Activation of the intracellular Ca++-AMPK pathway ameliorates apoptosis and improves autophagy in diabetic peripheral neuropathy

Abstract

A decrement of AMP-activated protein kinase-eNOS bioavailability is critical for the pathogenesis of diabetic peripheral neuropathy. We evaluated the neuro-protective effect of cinacalcet on DPN by activating the AMPK-eNOS pathway using db/db mice. The db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet or a diet containing cinacalcet. Mice were evaluated sciatic motor nerve conduction and tactile response. We performed Mason's trichrome staining and immunohistochemistry for type IV collagen and 8-hydroxy-deoxyguanosine, an oxidative DNA damage marker. For immunofluorescence double staining, apoptosis was detected by ApopTag Fluorescein In Situ Apoptosis Detection Kit. The sciatic nerve specimens were double stained and we measured unmyelinated fiber areas and axonal areas using NIH Image J under a transmission electron microscope. Western blot assay was performed total protein of sciatic nerve with specific antibodies for CaSR, CaMKK, total LKB1, phospho-Ser428 LKB1, total AMPK, phospho-Thr172 AMPK, total eNOS, phospho-Ser1177 eNOS, PGC-1. The db/db mice showed sensorimotor impairment, nerve fibrosis and inflammation, apoptosis, disorganized myelin with axonal shrinkage and degeneration, and fewer unmyelinated fibers in the sciatic nerve compared to db/m mice. Cinacalcet administration, without causing any changes in blood glucose and Ca++ concentrations, significantly increased the paw withdrawal mechanical threshold and decreased the motor conduction latency, and ameliorated the deterioration of sciatic nerve pathology, accompanied by increases in the expressions of calcium-sensing receptor-CaMKK and phosphorylation of AMPK-eNOS in diabetic mice. Cinacalcet may play an important role in the prevention and amelioration of DPN by amplifying AMPK signaling.