Biomedicine & Pharmacotherapy 60 (2006) 139–143 http://france.elsevier.com/direct/BIOPHA/ Original article The hydrolipidic ratio in age-related maturation of adipose tissues Isabella Cavallini a, Maria A. Marino b, Cristina Tonello b, Pasquina Marzola a, Elena Nicolato a, Paolo Francesco Fabene a, Laura Calderan a, Paolo Bernardi a, Roberto M. Asperio a, Enzo Nisoli b, Andrea Sbarbati a,* a Section of Anatomy and Histology, Department of Morphological and Biomedical Sciences, Medical Faculty, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy b Department of Preclinical Sciences, Center for Study and Research on Obesity, Luigi Sacco Hospital, University of Milan, Milan, Italy Received 4 November 2005; accepted 13 January 2006 Available online 28 February 2006 Abstract The hydrolipidic ratio (HLR) expresses the amount of water and fat in a tissue. HLR can be studied non-invasively in the living organism and can be mapped in different areas of the body with high spatial and temporal resolution. In the present work we have evaluated the HLR in different adipose tissue depots in young or adult rats using tissue arrays of fat fragments by 1H-spectroscopy. In young animals, the highest percentage of water (33%) was found in the interscapular brown adipose tissue (iBAT). Mesenteric fat (mWAT) also appeared highly hydrated (24%). The deposits composed of epididymal, retroperitoneal and pelvic white adipose tissue (eWAT, rWAT and pWAT, respectively) contained an amount of water ranging from 14% to 17%. In adult animals, a reduction of the water content was found in all the depots. In e/r/pWAT, the age-related maturation was characterized by large changes in adipocyte diameter accompanied by a small change in HLR. In the iBAT, the maturation was accompanied by small change in adipocyte diameter and a greater diminution of HLR. mWAT showed an intermediate pattern between e/r/pWAT and iBAT. In all the studied depots, an age-related increase in leptin expression was found. This increase was relatively low in iBAT (40%) and high in the e/r/pWAT (204–273%). The work expand the knowledge about the physiological significance of the HLR by 1Hspectroscopy. © 2006 Elsevier SAS. All rights reserved. Keywords: Magnetic resonance; Brown adipose tissue; Visceral fat; Aging; Rat 1. Introduction Findings in adult humans and in animals suggest that the inflammatory status associated with the increase of fat mass may be involved in the pathogenesis of obesity complications [1]. The inflammatory process seems to reside mainly in adipose tissue, which could regulate the process through cytokine production. Recent studies in mice documented leukocyte populations within adipose tissue, which are potentially involved in the development of the inflammatory status that is characteristic of obesity [2] and a significant infiltration of macrophages in adipose tissue with inflammation and macrophage-specific * Corresponding author. Tel.: +39 045 802 7155; fax: +39 045 802 7163. E-mail address: andrea.sbarbati@univr.it (A. Sbarbati). 0753-3322/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2006.01.007 genes up-regulated were found [3]. On the basis of evidence available mainly from animal models, the presence of inflammatory areas in the adipose tissue of obese individuals seems to have been proved. These data strength the need for methods capable to detect inflammatory (i.e. more hydrated areas in the fat). A parameter correlated to the hydration of the adipose tissue is the hydrolipidic ratio (HLR) that expresses the amount of water and fat in a tissue and can be obtained in vivo by proton magnetic resonance spectroscopy (1H-MRS). The variation of the HRL is not known in relation to adipocyte size or age. In previous work, we have demonstrated that HLR can be mapped at high spatial resolution in adipose tissues of living animals [4,5] and BAT revealed significantly higher amount of water with respect to WAT. These experiments suggested that HLR roughly mirrors the cytological composition of the tissue. The possibi- 140 I. Cavallini et al. / Biomedicine & Pharmacotherapy 60 (2006) 139–143 lity to obtain information about the adipocyte volume using MRS is interesting considering the importance of adipocyte volume in the diagnosis of the type of obesity and the feasibility of MRS in a clinical setting. If MRS can provide information about fat cellularity a virtual biopsy by MRS could be feasible in obese patients. We have faced the problem using an innovative approach of tissue array. The method is based on a standardized analysis of fat fragments by 1H-MRI. This approach allows examination of up to 36 specimens in the same acquisition to avoid artefact linked to partial volume effect that can be present studying small depots in small animals. 2. Material and methods home-built sample holder having 30 holes of 0.5 × 0.5 × 0.5 cm3. The sample holder was then placed in a 7.2 cm i.d. transmitter/receiver bird cage coil for images and spectra acquisitions. After a scout acquisition for localizing and optimizing sample position, a multi-echo multi-slice spin echo sequence was acquired with the following parameter: TR/TE = 3000/ 14.8 ms, FOV = 5 × 5 cm2, matrix size 192 × 256, zero-filled at 256 × 256, slice thickness = 1.74 mm, NEX = 1.Three coronal slices were acquired. Localized spectra were acquired in each of the holes containing fat tissue using a STEAM sequences with TR/TE = 2500/22 ms, NEX = 128, voxel size = 3 × 3 × 3 mm3. MRS data were analyzed using the XWin-NMR software (version 2.6). 2.1. Animals 2.3. Calibration of the MRS data Male Wistar rats (N = 16) were used. All rats were housed at constant temperature (20–24 °C) in 12-h light/dark cycle and fed standard mouse chow ad libitum. The sacrifice was performed under ether anesthesia by dislocation of the cervical vertebrae. Young animals were 1–4-month-old (mean age 2.3 months) while 10-month-old animals were considered adult. As representative of the lipid depot, we have removed a typical BAT (i.e. the interscapular brown adipose tissue (iBAT)), three depots of typical WAT, i.e. pelvic WAT (pWAT), epididymal WAT (eWAT) and retroperitoneal WAT (rWAT), and the mesenteric adipose tissue (mWAT). Morphologically, mWAT is different from the other depots being composed of unilocular adipocytes but contains some amount of connective tissue. The experiments were conducted following the principles of the NIH Guide for the Use and Care of Laboratory Animals and the European Community Council (86/609/EEC) directive. All efforts were made to minimize the number of animals used and to avoid their suffering. To test the validity of data from 1H-localized spectra, we have used phantoms of vegetable oil made by mixing known amount of water (doped with MnCl2 0.2 mM) and vegetable oil (extra virgin olive oil, Conad) containing 2% and 5%, in relation to the proportion between oil and water, of TWEEN80 (polyoxyethylene sorbitan monooleate, Sigma Aldrich). The mixture homogeneously mixed by ultrasonic disruption (sonopuls ultrasonic homogenizer, with a main voltage of 50 or 60 Hz transforms into high frequency voltage of 20 kHz, BANDELIN Elettrofor, Borsea, Italy) contained 20%, 40%, 80% and 100% of oil by volume. Suspensions, contained into 15 mm diameter plastic tubes, were placed longitudinally into the magnet. We analyzed phantoms with the same experimental protocol used in vivo; after a spin echo sequence to localize the phantom position we acquired a transversal T1-weighted image to localized the voxel useful for the 1H-MRS-localized spectra acquisition, that was a stimulated-echo sequence with TR = 2500 ms, TM = 8.9 ms, TE = 22 ms and a voxel size 3 × 3×3 mm3, NEX = 128. 2.2. Magnetic resonance spectroscopy 2.4. Cell diameter We have faced the problem of HLR calculation using an innovative approach of tissue array. The method is based on a standardized analysis of fat fragments in a predefined home-built apparatus by 1H-MRI. This approach allows examination of up to 36 specimens in the same acquisition to avoid artefact linked to partial volume effect that can be present studying small depots in small animals. The possibility to examine multiple specimens by the tissue array approach allowed us the sampling of whole deposits or at least a large part of them. In general, the evaluations were performed immediately after the sacrifice but we have also evaluated the possibility to store the fat at –20 °C. All MRI experiments were carried out using a Biospec Tomograph System (Bruker, Karlsruhe, Germany) equipped with a horizontal magnet (Oxford Ltd, Oxford, UK) having a 33 cm bore with a gradient intensity of 20 Gauss/cm. The software ParaVision 2.1.1 (Bruker, Karlsruhe, Germany) running on a Silicon Graphics O2 computer was used to analyze data. Fat samples were placed in a The mean adipocyte diameter has been evaluated in two different ways. In a first step, we have examined whole mount of tissue at light microscopy. Briefly, immediately after the sacrifice the tissue was removed, fixed in buffered formalin (Fluka) for 30 min, sectioned with a blade, mounted on a slide and observed unstained by an Olympus BX51 using a 10× objective. Opportune diaphragms were used to obtain sufficient contrast. The diameters were evaluated using the Image-Pro Plus software (Media Cybernetics, San Diego, CA). In each specimens three different areas were examined and in each area 20 elements selected by a systematized randomized protocol were analyzed. All values were expressed as mean ± S.D. The measures were then repeated at scanning electron microscopy (SEM). On the WATs the two methods provided similar results but the ultrastructural approach allowed us to better evaluate the brown adipocyte mean diameter. Briefly, for SEM, the tissues were fixed with glutaraldehyde 2% in 0.1 M PB, postfixed in 1% osmium tetroxide in the same buffer for 141 I. Cavallini et al. / Biomedicine & Pharmacotherapy 60 (2006) 139–143 1 hour, dehydrated in graded ethanols, critical point dried (CPD 030, Balzers, Vaduz, Liechtenstein), fixed to stubs with colloidal silver, sputtered with gold by an MED 010 coater (Balzers) and examined with a DSM 690 scanning electron microscope (Zeiss). 2.5. Leptin expression 2.6. Statistical analysis All data are expressed as mean ± S.D. and evaluated by a general linear model for repeated measures, followed by paired Student’s test (post-hoc). Pearson’s correlation coefficient was used to evaluate the relationships among variables. A P < 0.05 was considered statistically significant. 3. Results Total RNA from various rat adipose tissue was isolated using the RNeasy® Lipid Tissue Mini Kit (Qiagen S.p.A., Italy). One microgram of total RNA of various samples was treated for 1 h at 37 °C with 6 U ribonuclease (RNase)-free deoxyribonuclease I per μg RNA in 100 mM Tris–HCl, pH 7.5, and 50 mM MgCl2 in the presence of 2 U/μl placental RNase inhibitor and then it was reverse transcribed using iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Italy). Rat leptin and â-actin primers were designed using Beacon Designer 2.6 software of Premier Biosoft International (Palo Alto, CA, USA) and they were: rat leptin (NM_013076) forward primer 5′ ACC CTG GCA GTC TAT CAA C 3′ and reverse 5′ GTA GAG CGA GGC TTC CAG 3′; rat α-actin (NM_013144) forward primer 5′ TAA AGA CCT CTA TGC CAA CAC AGT 3′ and reverse 5′ CAC GAT GGA GGG GCC GGA CTC ATC 3′. Triplicate PCR reactions were carried out with the intercalating dye SybrGreen. Each sample (25 μl) contained 12.5 μl iQTM SybrGreenI SuperMix (Bio-Rad), 0.4 μM each primer and 1/50 of reverse transcriptase product. PCR cycles were programmed on an iCycler iQTM real time PCR detection system (Bio-Rad) and consisted of hot-start incubation (3 min at 95 °C) and amplification (10 s at 95 °C and 10 s at 60 °C, 40 repeats). A melting profile starting at 55 °C with increments of 0.5 °C every 10 s followed the amplification program to exclude the presence of non-specific fragments and/or primer-dimers formation. The cycle number at which analyzed transcripts were detectable (CT) was normalized to that of α-actin, referred to as .CT. The fold change of leptin expression relative to young rat was expressed as 2-(..CT), in which ..CT equals .CT of the old rat minus. CT of the young one. 3.1. Evaluation of the HLR by 1H-MRS In a first step, we have calculated the HLR in fragments of adipose tissue removed from different depots (Table 1). In young animals, the results showed significant differences between some of the studied depots. As expected, the higher percentage of water (33% of the protons in the tissue) was found in the iBAT. The mWAT also appeared highly hydrated (24%). The e/r/pWAT contained an amount of water ranging from 14 to 17%. The least hydrated tissue was the eWAT. The rWAT and pWAT showed not significant differences (16% and 17%). In adult animals, a significant reduction of the water content was found in all the depots as compared to young rats. This reduction was particularly evident in the tissue that in young animals were the most hydrated (i.e. iBAT and mWAT). The eWAT, was the most rich in lipid also in the adult. Table 1 HLR and adipocyte diameter in fragments of adipose tissue removed from different depots Deposit IBAT EWAT PWAT MWAT RWAT Young 33 ± 15 14 ± 4 17 ± 6 24 ± 8 16 ± 5 HLR ± S.D. Adult 19 ± 2 12 ± 2 15 ± 3 17 ± 2 15 ± 4 Fig. 1. Light microscopy appearance of the different WAT depots in young or adult rats. Diameter Young 37.5 ± 1.5 62.4 ± 5.2 79.5 ± 7.1 49.5 ± 4.1 70.4 ± 12 (μm) ± S.D. Adult 45.2 ± 4.2 104.7 ± 13.2 98.1 ± 11.3 73.8 ± 7.8 91.8 ± 13.8 142 I. Cavallini et al. / Biomedicine & Pharmacotherapy 60 (2006) 139–143 Light microscopy evaluation of the whole mount tissue (Fig. 1) clearly showed the increased diameter of the adipocytes in all the studied depots in the adult animals (Table 1). In young, quantitative evaluation, revealed the smallest adipocytes in the iBAT (37 μm). The highest values in the e/r/pWAT depots (ranging from 62 to 72 μm) and intermediate values in the mWAT depots (49 μm). In the adult, all the depots significantly increased the diameter of the adipocytes with the largest diameter found in the eWAT. The statistical evaluation pointed out a significant difference in the HLR between the considered fat tissue samples (P < 0.009) and between the age-related maturation effect (P < 0.03). A not relevant interaction between this two variables was found. In the iBAT (Table 1), the maturation is accompanied by small change in adipocyte diameter and by a significant (P < 0.014) diminution of HLR (i.e. water content of the tissue). The mWAT shows an intermediate pattern between e/r/ pWAT and iBAT, characterized by a significant decrease in the HLR in aging (Table 1; P < 0.046). 3.3. Leptin expression 4. Discussion In all the studied depots, we have found an age-related increase in leptin expression (Table 2). As expressed by vertical bars in Table 2, this increase was relatively low in iBAT (40%) and high in the e/r/pWAT (204–273%). An intermediate value was found in the mWAT. The study demonstrates that the water content of the adipose tissue is variable in function of the age and of the anatomical location. Higher values are present in the multilocular fat and in areas in which adipocytes are mixed with connective tissue. In typical unilocular WAT (i.e. e/r/pWAT depots), the water content is low. The IBAT is clearly differentiated from WAT and it is characterized by the most relevant age-related decrease of the HLR. It is due to the well-known transformation of the initially multilocular tissue in an unilocular WAT-like tissue [6]. This morphological transformation is paralleled by changes in endocrine activity and UCP1 expression [7,8]. Both HLR and diameter analysis demonstrates that also in adult animals, the tissue remains different from a typical WAT in particular for its high degree of hydration. Using this characteristic, in previous works, we have demonstrated the possibility to detect small depots of multilocular BAT in living organisms [9,10]. The present work, demonstrates that these depots can be also detected in their unilocular WAT-like form. This feature opens new perspectives in testing of drugs capable to modify the activity of the brown adipocytes or in evaluate collateral effects of drugs on brown adipocytes [11]. The mWAT revealed high values of HLR and small cell diameter. These values were intermediate between those of the typical WATs and those of the iBAT. Also the maturation pattern and the leptin expression suggested a similar intermediate feature. We have found that in adult animals the HLR fall to a value of about 17%. This value is similar to that found in typical WATs of young animals therefore it is probable that a further increase in fat content could cause a small change in HLR and a more relevant change in cell diameter. Among rWAT, pWAT and eWAT, the HLR differences are small and the maturation pattern of these depots is similar. During the aging of the tissue, the changes in HLR are not so marked as in cell diameter but the percentage of water can fall to 12%. The possibility to detect fat regions with similar low degree of hydration opens new perspectives on evaluation of the “maturation age” of single fat depots also in living organisms. This evaluation seems to be relevant also to predict the endocrine activity of these depots because our study demon- 3.2. Evaluation of the adipocyte diameter 3.4. Statistical evaluations In Fig. 2, HLR and adipocyte mean diameter are shown in young and adult animals. The values are inversely correlated both in young (–0.87512; P < 0.01) and in adult (–0.9396; P < 0.005) animals. In the HLR/diameter diagram (Fig. 2), e/r/pWAT, iBAT and mWAT are characterized by different patterns of maturation (Table 2). In e/r/pWAT, the fat age-related maturation is characterized by large changes in adipocyte diameter accompanied by non-significant change in HLR. Table 2 Numeric values expressing the patterns of maturation for BAT and WAT depots. The age-related modification in leptin expression, HLR and adipocyte diameters are reported. The slope of the lines A–E in the HLR/diameter diagram (Fig. 2), are also reported Deposit IBAT EWAT PWAT MWAT RWAT Slope –2.12 –0.02 –0.05 –0.16 –0.04 %Δ Leptin 40 204 220 80 273 ΔHRL 14 2 2 7 1 Δ Diameter 7.7 42.3 18.6 24.3 21.4 Fig. 2. Diagram showing HLR, mean diameter and variation of leptin expression in young or adult rats. The vertical bars show the increase in leptin expression for each depots. I. Cavallini et al. / Biomedicine & Pharmacotherapy 60 (2006) 139–143 strates that WATs display a relevant age-related increase in leptin expression. This methodological development could be used to test the hypothesis that the WAT is not homogeneous in all the body but that areas with low or high degree of hydration exist. The present work expand the knowledge about the significance of the HLR representing a further step toward the possibility to obtain a virtual biopsy by MRS with the aim to detect eventual inflammatory areas in the adipose tissue. References [1] Yudkin JS. Adipose tissue, insulin action and vascular disease: inflammatory signals. Int J Obes Relat Metab Disord 2003;27:S25–8. [2] Robker RL, Collins RG, Beaudet AL, Mersmann HJ, Smith CW. Leukocyte migration in adipose tissue of mice null for ICAM-1 and Mac-1 adhesion receptors. Obes Res 2004;12:936–40. [3] Xu H, Barnes GT, Yang Q, et al. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 2003;112:1821–30. 143 [4] Lunati E, Marzola P, Nicolato E, Sbarbati A. In vivo quantitative hydrolipidic map of perirenal adipose tissue by chemical shift imaging at 4.7 Tesla. Int J Obes Relat Metab Disord 2001;25:457–61. [5] Lunati E, Marzola P, Nicolato E, Fedrigo M, Villa M, Sbarbati A. In vivo quantitative lipidic map of brown adipose tissue by chemical shift imaging at 4.7 Tesla. J Lipid Res 1999;40:1395–400. [6] Sbarbati A, Morroni M, Zancanaro C, Cinti S. Rat interscapular brown adipose tissue at different ages: a morphometric study. Int J Obes 1991; 15:581–7. [7] Cancello R, Zingaretti MC, Sarzani R, Ricquier D, Cinti S. Leptin and UCP1 genes are reciprocally regulated in brown adipose tissue. Endocrinology 1998;139:4747–50. [8] Cinti S, Frederich RC, Zingaretti MC, De Matteis R, Flier JS, Lowell BB. Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue. Endocrinology 1997;138:797– 804. [9] Sbarbati A, Guerrini U, Marzola P, Asperio R, Osculati F. Chemical shift imaging at 4.7 Tesla of brown adipose tissue. J Lipid Res 1997;38:343– 7. [10] Sbarbati A, Baldassarri AM, Zancanaro C, Boicelli A, Osculati F. In vivo morphometry and functional morphology of brown adipose tissue by magnetic resonance imaging. Anat Rec 1991;231:293–7. [11] Sbarbati A, Leclercq F, Osculati F, Gresser I. Interferon alpha/beta-induced abnormalities in adipocytes of suckling mice. Biol Cell 1995;83: 163–7.