Nonalcoholic fatty liver disease (NAFLD) is a risk factor of type-2 diabetes and cardiovascular disease. Obesity induced by various high-fat diets from different sources results in different types of meta-inflammatory derangements, including NAFLD. Fats are a central part of healthy diets, however, the impact of various dietary fats, lacking in sucrose, on liver fat accumulation and expression of lipogenic and inflammatory markers remains unclear. To study this, C57BL/6J mice were fed sucrose-free HFDs comprising fat from diverse sources, including cocoa butter (c-HFD), sunflower oil (s-HFD), soybean oil (so-HFD), and fish oil (f-HFD). Mice fed c-HFD or so-HFD developed more severe liver steatosis, compared with those fed s-HFD or f-HFD. Liver histopathology displayed high levels of lobular inflammation in mice fed c-HFD, so-HFD, and s-HFD. Kupffer cell counts were higher in mice fed c-HFD and s-HFD. Hepatic fibrosis was seen in mice fed s-HFD or so-HFD. Of note, there was no hepatic fibrosis in mice fed f-HFD. None of the diet had a significant impact on total body weight. However, liver weight was slightly increased in mice fed c-HFD or s-HFD. Mice fed c-HFD, s-HFD, and so-HFD displayed insulin resistance. Expression of the key genes of glycolysis (Pklr), de novo lipogenesis (Acaca, Fasn, Scd1), fatty acid oxidation (Cpt1a, Ppar-α), inflammation (Tnf-α) was upregulated in mice fed c-HFD or s-HFD. No significant difference was seen regarding genes of fatty acid uptake (Cd36, Fabp2), and chemokine-associated inflammation (Ccl2). Interestingly, hepatic IL-1β and IL-6 were elevated only in mice fed c-HFD. Taken together, these findings indicate that mice fed the sucrose-free HFDs comprising lipids from various dietary sources may have differential effects on hepatic steatosis at levels of de novo lipogenesis, hepatic inflammation, and whole-body insulin resistance. Disclosure R. Ahmad: None. T.K. Jacob: None. S.P. Kochumon: None. R.S. Thomas: None. S. Shenouda: None. N. Akhter: None. A. Wilson: None. F. Bahman: None. A. Hasan: None. F. Alrashed: None. H. Arefanian: None. A. Al Madhoun: None. F. Almulla: None. S.T.K. Sindhu: None. Funding Kuwait Foundation for the Advancement of Sciences (RAAM-2016-007)
Obesity is marked by metabolic inflammation and by metabolic impairment caused by increased endotoxin, free fatty acids, and vascular endothelial growth factor (VEGF) levels; and involves the endoplasmic reticulum (ER) stress as well. However, it remains unclear whether the ER stress can induce/amplify VEGF expression in metabolically-stressed monocytic cells; and if so, by which mechanism(s). To test this, metabolic stress was induced in THP-1 monocytic cells by treating cells separately with lipopolysaccharide (LPS), palmitic acid (PA), and oleic acid (OA), in presence/absence of ER stressor thapsigargin (TG). Gene expression of VEGF/ER stress markers was assessed by qRT-PCR, protein expression by ELISA, ROS by DCFH-DA assay, phosphorylation of HIF-1α, NF-κB, ERK1/2, and p38 MAPK by immunoblotting, and the insulin response in stressed cells by glucose-uptake assay. Regarding clinical analyses, adipose VEGF gene and protein expression was detected using qRT-PCR and IHC, respectively, while plasma hs-CRP, TNF-α, MDA, and OX-LDL levels were measured by ELISA. The experimental data show that a cooperative interaction between the metabolic and ER stresses led to the expression of VEGF, ROS, CHOP, ATF6, SOD2, and NRF2 (P˂0.05), as well as stimulated the CHOP and NRF2 promoter activities in reporter cells (P˂0.05). However, the glucose uptake was not impaired. The VEGF expression was dependent on phosphorylation of HIF-1α, NF-κB, and p38 MAPK; and the inhibitors of NF-κB/MAPK pathways as well as antioxidants or ROS scavengers suppressed the VEGF production. Further, individuals with obesity showed increased VEGF expression which associated positively with plasma levels of hs-CRP, TNF-α, MDA, and OX-LDL (P≤0.05). Overall, our findings support a cooperativity model in which the ER and metabolic stresses interact to augment VEGF expression in monocytic cells via the mechanisms involving CHOP/ROS/HIF-1α/NRF2 and NF-κB/p38 MAPK pathways. Disclosure S.A.K.Sindhu: None. F.Alzaid: None. F.Almulla: None. R.Ahmad: None. N.Akhter: None. A.Wilson: None. H.Arefanian: None. A.Al madhoun: None. R.S.Thomas: None. S.P.Kochumon: None. F.Alrashed: None. F.Bahman: None. Funding Kuwait Foundation for the Advancement of Sciences (RA2015-027, RA2010-003, RAHM-2019-022)
Obesity induced chronic low-grade inflammation is a central risk factor for the development of metabolic syndrome. It has been well documented that high LDL-c induces inflammation. The proinflammatory cytokine Il-23 plays a pivotal role in the pathogenesis of inflammatory diseases. IL-23 and its relationship with LDL- c has not been reported yet. In this cross-sectional study we investigated whether adipose tissue expression of IL-23 associated with the other inflammatory mediators in individuals with high serum levels of low-density lipoprotein cholesterol (LDL-c) . Subcutaneous adipose samples were collected from 67 individuals and divided into two groups based on their serum LDL-c levels (LDL-c: < 2.9 or ≥2.9 mmol/L) . Expression of IL-23 and inflammatory markers was determined using real-time RT-PCR. Plasma lipid measurements included total cholesterol (TC) , triglyceride (TG) , high-density lipoprotein cholesterol (HDL-c) and LDL-c by standard methods, and serum adiponectin was measured by enzyme-linked immunosorbent assay (ELISA) . Individuals with increased serum levels of LDL-c showed high IL-23 expression levels in adipose tissue (p < 0.011) . AT IL-23 expression was correlated positively with LDL-c (r= 0.39, p < 0.0001) . IL-23 expression levels were positively correlated with macrophage markers (CD11c, CD68, CD86, CD127; (r ≥0.37, p≤ 0.02) , TLRs (TLR8, TLR10; (r ≥ 0.39, p ≤ 0.022) , IRF3 (r= 0.46, p< 0.01) , cytokines (TNF-α, IL-12, IL-18; (r ≥ 0.35, p ≤ 0.04) , chemokines (CXCL8, CCL3, CCL5, CCL15, CCL20; (r ≥0.43, p ≤ 0.01) . Notably, IL-23 is negatively correlated with adiponectin (r=-0.44, p < 0.03) in the individuals with high LDL-c. However, such association of IL-23 with inflammatory markers was not found in the individuals with low LDL-c. In conclusion, adipose tissue IL-23 may be a biomarker for inflammation progression in the individuals with high LDL-c and could be used as a therapeutic target for the treatment of metabolic syndrome. Disclosure R.Ahmad: None. S.P.Kochumon: None. A.Hasan: None. S.T.Sindhu: None. H.Arefanian: None. F.Alrashed: None. F.Almulla: None. Funding Kuwait Foundation for Advancement of Sciences (RA-2010-003)
Nonalcoholic fatty liver disease (NAFLD) represents a global healthcare challenge; it is the hepatic manifestation of metabolic syndrome and is strongly associated with developing type 2diabetes mellitus (T2DM). Liver fat accumulation is the first step of disease progression that triggers hepatic lipotoxicity. The role of ceramides in inducing deleterious effects on hepatic metabolism is now well-accepted. Yet, the specific role of stress responsive sphingomyelinase activation under lipotoxic conditions remains under-investigated. The complexity of NAFLD pathogenesis contributes to lack of proper investigatory model, limiting progression in developing and testing novel treatment and prevention strategies. Here, we first report a convenient in-vitro human cell-based model with great resemblance to in-vivo NAFLD hallmarks. Neutral Sphingomyelinase (nSMase2) expression and activity was found to be elevated in both the liver of high fat steatosis mouse models and in HepG2-steatosis cell models. Meanwhile, the functional inhibition of nSMase2 prevented hepatotoxicity-induced pathologies by significantly reducing intracellular lipid accumulation and prevented the upregulation of TNF-α triggered inflammation. Furthermore, inhibition of nSMase2 showed significant increase in PPARα at both gene and protein levels, while PPARα reduction was observed under the stimulation of nSMase2 activity by its agonist daunorubicin (DNR). Together the presented data highlight the role of nSMase2 in the pathogenesis of NAFLD and other disorders linked to hepatic steatosis, providing a novel therapeutic target. Disclosure F. Alrashed: None. H. Arefanian: None. S.T. Sindhu: None. F. Bahman: None. H. AlSaeed: None. A. Al Madhoun: None. F. Alzaid: None. F. Al-Mulla: None. R. Ahmad: None. Funding Kuwait Foundation for the Advancement of Sciences (RA0402021)
Background: ANGPTL8 has been recently identified as lipid metabolism and inflammation regulator. It interacts with ANGPTL3 and, more recently, ANGPTL4 to regulate LPL activity. It was also shown to modulate NF-κB activity through its interaction with ikkα/β. A genetic variant rs2278426 (R59W) in ANGPTL8 has been associated with reduced LDL and HDL in Hispanics and increased FBG in Arabs. The objective of the study was to investigate the impact of the R59W variant on LPL and the inflammatory activity of ANGPTL8. Methods: ANGPTL8 was genotyped in a discovery cohort of 738 Kuwaiti individuals. ANGPTL8 level, as well as inflammatory markers and LPL, were measured in plasma. Additionally, metabolite analysis, overexpression, luciferase binding assay, confocal microscopy, and cellular fractionation analyses were conducted in the HepG2 cell line to assess differences between the ANGPTL8 and its variant. Results: The ANGPTL8 rs2278426 variant was associated with an increase in circulatory levels of TNF-α and IL7 in our cohort. Carrier versus reference genotype of the studied variant showed differences in the following metabolites: Acylcarnitines, Phosphatidylcholine, and Cholesteryl Ester. These metabolites are implicated in modulating NF-κB and TNF-α levels. Our in vitro analyses showed that the activity levels of NF-κB p65 was higher in the R59W than the wild type, and it was further elevated with TNFα stimulations. The data was validated by the luciferase activity, confocal microscopy, and cellular fractionation. Conclusion: This study showed that ANGPTL8 variant R59W is associated with increased circulatory TNF-α and IL7 levels but not the LPL plasma levels. We have also demonstrated the pro-inflammatory role of the R59W variant in regulating the NF-κB pathway as reflected by genotyping, in vitro, metabolomics, and structural analyses. Further studies are needed to expand our understanding of other signaling pathways and possible crosstalk in the context of inflammation. Disclosure A.Mohammad: None. A.T.Thangavel: None. F.Almulla: None. J.Abubaker: None. M.Abu-farha: None. D.Madhu: None. P.T.Cherian: None. I.Al khairi: None. P.Hebbar: None. S.A.Kavalakatt: None. H.Arefanian: None. N.Alam-eldin: None. Funding Kuwait Foundation for the Advancement of Sciences
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