Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.

The transcriptional regulators CTBP-1 and PAG-3 are required for the development of AFT.Animals were treated with 0 mM and 400 mM exogenous ethanol. Relative speeds were calculated as treated over untreated speeds. Animals shown in the same graph were tested simultaneously on the same plates. a) Wild-type N2 animals develop AFT, and npr-1(ky13) animals are significantly faster developers of AFT. Compared with npr-1(ky13), npr-1(ky13) ctbp-1(eg613) animals have decreased initial sensitivity to ethanol; the decrease in development of AFT was not significantly different from npr-1(ky13). ctbp-1(eg613) animals in an npr-1(+) (N2) background develop significantly less AFT than N2 (n = 22). b) PAG-3 is required for the development of AFT. pag-3(n3098) animals do not develop AFT, and when tested in the sensitized background, pag-3(n3098) npr-1(ky13) animals have decreased initial sensitivity and are slow developers of AFT compared with npr-1(ky13) (n = 13). Note that the large decrease in speed in pag-3(n3098) animals at 30 minutes of ethanol exposure is largely driven by an increase in their untreated speed at 30 minutes, and may not reflect an actual increase in sensitivity to ethanol during that interval. We have not observed this kind of increase in untreated speed in other strains of animals tested, including in the npr-1(ky13) pag-3(n3098) double mutant. pag-3(n3098) animals do not increase their speed on ethanol over the course of 30 minutes, indicating that they are defective in AFT (at 400 mM: 10 minutes = 32.4 ± 2.6 µm/sec, 30 minutes = 33.7 ± 1.3 µm/sec; not significantly different t40 = 0.42, P = 0.67). Error bars are s.e.m. For indicated comparisons: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; for comparison to N2 at 10 minutes: ###, P < 0.001; for comparison to npr-1(ky13) at 10 minutes: †, P < 0.05; ††, P < 0.01; †††, P < 0.001.

The ctbp-1-regulated gene lips-7 modulates the rate of the development of AFT.Animals were tested on 0 mM and 400 mM exogenous ethanol. Relative speeds were calculated as treated over untreated speeds. All animals were tested simultaneously on the same plates. a) lips-7(ok3110) animals are resistant to ethanol at 10 minutes of exposure relative to wild-type N2, and they develop significantly more AFT than wild-type N2 animals (n = 13). b) The transcription factor sir-2.1 is a negative regulator that acts genetically upstream of ctbp-1 [21]; in a sir-2.1 mutant strain, loss of negative regulation of ctbp-1 should result in fast development of AFT. Compared with N2, sir-2.1(ok434) mutant animals were resistant to ethanol at 10 minutes of exposure but their apparent increase in development of AFT did not reach statistical significance (n = 13). Error bars are s.e.m. For indicated comparisons: n.s., not significant; *, P < 0.05; ***, P < 0.001; for comparison to N2 at 10 minutes: ###, P < 0.001.

Disruption of the plasma membrane through cholesterol starvation eliminates the ability to develop AFT.Worms require exogenous cholesterol for plasma membrane mediated processes such as clathrin-independent endocytosis. Young adult animals that were starved of exogenous cholesterol during their development were tested on 0 mM and 400 mM ethanol. Relative speeds were calculated as treated over untreated. Animals shown in the same graph were tested simultaneously on the same plates. a) Wild-type N2 animals develop AFT when reared on cholesterol-containing (NGM) plates, but do not develop AFT when starved of cholesterol (n = 14). b) npr-1(ky13) animals develop AFT when reared on NGM plates, but develop reduced levels of AFT when starved of cholesterol (n = 7). Together, these results suggest that the development of AFT requires cholesterol in the membrane. Error bars are s.e.m. For indicated comparisons: n.s., not significant; *, P < 0.05; **, P < 0.01; for comparison to N2 on NGM at 10 minutes: ###, P < 0.001; for comparison to npr-1(ky13) on NGM at 10 minutes: †, P < 0.05.

SLO-1 protein function is modulated by lips-7 function.a) Representative micrographs of crawling C. elegans, when the animal’s head is at the dorsal most point in the propagation of its waveform. Anterior is left, scale bar is 200 µm. Wild-type N2 demonstrates the normal sinusoidal body posture of a crawling worm. slo-1(eg142), lips-7(ok3110), and sir-2.1(ok434) demonstrate a similar exaggerated, loopy, body posture. In contrast, ctbp-1(eg613) has a flattened body posture. b) SLO-1 function is required for normal AFT. Worms were treated with 400 mM exogenous ethanol. slo-1(eg142) null mutant animals do not develop significant AFT, whereas slo-1(eg142); npr-1(ky13) double mutants do develop tolerance. The degree of recovered speed of slo-1(eg142); npr-1(ky13) did not achieve statistical significance compared with npr-1(ky13), although there was a strong trend for an attenuated response compared with the robust development of AFT by npr-1(ky13) (n = 14). These data suggest that SLO-1 is not the sole mediator of the development of AFT in the worm. c) Loss of lips-7 can modulate function of slo-1 gain-of-function alleles. slo-1(ky398gf) and slo-1(ky399gf) mutations increase the open probability or open time of the SLO-1 channel, resulting in a slow locomotion phenotype [29]. Loss of lips-7 significantly suppresses the slow movement phenotype of both slo-1(gf) alleles in the absence of ethanol (n = 6 for slo-1(ky389gf); n = 9 for slo-1(ky399gf)) indicating that the increase in TAGs in lips-7 can cause a change in the function of the membrane channel, SLO-1. These results suggest that lips-7 alters SLO-1 function through a plasma membrane mechanism. Error bars are s.e.m. For indicated comparisons: n.s., not significant; *, P < 0.05; ***, P < 0.001; for comparison to N2: #, P < 0.05; ##, P < 0.01; ###, P < 0.001.