Obesity and overweight have dramatically increased worldwide. In 2008, 35% of the worldwide population was overweight, while 11% was obese. It has been estimated that obesity will affect one-third of the population in 2030. Due to the high incidence and severity of obesity and its related disorders, it is highly desirable to develop new strategies to treat or even to prevent its development. The consumption of high-fat diets is directly involved in the obesity pathogenesis since it affects either central control of food intake and peripheral metabolism, resulting in increased body weight gain, insulin resistance, and other metabolic disturbances. Insulin resistance is a chronic condition in which the hormone insulin fails to activate its own signaling cascade, resulting in hyperglycemia. It has been highly correlated to visceral adiposity excess and increased white adipose tissue (WAT) expression of cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Most hypoglycemiants present undesirable side effects. It is highly desirable to discover new drugs and treatment methods due to the severity of insulin resistance progression. Ginkgo biloba extract (GbE) might have positive effects on hyperglycemia.
Ginkgo Biloba: A Potential Ally in Weight Management
Ginkgo biloba (G. biloba), commonly known as ginkgo, brings considerable benefit to common medicine, including weight loss effects, as well as antidiabetic, antihypertensive, and antilipidemic properties that could be effective in the treatment of Metabolic syndrome (MetS) associated with increased risk of cardiovascular disease events. Major compounds of G. biloba are terpene lactones (bilobalide and ginkgolides A, B, and C) and flavone glycosides (isorhamnetin, quercetin, and kaempferol).
We have previously described that prolonged GbE treatment significantly reduced food intake and body adiposity, prevented against hyperglycemia and dyslipidemia, while it increased insulin sensitivity evaluated by ITT (insulin tolerance test) in obese rats fed with lard-enriched hyperlipidic diet. In agreement to our previous findings, other studies proposed that GbE intake improved glycaemic profile of both healthy and T2D patients. The data above suggest beneficial effects of GbE on insulin resistance and obesity-related disorders. However, it is highly important to better describe the mechanisms by which GbE improves insulin action. In this context, the present study aimed to evaluate if a 14-day oral GbE treatment alters retroperitoneal WAT depot insulin and Toll-like receptors signaling cascades of diet-induced obese rats, a model of insulin resistance.
Animal Studies: Exploring the Effects of GbE on Obese Rats
Study Design and Methods
All procedures for the care of the animals used in this study were approved by The Committee on Animal Research Ethics of the Universidade Federal de São Paulo. All efforts were made to minimize suffering. 2-month-old rats were fed a highly fat-enriched diet which was prepared by adding 40% (w/w) standard chow plus 28% (w/w) lard, 2% (w/w) soy oil, 10% (w/w) sucrose, 20% (w/w) casein, in order to obtain the protein content of the control diet, and butylated hydroxytoluene in the amount of 0.02% (w/w) of the additional oil. This provided 19.5% of energy as carbohydrate, 23.2% as protein, and 57.3% as fat. After 8 weeks, animals were divided into two groups, according to the phytotherapy treatment described below.
Phytotherapy treatment was performed for a 14-day period. The obese animals were divided in two groups: O+V (Obese + Vehicle) and O+Gb (Obese + Ginkgo biloba). The O+Gb group was daily gavaged with 500 mg/kg of GbE diluted in 1 mL of 0.9% saline (vehicle) while the O+V was gavaged with 1 mL of vehicle.
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During the phytotherapy treatment period, 24-h food intake and body weight were daily measured. Body weight gain was calculated by the difference between final weight (last day of treatment) and initial weight (first day of treatment). Accumulated food and energy intakes were measured by the mean of the first 13 days of treatment. In the last day of treatment rats were kept overnight fasted.
Rats were anesthetized with sodium thiopental (80 mg/Kg of body wt, intraperitoneal) and decapitated after a 10-hour fasting period. Retroperitoneal white adipose tissue depot was removed and homogenized in 1.0 mL of lysis buffer (100 mM Tris, pH 7.5, 10 mM EDTA, and 0,1 mg/mL aprotinin; 2 mM PMSF; 10 mM sodium orthovanadate; 100 mM sodium fluoride; 10 mM sodium pyrophosphate; and 10% TritonX-100). Portal vein blood was also collected for the measurement of adiponectin by Milliplex MAP (Millipore).
Rats were deeply anesthetized with sodium thiopental (80 mg/Kg of body wt, intraperitoneal). The abdominal cavity was opened and negative control samples (O+V− and O+Gb−) were obtained from the left side retroperitoneal fat depot. After the collection, samples were immediately inserted into a vial containing 3.0 mL of lysis buffer (100 mM Tris, pH 7.5, 10 mM EDTA, and 0.1 mg/mL aprotinin; 2 mM PMSF; 10 mM sodium orthovanadate; 100 mM sodium fluoride; 10 mM sodium pyrophosphate; and 10% TritonX-100), homogenized, and centrifuged at 16000 g for 40 minutes at 4°C. Then, the portal vein was exposed and 10−5 M of insulin was injected intravenously (i.v.). Right side retroperitoneal fat depot was removed 90 seconds after the i.v. insulin injection (positive samples: O+V+ and O+Gb+) following the same protocol described above. To reduce the risk of nonspecific antibody binding, we evaluated the IR phosphorylation levels after immunoprecipitation with antibody against IR. To perform immunoprecipitation experiments, samples were overnight incubated with 10 µL primary antibody anti-IR (insulin Rβ sc-711) and proteins were precipitated by Protein A Sepharose (GE). After all, proteins were separated on 10% SDS-PAGE. Proteins were then transferred to nitrocellulose membranes by wet transfer apparatus (Bio-Rad). The membranes were preincubated for 1 hour in blocking buffer (5% bovine serum albumin [BSA], 1 M Tris, pH 7.5, 5 M NaCl, and 0.02% Tween-20). Membranes were overnight incubated at 4°C with the primary antibody against p-Tyr (Cell Signaling 8954). All membranes were then incubated with specific horseradish peroxidase-conjugated anti-rabbit IgG antibody (Cell Signaling 7074) followed by chemiluminescence detection (Amersham Biosciences). Since all samples were immunoprecipitated with IR antibody, we considered that bands with molecular weight of 95 kDa were related to the phosphorylated form of IR. To perform the total extract experiments, after the protein quantification, total proteins were then separated on 8% SDS-PAGE. All membranes were overnight incubated at 4°C with the primary antibody against phospho-Akt (Cell Signaling Ser 473-9271); Akt (Cell Signaling 9272), phospho-NF-κB p65 (Cell Signaling Ser 536-3033), NF-κB p65 (Cell Signaling 6956), MyD88 (Cell Signaling 4283), TLR4 (SC 293072), and β-tubulin (Cell Signaling 2146). All membranes were then incubated with specific horseradish peroxidase-conjugated anti mouse/rabbit IgG antibody (Cell Signaling 7076; Cell Signaling 7074, resp.) followed by chemiluminescence detection (Amersham Biosciences). Quantitative analysis was performed with Scion Image software (Scion Corporation, Frederick, MD, USA). In all experiments, at least one sample from each group was analyzed simultaneously and the results were expressed as percentage change relative to the basal levels.
In order to evaluate the gene expression of Adipo R1, Adipo R2, and IL-10, additional groups (O+V and O+Gb) of five rats each were performed. For total RNA extraction, two hundred mg of frozen retroperitoneal adipose tissue from each sample were homogenized by adding 1 mL of Trizol reagent (Invitrogen, USA). The samples were centrifuged at 16.000 g for 15 min at 4°C and the aqueous phase was removed and mixed with 0.5 mL of isopropyl alcohol. One microgram of RNA was reverse transcribed to cDNA using the High-Capacity cDNA kit (Applied Biosystems). Gene expression was evaluated by real-time qPCR using the Taqman PCR Assays. Reactions were performed in 96-well plates and carried out in triplicate. Amplification conditions consisted of 40 cycles of 50°C/2 min, 95°C/10 min, 95°C/15 s, and 60°C/1 min. The method 2−ΔΔCt was used to evaluate the relative quantification of amplification products.
Statistical analysis was performed using PASW Statistics version 19 (SPSS Inc, Chicago, IL, USA) with the level of statistical significance set at P < 0.05. Comparisons among two groups were performed by Student's t test.
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Key Findings
The GbE treatment promoted a significant reduction on both food/energy intake and body weight gain in comparison to the nontreated obese rats. In addition, a significant increase of both Adipo R1 and IL-10 gene expressions and IR and Akt phosphorylation was also observed, while NF-κB p65 phosphorylation and TNF-α levels were significantly reduced.
Accumulated food intake during the first 13 days of phytotherapy treatment is illustrated in Figure 1(a). It is interesting to note that O+Gb group ingested 6.3% less than O+V group (P = 0.031). The effect of GbE on body weight gain is presented in Figure 1(c). It can be seen that the O+Gb group had a significant reduction of 62% (P = 0.013) in comparison to O+V group.
A decrease of 36% (P = 0.014) on TNF-α was observed in the O+Gb in comparison to the O+V group. It can be observed in Figures 2(a) and 2(c) that the GbE treatment promoted a significant increase on gene expression of both Adipo R1 (33%; P = 0.013) and IL-10 (70%; P = 0.040), in comparison to the O+V group.
In relation to adiponectin serum levels, no differences were observed among O+V group (14.74 ± 0.92 µg/mL) and O+Gb group (12.96 ± 1.16 µg/mL).
In Figure 3(a) it can be observed that insulin-induced IR phosphorylation (O+V+) was impaired by the ingestion of high-fat diet, since no differences were observed in relation to basal levels (O+V−). Figure 4 illustrates that Akt phosphorylation was also stimulated by the GbE treatment.
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It can be seen in Figure 5 that GbE treatment did not modify the total protein levels of TLR4, MyD88, and NF-κB p65 (P = 0.900; P = 0.982; P = 0.163, resp.) in retroperitoneal fat depot.
Interpretation of Results
It has been considered that prolonged fat intake is the main predisposing risk factor for the development of obesity. High fat intake also impairs insulin action by reducing glucose uptake and both IR and Akt phosphorylation in brown and white adipose tissues. In our previous study it was demonstrated that prolonged treatment with GbE promoted a significant visceral adiposity loss, improvement of insulin sensitivity, reduction of dyslipidemia, and stimulation of insulin signaling cascade in gastrocnemius muscle. Similar to our previous study, the present data has demonstrated that GbE treatment significantly has decreased food/energy intake and, in addition, it has also reduced the body weight gain of diet-induced obese rats. Data on literature are scarce to demonstrate such effect. However, some studies demonstrated a potent anti-inflammatory effect of GbE especially via reduction of LPS-induced inflammatory cytokines or inhibition of the Toll-like receptors pathway. Since obesity is related to hypothalamic inflammation, it is possible that the treatment with GbE might have promoted a positive anti-inflammatory effect on hypothalamus, increasing anorexigenic peptides levels and/or reducing the orexigenic ones resulting in appetite suppression and weight loss. In the nontreated obese group insulin failed to stimulate the phosphorylation of both IR and Akt in retroperitoneal fat depot indicating that high fat intake impairs insulin signaling. Interestingly, in the obese group treated with GbE, the phosphorylation of both IR and Akt was significantly increased by 281% and 67%, respectively. Previous study of our laboratory showed that GbE improved insulin sensitivity evaluated by the insulin tolerance test while it did significantly improve insulin-induced Akt phosphorylation and IRS-1 levels with a concomitant reduction on PTP-1B levels in gastrocnemius muscle. In addition, other studies have shown that GbE reduces glycaemia and improves glucose intolerance. It is well described that adiponectin-an adipokyne expressed inversely to body adiposity-improves insulin signaling and reduces inflammation especially via Adipo R1 receptor. We failed to demonstrate an effect of GbE on the adiponectin serum levels. However, the present study has demonstrated a significant increase on the adiponectin receptor, Adipo R1, gene expression in retroperitoneal fat depot while no effect was observed on the Adipo R2, indicating that GbE might improve the signaling of adiponectin.
Ginkgo Biloba and Menopause-Related Weight Gain and Depression
Although menopause is recognized as a natural and physiological state, its consequences deserve attention and an adequate care. Particularly, menopause-related obesity and depression are considered important health problems, which affect negatively women’s quality of life. Women have been shown to have a twofold higher risk of developing depressive or anxious disorders in comparison to men. It has been estimated that, mostly due to the hormonal fluctuations of menopause, at least 20% of women will present some depressive or anxious symptoms. Depressive like-behaviours have been demonstrated in ovariectomized rodents, which have also exhibited alterations involved in depression, such as decrement of serotonergic activity and elevation of hippocampal levels of inflammatory mediators.
Obesity has been related to depression, physical inactivity and worsening of menopause troubles. A link between obesity-associated inflammation and depressive symptoms has recently been proposed. Excessive body weight and increased adiposity result in hypertrophy, lysis and necrosis of adipose tissue leading to the recruitment of macrophages and the secretion of pro-inflammatory factors. In a study with obese and non-obese individuals diagnosed with major depressive disorder (MDD), the boost of some cytokines such as TNF-alpha and C-reactive protein (CRP) seemed to be partially dependent on body mass index (BMI).
There is evidence that estrogen reduction might be involved in depression-related neuroinflammation. Ovariectomized rats have exhibited depressive-like behaviours associated with high hippocampal levels of interferon-γ, IL-6, Toll-like receptor-4, and pNF-κBp65, while serotonin levels were decreased. Moreover, important risk factors for obesity-associated cardiovascular diseases, including dyslipidaemia and leptin/adiponectin imbalance, have been reported in obese post-menopausal women and ovariectomized animals. In overweight middle-aged women leptin resistance has been associated with impairment of mental health. This agrees with the demonstrations of antidepressant and anxiolytic effects of the hormone administered to control rodents and to the behavioural impairment reported in animal models of leptin resistance. It is thus likely that leptin resistance may be associated with depression and anxiety in obese people, although the participation of leptin in mental disorders is not fully established in humans.
Adiponectin has also been associated with depression and anxiety disorders. A meta-analysis has concluded that depressive patients have lower serum adiponectin levels in comparison to healthy subjects. Obese people tend to present hypoadiponectinemia, which has been associated with an excessive production of proinflammatory cytokines, contributing to the neuroinflammation.
Data from our laboratory have demonstrated that ovariectomized rats developed obesity, transient hyperphagia, and impairment of the anorexigenic response to serotonin, as well as reduction of serotonin extracellular levels in the medial hypothalamus. The treatment with Ginkgo biloba extract (GbE) for 14 days decreased food and energy intake and restored serotonin hypophagia. In addition, GbE enhanced serotonin levels in the medial hypothalamus and reduced hypothalamic serotonin transporter density. These results led us to suggest a potential therapeutic action of GbE on the regulation of food intake in post-menopausal women, preventing excessive weight gain and ameliorating serotonin hypophagia.
Studying diet-induced obese rats, we have demonstrated both anti-oxidant and anti-inflammatory roles for GbE, along with improvement of insulin signaling in gastrocnemius muscle and retroperitoneal adipose tissue. GbE also had an anti-obesogenic effect and improved the lipid profile. Furthermore, GbE has been shown to modulate short and long-term memory and it has been indicated as an alternative treatment for psychiatric disorders such as anxiety, depression and schizophrenia. These data show that GbE represents a promising therapeutic agent for the treatment of the metabolic and psychological changes associated with menopause. Thus, the present study aimed at further investigating the effect of GbE therapy on both the obesity and the depressive/anxious-like behaviours induced by ovariectomy.
Impact of GbE on Anxiety and Depression in Ovariectomized Rats
Figure 1 illustrates the number of entries (1A), distance travelled (1B), and percentage of time spent (1C) in open and closed arms and in the centre platform of the elevated maze, as measured in the EPM test, as well as the calculated anxiety index (1D). No differences among the groups were observed neither in the entries in the open [F(3,38) = 2.060, p = 0.123] and in the closed arms [F(3,41) = 1.200, p = 0.323], nor in the distance travelled in the open [F (3,39) = 0.958, p = 0.423] and in the closed arms [F(3,38) = 0.329, p = 0.805]. Concerning the percentage of time spent in the open arms [F(3,40) = 4.212, p = 0.012], the OVX + GbE rats had a 333% increase in comparison to the OVX rats (p = 0.007). The OVX rats spent less time in the centre platform [F(3,40) = 7.270, p = 0.001] in comparison to the Sham (− 56%, p = 0.012) and the OVX + GbE (− 63%, p < 0.0001) groups. In addition, OVX animals presented the highest percentage of time spent in the closed arms [F(3,40) = 16.806, p < 0.0001] when compared to Sham (59%, p < 0.0001), Sham + GbE (60%, p < 0.0001) and OVX + GbE (143.5%, p < 0.0001) animals. Regarding the anxiety index [F(3,39) = 5.087, p = 0.005], the OVX rats showed an index higher than that of the Sham + GbE rats (15%, p = 0.048) while the OVX + GbE group presented a lower index in comparison to the OVX group (− 17%, p = 0.004).
The grooming (2A), rearing (2B), and head-dipping (2C) events are shown in Fig. 2. There was a significant elevation of the number of head-dipping occurrences [F(3,41) = 4.814, p = 0.006] in the OVX + GbE group in comparison to the OVX group (235%, p = 0.004). No differences were observed in grooming [F(3,40) = 0.580, p = 0.632] and rearing [F(3,40) = 0.825, p = 0.489] events among the groups.
The results obtained in the Modified Forced Swim test are shown in Fig. 3. The OVX group demonstrated a lower swimming frequency [F(3,37) = 7.777, p < 0.0001] than that of the Sham + GbE rats (43%, p < 0.0001). The OVX + GbE animals showed a non-significant increment of 42% (p = 0.075) in swimming frequency in relation to the OVX rats (Fig. 3A). Concerning the climbing frequency [F(3,38) = 2.540, p = 0.072], represented in Fig. 3B, the OVX group presented a strong tendency of a 43% reduction in relation to the Sham + GbE group (p = 0.059). The OVX group presented a higher immobility frequency (Fig. 3C) [F(3,37) = 10.125, p < 0.0001], in comparison to the Sham (117.5%, p = 0.001) and the Sham + GbE (132%, p < 0.0001) groups and a lower latency to immobility (Fig. 3D) [F(3,38) = 7.423, p = 0.001] than those of the Sham (57%, p = 0.004) and the Sham + GbE (58%, p = 0.001) groups. The OVX + GbE rats showed a reduction of immobility frequency (− 48%, p = 0.002) as well as an increase in the latency to immobility (148%, p = 0.003), in comparison to the OVX group. The number of diving events was similar among the groups (Fig. 3E) [F(3,37) = 0.958, p = 0.433].
GbE Improves Body Composition in Ovariectomized Rats
The effectiveness of ovariectomy was confirmed by the significant uterus atrophy of OVX and OVX + GbE rats (F(3,46) = 128.772, p < 0.0001) when compared to both Sham (p < 0.0001) and Sham + GbE (p < 0.0001) rats (Fig. 4A). Figure 4B depicts the elevated retroperitoneal fat pad [F(3,45) = 13.395, p < 0.0001] presented by the OVX group, in relation to both the Sham (65%; p < 0.0001) and the Sham + GbE (72%; p < 0.0001) groups. GbE restored retroperitoneal adipose tissue mass of OVX + GbE, promoting a reduction of 21% (p = 0.033), in comparison to OVX rats. No differences were observed in relation to mesenteric [F(3,48) = 1.674, p = 0.186] and gonadal [F(3,46) = 1.194, p = 0.323] fat pads among the groups. A significant elevation of the sum of adipose tissues masses was observed in the OVX group [F(3,45) = 6.459, p = 0.001] in relation to the Sham (33%, p = 0.006) and the Sham + GbE (41%, p = 0.001) groups. The OVX group exhibited 64% (p = 0.006) and 42% (p = 0.018) increases of carcass fat content [F (3,21) = 6.552, p = 0.003], in comparison to the Sham and the Sham + GbE groups, respectively (Fig. 4C). Considering carcass protein content (Fig. 4D), the OVX + GbE group had an increment [F(3,21) = 4.263, p = 0.019] of 26% (p = 0.034) and 25.5% (p = 0.022), in comparison to the Sham and the OVX groups, respectively. At the 14th day of the phytotherapy treatment, OVX rats tended to present a higher body weight [F(3,21) = 3.046, p = 0.041] in relation to Sham + GbE group (p = 0.057), but GbE did not restore this parameter in the OVX + GbE group (Fig. 4E).
Hormonal and Lipid Profile Improvements with GbE
The measured serum parameters are illustrated in Table 1. No significant differences among the groups were observed in glycemia [F(3,57) = 1.148, p = 0.338], fasting insulin levels [F(3,43) = 0.678, p = 0.571], HOMA-IR [F(3,44) = 0.417, p = 0.742], HOMA-β [F(3,42) = 0.570, p = 0.638] and TNF-α levels [F(3,44) = 0.076, p = 0.972]. However, leptin levels [F(3,44) = 4.109, p = 0.012] were 139% higher in the OVX group in comparison to the Sham + GbE group (p = 0.006). Additionally, the OVX + GbE group presented an increment of 115% on adiponectin levels [F(3,43) = 3.234, p = 0.032] in relation to the Sham group (p = 0.043).
In relation to the lipid profile (Fig. 5A-E), it can be noted that the OVX group presented hypercholesterolemia [F(3,47) = 5.288, p = 0.003], as evidenced by an increase of 40.5% (p = 0.002) on total cholesterol levels when compared to the Sham group as well as an elevation of 92% (p = 0.015) and 57% (p = 0.044) on LDL-Cholesterol levels [F(3,51) = 3.956, p = 0.013], in comparison to the Sham and the Sham + GbE groups, respectively. The treatment with GbE increased HDL-Cholesterol levels of the OVX + GbE group [F(3,55) = 5.875, p = 0.002], by 75% (p = 0.001) in relation to the Sham group, and by 43% (p = 0.028) in relation to the OVX group. Ovariectomy caused a decrement [F(3,54) = 7.944, p < 0.0001] of 31.3% of triacylglycerol levels, as compared to the Sham group (p = 0.004). The GbE therapy failed to modify this effect, leading to triacylglycerol levels 39% (p < 0.0001) and 26% (p = 0.046) lower than those of Sham and Sham + GbE animals, respectively. NEFA concentrations were 23.5% and 20.5% lower in the OVX + GbE [F(3,55) = 4.439, p = 0.007] group in comparison to those in the Sham + GbE (p = 0.006) and the OVX (p = 0.045) groups, respectively.
Correlations Between Body Composition, Hormones, and Behavior
The following parameters were included in the correlation analysis: body weight, retroperitoneal/mesenteric/gonadal adipose tissues masses, sum of fat depots masses, carcass fat and protein contents, all measured serum parameters and all measured parameters of the behavioural tests. Table 2 shows the variables with at least one significant correlation.
Considering depressive associated-behaviours evaluated by the forced swimming test, climbing events were negatively correlated with body weight (p = 0.026), retroperitoneal adipose tissue (p = 0.024), sum of fat depots (p = 0.001), and carcass fat (p = 0.014). The immobility frequency exhibited a positive correlation with retroperitoneal adipose tissue mass (p = 0.004), sum of fat depots masses (p = 0.044) and carcass fat (p = 0.023). The latency to immobility correlated negatively with body weight (p = 0.027), mesenteric adipose tissue (p = 0.031), sum of fat depots (p = 0.014), carcass fat (p = 0.010) and positively with uterus mass (p = 0.039).
In relation to anxiety related-behaviours evaluated in elevated plus maze, the anxiety index was negatively correlated with HDL-cholesterol levels (p = 0.025). The number of entries (p = 0.046), the distance travelled (p = 0.049) and the percentage of time spent in the open arms (p = 0.049) as well as the number of head dipping events (p = 0.047) were negatively correlated with retroperitoneal adipose tissue mass. In addition, the number of entries in the open arms was positively correlated with uterus mass (p = 0.031). The permanence in the open arms also showed a negative correlation with total cholesterol levels (p = 0.024). The percentage of time spent in the closed arms was positively correlated with leptin levels (p = 0.023) while the distance travelled in the closed arms demonstrated a positive correlation with body weight (p = 0.047).
Ginkgo Biloba in the Context of Metabolic Syndrome
This review indicated that G. biloba might be efficient in the improvement of MetS; however, more studies especially clinical trials are needed to evaluate safety and efficacy of G.