The human WD consists of highly palatable, high-salt, high-fat, and high-sugar, energy-dense foods 48. This diet imitated in rodent studies and often dubbed as a "cafeteria diet" results in rapid weight gain, glucose and insulin intolerance, with concomitant alterations in neuronal signaling involved in the control of feeding behavior 49, 50, 51, 52. While several “cafeteria diets” have been developed for rodents 53, to the best of our knowledge, no such diet has been developed for Drosophila. As a first step toward filling this knowledge gap, we have recently created a diet with increased fat, sugar, and salt for Drosophila called the Western diet (WD) 54.
For this study we used D. simulans, which was successfully used to characterize effects of WD previously 37.
3.1 Ancestral WD causes increase in offspring feeding behavior up to the fourth generation.
To begin to explore whether ancestral diet may alter offspring feeding behavior, paternal male flies were subjected to the WD or the WD plus exercise. Two other groups included sedentary flies on the CD and the CD plus exercise. The effect of ancestral diet on male and female flies of F1-F4 generations was studied using Fly Liquid-Food Interaction Counter (FLIC), which allows high-throughput, continuous analysis of feeding behaviors 38. Specifically, FLIC allows to measure feeding behaviors by detecting electronic signals, “licks,” when the fly proboscis touches the food. Flies were removed from their food environment and gently loaded onto Drosophila Feeding Monitor (DFM), using a flight aspirator. Feeding reservoirs were filled with 10% sucrose solution. Fly feeding behavior was monitored continuously for 24 h starting at 12 pm.
The offspring groups included control father offspring (CFO), exercised father offspring (EFO), WD father offspring (WFO), WD + exercise father offspring (WEFO). The assay showed that both male and female WFO offspring had significantly more licks than other groups. This increase was observed in F1-F4 male flies and F2-F4 female flies according to one-way ANOVA (Fig. 1. A, B). The significant increase in licks was observed in F2, F4 males and F3-4 females in comparison to CFO lineage. In F1, F3 males and F2 females the increase in feeding was significant in comparison to exercise lineages EFO and WEFO. In F4 females the increase in feeding was significant against WEFO. Paternal exercise led to decrease in feeding behavior in F3 males and F2 females, and to increase in F3, F4 females. Interestingly, combination of paternal WD and exercise in negated effect of WD in F1, F3, F4 males and F2, F4 females of WEFO lineage. The findings of this study suggest that ancestral WD causes transgenerational increase in offspring feeding behavior while ancestral exercise counterbalances this effect.
Increase in food consumption leads to obesity with concomitant increase in biochemical markers. In the following experiment we performed biochemical assays to assess if transgenerational increase in offspring feeding behavior altered level of triglycerides (Fig. 1C). The experiment revealed that level of triglycerides in whole body homogenate was the highest in WF flies and lowest in EF animals. Ancestral exercise significantly negated the impact of WD, reducing the triglycerides in WEF flies compared with control. Taken together, these data show that ancestral WD leads to obesity in offspring.
3.2 Ancestral WD negatively impacts locomotor activity and muscle and brain mitochondrial enzymes.
Generally, increased food consumption and triglycerides have a negative impact on physical activity, so next we asked if ancestral experiences affected the activity of offspring. The activity of male and female offspring was recorded over 5-day period using LAM25H locomotor activity monitors (TriKinetics). The experiments showed that in male WFO offspring the activity was significantly increased in the F1 but decreased in F2-F4 (Fig. 1D). In contrast, activity in EFO offspring was higher in F1-F3 and decreased in F4. The activity of WEFO offspring was significantly lower than WFO in F1-F3 but higher in F4.
In female offspring, the activity of WFO group was significantly decreased in F1-F4, while EFO activity was higher in F1 and lower in F3-F4 (Fig. 1E). WEFO activity was higher than WFO in F2-F4. Thus, according to these data, WD causes a transgenerational decline in locomotor activity in both sexes.
Locomotor activity relies on muscle bioenergetics including efficiency in oxidative phosphorylation (OXPHOS) efficiency, and activity of individual enzymes in the mitochondrial electron transport chain (ETC). To investigate whether offspring alterations in locomotor activity were due to deficiency in mitochondrial OXPHOS, we performed western blot analysis for Cox4 (cytochrome c oxidase or Complex IV) and ATP5A, a subunit of the catalytic portion of the ATPase, also known as Complex V. The experiments showed that both Cox4 and ATP5A were significantly downregulated in flight muscle of F1 WFO males, suggesting compromised mitochondrial function (Fig. 2A, B, SI Fig. 1). The brain western blots did not reveal any statistically significant differences (SI Fig. 1).
3.3 Decreased mitochondrial density in muscles and the brain due to ancestral WD
Ancestral diet could affect bioenergetic phenotype in several ways epigenetically, including reducing metabolic capacity through changes in mitochondrial density 55. A decrease in mitochondrial density would lead to a reduction in ATP-producing sites, making cells less energy efficient and susceptible to metabolic disorders 56. In the following experiment, we investigated whether ancestral WD affected mitochondrial copy number (MCN) using qPCR approach 57. MCN was studied in brain and fly muscle of parental and offspring flies. The experiments showed that MCN levels were significantly lower in brains and muscles of WD-fed fathers (WF) (Fig. 2C). Interestingly, exercise mitigated the negative effect of WD in WEF group and increased the amount of MCN in muscles from exercised fathers (EF). In offspring groups, the ancestral WD decreased MCN in both brains and muscles of F1-F4 WFO males(Fig. 2D, E). The decrease of MCN was less pronounced in female offspring reaching statistical significance in F2 brains and F1, F3 muscles. This data show that ancestral WD and exercise have transgenerational programming effects on brain and muscle mitochondrial density and consequently these tissues bioenergetics.
3.4 Paternal WD causes deregulation of F1 brain proteome.
Several lines of evidence indicate that obesity induces changes in brain proteomics 58. It is less well understood, however, how ancestors' diets might affect offspring's brain proteins. To begin to explore whether ancestral WD may impact offspring proteome, nLC-MS/MS was used to interrogate the proteome of F1 offspring brain. This approach yielded 2,802 proteins identified and quantified across all samples. Importantly, the top 10 most abundant proteins corresponded to known mitochondrial membrane and cytoskeletal proteins (ATP5a, betaTub56D, ATPsynβ, porin, alpha-Spec, Gs2, mAcon1, sesB, Adh, PyK). ATP5a synthase abundance was identical across offspring lineages, confirming equal total amounts of protein across samples (Fig. 3). Using a P value less than 0.01 cutoff we have identified 74 differentially expressed proteins comparing WFO to CFO in males, and 70 differentially expressed protein comparing WFO to CFO in females, 28 differentially expressed proteins comparing EFO to CFO in males, and 34 differentially expressed protein comparing EFO to CFO in females, and 125 differentially expressed proteins comparing WEFO to CFO in males, and 103 differentially expressed protein comparing WEFO to CFO in females (Fig. 4, SI Fig. 2 Heat map, SI Excel table1). Metascape Gene Analysis (https://metascape.org) showed a significant enrichment in terms related to translation and translation factors, small molecule metabolic process, purine metabolism, Rho GTPases, and carbohydrate metabolism (Fig. 4B, D, F, H, J, L). The TCA cycle and ETC were among downregulated pathways in both WFO and WEFO males (SI Fig. 2). In WFO females the downregulated proteins were associated with "small molecule metabolic process", "vesicle-mediated transport in synapse", and "cellular homeostasis". Upregulated proteins were related to "metabolism of RNA", "translation", and "heterochromatin organization"(SI Fig. 3).
Using a 2-fold cutoff, we discovered that Huntingtin (Htt) was downregulated in both WFO and WEFO female (see volcano plots Fig. 4C, K). Htt is the ortholog of human HTT. It encodes a scaffold protein involved in mitotic spindle orientation, chromatin regulation and axonal transport59, 60. Interestingly, dHYPK- an ortholog to human huntingtin interacting protein K was 3.6-fold down in WEFO males (volcano plot Fig. 4I). Among upregulated proteins, mei-P26 (meiotic P26) was elevated in both WFO and WEFO males and WEFO females (volcano plots, Fig. 4A, I, K). Recent observations indicate that mei-P26 regulates translational repression by inhibiting the microRNA pathway 61, 62.
3.5 The evolutionary conserved miRNA mir-10 is identified as a potential epigenetic regulator by miRNA target prediction
Growing evidence suggests that miRNAs play significant role in inheritance of transgenerational phenotype 63, 64. To determine if miRNA might be involved in regulating proteomic changes in the brain, web tool MIENTURNET (MicroRNA ENrichment TURned NETwork)65 was used for miRNA-target enrichment analysis. The tool uses TargetScanFly 7.2 to locate evolutionarily conserved microRNA binding sites. The analysis was performed on subset of proteins with significant enrichment for terms related to translation, small molecule metabolic processes, and carbohydrate metabolism. The results showed that mir- 277-3p, mir-10-3p and mir-927-5p had the highest number of predicted targets (Fig. 5A, B). In turn, mei-P26 contained the highest number of conserved sites for miRNAs including mir- 277-3p, mir-10-3p and mir-927-5p (Fig. 5C). To assess miRNA regulatory roles and identify controlled pathways, we used DIANA-tools mirPath v.3 (www.microrna.gr). Based on unbiased empirical distributions and meta-analysis statistics, this tool performs functional annotation of one or more miRNAs66. The MirPath analysis identified "Purine metabolism" as a pathway targeted by all three miRNAs: mir-277-3p, mir-10-3p, and mir-927-5p (Fig. 5D). We chose miR-10 for study because, among three, it is the only miRNA conserved in bilaterian animals, including humans 67. miR-10 has been implicated in variety of epigenetic processes including cancer, regulation of Hox translation 68 and control of cell differentiation 69.
3.6 The expression of Mir-10 in brain and spermatozoa is controlled by paternal WD
In several studies, sperm miRNA has been identified as a carrier of epigenetic information. 63, 64, 70. In order to investigate whether miR-10-3p could mediate transgenerational phenotype, we examined expression of mir-10 in brain and sperm by qRT-PCR.
As shown in Fig. 6A, several miRNAs including miR-10-3p were upregulated in brains of WF fathers and their offspring. Interestingly, miR-10-3p was increased in female offspring but not male offspring. qRT-PCR on purified spermatozoa revealed significant increase of several miRNAs including miR-10-3p in paternal and offspring spermatozoa (Fig. 6B). Several miRNAs including mir-277-3p were undetectable in spermatozoa. This data suggests that miR-10-3p could potentially mediate transgenerational phenotype in a fruit fly model.
3.7 Knock-down of miR-10 increases feeding behavior.
To begin to elucidate if miR-10-3p could be responsible for transmission of transgenerational phenotype we used the UASxGAL4 strategy to induce UAS-miR10 lines with dopa decarboxylase (Ddc)-GAL4 driver. Ddc-Gal4 encodes the promoter region of the DDC gene, which is involved in the serotonin and dopamine synthesis, and its expression is associated with dopaminergic and serotonergic neurons, which are responsible for both aversive and appetitive behaviors71. We used two RNAi lines (35014 and 61377) targeting miR-10, thus avoiding potential off-target effects associated with a particular construct. 35014 expresses dsRNA for RNAi of mir-10 (FBgn0262424) under UAS control in the VALIUM20 vector. 61377 expresses an antisense 'sponge' RNA under UAS control for knocking down mir-10 expression. For control, we used the parent lines or F1 cross to Canton-S (CS). The analysis of the of F1 feeding behavior using FLIC showed that both male and female flies had significant increase in feeding behavior (Fig. 6C). The biochemical assay revealed an increase in total body triglycerides in males and a strong trend in female offspring (Fig. 6D). qRT-PCR in the brains demonstrated a reduction in miR-10-3p in 7009x35014 confirming successful knockdown (Fig. 6E).
In sum, these findings suggest that miR-10 might act as an epigenetic factor modulating feeding behavior.