Effect of Recurrent Hypoglycemia on NRG1/ErbB Receptor Subtypes during Diabetes and its Implications in Motor and Cognitive Functions

Abstract

Background Glycemic discrepancies cause irreversible injury in several brain regions, culminating in improper function and dampen the regenerative efficiency. We intended to understand the influence of extreme disparity caused by glycemic levels on Neuregulin1, ErbB receptor subtypes, Ki67, Syntaxin-1 and Olig1 expression in relation to functional brain impairment as a consequence of cellular dysfunction/degeneration during diabetes. Methods Adult male wistar rats were divided into groups as Control(C), Diabetic(D), Insulin induced control hypoglycemia(C+IIH), Insulin induced diabetic Hypoglycemia(D+IIH). Diabetes was induced using streptozotocin (50mg/kg body-weight) in 0.1M citrate buffer, pH-4.5. Hypoglycemia was induced by insulin (Human Actrapid). C+IIH and D+IIH groups received daily two insulin doses - 1.5IU/kg body-weight and 10.0 IU/kg body-weight respectively for six weeks. Neuro-behavioral tests viz. grid walk test, elevated plus maze and ‘T’ maze tests were done to assess motor function, anxiety and spatial memory respectively. Prooxidant system was assessed by measuring SDH activity, ROS and MDA levels. Antioxidant system was evaluated by measuring specific activity of SOD, catalase and GPx expression. To understand the effect of glycemic stress on neurons and glial cells individually, hyperglycemia and hypoglycemia was induced in Neuro2A and C6 cells in vitro.NRG1 and ErbB receptors’ expression was assessed, both in vitro and in vivo by qPCR. Protein expression of NRG1, Olig1 and Ki67 was confirmed in vivo by immunohistochemistry. Results ROS, SDH activity and MDA levels were found to be elevated in sensory and motor cortex, cerebellum and brainstem of diabetic rat brains which were exuberated during hypoglycemia. Compromised activity of SOD, catalase and GPx was observed in sensory and motor cortex, and cerebellum of experimental rats. Also, in hippocampus and corpus callosum, altered ROS and GPx levels were observed. Defunct antioxidant system fails to counteract the oxidative stress leading to cellular degeneration. Vacuolations in motor and sensory cortex, and corpus callosum, central myelin of cerebellum, cellular alteration of Purkinje neurons, presence of axonal spheroids in the brainstem and disorganization of pyramidal neurons are suggestive of impairment to neural circuits during diabetes and hypoglycemia, impairing the resultant motor and cognitive functions. Elevated oxidative stress due to altered antioxidant system generates a non-conducible environment for regeneration which leads to functional deficits in brain. NRG1/ErbB network plays a critical role in regeneration, glucose homeostasis, attenuating oxidative stress and synaptic functions. Altered expression of NRG1/ErbB receptors was observed in brain, concomitant with expression of proliferativemarker-Ki67 and mature oligodendrocytes’ marker-Olig1. Hyperglycemia and hypoglycemia was induced in Neuro2A and C6 cells for understanding effect of glycemic fluctuations on neurons and glial cells individually. NRG1 and ErbB receptor subtypes were found to be down regulated in both N2A and C6 cells indicative of its compromised function in both neurons and glial cells during hyperglycemia and hypoglycemia. Moreover, down-regulation of Syntaxin-1 in N2A cells suggests poor synaptic stability during hyperglycemic and hypoglycemic conditions. Grid walk test, elevated plus maze test and ‘T’ maze test confirmed motor impairment, anxiogenic response and spatial memory deficits, respectively during diabetes which worsens with hypoglycemic insults. Conclusion Our findings suggest that hyperglycemia and hypoglycemia increases ROS levels concomitant with debilitated antioxidant system, dampening the regenerative potential mediated via NRG1/ErbB receptors in brain regions. Recurrent hypoglycemic events pose potential threat to maintenance of neural circuits needed for execution of cognitive and motor functions. Thus, hyperglycemia and hypoglycemia affect behavior by compromising proliferation and differentiation property of brain cells’ regeneration