Clinica Chimica Acta 438 (2015) 382–387
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Ethnic differences in pre-adipocyte intracellular lipid accumulation and alkaline phosphatase activity Aus T. Ali a,⁎, George Chirambo b, Clement Penny c, Janice E. Paiker b, Faisel Ikram d, George Psaras d, Nigel J. Crowther b a
Division of Chemical Pathology, National Health Laboratory Service, Tygerberg Hospital, University of Stellenbosch Medical School, South Africa Department of Chemical Pathology, National Health Laboratory Service, University of Witwatersrand Medical School, Parktown, South Africa Department of Internal Medicine, University of Witwatersrand Medical School, Parktown, South Africa d Department of Surgery, University of Witwatersrand Medical School, Parktown, South Africa b c
a r t i c l e
i n f o
Article history: Received 21 August 2014 Received in revised form 11 September 2014 Accepted 13 September 2014 Available online 1 October 2014 Keywords: Intracellular lipid accumulation Histidine Alkaline phosphatase
a b s t r a c t Alkaline phosphatase (ALP) increases lipid accumulation in human pre-adipocytes. This study was performed to assess whether ethnic differences in the prevalence of obesity in African and European females are related to differences in pre-adipocyte lipid accretion and ALP activity. Pre-adipocytes were isolated from 13 black and 14 white females. Adipogenesis was quantified using the lipid dye, Oil red O, whilst ALP activity was assayed in cell extracts on day zero and 12 days after initiating adipogenesis. Lipid levels (OD units/mg protein) were lower in preadipocytes from white than black females on day 0 (0.36 ± 0.05 versus 0.44 ± 0.03, respectively; p b 0.0005) and day 12 (1.18 ± 0.14 versus 1.80 ± 0.22, respectively; p b 0.0005), as was ALP activity (mU/mg protein) on day zero (36.5 ± 5.8 versus 136.4 ± 10.9, respectively; p b 0.0005) and day 12 (127 ± 16 versus 278 ± 27, respectively; p b 0.0005). Treatment of pre-adipocytes with histidine, an ALP inhibitor, blocked lipid accumulation. Thus, lipid uptake is higher in pre-adipocytes isolated from black compared to white females which parallels the obesity prevalence rates in these population groups. The reason for higher fat accumulation in pre-adipocytes isolated from black females may be related to higher ALP activity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Alkaline phosphatases (ALP; EC 3.1.3.1) are a class of enzymes that catalyse a number of different reactions involving phosphate groups and which include the hydrolysis of phosphate esters from organic molecules of low molecular mass [1], and phosphotransferase [2] and protein phosphatase reactions [3]. There are four alkaline phosphatase isoenzymes, each coded by a different gene and which are termed intestinal, placental and germ cell ALP (collectively known as the tissue specific ALPs) and tissue nonspecific ALP (TNALP). Tissue nonspecific ALP is also known as liver/bone/kidney ALP. The genes encoding the three tissue specific isoenzymes are on the long arm of chromosome 2, whilst the gene for TNALP is located on chromosome 1 [4]. The tissue specific and tissue nonspecific isoenzymes can be differentiated from each other, not only by their gene sequences, but also by their biochemical response to specific inhibitors. Thus, TNALP is inhibited by levamisole, histidine and homoarginine but not by L-phenylalanyl-glycyl-glycine (Phe-Gly-Gly), whilst the reverse is true for the tissue specific forms ⁎ Corresponding author at: Division of Chemical Pathology, National Health Laboratory Service, Tygerberg Hospital, University of Stellenbosch Medical School, 7505 Cape Town, South Africa. Tel.: +27 21 938 4166; fax: +27 21 938 4640. E-mail address: [email protected] (A.T. Ali).
http://dx.doi.org/10.1016/j.cca.2014.09.022 0009-8981/© 2014 Elsevier B.V. All rights reserved.
[5,6]. Despite the importance of ALP in a number of different biological processes including fat accumulation [7,8], tumorigenesis [9], and skeletal mineralization [10], little is known about its mode of action. Serum levels of the tissue specific ALPs may be elevated during pregnancy (placental ALP) or after food ingestion (intestinal ALP) or due to the presence of different cancer types including lung, ovarian, testicular and colorectal. The principal reasons for high levels of serum TNALP are hepatobiliary disease, liver and bone cancers and diseases involving heightened osteoblastic activity e.g. Paget's disease [11]. Besides being expressed in liver, bone and kidney, TNALP has also been detected in a number of different mammalian tissues, including the mammary gland [12], the adrenal gland [13] and the pancreas [14]. Studies within our laboratory have also shown that TNALP is present within the murine 3T3-L1 pre-adipocyte cell line [7] and human pre-adipocytes [8]. Preadipocytes mature into adipocytes by a process termed adipogenesis, the defining characteristic of which is the intra-cellular accumulation of membrane-bound lipid droplets. Tissue nonspecific ALP is located on the membrane of these intra-cellular lipid droplets [7], and intracellular lipid accumulation (ICLA) is inhibited by the treatment of preadipocytes with inhibitors of TNALP. Furthermore, the level of ICLA within pre-adipocytes correlates strongly with the cellular level of TNALP activity [8]. These studies suggest that TNALP is an important regulator of lipid storage and that its level of expression may influence
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
the ability of pre-adipocytes to accumulate lipid during the process of adipogenesis. Studies in adult populations have shown that the prevalence of obesity, particularly in females, is higher in African subjects resident in North America or Africa when compared to European subjects living in the same country [15,16]. The reasons for these ethnic differences in the prevalence of obesity may include cultural and socio-economic influences [15]. It is also possible that physiological factors are involved, most particularly the hypothalamic regulation of appetite and the level of hyperplasia and hypertrophy within adipose tissue. Thus, genomewide association studies have already demonstrated that polymorphisms within, or lying close to genes controlling appetite regulation and adipocyte function are related to the level of adiposity [17]. It is interesting to note that a polymorphism within the TNALP gene has been associated with waist-to-hip ratio [18], an effect that may be mediated by the ability of TNALP to regulate ICLA during adipogenesis in human pre-adipocytes [8]. There is evidence to suggest that the level of adipogenesis may influence body fat mass and this comes from genetic studies showing that some rare forms of monogenic obesity [19], as well as common polygenic obesity [20], are caused by DNA sequence variants that lie close to, or within genes involved in the regulation of adipogenesis. Therefore, the hypothesis of the current study is that ethnic differences in the prevalence of obesity may partly be due to ethnic differences in the level of pre-adipocyte adipogenesis. Thus, the principle aim of the present study was to determine whether the adipogenic potential of preadipocytes differed between two female population groups with different prevalence levels of obesity (European and African) and whether ethnic differences in pre-adipocytic TNALP activity mirror these differences. 2. Materials and methods 2.1. Reagents All tissue culture reagents were purchased from Invitrogen (Invitrogen Corporation, Paisley, Scotland) and all laboratory reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, Aston Manor, South Africa), unless otherwise stated. 2.2. Subjects and adipose tissue isolation Human adipose tissue samples were obtained from the mammary adipose tissue of 14 white and 13 black women. All subjects were undergoing elective surgical mammary gland reduction and all were healthy as assessed by clinical history and physical examination. Ethical approval for the use of the adipose tissue was obtained from the University of Witwatersrand, Faculty of Health Sciences Human Ethics Committee. 2.3. Isolation of pre-adipocytes from adipose tissue Adipose tissue was processed immediately after removal, and was transferred to the laboratory in human adipocyte isolation medium consisting of sterile Hanks balanced salt solution supplemented with 25 mmol/l HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 3% bovine serum albumin (BSA). Pre-adipocytes were isolated using a previously published method [8]. After excision of blood vessels, adipose tissue was minced into small pieces, washed once in Hank's balanced salt solution (HBSS), and centrifuged for 5 min at 380 g. Tissue was then decanted into isolation medium (0.7 g tissue/ml HBSS supplemented with 25 mmol/l HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 3% BSA, and 0.75 mg/ml collagenase) and digested for 1 h at 37 °C with constant shaking. Suspended cells were then filtered through a 250 μm metal filter and the filtrate spun for 10 min at 380 g. The pellet of stromavascular cells was re-suspended in 100 ml of red cell lysis buffer (0.154 mol/l ammonium chloride, 10 mmol/l
383
potassium bicarbonate, 0.1 mmol/l EDTA and 10% foetal bovine serum) and allowed to settle for 10 min at room temperature followed by 10 min centrifugation at 380 g. The cell pellet was resuspended in human pre-adipocyte tissue culture medium (DMEM-Ham's F12 medium containing 15 mmol/l HEPES, 2 mmol/l glutamine, 10% foetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin) and then filtered through a 20 μm nylon mesh and aliquoted into 6well tissue culture plates. The cells were cultured overnight at 37 °C in humidified atmosphere with 5% CO2 and the next day washed with DMEM-Ham's F12 medium. The medium was changed every third day until the cells were confluent. Pre-adipocytes were stimulated to differentiate over 12 days in differentiation medium supplemented with 100 nmol/l hydrocortisone, 66 nmol/l insulin (Novo-Nordisk, Copenhagen, Denmark), 0.5 mmol/l 3-isobuty-1methylxanthine (IBMX) and 1 nmol/l triiodo-1-thyronine. 2.4. Alkaline phosphatase inhibitors Pre-adipocytes were cultured in the absence or presence of ALP inhibitors, these being added with the differentiation medium and refreshed every third day. The inhibitors included the tissue non-specific ALP inhibitor, histidine (Merck, Darmstadt, Germany) and the tissue specific ALP inhibitor, Phe-Gly-Gly [5,6]. The concentrations of these inhibitors in the differentiation medium were 50 mmol/l and 20 mmol/l respectively. The concentrations chosen were based on a previous study using the murine 3T3-L1 pre-adipocyte cell line [7]. 2.5. Extraction of ALP from human pre-adipocytes Cellular extracts from pre-adipocytes were isolated at baseline and 12 days after initiation of adipogenesis using a previously published method [8]. Briefly, tissue culture medium was removed from the cells and 0.5 ml of an ice cold solution of 10 mmol/l Tris-HCl containing 1% Triton X-100 and 2 mmol/l phenylmethylsulfonyl-fluoride (pH 7.2) was added. The flasks were shaken to detach the cells and the suspension transferred to an Eppendorf tube and centrifuged for 10 min at 15,000 g. The supernatant was removed and immediately analysed for alkaline phosphatase activity using an automated colorimetric assay (Roche Diagnostics, Randburg, South Africa). The protein content of the supernatant was analysed using the Bradford method [21]. Alkaline phosphatase activity was calculated as mU of activity per mg protein. 2.6. Measurement of cellular lipid accumulation Intracellular lipid accumulation, which is the hallmark of adipogenesis was measured on day zero and day 12 using the Oil red O technique [22]. This technique depends on the ability of the lipid droplets in the adipocyte to collect the red stain (the stain only binds to triglycerides and cholesteryl oleate), which is then extracted from the cells using isopropyl alcohol, and the absorbance was measured at 510 nm [22]. Intracellular lipid accumulation was then expressed as OD units per mg of cellular protein. 2.7. Detection of polymorphisms in promoter region of TNALP gene Genomic DNA was isolated from buffy coat preparations taken from 6 white (BMI, 31.5 ± 4.5; age, 39.5 ± 11.9 years) and 6 black (BMI, 33.0 ± 4.6; age, 49.7 ± 6.3 years) females using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Primers were designed for PCR amplification of the promoter region of the human TNALP gene which provisionally lies within a 3780 bp region 5′ to the major transcription start site [23]. Primer design was accomplished using the Gene Runner software, version 3.05 (Hastings Software, Inc., Hastings, NY, USA). This region was amplified using 7 sets of overlapping PCR primers. The initial denaturation temperature was 95 °C for 11 min whilst the extension temperature was 72 °C for 10 min for all the PCR reactions. The cycling conditions for all reactions
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
2.8. Statistical analysis Data that was not normally distributed was log transformed before analysis. Differences in TNALP activity or triglyceride accumulation across ethnic groups or between different time points were analysed using Student's non-paired or paired t-test, respectively. Data in the text and figures are given as mean ± SD. Multivariable regression analysis was used to determine the effect of TNALP activity and ethnicity on intracellular lipid accumulation in pre-adipocytes. The standardized β value was used in all analyses. Statistical analyses were performed using Statistica v9 (StatSoft, Tulsa, OK, USA). 3. Results 3.1. Ethnic differences in TNALP activity and adipogenesis The mean BMIs (±SD) of the white and black women from whom adipose tissue was obtained were 24.7 ± 1.3 and 25.1 ± 1.6, respectively (p = 0.56). Their mean ages were 43.0 ± 4.6 and 45.5 ± 5.2 years, respectively (p = 0.23). The data in Fig. 1 demonstrates that intracellular lipid accumulation was higher in pre-adipocytes isolated from black women when compared to white women at both baseline (p b 0.0005) and after 12 day incubation with differentiation medium (p b 0.0005). This pattern was repeated for TNALP activity (p b 0.0005 at both baseline and after 12 day incubation with differentiation medium—see Fig. 1). When comparing the preadipocyte TNALP activity levels between white and black females it was observed that for the baseline levels there was no overlap in TNALP activity between the 2 groups with the range for white females being 3.56–66.1 mU/mg (median of 38.6) and for the black females, 70.2– 224.1 (median of 130.3 mU/mg). Similarly, for the TNALP activity levels after 12 days of exposure to differentiation medium the levels were much higher in the black females with a range of 107.6–493.2 (median of 289.8 mU/mg) compared to a range of 31.2–207.3 (median of 110.1 mU/mg) for the white females (see Fig. 1). 3.2. The relationship between TNALP activity and intracellular lipid accumulation The low N number of study participants ruled out the use of regression analysis to investigate the relationship between TNALP activity and lipid accretion within each ethnic group and at each of the two time points. Therefore, to increase the power of the analysis data from both ethnic groups was combined as was the data from the two time points to give N = 54. All the regression models included the Oil red O data (logged) as the dependent variable and the first model used TNALP activity (logged) as the sole independent variable and gave R2 = 0.58 (p b 0.0001) with β = 0.76 (p b 0.0001). The second model used ethnicity (white subjects coded as 1 and black subjects as 2) as the only independent variable and gave R2 = 0.12 (p b 0.05) with β = 0.35 (p b 0.05). The third model included both ethnicity and TNALP activity as independent variables and gave R2 = 0.61 (p b 0.0001) with β = − 0.22 (p = 0.06) for ethnicity and β = 0.90 (p b 0.0001) for TNALP
A) Cell lipid (OD units/mg protein)
were 95 °C for 40 s whilst the annealing time was 30 s. The reaction volume was 12 μl and contained 1.0 μl of buffer (SuperTherm Gold PCR buffer, JMR Holdings, Sevenoaks, Kent, UK), 0.8 mM of each dNTP (Bioline, Randolph, MA, USA), 0.075 U/l of SuperTherm Gold Taq polymerase (JMR Holdings, Sevenoaks, Kent, UK) and 1 μM of each of the primers and 2 μl of the DNA template. The PCR products were purified using the QIAquick purification kit (Qiagen, Hilden, Germany) and sequenced by Inqaba Biotec (Pretoria, South Africa) using a SpectruMedix SCE 2410 DNA sequence analyser (SpectruMedix LLC, State College, PA, USA). The DNA sequences were compared against that present within the GenBank database (accession code AB176449).
2.4
*** +++
2.0
1.6
*** 1.2
0.8
+++ 0.4
0.0 0
12
Days post induction of adipogenesis
B) 360 ** +++
320
ALP activity (mU/mg protein)
384
280 240 200
+++
160
**
120 80 40 0 0
12
Days post induction of adipogenesis Fig. 1. Intracellular lipid accumulation (A) and TNALP activity (B) in pre-adipocytes isolated from 14 white (grey bars) and 13 black (black bars) females. ++p b 0.005, +++p b 0.0005 vs. pre-adipocytes obtained from white females; **p b 0.005, ***p b 0.0005 vs. day zero. Each bar is a mean ± SD.
activity. These results suggest that the ethnic difference in intracellular lipid accumulation is due to the ethnic difference in TNALP activity.
3.3. Effect of ALP inhibitors The tissue nonspecific ALP inhibitor histidine was shown to block lipid accumulation in pre-adipocytes isolated from black females. Thus, Fig. 2A shows that after 12 day incubation with differentiation medium in the presence of histidine, intracellular lipid accumulation was significantly lower in cells treated with histidine than cells that were untreated, and this significant effect was observed in both ethnic groups. A similar pattern was observed for TNALP activity (Fig. 2B), with the activity at 12 days of histidine treatment being far lower than that observed in cells incubated for 12 days in the absence of histidine. In black females both intracellular lipid accumulation (Fig. 2A) and TNALP activity (Fig. 2B) were higher at day 0 and at day 12 (in the absence of His) than in white females. The tissue-specific ALP inhibitor Phe-Gly-Gly, did not show any effect on either intracellular lipid accumulation (Fig. 3A) or ALP activity (Fig. 3B) in pre-adipocytes isolated from black or white females. Both intracellular lipid accumulation (Fig. 3A) and ALP activity (Fig. 3B) were higher in black than white females at both time points and in the absence and presence of Phe-Gly-Gly.
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
A)
2.8
*** +++
2.4 2.0 1.6
**
1.2
#
*
0.8
##
**
+ 0.4
Cell lipid (OD units/mg protein)
Cell lipid (OD units/mg protein)
A)
385
2.8
*** ++
2.4
*** ++
2.0 1.6
*
1.2
*
0.8
+++ 0.4 0.0
0.0 0
12 Without His
0
12 With His
Days post induction of adipogenesis
12 Without Phe-Gly-Gly
12 With Phe-Gly-Gly
Days post induction of adipogenesis
B)
** ++
360 300 240 180
+++
*
120 60
#
##
**
ALP activity (mU/mg protein)
ALP activity (mU/mg protein)
B) 420
420
*** ++
** ++
360 300 240 180
+++
*
*
120 60
0 0
12 Without His
12 With His
Days post induction of adipogenesis Fig. 2. Histidine effects on lipid accumulation (A) and TNALP activity (B) in pre-adipocytes taken from mammary tissue of 4 black (black bars) and 4 white (grey bars) females. +p b 0.05, ++p b 0.005, +++p b 0.0005 vs. pre-adipocytes from white females; *p b 0.05, **p b 0.005, ***p b 0.0005 vs. day zero; #p b 0.05, ##p b 0.005 vs. without histidine. Each bar is a mean ± SD.
0 0
12 Without Phe-Gly-Gly
12 With Phe-Gly-Gly
Days post induction of adipogenesis Fig. 3. Phe-Gly-Gly effects on lipid accumulation (A) and TNALP activity (B) in preadipocytes taken from mammary tissue of 4 black (black bars) and 4 white (grey bars) females. ++p b 0.005, +++p b 0.0005 vs. pre-adipocytes from white females; *p b 0.05, **p b 0.005, ***p b 0.0005 vs. day zero. Each bar is a mean ± SD.
3.4. Sequence of the promoter region of the human tissue non-specific TNALP gene The profound difference in pre-adipocyte TNALP activity observed between white and black females suggested that either the catalytic activity, or the level of expression of the enzyme was much higher in the latter group. It was therefore decided to analyse the promoter region of the TNALP gene to determine if genomic sequence variation existed between the 2 ethnic groups. However, comparison of the sequences isolated from 6 white and 6 black females showed no polymorphic sites within the promoter region of the human ALP gene.
4. Discussion We demonstrate in the present study the significant differences in cellular fat accumulation between pre-adipocytes collected from black and white South African females. This is the first study to investigate and to show that ethnic differences in adipogenesis exist. Whilst the significance of this is currently unknown, the prevalence of obesity is however higher in black than white females in both South Africa [15] and the USA [16]. Furthermore, it is known that obesity involves both
adipocyte hypertrophy and hyperplasia [24]. These studies therefore suggest that it is possible that ethnic differences in adipogenesis may play a role in the higher prevalence of obesity in black than white females. It is known that visceral fat mass is lower in black than white subjects, when matched for BMI [25]. The higher level of adipogenesis in subcutaneous pre-adipocytes isolated from black compared to white females may explain this ethnic difference in body fat distribution. It is thought that the subcutaneous fat depot acts to buffer serum triglyceride levels removing these molecules from the circulation thus ensuring that serum levels do not rise too high [26]. This buffering system also ensures that triglyceride does not deposit within the visceral fat depot or at other ectopic sites [26]. Studies have shown that the insulin sensitizing agents, the thiazolidinediones increase lipid deposition in the subcutaneous depot leading to reduced visceral fat mass [27]. It is thought that this is accomplished by the ability of these drugs to increase adipogenesis more in subcutaneous than visceral pre-adipocytes [28]. Thus, if adipogenic capacity is greater in subcutaneous pre-adipocytes from black than white females, this may lead to lower triglyceride deposition in the visceral fat depot of the former population group.
386
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
The ethnic difference in pre-adipocyte maturation observed in the present study is not the only adipocyte-related functional difference observed between black and white subjects. Thus, studies have shown that lipolytic rate is greater in adipose tissue of black than white females [29] and that adipocytes isolated from black females are more resistant to the anti-lipolytic action of insulin than adipocytes from white females [30]. Also, serum levels of the adipocyte-derived hormone, adiponectin are lower in black than white subjects [31]. Therefore, ethnic differences in both pre-adipocyte and mature adipocyte functionality have now been observed, but whether these play any role in the ethnic differences observed in body fat distribution [25] or whole body insulin sensitivity [32] is still a matter of conjecture. Previous studies have shown that TNALP is expressed in adipocyte precursor cells found in human [33], murine [34] and avian [35] bone marrow and in rat adipose tissue [36] and rabbit adipocytes [37]. However, one study failed to find TNALP expression in mature human adipocytes [38]. This may be because in humans TNALP is important only during adipogenesis and is not required for mature adipocyte function. This theory is supported by the finding that in pre-adipocytes isolated from human bone marrow, TNALP activity was high in the small, multilobular pre-adipocytes but absent from the large, unilobular mature adipocytes [33]. The present study clearly shows that the ALP present in preadipocytes isolated from black and white females is the tissue nonspecific isoenzyme as it was unresponsive to Phe-Gly-Gly but was inhibited by histidine. Similar data was obtained for ALP activity present in the 3T3-L1 pre-adipocyte cell line [7]. Furthermore, a study has shown that although the serum level of liver TNALP was higher in obese than lean subjects, but no differences were observed for bone or intestinal ALP levels [39]. A second study in which serum total ALP levels were measured in 32,329 subjects demonstrated that females who were more than 15% overweight had a 20% higher ALP activity than those who were not overweight [40]. These data suggest that adipose tissue contributes to the serum level of ALP and that it is the liver form of the tissue nonspecific ALP that is present in adipose tissue. The inhibition of adipogenesis and TNALP activity by histidine suggests that TNALP may regulate adipogenesis. Histidine is a nonspecific inhibitor of TNALP activity and therefore it is possible that the inhibition of adipogenesis observed in the presence of histidine may be due to non-ALP related effects of the amino acid. However, a study using a more specific method for reducing TNALP activity i.e. small interfering (si) RNA, also demonstrated a blockage in intracellular lipid accumulation in the 3T3-L1 pre-adipocyte cell line [41]. The finding that TNALP activity is localised to the lipid droplets of both human [8] and 3T3-L1 [7] preadipocytes further suggests that TNALP may be involved in controlling intracellular lipid accumulation. Also, a study has shown that triglyceride transport into and across gut enterocytes involves surfactant-like particles that are composed of a membrane-bound lipid particle containing membrane-associated intestinal ALP [42]. Currently, the exact role played by TNALP in modulating adipogenesis is unknown. The principle aim of the current study was to determine whether ethnic differences exist in adipogenic rate and whether the level of pre-adipocyte TNALP activity parallels this variable. The study was not designed to uncover the mechanistic relationship between TNALP and adipogenesis. However, it is possible to hypothesise about the role played by TNALP in the control of pre-adipocyte intracellular lipid accumulation. Thus, because TNALP is able to dephosphorylate phosphoproteins (3) and with its known location on the lipid droplet it is possible that TNALP may act to control the phosphorylation state of molecules present on, or that interacts with, the lipid droplet. Possible candidate molecules include perilipin [43], caveolin [44] and vimentin [45], which are all proteins that are associated with the lipid droplet and which are phosphorylated. These molecules may not be the direct substrates of TNALP but their phosphorylation status could be modified by TNALP altering the phosphorylation level of other kinases or phosphatases that act on these molecules.
The profound ethnic difference in pre-adipocyte TNALP activity is a novel finding. Statistical analysis of this data suggests that the ethnic difference in adipogenesis is directly related to the differences in TNALP activity. The molecular mechanisms that underlie the ethnic difference in pre-adipocyte TNALP activity are not known. The analysis of the promoter region of the TNALP gene demonstrated no sequence differences between the 2 ethnic groups. Although the number of subjects studied was small, the very large difference in TNALP activity between the 2 groups would suggest that this difference would be due to a very common polymorphism. However, this was not observed and therefore we would suggest that further studies must be conducted on possible differences in TNALP mRNA stability, the detection of polymorphisms within the TNALP exons and the intracellular levels of transcription factors that control TNALP gene expression. With regards to transcription factors, it is known that retinoic acid can increase TNALP gene expression [23]. It is interesting to note that in a study of the molecular aetiology of uterine leiomyomata, differences existed in the ratio of uterine tumour:non-tumour gene expression levels of retinoic acid receptor α and retinoid X receptor α between African–American and Hispanic, Chinese–American and white females [46]. This in vitro study has shown that pre-adipocytes isolated from black women have higher basal and stimulated intracellular lipid accumulation than those collected from white women. The significant difference in lipid accumulation was paralleled by a much higher level of TNALP activity in pre-adipocytes obtained from black than white females. This novel finding linking higher TNALP activity with higher intracellular triglyceride accumulation in black compared to white females may partially explain the greater level of obesity observed in the former population group. Acknowledgements The authors would like to thank the members of the National Health Laboratory Service (NHLS) Chemical Pathology routine laboratory at the Charlotte Maxeke Academic Hospital, Johannesburg for performing the ALP assays. This study was funded by the NHLS, the South African MRC and the National Research Foundation (NRF) of South Africa. AT Ali received bursaries from the NHLS and the University of the Witwatersrand. References [1] McComb RB, Bowers GN, Posen S. Alkaline phosphatase. New York: Plenum Press; 1979. [2] Stinson RA, McPhee JL, Collier HB. Phosphotransferase activity of human alkaline phosphatases and the role of enzyme Zn2+. Biochim Biophys Acta 1987;913:272–8. [3] Chan JR, Stinson RA. Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases. J Biol Chem 1986;261:7635–9. [4] Henthorn PS, Raducha M, Kadesch T, Weiss MJ, Harris H. Sequence and characterization of the human intestinal alkaline phosphatase gene. J Biol Chem 1988;263:12011–9. [5] van Belle. Alkaline phosphatase. 1. Kinetics and inhibition by levamisole of purified isoenzymes from humans. Clin Chem 1978;22:972–6. [6] Mulivor RA, Plotkin LI, Harris H. Differential inhibition of the products of the human alkaline phosphatase loci. Ann Hum Genet 1978;42:1–13. [7] Ali AT, Penny CB, Paiker JE, et al. Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1. Clin Chim Acta 2005; 354:101–9. [8] Ali AT, Penny CB, Paiker JE, Psaras G, Ikram F, Crowther NJ. The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3-L1 cells and human preadipocytes. Anal Biochem 2006;354:247–54. [9] Prasad R, Lambe S, Kaler P, et al. Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma. Biochim Biophys Acta 2005;1741:240–5. [10] Millán JL. The role of phosphatases in the initiation of skeletal mineralization. Calcif Tissue Int 2013;93:299–306. [11] Price CP. Multiple forms of human serum alkaline phosphatase: detection and quantitation. Ann Clin Biochem 1993;30:355–71. [12] Leung CT, Maleeff BE, Farrell Jr HM. Subcellular and ultrastructural localization of alkaline phosphatase in lactating rat mammary glands. J Dairy Sci 1989;72:2495–509. [13] Hillman JR, Seliger WG, Epling GP. Histochemistry and ultrastructure of adrenal cortical development in the golden hamster. Gen Comp Endocrinol 1975;25:14–24. [14] Githens S. Localization of alkaline phosphatase and adenosine triphosphatase in the mammalian pancreas. J Histochem Cytochem 1983;31:697–705.
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387 [15] Puoane T, Steyn K, Bradshaw D, et al. Obesity in South Africa: the South African demographic and health survey. Obes Res 2002;10:1038–48. [16] Ogden CL, Carroll MD, Kit BK, Flegal KM. Prevalence of obesity among adults: United States, 2011–2012. NCHS Data Brief 2013;131:1–8. [17] Walley AJ, Asher JE, Froguel P. The genetic contribution to non-syndromic human obesity. Nat Rev Genet 2009;10:431–42. [18] Korostishevsky M, Cohen Z, Malkin I, Ermakov S, Yarenchuk O, Livshits G. Morphological and biochemical features of obesity are associated with mineralization genes' polymorphisms. Int J Obes 2010;34:1308–18. [19] Ristow M, Müller-Wieland D, Pfeiffer A, Krone W, Kahn CR. Obesity associated with a mutation in a genetic regulator of adipocyte differentiation. N Engl J Med 1998;339: 953–9. [20] Meyre D, Delplanque J, Chèvre JC, et al. Genome-wide association study for early-onset and morbid adult obesity identifies three new risk loci in European populations. Nat Genet 2009;41:157–9. [21] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein–dye binding. Anal Biochem 1976; 72:248–54. [22] Ramirez-Zacarias JL, Castro-Munozledo F, Kuri-Harcuch W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 1992;97:493–7. [23] Orimo H, Shimada T. Regulation of the human tissue-nonspecific alkaline phosphatase gene expression by all-trans-retinoic acid in SaOS-2 osteosarcoma cell line. Bone 2005; 36:866–76. [24] Couillard C, Mauriege P, Imbeault P, et al. Hyperleptinemia is more closely associated with adipose cell hypertrophy than with adipose tissue hyperplasia. Int J Obes Relat Metab Disord 2000;24:782–8. [25] Lovejoy JC, de la Bretonne JA, Klemperer M, Tulley R. Abdominal fat distribution and metabolic risk factors: effects of race. Metabolism 1996;45:1119–24. [26] van der Merwe MT, Crowther NJ, Schlaphoff GP, et al. Lactate and glycerol release from the subcutaneous adipose tissue of obese urban women from South Africa; important metabolic implications. J Clin Endocrinol Metab 1998;83:4084–91. [27] Frayn KN. Adipose tissue as a buffer for daily lipid flux. Diabetologia 2002;45: 1201–10. [28] Miyazaki Y, Mahankali A, Matsuda M, et al. Effect of pioglitazone on abdominal fat distribution and insulin sensitivity in type 2 diabetic patients. J Clin Endocrinol Metab 2002;87:2784–91. [29] Adams M, Montague CT, Prins JB, et al. Activators of peroxisome proliferatoractivated receptor gamma have depot-specific effects on human preadipocyte differentiation. J Clin Invest 1997;100:3149–53. [30] Buthelezi EP, van der Merwe MT, Lönnroth PN, Gray IP, Crowther NJ. Ethnic differences in the responsiveness of adipocyte lipolytic activity to insulin. Obes Res 2000;8:171–8.
387
[31] Ferris WF, Naran NH, Crowther NJ, Rheeder P, van der Merwe L, Chetty N. The relationship between insulin sensitivity and serum adiponectin levels in three population groups. Horm Metab Res 2005;37:695–701. [32] van der Merwe MT, Crowther NJ, Schlaphoff GP, Gray IP, Joffe BI, Lönnroth PN. Evidence for insulin resistance in black women from South Africa. Int J Obes Relat Metab Disord 2000;24:1340–6. [33] Bianco P, Costantini M, Dearden LC, Bonucci E. Alkaline phosphatase positive precursors of adipocytes in the human bone marrow. Br J Haematol 1988;68:401–3. [34] Kodama H, Koyama H, Sudo H, Kasai S. Adipose conversion of mouse bone marrow fibroblasts in vitro: their alkaline phosphatase activity. Cell Struct Funct 1983;8: 19–27. [35] Yoshida H, Yumoto T. Alkaline phosphatase-positive reticular cells of chicken bone marrow—in vivo and in vitro studies. Int J Cell Cloning 1987;5:35–44. [36] Wallach DP, Ko H. Some properties of an alkaline phosphatase from rat adipose tissue. Can J Biochem 1964;42:1445–7. [37] Lecoeur L, Ouhayoun JP. In vitro induction of osteogenic differentiation from nonosteogenic mesenchymal cells. Biomaterials 1997;18:989–93. [38] Okochi T, Seike H, Saeki K, Sumikawa K, Yamamoto T, Higashino K. A novel alkaline phosphatase isozyme in human adipose tissue. Clin Chim Acta 1987;162:19–27. [39] Ali AT, Paiker JE, Crowther NJ. The relationship between anthropometry and serum concentrations of alkaline phosphatase isoenzymes, liver enzymes, albumin and bilirubin. Am J Clin Pathol 2006;126:1–6. [40] Schiele F, Henny J, Hitz J, Petitclerc C, Gueguen R, Siest G. Total bone and liver alkaline phosphatase in plasma: Biological variations and reference limits. Clin Chem 1983; 29:634–41. [41] Chirambo GM, van Niekerk C, Crowther NJ. Post-translational silencing of the tissue non-specific alkaline phosphatase gene blocks intracellular lipid accumulation in a murine preadipocyte cell line, 3T3-L1 and in a human hepatocarcinoma cell line, HepG2. J Endocrinol Metab Diab S Afr 2010;15:25. [42] Zhang Y, Shao JS, Xie QM, Alpers DH. Immunolocalisation of alkaline phosphatase and surfactant-like particle proteins in rat duodenum during fat absorption. Gastroenterology 1996;110:478–88. [43] Greenberg AS, Egan JJ, Wek SA, Garty NB, Blanchette-Mackie EJ, Londos C. Perilipin, a major hormonally-regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J Biol Chem 1991;266:11341–6. [44] Mastick CC, Brady MJ, Saltiel AR. Insulin stimulates the phosphorylation of caveolin. J Cell Biol 1995;129:1523–31. [45] Brandes R, Arad R, Gaathon A, Bar-Tana J. Induction of adipose conversion in 3T3-L1 cells is associated with an early phosphorylation of a protein partly homologous with mouse vimentin. FEBS Lett 1993;333:179–82. [46] Wei JJ, Chiriboga L, Arslan AA, Melamed J, Yee H, Mittal K. Ethnic differences in expression of the dysregulated proteins in uterine leiomyomata. Hum Reprod 2006; 21:57–67.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Ethnic differences in pre-adipocyte intracellular lipid accumulation and alkaline phosphatase activity Aus T. Ali a,⁎, George Chirambo b, Clement Penny c, Janice E. Paiker b, Faisel Ikram d, George Psaras d, Nigel J. Crowther b a
Division of Chemical Pathology, National Health Laboratory Service, Tygerberg Hospital, University of Stellenbosch Medical School, South Africa Department of Chemical Pathology, National Health Laboratory Service, University of Witwatersrand Medical School, Parktown, South Africa Department of Internal Medicine, University of Witwatersrand Medical School, Parktown, South Africa d Department of Surgery, University of Witwatersrand Medical School, Parktown, South Africa b c
a r t i c l e
i n f o
Article history: Received 21 August 2014 Received in revised form 11 September 2014 Accepted 13 September 2014 Available online 1 October 2014 Keywords: Intracellular lipid accumulation Histidine Alkaline phosphatase
a b s t r a c t Alkaline phosphatase (ALP) increases lipid accumulation in human pre-adipocytes. This study was performed to assess whether ethnic differences in the prevalence of obesity in African and European females are related to differences in pre-adipocyte lipid accretion and ALP activity. Pre-adipocytes were isolated from 13 black and 14 white females. Adipogenesis was quantified using the lipid dye, Oil red O, whilst ALP activity was assayed in cell extracts on day zero and 12 days after initiating adipogenesis. Lipid levels (OD units/mg protein) were lower in preadipocytes from white than black females on day 0 (0.36 ± 0.05 versus 0.44 ± 0.03, respectively; p b 0.0005) and day 12 (1.18 ± 0.14 versus 1.80 ± 0.22, respectively; p b 0.0005), as was ALP activity (mU/mg protein) on day zero (36.5 ± 5.8 versus 136.4 ± 10.9, respectively; p b 0.0005) and day 12 (127 ± 16 versus 278 ± 27, respectively; p b 0.0005). Treatment of pre-adipocytes with histidine, an ALP inhibitor, blocked lipid accumulation. Thus, lipid uptake is higher in pre-adipocytes isolated from black compared to white females which parallels the obesity prevalence rates in these population groups. The reason for higher fat accumulation in pre-adipocytes isolated from black females may be related to higher ALP activity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Alkaline phosphatases (ALP; EC 3.1.3.1) are a class of enzymes that catalyse a number of different reactions involving phosphate groups and which include the hydrolysis of phosphate esters from organic molecules of low molecular mass [1], and phosphotransferase [2] and protein phosphatase reactions [3]. There are four alkaline phosphatase isoenzymes, each coded by a different gene and which are termed intestinal, placental and germ cell ALP (collectively known as the tissue specific ALPs) and tissue nonspecific ALP (TNALP). Tissue nonspecific ALP is also known as liver/bone/kidney ALP. The genes encoding the three tissue specific isoenzymes are on the long arm of chromosome 2, whilst the gene for TNALP is located on chromosome 1 [4]. The tissue specific and tissue nonspecific isoenzymes can be differentiated from each other, not only by their gene sequences, but also by their biochemical response to specific inhibitors. Thus, TNALP is inhibited by levamisole, histidine and homoarginine but not by L-phenylalanyl-glycyl-glycine (Phe-Gly-Gly), whilst the reverse is true for the tissue specific forms ⁎ Corresponding author at: Division of Chemical Pathology, National Health Laboratory Service, Tygerberg Hospital, University of Stellenbosch Medical School, 7505 Cape Town, South Africa. Tel.: +27 21 938 4166; fax: +27 21 938 4640. E-mail address: [email protected] (A.T. Ali).
http://dx.doi.org/10.1016/j.cca.2014.09.022 0009-8981/© 2014 Elsevier B.V. All rights reserved.
[5,6]. Despite the importance of ALP in a number of different biological processes including fat accumulation [7,8], tumorigenesis [9], and skeletal mineralization [10], little is known about its mode of action. Serum levels of the tissue specific ALPs may be elevated during pregnancy (placental ALP) or after food ingestion (intestinal ALP) or due to the presence of different cancer types including lung, ovarian, testicular and colorectal. The principal reasons for high levels of serum TNALP are hepatobiliary disease, liver and bone cancers and diseases involving heightened osteoblastic activity e.g. Paget's disease [11]. Besides being expressed in liver, bone and kidney, TNALP has also been detected in a number of different mammalian tissues, including the mammary gland [12], the adrenal gland [13] and the pancreas [14]. Studies within our laboratory have also shown that TNALP is present within the murine 3T3-L1 pre-adipocyte cell line [7] and human pre-adipocytes [8]. Preadipocytes mature into adipocytes by a process termed adipogenesis, the defining characteristic of which is the intra-cellular accumulation of membrane-bound lipid droplets. Tissue nonspecific ALP is located on the membrane of these intra-cellular lipid droplets [7], and intracellular lipid accumulation (ICLA) is inhibited by the treatment of preadipocytes with inhibitors of TNALP. Furthermore, the level of ICLA within pre-adipocytes correlates strongly with the cellular level of TNALP activity [8]. These studies suggest that TNALP is an important regulator of lipid storage and that its level of expression may influence
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
the ability of pre-adipocytes to accumulate lipid during the process of adipogenesis. Studies in adult populations have shown that the prevalence of obesity, particularly in females, is higher in African subjects resident in North America or Africa when compared to European subjects living in the same country [15,16]. The reasons for these ethnic differences in the prevalence of obesity may include cultural and socio-economic influences [15]. It is also possible that physiological factors are involved, most particularly the hypothalamic regulation of appetite and the level of hyperplasia and hypertrophy within adipose tissue. Thus, genomewide association studies have already demonstrated that polymorphisms within, or lying close to genes controlling appetite regulation and adipocyte function are related to the level of adiposity [17]. It is interesting to note that a polymorphism within the TNALP gene has been associated with waist-to-hip ratio [18], an effect that may be mediated by the ability of TNALP to regulate ICLA during adipogenesis in human pre-adipocytes [8]. There is evidence to suggest that the level of adipogenesis may influence body fat mass and this comes from genetic studies showing that some rare forms of monogenic obesity [19], as well as common polygenic obesity [20], are caused by DNA sequence variants that lie close to, or within genes involved in the regulation of adipogenesis. Therefore, the hypothesis of the current study is that ethnic differences in the prevalence of obesity may partly be due to ethnic differences in the level of pre-adipocyte adipogenesis. Thus, the principle aim of the present study was to determine whether the adipogenic potential of preadipocytes differed between two female population groups with different prevalence levels of obesity (European and African) and whether ethnic differences in pre-adipocytic TNALP activity mirror these differences. 2. Materials and methods 2.1. Reagents All tissue culture reagents were purchased from Invitrogen (Invitrogen Corporation, Paisley, Scotland) and all laboratory reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, Aston Manor, South Africa), unless otherwise stated. 2.2. Subjects and adipose tissue isolation Human adipose tissue samples were obtained from the mammary adipose tissue of 14 white and 13 black women. All subjects were undergoing elective surgical mammary gland reduction and all were healthy as assessed by clinical history and physical examination. Ethical approval for the use of the adipose tissue was obtained from the University of Witwatersrand, Faculty of Health Sciences Human Ethics Committee. 2.3. Isolation of pre-adipocytes from adipose tissue Adipose tissue was processed immediately after removal, and was transferred to the laboratory in human adipocyte isolation medium consisting of sterile Hanks balanced salt solution supplemented with 25 mmol/l HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 3% bovine serum albumin (BSA). Pre-adipocytes were isolated using a previously published method [8]. After excision of blood vessels, adipose tissue was minced into small pieces, washed once in Hank's balanced salt solution (HBSS), and centrifuged for 5 min at 380 g. Tissue was then decanted into isolation medium (0.7 g tissue/ml HBSS supplemented with 25 mmol/l HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 3% BSA, and 0.75 mg/ml collagenase) and digested for 1 h at 37 °C with constant shaking. Suspended cells were then filtered through a 250 μm metal filter and the filtrate spun for 10 min at 380 g. The pellet of stromavascular cells was re-suspended in 100 ml of red cell lysis buffer (0.154 mol/l ammonium chloride, 10 mmol/l
383
potassium bicarbonate, 0.1 mmol/l EDTA and 10% foetal bovine serum) and allowed to settle for 10 min at room temperature followed by 10 min centrifugation at 380 g. The cell pellet was resuspended in human pre-adipocyte tissue culture medium (DMEM-Ham's F12 medium containing 15 mmol/l HEPES, 2 mmol/l glutamine, 10% foetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin) and then filtered through a 20 μm nylon mesh and aliquoted into 6well tissue culture plates. The cells were cultured overnight at 37 °C in humidified atmosphere with 5% CO2 and the next day washed with DMEM-Ham's F12 medium. The medium was changed every third day until the cells were confluent. Pre-adipocytes were stimulated to differentiate over 12 days in differentiation medium supplemented with 100 nmol/l hydrocortisone, 66 nmol/l insulin (Novo-Nordisk, Copenhagen, Denmark), 0.5 mmol/l 3-isobuty-1methylxanthine (IBMX) and 1 nmol/l triiodo-1-thyronine. 2.4. Alkaline phosphatase inhibitors Pre-adipocytes were cultured in the absence or presence of ALP inhibitors, these being added with the differentiation medium and refreshed every third day. The inhibitors included the tissue non-specific ALP inhibitor, histidine (Merck, Darmstadt, Germany) and the tissue specific ALP inhibitor, Phe-Gly-Gly [5,6]. The concentrations of these inhibitors in the differentiation medium were 50 mmol/l and 20 mmol/l respectively. The concentrations chosen were based on a previous study using the murine 3T3-L1 pre-adipocyte cell line [7]. 2.5. Extraction of ALP from human pre-adipocytes Cellular extracts from pre-adipocytes were isolated at baseline and 12 days after initiation of adipogenesis using a previously published method [8]. Briefly, tissue culture medium was removed from the cells and 0.5 ml of an ice cold solution of 10 mmol/l Tris-HCl containing 1% Triton X-100 and 2 mmol/l phenylmethylsulfonyl-fluoride (pH 7.2) was added. The flasks were shaken to detach the cells and the suspension transferred to an Eppendorf tube and centrifuged for 10 min at 15,000 g. The supernatant was removed and immediately analysed for alkaline phosphatase activity using an automated colorimetric assay (Roche Diagnostics, Randburg, South Africa). The protein content of the supernatant was analysed using the Bradford method [21]. Alkaline phosphatase activity was calculated as mU of activity per mg protein. 2.6. Measurement of cellular lipid accumulation Intracellular lipid accumulation, which is the hallmark of adipogenesis was measured on day zero and day 12 using the Oil red O technique [22]. This technique depends on the ability of the lipid droplets in the adipocyte to collect the red stain (the stain only binds to triglycerides and cholesteryl oleate), which is then extracted from the cells using isopropyl alcohol, and the absorbance was measured at 510 nm [22]. Intracellular lipid accumulation was then expressed as OD units per mg of cellular protein. 2.7. Detection of polymorphisms in promoter region of TNALP gene Genomic DNA was isolated from buffy coat preparations taken from 6 white (BMI, 31.5 ± 4.5; age, 39.5 ± 11.9 years) and 6 black (BMI, 33.0 ± 4.6; age, 49.7 ± 6.3 years) females using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Primers were designed for PCR amplification of the promoter region of the human TNALP gene which provisionally lies within a 3780 bp region 5′ to the major transcription start site [23]. Primer design was accomplished using the Gene Runner software, version 3.05 (Hastings Software, Inc., Hastings, NY, USA). This region was amplified using 7 sets of overlapping PCR primers. The initial denaturation temperature was 95 °C for 11 min whilst the extension temperature was 72 °C for 10 min for all the PCR reactions. The cycling conditions for all reactions
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
2.8. Statistical analysis Data that was not normally distributed was log transformed before analysis. Differences in TNALP activity or triglyceride accumulation across ethnic groups or between different time points were analysed using Student's non-paired or paired t-test, respectively. Data in the text and figures are given as mean ± SD. Multivariable regression analysis was used to determine the effect of TNALP activity and ethnicity on intracellular lipid accumulation in pre-adipocytes. The standardized β value was used in all analyses. Statistical analyses were performed using Statistica v9 (StatSoft, Tulsa, OK, USA). 3. Results 3.1. Ethnic differences in TNALP activity and adipogenesis The mean BMIs (±SD) of the white and black women from whom adipose tissue was obtained were 24.7 ± 1.3 and 25.1 ± 1.6, respectively (p = 0.56). Their mean ages were 43.0 ± 4.6 and 45.5 ± 5.2 years, respectively (p = 0.23). The data in Fig. 1 demonstrates that intracellular lipid accumulation was higher in pre-adipocytes isolated from black women when compared to white women at both baseline (p b 0.0005) and after 12 day incubation with differentiation medium (p b 0.0005). This pattern was repeated for TNALP activity (p b 0.0005 at both baseline and after 12 day incubation with differentiation medium—see Fig. 1). When comparing the preadipocyte TNALP activity levels between white and black females it was observed that for the baseline levels there was no overlap in TNALP activity between the 2 groups with the range for white females being 3.56–66.1 mU/mg (median of 38.6) and for the black females, 70.2– 224.1 (median of 130.3 mU/mg). Similarly, for the TNALP activity levels after 12 days of exposure to differentiation medium the levels were much higher in the black females with a range of 107.6–493.2 (median of 289.8 mU/mg) compared to a range of 31.2–207.3 (median of 110.1 mU/mg) for the white females (see Fig. 1). 3.2. The relationship between TNALP activity and intracellular lipid accumulation The low N number of study participants ruled out the use of regression analysis to investigate the relationship between TNALP activity and lipid accretion within each ethnic group and at each of the two time points. Therefore, to increase the power of the analysis data from both ethnic groups was combined as was the data from the two time points to give N = 54. All the regression models included the Oil red O data (logged) as the dependent variable and the first model used TNALP activity (logged) as the sole independent variable and gave R2 = 0.58 (p b 0.0001) with β = 0.76 (p b 0.0001). The second model used ethnicity (white subjects coded as 1 and black subjects as 2) as the only independent variable and gave R2 = 0.12 (p b 0.05) with β = 0.35 (p b 0.05). The third model included both ethnicity and TNALP activity as independent variables and gave R2 = 0.61 (p b 0.0001) with β = − 0.22 (p = 0.06) for ethnicity and β = 0.90 (p b 0.0001) for TNALP
A) Cell lipid (OD units/mg protein)
were 95 °C for 40 s whilst the annealing time was 30 s. The reaction volume was 12 μl and contained 1.0 μl of buffer (SuperTherm Gold PCR buffer, JMR Holdings, Sevenoaks, Kent, UK), 0.8 mM of each dNTP (Bioline, Randolph, MA, USA), 0.075 U/l of SuperTherm Gold Taq polymerase (JMR Holdings, Sevenoaks, Kent, UK) and 1 μM of each of the primers and 2 μl of the DNA template. The PCR products were purified using the QIAquick purification kit (Qiagen, Hilden, Germany) and sequenced by Inqaba Biotec (Pretoria, South Africa) using a SpectruMedix SCE 2410 DNA sequence analyser (SpectruMedix LLC, State College, PA, USA). The DNA sequences were compared against that present within the GenBank database (accession code AB176449).
2.4
*** +++
2.0
1.6
*** 1.2
0.8
+++ 0.4
0.0 0
12
Days post induction of adipogenesis
B) 360 ** +++
320
ALP activity (mU/mg protein)
384
280 240 200
+++
160
**
120 80 40 0 0
12
Days post induction of adipogenesis Fig. 1. Intracellular lipid accumulation (A) and TNALP activity (B) in pre-adipocytes isolated from 14 white (grey bars) and 13 black (black bars) females. ++p b 0.005, +++p b 0.0005 vs. pre-adipocytes obtained from white females; **p b 0.005, ***p b 0.0005 vs. day zero. Each bar is a mean ± SD.
activity. These results suggest that the ethnic difference in intracellular lipid accumulation is due to the ethnic difference in TNALP activity.
3.3. Effect of ALP inhibitors The tissue nonspecific ALP inhibitor histidine was shown to block lipid accumulation in pre-adipocytes isolated from black females. Thus, Fig. 2A shows that after 12 day incubation with differentiation medium in the presence of histidine, intracellular lipid accumulation was significantly lower in cells treated with histidine than cells that were untreated, and this significant effect was observed in both ethnic groups. A similar pattern was observed for TNALP activity (Fig. 2B), with the activity at 12 days of histidine treatment being far lower than that observed in cells incubated for 12 days in the absence of histidine. In black females both intracellular lipid accumulation (Fig. 2A) and TNALP activity (Fig. 2B) were higher at day 0 and at day 12 (in the absence of His) than in white females. The tissue-specific ALP inhibitor Phe-Gly-Gly, did not show any effect on either intracellular lipid accumulation (Fig. 3A) or ALP activity (Fig. 3B) in pre-adipocytes isolated from black or white females. Both intracellular lipid accumulation (Fig. 3A) and ALP activity (Fig. 3B) were higher in black than white females at both time points and in the absence and presence of Phe-Gly-Gly.
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
A)
2.8
*** +++
2.4 2.0 1.6
**
1.2
#
*
0.8
##
**
+ 0.4
Cell lipid (OD units/mg protein)
Cell lipid (OD units/mg protein)
A)
385
2.8
*** ++
2.4
*** ++
2.0 1.6
*
1.2
*
0.8
+++ 0.4 0.0
0.0 0
12 Without His
0
12 With His
Days post induction of adipogenesis
12 Without Phe-Gly-Gly
12 With Phe-Gly-Gly
Days post induction of adipogenesis
B)
** ++
360 300 240 180
+++
*
120 60
#
##
**
ALP activity (mU/mg protein)
ALP activity (mU/mg protein)
B) 420
420
*** ++
** ++
360 300 240 180
+++
*
*
120 60
0 0
12 Without His
12 With His
Days post induction of adipogenesis Fig. 2. Histidine effects on lipid accumulation (A) and TNALP activity (B) in pre-adipocytes taken from mammary tissue of 4 black (black bars) and 4 white (grey bars) females. +p b 0.05, ++p b 0.005, +++p b 0.0005 vs. pre-adipocytes from white females; *p b 0.05, **p b 0.005, ***p b 0.0005 vs. day zero; #p b 0.05, ##p b 0.005 vs. without histidine. Each bar is a mean ± SD.
0 0
12 Without Phe-Gly-Gly
12 With Phe-Gly-Gly
Days post induction of adipogenesis Fig. 3. Phe-Gly-Gly effects on lipid accumulation (A) and TNALP activity (B) in preadipocytes taken from mammary tissue of 4 black (black bars) and 4 white (grey bars) females. ++p b 0.005, +++p b 0.0005 vs. pre-adipocytes from white females; *p b 0.05, **p b 0.005, ***p b 0.0005 vs. day zero. Each bar is a mean ± SD.
3.4. Sequence of the promoter region of the human tissue non-specific TNALP gene The profound difference in pre-adipocyte TNALP activity observed between white and black females suggested that either the catalytic activity, or the level of expression of the enzyme was much higher in the latter group. It was therefore decided to analyse the promoter region of the TNALP gene to determine if genomic sequence variation existed between the 2 ethnic groups. However, comparison of the sequences isolated from 6 white and 6 black females showed no polymorphic sites within the promoter region of the human ALP gene.
4. Discussion We demonstrate in the present study the significant differences in cellular fat accumulation between pre-adipocytes collected from black and white South African females. This is the first study to investigate and to show that ethnic differences in adipogenesis exist. Whilst the significance of this is currently unknown, the prevalence of obesity is however higher in black than white females in both South Africa [15] and the USA [16]. Furthermore, it is known that obesity involves both
adipocyte hypertrophy and hyperplasia [24]. These studies therefore suggest that it is possible that ethnic differences in adipogenesis may play a role in the higher prevalence of obesity in black than white females. It is known that visceral fat mass is lower in black than white subjects, when matched for BMI [25]. The higher level of adipogenesis in subcutaneous pre-adipocytes isolated from black compared to white females may explain this ethnic difference in body fat distribution. It is thought that the subcutaneous fat depot acts to buffer serum triglyceride levels removing these molecules from the circulation thus ensuring that serum levels do not rise too high [26]. This buffering system also ensures that triglyceride does not deposit within the visceral fat depot or at other ectopic sites [26]. Studies have shown that the insulin sensitizing agents, the thiazolidinediones increase lipid deposition in the subcutaneous depot leading to reduced visceral fat mass [27]. It is thought that this is accomplished by the ability of these drugs to increase adipogenesis more in subcutaneous than visceral pre-adipocytes [28]. Thus, if adipogenic capacity is greater in subcutaneous pre-adipocytes from black than white females, this may lead to lower triglyceride deposition in the visceral fat depot of the former population group.
386
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387
The ethnic difference in pre-adipocyte maturation observed in the present study is not the only adipocyte-related functional difference observed between black and white subjects. Thus, studies have shown that lipolytic rate is greater in adipose tissue of black than white females [29] and that adipocytes isolated from black females are more resistant to the anti-lipolytic action of insulin than adipocytes from white females [30]. Also, serum levels of the adipocyte-derived hormone, adiponectin are lower in black than white subjects [31]. Therefore, ethnic differences in both pre-adipocyte and mature adipocyte functionality have now been observed, but whether these play any role in the ethnic differences observed in body fat distribution [25] or whole body insulin sensitivity [32] is still a matter of conjecture. Previous studies have shown that TNALP is expressed in adipocyte precursor cells found in human [33], murine [34] and avian [35] bone marrow and in rat adipose tissue [36] and rabbit adipocytes [37]. However, one study failed to find TNALP expression in mature human adipocytes [38]. This may be because in humans TNALP is important only during adipogenesis and is not required for mature adipocyte function. This theory is supported by the finding that in pre-adipocytes isolated from human bone marrow, TNALP activity was high in the small, multilobular pre-adipocytes but absent from the large, unilobular mature adipocytes [33]. The present study clearly shows that the ALP present in preadipocytes isolated from black and white females is the tissue nonspecific isoenzyme as it was unresponsive to Phe-Gly-Gly but was inhibited by histidine. Similar data was obtained for ALP activity present in the 3T3-L1 pre-adipocyte cell line [7]. Furthermore, a study has shown that although the serum level of liver TNALP was higher in obese than lean subjects, but no differences were observed for bone or intestinal ALP levels [39]. A second study in which serum total ALP levels were measured in 32,329 subjects demonstrated that females who were more than 15% overweight had a 20% higher ALP activity than those who were not overweight [40]. These data suggest that adipose tissue contributes to the serum level of ALP and that it is the liver form of the tissue nonspecific ALP that is present in adipose tissue. The inhibition of adipogenesis and TNALP activity by histidine suggests that TNALP may regulate adipogenesis. Histidine is a nonspecific inhibitor of TNALP activity and therefore it is possible that the inhibition of adipogenesis observed in the presence of histidine may be due to non-ALP related effects of the amino acid. However, a study using a more specific method for reducing TNALP activity i.e. small interfering (si) RNA, also demonstrated a blockage in intracellular lipid accumulation in the 3T3-L1 pre-adipocyte cell line [41]. The finding that TNALP activity is localised to the lipid droplets of both human [8] and 3T3-L1 [7] preadipocytes further suggests that TNALP may be involved in controlling intracellular lipid accumulation. Also, a study has shown that triglyceride transport into and across gut enterocytes involves surfactant-like particles that are composed of a membrane-bound lipid particle containing membrane-associated intestinal ALP [42]. Currently, the exact role played by TNALP in modulating adipogenesis is unknown. The principle aim of the current study was to determine whether ethnic differences exist in adipogenic rate and whether the level of pre-adipocyte TNALP activity parallels this variable. The study was not designed to uncover the mechanistic relationship between TNALP and adipogenesis. However, it is possible to hypothesise about the role played by TNALP in the control of pre-adipocyte intracellular lipid accumulation. Thus, because TNALP is able to dephosphorylate phosphoproteins (3) and with its known location on the lipid droplet it is possible that TNALP may act to control the phosphorylation state of molecules present on, or that interacts with, the lipid droplet. Possible candidate molecules include perilipin [43], caveolin [44] and vimentin [45], which are all proteins that are associated with the lipid droplet and which are phosphorylated. These molecules may not be the direct substrates of TNALP but their phosphorylation status could be modified by TNALP altering the phosphorylation level of other kinases or phosphatases that act on these molecules.
The profound ethnic difference in pre-adipocyte TNALP activity is a novel finding. Statistical analysis of this data suggests that the ethnic difference in adipogenesis is directly related to the differences in TNALP activity. The molecular mechanisms that underlie the ethnic difference in pre-adipocyte TNALP activity are not known. The analysis of the promoter region of the TNALP gene demonstrated no sequence differences between the 2 ethnic groups. Although the number of subjects studied was small, the very large difference in TNALP activity between the 2 groups would suggest that this difference would be due to a very common polymorphism. However, this was not observed and therefore we would suggest that further studies must be conducted on possible differences in TNALP mRNA stability, the detection of polymorphisms within the TNALP exons and the intracellular levels of transcription factors that control TNALP gene expression. With regards to transcription factors, it is known that retinoic acid can increase TNALP gene expression [23]. It is interesting to note that in a study of the molecular aetiology of uterine leiomyomata, differences existed in the ratio of uterine tumour:non-tumour gene expression levels of retinoic acid receptor α and retinoid X receptor α between African–American and Hispanic, Chinese–American and white females [46]. This in vitro study has shown that pre-adipocytes isolated from black women have higher basal and stimulated intracellular lipid accumulation than those collected from white women. The significant difference in lipid accumulation was paralleled by a much higher level of TNALP activity in pre-adipocytes obtained from black than white females. This novel finding linking higher TNALP activity with higher intracellular triglyceride accumulation in black compared to white females may partially explain the greater level of obesity observed in the former population group. Acknowledgements The authors would like to thank the members of the National Health Laboratory Service (NHLS) Chemical Pathology routine laboratory at the Charlotte Maxeke Academic Hospital, Johannesburg for performing the ALP assays. This study was funded by the NHLS, the South African MRC and the National Research Foundation (NRF) of South Africa. AT Ali received bursaries from the NHLS and the University of the Witwatersrand. References [1] McComb RB, Bowers GN, Posen S. Alkaline phosphatase. New York: Plenum Press; 1979. [2] Stinson RA, McPhee JL, Collier HB. Phosphotransferase activity of human alkaline phosphatases and the role of enzyme Zn2+. Biochim Biophys Acta 1987;913:272–8. [3] Chan JR, Stinson RA. Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases. J Biol Chem 1986;261:7635–9. [4] Henthorn PS, Raducha M, Kadesch T, Weiss MJ, Harris H. Sequence and characterization of the human intestinal alkaline phosphatase gene. J Biol Chem 1988;263:12011–9. [5] van Belle. Alkaline phosphatase. 1. Kinetics and inhibition by levamisole of purified isoenzymes from humans. Clin Chem 1978;22:972–6. [6] Mulivor RA, Plotkin LI, Harris H. Differential inhibition of the products of the human alkaline phosphatase loci. Ann Hum Genet 1978;42:1–13. [7] Ali AT, Penny CB, Paiker JE, et al. Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1. Clin Chim Acta 2005; 354:101–9. [8] Ali AT, Penny CB, Paiker JE, Psaras G, Ikram F, Crowther NJ. The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3-L1 cells and human preadipocytes. Anal Biochem 2006;354:247–54. [9] Prasad R, Lambe S, Kaler P, et al. Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma. Biochim Biophys Acta 2005;1741:240–5. [10] Millán JL. The role of phosphatases in the initiation of skeletal mineralization. Calcif Tissue Int 2013;93:299–306. [11] Price CP. Multiple forms of human serum alkaline phosphatase: detection and quantitation. Ann Clin Biochem 1993;30:355–71. [12] Leung CT, Maleeff BE, Farrell Jr HM. Subcellular and ultrastructural localization of alkaline phosphatase in lactating rat mammary glands. J Dairy Sci 1989;72:2495–509. [13] Hillman JR, Seliger WG, Epling GP. Histochemistry and ultrastructure of adrenal cortical development in the golden hamster. Gen Comp Endocrinol 1975;25:14–24. [14] Githens S. Localization of alkaline phosphatase and adenosine triphosphatase in the mammalian pancreas. J Histochem Cytochem 1983;31:697–705.
A.T. Ali et al. / Clinica Chimica Acta 438 (2015) 382–387 [15] Puoane T, Steyn K, Bradshaw D, et al. Obesity in South Africa: the South African demographic and health survey. Obes Res 2002;10:1038–48. [16] Ogden CL, Carroll MD, Kit BK, Flegal KM. Prevalence of obesity among adults: United States, 2011–2012. NCHS Data Brief 2013;131:1–8. [17] Walley AJ, Asher JE, Froguel P. The genetic contribution to non-syndromic human obesity. Nat Rev Genet 2009;10:431–42. [18] Korostishevsky M, Cohen Z, Malkin I, Ermakov S, Yarenchuk O, Livshits G. Morphological and biochemical features of obesity are associated with mineralization genes' polymorphisms. Int J Obes 2010;34:1308–18. [19] Ristow M, Müller-Wieland D, Pfeiffer A, Krone W, Kahn CR. Obesity associated with a mutation in a genetic regulator of adipocyte differentiation. N Engl J Med 1998;339: 953–9. [20] Meyre D, Delplanque J, Chèvre JC, et al. Genome-wide association study for early-onset and morbid adult obesity identifies three new risk loci in European populations. Nat Genet 2009;41:157–9. [21] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein–dye binding. Anal Biochem 1976; 72:248–54. [22] Ramirez-Zacarias JL, Castro-Munozledo F, Kuri-Harcuch W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 1992;97:493–7. [23] Orimo H, Shimada T. Regulation of the human tissue-nonspecific alkaline phosphatase gene expression by all-trans-retinoic acid in SaOS-2 osteosarcoma cell line. Bone 2005; 36:866–76. [24] Couillard C, Mauriege P, Imbeault P, et al. Hyperleptinemia is more closely associated with adipose cell hypertrophy than with adipose tissue hyperplasia. Int J Obes Relat Metab Disord 2000;24:782–8. [25] Lovejoy JC, de la Bretonne JA, Klemperer M, Tulley R. Abdominal fat distribution and metabolic risk factors: effects of race. Metabolism 1996;45:1119–24. [26] van der Merwe MT, Crowther NJ, Schlaphoff GP, et al. Lactate and glycerol release from the subcutaneous adipose tissue of obese urban women from South Africa; important metabolic implications. J Clin Endocrinol Metab 1998;83:4084–91. [27] Frayn KN. Adipose tissue as a buffer for daily lipid flux. Diabetologia 2002;45: 1201–10. [28] Miyazaki Y, Mahankali A, Matsuda M, et al. Effect of pioglitazone on abdominal fat distribution and insulin sensitivity in type 2 diabetic patients. J Clin Endocrinol Metab 2002;87:2784–91. [29] Adams M, Montague CT, Prins JB, et al. Activators of peroxisome proliferatoractivated receptor gamma have depot-specific effects on human preadipocyte differentiation. J Clin Invest 1997;100:3149–53. [30] Buthelezi EP, van der Merwe MT, Lönnroth PN, Gray IP, Crowther NJ. Ethnic differences in the responsiveness of adipocyte lipolytic activity to insulin. Obes Res 2000;8:171–8.
387
[31] Ferris WF, Naran NH, Crowther NJ, Rheeder P, van der Merwe L, Chetty N. The relationship between insulin sensitivity and serum adiponectin levels in three population groups. Horm Metab Res 2005;37:695–701. [32] van der Merwe MT, Crowther NJ, Schlaphoff GP, Gray IP, Joffe BI, Lönnroth PN. Evidence for insulin resistance in black women from South Africa. Int J Obes Relat Metab Disord 2000;24:1340–6. [33] Bianco P, Costantini M, Dearden LC, Bonucci E. Alkaline phosphatase positive precursors of adipocytes in the human bone marrow. Br J Haematol 1988;68:401–3. [34] Kodama H, Koyama H, Sudo H, Kasai S. Adipose conversion of mouse bone marrow fibroblasts in vitro: their alkaline phosphatase activity. Cell Struct Funct 1983;8: 19–27. [35] Yoshida H, Yumoto T. Alkaline phosphatase-positive reticular cells of chicken bone marrow—in vivo and in vitro studies. Int J Cell Cloning 1987;5:35–44. [36] Wallach DP, Ko H. Some properties of an alkaline phosphatase from rat adipose tissue. Can J Biochem 1964;42:1445–7. [37] Lecoeur L, Ouhayoun JP. In vitro induction of osteogenic differentiation from nonosteogenic mesenchymal cells. Biomaterials 1997;18:989–93. [38] Okochi T, Seike H, Saeki K, Sumikawa K, Yamamoto T, Higashino K. A novel alkaline phosphatase isozyme in human adipose tissue. Clin Chim Acta 1987;162:19–27. [39] Ali AT, Paiker JE, Crowther NJ. The relationship between anthropometry and serum concentrations of alkaline phosphatase isoenzymes, liver enzymes, albumin and bilirubin. Am J Clin Pathol 2006;126:1–6. [40] Schiele F, Henny J, Hitz J, Petitclerc C, Gueguen R, Siest G. Total bone and liver alkaline phosphatase in plasma: Biological variations and reference limits. Clin Chem 1983; 29:634–41. [41] Chirambo GM, van Niekerk C, Crowther NJ. Post-translational silencing of the tissue non-specific alkaline phosphatase gene blocks intracellular lipid accumulation in a murine preadipocyte cell line, 3T3-L1 and in a human hepatocarcinoma cell line, HepG2. J Endocrinol Metab Diab S Afr 2010;15:25. [42] Zhang Y, Shao JS, Xie QM, Alpers DH. Immunolocalisation of alkaline phosphatase and surfactant-like particle proteins in rat duodenum during fat absorption. Gastroenterology 1996;110:478–88. [43] Greenberg AS, Egan JJ, Wek SA, Garty NB, Blanchette-Mackie EJ, Londos C. Perilipin, a major hormonally-regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J Biol Chem 1991;266:11341–6. [44] Mastick CC, Brady MJ, Saltiel AR. Insulin stimulates the phosphorylation of caveolin. J Cell Biol 1995;129:1523–31. [45] Brandes R, Arad R, Gaathon A, Bar-Tana J. Induction of adipose conversion in 3T3-L1 cells is associated with an early phosphorylation of a protein partly homologous with mouse vimentin. FEBS Lett 1993;333:179–82. [46] Wei JJ, Chiriboga L, Arslan AA, Melamed J, Yee H, Mittal K. Ethnic differences in expression of the dysregulated proteins in uterine leiomyomata. Hum Reprod 2006; 21:57–67.
