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. 2017 Nov 11;24(2):98–107. doi: 10.1111/cns.12776

Ginsenoside Rb1 improves leptin sensitivity in the prefrontal cortex in obese mice

Yizhen Wu 1, Xu‐Feng Huang 1,2,✉, Christopher Bell 1, Yinghua Yu 1,2,✉
PMCID: PMC6490040  PMID: 29130652

Summary

Aim

Obesity impairs leptin‐induced regulation of brain‐derived neurotrophic factor (BDNF) expression and synaptogenesis, which has been considered to be associated with the incidence of neuronal degenerative diseases, cognitive decline, and depression. Ginsenoside Rb1 (Rb1), a major bioactive component of ginseng, is known to have an antiobesity effect and improve cognition. This study examined whether Rb1 can improve central leptin effects on BDNF expression and synaptogenesis in the prefrontal cortex during obesity using an in vivo and an in vitro model.

Result

Ginsenoside Rb1 (Rb1) chronic treatment improved central leptin sensitivity, leptin‐JAK2‐STAT3 signaling, and leptin‐induced regulation of BDNF expression in the prefrontal cortex of high‐fat diet‐induced obese mice. In cultured prefrontal cortical neurons, palmitic acid, the saturated fat, impaired leptin‐induced BDNF expression, reduced the immunoreactivity and mRNA expression of synaptic proteins, and impaired leptin‐induced neurite outgrowth and synaptogenesis. Importantly, Rb1 significantly prevented these pernicious effects induced by palmitic acid.

Conclusion

These results indicate that Rb1 reverses central leptin resistance and improves leptin‐BDNF‐neurite outgrowth and synaptogenesis in the prefrontal cortical neurons. Thus, Rb1 supplementation may be a beneficial avenue to treat obesity‐associated neurodegenerative disorders.

Keywords: BDNF, ginsenoside Rb1, leptin, neurite outgrowth, prefrontal cortex, synaptogenesis

1. INTRODUCTION

Obesity increases the incidence of neuronal degenerative diseases, cognitive decline, and depression,1, 2, 3 which have been linked to a high intake of dietary fat.4, 5 In humans, an increased intake of dietary fat results in cognitive impairment in later life4 and is strongly associated with cognitive decline in women with type 2 diabetes.5 The prefrontal cortex is a major regulatory center for cognitive function.6 Impairment in the prefrontal cortex has been recognized as a feature of obesity.7 Positron emission tomography shows low postprandial activation in the dorsolateral prefrontal cortex in obese individuals.7, 8 Furthermore, an increased body mass has been linked to cognitive deficits and decreased prefrontal cortical activity.9

Brain‐derived neurotrophic factor (BDNF) promotes neurite outgrowth (ie, elongation and branching) and synaptogenesis, as well as the regulation of energy balance.10, 11 Aberrant BDNF signaling is associated with obesity, cognitive impairment, and other neurodegenerative diseases.12, 13, 14 Furthermore, leptin, secreted by adipocytes, elevates BDNF expression in the hypothalamus15 and brainstem,16 promoting negative energy balance. Leptin increases hippocampal BDNF in normal mice, but not in diet‐induced obese mice with depressive‐like behavior,3 suggesting central leptin resistance in the obese mice. Therefore, these evidences suggest that leptin‐induced activation of BDNF in the central nervous system may be involved in cognition and synaptogenesis, while brain leptin resistance could contribute to obesity‐related neurodegenerative diseases.

Ginseng, a traditional herbal medicine, is reported to be an adaptogen that enhances body stability against physical loads, and is also suggested to improve cognitive functions.17 Extracts of ginseng promote neurite outgrowth of spinal cord neurons.18 Ginsenoside Rb1 (Rb1) is one of the major bioactive saponins in ginseng.18, 19 Recent animal and cell models show that Rb1 has neuroprotective18, 20, 21, 22 and antiobesity effects.23 For example, Rb1 increases BDNF expression in the forebrain of rats with cerebral ischemia.20 In vitro, Rb1 reduces the neurotoxic effects of glutamate on neurite outgrowth in neurons.18 Our previous study has demonstrated that Rb1 improves leptin signaling in the hypothalamus of obese mice.24 The main aim of this study was to examine whether Rb1 can improve central leptin effects on BDNF expression and synaptogenesis in the prefrontal cortex during obesity using an in vivo and an in vitro model. Firstly, we investigated the effect of Rb1 on leptin signaling in the prefrontal cortex of high‐fat diet‐induced obese mice. Furthermore, we investigated the effect of Rb1 on neurite outgrowth and synaptogenesis in cultured primary prefrontal cortical neurons in response to leptin and palmitic acid, one of the most abundant saturated fatty acids in human diet.

2. MATERIALS AND METHODOLOGY

2.1. Experimental animals and Rb1 treatment

Sixty C57Bl/6J male mice (6 weeks old, body weight: 19.6 ± 1.4 g) were obtained from the Animal Resources Centre (Perth, WA, Australia), and housed in environmentally controlled conditions (temperature 22°C, 12 hour light/dark cycle). All mice were fed a lab chow diet (LC, 5% fat, Vella Stock Feeds, Doonside, NSW, Australia) to acclimatize for 1 week. Throughout the study, LC was served as the low fat control diet and was provided ad libitum except where noted. All procedures were approved by the Animal Ethics Committee, University of Wollongong, NSW, Australia, and complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. After acclimatization, the mice were fed a high‐saturated fat diet (40% fat in calories) for 16 weeks and developed obesity (average body weight 44.2 ± 2.6 g). Subsequently, the obese mice were randomized into two groups and intraperitoneally (ip) administered either ginsenoside Rb1 (Rb1, purity ≥98% [HPLC], purchased from Jilin University in China) at a dose of 14 mg/kg/d24 or vehicle (saline) for 21 days. Body weight and food intake for 24 hours ([weight of food placed in hopper]–[weight of food remaining in hopper]) were monitored every 2 days. Energy intake (Kcal/24 hours) was calculated as [(food intake) × (energy density of food in Kcal/g)]. Average energy intake was calculated as [(total energy intake measured)/(number of measurement)].

2.2. Intraperitoneal glucose tolerance test (IPGTT)

On day 18 of Rb1 treatment, the mice were fasted overnight and injected (ip) with glucose at a dose of 0.5 g/kg. Blood samples were taken from the tail vein, and blood glucose measured using a glucometer (Abbott Diabetes Care, Alameda, CA, USA) at 0 (fasting), 30, 60, and 120 minutes following glucose administration.

2.3. Central leptin sensitivity test

After Rb1 treatment for 21 days, the mice were anesthetized by isoflurane inhalation and placed in a stereotactic device. The mice were implanted with an intracerebroventricular (icv) cannula into the right lateral brain ventricle (0.25 mm posterior and 1.0 mm lateral relative to Bregma and 2.5 mm inferior to the surface of the skull).25 Five days after cannula implantation, the mice were fasted for 6 hours, and then administered with either leptin (0.1 μg/3 μL) or saline (3 μL) into the lateral ventricle through the cannula. Food intake was measured 1 and 4 hours after the leptin or vehicle injection. Body weight gain was recorded 24 hours after icv injection.

2.4. Blood and tissue collection

Following a 4 day interval after measuring central leptin sensitivity, the mice were fasted for 6 hours, and were then given an icv injection of leptin or vehicle. The mice were sacrificed one hour after icv injection. Blood samples were collected in EDTA‐coated tubes. Plasma was collected after centrifugation at 845 g for 15 minutes. Brain tissue was removed and immediately stored in liquid nitrogen. Epididymal fat (left and right sides) was dissected, weighed, and immediately stored in liquid nitrogen. Plasma, brain, and epididymal fat samples were then transferred to a −80°C freezer until further analysis.

2.5. Determination of plasma leptin and insulin

Plasma leptin and insulin were measured using the mouse metabolic magnetic bead panel kit (Merck Millipore, Billerica, MA, USA).

2.6. Western blotting

Prefrontal cortical tissue, equivalent to the prelimbic cortex, was identified and dissected at the level of Bregma 2.8‐1.98 mm according to a standard mouse brain atlas.25 Tissue protein was extracted in an NP‐40 lysis buffer as described in our previous study.26 The antibodies were used as follows: pJAK2 (sc‐21870) and BDNF (sc‐20981) from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA); and pSTAT3 (#9145) from Cell Signaling Technology (Beverly, MA, USA). Bands corresponding to the proteins of interest were scanned, and their density analyzed using the Quantity One automatic imaging analysis system (Bio‐Rad Laboratories, Hercules, CA, USA). All quantitative analyses were normalized to β‐actin, based on our previous study.26

2.7. Primary prefrontal cortical neuronal cultures and treatment

Mouse prefrontal cortical neuronal cultures were prepared from pups at postnatal day 1‐2 (C57Bl/6J mice, Perth, Western Australia) as described previously.27 Dissociated cells were seeded at an approximate final density of 1 × 105 cells/cm2 into a Poly‐D‐Lysine‐coated 24‐well plate (1.9 cm2/well) for qRT‐PCR measurement. For neuronal morphology measurement, the neurons were cultured on Poly‐D‐Lysine‐coated glass coverslips. At 7 days in vitro, Rb1 (40 μmol/L, refer to18) was used to treat the cultures simultaneously in the presence of palmitic acid (final concentration of 10 μmol/L, P5585, Sigma‐Aldrich, St Louis, MO, USA). Palmitic acid was dissolved following the method described previously.28 Four hours after palmitic acid and/or Rb1 pre‐exposure, leptin (100 ng/mL, refer to,29 Cat#: 450‐31, Peprotech, Rocky Hill, NJ, USA) or vehicle was added to the cultures for another 44 hours before the cells were harvested.

2.8. Immunofluorescence

For immunocytochemical staining, neurons were washed three times in phosphate‐buffered saline (PBS), and then fixed with 4% paraformaldehyde (Sigma‐Aldrich) for 30 minutes at room temperature. After rinsing with PBS, the neurons were permeabilized with 0.3% Triton‐X (Sigma‐Aldrich) in PBS (ie, PBST) for 10 minutes, and blocked with 5% goat serum in PBST for 1 hour at room temperature. Then anti‐BDNF antibody, antimicrotubule‐associated protein 2 (MAP2) antibody, antisynaptophysin (SYN) antibody, and antipostsynaptic density protein 95 (PSD 95) antibody were applied overnight at 4°C. MAP2 was visualized by goat anti‐mouse secondary antibody conjugated with Alexa Fluor 594. BDNF, SYN, and PSD95 were visualized with isotype‐specific donkey anti‐rabbit secondary antibody conjugated with Alexa Fluor 488. The details of origin and concentration of antibodies were given in the Table S1. A fluorescence microscope (Axiovert 200, Carl Zeiss, Oberkochen, Germany) with an attached digital camera was used to obtain MAP2 immunofluorescence images for neuronal morphology analysis. A confocal microscope (Leica TCS SP5 Advanced System, Wetzlar, Germany) equipped with a digital camera was used to obtain images for immunoreactivity analysis of BDNF, PSD95, and SYN.

2.9. Neurite length and branching analysis

The quantification of neuronal morphology was carried out using the NeuriteQuant program,30 including the following: average neurite length, neurite length per cell, neurite number per cell, branches per neurite, and branches per cell. Experiments were independently performed three times, with each time repeated in triplicate (n = 9). The image analysis was based on these nine repeats.

2.10. Immunoreactivity analysis for BDNF, PSD95, and SYN

Software Image J 1.40 g was used to quantify the immunoreactivity of BDNF, PSD95, and SYN. Images were converted into a 16‐bit scale for analysis. The mean intensity of the fluorescence of these biomarkers in the cell body was measured. Approximately, 1‐2 areas (around 10 × 10 μm2 per area) of the cell body per neuron were analyzed, and 7‐8 neurons per image were randomly selected. A range of 6‐7 images per group from three independent experiments was analyzed. For PSD95 and SYN, dendrite immunoreactivity was also analyzed. In each dendrite, 2‐4 areas (around 10 × 10 μm2 per area) were measured, and 2‐4 dendrites per neuron were analyzed.

2.11. RNA extraction and qRT‐PCR

Total RNA was extracted from the cultured cells using Aurum total RNA mini kits (Bio‐Rad Laboratories). Complementary DNA synthesis and quantitative real‐time PCR (qPCR) were performed as previously described.24 The primers used are listed in Table S2. Amplification was carried out with 45 cycles of: 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The mRNA expression levels for MAP2, PSD95, SYN, and BDNF were normalized to γ‐actin, which served as the internal control. Experiments were performed in triplicate.

2.12. Statistical analysis

Data were analyzed using the SPSS 19 statistical package (SPSS, Chicago, IL, USA). One‐way analysis of variance (ANOVA) followed by the post hoc Tukey‐Kramer honestly significant difference (HSD) test was used to analyze the body weight change, energy intake, epididymal fat, glucose tolerance test and leptin signaling of the LC, obese, and Rb1 treatment groups in response to icv leptin/saline, as well as biomarkers of the in vitro study and neuronal morphology data. A P‐value <0.05 was considered statistically significant. Data are presented as mean ± SEM.

3. RESULTS

3.1. Ginsenoside Rb1 reduced energy intake, prevented weight gain, fat deposition, and improved glucose tolerance in obese mice

As shown in Figure 1A, high‐saturated fat diet‐induced obese mice showed higher average energy intake than LC lean mice, while Rb1 treatment decreased the average energy intake. Meanwhile, Rb1 treatment significantly reduced body weight gain in obese mice from day 9 to the end of the treatment (Figure 1B). Ginsenoside Rb1 (Rb1) also reduced the epididymal fat mass in obese mice, although it was still higher than LC mice (Figure 1C). In the IPGTT, the Rb1‐treated group showed lower blood glucose at 30 and 60 minutes after glucose injection compared to the obese group without treatment (Figure 1D). However, at 0, 30, and 120 minutes, blood glucose in Rb1 group was still higher than the LC group.

Figure 1.

Figure 1

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Chronic Rb1 prevented weight gain, fat deposition, and improved glucose tolerance in obese mice fed a high‐saturated fat diet for 16 wk. Ginsenoside Rb1 (Rb1) treatment reduced average energy intake (A); prevented body weight gain (B) since day 9; reduced epididymal fat mass (C); as well as lowered blood glucose at time point 30 min and 60 min in the glucose tolerance test (D). LC, lab chow fed mice; Ob, high‐saturated fat diet‐induced obese mice without treatment; Ob + Rb1, high‐saturated fat diet‐induced obese mice chronic treated with ginsenoside Rb1. Data are presented as mean ± SEM (n = 6‐8). *P < 0.05 vs LC, # P < 0.05 vs Ob

3.2. Ginsenoside Rb1 restored central leptin sensitivity and significantly reduced hyperleptinemia in obese mice

In the LC lean mice, leptin significantly reduced energy intake for 4 hours after the icv leptin injection (all P < 0.05, Table S3). However, in the obese mice, the central leptin injection failed to suppress energy intake (all P > 0.05). Importantly, in the Rb1 treatment group, leptin significantly decreased food intake for 4 hours compared with saline in the obese group ([Ob + Rb1]/leptin vs [Ob + Rb1]/Saline, P < 0.05), suggesting Rb1 improved central leptin sensitivity in the obese mice.

Plasma leptin was significantly higher in the obese mice compared with the LC mice (Table S3). Ginsenoside Rb1 (Rb1) treatment lowered plasma leptin in the obese mice (Ob/saline: 16.9 ± 2.0 ng/mL, (Ob + Rb1)/saline: 8.9 ± 1.1 ng/mL, P < 0.05), but icv leptin injection did not further decrease plasma leptin.

3.3. Ginsenoside Rb1 improved leptin‐JAK2‐STAT3 signaling and leptin‐induced increase in BDNF in the prefrontal cortex of obese mice

We examined leptin‐JAK2‐STAT3 signaling in the prefrontal cortex of obese mice induced by high‐saturated fat diet with and without Rb1 treatment. Western blot showed that central leptin administration evoked pJAK2 (+40%, P < 0.05) in the prefrontal cortex of the LC lean mice, but not the obese mice (P > 0.05, Figure 2A). Importantly, after Rb1 treatment, leptin administration significantly increased pJAK2 compared with saline (+28%, P < 0.05) in the obese mice. There was a similar response in the pSTAT3, downstream step of leptin/pJAK2 signaling in the LC, obese, and Rb1‐treated obese mice (Figure 2B). Leptin significantly increased pSTAT3 by 68% in the prefrontal cortex of LC mice, while in the obese mice pSTAT3 only increased by 28% after the leptin injection. Following chronic Rb1 treatment, pSTAT3 increased by 81% in response to leptin compared with the saline injection, suggesting that Rb1 improved leptin‐pJAK2‐pSTAT3 signaling in the obese mice. We also determined the leptin‐stimulated expression of BDNF in the prefrontal cortex. Results showed that in the LC group, leptin significantly increased BDNF expression in the prefrontal cortex by +41% (Figure 2C), but not in the obese mice. Chronic Rb1 treatment not only increased BDNF expression in the prefrontal cortex, but also facilitated leptin's action in increasing BDNF expression (P < 0.05).

Figure 2.

Figure 2

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Chronic Rb1 treatment improved leptin‐pJAK2‐pSTAT3 signaling and leptin‐mediated brain‐derived neurotrophic factor (BDNF) expression in the prefrontal cortex of obese mice fed a high‐saturated fat diet for 16 wk. Protein expression level of pJAK2 (A), pSTAT3 (B), and BDNF (C) responding to icv leptin or saline in the prefrontal cortex in LC lean mice and high‐saturated fat diet‐induced obese mice with or without Rb1 treatment (14 mg/kg, ip) for 3 wk. LC, lab chow fed mice; Ob, high‐saturated fat diet‐induced obese mice without treatment; Ob + Rb1, high‐saturated fat diet‐induced obese mice chronic treated with ginsenoside Rb1. Data are presented as mean ± SEM (n = 7‐9). *P < 0.05 vs LC/saline, # P < 0.05 vs Ob/saline, + P < 0.05 vs (Ob+Rb1)/saline

3.4. Ginsenoside Rb1 increased leptin‐induced BDNF expression in the prefrontal cortical neurons

Using cultured primary prefrontal cortical neurons, the effect of Rb1 on the leptin‐stimulated BDNF expression under the circumstance of saturated fatty acid, palmitic acid exposure was examined. Leptin treatment significantly increased BNDF immunoreactivity by 65% (P < 0.001, Figure 3A,B). However, in neurons pretreated with palmitic acid, leptin failed to increase BDNF immunoreactivity (P > 0.05). Importantly, with Rb1 treatment, leptin increased BDNF immunoreactivity by 40% (P = 0.020) in palmitic acid pretreated neurons, suggesting that Rb1 is able to prevent palmitic acid‐induced leptin insensitivity. Consistent with the immunocytochemistry, a similar pattern of BDNF mRNA expression was observed (Figure 3C).

Figure 3.

Figure 3

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Ginsenoside Rb1 (Rb1) increased leptin‐induced brain‐derived neurotrophic factor (BDNF) immunoreactivity and mRNA expression in the prefrontal cortical neurons pretreated by palmitic acid (PA). A‐B, immunocytochemistry revealed that palmitic acid significantly reduced the BDNF immunoreactivity response to leptin, while Rb1 treatment reversed this reduction. A, Immunocytochemistry images taken by confocal microscope show the typical BDNF‐containing vesicles (indicated by white arrowheads). Upper: images after staining with anti‐BDNF antibody (green), scale bar = 10 μm. Bottom: images with double staining of anti‐BDNF (green) and anti‐MAP2 (red) antibodies. B, Quantification of BDNF immunoreactivity: the mean intensity of BDNF immunoreactivity of neurons was measured using Image J Software (n = 5‐6, ie, 5‐6 images obtained from three independent experiments). C, Rb1 reversed the palmitic acid‐induced reduction in leptin‐stimulated BDNF mRNA expression (n = 5‐7), measured with qRT‐PCR. Data are presented as mean ± SEM. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001

3.5. Palmitic acid reduced leptin‐stimulated neurite outgrowth in the prefrontal cortical neurons, which can be reversed by Rb1 treatment

Following examination of BDNF, we examined neurite outgrowth, including neurite branching and length, in cultured primary cortical neurons (Figure 4). Leptin treatment significantly increased neurite branching (branches per neurite: +77%, P < 0.001, Figure 4D; branches per cell: +80%, P < 0.001, Figure 4E) compared to the saline treatment in cortical neurons. However, palmitic acid pretreatment significantly suppressed leptin‐stimulated branching. Ginsenoside Rb1 (Rb1) significantly ameliorated palmitic acid‐induced impairment in leptin‐stimulated branching compared with the palmitic acid pretreatment (branches per neurite: +45%, P = 0.018, Figure 4D; branches per cell: +56%, P = 0.001, Figure 4E).

Figure 4.

Figure 4

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Ginsenoside Rb1 (Rb1) ameliorated palmitic acid (PA)‐induced impairment of leptin‐mediated neurite elongation and branching in the prefrontal cortical neurons. A, Cultured cortical neurons stained with MAP2 used for neuronal analysis; B, Neuronal analysis tracing generated by the NeuriteQuant toolkit. C, Represented MAP2 immunofluorescence staining images in cortical neurons; All parameters (D‐H) were automatically analyzed and calculated using the NeuriteQuant (n = 9); (D) Branches per neurite; (E) Branches per cell; (F) Neurite number per cell; (G) Average neurite length; (H) Neurite length per cell; (I) MAP2 mRNA expression measured by qRT‐PCR (n = 5‐7). Scale bar = 100 μm. Data are presented as mean ± SEM. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001

Ginsenoside Rb1 (Rb1) significantly reversed the palmitic acid‐induced decrease in average neurite length and total neurite length per cell (P = 0.009 and P = 0.045, respectively, Figure 4G,H). Firstly, leptin significantly increased average neurite length and total neurite length per cell (both P < 0.001) compared to the saline control group. However, after exposing cortical neurons to palmitic acid, leptin did not increase the average neurite length nor the total neurite length per cell (both P > 0.05). Ginsenoside Rb1 (Rb1) reversed this impairment, evidenced by the leptin increased average neurite length by 37% (P = 0.003, Figure 4G) and total neurite length per cell by 44% (P = 0.001, Figure 4H) in palmitic acid and Rb1 co‐treated cells compared to palmitic acid pretreated neurons alone. The mRNA expression of MAP2, an important marker of neurite outgrowth, was subsequently measured by qRT‐PCR. Ginsenoside Rb1 (Rb1) prevented palmitic acid from inhibiting the leptin‐induced stimulation of MAP2 mRNA (Figure 4I).

3.6. Palmitic acid reduced leptin‐mediated synaptogenesis in the prefrontal cortical neurons, which can be reversed by Rb1 treatment

We examined the effect of Rb1, in the presence of palmitic acid, on the postsynaptic density marker 95 (PSD95) in response to leptin in cortical neurons (Figure 5A,B, and Table S4). Leptin significantly increased PSD95 immunoreactivity (P < 0.001 both in dendrite and soma, Figure 5A,B) and mRNA expression (P < 0.001, Table S4) compared to the saline group. However, palmitic acid pretreatment decreased the leptin‐elevated PSD95 immunoreactivity (P < 0.001 both in dendrite and soma, Figure 5A,B) and mRNA expression (P = 0.007, Table S4). Importantly, Rb1 corrected the alteration of leptin‐stimulated PSD95 immunoreactivity (dendrite: P = 0.006; and soma: P < 0.001, Figure 5A,B) and mRNA expression (+26%, P = 0.009, Table S4) in the palmitic acid pretreated cortical neurons.

Figure 5.

Figure 5

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Ginsenoside Rb1 (Rb1) attenuated palmitic acid (PA)‐induced impairment of leptin‐mediated postsynaptic density marker 95 (PSD95) immunoreactivity in the prefrontal cortical neurons. A, Amplified represented immunofluorescence images of PSD95 in the cortical neurons, left column: control neurons without (upper panel) or with leptin (lower panel); middle column: PA‐exposure neurons without or with leptin; Right column: Rb1‐treated PA‐exposure neurons without or with leptin; scale bar = 25 μm, taken by confocal microscope (Leica, objective 63*1.4L oil). B, Quantification of PSD95 immunoreactivity: mean intensity of PSD95 fluorescence in dendrite and soma of cortical neurons were measured using Image J Software. A total of 6‐7 images per group and 7‐8 neurons per image, as well as 2‐4 dendrites per neuron were applied to the analysis. Data are presented as mean ± SEM (n = 6‐7, ie, 6‐7 images per group obtained from three independent experiments). *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001

We also examined the effects of Rb1, in the presence of palmitic acid, on the presynaptic marker, SYN, in response to leptin in cortical neurons (Figure 6A,B, and Table S4). The palmitic acid pretreatment decreased the leptin‐elevated SYN immunoreactivity in both dendrite and soma (both P < 0.001, Figure 6A,B) and mRNA expression (P = 0.004, Table S4). The Rb1 treatment ameliorated palmitic acid‐induced impairment on leptin‐stimulated SYN immunoreactivity in the dendrite (+55%, P = 0.011), but not in the soma (P > 0.05) of cortical neurons (Figure 6A,B).

Figure 6.

Figure 6

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Ginsenoside Rb1 (Rb1) attenuated palmitic acid (PA)‐induced impairment of leptin‐mediated presynaptic marker (synaptophysin, SYN) immunoreactivity in the prefrontal cortical neurons. A, Amplified represented immunofluorescence images of SYN in the cortical neurons, left column: control neurons without (upper panel) or with leptin (lower panel); middle column: PA‐exposure neurons without or with leptin; Right column: Rb1‐treated PA‐exposure neurons without or with leptin; scale bar = 25 μm, taken by confocal microscope (Leica, objective 63*1.4L oil). B, Quantification of SYN immunoreactivity: mean intensity of SYN fluorescence in dendrite and soma of cortical neurons were measured using Image J Software. A total of 6‐7 images per group and 7‐8 neurons per image, as well as 2‐4 dendrites per neuron were applied to the analysis. Data are presented as mean ± SEM (n = 6‐7, ie, 6‐7 images per group obtained from three independent experiments). *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001

4. DISCUSSION

This study showed that Rb1 can reverse the adverse effects of high‐saturated fat on leptin sensitivity in the prefrontal cortex. Chronic Rb1 treatment ameliorated hyperleptinemia, and improved leptin signaling via JAK2‐STAT3 and leptin‐induced BDNF expression in the prefrontal cortex of obese mice. Moreover, in the cultured primary prefrontal cortical neurons, palmitic acid impaired the effect of leptin on BDNF, neurite outgrowth, and synaptogenesis markers. Ginsenoside Rb1 (Rb1) treatment improved the leptin‐mediated neurite outgrowth (branching and length) and synaptic proteins (SYN and PSD95) in palmitic acid pretreated prefrontal cortical neurons.

In the present study, we found that leptin activates JAK2‐STAT3 signaling in the prefrontal cortex in LC lean mice. The prefrontal cortex is important for cognitive control,31 and high‐fat diet reduces synaptic plasticity in this region32 leading to learning and memory impairments.33 Furthermore, in the present study, we found that leptin‐JAK2‐STAT3 signaling was substantially impaired in the prefrontal cortex of the obese mice. In obese individuals, central leptin insensitivity also exists. The prevalence of dementia and other neurodegenerative diseases was higher in obese subjects with hyperleptinemia,34, 35, 36 which suggests that high plasma leptin levels unlikely activate signaling pathways in the brain of obese individuals. Therefore, central leptin resistance is a pathology of obesity.37 Importantly, in the present study, Rb1 treatment improved impairments of the leptin‐pJAK2‐pSTAT3 signaling pathway in the prefrontal cortex, restored central leptin sensitivity and reduced hyperleptinemia in obese mice. Previously, both clinical and animal studies have shown that ginseng extract or Rb1 treatment improves cognitive function.17, 38, 39, 40 Therefore, these findings, including ours, suggest that Rb1, in improving the leptin‐JAK2‐STAT3 pathway in the prefrontal cortex, may contribute to ginseng and Rb1 in improving cognition.

The present study found that leptin elevates BDNF in the prefrontal cortex, as illustrated by protein expression level in the in vivo study, and immunochemistry and mRNA in the in vitro study. High‐fat diet or palmitic acid altered the ability of leptin to elevate BDNF in the prefrontal cortex, suggesting that a high‐saturated fat diet can affect leptin function in prefrontal cortical neurons. In accordance with our results, the impairment of central leptin‐regulated BDNF has been demonstrated in obese mice, which is associated with depressive behavior.3 Moreover, mutation of the BDNF gene is associated with increasing early‐onset obesity and cognitive impairment in BDNF‐haploinsufficient patients.41 Brain‐derived neurotrophic factor (BDNF) in the prefrontal cortex promotes neuronal plasticity and neurogenesis, which are important for learning and memory.42, 43 Thus, the impairment of leptin‐regulated BDNF in the prefrontal cortex by saturated fat may play an important role in central nervous system dysfunction in obese subjects. Along with improving the leptin‐pJAK2‐pSTAT3 signaling pathway, our results demonstrated a beneficial effect of Rb1 on the upregulation of BDNF by leptin. These results suggest that Rb1‐induced activation of leptin signaling may modulate BDNF expression, enhancing neuronal plasticity in the prefrontal cortex.

In this study, we demonstrated the beneficial effect of leptin on neurite outgrowth and synaptogenesis in cultured primary prefrontal cortical neurons. We found leptin increases neurite branching and length, neurite marker MAP2 mRNA expression, and synaptic marker (PSD95 and SYN) immunoreactivity and mRNA expression. It has been reported that the expression of synaptogenesis markers, SYN and PSD95, depends on BDNF processing.44, 45 It is known that leptin signaling molecules, JAK2 and STAT3, distribute around the postsynaptic sites in the cerebral cortex.46 Therefore, leptin may promote neurogenesis and synaptic plasticity via activation of leptin signaling molecules and increasing BDNF, SYN, and PSD95 in cortical neurons. Furthermore, we found that high‐saturated fatty acid diet impaired leptin signaling JAK2 and STAT3 in the prefrontal cortex. We confirmed that saturated palmitic acid impaired leptin's action on neurite outgrowth and synaptogenesis in cultured prefrontal cortical neurons. These findings suggest that saturated fatty acids may impair leptin signaling and induce a leptin insensitivity effect on neurite outgrowth and synaptogenesis in the prefrontal cortical neurons. Importantly, our results demonstrated that this impairment induced by palmitic acid was prevented by the Rb1 treatment. Especially, Rb1 ameliorated palmitic acid‐induced impairment on leptin‐stimulated SYN immunoreactivity in the dendrite but not in the soma. It is possible that Rb1 may interact with the leptin receptor in the presynaptic dendrites of neurons and activate synaptophysin expression, although this requires further investigation. Clinical data show that ginseng treatment for 24 weeks improves cognitive function in patients with Alzheimer's disease.17 Overall, these findings suggest that Rb1 treatment in improving leptin‐regulated BDNF and synaptogenesis via JAK2‐STAT3 signaling may, at least partly, contribute to ginseng improvements in cognitive function.

Ginsenoside Rb1 belongs to steroidal saponins, which share structure features with steroid hormones.47 Due to the steroid‐like structure and the amphiphilic nature,47, 48 ginsenosides can intercalate into the plasma membrane replacing membrane cholesterol, which increases membrane fluidity and changes the immediate environment of cell membrane proteins, such as the GABA receptor.47, 48 Furthermore, ginsenoside may traverse cell membranes freely and activate intracellular membrane receptors, such as steroid receptors, which may regulate the transcription of target genes.47 This study demonstrated that Rb1 increased leptin sensitivity in the prefrontal cortex of obese mice, and in prefrontal cortical neurons treated with palmitic acid, as well as reversed the alterations of leptin downstream signaling molecules (pJAK2, STAT3, BDNF, and PSD95). However, it remains to be determined if Rb1 directly interacts with the leptin receptor. Furthermore, it is reported that hyperleptinemia is required for the development of leptin resistance in diet‐induced obese mice.49 In our study, Rb1 significantly attenuates hyperleptinemia in obese mice, which may reduce the overstimulation of the leptin receptor, thereby improving downstream signaling.

In summary, this study has shown ginsenoside Rb1 treatment ameliorates alteration of leptin‐pJAK2‐pSTAT3 and leptin‐BDNF in the prefrontal cortex of obese mice and improves hyperleptinemia. Treatment with Rb1 also promoted leptin's effect on neurite branching and elongation, and synaptogenesis in prefrontal cortical neurons. As high‐fat diet‐induced obesity has been implicated in the progression of neurodegenerative diseases, such as vascular dementia, Rb1 treatment may have therapeutic effects in attenuating the progression of cognitive decline in obese patients and reducing the risk of neurodegenerative diseases. This will be the next work to further validate using obese mice model.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

 

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ACKNOWLEDGMENTS

The authors wish to thank Ms Licai Cheng for her assistance in the animal work and Ms Linda Cohen for her editorial revision of the manuscript.

Wu Y, Huang X‐F, Bell C, Yu Y. Ginsenoside Rb1 improves leptin sensitivity in the prefrontal cortex in obese mice. CNS Neurosci Ther. 2018;24:98–107. 10.1111/cns.12776

Funding information

This work was supported by Diabetes Australia Research Trust Research Projects to Dr Yinghua Yu and the Schizophrenia Research Institute, utilizing infrastructure funding from NSW Health. YHY is supported by the National Health and Medical Research Council of Australia (NHMRC 573441) and the Schizophrenia Research Institute.

Contributor Information

Xu‐Feng Huang, Email: xhuang@uow.edu.au.

Yinghua Yu, Email: yinghua@uow.edu.au.

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