[1524] Activation of AMPK by AICAR Inhibits Palmitate-Induced Increases in Ceramide Mass, Endoplasmic Reticulum (ER) Stress, NF-kappaB, and Apoptosis in Cultured Bovine Retinal Pericytes (BRPC)

JM Cacicedo, E Chou, NB Ruderman, Y Ido. Boston University School of Medicine, Boston, MA

Background: We have previously shown that moderately high levels of the saturated fatty acid palmitate, but not the monounsaturated fatty acid oleate, induces apoptosis in cultured bovine retinal pericytes. An important observation as high levels of saturated fatty acids, in addition to high sugar, may be a causal factor leading to retinal pericyte loss, the hallmark of diabetic retinopathy (DR). In developed countries worldwide, DR is one of the leading causes of blindness to which there is no current preventative therapy. Finding a way to prevent, inhibit, or improve DR is therefore a worthy aim.
Design: Retinal pericytes were cultured from bovine retinas. These cultured cells were greater than 95% pure and only used from passages 3-6. Both palmitate and oleate were conjugated with bovine serum albumin at a 2:1 molar ratio. The cells were incubated in DMEM media with the indicated fatty acid and/or chemical agent for 24 hr and then tested for accumulation of ceramide mass, ER stress markers, NF-kappa B activity, and apoptosis (TUNEL and caspase assays).
Results: We discovered that activation of the stress activated kinase, AMP-activated protein kinase (AMPK), by the cell permeable AMPK activator AICAR (5-amino-4- imidazolecarboxamide riboside) or use of a constitutively active AMPK can prevent retinal pericytes from undergoing palmitate-induced cell death. Though the exact mechanism of palmitate-induced apoptosis is not currently known, we found that it may be brought on by several processes that cause cell death in the context of diabetes. We found that 1) ceramide mass was increased dose dependently by palmitate; 2) ER stress was upregulated as witnessed by a 2-4-fold increase in the ER stress markers BiP and CHOP at both the mRNA and protein levels; 3) NF-kappaB activity was increased as measured by reporter gene assay; and 4) that inhibition of any one of these processes was sufficient to rescue the BRPC from palmitate-induced cell death.
Conclusions: Activation of AMPK with AICAR was able to prevent all the processes studied herein, including apoptosis itself, therefore providing a potential therapeutic option for improving vascular homeostasis in diabetic retinopathy.
Category: Ophthalmic

Tuesday, March 10, 2009 9:30 AM

Poster Session III # 212, Tuesday Morning