To better understand the role of irisin in humans, we examined the effects of irisin in human primary adipocytes and fresh human subcutaneous white adipose tissue (scWAT). Human primary adipocytes derived from 28 female donors' fresh scWAT were used to examine the effects of irisin on browning and mitochondrial respiration, and preadipocytes were used to examine the effects of irisin on adipogenesis and osteogenesis. Cultured fragments of scWAT and perirenal brown fat were used for investigating signal transduction pathways that mediate irisin's browning effect by Western blotting to detect phosphorylated forms of p38, ERK, and STAT3 as well as uncoupling protein 1 (UCP1). Individual responses to irisin in scWAT were correlated with basal expression levels of brown/beige genes. Irisin upregulated the expression of browning-associated genes and UCP1 protein in both cultured primary mature adipocytes and fresh adipose tissues. It also significantly increased thermogenesis at 5 nmol/l by elevating cellular energy metabolism (OCR and ECAR). Treating human scWAT with irisin increased UCP1 expression by activating the ERK and p38 MAPK signaling. Blocking either pathway with specific inhibitors abolished irisin-induced UCP1 upregulation. However, our results showed that UCP1 in human perirenal adipose tissue was insensitive to irisin. Basal levels of brown/beige and FNDC5 genes correlated positively with the browning response of scWAT to irisin. In addition, irisin significantly inhibited adipogenic differentiation but promoted osteogenic differentiation. We conclude that irisin promotes "browning" of mature white adipocytes by increasing cellular thermogenesis, whereas it inhibits adipogenesis and promotes osteogenesis during lineage-specific differentiation. Our findings provide a rationale for further exploring the therapeutic use of irisin in obesity and exercise-associated bone formation.
Kinesin-2 motors power anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. However, it remains elusive how kinesin-2 motors are regulated in vivo. Here, we performed forward genetic screens to isolate suppressors that rescue the ciliary defects of OSM-3-kinesin (homolog of mammalian homodimeric kinesin-2 KIF17) mutants in Caenorhabditis elegans. We identified the C. elegans dyf-5 and dyf-18, which encode the homologs of mammalian male germ cell-associated kinase and cell cycle-related kinase, respectively. Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate elongated cilia and ectopic localization of the heterotrimeric kinesin-2 (kinesin-II) at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are important for stabilizing the interaction between IFT particles and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.
Objective Diffuse alveolar hemorrhage (DAH) in lupus patients is >50% fatal. The cause is unknown. The pathogenesis of DAH in C57BL/6 mice with pristane-induced lupus, a model of human lupus-associated DAH, was examined. Methods Clinical/pathological and immunological manifestations DAH in pristane-lupus were compared with human DAH. Tissue distribution of pristane was examined by mass spectrometry. Cell types responsible for disease were determined by in vivo depletion using clodronate liposomes (CloLip) and anti-neutrophil monoclonal antibodies (GR1). The effect of complement depletion with cobra venom factor (CVF) was examined. Results After i.p. injection, pristane migrated to the lung, causing cell death, small vessel vasculitis, and alveolar hemorrhage similar to human DAH. B-cell-deficient mice were resistant to induction of DAH, but susceptibility was restored by infusing IgM. C3-deficient and CD18-deficient mice also were resistant and DAH was prevented in wild-type mice by CVF. Induction of DAH was independent of TLRs, inflammasomes, and inducible nitric oxide (iNOS). Mortality was increased in IL-10-deficient mice and pristane treatment decreased IL-10 receptor expression in monocytes and Stat3 phosphorylation in lung macrophages. In vivo neutrophil depletion was not protective, whereas treatment with CloLip prevented DAH, suggesting that macrophage activation is central to DAH pathogenesis. Conclusion The pathogenesis of DAH involves opsonization of dead cells by natural IgM and complement followed by complement receptor-mediated lung inflammation. The disease is macrophage-dependent and IL-10 is protective. Complement inhibition and/or macrophage-targeted therapies may reduce mortality in lupus-associated DAH.
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