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.

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Hindlimb suspension (HS) in rats was used to study the effects of simulated microgravity on whole-body glucose tolerance and soleus muscle insulin signaling. Following short-term HS (1-day), glucose intolerance and whole-body insulin resistance developed. Insulin resistance was also identified at the skeletal muscle level, with the type I soleus showing more insulin resistance than the type II extensor digitorum longus (EDL). Examination of insulin signaling components in the soleus revealed a tendency towards a decrease in the expression of the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI3-K) in addition to a decrease in the basal phosphorylation levels of Akt. These changes may contribute to the observed insulin resistance in the soleus, but clearly other factors likely also contribute. The data from the whole-body and muscle studies after 1-day HS did not clearly identify the factors responsible for the observed glucose intolerance and reduced glucose uptake. However, because both the soleus and EDL exhibited decreased insulin-mediated glucose uptake, there may be a circulating factor responsible for the observed insulin resistance and glucose intolerance. The whole-body glucose intolerance and muscle insulin resistance was no longer apparent after 3-day HS. Interestingly, 7-day HS resulted in the development of enhanced whole-body insulin sensitivity. In both 3-day and 7-day HS soleus, insulin action on glucose transport was increased. In addition, GLUT-4 protein and activities of hexokinase and citrate synthase were increased with prolonged HS. Following 3-day HS, expression of insulin receptor substrate-1 (IRS-1) and -2 (IRS-2) were decreased. However, assessment of the functionality of signaling proteins indicated insulin-induced increases in tyrosine phosphorylation per unit IRS-1 protein, IRS-1 association with p85, and Akt phosphorylation. The PI3-kinase inhibitor wortmannin did not completely block the observed increases in insulin-mediated glucose transport in 3-day and 7-day HS soleus, indicating the small role of a P13-K independent mechanism. These results indicate that prolonged HS (3-7 days) can alter the functionality of specific insulin signaling components in the soleus muscle, accounting for most of the increase in insulin-stimulated glucose uptake induced by simulated weightlessness.

The coupling of oxygen delivery to oxygen consumption is thought to occur as the result of an increase in the production of vasodilating substances which diffuse from the tissue and act on the smooth muscle of arterioles, resulting in an increase in regional blood flow (metabolic regulation). A majority of the hypotheses regarding the nature of metabolic regulation of blood flow focus on the role of cytochrome oxidase and oxidative phosphorylation. The oxygen tension (PO₂) at which the rate of cytochrome oxidase is one-half maximal (K(m)) has been determined in isolated mitochondria but has not been determined in vivo and some evidence suggests that it may be higher than in vitro. In these experiments we determine the PO₂ at which NADH fluorescence began to increase in vivo and in vitro in response to oxygen limitation (critical PO₂). These changes in fluorescence are assumed to reflect oxygen limitation of oxidative phosphorylation and can be compared to the cytochrome oxidase K(m) values. This assumption was tested using imaging techniques on single striated myocytes to determine whether the change in NADH fluorescence that occurs with oxygen removal was associated with mitochondrial dense regions. Our findings support the use of NADH fluorescence to monitor oxidative phosphorylation. Using this technique the critical PO₂ was measured at tissue sites in post-capillary venular regions and in 20 micron diameter post-capillary venules. The critical PO₂ in the tissue was 2.4 mmHg and was higher in the venule (7.7 mmHg), consistent with predicted diffusion gradients for oxygen from vessel to tissue. The difference between the extracellular tissue PO₂ and the of Km cytochrome oxidase for oxygen (.05-0.05 mmHg) is consistent with theorized gradients existing between cell membrane and mitochondria and does not necessarily indicate a higher critical PO₂ in vivo. Using techniques similar to those used in vivo we measured the critical PO₂ from single isolated striated cells and determined it was significantly different from the critical PO₂ measured in vivo. This difference is likely due to the differences between in vivo and in vitro measurement conditions rather than real differences in the critical PO₂.

Apoptosis of granulosa cells is an early feature of atresia in bovine follicles. However, the mechanisms initiating apoptosis of granulosa cells have not been well characterized. In many cell types, apoptosis can be induced by oxidative stress and prevented by increased expression of endogenous oxidative stress response proteins. The transcription factor nuclear factor-kappaB (NF-κB) regulates genes involved in preventing oxidative stress and has been characterized as a mediator of cell survival. Studies have suggested that NF-κB activity may be regulated by estradiol-17β, a major determinant of follicular health. Therefore, these studies tested the hypotheses that oxidative stress is involved in the atresia of non-ovulatory dominant bovine follicles and NF-κB suppresses apoptosis in the healthy dominant follicle. Because the onset of atresia during the first wave of follicular development following ovulation has previously been characterized in cattle, it provides an excellent model to study the relationship between apoptosis, oxidative stress response proteins, NF-kappaB, and estradiol production. Decreased production of estradiol was accompanied by increased numbers of non-viable granulosa cells in dominant follicles between Days 4 and 6 of the first follicular wave in cattle. Unexpectedly, expression of genes encoding oxidative stress response proteins in granulosa cells increased on Day 8 of the follicular wave, but did not translate into increased expression of the corresponding proteins or greater enzyme activity. The decline in estradiol production observed on Day 6 of the 1st follicular wave is not due to lack of androgen substrate or down-regulated expression of the aromatase gene, but instead appears to be the direct result of decreased activity of the aromatase enzyme within granulosa cells. Follicular NF-κB activation was associated with estradiol production. However, direct inhibition of NF-κB activity suppressed apoptosis of granulosa cells in vitro. Therefore, whether NF-κB is playing a pro- or anti-apoptotic role in granulosa cells of bovine follicles remains to be elucidated. These experiments begin to elucidate the signals that trigger the sequence of events leading to apoptosis and atresia of bovine follicles and provide the framework for future experiments designed to further clarify the complex interplay of signals that determine a follicle's fate.

Using epifluorescence microscopy I have characterized the transport of the fluorescent organic cation NBD-TMA⁺, [2-(4-nitro-2,1,3-benzoxadiaxol-7-yl)aminoethyl]trimethylammonium. NBD-TMA⁺ has structural characteristics common to other secreted organic cations and it is fluorescent (λₑₓ = 458 nm; λ(em) = 530 nm). Transport of NBD-TMA⁺ by rabbit renal proximal tubules was time dependent, saturable, and inhibited by TEA⁺, cimetidine, and procainamide. These results provide strong evidence that the fluorescent organic cation, NBD-TMA⁺, is transported by the classical organic cation transporter and can be used to monitor organic cation transport in renal proximal tubules or cells expressing organic cation transporters. Additionally, a stable line of HeLa cells expressing hOCT1 was used to investigate the structural and chemical characteristics influencing substrate binding to the transporter. I found that the systematic rotation of a planar hydrophobic mass about a vertical axis aligned with the positively charged nitrogen reduced the binding affinity of substrate for hOCT1 suggesting that hOCT1 possesses a lateral steric limitation not seen in the apical organic cation transporter. Furthermore, the addition of supraplanar hydrophobic mass to a planar chemical structure was found to increase the binding affinity for a substrate substantially more that expected for the increased hydrophobicity suggesting that the binding site for hOCT1 is not simply planar in nature, but may be better described as a pocket.