Adaptive capacity to dietary Vitamin B12 levels is maintained by a gene‐diet interaction that ensures optimal life span

Abstract Diet regulates complex life‐history traits such as longevity. For optimal lifespan, organisms employ intricate adaptive mechanisms whose molecular underpinnings are less known. We show that Caenorhabditis elegans FLR‐4 kinase prevents lifespan differentials on the bacterial diet having higher Vitamin B12 levels. The flr‐4 mutants are more responsive to the higher B12 levels of Escherichia coli HT115 diet, and consequently, have enhanced flux through the one‐carbon cycle. Mechanistically, a higher level of B12 transcriptionally downregulates the phosphoethanolamine methyltransferase pmt‐2 gene, which modulates phosphatidylcholine (PC) levels. Pmt‐2 downregulation activates cytoprotective gene expression through the p38‐MAPK pathway, leading to increased lifespan only in the mutant. Evidently, preventing bacterial B12 uptake or inhibiting one‐carbon metabolism reverses all the above phenotypes. Conversely, supplementation of B12 to E. coli OP50 or genetically reducing PC levels in the OP50‐fed mutant extends lifespan. Together, we reveal how worms maintain adaptive capacity to diets having varying micronutrient content to ensure a normal lifespan.


Life span analysis
Gravid worms of desired strain, grown on E. coli OP50, were bleached and eggs were subjected to L1 synchronization in 1 x M9 for 16 hours at 20 o C before growing the worms on respective RNAi, vitamin B12 supplemented and/or choline supplemented plates. Once the worms reached late L4 stage, they were transferred to respective plates overlaid with 5-fluorodeoxyuridine (FUDR, final concentration 0.1 mg/ml of media). Life span scoring was started on 7 th day of adulthood and continued every alternate day. For statistical analyses of survivability rate, OASIS software (http://sbi.postech.ac.kr/oasis) was used and P-values were calculated by applying a log rank (Mantel-Cox method) test. Unhealthy worms were censored.

Osmotic stress assay
Worms were grown till L4 stage on NGM plates (containing 50 mM NaCl) seeded with different bacterial feeds (supplemented or RNAi). L4 stage worms were the placed on unseeded NGM (n≈75 and N=3) plates containing 350 mM sodium chloride for 9 minutes. Post 9 mins, the worms were transferred onto unseeded NGM plates containing 50 mM NaCl. The fraction of motile worms was calculated over a time period of 15 minutes to determine percentage recovery. Statistical analyses were performed using Graphpad 9.0. Propionate toxicity assay L1 synchronized worms were placed on propionate-supplemented (0, 110, 130, 150 mM) plates seeded with E. coli OP50 or E. coli HT115. Worm development was observed till 72 h post L1 to check for developmental arrest at each stage. Un-arrested worms were counted, and imaging was performed using Axiocam MRm (Carl Zeiss, Germany) camera attached to M205FA microscope (Leica, Germany).
Quantification of GFP expression was done using NIH ImageJ software. Statistical analyses were done using Graphpad 9.0.

RNA isolation
Synchronized L1 worms grown on supplemented, or RNAi plates were collected on Day 1 of adulthood (64-68 h post L1) in 1 x M9 buffer and further washed three times in 1 x M9 buffer. Trizol of approximately 4 times the volume of the worm pellet was added, and the worms lysed using three freeze thaw cycles. Further, the worms were subjected to vigorous vortexing. RNA was isolated using phenol:chloroform:isoamylalcohol extraction followed by isopropanol precipitation. For quantitative Reverse Transcriptase PCR (qRT-PCR) experiments, the RNA concentrations were estimated using NanoDrop 2000 (Thermo Scientific, USA) and the integrity of the ribosomal 28 S and 18 S was determined using denaturing agarose gel.

QRT-PCR analysis
Approximately 1 μg of RNA was converted to cDNA with the help of Superscript III Reverse Transcriptase enzyme and poly-T primers (Invitrogen, USA). To determine the relative gene expression levels, QRT-PCR analysis was performed using the 2X Brilliant III Ultra-Fast SYBR® Green QPCR mastermix (Agilent Technologies, USA) and Agilent AriaMx Real-time PCR System (Agilent Technologies, USA). Statistical analysis was completed using Graphpad 9.0. Expression levels were normalized to actin.

Sr. No
Gene Primer sequence For the quantification of p-PMK-1 and PMK-1 activity, the band intensities of total PMK-1 and pPMK-1 were quantified and compared with the basal intensity of beta-actin bands using ImageJ software (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/). The values obtained for pPMK-1 were divided by the values of total PMK-1 and provided as percentile representation. Statistical analysis was completed using Graphpad 9.0.
Measuring Intracellular Vitamin B12 concentrations of C. elegans Intracellular vitamin B12 was measured by a kit-based electrochemiluminescence immunoassay (ECLIA) using COBAS e411 Analyzer. Briefly, C. elegans pellet from each of the conditions were taken in a microcentrifuge tube, washed three times with water and gently pelleted. A 10 mg freeze dried weight of pellet was then suspended in 300 µl of 1 x PBS, followed by homogenization using a handheld homogenizer. The suspension was then centrifuged at 15,000 g at room temperature for 10 minutes. Supernatant was transferred to a fresh tube and subjected for vitamin B12 measurement. Vitamin B12 measurements were statistically analysed using GraphPad 9.0.

Measuring Intracellular Vitamin B12 concentrations of bacteria
Intracellular B12 was measured as above. Bacterial cells equivalent to OD600 150 (OD600 = 1 ~ 3x10 9 cells) were taken in a microcentrifuge tube, pelleted by centrifugation and washed three times with water. The pellet was then suspended in 500 µl of 1 x PBS, followed by lysis using a FastPrep-24 TM 5G bead beater (MP Biomedicals, CA, USA).
The suspension was then centrifuged at 15,000 g at 4 o C for 10 minutes. Supernatant was transferred to a fresh tube and subjected for vitamin B12 measurement. Vitamin B12 measurements were statistically analyzed was using Graphpad 9.0.

Metabolomics
Intracellular metabolites for MS-based targeted metabolomics were extracted using cold Actonitrile-methanol-water. Briefly, 10 mg dry weight of C. elegans pellet was collected by centrifugation in a microcentrifuge tube and washed three times with sterile water, then quenched with chilled Acetonitrile-methanol-water (3:5:2) (kept at −80°C), followed by sonication. The suspension was then transferred to a fresh tube and centrifuged at 15,000 g at 4°C for 10 min. Supernatant was vacuum dried and reconstituted in 50 μl of 50% methanol. The reconstituted mixture was centrifuged at 15,000 g for 10 min, and 5 μl was injected for LC-MS/MS analysis.
The data were acquired using a Sciex Exion LCTM analytical UHPLC system coupled with a triple quadrupole hybrid ion trap mass spectrometer (QTrap 6500; Sciex) in a positive mode. Samples were loaded onto an Acquity UPLC BEH HILIC (1.7 μm, 2.1 × 100 mm) column, with a flow rate of 0.3 ml/min. The mobile phases comprising of 10 mM ammonium acetate and 0.1% formic acid (buffer A) and 95% acetonitrile with 5 mM ammonium acetate and 0.2% formic acid (buffer B). The linear mobile phase was applied from 95% to 20% of buffer A. The gradient program was used as follows: 95% buffer B for 1.5 min, 80%-50% buffer B in next 0.5 min, followed by 50% buffer B for next 2 min, and then decreased to 20% buffer B in next 50 s, 20% buffer B for next 2 min, and finally again 95% buffer B for next 4 min. Data were acquired using five biological replicates, with three technical replicates, for each run. Relative quantification was performed using MultiQuantTM software v.2.1 (Sciex).
Cysteine and Homocysteine were measured by HPLC (Agilent 1290 Infinity II LC system). Briefly 10mg dry weight of worms were suspended in 100µl of autoclaved milliQ water and lysed by 5 rounds of vortex-mixing with glass beads for 2 min each. The Experiments were performed at 20 o C. Source data is provided as a source data file. Source data is provided as a source data file.  Source data is provided as a source data file.

B)
No difference in osmotic stress tolerance is observed in wild-type grown on control (HT115) RNAi, control (OP50) RNAi or on knocking down cka-1 and cept-1 using an OP50 RNAi system.

C)
No difference in life span is observed in wild-type grown on control (HT115) RNAi, control (OP50) RNAi or on knocking down cka-1 and cept-1 using an OP50 RNAi system.
One of three biologically independent replicates shown for all experiments. All experiments were performed at 20 o C. Life span summary is provided in Supplementary   Table 1.
Summary of osmotic tolerance assay is provided in Supplementary Table 2.
Source data is provided as a source data file. A) Quantification of fluorescence on pmt-1 RNAi in Figure 7B. Mean of three biologically independent experiments ± SEM. Two-way Annova. ns = non-significant B) Quantification of fluorescence on pmt-2 RNAi in Figure 7B. Mean of three biologically independent experiments ± SEM. Two-way Annova. ns = non-significant C-D) The osmotic tolerance of wild-type worms are unaffected on pmt-1 and pmt-2 RNAi. One of three biologically independent replicates shown.

E-F)
The life span of wild-type worms is unaffected on pmt-1 and pmt-2 RNAi. One of three biologically independent replicates shown for all experiments.
Experiments were performed at 20 o C. Summary of osmotic tolerance assay is provided in Supplementary Table 2. Source data is provided as a source data file.