The incidence of obesity is increasing throughout the western world 1,2. Obesity is associated with cardiac disease 3. Hence, cardiac disease in obese individuals is an increasing challenge for the healthcare system.
Triglyceride accumulation in the heart promotes the impairment of cardiac function 3. Thus, obese mice and mice with increased cardiac triglyceride stores due to genetic disorders in lipid metabolism display contractile cardiac dysfunction 4–7. In humans, cardiac lipid accumulation also leads to impaired cardiac function 8.
Elevated plasma concentrations of the natriuretic peptides ANP and BNP are diagnostic for cardiac dysfunction 9. Paradoxically, even though obesity is associated with cardiac dysfunction, plasma concentrations of atrial natriuretic peptide (ANP) and BNP are reduced in obese individuals 10. This is likely caused by impaired cardiac production 11, but increased clearance through natriuretic peptide receptor-C (NPR-C), as reported in obese individuals 12, could also contribute to the lower plasma levels.
Interestingly, ANP stimulates lipolysis in white adipose tissue through cGMP-dependent phosphorylation of hormone-sensitive lipase (HSL) through the NPR-A receptor 13,14. Both HSL and NPR-A are highly expressed in cardiac myocytes 15,16. Low plasma ANP concentration in the obese could reduce lipolysis and increase triglyceride stores in the obese heart. Hence, in theory, pharmacological stimulation of NPR-A might be a new target that could reduce both blood pressure and cardiac triglyceride stores in obesity.
To test this idea, we have examined the effects of ANP infusion and NPR-A receptor deficiency on cardiac lipid stores in mice.
Materials and methods
ob/ob mice (n=30), ob/+ mice (n=10), and C57Bl6 mice (n=20) were purchased from Taconic (Ry, Denmark). Heterozygote NPR-A knockout mice (NPR-A KO) (Stock Number: 004374; Jackson Laboratories, Bar Harbor, Maine, USA) were bred to obtain the desired genotypes. The mice were housed with free access to food and water under temperature-controlled conditions. Alzet osmotic minipumps (D1007) were purchased from Scanbur (Sollentuna, Sweden), and the murine ANP peptide (H2100) was purchased from Bachem (Bubendorf, Switzerland).
For the ANP-infusion experiments the mice were anesthetized with hyponorm/midazolam before subcutaneous implantation of the pump. Postoperative analgesia was obtained with subcutaneous bupivacaine injections. After 7 days of infusion, the mice were anesthetized with hypnorm/midazolam and blood samples were taken from the retro-orbital venous plexus before they were killed by cervical dislocation.
Blood pressure was determined in awake mice with the BP-2000 Blood Pressure Analysis System (Visitech Systems Inc., Apex, North Carolina, USA) as previously described 4.
NPR-A KO mice (n=9) and wild-type littermate controls (n=9) were fasted for 16 h before blood sampling and killed by cervical dislocation. A control group of fed mice [NPR-A KO mice (n=10) and wild-type littermate controls (n=11)] were examined at the same time as the fasted mice. The heart was removed immediately after they were killed, carefully cleansed for pericardial adipose tissue, divided into atria and left and right ventricles before being snap-frozen in liquid nitrogen, and kept at −80°C until analysis.
All animal experiments were approved by the Danish Animal Experiments Inspectorate (Dyreforsoegstilsynet).
HL-1 cells were incubated at 37°C and 5% CO2 in flasks precoated with a 0.02% gelatin/0.005 mg/ml fibronectin mix (Sigma-Aldrich, Brondby, Denmark) as described 17. Lipid accumulation was induced with 0.5 mmol/l oleic acid [bound to BSA 2 : 1 (mol : mol)] (Sigma). As control, cells were incubated in medium containing 0.25 mmol/l fatty acid-free BSA (Sigma). Before ANP stimulation HL-1 cells were incubated for 1 h in lipolysis medium (Claycomb medium without serum, norephedrine, and oleic acid but with 1% BSA). ANP (Bachem) was diluted in lipolysis medium to a final concentration of 10–7 pmol/l. Cells were incubated for 6 h before lipids were extracted and analyzed by TLC. Intracellular cGMP was measured with ELISA (CG200; Sigma) after 15 min of ANP stimulation according to the manufacturer’s instructions.
Blood samples were drawn into precooled Na2–EDTA tubes and centrifuged within 10 min. Plasma was snap-frozen in liquid nitrogen and kept at −80°C until analysis. ANP was measured with ELISA (Cat. No: EK-005-24; Phoenix Phamaceuticals, Karlsruhe, Germany or Cat. No: S-1132; Bachem). Plasma free fatty acids, cholesterol, and triglyceride concentrations were determined with enzymatic kits (Wako NEFA C kit; TriChem Aps, Frederikssund, Denmark, GPO-trinder; Sigma-Aldrich, and Chod-PAP; Roche, Hvidovre, Denmark, respectively). Insulin was measured with ELISA (Cat. No: EIA-3439, DRG, Germany).
mRNA was extracted with the Trizol reagent (Invitrogen, Taastrup, Denmark), and the quality of the mRNA was controlled with capillary gel electrophoresis on a 2100 Bioanalyzer (Agilent Technologies, Hoersholm, Denmark). cDNA was synthesized with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, California, USA). Real-time PCR analyses were performed using the ABI 7900 HT sequence detection system (Applied Biosystems).
Cell and tissue lipid analysis
Lipids were extracted using chloroform/methanol, dried under N2, redissolved in toluol (4 μl/mg wet weight of tissue), and quantified by thin-layer chromatography (TLC) 18. TLC plates (DC-Fertigplatten SIL G-25, 20×20 cm; Macherey-Nagel, Düren, Germany) were washed in acetone and activated at 110°C for 30 min. Samples and a dilution series of a standard were applied. The standard contained triglyceride, free cholesterol, and cholesterol esters. The plates were developed in a two-step procedure using hexan:diethylether:ethylacetate (70 : 30 : 2) and pure hexan. After development, the plates were placed in 10% cupric sulfate (w/v) in 8% phosphoric acid (v/v) for 10 s, dried with a hair dryer, and baked for 2 min at 200°C. For quantification, the TLC plates were scanned at 300×300 dpi with a flatbed scanner. The digitalized images were analyzed with Image-J software (Bethesda, Maryland, USA, http://imagej.nih.gov/ij/). All samples were analyzed at least twice on different TLC plates.
Tissue triglyceride uptake
Overnight fasted C57Bl6 mice were given a dose of radiolabelled triolin (PerkinElmer, Skovlunde, Denmark) dissolved in olive oil by oral gavage (300 μl/1.28×107 DPM). Blood samples were drawn before the gavage and after 30, 60, and 120 min. The mice were killed with cervical dislocation and perfused with sterile saline before the tissue was weighed, snap-frozen, and stored at −80°C until analysis. Aliquots (5 μl) were counted in a gamma counter (1470 Automatic Counter; PerkinElmer Danmark A/S, Skovlunde, Denmark) for 10 min. Tissues were incubated in Solvable (PerkinElmer) according to the manufacturer’s instructions and counted in a gamma counter (1470 Automatic Counter; PerkinElmer Danmark A/S).
Two-group comparison was performed with Student’s t-test or the nonparametric Mann–Whitney U-test when appropriate. P value less than 0.05 was considered statistically significant.
Low-dose ANP (125 ng/kg/min) in obese ob/ob mice led to a 15% nonsignificant increase in plasma ANP (P=0.06) and did not affect the blood pressure (Table 1). However, there was a modest reduction in plasma triglycerides and cholesterol in ANP-infused mice (Table 1). High-dose ANP (500 ng/kg/min) increased plasma ANP in obese (from 12±8 to 108±25 pmol/l, P=0.03) and lean mice (from 30±12 to 94±27 pmol/l, P=0.08). High-dose ANP infusion reduced both systolic and diastolic blood pressure by ∼16 mmHg (P<0.05) in obese mice and by ∼20 mmHg in lean mice (P<0.005) (Table 1). High-dose ANP infusion did not affect plasma lipids, insulin, nor glucose in obese mice. In fed lean mice, free fatty acids, triglycerides, glycerol, and glucose were increased in the ANP-infused group (Table 1).
Cardiac triglyceride stores were not affected by either low-dose nor high-dose ANP infusion (Fig. 1a and b).
Cardiac mRNA expression of NPR-A and NPR-C was unaffected by ANP infusion in both obese and lean mice (Supplementary Fig. 1, Supplemental digital content 1, http://links.lww.com/CAEN/A4). High-dose ANP infusion in OB/OB and OB/+ mice did not affect NPR-A, NPR-C, or markers of brown adipose tissue (BAT) in the epididymal fat pads (Supplementary Fig. 2, Supplemental digital content 2, http://links.lww.com/CAEN/A5), nor was uptake of triglyceride affected by high-dose ANP infusion in brown and white adipose tissue, liver, and skeletal muscle in lean mice (Supplementary Fig. 3, Supplemental digital content 3, http://links.lww.com/CAEN/A6).
When HL-1 cells were grown in oleic acid-supplemented culture media, the triglyceride content increased almost linearly in 24 h and the triglyceride stores could rapidly be mobilized after removal of oleic acid from the culture medium (Supplementary Fig. 4a and b, Supplemental digital content 4, http://links.lww.com/CAEN/A7). ANP (1×10–7 mol/l) led to an ∼19-fold increase in the intracellular cGMP of oleic acid-loaded HL-1 cells (P<0.05, Fig. 2a), but did not affect cellular triglyceride stores (Fig. 2b). We could not detect any effect of ANP on HSL mass or phosphorylation by western blot, but the amount of phosphorylated HSL was very low in HL-1 cells, even in cells treated with 8-bromoguanosine as a positive control (data not shown).
To examine whether impairment of the ANP signaling cascade induces cardiac triglyceride accumulation, male NPR-A KO mice and wild-type littermate controls were studied. Except for increased levels of plasma glycerol in NPR-A KO mice, we found no difference in plasma lipids compared with wild-type littermate controls both in the fed and fasted state (Table 2). NPR-A KO mice had enlarged hearts as previously described (Table 2) 19. In fed mice, cardiac triglyceride stores were similar in NPR-A KO mice and wild-type littermate controls. To induce an increase in cardiac lipid content similar to what is seen in obese mice, the NPR-A KO mice and wild-type controls were fasted for 16 h. This induced a similar increase in plasma free fatty acids in NPR-A KO mice and wild-type controls (Table 2), providing equal amounts of energy substrate for the heart. NPR-A KO mice accumulated significantly less triglycerides in the heart compared with wild-type control mice after fasting (Fig. 3a and b).
In the present study, we examined whether pharmacological stimulation of cardiac NPR-A might be a new target that could reduce both blood pressure and cardiac triglyceride stores in obesity. The receptor, intracellular kinases, and HSL are present in cardiac myocytes 13,15,16. ANP indeed caused cGMP formation in cultured cardiomyocytes but did not affect triglyceride stores in oleic acid-loaded HL-1 cells. Also, triglyceride stores in the heart were unaffected by in-vivo ANP stimulation in lean and obese mice. Notably, the lack of effect of ANP in vivo was not caused by a lack of physiological activity of the infused ANP peptide, as blood pressure was reduced at high-dose infusion. Recently, ANP and BNP have been suggested to induce differentiation of white adipose tissue into BAT through activation of the NPR-A receptor 20. If such a differentiation had a physiological impact, it would result in a faster triglyceride clearance 21. In the present study we found no change in triglyceride uptake in white or BAT, suggesting that this pathway is of minor importance in mice. This is also supported by the very low levels of gene expression of BAT markers found in white adipose tissue of ANP-infused mice.
Despite previous reports that ANP-induced lipolysis is confined to white adipose tissue in primates 22, we observed an increase in plasma glycerol, free fatty acids, and triglycerides in the lean ANP-infused ob/+ mice. This might be explained by the higher expression of NPR-A in white adipose tissue [although the difference did not reach significance (P=0.08), probably because of the low number of animals in each group] in lean compared with obese mice without any changes in adipose tissue NPR-C expression. This would result in a more favorable ratio between the signaling and the clearance receptor, and thus a higher sensitivity toward ANP 20.
The NPR-A KO mice accumulated less triglycerides in the heart than did their controls after prolonged fasting. The fasting-induced increase in plasma free fatty acids was similar in both NPR-A KO and wild-type mice. Hence, a difference in substrate availability could not explain our finding. Hypertensive heart failure causes downregulation of uptake 23 and utilization 24 of free fatty acids. NPR-A KO mice have enlarged hearts compared with wild-type controls – that is, they have hypertrophic cardiomyopathy due to hypertension 19. Hence, the reduced triglyceride accumulation after fasting may reflect decreased uptake secondary to the cardiomyopathy in NPR-A KO mice.
The fact that natriuretic peptides seemingly do not play a direct role in the cardiac lipid metabolism does not exclude a secondary role for these peptides in cardiac lipid metabolism in patients with heart failure. These patients have chronically elevated plasma levels of natriuretic peptides 25 and ANP stimulates peripheral lipolysis in humans 22,26. Hence, heart failure could predictively result in increased released of free fatty acids from adipose tissue. This could provide a surplus of free fatty acid to the heart. This possibility deserves attention as ANP and BNP mimetic drugs are now in trial as therapeutics for heart failure 27,28.
The authors thank Maria Kristensen for technical assistance and Dr Claycomb, New Orleans, LA, for HL-1 cells.
The study was supported by Weimanns Foundation, Aase and Ejnar Danielsen Foundation, The Novo Nordisk Foundation, and A.P. Møller Foundation for the Advancement of Medical Science.
Conflicts of interest
There are no conflicts of interest.
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