Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction.

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpnaflox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

While extensive investigations have been devoted to the study of genetic pathways related to fatty liver diseases, much less is known about epigenetic mechanisms underlying these disorders. DNA methylation is an epigenetic link between environmental factors (e.g., diets) and complex diseases (e.g., non-alcoholic fatty liver disease). Here, it is aimed to study the role of DNA methylation in the regulation of hepatic lipid metabolism. A dynamic change in the DNA methylome in the liver of high-fat diet (HFD)-fed mice is discovered, including a marked increase in DNA methylation at the promoter of Beta-klotho (Klb), a co-receptor for the biological functions of fibroblast growth factor (FGF)15/19 and FGF21. DNA methyltransferases (DNMT) 1 and 3A mediate HFD-induced methylation at the Klb promoter. Notably, HFD enhances DNMT1 protein stability via a ubiquitination-mediated mechanism. Liver-specific deletion of Dnmt1 or 3a increases Klb expression and ameliorates HFD-induced hepatic steatosis. Single-nucleus RNA sequencing analysis reveals pathways involved in fatty acid oxidation in Dnmt1-deficient hepatocytes. Targeted demethylation at the Klb promoter increases Klb expression and fatty acid oxidation, resulting in decreased hepatic lipid accumulation. Up-regulation of methyltransferases by HFD may induce hypermethylation of the Klb promoter and subsequent down-regulation of Klb expression, resulting in the development of hepatic steatosis.

Single ethanol binge causes severe liver injury in mice fed Western diet.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) and NAFLD often coexist in Western societies that consume energy-rich and cholesterol-containing Western diets. Increased rates of ALD mortality in young people in these societies are likely attributable to binge drinking. It is largely unknown how alcohol binge causes liver damage in the setting of Western diets. APPROACH AND RESULTS: In this study, we showed that a single ethanol binge (5 g/kg body weight) induced severe liver injury as shown by marked increases in serum activities of the 2 aminotransferases AST and ALT in C57BL/6J mice that have been fed a Western diet for 3 weeks. The Western diet plus binge ethanol-fed mice also displayed severe lipid droplet deposition and high contents of triglycerides and cholesterol in the liver, which were associated with increased lipogenic and reduced fatty acid oxidative gene expression. These animals had the highest Cxcl1 mRNA expression and myeloperoxidase (MPO)-positive neutrophils in the liver. Their hepatic ROS and lipid peroxidation were the highest, but their hepatic levels of mitochondrial oxidative phosphorylation proteins remained largely unaltered. Hepatic levels of several ER stress markers, including mRNAs for CHOP, ERO1A, ERO1B, BIM, and BIP, as well as Xbp1 splicing and proteins for BIP/GRP78 and IRE-α were also the highest in these animals. Interestingly, Western diet feeding for 3 weeks or ethanol binge dramatically increased hepatic caspase 3 cleavage, and the combination of the 2 did not further increase it. Thus, we successfully established a murine model of acute liver injury by mimicking human diets and binge drinking. CONCLUSIONS: This simple Western diet plus single ethanol binge model recapitulates major hepatic phenotypes of ALD, including steatosis and steatohepatitis characterized by neutrophil infiltration, oxidative stress, and ER stress.

Identification of Scoparone from Chinese Olive Fruit as a Modulator of Macrophage Polarization.

Chinese olive (Canarium album L.) has been highlighted for its remarkable health benefits. We previously showed that the ethyl acetate fraction of Chinese olive (COE) is an effective anti-inflammatory agent. In this study, we used a luciferase-based RAW 264.7 cell platform to detect the transcriptional activity of NF-κB, a key mediator of inflammation, and the promoter activity of its downstream target, COX-2. Through functional-oriented screening using these platforms, we further divided COE into several subfractions. Subsequently, we used silica gel column chromatography for purification, and the active compounds were separated and isolated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The structure of the resulting compound with high anti-inflammatory activity was then identified as scoparone. Our results showed that scoparone not only inhibited lipopolysaccharide (LPS)-induced secretion of nitric oxide and suppressed M1 macrophage markers (iNOS, Il-6, Ccl2, and Tnf-α) but also markedly decreased the production of pro-inflammatory cytokines (IL-6, CCL2, and TNF-α). Treatment with scoparone significantly reduced the protein level of TNF-α in LPS-treated bone-marrow-derived macrophages (BMDMs). In addition, scoparone promoted macrophages toward an M2 anti-inflammatory phenotype, as determined by the significantly increased gene expression of M2 macrophage markers (Arg1, Ym1, Mrc1, Il-10, and Cd206) and the protein level of Arg1. This study indicates that COE fruit has high therapeutic potential for various inflammatory diseases as a result of switching the macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2.

Adipose lipolysis is important for ethanol to induce fatty liver in the National Institute on Alcohol Abuse and Alcoholism murine model of chronic and binge ethanol feeding.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) pathologies include steatosis, inflammation, and injury, which may progress to fibrosis, cirrhosis, and cancer. The liver receives ~60% of fatty acids from adipose tissue triglyceride hydrolysis, but the role of this lipolytic pathway in ALD development has not been directly examined in any genetic animal models with selective inactivation of adipose lipolysis. APPROACH AND RESULTS: Using adipose-specific comparative gene identification-58 (CGI-58) knockout (FAT-KO) mice, a model of impaired adipose lipolysis, we show that mice deficient in adipose lipolysis are almost completely protected against ethanol-induced hepatic steatosis and lipid peroxidation when subjected to the National Institute on Alcohol Abuse and Alcoholism chronic and binge ethanol feeding model. This is unlikely due to reduced lipid synthesis because this regimen of ethanol feeding down-regulated hepatic expression of lipogenic genes similarly in both genotypes. In the pair-fed group, FAT-KO relative to control mice displayed increased hepatocyte injury, neutrophil infiltration, and activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in the liver; and none of these were exacerbated by ethanol feeding. Activation of STAT3 is associated with a marked increase in hepatic leptin receptor mRNA expression and adipose inflammatory cell infiltration. CONCLUSIONS: Our findings establish a critical role of adipose lipolysis in driving hepatic steatosis and oxidative stress during ALD development.

Analysis and identification of phenolic compounds with antiproliferative activity from Chinese olive (Canarium album L.) fruit extract by HPLC-DAD-SPE-TT-NMR.

Chinese olives (Canarium album L.) are rich in phenolic compounds, exhibiting a broad spectrum of potential clinical applications. This study is the first report on the isolation and elucidation of bioactive compounds with high antiproliferative activity from the ethyl acetate fraction of a Chinese olive fruit methanolic extract (CO-EtOAc). We used the WST-1 assay to determine which subfractions of CO-EtOAc had significant antiproliferative activity using the murine colon cancer cell line CT26. Subsequently, the functional compounds were characterized by the hyphenated technique and high-performance liquid chromatography-diode array detector-solid phase extraction-transfer tube-nuclear magnetic resonance (HPLC-DAD-SPE-TT-NMR). Thirteen phenolic constituents were identified from the antiproliferation-enhancing subfractions of CO-EtOAc, including two new compounds, 2,4-didehydrochebulic acid 1,7-dimethyl ester (5) and 1-hydroxybrevifolin (7), which were further purified and found to exhibit marked antiproliferative activity. Chebulic acid dimethyl ester (2), which was isolated from C. album for the first time, also possessed antiproliferative activity.

Evidence of islet CADM1-mediated immune cell interactions during human type 1 diabetes.

BACKGROUNDPathophysiology of type 1 diabetes (T1D) is illustrated by pancreatic islet infiltration of inflammatory lymphocytes, including CD8+ T cells; however, the molecular factors mediating their recruitment remain unknown. We hypothesized that single-cell RNA-sequencing (scRNA-Seq) analysis of immune cell populations isolated from islets of NOD mice captured gene expression dynamics providing critical insight into autoimmune diabetes pathogenesis.METHODSPancreatic sections from human donors were investigated, including individuals with T1D, autoantibody-positive (aAb+) individuals, and individuals without diabetes who served as controls. IHC was performed to assess islet hormones and both novel and canonical immune cell markers that were identified from unbiased, state-of-the-art workflows after reanalyzing murine scRNA-Seq data sets.RESULTSComputational workflows identified cell adhesion molecule 1-mediated (Cadm1-mediated) homotypic binding among the most important intercellular interactions among all cell clusters, as well as Cadm1 enrichment in macrophages and DCs from pancreata of NOD mice. Immunostaining of human pancreata revealed an increased number of CADM1+glucagon+ cells adjacent to CD8+ T cells in sections from T1D and aAb+ donors compared with individuals without diabetes. Numbers of CADM1+CD68+ peri-islet myeloid cells adjacent to CD8+ T cells were also increased in pancreatic sections from both T1D and aAb+ donors compared with individuals without diabetes.CONCLUSIONIncreased detection of CADM1+ cells adjacent to CD8+ T cells in pancreatic sections of individuals with T1D and those who were aAb+ validated workflows and indicated CADM1-mediated intercellular contact may facilitate islet infiltration of cytotoxic T lymphocytes and serve as a potential therapeutic target for preventing T1D pathogenesis.FUNDINGThe Johns Hopkins All Children's Foundation Institutional Research Grant Program, the National Natural Science Foundation of China (grant 82071326), and the Deutsche Forschungsgemeinschaft (grants 431549029-SFB1451, EXC2030-390661388, and 411422114-GRK2550).

Adipose Lipolysis Regulates Cardiac Glucose Uptake and Function in Mice under Cold Stress.

The heart primarily uses fatty acids as energy substrates. Adipose lipolysis is a major source of fatty acids, particularly under stress conditions. In this study, we showed that mice with selective inactivation of the lipolytic coactivator comparative gene identification-58 (CGI-58) in adipose tissue (FAT-KO mice), relative to their littermate controls, had lower circulating FA levels in the fed and fasted states due to impaired adipose lipolysis. They preferentially utilized carbohydrates as energy fuels and were more insulin sensitive and glucose tolerant. Under cold stress, FAT-KO versus control mice had >10-fold increases in glucose uptake in the hearts but no increases in other tissues examined. Plasma concentrations of atrial natriuretic peptide and cardiac mRNAs for atrial and brain-type natriuretic peptides, two sensitive markers of cardiac remodeling, were also elevated. After one week of cold exposure, FAT-KO mice showed reduced cardiac expression of several mitochondrial oxidative phosphorylation proteins. After one month of cold exposure, hearts of these animals showed depressed functions, reduced SERCA2 protein, and increased proteins for MHC-ß, collagen I proteins, Glut1, Glut4 and phospho-AMPK. Thus, CGI-58-dependent adipose lipolysis critically regulates cardiac metabolism and function, especially during cold adaptation. The adipose-heart axis may be targeted for the management of cardiac dysfunction.

Chinese olive (Canarium album L.) fruit regulates glucose utilization by activating AMP-activated protein kinase.

A growing body of evidence demonstrates obesity-induced insulin resistance is associated with the development of metabolic diseases. This study was designed to investigate ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc)-mediated attenuation of obesity and hyperglycemia in a mouse model. About 60% HFD-fed mice were treated intragastrically with CO-EtOAc for last 6 weeks, and body weight, blood biochemical parameters as well as hepatic inflammation response were investigated. Our results showed that CO-EtOAc treatment significantly reduced the formation of hepatic lipid droplets, body weight gain, blood glucose, and improved serum biochemical parameters in HFD-induced obese and insulin resistant mice. We further explored the molecular mechanism underlying the blood glucose modulating effect of CO-EtOAc using L6 myotubes model. We conclude that CO-EtOAc effectively increases the glycogen content and glucose uptake by stimulating the membrane translocation of glucose transporter 4. In addition, CO-EtOAc depolarizes the mitochondrial membrane and decreases the mitochondrial oxygen consumption, which may result in AMPK activation and the consequent mitochondrial fission. This study shows that CO-EtOAc prevents the development of obesity in mice fed with HFD and is also capable of stimulating glucose uptake. The possible mechanism might be due to the effects of CO-EtOAc on activation of AMPK and promotion of mitochondrial fission.

Identification and Structural Elucidation of Anti-Inflammatory Compounds from Chinese Olive (Canarium Album L.) Fruit Extracts.

Chinese olive (Canarium album L.), a rich source of polyphenols, can be used as a functional food ingredient. We previously showed that the ethyl acetate fraction of this extract (CO-EtOAc) is an effective anti-inflammatory agent. Therefore, here, we aimed to screen the bioactive fractions extracted from CO-EtOAc using different isolation techniques, and purify the bioactive compounds based on their cytotoxic and anti-inflammatory abilities. CO-EtOAc was fractionated using silica gel and Sephadex column chromatography, and the active compounds were isolated and purified by high-performance liquid chromatography (HPLC). The structures of the resulting compounds were identified using proton nuclear magnetic resonance (NMR) spectra. Activity-directed fractionation and purification were used to identify the following active compounds with anti-inflammatory effects using lipopolysaccharide (LPS)-stimulated mouse macrophages: sitoindoside I, amentoflavone, tetrahydroamentoflavone and protocatechuic acid. For the first time, sitoindoside I and tetrahydroamentoflavone were isolated from Chinese olive, and the anti-inflammatory compounds of CO-EtOAc were identified, suggesting its potential for used as a health food ingredient.

Chinese olive extract ameliorates hepatic lipid accumulation in vitro and in vivo by regulating lipid metabolism.

Chinese olive contains plenty of polyphenols, which possess a wide range of biological actions. In this study, we aimed to investigate the role of the ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc) in the modulation of lipid accumulation in vitro and in vivo. In cellular studies, CO-EtOAc attenuated oleic acid-induced lipid accumulation; we then elucidated the molecular mechanisms of CO-EtOAc in FL83B mouse hepatocytes. CO-EtOAc suppressed the mRNA levels of fatty acid transporter genes (CD36 and FABP) and lipogenesis genes (SREBP-1c, FAS, and ACC1), but upregulated genes that govern lipolysis (HSL) and lipid oxidation (PPARα, CPT-1, and ACOX). Moreover, CO-EtOAc increased the protein expression of phosphorylated AMPK, ACC1, CPT-1, and PPARα, but downregulated the expression of mature SREBP-1c and FAS. AMPK plays an essential role in CO-EtOAc-mediated amelioration of lipid accumulation. Furthermore, we confirmed that CO-EtOAc significantly inhibited body weight gain, epididymal adipose tissue weight, and hepatic lipid accumulation via regulation of the expression of fatty acid transporter, lipogenesis, and fatty acid oxidation genes and proteins in C57BL/6 mice fed a 60% high-fat diet. Therefore, Chinese olive fruits may have the potential to improve the metabolic abnormalities associated with fatty liver under high fat challenge.

Chinese Olive (Canarium album L.) Fruit Extract Attenuates Metabolic Dysfunction in Diabetic Rats.

Hyperglycemia and dysregulation of lipid metabolism play a crucial role in metabolic dysfunction. The aims of present study were to evaluate the ameliorative effect of the ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc) on high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic rats. CO-EtOAc, rich in gallic acid and ellagic acid, could markedly decreased the body weight and epididymal adipose mass. In addition, CO-EtOAc increased serum HDL-C levels, hepatic GSH levels, and antioxidant enzyme activities; lowered blood glucose, serum levels of total cholesterol (TC), triglycerides (TG), bile acid, and tumor necrosis factor alpha (TNFα); and reduced TC and TG in liver. We further demonstrated that CO-EtOAc mildly suppressed hepatic levels of phosphorylated IRS-1, TNF-α, and IL-6, but enhanced Akt phosphorylation. The possible mechanisms of cholesterol metabolism were assessed by determining the expression of genes involved in cholesterol transportation, biosynthesis, and degradation. It was found that CO-EtOAc not only inhibited mRNA levels of SREBP-2, HMG-CoAR, SR-B1, and CYP7A1 but also increased the expression of genes, such as ABCA1 and LDLR that governed cholesterol efflux and cholesterol uptake. Moreover, the protein expressions of ABCA1 and LDLR were also significantly increased in the liver of rats supplemented with CO-EtOAc. We suggest that Chinese olive fruit may ameliorate metabolic dysfunction in diabetic rats under HFD challenge.

The methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of the NF-κB signaling pathway.

Chinese olives (Canarium album L.) have historically been used for medicinal purposes rather than commercially for oil. In this report, we reveal that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits (MEO), of which ellagic acid accounted for 12%, exhibited profound anti-proliferative activities in the human colon cancer cell line, HCT116. Additionally, oral administration of MEO remarkably inhibited the tumor growth of subcutaneously implanted CT26 cells, a mouse colon carcinoma cell line, in BALB/c mice. Treatment with MEO induced a significant increase in the percentage of apoptotic cells and resulted in poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that MEO inhibits cancer cell proliferation by promoting apoptosis. Our study also showed that MEO exerted the most potent effect on the inhibition of NF-κB-mediated signaling among the partitioned fractions from Chinese olives. This process employed the use of reporter-based bio-platforms that are capable of detecting the activation of NF-κB. In addition, phosphorylation of NF-κB signaling-associated proteins, IKKα/ß, IκBα, and p65, was reduced in MEO-incubated cancer cells, indicating that MEO suppresses NF-κB activation. Moreover, MEO treatment significantly suppressed lipopolysaccharide (LPS)-induced cancer cell proliferation, demonstrating that MEO promotes cancer cell apoptosis through the inhibition of the NF-κB signaling pathway. In summary, our findings demonstrate that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of NF-κB signaling. Therefore, the Chinese olive fruit has promising potential in cancer treatment.