PAQR proteins and the evolution of a superpower: Eating all kinds of fats: Animals rely on evolutionarily conserved membrane homeostasis proteins to compensate for dietary variation.

Recently published work showed that members of the PAQR protein family are activated by cell membrane rigidity and contribute to our ability to eat a wide variety of diets. Cell membranes are primarily composed of phospholipids containing dietarily obtained fatty acids, which poses a challenge to membrane properties because diets can vary greatly in their fatty acid composition and could impart opposite properties to the cellular membranes. In particular, saturated fatty acids (SFAs) can pack tightly and form rigid membranes (like butter at room temperature) while unsaturated fatty acids (UFAs) form more fluid membranes (like vegetable oils). Proteins of the PAQR protein family, characterized by the presence of seven transmembrane domains and a cytosolic N-terminus, contribute to membrane homeostasis in bacteria, yeasts, and animals. These proteins respond to membrane rigidity by stimulating fatty acid desaturation and incorporation of UFAs into phospholipids and explain the ability of animals to thrive on diets with widely varied fat composition. Also see the video abstract here: https://youtu.be/6ckcvaDdbQg.

AdipoR2 recruits protein interactors to promote fatty acid elongation and membrane fluidity.

The human AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 are multipass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labeled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membrane-fluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified coimmunoprecipitated proteins using mass spectrometry. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely, with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids.

Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins.

The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele.

Paradigm shift: the primary function of the "Adiponectin Receptors" is to regulate cell membrane composition.

The ADIPOR1 and ADIPOR2 proteins (ADIPORs) are generally considered as adiponectin receptors with anti-diabetic properties. However, studies on the yeast and C. elegans homologs of the mammalian ADIPORs, and of the ADIPORs themselves in various mammalian cell models, support an updated/different view. Based on findings in these experimental models, the ADIPORs are now emerging as evolutionarily conserved regulators of membrane homeostasis that do not require adiponectin to act as membrane fluidity sensors and regulate phospholipid composition. More specifically, membrane rigidification activates ADIPOR signaling to promote fatty acid desaturation and incorporation of polyunsaturated fatty acids into membrane phospholipids until fluidity is restored. The present review summarizes the evidence supporting this new view of the ADIPORs, and briefly examines physiological consequences.

Palmitic acid causes increased dihydroceramide levels when desaturase expression is directly silenced or indirectly lowered by silencing AdipoR2.

BACKGROUND: AdipoR1 and AdipoR2 (AdipoRs) are plasma membrane proteins often considered to act as adiponectin receptors with a ceramidase activity. Additionally, the AdipoRs and their yeast and C. elegans orthologs are emerging as membrane homeostasis regulators that counter membrane rigidification by promoting fatty acid desaturation and incorporation of unsaturated fatty acids into phospholipids, thus restoring fluidity. METHODS: Using cultured cells, the effects of AdipoR silencing or over-expression on the levels and composition of several sphingolipid classes were examined. RESULTS: AdipoR2 silencing in the presence of exogenous palmitic acid potently causes increased levels of dihydroceramides, a ceramide precursor in the de novo ceramide synthesis pathway. Conversely, AdipoR2 over-expression caused a depletion of dihydroceramides. CONCLUSIONS: The results are consistent with AdipoR2 silencing leading to increased intracellular supply of palmitic acid that in turn leads to increased dihydroceramide synthesis via the rate-limiting serine palmitoyl transferase step. In agreement with this model, inhibiting the desaturase SCD or SREBF1/2 (positive regulators of SCD) also causes a strong increase in dihydroceramide levels.

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration.

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.

The adiponectin receptor AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 prevent membrane rigidification by exogenous saturated fatty acids.

Dietary fatty acids can be incorporated directly into phospholipids. This poses a specific challenge to cellular membranes since their composition, hence properties, could greatly vary with different diets. That vast variations in diets are tolerated therefore implies the existence of regulatory mechanisms that monitor and regulate membrane compositions. Here we show that the adiponectin receptor AdipoR2, and its C. elegans homolog PAQR-2, are essential to counter the membrane rigidifying effects of exogenously provided saturated fatty acids. In particular, we use dietary supplements or mutated E. coli as food, together with direct measurements of membrane fluidity and composition, to show that diets containing a high ratio of saturated to monounsaturated fatty acids cause membrane rigidity and lethality in the paqr-2 mutant. We also show that mammalian cells in which AdipoR2 has been knocked-down by siRNA are unable to prevent the membrane-rigidifying effects of palmitic acid. We conclude that the PAQR-2 and AdipoR2 proteins share an evolutionarily conserved function that maintains membrane fluidity in the presence of exogenous saturated fatty acids.

AdipoR1 and AdipoR2 maintain membrane fluidity in most human cell types and independently of adiponectin.

The FA composition of phospholipids must be tightly regulated to maintain optimal cell membrane properties and compensate for a highly variable supply of dietary FAs. Previous studies have shown that AdipoR2 and its homologue PAQR-2 are important regulators of phospholipid FA composition in HEK293 cells and Caenorhabditiselegans, respectively. Here we show that both AdipoR1 and AdipoR2 are essential for sustaining desaturase expression and high levels of unsaturated FAs in membrane phospholipids of many human cell types, including primary human umbilical vein endothelial cells, and for preventing membrane rigidification in cells challenged with exogenous palmitate, a saturated FA. Three independent methods confirm the role of the AdipoRs as regulators of membrane composition and fluidity: fluorescence recovery after photobleaching, measurements of Laurdan dye generalized polarization, and mass spectrometry to determine the FA composition of phospholipids. Furthermore, we show that the AdipoRs can prevent lipotoxicity in the complete absence of adiponectin, their putative ligand. We propose that the primary cellular function of AdipoR1 and AdipoR2 is to maintain membrane fluidity in most human cell types and that adiponectin is not required for this function.

Correction: Caenorhabditis elegans PAQR-2 and IGLR-2 Protect against Glucose Toxicity by Modulating Membrane Lipid Composition.

[This corrects the article DOI: 10.1371/journal.pgen.1005982.].

Caenorhabditis elegans PAQR-2 and IGLR-2 Protect against Glucose Toxicity by Modulating Membrane Lipid Composition.

In spite of the worldwide impact of diabetes on human health, the mechanisms behind glucose toxicity remain elusive. Here we show that C. elegans mutants lacking paqr-2, the worm homolog of the adiponectin receptors AdipoR1/2, or its newly identified functional partner iglr-2, are glucose intolerant and die in the presence of as little as 20 mM glucose. Using FRAP (Fluorescence Recovery After Photobleaching) on living worms, we found that cultivation in the presence of glucose causes a decrease in membrane fluidity in paqr-2 and iglr-2 mutants and that genetic suppressors of this sensitivity act to restore membrane fluidity by promoting fatty acid desaturation. The essential roles of paqr-2 and iglr-2 in the presence of glucose are completely independent from daf-2 and daf-16, the C. elegans homologs of the insulin receptor and its downstream target FoxO, respectively. Using bimolecular fluorescence complementation, we also show that PAQR-2 and IGLR-2 interact on plasma membranes and thus may act together as a fluidity sensor that controls membrane lipid composition.

Revisiting the membrane-centric view of diabetes.

Fundamental questions remain unresolved in diabetes: What is the actual mechanism of glucose toxicity? Why is there insulin resistance in type 2 diabetes? Why do diets rich in sugars or saturated fatty acids increase the risk of developing diabetes? Studying the C. elegans homologs of the anti-diabetic adiponectin receptors (AdipoR1 and AdipoR2) has led us to exciting new discoveries and to revisit what may be termed "The Membrane Theory of Diabetes". We hypothesize that excess saturated fatty acids (obtained through a diet rich in saturated fats or through conversion of sugars into saturated fats via lipogenesis) leads to rigid cellular membranes that in turn impair insulin signalling, glucose uptake and blood circulation, thus creating a vicious cycle that contributes to the development of overt type 2 diabetes. This hypothesis is supported by our own studies in C. elegans and by a wealth of literature concerning membrane composition in diabetics. The purpose of this review is to survey this literature in the light of the new results, and to provide an admittedly membrane-centric view of diabetes.

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway.

Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction.

PAQR-2 regulates fatty acid desaturation during cold adaptation in C. elegans.

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant.

Regulation of meiotic telomere dynamics through membrane fluidity promoted by AdipoR2-ELOVL2.

The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.

A Genome-Wide Association Study of Oxypurinol Concentrations in Patients Treated with Allopurinol.

Cohort studies have identified several genetic determinants that could predict the clinical response to allopurinol. However, they have not been commonly used for genome-wide investigations to identify genetic determinants on allopurinol metabolism and concentrations. We conducted a genome-wide association study of a prior cross-sectional investigation of patients from the Montreal Heart Institute Biobank undergoing allopurinol therapy. Four endpoints were investigated, namely plasma concentrations of oxypurinol, the active metabolite of allopurinol, allopurinol, and allopurinol-riboside, as well as allopurinol daily dosing. A total of 439 participants (mean age 69.4 years; 86.4% male) taking allopurinol (mean daily dose 194.5 mg) and who had quantifiable oxypurinol concentrations were included in the genome-wide analyses. Participants presented with multiple comorbidities and received concomitant cardiovascular medications. No association achieved the predefined genome-wide threshold values for any of the endpoints (all p > 5 × 10-8). Our results are consistent with prior findings regarding the difficulty in identifying genetic determinants of drug concentrations or pharmacokinetics of allopurinol and its metabolites, as well as allopurinol daily dosing. Given the size of this genome-wide study, collaborative investigations involving larger and diverse cohorts may be required to further identify pharmacogenomic determinants of allopurinol and measure their clinical relevance to personalize allopurinol therapy.

Females present higher dose-adjusted drug concentrations of metoprolol and allopurinol/oxypurinol than males.

Females present a higher risk of adverse drug reactions. Sex-related differences in drug concentrations may contribute to these observations but they remain understudied given the underrepresentation of females in clinical trials. The aim of this study was to investigate whether anthropometric and socioeconomic factors and comorbidities could explain sex-related differences in concentrations and dosing for metoprolol and oxypurinol, the active metabolite of allopurinol. We conducted an analysis of two cross-sectional studies. Participants were self-described "White" adults taking metoprolol or allopurinol selected from the Montreal Heart Institute Hospital Cohort. A total of 1007 participants were included in the metoprolol subpopulation and 459 participants in the allopurinol subpopulation; 73% and 86% of the participants from the metoprolol and allopurinol subpopulations were males, respectively. Females presented higher age- and dose-adjusted concentrations of both metoprolol and oxypurinol (both p < 0.03). Accordingly, females presented higher unadjusted and age-adjusted concentration:dose ratio of both metoprolol and allopurinol/oxypurinol compared to males (all p < 3.0 × 10-4 ). Sex remained an independent predictor of metoprolol concentrations (p < 0.01), but not of oxypurinol concentrations, after adjusting for other predictors. In addition to sex, age, daily dose, use of moderate to strong CYP2D6 inhibitors, weight, and CYP2D6 genotype-inferred phenotype were associated with concentrations of metoprolol (all p < 0.01). Daily dose, weight, estimated glomerular filtration rate (eGFR), and employment status were associated with oxypurinol concentrations (all p < 0.01). Females present higher dose-adjusted concentrations of metoprolol and oxypurinol than males. This suggests the need for sex-specific dosing requirements for these drugs, although this hypothesis should be validated in prospective studies.

Impact of amiodarone use on metoprolol concentrations, α-OH-metoprolol concentrations, metoprolol dosing and heart rate: A cross-sectional study.

Small studies suggest that amiodarone is a weak inhibitor of cytochrome P450 (CYP) 2D6. Inhibition of CYP2D6 leads to increases in concentrations of drugs metabolized by the enzyme, such as metoprolol. Considering that both metoprolol and amiodarone have ß-adrenergic blocking properties and that the modest interaction between the two drugs would result in increased metoprolol concentrations, this could lead to a higher risk of bradycardia and atrioventricular block. The primary objective of this study was to evaluate whether metoprolol plasma concentrations collected at random timepoints from patients enrolled in the Montreal Heart Institute Hospital Cohort could be useful in identifying the modest pharmacokinetic interaction between amiodarone and metoprolol. We performed an analysis of a cross-sectional study, conducted as part of the Montreal Heart Institute Hospital Cohort. All participants were self-described "White" adults with metoprolol being a part of their daily pharmacotherapy regimen. Of the 999 patients being treated with metoprolol, 36 were also taking amiodarone. Amiodarone use was associated with higher metoprolol concentrations following adjustment for different covariates (p = .0132). Consistently, the association between amiodarone use and lower heart rate was apparent and significant after adjustment for all covariates under study (p = .0001). Our results highlight that single randomly collected blood samples can be leveraged to detect modest pharmacokinetic interactions.

Cardiomyocyte-specific PCSK9 deficiency compromises mitochondrial bioenergetics and heart function.

AIMS: Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9-/- mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. METHODS AND RESULTS: Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9-/- mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9-/- hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9-/- mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9-/- mice. Circulating lipid levels were unchanged in CM-Pcsk9-/- mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9-/- mice had an increased number of mitochondria-endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. CONCLUSION: PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Elevated Adipocyte Membrane Phospholipid Saturation Does Not Compromise Insulin Signaling.

Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.