Levels of Cocaine- and Amphetamine-Regulated Transcript in Vagal Afferents in the Mouse Are Unaltered in Response to Metabolic Challenges

We have previously demonstrated that vagal afferent neurons switch neuropeptide expression depending on feeding state 1-4. In vivo, vagal afferent neurons of fasted animals express melanin concentrating hormone (MCH) 2, while refeeding reduces MCH expression and increases expression of cocaine and amphetamine regulated transcript (CART) 1,2. Contrary to our previous findings, Yuan et al 5 report constitutive CART expression and a complete absence of MCH expression in vagal afferent neurons in ad libitum fed compared to fasted animal.

Critically, the feeding paradigm used by Yuan et al 5 was inadequate to observe changes in CART or MCH expression in vagal afferent neurons. In this study mice and rats were either fasted overnight or fed ad libitum and euthanized 3-5 hours into the light phase. Given that fed ad libitum rodents consume most of their calories in the dark phase with almost no intake in the initial period of the light phase, the animals identified as fed are unlikely to have consumed any food for at least 3-5 hours. In contrast, we expose our animals to a longer 24-48h fast on wire-bottom cages to ensure that the stomach is empty, and re-feed half of the fasted animals ad libitum for 2 hours until satiated. This is important since the mechanisms that promote CART and depress MCH expression in vagal afferent neurons requires postprandial release of the gastrointestinal hormone, cholecystokinin (CCK). In support of this, CCK-A receptor encoding nodose ganglia neurons co-express with CART6 and MCH2. Furthermore, administration of exogenous CCK in fasted animals increases CART 1 and depresses MCH 2; while, CCK-A receptor antagonist injection in re-fed animals prevents postprandial increase in CART 1 and decrease in MCH 2 expression. In culture, CCK increases CART promoter activation and CART release 7 and within the same cultured vagal afferent neuron MCH expression can be switched to CART within a couple hours following CCK administration 1. Therefore it is likely that in Yuan et al 5 the CART staining in nodose ganglion looked identical in fed, fasted, and obese animals because all the animals were at various points on a fasted spectrum, and perhaps not fasted long enough for MCH to be expressed.

Yuan et al 5 frequently refer to the fact that their results are in agreement with a previous report by Broberger et al 6 showing no change in CART mRNA in response to food restriction or diet-induced obesity. However Broberger et al did not fast their animals but rather restricted rats to 10g of chow intake per day for 14 days with no indication of when the animals had last eaten prior to euthanasia6. Therefore this neither challenges nor supports the role of feeding and fasting in the control of vagal CART expression. Although Broberger et al found no effect of high fat diet feeding in rats on CART expression in vagal afferent neurons6, it should be noted that these rats were only kept on the diet for 2 weeks. At this time-point rats do not weigh significantly more than lean controls and can therefore not be referred to as diet-induced obese 8-11. Yuan et al quantified CART expression in the nodose ganglia of mice fed a 60% high fat diet for 4 weeks5. No information was provided about the weight gain of these animals in comparison to age matched lean controls. In our previous studies rats were fed an obesigenic diet composed of lower fat content (45% kcal from fat) and higher sucrose content for 8 weeks, at which time these rats weighed significantly more than control littermates maintained on chow 8,12. Therefore even if Yuan et al5 had used a re-feeding paradigm, it is unclear that a blunted CART expression would have been observed in mice maintained on a high-fat low-sugar diet for only 4 weeks.

It is inaccurate and misleading to refer to the change in CART and MCH expression in vagal afferent neurons as a unique event. Despite being a largely understudied tissue, there have been several examples of stimuli-mediated changes in neuropeptide expression in vagal afferent neurons. Allergic inflammation in guinea pig airways increases the expression of the neuropeptides substance P and CGRP within neurons of the nodose ganglia13. Axonal damage of the vagus nerve increases expression of the neuropeptides galanin, NPY, VIP and CCK, and decreases CCK-1 receptor expression while increasing expression of CCK2 and Y2 receptors in vagal afferent neurons 14-16. Furthermore, metabolic cues from the gut have been extensively reported to alter gene expression in vagal afferent neurons. Expression of clock genes in the nodose ganglia oscillate throughout the day, and are entrained by food intake 17. In lean rats, fasting increases expression of melanin concentrating hormone receptor 12, cannabinoid receptor 1 18, and ghrelin receptor 19. Re-feeding reduces expression of these receptors and promotes expression of Y2 receptor 20,21. Chronic consumption of high fat diets alters mRNA expression of ghrelin receptor 11,19, CB1 receptor 8,22, MCH1 receptor 8, orexin receptor 22, Y2 receptor 8, PPAR-gamma receptor 23, CCK-1 receptor 11,22, GPR4024, GPR41 24, and GPR1 20,24. Given the propensity of vagal afferent neurons to change their neurochemistry in response to peripheral signals, a change in neuropeptide expression in response to nutrient signaling is in keeping with previous findings.

In summary, the experimental protocol used in Yuan et al 5 is not appropriate to determine neuropeptidergic profile changes in vagal afferent neurons in response to feeding or metabolic state. Crucially the time of last meal and the quantity of food ingested was not controlled in these experiments. However the data by Yuan et al5 suggests that satiation itself is not sufficient to drive the change in neuropeptide expression, since CART and MCH expression are unchanged in animals that chose not to eat despite having access to food during the light phase. A hypothesis that unifies our previous data with those of Yuan et al is that changes in neuropeptide expression is driven by nutrient induced activation of vagal afferent neurons rather than a physiological state that arises from the nutrient consumption.

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