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Dietary syringic acid reduces fat mass in an ovariectomy-induced mouse model of obesity

Tanaka, Teruyoshi PhD1,2; Iwamoto, Kazuko PhD3; Wada, Maki BD3; Yano, Erika BD3; Suzuki, Toshiyuki PhD1; Kawaguchi, Nobuhisa MS4; Shirasaka, Norifumi PhD3; Moriyama, Tatsuya PhD3; Homma, Yoshimi PhD1,5

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doi: 10.1097/GME.0000000000001853
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Abstract

Postmenopausal women are at increased risk of metabolic diseases, such as obesity and diabetes,1,2 which in turn increase the risk of the development of more serious conditions, including cancers, neurological disorders, hypertension, and other cardiovascular diseases.3 Postmenopausal obesity and diabetes are induced by estrogen deficiency, which exacerbates insulin resistance.4,5 The number of postmenopausal patients with obesity and diabetes has increased in recent years owing to population ageing; therefore, dietary strategies to reduce obesity and diabetes risk in this regard have become a necessity with the end of improving postmenopausal health.

It has been demonstrated that soybean isoflavones, including daidzein, and genistein, can improve lipid metabolism in animal models of postmenopausal obesity and diabetes.6-9 Specifically, isoflavones have an affinity for estrogen receptors (ERs), specifically ERβ;10,11 however, they also promote the proliferation of ER-positive MCF-7 human breast cancer cells and enhance hyperplasia of the endometrium.11-15 Therefore, diets incorporating excessive levels of these isoflavones may exert breast and uterine oncogenic effects via ERs. For this reason, the identification of compounds that demonstrate beneficial effects on menopausal lipid metabolism, independent of ER-mediated pathways, is of great significance.

Syringic acid (SA) (Fig. 1) is a phenolic compound that is naturally present in many edible plants, such as the fruit of the assai palm, Euterpe oleracea,16 and the mycelium of the shiitake mushroom, Lentinula edodes.17 Importantly, this compound has no affinity for ERs18 and exhibits radical scavenging,19,20 antioxidative, and anti-inflammatory activities.17,19,20-22 In our previous study, we demonstrated that dietary SA improves murine bone mineral density and microstructure.23 Furthermore, Ham et al24 reported that a diet including SA mitigates high-fat diet-induced obesity in mice, and John et al25 demonstrated that SA suppresses adipogenesis in mouse 3T3-L1 preadipocytes. Furthermore, a recent study suggested that these effects are associated with decreases in reactive oxygen species (ROS) levels induced by SA.26 However, it is still unclear how SA affects estrogen deficiency-induced obesity.

FIG. 1
FIG. 1:
Chemical structure of syringic acid (SA).

Ovariectomised (OVX) animals are used to model postmenopausal obesity and diabetes,8,27,28 and OVX mice demonstrate glucose intolerance, insulin resistance, and increased body weight and fat mass. In this study, we investigated whether and how dietary SA affects these factors in OVX mice.

METHODS

Materials

Syringic acid (> 97% purity) was purchased from Tokyo Chemical Industry (Tokyo, Japan). All the other chemicals used in this study were of the highest purity available.

Animals and diets

All the animal experiments were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the guidelines specified by Kindai and Fukushima Medical University Animal Experimentation Regulations.

Experiment 1

Slc:ddY female mice were purchased from Japan SLC (Shizuoka, Japan). They were OVX or sham-operated (Sham) at 9 weeks. After 1 week, the Sham and OVX mice were divided into four groups as follows: Sham-control group (n = 7), Sham-SA group (n = 8), OVX-control group (n = 7), and OVX-SA group (n = 8). The control group was fed a commercial diet (MF; Oriental Yeast, Tokyo, Japan) as the control diet, while the SA groups received SA-containing diets (100 mg/kg body weight/d, corresponding to 3 mg of SA/d/30 g mouse in 4.5 g of diet), prepared by adding SA to MF powder, for 12 weeks. The efficacy of this dosage was preliminarily determined based on the elevation of serum glucose and triglyceride levels, after administration to five OVX mice for 4 weeks (data not shown). CT analysis was chronologically performed, and the mice were fed SA diets until there was a significant difference in fat mass between the OVX and OVX-SA groups. Mice were housed in individual cages under a 12-h/12-h light/dark cycle at 20 ± 2°C. The diet and tap water were supplied ad libitum. Further, during the experimental period, body weight and food intake were measured once a week. After 12 weeks, the mice were sacrificed, and the lung, liver, spleen, kidney, and uterus was harvested and weighed.

Experiment 2

Ten-week-old OVX mice were divided into the control (n = 8), SA10 (n = 8), SA50 (n = 8), and SA100 (n = 8) groups. Mice in the control group were fed normal diets, while those in the SA10, SA50, and SA100 groups received SA diets containing 10, 50, and 100 mg/kg body weight/d, respectively. The other experimental conditions were identical to those described in Experiment 1.

Experiment 3

Experiment 3 was performed to compare the effects of SA and E2 on OVX mice. Ten-week-old OVX mice were divided into the Sham (n = 8), OVX-control (n = 8), OVX-SA (n = 8), and OVX-E2 (n = 8) groups, and fed normal diets for 12 weeks. The mice in the OVX-SA group received an SA-containing diet (100 mg/kg body weight/d), while those in the OVX-E2 group were injected with 17β-estradiol, and those in the other groups were injected with vehicle from the abdominal cavity. The other experimental conditions were identical to those described in Experiment 1.

Experiment 4

Ten-week-old OVX mice were divided into the control (n = 10) and SA (n = 10) groups. The mice in the control group were fed normal diets, while those in the SA group received an SA-containing diet (100 mg/kg body weight/d). After 12 weeks, the mice were fasted for 18 h prior to the evaluation of their glucose tolerance based on the oral administration of glucose (1,000 mg/kg body weight). Blood samples were collected from the tail vein 0-120 minutes after glucose administration. The glucose tolerance of the mice was shown based on the increase in blood glucose level at 0 minute. Other experimental conditions were identical to those described in Experiment 1.

Experiment 5

Ten-week-old female mice were orally administered 100 mg/kg of SA suspended in 0.1% CMC-Na solution, while mice in the control group were administered the vehicle solution. Thereafter, blood samples were collected from the tail vein 0-720 minutes after the single oral administration of SA. The resulting serum samples were then stored at −80°C until further analysis.

Computed tomography

Mouse bodies were scanned using an X-ray computed tomography system (LCT-100; ALOKA, Tokyo, Japan). Tomograms were taken at 0.5-mm intervals from the diaphragm to the bottom of the abdominal cavity. Fat mass (visceral, subcutaneous, and total fat) was measured by analyzing the tomograms. Data were presented as the relative fat mass (per body weight × 100).

Histomorphometric analysis

At necropsy, adipose and hepatic tissues were fixed in a 3.7% formaldehyde solution using phosphate buffered saline. Thereafter, the tissues were embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan) and rapidly frozen in liquid nitrogen at −80°C until further analysis. Subsequently, the tissues were sectioned using a cryostat followed by staining with hematoxylin and eosin. The diameter of the adipocytes was determined using CellSens (Olympus, Tokyo, Japan).

Triglyceride, cholesterol, and glucose levels

Serum glucose, triglyceride, total cholesterol, and high-density lipoprotein (HDL) cholesterol levels were determined using triglyceride E, cholesterol E, and HDL-C kits (Wako Pure Chemicals, Osaka, Japan), respectively. Further, serum low-density lipoprotein (LDL) cholesterol levels were calculated from the total cholesterol, HDL-cholesterol, and triglyceride levels.

Liver lipids were extracted as previously described by Bligh and Dyer29 and evaporated under N2. The extract was dissolved in isopropanol prior to measuring liver triglyceride and cholesterol levels using the above-mentioned kits.

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) analysis

AST and ALT activities were determined using commercial kits (Transaminase C II-test Wako; Wako Pure Chemicals, Osaka, Japan).

Analysis of the translocation form of orally administered SA in blood and translocation amount of SA in the intact form via mass spectrometry

A fixed amount of sample was loaded into a mass spectrometer [Mass spectrometer: Orbitrap Elite, Thermo Fisher Scientific (Waltham, MA); Controller, XCALIBUR v2.2 (Thermo Fisher Scientific)] and analyzed in the negative ion mode. The ions generated from SA and their conjugation forms were then determined via selective reaction monitoring. The SA conjugates that translocated in blood were estimated from the molecular weights obtained using METWORKS 1.3 (Thermo Fisher Scientific), and the level of SA in the intact form in blood was calculated from the calibration curve obtained using a commercially available standard.

Serum insulin, leptin, adiponectin, and resistin levels

Serum insulin levels were measured using an enzyme-linked immunosorbent assay kit (Morinaga Institute of Biological Science, Kanagawa, Japan), while serum leptin levels were measured using a rat leptin enzyme-linked immunosorbent assay kit (Morinaga Institute of Biological Science, Kanagawa, Japan). Further, serum adiponectin and resistin levels were determined using commercial kits (R&D Systems, Minneapolis, MN).

Statistical analysis

Data are presented as mean ± standard error (SE). For multiple comparisons, one-way analysis of variance followed by a post-hoc Tukey test was used to examine differences between groups. For the analysis results shown in Figures 1A, B and 5, two-way analysis of variance was performed followed by a post-hoc Tukey test. The Mann-Whitney rank sum test or two-tailed t tests were also performed to determine significant differences between groups. Specifically, the Mann-Whitney rank sum test was used to calculate P values for highly skewed distributions, while the tow-tailed t test was used for Gaussian-like distributions. P < 0.05 was considered statistically significant.

RESULTS

Effects of dietary SA on body weight, food intake, fat mass, and adipocytes

In this study, Sham and OVX mice were fed a diet containing 100 mg/kg/d SA for 12 weeks. From Figure 2A, which shows the changes in mice body weights over the experimental period, it is evident that the SA diet did not significantly alter the body weights of Sham and OVX mice. The final body weights of the mice in the Sham-control, Sham-SA, OVX-control, and OVX-SA groups were 33.0 ± 0.6, 33.4 ± 0.8, 39.2 ± 1.9, and 38.5 ± 1.2 g, respectively. Further, as shown in Figure 2B, there were no significant differences among the four groups with respect to food intake. X-ray computed tomography analysis was performed to determine the fat mass of mice relative to their body weights. Even though there were no significant differences between the Sham-control and Sham-SA groups in this regard, the SA diet resulted in significantly decreased fat mass (visceral, subcutaneous, and total fat) in OVX mice (Fig. 2C, D). The relative total, visceral, and subcutaneous fat levels of mice in the OVX-SA group were 67.9, 69.3, and 68.5% of the corresponding values for the mice in the OVX-control group, respectively. Next, the staining of adipose tissues (micrographs shown in Fig. 2E) to evaluate changes induced by ovariectomy and the SA diet showed that the SA diet significantly decreased adipocyte diameter in OVX mice (76.2 ± 3.2 vs. 58.7 ± 2.1 μm, P < 0.05, Fig. 2F). However, the decrease in Sham mice was not significant. Additionally, the SA diet (10-100 mg/kg body weight) resulted in a decrease in white adipose tissue mass, but had no significant effect on body weight in OVX mice (Table 1). It was also observed that E2 treatment showed a stronger effect on white adipose tissue and body weight than the SA diets (Table 2).

FIG. 2
FIG. 2:
Effect of dietary syringic acid (SA) on: (A) body weight, (B) food intake, (C, D) fat mass, (E) adipocyte morphology, and (F, G) adipocyte size of mice in the Sham-control (n = 7), Sham-SA (100 mg/kg body weight/d, n = 8), OVX-control (n = 7), and OVX-SA (100 mg/kg body weight/d, n = 8) groups. Scale bar, 50 μm. Each value represents the mean ± SE. OVX, ovariectomized; Sham, sham-operated.
TABLE 1 - Effect of dietary syringic acid (SA) on serum parameters after 12 wks in the OVX-control (n = 8), OVX-SA10 (n = 8), OVX-SA50 (n = 8), and OVX-SA100 (n = 8) groups
Control SA10 SA50 SA100
Initial body weight (g) 33.4 ± 1.1 33.7 ± 1.7 33.5 ± 0.6 33.9 ± 1.9
Final body weight (g) 49.6 ± 5.8 46.8 ± 0.7 47.5 ± 6.4 46.9 ± 8.0
Serum triglyceride (mg/dL) 59.2 ± 8.3 57.5 ± 13.3 54.9 ± 9.2 43.9 ± 12.2 a
Serum glucose (mg/dL) 130.3 ± 27.2 102.6 ± 34.7 99.9 ± 26.5 a 93.2 ± 13.1 a
Liver weight (g) 1.4 ± 0.2 1.3 ± 0.1 1.3 ± 0.2 1.4 ± 0.1
Fat weight (g) 4.7 ± 1.3 4.1 ± 0.8 a 4.0 ± 1.6 a 3.6 ± 2.0 a
Uterus weight (g) 0.055 ± 0.01 0.062 ± 0.02 0.066 ± 0.02 0.066 ± 0.02
The control group was fed normal diets. The SA10, SA50, and SA100 groups received SA-10, 50, and 100 mg/kg body weight/d containing diets, respectively. Data are presented as mean ± standard error of the mean. OVX, ovariectomized.
aP < 0.05 vs. control group.

TABLE 2 - Comparison of effect of syringic acid (SA) and that of E2 after 12 wks in the Sham (n = 8), OVX-control (n = 8), OVX-SA (n = 8), and OVX-E2 (n = 8) groups
Sham OVX OVX-SA OVX-E2
Initial body weight (g) 28.5 ± 2.0 30.0 ± 2.4 29.9 ± 2.4 30.6 ± 2.9
Final body weight (g) 33.6 ± 2.7 43.4 ± 4.6 43.8 ± 3.4 35.3 ± 4.8 a
Serum triglyceride (mg/dL) 46.3 ± 4.5 63.6 ± 8.4 51.7 ± 6.0 a 50.9 ± 6.4 a
Serum glucose (mg/dL) 99.5 ± 14.3 138.2 ± 21.0 101.7 ± 15.3 a 102.0 ± 10.2 a
Liver weight (g) 1.5 ± 0.2 1.6 ± 0.2 1.6 ± 0.3 1.5 ± 0.3
Fat weight (g) 2.0 ± 0.4 3.8 ± 0.8 2.6 ± 0.9 a 2.3 ± 0.7 a
Uterus weight (g) 1.171 ± 0.466 0.039 ± 0.01 0.037 ± 0.015 0.071 ± 0.026 a
The Sham, OVX-control, and OVX-E2 groups were fed normal diets. The OVX-SA group received SA 100 mg/kg body weight/d containing diets. The mice of OVX-E2 group were injected with 17β-estradiol, while the mice of other groups were injected vehicle from abdominal cavity. Data are presented as mean ± standard error of the mean.
aP < 0.05 vs. control group.

Effects of SA diet on serum parameters

To further examine the effects of the SA diet on OVX mice, serum parameters (triglyceride, total cholesterol, and HDL- and LDL-cholesterol levels) were measured (Table 3). Serum triglyceride levels in mince in the OVX-SA group were significantly lower than those in mice in the OVX-control group. However, SA exerted no effect on Sham mice in this respect. Additionally, the SA diet dose-dependently (0-100 mg/kg body weight) brought about a decrease in serum triglyceride levels in OVX mice (Table 1). However, it did not alter serum levels of total, HDL-, and LDL-cholesterol in Sham or OVX mice. Further, the evaluation of the effect of the SA diet on serum glucose and insulin levels indicated that the SA diet significantly decreased serum glucose concentrations in OVX mice (Table 1); however, this parameter did not differ between the Sham-control and Sham-SA groups. Conversely, neither Sham nor OVX mice exhibited differences in insulin levels owing to dietary SA. This notwithstanding, the SA diet dose-dependently (0-100 mg/kg body weight) brought about a decrease in serum glucose levels (Table 1), suggesting that SA exerts beneficial effects on serum glucose levels without affecting serum insulin concentrations. Moreover, the effects of SA diets on serum glucose and triglyceride levels were similar to those of the E2 treatment (Table 2).

TABLE 3 - Effect of dietary syringic acid (SA) on serum parameters after 12 weeks in the Sham-control (n = 7), Sham-SA (n = 8), OVX-control (n = 7), and OVX-SA (n = 8) groups
Sham-control Sham-SA OVX OVX-SA
Triglyceride (mg/dL) 50.0 ± 3.5 52.5 ± 3.9 64.0 ± 4.0 47.9 ± 4.1 a
Total cholesterol (mg/dL) 133.7 ± 12.8 135.3 ± 11.0 198.1 ± 5.2 212.5 ± 10.1
HDL-cholesterol (mg/dL) 69.4 ± 9.0 59.8 ± 6.6 102.8 ± 7.2 114.5 ± 5.7
LDL-cholesterol (mg/dL) 58.6 ± 4.6 65.1 ± 6.9 80.1 ± 6.9 88.4 ± 6.4
Fasting glucose (mg/dL) 115 ± 6.5 101.6 ± 7.9 152.9 ± 14.0 104.5 ± 11.8 a
Insulin (ng/mL) 0.7 ± 0.04 0.8 ± 0.06 1.2 ± 0.18 1.2 ± 0.12
The control group was fed normal diets. The Sham-SA and OVX-SA groups received SA 100 mg/kg body weight/d containing diets. Data are presented as mean ± standard error of the mean.HDL, high-density lipoprotein; LDL, low-density lipoprotein; OVX, ovariectomized; Sham, sham-operated.
aP < 0.05 vs. control group.

Effects of SA diet on liver biology in OVX mice

To evaluate the effects of ovariectomy and the SA diet on the liver, hepatic tissues were stained and examined. The micrographs of these tissues are shown in Figure 3A, from which it is evident that a greater number of lipid droplets were observed in the hepatic tissue of OVX-control mice than in those of Sham-control mice. It was also observed that the SA diet led to a decreased presence of lipid droplets in OVX mice, even though the number of lipid droplets did not evidently vary between Sham-control and Sham-SA mice. Further, given that histological analysis indicated that the SA diet mitigated steatohepatitis in OVX mice, we next measured the levels of liver lipids (triglycerides and total cholesterol), and it was observed that dietary SA resulted in significantly lower liver triglyceride levels than the control treatment among OVX mice (14.1 ± 1.2 vs. 10.3 ± 0.7 mg/g liver, P < 0.05), but not Sham mice (Fig. 3B). Furthermore, the results indicated that the SA diet had no effect on total liver cholesterol levels in either Sham or OVX mice (Fig. 3C). The subsequent measurement of serum AST and ALT activity, as markers of liver inflammation, indicated no significant differences between the levels of these activities in the control and SA-fed groups (Fig. 3D, E). These results suggested that SA restricts the development of a fatty liver, without affecting cholesterol metabolism or inducing liver inflammation.

FIG. 3
FIG. 3:
Effect of dietary syringic acid (SA) on liver biology of mice in the Sham-control (n = 7), Sham-SA (100 mg/kg body weight/d, n = 8), OVX-control (n = 7), and OVX-SA (100 mg/kg body weight/d, n = 8) groups. (A) Liver tissue fixed in 3.7% formaldehyde solution using phosphate-buffered saline and stained with hematoxylin and eosin. Scale bar, 100 μm. (B) Triglyceride and (C) total cholesterol levels in liver of Sham and OVX mice. (D) Aspartate aminotransferase (AST) and (E) Alanine transaminase (ALT) activity based on serum analysis. Each value represents mean ± SE; P < 0.05. OVX, ovariectomized; Sham, sham-operated.

Effects of SA diet on serum adipocytokine levels in OVX mice

The investigation of the effects of the SA diet on serum adipocytokine (adiponectin, leptin, and resistin) levels showed that serum adiponectin concentrations were significantly higher in the OVX-SA group than the OVX-control group (7.7 ± 0.3 vs. 9.5 ± 0.6 μg/mL, P < 0.05, Fig. 4A). However, no significant differences were observed between the Sham-control and Sham-SA groups in this regard. Further, it was observed that the SA diet did not alter serum leptin and resistin levels in Sham or OVX mice (Fig. 4B, C). This implies that SA influences adiponectin secretion without altering the secretion of leptin and resistin, and possibly enhances lipid metabolism in OVX mice.

FIG. 4
FIG. 4:
Effect of dietary syringic acid (SA) on serum (A) Adiponectin, (B) Leptin, and (C) Resistin levels in Sham-control (n = 7), Sham-SA (100 mg/kg body weight/d, n = 8), OVX-control (n = 7), and OVX-SA (100 mg/kg body weight/d, n = 8) mice. Each value represents the mean ± SE; P < 0.05. OVX, ovariectomized; Sham, sham-operated.

Effects of SA diet on glucose tolerance in OVX mice

The results of glucose tolerance tests, which were performed to examine whether glucose absorption was altered by the SA diet, are shown in Figure 5 (time-dependent changes in serum glucose levels in OVX mice). From this figure, it is evident that in control mice on the control diet, the oral administration of glucose increased blood glucose levels by approximately 250 mg/dL from baseline, and the glucose levels decreased gradually until 120 minutes. Conversely, the SA diet significantly improved glucose tolerance in mice; serum glucose levels were significantly lower in SA-fed mice than in control mice at 60 and 90 minutes.

FIG. 5
FIG. 5:
Time-dependent changes in postprandial blood glucose levels after syringic acid (SA) intake for 12 weeks in ovariectomized (OVX)-control (n = 10) and OVX-SA (100 mg/kg body weight, n = 10) mice. After an overnight fast (18 h), a glucose solution (1,000 mg/kg body weight) was administered orally and blood samples were collected from the tail vein at the indicated time-points. Data are presented as mean ± SD. P < 0.05 vs. OVX-control group.

Identification of SA and SA metabolites in mice blood

As described above, we demonstrated that the SA diet reduces fat mass, serum parameters (triglyceride and glucose levels), and serum adiponectin levels in OVX mice. To clarify the potential mechanism, we examined the translocation forms of the orally administered SA in murine blood. From Table 4, which shows the translocation forms of SA in serum estimated from the molecular weights recorded 30 minutes after SA administration, the predominant translocation form of SA after 30 minutes of the oral administration was the sulfate conjugate (48.6%, area value ratio). Intact SA was identified as the second most predominant translocation form of SA in serum 30 minutes after administration (12.7%), followed by the glucuronate conjugate (12.6%), the glycine conjugate (6.9%), and other minor conjugates.

TABLE 4 - Deduced main compound in murine blood 30 minutes after administration of syringic acid (SA)
Deduced compound m/z Area value ratio (%)
M 197.05 12.7
M+SO3 277.15 48.6
M+C6H8O6 373.11 12.6
M-OH+C2H4NO2 254.07 6.9
M-C2H4 169.03 4.2
M+O 213.05 3.2
M-CH2 183.04 3.2
M, hydrogen donor of SA.

Next, we observed that the amount of intact SA that was transferred into blood as intact SA was predominant. Further, changes in serum SA levels after a single oral SA administration (100 mg/kg body weight) were examined to determine the time at which the concentration of SA was maximum (Tmax), the maximum SA concentration (Cmax), and the half-life (T1/2) of SA in serum. The time-dependent changes in serum SA levels are shown in Figure 6, from which it is evident that the Tmax of SA in serum was 30 minutes (Cmax: 346 ± 27 μg/mL). Further, the Cmax of SA was determined to be approximately 25% of the initially administered SA, suggesting a high SA absorption ratio in mice. After Tmax, the serum SA levels showed a time-dependent decrease, and finally were below the detection limit 720 minutes after administration. Furthermore, the T1/2 of SA in serum was determined to be 72 minutes. These findings suggested that SA administered orally is rapidly absorbed into blood circulation, and excreted via urine in mice.

FIG. 6
FIG. 6:
Time-dependent changes of serum syringic acid (SA) levels after a single oral administration of SA (100 mg/kg body weight) in mice. Blood samples were collected from the tail vein at 0, 30, 60, 90, 180, 360, and 720 minutes after a single oral administration of SA. Data are presented as mean ± SD (n = 5).

Effects of SA diet on major organ weight and anatomy in OVX mice

There were no significant differences in relative heart, lung, liver, spleen, or kidney weights between the study groups (Fig. 7A). However, a significant decrease in uterine weight was observed in OVX mice (Fig. 7B), ie, the SA diet did not bring about increases in uterine weight (Fig. 7A, B). Conversely, E2 treatment resulted in a recovery of the uterine weight (Table 2), suggesting that in this study, SA and E2 exerted different effects on uterine weight.

FIG. 7
FIG. 7:
Effect of dietary syringic acid (SA) on: (A) organ weights, (B) uteri, and (C) anatomy at 12 weeks in the Sham-control (n = 7), Sham-SA (100 mg/kg body weight/d, n = 8), OVX-control (n = 7), and OVX-SA (100 mg/kg body weight/d, n = 8) groups. Data are presented as mean ± SE; P < 0.05 vs. Sham-control group. OVX, ovariectomized; Sham, sham-operated.

DISCUSSION

Decreases in female hormone secretion increase insulin resistance.4,5 Therefore, in this study, we used OVX mice as a model of postmenopausal obesity and diabetes. Ovariectomy resulted in significant increases in weight gain, adipose tissue mass, and serum glucose levels compared with sham surgery, as previously described.8,30,31 Further, dietary SA significantly decreased serum glucose levels without affecting serum insulin concentrations. Moreover, the relative fat mass (visceral, subcutaneous, and total fat) in OVX mice fed with SA was lower than that in OVX-control mice, indicating that the SA diet attenuated the postmenopausal obesity phenotype by reducing serum glucose levels.

In recent years, the importance of preventive medicine, which is aimed at maintaining the body in a state of good health such that it is less likely to get sick, rather than curing it after getting sick, has been emphasized. In this study, the effects of SA on fat mass and triglycerides levels were weaker than those of E2. Even though it may be less effective as a therapeutic agent than E2, it can be expected that the daily consumption of SA present in foods from a normal diet can lead to the prevention of postmenopausal diabetes.

It can be considered that the SA diet regulates lipid metabolism in OVX mice given that it exerts estrogen-like effects; however, since it has no affinity for ER-α or ER-β, this possibility may be excluded.18 In our previous study, we reported that SA does not support the proliferation of MCF-7 cells, which express ERs and show E2-dependent growth.32 Further, there were no significant changes in uterine weight in the SA-fed mice compared with the control mice. In contrast, in this study, it was observed that E2 treatment resulted in a recovery of uterine weight. These results indicated that the actions of SA in OVX mice are different from those of E2. For estrogenic activity in vivo, a detailed assessment of the effects of SA on endometrial epithelial cell proliferation is needed.

Adiponectin decreases blood glucose levels and regulates lipid metabolism.33 Further, leptin, which exerts a hypophagic effect,34 is secreted from adipocytes in response to lipid accumulation,35 and reportedly, resistin regulates glucose metabolism as well as insulin sensitivity.36 While SA showed no effect on leptin and resistin concentrations, the SA diet increased serum adiponectin levels in OVX mice. Hotta et al37 demonstrated a relationship between decreased plasma adiponectin levels and the development of insulin resistance in type 2 diabetes. Therefore, increased adiponectin production by adipose tissue may constitute one of the mechanisms by which SA decreases serum glucose levels in OVX mice. Furthermore, in this study, it was observed that liver triglyceride levels were lower in OVX-SA mice than in OVX-control mice. It has been observed that high-glucose conditions stimulate the expression of genes that are involved in fatty acid synthesis in human hepatoma HepG2 cells,38 and the liberated fatty acids accumulate as triglycerides in the liver.39,40 Therefore, improved insulin resistance and reduced blood glucose levels can bring about a decrease in liver triglyceride synthesis.

The results of previous studies have also suggested that SA attenuates high-fat diet-induced obesity by improving insulin resistance in mice.24 Thus, in this study, we performed glucose tolerance tests to examine whether glucose uptake was altered owing to improved insulin resistance resulting from the consumption of the SA diet. The test results indicated that the SA diet significantly improved glucose tolerance. Mice blood glucose levels did not change when glucose (1,000 mg/kg body weight) was orally administered 30 minutes after the oral administration of SA (data not shown). This improvement in glucose resistance supports the suggestion that SA possibly mitigates insulin resistance.

The dysregulation of the expression of adipokines caused by adipocyte hypertrophy and dysfunction has been linked to chronic inflammation.41 Hypertrophic adipocytes secrete various pro-inflammatory cytokines, including TNFα, IFNγ, and IL-6, which cause various inflammatory symptoms in vivo.41 In the previous study, it was observed that the SA diet resulted in a reversal of the elevated serum TNFα, IFNγ, IL-6, and MCP-1 levels in high-fat diet-induced obese mice.24 Possibly, these anti-inflammatory effects of SA were also exerted in the adipose tissue of OVX mice. Further, when total RNA was extracted from the adipose tissue of SA-fed OVX mice and RNA-Seq was performed, a significant decrease in the expression of genes that are involved in anti-inflammatory action was observed (will be published). These findings notwithstanding, further studies are needed to elucidate the mechanism of the effects of SA on the adipose tissue of SA-fed mice.

The SA diet did not change relative heart, lung, liver, spleen, or kidney weights in OVX mice, suggesting that it could suppress fat accumulation without affecting other tissues. Further, the gross anatomy of the visceral organs in each mouse was also examined as a measure of the safety of the SA treatment. No abnormalities were observed; both Sham-SA and OVX-SA mice exhibited no abnormal behaviors or signs of adverse effects. Further, no changes in serum levels of thiobarbituric acid-reactive substances or markers of oxidative stress were observed owing to SA treatment (data not shown). These findings suggest that a diet containing 100 mg/kg/d SA safely decreases fat mass in OVX mice.

Potential clinical value

The current study indicates that dietary SA decreases fat mass without affecting the uterus or weights of other major organs in OVX mice. Even though further studies are necessary, SA has potential for use in the prevention of postmenopausal obesity and type 2 diabetes.

Limitations

This study had some limitations. For post-hoc analyses, we used multiple comparison correction for each parameter to perform comparison across groups. Overall, several parameters were compared; this presents the possibility of performing a different type of multiple comparison. Additionally, the number of mice in each group was pretty small. This might have prevented the identification of modest differences, and given that several parameters were evaluated, it is possible that the significant findings resulted from chance.

Furthermore, the study findings might be strengthened using aged mice rather than young 10-week-old OVX mice. Experiments with OVX mice older than 10 weeks and naturally aged female mice will be conducted in the future.

CONCLUSION

In conclusion, we demonstrated that dietary SA decreases fat mass without affecting the uterus or the weights of other major organs in OVX mice. Therefore, SA may be suitable for use in the prevention of postmenopausal obesity and diabetes. However, further studies are needed before it can be applied as a preventive agent in postmenopausal obesity and type 2 diabetes.

Acknowledgments

The authors thank the students of the Applied Cell Biology Laboratory at the Department of Applied Biological Chemistry, Graduate School of Agriculture, Kindai University for their support during the experiment.

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Keywords:

Diabetes; Lipid metabolism; Menopause; Syringic acid

© 2021 by The North American Menopause Society