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This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic β-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 β-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost β-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for β-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering β-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing β-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of β-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting β-cell functionality. [ABSTRACT FROM AUTHOR]

Obesity is characterized by chronic low-grade inflammation. Obese people have higher levels of caveolin-1 (CAV1), a structural and functional protein present in adipose tissues (ATs). We aimed to define the inflammatory mediators that influence CAV1 gene regulation and the associated mechanisms in obesity. Using subcutaneous AT from 27 (7 lean and 20 obese) normoglycemic individuals, in vitro human adipocyte models, and in vivo mice models, we found elevated CAV1 expression in obese AT and a positive correlation between the gene expression of CAV1, tumor necrosis factor-alpha (TNF-α), and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). CAV1 gene expression was associated with proinflammatory cytokines and chemokines and their cognate receptors (r ≥ 0.447, p ≤ 0.030), but not with anti-inflammatory markers. CAV1 expression was correlated with CD163, indicating a prospective role for CAV1 in the adipose inflammatory microenvironment. Unlike wild-type animals, mice lacking TNF-α exhibited reduced levels of CAV1 mRNA/proteins, which were elevated by administering exogenous TNF-α. Mechanistically, TNF-α induces CAV1 gene transcription by mediating NF-κB binding to its two regulatory elements located in the CAV1 proximal regulatory region. The interplay between CAV1 and the TNF-α signaling pathway is intriguing and has potential as a target for therapeutic interventions in obesity and metabolic syndromes. [ABSTRACT FROM AUTHOR]

Caveolin-1 (CAV1) is implicated in the pathophysiology of diabetes and obesity. Previously, we demonstrated an association between the CAV1 rs1997623 C > A variant and metabolic syndrome (MetS). Here, we decipher the functional role of rs1997623 in CAV1 gene regulation. A cohort of 38 patients participated in this study. The quantitative MetS scores (siMS) of the participants were computed. CAV1 transcript and protein expression were tested in subcutaneous adipose tissue using RT-PCR and immunohistochemistry. Chromatin immunoprecipitation assays were performed using primary preadipocytes isolated from individuals with different CAV1 rs1997623 genotypes (AA, AC, and CC). The regulatory region flanking the variant was cloned into a luciferase reporter plasmid and expressed in human preadipocytes. Additional knockdown and overexpression assays were carried out. We show a significant correlation between siMS and CAV1 transcript levels and protein levels in human adipose tissue collected from an Arab cohort. We found that the CAV1 rs1997623 A allele generates a transcriptionally active locus and a new transcription factor binding site for early B-cell factor 1 (EBF1), which enhanced CAV1 expression. Our in vivo and in vitro combined study implicates, for the first time, EBF1 in regulating CAV1 expression in individuals harboring the rs1997623 C > A variant. [ABSTRACT FROM AUTHOR]

Chronic low-grade inflammation induced by obesity is a central risk factor for the development of metabolic syndrome. High low-density lipoprotein cholesterol (LDL-c) induces inflammation, which is a common denominator in metabolic syndrome. IL-23 plays a significant role in the pathogenesis of meta-inflammatory diseases; however, its relationship with LDL-c remains elusive. In this cross-sectional study, we determined whether the adipose tissue IL-23 expression was associated with other inflammatory mediators in people with increased plasma LDL-c concentrations. Subcutaneous adipose tissue biopsies were collected from 60 people, sub-divided into two groups based on their plasma LDL-c concentrations (<2.9 and ≥2.9 mmol/L). Adipose expression of IL-23 and inflammatory markers were determined using real-time qRT-PCR; plasma concentrations of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c) and LDL-c were determined using the standard method; and adiponectin levels were measured by enzyme-linked immunosorbent assay (ELISA). Adipose IL-23 transcripts were found to be increased in people with high LDL-c, compared to low LDL-c group (H-LDL-c: 1.63 ± 0.10–Fold; L-LDL-c: 1.27 ± 0.09–Fold; p < 0.01); IL-23 correlated positively with LDL-c (r = 0.471, p < 0.0001). Immunochemistry analysis showed that AT IL-23 protein expression was also elevated in the people with H-LDL-c. IL-23 expression in the high LDL-c group was associated with multiple adipose inflammatory biomarkers (p ≤ 0.05), including macrophage markers (CD11c, CD68, CD86, CD127), TLRs (TLR8, TLR10), IRF3, pro-inflammatory cytokines (TNF-α, IL-12, IL-18), and chemokines (CXCL8, CCL3, CCL5, CCL15, CCL20). Notably, in this cohort, IL-23 expression correlated inversely with plasma adiponectin. In conclusion, adipose IL-23 may be an inflammatory biomarker for disease progression in people with high LDL-c. [ABSTRACT FROM AUTHOR]

In obesity, macrophage activation and infiltration in adipose tissue (AT) underlie chronic low-grade inflammation-induced insulin resistance. Although dectin-1 is primarily a pathogen recognition receptor and innate immune response modulator, its role in metabolic syndromes remains to be clarified. This study aimed to investigate the dectin-1 gene expression in subcutaneous AT in the context of obesity and associated inflammatory markers. Subcutaneous AT biopsies were collected from 59 nondiabetic (lean/overweight/obese) individuals. AT gene expression levels of dectin-1 and inflammatory markers were determined via real-time reverse transcriptase-quantitative polymerase chain reaction. Dectin-1 protein expression was assessed using immunohistochemistry. Plasma lipid profiles were measured by ELISA. AT dectin-1 transcripts and proteins were significantly elevated in obese as compared to lean individuals. AT dectin-1 transcripts correlated positively with body mass index and fat percentage (r ≥ 0.340, p ≤ 0.017). AT dectin-1 RNA levels correlated positively with clinical parameters, including plasma C-reactive protein and CCL5/RANTES, but negatively with that of adiponectin. The expression of dectin-1 transcripts was associated with that of various proinflammatory cytokines, chemokines, and their cognate receptors (r ≥ 0.300, p ≤ 0.05), but not with anti-inflammatory markers. Dectin-1 and members of the TLR signaling cascade were found to be significantly associated, suggesting an interplay between the two pathways. Dectin-1 expression was correlated with monocyte/macrophage markers, including CD16, CD68, CD86, and CD163, suggesting its monocytes/macrophage association in an adipose inflammatory microenvironment. Dectin-1 expression was independently predicted by CCR5, CCL20, TLR2, and MyD88. In conclusion, dectin-1 may be regarded as an AT biomarker of metabolic inflammation in obesity. [ABSTRACT FROM AUTHOR]

IL-6 was found to be overexpressed in the adipose tissue of obese individuals, which may cause insulin resistance. However, the regulation of IL-6 in adipocytes in obesity setting remains to be explored. Since IL-1β and TNFα are increased in obese adipose tissue and promote inflammation, we investigated whether cooperation between IL-1β and TNFα influences the production of IL-6. Our data show that IL-1β and TNFα cooperatively enhance IL-6 expression in 3T3L-1 adipocytes. Similar results were seen in human adipocytes isolated from subcutaneous and visceral fat. Although adipocytes isolated from lean and obese adipose tissues showed similar responses for production of IL-6 when incubated with IL-1β/TNFα, secretion of IL-6 was higher in adipocytes from obese tissue. TNFα treatment enhanced CREB binding at CRE locus, which was further enhanced with IL-1β, and was associated with elevated histone acetylation at CRE locus. On the other hand, IL-1β treatments mediated C/EBPβ binding to NF-IL-6 consensus, but not sufficiently to mediate significant histone acetylation. Interestingly, treatment with both stimulatory factors amplifies CREB binding and H3K14 acetylation. Furthermore, histone acetylation inhibition by anacardic acid or curcumin reduces IL-6 production. Notably, inhibition of histone deacetylase (HDAC) activity by trichostatin A (TSA) resulted in the further elevation of IL-6 expression in response to combined treatment of adipocytes with IL-1β and TNFα. In conclusion, our results show that there is an additive interaction between IL-1β and TNFα that depends on CREB binding and H3K14 acetylation, and leads to the elevation of IL-6 expression in adipocytes, providing interesting pathophysiological connection among IL-1β, TNFα, and IL-6 in settings such as obesity. [ABSTRACT FROM AUTHOR]

Repetitive intermittent hyperglycemia (RIH) is an independent risk factor for complications associated with type-2 diabetes (T2D). Glucose fluctuations commonly occur in T2D patients with poor glycemic control or following intensive therapy. Reducing blood glucose as well as glucose fluctuations is critical to the control of T2D and its macro-/microvascular complications. The interferon regulatory factor (IRF)-5 located downstream of the nutrient sensor toll-like receptor (TLR)-4, is emerging as a key metabolic regulator. It remains unclear how glucose fluctuations may alter the IRF5/TLR4 expression and inflammatory responses in monocytes/macrophages. To investigate this, first, we determined IRF5 gene expression by real-time qRT-PCR in the white adipose tissue samples from 39 T2D and 48 nondiabetic individuals. Next, we cultured THP-1 macrophages in hypo- and hyperglycemic conditions and compared, at the protein and transcription levels, the expressions of IRF5, TLR4, and M1/M2 polarization profile and inflammatory markers against control (normoglycemia). Protein expression was assessed using flow cytometry, ELISA, Western blotting, and/or confocal microscopy. IRF5 silencing was achieved by small interfering RNA (siRNA) transfection. The data show that adipose IRF5 gene expression was higher in T2D than nondiabetic counterparts (p = 0.006), which correlated with glycated hemoglobin (HbA1c) (r = 0.47/p < 0.001), homeostatic model assessment of insulin resistance (HOMA-IR) (r = 0.23/p = 0.03), tumor necrosis factor (TNF)-α (r = 0.56/p < 0.0001), interleukin (IL)-1β (r = 0.40/p = 0.0009), and C-C motif chemokine receptor (CCR)-2 (r = 0.49/p < 0.001) expression. IRF5 expression in macrophages was induced/upregulated (p < 0.05) by hypoglycemia (3 mM/L), persistent hyperglycemia (15 mM/L–25 mM/L), and RIH/glucose fluctuations (3–15 mM/L) as compared to normoglycemia (5 mM/L). RIH/glucose fluctuations also induced M1 polarization and an inflammatory profile (CD11c, IL-1β, TNF-α, IL-6, and monocyte chemoattractant protein (MCP)-1) in macrophages. RIH/glucose fluctuations also drove the expression of matrix metalloproteinase (MMP)-9 (p < 0.001), which is a known marker for cardiovascular complication in T2D patients. Notably, all these changes were counteracted by IRF5 silencing in macrophages. In conclusion, RIH/glucose fluctuations promote the M1 polarization and inflammatory responses in macrophages via the mechanism involving TLR4-IRF5 pathway, which may have significance for metabolic inflammation. [ABSTRACT FROM AUTHOR]

Metabolic inflammation is associated with increased expression of saturated free fatty acids, proinflammatory cytokines, chemokines, and adipose oxidative stress. Macrophage inflammatory protein (MIP)-1α recruits the inflammatory cells such as monocytes, macrophages, and neutrophils in the adipose tissue; however, the mechanisms promoting the MIP-1α expression remain unclear. We hypothesized that MIP-1α co-induced by palmitate and tumor necrosis factor (TNF)-α in monocytic cells/macrophages could be further enhanced in the presence of reactive oxygen species (ROS)-mediated oxidative stress. To investigate this, THP-1 monocytic cells and primary human macrophages were co-stimulated with palmitate and TNF-α and mRNA and protein levels of MIP-1α were measured by using quantitative reverse transcription, polymerase chain reaction (qRT-PCR) and commercial enzyme-linked immunosorbent assays (ELISA), respectively. The cognate receptor of palmitate, toll-like receptor (TLR)-4, was blunted by genetic ablation, neutralization, and chemical inhibition. The involvement of TLR4-downstream pathways, interferon regulatory factor (IRF)-3 or myeloid differentiation (MyD)-88 factor, was determined using IRF3-siRNA or MyD88-deficient cells. Oxidative stress was induced in cells by hydrogen peroxide (H2O2) treatment and ROS induction was measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. The data show that MIP-1α gene/protein expression was upregulated in cells co-stimulated with palmitate/TNF-α compared to those stimulated with either palmitate or TNF-α (P < 0.05). Further, TLR4-IRF3 pathway was implicated in the cooperative induction of MIP-1α in THP-1 cells, and this cooperativity between palmitate and TNF-α was clathrin-dependent and also required signaling through c-Jun and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Notably, ROS itself induced MIP-1α and could further promote MIP-1α secretion together with palmitate and TNF-α. In conclusion, palmitate and TNF-α co-induce MIP-1α in human monocytic cells via the TLR4-IRF3 pathway and signaling involving c-Jun/NF-κB. Importantly, oxidative stress leads to ROS-driven MIP-1α amplification, which may have significance for metabolic inflammation. [ABSTRACT FROM AUTHOR]