CX3CL1/CX3CR1 interaction protects against lipotoxicity-induced nonalcoholic steatohepatitis by regulating macrophage migration and M1/M2 status.

BACKGROUND AND OBJECTIVES: 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. METHODS AND RESULTS: The expression of Cx3cl1 and Cx3cr1 was markedly upregulated in the livers of lipotoxicity-induced NASH mice. CX3CR1 was predominantly expressed by F4/80+ macrophages and to a lesser degree by hepatic stellate cells or endothelial cells in the livers of NASH mice. Flow cytometry analysis revealed that, compared with chow-fed mice, NASH mice exhibited a significant increase in CX3CR1+ expression by liver macrophages (LMs), particularly M1 LMs. CX3CR1 deficiency caused a significant increase in inflammatory monocyte/macrophage infiltration and a shift toward M1 dominant macrophages in the liver, thereby exacerbating the progression of NASH. Moreover, transplantation of Cx3cr1-/- bone marrow was sufficient to cause glucose intolerance, inflammation, and fibrosis in the liver. In addition, deletion of CCL2 in Cx3cr1-/- mice alleviated NASH progression by decreasing macrophage infiltration and inducing a shift toward M2 dominant LMs. Importantly, overexpression of CX3CL1 in vivo protected against hepatic fibrosis in NASH. CONCLUSION: Pharmacological therapy targeting liver CX3CL1/CX3CR1 signaling might be a candidate for the treatment of NASH.

CC chemokine ligand 3 deficiency ameliorates diet-induced steatohepatitis by regulating liver macrophage recruitment and M1/M2 status in mice.

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 CC 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.

Molecular Mechanisms of Nonalcoholic Fatty Liver Disease (NAFLD)/Nonalcoholic Steatohepatitis (NASH).

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.

LECT2 as a hepatokine links liver steatosis to inflammation via activating tissue macrophages in NASH.

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.

DPP-4 Inhibition with Anagliptin Reduces Lipotoxicity-Induced Insulin Resistance and Steatohepatitis in Male Mice.

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.

Xanthine oxidase inhibition attenuates insulin resistance and diet-induced steatohepatitis in mice.

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 improves nonalcoholic steatohepatitis (NASH), a more advanced form of NAFLD, in terms of both liver inflammation and fibrosis. Here, we investigated the effects of febuxostat and allopurinol, two XO inhibitors clinically used for gout, on a mouse model of NASH. Furthermore, we conducted a single-arm, open-label intervention study with febuxostat for NAFLD patients with hyperuricemia. Despite a similar hypouricemic effect of the XO inhibitors on blood UA level, febuxostat, but not allopurinol, significantly decreased hepatic XO activity and UA levels in the NASH model mice. These reductions in hepatic XO activity and UA levels were accompanied by attenuation of 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 reductions in the serum levels of liver enzymes, alanine aminotransferase and aspartate aminotransferase. XO may represent a promising therapeutic target in NAFLD/NASH, especially in patients with hyperuricemia.

Adipose Tissue Macrophage Phenotypes and Characteristics: The Key to Insulin Resistance in Obesity and Metabolic Disorders.

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.

Circulating extracellular vesicle-encapsulated HULC is a potential biomarker for human pancreatic cancer.

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-ß 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.

Increment of plasma glucose by exogenous glucagon is associated with present and future renal function in type 2 diabetes:a retrospective study from glucagon stimulation test.

BACKGROUND: Glucagon 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. METHODS: A 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). RESULTS: In 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. CONCLUSION: Our 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.

Unusual manifestations of giant cell arteritis and granulomatosis with polyangiitis.

Giant cell arteritis (GCA) is a type of large vessel vasculitis, and it involves the aorta, large vessels and terminal branches of the external carotid artery, especially the temporal artery. Temporal artery biopsy is a simple tool for the diagnosis of vasculitis, however, the histopathological findings do not always differentiate between the small-vessel vasculitis and GCA. We report the case of 72-year-old male who initially had a clinical diagnosis of GCA, then in the course of treatment, diagnostic histopathological approach revealed the necrotizing vasculitis with bronchocentric granulomatosis in the inflammatory nodule of the lung. The manifestations of patients with systemic vasculitis represent the disorders of multiple organ systems thus are diverse and may vary through the course of the disease. Presentation of unexpected features such as insufficient response to antibiotics, sinusitis, runny nose, discomfort of frontal region or pachymeningitis which anticipates re-evaluation of systemic vasculitis that may lead us to an appropriate diagnosis and the treatment.

Lycopene Alleviates Obesity-Induced Inflammation and Insulin Resistance by Regulating M1/M2 Status of Macrophages.

SCOPE: Adipose tissue macrophage (ATM) recruitment and polarization are pivotal in the development of insulin resistance. However, treatment modalities targeting ATMs remain limited. The effects of lycopene, an antioxidant carotenoid compound, on adipose tissue inflammation and insulin resistance in high fat (HF)-diet-induced obese mice are examined. METHODS AND RESULTS: C57BL/6J mice are fed an HF diet or an HF diet containing lycopene (HF+LY) for 8 weeks. Lycopene attenuates HF-diet-induced glucose intolerance and hyperinsulinemia. Compared with HF mice, HF+LY mice exhibit attenuated adipocyte hypertrophy and macrophage infiltration in epididymal white adipose tissue (eWAT) and hepatic steatosis and inflammation. Flow cytometry analysis of ATMs demonstrates that lycopene attenuated the increased number of ATMs in HF diet-fed mice. In addition, HF+LY mice have 23% fewer M1-polarized ATMs and 60% more M2-polarized ATMs than HF mice, resulting in the predominance of M2 over M1 in the ATM population. M2-dominant polarization is also seen in hepatic macrophages in HF+LY mice. Moreover, lycopene promotes IL-4-induced M2 polarization by increasing the phosphorylation levels of STAT6 and Akt in Raw 264.7 macrophages. CONCLUSIONS: Lycopene facilitates M2-dominant polarization in ATM, thereby attenuating HF diet-induced inflammation and insulin resistance in eWAT and the liver.

Pirfenidone prevents and reverses hepatic insulin resistance and steatohepatitis by polarizing M2 macrophages.

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.

Role of vitamin E in nonalcoholic fatty liver disease.

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.

Impacts of Diabetes and an SGLT2 Inhibitor on the Glomerular Number and Volume in db/db Mice, as Estimated by Synchrotron Radiation Micro-CT at SPring-8.

BACKGROUND: Recent large-scale clinical studies demonstrate that sodium-glucose cotransporter 2 (SGLT2) inhibitors protect the diabetic kidney. However, clinical and animal studies have not shown the changes of the total glomeruli in the whole kidney treated with SGLT2 inhibitors. METHODS: We performed computed tomography (CT) imaging on mice using synchrotron radiation to investigate the impact of luseogliflozin, a SGLT2 inhibitor, on the number and volume of glomeruli in the whole kidney. FINDINGS: We did not observe a significant difference in the total glomerular number (Nglom) among mice. Luseogliflozin redistributed the number of glomeruli in different regions, accompanied by the normalization of diabetes-augmented renal volume (Vkidney). Diabetic db/db mice had a larger glomerular volume in the mid-cortex than did control db/m mice, and luseogliflozin increased the glomerular volume in all renal cortical zones of the whole kidney in db/db mice. According to the multivariate regression analysis, hemoglobin A1c level was the most relevant determinant of Vkidney, not Nglom or mean glomerular volume (Vglom), indicating that hyperglycemia induced renal (tubular) hypertrophy, but not glomerular enlargement. Luseogliflozin increased hypoxia in the juxtamedullary region, sustained upregulated renal renin expression and plasma renin activity, and failed to decrease albuminuria by downregulating megalin in db/db mice. INTERPRETATION: Based on our findings, SGLT2 inhibitors may alter glomerular distribution and size in addition to their glucose-lowering effects, presumably by affecting oxygen metabolism and humoral factors. FUND: Funding for this research was provided by The Japan Society for the Promotion of Science, the Japan Diabetes Foundation, and Asahikawa Medical University.

Glucoraphanin: a broccoli sprout extract that ameliorates obesity-induced inflammation and insulin resistance.

Obesity is a low-grade sustained inflammatory state that causes oxidative stress in different metabolic tissues, which leads to insulin resistance and nonalcoholic fatty liver disease (NAFLD). Particularly, obesity-induced metabolic endotoxemia plays an important role in the pathogenesis of insulin resistance and inflammation. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of antioxidant signaling that serves as a primary cellular defense against the cytotoxic effects of oxidative stress. Pharmacological stimulation of Nrf2 mitigates obesity and insulin resistance in mice; however, Nrf2 activators are not clinically available due to biosafety concerns. A recent study demonstrated that glucoraphanin, a precursor of the Nrf2 activator sulforaphane, ameliorates obesity by enhancing energy expenditure and browning of white adipose tissue, and attenuates obesity-related inflammation and insulin resistance by polarizing M2 macrophages and reducing metabolic endotoxemia. Thus, this review focuses on the efficiency and safety of glucoraphanin in alleviating obesity, insulin resistance, and NAFLD. Abbreviations: ALT, Alanine aminotransferase; AMPK, AMP-activated protein kinase; ATMs, Adipose tissue macrophages; BAT, Brown adipose tissue; CDDO-Im, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid-imidazolide; CDDO-Me, CDDO-methyl ester; DIO, High-fat-diet-induced obese; FFA, Free fatty acid; FGF, Fibroblast growth factor; GTP, Glutamyl transpeptidase; HFD, High-fat diet; IKKß, Inhibitor of κB-kinase ß; IL, Interleukin; JNK, C-Jun N-terminal kinase; KD, Knockdown; Keap1, Kelch-like ECH-associated protein 1; KO, Knockout; LPS, Lipopolysaccharide; NADPH, Nicotinamide adenine dinucleotide phosphate; NAFLD, Non-alcoholic fatty liver disease; NF-κB, Nuclear factor-κB; Nrf2, Nuclear factor E2-related factor 2; ROS, Reactive oxygen species; T2D, Type 2 diabetes; TLR, Toll-like receptor; TNF, tumor necrosis factor; UCP, Uncoupling protein; WAT, White adipose tissue.

Ursodeoxycholic acid potentiates dipeptidyl peptidase-4 inhibitor sitagliptin by enhancing glucagon-like peptide-1 secretion in patients with type 2 diabetes and chronic liver disease: a pilot randomized controlled and add-on study.

OBJECTIVE: We evaluated the effects of ursodeoxycholic acid (UDCA) on glucagon-like peptide-1 (GLP-1) secretion and glucose tolerance in patients with type 2 diabetes with chronic liver disease. RESEARCH DESIGN AND METHODS: Japanese patients with type 2 diabetes (glycated hemoglobin (HbA1c) levels ≥7.0%) and chronic liver disease were included in this study. Sixteen patients (HbA1c level, 7.2%±0.6%(55.2 mmol/mol)) were randomized to receive 900 mg UDCA for 12 weeks followed by 50 mg sitagliptin add-on therapy for 12 weeks (UDCA-first group; n=8) or 50 mg sitagliptin for 12 weeks followed by 900 mg UDCA add-on therapy for 12 weeks (sitagliptin-first group; n=8). All patients underwent a liquid high-fat meal test before and after 12 or 24 weeks of treatment. RESULTS: The baseline characteristics were similar between the UDCA-first and sitagliptin-first groups. There was a decrease in body weight (72.5±8.4 to 70.6±8.6 kg; P=0.04) and the HbA1c level (7.0%±0.3% to 6.4%±0.5%(53.0 to 46.4 mmol/mol); P=0.01) in the UDCA-first group. The HbA1c level decreased further after sitagliptin administration (6.4%±0.5% to 6.0%±0.4%(46.4 to 42.1 mmol/mol); P<0.01). Although there were no initial changes in the weight and HbA1c level in the sitagliptin-first group, the HbA1c level decreased after UDCA addition (7.1%±1.1% to 6.6%±0.9%(54.1 to 48.6 mmol/mol); P=0.04). UDCA alone increased the area under the curve0-30 for GLP-1 response (115.4±47.2 to 221.9±48.9 pmol·min/L; P<0.01), but not the glucose-dependent insulinotropic polypeptide response, in the UDCA-first group. CONCLUSIONS: UDCA treatment resulted in a greater reduction in HbA1c levels, and an increased early phase GLP-1 secretion. TRIAL REGISTRATION NUMBER: NCT01337440.

Extracellular vesicle-encapsulated miR-30e suppresses cholangiocarcinoma cell invasion and migration via inhibiting epithelial-mesenchymal transition.

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-ß (TGF-ß) 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-ß in CCA cells. Among these, miR-30e was highly downregulated by TGF-ß 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.

Emerging roles of SGLT2 inhibitors in obesity and insulin resistance: Focus on fat browning and macrophage polarization.

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.

Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

Sphingosine-1-phospate is a potent bioactive lipid metabolite that regulates cancer progression. Because sphingosine kinase 1 and sphingosine kinase 2 (SPHK 1/2) are both essential for sphingosine-1-phospate production, they could be a therapeutic target in various cancers. Peretinoin, an acyclic retinoid, inhibits post-therapeutic recurrence of hepatocellular carcinoma via unclear mechanisms. In this study, we assessed effects of peretinoin on SPHK expression and liver cancer development in vitro and in vivo. We examined effects of peretinoin on expression, enzymatic and promoter activity of SPHK1 in a human hepatoma cell line, Huh-7. We also investigated effects of SPHK1 on hepatocarcinogenesis induced by diethylnitrosamine using SPHK1 knockout mice. Peretinoin treatment of Huh-7 cells reduced mRNA levels, protein expression and enzymatic activity of SPHK1. Peretinoin reduced SPHK1 promoter activity; this effect of peretinoin was blocked by overexpression of Sp1, a transcription factor. Deletion of all Sp1 binding sites within the SPHK1 promoter region abolished SPHK1 promoter activity, suggesting that peretinoin reduced mRNA levels of SPHK1 via Sp1. Additionally, diethylnitrosamine-induced hepatoma was fewer and less frequent in SPHK1 knockout compared to wild-type mice. Our data showed crucial roles of SPHK1 in hepatocarcinogenesis and suggests that peretinoin prevents hepatocarcinogenesis by suppressing mRNA levels of SPHK1.

SGLT2 Inhibition by Empagliflozin Promotes Fat Utilization and Browning and Attenuates Inflammation and Insulin Resistance by Polarizing M2 Macrophages in Diet-induced Obese Mice.

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 16weeks. 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.