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, 151 (1), 96-110

TRPV4 Is a Regulator of Adipose Oxidative Metabolism, Inflammation, and Energy Homeostasis

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TRPV4 Is a Regulator of Adipose Oxidative Metabolism, Inflammation, and Energy Homeostasis

Li Ye et al. Cell.

Abstract

PGC1α is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor potential vanilloid), a family of ion channels, induced PGC1α expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1α, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.

Figures

Figure 1
Figure 1. Chemical screen identifies TRPVs as negative regulators of PGC1α expression
qPCR analysis of Pgc1α mRNA in 3T3-F442A adipocytes after 24-hour treatment of CB1 antagonists (A) or TRPV1 antagonists (B). All chemicals were used at 0.2, 2 and 20uM, except AM251 (20uM). (C) Normalized mRNA expression of Trpv1–4. aP2 (D) and Pgc1α (E) mRNA levels in adipocytes infected with scrambled (SCR), shTRPV1, shTRPV2 or shTRPV4 lentivirus. Data are presented as mean ± sd. See also Figure S1.
Figure 2
Figure 2. TRPV4 negatively regulates oxidative metabolism and respiration in adipocytes
(A) TRPV4 protein in 3T3-F442A adipocytes with shTRPV4 or shGFP. Adipose tissue lysate from WT and Trpv4−/− mice were used as controls. (B). Intracellular Ca2+ rose in response to hypotonicity (220mOsm, arrow) and to 100nM GSK1016790A (arrow head), only in cells with shGFP (n=48) but not in cells with shTRPV4 (n=49). (C) Representative current-voltage plots of endogenous whole-cell Trpv4 current measured in adipocytes with shGFP (right) and shTRPV4 (left) in Tyrode’s solution (grey) and upon stimulation with 100nM GSK1016790A (black). Voltage ramp protocol (500ms) shown in inset. (D) Mean current densities at −112 mV and +88 mV in adipocytes with shGFP (right, n = 6) and shTRPV4 (left, n = 6) in Tyrodes solution (grey) and upon stimulation with GSK1016790A (black). (E) Pgc1α and Ucp1 mRNA expression, with or without 100nM norepinephrine. (F) PGC1α protein. (G) mRNA expression of mitochondrial components. (H) Basal, uncoupled and FCCP-stimulated oxygen consumption rates. (I) mRNA expression of aP2, Pgc1a, Ucp1 and Cox8b, after 48 hours treatment of 100nM GSK1016790A. Data are presented as mean ± sd. See also Figure S2.
Figure 3
Figure 3. TRPV4 controls proinflammatory gene expression in adipocytes
qPCR analysis of mRNA encoding chemokines/cytokines (A) and genes involved in proinflammatory pathways (B) in 3T3-F442A adipocytes with shTRPV4 or shGFP. (C) mRNA expression of Mcp1, Mip1α, Rantes, Mcp3, Il6, Cxcl1, Timp1 and Tlr2, with or without 48 hours GSK1016790A treatment (100nM). (D) Protein concentrations of MCP1, MIP1α, CXCL1, IL6 and RANTES in culture medium from adipocytes in (C) were determined by ELISA. Data are presented as mean ± sd. * P<0.05, ** P<0.01, *** P<0.001, comparing adipocytes with shTRPV4 or shGFP; ##, P<0.01, ### P<0.001, comparing cells treated with DMSO or GSK1016790A. See also Figure S3.
Figure 4
Figure 4. ERK1/2 mediates the signal transduction from TRPV4 to gene expression
(A) 3T3-F442A adipocytes with shTRPV4 or shGFP were treated with 100nM GSK1016790A for the indicated time and cell lysates were analyzed by western blot. CL316243 (10uM, 20 minutes) was used as a positive control for the detection of p38 phosphorylation. (B) Adipocytes were exposed to 100nM GSK1016790A or 50ng/ml TNFα for 15 minutes in regular or calcium-free DMEM. (C) Adipocytes were exposed to 100nM GSK1016790A for 15 minutes, with 45-minute pretreatments of vehicle (GSK101+V), U0126 (GSK101+U) or SP600125 (GSK101+SP). (D) mRNA expression of Pgc1α, Mip1α and Cxcl1 were analyzed 48 hours after the treatment. Data are presented as mean ± sd.
Figure 5
Figure 5. Altered thermogenic and proinflammatory programs in Trpv4−/− adipose tissue
(A) Body weights of WT and Trpv4−/− mice on chow and HFD. mRNA expression of thermogenic genes (B), PGC1α protein (C) and UCP1 protein (D) in SubQ fat from chow -fed mice. (E) Representative images from immunohistochemistry for UCP1 (brown stain) in SubQ fat after 8 weeks of HFD. UCP1-expressing cells are indicated by arrows. (F) mRNA expression of chemokines in EPI fat were analyzed under three diet conditions. (G) Thermogenic gene expression and (H) Chemokines and Tnfα mRNA expression in Trpv4−/− and WT primary adipocytes. Data are presented as mean ± sem. See also Figure S4 and S5.
Figure 6
Figure 6. Trpv4−/− mice are protected from obesity, adipose inflammation and metabolic dysfunction with exposure to high fat diet
(A) Body composition in WT and Trpv4−/− mice. (B) Energy expenditure (as oxygen consumption) after 7 weeks HFD. (C) mRNA expression of macrophage markers. (D) H&E staining of EPI fat after 16 weeks of HFD, arrows indicates “crown like structures” (E) Western blot analysis of PPARγ serine-273 phosphorylation in EPI fat. (F) Tnfa mRNA expression in EPI fat. (G) Fasting and glucose (1g/kg) stimulated insulin levels. (H) IP-glucose tolerance test (1.0g/kg) and (I) IP-insulin tolerance test (1U/kg) in WT and Trpv4−/− mice after 12 weeks HFD. Data are presented as mean ± sem. See also Figure S6.
Figure 7
Figure 7. TRPV4 antagonist modulates an adipose gene program and improves glucose homeostasis in vivo
(A) Intracellular Ca2+ measurement in 3T3-F442A cells in response to 100nM GSK101 (Agonist, arrow head), with (n=13) or without (n=12) 10uM TRPV4 antagonist GSK205. (B) The mRNA expression of thermogenic and proinflammatory genes in GSK205 (5uM) treated 3T3-F442A adipocytes, and (C) in EPI fat from GSK205 treated animals. (D) IP-glucose tolerance test (1g/kg) in GSK205 or vehicle-treated DIO mice. Data are presented as mean ± sem. See also Figure S7.

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