RESEARCH ARTICLE

Intestinal epithelial cell caveolin 1 regulates fatty acid andlipoprotein cholesterol plasma levelsJessica P. Otis1, Meng-Chieh Shen1, Vanessa Quinlivan1,2, Jennifer L. Anderson1 and Steven A. Farber1,2,*

ABSTRACTCaveolae and their structural protein caveolin 1 (CAV1) have roles incellular lipid processing and systemic lipid metabolism. Globaldeletion of CAV1 in mice results in insulin resistance and increasesin atherogenic plasma lipids and cholesterol, but protects from diet-induced obesity and atherosclerosis. Despite the fundamental role ofthe intestinal epithelia in the regulation of dietary lipid processing andmetabolism, the contributions of CAV1 to lipid metabolism in thistissue have never been directly investigated. In this study the cellulardynamics of intestinal Cav1 were visualized in zebrafish and themetabolic contributions of CAV1 were determined with mice lackingCAV1 in intestinal epithelial cells (CAV1IEC-KO). Live imaging ofCav1–GFP and fluorescently labeled caveolae cargos showslocalization to the basolateral and lateral enterocyte plasmamembrane (PM), suggesting Cav1 mediates transport betweenenterocytes and the submucosa. CAV1IEC-KO mice are protectedfrom the elevation in circulating fasted low-density lipoprotein (LDL)cholesterol associated with a high-fat diet (HFD), but have increasedpostprandial LDL cholesterol, total free fatty acids (FFAs), palmitoleicacid, and palmitic acid. The increase in circulating FAs in HFDCAV1IEC-KO mice is mirrored by decreased hepatic FAs, suggesting anon-cell-autonomous role for intestinal epithelial cell CAV1 inpromoting hepatic FA storage. In conclusion, CAV1 regulatescirculating LDL cholesterol and several FA species via thebasolateral PM of enterocytes. These results point to intestinalepithelial cell CAV1 as a potential therapeutic target to lowercirculating FFAs and LDL cholesterol, as high levels are associatedwith development of type II diabetes and cardiovascular disease.

KEY WORDS: Caveolin 1, Caveolae, Zebrafish, Knockout mice, Freefatty acids, LDL cholesterol

INTRODUCTIONCaveolae are flask-shaped pits, 50 to 100 nm in diameter, which formin lipid-rich plasma membrane (PM) regions of most vertebrate cells(Parton and Simons, 2007). Caveolar vesicle structure is formed byoligomers of caveolin proteins; ∼144 caveolin proteins are present ina single caveola (Parton and Simons, 2007). CAV1 is synthesized inthe endoplasmic reticulum, transported to the Golgi, and upon exit,oligomerizes and associates with lipid-rich membrane regions

(Parton and Simons, 2007). A threshold level of membranecholesterol is required for caveolae to form (Rothberg et al., 1992)and CAV1 can directly bind cholesterol (Murata et al., 1995) and fattyacids (FAs) (Trigatti et al., 1999).

Historically recognized for their endocytic function, caveolaealso regulate cell-signaling pathways, internalization of cell-surfacereceptors and ligands, cell adhesion molecule expression,exocytosis, and transcytosis of caveolae cargos (Parton andSimons, 2007). Caveolae are also emerging players in lipidmetabolism. Global CAV1 knockout mice (CAV1KO) mice havesevere alterations in circulating lipids, including decreased fastingfree FAs (FFAs), increased postprandial FFAs (Razani et al., 2002),increased triglycerides (TGs) (Frank et al., 2008; Razani et al.,2002), and increased non-high-density lipoprotein (HDL) total, freeand esterified cholesterol (Frank et al., 2008; Heimerl et al., 2008;Razani et al., 2002; Valasek et al., 2005). CAV1KO mice are insulin-resistant (Cohen et al., 2003), but are protected from diet-inducedobesity (Razani et al., 2002) and atherosclerosis (Fernandez-Hernando et al., 2009; Frank et al., 2004). The plasma lipid andbody mass changes in CAV1KO mice have been proposed to resultfrom a variety of mechanisms including altered lipid dropletarchitecture, reduced adipocyte lipid droplet formation (Cohenet al., 2004), and impaired adipocyte metabolic flexibility(Asterholm et al., 2012), whereas protection from atherosclerosisis likely a result of decreased endothelial adhesion moleculeexpression.

The cells that line the intestinal epithelia, enterocytes, are highlyspecialized to bidirectionally absorb, transport and export largequantities of luminal contents and basolateral plasma components.However, the mechanisms by which dietary lipids are internalized,transported and externalized by enterocytes, as well as howenterocytes receive adequate lipids from adipose stores duringfasting, remain incompletely understood. The close association ofCAV1 with cholesterol, FAs, lipid droplets and lipid-rich PMregions suggests a role for CAV1 and caveolae in intestinal lipidmetabolism (Parton and del Pozo, 2013). CAV1 is expressed, andcaveolae form, in the enterocytes of several species includinghumans and mice (Badizadegan et al., 2000; Field et al., 1998;Marchiando et al., 2010; McConnell et al., 2011; Morroni et al.,2007; Nabeyama and Leblond, 1974; Siddiqi et al., 2013). Althoughit is known that CAV1 and caveolae are present in enterocytes,several basic aspects of their biology, including subcellularlocalization and metabolic functions, remain unclear.

Although intestinal cholesterol absorption is not disrupted inmice lacking CAV1 (Valasek et al., 2005), isolated intestinalcaveolae contain dietary FAs (Siddiqi et al., 2013) and intestinallipoprotein cholesterol export influences plasma cholesterol levels,so intestinal CAV1 might significantly impact cholesterolmetabolism. Cell culture studies have suggested that CAV1 PMlocalization might be asymmetric in polarized cells such asintestinal enterocytes; however, reports are conflicting. ForReceived 26 July 2016; Accepted 23 January 2017

1Carnegie Institution for Science, Department of Embryology, Baltimore, MD 21218,USA. 2Johns Hopkins University, Department of Biology, Baltimore, MD 21218,USA.

*Author for correspondence (farber@ciwemb.edu)

J.P.O., 0000-0003-3562-350X

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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example, in human intestinal cells CAV1 localizes asymmetrically,but the pattern varies by cell type: in human T84 colonicadenocarcinoma cells, CAV1 was observed only on lateralmembranes (Nusrat et al., 2000); in human intestinal biopsies,CAV1 was found only on the basolateral surface (Badizadeganet al., 2000); and in Caco2 cells, CAV1 localizes to the apicalplasma membrane (Field et al., 1998). This imprecise understandingof enterocyte CAV1 localization hampers understanding of itsfunctions in health and disease in the intestine.A shortage of studies in live, intact animal models has limited our

understanding of the contributions of intestinal CAV1 to enterocytecell biology and global lipid metabolism. Therefore, in this study weharnessed the genetic tractability and optical clarity of the larvalzebrafish (Danio rerio) to perform live imaging of intestinal Cav1and caveolae-mediated endocytosis for the first time. The zebrafishdigestive system is similar to that of the human, composed of a liver,gallbladder and intestine (Carten and Farber, 2009), and lipid andlipoprotein metabolism are highly conserved (Babin and Vernier,1989; Otis et al., 2015). Similar to humans and mice, the zebrafishgenome contains one cav1 gene, with two major splice transcripts,and is expressed in the intestine (Nixon et al., 2007). Zebrafish havepreviously enabled the elucidation of a role for Cav1 in embryonicorganogenesis (Fang et al., 2006; Nixon et al., 2007) and live

super-resolution imaging of Cav1 in the embryonic tail (Gaboret al., 2015). Additionally, we employed the power of the mousemodel to generate a tissue-specific Cav1 deletion and determine itscontribution to global lipid metabolism. Our transgenic zebrafishand knockout mice, combined with a innovative approach to assayenterocyte endocytosis in vivo, allows for an unprecedentedunderstanding of enterocyte CAV1 cell biology, the effects ofenterocyte CAV1 on systemic lipid metabolism, and how CAV1 inthe intestinal epithelia influence metabolic disease risk throughalterations in circulating lipids.

RESULTSCav1 localizes to the lateral and basolateral PM of zebrafishenterocytesWe generated Tg(hsp70l:cav1-eGFP) zebrafish that expresszebrafish Cav1 tagged with GFP and performed live imaging.Cav1 localizes to the basolateral and lateral PM of intestinalepithelial cells, but is excluded from the luminal brush border(Fig. 1A). Mean fluorescence intensity is 7.5-fold greater on thelateral PM (605.5 relative units) than the brush border (80.7 relativeunits) (one-way ANOVA, P<0.05; Fig. 1B).

As Cav1 protein can exist as either a monomer or an oligomerstructuring caveolae, we examined EM sections to determine where

Fig. 1. Cav1 and caveolae localize to the basolateral and lateral PMof enterocytes. (A) Live imaging of Tg(hsp70l:cav1-eGFP) (6 dpf) zebrafish larvae showslocalization of Cav1-eGFP to the lateral and basolateral plasma membranes (PM) of enterocytes, but not the luminal brush border. One enterocyte is outlined.(B) Mean fluorescence intensity, in relative units, of Cav1-eGFP in subcellular regions of larval enterocytes. Data is mean±s.e.m, n=3: nine fish per replicate, threeareas of each region per fish; groups with different letters are significantly different (one-way ANOVA, P<0.05). (C,D) Representative EM images of caveolavesicles observed on the basolateral and lateral PMs of larval (6 dpf) (C) and adult (D) zebrafish enterocytes. BB, brush border; L, lateral membrane; B,basolateral membrane; I, intracellular; c, caveolae. Scale bars: 100 nm.

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caveolae localize. Similar to the localization of Cav1, caveolae areobserved on both the basolateral and lateral membranes, but not onthe brush border (Fig. 1C,D).

Caveolae-mediated endocytosis occurs only on thebasolateral side of enterocytesThe asymmetric PM localization of zebrafish enterocyte Cav1suggests that caveolae-specific endocytosis occurs between theintestinal epithelia and submucosa, but not the intestinal lumen. Totest this hypothesis, we developed a technique to visualizeendocytosis in live zebrafish larvae based on cell culture studiesthat use fluorescently labeled endocytic cargos (Singh et al., 2006).We injected fluorescently labeled endocytic cargos that areinternalized specifically by caveolae [Alexa Fluor–albumin(Chanthick et al., 2016; Dobrinskikh et al., 2014; Ghitescu andBendayan, 1992; Ghitescu et al., 1986; Schubert et al., 2001) andBODIPY–d-LacCer (Singh et al., 2003, 2006)] or clathrin-coatedvesicles [BODIPY–l-LacCer (Singh et al., 2006)] into thebasolateral or luminal side of enterocytes. Live confocal imagingindicated if endocytosis took place from the basolateral/lateral PM(basolateral injection) or brush border (luminal injection) in theintestinal epithelia. Whereas BODIPY–l-LacCer, the cargotransported specifically by clathrin-coated vesicles, is internalizedfrom both enterocyte PM regions, the caveolae-specific cargosAlexa Fluor–albumin and BODIPY–d-LacCer, are onlyendocytosed from the basolateral PM (Fig. 2A). The fluorescentlylabeled caveolae-specific cargos localize to similar cellularlocations as endogenous Cav1 and Cav1–GFP (lateral andbasolateral PM), whereas the clathrin-specific cargo localizes todistinct cellular locations (brush border) (Fig. 2A). The meanfluorescence intensity of the lateral PM was 3.45-fold and 11.1-foldgreater following basolateral injection of Alexa Fluor–albumin andBODIPY–d-LacCer, respectively, relative to luminal injection(Student’s t-test, P=0.018 and P=0.003; Fig. 2B). Two lines ofzebrafish with mutations in cav1 have recently been published (Caoet al., 2016); we hypothesized that uptake of caveolar cargos wouldbe lost in the enterocytes of the fish. Basolateral albumin injectionswere repeated in the larvae; however, uptake of Alexa Fluor–albumin was not decreased in cav1PD1094, likely because total cav1mRNA is not decreased in this line. This experiment was alsoattempted in two clutches of cav1PD1104 larvae, and although somefish were viable to adulthood, homozygous mutant larvae did notsurvive the experimental treatment (data not shown; fish generouslyprovided by Michel Bagnat, Department of Cell Biology, DukeUniversity Medical Center, Durham, NC, USA). These resultssuggest that caveolae perform a polarized endocytic function inenterocytes, mediating transport between enterocytes and thesubmucosa, but not the intestinal lumen.

Generation of intestinal epithelial cell CAV1 knockout miceAlthough zebrafish are an ideal model to visualize Cav1 andendocytosis, it is extremely challenging to perform tissue-specificgene deletion and measurements of plasma metabolites. To addressthis challenge, mice lacking CAV1 specifically in intestinalepithelial cells (CAV1IEC-KO) were generated by crossing micewith floxed Cav1 (Cav1fl/fl) (Cao et al., 2003) with Villin-Cre mice(Madison et al., 2002) (Fig. 3A).PCR of gDNA from whole jejunum (a mixed tissue sample

containing non-genetically modified smooth muscle and vasculatureendothelial cells in addition to intestinal epithelial cells lackingCAV1) shows evidence that Cre recombination occurs in the intestineof CAV1IEC-KO mice but not in wild-type (WT) Cav1fl/fl littermates

(Fig. 3B). Real-time PCR of jejunum (tissue includes epithelial cellsas well as submucosa and muscle) demonstrates that Cav1 mRNA isdecreased ∼70% in CAV1IEC-KO mice relative to Cav1fl/fl WT(Student’s t-test, P=0.01; Fig. 3C) and not expressed in the jejunumof negative control CAV1KO mice (data not shown). Cav2 mRNA isalso decreased in CAV1IEC-KO mice, which have 75% less Cav2mRNA relative to Cav1fl/fl WT mice (Student’s t-test, P=0.01;Fig. 3D) (Drab et al., 2001; Razani et al., 2001). Western blot showsCAV1 protein expression is 55% lower in CAV1IEC-KO mousejejunum than in Cav1fl/fl WT littermates (Fig. 3E) and that CAV1 islost in the jejunum of negative control CAV1KO mice (Fig. 3F).

Loss of intestinal epithelial CAV1 does not affect body massor glucose metabolismHigh-fat diet (HFD) increases the body mass of male and femalemice relative to mice fed a low-fat diet (LFD), but CAV1IEC-KO doesnot protect against diet-induced obesity (16 weeks on diet,22 weeks old, linear regression; males: F=37.7689, P<0.0001 fordiet; females: F=33.8792, P<0.0001 for diet; Fig. 3G,H). Fastingplasma glucose (Fig. S1A), glucose tolerance (Fig. S1B,C) andinsulin tolerance (Fig. S1D) are similarly unaffected by genotype.Thus, neither body mass nor glucose metabolism influence thefollowing changes in lipid metabolism.

Deletion of intestinal epithelial CAV1 alters cholesterollevelsDeletion of CAV1 in the intestinal epithelia results in changes infasting and postprandial circulating total and lipoprotein cholesterol,especially when mice were challenged by 16 weeks HFD.Compared with Cav1fl/fl WTs, male CAV1IEC-KO mice have agreater increase in total and esterified cholesterol upon HFDtreatment relative to LFD controls (increase in total cholesterol:62.9 mg/dl CAV1IEC-KO vs 24.3 mg/dl WT, two-way ANOVA,effect of diet: F1,24=8.055, P=009; increase in esterified cholesterol:54.4 mg/dl CAV1IEC-KO vs 19.5 mg/dl WT, two-way ANOVA,effect of diet: F1,23=7.610, P=0.01; Fig. 4A). A similar trend forgreater total cholesterol is observed in postprandial male micemaintained on chow diet (CD), but only free cholesterol issignificantly elevated by these conditions (34.6 mg/dl WT vs49.1 mg/dl CAV1IEC-KO increase, Student’s t-test, P<0.05; Fig. 4C).It is well established that plasma lipids can show sexualdimorphism, and here, the effects of intestinal CAV1 on plasmacholesterol are sexually dimorphic: Cav1fl/fl WT females havegreater HFD-associated elevations in total cholesterol (10.8 mg/dlCAV1IEC-KO vs 55.1 mg/dl WT, two-way ANOVA, effect of diet:F1,26=5.881, P=0.02) free cholesterol (1.7 mg/dl CAV1IEC-KO vs10.9 mg/dl WT, two-way ANOVA, effect of diet: F1,26=5.075,P=0.03), and esterified cholesterol compared with CAV1IEC-KO

females (9.2 mg/dl CAV1IEC-KO vs 44.3 mg/dl WT, two-wayANOVA, effect of diet: F1,26=4.707, P=0.04; Fig. 4B).

Intestinal epithelial CAV1 deletion protects against HFD-induced LDL cholesterol increaseMale CAV1IEC-KO mice are protected against the HFD-associatedincrease in fasted (4 h) plasma low-density lipoprotein cholesterol(LDL-C) seen in Cav1fl/fl WT littermates (HFD WT: 35.5 mg/dl,5.5 mg/dl greater than LFD; HFD CAV1IEC-KO: 23.7, 6.3 mg/dl lessthan LFD; two-way ANOVA, significant interaction between diet andgenotype, F1,24=5.261, P=0.03; Fig. 5A), though female mice do notexhibit this protection (Fig. 5B). Conversely, postprandial LDL-C iselevated in male CAV1IEC-KO mice (23.4 mg/dl) compared with WT(16.1 mg/dl; Student’s t-test, P<0.05; Fig. 5C). HDL-C is also altered

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in CAV1IEC-KO mice, as female mice have a greater HFD-inducedincrease in HDL-C relative to LFD in CAV1IEC-KO mice (13.3 mg/dlfor males, 17.1 mg/dl females) compared with WT (9.6 mg/dl formales, 0.2 mg/dl for females; two-way ANOVA, effect of diet:F1,28=9.516, P=0.005, and interaction between diet and genotypeF1,28=5.684, P=0.024; Fig. 5A,B).

Intestinal epithelial CAV1 deletion increases plasma freefatty acidsLoss of intestinal epithelial CAV1 increases plasma non-esterified fattyacids (NEFAs) in fasted male HFD CAV1IEC-KO mice (1.43 mmol/l)compared with HFD Cav1fl/fl WT littermates (0.94 mmol/l; two-wayANOVA, effect of diet: F1,23=13.15, P=0.0014; Fig. 6A). NEFAsare also greater in postprandial CD-fed male CAV1IEC-KO mice(0.42 mmol/l) than WT mice (0.27 mmol/l; Student’s t-test, P<0.05;Fig. 6C). Moreover, serum NEFAs decrease less in CD-fed maleCAV1IEC-KO mice upon feeding (0.44-fold decrease) than WT malemice (0.83-fold decrease; Student’s t-test, P<0.05).

HPLC lipidomicsTo investigate changes in the levels of specific species ofplasma NEFAs and cholesteryl esters we performed a lipidomicsstudy. Using HPLC charged aerosol detection (HPLC-CAD), wemeasured the relative levels of several major plasma lipids includingeight FA species, free cholesterol, two cholesteryl esters (cholesteryloleate and palmitate), and five putative lysophospholipids in theplasma of CAV1IEC-KO and Cav1fl/fl WTmale mice (16 weeks HFDor LFD, 26 weeks old, 4 h fast). Strikingly, palmitoleic acid (16:1)is altered in the same pattern by diet and genotype as total NEFAs(two-way ANOVA, effect of diet: F1,20=41.70, P<0.0001; effect ofgenotype: F1,20=5.438, P=0.03; Fig. 6E). Similarly, palmitic acid(16:0) is significantly affected by both diet and the interactionbetween diet and genotype (two-way ANOVA, effect of diet:F1,20=10.77, P=0.0037; interaction effect: F1,20=5.388, P=0.031;Fig. 6E). Stearic acid (18:0) is significantly elevated by HFDcompared with LFD in CAV1IEC-KO mice (two-way ANOVA,F1,20=11.95, P=0.0025; Fig. 6E). Oleic acid (18:1) and linolenic

Fig. 2. Fluorescently labeled endocytic cargos enable imaging of caveolar endocytosis in the intact zebrafish intestine. (A) Representative images showthat the caveolar-specific cargos Alexa Fluor–albumin and BODIPY–d-LacCer are internalized from the basolateral PM of enterocytes, but not the intestinallumen. In contrast, the cargo transported specifically by clathrin-coated vesicles, BODIPY–l-LacCer, is transported into enterocytes from both the basolateral andluminal PMs. BB, brush border; L, lateral membrane; B, basolateral membrane; I, intracellular; N, nucleus; arrowhead, intracellular puncta. (B) The meanfluorescence intensity of Alexa Fluor–albumin and BODIPY–d-LacCer on the lateral PM of enterocytes is significantly greater following basolateral injectioncompared to luminal injection. In contrast, the mean fluorescence intensity of BODIPY–l-LacCer on the lateral PM of enterocytes is the same following basolateraland luminal injection. Data is presented relative to lateral PM mean fluorescence intensity following luminal injection. Mean±s.e.m, n=3: nine fish per replicate,three areas of each region per fish; Student’s t-test; *P<0.05.

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Fig. 3. Deletion of Cav1 from mouse intestinal epithelial cells (CAV1IEC-KO). (A) Schematic representation of deletion of Cav1 in the intestinal epithelium.(B) PCRof genomic DNA fromwholemouse jejunum shows that Cre recombination ofCav1 has occurred in CAV1IEC-KO jejunum but not inCav1fl/flWT littermates.(C) Cav1 mRNA is decreased 68% in CAV1IEC-KO mouse jejunum as evidenced by RT-PCR (mean, Student’s t-test, *P=0.01, n=10). (D) Cav2 mRNA isdecreased 75% in CAV1IEC-KO mouse jejunum as evidenced by RT-PCR (mean, Student’s t-test, *P=0.01, n=10). (E,F) CAV1 protein is reduced in the jejunum ofCAV1IEC-KOmice asmeasured by western blot (F) and normalized to α-tubulin. (E) Data are expressed relative toCav1fl/flWTCAV1 protein, n=3western blots, fiveWT and five CAV1IEC-KO mice per blot, Student’s t-test; *P<0.05. (G,H) Body mass of male (G) and female (H) mice; mice were fed a low-fat (10%) or high-fat(60%) diet starting at 10 weeks (n=10-15). HFD mice had significantly higher body mass than LFD mice, but loss of intestinal epithelial cell CAV1 did not affectbody mass. Mean±s.e.m, linear regression; *P<0.05.

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acid (18:3) are both higher in LFD than HFD fed mice (diet effectfound by two-way ANOVA, oleic acid: F1,20=7.664, P=0.0119;linolenic acid: F1,20=28.72, P<0.0001; Fig. 6E). Neither diet norgenotype affects any of the other FAs measured (22:6, 20:4 and18:2). In sum, palmitoleic, palmitic, stearic and oleic acid mightcontribute to the observed elevation of total NEFAs in LFD micecompared with HFD mice.As described above, cholesterol assay kits found that diet affects

total and esterified plasma cholesterol in CAV1IEC-KO mice (Fig. 4).HPLC analysis identified a direct contribution of free cholesterol(two-way ANOVA, effect of diet: F1,20=8.693, P=0.008) andcholesteryl palmitate (two-wayANOVA, effect of diet:F1,20=4.800,

P=0.04), which are both increased by HFD (Fig. 6E). Finally, oneputative lysophospholipid (peak 10) that could not be identifiedwith standards is also increased by HFD (two-way ANOVA, effectof diet: F1,20=33.25, P<0.0001; Fig. 6E).

To investigate the mechanism underlying the elevation in plasmaNEFAs in male HFD CAV1IEC-KO relative to male HFD WT mice,liver and white adipose lipids were also measured by HPLC in maleHFD mice. Four of the FAs that were decreased in CAV1IEC-KO

plasma – palmitoleic, palmitic, stearic, oleic and linolenic acids –were also significantly decreased in the liver (Fig. 6F). It is strikingthat all of the FAs that are decreased in the liver of CAV1IEC-KO

mice, with the exception of stearic acid, have a trend to be increased

Fig. 4. Loss of CAV1 in the intestinal epithelia alters plasma cholesterol levels. (A) In male mice, total and esterified plasma cholesterol are elevated by HFDcompared with LFD in WT, but not CAV1IEC-KO (IKO), mice following a 4 h fast (n=5-8). (B) Conversely, for female mice, total, free, and esterified plasmacholesterol are elevated by HFD compared with LFD in 4 h fasted CAV1IEC-KO, but not WT, mice (n=8-9). (C) Postprandial male CAV1IEC-KO mice fed CD havegreater plasma-free cholesterol mice than WT (n=6-8). Data are mean±s.e.m, two-way ANOVA, *P<0.05; groups with different brackets show an effect of diet,groups with different letters are significantly different by post hoc testing.

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in the plasma where total NEFAs are increased. Additionally,hepatic cholesterol is lower in HFD CAV1IEC-KO mice (Fig. 6F). Nodifferences were observed in triglycerides, cholesterol esters, or,similar to plasma, 22:6, 20:4 or 18:2 in the liver of HFD mice (datanot shown). Of all these lipids, only cholesteryl oleate varied in thewhite adipose tissue, showing a decrease in CAV1IEC-KO mice(Fig. 6G; data for other lipids not shown).

DISCUSSIONThe global obesity epidemic has caused an explosion in theprevalence of metabolic diseases such as type II diabetes andcardiovascular disease. Intensive efforts have focused on theidentification of therapeutic targets to better prevent and treatmetabolic syndrome. Previous work had identified functional rolesof adipocyte and endothelial CAV1 in susceptibility to diet-inducedobesity (Razani et al., 2002), insulin resistance (Cohen et al., 2003)and atherosclerosis (Frank et al., 2004). In this study, we expandedupon these findings by using zebrafish larvae to visualize thelocalization of enterocyte Cav1 and caveolar endocytosis, and aknockout mouse model to identify a role for CAV1 in the intestinalepithelia in the regulation of plasma FAs and LDL cholesterol,

lipids that contribute to the development of several metabolicdiseases.

Although it is known that CAV1 is expressed in the intestinalepithelium (Field et al., 1998), the intracellular localization of thisprotein has remained ambiguous: lateral PM localization in Caco-2colon-derived cells (Vogel et al., 1998); either the brush border orlateral and basolateral PM localization in Caco-2 cells depending onthe fix, permeabilization method, and antibody used (Field et al.,1998); lateral membrane of T84 colon-derived cells (Badizadeganet al., 2000); cytoplasmic vesicles and lateral PM at adherens andtight junctions in mouse jejunum (Marchiando et al., 2010); lowlevels on the mouse small intestine brush border (McConnell et al.,2011); cytoplasmic vesicles in mouse colon (Nabeyama andLeblond, 1974); deep apical tubules in pig small intestine(Hansen et al., 2003); human small intestine cytoplasmic vesicles(Morroni et al., 2007); and apical PM and cytoplasmic vesicles inCaenorhabditis elegans intestine (Parker et al., 2009). Here, ourin vivo study reveals that Cav1 localizes asymmetrically to lateraland basolateral enterocyte PM in larval zebrafish. CAV1 is aFA- and cholesterol-binding protein and caveolae can only form inlipid-rich PM regions. Although these lipid-rich regions do form on

Fig. 5. Lipoprotein cholesterol levelsare affected by loss of CAV1 in theintestinal epithelia. (A,B) MaleCAV1IEC-KO (IKO) mice are protected fromHFD-induced increase in fasted plasmaLDL cholesterol (n=6-10, mean±s.e.m,two-way ANOVA, significant interactionbetween diet and genotype) (A), butfemale mice are not (n=8-9) (B). FemaleCAV1IEC-KO, but notWT, mice have higherfasted plasma HDL cholesterol on HFDthan LFD. Mean±s.e.m, two-way ANOVA,groups with different letters aresignificantly different by post hoc testing.(C) CAV1IEC-KO mice on CD have higherpostprandial plasma LDL cholesterol thanWT mice. Mean±s.e.m, Student’s t-test,*P<0.05, n=6-9.

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Fig. 6. See next page for legend.

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the enterocyte brush border (Hansen et al., 2001), this membranehas a lipid composition distinct from the lateral and basolateral PM,with more glycolipids and less cholesterol and sphigomyelin(Danielsen and Hansen, 2006). It is possible that the relative scarcityof cholesterol in the brush border excludes CAV1 and caveolae fromthis PM. Unfortunately, the cellular localization of mouse CAV1 inthe intestinal epithelia could not be determined because of non-specific antibody binding that we observed in enterocytes.Fluorescently labeled endocytic cargos are valuable tools to

visualize various types of endocytosis in cultured cells (Singh et al.,2006, 2007). Here, we extended this technology to image theenterocyte PM regions that likely perform caveolae-mediatedendocytosis in vivo. The results suggesting that caveolae-mediatedendocytic activity in enterocytes is asymmetric points to a role forcaveolae in vesicular transport between enterocytes and the body, butnot the intestinal lumen. The optical clarity of larval zebrafish andadvances inmouse vital imaging present the opportunity to extend thistechnique to investigations of caveola- and clathrin-mediatedendocytosis in amultitude of tissues in the context of health or disease.Prior studies have reported increased fasting plasma cholesterol in

male CAV1KO mice, largely consisting of elevations in VLDL andLDL cholesterol resulting from adipocyte lipid storage defects anddecreased aortic and hepatic LDL cholesterol uptake (Frank et al.,2008; Valasek et al., 2005). Our studies find that the intestinalepithelium does not mediate these changes, as we did not observedifferences in plasma cholesterol or cholesterol palmitate inCAV1IEC-KO mice. However, we did find that LDL cholesterollevels were increased postprandially and decreased duringfasting (when hepatic cholesterol is also decreased) in male HFDCAV1IEC-KO mice; this is opposite of the pattern observed inCAV1KOmice. Another striking observation is that male CAV1IEC-KO

mice have increased fasting plasma NEFAs (HFD), mirrored bydecreases in several hepatic FAs, and decreased postprandial NEFAs(CD). The literature regarding the effect of total body CAV1 deletionon circulating FFAs is conflicting, with reports of higher fasting andpostprandial FFAs (Asterholm et al., 2012; Razani et al., 2002),decreased fasting FFAs (Cohen et al., 2004), or no changes (Valaseket al., 2005).First, we hypothesized that the changes in LDL cholesterol and

FFAs could be mediated by the cluster of differentiation 36scavenger receptor (CD36). CD36 delays LDL cholesterol clearance(Luangrath et al., 2008), so impairment of its function in CAV1IEC-KO

mice could accelerate LDL cholesterol clearance, causing theobserved decrease in fasting LDL cholesterol. Increasedpostprandial LDL cholesterol could also be explained, as CD36facilitates cellular cholesterol uptake (Nassir et al., 2007).Additionally, FFAs could be increased due to impaired CD36

localization and function, as CD36 facilitates FA uptake (Nassiret al., 2007; Pepino et al., 2014) and its deletion increases serumFFAs (Febbraio et al., 1999). CD36 is highly expressed in theproximal intestine (Poirier et al., 1996) and CAV1 is necessary forCD36 to properly localize to the PM of mouse embryonicfibroblasts (Ring et al., 2006). Therefore, we speculated thatCD36 PM localization and or expression might be disregulated inCAV1IEC-KO mice. However, contrary to the previous findings inmouse embryonic fibroblasts, no change in CD36 localization wasobserved in the intestinal epithelia of HFD-treated CAV1IEC-KO

mice (Fig. S2A) and no decreases in mRNA or protein expressionwere observed (data not shown). In support of this conclusion, itwould be expected that hepatic cholesterol would increase if theCD36-medated LDL cholesterol clearance delay was perturbed, butinstead HPLC analysis found a decrease in hepatic cholesterol.

Second, changes in circulating FFAs in CAV1IEC-KO mice couldbe mediated by changes in intestinal albumin uptake. CirculatingFFAs are transported by albumin, as much as 18% of which isabsorbed and catabolized by the intestine (Yedgar et al., 1983).Indeed, we showed that Alexa Fluor–albumin is internalized bycaveolae on the basolateral PM of enterocytes. If this were true aconcomitant increase in plasma albumin would be expected;however, this was not observed (Fig. S2B). As circulatingalbumin levels are tightly regulated, this hypothesis cannot beruled out because of a lack of direct measurement of albumin fluxfrom the liver. Nonetheless, in further support of our conclusion,increased postprandial plasma lipids likely do not result from alteredintestinal processing because no changes in serum NEFAs or TGswere observed by an oral lipid tolerance test (Fig. S3).

Third, we hypothesized that CAV1IEC-KO mice might havedecreased intestinal insulin signaling, and thus insulin stimulatedplasma FA uptake, underlying the observed increase in circulatingNEFAs. This hypothesis is supported by the fact that insulinsignaling is present in the intestine (Veilleux et al., 2014), theinsulin receptor localizes to caveolae (Gustavsson et al., 1999), andthat global CAV1 knockout mice are insulin resistant (Cohen et al.,2003). Substantiating this hypothesis, insulin receptor mRNA issignificantly decreased in the jejunum of CAV1IEC-KO micecompared with controls (Fig. S4), but no change in insulinreceptor subunit β protein was observed by western blot (data notshown). Importantly, there might be a non-IEC-autonomousmechanism (insulin signaling or otherwise) by which hepatic FAuptake is decreased or secretion is increased leading to the observeddecrease in several hepatic FAs and corresponding increase incirculating NEFAs. Similarly, loss of CAV1 in IEC might underlie anon-cell-autonomous mechanism by which hepatic cholesterol isdecreased, limiting the amount of LDL cholesterol that can besecreted, causing the decrease in LDL cholesterol observed inCAV1IEC-KO mice.

HPLC determined that palmitoleic and palmitic acid contribute tothe overall increase in fasting NEFAs observed in HFDCAV1IEC-KO

mice. Palmitoleic acid is an omega-7 mono-unsaturated FA that isenriched in endothelial caveolae (Gafencu et al., 1998). Ifpalmitoleic acid is also preferentially carried in enterocytecaveolae, it follows that loss of CAV1 could result in decreasedpalmitoleic acid uptake, and the observed plasma elevation. Furtherinvestigation of the detailed mechanism would be of interest aspalmitoleic acid is a bioactive lipid; dietary supplementation hasplasma-lipid-lowering, anti-diabetic, and anti-inflammatory activity(Cao et al., 2008), but elevated plasma palmitoleic acid is correlatedwith increased heart failure (Djousse et al., 2012) and non-alcoholicfatty liver disease (Puri et al., 2009). It is possible that no influence

Fig. 6. Intestinal CAV1 deletion alters circulating FFAs but not TGs. (A) In4 h fasted male mice, plasma NEFAs are higher in HFD-fed CAV1IEC-KO (IKO)mice than WT mice. Mean±s.e.m, n=5-8; two-way ANOVA, means withdifferent letters are significantly different by post hoc testing. (B) Female miceshowed no changes in fasted plasma TGs or NEFAs (n=8-9). (C,D) NEFAs arealso higher in postprandial plasma of male CDCAV1IEC-KOmice comparedwithWT controls (C) and show a greater fold decrease upon feeding relative tofasting (D). Mean±s.e.m, n=6-8; Student’s t-test, *P<0.05. (E) Lipids measuredby HPLC in plasma of male mice fasted 4 h. There are significant effects of dietfor all and, for palmitoleic and palmitic acid, the interaction between diet andgenotype. Mean±s.e.m, n=6; two-way ANOVA, means with different letters aresignificantly different by post hoc testing, means with different brackets showonly a diet effect. (F) Lipids measured by HPLC in liver of male mice fasted 4 h.Mean±s.e.m, n=5; Student’s t-test, *P<0.05. (G) Cholesteryl oleate measuredby HPLC in white adipose tissue of male mice fasted 4 h. Mean±s.e.m, n=5;Student’s t-test, *P<0.05.

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of genotype on plasma palmitoleic acid levels in LFD mice wasfound simply because it comprises a much smaller proportion of thediet (0.04% of total LFD vs. 0.44% of total HFD by mass).Caveolae are also enriched in palmitic acid (Cai et al., 2013) soplasma palmitic acid might in higher in CAV1IEC-KO than WT micedue to the mechanism proposed above. As CAV1 is palmitoylated(Dietzen et al., 1995), decreased activity in this pathway mightcontribute to the plasma palmitic acid elevation.Finally, the sexual dimorphism observed in the changes in plasma

lipids in CAV1IEC-KO mice must be addressed. It is well establishedthat male and female animals have metabolic differences; inC57BL/6J mice, this includes differences in lipid and FAmetabolism gene expression in multiple tissues (Yang et al.,2006). Moreover, sex-specific metabolic differences have beenobserved previously in global CAV1 KO mice; decreased bodymass on chow diet compared with WT in male, but not female, miceand increased food intake relative to WT in female, but not male,mice (Razani et al., 2002). However, neither sex had increasedcirculating free FAs compared with WT, and LDL cholesterol wasonly measured in male mice, where it was increased compared withWT (Razani et al., 2002). In contrast to this previous study, wereport that CAV1IEC-KO male, but not female, mice were protectedfrom the HFD-induced increase in plasma LDL cholesterolobserved in WT. Although the mechanism for this difference isunknown, it is possible that sexual dimorphism in estrogen receptorsignaling underlies this difference. When estrogen binds theestrogen receptor α (ERα) transcription factor, ERα activatestranscription of the LDL receptor (LDLR), which takes up LDLcholesterol from circulation (Cooper et al., 1987). CAV1 has beenshown to potentiate the action of ERα in cultured cells (Schlegelet al., 1999). Additionally, female mice of mixed geneticbackground (129/Pas×C57BL/6J, ∼90% C57BL/6J) have higherLdlr and Cd36 hepatic gene expression compared with males(Lorbek et al., 2013). Therefore, because estrogen levels are higherin females, differences in estrogen-stimulated LDLR transcriptionand subsequent cellular uptake might underlie the observed sex-specific differences in LDL cholesterol in CAV1IEC-KO mice.Furthermore, female CAV1IEC-KO mice had significantly increasedHDL cholesterol on HFD compared with LFD; this difference wasnot observed in WT females, and the trend was non-significant inmales. These findings are consistent with Link et al.’s report on therole of sex chromosomes that mice with female (XX) chromosomeshave higher HDL cholesterol levels than male (XY) mice (Linket al., 2015). The observed sexual dimorphism does not precludeintestinal epithelial CAV1 as a potential therapeutic target formetabolic disease as many pharmaceuticals currently in use havesexually dimorphic actions, including the LDL cholesterol-loweringdrug fenofibrate (Yoon et al., 2002). Nor does the modest decreasein LDL cholesterol preclude intestinal epithelial CAV1 as atherapeutic target, as even a 1% reduction in LDL cholesterolleads to a 1% decrease in cardiovascular disease risk (Baigent et al.,2005).In conclusion, this study demonstrated that CAV1 localizes to,

and caveolar endocytosis occurs on, the lateral and basolateral PMof intestinal enterocytes. Although the detailed cellular mechanismremains to be elucidated, it is clear that CAV1 on enterocytebasolateral membranes influences circulating levels of LDLcholesterol and NEFAs, specifically palmitoleic and palmiticacid. Elevated plasma FAs and LDL cholesterol are associatedwith metabolic disease, including type II diabetes andcardiovascular disease. The results of this study indicate thatCAV1 in the intestinal epitheliummight serve as a therapeutic target

to lower circulating FFAs and LDL cholesterol and prevent diseasein males.

MATERIALS AND METHODSGeneration of transgenic zebrafishZebrafish research was approved by the Carnegie Institution Department ofEmbryology IACUC Committee (protocol #139). Zebrafish were housed at28°C with a 14 h:10 h light:dark cycle. Tg(hsp70l:cav1-eGFP) zebrafishwere created with the tol2-Gateway system (Kwan et al., 2007). Zebrafishcav1 was cloned from Image Consortium plasmid #3719638 (fb95c12) andGateway cloning constructed hsp70l:cav1-eGFP: the zebrafish heat shockcognate 70-kd protein, like (hsp70l) promoter driving cav1 tagged witheGFP [provided by Chi-bin Chien (Kwan et al., 2007)]. This plasmid wasinjected with tol2 transposase for genome integration with a microforgedglass needle (P-97 Flaming/Brown micropipette puller, Stutter Instruments,Novato, CA) connected to a nitrogen gas pressure injector (PLI 100,Harvard Apparatus, Cambridge, MA) into 1-2-cell zebrafish embryos (ABbackground). F0 larvae were heat shocked [45 min, 37°C, in 15 ml embryomedia (EM)] at 6 days post-fertilization (6 dpf) and screened for mosaicCav1-eGFP expression 4-6 h later. Three independent stable lines wereestablished and all further experimentation was undertaken in stabletransgenic animals.

Live imaging of zebrafish larvae and quantification offluorescenceApproximately 24 h prior to imaging, larvae were heat shocked as describedabove to induce Cav1-eGFP expression. Larvae were anesthetized withtricaine (Sigma-Aldrich, St Louis, MO) and mounted in 3% methylcellulose (Sigma-Aldrich) under a coverslip (Carten et al., 2011). LiveTg(hsp70:cav1-eGFP) larvae were imaged on a SP-2 confocal microscope(Leica Microsytems, Deerfield, IL) with an argon laser under a 63× oilimmersion objective. Images were collected as 12 bit and analyzed withMetamorph software (Molecular Devices, Sunnyvale, CA). Regions of thebrush border, lateral and basolateral PM, as well as intracellular regions,were outlined for quantification of mean fluorescence intensity (threeregions of interest for each cellular area, three images per fish, three fish perexperiment, for a total of three experiments or nine fish).

Electron microscopy and immunofluorescenceLarval (6 dpf) zebrafish and intestines from adult zebrafish and werecollected for EM. Prior to euthanasia adult fish were fed on a lipid-rich,hard-boiled chicken egg yolk for 1 h ad lib; larvae studied werelecithotrophic (6 dpf) and thus were not provided exogenous food. EMsamples were fixed in 3% glutaraldehyde (Electron Microscopy Sciences,Hatfield, PA) and 1% formaldehyde, post-fixed in reduced osmium(Electron Microscopy Sciences), stained with uranyl acetate (FisherScientific), embedded in Epon 812 resin (Ladd Research Industries,Williston, VT), and imaged on a Technai-12 electron microscope (FEI,Hillsboro, OR) with a 794 multiscan camera (Gatan, Pleasanton, CA).

Mouse CAV1 IF was attempted on paraffin sections and cryosections ofjejunum collected after a 4 h fast, but non-specific fluorescence wasobserved in enterocytes, even in the negative control, global CAV1 KOmice. The antibodies tested at a range of dilutions were: BD Biosciences/Transduction Labs #610059, 610057 and 610406, Santa CruzBiotechnology #sc-894, Abcam #ab2910, and Cell Signaling #3238 s.The antigen retrieval methods tested were Tris-EDTA buffer, sodium citratebuffer, and Diva Decloaker (Biocare Medical, Concord, CA).

Imaging fluorescently labeled endocytic cargos in vivoWT Larvae (6 dpf) were anesthetized in tricaine and mounted in 1.2% low-melt agarose (Sigma-Aldrich) in EM. A microforged glass needle was loadedwith 4,4-difluoro-4-bora-3a, 4a-diaza-S-indacene-l-threo-lactosylceramide(BODIPY–l-LacCer) (provided by David Marks, Thoracic DiseaseResearch Unit, Mayo Clinic and Foundation, Rochester, MN, USA) orBODIPY–d-erythro-LacCer (#895279, Invitrogen) at a concentration of2.5 μg/μl in 30% ethanol and 70% embryo media, or Alexa Fluor 594–albumin (#A13101, Fisher Scientific, Pittsburg, PA) at a concentration of

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5 μg/μl in PBS. A nitrogen-pressured injection rig was used to inject 2 nl tothe basolateral side of the intestine or into the intestinal lumen. Larvae werefreed from the agarose, allowed to recover for 30 min (LacCer) or 1 h(albumin) while swimming freely, re-anesthetized, mounted in 3% methylcellulose under a coverslip, placed on ice to stop endocytosis (Bourseau-Guilmain et al., 2016), imaged, and the same sample size was analyzed withMetamorph software as described above.

Breeding CAV1IEC-KO miceMouse research was approved by the Carnegie Institution Department ofEmbryology IACUC Committee (protocol #156). Mice were housed at20-21°C with a 12 h:12 h light cycle. Mice with a floxed Cav1 allele(provided byMichael Elliot; Cao et al., 2003) were crossed with Tg(Vil-cre)997Gum/J mice (#004586, Jackson Labs, Bar Harbor, ME), which expressCre recombinase specifically in the intestinal epithelium, producing micelacking Cav1 specifically in the intestinal epithelium (CAV1IEC-KO, backcrossed to C57BL/6 6 times; Fig. 1A). All experiments were performed withCAV1IEC-KO versus Cav1fl/fl (WT) littermates.

Mice were genotyped for floxed Cav1 (350 bp band following PCR withF1: TTC TGT GTG CAA GCC TTT CC and R1: GTG TGC GCG TCATAC ACT TG) and Vil-Cre PCR (1100 bp band following PCR with F:GTG TGG GAC AGA GAACAA ACC and R: ACATCT TCA GGT TCTGCG GG). The occurrence of Cre recombination in the intestine ofCAV1IEC-KO mice was verified by the presence of a 350 bp band (F1 primerabove, R2: GGG GAG GAG TAG AAG GTG GC; Cao et al., 2003) ongenomic DNA isolated from whole jejunum segments by NaOH extraction(Truett et al., 2000). Cav1tm1Mls (CAV1KO) mice lacking functional Cav1 inall tissues were used as controls (#007083, Jackson Labs, Bar Harbor, ME).

Real-time PCRCav1 andCav2mRNA levels were measured in whole jejunum by RT-PCR.mRNA was isolated with Trizol (Invitrogen), cDNA was synthesized withAMV reverse transcriptase (New England Biolabs, Ipswich, MA), andRT-PCR was run with the PrimePCR SYBR Green assay for mouse Cav1and Cav2 according to the manufacturer’s instructions (#100-25636,Bio-Rad Laboratories, Hercules, CA).

Western blotCAV1 protein was measured by western blot as previously described (Otiset al., 2011). Briefly, cytosolic fractions of jejunum were run on denaturingSDS-PAGE gels with a Mini-Protean system (Bio-Rad Laboratories),transferred to nitrocellulose, blocked with blocking grade blocker (Bio-RadLaboratories), and probed with antibodies to CAV1 (#610059, BDTransduction Labs, Lexington, KY; 1:5000 dilution), α-tubulin (#T6199,Sigma-Aldrich; 1:10,000 dilution), goat anti-rabbit IgG–HRP (#170-6515,Bio-Rad Laboratories; 1:5000 dilution), and goat anti-mouse IgG–HRP(#170-6516, Bio-Rad Laboratories; 1:10,000 dilution). Protein expressionwas imaged by chemiluminescence (SuperSignal West PicoChemiluminescent Substrate, Pierce, Rockford, IL). Data from threewestern blots, with five WT and five CAV1IEC-KO mice each, wereanalyzed with ImageJ software (NIH, Bethesda, MD).

Experimental dietsUpon weaning, mice were fed CD ad libitum (#7012, Harlan Teklad,Fredrick, MD). At 10 weeks of age, mice were separated randomly intogroups and either continued on CD, or fed a 60% HFD or 10% fat, nutrient-matched LFD (#D12492 and #D12450J respectively, Research Diets, NewBruswick, NJ) ad libitum. Mice fed HFD and LFD were weighed weeklyuntil euthanasia at 26 weeks; n=10-15 mice per diet.

Plasma cholesterol and lipid analysisMice were euthanized by CO2 inhalation and blood was collected by cardiacpuncture. Serum was collected by centrifugation (15 min, 2000 g, 4°C) afterallowing blood to clot on ice. Plasmawas collected by centrifugation (15 min,2000 g, 4°C) in EDTA-treated tubes (Sarstedt, Numbrecht, Germany). Allserum and plasma samples were snap frozen and stored at −80°C. Kits wereused to measure total cholesterol (Infinity Total Cholesterol, FisherScientific), free cholesterol (Free Cholesterol E, Wako Diagnostics,

Richmond, VA), HDL cholesterol (HDL-C E, Wako Diagnostics), LDLcholesterol (LDL-C Reagent L-type, Wako Diagnostics), NEFAs [HR SeriesNEFA-HR(2), Wako Diagnostics], and TGs (Infinity Triglycerides, FisherScientific) according to the manufacturer’s directions. For HFD and LFDmale mice, n=5-8; for HFD and LFD female mice, n=8-9; and for CD malemice, n=6-8.

High-performance liquid chromatographyHPLC-CAD was implemented to measure levels of specific plasma lipids.Lipids were extracted from plasma (50 μl; n=6), liver (10-50 mg; n=5), andwhite adipose tissue (15-40 mg; n=5) using a modified Bligh–Dyerprocedure (Carten et al., 2011), dried under vacuum, and resuspendedin the injection solvent HPLC-grade 2-propanol. The investigator thatextracted the lipids blinded the samples from the investigator whocompleted the HPLC experimentation and analysis.

HPLC-CAD was performed using a LPQ-3400RS quaternary pump,WPS-3000TRS autosampler, TCC-3000RS column oven, Accucore C18column (150×3.0 mm, 2.1 μmparticle size) andDionex Corona Veo chargedaerosol detector (all from Thermo Scientific). Lipids were separated over80 min in a multistep gradient as follows: 0-5 min at 0.8 ml/min in 98.0%mobile phase A (methanol:water:acetic acid, 750:250:4) and 2.0% mobilephase B (acetonitrile:acetic acid, 1000:4); 5-35 min at 0.8-1.0 ml/min in30-98% A, 2-65% B and 0-5% mobile phase C (2-propanol); 35-45 min at1.0 ml/min in 0-30% A, 65-95% B and 5% C; 45-73 min at 1.0 ml/min in60-95% B and 5-40% C; and 73-80 min at 1.0 ml/min in 60% B and 40% C(adapted from personal correspondence with Marc Plante, ThermoScientific). Following the analytical portion of the gradient, the columnwas washed for 20 min with 100% mobile phase C at 0.4 ml/min, thenreturned to 98% A and 2% B at 0.8 ml/min for re-equilibration. Injectionvolumewas between 5 and 25 μl andwas adjusted for each sample to produceoptimum peak shape for quantitation. The autosampler tray was maintainedat 20.0°C and the column oven temperature at 40.0°C.

Chromatographic peaks were identified by comparison with standards(Sigma-Aldrich). Early-eluting peaks not identified as FAs were confirmedas polar lipids by acetone precipitation (adapted from Morris, 1972), andbased on their retention times when compared with those of polar lipidstandards, were determined to most likely be lysophospholipids (data notshown). Quantitative comparison of the relative amounts of lipid species ofinterest was performed blinded using Chromeleon 7.2 (Dionex, Sunnyvale,CA). Peak baselines were drawn manually and areas [in picoamperes(pA) min] were determined automatically. For plasma samples, all peakareas were divided by the area of the second lysophospholipid peak in thesame sample. This lipid was chosen as it varied the least among all of thesamples after accounting for injection volume. For comparison betweensamples, lipid amounts are expressed as (peak area)/(area of peak 2), bothmeasured in pA×min. For liver and white adipose samples, all peak areaswere normalized to percent of total signal to control for unequal extractionefficiencies and differences in starting tissue mass between samples.

Glucose metabolismBlood glucose was measured in mice fasted 4 h by tail bleed using aglucometer (AlphaTRAK glucose meter, VWR, Radnor, PA) just prior toeuthanasia (n=6-10). For intraperitoneal (IP) glucose tolerance test (GTT),mice fasted 4 h were IP-administered 2 g/kg sterilized glucose in water, andblood glucose was measured from the tail vein by glucometer at 0, 15, 30, 60and 120 min (n=9-14). For oral GTT, the same dose of sterile glucose wasadministered by gavage, and blood glucose was monitored as described(n=7-9). Insulin tolerance test (ITT) was performed by administering miceIP insulin (#12585-014, Gibco, Gaithersburg, MD) at a dose of 0.76 U/kgfor LFD mice and 1 U/kg for HFD mice; blood glucose was measured asdescribed for GTT (n=7-13). GTT and ITT results were analyzed by two-way, and oral GTT by one-way repeated measures ANOVA followed byTukey’s post hoc test.

Lipid tolerance testMale and female CAV1IEC-KO and WT littermates were housed on CD until12 weeks old, fasted 16 h, and gavaged with 10 μl/g body mass 20%emulsified Intralipid (soybean oil) (Sigma). Serum was collected from tail

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bleed at 0, 1, 2, 3 and 4 h post gavage. TGs and NEFAs were assayed asdescribed in ‘Plasma cholesterol and lipid analysis’ above.

StatisticsDifferences in Cav1-eGFP fluorescence intensities were compared with aone-way ANOVA followed by Tukey’s post hoc test. Student’s t-test wasused to compare endocytic cargo membrane fluorescence and RT-PCRmeasurements of jejunal mRNA. Western blot data from each of three blotswere adjusted by expression of the loading control and expressed relative toWT levels, averaged together, and compared by Student’s t-test. Differencesin body mass were compared by linear regression. Plasma lipids identifiedby HPLC were compared by two-way ANOVA followed by Tukey’s posthoc test. Liver and white adipose lipids measured by HPLC were comparedby Student’s t-test. All data analyzed by ANOVA were first confirmed forhomogeneity of variance by Bartlett’s or Brown–Forsythe tests. Statisticswere performed with Prism software (GraphPad, La Jolla, CA).

AcknowledgementsThe authors are grateful to Michael Elliot, David Marks, Michel Bagnat, and the lateChi-bin Chien for experimental reagents and animals, to Michael Wolfgang, GuangWilliam Wong, and Alex Bortvin for consultation on experimental design, to BlakeCaldwell, Eugenia Dikovsky, Amy Kowalski, Mike Sepanski, and Erin Zeituni fortechnical assistance, and to Nada Abumrad for IF advice.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: J.P.O and S.A.F.; Methodology: J.P.O., V.Q.R. and S.A.F.;Formal analysis: J.P.O, J.L.A. and V.Q.R.; Investigation: J.P.O., M.-C.S. and V.Q.R.;Writing−original draft: J.P.O. and V.Q.R.; Writing−review and editing: J.P.O., J.L.A.,S.A.F, M.C.S. and V.Q.R.; Funding acquisition: J.P.O. and S.A.F.

FundingThis study was funded by National Institute of Diabetes and Digestive and KidneyDiseases (F32DK096786 to J.P.O.; RO1DK093399 to S.A.F.; and RO1GM63904 toThe Zebrafish Functional Genomics Consortium: PI Stephen Ekker and Co-PIS.A.F). This content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institutes of Health. Additionalsupport for this work was provided by the Carnegie Institution of Washingtonendowment and the G. Harold and Leila Y. Mathers Charitable Foundation (S.A.F).

Supplementary informationSupplementary information available online athttp://dmm.biologists.org/lookup/doi/10.1242/dmm.027300.supplemental

ReferencesAsterholm, I. W., Mundy, D. I., Weng, J., Anderson, R. G. and Scherer, P. E.(2012). Altered mitochondrial function and metabolic inflexibility associated withloss of caveolin-1. Cell Metab. 15, 171-185.

Babin, P. J. and Vernier, J. M. (1989). Plasma lipoproteins in fish. J. Lipid Res. 30,467-489.

Badizadegan, K., Dickinson, B. L., Wheeler, H. E., Blumberg, R. S., Holmes,R. K. and Lencer, W. I. (2000). Heterogeneity of detergent-insoluble membranesfrom human intestine containing caveolin-1 and ganglioside G(M1).Am. J. Physiol. Gastrointest. Liver Physiol. 278, G895-G904.

Baigent, C., Keech, A., Kearney, P. M., Blackwell, L., Buck, G., Pollicino, C.,Kirby, A., Sourjina, T., Peto, R., Collins, R. et al. (2005). Efficacy and safety ofcholesterol-lowering treatment: prospective meta-analysis of data from 90,056participants in 14 randomised trials of statins. Lancet 366, 1267-1278.

Bourseau-Guilmain, E., Menard, J. A., Lindqvist, E., Indira Chandran, V.,Christianson, H. C., Cerezo Magan a, M., Lidfeldt, J., Marko-Varga, G.,Welinder, C. and Belting, M. (2016). Hypoxia regulates global membrane proteinendocytosis through caveolin-1 in cancer cells. Nat. Commun. 7, 11371.

Cai, Q., Guo, L., Gao, H. and Li, X. A. (2013). Caveolar fatty acids and acylation ofcaveolin-1. PLoS ONE 8, e60884.

Cao, G., Yang, G., Timme, T. L., Saika, T., Truong, L. D., Satoh, T., Goltsov, A.,Park, S. H., Men, T., Kusaka, N. et al. (2003). Disruption of the caveolin-1 geneimpairs renal calcium reabsorption and leads to hypercalciuria and urolithiasis.Am. J. Pathol. 162, 1241-1248.

Cao, H., Gerhold, K., Mayers, J. R., Wiest, M. M., Watkins, S. M. andHotamisligil, G. S. (2008). Identification of a lipokine, a lipid hormone linkingadipose tissue to systemic metabolism. Cell 134, 933-944.

Cao, J., Navis, A., Cox, B. D., Dickson, A. L., Gemberling, M., Karra, R., Bagnat,M. and Poss, K. D. (2016). Single epicardial cell transcriptome sequencingidentifies Caveolin 1 as an essential factor in zebrafish heart regeneration.Development 143, 232-243.

Carten, J. D. and Farber, S. A. (2009). A newmodel system swims into focus: usingthe zebrafish to visualize intestinal metabolism in vivo. Clin. Lipidol. 4, 501.

Carten, J. D., Bradford, M. K. and Farber, S. A. (2011). Visualizing digestive organmorphology and function using differential fatty acid metabolism in live zebrafish.Dev. Biol. 360, 276-285.

Chanthick, C., Kanlaya, R., Kiatbumrung, R., Pattanakitsakul, S. N. andThongboonkerd, V. (2016). Caveolae-mediated albumin transcytosis isenhanced in dengue-infected human endothelial cells: a model of vascularleakage in dengue hemorrhagic fever. Sci. Rep. 6, 31855.

Cohen, A. W., Combs, T. P., Scherer, P. E. and Lisanti, M. P. (2003). Role ofcaveolin and caveolae in insulin signaling and diabetes. Am. J. Physiol.Endocrinol. Metab. 285, E1151-E1160.

Cohen, A. W., Razani, B., Schubert, W., Williams, T. M., Wang, X. B., Iyengar, P.,Brasaemle, D. L., Scherer, P. E. and Lisanti, M. P. (2004). Role of caveolin-1 inthe modulation of lipolysis and lipid droplet formation. Diabetes 53, 1261-1270.

Cooper, A. D., Nutik, R. and Chen, J. (1987). Characterization of the esterogen-induced lipoprotein receptor in rat liver. J. Lipid Res. 28, 59-68.

Danielsen, E. M. and Hansen, G. H. (2006). Lipid raft organization and function inbrush borders of epithelial cells. Mol. Membr. Biol. 23, 71-79.

Dietzen, D. J., Hastings, W. R. and Lublin, D. M. (1995). Caveolin is palmitoylatedon multiple cysteine residues. Palmitoylation is not necessary for localization ofcaveolin to caveolae. J. Biol. Chem. 270, 6838-6842.

Djousse, L., Weir, N. L., Hanson, N. Q., Tsai, M. Y. and Gaziano, J. M. (2012).Plasma phospholipid concentration of cis-palmitoleic acid and risk of heart failure.Circ. Heart. Fail. 5, 703-709.

Dobrinskikh, E., Okamura, K., Kopp, J. B., Doctor, R. B. and Blaine, J. (2014).Human podocytes perform polarized, caveolae-dependent albumin endocytosis.Am. J. Physiol. Renal. Physiol. 306, F941-F951.

Drab, M., Verkade, P., Elger, M., Kasper, M., Lohn, M., Lauterbach, B., Menne,J., Lindschau, C., Mende, F., Luft, F. C. et al. (2001). Loss of caveolae, vasculardysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science293, 2449-2452.

Fang, P.-K., Solomon, K. R., Zhuang, L., Qi, M., McKee, M., Freeman, M. R. andYelick, P. C. (2006). Caveolin-1alpha and -1beta perform nonredundant roles inearly vertebrate development. Am. J. Pathol. 169, 2209-2222.

Febbraio, M., Abumrad, N. A., Hajjar, D. P., Sharma, K., Cheng, W., Pearce,S. F. A. and Silverstein, R. L. (1999). A null mutation in murine CD36 reveals animportant role in fatty acid and lipoprotein metabolism. J. Biol. Chem. 274,19055-19062.

Fernandez-Hernando, C., Yu, J., Suarez, Y., Rahner, C., Davalos, A.,Lasuncion, M. A. and Sessa, W. C. (2009). Genetic evidence supporting acritical role of endothelial caveolin-1 during the progression of atherosclerosis.Cell Metab. 10, 48-54.

Field, F. J., Born, E., Murthy, S. and Mathur, S. N. (1998). Caveolin is present inintestinal cells: role in cholesterol trafficking? J. Lipid Res. 39, 1938-1950.

Frank, P. G., Lee, H., Park, D. S., Tandon, N. N., Scherer, P. E. and Lisanti, M. P.(2004). Genetic ablation of caveolin-1 confers protection against atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 24, 98-105.

Frank, P. G., Pavlides, S., Cheung, M. W.-C., Daumer, K. and Lisanti, M. P.(2008). Role of caveolin-1 in the regulation of lipoprotein metabolism.Am. J. Physiol. Cell Physiol. 295, C242-C248.

Gabor, K. A., Kim, D., Kim, C. H. and Hess, S. T. (2015). Nanoscale imaging ofcaveolin-1 membrane domains in vivo. PLoS ONE 10, e0117225.

Gafencu, A., Stanescu, M., Toderici, A. M., Heltianu, C. and Simionescu, M.(1998). Protein and fatty acid composition of caveolae from apical plasmalemmaof aortic endothelial cells. Cell Tissue Res. 293, 101-110.

Ghitescu, L. and Bendayan, M. (1992). Transendothelial transport of serumalbumin: a quantitative immunocytochemical study. J. Cell Biol. 117, 745-755.

Ghitescu, L., Fixman, A., Simionescu, M. and Simionescu, N. (1986). Specificbinding sites for albumin restricted to plasmalemmal vesicles of continuouscapillary endothelium: receptor-mediated transcytosis. J. Cell Biol. 102,1304-1311.

Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M.,Lindroth, M., Peterson, K. H., Magnusson, K. E. and Stralfors, P. (1999).Localization of the insulin receptor in caveolae of adipocyte plasma membrane.FASEB J. 13, 1961-1971.

Hansen, G. H., Immerdal, L., Thorsen, E., Niels-Christiansen, L. L., Nystrøm,B. T., Demant, E. J. and Danielsen, E. M. (2001). Lipid rafts exist as stablecholesterol-independent microdomains in the brush border membrane ofenterocytes. J. Biol. Chem. 276, 32338-32344.

Hansen, G. H., Niels-Christiansen, L. L., Immerdal, L. and Danielsen, E. M.(2003). Scavenger receptor class B type I (SR-BI) in pig enterocytes: traffickingfrom the brush border to lipid droplets during fat absorption. Gut 52, 1424-1431.

Heimerl, S., Liebisch, G., Le Lay, S., Bottcher, A., Wiesner, P., Lindtner, S.,Kurzchalia, T. V., Simons, K. and Schmitz, G. (2008). Caveolin-1 deficiency

294

RESEARCH ARTICLE Disease Models & Mechanisms (2017) 10, 283-295 doi:10.1242/dmm.027300

Disea

seModels&Mechan

isms

alters plasma lipid and lipoprotein profiles in mice. Biochem. Biophys. Res.Commun. 367, 826-833.

Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell,D. S., Parant, J. M., Yost, H. J., Kanki, J. P. andChien, C.-B. (2007). The Tol2kit:a multisite gateway-based construction kit for Tol2 transposon transgenesisconstructs. Dev. Dyn. 236, 3088-3099.

Link, J. C., Chen, X., Prien, C., Borja, M. S., Hammerson, B., Oda, M. N., Arnold,A. P. and Reue, K. (2015). Increased high-density lipoprotein cholesterol levels inmice with XX versus XY sex chromosomes. Arterioscler. Thromb. Vasc. Biol. 35,1778-1786.

Lorbek, G., Perse, M., Horvat, S., Bjorkhem, I. and Rozman, D. (2013). Sexdifferences in the hepatic cholesterol sensing mechanisms in mice.Molecules 18,11067-11085.

Luangrath, V., Brodeur, M. R., Rhainds, D. andBrissette, L. (2008). Mouse CD36has opposite effects on LDL and oxidized LDL metabolism in vivo. Arterioscler.Thromb. Vasc. Biol. 28, 1290-1295.

Madison, B. B., Dunbar, L., Qiao, X. T., Braunstein, K., Braunstein, E. andGumucio, D. L. (2002). Cis elements of the villin gene control expression inrestricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axesof the intestine. J. Biol. Chem. 277, 33275-33283.

Marchiando, A. M., Shen, L., Graham, W. V., Weber, C. R., Schwarz, B. T.,Austin, J. R., II, Raleigh, D. R., Guan, Y., Watson, A. J. M., Montrose, M. H.et al. (2010). Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo. J. Cell Biol. 189, 111-126.

McConnell, R. E., Benesh, A. E., Mao, S., Tabb, D. L. and Tyska, M. J. (2011).Proteomic analysis of the enterocyte brush border. Am. J. Physiol. Gastrointest.Liver Physiol. 300, G914-G926.

Morris, K. (1972). Techniques of Lipidology: Isolation, Analysis, and Identification ofLipids. Elsevier: St Louis, MO.

Morroni, M., Cangiotti, A. M. and Cinti, S. (2007). Brush cells in the humanduodenojejunal junction: an ultrastructural study. J. Anat. 211, 125-131.

Murata, M., Peranen, J., Schreiner, R., Wieland, F., Kurzchalia, T. V. andSimons, K. (1995). VIP21/caveolin is a cholesterol-binding protein. Proc. Natl.Acad. Sci. USA 92, 10339-10343.

Nabeyama, A. and Leblond, C. P. (1974). “Caveolated cells” characterized by deepsurface invaginations and abundant filaments in mouse gastro-intestinal epithelia.Am. J. Anat. 140, 147-165.

Nassir, F., Wilson, B., Han, X., Gross, R. W. and Abumrad, N. A. (2007). CD36 isimportant for fatty acid and cholesterol uptake by the proximal but not distalintestine. J. Biol. Chem. 282, 19493-19501.

Nixon, S. J., Carter, A., Wegner, J., Ferguson, C., Floetenmeyer, M., Riches, J.,Key, B., Westerfield, M. and Parton, R. G. (2007). Caveolin-1 is required forlateral line neuromast and notochord development. J. Cell Sci. 120, 2151-2161.

Nusrat, A., Parkos, C. A., Verkade, P., Foley, C. S., Liang, T. W., Innis-Whitehouse, W., Eastburn, K. K. and Madara, J. L. (2000). Tight junctions aremembrane microdomains. J. Cell Sci. 113, 1771-1781.

Otis, J. P., Sahoo, D., Drover, V. A., Yen, C.-L. E. and Carey, H. V. (2011).Cholesterol and lipoprotein dynamics in a hibernating mammal. PLoS ONE 6,e29111.

Otis, J. P., Zeituni, E. M., Thierer, J. H., Anderson, J. L., Brown, A. C., Boehm,E. D., Cerchione, D. M., Ceasrine, A. M., Avraham-Davidi, I., Tempelhof, H.et al. (2015). Zebrafish as a model for apolipoprotein biology: comprehensiveexpression analysis and a role for ApoA-IV in regulating food intake. Dis. Model.Mech. 8, 295-309.

Parker, S., Walker, D. S., Ly, S. and Baylis, H. A. (2009). Caveolin-2 is required forapical lipid trafficking and suppresses basolateral recycling defects in the intestineof Caenorhabditis elegans. Mol. Biol. Cell 20, 1763-1771.

Parton, R. G. and del Pozo, M. A. (2013). Caveolae as plasmamembrane sensors,protectors and organizers. Nat. Rev. Mol. Cell Biol. 14, 98-112.

Parton, R. G. and Simons, K. (2007). Themultiple faces of caveolae.Nat. Rev. Mol.Cell Biol. 8, 185-194.

Pepino, M. Y., Kuda, O., Samovski, D. and Abumrad, N. A. (2014). Structure-function of CD36 and importance of fatty acid signal transduction in fatmetabolism. Annu. Rev. Nutr. 34, 281-303.

Poirier, H., Degrace, P., Niot, I., Bernard, A. and Besnard, P. (1996). Localizationand regulation of the putative membrane fatty-acid transporter (FAT) in the small

intestine. Comparison with fatty acid-binding proteins (FABP). Eur. J. Biochem.238, 368-373.

Puri, P., Wiest, M. M., Cheung, O., Mirshahi, F., Sargeant, C., Min, H.-K., Contos,M. J., Sterling, R. K., Fuchs, M., Zhou, H. et al. (2009). The plasma lipidomicsignature of nonalcoholic steatohepatitis. Hepatology 50, 1827-1838.

Razani, B., Engelman, J. A., Wang, X. B., Schubert, W., Zhang, X. L., Marks,C. B., Macaluso, F., Russell, R. G., Li, M., Pestell, R. G. et al. (2001). Caveolin-1null mice are viable but show evidence of hyperproliferative and vascularabnormalities. J. Biol. Chem. 276, 38121-38138.

Razani, B., Combs, T. P., Wang, X. B., Frank, P. G., Park, D. S., Russell, R. G., Li,M., Tang, B., Jelicks, L. A., Scherer, P. E. et al. (2002). Caveolin-1-deficientmice are lean, resistant to diet-induced obesity, and show hypertriglyceridemiawith adipocyte abnormalities. J. Biol. Chem. 277, 8635-8647.

Ring, A., Le Lay, S., Pohl, J., Verkade, P. and Stremmel, W. (2006). Caveolin-1 isrequired for fatty acid translocase (FAT/CD36) localization and function at theplasma membrane of mouse embryonic fibroblasts. Biochim. Biophys. Acta 1761,416-423.

Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R. andAnderson, R. G. (1992). Caveolin, a protein component of caveolae membranecoats. Cell 68, 673-682.

Schlegel, A., Wang, C., Katzenellenbogen, B. S., Pestell, R. G. and Listanti,M. P. (1999). Caveolin-1 potentiates estrogen receptor alpha (ERalpha) signaling.caveolin-1 drives ligand-independent nuclear translocation and activation ofERalpha. J. Biol. Chem. 274, 33551-33556.

Schubert, W., Frank, P. G., Razani, B., Park, D. S., Chow, C.-W. and Lisanti, M. P.(2001). Caveolae-deficient endothelial cells show defects in the uptake andtransport of albumin in vivo. J. Biol. Chem. 276, 48619-48622.

Siddiqi, S., Sheth, A., Patel, F., Barnes, M. and Mansbach, C. M.II. (2013).Intestinal caveolin-1 is important for dietary fatty acid absorption. Biochim.Biophys. Acta 1831, 1311-1321.

Singh, R. D., Puri, V., Valiyaveettil, J. T., Marks, D. L., Bittman, R. and Pagano,R. E. (2003). Selective caveolin-1-dependent endocytosis of glycosphingolipids.Mol. Biol. Cell 14, 3254-3265.

Singh, R. D., Liu, Y., Wheatley, C. L., Holicky, E. L., Makino, A., Marks, D. L.,Kobayashi, T., Subramaniam, G., Bittman, R. and Pagano, R. E. (2006).Caveolar endocytosis and microdomain association of a glycosphingolipid analogis dependent on its sphingosine stereochemistry. J. Biol. Chem. 281,30660-30668.

Singh, R. D., Holicky, E. L., Cheng, Z. J., Kim, S. Y., Wheatley, C. L., Marks, D. L.,Bittman, R. and Pagano, R. E. (2007). Inhibition of caveolar uptake, SV40infection, and beta1-integrin signaling by a nonnatural glycosphingolipidstereoisomer. J. Cell Biol. 176, 895-901.

Trigatti, B. L., Anderson, R. G. W. and Gerber, G. E. (1999). Identification ofcaveolin-1 as a fatty acid binding protein. Biochem. Biophys. Res. Commun. 255,34-39.

Truett, G. E., Heeger, P., Mynatt, R. L., Truett, A. A., Walker, J. A. and Warman,M. L. (2000). Preparation of PCR-quality mouse genomic DNA with hot sodiumhydroxide and tris (HotSHOT). Biotechniques 29, 52-54.

Valasek, M. A.,Weng, J., Shaul, P.W., Anderson, R. G.W. and Repa, J. J. (2005).Caveolin-1 is not required for murine intestinal cholesterol transport. J. Biol. Chem.280, 28103-28109.

Veilleux, A., Grenier, E., Marceau, P., Carpentier, A. C., Richard, D. and Levy, E.(2014). Intestinal lipid handling: evidence and implication of insulin signalingabnormalities in human obese subjects. Arterioscler. Thromb. Vasc. Biol. 34,644-653.

Vogel, U., Sandvig, K. and van Deurs, B. (1998). Expression of caveolin-1 andpolarized formation of invaginated caveolae in Caco-2 and MDCK II cells. J. CellSci. 111, 825-832.

Yang, X., Schadt, E. E., Wang, S., Wang, H., Arnold, A. P., Ingram-Drake, L.,Drake, T. A. and Lusis, A. J. (2006). Tissue-specific expression and regulation ofsexually dimorphic genes in mice. Genome Res. 16, 995-1004.

Yedgar, S., Carew, T. E., Pittman, R. C., Beltz, W. F. and Steinberg, D. (1983).Tissue sites of catabolism of albumin in rabbits. Am. J. Physiol. 244, E101-E107.

Yoon, M., Jeong, S., Nicol, C. J., Lee, H., Han, M., Kim, J. J., Seo, Y. J., Ryu, C.and Oh, G. T. (2002). Fenofibrate regulates obesity and lipid metabolism withsexual dimorphism. Exp. Mol. Med. 34, 481-488.

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