Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1alpha stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1alpha acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-alpha. Although both clones expressed receptor activator NFkappaB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1alpha stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1alpha also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-alpha. These findings provide strong evidence to show that MIP-1alpha acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1alpha significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1alpha in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.
T Watanabe, T Kukita, A Kukita, N Wada, K Toh, K Nagata, H Nomiyama and T Iijima
M Iitaka, S Kakinuma, S Fujimaki, I Oosuga, T Fujita, K Yamanaka, S Wada and S Katayama
Zinc at concentrations of 150, microM or higher induced necrosis as well as apoptosis in thyroid cancer cell lines. Necrosis was induced by zinc in a dose-dependent manner, whereas apoptosis did not increase at higher concentrations of zinc. The expression of the antiapoptotic protein phosphorylated Bad was markedly increased, whereas the expression of the proapoptotic proteins Bax and Bad decreased following Zn(2+) exposure. Zn(2+) induced rapid degradation of IkappaB, and an increase in the binding of nuclear transcription factor-kappaB (NF-kappaB). These observations indicate that antiapoptotic pathways were activated in thyroid cancer cells following exposure to Zn(2+). This may be a self-defence mechanism against apoptosis and may underlie the general resistance of thyroid cancer cells to apoptotic stimuli. Zinc may be a potential cytotoxic agent for the treatment of thyroid cancer.
S Otabe, N Wada, T Hashinaga, X Yuan, I Shimokawa, T Fukutani, K Tanaka, T Ohki, S Kakino, Y Kurita, H Nakayama, Y Tajiri and K Yamada
We previously reported that transgenic (Tg) expression of adiponectin significantly prolonged the lifespan of normal mice. The aim of this study was to elucidate the mechanism involved in the longevity effects of adiponectin using KK/Ta mice, a murine model of metabolic syndrome. We established a Tg line of KK/Ta (Tg-KK/Ta) mice expressing human adiponectin in the liver, and assessed their lifespan. The cause of death was determined by macroscopic and microscopic examinations immediately after death. The expressions of SIRT1, C-reactive protein (CRP), inflammatory cytokines, AMPK, and AKT were measured by quantitative real-time PCR, ELISAs, and/or western blotting. KK/Ta mice had lower serum adiponectin levels and shorter lifespan (57.6±13.9 vs 106.5±18.3 weeks, P<0.0001) than C57BL/6N mice. Tg adiponectin expression significantly extended the lifespan of KK/Ta mice (73.6±16.6 weeks, P<0.001) without affecting body weight, daily food consumption, or plasma glucose levels. Neoplasms were observed in only three of 22 KK/Ta mice that died spontaneously because of tumors. Atherosclerotic lesions were not detected in any mice. SIRT1 levels were not significantly different between KK/Ta and Tg-KK/Ta mice. Gene expressions of Crp, Tnf α, Il6, and Nf κ b were increased in KK/Ta mice, but they were significantly attenuated in Tg-KK/Ta mice. Phosphorylated AMPK levels were increased and phosphorylated AKT levels were decreased in Tg-KK/Ta mice. The anti-inflammatory effects of adiponectin, achieved by inhibiting the AKT signaling pathway, may explain how adiponectin slows the accelerated aging process associated with the metabolic syndrome.
T Ishizuka, A Miura, K Kajita, K Yamada, H Wada, S Itaya, Y Kanoh, M Ishizawa, M Kimura and K Yasuda
The Otsuka Long-Evans Tokushima fatty (OLETF) rat is a new spontaneous non-insulin-dependent diabetes mellitus (NIDDM) model rat strain developed in Tokushima, Japan. After 18 weeks of age, decreases of 45% and 40% respectively in insulin- and phorbol ester-stimulated [3H]2-deoxyglucose (DOG) uptake were observed, compared with those in Long-Evans Tokushima (LETO) rats (control). Insulin-specific binding and 95 kDa autophosphorylation of insulin receptor in OLETF rats were not different from those in LETO rats. Insulin-induced diacylglycerol (DG) production and Mono Q column-purified protein kinase C (PKC) translocation in adipocytes of OLETF rats were decreased compared with those of LETO rats. Insulin-induced PKC beta translocation from cytosol to membrane was also decreased in adipocytes of OLETF rats. Increases of the PKC beta I, beta II, epsilon and zeta isoforms in membranes of OLETF rats were markedly smaller than those of LETO rats. Analysis of mRNA levels of PKC isoforms in adipocytes of OLETF rats showed decreases of basal level and insulin-induced delayed responses of PKC beta I, beta II, epsilon and zeta mRNA in OLETF rats. On the other hand, insulin- or phorbol ester-induced phosphatidylinositol 3-kinase (PI 3-kinase) activation was decreased in adipocytes of OLETF rats compared with those of LETO rats. These results suggest that insulin resistance in OLETF rats, a spontaneous NIDDM model rat, may be associated with deterioration of insulin-induced DG-PKC signaling and subsequent decrease in PI 3-kinase activation.
T Akamizu, T Murayama, S Teramukai, K Miura, I Bando, T Irako, H Iwakura, H Ariyasu, H Hosoda, H Tada, A Matsuyama, S Kojima, T Wada, Y Wakatsuki, K Matsubayashi, T Kawakita, A Shimizu, M Fukushima, M Yokode and K Kangawa
Aging is associated with a decrease in growth hormone (GH) secretion, appetite and energy intake. As ghrelin stimulates both GH secretion and appetite, reductions in ghrelin levels may be involved in the reductions in GH secretion and appetite observed in the elderly. However, only preliminary studies have been performed on the role of ghrelin in elderly subjects. In this study, we sought to clarify the physiologic implications of the age-related alterations in ghrelin secretion by determining plasma ghrelin levels and other clinical parameters in healthy elderly subjects. Subjects were ≥ 65 years old, corresponding to the SENIEUR protocol, had not had a resection of the upper gastrointestinal tract and had not been treated with hormones. One hundred and five volunteers (49 men and 56 women) were admitted to this study (73.4 ± 6.3 years old). Plasma levels of acylated ghrelin in elderly female subjects positively correlated with serum IGF-I levels and bowel movement frequency and negatively with systolic blood pressure. In elderly men, desacyl ghrelin levels correlated only weakly with bowel movement frequency. These findings suggest that the plasma levels of the acylated form of ghrelin may influence the age-related alterations in GH/IGF-I regulation, blood pressure and bowel motility. These observational associations warrant further experimental studies to clarify the physiologic significance of these effects.