Correlation Profile of Suppression of Tumorigenicity 2 and/or Interleukin-33 with Biomarkers in the Adipose Tissue of Individuals with Different Metabolic States

Purpose: The suppression of tumorigenicity 2 (ST2) has two main splice variants including a membrane bound (ST2) form, which activates the myeloid differentiation primary response 88 (MyD88)/nuclear factor-kappa B (NF-kappa B) signaling pathway, and a secreted soluble form (sST2), which acts as a decoy receptor for ST2 ligand, interleukin (IL)-33. The IL-33/ST2 axis is protective against obesity, insulin resistance, and type 2 diabetes (T2D). In humans, adipose tissue IL-33 displays distinct correlation profiles with glycated hemoglobin, ST2, and other immunometabolic mediators, depending on the glycemic health of the individuals. We determined whether adipose tissue ST2 displays distinct correlation profiles with immunometabolic mediators and whether ST2 and/or IL-33 are correlated with intracellular signaling molecules. Patients and Methods: A total of 91 adults with normal glycemia, prediabetes, and T2D were included. After measuring their anthropometric and biochemical parameters, subcutaneous adipose tissues were isolated and mRNA expression of biomarkers was measured. Results: In individuals with normal glycemia, adipose tissue ST2 was directly correlated with chemokine (C-C motif) ligand (CCL)-2, CCL5, IL-12, fibrinogen-like protein 2 (FGL2) and interferon regulatory factor (IRF)-4, but inversely correlated with cytochrome C oxidase subunit 7A1. IL-33 and ST2 were directly correlated with tumor necrosis factor receptorassociated factor 6 (TRAF6), NF-kappa B, and nuclear factor of activated T-cells 5 (NFAT5). In individuals with prediabetes, ST2 was inversely correlated with IL-5, whereas IL-33 but not ST2 was directly correlated with MyD88 and NF-kappa B. In individuals with T2D, ST2 was directly correlated with CCL2, IL-1 beta, and IRF5. IL-33 and ST2 were directly correlated with MyD88, TRAF6, and NF-kappa B. Conclusion: Adipose tissue ST2 and IL-33 show different correlation profiles with various immunometabolic biomarkers depending on the metabolic state of the individuals. Therefore, targeting the IL-33/ST2 axis might form the basis for novel therapies to combat metabolic disorders.Peer reviewe

Increased expression of the interleukin-1 receptor-associated kinase (IRAK)-1 is associated with adipose tissue inflammatory state in obesity

Figure S2. Comparison of IRAK-1 gene and protein expression in the adipose tissue. The gene and protein expression of IRAK-1 in non-diabetic obese, overweight, and lean adipose tissue samples, 5 each, were determined by using real-time RT-PCR and immunohistochemistry, respectively, as described in Patients and Methods. The relative mRNA expression was measured as fold expression over average of control gene expression taken as 1. The protein expression was measured as intensity which was calculated by using Aperio positive pixel count algorithm software (version 9)

Expression of Steroid Receptor RNA Activator 1 (SRA1) in the Adipose Tissue Is Associated with TLRs and IRFs in Diabesity

Steroid receptor RNA activator gene (SRA1) emerges as a player in pathophysiological responses of adipose tissue (AT) in metabolic disorders such as obesity and type 2 diabetes (T2D). We previously showed association of the AT SRA1 expression with inflammatory cytokines/chemokines involved in metabolic derangement. However, the relationship between altered adipose expression of SRA1 and the innate immune Toll-like receptors (TLRs) as players in nutrient sensing and metabolic inflammation as well as their downstream signaling partners, including interferon regulatory factors (IRFs), remains elusive. Herein, we investigated the association of AT SRA1 expression with TLRs, IRFs, and other TLR-downstream signaling mediators in a cohort of 108 individuals, classified based on their body mass index (BMI) as persons with normal-weight (N = 12), overweight (N = 32), and obesity (N = 64), including 55 with and 53 without T2D. The gene expression of SRA1, TLRs-2,3,4,7,8,9,10 and their downstream signaling mediators including IRFs-3,4,5, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), and nuclear factor-κB (NF-κB) were determined using qRT-PCR and SRA1 protein expression was determined by immunohistochemistry. AT SRA1 transcripts’ expression was significantly correlated with TLRs-3,4,7, MyD88, NF-κB, and IRF5 expression in individuals with T2D, while it associated with TLR9 and TRAF6 expression in all individuals, with/without T2D. SRA1 expression associated with TLR2, IRAK1, and IRF3 expression only in individuals with obesity, regardless of diabetes status. Furthermore, TLR3/TLR7/IRAK1 and TLR3/TLR9 were identified as independent predictors of AT SRA1 expression in individuals with obesity and T2D, respectively. Overall, our data demonstrate a direct association between the AT SRA1 expression and the TLRs together with their downstream signaling partners and IRFs in individuals with obesity and/or T2D

Urocortin Neuropeptide Levels Are Impaired in the PBMCs of Overweight Children

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

Differential effects of fish-oil and cocoa-butter based high-fat/high-sucrose diets on endocrine pancreas morphology and function in mice

IntroductionA high-fat/high-sucrose diet leads to adverse metabolic changes that affect insulin sensitivity, function, and secretion. The source of fat in the diet might inhibit or increase this adverse effect. Fish oil and cocoa butter are a significant part of our diets. Yet comparisons of these commonly used fat sources with high sucrose on pancreas morphology and function are not made. This study investigated the comparative effects of a fish oil-based high-fat/high-sucrose diet (Fish-HFDS) versus a cocoa butter-based high-fat/high-sucrose diet (Cocoa-HFDS) on endocrine pancreas morphology and function in mice.MethodsC57BL/6 male mice (n=12) were randomly assigned to dietary intervention either Fish-HFDS (n=6) or Cocoa-HFDS (n=6) for 22 weeks. Intraperitoneal glucose and insulin tolerance tests (IP-GTT and IP-ITT) were performed after 20-21 weeks of dietary intervention. Plasma concentrations of c-peptide, insulin, glucagon, GLP-1, and leptin were measured by Milliplex kit. Pancreatic tissues were collected for immunohistochemistry to measure islet number and composition. Tissues were multi-labelled with antibodies against insulin and glucagon, also including expression on Pdx1-positive cells.Results and discussionFish-HFDS-fed mice showed significantly reduced food intake and body weight gain compared to Cocoa-HFDS-fed mice. Fish-HFDS group had lower fasting blood glucose concentration and area under the curve (AUC) for both GTT and ITT. Plasma c-peptide, insulin, glucagon, and GLP-1 concentrations were increased in the Fish-HFDS group. Interestingly, mice fed the Fish-HFDS diet displayed higher plasma leptin concentration. Histochemical analysis revealed a significant increase in endocrine pancreas β-cells and islet numbers in mice fed Fish-HFDS compared to the Cocoa-HFDS group. Taken together, these findings suggest that in a high-fat/high-sucrose dietary setting, the source of the fat, especially fish oil, can ameliorate the effect of sucrose on glucose homeostasis and endocrine pancreas morphology and function

Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

Increased circulatory and adipose tissue expression of macrophage inflammatory protein (MIP)-1α (CC motif chemokine ligand-3/CCL3) and its association with inflammation in the state of obesity is well documented. Since obesity is associated with increases in both stearic acid and tumor necrosis factor α (TNF-α) in circulation, we investigated whether stearic acid and TNF-α together could regulate MIP-1α/CCL3 expression in human monocytic cells, and if so, which signaling pathways were involved in MIP-1α/CCL3 modulation. Monocytic cells were treated with stearic acid and TNF-α resulted in enhanced production of MIP-1α/CCL3 compared to stearic acid or TNF-α alone. To explore the underlying mechanisms, cooperative effect of stearic acid for MIP-α/CCL3 expression was reduced by TLR4 blocking, and unexpectedly we found that the synergistic production of MIP-α/CCL3 in MyD88 knockout (KO) cells was not suppressed. In contrast, this MIP-α/CCL3 expression was attenuated by inhibiting TBK1/IRF3 activity. Cells deficient in IRF3 did not show cooperative effect of stearate/TNF-α on MIP-1α/CCL3 production. Furthermore, activation of IRF3 by polyinosinic-polycytidylic acid (poly I:C) produced a cooperative effect with TNF-α for MIP-1α/CCL3 production that was comparable to stearic acid. Individuals with obesity show high IRF3 expression in monocytes as compared to lean individuals. Furthermore, elevated levels of MIP-1α/CCL3 positively correlate with TNF-α and CD163 in fat tissues from individuals with obesity. Taken together, this study provides a novel model for the pathologic role of stearic acid to produce MIP-1α/CCL3 in the presence of TNF-α associated with obesity settings

FSL-1 Induces MMP-9 Production through TLR-2 and NF-κB /AP-1 Signaling Pathways in Monocytic THP-1 Cells

Background: Matrix metalloproteinase-9 (MMP-9) is known to be implicated in the pathogenesis of many inflammatory disorders. FSL-1 (fibroblast-stimulating lipopeptide-1) induces cytokine production by monocytes/macrophages. However, it is unclear whether FSL-1 is also able to induce MMP-9 production. Herein, we determined whether FSL-1 could induce MMP-9 production, and if so, which signal transduction pathway(s) were involved. Methods: MMP-9 expression was assessed with real-time qPCR and ELISA. Signaling pathways were studied by using THP1-XBlue™ cells, THP1-XBlue™-defMyD cells, anti-TLR2 mAb and pharmacological inhibitors. Phospho and total proteins were determined by Western blotting. Results: FSL-1 induces MMP-9 expression (PP-/- THP-1 cells did not express MMP-9 in response to FSL-1 treatment. By small interfering RNA-mediated knockdown, we also show that FSL-1-induced up-regulation of MMP-9 requires MyD88. Pre-treatment of THP-1 cells with inhibitors of JNK (SP600125), MEK/ERK (U0126; PD98056; XMD 8-92), p38 MAPK (SB203580) and NF-κB (BAY11-7085, Triptolide, Resveratrol) significantly suppressed (PConclusion: These findings provide the first evidence that FSL-1 induces TLR-2-dependent MMP-9 gene expression which requires the recruitment of MyD88 and leads to activation of MEK1/2 /ERK 1/2, MEK5/ERK5, JNK, p38 MAPK and NF-κB/AP-1

Pam3CSK4 Induces MMP-9 Expression in Human Monocytic THP-1 Cells

Background: Matrix metalloproteinase (MMP)-9 is known to degrade the extracellular matrix and increased MMP-9 levels are related with the pathogenesis of many inflammatory conditions including obesity. Pam3CSK4 is a synthetic triacylated lipopeptide (LP) which is a potent activator of immune cells and induces cytokine production. However, it is unclear whether Pam3CSK4 is able to induce MMP-9 expression in monocytic cells. We, therefore, determined MMP-9 production by Pam3CSK4-treated THP-1 cells and also investigated the signal transduction pathway(s) involved. Methods: MMP-9 expression was determined by real-time qPCR and ELISA. MMP-9 activity was assessed by zymography. THP-1 cells, THP1-XBlueTM cells, THP1-XBlueTM-defMyD cells, anti-TLR2 mAb and selective pharmacological inhibitors were used to study signaling pathways involved. Phosphorylated and total proteins were detected by western blotting. Results: Pam3CSK4 induced MMP-9 expression (P<0.05) at both mRNA and protein levels in human monocytic THP-1 cells. Increased NF-κB/AP-1 activity was detected in Pam3CSK4-treated THP-1 cells and MMP-9 production in these cells was significantly suppressed by pre-treatment with anti-TLR2 neutralizing antibody or by inhibition of clathrin-dependent endocytosis. Also, MyD88-/- THP-1 cells did not express MMP-9 following treatment with Pam3CSK4. Inhibition of JNK, MEK/ERK, p38 MAPK and NF-κB significantly suppressed MMP-9 gene expression (P<0.05). Conclusion: Pam3CSK4 induces MMP-9 production in THP-1 cells through the TLR-2/MyD88-dependent mechanism involving MEK/ERK, JNK, p38 MAPK and NF-κB/AP-1 activation