FoxO6-Mediated TXNIP Induces Lipid Accumulation in the Liver through NLRP3 Inflammasome Activation.

BACKGRUOUND: Hepatic steatosis, which involves the excessive accumulation of lipid droplets in hepatocytes, presents a significant global health concern due to its association with obesity and metabolic disorders. Inflammation plays a crucial role in the progression of hepatic steatosis; however, the precise molecular mechanisms responsible for this process remain unknown. METHODS: This study investigated the involvement of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome and the forkhead box O6 (FoxO6) transcription factor in the pathogenesis of hepatic steatosis. We monitored the NLRP3 inflammasome and lipogenesis in mice overexpressing the constitutively active (CA)-FoxO6 allele and FoxO6-null mice. In an in vitro study, we administered palmitate to liver cells overexpressing CA-FoxO6 and measured changes in lipid metabolism. RESULTS: We administered palmitate treatment to clarify the mechanisms through which FoxO6 activates cytokine interleukin (IL)-1ß through the NLRP3 inflammasome. The initial experiments revealed that dephosphorylation led to palmitate-induced FoxO6 transcriptional activity. Further palmitate experiments showed increased expression of IL-1ß and the hepatic NLRP3 inflammasome complex, including adaptor protein apoptotic speck protein containing a caspase recruitment domain (ASC) and pro-caspase-1. Furthermore, thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox conditions upstream of the NLRP3 inflammasome, was induced by FoxO6 in the liver and HepG2 cells. CONCLUSION: The findings of this study shed light on the molecular mechanisms underpinning the FoxO6-NLRP3 inflammasome axis in promoting inflammation and lipid accumulation in the liver.

Protective effect of gomisin N on benzyl butyl phthalate-induced dysfunction of testosterone production in TM3 Leydig cells.

BACKGROUND: Exposure to benzyl butyl phthalate (BBP) may induce disorders in the male reproductive system. However, the molecular mechanisms remain unknown. Here we investigated the effect of BBP on testosterone production and its molecular mechanisms. Furthermore, we also investigated the role of gomisin N (GN) from Schisandra chinensis (S. chinensis) in testosterone synthesis in TM3 Leydig cells. METHOD AND RESULTS: First, we examined the effects of BBP on expression levels of testosterone biosynthesis-related genes (StAR, CYP11α1, CYP17α1, 3ßHSD, and 17ßHSD) and attenuation-related genes (CYP1ß1, CYP19α1, and Srd5α1-3). Although testosterone biosynthesis-related genes did not change, attenuation-related genes such as CYP1ß1 and CYP19α1 were upregulated with ROS generation and testosterone level attenuation in the presence of 50 µM of BBP. However, the compound with the highest ROS and ONOO- scavenging activity from S. chinensis, GN, significantly reversed the expression of BBP-induced testosterone attenuation-related gene to normal levels. Subsequently, GN improved the testosterone production levels in TM3 Leydig cells. These events may be regulated by the antioxidant effect of GN. CONCLUSIONS: On conclusion, our study suggests, for the first time, that BBP impairs testosterone synthesis by the modulation of CYP1ß1 and CYP19α1 expression in TM3 cells; GN could potentially minimize the BBP-induced dysfunction of TM3 cells to produce testosterone by suppressing CYP19α1 expression.

Schisandrol A and gomisin N from Schisandra chinensis extract improve hypogonadism via anti-oxidative stress in TM3 Leydig cells.

BACKGROUND/OBJECTIVES: Male hypogonadism is a condition where the body does not produce enough testosterone and significantly impacts health. Age, obesity, genetics, and oxidative stress are some physiological factors that may contribute to testosterone deficiency. Previous studies have shown many pharmacological benefits of Schisandra chinensis (S. chinensis) Baillon as an anti-inflammatory and antioxidant. However, the molecular mechanism of attenuating hypogonadism is yet to be well established. This research was undertaken to study the effects of S. chinensis extract (SCE) on testosterone deficiency. MATERIALS/METHODS: S. chinensis fruit was pulverized and extracted using 60% aqueous ethanol. HPLC analysis was performed to analyze and quantify the lignans of the SCE. RESULTS: The 2,2-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging assays confirmed that the SCE and its major lignans (schisandrol A and gomisin N) inhibit oxidative stress. Effects of SCE analysis on the testosterone level under oxidative stress conditions revealed that both schisandrol A and gomisin N were able to recover the lowered testosterone levels. Through mRNA expression of TM3 Leydig cell, we observed that the SCE lignans were able to induce the enzymes involved in testosterone biosynthesis-related genes such as 3ß-HSD4 (P < 0.01 for SCE, and P < 0.001 for schisandrol A and gomisin N), 17ß-HSD3 (P < 0.001 for SCE, schisandrol A and gomisin N), and 17, 20-desmolase (P < 0.01 for schisandrol A, and P < 0.001 for SCE and gomisin N). CONCLUSIONS: These results support that SCE and its active components could be potential therapeutic agents for regulating and increasing testosterone production.

Schisandrin C: an active compound from the fruit of Schisandra chinensis in anti-inflammation and anti-oxidation.

Inflammatory responses are involved in various diseases, such as insulin resistance, atherosclerosis, and hypogonadism. This study investigates the effects of SCE on anti-inflammation and molecular mechanisms in LPS-induced macrophages. RAW 264.7 macrophage cells were treated with LPS for 24 hr, followed by SCE, schisandrin C (Sch C) (1, 10, and 100 µM), and gomisin N (GN) (1, 10, and 100 µM) for 24 hr. Gene expression levels of pro-inflammatory cytokines were measured by qPCR. Protein expression of NLPR3 inflammasome was examined by western blot analysis. The expression levels of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNFα, were significantly reduced after SCE treatment. Sch C significantly inhibits these pro-inflammatory cytokines, while GN suppresses only IL6. Furthermore, Sch C significantly prevented the activation of NLRP3 inflammasome complexes such as NLRP3 and caspase-1. Sch C is the major active compound of SCE on anti-inflammation through attenuation of NLRP3 inflammasome.

LncRNA DLEU2 regulates sirtuins and mitochondrial respiratory chain complex IV: a novel pathway in obesity and offspring's health.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a rapidly expanding area of interest in chronic diseases. They are mostly unknown for roles in metabolic regulation. Sirtuins, an epigenetic modulator class, regulate metabolic pathways. However, how sirtuins are regulated via lncRNA is unknown. We hypothesized that a high-fat high-fructose diet (HFD-HF) during pregnancy would increase the risk for obesity via lncRNA-Sirtuin pathways. METHODS: Female C57Bl/6 mice (F0) were fed either chow diet (CD) or HFD-HF for 6 weeks till birth. The pups (F1) were fed either CD or HFD-HF for 20 weeks. Expression of Dleu2, sirtuins, mitochondrial respiratory complexes, and oxidative stress were investigated in the F1 livers. Fasting blood glucose, insulin sensitivity, glucose tolerance, body and tissues weight were measured. A mechanistic interaction was then carried out using a DLEU2 knockdown experiment in the HepG2 cell. RESULTS: Dleu2 and sirtuins were both significantly decreased in the livers of HFD-HF fed male F1 whose mothers were either fed CD or HFD-HF during reproductive and pregnancy windows. Confirming this connection, upon silencing DLEU2, transcription levels of SIRT1 through 6 and translational levels of SIRT1, 3, 5, and 6 were significantly downregulated. Knockdown of DLEU2 significantly decreased the protein level of cytochrome-c oxidase (complex IV, MTCO1) without altering other mitochondrial complexes, decreased mitochondrial membrane potential, decreased ATP, and increased reactive oxygen species. Interestingly, in F1 livers, the protein level of MTCO1 was also significantly decreased under an HFD-HF diet or even under chow diet if the mother was exposed to HFD-HF. CONCLUSION: Our findings reveal for the first time that one lncRNA can regulate sirtuins and a specific mitochondrial complex. Furthermore, diet or maternal diet can modulate Dleu2 and its downstream regulators in offspring, suggesting a potential role of DLEU2 in metabolic disorders over one or more generations.

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters.

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.

A moderate physiological dose of benzyl butyl phthalate exacerbates the high fat diet-induced diabesity in male mice.

Exposure to endocrine disrupting chemicals (EDCs) used in plastic manufacturing processes may be contributing to the current increase in metabolic disorders. Here, we determined that benzyl butyl phthalate (BBP), a common EDC and food packaging plasticizer, mixed into chow diet (CD) and high fat diets (HFD) at varying concentrations (4 µg/kg body weight (bw)/day, 169 µg/kg bw/day, 3 mg/kg bw/day, 50 mg/kg bw/day) produced a number of detrimental and sex-specific metabolic effects in C57BL/6 male and female mice after 16 weeks. Male mice exposed to moderate (3 mg/kg bw/day) concentrations of BBP in an HFD were especially affected, with significant increases in body weight due to significant increases in weight of liver and adipose tissue. Other doses did not show any significant changes when compared to only CD or HFD alone. HFD in the presence of 3 mg/kg bw/day BBP showed significant increases in fasting blood glucose, glucose intolerance, and insulin intolerance when compared to HFD alone. Furthermore, this group significantly alters transcriptional regulators involved in hepatic lipid synthesis and its downstream pathway. Interestingly, most of the BBP doses had no phenotypic effect when mixed with CD and compared to CD alone. The female mice did not show a similar response as the male population even though they consumed a similar amount of food. Overall, these data establish a dose which can be used for a BBP-induced metabolic research model and suggest that a moderate dosage level of EDC exposure can contribute to widely ranging metabolic effects.

Senoinflammation: A major mediator underlying age-related metabolic dysregulation.

Chronic inflammation is a complex and unresolved inflammatory response with low-grade multivariable patterns that aggravate systemic pathophysiological conditions and the aging process. To redefine and delineate these age-related complex inflammatory phenomena at the molecular, cellular, and systemic levels, the concept of "Senoinflammation" was recently formulated. In this review, we describe the accumulated data on both the multiphase systemic inflammatory process and the cellular proinflammatory signaling pathway. We also describe the proinflammatory mechanisms underlying the metabolic molecular pathways in aging. Additionally, we review age-related lipid accumulation, the role of the inflammatory senescence-associated secretory phenotype (SASP), the involvement of cytokine/chemokine secretion, endoplasmic reticulum (ER) stress, insulin resistance, and autophagy. The last section of the review highlights the modulation of the senoinflammatory process by the anti-aging and anti-inflammatory action of calorie restriction (CR). Evidence from aging and CR research strongly suggests that SASP from senescent cells may be the major source of secreted cytokines and chemokines during aging. A better understanding of the mechanisms underpinning the senoinflammatory response and the mitigating role of CR will provide insights into the molecular mechanisms of chronic inflammation and aging for potential interventions.

Mono-(2-Ethylhexyl)phthalate Regulates Cholesterol Efflux via MicroRNAs Regulated m6A RNA Methylation.

Mono-(2-ethylhexyl)phthalate (MEHP) is a major bioactive metabolite which occurs from the widely used industrial plasticizer di(2-ethylhexyl)phthalate, which has been found to be toxic to several human physiological systems, including the cardiometabolic system. Recently, RNA methylation has been shown to be involved in cardio-metabolic regulation. Despite the importance of m6A mRNA methylation in physiological processes, studies of RNA methylation associated with endocrine disrupting chemicals are lacking. Here, we investigated the effects of MEHP in a cholesterol efflux pathway and the roles of m6A methylation using murine macrophage Raw 264.7 cells. MEHP exposure significantly reduced (P < 0.01 for 50 µM) m6A mRNA methylation with decreases in both mRNA (P < 0.01 for 5 and 50 µM) and protein (P < 0.05 for 0.5 µM; P < 0.01 for 5 µM; and P < 0.001 for 50 µM) expression levels of METTL14, a component of the methyltransferase complex. However, m5C RNA methylation remained unchanged. MEHP significantly reduced the expression of Scavenger Receptor B type 1 (SR-B1) (P < 0.01 for 5 µM and P < 0.05 for 50 µM). Additionally, we demonstrated that silencing METTL14 with MEHP decreased SR-B1 gene expression compared to the MEHP treatment (P < 0.01) or silencing METTL14 alone (P < 0.05). Furthermore, MEHP significantly promoted the m6A modification in SR-B1 (P < 0.001) and activated miRNAs which are predicted to regulate METTL14, such as miR16-1-3p (P < 0.05 for 50 µM MEHP), miR101a-3p (P < 0.05 for 5 and 50 µM MEHP), miR362-3-5p (P < 0.05 for 50 µM MEHP), miR501-5p (P < 0.01 for 50 µM MEHP), miR532-3p (P < 0.05 for 50 µM MEHP), and miR542-3p (P < 0.05 for 50 µM MEHP). Together, these results reveal for the first time that MEHP can modulate RNA methylation to regulate SR-B1 in a cholesterol efflux pathway.

Mono-(2-ethylhexyl) Phthalate Aggravates Inflammatory Response via Sirtuin Regulation and Inflammasome Activation in RAW 264.7 Cells.

Artificial environmental endocrine disrupting chemicals (EDCs) exert public health concerns. Exposure to EDCs may induce various disorders in the cardiometabolic system. However, the underlying mechanisms remain largely unknown. Over the past decade, an abundance of evidence has emerged demonstrating a close link between cardiometabolic disorders and inflammation. The aim of the present study was to evaluate the immunological effects on macrophages from six EDCs via sirtuin (SIRT) regulation using the murine macrophage RAW 264.7 cell. We studied first the effects of these EDCs, including a series of doses of benzyl butyl phthalate (BBP), bisphenol A (BPA), diethylhexyl phthalate (DEHP), mono-(2-ethylhexyl)phthalate (MEHP), perfluorooctanoate (PFOA), or perfluorooctanesulfonate (PFOS), on SIRT1-7 transcriptional level. Among these EDCs, MEHP significantly decreased all sirtuin genes' expression in a dose-dependent manner. Under MEHP treatment, SIRT activity and protein expression were significantly decreased, while the protein expression of acetylated NF-κB was significantly increased along with significant increases in IL-1ß transcription. These results indicate that MEHP may induce the inflammatory response via SIRT-mediated acetylation of NF-κB. Additionally, the enhanced IL-1ß secretion in the presence of 50 µM MEHP ( P < 0.01) also supports inflammasome activation (significant ASC and NLRP3 protein augmentation). Both events may be regulated by MEHP induced reactive oxygen species ( P < 0.01). In conclusion, our study suggests for the first time that EDCs differentially modulate sirtuins' gene expression levels in macrophages and that a specific phthalate MEHP can lead to an increased inflammatory response by impairing vital epigenetic regulators and inflammasome activation.

Anti-inflammatory action of ß-hydroxybutyrate via modulation of PGC-1α and FoxO1, mimicking calorie restriction.

ß-Hydroxybutyrate (HB) is a ketone body used as an energy source that has shown anti-inflammatory effects similar to calorie restriction (CR); Here, PGC-1α, an abundantly expressed co-factor in the kidney, was reported to interact with both FoxO1 and NF-κB although the definitive interactive mechanism has not yet been reported. In this study, we investigated whether renal aging-related inflammation is modulated by HB. We compared aged rats administered with HB to calorie restricted rats and examined the modulation of FoxO1 and the NF-κB pathway through interactions with PGC-1α. We found that in aged rats treated with HB, pro-inflammatory signaling changes were reversed and showed effects comparable to CR. As FoxO1 and its target genes catalase/MnSOD were upregulated by HB treatment and PGC-1α selectively interacted with FoxO1, not with NF-κB, and ameliorated the renal inflammatory response. These findings were further confirmed using FoxO1 overexpression and siRNA transfection in vitro. Our findings suggest that HB suppressed aging-related inflammation as a CR mimetic by enabling the co-activation and selective interaction between FoxO1 and PGC-1α. This study demonstrates the potential therapeutic role of HB as a CR mimetic, which ameliorates inflammation by a novel mechanism where FoxO1 outcompetes NF-κB by interacting with PGC-1α in aging kidneys.

Hepatoprotective Effects of MHY3200 on High-Fat, Diet-Induced, Non-Alcoholic Fatty Liver Disease in Rats.

This study investigated the effects of 2-(4-(5-chlorobenzo[d]thiazol-2-yl)phenoxy)-2,2-difluoroacetic acid (MHY3200) on high-fat diet (HFD)-induced hepatic lipid accumulation and inflammation. The measurement of peroxisome proliferator-activated receptor (PPAR)α activity by using a luciferase assay indicated that MHY3200 was more potent than a known PPARα agonist, WY14643, in AC2F cells. Six-month-old male SD rats were fed chow or HFD for 1 month, and after, with or without added MHY3200 (1 or 2 mg/kg/day) for 4 weeks. The oral administration of MHY3200 caused a significant decrease in serum triglyceride (TG), glucose, alanine aminotransferase, and insulin, as well as a slight decrease in the level of free fatty acid and aspartate transaminase. No weight gain was detected when compared with HFD rats, and hepatic TG content was also attenuated by the administration of MHY3200. Furthermore, phosphorylation of the ER stress marker, inositol-requiring kinase 1 and its downstream gene, c-Jun N-terminal kinase, in addition to serine phosphorylation of insulin receptor substrate 1 were suppressed by MHY3200. Consistent with these results, MHY3200 administration reduced the levels of activation of protein-1, cyclooxygenase-2, and inducible nitric oxide synthase. Our results suggested that MHY3200 ameliorated HFD-induced hepatic lipid accumulation and inflammation, and improved insulin resistance through PPARα activation.

Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming.

Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr-/- mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3-/-/Ldlr-/- mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.

HDL Cholesterol Efflux Capacity in Newly Diagnosed HIV and Effects of Antiretroviral Therapy.

Context: In the general population, high-density lipoprotein (HDL) cholesterol efflux capacity (HCEC) relates inversely to incident cardiovascular events. Previous studies have suggested that HCEC is decreased in HIV and that antiretroviral therapy (ART) initiation might improve HCEC. Objective: To evaluate HCEC in the context of ART initiation and immune activation in HIV. Design and Outcome Measures: Baseline HCEC from 10 ART-naive HIV-infected males and 12 prospectively matched non-HIV-infected males were analyzed. In the HIV cohort, HCEC 6 months after elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (E/C/F/TDF) therapy was evaluated. HCEC served as the primary outcome and was measured by the ability of J774 mouse macrophages to efflux cholesterol. Our ex vivo assay used two cholesterol acceptors [apolipoprotein B (apoB)-depleted sera or purified HDL] and modulation of cellular efflux pathways using a liver X receptor (LXR) agonist. Results: The median age was 34 years [interquartile range (IQR), 27 to 51], and baseline HDL was 46 mg/dL (IQR, 38 to 61). HCEC was significantly greater in the non-HIV-infected subjects than in the HIV-infected subjects at baseline. HCEC, assessed using apoB-depleted sera, significantly increased after ART (no LXR agonist, baseline: median, 8.1%; IQR, 7.0% to 11.9%; after ART: median, 12.9%; IQR, 10.4% to 21.1%; P = 0.006; LXR agonist, baseline, 1.3% ± 1.3%; after ART, 2.5% ± 1.0%; P = 0.02), although not to the levels in the non-HIV-infected subjects (no LXR agonist: median, 14.9%; IQR, 11.5% to 19.1%; LXR agonist: 5.8% ± 1.3%). HCEC, assessed using purified HDL, did not significantly increase after ART. The change in HCEC with ART related inversely to the change in the percentage of CD14-CD16+ (nonclassical) monocytes (ρ = -0.74, P = 0.04) and directly to the change in the percentage of CD14+CD16- (classical) monocytes (ρ = 0.72, P = 0.045). Conclusions: Our data suggest improvement of HCEC with E/C/F/TDF and a relationship between the ART-induced decrease in immune activation and ART-induced improvement in HCEC.

PPARα activation by MHY908 attenuates age-related renal inflammation through modulation of the ROS/Akt/FoxO1 pathway.

2-[4-(5-Chlorobenzothiazothiazol-2-yl)phenoxy]-2-methyl-propionic acid (MHY908) has been shown to prevent insulin resistance-induced hyperinsulinemia in aged rats. However, the mechanism underlying MHY908-mediated amelioration of renal inflammation with insulin resistance during aging remains unknown. This study investigated the effects of MHY908 on age-related changes in the IRS/Akt/forkhead box (FoxO) 1 signaling pathway in the kidneys of aged rats and HEK293T cells. Experiments were performed in young, old, and MHY908-fed old rats (1mg or 3mg/kg/day MHY908 for 4 weeks). We found that MHY908-fed old rats suppressed phosphorylation of IRS/Akt and induced FoxO1 activation, leading to increased expression of MnSOD and catalase. In addition, in insulin-treated cells, MHY908 prevented the FoxO1 inactivation and increased the expression of MnSOD and catalase by inactivating IRS and Akt. In contrast, NF-κB signaling pathway decreased with MHY908 treatment in insulin-treated cells. Furthermore, MHY908 exclusively activated peroxisome proliferator-activated receptor (PPAR) α in the kidneys, leading to the inhibition of insulin-induced NADPH oxidase subunit 4 (NOX4)-derived reactive oxygen species (ROS) generation and FoxO1 inactivation. In conclusion, MHY908 improved the hyperinsulinemia-induced pro-inflammatory response through NF-κB inactivation and FoxO1 activation in aged rat kidneys. These phenomena suggest that PPARα activation by MHY908 attenuates NOX4-derived ROS generation in response to insulin.

Physiological characterization of a novel PPAR pan agonist, 2-(4-(5,6-methylenedioxybenzo[d]thiazol-2-yl)-2-methylphenoxy)-2-methylpropanoic acid (MHY2013).

Recently, agonists targeting multiple peroxisome proliferator-activated receptors (PPARs) have been developed to improve metabolic disorders and minimize the side effects of selective PPAR agonists such as weight gain and dyslipidemia. We newly synthesized six 2-methyl-2-(o-tolyloxy)propanoic acid derivatives based on the structure of a well-known PPAR pan agonist, bezafibrate. Of six compounds, MHY2013 was screened as the strongest activator of three PPAR subtypes based on protein docking simulation and luciferase assays. When treated orally in db/db mice, MHY2013 ameliorated obesity-induced insulin resistance, dyslipidemia, and hepatic steatosis without changes of the body weight and levels of liver and kidney injury markers. MHY2013 decreased the serum triglyceride and fatty acid levels, which is associated with an increase in fatty acid oxidation signaling in the liver and thermogenic signaling on white adipose tissue, respectively. Furthermore, MHY2013 markedly increased serum levels of insulin-sensitizing hormones including fibroblast growth factor 21 (FGF21) and adiponectin. In conclusion, this study suggests that, MHY2013 is a novel PPAR pan agonist that improves obesity-induced insulin resistance, dyslipidemia and hepatic steatosis and elevates insulin-sensitizing hormones in the blood.

ß-Hydroxybutyrate suppresses inflammasome formation by ameliorating endoplasmic reticulum stress via AMPK activation.

ß-Hydroxybutyrate, a ketone body that is used as an energy source in organs such as the brain, muscle, and heart when blood glucose is low, is produced by fatty acid oxidation in the liver under the fasting state. Endoplasmic reticulum (ER) stress is linked with the generation of intracellular reactive oxygen species and the accumulation of misfolded protein in the ER. ER stress is known to induce the NOD-like receptor protein 3 inflammasome, which mediates activation of the proinflammatory cytokine interleukin-1ß, whose maturation is caspase-1-dependent. We investigated whether ß-hydroxybutyrate modulates ER stress, inflammasome formation, and insulin signaling. Sprague Dawley rats (6 and 24 months of age) that were starved for 3 d and rats treated with ß-hydroxybutyrate (200 mg·kg-1·d-1 i.p., for 5 d) were used for in vivo investigations, whereas human hepatoma HepG2 cells were used for in vitro studies. Overexpression of AMPK in cultured cells was performed to elucidate the molecular mechanism. The starvation resulted in increased serum ß-hydroxybutyrate levels with decreased ER stress (PERK, IRE1, and ATF6α) and inflammasome (ASC, caspase-1, and NLRP3) formation compared with non-fasted 24-month-old rats. In addition, ß-hydroxybutyrate suppressed the increase of ER stress- and inflammasome-related marker proteins. Furthermore, ß-hydroxybutyrate treatment increased the expression of manganese superoxide dismutase and catalase via the AMP-activated protein kinase-forkhead box protein O3α transcription factor pathway both in vivo and in vitro. The significance of the current study was the discovery of the potential therapeutic role of ß-hydroxybutyrate in suppressing ER-stress-induced inflammasome formation.

Molecular Mechanism of Betaine on Hepatic Lipid Metabolism: Inhibition of Forkhead Box O1 (FoxO1) Binding to Peroxisome Proliferator-Activated Receptor Gamma (PPARγ).

Betaine is a major water-soluble component of Lycium chinensis. Although there are reports about the protective effects of betaine on hepatic steatosis, the underlying mechanisms are unclear. We used db/db mice and HepG2 cells to examine the mechanism underlying betaine-mediated protection against hepatic steatosis. Here, we showed increased hepatic lipid accumulation in db/db mice, which is associated with increased activation of lipogenic transcription factors including forkhead box O1 (FoxO1) and peroxisome proliferator-activated receptor gamma (PPARγ), whereas betaine administration by oral gavage reversed these characteristics. We investigated whether betaine ameliorates hepatic steatosis by inhibiting FoxO1/PPARγ signaling in HepG2 cells. Although adenovirus-mediated FoxO1 overexpression notably increased mRNA expression levels of PPARγ and its target genes including FAS and ACC, betaine treatment reversed them. Furthermore, betaine inhibited FoxO1 binding to the PPARγ promoter and PPARγ transcriptional activity in HepG2 cells, which was previously shown to induce hepatic steatosis. We concluded that betaine ameliorates hepatic steatosis, at least in part, by inhibiting the FoxO1 binding to PPARγ and their downstream lipogenic signaling cascade.

RNA-Seq analysis reveals new evidence for inflammation-related changes in aged kidney.

Age-related dysregulated inflammation plays an essential role as a major risk factor underlying the pathophysiological aging process. To better understand how inflammatory processes are related to aging at the molecular level, we sequenced the transcriptome of young and aged rat kidney using RNA-Seq to detect known genes, novel genes, and alternative splicing events that are differentially expressed. By comparing young (6 months of age) and old (25 months of age) rats, we detected 722 up-regulated genes and 111 down-regulated genes. In the aged rats, we found 32 novel genes and 107 alternatively spliced genes. Notably, 6.6% of the up-regulated genes were related to inflammation (P < 2.2 × 10-16, Fisher exact t-test); 15.6% were novel genes with functional protein domains (P = 1.4 × 10-5); and 6.5% were genes showing alternative splicing events (P = 3.3 × 10-4). Based on the results of pathway analysis, we detected the involvement of inflammation-related pathways such as cytokines (P = 4.4 × 10-16), which were found up-regulated in the aged rats. Furthermore, an up-regulated inflammatory gene analysis identified the involvement of transcription factors, such as STAT4, EGR1, and FOSL1, which regulate cancer as well as inflammation in aging processes. Thus, RNA changes in these pathways support their involvement in the pro-inflammatory status during aging. We propose that whole RNA-Seq is a useful tool to identify novel genes and alternative splicing events by documenting broadly implicated inflammation-related genes involved in aging processes.