Abscisic acid synergizes with rosiglitazone to improve glucose tolerance and down-modulate macrophage accumulation in adipose tissue: possible action of the cAMP/PKA/PPAR γ axis

Abstract

Background & aims: Abscisic acid (ABA) is effective in preventing insulin resistance and obesity-related inflammation through a PPAR γ-dependent mechanism. The objective of this study was to assess the efficacy ABA in improving glucose homeostasis and suppress inflammation when administered in combination with rosiglitazone (Ros) and to determine whether PPAR γ activation by ABA is initiated via cAMP/protein kinase A (PKA) signaling.

Methods: Obese db/db mice were fed high-fat diets containing 0, 10, or 70 mg/kg Ros with and without racemic ABA (100 mg/kg) for 60 days. Glucose tolerance and fasting insulin levels were assessed at 6 and 8 weeks, respectively, and adipose tissue macrophage (ATM) infiltration was examined by flow cytometry. Gene expression was examined on white adipose tissue (WAT) and stromal vascular cells (SVCs) cultured with ABA, Ros, or an ABA/Ros combination.

Results: Both Ros and ABA improved glucose tolerance, and ABA decreased plasma insulin levels while having no effect on Ros-induced weight gain. ABA in combination with low-dose Ros (10 mg/kg; Roslo) synergistically inhibited ATM infiltration. Treatment of SVCs with Ros, ABA or ABA/Ros suppressed expression of the M1 marker CCL17. ABA and Ros synergistically increased PPAR γ activity and pretreatment with a cAMP-inhibitor or a PKA-inhibitor abrogated ABA-induced PPAR γ activation.

Conclusions: ABA and Ros act synergistically to modulate PPAR γ activity and macrophage accumulation in WAT and ABA enhances PPAR γ activity through a membrane-initiated mechanism dependent on cAMP/PKA signaling.

Figure 1

Effect of abscisic acid (ABA) and rosiglitazone (Ros) on glucose tolerance and fasting insulin. Obese db/db mice were fed high-fat diets containing 0, 15, or 70 mg/kg diet rosiglitazone maleate (control, Ros^lo, and Ros^hi, respectively) with and without racemic ABA (100 mg/kg diet). On day 42 mice underwent an intraperitoneal glucose tolerance test (IPGTT) (A). Areas under the curve (B) were calculated for each treatment. On day 55 fasting insulin levels were measured for mice on each diet (C). Data are represented as mean ± standard error. Points with different subscripts are significantly different from each other (P<0.05).

Figure 2

Effect of abscisic acid (ABA) and rosiglitazone (Ros) on immune cell infiltration into white adipose tissue. Obese db/db mice were fed high-fat diets containing 0, 15, or 70 mg/kg diet rosiglitazone maleate (control, Roslo, and Roshi, respectively) with and without racemic ABA (100 mg/kg diet). On day 60 the percent of F4/80+CD11b+ in the stromal vascular fractions of abdominal white adipose tissue (Ab. WAT) (A) and subcutaneous WAT (B) were assessed by flow cytometry. The expressions of the M1 marker CCL17 (C) and peroxisome proliferator activated receptor γ (PPAR γ) (D) in Ab. WAT were calculated as a ratio to the housekeeping gene β-actin. Data are represented as mean ± standard error. Points with different subscripts are significantly different from each other (P<0.05).

Figure 3

Effect of abscisic acid (ABA) and rosiglitazone (Ros) on blood immune cells. Obese db/db mice were fed high-fat diets containing 0, 15, or 70 mg/kg diet rosiglitazone maleate (control, Roslo, and Roshi, respectively) with and without racemic ABA (100 mg/kg diet). The percent of CD11b+CCR2+ monocytes (A) and CD4+CD25+FoxP3+ Tregs (B) were assessed in whole blood. Data are represented as mean ± standard error. Points with different subscripts are significantly different from each other (P<0.05).

Figure 4

Effect of abscisic acid (ABA) and rosiglitazone combination on gene expression in lipopolysaccharide (LPS)-treated stromal vascular cells (SVCs). SVCs from abdominal white adipose tissue were isolated from db/db mice fed high-fat control diet for 60 days. Cells were seeded into 24-well plates at 1 × 10⁶ cells/well and treated with LPS (100 ng/mL) with and without ABA (10 μM), Ros (1 μM), or ABA and Ros (ABA/Ros). The relative expressions of genes peroxisome proliferator activated receptor γ (PPAR γ) (A), CCL17 (B), monocyte chemoattractant protein 1 (MCP-1) (C), and mannose receptor (D) were calculated as a ratio to the housekeeping gene β-actin. Data are represented as mean ± standard error. Points with an asterisk are significantly different the control treatment (P<0.05).

Figure 5

Effect of abscisic acid (ABA) and rosiglitazone (Ros) combination on PPAR γ transactivation in 3T3-L1 pre-adipocytes and RAW 264.7 macrophages. 3T3-L1 preadipocytes and RAW 264.7 macrophages were seeded into the lower and upper chambers, respectively, of a 24-transwell plate before being transfected with a pTK.PPRE3x luciferase reporter plasmid driven by the PPRE-containing Acyl-CoA oxidase promoter and a pRL control plasmid. Cells were treated for 24 with ABA (10 μM), Ros (1 or 10 μM), or their combination. Luciferase activity was normalized to pRL activity in the cell extracts and relative luciferase activity was calculated a ratio of the activity in the treatment wells to control wells. Data are represented as mean ± standard error. Points with an asterisk indicate that a treatment is significantly different from control, non-ABA treated well (P<0.05).

Figure 6

Effect of cAMP/PKA inhibition on abscisic acid (ABA)-induced PPAR γ activation in 3T3-L1 preadipocytes. Cells were transfected with a pTK.PPRE3x luciferase reporter plasmid driven by the PPRE-containing Acyl-CoA oxidase promoter and were treated for 6-h with vehicle (DMSO) or Ros (1 μM) with ABA (10 μM), the cAMP-inhibitor 2’5’ dideoxyadenosine (dideoxy, 10 μM), or 14-22 myristolated PKA inhibitor fragment (PKAi, 30 μM) (A). PPAR γ-transfected 3T3-L1 pre-adipocytes were also treated with cAMP activator forskolin (forks, 10 μM) with and without PKAi (B). Luciferase activity was normalized to pRL activity in the cell extracts and relative luciferase activity was calculated a ratio of the activity in the treatment wells to control wells. Data are represented as mean ± standard error. Points with an asterisk indicate that a treatment is significantly different from its respective ‘without ABA’ control (P<0.05).