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Article

4-Galactosylkojibiose Extends the Lifespan of Drosophila melanogaster

by
Haruki Kato
1,†,
Akari Hara
2,†,
Rinka Ota
2,†,
Riho Kobayashi
1,‡,
Ryo Miyake
1,
Rabia Garibağaoğlu
1,
Jun Tomita
1,
Misato Tsuboi
3,4,*,
Chisato Oba
4,
Kentaro Nakamura
4 and
Kazuhiko Kume
1,*
1
Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
2
Faculty of Pharmacy, Nagoya City University, Nagoya 467-8603, Japan
3
Wellness Science Labs, Meiji Holdings Co., Ltd., Tokyo 192-0919, Japan
4
R&D Division, Meiji Co., Ltd., Tokyo 192-0919, Japan
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
Current address: Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Nutrients 2026, 18(4), 604; https://doi.org/10.3390/nu18040604
Submission received: 8 January 2026 / Revised: 5 February 2026 / Accepted: 6 February 2026 / Published: 12 February 2026
(This article belongs to the Section Carbohydrates)
Browse Figures
Review Reports Versions Notes

Abstract

Background/Objectives: Oligosaccharides, such as fructooligosaccharides (FOS), have long been used to promote human health due to their beneficial effects on the intestinal environment and their anti-inflammatory properties. Recent advances in manufacturing technologies have enabled the production of novel oligosaccharides derived from rare sugars. These compounds may exert unique health benefits; however, their physiological functions remain largely unexplored. Because sleep is a conserved, lifespan-linked physiological phenotype governed by metabolic and stress-response pathways that oligosaccharides can influence, we evaluated sleep alongside lifespan to capture systemic functional effects. Methods: Using the model organism Drosophila melanogaster, we investigated the effects of 4-galactosylkojibiose (4-GK), a promising new oligosaccharide, on sleep and lifespan. We also conducted RNA sequencing following 4-GK or FOS application. Results: Our results demonstrated that both 4-GK and FOS extended lifespan, with 4-GK showing comparable or numerically greater efficacy than FOS. While the addition of 4-GK or FOS to sucrose diet did not affect overall sleep or activity levels, 4-GK alone without sucrose decreased sleep compared to sucrose alone. Furthermore, RNA-seq analysis revealed upregulation of gene groups associated with longevity in both the 4-GK and FOS treatment groups, particularly genes encoding heat shock proteins. Conclusions: 4-GK promotes longevity in D. melanogaster and activates stress-response programs, suggesting a health-promoting role comparable to FOS. Sleep effects were diet-context dependent, unchanged when added to sucrose but reduced when given without sucrose. These findings suggest a novel potential role for 4-GK in promoting longevity.

1. Introduction

Oligosaccharides, such as fructooligosaccharides (FOS), have been reported to exert various health benefits, including antioxidant [1] and anti-inflammatory effects [2,3], as well as improvement to the intestinal environment [4]. They have been used to support human health for many years. In recent years, advancements in manufacturing technologies have enabled the development of novel prebiotic candidates containing rare sugars [5], offering the potential for new health benefits. However, their functional properties and differences from conventional oligosaccharides remain incompletely understood. One such candidate is 4-galactosylkojibiose (a-D-glucopyranosyl-(1⟶2)-[b-D-galactopyranosyl-(1⟶4)-]D-glucopyranoside; 4-GK). 4-GK is a trisaccharide composed of galactose linked to the rare disaccharide kojibiose. Together with glucosyl-galactosylkojibiose (GGK), a tetrasaccharide consisting of one additional glucose molecule attached to 4-GK, these compounds are referred to as “glucolacto-oligosaccharides.” 4-GK has been reported to support intestinal health, exhibiting strong bifidogenic activity [6] and enhancing iron absorption [7]; however, its other physiological functions remain undefined. Given that certain oligosaccharides [8] have been shown to influence lifespan, possibly by affecting metabolic homeostasis and organismal stress resistance [9], we hypothesized that 4-GK might similarly modulate stress resistance and related homeostatic processes and therefore investigated whether 4-GK likewise influence lifespan and concurrently evaluated sleep as a conserved, lifespan-associated physiological phenotype. Drosophila melanogaster is a well-established model organism for studying physiological processes, including the relationship between sleep and lifespan. It has also been widely used to investigate the biological effects of sugars, nutrients, and dietary supplements. For example, recent studies have shown that the fatty acid triptamide, derived from cacao, extends the lifespan of Drosophila [10]. It has also been used to study the effects of lactic acid bacteria on sleep promotion [11].
In this study, we investigated the effects of 4-GK, a promising new oligosaccharide, on sleep and lifespan in Drosophila, in comparison to the well-studied FOS.

2. Materials and Methods

2.1. Saccharides

4-GK was synthesized by Meiji Co. Ltd. (Tokyo, Japan) [12]. The production process, in brief, was as follows: a mixture containing lactose and sucrose was reacted with glucansucrase and then purified by carbon-Celite column chromatography. The final material was obtained as an amorphous powder with a relative sweetness of approximately 0.3 (vs. sucrose). The content of 4-GK was more than 95%. The short-chain FOS was used (Orafti® P95, Beneo, Tienen, Belgium) and it consisted of approximately 95% oligofructose with a degree of polymerization of 2–8.

2.2. Drosophila Stock and Maintenance

In all experiments, the male D. melanogaster strain w1118 (DGRC: 150534) was used, obtained from the Kyoto Drosophila Stock Center. Flies were maintained at 25 °C, 50–60% humidity, and a 12 h light (natural white fluorescnet light about 200–500 lux)/12 h dark cycle, as previously described [13]. Standard cornmeal-based food was used for rearing, containing 10% glucose, 5% cornmeal, 3% dry yeast, 1.25% wheat germ, and 1% agar with 0.1% butyl parahydroxybenzoate and 0.3% propionic acid as preservatives. Experimental diets were based on a simplified medium containing 5% sucrose and 1% agar (control), to which 1% or 5% sucrose (1% sucrose, 5% sucrose, respectively), 1% or 5% 4-GK (1% 4-GK and 5% 4-GK, respectively), or 1% or 5% FOS (1% FOS and 5% FOS, respectively) was added.

2.3. Measurement of Activity, Sleep, and Survival

Males and females were separated under carbon dioxide anesthesia at least 1 day after eclosion and maintained for at least one additional day before testing. To measure the activity and longevity, each fly was placed in a glass tube (3 mm inner diameter, 65 mm length). One end of the tube was filled with control or test food, and the other end was covered with a cotton plug. The tubes were placed in a Drosophila Activity Monitor (DAM; Trikinetics, Waltham, MA, USA), and activity was recorded as the number of times each fly crossed a central infrared beam, measured every minute. All experiments were conducted under a 12 h light/dark cycle at 25 °C, consistent with rearing conditions. Food was replaced once per week by transferring flies to new tubes. Monitoring was continued until all flies died, and survival was recorded. Fly behavior was observed every hour after the start of measurements, with activity data summed over 1 h intervals, and the time of death was defined as the last recorded activity; based on previous observations and direct confirmation in some flies, individuals rarely survived more than 24 h after their final activity, indicating that this definition introduces minimal error on a 24 h timescale. Based on previous studies, sleep was defined as a period of immobility lasting more than 5 min [14]. To measure survival during vial rearing, 20 males aged 3–7 days were placed in individual 30 mm diameter vials. Flies were transferred to new vials three times a week (every 2–3 days), and the number of dead flies was counted to measure longevity. Observations were continued for up to 42 days, and flies that remained alive at this time point were treated as censored.

2.4. RNA Sequencing (RNA-Seq) Analysis

Male flies aged 3–7 days were reared for three weeks on either a control diet (5% sucrose + 1% agar) or a diet containing 5% of the test sugar. For each condition, three biological replicates were prepared, each consisting of 15–20 fly heads. Total RNA was extracted using RNAiso Plus (Takara, Kyoto, Japan). Due to the small number of flies surviving in the three-week-old control group, all surviving flies were pooled and divided into three samples for RNA extraction. The extracted RNA was further purified using phenol–chloroform extraction and ethanol precipitation with 70% ethanol and 0.3 M sodium acetate. RNA-seq and succeeding analysis were outsourced to Novogene (https://www.novogene.com/) (accessed on 29 January 2024). Here, strand-specific libraries were prepared, sequencing was conducted using the Illumina Novaseq6000 platform (Illumina, San Diego, CA, USA), and gene expression quantification was performed using featureCounts. Gene Set Enrichment Analysis (GSEA) was performed using gene sets from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify significantly enriched pathways. Terms with adjusted p-values (padj) < 0.05 were considered significant. All libraries were prepared and sequenced in a single batch at Novogene. Per-sample QC metrics (total reads, total map, unique map, proper map, splice map and unsplice map) were comparable across groups, indicating minimal risk of batch effects; therefore, no batch correction was applied.

2.5. Data Processing and Statistical Analysis

Data processing and statistical analyses were performed using Microsoft Office, R (https://www.r-project.org/) (accessed on 29 January 2024), and Python 3.11.5. The specific statistical tests as well as the sample size used in each experiment are detailed in the figure legends. p value less than 0.05 was considered statistically significant. In details, Otherwise, we wrote detailed in figure legends. Survival curves were compared using the log-rank test implemented in the lifelines Python package. For sleep measurements, data normality was assessed using the Shapiro–Wilk test. Depending on the outcome of this test, statistical comparisons were performed using either one-way ANOVA or the Kruskal–Wallis test.

3. Results

3.1. 4-GK Prolongs the Lifespan of Drosophila

We measured the lifespan of male flies fed a control diet (5% sucrose) supplemented with different sugars. Adjusted p values for all comparisons are provided in Table S1. In Figure 1a, either 4-GK or FOS was added at concentrations of 1% and 5%. Compared to the control group (median lifespan: 19.1 days), the 4-GK-fed groups exhibited a significantly extended lifespan (Figure 1a). This effect was observed even at 1% (median lifespan: 1% 4-GK; 23.3 days, 5% s 5% 4-GK; 25.0 days). FOS also significantly extended lifespan at 5% (median lifespan: 23.6 days).
To control for total sugar, we next examined the effect of adding an equal amount of sucrose to each treatment. In Figure 1b, survival rates were compared among groups supplemented with 1% sucrose, 1% FOS, or 1% 4-GK, keeping the total sugar concentration at 6% in all groups. The results showed that flies fed 1% 4-GK exhibited significantly prolonged survival (median lifespan: 22.6 days) compared to that of the 1% sucrose group (median lifespan: control; 18.4 days, 1% sucrose; 18.2 days), while the 1% FOS group showed a similar trend (median lifespan: 21.1 days), although it was not statistically significant (Figure 1b).
Subsequent experiments using standard rearing vials with larger sample sizes also confirmed the lifespan-extending effect (Figure 1c). Collectively, these findings indicate that both 4-GK and FOS prolong the lifespan of Drosophila (median lifespan: 1% sucrose; 19.0 days, 1% FOS; 19.0 days, 1% 4-GK; 21.5 days).

3.2. 4-GK Alters Gene Expression in the Longevity-Regulating Pathway

To investigate the mechanism by which 4-GK affects longevity, we performed RNA-seq on flies fed a diet supplemented with 4-GK. Based on the substantial differences in survival rates observed in vial-reared flies (Figure 1c), gene expression analysis was conducted using flies maintained for three weeks in vials. At 21 d after the start of the experiment, survival was notably low in the control group (8.8%), whereas the survival rates were 52.5%, 78.1%, and 75.0% in the 5% sucrose, 5% FOS, and 5% 4-GK, respectively. These results indicate that the FOS and 4-GK groups showed higher survival than both the control and 5% sucrose groups (Table 1).
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (Figure 2a,b, Table S2) showed no significant differences in the longevity-regulating pathway between the control and 5% sucrose (p = 0.51, padj = 0.89 vs. control; Figure 2a) groups. In contrast, the 5% 4-GK group showed nominal evidence of higher pathway-level expression relative to the control and 5% sucrose groups; however, the enrichments did not reach adjusted significance (p = 0.0090, padj = 0.12, vs. control; p = 0.011, padj = 0.11, vs. 5% sucrose; Figure 2b). A similar trend was observed in the 5% FOS group (p = 0.0045, padj = 0.088 vs. control; p = 0.058, padj = 0.21 vs. 5% sucrose). Among the representative genes in the longevity-regulating pathway, as well as associated molecular species, the expression of several heat shock proteins was markedly upregulated in both the 4-GK- and FOS-fed groups (Table S3).

3.3. 4-GK Does Not Affect Activity or Sleep Quantity in Drosophila After Short- or Long-Term Administration

To examine the effects of 4-GK on activity and sleep following short- and long-term administration, data from the experiment shown in Figure 1a was analyzed. Specifically, activity and sleep levels were calculated from days 1–3 (immediately after the start of the experiment) and from days 21–23 (in the third week). As shown in Figure 3, no significant changes in circadian patterns, sleep duration, or locomotor activity were observed in either young (day 1–3, Figure 3a, day 1–7, Figure 3c,e) or old (day 26–28, Figure 3b, day 22–28, Figure 3d,f) flies, regardless of the supplied concentration of FOS or 4-GK. As expected, aged flies exhibited reduced activity and diminished amplitude of activity rhythms compared to young flies; however, these age-related changes were not influenced by supplementation of either FOS or 4-GK. These findings suggest that while supplementation of 4-GK extends lifespan, it does not affect overall activity or sleep under standard conditions. Adjusted p values for all comparisons in Figure 3 are provided in Table S1.
Next, we examined the effects of replacing the base 5% sucrose with 5% 4-GK (Figure S1). Even when 4-GK was the sole carbohydrate source, the flies remained viable: 15 out of 16 flies survived the 3 d activity monitoring period, and 13 survived for more than one week. In this replacement experiment, total sleep duration was reduced, particularly during the nighttime, where a significant decrease in sleep was observed (Figure S1a,c). In contrast, no significant changes in activity were observed (Figure S1b,d).

4. Discussion

To the best of our knowledge, this study is the first to evaluate the physiological effects of 4-GK in comparison to FOS using Drosophila as a model organism. Our findings demonstrate that oral administration of both 4-GK and FOS significantly extends the lifespan of Drosophila without affecting sleep duration or activity levels. Notably, 4-GK exhibited a nominally stronger lifespan-extended effect than that by FOS, which has been more extensively studied. Transcriptomic analysis indicated that lifespan extension in both groups was accompanied by upregulation of HSP genes. Together, these findings suggest that 4-GK may offer comparable or numerically greater health benefits than conventional oligosaccharides, potentially with fewer gastrointestinal side effects associated with some oligosaccharides.
Oral administration of 4-GK extended the lifespan of Drosophila, suggesting that its effect may be comparable or numerically greater than that of FOS. 4-GK significantly extended lifespan at a concentration of 1% compared to the control under all tested conditions. Furthermore, under individual feeding conditions with 5% sucrose as the base diet, 1% 4-GK significantly extended lifespan compared to control. In contrast, FOS significantly extended lifespan only at 5%, suggesting that 4-GK might act at a lower dose than FOS. Oligosaccharides are generally known to cause gastrointestinal side effects such as bloating and discomfort [15,16]. Therefore, if 4-GK is effective even at lower concentrations, it may reduce the incidence of such side effects while maintaining efficacy, positioning it as a promising new prebiotic.
Our findings suggest that the lifespan-extending effect of 4-GK in Drosophila may be mediated by changes in the expression of genes encoding heat shock proteins. To investigate which components of the longevity-regulating pathway were involved, we examined the expression of individual genes. Notably, InR [17,18], a gene involved in energy metabolism, and sirtuins such as dsir2 [19], dsir4 [20], and dsir6 [21], which are involved in lifespan regulation, were not upregulated in Drosophila following oral administration of 4-GK. These results suggest that the lifespan-extending effect is mediated by heat shock proteins, not by InR or sirtuin. Heat shock proteins reported to be associated with lifespan include hsp70 [22,23] and hsp68 [24], as well as small heat shock proteins such as hsp27 [25,26], hsp26 [25,26,27], hsp23 [28,29], and hsp22 [22,28,30,31]. Increased levels of these RNAs have been reported to correlate with lifespan extension. In the present study, oral administration of 4-GK increased the expression of these genes by more than 2-fold in log2 fold change compared to controls, consistent with previous reports that upregulation of these genes extends lifespan. However, we did not further validate HSP genes using qPCR. Therefore, validation of key HSP genes by qPCR will be necessary in future studies to more precisely assess their expression changes. The absence of significant differences in the expression of heat shock proteins between the 4-GK and FOS groups may reflect the shared lifespan-prolonging effects of these oligosaccharides. We propose two non-mutually exclusive hypotheses for HSP upregulation: (i) a microbiota-mediated mechanism, whereby 4-GK and FOS alter gut microbial communities (e.g., Lactobacillus and Clostridium species [32]) and their metabolites to induce HSP expression; and (ii) a direct gut-signaling mechanism (e.g., short-chain fatty acids [33], arginine [34], and glutamine [35]). Our current data do not distinguish between these mechanisms. Further studies are warranted to clarify the differences in effective concentrations and mechanisms of action between 4-GK and FOS. These should include comparative analysis of gene expression at lower concentrations, profiling of gut microbiota, and quantification of HSP expression in the intestinal tract.
The addition of 4-GK did not affect sleep or activity levels. As oligosaccharides have been reported to improve sleep [36] and mental health [37], we hypothesized that 4-GK would influence sleep and activity levels. However, no significant effects were observed. Similarly, FOS supplementation had no effect on sleep or activity levels. Therefore, these oligosaccharides had no impact on Drosophila during undisturbed sleep or stress. Gene expression analysis also revealed no consistent changes in the expression of sleep-related genes. In contrast, the administration of 4-GK as the sole nutrient source reduced sleep duration compared to sucrose alone. Previous studies have shown that sleep is reduced when sorbitol, a sugar with a low sweetness, is administered [38]. Although these observations may suggest that 4-GK provides nutritional value while lacking sucrose-like sweetness, palatability was not directly assessed in this study, and this interpretation remains speculative. In addition, starvation is known to reduce sleep [38]. Thus, insufficient energy availability under the 4-GK-only condition may also contribute to the reduced sleep phenotype. Sleep duration in Drosophila is influenced by various nutrients. For example, our previous studies have demonstrated that D-serine increases sleep through N-methyl-D-aspartate receptors [39,40]. Further research is needed to clarify the distinct effects of 4-GK relative to other sugars using other experimental systems.
As for general limitations of the Drosophila experiments, two points should be noted. First, only male flies were used in this study. Female flies were excluded because oviposition and the emergence of larvae can interfere with longevity and locomotor assays; therefore, potential sex-specific differences could not be evaluated. Second, the experiments were not intentionally randomized. This approach was adopted for several reasons. The flies used had highly congenic genetic backgrounds and were reared under uniform environmental conditions. In addition, each experimental condition was always conducted in parallel with its corresponding control, which served to monitor inter-experimental variability. Furthermore, flies were collected, pooled, and then allocated to each experimental condition, and the number of flies analyzed was sufficient to minimize individual variability. Although this procedure is not typically described as “randomization” in Drosophila research, it may be functionally equivalent to randomization practices used in other research fields. Furthermore, the mechanism of action of 4-GK may involve alterations in the intestinal microbiota, which were not evaluated here, and thus we cannot determine whether the observed effects are directly effect- or microbiota-mediated. To address this, studies employing axenic or antibiotic-treated flies and defined microbiota (gnotobiotic) reconstructions, together with 16S/metagenomic and metabolomic profiling, are warranted. Additionally, although flies have a short life cycle and lifespan enabling rapid assessment compared with mammalian models such as mice, the gut microbiota of Drosophila is considerably simpler than that of mammals [41]. Therefore, additional studies using mammalian models are warranted to test microbiota-dependent mechanisms in a more complex community context. Furthermore, a potential survivorship bias may have affected our transcriptomic analyses. Due to the substantial mortality in the control group at the sampling time point, RNA-seq libraries were generated from the surviving individuals only. As a result, the gene expression data may be enriched for flies with relatively high stress resistance or longevity. Therefore, these profiles should be interpreted as reflecting molecular characteristics of surviving flies. This limitation should be considered when interpreting the results. Additional investigations are required to more precisely delineate the magnitude, dose–response, and mechanistic basis of 4-GK’s effects, including comprehensive profiling of the gut microbiota, experiments in germ-free (axenic) and antibiotic-treated flies to parse microbiota-mediated effects. In addition, after validating key HSPs by qPCR, further analysis such as genetic perturbation using RNAi/overexpression lines will be necessary to test the involvement of HSPs and the heat shock transcription factor.

5. Conclusions

In conclusion, 4-GK extended the lifespan of Drosophila without affecting sleep or activity. Our findings suggest that 4-GK elicits effects comparable to those of FOS, a conventional oligosaccharide, with numerically greater responses and trend toward efficacy at lower doses. These findings demonstrate the effects of 4-GK in the Drosophila model and provide a basis for future studies to evaluate its potential relevance as a prebiotic in other systems.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nu18040604/s1, Figure S1: Effects of 4-GK feeding without sucrose on sleep–wake behavior; Table S1: Adjusted p values for statistical comparisons shown in Figure 1 and Figure 3. Table S2: RNA-seq analysis: KEGG pathway enrichment. Table S3: RNA-seq analysis: Expression of each representative gene.

Author Contributions

Conceptualization: H.K., R.K., J.T., M.T., C.O., K.N. and K.K.; Methodology: H.K., A.H., R.O., R.K., R.M., R.G., J.T., M.T., C.O., K.N. and K.K.; Software: H.K., A.H., R.O., R.K. and K.K.; Formal analysis: H.K., A.H., R.O., R.K., R.M., R.G., J.T., M.T. and K.K.; Investigation: H.K., A.H., R.O., R.K., R.M., R.G., J.T., M.T. and K.K.; Resources: M.T., C.O., K.N. and K.K.; Writing—Original Draft: H.K., M.T. and K.K.; Writing—Review and Editing: H.K., A.H., R.O., R.K., R.M., R.G., J.T., M.T., C.O., K.N. and K.K.; Supervision: H.K., R.K., J.T., M.T., C.O., K.N. and K.K.; Project administration: M.T., C.O., K.N. and K.K.; Funding acquisition: J.T., M.T., C.O., K.N. and K.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are not publicly available due to institutional data-sharing policy restrictions. The raw sequencing data generated and analyzed during this study are available from the corresponding authors on reasonable request.

Acknowledgments

We are grateful to the members of the Kume Lab for their help in performing this work.

Conflicts of Interest

This work was carried out as part of a collaboration between Meiji Co., Ltd., and Nagoya City University. M.T., C.O., and K.N. are employees of Meiji Co., Ltd. M.T. is also affiliated with Meiji Holdings Co., Ltd. Meiji Holdings had no role in the study design; data collection, analysis, or interpretation; or in the preparation of the manuscript.

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Nutrients 18 00604 g001
Figure 1. Effects of 4-galactosylkojibiose (4-GK) and fructooligosaccharides (FOS) feeding on Drosophila melanogaster longevity. Flies were fed a diet containing 5% sucrose and 1% agar, supplemented with various concentrations of 4-GK, FOS, or sucrose. (a,b) Survival of individually housed flies in glass vials was measured using a Drosophila Activity Monitor. Survival rates were plotted hourly (n = 30). (c) Survival of flies housed in groups in vials was measured and plotted daily. Each plot represents the mean survival rate ± standard error of the mean (SEM) at each time point (n = 5; 10 flies per group). *** p < 0.001, ** p < 0.01, * p < 0.05; log-rank test with Holm correction.
Figure 1. Effects of 4-galactosylkojibiose (4-GK) and fructooligosaccharides (FOS) feeding on Drosophila melanogaster longevity. Flies were fed a diet containing 5% sucrose and 1% agar, supplemented with various concentrations of 4-GK, FOS, or sucrose. (a,b) Survival of individually housed flies in glass vials was measured using a Drosophila Activity Monitor. Survival rates were plotted hourly (n = 30). (c) Survival of flies housed in groups in vials was measured and plotted daily. Each plot represents the mean survival rate ± standard error of the mean (SEM) at each time point (n = 5; 10 flies per group). *** p < 0.001, ** p < 0.01, * p < 0.05; log-rank test with Holm correction.
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Nutrients 18 00604 g002
Figure 2. RNA sequencing (RNA-seq) analysis of flies fed 4-GK. (a,b) Results of RNA-seq analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis at three-week-old: comparison of 5% sucrose-supplemented vs. control groups (a) and 5% sucrose-supplemented vs. 5% 4-GK-supplemented groups (b). See Supplementary Tables S2 and S3 for additional details.
Figure 2. RNA sequencing (RNA-seq) analysis of flies fed 4-GK. (a,b) Results of RNA-seq analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis at three-week-old: comparison of 5% sucrose-supplemented vs. control groups (a) and 5% sucrose-supplemented vs. 5% 4-GK-supplemented groups (b). See Supplementary Tables S2 and S3 for additional details.
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Figure 3. Effects of 4-GK and FOS on sleep–wake behavior in young and aged flies. Age-related changes in locomotor activity and sleep in 4-GK and control groups. (a,b) Average sleep profiles in 30 min intervals over 3 d for young (a) and aged (b) flies. (c,d) Total daily sleep duration in young (c) and old (d) flies. (e,f) Total daily activity counts in young (e) and old (f) flies. (a–f) Each diet contained 5% sucrose and 1% agar, supplemented with the indicated concentrations of 4-GK, or FOS. (c–f) Data are presented as mean ± SEM (n = 29, 30, 32, 29, and 30 for young flies; n = 8, 18, 23, 21, and 25 for old flies, respectively). No significant differences were observed among groups using Kruskal–Wallis test (p = 0.27 for (c); p = 0.17 for (e)) or one-way analysis of variance (p = 0.47 for (d); p = 0.26 for (f)) depending on the result of Shapiro–Wilk test.
Figure 3. Effects of 4-GK and FOS on sleep–wake behavior in young and aged flies. Age-related changes in locomotor activity and sleep in 4-GK and control groups. (a,b) Average sleep profiles in 30 min intervals over 3 d for young (a) and aged (b) flies. (c,d) Total daily sleep duration in young (c) and old (d) flies. (e,f) Total daily activity counts in young (e) and old (f) flies. (a–f) Each diet contained 5% sucrose and 1% agar, supplemented with the indicated concentrations of 4-GK, or FOS. (c–f) Data are presented as mean ± SEM (n = 29, 30, 32, 29, and 30 for young flies; n = 8, 18, 23, 21, and 25 for old flies, respectively). No significant differences were observed among groups using Kruskal–Wallis test (p = 0.27 for (c); p = 0.17 for (e)) or one-way analysis of variance (p = 0.47 for (d); p = 0.26 for (f)) depending on the result of Shapiro–Wilk test.
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Table 1. Number of flies and survival rate.
Table 1. Number of flies and survival rate.
n (Flies)Survival Ratep-Value
(vs. Control)
At 0-Week-OldAt 3-Week-Old
Control160148.8%-
5% sucrose1608452.5%<0.01
5% FOS16012578.1%<0.01
5% 4-GK16012075.0%<0.01
The control group was fed 5% sucrose alone, while the other groups were fed the control diet supplemented with an additional 5% sucrose, 5% FOS, or 5% 4-GK, respectively. Survival rates at 3 weeks were compared with the control using chi-square tests. p values were adjusted for multiple comparisons using the Holm method.
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MDPI and ACS Style

Kato, H.; Hara, A.; Ota, R.; Kobayashi, R.; Miyake, R.; Garibağaoğlu, R.; Tomita, J.; Tsuboi, M.; Oba, C.; Nakamura, K.; et al. 4-Galactosylkojibiose Extends the Lifespan of Drosophila melanogaster. Nutrients 2026, 18, 604. https://doi.org/10.3390/nu18040604

AMA Style

Kato H, Hara A, Ota R, Kobayashi R, Miyake R, Garibağaoğlu R, Tomita J, Tsuboi M, Oba C, Nakamura K, et al. 4-Galactosylkojibiose Extends the Lifespan of Drosophila melanogaster. Nutrients. 2026; 18(4):604. https://doi.org/10.3390/nu18040604

Chicago/Turabian Style

Kato, Haruki, Akari Hara, Rinka Ota, Riho Kobayashi, Ryo Miyake, Rabia Garibağaoğlu, Jun Tomita, Misato Tsuboi, Chisato Oba, Kentaro Nakamura, and et al. 2026. "4-Galactosylkojibiose Extends the Lifespan of Drosophila melanogaster" Nutrients 18, no. 4: 604. https://doi.org/10.3390/nu18040604

APA Style

Kato, H., Hara, A., Ota, R., Kobayashi, R., Miyake, R., Garibağaoğlu, R., Tomita, J., Tsuboi, M., Oba, C., Nakamura, K., & Kume, K. (2026). 4-Galactosylkojibiose Extends the Lifespan of Drosophila melanogaster. Nutrients, 18(4), 604. https://doi.org/10.3390/nu18040604

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