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)
Dectin-1 is a pathogen recognition receptor as well as an innate immune response modulator; its function in metabolic disorders is yet unclear. Previously, we identified dectin-1 as a biomarker of metabolic inflammation in obesity. In this study, we sought to identify potential signaling pathways that could modify the expression of the dectin-1 gene and assess the expression of dectin-1 in the adipose tissue (AT) of obese patients based on their diabetic status. The study cohort included 95 obese individuals split into two groups: prediabetics with moderate glycemia (Hb1Ac 6.5%, n = 49) and diabetics with hyperglycemia (Hb1Ac 6.5%, n = 46). Dectin-1 expression was assessed using immunohistochemistry. Gene expression and inflammatory markers were determined via qRT-PCR. We found a significant positive correlation between dectin-1 expression and HbA1C levels in AT isolated from obese individuals with HbA1C levels of 6.5% or higher. Dectin-1 gene expression was significantly correlated with several inflammatory markers; however, glycemic-dependent associations were also observed. Dectin-1 and TNF-α were found to be significantly correlated in AT from individuals with Hb1Ac 6.5%, indicating a possible mechanism of gene regulation between these two factors. As a result, we investigated the observed dectin-1/TNF-α crosstalk using in vitro cell culture and animal studies. Unlike wild-type animals, mice lacking TNF-α exhibited reduced levels of dectin-1 gene and protein expression in their AT, which were restored by injecting exogenous TNF-α. ChIP studies showed that TNF-α induced dectin-1 gene transcription by mediating NF-kB binding to newly identified regulatory elements located in the dectin-1 proximal regulatory region. The interplay between dectin-1 and TNF-α signaling pathways is intriguing and has the potential to be a therapeutic target in obesity and diabetes. Furthermore, dectin-1 could be a potential marker for the onset of hyperglycemia and diabetes. Disclosure A.Al madhoun: None. D.Haddad: None. S.P.Kochumon: None. F.Alrashed: None. R.S.Thomas: None. L.P.Miranda: None. S.T.K.Sindhu: None. R.Ahmad: None. F.Almulla: None.
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)
We Have previously established that caveolin-1 (CAV1) rs1997623 C/A variant is associated with metabolic syndrome in obese children (PMID: 30622557) and adults (ADA 82) . Here, we evaluated the functional role of the identified variant in association with obesity. Sequence analysis revealed that the variant is located at CAV1 regulatory region and creates a site for EBF Transcription Factor 1 (EBF1) . DNA fragment (240pb) flanking the variant was cloned upstream luciferase reporter gene. Wildtype, CAV1-C-Luc, and the generated mutant, CAV1-A-Luc, were individually transfected into pre-adipocytes. CAV1-A-luc significantly upregulated Luciferase activity indicating a prospective functional role of CAV1-A allele in CAV1 regulation. The reporter gene activity was differential regulated in lean versus obese pre-adipocytes. Co-transfections with EBF1 construct resulted in a significant upregulation in CAV1-A Luc activity, particularly in pre-adipocytes from obese individuals. The role of EBF1 was further evaluated in vivo using ChIP assays. PCR analysis using specific primers flanking the CAV1 variant and anti-bodies against EBF1 showed a significant elevated enrichment of chromatins from obese individuals, which was also associated with an enrichment for H3K27ac at the same locus, indicating active transcription. Moreover, CAV1 expression was differentially regulated in response to EBF1 siRNA/overexpression in pre-adipocytes harboring CAV1 A/A or A/C alleles relative to that with C/C allele. Furthermore, a significant reduction of DNA methylation at the variant locus was observed particularly in preadipocytes with A/A alleles. Finally, RNA isolated from adipose tissue of lean and obese individuals showed a significant correlation between CAV1 and cytokines transcripts in healthy obese individuals, at least in part, is due to CAV1 rs1997623 variant as verified by sequence analysis. Taken together, our study delineates the role of CAV1 rs1997623 variant in the context of obesity and MetS. Disclosure A.Al madhoun: None. D.Haddad: None. S.P.Kochumon: None. R.Nizam: None. S.Jacob: None. S.T.Sindhu: None. R.Ahmad: None. F.Almulla: None. Funding Kuwait Foundation for the Advancement of Sciences (KFAS) and Dasman Diabetes Institute, Project RA CB-2021-007
The onset of obesity is vastly associated with low levels of high-density lipoprotein cholesterol (HDL-C) , which predisposes to cardiovascular diseases. Recently, mounting evidence indicates that IL-6 plays a key role in metabolism, especially in lipid metabolic homeostasis. Yet, the precise nature of the HDL/IL-6 dynamics has not been fully elucidated. Therefore, we evaluated the expression of IL-6 in circulatory monocytes of the individuals with various body mass index and we also determined their lipid profile. Our data show that monocytic IL-6 expression was found to be negatively associated with HDL-C levels in obese individuals and positively associated with C-reactive protein and other monocytic pro-inflammatory markers. Mechanistically, chemical inhibition or genetic silencing of IL-6 receptor gp130/IL-6ST gene induced severe upregulation of intracellular lipid accumulation in THP-1 transformed macrophages that was found to be further augmented under fatty acid rich culture conditions. Moreover, analysis of the genes involved in lipid and cholesterol metabolism showed up-regulation of the LDLR along with a remarkable inhibition of ATP Binding Cassette (ABC) Subfamily transporters ABCG1 and ABCB1. We also found that the macrophages lacking gp130/IL-6 receptors had very low beta-oxidation genes expression (CPT1A and CPT2) , however, with no effect on triglyceride synthesis genes expression (DGAT, ACACA, FASN and Srebp1c) . In conclusion, our data support a role for IL-6 receptor signaling pathways in lipid alteration and cholesterol efflux which may have potential as a therapeutic target for metabolic syndrome. Disclosure F.Alrashed: None. F.Alzaqqah: None. A.Al madhoun: None. R.Alqabandi: None. S.T.Sindhu: None. F.Almulla: None. R.Ahmad: None. Funding (KFAS) (Grant #: RA AM 2016-007)
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