Fundamental to life is the ability to acquire and assimilate nutrients. Individual cell types exhibit preferences for different nutrients, but only certain cells utilize nutrients as signaling molecules. The most intensely studied nutrient signaling system is the pancreatic beta cell, which secretes insulin in response to changes in blood glucose. Another glucose sensing system is found in the neurons within the hypothalamus of the brain. To study how single cells sense changes in glucose, a sensitive marker for secretion is required. To this end the human Growth Hormone (hGH) gene was fused to the 5' end of the enhanced Green Fluorescent Protein (GFP) gene and expressed in the RIN-1038 beta (β)-cell line. The hGH-GFP fusion protein was targeted to secretory granules and its secretion into culture media was detected from cell populations. At stimulatory levels of glucose (5 mM), hGH-EGFP secretion doubled, and potentiators of insulin secretion enhanced glucose-induced hGH-EGFP release. However, at the single cell level, hGH-EGFP fluorescence acted as a sensor for changes in secretory granule pH. Glucose induced granule acidification by increasing activity of the V-type proton ATPase resident in the vesicular membrane. Moreover, potentiators of secretion elicited alkalinization of the vesicle lumen suggesting a mechanism by which they enhance release. To compare components of the glucose-sensing mechanism of hypothalamic neurons to those described for the beta cell, RT-PCR analysis was performed on RNA samples taken from the Arcuate nucleus (ARC), Lateral Hypothalamus (LH), Paraventricular Hypothalamus (PVH) and Ventromedial Hypothalamus (VMH). Tissue-specific expression of Glucokinase (GK), Glucokinase Regulatory Protein (GKRP), Glucose transporter isoforms (GLUT) 1, 2, 3 and X1 genes were determined. GK gene expression was found in all hypothalamic regions, with highest levels in the ARC. Enzymatic activity assays show that GK activity accounts for approximately 20% of the total soluble hexokinase activity in pooled samples of ARC and VMH. All regions also express GLUT 1, 3 and X1. However, no GLUT-2 or GKRP mRNA was detected in any sample. Because the expression of the low K(m) GLUTS predominates and GKRP expression is absent, the hypothalamic glucose-sensing mechanism is adapted to the lower levels of glucose present in the cerebrospinal fluid compared to blood levels.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/279863 |
| Date | January 2000 |
| Creators | Tompkins, Linda Suzanne |
| Contributors | Lynch, Ronald M. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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