Drosophila Lipophorin Receptors Mediate the Uptake of Neutral Lipids in Oocytes and Imaginal Disc Cells by an Endocytosis-Independent Mechanism

Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR–like proteins in this process

Cardiomyocyte Regulation of Systemic Lipid Metabolism by the Apolipoprotein B-Containing Lipoproteins in Drosophila

We thank our colleagues Linda Thompson and Luke Szweda at OMRF, and Gary Struhl at Columbia University for critical comments on the manuscript. We are grateful to Rolf Bodmer (Sanford-Burnham-Presby Medical Discovery Institute), Joquim Culi (CSIC-UPO), Susan Abmayr (Stowers Institute) and Laurent Perrin (TAGC) for fly stocks and antibodies. We also thank the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and the TRiP at Harvard Medical School for fly stocks. We acknowledged the Imaging Core Facility at OMRF for excellent technical assistance.Author Summary The heart is increasingly recognized to serve an important role in the regulation of whole-body lipid homeostasis; however, the underlying mechanisms remained poorly understood. Here, our study in Drosophila reveals that cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body (insect liver and adipose tissue). We found that apoB-lipoproteins generated by the Drosophila cardiomyocytes serve an equally significant role as their fat body-derived counterparts in maintaining systemic lipid homeostasis on normal food diet. Importantly, on high fat diet (HFD), the cardiomyocyte-derived apoB-lipoproteins are the major determinants of whole-body lipid metabolism, a role which could be attributed to the HFD-induced up-regulation of apoB-lipoprotein biosynthesis genes in the cardiomyocytes and their down-regulation in the fat body. Taken together, our results reveal that apoB-lipoproteins are new players in mediating the heart control of lipid metabolism, and provide first evidence supporting the notion that HFD-induced differential regulation of apoB-lipoprotein biosynthesis genes could alter the input of different tissue-derived apoB-lipoproteins in systemic lipid metabolic control.Yeshttp://www.plosgenetics.org/static/editorial#pee

Drosophila lipophorin receptors recruit the lipoprotein LTP to the plasma membrane to mediate lipid uptake

This is an open access article distributed under the terms of the Creative Commons Attribution License.Lipophorin, the main Drosophila lipoprotein, circulates in the hemolymph transporting lipids between organs following routes that must adapt to changing physiological requirements. Lipophorin receptors expressed in developmentally dynamic patterns in tissues such as imaginal discs, oenocytes and ovaries control the timing and tissular distribution of lipid uptake. Using an affinity purification strategy, we identified a novel ligand for the lipophorin receptors, the circulating lipoprotein Lipid Transfer Particle (LTP). We show that specific isoforms of the lipophorin receptors mediate the extracellular accumulation of LTP in imaginal discs and ovaries. The interaction requires the LA-1 module in the lipophorin receptors and is strengthened by a contiguous region of 16 conserved amino acids. Lipophorin receptor variants that do not interact with LTP cannot mediate lipid uptake, revealing an essential role of LTP in the process. In addition, we show that lipophorin associates with the lipophorin receptors and with the extracellular matrix through weak interactions. However, during lipophorin receptor-mediated lipid uptake, LTP is required for a transient stabilization of lipophorin in the basolateral plasma membrane of imaginal disc cells. Together, our data suggests a molecular mechanism by which the lipophorin receptors tether LTP to the plasma membrane in lipid acceptor tissues. LTP would interact with lipophorin particles adsorbed to the extracellular matrix and with the plasma membrane, catalyzing the exchange of lipids between them.This work was funded by a grant (BFU2011-29296) from the Spanish Ministerio de Ciencia e Innovación (www.idi.mineco.gob.es/) to JC. Centro Andaluz de Biología del Desarrollo is institutionally supported by CSIC, Universidad Pablo de Olavide, and Junta de Andalucía.Peer Reviewe

Lpr2E-mediated lipophorin extracellular stabilization requires LTP.

<p>(A and B) Wing imaginal discs expressing <i>UAS-lpr2E</i> in the posterior compartment, driven by <i>hh-gal4</i>. Lpr2E-HA is shown in red and extracellular lipophorin in green and also in a separate channel (A' and B'). (A) Control disc. (B) <i>apoLTP</i> was silenced by the expression of <i>UAS-apoLTPi</i> in the fat body driven by <i>Cg-gal4</i> for 2 days prior dissection. Temporal control was provided by a <i>tub-gal80</i>^<i>ts</i> transgene. A strong reduction in lipophorin accumulation was observed. Note that for technical reasons, the complete genotype of <i>apoLTPi</i> animals shown in (B) was <i>Cg-gal4</i>,<i>tub-gal80</i>^<i>ts</i>/<i>UAS-Lpr2E</i>;<i>hh-gal4</i>/<i>UAS-apoLTPi</i>. Thus, <i>UAS-lpr2E</i> and <i>UAS-apoLTPi</i> were co-expressed both in the fat body and in imaginal discs. However, this does not have unintended effects, as shown in the next panel and in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005356#pgen.1005356.s009" target="_blank">S9 Fig</a>). Scale bar: 10μm. (C) Western blot of hemolymph samples from wild type (wt, lanes 2–4) and the experimental animals (exp., lanes 5–7) described in (B). LTP and lipophorin were detected using the indicated antibodies. Three biological replicates were analyzed for each genotype. Total proteins stained with colloidal coomassie are shown as loading control at the bottom. Molecular weight markers were loaded in lanes 1 and 8. Circulating LTP levels are undetectable in the experimental animals whereas lipophorin levels are equivalent to the control.</p

The LA-1 and ED domains of the lipophorin receptors are required for robust lipophorin stabilization in imaginal discs.

<p>(A) Optical cross-sections of wing imaginal discs expressing <i>UAS-lpr2E-myc</i> and <i>UAS-lpr2F-myc</i> in the posterior compartment, as indicated. Extracellular proteins were detected by an immunohistochemical protocol performed without cell permeabilization. Lpr2E and Lpr2F accumulate at similar levels in two domains, apical cell membranes and a basal region. (B-G) Wing imaginal discs expressing different lipophorin receptor isoforms and chimeras in the posterior compartment, as indicated. All images were taken at a basal plane of the imaginal discs. Its approximate location is indicated by arrows in A. Lipophorin (green and also in a separate channel) and the overexpressed lipophorin receptors and chimeras (red, detected with α-HA) are shown. All immunostainings were performed after blocking endocytosis for 2.5 hours. (A-G) Shown at the same magnification. Scale bar: 10μm.</p

LTP is a ligand for a subset of lipophorin receptor isoforms.

<p>(A) Co-IP of LTP (top) and lipophorin (middle) with Lpr2F, Lpr2E or empty beads (control), analyzed by western blot. Lpr2E and Lpr2F, both containing an HA tag, were purified from transfected <i>Drosophila</i> S2 cells, incubated with diluted hemolymph and immunoprecipitated with anti-HA. Eluates were analyzed for the presence of lipophorin receptors, shown in the lower panel, of LTP, in the upper panel and of lipophorin, in the middle panel. Last lane contains 0.13 μl of diluted hemolymph. (B-E) Wing imaginal discs expressing <i>UAS-lpr2E</i> (red, detected with α-HA) in the posterior compartment driven by <i>hh-gal4</i>. Optical sections through the basal domain of imaginal discs (B and D) and cross-sections (C and E, apical domain at the top) are shown. LTP (green, also shown in a separate channel) accumulates at higher levels in the posterior compartment where Lpr2E is overexpressed (B and B'). In this region, LTP is detected at higher levels in basolateral membranes (bracket) as well as in vesicles (arrows), colocalizing with Lpr2E (C and C'). An immunostaining technique that solely detects extracellular proteins showed LTP (green, also in a separate channel) in basolateral membranes through the wing pouch area and at higher levels in the posterior compartment, where Lpr2E was overexpressed (D, D', E and E'). (F and G) Wing imaginal discs expressing <i>UAS-lpr2E</i> and <i>UAS-rab5-GFP</i> (F) or <i>UAS-lamp1-GFP</i> (G), shown in cross-section through the wing pouch area. LTP (green) is found in endocytic vesicles (arrows) partially colocalizing with Rab5 (red, F) and Lamp1 (red, G). (H) Wing imaginal disc expressing <i>UAS-lpr2F</i> (red) in the posterior compartment driven by <i>hh-gal4</i>. LTP distribution (green, also in a separate channel) imaged at a basal plane, is not modified by Lpr2F overexpression. B, D and H shown at the same magnification. Scale bar: 200 μm. C, E, F and G at the same magnification. Scale bar: 10 μm.</p

Lpr2E-mediated lipophorin association with cells is transient.

<p>(A-F) Basal optical sections of imaginal discs expressing <i>UAS-lpr2E</i> in the posterior compartment (red). Endocytosis was inhibited for 3 hours and the discs were either fixed immediately (A and D) or washed for 30 minutes (B and E) or for 60 minutes (C and F) in ice-cold cell culture media, as indicated. The distribution of Lipophorin (A-C) and of LTP (D-F) is shown in green, also in separate channels as indicated. Scale bar: 50μm.</p

LTP is required for neutral lipid accumulation in ovaries.

<p>(A-C) Ovarioles containing egg chambers of progressive stages of development, with most mature egg chambers to the right. Neutral lipids are revealed by Nile red staining in yellow, nuclei (DAPI) in blue. Asterisks indicate vitellogenic egg chambers of an equivalent developmental stage (10b). (A) Wild type. (B) <i>apoLTP</i> was silenced in the fat body for two days prior dissection of the ovaries by the expression of <i>UAS-apoLTPi</i> driven by <i>Cg-gal4</i>. Temporal control was provided by a <i>tub-gal80</i>^<i>ts</i> transgene. (C) <i>apolipophorin</i> was similarly silenced in the fat body for six days prior to dissection. Scale bar: 100μm.</p