Research ArticleMetabolism

Enhancing natriuretic peptide signaling in adipose tissue, but not in muscle, protects against diet-induced obesity and insulin resistance

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Science Signaling  25 Jul 2017:
Vol. 10, Issue 489, eaam6870
DOI: 10.1126/scisignal.aam6870
  • Fig. 1 Nprc is increased at the mRNA and protein levels in adipose tissue by HFD feeding.

    (A and B) Nprcfl/fl and NprcAKO mice were fed with a chow diet (CD) or an HFD for 12 weeks. qRT-PCR for the expression of Nprc mRNA relative to 36B4 in gonadal WAT (gWAT) (A) and BAT (B) of CD-fed (n = 3) and HFD-fed (n = 5) Nprcfl/fl mice. (C) Western blotting analysis for NPRC protein performed on the lysates from gWAT and BAT of NprcAKO and Nprcfl/fl mice. Blots are representative of three separate cohorts. ***P < 0.001, unpaired two-tailed Student’s t test.

  • Fig. 2 HFD-fed Nprc−/− mice are leaner and exhibit improved glucose tolerance.

    (A) Image of Nprc−/− and wild-type (WT) mice after 12 weeks on HFD. (B to E) Body weights (BWs) (B), fat mass (C), lean mass (D), and tissue weights (E) of Nprc−/− (n = 5) and WT (n = 6) mice after 12 weeks on HFD. (F) Daily food intake of Nprc−/− (n = 3) and WT (n = 4) mice on HFD. (G) Representative images and hematoxylin and eosin (H&E) staining of BAT, inguinal WAT (iWAT), gWAT, and liver from WT (n = 3) and Nprc−/− (n = 3) mice after 12 weeks on HFD. Scale bars, 100 μm. (H) Immunostaining and quantification of UCP1 in the BAT of WT (n = 2) and Nprc−/− (n = 2) mice after 12 weeks on HFD, as described in Materials and Methods. Scale bar, 200 μm. A.U., arbitrary units. (I and J) Plasma glucose concentration and area under the curve (AUC) during intraperitoneal glucose tolerance test (GTT) (I) and insulin tolerance test (ITT) (J) of Nprc−/− (n = 12) and WT (n = 8) mice after 12 weeks on HFD. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired two-tailed Student’s t test.

  • Fig. 3 HFD-fed NprcMKO and Nprcfl/fl mice show comparable body weight, energy expenditure, and glucose tolerance.

    (A) Image of NprcMKO and Nprcfl/fl mice after 12 weeks on HFD. (B to D) Body weights (B), body composition (C), and tissue weights (D) of NprcMKO (n = 7) and Nprcfl/fl (n = 6) mice after 12 weeks on HFD. QU, quadriceps; GA, gastrocnemius; PLA, plantaris; TA, tibialis anterior; EDL, extensor digitorum longus; SO, soleus. (E to G) O2 consumption (VO2), CO2 production (VCO2), energy expenditure (EE) (E), food intake (F), and physical activity (G) of NprcMKO (n = 7) and Nprcfl/fl (n = 6) mice measured by indirect calorimetry using CLAMS (Comprehensive Lab Animal Monitoring System) after 6 weeks on HFD. Data in (E) were normalized to lean body mass (LBM). See fig. S3 for data calculated per body weight and per mouse. (H and I) Plasma glucose concentration and AUC of NprcMKO (n = 7) and Nprcfl/fl (n = 6) mice after 12 weeks on HFD during intraperitoneal GTT (H) and ITT (I).

  • Fig. 4 HFD-fed NprcAKO mice gain less body weight and exhibit higher energy expenditure and improved glucose tolerance.

    (A) Image of male NprcAKO and Nprcfl/fl mice after 12 weeks on HFD. (B to D) Body weights (B), fat mass (C), and lean mass (D) of NprcAKO (n = 14) and Nprcfl/fl (n = 7) mice after 12 weeks on HFD. (E to G) O2 consumption, CO2 production, energy expenditure (E), food intake (F), and physical activity (G) of NprcAKO (n = 8) and Nprcfl/fl (n = 6) mice measured by indirect calorimetry using CLAMS after 5 weeks on HFD. Data in (E) were normalized to lean body mass. See fig. S3 for data calculated per body weight and per mouse. (H) Fasting plasma insulin concentration of NprcAKO (n = 7) and Nprcfl/fl (n = 3) mice after 12 weeks on HFD. (I and J) Plasma glucose concentration and AUC during intraperitoneal GTT (I) and ITT (J) in NprcAKO (n = 14) and Nprcfl/fl (n = 7) mice after 12 weeks on HFD. *P < 0.05; **P < 0.01, unpaired two-tailed Student’s t test.

  • Fig. 5 Glucose uptake and expression of thermogenesis markers are increased in the BAT of HFD-fed NprcAKO mice.

    (A) Glucose infusion rate of NprcAKO (n = 10) and Nprcfl/fl (n = 8) mice during a hyperinsulinemic-euglycemic clamp experiment performed at 12 weeks of HFD feeding. (B and C) Rates of glucose disposal (Rd) (B) and endogenous glucose appearance (EndoRa) (C) in NprcAKO (n = 10) and Nprcfl/fl (n = 8) mice under basal and clamp states. (D) Rate of glucose uptake (Rg) in the BAT, iWAT, gWAT, gastrocnemius (GA), superficial vastus lateralis (SVL), soleus (SO), heart, and brain of NprcAKO (n = 10) and Nprcfl/fl (n = 8) mice at the end of the clamp experiment. (E) Western blot analysis of thermogenic and mitochondrial proteins in the BAT of NprcAKO and Nprcfl/fl mice after 12 weeks on HFD. Blots are representative of three independent experiments. COX4, cytochrome c oxidase subunit 4; CYTOC, cytochrome c; NDUFS4, NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase ubiquinone iron-sulfur protein 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Immunostaining and quantification of UCP1 in the BAT of NprcAKO (n = 2) and Nprcfl/fl (n = 2) mice after 12 weeks on HFD, as described in Materials and Methods. Scale bar, 200 μm. (G) qRT-PCR for the expression of genes coding thermogenic, mitochondrial, fatty acid oxidation (FAOx) markers, and BAT-derived adipokines in the BAT of NprcAKO (n = 14) and Nprcfl/fl (n = 7) mice after 12 weeks on HFD. Data were relative to 36B4. *P < 0.05; **P < 0.01; ***P < 0.001. P values of glucose infusion rate were obtained by one-tailed Student’s t test. P values of Rd, Ra, Rg, and relative mRNA expression were calculated using unpaired two-tailed Student’s t tests.

  • Fig. 6 HFD-fed NprcAKO mice are protected from hepatic steatosis and adipose tissue inflammation.

    (A) Tissue weight of NprcAKO (n = 14) and Nprcfl/fl (n = 7) mice after 12 weeks on HFD. (B) Representative images and H&E staining of BAT, iWAT, gWAT, and liver from Nprcfl/fl (n = 3) and NprcAKO (n = 3) mice. Scale bars, 100 μm. (C) qRT-PCR for the expression of markers of de novo lipogenesis and fatty acid uptake in the liver of HFD-fed NprcAKO (n = 8) and Nprcfl/fl (n = 7) mice. Data were relative to 36B4. (D) Immunostaining of F4/80 in gWAT sections from Nprcfl/fl (n = 2) and NprcAKO (n = 2) mice. Crown-like structures are indicated by arrows. Scale bars, 100 μm (top) and 50 μm (bottom). (E and F) qRT-PCR for the expression of genes coding macrophage markers and inflammatory cytokines in gWAT (E) and iWAT (F) of HFD-fed NprcAKO (n = 14) and Nprcfl/fl (n = 7) mice. Data were relative to 36B4. (G and H) Picro-sirius staining of gWAT (G) and liver (H) sections from Nprcfl/fl (n = 2) and NprcAKO (n = 2) mice. Collagen fibrils were stained red (H; arrows). Scale bars, 100 μm (top) and 50 μm (bottom). (I) Plasma concentrations of adiponectin of HFD-fed NprcAKO (n = 8) and Nprcfl/fl (n = 7) mice. *P < 0.05; **P < 0.01, unpaired two-tailed Student’s t test.

  • Fig. 7 Insulin signaling, de novo lipogenesis, adipogenesis, lipolysis, and PKG activity in adipose tissues of HFD-fed NprcAKO mice.

    (A to C) Western blotting analysis for proteins involved in insulin signaling (p-Ser473 AKT, p-Thr308 AKT, and GLUT4), de novo lipogenesis (ACC and FASN), adipogenesis (PPARγ, C/EBPα, and ADIPOQ), PKG activity (p-Ser239 VASP), and lipolysis (p-Ser563 HSL and ATGL) performed on lysates from gWAT (A), iWAT (B), and BAT (C) of NprcAKO and Nprcfl/fl mice after 12 weeks on HFD. p, phosphorylated. Blots are representative of three independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/489/eaam6870/DC1

    Fig. S1. Blood pressures and circulating NP concentrations of Nprc−/−, NprcMKO, and NprcAKO mice.

    Fig. S2. Expression of Nprc and Npra in skeletal muscle and adipose tissue.

    Fig. S3. Alternative representation of CLAMS indirect calorimetry data of HFD-fed NprcMKO and NprcAKO mice.

    Fig. S4. HFD-fed NprcAKO and Nprcfl/fl mice show comparable expression of thermogenic-related genes in gWAT and iWAT.

    Fig. S5. Adipocyte size distribution in iWAT and gWAT of HFD-fed Nprcfl/fl and NprcAKO mice.

    Fig. S6. NPRC deficiency enhances NP signaling.

    Table S1. PCR primer sequences.

  • Supplementary Materials for:

    Enhancing natriuretic peptide signaling in adipose tissue, but not in muscle, protects against diet-induced obesity and insulin resistance

    Wei Wu, Fubiao Shi, Dianxin Liu, Ryan P. Ceddia, Robert Gaffin, Wan Wei, Huafeng Fang, E. Douglas Lewandowski, Sheila Collins*

    *Corresponding author. Email: scollins{at}sbpdiscovery.org

    This PDF file includes:

    • Fig. S1. Blood pressures and circulating NP concentrations of Nprc−/−, NprcMKO, and NprcAKO mice.
    • Fig. S2. Expression of Nprc and Npra in skeletal muscle and adipose tissue.
    • Fig. S3. Alternative representation of CLAMS indirect calorimetry data of HFDfed NprcMKO and NprcAKO mice.
    • Fig. S4. HFD-fed NprcAKO and Nprcfl/fl mice show comparable expression of thermogenic-related genes in gWAT and iWAT.
    • Fig. S5. Adipocyte size distribution in iWAT and gWAT of HFD-fed Nprcfl/fl and NprcAKO mice.
    • Fig. S6. NPRC deficiency enhances NP signaling.
    • Table S1. PCR primer sequences.

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    Citation: W. Wu, F. Shi, D. Liu, R. P. Ceddia, R. Gaffin, W. Wei, H. Fang, E. D. Lewandowski, S. Collins, Enhancing natriuretic peptide signaling in adipose tissue, but not in muscle, protects against diet-induced obesity and insulin resistance. Sci. Signal. 10, eaam6870 (2017).

    © 2017 American Association for the Advancement of Science

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