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RXFP3 (relaxin-family peptide 3 receptor) is the cognate G-protein-coupled receptor for the neuropeptide, relaxin-3. RXFP3 is expressed widely throughout the brain, including the hypothalamus, where it has been shown to modulate feeding behavior and neuroendocrine activity in rodents. In order to better characterize its potential mechanisms of action, this study determined whether RXFP3 is expressed by dopaminergic neurons within the arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), in addition to the ventral tegmental area (VTA). Neurons that express RXFP3 were visualized in coronal brain sections from RXFP3-Cre/tdTomato mice, which express the tdTomato fluorophore within RXFP3-positive cells, and dopaminergic neurons in these areas were visualized by simultaneous immunohistochemical detection of tyrosine hydroxylase-immunoreactivity (TH-IR). Approximately 20% of ARC neurons containing TH-IR coexpressed tdTomato fluorescence, suggesting that RXFP3 can influence the dopamine pathway from the ARC to the pituitary gland that controls prolactin release. The ability of prolactin to reduce leptin sensitivity and increase food consumption therefore represents a potential mechanism by which RXFP3 activation influences feeding. A similar proportion of DMH neurons containing TH-IR expressed RXFP3-related tdTomato fluorescence, consistent with a possible RXFP3-mediated regulation of stress and neuroendocrine circuits. In contrast, RXFP3 was barely detected within the VTA. TdTomato signal was absent from the ARC and DMH in sections from Rosa26-tdTomato mice, suggesting that the cells identified in RXFP3-Cre/tdTomato mice expressed authentic RXFP3-related tdTomato fluorescence. Together, these findings identify potential hypothalamic mechanisms through which RXFP3 influences neuroendocrine control of metabolism, and further highlight the therapeutic potential of targeting RXFP3 in feeding-related disorders.

TRPA1 is a ligand-activated cation channel found in the intestine and other tissues. Components of food that stimulate TRPA1 receptors (phytonutrients) include allyl isothiocyanate, cinnamaldehyde and linalool, but these may also act at other receptors. Cells lining the intestinal mucosa are immunoreactive for TRPA1 and Trpa1 mRNA occurs in mucosal extracts, suggesting that the TRPA1 receptor is the target for these agonists. However, in situ hybridisation reveals Trpa1 expression in 5-HT containing enteroendocrine cells, not enterocytes. TRPA1 agonists evoke mucosal secretion, which may be indirect (through release of 5-HT) or direct by activation of enterocytes. We investigated effects of the phytonutrients on transmucosal ion currents in mouse duodenum and colon, and the specificity of the phytonutrients in cells transfected with Trpa1, and in Trpa1-deficient mice. The phytonutrients increased currents in the duodenum with the relativepotencies: allyl isothiocyanate (AITC) > cinnamaldehyde > linalool (0.1 to 300 µM). The rank order was similar in the colon, but linalool was ineffective. Responses to AITC were reduced by the TRPA1 antagonist HC-030031 (100 µM), and were greatly diminished in Trpa1-/- duodenum and colon. Responses were not reduced by tetrodotoxin, 5-HT receptor antagonists, or atropine, but inhibition of prostaglandin synthesis reduced responses. Thus, functional TRPA1 channels are expressed by enterocytes of the duodenum and colon. Activation of enterocyte TRPA1 by food components has the potential to facilitate nutrient absorption.

AIM: To determine if manipulation of dietary advanced glycation end product (AGE), intake affects non-alcoholic fatty liver disease (NAFLD) progression and whether these effects are mediated via RAGE. METHODS: Male C57Bl6 mice were fed a high fat, high fructose, high cholesterol (HFHC) diet for 33 wk and compared with animals on normal chow. A third group were given a HFHC diet that was high in AGEs. Another group was given a HFHC diet that was marinated in vinegar to prevent the formation of AGEs. In a second experiment, RAGE KO animals were fed a HFHC diet or a high AGE HFHC diet and compared with wildtype controls. Hepatic biochemistry, histology, picrosirius red morphometry and hepatic mRNA were determined. RESULTS: Long-term consumption of the HFHC diet generated significant steatohepatitis and fibrosis after 33 wk. In this model, hepatic 4-hydroxynonenal content (a marker of chronic oxidative stress), hepatocyte ballooning, picrosirius red staining, α-smooth muscle actin and collagen type 1A gene expression were all significantly increased. Increasing the AGE content of the HFHC diet by baking further increased these markers of liver damage, but this was abrogated by pre-marination in acetic acid. In response to the HFHC diet, RAGE(-/-) animals developed NASH of similar severity to RAGE(+/+) animals but were protected from the additional harmful effects of the high AGE containing diet. Studies in isolated Kupffer cells showed that AGEs increase cell proliferation and oxidative stress, providing a likely mechanism through which these compounds contribute to liver injury. CONCLUSION: In the HFHC model of NAFLD, manipulation of dietary AGEs modulates liver injury, inflammation, and liver fibrosis via a RAGE dependent pathway. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD.