Functional characterization of all missense variants in , and genes arising from single-nucleotide variants.

Objective: Hyperphagia and early-onset, severe obesity are clinical characteristics of rare melanocortin-4 receptor (MC4R) pathway diseases due to loss-of-function (LOF) variants in genes comprising the MC4R pathway. In vitro functional characterization of 12,879 possible exonic missense variants from single-nucleotide variants (SNVs) of , and was performed to determine the impact of these variants on protein function.

Methods: SNVs of the three genes were transiently transfected into cell lines, and each variant was subsequently classified according to functional impact.

A rapidly acting glutamatergic ARC→PVH satiety circuit postsynaptically regulated by α-MSH.

Arcuate nucleus (ARC) neurons sense the fed or fasted state and regulate hunger. Agouti-related protein (AgRP) neurons in the ARC (ARC neurons) are stimulated by fasting and, once activated, they rapidly (within minutes) drive hunger. Pro-opiomelanocortin (ARC) neurons are viewed as the counterpoint to ARC neurons.

Dynamic GABAergic afferent modulation of AgRP neurons.

Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) promote homeostatic feeding at times of caloric insufficiency, yet they are rapidly suppressed by food-related sensory cues before ingestion. Here we identify a highly selective inhibitory afferent to AgRP neurons that serves as a neural determinant of this rapid modulation. Specifically, GABAergic projections arising from the ventral compartment of the dorsomedial nucleus of the hypothalamus (vDMH) contribute to the preconsummatory modulation of ARC neurons.

A neural basis for melanocortin-4 receptor-regulated appetite.

Pro-opiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus (ARC) are oppositely regulated by caloric depletion and coordinately stimulate and inhibit homeostatic satiety, respectively. This bimodality is principally underscored by the antagonistic actions of these ligands at downstream melanocortin-4 receptors (MC4R) in the paraventricular nucleus of the hypothalamus (PVH). Although this population is critical to energy balance, the underlying neural circuitry remains unknown.

A parabrachial-hypothalamic cholecystokinin neurocircuit controls counterregulatory responses to hypoglycemia.

Hypoglycemia engenders an autonomically mediated counterregulatory (CR)-response that stimulates endogenous glucose production to maintain concentrations within an appropriate physiological range. Although the involvement of the brain in preserving normoglycemia has been established, the neurocircuitry underlying centrally mediated CR-responses remains unclear. Here we demonstrate that lateral parabrachial nucleus cholecystokinin (CCK(LPBN)) neurons are a population of glucose-sensing cells (glucose inhibited) with counterregulatory capacity.

MC4R-expressing glutamatergic neurons in the paraventricular hypothalamus regulate feeding and are synaptically connected to the parabrachial nucleus.

Activation of melanocortin-4 receptors (MC4Rs) restrains feeding and prevents obesity; however, the identity, location, and axonal projections of the neurons bearing MC4Rs that control feeding remain unknown. Reexpression of MC4Rs on single-minded 1 (SIM1)(+) neurons in mice otherwise lacking MC4Rs is sufficient to abolish hyperphagia. Thus, MC4Rs on SIM1(+) neurons, possibly in the paraventricular hypothalamus (PVH) and/or amygdala, regulate food intake.

An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger.

Hunger is a hard-wired motivational state essential for survival. Agouti-related peptide (AgRP)-expressing neurons in the arcuate nucleus (ARC) at the base of the hypothalamus are crucial to the control of hunger. They are activated by caloric deficiency and, when naturally or artificially stimulated, they potently induce intense hunger and subsequent food intake.

Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators GABA, NPY, and AgRP.

Agouti-related peptide (AgRP) neurons of the hypothalamus release a fast transmitter (GABA) in addition to neuropeptides (neuropeptide Y [NPY] and Agouti-related peptide [AgRP]). This raises questions as to their respective functions. The acute activation of AgRP neurons robustly promotes food intake, while central injections of AgRP, NPY, or GABA agonist results in the marked escalation of food consumption with temporal variance.

Activation of oral trigeminal neurons by fatty acids is dependent upon intracellular calcium.

The chemoreception of dietary fat in the oral cavity has largely been attributed to activation of the somatosensory system that conveys the textural properties of fat. However, the ability of fatty acids, which are believed to represent the proximate stimulus for fat taste, to stimulate rat trigeminal neurons has remained unexplored. Here, we found that several free fatty acids are capable of activating trigeminal neurons with different kinetics.

Targeted biodegradable nanoparticles for drug delivery to smooth muscle cells.

Targeted delivery of therapeutic agents to prevent smooth muscle cell (SMC) proliferation is important in averting restenosis (a narrowing of blood vessels). Since platelet derived growth factor (PDGF) receptors are over-expressed in proliferating SMCs after injury from cardiovascular interventions, such as angioplasty and stent implantation, our hypothesis is that conjugation of PDGF-BB (platelet-derived growth factor BB (homodimer)) peptides to biodegradable poly (D,L-lactic-co-glycolide) (PLGA) nanoparticles (NPs) would exhibit an increased uptake of these NPs by proliferating SMCs. In this study, poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles containing dexamethasone were formulated and conjugated with PDGF-BB peptides.

Fasting activation of AgRP neurons requires NMDA receptors and involves spinogenesis and increased excitatory tone.

AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons.

TRPM5 is critical for linoleic acid-induced CCK secretion from the enteroendocrine cell line, STC-1.

Fatty acid-induced stimulation of enteroendocrine cells leads to release of the hormones such as cholecystokinin (CCK) that contribute to satiety. Recently, the fatty acid activated G protein-coupled receptor GPR120 has been shown to mediate long-chain unsaturated free fatty acid-induced CCK release from the enteroendocrine cell line, STC-1, yet the downstream signaling pathway remains unclear. Here we show that linoleic acid (LA) elicits membrane depolarization and an intracellular calcium rise in STC-1 cells and that these responses are significantly reduced when activity of G proteins or phospholipase C is blocked.

Transient receptor potential channel type M5 is essential for fat taste.

Until recently, dietary fat was considered to be tasteless, and its primary sensory attribute was believed to be its texture (Rolls et al., 1999; Verhagen et al., 2003).