Introduction

The lifestyle of the current world population has changed. Stress and lack of physical activities, coupled with heightened consumption of fat-rich fast food, are the primary culprits of the emergence of obesity and early onset of various age-associated physiological disorders. Numerous studies have shown that administration of high-fat diet in model organisms affected overall metabolism by increased lipid accumulation, altered gut-microbiome, loss of intestinal health, mal-absorption of nutrients, loss of cardiac health, renal injury, induction of non-alcoholic fatty liver, to name a few (Guida et al. 2019; J. Li et al. 2020; Lian et al. 2020; Liu et al. 2021; Sun et al. 2020; Wali et al. 2020). Cumulative effects of such altered metabolism could drastically impact lifespan, as evident in studies where high-fat diet feed significantly reduced longevity in model organisms like Drosophila melanogaster (Gáliková and Klepsatel 2018; Guida et al. 2019). The past decade has witnessed a surge in the exploration of various interventions that impede high-fat diet-induced aging (Colpo et al. 2018; Kapahi et al. 2017; Landis et al. 2021; Wang et al. 2016). This study aimed to assess the efficacy of α-lipoic acid on longevity in Drosophila melanogaster fed with a high-fat diet.

α-lipoic acid (1,2-dithiolane-3-pentanoic acid) is a sulfur-containing vitamin-like compound gaining popularity as a nutritional supplement to combat obesity and age-associated complications (Shay et al. 2009; Park et al. 2014; Salehi et al. 2019). Discontinuous treatment of Sprague–Dawley rats with α-lipoic acid at 50 mg/kg body weight prevented gain in body weight and had a protective efficacy on the cardiovascular system by decreasing metabolic and cardiac perturbations (Pop et al. 2020). In another study, chronic treatment of diet-induced obese mice with 250 mg/kg of α-lipoic acid in combination with β3-AR agonist CL 316243 improved body mass composition, reduced body fat percentage by 9%, and improved systemic and epididymal white adipose tissue inflammation (Abdul Sater et al. 2022). C57Bl/6 J mice fed with a high-fat diet and administered with α-lipoic acid at 2.5 g/kg of food in conjunction with exercise on a treadmill showed improved fatty acid oxidation and reduced visceral adipose tissue (Le Garf et al. 2021). Murine hypertrophic 3T3-L1 adipocytes exposed to palmitic acid and treated with α-lipoic acid at 1 µM showed decreased adipocyte hypertrophy, reduced intracellular lipid accumulation by reducing gene expression levels of adipogenic markers like PPAR-γ and FABP4, and improved insulin sensitization (Molonia et al. 2024).

In Drosophila melanogaster, α-lipoic acid supplementation reversed age-associated intestinal dysfunction via activation of the endocytic-autophagy network (Du et al. 2020). A study showed that α-lipoic acid in a dose range of 0.001–0.025% w/w of diet could rescue behavioral deficits in ERUPR (h-tau induced ER unfolded protein response) fruit-flies, a transgenic model for Alzheimer’s disease. In contrast, low doses of α-lipoic acid exerted neuroprotective effects in young adults of this mode; a higher dose of α-lipoic acid was required to ameliorate behavioral deficits, thereby indicating a dose-dependent efficacy of α-lipoic acid (Zarini-Gakiye et al. 2021). Another study showed that a conjugate of lipoic acid and sesamol at 30 µM and 60 µM could improve the longevity of female fruit flies by 18% and of male fruit flies by 15% (Jayaraj et al. 2022). Data from these studies prompted us to hypothesize that administration of lipoic acid could negate the detrimental impact of a high-fat diet on longevity in Drosophila melanogaster. Furthermore, we also hypothesized that if lipoic acid indeed positively influenced lifespan in flies, it could also exert similar efficacies on physiological parameters like fecundity, locomotion, and body weight in flies. Lastly, improvement in longevity is also associated with improvement in resistance to various stresses; we also hypothesized that lipoic acid’s positive impact on lifespan in flies could also translate into improved stress resistance in flies fed with a high-fat diet. For this, we performed survival analysis and physiological assays of fecundity, locomotion function, and body weight. We also analyzed the possible protective efficacy of α-lipoic acid against oxidative, heat shock, cold shock, and starvation stresses, respectively. Moreover, various studies have shown that mating status significantly impacts longevity in fruit flies (Branco et al. 2017; Lee et al. 2013; Markow 2011; Vermeulen et al. 2006). Since high-fat diet consumption is known to reduce the mean lifespan in flies, we further wished to analyze the probable impact of a high-fat diet on virgin versus mated flies. For this, we subjected flies to two experimental groups—one in which male and female flies were housed together and another in which virgin male and female flies were housed separately. Our aim was not only to analyze the probable efficacies of lipoic acid on longevity and physiological parameters in flies fed with a high-fat diet but also to observe whether mating status had any outcome on this probable longevity-promoting efficacy.

Materials and methods

Maintenance and age-matching of flies

Wild-type Drosophila melanogaster Canton-S flies were procured from the Drosophila Stock Centre at the University of Mysore, Karnataka, India, and reared at 25 °C ± 2 °C in 12:12 h light and dark cycle under standard fly rearing conditions. Before performing assays, flies were reared and maintained on a semolina-jaggery (SJ) diet [10% semolina (w/v), 10% jaggery (w/v), 0.3% propionic acid (v/v) and 3% methylparaben (v/v)]. To prepare 1000 ml of SJ diet, semolina and jaggery were first thoroughly boiled in 600 ml of distilled water to allow the semolina to be cooked entirely. Since semolina tended to thicken progressively, adding 50 ml of distilled water at regular intervals was required to maintain the consistency of the diet. Once approximate pouring consistency was reached, boiling was stopped, and the diet was allowed to cool down to 50–55 °C. Propionic acid and methylparaben were thoroughly mixed into the diet at this stage. The final volume was adjusted to 1000 ml with distilled water, mixed, and immediately dispensed into 300 ml glass bottles with 30 ml diet per bottle. Care was taken not to pour diet along the inner walls of bottles. Each of these bottles housed 15 male flies and 15 female flies. Mating was allowed for 12 h between 7 days old male and female flies to procure age-matched progeny. All male flies were removed from bottles post-mating. Female flies were allowed to lay eggs in the same bottles for the next 12 h, after which female flies were removed. Eggs from such bottles eventually hatched, pupated, and eclosed as age-matched flies.

Fly treatment with HFD and α-lipoic acid

For all assays, flies were transferred to high-fat diet (HFD) [10% sucrose (w/v), 10% yeast extract (w/v); 2% palmitic acid (w/v), 1% Tween—80 (v/v), 1% agar (w/v), 0.3% propionic acid (v/v) and 3% methylparaben (v/v); HiMedia Laboratories, India]. To prepare 1000 ml of HFD, sucrose and yeast extract were mixed in 750 ml of warm distilled water until the yeast extract was completely dissolved. Separately, palmitic acid was first emulsified in 10 ml of Tween-80, mixed in 140 ml of warm distilled water, and added to the solution with sucrose and yeast extract. Since palmitic acid floated on top as a layer of oil when warmed, rigorous mixing was required to incorporate palmitic acid into the diet solution thoroughly. Following this, any remnant floating layer of palmitic acid was removed using Whatman filter paper. This step was crucial since the wings of flies tended to stick to this layer of palmitic acid and interfered with assay performance. Finally, agar was added to this solution, which consisted of sucrose, yeast extract, and palmitic acid, and thoroughly boiled. The diet was allowed to cool down to 50–55 °C. At this stage, propionic acid and methylparaben were thoroughly mixed into the diet; the final volume was adjusted to 1000 ml with distilled water and immediately dispensed into 50 ml plastic tubes with 10 ml of HFD per bottle.

HFD was supplemented with α-lipoic acid to prepare treatment tubes at a final dosage range of 0.5—2.5 mM. A stock solution of 10 mM α-lipoic acid (HiMedia Laboratories, India) was prepared by first dissolving α-lipoic acid in 100 µL of 75% ethanol, with the final volume being made up with water. Treatment tubes were prepared by dissolving the stock solution of α-lipoic acid to final concentrations of 0.5 mM, 1 mM, 1.5 mM, 2 mM, and 2.5 mM in 10 ml each of HFD. Control tubes consisted of an HFD diet supplemented with water and 100 µL of 75% ethanol. For all assays, 1-day-old, age-matched flies were used; flies were divided into two experimental groups. The first group had newly eclosed, age-matched male and female flies housed within the same tube with HFD supplemented with or without α-lipoic acid (labeled as flies housed together). The second group consisted of virgin, newly eclosed, and age-matched male and female flies housed separately in tubes having HFD supplemented with or without α-lipoic acid (labeled as flies housed separately). For all assays, flies were housed in 50 ml plastic tubes with 10 ml of HFD with or without respective doses of α-lipoic acid.

Longevity assay

To measure fly longevity, flies were transferred to tubes containing HFD with or without different doses of α-lipoic acid. Flies were transferred to tubes with fresh food every 2 days, during which the numbers of dead flies were recorded. The assay was continued till all flies were dead. For the first group, five replicate tubes were set up for each treatment, with fifteen male flies and fifteen female flies housed together (a total of thirty flies) in each replicate tube. For the second group, five replicate tubes were set up for each treatment, with thirty virgin male flies and thirty virgin female flies housed separately for each replicate.

Measurement of fly fecundity

Before performing the fecundity assay, flies from the first and second experimental groups were reared as described in tubes with HFD with or without different doses of α-lipoic acid for 2 weeks. To estimate fly fecundity, two male flies and two female flies were randomly selected from respective experimental groups per treatment tube and transferred to fresh HFD tubes with or without α-lipoic acid. This was marked as 1st day of measurement of fly fecundity. Flies were transferred to fresh tubes with HFD with or without α-lipoic acid every day for a total of 7 days. A total number of eggs laid in each tube per day was recorded. Five replicate tubes were set up for each treatment to perform the fecundity assay for respective groups, with two male flies and two female flies for each replicate tube.

Measurement of climbing efficiency in flies (Negative Geotaxis Assay)

For 2 weeks, flies from respective experimental groups were reared in tubes containing HFD with or without different doses of α-lipoic acid. Following treatment, flies were randomly pooled from respective treatment tubes in batches of ten flies and transferred to empty tubes (15 cm long), and climbing efficiency was measured according to Chambers et al. (2013) with modifications. The tubes were gently tapped to gather the flies at the bottom and then allowed to climb a length of 13 cm (measured with a scale and marked on the side of the tubes). Climbing time was recorded on a stopwatch until all the flies (as a group) in each tube had climbed the 13 cm mark. The assay was repeated thrice for each treatment tube per replicate, with thirty minutes of recovery time in between. For the first group, five replicate tubes were set up for each treatment, with fifteen male flies and fifteen female flies housed together (a total of thirty flies) in each replicate tube. For the second group, five replicate tubes were set up for each treatment, with thirty virgin male flies and thirty virgin female flies housed separately for each replicate.

Measurement of body weight

For 2 weeks, flies from respective experimental groups were reared in tubes containing HFD with or without different doses of α-lipoic acid. Following treatment, male and female flies per treatment tube were randomly pooled, and body weight was measured in batches of ten flies per measurement per sex (Shimadzu ATY224 UniBloc analytical balance). Flies were starved for 2 h before performing the assay. For the first group, five replicate tubes were set up for each treatment, with fifteen male flies and fifteen female flies housed together (a total of thirty flies) in each replicate tube. For the second group, five replicate tubes were set up for each treatment, with thirty virgin male flies and thirty virgin female flies housed separately for each replicate.

Stress resistance assays

Oxidative stress resistance

For 2 weeks, flies from respective experimental groups were housed in tubes containing HFD with or without different doses of α-lipoic acid. Following this, flies were subjected to an oxidative challenge, according to Weber et al. with modifications (Weber et al. 2012). Briefly, 5% H2O2 (HiMedia Laboratories, India) and 1% sucrose solution were added on filter paper strips placed inside tubes with 1% agar. The assay was continued till all flies were dead. For the first group, five replicate tubes were set up for each treatment, with five male flies and five female flies housed together (a total of ten flies) in each replicate tube. For the second group, five replicate tubes were set up for each treatment, with ten virgin male flies and ten virgin female flies housed separately, for each replicate.

Heat shock resistance

Heat shock resistance assay was performed according to Sorensen et al. with modifications (Sørensen et al., 2006). For 2 weeks, flies from respective experimental groups were reared in tubes containing HFD with or without different doses of α-lipoic acid. Following this, tubes with flies were kept inside an incubator at 37 °C, with a 12:12 light–dark cycle. Flies were transferred to fresh tubes with HFD with or without different doses of α-lipoic acid every 2 h, during which number of dead flies was recorded during each transfer. The assay was continued till all flies were dead. For the first group, five replicate tubes were set up for each treatment, with five male flies and five female flies housed together (a total of ten flies) in each replicate tube. For the second group, five replicate tubes were set up for each treatment, with ten virgin male flies and ten virgin female flies housed separately for each replicate.

Cold shock recovery

Cold shock recovery assay was performed according to Czajka et al. with modifications (Czajka et al. 1990). For 2 weeks, flies from respective experimental groups were reared in tubes containing HFD with or without different doses of α-lipoic acid. Following this, tubes with flies were exposed to 4 °C inside a refrigerator for 1 to 2 min until all flies ceased to move. The tubes were then returned to room temperature, and the recovery time of the flies was recorded on a stopwatch. Recovery time was measured until all flies in each tube regained activity. For the first group, five replicate tubes were set up for each treatment, with five male flies and five female flies housed together (a total of ten flies) in each replicate tube. For the second group, five replicate tubes were set up for each treatment, with ten virgin male flies and ten virgin female flies housed separately for each replicate.

Starvation resistance

Starvation resistance assay was performed according to Li et al., with modifications (Li et al. 2016). For 2 weeks, flies from respective experimental groups were reared in tubes containing HFD with or without different doses of α-lipoic acid. Following treatment, flies were transferred to tubes containing only 1% agar. To prevent desiccation, tubes were provided with filter paper discs dipped in sterile distilled water. Flies were transferred to fresh 1% agar tubes every 2 h, during which the number of dead flies was recorded during each transfer. For the first group, five replicate tubes were set up for each treatment, with five male flies and five female flies housed together (a total of ten flies) in each replicate tube. For the second group, five replicate tubes were set up for each treatment, with ten virgin male flies and ten virgin female flies housed separately for each replicate.

Statistical analysis

Data from lifespan assays were analyzed with OASIS software via Kaplan–Meier survival analysis, and survival curves were generated using GraphPad Prism version 5 (Yang et al. 2011). Data from other assays were analyzed using the GraphPad Prism version 5. Two-way ANOVA with Bonferroni post-hoc test was used to analyze data of all assays except the fecundity assay. One-way ANOVA with Dunnet’s post-hoc tests was used for the fecundity assay. p-values for levels of significance are represented as * < 0.05, ** < 0.01, *** < 0.001 and **** < 0.0001.

Results

Efficacy of α-lipoic acid on mean lifespan

To assess the effect of α-lipoic acid on longevity, flies were fed HFD with α-lipoic acid at doses of 0.5 mM, 1 mM, 1.5 mM, 2 mM, and 2.5 mM, respectively. In the experimental group that comprised of male and female flies housed together, female flies showed no significant improvement in mean lifespan when fed with HFD and treated with α-lipoic acid at 0.5 mM (19.2 days), 1 mM (19.6 days) and 1.5 mM (20.4 days) as compared to only HFD fed flies (18.8 days). A significant increase in mean lifespan was observed in female flies fed with HFD and α-lipoic acid at 2 mM (22 days, 22% increase) and 2.5 mM (22.4 days, 24% increase), respectively, when compared to flies fed only with HFD [Fig. 1a, Fig. 1c]. In male flies fed with HFD, no significant improvement in mean lifespan was observed with α-lipoic acid at 0.5 mM (17.2 days), 1 mM (18 days), 1.5 mM (18.8 days) and 2 mM (19.2 days) respectively, as compared to flies fed with only HFD (17.2 days); a significant improvement was observed with α-lipoic acid only at 2.5 mM (20.4 days, 19% increase) as compared to male flies fed only with HFD [Fig. 1b, Fig. 1c].

Fig. 1
figure 1

Efficacy of α-lipoic acid on mean lifespan. I—Flies housed together: a Effect of α-lipoic acid on percentage survival in female Canton-S flies. b Effect of α-lipoic acid on percentage survival in male Canton-S flies. c Effect of α-lipoic acid on mean survival of male and female flies. II—Flies housed separately: d Effect of α-lipoic acid on percentage survival in female Canton-S flies. e Effect of α-lipoic acid on percentage survival in male Canton-S flies. f Effect of α-lipoic acid on mean survival of male and female flies. No significant interaction existed between treatment and gender of flies of both groups, respectively. Data represented as mean lifespan (in days) with error bars denoting SD. Symbols represented as * p < 0.05, ** p < 0.01 and *** p < 0.001, two-way ANOVA, Bonferroni post-hoc test (HFD control vs. α-lipoic acid supplemented diets). For the first group, for each treatment, replicates = 5, n = 30 flies (15 males + 15 females) for each replicate. For the second group, for each sex and treatment, replicates = 5, n = 30 male flies and 30 female flies, separately for each replicate

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In the experimental group of male and female flies housed separately, the mean lifespan of female flies administered with α-lipoic acid at 0.5 mM (19.6 days) was comparable to flies fed with only HFD (16.8 days). Significant improvement was observed in female flies fed with α-lipoic acid at 1 mM (20.4 days, 21% increase), 1.5 mM (23.6 days, 40% increase), 2 mM (26 days, 55% increase) and 2.5 mM (25.6 days, 52% increase) respectively, as compared to female flies fed with only HFD [Fig. 1d, Fig. 1f]. Male flies fed with HFD showed no significant increase in mean lifespan with α-lipoic acid at 0.5 mM (15.6 days) and 1 mM (16 days), respectively, as compared to male flies fed with only HFD (15.2 days). A significant increase in mean lifespan was observed with male flies fed with HFD and treated with α-lipoic acid at 1.5 mM (22 days, 45% increase), 2 mM (22.4 days, 47% increase), and 2.5 mM (23.2 days, 53% increase) respectively, as compared to male flies fed with only HFD [Fig. 1e, Fig. 1f]. To summarize, α-lipoic acid improved longevity in flies of both experimental groups, which was significantly impacted by mating status, dose, and gender of flies.

Effect of α-lipoic acid on fecundity in female flies

To test efficacy on female reproductive output, female flies from the first and second groups were housed on HFD with or without different doses of α-lipoic acid and assessed for the number of eggs laid over 7 days. In female flies belonging to the first group, no significant changes in mean eggs laid per female were observed with α-lipoic acid at 0.5 mM (215 eggs), 1 mM (212 eggs), and 1.5 mM (213 eggs), respectively, as compared to female flies reared only in HFD (220 eggs) [Fig. 2a]. A significant decrease in mean eggs laid per female was observed with α-lipoic acid at 2 mM (206 eggs, 6.36% decrease) and 2.5 mM (201 eggs, 8.64% decrease), respectively, as compared to female flies reared only in HFD. In female flies belonging to second group, fecundity of female flies decreased with increasing dosage of α-lipoic acid at 1 mM (179 eggs, 15.57% decrease), 1.5 mM (180 eggs, 15.09% decrease), 2 mM (161 eggs, 24.1% decrease) and 2.5 mM (144 eggs, 32.08% decrease) as compared to female flies reared only in HFD (212 eggs); female flies administered with α-lipoic acid at 0.5 mM showed decrease in mean eggs laid (205 eggs) which was non-significant [Fig. 2b]. Collectively, α-lipoic acid reduced fecundity in flies of both groups, but the effect was more prominent in flies housed separately.

Fig. 2
figure 2

Effect of α-lipoic acid on fecundity in female flies, a flies housed together and b flies housed separately. Data represented mean number of eggs laid by a female for 7 days, with error bars denoting SD. Symbols represented as ** p < 0.01 and *** p < 0.001, one-way ANOVA, Dunnet’s post-hoc test (HFD control vs. α-lipoic acid supplemented diets per day). 5 replicate vials were set up for each treatment, with two males and two females in each replicate

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Effect of α-lipoic acid on climbing efficiency in flies

Male and female flies of both experimental groups were assessed for their climbing efficiency when reared in HFD administered with or without α-lipoic acid. In the first group, female flies exhibited significant improvement in climbing efficiency when administered with α-lipoic acid at 1.5 mM (14 s), 2 mM (12 s), and 2.5 mM (10 s), respectively, as compared to female flies reared in HFD alone (19 s). Male flies exhibited significant improvement in climbing efficiency when administered with α-lipoic acid at 2 mM (15 s) and 2.5 mM (13 s), respectively, as compared to male flies reared in HFD alone (19 s) [Fig. 3a]. In the second group, female flies exhibited significant improvement in climbing efficiency at 1 mM (18 s), 1.5 mM (14 s), 2 mM (11 s), and 2.5 mM (11 s), respectively, as compared to female flies reared only in HFD (20 s). In male flies, lower doses of α-lipoic acid at 0.5 mM (20 s) and 1 mM (19 s) non-significantly improved climbing efficiency; significant improvement in climbing efficiency was observed at 1.5 mM (18 s), 2 mM (13 s) and 2.5 mM (11 s) respectively, as compared to male flies reared only in HFD [Fig. 3b]. To infer, α-lipoic acid improved climbing efficiency in flies of both groups irrespective of mating status.

Fig. 3
figure 3

Effect of α-lipoic acid on climbing efficiency in flies in a flies housed together and b flies housed separately. Data represented as mean time taken by 10 flies per replicate per treatment (in s) to climb 13 cm, with error bars denoting SD. Symbols represented as * p < 0.05 and *** p < 0.001, two-way ANOVA, Bonferroni post-hoc test (HFD control vs. α-lipoic acid supplemented diets). No significant interaction existed between treatment and gender of flies. For the first group, for each treatment, replicates = 5, n = 30 flies (15 males + 15 females) in each replicate. For the second group, for each sex and treatment, replicates = 5, n = 30 male flies and 30 female flies, separately for each replicate

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Effect of α-lipoic acid on body weight in flies

The effect of α-lipoic acid on body weight in flies from both groups fed with HFD was assessed. In the first group, a significant decrease in mean body weight was observed in female flies housed in HFD and administered with α-lipoic acid at 2 mM (9.68 mg, 16.83% decrease) and 2.5 mM (9.16 mg, 21.30% decrease) respectively, as compared to female flies reared in HFD alone (11.64 mg). Similarly, male flies also exhibited a significant decrease in mean body weight when reared in HFD and administered with α-lipoic acid at 2 mM (6.18 mg, 19.10% decrease) and 2.5 mM (5.82 mg, 23.82% decrease), respectively, as compared to male flies housed in HFD alone (7.64 mg) [Fig. 4a]. In the second group, a significant decrease in mean body weight of female flies was observed with α-lipoic acid at 1 mM (11.46 mg, 11.84% decrease), 1.5 mM (11.14 mg, 14.30% decrease), 2 mM (10.7 mg, 17.69% decrease) and 2.5 mM (10.02 mg, 22.92% decrease) respectively, as compared to female flies housed only in HFD (13 mg). In male flies fed with HFD, a significant decrease in mean body weight was observed with α-lipoic acid at 1.5 mM (7.4 mg, 12.32% decrease), 2 mM (6.52 mg, 22.74% decrease) and 2.5 mM (6 mg, 28.90% decrease) respectively, as compared to flies housed only in HFD (8.44 mg). Lower doses of α-lipoic acid (0.5 mM in female flies; 0.5 mM and 1 mM in male flies) did not have any significant influence on body weight in flies of this group [Fig. 4b]. To summarize, α-lipoic acid reduced body weight in flies of both groups, but the reduction was more remarkable in flies housed separately and probably could be correlated to climbing efficiency in flies.

Fig. 4
figure 4

Effect of α-lipoic acid on body weight in flies in a flies housed together and b flies housed separately. Data represented as mean body weight of 10 flies per replicate per treatment with error bars denoting SD. Symbols represented as * p < 0.05, ** p < 0.01 and *** p < 0.001, two-way ANOVA, Bonferroni post-hoc test (HFD control vs. α-lipoic acid supplemented diets). No significant interaction existed between treatment and gender of flies. For the first group, for each treatment, replicates = 5, n = 30 flies (15 males + 15 females) in each replicate. For the second group, for each sex and treatment, replicates = 5, n = 30 male flies and 30 female flies, separately for each replicate

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Effect of α-lipoic acid on stress tolerance in flies

Male and female flies from both groups housed in HFD with or without α-lipoic acid were assessed for tolerance to oxidative, heat shock, cold shock, and starvation stresses, respectively. When exposed to oxidative stress, female flies from the first group exhibited a significant increase in mean lifespan with α-lipoic acid only at 2.5 mM (16 h, 38% increase) as compared to female flies reared only on HFD (11.6 h); male flies did not exhibit any significant increase in mean lifespan at any doses of α-lipoic acid as compared to flies housed only in HFD [Fig. 5a]. In female flies from the second group, an increased mean lifespan was observed with α-lipoic acid at 2 mM (19.6 h, 40% increase) and 2.5 mM (19.2 h, 37% increase), respectively, as compared to flies housed only in HFD (14 h). Male flies exhibited a significant increase in mean lifespan with α-lipoic acid only at 2.5 mM (20.4 h, 50% increase) as compared to male flies housed only in HFD (13.6 h) [Fig. 5b].

Fig. 5
figure 5

Efficacy of α-lipoic acid on resistance to oxidative stress. I—Flies housed together: a Effect of α-lipoic acid on percentage survival in female flies. b Effect of α-lipoic acid on percentage survival in male flies. c Effect of α-lipoic acid on mean survival of male and female following oxidative stress. II—Flies housed separately: d Effect of α-lipoic acid on percentage survival in female flies. e Effect of α-lipoic acid on percentage survival in male flies. f Effect of α-lipoic acid on mean survival of male and female flies following oxidative stress. Data represented as mean survival (in h) following exposure to oxidative stress in flies, with error bars denoting SD. No significant interaction existed between treatment and gender of flies. Symbols represented as * p < 0.05, ** p < 0.01 and *** p < 0.001, two-way ANOVA, Bonferroni post-hoc test (HFD control vs. α-lipoic acid supplemented diets). For the first group, for each treatment, replicates = 5, n = 10 flies (5 males + 5 females) in each replicate. For the second group, for each sex and treatment, replicates = 5, n = 10 male flies and 10 female flies, separately for each replicate

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Next, male and female flies from both groups were assessed for tolerance to heat shock stress when housed in HFD with or without α-lipoic acid. Following exposure to heat shock stress, female flies in the first group showed a mildly significant increase in mean lifespan with α-lipoic acid only at higher doses of 2 mM (41 h, 29% increase) and 2.5 mM (40 h, 38% increase), respectively, as compared to female flies fed with HFD alone (29 h). Male flies showed a mild significant increase in mean lifespan only at the highest dose of α-lipoic acid at 2.5 mM (45 h, 32% increase) as compared to only the fed group (34 h) [Fig. 6a]. Male and female flies of the second group did not have any significant changes in mean lifespan following administration with different doses of α-lipoic acid and exposure to heat shock stress [Fig. 6b].

Fig. 6
figure 6

Effect of α-lipoic acid on resistance to heat shock stress in flies. I—Flies housed together: a Effect of α-lipoic acid on percentage survival in female flies. b Effect of α-lipoic acid on percentage survival in male flies. c Effect of α-lipoic acid on mean survival of male and female following heat shock stress. II—Flies housed separately: d Effect of α-lipoic acid on percentage survival in female flies. e Effect of α-lipoic acid on percentage survival in male flies. f Effect of α-lipoic acid on mean survival of male and female flies following heat shock stress. Data represented as mean survival (in min) following exposure to heat shock stress in flies, with error bars denoting SD. Data represented as mean survival (in min) following exposure to heat shock stress in flies, with error bars denoting SD. No significant interaction existed between treatment and gender of flies. Symbol represented as * p < 0.05, two-way ANOVA, Bonferroni post-hoc test (HFD control vs. α-lipoic acid supplemented diets). For the first group, for each treatment, replicates = 5, n = 10 flies (5 males + 5 females) in each replicate. For the second group, for each sex and treatment, replicates = 5, n = 10 male flies and 10 female flies, separately for each replicate

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Male and female flies from both groups were then assessed for tolerance to cold shock stress when housed in HFD with or without α-lipoic acid. In the first group, no significant changes in recovery time were observed in male and female flies following exposure to cold shock at any dose of α-lipoic acid as compared to flies fed with only HFD [Fig. 7a]. In the second group, only female flies exhibited a mild but significant improvement in mean recovery time following exposure to cold shock with α-lipoic acid only at the highest dose of 2.5 mM (5 s), as compared to female flies reared in HFD alone (7 s). Male flies exhibited no significant changes in mean recovery time following cold shock exposure at any dose of α-lipoic acid as compared to male flies housed only in HFD [Fig. 7b].

Fig. 7
figure 7

Effect of α-lipoic acid on resistance to cold shock stress in a flies housed together and b flies housed separately. Data represented as mean recovery time (in min) following exposure to cold shock stress in flies, with error bars denoting SD. No significant interaction existed between treatment and gender of flies. Symbol represented as * p < 0.05, two-way ANOVA, Bonferroni post-hoc test (HFD control vs. α-lipoic acid supplemented diets). For the first group, for each treatment, replicates = 5, n = 10 flies (5 males + 5 females) in each replicate. For the second group, for each sex and treatment, replicates = 5, n = 10 male flies and 10 female flies, separately for each replicate

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Lastly, male and female flies from both groups were assessed for their tolerance to starvation stress when housed with HFD with or without α-lipoic acid. No significant changes in mean lifespan were observed in male and female flies of either group at any dose of α-lipoic acid as compared to flies reared in HFD alone [Fig. 8a, Fig. 8b]. To collectively infer, α-lipoic acid improved resistance to oxidative stress, but the efficacy was selective and depended on mating status in flies. α-lipoic acid improved resistance to heat shock in flies housed together, whereas no such effect was seen in flies housed separately. α-lipoic acid improved resistance to cold shock in only female flies housed separately, whereas no change was observed in flies housed together. No change in resistance to starvation stress was observed in flies of either group when administered with α-lipoic acid.

Fig. 8
figure 8

Effect of α-lipoic acid on resistance to starvation stress in flies. I—Flies housed together: a Effect of α-lipoic acid on percentage survival in female flies. b Effect of α-lipoic acid on percentage survival in male flies. c Effect of α-lipoic acid on mean survival of male and female following starvation stress. II—Flies housed separately: d Effect of α-lipoic acid on percentage survival in female flies. e Effect of α-lipoic acid on percentage survival in male flies. f Effect of α-lipoic acid on mean survival of male and female flies following starvation stress. Data represented as mean survival (in h) following exposure to starvation stress in flies, with error bars denoting SD, two-way ANOVA, Bonferroni post-hoc test (HFD control vs. α-lipoic acid supplemented diets). No significant interaction existed between treatment and gender of flies. For the first group, for each treatment, replicates = 5, n = 10 flies (5 males + 5 females) in each replicate. For the second group, for each sex and treatment, replicates = 5, n = 10 male flies and 10 female flies, separately for each replicate

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Discussion

Efficacy of α-lipoic acid to prolong lifespan in flies was dose-dependent and probably depended on mating status

In this study, we reared D. melanogaster flies in HFD and subjected them to two different housing conditions: the first group, in which both male and female flies were housed together, and the second group, in which virgin male and female flies were housed separately. Treatment with α-lipoic acid overall increased mean lifespan in male and female flies from both these groups. However, this improvement in lifespan was more pronounced in the second group, where male and female flies were housed separately. In this group, the pro-longevity effect of α-lipoic acid was observed even at a lower dose of 0.5 mM in female flies. In contrast, male flies had an increased lifespan with α-lipoic acid at 1.5 mM dosage onwards. In the first group, where male and female flies were housed together, increased mean lifespan in female flies was observed with α-lipoic acid at higher doses of 2 mM and 2.5 mM. In contrast, an increased lifespan was observed only at the highest dose of 2.5 mM with male flies. Interestingly, the mean lifespan of male and female flies fed with only HFD (control flies) was higher in the first group than in the second group.

Nutrition plays a significant role in the longevity of flies. Rearing flies on a high-fat diet has been shown to negatively impact lifespan in flies by reducing median and maximum lifespan (Liao et al. 2021). Incorporation of palmitic acid in fly feed has been shown to significantly impact fly longevity by as much as 20%, with higher fat percentages (5% and 10%, respectively) incurring higher mortality rates in flies. Eickelberg et al. 2022 showed that increasing dietary fat content in the 3–12% range negatively impacted lifespan in male and female flies in a dose-dependent manner. One probable reason for this reduced longevity might be attributed to the progressive accumulation of lipid peroxides associated with HFD consumption. Supplementation with α-lipoic acid has been shown to reduce oxidative stress related to high-fat consumption. In a study by Sztolsztener et al., male Wistar rats fed with a high-fat diet (60% fat content) and administered with α-lipoic acid at 500 mg/kg body weight showed reduced expression of oxidative stress markers COX2, 5LOX, and lipid peroxidation markers (Sztolsztener et al. 2022). In a similar study by Dajnowicz et al., male Wistar rats fed with a high-fat diet (60% fat content) and administered with α-lipoic acid at 30 mg/kg body weight also showed reduced levels of lipid hydroperoxides, advanced oxidation protein products, and proinflammatory markers (Dajnowicz-Brzezika et al. 2022). The efficacy of pro-longevity interventions is also significantly dependent on the fat percentage being administered. For instance, in a study by Li et al., green tea catechins and broccoli extract improved lifespan only in flies fed with 5% fat in HFD as compared to flies fed with 10% fat in HFD (Y. M. Li et al. 2008). Our study incorporated a relatively moderate dose of 2% palmitic acid. Even at this moderate fat percentage, not all doses of α-lipoic acid negated detrimental effects associated with palmitic acid consumption in both groups’ flies. This suggests a possible dose-dependent efficacy of α-lipoic acid. However, if dose-dependent efficacy was the sole factor, then flies belonging to the second group would also have exhibited a similar trend. Since this was not the case, there appear to be other contributing factors that affect the efficacy of α-lipoic acid.

Housing conditions impacted the pro-longevity efficacy of α-lipoic acid and was dependent on mating status in flies

Fly longevity is gender-specific and greatly influenced by mating behaviors. Reproduction has a considerable energy burden on lower-model organisms. Studies have shown that interventions that reduced fecundity in flies also improved their lifespan by conserving and channeling energy earmarked for reproductive output (De Loof 2011; Hoffman et al. 2021). The efficacy of α-lipoic acid observed in our study plausibly depended on housing conditions and the gender of flies fed with HFD. This is evident from our data from fecundity assay wherein higher doses of α-lipoic acid (2 mM and 2.5 mM) reduced reproductive output in female flies from the first group; these were interestingly also the same doses at which longevity of female flies was increased. As flies in the first group were housed together, there were no restrictions on mating behavior. Thus, a higher dose of α-lipoic acid might have been required to reduce reproductive output in such vigorously mating flies. This reduced fecundity in flies administered with higher doses of α-lipoic acid could be due to the inherent efficacy of α-lipoic acid to prolong lifespan by redirecting reproductive energy to be invested for lifespan extension in flies. This is further emphasized by data on mean lifespan and female fecundity in flies from the second group. Female flies from the second group exhibited a progressive reduction in reproductive output at even lower doses of α-lipoic acid (1 mM onwards), which were incidentally also doses at which lifespan had increased in female flies of this group. When housed separately, flies in the second group were deprived of natural mating responses. Studies have shown that reproductive health in fruit flies, as with most organisms, is most robust during the initial weeks of life and gradually starts declining with progressive aging (Promislow et al. 2022). Hence, in this study, when flies were eventually allowed to mate during fecundity assay post-treatment with different doses of α-lipoic acid in HFD for 15 days, the reproductive health of flies in this group might have already started to lose robustness. Additionally, flies might have required time to overcome a lag in reproduction due to being housed separately. Treatment with α-lipoic acid further reduced reproductive output in this group’s female flies, thereby reiterating that α-lipoic acid might perturb natural reproductive behavior to increase lifespan in flies. The day-wise egg-laying trend of female flies in the second group also reaffirmed a dose-dependent efficacy wherein higher doses of α-lipoic acid (1 mM onwards) progressively reduced the number of eggs laid by female flies observed as early as day 1 of fecundity assay; reduction in day-wise egg laying trend in flies of the first group was not as drastic and was observed only with higher doses of α-lipoic acid (2 mM and 2.5 mM) [Supplementary data, Fig a, Fig b]. Thus, the combined effects of housing conditions and treatment with α-lipoic acid might have had a conjunctive effect on increasing mean lifespan in flies fed with HFD, which in turn probably depended heavily on the reduction of reproductive output in female flies.

Treatment with α-lipoic acid improved climbing efficiency in flies

Loss of locomotion function is a common outcome of progressive aging (Magwere et al. 2006). Aging coupled with a high-fat feed might aggravate the loss of locomotion function in flies. This is validated in a study by Moghadam et al., where inhibition of fat accumulation in flies improved lifespan and locomotion (Nasiri Moghadam et al. 2015). A study by Nayak and Mishra showed that the incorporation of 2% coconut oil in fly feed decreased both day and night time activities in flies fed with HFD compared to control flies (Nayak and Mishra 2021). Data from Eickelberg et al. showed that supplementation with 12% butterfat significantly reduced climbing efficiency in flies, with female flies being more affected than male flies (Eickelberg et al. 2022). Based on these data, we hypothesized that doses of α-lipoic acid that promoted lifespan extension might also improve locomotion function. Also, a study by Isaac et al. showed that mating changed locomotion function in flies, and mated females were more active than their non-mated counterparts (Isaac et al. 2010). Since α-lipoic acid reduced fecundity in flies of both groups, we wondered whether mating status would impact locomotion in flies administered with α-lipoic acid. Data from our negative geotaxis assay showed that α-lipoic acid progressively improved vertical movement in flies in both groups. However, improvement in locomotion function was restricted to only higher doses of α-lipoic acid in both groups of flies, thereby highlighting that beneficial efficacies were consistent only with higher doses of α-lipoic acid. Moreover, this improvement was observed in both groups of flies, implying that locomotion function improvement by α-lipoic acid was probably independent of mating status in flies. Nonetheless, α-lipoic acid might have successfully rescued muscle function in flies by eliminating the burden of increased body weight due to the consumption of HFD.

Pro-longevity efficacy of α-lipoic acid depended partly on the reduction of body weight in flies

Data from the assessment of mean body weight highlighted that α-lipoic acid reduced mean body weight in flies of both groups. Moreover, this reduction in body weight followed a trend similar to that of the longevity assay. In flies of the first group, only higher doses of α-lipoic acid (2 mM and 2.5 mM) significantly reduced mean body weight in both male and female flies. In the second group of flies, even lower doses of α-lipoic acid (1 mM in female flies, 1.5 mM in male flies) significantly reduced mean body weight. Incidentally, these were also similar doses that positively influenced lifespan extension in flies. α-lipoic acid has been shown to improve metabolism and mobilization of fat stores in model organisms (Erickson et al. 2020). Higher fat stores in adult flies have been associated with loss of locomotion function. This could be attributed to increased body weight hindering efficient and smooth movement in flies fed with HFD. Our study’s body weight analysis showed that α-lipoic acid could reduce body weight in flies of both groups, albeit at different doses. Recently, a fascinating study by Rodrigues et al. has shown that lipid accumulation is governed by the status of reproduction, where germline-ablated groups of flies had lesser lipid accumulation than the fertile group (Rodrigues et al. 2024). Our study observed a similar trend where flies housed separately had reduced body weight with α-lipoic acid at lower doses than flies housed together. This reduction in body weight in flies could result from the ability of α-lipoic acid to deplete fat stores in flies and could be a probable contributing factor to the efficacy of α-lipoic acid in promoting longevity in flies. Also, depleted reproductive requirements in flies housed separately might have reduced their requirement to store more lipids. Another extrapolated factor could be the feeding behavior of flies of both groups. Since flies of the first group were allowed to mate and reproduce, it could be hypothesized that their feeding requirement was higher than that of flies of the second group, which were denied mating. Due to this high feeding requirement, flies of the first group would have fed more on HFD than the second group and, as such, had more overall exposure to HFD. Thus, lower doses of α-lipoic acid would not have sufficed to induce any positive influence in flies of the first group. However, data on feeding behavior would be required to substantiate this hypothesis further.

Pro-longevity efficacy of α-lipoic acid was associated with improvement in sensitivity to various stresses

Aging in flies is associated with progressive loss of cellular homeostasis and perturbed adaptive responses (Belyi et al. 2020; Vermeulen and Loeschcke 2007). This, in turn, increases sensitivity to various forms of stress. Additionally, feeding flies with a high-fat diet has been shown to induce significant oxidative stress in flies by increasing the accumulation of reactive oxygen species (Heinrichsen et al. 2014; Heinrichsen and Haddad 2012; Magwere et al. 2006). A study by Paula et al. showed that the incorporation of 20% coconut oil in fly feed resulted in a 47% increase in the production of reactive oxygen species, a 19% increase in lipid peroxidation, and increased activities of antioxidant enzymes catalase and superoxide dismutase (Trindade de Paula et al. 2016). Another interesting study by Koliada et al. showed that mating status significantly impacted the status of oxidative stress in flies; data from this study showed that flies with multiple mating partners had increased SOD activity, increased total peroxidases activities, and higher levels of advanced glycation end products (Koliada et al. 2020).

Data from the fecundity assay in this study showed that female flies of the first group exhibited reduced reproductive output only at higher doses of α-lipoic acid. Flies of the first group were exposed to a more natural rearing environment than flies of the second group. Hence, it could be possible that normal physiological responses, mating status, for instance, could induce a higher oxidative load in flies of the first group. In conjunction with a high-fat diet feed, this could have caused more significant oxidative stress in flies of the first group, which was impossible to facilitate with lower doses of α-lipoic acid. Therefore, only a slight improvement in survival following oxidative stress exposure could be observed in female flies of the first group. The absence of mating responses in the second group of flies could have resulted in a lesser accumulation of oxidative load. Hence, even lower doses of α-lipoic acid could improve resistance to oxidative stress in flies of the second group.

Lipid stores in adult fly bodies modulate various neuro-endocrine signaling in flies. Studies have shown that increased body lipid content in flies influenced resistance to cold shock and starvation stresses by modulating water balance and ion homeostasis (Sinclair et al. 2007). A fascinating ng study by Stobdan et al. showed that a high-fat diet downregulated expression levels of Turandot genes like TotA, TotC, and TotM, which are usually responsible for tolerance to various stresses in Drosophila melanogaster; this study also showed that a high-fat diet resulted in differential expression of fat body-specific genes in Drosophila melanogaster that regulated lipid metabolism (Stobdan et al. 2019). Another study by Sisodia et al. showed that nutrients in diet impacted the resistance of Drosophila anannassae to multiple stresses (Sisodia and Singh 2012). Data from our studies prompted us to hypothesize that decreased body weight in flies fed with a high-fat diet and administered with α-lipoic acid altered lipid stores within flies, thereby modulating resistance to various stresses. In our study, when treated with different doses of α-lipoic acid, only the flies of the first group had improved tolerance to heat shock, whereas no change in heat shock stress response was observed in the flies of the second group. One probable reason could be the increased expression of heat shock proteins by α-lipoic acid in the first group of flies. Since α-lipoic acid showed no improvement in heat shock stress in flies of the second group, it could be inferred that mating negated the expression of genes, which had improved heat shock stress resistance in flies of the first group. This intriguing hypothesis of this study warrants further investigation. Alteration in lipid stores mediated by α-lipoic acid could also have impacted genes like Turandot in flies of both groups, which in turn failed to improve resistance to starvation or cold shock stresses significantly. Also, as discussed previously, mating status could significantly impact lipid stores in flies, with mated and fertile flies having an increased requirement to store more lipids than non-mated flies (Rodrigues et al. 2024). Thus, it could be implied that α-lipoic acid, together with mating status in flies, could have led to altered lipid stores within flies, ultimately leading to altered response to various stresses. To summarize, data from stress resistance assays showed that α-lipoic acid selectively influenced stress resistance which again depended considerably on the mating status of flies. This also highlights that interventions that improve lifespan might not have a universal effect across all aspects of aging physiology.

Conclusion—overall impact of α-lipoic acid and mating status on flies fed with a high-fat diet

Aging is a complex and holistic process that gradually and eventually interferes with the smooth functioning and homeostasis of the body. Diverse factors affect and modulate aging; it is increasingly difficult to identify one single cause of aging and even more challenging to control. Given the complexity and diversity of the aging process, it is quite understandable to have knowledge gaps in biogerontology. In his article, Suresh Rattan identified evolutionary knowledge, biological survival and death aspects, and heterogeneity in aging progression and phenotype as three significant categories of knowledge gaps in the biology of aging research that need to be addressed (Rattan 2024). Keeping this in mind, we attempted to dissect whether this study could contribute, even in a minuscule, towards bridging this gap. The list will be exhaustive if we scan through studies on dietary interventions for aging, and our study also shares similar aspects of many of such investigations. We investigated whether α-lipoic acid could influence longevity, physiology, and stress resistance in flies fed with a high-fat diet and housed as virgin or mated flies. Our study primarily attempted to screen for a dietary intervention for aging. Data from this study suggested that α-lipoic acid increased lifespan, positively influenced physiology, and improved fly stress resistance when flies were fed a high-fat diet. However, these efficacies significantly depended on the gender of the flies, their status of mating, and the dose of α-lipoic acid, with the status of mating and diet nutrients being the most essential factors. Mating status can crucially govern lipid metabolism in flies with higher lipid levels affecting longevity, locomotion, body weight, and resistance to various stresses. Nutrients in the diet alone could dramatically impact the outcome of such studies as it did with our research, where α-lipoic acid failed to influence mean lifespan in flies fed with a 10% sugar-yeast (SY) diet (unpublished data) but significantly increased mean lifespan in flies when fed with a high-fat diet. We feel that data from this study merely scratches at the tip of the massive iceberg of aging, and the investigation has to transcend the levels of expression patterns of genes and proteins to regulatory molecules that control aging down to epigenetic modifications. Only then we would genuinely be able to understand and appreciate the impact that α-lipoic acid and similar dietary interventions might have on aging.