Leptin, a hormone produced in white adipose tissue, acts in the brain to communicate fuel status, suppress appetite following a meal, promote energy expenditure, and maintain blood glucose stability1,2. Dysregulations of leptin or its receptors (LepR) result in severe obesity and diabetes3–5. Although intensive studies on leptin have transformed obesity and diabetes research2,6, clinical applications of the molecule are still limited7 which, at least in part, is due to the complexity and our incomplete understanding of the underlying neural circuits. The hypothalamic neurons expressing agouti-related peptide (AgRP) and proopiomelanocortin (POMC) were posited as the first-order leptin-responsive neurons. Selective deletion of LepR in these neurons with Cre-loxP system, however, failed to or marginally recapitulated obesity and diabetes in LepR-deficient Leprdb/db mice, suggesting that AgRP or POMC neurons are not directly required8–10. The primary neural targets for leptin are thus still unclear. Here, we conduct a systematic, unbiased survey of leptin-responsive neurons in streptozotocin (STZ)-induced diabetic mice and exploit CRISPR/Cas9-mediated genetic ablation of LepR in vivo. Unexpectedly, we find that AgRP neurons but not POMC neurons integrate the primary action of leptin to regulate both energy balance and glucose homeostasis. Leptin deficiency disinhibits AgRP neurons, and their chemogenetic inhibition reverses both diabetic hyperphagia and hyperglycemia. In sharp contrast with prior studies, we show that CRISPR-mediated deletion of LepR in AgRP neurons causes severe obesity and diabetes, fatefully replicating the phenotype of Leprdb/db mice. We also uncover divergent mechanisms underlying leptin’s acute and chronic inhibition of AgRP neurons (i.e., presynaptic potentiation of GABAergic neurotransmission and postsynaptic activation of ATP-sensitive potassium channels, respectively). Our findings provide the framework underlying the neurobiological mechanisms of leptin and associated metabolic disorders.
Abstract. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8 ns and that of interaction with the duplex structure of a calf thymus is ∼1.2 ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. We have introduced a G-quadruplex (G4) ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide, to monitor the cellular uptake of naked GROs and map their intracellular localizations in living cells by using confocal microscopy. The GROs that form parallel G4 structures, such as PU22, T40214 and AS1411, are detected mainly in the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary, the GROs that form non-parallel G4 structures, such as human telomeres (HT23) and thrombin binding aptamer (TBA), are rarely detected in the lysosome, but found mainly in the mitochondria. Moreover, the fluorescence resonant energy transfer studies of fluorophore-labeled GROs show that the parallel G4 structures can be retained in CL1-0 cells, whereas the non-parallel G4 structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G4 structure of HT23 from the non-parallel G4 structure can reform to a probable parallel G4 structure induced by a G4 ligand in CL1-0 living cells. These findings are valuable to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs.
Drosophila is one of the most valuable model organisms for studying genetics and developmental biology. The fat body in Drosophila, which is analogous to the liver and adipose tissue in human, stores lipids that act as an energy source during its development. At the early stages of metamorphosis, the fat body remodeling occurs involving the dissociation of the fat body into individual fat cells. Here we introduce a combination of coherent anti-Stokes Raman scattering (CARS) and two-photon excitation autofluorescence (TPE-F) microscopy to achieve label-free imaging of Drosophila in vivo at larval and pupal stages. The strong CARS signal from lipids allows direct imaging of the larval fat body and pupal fat cells. In addition, the use of TPE-F microscopy allows the observation of other internal organs in the larva and autofluorescent globules in fat cells. During the dissociation of the fat body, the findings of the degradation of lipid droplets and an increase in autofluorescent globules indicate the consumption of lipids and the recruitment of proteins in fat cells. Through in vivo imaging and direct monitoring, CARS microscopy may help elucidate how metamorphosis is regulated and study the lipid metabolism in Drosophila.
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