Characterization of the dynamics of fat cell turnover in different bovine adipose tissue depots

Research in Veterinary Science 95 (2013) 1142–1150

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Characterization of the dynamics of fat cell turnover in different bovine adipose tissue depots S. Häussler a,⇑, D. Germeroth a, K. Friedauer a, S.H. Akter a,b, S. Dänicke c, H. Sauerwein a a

Institute of Animal Science, Physiology and Hygiene Group, University of Bonn, 53115 Bonn, Germany Faculty of Veterinary Science, Department of Anatomy and Histology, Bangladesh Agricultural University, Mymensing 2202, Bangladesh c Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, 38116 Braunschweig, Germany b

a r t i c l e

i n f o

Article history: Received 26 September 2012 Accepted 8 July 2013

Keywords: Adipose tissue Apoptosis Cattle Cell proliferation Conjugated linoleic acids Preadipocytes

a b s t r a c t In many but not all high producing cows, the energy requirements for milk yield and maintenance exceed energy intake by voluntary feed intake during early lactation. Prioritizing milk secretion, body reserves mainly from adipose tissue are mobilized and imply an increased risk for metabolic diseases. Reducing the energy output via milk by decreasing the milk fat content through feed supplements containing conjugated linoleic acids (CLAs) may attenuate the negative energy balance during this period. In two separate trials, variables characterizing fat cell turnover were investigated in different subcutaneous and visceral fat depots from primiparous heifers (n = 25) during early lactation, and subcutaneous fat from non-lactating, over-conditioned heifers (n = 12) by immunohistochemistry. The portion of apoptotic adipocytes was consistently greater than that of proliferating cells and preadipocytes; the sporadically observed effects of CLA were limited to visceral fat. Lactating heifers had more apoptosis and less preadipocytes than non-lactating heifers. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Early lactation is classified as the most metabolically stressful period in dairy cows, because the energetic needs for the rapidly increasing milk synthesis cannot be covered by voluntary feed intake, resulting in a state of negative energy balance (NEB; Grummer, 1995). During this period, high-yielding dairy cows are susceptible to metabolic disorders, compromised immune response and reduced fertility (Butler and Smith, 1989; Mallard et al., 1998). The energy deficit during early lactation is accompanied by fat mobilization from adipose tissue (AT): during the NEB lipogenesis is decreased and lipolysis is increased (McNamara and Hillers, 1989). When the energy balance reaches positive values, the energy stores in AT are increasingly refilled by lipogenesis and possibly also by adipogenesis thus preparing the organism for the upcoming energy deficit in the subsequent lactation. The cellular components of AT comprise not only mature adipocytes but also other cell types e.g. endothelial cells, adipocyte progenitors (preadipocytes), fibroblasts, and immune cells, that are commonly summarized as the stromal vascular fraction (SVF; Eto et al., 2009). The major processes involved in adipogenesis are proliferation and differentiation of preadipocytes into mature adipocytes (Hausman ⇑ Corresponding author. Address: Institute of Animal Science, Physiology and Hygiene Group, University of Bonn, Katzenburgweg 7 – 9, 53115 Bonn, Germany. Tel.: +49 228739669; fax: +49 228737938. E-mail address: [email protected] (S. Häussler). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.07.004

et al., 2009), whereby the dynamic balance between both processes causes changes in adipocyte size (Prins and O‘Rahilly, 1997). In dairy cows, changes in adipocyte size generally appear after 120 days of lactation (Smith and McNamara, 1989). Adipocyte homeostasis and turnover processes include cell proliferation, differentiation of preadipocytes as well as programmed cell death (apoptosis). Conjugated linoleic acids (CLA) designate a group of positional and geometric isomers of linoleic acid. CLA has been reported to have many health benefits, including anti-obesity, anti-carcinogenic, anti-atherogenic, anti-diabetogenic, and immunomodulating properties in certain animal models (Belury, 2002; McLeod et al., 2004). In dairy cows, dietary CLA supplementation alters lipid metabolism and causes a decrease in milk fat secretion during lactation (Loor and Herbein, 1998). Commercially available CLA preparations for supplementing cows‘ diets mainly contain the cis-9, trans-11 (c9,t11) and the trans-10, cis-12 (t10,c12) isomer in equal portions. In cultured adipocytes and AT from rodents, antilipogenic and/or lipolytic effects of dietary CLA are described (Brodie et al., 1999; Azain et al., 2000; Miner et al., 2001). The milk fat reducing effect of CLA is mainly attributed to the t10,c12 isomer (Baumgard et al., 2001), whereas the c9,t11 isomer had less effects (Perfield et al., 2007). Furthermore, body fat accretion in growing animals was decreased through decreasing de novo lipogenesis by the t10,c12 isomer (Bauman et al., 2000). In dairy cattle, we recently observed that adipocyte size is decreased at varying degrees in different fat depots from animals receiving a 1:1 mixture of the t10,c12 and the c9,t11 isomer when compared with a control group

S. Häussler et al. / Research in Veterinary Science 95 (2013) 1142–1150

that received a fat supplement in which CLA was substituted by stearic acid (Akter et al., 2011), indicating lipolytic and/or adipogenic effects of CLA on bovine AT. To elucidate the underlying processes for the observed CLA-induced reduction of adipocyte size but also for the changes related to time of lactation, our objective was to characterize the dynamics of fat cell turnover in different AT depots of early-lactating dairy heifers treated with or without CLA. In addition, the studies were extended to non-lactating, over-conditioned heifers in which more large adipocytes are expected, to characterize the effects of a moderate weight loss induced by feed restriction. The variables characterizing fat cell turnover were tested for relationships with the serum concentrations of metabolic hormones and non-esterified fatty acids (NEFA) released during lipolysis.

2. Materials and methods 2.1. Animals, experimental design and diets The experimental design of the trials described below is illustrated in Fig. 1, which has in parts been already published previously (Akter et al. 2011; von Soosten et al., 2011). Trial 1: Early-lactating heifers. All steps of the animal experiment were approved by the animal welfare commission (Lower Saxony State Office for Customer Protection and Food Safety (LAVES, File No. 33.11.42502-04-071/07, Oldenburg, Germany). In brief, 25 primiparous German Holstein Friesian (HF) heifers were kept in a free-stall barn at the Experimental Station of the Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Braunschweig, Germany. Body condition of the heifers was scored according to the 5 scale system by Edmonson et al. (1989) in which 5 defines a status of over-condition. At the day of slaughter, earlylactating heifers in the present study had a mean body condition

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score (BCS) of 3.0; at slaughter their average age was 25.5 ± 1.3 months. The animals had access to water for ad libitum intake. Before parturition, heifers received a diet consisting of 60% corn silage and 40% grass silage (6.7 MJ NEL/kg dry matter (DM)) on a DM basis ad libitum and 2 kg concentrate/d (6.7 MJ NEL/kg DM). After parturition, they were fed a partial mixed ration (PMR) consisting of 25% grass silage, 38% corn silage and 37% concentrate on a DM basis for ad libitum intake (7.5 MJ NEL/kg DM). In addition, they received 4 kg concentrate/d (average: 8.8 MJ NEL/kg DM) from parturition until slaughter. Animals were randomly allotted to a CON (n = 15) or a CLA (n = 10) group. From 1 d post partum (p.p.) until slaughter, CLA heifers received 100 g/d CLA supplement (LutrellÒ Pure, BASF SE, Ludwigshafen, Germany) containing 6.0 g each of the c9,t11 and the t10,c12 isomer (calculated portion in the CLA-concentrate). CON heifers received 100 g/d of a control fat supplement (SilafatÒ, BASF SE) in which CLA were substituted by stearic acid in addition to the standard diet. All diets were following the recommendations of GfE (2001) for late pregnant and early-lactating heifers, respectively. The CON heifers (n = 5 each) were slaughtered at 1, 42, and 105 d p.p., whereas the CLA heifers (n = 5 each) were slaughtered at 42 and 105 d pp, respectively. Energy balance was calculated previously (von Soosten et al., 2011) according to the recommendations of the German Society of Nutrition Physiology (GfE, 2001; see Fig. 2). Trial 2: Non-pregnant, over-conditioned heifers. The experiment was approved by the German animal welfare commission (competent authority: LANUV NRW 8.87-51.05.20.10.103, Recklinghausen, Germany). Twelve Simmental (SM) heifers with a mean BCS of 5.0 were housed in tie-stall barns on straw at the research farm Frankenforst of the Faculty of Agriculture, University Bonn, Germany. The average age of the animals was 42.2 ± 2.7 months at the first biopsy. Fig. 1 shows the experimental design. The animals were continuously fed with grass silage for ad libitum intake until they were allocated to 2 groups of similar

Fig. 1. Experimental designs of trial 1 [early lactating Holstein heifers fed diets of control (CON) and conjugated linoleic acid (CLA) supplements; n = 25] and trial 2 (nonpregnant, over-conditioned Simmental heifers fed diets of varying energy density; n = 12). The animals from trial 1 slaughtered from the CON and the CLA groups at each sampling day (n = 5) per group are depicted as hatched bar sections (already published by Akter et al. (2012)).

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S. Häussler et al. / Research in Veterinary Science 95 (2013) 1142–1150

Fig. 2. Energy balance of control (CON) and conjugated linoleic acid (CLA) supplemented dairy cows during the first 105 days post partum. Each group represents the mean ± standard error of the means of 5 animals. ⁄⁄⁄p < 0.001. Net energy balance (MJ of NEL/d) = energy intake (MJ of NEL/d) [NEM(MJ of NEL/ d) + NEL (MJ of – NEL/d)]. 1 These data has been recently published by Akter et al. (2011) and von Soosten et al. (2011).

mean BW (692 ± 43 kg). To investigate the effects of a moderate weight loss, half of the heifers was randomly allotted to a ration in which the grass silage was blended with a hay/straw mixture (ratio 37:63 on a DM basis, n = 6, LE group), whereas the other half of the heifers remained on the grass silage feeding (HE group). The grass silage contained 10.6% crude protein (CP), 54.8% neutral detergent fiber (NDF, ash-free), and 34.6% acid detergent fiber (ADF, ash-free), and the hay/straw-silage mixture contained 8.5% CP, 63.2% NDF (ash-free), and 37.0% ADF (ash-free) on a DM basis. The estimated ME concentrations of the grass silage and hay/straw mixture were 9.5 and 8.1 MJ/kg of DM, respectively. After 4 weeks of differential feeding, all animals were fed with grass silage for further 3 weeks. To avoid potential effects of divergent stages of estrus cycle, all animals received a progesterone-releasing intravaginal device (PRID-alphaÒ, Ceva Sante Animale, Libourne, France) that was renewed every third week. Serum progesterone concentrations, with means values of 3.57 ± 0.4 ng/mL, were analyzed by ELISA as described by Sauerwein et al. (2006).

disinfected. After local sc lumbar anesthesia with 2% procaine hydrochloride (Procasel 2%, Selectavet, Dr. Otto Fischer, WeyarnHolzolling, Germany), an approximately 5 cm incision was done to remove sc AT. Tissues were rinsed with 0.9% sodium chloride and immediately snap frozen in liquid nitrogen and stored at 80 °C until analyses. Samples (n = 36) were collected by repeated surgical biopsies from alternate sites as described above, i.e. one sampling each at the end of the consistent grass silage feeding (start/0 wk), the differential feeding (4 wk) and the final (re)feeding period (7 wk), as shown in Fig. 1. Blood samples were collected by venipuncture of the Vena coccygea using monovettes (Sarstedt, Nümbrecht, Germany) before biopsy sampling procedure was started. Serum was prepared by centrifugation (20 min, 1200g, 4 °C) and analyzed for NEFA, IGF-1 and leptin. Serum NEFA concentrations were determined by enzymatic-colorimetric method using a commercial test kit (Free fatty acids, Half-micro test, Cat. No. 11383175, Roche Diagnostics, Mannheim, Germany). Leptin was analyzed by ELISA as described by Sauerwein et al. (2004), while IGF-1 was measured by a commercial test kit (Mediagnost, Reutlingen, Germany). 2.3. Histological and immunohistochemical techniques Either paraffin embedded or frozen tissue sections were used. After fixation for 48 h in paraformaldehyde (Roth, Karlsruhe, Germany), samples were dehydrated and embedded in paraffin (Romeis, 2010). Sections from bovine AT (12–14 lm) sampled within the described trials, as well as tissues from cows slaughtered at a local abattoir which served as control tissue: liver (6 lm), lymph node (6 lm), adrenal cortex and fetal placenta (6 lm), were cut either with a rotation microtome (SLEE, Mainz, Germany) or a cryostat (Leica, Microsystems, Wetzlar, Germany). Paraffin embedded sections were deparaffinized in RotihistolÒ (Roth), rehydrated through descending grades of isopropanol (Roth) and heated in a microwave 5 times (700 W) each for 5 min in 0.01 M citrate buffer (pH 6.0) for antigen retrieval. Cryosections were fixed in ice-cold acetone for 10 min followed by 3  5 min hot PBS-washing (pH 7.2, 700 W, for Ki67) or a methanol/chloroform mixture (1:1, vol/vol) for 4 h, dried at room temperature (RT) for 1 h and rehydrated with PBS (pH 7.4) for 5 min (Pref-1). 2.4. Cell proliferation (Ki67) and preadipocytes (Pref-1)

2.2. Sample collection and measurements For trial 1, AT samples from different visceral (vc; retroperitoneal, omental, and mesenteric) and subcutaneous (sc; tailhead, withers, and sternum) depots were obtained immediately after slaughter. Fat tissue (1 cm3) was removed and fixed immediately either in 4% paraformaldehyde (Roth, Karlsruhe, Germany) until further embedding or was snap frozen in liquid nitrogen and stored at 80 °C until analyses. Blood was collected in Vacutainer tubes containing sodium heparin or potassium EDTA (Becton Dickinson VacutainerÒ Systems USA, Rutherford, NJ) by venipuncture 30 min before slaughter. Blood samples were analyzed for plasma concentrations of non-esterified fatty acids (NEFA) and b-hydroxybutyrate (BHBA) with commercial kits (NEFA-C, Wako Chemicals GmbH, Neuss and RANBUT, Randox Laboratories GmbH, Wulfrath, Germany). Leptin serum concentrations were assayed by ELISA as described by Sauerwein et al. (2004). IGF-1 plasma concentrations were analyzed in duplicates by a commercially available two-site immunoradiometric assay (IRMA, DSL-5600 Active IGF-1-IRMA; Diagnostic Systems Laboratories, Inc., Webster, TX). For trial 2, tissue biopsies were removed from the area of the sc tailhead AT as described earlier in detail by Smith and McNamara (1989). In brief, the region of sampling was cleaned and

The detection of cell proliferation was based on a monoclonal mouse antibody against the proliferation marker Ki67 (1:25; clone MM1; LS-C95454; LifeSpan BioSciences; Biozol, Eching, Germany) on frozen sections. Preadipocytes were localized on either paraffin (HF heifer samples) or frozen (SM heifer samples) sections with polyclonal antibodies raised in goat against the human preadipocyte factor-1 (Pref-1; 1:100; sc-8624; Santa Cruz Biotechnology, Santa Cruz, CA). Samples were treated either with 3% (paraffin sections) or 0.3% (frozen sections) H2O2 to quench endogenous peroxidase activity. Unspecific binding was blocked with normal serum (1:10 in PBS; Ki67: horse serum; Pref-1: rabbit serum) at RT for 20 min. Incubation with the specific antibodies was done either for 1 h at 37 °C (paraffin sections) or overnight at RT (frozen sections). Afterwards, the sections were washed and incubated with the corresponding biotinylated antibodies for 30 min at RT (Ki67: horse-anti-mouse; 1:50 in PBS; Vectastain Kit, Vector Laboratories, Burlingame, CA; Pref-1: rabbit anti-goat; 1:200 in PBS; Southern Biotech, Birmingham, AL, containing 5% bovine serum), followed by incubation with streptavidin (1:1000 in PBS) for 30 min at RT. Immunoreaction was visualized with 3-amino-9-ethylcarbazol (Biozol) as a substrate, counterstaining was done with hematoxylin (Merck, Darmstadt, Germany). For positive controls, liver sections

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(Ki67), placental chorion (Pref-1; paraffin section) and adrenal cortex (Pref-1; frozen sections) from mature cows were used. For negative controls, the primary antibodies were replaced by PBS to exclude unspecific binding of the secondary antibody.

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NEFA, BHBA, leptin and IGF-1 as well as energy balance (for trial 1), regression coefficients were calculated and considered as biologically meaningful at r > 0.316 (regression above 10%). Statistical significance was declared at P < 0.05; a trend was defined for 0.05 < P < 0.1.

2.5. TUNEL assay Apoptosis was identified by a modified terminal deoxynucleotidyl transferase mediated dUTP nick-end-labelling (TUNEL) assay (Gavrieli et al., 1992) for staining, apoptosis-specific DNA fragments. In brief, sections were treated with recombinant Proteinase K (1:100; Roche Diagnostics, Mannheim, Germany) for 10 min. Endogenous peroxidase was blocked using 0.3% H2O2, followed by a 10 min incubation with terminal deoxynucleotidyl-transferase buffer (pH 7.2) for TUNEL staining. Terminal transferase (16 U/ll; Roche Diagnostics, Mannheim, Germany) and 11-dUTPbiotin (Applichem, Darmstadt, Germany) were then incubated in a humidifying chamber at 37 °C for 1 h. After rinsing in TB-buffer (0.3 M NaCl + 0.03 M NaCitrat pH 8.0) and TBS (pH 7.6), the sections were incubated with 3% bovine serum albumin in TBS and streptavidin-peroxidase-complex (Southern Biotech) at RT for 30 min. Staining was done with 3-amino-9-ethylcarbazol (Biozol), counterstaining was done with hematoxylin (Merck). Lymph node samples from mature cows served as internal control. For positive controls, sections were treated with DNase I (1 U/ll; Roche Diagnostics) for 10 min before blocking endogenous peroxidase to provoke DNA strand breaks. Negative controls were treated in an identical manner to the normal slides, without the addition of the transferase solution. 2.6. Histological analyses The sections were examined by light microscopy (Leica DMR, Leica Microsystems, Wetzlar, Germany) at 200-fold magnification. From each section, 10 randomly selected different fields were evaluated using a grid (350  450 lm). The number of positive cells as well as the total number of cells was manually counted within the different fields; subsequently the mean out of the evaluated fields was calculated and finally presented as the percentage of total number of cells. For positive cells, mature adipocytes and cells belonging to the SVF were distinguished; the latter included all cells without large lipid droplets i.e. without mature adipocytes. Adipocyte areas (lm2) of 100 adipocytes per section from HF heifers and SM heifers were measured as described by Akter et al. (2011).

3. Results 3.1. Histological and immunohistochemical stainings Ki67-positive nuclei, representing cell proliferation in AT, and cells stained for Pref-1 in the cytoplasma (preadipocytes) were exclusively found in the SVF of AT, whereas TUNEL-positive stainings (apoptosis) were exclusively observed in the nucleus of mature adipocytes. Apoptosis, proliferating cells and preadipocytes were found in almost all fat depots of early-lactating heifers and in sc tailhead fat of SM heifers. Examples for histological and immunohistochemical stainings are provided in Fig. 3. 3.2. Variables characterizing cell turnover in adipose tissues Trial 1. Irrespective of energy balance (changes are summarized in Fig. 2), time of lactation or CLA treatment, a mean cell proliferation of 0.18 ± 0.14% per total cell number was observed in all fat depots of the HF heifers (Table 1). In all fat depots of HF heifers 1.04 ± 0.11% preadipocytes per total cell number were observed and were neither affected by time of lactation nor CLA supplementation in all depots except retroperitoneal tissue. In this depot 25times more preadipocytes were detected in CON animals at 42 d p.p. compared to animals at 105 d p.p.. In the retroperitoneal tissue, less apoptosis (4.97 ± 1.55%) was detected compared to all other five fat depots (13.9 ± 3.23%), independent of lactation period and treatment. In CON heifers, about one third less apoptosis was observed in retroperitoneal fat at 42 d p.p. compared to mesenteric and sc tailhead fat. Only in the sc sternum depot apoptosis of adipocytes changed during lactation, where the portion was more than 3times greater at 42 and 105 d p.p. compared to 1 d p.p. (Table 1). After CLA supplementation apoptosis decreased by about 50% in mesenteric fat at 42 d p.p.. Moreover, at 105 d p.p. 16-times more preadipocytes were observed in retroperitoneal tissue from CLAfed heifers than in CON heifers. Comparing the different fat depots from CLA supplemented heifers, about 30% less apoptosis was observed in the retroperitoneal fat compared to the sc fat from sternum and about 25% less in sc withers fat in CLA heifers at 105 d p.p..

2.7. Statistical analyses For statistical analyses, the samples were tested for normal distribution using the Kolmogorov–Smirnov test and for homogeneity of variances with the Levene’s test. Parametric and non-parametric tests were used (SPSS Statistics 19 Inc., Chicago, IL). All data are given as the arithmetic mean ± SEM. For trial 1, day of lactation was considered as main effect and the General linear model (GLM) followed by a Bonferroni post hoc test was used for apoptosis, Pref-1, Ki67 and the adipocyte area in samples obtained after slaughter. To assess potential differences between CON and CLA treatment, the Students t-test was used. In trial 2, the Friedman test combined with the Mann–Whitney U-test for pair wise comparisons was used to compare repeated measurements between sampling days (0, 4 and 7 wk). For comparisons between the treatment and the sampling days, the threshold for statistical significance was defined as P < 0.017 after the Bonferroni a-correction for multiple comparisons (n = 3; a = 0.05/3 = 0.017). To examine the relation between the variables characterizing fat cell turnover and adipocyte area, plasma concentrations of

3.3. Variables characterizing fat cell turnover and adipocyte area in different fat depots Trial 1. Adipocyte areas were related to the variables characterizing fat cell turnover. The adipocyte sizes of different AT from HF heifers used herein were published earlier (Akter et al., 2011) and are presented here in brief to allow for comparisons (Table 1). Over all, adipocyte size and cell proliferation as well as preadipocyte numbers were not related. In contrast, apoptosis of adipocytes in fat from CLA treated animals was significantly associated with adipocyte size, albeit regression was less than 10%. Similarly, the relation between fat mass and apoptotic rate -irrespective of CON or CLA- was significant, but with r < 0.316 (Fig. 4). 3.4. Relationships between cell turnover in adipose tissue and the blood concentrations of metabolic hormones, NEFA and BHBA Trial 1. The circulating plasma concentrations of IGF-1, leptin, NEFA and BHBA of HF heifers are represented in Table 2. Significant

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Fig. 3. Localization of variables characterizing fat cell turnover. Examples of the histological and immunohistochemical stainings. A & B1,2: cell proliferation (Ki67 positive cells) in retroperitoneal fat tissue (A) and bovine liver served as positive (B1) and negative control (B2); 400-fold magnification. C & D1,2: preadipocytes (Pref-1 positive cells) in retroperitoneal fat tissue (C); 400-fold magnification, bovine placental chorion served as positive (D1) and negative (D2) control; 200-fold magnification; E & F1,2: Apoptosis (TUNEL assay) in retroperitoneal fat tissues (E); 400-fold magnification, bovine lymph nodes served as positive (F1) and negative (F2) control; 200-fold magnification. Arrow heads mark the positive cells and nuclei. Scale bars represent 20 lm.

relationships were observed between apoptosis of adipocytes over all fat depots and plasma NEFA as well as IGF-1 concentrations but r was less than 0.316 (Fig. 4). All variables used to characterize fat cell turnover were neither related to leptin nor BHBA concentrations. 3.5. Variables characterizing and regulating cell turnover in adipose tissues of non-lactating, over-conditioned heifers Trial 2. None of the variables characterizing cell turnover in AT from non-lactating, over-conditioned heifers was influenced by the different feeding, therefore the data from the HE and LE feeding groups were combined (Table 3). The mean portions for cell proliferation, preadipocytes and apoptosis were 0.32 ± 0.10%, 3.54 ± 0.82% and 0.88 ± 0.26%, respectively. The feeding aiming to induce a moderate weight loss was not effective: neither body weight nor the serum concentrations of NEFA, IGF-1 and leptin were affected and therefore we pooled the data from both groups (Table 4). 4. Discussion 4.1. Cell turnover in bovine adipose tissue Adipose tissue mass is determined by balancing lipolysis, lipogenesis, and adipocyte proliferation (Della-Fera et al., 2001). To understand the detailed mechanisms of cellular dynamics in different fat depots of dairy cattle, we investigated three different variables to characterize fat cell turnover in AT. Determination of cell

proliferation, preadipocytes and apoptosis are key variables to define cell turnover within AT, as shown by several authors (Prins and O‘Rahilly, 1997; Corino et al., 2005; Tchoukalova et al., 2010). The localization of these key variables either to the nucleus or the cytoplasm corresponded to previous studies (Smas et al., 1997; Baldwin et al., 2004; Monteiro et al., 2008). Fat depots are located in defined areas in the body and are distinguished into visceral (vc) and subcutaneous (sc) fat with different metabolic functions (Rosen et al., 2000). Our results support that variables characterizing cell turnover for bovine AT are also depot specific. Both, depot specificity as well as age are strong factors influencing fat cell turnover: Perirenal AT is the first fat depot appearing in fetal development followed by subcutaneous fat, however, hyperplasia of perirenal fat stagnate postnatal (Bonnet et al., 2010). Retroperitoneal AT is the most metabolically active depot in cattle (von Soosten et al., 2011), which implies a high lipolytic and lipogenic activity in this fat depot. In the present study, apoptosis was significantly lower in reptroperitoneal AT when compared to other vc and sc depots. In addition, adipocyte sizes in retroperitoneal AT were mostly larger compared to adipocytes from other sites (Akter et al., 2011). We thus assume that more lipids can be stored and eventually mobilized in the retroperitoneal adipocytes. In the present study, apoptosis was increased in fat depots with smaller adipocytes; however the total number of adipocytes per depot could not be determined. The reason why other fat depots exhibit more apoptosis as compared to retroperitoneal fat might be that smaller adipocytes lose their function as lipid storage once having reached a critical (small) size, consequently cells might be undernourished which finally induce apoptosis.

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Table 1 Portions (%) of proliferating cells (Ki67), preadipocytes (Pref-1), apoptotic cells (TUNEL) and adipocyte area (lm2, already published by Akter et al. (2011)) in different fat depots of Holstein Friesian heifers supplemented with control fat (CON, n = 15) or with conjugated linoleic acids (CLA, n = 15) during the first 105 days post partum (p.p.); means ± SEM. d.p.p.

Subcutaneous depots Ki67 Tailhead Withers Sternum

CON

CLA

1

42

105

42

105

0.66 ± 0.34 n.d. 0.11 ± 0.06

n.d. 0.04 ± 0.03 n.d.

0.02 ± 0.02 n.d. 0.10 ± 0.06

n.d. 0.62 ± 0.50 n.d.

n.d. 0.01 ± 0.01 0.89 ± 0.82

Pref-1 Tailhead Withers Sternum

1.75 ± 0.79 0.65 ± 0.47 0.19 ± 0.17

1.21 ± 0.81 1.07 ± 0.45 1.04 ± 0.66

2.07 ± 0.44 0.49 ± 0.30 0.62 ± 0.23

1.98 ± 0.39 0.38 ± 0.14 0.61 ± 0.25

1.81 ± 0.84 0.77 ± 0.15 1.49 ± 0.64

TUNEL Tailhead Withers Sternum

15.0 ± 6.46 12.0 ± 3.57 5.99 ± 2.03a

13.3 ± 0.782 13.1 ± 4.331,2 20.3 ± 3.58b/1,2

9.60 ± 2.55 22.9 ± 8.02 19.0 ± 2.50b

10.8 ± 0.95 14.9 ± 4.81 12.2 ± 2.71

14.8 ± 2.821,2 22.7 ± 3.362 16.2 ± 1.282

Adipocyte area Tailhead Withers Sternum Visceral depots

6300 ± 4481,2 6258 ± 4511,2 5782 ± 2431

5442 ± 408 5110 ± 407 5180 ± 314

6103 ± 325 4521 ± 390 4831 ± 454

4573 ± 2911,2/a 3976 ± 3321 3959 ± 871#

3233 ± 127b# 3021 ± 583 3452 ± 329

Ki67 Omental Mesenterial Retroperitoneal

0.04 ± 0.03 0.09 ± 0.08 0.16 ± 0.15

0.03 ± 0.02 0.09 ± 0.05 0.05 ± 0.04

0.17 ± 0.07 n.d. 0.10 ± 0.01

0.06 ± 0.05 0.08 ± 0.07 n.d.

Pref-1 Omental Mesenteric Retroperitoneal

0.48 ± 0.31 0.56 ± 0.31 1.07 ± 0.35a,b

0.56 ± 0.18 0.43 ± 0.26 2.04 ± 0.46a

1.30 ± 0.31 1.10 ± 0.79 0.08 ± 0.04b

1.47 ± 0.89 0.86 ± 0.34 1.32 ± 0.47

2.27 ± 0.54 0.29 ± 0.27 1.33 ± 0.32

TUNEL Omental Mesenteric Retroperitoneal

10.2 ± 3.30 16.8 ± 4.94 4.57 ± 1.23

12.6 ± 4.181,2 17.5 ± 2.652 5.16 ± 1.321

10.2 ± 2.08 12.0 ± 2.40 3.71 ± 0.92

9.84 ± 2.33 8.35 ± 2.07# 5.59 ± 2.76

10.9 ± 4.191,2 16.7 ± 2.981,2 5.80 ± 1.521

Adipocyte area Omental Mesenteric Retroperitoneal

6381 ± 3721,2 6172 ± 2941,2 7945 ± 2082/a

6077 ± 289 6222 ± 267 6634 ± 234a,b

5851 ± 286 5784 ± 436 6500 ± 414b

4750 ± 2771,2 4235 ± 2961,2# 5552 ± 4072

0.29 ± 0.12 0.30 ± 0.16 0.37 ± 0.28#

3997 ± 410# 3902 ± 207# 4316 ± 335#

1,2

Within a column, means without a common superscript differ within one variable/day (P < 0.05). Within a row, means without a common superscript differ during lactation (P < 0.05). #: CLA effect, p < 0.05 when compared against CON of the same day of lactation. n = Number of heifers per treatment. n.d.: not detected.

a,b

In the present study, the dynamic observed in fat cell turnover of HF heifers was based on apoptosis of adipocytes and renewing of adipocytes as indicated by the number of preadipocytes. In contrast, cell proliferation was only marginal in the different fat depots of early-lactating heifers. In addition, in the over-conditioned state, more preadipocytes than proliferating cells were observed. Stimulating a moderate weight loss in AT of non-lactating, overconditioned heifers was aiming to induce fat mobilization. However, in the present study, 4 weeks of feeding a diet with reduced energy content were not sufficient to induce a significant weight loss. Assumingly the number of fat cells in cattle is set in early development showing a remarkable turnover within the static cell population in adults, as postulated for humans (Spalding et al., 2008). In dairy cattle, shrinkage of adipocyte size is accompanied by a stable number of adipocytes in early lactation (Smith and McNamara, 1989). In addition, increased fat mobilization (Bauman and Elliot, 1983) as well as a decrease in lipid synthesis in early lactation has been observed repeatedly (Smith and McNamara, 1989; McNamara, 2012). As described earlier, body fat continues to accumulate and new fat cells appear in later lactation (Smith and McNamara, 1989). Newly formed mature adipocytes arise from preadipocytes, which in turn proliferate from progenitor cells (DiGirolamo et al., 1998). Besides their role as preadipocytes in the process of adipocyte differentiation, progentitor cells, i.e. stem cells

and adipoblasts are part in the process of lineage determination (Koutnikova and Auwerx, 2001). This implies that progenitor cells are the ones being set, whereas the preadipocytes are underlying turnover. However, the cell turnover represented by cell proliferation, preadipocytes and apoptosis within different bovine fat depots has not been investigated so far. 4.2. Effects of CLA supplementation on variables characterizing fat cell turnover Adipocyte size decreased in vc and at least in one sc fat depot due to diets enriched with CLA (Akter et al., 2011), although fat mass and fat mobilization of the individual AT depots were largely not affected by CLA supplementation (von Soosten et al., 2011). Therefore, we aimed to investigate whether CLA induced fat cell size reduction might be related to alterations of apoptosis, preadipocytes and/or cell proliferation in different AT depots. When investigating the effects of CLA on variables characterizing cell turnover in AT, apoptosis in mesenteric fat at 42 d p.p. was decreased in CLA heifers compared to the CON group and more preadipocytes at 105 d p.p. were observed in retroperitoneal fat of CLA supplemented heifers. CLA thus seems to increase the number of functional adipocytes at least for these two vc depots. CLA reduces adipocyte volume without effect on adipose tissue mass,

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Fig. 4. Relationships between apoptosic cells (%) and (A) plasma NEFA concentrations (lM), (B) fat mass (kg), (C) plasma IGF-1 concentrations (ng/mL), and (D) adipocyte area (lm2). Regressions were calculated either for both control and conjugated linoleic acid (CLA) treatment (d) or separately for CLA treated animals (N). Statistical significance was defined as P < 0.05.

Table 2 Plasma concentrations of IGF-1 (ng/mL), Leptin (ng/mL), NEFA (lM) and BHBA (lM) of Holstein Friesian heifers supplemented with control fat (CON) or with conjugated linoleic acids (CLA) during the first 105 days post partum (p.p.); means ± SEM. Concentrations

1 d p.p.

42 d p.p.

105 d p.p.

CON (n = 15) IGF-1 (ng/mL) Leptin (ng/mL) NEFA (lM) BHBA (lM)

33.0 ± 2.2a 4.83 ± 0.48 710 ± 93.9a 516 ± 33.6

105 ± 14.7b 4.88 ± 0.50 292 ± 51.0b 593 ± 77.9

119 ± 14.3b 8.21 ± 2.49 268 ± 64.2b 548 ± 30.6

100 ± 8.4 5.32 ± 0.68 274 ± 48.1 573 ± 94.5

110 ± 14.0 5.11 ± 0.41 182 ± 40.9 548 ± 74.5

Items

CLA (n = 10) IGF-1 (ng/mL) Leptin (ng/mL) NEFA (lM) BHBA (lM) a,b

Within a row, means without a common superscript differ (P < 0.017). n = Number of heifers per treatment.

Table 3 Portions (%) of proliferating cells (Ki67), preadipocytes (Pref-1), apoptotic cells (TUNEL) and adipocyte area (lm2) in the subcutaneous tailhead depot of Simmental heifers on three different sampling points (start/0 wk, 4 wk, 7 wk), irrespective of the different feeding groups (high and low energetic grass silage); means ± SEM; n = number of heifers per treatment. Items

Heifers (n = 12) Ki67 Pref-1 TUNEL Adipocyte area

Table 4 Body weight (kg), serum NEFA (lM), IGF-1 (ng/mL) and leptin (ng/mL) concentrations of Simmental heifers fed with high (HE) and low (LE) energetic grass silage on three different sampling points (start/0 wk, 4 wk, 7 wk); means ± SEM; n = number of heifers per treatment.

Sampling points Start/0 wk

4 wk

7 wk

0.35 ± 0.14 4.33 ± 0.93 0.80 ± 0.24 8045 ± 380

0.16 ± 0.08 3.02 ± 1.08 1.25 ± 0.38 7954 ± 385

0.46 ± 0.09 3.27 ± 0.44 0.60 ± 0.17 8682 ± 589

probably because CLA increase the number of preadipocytes poised to differentiate into adipocytes as already proposed in rats (Poulos et al., 2001) and pigs (Corino et al., 2005). Azain et al. (2000) reported that CLA reduced adipocyte size in rats, and Poulos et al. (2001) found that CLA increased the portion of smaller cells in

Heifers (n = 12) Body weight (kg) NEFA (lM) IGF-1 (ng/ml) Leptin (ng/ml)

Sampling points Start/0 wk

4 wk

7 wk

730 ± 12.4 74.0 ± 0.00 202 ± 18 8.83 ± 1.39

722 ± 11.1 115 ± 12 261 ± 20 9.04 ± 1.46

740 ± 11 75.4 ± 0.71 307 ± 28 9.54 ± 1.48

rat pups. However, in all these studies the supplemented amount of CLA isomers was more than 8-times greater compared to our study. Besides its dose-dependency, CLA effects depend on the isoform used as reported recently for t10,c12 CLA reducing milk fat yield but not milk yield and other milk components in dairy cows (Harvatine et al., 2009). The markers of cell turnover we used herein were not as consistently altered as was adipocyte size in the present study, however, the detachedly observed differences between CON and CLA-treated animals indicate that the increased frequency of smaller adipocytes during CLA supplementation might be due to both reduced apoptosis and increased portion of preadipocytes. Possibly, using the individual CLA isomers separately and/or a higher dose would have yielded different results.

4.3. Regulation of cell turnover in bovine adipose tissue Several regulators are regarded as important for controlling adipocyte number and fat cell turnover including insulin, ligands for the peroxisome proliferator activated receptor c, e.g. CLA, retinoids, corticosteroids and tumor necrosis factor a (Prins and O‘Rahilly, 1997). Changes in AT, such as adipokine expression or oxygen content, may effect preadipocyte replication and differentiation (Rigamonti et al., 2011). In the current study, significant

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regressions (below 10%) between variables characterizing fat cell turnover, i.e. apoptosis with plasma NEFA and IGF-1 concentrations were established. Furthermore, both the decrease of adipocyte size (Akter et al., 2011) as well as apoptosis of adipocytes were related, albeit only weakly (regression below 10%) to the loss of fat mass during early lactation. Therefore, we assume that small adipocytes (showing more apoptosis) have less lipids to mobilize and thus release less NEFA. In sheep, high concentrations of unesterified fatty acids, indicating lipolysis, were accompanied by decreased adipocyte mean volume during late pregnancy and early lactation (Vernon et al., 1981). In general, mobilization of body reserves increases plasma NEFA concentrations and may result in an increased fatty acid accumulation in the liver (Grummer, 1995). In dairy cows, increased AT lipolysis is generally reported in late pregnancy (McNamara and Hillers, 1989, Sumner and McNamara, 2007), pursuing in the period of NEB in early lactation (Chilliard et al., 2000) and also occurring during chronic malnutrition (Dunshea et al., 1988). Furthermore, the marginal rate of apoptosis observed in fat depots of nonlactating, non-pregnant heifers was not related to NEFA concentrations, given that the reduced energy content of the ration was not sufficient to increase lipolysis. IGF-1 is a mitogenic factor, stimulating cell proliferation and preadipocyte numbers. IGF-1 promotes cell survival in many tissues and cell proliferation in some tissues (Raff, 1996). It is known to have an anti-apoptotic effect, and to contribute to cell survival (Gehmert et al., 2008). In addition, IGF-1 enhanced TNF-a induced apoptosis in 3T3-L1 preadipocytes (Niesler et al., 2000). However, in the present study, the relevance of IGF-1 on variables characterizing cell turnover in AT remain unknown. The relations between apoptotic cell portion and circulating IGF-1 observed in the current study were only small (r < 0.316). Leptin is a peptide hormone produced by adipocytes; it reduces food intake and increases metabolic rate (Houseknecht and Portocarrero, 1998). The importance of leptin in regulation of AT mass has been reported previously (Prins and O‘Rahilly, 1997), nevertheless, in the present study the hormone was only weakly correlated with fat mass (r < 0.316). Leptin has been demonstrated to reduce fat mass not only by increasing lipolysis, but also by stimulating apoptosis in AT (Qian et al., 1998; Gullicksen et al., 2003), we therefore assumed that apoptosis in bovine AT and plasma leptin concentrations might be related. However, in the present study, no relation between variables determining fat cell turnover and plasma leptin concentrations was observed in early lactating heifers. Indeed, when comparing both trials, over-conditioned heifers with higher leptin concentrations showed less apoptosis than early-lactating heifers. An inhibitory effect of leptin on adipogenesis is known: leptin does not act directly to induce apoptosis but can directly inhibit maturation of preadipocytes (Ambati et al., 2007). However, no relationship was observed between preadipocytes and leptin concentrations. From the available results, the effects of leptin on adipogenesis still remain unclear, stimulatory or inhibitory effects depend on the study and/or the cell types being investigated. In summary, in early lactating heifers fat cell turnover was mainly influenced by apoptosis of fat cells. Apoptosis was neither balanced by cell proliferation nor by the amount of preadipocytes. However, due to the complexity of fat cell turnover, besides preadipocytes, stem cells and/or adipoblasts might be involved in balancing apoptosis. Comparing apoptotic rate and adipocyte area from subcutaneous fat irrespective of breed, physiological status and treatment, a significant relation between adipocyte size and apoptotic portion was observed (r = 0.616), indicating that generally the rate of apoptosis depends on adipocyte size in cattle. However, when comparing the numbers across different fat depots, different depot-specific relationships are suggested since the biggest

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adipocyte sizes in retroperitoneal tissue had the lowest apoptotic activity. Conflict of interest None of the authors of this paper has a financial and personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements We thank the German Research Foundation (Grant No: HA 6026/1-1 and PAK 286/1, SA 432/10-1) as well as the H. Wilhelm Schaumann Foundation (Grant for D. Germeroth) and the Islamic Development Bank (Grant for H. Akter) for the financial support of this study. We also thank B. Heitkönig, B. Mielenz, and K. Strack for their excellent technical assistance as well as I. Gockel-Böhner for assistance in tissue sampling. In terms of animal care and sampling in trial 2, we thank Dr. J. Griese and Dr. M. Hoelker and the staff of the research farm Frankenforst of the Agricultural Faculty, University Bonn. We would like to thank Prof. Dr. K.-H. Südekum, Bonn for providing the diet data from trial 2 and Prof. Dr. C. Wrenzycki, Giessen for making IGF-1 data from trail 1 available. References Akter, S.H., Häussler, S., Dänicke, S., Müller, U., von Soosten, D., Rehage, J., Sauerwein, H., 2011. Physiological and conjugated linoleic acid-induced changes of adipocyte size in different fat depots of dairy cows during early lactation. Journal of Dairy Science 94, 2871–2882. Akter, S.H., Häussler, S., Germeroth, D., von Soosten, D., Dänicke, S., Südekum, K.-H., Sauerwein, H., 2012. Immunohistochemical characterization of phagocytic immune cell infiltration into different adipose tissue depots of dairy cows during early lactation. Journal of Dairy Science 95, 3032–3044. Ambati, S., Kim, H.-K., Yang, J.-Y., Li, J., Della-Ferra, M.A., Baile, C.A., 2007. Effects of leptin on apoptosis and adipogenesis in 3T3-L1 adipocytes. Biochemical Pharmacology 73, 378–384. Azain, M.J., Hausman, D.B., Sisk, M.B., Flatt, W.P., Jewell, D.E., 2000. Dietary conjugated linoleic acid reduces rat adipose tissue cell size rather than cell number. Journal of Nutrition 130, 1548–1554. Baldwin VI, R.L., McLeod, K.R., Capuco, A.V., 2004. Visceral tissue growth and proliferation during the bovine lactation cycle. Journal of Dairy Science 87, 2977–2986. Bauman, D.E., Elliot, J.M., 1983. Control of nutrient partitioning in lactating ruminants. In: Mepham, T.B. (Ed.), Biochemistry of Lactation. Elsevier Science Publishing, Amsterdam, the Netherlands, pp. 437–468. Bauman, D.E., Baumgard, L.H., Corl, B.A., Griinari, J.M., 2000. Biosynthesis of conjugated linoleic acids in ruminants. Journal of Animal Sciences 77 (Suppl. E), 1–15. Baumgard, L.H., Corl, B.A., Dwyer, D.A., Bauman, D.E., 2001. Effects of conjugated linoleics (CLA) on tissue response to homeostatic signals and plasma variables associated with lipid metabolism in lactating dairy cows. Journal of Animal Science 80, 1285–1293. Belury, M.A., 2002. Dietary conjugated linoleic acid in health: Physiological effects and mechanisms of action. Annual Reviews in Nutrition 22, 505. Bonnet, M., Cassar Malek, I., Chillard, Y., Picard, B., 2010. Ontogenesis of muscle and adipose tissues and their interactions in ruminants and other species. Animal 4, 1093–1109. Brodie, A.E., Manning, V.A., Fagertun, H., Thom, E., Wadstein, J., Gudmundsen, O., 1999. Conjugated linoleic acid reduces body fat mass in overweight and obese humans. Journal of Nutrition 130, 2943–2948. Butler, W.R., Smith, D., 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. Journal of Dairy Science 72, 767–783. Chilliard, Y., Ferlay, A., Faulconnier, Y., Bonnet, M., Rouel, J., Bocquier, F., 2000. Adipose tissue metabolism and its role in adaptions to undernutrition in ruminants. Proceedings of the Nutrition Society 59, 127–134. Corino, C., Di Giancamillo, A., Rossi, R., Domeneghini, C., 2005. Dietary conjugated linoleic acid affects morphofunctional and chemical aspects of subcutaneous adipose tissue in heavy pigs. Journal of Nutrition 135, 1444–1450. Della-Fera, M.A., Qian, H., Baile, C.A., 2001. Adipocyte apoptosis in the regulation of body fat mass by leptin. Diabetes Obesity & Metabolism 3, 299–310. DiGirolamo, M., Fine, J.B., Tagra, K., Rossmanith, R., 1998. Qualitative regional differences in adipose tissue growth and cellularity in male Wistar rats fed ad libitum. American Journal of Physiology 274, R1460–R1467. Dunshea, F.R., Bell, A.W., Trigg, T.E., 1988. Relations between plasma non-esterified fatty acid metabolism and body tissue mobilization during chronic undernutrition in goats. British Journal of Nutrition 60, 633–644.

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