Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes

Abstract

The ability of mammals to resist body fat accumulation is linked to their ability to expand the number and activity of "brown adipocytes" within white fat depots. Activation of β-adrenergic receptors (β-ARs) can induce a functional "brown-like" adipocyte phenotype. As cardiac natriuretic peptides (NPs) and β-AR agonists are similarly potent at stimulating lipolysis in human adipocytes, we investigated whether NPs could induce human and mouse adipocytes to acquire brown adipocyte features, including a capacity for thermogenic energy expenditure mediated by uncoupling protein 1 (UCP1). In human adipocytes, atrial NP (ANP) and ventricular NP (BNP) activated PPARγ coactivator-1α (PGC-1α) and UCP1 expression, induced mitochondriogenesis, and increased uncoupled and total respiration. At low concentrations, ANP and β-AR agonists additively enhanced expression of brown fat and mitochondrial markers in a p38 MAPK-dependent manner. Mice exposed to cold temperatures had increased levels of circulating NPs as well as higher expression of NP signaling receptor and lower expression of the NP clearance receptor (Nprc) in brown adipose tissue (BAT) and white adipose tissue (WAT). NPR-C(-/-) mice had markedly smaller WAT and BAT depots but higher expression of thermogenic genes such as Ucp1. Infusion of BNP into mice robustly increased Ucp1 and Pgc-1α expression in WAT and BAT, with corresponding elevation of respiration and energy expenditure. These results suggest that NPs promote "browning" of white adipocytes to increase energy expenditure, defining the heart as a central regulator of adipose tissue biology.

Figure 1. Mice lacking NPRC have reduced fat mass.

(A) Ratio between weight of isolated tissues and total body weight of NPR-C^+/+ mice compared with that of NPR-C^–/– mice. eWAT, epididymal WAT; rpWAT, retroperitoneal WAT; iWAT, inguinal WAT. **P < 0.01, ***P < 0.001 versus control. (B) Representative images of BAT from NPR-C^+/+ mice compared with those from NPR-C^–/– mice. (C and D) Representative hematoxylin-eosin staining of iBAT and epididymal WAT sections, respectively, taken from NPR-C^+/+ mice compared with those from NPR-C^–/– mice. Original magnification, ×20 (left column); ×40 (right column).

Figure 2. Adipose tissues from NPR-C^–/– mice express higher levels of brown adipocyte marker genes as well as NPRA.

Gene expression analysis of Ucp1, Pgc-1α, Cycs, and NPRA in (A–D) BAT, (E–H) epididymal WAT, and (I–L) inguinal WAT from NPR-C^+/+ and NPR-C^–/– mice. Typical cycle threshold values for NPR-C^+/+ samples are presented above each bar. *P < 0.05 versus NPR-C^+/+; **P < 0.01 versus NPR-C^+/+.

Figure 3. ANP increases brown adipocyte marker genes in hMADS cells.

Cells were treated with ANP (1–100 nM) for 6 hours and levels of UCP1, PGC-1α, CYCS, and PRDM16 gene expression were measured. *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated. Typical cycle threshold values in untreated cells are as follows: UCP1, 27; PGC-1α, 23; cytochrome c, 20; and PRDM16, 29.

Figure 4. NPs induce UCP1, PGC-1α, and cytochrome c levels in human adipocytes through PKG.

(A) hMADS cells were treated or not with ANP, Iso, or the β3-AR agonist L755 (100 nM each), and protein levels were measured by Western blot with GAPDH as the internal standard, as described in Methods. CYTO C, cytochrome c; C, control. (B) As in A after treatment or not with 100 nM BNP. (C) hMADS cells were pretreated or not for 30 minutes with 500 μM PKGi followed by ANP (100 nM, 6 hours), and mRNA levels for indicated genes were measured. Typical cycle threshold values in untreated cells are as follows: UCP1, 27; PGC-1α, 23; CYCS, 20. (D) Human subcutaneous adipocytes were treated as in A and C, and samples were analyzed for protein levels by Western blotting or (E) mRNA levels of indicated genes. Typical cycle threshold values in untreated cells are as follows: UCP1, 29; PGC-1, 24; and cytochrome c, 23. Results are mean ± SD of 3 to 5 independent experiments. **P < 0.01, ***P < 0.001 ANP versus untreated controls; ^††P < 0.01 PKGi versus untreated controls.

Figure 5. ANP promotes mitochondrial biogenesis in human adipocytes.

(A) Heat map of mitochondrial PCR array results from hMADS adipocytes that were treated or not with 100 nM ANP for 6 hours. The range of fold change versus basal was 3.1 to –1.4. (B) Validation of response to ANP and effect of PKGi on select genes from PCR array data. SIRT3 was also measured. **P < 0.01, ***P < 0.001 for ANP versus untreated cells; ^†P < 0.05, ^††P < 0.01 PKGi versus untreated control. (C) Phase-contrast and MitoTracker fluorescence images of hMADS cells that were treated or not with 100 nM ANP, as described in Methods. Mitotracker is identified by the orange-red color. DAPI-stained nuclei are blue (original magnification, ×20). (D) mtDNA content was quantified by qPCR, as described in Methods. Bars represent mean ± SEM. *P < 0.05 compared with control.

Figure 6. Respiration and uncoupling are increased by β-agonists and ANP.

(A) Representative measurements of the percentage increase in OCRs and relative baseline rates in hMADS cells in response to ANP, Iso, or L755, and effects of respiratory chain modulators. OCR measurements before drug injections (i) were set as 100%. At the time points indicated in the figures, the following were injected sequentially: (ii) oligomycin (ATP synthase inhibitor), (iii) FCCP, and (iv) rotenone (complex I inhibitor). Each data point is a mean of 9 to 10 wells. Supplemental Figure 1 shows histograms summarizing the average maximal percentage increase of OCRs or ECARs over their baseline rates. (B) Representative measurements of the percentage change in OCRs in hMADS cells after injection of oligomycin, as an index of uncoupled respiration. The OCR before oligomycin injection was set as 100%. Each data point is the mean of 9 to 10 wells. (C) Basal levels of percentage OCR in hMADS cells before treatment overnight with 100 nM ANP or ISO. The OCR measured in control cells (no pretreatment) was set as 100%. ***P < 0.001 versus control. (D) Representative measurements of OCRs in hMADS cells that had been pretreated (Prtxt) or not treated (–) overnight with ANP or Iso (100 nM) and analyzed as in A. At the times indicated, acute injections of drugs (i) and of the respiratory chain modulators (ii–iv) were provided. (E) The percentage of oligomycin-insensitive OCRs in pretreated and acutely treated hMADS cells as in B.

Figure 7. Activation of brown adipocyte marker genes by ANP and PKG requires p38 MAPK.

(A) hMADS adipocytes were pretreated or not for 30 minutes with p38 inhibitor SB (10 μM) or PKGi (500 μM) as indicated, followed by 100 nM ANP for 1 hour. Cell lysates were processed for Western blotting, as described in Methods, to detected phosphorylated and total p38 MAPK, MK2, and ATF2, with GAPDH as the internal standard. (B) hMADS adipocytes were treated as in A for 6 hours. Cell lysates were prepared, and UCP1, PGC-1α, cytochrome c, and GAPDH were measured by immunoblotting using specific antisera, as indicated in Methods.

Figure 8. ANP signaling increases transcriptional activity and transcription factor recruitment to the UCP1 enhancer.

(A) Schematic representation of the human UCP1 gene with PPRE and CRE2 indicated. (B) UCP1 enhancer reporter gene activity in transfected hMADS cells treated or not with ANP (100 nM), as described in Methods. Results are mean ± SEM. ***P < 0.001 versus untreated cells (n = 6). (C) ChIP assay. hMADS adipocytes were pretreated or not with PKGi or SB, followed by ANP, as described in Methods. Chromatin was prepared and immunoprecipitated with antisera against PGC-1α or ATF2. The final DNA extraction was amplified by PCR using a primer encompassing the UCP1 enhancer region or exon 2 as a control region (ENSG00000109424).

Figure 9. Combining β-agonist and ANP treatment augments their individual capacities to induce brown adipocyte markers.

hMADS adipocytes were treated with (A–C) 1 nM or (D–F) 10 nM of ANP or Iso, both alone and together (ANP+Iso), for 6 hours, and mRNA levels were measured for (A and D) UCP1, (B and E) PGC-1α, and (C and F) CYCS. *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated cells; ^#P < 0.01 for ANP plus Iso versus ANP or Iso treatment alone. (G) In a sample experiment, similarly treated hMADS adipocytes were harvested, cell lysates were prepared, and Western blotting using specific antisera was performed, as indicated in Methods, and normalized for GAPDH (values are reported under each band.

Figure 10. Physiological modulation of the NP system in C57BL/6J mice after 6 hours of cold exposure (4°C; n = 9) compared with control mice maintained at room temperature (25°C; n = 8).

(A) BNP circulating plasma levels. (B and C) Gene expression levels of ANP and BNP in the heart. *P < 0.05, **P < 0.01 versus 25°C. (D and E) NPRA/NPR-C gene expression ratio in iBAT and epididymal WAT, respectively. (F) Representative NPRC protein levels in WAT of C57BL/6 mice maintained at 25°C or at 4°C for 6 hours. GAPDH was used as internal control. Samples from NPR-C^–/– mice were used as controls for NPRC antibody specificity.

Figure 11. BNP infusion increased oxygen consumption, energy expenditure, and expression of brown adipocyte markers.

C57BL/6 mice were treated for 7 days with saline (0.9% NaCl) or with BNP (2 ng/kg/ml) via Mini-pump. Food intake, physical activity, and respiration were measured by the Oxymax Comprehensive Lab Animal Monitoring System. (A) Plasma BNP levels measured on day 7. (B) Oxygen consumption and (C) energy expenditure (EE) during the light and dark periods. *P < 0.05; **P < 0.01; ***P < 0.001. Western blot in (D) iBAT (BAT) and (E) inguinal WAT for UCP1, PGC-1α, and cytochrome c protein levels. GAPDH was used as internal standard.

Figure 12. Model for parallel β-AR and NPRA activation of p38 MAPK to trigger expression of the brown fat thermogenic gene program.

Catecholamines bind the heptahelical β-ARs on adipocytes to activate the G protein Gs and increase cAMP (pink ovals). cAMP binds the regulatory (R) subunits of PKA. The released catalytic (C) subunits (purple ovals) can then phosphorylate targets, including HSL and perilipin (Peri A), to allow lipolysis of stored triglycerides. Lipolysis can also be activated by NPs. ANP and BNP bind to guanylyl cyclase (GC) receptor NPRA to increase cGMP (yellow circles). Adipocytes also express NPRC that mainly removes NPs from circulation. The cGMP produced by NPRA activates PKG (α and β subunits are represented as thin green and yellow ovals, respectively), whose substrate specificity for phosphorylation closely overlaps that of PKA. Thus, PKG can phosphorylate the same targets as PKA to elicit lipolysis. β-ARs and PKA can also activate a protein kinase cascade, culminating in the activation of p38α MAPK (p38 MK). Now, we can add NPs and PKG as parallel activators of p38α MAPK. Thus, in response to β-agonist or NPs, p38α MAPK phosphorylates (light blue ovals) the transcriptional regulators ATF2 and PGC-1α. PGC-1α interacts with PPARγ and RXRα. These phosphorylated and activated factors are recruited to specific motifs within the UCP1 enhancer (PPRE, CRE2) to increase its gene expression (blue arrow). ATF2 also binds the CRE in the PGC-1α promoter to increase transcription (blue arrow) and increase the amount of PGC-1α.