Diet‐MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan

Abstract The number, size, and composition of lipid droplets can be influenced by dietary changes that shift energy substrate availability. This diversification of lipid droplets can promote metabolic flexibility and shape cellular stress responses in unique tissues with distinctive metabolic roles. Using Drosophila, we uncovered a role for myocyte enhancer factor 2 (MEF2) in modulating diet‐dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle‐specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester‐enriched intramuscular lipid droplets in response to high calorie, carbohydrate‐sufficient diets. The diet‐dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up‐regulation of Cyclin E is required for diet‐ and MEF2‐dependent diversification of intramuscular lipid droplets. The integration of MEF2‐regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation.

min to inactivate endogenous enzymes. Samples were centrifuged at 4000 rpm for 3 min at 4 °C to clear homogenate. To prepare hemolymph for metabolic measurements, the thoraces of 40 flies were carefully pierced with a tungsten needle and then placed in a perforated 0.5 ml Eppendorf tube within a 1.5 ml Eppendorf tube. Then, the pierced flies were centrifuged at 4000 rpm for 3 min at 4 °C. The supernatant was carefully collected to avoid debris. Collected hemolymph was centrifuged again at 4000 rpm for 3 min to precipitate debris. The collected hemolymph was used measure metabolites immediately.
Ten microliters of cleared extract were used to measure triglycerides (TAGs, StanBio Liquicolor Triglycerides Kit) or glucose (Glucose Assay Kit,  or trehalose (Megazyme) or protein concentrations (Bio-Rad Protein Assay) according to the manufacturer instructions. TAGs or glucose or trehalose levels were normalized to weight (for whole animal, measured using MT XS64 scale) or protein level (for thorax and hemolymph). Note: The kit measures glycerol cleaved from TAG and diacylglycerol (DAG), as well as minimal amounts of free glycerol; the majority of neutral lipids extracted from whole flies are TAG (Carvalho et al. 2012;Palm et al. 2012).

Cholesterol measurements
Total and free cholesterol were measured using the Amplex Red Cholesterol Assay Kit (Invitrogen) according to the manufacturer's instructions. Briefly, thoraces from 10 female flies were homogenized in 200 μL PBST (PBS, 0.1% Tween 20), then heat treated for 5 min at 70 ℃ to inactivate endogenous enzymes. After centrifuging for 3 min at 4,000 rpm, 50 μL of supernatant was added to an equal volume of working solution with or without cholesterol esterase. After 30 min incubation at 37 °C, the fluorescence intensity (λex = 530/λem = 590 nm) was measured on a CLARIOstar microplate reader (BMG LABTECH). Total cholesterol and cholesterol ester(s) (total subtracted of free cholesterol values) were determined by normalization to protein levels by using the BCA Protein Assay Kit (Pierce).

Lactate measurements
Lactate concentrations were measured using the Lactate Assay Kit (Sigma-Aldrich) according to the manufacturer's instructions. In brief, thoraces from 10 female flies were homogenized in 200 μL PBST (PBS, 0.1% Tween 20), then heat treated for 5 min at 70 ℃ to inactivate endogenous enzymes. Four volumes of the lactate assay buffer were added to the sample, and centrifuged at 13,000g for 10 min to remove insoluble material. The supernatant was deproteinized with 10 kDa MWCO spin filter (Life Technologies) to remove lactate dehydrogenase. Samples were brought to 50 μL per well with lactate assay buffer, and incubated for 30 min at room temperature. Absorbance (570 nm [A570]) was obtained using an Epoch Microplate Spectrophotometer (BioTek Instruments).
Lactate levels were normalized to total protein levels using the BCA Protein Assay Kit (Pierce).

Oil Red O staining
Intact intestines (midguts) and carcasses (with all of the eggs and intact intestines removed) were dissected in PBS and fixed in 4% paraformaldehyde for 20 min, then washed twice with PBS, incubated for 20 min in fresh Oil Red O solution (6 ml of 0.1% Oil Red O in isopropanol and 4 ml distilled water, and passed through a 0.45 µm syringe), followed by rinsing with distilled water.
To visualize LD-GFP tagged lipid droplets in muscle, longitudinal thorax muscle segments were dissected in PBS and fixed with 4% paraformaldehyde for 20 min at room temperature, washed 3 times with PBST for 10 min each, and then stained with DAPI (1:500) and Alexa Fluor 555 Phalloidin (Thermo Fisher Scientific, 1:500).
Confocal images were collected using a Nikon Eclipse Ti confocal system and processed using the Nikon software and Adobe Photoshop.

Nile Red staining
Longitudinal thorax muscle segments were dissected in PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, washed 3 times with PBS for 10 min each, and then incubated with fresh Nile Red solution (2µl of 0.004% Nile Red Solution in 500 µl PBS) for 2 h at room temperature, followed by rinsing with PBS and then staining with DAPI (1:500). Identical protocols were used to visualize Nile Red and LD-GFP positive lipid droplets in muscle segments.
Confocal images were collected using a Nikon Eclipse Ti confocal system (utilizing a single focal plane) and processed using the Nikon software and Adobe Photoshop.

Ex vivo CholEsteryl BODIPY staining
CholEsteryl (CE) BODIPY™ 542/563 C11 (Thermo Fisher Scientific) was used to trace cholesteryl esters in thorax muscle. Briefly, longitudinal thoracic muscle segments were isolated/dissected in PBS and then incubated/stained with CholEsteryl BODIPY (2 μM in PBST) for 1 h at room temperature ( Fig. 2E) and imaged live (unfixed). In order to monitor co-staining of CE BODIPY with LD-GFP+ lipid droplets (Fig. 2F), muscle segments had to be fixed minimally in 4% PFA (for 5 min.), before incubation with CE BODIPY.
If needed, DAPI (1:500) and Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific, 1:500) were added to stain DNA and F-actin filaments, respectively. Confocal images were collected immediately after CE BODIPY incubation/staining using a Nikon Eclipse Ti confocal system and processed using the Nikon software and Adobe Photoshop.

Food (Calorie) intake / Feeding behavior analysis
For measuring food intake/calorie intake ( Fig. 1A and previously described in (Skorupa et al. 2008)); feeding assays on blue dye-labeled food were done as follows: 30 flies were transferred from various diets (high calorie or HS-LY or HY-LS) to vials filled with identical dietary media containing 0.5% brilliant blue (dye). Feeding was interrupted after 1h and 5 flies each were transferred to 50 µl 1 x PBS containing 0.1% Triton X-100 (PBST) and homogenized immediately.
Blue dye consumption was quantified by measuring absorbance of the supernatant at 630 nm (A630). Various amounts of dye-containing food were weighed, homogenized in PBST, and measured (A630) in order to create a standard curve used to quantify blue dye food consumption.
For measuring food intake/feeding behavior (Fig. S5); the CAFE assay (Deshpande et al. 2014) was done as follows: Briefly, a single fly was transferred to vials filled with 5 ml of 1.5% agar that maintains internal humidity and serves as a water source. Flies were fed with 5% sucrose solution maintained in 5 ul capillaries (VWR, #53432-706). After twelve hours habituation, the old capillaries were replaced with a new one at the start of the assay. The amount of liquid food consumed was recorded after 24 hr and corrected on the basis of the evaporation (typically < 10% of ingested volumes) observed the identical vials without flies. 5 flies were weighed in order to normalize samples.

RNA-Seq analysis
Intact fly thoraces (8) were dissected in PBS. Total RNA was extracted using Trizol reagent and used as template to generate sample libraries for RNA sequencing (using the TruSeq Stranded Total RNA Library Prep Kit). Sample libraries were sequenced using the Illumina NextSeq 500.
Sequence cluster identification, quality pre-filtering, base calling and uncertainty assessment were done in real time using Illumina's HCS and RTA software with default parameter settings. Between 8 and 10 million (2X150) base pair reads were generated per library and mapped to the Drosophila genome (Release 6). Expression was recorded as TPM (transcripts per kbp per million reads) followed by Log2 transformation. Gene Ontology clustering analysis was performed using FlyMine.
FASTQ data files representing unique libraries were deposited in the NCBI Gene Expression Omnibus database (GSE147676).

de novo lipid synthesis analysis
After ten days of feeding on indicated diets, files were transferred to a blue dye (0.5% brilliant blue)labeled diet with 2µCi of 14C-labeled glucose for 2h. Files with blue dye were then transferred to fresh diet without 14C-labeled glucose for 5h, thoraces (5) were dissected in PBS and total lipid was extracted. For extraction of total lipid, 10 thoraces or 10 carcasses for each sample were homogenized in 2 ml Folch reagent (CHCl3: MeOH 1:1 v/v). Add 0.4 ml cold 0.1 M KCl, vortex 1 min and then spin at 3000 rpm at 4°C for 5 min, then transferred lower phase to new glass tube and dried down. The dried lipid was re-suspended in scintillation fluid, and counted (CPM). Zerohour samples indicate the rate of incorporation of glucose into fatty acids and 24-hour samples indicate the breakdown of the labeled fatty acids.

Quantification of LD-GFP positive lipid droplets and poly-Ubiquitin
For the quantification of lipid droplet size/area and numbers, about 6-18 randomly selected confocal images from different samples were processed by using ImageJ software (version 1.52, NIH, https://imagej.nih.gov/ij/). Lipid droplets were marked as a region of interest (ROI) by free hand selection tool, then assessed their size and numbers. An identical protocol was used to quantify poly-Ubiquitin.

Climbing assay
Twenty flies were placed into the empty vials, tapped to the bottom, and then given 30 sec. to climb a distance of 6 cm. Flies that successfully climbed 6 cm or beyond in 30 sec were counted. At least 100 total flies (5 cohorts) were used for each genotype tested.

Lifespan analysis
Fifty virgins were crossed to 10 males in bottles for all lifespan experiments unless otherwise indicated. The mated parental files were kept in bottles for 2-3 days to lay enough eggs. Wet folded filter (GE healthcare, CAT No.10311843) were inserted in bottles after parental flies removed.
Progeny of crosses was collected for 3-4 d after initial eclosion. Progeny were then transferred to new bottles to mate for 2 d. Around 100 flies were then separated according to sex and genotype into each cage and aged at 25 °C with constant humidity (approximately 65%).
For all independent populations, plastic cages (175-ml volume, 5-cm diameter from Greiner Bio-One) were used for lifespan experiments. Food, changed every 2-3 d, was provided in vials inserted into a foam plug (4.9 cm in diameter, 3-cm thick from Greiner Bio-One). RU486 or ethanol was mixed with food, resulting in a 100 μM concentration of RU486 in the food (unless otherwise indicated for dose response experiments). Dead flies were counted every 2-3 d. lifespan data were analyzed using Prism statistical software. Table   S1, and raw data for all lifespan analysis (unique populations) is included in Table S3.

Mortality estimation
Mortality rate was calculated as described (Mair et al. 2003). N0 is the number of individuals in the initial cohort, as well as Nx, the number alive at the start of each day. The probability of surviving from age x to age x + 1, given the individual is alive at the start of age x, is Px = Nx + 1/Nx. The age-specific rate of mortality is estimated as μx = −ln(Px). The mortality rate is plotted as ln(μx).

Analysis of gene expression
Total RNA from intact fly thorax (8) were extracted using Trizol and complementary DNA synthesized using Superscript Ⅲ (Invitrogen). Real-time PCR was performed using SYBR Green, the Applied Biosystems StepOnePlus Real-Time PCR systems, and the primers pairs described in the extended experimental procedures (Table S2). Results are average ± standard error of at least three independent samples, and quantification of gene expression levels calculated using the ΔCt method and normalized to actin5C expression levels.
Ultrathin sections (85 nm) were cut at various levels of each block and stained with 2% uranyl acetate and lead citrate. Images were captured with a FEI TECNAI G2 F20 FE-TEM transmission electron microscope.

Statistical analysis
For all experiments, the data is represented as mean ± SE. All p-values were calculated using the Student's t-test excluding the lifespan analysis. Lifespan data was analyzed using Prism statistical software, and Log-rank and Wilcoxin Chi Square analysis were used to calculate p-values.