No abstract

Objective Leptin (Lep) plays a crucial role in controlling food intake and energy expenditure. Defective Lep/LepRb-signaling leads to fat accumulation, massive obesity, and the development of diabetes. We serendipitously noticed spontaneous development of obesity similar to LepR-deficient (db/db) mice in offspring from a C57BL/6J breeding and transmittance of the phenotype in a Mendelian manner. Candidate gene sequencing revealed a spontaneous point mutation in the LepRb gene. We investigated leptin responsiveness, leptin receptor signaling and metabolic phenotype of this novel LepRb mutant mouse variant. Methods Overexpression and functional tests of the mutant LepRb in 3T3 cells. Measurement of leptin responsiveness in hypothalamic nuclei, glucose tolerance, food uptake and energy expenditure in the mutant mice. Results The mutation results in the exchange of a glycine for serine (G506S) and introduces an alternative splice acceptor which, when used, encodes for a protein with a 40aa deletion that is retained in the cytoplasm. LepRb signaling was abrogated in the hypothalamic ventromedial nucleus (VMN) and dorsomedial nucleus (DMN), but only partially reduced in the hypothalamic arcuate nucleus (ARC) of LepRb G506S/G506S mice, most likely due to differential splicing in neurons located in the respective regions of the hypothalamus. Extensive metabolic characterization of these mice revealed interesting differences in the control of food intake, glucose tolerance, energy expenditure, and fat accumulation in LepRb G506S/G506S compared with LepRb-deficient db/db mice. Conclusions This study provides further insight into differences of the leptin responsiveness in VMN, DMN, and ARC and its metabolic consequences.

Nuclear factor-κB (NF-κB) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-κB activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-κB reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-κB, the latter have the potential to allow the analysis of NF-κB activity at single-cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knock-in NF-κB reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-κB transcriptional activity at the single-cell level of various cell types during inflammatory and infectious diseases.

Nuclear factor-kB (NF-kB) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-kB activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-kB reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-kB, the latter have the potential to allow the analysis of NF-kB activity at single cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knockin NF-kB reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-kB activity at the single-cell level of various cell types during inflammatory and infectious diseases.