Fibroblast growth factor 21 (FGF21) is the first known endocrine signal activated by protein restriction. Although FGF21 is robustly elevated in low-protein environments, increased FGF21 is also seen in various other contexts such as fasting, overfeeding, ketogenic diets, and high-carbohydrate diets, leaving its nutritional context and physiological role unresolved and controversial. Here, we use the Geometric Framework, a nutritional modeling platform, to help reconcile these apparently conflicting findings in mice confined to one of 25 diets that varied in protein, carbohydrate, and fat content. We show that FGF21 was elevated under low protein intakes and maximally when low protein was coupled with high carbohydrate intakes. Our results explain how elevation of FGF21 occurs both under starvation and hyperphagia, and show that the metabolic outcomes associated with elevated FGF21 depend on the nutritional context, differing according to whether the animal is in a state of under- or overfeeding.

BACKGROUNDG protein-coupled inwardly rectifying potassium (KIR3) channels are important proteins that regulate numerous physiological processes including excitatory responses in the CNS and the control of heart rate. Flavonoids have been shown to have significant health benefits and are a diverse source of compounds for identifying agents with novel mechanisms of action. EXPERIMENTAL APPROACHThe flavonoid glycoside, naringin, was evaluated on recombinant human KIR3.1-3.4 and KIR3.1-3.2 expressed in Xenopus oocytes using two-electrode voltage clamp methods. In addition, we evaluated the activity of naringin alone and in the presence of the KIR3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant KIR3.1-3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1-M2 loop of the KIR3.1 F137S mutant channel important for naringin's activity. KEY RESULTSNaringin (100 mM) had minimal effect on uninjected oocytes but activated KIR3.1-3.4 and KIR3.1-3.2 channels. The activation by naringin of KIR3.1-3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the KIR3.1 F137S mutant channel, replacing one by one aromatic amino acids within the M1-M2 loop, identified tyrosines 148 and 150 to be significantly contributing to the affinity of naringin as these mutations reduced the activity of naringin by 20-and 40-fold respectively. CONCLUSIONS AND IMPLICATIONSThese results show that naringin is a direct activator of KIR3 channels and that tertiapin-Q shares an overlapping binding site on the KIR3.1-3.4. This is the first example of a ligand that activates KIR3 channels by binding to the extracellular M1-M2 linker of the channel. AbbreviationsCGP36742 or SGS742, 3-aminopropyl-n-butylphosphinic acid; GPCRs, G protein-coupled receptors; KIR3, G proteincoupled inwardly rectifying potassium channels; LPA, lysophosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; r KIR1.1, rat renal outer medullary potassium; TPN-Q, tertiapin-Q

Background and Purpose: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties.Experimental Approach: We used the Scn1a +/À mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a +/À mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets.

Introduction: Cannabis sativa produces hundreds of bioactive compounds, including cannabinoids and terpenoids. It has been proposed that cannabinoids act in synergy with terpenoids to produce the entourage effect, a concept used to explain the therapeutic benefits of medicinal cannabis. One molecular explanation for the entourage effect is that the terpenoids augment the actions of cannabinoids at their molecular drug targets in cells. We recently reported that terpenoids commonly found in cannabis do not influence the functional effects of D 9 -tetrahydrocannabinol (D 9 -THC) on cannabinoid 1 and cannabinoid 2 receptors. The present study aimed to extend on this research by examining whether terpenoids influence the effects of phytocannabinoids and endocannabinoids on human transient receptor potential ankyrin 1 (hTRPA1) and human transient receptor potential vanilloid 1 (hTRPV1) channels heterologously expressed in mammalian cells. Materials and Methods: The activity of terpenoids, phytocannabinoids, and endocannabinoids was assessed in inducible HEK Flp-In T-Rex cells transfected with hTRPA1 and hTRPV1 channels, respectively. Real-time changes in intracellular calcium ([Ca] i ) were measured using the Calcium 5 dye and a FlexStation 3 plate reader. Results: a-pinene, b-pinene, b-caryophyllene, linalool, limonene, b-myrcene or a-humulene did not affect [Ca] i in hTRPA1 and hTRPV1 overexpressing cells. Cinnamaldehyde (CA), D 9 -THC, and 2-arachidonoylglycerol (2-AG) activated TRPA1 receptors with high efficacy and similar potency (EC 50 s of *10 lM). Capsaicin and anandamide (AEA) activated TRPV1 receptors with an EC 50 of 61 nM and 4.3 lM, respectively, but TRPV1 showed no response to D 9 -THC, cannabidiol, and other minor cannabinoids. Terpenoids did not significantly affect the responses of TRPA1 and TRPV1 receptors to submaximal and maximal concentrations of CA and D 9 -THC or the endocannabinoids AEA and 2-AG. Discussion: We could not find any evidence that the terpenoids tested here activate TRPA1 and TRPV1 channels or modulate their activation by D 9 -THC and other agonists, including endocannabinoids.