This study gives insight into a Ca2+
entry mechanism of adipocytes. Molecular components, TRPC1 and TRPC5, were up-regulated as mature adipocytes formed, leading to constitutively-active heteromeric Ca2+
-permeable channels. The arising Ca2+
influx inhibited the generation of adiponectin, without effect on leptin. Most assays showed about 25 % increase in the generation of adiponectin when the TRPC channels were inhibited. While TRP channels in general have been found to be chemically-activated, the constitutive nature of the adipocyte channels conferred significance to chemical inhibition. Dietary ω-3 fatty acids were identified as inhibitors with strong relevance to adipocyte biology, metabolic syndrome, and cardiovascular disease. The findings of the study are summarised schematically in Online Figure IX
TRPC1 and TRPC5 have multiple functions in addition to those in adipocytes, including roles in vascular and cardiac remodelling24, 25
. Striking vascular up-regulation has been observed in metabolic syndrome, with protection conferred by exercise26
. Channel activity has been shown to be stimulated acutely by factors associated with cardiovascular disease, such as oxidised phospholipids18
. Therefore, suppression of adiponectin by TRPC channels may be part of a general effect of the channels as drivers or facilitators of inflammatory responses such as those occurring in the metabolic syndrome.
The fatty acids identified as TRPC inhibitors included the ω-3 polyunsaturated fatty acids that derive primarily from the diet. α-Linolenic acid is found mostly in vegetable oils, including those from rapeseed and soybean. DHA and EPA are in oily fishes that consume marine microorganisms. Depending on the diet, ω-3 fatty acids occur at plasma concentrations of 1-100 μmole/L20, 21
, which would be sufficient to affect TRPC1/TRPC5-containing channels. Large-scale trials suggest that ω-3 fatty acids decrease the risk of major diseases or disease-related events, including coronary heart disease, insulin resistance, myocardial infarction, atrial fibrillation, and heart failure22, 27
. ω-3 fatty acid therapy shows promise for disease prevention22, 28
Our data suggest that ω-3 fatty acids elevate adiponectin substantially by acting through a mechanism that depends on TRPC1/TRPC5-containing channels. Molecular targets of ω-3 fatty acids are not, however, restricted to TRPC channels. They bind or indirectly affect PPAR-γ, the GPR120 receptor, voltage-dependent Na+
channels, and TRPV1 channels29-31
. The mechanism by which ω-3 fatty acids suppress TRPC channels has not been elucidated but it was not a transcriptional effect (because the effect occurred within a few minutes) and is unlikely to have occurred through GPR120 because this receptor couples via Gq/11
, which stimulates TRPC channel activity31, 32
. TRPV1 modulation by ω-3 fatty acids was suggested to occur via protein kinase C33
, which inhibits TRPC534
. Therefore, protein kinase C is a putative transduction mechanism. More direct effects are possible, although lipid effects on TRPC5 have previously been found to be stimulatory35
. Intriguingly, the Drosophila
TRP channel is activated directly by polyunsaturated fatty acids36
; our data indicate that mammalian orthologues (i.e. TRPC1/TRPC5) are also sensitive to such fatty acids but that the functional consequence is the opposite (i.e. inhibition). Substantial sequence differences between the mammalian and Drosophila
channels make it difficult to predict which residues are responsible for the reversal of polarity.
Fatty acid inhibitors of TRPC1-TRPC5 channels are predicted to oppose the adverse effects of TRPC channel activation in inflammation and cardiovascular disease. There may be additional inhibitory factors acting similarly on TRPCs, such as resveratrol, vitamin C, and gallic acid37
(Online Figure IX
). These factors are exogenous to the body, suggesting that a general function of TRPC channels may be to enable coupling between external chemicals and the internal biology of the body. Previously studies have focused on TRP channels other than TRPCs as integrators of cells with external signals10
The study used 3T3-L1 cells as a foundation, but data obtained using human tissue and mouse samples and through genetic manipulation in vivo supported the 3T3-L1 findings, and studies of over-expressed TRPCs supported the conclusion that the specified channel is a target of ω-3 fatty acids. There was technical difficulty in measuring intracellular Ca2+ in the mature adipocytes, but independent electrophysiological studies supported the data obtained with the fluo-4 Ca2+ indicator.
This study identified a Ca2+-permeable cationic channel (TRPC1/5) mechanism of adipocytes. Inhibition of the mechanism raised circulating adiponectin levels and would thus be expected to confer cardiovascular protection. Constitutive activity of the channels was significant, suggesting that inhibitors are likely to be important even in the absence of an activator. Novel inhibitors of the channels were identified (i.e. ω-3 fatty acids), adding to previously identified TRPC inhibitors which are associated with protection against major cardiovascular diseases.