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Alpha Alex, Mohammed Ali, Qianyi Luo, Ashay D Bhatwadekar; Circadian rhythm disruption leads to an abnormal Kir4.1 expression due to AMPK inactivation in retinal Müller cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5204.
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© ARVO (1962-2015); The Authors (2016-present)
The Müller cell, a major retinal glial spanning across the retina plays an integral role in potassium homeostasis via inwardly rectifying potassium channel, Kir4.1. In diabetes Kir4.1 is downregulated resulting in a Müller cell dysfunction and swelling. The 5' adenosine monophosphate-activated protein kinase (AMPK) regulates glucose and lipid metabolism. The AMPK also acts as a cellular circadian metabolic regulator which phosphorylates and destabilizes circadian locomotor output cycles kaput (clock) genes. Circadian rhythm disruption (CRD) is known to induce insulin resistance and AMPK imbalance, however, it remains unknown whether CRD affects the Kir4.1 expression. Therefore, we hypothesize that CRD will promote AMPK inactivation with a subsequent reduction in Kir4.1 retinal Müller cells.
We exposed type 2 diabetic (db/db) or control (db/m) mice to both a constant dark (DD) or 12hr light and 12hr dark (LD) cycle for 14 days. Retinas were harvested to determine AMPKα1/2 and KCNJ10 (gene for Kir4.1) mRNA expression. To understand the role of AMPK in clock gene regulation, cultured rat Müller cells (rMC-1) were transfected with 50nM AMPKα1 siRNA or negative control siRNA and the mRNA expression of KCNJ10, and Kir4.1 protein expression was determined using Western blot. In parallel, the rMC-1 cells were treated with 500μM 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), a specific agonist of AMPK and the KCNJ10 mRNA expression were evaluated.
The CRD resulted in a significantly lower AMPKα1 mRNA expression compared to the mice maintained under regular LD cycle (0.5-fold; p<0.01). Furthermore, both AMPKα2 (0.6-fold; p<0.05) and KCNJ10 (0.4-fold; p<0.05) mRNA expression were significantly decreased in LD-db/db and DD-db/m compared to control, LD-db/m. The AMPKα1 silencing resulted in a profound decrease in KCNJ10 (0.8-fold; p<0.05) mRNA expression with a prominent decrease in Kir4.1 expression (0.7-fold; p<0.05). Evidently, AICAR exhibited a 3-fold increase in KCNJ10 mRNA expression (p<0.05).
Our findings demonstrate that CRD is detrimental to normal Kir4.1 expression due to inactivation of AMPK in retinal Müller cells. In conclusion, AMPK is a critical pharmacological target in preventing Müller cell dysfunction in type 2 diabetes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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