Abstract
Dehydroepiandrosterone (DHEA) is reported to exert beneficial effects, such as protection from cardiovascular risk and lowering serum insulin levels. Adipose tissue (AT) is a target for DHEA actions, and the hormone can also affect hepatic fatty acid (FA) metabolism. FAs are involved in the development of insulin resistance; thus, there might be a relationship between DHEA, FA, and insulin. However, few data are available regarding DHEA and FA composition, especially concerning AT. Seventeen-month old female Sprague–Dawley rats (n=11; controls: n=10) were treated with DHEA (0.5% w/w in the diet) for 13 weeks, after which serum, periovarian, mesenteric, s.c., and brown AT were analyzed for FA composition. DHEA treatment resulted in significant changes in FA profiles in serum and adipose depots, like reduced 16:1n-7 (s.c. and brown AT; P<0.01), elevated n-9 monounsaturated FA (serum and s.c. AT; P<0.05), diminished n-6 polyunsaturated FA (PUFA; general; P<0.05) and increased n-3 PUFA (brown AT; P<0.01), along with lower n-6/n-3 ratios (s.c. and brown AT; P<0.05, P<0.01 respectively). DHEA modified estimates of desaturase activities, decreasing stearoyl-CoA-desaturase markers in s.c., and brown AT (P<0.05) and increasing those of delta-6-desaturase in serum and AT (P<0.05). In addition, DHEA-treated rats showed lower serum insulin levels (P<0.05). We have demonstrated for the first time that DHEA induces significant modifications in AT fatty acid composition in vivo, mainly concerning unsaturated FAs, and changes occurred in a tissue-dependent manner. We propose that these changes may be related to the capacity of DHEA to lower serum insulin levels.
Introduction
The steroid hormone dehydroepiandrosterone (DHEA) and its sulphated form (DHEA-S) are known as precursors for most sexual hormones, both androgens and estrogens (Regelson et al. 1994), but they have also been shown to exert several positive effects themselves, independently of their conversion into androgens or estrogens, and there is even evidence for specific receptors for DHEA (Liu & Dillon 2004).
DHEA and DHEA-S show a marked decline in their serum levels with advancing age (Arlt 2004). Besides, low serum concentrations of these hormones, they have been associated with certain pathological states that increase their prevalence during ageing. Among the beneficial effects of DHEA, we can find cancer prevention (Ratko et al. 1991, Kawai et al. 1995), amelioration of cognitive function (Yanase et al. 1996), or protection against cardiovascular disease (Ebeling & Koivisto 1994), atherosclerosis (Nestler et al. 1992), or obesity (Richards et al. 2000, Abadie et al. 2001, de Heredia et al. 2007). Improvement of peripheral insulin sensitivity has also emerged as one of DHEA's relevant actions; indeed, we and other authors have previously observed that DHEA administration to rodents reduced significantly serum insulin concentrations (Richards et al. 2000, Abadie et al. 2001, Sánchez et al. 2008).
Adipose tissue (AT) seems to be one of the main targets in DHEA and DHEA-S actions. It has been reported that these steroids reduce AT cellularity (Ryu et al. 2003), inhibit preadipocyte differentiation in vitro (Lea-Currie et al. 1998), stimulate lipolysis (Tagliaferro et al. 1995, Hernández-Morante et al. 2008), and alter AT gene expression (Kajita et al. 2003, Sánchez et al. 2008). In addition, DHEA-S can modulate fatty acid (FA) metabolism, through up-regulation of hepatic enzymes involved in β-oxidation (Waxman 1996, Depreter et al. 2002).
The relevance of FAs is far beyond their role as energy storage molecules in AT, and they are recognized as important metabolic effectors (Yaqoop 2002, Drevon 2005, Steinberg 2007). FAs act as transcriptional modulators, regulating the expression of genes involved in energy metabolism or AT function (Raclot et al. 1997, Wilding 2001). Of particular concern is their impact on the development of insulin resistance (Kraegen et al. 2001, Russell 2004, Saravanan et al. 2005), and research is constantly demonstrating that the nature of FAs present in the different tissues is as important as their total amount in relation to the development of metabolic alterations (Garaulet et al. 2001). Consequently, the proportions in which FAs appear in AT may have a great relevance in the physiology of this tissue, in particular regarding the sensitivity to insulin stimulation.
The relationships among DHEA, FAs, and insulin sensitivity suggest that FAs could be mediators in the insulin-sensitizing actions of the hormone. Despite this fact, little is known about the effects of DHEA on FA composition in different tissues (Abadie et al. 2001, Imai et al. 2001, Gómez et al. 2002), and no studies to our knowledge have shown significant changes in AT fatty acid profile due to DHEA action.
Considering all this information, we aimed to study the effect of a pharmacological dose of DHEA on FA composition in serum and different AT depots in an animal model prone to metabolic disturbances, i.e., ageing rats fed on a high-energy saturated-fat diet. In the present work, we found that the hormonal treatment resulted in significant changes in the FA profiles of serum, of visceral and s.c. white AT and of brown AT, and propose that these changes can be related to the insulin-sensitizing action of DHEA.
Materials and Methods
Animals and conditions
Twenty-one female Sprague–Dawley rats, provided by the animal care facilities of the University of Murcia, were kept in a temperature-controlled room (24±2 °C) in a 12 h light: 12 h darkness schedule with lights on at 0800 h. Water and food were always provided ad libitum. The animal protocol followed in this study was reviewed and approved by the bioethical committee of our university and guidelines for the use and care of laboratory animals of the university were followed.
From 7 weeks of age, animals were fed on a hypercaloric diet that consisted on 200 g/kg casein (Hero, Murcia, Spain), 245 g/kg corn starch (Hero), 244 g/kg sucrose (local market), 200 g/kg palm oil (Croexsa, Barcelona, Spain), 50 g/kg cellulose (Avicel, Barcelona, Spain), 2 g/kg choline HCl (J Escuder, Barcelona, Spain), and 4 g/kg methionine (J Escuder); vitamin (10 g/kg) and mineral (45 g/kg) mixes were formulated according to AIN-93 guidelines (Reeves et al. 1993) and were supplied by Tegasa (Valencia, Spain) and Sigma. This diet contained (expressed as a percentage of fresh mass) 18.4% protein, 50.4% carbohydrates (50% of which was sucrose), and 19.6% fat, with an energy content of 1880 kJ/100 g; in terms of energy fat accounted for 39% total dietary energy. FA composition of dietary fat was 53.6% saturated FAs (SFA), 36.3% monounsaturated FAs (MUFA), 9.5% polyunsaturated FAs (PUFA) and 0.6% trans FAs (Pérez de Heredia et al. 2008). The diet was prepared once a week in our laboratory and stored at 5 °C in order to avoid rancidity, and food was freshly dispensed every other day.
DHEA treatment
At the age of 17 months, rats were divided into two groups: one received the diet supplemented with DHEA (Roig Farma, S.A., Terrasa, Barcelona, 99.5% purity) at a concentration of 0.5% (5 g DHEA/kg diet) (DHEA group, n=11), while the rest of the animals continued without changes and served as controls (control group, n=10). The hormonal treatment lasted for 13 weeks, when rats were 20 months old.
Sample collection
In the beginning of the light phase and following overnight fast, rats were weighed and killed under anesthesia (ether). Blood samples were collected by cardiac puncture, immediately centrifuged 15 min at 1500 g and 4 °C, and serum was stored at −80 °C until analyzed. White AT from visceral (periovarian and mesenteric) and s.c. (abdominal region) depots, and interscapular brown AT were removed, weighed, immediately frozen in liquid nitrogen, and stored at −80 °C.
In serum, insulin levels and the homeostatic model assessment (HOMA-IR index) were determined as previously described (Sánchez et al. 2008). Briefly, serum insulin was measured by RIA, using a commercial kit (Linco, St Charles, MO, USA), and serum glucose was measured by spectrophotometry using an enzymatic kit with glucose-oxidase and peroxidase. The HOMA-IR index was then calculated with the following formula (Matthews et al. 1985): HOMA-IR=Fasting glucose (mmol/l)×fasting insulin (mIU/l)/22.5.
Determination of FA composition
Total lipids from serum and AT samples were extracted according to the method of Folch et al. (1957), previous addition of an internal standard (tridecanoic acid, C13:0). FA methyl esters (FAME) were prepared with methanol HCl 3 M (Supelco, Bellefonte, PA, USA) at 100 °C for 1 h and dissolved in hexane. FAME were analyzed in a gas chromatograph HP-6890 (Agilent Technologies, Inc. Palo Alto, CA, USA), using a column SP-2560 of 60 m×0.25 mm id×0.15 μm (Supelco, SIGMA–Aldrich). The oven temperature was programmed at an initial temperature of 175 °C and after 30 min it was increased at a rate of 7 °C/min to 230 °C for 20 min. The injector and detector were set at 240 °C. Helium was used as the carrier gas at a pressure of 290 kPa. Peaks were identified by comparison of their retention times with appropriate FAME standards purchased from Sigma Chemical (SIGMA–Aldrich).
The amount of each individual FA was calculated and expressed as a percentage of total lipids FAs present in the samples. Then, total SFA were calculated as the sum of 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, 21:0, 22:0, 23:0, and 24:0; total MUFA were calculated as the sum of 14:1 n-9, 16:1 n-9, 17:1 n-9, 18:1 n-9, 20:1 n-9, 22:1 n-9, 24:1 n-9, 16:1 n-7, and 18:1 n-7; n-6 PUFA were obtained from 18:2 n-6, 18:3 n-6, 20:2 n-6, 20:3 n-6, 20:4 n-6, 22:2 n-6, 22:4 n-6, and 22:5 n-6; n-3 PUFA were calculated from 18:3 n-3, 18:4 n-3, 20:3 n-3, 20:5 n-3, 22:5 n-3, and 22:6 n-3; finally, trans FAs were calculated as the sum of C16:1 trans, C18:1 trans, C18:2 cis, trans, C18:2 trans, cis and, C18:2 trans, trans.
Statistical analysis
Results are presented as mean±s.e.m. Comparisons of FA proportions between control and DHEA groups were analyzed by the Student's t-test. To determine whether the effect of DHEA treatment on FA composition was tissue-specific, a two-way ANOVA test was also performed for AT depots, followed by Bonferroni post-hoc correction. Significance was always set at P<0.05. All analyses were performed using the statistic software SPSS v13.0 (SPSS Inc, Chicago, IL, USA).
Results
According to the average food intake, rats in the DHEA group received an estimated average dose of 6.8 mg DHEA/rat per day. After 13 week treatment, these animals had significantly higher serum DHEA-S levels than their controls (829.6±93.3 and 71.8±26.0 ng/ml respectively; P<0.001). In addition, DHEA-treated rats showed lower serum insulin levels than control ones (42.2±7.5 and 79.3±16.4 pmol/l respectively, P=0.047), and a tendency to a lower HOMA-IR index (1.9±0.3 and 3.5±1.00 respectively, P=0.154).
In serum from DHEA-treated rats, MUFA percentages were higher, while n-6 and n-3 PUFA were lower than in the control group (Table 1). Trans FAs were similar between groups, and so were total SFA, although with increased 16:0 and decreased 18:0 proportions in the DHEA group, which explain the apparent stability of total SFA (Table 1).
Selected fatty acids in serum from experimental groups
Control (n=10) | DHEA (n=10) | P | |
---|---|---|---|
Fatty acids (%) | |||
12:0 | 0.21±0.102 | 0.32±0.208 | NS |
14:0 | 0.83±0.452 | 0.48±0.081 | NS |
16:0 | 18.20±1.278 | 22.72±1.089 | 0.015 |
18:0 | 12.97±0.658 | 8.72±0.428 | <0.001 |
16:1 n-9 | 0.25±0.016 | 0.42±0.041 | 0.003 |
16:1 n-7 | 1.03±0.096 | 0.78±0.077 | 0.055 |
18:1 n-9 | 15.14±1.000 | 22.15±1.395 | 0.001 |
18:1 n-7 | 1.40±0.101 | 1.51±0.080 | NS |
18:2 n-6 | 7.31±0.207 | 5.85±0.446 | 0.008 |
18:3 n-6 | 0.48±0.036 | 0.59±0.045 | 0.056 |
20:2 n-6 | 0.19±0.062 | 0.49±0.129 | 0.053 |
20:3 n-6 | 0.33±0.039 | 0.56±0.059 | 0.006 |
20:4 n-6 | 32.74±1.549 | 27.41±1.534 | 0.025 |
22:5 n-6 | 1.93±0.204 | 0.80±0.080 | <0.001 |
18:3 n-3 | 0.41±0.179 | 0.44±0.181 | NS |
18:4 n-3 | 0.11±0.025 | 0.10±0.031 | NS |
20:5 n-3 | 0.18±0.052 | 0.20±0.056 | NS |
22:6 n-3 | 2.03±0.115 | 1.03±0.091 | <0.001 |
18:1 trans | 0.32±0.070 | 0.43±0.103 | NS |
18:2 trans, trans | 0.08±0.024 | 0.01±0.010 | 0.029 |
SFA | 34.13±1.235 | 34.70±1.091 | NS |
MUFA | 18.42±1.130 | 25.48±1.44 | 0.001 |
N-6 PUFA | 43.83±1.883 | 36.64±1.450 | 0.007 |
N-3 PUFA | 3.00±0.167 | 2.32±0.250 | 0.037 |
N-6/n-3 PUFA | 14.9±0.89 | 17.8±2.21 | NS |
TRANS FA | 0.62±0.078 | 0.86±0.147 | NS |
Data are presented as mean±s.e.m. Comparisons were performed by the Student's t-test. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data in bold represents statistical significance, P<0.05
Tables 2 and 3 contain the FA profile of the periovarian and mesenteric ATs -both visceral depots- respectively. N-6 PUFA proportions were significantly lower in rats that received DHEA, mainly due to a reduction on 18:2 n-6, while 20:4 n-6 was increased, pointing toward a higher delta-6-desaturase activity.
Selected fatty acids in periovarian adipose tissue from experimental groups
Control (n=10) | DHEA (n=11) | P | |
---|---|---|---|
Fatty acids (%) | |||
12:0 | 0.32±0.090 | 0.30±0.034 | NS |
14:0 | 0.92±0.087 | 0.93±0.028 | NS |
16:0 | 30.35±0.583 | 30.97±0.602 | NS |
18:0 | 2.48±0.103 | 2.68±0.113 | NS |
16:1 n-9 | 0.62±0.047 | 0.83±0.044 | 0.004 |
16:1 n-7 | 2.67±0.232 | 2.41±0.245 | NS |
18:1 n-9 | 48.81±0.400 | 47.60±1.523 | NS |
18:1 n-7 | 3.01±0.170 | 4.44±1.525 | NS |
18:2 n-6 | 8.46±0.205 | 7.29±0.094 | <0.001 |
18:3 n-6 | 0.05±0.004 | 0.11±0.009 | <0.001 |
20:3 n-6 | 0.03±0.006 | 0.05±0.008 | 0.055 |
20:4 n-6 | 0.26±0.023 | 0.45±0.023 | <0.001 |
18:3 n-3 | 0.20±0.017 | 0.19±0.020 | NS |
18:4 n-3 | 0.05±0.008 | 0.10±0.007 | <0.001 |
20:5 n-3 | 0.01±0.006 | 0.03±0.013 | NS |
22:6 n-3 | 0.04±0.006 | 0.04±0.002 | NS |
18:1 trans | 0.18±0.044 | 0.12±0.011 | NS |
18:2 cis, trans | 0.16±0.010 | 0.27±0.014 | <0.001 |
SFA | 34.91±0.632 | 35.52±0.543 | NS |
MUFA | 55.27±0.571 | 55.44±0.531 | NS |
N-6 PUFA | 8.99±0.189 | 8.07±0.083 | 0.001 |
N-3 PUFA | 0.33±0.034 | 0.41±0.033 | NS |
N-6/n-3 PUFA | 30.0±3.62 | 21.4±2.58 | 0.063 |
TRANS FA | 0.50±0.057 | 0.55±0.019 | NS |
Data are presented as mean±s.e.m. Comparisons were performed by the Student's t-test. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data in bold represents statistical significance, P<0.05
Selected fatty acids in mesenteric adipose tissue from experimental groups
Control (n=10) | DHEA (n=11) | P | |
---|---|---|---|
Fatty acids (%) | |||
12:0 | 0.09±0.020 | 0.11±0.051 | NS |
14:0 | 0.76±0.039 | 0.87±0.055 | NS |
16:0 | 30.99±0.384 | 31.57±0.495 | NS |
18:0 | 3.17±0.210 | 3.41±0.128 | NS |
16:1 n-9 | 0.52±0.029 | 0.78±0.041 | <0.001 |
16:1 n-7 | 2.39±0.222 | 2.00±0.240 | NS |
18:1 n-9 | 47.72±0.598 | 48.95±0.505 | NS |
18:1 n-7 | 2.59±0.082 | 1.89±0.076 | <0.001 |
18:2 n-6 | 8.43±0.161 | 7.22±0.107 | <0.001 |
18:3 n-6 | 0.05±0.008 | 0.12±0.005 | <0.001 |
20:3 n-6 | 0.03±0.004 | 0.07±0.004 | <0.001 |
20:4 n-6 | 0.27±0.043 | 0.52±0.035 | <0.001 |
18:3 n-3 | 0.22±0.016 | 0.21±0.010 | NS |
18:4 n-3 | 0.09±0.012 | 0.12±0.007 | 0.012 |
20:5 n-3 | 0.02±0.006 | 0.04±0.005 | NS |
22:6 n-3 | 0.12±0.082 | 0.04±0.006 | NS |
18:1 trans | 0.28±0.062 | 0.19±0.031 | NS |
18:2 cis, trans | 0.20±0.006 | 0.31±0.007 | <0.001 |
SFA | 36.26±0.787 | 36.84±0.516 | NS |
MUFA | 53.50±0.757 | 53.81±0.513 | NS |
N-6 PUFA | 8.98±0.179 | 8.12±0.476 | 0.001 |
N-3 PUFA | 0.53±0.073 | 0.48±0.023 | NS |
N-6/n-3 PUFA | 18.7±1.58 | 17.2±0.83 | NS |
TRANS FA | 0.72±0.060 | 0.75±0.034 | NS |
Data are presented as mean±s.e.m. Comparisons were performed by the Student's t-test. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data in bold represents statistical significance, P<0.05
In the s.c. AT, changes were more pronounced (Table 4). Total proportions of n-9 MUFA, trans FAs and 20:5 n-3 were higher in the DHEA group, while n-6 PUFA were again lower, leading to significantly diminished n-6/n-3 PUFA ratio. It is important to remark the reduction in the percentages of 16:1 n-7 by DHEA treatment, since it has been suggested as a potential marker of metabolic syndrome (Gil-Campos et al. 2008, Paillard et al. 2008). This decrease in 16:1 n-7 was also observed in the interscapular brown AT of DHEA-treated rats (Table 5), where we found again a lower n-6/n-3 PUFA ratio, in this case caused mainly by significantly higher n-3 PUFA levels.
Selected fatty acids in subcutaneous adipose tissue from experimental groups
Control (n=10) | DHEA (n=11) | P | |
---|---|---|---|
Fatty acids (%) | |||
12:0 | 0.34±0.044 | 0.22±0.029 | 0.039 |
14:0 | 1.04±0.052 | 0.87±0.030 | 0.010 |
16:0 | 30.07±0.604 | 29.72±1.167 | NS |
18:0 | 2.60±0.272 | 3.45±0.132 | 0.009 |
16:1 n-9 | 0.57±0.044 | 0.73±0.045 | 0.020 |
16:1 n-7 | 3.54±0.524 | 1.33±0.107 | 0.002 |
18:1 n-9 | 47.53±0.601 | 50.08±1.033 | 0.052 |
18:1 n-7 | 2.94±0.207 | 2.37±0.133 | 0.029 |
18:2 n-6 | 8.71±0.082 | 7.75±0.136 | <0.001 |
18:3 n-6 | 0.06±0.005 | 0.09±0.008 | 0.001 |
20:3 n-6 | 0.04±0.005 | 0.08±0.006 | <0.001 |
20:4 n-6 | 0.50±0.042 | 0.73±0.031 | <0.001 |
18:3 n-3 | 0.17±0.047 | 0.19±0.030 | NS |
18:4 n-3 | 0.05±0.007 | 0.12±0.007 | <0.001 |
20:5 n-3 | 0.03±0.004 | 0.05±0.009 | 0.026 |
22:6 n-3 | 0.05±0.008 | 0.05±0.004 | NS |
18:1 trans | 0.18±0.021 | 0.22±0.030 | NS |
18:2 cis, trans | 0.17±0.009 | 0.26±0.012 | <0.001 |
SFA | 35.20±0.960 | 35.53±1.247 | NS |
MUFA | 55.46±1.063 | 55.59±1.155 | NS |
N-9 MUFA | 48.42±0.610 | 51.18±1.073 | 0.043 |
N-6 PUFA | 9.56±0.097 | 8.93±0.139 | 0.002 |
N-3 PUFA | 0.35±0.047 | 0.48±0.045 | 0.061 |
N-6/n-3 PUFA | 29.9±2.28 | 19.9±1.52 | 0.001 |
TRANS FA | 0.54±0.033 | 0.68±0.040 | 0.011 |
Data are presented as mean±s.e.m. Comparisons were performed by the Student's t-test. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data in bold represents statistical significance, P<0.05
Selected fatty acids in interscapular brown adipose tissue from experimental groups
Control (n=10) | DHEA (n=11) | P | |
---|---|---|---|
Fatty acids (%) | |||
12:0 | 0.04±0.007 | 0.03±0.017 | NS |
14:0 | 0.95±0.048 | 0.85±0.048 | NS |
16:0 | 25.28±0.254 | 24.82±0.379 | NS |
18:0 | 5.59±0.439 | 6.66±0.234 | 0.050 |
16:1 n-9 | 0.53±0.028 | 0.63±0.024 | 0.019 |
16:1 n-7 | 1.53±0.170 | 0.80±0.077 | 0.002 |
18:1 n-9 | 46.81±0.410 | 47.45±0.234 | NS |
18:1 n-7 | 7.30±0.274 | 6.84±0.532 | NS |
18:2 n-6 | 8.88±0.134 | 7.67±0.130 | <0.001 |
18:3 n-6 | 0.07±0.003 | 0.12±0.007 | <0.001 |
20:3 n-6 | 0.10±0.008 | 0.20±0.014 | <0.001 |
20:4 n-6 | 1.19±0.121 | 1.55±0.171 | NS |
18:3 n-3 | 0.10±0.038 | 0.16±0.049 | NS |
18:4 n-3 | 0.04±0.005 | 0.14±0.011 | <0.001 |
20:5 n-3 | 0.03±0.003 | 0.06±0.010 | 0.022 |
22:6 n-3 | 0.06±0.006 | 0.08±0.008 | NS |
18:1 trans | 0.06±0.007 | 0.07±0.012 | NS |
18:2 cis, trans | 0.09±0.005 | 0.10±0.004 | 0.035 |
18:2 trans, cis | 0.07±0.005 | 0.15±0.007 | <0.001 |
SFA | 32.41±0.282 | 33.06±0.354 | NS |
MUFA | 56.55±0.460 | 56.21±0.408 | NS |
N-6 PUFA | 10.57±0.253 | 9.95±0.228 | NS |
N-3 PUFA | 0.25±0.046 | 0.47±0.057 | 0.009 |
N-6/n-3 PUFA | 54.4±8.0 | 23.9±2.54 | 0.004 |
TRANS FA | 0.22±0.009 | 0.32±0.015 | <0.001 |
Data are presented as mean±s.e.m. Comparisons were performed by the Student's t-test. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data in bold represents statistical significance, P<0.05
DHEA treatment also altered the estimates of desaturase activity calculated by their product/substrate ratios (Fig. 1). The ratios calculated with serum FAs are commonly used as estimates of hepatic desaturase activities (Sjögren et al. 2008). In serum from DHEA-treated rats, the ratio 16:1 n-7/16:0 was reduced while 18:1 n-9/18:0 was increased (both are markers of stearoyl-CoA desaturase – SCD- activity); by contrary, in the s.c. and interscapular brown ATs both indices were lower (Fig. 1A and B). By contrast, the markers of delta-6-desaturase activity in n-6 PUFA were increased, with significantly higher 18:3/18:2 n-6 and 20:4/18:2 n-6 ratios (Fig. 1C and D).
When FA data from AT samples were analyzed by means of the two-way ANOVA test, the interactions DHEA×tissue were non-significant for several FAs, which means that in these cases all fat depots responded to DHEA treatment in a similar way. However, other changes were indeed tissue-specific. For instance, variations in total n-3 PUFA were specific for s.c. and brown AT (Pinteraction=0.047); similarly, reductions in the proportions of 16:1 n-7 and in the n-6/n-3 PUFA ratio were more pronounced in these two depots (Pi=0.001 for both variables). Other FAs that varied in a tissue-specific manner were 14:0 (Pi=0.048), 20:3 n-6 (Pi<0.001), 18:4 n-3 (Pi=0.001), 18:2 cis, trans (Pi<0.001), and 18:2 trans, trans (Pi<0.001).
Discussion
We had previously reported that DHEA administration to our rats led to significant reductions in food intake, body weight, total body fat content, the weight of different adipose depots, and adipocyte size (de Heredia et al. 2007). In addition, we found that DHEA-treated rats had significantly lower serum insulin concentrations and a tendency to lower HOMA-IR index (Sánchez et al. 2008). The present work has revealed that DHEA treatment also induced significant changes in FA profile in serum and AT from those rats, and that these changes varied according to the tissue studied. We found elevated n-9 MUFA in serum and s.c. AT, reduced n-6 PUFA in serum and white adipose depots, and increased n-3 PUFA (18:4 n-3 in all adipose regions, 20:5 n-3 in s.c. tissue and total n-3 PUFA in interscapular brown AT). Besides, DHEA treatment altered the surrogate estimates of desaturase activities, again in a tissue-specific manner.
This is to our knowledge, the first work that demonstrates a significant effect of DHEA on AT fatty acid composition. There is a previous work that reported no changes in rat AT fatty acids after 7 days of DHEA administration (Abadie et al. 2001). However, due to the lower lipid turn-over rate of this tissue, a 7-day period may not be long enough to appreciate variations in FA profile in AT. By contrast, our experiment spanned a 13-week period that allowed us to observe a significant effect of DHEA on AT composition. Similarly to us, other authors have reported differences in FAs in muscle (Abadie et al. 2001), liver (Imai et al. 2001), and 3T3-L1 adipocytes (Gómez et al. 2002) due to DHEA administration.
In our study, changes in serum 16:0 (increased) and 18:0 (decreased) observed in the DHEA group were not reflected in AT; on the contrary, individual SFA did not varied in the visceral depots, while 18:0 was higher in s.c. and brown ATs of DHEA-treated rats. The increment in serum 16:0 after DHEA administration is somehow surprising, since this FA induces peripheral insulin resistance (Chávez & Summers 2003, Powell et al. 2004), but we observed lower insulin concentrations in DHEA-treated rats (Sánchez et al. 2008). However, it seems that impairment of peripheral insulin sensitivity by 16:0 is produced by conversion of this FA into ceramides (Chávez & Summers 2003, Powell et al. 2004), and DHEA could be counteracting this process somehow.
Other FAs associated with insulin resistance are trans FAs (Saravanan et al. 2005). In the present work, total trans FAs practically did not change because of DHEA in the different fat depots. We found lower proportions of 18:2 trans, trans in serum and higher percentages of 18:2 cis, trans and 18:2 trans, cis in AT of DHEA-treated rats, but these are minority trans isomers and even their quantification is very difficult. The main trans isomer in diet and tissues is 18:1 trans that were practically not modified by the hormonal treatment.
More remarkable were the changes in n-9 MUFA. These FAs, of which oleic (18:1 n-9) is the most representative, were significantly higher in the serum and s.c. AT from the DHEA group than from the control one. Quite the opposite, palmitoleic acid (16:1 n-7) was reduced in s.c. and brown AT (and in serum, with marginal significance). These results are in agreement with those obtained by Imai et al. (2001), who observed that DHEA stimulates oleate synthesis, resulting in increased 18:1 n-9 contents in liver, and with Gómez et al. (2002), who also reported reduced 16:1 n-7 and elevated 18:1 n-9 in 3T3-L1 adipocytes treated with DHEA.
Different authors have reported that oleic acid is negatively associated with features of the metabolic syndrome (Garaulet et al. 2001, Carluccio et al. 2007), and positively with enhanced peripheral glucose uptake (Vessby et al. 1994, Dimopoulos et al. 2006). Regarding palmitoleic acid, it also has been shown to increase glucose uptake in vitro (Dimopoulos et al. 2006), whereas other authors have proposed it as a marker for metabolic risk (Gil-Campos et al. 2008, Paillard et al. 2008). Although it remains to be determined, whether increments in palmitoleic acid indicate causal or compensatory events for insulin resistance, higher levels of this FA are related to metabolic alterations. In light of these data, it could be suggested that the insulin-lowering effect of DHEA that we observed may be related to the higher oleic acid and the reduced palmitoleic acid proportions in the tissues of DHEA-treated rats.
PUFA were also affected by DHEA treatment in our study. In serum, the DHEA group showed a significant reduction of linoleic (18:2 n-6) and arachidonic (20:4 n-6) acids. In AT, linoleic acid was also lower while arachidonic acid was higher, in agreement with observations in rat liver (Imai et al. 2001). Some authors have reported negative associations between n-6 PUFA and insulin sensitivity (Vessby et al. 1994, Aldámiz-Echevarría et al. 2007), while others found positive ones (Nugent et al. 2001, Dimopoulos et al. 2006). The relation between n-6 PUFA and insulin sensitivity could depend on the localization of the FAs, so that high levels in serum would be related to insulin resistance, while in AT or skeletal muscle they would help to improve sensitivity. If this was the case, the reduction in serum n-6 PUFA in DHEA-treated rats observed in our work might be related to the lower insulin concentrations found in these animals. By contrast to n-6 FAs, our results show increased proportions of n-3 PUFA in brown AT in rats receiving DHEA. It has been proposed that n-3 PUFA in brown AT are related to UCP-1 levels (Takahashi & Ide 2000), and DHEA has been shown to increase brown fat UCP-1 levels (Ryu et al. 2003); therefore, we hypothesize that DHEA treatment could enhance brown fat thermogenesis, at least in part, through increments in n-3 PUFA contents.
Of note, the changes in PUFA proportions led to significant reductions in n-6/n-3 PUFA ratios in s.c. and brown ATs–-and the same trend was followed by the other adipose regions analyzed- of DHEA-treated rats. It has been proposed that high n-6/n-3 PUFA ratios are associated with greater body fat accumulation, cardiovascular risk, and inflammatory processes (Ghafoorunissa et al. 2005, Ailhaud et al. 2006, Riediger et al. 2008); therefore, the lower n-6/n-3 ratios observed in the present work could be a part of the protective actions attributed to DHEA.
It is interesting to highlight that the effects of DHEA on AT seem to be depot-specific, since the changes observed varied among the different tissues analyzed. It appeared that the s.c. white AT and the interscapular brown AT were more affected by the treatment, while the changes in the visceral depots were less pronounced. These results could be related, among other possible causes, to the fact that this experiment was conducted in female rats, since previous work performed in humans showed a gender-specific action of DHEA on AT, its effects being more evident in visceral fat among men and in s.c. fat among women (Hernández-Morante et al. 2008). Further studies should be carried out in order to confirm this hypothesis.
Finally, in our experiment, DHEA administration also modified significantly the estimates of desaturase activities. In serum of DHEA-treated rats, the marker of SCD activity on C18 (ratio 18:1/18:0) was increased, while SCD on C16 (ratio 16:1/16:0) was reduced. These indices are commonly used as estimates of hepatic desaturase activity, based on the fact that in the fasting state most serum FAs are derived from the liver (Sjögren et al. 2008). Some authors have suggested that higher SCD activity leading to increased synthesis of palmitoleic acid might be involved in metabolic alterations associated with obesity (Gil-Campos et al. 2008, Paillard et al. 2008). Therefore, these inverse changes observed in serum SCD markers that imply decreased palmitoleic and increased oleic synthesis could partly mediate the beneficial effects of DHEA. In AT, in contrast, the two markers of SCD activity were lower in the DHEA group than in the control one, the difference being significant in the s.c. and brown depots. It is worth highlighting that our results show opposite trends in serum and AT 18:1/18:0 in response to DHEA treatment. These changes are in agreement with the findings by Imai et al. and Gómez et al. The former observed that DHEA induces SCD in liver to increase 18:1 synthesis (Imai et al. 2001), which would explain the higher 18:1/18:0 ratio found in serum in our work, while the latter found that DHEA inhibits SCD expression in adipocytes in vitro (Gómez et al. 2002), leading to the lower 18:1/18:0 ratio in AT, mainly in the s.c. and brown depots. In addition, we found a general increment in the markers of delta-6-desaturase activity in the DHEA group. To our knowledge, no previous data are available on the effects of DHEA on delta-6-desaturase activity. Considering that SCD activity in AT has been associated with insulin resistance (Dobrzyn & Ntambi 2005, Sjögren et al. 2008), and that delta-6-desaturase has been reported to be involved in insulin-stimulated glucose uptake (Das 2005), this DHEA-related reduction in SCD markers in AT together with the increased delta-6-desaturase estimates observed in our work could reflect another mechanism by which DHEA improves insulin sensitivity.
It must be kept in mind that other major tissues, such as liver and muscle, play an important role in insulin resistance and they also seem to be targets for DHEA; therefore, they should be taken into account when dealing with systemic insulin sensitivity. Nevertheless, the contribution of AT should not be underestimated, considering that it is one of the main sites for insulin action and the principal source for circulating FAs in the body.
In summary, we have demonstrated for the first time that pharmacological administration of DHEA modifies significantly the FA profiles in serum and different AT depots in rats, and that changes vary according to the tissue studied. The hormone affected mainly n-9 MUFA, palmitoleic acid, n-6 PUFA, and n-3 PUFA, and also the markers of desaturase activities. Although the relationships among DHEA, FAs and insulin sensitivity are still poorly understood, we hypothesize that changes in FAs due to DHEA treatment may be related to DHEA's insulin-lowering effect, and speculate that the lower insulin levels might be accompanied by improved insulin sensitivity.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Seneca Foundation from the Government of Murcia (project 02934/PI/05 to M G).
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