The Mechanism Of Obesity Prevention Through Activating Cold Sensitive Channels(TRPM8) In Brown Adipose Tissue

Background and purpose:The morbidity of obesity has been growing worldwide with the gradual living standards improvement. More energy intake compared with consumption is regarded as the key point for obesity generation. Therefore, consuming energy effectively has become a new strategy for obesity prevention and treatment. Human body has two kinds of adipose tissues, one is the white adipose tissue(WAT) and the other is brown adipose tissue(BAT). WAT is mainly responsible for energy storage, while BAT is key to energy consumption. Apart from rodents and human babies, functional BAT has been detected in adult in recent years. Meanwhile, it has been demonstrated that chronic cold stimulation c ould improve heat production and decease fat accumulation through upregulating the uncoupling mitochondrial protein 1(UCP1). Therefore, the strategy and molecular mechanism of BAT in regulating heat production have become meaningful in obesity prevention.Thermosensitive Transient Receptor Potential TRP channels(Thermo-TRPs) feeling ambient temperature changes exist in mammalian body. Transient receptor potential melastatin 8(TRPM8) can be activated by low temperature below 27℃ and menthol, also known as the “ Cold Channel ”. It has the characteristics of nonselective cation channel and sensitive to calcium and sodium ions. Apart from temperature and pain sensory neurons, TRPM8 has been detected in BAT of mice in recent years. TRPM8-knockout mice is not sensitive to cold stimulation, suggesting this "cold channel" in mammals plays an important role to sensing the ambient temperature. Furthermore, menthol is a common spice and additive, as well as the TRPM8 natural agonist. Our previous research found that TRPM8 was expressed in vascular tissue, TRPM8 activation by menthol could inhibit vasoconstriction of blood vessels and control blood pressure. It has also been reported that BAT also have TRPM8 expression, but its physiological function is not clear yet. Thus, we speculate that TRPM8 activation in BAT may be associated with energy metabolism and heat production. UCP1 in BAT mitochondria play vital roles in respiratory chain uncoupling and thermogenesis of BAT. So we put forward the hypothesis that menthol influence the expression of UCP1 and its related molecules by activating TRPM8 and regulating intracellular Ca2+, thus increasing BAT thermogenesis and preventing obesity.In order to verify the above hypothesis, our work is divided into three parts. The first part: We confirmed the expressions and distributions of TRPM8 and UCP1 in mice BAT and brown adipocytes through Western blot and immunofluorescence staining, and then clarified the influence of menthol intervention on their expressions. Verifying whether the TRPM8 channel on the brown adipocytes has physiological function through detecting intracellular calcium concentration([Ca2+]i) activated by menthol. The second part: We observed the influence of TRPM8 activation on phenotypes related to energy metabolism, such as autonomic activity, core body temperature, dynamic heart rate and weight of mice, etc, through long-term menthol diet intervention on C57 / BL6 J wild type mice and TRPM8-/-mice. We confirmed that long-term menthol intervention influenced the energy metabolism and the glucose tolerance on the obese mice models induced by high-fat diet. Furthermore, we detected whether TRPM8 channel play a key role in regulating BAT thermogenesis. The third part: We clarified the concrete mechanism of menthol activated TRPM8 in regulating UCP1 thermogenesis in BAT through intervening heat production-related protein kinase(PKA, AMPK, PI3K), BAT proliferation and differentiation-related transcription factors(PGC-1, BMP7, PPARγ),and [Ca2+]i in brown adipocytes.Materials and methods:The whole research includes two parts(in vitro experiments and animal experiments). The culture of primary brown adipose cells of wild type(WT) and gene knockout mice were considered as the main part in vitro experiments. WT, TRPM8-/- and UCP1-/- mice were used as the main research object in animal experiments. These mice was feed with Chow, Chow+Menthol(Chow+M), high fat feed(HFD) and high fat feed +menthol feed(HFD+M), respectively. Dietary intervention duration was 28 weeks, then we study on the influence of menthol on body weight and metabolism of mice.Method:1. Measurement for mice weight, food intake, faeces excretion, rectal temperature, glucose tolerance and insulin tolerance, and so on.2. The implanted wireless telemetry for 24 h autonomic activity, core body temperature, 24-hours telemetry heart rates and mean heart rates in mice.3. Test the protein expression of TRPM8、UCP1、PGC-1、p PKA、PPARγ and BMP7 in BAT and the original cultivation brown adipose cells of WT and gene knockout mice with protein immunoblot method.4. Detect the [Ca2+]i in brown adipose cells of WT and gene knockout mice after the intervention by menthol with Ratio Fluorescence Imaging System.5. Detect the resting oxygen consumption in mice with the little animals breath monitoring system.6. Immunofluorescence detection of TRPM8 and UCP1 expression in brown adipose tissue and cells.7. The serum thyroid hormone, insulin levels of WT mice was tested by ELISA.Results:1. TRPM8 and UCP1 co-expressed in brown adipose cells after intervened by menthol.2. Menthol activated TRPM8 in brown adipose cells, increased [Ca2+]i, but there wasn’t significant effect on TRPM8-/- mice.3. Menthol upregulated UCP1 expression in dose-dependent manner in brown adipose cells, but there wasn’t significant effect on TRPM8-/- mice.4. Twenty-four hours autonomic activity, rectal temperature, 24 h core temperature and oxygen consumption were increased in long-term menthol fed on WT mice, but there wasn’t significant effect on TRPM8-/- mice.5. Body weight was decreased, 24 h core body temperature, oxygen consumption were significantly increased and glucose tolerance and insulin tolerance were improved in long-term menthol raised high-fat diet induced obesity WT mice, but there wasn’t significant effect on TRPM8-/- mice.6. UCP1 expression in BAT was increased in long-term menthol fed on WT mice and high-fat diet-induced WT mice, but there wasn’t significant effect on TRPM8-/- mice.7. The long-term menthol-fed UCP1-/- mice has no influence on the 24 h core body temperature.8. Twenty-four hour autonomic activity, plasma insulin levels, 24 h heart rate and thyroid hormone levels in long-term menthol-dietary WT mice on a high-fat diet has no influence on.9. Food intake and defecation of mice were not affected by long-term menthol dietary.10. The expression of PGC-1, BMP7, PPARγ were not affected by long-term menthol dietary.11. Menthol promoted PKA phosphorylation and increased UCP1 expression.12. The influence of menthol on UCP1 action could be significantly inhibited by the calcium ion chelating agent, but there wasn’t any effect of beta adrenergic receptors.Conclusion:1. The TRPM8 channel exists in BAT,Menthol activation of TRPM8 increases intracellular [Ca2+]i, upregulates expression of UCP1 in brown adipocytes from WT mice.2. Upon mimicking long-term cold exposure with chronic dietary menthol application, significantly increases 24 h core body temperature, basal metabolic and autonomic activity WT in mice. But these effects were absent in both TRPM8-/- and UCP1-/- mice.3. Long-term dietary menthol prevents WT mice from a high-fat diet-induced obesity and abnormal glucose tolerance. Menthol activates TRPM8 and regulates UCP1 expression of BAT by Ca2+-mediated PKA phosphorylation, indicating that activation of TRPM8 could promote heat production...