Glucose and cholesterol induce abnormal cell divisions via DAF-12 and MPK-1 in C. elegans

High glucose and cholesterol levels induce neuronal Q-cell divisions during L1 arrest

L1-arrested C. elegans larva pause all development events, including cell divisions, which makes them a perfect model to dissect the physiological or pathological effects of external variables. The two embryonic neuronal Q-cells stop dividing in L1-arrested worms (Figure 1A), but we found that treatment with high glucose caused the neuronal Q-cells to start dividing in L1-arrested worms (Figure 1B, 1E, and dosage treatment results in Supplementary Figure 1). Glucose was reported to regulate IIS pathway [7, 19] and daf-2, an IIS receptor gene, mutants could significantly suppressed Q-cell divisions during L1 arrest [17]. We speculated that glucose might serve to alter the function of IIS signaling in L1-arrested worms. Next, we tested if glucose could work through the IIS receptor (DAF-2) to induce Q-cell divisions, and we found that daf-2 mutants suppressed the Q-cell divisions induced by glucose treatments in L1-arrested worms (Figure 1H). We also observed that treatment with high cholesterol (Figure 1C and 1F) or Δ⁷-dafachronic acid (an nuclear hormone receptor, NHR, ligand) (Figure 1D and 1G) caused the Q-cells to divide. The Q-cell divisions were further confirmed by using another marker zdIs5 (Supplementary Figure 2). Cholesterol is a steroid precursor and is involved in the synthesis of NHR ligands. Two genes, daf-36 and daf-9, control the conversion of cholesterol to NHR ligands in C. elegans [20], and we found that both daf-9 and daf-36 mutants suppressed the Q-cell divisions caused by cholesterol treatment (Figure 1I). These results suggested that glucose and cholesterol affected the IIS and steroidogenic pathways to induce abnormal neuronal cell divisions in L1-arrested worms.

Figure 1. Glucose, cholesterol, and Δ⁷-dafachronic acid promote neuronal Q-cell divisions in L1-arrested worms. (A) Normal L1-arrested wild-type worms. The wild-type L1-arrested worms only have two Q-cells (QR/L). Wild-type L1-arrested worms treated with 20 mg/mL glucose (B, E), 1 mg/mL cholesterol (C, F), or 1 μg/mLΔ⁷-dafachronic acid (D, G). Q-cell divisions occurred in these worms, as at least four Q-cells were observed under these conditions. See the dosage treatment results in Supplementary Figure 1, and the Q-cell final descendants test in Supplementary Figure 2. (H) Q-cell divisions induced by glucose or insulin treatments can be suppressed by daf-2 mutants. (I) Q-cell divisions induced by cholesterol treatment can be suppressed by daf-9 and daf-36 mutants. White bar: 50 μm. Data are the average of three independent experiments. Error bars: Standard Deviation (SD). ***: P<0.001.

Glucose and cholesterol induce cell divisions independent of their effects on survival extension

Our results showed that glucose could induce Q-cell divisions through IIS signal pathway during L1 arrest. Previously, we found that manipulating IIS pathway, such as overexpressing agonist insulin like protein genes (such as ins-6) or disruption IIS pathway negative regulator daf-18/pten, all can induce Q-cell divisions, and also make these worms have a very short L1 lifespan [17, 18, 21]. We found that glucose could induce neuronal Q-cell divisions in L1-arrested worms; however, a recent study has shown that glucose can also promote the survival of wild-type L1 arrested worms [7]. With this in mind, we tested whether glucose could promote the survival of ins-6 (oe) and daf-18 mutants. We found that the longevity of ins-6 (oe) and daf-18 L1-arrested worms was significantly extended following glucose treatment (Figure 2A and 2B). High glucose levels can lead to increased production of glycogen, and the metabolic shift from glycogen to trehalose can make worms live longer [19]. We tested the role of trehalose on Q-cell divisions and L1 survival and found that trehalose promoted L1 survival in ins-6 (oe) and daf-18 L1 mutants (Figure 2C and 2D). However, trehalose failed to suppress the Q-cell divisions in these worms; moreover, it was observed that treatment with trehalose could induce Q-cell divisions in wild-type L1-arrested worms (Figure 2E). Additionally, gsy-1 plays a key role in the process of glycogen synthesis under high-glucose conditions; however, we observed that gsy-1 mutant L1-arrested worms had a normal longevity (Figure 2F) and presented with no aberrant cell divisions. The conversion of external trehalose into glucose and back again to trehalose is necessary to produce the observed physiological effects (i.e., survival extension) [22]. tps-1/2 are the key genes that control trehalose synthesis, but we found that tps-1;tps-2 worms had no effect on the glucose-induced cell divisions. tps-1; tps-2 also failed to block the aberrant cell divisions in ins-6 (oe) or daf-18 L1 mutants (Figure 2G). These results suggested that high glucose levels could regulate Q-cell divisions, and this effect might be physiologically distinct from glucose metabolism and its role in survival. As hormonal steroids induced by high levels of cholesterol also promote Q-cell divisions in L1-arrested worms, we also assessed the role of cholesterol in regard to L1 survival. We found that cholesterol and Δ⁷-dafachronic acid could also extend the survival of wild-type L1 worms (Figure 2H). Together, these results suggested that high glucose and hormonal steroids could affect the Q-cell divisions; however, their effects on L1 survival involved distinct mechanisms (All survival data details in Supplementary Table 1).

Figure 2. The effects of glucose, trehalose, cholesterol, and Δ⁷-dafachronic acid on the survival of L1-arrested worms. Glucose extends the longevity of L1-arrested ins-6 (oe) (A) and daf-18 mutants (B). Trehalose extends the longevity of L1-arrested ins-6 (oe) (C) and daf-18 mutants (D). (E) Trehalose has no suppression function on Q-cell proliferation; in contrast, it can induce Q-cell proliferation in wild-type L1-arrested worms. Data are the average of at least three independent experiments. Error bars: Standard Deviation (SD).***: P<0.001. (F) The glycogen synthesis controlling gene gsy-1 has no effect on the survival of L1-arrested worms. (G) The glycogen and trehalose synthesis controlling genes gsy-1 and tps-1/2 have no effect on the Q-cell divisions in daf-18 (-), ins-6 (oe), and wild-type L1-arrested worms. (H) Cholesterol and dafchronic acid can extend the survival of wild-type L1-arrested worms. Survival of these worms was checked every day, and the mean survival rate was calculated using the Kaplan-Meier method, and any significant difference in overall survival rates was determined using the log-rank test (P-value; see Supplementary Table 1).

DAF-12 and MPK-1 block Q-cell divisions in glucose- and cholesterol-treated L1-arrested worms

Glucose has been reported to influence a worm’s longevity and stress resistance via the transcription factor DAF-16 of the IIS signaling pathway [22–24]; however, we found that disruption of the daf-16 gene did not alter the Q-cell divisions occurring in glucose-, trehalose-, or cholesterol-treated worms (Figure 3A). These results suggested that the IIS pathway uses distinct downstream signals (DAF-16 and DAF-16-independent) to regulate survival and cell divisions during L1 arrest. Previously, we observed that mpk-1/MAPK played a key role in controlling Q-cell divisions in daf-18 L1-arrested mutants [17]. Based on this result, we treated mpk-1 mutants with high concentrations of glucose, trehalose, cholesterol, or Δ⁷-dafachronic acid, and the results indicated that the Q-cell divisions were suppressed in the mpk-1 mutant worms (Figure 3B). Hormonal steroids and Δ⁷-dafachronic acid mainly function by way of the DAF-12 hormone receptor in C. elegans. As such, we tested the role of daf-12 on Q-cell divisions and found that these cell divisions in glucose-, trehalose-, cholesterol-, or Δ⁷-dafachronic acid-treated worms and daf-18 (-) mutants were suppressed by disruption of daf-12 (Figure 3C). Glucose and trehalose might affect insulin-related gene expression to regulate the IIS pathway and control the Q-cell divisions. As such, we also tested ins-6 (oe) and daf-18 mutants and found that disruption of mpk-1 or daf-12 indeed suppressed the Q-cell divisions in these two strains (Figure 3D). In order to further confirm the functions of daf-12 and mpk-1, more alleles of these gene mutations were tested and showed the similar results (Supplementary Figure 3). These results suggested that DAF-12 and MPK-1 work together to regulate the Q-cell divisions induced by glucose and cholesterol in L1-arrested worms.

Figure 3. Neuronal Q-cell divisions in glucose-, trehalose-, cholesterol-, and Δ⁷-dafachronic acid-treated L1-arrested worms are controlled by daf-12 and mpk-1. (A) Q-cell divisions resulting from glucose, trehalose, cholesterol, and Δ⁷-dafachronic acid treatments are not dependent on daf-16. Disruption of mpk-1 (B) and daf-12 (C) can suppress the Q-cell divisions in glucose-, trehalose- and steroid-treated wild-type, ins (oe), and daf-18 (-) (D) L1-arrested worms. More alleles of daf-12 and mpk-1 were tested to further confirm these results (Supplementary Figure 3). Data are the average of at least three independent experiments. Error bars: Standard Deviation (SD). ***: P<0.001.

dpMPK-1 plays a major role in neuronal Q-cell divisions induced by glucose and cholesterol treatments during L1 arrest

MPK-1 is reported to be the main regulator controlling Q-cell proliferation during L1 arrest [17], and dpMPK-1 is the functional form of MPK-1 that acts during cell proliferation [17, 25–27]. With this in mind, we tested whether high glucose or cholesterol treatments could affect dpMPK-1 levels. We found that glucose- and cholesterol-treated L1-arrested worms both had high dpMPK-1 levels during cell divisions (Figure 4A, 4C, and 4E). DAF-12 and MPK-1 can both suppress the Q-cell divisions during L1 arrest. Moreover, DAF-12 is a nuclear hormone receptor, and MPK-1 can translocate into the nucleus. As such, we assess dpMPK-1 levels in daf-12 mutants and found that the dpMPK-1 levels, which were up-regulated by glucose and cholesterol, were rescued by mutation of daf-12 (Figure 4B, 4D, and 4E). These results suggest that the function of dpMPK-1 on Q-cell divisions requires the nuclear hormone receptor DAF-12.

Figure 4. dpMPK-1 is activated by glucose and cholesterol, and daf-12 is needed to induce Q-cell divisions. The activated form of MPK-1 (dpMPK-1) is detected in glucose- (A) and cholesterol- (C) treated L1-arrested worms. daf-12(-) can suppress Q-cell divisions and block dpMPK-1 formation in glucose- (B) and cholesterol- (D) treated L1-arrested worms. (E) Worms with and without dpMPK-1 fluorescence were counted, and the percentage of worms expressing dpMPK-1 was calculated. Data are the average of at least ten independent experiments (sample size >50 in each experiment). Error bars: Standard Deviation (SD). ***: P<0.001. White bar: 50 μm. (F) The glucose and cholesterol treatment is only performed in F0-F1 culture period. F1 and F2 worms were collected and assessed for Q-cell divisions. (G) The F1 progeny of glucose- and cholesterol-treated worms also present aberrant Q-cell divisions, but the F2 generation shows no abnormal Q-cell division. Data are the average of at least three independent experiments (sample size >50 in each experiment). Error bars: Standard Deviation (SD). (H) The survival of the F1 and F2 progeny of F0 worms treated with glucose and cholesterol. (I) Working model: cholesterol and glucose can work through DAF-12 and DAF-16 independent of the IIS pathway to activate MPK-1 to induce neuronal Q-cell divisions in L1-arrested worms, and these effects can also affect the F1 generation.

Mothers who experience starvation have high blood insulin concentrations and are also reported to have babies with poor health who have a high chance of developing diseases, including cancer [2, 3, 28]. We wanted to determine if high glucose or steroid levels could influence subsequent generations of worms. To this end, we tested whether the decedents of high glucose- or cholesterol-treated worms still underwent Q-cell divisions during L1 arrest. We found the F1 L1-arrested worms also presented with aberrant Q-cell divisions (Figure 4F and 4G) and a shortened survival time (Figure 4H), but the F2 generation of worms was essentially wild type (Figure 4G and 4H). These results suggested that the effects of high glucose or steroid treatments in regard to these cell divisions could be passed on to the F1 generation, though this was absent in the F2 generation.

Strains

The strains used in this study were acquired from the Caenorhabditis Genetics Center (CGC) and were crossed into Pegl-17-mCherry (rdvIs1) to assess Q-cell divisions. Standard culture methods were used as previously described [46]. Strains were maintained on worm NGM plates and cultured with OP50 Escherichia coli at 20°C. The strains used in this study were RDV55: rdvIs1, RB712: daf-18(ok480), SD420: mpk-1(ga119)/dpy-17(e164)unc-79(e1068), VC255: tps-1(ok373), AA1: daf-12(rh257), CB1370: daf-2 (e1370), DR2281: daf-9 (m540), and AA292: daf-36 (k114).

Chemicals and treatments

Glucose, trehalose, Δ⁷-dafachronic acid and cholesterol were bought from Sigma-Aldrich (USA). Glucose and trehalose were dissolved in water, while the other compounds were dissolved in DMSO. Serial dilutions of each chemical were added into M9 right after the embryos were prepared, so the worms were hatched in a drug-treatment environment and influenced by the chemical prior to L1 arrest.

Q-cell division analyses

Normally fed, mixed-staged worms were harvested and bleached to prepare the embryos, as previously described [17]. In brief, embryos were maintained and hatched in sterile M9 and incubated at 20°C with low-speed rocking to initiate L1 arrest. Q-cell descendants were observed under an Axioplan fluorescent microscope (Zeiss) after 12 hours in L1 arrest. A total of 50-200 μL M9 containing L1-arrested worms were removed from the culture each time to make sure the sample was larger than 50 worms. The total number of worms and the worms presenting with Q-cell divisions were counted. Q-cell divisions were confirmed via lineage analysis using the Q-cell marker Pegl-17::mCherry (rdvIs1). For transgenic strains, only the worms with the injection marker (odr-1::gfp) were counted and analyzed.

L1 longevity analysis

Life span was assessed in liquid medium. L1 worms were cultured in 1 mL M9, and 50-100 μL was taken to ensure the sample size was larger than 50 worms. The worms were scored every day by counting the number of worms that were moving (alive) and then dividing that number by the total number of worms in the aliquot. To compare the survival rates between strains, the assay was carried out in triplicate with at least 100 L1s, and the mean survival rate was calculated using the Kaplan-Meier method [21] to determine the fraction of living animals over the time course of the experiment. Any significant difference in the overall survival rates was determined using the log-rank test.

Transgenic strains

For the INS-6 overexpression strains, the gene sequence was amplified from C. elegans genomic DNA and placed under the control of the pan-neuronal promoter Prgef-1. A plasmid with the injection marker odr-1::rfp was injected into Pegl-17::mCherry (rdvIs1) worms using standard microinjection methods (Mello et al., 1991), and at least three stable lines were selected for each injected strain.

Primer sequences used for ins-6 amplification: Forward 5’-aattgctagcATGAACTCTGTCTTTACTATCATCTTCG-3’, Reverse 5’-aattggtaccTCATGGACAACAAGCAGATCTTATG-3’.

Antibody staining

Antibody staining was performed as previously described [47]. In brief, L1 worms were collected in 100 μl M9, fixed with 200 μl of cold 2X witches brew and 100 μl 10% paraformaldehyde, and then incubated at 4°C for 30 min to overnight. The worms were washed twice in Tris-Triton buffer, incubated in 1% ßME/Tris-Triton for 1-2 hours at 37 °C, and then washed in 1X Borate buffer. Subsequently, the worms were incubated in 10 mM DTT/1X Borate buffer for 15 min at room temperature, washed in 1X Borate buffer, incubated in 0.3% H₂O₂/1X Borate buffer for 15 min, incubated for 15 min in PBST-B, and then washed with PBST-A. The worms were then visualized using an Axioplan fluorescent microscope (Zeiss, Germany).

For dpMPK-1 observation and antibody staining, the L1-arrested worms were collected at 10, 20, 40, and 48 hours after the embryos were prepared. We found that 40 hours after the embryos were placed in M9 buffer was the best time to detect dpMPK-1. Worms with more than two cells detected using anti-dpMPK-1 were scored. The anti-dpMPK-1 [26] and all secondary antibodies were purchased from Sigma-Aldrich (USA).

RNAi

An RNAi bacterial strain (HT115) expressing a double-stranded target RNA (vector, L4440) was cultured and used to inactivate the target gene function. Eggs from RNAi fed worms were transferred to fresh NGM plates containing the same bacteria and allowed to grow at 15°C for 3 days. Stage 4 larva were then transferred to the same RNAi NGM plates and cultured at 20°C for 24 hours. The RNAi NGM plates contained 1 mM isopropyl-B-D-thiogalactopyranoside (IPTG) for the induction of the double-stranded RNA. The L4440 carrying the empty RNAi plasmad was used as a negative control in RNAi experiments.