Ginsenoside Rf improves glucose metabolism via the IRS/PI3K/Akt and PPARα/PGC1α signaling pathways in insulin-resistant AML12 cells

Abstract

Background

Insulin resistance (IR) is a key component of type 2 diabetes mellitus and other metabolic disorders. Persistent hyperglycemia impairs insulin signaling and mitochondrial function, thereby contributing to IR progression. Ginsenoside Rf (G-Rf), a bioactive component of Panax ginseng (ginseng), possesses various pharmacological properties. However, its role in glucose metabolism under IR conditions remains largely unexplored.

Methods

High-glucose and insulin treatment was used to induce IR in AML12 hepatocytes. G-Rf cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays. Glucose metabolism was evaluated by measuring glucose consumption and Glut2 expression. Western blot analysis and real-time quantitative polymerase chain reaction were used to analyze the key proteins in the IRS1/PI3K/Akt and PPARα/PGC1α signaling pathways. Mitochondrial function was assessed through mitochondrial DNA (mtDNA) quantification and intracellular adenosine triphosphate (ATP) measurements. In parallel, liquid chromatography–mass spectrometry (LC–MS) analysis was conducted to quantify the G-Rf content in mature and sprouted ginseng.

Results

No cytotoxicity was observed at G-Rf concentrations up to 10 µM. G-Rf treatment significantly improved glucose uptake and Glut2 expression in IR-AML12 cells and restored IRS1 phosphorylation, PI3K expression, and activation of downstream Akt/GSK-3β signaling, promoting glycogen synthesis. Moreover, it upregulated PPARα and PGC1α expression and increased mtDNA content and ATP production, indicating improved mitochondrial function. LC–MS/MS analysis revealed that mature and sprouted ginseng contained G-Rf, with higher concentrations in underground parts. Importantly, substantial G-Rf levels were found in the edible aerial parts of sprouted ginseng.

Conclusion

G-Rf improves glucose metabolism in IR hepatocytes by activating the IRS1/PI3K/Akt and PPARα/PGC1α signaling pathways and enhancing mitochondrial function. Thus, G-Rf is a promising candidate for the dietary or therapeutic management of IR. Furthermore, sprouted ginseng may serve as a sustainable source of G-Rf, supporting its application as a functional food.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-025-05091-7.

Introduction

Insulin resistance (IR) is a condition characterized by impaired insulin signaling and reduced glucose uptake in peripheral tissues, resulting in chronic hyperglycemia [1]. IR is the fundamental pathogenic mechanism underlying type 2 diabetes mellitus (T2DM) and metabolic syndrome [2]. Under physiological conditions, elevated postprandial blood glucose levels stimulate insulin secretion from pancreatic β-cells, which promotes glucose uptake and storage in insulin-responsive organs, including the liver, skeletal muscle, and adipose tissue [3]. However, in IR conditions, hepatic insulin signaling is attenuated, leading to insufficient suppression of hepatic gluconeogenesis and compromised glucose clearance [4]. This persistent hyperglycemia exacerbates metabolic dysregulation by promoting de novo lipogenesis, hepatic lipid accumulation, and chronic low-grade inflammation, thereby intensifying IR and contributing to the development of comorbidities, including dyslipidemia and nonalcoholic fatty liver disease [5]. Therefore, early therapeutic interventions aimed at restoring insulin sensitivity are critical to stop the progression of metabolic disorders.

In hepatocytes, insulin facilitates glucose uptake and promotes glucose utilization through storage as glycogen or conversion into lipids while simultaneously inhibiting gluconeogenesis and glycogenolysis [6]. These metabolic effects are predominantly mediated by the insulin receptor substrate (IRS)-dependent signaling cascade [6]. IRS proteins are activated upon insulin binding and subsequently recruit and activate phosphatidylinositol 3-kinase (PI3K), which stimulates the downstream effector protein kinase B (Akt) [7]. Akt plays an important role in suppressing hepatic glucose production by modulating the transcription of key gluconeogenic enzymes [8]. Moreover, Akt promotes hepatic glycogen synthesis by phosphorylating and inactivating glycogen synthase kinase 3 (GSK3), thereby relieving its inhibitory effect on glycogen synthase (GYS) [9]. Because the IRS/PI3K/Akt signaling axis plays an important role in hepatic glucose regulation, pharmacological or nutritional interventions targeting this pathway hold significant promise for enhancing insulin sensitivity and treating metabolic disorders, including T2DM.

Chronic IR is also closely associated with mitochondrial dysfunction in hepatocytes, further aggravating hepatic IR [10]. Mitochondrial impairments not only disrupt insulin signaling pathways but also contribute to β-cell dysfunction, resulting in impaired insulin secretion and reduced insulin-stimulated glucose uptake [11]. Oxidative phosphorylation (OXPHOS), an important process governing mitochondrial energy metabolism, is strongly associated with insulin sensitivity [12]. According to previous studies, OXPHOS activity is significantly decreased under IR conditions, underscoring the potential of mitochondrial-targeted interventions to restore metabolic homeostasis [13]. Thus, enhancing mitochondrial function, particularly adenosine triphosphate (ATP) production, represents a viable strategy for alleviating IR.

Panax ginseng (Ginseng) is a traditional Asian medicinal herb widely known for its pharmacological properties, particularly in metabolic regulation [14]. Its primary bioactive components are ginsenosides—a class of steroidal saponins with diverse therapeutic effects [15]. Among them, ginsenoside Rf (G-Rf), a protopanaxatriol-type saponin, exhibits neuroprotective, anti-allergic, and anti-inflammatory activities [16]. However, its role in hepatic glucose metabolism under IR conditions remains largely unexplored. Therefore, the present study aimed to investigate the potential metabolic benefits of G-Rf in insulin-resistant AML12 (IR-AML12) hepatocytes, which were induced by high-glucose exposure. Specifically, we determined whether G-Rf modulates glucose metabolism by activating the IRS signaling cascade and mitochondrial regulatory pathways, thereby evaluating its potential as a therapeutic candidate for IR-related metabolic diseases, including T2DM.

Materials and methods

Chemical and reagents

G-Rf (Fig. 1A) was purchased from MedChemExpress (#HY-N0601, Monmouth Junction, NJ, USA). Fetal bovine serum (FBS, S001-01) was obtained from Welgene (Gyeongsan, South Korea). Dulbecco’s Modified Eagle Medium/F-12 (DMEM/F12), penicillin–streptomycin solution (10,000 U/mL), and insulin–transferrin–selenium (ITS) solution were purchased from Gibco (Carlsbad, CA, USA). Antibodies against phospho-IRS-1 (Ser1101) (#2385, Cell Signaling, Danvers, MA, USA), IRS-1 (#sc-8038, Santa Cruz Biotechnology, CA, USA), PI3K p110α (#4249, Cell Signaling), phospho-Akt (Ser473) (#4060, Cell Signaling), Akt (#9272, Cell Signaling), phospho-GSK-3β (Ser9) (#5558, Cell Signaling), GSK-3β (#12456, Cell Signaling), phospho-GYS (Ser641) (#3891, Cell Signaling), GYS (#sc-81173, Santa Cruz Biotechnology), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α, #66369-1-Ig, Proteintech, Chicago, IL, USA), peroxisome proliferator-activated receptor α (PPARα, #ab3484, Abcam, Cambridge, MA, USA), α/β-tubulin (#2148, Cell Signaling), and GAPDH (#2118, Cell Signaling) were used for Western blot analysis.

Fig. 1.

Effects of ginsenoside Rf (G-Rf) on cell viability and glucose metabolism in insulin-resistant AML12 (IR-AML12) cells. A Chemical structure of G-Rf. AML12 cells were cultured under IR conditions as described in the Materials and Methods section. Normal and IR-AML12 cells were treated with various concentrations of G-Rf (0–10 µM) for 24 h. B and C The cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium assay. D and E The glucose consumption and glucose uptake was quantified. F Representative fluorescence images of 2-NBDG (green; glucose analogue) and DAPI (blue; nuclei). Scale bars, 20 μm. G Quantitative analysis of 2-NBDG fluorescence intensity. H The messenger RNA (mRNA) expression levels of glucose transporter type 2 (Glut2) were analyzed by real-time quantitative polymerase chain reaction. Data are expressed as mean ± standard deviation (n ≥ 3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s post hoc test. Different letters indicate significant differences (p < 0.05). “ns” denotes nonsignificant differences

Cell experiment

AML12 cells were obtained from the American Type Culture Collection and cultured in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin–streptomycin, 1% ITS solution, and 100 nM dexamethasone. The culture medium was replaced with DMEM/F12 containing 2% FBS to induce IR. After 24 h of incubation, 10 mM D-(+)-glucose (Sigma-Aldrich, St. Louis, MO, USA) and ITS solution were added to the medium and incubated for an additional 24 h, with or without G-Rf treatment, as described in a previous study [17].

Cell viability assay

The cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well. They were exposed to the indicated G-Rf concentrations from 0 μM to 10 μM to evaluate G-Rf cytotoxicity. After 24 h of incubation, the cells were treated with 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide solution (5 mg/mL, Sigma-Aldrich) and then incubated for another 2 h. After removing the medium, the resulting formazan crystals were dissolved in 100 μL of dimethylsulfoxide. The absorbance was measured at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Glucose consumption assays

Glucose concentrations were measured using a Glucose Fluorometric Assay Kit (eEnzyme, Gaithersburg, MD, USA; #CA-G005). Briefly, supernatants were diluted with assay buffer and then mixed with an equal volume of the reaction buffer. The reaction mixtures were incubated at 37 °C for 10 min. Fluorescence intensity was measured using a microplate reader (Molecular Devices) at excitation/emission wavelengths of 540/590 nm.

Glucose uptake assay

Glucose uptake was measured using the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)−2-deoxyglucose (2-NBDG) in conjunction with flow cytometry (Beckman Coulter, Brea, CA, USA). The cells were seeded in six-well plates and cultured for 24 h. After incubation, the cells were treated with the designated experimental conditions, followed by insulin stimulation (10 μg/mL). Subsequently, the cells were incubated with 2-NBDG (100 μM) for 40 min at 37 °C. After incubation, the cells were washed twice with cold serum-free medium to remove unincorporated 2-NBDG, harvested by trypsinization, and resuspended in phosphate-buffered saline containing 2% FBS. The fluorescence intensity of 2-NBDG was analyzed using a flow cytometer (excitation/emission = 488/530 nm). For fluorescence imaging, cells were fixed and counterstained with DAPI for 5 min following 2-NBDG (100 μM) staining. Fluorescence images were captured using a fluorescence microscope, and the fluorescence intensity of 2-NBDG was quantified using ImageJ software (version 1.54d).

Glycogen staining assays

Glycogen staining assay was performed using Periodic acid-Schiff (PAS) staining kit (Abcam, #ab150680). After washing, the cells were fixed with 4% paraformaldehyde and stained with the PAS kit according to the manufacturer’s instructions.

RNA analysis

Total RNA was isolated using the RNeasy Mini Kit (Qiagen Hilden, Germany) in accordance with the manufacturer’s instructions. Total RNA was converted to complementary DNA (cDNA) using a cDNA reverse transcription kit (TOYOBO Co., Ltd., Osaka, Japan). Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted using equal amounts of cDNA, FastStart Universal SYBR Green (Roche, Basel, Switzerland), and primers on the CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The following RT-qPCR primers (forward and reverse) were used: glucose transporter type 2 (Glut2): 5′-GTGTCTGCTACTGCTCTTCTGTC-3′ and 5′-GACATCCTCAGTTCCTCTTAGTCTC-3′; Gys2: 5′-TGGGTCTTTAACTGCCTGGTTC-3′ and 5′-TGTTTACCGTCTGCGTGGTC-3′; Pgc1a: 5′-AAGTGGTGTAGCGACCAATCG-3′ and 5′-AATGAGGGCAATCCGTCTTCA-3′; Pparα: 5′-AGAGCCCCATCTGTCCTCTC-3′ and 5′-ACTGGTAGTCTGCAAAACCAAA-3′; and GAPDH: 5′-AGTATGACTCCACTCACGGCAAAT-3′ and 5′-GTCTCGCTCCTGGAAGATGGT-3′. Relative messenger RNA (mRNA) quantification was performed using the ΔΔCt method and normalized to endogenous control genes, including GAPDH.

Protein analysis

The cells were lysed with lysis buffer (Cell Signaling) containing protease and phosphatase inhibitor cocktails (Roche). After centrifuging the lysate, an equal amount of protein was mixed with 5 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Biosesang, Yongin, Korea), electrophoresed on SDS-PAGE, and transferred to polyvinylidene fluoride or nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After treatment with a blocking buffer (Welgene), the membranes were incubated with the primary antibodies overnight at 4 °C. Following washing with TBST, the membranes were further incubated with the appropriate secondary antibodies for 1 h. The proteins were detected using a chemiluminescent substrate (Thermo Fisher Scientific, Sunnyvale, CA, USA), and densitometric analysis was performed using ImageJ software (version 1.54d).

Mitochondrial DNA (mtDNA) quantification

DNA was isolated from the cells using a DNA purification kit (Bioneer Corp., Dae-jeon, Korea). The ratio of mtDNA to genomic DNA was determined by qPCR. The following RT-qPCR primers (forward and reverse) were used: mitochondrially encoded cytochrome b (mt-Cytb): 5′-ACGCAAACGGAGCCTCAATA-3′ and 5′-TGTGGCTATGACTGCGAACA-3′; mitochondrially encoded NADH dehydrogenase 1 (mt-Nd1): 5′-TCCGAGCATCTTATCCACGC-3′ and 5′-GTATGGTGGTACTCCCGCTG-3′; and nuc-Beta-2-microglobulin (B2m): 5′-TTCTGGTGCTTGTCTCACTGA-3′ and 5′-CAGTATGTTCGGCTTCCCATTC-3′. All primer pairs were run in individual reactions. The final mtDNA/nuclear DNA ratio for each sample was calculated by averaging the ratios obtained from each primer pair. The expression data were analyzed using the 2^−ΔΔCT method.

ATP production assays

Intracellular ATP levels were measured using the ATP Assay Kit (Abcam, #ab83355) in accordance with the manufacturer’s instructions. Briefly, the cells (1 × 10⁴ per well) were seeded in a 96-well plate in complete medium and incubated for 24 h at 37 °C. Following incubation, the culture medium was removed, and the reaction mixture was directly added to each well. Fluorescence was measured within 1 min using a microplate reader at excitation/emission wavelengths of 535/587 nm, respectively. The ATP levels were normalized to the total protein content.

Extraction and quantification of G-Rf from ginseng

One-year-old ginseng (sprouted ginseng) cultivated in Danyang-gun (Chungcheongbuk-do, Republic of Korea) and five-year-old ginseng (mature ginseng) obtained from Geumsan-gun (Chungcheongnam-do, Republic of Korea) were used to extract and quantify G-Rf. Each ginseng sample was separated into aerial parts (leaves and stems) and underground parts (roots) and then subjected to ethanol and hot water extraction. Before extraction, the samples were thoroughly washed and dried at room temperature. For ethanol extraction, 1 g of dried ginseng sample was extracted twice with 70% ethanol (100 g/L) at 50 °C for 3 h under reflux conditions. The combined extracts were filtered, concentrated under reduced pressure using a rotary evaporator, and subsequently freeze-dried to yield powdered extracts. For hot water extraction, 1 g of dried ginseng sample was first pulverized and extracted twice with distilled water at 20 times the sample weight at 50 °C for 3 h under reflux. Thereafter, the aqueous extracts were filtered and freeze-dried. All dried extracts were stored at − 20 °C until further analysis.

To prepare samples for liquid chromatography tandem mass chromatography (LC–MS/MS), 10 mg of each freeze-dried extract was dissolved in methanol and sonicated for 3 h at 40 °C. The resulting solutions were filtered through a 0.22 μm syringe filter and injected into a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA) equipped with an electrospray ionization source operating in the negative ion mode. The capillary voltage was set to 3.5 kV, with the source and desolvation temperatures maintained at 150 °C and 350 °C, respectively. The desolvation and cone gas flow rates were set to 650 and 150 L/h, respectively, whereas the nebulizer gas pressure was maintained at 7.00 bar. Chromatographic separation was performed using a reversed-phase column under a gradient elution system, with mobile phase A comprising 0.1% formic acid in water and mobile phase B comprising 0.1% formic acid in acetonitrile. The flow rate was maintained at 0.6 mL/min. The gradient program was as follows: initial composition of 85% A and 15% B; 0–2.0 min, 65% A/35% B; 2.0–4.0 min, Linear gradient to 55% A/45% B; 4.0–7.0 min, linear gradient to 50% A/50% B; 7.0–7.5 min, linear gradient to 30% A/70% B; 7.5–11.0 min, linear gradient to 0% A/100% B; 11.0–12.5 min, returned to 85% A/15% B; and 12.5–15.0 min, held at the initial conditions for re-equilibration. G-Rf quantification was performed in the multiple reaction monitoring (MRM) mode, monitoring the transition m/z 799.6 [M − H]⁻ → 101.1 for quantification and m/z 799.6 [M − H]⁻ → 475.5 [M − H]⁻ for qualification. Standard solutions of G-Rf at concentrations ranging from 10 µg/L to 4,000 µg/L were used to construct calibration curves. The calibration curve exhibited excellent linearity, with the regression equation y = 2106.01x–2.98987 and a coefficient of determination (R²) of 0.996. All measurements were performed in triplicate. Data acquisition and analysis were performed using the MassLynx software (version 4.1, Waters).

Statistical analysis

Data are presented as the mean ± standard deviation of at least triplicate experiments. Statistical analysis was performed using one-way analysis of variance, followed by Tukey’s post hoc test in Prism (GraphPad Software, La Jolla, CA, USA). Statistical differences are indicated with different letters, and statistical significance was set at p < 0.05.

Results

Effects of G-Rf on AML12 cell viability and glucose metabolism

A cell viability assay was performed in normal AML12 hepatocytes to assess G-Rf cytotoxicity. G-Rf treatment at concentrations up to 10 µM did not significantly affect cell viability compared with the untreated control (Fig. 1B). Subsequently, IR-AML12 cells were established by high-glucose and insulin treatment following serum starvation, as previously described [17]. G-Rf exhibited no cytotoxicity in IR-AML12 cells at concentrations ≤ 10 µM (Fig. 1C).

Next, we investigated whether G-Rf influences IR-AML12 cell glucose metabolism. IR induction resulted in a marked reduction in glucose consumption and Glut2 mRNA expression, indicating impaired glucose uptake (Fig. 1D-H). Notably, G-Rf treatment significantly and dose-dependently increased glucose consumption at concentrations up to 2 µM (Fig. 1D-G). Furthermore, G-Rf treatment significantly reversed the IR-induced downregulation of Glut2 expression (Fig. 1H). These results suggest that G-Rf is a nontoxic compound that can enhance glucose metabolism in IR hepatocytes.

Effects of G-Rf on IRS1/PI3K signaling in IR-AML12 cells

We analyzed the key proteins involved in the IRS1/PI3K signaling cascade to investigate the role of G-Rf in modulating insulin signaling in IR hepatocytes. As expected, IR markedly reduced IRS1 (Ser1101) phosphorylation compared with normal AML12 cells (Fig. 2A–C). G-Rf treatment significantly restored IRS1 phosphorylation and upregulated PI3K expression in a dose-dependent manner in IR-AML12 cells. These results suggest that G-Rf enhances insulin signaling by activating the IRS1/PI3K pathway, thereby potentially improving the glucose metabolic regulation in IR hepatocytes.

Fig. 2.

Effects of ginsenoside Rf (G-Rf) on IRS/PI3K signaling in insulin-resistant (IR) AML12 cells. AML12 cells were cultured under IR conditions as described in the Materials and Methods section. A The protein expression levels of phosphorylated IRS1 (p-IRS1, Ser1101), total IRS1 (t-IRS1), PI3K p100α, and α/β-tubulin were analyzed by Western blot analysis. B and C Quantification of the relative protein expression levels of p-IRS1 (Ser1101)/t-IRS1 and PI3K p110α/α/β-tubulin, presented as bar graphs. Data are expressed as mean ± standard deviation (n ≥ 3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s post hoc test. Different letters indicate statistically significant differences (p < 0.05)

Effects of G-Rf on Akt/GSK-3β signaling in IR-AML12 cells

We examined the downstream components of the IRS/PI3K signaling pathway to further elucidate the mechanisms underlying the metabolic effects of G-Rf. G-Rf treatment significantly increased Akt (Ser473) and GSK-3β (Ser9) phosphorylation levels in a dose-dependent manner in IR-AML12 cells (Fig. 3A, B, and C). Additionally, G-Rf treatment led to a reduction in GYS (glycogen synthase) phosphorylation at Ser641 (Fig. 3D). RT-qPCR analysis also revealed that G-Rf significantly elevated the mRNA expression of Gys2, a key gene involved in glycogen synthesis, under IR conditions (Fig. 3E). Consistently, G-Rf treatment enhanced glycogen accumulation in IR-AML12 cells (Fig. 3F). These findings indicate that G-Rf promotes glycogen synthesis by activating the Akt/GSK-3β signaling axis, thereby contributing to the improvement of hepatic glucose metabolism under IR conditions.

Fig. 3.

Effects of ginsenoside Rf (G-Rf) on Akt/GSK-3β signaling in insulin-resistant (IR) AML12 cells. AML12 cells were cultured under IR conditions as described in the Materials and Methods section. A The protein expression levels of phosphorylated Akt (p-Akt, Ser473), total Akt (t-Akt), phosphorylated GSK-3β (p-GSK-3β, Ser9), total GSK-3β (t-GSK-3β), phosphorylated GYS (p-GYS, Ser641), total GYS (t-GYS), and GAPDH were analyzed by Western blot analysis. B, C and D Quantification of the relative protein expression levels of p-Akt (Ser473)/t-Akt, p-GSK-3β/t-GSK-3β, and p-GYS/t-GYS, presented as bar graphs. E The messenger RNA (mRNA) expression levels of Gys2 were analyzed by real-time quantitative polymerase chain reaction. F Representative images of PAS staining. Scale bars, 50 μm. Data are expressed as mean ± standard deviation (n ≥ 3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s post hoc test. Different letters indicate statistically significant differences (p < 0.05)

Effects of G-Rf on the mitochondrial signaling pathway in IR-AML12 cells

We examined the expression of the key regulators of mitochondrial biogenesis, PPARα and PGC1α, to investigate whether G-Rf modulates mitochondrial function in IR hepatocytes. The mRNA and protein levels of PPARα and PGC1α were significantly reduced under IR conditions (Fig. 4A–E). G-Rf treatment dose-dependently restored the expression of these genes at the transcriptional and protein levels, suggesting enhanced activation of the PPARα/PGC1α signaling axis. Furthermore, we quantified the expression of mtDNA-encoded genes (Cytb and Nd1), which were also significantly upregulated in response to G-Rf treatment in IR-AML12 cells, to assess mitochondrial function (Fig. 5A and B). Following G-Rf treatment, intracellular ATP levels were markedly increased under IR conditions (Fig. 5C). These results collectively suggest that G-Rf enhances mitochondrial quality and function by activating the PPARα/PGC1α pathway, thereby contributing to IR alleviation in hepatocytes.

Fig. 4.

Effects of ginsenoside Rf (G-Rf) on PPARα/PGC1α signaling in insulin-resistant (IR) AML12 cells. AML12 cells were cultured under IR conditions as described in the Materials and Methods section. A and B The messenger RNA (mRNA) expression levels of Pgc1α and Pparα were analyzed by real-time quantitative polymerase chain reaction. C The protein expression levels of PGC1α, PPARα, and GAPDH were analyzed by Western blot analysis. D and E Quantification of the relative protein expression levels of PGC1α/GAPDH and PPARα/GAPDH, presented as bar graphs. Data are presented as mean ± standard deviation (n ≥ 3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s post hoc test. Different letters indicate statistically significant differences (p < 0.05)

Fig. 5.

Effects of ginsenoside Rf (G-Rf) on mitochondrial function in insulin-resistant (IR) AML12 cells. AML12 cells were cultured under IR conditions as described in the Materials and Methods section. A The relative mitochondrial DNA (mtDNA) content was analyzed by real-time quantitative polymerase chain reaction and expressed as the ratio of mtDNA to nuclear DNA. B Intracellular adenosine triphosphate levels were quantified as described in the Materials and Methods section. Data are presented as mean ± standard deviation (n ≥ 3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s post hoc test. Different letters indicate statistically significant differences (p < 0.05)

LC–MS analysis of G-Rf from Panax ginseng

We performed a quantitative analysis using ultra-high-performance LC–MS to evaluate the natural abundance of G-Rf. Hot water and ethanol extracts were prepared from the aerial and underground parts of sprouted and mature ginseng. The standard peak for G-Rf was clearly detected at a retention time of 2.42 min under the MRM mode (Fig. 6A). The quantitative results revealed that the underground parts of ginseng contained higher G-Rf levels compared with the aerial parts, in both extraction methods (Supplementary Fig. 1 and Fig. 6B). Moreover, mature ginseng exhibited significantly greater G-Rf content than sprouted ginseng. These findings demonstrate that G-Rf is naturally present in commonly consumed ginseng preparations, supporting its potential role in mediating the anti-IR effects attributed to ginseng.

Fig. 6.

Quantitative analysis of ginsenoside Rf (G-Rf) from Panax ginseng. A Representative ultra-high-performance liquid chromatogram of ginsenoside Rf using multiple reaction monitoring (MRM) with a transition of m/z 799.6 [M − H]⁻ → 475.5 [M − H]⁻. B Quantification of the ginsenoside Rf content in various ginseng extracts. Data are presented as mean ± standard deviation (n ≥ 3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s post hoc test. Different letters indicate statistically significant differences (p < 0.05)

Discussion

Persistent hyperglycemia not only exacerbates metabolic dysfunction but also promotes systemic inflammation and mitochondrial impairment, further aggravating IR [18]. Therefore, restoring insulin sensitivity remains an important therapeutic strategy in preventing and managing IR-associated metabolic disorders, including T2DM. Recently, natural bioactive compounds that can enhance insulin signaling and regulate glucose metabolism have attracted significant attention as safer, long-term alternatives to conventional pharmacological treatments [19]. In the present study, we demonstrated that G-Rf effectively attenuates high-glucose-induced IR in AML12 hepatocytes. Specifically, G-Rf improved glucose metabolic function by activating the IRS/PI3K/Akt and PPARα/PGC1α signaling pathways and promoting glycogenesis. These beneficial effects may be attributed, at least in part, to the enhanced mitochondrial oxidative function, ultimately resulting in improved insulin sensitivity (Fig. 7).

Fig. 7.

A proposed model illustrating the mechanism by which ginsenoside Rf (G-Rf) enhances insulin sensitivity in insulin-resistant hepatocytes. G-Rf activates key components of the insulin signaling pathway—including IRS1, PI3K, and Akt—resulting in increased glucose uptake and stimulation of glycogen synthesis. Furthermore, G-Rf promotes mitochondrial biogenesis by upregulating the PPARα/PGC1α signaling axis, thereby improving mitochondrial function. Collectively, these effects contribute to the restoration of insulin sensitivity in insulin-resistant AML12 cells. This schematic model was conceptualized and created by the authors

Under physiological conditions, glucose absorption stimulates insulin secretion from pancreatic β-cells, which subsequently inhibits hepatic gluconeogenesis and promotes glucose uptake and storage in peripheral tissues, including the liver [20]. Hepatocytes express insulin receptors that activate receptor tyrosine kinase activity upon insulin binding, leading to the autophosphorylation and recruitment of insulin receptor substrates (IRS1/IRS2) [21]. These phosphorylated substrates activate PI3K, which then stimulates the downstream kinase Akt [22]. The IRS/PI3K/Akt signaling cascade regulates multiple aspects of glucose metabolism, including facilitating glucose uptake via Glut2 and promoting glycogen synthesis through GSK3 inhibition [23]. Glucose taken up by hepatocytes is metabolized via glycolysis and the tricarboxylic acid cycle to generate ATP [24]. However, this signaling pathway is impaired in IR conditions, resulting in decreased glucose uptake, increased gluconeogenesis, and hyperglycemia development [25]. Our findings demonstrate that G-Rf effectively reactivates this pathway in IR-AML12 cells by increasing IRS1, Akt, and GSK3β phosphorylation, thereby enhancing glucose uptake and glycogen synthesis.

The PPARα/PGC1α signaling axis is an important regulatory pathway in hepatic metabolism, particularly in mitochondrial biogenesis and OXPHOS. PPARα (a nuclear receptor) plays a central role in maintaining lipid and glucose homeostasis and is known to be dysregulated in conditions such as IR and T2DM [26]. Upon activation, PPARα enhances mitochondrial gene expression in cooperation with its transcriptional coactivator (PGC1α) [27]. Given the pivotal role of mitochondria in cellular energy production, mitochondrial dysfunction is closely associated with the pathogenesis of metabolic diseases [28]. Decreased mitochondrial protein expression and structural integrity can exacerbate IR and impair glucose regulation [29]. Thus, pharmacological strategies aimed at activating the PPARα/PGC1α pathway hold considerable promise for improving mitochondrial quality and mitigating metabolic dysfunction. Previous studies have demonstrated the therapeutic relevance of this axis. For example, ginsenoside Rg5 reportedly improves glucose metabolism in IR hepatocytes by upregulating PGC1α-mediated mitochondrial activity [30]. Similarly, baicalein (5,6,7-trihydroxyflavone) enhances glucose uptake in IR hepatocytes by increasing PGC1α mRNA and protein expression levels [31]. Dihydromyricetin (a natural flavonoid) ameliorates hepatic IR and increases Glut2 expression by activating the PPARα/PGC1α-dependent pathway [32].

In our study, IR-induced AML12 hepatocytes exhibited significant downregulation of PPARα and PGC1α at the transcriptional and protein levels. However, G-Rf treatment markedly restored the expression of these key regulators. Furthermore, G-Rf significantly increased the mtDNA content and intracellular ATP levels, indicating enhanced mitochondrial biogenesis and function. These findings suggest that G-Rf improves insulin sensitivity, at least in part, by activating the mitochondrial regulatory pathways involving PPARα and PGC1α.

Although G-Rf demonstrated significant pharmacological effects at concentrations up to 2 µM in our in vitro experiments, the levels of G-Rf present in commercially available ginseng preparations or dietary supplements may not be sufficient to achieve such pharmacologically relevant concentrations. Previous studies have reported that the oral bioavailability of ginsenosides, such as Rb1, is less than 0.8% in rodents [33]. This poor absorption is primarily attributed to their low intestinal permeability, owing to their large molecular weight, high hydrogen-bonding potential, and structural flexibility [34]. Furthermore, ginsenosides are subject to degradation or biotransformation by gastric acid and gut microbiota [35]. Pharmacokinetic data specifically for G-Rf remain limited. Therefore, further research is warranted to improve G-Rf bioavailability through advanced formulation strategies, explore the role of gut microbiota in its metabolic activation, and develop effective delivery systems that enhance its intestinal absorption.

In parallel, the mitochondrial quality control (MQC) system—including mitochondrial dynamics (fusion and fission) and mitophagy—has emerged as a critical regulatory mechanism in maintaining cellular energy homeostasis and mitigating metabolic diseases such as T2DM [36]. For example, ginsenoside Rg3 has been reported to ameliorate IR in C2C12 skeletal muscle cells by increasing ATP production and oxygen consumption rate, alongside upregulation of mitochondrial biogenesis markers such as PGC1α, NRF-1, and complexes IV and V [37]. Therefore, future studies should explore whether G-Rf exerts its metabolic effects by modulating mitochondrial energy metabolism through MQC-related mechanisms, including mitophagy and mitochondrial dynamics. Moreover, to improve the translational relevance of these findings, studies utilizing human hepatocyte models—such as human-induced pluripotent stem cell (hiPSC)-derived liver cells—are necessary to further elucidate the therapeutic potential and mechanistic actions of G-Rf in IR conditions.

Aside from its pharmacological effects, the source and accessibility of G-Rf are critical for its practical application. Traditional ginseng cultivation is time-consuming and labor-intensive, often requiring four to six years before harvest [38]. Furthermore, ginseng is highly susceptible to pests and diseases, necessitating pesticide use. To address these limitations, a more sustainable approach (i.e., sprouted ginseng production) has been developed. This method involves the hydroponic cultivation of one-year-old ginseng seedlings over a short period (6–8 days) in controlled environments [39]. Sprouted ginseng offers several advantages: drastically reduced cultivation time; year-round indoor production; pesticide-free growth; and the ability to consume the entire plant, including aerial and underground parts [40]. According to previous studies, sprouted ginseng contains comparable ginsenoside levels to those found in mature ginseng roots [41, 42]. However, the presence and distribution of G-Rf in sprouted ginseng had not been previously examined. Our quantitative LC–MS analysis revealed that mature (five-year-old) and sprouted (one-year-old) ginseng contained detectable G-Rf levels. Importantly, G-Rf was more abundant in the underground parts than in the aerial parts of both plant types, with approximately one-fourth of the root concentration detected in the aerial tissues of sprouted ginseng. Given that the entire sprouted plant is typically consumed soon after harvest (unlike mature ginseng, which often undergoes further processing), this distribution suggests that G-Rf may contribute meaningfully to the bioactivity of sprouted ginseng. These findings provide new insights into the functional potential of sprouted ginseng and support its application as a dietary source of G-Rf in preventing or managing IR and related metabolic diseases.

Conclusion

G-Rf effectively ameliorates high-glucose-induced IR in AML12 hepatocytes by enhancing glucose metabolism through the activation of the IRS/PI3K/Akt and PPARα/PGC1α signaling pathways. G-Rf promotes glycogen synthesis and mitochondrial biogenesis, contributing to improved insulin sensitivity and cellular energy homeostasis. Furthermore, LC–MS/MS analysis confirmed the presence of G-Rf in mature and sprouted ginseng, with notable levels found in the edible aerial and underground parts of sprouted ginseng. These findings not only highlight the therapeutic potential of G-Rf as a functional component for managing IR and related metabolic disorders but also suggest that sprouted ginseng may serve as a practical, sustainable dietary source of G-Rf.

Abbreviations

IR

Insulin resistance

T2DM

Type 2 diabetes mellitus

IRS

Insulin receptor substrate

PI3K

Phosphatidylinositol 3-kinase

Akt

Protein kinase B

GSK3

Glycogen synthase kinase 3

GYS

Glycogen synthase

OXPHOS

Oxidative phosphorylation

ATP

Adenosine triphosphate

Ginseng

Panax ginseng

G-Rf

Ginsenoside Rf

IR-AML12

Insulin-resistant AML12

FBS

Fetal bovine serum

DMEM/F12

Dulbecco’s Modified Eagle Medium/F-12

ITS

Insulin–transferrin–selenium

PGC1α

Peroxisome proliferator-activated receptor gamma coactivator 1-α

PPARα

Peroxisome proliferator-activated receptor α

2-NBDG

2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose

cDNA

Complementary DNA

RT-qPCR

Real-time quantitative polymerase chain reaction

Glut2

Glucose transporter type 2

mRNA

Messenger RNA

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

mtDNA

Mitochondrial DNA

mt-Cytb

Mitochondrially encoded cytochrome b

mt-Nd1

Mitochondrially encoded NADH dehydrogenase 1

LC–MS/MS

Liquid chromatography tandem mass chromatography

MRM

Multiple reaction monitoring

Authors’ contributions

J.-H.P. and Y.G.L. conceived and designed the study. S.H. prepared the original draft. S.H. and J.L., performed the formal analysis. S.Y.C. performed the extraction experiments for ginseng. J.-H.P. acquired the funding. Y.G.L. supervised the project and also reviewed and edited the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by grants from the Main Research Program (E0210400 and E0210601) of the Korea Food Research Institute funded by the Ministry of Science, ICT, and Future Planning.

Data availability

All relevant data generated or analyzed during this study are included in this published article. Additional information is available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.