Final Report Summary - SPICES, GUT & BRAIN (TRP channels in gut and brain - function, role and ligand crosstalk)
The SPICES, GUT & BRAIN project investigates the physiological mechanism of food perception. Any food when consumed enters into contact with millions of active cells and receptors in the body, either as whole food components or as molecules. The resulting interactions can lead to sensations of pleasure or displeasure but also to good health or food-related health problems.
More specifically, we study the interaction at molecular level of spice molecules with transient receptor potential (TRP) channels expressed in gastrointestinal tract and brain searching possible health benefits. Our objectives were:
- Firstly, to determine the expression and co-localisation of selected TRP channels (TRPA1, TRPM8 and TRPV1) in the mouse gastrointestinal tract and selected regions of the mouse brain.
- Secondly, to investigate physiological responses to TRPA1, M8 and V1 agonists after ingestion or direct stimulation on neuron in brain slices.
During this two years project the most relevant results obtained are:
GUT
(1) Co-localisation of ghrelin secreting cells and TRPA1
During the determination of TRP channels through the gut, a combination of immune-fluorescence studies to localise ghrelin secreting cells with in situ hybridisation to localise TRPA1 revealed that endocrine cells positive for ghrelin displayed up to 65 % co-localisation with TRPA1 in duodenum. Furthermore, the expression pattern of TRPA1 is very similar and proportional to the ghrelin expression pattern.
(2) Cinnamaldehyde (CIN) decreased food intake and gastric emptying
Gavage with CIN, a potent TRPA1 agonist shows a significant decrease in food intake two hours after most likely based upon a strong observed decrease in gastric emptying. These results also demonstrate a positive effect of CIN increasing satiation.
(3) Anti-obesity and anti-hyperglycemic effect of CIN
After a chronic treatment with high fat diet supplemented with 0.2 % CIN in obese induced mice we found a decreased in cumulative body weight gain and no effect in daily food intake. Furthermore, at the end of the treatment we found fat mass reduction and improvement in glucose tolerance. This enhancement in plasma glucose levels could be partially due to better mobilisation of glucose by peripheral tissues and better insulin sensitivity.
Quantitative real-time polymerase chain reaction (RT-PCR) analysis showed that messenger ribonucleic acid (mRNA) levels for several GLUT glucose transporters in white adipose tissue (GLUT 1, 8 and 12), Cpt 1a and Ins R were significantly increased in CIN fed mice. This set of genes play an important role in lipid storage and regulation of glucose homeostasis. In brown adipose tissue we found up-regulation of Acs reported to activate fatty acids destined for oxidation. (Ellis et al., 2010).
(4) Potential pharmacological target to decrease ghrelin secretion
We used a recently produced cell line named MGN3-1 cells. This cell line was a useful tool for studying the production and secretion of ghrelin and screening of ghrelin-modulating drugs. These cells are the first cell line derived from a gastric ghrelin-producing cell preserving secretion of substantial amounts of ghrelin under physiological regulation. Using MGN3-1 cells we could first confirm the co-expression of TRPA1 and ghrelin; and second discovered that CIN up-regulates expression of InsR and TRPA1 genes. We also found that CIN decrease ghrelin secreted by this model cell. This is a key finding since modulation of endogenous ghrelin levels by tastants such as CIN may provide novel therapeutic applications for the treatment of weight and gastrointestinal motility disorders.
In conclusion, TRPA1 could represent a new interesting pharmacological target to modulate ghrelin secretion.
The participation of ghrelin in obesity and the better knowledge of ghrelin biology have helped to identify pharmacological targets and the development of pharmacological compounds for the treatment of obesity and related diseases. To date, pharmacological compounds have been designed to target Ghrelin O-acyltransferase (GOAT), ghrelin, and GHS-R1a. We suggest that TRPA1 could also represent an interesting target in the context of ghrelin and the treatment of obesity and diabetes.
BRAIN
(1) TRPA1, M8, V1 agonists modulate neuronal activity in the brain somatonsensory cortex. To assess the effect of specific ligands (extract of spices) for different TRP channels on brain cells, we performed whole-cell patch-clamp analysis (current clamp modality) in pyramidal cells of layer 5 in the somato-sensory cortex (glutamate-ergic excitatory neurons). The results obtained are summarised in the following table where we also suggest potential areas of translation.
(2) Expression of TRPA1, M8 and V1 in the brain
TRPA1, M8 and V1 are expressed in the brain but at lower levels as compared with trigeminal root ganglia. Despite TRPM8 being the lowest, we were able to detect a high degree of co-localisation with a neuronal marker in many different areas of the brain as detected by quantitative RT (qRT)-PCR.
(3) Both capsaicin and menthol depress neuronal activity. Capsaicin (TRPV1 agonist) and menthol (TRPM8 agonist) depress neural activity by reducing action potential firing frequency in the lateral amygdala, the substantia Nigra pars compacta and hippocampal pyramidal cells CA1.
(4) The menthol effect in neuronal activity is partially mediated by TRPM8 channels. Using a different TRPM8 agonist we observed the same effect on neuronal activity in somatosensory cortex, hypothalamus and amygdala.
Using different TRPM8 antagonists and intracellular Ca2+ chelators we found that at least 60 and 50 % of menthol and Icilin effects are mediated by TRPM8 channels.
These results support our hypothesis that the menthol effect in neuronal activity is partially mediated by TRPM8.
We conclude that TRPM8 receptor could be a good target to prevent or treat epilepsy. Indeed, an independent observation showed that menthol does have a potential alleviating effect (Zhang et al. 2008).
References:
(1) Ellis, J. M., et al. 2010. Adipose acyl-CoA synthetase-1 (ACSL1) directs fatty acids towards _-oxidation and is required for cold thermogenesis. Cell Metab. 12:53-64.
(2) Zhang XB et al. 2008. A-type gamma-aminobutyric acid (GABA) receptor as a central target of TRPM8 agonist menthol. PLoS ONE, 3(10):e3386.
More specifically, we study the interaction at molecular level of spice molecules with transient receptor potential (TRP) channels expressed in gastrointestinal tract and brain searching possible health benefits. Our objectives were:
- Firstly, to determine the expression and co-localisation of selected TRP channels (TRPA1, TRPM8 and TRPV1) in the mouse gastrointestinal tract and selected regions of the mouse brain.
- Secondly, to investigate physiological responses to TRPA1, M8 and V1 agonists after ingestion or direct stimulation on neuron in brain slices.
During this two years project the most relevant results obtained are:
GUT
(1) Co-localisation of ghrelin secreting cells and TRPA1
During the determination of TRP channels through the gut, a combination of immune-fluorescence studies to localise ghrelin secreting cells with in situ hybridisation to localise TRPA1 revealed that endocrine cells positive for ghrelin displayed up to 65 % co-localisation with TRPA1 in duodenum. Furthermore, the expression pattern of TRPA1 is very similar and proportional to the ghrelin expression pattern.
(2) Cinnamaldehyde (CIN) decreased food intake and gastric emptying
Gavage with CIN, a potent TRPA1 agonist shows a significant decrease in food intake two hours after most likely based upon a strong observed decrease in gastric emptying. These results also demonstrate a positive effect of CIN increasing satiation.
(3) Anti-obesity and anti-hyperglycemic effect of CIN
After a chronic treatment with high fat diet supplemented with 0.2 % CIN in obese induced mice we found a decreased in cumulative body weight gain and no effect in daily food intake. Furthermore, at the end of the treatment we found fat mass reduction and improvement in glucose tolerance. This enhancement in plasma glucose levels could be partially due to better mobilisation of glucose by peripheral tissues and better insulin sensitivity.
Quantitative real-time polymerase chain reaction (RT-PCR) analysis showed that messenger ribonucleic acid (mRNA) levels for several GLUT glucose transporters in white adipose tissue (GLUT 1, 8 and 12), Cpt 1a and Ins R were significantly increased in CIN fed mice. This set of genes play an important role in lipid storage and regulation of glucose homeostasis. In brown adipose tissue we found up-regulation of Acs reported to activate fatty acids destined for oxidation. (Ellis et al., 2010).
(4) Potential pharmacological target to decrease ghrelin secretion
We used a recently produced cell line named MGN3-1 cells. This cell line was a useful tool for studying the production and secretion of ghrelin and screening of ghrelin-modulating drugs. These cells are the first cell line derived from a gastric ghrelin-producing cell preserving secretion of substantial amounts of ghrelin under physiological regulation. Using MGN3-1 cells we could first confirm the co-expression of TRPA1 and ghrelin; and second discovered that CIN up-regulates expression of InsR and TRPA1 genes. We also found that CIN decrease ghrelin secreted by this model cell. This is a key finding since modulation of endogenous ghrelin levels by tastants such as CIN may provide novel therapeutic applications for the treatment of weight and gastrointestinal motility disorders.
In conclusion, TRPA1 could represent a new interesting pharmacological target to modulate ghrelin secretion.
The participation of ghrelin in obesity and the better knowledge of ghrelin biology have helped to identify pharmacological targets and the development of pharmacological compounds for the treatment of obesity and related diseases. To date, pharmacological compounds have been designed to target Ghrelin O-acyltransferase (GOAT), ghrelin, and GHS-R1a. We suggest that TRPA1 could also represent an interesting target in the context of ghrelin and the treatment of obesity and diabetes.
BRAIN
(1) TRPA1, M8, V1 agonists modulate neuronal activity in the brain somatonsensory cortex. To assess the effect of specific ligands (extract of spices) for different TRP channels on brain cells, we performed whole-cell patch-clamp analysis (current clamp modality) in pyramidal cells of layer 5 in the somato-sensory cortex (glutamate-ergic excitatory neurons). The results obtained are summarised in the following table where we also suggest potential areas of translation.
(2) Expression of TRPA1, M8 and V1 in the brain
TRPA1, M8 and V1 are expressed in the brain but at lower levels as compared with trigeminal root ganglia. Despite TRPM8 being the lowest, we were able to detect a high degree of co-localisation with a neuronal marker in many different areas of the brain as detected by quantitative RT (qRT)-PCR.
(3) Both capsaicin and menthol depress neuronal activity. Capsaicin (TRPV1 agonist) and menthol (TRPM8 agonist) depress neural activity by reducing action potential firing frequency in the lateral amygdala, the substantia Nigra pars compacta and hippocampal pyramidal cells CA1.
(4) The menthol effect in neuronal activity is partially mediated by TRPM8 channels. Using a different TRPM8 agonist we observed the same effect on neuronal activity in somatosensory cortex, hypothalamus and amygdala.
Using different TRPM8 antagonists and intracellular Ca2+ chelators we found that at least 60 and 50 % of menthol and Icilin effects are mediated by TRPM8 channels.
These results support our hypothesis that the menthol effect in neuronal activity is partially mediated by TRPM8.
We conclude that TRPM8 receptor could be a good target to prevent or treat epilepsy. Indeed, an independent observation showed that menthol does have a potential alleviating effect (Zhang et al. 2008).
References:
(1) Ellis, J. M., et al. 2010. Adipose acyl-CoA synthetase-1 (ACSL1) directs fatty acids towards _-oxidation and is required for cold thermogenesis. Cell Metab. 12:53-64.
(2) Zhang XB et al. 2008. A-type gamma-aminobutyric acid (GABA) receptor as a central target of TRPM8 agonist menthol. PLoS ONE, 3(10):e3386.