Inhibitory Effects of Toll-Like Receptor 4, NLRP3 Inflammasome, and Interleukin-1β on White Adipocyte Browning

Adipose tissue expansion is accompanied by infiltration and accumulation of pro-inflammatory macrophages, which links obesity to pathologic conditions such as type 2 diabetes. However, little is known regarding the role of pro-inflammatory adipose tissue remodeling in the thermogenic activation of brown/beige fat. Here, we investigated the effect of pattern recognition receptors (PRR) activation in macrophages, especially the toll-like receptor 4 (TLR4) and Nod-like receptor 3 (NLRP3), on white adipocyte browning. We report that TLR4 activation by lipopolysaccharide repressed white adipocyte browning in response to β3-adrenergic receptor activation and caused ROS production and mitochondrial dysfunction, while genetic deletion of TLR4 protected mitochondrial function and thermogenesis. In addition, activation of NLRP3 inflammasome in macrophages attenuated UCP1 induction and mitochondrial respiration in cultures of primary adipocytes, while the absence of NLRP3 protected UCP1 in adipocytes. The effect of NLRP3 inflammasome activation on browning was mediated by IL-1β signaling, as blocking IL-1 receptor in adipocytes protected thermogenesis. We also report that IL-1β interferes with thermogenesis via oxidative stress stimulation and mitochondrial dysfunction as we observed a statistically significant increase in ROS production, decrease in SOD enzyme activity, and increase in mitochondrial depolarization in adipocytes treated with IL-1β. Collectively, we demonstrated that inflammatory response to obesity, such as TLR4 and NLRP3 inflammasome activation as well as IL-1β secretion, attenuates β3-adrenoreceptor-induced beige adipocyte formation via oxidative stress and mitochondrial dysfunction. Our findings provide insights into targeting innate inflammatory system for enhancement of the adaptive thermogenesis against obesity

The cysteine protease legumain is upregulated by vitamin D and regulates vitamin D metabolism

Legumain is a lysosomal asparaginyl endopeptidase involved in different biological processes and pathogenesis of several malignant and non-malignant diseases. Although an increasing number of proteins have been identified as substrates of legumain, the upstream mechanisms regulating the expression and function of legumain are not well-understood. Here, we provide in vitro and in vivo data showing that vitamin D3 (VD3) enhances legumain expression and function. In turn, legumain alters VD3 bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP) and thereby playing a role in the regulation of VD3 metabolism. Active VD3 (1,25(OH)2D3) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Positive regulation of legumain was also observed in vivo, evidenced by increased expression of legumain mRNA in liver and spleen, as well as increased legumain activity in the kidneys, and increased level of circulating legumain in serum from mice treated with 25(OH)D3 (50 µg/kg, subcutaneous) for 8 days, as compared to vehicle-treated mice. We further showed that legumain cleaved VDBP forming a 45 kDa VDBP fragment in vitro when purified VDBP (55 kDa) was incubated with purified active legumain. In vivo, no VDBP cleavage was found in kidneys or liver from legumain deficient mice (Lgmn-/-), whereas VDBP was cleaved in wild type control mice (Lgmn+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D3 and total VD3 and altered expression of key renal enzymes involved in VD3 metabolism (CYP24A1 and CYP27B1). Taken together, our data suggest existence of a regulatory interplay between VD3 and legumain

The stimulus effect of BMP7 and the inhibitory effect of TLR4 on adaptive thermogenesis

Adaptive thermogenesis is a cellular process that converts excess energy into heat via uncoupled respiration. Adaptive thermogenesis is mediated by an emergence of brown-like adipocytes (beige) within white adipose tissue (WAT), in a process called ‘WAT browning’. Adult humans retain a substantial amount of beige fat that increases energy expenditure upon various inducers such as cold exposure. Therefore, activation of brown/beige adipocytes is an attractive strategy to facilitate weight loss and improve metabolic health. However, obese subjects are less responsive to thermogenic signaling cues compared to lean subjects. To enhance WAT browning, either increasing brown stimulatory signaling cues, or decreasing inhibitory signaling is necessary. BMP7 stimulates brown adipocyte formation, especially in rodents. However, less is known about its role in humans. In addition, the characteristic of the BMP7-induced brown adipocytes, whether they are beige or classical brown fat cells, is undetermined. In this dissertation, I aimed to investigate the effect of BMP7 on human brown adipocyte differentiation and metabolic activities. I found that BMP7 stimulates the differentiation of human stem cells to brown adipocytes expressing beige-specific markers. BMP7-derived beige adipocytes were metabolically active and their oxygen consumption rate was higher than non-BMP7 treated cells. These findings can facilitate the discovery of new compounds that can affect thermogenesis as BMP7-derived beige cells can be used as a human model of beige adipocytes. TLR4 activation, in response to elevated endotoxins and/or free fatty acids, is well documented in obese subjects and can lead to chronic inflammation. However, whether TLR4 activation plays a role in the adaptive thermogenesis impairment observed in obesity has not been established yet. In the second part of my dissertation, I aimed to identify the role of TLR4-induced inflammation on WAT browning. TLR4 activation by a high-fat diet or LPS, a TLR4 ligand, attenuated adaptive thermogenesis in mice, whereas adaptive thermogenesis in TLR4 knockout mice was protected. Chronic TLR4 activation increased ROS production and ER stress. Blunting ER stress by pharmacological inhibitors or genetic deletion of upstream regulator CHOP protected from TLR4-induced WAT browning inhibition. These results indicate that 1) TLR4 activation is a pivotal contributor in thermogenic impairment observed in obesity, and 2) targeted inhibition of TLR4/ER stress signaling axis might be efficacious in promoting adaptive thermogenesis

Ellagic acid inhibits adipocyte differentiation through coactivator-associated arginine methyltransferase 1-mediated chromatin modification

Chromatin remodeling is a key mechanism in adipocyte differentiation. However, it is unknown whether dietary polyphenols are epigenetic effectors for adiposity control. Ellagic acid (EA) is a naturally occurring polyphenol in numerous fruits and vegetables. Recently, EA-containing foods have been reported to reduce adiposity. In the present study, we sought to determine whether EA inhibits adipogenesis by modifying chromatin remodeling in human adipogenic stem cells (hASCs). qPCR microarray of chromatin modification enzymes revealed that 10 μmol/L of EA significantly inhibits histone deacetylase (HDAC)9 downregulation. In addition, EA was associated with up-regulation of HDAC activity and a marked reduction of histone acetylation levels. However, chemical inhibition of HDAC activity or depletion of HDAC9 by siRNA were not sufficient to reverse the antiadipogenic effects of EA. Intriguingly, EA treatment was also associated with reduced histone 3 arginine 17 methylation levels (H3R17me2), implying the inhibitory role of EA in coactivator-associated arginine methyltransferase 1 (CARM)1 activity during adipogenesis. Boosting CARM1 activity by delivering cell-penetrating peptides of CARM1 not only recovered H3R17me2 but also restored adipogenesis evidenced by H3 acetylation at lysine 9, HDAC9 down-regulation, PPARγ expression and triglyceride accumulation. Taken together, our data suggest that reduced CARM1 activity by EA results in a decrease of H3R17me2 levels, which may interrupt consecutive histone remodeling steps for adipocyte differentiation including histone acetylation and HDAC9 dissociation from chromatin. Our work provides the mechanistic insights into how EA, a polyphenol ubiquitously found in fruits and vegetables, attenuates human adipocyte differentiation by altering chromatin remodeling

Inhibitory Effects of Toll-Like Receptor 4, NLRP3 Inflammasome, and Interleukin-1β on White Adipocyte Browning

Adipose tissue expansion is accompanied by infiltration and accumulation of pro-inflammatory macrophages, which links obesity to pathologic conditions such as type 2 diabetes. However, little is known regarding the role of pro-inflammatory adipose tissue remodeling in the thermogenic activation of brown/beige fat. Here, we investigated the effect of pattern recognition receptors (PRR) activation in macrophages, especially the toll-like receptor 4 (TLR4) and Nod-like receptor 3 (NLRP3), on white adipocyte browning. We report that TLR4 activation by lipopolysaccharide repressed white adipocyte browning in response to β3-adrenergic receptor activation and caused ROS production and mitochondrial dysfunction, while genetic deletion of TLR4 protected mitochondrial function and thermogenesis. In addition, activation of NLRP3 inflammasome in macrophages attenuated UCP1 induction and mitochondrial respiration in cultures of primary adipocytes, while the absence of NLRP3 protected UCP1 in adipocytes. The effect of NLRP3 inflammasome activation on browning was mediated by IL-1β signaling, as blocking IL-1 receptor in adipocytes protected thermogenesis. We also report that IL-1β interferes with thermogenesis via oxidative stress stimulation and mitochondrial dysfunction as we observed a statistically significant increase in ROS production, decrease in SOD enzyme activity, and increase in mitochondrial depolarization in adipocytes treated with IL-1β. Collectively, we demonstrated that inflammatory response to obesity, such as TLR4 and NLRP3 inflammasome activation as well as IL-1β secretion, attenuates β3-adrenoreceptor-induced beige adipocyte formation via oxidative stress and mitochondrial dysfunction. Our findings provide insights into targeting innate inflammatory system for enhancement of the adaptive thermogenesis against obesity

Activation of autophagy and AMPK by gamma-tocotrienol suppresses the adipogenesis in human adipose derived stem cells

Scope: This study investigated the mechanistic details by which gamma-tocotrienol (γ-T3) manipulates adipocyte differentiation in human adipose derived stem cells (hASCs). Methods and results: γ-T3 specifically inhibited the early stage of adipocyte differentiation by acting on downstream of C/EBP-β but upstream of C/EBP-α in hASCs. In searching a potential mechanism, we identified that γ-T3 promoted two catabolic signaling pathways: (i) AMP kinase (AMPK), and (ii) enhanced autophagy, as assessed by autophagic flux and cytosolic autophagosome (LC3II) accumulation. In addition, chronic exposure of γ-T3 induced caspase3-mediated apoptotic cell death. The blockage of AMPK by a dominant negative mutant of AMPK was insufficient to normalize γ-T3-mediated autophagy, suggesting that enhanced autophagic activity of γ-T3 is independent of AMPK activation. Intriguingly, AMPK inhibition significantly restored PPAR-γ activation, but marginally rescued lipid-loaded adipocyte morphology due to, at least partly, a lack of lipid droplet-coating protein. These data suggest that γ-T3 activates AMPK and autophagy signaling, which synergistically contributes to the suppression of adipogenic conversion of hASCs into adipocytes. Conclusion: These results provide a novel insight into the molecular mechanism involved in anti-adipogenic action of γ-T3 in humans via AMPK and autophagy activation. Thus, γ-T3may constitute a new dietary avenue to attenuate hyperplastic obesity in humans

Suppression of NLRP3 inflammasome by γ-tocotrienol ameliorates type 2 diabetes

The Nod-like receptor 3 (NLRP3) inflammasome is an intracellular sensor that sets off the innate immune system in response to microbial-derived and endogenous metabolic danger signals. We previously reported that γ-tocotrienol (γT3) attenuated adipose tissue inflammation and insulin resistance in diet-induced obesity, but the underlying mechanism remained elusive. Here, we investigated the effects of γT3 on NLRP3 inflammasome activation and attendant consequences on type 2 diabetes. γT3 repressed inflammasome activation, caspase-1 cleavage, and interleukin (IL) 1β secretion in murine macrophages, implicating the inhibition of NLRP3 inflammasome in the anti-inflammatory and antipyroptotic properties of γT3. Furthermore, supplementation of leptin-receptor KO mice with γT3 attenuated immune cell infiltration into adipose tissue, decreased circulating IL-18 levels, preserved pancreatic β-cells, and improved insulin sensitivity. Mechanistically, γT3 regulated the NLRP3 inflammasome via a two-pronged mechanism: 1) the induction of A20/TNF-α interacting protein 3 leading to the inhibition of the TNF receptor-associated factor 6/nuclear factor κB pathway and 2) the activation of AMP-activated protein kinase/autophagy axis leading to the attenuation of caspase-1 cleavage. Collectively, we demonstrated, for the first time, that γT3 inhibits the NLRP3 inflammasome thereby delaying the progression of type 2 diabetes. This study also provides an insight into the novel therapeutic values of γT3 for treating NLRP3 inflammasome-associated chronic diseases

Activation of Toll-like Receptor 4 (TLR4) Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress

Background: Human obesity is associated with defective brown adipose tissue (BAT) activation. Results: Toll-like receptor 4(TLR4) activation by high fat diet or lipopolysaccharide impairs adaptive thermogenesis. Conclusion: Obesity-mediated TLR4 activation represses adaptive thermogenesis through endoplasmic reticulum (ER) stress-mediated mitochondrial dysfunction. Significance: Inhibition of TLR4/ER stress axis is a novel target to augment BAT activity. Abstract: Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study we hypothesized that Toll-like receptor 4 (TLR4) activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by a high fat diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of endoplasmic reticulum (ER) stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced upregulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone conferred a resistance to the LPSinduced suppression of adaptive thermogenesis. Collectively, our data indicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective brown adipose tissue activatio