FSH is known to activate not only protein kinase A and p38MAPK pathways in both cell types but also activates protein kinase C (PKC). Because PKC-induced association of cellular-Sarcoma (c-Src) and TACE/ADAM17 is required for TACE/ADAM17 enzyme activation in some cancer cells, we hypothesized that PKC and c-Src impact TACE/ADAM17-mediated activation of EGFR signaling pathway in porcine granulosa and cumulus cells. When granulosa cells or cumulus cell-oocyte complexes were cultured with FSH, PKC activity and c-Src phosphorylation increased and were associated with increased TACE/ADAM17 enzyme activity. The PKC inhibitor calphostin C (CalC) and the c-Src inhibitor (4 amino 5 (4 chlorophenyl) 7 (t butyl)pyrazolo[3,4 d]pyrimidine [PP2]) suppressed TACE/ADAM17 enzyme activity, whereas these inhibitors did not affect Tace/Adam17 mRNA expression. Immunoprecipitation analysis showed that FSH mediated the association of c-Src with TACE/ADAM17 via a PKC-dependent mechanism. Either CalC or PP2 suppressed EGFR downstream signaling pathway (MAPK3/1) in these ovarian cell types and reduced cumulus expansion, meiotic maturation of oocytes, and progesterone production. The negative effects were overcome by the addition of amphiregulin. Collectively, these results indicate that activation of TACE/ADAM17 via a PKC-induced c-Src-dependent manner mediates proteolytic activation of the EGF-like factors that are involved in the induction of granulosa cell differentiation, cumulus expansion, and meiotic maturation of porcine oocytes in vitro.

Positive feedback loop between prostaglandin E2 and EGF-like factors is essential for sustainable activation of MAPK3/1 in cumulus cells during in vitro maturation of porcine cumulus oocyte complexes.

During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.