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Background: Angiopoietin-like protein 8 (ANGPTL8) is known to regulate lipid metabolism and inflammation. It interacts with ANGPTL3 and ANGPTL4 to regulate lipoprotein lipase (LPL) activity and with IKK to modulate NF-κB activity. Further, a single nucleotide polymorphism (SNP) leading to the ANGPTL8 R59W variant associates with reduced low-density lipoprotein/high-density lipoprotein (LDL/HDL) and increased fasting blood glucose (FBG) in Hispanic and Arab individuals, respectively. In this study, we investigate the impact of the R59W variant on the inflammatory activity of ANGPTL8. Methods: The ANGPTL8 R59W variant was genotyped in a discovery cohort of 867 Arab individuals from Kuwait. Plasma levels of ANGPTL8 and inflammatory markers were measured and tested for associations with the genotype; the associations were tested for replication in an independent cohort of 278 Arab individuals. Impact of the ANGPTL8 R59W variant on NF-κB activity was examined using approaches including overexpression, luciferase assay, and structural modeling of binding dynamics. Results: The ANGPTL8 R59W variant was associated with increased circulatory levels of tumor necrosis factor alpha (TNFα) and interleukin 7 (IL7). Our in vitro studies using HepG2 cells revealed an increased phosphorylation of key inflammatory proteins of the NF-κB pathway in individuals with the R59W variant as compared to those with the wild type, and TNFα stimulation further elevated it. This finding was substantiated by increased luciferase activity of NF-κB p65 with the R59W variant. Modeled structural and binding variation due to R59W change in ANGPTL8 agreed with the observed increase in NF-κB activity. Conclusion: ANGPTL8 R59W is associated with increased circulatory TNFα, IL7, and NF-κB p65 activity. Weak transient binding of the ANGPTL8 R59W variant explains its regulatory role on the NF-κB pathway and inflammation.

Abstract Background Angiopoietin-like proteins (ANGPTL), primarily 3, 4, and 8, play a major role in maintaining energy homeostasis by regulating triglyceride metabolism. This study evaluated the level of ANGPTL3, 4, and 8 in the liver, brown adipose tissue (BAT), and subcutaneous white adipose tissue (SAT) of mice maintained under acute and chronic cold conditions. Methods C57BL/6J mice were exposed to cold temperature (4 °C) for 10 days with food provided ad libitum. Animal tissues were harvested at Day 0 (Control group, n = 5) and Days 1, 3, 5, and 10 (cold treatment groups, n = 10 per group). The expression levels of various genes were measured in the liver, SAT, and BAT. ANGPTL3, 4, and 8 expressions were measured in the liver. ANGPTL4, 8, and genes involved in browning and lipid metabolism [uncoupling protein 1 (UCP1), lipoprotein lipase (LPL), and adipose triglyceride lipase (ATGL)] were measured in SAT and BAT. Western blotting (WB) analysis and immunohistochemistry (IHC) were performed to confirm ANGPTL8 expression in these tissues. Results The expressions of ANGPTL3 and 8 mRNA were significantly reduced in mouse liver tissues after cold treatment (P

Abstract The neuropeptide urocortin 3 (UCN3) has a beneficial effect on metabolic disorders, such as obesity, diabetes, and cardiovascular disease. It has been reported that UCN3 regulates insulin secretion and is dysregulated with increasing severity of obesity and diabetes. However, its function in the adipose tissue is unclear. We investigated the overexpression of UCN3 in 3T3-L1 preadipocytes and differentiated adipocytes and its effects on heat shock response, ER stress, inflammatory markers, and glucose uptake in the presence of stress-inducing concentrations of palmitic acid (PA). UCN3 overexpression significantly downregulated heat shock proteins (HSP60, HSP72 and HSP90) and ER stress response markers (GRP78, PERK, ATF6, and IRE1α) and attenuated inflammation (TNFα) and apoptosis (CHOP). Moreover, enhanced glucose uptake was observed in both preadipocytes and mature adipocytes, which is associated with upregulated phosphorylation of AKT and ERK but reduced p-JNK. Moderate effects of UCN3 overexpression were also observed in the presence of 400 μM of PA, and macrophage conditioned medium dramatically decreased the UCN3 mRNA levels in differentiated 3T3-L1 cells. In conclusion, the beneficial effects of UCN3 in adipocytes are reflected, at least partially, by the improvement in cellular stress response and glucose uptake and attenuation of inflammation and apoptosis.

Abstract Fetuin-A (Fet-A) was one of the first hepatokines to be reportedly linked to metabolic diseases. Fet-A was also suggested to be an adipokine, but its expression in the adipose tissue remains debatable. Here we compared the expression of Fet-A between human and mice adipose tissue biopsies as well as among human subcutaneous tissue and visceral adipose tissue primary cells, and mouse 3 T3-L1 cells at various stages of differentiation. Fet-A was expressed in mice biopsies and cells but not in human biopsies and cells, except in visceral adipose tissue primary cells following differentiation. Although the marginal expression of Fet-A in human visceral adipose tissue, a major contribution of Fet-A expression in human adipose tissue to systemic Fet-A levels is discounted, but it could indicate specific local Fet-A action in the visceral adipose tissue.

Abstract Background The hepatokine fetuin-A is linked to obesity and type 2 diabetes, but its presence and expression in adipose tissue remain unclear. In this study, we aimed to assess the circulating levels of fetuin-A and its expression in subcutaneous adipose tissue (SAT) from diabetic and non-diabetic obese subjects and their modulation by exercise. Methods SAT and blood were obtained from adults obese (diabetic, n=118 and non-diabetic, n=166) before and after a 3-month exercise program (diabetic, n=40 and non-diabetic, n=36, respectively). Plasma fetuin-A was assayed using ELISA. The presence and expression of fetuin-A in SAT, peripheral blood mononuclear cells (PBMCs) and cell lines (3T3-L1, THP-1, HepG2, RAW 264.7) were analysed using confocal microscopy, immunoblotting and qRT-PCR. Results Plasma fetuin-A level did not significantly differ between diabetic and non-diabetic obese subjects. However, when the non-diabetic group was divided into metabolically healthy and unhealthy phenotypes, significantly higher fetuin-A level was observed in the unhealthy sub-group. Circulating fetuin-A was mainly associated with glycaemic markers. In SAT, fetuin-A protein level was significantly higher in the diabetic obese subjects but its mRNA was not detected. Similarly, fetuin-A protein was detected in PBMCs, but its mRNA was not. In line with this, the use of various cell lines and culture media indicated that the presence of fetuin-A in SAT and PBMCs was due to its uptake from circulation rather than its endogenous expression. Finally, physical exercise decreased fetuin-A levels in both plasma and SAT in both groups. Conclusions Fetuin-A levels increased in association with diabetes in SAT but not in circulation in the obese subjects. Moreover, physical exercise decreased fetuin-A level. Fetuin-A potentially acts as a hepatokine taken up by other tissues, such as adipose tissue.

The corticotropin-releasing hormone (CRH) and urocortins (UCNs) have been implicated in energy homeostasis and the cellular stress response. However, the expression of these neuropeptides in children remains unclear. Therefore, we determined the impact of obesity on their expression in 40 children who were normal weight, overweight, and had obesity. Peripheral blood mononuclear cells (PBMCs) and plasma were used to assess the expression of neuropeptides. THP1 cells were treated with 25 mM glucose and 200 µM palmitate, and gene expression was measured by real-time polymerase chain reaction (RT-PCR). Transcript levels of neuropeptides were decreased in PBMCs from children with increased body mass index as indicated by a significant decrease in UCN1, UCN3, and CRH mRNA in overweight and obese children. UCN3 mRNA expression was strongly correlated with UCN1, UCN2, and CRH. Exposure of THP1 cells to palmitate or a combination of high glucose and palmitate for 24 h increased CRH, UCN2, and UCN3 mRNA expression with concomitant increased levels of inflammatory and endoplasmic reticulum stress markers, suggesting a crosstalk between these neuropeptides and the cellular stress response. The differential impairment of the transcript levels of CRH and UCNs in PBMCs from overweight and obese children highlights their involvement in obesity-related metabolic and cellular stress.

Cardiovascular disease (CVD) risks persist in patients despite treatment. CVD susceptibility also varies with sex and ethnicity and is not entirely explained by conventional CVD risk factors. The aim of the present study was to identify novel CVD candidate markers in circulating Peripheral blood mononuclear cells (PBMCs) and plasma from Arab obese subjects with and without CVD using proteomic approaches. Human adults with confirmed CVD (n = 208) and matched non-CVD controls (n = 152) living in Kuwait were examined in the present cross-sectional study. Anthropometric and classical biochemical parameters were determined. We employed a shotgun proteomic profiling approach on PBMCs isolated from a subset of the groups (n = 4, each), and differentially expressed proteins selected between the two groups were validated at the mRNA level using RT-PCR (n = 6, each). Plasma levels of selected proteins from the proteomics profiling: Proteinase-3 (PR3), Annexin-A3 (ANX3), Defensin (DEFA1), and Matrix Metalloproteinase-9 (MMP9), were measured in the entire cohort using human enzyme-linked immunosorbent assay kits and were subsequently correlated with various clinical parameters. Out of the 1407 we identified and quantified from the proteomics profiling, 47 proteins were dysregulated with at least twofold change between the two subject groups. Among the differentially expressed proteins, 11 were confirmed at the mRNA levels. CVD influenced the levels of the shortlisted proteins (MMP9, PR3, ANX3, and DEFA1) in the PBMCs and plasma differentially. Despite the decreased levels of both protein and mRNA in PBMCs, PR3 circulating levels increased significantly in patients with CVD and were influenced by neither diabetes nor statin treatment. No significant changes were; however, observed in the DEFA1, MMP9, and ANX3 levels in plasma. Multivariate logistic regression analysis revealed that only PR3 was independently associated with CVD. Our results suggest that the dysregulation of PR3 levels in plasma and PBMCs reflects underlying residual CVD risks even in the treated population. More prospective and larger studies are required to establish the role of PR3 in CVD progression.

Urocortin3 (UCN3) regulates metabolic functions and is involved in cellular stress response. Although UCN3 is expressed in human adipose tissue, the association of UCN3 with obesity and diabetes remains unclear. This study investigated the effects of Type 2 diabetes (T2D) and increased body weight on the circulatory and subcutaneous adipose tissue (SAT) levels of UCN3 and assessed UCN3 modulation by a regular physical exercise. Normal-weight (n = 37) and overweight adults with and without T2D (n = 98 and n = 107, respectively) were enrolled in the study. A subset of the overweight subjects (n = 39 for each group) underwent a supervised 3-month exercise program combining both moderate intensity aerobic exercise and resistance training with treadmill. UCN3 levels in SAT were measured by immunofluorescence and RT-PCR. Circulatory UCN3 in plasma was assessed by ELISA and was correlated with various clinical and metabolic markers. Our data revealed that plasma UCN3 levels decreased in overweight subjects without T2D compared with normal-weight controls [median; 11.99 (0.78–86.07) and 6.27 (0.64–77.04), respectively; p < 0.001], whereas plasma UCN3 levels increased with concomitant T2D [median; 9.03 (0.77–104.92) p < 0.001]. UCN3 plasma levels were independently associated with glycemic index; fasting plasma glucose and hemoglobin A1c (r = 0.16 and r = 0.20, p < 0.05, respectively) and were significantly different between both overweight, with and without T2D, and normal-weight individuals (OR = 2.11 [1.84–4.11, 95% CI] and OR = 2.12 [1.59–3.10, 95% CI], p < 0.01, respectively). Conversely, the UCN3 patterns observed in SAT were opposite to those in circulation; UCN3 levels were significantly increased with body weight and decreased with T2D. After a 3-month supervised exercise protocol, UCN3 expression showed a significant reduction in SAT of both overweight groups (2.3 and 1.6-fold change; p < 0.01, respectively). In conclusion, UCN levels are differentially dysregulated in obesity in a tissue-dependent manner and can be mitigated by regular moderate physical exercise.

Epoxide hydrolase 2 (EPHX2) is an emerging therapeutic target in several immunometabolic disorders. EPHX2 metabolizes anti-inflammatory epoxyeicosatrienoic acids into pro-inflammatory diols. The contribution of EPHX2 activity to human obesity remains unexplored. We compared the expression of EPHX2 between lean and obese humans (n = 20 each) in subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs) using RT-PCR, Western Blot analysis, immunohistochemistry, and confocal microscopy before and after a 3-month physical activity regimen. We also assessed EPHX2 levels during preadipocyte differentiation in humans and mice. EPHX2 mRNA and protein expression were significantly elevated in obese subjects, with concomitant elevated endoplasmic reticulum (ER) stress components (the 78-kDa glucose-regulated protein; GRP78, and the Activating transcription factor 6; ATF6) and inflammatory markers (Tumor necrosis factor-α; TNFα, and Interleukin 6; IL6) as compared to controls (p < 0.05). EPHX2 mRNA levels strongly correlated with adiposity markers. In obese individuals, physical activity attenuated EPHX2 expression levels in both the SAT and PBMCs, with a parallel decrease in ER stress and inflammation markers. EPHX2 expression was also elevated during differentiation of both human primary and 3T3-L1 mouse preadipocytes. Mediators of cellular stress (palmitate, homocysteine, and macrophage culture medium) also increased EPHX2 expression in 3T3-L1 preadipocytes. Our findings suggest that EPHX2 upregulation is linked to ER stress in adiposity and that physical activity may attenuate metabolic stress by reducing EPHX2 expression.

ANGPTL7 is a member of the Angiopoietin-like (ANGPTL) protein family that is composed of eight proteins (1-8). Increasing evidence is associating ANGPTL proteins to obesity and insulin resistance. The biological role of ANGPTL7 is yet to be understood except for a recently proposed role in the pathophysiology of glaucoma. This study was designed to shed light on the function of ANGPTL7 in obesity and its modulation by physical exercise as well as its potential association with lipid profile.A total of 144 subjects were enrolled in this study and finished three months of physical exercise. The participants were classified based on their BMI, 82 subjects were non-obese and 62 obese. ANGPTL7 levels in plasma and adipose tissue were measured by ELISA, RT-PCR and immunohistochemistry.In this study, we showed that ANGPTL7 level was increased in the plasma of obese subjects (1249.05± 130.39 pg/mL) as compared to non-obese (930.34 ± 87.27 pg/mL) (p-Value = 0.032). ANGPTL7 Gene and protein expression levels in adipose tissue also showed over two fold increase. Physical exercise reduced circulating level of ANGPTL7 in the obese subjects to 740.98± 127.18 pg/mL, (p-Value = 0.007). ANGPTL7 expression in adipose tissue was also reduced after exercise. Finally, ANGPTL7 circulating level showed significant association with TG level in the obese subjects (R2 = 0.183, p-Value = 0.03).In conclusion, our data shows for the first time that obesity increases the level of ANGPTL7 in both plasma and adipose tissue. Increased expression of ANGPTL7 might play a minor role in the regulation of TG level in obese subjects either directly or through interaction with other ANGPTL protein members. Physical exercise reduced the level of ANGPTL7 highlighting the potential for targeting this protein as a therapeutic target for regulating dyslipidemia.