Oestrogen replacement fails to fully revert ovariectomy-induced changes in adipose tissue monoglycerides, diglycerides and cholesteryl esters of rats fed a lard-enriched diet

Menopause may be accompanied by abdominal obesity and inflammation, conditions accentuated by high-fat intake, especially of saturated fat (SFA)-rich diets. We investigated the consequences of high-SFA intake on the fatty acid (FA) profile of monoglycerides, diglycerides and cholesteryl esters from retroperitoneal white adipose tissue (RET) of rats with ovariectomy-induced menopause, and the effect of oestradiol replacement. Wistar rats were either ovariectomized (Ovx) or sham operated (Sham) and fed either standard chow (C) or lard-enriched diet (L) for 12 weeks. Half of the Ovx rats received 17β-oestradiol replacement (Ovx + E2). Body weight and food intake were measured weekly. RET neutral lipids were chromatographically separated and FAs analysed by gas chromatography. Ovariectomy alone increased body weight, feed efficiency, RET mass, leptin and insulin levels, leptin/adiponectin ratio, HOMA-IR and HOMA-β indexes. OvxC + E2 showed attenuation in nearly all blood markers. HOMA-β index was restored in OvxL + E2. OvxC showed significantly disturbed SFA and polyunsaturated FA (PUFA) profile in RET cholesteryl esters (CE). OvxC also showed increased monounsaturated FA (MUFA) in the monoglyceride diglyceride (Mono–Di) fraction. Similar changes were not observed in OvxL, although increased SFA and decreased PUFA was observed in Mono–Di. Overall, HRT was only partially able to revert changes induced by ovariectomy. There appears to be increased mobilization of essential FA in Ovx via CE, which is a dynamic lipid species. The same results were not found in Mono–Di, which are more inert. HRT may be helpful to preserve FA profile in visceral fat, but possibly not wholly sufficient in reverting the metabolic effects induced by menopause.


Results
High-fat diet intake further exacerbates the deleterious effects of ovariectomy. As shown in Table 1, the success of ovariectomy was confirmed by the lower uterus weight in the Ovx groups. Body weight at the beginning of the study was similar amongst the six groups, whilst the four ovariectomized groups (OvxC, OvxC + E2, OvxL, OvxL + E2) showed increased body weight, feed efficiency and RET fat mass at the end of the 12 weeks period. OvxL and OvxL + E2 showed further increased body weight and RET mass in comparison to OvxC and OvxC + E2. Ovariectomy alone or in combination with the high-fat diet significantly increased leptin and insulin levels, leptin/adiponectin ratio, HOMA-IR and HOMA-β indexes. Oestradiol replacement therapy was able to attenuate the effects of ovariectomy in the control diet group while only restoring the HOMA-β index in the lard-fed group. Total cholesterol, HDL-cholesterol and triglycerides levels did not differ significantly amongst the groups.
Ovariectomy modifies RET monoglyceride, diglyceride and cholesteryl ester FA composition, which is marginally restored by oestrogen replacement therapy. The FA composition of RET CE and Mono-Di fractions from rats fed the control diet are presented in Tables 2 and 3. Regarding the CE fraction (Table 2), the sum of SFA (∑SFA) was significantly decreased in OvxC in comparison to ShamC, mainly as a consequence of the significantly decreased C16:0 and C18:0. OvxC + E2 showed partial restoration of the ∑SFA, but which did not reach statistical significance in relation to OvxC. The C18/C18:1 ratio was decreased in OvxC compared to ShamC, but this statistically significant difference disappeared when comparing OvxC + E2 versus ShamC. The sum of monounsaturated fatty acids (∑MUFA) was statistically similar amongst ShamC, OvxC and OvxC + E2.

Discussion
Menopause is an important risk factor for the development of obesity, which becomes further exacerbated when associated with the consumption of energy dense diets 33 . Our group has recently demonstrated that ovariectomy modified the fatty acid profile of RET total lipid extract, which was marginally normalized by oestrogen replacement 30 . Those results confirm that the loss of ovarian hormones, combined or not with the consumption of a lard-enriched diet, could lead to impaired lipid and fatty acid metabolism in visceral adipose tissue. In order to further examine our hypothesis, we investigated the fatty acid composition of cholesteryl ester (CE) and monoglyceride diglyceride (Mono-Di) lipid fractions extracted from retroperitoneal white adipose tissue, firstly to determine to what extent lipids are affected by ovarian losses, and secondly to evaluate how efficient oestrogen replacement is at restoring any observed alteration.
As our most recently published results 30 most likely reflect the predominant triglyceride portion of the RET adipocyte fatty acid composition, in order to further examine our hypothesis, we investigated the fatty acid composition of cholesteryl ester (CE) and monoglyceride diglyceride (Mono-Di) lipid fractions extracted from retroperitoneal white adipose tissue, firstly to determine to what extent these dynamic lipid fractions are affected by ovarian losses, and secondly to evaluate how efficient oestrogen replacement is at restoring any observed alteration.
Ovariectomy alone increased body weight gain and adiposity due to increased feed efficiency, as food intake was not increased (Table 1). Insulin and leptin levels, leptin/adiponectin ratio, and HOMA indexes also increased after ovariectomy. The observed alterations were further exacerbated by high-fat diet ingestion. Our findings are consistent with the demonstration that impaired glucose homeostasis influences adipose tissue inflammation during high-fat intake 34 . In our study, whilst oestradiol replacement was able to attenuate the serum parameters altered by ovariectomy alone, the same was not observed when in combination with high-fat diet ingestion. We Table 2. Fatty acid composition of RET cholesteryl ester lipids obtained from ovariectomized (Ovx) or Sham operated rats, fed a control (C) diet, subjected or not to oestrogen replacement therapy (E2). Data presented as means ± standard error (SE) of the % of total FAs. n = 6 for each group. SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids. *p < 0.05 vs ShamC; #p < 0.05 OvxC vs OvxC + E2. www.nature.com/scientificreports/ suggest that due to the much poorer metabolic background of the lard diet-fed ovariectomized rats in comparison to the control diet-fed ovariectomized rats, the present oestradiol replacement therapy of 2.8 µg/day was possibly not sufficient to induce positive metabolic effects. Our suggestion agrees with a similar experiment in ovariectomized mice fed a lard-enriched diet, in which an oestradiol dose of 1.7 µg/day protected the mice from insulin resistance 35 . The oestrogen dose to mice in the study of Riant was relatively much higher in comparison to our study in rats, although the oestrogen replacement dose chosen for our study appears to be compatible with the human dosage of the average transdermal replacement therapy commonly used for postmenopausal women 36,37 . The CE FA analyses from rats fed the control diet showed that SFA decreased in OvxC in comparison to ShamC, while both n-3 and n-6 PUFA levels increased and MUFAs remained unchanged (Table 2). However, the CE FA analyses from rats fed the lard diet showed similar SFA, MUFAs and PUFAs levels, with no further alterations than the ones caused by ovariectomy alone (Table 4). These results agree with CE being a dynamic fraction and a reflection of global metabolic status, playing a major role as an integral component of membrane lipid rafts and as vehicle for the exportation of FA stored intracellularly in adipocytes 38,39 .
The decreased SFA levels observed in ovariectomized rats, particularly palmitic acid, was previously reported in the visceral adipose tissue of postmenopausal women, where a decreased ratio of saturation/unsaturation was identified 40 . Whilst the study of Garaulet 40 suggests that dietary factors combined with the age of subjects may play an important role in this phenomenon, our results suggest that ovariectomy alone may have had an effect, once SFA content decreased from 30.37% in ShamC down to 19.15% in OvxC, whilst ShamL showed 19.04% SFA, a content similar to 19.01% in OvxL (Table 2).
Decreased levels of SFA in serum phospholipids in postmenopausal women were observed previously 41 , whilst another report showed increased PUFA levels in plasma CE in postmenopausal women, alongside a positive association with adiposity 42 . Our findings that CE PUFAs were significantly higher in OvxC as compared to ShamC Table 3. Fatty acid composition of RET monoglyceride diglyceride lipids obtained from ovariectomized (Ovx) or Sham operated rats, fed a control (C) diet, subjected or not to oestrogen replacement therapy (E2). Data presented as means ± standard error (SE) of the % of total FAs. n = 6 for each group. SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids. *p < 0.05 vs ShamC; #p < 0.05 OvxC vs OvxC + E2.  (Table 4), such differences did not reach the statistical significance observed between OvxC versus ShamC (Table 2). Nonetheless, the Mono-Di ∑PUFA content was significantly lower in OvxL (23.46%) as compared to ShamL (31.92%) ( Table 5).
The lard-fed rats in the present study were exposed to an EFA-deficient diet. The deleterious impact of saturated fat-rich diets upon peripheral tissue fatty acid profile has been previously demonstrated by us 43 and others 44,45 . In a chronically deficient EFA diet, it may be possible that the biochemically harsh conditions prevented the RET CE from further adapting in ovariectomy, but it appears the Mono-Di fraction, which is located intracellularly in abundance, was able to buffer some of those unfavourable conditions, confirming the altruist role of the adipose tissue in protecting the body 46 .
Oestrogen replacement was able to partially attenuate the CE FA alterations observed in ovariectomy, in which it appears to return the levels of some FA, including behenic, AA, EPA and DHA levels, to levels similar to those found in Sham rats. It has been documented that hormone replacement therapy was able to decrease the levels of behenic acid in serum phospholipids 41 as well as decreased AA levels in whole blood of postmenopausal women 47 . In the particular case of AA, we acknowledge the disagreement of our findings with previous studies showing that hormone replacement increase AA levels, mostly due to alterations in Δ6-desaturase activity caused Table 4. Fatty acid profile of RET cholesteryl ester lipids obtained from ovariectomized (Ovx) or Sham operated rats, fed a lard-enriched (L) diet, subjected or not to oestrogen replacement therapy (E2). Data presented as means ± standard error (SE) of the % of total FAs. n = 6 for each group. SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids. *p < 0.05 vs ShamL; #p < 0.05 OvxL vs OvxL + E2. www.nature.com/scientificreports/ by oestrogen 41,48 . It is worth noting however that whilst those results refer to whole blood, in which there is transport of EFAs by phospholipids for the brain and other prime tissues, in our study the results were observed in retroperitoneal white adipose tissue neutral lipid species. As compared to ShamC, the OvxC group showed in the Mono-Di fraction no differences in SFA, but the ∑MUFA was significantly increased in OvxC, mainly attributed to higher oleic acid levels ( Table 3). The stearoyl-CoA desaturase, also known as Δ9-desaturase, is the intracellular enzyme that catalyses the rate-limiting conversion of palmitoyl-CoA and stearoyl-CoA to palmitoleic and oleic acids 49 . Oleic acid is the predominant FA stored in triglycerides of adipose tissue 50 , with the formation of oleic acid being a direct product of Δ9 desaturase activity on fatty acyl-CoA substrates 51 . Alessandri and colleagues 52 reported increased Δ9-desaturase levels following ovariectomy in rats, whilst oestrogen has been shown to suppress its expression in liver and adipose tissue 53,54 . Overall, such findings may explain the increased oleic acid levels in Mono-Di following ovariectomy, with subsequent tendency for reduction following oestrogen replacement.
DPA and DHA were significantly decreased in Mono-Di OvxC (Table 3), and the reduced levels of the n-6 counterparts AA and docosatetraenoic (DTA) did not reach statistically significant differences, even though a tendency was clear. Our findings that the Mono-Di fraction contained more MUFA and less PUFA agree with previous reports showing that Mono-Di molecules have an important role as intracellular storage 55,56 , and, as opposed to CE, Mono-Di are relatively more inert. The alternate increase and decrease of PUFA levels in CE and Mono-Di respectively may suggest the transfer of PUFA from within the cell to CE for membrane utilization and exportation, resulting in Mono-Di left with higher MUFAs.
Our suggestion of increased PUFA exportation through CE, but not SFA or MUFA, in OvxC rats may be attributed to the ability of peripheral tissues to synthesise SFA and MUFA but not essential PUFA. Interestingly, Belkaid and colleagues reported that 17β-oestradiol induces stearoyl-CoA desaturase-1 expression in oestrogenresponding cancer cells 57 , whilst in the opposite direction, Alessandri and colleagues 52 reported increased hepatic Δ9-desaturase levels following ovariectomy in rats. In our study, the ratio C18/C18:1 was significantly decreased Table 5. Fatty acid profile of RET monoglyceride diglyceride lipids obtained from ovariectomized (Ovx) or Sham operated rats, fed a lard-enriched (L) diet, subjected or not to oestrogen replacement therapy (E2). Data presented as means ± standard error (SE) of the % of FAs. n = 6 for each group. SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids. *p < 0.05 vs ShamL; #p < 0.05 OvxL vs OvxL + E2. www.nature.com/scientificreports/ in CE of OvxC as compared to ShamC (Table 2), and significantly increased in the Mono-Di fraction of OvxL versus ShamL (Table 3). We also found more MUFA in Mono-Di in OvxC as compared to ShamC, but no MUFA changes in CE between OvxC and ShamC. We have not measured stearoyl-CoA desaturase activity in our study; however, as a higher C18/C18:1 ratio denotes proportionally more saturated than monounsaturated species, we further speculate that there is transfer of fatty acids from one compartment to another. The association of lard diet with ovariectomy (OvxL) did not trigger differences in MUFA content in the Mono-Di fraction; however, it significantly increased ∑SFA and decreased ∑PUFA in relation to ShamL (Table 5). Differently from OvxC, the ∑SFA increase in OvxL may be traced to an effect of the diet alone. Our view is corroborated by a report on rats that showed a high-fat diet regimen containing 60% of kcal from fat increased the content of diglycerides in the liver, associated with systemic insulin resistance 58 . Additionally, it has been shown that a regimen of n-3 FA supplementation improved insulin sensitivity and decreased the content of diglycerides in visceral adipose tissue of rats fed a high-fat diet 59 .
OvxC + E2 showed partial restoration of ∑MUFA levels in Mono-Di, whist showing little effect in PUFA levels. Interestingly, ∑SFA and ∑PUFA were partially restored in Mono-Di in OvxL + E2. We believe oestrogen replacement may be responsible for partial attenuation of the changes observed in ovariectomy, alone or in combination with a lard diet. Our results agree with previous findings that oestrogen replacement ameliorated the overall lipid metabolism in ovariectomy alone 60 as well as in association of ovariectomy with a high-fat diet 61 .
The limitation of this study was to not include triglycerides, which represents a predominant fraction of neutral lipids within adipose tissue. Our choice to exclude triglycerides was based on data from our previous study where we demonstrated that ovariectomy leads to a disruption of the fatty acid composition of total lipid extract, which includes, in its majority, the triglyceride portion of neutral lipids 30 . By choosing to focus on cholesteryl esters, monoglycerides and diglycerides we aimed to investigate the more subtle and dynamic portions of the neutral lipid synthesis pathway within the adipose tissue that would confirm any metabolic changes taking place. Nevertheless, we acknowledge that including the triglycerides in this study could add to further its relevance, and deserves attention for future studies.
In conclusion, the present study showed that ovariectomy induced dramatic changes in the cholesteryl ester and monoglyceride/diglyceride fatty acid profiles of retroperitoneal white adipose tissue. Such changes appear to have been more dramatic in rats that received the control diet, as compared to the rats that received a diet enriched with lard. The control diet-fed rats may have shown greater capacity to adapt to hormone deficiency possibly due to a better fatty acid profile and a lower mild chronic pro-inflammatory background, as compared to the rats that chronically received the lard diet. We have also found that hormone replacement therapy tended to restore the level of some of the fatty acid families, but such findings were not consistently significant across all lipid families analysed in the present study. We speculate that hormone replacement therapy alone may not be sufficient to restore fatty acid profile changes observed in ovariectomy. Further studies are necessary to investigate whether hormone replacement therapy combined with positive nutrition interventions could promote better outcomes for menopaused women, particularly those subjected to nutrient-deficient diets.

Material and methods
Experimental procedures. All experiments were conducted in accordance with the Committee in Research Ethics of the Universidade Federal de São Paulo (CEUA no.: 2172030315/2016), through the guidelines of the Conselho Nacional de Controle de Experimentação Animal (CONCEA), Ministry of Science and Technology, Brazil. Detailed experimental procedures, body composition analyses and biochemical measurements adopted in the present study have been published previously 6 .
The lard-enriched diet was prepared by adding to the powdered standard chow 18% lard (w/w) (Cooperativa Central Aurora de Alimentos, Chapecó, Santa Catarina, Brazil), 2% soybean oil (w/w), 10% sucrose (w/w), 20% casein (w/w) to obtain the protein content of the control diet, and 0.02% (w/w) butylated hydroxytoluene (BHT). The mixture was mechanically mixed with lukewarm water for thorough homogenization of all ingredients, passed through a milling machine to produce pellets and dried in a forced ventilation oven for 24 h at 60 °C. The diet was stored at -20 °C and offered in standardized portions. The leftovers were weighted and discarded. Body weight and 24 h food mass intake were measured weekly. Feed efficiency was calculated as (body weight gain / energy intake) × 100.
Twelve weeks after surgery, rats were fasted for 24 h and sacrificed by decapitation under thiopental anaesthesia (80 mg/kg intraperitoneally). Trunk blood was collected. RET were dissected, weighed, snap-frozen in liquid nitrogen and stored at -80 ºC. The uteri were weighed for confirmation of completeness of ovariectomy.
Bodily measurements and serum biomarkers. Initial and final body weight, uterus and RET mass, serum leptin, adiponectin, glucose, insulin, total cholesterol, HDL cholesterol and triglycerides were quantified. The HOMA index was calculated as previously described 6 . The sensitivity, intra-assay and inter-assay varia- www.nature.com/scientificreports/ tions of the ELISA kits used to determine the serum levels of leptin, insulin and adiponectin were, respectively, 0.08 ng/mL; 2.49% and 3.93% for leptin; 0.1 ng/mL; 1.33% and 6.71% for insulin; 0.4 ng/mL; 1.18% and 7.34% for adiponectin.
RET lipid extraction, solid-phase chromatographic separation and fatty acid analysis. Aliquots of 1000 mg of RET was homogenized and extracted in hexane/isopropanol (3:2 v/v) containing 0.01% BHT. After addition of chloroform/methanol/water (2:1:1 v/v/v), the samples were centrifuged at 10,000g for 10 min. The organic layer was separated and evaporated to complete dryness with oxygen-free nitrogen (OFN). The lipids were partitioned again in chloroform/methanol/water (8:4:3 v/v/v), the chloroform layer was obtained, dried under OFN and kept in airtight glass vials under OFN at -20 ºC. CE and Mono-Di fractions were chromatographically obtained according to a previously established protocol 31 . Briefly, sample lipid residues were extracted twice in succession with 0.5 ml of isooctane-ethyl acetate (80:1 v/v) and applied to previously prepared silica gel columns (Thermo Scientific HyperSep silica columns; 100 mg bed weight) gravimetrically. CE were eluted with 5 ml of isooctane-ethyl acetate (20:1 v/v), followed by the Mono-Di fraction elution with 4.5 ml of isooctane-ethyl acetate (75:25 v/v). The collected fractions were immediately dried under OFN and kept in airtight glass vials under OFN at -20 ºC until derivatization.
FA analysis was performed as previously described by our group 32 . Briefly, fatty acid methyl esters (FAMEs) were obtained by heating lipid samples in sealed glass tubes at 70 ºC for 3 h with 15% acetyl chloride in dry methanol under OFN. The reaction was stopped with 5% NaCl solution at room temperature and FAMEs were extracted with three washes of petroleum spirit containing 0.01% BHT. Extracted FAMEs were analysed by gas chromatography with flame ionization detector (GC2010 Plus, Shimadzu, Kyoto, Japan) equipped with a Trace TR-FAME capillary column (60 m × 0.32 mm × 0.25 µm, Thermo Scientific, Rockford, IL, USA). FAMEs were identified by comparison with the retention times of previously injected authentic standards (Sigma-Aldrich, United Kingdom). Peak areas were analysed using Shimadzu software LabSolutions (Shimadzu, Kyoto, Japan).
Statistical analyses. Body weight, RET mass and serum parameters were tested for normality (Shapiro-Wilk test) and homoscedasticity (Levene's test). Normally distributed variables (means ± standard error of the mean) were analysed by two-way ANOVA and Tukey post hoc test. Non-parametric variables (median and interquartile range) were analysed by Kruskal-Wallis followed by multiple comparisons. Tests were performed with Statistica 12 Software (StatSoft, Tulsa, OK, USA).
CE and Mono-Di FA composition results were compared by one-way ANOVA, with groups separated according to their diet. Tests were performed by SPSS software (IBM, Chicago, IL, USA). Fatty acid results are presented as mean and standard deviation of the mean, and the level of statistical significance was set at p < 0.05. www.nature.com/scientificreports/