Search

Background: High-fat diets cause gut dysbiosis and promote triglyceride accumulation, obesity, gut permeability changes, inflammation, and insulin resistance. Both cocoa butter and fish oil are considered to be a part of healthy diets. However, their differential effects on gut microbiome perturbations in mice fed high concentrations of these fats, in the absence of sucrose, remains to be elucidated. The aim of the study was to test whether the sucrose-free cocoa butter-based high-fat diet (C-HFD) feeding in mice leads to gut dysbiosis that associates with a pathologic phenotype marked by hepatic steatosis, low-grade inflammation, perturbed glucose homeostasis, and insulin resistance, compared with control mice fed the fish oil based high-fat diet (F-HFD). Results: C57BL/6 mice (5–6 mice/group) were fed two types of high fat diets (C-HFD and F-HFD) for 24 weeks. No significant difference was found in the liver weight or total body weight between the two groups. The 16S rRNA sequencing of gut bacterial samples displayed gut dysbiosis in C-HFD group, with differentially-altered microbial diversity or relative abundances. Bacteroidetes, Firmicutes, and Proteobacteria were highly abundant in C-HFD group, while the Verrucomicrobia, Saccharibacteria (TM7), Actinobacteria, and Tenericutes were more abundant in F-HFD group. Other taxa in C-HFD group included the Bacteroides, Odoribacter, Sutterella, Firmicutes bacterium (AF12), Anaeroplasma, Roseburia, and Parabacteroides distasonis. An increased Firmicutes/Bacteroidetes (F/B) ratio in C-HFD group, compared with F-HFD group, indicated the gut dysbiosis. These gut bacterial changes in C-HFD group had predicted associations with fatty liver disease and with lipogenic, inflammatory, glucose metabolic, and insulin signaling pathways. Consistent with its microbiome shift, the C-HFD group showed hepatic inflammation and steatosis, high fasting blood glucose, insulin resistance, increased hepatic de novo lipogenesis (Acetyl CoA carboxylases 1 (Acaca), Fatty acid synthase (Fasn), Stearoyl-CoA desaturase-1 (Scd1), Elongation of long-chain fatty acids family member 6 (Elovl6), Peroxisome proliferator-activated receptor-gamma (Pparg) and cholesterol synthesis (β-(hydroxy β-methylglutaryl-CoA reductase (Hmgcr). Non-significant differences were observed regarding fatty acid uptake (Cluster of differentiation 36 (CD36), Fatty acid binding protein-1 (Fabp1) and efflux (ATP-binding cassette G1 (Abcg1), Microsomal TG transfer protein (Mttp) in C-HFD group, compared with F-HFD group. The C-HFD group also displayed increased gene expression of inflammatory markers including Tumor necrosis factor alpha (Tnfa), C-C motif chemokine ligand 2 (Ccl2), and Interleukin-12 (Il12), as well as a tendency for liver fibrosis. Conclusion: These findings suggest that the sucrose-free C-HFD feeding in mice induces gut dysbiosis which associates with liver inflammation, steatosis, glucose intolerance and insulin resistance. [ABSTRACT FROM AUTHOR]

Background/Aims: Innate immune toll-like receptors (TLRs) are emerging as nutrient sensors. Oxidative stress in the adipose tissue in obesity acts as a critical early trigger of altered pathophysiology. TLR2/TLR4 adipose upregulation has been associated with insulin resistance in humans; however, it remains unclear whether oxidative stress can modulate expression of TLR2/4 and related immune-metabolic regulators (IRF3/5) in immune cells. We, therefore, assessed their expression along with proinflammatory cytokines in the human PBMC following induction of oxidative stress. Methods: PBMC were isolated from blood of healthy donors using Ficoll-Paque method and cells were treated with H2O2 to induce oxidative stress. ROS was measured by DCFH-DA assay. Target gene and protein expression was determined using real-time RT-PCR and flow cytometry/confocal microscopy, respectively. TLR2/4 expression by H2O2 in presence of ROS-inhibitors or leptin/LPS/fatty acids was also assessed. Expression of phosphorylated/total ERK1/2, c-Jun, p38, and NF-κB was determined by western blotting. The data (mean±SEM) were compared using unpaired student's t-test or ANOVA; all P-values <0.05 were considered significant. Results: TLR2/4 mRNA/protein expression was elevated by oxidative stress in PBMC compared to controls (P<0.001). This induction was abrogated by apocynin/N-acetyl cysteine treatments (P<0.01). H2O2-induced TLR2/4 gene expression was further enhanced by leptin, LPS, oleate, or palmitate (P<0.05). Oxidative stress also promoted expression of IRF3/5 and proinflammatory cytokines including IFN-γ, IL-1β, IL-6, TNF-α, and MCP-1/CCL2. This oxidative stress in PBMC involved MAPK/NF-κB dependent signaling. Conclusion: Taken together, oxidative stress upregulates expression of TLR2/4, IRF3/5 and signature proinflammatory cytokines in PBMC, involving MAPK/NF-κB dependent signaling, all of which may have implications for metabolic inflammation. [ABSTRACT FROM AUTHOR]

Background/Aims: MIP-1α (macrophage inflammatory protein 1α)/CCL3 chemokine is associated with the adipose tissue inflammation in obesity. Both MIP-1α and free fatty acids are elevated in obesity/T2D. We asked if free fatty acid palmitate could modulate MIP-1α expression in the human monocytic cells. Methods: Human monocytic THP-1 cells and macrophages were stimulated with palmitate and TNF-α (positive control). MIP-1α expression was measured with real time RT-PCR, Flow Cytometry and ELISA. Signaling pathways were identified by using THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, anti-TLR4 mAb and TLR4 siRNA. Results: Our data show that palmitate induced significant increase in MIP-1α production in monocytic THP-1 cells/macrophages. MIP-1α induction was significantly suppressed when cells were treated with anti-TLR4 antibody prior stimulation with palmitate. Using TLR4 siRNA, we further demonstrate that palmitate-induced MIP-1α expression in monocytic cells requires TLR4. Moreover, THP1 cells defective in MyD88, a major adaptor protein involved in TLR4 signaling, were unable to induce MIP-1α production in response to palmitate. Palmitate-induced MIP-1α expression was suppressed by inhibition of MAPK, NFkB and PI3K signaling pathways. In addition, palmitate-induced NF-kB/AP-1 activation was observed while production of MIP-1α. However, this activation of NF-kB/AP-1 was abrogated in MyD88 deficient cells. Conclusion: Overall, these results show that palmitate induces TLR4-dependent MIP-1α expression requiring the MyD88 recruitment and activation of MAPK, NF-kB/AP-1 and PI3K signaling. It implies that the increased systemic levels of free fatty acid palmitate in obesity/T2D may contribute to metabolic inflammation through excessive production of MIP-1α. [ABSTRACT FROM AUTHOR]

IL-6 concentrations are positively correlated with the level of obesity and associated proinflammatory state that promotes the development of insulin resistance. The metabolic stimuli that regulate IL-6 production has not been fully revealed. Since metabolic concentrations of plasma LPS and palmitate (free fatty acid) are increased in obesity and are considered as potent inducers of IL-6. Therefore, we have examined whether interaction of palmitate with LPS trigger the production of IL-6 in human monocytic cells. THP-1 and primary human monocytic cells incubated with palmitate and LPS together produced more biological active IL-6 compared to monocytes incubated with palmitate or LPS alone. Synergistic IL-6 induction was blocked either by silencing the expression of TLR4 or MyD88. However, IRF3 did not have impact on this synergism. Non-metabolizable analog as well as specific metabolic pathway inhibitors of palmitate showed that esterification of palmitate with coenzyme A (CoA) involved, in part, in this synergism. Furthermore, synergistic upregulation of IL-6 was suppressed by histone acetyltransferase inhibitor (anacardic acid). Histone deacetylase (HDAC) inhibitor (sodium butyrate) enhanced IL-6 production. In conclusion, our results show that an interaction between palmitate and LPS depends on TLR4/MyD88, lipid metabolism and epigenetic signaling pathways, which leads to the elevation of IL-6, providing interesting pathophysiological connections among FFAs, LPS, and IL-6 in settings such as obesity or metabolic inflammation. Disclosure: R. Ahmad: None. M.S. Koshy: None. S.P. Kochumon: None. R.S. Thomas: None. F. Al-Mulla: None. Funding: Kuwait Foundation for the Advancement of Science [ABSTRACT FROM AUTHOR]