Michiko Itoh's research while affiliated with Kanagawa Institute of Technology and other places

Background NAFLD caused by abnormalities in hepatic lipid metabolism is associated with an increased risk of developing HCC. The molecular mechanisms underlying the progression of NAFLD-related HCC are not fully understood. We investigated the molecular mechanism and role of KDM6B downregulation in NAFLD-related HCC after the KDM6B gene was identified using microarray analysis as commonly downregulated in mouse NAFLD-related HCC and human nonhepatitis B and nonhepatitis C viral-HCC. Methods The 5-hydroxymethylcytosine levels of KDM6B in HCC cells were determined using glycosylated hydroxymethyl-sensitive PCR. Microarray and chromatin immunoprecipitation analyses using KDM6B -knockout (KO) cells were used to identify KDM6B target genes. Lipotoxicity was assessed using a palmitate-treated cell proliferation assay. Immunohistochemistry was used to evaluate KDM6B expression in human HCC tissues. Results KDM6B expression levels in HCC cells correlated with the 5-hydroxymethylcytosine levels in the KDM6B gene body region. Gene set enrichment analysis revealed that the lipid metabolism pathway was suppressed in KDM6B -KO cells. KDM6B -KO cells acquired resistance to lipotoxicity ( p < 0.01) and downregulated the expression of G0S2 , an adipose triglyceride lipase/patatin like phospholipase domain containing 2 (ATGL/PNPLA2) inhibitor, through increased histone H3 lysine-27 trimethylation levels. G0S2 knockdown in KDM6B-expressed HCC cells conferred lipotoxicity resistance, whereas ATGL/PNPLA2 inhibition in the KDM6B -KO cells reduced these effects. Immunohistochemistry revealed that KDM6B expression was decreased in human NAFLD-related HCC tissues ( p < 0.001), which was significantly associated with decreased G0S2 expression ( p = 0.032). Conclusions KDM6B-disrupted HCC acquires resistance to lipotoxicity via ATGL/PNPLA2 activation caused by epigenetic downregulation of G0S2 expression. Reduced KDM6B and G0S2 expression levels are common in NAFLD-related HCC. Targeting the KDM6B-G0S2-ATGL/PNPLA2 pathway may be a useful therapeutic strategy for NAFLD-related HCC.

Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. β-cyclodextrin polyrotaxane (βCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with βCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes are interacting comorbidities of obesity, and increased hepatic de novo lipogenesis (DNL), driven by hyperinsulinemia and carbohydrate overload, contributes to their pathogenesis. Fatty acid synthase (FASN), a key enzyme of hepatic DNL, is upregulated in association with insulin resistance. However, the therapeutic potential of targeting FASN in hepatocytes for obesity-associated metabolic diseases is unknown. Here, we show that hepatic FASN deficiency differentially affects NAFLD and diabetes depending on the etiology of obesity. Hepatocyte-specific ablation of FASN ameliorated NAFLD and diabetes in melanocortin 4 receptor-deficient mice but not in mice with diet-induced obesity. In leptin-deficient mice, FASN ablation alleviated hepatic steatosis and improved glucose tolerance but exacerbated fed hyperglycemia and liver dysfunction. The beneficial effects of hepatic FASN deficiency on NAFLD and glucose metabolism were associated with suppression of DNL and attenuation of gluconeogenesis and fatty acid oxidation, respectively. The exacerbation of fed hyperglycemia by FASN ablation in leptin-deficient mice appeared attributable to impairment of hepatic glucose uptake triggered by glycogen accumulation and citrate-mediated inhibition of glycolysis. Further investigation of the therapeutic potential of hepatic FASN inhibition for NAFLD and diabetes in humans should thus consider the etiology of obesity.

The molecular pathogenesis of nonalcoholic steatohepatitis (NASH) includes a complex interaction of metabolic stress and inflammatory stimuli. Considering the therapeutic goals of NASH, it is important to determine whether the treatment can prevent the progression from NASH to hepatocellular carcinoma. Taxifolin, also known as dihydroquercetin, is a natural bioactive flavonoid with antioxidant and anti-inflammatory properties commonly found in various foods and health supplement products. In this study, we demonstrated that Taxifolin treatment markedly prevented the development of hepatic steatosis, chronic inflammation, and liver fibrosis in a murine model of NASH. Its mechanisms include a direct action on hepatocytes to inhibit lipid accumulation. Taxifolin also increased brown adipose tissue activity and suppressed body weight gain through at least two distinct pathways: direct action on brown adipocytes and indirect action via fibroblast growth factor 21 production in the liver. Notably, the Taxifolin treatment after NASH development could effectively prevent the development of liver tumors. Collectively, this study provides evidence that Taxifolin shows pleiotropic effects for the treatment of the NASH continuum. Our data also provide insight into the novel mechanisms of action of Taxifolin, which has been widely used as a health supplement with high safety.

Chronic inflammation is currently considered as a molecular basis of metabolic syndrome. Particularly, obesity-induced inflammation in adipose tissue is the origin of chronic inflammation of metabolic syndrome. Adipose tissue contains not only mature adipocytes with large lipid droplets, but also a variety of stromal cells including adipocyte precursors, vascular component cells, immune cells, and fibroblasts. However, crosstalk between those various cell types in adipose tissue in obesity still remains to be fully understood. We focus on two innate immune receptors, Toll-like receptor 4 (TLR4) and macrophage-inducible C-type lectin (Mincle). We provided evidence that adipocyte-derived saturated fatty acids (SFAs) activate macrophage TLR4 signaling pathway, thereby forming a vicious cycle of inflammatory responses during the development of obesity. Intriguingly, the TLR4 signaling pathway is modulated metabolically and epigenetically: SFAs augment TLR4 signaling through the integrated stress response and chromatin remodeling, such as histone methylation, regulates dynamic transcription patterns downstream of TLR4 signaling. Another innate immune receptor Mincle senses cell death, which is a trigger of chronic inflammatory diseases including obesity. Macrophages form a histological structure termed “crown-like structure (CLS)”, in which macrophages surround dead adipocytes to engulf cell debris and residual lipids. Mincle is exclusively expressed in macrophages forming the CLS in obese adipose tissue and regulates adipocyte death-triggered adipose tissue fibrosis. In addition to adipose tissue, we found a structure similar to CLS in the liver of nonalcoholic steatohepatitis (NASH) and the kidney after acute kidney injury. This review article highlights the recent progress of the crosstalk between immune and metabolic systems in metabolic syndrome, with a focus on innate immune receptors.

Recently, phenylboronic acid (PBA) gel containing microneedle (MN) technology with acute and sustained glucose-sensitive functionality has attracted significant research attention. Herein, we report a polyvinyl alcohol(PVA)-coated MNs patch with an interconnected porous gel drug reservoir for enhanced skin penetration efficiency and mechanical strength. The hybrid MNs patch fabricated with a novel, efficient method displayed a “cake-like” two-layer structure, with the tip part being composed of boronate-containing smart gel attached to a porous gel layer as a drug reservoir. The porous structure provides the necessary structural support for skin insertion and space for insulin loading. The mechanical strength of the hybrid MNs patch was further enhanced by surface coating with crystallized PVA. Compared with MNs patches attached to hollow drug reservoirs, this hybrid MNs patch with a porous gel reservoir was shown to be able to penetrate the skin more effectively, and is promising for on-demand, long-acting transdermal insulin delivery with increased patient compliance.

Background: Diabetes and obesity contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, how diabetes and obesity accelerate liver tumorigenesis remains to be fully understood. Moreover, to verify the therapeutic potential of anti-diabetic drugs, there exists a strong need for appropriate animal models that recapitulate human pathophysiology of NASH and HCC. Methods: We established a novel murine model of NASH-associated liver tumors using genetically obese melanocortin 4 receptor-deficient mice fed on Western diet in combination with a chemical procarcinogen, and verified the validity of our model in evaluating drug efficacy. Findings: Our model developed multiple liver tumors together with obesity, diabetes, and NASH within a relatively short period (approximately 3 months). In this model, sodium glucose cotransporter 2 inhibitor Tofogliflozin prevented the development of NASH-like liver phenotypes and the progression of liver tumors. Tofogliflozin attenuated p21 expression of hepatocytes in non-tumorous lesions in the liver. Interpretation: Tofogliflozin treatment attenuates cellular senescence of hepatocytes under obese and diabetic conditions. This study provides a unique animal model of NASH-associated liver tumors, which is applicable for assessing drug efficacy to prevent or treat NASH-associated HCC.

Nonalcoholic fatty liver disease (NAFLD) is a clinical spectrum that encompasses simple steatosis to nonalcoholic steatohepatitis (NASH), the latter of which is characterized by chronic inflammation and fibrosis. NASH is now becoming the leading cause of cirrhosis and hepatocellular carcinoma worldwide. The pathophysiology of NASH is multifactorial and, therefore, not yet completely understood, although it is pointed out that hepatocyte death and subsequent inflammation play a central roles in disease pathogenesis. Since stromal cells dramatically change their cellular components and activation status as liver fibrosis develops, it is important to reveal the subset responsible for the disease development in each etiology. Macrophages foam crown-like structures (CLS), in which CD11c-positive macrophages surround dead hepatocytes induced by lipotoxic injury in mouse and human NASH. Hepatic CLS-constituting macrophages exhibit gene expression profiles distinct from other scattered macrophages in the liver, suggesting NASH-specific macrophages represent a subset that drives metabolic stress-induced liver fibrosis. Moreover, cancer-associated pathways are upregulated in activated fibroblasts from the liver of a mouse NASH model, suggesting that fibroblasts provide the microenvironment that promotes tumor progression. A better understanding of the upstream signals and regulatory mechanisms that drive the generation of NASH-specific macrophage and fibroblast subsets is crucial for the development of novel diagnostic and therapeutic strategies.

Eradication of cancer stem cells (CSCs) is an ultimate goal in cancer chemotherapy. Although a ligand-assisted targeting approach seems rational, the existence of subpopulations of CSCs and their discrimination from those present on healthy sites makes it a severe challenge. Some boronic acid (BA) derivatives are known for the ability to bind with glycan-terminal sialic acid (SA), in a manner dependent on the acidification found in hypoxic tumoral microenvironment. Taking advantage of this feature, here we show that the BA-ligand fluorescence conjugate can effectively target multiple CSC subpopulations in parallel, which otherwise must be independently aimed when using antibody--ligands.

Although recent evidence suggests the involvement of iron accumulation in the pathogenesis of nonalcoholic steatohepatitis (NASH), the underlying mechanisms remain poorly understood. Previously, we reported a unique histological structure termed “crown-like structure (CLS)”, where liver-resident macrophages (Kupffer cells) surround dead hepatocytes, scavenge their debris, and induce inflammation and fibrosis in NASH. In this study, using magnetic column separation, we show that iron-rich Kupffer cells exhibit proinflammatory and profibrotic phenotypic changes during the development of NASH, at least partly, through activation of MiT/TFE transcription factors. Activation of MiT/TFE transcription factors is observed in Kupffer cells forming CLSs in murine and human NASH. Iron chelation effectively attenuates liver fibrosis in a murine NASH model. This study provides insight into the pathophysiologic role of iron in NASH. Our data also shed light on a unique macrophage subset rich in iron that contributes to CLS formation and serves as a driver of liver fibrosis.