The Peroxisome Proliferator-Activated Receptor (PPAR)-γ Antagonist 2-Chloro-5-Nitro-N-Phenylbenzamide (GW9662) Triggers Perilipin 2 Expression via PPARδ and Induces Lipogenesis and Triglyceride Accumulation in Human THP-1 Macrophages.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ SIGNIFICANCE STATEMENT: Peroxisome proliferator-activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.

Mutation analysis of familial GJB2-related deafness in Iranian Azeri Turkish patients.

AIMS: Mutations in the GJB2 gene-encoding connexin 26 protein are the main cause for autosomal recessive nonsyndromic hearing loss worldwide. In this study, we assessed the contributions made by GJB2 and del(GJB6-D13S1830) mutations to the autosomal recessive nonsyndromic deafness genetic load in Iranian Azeri Turkish patients. RESULTS: Probands from 209 different nuclear families were investigated. GJB2 mutations were found in 28% of the patients. Among these patients 44 families had 35delG mutation. The following GJB2 variants, R184P, DelE120, V27I+E114G, W24 x, DelE119, R127H, 235DelC, 290-291 insA, Y155 x, A171T, E147 x, 35insG, G158S, R32H, R143Q, T123N, R143W, H16R, V153I, V27I, M163V, and F154F (a new variant), were identified in 126 of the 418 chromosomes. R143Q mutation was identified as compound heterozygous with 35delG in one profoundly deaf patient. Both parents of this patient were healthy, and one normal sister of this patient was also a carrier for the R143Q, indicating that this mutation has incomplete penetrance. CONCLUSIONS: Our results show that GJB2 mutations are responsible for about 28% of the autosomal recessive nonsyndromic hearing loss in this ethnic group. 35delG is the most prevalent GJB2 mutation accounting for 64.5% of the GJB2 mutations.