Nanoliposomal minocycline for ocular drug delivery.

Nanoliposomal technology is a promising drug delivery system that could be employed to improve the pharmacokinetic properties of clearance and distribution in ocular drug delivery to the retina. We developed a nanoscale version of an anionic, cholesterol-fusing liposome that can encapsulate therapeutic levels of minocycline capable of drug delivery. We demonstrate that size extrusion followed by size-exclusion chromatography can form a stable 80-nm liposome that encapsulates minocycline at a concentration of 450 ± 30 µM, which is 2% to 3% of loading material. More importantly, these nontoxic nanoliposomes can then deliver 40% of encapsulated minocycline to the retina after a subconjunctival injection in the STZ model of diabetes. Efficacy of therapeutic drug delivery was assessed via transcriptomic and proteomic biomarker panels. For both the free minocycline and encapsulated minocycline treatments, proinflammatory markers of diabetes were downregulated at both the messenger RNA and protein levels, validating the utility of biomarker panels for the assessment of ocular drug delivery vehicles. FROM THE CLINICAL EDITOR: Authors developed a nano-liposome that can encapsulate minocycline for optimized intraocular drug delivery. These nontoxic nanoliposomes delivered 40% of encapsulated minocycline to the retina after a subconjunctival injection in a diabetes model.

Circulating sphingolipid biomarkers in models of type 1 diabetes.

Alterations in lipid metabolism may contribute to diabetic complications. Sphingolipids are essential components of cell membranes and have essential roles in homeostasis and in the initiation and progression of disease. However, the role of sphingolipids in type 1 diabetes remains largely unexplored. Therefore, we sought to quantify sphingolipid metabolites by LC-MS/MS from two animal models of type 1 diabetes (streptozotocin-induced diabetic rats and Ins2(Akita) diabetic mice) to identify putative therapeutic targets and biomarkers. The results reveal that sphingosine-1-phosphate (So1P) is elevated in both diabetic models in comparison to respective control animals. In addition, diabetic animals demonstrated reductions in plasma levels of omega-9 24:1 (nervonic acid)-containing ceramide, sphingomyelin, and cerebrosides. Reduction of 24:1-esterfied sphingolipids was also observed in liver and heart. Nutritional stress via a high-fat diet also reduced 24:1 content in the plasma and liver of mice, exacerbating the decrease in some cases where diabetes was also present. Subcutaneous insulin corrected both circulating So1P and 24:1 levels in the murine diabetic model. Thus, changes in circulating sphingolipids, as evidenced by an increase in bioactive So1P and a reduction in cardio- and neuro-protective omega-9 esterified sphingolipids, may serve as biomarkers for type 1 diabetes and represent novel therapeutic targets.

Insulin signaling in retinal neurons is regulated within cholesterol-enriched membrane microdomains.

Neuronal cell death is an early pathological feature of diabetic retinopathy. We showed previously that insulin receptor signaling is diminished in retinas of animal models of diabetes and that downstream Akt signaling is involved in insulin-mediated retinal neuronal survival. Therefore, further understanding of the mechanisms by which retinal insulin receptor signaling is regulated could have therapeutic implications for neuronal cell death in diabetes. Here, we investigate the role of cholesterol-enriched membrane microdomains to regulate PKC-mediated inhibition of Akt-dependent insulin signaling in R28 retinal neurons. We demonstrate that PKC activation with either a phorbol ester or exogenous application of diacylglycerides impairs insulin-induced Akt activation, whereas PKC inhibition augments insulin-induced Akt activation. To investigate the mechanism by which PKC impairs insulin-stimulated Akt activity, we assessed various upstream mediators of Akt signaling. PKC activation did not alter the tyrosine phosphorylation of the insulin receptor or IRS-2. Additionally, PKC activation did not impair phosphatidylinositol 3-kinase activity, phosphoinositide-dependent kinase phosphorylation, lipid phosphatase (PTEN), or protein phosphatase 2A activities. Thus, we next investigated a biophysical mechanism by which insulin signaling could be disrupted and found that disruption of lipid microdomains via cholesterol depletion blocks insulin-induced Akt activation and reduces insulin receptor tyrosine phosphorylation. We also demonstrated that insulin localizes phosphorylated Akt to lipid microdomains and that PMA reduces phosphorylated Akt. In addition, PMA localizes and recruits PKC isotypes to these cholesterol-enriched microdomains. Taken together, these results demonstrate that both insulin-stimulated Akt signaling and PKC-induced inhibition of Akt signaling depend on cholesterol-enriched membrane microdomains, thus suggesting a putative biophysical mechanism underlying insulin resistance in diabetic retinopathy.

Diabetes diminishes phosphatidic acid in the retina: a putative mediator for reduced mTOR signaling and increased neuronal cell death.

PURPOSE: We demonstrated previously that pro-survival insulin receptor, PI3K-Akt, and p70 S6K signaling is diminished in models of diabetic retinopathy. As mammalian target of rapamycin (mTOR), an upstream activator of p70 S6Kinase is, in part, regulated by lipid-derived second messengers, such as phosphatidic acid (PA), we sought to determine if diminished mTOR/p70 S6Kinase signaling in diabetic retinas may reflect diminished PA levels. METHODS: Alterations in PA mass from retinas of control and streptozotocin-induced diabetic rats were determined by mass spectrometry. The biochemical and biophysical mechanisms underlying the actions of PA on insulin-activated mTOR/p70 S6Kinase signaling were determined using R28 retinal neuronal cells. RESULTS: We demonstrate a significant decrease in PA in R28 retinal neuronal cells exposed to hyperglycemia as well as in streptozotocin-induced diabetic rat retinas. Exogenous PA augmented insulin-induced protection from interleukin-1ß-induced apoptosis. Moreover, exogenous PA and insulin cooperatively activated mTOR survival pathways in R28 neuronal cultures. Exogenous PA colocalized with activated mTOR/p70 S6kinase signaling elements within lipid microdomains. The biochemical consequences of this biophysical mechanism is reflected by differential phosphorylation of tuberin at threonine 1462 and serine 1798, respectively, by PA and insulin, which reduce this suppressor of mTOR/S6Kinase signaling within lipid microdomains. CONCLUSIONS: These results identify PA-enriched microdomains as a putative lipid-based signaling element responsible for mTOR-dependent retinal neuronal survival. Moreover, diabetic retinal neuronal apoptosis may reflect diminished PA mass. Elevating PA concentrations and restoring mTOR signaling may be an effective therapeutic modality to reduce neuronal cell death in diabetic retinopathy.