Browsing by Subject "Insulin"

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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.
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.
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.
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.
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.
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, Krishnendu
To 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.
Physical inactivity does not impair the insulin-lowering effects of moderate-intensity exercise, yet it does impair fat metabolism
(2020-08-17) Dial, Michael; Coyle, Edward F., 1952-
Acute exercise and physical activity improve insulin sensitivity, glucose tolerance, and postprandial lipemia, although recent research suggests that physical inactivity may attenuate some of these healthy metabolic benefits of exercise. This study aimed to determine how two days of physical inactivity and physical activity affected exercise-induced changes in plasma insulin, glucose, and triglyceride concentrations during an oral glucose tolerance test (OGTT) performed the next morning. Five untrained men (n=2) and women (n=3) completed three five-day trails in a randomized crossover design. Each trial began with two days of normal activity levels, followed by two intervention days. Two days of physical inactivity (3,666 ± 100 steps) without exercise (SIT) were compared to two days of physical inactivity (3,077 ± 141 steps) with a 1-hr bout of moderate intensity cycling at 65% of VO2peak (SIT + EX). Finally, two intervention days of high activity (12,270 ± 408 steps) were performed with a 1-hour bout of moderate cycling (ACTIVE + EX). The following morning, subjects completed a 120-min OGTT, during which plasma was collected and analyzed for glucose, insulin, and triglycerides. No changes were observed in plasma glucose. Compared to SIT, insulin total area under the curve (AUCT) was 39% lower in SIT+EX (p=0.18) and 21% lower in ACTIVE+EX (p=0.48), demonstrating that exercise had an insulin-lowering effect. Plasma triglyceride AUCT in ACTIVE+EX was 26% lower than SIT (p=0.10) and 28% lower than SIT+EX (p=0.03), and fasting plasma triglyceride concentration in ACTIVE+EX was 34% lower than SIT (p=0.06) and 20% lower than SIT+EX (p=0.43). These data indicate that the insulin-lowering effect of physical activity is influenced to a greater extent by an acute bout of moderate exercise (65% VO2peak) than the background daily steps. Conversely, the triglyceride lowering effect of physical activity appears to be contingent upon higher physical activity (daily step count) and not acute moderate-intensity exercise. This also was the case with postprandial whole-body fat oxidation as ACTIVE+EX was 26% higher when compared to SIT+EX. Taken together, insulin sensitivity appears to be improved by prolonged moderate-intensity exercise (i.e. 65% of VO2peak), while postprandial triglyceride concentration and fat oxidation are improved by increasing daily step count (i.e.; from < 4,000 to >11,000 per day).
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.