Browsing by Subject "Insulin"
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Item An amino acid mixture enhances insulin-stimulated glucose uptake in isolated epitrochlearis muscle(2010-08) Kleinert, Maximilian; Ivy, John, 1945-; Farrar, Roger P.Amino acids are important modulators of skeletal muscle metabolism, but their impact on glucose uptake by skeletal muscle remains unclear. To address the effect of an amino acid (AA) mixture consisting predominately of isoleucine on glucose uptake we first conducted a dose-response experiment, investigating how different concentrations of the AA mixture affect glucose uptake by isolated rat epitrochlearis muscle. In a subsequent experiment we examined how the AA mixture affects insulin-stimulated glucose uptake by isolated rat epitrochlearis muscle. It was found that the AA mixture with as little as 0.5 mM Ile increases [H3]2-deoxy-D-glucose (2-DG) uptake by 76% compared to basal glucose uptake. The AA mixtures with 1, 2 or 4 mM Ile provided no significant additional effect. Next we combined the AA mixture consisting of 2 mM Ile, 0.012 mM Cys, 0.006 mM Val and 0.014 mM Leu with physiological levels (75 μU/ml, sINS) and maximally-stimulating levels (2 mU/ml, mINS) of insulin. The AA mixture only, sINS and mINS significantly increased 2-DG uptake compared to basal by 63, 79 and 298%, respectively. When the AA mixture was combined with sINS and mINS 2-DG uptake was further increased significantly by 26 and 14%, respectively. Western blotting analysis revealed that compared to basal the AA mixture increased AS160 phosphorylation, while phosphorylation of Akt and mTOR did not change. Combining the AA mixture with sINS resulted in no additional phosphorylation compared to sINS alone. Interestingly, addition of the AA mixture to mINS resulted in increased phosphorylation of mTOR, Akt and AS160 compared to mINS alone. Our results suggest that certain AAs (1) increase glucose uptake in the absence of insulin and (2) augment insulin-stimulated glucose uptake in an additive manner. These effects on glucose uptake appear to be mediated via a molecular pathway that is partially independent from the canonical insulin signaling cascade.Item Characterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis(2004-05-04) Hays, Lori Beth; Roth, MichaelInsulin exocytosis is a complex, regulated process involving numerous exocytotic proteins to coordinate the release of insulin. Syncollin has been implicated in zymogen granule exocytosis in acinar cells. It was hypothesized that either syncollin or a ‘syncollin-like’ protein may be expressed in β-cells and influence insulin exocytosis. Adenoviral mediated expression of either long or short forms of syncollin in isolated islets and INS-1 cells showed both forms underwent N-terminal signal peptide cleavage to yield the same 14kD mature protein. Immunofluorescence revealed that adenovirally-expressed syncollin was specifically targeted to the ß-granule lumen. In perifused islets, syncollin expression significantly inhibited first-phase glucose-induced insulin secretion compared to AdV-GFP infected islets. GLP-1 and glyburide potentiation of insulin secretion was inhibited; whereas constitutive secretion and insulin content were normal in syncollin-infected islets indicating syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production or secondary stimulus-coupling signals. Thus, syncollin likely inhibited the distal stages of insulin exocytosis providing the first evidence that an intragranular protein is capable of influencing regulated insulin secretion. Syncollin fluorescent fusion proteins were localized to ß-granules, but did not influence insulin secretion implicating these chimeras as ß-granule specific markers for emerging imaging technology. Real-time confocal microscopy demonstrated syncollin-GFP could be used to examine spatiotemporal dynamics of exocytosis. Furthermore, consecutive infection of syncollin-GFP and syncollin-dsRFP labeled distinct pools of β-granules. Expression of syncollin was not identified in β-cells; however, a 10Kd ‘syncollin-like’ protein was expressed, which when sequenced corresponded to FXYD6. Comparison of syncollin and FXYD6 protein structure revealed several conserved domains, indicating syncollin is likely a pseudo-FXYD family member. FXYD6 was the only FXYD protein endogenously expressed in β-cells, which localized to distinct regions of the plasma membrane. Overexpression of FXYD6-Myc enhanced β-granule transport to distinct regions of the plasma membrane that also expressed FXYD6; however, there was no significant effect on glucose-stimulated insulin secretion in isolated islets. SiRNA-mediated reduction of FXYD6 resulted in no obvious changes in β-granule distribution; however, β-granule movement during glucose stimulation was erratic and misdirected. These data implicate FXYD6 as a molecular beacon on the plasma membrane guiding β-granules to the active site of exocytosis.Item Effect of Nutrition on In Vitro Biofilm Formation of Gastrointestinal Associated Microbes(2013-04-28) Hokazono, AsukaGastrointestinal (GI) health is an important contributor to one?s overall well-being. In the past decade, the focus of this aspect of health has been on the organisms that inhabit the intestines: gut microbes. A concept central to understanding bacterial behavior for health or disease promotion is biological film (biofilm) formation. The predominant form of growth for bacteria is biofilm formation, an as yet poorly described phenomenon for gut microbes. In order to better understand bacterial behavior in response to nutrients that pass through the GI system, a high throughput system to assess biofilm formation was developed. Gastrointestinal-associated microbes, Escherichia coli and Enterococcus faecalis, were assayed for biofilm formation in 96-well plates after 24 hours of incubation. Nutrients, inter-and intrakingdom signaling molecules such as monosaccharides, calcium, insulin, endocannabinoids, and AI-1, AI-2 like signaling compounds, respectively, were added to cultures in order to test their effects on biofilm formation. Biofilm was quantified spectrophotometrically by the measurement of optical density of each well measured at 580nm following crystal violet staining of adherent biofilm. Values were expressed as means ? standard error of the mean (SEM); differences between means were assessed using t-testing and ANOVA using GraphPad Prism, with mean differences considered significant at P < 0.05. Results showed that biofilm formation by E. coli was enhanced by glucose, galactose, lactose, AI-1 like signaling compound and insulin at 50 and 100?U/ml, while AI-2 like compound and calcium inhibited biofilm formation. Biofilm formation by E. faecalis was also enhanced by AI-1 like compound and insulin at 50?U/ml in RPMI medium and inhibited in cultures grown in BHI medium or with added calcium. We conclude that gastrointestinal-associated microbes are influenced by nutrients as well as various factors, including the culture medium, signaling compounds, as well as host-signaling compounds such as insulin and calcium. This study provides a platform required for future studies involving nutrient effect on biofilm formation.Item The effects of an amino acid mixture beverage on glucose tolerance, glycogen replenishment, muscle damage, and anaerobic exercise performance(2011-08) Wang, Bei, doctor of kinesiology; Ivy, John, 1945-; Farrar, Roger P.; Wilcox, Richard E.; Griffin, Lisa; Jolly, Christopher A.Recent research suggests that amino acids, such as leucine and isoleucine, can improve glucose tolerance in vivo and in vitro animal models by accelerating glucose uptake in peripheral tissues and stimulate glycogen synthesis in vitro in the absence of insulin. Our laboratory recently found that gavaging normal Sprague-Dawley rats with an amino acid mixture, composed of isoleucine, leucine, cystine, methionine, and valine, improved blood glucose response during an oral glucose challenge without an increase in the plasma insulin response. The blood glucose-lowering effect of the amino acid mixture was due to an increase in skeletal muscle glucose uptake. These results suggest that this amino acid supplement acutely improves muscle insulin sensitivity and blood glucose homeostasis. However, the effect of this amino acid mixture on glucose tolerance and muscle glycogen synthesis in humans has not been investigated. Some studies have also shown that daily supplementation or acute ingestion of amino acids may prevent muscle damage that occurs as a result of a prolonged, intense endurance exercise or strength training and therefore improves force production and exercise performance. However, the effects of the addition of an amino acid mixture to carbohydrate supplement on muscle damage after a prolonged endurance exercise, as well as on the subsequent anaerobic exercise performance, have not been characterized. Therefore, in this series of two studies, the effects of an amino acid mixture, composed of isoleucine, leucine, cyctine, methionine, and valine, on glucose tolerance, muscle glycogen resynthesis, muscle damage, and anaerobic exercise performance were investigated. Study 1 demonstrated that our amino acid mixture lowered the glucose response to an OGTT in healthy overweight/obese subjects in an insulin-independent manner. Study 2 demonstrated that both high and low dosages of amino acid mixture were effective in lowering blood glucose response to a carbohydrate bolus in athletes postexercise. High dosage of amino acid mixture was more potent in glucose regulation by providing a higher insulin response and amino acid effect. However, our amino acid mixture had no effects on post exercise muscle glycogen synthesis, exercise-induced muscle damage or subsequent anaerobic performance. Taken together, the results of this research series suggest that an amino acid mixture, composed of isoleucine and 4 additional amino acids, attenuates the glucose response to a glucose bolus in an insulin-independent manner, but does not enhance muscle glycogen restoration following exercise or prevent exercise-induced muscle damage.Item Effects of prolonged sitting and walking for two days on postprandial triglycerides in men : interaction with energy intake(2011-05) Park, Sanghee; Coyle, E. F. (Edward F.), 1952-; Brothers, Robert M.Postprandial hypertriglyceridemia (PPHT), an independent risk factor for atherosclerosis (Smyth and Heron 2006; Nordestgaard, Benn et al. 2007), is strongly associated with metabolic syndrome and cardiovascular diseases (CVD) (Kolovou, Anagnostopoulou et al. 2005). It has been proposed that elevated triglycerides after a high-fat meal may be a postprandial phenomenon (Zilversmit 1979). PPHT are commonly concurrent with sedentary behaviors, such as extended sitting, which amplify PPHT (Levine, Vander Weg et al. 2006). The purpose of this study was to examine the effects of prolonged sitting with or without a balanced caloric diet and walking with a balanced diet on postprandial triglycerides (PPTG). Seven healthy, young men (age, 25.6 ± 3.7 y; height, 174 ± 5 cm; weight, 71.4 ± 6.2kg; VO2max, 49.3 ± 7.7 ml/kg/min) were recruited from a college and from within the Austin community. After 2 days of food and activity control (D1and D2), subjects performed one of three trials in a randomized, cross-over design for 2 days (D3 and D4); (1) active walking with a balanced diet (WB), (2) prolonged sitting with a hyper-caloric diet (SH), and (3) prolonged sitting with a balanced diet (SB). High fat tolerance tests (HFTT) were conducted on the following day, (D5), after 13 hour over-night fasting. Blood samples were obtained in the fasting state and every hour for 6 hours after subjects had eaten a high fat test meal consisting of 1.2 g fat, 1.1 g CHO, 0.2 g protein/kg body mass. All food was provided during the 5-day duration of the study. Body postures, heart rate, and daily steps were monitored. In both sit trials (SH and SB), subjects sat ~320 minutes longer and took 10 times fewer steps than WB. In WB, the total area under the curves for plasma triglycerides (AUC[subscript T] TG) was lower, compared to SH by 21.3% (p<0.001) and to SB by 19.7% (N.S.; p = 0.055), respectively. In WB, the incremental AUC TG (AUC[subscript I] TG), an index of postprandial response, was lower than both SH by 17.4% (p <0.005) and SB by 20.1% (p <0.05), respectively. Postprandial plasma insulin concentration was lower in WB, compared to SH by 19.4% (p <0.005) in AUC[scubscript T] and 18.8 % (p < 0.05) in AUC[subscript I]. No differences were shown in the metabolic responses between SB and SH despite the diet modifications. These findings indicate that two days of prolonged sitting significantly amplifies PPTG and suppresses insulin action.Item Factors influencing dietary compliance in a study examining marital interaction and diet change in insulin-dependent diabetics(Texas Tech University, 1984-08) Trodahl, Susan ElaineNot availableItem FOXO is the Mediator Linking Temporal Differentiation and the Insulin Signaling Pathway(2005-12-20) Lah, Carol Joonhyun; Cameron, ScottThe timing of differentiation is crucial for the correct development of an organism, because specific pathways can be used reiteratively to differentiate cells. Until recently, the molecular mechanism behind the temporal control of differentiation has remained elusive. Bateman and McNeill (2004) revealed a novel role for the insulin/insulin-like growth factor receptor (InR) pathway in regulating the timing of differentiation in neuronal photoreceptor cells in the Drosophila compound eye. The link between the InR pathway and temporal differentiation is significant, because of the implication that external factors, e.g. nutrition, are tightly coupled to the timing of differentiation. This proposal tests the hypothesis that FOXO, a crucial component of the InR pathway, mediates the regulation of developmental timing. The aims are the following: 1. Observe if dFOXO mutants affect temporal differentiation in the Drosophila eye. 2. Perform epistasis experiments to determine if dFOXO is downstream of other insulin signaling components. 3. Analyze the downstream targets of dFOXO that may play a role in neuronal differentiation.Item Glucose, insulin and lipase activity of rats fed chromium from beef and soy(Texas Tech University, 1983-08) Keim, Kathryn Sarah HochsprungNot availableItem Influence of dietary energy source on in vitro substrate utilization and insulin sensitivity of muscle and adipose tissue of beef cattle(2009-05-15) Rhoades, Ryan D.Beef carcass value is influenced by the quantity and distribution of adipose tissue. Elucidation of metabolic controls of caloric partitioning between adipose depots could lead to development of production solutions that enhance beef carcass value. Historical trends in Choice and Select beef supply and short-term demand structures for Choice and Select boxed beef were explored. Recent stabilization in slaughter mix may suggest an optimum is being approached. Evaluation of short-run demand supports this premise, and suggests that Choice and Select products may not be strong substitutes. Growth-based prediction equations relating carcass traits to growth traits using ultrasound measurements as the basis of projections under different growing systems were explored. Accuracy of carcass fat predictions from growth-based equations is influenced by weight gain between ultrasound and endpoint, breed, and gender; scans out to 120 d pre-harvest may be accurate. Angus steers were used to test effects of dietary energy source on muscle and adipose tissue metabolism and insulin sensitivity. Results suggest that feeding hay limited both glucose supply and tissue capacity to increase glucose utilization in response to insulin without altering acetate conversion to fatty acids. Because subcutaneous (s.c.) adipose tissue consistently utilized more acetate and oxidized more glucose than intramuscular (i.m.), these results suggest that hay-based diets may alter i.m. adipose tissue metabolism with less impact on s.c. adipose tissue. Additionally, s.c. adipose tissue may become resistant to insulin in steers fed to an excessive s.c. fat thickness. A final experiment was designed to test the effects of dietary energy source during backgrounding and compositional endpoint on adipose tissue metabolism and insulin sensitivity. Feeding hay during backgrounding may have differential effects on tissue lipogenesis. Feeding hay increased both glucose oxidation and incorporation of acetate into fatty acids; in i.m. insulin failed to stimulate glucose conversion to lipid. As physiological maturity increases, glucose conversion to CO2 and lactate increased, but the ability of insulin to stimulate lipid synthesis from glucose may be reduced. These data provide foundation for a hypothesis regarding diet-mediated regulation of differential adipose tissue metabolism. Validation of these hypotheses could generate nutritional strategies that alter the rate and site of adipose deposition.Item Insulin-Mediated Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cell Cultures(Texas Tech University, 1984-05) Ehlers, Susan ElizabethInsulin lowers basal levels of tyrosinase activity and inhibits the MSH-stimulated rise in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. These cultures are very sensitive to insulin. A concentration of insulin as low as 5 x 10 M insulin produces optimum inhibition. At maximum inhibition, tyrosinase activity is reduced to approximately 50% of control levels. Insulin inhibits cellular proliferation in melanoma cells; however, inhibition of tyrosinase activity precedes this effect. Insulin also inhibits the (Bu)2 cAMP and theophylline stimulated rise in enzyme activity. This finding suggests that insulin exerts its effects at a site distal to cAMP production. Insulin, in fact, does not lower cAMP levels in melanoma cells, nor does it alter the MSH-stimulated rise of cAMP. The inhibitory effect of insulin on tyrosinase activity could not be mimicked by either (Bu)2 cGMP or 8-bromo-cGMP, suggesting that insulin does not exert its effects by altering cellular levels of this nucleotide. Insulin decreases the incorporation of [3H]-leucine into trichloracetic acid insoluble material by 50%, an inhibition which corresponds well with the observed level of reduction of tyrosinase activity. This finding suggests that the inhibition of tyrosinase activity may be caused by a general reduction in protein synthesis due to insulin treatment.Item The modification of insulin to enhance oral delivery systems(2009-05) Kanzelberger, Melissa Ann; Peppas, Nicholas A., 1948-While a number of PEGylated proteins have been studied for injectable applications and reviewers have used this data to speculate possible oral delivery improvements, a detailed investigation of PEGylated insulin for oral delivery and the development of an optimized pH-sensitive carrier for PEGylated insulin conjugates had yet to be accomplished. In order to proceed with oral delivery study, improvements in yield, with respect to previous PEGylation methods were necessary to enable the completion of high throughput drug delivery studies. Subsequently, a reaction scheme for the covalent attachment of PEG to insulin using nitrophenyl carbonate-PEG was developed. It was demonstrated that this reaction occurred at a 1:1 ratio and was site specific at the B29Lys position. A P(MAA-g-EG) hydrogel carrier was developed to optimize loading and release behavior for PEGylated insulin. It was demonstrated that the density and length of polymer grafts affected both loading and release behavior of PEGylated insulin. The best performing grafted polymers had a 3:1 methacrylic acid: ethylene glycol (MAA:EG) ratio and achieved loading efficiencies from 96% to nearly 100%. With respect to release, polymer particles containing fewer, but longer grafts shown to release faster than polymers with shorter grafts with the same MAA:EG ratio. Finally, the effects of PEGylation on intestinal absorption was investigated using an intestinal epithelial model as well as a rat model. It was demonstrated that PEGylated insulin in the presence of P(MAA-g-EG) microparticles did not significantly alter the tight junctions over unmodified insulin. However, the conjugate permeabilities across the membrane were reduced. The pharmacological availability (PA) was then verified by injecting the insulin conjugates subcutaneously in fasted Sprague-Dawley rats. It was determined that PEG 1000 insulin (1KPI) had a PA roughly equivalent to insulin, while it was reduced by 59% for 2KPI and by 81% for 5KPI. The effectiveness of utilizing PEGylated insulin as an oral drug delivery candidate was evaluated with a closed loop intestinal study, in which PEGylated insulin or insulin in solution was delivered directly to the jejunum. It was shown that 1KPI and insulin performed identically; with a pharmacological availability of 0.56%. 2KPI, however improved the pharmacological availability of insulin by 2.8 times. These results demonstrate that PEGylation holds promise for improving the oral delivery of proteins.Item Molecular design of advanced oral protein delivery systems using complexation hydrogels(2006) Wood, Kristy Marie; Peppas, Nicholas A., 1948-A novel class of pH sensitive complexation hydrogels composed of methacrylic acid and functionalized poly(ethylene glycol) tethers, referred to as P(MAA-g-EG) WGA, was investigated as an oral protein delivery system. The PEG tethers were functionalized with wheat germ agglutinin (WGA), a lectin that can bind to carbohydrates in the intestinal mucosa, to improve residence time of the carrier and absorption of the drug at the delivery site. P(MAA-g-EG) WGA created a specific mucoadhesive interaction between mucin and WGA in in vitro experiments. In addition, it improved the overall adhesion of the carrier by 17% to a cellular monolayer, as compared to P(MAA-g-EG). Administration of P(MAA-g-EG) WGA to a rat small intestine demonstrated that 99% of the microparticles still remained in the rat small intestine after 1 hour. These results confirmed that functionalizing P(MAA-g-EG) with WGA improved the mucoadhesive properties of the carrier. Insulin was effectively entrapped within the polymer network with a loading efficiency of 74%. Release studies with insulin-loaded P(MAA-g-EG) WGA showed that the carrier released less than 10% of the insulin at pH 3.2 after 60 minutes and 70% of the insulin at pH 7.0 after 60 minutes. These studies confirmed that P(MAA-g-EG) WGA can protect insulin in the low pH of the stomach and that the pH change between the stomach and the small intestine can be used as a physiologic trigger to quickly release insulin. The ability of P(MAA-g-EG) WGA to improve insulin absorption was investigated in two different intestinal epithelial models and an animal model. In the Caco-2 cells, P(MAA-g-EG) WGA improved insulin permeability by 9-fold as compared to an insulin only solution. P(MAA-g-EG) WGA was also evaluated in a mucussecreting culture that contained Caco-2 and HT29-MTX cells. Insulin permeability was increased by 5-fold in the presence of P(MAA-g-EG) WGA. The final study determined bioavailability of insulin-loaded P(MAA-g-EG) WGA when administered to a rat small intestine. Bioavailability of insulin was 11.9% for insulin-loaded P(MAA-g-EG) WGA, which is a vast improvement over the 0.5% bioavailability of an insulin only solution. Overall, it is clear that P(MAA-gEG) WGA holds great promise as an oral insulin delivery system.Item Mucoadhesive films for the buccal delivery of insulin(2012-12) Morales, Javier Octavio; Williams, Robert O., 1956-; McConville, Jason Thomas; Smyth, Hugh D; Cui, Zhengrong; Roy, KrishnenduTo address the need of a patient friendly and therapeutically effective method of administration of insulin (Ins) we sought to develop mucoadhesive films for delivery through the buccal mucosa. Ins is a labile molecule exhibiting limited activity and stability in solid solutions in films and other solid delivery devices. Early investigations outlined in Chapter 3 revealed the need for a certain particle size (below the one micrometer) for the addition of particulate material in films. In Chapter 4 a novel method for the manufacture of protein-coated nanoparticles (PCNP) is depicted. Successful particle batches were achieved in terms of size, uniformity, stability and activity and these particles were further investigated for their inclusion on films for buccal delivery. The method of manufacture of particles was based on an antisolvent co-precipitation process that immobilized macromolecules to the surface of crystalline core particles resulting in high yields and highly active protein loaded particles. Films loaded with PCNP were developed and characterized in Chapter 5. Lysozyme was utilized as a model macromolecule and high yields and activity were obtained after manufacture, demonstrating that after all the processing the protein is subjected to, activity is preserved. Using Eudragit® RLPO (ERL) as the matrix forming polymer, films with excellent mucoadhesion were developed. Here is described a high mucoadhesion for ERL that was even further increased by the addition of the water soluble PCNP. This occurred by the water movement into the ERL matrix that the solubilizing particles generate. Finally, films containing Ins were developed and assayed for permeation through buccal mucosa. By adapting the method of manufacture, Ins-coated nanoparticles were obtained and embedded in films. ERL films corroborated previous findings by exhibiting excellent performance. Investigations on the permeation of Ins through buccal mucosa revealed that the inclusion of Ins in films enhanced its permeation in comparison with a control Ins solution. Thus here is described the successful development of mucoadhesive films for the buccal delivery of Ins.Item Oral delivery of proteins: preparation and characterization of an oral dual-controlled release formulation of insulin and ovomucoid(Texas Tech University, 2001-05) Agarwal, VikasOral bioavailability of proteins is extremely low due to extensive degradation in the gastrointestinal tract and low epithelial permeability. Further, the structure and conformation of proteins are easily altered when exposed to formulation and process conditions leading to a loss of biological activity. Oral delivery of proteins may be enhanced by the use of absorption modifiers such as enzyme inhibitors and permeation enhancers. In the present study, the role of ovomucoids has been investigated as possible absorption modifiers in the oral delivery of a model protein, insulin. Ovomucoids are enzyme inhibitors isolated from egg white of avian species. They have protease inhibitory activity and also bind to lectins on the cell surfaces through their carbohydrate moeity. However, their role in the oral delivery of proteins has not been investigated. The purpose of this study was to develop a dual controlled release formulation of insulin and ovomucoid. Enzymatic degradation studies revealed that insulin is degraded extensively in the presence of trypsin and a-chymotrypsin. Duck ovomucoid (DkOVM) stabilized insulin against degradation in the presence of trypsin and a-chymotrypsin for an hour. In contrast, chicken ovomucoid (CkOVM) was only effective against trypsin mediated degradation of insulin. Permeability studies of insulin from rat intestinal segments revealed that insulin is absorbed more from the jejunum and ileum than from the duodenum. In the presence of CkOVM and DkOVM, the permeability of insulin decreased, which may be explained in part, by the action of insulin by adipocytes. A coprecipitation technique was successfully applied to prepare microcapsules of insulin with high encapsulation efficiency. Dissolution stability studies of insulin microcapsules in the presence of enzymes revealed considerable improvement in the availability of insulin with ovomucoids even at the end of 6 hours. Characterization of insulin in the microcapsules using DSC, FT-IR, powder x-ray diffraction and size exclusion chromatography revealed that the structure of insulin was conserved after subjecting it to formulation and process conditions. A three-factor, three-level optimization design was used to evaluate the effect of critical process variables including the rate of addition of polymeric solution, compression pressure and volume of water with respect to polymeric solution. Mathematical relationships, contour plots and response surface methods were employed with constrained optimization to predict levels of factors that provide optimum response. The predicted and observed values were in close agreement. The release of DkOVM was delayed from the formulation. The novel formulation incorporates controlled release characteristics of both protein and inhibitor that may enhance the stability and availability of proteins with less potential for inhibitor related toxicity.Item Regulation of Insulin and CHOP Gene Expression in Pancreatic Beta Cells(2009-01-14) Shao, Chunli; Cobb, MelanieInsulin is a major hormone in maintaining glucose homeostasis. It is essential to understand the mechanisms by which insulin gene expression is regulated in pancreatic beta cells. In addition to examining histone modifications on the insulin gene promoter, I focused on the effect of MafA modification on insulin expression. MafA is a transcriptional activator of the insulin gene via binding to the RIPE3b/C1 (rat insulin promoter element 3b) element. Mutagenesis showed that MafA was post-translationally modified by SUMO-1/2 (small ubiquitin-like modifier) mainly at lysine 32. Low glucose starvation or hydrogen peroxide stimulation increased sumoylation of MafA. Forced sumoylation of MafA reduced its transcriptional activity towards the insulin gene promoter and increased its suppression of the CHOP (C/EBP homologous protein) gene promoter. However, sumoylation of MafA did not alter its nuclear localization, protein stability, or apparently its DNA binding to the insulin promoter in beta cells. These studies suggest that MafA sumoylation modulates gene transcription in beta cells. In type I diabetes, beta-cell apoptosis is the major reason for immune-mediated pancreatic beta-cell death. IL-1beta (interleukin 1beta), a proinflammatory cytokine, induces ER (endoplasmic reticulum) stress and activates proapoptotic networks in beta cells, such as NF-kappaB (nuclear factor-kappaB) and JNK (c-Jun N-terminal kinase) signaling pathways. The second project focused on the mechanisms by which JNK and NF-kappaB regulate the expression of CHOP, a mediator of ER stress-induced apoptosis, upon IL-1beta stimulation. Exposure of beta cells to IL-1beta markedly increased CHOP messenger RNA and protein. Electrophoretic mobility shift assays showed that IL-1beta-activated NF-kappaB bound to the CHOP promoter. Furthermore, immunoblot data indicated that expression of c-Jun was strongly increased, and that multiple residues on c-Jun were phosphorylated after IL-1beta treatment. IL-1beta also increased c-Fos expression in beta cells. These data suggest that IL-1beta-induced activation of NF-kappaB and JNK controls CHOP gene expression in pancreatic beta cells, and that IL-1beta influences beta-cell function through a variety of signaling pathways.Item Safety and efficacy of inhaled insulin formulated with alkylglycosides(2005-12) Hussain, Alamdar; Ahsan, Fakhrul; Allen, David D.; Klein, Jochen; Mehvar, Reza; Youan, Bi-Botti CelestinThe pulmonary route has shown a tremendous promise for delivery of large molecular weight therapeutic agents. Although the respiratory route has a larger surface area compared to other mucosal routes of administration, a major impediment to deliver proteins and macromolecules by this route is their poor absorption through the lung epithelium. To obtain a therapeutically acceptable bioavailability after pulmonary administration, drugs are co-administered with absorption promoters. Agents that have been tested as absorption promoters include protease inhibitors, surfactants, lipids and polymers. Unfortunately, none of the absorption promoters satisfy requirement of safety and efficacy. Recently it has been shown that alkylglycosides could be used at an extremely low concentration to enhance nasal and ocular absorption of peptide and protein drugs. Alkylglycosides are disaccharides such as sucrose and maltose attached to alkyl chains of variable lengths. Currently, it is not known if alkylglycosides enhance pulmonary absorption of peptide and protein drugs. Further, the safety of these excipients also remains unknown. The purpose of the present dissertation is to test the hypothesis that alkylglycosides safely and effectively enhance pulmonary absorption of insulin. This research project is the first to document the safety and efficacy of alkylglycosides in enhancing pulmonary absorption of peptides. The safety of insulin formulated with alkylglycosides was tested in rats by bronchoalveolar lavage fluid analysis and studying the effect of formulation on mucociliary transport rate in a frog palate model. The efficacy of alkylglycoside based insulin preparations was tested in rats by monitoring the increase in plasma insulin and decrease in plasma glucose in a transiently hyperglycemic rat model. The study suggests that octylmaltoside, an alkylglycoside containing 8-carbon alkyl chain length, was the safest of all agents for the duration tested. Dry powder pulmonary preparation of insulin formulated with octylmaltoside showed a bioavailability of 48±7 %. The optimum dry powder insulin formulation contained 0.375 U of insulin plus 4.6 mmol of octylmaltoside per 5 mg of the dose. Further studies in higher laboratory animals such as dogs and monkeys are required to establish the safety and efficacy of pulmonary formulation of insulin with octylmaltoside before the preparation can be tested in humans.Item Safety and efficacy of inhaled insulin formulated with alkylglycosides(Texas Tech University, 2005-12) Hussain, Alamdar; Ahsan, Fakhrul; Allen, David D.; Klein, Jochen; Mehvar, Reza; Youan, Bi-Botti CelestinThe pulmonary route has shown a tremendous promise for delivery of large molecular weight therapeutic agents. Although the respiratory route has a larger surface area compared to other mucosal routes of administration, a major impediment to deliver proteins and macromolecules by this route is their poor absorption through the lung epithelium. To obtain a therapeutically acceptable bioavailability after pulmonary administration, drugs are co-administered with absorption promoters. Agents that have been tested as absorption promoters include protease inhibitors, surfactants, lipids and polymers. Unfortunately, none of the absorption promoters satisfy requirement of safety and efficacy. Recently it has been shown that alkylglycosides could be used at an extremely low concentration to enhance nasal and ocular absorption of peptide and protein drugs. Alkylglycosides are disaccharides such as sucrose and maltose attached to alkyl chains of variable lengths. Currently, it is not known if alkylglycosides enhance pulmonary absorption of peptide and protein drugs. Further, the safety of these excipients also remains unknown. The purpose of the present dissertation is to test the hypothesis that alkylglycosides safely and effectively enhance pulmonary absorption of insulin. This research project is the first to document the safety and efficacy of alkylglycosides in enhancing pulmonary absorption of peptides. The safety of insulin formulated with alkylglycosides was tested in rats by bronchoalveolar lavage fluid analysis and studying the effect of formulation on mucociliary transport rate in a frog palate model. The efficacy of alkylglycoside based insulin preparations was tested in rats by monitoring the increase in plasma insulin and decrease in plasma glucose in a transiently hyperglycemic rat model. The study suggests that octylmaltoside, an alkylglycoside containing 8-carbon alkyl chain length, was the safest of all agents for the duration tested. Dry powder pulmonary preparation of insulin formulated with octylmaltoside showed a bioavailability of 48±7 %. The optimum dry powder insulin formulation contained 0.375 U of insulin plus 4.6 mmol of octylmaltoside per 5 mg of the dose. Further studies in higher laboratory animals such as dogs and monkeys are required to establish the safety and efficacy of pulmonary formulation of insulin with octylmaltoside before the preparation can be tested in humans.Item Spectroscopic and calorimetric studies of aggregated macromolecules(2007) Kitts, Catherine Carter, 1979-; Vanden Bout, David A.Different optical and calorimetric techniques were utilized to gain a better understanding of aggregated macromolecules. This research looked at two different macromolecules: poly(9,9'-dioctylfluorene), a conjugated polymer that forms aggregates in organic solvents; and bovine insulin, which forms amyloid fibrils. Conjugated polymers are of increasing interest due to their thermal stability and ease of solution processing for use in devices. A member of the polyfluorene family, poly(9,9'-dioctylfluorene) (PFO), has been studied due to its blue-emitting spectral properties. However, PFO has been found to form aggregates in solution, which is detected by the presence of a red-shifted absorption peak. This peak is caused when a section of the backbone planarizes forming the [beta]-phase. The [beta]-phase can be removed from the solution upon heating and will not return until the solution is cooled, making it a non-equilibrium process. The dissolution and reformation of the -phase were monitored using absorption spectroscopy and differential scanning calorimetry. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were able to probe the aggregates in films. It is important to understand polymer properties in solution in order to understand film morphology. Amyloid fibrils contribute to over 20 different neurodegenerative diseases, in which cures have yet to be found. The fibrils form when a soluble protein misfolds and self-assembles to form insoluble protein aggregates, and the cause of the fibril formation in vivo has still yet to be determined. Spectroscopy studies have been made possible with the use of fluorescent dyes: thioflavin T (ThT), BTA-2, and Congo red (CR). These dyes bind to amyloid fibrils and exhibit changes in their spectral properties. However, the exact mechanism for the binding of these dyes has only recently been studied. Through the use of calorimetry, the forces involved with binding of ThT and CR to amyloid fibrils can be determined. Absorption and fluorescence spectroscopy techniques were employed to study the spectral properties of these dyes. Polarized NSOM was used to determine the ThT or BTA-2's orientation with an individual fibril. Understanding how these dyes bind to fibrils will enable researchers to use spectroscopy to study the early stages of fibril formation.Item The Effect of Glucose Utilization and Feed Efficiency on Beef Cattle Production(2012-02-14) Bradbury, BrookFeed efficiency and metabolism affect profitability of the various components of the beef industry by modulating distribution and use of nutrients within cattle. Separate studies were conducted to determine the 1) repeatability of feed efficiency measurements over time as beef heifers mature into cows, and 2) whether the production and regulation of glucose in heifers is affected by temperament. The influence of temperament on glucoregulatory hormones was studied in Angus crossbred heifers and Brahman heifers whose temperament was determined at weaning. The 6 most calm and 6 most temperamental heifers of each breed were fitted with jugular cannulas. Blood was collected at cannulation and then via the cannula during a 90-min rest period. Following 90 min, dextrose was infused (0.5 mg/kg BW) and blood samples were collected at specific intervals for 3 h total. In the crossbred heifers cortisol (P = 0.0560) and glucose (P = 0.0485) concentrations during the challenge were higher in temperamental relative to calm crossbred heifers. Insulin concentrations tended (P = 0.0737) to be higher in temperamental crossbred heifers. Cortisol (P = 0.0282) and glucose (P = 0.0011) concentrations were significantly higher in temperamental Brahman heifers. Insulin concentrations tended (P = 0.0793) to be greater for calm Brahman heifers. Temperamental cattle had a greater HPA axis response, which led to greater concentrations of cortisol and glucose, possibly because the glucose was being utilized differently by the temperamental cattle. Mature Brahman cow feed efficiency data was collected over two years, on two different cohorts of cows that had previous residual feed intake data as post-weaning heifers. In 2009 and 2010, 37 and 41 cows, respectively, in their first trimester of gestation were evaluated for RFI via the Calan gate system. Cows were fed 2.6% BW for 70 d with BW recorded weekly. Cows were classified according to their RFI values as either efficient or inefficient. Heifer RFI was not correlated to mature cow RFI based on assessment of the Pearson?s correlation coefficient (r = -0.06, P = 0.57). This study indicates that establishment of RFI in heifers may not accurately predict their feed efficiency as mature cows.Item The Investigation of the Effects of Low Light Laser Therapy on Insulin Secretion in Porcine Islets, a Pilot StudyRaptis, Darren James; Huang, Floyd; Mason, Carolyn; Moore, KellyLED light technology is used in this pilot study to demonstrate the ability to create a cost effective light apparatus using 1-watt LEDs with wavelengths of 740nm,850nm, and 940nm to test for an increased insulin response using porcine islet of Langerhans cells. This is building on recent research showing increased insulin response in rat islet cells when exposed to LED light. However, due to the high variance and low sample size, statistically significant changes were not measured when irradiating cells with 740nm, 850nm, and 940nm LED light using 7.9 J/cm2 and 15.8 J/cm2 dosages along with low and high glucose conditions. There are however possible trends of increased insulin secretion that may become significant with increased sample size. There is a need to repeat this study to definitively determine if increased insulin secretion does occur in porcine islet of Langerhans cells along with any potential negative effects and what the optimal wavelengths and dosages would be. Using the cost effective light apparatus, additional wavelengths can be experimented with to cover between 300nm to over 1600nms. The information garnered from this research has the potential to improve islet of Langerhans cell transplantation for type one diabetes (T1D) patients by decreasing the number of cells needed to be therapeutic and increasing insulin release.