Abstract
Appetite loss or anorexia substantially deteriorates quality of life in various diseases, and stand upstream of frailty. Neuropeptide Y (NPY) in the hypothalamic arcuate nucleus (ARC) and ghrelin released from stomach are potent inducers of appetite. We previously reported that Ninjin'yoeito, a Japanese kampo medicine comprising twelve herbs, restores food intake, and body weight in cisplatin-treated anorectic mice. Furthermore, Ninjin'yoeito increased cytosolic Ca2+ concentration ([Ca2+]i) in not only ghrelin-responsive but ghrelin-unresponsive NPY neurons in ARC. The cellular lineage/differentiation of ghrelin-unresponsive neuron is less defined but might alter along with aging and diet. This study examined the occupancy of ghrelin-unresponsive neurons among ARC NPY neurons in adult mice fed normal chow, and explored the mechanisms underlying Ninjin'yoeito-induced [Ca2+]i increases in ghrelin-unresponsive vs. ghrelin-responsive NPY neurons. Single ARC neurons were subjected to [Ca2+]i measurement and subsequent immunostaining for NPY. Ghrelin failed to increase [Ca2+]i in 42% of ARC NPY neurons. Ninjin'yoeito (10 μg/ml)-induced increases in [Ca2+]i were abolished in Ca2+ free condition in ghrelin-responsive and ghrelin-unresponsive ARC NPY neurons. Ninjin'yoeito-induced [Ca2+]i increases were inhibited by N-type Ca2+ channel blocker ω-conotoxin in the majority (17 of 20), while by L-type Ca2+ channel blocker nitrendipine in the minority (2 of 23), of ghrelin-responsive neurons. In contrast, Ninjin'yoeito-induced [Ca2+]i increases were inhibited by nitrendipine in the majority (14 of 17), while by ω-conotoxin in the minority (8 of 24), of ghrelin-unresponsive neurons. These results indicate that ghrelin-unresponsive neurons occur substantially among NPY neurons of ARC in adult mice fed normal chow. Ninjin'yoeito preferentially target N-type and L-type Ca2+ channels in the majority of ghrelin-responsive and ghrelin-unresponsive neurons, respectively, to increase [Ca2+]i. We suggest ARC N- and L-type Ca2+ channels as potential targets for activating, respectively, ghrelin-responsive, and unresponsive NPY neurons to treat anorexia.
Highlights
Reduced appetite and body weight are associated with cancer, sarcopenia and frailty [1, 2], and deteriorate the quality of life (QOL) [3, 4]
Ninjin’yoeito-induced [Ca2+]i increases were inhibited by N-type Ca2+ channel blocker ω-conotoxin in the majority (17 of 20), while by L-type Ca2+ channel blocker nitrendipine in the minority (2 of 23), of ghrelin-responsive neurons
Among 43 arcuate nucleus (ARC) Neuropeptide Y (NPY) neurons, 25 (58%) neurons responded to ghrelin and 18 (42%) neurons did not respond to ghrelin (Figure 1C)
Summary
Reduced appetite and body weight are associated with cancer, sarcopenia and frailty [1, 2], and deteriorate the quality of life (QOL) [3, 4]. Effective means to promote appetite have been awaited. Ninjin’yoeito, a Japanese traditional Kampo medicine, has been used clinically and demonstrated to be effective to treat anorexia, fatigue, anemia, cold limbs, persistent cough, mental disequilibrium, and to promote recovery from disease [5,6,7]. Ninjin’yoeito comprises 12 crude drugs (Table 1). Some of these crude drugs are known to pass through the blood brain barrier (BBB) and possibly access to the hypothalamus including the arcuate nucleus (ARC), while ghrelin, an orexigenic gut hormone, freely accesses to the ARC neurons without crossing the BBB [8]. Oral administration of Ninjin’yoeito could act on ARC via one or both of these routes
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