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Background Skeletal muscle is mainly responsible for insulin‐stimulated glucose disposal. Dysfunction in skeletal muscle metabolism especially during obesity contributes to the insulin resistance. Astaxanthin (AX), a natural antioxidant, has been shown to ameliorate hepatic insulin resistance in obese mice. However, its effects in skeletal muscle are poorly understood. The current study aimed to investigate the molecular target of AX in ameliorating skeletal muscle insulin resistance. Methods We fed 6‐week‐old male C57BL/6J mice with normal chow (NC) or NC supplemented with AX (NC+AX) and high‐fat‐diet (HFD) or HFD supplemented with AX for 24 weeks. We determined the effect of AX on various parameters including insulin sensitivity, glucose uptake, inflammation, kinase signaling, gene expression, and mitochondrial function in muscle. We also determined energy metabolism in intact C2C12 cells treated with AX using the Seahorse XFe96 Extracellular Flux Analyzer and assessed the effect of AX on mitochondrial oxidative phosphorylation and mitochondrial biogenesis. Results AX‐treated HFD mice showed improved metabolic status with significant reduction in blood glucose, serum total triglycerides, and cholesterol (p< 0.05). AX‐treated HFD mice also showed improved glucose metabolism by enhancing glucose incorporation into peripheral target tissues, such as the skeletal muscle, rather than by suppressing gluconeogenesis in the liver as shown by hyperinsulinemic–euglycemic clamp study. AX activated AMPK in the skeletal muscle of the HFD mice and upregulated the expressions of transcriptional factors and coactivator, thereby inducing mitochondrial remodeling, including increased mitochondrial oxidative phosphorylation component and free fatty acid metabolism. We also assessed the effects of AX on mitochondrial biogenesis in the siRNA‐mediated AMPK‐depleted C2C12 cells and showed that the effect of AX was lost in the genetically AMPK‐depleted C2C12 cells. Collectively, AX treatment (i) significantly ameliorated insulin resistance and glucose intolerance through regulation of AMPK activation in the muscle, (ii) stimulated mitochondrial biogenesis in the muscle, (iii) enhanced exercise tolerance and exercise‐induced fatty acid metabolism, and (iv) exerted antiinflammatory effects via its antioxidant activity in adipose tissue. Conclusions We concluded that AX treatment stimulated mitochondrial biogenesis and significantly ameliorated insulin resistance through activation of AMPK pathway in the skeletal muscle.

Recently, obesity-induced insulin resistance, type 2 diabetes, and cardiovascular disease have become major social problems. We have previously shown that Astaxanthin (AX), which is a natural antioxidant, significantly ameliorates obesity-induced glucose intolerance and insulin resistance. It is well known that AX is a strong lipophilic antioxidant and has been shown to be beneficial for acute inflammation. However, the actual effects of AX on chronic inflammation in adipose tissue (AT) remain unclear. To observe the effects of AX on AT functions in obese mice, we fed six-week-old male C57BL/6J on high-fat-diet (HFD) supplemented with or without 0.02% of AX for 24 weeks. We determined the effect of AX at 10 and 24 weeks of HFD with or without AX on various parameters including insulin sensitivity, glucose tolerance, inflammation, and mitochondrial function in AT. We found that AX significantly reduced oxidative stress and macrophage infiltration into AT, as well as maintaining healthy AT function. Furthermore, AX prevented pathological AT remodeling probably caused by hypoxia in AT. Collectively, AX treatment exerted anti-inflammatory effects via its antioxidant activity in AT, maintained the vascular structure of AT and preserved the stem cells and progenitor’s niche, and enhanced anti-inflammatory hypoxia induction factor-2α-dominant hypoxic response. Through these mechanisms of action, it prevented the pathological remodeling of AT and maintained its integrity.

Muscle regeneration requires the coordination of muscle stem cells, mesenchymal fibro-adipogenic progenitors (FAPs), and macrophages. How macrophages regulate the paracrine secretion of FAPs during the recovery process remains elusive. Herein, we systemically investigated the communication between CD206

Meflin (Islr) expression has gained attention as a marker for mesenchymal stem cells, but its function remains largely unexplored. Here, we report the generation of Meflin-CreERT2 mice with CreERT2 inserted under the Meflin gene promoter to label Meflin-expressing cells genetically, thereby enabling their lineages to be traced. We found that in adult mice, Meflin-expressing lineage cells were present in adipose tissue stroma and had differentiated into mature adipocytes. These cells constituted Crown-like structures in the adipose tissue of mice after high-fat diet loading. Cold stimulation led to the differentiation of Meflin-expressing lineage cells into beige adipocytes. Thus, the Meflin-CreERT2 mouse line is a useful new tool for visualizing and tracking the lineage of Meflin-expressing cells.

Obesity and insulin resistance are associated with dysbiosis of the gut microbiota and impaired intestinal barrier function. Herein, we report that Bofutsushosan (BFT), a Japanese herbal medicine, Kampo, which has been clinically used for constipation in Asian countries, ameliorates glucose metabolism in mice with diet–induced obesity. A 16S rRNA sequence analysis of fecal samples showed that BFT dramatically increased the relative abundance of Verrucomicrobia, which was mainly associated with a bloom of Akkermansia muciniphila (AKK). BFT decreased the gut permeability as assessed by FITC-dextran gavage assay, associated with increased expression of tight-junction related protein, claudin-1, in the colon. The BFT treatment group also showed significant decreases of the plasma endotoxin level and expression of the hepatic lipopolysaccharide-binding protein. Antibiotic treatment abrogated the metabolic effects of BFT. Moreover, many of these changes could be reproduced when the cecal contents of BFT-treated donors were transferred to antibiotic-pretreated high fat diet-fed mice. These data demonstrate that BFT modifies the gut microbiota with an increase in AKK, which may contribute to improving gut barrier function and preventing metabolic endotoxemia, leading to attenuation of diet-induced inflammation and glucose intolerance. Understanding the interaction between a medicine and the gut microbiota may provide insights into new pharmacological targets to improve glucose metabolism.

Due to the increasing incidence of metabolic syndrome, the development of new therapeutic strategies is urgently required. One promising approach is to focus on the predisease state (so-called Mibyou in traditional Japanese medicine) before metabolic syndrome as a preemptive medical target. We recently succeeded in detecting a predisease state before metabolic syndrome using a mathematical theory called the dynamical network biomarker (DNB) theory. The detected predisease state was characterized by 147 DNB genes among a total of 24,217 genes in TSOD (Tsumura-Suzuki Obese Diabetes) mice, a well-accepted model of metabolic syndrome, at 5 weeks of age. The timing of the predisease state was much earlier than the onset of metabolic syndrome in TSOD mice reported to be at approximately 8–12 weeks of age. In the present study, we investigated whether the predisease state in TSOD mice can be inhibited by the oral administration of a Kampo formula, bofutsushosan (BTS), which is usually used to treat obese patients with metabolic syndrome in Japan, from 3 to 7 weeks of age. We found the comprehensive suppression of the early warning signals of the DNB genes by BTS at 5 weeks of age and later. Specifically, the standard deviations of 134 genes among the 147 DNB genes decreased at 5 weeks of age as compared to the nontreatment control group, and 80 of them showed more than 50% reduction. In addition, at 7 weeks of age, the body weight and blood glucose level were significantly lower in the BTS-treated group than in the nontreatment control group. The results of our study suggest a novel mechanism of BTS; it suppressed fluctuations of the DNB genes at the predisease state before metabolic syndrome and thus prevented the subsequent transition to metabolic syndrome. In conclusion, this study demonstrated the preventive and preemptive effects of a Kampo formula on Mibyou before metabolic syndrome for the first time based on scientific evaluation.

Beige adipocytes are an inducible form of thermogenic adipocytes that become interspersed within white adipose tissue (WAT) depots in response to cold exposure. Previous studies have shown that type 2 cytokines and M2 macrophages induce cold-induced browning in inguinal WAT (ingWAT) by producing catecholamines. Exactly how the conditional and partial depletion of CD206+ M2-like macrophages regulates the cold-induced browning of ingWAT, however, remains unknown. We examined the role of CD206+ M2-like macrophages in the cold-induced browning of WAT using genetically engineered CD206DTR mice, in which CD206+ M2-like macrophages were conditionally depleted. The partial depletion of CD206+ M2-like enhanced UCP1 expression in ingWAT, as shown by immunostaining, and also upregulated the expression of Ucp1 and other browning-related marker genes in ingWAT after cold exposure. A flow cytometry analysis showed that the partial depletion of CD206+ M2-like macrophages caused an increase in the number of beige progenitors in ingWAT in response to cold. Thus, we concluded that CD206+ M2-like macrophages inhibit the proliferation of beige progenitors and that the partial depletion of CD206+ M2-like macrophages releases this inhibition, thereby enhancing browning and insulin sensitivity.

Objective Expansion of adipose tissue during obesity through the recruitment of newly generated adipocytes (hyperplasia) is metabolically healthy, whereas that through the enlargement of pre-existing adipocytes (hypertrophy) leads to metabolic complications. Accumulating evidence from genetic fate mapping studies suggests that in animal models receiving a high-fat diet (HFD), only adipocyte progenitors (APs) in gonadal white adipose tissue (gWAT) have proliferative potential. However, the proliferative potential and differentiating capacity of APs in the inguinal WAT (iWAT) of male mice remains controversial. The objective of this study was to investigate the proliferative and adipogenic potential of APs in the iWAT of HFD-fed male mice. Methods We generated PDGFRα-GFP-Cre-ERT2/tdTomato (KI/td) mice and traced PDGFRα-positive APs in male mice fed HFD for 8 weeks. We performed a comprehensive phenotypic analysis, including the histology, immunohistochemistry, flow cytometry, and gene expression analysis, of KI/td mice fed HFD. Results Contrary to the findings of others, we found an increased number of newly generated tdTomato+ adipocytes in the iWAT of male mice, which was smaller than that observed in the gWAT. We found that in male mice, the iWAT has more proliferating tdTomato+ APs than the gWAT. We also found that tdTomato+ APs showed a higher expression of Dpp4 and Pi16 than tdTomato− APs, and the expression of these genes was significantly higher in the iWAT than in the gWAT of mice fed HFD for 8 weeks. Collectively, our results reveal that HFD feeding induces the proliferation of tdTomato+ APs in the iWAT of male mice. Conclusion In male mice, compared with gWAT, iWAT undergoes hyperplasia in response to 8 weeks of HFD feeding through the recruitment of newly generated adipocytes due to an abundance of APs with a high potential for proliferation and differentiation., Graphical abstract Image 1, Highlights • High-fat diet (HFD) increases proliferative APs in iWAT of male mice. • HFD promotes the recruitment of smaller adipocytes in the iWAT of male mice. • iWAT expands by recruiting newly-generated adipocytes (hyperplasia) in male mice. • gWAT expands by enlargement of pre-existing adipocytes (hypertrophy) in male mice.

Adipose tissue resident macrophages have important roles in the maintenance of tissue homeostasis and regulate insulin sensitivity for example by secreting pro-inflammatory or anti-inflammatory cytokines. Here, we show that M2-like macrophages in adipose tissue regulate systemic glucose homeostasis by inhibiting adipocyte progenitor proliferation via the CD206/TGFβ signaling pathway. We show that adipose tissue CD206+ cells are primarily M2-like macrophages, and ablation of CD206+ M2-like macrophages improves systemic insulin sensitivity, which was associated with an increased number of smaller adipocytes. Mice genetically engineered to have reduced numbers of CD206+ M2-like macrophages show a down-regulation of TGFβ signaling in adipose tissue, together with up-regulated proliferation and differentiation of adipocyte progenitors. Our findings indicate that CD206+ M2-like macrophages in adipose tissues create a microenvironment that inhibits growth and differentiation of adipocyte progenitors and, thereby, control adiposity and systemic insulin sensitivity. Adipose tissue contains macrophages that can influence both local and systemic metabolism via the secretion of cytokines. Here, Nawaz et al. report that M2-like macrophages, present in adipose tissue, create a microenvironment that inhibits proliferation of adipocyte progenitors due to the secretion of TGF-β1

Adipose tissue not only functions as the major energy-storing tissue, but also functions as an endocrine organ that regulates systemic metabolism by releasing various hormones called adipokines. Macrophages play a critical role in maintaining adipocyte health in a lean state and in remodeling during the progression of obesity. Large numbers of classically activated (M1) macrophages accumulate in adipose tissue as adipocytes become larger because of excessive energy conditions, and they adversely affect insulin resistance by triggering local and systemic inflammation. In contrast, alternatively activated (M2) macrophages seem to maintain the health of adipose tissues in a lean state. In addition, they play a role in adapting to excess energy states, because M2 macrophage dysfunction caused by genetic disruption of the M2 gene results in metabolic disorders under high-fat-fed conditions that are probably attributable to their anti-inflammatory functions. Nonetheless, how M2 macrophages contribute to maintaining the health of adipose tissue and therefore to insulin sensitivity is largely unknown. In this article, we review the literature on the role of M1 and M2 macrophages in metabolism, with a special focus on the role of M2 macrophages in adipose tissue. Likewise, we raise topics of M2 macrophages in non-adipose tissues to expand our understanding of macrophage heterogeneity.

Adipose tissue hypoxia is an important feature of pathological adipose tissue expansion. Hypoxia-inducible factor-1α (HIF-1α) in adipocytes reportedly induces oxidative stress and fibrosis, rather than neoangiogenesis via vascular endothelial growth factor (VEGF)-A. We previously reported that macrophages in crown-like structures (CLSs) are both hypoxic and inflammatory. In the current study, we examined how macrophage HIF-1α is involved in high-fat diet (HFD)–induced inflammation, neovascularization, hypoxia, and insulin resistance using mice with myeloid cell–specific HIF-1α deletion that were fed an HFD. Myeloid cell–specific HIF-1α gene deletion protected against HFD-induced inflammation, CLS formation, poor vasculature development in the adipose tissue, and systemic insulin resistance. Despite a reduced expression of Vegfa in epididymal white adipose tissue (eWAT), the preadipocytes and endothelial cells of HIF-1α–deficient mice expressed higher levels of angiogenic factors, including Vegfa, Angpt1, Fgf1, and Fgf10 in accordance with preferable eWAT remodeling. Our in vitro study revealed that lipopolysaccharide-treated bone marrow–derived macrophages directly inhibited the expression of angiogenic factors in 3T3-L1 preadipocytes. Thus, macrophage HIF-1α is involved not only in the formation of CLSs, further enhancing the inflammatory responses, but also in the inhibition of neoangiogenesis in preadipocytes. We concluded that these two pathways contribute to the obesity-related physiology of pathological adipose tissue expansion, thus causing systemic insulin resistance.

Increased incidence and prevalence of pediatric inflammatory bowel disease (IBD) have been reported in Western countries. Changes in the prevalence of pediatric IBD in Asian countries, however, remain unclear. We evaluated the changes in the prevalence of IBD among Japanese adults and children from 2004 to 2013, by using the Japanese national registry data of patients receiving financial aid. Data from children (ages 0-19 years) were compared with those from young adults (ages 20-39 years). In 2004, age-standardized prevalences of Crohn disease (CD) and ulcerative colitis (UC) among children were 4.2 of 100,000 and 11.0 of 100,000, respectively. The corresponding prevalences among young adults were 41.0 of 100,000 and 89.8 of 100,000, respectively. In 2013, age-standardized prevalences of pediatric CD and UC were 7.2 of 100,000 and 15.0 of 100,000, respectively. During this period, prevalence of pediatric CD increased by 73.8% among children and by 49.0% in young adults. The prevalence of UC increased by 45.0% among children, and by 73.7% among young adults.

The establishment of new therapeutic strategies for metabolic syndrome is urgently needed because metabolic syndrome, which is characterized by several disorders, such as hypertension, increases the risk of lifestyle-related diseases. One approach is to focus on the pre-disease state, a state with high susceptibility before the disease onset, which is considered as the best period for preventive treatment. In order to detect the pre-disease state, we recently proposed mathematical theory called the dynamical network biomarker (DNB) theory based on the critical transition paradigm. Here, we investigated time-course gene expression profiles of a mouse model of metabolic syndrome using 64 whole-genome microarrays based on the DNB theory, and showed the detection of a pre-disease state before metabolic syndrome defined by characteristic behavior of 147 DNB genes. The results of our study demonstrating the existence of a notable pre-disease state before metabolic syndrome may help to design novel and effective therapeutic strategies for preventing metabolic syndrome, enabling just-in-time preemptive interventions.

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Sirt1 plays an important role in regulating glucose and lipid metabolism in obese animal models. Impaired adipose tissue angiogenesis in the obese state decreases adipogenesis and thereby contributes to glucose intolerance and lipid metabolism. However, the mechanism by which Sirt1 activation affects obesity-associated impairments in angiogenesis in the adipose tissue is not fully understood. Here, we show that SRT1720 treatment induces angiogenic genes in cultured 3T3-L1 preadipocytes and ex vivo preadipocytes. siRNA-mediated knockdown of Sirt1 in 3T3-L1 preadipocytes downregulated angiogenic genes in the preadipocytes. SRT1720 treatment upregulated metabolically favorable genes and reduced inflammatory gene expressions in the adipose tissue of diet-induced obese (DIO) mice. Collectively, these findings suggest a novel role of SRT1720-induced Sirt1 activation in the induction of angiogenic genes in preadipocytes, thereby reducing inflammation and fibrosis in white adipose tissue (WAT) and promoting insulin sensitivity.

We have previously shown that CD2M2-like macrophages constitute a microenvironment for adipocyte progenitors (APs), in a Tgfβ-dependent manner, to retain systemic insulin sensitivity by tuning the quiescence/proliferation balance of APs to adapt to changes in nutritional status. Previously we generated CD206DTR transgenic mice and CD206-CreERT2/ Tgfβ1flox/flox (Tgfβ1 KO) mice to determine the role of CD2M2-like macrophages a role of CD206+ cell specific Tgfβ1 respectively, in the APs proliferation. Previous reports have shown that M2-like macrophages play a key role in the browning of WAT via activation of type 2 cytokines production during cold exposure. These findings revealed the relationship between the occurrence of beige adipocytes and M2-like macrophage recruitment. In order to investigate the role of CD2M2-like macrophages in the induction of browning into the WAT, we extended our study to see the browning phenomenon by using CD206DTR and Tgfβ1 KO mice. Here, we show that depletion of CD2M2-like macrophages promotes the browning of WAT. We report that cold stimulation resulted in the generation of smaller adipocytes, upregulation of UCP1 and CD137, a marker for beige progenitors, in the WAT of CD206-ablated mice. Flow cytometry analysis further confirmed enhanced beige progenitors in the WAT. Since blocking of Tgfβ is known to promote browning of the WAT. Mechanistically, we assume that enhanced browning into the WAT of CD206DTR mice might be due to the deletion of Tgfβ1 from CD206-M2-like macrophages. Taken together, we concluded that CD2M2-like macrophages induce the proliferation of beige progenitors in the WAT, which may serve as effective therapeutic tools for the prevention and treatment of obesity. Disclosure A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. I. Usui: None. K. Yagi: None. T. Nakagawa: None. T. Kado: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura & Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. Y. Igarashi: None. K. Okabe: None. K. Saeki: None.

Bofutsushosan (BFT), an oriental herbal medicine, has been clinically used for obese patients. To explore the impact of BFT on glucose metabolism, male C57BL6 mice were fed on high-fat diet (HFD) for 12 weeks and administered either BFT (25 mg /day) or saline for the last 8 weeks. Oral glucose tolerance test and insulin tolerance test revealed improved glucose metabolism with improved insulin sensitivity in BFT treated mice, which was associated with decreased inflammatory gene expressions and improved insulin signaling in the adipose tissue. Whereas the weight of liver and adipose tissue was not changed, cecum weight was significantly increased by BFT. 16S rRNA sequence analysis of fecal samples showed that microbial composition was markedly changed. BFT reduced the relative abundance of Bacteroidetes from 52% to 34%, whereas it increased Verrucomicrobia from 3.4% to 24%. An increase in Verrucomicrobia was mainly associated with Akkermansia mucuniphila (Akk). The bloom of Akk was observed at one week of BFT treatment. Consistent with the previous reports that Akk improves gut barrier function and prevents from metabolic endotoxemia in obese subjects, BFT decreased the gut permeability as assessed by FITC-dextran gavage assay, which was associated with increased claudin-1 protein in the colon. Furthermore, plasma endotoxin level and hepatic lipopolysaccharide binding protein expression were significantly decreased in BFT group. Antibiotic treatment canceled the metabolic effect of BFT. Moreover, when the gut microbiota of BFT-treated mice were transferred to HFD-fed mice, glucose metabolism was significantly improved with decreased diet -induced inflammation with AKK bloom. These data demonstrate that BFT increases Akk in the gut, which may contribute to improving gut barrier function and preventing metabolic endotoxemia, leading to improved diet-induced inflammation, thereby controlling glucose metabolism. Disclosure S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. I. Usui: None. A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. Y. Igarashi: None. T. Kado: None. K. Okabe: None. K. Yagi: None. T. Nakagawa: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura & Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co.

During cell differentiation, intracellular energetic pathways are drastically reorganized. Although this alteration had been considered as a passive process to adapt to environmental change during differentiation, recent studies suggested that the metabolic alteration rather actively regulates the differentiation process. It is known that the dynamic metabolic reprogramming occurs during differentiation of preadipocytes, however its implication in adipogenesis is unknown. In this study, we employed mass spectrometry-based metabolomics and investigated the metabolic change during differentiation of 3T3-L1 preadipocytes. We found that the level of nicotinamide adenine dinucleotide (NAD), a cofactor mediating redox reaction and protein modification, was significantly increased during the differentiation. Consistently Nampt, a rate-limiting enzyme of NAD synthesis, was also upregulated in the early phase of adipogenesis. Further, we found that pharmacological or genetic inhibition of Nampt reduced NAD synthesis and repressed the metabolic reprogramming during adipogenesis. Notably, inhibition of Nampt blocked the gene expression of Pparg and impeded the preadipocyte differentiation. The effect of Nampt inhibition was cancelled by the supplementation of nicotinamide mononucleotide, a precursor of NAD. Our metabolomic analysis also revealed that TCA cycle intermediate, α-ketoglutarate (αKG) was upregulated during the differentiation and Nampt inhibition disturbed the rise of αKG. Interestingly, αKG is known as a cofactor of the demethylation of histones. The ChIP assay revealed that αKG mediated the demethylation of H3K9me3 on Pparg promoter region and promote the gene expression of Pparg during adipogenesis. This process was inhibited by Nampt inhibitor treatment, and was reversed by the supplementation of αKG. Altogether, our data indicated that Nampt-mediated NAD synthesis is necessary for differentiation of preadipocyte. Disclosure K. Okabe: None. I. Usui: None. A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. T. Kado: None. Y. Igarashi: None. K. Yagi: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura & Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. T. Nakagawa: None.

Influenza-associated encephalitis and encephalopathy (IAE) is a severe complication of influenza infection with high morbidity and mortality. We present the case of a patient with IL-10RA mutation who developed encephalopathy after influenza infection. A 10-day-old boy developed recurrent fever and anal fistula. Growth failure gradually became apparent. He had been treated with antibiotics and elemental nutrition. However, the patient did not respond to the treatments. At 11 months, he suddenly developed shock with encephalopathy and multiple organ failures. He was then diagnosed with IAE. A cytokine study revealed elevated levels of IL-1 receptor antagonist, IL-2, IL-6, IL-8, IP-10, eotaxin, G-CSF, MCP-1, and IL-10. These cytokines are normally downregulated by IL-10. Genetic testing revealed a IL-10RA mutation at the 3′ end of exon 4 (c.537G→A). These findings might reflect an increased risk of severe IAE in patients with IL-10RA mutation.

The objectives of this study were to determine the effects of deoxyshikonin on lymphangiogenesis. Deoxyshikonin enhanced the ability of human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) to undergo time-dependentin vitrocord formation. Interestingly, an opposite result was observed in cells treated with shikonin. The increased cord formation ability following deoxyshikonin treatment correlated with increased VEGF-C mRNA expression to higher levels than seen for VEGF-A and VEGF-D mRNA expression. We also found that deoxyshikonin regulated cord formation of HMVEC-dLy by increasing the HIF-1αmRNA level, HIF-1αprotein level, and the accumulation of HIF-1αin the nucleus. Knockdown of the HIF-1αgene by transfection with siHIF-1αdecreased VEGF-C mRNA expression and cord formation ability in HMVEC-dLy. Deoxyshikonin treatment could not recover VEGF-C mRNA expression and cord formation ability in HIF-1αknockdown cells. This indicated that deoxyshikonin induction of VEGF-C mRNA expression and cord formation in HMVEC-dLy on Matrigel occurred mainly via HIF-1αregulation. We also found that deoxyshikonin promoted wound healingin vitroby the induction of HMVEC-dLy migration into the wound gap. This study describes a new effect of deoxyshikonin, namely, the promotion of cord formation by human endothelial cells via the regulation of HIF-1α. The findings suggest that deoxyshikonin may be a new drug candidate for wound healing and treatment of lymphatic diseases.

Adipose tissue resident macrophages have important roles in the maintenance of tissue homeostasis and regulate insulin sensitivity for example by secreting pro-inflammatory or anti-inflammatory cytokines. Here, we show that M2-like macrophages in adipose tissue regulate systemic glucose homeostasis by inhibiting adipocyte progenitor proliferation via the CD206/TGFβ signaling pathway. We show that adipose tissue CD206+ cells are primarily M2-like macrophages, and ablation of CD206+ M2-like macrophages improves systemic insulin sensitivity, which was associated with an increased number of smaller adipocytes. Mice genetically engineered to have reduced numbers of CD206+ M2-like macrophages show a down-regulation of TGFβ signaling in adipose tissue, together with up-regulated proliferation and differentiation of adipocyte progenitors. Our findings indicate that CD206+ M2-like macrophages in adipose tissues create a microenvironment that inhibits growth and differentiation of adipocyte progenitors and, thereby, control adiposity and systemic insulin sensitivity., Adipose tissue contains macrophages that can influence both local and systemic metabolism via the secretion of cytokines. Here, Nawaz et al. report that M2-like macrophages, present in adipose tissue, create a microenvironment that inhibits proliferation of adipocyte progenitors due to the secretion of TGF-β1

Colorectal cancer (CRC) is a typical lifestyle-related disease, and it metastasizes mostly to the liver. It is important to understand the molecular mechanisms of CRC metastasis in order to design new and effective treatments for CRC patients. Chemokines are known to have antitumor effects as their chemoattractant properties stimulate the accumulation of infiltrating immune cells (TILs) in tumors. Chemokine (C-X-C motif) ligand 16 (CXCL16), also known as SR-PSOX, is a unique membrane-bound chemokine that induces the expression of its specific receptor CXCR6. We previously reported that the expression of CXCL16 by cancer cells enhances the recruitment of TILs, thereby improving the prognosis of CRC. It has since been reported that CXCL16/CXCR6 expression is involved in the metastasis of various types of cancer. However, there is no report of the association between CXCL16 expression and liver metastasis in CRC. In this study, we investigated the role of cancer-derived CXCL16 and the possibility of gene therapy using CXCL16. Therefore, we examined the metastasis of colon 38 SL4 cells to the liver in an experimental model. Following injection of cancer cells into the intraportal vein, CXCL16-expressing CRC cells drastically inhibited liver metastasis. We also found that CD8 T cells and natural killer T (NKT) cells, known as CXCR6-expressing cells, increased in CXCL16-expressing metastatic tissue. Collectively, the inhibitory effect on metastasis to the liver by CXCL16 was observed in NKT cell-depleted mice but not in CD8 T cell-depleted mice. These results demonstrate the inhibitory effect of CXCL16 on liver metastasis via NKT cells in CRC.

Background Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients. Methods We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages. Results CXCL16 expression enhanced TNF-α-induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-κB-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF-α-induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF-α-induced apoptosis through the induction of M1 macrophages, which released TNF-α. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis. Conclusions Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-949) contains supplementary material, which is available to authorized users.

The objectives of this study were to determine the effects of deoxyshikonin on lymphangiogenesis. Deoxyshikonin enhanced the ability of human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) to undergo time-dependent in vitro cord formation. Interestingly, an opposite result was observed in cells treated with shikonin. The increased cord formation ability following deoxyshikonin treatment correlated with increased VEGF-C mRNA expression to higher levels than seen for VEGF-A and VEGF-D mRNA expression. We also found that deoxyshikonin regulated cord formation of HMVEC-dLy by increasing the HIF-1 α mRNA level, HIF-1 α protein level, and the accumulation of HIF-1 α in the nucleus. Knockdown of the HIF-1 α gene by transfection with siHIF-1 α decreased VEGF-C mRNA expression and cord formation ability in HMVEC-dLy. Deoxyshikonin treatment could not recover VEGF-C mRNA expression and cord formation ability in HIF-1 α knockdown cells. This indicated that deoxyshikonin induction of VEGF-C mRNA expression and cord formation in HMVEC-dLy on Matrigel occurred mainly via HIF-1 α regulation. We also found that deoxyshikonin promoted wound healing in vitro by the induction of HMVEC-dLy migration into the wound gap. This study describes a new effect of deoxyshikonin, namely, the promotion of cord formation by human endothelial cells via the regulation of HIF-1 α . The findings suggest that deoxyshikonin may be a new drug candidate for wound healing and treatment of lymphatic diseases.

beta2-Glycoprotein I (beta2-GPI) consists of five repeats of a homologous domain. We designed a series of human beta2-GPI mutant genes, ie, three mutant genes lacking the domain(s) present in the NH2- terminal region and two of those present in the COOH-terminal region. These mutant genes were expressed in Spodoptera frugiperda insect cells (Sf9) infected with recombinant baculoviruses and the mutant proteins were secreted into the culture medium. The molecular mass of the purified mutant proteins, estimated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, was fairly consistent with the size calculated from their nucleotide sequences. Binding of beta2-GPI to solid-phase cardiolipin (CL) was diminished by the deletion of the fifth domain (domain V) from its complete structure. Thus, the phospholipid binding site of beta2-GPI is located on its domain V. Monoclonal anti-CL antibodies (aCL) derived either from NZW x BXSB (WB) F1 mice or from patients with antiphospholipid syndrome bound directly to the domain V-deleted mutant protein (DI-IV) absorbed not only on an oxygenated but also on a plain polystyrene surface. We conclude from this study that the epitope for aCL is exposed on a conformationally changed structure of beta2-GPI by interacting with negatively charged phospholipid or on the mutant protein, DI-IV.

Anticardiolipin antibodies (aCL) derived from the sera of individuals exhibiting the antiphospholipid syndrome (APS) directly bind to beta 2-glycoprotein I (beta 2-GPI), which is adsorbed to an oxidized polystyrene surface. Oxygen atoms were introduced on a polystyrene surface by irradiation with electron or gamma-ray radiation. X-ray photoelectron spectroscopy revealed the irradiated surfaces were oxidized to generate C-O and C = O moieties. aCL derived from either APS patients or (NZW x BXSB)F1 mice bound to beta 2-GPI coated on the irradiated plates, depending on the radiation dose. Antibody binding to beta 2-GPI on the irradiated plates was competitively inhibited by simultaneous addition of cardiolipin (CL)-coated latex beads mixed together with beta 2-GPI but were unaffected by addition of excess beta 2-GPI, CL micelles, or CL-coated latex beads alone. There was a high correlation between binding values of aCL in sera from 40 APS patients obtained by the anti-beta 2-GPI enzyme-linked immunosorbent assay (ELISA) using the irradiated plates and those by the beta 2-GPI-dependent aCL ELISA. Therefore, aCL have specificity for an epitope on beta 2-GPI. This epitope is expressed by a conformational change occurring when beta 2-GPI interacts with an oxygen-substituted solid phase surface.

SUMMARY A full-length cDNA coding a human β2-glycoprotein I (β2-GPI) was introduced into the baculovirus genome to construct a recombinant baculovirus. Spodoptera frugiperda (Sf9) cells were infected with the recombinant baculovirus. A protein (mol. wt 43000) reactive with anti-β2-GPI antisera was produced in the insect cells and secreted into the culture medium. The recombinant β2-GPI was purified from the culture supernatant by sequential cardiolipin (CL)-affinity column chromatography and gel filtration. The N-terminal amino acid sequence of the protein was identical to that of the native β2:-GPI purified from human sera, and a putative signal peptide was cleaved from the secreted form of the recombinant protein. The purified recombinant protein had a cofactor activity which enhances CL binding of anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE) patients, as well as the native β2-GPI. Thus, the β2-GPI expressed in insect cells is an immunologically active cofactor.

Human beta 2-glycoprotein I (beta 2-GPI) is involved in cardiolipin (CL) binding of anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE). We examined the inter-species differences of beta 2-GPI in alternation of CL binding of aCL. beta 2-GPI preparations were obtained from human, bovine, and rat sera by sequential CL--polyacrylamide affinity, DEAE--cellulose, and anti-human IgG-conjugated Sepharose CL-4B column chromatography, and they had apparent molecular weights of 50, 53, and 55 kDa respectively. Human beta 2-GPI not only enhanced CL binding by aCL in SLE but also depressed it by those in syphilis. Either bovine and rat beta 2-GPI exerted no or quite small inhibition of the binding of syphilitic aCL compared with human beta 2-GPI whereas all three species of beta 2-GPI generated binding of aCL in SLE to a similar degree. Further, a complete cDNA clone, p beta 2-GPI, was isolated from a human hepatoma cell line, HepG2, and its nucleotide sequence was analyzed. The sequences of bovine and rat counterpart molecules (beta 2-GPI) are highly homologous to that of the deduced sequence, and their corresponding regions are 84.0 and 82.5% identical to the complete domain and to the amino acid sequence 53-326 of human beta 2-GPI respectively. One of major differences appears at position 154 in human beta 2-GPI, and might be associated with the inhibitory effect on the binding of syphilitic aCL. The sequencing analysis of these beta 2-GPI proteins might provide leads to functional sites of domains which would be associated with such serological phenomena.