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International Journal of Obesity (2008) 32, 259–267 & 2008 Nature Publishing Group All rights reserved 0307-0565/08 $30.00 www.nature.com/ijo

ORIGINAL ARTICLE Angiotensin II stimulates and atrial natriuretic peptide inhibits human visceral adipocyte growth R Sarzani1, P Marcucci1, F Salvi1, M Bordicchia1, E Espinosa1, L Mucci1, B Lorenzetti1, D Minardi2, G Muzzonigro2, P Dessı`-Fulgheri1 and A Rappelli1 1 Department of Internal Medicine, University of AnconaF‘Politecnica delle Marche’, Ancona, Italy and 2Department of Urology, University of AnconaF‘Politecnica delle Marche’, Ancona, Italy

Objective: Cardiovascular peptides such as angiotensin II (Ang II) and atrial natriuretic peptide (ANP) have metabolic effects on adipose cells. These peptides might also regulate adipocyte proliferation and visceral adipose tissue (VAT) expansion. Welldifferentiated and stabilized primary cultures of human visceral mature adipocytes (MA) and in vitro-differentiated preadipocytes (DPA) were used as a model to study regulation of VAT expansion. Methods: Adipocyte differentiation was evaluated by Oil Red O staining and antiperilipin antibodies. MA and DPA from intraand retro-peritoneal depots were treated with increasing Ang II (with or without valsartan, a highly selective, competitive, ‘surmountable’ AT1 antagonist devoid of peroxisome proliferator-activated receptor g agonistic activity) or ANP concentrations. Cell counts and bromodeoxyuridine incorporation were used to evaluate proliferation. Apoptosis was evaluated by Hoechst 33342 staining. 8-Bromo cyclic guanosine monophosphate (8Br-cGMP) was used to investigate ANP effects, and real-time PCR to evaluate Ang II and ANP receptors’ expression. Results: Cell proliferation was progressively stimulated by increasing Ang II concentrations (starting at 1011 M) and inhibited by ANP (already at 1013 M) in both MA and DPA. Co-incubation with increasing Ang II concentrations and valsartan indicated that Ang II effects were AT1-mediated. Indeed, AT2 receptors were not expressed. Valsartan alone slightly inhibited basal proliferation indicating an autocrine/paracrine growth factor-like effect of endogenous, adipocyte-derived Ang II. 8Br-cGMP experiments indicated that the effects of ANP were mediated by the guanylyl cyclase type A receptor. Conclusion: A cell-culture model to study VAT growth showed stimulation by Ang II and inhibition by ANP at physiological concentrations. Because similar effects are likely to occur in vivo, Ang II and ANP might be important modulators of VAT expansion and associated metabolic and cardiovascular consequences. International Journal of Obesity (2008) 32, 259–267; doi:10.1038/sj.ijo.0803724; published online 18 September 2007 Keywords: adipocytes; visceral adipose tissue; angiotensin II; atrial natriuretic peptide; human

Introduction Obesity is a common condition associated with metabolic syndrome, hypertension and increased global cardiovascular risk.1 Increased visceral fat is a key factor in the metabolic and cardiovascular abnormalities associated with obesity.2 The pathophysiological link between visceral adiposity and cardiovascular diseases is complex.3 Recently, many studies have suggested that two antagonistic regulatory systems, the renin–angiotensin–aldosterone system and

Correspondence: Professor R Sarzani, Department of Internal Medicine, University of Ancona Politecnica delle Marche, Clinica Medicina Interna, Via Conca n. 71, Ancona 60020, Italy. E-mail: [email protected] Received 7 March 2007; revised 8 August 2007; accepted 10 August 2007; published online 18 September 2007

cardiac natriuretic peptides (NP) are not only involved in the regulation of sodium retention and blood pressure but also in the metabolic aspects of obesity.3–6 Adipose tissue has the full molecular machinery required for angiotensin II (Ang II) synthesis and signal transduction. Ang II has been shown to stimulate lipogenesis7 and preadipocyte differentiation.8 Moreover, Ang II is a trophic factor for adipose tissue growth and development through type 1 receptor (AT1).9,10 It is able to induce G1-phase progression of the cell cycle of human preadipocytes through AT1 receptor,9 confirming the well-known growth factor-like effects of Ang II. Visceral adipose tissue (VAT) expansion is a process characterized by an increase of the number as well as size of adipocytes. For more than 20 years it was clearly shown that in vitro-cultured mature unilocular adipocytes are able to become multilocular adipocytes and to undergo cell replication,11 as it was confirmed later.12 Because Ang II appears to

Ang II, ANP and human adipocyte proliferation R Sarzani et al

260 have a trophic role in adipose tissue expansion, it may also contribute to the development of insulin resistance and type 2 diabetes. The improved insulin sensitivity and the reduced new-onset diabetes incidence in patients treated with angiotensin-converting enzyme inhibitors or AT1 receptor blockers13,14 may be due to a ‘brake’ on the rate of VAT expansion. Despite extensive research, the effects of Ang II on adipocytes is still controversial, probably because of the different models and methodologies used.15 Contrasting data were obtained with preadipocytes during in vitro differentiation,16 but human adult adipose tissue is largely composed of well-differentiated adipocytes. Moreover, most human studies have been performed using subcutaneous adipocytes, whereas visceral adiposity is associated with metabolic complications. Therefore, in the present study, human, visceral, mature adipocytes (MA), as well as in vitro-differentiated preadipocytes (DPA) were used. Stabilized ‘well-fed’ culture conditions without fetal calf serum (FCS) resemble, as close as possible, visceral adipose cells in vivo. As reported below and shown previously by others,11,12 we have found that MA and DPA are able to undergo cell division in these conditions. The cardiac hormones atrial NP (ANP) and B-type NP (BNP) induce natriuresis, diuresis, and antagonize vasoconstriction by cGMP, their second intracellular messenger.17 NP have also well-known antiproliferative activity on cardiovascular cells.18 Moreover, physiological concentrations of NP induce a potent and specific lipolytic and lipid-mobilizing response in primate adipocytes, but not in rodents.5,6,19 This study aimed at evaluating the effects of increasing concentrations of Ang II or ANP on visceral adipocyte proliferation that is likely to be involved, in vivo, in the maintenance and the expansion of human VAT. Such investigations were extended on both intra- and extraperitoneal VAT depots.

Methods Subjects and adipose tissue samples Sixteen samples of perirenal (retro-peritoneal adipocytes) and nine of omental adipose tissue (intra-peritoneal adipocytes) were obtained during elective abdominal surgery for localized renal or bladder carcinoma. Patients were 18 men and 7 postmenopausal women (mean age 67.2711.8 years (range 30– 80) for perirenal and 69.3710.2 years (range 50–82) for omental tissue) admitted to the Ancona University Hospital. The study protocol was approved by the Ethics Committee and all patients gave their written informed consent.

Adipocyte and preadipocyte isolation, primary cultures and initial differentiation Two to eight grams of omental or perirenal adipose tissue were incubated (45 min at room temperature) in Hank’s balanced salt solution (Euroclone-Ltd, Milan, Italy) with 3% human albumin (Sigma, Milan, Italy). Centrifugation at International Journal of Obesity

800 r.p.m. for 7 min removed blood cells and tissue fragments. The floating tissue was collected from the supernatant, minced into small pieces, transferred into 50 ml tubes, digested at 37 1C for 2–3 h in Hank’s balanced salt solution with 2 mg ml1 Type I collagenase (Gibco, Invitrogen, Milan, Italy), and then filtered through a sterile gauze and 250 mm nylon mesh. Centrifugation (1000 r.p.m. for 10 min) separated the stromal part from free adipocytes. The floating fraction containing only MA was layered onto 25 cm2 tissue culture flasks or 100  20 mm dishes (Orange Scientific, M-medical, Milan, Italy), incubated at 37 1C in 5% CO2 and allowed to adhere. The next day they were washed and then incubated with culture medium (DMEM/F12, 10% FCS, penicillin 100 U ml1 streptomycin 100 mg ml1). After 4 days of culture, 33 mM biotin, 18 mM panthotenate, 10 mg ml1 transferrin, 10 mg ml1 insulin, 106 M dexametasone and 0.5 mM IBMX (3-isobutyl-1-methyl-xanthine) (Sigma) were added for 3 days in the absence of FCS to enhance triglyceride accumulation and retain MA features (differentiation–maintenance medium). After collagenase digestion step, the pelleted stromal component containing preadipocytes was again washed with erythrocyte lysing buffer and centrifuged. The new pellet was resuspended, washed in culture medium, layered onto plastic support and allowed to adhere. The next day, after removal of the non-attached cells by aspiration, subconfluent cells were trypsinized and plated in 96 multi-well plates. The differentiation of preadipocytes was induced by the same differentiating mix used for MA (Figures 1a and b). We used a modified Oil Red O staining (Sigma) and an antiperilipin polyclonal antibody (Ab. PREK)20 as markers of differentiation and to visualize intracellular lipid accumulation. Cells stained with Oil Red O were easily detected. The ratio of differentiated cells were indicated as percentage of stained cell vs total cell number seen at the end of differentiation (for preadipocytes) or after 3 day of culture (for MA). There were 9075% of differentiated lipid-laden cells both for MA and DPA cultures. Moreover, after fixation of control adipocytes cultured on a microscope slide (in 4% paraformaldehyde in 0.1 M phosphate buffer for 1 h), immunohistochemical demonstration of perilipin was performed with the avidin–biotin complex as detection system. Incubation steps were performed as described previously,21 using ‘PREK’, a rabbit antiperilipin polyclonal antibody at 1:300 final dilution.20 Before adding vehicle or peptides, viability was determined using Trypan Blue (Sigma). The degree of cell viability was indicated as percentage of stained cell (9574%) to the total cells seen.

Stabilization of differentiated adipocytes and treatment with Ang II or ANP After preadipocyte differentiation and with MA in stabilized, lipid-rich conditions, cell cultures underwent serum starvation for 10 days, maintaining both vitality and adipocyte

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Figure 1 (a) Primary culture of human mature adipocytes (MA) kept well differentiated in vitro with many large lipid droplets (  200). (b) Primary cultures of human preadipocytes before differentiation (  100). (c) Differentiated preadipocytes growing on a microscope slide (perilipin immunohistochemistry) (  400). White arrows: lipid droplets. Black arrows: adipocyte nuclei. (d) Human visceral multiloculate MA during cell replication in vitro. Continuous line: cell membrane. Outlined line: line of cellular division. Black arrows: neoformed lipidic droplets in each cell. White arrows: nuclei during cell division.

features. At this point, increasing concentrations of human synthetic Ang II or ANP (Sigma) were added with fresh medium (without FCS) and incubated for 24 h. Ang II was used from 1012 to 108 M, considering 1012 M as a normal plasma concentration, 1010 M as ‘pathophysiological’ and from 109 M as pharmacological concentrations. IBMX in the differentiation mix is known to antagonize some Ang II effects by increasing cAMP.22 As such, we used Ang II at slightly higher concentrations compared to physiological plasma levels. ANP was used from 1013 to 109 M, considering 1011 M as normal plasma concentration, 1010 M as ‘pathophysiological’ and 109 M as ‘pharmacological’.6,23,24 To investigate whether Ang II effects were mediated by AT1 receptor, we performed experiments incubating DPA with 1010 M valsartan alone or together with increasing Ang II concentrations (1012, 1010 and 108 M). Valsartan is a highly selective, competitive, ‘surmountable’ AT1-receptor blocker devoid of peroxisome proliferator-activated receptor g (PPARg) agonistic activity. Valsartan was provided generously by Novartis Pharmaceuticals Corporation. Finally, to study whether the ANP effects were mediated by the guanylyl cyclase type A NP receptor (NPRA), we performed experiments on DPA with 8-bromo cyclic guanosine monophosphate (8Br-cGMP) from 109 to 104 M.

Evaluation of cell proliferation Cells were seeded into six-well plastic supports at a density of 15 000 cells cm2. Adipocyte proliferation was assessed by

counting the number of differentiated cells after 24 h of Ang II or ANP incubation, in comparison to control vehicletreated cells from the same patient. The count was performed under microscope, in Burker chamber. A single investigator (PM) performed all cell counts three times, with an intraclass correlation coefficient of 0.996 (95% IC 0.996– 0.997; Po0.0001). The bromodeoxyuridine (BrdU)-based colorimetric immunoassay (Roche, Penzberg, Germany), expressed as optical density, was also used to assess proliferation.25 Apoptosis was detected by Hoechst 33342 (Molecular Probes, Invitrogen, Eugene, Oregon, USA), and apoptotic nuclei, identified from the altered chromatin, were counted.

Gene expression in cultured adipocytes Total RNA was extracted from floating MA and in vitro-DPA using Qiagen RNeasy MiniKit (including the RNase-free DNase set; Qiagen, Hilden, Germany). RNA was also extracted from control tissues (normal kidney cortex and whole VAT) after homogenization in guanidine thiocyanate buffer and CsCl gradient modified as reported previously.26 Reverse transcription of 1.5 mg RNA was performed with high-Capacity cDNA reverse Transcription Kits with RNase Inhibitor (Applied Biosystems, Warrington, Cheshire, UK). AT1 and type 2 Ang II receptor (AT2), NPRA and the NP clearance receptor (NPRC) gene expression were measured with TaqMan Gene Expression Assay (AT1, Hs00258938_m1; International Journal of Obesity

Ang II, ANP and human adipocyte proliferation R Sarzani et al

262 AT2, Hs00169126_m1; NPRA, Hs00181445_m1; NPRC, Hs00168558_m1) (Applied Biosystems). Gene expression was measured using an ABI 7300 for real-time PCR (Applied Biosystems) with the standard curve method. AT1, AT2, NPRA and NPRC mRNA expression were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (housekeeping gene).

Statistical analysis From each subject, we established three distinct primary cultures. Therefore, all the experiments were performed in triplicate. The effects of increasing Ang II and ANP concentrations on cell proliferation were analyzed by a nonparametric test for multiple related samples (Friedman’s test). A nonparametric test for two related samples (Wilcoxon’s signed ranks test) was used to identify difference between each treated group and controls. Data are presented as boxes and whisker plots (with median, 95% intervals of confi-

dence, and extremes). Statistical analysis was performed with SPSS 11.0 statistical software (SPSS Inc., Chicago, IL, USA) and a level of Po0.05 was considered as significant.

Results From each patient and each visceral intra- and retroperitoneal depots, we successfully obtained primary cultures from both MA (Figure 1a) and preadipocytes (Figure 1b). We were able to confirm that DPA (Figure 1c) and MA are still able to undergo cell division in vitro, in the absence of FCS (Figure 1d). In serum-free conditions, Ang II was able to stimulate, dose-dependently, cell proliferation (Po0.0001, starting at 1012 M) of both omental (Figure 2a) and perirenal (Figure 2b) MA, as well as of omental (Figure 2c) and perirenal (Figure 2d) DPA.

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Figure 2 Influence of 24-h incubation with increasing angiotensin II (Ang II) concentrations, compared to untreated controls, on proliferation of human visceral mature adipocytes and in vitro-differentiated preadipocytes evaluated by cell count. (a) Omental and (b) perirenal adipocytes; (c) omental and (d) perirenal preadipocytes. Data are presented as the median, 95% intervals of confidence, and range; circles represent out of range values (n ¼ 7). *P ¼ 0.02 vs control.

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263 The Ang II ‘antagonistic’ peptide ANP, dose-dependently, inhibited cell proliferation (Po0.0001) already at 1013 M of both omental (Figure 3a) and perirenal (Figure 3b) MA. Similar inhibition was found on omental (Figure 3c) and perirenal (Figure 3d) DPA. ANP did not reduce cell number by apoptosis; apoptotic nuclei were less than 1% in both treated and control adipocytes (data not shown). To confirm the results obtained by cell counting, we used BrdU incorporation, a microscope-independent methodology, both MA and DPA in seven perirenal samples. BrdU incorporation confirmed both Ang II stimulation and ANP inhibition of the basal level of adipocyte proliferation (Figures 4a–d). To define if the Ang II-induced proliferation was mediated by AT1 receptor, we performed experiments with valsartan. In vitro-DPA were incubated with Ang II (1010 M), valsartan (1010 M) and combination of fixed dose of valsartan (1010 M) and increasing Ang II concentrations (1012, 1010 and 108 M). The results are shown in Figure 5a (cell

counting) and 5B (BrdU). Valsartan alone significantly reduced cell number but with BrdU statistical significance was not reached. Co-incubation of Ang II and valsartan clearly showed that the proliferative effect of Ang II was mediated by AT1 receptors. We also evaluated mRNA expression levels of AT1 and AT2 receptors in DPA, whole VAT and renal cortex (as control tissue). AT1 mRNA expression levels are shown in Figure 6. We did not find AT2 expression in DPA (after 39 cycles); very low levels of AT2 were expressed in renal cortex and VAT (detectable after near 31 and X33 cycles, respectively). Both ANP receptors, NPRA and NPRC, were well expressed in DPA (data not shown). To explain the intracellular mechanism of ANP-induced inhibition, we treated perirenal and omental DPA with 8Br-cGMP for 24 h. The results obtained confirmed that the inhibitory effect of ANP was mediated by NPRA through its second-messenger cGMP (Figure 7).

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Figure 3 Influence of 24-h incubation with increasing atrial natriuretic peptide (ANP) concentrations, compared to untreated controls, on proliferation of human visceral mature adipocytes and in vitro-differentiated preadipocytes evaluated by cell count. (a) Omental and (b) perirenal adipocytes; (c) omental and (d) perirenal preadipocytes. Data are presented as the median, 95% intervals of confidence, and range; circles represent out of range values (n ¼ 7). *P ¼ 0.02 vs control.

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Ang II, ANP and human adipocyte proliferation R Sarzani et al

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Figure 4 Influence of 24-h incubation with increasing angiotensin II (Ang II) and atrial natriuretic peptide (ANP) concentrations, compared to untreated controls, on human visceral mature adipocytes and in vitro-differentiated preadipocytes proliferation evaluated by bromodeoxyuridine. (a) Perirenal adipocytes and (b) preadipocytes (Ang II); (c) perirenal adipocytes and (d) preadipocytes (ANP). Data are presented as the median, 95% intervals of confidence, and range; circles represent out of range values (n ¼ 7). *P ¼ 0.02 vs control.

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Figure 5 Influence of 24-h incubation with angiotensin II (Ang II) (1010 M) or valsartan (1010 M), and co-incubation with increasing Ang II concentrations and valsartan (1010 M), compared to untreated controls, on human visceral in vitro-differentiated preadipocytes proliferation evaluated by cell counting (a) and bromodeoxyuridine (b). Data are presented as the median, 95% intervals of confidence and range (n ¼ 4). *P ¼ 0.02 vs control.

Discussion Using traditional, ‘standard’ adipocyte cultures and protocols (with IBMX and without PPARg agonists), we found that human differentiated visceral adipocytes and preadipocytes retain the ability to undergo cell division in serum-free International Journal of Obesity

conditions. Moreover, Ang II (stimulatory) and ANP (inhibitory) have opposite effects on the proliferation of human MA and DPA from two different VAT depots. Valsartan alone somewhat reduced the basal rate of DPA proliferation (significance with BrdU test was not reached), suggesting an autocrine/paracrine role for endogenous Ang II. Increasing

Ang II, ANP and human adipocyte proliferation R Sarzani et al

265 Ang II concentrations overcame this competitive AT1 blocker. Thus, the Ang II-induced proliferation was AT1mediated. Indeed, VAT and DPA do not express AT2 receptors, as also reported previously.27 Finally, ANP effect is mediated by NPRA because NPRA are expressed and 8BrcGMP induced similar inhibition of growth. The cell-culture model we used has been widely utilized to study metabolic aspects of human adipocytes, with cells kept under stimulation to store triglycerides into lipid droplets. These stable ‘well-fed’ primary cultures are likely to resemble VAT adipocytes, which in vivo are stimulated to accumulate triglycerides and to expand in obesity. In these stabilized, lipid-rich conditions, without FCS for many days, mature multiloculate adipocytes (Figure 1a) are able to proliferate (Figure 1d), confirming previous suggestions and evidences.9,11,12 Unilocular adipocytes are not terminally

differentiated cells but can revert to multilocular and undergo cell division.11 Our findings were confirmed in cultures of floating, mature, paucilocular adipocytes, free from contamination of other cell types (for example, fibroblasts or mesenchimal stem cells) that could affect the results. In any case, even if a subpopulation of cells could have a less-differentiated, proliferation-prone phenotype, the general interpretation of the results do not change because VAT expansion in vivo is very likely to depend also on these cells to expand. Conflicting results have been published previously about the control of adipocyte proliferation and differentiation. Using cultured preadipocytes from human subcutaneous mammary adipose tissue during in vitro differentiation, it has been hypothesized that Ang II might inhibit preadipocyte differentiation reducing formation of new smaller adipocytes, believed to be less insulin-resistant than the larger, older ones.16 In contrast, starting from mesenchymal stem cells treated with Ang II in the presence of valsartan and/or PD123319, Matsushita et al.15 showed that differentiation to adipocytes was stimulated by Ang II through AT1 and inhibited through AT2 receptors. Finally, when undifferentiated9 and fully DPA or MA (our present data) were considered, Ang II showed an AT1-mediated proliferative effect, whereas AT2 resulted in no expression at all. Several considerations may help to explain these discrepancies. First, expression of AT2 receptors is likely to be crucial to explain these contrasting findings. Human mesenchymal stem cells express both AT1 and AT2 receptors and, using this model, Ang II seems to induce adipocyte differentiation through AT1, whereas AT2 play a counteracting action.15 Instead, human undifferentiated, differentiating and differentiated preadipocytes and MA showed no AT2 expression when evaluated both by binding27 and realtime PCR (Janke et al.16 and Figure 6). The reported AT2 expression in human MA by Jones et al.7 was based on northern blot, which is a less sensitive and specific

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Figure 6 RNA expression analysis of AT1 gene in renal cortex, visceral adipose tissue (VAT) and visceral differentiated preadipocytes (DPA). Data are presented as the median, 95% intervals of confidence, and range (n ¼ 3). *Po0.05 vs renal tissue.

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Figure 7 Influence of 24-h incubation with increasing 8-bromo cyclic guanosine monophosphate (8Br-cGMP) concentrations compared to untreated controls on proliferation of human in vitro-differentiated preadipocytes evaluated by cell count. (a) Omental and (b) perirenal preadipocytes. Data are presented as the median, 95% intervals of confidence and range (n ¼ 3).

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Ang II, ANP and human adipocyte proliferation R Sarzani et al

266 methodology prone to errors because of cross-hybridization. Moreover, no quantification of AT2 expression levels was reported.9 Although we cannot exclude extremely low levels of AT2 gene expression in MA, binding studies showed that AT2 receptor in these cells was practically undetectable.27 Second, human preadipocytes during differentiation, as studied by Janke et al.,16 appear to be a really complex model. Serum-free cultured preadipocytes exhibit a higher rate of adipose conversion than cells exposed to serum because of a marked stimulation of cell proliferation and a dramatic decrease in adipose differentiation.28 Like serum, Ang II can act as a mitogen.9 Incubating preadipocytes with very high Ang II concentrations (from 108 to 105 M) and without IBMX, which counteract some Ang II effects, overstimulated proliferation and blocked/delayed differentiation is very likely to occur.16 Moreover, MA co-cultured with differentiating preadipocytes could inhibit preadipocyte differentiation through a paracrine effect of Ang II that stimulates proliferation. Our results have been obtained with IBMX and much lower Ang II concentrations (more likely to exist in vivo, see Schling and Schafer22) on mature or welldifferentiated adipocytes. It is also important to note that high concentrations of the AT1-receptor blocker irbesartan induced adipogenesis.16 The authors suggested that an inhibitory effect of Ang II was present and AT1-mediated. Notably, at these high concentrations, irbesartan acts as a partial agonist of PPARg and it is able to induce adipocyte differentiation, as also shown by the same authors.29 Thus, the effect of irbesartan could have been due to the PPARg agonist activity of that AT1 antagonist used, and not due to an inhibition of the ‘antidifferentiation’ effects of Ang II. Our results were obtained using a highly selective, competitive, ‘surmountable’ AT1 antagonist (valsartan) devoid of agonistic activity on PPARg. Taken together, these data and considerations make unlikely the hypothesis by Sharma et al.30 that Ang II inhibits the formation of new adipocytes. On the contrary, Ang II is more likely to have a growth factor-like activity on DPA, similar to what happens in other cell types.31 Indeed, Ang II may inhibit the metabolic actions of insulin via the phosphatidylinositol 3 kinase pathway but synergistically promote its proliferative effects via the mitogen-activated protein kinase pathway.32 This link between insulin and Ang II could in part explain the proliferation we observed in our adipocytes cultured with insulin. Regarding the other relevant results of the present paper, recent studies5,33,34 have shown multiple interactions between NPs and adipose tissue. NPRA, which mediates NP biological effects through cGMP, and NPRC are abundant in human adipose tissue.35 Moreover, the ratio between NPRA and NPRC expression is decreased in subcutaneous adipose tissue of obese hypertensive patients,36 suggesting an increased NP clearance in obesity, coupled with lower lipolysis and lipomobilization. The ‘ancestral’ variant of a functional region of NPRC gene promoter37 was associated with increased abdominal circumference and likelihood of International Journal of Obesity

developing obesity in unselected male workers of the Olivetti factories.38 These previous findings, together with those of the present study, suggest that increased NPRC expression in adipose tissue could modify visceral adiposity by reducing the inhibitory effect of ANP/BNP on adipocyte and preadipocyte growth. Therefore, we hypothesize that ANP/BNP might have an inhibitory role on adipose tissue volume and related metabolic complications in vivo, at physiological or pathophysiological concentrations. Indeed, the ANP antiproliferative activity is already well known and described in several cellular types of mesenchymal origin.17,18 In summary, the opposing regulatory activities of Ang II and ANP on visceral adipocytes resemble the opposite role that these peptides have on the cardiovascular system.39 If so, our findings could have a biological relevance that goes beyond the model we used to assess adipocyte growth. A further support of this hypothesis is coming from a recent study that shows an AT1-mediated inhibitory effect of Ang II on lipolysis in human adipocytes,40 again the opposite effect of ANP. In conclusion, the antagonistic effects of Ang II and ANP on the proliferation of visceral differentiated adipocytes are likely to be present in human VAT in vivo, suggesting that ANP and Ang II may have important regulatory roles on visceral adipose organ expansion.

Acknowledgements This study was supported by research grants from the Italian University and Research Ministry (MiUR) and the University of Ancona ‘Politecnica delle Marche’. We are indebted to Dr Liana Spazzafumo, from the Statistic and Biometry Center, Department of Gerontological Research, National Institute for Research and Care on Aging (INRCA), Ancona, Italy, for statistical advice and with Professor Saverio Cinti and Dr Maria Cristina Zingaretti, Institute of Normal Human Morphology, University of AnconaF‘Politecnica delle Marche’, Ancona, Italy, for the help in the immunohistochemistry procedures. We also thank Mr Giorgio Tesei and Mrs Leda Rinaldi, in the year of their retirement, for the technical support in this as well as in many other studies performed in the last 20 years.

References 1 Poirier P, Giles TD, Bray GA, Hong Y, Stern JS, Pi-Sunyer FX, et al., American Heart Association; Obesity Committee of the Council on Nutrition, Physical Activity, and Metabolism. Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss: an update of the 1997 American Heart Association Scientific Statement on Obesity and Heart Disease from the Obesity Committee of the Council on Nutrition, Physical Activity, and Metabolism. Circulation 2006; 113: 898–918. 2 Carr DB, Utzschneider KM, Hull RL, Kodama K, Retzlaff BM, Brunzell JD et al. Intra-abdominal fat is a major determinant of

Ang II, ANP and human adipocyte proliferation R Sarzani et al

267 3 4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

the National Cholesterol Education Program Adult Panel III criteria for the metabolic syndrome. Diabetes 2004; 53: 2087–2094. Aneja A, El-Atat F, McFarlane SI, Sowers JR. Hypertension and obesity. Recent Progr Horm Res 2004; 59: 169–205. Engeli S, Negrel R, Sharma AM. Physiology and pathophysiology of the adipose tissue renin–angiotensin system. Hypertension 2000; 35: 1270–1277. Lafontan M, Moro C, Sengenes C, Galitzky J, Crampes F, Berlan M. An unsuspected metabolic role for atrial natriuretic peptides: the control of lipolysis, lipid mobilization, and systemic nonesterified fatty acids levels in humans. Arterioscler Thromb Vasc Biol 2005; 25: 2032–2042. Birkenfeld AL, Boschmann M, Moro C, Adams F, Heusser K, Franke G et al. Lipid mobilization with physiological atrial natriuretic peptide concentrations in humans. J Clin Endocrinol Metab 2005; 90: 3622–3628. Jones BH, Standridge MK, Moustaid N. Angiotensin II increases lipogenesis in 3T3-L1 and human adipose cells. Endocrinology 1997; 138: 1512–1519. Darimont C, Vassaux G, Ailhaud G, Negrel R. Differentiation of preadipose cells: paracrine role of prostacyclin upon stimulation of adipose cells by angiotensin-II. Endocrinology 1994; 135: 2030–2036. Crandall DL, Armellino DC, Busler DE, McHendry-Rinde B, Kral JG. Angiotensin II receptors in human preadipocytes: role in cell cycle regulation. Endocrinology 1999; 140: 154–158. Saint-Marc P, Kozak LP, Ailhaud G, Darimont C, Negrel R. Angiotensin II as a trophic factor of white adipose tissue: stimulation of adipose cell formation. Endocrinology 2001; 142: 487–492. Sugihara H, Yonemitsu N, Miyabara S, Toda S. Proliferation of unilocular fat cells in the primary culture. J Lipid Res 1987; 28: 1038–1045. Zhang HH, Kumar S, Barnett AH, Eggo MC. Ceiling culture of mature human adipocytes: use in studies of adipocyte functions. J Endocrinol 2000; 164: 119–128. Gillespie EL, White CM, Kardas M, Lindberg M, Coleman CI. The impact of ACE inhibitors or angiotensin II type 1 receptor blockers on the development of new-onset type 2 diabetes. Diabetes Care 2005; 28: 2261–2266. Abuissa H, Jones PG, Marso SP, O’Keefe Jr JH. Angiotensinconverting enzyme inhibitors or angiotensin receptor blockers for prevention of type 2 diabetes: a meta-analysis of randomized clinical trials. J Am Coll Cardiol 2005; 46: 821–826. Matsushita K, Wu Y, Okamoto Y, Pratt RE, Dzau VJ. Local renin angiotensin expression regulates human mesenchymal stem cell differentiation to adipocytes. Hypertension 2006; 48: 1095–1102. Janke J, Engeli S, Gorzelniak K, Luft FC, Sharma AM. Mature adipocytes inhibit in vitro differentiation of human preadipocytes via angiotensin type 1 receptors. Diabetes 2002; 51: 1699–1707. Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006; 27: 47–72. Silberbach M, Roberts Jr CT. Natriuretic peptide signaling: molecular and cellular pathways to growth regulation. Cell Signal 2001; 13: 221–231. Sengenes C, Zakaroff-Girard A, Moulin A, Berlan M, Bouloumie A, Lafontan M et al. Natriuretic peptide-dependent lipolysis in fat cells is a primate specificity. Am J Physiol Regul Integr Comp Physiol 2002; 283: R257–R265. Souza SC, de Vargas LM, Yamamoto MT, Lien P, Franciosa MD, Moss LG et al. Overexpression of perilipin A and B blocks the ability of tumor necrosis factor aloha to increase lipolysis in 3T3-L1 adipocytes. J Biol Chem 1998; 273: 24665–24669. Cinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Faloia E et al. Adipocyte death defines macrophage localization and function in

22

23

24

25

26

27

28

29

30

31 32

33

34

35

36

37

38

39

40

adipose tissue of obese mice and humans. J Lipid Res 2005; 46: 2347–2355. Schling P, Schafer T. Human adipose tissue cells keep tight control on the angiotensin II levels in their vicinity. J Biol Chem 2002; 277: 48066–48075. Richards AM, McDonald D, Fitzpatrick MA, Nicholls MG, Espiner EA, Ikram H et al. Atrial natriuretic hormone has biological effects in man at physiological plasma concentrations. J Clin Endocrinol Metab 1988; 67: 1134–1139. Dessi-Fulgheri P, Sarzani R, Serenelli M, Tamburrini P, Spagnolo D, Giantomassi L et al. Low calorie diet enhances renal, hemodynamic, and humoral effects of exogenous atrial natriuretic peptide in obese hypertensives. Hypertension 1999; 33: 658–662. Gratzner HG. Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 1982; 218: 474–475. Sarzani R, Fallo F, Dessi-Fulgheri P, Pistorello M, Lanari A, Rappelli A et al. Local renin–angiotensin system in human adrenals and aldosteronomas. Hypertension 1992; 19: 702–707. Crandall DL, Herzlinger HE, Saunders BD, Armellino DC, Kral JG. Distribution of angiotensin II receptors in rat and human adipocytes. J Lipid Res 1994; 35: 1378–1385. Entenmann G, Hauner H. Relationship between replication and differentiation in cultured human adipocyte precursor cells. Am J Physiol 1996; 270: C1011–C1016. Janke J, Schupp M, Engeli S, Gorzelniak K, Boshmann M, Sharma AM et al. Angiotensin type 1 receptor antagonists induce human in-vitro adipogenesis through peroxisome proliferator-activated receptor-g activation. J Hypertens 2006; 24: 1809–1816. Sharma AM, Janke J, Gorzelniak K, Engeli S, Luft FC. Angiotensin blockade prevents type 2 diabetes by formation of fat cells. Hypertension 2002; 40: 609–611. Bouzegrhane F, Thibault G. Is angiotensin II a proliferative factor of cardiac fibroblasts? Cardiovasc Res 2002; 53: 304–312. Velloso LA, Folli F, Sun XJ, White MF, Saad MJ, Kahn CR. Crosstalk between the insulin and angiotensin signaling systems. Proc Natl Acad Sci USA 1996; 93: 12490–12495. Wang TJ, Larson MG, Levy D, Benjamin EJ, Leip EP, Wilson PW et al. Impact of obesity on plasma natriuretic peptide levels. Circulation 2004; 109: 594–600. Dessi-Fulgheri P, Sarzani R, Rappelli A. Role of the natriuretic peptide system in lipogenesis/lipolysis. Nutr Metab Cardiovasc Dis 2003; 13: 244–249. Sarzani R, Dessi-Fulgheri P, Paci MV, Espinosa E, Rappelli A. Expression of natriuretic peptide receptors in human adipose and other tissues. J Endocrinol Invest 1996; 19: 581–585. Dessi-Fulgheri P, Sarzani R, Tamburrini P, Moraca A, Espinosa E, Cola G et al. Plasma atrial natriuretic peptide and natriuretic peptide receptor gene expression in adipose tissue of normotensive and hypertensive obese patients. J Hypertens 1997; 15: 1695–1699. Sarzani R, Dessi-Fulgheri P, Salvi F, Serenelli M, Spagnolo D, Cola G et al. A novel promoter variant of the natriuretic peptide clearance receptor gene is associated with lower atrial natriuretic peptide and higher blood pressure in obese hypertensives. J Hypertens 1999; 17: 1301–1305. Sarzani R, Strazzullo P, Salvi F, Iacone R, Pietrucci F, Siani A et al. Natriuretic peptide clearance receptor alleles and susceptibility to abdominal adiposity. Obes Res 2004; 12: 351–356. Johnston CI, Hodsman PG, Kohzuki M, Casley DJ, Fabris B, Phillips PA. Interaction between atrial natriuretic peptide and the renin angiotensin aldosterone system: endogenous antagonists. Am J Med 1989; 87: 24S–28S. Goossens GH, Blaak EE, Arner P, Saris WH, van Baak MA. Angiotensin II: a hormone that affects lipid metabolism in adipose tissue. Int J Obes (Lond) 2007; 31: 382–384.

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