β-hydroxy- β-methylbutyrate Attenuates Age-Dependent Loss of Flight Ability and Extends Lifespan in Drosophila

Abstract

β-hydroxy-β-methylbutyrate (HMB) has been shown to enhance muscle function and strength in older humans and rodents after periods of consumption extending for several weeks. We investigated the feasibility of utilizing Drosophila as a model organism to study the biological effects of HMB on aging muscle when consumed throughout adult life. Using flight ability as an index of flight muscle function, we found that HMB attenuates the age-dependent decline in flight ability. Male and female flies fed a diet supplemented with 10 mg/mL HMB had significantly higher flight scores from median age until the onset of flight senescence than control flies fed a standard diet. HMB supplementation also resulted in improved flight scores in males before median age and delayed the onset of flight senescence in females. Notably, the consumption of HMB throughout adult life increased the rate of survival and extended lifespan. The effect on lifespan did not result from changes in food consumption or body weight. Old flies on the HMB-supplemented diet retained a higher proportion of flight muscle mitochondria whose morphology resembled that of young flies than the control diet group. Together, these results suggest that HMB attenuates the age-dependent decline in flight ability and prolongs lifespan by enhancing muscle health.

Keywords: Drosophila; HMB; aging; flight muscle; lifespan; mitochondria.

Figure A1

Schematic of flight testing and scoring is depicted in this image. Briefly, a fly is introduced into the chamber which is 30 cm × 30 cm × 40 cm, and kept in a dark room. On top there is a light source. Depending on the flight path of the fly, score is assigned. Image created using BioRender. https://BioRender.com/d89t238 accessed on 12 January 2025.

Figure 1

Effect of HMB supplementation on male flight ability. Long-term HMB supplementation attenuates aging-associated loss of flight ability by significantly improving flight ability in median-aged males. (A) Flight scores of HMB-supplemented (red) and control diet (blue) flies compared within age groups. All flight scores were evaluated with Student’s t-test (95% CI, two-tailed distribution) and error bars represent SE, without any corrections. (B): Flight scores of groups within the same diet, comparing flight scores across age groups. HMB-supplemented males (right panel) show delayed onset (at 5 weeks) of the significant decline in flight ability when compared to their normal-diet-reared counterparts, who showed a significant decline at week 4 onwards (left panel). * = (p < 0.05), ** = (p < 0.01),*** = (p <0.001), **** = (p < 0.0001).

Figure 2

Effect of HMB supplementation on female flight ability: Long-term HMB supplementation in adults attenuates aging-associated loss of flight ability by significantly improving flight ability in median-aged females. (A) Flight scores of HMB-supplemented (red) and control diet (blue) female flies, compared within age groups. All flight scores were evaluated with Student’s t-test (95% confidence interval (CI), with two-tailed distribution) and error bars represent SE, without any corrections. (B): Flight scores of groups within the same diet, comparing flight scores across age groups. HMB-supplemented females show a delayed onset of significant decline in flight ability when compared to their normal-diet-reared counterparts, who showed a significant decline from week 4 onwards. * = (p < 0.05), ** = (p < 0.01),*** = (p < 0.001), **** = (p < 0.0001).

Figure 3

Effect of HMB supplementation on rate of survival: both males and females exhibit a significantly improved lifespan when supplemented with HMB. (A): Kaplan–Meier curves of female flies raised on normal and HMB-supplemented diets, shown with 95% Confidence interval (CI). p-value calculated with Log rank test. (B): Kaplan–Meier curves of male flies raised on normal and HMB-supplemented diets shown with 95% CI. p-value calculated with Log rank test. Dashed lines indicate median survival.

Figure 4

HMB supplementation does not alter food consumption: food consumption showed no significant difference within age groups in both females (A) and males (B). Food intake was normalized to number of the flies, and corrected for evaporation, n = 5 for each age, diet, and sex group, with three replicates of each. Flies were allowed to acclimate for 24 h before the measurements were taken. Aging led to a significant decline in food intake in both sexes (p-value < 0.001, post-hoc tukeyHSD test).

Figure 5

Effect of diet on body weight: HMB supplementation had no effect on adult body weight in either wet (A) or dry (B) body weight measurements. Three-way Anova indicated that flies show significant differences in body weights according to sex and age, with females being significantly heavier than males (p < 0.01, pairwise t-test with Bonferroni correction post hoc test) in both dry and wet measurements. Age caused a significant reduction in the wet body weight of females and males (p-value < 0.05, pairwise t-test with bonferroni correction post-hoc test).

Figure 6

Effect of diet and age on resistance to paraquat: the log rank test on survival curves shows no beneficial effect of HMB supplementation on resistance to paraquat-induced oxidative stress. In males, a significant increase in rate of survival was seen at week 4 in HMB-supplemented flies only, while females raised on an HMB diet showed a significantly higher rate of survival at week 3 only.

Figure 7

Effect of diet and age on resistance to hydrogen peroxide: the log rank test shows no beneficial effect of HMB supplementation on resistance to hydrogen peroxide-induced oxidative stress in males or females at young and middle age.

Figure 8

Electron Micrographs of IFM: long-term HMB supplementation improves the prevalence of smaller mitochondria. The top panel shows the EM image IFM of females supplemented with/without HMB at various ages, and bottom panel shows the EM image IFM of males supplemented with/without HMB at various ages. In both sexes, smaller mitochondria (examples of which are indicated with white stars) and an overall higher density of mitochondria were observed. scale bar = 2 μm.

Figure 9

Effect of HMB supplementation on IFM mitochondrial area: long-term HMB supplementation improves the prevalence of smaller mitochondria. (A) The distribution of mitochondrial area in female flies fed different diets across the two age groups. Female flies fed an HMB-supplemented diet show a higher prevalence of smaller mitochondria and a significant (**** p < 0.005) overall reduction in average mitochondrial area at 6 weeks of age. (B): The distribution of mitochondrial area in males flies fed different diets across two age groups. Male flies fed an HMB-supplemented diet show a higher prevalence of smaller mitochondria at six weeks of age and a significant (**** p < 0.005) overall reduction in average mitochondrial area with age.

Figure 10

Effect of HMB supplementation on axes ratio: HMB supplementation significantly alters the mitochondrial morphology, which is evident in the differences in axes ratios. (A) In females, the ratio in young flies raised on a normal diet is 2.05 ± 0.87 and 1.9 ± 0.69 at six weeks, while in flies supplemented with HMB, the ratio is 1.46 ± 0.59 in young flies and remains relatively constant at 1.45 ± 0.707 at six weeks. The ratio is significantly different (**** p < 0.0001, and multiple pairwise t-test with BH correction, two tailed distribution). (B) In males, HMB supplementation leads to a significantly smaller ratio between the major and minor axis, reflecting a preponderance of rounder mitochondria. The ratio is significantly different (** p < 0.01, **** p < 0.0001 and, multiple pairwise t-test with BH correction, two tailed distribution) in young age, a difference that is maintained in old age. The ratio in young flies raised on a normal diet was 2.27 ± 0.58 and increased to 2.56 ± 1.49 at six weeks, while in HMB-fed flies the ratio was 1.51 ± 0.58 in young flies and increased to 2.02 ± 0.89 at six weeks.

Figure 11

Effect of HMB supplementation on citrate synthase activity: long-term HMB supplementation was not shown to lead to a significant improvement in citrate synthase activity levels in females (left panel) or males (right panel). All samples were evaluated using two-way ANOVA.