Visceral adipocytes and associated macrophages produce and release excessive amounts of biologically active inflammatory cytokines via the portal and systemic vascular system, which induce insulin resistance in insulin target tissues such as fat, liver, and muscle. Free fatty acids (FFAs) absorbed via the portal system or released from adipocytes also induce insulin resistance. In this report, we show that phenylmethimazole (C10) blocks basal IL6 and leptin production as well as basal Socs-3 expression in fully differentiated 3T3L1 cells (3T3L1 adipocytes) without affecting insulin-stimulated AKT signaling. In addition, C10 inhibits palmitate-induced IL6 and iNos up-regulation in both 3T3L1 adipocytes and RAW 264.7 macrophages, LPS-induced NF-κB and IFN-β activation in 3T3L1 cells, and LPS-induced iNos, Ifn- β, Il1 β, Cxcl10, and Il6 expression in RAW 264.7 macrophages. C10 also blocks palmitate-induced Socs-3 up-regulation and insulin receptor substrate-1 (IRS-1) serine 307 phosphorylation in 3T3L1 adipocytes. Additionally, we show for the first time that although palmitate increases IRS-1 serine 307 phosphorylation in 3T3L1 adipocytes, AKT serine 473 phosphorylation is enhanced, not reduced, by palmitate. These results suggest that through inhibition of FFA-mediated signaling in adipocytes and associated macrophages, as well as possibly other insulin target cells/tissues (i.e. non-immune cells), C10 might be efficacious to prevent or reverse cytokine-induced insulin resistance seen in obesity-related insulin resistance and type 2 diabetes mellitus.

Wnt binding to cell surface receptors can activate a ‘canonical’ pathway that increases cellular β-catenin or a ‘noncanonical’ Ca⁺⁺ pathway which can increase protein kinase C (PKC) activity. Although components of both Wnt/β-catenin-signaling pathways exist in thyrocytes, their biological role is largely unknown. In evaluating the biological role of Wnt signaling in differentiated FRTL-5 thyroid cells, we showed that TSH increased canonical Wnt-1 but, surprisingly, decreased the active form of β-catenin. Transient overexpression of Wnt-1 or β-catenin in FRTL-5 cells increased active β-catenin (ABC), decreased thyroperoxidase (TPO) mRNA, and suppressed TPO-promoter activity. The target of β-catenin suppressive action was a consensus T cell factor/lymphoid enhancing factor (TCF/LEF)-binding site 5′-A/T A/T CAAAG-3′, −137 to −129 bp on the rat TPO promoter. β-Catenin overexpression significantly increased complex formation between β-catenin/TCF-1 and an oligonucleotide containing the TCF/LEF sequence, suggesting that the β-catenin/TCF-1 complex acts as a transcriptional repressor of the TPO gene. Stable over-expression of Wnt-1 in FRTL-5 cells significantly increased the growth rate without increasing β-catenin levels. Increased growth was blunted by a PKC inhibitor, staurosporin. Wnt-1 overexpression increased serine phosphorylation, without affecting tyrosine phosphorylation, of signal transducers and activators of transcription 3 (STAT3) protein. In addition, these final results suggest that TSH-induced increase in Wnt-1 levels in thyrocytes contributes to enhanced cellular growth via a PKC pathway that increases STAT3 serine phosphorylation and activation, whereas TSH-induced decrease in activation of β-catenin simultaneously relieves transcriptional suppression of TPO. We hypothesize that Wnt signaling contributes to the ability of TSH to simultaneously increase cell growth and functional, thyroid-specific, gene expression.

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.