Galectin-8 as a novel regulator of primary cilium and energy balance

Abstract

Primary cilium (PC) is cell surface antenna-like organelle that contribute to elaborate and integrate signaling pathways triggered by a variety of extracellular stimuli. In neurons of the hypothalamic region, the PC plays a crucial role in metabolism regulation by signals such as leptin and insulin. Loss of cilia in these neurons leads to leptin and insulin resistance and obesity, which are main public health problems. Therefore, exploring brain-dependent factors and conditions that regulate PC function in hypothalamic neurons is necessary to improve our understanding of metabolic physiology and its disorders. The leptin-melanocortin circuit in the hypothalamus depends on PC function to control energy balance and food intake by enhancing anorexigenic signals and energy expenditure in response to leptin. Two antagonistic populations of neurons are involved in this circuit: AgRP neurons that express neuropeptide-Y/agouti related peptide and POMC neurons that express pro-opiomelanocortin. During fasting, AgRP neurons inhibit POMC neurons through AgRP and GABA neurotransmitters, thus generating appetite and low energy expenditure signals. After food intake, leptin secreted into the bloodstream inhibits AgRP neurons and activates POMC neurons, which now trigger satiety and energy expenditure signals. Galectin-8 (Gal-8) is a beta-galactosidase binding protein that modulates various cellular processes by interacting with different glycoproteins, both intracellularly and extracellularly. In the central nervous system, Gal-8 is expressed at different levels in several regions, including the hypothalamus. It is highly expressed in the choroid plexus and is found in the cerebrospinal fluid (CSF), which permeates the entire brain. In the brain, Gal-8 has been shown to play neuroprotective and immunosuppressive roles. Proinflammatory conditions, such as those observed in obesity and diabetes, upregulate Gal-8 expression. Studies in platelets suggest that Gal-8 can induce calcium influx in certain cellular systems, although the underlying mechanism remains unknown. Gal-8 binds and activates β1 integrins, which are major receptors for this lectin and have been shown to induce calcium influx through L-type calcium channels. In this study, we investigate the role of Gal-8 on primary cilia biogenesis, leptin signaling, and calcium homeostasis related to the metabolic function of hypothalamic neurons. Hypothesis: Galectin-8 induces disassembly of primary cilia in Clu-177 hypothalamic cells, affecting leptin signaling and involving calcium influx through L type calcium channels. The results demonstrate that Gal-8 induces loss of primary cilia in the hypothalamic Clu-177 cell line, in a time- and concentration-dependent manner. Treatment with 30nM Gal-8 reduced the number of ciliated cells by ~20% and the length of primary cilia by 22.5% involving the AurkA/HDAC6 axis. Pulldown assays showed that Gal 8 binds α3β1 and α5β1 integrins. Accordingly, Gal-8 treatment induced activation of β1-integrin downstream elements such as src and FAK kinases. Inhibition of these kinases prevented cilia loss. Gal-8 treatment also induced calcium influx through L type calcium channels. Blocking these channels, as well as chelating extracellular calcium, prevented calcium influx and loss of primary cilia. Gal-8 treatment reduced leptin signaling in hypothalamic Clu-177 cells. In vivo experiments show that Gal-8 knockout (Gal-8-KO) mice exhibit lower weight and food intake, as well as higher locomotor activity and respiratory exchange ratio (RER), compared to wild-type (WT) mice. Moreover, intranasal administration of Gal-8 in Gal-8-KO animals restored the RER to the WT phenotype. All these results are consistent with a potential role of Gal-8 in brain contributing to control energy balance through the PC signaling function in the hypothalamus. The Gal-8-dependent signaling pathway that includes integrin/FAK/Src activation, calcium influx through L-type calcium channel Cav1.3 and downstream AurKA/HDAC6 axis, impacting upon PC biogenesis and structure in hypothalamic neurons, may provide new elements amenable to metabolic alteración and therapeutic opportunities.