Stepwise cell fate decision pathways during osteoclastogenesis at single-cell resolution.

Osteoclasts are the exclusive bone-resorbing cells, playing a central role in bone metabolism, as well as the bone damage that occurs under pathological conditions1,2. In postnatal life, haematopoietic stem-cell-derived precursors give rise to osteoclasts in response to stimulation with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, both of which are produced by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes1-3. However, the precise mechanisms underlying cell fate specification during osteoclast differentiation remain unclear. Here, we report the transcriptional profiling of 7,228 murine cells undergoing in vitro osteoclastogenesis, describing the stepwise events that take place during the osteoclast fate decision process. Based on our single-cell transcriptomic dataset, we find that osteoclast precursor cells transiently express CD11c, and deletion of receptor activator of nuclear factor-κB specifically in CD11c-expressing cells inhibited osteoclast formation in vivo and in vitro. Furthermore, we identify Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) as the molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Together, the results of this study provide a detailed molecular road map of the osteoclast differentiation process, refining and expanding our understanding of the molecular mechanisms underlying osteoclastogenesis.

PHD3 regulates glucose metabolism by suppressing stress-induced signalling and optimising gluconeogenesis and insulin signalling in hepatocytes.

Glucagon-mediated gene transcription in the liver is critical for maintaining glucose homeostasis. Promoting the induction of gluconeogenic genes and blocking that of insulin receptor substrate (Irs)2 in hepatocytes contributes to the pathogenesis of type 2 diabetes. However, the molecular mechanism by which glucagon signalling regulates hepatocyte metabolism is not fully understood. We previously showed that a fasting-inducible signalling module consisting of general control non-repressed protein 5, co-regulator cAMP response element-binding protein binding protein/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2, and protein kinase A is required for glucagon-induced transcription of gluconeogenic genes. The present study aimed to identify the downstream effectors of this module in hepatocytes by examining glucagon-induced potential target genes. One of these genes was prolyl hydroxylase domain (PHD)3, which suppressed stress signalling through inhibition of the IκB kinase-nuclear factor-κB pathway in a proline hydroxylase-independent manner to maintain insulin signalling. PHD3 was also required for peroxisome proliferator-activated receptor γ coactivator 1α-induced gluconeogenesis, which was dependent on proline hydroxylase activity, suggesting that PHD3 regulates metabolism in response to glucagon as well as insulin. These findings demonstrate that glucagon-inducible PHD3 regulates glucose metabolism by suppressing stress signalling and optimising gluconeogenesis and insulin signalling in hepatocytes.

Cyanidin-3-glucoside enhances mitochondrial function and biogenesis in a human hepatocyte cell line.

Mitochondrial dysfunction has been identified as one of the primary factors contributing to liver diseases. Pathways that control mitochondrial biogenesis are potential therapeutic targets for the amelioration of hepatocyte dysfunction and liver disease. Research on natural pharmacological agents that ameliorate liver diseases has intensified over the last two decades. Cyanidin-3-glucoside (Cy3g), a dietary flavonoid compound extracted from a wide variety of fruits and vegetables, reportedly has several beneficial health effects. In this study, we used an adult human hepatoma cell line (HuH7) to investigate the effects of the Cy3g polyphenolic compound on mitochondrial function and biogenesis in vitro. An increase in intracellular mitochondrial reductase levels was observed after treatment with Cy3g, but cytotoxicity was not induced. In addition, mitochondrial membrane potential and ATP production were increased following Cy3g treatment. Cy3g treatment also resulted in a dose- and time-dependent upregulation of the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a transcription factor considered a master regulator of mitochondrial biogenesis and metabolism. Additionally, the expression of sirtuin 1 (SIRT1), which plays a key role in deacetylating PGC-1α, was also increased in a dose- and time-dependent manner. Cy3g treatment also increased the expression of downstream PGC-1α genes, nuclear respiratory factor 1 and mitochondrial transcription factor A (TFAM). Our results suggest that Cy3g has potential as a hepatoprotective therapeutic agent that enhances mitochondrial function and biogenesis in hepatocytes.

Comparison of physiological functions of antagonistic insulin-like peptides, INS-23 and INS-18, in Caenorhabditis elegans.

In Caenorhabditis elgans, insulin-like peptides have significant roles in modulating larval diapause and adult lifespan via the insulin/IGF-1 signaling (IIS) pathway. Although 40 insulin-like peptides (ILPs) have been identified, it remains unknown how ILPs act as either agonists or antagonists for their sole receptor, DAF-2. Here we found 1) INS-23 functions as an antagonistic ILP to promote larval diapause through the IIS pathway like a DAF-2 antagonist, INS-18, 2) INS-23 and INS-18 have similar biochemical functions. In addition, our molecular modeling suggests that INS-23 and INS-18 have characteristic insertions in the B-domain, which are crucial for the recognition of the insulin receptor, when compared with DAF-2 agonists. These characteristic insertions in the B-domain of INS-23 and INS-18 would modulate their intermolecular interactions with the DAF-2 receptor, which may lead these molecules to act as antagonistic ligands. Our study provides new insight into the function and structure of ILPs.

Upregulation of skeletal muscle PGC-1α through the elevation of cyclic AMP levels by Cyanidin-3-glucoside enhances exercise performance.

Regular exercise and physical training enhance physiological capacity and improve metabolic diseases. Skeletal muscles require peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) in the process of their adaptation to exercise owing to PGC-1α's ability to regulate mitochondrial biogenesis, angiogenesis, and oxidative metabolism. Cyanidin-3-glucoside (Cy3G) is a natural polyphenol and a nutraceutical factor, which has several beneficial effects on human health. Here, the effect of Cy3G on exercise performance and the underlying mechanisms involved were investigated. ICR mice were given Cy3G (1 mg/kg, orally) everyday and made to perform weight-loaded swimming exercise for 15 days. The endurance of mice orally administered with Cy3G was improved, enabling them to swim longer (time) and while the levels of exercise-induced lactate and fatigue markers (urea nitrogen, creatinine and total ketone bodies) were reduced. Additionally, the expression of lactate metabolism-related genes (lactate dehydrogenase B and monocarboxylate transporter 1) in gastrocnemius and biceps femoris muscles was increased in response to Cy3G-induced PGC-1α upregulation. In vitro, using C2C12 myotubes, Cy3G-induced elevation of intracellular cyclic AMP levels increased PGC-1α expression via the Ca2+/calmodulin-dependent protein kinase kinase pathway. This study demonstrates that Cy3G enhances exercise performance by activating lactate metabolism through skeletal muscle PGC-1α upregulation.

Increasing cAMP levels of preadipocytes by cyanidin-3-glucoside treatment induces the formation of beige phenotypes in 3T3-L1 adipocytes.

Obesity is a serious health problem and a major risk factor for the onset of several diseases such as heart disease, diabetes, stroke and cancer. The conversion of white adipocytes to brown-like adipocytes, also called beige or brite adipocytes, by pharmacological and dietary compounds has gained attention as an effective treatment for obesity. Cyanidin-3-glucoside (Cy3G), a polyphenolic compound contained in black soybean, blueberry and grape, has several antiobesity effects. However, there are no reports on the role of Cy3G in the induction of differentiation of preadipocytes to beige adipocytes and corresponding phenotypes. Here, the formation of beige adipocyte phenotypes following treatment with Cy3G was evaluated using 3T3-L1 adipocytes. Cy3G induced phenotypic changes to white adipocytes, such as increased multilocular lipid droplets and mitochondrial content. Additionally, the expression of mitochondrial genes (TFAM, SOD2, UCP-1 and UCP-2), UCP-1 protein and beige adipocyte markers (CITED1 and TBX1) in 3T3-L1 adipocytes was increased by Cy3G. Furthermore, Cy3G promoted preadipocyte differentiation by up-regulating of C/EBPß through the elevation of the intracellular cAMP levels. These results indicated that Cy3G elevates the intracellular cAMP levels, which induces beige adipocyte phenotypes. This is the first report on the effect of Cy3G on induction of differentiation of preadipocytes into beige adipocyte phenotypes.

Cyanidin-3-glucoside derived from black soybeans ameliorate type 2 diabetes through the induction of differentiation of preadipocytes into smaller and insulin-sensitive adipocytes.

Black soybean is a health food has been reported to have antidiabetes effect. The onset of diabetes is closely associated with adipocyte differentiation, and at present, the effect of black soybean on adipocyte differentiation is unknown. Here, we investigated the antidiabetes effect of black soybean, and its anthocyanin cyanidin-3-glucoside (Cy3G), on adipocyte differentiation. Orally administered black soybean seed coat extract (BSSCE) reduced the body and white adipose tissue (WAT) weight of db/db mice accompanied by a decrease in the size of adipocytes in WAT. Furthermore, 3T3-Ll cells treated with BSSCE and Cy3G were observed to differentiate into smaller adipocytes which correlated with increased PPARγ and C/EBPα gene expressions, increased adiponectin secretion, decreased tumor necrosis factor-α secretion, activation of insulin signalling and increased glucose uptake. C2C12 myotubes cultured with conditioned medium, obtained from 3T3-L1 adipocyte cultures treated with Cy3G, also showed significantly increased expression of PGC-1α, SIRT1 and UCP-3 genes. Here we report that BSSCE, as well as its active compound Cy3G, has antidiabetes effects on db/db mice by promoting adipocyte differentiation. This notion is supported by BSSCE and Cy3G inducing the differentiation of 3T3-L1 preadipocytes into smaller, insulin-sensitive adipocytes, and it induced the activation of skeletal muscle metabolism. This is the first report on the modulation effect of Cy3G on adipocyte differentiation.