Youde Jiang

Compound 49b Prevents Diabetes-Induced Apoptosis through Increased IGFBP-3 Levels

PURPOSE To determine whether Compound 49b, a novel PKA-activating drug, can prevent diabetic-like changes in the rat retina through increased insulin-like growth factor binding protein-3 (IGFBP-3) levels. METHODS For the cell culture studies, we used both human retinal endothelial cells (REC) and retinal Müller cells in either 5 mM (normal) or 25 mM (high) glucose. Cells were treated with 50 nM Compound 49b alone of following treatment with protein kinase A (PKA) siRNA or IGFBP-3 siRNA. Western blotting and ELISA analyses were done to verify PKA and IGFBP-3 knockdown, as well as to measure apoptotic markers. For animal studies, we used streptozotocin-treated rats after 2 and 8 months of diabetes. Some rats were treated topically with 1 mM Compound 49b. Analyses were done for retinal thickness, cell numbers in the ganglion cell layer, pericyte ghosts, and numbers of degenerate capillaries, as well as electroretinogram and heart morphology. RESULTS Compound 49b requires active PKA and IGFBP-3 to prevent apoptosis of REC. Compound 49b significantly reduced the numbers of degenerate capillaries and pericyte ghosts, while preventing the decreased retinal thickness and loss of cells in the ganglion cell layer. Compound 49b maintained a normal electroretinogram, with no changes in blood pressure, intraocular pressure, or heart morphological changes. CONCLUSIONS Topical Compound 49b is able to prevent diabetic-like changes in the rat retina, without producing systemic changes. Compound 49b is able to prevent REC apoptosis through increasing IGFBP-3 levels, which are reduced in response to hyperglycemia.

TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNFα leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNFα, TNFα siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNFα and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNFα and SOCS3 protein levels. Knockdown of TNFα and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNFα leads to decreased phosphorylation of IRS-1(Ser307), which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IR(Tyr960), both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNFα inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1(Ser307), 2) increased SOCS3 levels to decrease total IRS-1 and increase IR(Tyr960), both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNFα levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling.

Application of Isoproterenol Inhibits Diabetic-Like Changes in the Rat Retina

Diabetic retinopathy is the leading cause of blindness to working-age adults. We have recently shown that surgical removal or genetic manipulations to eliminate sympathetic neurotransmission produces many of the retinal changes similar to rodent diabetic retinopathy with normal glucose levels. We hypothesized that application of a beta-adrenergic receptor agonist, isoproterenol, could reach the retina to elicit normal cellular signaling and inhibit the functional and morphological markers of early stage diabetic retinopathy in the rat. Rats were made diabetic by injection of 60 mg/kg streptozotocin. Within 3 days of diabetes-induction, rats were placed into 1 of 3 groups (control, diabetes, or diabetic + isoproterenol). Dose and time course studies were done for isoproterenol using a PKA ELISA and CREB analyses. Once the optimal dose and time course were established, electrical activity of the retina was analyzed by electroretinogram each month for the 8-month study. Western blotting was done for insulin receptor signaling and Akt and ELISA analyses for TNFalpha concentration and cleavage of caspase 3 at 2- and 8-months of diabetes. Diabetes-induced degeneration of neural cells and retinal thickness were assessed at 2 months, while degenerate capillaries were quantitated at 8 months of treatment. Daily application of 50 mM isoproterenol was effective in inhibiting the diabetes-induced loss of a- and b-wave and oscillatory potential amplitudes in the electroretinogram. Isoproterenol blocked the increase in TNFalpha and apoptosis in the diabetic retina. The numbers of degenerate capillaries were also reduced in the treated + diabetes retina. These data strongly suggest that loss of beta-adrenergic receptor signaling may be a key factors in early stage diabetic retinopathy. Resolution of this loss of adrenergic receptor signaling can inhibit some of the hallmarks of diabetic retinopathy in the retina.

Systemic propranolol reduces b-wave amplitude in the ERG and increases IGF-1 receptor phosphorylation in rat retina.

PURPOSE To determine whether systemic application of propranolol, a nonselective beta-adrenergic receptor antagonist, with an osmotic pump will decrease the b-wave amplitude of the electroretinogram (ERG) and increase insulin-like growth factor (IGF)-1 receptor signaling. METHODS Young rats at 8 weeks of age were treated with saline, phentolamine, a nonselective alpha-adrenergic receptor antagonist, or propranolol, a nonselective beta-adrenergic receptor antagonist, delivered by osmotic pumps for 21 days. On the 21st day, all rats underwent electroretinographic analyses followed by collection of the retinas for protein assessment using Western blot analysis for IGF binding protein 3 (IGFBP3), IGF-1 receptor (IGF-1R), Akt, extracellular signal-related kinases 1 and 2 (ERK1/2), and vascular endothelial cell growth factor (VEGF). RESULTS Data indicate that 21 days of propranolol significantly decreased the b-wave amplitude of the ERG. The decrease in the b-wave amplitude occurred concurrently with a decrease in IGFBP3 levels and an increase in tyrosine phosphorylation of IGF-1 receptor on 1135/1136. This phosphorylation of IGF-1 receptor led to increased phosphorylation of Akt and ERK1/2. VEGF protein levels were also increased. CONCLUSIONS Overall, beta-adrenergic receptor antagonism produced a dysfunctional ERG, which occurred with an increase in IGF-1R phosphorylation and activation of VEGF. Systemic application of beta-adrenergic receptor antagonists may have detrimental effects on the retina.

IGFBP-3 and TNF-α regulate retinal endothelial cell apoptosis.

PURPOSE We hypothesized that loss of insulin-like growth factor binding protein 3 (IGFBP-3) signaling would produce neuronal changes in the retina similar to early diabetes. METHODS To understand better the role of IGFBP-3 in the retina, IGFBP-3 knockout (KO) mice were evaluated for neuronal, vascular, and functional changes compared to wild-type littermates. We also cultured retinal endothelial cells (REC) in normoglycemia or hyperglycemia to determine the interaction between IGFBP-3 and TNF-α, as data indicate that both proteins are regulated by β-adrenergic receptors and respond antagonistically. We also treated some cells with Compound 49b, a novel β-adrenergic receptor agonist we have reported previously to regulate IGFBP-3 and TNF-α. RESULTS Electroretinogram analyses showed decreased B-wave and oscillatory potential amplitudes in the IGFBP-3 KO mice, corresponding to increased apoptosis. Retinal thickness and cell numbers in the ganglion cell layer were reduced in the IGFBP-3 KO mice. As expected, loss of IGFBP-3 was associated with increased TNF-α levels. When TNF-α and IGFBP-3 were applied to REC, they worked antagonistically, with IGFBP-3 inhibiting apoptosis and TNF-α promoting apoptosis. Due to their antagonistic nature, results suggest that apoptosis of REC may depend upon which protein (IGFBP-3 versus TNF-α) is active. CONCLUSIONS Taken together, loss of IGFBP-3 signaling results in a phenotype similar to neuronal changes observed in diabetic retinopathy in the early phases, including increased TNF-α levels.

β2-Adrenergic Receptor Knockout Mice Exhibit A Diabetic Retinopathy Phenotype

There is considerable evidence from our lab and others for a functional link between β-adrenergic receptor and insulin receptor signaling pathways in retina. Furthermore, we hypothesize that this link may contribute to lesions similar to diabetic retinopathy in that the loss of adrenergic input observed in diabetic retinopathy may disrupt normal anti-apoptotic insulin signaling, leading to retinal cell death. Our studies included assessment of neural retina function (ERG), vascular degeneration, and Müller glial cells (which express only β1 and β2-adrenergic receptor subtypes). In the current study, we produced β2-adrenergic receptor knockout mice to examine this deletion on retinal neurons and vasculature, and to identify specific pathways through which β2-adrenergic receptor modulates insulin signaling. As predicted from our hypothesis, β2-adrenergic receptor knockout mice display certain features similar to diabetic retinopathy. In addition, loss of β2-adrenergic input resulted in an increase in TNFα, a key inhibitor of insulin receptor signaling. Increased TNFα may be associated with insulin-dependent production of the anti-apoptotic factor, Akt. Since the effects occurred in vivo under normal glucose conditions, we postulate that aspects of the diabetic retinopathy phenotype might be triggered by loss of β2-adrenergic receptor signaling.

Beta-adrenergic receptor regulation of growth factor protein levels in human choroidal endothelial cells

Remodeling of the choroidal vasculature is a prominent factor in age-related macular degeneration. While many of the growth factors involved in this vascular remodeling are known, their regulation remains much less so. The hypothesis of the present study was that stimulation of human choroidal endothelial cells with the beta-adrenergic receptor agonist isoproterenol would lead to an increase in pigment epithelial derived factor (PEDF) and angiopoietin 1 (Ang1), markers of a stable vasculature. Protein levels of PEDF and Ang1 were significantly increased following stimulation with isoproterenol. However, isoproterenol also significantly increased protein levels of vascular endothelial cell growth factor, which is active during vasculature remodeling. These data suggest that beta-adrenergic receptor agonists are likely upstream of a number of growth factors implicated in ocular disease and have multiple effects on choroidal endothelial cells. Modulation of this signaling in the choroid may offer a new avenue for therapeutics.

Pioglitazone normalizes insulin signaling in the diabetic rat retina through reduction in tumor necrosis factor α and suppressor of cytokine signaling 3

Background: Tumor necrosis factor α (TNFα) impairs insulin signaling in the retina. Results: Pioglitazone reduced TNFα- and SOCS3-activated insulin resistance pathways in retinal cells as well as in lysates from whole rat retina. Conclusion: PPARγ regulates insulin signaling in retina. Significance: Increased understanding of retinal insulin signaling may lead to new therapies for type 2 diabetes. Dysfunctional insulin signaling is a key component of type 2 diabetes. Little is understood of the effects of systemic diabetes on retinal insulin signaling. A number of agents are used to treat patients with type 2 diabetes to normalize glucose levels and improve insulin signaling; however, little has been done to investigate the effects of these agents on retinal insulin signal transduction. We hypothesized that pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, would normalize retinal insulin signal transduction through reduced tumor necrosis factor α (TNFα) and suppressor of cytokine signaling 3 (SOCS3) activities in whole retina and retinal endothelial cells (REC) and Müller cells. To test this hypothesis, we used the BBZDR/Wor type 2 diabetic rat model, as well as REC and Müller cells cultured in normoglycemia and hyperglycemic conditions, to investigate the effects of pioglitazone on TNFα, SOCS3, and downstream insulin signal transduction proteins. We also evaluated pioglitazones effects on retinal function using electroretinogram and markers of apoptosis. Data demonstrate that 2 months of pioglitazone significantly increased electroretinogram amplitudes in type 2 diabetic obese rats, which was associated with improved insulin receptor activation. These changes occurred in both REC and Müller cells treated with pioglitazone, suggesting that these two cell types are key to insulin resistance in the retina. Taken together, these data provide evidence of impaired insulin signaling in type 2 diabetes rats, which was improved by increasing PPARγ activity. Further investigations of PPARγ actions in the retina may provide improved treatment options.

Intravitreal Injection of IGFBP-3 Restores Normal Insulin Signaling in Diabetic Rat Retina

Diabetes-induced changes in growth factor binding protein 3 (IGFBP-3) and tumor necrosis factor alpha (TNFα) have been linked to decreased insulin receptor signaling in diabetic retinopathy. Our previous studies in retinas of diabetic rats have shown that Compound 49b, a novel β-adrenergic receptor agonist, prevented diabetic changes by increasing IGFBP-3 and decreasing TNFα, thus restoring insulin signaling and protection against diabetic retinopathy. The current study was designed to determine whether boosted expression of IGFBP-3 NB (a non-IGF-1 binding form of IGFBP-3) alone is sufficient to mimic the full actions of Compound 49b in protecting against diabetic retinopathy, as well as testing whether IGFBP-3 NB is linked to a restoration of normal insulin signal transduction. Two months after initiation of streptozotocin-induced diabetes, rats received a single intravitreal injection of IGFBP-3 NB plasmid in the right eye. Four days after injection, electroretinogram (ERG) analyses were performed prior to sacrifice. Whole retinal lysates from control, diabetic, diabetic + control plasmid, and diabetic+ IGFBP-3 NB were analyzed for IGFBP-3, TNFα, suppressor of cytokine signaling 3 (SOCS3), and insulin receptor signaling partners using Western blotting or ELISA. Data show that a single intraocular injection of IGFBP-3 NB in diabetic animals significantly reduced TNFα levels, concomitant with reductions in IRS-1Ser307, SOCS3, and pro-apoptotic markers, while restoring insulin receptor phosphorylation and increasing anti-apoptotic marker levels. These cellular changes were linked to restoration of retinal function. Our findings establish IGFBP-3 as a pivotal regulator of the insulin receptor/TNFα pathway and a potential therapeutic target for diabetic retinopathy.

β-Adrenergic receptor agonist, compound 49b, inhibits TLR4 signaling pathway in diabetic retina

Diabetic retinopathy has recently become associated with complications similar to chronic inflammatory diseases. Although it is clear that tumor necrosis factor‐α is increased in diabetes, the role of innate immunity is only recently being investigated. As such, we hypothesized that diabetes would increase Toll‐like receptor 4 (TLR4) signaling, which could be inhibited by a β‐adrenergic receptor agonist (Compound 49b) previously shown to have anti‐inflammatory actions. In order to investigate β‐adrenergic receptor signaling and TLR4 in the diabetic retina, streptozotocin‐injected diabetic mice, as well as human primary retinal endothelial cells (RECs) and rat retinal Müller cells (rMC‐1) exposed to high glucose (25 mm), were treated with a novel β‐adrenergic receptor agonist, Compound 49b (50 nm), or phosphate‐buffered saline (control). TLR4 and its downstream signaling partners (MyD88, IL‐1 receptor‐associated kinase 1, TNF receptor‐associated factor 6 and total and phosphorylated nuclear factor‐κB) were examined. In addition, we assessed high‐mobility group box 1 (HMGB1) protein levels. Our data showed that diabetes or high‐glucose culture conditions significantly increased TLR4 and downstream signaling partners. Compound 49b was able to significantly reduce TLR4 and related molecules in the diabetic animal and retinal cells. HMGB1 was significantly increased in RECs and Müller cells grown in high‐glucose culture conditions, which was subsequently reduced with Compound 49b treatment. Our findings suggest that high glucose may increase HMGB1 levels that lead to increased TLR4 signaling. Compound 49b significantly inhibited this pathway, providing a potential mechanism for its protective actions.