Michiko Itoh

Role of the Toll-like Receptor 4/NF-κB Pathway in Saturated Fatty Acid–Induced Inflammatory Changes in the Interaction Between Adipocytes and Macrophages

Objective—Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)-α derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes. Methods and Results—Coculture of adipocytes and macrophages resulted in the activation of nuclear factor-&kgr;B (NF-&kgr;B), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF-&kgr;B markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNFα production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the β3-adrenergic stimulation, resulted in the activation of the TLR4/NF-&kgr;B pathway. Conclusion—These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF-&kgr;B activation.

Increased Adiponectin Secretion by Highly Purified Eicosapentaenoic Acid in Rodent Models of Obesity and Human Obese Subjects

Objectives—Fish oil rich in n-3 polyunsaturated fatty acids (PUFAs) or n-3 PUFAs have been shown to reduce the incidence of coronary heart disease. Here we investigated the effect of highly purified eicosapentaenoic acid (EPA) on production of adiponectin, the only established antiatherogenic and antiinflammatory adipocytokine, in rodent models of obesity and human obese subjects. Methods and Results—We demonstrated that EPA increases adiponectin secretion in genetically obese ob/ob mice and high-fat diet–induced obese mice. In the in vitro coculture of adipocytes and macrophages, EPA reversed the coculture-induced decrease in adiponectin secretion at least in part through downregulation of tumor necrosis factor-&agr; in macrophages. We also showed significant increase in plasma adiponectin concentrations in human obese subjects after a 3-month treatment with EPA (1.8 g daily). Multivariate regression analysis revealed that EPA treatment is the only independent determinant of plasma adiponectin concentrations. Conclusion—This study demonstrates that EPA increases adiponectin secretion in rodent models of obesity and human obese subjects, possibly through the improvement of the inflammatory changes in obese adipose tissue. Because EPA has reduced the risk of major coronary events in a large-scale, prospective, randomized clinical trial, this study provides important insight into its therapeutic implication in obesity-related metabolic sequelae.

Antiobesity effect of eicosapentaenoic acid in high-fat/high-sucrose diet-induced obesity: importance of hepatic lipogenesis.

OBJECTIVE Given the pleiotropic effect of eicosapentaenoic acid (EPA), it is interesting to know whether EPA is capable of improving obesity. Here we examined the anti-obesity effect of EPA in mice with two distinct models of obesity. RESEARCH DESIGN AND METHODS Male C57BL/6J mice were fed a high-fat/high-sucrose diet (25.0% [w/w] fat, 32.5% [w/w] sucrose) (HF/HS group) or a high-fat diet (38.1% [w/w] fat, 8.5% [w/w] sucrose) (HF group) for 4–20 weeks. A total of 5% EPA was administered by partially substituting EPA for fat in the HF/HS + EPA and HF + EPA groups. RESULTS Both the HF/HS and HF groups similarly developed obesity. EPA treatment strongly suppresses body weight gain and obesity-related hyperglycemia and hyperinsulinemia in HF/HS-fed mice (HF/HS + EPA group), where hepatic triglyceride content and lipogenic enzymes are increased. There is no appreciable effect of EPA on body weight in HF-fed mice (HF + EPA group) without enhanced expression of hepatic lipogenic enzymes. Moreover, EPA is capable of reducing hepatic triglyceride secretion and changing VLDL fatty acid composition in the HF/HS group. By indirect calorimetry analysis, we also found that EPA is capable of increasing energy consumption in the HF/HS + EPA group. CONCLUSIONS This study is the first demonstration that the anti-obesity effect of EPA in HF/HS-induced obesity is associated with the suppression of hepatic lipogenesis and steatosis. Because the metabolic syndrome is often associated with hepatic lipogenesis and steatosis, the data suggest that EPA is suited for treatment of the metabolic syndrome.

fect of Eicosapentaenoic Acid in High-fat/High-sucrose Diet-induced Obesity: Importance of Hepatic Lipogenesis

OBJECTIVE Given the pleiotropic effect of eicosapentaenoic acid (EPA), it is interesting to know whether EPA is capable of improving obesity. Here we examined the anti-obesity effect of EPA in mice with two distinct models of obesity. RESEARCH DESIGN AND METHODS Male C57BL/6J mice were fed a high-fat/high-sucrose diet (25.0% [w/w] fat, 32.5% [w/w] sucrose) (HF/HS group) or a high-fat diet (38.1% [w/w] fat, 8.5% [w/w] sucrose) (HF group) for 4–20 weeks. A total of 5% EPA was administered by partially substituting EPA for fat in the HF/HS + EPA and HF + EPA groups. RESULTS Both the HF/HS and HF groups similarly developed obesity. EPA treatment strongly suppresses body weight gain and obesity-related hyperglycemia and hyperinsulinemia in HF/HS-fed mice (HF/HS + EPA group), where hepatic triglyceride content and lipogenic enzymes are increased. There is no appreciable effect of EPA on body weight in HF-fed mice (HF + EPA group) without enhanced expression of hepatic lipogenic enzymes. Moreover, EPA is capable of reducing hepatic triglyceride secretion and changing VLDL fatty acid composition in the HF/HS group. By indirect calorimetry analysis, we also found that EPA is capable of increasing energy consumption in the HF/HS + EPA group. CONCLUSIONS This study is the first demonstration that the anti-obesity effect of EPA in HF/HS-induced obesity is associated with the suppression of hepatic lipogenesis and steatosis. Because the metabolic syndrome is often associated with hepatic lipogenesis and steatosis, the data suggest that EPA is suited for treatment of the metabolic syndrome.

In Vivo and In Vitro Inhibition of Monocyte Adhesion to Endothelial Cells and Endothelial Adhesion Molecules by Eicosapentaenoic Acid

Objective—A large-scale, prospective, randomized clinical trial has recently revealed that the addition of highly purified eicosapentaenoic acid (EPA) to low-dose statin therapy significantly reduces the incidence of major coronary events. Here we investigated in vivo and in vitro effect of EPA on monocyte adhesion to endothelial cells and adhesion molecules. Methods and Results—A new en face immunohistochemistry of endothelial surface in combination with confocal microscopy revealed marked reduction of lipopolysaccharide (LPS)-induced monocyte adhesion to the aortic endothelium in parallel with the suppression of vascular cell adhesion molecule 1 (VCAM-1) and nuclear translocation of nuclear factor-&kgr;B p65 in EPA-treated mice relative to vehicle-treated groups. In an in vitro adhesion assay system under physiological flow conditions, EPA inhibited LPS-induced monocyte adhesion and endothelial adhesion molecules. We found significant decrease in plasma concentrations of soluble intercellular adhesion molecule 1 (sICAM-1) and sVCAM-1 in patients with the metabolic syndrome after a 3-month administration of highly purified EPA (1.8 g daily). Multivariate regression analysis revealed that EPA administration is the only independent determinant of sICAM-1 and sVCAM-1. Conclusions—This study provides evidence that EPA inhibits monocyte adhesion to endothelial cells in parallel with the suppression of endothelial adhesion molecules in vivo and in vitro.

Adipose Tissue Remodeling as Homeostatic Inflammation

Evidence has accumulated indicating that obesity is associated with a state of chronic, low-grade inflammation. Obese adipose tissue is characterized by dynamic changes in cellular composition and function, which may be referred to as “adipose tissue remodeling”. Among stromal cells in the adipose tissue, infiltrated macrophages play an important role in adipose tissue inflammation and systemic insulin resistance. We have demonstrated that a paracrine loop involving saturated fatty acids and tumor necrosis factor-α derived from adipocytes and macrophages, respectively, aggravates obesity-induced adipose tissue inflammation. Notably, saturated fatty acids, which are released from hypertrophied adipocytes via the macrophage-induced lipolysis, serve as a naturally occurring ligand for Toll-like receptor 4 complex, thereby activating macrophages. Such a sustained interaction between endogenous ligands derived from parenchymal cells and pathogen sensors expressed in stromal immune cells should lead to chronic inflammatory responses ranging from the basal homeostatic state to diseased tissue remodeling, which may be referred to as “homeostatic inflammation”. We, therefore, postulate that adipose tissue remodeling may represent a prototypic example of homeostatic inflammation. Understanding the molecular mechanism underlying homeostatic inflammation may lead to the identification of novel therapeutic strategies to prevent or treat obesity-related complications.

Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.

Although macrophages are thought to be crucial for the pathogenesis of chronic inflammatory diseases, how they are involved in disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is poorly understood. Here we report the unique histological structure termed “hepatic crown-like structures (hCLS)” in the mouse model of human NASH; melanocortin-4 receptor deficient mice fed a Western diet. In hCLS, CD11c-positive macrophages aggregate to surround hepatocytes with large lipid droplets, which is similar to those described in obese adipose tissue. Histological analysis revealed that hCLS is closely associated with activated fibroblasts and collagen deposition. When treatment with clodronate liposomes effectively depletes macrophages scattered in the liver, with those in hCLS intact, hepatic expression of inflammatory and fibrogenic genes is unaffected, suggesting that hCLS is an important source of inflammation and fibrosis during the progression of NASH. Notably, the number of hCLS is positively correlated with the extent of liver fibrosis. We also observed increased number of hCLS in the liver of non-alcoholic fatty liver disease/NASH patients. Collectively, our data provide evidence that hCLS is involved in the development of hepatic inflammation and fibrosis, thereby suggesting its pathophysiologic role in disease progression from simple steatosis to NASH.

Eicosapentaenoic Acid Ameliorates Non-Alcoholic Steatohepatitis in a Novel Mouse Model Using Melanocortin 4 Receptor-Deficient Mice

Many attempts have been made to find novel therapeutic strategies for non-alcoholic steatohepatitis (NASH), while their clinical efficacy is unclear. We have recently reported a novel rodent model of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice, which exhibit the sequence of events that comprise hepatic steatosis, liver fibrosis, and hepatocellular carcinoma with obesity-related phenotypes. In the liver of MC4R-KO mice, there is a unique histological feature termed hepatic crown-like structures (hCLS), where macrophages interact with dead hepatocytes and fibrogenic cells, thereby accelerating inflammation and fibrosis. In this study, we employed MC4R-KO mice to examine the effect of highly purified eicosapentaenoic acid (EPA), a clinically available n-3 polyunsaturated fatty acid, on the development of NASH. EPA treatment markedly prevented the development of hepatocyte injury, hCLS formation and liver fibrosis along with lipid accumulation. EPA treatment was also effective even after MC4R-KO mice developed NASH. Intriguingly, improvement of liver fibrosis was accompanied by the reduction of hCLS formation and plasma kallikrein-mediated transforming growth factor-β activation. Moreover, EPA treatment increased the otherwise reduced serum concentrations of adiponectin, an adipocytokine with anti-inflammatory and anti-fibrotic properties. Collectively, EPA treatment effectively prevents the development and progression of NASH in MC4R-KO mice along with amelioration of hepatic steatosis. This study unravels a novel anti-fibrotic mechanism of EPA, thereby suggesting a clinical implication for the treatment of NASH.

Antifibrotic effect of pirfenidone in a mouse model of human nonalcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis with lobular inflammation and hepatocyte injury. Pirfenidone (PFD) is an orally bioavailable pyridone derivative that has been clinically used for the treatment of idiopathic pulmonary fibrosis. However, it remains unknown whether PFD improves liver fibrosis in a mouse model with human NASH-like phenotypes. In this study, we employed melanocortin 4 receptor-deficient (MC4R-KO) mice as a mouse model with human NASH-like phenotypes to elucidate the effect and action mechanisms of PFD on the development of NASH. PFD markedly attenuated liver fibrosis in western diet (WD)-fed MC4R-KO mice without affecting metabolic profiles or steatosis. PFD prevented liver injury and fibrosis associated with decreased apoptosis of liver cells in WD-fed MC4R-KO mice. Pretreatment of PFD inhibited the tumor necrosis factor-α (TNF-α)-induced liver injury and fibrogenic responses associated with decreased apoptosis of liver cells in wild-type mice. PFD also prevented TNF-α-induced hepatocyte apoptosis in vitro with reduced activation of caspase-8 and -3. This study provides evidence for the antifibrotic effect of PFD in a mouse model of human NASH. The data of this study highlight hepatocyte apoptosis as a potential therapeutic target, and suggest that PFD can be repositioned as an antifibrotic drug for human NASH.

YAP determines the cell fate of injured mouse hepatocytes in vivo

The presence of senescent, transformed or damaged cells can impair tissue function or lead to tumorigenesis; therefore, organisms have evolved quality control mechanisms to eliminate them. Here, we show that YAP activation induced by inactivation of the Hippo pathway specifically in damaged hepatocytes promotes their selective elimination by using in vivo mosaic analysis in mouse liver. These damaged hepatocytes migrate into the hepatic sinusoids, undergo apoptosis and are engulfed by Kupffer cells. In contrast, YAP activation in undamaged hepatocytes leads to proliferation. Cellular stresses such as ethanol that damage both liver sinusoidal endothelial cells and hepatocytes switch cell fate from proliferation to migration/apoptosis in the presence of activated YAP. This involves the activation of CDC42 and Rac that regulate cell migration. Thus, we suggest that YAP acts as a stress sensor that induces elimination of injured cells to maintain tissue and organ homeostasis.