TNFα-induced NLRP3 inflammasome mediates adipocyte dysfunction and activates macrophages through adipocyte-derived lipocalin 2.

BACKGROUND AND AIMS: Obesity is a state of chronic low-grade systemic inflammation. Recent studies showed that NLRP3 inflammasome initiates metabolic dysregulation in adipose tissues, primarily through activation of adipose tissue infiltrated macrophages. However, the mechanism of NLRP3 activation and its role in adipocytes remains elusive. Therefore, we aimed to examine the activation of TNFα-induced NLRP3 inflammasome in adipocytes and its role on adipocyte metabolism and crosstalk with macrophages. METHODS: The effect of TNFα on adipocyte NLRP3 inflammasome activation was measured. Caspase-1 inhibitor (Ac-YVAD-cmk) and primary adipocytes from NLRP3 and caspase-1 knockout mice were utilized to block NLRP3 inflammasome activation. Biomarkers were measured by using real-time PCR, western blotting, immunofluorescence staining, and enzyme assay kits. Conditioned media from TNFα-stimulated adipocytes was used to establish the adipocyte-macrophage crosstalk. Chromatin immunoprecipitation assay was used to identify the role of NLRP3 as a transcription factor. Mouse and human adipose tissues were collected for correlation analysis. RESULTS: TNFα treatment induced NLRP3 expression and caspase-1 activity in adipocytes, partly through autophagy dysregulation. The activated adipocyte NLRP3 inflammasome participated in mitochondrial dysfunction and insulin resistance, as evidenced by the amelioration of these effects in Ac-YVAD-cmk treated 3T3-L1 cells or primary adipocytes isolated from NLRP3 and caspase-1 knockout mice. Particularly, the adipocyte NLRP3 inflammasome was involved in glucose uptake regulation. Also, TNFα induced expression and secretion of lipocalin 2 (Lcn2) in a NLRP3-dependent manner. NLRP3 could bind to the promoter and transcriptionally regulate Lcn2 in adipocytes. Treatment with adipocyte conditioned media revealed that adipocyte-derived Lcn2 was responsible for macrophage NLRP3 inflammasome activation, working as a second signal. Adipocytes isolated from high-fat diet mice and adipose tissue from obese individuals showed a positive correlation between NLRP3 and Lcn2 gene expression. CONCLUSIONS: This study highlights the importance of adipocyte NLRP3 inflammasome activation and novel role of TNFα-NLRP3-Lcn2 axis in adipose tissue. It adds rational for the current development of NLRP3 inhibitors for treating obesity-induced metabolic diseases.

Multiple signaling pathways converge to regulate bone-morphogenetic-protein-dependent glial gene expression.

A fundamental problem in developmental neuroscience is understanding how extracellular cues link to complex intracellular signaling pathways to drive stage-specific developmental decisions. During the formation of the mammalian peripheral nervous system, bone morphogenetic proteins (BMPs) promote neuronal differentiation. BMPs also maintain the expression of early glial genes such as GFAP, while blocking the acquisition of a mature, myelinating Schwann cell phenotype. We investigated the BMP-activated signaling pathways that contribute to early glial gene expression to address the question of how specific signaling interactions contribute to cell fate decisions in neural crest lineages. Using a neural-crest-derived cell line that exhibits the characteristics of immature Schwann cells, we found that BMP2 promotes GFAP expression using Smad signaling as well as the phosphoinositide-3 kinase (PI3K) and mitogen-activated protein kinase1/2extracellular signal-regulated kinase- (MEK1/2/ERK) pathways. The GFAP promoter does not contain known Smad consensus sites, suggesting that Smads may act indirectly to promote GFAP expression. We provide evidence that this indirect effect may be mediated via induction of immediate early genes and the transcription factor Sp1 by demonstrating that these transcriptional regulators are induced by BMP2 and contribute to GFAP promoter activity. These findings demonstrate new roles for intracellular kinase pathways in mediating the effects of BMPs during the early stages of glial differentiation and suggest that differential contributions by signaling and transcriptional networks may contribute to the range of effects of BMPs on neuronal and glial development during the formation of the peripheral nervous system.

Novel Longitudinal and Propensity Score Matched Analysis of Hands-On Cooking and Nutrition Education versus Traditional Clinical Education among 627 Medical Students.

Background. Physicians are inadequately equipped to respond to the global obesity and nutrition-associated chronic disease epidemics. We investigated superiority of simulation-based medical education with deliberate practice (SBME-DP) hands-on cooking and nutrition elective in a medical school-based teaching kitchen versus traditional clinical education for medical students. Materials and Methods. A 59-question panel survey was distributed to an entire medical school twice annually from September 2012 to May 2014. Student diet and attitudes and competencies (DACs) counseling patients on nutrition were compared using conditional multivariate logistic regression, propensity score-weighted, and longitudinal panel analyses. Inverse-variance weighted meta-analysis (IVWM) was used for planned subgroup analysis by year and treatment estimates across the three methods. Results. Of the available 954 students, 65.72% (n = 627) unique students were followed to produce 963 responses. 11.32% (n = 109) of responses were from 84 subjects who participated in the elective. SBME-DP versus traditional education significantly improved fruit and vegetable diet (OR = 1.38, 95% CI: 1.07-1.79, p = 0.013) and attitudes (OR = 1.81, 95% CI: 1.40-2.35, p < 0.001) and competencies (OR = 1.72, 95% CI: 1.54-1.92, p < 0.001). Conclusions. This study reports for the first time superiority longitudinally for SBME-DP style nutrition education for medical students which has since expanded to 13 schools.

Antiangiogenic effect of TW37, a small-molecule inhibitor of Bcl-2.

Bcl-2 is an antiapoptotic protein that is up-regulated in several tumor types, and its expression levels have strong correlation to development of resistance to therapy and poor prognosis. We have shown recently that Bcl-2 also functions as a proangiogenic signaling molecule that activates a nuclear factor-kappaB-mediated pathway resulting in up-regulation of the angiogenic chemokines CXCL1 and CXCL8 by neovascular endothelial cells. Here, we evaluate the antiangiogenic effect of the novel small-molecule inhibitor of Bcl-2 (TW37) developed using a structure-based design strategy. We observed that TW37 has an IC(50) of 1.8 mumol/L for endothelial cells but showed no cytotoxic effects for fibroblasts at concentrations up to 50 mumol/L. The mechanism of TW37-induced endothelial cell death was apoptosis, in a process mediated by mitochondrial depolarization and activation of caspase-9 and caspase-3. The effect of TW37 on endothelial cell apoptosis was not prevented by coexposure to the growth factor milieu secreted by tumor cells. Inhibition of the angiogenic potential of endothelial cells (i.e., migration and capillary sprouting assays) and expression of the angiogenic chemokines CXCL1 and CXCL8 were accomplished at subapoptotic TW37 concentrations (0.005-0.05 micromol/L). Notably, administration of TW37 i.v. resulted in a decrease in the density of functional human microvessels in the severe combined immunodeficient mouse model of human angiogenesis. In conclusion, we describe functionally separate proapoptotic and antiangiogenic mechanisms for a small-molecule inhibitor of Bcl-2 and show the potential for Bcl-2 inhibition as a target for antiangiogenic therapy.