Association of fat mass profile with natriuretic peptide receptor alpha in subcutaneous adipose tissue of medication-free healthy men: A cross-sectional study [version 2; peer review: 2 approved]

Background: Atrial natriuretic peptide increases lipolysis in human adipocytes by binding to natriuretic peptide receptor-A (NPRA). The aim of the current study was to examine the associations of NPRA mRNA of subcutaneous adipose tissue with fat mass, fat-free mass, body mass index (BMI) and arterial blood pressure in medication-free healthy men. Method: Thirty-two volunteers [age (years): 36.06±7.36, BMI: 27.60±4.63 (kg/m 2 )] underwent assessments of body height/weight, % fat mass, fat-free mass (kg), blood pressure, and a subcutaneous adipose tissue biopsy via a surgical technique. Results: We found that NPRA mRNA was negatively associated with % fat mass (r=-0.40, R 2 =0.16, p=0.03) and BMI (r=-0.


Introduction
Atrial natriuretic peptide (ANP) lowers arterial pressure to maintain fluid volume homeostasis, thus protecting against renal and cardiac pathogenesis 1 . ANP also increases lipolysis in human adipocytes 2 by binding to natriuretic peptide receptor-A (NPRA) 3 . NPRA is less expressed in subcutaneous adipose tissue (SAT) in obese individuals and type 2 diabetes patients than in normal glucose tolerant individuals 4 . Also, NPRA signalling in skeletal muscle may enhance long-term insulin sensitivity 5 . Collectively, NPRA may potentially treat obesity-related disorders while ANP may play a role in the therapeutic mechanisms of betaadrenoceptor antagonists in the mitigation of heart dysfunction and utilization of lipid mobilization 6 . However, the role of ANP in lipolysis has been primarily investigated mainly in vitro models [7][8][9] , in human blood cells from individuals under medication treatment 10 , and in animal models 9 . To our knowledge, no such information is currently available in relation to the role of its receptor (NPRA) on the adipocytes of healthy individuals. Therefore, the aim of the current study was to examine the associations of NPRA mRNA of SAT with fat mass, fat-free mass (FFM), body mass index (BMI) and arterial blood pressure (BP) in medication-free healthy men.

Methods
The study was approved by the Ethics Committee of the University of Thessaly (protocol no. 698/2013). The inclusion criteria were: healthy adult men, non-smokers, no chronic disease and/or being under medication treatment. The participants were recruited by advertisements in a local newspaper in Trikala, Thessaly, Greece and the data collection started in July 2013 and ended in June 2014. Written consent was obtained from the 32 healthy men recruited [age (years): 36.06±7.36, BMI: 27.60±4.63 (kg/m 2 )].
To avoid misleading results, the participants refrained from exercise, alcohol, and passive smoking 72h prior the measurements, while they followed an overnight fast before they visit the physiology laboratory in the School of Exercise Science between 07:00-09:00 am. PCD and DP performed the following measurements: body height using a Seca 220 (Hamburg, Germany) stadiometer, body weight using a precision scale (KERN & Sohn GmbH, Version 5.3, Germany) and blood pressure (BP) using a Standard Aneroid sphygmomanometer (Medisave, UK) according to standard guidelines 11 . Briefly, participants were instructed to empty their bladders and sit for five minutes in a relaxed back rest position without talking. BP readings were taken twice, each two minutes apart, while the mean of the two BP readings was considered as the final BP values. Fat mass percentage (%FM) and FFM were measured via bioelectrical impedance using a body composition monitor (Fresenius Medical Care AG & Co. KGaA D-61346 Bad Hamburg, Germany).
Following the aforementioned measurements, the participants underwent a SAT biopsy in the physiology laboratory by a trained physician, as previously described 12 . Briefly, the site of the incision was disinfected and a 10 ml of 2% xylocaine (no adrenaline) was injected for local anaesthesia. An incision of 2-2.5 cm was made 3-5 cm to the left of the navel. Nearly 500 mg of subcutaneous adipose tissue was captured and removed. The NPRA mRNA analysis of SAT samples is described elsewhere 13 . Briefly, total RNA was extracted using RNeasy Lipid Tissue mini kit (QIAGEN). First-strand cDNAs were synthesized from equal amounts of total RNA using random primers and M-MLV reverse transcriptase (Promega). Quantitative real-time polymerase chain reaction was performed using Sybr Green fluorophore. 18S rRNA gene was used as a reference for normalization.
Following previous methodology, we removed two mean values (i.e. outliers) of NPRA mRNA that were at a distance of more than two standard deviations from the mean of the distribution 14,15 . Also, there were three missing values in the NPRA mRNA analysis of SAT samples due to failure to extract RNA from adipose tissue. Eventually, 27 NPRA mRNA values were included in the statistical analysis using SPSS (version 24; SPSS Inc., Chicago, IL, USA). Normal distribution was determined using Shapiro-Wilk test, whereas Pearson's correlation coefficient, linear regression, and Cohen's f 2 effect size (R 2 /1-R 2 ) 16 were used to detect associations between NPRA mRNA, %FM, FFM (kg), BMI, and BP. We also used one-way analysis of variance (ANOVA) with Bonferroni post-hoc tests, and Cohen's d effect size analyses to explore the mean differences of NPRA mRNA across different BMI categories [normal weight <25 kg/m 2 (n=9); overweight 25-30 kg/m 2 (n=9); obese >30 kg/m 2 (n=9)]. The level of significance was set at p≤0.05.

Amendments from Version 1
We have added in the limitations section information regarding the link between NPRA and levels of triglycerides, cholesterol, ANP and BNP. Similarly, we have included in our limitations and discussion section possible connections between NPRA mRNA and hormone sensitive lipase, adipocytes' size as well as the ANP-BNP/NPRA signalling axis. We put all the above information in context with the obesity phenomenon, while we have listed all the necessary references. Finally, we have added further details about blood pressure registers, providing a relevant reference, while we acknowledge the relevance of our results in context with the ethnicity and the gender of the participants in our study.

Discussion and conclusions
We have shown that the NPRA mRNA is negatively associated with %FM and BMI in medication-free healthy men and that it is expressed less in obese compared to lean individuals. Previous evidence showed that NPRA mRNA is expressed less in normal glucose tolerant individuals than in type 2 diabetes patients 4 , while it is positively associated with insulin sensitivity 4 . Given that insulin sensitivity is negatively associated with excessive FM in humans 17,18 the inverse association of NPRA mRNA with %FM and BMI observed in the current study suggests a possible role of NPRA in lowering FM in humans. Indeed, it has been established that natriuretic peptides by binding to NPRA, increase cyclic guanosine monophosphate -a well-known intracellular second messenger -which phosphorylates protein kinase G leading to activation of hormone sensitive lipase 19,20 . This process mediates triglyceride degradation (i.e. lipolysis), which subsequently increases fatty acid availability 19 . This lipolytic effect of NPRA however, appears to be supressed by NPRC action in obese individuals and clearance of NPR3 gene -coding for NPRC -in animals showed to restore NPRA action 9 , which should be considered in future studies. Furthermore, findings in mice showed that the lack of NPRA gene may increase FM 9 . Finally, NPRA signalling as part of ANP/NPRA axis may induce a browning of white adipocytes, indicating increased energy expenditure and thus, a potential to mitigate obesity 21 .
The current study may be affected by methodological limitations such as the lack of insulin sensitivity measurements and a priori power calculation to determine the sample size. However, a post-measurements power calculation was conducted using an online software (DSS Research) to test statistical power. This revealed 89% of statistical power for the 27 available samples, based on the NPRA mRNA value (1.02±0.38) we detected in our study and expected NPRA mRNA value (0.81±0.08) from a previous similar study that examined NPRA in SAT in humans 4 . Another limitation was the lack of triglyceride, cholesterol and ANP plasma levels, to determine whether there is a link with NPRA mRNA, given the association of the aforementioned factors with lipolysis 2,19 . Previous evidence also suggests that large adipocytes express higher NPRA mRNA than small adipocytes, indicating enhanced ANP-stimulated lipolysis in large adipocytes 22 . However, we did not determine the size of the examined adipocytes and its possible association with NPRA mRNA. Furthermore, brain natriuretic peptide (BNP) may alter expression of NPRA to release free fatty acids from adipose tissue, while obesity is inversely associated with circulating BNP, a situation known as "natriuretic handicap" 23,24 . Circulating BNP was not measured in our study to examine whether there is an association with NPRA mRNA. Also, ANP may inhibit the secretion of adipokines and cytokines from adipose tissue and, thus, may attenuate chronic inflammation and insulin resistance 25,26 , a mechanism that was not investigated in our study. Given the action of ANP/BNP on lipolytic hormones, it would have been interesting to determine hormone sensitive lipase in the current study 2,6 . Finally, it is important to note that the current results are limited to the Greek population and male participants, therefore, they should be treated with caution when applied to other ethnicities and females.
In conclusion, NPRA mRNA is negatively associated with %FM and BMI in medication-free healthy men, suggesting a possible role of ANP/NPRA axis in the control of FM accumulation. To date, the investigation of NPRA has mainly focused either on circulating and muscle NPRA 27-29 or on medicationdependent NPRA measurements 4,10 . Our study indicates that NPRA may also play role in FM profile of healthy individuals, which should be further explored in a cause-and-effect research setting.

Competing interests
No competing interests were disclosed. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Open Peer Review
Interestingly, ANP-BNP/NPRA signaling axis has been shown to induce a browning of white adipocytes in humans, which seems to be physiologically significant (Enerback, 2010 10 ; Collins et al., 2014 11 ; Liu et al., 2018 12 ). A brief discussion on this aspect of ANP/NPRA signaling mechanisms might be helpful.

4.
The previous findings have suggested that NPRA acts as a determinant of insulin sensitivity; however, the upregulation of natriuretic peptide receptor-C (NPRC) decreases the glucose tolerance in obase subjects, which seems to repress ANP-BNP/NPRA signaling axis and thereby the lipolytic effects of ANP was completely rescued in Npr3 (coding for NPRC) geneknockout mice (Bordicchia et al., 2012 13 ). The significance of these previous findings and the relationship to the current work could be discussed in the current manuscript.

5.
The conclusions of the present study are based on the findings in male gender. The limitations on the inclusion of female gender should be mentioned.

6.
I have read and reviewed the manuscript and believe that I have the expertise to evaluate and state that the current work is of an acceptable scientific quality. Indeed, there are some concerns in the manuscript, which are summarized above.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly interesting if the authors had determined the NPRA mRNA according to the size of the adipocyte. This issue should be considered in the discussion section and mentioned as a limitation of this study. (see also Laurencikiene et al. 2

)
It has been reported that obesity is associated with low circulating levels of BNP and recent evidences suggest an altered expression of BNP receptors-both the signaling receptors (NPR)-A and the clearance NPR-C receptor-in Pivovarova et al. 3 ; Gentili et al. 4 ). Moreover, increased tissue secretion of adipokines and cytokines has been implicated in the chronic low-grade state associated with obesity (Schindler et al. 5 ; Moro et al. 6 ). This issue should be considered in the discussion section and mentioned as a limitation of this study.

4.
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