ABSTRACT
Objective
Perivascular adipose tissue (PVAT) surrounds most peripheral blood vessels and exerts an anti‐contractile influence through paracrine mediators. Although numerous vasoactive factors have been identified, the mechanisms linking adipocyte metabolism to PVAT‐dependent modulation of vascular tone remain poorly defined. Because adipocytes store energy as triglycerides hydrolyzed by adipose triglyceride lipase (ATGL) and hormone‐sensitive lipase (HSL) to generate free fatty acids, we hypothesized that lipolysis‐derived fatty acids may contribute to PVAT's anti‐contractile actions through activation of long‐chain fatty acid‐sensing G protein‐coupled receptors, Gpr40 and/or Gpr120.
Methods
Mesenteric resistance arteries (MRAs) from adult Wistar rats were studied using wire myography, with or without PVAT, and pharmacological agonists/antagonists and endothelial denudation were used to study signaling. To examine changes in hypertension, PVAT and MRAs from spontaneously hypertensive rats (SHRs) were analyzed by western blotting, and plasma from non‐fasting or fasting SHR was assessed by untargeted lipidomics.
Results
In Wistar rats, inhibition of ATGL, but not HSL, abolished PVAT's anti‐contractile effect, and blockade of Gpr40, but not Gpr120, similarly diminished this response, identifying ATGL and Gpr40 as important mediators. Activation of Gpr40 in PVAT‐ and endothelium‐denuded MRAs further recapitulated the anti‐contractile effect in a β‐arrestin‐dependent manner. In SHR, PVAT ATGL expression was significantly upregulated and MRA Gpr40 expression tended to increase. However, circulating Gpr40 ligand abundance was largely unchanged between strains, suggesting that impaired ligand availability is unlikely to underlie PVAT dysfunction in hypertension.
Conclusions
These findings identify a previously unrecognized ATGL‐Gpr40 signaling axis linking adipocyte triglyceride metabolism to PVAT‐mediated regulation of vascular tone.
Keywords: ATGL, free fatty acids, hypertension, lipolysis, PVAT, vascular smooth muscle
1. Introduction
Perivascular adipose tissue (PVAT) is an adipose depot surrounding most blood vessels, with the notable exception of those in the cerebral circulation [1]. It is sometimes described as the fourth layer of the vascular wall (tunica adiposa). PVAT is composed of adipocytes, stem cells, immune cells, and nerves [2, 3], and emerging evidence suggests that it arises from its own distinct precursors, indicating a unique developmental and functional relationship with the vasculature [4]. PVAT also exhibits striking depot‐specific heterogeneity [5]; for instance, white adipocytes predominate in mesenteric PVAT, whereas brown adipocytes are more abundant around the thoracic aorta. Beyond providing structural support, PVAT actively regulates vascular tone through paracrine release of multiple vasoactive mediators and, under physiological conditions, exerts a robust anti‐contractile influence mediated by adipokines, nitric oxide, potassium ions, hydrogen sulfide, hydrogen peroxide, and angiotensin‐(1–7) [6, 7, 8]. Loss of these anti‐contractile mediators, coupled with increased production of pro‐contractile factors, is widely recognized as a key mechanism underlying the pro‐contractile PVAT phenotype observed in conditions such as obesity [9], hypertension [10], and heart failure [11].
A fundamental role of adipocytes within PVAT, and other adipose tissue stores in the body, is the storage of energy as triglycerides in cytosolic lipid droplets. When energy is needed, metabolic and physiological cues trigger triglyceride hydrolysis, liberating free fatty acids. This lipolytic process is initiated by adipose triglyceride lipase (ATGL), the rate‐limiting enzyme responsible for converting triglycerides into diacylglycerol and free fatty acids and is further propagated by hormone‐sensitive lipase (HSL), which hydrolyzes diacylglycerol into monoacylglycerol [12]. This tightly regulated cascade not only controls lipid turnover within adipocytes but also generates fatty acids that may function as paracrine mediators capable of influencing vascular tone. Thus, triglyceride metabolism within PVAT is well positioned to contribute directly to PVAT‐dependent regulation of vascular function.
One potential mechanism by which PVAT‐derived fatty acids may modulate vascular tone is through activation of free fatty acid receptors (FFARs) expressed on endothelial and vascular smooth muscle cells. FFARs are lipid‐sensing G protein‐coupled receptors (GPCRs) that detect medium‐ and long‐chain fatty acids and translate changes in extracellular lipid availability into intracellular signaling responses [13]. Among these, Gpr40 (FFAR1) and Gpr120 (FFAR4) are the most widely studied. Both receptors are activated by unsaturated fatty acids, including oleic acid (18:1), linoleic acid (18:2), arachidonic acid (20:4), and docosahexaenoic acid (22:6), and regulate diverse physiological processes such as hormone secretion, inflammation, and systemic metabolism [14]. Within adipose depots, FFARs serve as molecular transducers that link local fatty acid bioavailability to downstream cellular responses, suggesting an important role in PVAT‐mediated vascular signaling.
Despite extensive study of PVAT‐derived paracrine factors, the mechanisms that connect the core metabolic function of adipocytes, lipid storage and mobilization, to PVAT's anti‐contractile effects remain poorly understood. Based on this gap in knowledge, we formulated three hypotheses for the current investigation: (1) inhibition of ATGL and HSL would abolish the anti‐contractile effects of PVAT in resistance arteries from healthy rats; (2) long‐chain fatty acid‐sensing GPCRs, Gpr40 and Gpr120, would mediate PVAT‐driven anti‐contractile signaling; and (3) ATGL/HSL and Gpr40/Gpr120 expression and/or activity would be altered in hypertensive animals. Through our experiments, we identify PVAT ATGL and vascular smooth muscle Gpr40 as central mediators of this lipolytic‐ and free fatty acid‐dependent anti‐contractile pathway in mesenteric resistance arteries (MRA) of healthy rats.
2. Materials and Methods
2.1. Animals
Male and female Wistar rats and spontaneously hypertensive rats (SHR) were obtained from Charles River Laboratories and used at 11–15 weeks of age. The sample sizes reported in the figure legends represent the number of independent rats used per group. Rats were housed two per cage and maintained on a 12:12 h light–dark cycle with ad libitum access to standard chow and water. For plasma lipidomics, chow was removed from a subset of Wistar and SHR animals 24 h prior to euthanasia to generate fasted samples. All procedures adhered to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of South Carolina (protocol #: 2762‐102 015‐050225). Euthanasia was performed using an overdose of isoflurane delivered via nose cone (5% in 100% O2), followed by thoracotomy and exsanguination via cardiac puncture. In designated Wistar and SHR, blood was collected from the abdominal aorta prior to thoracotomy. Plasma was isolated by centrifugation (1500 RCF, 15 min, 4°C) and snap‐frozen in liquid nitrogen for subsequent lipidomic analysis.
2.2. Vascular Reactivity
The mesenteric vascular bed was excised and immediately placed in an ice‐cold Krebs buffer (mmol/L: 118.0 NaCl, 4.7 KCl, 25.0 NaHCO3, 2.5 CaCl2‐2H2O, 1.2 KH2PO4, 1.2 MgSO4‐7H2O, 5.5 glucose and 0.01 EDTA). Third‐order MRA segments (2 mm), with (+PVAT) or without perivascular adipose tissue (‐PVAT), were mounted on DMT 620 M wire myographs for isometric force measurements. Vessels equilibrated for 30 min in Krebs buffer continuously aerated with 5% CO2 and 95% O2 to maintain a pH of 7.4. The relationship between resting wall tension and internal circumference was established according to the Law of Laplace [15]. Following diameter normalization, arterial integrity was verified by contraction to KCl (120 mmol/L) and by endothelium‐dependent relaxation to acetylcholine (ACh, 1 μmol/L) after constriction with the α1‐adrenergic agonist, phenylephrine (PE, 1 μmol/L). Vessels demonstrating > 90% relaxation to this ACh bolus were deemed suitable for experimentation. For studies requiring endothelial denudation (E−), the endothelium was removed by vigorously rubbing the MRA lumen with a hair shaft. Successful removal was defined by a bolus PE contraction of > 10 mN and < 20% relaxation to ACh. Concentration‐response curves were subsequently generated for PE (10−8‐3 × 10−5 mol/L) and KCl (10–80 mmol/L). Pharmacological modulators used in this study included: ATGL inhibitor Atglistatin (Millipore Sigma; 10 μmol/L), HSL inhibitor BAY 59‐9435 (MedChemExpress; 100 nmol/L), CPT1 inhibitor Etomoxir (Millipore Sigma; 10 μmol/L), Gpr40 antagonist GW 1100 (Millipore Sigma; 10 μmol/L), Gpr120 antagonist AH 7614 (Millipore Sigma; 10 μmol/L), Gpr40 agonist LY2922470 (MedChemExpress; 100 nmol/L), dual Gpr40/Gpr120 agonist GW‐9508 (Tocris; 10 μmol/L), and the β‐arrestin/β2‐adaptin interaction inhibitor Barbadin (MedChemExpress; 50 μmol/L). Vehicle was an equivalent volume of DMSO (5–10 μL). Drugs were incubated for 30 min; when two agents were co‐incubated, the first drug was applied 10 min before the second, yielding a total incubation time of 40 min. Contractile responses are expressed in absolute units (mN), as normalization to KCl contraction would eliminate the well‐established anti‐contractile effect of PVAT. Excess PVAT and MRA after vascular reactivity preparation were collected and snap frozen in liquid nitrogen for western blot analysis.
2.3. Western Blot Analysis
PVAT or MRA samples were homogenized in T‐PER lysis buffer supplemented with protease and phosphatase inhibitors. Protein extracts (100 μg for PVAT and 40 μg for MRA) were separated on 10 or 12% SDS‐PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 3% BSA and incubated overnight at 4°C with primary antibodies (1:1000). For PVAT samples, the following antibodies were used: ATGL (ABclonal, Catalog A5126, Lot 4 000 001 192; α‐rabbit secondary), ABHD5 (Novus, Catalog H00051099‐M01, Lot O4121‐1F3; α‐mouse secondary), Perilipin 5 (Novus, Catalog NBP3‐16013, Lot 5 500 006 358; α‐rabbit secondary). For MRA samples, membranes were probed with: Gpr40 (Novus, Catalog NBP3‐12467, Lot 2435.PLD.AP; α‐rabbit secondary). After three washes in TBS‐T, membranes were incubated for 90 min with horseradish peroxidase (HRP)‐conjugated secondary antibodies (1:1000). Chemiluminescent signals were visualized using a GeneGnome XRQ system (Syngene). Loading controls included Ponceau or Direct Blue 71 total protein staining for PVAT samples and HRP‐conjugated β actin (Millipore Sigma, Catalog A3854, Lot 0000239225) for MRA samples. Densitometric analyses were performed using ImageJ (Version 1.52), and data are presented as the ratio of target protein to loading control.
2.4. Lipidomics
Lipidomics was performed by Creative Proteomics on plasma collected from non‐fasting and fasting (24 h) male Wistar and SHR. Thawed plasma (50 μL) was added to 1.5 mL chloroform: methanol (2:1, v/v). Samples were vortexed (1 min), mechanically ground (180 s at 65 Hz), mixed with 0.5 mL ultrapure water, sonicated for 30 min at 4°C, and centrifuged (3000 rpm, 10 min, 4°C). The lower organic phase was collected, dried under nitrogen, and reconstituted in 200 μL isopropanol: methanol (1:1, v/v) containing the internal standard LPC (12:0) (5 μL, 0.14 mg/mL). After centrifugation (12 000 rpm, 10 min, 4°C), supernatants were subjected to UPLC‐MS using an ACQUITY BEH C18 column (100 × 2.1 mm, 1.7 μm) coupled to a Q Exactive mass spectrometer (Thermo Fisher). Lipids were separated with solvents A (60% acetonitrile, 40% water, 10 mM ammonium formate) and B (10% acetonitrile, 90% isopropanol, 10 mM ammonium formate) using a linear gradient from 30% to 100% B (flow 0.3 mL/min, 40°C column, 4°C autosampler). Data were acquired in ESI+ and ESI− modes with the following settings: heater 300°C, sheath gas 45 arb, auxiliary gas 15 arb, sweep gas 1 arb, capillary 350°C, spray voltage 3.0 kV (ESI+) or 3.2 kV (ESI−), S‐Lens RF 30% (ESI+) or 60% (ESI−). Lipidomic comparisons included non‐fasting Wistar versus non‐fasting SHR and fasting Wistar versus. fasting SHR.
2.5. Statistical Analysis
Data are expressed as mean ± SEM. Concentration‐response curves were analyzed using non‐linear regression to determine E max and LogEC50 values. Strain comparisons were assessed using unpaired t‐tests, and statistical significance was accepted at p < 0.05. Analyses were performed in GraphPad Prism 10.6.1.
Raw lipidomic data were processed and aligned in LipidSearch (Thermo Fisher) based on m/z and retention time, and features from ESI+/ESI− modes were merged and imported into SIMCA‐P (v14.1) for multivariate analysis. Principal Components Analysis (PCA) was used for unsupervised data visualization and outlier detection, followed by supervised modeling with PLS‐DA or OPLS‐DA to identify discriminant lipid species. Lipid species were additionally screened for putative Gpr40 ligands. Lipids were classified as candidate Gpr40 ligands if any acyl chain (FA1, FA2, or FA3) contained a known long‐chain fatty acid agonist of Gpr40, specifically: 18:1 (oleate), 18:2 (linoleate), 20:4 (arachidonate), or 22:6 (DHA). This criterion was applied independent of lipid class (e.g., FFA, LPC, PC, PG, TG, sphingolipids) because Gpr40 signaling depends on fatty acid structure rather than lipid headgroup identity. Differential abundance between WKY and SHR was subsequently determined using Log2(FC) and p‐value, with p < 0.05 defining statistical significance.
3. Results
To determine whether adipose tissue lipases contribute to the anti‐contractile effect of mesenteric PVAT, we first incubated isolated MRA from male Wistar rats with the ATGL inhibitor Atglistatin and the HSL inhibitor BAY 59‐9435. As expected, MRA with intact PVAT displayed significantly attenuated PE‐induced contraction compared with PVAT‐denuded MRA (Figure 1A). Notably, Atglistatin, but not BAY 59–9435, significantly increased contraction in MRA with PVAT (Figure 1B,C). Neither inhibitor altered contraction in PVAT‐denuded MRA, indicating that the effect of Atglistatin were PVAT‐dependent (Figure 1D,E). Similarly, Atglistatin‐ and PVAT‐specific effects were observed in non‐GPCR‐induced contractions to KCl (Figure S1) and in MRA with intact PVAT from female Wistar rats (Figure S2). Together, these findings identify ATGL as a novel contributor to the anti‐contractile effect of mesenteric PVAT.
FIGURE 1.
Adipose triglyceride lipase (ATGL), but not hormone‐sensitive lipase (HSL), contributes to the anti‐contractile effect of PVAT. Concentration‐response curves to phenylephrine (PE), with or without Atglistatin (10 μmol/L) or BAY 59–9435 (100 nmol/L), in PVAT intact (+PVAT) or PVAT denuded (‐PVAT) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 4–12. Non‐linear regression analysis (E max): **p < 0.01, ***p < 0.001.
Next, we investigated whether mitochondrial β‐oxidation of ATGL‐derived fatty acids contributed to the anti‐contractile effect of PVAT. MRA were incubated with Etomoxir, an irreversible inhibitor of CPT1, the rate‐limiting enzyme in fatty acid oxidation. In PVAT‐intact MRA, Etomoxir modestly reduced contraction (Figure S3A), whereas no effects were observed in PVAT‐denuded MRA (Figure S3B). These results indicate that oxidation of liberated fatty acids does not mediate the anti‐contractile influence of PVAT.
Because long‐chain fatty acids can activate the GPCRs Gpr40 [16] and Gpr120 [17], we tested whether these receptors contribute to PVAT‐dependent anti‐contraction. Incubation of MRA with intact PVAT and the Gpr40 antagonist GW 1100 significantly increased PE‐induced contraction, whereas inhibition of Gpr120 with AH 7614 had no effect (Figure 2A,B). In PVAT‐denuded MRA, GW 1100 did not alter contraction (Figure 2C), while AH 7614 increased contractility (Figure 2D). These findings suggest that Gpr40, but not Gpr120, is a mediator of the PVAT‐dependent anti‐contractile response.
FIGURE 2.
Long‐chain fatty acid receptor, Gpr40, but not Gpr120, contributes to the anti‐contractile effect of PVAT. Concentration‐response curves to phenylephrine (PE), with or without GW‐1100 (10 μmol/L) or AH 7614 (10 μmol/L), in PVAT intact (+PVAT) or PVAT denuded (‐PVAT) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 4–12. Non‐linear regression analysis (E max): **p < 0.01, ***p < 0.001.
To narrow down whether Gpr40 activation alone can modulate vascular tone independent of PVAT, MRA (‐PVAT) were incubated with the selective Gpr40 agonist LY2922470 or the dual Gpr40/Gpr120 agonist GW‐9508. Both agonists significantly reduced PE‐induced contraction in endothelium‐denuded MRA (Figure 3A, Figure S4A), with no effects in vessels with intact endothelium (Figure 3B, Figure S4B). These data suggest that vascular smooth muscle Gpr40 activation reduces contractility in the absence of PVAT.
FIGURE 3.
Specific activation of vascular smooth muscle Gpr40 decreases contractile responses. Concentration‐response curves to phenylephrine (PE), with or without LY2922470 (100 nmol/L), in endothelium denuded (E−) or endothelium intact (E+) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 5–14. Non‐linear regression analysis (E max): ****p < 0.0001.
Because LY2922470 exhibits β‐arrestin‐biased signaling [18], we next examined whether β‐arrestin is required for its anti‐contractile effect. Endothelium‐denuded MRA (‐PVAT) were co‐incubated with LY2922470 and Barbadin, a selective β‐arrestin/β2‐adaptin interaction inhibitor. Barbadin significantly increased PE‐induced contraction, preventing the reduction induced by LY2922470 (Figure 4A), while having no effect under vehicle conditions (Figure 4B). Thus, Gpr40‐mediated reduction in vascular contractility is likely to occur through a β‐arrestin‐dependent pathway.
FIGURE 4.
Inhibition of β‐arrestin prevents the anti‐contractile effect of Gpr40 activation. Concentration‐response curves to phenylephrine (PE), with or without LY2922470 (100 nmol/L), and co‐incubated with Barbadin (50 μmol/L) in endothelium‐denuded (E−) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 5–14. Non‐linear regression analysis (E max): ***p < 0.001, ****p < 0.0001.
Given that the physiological anti‐contractile role of PVAT is impaired in hypertension [10], we assessed whether ATGL‐Gpr40 signaling is altered in SHR. Surprisingly, ATGL expression was increased in SHR PVAT (Figure 5A), and Gpr40 expression in MRA showed a trend toward elevation (Figure 5B). Expression of ATGL regulatory proteins, including ABHD5 (co‐activator) and Perilipin 5 (OXPAT; scaffold), were unchanged (Figure 5C,D). To evaluate endogenous Gpr40 ligands, we performed plasma lipidomics in Wistar and SHR rats, applying a composition‐based screen for fatty acids known to activate Gpr40 (18:1, 18:2, 20:4, and 22:6). Lipid species were retained if any fatty acyl chain matched a known Gpr40‐activating fatty acid, independent of lipid class. This generated a focused list of biologically plausible Gpr40 agonists. Plasma from both Wistar and SHR contained numerous lipid species with established Gpr40‐activating acyl chains. In the non‐fasting state, a subset of ligands differed between strains, with SHR showing higher levels of several phospholipid‐bound species (e.g., LPC, PG, and PC) and lower levels of others (Table 1), indicating selective lipid remodeling rather than uniform enrichment or depletion. However, the majority of Gpr40‐active ligands, spanning lysophospholipids, diacyl phospholipids, MLCL species, triglycerides, and free fatty acids, were present at comparable levels in both strains and did not differ statistically (Table S1). Fasting plasma similarly contained a broad array of Gpr40‐active ligands in both Wistar and SHR. As in the non‐fasting state, a subset of ligands differed between strains, with SHR displaying higher levels of specific phospholipid‐bound species (e.g., PG, PC, and PIP) and lower levels of select LPC, CerP, MLCL, and triglyceride species (Table 2). Nonetheless, the majority of fasting ligands across phospholipids, lysophospholipids, MLCL species, triglycerides, and free fatty acids were not statistically different between strains (Table S2). Collectively, these findings demonstrate that under both non‐fasting and fasting conditions, systemic Gpr40 ligand abundance remains largely comparable between Wistar and SHR. Thus, altered circulating Gpr40 ligand availability is unlikely to account for the impaired anti‐contractile effect of PVAT in hypertension.
FIGURE 5.
Adipose triglyceride lipase (ATGL) and Gpr40 expression were increased in hypertension. Protein expression analysis was performed for ATGL in PVAT (A), Gpr40 in MRA (B), ABHD5 in PVAT (C), and Perilipin 5 in PVAT (D), from Wistar and SHR. Left/above, representative images of immunoblots; right/below, densitometric analysis. Mean ± SEM. n = 5–6. t‐test: *p < 0.05.
TABLE 1.
Plasma lipid species containing established Gpr40‐activating fatty acids (18:1, 18:2, 20:4, or 22:6) that are significantly different between non‐fasted Wistar and SHR. The table includes lipid class annotations, fatty acyl compositions, group mean abundance values, fold‐change calculations, and statistical comparisons between strains. All lipids were selected based solely on the presence of at least one Gpr40‐activating fatty acid, independent of lipid class (n = 5; p < 0.05).
| Lipid species | Class | FA chains | AVG (WKY) | AVG (SHR) | Log2FC (SHR/WKY) | p‐value |
|---|---|---|---|---|---|---|
| LPC(18:1)+HCOO | LPC | 18:01 | 20.37 | 4.03 | −2.34 | 0.00022 |
| MLCL(12:1_22:4_18:2)−H | MLCL | 12:1/22:4/18:2 | 56.96 | 16.67 | −1.77 | 0.00012 |
| PC(18:1_18:1)+HCOO | PC | 18:1/18:1 | 4.94 | 1.62 | −1.61 | 0.00297 |
| LPC(18:1)+H | LPC | 18:01 | 22.51 | 9.40 | −1.26 | 0.00138 |
| CerP(m19:1_20:4)+NH4 | CerP | m19:1/20:4 | 15.75 | 7.23 | −1.12 | 0.00701 |
| LPC(18:1)+H | LPC | 18:01 | 12.73 | 6.64 | −0.94 | 0.0013 |
| FA(20:4)−H | FA | 20:04 | 7059.60 | 4362.69 | −0.69 | 0.01229 |
| PG(16:1_22:6)−H | PG | 16:1/22:6 | 146.86 | 278.73 | 0.92 | 0.00492 |
| TG(4:0_6:0_18:1)+Na | TG | 4:0/6:0/18:1 | 23.90 | 60.58 | 1.34 | 0.00007 |
| LPC(20:4)+H | LPC | 20:04 | 3.48 | 9.61 | 1.46 | 0.00076 |
| PMe(18:1+2O_22:6)−H | PMe | 18:1+2O/22:6 | 73.56 | 253.97 | 1.79 | 0.00782 |
| CerP(m19:1_20:4)+NH4 | CerP | m19:1/20:4 | 4.01 | 15.02 | 1.91 | 0.01931 |
| PC(18:2_20:4)+HCOO | PC | 18:2/20:4 | 7.40 | 30.62 | 2.05 | 0.04671 |
| PG(11:0_18:1)−H | PG | 11:0/18:1 | 8.29 | 50.19 | 2.60 | 0.00007 |
| LPC(18:1)+H | LPC | 18:01 | 2.38 | 24.63 | 3.37 | 0.00033 |
| PC(18:2_18:2)+HCOO | PC | 18:2/18:2 | 2.16 | 22.91 | 3.41 | 0.03548 |
Abbreviations: CerP, ceramide‐1‐phosphate; FA, free fatty acid; LPC, lysophosphatidylcholine; MLCL, monolysocardiolipin; PC, phosphatidylcholine; PG, phosphatidylglycerol; PMe, methoxylated phosphatidylcholine; TG, triacylglycerol.
TABLE 2.
Plasma lipid species containing established Gpr40‐activating fatty acids (18:1, 18:2, 20:4, or 22:6) that are significantly different between fasted Wistar and SHR. The table includes lipid class annotations, fatty acyl compositions, group mean abundance values, fold‐change calculations, and statistical comparisons between strains. All lipids were selected based solely on the presence of at least one Gpr40‐activating fatty acid, independent of lipid class (n = 5; p < 0.05).
| Lipid species | Class | FA chains | AVG (WKY) | AVG (SHR) | Log2FC (SHR/WKY) | p‐value |
|---|---|---|---|---|---|---|
| PC(18:1_18:1)+HCOO | PC | 18:1/18:1 | 17.69 | 6.10 | −1.96 | 0.0126 |
| LPC(18:1)+HCOO | LPC | 18:01 | 41.64 | 11.00 | −1.83 | 0.0041 |
| PE(P‐18:0_22:6)−H | PIP | 18:0/22:6 | 4.27 | 1.31 | −1.71 | 0.0468 |
| LPC(18:1)+H | LPC | 18:01 | 45.07 | 17.34 | −1.38 | 0.0035 |
| CerP(m19:1_20:4)+NH4 | CerP | m19:1/20:4 | 6.82 | 2.74 | −1.31 | 0.0172 |
| FA(20:4)−H | FA | 20:04 | 5734.13 | 2537.45 | −1.18 | 0.0023 |
| MLCL(12:1_22:4_18:2)−H | MLCL | 12:1/22:4/18:2 | 12.94 | 6.27 | −1.04 | 0.0362 |
| TG(16:0_16:0_18:1)+Na | TG | 16:0/16:0/18:1 | 21.34 | 11.27 | −0.99 | 0.0413 |
| TG(17:0_18:2_18:2)+NH4 | TG | 17:0/18:2/18:2 | 150.85 | 247.58 | 0.63 | 0.0274 |
| PC(16:0_18:2)+HCOO | PC | 16:0/18:2 | 3.59 | 6.78 | 0.92 | 0.0479 |
| PIP(18:1_22:6)−H | PIP | 18:1/22:6 | 2.84 | 5.81 | 1.03 | 0.0465 |
| PG(16:1_22:6)−H | PG | 16:1/22:6 | 124.61 | 271.64 | 1.13 | 0.0019 |
| PC(18:2_20:4)+HCOO | PC | 18:2/20:4 | 1.58 | 3.65 | 1.21 | 0.0495 |
| PG(18:1_22:6)−H | PG | 18:1/22:6 | 6.58 | 17.55 | 1.42 | 0.008 |
| PC(18:0_22:6)+HCOO | PC | 18:0/22:6 | 3.69 | 10.09 | 1.45 | 0.0438 |
| PG(18:0_20:4)−H | PG | 18:0/20:4 | 2.92 | 8.43 | 1.53 | 0.0451 |
| PC(18:1_18:2)+HCOO | PC | 18:1/18:2 | 39.83 | 114.94 | 1.53 | 0.0269 |
| PC(18:2_18:2)+HCOO | PC | 18:2/18:2 | 0.66 | 2.02 | 1.60 | 0.0026 |
| TG(18:1_18:1_18:1)+NH4 | TG | 18:1/18:1/18:1 | 10.40 | 33.01 | 1.67 | 0.0036 |
| PMe(18:1+2O_22:6)−H | PMe | 18:1+2O/22:6 | 25.25 | 115.71 | 2.20 | 0.0175 |
Abbreviations: CerP, ceramide‐1‐phosphate; FA, free fatty acid; LPC, lysophosphatidylcholine; MLCL, monolysocardiolipin; PC, phosphatidylcholine; PG, phosphatidylglycerol; PIP, phosphatidylinositol phosphate; PMe, methoxylated phosphatidylcholine; TG, triacylglycerol.
4. Discussion
PVAT is well established as an active metabolic and signaling organ that modulates vascular tone, but little is known about the molecular link between adipocyte lipid metabolism and vascular anti‐contraction. Classically, PVAT's inhibitory effect on vascular contraction has been attributed to anti‐contractile mediators such as adipokines (e.g., adiponectin) and hyperpolarizing factors such as nitric oxide, potassium channels, hydrogen sulfide, and hydrogen peroxide [6, 8]. Our findings extend this paradigm by showing that lipolytic triglyceride turnover, specifically through ATGL, is an important determinant of the PVAT anti‐contractile phenotype. Inhibition of ATGL, but not HSL, abolished PVAT‐dependent anti‐contraction, indicating that the initial hydrolysis step of triglycerides within lipid droplets can help generate vasoactive long‐chain fatty acids.
Classically, ATGL is widely expressed and active in conventional adipose depots [19], with comparable mRNA levels in visceral and subcutaneous fat [20], and expression generally increasing in obesity as an adaptive response to excess triglyceride storage [21]. ATGL‐mediated lipolysis is essential for systemic metabolic homeostasis, supporting lipid turnover and preventing excessive adipocyte hypertrophy [19]. However, its cardiovascular consequences appear highly context‐dependent. Appropriate lipolysis maintains lipid flux, whereas dysregulated or excessive fatty acid release may promote ectopic lipid deposition in non‐adipose organs, inflammation, oxidative stress, and tissue injury [22]. This bidirectional potential likely reflects a physiological balance between lipid storage and mobilization, differences between adipose tissue depots, and the interplay between metabolic demand and systemic stress.
Our findings directly contrast a recent publication showing that genetic deficiency of adipocyte ATGL reduced vascular contractility, and adipocyte‐specific rescue of ATGL partially restored contractile tone [23]. A key distinction between the two studies is experimental strategy: our design employed acute (30 min) pharmacologic inhibition of ATGL using Atglistatin ex vivo, whereas Schrammel and colleagues used chronic, adipocyte‐specific genetic deletion in vivo, which likely induces long‐term metabolic and transcriptional adaptations. Notably, in their model, reduced contractility to PE occurred both in the presence and absence of PVAT, indicating that adipocyte‐specific ATGL deletion modulated vascular contractility through PVAT‐independent mechanisms, potentially involving vascular smooth muscle and/or endothelial signaling. The identity of the vasoactive mediator responsible for reduced contractility, or the pro‐contractile factor downstream of ATGL activity, was not determined. Additional methodological differences may also contribute to the divergent outcomes. We studied mesenteric resistance arteries, which are enveloped by predominantly white adipose tissue (WAT), whereas Schrammel et al. [23] assessed the aorta, which is predominantly covered by brown adipose tissue (BAT). BAT is highly vascularized, metabolically active, and specialized for thermogenic energy expenditure, whereas WAT is less vascularized and functions primarily as a lipid storage depot [6]. Given these differences in cellular composition and metabolic activity, the vasoactive mediators released by BAT and WAT are unlikely to be equivalent, and thus the vascular consequences of ATGL modulation may differ between vascular beds [24].
We suggest that Gpr40 is the receptor responsible for sensing long‐chain free fatty acids liberated by ATGL. Specifically, inhibition of Gpr40 prevented the anti‐contractile effect of PVAT, whereas Gpr120 blockade did not, highlighting receptor specificity despite overlapping ligand profiles. Importantly, direct Gpr40 activation reduced contractile tone even in the absence of PVAT and required endothelial denudation, indicating that Gpr40 primarily signals through vascular smooth muscle rather than through endothelium‐dependent pathways. The anti‐contractile effect of Gpr40 agonism depended on β‐arrestin, demonstrating that biased, non‐canonical signaling is functionally important. This signaling is notable because classical Gpr40 activation couples to phospholipase C (PLC), which hydrolyzes phosphatidylinositol‐4,5‐bisphosphate (PIP₂) to generate inositol‐1,4,5‐trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ then binds to its receptor on the endoplasmic reticulum, triggering Ca2+ release and elevating intracellular Ca2+ concentration ([Ca2+]ᵢ) [25]. Therefore, under classical Gpr40 signaling, we would have expected enhanced vascular contraction in endothelium‐denuded MRA treated with the Gpr40 agonists LY2922470 or GW‐9508. Instead, we consistently observed reduced contractile responses to both agonists. This implies activation of a non‐calcium‐dependent signaling pathway and is consistent with prior work identifying LY2922470 as a β‐arrestin‐biased ligand [18]. Notably, LY2922470, but not Gpr40 agonist TAK‐875, has been shown to attenuate inflammatory signaling in endothelial cells, suggesting that these anti‐inflammatory effects were similarly dependent on β‐arrestin signaling [26]. Moreover, Gpr40 can function as a low‐affinity receptor for epoxyeicosatrienoic acids (EETs) in endothelial cells, vascular smooth muscle cells, and intact arteries [27], suggesting a mechanism of a purported endothelium‐derived hyperpolarizing factor (EDH). Together, these findings highlight that Gpr40 signaling in vascular cells is both ligand‐ and context‐dependent: distinct agonists may engage different signaling pathways and produce divergent outcomes on vascular tone, consistent with biased agonism [28].
To determine whether altered ATGL‐Gpr40 signaling contributes to the loss of PVAT‐mediated anti‐contractility in hypertension, we first assessed expression of key pathway components in SHR and found that PVAT ATGL expression was increased, while vascular Gpr40 expression showed a modest upward trend. These findings prompted us to examine whether endogenous Gpr40 ligand availability might also be altered. Accordingly, we performed untargeted lipidomics on plasma from non‐fasting and fasting rats. Systemic levels of established Gpr40 ligands were largely preserved across strains under both nutritional states, indicating that impaired circulating ligand supply is unlikely to account for dysfunctional PVAT signaling in SHR. Instead, selective remodeling of discrete phospholipid species suggests altered lipid handling rather than global depletion of Gpr40‐dependent vasoactive fatty acids. Notably, the apparent upregulation of ATGL and Gpr40 contrasts with the impaired anti‐contractile function of PVAT in hypertension [10]. One possible explanation is that these changes reflect a compensatory response rather than enhanced signaling capacity. In this context, increased expression may not translate into functional signaling. Thus, despite increased expression of pathway components, the ATGL‐Gpr40 axis may be uncoupled or dysregulated in hypertension, contributing to the loss of PVAT‐mediated anti‐contraction.
While this work reveals a novel ATGL‐GPR40 signaling axis that links PVAT lipolysis to microvascular function, we would like to acknowledge the following limitations with our work: (1) We did not directly quantify long chain fatty acid or Gpr40 ligand release from PVAT; thus, the proposed link between ATGL‐dependent lipolysis and GPR40 activation is inferred from complementary pharmacological and myography data. (2) Our conclusions regarding Gpr40 involvement are based primarily on acute pharmacological modulation. Although our multiple complementary approaches support receptor specificity, independent genetic or molecular validation would further strengthen causal inference. (3) While our myograph data in endothelium‐denuded mesenteric resistance arteries are suggestive that Gpr40 signaling in vascular smooth muscle is responsible for diminished contraction, we did not directly visualize receptor localization within the vessel wall. (4) Lipidomic analysis was performed in systemic plasma rather than PVAT tissue. Given the likely paracrine nature of this signaling axis, direct assessment of Gpr40 ligands within PVAT would have provided more relevant mechanistic insight. Collectively, these limitations highlight areas for further refinement, but do not diminish our overall conclusion that ATGL‐dependent lipolysis and Gpr40 signaling represent an important pathway linking PVAT metabolism to microvascular function, and their investigation should be pursued further.
5. Perspectives
In summary, this work has started to reveal a previously unrecognized lipolytic signaling axis linking PVAT triglyceride metabolism to vascular smooth muscle anti‐contraction. We demonstrate that ATGL‐dependent liberation of long‐chain fatty acids is required for the anti‐contractile function of PVAT and that these lipolytic products act predominantly through vascular smooth muscle Gpr40 via β‐arrestin‐biased signaling. This mechanism extends classical paradigms of PVAT‐derived paracrine mediators by suggesting that lipid flux is a dynamic regulator of vascular tone, although its contribution to PVAT dysfunction in hypertension remains incompletely understood. Nevertheless, taken together, these findings position adipocyte lipolysis and biased GPCR signaling as important regulators of PVAT function and highlight a framework through which metabolic dysregulation may translate into vascular dysfunction.
Funding
This work was supported by the NIH (R01HL149762, R21AG085331 to CFW; R00HL151889, R56HL169223 to CGM), the Alzheimer's Association (AARG‐NTF‐23‐1 145090 to CFW), the USC Columbia Research Institutes Funding Program (CFW, CGM), a USC ASPIRE Award (CGM), the National Council for Scientific and Technological Development (CNPq; 310 719/2023‐2 to FAA and DFS), and the Foundation for Research Support in the State of Bahia (FAPESB; APP 0024/2023 to FAA and DFS).
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Figure S1: Adipose triglyceride lipase (ATGL) contributes to the anti‐contractile effect of PVAT, independent of a GPCR agonist. Concentration‐response curves to potassium chloride (KCl), with or without Atglistatin (10 μmol/L), in PVAT intact (+PVAT) or PVAT denuded (‐PVAT) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 5–6. Non‐linear regression analysis (E max): *p < 0.05, **p < 0.01.
Figure S2: Adipose triglyceride lipase (ATGL) contributes to the anti‐contractile effect of PVAT from female rats. Concentration‐response curves to phenylephrine (PE) with or without Atglistatin (10 μmol/L), in PVAT intact (+PVAT) or PVAT denuded (‐PVAT) mesenteric resistance arteries from female Wistar rats. Mean ± SEM; n = 7–8. Non‐linear regression analysis (LogEC50): **p < 0.01, ****p < 0.0001.
Figure S3: The anti‐contractile effect of PVAT is not related to mitochondrial β oxidation. Concentration‐response curves to phenylephrine (PE) with or without Etomoxir (10 μmol/L), in PVAT intact (+PVAT) or PVAT denuded (−PVAT) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 4–12.
Figure S4: Non‐specific activation of vascular smooth muscle Gpr40 decreases contractile responses. Concentration‐response curves to phenylephrine (PE), with or without GW‐9508 (1 μmol/L), in endothelium denuded (E−) or endothelium intact (E+) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 5–14. Non‐linear regression analysis (E max): *p < 0.05.
Table S1: Plasma lipid species containing established Gpr40‐activating fatty acids (18:1, 18:2, 20:4, or 22:6) that are not significantly different between non‐fasted Wistar and SHR. The table includes lipid class annotations, fatty acyl compositions, group mean abundance values, fold‐change calculations, and statistical comparisons between strains. All lipids were selected based solely on the presence of at least one Gpr40‐activating fatty acid, independent of lipid class (n = 5; p > 0.05).
Table S2: Plasma lipid species containing established Gpr40‐activating fatty acids (18:1, 18:2, 20:4, or 22:6) that are not significantly different between fasted Wistar and SHR. The table includes lipid class annotations, fatty acyl compositions, group mean abundance values, fold‐change calculations, and statistical comparisons between strains. All lipids were selected based solely on the presence of at least one Gpr40‐activating fatty acid, independent of lipid class (n = 5; p > 0.05).
Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request. Lipidomics datasets will be made publicly available in an appropriate repository upon acceptance of the manuscript.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1: Adipose triglyceride lipase (ATGL) contributes to the anti‐contractile effect of PVAT, independent of a GPCR agonist. Concentration‐response curves to potassium chloride (KCl), with or without Atglistatin (10 μmol/L), in PVAT intact (+PVAT) or PVAT denuded (‐PVAT) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 5–6. Non‐linear regression analysis (E max): *p < 0.05, **p < 0.01.
Figure S2: Adipose triglyceride lipase (ATGL) contributes to the anti‐contractile effect of PVAT from female rats. Concentration‐response curves to phenylephrine (PE) with or without Atglistatin (10 μmol/L), in PVAT intact (+PVAT) or PVAT denuded (‐PVAT) mesenteric resistance arteries from female Wistar rats. Mean ± SEM; n = 7–8. Non‐linear regression analysis (LogEC50): **p < 0.01, ****p < 0.0001.
Figure S3: The anti‐contractile effect of PVAT is not related to mitochondrial β oxidation. Concentration‐response curves to phenylephrine (PE) with or without Etomoxir (10 μmol/L), in PVAT intact (+PVAT) or PVAT denuded (−PVAT) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 4–12.
Figure S4: Non‐specific activation of vascular smooth muscle Gpr40 decreases contractile responses. Concentration‐response curves to phenylephrine (PE), with or without GW‐9508 (1 μmol/L), in endothelium denuded (E−) or endothelium intact (E+) mesenteric resistance arteries from male Wistar rats. Mean ± SEM; n = 5–14. Non‐linear regression analysis (E max): *p < 0.05.
Table S1: Plasma lipid species containing established Gpr40‐activating fatty acids (18:1, 18:2, 20:4, or 22:6) that are not significantly different between non‐fasted Wistar and SHR. The table includes lipid class annotations, fatty acyl compositions, group mean abundance values, fold‐change calculations, and statistical comparisons between strains. All lipids were selected based solely on the presence of at least one Gpr40‐activating fatty acid, independent of lipid class (n = 5; p > 0.05).
Table S2: Plasma lipid species containing established Gpr40‐activating fatty acids (18:1, 18:2, 20:4, or 22:6) that are not significantly different between fasted Wistar and SHR. The table includes lipid class annotations, fatty acyl compositions, group mean abundance values, fold‐change calculations, and statistical comparisons between strains. All lipids were selected based solely on the presence of at least one Gpr40‐activating fatty acid, independent of lipid class (n = 5; p > 0.05).
Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request. Lipidomics datasets will be made publicly available in an appropriate repository upon acceptance of the manuscript.