Ibuki Shirakawa

Activating Transcription Factor 3 Constitutes a Negative Feedback Mechanism That Attenuates Saturated Fatty Acid/Toll-Like Receptor 4 Signaling and Macrophage Activation in Obese Adipose Tissue

Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid–stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor-α production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor-α promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKAy mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation.

Macrophage-inducible C-type lectin underlies obesity-induced adipose tissue fibrosis

In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation.

The role of circulating precursors in vascular repair and lesion formation

The accumulation of smooth muscle cells (SMCs) plays a principal role in atherogenesis, post‐angioplasty restenosis and transplantation‐associated vasculopathy. Therefore, much effort has been expended in targeting the migration and proliferation of medial smooth muscle cells to prevent occlusive vascular remodeling. Recent evidence suggests that bone marrow‐derived circulating precursors can also give rise to endothelial cells and smooth muscle cells that contribute to vascular repair, remodeling, and lesion formation under physiological and pathological conditions. This article overviews recent findings on circulating vascular progenitor cells and describes potential therapeutic strategies that target these cells to treat occlusive vascular diseases.

Increased Expression of Macrophage-Inducible C-type Lectin in Adipose Tissue of Obese Mice and Humans

OBJECTIVE We have provided evidence that saturated fatty acids, which are released from adipocytes via macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for the Toll-like receptor (TLR) 4 complex in macrophages, thereby aggravating obesity-induced adipose tissue inflammation. The aim of this study was to identify the molecule(s) activated in adipose tissue macrophages in obesity. RESEARCH DESIGN AND METHODS We performed a cDNA microarray analysis of coculture of 3T3-L1 adipocytes and RAW264 macrophages. Cultured adipocytes and macrophages and the adipose tissue of obese mice and humans were used to examine mRNA and protein expression. RESULTS We found that macrophage-inducible C-type lectin (Mincle; also called Clec4e and Clecsf9), a type II transmembrane C-type lectin, is induced selectively in macrophages during the interaction between adipocytes and macrophages. Treatment with palmitate, a major saturated fatty acid released from 3T3-L1 adipocytes, induced Mincle mRNA expression in macrophages at least partly through the TLR4/nuclear factor (NF)-κB pathway. Mincle mRNA expression was increased in parallel with macrophage markers in the adipose tissue of obese mice and humans. The obesity-induced increase in Mincle mRNA expression was markedly attenuated in C3H/HeJ mice with defective TLR4 signaling relative to control C3H/HeN mice. Notably, Mincle mRNA was expressed in bone-marrow cell (BMC)-derived proinflammatory M1 macrophages rather than in BMC-derived anti-inflammatory M2 macrophages in vitro. CONCLUSIONS Our data suggest that Mincle is induced in adipose tissue macrophages in obesity at least partly through the saturated fatty acid/TLR4/NF-κB pathway, thereby suggesting its pathophysiologic role in obesity-induced adipose tissue inflammation.

Activating Transcription Factor 4 Links Metabolic Stress to Interleukin-6 Expression in Macrophages

Chronic inflammation is a molecular element of the metabolic syndrome and type 2 diabetes. Saturated fatty acids (SFAs) are considered to be an important proinflammatory factor. However, it is still incompletely understood how SFAs induce proinflammatory cytokine expression. Hereby we report that activating transcription factor (ATF) 4, a transcription factor that is induced downstream of metabolic stresses including endoplasmic reticulum (ER) stress, plays critical roles in SFA-induced interleukin-6 (Il6) expression. DNA microarray analysis using primary macrophages revealed that the ATF4 pathway is activated by SFAs. Haploinsufficiency and short hairpin RNA–based knockdown of ATF4 in macrophages markedly inhibited SFA- and metabolic stress–induced Il6 expression. Conversely, pharmacological activation of the ATF4 pathway and overexpression of ATF4 resulted in enhanced Il6 expression. Moreover, ATF4 acts in synergy with the Toll-like receptor-4 signaling pathway, which is known to be activated by SFAs. At a molecular level, we found that ATF4 exerts its proinflammatory effects through at least two different mechanisms: ATF4 is involved in SFA-induced nuclear factor-κB activation; and ATF4 directly activates the Il6 promoter. These findings provide evidence suggesting that ATF4 links metabolic stress and Il6 expression in macrophages.

Fluvastatin accelerates re-endothelialization impaired by local sirolimus treatment

Sirolimus-eluting stent reduces restenosis after percutaneous coronary intervention. However, accumulating evidence suggests that sirolimus potentially affects re-endothelialization, leading to late thrombosis. Statins have protective effects on endothelium. Recently, statins are reported to increase the number of circulating endothelial progenitor cells (EPCs) and accelerate re-endothelialization after vascular injury. Here, we tested the hypothesis that fluvastatin has beneficial effect on re-endothelialization after local sirolimus treatment. We performed wire-mediated vascular injury to both sides of femoral arteries of wild-type mice and bone marrow chimeric mice. Either sirolimus (100 microg) or DMSO was administered locally to the perivascular area of the injured arteries. All mice received either fluvastatin (5 mg/kg/day) or vehicle by gavage starting at one week before the surgery until sacrifice. At 4 weeks after the surgery, re-endothelialization of the sirolimus-treated artery was significantly less than that of DMSO-treated one in the vehicle-treated mice as determined by the percentage of CD31-positive area (P<0.05). Systemic administration of fluvastatin accelerated the re-endothelialization in the sirolimus-treated artery to the similar degree of that in the DMSO-treated artery (P=NS). Contribution of bone marrow-derived cells to re-endothelialization was seldom observed in bone marrow chimeric mice regardless of fluvastatin administration. Fluvastatin significantly ameliorated proliferation (2.5-folds) and migration activities (2.3-folds) of mature endothelial cells impaired by sirolimus treatment (P<0.05, respectively). Fluvastatin increased endothelial nitric oxide synthase expression and decreased plasminogen activator inhibitor-1 expression in mature endothelial cell in the presence of sirolimus (P<0.05, respectively). Our findings suggest that fluvastatin has protective effects against impaired re-endothelialization after sirolimus treatment.

Evidence for load-bearing structures specialized for the catch state in Mytilus smooth muscle

Abstract To give information about the catch state, i.e. a prolonged tonic contraction maintained with very little energy expenditure, sinusoidal vibrations (peak-to-peak amplitude, 0.5% of L 0 , frequency, 10 Hz) and ramp releases (≤2% of slack length, L 0 , complete in 4 ms) were applied to the anterior byssal retractor muscle (ABRM) fibers of a bivalve mollusc Mytilus edulis in various states. The mean force level of force responses to sinusoidal vibration decreases with time during the force development with 10 −3 M acetylcholine (ACh), but remained unchanged during the catch state attained after removal of ACh and during the accelerated relaxation with 10 −6 M 5-hydroxytryptamine (5-HT). Muscle fiber stiffness, determined from the amplitude of the force response to sinusoidal vibration, approached a finite value (20–25% of the maximum value) as the isometric force decreased towards zero during the catch state. The force–extension curves of the series elastic component (SEC) during the catch state was steeper at low force levels (≤50% of the maximum isometric force) than those during the force development and the accelerated relaxation. These results can be taken as evidence for the load-bearing system specialized for the catch state.

Synthetic “smart gel” provides glucose-responsive insulin delivery in diabetic mice

A synthetic polymer gel–based insulin delivery device provides an artificial pancreas–like function in healthy and diabetic mice. Although previous studies have attempted to create “electronics-free” insulin delivery systems using glucose oxidase and sugar-binding lectins as a glucose-sensing mechanism, no successful clinical translation has hitherto been made. These protein-based materials are intolerant of long-term use and storage because of their denaturing and/or cytotoxic properties. We provide a solution by designing a protein-free and totally synthetic material–based approach. Capitalizing on the sugar-responsive properties of boronic acid, we have established a synthetic polymer gel–based insulin delivery device confined within a single catheter, which exhibits an artificial pancreas–like function in vivo. Subcutaneous implantation of the device in healthy and diabetic mice establishes a closed-loop system composed of “continuous glucose sensing” and “skin layer”–regulated insulin release. As a result, glucose metabolism was controlled in response to interstitial glucose fluctuation under both insulin-deficient and insulin-resistant conditions with at least 3-week durability. Our “smart gel” technology could offer a user-friendly and remarkably economic (disposable) alternative to the current state of the art, thereby facilitating availability of effective insulin treatment not only to diabetic patients in developing countries but also to those patients who otherwise may not be strongly motivated, such as the elderly, infants, and patients in need of nursing care.

Effect of deuterium oxide on actomyosin motility in vitro

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D(2)O) and water (H(2)O) to examine the effect of D(2)O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D(2)O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H(2)O assay solution, but the maximum velocity (4.1+/-0.5 microm/s, n=11) at pD 8.5 in D(2)O was about 60% of that (7.1+/-1.1 microm/s, n=11) at pH 8.5 in H(2)O. The K(m) values of 95 and 80 microM and V(max) values of 3.2 and 5.1 microm/s for the D(2)O and H(2)O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The K(m) value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 microM [actin] in H(2)O to 33 microM [actin] in D(2)O without any significant changes in V(max) (9.4 s(-1) in D(2)O and 9.3 s(-1) in H(2)O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361+/-26 s(-1) (n=27) in D(2)O and 512+/-39 s(-1) (n=27) in H(2)O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D(2)O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D(2)O.

In vitro actomyosin motility in deuterium oxide.

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D2O) and water (H2O) to examine the effect of D2O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D2O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H2O assay solution, but the maximum velocity (4.1 +/- 0.5 microm/sec, n=11) at pD 8.5 in D2O was about 60% of that (7.1 +/- 1.1 microm/sec, n=11) at pH 8.5 in H2O. The Km values of 95 and 80 microM and Vmax values of 3.2 and 5.1 microm/sec for the D2O and H2O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The Km value of actin-activated Mg-ATPase activity of myosin subfragment 1(S1) was decreased from 50 microM[actin] in H2O to 33 microM[actin] in D2O without any significant changes in Vmax (9.4 s(-1) in D2O and 9.3 s(-1) in H2O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361 +/- 26 s(-1) (n=27) in D2O and 512 +/- 39 s(-1) (n=27) in H2O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D2O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D2O.