The brain and peripheral nervous system have an essential role in controlling energy homeostasis. Multiple neuronal mechanisms control various parameters of metabolism, such as appetite, food intake, food preference, energy expenditure, insulin secretion, glucose production or glucose/lipid metabolism, with many of these processes requiring interactions between different brain regions or between the brain and peripheral organs. This highly coordinated interactions are mediated via secretion of signaling peptides from neurons, termed neuropeptides. It is well documented that inherited or acquired alterations of these neuropeptides have a crucial role in development of obesity or obesity-related metabolic disorders.
Neuromedins are a group of neuropeptides consisting of several different peptides with varying degrees of structural similarity. It has been reported that neuromedins are involved in a wide range of physiological processes, including smooth muscle contraction, immunity, stress responses, breathing, nociception and energy homeostasis. Due to their potential role in controlling weight gain, some of these neuropeptides have been suggested as anti-obesity strategies. Neuromedin B (NMB) belongs to the bombesin-like neuromedin subgroup. In humans NMB is highly expressed in the hypothalamus, cerebral cortex and hippocampus, with lower expression in intestine, pancreas, and adrenal glands. High NMB expression has also been reported in the olfactory bulb of pigs, rats and mice. Neuromedin B receptor (NMBR) is a member of the G protein-coupled receptor (GPCR) family with broad expression in the CNS (caudate nucleus, amygdala, thalamus, hippocampus, brain stem, hypothalamus, spinal cord and olfactory region) as well as peripheral tissues such as testis, urogenital smooth muscles, gastrointestinal system, esophagus and adipose tissues, suggesting that its activation upon NMB binding might regulate multiple physiological functions.
Several human GWAS studies of obese patients have proposed a link between single nucleotide polymorphisms in the coding region of NMB to increased body weight, due to alterations in appetite control and food preference. Moreover, in vitro studies have shown that NMB stimulates insulin release from perfused rat and canine pancreata. These data suggested that NMB might act as an important modulator of body weight gain and metabolism, and thus might be considered as a promising therapeutic option for metabolic diseases. Given that a detailed functional in vivo study addressing these potential functions had never been performed and that NMB is predominantly expressed in the nervous system, we therefore generated neuron-specific Nmb mutant mice (NestinCre-Nmb flox/flox). We hypothesized that in these mice, loss of Nmb would alter their feeding behavior causing obesity or metabolic disease, as proposed by human GWAS studies.
To investigate the impact of loss of NMB in neurons, we initially generated Nmb flox/flox mice using the CRISPR-Cas9 system. Six founders were identified and the correct integration of LoxP sites was confirmed by PCR analysis and Sanger sequencing (not shown). To further generate Nmb global neuronal mutant mice, the Nmb flox/flox mice were crossed with Nestin-Cre transgenic mice, as this Cre driver has high global deletion efficiency in neurons. In NestinCre -Nmb flox/flox animals, Nmb expression was undetectable in neuronal tissues known to have high Nmb expression, such as olfactory bulb, hippocampus and hypothalamus, thus confirming the successful deletion of Nmb in neuronal tissues. We have also observed efficient deletion of Nmb in dorsal root ganglia (DRG) and spinal cord neurons (Supplementary Fig.1B).
To assess the role of NMB in weight regulation, we first monitored NestinCre-Nmb KO termed NestinCre-Nmb flox/flox) mice for weight gain under standard diet (STD). Adult 6 month old NestinCre-Nmb KO mice were found to be slightly smaller than their Nmb expressing littermates (C56BL/6 J 29.4 ± 2.64; Nmb flox/flox 29.44 ± 2.25 g; NestinCre -Nmb+/+ 26.29 ± 1.49 g; NestinCre-Nmb flox/flox 25.96 ± 1.42 g).
