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.
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)
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)
Diabetes is associated with several complications, including neuropathic pain, which is challenging to manage with currently available drugs. Descending noradrenergic neurons possess anti-nociceptive activity; however their involvement in diabetic neuropathic pain remains to be explored. To infer the regulatory role of this system, we examined in the pons, a part of the brainstem, lumbar nerves of the spinal cord and dorsal root ganglia of streptozotocin (STZ) -treated rats, a model for type 1 diabetes (T1D) , the localization (immunofluorescence) and the mRNA (qRT-PCR) and protein (Western blotting) expression of alpha-2A adrenoceptor (ADRA2A) . The data revealed that presynaptic SNAP-25 labeled ADRA2A in the central and peripheral nervous system of STZ diabetic rats were up-regulated both at the mRNA and protein levels. Interestingly, the levels of PSD-95 labeled postsynaptic neuronal ADRA2A remain unaltered as a function of diabetes. These biochemical abnormalities in the noradrenergic system of diabetic animals were associated with increased pain sensitivity as typified by the presence of hyperalgesia and cold/mechanical allodynia. The latter pain-related behaviors were assessed using Hargreaves apparatus, cold-plate and dynamic plantar aesthesiometer. Chronically administered guanfacine, a selective ADRA2A agonist, to diabetic animals downregulated the upregulation of neuronal presynaptic ADRA2A and ameliorated the hyperalgesia and the cold/mechanical allodynia in these animals. Together, these findings demonstrate that guanfacine may function as a potent analgesic and highlight ADRA2A, a key component of the descending neuronal autoinhibitory pathway as a potential therapeutic target in the treatment of diabetic neuropathic pain. Disclosure N. Munawar: None. A. Al madhoun: None. J. Nader: None. W. Al-ali: None. W. Masocha: None. F. Al-mulla: None. M. S. Bitar: None. Funding Grant YM05/19 from Kuwait University Research Sector.
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