A salt-induced kinase (SIK) is required for the metabolic regulation of sleep

Many lines of evidence point to links between sleep regulation and energy homeostasis, but mechanisms underlying these connections are unknown. During C. elegans sleep, energetic stores are allocated to non-neural tasks with a resultant drop in the overall fat stores and energy charge. Mutants lacking KIN-29, the C. elegans homolog of a mammalian Salt-Inducible Kinase (SIK) that signals sleep pressure, have low ATP levels despite high fat stores, indicating a defective response to cellular energy deficits. Liberating energy stores corrects adiposity and sleep defects of kin-29 mutants. kin-29 sleep and energy homeostasis roles map to a small number of sensory neurons that act upstream of fat regulation as well as of central sleep-controlling neurons, suggesting hierarchical somatic/neural interactions regulating sleep and energy homeostasis. Genetic interaction between kin-29 and the histone deacetylase hda-4 coupled with subcellular localization studies indicate that KIN-29 acts in the nucleus to regulate sleep. We propose that KIN-29/SIK acts in nuclei of sensory neuroendocrine cells to transduce low cellular energy charge into the mobilization of energy stores, which in turn promotes sleep. Highlights Sleep is associated with fat mobilization and low ATP levels Metabolic regulation of sleep requires the salt-induced kinase (SIK) homolog KIN-29 KIN-29 acts in sensory neurons upstream of sleep-promoting neurons Nuclear localization of KIN-29 is required for the metabolic regulation of sleep A type 2 histone deacetylase acts down stream of KIN-29 in the regulation of sleep. Liberation of energy from fat-storage cells promotes sleep. Beta-oxidation promotes sleep.

483 and movement quiescence was measured continuously using the WorMotel device for 8 hours 484 after UV exposure.
485 For the assay of microsphere accumulation in the absence of food, worms were exposed to 486 fluorescent polystyrene microspheres of 1.0 μm diameter (Polysciences) as described [67]. In 487 brief, a 100 μl microsphere suspension was mixed with 100 μl S-basel buffer, spread on a 3 cm 488 NMG-agar plate, and left at room temperature for ~60 min for liquid absorption. An age-489 synchronized population was grown until the adult stage. First day-old adult worms were 490 washed three times in S-basal buffer, transferred to the microsphere plates, and incubated ~15 491 min for uptake of the microspheres. After incubation, worms were quickly washed with M9 buffer 492 to remove excess microspheres, mounted on 2% agar pads containing the anesthetic Na-azide 493 (NaN 3 ), and imaged on a Leica DM5500 Nomarski microscope equipped with a Hamamatsu 494 Orca II camera. The fluorescence intensity of microspheres accumulated in the worm gut was 495 quantified using Volocity software (PerkinElmer)

SIS induction by UV irradiation and heat shock
497 UV induced sleep assays [43] were performed by exposing first day-old adult worms to 1500 498 J/m 2 UVC irradiation (254 nm) in a Spectrolinker™ XL-1500 (Spectroline). For the UV exposure, 499 the worms were housed either in a WorMotel chip placed in an uncovered plastic 10 cm Petri 500 dish or on the agar surface of an uncovered 5.5 cm Petri dish filled with NGM agar. A thin layer 501 of E. coli DA837 or OP50 bacteria was spread onto the surface of the NGM agar immediately 502 before the experiment in order to minimize growth of the bacteria, which could act to screen the 503 worms from the UV. Heat shock induced sleep assays were performed by submerging first day-504 old day adult animals in a circulating water bath to 35°C for 30 minutes. During the submersion, 505 the worms were housed on the agar surface of a 5.5 cm diameter Petri dish containing 11 mL 506 NGM agar or of a WorMotel placed in an empty plastic 10 cm Petri dish sealed with Parafilm. 514 Assessment of total body fat stores 515 Oil-Red O fixative staining was performed as described [108]. Briefly, well-fed worms were age-516 synchronized by the bleaching method and grown at 20°C on NGM plates seeded with E. coli 517 OP50. L4-staged worms were collected with dH 2 O and washed over a 15 μm nylon mesh filter 518 to remove any bacteria. Worms were transferred to 1.5 ml tube and excess water was aspirated 519 off. 600 μl of 60% isopropanol was added to fix animals and centrifugated at 1,200 relative 520 centrifugal force (rcf) to pellet worms. The supernatant was removed and 600 μl of Oil Red O 521 solution was added to each tube with pelleted worms. The Oil Red O solution was made using 522 0.5 g Oil Red O (Sigma, Cat # O0625) in 100 ml of 100% isopropanol, filtered through a 0.20 523 μm PVDF filter and allowed to equilibrate overnight with agitation at room temperature. Tubes 524 were placed in a wet chamber and worms were stained for six hours at 25°C. After staining, 525 worms were centrifugated at 1,200 rcf, washed twice, and re-suspended in 0.01% Triton X-100 526 in S-buffer. Worms were imaged on a 2% agar pad using a Leica DMI 3000-B inverted 527 microscope coupled to a Leica DFC295 color camera. Oil-Red intensity was quantified using the 528 Image J software (NIH). Pixel intensity was measured in the green color channel of the images.
529 The region of the intestine measured on each animal was from the anterior part of the intestine 530 (first cell) to region of the intestine in the mid-body at the same AP location as the vulva. Each 531 worm was analyzed using an equivalently sized window.
23 532 Fixative Nile Red staining was performed on transgenic and non-transgenic worms as described 533 [108]. Briefly, well-fed worms were age-synchronized by the bleaching method, and 500-1,000 534 L4-staged worms were washed from NGM plates seeded with E. coli OP50 using PBS 535 containing 0.01% Triton X-100. Worms were allowed to settle by gravity and washed once with 536 PBS. Excess PBS was removed and 200 μl of 40% isopropanol was added to fix animals for 3 537 minutes. Next, the supernatant was removed, and 150 μl of a Nile Red (Sigma, Cat # 19123) 538 solution in isopropanol was added to the fixed animals and allowed to stain for 30 min in the 539 dark with agitation. After staining, worms were allowed to settle and washed once with 1x M9 540 buffer and kept in the dark at 4°C before visualization. Stained worms were mounted on 2% 541 agar pads and imaged on a Leica DM5500 Nomarski microscope equipped with a Hamamatsu 542 Orca II camera.
544 Worms were age-synchronized by the bleaching method and grown at 20°C until the L4 larval 545 stage on NGM plates seeded with E. coli OP50. Worms were collected and washed with S-546 basal solution. A 5% Triton X-100 solution with 1x protease inhibitors (Roche Complete Mini, 547 EDTA free) was added 1:1 to a 50 μl worm pellet, and worms were sonicated with a water bath 548 sonicator (Branson). Lipids were dissolved twice by heating the lysate to 90°C for 5 min followed 549 by vortexing. Following centrifugation, the supernatant was used to determine the total TAG 550 levels according to the manufacturers protocol. TAG concentrations were normalized to the total 551 protein content as determined by a Micro BCA protein assay kit (ThermoFisher, Cat # 23235).
552 Each assay was done in triplicate and the average TAG level (μg TAG/ μg protein) was 553 calculated. 574 capturing images at a rate of 0.5 frames/s. Video files were then imported into Volumetry, and 575 each frame was converted into an 8-bit grayscale image for subsequent analysis. To quantify 576 food-leaving behavior, we generated a binary image containing only white pixels when the 577 grayscale value was above a user defined threshold that approached the maximum (intensity) 578 grayscale value (255). This binary image identified the worms in each frame. We then collapsed 579 the resulting frames into a single image to visualize worm tracks outside of the bacterial lawn for 580 each 12-hour video. Worm track images were imported into the ImageJ software (NIH) and the 581 average number of pixels representing worm tracks outside of the bacterial lawn was quantified 25 582 Measurements of ATP levels 583 ATP levels in whole worms were determined as described [49]. For DTS experiments, ~6,000-584 7000 worms were age-synchronized using the double-bleaching method [112], transferred to 585 NGM agar surface (10 cm diameter) that was fully covered with a lawn E. coli OP50 and grown 586 at 20°C. L1 animals were washed off the agar surface using a pipette filled with 5 ml of M9 587 buffer. The worm and bacterial suspension was allowed to settle through a 5 μm nylon mesh 588 filter, which passes bacteria but traps the worms. The worms trapped by the filter were then 589 flash frozen in liquid N 2 and stored at -80°C until analysis. Worms were collected every hour on 590 the hour between 12 hours and 21 hours after feeding developmentally-arrested L1 animals. 591 Because at 20°C, lethargus occurs between 16.5-18.5 hours, these sampling times including 592 animals before, during, and after L1 lethargus. For SIS experiments, one-day old adult worms 593 were exposed while on an agar surface without peptone in the presence of a thin layer of 594 bacteria to 1500 J/m 2 UVC irradiation, 254 nm, ultraviolet irradiation. Following irradiation, worm 595 samples were collected hourly on the hour between time 0 and 5 hours after irradiation. 30-40 596 adults were collected in a 1.5 mL microfuge tube under stereomicroscopal observation using a 597 platinum wire. The samples were flash frozen in liquid N 2 and stored at -80°C until analysis. For 598 time-course experiments, sleep was identified by measuring the fraction of non-pumping L1 599 worms for DTS, and the minutes per hour of body movement quiescence for SIS. For measuring 600 ATP levels in L4 animals, worms were grown until the mid L4 larval stage, and 50 animals per 601 sample were collected in a 1.5 mL microfuge tube, flash frozen in liquid N 2 and stored at -80°C 602 until analysis. 603 All samples for ATP determination were treated identically. Following collection off the agar 604 surface using water and a nylon mesh into 1.5 mL microfuge tubes, the worms were flash frozen 605 in liquid nitrogen within 8-12 min of preparation time. In preliminary experiments we found that, 606 while there was some time-dependent degradation of ATP in the first five minutes, the levels