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The number of individuals diagnosed with type 2 diabetes mellitus, which is caused by insulin resistance and/or abnormal insulin secretion, is increasing worldwide, creating a strong demand for the development of more effective anti-diabetic drugs. However, animal-based screening for anti-diabetic compounds requires sacrifice of a large number of diabetic animals, which presents issues in terms of animal welfare. Here, we established a method for evaluating the anti-diabetic effects of compounds using an invertebrate animal, the silkworm, Bombyx mori. Sugar levels in silkworm hemolymph increased immediately after feeding silkworms a high glucose-containing diet, resulting in impaired growth. Human insulin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, decreased the hemolymph sugar levels of the hyperglycemic silkworms and restored growth. Treatment of the isolated fat body with human insulin in an in vitro culture system increased total sugar in the fat body and stimulated Akt phosphorylation. These responses were inhibited by wortmannin, an inhibitor of phosphoinositide 3 kinase. Moreover, AICAR stimulated AMPK phosphorylation in the silkworm fat body. Administration of aminoguanidine, a Maillard reaction inhibitor, repressed the accumulation of Maillard reaction products (advanced glycation end-products; AGEs) in the hyperglycemic silkworms and restored growth, suggesting that the growth defect of hyperglycemic silkworms is caused by AGE accumulation in the hemolymph. Furthermore, we identified galactose as a hypoglycemic compound in jiou, an herbal medicine for diabetes, by monitoring its hypoglycemic activity in hyperglycemic silkworms. These results suggest that the hyperglycemic silkworm model is useful for identifying anti-diabetic drugs that show therapeutic effects in mammals.

Autoantibody production and immune complex (IC) formation are frequently observed in autoimmune diseases associated with bone loss. However, it has been poorly understood whether ICs regulate bone metabolism directly. Here we show that the level of osteoclastogenesis is determined by the strength of FcRγ signalling, which is dependent on the relative expression of positive and negative FcγRs (FcγRI/III/IV and IIB, respectively) as well as the availability of their ligands, ICs. Under physiological conditions, unexpectedly, FcγRIII inhibits osteoclastogenesis by depriving other osteoclastogenic Ig-like receptors of FcRγ. Fcgr2b −/− mice lose bone upon the onset of a hypergammaglobulinemia or the administration of IgG1 ICs, which act mainly through FcγRIII. The IgG2 IC activates osteoclastogenesis by binding to FcγRI and FcγRIV, which is induced under inflammatory conditions. These results demonstrate a link between the adaptive immunity and bone, suggesting a regulatory role for ICs in bone resorption in general, and not only in inflammatory diseases. Bone and the immune system are functionally intertwined. This study shows that osteoclastogenesis is modulated by the intensity of Fcγ receptor signalling, which is shaped by the balance between the positive and negative Fcγ receptors expressed on osteoclasts and the availability of their ligands, immune complexes.

We previously reported that sugar levels in the silkworm hemolymph, i.e., blood, increase immediately (within 1 h) after intake of a high-glucose diet and that the administration of human insulin decreases elevated hemolymph sugar levels in silkworms. In this hyperglycemic silkworm model, however, administration of pioglitazone or metformin, drugs used clinically for the treatment of type II diabetes, have no effect. Therefore, here we established a silkworm model of type II diabetes for the evaluation of anti-diabetic drugs such as pioglitazone and metformin. Silkworms fed a high-glucose diet over a long time-period (18 h) exhibited a hyperlipidemic phenotype. In these hyperlipidemic silkworms, phosphorylation of JNK, a stress-responsive protein kinase, was enhanced in the fat body, an organ that functionally resembles the mammalian liver and adipose tissue. Fat bodies isolated from hyperlipidemic silkworms exhibited decreased sensitivity to human insulin. The hyperlipidemic silkworms have impaired glucose tolerance, characterized by high fasting hemolymph sugar levels and higher hemolymph sugar levels in a glucose tolerance test. Administration of pioglitazone or metformin improved the glucose tolerance of the hyperlipidemic silkworms. These findings suggest that the hyperlipidemic silkworms are useful for evaluating the hypoglycemic activities of candidate drugs against type II diabetes.

Cell fate determination is tightly regulated by transcriptional activators and repressors. Leukemia/lymphoma-related factor (LRF; encoded by Zbtb7a), known as a POK (POZ/BTB and Krüppel) family transcriptional repressor, is induced during the development of bone-resorbing osteoclasts, but the physiological significance of LRF in bone metabolism and the molecular mechanisms underlying the transcriptional regulation of osteoclastogenesis by LRF have not been elucidated. Here we show that LRF negatively regulates osteoclast differentiation by repressing nuclear factor of activated T cells c1 (NFATc1) induction in the early phase of osteoclast development, while positively regulating osteoclast-specific genes by functioning as a coactivator of NFATc1 in the bone resorption phase. The stage-specific distinct functions of LRF were demonstrated in two lines of conditional knockout mice in which LRF was deleted in the early or late phase of osteoclast development. Thus, this study shows that LRF plays stage-specific distinct roles in osteoclast differentiation, exemplifying the delicate transcriptional regulation at work in lineage commitment.

Autoantibody production and immune complex (IC) formation are frequently observed in autoimmune diseases associated with bone loss. However, it has been poorly understood whether ICs regulate bone metabolism directly. Here we show that the level of osteoclastogenesis is determined by the strength of FcRγ signalling, which is dependent on the relative expression of positive and negative FcγRs (FcγRI/III/IV and IIB, respectively) as well as the availability of their ligands, ICs. Under physiological conditions, unexpectedly, FcγRIII inhibits osteoclastogenesis by depriving other osteoclastogenic Ig-like receptors of FcRγ. Fcgr2b-/- mice lose bone upon the onset of a hypergammaglobulinemia or the administration of IgG1 ICs, which act mainly through FcγRIII. The IgG2 IC activates osteoclastogenesis by binding to FcγRI and FcγRIV, which is induced under inflammatory conditions. These results demonstrate a link between the adaptive immunity and bone, suggesting a regulatory role for ICs in bone resorption in general, and not only in inflammatory diseases.

The PCR-based gene disruption strategy originally devised by Baudin et al. is widely used for gene targeting in Saccharomyces cerevisiae. An advantage of this strategy is its simplicity in making targeting constructs. The efficiencies of the targeted disruption are highly variable from locus to locus, however, and often very low. In this report, a method for improving the gene deletion efficiency is described.

Saccharomyces cerevisiae, for centuries the yeast that has been the workhorse for the fermentative production of ethanol, is now also a model system for biological research. The recent development of chromosome-splitting techniques has enabled the manipulation of the yeast genome on a large scale, and this has allowed us to explore questions with both biological and industrial relevance, the number of genes required for growth and the genome organization responsible for the ethanol production. To approach these questions, we successively deleted portions of the yeast genome and constructed a mutant that had lost about 5% of the genome and that gave an increased yield of ethanol and glycerol while showing levels of resistance to various stresses nearly equivalent to those of the parental strain. Further systematic deletion could lead to the formation of a eukaryotic cell with a minimum set of genes exhibiting appropriately altered regulation for enhanced metabolite production.