Oxygen-sensing neurons reciprocally regulate peripheral lipid metabolism via neuropeptide signaling in <i>Caenorhabditis elegans</i>

<div><p>The mechanisms by which the sensory environment influences metabolic homeostasis remains poorly understood. In this report, we show that oxygen, a potent environmental signal, is an important regulator of whole body lipid metabolism. <i>C</i>. <i>elegans</i> oxygen-sensing neurons reciprocally regulate peripheral lipid metabolism under normoxia in the following way: under high oxygen and food absence, URX sensory neurons are activated, and stimulate fat loss in the intestine, the major metabolic organ for <i>C</i>. <i>elegans</i>. Under lower oxygen conditions or when food is present, the BAG sensory neurons respond by repressing the resting properties of the URX neurons. A genetic screen to identify modulators of this effect led to the identification of a BAG-neuron-specific neuropeptide called FLP-17, whose cognate receptor EGL-6 functions in URX neurons. Thus, BAG sensory neurons counterbalance the metabolic effect of tonically active URX neurons via neuropeptide communication. The combined regulatory actions of these neurons serve to precisely tune the rate and extent of fat loss to the availability of food and oxygen, and provides an interesting example of the myriad mechanisms underlying homeostatic control.</p></div

C. elegans Body Cavity Neurons Are Homeostatic Sensors that Integrate Fluctuations in Oxygen Availability and Internal Nutrient Reserves

SummaryIt is known that internal physiological state, or interoception, influences CNS function and behavior. However, the neurons and mechanisms that integrate sensory information with internal physiological state remain largely unknown. Here, we identify C. elegans body cavity neurons called URX(L/R) as central homeostatic sensors that integrate fluctuations in oxygen availability with internal metabolic state. We show that depletion of internal body fat reserves increases the tonic activity of URX neurons, which influences the magnitude of the evoked sensory response to oxygen. These responses are integrated via intracellular cGMP and Ca2+. The extent of neuronal activity thus reflects the balance between the perception of oxygen and available fat reserves. The URX homeostatic sensor ensures that neural signals that stimulate fat loss are only deployed when there are sufficient fat reserves to do so. Our results uncover an interoceptive neuroendocrine axis that relays internal state information to the nervous system

The BAG neuropeptide FLP-17 controls URX baseline responses.

<p>(A-D) Measurements of neuronal activity by Ca²⁺ imaging of URX neurons for each genotype. The number of animals used for each condition is shown in the figure. We conducted Ca²⁺ imaging experiments in URX neurons in living wild-type animals and <i>flp-17</i> mutants bearing GCaMP5K under the control of the <i>flp-8</i> promoter. Oxygen concentrations in the microfluidic chamber were 10% (low) and 21% (high) as indicated. (A-B) For each genotype, black traces show the average percent change of GCaMP5K fluorescence (ΔF/F₀) and gray shading indicates SEM. (C-D) Individual URX responses are shown for each genotype; each row represents one animal. (E) Maximum ΔF/F₀ values are shown for individual wild-type animals and <i>flp-17</i> mutants. Bars indicate the average value within each genotype. *, p<0.05 by Student’s t-test. (F) Individual baseline fluorescence (F₀) values at 10% (low) oxygen are shown for individual wild-type animals and <i>flp-17</i> mutants. Bars indicate the median value within each genotype. *, p<0.05 by Student’s t-test. (G) We imaged mCherry fluorescence in wild-type animals and <i>flp-17</i> mutants expressing both GCaMP5K and mCherry under the control of the <i>flp-8</i> promoter. Images were taken in animals exposed to 10% (low) oxygen. For each genotype, the fluorescence intensity was imaged at the same exposure, determined to be within the linear range. Fluorescence intensity was quantified and expressed as an average + SEM (n = 20). NS, not significant by Student’s t-test. (H) The background-subtracted maximum fluorescence (max FL) at 21% (high) oxygen is shown for each wild-type animal and <i>flp-17</i> mutant. Bars indicate the median value within each genotype. NS, not significant by Student’s t-test.</p

EGL-6 functions in URX neurons to regulate oxygen-dependent fat loss.

<p>(A) Worms from a transgenic line bearing <i>egl-6</i> expression using the <i>flp-8</i> promoter were subjected to the oxygen-dependent fat loss assay. Non-transgenic animals (-) and transgenic animals (+) are shown. Relative to non-transgenic controls (light gray bars), transgenic animals (dark gray bars) restore body fat content at 10% (low) oxygen to that seen in wild-type animals. Data are expressed as a percentage of body fat in wild-type animals + SEM (n = 16–20). NS, not significant and *, p<0.05 by one-way ANOVA. (B) Representative fluorescent images showing the expression of P<i>egl-6</i>::<i>GFP</i> (left panels), P<i>flp-8</i>::<i>mCherry</i> (central panels), and their co-localization in URX (merge, right panels). <i>egl-6</i> expression was also observed in DVA, SDQL, SDQR, HSN (not depicted) and additional head neurons. (C) The indicated genotypes were subjected to the oxygen-dependent fat loss assay. Fat content was quantified for each genotype and condition. White bars, fed; gray bars, fasted at 21% oxygen and black bars, fasted at 10% oxygen. Data are expressed as a percentage of body fat in wild-type fed controls + SEM (n = 16–20). NS, not significant and *, p<0.05 by one-way ANOVA.</p

BAG neurons control the resting-state Ca²⁺ concentrations in URX neurons.

<p>(A-D) Measurements of neuronal activity by Ca²⁺ imaging of URX neurons for each genotype. The number of animals used for each condition is shown in the figure. We conducted Ca²⁺ imaging experiments in URX neurons in living wild-type and BAG-ablated animals bearing GCaMP5K under the control of the <i>flp-8</i> promoter. Oxygen concentrations in the microfluidic chamber were 10% (low) and 21% (high) as indicated. (A-B) For each genotype, black traces show the average percent change of GCaMP5K fluorescence (ΔF/F₀) and gray shading indicates SEM. (C-D) Individual URX responses are shown for each genotype; each row represents one animal. (E) Maximum ΔF/F₀ values are shown for individual wild-type and BAG ablated animals. Bars indicate the average value within each genotype. ***, p<0.001 by Student’s t-test. N.B. Log scale. (F) Individual baseline fluorescence (F₀) values at 10% (low) oxygen are shown for individual wild-type animals and <i>gcy-33</i> mutants. Bars indicate the median value within each genotype. **, p<0.01 by Student’s t-test. N.B. Log scale. (G) We imaged mCherry fluorescence in wild-type and BAG ablated animals expressing both GCaMP5K and mCherry under the control of the <i>flp-8</i> promoter. Images were taken in animals exposed to 10% (low) oxygen. For each genotype, the fluorescence intensity was imaged at the same exposure, determined to be within the linear range. Fluorescence intensity was quantified and expressed as an average + SEM (n = 20). NS, not significant by Student’s t-test. (H) The background-subtracted maximum fluorescence (max FL) at 21% (high) oxygen is shown for each wild-type and BAG ablated animal. Bars indicate the median value within each genotype. NS, not significant by Student’s t-test.</p

GCY-33 controls the resting-state Ca²⁺ concentrations in URX neurons.

<p>(A-D) Measurements of neuronal activity by Ca²⁺ imaging of URX neurons for each genotype. The number of animals used for each condition is shown in the figure. We conducted Ca²⁺ imaging experiments in URX neurons in living wild-type animals and <i>gcy-33</i> mutants bearing GCaMP5K under the control of the <i>flp-8</i> promoter. Oxygen concentrations in the microfluidic chamber were 10% (low) and 21% (high) as indicated. (A-B) For each genotype, black traces show the average percent change of GCaMP5K fluorescence (ΔF/F₀) and gray shading indicates SEM. (C-D) Individual URX responses are shown for each genotype; each row represents one animal. (E) Maximum ΔF/F₀ values are shown for individual wild-type animals and <i>gcy-33</i> mutants. Bars indicate the average value within each genotype. *, p<0.05 by Student’s t-test. (F) Individual baseline fluorescence (F₀) values at 10% (low) oxygen are shown for individual wild-type animals and <i>gcy-33</i> mutants. Bars indicate the median value within each genotype. *, p<0.05 by Student’s t-test. (G) We imaged mCherry fluorescence in wild-type animals and <i>gcy-33</i> mutants expressing both GCaMP5K and mCherry under the control of the <i>flp-8</i> promoter. Images were taken in animals exposed to 10% (low) oxygen. For each genotype, the fluorescence intensity was imaged at the same exposure, determined to be within the linear range. Fluorescence intensity was quantified and expressed as an average + SEM (n = 20). NS, not significant by Student’s t-test. (H) The background-subtracted maximum fluorescence (max FL) at 21% (high) oxygen is shown for each wild-type animal and <i>gcy-33</i> mutant. Bars indicate the median value within each genotype. NS, not significant by Student’s t-test.</p

GCY-33 acts upstream of the URX-cGMP signaling pathway in fat regulation.

<p>(A) Schematic depiction of the oxygen-dependent fat loss assay. Worms were washed off food, and then fasted at either 21% (high) oxygen or 10% (low) oxygen. At the end of the fasting period, worms were fixed and stained with Oil Red O. (B) Worms of the indicated genotypes were subjected to the oxygen-dependent fat loss assay described in (A). White bars, fed; gray bars, fasted at 21% oxygen and black bars, fasted at 10% oxygen. Fat content was quantified for each genotype and condition. Data are expressed as a percentage of body fat in wild-type fed controls + SEM (n = 16–20). NS, not significant and *, p<0.05 by one-way ANOVA. (C) Worms of the indicated genotypes were subjected to the oxygen-dependent fat loss assay. Fat content was quantified for each genotype and condition. Data are expressed as a percentage of body fat in wild-type fed controls + SEM (n = 18–20). NS, not significant and *, p<0.05 by one-way ANOVA. (D) Oxygen consumption rate (OCR) in wild-type, <i>gcy-33</i>, <i>gcy-36</i>, and <i>gcy-33;gcy-36</i> mutants. Data are presented as pmol/min/worm + SEM (n = 5–15). *, p<0.05 by two-way ANOVA.</p

The oxygen sensor GCY-33 acts in BAG neurons to regulate lipid metabolism in intestinal cells.

<p>(A) Animals were fixed and stained with Oil Red O. Fat content was quantified for each genotype and is expressed as a percentage of wild-type animals + SEM (n = 12–20). *, p<0.05 by one-way ANOVA. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007305#pgen.1007305.s001" target="_blank">S1 Fig</a>. (B) Total triglyceride levels in wild-type and <i>gcy-33</i> mutants. Data are presented as μmol per mg of protein + SEM (n = 3). *, p<0.05 by Student’s t-test. (C) Food intake in <i>gcy-33</i> mutants expressed as a percentage of wild-type + SEM (n = 10). NS, not significant by Student’s t-test. (D) For each transgenic line bearing <i>gcy-33</i> expression in the indicated pair of neurons, non-transgenic animals (-) and transgenic animals (+) are shown. Relative to non-transgenic controls (light gray bars), transgenic animals (dark gray bars) bearing <i>gcy-33</i> expression in BAG/URX or BAG only neurons restore body fat content to that seen in wild-type animals. Conversely, transgenic animals expressing <i>gcy-33</i> in only URX neurons fail to restore body fat relative to wild-type animals. Data are expressed as a percentage of body fat in wild-type animals + SEM (n = 18–20). NS, not significant and *, p<0.05 by one-way ANOVA including the Kruskal-Wallis correction. (E) Representative fluorescent image showing expression of <i>gcy-33</i>::<i>GFP</i> observed in BAG (closed arrowheads) and URX (open arrowheads) neurons. (F) Fat content was quantified in wild-type controls and worms with BAG neurons ablated. Data are expressed as a percentage of body fat in wild-type controls + SEM (n = 18–20). *, p<0.05 by Student’s t-test.</p

Model depicting the role of the oxygen-sensing neurons in intestinal lipid metabolism.

<p>When animals are exposed to high (21%) oxygen (left panel), the URX neurons are activated via the soluble guanylate cyclase GCY-36, which promotes fat loss in the intestine. Under low (10%) oxygen (right panel), signaling via GCY-33 in the BAG neurons promotes the release of the neuropeptide FLP-17, which is detected by the EGL-6 GPCR in the URX neurons. Communication from BAG neurons to the URX neurons, mediated by FLP-17/EGL-6 signaling dampens URX neuron activity, thus inhibiting fat loss. Our model provides a mechanism by which tonic activity of the URX neurons is controlled, and the symbiotic relationship between the BAG neurons and the URX neurons that converts the perception of oxygen to tune the rate and extent of fat loss in the intestine.</p