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L-alanine is a good indicator of dietary protein intake as it is abundant in most protein rich foods and its concentration increases rapidly after a meal in both blood plasma and the hypothalamus (Choi et al. 1999, Choi et al. 2000, Choi et al. 2001). The results of the food intake and metabolism experiments described in this thesis point towards a potential role for L-alanine detection in the control of energy metabolism.

Although the most direct experiment on the effects of L-alanine on food intake, where rats were supplemented with Ala after a period of fasting and their food consumption was monitored, showed no involvement of Ala in refeeding or weight gain, the other parts of the study provided several instances when the importance of Ala could be established. Firstly, both fasting and essential amino acid starvation increased tanycyte sensitivity to Ala; secondly, mice starved of essential amino acids reduced their Ala intake; and lastly, a diet enhanced in Ala reduced light phase feeding and increased metabolic rate, but mice that had been starved of essential amino acids exhibited these effects to a lesser extent. Increased tanycyte sensitivity to Ala could have mediated the latter two effects, or, otherwise, all the observed effects may have been influenced by the same factor. For example, a global upregulation of mGluR4 would affect both tanycyte responses to Ala and oral Ala intake due to perceived stronger taste. Either way, combined with the fast rate of post-meal changes in blood and brain Ala levels, these results clearly demonstrate that Ala is an important dietary signal

The strong Ala-associated satiety signal is not necessarily a positive one; the reduction in Ala intake observed in the above described experiments, as well as

Tas1r1-null avoidance of Ala-enriched water seen in the second 2-bottle preference test, suggest an aversive effect. Indeed, in both rodents and

humans, extensive satiety eventually leads to leptin resistance (Frederich et al. 1995). Moreover, leptin receptor and its downstream signalling pathway share a lot of similarities with detection mechanisms for some inflammatory markers (Anesten et al. 2017). Satiety also reduces motivation and inhibits the reward pathway (Hommel et al. 2006), which is not beneficial for animals. While satiety induced by high Ala sensitivity most likely does not involve increased leptin secretion, it is likely that rodents could associate feeling excessively satiated with the negative effects of leptin, and therefore would learn to avoid Ala. Although humans and rodents have different dietary needs, feeding patterns and amino acid receptor sensitivity profiles, their neuroendocrine systems responsible for energy homeostasis are regarded as remarkably similar. Interestingly, one human study has shown that L-alanine supplementation reduced subsequent food intake in healthy men (Rogers and Blundell 1994). These findings have unfortunately not received much attention and therefore were never repeated, so the exact role of L-alanine in human energy

homeostasis is yet to be revealed.

5.4. Potential role for amino acid sensing in tanycyte neurogenesis

Hypothalamic tanycytes form one of the three currently known neurogenic niches in the adult rodent brain (Lee et al. 2012). Changes in diet have been repeatedly reported to influence hypothalamic neurogenesis (Kokoeva et al. 2005, Li et al. 2014). The newly produced neurons migrate from the wall of 3V into the arcuate nucleus where they are integrated in the POMC/CART and NPY/AgRP circuits (Pierce and Xu 2010).

Neural stem cell proliferation can be induced by P2Y receptor signalling (Lin et al. 2007). The results acquired in the current project and a previous study in our laboratory show that P2Y1 is one of the receptors in tanycytes that is activated

could be speculated that chronic tanycyte activation with amino acids or glucose over a long time could lead to a higher probability of cell proliferation. Receiving a high-fat diet for an extended period of time leads to an increase in POMC neuron generation in the male mouse hypothalamus, which may be associated with the G-protein-coupled receptor 40 (Nascimento et al. 2016). In female mice, the effects of high-fat and low-protein diets on neurogenesis are region-specific: tanycyte proliferation is reduced in the arcuate nucleus, while the number of new cells is increased in the median eminence (Lee et al. 2014). It is not yet clear how a low-protein diet could affect tanycyte proliferation in female mice only. However, considering the data presented in this thesis, it could be speculated that tanycyte amino acid sensing via umami taste receptors could be involved in the maintenance of normal levels of neurogenesis in the hypothalamus, and that a reduction in protein content in the diet would alter the number of newly generated neurons in order to adapt to such changes.

Direct tanycyte-to-neuron signalling about amino acid availability together with amino acid and ATP-induced tanycyte proliferation into arcuate neurons could represent a short-term and a long-term role for tanycytes in the control of energy homeostasis.