Bloemink, M.

Harvey, S.

Stastna, J.

Strudwick, A.

Tumbapo, S.

English

Canterbury Research and Theses Environment

Effect of Caloric Restriction and Omega-3 EPA Supplementation on Sarcopenia in C. elegansSobha Tumbapo, Adam Strudwick, Jana Stastna, Simon Harvey and Marieke Bloemink,   School of Psychology and Life Sciences, Canterbury Christ Church University, Canterbury CT1 1QU, UKBackgroundSarcopenia, a progressive decrease of skeletal muscle mass and strength, is one ofthe primary changes associated with ageing and a major cause of physical frailty inthe elderly. Despite extensive research, sarcopenia is still not well understood at themolecular level, although multiple factors have been found to influence thedevelopment of sarcopenia, including physical inactivity, and an unbalanced diet.Using C. elegans, this study determines the effect of dietary interventions, like caloricrestriction and dietary supplementation, on the rate of muscle loss. The specific C.elegans strain used (unc54::GFP) has its major myosin isoform (UNC-54) in body wallmuscle fluorescently labelled, allowing UNC-54 myosin density to be measuredthroughout C. elegans’ lifespan. Average lifespan, motility, and intestinal barrierfunction (IBF) were also determined.Figure 11: Uptake of supplemented EPA by E. coli OP50 (A) and C. elegans (B). OP50 and worms were harvested after 5 days, and the extracted FAMEs were analyzed with GC-MS. Data are the mean ± SD of three independent assays (One-way ANOVA, *P<0.05).Figure 10: Age-related breakdown of intestinal barrier function is reduced in EPA from algae-oil supplemented worms. Proportion of worms showing loss of intestinal integrity as a function of age. Data are the mean ± SD of three independent assays (Two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001).Figure 6: Microscopy images showing loss of intestinal integrity in nematodes grown under control and algae oil supplementation. During early adulthood, dye is restricted to the intestine but during middle age, dye is seen throughout thebody cavity.Lifespan Assay and media:Worms were maintained on NGM plates withOP50 E.coli as standard food. Lifespan assayswere done at 20°C on varying concentration of DR(figure 2) or EPA-supplemented plates (0.1mMand 0.3mM EPA) and transferred to new platesevery other day. Peptone was removed from theDR media to prevent bacterial growth on theplates (5). FUDR was used to supressreproduction.• Our data show that C. elegans exposed to mild and medium caloric restriction (CR)display an increase in lifespan and heath span, with a delayed onset of sarcopenia.• Supplementation with sources of omega-3 EPA (eicosapentaenoic acid), has asimilar effect, with improved lifespan and delayed sarcopenia.• Age-related intestinal barrier function was also improved in EPA-supplementedworms, with GC-MS confirming the uptake of omega-3 EPA.• These results show that dietary intervention can delay the onset of age-relateddiseases such as sarcopenia and intestinal barrier function in C. elegans.Motility Assay:Motility was assessed daily by subjecting the worms to gentle stroking and their response to the stimulus was grouped into one of three motility classes (2).Fluorescence Microscopy:Fluorescence images were measured using arandom sample of five worms from eachpopulation. Average pixel intensity of whole-bodyimages was determined for quantification of GFPfluorescence using ImageJ software. Allquantifications were calibrated using GFP-labelledbeads as an internal standard and corrected forbackground fluorescence.Intestinal Barrier Function (Smurf Assay):Worms were raised with varying concentration (0.1mM and 0.3 mM) of omega-3 Eicosapentaenoic acid(EPA) in comparison with control. 10 worms wereremoved from the experimental plates andsuspended for 3 h in liquid cultures of OP50 E.colimixed with Erigolaucine disodium salt. Worms wereexamined for the presence of blue dye in the bodycavity and scored manually (Olympus IX83microscope, 10x magnification).Methods + ResultsMass Spectrometry:To quantify the uptake of EPA, synchronized L4larvae were harvested after five days growth.FAMEs were generated after 1 hour incubation at70 °C in 2.5% H2SO4 in MeOH and extraction intoa hexane/water (). The hexane layer was thentransferred to a GC vial insert for GC-MS analysisusing established methods ().The EPA-rich algae oil was kindly provided by AlgaeCytes (Discovery Park, Sandwich). ResultsCR + EPA supplementation increases lifespan of C. elegansFigure 7. Effect of caloric restriction on life span in C.elegans. Mild and medium CR show a significant increase in mean lifespan compared to control (p < 0.001 and p = 0.011). Severe CR results in a reduced mean lifespan (p = 0.026).(Kaplan-Meier analysis)Figure 8. Effect of EPA-supplementation on life span of C.elegans. 0.1mM and 0.3mM EPA-supplementation increased themean lifespan 34% and 38%, respectively, compared to control(p < 0.0001).Age-related decline in motility is delayed with CR + EPA Class I – Continuous smooth movement, fast movement when stimulated; Class II – Slow halting movement, smooth movement when stimulated; Class III – Small movement of head or tail, very slow movement when stimulated.C1-2(days)C2-3 (days) ΔC2 (days) Mean life span (days)Control 6.9 ±0.2 12 ±3 5.1 15.5±0.7Mild 8.6 ±0.1 15.4 ±0.3 6.8 19.9±0.8Medium 8.5 ±0.4 14.3 ±0.4 5.8 18.3±0.8Severe 6.9 ±0.1 10.0 ±0.5 3.1 13.0±0.80.1mM EPA 11.05 ±0.04 16.3±0.2 5.3 18.6±0.60.3mM EPA 11.15 ±0.03 17.0±0.1 5.9 19.4±0.6Table 1: Age-related changes in motility. Average nr of days for 50% of initial population to move from fitness class 1 to class 2 (C1-2) and for 50 % of the class 2 population to move to class 3 (C2-3). Difference between two mid-points (ΔC2) is average time spent in class 2.Age-related muscle loss is delayed under CR and EPA supplFigure 1: C. elegans is used as a model for sarcopenia: Left: one day old C. elegans nematode; Middle: 18 days old C. elegans nematode; Right: C. elegans with one of its main myosin muscle proteins (UNC54) labelled with GFP (UNC54::GFP).2 4 6 8 10 12 14 16 1805×1061×1071.5×1072×1072.5×107 Control0.1mM EPA0.3mM EPAAge (days of adulthood)Fluorescence Intensity✱✱ ✱Figure 9: Average fluorescence intensity of whole-body images under different CR conditions (left) and EPA supplementation from algae oil (right). Error bars show SD. *P < 0.05 by 2-way ANOVA.Age-related breakdown of intestinal barrier function is reduced for EPA-supplemented C. elegansFigure 3. algae oil was used as a source of EPAFigure 2: Set-up of CR assay using C. elegans expressingUNC54-GFP) Figure 4: Fluorescence microscopy images show early onset of sarcopenia for worms under control and severe DR, compared to mild and medium CR. Figure 5: GC/MS of C. elegans supplemented with 0.1mM EPA (bottom) and 0.3mM EPA (middle) compared to control (top).Figure 9: EPA-supplementation reduces age-related decline in intestinal barrier function. Representative images (10X)  of control worms after soaking in dye for 3 hours  on day 2 (left), day 8 (middle ) and day 14 (right) of adulthood. ConclusionsAcknowledgementsThis work was supported by CCCU  (QR research fund and staff development fund) to MJB, a CCCU PhD scholarship to ST and a summer internship grant from RED to AS. We thank Dr András Málnási-Csizmadi (Eötvös Loránd University, Budapest, Hungary) for generously providing the C.elegans strain with GFP-labelled myosin (unc54::GFP) and Algaecytes(Discovery Park, Sandwich) for providing the algae oil as a source of EPA. 

Effect of caloric restriction and omega-3 EPA supplementation on sarcopenia in C. elegans

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Effect of caloric restriction and omega-3 EPA supplementation on sarcopenia in C. elegans

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