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BackgroundAngiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and β-sheet regions among species; however, their functions have not been studied.MethodsAdeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), β-sheet (AAV.βsheet-Mut), or both regions (AAV.loop/βsheet-Mut) were injected into male hepatocyte-specific AGT deficient (hepAGT-/-) mice in an LDL receptor –/– background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a Western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells.ResultsIn hepAGT-/– mice infected with AAV.loop-Mut or βsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Surprisingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/– mice infected with AAV.loop/βsheet-Mut were not different from mice infected with AAV.null. In contrast, hepaticAgtmRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated trol and AAV containing wild-type mouse AGT as a positive control. We have demonstra ted consistently in this and previous studies tht located in the endoplasmic reticulum.ConclusionsThe conserved sequences in either the loop or β-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.HIGHLIGHTSThe loop and β-sheet regions in the distal face of angiotensinogen (AGT) have highly conserved sequences across species.Mutations on either the loop or β-sheet regions do not affect plasma AGT concentrations, blood pressure, and atherosclerosis in hypercholesterolemic mice.The conserved sequences in the loop and β-sheet regions regulate the secretion of AGT from hepatocytes synergistically in vivo and in cultured cells.

Obesity is associated with alterations in hepatic lipid metabolism. We previously identified the prorenin receptor (PRR) as a potential contributor to liver steatosis. Therefore, we aimed to determine the relative contribution of PRR and its soluble form, sPRR, to lipid homeostasis. PRR-floxed male mice were treated with an adeno-associated virus with thyroxine-binding globulin promoter-driven Cre to delete PRR in the liver [liver PRR knockout (KO) mice]. Hepatic PRR deletion did not change the body weight but increased liver weights. The deletion of PRR in the liver decreased peroxisome proliferator-activated receptor gamma (PPARγ) and triglyceride levels, but liver PRR KO mice exhibited higher plasma cholesterol levels and lower hepatic low-density lipoprotein receptor (LDLR) and Sortilin 1 (SORT1) proteins than control (CTL) mice. Surprisingly, hepatic PRR deletion elevated hepatic cholesterol, and up-regulated hepatic sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA-R) genes. In addition, the plasma levels of sPRR were significantly higher in liver PRR KO mice than in controls. In vitro studies in HepG2 cells demonstrated that sPRR treatment upregulated SREBP2, suggesting that sPRR could contribute to hepatic cholesterol biosynthesis. Interestingly, PRR, total cleaved and noncleaved sPRR contents, furin, and Site-1 protease (S1P) were elevated in the adipose tissue of liver PRR KO mice, suggesting that adipose tissue could contribute to the circulating pool of sPRR. Overall, this work supports previous works and opens a new area of investigation concerning the function of sPRR in lipid metabolism and adipose tissue–liver cross talk. NEW & NOTEWORTHY Hepatic PRR and its soluble form, sPRR, contribute to triglyceride and cholesterol homeostasis and hepatic inflammation. Deletion of hepatic PRR decreased triglyceride levels through a PRR-PPARγ-dependent mechanism but increased hepatic cholesterol synthesis through sPRR-medicated upregulation of SREBP-2. Our study highlighted a new paradigm of cross talk between the liver and the adipose tissue involving cholesterol and sPRR.

Background: ABCG5/ABCG8 (G5/G8) is the primary biliary cholesterol transporter and accounts for >70% of biliary cholesterol secretion. Mice and humans maintain fecal cholesterol excretion under a variety of conditions that disrupt biliary cholesterol secretion, indicating the presence of a non-biliary route for cholesterol elimination. Cholesterol can be actively secreted from the intestine, a pathway termed transintestinal cholesterol excretion (TICE). The present study investigates rates of intestinal cholesterol secretion in mice lacking G5/G8 to determine if adaptive responses within the gut facilitate cholesterol excretion in the absence of the primary biliary cholesterol transporter. Methods: Wild-type (WT) and whole body Abcg5/Abcg8 deficient mice (G5/G8 KO) were maintained on a plant sterol free (PSF) diet to prevent the development of experimental sitosterolemia and potential confounding effects of biologically active phytosterols. Mice were anesthetized, the gall bladder cannulated, and the intestine perfused with model bile to simultaneously determine biliary and intestinal cholesterol secretion rates. Sterol balance studies were conducted in mice sequentially maintained on PSF diet, a PSF diet supplemented with 0.2% cholesterol, and standard rodent chow. Mice were allowed to acclimate to each diet for 14 days, then placed on wire-bottom cages for fecal collection. At termination of the experiment, basal bile, plasma, and tissues were collected. Fecal neutral sterols (FNS) were analyzed by GC-MS. Results: Despite a substantial reduction in biliary cholesterol secretion in G5/G8 KO mice, differences in intestinal cholesterol secretion rates were only observed in males, with KO mice excreting less cholesterol into the intestinal lumen. FNS did not differ between genotypes when maintained on the PSF diet. On chow diet, FNS increased 2x in both genotypes, but did not differ across genotypes. When PSF diet was supplemented with 0.2% cholesterol, FNS increased by 3x in WT mice, but were unchanged in G5/G8 KO mice, relative to PSF diet levels. Conclusions: Abcg5/Abcg8 is essential to maintain sterol balance when cholesterol is present in the diet. TICE is unable to compensate for Abcg5/Abcg8 deficiency in cholesterol fed mice.

Background: ABCG5 and ABCG8 form a heterodimer that promotes biliary secretion and opposes intestinal absorption of cholesterol and phytosterols. Whole-genome and exome sequencing has revealed over 2000 variants of ABCG5 and ABCG8, many of which are predicted to be pathogenic or likely pathogenic. Missense mutations in clinically confirmed cases of Sitosterolemia are of particular interest for their potential to reveal structure-function relationships in ABCG5 ABCG8 and other ABC transporter family members. Mutations in ABCG5 ABCG8 with clinically confirmed Sitosterolemia generally cluster within the ATP binding cassette and transmembrane spanning segments that form the substrate-binding domain. However, four disease-causing mutations occur in the short extracellular loop in ABCG8, suggesting that these mutations may reveal novel elements of the ABCG5 ABCG8 function. Methods: Mutants of ABCG8 were generated by site-directed mutagenesis. Plasmids encoding normal ABCG5 and normal or mutant ABCG8s were co-transfected into human Huh7 hepatocytes, lysates prepared, and analyzed by SDS-PAGE and immunoblot analysis. Maturation and trafficking of the ABCG5 ABCG8 heterodimer beyond the endoplasmic reticulum was assessed by the appearance of the higher molecular weight, mature form of each glycoprotein. Results: Mutants G574R, G575D, and L596R failed to support trafficking of ABCG5 ABCG8 and were designated as Class II mutants causative in Sitosterolemia. Only G574E supported maturation of the ABCG5 ABCG8 transporter in cultured human hepatocytes and remains unclassified. Conclusion: Formation and trafficking of the ABCG5 ABCG8 heterodimer is dependent on key residues within the third extracellular loop in ABCG8. Maturation of ABCG5 ABCG8 is tolerant of substitutions in glycine 574, including the addition of a larger and negatively charged residue. However, neither substitution forms a functional ABCG5 ABCG8 transporter.

Background: Metabolic syndrome (MetS) is a cluster of risk factors that predisposes individuals to develop atherosclerotic cardiovascular disease, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. Dyslipidemia is an essential element of MetS. Mice lack cholesterol ester transfer protein (CETP) and rapidly clear ApoB-containing lipoprotein and are resistant to the development of dyslipidemia and clinically relevant metabolic phenotypes when challenged with high fat, high cholesterol Western-type diet (WTD). We hypothesized that the presence of CETP and ApoB-containing lipoproteins would sensitize mice to the development of dyslipidemia and exacerbate metabolic phenotypes. Approach and Results: Male and female (n=7-11) C57Bl6/J mice harboring a human CETP transgene, a human ApoB100 transgene, or both transgenes were placed on a WTD (42% kCal fat, 0.2% cholesterol). Adiposity, insulin sensitivity, glucose tolerance and plasma cholesterol were assessed at the midpoint and termination of the study. Mice were anesthetized, basal bile was collected (30 min), euthanized via cardiac puncture, and tissues dissected and snap frozen. One lobe of the liver was additionally fixed for histological analysis. In both females and males, CETP/ApoB100 and ApoB100 transgenic mice showed a robust increase in total plasma cholesterol and LDL-C. The presence of CETP further increased LDL-C in ApoB100 expressing mice and modestly reduced HDL-C. Differences in body weight were not observed across genotypes. However, ApoB100 expression reduced adiposity in females, but not males. Conversely, the presence of either ApoB100 or CETP impaired insulin sensitivity in male, but not female mice. Liver histology showed increased hepatocyte ballooning in ApoB100 and CETP/ApoB100 transgenic mice, but other measures of NAFLD were unaffected. Conclusions: Mice expressing CETP/ApoB100 transgenes develop dyslipidemia more consistent with human lipoprotein profiles. The humanized lipoprotein profile reveals sexually dimorphic responses to WTD.

Background: Sitosterolemia is a rare form of Familial Hypercholesterolemia (FH) which is unique from other forms of FH due to the accumulation of phytosterols in the plasma and tissues. Sitosterolemia is autosomal recessive and caused by mutations in either the ABCG5 or ABCG8 gene. ABCG5 and ABCG8 form a heterodimer (ABCG5/ABCG8) that functions at the apical surface of hepatocytes and enterocytes to promote cholesterol and phytosterol excretion. Small molecule modulators classified as either correctors or potentiators have therapeutic benefit when used to treat mutants of ABCB4 (PFIC3) or ABCC7 (Cystic Fibrous) that have impaired folding, stability, or activity. There are over 40 missense mutations in ABCG5/ABCG8 which have been clinically confirmed in patients with Sitosterolemia. Methods: We developed a classification system for ABCG5 and ABCG8 mutations based on the underlying molecular defect for ABCG5/ABCG8 dysfunction (maturation, trafficking, activity, etc.). We used site directed mutagenesis to introduce ABCG8 missense mutations located in the N-terminal, cytosolic domain of human ABCG8. Normal and mutant ABCG5/ABCG8 constructs were transiently transfected into a human hepatocyte cell line, Huh-7, and analyzed for heterodimerization, trafficking beyond the ER, and localization at the cell surface. Results: Of the ABCG8 cytosolic mutants studied, R184H, L196Q, L228P, and R263Q failed to traffic beyond the ER to form a stable heterodimer (class II) while P231T, T400K, N409D, N409I, and P415H were trafficking competent. Conclusions: Forty four percent (4 of 9) of cytosolic, ABCG8 mutations that cause Sitosterolemia are due to failures in complex formation and trafficking beyond the ER. Fifty six (5 of 9) percent were trafficking competent, indicating a stable ABCG5/G8 complex, but loss of activity due to impaired ATP hydrolysis or substrate binding. ABCG8 mutants may be potentially restored by proteostatic regulators and/or potentiators & correctors that have shown to be effective in disease-causing mutations in other ABC transporters.

Background: Nonalcoholic fatty liver disease (NAFLD) affects almost 1 billion people worldwide and is associated with risk factors such as obesity and dyslipidemia. Studies show linked variants and methylation status of carnitine palmitoytransferase 1a (CPT1a) to disrupt very low-density lipoprotein (VLDL) cholesterol and triglyceride (TG) levels. We exhibit liver-specific deletion of CPT1a in mice lowers plasma cholesterol and TG while exacerbating NAFLD and inflammation. Methods: Eight-week old Cpt1a floxed mice with the human apoB100 transgene (Cpt1a fl/fl /B100 Tg ) were administered control adenoassociated virus (AAV) or AAV encoding Cre-recombinase under control of a liver specific promoter (TBG-Cre). Control and Cpt1a liver-specific knock out (LKO) mice were placed on low-fat control or western-type diet (WTD; 42% kcal fat, 0.2% cholesterol) for 16 weeks. Body weights were recorded weekly and body composition by MRI was performed at the study midpoint and end. Tissues and plasma were collected and analyzed for lipid composition and gene and protein expression by QPCR and immunoblotting, respectively. Results: CPT1a LKO mice had lower plasma cholesterol and TG irrespective of diet. The reduction in plasma cholesterol was limited to the LDL pool in FPLC-fractionated plasma. Hepatic TG was elevated in mice fed WTD and LKO Cpt1a mice. Loss of hepatic Cpt1a had no effect on hepatic cholesterol in male mice, but increased total and free cholesterol by 2- and 2.5-fold, respectively, in females. The rise in hepatic free cholesterol in female Cpt1a LKO mice associated with increases in Kupffer cell ( Clec4f ) and collagen ( Col1a1 ) gene expression. Free cholesterol levels were not related to differences in transcripts for enzymes involved in cholesterol synthesis or secretion ( Srebp2, Hmgcr, Hmgcs, Soat2, Mttp ), hepatic clearance ( Ldlr ), metabolism ( Cpy7a1, Cyp8b1 ), or biliary secretion ( Abcg5, Abcg8 ). Conclusions: Liver-specific deletion of CPT1a reduces plasma LDL-cholesterol and increases cholesterol levels in female, but not male mice. The increase in hepatic free cholesterol did not alter expression of cholesterol-responsive genes, suggesting cholesterol accumulation outside of the regulatory pool.

Background: Insulin signaling regulates a variety of features of hepatic lipid and lipoprotein metabolism. Liver-specific deletion of insulin receptor (LIRKO) reduces plasma HDL, an effect attributed to a reduction in deiodinase 2 expression and apolipoprotein (Apo) AI mRNA expression. We report a concomitant accumulation of ApoAI protein in the liver of LIRKO mice in a yet to be fully characterized compartment within hepatocytes. Methods: Mice harboring loxP sites within the insulin receptor gene (IR fl/fl ) were administered a control adenoassociated viral vector (AAV-Empty) or an AAV encoding Cre recombinase under the control of a liver-specific promoter (AAV_TBG-Cre) at 8 weeks of age to generate Control and LIRKO mice. Male and female mice were evaluated for the abundance of ApoAI in liver and total and FPLC-fractionated plasma. ApoAI secretion rates were evaluated in primary hepatocytes from both genotypes. Subcellular localization of ApoAI in liver of Control and LIRKO mice was evaluated by immunofluorescence microscopy and biochemical fractionation. Results: The loss of hepatic insulin receptor and signaling was confirmed by rtPCR, immunoblot analysis and glucose tolerance test. Whereas ApoAI was unaffected in LIRKO mice compared to controls, protein levels were reduced in plasma and elevated in liver. In contrast, no changes were observed for ApoB or ApoE in either liver or plasma. FPLC fractionation of plasma indicates a shift in HDL size and an accumulation of ApoM in the HDL fractions. Secretion rates of newly synthesized ApoAI were modestly reduced in primary hepatocytes isolated from LIRKO mice. Immunolocalization of ApoAI demonstrated accumulation in large, membrane-bound organelles located peripherally in cells. These organelles stained positively for LampI and other markers of the endosomal-lysosomal system. However, markers of the endosomal-lysosomal compartment in perinuclear regions of the cells were negative for ApoAI. Conclusions: Insulin signaling is a regulator of ApoAI secretion that alters the intracellular itinerary of ApoAI in the secretory and endocytic pathways and alters the plasma HDL proteome in mice.

Background: The proteostasis network is essential for the maintenance of cellular integrity by ensuring the correct folding of newly synthesized proteins. However, the high fidelity of this system contributes to diseases such as Cystic Fibrosis (CF, ABCC7), Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3, ABCB4), and Sitosterolemia (ABCG5/ABCG8). Interference with proteostasis and allowing sub-optimally folded ATP-Binding Cassette (ABC) transporters to transit to the cell surface partially restores function, with clinical benefit for CF and PFIC3. We sought to delineate the molecular underpinnings of Sitosterolemia, an autosomal recessive form of Familial Hypercholesterolemia (FH), characterized by accumulation of phytosterols in the plasma and tissues. ABCG5 ABCG8 form an obligate heterodimer at the surface of the liver and small intestine, and mediates sterol transport into bile and the intestinal lumen. Case studies reveal 57 loss of function and over 40 missense mutations of ABCG5 associated with Sitosterolemia. Recent pharmaceutics show promise with rescue of mutations in CF and PFIC3 with small molecule chaperones and potentiators. Through our analysis of ABCG5 missense mutations causing Sitosterolemia, we anticipate rescue by these compounds as well. Methods: We established a classification system for missense mutations of ABCG5, similar to CF and PFIC3. Mutations were generated by site-directed mutagenesis, and confirmed through Sanger sequencing. Native and mutant ABCG5 were co-transfected with native ABCG8 into human hepatocytes and evaluated for protein abundance and trafficking beyond the endoplasmic reticulum (ER), by SDS-PAGE and immunoblot analysis. Results: Nine mutants of ABCG5 have been generated. Co-expression of ABCG5 with ABCG8 i n vitro demonstrated I68N, A98G, E146Q, and R419P successfully traffic while R284S, T305R, R389H, R419H, and N437K were arrested within the proteostasis network. Conclusions: Similar to CF and PFIC3, Sitosterolemia-associated mutations result in compromised heterodimer formation and trafficking to the apical surface. Small molecule chaperones and potentiators may partially rescue the mutants and provide clinical benefit for Sitosterolemia.

The ABCG5/G8 heterodimer is the primary neutral sterol transporter in hepatobiliary and transintestinal cholesterol excretion. Inactivating mutations on either the ABCG5 or ABCG8 subunit cause Sitosterolemia, a rare genetic disorder. In 2016, a crystal structure of human ABCG5/G8 in an apo state showed the first structural information on ATP-binding cassette (ABC) sterol transporters and revealed several structural features that were observed for the first time. Over the past decade, several missense variants of ABCG5/G8 have been associated with non-Sitosterolemia lipid phenotypes. In this review, we summarize recent pathophysiological and structural findings of ABCG5/G8, interpret the structure-function relationship in disease-causing variants and describe the available evidence that allows us to build a mechanistic view of ABCG5/G8-mediated sterol transport.

Objective: Determine the impact of CETP (cholesteryl ester transfer protein) on the route of cholesterol elimination in mice. Approach and Results: We adapted our protocol for biliary cholesterol secretion with published methods for measuring transintestinal cholesterol elimination. Bile was diverted and biliary lipid secretion maintained by infusion of bile acid. The proximal small bowel was perfused with bile acid micelles. In high-fat, high-cholesterol–fed mice, the presence of a CETP transgene increased biliary cholesterol secretion at the expense of transintestinal cholesterol elimination. The increase in biliary cholesterol secretion was not associated with increases in hepatic SR-BI (scavenger receptor BI) or ABCG5 (ATP-binding cassette G5) ABCG8. The decline in intestinal cholesterol secretion was associated with an increase in intestinal Niemann-Pick disease, type C1, gene-like 1 mRNA. Finally, we followed the delivery of HDL (high-density lipoprotein) or LDL (low-density lipoprotein) cholesteryl esters (CE) from plasma to bile and intestinal perfusates. HDL-CE favored the biliary pathway. Following high-fat feeding, the presence of CETP directed HDL-CE away from the bile and towards the intestine. The presence of CETP increased LDL-CE delivery to bile, whereas the appearance of LDL-CE in intestinal perfusate was near the lower limit of detection. Conclusions: Biliary and intestinal cholesterol secretion can be simultaneously measured in mice and used as a model to examine factors that alter cholesterol elimination. Plasma factors, such as CETP, alter the route of cholesterol elimination from the body. Intestinal and biliary cholesterol secretion rates are independent of transhepatic or transintestinal delivery of HDL-CE, whereas LDL-CE was eliminated almost exclusively in the hepatobiliary pathway.

Background Developing an understanding of the biochemistry of aging in both sexes is critical for managing disease throughout the lifespan. Lipidomic associations with age and sex have been reported, but prior studies are limited by measurements in serum rather than plasma or by participants taking lipid-lowering medications. Methods Our study included lipidomic data from 980 participants aged 18–87 years old from the Genetics of Lipid-Lowering Drugs and Diet Network (GOLDN). Participants were off lipid-lowering medications for at least 4 weeks, and signal intensities of 413 known lipid species were measured in plasma. We examined linear age and sex associations with signal intensity of (a) 413 lipid species; (b) 6 lipid classes (glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, fatty acids, and acylcarnitines); and (c) 15 lipid subclasses; as well as with the particle sizes of three lipoproteins. Results Significant age associations were identified in 4 classes, 11 subclasses, 147 species, and particle size of one lipoprotein while significant sex differences were identified in 5 classes, 12 subclasses, 248 species, and particle sizes of two lipoproteins. For many lipid species (n = 97), age-related associations were significantly different between males and females. Age*sex interaction effects were most prevalent among phosphatidylcholines, sphingomyelins, and triglycerides. Conclusion We identified several lipid species, subclasses, and classes that differ by age and sex; these lipid phenotypes may serve as useful biomarkers for lipid changes and associated cardiovascular risk with aging in the future. Future studies of age-related changes throughout the adult lifespan of both sexes are warranted. Trial registration ClinicalTrials.gov NCT00083369; May 21, 2004.

Sitosterolemia is a lipid disorder characterized by the accumulation of dietary xenosterols in plasma and tissues caused by mutations in either ABCG5 or ABCG8. ABCG5 ABCG8 encodes a pair of ABC half transporters that form a heterodimer (G5G8), which then traffics to the surface of hepatocytes and enterocytes and promotes the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. We review the literature from the initial description of the disease, the discovery of its genetic basis, current therapy, and what has been learned from animal, cellular, and molecular investigations of the transporter in the twenty years since its discovery. The genomic era has revealed that there are far more carriers of loss of function mutations and likely pathogenic variants of ABCG5 ABCG8 than previously thought. The impact of these variants on G5G8 structure and activity are largely unknown. We propose a classification system for ABCG5 ABCG8 mutants based on previously published systems for diseases caused by defects in ABC transporters. This system establishes a framework for the comprehensive analysis of disease-associated variants and their impact on G5G8 structure–function.

Obesity is associated with alterations in hepatic lipid metabolism. We previously identified the prorenin receptor (PRR) as a potential contributor to liver steatosis. Therefore, we aimed to determine the relative contribution of PRR and its soluble form, sPRR, to lipid homeostasis. PRR-floxed male mice were treated with an adeno-associated virus with thyroxine-binding globulin promoter driven Cre to delete specifically PRR in hepatocytes (Liver PRR KO mice). Hepatic PRR deletion did not change the body weight but increased liver weights. Liver PRR KO mice exhibited higher plasma cholesterol levels and lower hepatic LDLR protein than control mice. Surprisingly, hepatic PRR deletion elevated hepatic cholesterol, and up-regulated hepatic SREBP2 and HMG CoA-R genes. In addition, hepatic PRR deletion increased plasma sPRR levels.In vitrostudies in Hep-G2 cells demonstrated that sPRR treatment up-regulated SREBP2 suggesting that elevated plasma sPRR could contribute to hepatic cholesterol biosynthesis. Interestingly, PPARγ, PRR and total sPRR were elevated in the adipose tissue of Liver PRR KO mice suggesting that elevated plasma sPRR originated from the adipose tissue. In 3T3-L1 cells, sPRR treatment up-regulated PPARγ indicating that sPRR stimulates master regulator of adipocyte differentiation. Overall, this work support a new role for sPRR in lipid metabolism and adipose tissue – liver crosstalk.

The elucidation of the molecular basis of the rare disease, sitosterolemia, has revolutionized our mechanistic understanding of how dietary sterols are excreted and how cholesterol is eliminated from the body. Two proteins, ABCG5 and ABCG8, encoded by the sitosterolemia locus, work as obligate dimers to pump sterols out of hepatocytes and enterocytes. ABCG5/ABCG8 are key in regulating whole-body sterol trafficking, by eliminating sterols via the biliary tree as well as the intestinal tract. Importantly, these transporters keep xenosterols from accumulating in the body. The sitosterolemia locus has been genetically associated with lipid levels and downstream atherosclerotic disease, as well as formation of gallstones and the risk of gallbladder cancer. While polymorphic variants raise or lower the risks of these phenotypes, loss of function of this locus leads to more dramatic phenotypes, such as premature atherosclerosis, platelet dysfunction, and thrombocytopenia, and, perhaps, increased endocrine disruption and liver dysfunction. Whether small amounts of xenosterol exposure over a lifetime cause pathology in normal humans with polymorphic variants at the sitosterolemia locus remains largely unexplored. The purpose of this review will be to summarize the current state of knowledge, but also highlight key conceptual and mechanistic issues that remain to be explored.

Despite advances in healthcare, cardiovascular disease (CVD) remains the leading cause of death in the United States. Elevated levels of plasma cholesterol are highly predictive of CVD and stroke and are the principal driver of atherosclerosis. Unfortunately, current cholesterol lowering agents, such as statins, are not known to reverse atherosclerotic disease once it has been established. In preclinical models, agonists of nuclear receptor, LXR, have been shown to reduce and reverse atherosclerosis. Phytosterols are bioactive non-cholesterol sterols that act as LXR agonists and regulate cholesterol metabolism and transport. We hypothesized that stigmasterol would act as an LXR agonist and alter intestinal cholesterol secretion to promote cholesterol elimination. Mice were fed a control diet, or a diet supplemented with stigmasterol (0.3% w/w) or T0901317 (0.015% w/w), a known LXR agonist. In this experiment we analyzed the sterol content of bile, intestinal perfusate, plasma, and feces. Additionally, the liver and small intestine were analyzed for relative levels of transcripts known to be regulated by LXR. We observed that T0901317 robustly promoted cholesterol elimination and acted as a strong LXR agonist. Stigmasterol promoted transintestinal cholesterol secretion through an LXR-independent pathway.

High-throughput small RNA sequencing (sRNA-seq) has facilitated the discovery of many classes of small RNAs (sRNA) and helped establish the field of extracellular RNA (exRNA). Although several tools are available for sRNA-seq analysis, exRNAs present unique analytical challenges that are not met by current software. Therefore, we developed a novel data analysis pipeline specifically for exRNAs entitled, Tools for Integrative Genome analysis of Extracellular sRNAs (TIGER). To demonstrate the power of this tool, sRNA-seq was performed on high-density lipoproteins (HDL), apolipoprotein B-containing particles (APOB), bile, urine, and liver samples collected from wild-type (WT) and scavenger receptor BI knockout (SR-BI KO) mice. TIGER was able to account for approximately 60% of reads on lipoproteins and >85% of reads in liver, bile, and urine, a significant advance compared to existing software, largely due to the identification of non-host sRNAs in these datasets. A key advance for the TIGER pipeline is the ability to analyze host and non-host sRNAs across many classes at the genome, parent RNA, and individual fragment levels. Moreover, disparate sample types were compared at each level using hierarchical clustering, correlations, betadispersions, principal coordinate analysis, and permutational multivariate analysis of variance. TIGER analysis was also used to quantify distinct features of exRNAs, including 5-prime microRNA (miRNA) variants, 3-prime miRNA non-templated additions, parent RNA positional coverage, and length distributions by RNA class. Results suggest that the majority of sRNAs on lipoproteins are non-host sRNAs derived from bacterial sources in the microbiome and environment, specifically rRNA-derived sRNAs from proteobacteria. Here, we report novel discoveries of lipoprotein sRNAs that were facilitated by the new sRNA-seq analysis pipeline, TIGER, which has tremendous applicability for the field of exRNA.

Objective: The ABCG5 ABCG8 (G5G8) sterol transporter is the primary mechanism for biliary cholesterol secretion, but mice maintain fecal sterol excretion in its absence. The mechanism by which mice maintain sterol excretion in the absence of this pathway is not known. Transintestinal cholesterol excretion (TICE) is an alternative pathway to hepatobiliary secretion. We investigated the impact of G5G8 deficiency on TICE in the absence of Sitosterolemia. Methods and Results: We compared both hepatobiliary and transintestinal cholesterol excretion rates in wild-type (WT) and G5G8 deficient mice of both sexes. WT and G5G8 were maintained on a plant-sterol free diet from the time of weaning to prevent the development of secondary phenotypes associated with Sitosterolemia. Biliary and intestinal cholesterol secretion rates were determined by biliary diversion with simultaneous perfusion of the proximal 10 cm of the small bowel. Among WT mice, biliary cholesterol secretion was greater in female mice compared to males. Conversely, male mice exhibited greater rates of TICE than females. As expected, WT mice had higher biliary cholesterol secretion rates than their G5G8 deficient littermates. However, the decline in biliary cholesterol secretion was far less in male mice compared to females in the absence of G5G8. In female mice, the absence of G5G8 resulted in a two-fold increase in TICE, whereas males were unaffected. Conclusion: Female mice are more dependent upon the biliary pathway for cholesterol excretion, whereas males are more dependent upon TICE. G5G8 independent pathways are present for both biliary and intestinal cholesterol secretion. Female and male mice differ in their adaptation to G5G8 deficiency in order to maintain fecal sterol excretion.

The aim of the study was to assess the effect of insulin receptor (IR) signaling on hepatic apoA-I metabolism. The experimental approach was to compare hepatic apoA-I expression, secretion and cellular localization in IRfl/fl mice and LIRKO mice in which hepatic insulin receptors were specifically deleted by AAV delivered Cre-recombinase. Results showed that IR mRNA and protein levels were markedly reduced in the livers of LIRKO mice compared to control IRfl/fl mice. As expected, LIRKO mice exhibited decreased glucose tolerance and reduced hepatic insulin signaling. Knockdown of hepatic IR decreased plasma HDL cholesterol and apoA-I levels. Whereas apoA-I mRNA levels were similar in LIRKO and control hepatocytes, apoA-I protein levels were increased in both the liver and primary hepatocytes isolated from LIRKO mice. In contrast to apoA-I, apoE and apoB protein levels in the liver and in cultured hepatocytes, as well as in the plasma, were similar in LIRKO and control mice. ApoA-I accumulation in LIRKO hepatocytes was associated with a decreased rate of apoA-I secretion. Immunofluorescence staining demonstrated that apoA-I accumulated in LIRKO hepatocytes in membrane bound inclusions. These inclusions shared markers characteristic of early, late and recycling endosomes, and of lysosomes. We conclude that IR-mediated insulin signaling plays an important role in hepatic apoA-I secretion and consequent nascent HDL formation. Reduced apoA-I secretion from liver into the circulation may contribute to the lower HDL levels typically associated with insulin resistance.

Objective— Recent studies support a role for cholesterol in the development of obesity and nonalcoholic fatty liver disease. Mice lacking the ABCG5 ABCG8 (G5G8) sterol transporter have reduced biliary cholesterol secretion and are more susceptible to steatosis, hepatic insulin resistance, and loss of glycemic control when challenged with a high-fat diet. We hypothesized that accelerating G5G8-mediated biliary cholesterol secretion would correct these phenotypes in obese mice. Approach and Results— Obese ( db / db ) male and their lean littermates were administered a cocktail of control adenovirus or adenoviral vectors encoding ABCG5 and ABCG8 (AdG5G8). Three days after viral administration, measures of lipid and glucose homeostasis were determined, and tissues were collected for biochemical analyses. AdG5G8 increased biliary cholesterol and fecal sterol elimination. Fasting glucose and triglycerides declined, and glucose tolerance improved in obese mice expressing G5G8 compared with mice receiving control adenovirus. These changes were associated with a reduction in phosphorylated eukaryotic initiation factor 2α and c-Jun N-terminal kinase in liver, suggesting alleviation of endoplasmic reticulum stress. Phosphorylated insulin receptor and protein kinase B were increased, indicating restored hepatic insulin signaling. However, there was no reduction in hepatic triglycerides after the 3-day treatment period. Conclusions— Accelerating biliary cholesterol secretion restores glycemic control and reduces plasma triglycerides in obese db / db mice.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Concentrations of the major LPA species in mouse plasma decreased uniformly following administration of a potent selective inhibitor of the LPA-generating lysophospholipase D autotaxin, identifying an active mechanism for removal of LPA from the circulation. LPA, akylglycerol phosphate (AGP), sphingosine 1-phosphate (S1P), and a variety of structural mimetics of these lipids, including phosphatase-resistant phosphonate analogs of LPA, were rapidly eliminated (t1/2 < 30 s) from the circulation of mice following intravenous administration of a single bolus dose without significant metabolism in situ in the blood. These lipids accumulated in the liver. Elimination of intravenously administered LPA was blunted by ligation of the hepatic circulation, and ∼90% of LPA administered through the portal vein was accumulated by the isolated perfused mouse liver at first pass. At early times following intravenous administration, more LPA was associated with a nonparenchymal liver cell fraction than with hepatocytes. Primary cultures of nonparenchymal liver cells rapidly assimilated exogenously provided LPA. Our results identify hepatic uptake as an important determinant of the bioavailability of LPA and bioactive lysophospholipid mimetics and suggest a mechanism to explain changes in circulating LPA levels that have been associated with liver dysfunction in humans.

Selective lipid uptake (SLU) is known to be a major pathway of lipoprotein cholesterol metabolism in experimental animals and humans, but remains poorly understood. This review provides a brief overview of SLU mediated by the HDL receptor scavenger receptor B-type I (SR-BI), and highlights several surprising new findings related to the impact of SLU pathways in cholesterol homeostasis.Under certain conditions, SR-BI-mediated SLU contributes to reverse cholesterol transport (RCT) independently of ABCG5/G8-mediated biliary cholesterol secretion, implying a novel trafficking mechanism. Hepatic SR-BI expression and RCT are decreased in diabetic mice. Farnesoid X receptor (FXR) and the microRNAs miR-185, miR-96 and miR-223 are emerging therapeutic targets for increasing SR-BI expression. SR-BI-independent selective cholesteryl ester uptake is a newly characterized pathway in macrophage foam cells.New findings underscore the importance of SR-BI-mediated SLU in hepatic SLU and RCT, while indicating that further investigation is needed to define SLU pathways, including SR-BI-independent macrophage selective cholesteryl ester uptake. The intracellular trafficking of cholesterol in these pathways appears to be critical to their normal function and is a major subject of ongoing studies.

Background: Elevated hepatic cholesterol is thought to contribute to the development of nonalcoholic fatty liver disease, a condition highly associated with cardiovascular risk factors. The ABCG5 and ABCG8 (G5G8) heterodimer is responsible for up to 90% of biliary cholesterol secretion and is a potential therapeutic target for promoting cholesterol elimination. We have previously demonstrated that ursodiol (UDCA), a pharmacologic bile acid, increases G5G8 protein expression and biliary cholesterol secretion. However, whole body cholesterol elimination is minimized likely due to simultaneous suppression of bile acid synthesis through upregulation of FGF15/19. The objectives of this study are to determine whether FGF15/19 regulates G5G8 and determine whether UDCA requires FGF15/19 signaling in order to upregulate G5G8. Methods: Mice were injected with two doses of FGF19 or carrier (PBS) 1μg/g body weight within an 8-hour treatment window. A separate group of wild type (WT) and G5G8 knockout (KO) mice were similarly injected with FGF19. In another experiment, WT mice were fed chow or UDCA-supplemented diet in the absence or presence of FGF15/19 signaling inhibition which was achieved by FGFR4 antisense oligonucleotide (ASO) supplied by Ionis Pharmaceuticals. In all experiments, body weight, liver weight, bile flow rate and plasma, hepatic and biliary lipids were measured. Immunoblotting of G5G8 and real-time PCR of genes involved in cholesterol metabolism were also conducted. Results: Mice injected with FGF19 had increased biliary lipids (PBS: 5.287±0.5720, FGF19: 8.098±0.6114, n=6), decreased Cyp7a1 (PBS: 1.021±0.1064 FGF19: 0.07787±0.01345 n=5-7) and Cyp8b1 (PBS: 1.018±0.09846, FGF19: 0.2647±0.05609, n=5-7) expression, and increased G5G8 protein expression compared to mice injected with PBS. In G5G8 KO mice injected with FGF19, there was only a small increase in plasma free cholesterol (WT: 51.96±2.098, KO: 62.24±2.562, n=4) and no other significant changes in cholesterol metabolism compared to wild type mice injected with FGF19. Conclusion: In conclusion, FGF15/19 suppresses bile acid synthesis and post-transcriptionally upregulates G5G8. However, in the absence of G5G8, FGF15/19 did not disrupt cholesterol metabolism.

ABCG5 and ABCG8 form a complex (G5G8) that opposes the absorption of plant sterols but is also expressed in liver where it promotes the excretion of cholesterol into bile. Hepatic G5G8 is transcriptionally regulated by a number of factors implicated in the development of insulin resistance and nonalcoholic fatty liver disease. Therefore, we hypothesized that G5G8 may influence the development of diet-induced obesity phenotypes independently of its role in opposing phytosterol absorption. G5G8 knock-out (KO) mice and their wild type (WT) littermates were challenged with a plant sterol-free low fat or high fat (HF) diet. Weight gain and the rise in fasting glucose were accelerated in G5G8 KO mice following HF feeding. HF-fed G5G8 KO mice had increased liver weight, hepatic lipids, and plasma alanine aminotransferase compared with WT controls. Consistent with the development of nonalcoholic fatty liver disease, macrophage infiltration, the number of TUNEL-positive cells, and the expression of proinflammatory cytokines were also increased in G5G8 KO mice. Hepatic lipid accumulation was associated with increased peroxisome proliferator activated receptor γ, CD36, and fatty acid uptake. Phosphorylation of eukaryotic translation initiation factor 2α (eiF2α) and expression of activating transcription factor 4 and tribbles 3 were elevated in HF-fed G5G8 KO mice, a pathway that links the unfolded protein response to the development of insulin resistance through inhibition of protein kinase B (Akt) phosphorylation. Phosphorylation of Akt and insulin receptor was reduced, whereas serine phosphorylation of insulin receptor substrate 1 was elevated.

Lactation is associated with increased expression of bile acid transporters and an increased size and hydrophobicity of the bile acid pool in rats. ATP-binding cassette (ABC) transporters multidrug resistance protein 2 (Mdr2), Abcb11 [bile salt export pump (Bsep)], and Abcg5/Abcg8 heterodimers are essential for the biliary secretion of phospholipids, bile acids, and cholesterol, respectively. We investigated the expression of these transporters and secretion of their substrates in female control and lactating Sprague Dawley rats and C57BL/6 mice. Expression of Abcg5/Abcg8 mRNA was decreased by 97 and 60% by midlactation in rats and mice, respectively; protein levels of Abcg8 were below detection limits in lactating rats. Mdr2 mRNA expression was decreased in lactating rats and mice by 47 and 59%, respectively. Despite these changes in transporter expression, basal concentrations of cholesterol and phospholipid in bile were unchanged in rats and mice, whereas increased Bsep mRNA expression in early lactation coincided with an increased basal biliary bile acid concentration in lactating mice. Following taurocholate infusion, coupling of phospholipid and taurocholate secretion in bile of lactating mice was significantly impaired relative to control mice, with no significant changes in maximal secretion of cholesterol or bile acids. In rats, taurocholate infusion revealed a significantly impaired coupling of cholesterol to taurocholate secretion in bile in lactating vs. control animals. These data reveal marked utilization of an Abcg5/Abcg8-independent mechanism for basal biliary cholesterol secretion in rats during lactation, but a dependence on Abcg5/g8 for maximal biliary cholesterol secretion.

PurposeThe purpose of this paper is to investigate design synthesis methods for designing lattice cellular structures to achieve desired stiffnesses. More generally, to find appropriate design problem formulations and solution algorithms for searching the large, complex design spaces associated with cellular structures.Design/methodology/approachTwo optimization algorithms were tested: particle swarm optimization (PSO) and Levenburg‐Marquardt (LM), based on a least‐squares minimization formulation. Two example problems of limited complexity, specifically a two‐dimensional cantilever beam and a two‐dimensional simply‐supported plate, were investigated. Computational characteristics of the algorithms were reported for design problems with hundreds of variables. Constraints from additive manufacturing processes were incorporated to ensure that resulting designs are realizable.FindingsBoth PSO and LM succeeded in searching the design spaces and finding good designs. LM is one to two orders of magnitude more efficient for this class of problems.Research limitations/implicationsThree‐dimensional problems are not investigated in this paper.Practical implicationsLM appears to be a viable algorithm for optimizing structures of complex geometry for minimum weight and desired stiffness.Originality/valueThe testing of design synthesis methods (problem formulations and algorithms) for lattice cellular structures, and the testing of PSO and LM algorithms, are of particular value.

Purpose of review This review summarizes recent developments in the activity, regulation, and physiology of the ABCG5 ABCG8 (G5G8) transporter and the use of its xenobiotic substrates, phytosterols, as cholesterol lowering agents in the treatment of cardiovascular disease. Recent progress has significant implications for the role of G5G8 and its substrates in complications associated with features of the metabolic syndrome. Recent findings Recent reports expand the clinical presentation of sitosterolemia to include platelet and adrenal dysfunction. The G5G8 sterol transporter is critical to hepatobiliary excretion of cholesterol under nonpathological conditions and has been linked to the cholesterol gallstone susceptibility. Finally, the cardiovascular benefits of cholesterol lowering through the use of phytosterol supplements were offset by vascular dysfunction, suggesting that alternative strategies to reduced cholesterol absorption offer greater benefit. Summary Insulin resistance elevates G5G8 and increases susceptibility to cholesterol gallstones. However, this transporter is critical for the exclusion of phytosterols from the absorptive pathways in the intestine. Challenging the limits of this protective mechanism through phytosterol supplementation diminishes the cardioprotective benefits of cholesterol lowering in mouse models of cardiovascular disease.

In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

ABGG5 (G5) and ABCG8 (G8) are ABC half-transporters that dimerize within the endoplasmic reticulum, traffic to the cell surface, and mediate cholesterol excretion into bile. Mice harboring defects in the leptin axis (db/db and ob/ob) have reduced biliary cholesterol concentrations. Rapid weight loss brought about by administration of leptin or dietary restriction increases biliary cholesterol excretion. We hypothesized that the reduction in biliary cholesterol in mice harboring defects in the leptin axis is associated with a reduction in G5G8 transporters and that levels of the transporter would increase with leptin administration and dietary restriction. We examined mRNA and protein levels for G5 and G8 in db/db and ob/ob mice. In both models G5 and G8 protein levels were reduced. In ob/ob mice, both leptin administration and dietary restriction increased G5 and G8 protein and biliary cholesterol concentrations. Finally, we examined the effects of tauroursodeoxycholate, which has been shown to increase biliary cholesterol excretion and function as a molecular chaperone. Tauroursodeoxycholate increased G5 and G8 protein and biliary cholesterol concentrations in both wild-type and db/db mice. Our results indicate that the mechanism for reduced biliary cholesterol excretion in db/db and ob/ob mice involves reductions in G5 and G8 protein levels and that this may occur at the level of G5G8 heterodimer assembly within the endoplasmic reticulum.

Objective Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant, and have reduced levels of biliary cholesterol due, in part, to reduced levels of hepatic G5G8. Chronic leptin replacement restores G5G8 abundance and increases biliary cholesterol concentrations, but the molecular mechanisms responsible for G5G8 regulation remain unclear. In the current study, we used a series of mouse models to address potential mechanisms for leptin-mediated regulation of G5G8. Methods and Results We acutely replaced leptin in ob/ob mice and deleted hepatic leptin receptors in lean mice. Neither manipulation altered G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy had no effect on G5G8. Alternatively, G5G8 may be decreased in ob/ob and db/db mice due to ER dysfunction, the site of G5G8 complex assembly. Overexpression of the ER chaperone GRP78 using an adenoviral vector restores ER function and reduces steatosis in ob/ob mice. Therefore, we determined if AdGRP78 could rescue G5G8 in db/db mice. As in ob/ob mice, AdGRP78 reduced expression of lipogenic genes and plasma triglycerides in the db/db strain. Both G5 and G8 protein levels increased as did total biliary cholesterol, but in the absence of changes in G5 or G8 mRNAs. The increase in G5G8 was associated with increases in a number of proteins, including the ER lectin chaperone, calnexin, a key regulator of G5G8 complex assembly. Conclusions Leptin signaling does not directly regulate G5G8 abundance. The loss of G5G8 in mice harboring defects in the leptin axis is likely associated with compromised ER function.

Objective: Transintestinal cholesterol excretion (TICE) is an alternative pathway to hepatobiliary secretion for cholesterol elimination. The cholesterol donors contributing to this pathway in plasma remain unclear, but appear to include both ApoA and ApoB lipoproteins. Cholesteryl ester transfer protein (CETP) facilitates the transport of cholesteryl esters and triglycerides between lipoproteins in plasma. Our study was aimed at determining the impact of CETP on cholesterol elimination pathways in mice. Methods and Results: We compared both hepatobiliary and intestinal cholesterol secretion rates in male wild-type (WT) and CETP transgenic (TG) mice at age of 12 weeks. WT and TG mice did not differ either biliary or intestinal cholesterol secretion rates when maintained on a standard chow diet. However, TG mice showed increased biliary cholesterol secretion rates and decreased intestinal cholesterol secretion rates compared to the WT group in response to high fat, high cholesterol Western diet. We next determined the effect of CETP on the delivery of radiolabeled HDL-cholesterol ester to the bile and intestinal lumen. Unlike bulk cholesterol secretion, HDL-derived cholesterol esters were preferentially delivered to the intestine in CETP transgenic mice. To further explore the mechanism, we injected radiolabeled HDLs from both WT mice (WT-HDL) and TG mice (CETP-HDL) back into male WT mice. Although CETP did not alter HDL-CE delivery to bile, cholesterol from HDL isolated from CETP TG mice were secreted into the intestinal lumen at a greater rate than WT mice. Conclusion: The data suggest that CETP alters routes of total and HDL cholesterol elimination from the body in mice.

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.

The ATP-binding cassette half-transporters ABCG5 (G5) and ABCG8 (G8) promote secretion of neutral sterols into bile, a major pathway for elimination of sterols. Mutations in either ABCG5 or ABCG8 cause sitosterolemia, a recessive disorder characterized by impaired biliary and intestinal sterol secretion, sterol accumulation, and premature atherosclerosis. The mechanism by which the G5G8 heterodimer couples ATP hydrolysis to sterol transport is not known. Here we examined the roles of the Walker A, Walker B, and signature motifs in the nucleotide-binding domains (NBD) of G5 and G8 using recombinant adenoviruses to reconstitute biliary sterol transport in G5G8-deficient mice. Mutant forms of each half-transporter were co-expressed with their wild-type partners. Mutations at crucial residues in the Walker A and Walker B domains of G5 prevented biliary sterol secretion, whereas mutations of the corresponding residues in G8 did not. The opposite result was obtained when mutations were introduced into the signature motif; mutations in the signature domain of G8 prevented sterol transport, but substitution of the corresponding residues in G5 did not. Taken together, these findings indicate that the NBDs of G5 and G8 are not functionally equivalent. The integrity of the canonical NBD formed by the Walker A and Walker B motifs of G5 and the signature motif of G8 is essential for G5G8-mediated sterol transport. In contrast, mutations in key residues of the NBD formed by the Walker A and B motifs of G8 and the signature sequence of G5 did not affect sterol secretion.

ATP-binding cassette transporter D2 (D2) is an ABC half transporter that is thought to promote the transport of very long-chain fatty acyl-CoAs into peroxisomes. Both D2 and peroxisomes increase during adipogenesis. Although peroxisomes are essential to both catabolic and anabolic lipid metabolism, their function, and that of D2, in adipose tissues remain largely unknown. Here, we investigated the D2 localization and the proteome of D2-containing organelles, in adipose tissue. Centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but deficient in peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with a dense matrix and a diameter of ∼200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment confirmed the scarcity of PEX19 and proteomic profiling revealed the presence of proteins associated with peroxisome, endoplasmic reticulum (ER), and mitochondria. D2 is localized to a distinct class of peroxisomes that lack many peroxisome proteins, and may associate physically with mitochondria and the ER.

Fenofibrate is a peroxisome proliferator-activated receptor (PPAR) α ligand that has been widely used as a lipid-lowering agent in the treatment of hypertriglyceridemia. ABCD2 (D2) is a peroxisomal long-chain acyl-CoA transporter that is highly induced by fenofibrate in the livers of mice. To determine whether D2 is a modifier of fibrate responses, wild-type and D2-deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPARα signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knockdown of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of diet-induced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes.

In healthy individuals, acute changes in cholesterol intake produce modest changes in plasma cholesterol levels. A striking exception occurs in sitosterolemia, an autosomal recessive disorder characterized by increased intestinal absorption and decreased biliary excretion of dietary sterols, hypercholesterolemia, and premature coronary atherosclerosis. We identified seven different mutations in two adjacent, oppositely oriented genes that encode new members of the adenosine triphosphate (ATP)–binding cassette (ABC) transporter family (six mutations inABCG8and one inABCG5) in nine patients with sitosterolemia. The two genes are expressed at highest levels in liver and intestine and, in mice, cholesterol feeding up-regulates expressions of both genes. These data suggest that ABCG5 and ABCG8 normally cooperate to limit intestinal absorption and to promote biliary excretion of sterols, and that mutated forms of these transporters predispose to sterol accumulation and atherosclerosis.

Caveolae were originally identified as flask-shaped invaginations of the plasma membrane in endothelial and epithelial cells (14). Prior to the development of biochemical methods for their purification, caveolae were thought to principally mediate the transcellular movement of molecules (101, 145). Recently, the development of novel purification procedures has greatly expanded our knowledge regarding the putative functions of caveolae in vivo. In this review, we seek to update the working definition of caveolae, describe the functional roles of the caveolin gene family, and summarize the evidence that supports a role for caveolae as mediators of a number of cellular signaling processes.