Momordica charantia Extract Treatment Extends the Healthy Lifespan of Aging Mice via the Bitter Taste Receptor/mTOR Pathway

Abstract

We live in a society where extending one’s healthy lifespan is becoming increasingly important. Momordica charantia (MC) extract contains many bioactive substances, such as vitamin D, phytosterols, glycosides, saponins, alkaloids, and triterpenes, and has various health-promoting effects, but its effect on extending a healthy lifespan is unknown. This study investigated the effects of MC extract on a healthy lifespan, focusing on bitter taste receptors and the mammalian target of rapamycin (mTOR). Male and female mice from the Institute of Cancer Research (ICR) were divided into control and MC-extract-treated groups, with the latter receiving oral doses of MC extract three times a week for two years. In aged male mice, MC extract increased the muscle mass and grip strength and prolonged the time to exhaustion. MC extract also enhanced the signaling from taste receptor type 2 member 1 (T2R1) to mTOR in muscle in both sexes, elevating the ribosomal protein S6 kinase beta-1 and ribosomal protein S6 levels. This T2R1/mTOR pathway works in protein synthesis and is important for increasing muscle mass. Conversely, the levels of eukaryotic translation initiation factor 4E-binding protein 1 and microtubule-associated protein light chain 3 decreased in both aged male and female mice after MC extract administration. These findings suggest that the administration of MC extract may extend the healthy lifespan of male mice, with bitter taste receptors and mTOR signaling playing key roles in this process.

1. Introduction

The modern world is witnessing an unprecedented rise in life expectancy. However, as people age, they undergo various mental and physical changes, leading to declines in motor skills, memory, and learning abilities [1]. In our long-lived society, extending a healthy lifespan has become a major objective. The musculoskeletal system is often the first to show signs of aging, with bone density decreasing, which weakens the bones and makes them more susceptible to fractures [2], followed by a loss of muscle mass. Aging has been attributed to factors such as telomere shortening, which hampers cell division [3], and cell damage caused by increased oxidative stress [4]. This cell reduction is particularly evident in the testes, ovaries, liver, and kidneys [5]. Chronic fatigue is a significant factor associated with aging. Senescent cells release inflammatory cytokines, chemokines, and matrix-degrading enzymes, contributing to the senescence-associated secretory phenotype (SASP) [6,7], a process linked to chronic inflammation and carcinogenesis [8,9,10]. Calorie restriction is effective in extending the lifespan [11], with insulin-like growth factor 1 (IGF-1) being regulated by calorie intake in mice and worms, thereby affecting lifespan [12]. However, the benefits and drawbacks of calorie restriction for lifespan extension are still unclear [13]. While many studies have focused on how it extends the lifespan, few have explored it from the perspective of enhancing a healthy lifespan.

We previously reported that Momordica charantia (MC) extract improves the natural aging of the skin [14]. Commonly known as bitter melon, MC is a perennial vine that produces long, narrow fruits with knots on their surface and a distinctive bitter taste, attributed to its bitter components, momordicin and cucurbitacin. MC has various health benefits, including anti-hyperglycemic [15,16], anti-gastric ulcer [17,18], wound-healing [19], and anti-aging properties [20]. Furthermore, cucurbitacin and momordicin offer additional advantages, including anti-inflammatory, whitening [21], and anticancer effects [22]. Momordicin in particular promotes appetite [23], stabilizes blood pressure, and regulates blood sugar levels. However, its potential role in extending healthy life expectancy remains largely unexplored.

Furthermore, MC extract contains many bitter components, which have a significant effect on bitter taste receptors. Bitter taste receptors play various roles in maintaining health. In recent studies, bitter taste receptors have been reported to induce a defensive response against invading bacteria [24]. Bitter taste receptors expressed in the stomach and oral cavity are involved in gastric acid secretion and regulating stomach pH [25]. Furthermore, bitter taste receptors expressed in white adipocytes are involved in the differentiation of adipocytes, and their overexpression reduces fat accumulation [26].

In this study, we administered MC extract to mice over two years to assess its impact on extending a healthy lifespan. Muscle strength influences the survival rate [27], and aging typically results in a more significant decline in muscle mass in the lower limbs than in the upper limbs [28]. Based on these observations, we examined the mechanical and biochemical changes occurring in skeletal muscle, specifically in the quadriceps, as indicators of healthy lifespan extension. Furthermore, we investigated the role of bitter taste receptors in this process, focusing on the signal transduction pathways (especially the bitter taste receptor/mammalian target of rapamycin (mTOR) pathway involved in protein synthesis) associated with these receptors. Our findings demonstrated that MC extract treatment significantly increased muscle mass, grip strength, and time to fatigue in aged male mice, suggesting an extension of their healthy lifespan. Additionally, the study revealed that the MC extract enhanced signaling through bitter taste receptors and the mTOR pathway, leading to increased protein synthesis and reduced autophagy in skeletal muscle, thereby highlighting the crucial role of this pathway in promoting healthy aging.

2. Materials and Methods

2.1. Animals and Experimental Design

Specific pathogen-free (SPF) male and female Institute of Cancer Research (ICR) mice (SLC, Hamamatsu, Shizuoka, Japan) aged 8 weeks were used. Mice were housed in cages in an air-conditioned room at 23 °C ± 1 °C under SPF conditions and a 12 h light/12 h dark cycle (lights were turned on at 8:00 AM). Food and water were provided ad libitum. The animals were then randomly divided into three groups according to sex: control, solvent (water)-treated, and MC extract-treated (n = 5/group) groups. The animals underwent laparotomy under general anesthesia induced using pentobarbital. This study was approved by the Suzuka University of Medical Sciences Animal Experimentation Ethics Committee on 25 September 2014 and was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals at Suzuka University of Medical Sciences (Approval No: 34). All surgeries were conducted under pentobarbital anesthesia, and efforts were made to minimize animal suffering.

2.2. MC Extract Treatment

The MC extract was provided by ChromaDex Inc. (Irvine, CA, USA). The purchased dried MC powder sample was added with approximately 20 times the amount of 80% (v/v) ethanol and extracted by stirring overnight at 4 °C. Insoluble matter was then removed by centrifugation and suction filtration, after which the mixture was concentrated in an evaporator and freeze-dried to obtain an ethanol extract [29]. Approximately 50 mg/kg body weight of the MC extract in 0.1% dimethyl sulfoxide (DMSO) was orally injected into mice three times per week for 2 years. This dose is the minimum concentration found to be most effective. The solvent-administered animals were administered DMSO alone [30]. The reason for using DMSO was that the MC extract was extracted with ethanol and was poorly soluble in water, so it was dissolved in DMSO for uniform administration.

2.3. Measurement of Muscle, Bone, and Fat Tissue Mass in Mice Using a CT Scan

Overall, 10-week-old, 24-week-old water-fed, and MC-extract-treated mice were intraperitoneally anesthetized with 100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride. The anesthetized mice underwent whole-body tomographic imaging using a 3D micro X-ray micro-CT system (Rigaku, Tokyo, Japan) under the following conditions: tube voltage of 90 kV, tube current of 150 μA, imaging time of 17 s, field of view (FOV)/image size of φ9.6 × H9.6 mm, and resolution/voxel size of 20 × 20 × 20 μm. Tomographic data were acquired from the upper body of the mice, from the neck to the third lumbar vertebra, and from the lower body, from the third lumbar vertebra to the caudal vertebra, including the sciatic and pubic bones, to measure the muscle, bone, and adipose tissue volumes. The tomographic data obtained were analyzed using the trial version of Analyze 12.0, an image analysis software version 12.0 from Analyze Direct Inc. (Overland Park, KS, USA).

2.4. Skeletal Muscle Performance

2.4.1. Grip Force Test

Forelimb grip strength was measured by using a strain gauge (GPM-100; Melquest, Toyama, Japan). The mouse forepaws grasped a horizontal bar attached to the gauge, and the experimenter slowly pulled their tail back. The tension value was recorded using the gauge when the mouse released its forepaw from the horizontal bar. The measurements were repeated three times, and the maximum tension value obtained from the three measurements was used for the analysis.

2.4.2. Time to Exhaustion

Before exercising, the mice from each group were habituated to a treadmill (MK-680; Muromachi Kikai, Tokyo, Japan). Male Keap1-KD mice underwent adaptation training on day 1 (starting at 10 m/min for 10 min, followed by 20 m/min for 15 min). Treadmill testing was performed on day 2. The starting speed was 5 m/min for 3 min, and then the speed was increased by 5 m/min every 3 min until it reached 28 m/min. The time to exhaustion was measured at a maximum speed of 28 m/min. Mice were considered fatigued if they remained in the grid area for more than 10 s.

2.5. Preparation and Staining of the Skeletal Muscle

Skeletal muscle samples were collected under anesthesia on the last day of the experiment, fixed in 4% phosphate-buffered paraformaldehyde, embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan), and cryo-sectioned. These sections were stained with antibodies for immunohistological analysis, as previously described [31]. Skeletal muscle specimens were incubated with rabbit polyclonal anti-taste receptor type 2 member 1 (T2R1) (1:100; Bioworld Technology, Inc., St. Louis Park, MN, USA), rabbit monoclonal anti-mammalian target of rapamycin (mTOR; 1:100; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-ribosomal protein S6 (rpS6; 1:100; Gene Tex, Inc., Alton Pkwy Irvine, CA, USA), rabbit monoclonal anti-ribosomal protein S6 kinase beta-1 (S6K1; 1:100; Abcam, Cambridge, MA, USA), rabbit monoclonal anti-eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 1:100; Abcam), rabbit monoclonal anti-eukaryotic translation initiation factor 4E (eIF4E; 1:100; Abcam), or mouse monoclonal anti-microtubule-associated protein light chain 3 (LC3; marker of autophagy; 1:100; MEDICAL & BIOLOGICAL Lab., Co. Ltd., Minato-ku, Tokyo, Japan) primary antibodies. Sections were subsequently incubated with fluorescein isothiocyanate-conjugated anti-rabbit or anti-mouse secondary antibodies (1:30; Daco Cytomation, Glostrup, Denmark). The intensities corresponding to T2R1, mTOR, rpS6, S6K1, eIF4EBP1, eIF4E, and LC3 were calculated based on six random visual fields with the same area using ImageJ software version 1.53 (National Institutes of Health, Bethesda, MD, USA). Briefly, the original files were converted to monochrome 8-bit files. Then, a luminous intensity threshold was established. The areas above the threshold, which were defined as “intensity”, were measured for each sample.

2.6. Western Blotting Analysis of the Skeletal Muscle

Skeletal muscle samples were homogenized in lysis buffer (Kurabo Industries, Osaka, Japan) and centrifuged to obtain the supernatants. Western blotting was performed as previously described [32]. After electrophoresis, the membranes were incubated with primary antibodies against pyruvate dehydrogenase kinase isoform 1 (PDK1) (1:1000; Proteintech Group, Rosemont, IL, USA), protein kinase B (Akt) (1:1000; Cell Signaling Technology, Inc.), sterol regulatory element-binding protein (SREBP) (1:1000; Proteintech Group), and β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. β-actin was used as a loading control. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako Pure Chemical Industries, Osaka, Japan), and images were acquired using Multi Gauge software (Fujifilm, Greenwood, SC, USA).

2.7. Measurement of the IP3 Levels in Skeletal Muscle

Skeletal muscle samples were collected on the final day of the experiment. The skin muscle was isolated and homogenized in lysis buffer (Kurabo). The tissue extracts were centrifuged at 10,000 rpm (Tomy MX-201), and the supernatants were collected for the assay. The levels of inositol trisphosphate (IP3) in skeletal muscle were determined using commercial ELISA kits (Abcam) according to the manufacturer’s instructions. The optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

2.8. Statistical Analysis

All data are presented as the mean ± standard deviation (SD). Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA) was used to analyze the statistical significance of the data, along with one-way analysis of variance, followed by Tukey’s post hoc test, using SPSS version 20 (SPSS Inc., Chicago, IL, USA). Results with p-values < 0.05 (*) and <0.01 (**) were considered statistically significant.

3. Results

3.1. Effects of MC Extract Treatment on the Body Weight, and Muscle, Bone, and Adipose Tissue Mass in Aged Mice

MC extract (50 mg/kg) was administered orally three times a week for 2 years. By the end of the experiment, no changes in body weight were observed in either female or male aged mice following MC extract treatment (Figure 1A). Old female mice who received MC extract showed the same muscle mass as young mice and increased muscle mass compared to old mice who did not receive MC extract. Muscle mass increased in old male mice administered MC extract (Figure 1B,C). Bone mass did not differ between males and females (Figure 1B,D). Notably, adipose tissue mass was reduced in aged female mice; however, no significant difference was observed in aged male mice (Figure 1B,E).

3.2. Effect of the MC Extract Treatment on Muscle Strength in Aged Mice

Grip strength and fatigue tests were conducted to assess muscle strength. In aged female mice, grip strength remained unchanged following MC extract treatment, while it increased in aged male mice (Figure 2A). Similarly, the time to exhaustion on the treadmill did not change in aged female mice but was extended in aged male mice after the MC extract treatment (Figure 2B).

3.3. Effect of the MC Extract on the Levels of T2R1, IP3, PDK-1, AKT, and mTOR in Skeletal Muscle

We examined the expression levels of T2R1, a bitter taste receptor expressed in skeletal muscle, along with IP3, PDK-1, AKT, and mTOR, which are downstream of its signal transduction. MC extract treatment significantly increased the expression of T2R1, IP3, PDK-1, AKT, and mTOR in both aged female and male mice (Figure 3A–F).

3.4. Effect of the MC Extract on the Expression of S6K1, rpS6, eIF4EBP1, eIF4E, and LC3 in Skeletal Muscle

Then, we examined the signal transduction pathways related to protein synthesis and autophagy inhibition induced by mTOR. Among the mTOR-activated proteins, rpS6, S6K1, and eIF4EBP1, the expression of S6K1 and rpS6 was significantly increased following MC extract administration in both aged male and female mice (Figure 4A,B). In contrast, the expression of eIF4EBP1 was significantly decreased by the MC extract treatment (Figure 4C). Additionally, the expression of eIF4E, which is normally suppressed by eIF4EBP1, was significantly increased in both aged male and female mice after the MC extract treatment (Figure 4D). The treatment also led to a decrease in LC3 expression in both aged male and female mice (Figure 4E).

3.5. Effect of the MC Extract on the Levels of SREBP in Skeletal Muscle

We investigated the activation of SREBP by mTOR through various pathways. The SREBP levels in skeletal muscle were not affected by the MC extract treatment in either aged female or male mice (Figure 5A,B).

4. Discussion

In this study, we found that the MC extract treatment increased muscle mass, grip strength, and time to fatigue in aged male mice, suggesting that it extended their healthy lifespan. Furthermore, the expression of bitter taste receptors T2R1, PDK1, and mTOR in the skeletal muscle of MC extract-treated aged mice was higher than that in the control mice. The treatment also led to increased expression levels of S6K1, rpS6, and eIF4EBP1 in skeletal muscle, while eIF4E expression decreased. Moreover, SREBP expression was increased following MC extract treatment.

This study demonstrated that MC extract, which contains bitter components, increased the expression of bitter taste receptors in skeletal muscle (Figure 3A). When a taste stimulus binds to a taste receptor, it triggers taste transmission, leading to the depolarization of taste cells, calcium influx, and the release of neurotransmitters to taste-afferent neurons [33]. Complex stimuli such as bitter compounds in particular bind to G protein-coupled receptors and initiate intracellular signaling, with IP3 playing a central role in bitter taste transduction [34]. Furthermore, bitter compounds increase the IP3 levels [35,36,37,38]. Consistent with this, our study showed that the MC extract treatment led to an increase in both bitter taste receptors and IP3 (Figure 3A,B). IP3 is crucial for releasing Ca²⁺ from the intracellular stores [39], and the resulting rise in the cytosolic Ca²⁺ level activates Ca²⁺-dependent kinases and transcription factors. IP3 also activates the transcription factor NF-kB [40], which in turn increases the activity of PDK-1, Akt, and mTOR [41,42,43].

In cells, mTOR functions as an mTOR complex composed of mTORC1 and mTORC2, which activate downstream proteins, including S6K1, thereby promoting protein synthesis [44,45]. Moreover, mTOR regulates the expression of eIF4E, a protein that binds to the 5’ cap structure of mRNA and initiates translation by recruiting ribosomes. In contrast, eIF4EBP1, a combination of eIF4E and 4E-BB, inhibits translation by preventing the ribosomes from attaching to the mRNA [46]. In this study, the treatment with MC extract increased eIF4E expression and decreased the eIF4EBP1 levels, suggesting that it enhances protein synthesis.

Furthermore, mTOR functions as a sensor that detects nutrient and energy levels in various intracellular and extracellular environments. A decrease in mTOR activity triggered by nutrient depletion induces autophagy [47]. mTOR regulates autophagy via the ULK complex, which includes three serine/threonine kinases: UNC-51-like kinase-1, -2, and -3 (ULK1, ULK2, and ULK3), which act downstream of mTOR [48]. Under nutrient starvation conditions, autophagy acts as a survival mechanism; however, excessive autophagy can lead to cell death. mTORC1 phosphorylates ULK1 and autophagy-related protein 13 (ATG13), initiating autophagy and thereby suppressing autophagosome formation to prevent cell loss [49,50]. In our study, we examined the expression of LC3, an autophagy marker (Figure 4E), and found that autophagy was suppressed by the MC extract treatment. These findings suggest that the regulation of translation by 4EBP and autophagy work in concert to regulate protein synthesis.

Furthermore, mTOR regulates glucose metabolism, lipid metabolism, and mitochondrial function not only through signaling pathways, including 4EBP and S6K, but also via transcription factors, such as hypoxia-inducible factor (HIF)-1a, peroxisome proliferator-activated receptor-g co-activator-1a (PGC-1a), and SREBP [51]. In this study, we focused on SREBP, a protein involved in lipid synthesis that is activated by mTOR. The body weights of both male and female aged mice increased significantly compared to those of younger mice; however, no significant changes were observed in either sex following MC extract treatment. Nonetheless, MC extract treatment did reduce the adipose tissue mass in aged female mice, while no change was noted in aged male mice. Generally, increased mTORC1 expression activates SREBP, leading to increased lipid synthesis [52]. Although MC extract administration increased the mTOR levels, no change was observed in the SREBP levels. The MC extract treatment may have negatively regulated the increase in the SREBP level, either directly or via bitter taste receptors, warranting further investigation.

This study demonstrated that signal transduction from bitter taste receptors via mTOR plays a crucial role in extending the healthy lifespan of mice (Figure 6). However, we did not differentiate between the mTOR complexes 1 and 2 in our analysis. Each of these complexes should be examined separately. Moreover, the mTORC1-4EBP signaling pathway regulates mitochondrial function and the glycolytic system [46,52], making it essential to explore this aspect further. Given the diversity of mTOR-derived signaling systems, considering the entire bitter taste receptor/mTOR complex cascade is essential to fully understand its impact.

This study was an animal experiment using mice. There are differences between humans and animals, and it is thought that these results cannot be generally applied to humans. However, the effectiveness of MC extract has also been confirmed in humans, and it is highly likely that the results of this study can be applied to humans. At a time when healthy life expectancy has become an important issue, this will help solve this problem. Therefore, clinical trials are necessary.