Mitsuko Nonaka

Modulatory effect of macrolide antibiotics on the Th1- and Th2-type cytokine production.

The effect of the macrolide antibiotics, clarithromycin, midecamycin acetate and josamycin, on the generation of Th1- and Th2-type cytokines by mitogen-stimulated human T lymphocytes was compared with that of fosfomycin. The following results were obtained. These drugs demonstrated potent inhibitory activity on the release and gene expression of TNF-alpha and IL-2. Their inhibitory effect on IFN-alpha, IL-4, IL-5, IL-6 was less marked. The release of IL-10 was poorly suppressed. Clarithromycin had the most potent inhibitory effect of the drugs used. The present results suggested that anti-bacterial agents might modify the hosts immunological response by interfering with the activity of T helper cells.

Naringenin and Hesperetin Induce Growth Arrest, Apoptosis, and Cytoplasmic Fat Deposit in Human Preadipocytes

Citrus flavonoids are reported to be promising bioactive compounds against hyperlipidemia and lipid biosynthesis. However, the mechanism of the lipid lowering effect by flavonoids remains unknown. The present study examines the effect of some flavanones on the adipocytic conversion of the human preadipocyte cell line, AML-I. Among four structure-related flavanones including naringenin, naringenin-7-rhamnoglucoside (naringin), hesperetin, and hesperetin-7-rhamnoglucoside (hesperidin), the aglycones such as naringenin and hesperetin exhibited the growth arrest of AML-I cells. When the cells were examined by Annexin V-FITC staining method, it was noticed that growth arrest was induced by apoptotic cell death. In the study of apoptosis-related protein in the naringenin-treated cells, anti-apoptotic proteins such as p-Akt, NF-kappaB, and Bcl-2 were decreased, and pro-apoptotic protein Bad was accumulated by Western blot analysis. Interestingly, exposure of AML-I cells to naringenin or hesperetin during short-term cultures increased cytoplasmic lipid droplets by Sudan Black B staining. Furthermore, expression of fatty acid synthase (FAS) and peroxisome proliferator activated receptor (PPAR)-gamma was enhanced in naringenin-treated cells. These data suggest that apoptosis by flavanones does not inhibit the adipocytic conversion of AML-I preadipocytes. The result also indicates that adipocyte may not be a direct target for the lipid-lowering activity of the flavanones.

Effect of genistein and daidzein on the proliferation and differentiation of human preadipocyte cell line.

Isoflavones are known to have several biological activities, including a hypolipidemic effect. However, the mechanism of the lipid lowering effect of genistein remains to be elucidated. There is conflicting evidence on the effect of genistein for the deposition of adipocyte tissues. We examined the effect of the isoflavones on the growth and differentiation of human preadipocyte cells, AML-I. Growth arrest accompanied by the appearance of characteristics of apoptosis was observed by genistein or daidzein treatment under the adipogenic stimulation. The expressions of apoptosis-related proteins, Bad, Akt, and p-Akt, were modulated in the genistein-treated cells by Western blot analysis. On the other hand, exposure of AML-I to the isoflavones increased accumulation of cytoplasmic lipid droplets. Actually, the cytoplasmic expressions of fatty acid synthase (FAS) and peroxisome proliferator-activated receptor (PPAR)-gamma were increased in the genistein-treated cells. Glycosylated forms of the isoflavones genistein and puerarin did not have such activities. These results suggested that only aglycon forms of isoflavones induced not only apoptosis but also lipogenesis in the preadipocyte cell line AML-I. The possible mechanism of these phenomena has been discussed in the text.

Epigallocatechin gallate-induced apoptosis does not affect adipocyte conversion of preadipocytes.

In the present study, we examined the effect of epigallocatechin gallate (EGCG) on the growth and differentiation of human preadipocyte cells, AML‐I. EGCG exhibited cytotoxic activity on AML‐I cells, accompanied by the appearance of characteristics of apoptosis by Annexin V—FITC staining method. Among apoptosis‐related proteins examined, loss of NF‐κB and p‐Akt, and accumulation of Bad were displayed in EGCG‐treated cells by Western blot analysis. Among 6 structure‐related catechins including catechin (C), epicatechin (EC), catechin gallate (CG), epigallocatechin (EGC), epicatechin gallate (ECG) and EGCG, the catechins containing galloyl moiety exhibited apoptotic capacity. Interestingly, exposure of AML‐I to EGCG increased the amounts of cytoplasmic lipid droplets as well as the expression of fatty acid synthase and peroxisome proliferator activated receptor‐γ proteins. Our results suggest that EGCG induces growth arrest and apoptosis, but does not affect adipocyte conversion of preadipocytes.

Modulatory effect of fosfomycin on acute inflammation in the rat air pouch model.

We examined the effect of fosfomycin (FOM) on the inflammatory response induced by carrageenan in the rat. Air pouches were induced subcutaneously on the backs of rats and injected with carrageenan. The rats were treated with either vehicle or FOM at a dose of 100 mg/kg 1 h before carrageenan challenge. After carrageenan challenge (48 h), the air pouches were removed and analyzed. The volume, protein amounts and cell counts in the exudate obtained from FOM-treated animals were significantly reduced compared with that from vehicle-treated animals. The contents of PGE(2) and TNF-alpha, and mRNA for cyclooxygenase-2 were also markedly suppressed in FOM-treated rats. Histological examination showed suppression of the inflammatory response in the pouch tissues from FOM-treated rats.

Induction of apoptosis and lipogenesis in human preadipocyte cell line by n-3 PUFAs.

We examined the effect of n −3 PUFAs (polyunsaturated fatty acids) on the growth and maturation of human preadipocyte cell line AML‐I. On day 3 of the culture, n −3 fatty acids such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), but not n −6 fatty acid LA (linoleic acid), induced growth arrest accompanied by the appearance of characteristics of apoptosis in AML‐I cells at concentrations between 250 and 500 μM by Annexin V‐FITC staining. In Western blotting analysis, the loss of NF‐κB, Bcl‐2 and p‐Akt and the accumulation of Bad and Akt were observed in the cytoplasmic protein from the EPA‐treated cells. Exposure of AML‐I to EPA or DHA increased the cytoplasmic lipid accumulation compared with the vehicle‐treated cells in a time‐dependent manner during 4 and 6 days culture period by Oil Red O staining. The expression of FAS (fatty acid synthase) and PPAR‐γ (peroxisome proliferator‐activated receptor‐γ) were increased in EPA‐treated cells. These results suggest that EPA and DHA promote differentiation, inhibit proliferation and induce apoptosis in preadipocyte cell line AML‐I.

All-trans retinoic acid displays multiple effects on the growth, lipogenesis and adipokine gene expression of AML-I preadipocyte cell line

Adipose tissue is a potential site of retinoic acid (RA) action, but its physiological significance remains to be clarified. We have examined the effect of all‐trans retinoic acid (ATRA) on growth and differentiation of preadipocytes, and on adipokine gene expression in mature adipocytes using human preadipocyte cell model, AML‐I. Both ATRA and 9‐cis RA induced growth arrest in AML‐I preadipocyte at between 50 and 100 µM, which was accompanied by apoptosis. Western blotting showed a loss of NF‐κB, Bcl‐2 and p‐Akt, and the accumulation of Bad and Akt in cytoplasm of ATRA‐treated AML‐I preadipocytes. Exposure of AML‐I to ATRA or 9‐cis RA increased intracellular lipid accumulation in a time‐dependent manner compared to vehicle‐treated cells. Expression of fatty acid synthase (FAS) and peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) proteins was increased in ATRA‐treated cells. Thus, both ATRA and 9‐cis RA promoted differentiation, inhibited proliferation and induced apoptosis in AML‐I preadipocytes. ATRA also modulated adipokine expression by increasing the mRNA level of adipocytokines (adiponectin, leptin and LPL), and by inhibiting PAI‐1 mRNA expression in mature AML‐I adipocytes. The data suggest that ATRA exerts a wide range of effects—growth arrest, apoptosis, lipogenesis and modulation of adipokine gene expression—during the maturation of preadipocytes into adipocytes.