Wight gain with advancing age is controlled by the muscarinic acetylcholine receptor M4 in male mice

Obesity is characterized by the excessive accumulation of adipose tissue and considered to be a serious global health issue. Therefore, understanding the pathology of obesity is important for identifying a signal. Here we used knockout (KO) mice to delete chrm4, so far well known as regulation of key functions of the central and peripheral nervous system. M4-KO male mice resulted in weight gain and higher accumulation of white adipose (WAT) tissue with advancing age compared to wild-type (WT) mice. Chrm4 mutants showed up-regulation of leptin gene and its protein relative to WT mice. RNA-sequencing and quantitative PCR of subcutaneous adipose tissues revealed that the WAT marker genes were significantly enhanced in M4-KO mice. In contrast, brown adipose tissue and/or beige adipose tissue markers were strongly decreased in the animals. We also generated Chrm4-mScarlet knock-in (M4-KI) reporter mice and examined the localization of mScarlet fluorescent signals in subcutaneous tissues. The signals could be detected in WAT cells in the KI mice. Moreover, acetylcholine (ACh) could be quantitatively measured with LC-MS/MS in subcutaneous tissues. Collectively, our data provide evidence that endogenous ACh released from subcutaneous tissue cells maintains homeostasis of adipose cell growth and differentiation via M4 in male mice.