Increased tumor necrosis factor-α, cleaved caspase 3 levels and insulin receptor substrate-1 phosphorylation in the β₁-adrenergic receptor knockout mouse

Abstract

Purpose: To investigate the role of β1-adrenergic receptors on insulin like growth factor (IGF)-1 receptor signaling and apoptosis in the retina using β1-adrenergic receptor knockout (KO) mice.

Methods: Western blotting and enzyme-linked immunosorbent assay analyses were done on whole retinal lysates from β1-adrenergic receptor KO mice and wild-type littermates. In addition, vascular analyses of degenerate capillaries and pericyte ghosts were done on the retina of the β1-adrenergic receptor KO mice versus littermates.

Results: Lack of β1-adrenergic receptors produced a significant increase in both degenerate capillaries and pericyte ghosts. This was accompanied by an increase in cleaved caspase 3 and tumor necrosis factor α levels. IGF-1 receptor phosphorylation was not changed; however, protein kinase B (Akt) phosphorylation was significantly decreased. The decrease in Akt phosphorylation is likely caused by increased insulin receptor substrate-1 serine 307 (IRS-1(Ser307)) phosphorylation, which is inhibitory to IGF-1 receptor signaling.

Conclusions: These studies further support the idea that maintenance of β-adrenergic receptor signaling is beneficial for retinal homeostasis. Loss of β1-adrenergic receptor signaling alters tumor necrosis factor α and apoptosis levels in the retina, as well as Akt and IGF-1 receptor phosphorylation. Since many of these same changes are observed in the diabetic retina, these data support that novel β-adrenergic receptor agents may provide additional avenues for therapeutics.

Figure 1

Degenerate capillaries and pericyte ghosts in the β1-adrenergic receptor retina A: This panel is a bar graph of the number of degenerate capillaries in the β1-adrenergic receptor knockout (KO) mice versus wild-type (WT) littermates. B: This panel is a bar graph of the number of pericyte ghosts in the same samples of retina from β1-adrenergic receptor KO and their WT littermates. *p<0.05 versus WT. n=5 for each group.

Figure 2

Cleaved caspase 3 is increased in knockout animals. Enzyme-linked immunosorbent assay (ELISA) analyses of whole retinal lysates from β1-adrenergic receptor knockout (KO) animals versus their wild-type (WT) littermates. Caspase 3 levels are increased in the knockout mice KO animals. *Significance was found at p<0.05 versus wildtype samples. Animal numbers (N) is equal to 6.

Figure 3

Retina from β1-adrenergic receptor has reduced insulin-like growth factor binding protein (IGFBP)-3 levels. Representative blots and bar graph of IGFBP-3 protein levels in whole retinal lysates in the β1-adrenergic receptor knockout (KO) mice and their wild-type (WT) controls. *Significance was found at p<0.05 versus wildtype samples. Animal numbers (N) is equal to 6.

Figure 4

Insulin like growth factor-1 (IGF-1) receptor phosphorylation does not change in β1-adrenergic receptor knockout retina. Representative blots and bar graph of phosphorylated IGF-1 receptor (Tyr 1135/1136) and total IGF-1 receptor protein levels in retinal lysates in the β1-adrenergic receptor knockout (KO) mice and the wild-type (WT) controls. Bar graph is the ratio of phosphorylated protein to total protein levels. *Significance was found at p<0.05 versus wildtype samples. Animal numbers (N) is equal to 6.

Figure 5

Despite no change in insulin like growth factor-1 receptor phosphorylation, Akt phosphorylation is reduced in retina from β1-adrenergic receptor knockout mice. Representative blots and bar graph of the ratio of phosphorylated Akt (Ser 473) to total Akt protein levels in retinal lysates in the β1-adrenergic receptor knockout (KO) mice and the wild-type (WT) controls. *p<0.05 versus WT. n=5.

Figure 6

Enzyme linked immunosorbent assay results demonstrate that lack of β1-adrenergic receptors increases tumor necrosis factor a levels. Significance demonstrates that p<0.05 versus wildtype samples. Seven animals in each group were used for these studies.

Figure 7

Insulin receptor substrate-1 serine 307 (IRS-1^Ser307) is increased in retinal lysates from β1-adrenergic receptor knockout mice. Western blot image and bar graph of the ratio of phosphorylated IRS-1^Ser307 to total IRS-1 in the retina of β1-adrenergic receptor knockout (KO) mice versus wild-type (WT) littermates. *p<0.05 versus WT, n=4.