Search

It remains unclear how hepatic steatosis links to inflammation. Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine that senses fat in the liver and is upregulated prior to weight gain. The aim of this study was to investigate the significance of LECT2 in the development of nonalcoholic steatohepatitis (NASH). In human liver biopsy samples, elevated LECT2 mRNA levels were positively correlated with body mass index (BMI) and increased in patients who have steatosis and inflammation in the liver. LECT2 mRNA levels were also positively correlated with the mRNA levels of the inflammatory genes CCR2 and TLR4. In C57BL/6J mice fed with a high-fat diet, mRNA levels of the inflammatory cytokines Tnfa and Nos2 were significantly lower in Lect2 KO mice. In flow cytometry analyses, the number of M1-like macrophages and M1/M2 ratio were significantly lower in Lect2 KO mice than in WT mice. In KUP5, mouse kupffer cell line, LECT2 selectively enhanced the LPS-induced phosphorylation of JNK, but not that of ERK and p38. Consistently, LECT2 enhanced the LPS-induced phosphorylation of MKK4 and TAB2, upstream activators of JNK. Hepatic expression of LECT2 is upregulated in association with the inflammatory signature in human liver tissues. The elevation of LECT2 shifts liver residual macrophage to the M1-like phenotype, and contributes to the development of liver inflammation. These findings shed light on the hepatokine LECT2 as a potential therapeutic target that can dissociate liver steatosis from inflammation.

Chemokine (C-X3-C motif) ligand 1 (CX3CL1) and its receptor CX3CR1 regulate the migration and activation of immune cells and are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the mechanism remains elusive. Here, the roles of CX3CL1/CX3CR1 in the macrophage migration and polarization in the livers of NASH mice were investigated.The expression of Cx3cl1 and Cx3cr1 was markedly upregulated in the livers of lipotoxicity-induced NASH mice. CX3CR1 was predominantly expressed by F4/80Pharmacological therapy targeting liver CX3CL1/CX3CR1 signaling might be a candidate for the treatment of NASH.

Although excessive consumption of deep-fried foods is regarded as 1 of the most important epidemiological factors of lifestyle diseases such as Alzheimer's disease, type 2 diabetes, and obesity, the exact mechanism remains unknown. This review aims to discuss whether heated cooking oil-derived peroxidation products cause cell degeneration/death for the occurrence of lifestyle diseases. Deep-fried foods cooked in ω-6 PUFA-rich vegetable oils such as rapeseed (canola), soybean, sunflower, and corn oils, already contain or intrinsically generate “hydroxynonenal” by peroxidation. As demonstrated previously, hydroxynonenal promotes carbonylation of heat-shock protein 70.1 (Hsp70.1), with the resultant impaired ability of cells to recycle damaged proteins and stabilize the lysosomal membrane. Until now, the implication of lysosomal/autophagy failure due to the daily consumption of ω-6 PUFA-rich vegetable oils in the progression of cell degeneration/death has not been reported. Since the “calpain-cathepsin hypothesis” was formulated as a cause of ischemic neuronal death in 1998, its relevance to Alzheimer's neuronal death has been suggested with particular attention to hydroxynonenal. However, its relevance to cell death of the hypothalamus, liver, and pancreas, especially related to appetite/energy control, is unknown. The hypothalamus senses information from both adipocyte-derived leptin and circulating free fatty acids. Concentrations of circulating fatty acid and its oxidized form, especially hydroxynonenal, are increased in obese and/or aged subjects. As overactivation of the fatty acid receptor G-protein coupled receptor 40 (GPR40) in response to excessive or oxidized fatty acids in these subjects may lead to the disruption of Ca(2+) homeostasis, it should be evaluated whether GPR40 overactivation contributes to diverse cell death. Here, we describe the molecular implication of ω-6 PUFA-rich vegetable oil-derived hydroxynonenal in lysosomal destabilization leading to cell death. By oxidizing Hsp70.1, both the dietary PUFA- (exogenous) and the membrane phospholipid- (intrinsic) peroxidation product “hydroxynonenal,” when combined, may play crucial roles in the occurrence of diverse lifestyle diseases including Alzheimer's disease.

The role of long noncoding RNAs (lncRNAs) in the epithelial‐mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) is unclear. Some lncRNAs can be transferred by extracellular vesicles (EVs) and have potential as biomarkers. Here, we identify an lncRNA that could serve as a biomarker for PDAC and show the functional roles of the lncRNA. Expression profiling of lncRNAs revealed that highly upregulated in liver cancer (HULC) was highly expressed, and induced, by transforming growth factor‐β in PDAC cells and their EVs. Knockdown of HULC decreased PDAC cell invasion and migration by inhibiting the EMT. Thus, HULC could be transferred by EVs, and promote EMT, invasion, and migration in recipient PDAC cells. To assess the roles of HULC, PDAC cell xenografts in nude mice were established. Knockdown of HULC in PDAC cells implanted in mice inhibited tumor growth. Moreover, microRNA‐133b suppressed PDAC cell invasion and migration by inhibiting the EMT through targeting HULC. Furthermore, serum samples were obtained from 20 PDAC and 22 intraductal papillary mucinous neoplasm (IPMN) patients, as well as 21 healthy individuals. Analysis of serum EV HULC expression by digital PCR showed that HULC expression was significantly increased in PDAC patients compared to healthy individuals or IPMN patients. Additionally, HULC showed good predictive performance for discriminating PDAC, suggesting that the analysis of EV‐encapsulated HULC would contribute to the diagnosis for human PDAC. Extracellular vesicle‐transported HULC promotes cell invasion and migration by inducing the EMT, and microRNA‐133b suppresses the EMT by targeting HULC. Extracellular vesicle‐encapsulated HULC could be a potential circulating biomarker for human PDAC., Extracellular vesicle HULC can regulate cell invasion and migration through induction of epithelial‐mesenchymal transition. Extracellular vesicle HULC could compare favorably with CA19‐9 as a novel biomarker in liquid biopsy for human pancreatic ductal adenocarcinoma.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. NAFLD manifests as hepatic lipid accumulation, insulin resistance, and inflammation, and can progress to nonalcoholic steatohepatitis (NASH) and cirrhosis. However, the underlying mechanisms of NAFLD, including those that drive its progression, are unclear. Both liver-resident (Kupffer cells) and recruited macrophages play a crucial role in the development of insulin resistance and NASH. Therefore, NALFD could potentially be ameliorated by modifying the polarization of macrophages/Kupffer cells. Reactive oxygen species induce oxidative stress, which is implicated in the progression of NASH. Micronutrients, including vitamins, are potent antioxidants that exert anti-inflammatory effects, and are used in the treatment of NAFLD. We review here the molecular mechanisms of the pathogenesis of NAFLD and the potential utility of vitamin E in its prevention and/or treatment. © 2018 IUBMB Life, 71(4):516-522, 2019.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide and has garnered increasing attention in recent decades. NAFLD is characterized by a wide range of liver changes, from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. The pathogenesis of NAFLD/NASH is very complicated and involves lipid accumulation, insulin resistance, inflammation, and fibrogenesis. In addition, NAFLD is closely associated with complications such as obesity, dyslipidemia, and type 2 diabetes. In particular, the clinical spectrum, pathophysiology, and therapeutic options of NAFLD share many things in common with diabetes. Insulin resistance is an underlying basis for the pathogenesis of diabetes and NAFLD. This chapter focuses on the molecular mechanism involved in the pathogenesis of insulin resistance, diabetes, and NASH/NAFLD including those that drive disease progression such as oxidative stress, genetic and epigenetic mechanisms, adiponectin, cytokines, and immune cells.

Metabolic disorders, such as lipid accumulation, insulin resistance, and inflammation, have been implicated in the pathogenesis of NAFLD/NASH. Both innate and recruited immune cells mediate the development of insulin resistance and NASH. Oxidative stress is also pivotal for the progression of NASH. Astaxanthin is a natural carotenoid mainly derived from microorganisms and marine organisms. Due to its special chemical structure, astaxanthin has strong antioxidant activity. β-Cryptoxanthin is a xanthophyll carotenoid specifically found in the Satsuma mandarin. β-Cryptoxanthin is readily absorbed and relatively abundant in human plasma, together with α-carotene, β-carotene, lycopene, lutein, and zeaxanthin. Considering the unique chemical properties of astaxanthin and β-cryptoxanthin and the complex pathogenic mechanism of NASH, astaxanthin and β-cryptoxanthin are regarded as a considerable compound for the prevention and treatment of NASH. This chapter comprehensively describes the mechanism of the application for astaxanthin and β-cryptoxanthin on the prevention and treatment of NASH from the aspects, including antioxidative stress, inhibition of inflammation and promotion of M2 macrophage polarization, improvement of mitochondrial oxidative respiration, amelioration of insulin resistance, and suppression of fibrosis.

The CX3CL1-CX3CR1 system plays an important role in disease progression by regulating inflammation both positively and negatively. We reported previously that C-C chemokine receptors 2 and 5 promote obesity-associated adipose tissue inflammation and insulin resistance. Here, we demonstrate that CX3CL1-CX3CR1 signaling is involved in adipose tissue inflammation and insulin resistance in obese mice via adipose tissue macrophage recruitment and M1/M2 polarization. Cx3cl1 expression was persistently decreased in the epididymal white adipose tissue (eWAT) of high-fat diet-induced obese (DIO) mice, despite increased expression of other chemokines. Interestingly, in Cx3cr1−/− mice, glucose tolerance, insulin resistance, and hepatic steatosis induced by DIO or leptin deficiency were exacerbated. CX3CL1-CX3CR1 signaling deficiency resulted in reduced M2-polarized macrophage migration and an M1-dominant shift of macrophages within eWAT. Furthermore, transplantation of Cx3cr1−/− bone marrow was sufficient to impair glucose tolerance, insulin sensitivity, and regulation of M1/M2 status. Moreover, Cx3cl1 administration in vivo led to the attenuation of glucose intolerance and insulin resistance. Thus, therapy targeting the CX3CL1-CX3CR1 system may be beneficial in the treatment of type 2 diabetes by regulating M1/M2 macrophages.

Objective Impaired circadian clocks can cause obesity, but their pathophysiological role in brown adipose tissue (BAT), a major tissue regulating energy metabolism, remains unclear. To address this issue, we investigated the effects of complete disruption of the BAT clock on thermogenesis and energy expenditure. Methods Mice with brown adipocyte-specific knockout of the core clock gene Bmal1 (BA-Bmal1 KO) were generated and analyzed. Results The BA-Bmal1 KO mice maintained normal core body temperatures by increasing shivering and locomotor activity despite the elevated expression of thermogenic uncoupling protein 1 in BAT. BA-Bmal1 KO disrupted 24 h rhythmicity of fatty acid utilization in BAT and mildly reduced both BAT thermogenesis and whole-body energy expenditure. The impact of BA-Bmal1 KO on the development of obesity became obvious when the mice were fed a high-fat diet. Conclusions These results reveal the importance of the BAT clock for maintaining energy homeostasis and preventing obesity., Graphical abstract Image 1, Highlights • The circadian clock in brown adipocytes regulates the rhythmicity of lipid utilization. • Disruption of the brown adipocyte clock impairs thermogenesis. • Muscle thermogenesis somewhat compensates for impaired brown adipocyte function. • BA-Bmal1 KO mice are more prone to diet-induced obesity.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide and has garnered increasing attention in recent decades. NAFLD is characterized by a wide range of liver changes, from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. The pathogenesis of NAFLD/NASH is very complicated and involves lipid accumulation, insulin resistance, inflammation, and fibrogenesis. In addition, NAFLD is closely associated with complications such as obesity, dyslipidemia, and type 2 diabetes. In particular, the clinical spectrum, pathophysiology, and therapeutic options of NAFLD share many things in common with diabetes. Insulin resistance is an underlying basis for the pathogenesis of diabetes and NAFLD. This chapter focuses on the molecular mechanism involved in the pathogenesis of insulin resistance, diabetes, and NASH/NAFLD including those that drive disease progression such as oxidative stress, genetic and epigenetic mechanisms, adiponectin, cytokines, and immune cells.

Metabolic disorders, such as lipid accumulation, insulin resistance, and inflammation, have been implicated in the pathogenesis of NAFLD/NASH. Both innate and recruited immune cells mediate the development of insulin resistance and NASH. Oxidative stress is also pivotal for the progression of NASH. Astaxanthin is a natural carotenoid mainly derived from microorganisms and marine organisms. Due to its special chemical structure, astaxanthin has strong antioxidant activity. β-Cryptoxanthin is a xanthophyll carotenoid specifically found in the Satsuma mandarin. β-Cryptoxanthin is readily absorbed and relatively abundant in human plasma, together with α-carotene, β-carotene, lycopene, lutein, and zeaxanthin. Considering the unique chemical properties of astaxanthin and β-cryptoxanthin and the complex pathogenic mechanism of NASH, astaxanthin and β-cryptoxanthin are regarded as a considerable compound for the prevention and treatment of NASH. This chapter comprehensively describes the mechanism of the application for astaxanthin and β-cryptoxanthin on the prevention and treatment of NASH from the aspects, including antioxidative stress, inhibition of inflammation and promotion of M2 macrophage polarization, improvement of mitochondrial oxidative respiration, amelioration of insulin resistance, and suppression of fibrosis.

Intestinal mucosal barrier dysfunction is closely related to the pathogenesis of nonalcoholic steatohepatitis (NASH). Gut immunity has been recently demonstrated to regulate gut barrier function. The Lactobacillus pentosus strain S-PT84 activates helper T cells and natural killer/natural killer T cells. In this study, we examined the effect of S-PT84 on NASH progression induced by high-cholesterol/high-fat diet (CL), focusing on the immune responses involved in gut barrier function. C57BL/6 mice were fed a normal chow or CL diet with or without 1 × 1010 S-PT84 for 22 weeks. S-PT84 administration improved hepatic steatosis by decreasing triglyceride and free fatty acid levels by 34% and 37%, respectively. Furthermore, S-PT84 inhibited the development of hepatic inflammation and fibrosis, suppressed F4/80+ macrophage/Kupffer cell infiltration, and reduced liver hydroxyproline content. Administration of S-PT84 alleviated hyperinsulinemia and enhanced hepatic insulin signalling. Compared with mice fed CL diet, mice fed CL+S-PT84 had 71% more CD11c-CD206+ M2 macrophages, resulting in a significantly decreased M1/M2 macrophage ratio in the liver. Moreover, S-PT84 inhibited the CL diet-mediated increase in intestinal permeability. Additionally, S-PT84 reduced the recruitment of interleukin-17-producing T cells and increased the levels of intestinal tight junction proteins, including zonula occludens-1, occludin, claudin-3, and claudin-7. In conclusion, our findings suggest that S-PT84 attenuates diet-induced insulin resistance and subsequent NASH development by maintaining gut permeability. Thus, S-PT84 represents a feasible approach to prevent the development of NASH.

Excessive hepatic lipid accumulation drives the innate immune system and aggravates insulin resistance, hepatic inflammation, and fibrogenesis, leading to nonalcoholic steatohepatitis (NASH). Dipeptidyl peptidase-4 (DPP-4) regulates glucose metabolism and is expressed in many different cell types, including the cells of the immune system. In addition, DPP-4 may be involved in macrophage-mediated inflammation and insulin resistance. This study investigated the effects of anagliptin (Ana), an inhibitor of DPP-4, on macrophage polarity and phenotype in the livers of mice with steatohepatitis. We investigated the effects of Ana on steatohepatitis induced via a high-cholesterol high-fat (CL) diet or a choline-deficient L-amino acid-defined, high-fat (CDAHF) diet. DPP-4 activity, liver histology, and insulin sensitivity were evaluated, and liver DPP-4+ macrophages were quantified using fluorescence-activated cell sorting (FACS). Liver and plasma DPP-4 activity increased significantly in mice on both diets. FACS revealed that, compared with chow-fed mice, the CL-fed mice exhibited a significant increase in the proportion of DPP-4+ liver macrophages, particularly the M1-type macrophages. Ana decreased hepatic lipid and M1 macrophage accumulation and stimulated M2 macrophage accumulation in the liver, thereby attenuating insulin resistance, steatohepatitis, and fibrosis. Importantly, Ana alleviated hepatic fibrosis and steatohepatitis in mice fed CL diet and CDAHF diet. Using Ana to inhibit DPP-4 reduced lipotoxicity-induced hepatic insulin resistance through regulating the M1/M2 macrophage status.

Background: Hyperuricemia drives the development of nonalcoholic fatty liver disease (NAFLD). Pharmacological inhibition of xanthine oxidase (XO), a rate-limiting enzyme for uric acid (UA) production, has been demonstrated to improve hepatic steatosis in diet-induced obese mice. However, it remains unclear whether inhibition of XO could improve nonalcoholic steatohepatitis (NASH), a more advanced form of NAFLD, in terms of both liver inflammation and fibrosis. Methods: To investigate the effects of febuxostat and allopurinol, XO inhibitors clinically used for gout, on diet-induced NASH, C57BL/6J mice were fed a high-fat, high-cholesterol, and cholate diet (CL diet) with or without either 0.001% febuxostat or 0.003% allopurinol for 18 weeks. Systemic glucose tolerance and insulin sensitivity, liver histology, inflammation, and oxidative stress were tested. Furthermore, we conducted a single-arm, open-label intervention study with febuxostat for NAFLD patients with hyperuricemia. Findings: Despite a similar hypouricemic effect of the XO inhibitors on blood UA level, febuxostat, but not allopurinol, significantly decreased hepatic UA levels in CL diet-fed mice. This reduction in hepatic UA levels was accompanied by alleviated insulin resistance, lipid peroxidation, and classically activated M1-like macrophage accumulation in the liver. Furthermore, in NAFLD patients with hyperuricemia, treatment with febuxostat for 24 weeks decreased the serum UA level, accompanied by a reduction in serum levels of liver enzymes, alanine aminotransferase and aspartate aminotransferase. Interpretation: XO may represent a promising therapeutic target in NAFLD/NASH, especially in patients with hyperuricemia. Trial Registration: The study has been registered with UMIN-CTR (#UMIN000008686). Funding Statement: This work was supported by Grants-in-aid for Scientific Research (B) (25282017) and Challenging Exploratory Research (15K12698) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Japan Diabetes Foundation (to T.O.). This study was also supported by Teijin Pharma Limited. Declaration of Interests: The authors declare no conflict of interest associated with this manuscript. Ethics Approval Statement: The study was approved by the Ethics Committee of Kanazawa University Hospital. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University, Japan.

Creative Commons Attribution 3.0 License, Early-staged cholangiocarcinoma (CCA) is difficult to diagnose due to its high potential for invasion and metastasis. Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor-β (TGF-β) in a process thought to be important for invasion and metastasis in several cancers, including CCA. Although microRNAs (miRNAs) have been implicated in the pathogenesis of several malignancies, their roles to CCA are not clearly understood. Some miRNAs were reported to be included in extracellular vesicles (EVs) and transferred from their donor cells to other cells, modulating recipient cell behaviors. In this study, the involvement and functional roles of EV-contained miRNAs during EMT in human CCA were determined. Expression profiling identified a subset of miRNAs that were reduced by TGF-β in CCA cells. Among these, miR-30e was highly downregulated by TGF-β and predicted to target Snail, which is an EMT-inducible transcription factor. MiR-30e overexpression suppressed cell invasion and migration via inhibiting EMT, whereas miR-30e inhibition promoted EMT, cell invasion and migration. Moreover, miR-30e was enriched in EVs derived from CCA cells after miR-30e overexpression, and miR-30e intercellular transfer through EVs suppressed EMT, cell invasion and migration in recipient CCA cells. Together, our results suggest that EV-mediated miR-30e transfer could inhibit EMT via directly targeting Snail, which subsequently suppresses CCA cell invasion and migration. These findings provide several new insights into regulatory mechanisms of tumor invasion and metastasis in human CCA.

金沢大学医薬保健研究域医学系, Nonalcoholic fatty liver disease (NAFLD) is caused by ectopic fat accumulation in the liver. NAFLD is associated with hepatic inflammation and oxidative stress, resulting in nonalcoholic steatohepatitis (NASH) with advanced fibrosis. Placental extracts have been used to treat various chronic diseases due to their antioxidative effect. However, the effects of the extracts on the development of NASH have yet to be elucidated. Here, we demonstrated that supplementation with an oral porcine placental extract (PPE) attenuated lipid accumulation and peroxidation, insulin resistance, inflammatory and stress signaling, and fibrogenesis in the liver of NASH model mice fed a high-cholesterol and high-fat diet. The PPE reduced the number of M1-like liver macrophages, but increased the number of anti-inflammatory M2-like macrophages, resulting in a predominance of M2 over M1 macrophage populations in the liver of NASH mice. Accordingly, the PPE suppressed lipopolysaccharide-induced M1 polarization in isolated murine peritoneal macrophages, whereas it facilitated interleukin 4-induced M2 polarization. Furthermore, the PPE reduced the hepatic stellate cell (HSC) activation associated with the attenuated transforming growth factor-β/Smad3 signaling, both in the liver of NASH mice and in RI-T cells, a HSC line. The PPE may be a potential approach to prevent NASH by limiting lipid peroxidation, promoting M2 macrophage polarization, and attenuating HSC activation. © Xu et al.

Oxidative stress caused free radical and mitochondrial damage plays a critical role in the progression of aging and age-related damage at the cellular and tissue levels. Antioxidant supplementation has received growing attention and the effects of antioxidant on aging are increasingly assessed in both animal and human studies. However, additional and more promising treatments that contribute to the expansion of anti-aging therapies are needed. Astaxanthin, a super antioxidant carotenoid and free radical scavenger, inhibits lipid peroxidation more potently than vitamin E. In the present study, we investigated the preventative effects of astaxanthin on aging using an accelerated aging model: mice chronically treated with a combination of D-galactose and jet lag. After 6 weeks of treatment, astaxanthin administration tended to protect the liver weight loss in aged mice. It is probably by upregulating the mRNA expression of galactose-1-phosphate uridyltransferase, which contribute to the enhancement of D-galactose metabolism. Astaxanthin supplementation also improved muscle endurance of aged mice in a swimming test. These results were associated with reduced oxidative stress in serum and increased anti-oxidative enzymes activities and mRNA expression in vivo. Moreover, astaxanthin reversed the dysregulation of aging-related gene expression caused by the combination of D-galactose and jet lag in the liver and kidney of mice. In conclusion, astaxanthin prevents liver weight loss, ameliorates locomotive muscular function, exerts significant anti-aging effects by reducing oxidative stress and improving the expression of age-related genes in D-galactose and jet lag-induced aging model.

Background and aims The global prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing. Chemokines and their receptors have potential as therapeutic targets of NAFLD. We investigated the role of C C chemokine ligand 3 (CCL3) in the development of murine and human NAFLD. Methods CCL3-knockout mice (CCL3−/−) and littermate CCL3 wild-type control mice (WT) were fed a high-cholesterol and high-fat (CL) diet for 16 weeks to induce NAFLD. We investigated the impact of CCL3 gene deletion in bone marrow cells and leptin-deficient ob/ob mice on CL diet-induced steatohepatitis. We assayed the serum CCL3 levels in 36 patients with biopsy-proven NAFLD and nine healthy control subjects. Results Compared with normal chow (NC), the CL diet induced steatohepatitis and hepatic fibrosis and elevated the plasma CCL3 level. In the liver, CCL3 protein colocalized with F4/80+ macrophages, especially CD11c+ M1-like macrophages, rather than other cell types. CCL3−/− attenuated CL diet-induced steatohepatitis and fibrosis associated with M2-dominant liver macrophages compared with the WT. The reconstitution of bone marrow (BM) cells from CCL3−/− attenuated steatohepatitis in WT mice fed a CL diet. Furthermore, crossing CCL3−/− onto the ob/ob background prevented CL diet-induced NAFLD in ob/ob mice, which was associated with a lesser inflammatory phenotype of liver macrophages. Also, the serum and hepatic levels of CCL3 were significantly increased in patients with non-alcoholic steatohepatitis (NASH) compared to those with simple fatty liver (NAFL) and healthy subjects. Conclusion Our data indicate that CCL3 facilitates macrophage infiltration into the liver and M1 polarization in the progression of steatohepatitis and highlight the need for further studies to determine the effect of CCL3-CCR1 and -CCR5 signaling blockade on the treatment of NAFLD.

Obesity-associated low-grade inflammation underlies insulin resistance and associated metabolic comorbidities, such as type 2 diabetes (T2D) and nonalcoholic fatty liver disease. Excessive ectopic fat deposition in obesity causes disorders of energy homeostasis and low-grade chronic inflammation in metabolic tissues. In particular, obesity-induced recruitment and activation of adipose tissue macrophages play a key role in the pathogenesis of insulin resistance and T2D. Therefore, treatment options for energy metabolism and macrophage polarization in obese subjects are needed. Sodium-glucose cotransporter (SGLT) 2 inhibitors increase urinary glucose excretion by inhibiting renal glucose reabsorption, thereby having subsequent anti-hyperglycemic effects and reducing body weight. We recently reported that the SGLT2 inhibitor empagliflozin increases fat utilization and browning in white adipose tissue and attenuates obesity-induced inflammation and insulin resistance by activating M2 macrophages. Thus, this review focuses on the beneficial effects of empagliflozin in energy homeostasis and obesity-related inflammation and insulin resistance.

The immune response and metabolic regulation are highly integrated, and their interface maintains a homeostatic system. Their dysfunction can cause obesity and its comorbidities, including insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). Endoplasmic reticulum (ER) stress is a central abnormality linking obesity, insulin resistance, and NAFLD. ER stress in response to increased hepatic lipids may decrease the ability of the liver to secrete triglyceride by limiting apolipoprotein B secretion, thereby worsening fatty liver. Overnutrition or obesity activates the innate immune system, with the subsequent recruitment of immune cells that contributes to the development of insulin resistance. A significant advance in our understanding of obesity-induced inflammation and insulin resistance has been a recognition of the critical role of adipose tissue macrophages. A role for chemokines, small proteins that direct the trafficking of immune cells to sites of inflammation, has also been demonstrated. Chemokines activate the production of inflammatory cytokines through specific chemokine receptors. This review highlights the chemokine systems linking obesity to inflammation and insulin resistance. Treatment options that target immune cells with the aim of halting the development of insulin resistance and type 2 diabetes remain limited. DPP-4 inhibitors or micronutrients may contribute to the immune regulation of glucose and lipid metabolism by regulating macrophage polarization, thereby reducing insulin resistance and preventing the progression of NAFLD. A detailed understanding of the immune regulation of glucose and lipid homeostasis can lead to the development of a novel therapy for insulin resistance, type 2 diabetes, and NAFLD.

金沢大学医薬保健研究域医学系, Sodium-glucose cotransporter (SGLT) 2 inhibitors increase urinary glucose excretion (UGE), leading to blood glucose reductions and weight loss. However, the impacts of SGLT2 inhibition on energy homeostasis and obesity-induced insulin resistance are less well known. Here, we show that empagliflozin, a SGLT2 inhibitor, enhanced energy expenditure and attenuated inflammation and insulin resistance in high-fat-diet-induced obese (DIO) mice. C57BL/6J mice were pair-fed a high-fat diet (HFD) or a HFD with empagliflozin for 16 weeks. Empagliflozin administration increased UGE in the DIO mice, whereas it suppressed HFD-induced weight gain, insulin resistance, and hepatic steatosis. Moreover, empagliflozin shifted energy metabolism towards fat utilization, elevated AMP-activated protein kinase and acetyl-CoA carbolxylase phosphorylation in skeletal muscle, and increased hepatic and plasma fibroblast growth factor 21 levels. Importantly, empagliflozin increased energy expenditure, heat production, and the expression of uncoupling protein 1 in brown fat and in inguinal and epididymal white adipose tissue (WAT). Furthermore, empagliflozin reduced M1-polarized macrophage accumulation while inducing the anti-inflammatory M2 phenotype of macrophages within WAT and liver, lowering plasma TNFα levels and attenuating obesity-related chronic inflammation. Thus, empagliflozin suppressed weight gain by enhancing fat utilization and browning and attenuated obesity-induced inflammation and insulin resistance by polarizing M2 macrophages in WAT and liver.

Low-grade sustained inflammation links obesity to insulin resistance and nonalcoholic fatty liver disease (NAFLD). However, therapeutic approaches to improve systemic energy balance and chronic inflammation in obesity are limited. Pharmacological activation of nuclear factor (erythroid-derived 2)–like 2 (Nrf2) alleviates obesity and insulin resistance in mice; however, Nrf2 inducers are not clinically available owing to safety concerns. Thus, we examined whether dietary glucoraphanin, a stable precursor of the Nrf2 inducer sulforaphane, ameliorates systemic energy balance, chronic inflammation, insulin resistance, and NAFLD in high-fat diet (HFD)–fed mice. Glucoraphanin supplementation attenuated weight gain, decreased hepatic steatosis, and improved glucose tolerance and insulin sensitivity in HFD-fed wild-type mice but not in HFD-fed Nrf2 knockout mice. Compared with vehicle-treated controls, glucoraphanin-treated HFD-fed mice had lower plasma lipopolysaccharide levels and decreased relative abundance of the gram-negative bacteria family Desulfovibrionaceae in their gut microbiomes. In HFD-fed mice, glucoraphanin increased energy expenditure and the protein expression of uncoupling protein 1 (Ucp1) in inguinal and epididymal adipose depots. Additionally, in this group, glucoraphanin attenuated hepatic lipogenic gene expression, lipid peroxidation, classically activated M1-like macrophage accumulation, and inflammatory signaling pathways. By promoting fat browning, limiting metabolic endotoxemia-related chronic inflammation, and modulating redox stress, glucoraphanin may mitigate obesity, insulin resistance, and NAFLD.

BackgroundGlucagon stimulation test (GST) is often employed to assess the insulin reserve of the pancreatic beta cells in diabetic subjects. The clinical significance of the increment of plasma glucose (Δglucose) by exogenous glucagon during GST has not been elucidated. We investigated the relationship between Δglucose and clinical parameters including the liver and renal function in type 2 diabetic subjects, since we hypothesized that Δglucose is associated with the liver and renal function reflecting the capacity for gluconeogenesis in the organs.MethodsA total of 209 subjects with type 2 diabetes who underwent GST during admission were included in this cross-sectional study. We defined the difference between plasma glucose at fasting and 6 min after intravenous injection of 1 mg glucagon as Δglucose. We assessed correlations between Δglucose and clinical parameters such as diabetic duration, BMI, HbA1c, beta cell function, serum free fatty acids (FFA) which is known to stimulate gluconeogenesis, liver function, the indices of liver function, renal function, and urinary albumin excretion (UAE).ResultsIn correlation analysis, Δglucose positively correlated to FFA and estimated glomerular filtration rate (eGFR), but inversely to serum creatinine and cystatin C, although Δglucose showed no correlation with both liver function and the indices of residual liver function. Multiple regression analysis revealed that Δglucose was an independent determinant for the eGFR after 1 year, equally BMI, HbA1c, serum lipids, and UAE, which are known as the predictors for the development of chronic kidney disease.ConclusionOur results suggest that Δglucose during GST might be related to gluconeogenesis in the kidney and could be the determinant of future renal function in type 2 diabetes.

Previous studies have demonstrated intrarenal hypoxia in patients with diabetes. Hypoxia-inducible factor (HIF)-1 plays an important role in hypoxia-induced tubulointerstitial fibrosis. Recent clinical trials have confirmed the renoprotective action of SGLT2 inhibitors in diabetic nephropathy. We explored the effects of an SGLT2 inhibitor, luseogliflozin on HIF-1α expression in human renal proximal tubular epithelial cells (HRPTECs). Luseogliflozin significantly inhibited hypoxia-induced HIF-1α protein expression in HRPTECs. In addition, luseogliflozin inhibited hypoxia-induced the expression of the HIF-1α target genes PAI-1, VEGF, GLUT1, HK2 and PKM. Although luseogliflozin increased phosphorylated-AMP-activated protein kinase α (p-AMPKα) levels, the AMPK activator AICAR did not changed hypoxia-induced HIF-1α expression. Luseogliflozin suppressed the oxygen consumption rate in HRPTECs, and subsequently decreased hypoxia-sensitive dye, pimonidazole staining under hypoxia, suggesting that luseogliflozin promoted the degradation of HIF-1α protein by redistribution of intracellular oxygen. To confirm the inhibitory effect of luseogliflozin on hypoxia-induced HIF-1α protein in vivo, we treated male diabetic db/db mice with luseogliflozin for 8 to 16 weeks. Luseogliflozin attenuated cortical tubular HIF-1α expression, tubular injury and interstitial fibronectin in db/db mice. Together, luseogliflozin inhibits hypoxia-induced HIF-1α accumulation by suppressing mitochondrial oxygen consumption. The SGLT2 inhibitors may protect diabetic kidneys by therapeutically targeting HIF-1α protein.

Excessive fatty acid uptake-induced oxidative stress causes liver injury and the consecutive recruitment of inflammatory immune cells, thereby promoting the progression of simple steatosis to nonalcoholic steatohepatitis (NASH). Lycopene, the most effective singlet oxygen scavenger of the antioxidant carotenoids, has anti-inflammatory activity. Here, we investigated the preventive and therapeutic effects of lycopene in a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat diet. Lycopene alleviated excessive hepatic lipid accumulation and enhanced lipolysis, decreased the proportion of M1-type macrophages/Kupffer cells, and activated stellate cells to improve hepatic inflammation and fibrosis, and subsequently reduced the recruitment of CD4+ and CD8+ T cells in the liver. Importantly, lycopene reversed insulin resistance, as well as hepatic inflammation and fibrosis, in pre-existing NASH. In parallel, lycopene decreased LPS-/IFN-γ-/TNFα-induced M1 marker mRNA levels in peritoneal macrophages, as well as TGF-β1-induced expression of fibrogenic genes in a stellate cell line, in a dose-dependent manner. These results were associated with decreased oxidative stress in cells, which might be mediated by the expression of NADPH oxidase subunits. In summary, lycopene prevented and reversed lipotoxicity-induced inflammation and fibrosis in NASH mice by reducing oxidative stress. Therefore, it might be a novel and promising treatment for NASH.

Obesity is one of the most serious global health problems, with an incidence that increases yearly and coincides with the development of a variety of associated comorbidities (e.g., type 2 diabetes, nonalcoholic fatty liver disease, some immune-related disorders). Although many studies have investigated the pathogenesis of overweight and obesity, multiple regulatory factors underlying the onset of obesity-related metabolic disorders remain elusive. Macrophages contribute to modulation of obesity-related inflammation and insulin resistance (IR); adipose tissue macrophages are particularly important in this context. Based on newly identified links between the chemokine system and obesity, macrophage polarization has become an essential target of new therapies for obesity-related IR. The findings of multiple studies imply that variations in gut microbiota and its metabolites might contribute to the regulation of obesity and related metabolic disorders. Recently, several novel antidiabetic drugs, applied as treatment for weight loss, were shown to be effective for obesity-induced IR and other comorbidities. The present review will discuss the properties and functions of macrophages in adipose tissue under conditions of obesity from three perspectives: the chemokine system, the gut microbiota, and antidiabetic drug application. It is proposed that macrophages might be a key therapeutic target for obesity-induced complications.

Hepatic lipid accumulation drives innate immunity with recruitment of bone marrow-derived macrophages and activation of liver-resident Kupffer cells, leading to insulin resistance and nonalcoholic steatohepatitis (NASH) development. However, treatment options that target macrophage/Kupffer cells recruitment or activation to halt the insulin resistance and NASH remain limited. A C-C chemokine receptor 2 (CCR2) plays a central role in macrophage recruitment. In addition, we reported that a different C-C chemokine receptor, CCR5, promotes insulin resistance and hepatic steatosis by regulating macrophage M1/M2 status. Here, we investigated the effect of cenicriviroc (CVC), a dual CCR2 and CCR5 receptor antagonist in a lipotoxic model of NASH with hepatic fibrosis and insulin resistance. Eight-week-old C57BL/6 mice were fed a high-cholesterol, high-fat (CL) diet or a CL diet containing 0.015% CVC (CL+CVC) for 12 weeks. The liver histology, insulin sensitivity, and fibrogenesis were examined. The CVC administration decreased the increase in plasma ALT levels as well as liver triglyceride, cholesterol, and TBARS levels caused by the CL diet. In addition, CVC improved glucose intolerance and hyperinsulinemia in the CL group and augmented the insulin signal, assessed by IRβ and Akt phosphorylation in the liver. Flow cytometry analysis revealed that mice fed CL+CVC had 72% fewer CD11c+CD206- M1-like macrophages, but 94% more CD11c-CD206+M2-like macrophages than CL group, indicating that CVC caused an M2-dominant shift in macrophages/Kupffer cells in the liver. Furthermore, CVC attenuated hepatic fibrosis by repressing the activation of hepatic stellate cells (HSCs) and decreasing hydroxyproline content. Thus, CVC ameliorated lipotoxicity-induced insulin resistance and NASH with hepatic fibrosis by polarizing M2 macrophage and attenuating HSC activation. Disclosure T. Ota: None. G. Chen: None. M. Nagashimada: None. Funding Japan Ministry of Education, Culture, Sports, Science and Technology

The glomerular number and size are major factors in predicting outcomes in renal disease. We have recently established synchrotron radiation microcomputed tomography (SRµCT) imaging of the glomeruli in perfused kidneys, and reported the glomerular and renal hypertrophy, but not glomerular loss in obese type 2 diabetic db/db mice. To visualize pathophysiological basis of diabetic kidney of type 1 diabetes, we here explored SRµCT imaging of the glomeruli in streptozotocin-induced type 1 diabetic male mice (STZ mice) and of control male C57BL/6J mice (control mice). STZ mice at the age of 22 weeks significantly showed hyperglycemia and elevated urinary albumin excretion, but not hypertension or body weight changes compared to control mice. There was approximately 22% loss of the total glomerular number (Nglom) of STZ mice compared to that of control mice (p Disclosure Y. Takiyama: None. T. Sera: None. M. Nakamura: None. R. Bessho: None. T. Takiyama: None. T. Ota: None. M. Haneda: None.

We previously reported that the short-form gastric inhibitory polypeptide (GIP) 1-30 is released from islet alpha cells and promotes insulin secretion in a paracrine manner in vitro. However, the role played by GIP 1-30 in glucose metabolism in vivo remains unclear. We developed an enzyme-linked immunosorbent assay (ELISA) specific for GIP 1-30 and measured GIP 1-30 secretion in nondiabetic subjects (ND, n = 8) and patients with type 2 diabetes (T2D, n = 9). We developed a sandwich ELISA by combining a novel antibody to the GIP C-terminus with the N-terminal anti-GIP 1-42 antibody that is already available. We explored cross-reactivities with incretins and glucagon-related peptides. We next subjected ND and T2D subjects to the cookie meal test (CMT: carbohydrates 75 g, lipids 28.5 g, proteins 8.5 g) and measured GIP 1-30 blood levels. Absorbance increased in a dose-dependent manner on addition of the GIP 1-30 amide but not GIP 1-42, GLP-1, or glucagon. Post-CMT loading, GIP 1-30 concentrations increased in both ND and T2D subjects; however, in ND subjects, the increases were much lower than those of GIP 1-42 (GIP 1-30, before loading: 1.4 ± 0.5 pmol/L, after: 1.9 ± 0.6 pmol/L; GIP 1-42, before: 8.2 ± 0.1 pmol/L, after: 204.0 ± 35.2 pmol/L, P In conclusion, we developed a novel ELISA that is highly specific for GIP 1-30, the secretion of which was promoted by a mixed meal to a blood level lower than that of GIP 1-42. As is also true of the incretins, GIP 1-30 levels increased upon administration of DPP-4 inhibitor; GIP 1-30 secretion may differ between ND and T2D subjects. Disclosure Y. Takeda: None. Y. Fujita: None. T. Yanagimachi: None. N. Maruyama: Employee; Self; Immuno-Biological Laboratoties Co.,Ltd. R. Bessho: None. H. Sakagami: None. M. Haneda: None. T. Ota: None. Funding Japan Society for the Promotion of Science

The renoprotective mechanism of SGLT2 inhibitors has not been fully elucidated. Tubular hypoxia is major driving force for proximal tubulopathy in diabetic kidney. In addition, diabetes causes dysregulation of AMP-activated protein kinase (AMPK) in kidney cortex. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on hypoxia inducible factor-1α (HIF-1α) expression and AMPK phosphorylation in cultured human renal proximal tubular epithelial cells (HRPTECs) and on tubulointerstitial pathological changes in diabetic mice. Luseogliflozin inhibited hypoxia (1%O2, 24h)-induced HIF-1α protein expression in HRPTECs in a dose-dependent manner (1-100µM). In addition, luseogliflozin increased AMPKα protein phosphorylation (Th172) in normoxic and hypoxic condition. Intriguingly, luseogliflozin suppressed oxygen consumption rate (OCR) measured by oxygen quenching phosphorescent probe by 69% in HRPTECs from normoxic condition. Hypoxia decreased OCR by 52% and luseogliflozin further decreased OCR by 33% from hypoxic condition. Moreover, luseogliflozin rescued hypoxic state in HRPTECs even under hypoxic conditions assessed by a hypoxia-sensitive dye, pimonidazole. These data indicate luseogliflozin inhibits HIF-1α expression through suppression of mitochondrial OCR, which leads to restore intracellular hypoxia. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin ameliorated tubular injury compared to non-treated db/db mice. Furthermore, immunostaining of HIF-1α and fibronectin in cortical tubules revealed that luseogliflozin decreased HIF-1α and fibronectin expression in db/db mice. In conclusion, luseogliflozin decreases mitochondrial OCR and ameliorates tubular fibrosis at least partly by suppressing HIF-1α expression and activating AMPK in renal proximal tubules of diabetic mice. These results may provide a novel renoprotective target of SGLT2 inhibitor. Disclosure R. Bessho: None. Y. Takiyama: None. T. Takiyama: None. T. Ota: None.

Nonalcoholic steatohepatitis (NASH) is associated with lipotoxic liver injury, leading to insulin resistance, inflammation, and fibrosis. Despite its increased global incidence, very few promising treatments for NASH are available. Pirfenidone is an antifibrotic agent used to treat pulmonary fibrosis; it suppresses the pulmonary influx of T cells and macrophages. Here, we investigated the effect of pirfenidone in a mouse model of lipotoxicity-induced NASH via a high-cholesterol and high-fat diet. After 12 weeks of feeding, pirfenidone administration attenuated excessive hepatic lipid accumulation and peroxidation by reducing the expression of genes related to lipogenesis and fatty acid synthesis and enhancing the expression of those related to fatty acid oxidation. Flow cytometry indicated that pirfenidone reduced the number of total hepatic macrophages, particularly CD11c+CD206-(M1)-type macrophages, increased the number of CD11c-CD206+(M2)-type macrophages, and subsequently reduced T-cell numbers, which helped improve insulin resistance and steatohepatitis. Moreover, pirfenidone downregulated the lipopolysaccharide (LPS)-induced mRNA expression of M1 marker genes and upregulated IL-4-induced M2 marker genes in a dose-dependent manner in RAW264.7 macrophages. Importantly, pirfenidone reversed insulin resistance, hepatic inflammation, and fibrosis in mice with pre-existing NASH. These findings suggest that pirfenidone is a potential candidate for the treatment of NASH.

Recent clinical trials have demonstrated the renoprotective effects of SGLT2 inhibitors in diabetic nephropathy (DN). However, the mechanisms of SGLT2 inhibitors on prevention of DN have not been fully elucidated. Hypoxia-induced tubulointerstitial fibrosis is considered to be a common pathway for various progressive kidney diseases including DN. Hypoxia inducible factor (HIF)-1α plays an important role in these pathological processes. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on HIF-1α expression in both cultured human renal proximal tubular epithelial cells (HRPTECs) and kidney of diabetic mice. Hypoxia (1%O2 for 24h) markedly increased HIF-1α protein in HRPTEC, whereas luseogliflozin inhibited expression of hypoxia-induced HIF-1α protein. In addition, luseogliflozin inhibited mRNA expression for HIF-1α-targeted genes, PAI-1, VEGF and GLUT-1 under hypoxia. The inhibitors of mitochondrial respiratory complex I, rotenone or complex III, antimycin A equally suppressed hypoxia-induced HIF-1α expression. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin administration lowered plasma glucose levels and decreased glomerular mesangial matrix expansion as well as glomerular and interstitial fibronectin accumulation in db/db mice. Electron microscopic study revealed that luseogliflozin attenuated the thickening of glomerular basement membrane in db/db mice. Intriguingly, luseogliflozin decreased the length of mitochondria in the renal proximal tubules of the S1 segment. Furthermore, luseogliflozin attenuated diabetes-induced HIF-1α accumulation in the kidney of db/db mice. These results suggest that luseogliflozin inhibits hypoxia-induced HIF-1α accumulation at least partly by suppressing mitochondrial respiration. SGLT2 inhibitors may protect diabetic kidney from hypoxia-induced renal fibrosis by attenuating the expression of HIF-1α and profibrotic molecules. Disclosure R. Bessho: None. Y. Takiyama: None. T. Ota: None.