Insulin signaling is highly conserved across animals, and is known for its ubiquitous function in all aspects of animal physiology. Despite its relatively well-studied role in metabolism and energy expenditure, how it is involved in learning and memory remains a mystery, due to the complex nature of the nervous system. In this thesis, I have used C. elegans, a tractable model organism with a sophisticated behavioral repertoire, to investigate molecular and cellular mechanisms of insulin signaling in learning.

Chromium has been reported to increase glucose clearance in insulin resistant and diabetic populations. Skeletal muscle is the tissue primarily responsible for glucose clearance. We therefore tested the effect of chromium chloride (CrCl3) on skeletal muscle glucose uptake both in the absence and presence of a submaximal level of insulin via the rat hindlimb perfusion technique. 0.096 μM CrCl3 was used with and without 200 μU/ml insulin. Our testing showed that insulin significantly increased [H3]-2 deoxyglucose (2-DG) uptake in both the gastrocnemius and quadriceps muscles. Additionally, the combination of CrCl3 and insulin (Cr-sIns) led to greater amounts of 2-DG uptake than insulin alone (sIns) in both the gastrocnemius (Cr-sIns 6.49±0.75 μmol/g/h, sIns 4.83±0.42 μmol/g/h) and quadriceps (Cr-sIns 6.74±0.62 μmol/g/h, sIns 4.54±0.43 μmol/g/h). However, CrCl3 without insulin (Cr) had no affect on 2-DG uptake above basal (Bas) in both the gastrocnemius (Cr 1.45±0.14 μmol/g/h, Bas 1.61±30 μmol/g/h) and the quadriceps (Cr 1.35±0.15 μmol/g/h, Bas 1.27±0.13 μmol/g/h). It has been speculated that chromium works to increase glucose uptake by increasing insulin signaling. To examine this, we used western blotting analysis to test both Akt and AS160 phosphorylation in the mixed gastrocnemius. We found that insulin increased Akt and AS160 phosphorylation, but chromium had no affect on Akt (Cr-sIns 25%±2%, sIns 22%±4%) or AS160 (Cr-sIns 35%±5%, sIns 36%±4%) phosphorylation in the absence or presence of insulin. Our results suggest that supplementation with CrCl3 can lead to an increase in glucose uptake in skeletal muscle, but only in the presence of insulin. However, this effect of CrCl3 does not appear to be a result of enhanced insulin signaling. / text

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This project focused on determining the effect of lithium on glucose uptake, glycogen synthesis, and insulin signaling proteins, protein kinase B (Akt1) and GSK-3, in isolated soleus muscle from female lean Zucker rats. We also investigated the role of the stress-activated p38 MAPK in the action of lithium to activate skeletal muscle glucose transport. In the absence of insulin, lithium (10 mM LiCl) increased basal glucose transport by 62% (p<0.05) and glycogen synthesis by 112%. Lithium did not alter phosphorylation of Akt ser473, but enhanced GSK-3β ser9 phosphorylation by 41%. Lithium further enhanced the effect of insulin on glucose transport (42%), glycogen synthesis (44%), and GSK-3ß phosphorylation (13%). Lithium increased phosphorylated p38 MAPK 31% without and 19% with insulin. Moreover, a selective p38 MAPK inhibitor, A304000, completely prevented the lithium-induced enhancement of glucose transport revealing the critical involvement of p38 MAPK phosphorylation in lithium-induced glucose transport in isolated skeletal muscle.

Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting.

The aim of this study was to prepare biologically active insulin-loaded alginate microparticles by spray drying. Particles were produced from three alginate feed concentrations of 1, 1.5 and 2% w/v, with respective insulin loadings of 11.8, 7.8 and 5.8 mg/g of alginate and investigated in terms of mass yield, moisture content, particle size, morphology and encapsulation efficiency. The mass yield of the system was determined to be between 15 and 30%, with approximately 3% of the initial dry mass ending up in the exhaust filter. The moisture content of the particles was found to be between 4.9 and 11.1% and the mean size ranged between 1.2 and 1.6 μm. Particulate morphologies were observed to be mostly spherical with some ‘divots’ present on the surface. Lastly, the encapsulation efficiency determined by absorbance assay was approximately 40%. Particles produced from a 2% alginate feed were further assayed by determining the release of insulin in simulated gastrointestinal conditions and looking at the insulin and alginate distribution within spray dried particles. A steep release profile was observed in the first 120 min of the simulation in a gastric pH of 1.2 and a longer, more sustained release is observed in intestinal conditions, where an additional 20% of the total insulin in the particles is released over 600 min. Fluorescent labels revealed that insulin and alginate are concentrated towards the periphery of the particles. The residual bioactivity of insulin was assessed by an in vitro bioactivity assay, which was developed using Fast Activated Cell Based ELISA (FACE™) AKT kits specific for phosphylated AKT. The bioactivity of insulin in the particles after spray drying was determined to be 87.9 ± 15.3%. / Thesis (Master, Chemical Engineering) -- Queen's University, 2009-09-20 20:32:29.103