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PAQR4 regulates adipocyte function and systemic metabolic health by mediating ceramide levels

Nature Metabolism volume 6, pages 1347–1366 (2024)Cite this article

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Abstract

PAQR4 is an orphan receptor in the PAQR family with an unknown function in metabolism. Here, we identify a critical role of PAQR4 in maintaining adipose tissue function and whole-body metabolic health. We demonstrate that expression of Paqr4 specifically in adipocytes, in an inducible and reversible fashion, leads to partial lipodystrophy, hyperglycaemia and hyperinsulinaemia, which is ameliorated by wild-type adipose tissue transplants or leptin treatment. By contrast, deletion of Paqr4 in adipocytes improves healthy adipose remodelling and glucose homoeostasis in diet-induced obesity. Mechanistically, PAQR4 regulates ceramide levels by mediating the stability of ceramide synthases (CERS2 and CERS5) and, thus, their activities. Overactivation of the PQAR4–CERS axis causes ceramide accumulation and impairs adipose tissue function through suppressing adipogenesis and triggering adipocyte de-differentiation. Blocking de novo ceramide biosynthesis rescues PAQR4-induced metabolic defects. Collectively, our findings suggest a critical function of PAQR4 in regulating cellular ceramide homoeostasis and targeting PAQR4 offers an approach for the treatment of metabolic disorders.

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Fig. 1: PAQR4 is an important player in regulating adipose tissue function.
Fig. 2: Paqr4-overexpressing mice display insulin resistance despite reduced weight upon HFD feeding.
Fig. 3: Amelioration of PAQR4-induced metabolic dysfunction with adipose tissue transplants or leptin administration.
Fig. 4: Adipocyte-specific deletion of Paqr4 improves glucose homoeostasis in obesity.
Fig. 5: PAQR4 promotes adipose remodelling and adipocyte de-differentiation.
Fig. 6: Blocking ceramide de novo biosynthesis improves PAQR4-induced metabolic dysfunction.
Fig. 7: PAQR4 regulates ceramide levels by mediating ceramide synthase activity.

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Data availability

scRNA-seq data are available in the Gene Expression Omnibus (GSE246712). All other data from this study are available with this paper. Source data are provided with this paper.

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Acknowledgements

We thank staff at the UTSW Animal Resource Center, Histology Core, Metabolic Phenotyping Core, the Live Cell Imaging Core, Transgenic Core, Proteomics Core and Flow Cytometry Facility for their excellent assistance with experiments performed here. We thank Helmholtz Zentrum München for their support and Matthias Tschöp for helpful discussions. We also thank Shimadzu Scientific Instruments for the collaborative efforts in mass spectrometry technology resources. This study was supported by US National Institutes of Health (NIH) grants RC2-DK118620, R01-DK55758, R01-DK099110, R01-DK127274, P01 AG051459 and R01-DK131537 to P.E.S., as well as NIDDK-NORC P30-DK127984; NIH grant R00-AG068239, R01-DK138035 and a Voelcker Fund Young Investigator Award to S.Z., DFG Walter Benjamin Fellowship 444933586 to L.G.S. and AHA Career Development Award 855170 to Q.Z.

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Authors and Affiliations

  1. Touchstone Diabetes Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Qingzhang Zhu, Shiuhwei Chen, Jan-Bernd Funcke, Leon G. Straub, Qian Lin, Shangang Zhao, Chanmin Joung, Zhuzhen Zhang, Dae-Seok Kim, Na Li, Christy M. Gliniak, Line Pedersen, Ruth Gordillo, Christine M. Kusminski & Philipp E. Scherer

  2. Sam and Ann Barshop Institute for Longevity and Aging Studies, Division of Endocrinology, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Shangang Zhao

  3. Center for Hypothalamic Research, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Charlotte Lee

  4. Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany

    Alberto Cebrian-Serrano

  5. German Center for Diabetes Research (DZD), Neuherberg, Germany

    Alberto Cebrian-Serrano

  6. Department of Biomedicine, University of Bergen, Bergen, Norway

    Line Pedersen & Nils Halberg

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  1. Qingzhang Zhu
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Contributions

Q.Z. and P.E.S. conceptualized the study and designed experiments. Q.Z., S.C., J.-B.F., L.G.S., Q.L., S.Z., C.J., Z.Z., D.K., N.L., C.M.G., C.L., R.G., A.C.-S. and C.M.K. conducted experiments. N.H., L.P. and C.M.K. were involved in study design and data flow in the paper. All authors analysed and interpreted data. Q.Z. wrote the manuscript and C.M.K. and P.E.S. revised it.

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Correspondence to Philipp E. Scherer.

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Extended data

Extended Data Fig. 1 PAQR4 is an important player in regulating adipose tissue function.

Gene expression of Class I Paqr’s in less expanded adipose tissues (AT), including gonadal (gWAT), subcutaneous (sWAT), mesenteric white adipose tissues (mWAT), and brown fat (BAT) from obese mice, compared to massively expanded AT from morbidly obese mice, in males (a) and females (b) (n = 3). -Log2 (FC) and P-values are shown. FC, fold change. (c) Tissue expression of Paqr4 in chow-fed mice (n = 6). (d) HFD feeding increases Paqr4 expression in adipose tissues but not heart (n = 10). (e) Paqr4 is specifically induced in adipose tissues in Paqr4ad mice fed dox chow for 1 week (n = 4). (f-g) Time course of lean mass and percentage of lean mass (WT, n = 8; Paqr4ad, n = 10). (h-i) CT scan of WT and Paqr4ad mice. ROI of gWAT, sWAT and BAT were highlighted, and the fat pad volume was measured (n = 5). (j) Cold-acclimated Paqr4ad mice display hypothermia upon fasting (WT, n = 7; Paqr4ad, n = 6). (k) Overlapping glucose tolerance test prior to dox induction (WT, n = 7; Paqr4ad, n = 6). (l-m) Impaired glucose tolerance and insulin sensitivity in Paqr4ad mice (WT, n = 7; Paqr4ad, n = 6). Data shown as mean ± SEM and analysed by two-tailed unpaired t-test (d-e, i) and two-way ANOVA (f-g, j-m).

Source data

Extended Data Fig. 2 Paqr4 overexpressing in adipocytes reduced weight gain upon HFD feeding.

Mice were fed dox-HFD (600 mg/kg except in the dox-response assays). (a) Dox-dose dependent of Paqr4 induction in gonadal fat pads in Paqr4ad mice (WT, n = 6; Paqr4ad, n = 7). (b) Paqr4ad mice are smaller. (c-f) Body weight, fat mass, lean mass, percentage of lean mass and tissue mass of WT and Paqr4ad mice in response to different dox doses (WT, n = 6; Paqr4ad, n = 7). (g) Elevated serum ALT levels in Paqr4ad mice at week 10 (n = 6). (h) Respiratory exchange ratio (RER) assessed in mice fed dox-HFD for 2 weeks (n = 6). (i-j) Substrate oxidation calculated from RER and VO2 (n = 6). (k-o) VO2, VCO2, energy expenditure (EE), physical activity and food intake assessed in mice fed dox-HFD for 2 weeks (n = 6). Data shown as mean ± SEM and analysed by one-way ANOVA followed by Holm-Sidak multiple-comparison test (a), two-way ANOVA (c), and two-tailed unpaired t-test (d-g, h-n).

Source data

Extended Data Fig. 3 Paqr4 overexpressing in adipocytes induces insulin resistance upon HFD feeding.

(a) Immunofluorescence staining of Perilipin (red) and Mac2 (macrophage marker, green) in different fat pads (n = 3). Scale bar 200 µm. (b) Inflammatory gene expressions in BAT at week 3 (WT, n = 12; Paqr4ad, n = 14). (c) Trichrome staining indicated enhanced adipose fibrosis in Paqr4ad mice at week 10 (n = 3). Scale bar 200 µm. (d-e) Dox-dose dependent effects of hyperglycaemia and hyperinsulinaemia in Paqr4ad mice (WT, n = 6; Paqr4ad, n = 7). (f-g) Impaired glucose tolerance and insulin−stimulated glucose disposal in Paqr4ad mice at week 2-3 (WT, n = 14; Paqr4ad, n = 12). (h-i) Dox-dose dependent effects of glucose tolerance in Paqr4ad mice (WT, n = 6; Paqr4ad, n = 7). (j-k) Impaired insulin signalling as accessed by phospho-Akt (Ser473) in various tissues from Paqr4ad mice fed dox-HFD for 6 weeks (WT, n = 3; Paqr4ad, n = 4). Data shown as mean ± SEM and analysed by two-tailed unpaired t-test (b, d-e, i) and two-way ANOVA followed by Holm-Sidak multiple-comparison test (f-h, k).

Source data

Extended Data Fig. 4 Amelioration of PAQR4-induced metabolic defects by adipose transplants or leptin treatment.

Mice receiving adipose transplants (sWAT) were fed dox chow for 10 weeks. (a-b) Transplants do not rescue body weight and liver/body weight ratio (Sham, n = 8 per group; Transplant, n = 5 per group). (c) H&E staining of liver (n = 3). Scale bar 200 µm. (d) Transplants do not rescue serum adiponectin levels (Sham, n = 8 per group; Transplant, n = 5 per group). (e-f) Two weeks of leptin perfusion does not rescue body weight and adipose tissue mass, but reduces liver weight in Paqr4ad mice (WT, n = 7; Paqr4ad, n = 5 per group). (g) Unaltered food intake (WT, n = 7; Paqr4ad, n = 5 per group). (h) Leptin improves adipose health and fatty liver in Paqr4ad mice (WT, n = 3; Paqr4ad, n = 4 per group). Scale bar 200 µm. (i) Reduced body weight in ob/ob:Paqr4ad mice (ob/ob, n = 8; ob/ob:Paqr4ad, n = 4; Ob/ob and Paqr4ad, n = 5). (j) Food intake is reduced transiently upon dox induction and recovered thereafter (ob/ob, n = 8; ob/ob:Paqr4ad, n = 4; Ob/ob and Paqr4ad, n = 5). (k) Further aggravated glucose intolerance in ob/ob:Paqr4ad mice (ob/ob, n = 8; ob/ob:Paqr4ad, n = 4; Ob/ob and Paqr4ad, n = 5). (l) Insulin release during glucose tolerance test (ob/ob, n = 8; ob/ob:Paqr4ad, n = 4; Ob/ob and Paqr4ad, n = 5). Data shown as mean ± SEM and analysed by two-way ANOVA (a-b, d-e, g, i-l) and one-way ANOVA followed by Holm-Sidak multiple-comparison test (f).

Source data

Extended Data Fig. 5 Adipocyte-specific deletion of Paqr4 improves glucose homoeostasis in obesity.

(a-b) Specific recombination of Paqr4 (representative images from 3 independent assays with similar results) and downregulation in gene expression levels in Paqr4iAKO mice fed dox chow for 2 weeks (n = 6). (c-d) Comparable body weight and body composition in Paqr4iAKO mice fed dox chow for 20 weeks (Paqr4fl/fl, n = 7; Paqr4iAKO, n = 6). (e) Tissue weights in Paqr4iAKO mice fed dox chow for 20 weeks (n = 6). (f-g) Comparable glucose tolerance and insulin sensitivity after 8-9 weeks of dox chow feeding (Paqr4fl/fl, n = 7; Paqr4iAKO, n = 6). (h-j) Moderately improved glucose tolerance and insulin-mediated glucose disposal after 19-20 weeks of dox chow feeding (Paqr4fl/fl, n = 7; Paqr4iAKO, n = 6). (k-p) Comparable food intake, RER, VO2, VCO2, energy expenditure (EE), and physical activity after 15 weeks of dox-HFD feeding (n = 6). (q-r) Body composition and tissue weights after 20 weeks of dox-HFD feeding (Paqr4fl/fl, n = 16; Paqr4iAKO, n = 22). (s-u) Slightly improved glucose tolerance in Paqr4iAKO mice after 2 and 8 weeks and insulin-mediated glucose disposal after 9 weeks of dox-HFD feeding (Paqr4fl/fl, n = 16; Paqr4iAKO, n = 22). Data shown as mean ± SEM and analysed by two-tailed unpaired t-test (b, d-e, l-r) and two-way ANOVA (c, f-k, s-u).

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Extended Data Fig. 6 PAQR4 suppresses adipogenesis.

(a) PAQR4 decreases adipocyte marker expressions during adipogenesis (Group day 4, n = 6; other groups, n = 4) and reduces lipid accumulation as assessed by Oil Red O staining (n = 3) in stromal vascular fraction (SVF) cells. Scale bar 200 µm. (b) Expression of adipocyte markers (n = 6) and Oil Red O staining (n = 3) during adipogenesis upon Paqr4 deletion. Scale bar 200 μm. (c) Minor changes of cell cycle by PAQR4 and C2-ceramide at day 2 of adipogenesis (n = 3 biological samples). (d) H&E staining in gWAT and sWAT during adipose development of mice fed dox chow from E13 onwards (n = 4 mice per group). E18, embryotic day 18; P1 and P7, postnatal day 1 and 7. Scale bar 100 µm. (e) Immunofluorescence staining of Perilipin (red) and Mac2 (green) in sWAT during adipose development of mice fed dox chow from E13 onwards (n = 4). Scale bar 50 µm. (f) H&E staining in BAT during adipose development of mice fed dox chow from E13 onwards (n = 4). Scale bar 100 µm. (g) Smaller size of BAT and sWAT on P7 in Paqr4ad mice fed dox chow from E13 onwards. (h) Body composition at week 6 in mice fed dox chow from E13 onwards (WT, n = 9; Paqr4ad, n = 6). Data shown as mean ± SEM and analysed by two-way ANOVA (a), one-way ANOVA followed by Holm-Sidak multiple-comparison test (c), and two-tailed unpaired t-test (b, h).

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Extended Data Fig. 7 Effects of PAQR4 on sphingolipid levels in adipose tissues.

(a-c) Sphingolipid profiles in sWAT and gWAT of Paqr4ad mice (WT, n = 10; Paqr4ad, n = 6) or Paqr4iAKO mice (Paqr4fl/fl, n = 8; Paqr4iAKO, n = 15) fed dox-HFD. Data shown as mean ± SEM and analysed by two-tailed unpaired t-test (a-c).

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Extended Data Fig. 8 Blocking ceramide synthesis improves PAQR4-induced metabolic defects.

(a) Oil Red O staining (n = 3) and adipocyte markers (n = 4 biological samples) indicate that C2-Ceramide (C2-Cer) blocks adipogenesis in sWAT stromal vascular fraction (SVF) cells. Scale bar 200 µm. (b) C2-Cer causes de-differentiation of adipocytes with expression of fibroblast marker S100A4 and decreased expression of adipocyte markers (n = 6 biological samples). Adipocytes were treated with C2-Cer post-differentiation (PD) for the indicated days. Scale bar 100 µm. (c) Expression of adipogenic markers in SVF cells that were differentiated in the absence or presence of 5 µM C2-Cer or 10 µM myriocin (Myr) at day 4 of post-differentiation (n = 6 biological samples). (d-e) Body weight and weight gain upon Myr treatment in mice priorly fed dox-HFD for 12 weeks (n = 6). (f) H&E staining of adipose tissues and liver after 5 weeks of Myr treatment (WT, n = 3; Myr, n = 4). Scale bar 200 µm. (g) Tissue weights after 5 weeks of Myr treatment (n = 6). (h) Myr treatment reduced liver triglyceride content in Paqr4ad mice (n = 6). (i) Food intake upon Myr treatment in mice priorly fed dox-HFD for 12 weeks (n = 6). * indicates comparisons between groups of WT-Veh and WT-Myr; # indicates comparisons between groups of Paqr4ad-Veh and Paqr4ad-Myr. Data shown as mean ± SEM and analysed by two-way ANOVA followed by Holm-Sidak multiple-comparison test (a, c-e, g-i) and two-tailed unpaired t-test (b).

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Extended Data Fig. 9 PAQR4 regulates ceramide synthase activity.

(a-c) Fluorescent activity assays indicate PAQR4 itself does not have ceramide synthase or ceramidase activity. Representative images from 2 independent assays with similar results. (d) Consumption of D7-spingosine that originally comes from D7-sphinganine during the CERS activity assay (n = 3). (e-f) PAQR4 further increases CERS2 or CERS5-induced ceramide levels in HEK293T cells (n = 3). Ceramide species are measured in lysates of cells overexpressing Myc-Cers2 (C2), Myc-Cers5 (C5), and FLAG-Paqr4 (P4) alone or in combination of the two individual lysates (C2P4 and C5P4) as indicated. (g-h) Enzymatic activities of CERS2 and CERS5 are determined in individual cell lysates or in combination of the two individual lysates as indicated. Products of stable isotope labelled C24:1 and C16:0 D7-dehydroceamide (D7-DhCer) that are generated from the substrates D7-sphinganine and C24:1 or C16:0 acyl-CoA, which are mainly utilized by CERS2 and CERS5, respectively, reflecting their enzymatic activities (n = 3). Data shown as mean ± SEM and analysed by one-way ANOVA followed by Holm-Sidak multiple-comparison test (d-h).

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Extended Data Fig. 10 PAQR4 promotes ceramide synthase stability.

(a) Cers expressions during SVF-derived adipocyte differentiation (Day 4 group, n = 6; other groups, n = 4). (b) Cers expressions in sWAT of Paqr4ad and WT mice fed dox chow for 2 weeks (WT, n = 12; Paqr4ad, n = 18). (c) Representative staining of ER marker KDEL and CERS2 in sWAT and gWAT from 2 weeks of dox chow-fed Paqr4ad and WT mice (n = 3). Scale bar 50 µm. (d) Quantification analysis of Myc-CERS2 (C2, normalized by β-Tubulin) during the treatment of MG132 or bafilomycin A1 (BFA) (n = 3). (e) PAQR4 prevents CERS2 lysosomal degradation. HEK293A cells were transfected with Halo-Cers2 and SNAP-Paqr4 vectors for 24 h, and then treated with BFA for 8 h. CERS2 was labelled with HaloTag TMR ligand (red) and PAQR4 was labelled with SNAP-Cell Oregon Green (green). Cells were then fixed and stained with lysosomal marker LAMP1. Scale bar, 50 μm. Representative images from 2 independent assays with similar results. (f-g) ‘Pulse–chase’ indicates PAQR4 stabilization of CERS2. CERS2 or PAQR4 were first labelled with HaloTag TMR ligand (red) or SNAP-Cell Oregon Green (green), respectively, cells were then imaged at the indicated time points. About 20 cells of each condition were analysed. (n = 3 biological samples for each group.) Scale bar 10 μm. (h) Effect of PAQR4 on CERS5 protein levels in the presence of cycloheximide (CHX), MG132, or BFA (n = 2). (i) PAQR4 prevents CERS5 lysosomal degradation in HEK293A cells. Cells were transfected with Halo-Cers5 and SNAP-Paqr4 vectors for 24 h, and then treated as in (e). Representative images from 2 independent assays with similar results. Scale bar 50 μm. Data shown as mean ± SEM and analysed by two-way ANOVA followed by Holm-Sidak multiple-comparison test (a, g) and two-tailed unpaired t-test (b).

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Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figs. 1–13 and Supplementary Table 1.

Reporting Summary

Supplementary Video 1

CT imaging of WT mice.

Supplementary Video 2

CT imaging of Paqr4-transgenic mice.

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Zhu, Q., Chen, S., Funcke, JB. et al. PAQR4 regulates adipocyte function and systemic metabolic health by mediating ceramide levels. Nat Metab 6, 1347–1366 (2024). https://doi.org/10.1038/s42255-024-01078-9

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