Activation or inhibition of GLP-1 pathways

Abstract

This disclosure describes methods of increasing or decreasing food intake, including regulation of glucagon-like peptide 1 (GLP-1) pathways. Also disclosed are methods of increasing or decreasing food intake, preventing food aversion reactions, and treating various diseases and disorders including obesity and eating disorders. Also described herein are methods of evaluating C. elegans to identify means to regulate food intake.

Description

ACTIVATION OR INHIBITION OF GLP-1 PATHWAYSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 636,947, filed April 22, 2024. the disclosure of which is hereby incorporated by reference in its entirety .ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing in computer readable form entitled “Oi l 23-0019-00PCT.xml’', created March 31, 2025, having a size of 50,159 bytes, which is incorporated by reference herein.DESCRIPTION

[0003] This invention was made with government support under Research Grant No. R21AG071436 awarded by the National Institutes of Health / National Institute of General Medical Sciences and Research Grant Nos. R35GM122610 and R01AG054215 awarded by the National Institutes of Health / National Institute on Aging. The government has certain rights in the invention.FIELD

[0004] This disclosure includes, but is not limited to, methods of increasing or decreasing food intake, preventing food aversion reactions, and methods of treating various diseases and disorders including obesity and eating disorders. The specification, figures, and claims provide the full scope of the disclosure.BACKGROUND

[0005] It is largely an open question how intrinsic metabolic cues might influence food consumption. Caloric insufficiency induces feeding1, but the metabolic signals involved remain to be fully elucidated. It is also unknown whether the availability of critical nutrients might affect food consumption. Obesity can be treated through glucagon-like peptide- 1 receptor (GLP-1R) targeting, but it is unclear how this signaling is controlled physiologically2.

[0006] An understanding of mechanisms that determine hunger, satiety7, and satisfaction with food could yield improved solutions for the obesity7epidemic or conditions of inadequate food intake such as reduced appetite in the elderly and anorexia nervosa. Much has beenlearned about signals that indicate fat levels, gastric fullness, and the presence of nutrients in food, and how these signals are transmitted to and within the brain to regulate feeding1-4,5. Feedback regulation by energy reserves was revealed by the landmark finding that feeding is inhibited by the peptide hormone leptin, which is produced in adipose tissue in response to fat stores1,6. It is less clear whether metabolic parameters might affect food consumption more directly. A likely signal of caloric insufficiency is the AMP-activated kinase (AMPK), which senses low ATP levels3. In mice AMPK acts in hypothalamic regions to increase feeding and regulate the dietary preference for fat or carbohydrates7'9, but it is largely a mystery how AMPK exerts these effects.

[0007] Peptides that target the GLP-1R, alone or together with other secretin-like (SCT-like) G-protein coupled receptors (GPCRs)13. are remarkably effective at reducing food cravings and consumption, as well as inducing weight loss14. These effects are mediated through the action of these agents in the brain14. This exciting advance has made it possible to treat obesity as a modifiable condition, although these agents may induce side effects that include nausea, diarrhea, and aversion to food2. Further, these agents are not always as effective as would be desired and have limited half-lives, and must be delivered as peptide agonists requiring injection by the patient.

[0008] The present disclosure identifies metabolic mechanisms that regulate feeding behaviors in Caenorhabditis elegans. which is highly amenable to unbiased genetic analyses. This organism also lacks leptin signaling, and thus provides a platform for identifying ancestral pathways of feeding control. Unexpectedly, the neuropeptide pigment-dispersing factor-1 (PDF-1) and its G-protein coupled receptor (GPCR) PDFR-1 (PDF-1 / PDF-1R) signaling pathway in C. elegans was found to be orthologous at the sequence level to the GLP-1 / GLP-1R system, as well as having similar function in inhibiting eating and inducing food dissatisfaction, allowing description of the underlying mechanisms of how GLP-l / GLP- 1R signaling is controlled in different tissues to influence feeding using the powerful model of C. elegans genetic mutation and behavior. Also, the present disclosure highlights that PDF-1 / PDF-1R signaling in C. elegans corresponds to signaling through a number of human pathways, as shown in FIG. 2G and FIG. 20A, including CALCR (encoding the calcitonin receptor), GLP-1R, GCGR (encoding the glucagon receptor), and GIPR (encoding gastric inhibitory polypeptide receptor) signaling pathways.

[0009] As described herein, behaviors corresponding to eating and food satisfaction are regulated by the ratio of available monounsaturated to saturated fatty acids. These fatty acids are sensed in the nervous system and periphery at the endoplasmic reticulum, through incorporation into membrane lipids and signaling by the membrane sensor inositol-requiring enzyme 1 (IRE-1). The low-energy signal transducer AMP-activated kinase3regulates feeding through this pathway, which controls behavior by acting on serotonergic neurons and the glucagon-like peptide- 1 receptor counterpart. The latter signaling decreases eating- related behavior and food satisfaction, analogously to its actions in humans. These findings show that feeding behaviors respond to deficits in unsaturated fatty' acids, revealing a paradigm in which metabolic conditions in the brain profoundly affect feeding regulation, and emphasizing the critical importance of these lipids. By unexpectedly linking energy and fatty acid sensing to glucagon-like peptide-1 signaling, the present disclosure highlights models for future therapy development. In addition, the PDF-1 / PDF-1R signaling pathway in C. elegctns (which is the GLP-1 / GLP-1R ortholog) provides robust new targets to developing novel therapeutics for human eating disorders, such as IRE-1.

[0010] The present disclosure has also identified mechanisms through which these critical fatty acids are sensed at the endoplasmic reticulum (ER) to initiate signaling that controls these behaviors. An ER-based mechanism was identified involving lipid droplet formation and membrane phospholipid biosynthesis sensing the availability of specific fatty’ acids to control activity of C. elegctns ’ version of GLP-1 and its receptor (PDF-l / PDFR-1). This mechanism is triggered by a deficit in unsaturated fatty acids (UFAs) or a surplus of saturated FAs (SFAs), either of which activates serotonin signaling in C. elegctns that in turn acts on the GLP-1R counterpart. While serotonin signaling is part of the PDF-l / PDFR-1 pathway in C. elegans. it is unclear whether this is also true in humans. In other words, the metabolic relationship between lipid signaling and GLP-1 R and PDFR-1 activity is clear from the present data, but more work will be needed to understand the extent of overlap in the full signaling pathways, including whether human signaling downstream of the GLP-1 R also involves serotonin or might involve serotonin signaling in a different way.

[0011] When triggered by excess SFAs, this signaling can be suppressed by specific UFAs. These UFAs are the monounsaturated fatty acids (MUFAs) oleic and palmitoleic acid and the PUFA linoleic acid. Thus, the present disclosure defines these UFAs as nutrients so critical that they govern food consumption and affinity for food, presumably because of theirimportance for membrane and triglyceride synthesis. This mechanism reports not only on lipid metabolism directly but also indirectly on energy status (i.e. caloric need), as indicated by AMPK signaling. The readout from this signaling controls eating behaviors by acting through serotonin biosynthesis and production of the PDF-1 ligand, each of which represents a biomarker of pathway activity.

[0012] The present disclosure also identifies and validates multiple druggable metabolic and signaling targets upstream of GLP-1R signaling. Those targets include any mechanisms that are involved in lipid droplet formation or function or that affect synthesis of triglycerides and membrane phospholipids. Mechanisms also include the ER membrane receptor IRE1, which we determined is required for signaling from UFAs that indicates inadequate food, driving the desire to eat but also to find ‘"better” food. IRE-1 is also required for UFA-driven signaling in the brain that increases eating (dwelling). Inhibitors and activators of IRE1 could be employed to modulate behavior through the GLP-1R pathway. Further, the present disclosure also identifies multiple biomarkers for screening that would identify additional molecular targets for pharmacological intervention.

[0013] Further, as described herein, activation of PDFR-1 signaling downstream of this FA- based signaling system reduces eating, and at the same time induces food leaving and an indirect effect on eating caused by food aversion. Increasing the SFA / UFA ratio in the brain acts overcomes the background of PDFR-1 signaling and induces eating. By contrast, perturbing lipid metabolism by increasing the SFA / UFA ratio in peripheral tissues stimulates dissatisfaction with food that is manifested as food avoidance. This signaling is mediated by the PDFR-1 pathway, and this behavior may mimic nausea and aversion to food in humans. In summary', in C. elegans PDFR-1 signaling decreases feeding / eating in two ways, by acting in the brain to suppress eating (appetite) directly, and in the periphery’ to induce an aversion to food. In other words, activating PDFR-1 in the brain can reduce eating, while inhibiting PDFR-1 in the periphery' can reduce food aversion. Supplementation with specific UFAs suppresses each of these PDFR- 1 -induced behaviors, and in the brain this signal is mediated through alteration of phospholipid homeostasis and lipid droplet formation. Stimulating endogenous PDFR-1 and related signaling in neurons by modulating IRE-1 and / or AMPK in C. elegans in turn stimulates a serotonin pathway (through MOD- 1 / SER-4) that counteracts PDFR-1 signaling. These unexpected relationships present the opportunity’ to preferentiallytarget increasing food intake versus the side-effect of food aversion using agents that preferentially do or do not cross the blood-brain barrier efficiently.SUMMARY

[0014] Described herein are results identifying mediators of hunger and satiety perception that ultimately control food consumption by influencing feeding behaviors. Also described herein are drug targets and screening platforms for development of improved agents that would target the GLP-1R pathway in ways that greatly expand possibilities for therapeutic interventions. Each of the following embodiments are provided:

[0015] Embodiment 1. A method of decreasing food intake in a subject in need of treatment, comprising decreasing activity of Inositol-Requiring Enzyme 1 (IRE-1) and / or AMP- Activated Protein Kinase (AMPK) or a signaling molecule downstream thereof; and / or increasing activity of one or more of adipose triglyceride lipase (ATGL1 ), diglyceride acyltransferase 1 (DGAT1), perilipin-1 (PLIN1), fat storage-inducing transmembrane protein 2 (FITM2). or one or more protein involved in endoplasmic reticulum (ER)-associated degradation (ERAD).

[0016] Embodiment 2. The method of claim 1, wherein the subject has obesity, prediabetes, type 2 diabetes, and / or a metabolic syndrome.

[0017] Embodiment 3. A method of increasing food intake in a subject in need of treatment, comprising increasing activity of IRE- 1 and / or AMPK or a signaling molecule downstream thereof; and / or decreasing activity of one or more of Lipinl, ATGL1. DGAT1. PL1N1. FITM2, or one or more proteins involved in ERAD.

[0018] Embodiment 4. The method of claim 3, wherein the administering decreases signaling through the endogenous glucagon-like peptide- 1 receptor (GLP-1R) or another receptor in FIG. 2G and / or FIG. 20 A in the brain of the subject.

[0019] Embodiment 5. The method of claim 3 or claim 4, wherein the subject has anorexia nervosa and / or avoidant restrictive food intake disorder (ARFID).

[0020] Embodiment 6. The method of any one of claims 1-5, wherein the agent is modified to cross the blood brain barrier (BBB).

[0021] Embodiment 7. The method of claim 6, wherein the activator or inhibitor is modified by a modification to increase lipid solubility, decrease molecular weight, and / or decrease polarity.

[0022] Embodiment 8. The method of claim 7, wherein the modification is capable of increasing the ability to cross the BBB by 10% or more, 25% or more, or 50% or more.

[0023] Embodiment 9. The method of any one of claims 1-8, wherein the signaling molecule downstream of IRE1 and / or AMPK is an RNA acted on by IRE-1, p38, SKN-l(Nrf2), and / or a c-Jun N-terminal kinase (JNK).

[0024] Embodiment 10. The method of claim 9. wherein the RNA acted on by IRE-1 is xbp-1 or splicing of xbp-1 RNA.

[0025] Embodiment 11. The method of any one of claims 1-10, wherein activity of IRE- 1 is measured by regulated IRE 1 -dependent decay (RIDD) of one or more mRNAs; levels of an RNA acted on by IRE-E activation of p38 and SKN-l(Nrf2); IRE-1 autophosphorylation; and / or the JNK signaling cascade.

[0026] Embodiment 12. The method of claim 1 1, wherein the RNA acted on by IRE-1 is xbp-1 or splicing of xbp-1 RNA.

[0027] Embodiment 13. The method of any one of claims 1-12. wherein the one or more proteins involved in ERAD comprise one or more selected from osteosarcoma amplified 9, endoplasmic reticulum lectin (OS9), endoplasmic reticulum lectin 1 (ERLEC 1), E3 ubiquitin ligase HRD1, and / or SEL1L adaptor subunit Of ERAD E3 ubiquitin ligase (SEL1L).

[0028] Embodiment 14. A method of decreasing food aversion and / or nausea in a subject taking a GLP-1 -based therapy comprising administering an agent modified to not cross the BBB, wherein the agent is an inhibitor of IRE-1 or AMPK. and / or an activator of Lipinl, ATGL1, DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD.

[0029] Embodiment 15. The method of claim 14, wherein the activator or inhibitor is modified by a modification to reduce lipid sol ubili ty, increase molecular weight, and / or increase polarity.

[0030] Embodiment 16. The method of claim 15, wherein the modification is capable of reducing the ability to cross the BBB by 90% or more, 95% or more, or 99% or more.

[0031] Embodiment 17. The method of any one of claims 14-16, wherein the GLP-l-based therapy comprises a GLP-1 receptor agonist, dual-acting GLP-1 receptor agonist and glucosedependent insuhnotropic polypeptide (GIP) receptor agonist, amylin agonist, and / or a dualacting GLP-1 receptor and glucagon receptor (GCGR) agonist.

[0032] Embodiment 18. The method of claim 17, wherein the GLP-1 receptor agonist is exenatide, lixisenatide. liraglutide. albiglutide, dulaglutide, and / or semaglutide.

[0033] Embodiment 19. The method of claim 17, wherein the dual agonist for GLP-1 receptor and GCGR is BI 456906.

[0034] Embodiment 20. The method of claim 17, wherein the amylin agonist is pramlintide and / or cagrilintide.

[0035] Embodiment 21. The method of claim 17, wherein the dual-acting GLP-1 receptor agonist and GIP receptor agonist is tirzepatide.

[0036] Embodiment 22. A method of decreasing food intake in a subject in need of treatment by increasing the levels of monounsaturated fatty acids (MUFAs) relative to saturated Patty acids (SFAs).

[0037] Embodiment 23. The method of claim 22, wherein the increasing occurs in the brain.

[0038] Embodiment 24. The method of claim 22 or 23, wherein the MUFAs comprise oleic acid and / or palmitoleic acid.

[0039] Embodiment 25. The method of any one of claims 22-24, wherein the levels of MUFAs are increased relative to SFAs by treatment with one or more agent to increase activity of stearoyl-CoA desaturase (SCD) and / or elongation of very long chain fatty acids protein 6 (ELOVL6).

[0040] Embodiment 26. The method of any one of claims 22-25, wherein increasing the levels of MUFAs relative to SFAs decreases activity' of IRE-1 and / or AMPK and / or increases activity of one or more of Lipinl, ATGL1, DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD.

[0041] Embodiment 27. A method of identifying a drug target and / or evaluating an agent as a treatment for one or more disorder comprising evaluating pigment dispersing factor (PDF-1) and / or pigment dispersing factor receptor (PDFR-1) activity in C. elegans.

[0042] Embodiment 28. The method of claim 27, wherein the activity of PDF-1 and / or PDFR-1 is measured using promoter activity of PDF- 1 and / or behavior of C. elegans.

[0043] Embodiment 29. The method of claim 27, wherein the behavior of C. elegans is mobility' and / or feeding.

[0044] Embodiment 30. The method of claim 27, wherein the promoter activity is assessed using a fluorescent reporter assay.

[0045] Embodiment 31. The method of any one of claims 27-30, wherein the agent is an agonist of any receptor shown in FIG. 2G and / or FIG. 20A.

[0046] Embodiment 32. The method of claim 31, wherein the agent is a GLP-1 receptor agonist, GIP receptor agonist, amylin agonist, and / or GCGR agonist.

[0047] Embodiment 33. The method of claim 32, wherein the agent binds specifically to more than one of the receptors shown in FIG. 2G and / or FIG. 20A.

[0048] Embodiment 34. The method of claim 33, wherein the agent binds specifically to more than one of the GLP-1 receptor, GIP receptor, amylin receptor, and glucagon receptor.

[0049] Embodiment 35. A method of treating a disorder in a subject comprising administering one or more agent capable of modulating PDF-1 and / or PDFR-1 activity in C. elegans.

[0050] Embodiment 36. The method of claim 35, wherein the disorder is anorexia nervosa and / or ARFID.

[0051] Embodiment 37. The method of claim 35, wherein the disorder is obesity, prediabetes, type 2 diabetes, or a metabolic syndrome.

[0052] In some instances, in each of the embodiments, the subject is human.

[0053] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0054] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0055] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, sen e to explain the principles described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGs. 1 A-1L show metabolic conditions regulate feeding behaviors and food consumption. FIG. 1A Genes that affect consumption of bacterial food, identified by RNAi screening of a library' of more than 1100 transcription factors (see, FIG. 6A). These confirmed hits met two criteria: (i) knockdown did not impair development, and (ii) food consumption was altered by at least 20% compared to the empty vector (EV) control. Loss of DAF-1 was previously shown to increase food consumption16. Data were derived from two independent screens and two follow-up analyses of positives, each wi th at least five replicates per condition. Statistical comparisons were conducted against the EV control. Knockdown ofpositives where indicated induced food-leaving, (see, FIG. 7B)22. FIG. IB Schematic of the food-leaving assay. FIG. 1C Food-leaving behavior induced by crh-1 and sbp-1 RNAi since LI stage, statistical comparisons are relative to EV control at 54 hours. FIG. ID Simplified schematic of the square assay evaluating the proportion of dwelling versus roaming on food. The grid has 109 squares in total. FIG. IE RNAi-induced increases in dwelling, as detected by fewer squares entered over 16 hours by individual L4-stage animals, crh-1 or sbp-1 RNAi were compared to EV control. Data are from three biologically independent experiments, wherein at least 30 animals are assayed each condition. Each assay point represents a single animal. Comparisons against empty vector control are indicated, f, g, Induction of food-leaving by crh-1 or sbp-1 RNAi in peripheral tissues. The RNAi defective mutations sid-1 (qt9) (FIG. IF) and rde-l(ne219) (FIG. 1G) were rescued by tissue-specific transgenic expression. FIG. 1H Increased dwelling, as indicated by fewer squares entered, induced by crh-1 or sbp-1 RNAi specifically in neurons. FIG. II Effects of tissue-specific crh-1 or sbp-1 RNAi on food consumption. FIG. 1 J A model illustrating the regulation of food affinity behavior and food intake consumption by metabolic perturbation. Data represented as Mean + SEM, P-values were calculated by unpaired, two-sided t-test with Welch correction. FIG. IK shows effects of tissue-specific crh-1 or sbp-1 RNAi on food consumption. FIG. IL shows a model illustrating metabolic regulation of food affinity behaviors.

[0057] FIGs. 2A-2K show metabolic regulation of food-affinity’ behaviors through serotonin and SCT-like receptor signaling. FIG. 2A Dependence of crh-1 RNAi-induced dwelling on serotonin biosynthesis in the NSM neurons. The tph-1 mutation was rescued by tph-1 expression constructs that allowed tph-1 to be deleted specifically in the ADF or NSM neurons. n>18 animals in each of at least two independent experiments. FIG. 2B Dependence of crh-1 RNAi-induced food-leaving on serotonin biosynthesis in the NSM neurons. Statistical comparisons are relative to EV control at 56h of grow th on RNAi bacteria. FIG. 2C Food leaving evoked by low power (0.25mw7cm2) Chrimson activation in the NSM neurons. ATR: all-trans-retinal; no-ATR: lacking essential opsin co-factor, all-trans-retinal. Data represent three biologically independent experiments, with at least 30 animals assayed under each condition. FIG. 2D Dependence of sbp-1 RNAi-induced dwelling on the serotonin receptors SER-4 and MOD-1. FIG. 2E Dependence of sbp-1 or crh-1 RNAi-induced foodleaving on the SER-5 receptor. FIG. 2F Failure of crh-1 RNAi to increase dw elling in pdfr-1and tph-1 mutants. FIG. 2G Maximum likelihood phylogenetic tree comparison of SCT-like GPCRs in humans and C. elegans. The receptor amino acid sequences of human (hu) and C. elegans (ce), were aligned using MUSCLE, and a tree was constmcted using mega software of version 11. Gene IDs used for this analysis are shown in Table 4 below in the Materials and Methods example. FIG. 2H Food leaving rate changes of wild type animals and the pdfr- l(ok3425) mutant exposed to compound 2 (Sigma G8048, a GLP-1R agonist in humans60, 61) shortly. FIG. 21 Percentage of squares of wild type animals entered during 6 hours with or without compound 2 exposure. FIG. 2J Total food consumption of wild type animals in 5 days with or without compound 2 exposure. FIG. 2K A model for metabolic regulated food leaving and food dwelling resulted long-term food consumption in C. elegans. Dots in (FIGs. 2A, D, F. and I) represent individual animals assayed. In box plots, the center line shows the median, whisker extend to minimum and maximum values. Data represents Mean + SEM from at least 3 independent experiments, each with 4-5 biological replicates at least. P-values were calculated by unpaired, two-sided t-test with Welch correction.

[0058] FIGs. 3A-3I show specific fatty acid regulate food affinity responses. FIG. 3A Oleic acid supplementation suppress food-leaving behavior and FIG. 3B food-dw elling behavior induced by crh-1 and sbp-1 RNAi in wild-type animals. Dots in FIG. 3B represent individual animals assayed. In box plots, the center line show s the median, whisker extend to minimum and maximum values. FIG. 3C Conserved De Novo lipid synthesis pathway. FIG. 3D Oleic acid supplementation suppress food-leaving and FIG. 3E dwelling behavior of the double mutant fat-6(tm331),-fat-7(wa36), which redundantly mediate the synthesis of oleic aci44. FIG. 3F Food leaving rate changes of wild type animals treated with ImM palmitic acid, ImM oleic acid or ImM palmitic acid and ImM oleic acid. FIG. 3G Percentage of squares entered of wild type animal treated with ImM palmitic acid, ImM oleic acid or ImM palmitic acid and ImM oleic acid. FIG. 3H Food leaving rate of wild type, tph-l(mg280) mutant and ser-5(ok3087) mutant on the assay plates with or without PA treatment. FIG. 31 Dependence of PA induced food leaving on PDFR-1. In (FIGs. 3 A, D, F, and I) Data represents Mean + SEM from at least 3 independent experiments, each with 4-5 biological replicates at least. P-values were calculated by unpaired, two-sided t-test with Welch correction.

[0059] FIGs. 4A-4M show' that MUFAs modulate food affinity' behaviors at the endoplasmic reticulum within the nervous system. FIG. 4A schematic of the short-term food leaving assay.FIG. 4B Food leaving rate changes of wild type animals treated with or without crh-1 RNAi post-treated with or without OA. FIG. 4C Food leaving rate changes of w ild type animals maintained on plates with or without PA for 48h from LI stage, and then exposed to PA or OA for another 8h. FIG. 4D The tph-l::gfp level changes in the NSMs that maintained on control, OA, or PA compared to further exposure to PA or OA, as indicated respectively, tph- l:gfp value was quantified for each genotype by Image J, and expressed as mean ± SEM value (n=15). P-values were calculated by unpaired, two-sided t-test with Welch correction. FIG. 4E Percentage of squares entered by a single animal bearing atgl-1 RNA knockdown or neuron-specific atgl-1 knockdown with or without dietary OA supplementation. FIG. 4F Percentage of squares entered by a single animal bearing dgat-2 RNA knockdown or neuronspecific dgat-2 knockdown with or without dietary OA supplementation. FIG. 4G Food leaving rate changes of wild type animals treated with lpin-1, atgl-1, and dgat-2 respectively with or without dietary' OA. FIG. 4H Schematic of genetic and dietary intervention that involved in oleic acid induced lifespan extension. Mammalian gene names are indicated. Created by Biorender.com. FIG. 41 Percentage of squares entered by a single animal bearing lipin- 1 RNA knockdown or neuron-specific lipin-1 knockdown with or without dietary OA supplementation. FIG. 4J lipidomic results show the ratio of C16:0 to C 18: 1 in variety lipids under metabolic deficit with or without oleic acid.. FIG. 4K Dependence of PA induced food dwelling on ire-1. FIG. 4L Dependence of PA induced food leaving on ire-1. FIG. 4MThe UPR ER reporter hsp-4::GPP induced by crh-1 and sbp-1 RNAi is suppressed by oleic acid.

[0060] FIGs. 5A-5R show FA-mediated regulation of food-affinity behaviors by AMPK.FIG. 5A Constitutive whole-body AMPK expression reduces food consumption. Two transgenic CA-AAK-2 lines were compared to a crh-1 loss-of-function mutant. Data represented as Mean + SEM, P-values were calculated by unpaired, two-sided t-test with Welch correction. CA-AAK-2 is expressed trans genetically from its own promoter53. FIG. 5B Whole-body CA-AAK-2 expression increases dwelling, partially dependent upon tph-1. FIG. 5C Whole-body CA-AAK-2 expression induces / / ?- / -dependent food-leaving. FIG. 5D Robust increase in dwelling induced by neuronal expression of CA-AAK-2 (N-CA-AAK-2). compared to whole-body CA-AAK-2 expression. OA supplementation suppressed dwelling in each case. FIG. 5E OA suppression of food-leaving induced by whole-body' CA-AAK-2 expression. FIG. 5F Effects of tissue-specific CA-AAK-2 expression on food consumption. FIG. 5G peripheral constitutive expression of CA-AAK-2 increases food leaving cannot besuppressed by OA supplementation. FIG. 5H Quantification of the Oil red O staining level in the wild type and CA-AAK-2 animals on the regular or full lawn plates respectively. FIG. 51 Whole body increased food dwelling is suppressed by beta-oxidation genes knockdown. FIG. 5J whole body CA-AAK-2 increased food leaving is suppressed by dhs-28 RNAi. FIG. 5K-L OA suppresses CA-AAK-2-induced dwelling (FIG. 5K) and food-leaving (FIG. 5L). FIG. 5M ire-1 knockdown partially suppresses dwelling induced by N-CA-AAK-2. FIG. 5N ire-1 knockdown suppresses whole-body CA-AAK-2 -induced food leaving. FIG. 50 Knockdown of genes involved in peroxisomal beta-oxidation15suppresses CA-AAK-2-induced dwelling. FIG. 5P Constitutive whole-body AMPK activation increases food leaving on E. coli strains OP50 and HT115. FIG. 5Q Growth on DA837 increases food-leaving comparably to constitutive whole-body AMPK activation. In each case food-leaving is suppressed by OA. FIG. 5R OA suppression of hsp-4p: :GFP activation that is induced by growth on DA837. Data are mean ± SEM. P values are two-way repeated-measures ANOVA with Holm-Sidak's multiple comparisons test in (FIG. 5 K, M, O), two-way repeated-measures ANOVA with Bonferroni's multiple comparisons test in (FIG. 5 L. N. P),and two-tailed Mann-Whitney test in (FIG. 5Q), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.$denotes comparison between WT and CA-AAK-2 without OA treatment.#denotes comparison between CA-AAK-2 food leaving with or without OA supplementation.

[0061] FIGs. 6A-6F show identification of regulators that affect feeding behavior. FIG. 6A Workflow of bacterial clearance assay15 16. After development on RNAi plates, L4 stage animals were plated in wells, with 5 wells plated per condition. Bacterial clearance was assayed by optical density (OD) 5 days later. See Methods for additional information. FIG. 6B Volcano plot showing a representative of two independent rounds of screening for altered food consumption compared to EV control. FIG. 6C Average pharyngeal pumping rate of individual day-1 animals, comparing EV, crh-1, and sbp-1 RNAi. P value derived from two- tailed unpaired t test, n.s, not significant. Each dot represents an animal. FIGs. 6D-6F Multiple-worm tracking analysis of individual L4 stage animals over 90 mins after treatment with EV (FIG. 6D), crh-1 (FIG. 6E), or sbp-1 RNAi (FIG. 6F). Knockdown of crh-1 or sbp-1 reduced crawling speed and increased high-angle turning. The average speed and curvature, along with trajectory', were automatically truncated into 10s intervals and analyzed by Worm Lab software. The clustering boundary' line separating dwelling and roaming was defined for WT worms under standard conditions, then used for all genotypes.

[0062] FIGs. 7A-7K show that metabolically induced food-leaving is distinct from food aversion induced by pathogen or toxin-mimic responses, a-c, Knockdown of crh-1 or sbp-1 induced food-leaving in (FIG. 7A) npr-l(ad609), and (FIG. 7B) daf-7(ok3125) mutants. NPR-1 is involved in aerotaxis behavior that is needed for pathogen avoidance. DAF-7 is necessary to induce protective avoidance behavior in response to pathogens15. FIG. 7C Unlike pathogen exposure15, metabolic perturbation from sbp-1 RNAi does not induce daf-7 expression in the AS J neurons. Representative images are shown of daf-7p::GFP in adult hermaphrodites, comparing control with RNAi against sbp-1. d-f, Knockdown of crh-1 or sbp-1 induced food-leaving comparably in WT animals and the JNK / p38 MAPK pathway mutants (FIG. 7D) mlk-l(ok2471), (FIG. 7E) pmk-l(km25), and (FIG. 7F) mek-l(KS54), in contrast to pathogen avoidance or many toxin-mimic responses22. FIG. 7G Representative images of the pathogen reporter clec-60::GFP and the detoxification reporter irg-l::GFP in day-1 adult animals after the indicated RNAi treatments. FIG. 7H Pathogen exposure does not increase dwelling. Worms were placed on either OP50 control or Pseudomonas aeruginosa bacteria after the indicated RNAi treatments as in Fig. ID. i, Exposure to the proteasome inhibitor bortezomib (BTZ) does not induce dwelling. Dots in (FIGs. 7H-I) represent individual animals assayed. In box plots, the center line shows the median, whisker extend to minimum and maximum values. Data represents Mean + SEM from at least 3 independent experiments, each with 4-5 biological replicates at least. P values are derived from unpaired, two-sided t-test with Welch correction. FIG. 7J Pathogen exposure but not crh-1 RNAi increased daf-7p::GFP expression in the ASJ neurons1. FIG. 7K Representative images of the pathogen-induced reporters hsp-6p::GFP and cyp-14A4::GFP in day-1 adult animals after the indicated RNAi treatments.

[0063] FIGs. 8A-8I show that serotonin circuits mediating metabolic perturbation regulated food affinity behaviors. FIG. 8 A Representative image of tph-l::gfp expression in adult hermaphrodites exposed to OP50 or PA14 / OP50 mix for 4-6 hours. Arrows indicate neuronal cell bodies of the NSM and ADF respectively. Scale bars (applicable to images in the same row). lOum. FIG. 8B Quantification of tph-l::gfp signal in NSM and ADF neurons under indicated conditions of (FIG. 8 A). FIG. 8C Representative image of tph-l::gfp expression in adult hermaphrodites treated with crh-1 or sbp-1 RNAi from LI stage. Arrows indicate neuronal cell bodies of the NSM and ADF respectively. Scale bars (applicable to images in the same row), 20um. Also shown is quantification of lph-l::gfp signal in NSM neuronsunder indicated conditions of FIG. 8C). Dots in FIGs. 8B-C represent signals of individual neurons. P values were calculated by unpaired, two-sided t-test with Welch correction. FIGs. 8D-F Food leaving rate changes of wild type, and two tph-1 allele tph-l(mg280) (FIG. 8E) and tph-1 (n4622) (FIG. 8F) treated with crh-1 or sbp-1 RNAi after 56 hours since LI stage. Failure of crh-1 or sbp-1 RNAi to increase dwelling in the two tph-1 allele tph-l(mg280) (FIG. 8F) and tph-1 (n.4622) (FIG. 8G). FIG. 8H Schematic pathway indicates food leaving and food dwelling resulted from metabolic signal is mediated by independent 5-HT neuro circuits. FIG. 81 Low intensity (0.25mW / cm2) Chrimson activation in the NSM neurons do not increase dwelling. Dots in (g and h) represent individual animals assayed. In box plots, the center line shows the median, whisker extend to minimum and maximum values. P values were calculated by unpaired, two-sided t-test with Welch correction.

[0064] FIGs. 9A-9M show that distinct serotonin circuits mediating food-leaving and dwelling behavior. FIGs. 9A-F Percentage of squares entered of (FIG. 9A) serotonin receptor null mutant and serotonin receptor mutant ser-1 (ok345); ser-7(tm!944) (FIG. 9B), and ser- 4(ok512) (FIG. 9C), and ser-5 (ok3087) (FIG. 9D), and mod-1 (ok!03) (FIG. 9E), Igc- 50(flv8) (FIG. 9F), following treating with control or sbp-1 RNAi, respectively. FIGs. 9G-L Food leaving rate changes of serotonin receptor ser-l(ok345) (FIG. 9G), and ser-7(okl944) (FIG. 9H), ser-4(ok512) (FIG. 91), ser-l(ok345); ser-7(tm!944) (FIG. 9J), and mod-1 (okl03) (FIG. 9K), and, lgc-50(flv8) (FIG. 9L), following treated with the control, crh-1 or sbp-1 RNAi from LI to L4 stage, respectively, m. Food avoidance rate of wild type or ser- 5(ok3087) mutant animals exposed to pathogen bacteria PAI 4. Dots in FIGs. 9A-F represent individual animals assayed, n>15 in each condition, at least twice independent experiment are done. In box plots, the center line shows the median, whisker extend to minimum and maximum values. FIGS. 9G-L Data represent Mean + SEM, each with 4-5 biological replicates at least. P-values were calculated by unpaired, two-sided t-test with Welch correction.

[0065] FIGs. 10A-10D show amino acid sequence of human GLP-1R (SEQ ID NO: 51) and C. elegans PDFR-1 (SEQ ID NO: 52). a, The amino acid sequence of human GLP-1R and C. elegans PDFR-1 are compared. The a-helix, P-sheets, and TMH domains of GLP-1R are indicated. FIG. 10B The amino acid sequence of human GLP-1R and C. elegans PDFR-1 comparison within the TM5 area, highlighted residues are conserved between human and C. elegans. FIG. 10C The 3D structure of full length of human GLP-1R (PDB:6LN2) and C.elegans PDFR-1 (Alpha fold prediction, PDB: Q09460). The similarity between GLP-1R and PDFR-1 structure is calculated by PyMOL, the root mean square deviation (RMSD) equals to 2.757 for this sequence of GLP-1R (SEQ ID NO: 53) and PDFR-1 (SEQ ID NO: 54). FIG. 10D Sequence alignments of full sequences of GCGR (SEQ ID NO: 49), GIPR (SEQ ID NO: 50), GLP-1R (SEQ ID NO: 51), and PDFR-1 (SEQ ID NO: 52).

[0066] FIGs. 11A-1 IF show metabolic regulation of food dwelling behaviors through SCT- like receptor signaling. FIG. 11A Food leaving rate changes of wild type, pdfr-l(ok3425) mutant and tph-l(mg280) mutant animals treated with control, crh-1 and sbp-1 RNAi, respectively, from LI to L4 stage. FIG. 1 IB Percentage of squares entered of wild ty pe, pdfr- l(ok3425) mutant and pdf-1 (tml966) mutant exposed to OP50 and PA14 bacteria. FIG. 11C Food leaving rate of animals against control, crh-1 RNAi or the toxin-mimic R53.4 RNAi in wild type and pdfr-1 mutant. FIG. 1 ID Food leaving rate of PDF overexpression compared to wild ty pe animal. FIG. HE Percentage of squares entered of wild type animals and the PDF- 1 overexpression animal. FIG. 1 IF Food leaving evoked by compound 2 (a GLP-1R agonist in humans) in wild ty pe animals and the ser-5 mutant. Dots in FIGs. 1 IB and E represent individual animals assayed. In box plots, the center line shows the median, whisker extend to minimum and maximum values. In FIG. 11 A, C, D, and F, data represent Mean + SEM from at least 3 independent experiments, each with 4-5 biological replicates at least. P-values were calculated by unpaired, two-sided t-test with Welch correction.

[0067] FIGS. 12A-12K show that specific fatty acids regulate food affinity responses. FIG. 12A Representative images of oleic acid supplementation abrogated food-leaving behavior induced by crh-1 and sbp-1 RNAi in wild-ty pe. FIG. 12B Food-leaving changes of wild type animals exposed to solvent control (0.01% DMSO) or, bortezomib (60mg / ml BTZ), or Tunicamycin with or without dietary’ oleic acid supplementation. FIG. 12C Food-leaving changes of animals treated with indicated RNAi, which impair the FAs respectively. FIG. 12D Food-leaving rate of the wild type animals, three PUFA biosynthesis mutant, fat-l(wa9), fat-3(wa22), fat-4(wal4) at 56-hour culture on the regular food lawn. FIG. 12E Food-leaving rate changes of the fat-3(wa22) mutant treated with crh-1 RNAi or sbp-1 RNAi with or w ithout dietary oleic acid supplementation. FIG. 12F Food-leaving rate changes of the fat- 1 (wa9) mutant treated w i th crh-1 RNAi or sbp-1 RNAi wi th or without dietary’ oleic acid supplementation respectively. FIG. 12G Food-leaving changes of wild ty pe animals treated with MUFA synthesis genes fat-5, fat-6 / 7, sbp-1 RNAi with or without dietary oleic acid orPalmitotate acid supplementation respectively. FIG. 12H Food-leaving changes of wild type animals treated with crh-1 RNAi with or without cis-vaccenic acid, the POA derivative. FIG. 121 Food-leaving changes of wild type animals treated with MUFA synthesis genes fat-5, fat- 6 / 7, sbp-1 RNAi with or without one of the PUFAs, linoleic acid supplementation. FIG. 12J Food-leaving changes of wild type animals treated with indicated ratio of SFAs relative to MUFAs. FIG. 12K Food-leaving changes of wild type animals treated with indicated MUFA synthesis genes exposed to extra saturated fatty acid (SFA) at 56h. In all food-leaving assay, data represents Mean + SEM from at least 2 independent experiments, each with 4-5 biological replicates at least. P-values were calculated by unpaired, two-sided t-test with Welch correction.

[0068] FIGs. 13A-13F show that food leaving behavior results in decreased feeding that affects fat accumulation. FIG. 13 A Representative images of C. elegans treated with crh-1 or sbp-1 RNAi on regular or full plates respectively stained by Oil Red O. scale bar: 100um. FIG. 13B Quantification of the Oil red O staining level of (FIG. 13 A). FIG. 13C Representative images of wild type C. elegans treated with crh-1 RNAi on regular or on full plates with or without dietary OA treatment respectively stained by Oil Red O. scale bar: 100pm. FIG. 13D The representative images of animals defects in ADF 5-HT synthesis and defects in NSM 5-HT synthesis treated with crh-1 or sbp-1 RNAi on the regular plates stained by Oil Red O. scale bar: 200um. e, Quantification of the Oil red O staining level of (FIG. 13C). FIG. 13F Quantification of the Oil red O staining level of (FIG. 13C). Fat content was quantified for each genotype by Image J, and expressed as mean ± SEM value (n=10-15). P-values were calculated by unpaired, two-sided t-test with Welch correction. FIGs. 14A-14P show that MUFAs signal regulate food affinity behavior rapidly. FIG. 14A The tph-l::gfp expression level in the NSMs neurons changes in response to ImM PA exposure. FIG. 14B Diagram of saturated palmitate accumulation induced toxicity stress response. FIG. 14C acl4 / 5 or pbo-1 RNAi do not suppress PA induced food leaving. FIG. 14D Diagram of mono-unsaturated fatty acid induced across-meal satiety pathway. FIG. 14E OEA does not suppress crh-1 or sbp-1 RNAi induced food leaving. FIG. 14F Independence of OA rescuing effect of crh-1 or sbp-1 RNAi induced food leaving on OEA synthesis. FIG. 14G OEA supplement does not suppress sbp-1 RNAi induced food dwelling. FIG. 14H Foodleaving changes of fat-6(tm331)-. fat-7(wa6) treated with control, OEA supplementation failed to suppress food-leaving or dwelling induced by sbp-1 or crh-1 RNAi, and interference withOEA production did not block the suppressive effects of OA. FIG. 141 Model of dietary oleic acid extend C. elegans lifespan by SKN-1A-ERAD homeostasis. FIG. 14J OA suppress crh-1 or sbp-1 RNAi induced food leaving independent on skn-la(mg570). FIG. 14K hrd-1 RNAi induced food leaving is suppressed by OA supplement. FIG. 14L OA partially rescues lipid droplet related genes knockdown induced food leaving. FIG. 14M OA suppresses neuro RNAi of crh-1 or sbp-1 induced food dwelling. FIG. 14N Failure of OA to fully suppress food leaving induced by disruption of LD-ER homeostasis62, including knockdown offltm-2, which affects the shape and composition of the ER membrane and is important for LD budding, and the lipid droplet biogenesis genes plin-1, (perilipin) dnc-1 and acs-4, as well as the critical phosphatidylcholine biosynthesis gene cepl-1 (FIG. 4H). FIG. 140 Failure of OA to fully suppress food leaving induced by knocking down the lipid phospholipid biosynthesis gene pisy-I. (FIG. 4H). FIG. 14P OA suppresses food-leaving induced by ERAD disruption. The ubiquitin ligase HRD-1 is the channel through which proteins are extruded from the ER by the ERAD mechanism15.

[0069] FIGs.l5A-15H show LC-MS / MS lipid analysis of metabolic deficit animals. FIG. 15A Schematic of the untargeted lipidomic analysis on whole worms treated with sbp-1 RNAi on the full lawn plates with or without dietary OA supplement using LC-MS / MS. FIG. 15B The total lipids abundance in day-1 adult worms treated with sbp-1 RNAi with or without OA supplementation on the full lawn plates. FIG. 15C The total TAG abundance, and the total FIG. 15D DAG abundance among all lipids in day-1 adult worms treated with sbp-1 RNAi with or without OA supplementation on the full lawn plates. FIG. 15E-G Lipidomic analysis of the ratio of C16:0 / C18: l in membrane lipids (phospholipids) of PS, PE, or PI. Each dot represents a biological replicate; n=5 independent biological replicates examined in one experiment. / ?- values were calculated by unpaired, two-sided t-test with Welch correction. FIG. 15H Principal component (PC) analysis of the lipidome indicates separation of sbp-1 RNAi from 619 control conditions (EV), with OA supplementation substantially shifting both conditions.FIGs. 16A-16P show that AMPK regulates food affinity behavior through lipid signal. FIG. 16A Percentage of squares entered in n= 44, 44 and 18 of wild type, CA-AAK-2 and tph- l(mg280) mutant, respectively, each dot represents a single worm. FIG. 16B multiple-worm tracking analysis during 90 mins of L4 stage wild-type, CA-AAK-2 and neuro-constitutive AMPK (N-CA-AAK-2). The average speed and curvature, along with the trajectory, werethen automatically truncated into IOS intervals and analyzed by the Worm Lab software. The clustering boundary (Line A FIG. ID) was defined for wild A pe worms in standard conditions and was then used for all genotypes and conditions. FIG. 16C Average pharyngeal pumping rate of a single day-1 wild-ty pe animal and CA-AAK-2. P value is derived from two-tailed unpaired t test, n.s, not significant. FIG. 16D Oleic acid suppresses whole body CA-AAK-2 induced food leaving rapidly. FIG. 16E constitutive CA-AAK-2 in individual tissue does not induce food leaving. FIG. 16F whole body CA-AAK-2 induced food dwelling on both OP50 and HT115. FIG. 16G whole body CA-AAK-2 induced food dwelling on OP50, DA837, and HB101. FIG. 16H whole body CA-AAK-2 and neuro-CA-AAK-2 do not increase dwelling on pathogen bacteria PA14. FIG. 161 The decreased food consumption of CA-AAK-1 is suppressed by CRTC-I mutation in the neuro. FIG. 16J crh-I or sbp-1 RNAi induced food leaving is independent on AMPK pathway. FIG. 1 K The increased food dwelling in whole body CA-AAK-2 is suppressed by knocking the AMPK signaling in the brain. FIG. 16L Relative mRNA expression of lipid related genes and beta-oxidation related genes. FIG. 16 M- N) show analysis of CA-AAK-2 effects on fat storage in animals grown on regular or full lawn plates. FIG. 16M shows representative Oil Red O-stained images of WT and CA-AAK-2 animals that were cultured on regular or full-lawn plates. FIG. 1 N shows quantification of Oil Red O staining levels in (FIG. 16M). In contrast to crh-1 or sbp-1 RNAi, whole-body CA-AAK-2 expression reduced TAG stores in animals cultured on a full lawn, and therefore independently of effects on food-leaving and its possible effects on food consumption. This suggests that CA-AAK-2 reduces fat stores metabolically. FIG. 16O-P OA supplementation failed to suppress the reduction in fat storage induced by whole-body CA-AAK-2 expression. FIG. 160 provides representative Oil Red O-stained images of WT and CA-AAK-2 animals with or without OA treatment. FIG. 16P shows quantification of Oil Red O staining levels in (FIG. 160). Data are mean ± SEM, P values are determined using two-way ANOVA with Bonferroni multiple comparisons test in (FIG. 16N and P). *P<0.05, **P<0.01, ***P<0.001, ****P<O.OOOI.

[0070] FIGs. 17A-17K show effects of IRE-I on feeding behaviors. FIG. 17A Dependence of PA-induced food leaving on ire-1. FIG. 17B Dependence of PA-induced dwelling on ire-1. FIG. 17C Neuronal-specific ire-1 knockdown blocks PA-induced dwelling, indicating cell- autonomous signaling. FIG. 17D Average fold change of xbp-l(xbp-lu), spliced xbp-l(xbp- 1s) mRNA levels in control (EV), crh-1, and sbp-1 RNAi. Total xbp-1 RNA levels areincreased but not the spliced / unspliced ratio. FIG. 17E Representative images show that crh-1 and sbp-1 RNAi activate hsp-4p::gfp, with suppression by OA supplementation. HSP-4 (BiP) is a marker of unfolded protein response activation through IRE-1. FIG. 17F Representative images show that PA activates hsp-4p::gfp, with suppression by ire-1 RNAi. FIG. 17G PA- induced tph-1 expression (proxy for serotonin synthesis) in the NSM neurons depends on ire- 1, as predicted. FIG. 17H quantification of FIG. 17G. FIG. 171 Constitutive neuronal expression of the spliced XBP-1 protein induces dwelling, with only mild suppression by OA. This is consistent with our model that ER signaling and XBP-1 function downstream of OA. Dots in FIGs. 17A and D each represent one experiment, in FIGs. 17B, C, and I represent each animal assayed, in FIG. 17H represent one single neuron in each animal. P- values were calculated by two-way ANOVA, multiple comparison followed with Benjamin correction. P value style, 0.0332(*), 0.0021 (*), 0.0002(*), <0.0001 (****). FIG. 17J shows robust OA suppression of hsp-4p::GFP activation induced by sbp-1 RNAi but not by knockdown of TAG / phospholipid homeostasis network genes. FIG. 17K shows a model for how metabolic perturbations in peripheral tissues (intestine, hypodermis) that alter the SFA / MUFA ratio act through the ER-based mechanisms we have identified and signaling to the nervous system to induce food-leaving.

[0071] FIGs. 18A-18L show metabolic regulation of food affinity behaviors through serotonin signaling. FIG. 18A shows dependence of crh-1 RNAi-induced food leaving on serotonin biosynthesis in the NSM neurons. The tph-1 mutation was rescued by tph-1 expression constructs that allowed tph-1 to be deleted specifically in the ADF or NSM neurons6. FIG. 18B shows dependence of crh-1 RNAi-induced dwelling on serotonin biosynthesis in the NSM neurons. The tph-1 mutation was rescued by tph-1 expression constructs that allowed tph-1 to be deleted specifically in the ADF or NSM neurons6. Deletion from ADF had no effect, in contrast to NSM deletion. n>18 animals in each of at least two independent experiments. FIG. 18C Dependence of AMPK-induced dwelling on tph-1. FIG. 18D Dependence of neuronal-specific AMPK-induced dwelling on tph-1. FIG. 18E Representative images of tph-lp::GFP in the NSM neurons after crh-1, sbp-1 or dgat-2 RNAi with or without OA supplementation. FIG. 18F Quantification of / p / i-7::GFP levels in the NSM neurons in (FIG. 18E). FIG. 18G IRE- 1 -dependence of tph-l::GFP activation by PA. Representative images of tph-1: :GFP after PA supplementation with or without ire-1 RNAi. FIG. 18H Quantification of the lph-l::GFP levels in (FIG. 18G). FIG. 181Representative images and the quantification of tph-lp::GFP in the NSM neurons with or without N-CA-AAK-2. Scale bar: 50pm. FIG. 18 J Failure oi sbp-1 RNAi to increase dwelling in the ser-4(flv7); mod-l(ok!03) double mutant. FIG. 18K Failure of crh-1 or sbp-1 RNAi to increase food leaving in ser-5 (ok3087) mutants. FIG. 18L Model for how disruption of ER membrane lipid homeostasis in the brain induces dwelling through serotonin signaling from the NSM neurons. Data are mean ± SEM. P values are determined by Tw o- way ANOVA with Bonferroni multiple comparisons test in (FIGs. 18 B, G, I) and two-way repeated-measures ANOVA to assess main effects of treatment in (FIG. 18K), Two-way ANOVA with Holm-Sidak's multiple comparisons test in (FIGs. 18 A, J), one-way ANOVA in (FIG. 18C). *P<0.05, **P<0.01, ***P<0.00I, ****P<0.0001.

[0072] FIGs. 19A-19K show Regulation of food-affinity behaviors through serotonin signaling.FIG. 19A Pathogen induction of tph-1 expression in the ADF neurons. Representative image of tph-lp::GFP expression in adult hermaphrodites exposed to OP50 or P. aeruginosa PA14 / OP50 mix for 4-6h. Arrows indicate the NSM and ADF neuronal cell bodies. FIG. 19B Quantification of tph-lp::GFP signal in the NSM and ADF neurons under the conditions shown in (FIG. 19A). FIGs. 19C-D Failure of crh-1 or sbp-1 RNAi to increase food-leaving (FIG. 19C), and dwelling (FIG. 19D) in tph-l(n4622) mutants. FIG. 19E western blots show the tph-lp::GFP level increased in against to crh-1 and sbp-1 RNAi. FIG. 19F Quantification of the relative GFP level in (FIG. 19E). FIG. 19G Representative image of tph-1 ::GFP expression in day-one adult hermaphrodites treated with crh-1 or sbp-1 RNAi since the LI stage. FIG. 19H Quantification of the tph-l::GFP signal in the NSM neurons under the conditions in (FIG. 19E). FIG. 191 Representative images of tph-1: :GFP expression in adult hermaphrodites treated with PA or OA. FIG. 19J Quantification of the GFP levels in (FIG. 191). FIG. 19K Model for ER membrane lipid metabolism perturbation in peripheral tissues (gut, hypodermis) signaling to the brain to induce food leaving through NSM activation, and the SER-5 receptor. Data represent Mean ± SEM, P values are determined by using one-way ANOVA with Dunnett’s multiple comparisons test in (FIGs. 19 F and H), and two-way repeated-measures ANOVA with Holm-Sidak's multiple comparisons test in (FIG. 19D), two-way repeated-measures ANOVA with Bonferroni's multiple comparisons test in (FIGs. 19 B and C),and two-tailed Mann-Whitney test in (FIG. 19J), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0073] FIGs. 20A-R show regulation of food-affinity behaviors through GLP-l-like signaling. FIG. 20A Maximum likelihood phylogenetic tree comparison of PDFR-1 (ce) with human (hit) SCT-like GPCRs. Stars highlight PDFR-1, GLP-1R, and other receptors that have been targeted along with GLP-1R for obesity treatment using dual receptor agonists28. The amino acid sequences of these receptors were aligned using MUSCLE, and a tree was constructed using mega software version 1129. FIG. 20B Structural alignment of PDFR-1 (by AlphaFold 3) with the inactive human GLP-1R (PDB:6LN2). The root mean square deviation (RMSD) between the two structures is 1.104 angstroms. FIG. 20C Dependence of PA-induced food leaving on pdfr-1. FIG. 20D Dependence of sbp-1 RNAi induced food leaving on pdfr-1. FIG. 20E Representative image of PDF-1 ::GFP expression induced by crh- 1 or sbp-1 RNAi. FIG. 20F Quantification of PDF-1 ::GFP expression in (FIG. 20E). FIG. 20G Representative image of IRE- 1 -dependence PDF-1 ::GFP expression induced by sbp-1 RNAi. FIG. 20H Quantification of PDF-1 ::GFP expression in (FIG. 20G). FIG. 20I-J Dependence of sbp-1 RNAi-induced PDF-1 ::GFP expression on tph-1. FIG. 20K Dependence of crh-1 RNAi-induced food dwelling on pdfr-1 and tph-1. FIG. 20L Dependence of N-CA- AAK-2 suppressed PDF-1 ::GFP on tph-1. FIG. 20M PDF-1 overexpression decreases dwelling. FIG. 20N PDF-1 overexpression increases food leaving. FIG. 200 GLP-1RA administration reduces dwelling that is pdfr-1 -dependent. FIG. 20P GLP-1RA administration reduces food leaving that is / % / / / '- / -dependent. FIG. 20Q GLP-1RA administration reduces food consumption dependent upon PDF-1 and PDFR-1. FIG. 20R Model for how disruption of ER membrane lipid homeostasis in the brain induces dwelling by activating serotonin signaling from the NSM neurons that overcomes PDF-l / PDFR-1 signaling. Data are mean ± SEM. P values are determined by Two-way ANOVA with Bonferroni multiple comparisons test in (FIG. 20 D, P), Two-way ANOVA with Holm-Sidak's multiple comparisons test in (FIG. 20 C, K, O), Two-way ANOVA with Tukey's multiple comparisons test in (FIG. 20 F, H, Q), two-tailed Mann- Whitney t-test in (J, M). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0074] FIGs. 21A-21L show how GLP-l-like signaling regulates food affinity behaviors. FIG. 21A Structural alignments (AlphaFold 3) of PDFR-1 with the human gastric inhibitory polypeptide receptor (GIPR) (PDB:8WA3), calcitonin receptor (CALCR) (PDB: 6E3Y), and glucagon-like polypeptide-2 receptor (GLP-2R) (7D68), with the RMSD value for each indicated in angstroms. FIG. 21B Sequence alignment of the N-terminal domain of humanGLP-1R (SEQ ID NO: 51) and C. elegans PDFR-1 (SEQ ID NO: 52). The conserved amino acid motifs are boxed. The seven transmembrane domains are annotated. Accession numbers of GLP-1R HUMAN (P43220), PDFR-1 CE (Q09460-1). The sequence alignment was performed by using Clustal Omega Multiple Sequence Alignment (MSA) with default parameters. FIG. 21C Food-leaving induced by the knockdown of the ATP synthase membrane subunit R53.4, which mimics a toxin attack6, occurs independently of PDFR-1. FIG. 21 D PDFR-1 is not required for food-leaving induced by exposure to Pseudomonas aeruginosa (PA14), consistent with previously reported results44. FIG. 21E-F Dependence of PA induced PDF-1: :GFP expression on IRE-1. FIG. 21G-H Representative western blots shown TPH- 1 -dependence of PDF-1: :GFP level by sbp-1 RNAi. FIG. 211 Pathogen (PAI 4) exposure suppresses the increased dwelling that is characteristic of pdf-1 and pdfr-1 mutants. FIG. 21 J GLP-1RA reduces dwelling dependent upon PDF-1. FIG. 21K GLP-1RA increases food leaving dependent upon PDF-1. FIG. 21L Model for how disruption of ER membrane lipid homeostasis in the periphery activates PDF-1 / PDFR-1 signaling, thereby inducing food leaving. Data are mean ± SEM. P values are determined by using Two-way ANOVA with Bonferroni multiple comparisons test in (FIGs. 21 C, D, K) and with Holm-Sidak's multiple comparisons test in (FIGs. 21 I, J), using Two-way ANOVA Tukey's multiple comparison test in (FIGs. 21 F, H), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.DESCRIPTION OF THE SEQUENCES

[0075] Table 1 provides a listing of certain sequences referenced herein.DESCRIPTION OF THE EMBODIMENTSI. Methods of increasing or decreasing food intake

[0076] Described herein are methods of increasing or decreasing food intake. These methods of altering food intake can be used treat subjects with lowered food intake (such as subjectswith anorexia nervosa and / or avoidant restrictive food intake disorder (ARFID) or treat subjects with increased food intake (such as subjects with obesity, prediabetes, type 2 diabetes, and / or a metabolic syndrome).

[0077] In some embodiments, these methods involve modulating activity’ of Inositol - Requiring Enzy me 1 (IRE-1) and / or AMP- Activated Protein Kinase (AMPK). In some embodiments, these methods involve modulating activity of Lipinl, adipose triglyceride lipase (ATGL1), diglyceride acyltransferase 1 (DGAT1), perilipin-1 (PLIN1), fat storageinducing transmembrane protein 2 (FITM2), or one or more protein involved in endoplasmic reticulum (ER)-associated degradation (ERAD).

[0078] As used herein. AMPK refers to AMP-activated kinase. AMPK is also known as Protein Kinase, AMP-Activated: 5’ AMP-activated protein kinase; AMP-Activated Protein Kinase; 5'-AMP-Activated Protein Kinase: EC 2.7.1 1 : AMPKal ; Hydroxymethylglutaryl- CoA Reductase Kinase; Acetyl-CoA Carboxylase Kinase; Tau-Protein Kinase PRKAA1; ACACA Kinase; or HMGCR Kinase. AMPK is a heterotrimeric protein complex that is formed by a, [3, and y subunits, wherein each of these three subunits takes on a specific role in both the stability and activity' of AMPK. AMPK is comprised of the a subunits PRKAA1 (UniProt ID: QI 3131) and PRKAA2 (UniProt ID: P54646); |3 subunits PRKAB1 UmProt ID: Q9Y478) and PRKAB2 (UniProt ID: 043741); and y subunits PRKAG1 (UniProt ID: P54619), PRKAG2 (UniProt ID: Q9UGJ0), and PRKAG3 (UniProt ID: Q9UGI9).

[0079] As used herein. IRE-1 refers to Inositol-Requiring Enzyme 1 (UniProt ID: 075460). IRE-1 is also known as ERN1; Endoplasmic Reticulum To Nucleus Signaling 1; Serine / Threonine-Protein Kinase / Endoribonuclease IRE1; IRE1P; Inositol-Requiring Protein; Inositol-Requiring Enzyme; ER To Nucleus Signaling; Irel-Alpha; HIRElp; IREla 3; Endoplasmic Reticulum-To-Nucleus Signaling; and EC 2.7.11. IRE-1 is a serine / threonine kinase acting as one of three branches of the Unfolded Protein Response (UPR) signaling pathway, which is activated upon endoplasmic reticulum (ER) stress conditions. Activation of IRE- 1 occurs when the ER chaperone BiP is released from its luminal domain when a build-up of unfolded proteins arises in the ER. Once IRE-1 is activated, its endoribonuclease domain initiates splicing of the mRNA encoding the transcription factor XBP-1 in mammals. Methods of assessing IRE-1 activity' are known in the literature’8-59. In some embodiments, activity7of IRE-1 is measured by (a) regulated IRE 1 -dependent decay (RIDD) of one or more mRNAs; (b) total xbp-1 RNA levels and / or splicing of xbp-1 mRNA; (c) activation of p38and SKN-l(Nrf2); (d) IRE-1 autophosphorylation; and / or (e) the c-Jun N-terminal kinases (JNK) signaling cascade.

[0080] As used herein, Lipinl (UniProt ID: Q14693) also refers to Lipin; Phosphatidate Phosphatase LPIN; KIAA0188; EC 3.1.3.18; EC 3. 1.3.4; EC 5.4.2.7; Lipin-1; or PAPL Lipin-1 is a central regulator of lipid homeostasis, acting either as an enzyme or as a coregulator of transcription.

[0081] As used herein, ATGL1 (UniProt ID: Q96AD5) refers to Adipose Triglyceride Lipase, also known as Patatin Like Phospholipase Domain Containing; Desnutrin; ATGL; IPLA2zeta; TTS; FP17548; Patatin-Like Phospholipase Domain-Containing Protein; Pigment Epithelium-Derived Factor Receptor; Calcium-Independent Phospholipase A2-Zeta; IPLA2- Zeta; EC 3. 1.1.3; PEDF-R; TTS2.2; TTS2; Mutant Patatin-Like Phospholipase Domain Containing; Patatin-Like Phospholipase Domain Containing; Calcium-Independent Phospholipase A2; Pigment Epithelium-Derived Factor; Transport-Secretion Protein 2.2; Transport-Secretion Protein 2 4; Triglyceride Hydrolase; 1110001C14Rik; or EC 3. 1.1.4. ATGL1 is a lipolytic enzyme.

[0082] As used herein, DGAT1 (UniProt ID: 075907) refers to Diacylglycerol O- Acyltransferase, also known as ARGP1; DGAT; Acyl-CoA Retinol O-Fatty -Acyltransferase; Diglyceride Acyltransferase; EC 2.3.1.20; ARAT; Acyl Coenzy me A:Cholesterol Acyltransferase Related Gene; Diacylglycerol O-Acyltransferase (Mouse) Homolog; AcyL CoA:Diacylglycerol Acyltransferase; Retinol O-Fatty-Acyltransferase; ACAT Related Gene Product; ACAT-Related Gene Product; EC 2.3.1.76; EC 2.3.1; DIAR7; or AGRP1. DGAT1 catalyzes the conversion of diacylglycerol and fatty7acyl CoA to triacylglycerol.

[0083] As used herein. PLIN1 (UniProt ID: 060240) refers to Perilipin, also known as PLIN; Lipid Droplet-Associated Protein; Perilipin-1; PERI; and FPLD4. PLIN1 is a modulator of adipocyte lipid metabolism that coats lipid storage droplets to protect them from breakdown.

[0084] As used herein, FITM2 (UniProt ID: Q8N6M3) refers to Fat Storage Inducing Transmembrane Protein 2, also known as FIT2; DJ881L22.2; C20orfl42; Acyl-Coenzyme A Diphosphatase FITM2; Fat-Inducing Protein 2; Chromosome 20 Open Reading Frame 142; Fat Inducing Transcript 2; EC 3.6.1; and SIDDIS. FITM2 is a fatty acyl-coenzyme A (CoA) diphosphatase that preferentially hydrolyzes unsaturated long-chain acyl-CoA substrates in the ER lumen.

[0085] The ER is a membrane-bound organelle that is responsible for folding and synthesis of secretory and organelle-bound proteins. These processes are sensitive to perturbation, such as a build-up of unfolded proteins. Cells have a response mechanism to prevent further accumulation of unfolded proteins, know n as the unfolded protein response (UPR). As used herein, a protein involved in ER-associated degradation (ERAD) refers to any protein involved in the removal of misfolded or slowly folding proteins from the ER. ERAD requires three steps, substrate transportation from the ER to the cytoplasm (dislocation), ubiquitination by specific ubiquitin ligases, and proteolysis by proteasome in cytoplasm, and a protein involved in ERAD can be a protein involved in any of these processes. In some embodiments, the one or more proteins involved in ERAD comprise one or more selected from osteosarcoma amplified 9, endoplasmic reticulum lectin (OS9), endoplasmic reticulum lectin 1 (ERLEC 1), E3 ubiquitin ligase HRD1 , and / or SEL1L adaptor subunit Of ERAD E3 ubiquitin ligase (SEL1L).A. Decreasing food intake

[0086] In some embodiments, a method of decreasing food intake in a subject in need of treatment, comprises (a) decreasing activity of IRE- 1 and / or AMPK or a signaling molecule downstream thereof; and / or (b) increasing activity of one or more of adipose triglyceride lipase (ATGL1), diglyceride acyltransferase 1 (DGAT1), perilipin-1 (PLIN1), fat storageinducing transmembrane protein 2 (FITM2), or one or more protein involved in endoplasmic reticulum (ER)-associated degradation (ERAD). In some embodiments, the signaling molecule downstream of IRE1 and / or AMPK is an RNA acted on by IRE-1, p38, SKN- l(Nrf2), and / or a c-Jun N-terminal kinase (JNK). In some embodiments, the RNA acted on by IRE-1 is xbp-1 or splicing of xbp-1 RNA.

[0087] In some embodiments, a subject in need of treatment to decrease food intake or in need of decreasing food intake is any individual who has a high body mass index (BMI). In some embodiments, the subject is overweight. In some embodiments, an overweight subject has a BMI of 25-29.9. In some embodiments, the subject is obese. In some embodiments, an obese subject has a BMI of 30 or greater. In some embodiments, a subject in need of decreased food intake has been diagnosed with prediabetes, type 2 diabetes of a metabolic syndrome. As used herein, prediabetes refers to a subject having hemoglobin A1C (HbAlc) is between 5.7% -6.4%. As used herein, type 2 diabetes refers to HbAlc of 6.5% or more. The risk of diabetes and prediabetes is increased with higher BMI, and reductions of 5% ormore in body mass of overweight individuals have been associated with a reduced risk of diabetes.

[0088] As used herein, metabolic syndrome refers to a group of conditions that together raise the risk of coronary heart disease, diabetes, stroke, and other serious health problems. Metabolic syndrome may also be referred to as insulin resistance syndrome. Generally, metabolic syndrome refers to a subject with abdominal obesity or BMI over 30 and one or more of prediabetes or diabetes, high normal blood pressure hypertension, and elevated non- HDL cholesterol levels.

[0089] In some embodiments, decreased activity7of IRE- 1 is measured by decreased RIDD of one or more mRNAs; levels of an RNA acted on by IRE-1; activation of p38 and SKN- l(Nrf2); IRE-1 autophosphorylation; and / or the c-Jun N-terminal kinases (JNK) signaling cascade. In some embodiments, the RNA acted on by IRE-1 is xbp-1 RNA levels and / or splicing of xbp-1 mRNA.

[0090] In some embodiments, the one or more proteins involved in ERAD comprise one or more selected from osteosarcoma amplified 9. endoplasmic reticulum lectin (OS9), endoplasmic reticulum lectin 1 (ERLEC 1), E3 ubiquitin ligase HRD1, and / or SEL1L adaptor subunit Of ERAD E3 ubiquitin ligase (SEL1L).

[0091] In some embodiments, design of agents that are more preferentially targeted to the brain minimizes the side effect profile driven by peripheral effects of agents. In some embodiments, GLP-1 agonists that are designed to act in the brain to promote UFA incorporation into phospholipid biosynthesis or lipid droplet formation, or decreasing activity of IRE-1 and / or AMPK, will metabolically decrease food intake by acting through this system, without inducing these side effects. In some embodiments, inhibitors of IRE-1 and / or AMPK designed to act in the brain work decrease food intake.

[0092] As described herein, decreases in food intake may be preferentially mediated by central effects. In some embodiments, a method of decreasing food intake in a subject in need of treatment, comprises administering an agent modified to cross the blood brain barrier (BBB). wherein the agent is an inhibitor of IRE- 1 and / or AMPK or a signaling molecule downstream thereof; and / or an activator of Lipinl, ATGL1, DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD. In some embodiments, the activator or inhibitor is modified by a modification to increase lipid solubility7, decrease molecular weight, and / or decrease polarity. In some embodiments, the modification is capable of increasing the abilityto cross the BBB by 10% or more, 25% or more, or 50% or more. In some embodiments, the agent is not modified to cross the BBB. In some embodiments, the agent is not modified to cross the BBB.B. Increasing food intake

[0093] In some embodiments, a method of increasing food intake in a subject in need of treatment, comprises (a) increasing activity of Inositol-Requiring Enzyme 1 (IRE-1) and / or AMP- Activated Protein Kinase (AMPK) or a signaling molecule downstream thereof; and / or (b) decreasing activity of one or more of Lipinl, adipose triglyceride lipase (ATGL1), diglyceride acyltransferase 1 (DGAT1), perilipin-1 (PLIN1), fat storage-inducing transmembrane protein 2 (FITM2), or one or more protein involved in endoplasmic reticulum (ER)-associated degradation (ERAD). In some embodiments, the signaling molecule downstream of IRE1 and / or AMPK is an RNA acted on by IRE-1, p38, SKN-l (Nrf2), and / or a c-Jun N-terminal kinase (JNK). In some embodiments, the RNA acted on by IRE-1 is xbp-1 or splicing of xbp-1 RNA.

[0094] In some embodiments, the administering decreases signaling through the endogenous glucagon-like peptide-1 receptor (GLP-1R) or another receptor in FIG. 2G and / or FIG. 20A in the brain of the subject. In some embodiments, reducing endogenous GLP-1R signaling leads to increased appetite in the subject. In some embodiments, reducing endogenous GLP- 1R signaling leads to increased food intake in the subject.

[0095] In some embodiments, a subject in need of treatment to increase food intake or in need of increasing food intake is any individual who has a low body mass index (BMI). In some embodiments, the subject is underweight. In some embodiments, the subject has a BMI of 18.5 or lower, 15 or lower, or 13 or lower. In some embodiments, a subject in need of increased food intake has been diagnosed with anorexia nervosa and / or avoidant restrictive food intake disorder (ARFID). As used herein, anorexia or anorexia nervosa refers to an eating disorder characterized by relentless drive for thinness with a fear of gaining body weight associated with self-induced behaviors tow ards thinness. As used herein, avoidant restrictive food intake disorder or ARFID refers to an eating disorder wherein a subject limits the amount or type of food eaten, without fear or concern about body shape or size. In both anorexia and ARFID, a subject may not consume enough calories to support basic body function.

[0096] In some embodiments, reduced activity of IRE- 1 is measured by (a) increased RIDD of one or more mRNAs; increased levels of an RNA acted on by IRE-1; increased activation of p38 and SKN-l(Nrf2); increased IRE-1 autophosphorylation; and / or increased c-Jun N- terminal kinases (JNK) signaling. In some embodiments, the RNA acted on by IRE-1 is total xbp-1 RNA levels and / or splicing of xbp-1 mRNA.

[0097] In some embodiments, the one or more proteins involved in ERAD comprise one or more selected from osteosarcoma amplified 9, endoplasmic reticulum lectin (OS9), endoplasmic reticulum lectin 1 (ERLEC1), E3 ubiquitin ligase HRD1, and / or SEL1L adaptor subunit Of ERAD E3 ubiquitin ligase (SEL1L).

[0098] In some embodiments, the agent to increase food intake is modified to cross the blood brain barrier (BBB). In some embodiments, the activator or inhibitor is modified by a modification to increase lipid solubility, decrease molecular weight, and / or decrease polarity. In some embodiments, the modification is capable of increasing the ability to cross the BBB by 10% or more, 25% or more, or 50% or more.II. Peripherally active agents for reducing side effects

[0099] Current GLP-1R agonists induce side effects (nausea, diarrhea, and aversion to food) that the present results indicate result from action in peripheral tissues (gut and liver counterparts), and not due to activity in the brain. The present disclosure, for example, identifies that central effects of PDF-l / PDFR-1 are followed by subsequent signaling from the periphery to the brain.

[0100] As described herein, the appetite suppressant activity of GLP-1 agonists can be separated from their side effects by designing agents that act primarily within the brain. In some embodiments, blocking signaling to the brain from peripheral tissues after GLP-l / GLP- IR activation reduces side effects of nausea or food aversion.

[0101] In some embodiments, a method of decreasing food aversion and / or nausea in a subject taking a glucagon-like peptide-1 (GLP-l)-based therapy comprises administering an agent modified to not cross the BBB, wherein the agent is (a) an inhibitor of IRE- 1 or AMPK, and / or (b) an activator of Lipin 1, ATGL1, DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD. In some embodiments, the activator or inhibitor is modified by a modification to reduce lipid solubility, increase molecular weight, and / or increase polarity. In some embodiments, the modification is capable of reducing the ability7to cross the BBB by 90% or more, 95% or more, or 99% or more. In some embodiments, administering theinhibitor or activator modified to not cross the BBB decreases side effects of GLP-1 -based therapy so that the subject can take a higher dose of a GLP-l-based therapy or can avoid discontinuation of a GLP-l-based therapy due to side effects.

[0102] In some embodiments, the GLP-l-based therapy comprises a GLP-1 receptor agonist, dual-acting GLP-1 receptor agonist and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist, amylin agonist, and / or a dual-acting GLP-1 receptor and glucagon receptor (GCGR) agonist.

[0103] In some embodiments, the GLP-1 receptor agonist is exenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, and / or semaglutide. In some embodiments, the dual agonist for GLP-1 receptor and GCGR is BI 456906. In some embodiments, the amylin agonist is pramlintide and / or cagrilintide. In some embodiments, the dual-acting GLP-1 receptor agonist and GIP receptor agonist is tirzepatide.III. Methods of treatment by increasing levels of monounsaturated fatty acids

[0104] Also described herein are methods to decrease food intake in a subject in need of treatment by altering levels of certain fatty acids in relation to other fatty acids. In some embodiments, a method of decreasing food intake in a subject in need of treatment comprises increasing the levels of monounsaturated fatty acids (MUFAs) relative to saturated fatty acids (SFAs). In some embodiments, the increasing occurs in the brain. In some embodiments, the MUFAs comprise oleic acid and / or palmitoleic acid.

[0105] In some embodiments, the levels of MUFAs are increased relative to SFAs by treatment with one or more agent to increase activity of stearoyl-CoA desaturase (SCD) and / or elongation of very long chain fatty acids protein 6 (ELOVL6).

[0106] As used herein, SCD (UniProt ID: 000767) refers to Stearoyl-CoA Desaturase, also known as FADS5; SCD1; Fath’ Acid Desaturase; Acyl-CoA Desaturase; SCDOS; Stearoyl-CoA Desaturase (Delta-9-Desaturase); Stearoyl-CoA Desaturase Opposite Strand; Delta(9)-Desaturase; EC 1.14.19.1; HSCD1; Predicted Protein Of HQ0998; Delta-9- Desaturase; Delta-9 Desaturase; and MSTP008. SCD is an enzy me involved in fatty acid biosynthesis, primarily the synthesis of oleic acid, which belongs to the fatty acid desaturase family and is an integral membrane protein located in the endoplasmic reticulum.

[0107] As used herein, ELOVL6 (UniProt ID: Q9H5J4) refers to long chain fatty7acids protein 6, also known as LCE; ELOVL Family Member 6, Elongation Of Long Chain Fatty Acids (FEN1 / Elo2, SUR4 / Elo3-Like, Yeast); Elongation Of Very Long Chain FattyAcids Protein; Very Long Chain 3-Ketoacyl-CoA Synthase; Very Long Chain 3-Oxoacyl- CoA Synthase; 3-Keto AcyLCoA Synthase ELOVL6; Long-Chain Fatty-Acyl Elongase; Fatty Acyl-CoA Elongase; Fatty Acid Elongase; ELOVL FA Elongase; FLJ23378; MGC5487 2 5; HELO2; FACE; EC 2.3.1.199; and FAE. ELOVL6 catalyzes the first and rate-limiting reaction of the four reactions that constitute the long-chain fatty acids elongation cycle, and this endoplasmic reticulum-bound enzymatic process allows the addition of 2 carbons to the chain of long- and very long-chain fatty acids (VLCFAs) per cycle.

[0108] In some embodiments, increasing the levels of MUFAs relative to SFAs decreases activity7of IRE-1 and / or AMPK or a signaling molecule dow nstream thereof and / or increases activity of one or more of Lipinl, ATGL1, DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD. In some embodiments, increasing the levels of SFAs increases activity7of IRE-1 and / or AMPK or signaling molecule downstream thereof and / or decreases increases activity’ of one or more of Lipinl, ATGL1, DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD.IV. Screening assays to identify drug targets and agents modulating food intake

[0109] Described herein are methods to analyze and screen novel compounds and genes directly for effects on feeding behavior. In some embodiments, screening assays identify a drug target and / or evaluate an agent as a treatment for one or more disorder. In some embodiments, feeding behavior in C. elegans is regulated by the GLP-lR-like pathway. The present description of an assay for food-leaving, for instance in C. Elegans (See e.g. Example 1 and Example 8), provides a powerful strategy to identify mechanisms that act on this pathway. Currently, understanding of how GLP-1R signaling suppresses appetite is essentially a black box; however, mutational or RNAi-based genetic screening that alters the efficiency of FA-induced food aversion, as described herein, can provide an entry point into answering this question.

[0110] In some embodiments, a "drug target” refers to a biological molecule that may be used to develop a treatment. Such biological molecules that may be a drug target include proteins, mRNAs, and DNA. In some embodiments, the drug target is a protein, such as an enzyme or transcription factor. In some embodiments, a screen in C. elegans identifies biological molecules that were previously unknown to impact food intake. For example, in some embodiments, an RNAi-based genetic knockdown screen can be used to identify genesassociated with an increase or decrease in food intake (see e.g., Example 8 - Food intake screen).

[0111] In some embodiments, once a drug target is identified, further screening can then identify agents that bind to said drug target. As described herein, such a screening assay identified IRE-1 and other proteins as previously unknown drug targets that can affect food intake. In some embodiments, a screening assay with C. elegans allows for an unbiased screen to identify- biological molecules that impact feeding. Such screening may make use of genetic mutants of C. elegans and / or knockdow ns of various rnRNA or proteins.

[0112] In some embodiments, a screening assay in C. elegans identifies mutations / genes that act upstream, downstream, or in parallel to GLP-1R signaling. In some embodiments, a screening assay in C. elegans identifies new targets downstream of or parallel to GLP-1R that are more amenable to small molecule design than GLP-1 R itself. For example, some targets identified by screening assays may allow for development of orally active compounds that affect food intake.

[0113] In some embodiments, targets identified using screening assays described herein can facilitate activation of the pathway or its suppression in the setting of eating disorders, including identification of gut- or brain-specific strategies. In some embodiments, mechanisms identified with the present screening assay can then examined at smaller scale (hundreds of genes) for effects on eating behavior.

[0114] In some embodiments, a screening assay identifies genes that affect activation or inhibition of the peptide (PDF-1) that stimulates C. elegans GLP-1R (PDFR-1). In some embodiments, such an assay allows for a very simple screen to identify new drug targets in these pathways as well as strategies for altering the activity of this system in the brain or periphery. In some embodiments, screening assays described herein can identify new therapeutic targets upstream and downstream of the receptor / ligand interaction of PDFR- 1 / PDF-l. In some embodiments, these targets may be more amenable to pharmacological regulation and may reduce side-effect profile compared to presently known GLP-1 focused agents.

[0115] In particular, an assay to identify a drug target may in some embodiments comprise a method of reducing expression of one or more genes in C. elegans by a method such as RNAi, and assessing the impact of the reduced expression on mobility and / or feeding of C. elegans, for example using a food intake, dwelling, and / or leaving assay as describedherein. A gene or its respective mRNA or protein may be identified as a drug target in such an assay if reducing expression of the gene results in an increase or decrease in mobility and / or feeding of the C. elegans in the assay compared to C. elegans in which the expression is not reduced. In some embodiments, the gene to be assayed is believed to be in the pathway of PDFR-1 and / or PDF- 1.

[0116] In some embodiments, a method of identifying a drug target for one or more disorder comprises evaluating pigment dispersing factor (PDF-1) and / or pigment dispersing factor receptor (PDFR-1) activity in C. elegans. For example, a screen to identify new drug targets may comprise reducing the expression (e.g., by RNAi based methods as described herein) of one or more candidate genes, for example genes encoding enzymes or transcription factors. The effect of reduced expression of one or more candidate genes on the activity of PDF-1 and / or PDFR-1 can be assessed in some embodiments by measuring whether there is a quantitative increase or decrease in gene and / or protein expression of PDF-1 and / or PDFR-1 or of PDF-1 and / or PDFR-1 activity in C. elegans compared to an empty vector (EV) control. Thus, in some embodiments, a screen for a drug target comprises reducing the expression of a candidate drug target gene in C. elegans, e.g., by RNAi, and determining the gene and / or protein expression and / or the activity of PDF-1 and / or PDFR-1. If the reduced expression of the candidate drug target gene increases or decreases the expression and / or activity of PDF-1 and / or PDFR-1, the candidate is considered a drug target in the assay.

[0117] In other embodiments, screens may be performed to identify agents that affect the activity of drug targets that have already been identified by methods described herein. For example, experiments herein have identified the crh 1 and spb 1 genes as example drug targets in that they lead to reduced food intake in C. elegans when their expression is reduced. In other embodiments, assays in C. elegans may be used to identify potential agents that affect the expression and / or activity of PDF-1 and / or PDFR-1. In some embodiments, a method for evaluating an agent as a treatment for one or more disorder comprises evaluating PDF-1 and / or PDFR-1 activity in C. elegans in the presence or absence of said agent. For example, in some embodiments, candidate agents (e.g., small molecules and / or biologies such as proteins) can be administered to C. elegans and the subsequent effects on gene and / or protein expression of PDF-1 and / or PDFR-1 can be measured in comparison to no agent and / or a carrier control.

[0118] In some embodiments, the increase or decrease in PDF-1 and / or PDFR-1 gene and / or protein expression can be measured using methods known in the art for measuring gene and / or protein expression, e.g., quantitative PCR, Western blotting, or the like. In some embodiments, the activity of PDF-1 and / or PDFR-1 is measured using promoter activity of PDF-1 using a fluorescent reporter assay (e.g., PDF-1::GFP) as described herein (see e.g., Example 7).

[0119] In other embodiments, an agent is evaluated in an assay that utilizes the behavior of C. elegans. In some embodiments, for example, mobility and / or feeding using for instance the food intake, dwelling, and leaving assays as described herein (see e.g., Example 8 for description of methods for measuring C. elegans mobility and / or feeding) is measured in the presence of a candidate agent, and compared to the behavior in the absence of such a candidate agent. A candidate agent whose presence alters the mobility and / or feeding behavior of C. elegans compared to its absence is identified as a drug agent in such an assay herein.

[0120] In some embodiments, reduced expression of a candidate drug target or presence of a candidate agent may increase or decrease food intake in C. elegans, which can be measured as food intake per animal as described herein over the time course of a food intake assay (see e.g., Example 8 - Food intake screen). In some embodiments, reduced expression of a candidate drug target or presence of a candidate agent may increase or decrease mobility of C. elegans. One measure of mobility is dwelling of C. elegans on a bacterial food lawn. An example assay for measuring dwelling is described herein in Example 8. In this exemplary assay, the movement of C. elegans in a food lawn is tracked visually and quantified by overlaying a grid of 3 mm squares on top of the culture plate and measuring the number of squares entered by the animal during the course of the assay.Increased dwelling is defined as reduced percent squares entered, indicating that the animal is less mobile on the food lawn, while decreased dw elling is defined as increased percent squares entered by the animal in the assay, indicating greater mobility (see e.g., Example 8 - Food dwelling assay). In some embodiments, reduced expression of a candidate drug target or presence of a candidate agent may increase or decrease food leaving of C. elegans. An exemplary assay for assessing food leaving in C. elegans is described herein in Example 8. In such an assay, food leaving is measured by an increase or decrease in the food leaving ratio, which can be calculated as the number of the animals off the food lawn (Noff ) divided by thetotal number of animals (Ntotai) at each time point from L4 to young adult during treatment with the genetic knockdown and / or therapeutic agent see e.g., Example 8 - Food leaving assay).

[0121] As described herein, for example, increased PDF-1 activity is coupled with decreased food dwelling (FIG. 20M) and increased food leaving (FIG. 20N) as demonstrated through overexpression of PDF-1. Thus, activity of PDF-1 measured by gene expression, protein expression, or a fluorescent reporter assay described herein can be correlated to food affinity behavior in C. elegans. Accordingly, candidate drug targets and / or candidate therapeutic agents that increase PDF-1 activity in C. elegans either as measured directly based on gene or protein expression or a protein activity assay as described above or as measured indirectly via a C. elegans feeding assay as described above, should decrease appetite and candidate drug targets and / or candidate therapeutic agents that decrease PDF-1 activity in C. elegans should increase appetite. Thus, candidate drug targets or candidate agents can be chosen based on w hether increasing or decreasing appetite is desired based on the disease to be treated.

[0122] As shown in FIGs. 2G and 20 A, PDFR-1 was identified as the closest C. elegans counterpart to GLP-1R and other members of the secretin-like (SCT) GPCR family. In some embodiments, a screening assay identifies an agent that binds specifically to more than one of the receptors shown in FIG. 2G and / or FIG. 20 A. Receptor binding can be measured by methods known in the art for protein binding kinetics including but not limited to surface plasmon resonance (SPR), and biolayer interferometry (BLI). Hence, in yet further embodiments, a candidate agent is assessed to determine if the agent binds to PDFR-1 or one or more of the receptors shown in FIG. 2G and / or FIG. 20A. Binding to such a receptor may further be used to identify a candidate agent as a therapeutic agent.

[0123] Thus, while the present disclosure identifies PDFR-1 as the closest C. elegans counterpart to mammalian GLP-1R, screening for PDFR-1 activity as described above may also identify agents that act at more than one member of the SCT GPCR family (including the GLP-1 receptor, GLP-2 receptor, calcitonin receptor, corticotropin-releasing factor receptors, parathyroid hormone receptors, vasoactive intestinal peptide receptors, pituitary adenylate cyclase-activating polypeptide type I receptor, grow th-hormone-releasing hormone receptor, GIP receptor, amylin receptor, and glucagon receptor).

[0124] In some embodiments, a screen in C. elegans identifies an agent as an agonist of any receptor shown in FIG. 2G and / or FIG. 20A. In some embodiments, a screen in C. elegans identifies an agent as a GLP-1 receptor agonist, GIP receptor agonist, amylin agonist, and / or GCGR agonist.

[0125] In some embodiments, an agent identified using evaluation of PDF-1 and / or PDFR-1 activity in C. elegans can bind specifically to a single receptor. In some embodiments, a screen in C. elegans identifies an agent as a GLP-1 receptor agonist, GIP receptor agonist, amylin agonist, or GCGR agonist. In some embodiments, the agent identified binds specifically to the GLP-1 receptor, GIP receptor, amylin receptor, or glucagon receptor.

[0126] In some embodiments, an agent identified using evaluation of PDF-1 and / or PDFR-1 activity in C. elegans binds specifically to more than one receptor. In some embodiments, an agent identified using evaluation of PDF-1 and / or PDFR-1 activity in C. elegans binds specifically to more than one receptor shown in FIG. 2G and / or FIG. 20A.

[0127] In some embodiments, the agent identified binds specifically to more than one of the GLP-1 receptor, GIP receptor, amylin receptor, or glucagon receptor. In some embodiments, the agent identified binds specifically to two receptors selected from the GLP- 1 receptor, GIP receptor, amylin receptor, and glucagon receptor. In some embodiments, the agent identified binds specifically to three receptors selected from the GLP-1 receptor, GIP receptor, amylin receptor, and glucagon receptor. In some embodiments, the agent identified binds specifically to the GLP-1 receptor, GIP receptor, amylin receptor, and glucagon receptor.

[0128] Once an agent has been identified as being able to modulate (e.g., increase or decrease) PDF-1 and / or PDFR-1 activity in C. elegans as described herein, this agent is also predicted to have efficacy in modulating feeding behavior in humans. In some embodiments, a method of treating a disorder in a subject comprises administering one or more agent capable of modulating PDF-1 and / or PDFR-1 activity in C. elegans. In some embodiments, the disorder is anorexia nervosa and / or ARFID. In some embodiments, the disorder is obesity, prediabetes, type 2 diabetes, or a metabolic syndrome.EXAMPLESExample 1. Metabolic conditions regulate feeding behavior and food consumption in C. elegans

[0129] No information has been published or disclosed describing any processes or mechanisms that act upstream of GLP-1 to regulate its action in the context of appetite regulation, including the idea that this hormone might be part of a pathway that is subject to metabolic regulation. The physiological functions of the GLP-1 pathway with respect to appetite regulation have thus remained essentially a “black box". In addition, the tissue sites of action for GLP-1 for modulating appetite have also not been definitively established. No strategies therefore exist for developing therapeutic targets within this pathway aside from peptides acting on receptor(s) for GLP-1 or related peptide hormones, or for teasing apart its food aversion side effect from action directly on appetite. Currently it is similarly unclear whether it might be possible to reset this pathway metabolically and avoid the need for direct peptide hormone therapy. The present studies in C. elegans were designed to provide answers to these outstanding questions on GLP-1 activity.

[0130] A bacterial food clearance assay9,10was developed to perform high-throughput screening for genes affecting food consumption. Following RNA interference (RNAi) screening of >1100 transcription factors (FIG. 1 A, FIGs. 6A and 6B, and Table 2), detailed analyses was performed of two genes for which knockdown reduced food consumption, sbp- 1 (SREBP) and crh-1 (CREB), each of which is associated with lipid metabolism17-20.* Gene shows food aversion in published paper

[0131] C. elegans ingests bacteria through phary ngeal pumping21. Surprisingly, however, neither crh-1 nor sbp-1 RNAi altered pumping rates (FIG. 6C). leading to investigation of whether these genes might influence food intake by affecting behavior. For eight positives associated with reduced food consumption, including crh-1 and sbp- , RNAi increased the frequency at which animals explore away from the edge of a food lawn22, a behavior referred to herein as "‘food-leaving” (FIGs. 6B and C). Increased food-leaving should lower food intake by reducing the time spent on the food. C. elegans exhibits foodleaving when cultured on a bacterial pathogen23-24, or after toxin exposure or gene knockdowns that mimic cellular effects of bacterial toxins22How ever, sbp-1 and crh-1 RNAi induced food-leaving independently of mechanisms that are required for pathogen avoidance25’26(FIGs. 7A-D, FIGs. 7J-K) or toxin-mimic responses (FIGs. 7A-F) and activate detoxification genes (FIG. 7G)22. A description of parameters associated with food leaving is further provided in Table 3. Thus, sbp-1 and crh-1 RNAi may promote food-leaving by perturbing metabolism rather than mimicking a pathogen attack.Table 3: Comparison of parameters associated with food-leaving induction.

[0132] It was also investigated whether crh-1 or sbp-1 knockdown affects how C. elegans behaves within a food lawn. While crawling on a lawn, worms spontaneously alternate between "roaming", a state of rapid crawling, and a slow locomotion state termed “dwelling” in which they pause and turn more frequently, staying within a smaller area27'29. Food consumption may be higher during dwelling, because food access is increased29. The proportion of time spent roaming versus dwelling can be evaluated by assessing the area an animal covers within a food lawn that extends over an entire plate (full lawn)21(FIG. ID). Knockdown of sbp-1 or crh-1 markedly reduced plate coverage (FIG. IE), indicating a higher proportion of dwelling, a conclusion confirmed by worm tracking analysis (FIG. 6D). Bycontrast, pathogen or toxin exposure did not increase dwelling (FIGs. 7H and 71, and Table 3). Thus, knockdown of these metabolic genes increased the frequency of two behaviors that are predicted to have opposite effects on food consumption: exploration away from the edge of a bacterial lawn (food-leaving) and dwelling within a law n.

[0133] It was investigated whether these behaviors can be induced by disrupting sbp- 1 or crh-1 expression in specific tissues30. Food-leaving was increased by sbp-1 or crh-1 knockdown in either the hypodermis or intestine (gut), each of which has important metabolic functions31, but not in body-w all muscle or neurons (FIGs. 1F-G). A C. elegans hermaphrodite has 302 neurons32, which will be referred to collectively herein as the brain. In striking contrast to food-leaving, dwelling was increased by knocking down sbp-1 or crh-1 specifically in the brain, but not intestine or muscle (FIG. 1H). Food-leaving and dw elling are therefore induced by deficits in sbp-1 or crh-1 in different tissues.

[0134] By inducing food-leaving and dwelling independently, how these behaviors each influence food consumption was evaluated. Knockdown of sbp-1 or crh-1 in the gutdecreased food intake (FIGS. II and IK), as would be expected because food-leaving is increased (FIG. IF). By contrast, knockdown in the brain increased food intake (FIG. II), suggesting that when dwelling is increased, food intake is elevated. The effects on behaviors observed on plates thus appear to carry over into the liquid culture feeding assay, in which the bacterial food rests at the bottom of a well (data not shown). Thus, for whole-body sbp-1 or crh-1 knockdown, food consumption was determined by the sum of effects on foodleaving and dwelling (FIG. 1C-E), which together resulted in a net reduction (FIG. 1 A).

[0135] Gut or hypodermal sbp-1 or crh-1 knockdown induces food dissatisfaction, exploration away from the lawn. When these genes are knocked down in the brain, a perturbation that would presumably be more acute nutritionally, dwelling and therefore food consumption are increased. For simplicity, these responses will be referred to herein as “food affinity” behaviors because they each affect how much time the animal spends with access to its food.Example 2. Metabolic regulation of food-affinity behaviors through serotonin and SCT- like receptor signaling

[0136] The neuronal signaling circuits that control food affinity behaviors were examined. The neurotransmitter 5-hydroxytryptamine (serotonin) is associated with feeding regulation throughout evolution33-14. In C. elegans serotonin production occurs in three neuron pairs, ADF, NSM, and HSN35, and is largely dependent upon the enzyme tryptophan hydroxylase (tph-1)33’36. While pathogen avoidance response requires induction of serotonin biosynthesis in the ADF neurons37(FIGS. 8A-B, 19A-B), the background level of dwelling seen on a continuous food lawn requires serotonin synthesis by the NSM neurons27, which are considered enteric because they sense food through a dendrite that extends into the pharyngeal lumen38. Food-leaving and dwelling induced by sbp-1 or crh-1 RNAi also required induction of tph-1 expression in the NSM but not ADF neurons (FIGs. 2A-B, FIGs. 8C-G). Thus, serotonin synthesis by the NSM neurons mediates each of these food-affinity behaviors.

[0137] These data raise an intriguing question: how does serotonin synthesis in the NSM neurons induce distinct behaviors, dwelling and food-leaving, that are regulated independently (FIG. 8H). Optogenetic depolarization of the NSM neurons through a short pulse (several seconds) of high-intensity Chrimson activation (15mW / cm2) induces dwelling on food (data not shown). By contrast, low-intensity (0.25mW / cm2) NSM activation over 2hours induced 20% of animals to leave the food lawn without inducing dwelling (FIG. 2C and FIG. 81). Thus, whether an NSM serotonin signal induces dwelling or food-leaving maybe determined in part by the intensity and duration of NSM neuron activation.

[0138] Given that dwelling and food-leaving involve distinct locomolory effects, they might be mediated through different serotonergic circuits. Six serotonin receptors have been identified in C. elegans, five of which are thought to influence locomotion (MOD-1, SER-1, SER-4, SER-5, LGC-50)39. These receptors are expressed in distinct but overlapping sets of neurons39. Analysis of mutants that lack individual, subsets of, or all of these receptors revealed that SER-4 and MOD-1 are required redundantly for dwelling to be increased by sbp-1 RNAi (, FIG. 2D, FIGs. 9A-F and FIG. 18J), consistent with a previous analysis of the background dwelling frequency39. By contrast, the SER-5 receptor was required for sbp-1 or crh-1 knockdown to induce food-leaving ( FIG. 2E, FIGS. 9G-L, and FIG.18K). SER-5 also mediates an effect of the ADF neurons on pumping40but is dispensable for pathogen avoidance (FIG. 9M). Thus, metabolically-induced serotonin signals from NSM increase the frequency of dwelling or food leaving by acting through different neuronal networks (FIG. 18L and FIG.19K).

[0139] Serotonin signaling promotes dwelling by opposing the action of the neuropeptide pigment-dispersing factor- 1 (PDF-1) and its G-protein coupled receptor (GPCR) PDFR-121, which is expressed in multiple neurons27. It was determined that PDFR-1 is orthologous to the mammalian Secretin-like (SCT) GPCR family (FIG. 2G). and most 15 members of the SCT GPCR family have functions in endocrine or vascular regulation that are not present in C. e / egans] :'A]. However, PDFR-1 is the closest C. elegans counterpart to mammalian glucagon-like peptide 1 receptor (GLP-1R) (FIG. 2G), with which it shares extensive sequence homology and structural features (FIGs. 10A-D), suggesting that PDFR-1 and GLP-1R might have conserved functions in feeding regulation.

[0140] The role of PDF- 1 / PDFR-1 signaling was investigated in dwelling and foodleaving induced by sbp-1 or crh-1 knockdown. While pdfr-1 mutation dramatically increased dwelling (FIG. 2F) and prevented food-leaving induction (FIG. 11 A), overexpression of the PDFR-1 ligand PDF-1 reduced dwelling and increased food-leaving (FIGs. 1 1D-E). Thus, while PDF- 1 / PDFR-1 signaling must be overcome by serotonin signaling for dwelling to occur, PDF-l / PDFR-1 and a serotonin signal through SER-5 act in concert to drive metabolically induced food-leaving (FIG. 2H). Impairment of PDF-l / PDFR-1 signaling hasbeen implicated previously in certain exploratory behaviors42,43. However, PDF-l / PDFR-1 signaling was not required for pathogen exposure to reduce dwelling42, or for food-leaving induced by toxin mimicry (FIGs. 11B-C), indicating that this signaling is not generally required for roaming activity.

[0141] PDF-l / PDFR-1 signaling therefore responds to the metabolic perturbations induced by sbp-1 and crh-1 RNAi to induce two behaviors that reduce affinity for food and food consumption: roaming on a food lawn (decreased dwelling / eating) and food- leaving / avoidance. A small molecule agonist of mouse GLP-1R reduced dwelling (FIG. 21), increased food-leaving dependent upon SER-5 (FIG. 2H and FIG. 1 IF), and reduced food consumption dependent upon PDFR-1 (FIG. 2J). These data suggest that suppression of feeding and affinity for food are ancient, conserved functions of SCT-like receptor signaling.Example 3. Specific fatty acids regulate food affinity responses

[0142] It should be possible to suppress sbp-1 or crh-1 RNAi -induced dwelling and food leaving by correcting the underlying metabolic perturbations. Knockdown of sbp-1 markedly decreases fat stores. This can be rescued by supplementation with the monounsaturated fatty acid (MUFA) oleic acid (OA), which is produced by SBP-1 -regulated FA desaturases (FAT-6 / 7) (FIG. 3C)44and facilitates SFA storage in TAGs45. OA suppressed the increases in dwelling and food leaving that resulted from knockdown of not only sbp-1. but also crh-1 (FIGs. 3A-B, and FIG. 12A), but failed to inhibit toxin-induced food avoidance (FIG. 12B), identifying OA as a regulator of metabolically related foodaffinity behaviors.

[0143] Knockdown of crh-1 or sbp-1 decreased fat levels comparably (FIGs. 13A-B), suggesting that this dwelling and food-leaving might be induced when fat reserves are low. Surprisingly, this fat reduction derives largely from the reduced food consumption induced by sbp-1 or crh-1 RNAi (FIGs. 13C-F), suggesting that food affinity behaviors are regulated by more specific signals.

[0144] It was investigated whether interference with biosynthesis of OA or other FAs (FIG. 3C) might phenocopy behavioral effects of sbp-1 or crh-1 knockdown. Loss of fat-6 / 7 induced dwelling and food leaving that could be rescued by OA (FIGs. 3D-E), indicating OA levels are crucial. To investigate FA effects further, experiments concentrated primarily on food-leaving, which is more amenable to high-throughput analysis. Food-leaving was induced when biosynthesis of OA precursors or the MUFA palmitoleic acid (POA) wasimpaired (FIG. 3C and FIG. 12C). Unlike most animals, C. elegans encodes FA desaturases and elongases that convert OA to polyunsaturated FAs (PUFAs) (FIG. 3C)18. Blocking PUFA biosynthesis failed to induce food leaving (FIGs. 12C-D) or prevent OA rescue of food-leaving from sbp-1 or crh-1 RNAi (FIGs. 12E-F). Thus, this food affinity behavior is induced by deficits in the biosynthesis of MUFAs OA or POA, but not PUFAs.

[0145] The food-leaving induced by fat-5 mutation (FIG. 12C) suggests that its MUFA products might have rescuing activity (FIG. 3C). Supplementation with either POA or OA rescued food leaving induced by sbp-1 RNAi, or by genetically induced deficits in the other MUFA (FIG. 12G). The POA derivative vaccenic acid also exhibited rescuing activity (FIG. 12H). Surprisingly, although knockdown of the FAT-2 desaturase did not induce food leaving (FIG. 12C), its PUFA product linoleic acid (LA) rescued food-leaving induced by knockdown of sbp-1, fat-5, or fat-6 / 7 (FIG. 121). Thus, while food-leaving can be induced by impairing synthesis of particular MUFAs, any of these MUFAs or LA can suppress this behavior (FIG. 3C).

[0146] In striking contrast to these unsaturated FAs. supplementation with the POA / OA precursor SFA palmitic acid (PA) induced both food leaving and dwelling (FIGs. 3F-G). As would be predicted, the former behavior required SER-5 and SCT-like receptor signaling (FIGs. 3H-I). Supplementation with the immediate OA precursor stearic acid (SA, FIG. 3C) also induced food-leaving. Evidently, neither PA nor SA can be converted to MUFAs efficiently enough to prevent these SFAs from inducing food leaving or dwelling. Importantly, OA co-supplementation suppressed PA-induced food-affinity behaviors (FIGs. 3F-G) in a dose-dependent manner (FIG. 12J). Thus, food affinity behaviors can be triggered by MUFA deficits or an increase in the ratio of available SFAs relative to MUFAs. These data together suggest that these behaviors are likely regulated by a mechanism that responds to properties of these MUFAs and SFAs, and not by multiple specific receptors.Example 4. MUFAs and SFAs control food affinity behaviors through lipid metabolism and ER signaling

[0147] The time courses through which PA and OA alter food-affinity behaviors were examined. Two hours of OA-feeding rescued food-leaving that was induced by PA feeding of crh-1 knockdown (FIG. 4A and FIGs. 14A-B). PA supplementation increased tph-l::GFP levels in the NSM neurons w ithin only 20 minutes (FIG. 14C), and the tph-l: :GFP signal induced by PA could be suppressed by OA within 90 minutes (FIG. 4B). Thus, these FAsaffect serotonin production and behavior on a rapid trajectory, suggesting that they act through signaling and not chronic metabolic effects.

[0148] A model was evaluated that can explain the finding that multiple unsaturated FAs can suppress the effects of SFAs on food-affinity behaviors, wherein this is mediated through incorporation of these FAs into membrane phospholipids or storage TAGs at the endoplasmic reticulum (ER) (FIG. 4H). OA and other MUFAs extend C. elegans lifespan through a network of processes that mediate membrane phospholipid and TAG biosynthesis, as well as LD formation46,47. OA acts on this network by being incorporated into these complex lipids and increasing LD formation46,47. For simplicity7, these processes will be referred to herein as the TAG / phospholipid homeostasis network. OA thereby enhances ER- associated degradation (ERAD)46(FIG. 141), a process through which incompletely folded proteins are extruded from the ER48. ERAD enhancement in turn increases the processing and levels of the ER-resident transcription factor SKN-IA / Nrfl, which drives OA-induced lifespan extension46.

[0149] If a component of the TAG / phospholipid homeostasis network is required forMUFAs to reduce food-leaving and dwelling, interference with its expression should induce these behaviors and block their suppression by OA. SKN-laA did not affect food-leaving, but ERAD impairment induced food-leaving that was OA-suppressible (FIGs. 14L-M). This suggests that food-leaving responds to ER conditions (ERAD impairment), but also that ERAD is not required to transduce the OA signal. RNAi against the LD proteins plin-1, fltm- 2 and dnc-1, the phospholipid biosynthesis enzy mes PISY-1 and CEPT-1, and the LD biosynthesis factor FITM-245,63induced food-leaving that was partially rescued by OA (FIGs 14L and 14N-P), suggesting that LD biogenesis is involved in OA behavioral signaling. Importantly, knockdown of the TAG and PL biosynthesis and turnover enzymes lpin-1 (LPIN), atgl-1 (ATGL-1) or dgat-2 (DGAT-2) increased food leaving and dwelling that could not be significantly rescued by OA (FIG. 4G- J). The data suggest that food affinity behaviors and their suppression by OA are regulated through the TAG / phospholipid homeostasis, but not ERAD or SKN-1A. ERAD is critical for maintaining ER luminal protein homeostasis and is an essential mediator of OA-induced lifespan extension. Thus, while food-leaving can be induced by a disruption of ER lumenal conditions, this disruption does not interfere with its metabolic rescue. The suppression of food affinity behaviors by MUFAs therefore depends specifically upon the TAG / phospholipid homeostasis network.

[0150] This model predicts that metabolic perturbations that trigger food-affinity behaviors would alter incorporation of SFAs and MUFAs into TAGs and / or PLs. To test this idea, gas chromatography-mass spectrometry (GC-MS) lipidomics was performed in sbp-1 knockdown animals that could not leave their food (FIG. 15 A). Knockdown of sbp-1 R increased the ratio of C16:0 / C18: l (PA / OA) incorporation within DAG and the membrane phospholipids PS, PE, PI, PC, LPC, and LPE, indicating increased membrane lipid saturation (FIG. 4H and FIGs. 15E-G). OA supplementation reversed this increase significantly for PE and induced a trend towards reducing this ratio for DAG, PS and PI (FIG. 4H and FIGs. 15E- H). Consistent with our findings, previous w ork described membrane lipid saturation being increased by knockdown of the SBP-1 co-factor MDT-15, and decreased when OA levels are elevated. These observations are consistent with the present model that the SFA / MUFA ratio is a key regulator of feeding affinity behaviors, and suggest that FA incorporation into membrane PLs may mediate transduction of this FA signal at the ER.

[0151] How ould effects on ER membranes be transduced rapidly into a signal that affects behavior? The unfolded protein response (UPR), which induces ER expansion and remodeling, is activated not only by accumulation of misfolded proteins in the ER lumen, but also by ER membrane perturbations that include increased lipid saturation49'51. Under these conditions the UPR transmembrane sensor inositol-responsive enzyme 1 (IRE-1) is activated through dimerization, resulting in post-transcriptional splicing of the mRNA encoding the UPR transcription factor XBP-1. This signaling through IRE-1 has been induced independently of luminal protein misfolding by PA suppl ementati on in single-cell models, and in C. elegans by knockdow n of the SBP-1 co-factor MDT-15, in the latter case accompanied by an increase in saturated FAs within the membrane lipid phosphatidyl choline51.

[0152] IRE-1 mediates the signaling through which elevations in saturated FAs stimulate food-affinity behaviors. IRE-1 knockdown prevented PA supplementation from inducing either food-leaving or dw elling (FIGs. 17A-B). Neuronal-specific ire-1 RNAi also prevented PA from inducing dwelling (FIG. 17C), indicating tissue-autonomous regulation of this FA-sensing response in the brain. Knockdown of crh-1 or xbp-1 increased levels of the spliced (active) form of XBP-1 mRNA (FIG. 17D). Furthermore, a C. elegans marker of IRE- 1 -induced UPR signaling (hsp-4: :GFP)51w as upregulated by crh-1 or sbp-1 RNAi, and by PA feeding, w ith suppression by OA or ire-1 RNAi (FIG. 17E-F). Knockdown of thelipid metabolism genes acs-4 (fatty' acid coA synthetase family, located in lipid droplet surface, not shown), cept-1 (CEPT), atgl-1 and dgat-2 (FIG. 4H) also induced this UPR signaling, with no or minimal suppression by OA (FIG.4H, FIGs. 14N-P), consistent with IRE-l-based signaling transducing these lipid biosynthetic perturbations. IRE-1 was also required for PA activation of tph-l::GFP in the NSM neurons (FIG. 17G-H). indicating that IRE-1 is needed to initiate the serotonin-induced behavioral response to metabolic perturbation. Finally, neuronal-specific expression of the spliced form of XBP-1 (XBP-ls), a major effector of IRE-1 signaling, induced dwelling that was only weakly suppressed by OA (FIG. 171). The latter finding is consistent with XBP-1 functioning downstream of OA to regulate these behaviors, as predicted. Therefore, the ratio of available saturated and unsaturated FAs is sensed at the ER through the TAG / phospholipid homeostasis pathway and effects on membrane conditions, and this information is transduced as a signal by IRE-1 to regulate food-affinity behaviors.

[0153] Together, these results indicate that (1) the relative levels of saturated and unsaturated FAs are sensed through their incorporation into membrane lipids by the TAG / phospholipid homeostasis network and the resulting effects on membrane conditions, (2) that this information is transduced by IRE-1 to regulate food affinity behaviors, and (3) that this pathway functions autonomously within the brain to regulate dwelling (FIG. 17K). IRE- 1 signaling is also required for food-leaving to be induced by a peripheral MUFA deficit, but this involves a more complex set of mechanisms that include tissue-tissue signaling (FIG.17 J).Example 5. Fatty acid-mediated regulation of food-affinity behaviors by AMPK and diet

[0154] Other metabolic signals might alter food-affinity behaviors through the FA- based pathway. AMPK was examined because of its role in regulating mammalian food consumption8’9-52. In C. elegans. transgenic expression of a constitutively active AMPKa2 catalytic subunit (AAK-2) results in AMPK activation throughout the body53. CA-AAK-2 phenocopied the effects of sbp-1 or crh-1 knockdown on food-affinity behaviors, increasing dwelling and food-leaving, and reducing food consumption (FIGs. 5A-C and FIGs. 16A-B). AMPK was not required for sbp-1 or crh-1 knockdown to induce food-leaving (FIG. 16J), and CA-AAK-2-induced food-leaving and dwelling could be suppressed by serotoninsignaling impairment or OA feeding (FIGs. 5B-E). This suggests that AMPK regulates foodaffinity behaviors by acting through the FA-regulated pathway identified herein.

[0155] It seems paradoxical that whole-body CA-AAK-2 expression reduces food intake, whereas hypothalamic AMPK activation increases feeding in mice8,54. However, CA- AAK-2 overexpression specifically within the brain exhibited similar effects to neuronal sbp- 1 or crh-1 RNAi (FIG. 1F-G), increasing dw elling but not food leaving (FIG. 5D and FIG. 16E), and food consumption (FIG. 5F). This suggests that the action of AMPK in the brain to increase feeding is evolutionarily conserved. Food-leaving was not increased by CA-AAK-2 expression in any individual tissue tested (FIG. 16E). However, neuronal aak-2 knockdown in a strain expressing CA-AAK-2 throughout the body reduced dwelling as expected but not food-leaving (FIG. 5G and FIG. 16L) suggesting that simultaneous activation of AMPK in multiple peripheral tissues is sufficient to induce food-leaving. Although aak-2 mutation abrogates an increase in pharyngeal pumping induced by exogenous serotonin54, CA-AAK-2 did not detectably affect pumping frequency (FIG. 16C). Thus, AMPK activation affects food consumption largely by influencing dwelling and food-leaving.

[0156] Food-leaving and dwelling that were induced by CA-AAK-2 could be suppressed by either OA supplementation or ire-1 knockdown (FIG. 5K-N), indicating a requirement for FA and ER signaling. Also consistent with AMPK functioning upstream of these signals, the catalytic AMPK subunit was not required for sbp-1 or crh-1 knockdown to induce food-leaving ( FIG. 16J). The data suggest that AMPK regulates food affinity behaviors by acting through the FA-regulated pathway identified herein.

[0157] AMPK regulates numerous cellular processes. AMPK increases C. elegans lifespan dependent upon its inhibiting the CRH-1 co-activator CRTC-153, but reduced food intake independently this mechanism (Fig. 16j). AMPK signaling also inhibits lipid biosynthesis and increases lipid oxidation to mobilize energy55, mechanisms that could affect a lipid-based signal. Accordingly, CA-AAK-2 reduced fat storage in animals grown on a full lawn (FIG. 5H), downregulated FA biosynthesis genes, and increased p-oxidation gene expression (FIG. 16N). In contrast to sbp-1 or crh-1 RNAi, whole-body CA-AAK-2 reduced fat storage independently of its effects on food-leaving, suggesting that this reduction derived from altered lipid metabolism and not reduced food consumption (FIGs. 16M-P). Disruption of peroxisomal -oxidation by dhs-28. dhs-2. or prx-5 RNAi suppressed CA-AAK-2-induced dwelling (FIG. 51). and dhs-28 knockdown modestly induced food-leaving but suppressedfood-leaving induction by CA-AAK-2 (FIG. 5J), suggesting that lipid oxidation contributes to AMPK induction of these behaviors. The data suggest that AMPK acts on the TAG- phospholipid homeostasis pathway to regulate food-affinity behaviors and food consumption by modulating lipid and FA levels.

[0158] Next it was investigated whether the metabolically regulated mechanisms described above are affected by food quality. Previous work revealed that at the LI stage C. elegans larvae avoid poor-quality food64-66. The E. coli strain DA837 is considered to be poor quality because it does not efficiently support larval development64. This strain is comparatively difficult for developing larvae to ingest64, and differs in nutritional content from the OP50 and HT115 strains used here67. Culture on DA837 induced food-leaving at the L4 stage under our experimental conditions, to an extent that was comparable to CA- AAK-2 expression (FIGs. 5P-Q). DA837 feeding also dramatically increased hsp-4p::GFP expression (FIG. 5R). Importantly, these DA837-associated phenotypes were suppressed by OA (FIG. 5R), suggesting that the energy / FA-sensing pathway we have uncovered mediates this food leaving and influences C. elegans" affinity for its bacterial food.

[0159] Thus, two food-affinity behaviors, dwelling and food-leaving, alter food consumption in C. elegans and are controlled by metabolic cues from particular FAs.Example 6. Serotonin signaling mediates metabolic regulation of food affinity behaviors

[0160] A Cre-Lox deletion of / / ? / 7- / 27demonslrated that expression of tph-1 in the NSM but not ADF neurons was required for sbp-1 or crh-1 RNAi to induce food-leaving and partially required for dwelling induction (FIGs. 18A-B and FIGs. 19C-D), consistent with most serotonin synthesis being / A / -dependent. Expression of tph-1 was also largely required for dwelling to be induced by AMPK activation (FIGs. 18C-D), as would be predicted by the model that AMPK regulates food affinity behaviors by acting through the same FA- and ER-based pathway as sbp-1 or crh-1 RNAi (FIGs. 5K-N). The levels of tph- lp::GFP in the NSM neurons were increased by crh-1, sbp-1, or dgat-2 RNAi, suggesting that metabolic stimuli that stimulate food leaving upregulate serotonin biosynthesis (FIGs. 18E-F and FIGs. 19E-H). Supplementation with OA suppressed this upregulation for knockdown of crh-1 or sbp-1 but not dgat-2 (FIGs. 18E-F), consistent with DGAT-2 being critical in the TAG / Phospholipid homeostasis network (Example 4). Feeding of PA rapidly induced tph-lp::GFP expression that could be suppressed by ire-1 knockdown or OA supplementation (FIGs. 18G-H and FIGs. 19I-J), further supporting the model that in thisfeeding regulatory pathway tph-1 expression is induced by SFA-induced ER signaling. Finally, neuronal specific CA-AAK-2 expression, which induces dwelling, also upregulated tph-lp::GFP expression in the NSM neurons, consistent with the genetic requirement for tph- 1 in those neurons (FIG. 181). This tph-lp::GFP expression required ire-1, implicating ER signaling as the models herein predict (FIG. 19K). These multiple lines of evidence indicate that the ER-based energ / FA sensing mechanisms identified herein control food affinity behaviors by regulating serotonin expression in the NSM and not ADF neurons, further supporting the idea that this mechanism is distinct from those regulating pathogen avoidance (Table3).Example 7. GLP-l-like signaling establishes metabolic regulation of food affinity behaviors

[0161] Signaling through the neuropeptide pigment-dispersing factor (PDF-1 / 2) and its G-protein coupled receptor (GPCR) PDFR-1 has been implicated in some exploratory behaviors27,68, thus it was investigated whether this signaling might be involved in metabolic regulation of food affinity behaviors. As described above, PDFR-1 is the closest C. elegans relative to mammalian GLP-1R, and shares extensive sequence and structural features with GLP-1R (FIGS. 20A-B and FIGs. 21 A-B). While the closest mammalian relative of PDFR-1 is the calcitonin receptor, PDFR-1 is orthologous to the entire family of closely-related mammalian receptors, which have been designated the secretin-like (SCT-like) GPCRs (FIGs. 20 A-B)13. Most members of this receptor family function in processes that are not present in C. elegans, but some have been targeted for obesity treatment along with GLP-1R by poly agonists (FIG. 20 A)2. PDFR-1 therefore represents a C. elegans counterpart to GLP- 1R and the SCT-like receptor family (FIG.20A).

[0162] It was next investigated whether signaling through PDFR-1 is required for conditions associated with excess SFAs to induce food-leaving. Although PDFR-1 was dispensable for food-leaving to be induced by either toxin mimicry or pathogen exposure (FIGs. 21C-D), neither whole-body sbp-1 RNAi nor PA supplementation induced foodleaving in pdfr-1 mutants (FIGs. 20C-D), indicating that PDFR-1 is required for food-leaving to be induced by these metabolic stimuli. PDFR-1 and its major ligand PDF-1 are expressed in specific subsets of neurons, and PDF-1 expression can be readily visualized as a rescuing translational fusion with GFP27. PDF-1 ::GFP expression was increased within the brain in an / 'c- / -dependent manner by either sbp-1 or crh-1 RNAi, or PA supplementation (FIGs. 20E-H and FIGs. 21E-F). Furthermore, the increase in PDF-1 ::GFP that was induced by sbp-1 RNAi depended upon tph-1 (FIG. 20I-J and FIG. 21G-H). Thus, metabolic stimuli that result in excess SFAs being present in peripheral tissues act successively through serotonin and PDF-l / PDFR-1 signaling to induce food-leaving (FIG. 20L).

[0163] The typical levels of dwelling that are characteristic of WT animals raised under standard conditions are dependent upon serotonin signaling that opposes PDF-l / PDFR- 1 signaling, although an epistatic relationship between these pathways has not been established previously27. The models provided herein predict that metabolic perturbations that act in the brain to promote dwelling would need to overcome PDF-l / PDFR-1 signaling, possibly by reducing PDF-1 expression, and that this would require serotonin signaling (FIG. 18L). Within a food lawn pdfr-1 mutants exhibited dramatically elevated levels of dwelling that were not increased by crh-1 knockdown but could be reversed readily by pathogen exposure, further distinguishing responses to metabolic and pathogenic stimuli (FIGs. 20K and 211 and Table 3). To investigate the effects of altering metabolic conditions specifically within the brain, and therefore to induce dwelling but not food-leaving, animals that express CA-AAK-2 specifically in neurons were examined. These animals exhibit markedly elevated levels of dw elling that are suppressed by ire-1 knockdow n (FIG. 5N) and associated with elevated tph-lp.'.G P expression in the NSM neurons (FIG. 18E). Importantly, neuronal CA- AAK-2 reduced the levels and extent of PDF-1::GFP expression within the brain, an effect that was dependent upon tph-1 (FIG. 20L). Thus, metabolic stimuli that induce dwelling do so by acting within the brain to increase serotonin signaling, and thereby inhibit PDF- l / PDFR-1 signaling (FIG. 20R).

[0164] The models provided herein predict that activation of PDF-l / PDFR-1 signaling might be sufficient to regulate food affinity behaviors. Supporting this idea, transgenic PDF-1 overexpression reduced dwelling and increased food-leaving (FIGs. 20M- N). Furthermore, a small molecule allosteric agonist of human GLP-1R reduced dwelling (FIGs. 200 and 21J), increased food-leaving (FIGs. 20P and 21K), and decreased food consumption (FIG. 20Q), dependent upon PDF-l / PDFR-1. In conclusion, PDF-l / PDFR-1 signaling responds to MUFA insufficiency to induce two behaviors that reduce affinity for food and food consumption: roaming on a food lawn (decreased dwelling / eating) and food- leaving / aversion (FIGs. 20O-P and 21J-K). These findings suggest that the feedingregulatory functions of GLP-1 receptor-like signaling have been preserved between humans and roundworms, and that suppression of feeding and food satisfaction in response to metabolic signals is an ancient, conserved function of this signaling.

[0165] Also described herein are neuronal circuits and molecular pathways that mediate this regulation, demonstrating that feeding behavior and food consumption can be controlled by sensing of critical nutrient metabolites. Both food-leaving and dwelling are regulated by these FAs, demonstrating that not all calories are the same with respect to hunger and satiety. This is revealed most strikingly by the finding that MUFA supplementation can reduce dwelling, a behavior associated with increased food consumption, and by the opposing effects of MUFAs and SFAs on dwelling and foodleaving. The evidence that the available MUFA / SFA ratio is sensed at the ER in neurons through mechanisms that mediate LD and PL homeostasis suggests clues as to why these FAs are such important feeding regulators. Bilayer membranes are critical to cellular functions, particularly in neurons. FA incorporation profoundly affects TAG formation and membrane properties such as thickness and fluidity, with SFA content of phospholipids decreasing the latter. Regulation of these feeding behaviors by the MUFA / SFA ratio might have evolved as a strategy for controlling the accumulation of these FAs and optimizing their relative levels in cellular structures.

[0166] The essential role of IRE-1 in transducing this FA-based signaling from the ER was also revealed. Both PA-induced food leaving and PA-induced dwelling were dependent on ire-1, while neuronal-specific knockdown of ire-1 blocks PA-induced dwelling (FIGs. 17A-C). Further crh-1 and sbp-1 RNAi increased total xbp-1 RNA levels, activated HSP-4, and regulated tph-1 expression (FIGs. 17D-H). These results demonstrate the key role of IRE- 1 in controlling feeding behaviors in C. elegans.

[0167] If these pathways are evolutionarily conserved, as appears likely given the conservation of the molecular mechanisms involved, it may be feasible to maximize satiety by increasing the relative dietary consumption of MUFAs or possibly other unsaturated FAs.

[0168] This FA-sensing mechanism also responds to AMPK, an indicator of low energy availability, suggesting that this sensing mechanism transduces the effects of reduced energy and caloric intake on feeding and might detect other metabolic perturbations that affect FA levels.

[0169] GLP-1R agonists were developed to increase insulin resistance in peripheral tissues, and it has been unclear why they influence food consumption. It is remarkable that in C. elegans the SCT-like GLP-1R counterpart (PDFR-1) acts neuronally to effect the metabolic FA sensing uncovered herein, in each case driving an anorectic behavior (roaming vs. dwelling and food-leaving). These food-affinity behaviors are reminiscent of the effects of GLP-1R agonists in humans: reduced food consumption and the side effect of food aversion, respectively. By placing this receptor signaling downstream of a metabolic FA and energy sensing mechanism, the present results suggest paradigms for understanding the role of endogenous GLP-1 -related signaling in appetite regulation. This in turn could lead to the development of new agents for combating obesity and possibly disorders of decreased appetite, and strategies for minimizing side effects.References1 Andermann, M. L. & Lowell, B. B. 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[0170] The C. elegans strains were maintained under standard conditions at 20-22 °C on Nematode Grow th Medium plates containing E. coli OP50 bacterial lawns. The strains used in this study include: wild-type Bristol N2. rol-6, WM27 rde-l(ne219), VP303: rde- l(ne219) V; kbls7 [nhx-2p::rde-l + rol-6], FK171 mek-l(ks54), TK233 egl-4(ks60). DA609 npr-l(ad609f LD1900 zc!sl3[hsp-6::GFP , LD1807 agls219 [T24B8.5p::GFP::unc-54 3' UTR + ttx-3p::GFP::unc-54 3' UTR], DA2109 ser-7(tm!325) ser-l(ok345), LD1882: ser- l(ok345) X, D1103 fat-6(tm331); fat-7(wa36), LD I 790 fat-l(wa9) , LD I 79 I fat-2(wa!7f LD I 792 fat-3(wa22), LD I 793 fat-4(wa!4f YT17 crh-l(tz2\ TM1944 aak-7(7m79- / 4).RB754 aak-2(ok524f TG38 aak-2(gt33f NB245 aak-l(tm!944) : aak-2(gt33), AMJ345 rde- l(ne219) V;jamSi2 [mex-5p::rde-l(+)], QK52 rde-l(ne219);Is[wrt-2p::RDE-l::unc54 3'utr; myo2p::RFP3], RB1908 mlk-l(ok2471), KU25 pmk-l(km25), AX1295 gcy-35(ok769), RB2302 daf-7(ok3125), FK181 ksls2 [daf-7p::GFP + rol-6(sul006)]. AU133 aglsl7 [myo- 2p::mCherry + irg-lp::GFPJ, JIN810 agls26 [clec-60p::GFP + myo-2p::mCherry], GR2250 mgls73 [cyp-14A4p::gfp::cyp-14A4, 3’UTR + myo-2p::mCherry], MT15434 tph-l(mg280), MT14984 tph-l(n4622), GR1366 mgls42 [tph-l::GFP + rol-6(sul006)],CX14295 pdfr- l(ok3425), LSC90 pdf-l(tml996);lstlsl [pdf-lp::pdf-l::3'UTR + elt-2p::GFP], LSC84 lstEx2[pdf- Ip: :pdf-l:: 3'UTR + elt-2p::GFP],RB1622 ser-3(okl995), RB745 ser-4(ok512), RB2277 ser-5(ok3087). RB1585 ser-7(ok 19444). MT9668 mod-l(okl03). AGD638 sid-l(qt9); uthIs206[rab-3p::tomato::unc-54-3’UTR; rab-3p::sid-l::unc-54-3’UTR], AGD677[rgef- lp::tomato::unc-54-3’UTR; rgef-lp::sid-l::unc-54-3’UTR], AGD745:sid-l(qt9) V; uthls236[gly-19p::tdtomato + gly-19p: :sid-l], AGD855 sid-l(qt9); uthls236[gly- 19p::tdtomato + gly-19p: :sid- 1], MAH677:sid-l(qt9) V; sqls71 [rgef-lp: :gfp+rgef-lp: :sid- 1], HC196 sid-1 (qt9) V, CX13228 tph-l(mg280) II; kySi56[floxed tph-1 genomic rescue], CX13571 tph-l(mg280); kySi56; kyEx4077[srh-142::nCre], CX13572 tph-l(mg280); kySi56; kyEx4057[ceh-2::nCre], CX14385 pdf-l(tml996) ; kyEx4583[pdf-l::pdf-lb-sl2- GFP], CX14388 pdf-l(tml996); kyEx4586[pdf-l::pdf-la-sl2-GFP], CX15018 kyEx4984[pdfr-l::BlaC-sl2-GFP], CX15187 kyEx5091[pdfr-l::BlaC(D265K) -S12-GFP], CX15660 kyEx5273[tph-l::ChR2*(C128S)::GFP], WBM60 uthls248 [aak-2p::aak- 2(genomic aal-321)::GFP::unc-54 3'UTR + myo-2p::tdTOMATO], WBM181wbmEx66[rab- 3p::aak-2 (aal-321 cDNA)::tdTomato::unc-54 3'UTR], WBM182 wbmEx67[gly-19p::aak-2 (aal-321 cDNA)::tdTomato::unc-543'UTR], WBM183 wbmEx68[myo-3p::aak-2 (aal-321 cDNA)::tdTomato::unc-54 3'UTR], AGD383 wbmEx68[myo-3p::aak-2 (aal-321 cDNA)::tdTomato::unc-54 3'UTR], WBM34 uthIs205 [crtc-lp::crtc-l::tdTOMATO::unc-54 3'UTR + rol-6(sul006)], WBM132 wbmEx49 [rab-3p::crtc-l(S76A, S179A)::tdTomato::unc- 54 UTR], WBM184 wbmEx69 [ges-lp::crtc-l(cDNA S76A, S179A)::tdTOMATO::unc-54 3'UTR], SWF117: flvls2 [tph-lp (short): :Chrimson + elt-2p::mCherry], SWF250: mod- l(okl03); ser-4(flv7); flvls2 [tph-1 (short) ::Chrimson, elt-2::mCherry], SWF380: ser- 5(tm2647) I; ser-4(flv7) lgc-50(flv8) III; mod-l(okl03) V; ser-7(tml325) ser-l(ok345) X; flvls2, LD1960: pdf-l(tml996) III; kyEx4586[pdf-l:;pdf-la-s!2-GFP]; tph-l(mg280) IE LD1947: pdf-l(tml996) III; kyEx4586[pdf-l::pdf-la-sl2-GFP]; wbmEx66[rab-3p::aak-2 (aal-321 cDNA)::tdTomato::unc-543'utr], LD1970: pdf-l(tml996) III; kyEx4586[pdf- l::pdf-la-sl2-GFP]; tph-l(mg280) II; wbmEx66[rab-3p::aak-2 (aal-321 cDNA)::tdTomato::unc-54 3'utr], LD1951: mgls42 [tph-l::GFP + rol-6(sul006)]; wbmEx66[rab-3p::aak-2 (aal-321 cDNA)::tdTomato::unc-543'utr], and LD1948: tph- l(mg280) II; wbmEx66[rab-3p::aak-2 (aal-321 cDNA)::tdTomato::unc-54 3'utr], Peripheral constitutive overexpression of AMPK strain is made by crossing the neuronal RNAi strain AGD677[rgef-lp::tomato::unc-54-3’UTR; rgef-lp::sid-l::unc-54-3’UTR] and the wholebody constitutive overexpression of AMPK strain WBM60 uthls248 [aak-2p::aak-2(genomicaal-321)::GFP::unc-54 3'UTR + myo-2p::tdTOMATO]. All experiments were performed with hermaphrodite worms. All the RNAi clones were obtained from the Vidal RNAi library except fard-1 and confirmed by sequencing. As a control, empty vector L4440 was used. For fluorescence imaging, worms were immobilized on 2% agarose pads in 20mM sodium azide buffer and imaged using a Zeiss imager M2 fluorescence microscope.Food intake screen

[0171] To screen for new food intake regulators, a bacterial depletion assay1was used with small modifications. To perform the assay, synchronized LI stage worms were grown on RNAi bacteria under the standard conditions for 46~48h, washed with S-basal buffer (5 mM KPO4 pH 6.0, 1 mM CaC12 and 1 mM MgSO4) twice, 6-10 animals were transferred to the 96-well plates containing 120ul growth-arrested OP50. At least 6 replicates for each condition, read the ODeoo as ODeoodayo. After the indicated days, take out the 96-well plate from the incubator, use multi-pipette with non-stick pipette sticks to resuspend 5 times and read ODeoo immediately as ODeooday3. The food consumption of each animal during the days is calculated by Food intake / worm=[OD6oodayo-ODeooday3] / [Xo]. After two rounds repeats screen for the whole transcription factor library, all the positive and negative regulators are subjected to perform food intake assay in the 24-well plates same as described previously.Food leaving assay

[0172] Chronic food leaving assay was performed as previously described2. Briefly, HT115 RNAi bacteria cultured in the way described above. 45pl aliquots were dropped to 60mm RNAi plates and left on the benchtop for 72 hours before use. Then around 45 synchronized LI stage worms were seeded directly in the middle of the lawn and left to dry for 10 mins, and then kept under standard conditions at 20-22 °C incubator. The food-leaving ratio for each condition was calculated as the number of the worms off the lawn Non divided by the total number of worms Ntotai at each time point from L4 to young adult during RNAi treatment.

[0173] Acute food-leaving assay is performed with small modification to study the effect of specific nutrients on feeding related behaviors. To perform the acute food leaving assay, worms grown on RNAi bacteria or drug treated E coli OP50 under standard conditions were collected at young adult stage, washed with S-basal buffer twice, transferred 20-40 animals in the middle of the food lawn of the assay plates with or without specific nutrients or drugs as indicated. Scoring was started 2 hours afterwards and lasted as long as 16 hours atleast four replicates in each condition with three or more independent experiments. The investigators were not blinded to the condition and genotype of the worm.Food dwelling assay

[0174] Food dwelling assays are known as food exploration assays were performed as reported previously3. Single OP50 E. coli colonies were inoculated in 500 mL of LB and incubated at 37 °C shaking at 220rpm overnight, followed by centrifuge at 3000 rpm for lOmins, resuspend with fresh LB buffer with streptomycin (1000X dilution), and adjustment of OD600 to OD=14. Then 200ul OP50 was evenly spread on the 35-mm NGM plates one day before the assay. This protocol was used to make full lawn food density consistent with the regular lawn food density in the food-leaving assay, around 1.5X108cells per cm2.Worms were grown starts from L I stage unless otherwise specified on standard conditions to L4 stage. Individual L4 animals were then placed in the center of 35-mm NGM assay plates uniformly seeded with 200 pL of OP50 E. coli bacteria with or without specific nutrients or drugs as indicated in the figure legends. After a 16-hour period of exploration at 20 °C, the worms were removed, plates were superimposed on a grid of 3-mm squares, and the number of squares entered by worm tracks w as counted manually. Tracks could enter a maximum of 109 squares. The investigators w ere not alw ays blinded to genotype of the animals, and experiments were repeated at least three times.Optogenetics

[0175] Food leaving during optogenetic stimulation was conducted using previously described methods. L4 animals were picked to a 6cm plate seeded with 200ul OP50 plus 50um all trans retinal (ATR) 16-24 hours in advance. The day of experiments, young adult animals were w ashed with 1ml of M9 buffer twice and then placed onto a 6cm NGM plates with typical food leaving lawn present.Pathogen aversive behavior and toxin response aversive behavior

[0176] Exposure to pathogenic bacteria P. aeruginosa PA14 and low-quality food source DA837 or benign bacteria HT115 and OP50 induced behavior w as performed similarly as previously described unless otherwise specified. The worms were cultivated under standard conditions on OP50 from LI stage to young adult stage, and then washed 3 times by S-basal before were randomly split into groups, and transferred to plates that contain overnight grown PAM or OP50. Food aversion rate is scoring started after 2h and continued to 16h at 20 °C. For toxin induced food aversion experiment, 60ug / ml BTZ or 50ug / mlTunicamycin were used to only cover the bacteria lawn 2 hours before the experiment. Scoring was initiated after 2h and continued to 16h. For long-term exposure, LI stages worms were placed on NGM plates containing OP50 or DA837 with or without fatty acid supplement. Scoring was started at L4 stage to young adult.Multi-worm tracking analysis

[0177] The WormLab and the WormLab imaging system from MBF bioscience® was used to automated detection and quantification multiple freely moving young adult worms’ locomotion behavior. On average, 12 young adult animals transferred to a 35 mm NGM plate spread with uniform OP50 as described above, were recorded for 90 mins at 7.5 frames per second (fps). Worm trajectories were extracted from videos and automatically truncated into 10 second intervals. The animals average speed and curvature are analyzed by the Worm Lab software and exported as excel with speed and curvature plots. Two clusters were defined: the roaming zone and the dwelling zone. The clustering boundary7(see, Line A FIG. ID) was defined for wild type worms in standard conditions and was then used for all genotypes and conditions.Pumping rate assays

[0178] Pumping rates of wild type (N2) worms and constitutive AMPK CA-AAK-2 worms on the indicated RNAi bacteria were determined by counting pumps of the terminal pharyngeal bulb for 15 second intervals to determine pumps per min under a Zeiss Stereo Discovery. V12 microscope at lOOx magnification. The pumping rates of 20 animals per condition were determined and averaged to determine the rates indicated in the figures.Phylogenetic analysis of SCT family receptors

[0179] The amino acid sequences of SCT-like family peptides and receptors were downloaded from the GenBank database (www.blast.ncbi.nlm.nih.gov / Blast.cgi), as shown in Table 4. The full set of SCT-like receptor sequences were aligned using MUSCLE as implemented in MEGA vl l.l. The default alignment parameters were applied. And alignments were bootstrapped 1.000 times, and a maximum likelihood phylogenetic tree was constructed using the Jones-Taylor-Thornton model. Trees were unrooted and plotted using MEGA vl l. l.

[0180] To perform protein structure alignment in Chimera X, the C. elegans protein PDFR-1 sequence was submitted to by Alpha Fold3, the prediction confidence value that below 70 was pruned out for future alignment. The command ‘"Matchmaker” tool was used,which superimposes structures by first creating a sequence alignment between the proteins and then fitting the aligned residues based on their spatial positions, typically using the alpha carbon atoms as reference points. The PDB ID for each alignment was indicated in FIG. 21 A.Oil Red O staining

[0181] Oil Red O staining was performed as previously reported with small modification4’5. Briefly, 200-300 dayl adult worms were washed three times with PBS (PH 7.4) and resuspended in 60pl of 1 x PBS. Worms were kept on ice for 10 minutes to stop the pharyngeal activity before fixation. To better fix the worms, 120 pl of 2* MRWB buffer, which contains [160 mM KC1, 40 mM NaCl, 14 mM Na2-EGTA, 1 mM spermidine-HCl, 0.4 mM spermine, 30 mM Na-PIPES [Na-piperazine-N, N'-bis (2-ethane sulfonic acid); pH 7.4], 0.2% P-mercaptoethanol], were added along with 60 pl of 4% paraformaldehyde. Samples were gently rocked for Ih at room temperature and follow ed by three times freeze-thawed circles and washed with PBS. The worms were then dehydrated in 60% isopropyl alcohol for 10 min at room temperature and stained with Oil Red O solution. All Oil Red O images from the same experiment w ere acquired under identical settings and exposure times for direct and fair comparisons. Relative Oil Red O intensities w ere quantified using ImageJ software(NIH). Over 20 worms from each group were randomly selected for quantification. Anterior intestinal cell areas were selected to measure Oil Red O intensities6.Fatty acid supplementation

[0182] The fatty acid supplementation protocol was adapted from a previous study7with several modifications. To facilitate fatty7acid dissolution, the detergent Tergitol (ty pe NP-40, Sigma- Aldrich) was added to a final concentration of 0.001% in liquid NGM agar medium for both non- supplemented and supplemented plates immediately after autoclaving. NGM plates for RNAi experiments were prepared as described, and HT115 bacteria were used for all supplementation experiments unless indicated. A final concentration of ImM was used for all fatty acids in subsequent supplementation experiments. HT115 bacteria expressing the empty vector, or the appropriate RNAi were seeded onto plates containing the respective fatty7acids at room temperature for 24-48 hours before the addition of worms to ensure incorporation of fatty7acids into feeding bacteria.RNA isolation and RT-qPCR in C. elegans

[0183] Worms were collected from a 910cm plate and total RNA was isolated using Trizol (Invitrogen). RNA was quantified by spectrophotometry and lOOng of total RNA was reverse transcribed using a High-Capacity7cDNA Reverse Transcription kit (Applied Biosystems). For each condition, three independent samples were prepared. qPCR was performed using a Step One Plus Real-Time PCR system (Applied Biosystems) with the following PCR conditions: 3 mm at 95 °C. followed by 40 cycles of 5 s at 95 °C and 15 s at 60 °C. Amplified products were detected using SYBR Green (Brilliant III Ultra-Fast SYBR Green qPCR Master Mix, Agilent Technologies). Relative quantification was performed against GAPDH. The primers used are SEQ ID NOs: 1-48.Sample preparation for lipidomics

[0184] To analyze lipid composition using mass spectrometry, wild-type hermaphrodites were treated with control (empty vector) and sbp-1 RNAi full lawn (avoid food leaving behavior) until adult day 1. Each condition consists of 5 biological replicates. The adult worms w ere washed and collected in M9 buffer in a 15-ml tube and allowed to settle to the bottom of the tube. The supernatant w as removed as much as possible, and the samples were snap frozen in the liquid nitrogen and then stored at -80°C for later processing. LC-MS w ater was added to the sample, follow ed by adding around 15 mid-sized bead beater beads to each sample and homogenizing samples with the bead beater at hard mode. Sampleswere mixed well and 400pl homogenized solution was put into an 8ml wide-mouth glass vial. lOul SPLASH® LIPIDOMIX® Mass Spec Standard was added, followed with 1.54 ml methanol, and mixed well. Then 5.16 ml MTBE was added and shaken for 1 hour at room temperature to exact lipids. 880pl water was added and vortexed again for 1 minute. Spinning was performed for 10 minutes at 1,000g. The upper MTBE liquid phase containing the nonpolar lipids was transferred into a new 8ml glass vial. The MTBE extract was dried under a nitrogen stream.LC-MS analysis of lipids profiles

[0185] The samples were re-dissolved in 220 pL of IPA:MeOH (1: 1), and 2 pL was injected to run LC-MS. The experiments were performed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer equipped with Ion Max API source housing with HESI-II probe and Vanquish UHPLC System (ThermoFisher Scientific). The analytes were separated on a Accucore™ Vanquish™ C18+ UHPLC Column (2.1 x 150 mm, 1.5 pm) column operated at 55 °C and a flow rate of 150 pL / min using a 52 min gradient. The mobile phase A was 60:40 (v:v) acetonitrile / water with 10 mM ammonium formate and 0. 1% formic acid and mobile phase B was 90: 10 (v:v) IPA / acetonitrile with 10 mM ammonium formate and 0.1% formic acid1. The raw files were analyzed by LipidSearch5 (Thermo Fisher Scientific). The lipids were identified by matching product ion spectra to the LipidSearch library. Both precursor and product ion mass tolerance were set at 5 ppm and aligned based on retention time tolerance 0.05 min. The data of positive mode were normalized to the internal reference of 18: l(d7) Lyso PC in each sample. The data of negative mode were normalized to the internal references accordingly: The lipid subclass of Cer, CL, FA, HexCer, PA, PG, PI were normalized to the internal reference of 15:0-18: 1 (d7) PG in each sample, the lipid subclass of LPE. PE. PS were normalized to the internal reference of 15:0-18: l(d7) PE in each sample, the lipid subclass of PC, LPC were normalized to the internal reference of 15: 0- 18: 1 (d7) PC in each sample, the lipid subclass of SM were normalized to the internal reference of dl 8 : 1- 18: 1 (d9) SM in each sample3-4. For each sample, the FAME concentration (pg ml-1) was normalized to protein concentration (mg ml-1) as microgram of fatty acid detected per milligram of protein (pgmg-1). In total, 4068 lipids were identified by LipidSearch5. After manually removing the duplicates and checking the peak and product ions, to minimize the false positive results, the final dataset with 2194 lipids was used for further analysis2, with the lipids belonging to 20 lipid classes. Changes in the most abundant lipid classes -triacylglycerol (TG), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) — as well as differences in fatty acyl chain abundances were analyzed. Statistically significant differences between samples were assessed using the two- tailed Wilcoxon test with the Benjamini-Hochberg test for multiple hypothesis correction.Quantification and statistical analysis

[0186] Statistics were computed with GraphPad Prism 9 softw are using two-way ANOVA followed by the Holm-Sidak post-hoc test for dwelling assay or followed by the Bonferroni's multiple comparisons test for the food-leaving assay. Unpaired Student’s t test for comparisons between two groups. For lipidomics analyses, the ratio changes between experimental groups were computed by two-way ANOVA followed by Benjamini, Krieger and Yekutieli post-hoc test. All the data are presented as mean ± s.e.m. p values are denoted in the figures.References for materials and methods1 Wu, Z. et al. Dietary Restriction Extends Lifespan through Metabolic Regulation of Innate Immunity. Cell Metab 29, 1192-1205 el I98, doi: 10.1016 / j.cmet.2019.02.013 (2019).2 Melo, J. A. & Ruvkun. G. Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses. Cell 149, 452-466, doi: 10. 1016 / j.cell.2012.02.050 (2012).3 Flavell, S. W. et al. Serotonin and the neuropeptide PDF initiate and extend opposing behavioral states in C. elegans. Cell 154, 1023-1035, doi: 10. 1016 / j. cell.2013.08.001 (2013).4 Noble, T., Stieglitz, J. & Srinivasan, S. An integrated serotonin and octopamine neuronal circuit directs the release of an endocrine signal to control C. elegans body fat. Cell Metab 18, 672-684, doi:10.1016 / j.cmet.2013.09.007 (2013).5 O'Rourke, E. J., Soukas, A. A., Carr. C. E. & Ruvkun. G. <em>C. elegans< / em> Major Fats Are Stored in Vesicles Distinct from Lysosome-Related Organelles. Cell Metabolism 10, 430-435, doi: 10.1016 / j.cmet.2009.10.002 (2009).6 Lee, J. H. et al. Lipid droplet protein LID-1 mediates ATGL-1 -dependent lipolysis during fasting in Caenorhabditis elegans. Mol Cell Biol 34, 4165-4176, doi: 10. 1128 / MCB.00722-14 (2014).7 Castillo-Quan, J. I. et al. An antisteatosis response regulated by oleic acid through lipid droplet-mediated ERAD enhancement. Science Advances 9, eadc8917, doi:doi: 10.1126 / sciadv.adc8917 (2023).EQUIVALENTS

[0187] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0188] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g.. + / -5- 10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:

1. A method of decreasing food intake in a subject in need thereof comprising:(a) decreasing activity of Inositol-Requiring Enzyme 1 (IRE-1) and / or AMP- Activated Protein Kinase (AMPK) or a signaling molecule downstream thereof; and / or(b) increasing activity of one or more of adipose triglyceride lipase (ATGL1), diglyceride acyltransferase 1 (DGAT1), perilipin-1 (PLIN1), fat storage-inducing transmembrane protein 2 (FITM2), or one or more protein involved in endoplasmic reticulum (ER)-associated degradation (ERAD).

2. The method of claim 1, wherein the subject has obesity, prediabetes, type 2 diabetes, and / or a metabolic syndrome.

3. A method of increasing food intake in a subject in need thereof, comprising:(a) increasing activity of IRE- 1 and / or AMPK or a signaling molecule downstream thereof; and / or(b) decreasing activity of one or more of Lipin 1, ATGLL DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD.

4. The method of claim 3, wherein the administering decreases signaling through the endogenous glucagon-like peptide- 1 receptor (GLP-1R) or another receptor in FIG. 2G and / or FIG. 20A in the brain of the subject.

5. The method of claim 3 or claim 4, wherein the subject has anorexia nervosa and / or avoidant restrictive food intake disorder (ARFID).

6. The method of any one of claims 1-5, wherein the agent is modified to cross the blood brain barrier (BBB).

7. The method of claim 6, wherein the activator or inhibitor is modified by a modification to increase lipid solubility, decrease molecular weight, and / or decrease polarity.

8. The method of claim 7, wherein the modification is capable of increasing the ability' to cross the BBB by 10% or more. 25% or more, or 50% or more.

9. The method of any one of claims 1-8, wherein the signaling molecule downstream of IRE1 and / or AMPK is an RNA acted on by IRE-1, p38, SKN-l(Nrf2), and / or a c-Jun N- terminal kinase (JNK).

10. The method of claim 9, wherein the RNA acted on by IRE-1 is xbp-1 or splicing of xbp-1 RNA.

11. The method of any one of claims 1-10, wherein activity of IRE-1 is measured by:(a) regulated IRE 1 -dependent decay (RIDD) of one or more mRNAs;(b) levels of an RNA acted on by IRE- 1;(c) activation of p38 and SKN-l(Nrl2);(d) IRE-1 autophosphorylation; and / or(e) the JNK signaling cascade.

12. The method of claim 11, wherein the RNA acted on by IRE-1 is xbp-1 or splicing of xbp-1 RNA.

13. The method of any one of claims 1-12, wherein the one or more proteins involved in ERAD comprise osteosarcoma amplified 9, endoplasmic reticulum lectin (OS9), endoplasmic reticulum lectin 1 (ERLEC1), E3 ubiquitin ligase HRD1, and / or SEL1L adaptor subunit Of ERAD E3 ubiquitin ligase (SEL1L).

14. A method of decreasing food aversion and / or nausea in a subject taking a GLP-1- based therapy, comprising administering an agent modified to not cross the BBB, wherein the agent is:(a) an inhibitor of IRE- 1 or AMPK, and / or(b) an activator of Lipinl, ATGL1, DGAT1, PLIN1, FITM2, or one or more proteins involved in ERAD.

15. The method of claim 14, wherein the activator or inhibitor is modified by a modification to reduce lipid solubility, increase molecular weight, and / or increase polarity7.

16. The method of claim 15, wherein the modification is capable of reducing the ability to cross the BBB by 90% or more, 95% or more, or 99% or more.

17. The method of any one of claims 14-16, wherein the GLP-l-based therapy comprises a GLP-1 receptor agonist, dual-acting GLP-1 receptor agonist and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist, amylin agonist, and / or a dual-acting GLP-1 receptor and glucagon receptor (GCGR) agonist.

18. The method of claim 17, wherein the GLP-1 receptor agonist is exenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, and / or semaglutide.

19. The method of claim 17, wherein the dual agonist for GLP-1 receptor and GCGR isBI 456906.

20. The method of claim 17, wherein the amylin agonist is pramlintide and / or cagrilintide.

21. The method of claim 17, wherein the dual-acting GLP-1 receptor agonist and GIP receptor agonist is tirzepatide.

22. A method of decreasing food intake in a subject in need thereof, comprising increasing the levels of monounsaturated fatty acids (MUFAs) relative to saturated fatty acids (SFAs).

23. The method of claim 22, wherein the increasing occurs in the brain.

24. The method of claim 22 or 23, wherein the MUFAs comprise oleic acid and / or palmitoleic acid.

25. The method of any one of claims 22-24, wherein the levels of MUFAs are increased relative to SFAs by treatment with one or more agent to increase activity of stearoyl-CoA desaturase (SCD) and / or elongation of very long chain fatty acids protein 6 (ELOVL6).

26. The method of any one of claims 22-25, wherein increasing the levels of MUFAs relative to SFAs decreases activity of IRE-1 and / or AMPK and / or increases activity' of one or more of Lipin 1, ATGL1. DGAT1. PLIN1, FITM2, or one or more proteins involved in ERAD.

27. A method of identifying a drug target for one or more disorder, comprising evaluating pigment dispersing factor (PDF-1) and / or pigment dispersing factor receptor (PDFR-1) activity in C. elegans.

28. The method of claim 27. wherein the method comprises reducing the expression of a candidate drug target in C. elegans, optionally by RNA interference (RNAi), and determining the expression and / or activity of PDF-1 and / or PDFR-1, wherein increased or reduced expression and / or activity of PDF-1 and / or PDFR-1 where the expression of the candidate drug target is reduced compared to the expression and / or activity where the expression of the candidate drug target is not reduced indicates that the candidate drug target is a drug target for one or more disorder.

29. The method of claim 27 or 28, wherein the activity' of PDF-1 and / or PDFR-1 is measured using promoter activity of PDF-1 and / or behavior of C. elegans.

30. The method of claim 29, wherein the promoter activity is assessed using a fluorescent reporter assay.

31. The method of claim 29, wherein the behavior of C. elegans is mobility' and / or feeding (e.g.. food intake, food leaving, and / or dwelling).

32. The method of claim 31, wherein the method comprises reducing the expression of a candidate drug target in C. elegans, optionally by RNA interference (RNAi), and determining food intake, food leaving, and / or dwelling activity of the C. elegans, wherein increased or reduced food intake, food leaving, and / or dwelling activity of the C. elegans where the expression of the candidate drug target is reduced compared to the food intake, food leaving, and / or dwelling activity where the expression of the candidate drug target is not reduced indicates that the candidate drug target is a drug target for one or more disorder.

33. A method of evaluating an agent as a treatment for one or more disorder, comprising evaluating pigment dispersing factor (PDF-1) and / or pigment dispersing factor receptor (PDFR-1) activity in C. elegans.

34. The method of claim 33. wherein the method comprises administering a candidate agent to C. elegans, and determining the expression and / or activity of PDF-1 and / or PDFR-1 , wherein increased or reduced expression and / or activity of PDF-1 and / or PDFR-1 in the presence of the agent compared to the expression and / or activity' in the absence of the agent indicates that the candidate agent is an agent for treatment of one or more disorder.

35. The method of claim 33 or 34, wherein the activity of PDF-1 and / or PDFR-1 is measured using promoter activity of PDF-1 and / or behavior of C. elegans.

36. The method of claim 35, wherein the promoter activity' is assessed using a fluorescent reporter assay.

37. The method of claim 35. wherein the behavior of C. elegans is mobility and / or feeding (e g., food intake, food leaving, and / or dwelling).

38. The method of claim 37, wherein the method comprises administering a candidate agent to C. elegans, and determining food intake, food leaving, and / or dwelling activity' of the C. elegans. wherein increased or reduced food intake, food leaving, and / or dwelling activity in the presence of the agent compared to the food intake, food leaving, and / or dwelling activity' in the absence of the agent indicates that the candidate agent is an agent for treatment of one or more disorder.

39. The method of any one of claims 33-38, wherein the agent is a small molecule or protein.

40. The method of any one of claims 33-39, wherein the agent is an agonist of any receptor show n in FIG. 2G and / or FIG. 20 A.

41. The method of claim 39 or 40, wherein the agent is a GLP-1 receptor agonist, GIP receptor agonist, amylin agonist, and / or GCGR agonist.

42. The method of claim 39, 40, or 41, wherein the agent binds specifically to more than one of the receptors shown in FIG. 2G and / or FIG. 20A.

43. The method of any one of claims 39-42, wherein the agent binds specifically to more than one of the GLP-1 receptor, GIP receptor, amylin receptor, and glucagon receptor.

44. A method of treating a disorder in a subject, comprising administering one or more agent capable of modulating PDF-1 and / or PDFR-1 activity in C. elegans to the subject, optionally wherein the agent is identified by a method as described in any one of claims 33- 43.

45. The method of claim 44. wherein the disorder is anorexia nervosa and / or ARFID.

46. The method of claim 44, wherein the disorder is obesity, prediabetes, ty pe 2 diabetes, or a metabolic syndrome.

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