Transient receptor potential cation channel 6 deficiency leads to increased body weight and metabolic dysfunction

Abstract

Transient receptor potential cation channel 6 (TRPC6), a member of the TRPC family, is expressed in the hypothalamus and modulates cell Ca²⁺ influx. However, the role of TRPC6 in controlling metabolic and cardiovascular functions under normal conditions has not been previously determined. Thus the impacts of TRPC6 deletion on energy balance, metabolic, and cardiovascular regulation as well as the anorexic responses to leptin and melanocortin 3/4 receptor (MC3/4R) activation were investigated in this study. Extensive cardiometabolic phenotyping was conducted in male and female TRPC6 knockout (KO) and control mice from 6 to 24 wk of age to assess mechanisms by which TRPC6 influences regulation of energy balance and blood pressure (BP). We found that TRPC6 KO mice are heavier with greater adiposity, are hyperphagic, and have reduced energy expenditure, impaired glucose tolerance, hyperinsulinemia, and increased liver fat compared with controls. TRPC6 KO mice also have smaller brains, reduced proopiomelanocortin mRNA levels in the hypothalamus, and impaired anorexic response to leptin but not to MC3/4R activation. BP and heart rate, assessed by telemetry, were similar in TRPC6 KO and control mice, and BP responses to air-jet stress were attenuated in TRPC6 KO mice despite increased body weight and metabolic disorders that normally raise BP and increase BP responses to stress. Our results provide evidence for a novel and important role of TRPC6 in controlling energy balance, adiposity, and glucose homeostasis, which suggests that normal TRPC6 function may be necessary to link weight gain and hyperleptinemia with BP responses to acute stress.

INTRODUCTION

Transient receptor potential cation channel C (TRPC) represents a subfamily of TRP channels widely expressed in the cell membranes of many cell types, including neurons (1). The TRPC family consists of seven members, TRPC1–7, which are nonselective cation channels that increase cytoplasm Ca²⁺ influx to regulate downstream cellular responses, including membrane depolarization, metabolic activation, gene expression, apoptosis, and cell proliferation (2). In the brain, some subunits of TRPC can be activated by G protein-coupled receptors such as serotonin receptors and receptor tyrosine kinases (e.g., leptin receptors) through the phospholipase C-phosphatidylinositol 4,5-bisphosphate-diacylglycerol pathway (3). Although all members of TRPC channels are expressed in the brain, the spatial and temporal expression patterns of each TRPC subunit are unclear.

TRPC6 activation can be receptor mediated or by mechanically and osmotically induced membrane stretch, leading to Ca²⁺ influx (4). A growing interest in the potential role of TRPC6 channels in kidney injury emerged after discovering that gain-of-function mutations in TRPC6 cause focal segmental glomerulosclerosis in humans (5). Preclinical studies have examined the role of TRPC channels in diverse animal models of kidney disease (6) and found important roles of TRPC6 in maintaining normal physiological function of the kidney. In our preliminary studies designed to assess the role of TRPC6 in mediating chronic kidney disease, we observed that TRPC6 knockout (KO) mice were heavier than wild-type (WT) controls when fed a normal diet. To our knowledge, there have been no previous reports on the role of TRPC6 in regulating body weight, energy balance, and other metabolic functions, including leptin’s anorexic effect. Therefore, to confirm our preliminary findings and investigate potential mechanisms responsible for the obese phenotype of TRPC6 KO mice, we assessed their daily food intake from weaning until 16 wk of age, as well as fasting/refeeding response, energy expenditure, and motor activity. We also examined anorexic responses of TRPC6 KO mice to leptin and melanocortin 4 receptor (MC4R) activation as well as proopiomelanocortin (POMC) and agouti-related peptide (AgRP) gene expression in hypothalamus and hindbrain. Additionally, we measured their response to an oral glucose tolerance test (GTT) and their cardiovascular function, including blood pressure (BP), heart rate (HR), and BP response to acute pressor stress.

Our results indicate that TRPC6 plays an important role in regulating body weight, appetite, energy expenditure, and glucose homeostasis and in modulating the anorexic effects of leptin but not to MC3/4R activation. TRPC6 also appears to be important in cardiovascular regulation since TRPC6 KO mice had normal BP and HR and attenuated BP responses to stress despite obesity and associated metabolic disorders. These observations demonstrate a novel pathway for metabolic and cardiovascular regulation by TRPC6.

MATERIALS AND METHODS

Animals

The experimental procedures described in this study followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. TRPC6 knockout (KO) mice (B6;129S-Trpc6tm1Lbi/Mmjax) and background control B6/129s control mice from Jackson Laboratories were bred in our animal facility. The B6/129s hybrid mice used in these studies were the offspring of more than four generations of an F1 x F1 mating cross between C57BL/6J females (B6) and 129S1/SvImJ males (129S) and served as controls for TRPC6 KO mice that were generated with 129-derived embryonic stem cells and maintained on a mixed B6/129 background.

Validation of TRPC6 Gene Deletion in the Hypothalamus of TRPC6 KO Mice

Deletion of TRPC6 in mice has been previously validated (7). To confirm deletion of TRPC6 gene in TRPC6 KO mice in our study, DNA was isolated from the hypothalamus of 6-wk-old TRPC6 KO and control B6129s mice. Standard PCR assays were performed based on the Jackson Laboratories Genotyping Protocol 26638. Primer sets used for genotyping were as follows: WT: forward 5′-TCTTTATGCAATCGCTGT GG and reverse 5′-GCTAGTCTTCCTGCAATCCA; and mutant: forward 5′-TCTATTAACACTCAACTGGCACCT and reverse 5′-GCCAGAGGCCACTTGTGTAG.

Expression of AgRP, POMC, and TRPC Family Members in the Hypothalamus

We performed quantitative (q)RT-PCR to examine mRNA expression levels of AgRP and POMC in the hypothalamus and the nucleus tractus solitarius (NTS) and area postrema (AP) areas of the hindbrain. We also examined mRNA expression levels of TRPC6 and other TRPC family members including TRPC1, 3, 4, and 5 in the hypothalamus of 8-wk-old TRPC6 KO and control mice. The mice (n = 8–14/group, males and females combined) were euthanized, and the hypothalamus and NTS/AP area of the hindbrain were quickly removed and frozen immediately by immersion in liquid nitrogen and stored at −80°C. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol and quantified by spectrophotometry. Total RNA was reverse transcribed by SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA). qRT-PCR was performed on 1 ng of RNA using a StepOne Plus qRT-PCR system with PowerUP SYBR green master mix (Thermo Fisher Scientific). The following primer pairs were used to amplify mouse AgRP: forward 5′-AGTCACGTGTGGACCCTTTA and reverse 5′-ACTCAGCAACATTGCAGTCA; POMC: forward 5′-CATCTTTGTCCCCAGAGAGC and reverse 5′-GCACCAGCTCCACACATCTA; TRPC1: forward 5′-GCCATCTTTGTCACCAGGTT and reverse 5′-GCTCGAGCAAACTTCCATTC; TRPC3: forward 5′-GTAGTGGGTGTGCTGGACCT and reverse 5′-AAAAGCTGCTGTTGGCAGTT; TRPC4: forward 5′-TGAGAAGGAAGCCAGAAAGCTTCG and reverse 5′-CCTTAACATTCTCCTCCGTCAAGCC; TRPC5: forward 5′-GTTCACAGCCAACTCCCATT and reverse 5′-GGATCCCCTTGCAGTTGTTA; and TRPC6: forward 5′-GGCGCAAAACAGAATGAAGC and reverse 5′-CACGTCCGCATCATCCTCAA. Mouse 18S rRNA was used as an internal control to normalize expression levels of target genes and the delta delta cycle threshold (^Δ-ΔC_T) method was used to calculate the fold changes of target genes in TRPC6 KO compared with control mice.

In Vivo Study Protocols

Between 6 and 16 wk of age, male and female TRPC6 KO and B6129s control mice (n = 8/group per sex) were individually housed and fed a normal diet (Envigo Teklad Custom Diets, Madison, WI, CA-170955; 4.0 kcal/g, 66% kcal from carbohydrate, 16% kcal from fat, and 18% kcal from protein with 0.24–0.25% Na⁺ and 1% K⁺). Food intake and body weight were measured twice a week, and weekly changes in body composition were analyzed using magnetic resonance imaging (4-in-1 EchoMRI-900, Echo Medical System, Houston, TX).

Energy Expenditure and Motor Activity Measurements

After food intake and body weight measurements, 16-wk-old male and female TRPC6 KO and control mice (n = 6–8/group per sex) were placed individually in metabolic cages (Promethion Metabolic and Behavioral System, Sable Systems International, Las Vegas, NV) to measure oxygen consumption, energy expenditure (EE), respiratory quotient (RQ), and motor activity. Mice were acclimatized to the new environment for 3 days followed by 5 consecutive days of recordings.

Fasting/Refeeding Protocol and Food Intake Responses to Acute Leptin Injection

At 18 wk of age, daily food intake and body weight were measured in male and female TRPC6 KO and sex-matched control mice (n = 7–8/group per sex) for 3 consecutive days at baseline followed by a 24-h fast. After the fasting period, the mice were given food ad libitum for 3 consecutive days while daily food consumption was recorded.

One week after recovery from the fasting/refeeding protocol, the mice (n = 7–8/group per sex) were injected with leptin (5 mg/kg ip) or saline vehicle (0.2 mL) 1 h before lights out (5:00 PM) and food intake was measured 2, 4, 15, and 24 h postinjection. A within-subjects design was used in this study. Animals were randomly selected to receive saline or leptin injections during the first round and subjected to opposite injections 1 wk later. Changes in food intake following leptin injection were compared with saline injection in the same animal.

Food Intake Responses to Melanocortin 4 Receptor Agonist Injection

One week after recovery from the acute leptin or saline injections, the mice (n = 7–8/group per sex) were injected with an MC3/4R agonist, melanotan II (MTII; 4 mg/kg ip), or saline vehicle (0.2 mL) 1 h before lights out (5:00 PM), and food intake was measured 2, 4, 15, and 24 h postinjection. The same within-subjects design and injection protocol were used as described for acute leptin injections. Changes in food intake following MTII injection were compared with saline injection in the same animals.

Oral Glucose Tolerance Test

d-Glucose was administered by gavage (3 mg/kg of lean tissue plus 1 mg/kg of fat mass) after a 5-h fast in 22-wk-old male and female TRPC6 KO and control mice (n = 7–8/group per sex). Blood samples were collected by tail snip, and blood glucose was measured using glucose strips (ReliOn) at baseline, 15, 30, 60, 90, and 120 min after glucose administration.

Fasting Glucose, Leptin, and Insulin Measurements

At 24 wk of age, male and female TRPC6 KO and control mice (n = 7–10/group per sex) were fasted for 5 h from 8:00 AM to 1:00 PM. Around 160 µl of blood were drawn from the tail vein to measure plasma leptin and insulin concentrations with ELISA kits (R&D Systems and Crystal Chemical, Inc., respectively). Plasma glucose concentrations were determined using glucose strips (ReliOn).

Baseline Blood Pressure and Heart Rate Measurements

Additional groups of 13-wk-old TRPC6 KO and control mice (n = 11 in males; n = 6 in females) were implanted with telemeters for measurement of BP and heart rate (HR). Briefly, mice were anesthetized with 2% isoflurane and a telemetry probe (TA11PA-C10, Data Science International, St. Paul, MN) was implanted in the left carotid artery and advanced into the aorta for measurement of BP and HR, 24 h/day. The measurements were started after 10 days of recovery from surgery and continued for 5 consecutive days of stable BP and HR recordings. Mean, systolic, and diastolic BP were recorded for 30 s every 10 min 24 h/day (8).

Acute Air-Jet Stress Test

After 5 days of stable 24 h/day baseline BP and HR measurements, 16-wk-old male and female TRPC6 KO and control mice (n = 5–6/group per sex) were placed in special cages used for air-jet stress testing. Briefly, after 2 h of acclimation to the cage, BP and HR were continuously measured by telemetry for 30 min before the air-jet stress test was applied. The air-jet stress test consisted of 2-s pulses of air delivered every 5 s for 5 consecutive minutes aimed at the forehead of the mice at an approximate distance of 5 cm using a 14-gauge needle opening at the front of the tube connected to compressed air. After the 5-min air-jet stress, BP and HR were measured continuously for an additional 30-min recovery period. Changes in BP and HR responses during the air-jet stress and recovery period were calculated compared with the baseline period (average of the last 10 min of baseline period before air-jet stress was initiated). We also calculated the areas under the curves (AUCs) of the MAP and HR during the air-jet stress and recovery period using the following parameters: average change in MAP for each minute during the 5-min air-jet stress test and every 5 min during the 30-min recovery period.

Tissue Weight and Liver Fat/Lean Composition

Male and female TRPC6 and control mice (n = 6/group per sex) were euthanized at 27 wk of age. Body length and tibia length as well as weights of the heart, left and right kidneys, liver, epididymal fat, ovarian fat, brown adipose, and brain were measured (Table 1). Whole livers were then analyzed for fat and lean mass content using the 4-in-1 EchoMRI-900 analyzer.

Table 1.

Body weight, body length, and tissue weights in male and female TRPC6 KO and control mice at 27 wk of age

Parameter	B6/129s (Male)	TRPC6 KO (Male)	B6/129s (Female)	TRPC6 KO (Female)
Body weight, g	39.4 ± 1.2	43.1 ± 1.1*	28.6 ± 1.1	35.5 ± 0.8*
Body length, cm	10.0. ± 0.1	10.1 ± 0.1	9.4 ± 0.1	9.8 ± 0.1*
Tibia length, cm	2.0 ± 0.1	2.0 ± 0.0	1.9 ± 0.0	2.1 ± 0.0*
Heart weight, g	0.19 ± 0.01	0.22 ± 0.01*	0.15 ± 0.01	0.18 ± 0.01*
Heart/body weight, g of organ/g of total body weight	0.005 ± 0.0003	0.005 ± 0.0002	0.005 ± 0.0004	0.005 ± 0.0002
Liver weight, g	1.62 ± 0.08	2.31 ± 0.12*	1.17 ± 0.05	1.57 ± 0.09*
Liver/body weight, g of organ/g of total body weight	0.040 ± 0.0002	0.054 ± 0.0002*	0.041 ± 0.0002	0.051 ± 0.0003*
Liver fat mass, g	0.26 ± 0.02	0.50 ± 0.06*	0.16 ± 0.01	0.27 ± 0.04*
Liver lean mass, g	1.49 ± 0.03	1.84 ± 0.07*	0.96 ± 0.09	1.31 ± 0.06*
Brain weight, g	0.50 ± 0.01	0.44 ± 0.01*	0.51 ± 0.01	0.46 ± 0.01*
Brain/body weight, g of organ/g of total body weight	0.013 ± 0.0004	0.010 ± 0.0002*	0.018 ± 0.0001	0.013 ± 0.0003*
Kidney weight (right), g	0.21 ± 0.08	0.27 ± 0.01*	0.14 ± 0.01	0.17 ± 0.01*
Kidney/body weight, g of organ/g of total body weight	0.005 ± 0.0002	0.006 ± 0.0003*	0.005 ± 0.0003	0.005 ± 0.0001
Brown adipose, g	0.35 ± 0.04	0.47 ± 0.06*	0.13 ± 0.01	0.20 ± 0.01*
Epididymal fat in males; ovarian fat in female, g	1.47 ± 0.06	1.27 ± 0.07*	1.12 ± 0.16	2.35 ± 0.13*

Data are expressed as means ± SE. TRPC6, transient receptor potential cation channel; KO, knockout. *P < 0.05, compared with sex-matched control mice.

Immunofluorescence Staining

Mice fasted from 10:00 AM to 2:00 PM were deeply anesthetized with isoflurane and flushed with 20 mL 1× PBS through the left ventricle to remove blood. Fixative perfusion was performed with 4% paraformaldehyde (PFA), pH 6.0, in 1× PBS for 8–10 min. Subsequently, brains were removed from the skull and fixed in 4% PFA in PBS at 4°C for 24 h. Brains were then washed in 1× PBS and transferred to 30% sucrose in PBS for 48 h. Free-floating, coronal brain sections (25 μm) were obtained using a vibratome (VT1000S, Leica Microsystems) and stored in 1× PBS at 4°C.

TRPC6 was detected using rabbit antibody (ACC-120, Alomone Labs) diluted at 1:50; validation of the specificity of TRPC6 antibody is described in Supplemental Fig. S1 (all Supplemental material is available at https://doi.org/10.6084/m9.figshare.16543329). POMC was detected using rabbit antibody (H-029-30, Phoenix Pharmaceuticals) diluted at 1:1,000. The following secondary antibodies and dilution factors were used: goat anti-rabbit Alexa Fluor 594 IgG at 1:500; Alexa Fluor 488 Fab fragment goat anti-rabbit IgG at 1:500; Fab Fragment goat anti-rabbit IgG at 1:50; Biotin-SP conjugated Fab fragment goat anti-rabbit IgG at 1:500; and Alexa Fluor 488 streptavidin at 1:1,000 (Jackson ImmunoResearch Laboratories).

Free-floating sections were blocked and permeabilized with 1× PBS with 0.3% Triton X-100 and 5% normal goat serum for 1 h. Primary and secondary antibodies were diluted in 1× PBS containing 0.3% Triton X-100 and 1% BSA. For staining of TRPC6 colocalized with POMC, brain slices were first incubated with TRPC6 primary antibody for 24 h at 4°C and then incubated with Biotin-SP conjugated Fab fragment Goat anti-rabbit IgG, followed by Alexa Fluor 488 Streptavidin Secondary antibodies. After TRPC6 staining, the brain slices were blocked again and incubated with POMC primary antibodies for 24 h at 4°C and then were incubated with goat anti-rabbit Alexa 594 IgG for 1 h at room temperature. Brain slices were finally mounted on SuperfrostPlus glass slides using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) to counterstain the nucleus. Negative control staining without adding specific primary TRPC6 antibodies and staining for the TRPC6 KO mice are shown in Supplemental Figs. S2 and S3.

Analysis of POMC Neuron Fiber Density in the Arcuate Nucleus

Brain sections from TRPC6 KO and control mice were obtained and regions of interest in the arcuate nucleus (ARC) of the hypothalamus from bregma −1.2 to −2.7 mm were sliced in a thickness of 30 μm using a vibratome. Brain sections were obtained every 50 μm and stained with POMC antibody. The POMC antibody was generated from the epitope in the NH₂-terminal of the POMC precursor polypeptide that allows positive staining for POMC neuron bodies and axon projections. Images (∼20 pictures for each animal) were taken, and the relative density of POMC-immunoreactive axonal fibers in the ARC was measured. For each image, three 500-μm lines were randomly drawn in the ARC area. The average number of POMC-containing fibers crossing each line was calculated. Data are represented as an average of 20 images from each sample. The POMC neuron numbers were counted as the sum of each image with the same measuring area in the ARC.

Body Weight and Food Intake Measurement during 7 Days of Infusion of SAR 7334, a TRPC6 Inhibitor

A subgroup of 16-wk-old male B6/129s (n = 8–9) mice were placed individually in cages and fed a normal diet (Envigo Teklad Rodent Diets, Madison, WI, 8640; 3 kcal/g, 54% kcal from carbohydrate, 17% kcal from fat, and 29% kcal from protein with 0.4% Na⁺ and 1% K⁺). Mice were acclimatized to the new environment for ∼2–3 days, followed by 4 consecutive days of recording for baseline measurements. Then, a 7-day osmotic minipump (model 1007 D, Azet) was placed subcutaneously to deliver the specific TRPC6 inhibitor SAR 7334 (10 mg/kg/day) or PBS vehicle for 7 days. Body weight and food intake data were recorded daily for the 7 days during SAR 7334 or vehicle (PBS) infusion and 3 days after infusion was stopped. Cumulative food take was measured as the sum of daily food intake changes compared with baseline. Changes of daily food take were measured as the difference of daily food intake compared with average baseline values.

Statistical Analyses

The number of animals used was based on our previous studies and power analysis to predict a 20% difference among groups. Data are expressed as means ± SE. Significant differences between two groups were determined by unpaired Student’s t test. Significant differences between two groups over time were determined by two-way ANOVA with repeated measures. Bonferroni post hoc multiple comparisons were used to compare the means between two different groups at the same time point after two-way ANOVA test. Statistical significance was accepted at a level of P < 0.05. Three-way ANOVA tests were used to determine whether individual and interaction of different variables (time, strain, and sex) affect outcomes for body weight, body fat/lean mass, food intake, energy expenditure, RQ, and motor activity.

RESULTS

Validation of TRPC6 Deletion in KO Mice

Genotyping of TRPC6 KO mice was confirmed by PCR to examine DNA samples isolated from the brains of TRPC6 KO and control mice. To further confirm TRPC6 gene deletion from the hypothalamus of brains in TRPC6 KO mice, total mRNA was isolated from the hypothalamus of TRPC6 KO and control mice. qPCR results demonstrated no measurable TRPC6 mRNA transcription in the hypothalamus of TRPC6 KO mice compared with control mice. TRPC1, TRPC3, TRPC4, and TRPC5 mRNA levels were also examined in the hypothalamus of TRPC6 KO and control mice to determine whether TRPC6 deficiency stimulates overexpression of other isoforms of the TRPC family. The results showed no differences in mRNA expression levels of TRPC1, 3, 4, and 5 between the TRPC6 KO and control mice (Fig. 1), suggesting that global deletion of TRPC6 did not induce compensatory mRNA expression of other TRPC channels in the hypothalamus. Since TRPC3 is the sequencing homolog of TRPC6 and can generate heterotetramer with TRPC6, we also measured TRPC3 protein expression levels in the hypothalamus of TRPC6 KO and WT mice by Western blot. The results (Supplemental Fig. S1) show no significant differences in TRPC3 expression in the hypothalamus between WT and TRPC6 KO mice.

Figure 1.

Gene expression levels in the hypothalamus of transient receptor potential cation channel 6 (TRPC6) knockout (KO) and control mice. TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA levels were quantified by quantitative RT-PCR in the hypothalamus of TRPC6 KO and control mice (n = 8 per group; *P < 0.05, compared with B6/129s control mice by t test).

TRPC6 KO Mice Are Heavier with Greater Adiposity than Control Mice

TRPC6 KO male and female mice had significantly higher body weight compared with sex-matched control mice from 6 to 16 wk of age. (Fig. 2, A and B). At 16 wk of age, body weight was ∼21% greater in male TRPC6 KO mice compared with age-matched control mice (39.5 ± 1.1 vs. 32.6 ± 1.7 g) and 35% higher in TRPC6 KO females compared with controls (31.0 ± 0.9 vs. 23.0 ± 0.6 g).

Figure 2.

Body weight growth curves and fat/lean mass distribution in male and female transient receptor potential cation channel 6 (TRPC6) knockout (KO) and sex-matched control mice. A: body weight from 6 to 16 wk of age in male TRPC6 KO and control mice. B: body weight from 6 to 16 wk of age in female TRPC6 KO and control mice. C: body fat mass from 8 to 16 wk of age in male TRPC6 KO and control mice. D: body fat mass from 8 to 16 wk of age in female TRPC6 KO and control mice. E: body lean mass in male TRPC6 KO and control mice from 8 to 16 wk of age. F: body lean mass in female TRPC6 KO and control mice from 8 to 16 wk of age (n = 8/group per sex; *P < 0.05, between groups by two-way ANOVA; #P < 0.05, between groups at the same time point by Bonferroni post hoc test).

EchoMRI scans were performed from 8 to 16 wk of age to examine fat and lean mass distribution in TRPC6 KO and control mice. The higher body weights of male and female TRPC6 KO mice mainly were due to higher body fat content compared with controls (11.4 ± 0.8 vs. 7.3 ± 0.8 g in males and 9.9 ± 0.6 vs. 4.8 ± 0.4 g in females, at 16 wk of age) (Fig. 2, C and D). As shown in Fig. 2E, male TRPC6 KO mice and age-matched male controls had similar lean mass from 8 to 15 wk of age, with lean mass becoming significantly greater in TRPC6 KO mice at 16 wk of age. In female TRPC6 KO mice, however, lean mass was higher compared with female controls since the first measurement at 8 wk of age (Fig. 2F).

TRPC6 KO Mice Have Increased Daily Food Intake and Refeeding Responses after Fasting

Food intake was significantly increased in male and female TRPC6 KO mice compared with controls from 6 to 16 wk of age (Fig. 3, A and B). We also observed a trend for food intake to be higher during the first few weeks after weaning (weeks 6–9) compared with the weeks 13–16 in both groups of male and female mice; however, this trend was more pronounced in TRPC6 KO mice (Fig. 3, A and B). To further examine whether food intake regulation was altered in TRPC6 KO mice, we measured refeeding responses after 24-h fasting in 18 wk of age male and female TRPC6 KO and control mice. Compared with control mice, food intakes in male and female TRPC6 KO mice were significantly higher at baseline (3 days average food intake before 24-h fasting) as well as during the second and third days of refeeding (Fig. 3, C and D). Surprisingly, food intake on the first day of refeeding was similar in both groups (Fig. 3, C and D). Since food intake was different at baseline in TRPC6 KO and control mice, we also calculated the net cumulative food intake during the second and third days of refeeding after subtracting baseline food intake. The results showed that net cumulative food intakes during refeeding days 2 and 3 were greater in male and female TRPC6 KO mice compared with age-matched controls (Fig. 3, E and F).

Figure 3.

Average daily food intake and fasting-refeeding test in male and female transient receptor potential cation channel 6 (TRPC6) knockout (KO) and sex-matched control mice. A: average daily food intake from 6 to 16 wk of age in male TRPC6 KO and control mice. B: average daily food intake from 6 to 16 wk of age in female TRPC6 KO and control mice (n = 8/group per sex; *P < 0.05, between groups by two-way ANOVA; #P < 0.05, between groups at the same time point by Bonferroni post hoc test). C: daily food intake at baseline and 24, 48, and 72 h after refeeding in males. D: daily food intake at baseline and 24, 48, and 72 h after refeeding in females (n = 7–8/group per sex; *P < 0.05, compared with control mice at different time points by t test). E: cumulative food intake during days 2 and 3 of refeeding period in male TRPC6 KO and control mice. F: cumulative food intake during days 2 and 3 of refeeding period in female TRPC6 KO and control mice (n = 7–8/group per sex; *P < 0.05, compared with control mice by t test).

TRPC6 KO Mice Have Decreased Energy Expenditure and Reduced RQ

Since body weight reflects the balance between food intake and energy expenditure, we also tested if the obese phenotype of TRPC6 KO mice was accompanied by reduced energy expenditure. After 3 days of acclimation in the metabolic cages (Promethion metabolic and behavioral system), energy expenditure and respiratory quotient (RQ) were measured for 5 consecutive days in TRPC6 mice and control mice at 16 wk of age. Male and female TRPC6 KO mice had significantly lower (∼22%) daytime and nighttime energy expenditure when normalized for body weight (Fig. 4, A and B). Male and female TRPC6 KO mice also exhibited reduced daytime and nighttime RQs compared with controls (Fig. 4, C and D). Motor activity in male TRPC6 KO mice was significantly lower than in control WT mice. However, female TRPC6 KO mice had similar motor activity to controls (Fig. 4, E and F). Detailed statistical analyses of two-way ANOVA in Figs. 2, 3A, and 4 are listed in Table 2. We also used three-way ANOVA to examine how different variables (time during measurement, strain between control and TRPC6 KO, and sex difference) affect the outcome in body weight, body fat/lean mass, food intake, energy expenditure, RQ, and motor activity (Table 3). We found that the interaction of time with sex and interaction of time with strain significantly contribute to differences in metabolic parameters that we measured.

Figure 4.

Daytime and nighttime energy expenditure, respiratory quotient (RQ), and motor activity measurement in male and female transient receptor potential cation channel 6 (TRPC6) knockout (KO) and sex-matched control mice. A: daytime and nighttime energy expenditure normalized for body weight in male TRPC6 KO and control mice. B: daytime and nighttime energy expenditure normalized for body weight in female TRPC6 KO and control mice. C: daytime and nighttime RQ in male mice. D: daytime and nighttime RQ in female mice. E: daytime and nighttime motor activity in male mice. F: daytime and nighttime motor activity in male mice (n = 6–8/group per sex; *P < 0.05, between groups by two-way ANOVA; #P < 0.05, between groups at the same time point by Bonferroni post hoc test).

Table 2.

Two-way ANOVA analysis of strain and time difference contribution to body weight, body fat/lean mass, food intake, energy expenditure, respiratory quotient, and motor activity in male and female control and TRPC6 KO mice

Measurements/Sex	Strain	Time	Strain × Time
Body weight
Male	P = 0.0002; F = 20.73	P < 0.0001; F = 104.10	P < 0.0001; F = 8.17
Female	P < 0.0001; F = 87.24	P < 0.0001; F = 71.82	P < 0.0001; F = 4.01
Body fat mass
Male	P < 0.0001; F = 32.55	P < 0.0001; F = 88.27	P < 0.0001; F = 5.06
Female	P < 0.0001; F = 35.62	P < 0.0001; F = 23.10	NS; F = 1.63
Body lean mass
Male	NS; F = 2.06	P < 0.0001; F = 49.60	P < 0.0001; F = 23.10
Female	P < 0.0001; F = 37.37	P < 0.0001; F = 38.27	P = 0.0239; F = 2.33
Food intake
Male	P < 0.0001; F = 26.58	P < 0.0001; F = 20.73	P = 0.0325; F = 2.07
Female	P < 0.0001; F = 88.32	P < 0.0001; F = 22.56	P < 0.0001; F = 8.88
Energy expenditure
Male	P < 0.0001; F = 36.76	NS; F = 1.17	NS; F = 0.41
Female	P = 0.0017; F = 15.06	P < 0.0001; F = 31.09	P < 0.0001; F = 4.43
Respiratory quotient
Male	P < 0.0001; F = 56.63	P < 0.0001; F = 6.78	NS; F = 0.53
Female	P < 0.0001; F = 139.40	P < 0.0001; F = 18.56	NS; F = 1.42
Motor activity
Male	P = 0.0007; F = 12.18	P < 0.0001; F = 20.37	NS; F = 0.63
Female	NS; F = 0.36	P < 0.0001; F = 43.48	NS; F = 0.97

TRPC6, transient receptor potential cation channel 6; KO, knockout; NS, not significant.

Table 3.

Three-way ANOVA analysis indicating how time, strain, and sex affected body weight, body fat/lean mass, food intake, energy expenditure, respiratory quotient, and motor activity in male and female control and TRPC6 KO mice

Measurements/Factors	Time	Strain	Sex	Time × Strain	Time × Sex	Time × Strain × Sex
Body weight	P < 0.0001; F = 160.60	P < 0.0001; F = 70.04	P < 0.0001; F = 106.20	P < 0.0001; F = 9.97	P < 0.0001; F = 9.11	NS; F = 1.18
Body fat mass	P < 0.0001; F = 80.80	P < 0.0001; F = 68.13	P = 0.0446; F = 4.46	P = 0.0315; F = 2.16	P < 0.0001; F = 7.06	P = 0.0017; F = 3.25
Body lean mass	P < 0.0001; F = 87.59	P < 0.0005; F = 15.65	P < 0.0001; F = 163.20	P < 0.0001; F = 17.31	P < 0.0001; F = 5.40	P < 0.0001; F = 9.02
Food intake	P < 0.0001; F = 41.82	P < 0.0001; F = 78.20	P < 0.0001; F = 34.24	P < 0.0001; F = 9.49	NS; F = 1.25	NS; F = 1.28
Energy expenditure	P < 0.0001; F = 47.33	P = 0.0014; F = 12.82	P < 0.0001; F = 30.06	P < 0.0001; F = 11.00	P < 0.0001; F = 4.52	NS; F = 0.58
Respiratory quotient	P < 0.0001; F = 38.80	P < 0.0001; F = 33.82	P = 0.0485; F = 4.30	NS; F = 1.04	P = 0.0173; F = 2.30	P = 0.0271; F = 2.14
Motor activity	P < 0.0001; F = 73.35	NS; F = 2.06	NS; F = 1.07	NS; F = 0.89	P = 0.0260; F = 2.16	NS; F = 1.23

TRPC6, transient receptor potential cation channel 6; KO, knockout; NS, not significant.

Immunofluorescence Staining of TRPC6 is Colocalized with POMC in the Hypothalamus

The expression pattern of TRPC6 and colocalization with POMC were examined in the hypothalamus of B6/129s control mice. Positive TRPC6 staining was found in the neuronal terminals of the median eminence (ME), arcuate nucleus (ARC), and ventral medial hypothalamus (VMH) areas of the hypothalamus (Fig. 5, A and B). There was no positive TRPC6 staining in the paraventricular nucleus (PVN) and very weak staining in the lateral hypothalamus. To examine the colocalization of TRPC6 with POMC, antibodies against POMC were used to stain the neuron bodies and axons at different areas of the hypothalamus. Our results showed that TRPC6 was highly colocalized with POMC in the ME and partially in ARC and VMH. (Fig. 5B).

Figure 5.

Transient receptor potential cation channel 6 (TRPC6) expression and colocalization with proopiomelanocortin (POMC) in the hypothalamus of B6/129s control mice. Representative immunofluorescence images were shown with the bregma −1.70 to −2.06 mm. A: hypothalamic staining of nucleus (blue), TRPC6 (green), POMC (red), and merged images with all 3 colors. Scale bars = 100 μm. B: high-magnification view of ME, ARC, VMH, and PVN in hypothalamus staining with nucleus (blue), TRPC6 (green), POMC (red), and merged image. ARC, arcuate nucleus; DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; PVN, paraventricular nucleus; ME, median eminence; 3V, third ventricle. Scale bars = 10 μm.

TRPC6 KO Mice Have Reduced POMC mRNA Expression in the Hypothalamus and Hindbrain

To determine the impact of TRPC6 deletion on hypothalamic and hindbrain gene expression of two major neuropeptides that control food intake and energy balance, qRT-PCR was performed to examine mRNA transcriptional levels of agouti-related protein (AgRP), and POMC. We first compared 18S rRNA and β-actin expression levels between control and TRPC6 KO mice and decided to use 18S rRNA as an internal control for its constant expression between two strains (C_T = 7.4 ± 0.1 vs. 7.2 ± 0.1 in control and TRPC6 KO mice, respectively). The average C_T values in hypothalamus and hindbrain are 17 and 21 for POMC, and 19 and 26 for AgRP, respectively. Results showed that POMC mRNA expression was reduced by 51% in TRPC6 KO mice compared with control mice (Fig. 6A). AgRP expression levels were not different between groups (Fig. 6A). These results suggest that TRPC6 deficiency alters the balance of POMC/AgRP expression in the hypothalamus, favoring hyperphagia and increased activation of certain neurons in this area. In the NTS and AP of the hindbrain, both AgRP and POMC mRNA expression levels were significantly reduced in TRPC6 KO mice compared with WT control mice (Fig. 6B).

Figure 6.

Proopiomelanocortin (POMC) and agouti-related peptide (AgRP) mRNA expression in hypothalamus and nucleus tractus solitarius (NTS) and area postrema (AP) of the hindbrain. A: AgRP and POMC mRNA levels were quantified by quantitative RT-PCR in the hypothalamus of transient receptor potential cation channel 6 (TRPC6) knockout (KO) and control mice. B: AgRP and POMC mRNA levels in the NTS/AP areas of hindbrain in TRPC6 KO and control mice (n = 8–14 per group; *P < 0.05, compared with B6129s control mice by t test).

TRPC6 KO Mice Exhibit Decreased POMC Neuronal Projections

To further assess POMC neurons in TRPC6 KO mice, we examined neuron projection density and POMC neuron numbers in the ARC of TRPC6 KO and control mice at 27 wk of age. Compared with control mice, TRPC6 KO mice (Fig. 7, A–D) exhibited significant reductions (Fig. 7K) in the density of POMC neuronal axon projections in the ARC (Fig. 7, E–H and J). However, the numbers of POMC neurons in the ARC of TRPC6 KO and control mice were not significantly different (383 ± 44 in TRPC6 KO vs. 439 ± 34 in controls, average positive staining cells in 20 slides per animal).

Figure 7.

Reduced proopiomelanocortin (POMC) neuron projections in transient receptor potential cation channel 6 (TRPC6) knockout (KO) mice. Immunohistochemical labeling of POMC neuron axon projections was performed in TRPC6 KO and control mice. A–D: representative images of POMC staining in ARC from bregma −1.6 mm to −2.5 mm in TRPC6 KO mice. E–H: POMC staining in ARC from the same brain locations in B6/129s control mice. 3V, third ventricle. The squares in C and G show the location in the ARC used for quantification, illustrated by representative high-magnification images in I and J. Arrows indicate the neuron terminal fibers. K: quantification of POMC neuron fiber density in TRPC6 KO and control mice. Scale bar = 100 μm (n = 5/group; data are expressed as means ± SE of relative fiber density; *P < 0.05, compared with control mice by t test).

TRPC6 KO Mice Have Reduced Anorexic Responses to Leptin but Intact Responses to MC3/4R Activation

POMC neurons are important for regulation of energy balance, and their activity is modulated by leptin. To test if reduced POMC expression and impaired neuronal projections in TRPC6 KO mice are associated with blunted modulation of food intake by the leptin-POMC pathway in these mice, we tested the acute anorexic effects of leptin and the MC3/4R agonist melanotan II (MTII) in TRPC6 KO and control mice. Leptin injection caused significant reductions in food intake in control mice of both sexes, although the anorexic response to leptin was significantly blunted in male and female TRPC6 KO mice (Fig. 8, A and B). Leptin injection reduced 24-h food intake by 41% and 32% in male and female control mice compared with saline injection, while reducing food intake by only 8% and 18% in male and female TRPC6 KO mice, respectively.

Figure 8.

Food intake responses to leptin or melanotan II (MTII) injections in male and female transient receptor potential cation channel 6 (TRPC6) knockout (KO) and sex-matched control mice. A: change of food intake compared with saline injection at 2, 4, 15, and 24 h after leptin injection (5 mg/kg ip) in male TRPC6 KO and control mice. B: change of food intake compared with saline injection at 2, 4, 15, and 24 h after leptin injection (5 mg/kg ip) in female TRPC6 KO and control mice. C: change of food intake compared with saline injection at 2, 4, 15, and 24 h after MTII injection (4 mg/kg ip) in male TRPC6 KO and control mice. D: change of food intake compared with saline injection at 2, 4, 15, and 24 h after MTII injection (4 mg/kg ip) in female TRPC6 KO and control mice (n = 7–8/group per sex; *P < 0.05 compared with control mice at different time points by t test).

We also examined pSTAT3 by immunofluorescence staining in the ARC as an indicator of leptin receptor activation. However, there were no significant differences in pSTAT3 activation in the arcuate nucleus of control mice and TRPC6 KO mice 45 min after 5 mg/kg of acute leptin intraperitoneal injection (Supplemental Fig. S4).

One important component of leptin’s anorexic action is activating POMC neurons. These neurons, when activated, reduce food intake by releasing α-melanocyte-stimulating hormone (α-MSH) that stimulates MC3/4R in downstream second-order neurons to inhibit food intake and increase energy expenditure (9). Therefore, we also tested whether the anorexic response to injections of MTII is attenuated in TRPC6 KO mice. In contrast to the attenuated responses to leptin, the acute anorexic effects of MTII were not significantly affected by TRPC6 deficiency and MTII caused similar reductions in food intake in male TRPC6 KO and control mice while causing a more pronounced reduction in food intake in female TRPC6 KO compared with female controls (Fig. 8, C and D).

TRPC6 KO Mice Have Impaired Glucose Tolerance and Higher Fasting Plasma Glucose, Leptin, and Insulin Levels

Male and female TRPC6 KO mice were less tolerant to an acute load of glucose compared with sex-matched control mice (Fig. 9, A and B). After an oral GTT, the area under the glucose concentration curve (AUC) in TRPC6 KO mice was significantly higher than in control mice of both sexes (Fig. 9C). Fasting plasma leptin, insulin, and blood glucose concentrations were significantly higher in male and female TRPC6 KO mice compared with control mice (Fig. 9, D–F).

Figure 9.

Glucose tolerance tests and fasting plasma leptin, insulin and glucose concentrations in male and female transient receptor potential cation channel 6 (TRPC6) knockout (KO) and sex-matched control mice. A: blood glucose in male TRPC6 KO and control mice measured over 120 min postglucose gavage. B: blood glucose in female TRPC6 KO and control mice measured over 120 min postglucose gavage. C: blood glucose area under the curve (AUC) during a 120-min oral glucose tolerance test in male and female TRPC6 KO mice and control mice (n = 7–8/group per sex; *P < 0.05, compared with the sex-matched control mice by t test; #P < 0.05, between groups by two-way ANOVA). D: plasma leptin concentrations in male and female TRPC6 KO and control mice. E: plasma insulin concentrations in male and female TRPC6 KO and control mice. F: glucose concentrations in male and female TRPC6 KO and control mice (n = 7–10/group per sex; *P < 0.05, compared with the sex-matched control mice by t test).

TRPC6 KO Mice Have Normal BP and HR and Attenuated BP Responses to Acute Air-Jet Stress despite Obesity

There were no significant differences in mean arterial pressure (MAP) or HR, measured by telemetry 24 h/day for 5 consecutive days, in male or female TRPC6 KO and control mice at baseline (Fig. 10, A and B). However, peak increases of BP in response to air-jet stress were significantly lower in male and female TRPC6 KO mice compared with sex-matched controls (Fig. 10C). The AUCs of MAP during the stress stimulus and poststress period were also significantly reduced in male and female TRPC6 KO compared with control mice (Fig. 10D). The HR changes in response to air-jet stress were similar between control and TRPC6 KO mice (Fig. 10E).

Figure 10.

Baseline mean arterial pressure (MAP), heart rate (HR), and blood pressure and changes during air-jet stress test in male and female transient receptor potential cation channel 6 (TRPC6) knockout (KO) and sex-matched control mice. A: 5-day average MAP at baseline in male and female mice. B: 5-day average HR at baseline in male and female mice (n = 6–11/group per sex; *P < 0.05 compared with sex-matched control mice by t test) C: maximum MAP increases during air-jet stress compared with baseline in male and female TRPC6 KO and control mice. D: area under the curve (AUC) of MAP for male and female TRPC6 KO and control mice during 5 min of air-jet and 30 min of recovery after air-jet (n = 5–6/group per sex; *P < 0.05, compared with sex-matched control mice by t test). E: AUC of HR for male and female TRPC6 KO and control mice during 5 min of air-jet and 30 min of recovery after air-jet.

We also compared the blood pressure and heart rates in male control and TRPC6 KO mice at baseline with a normal diet to 1 and 6 wk after a 45% high-fat diet (HFD). Results showed that HFD increased blood pressure and heart rate in both control and TRPC6 KO mice. However, increased blood pressure in TRPC6 KO mice (5.7 ± 0.4 mmHg) was slightly but significantly lower than in control mice (8.0 ± 0.9 mmHg) after 6 wk of HFD (P < 0.05). Increased HR in TRPC6 KO mice (19 ± 4 beats/min) was also lower than in control mice (26 ± 4 beats/min) but did not reach statistical significance (P = 0.12). (Supplemental Fig. S5).

TRPC6 KO Mice Have Reduced Brain Weight and Increased Weight of Heart, Kidneys, and Liver

As shown in Table 1, male and female TRPC6 KO mice had significantly higher body weight and tissue weights for heart, liver, liver fat/lean mass, kidneys, and brown adipose tissue than sex-matched control mice. Liver fat mass was also significantly increased by ∼92% and 69% in male and female TRPC6 KO mice, respectively, compared with sex-matched control mice. In contrast to other organs, brain weights in male and female TRPC6 KO mice were significantly lower than in male and female control mice (0.44 ± 0.01 vs. 0.50 ± 0.01 g in males and 0.46 ± 0.01 vs. 0.51 ± 0.01 g in females, P < 0.05). We observed some sex differences in body length and tibia length in TRPC6 KO mice. Female TRPC6 KO mice had longer body and tibia lengths than female controls, while no differences were observed between males of both groups. There was less epididymal fat in male TRPC6 KO mice compared with male controls but more ovarian fat in female TRPC6 KO mice compared with female controls (Table 1).

Effects of Administration of the TRPC6 Inhibitor SAR 7334 in Male B6/129s Control Mice on Body Weight

Body weight and food intake were similar at baseline between PBS vehicle and SAR 7334 treated mice. During 7 days of infusion, daily food intake and body weight (Fig. 11, A and B) in the mice receiving SAR 7334 infusion were slightly but significantly increased compared with vehicle infusion mice (P = 0.047, F = 2.23 in food intake and P = 0.020, F = 2.67 in body weight by two-way ANOVA of time interaction with treatment). Also, mice that received SAR 7334 had higher changes of daily food intake, and greater body weight gain (Fig. 11, C and D) than vehicle-treated mice (P = 0.045, F = 2.26 and P = 0.022, F = 2.61, respectively, by two-way ANOVA of time interacts with treatment). Mice with SAR 7334 infusion showed a trend of increased cumulative food intake (Fig. 11E) compared with the vehicle group but did not reach a significant difference (P = 0.35, F = 1.14) level.

Figure 11.

Changes in body weight and food intake in control mice infused with transient receptor potential cation channel 6 (TRPC6) inhibitor SAR 7334 or vehicle. A: food intake. B: body weight. C: changes of daily food intake. D: change of body weight compared with baseline. E: cumulative food intake in male B6/129s mice when infused with SAR 7334 or vehicle by 7 days of minipump (n = 8–9 per group; *P < 0.05, between groups by two-way ANOVA to analyze the effect of time interacts with treatment).

DISCUSSION

There are four important new findings in this study: 1) TRPC6 deficiency significantly increased body weight and fat mass associated with increased daily food intake and reduced energy expenditure in male and female mice; 2) TRPC6 KO mice had reduced POMC mRNA expression in the hypothalamus and NTS and AP of the hindbrain, reduced numbers of POMC neuron projections, smaller brain weight, and impaired anorexic responses to leptin, but intact anorexic responses to MC3/4R activation; 3) TRPC6 KO mice had impaired glucose tolerance, hyperinsulinemia, and increased liver fat; and 4) TRPC6-deficient mice had normal BP and HR and attenuated BP responses to stress, compared with control mice, despite obesity and metabolic disorders that normally raise BP and increase BP responses to stress.

Role of TRPC Family, Including TRPC6, in Regulating Body Weight and Energy Balance

Although the role of TRPC6 in regulating body weight has not, to our knowledge, been previously assessed, a potential role for the TRPC family in food intake regulation was introduced by Cowley et al. (10). They suggested that leptin may reduce food intake by activating nonselective cation channels in POMC neurons, which are neurons recognized to mediate, at least in part, the effects of leptin on energy balance. Qiu et al. (11, 12) also reported that TRPC subunits might contribute to the activation of POMC neurons by leptin. Gao et al. (13) highlighted the role of TRPC5 in mediating the effects of leptin and insulin to stimulate POMC neurons and suggested that serotonin 5-hydroxytryptamine (5-HT)_2C receptors, G_q-subunit-coupled G protein-coupled receptors, may activate TRPC5 in POMC neurons. In the study of Gao et al. (13), male mice with selective deletion of TRPC5 in POMC neurons showed decreased energy expenditure, increased food intake and body weight, and blunted acute anorexic responses to leptin and 5-HT_2C receptor activation. Surprisingly, global TRPC5 deficiency did not alter body weight (14). TRPC3 is another member of the TRPC family expressed in the hypothalamus. Chrétien et al. (15) studied mice with global or conditional TRPC3 KO in the mediobasal hypothalamus and found increased body weight, food intake, and glucose intolerance in global TRPC3 KO mice, whereas mice with TRPC3 deletion only in the mediobasal hypothalamus exhibited increases in body weight and food intake but were not glucose intolerant. These studies suggest that TRPCs may contribute to regulation of body weight, energy balance, and glucose levels.

To our knowledge, no previous study has examined the importance of TRPC6 for normal regulation of body weight, adiposity, cardiovascular function and anorexic responses to leptin and MC3/4R activation. TRPC3 and TRPC6 subunits share 70 to 80% amino acid sequence homology (16). They form either homotetramers or heterotetramers with variable Ca²⁺ permeabilities (17) and have different affinities for Ca²⁺. TRPC3 functionally regulates basal Ca²⁺ influx, whereas TRPC6 is described as a receptor-operated Ca²⁺ channel (18, 19). Our results showed that the obese phenotype of TRPC6 KO mice is similar to that of TRPC3 KO mice previously reported despite no alterations in hypothalamic TRPC3 mRNA and protein expression level. This finding suggests that the obesity observed in TRPC6 KO mice cannot be explained by reduced TRPC3 levels and that TRPC3 and TRPC6 may independently regulate energy balance and glucose homeostasis.

Our results indicate that as early as 6 wk of age, TRPC6 KO mice are heavier than controls and this difference increases as they age. To examine potential mechanisms of this mild obesity in TRPC6 KO mice, we measured food intake from 6 to 16 wk of age and energy expenditure. In addition to exhibiting increased daily food intake, we found that male and female TRPC6 KO mice showed reduced energy expenditure compared with controls. TRPC6 deficiency was also associated with increased refeeding response to prolonged fasting, suggesting attenuated effectiveness of satiety mechanisms.

Effects of TRPC6 Deficiency on POMC Neurons and Metabolic Regulation

Although TRPC6 mRNA expression has been detected in POMC neurons, the expression pattern of TRPC6 in the hypothalamus has not, to our knowledge, been previously reported. Our study shows that TRPC6 is expressed in the ME, ARC, and VMH and is colocalized with POMC neuron bodies and axons. This observation, along with our finding that TRPC6 KO mice have reduced hypothalamic POMC mRNA expression and attenuated anorexic responses to leptin, suggests that TRPC6 may have an important role in leptin signaling in POMC neurons. While the precise mechanisms leading to reduced POMC expression in the hypothalamus of TRPC6 KO mice are still unclear, we observed no difference in pSTAT3 staining between WT and TRPC6 KO mice 45 min after an acute leptin injection. This result suggests that TRPC6 deletion may not directly affect leptin signaling via the JAK/Stat pathway, or at least the STAT3 component. TRPC6 deficiency may reduce Ca²⁺ influx and the interaction of Ca²⁺ and leptin receptor-mediated phosphoinositide 3-kinase signaling in POMC neurons to suppress POMC mRNA expression (20, 21). Reduced POMC expression in the hypothalamus in TRPC6 KO mice may impair leptin signaling transduction and alter the balance between orexigenic and anorexigenic systems toward a positive energy balance.

Another important finding of the present study is that TRPC6 KO mice had reduced numbers of POMC axon projections in the ARC compared with control mice. Thus TRPC6 deficiency may impair POMC neuron morphogenesis, reducing transmission of neurotransmitters/neuropeptides to second-order neurons. Other studies also support the possibility that TRPC6 has an important role in neuronal morphogenesis and growth (22, 23).

Leptin stimulates expression of POMC in a population of arcuate neurons that process the peptide precursor to α-MSH, which then acts on central MC3R and MC4R to inhibit food intake and increase energy expenditure (24). Although anorexic responses to leptin were attenuated in TRPC6 KO mice, the anorexic responses to MC3/4R activation with MTII remained intact and may have been even more pronounced than in control mice. Thus TRPC6 deficiency does not reduce the sensitivity of MC3/4R-expressing neurons to agonist-mediated activation, consistent with our observation that minimal TRPC6 expression was detected in the PVN where MC4R-expressing neurons are located.

Overall, our results suggest an important role of TRPC6 for leptin signaling in POMC neurons and metabolic regulation. However, one limitation in our study is that the role of AgRP signaling in contributing to the energy balance in TRPC6 KO mice was not fully examined. Although our results show that AgRP mRNA expression level was not significantly different in the hypothalamus of control and TRPC6 KO mice, whether TRPC6 may affect AgRP neuron activity is still unknown. The precise mechanisms by which TRPC6 contributes to the leptin-mediated activation of POMC neurons and whether TRPC6 in other leptin receptor-expressing neurons, such as AgRP neurons, may also regulate energy balance and glucose metabolism warrant further investigation.

TRPC6 and Overall Brain Development

Our study shows that TRPC6 KO mice had significantly higher weight of several major organs such as the heart, kidneys, and liver, as expected for obese animals with increased body weight. In contrast to other organs, brain weight was significantly reduced in TRPC6 KO mice compared with controls. This phenotype is similar to leptin-deficient ob/ob mice, which also exhibit smaller brains and heavier organs when compared with wild-type controls (25), although TRPC6 KO mice are not as obese as ob/ob mice.

Previous studies have shown that leptin plays an important role in neurogenesis, axon growth, dendrite proliferation, and synapse formation in the developing brain (26, 27). Although TRPC6 has been reported to contribute to neuronal development induced by brain-derived neurotrophic factor (28), whether the smaller brain weight observed in TRPC6 KO mice is mediated, at least in part, via abnormal signaling of leptin and/or brain-derived neurotrophic factor is unclear and merits further investigation. Whether deletion of TRPC6 may affect neuronal development during prenatal or early postnatal ages is also unclear and warrants further study.

A limitation of our study is that global deletion of TRPC6 may affect neuronal development during prenatal or early postnatal ages. Although we did not observe significant differences in the lean mass between WT and TRPC6 KO mice at an early age, we cannot rule out the possibility that some of the metabolic phenotypes were at least partly due to developmental effects or secondary to development of body weight gain.

Potential Peripheral Effects of TRPC6 in Contributing to Increased Adiposity

In the present study, we observed significant increases in overall adiposity in male and female TRPC6 KO mice compared with controls. This increased adiposity may be secondary to greater caloric intake and/or reduced energy expenditure due to impaired central nervous system (CNS) actions of leptin, as discussed previously. However, TRPC6 is also expressed in adipose tissue and differential expression of TRPC6 in preadipocytes and adipocytes suggests a potential role for TRPC6 in adipogenesis. Tan et al. (29) showed that 3T3L1 preadipocyte cells treated with the TRPC6 inhibitor SAR 7334 showed increased differentiation and lipid accumulation.

To overcome the limitation of the global KO in development and functional compensation of other TRP channels, we examined food intake and body weight in WT control mice treated with pharmacological TRPC6 inhibitor SAR 7334 for 7 days. We observed a slightly but significantly increased food intake and body weight during 7 days infusion of SAR 7334 compared with vehicle-treated mice, suggesting that the chronic inhibition of TRPC6 in adult age may also affect energy balance and metabolic regulation. Future studies are needed to test multiple doses and more extended treatment periods with specific TRPC6 antagonists to determine whether the effects of SAR 7334 are due to CNS or peripheral effects. Endogenous TRPC blockers, such as 20-HETE (30), could also be used to target the impact of CNS TRPC6 inhibition on body weight homeostasis.

Cardiovascular Regulation in TRPC6 KO Mice

Diet-induced obesity in humans and rodents is associated with increased risk of hypertension (31, 32). Previous studies from our laboratory and others suggest that elevated leptin levels may increase BP by increasing sympathetic nervous system (SNS) activity (33, 34). We previously showed that leptin receptors in POMC neurons mediate much of the chronic effects of leptin to raise BP (35–38). For example, leptin receptor deficiency, specifically in POMC neurons, completely abolished the rise in BP that occurred in control mice during 7 days of leptin infusion (38). Also, the BP responses to leptin were abolished in MC4R-deficient mice, which become severely obese but do not have hypertension.

Although we did not specifically test the chronic effects of leptin on BP regulation in the present study, TRPC6 mice were obese and had increased plasma concentrations of leptin. However, BP and HR in TRPC6 KO mice were not elevated despite increased body weight gain, hyperleptinemia, impaired glucose tolerance, and hyperinsulinemia. These results are consistent with previous studies which reported normal BP, as assessed by telemetry, in TRPC6 mice (39). In addition, the BP response to acute stress and HFD were both attenuated by TRPC6 deficiency. Based on our finding that leptin’s anorexic effects were attenuated on TRPC6 KO mice, it is possible that TRPC6 also plays a role in mediating the effects of leptin to stimulate SNS activity and raise BP; this possibility is supported by our finding that the increases in BP and HR after 6 wk of a HFD were attenuated in TRPC6 KO mice. However, TRPC6 is also expressed in vascular smooth muscle cells, which may affect vascular resistance and BP regulation (40). Further studies are needed to assess the importance of the central nervous system versus peripheral vascular TRPC6 in long-term regulation of BP.

Perspectives and Significance

TRPC6 appears to play critical roles in brain development (41), neuronal survival (28), and synaptic formation (42). Interruption of normal TRPC6 expression may increase susceptibility to ischemic stroke (43) and Alzheimer’s disease (44, 45), suggesting an essential role for TRPC6 in normal brain functions. Our results provide evidence for a novel and important role of TRPC6 in controlling energy balance and adiposity, as well as glucose homeostasis and in contributing to the anorexic actions of leptin. Although TRPC6 deficiency caused mild obesity, hyperleptinemia, insulin resistance, and glucose intolerance, BP and HR were not increased, suggesting that TRPC6 may also be important in linking increased body weight, hyperleptinemia, and SNS activation with increased blood pressure. An important question is whether the results from our studies in rodents can be translated to humans. There are interspecies differences in the role of some TRP channels in controlling various physiological actions (46, 47), and further studies are needed to determine the importance of TRPC6 channels in regulating energy balance in humans. Our observations provide rationale for future studies to assess the role of TRPC6 in specific neuronal populations of the hypothalamus and other brain regions to more fully understand its potential roles in neuronal development as well as cardiovascular and metabolic regulation.

SUPPLEMENTAL DATA

Supplemental Figs. S1–S5: https://doi.org/10.6084/m9.figshare.16543329.

GRANTS

This authors’ research was supported by National Heart, Lung, and Blood Institute Grant P01 HL51971 (to J. E. Hall), National Institute of Diabetes and Digestive and Kidney Diseases Grants R00DK113280 (to Z. Wang) and R01 DK121411 (to J. M. do Carmo), and National Institute of General Medical Sciences Grants P20 GM104357 (to J. E. Hall) and U54 GM115428 (to J. E. Hall).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Z.W. and J.E.H. conceived and designed research; Z.W., Y.F., L.T.J, J.S., and B.P.X. performed experiments; Z.W., Z.W., Y.F., L.T.J, J.S., and B.P.X. analyzed data; Z.W. and Y.F. interpreted results of experiments; Z.W. prepared figures; Z.W. drafted manuscript; Z.W., J.M.d.C., A.A.d.S., Y.F., L.T.J., J.S., X.L., A.J.M., A.C.M.O., B.P.X., and J.E.H. edited and revised manuscript; Z.W., J.M.d.C, A.A.d.S., Y.F., B.P.X., and J.E.H. approved final version of manuscript.