PPAR-γ regulates carnitine homeostasis and mitochondrial function in a lamb model of increased pulmonary blood flow

Abstract

Objective: Carnitine homeostasis is disrupted in lambs with endothelial dysfunction secondary to increased pulmonary blood flow (Shunt). Our recent studies have also indicated that the disruption in carnitine homeostasis correlates with a decrease in PPAR-γ expression in Shunt lambs. Thus, this study was carried out to determine if there is a causal link between loss of PPAR-γ signaling and carnitine dysfunction, and whether the PPAR-γ agonist, rosiglitazone preserves carnitine homeostasis in Shunt lambs.

Methods and results: siRNA-mediated PPAR-γ knockdown significantly reduced carnitine palmitoyltransferases 1 and 2 (CPT1 and 2) and carnitine acetyltransferase (CrAT) protein levels. This decrease in carnitine regulatory proteins resulted in a disruption in carnitine homeostasis and induced mitochondrial dysfunction, as determined by a reduction in cellular ATP levels. In turn, the decrease in cellular ATP attenuated NO signaling through a reduction in eNOS/Hsp90 interactions and enhanced eNOS uncoupling. In vivo, rosiglitazone treatment preserved carnitine homeostasis and attenuated the development of mitochondrial dysfunction in Shunt lambs maintaining ATP levels. This in turn preserved eNOS/Hsp90 interactions and NO signaling.

Conclusion: Our study indicates that PPAR-γ signaling plays an important role in maintaining mitochondrial function through the regulation of carnitine homeostasis both in vitro and in vivo. Further, it identifies a new mechanism by which PPAR-γ regulates NO signaling through Hsp90. Thus, PPAR-γ agonists may have therapeutic potential in preventing the endothelial dysfunction in children with increased pulmonary blood flow.

Figure 1. PPAR-γ gene silencing decreases PPAR-γ protein levels and activity in ovine pulmonary arterial endothelial cells.

Protein extracts (20 µg) prepared from PAEC transfected with a PPAR-γ siRNA or a scrambled control for 48 h, were analyzed by Western blot analysis and a significant decrease in PPAR-γ protein levels was observed (A). The decrease in PPAR-γ binding activity was reversed after treatment with PPAR-γ agonist, rosiglitazone (10 µM; 24 h) (B). Values are mean ± SE; n = 6–12. *P<0.05 vs scrambled siRNA, †P<0.05 vs PPAR-γ siRNA.

Figure 2. Inhibition of PPAR-γ signaling disrupts carnitine homeostasis in ovine pulmonary arterial endothelial cells.

PAEC were transiently transfected with a PPAR-γ siRNA or a scrambled siRNA for 48 h. HPLC analysis was then performed to determine cellular carnitine levels. There was a significant increase in both acyl carnitine levels (A) and the acyl carnitine to free carnitine (AC∶FC) ratio (B) in PPAR-γ siRNA transfected cells, indicating disruption of carnitine homeostasis. Western blot analysis identified a significant decrease in CPT1 (D), CPT2 (F), and CrAT (H) protein levels in the PPAR-γ siRNA transfected cells. The decrease in CrAT protein levels correlated with a significant decrease in CrAT activity (I). The mRNA levels of CPT2 (E) decreased significantly whereas there was no change in CPT1 (C) and CrAT (G) mRNA levels. Values are mean ± SE; n = 5–12. *P<0.05 vs scrambled siRNA.

Figure 3. Decreased PPAR-γ signaling induces mitochondrial dysfunction in ovine pulmonary arterial endothelial cells.

PAEC were transiently transfected with a PPAR-γ siRNA or a scrambled siRNA for 24 h then exposed or not to the PPAR-γ agonist, rosilglitazone (10 µM) for a further 24 h. The MitoSOX red mitochondrial ROS indicator was then added. Representative images after MitoSOX staining are shown (A, top). Images of 20 random fields were quantified to determine the mean fluorescence intensity of each sample. PPAR-γ inhibition significantly increased mitochondrial ROS levels and this was reversed by rosiglitazone (A). Mitochondrial membrane potential (MMP) was also determined using the DePsipher mitochondrial potential assay kit. Representative images after DePsipher staining are shown (B, top). PPAR-γ inhibition significantly decreased mitochondrial membrane potential and this was reversed by rosiglitazone (B). Total mitochondrial number was evaluated by fluorescent microscopy (C) and flow cytometry (D) in scrambled and PPAR-γ siRNA transfected PAEC stained with Mitotracker green. PPAR-γ gene silencing had no significant affect on mitochondrial number as evaluated by either method. There was also a significant reduction in ATP levels after PPAR-γ siRNA transfection (E). Values are mean ± SE; n = 7–12. *P<0.05 vs scrambled siRNA.

Figure 4. Decreased PPAR-γ signaling increases eNOS uncoupling in ovine pulmonary arterial endothelial cells.

The levels of eNOS (A) and Hsp90 (B) protein levels were unchanged between scrambled and PPAR-γ siRNA transfected PAEC. The interaction of eNOS with Hsp90 was determined by immunoprecipitation (IP) using a specific antiserum raised against eNOS followed by Western blot (IB) analysis with an anti-Hsp90 antibody. The membrane was reprobed for eNOS to normalize for immunoprecipitation efficiency. There was a significant decrease in the association of eNOS with Hsp90 in PPAR-γ siRNA transfected cells (C). The effect of PPAR-γ inhibition on eNOS uncoupling was determined. PAEC were treated or not with eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). Superoxide and NO levels were then determined in the presence or absence of acute laminar shear stress (20dyn/cm², 15 min). PPAR-γ siRNA transfection significantly increased eNOS-derived superoxide levels (D) and decreased NO levels (E) but only under shear-stimulated conditions. Values are mean ± SE; n = 6. *P<0.05 vs scrambled siRNA, no shear; †P<0.05 vs scrambled siRNA, shear.

Figure 5. Rosiglitazone treatment preserves carnitine homeostasis in lambs with increased pulmonary blood flow.

Protein extracts (50 µg) were prepared from peripheral lung tissues from vehicle- or rosiglitazone-treated Shunt lambs and age-matched controls. CPT1, CPT2 and CrAT protein levels were then determined by Western blot analyses. There was a significant decrease in CPT1 (A) and CrAT (C) protein levels in the vehicle-treated Shunt lambs. Rosiglitazone treatment prevented the decrease in the protein levels of CrAT enzyme (C). There was no significant change in the CPT2 protein levels (B) in any of the 3- groups. There was also a significant decrease in CrAT activity in vehicle-treated Shunt lambs, which was prevented by rosiglitazone (D). Acyl carnitines (E) and the AC∶FC ratio (F) were found to be significantly higher in the vehicle-treated Shunt lambs, indicating disruption of carnitine homeostasis. However, carnitine homeostasis was preserved by rosiglitazone treatment (E & F). Values are mean ± SE; n = 4–6 for each group. *P<0.05 vs control; †P<0.05 vs shunt.

Figure 6. Rosiglitazone treatment preserves mitochondrial function in lambs with increased pulmonary blood flow.

Protein extracts (50 µg) were prepared from peripheral lung tissues from vehicle- or rosiglitazone-treated Shunt lambs and age-matched controls. UCP-2 protein levels were then determined using Western blot analysis. There was a significant increase in UCP-2 protein levels in the vehicle-treated Shunt lambs (A). The increase in UCP-2 was not observed in Shunt lambs treated with rosiglitazone (A). There was also a significant reduction in lactate∶pyruvate ratio (B) and ATP levels (C) in vehicle-treated Shunt lambs. However, both the lactate∶pyruvate ratio and ATP levels were preserved by rosiglitazone and were unchanged compared to age-matched control lambs (B & C). Values are mean ± SE; n = 5–7 for each group. *P<0.05 vs control; †P<0.05 vs shunt.

Figure 7. Rosiglitazone treatment preserves NO signaling in lambs with increased pulmonary blood flow.

The interaction of eNOS with Hsp90 was determined by immunoprecipitation using specific antiserum raised against eNOS using peripheral lung extracts prepared from vehicle- or rosiglitazone-treated Shunt lambs and age-matched controls. Immunoprecipitated extracts were analyzed using antisera against either eNOS or Hsp90. The levels of eNOS protein associated with Hsp90 relative to total eNOS protein were calculated. There is a significant decrease in the association of eNOS with Hsp90 in vehicle-treated Shunt lambs, which is prevented by rosiglitazone (A). Further, there is an increase in NOS-derived superoxide generation (B) and decreased NO_x levels (C) in vehicle-treated Shunt lambs, indicating increased eNOS uncoupling. Rosiglitazone treatment preserved eNOS coupling and both NOS-derived superoxide (B) and NO_x (C) were unchanged to observed in age-matched control lambs. Values are mean ± SE; n = 4–8 for each group. *P<0.05 vs control; †P<0.05 vs shunt.