Combinatory high-resolution microdissection/ultra performance liquid chromatographic-mass spectrometry approach for small tissue volume analysis of rat brain glycogen

Abstract

Cyto-architectural diversity of brain structures emphasizes need for analytical tools for discriminative investigation of distinctive neural structures. Glycogen is the major energy reserve in the brain. There is speculation that brain utilization of this fuel source may affect detection of hypoglycemia. To evaluate sex-specific regulation of glycogen mass and mobilization in the glucose-sensory ventromedial hypothalamic nucleus (VMN), current research coupled UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) analysis capabilities with novel derivatization protocols for high-sensitivity measurement of glucose and glycogen in small-volume neural tissue samples. This work also sought to demonstrate utility of pairing this approach with optimized Western blot methods for measurement of glycogen metabolic enzyme protein expression. Here, high-resolution micropunch dissection tools for discriminative isolation of VMN tissue were used in conjunction with newly developed glycogen analytical methods and an experimental treatment paradigm for intra-cranial hindbrain-targeted administration of estrogen receptor-alpha (ERα) or -beta (ERβ) receptor antagonists to address the hypothesis that estradiol activates one or both hindbrain ER populations to exert sex-specific regulatory effects on VMN glycogen mass and hypoglycemia-associated mobilization. Outcomes validate a novel multi-analytical platform for investigation of in vivo sex-dimorphic regulation of glycogen metabolism in precisely-defined brain elements under conditions of energy balance versus imbalance. This combinatory approach will facilitate ongoing efforts to elucidate effects of acute versus chronic hypoglycemia on glycogen metabolism in characterized brain glucose-sensory loci and determine effects local glycogen mass and/or mobilization adaptions on sensory monitoring and signaling of recurring hypoglycemia in each sex.

Keywords: 1-Phenyl 3-methyl 5-pyrazolone; Estrogen receptor; Glycogen; Insulin-induced hypoglycemia; Ventromedial hypothalamic nucleus; Western blot.