Design and Synthesis of l-1'-Homologated Adenosine Derivatives as Potential Anti-inflammatory Agents.

Inflammatory responses are fundamental protective warning mechanisms. However, in certain instances, they contribute significantly to the development of several chronic diseases such as cancer. Based on previous studies of truncated 1'-homologated adenosine derivatives, l-nucleosides and their nucleobase-modified quinolone analogues were designed, synthesized, and evaluated for anti-inflammatory activities. The target molecules were synthesized via the key intramolecular cyclization of monotosylate and Mitsunobu condensation from the natural product, d-ribose. All compounds tested and showed potent anti-inflammatory activities, as indicated by their inhibition of LPS-induced IL-1ß secretion from the RAW 264.7 macrophages. Gene expressions of pro-inflammatory cytokines showed that all compounds, except 3a and 3b, significantly reduced LPS-induced IL-1ß and IL-6 mRNA expressions. The half-maximal inhibitory concentrations (IC50) of 2g and 2h against IL-1ß were 1.08 and 2.28 µM, respectively. In contrast, only 2d, 2g, and 3d effectively reversed LPS-induced TNFα mRNA expression. Our mechanistic study revealed that LPS-induced phosphorylation of NF-κB was significantly downregulated by all compounds tested, providing evidence that the NF-κB signaling pathway is involved in their anti-inflammatory activities. Among the compounds tested, 2g and 2h had the most potent anti-inflammatory effects, as shown by the extent of decrease in pro-inflammatory gene expression, protein secretion, and NF-κB phosphorylation. These findings suggest that the l-truncated 1'-homologated adenosine skeleton and its nucleobase-modified analogues have therapeutic potential as treatments for various human diseases by mediating inflammatory processes.

TNFα-induced NLRP3 inflammasome mediates adipocyte dysfunction and activates macrophages through adipocyte-derived lipocalin 2.

BACKGROUND AND AIMS: Obesity is a state of chronic low-grade systemic inflammation. Recent studies showed that NLRP3 inflammasome initiates metabolic dysregulation in adipose tissues, primarily through activation of adipose tissue infiltrated macrophages. However, the mechanism of NLRP3 activation and its role in adipocytes remains elusive. Therefore, we aimed to examine the activation of TNFα-induced NLRP3 inflammasome in adipocytes and its role on adipocyte metabolism and crosstalk with macrophages. METHODS: The effect of TNFα on adipocyte NLRP3 inflammasome activation was measured. Caspase-1 inhibitor (Ac-YVAD-cmk) and primary adipocytes from NLRP3 and caspase-1 knockout mice were utilized to block NLRP3 inflammasome activation. Biomarkers were measured by using real-time PCR, western blotting, immunofluorescence staining, and enzyme assay kits. Conditioned media from TNFα-stimulated adipocytes was used to establish the adipocyte-macrophage crosstalk. Chromatin immunoprecipitation assay was used to identify the role of NLRP3 as a transcription factor. Mouse and human adipose tissues were collected for correlation analysis. RESULTS: TNFα treatment induced NLRP3 expression and caspase-1 activity in adipocytes, partly through autophagy dysregulation. The activated adipocyte NLRP3 inflammasome participated in mitochondrial dysfunction and insulin resistance, as evidenced by the amelioration of these effects in Ac-YVAD-cmk treated 3T3-L1 cells or primary adipocytes isolated from NLRP3 and caspase-1 knockout mice. Particularly, the adipocyte NLRP3 inflammasome was involved in glucose uptake regulation. Also, TNFα induced expression and secretion of lipocalin 2 (Lcn2) in a NLRP3-dependent manner. NLRP3 could bind to the promoter and transcriptionally regulate Lcn2 in adipocytes. Treatment with adipocyte conditioned media revealed that adipocyte-derived Lcn2 was responsible for macrophage NLRP3 inflammasome activation, working as a second signal. Adipocytes isolated from high-fat diet mice and adipose tissue from obese individuals showed a positive correlation between NLRP3 and Lcn2 gene expression. CONCLUSIONS: This study highlights the importance of adipocyte NLRP3 inflammasome activation and novel role of TNFα-NLRP3-Lcn2 axis in adipose tissue. It adds rational for the current development of NLRP3 inhibitors for treating obesity-induced metabolic diseases.

Structure-Activity Relationships of Truncated 1'-Homologated Carbaadenosine Derivatives as New PPARγ/δ Ligands: A Study on Sugar Puckering Affecting Binding to PPARs.

Peroxisome proliferator-activated receptors (PPARs) are associated with the regulation of metabolic homeostasis. Based on a previous report that 1'-homologated 4'-thionucleoside acts as a dual PPARγ/δ modulator, carbocyclic nucleosides 2-5 with various sugar conformations were synthesized to determine whether sugar puckering affects binding to PPARs. (S)-conformer 2 was synthesized using Charette asymmetric cyclopropanation, whereas (N)-conformer 3 was synthesized using stereoselective Simmons-Smith cyclopropanation. All synthesized nucleosides did not exhibit binding affinity to PPARα but exhibited significant binding affinities to PPARγ/δ. The binding affinity of final nucleosides to PPARγ did not differ significantly based on their conformation, but their affinity to PPARδ depended greatly on their conformation, correlated with adiponectin production. (N)-conformer 3h was discovered to be the most potent PPARδ antagonist with good adiponectin production, which exhibited the most effective activity in inhibiting the mRNA levels of LPS-induced IL-1ß expression in RAW 264.7 macrophages, implicating its anti-inflammatory activity.

Inhibition of autophagy with chloroquine dysregulates mitochondrial quality control and energetics in adipocytes.

Autophagy is a complex degradation pathway through which damaged or dysfunctional proteins and organelles are removed. Its pharmacological modulators have been extensively used in a wide range of basic research and preclinical studies. However, the effects of these inhibitors on metabolism, in addition to autophagy inhibition, are not fully elucidated. Chloroquine is a clinically relevant compound that inhibits autophagy by preventing the fusion of autophagosomes with lysosomes. In this study, we aimed to examine the effect of chloroquine on mitochondrial quality control and respiratory function by utilizing 3T3-L1 mouse adipocytes treated with chloroquine at various time points. We found that chloroquine could disturb genes related to mitochondrial fission, biogenesis, and mitophagy, leading to mitochondrial DNA damage. Although the inhibition of autophagy by chloroquine resulted in an increased prohibitin expression, respiratory function was downregulated in a time-dependent manner. Moreover, chloroquine treatment induced oxidative stress, apoptosis, and metabolic dysregulation. These data demonstrated that chloroquine significantly affected mitochondrial respiratory function and metabolism, which was consistent with impaired mitochondrial quality associated with autophagy inhibition.

Gasdermin D Deficiency Does Not Protect Mice from High-Fat Diet-Induced Glucose Intolerance and Adipose Tissue Inflammation.

The adipose tissue NLRP3 inflammasome has recently emerged as a contributor to obesity-related metabolic inflammation. Recent studies have demonstrated that the activation of the NLRP3 inflammasome cleaves gasdermin D (GSDMD) and induces pyroptosis, a proinflammatory programmed cell death. However, whether GSDMD is involved in the regulation of adipose tissue function and the development of obesity-induced metabolic disease remains unknown. The aim of the present study was to investigate the role of GSDMD in adipose tissue inflammation as well as whole-body metabolism using GSDMD-deficient mice fed a high-fat diet (HFD) for 30 weeks. The effects of GSDMD deficiency on adipose tissue, liver, and isolated macrophages from wild-type (WT) and GSDMD knockout (KO) mice were examined. In addition, 3T3-L1 cells were used to examine the expression of GSDMD during adipogenesis. The results demonstrate that although HFD-induced inflammation was partly ameliorated in isolated macrophages and liver, adipose tissue remained unaffected by GSDMD deficiency. Compared with the WT HFD mice, GSDMD KO HFD mice exhibited a mild increase in HFD-induced glucose intolerance with increased systemic and adipose tissue IL-1ß levels. Interestingly, GSDMD deficiency caused accumulation of fat mass when challenged with HFD, partly by suppressing the expression of peroxisome proliferator-activated receptor gamma (PPARγ). The expression of GSDMD mRNA and protein was dramatically suppressed during adipocyte differentiation and was inversely correlated with PPARγ expression. Together, these findings indicate that GSDMD is not a prerequisite for HFD-induced adipose tissue inflammation and suggest a noncanonical function of GSDMD in regulation of fat mass through PPARγ.

Exercise-Induced Irisin Decreases Inflammation and Improves NAFLD by Competitive Binding with MD2.

Non-alcoholic fatty liver disease (NAFLD) is a global clinical problem. The MD2-TLR4 pathway exacerbates NAFLD progression by promoting inflammation. Long-term exercise is considered to improve NAFLD but the underlying mechanism is still unclear. In this study, we examined the protective effect and molecular mechanism of exercise on high-fat diet (HFD)-induced liver injury. In an HFD-induced NAFLD mouse model, exercise training significantly decreased hepatic steatosis and fibrosis. Interestingly, exercise training blocked the binding of MD2-TLR4 and decreased the downstream inflammatory response. Irisin is a myokine that is highly expressed in response to exercise and exerts anti-inflammatory effects. We found that circulating irisin levels and muscle irisin expression were significantly increased in exercised mice, suggesting that irisin could mediate the effect of exercise on NAFLD. In vitro studies showed that irisin improved lipid metabolism, fibrosis, and inflammation in palmitic acid (PA)-stimulated AML12 cells. Moreover, binding assay results showed that irisin disturbed MD2-TLR4 complex formation by directly binding with MD2 but not TLR4, and interfered with the recognition of stimuli such as PA and lipopolysaccharide with MD2. Our study provides novel evidence that exercise-induced irisin inhibits inflammation via competitive binding with MD2 to improve NAFLD. Thus, irisin could be considered a potential therapy for NAFLD.

Exercise Inhibits NLRP3 Inflammasome Activation in Obese Mice via the Anti-Inflammatory Effect of Meteorin-like.

Obesity is associated with chronic low-grade inflammation. The benefits of exercise are partly attributed to its anti-inflammatory effect, but whether exercise can regulate NLRP3 inflammasome activation in obese adipose tissue remains unknown. Meteorin-like (METRNL), a recently discovered myokine, has been implicated in mediating the effect of exercise on metabolism. Herein, we examined the effect of exercise and METRNL on NLRP3 inflammasome activation. High-fat diet (HFD)-induced obese mice were subjected to treadmill exercise for 8 weeks. A subgroup of HFD mice was switched to normal chow with the exercise intervention. Exercise and diet attenuated weight gain, fat accumulation, and insulin resistance in obese mice. In addition, exercise downregulated gene and protein levels of inflammasome markers, including NLRP3 and caspase-1, in adipose tissue. In isolated bone marrow-derived macrophages, activation of NLRP3 inflammasome was suppressed in the exercise group, as confirmed by the downregulation of IL-1ß and IL-18. Exercise significantly enhanced the expression of METRNL in various muscle depots, and further in vitro analysis revealed that recombinant METRNL treatment inhibited IL-1ß secretion in macrophages. In conclusion, exercise exerts its anti-inflammatory action by suppressing adipose tissue NLRP3 inflammasome, and this is, in part, associated with METRNL induction in muscle and its anti-inflammatory effects in macrophages.

Fibroblast growth factor 2 exacerbates inflammation in adipocytes through NLRP3 inflammasome activation.

Chronic inflammation in adipose tissue is the hallmark of obesity and a major risk factor for the development of obesity-induced insulin resistance. NLRP3 inflammasome regulates the maturation and secretion of pro-inflammatory cytokines, such as IL-1ß and IL-18, and was recently discovered to be involved in obesity-related metabolic diseases. Fibroblast growth factors (FGFs) such as FGF1, FGF10, and FGF21 are adipokines that regulate adipocyte development and metabolism, but reports on the effect of other FGFs on adipocytes are lacking. In the present study, the novel role of FGF2 in NLRP3 inflammasome activation was elucidated. Our results showed that FGF2 levels were increased during adipocyte differentiation and in the adipose tissue of high-fat diet (HFD)-induced obese mice. Recombinant FGF2 treatment upregulated inflammasome markers such as NLRP3, which was further exaggerated by TNF-ɑ treatment. Interestingly, ß-Klotho, a co-receptor of FGF21, was significantly decreased by FGF2 treatment. Results from mice confirmed the positive correlation between FGF2 and NLRP3 expression in epididymal and subcutaneous adipose tissue, while exercise training effectively reversed HFD-induced NLRP3 expression as well as FGF2 levels in both adipose depots. Our results suggest that FGF2 is an adipokine that may exacerbate the inflammatory response in adipocytes through NLRP3 inflammasome activation.

Bio-transformation of green tea infusion with tannase and its improvement on adipocyte metabolism.

Catechins in green tea possess various health benefits. Enzymatic treatment improves physiological activities by inducing bioconversion of catechins. Here, we investigated the effect of green tea infusion (GT) after tannase treatment, which transforms (-)-epigallocatechin gallate (EGCG) to gallic acid (GA) and (-)-epigallocatechin (EGC), on adipocyte differentiation and mature adipocyte metabolism. The optimal conditions for tannase-mediated improvement in GA and EGC yields in GT were investigated using response surface methodology. Yields of GA and EGC were 43-fold (0.43 mg/mL) and 1.66-fold higher (1.11 mg/mL), respectively, compared to GT without tannase treatment. The optimal reaction conditions for tannase-mediated biotransformation were observed on 0.54 mg mL-1 of tannase, reaction time (86.79 min), and reaction temperature at 22.59 °C. GT and tannase-treated GT (TANs) upregulated adiponectin, uncoupling protein 1, adipose triglyceride lipase, and hormone-sensitive lipase gene expression in differentiated 3T3-L1 adipocytes, with TAN inducing better effects than GT, which implies that tannase treatment improved the beneficial effect of GT on adipocyte metabolism. Thus, tannase-mediated bio-transformation is an attractive candidate for preparing GT with enhanced anti-obesity properties.