Effect of licorice flavonoid oil on cholesterol metabolism in high fat diet rats.

Dietary licorice fravonoid oil (LFO) significantly decreased hepatic cholesterol and plasma lipoprotein cholesterol levels in high-fat diet rats. It significantly suppressed hydroxymethylglutaryl-CoA synthase activity and increased cholesterol 7α-hydroxylase activity. The low density lipoprotein receptor mRNA level was significantly increased by LFO. These results suggest that dietary LFO improves cholesterol metabolism in obese animals.

A comparative study of the central effects of melanocortin peptides on food intake in broiler and layer chicks.

Broiler chicks eat more food than layer chicks. However, the causes of the difference in food intake in the neonatal period between these strains are not clear. In this study, we examined the involvement of proopiomelanocortin (POMC)-derived melanocortin peptides α-, ß- and γ-melanocyte-stimulating hormones (MSHs) in the difference in food intake between broiler and layer chicks. First, we compared the hypothalamic mRNA levels of POMC between these strains and found that there was no significant difference in these levels between broiler and layer chicks. Next, we examined the effects of central administration of MSHs on food intake in these strains. Central administration of α-MSH significantly suppressed food intake in both strains. Central administration of ß-MSH significantly suppressed food intake in layer chicks, but not in broiler chicks, while central administration of γ-MSH did not influence food intake in either strain. It is therefore likely that the absence of the anorexigenic effect of ß-MSH might be related to the increased food intake in broiler chicks.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells.

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 µM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Neuropeptide Y effect on food intake in broiler and layer chicks.

Broiler chicks eat more food than layer chicks. In this study, we examined the involvement of orexigenic peptide neuropeptide Y (NPY) in the difference in food intake between broiler and layer chicks (Gallus gallus). First, we compared the hypothalamic mRNA levels of NPY and its receptors (Y1 and Y5 receptors) between these strains at 1, 2, 4, and 8 days of age. Daily food intake was significantly higher in broiler chicks than layer chicks after 2 days of age. However, the hypothalamic NPY mRNA level was significantly lower in broiler chicks than layer chicks except at 8 days of age. In addition, the mRNA levels of NPY receptors were also significantly lower in broiler chicks than layer chicks at 2 and 4 days of age (Y1 receptor) or 2 days of age (Y5 receptor). These results suggest that the differences in the expressions of hypothalamic NPY and its receptors do not cause the increase in food intake in broiler chicks. To compare the orexigenic effect of NPY between broiler and layer chicks, we next examined the effects of central administration of NPY on food intake in these strains. In both strains, central administration of NPY significantly increased food intake at 2, 4 and 8 days of age. All our findings demonstrated that the increase in food intake in broiler chicks is not accompanied with the over-expression of NPY or its receptor.

Harmine, a ß-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo.

Bone homeostasis is controlled by the balance between osteoblastic bone formation and osteoclastic bone resorption. Excessive bone resorption is involved in the pathogenesis of bone-related disorders such as osteoporosis, arthritis and periodontitis. To obtain new antiresorptive agents, we searched for natural compounds that can inhibit osteoclast differentiation and function. We found that harmine, a ß-carboline alkaloid, inhibited multinucleated osteoclast formation induced by receptor activator of nuclear factor-κB ligand (RANKL) in RAW264.7 cells. Similar results were obtained in cultures of bone marrow macrophages supplemented with macrophage colony-stimulating factor and RANKL, as well as in cocultures of bone marrow cells and osteoblastic UAMS-32 cells in the presence of vitamin D(3) and prostaglandin E(2). Furthermore, harmine prevented RANKL-induced bone resorption in both cell and bone tissue cultures. Treatment with harmine (10 mg/kg/day) also prevented bone loss in ovariectomized osteoporosis model mice. Structure-activity relationship studies showed that the C3-C4 double bond and 7-methoxy group of harmine are important for its inhibitory activity on osteoclast differentiation. In mechanistic studies, we found that harmine inhibited the RANKL-induced expression of c-Fos and subsequent expression of nuclear factor of activated T cells (NFAT) c1, which is a master regulator of osteoclastogenesis. However, harmine did not affect early signaling molecules such as ERK, p38 MAPK and IκBα. These results indicate that harmine inhibits osteoclast formation via downregulation of c-Fos and NFATc1 induced by RANKL and represses bone resorption. These novel findings may be useful for the treatment of bone-destructive diseases.

Honokiol inhibits osteoclast differentiation and function in vitro.

Honokiol, a neolignan, is a physiologically active component of kouboku (Magnolia obovata), a herb used in traditional Chinese medicine. This study investigated the effects of honokiol on the differentiation and function of osteoclasts induced by receptor activator of nuclear factor-kappaB ligand (RANKL). Honokiol markedly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP-positive multinucleated cells in both bone marrow-derived monocytes and RAW264 cells. In experiments to elucidate its mechanism of action, honokiol was found to suppress RANKL-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The RANKL-induced expressions of c-Fos and nuclear factor of activated T cells-c1 (NFATc1), which are crucial transcriptional factors for osteoclastogenesis, were also reduced by treatment with honokiol. Furthermore, honokiol induced disruption of the actin rings in mature osteoclasts (mOCs) without affecting the cell viability and suppressed osteoclastic pit formation on dentin slices. Taken together, these results suggest that honokiol inhibits osteoclast differentiation by suppressing the activation of MAPKs (p38 MAPK, ERK and JNK), decreasing the expressions of c-Fos and NFATc1, and attenuates bone resorption by disrupting the actin rings in mOCs. Therefore, honokiol could prove useful for the treatment of bone diseases associated with excessive bone resorption.

Corticotropin-releasing factor is a downstream mediator of the beta-melanocyte-stimulating hormone-induced anorexigenic pathway in chicks.

Proopiomelanocortin (POMC, a precursor of anorexigenic neuropeptides) neurons in hypothalamus suppresses food intake in both mammals and chickens. In mammals, several lines of evidence suggest that POMC-derived anorexigenic peptides upregulate mRNA levels of anorexigenic peptides such as corticotropin-releasing factor (CRF) and thyrotropin-releasing factor and downregulate mRNA levels of orexigenic peptides such as orexin and melanin-concentrating hormone. However, the POMC-induced anorexigenic pathway in chickens has not been well characterized. In the present study, we investigated how POMC neurons regulate mechanisms of food intake using an anorexigenic peptide, beta-melanocyte-stimulating hormone (beta-MSH), derived from the post-transcriptional cleavage of POMC. Central administration of beta-MSH in chicks significantly suppressed food intake, and importantly, this suppression was accompanied by a significant upregulation of CRF mRNA levels. Furthermore, the CRF type 2 receptor antagonist alpha-helical CRF significantly reversed the anorexigenic action of beta-MSH. These findings indicate that CRF and its receptor, CRF type 2 receptor, act as the major mediators in beta-MSH-induced anorexigenic action in chicks. beta-MSH significantly increased orexin mRNA levels and did not alter mRNA levels of thyrotropin-releasing factor and melanin-concentrating hormone in chicks, suggesting that the beta-MSH-induced anorexigenic pathway in chicks is different from that in mammals. Increases in orexin mRNA levels were accompanied by significant decreases in plasma glucose concentration, suggesting that orexin mRNA might be stimulated by beta-MSH-induced hypoglycemia. Thus, this study demonstrates the direct evidence that CRF is a critical downstream target in the beta-MSH-induced anorexigenic pathway in chicks.

The molecular mechanism underlying the reduction in abdominal fat accumulation by licorice flavonoid oil in high fat diet-induced obese rats.

Licorice (Glycyrrhiza glabra) has been widely used in traditional medicines, and its flavonoid oil (LFO) decreases abdominal adipose tissue weight in mammals. In the present study, we investigated the molecular mechanisms underlying the decrease in abdominal adipose tissue weight by LFO. LFO significantly decreased the mRNA levels of rate-limiting enzymes in the hepatic fatty acid synthetic pathway, whereas LFO significantly increased the mRNA levels of a rate-limiting enzyme in the hepatic fatty acid oxidative pathway. LFO significantly decreased the mRNA levels of sterol regulatory element-binding protein-1c (SREBP-1c) (a transcription factor that promotes hepatic fatty acid synthesis), whereas the mRNA levels of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) (a transcription factor that promotes hepatic fatty acid oxidation) was significantly increased. All our findings suggest that the decrease in abdominal adipose tissue weight by LFO is mediated by the transcriptional regulation of SREBP-1c and PPAR-alpha in the liver. Thus, we infer that the natural ingredient LFO is a promising candidate for use as a feed additive to reduce abdominal fat accumulation in domestic animals.

Investigation of the anti-obesity action of licorice flavonoid oil in diet-induced obese rats.

Licorice flavonoid oil (LFO), which contains hydrophobic flavonoids from Glycyrrhiza glabra LINNE, is a new ingredient for functional foods. In this study, we investigated the anti-obesity action of LFO in diet-induced obese rats. The addition of 2% LFO in a high-fat diet significantly decreased the weight of abdominal adipose tissue and the levels of hepatic and plasma triglycerides. We found that the enzymatic activities of acetyl-CoA carboxylase and fatty acid synthase, the rate-limiting enzymes in the fatty acid synthetic pathway, were significantly decreased by LFO, whereas the enzymatic activity of acyl-CoA dehydrogenase, the rate-limiting enzyme in the fatty acid oxidative pathway, was significantly increased. All our findings suggest that the anti-obesity action of LFO is controlled by regulation of the rate-limiting enzymes in the fatty acid synthetic and oxidative pathways in the liver.

Central administration of glucagon suppresses food intake in chicks.

Food intake in chickens is regulated in a manner similar to that in mammals. Corticotropin-releasing factor (CRF), which increases the plasma corticosterone concentration, plays an important role as a mediator of many appetite-suppressive peptides in the central nervous system in both species. Central administration of glucagon suppresses food intake in rats. However, the anorexigenic action of glucagon in chicks has not yet been identified. In the present study, we investigated the effects of central administration of glucagon on food intake in chicks. Intracerebroventricular administration of glucagon in chicks significantly suppressed food intake and significantly induced hyperglycemia. In contrast, peripheral administration of the same dose of glucagon did not influence food intake and plasma glucose concentration. These results suggest that glucagon functions in chicks as an appetite-suppressive peptide in the central nervous system. Intracerebroventricular administration of glucagon in chicks also significantly increased CRF mRNA expression and plasma corticosterone concentration, suggesting that CRF acts as a downstream molecule for a glucagon-induced appetite-suppressive pathway in chicks. It is likely that the induction of hyperglycemia by central administration of glucagon is involved in its anorexigenic action, because peripheral administration of glucose in chicks suppressed food intake. These results suggest that CRF- and/or hyperglycemia-mediated pathways are involved in the anorexigenic action of glucagon in chicks.

Further studies on the anticancer activity of citrus limonoids.

Research in this laboratory has shown that some citrus limonoids can inhibit the development of 7,12-dimethylbenz[a]anthracene-induced oral tumors. The data from these studies have suggested that certain rings in the limonoid nucleus may be critical to antineoplastic activity. Using the hamster cheek pouch model, three new limonoids (ichangensin, deoxylimonin, and obacunone) have now been tested for cancer chemopreventive activity. In the first experiment, it was found that the treatments with ichangensin had no effect on tumor number or burden. In the second experiment, obacunone reduced tumor number and burden by 25 and 40%, respectively, whereas deoxylimonin reduced tumor number and burden by 30 and 50%, respectively. The results with deoxylimonin were significant, p < 0.05. Overall, the data indicated that changes in the A ring of the limonoid nucleus can lead to a loss of anticancer activity, whereas changes in the D ring can be tolerated without any apparent loss of biological activity.

The hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 in chicks.

The present study was carried out to investigate whether the hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 (GLP-1) in chicks. To examine this, Fos expression in the chick hypothalamus were immunohistochemically detected after intracerebroventricular (ICV) injection of 30-pmol GLP-1. ICV injection of GLP-1 stimulated the expression of Fos-like immunoreactive (FLI) cells in the ventromedial hypothalamic nucleus (VMN). When 15-pmol GLP-1 was directly injected into the chick VMN, the chick's food intake was significantly decreased compared with the control treatment. Microinjection of GLP-1 into the (LHA) also inhibited feeding in chicks, although ICV injection of GLP-1 did not stimulate FLI expression in the brain area. These results suggest that VMN and some brain regions are involved in the anorexic effect of GLP-1 in chicks.