ApoA1: mimetic peptide reverses adipocyte dysfunction in vivo and in vitro via an increase in heme oxygenase (HO-1) and Wnt10b

Abstract

Insulin resistance is a risk factor in the development of type 2 diabetes and is a major cause of atherosclerosis. Reduction in heme oxygenase (HO-1) has been shown to exacerbate vascular dysfunction and insulin resistance in obese mice and involves a decrease in adiponectin levels. Adiponectin is released from mesenchymal stem cell (MSC)-derived adipocytes, its levels are decreased in type 2 diabetes. We hypothesized that the apoA1 mimetic peptide, L-4F, will target the expression of the HO-1-adiponectin axis and reverse adipocyte dysfunction both in vivo and in vitro. The administration of L-4F [2 mg/Kg/daily (i.p.) for 4-week to 8-week-old obese (ob) mice restored adipocyte function, increased adiponectin release (p < 0.05) and decreased the levels of IL-1 and IL-6 (p < 0.05)]. These perturbations were associated with an increase in insulin sensitivity (p < 0.01 vs. untreated ob mice) and decreased glucose levels (309 + 42 vs. 201 + 8 mg/d after L-4F treatment). Treatment of both mesenchymal stem cell (MSC)-derived adipocytes with L-4F (50 μg/ml) increased adiponectin (p < 0.05), decreased IL-1 and IL-6 (p < 0.05) levels and increased MSC-derived adipocyte cell numbers by 50% in S phase (p < 0.05). MSC-derived adipocytes treated with L-4F increased WNT10b and decreased Peg 1/Mest. Inhibition of HO activity reversed the decrease in the adipogenic response gene, Peg 1/Mest. An increase of HO-1 expression by L-4F increased insulin-receptor phosphorylation. These findings support the hypothesis that L-4F increases early adipocyte markers, HO-1-adiponectin, WNT10b and decreases Peg1/Mest, negative regulators of adipocyte differentiation.

Figure 1

Effect of L-4F on body weight, blood glucose levels, adiponectin, IL-1 and IL-6 in ob mice. (A–E) respectively. (A) Body weight is shown. (B) Fasting blood glucose levels (*p < 0.001). (C) Plasma adiponectin levels (*p < 0.05). (D) Plasma IL-1 levels (*p < 0.01). (E) Plasma IL-6 levels (*p < 0.01). (F) Blood glucose levels after insulin administration (*p < 0.05). The results are mean ± SE, n = 6 in each group.

Figure 2

Determination of MSCs phenotype and cell cycle measurement. (A) Membrane antigen expressions of CD45, CD4, CD90, CD166 and on hMSCs were analyzed by FACS analysis. As positive markers CD90 (Thy-1) and CD166 (ALCAM, activated leukocyte cell adhesion molecule) were 87.72% and 77% respectively. As negative markers, CD45 (common lymphocytes antigen) and CD34, an hematopoietic stem cell marker, were shown to be expressed in less than 1% of cells. (B) Effect on cell cycle and Growth. Cells were treated Vinblastin to arrest mitosis then we measured DNA distribution by DAPI staining. Cell cycle analysis revealed that 15.5% of MSCs were in the S phase when the cells were cultured in L-4F.

Figure 3

Effect of L-4F on HO-1 expression and activity. (A) Western blot of HO-1 proteins in MSC-derived adipocytes treated with L-4F and SnMP. Representative immunoblots are shown (n = 4). MSCs were cultured in adipogenic differentiation media and L-4F was added every 3 days. Quantitative densitometry evaluation of HO-1 and actin proteins ratio was determined. Data are expressed as means ± SD (*p < 0.05 vs. day 12). (B) Effect of L-4F on HO activity as measured by bilirubin generation. Bilirubin formed in cellular homogenates in the presence of heme and NADPH was measured (*p < 0.001 vs. day 12). Combination of L-4F with SnMP reduces HO activity (#p < 0.001 vs. day 12 + L-4F).

Figure 4

Effect of L-4F on Wnt10b, β-catenin and PPARγ levels. Western blot of Wnt10b, β-catenin, PPARγ and actin proteins in MSC-derived adipocytes treated with L-4F alone or in combination with SnMP. Representative immunoblots are shown (n = 4). Quantitative densitometry evaluation of Wnt10b β-catenin and actin proteins ratio was determined. Data are expressed as means ± SD (*p < 0.05 vs. day 5, ^#p < 0.05 vs. day 12, ⁺p < 0.05 vs. day5 + L4F).

Figure 5

Effect of L-4F on Mest and IRP-Tyr1146. Western blot of Mest, IRP-Tyr1146 and actin proteins in MSC-derived adipocytes treated with L-4F alone or in combination with SnMP. Representative immunoblots are shown (n = 4). Quantitative densitometry evaluation of Mest, IRP-Tyr1146 and actin proteins ratio was determined. Data are expressed as means ± SD (*p < 0.05 vs. day 5, ^#p < 0.05 vs. day 12, ⁺p < 0.05 vs. day 5 + L4F).

Figure 6

Effect of L-4F on adipogenesis and adipocyte size. (A) Scan of lipid size of a representative experiment. Adipogenesis was measured as the relative absorbance of Oil Red O at day 12 after inducing adipogenesis as described in Materials and Methods. (B) Measurement of diameter of lipid droplet size. Results are mean ± SD (*p < 0.05 vs. L-4F, ^#p < 0.05 vs. L-4F + SnMP).

Figure 7

Hypertrophy adipocytes are associated with dysfunctional adipogenesis with resultant contributions toward metabolic imbalance. The proposed mechanism demonstrate that an upregulation of HO-1 is directly involved in the regulation of adipogenic markers by increasing Wnt10b protein levels, decreasing Peg/Mest and improving insulin sensitivity.