Hyperglycaemia is a common condition in the very low birth weight infant and is linked to mortality and increased risks of morbidities such as sepsis and retinopathy of prematurity. The preterm neonate is in a state of transition from complete dependence on the mother to physiological independence. Many metabolic regulation systems are under-developed, attenuating the natural metabolic hormonal control response. Tight regulation of glucose levels can significantly reduce the negative outcomes associated with hyperglycaemia, but achieving it remains clinically elusive for the neonate. Glucose control in adult critical care is a highly researched topic, and several studies have demonstrated significantly improved outcomes with protocols that modulate the insulin and/or nutrition inputs into the patient. Despite the potential, no standard protocol exists for neonates. Glucose restriction is often used as a treatment for neonatal hyperglycaemia, however this deprives the infant of much needed energy for growth. Limited trials of insulin infusions have been reported, based on fixed protocols or ad-hoc clinical decisions that do not objectively account for an individual patient's metabolic state. Model-based methods can deliver control that is patient-specific and adaptive to handle highly dynamic patients. A physiological model of the glucose-insulin regulatory system is presented in this thesis, adapted from adult critical care. This model has three compartments for glucose utilisation, effective interstitial insulin and its transport, and insulin kinetics in blood plasma, with emphasis on clinical applicability. The predictive control for the model is driven by the patient-specific and time-varying insulin sensitivity parameter. A novel integral-based parameter identification enables fast and accurate real-time model adaptation to individual patients and patient condition. Validation on retrospective clinical data demonstrated the model's ability to capture the major dynamics of the glucose-insulin system in the critically ill neonate. Model fit and prediction performance analysis resulted in a similar level of performance as adult intensive care models and thus suitable for model-based targeted control. Comparison of insulin sensitivity profiles with adult critical care patients highlighted the glycaemic control problem as one of managing inter- and intra-patient variability. Stochastic models and time-series methods for forecasting future insulin sensitivity are presented in this thesis. These methods can deliver probability intervals to support clinical control interventions. The risk of adverse glycaemic outcomes given observed variability from cohort-specific and patient-specific forecasting methods can be quantified to inform clinical staff. Hypoglycaemia can thus be further avoided with the probability interval guided intervention assessments. Simulation studies of clinical control trials on `virtual patients' derived from retrospective clinical data provided a framework to optimise control protocol design in-silico. Comparisons with retrospective control showed substantial improvements in glycaemia within the target 4 - 7 mmol/L range by optimising the infusions of insulin. The simulation environment allowed experimentation with controller parameters to arrive at a protocol that operates within the constraints imposed by the clinically fragile state of the preterm infant. The resulting control system was piloted in seven 12-24 hour clinical trials at the Christchurch Women's Neonatal Department. Glucose levels were tightly controlled in all cases over a trial cohort that represented a wide range of patient conditions and severity of illness. Model predictive performance agreed with simulation results and the stochastic model forecast bounds maintained patient safety. Overall, the research presented takes model-based neonatal glycaemic control from concept to proof-of-concept clinical pilot trials. The thesis develops the full range of models, tools and methods to optimise the protocol design and problem solution. This research thus provides a template for model-based glycaemic control development in general that could be extended to other glycaemic control and similar problems.

Transplantation of the islets of Langerhans is a promising technique for restoring the impairedinsulin production in brittle type 1 diabetics. The downside is that the patient will have to takeimmunosuppressant drugs in order to protect the islet cells from the immune system. Donorsare also sparse, making the quest of finding sufficient amounts of islets for transplantationhard. Encapsulation of the islets of Langerhans has been proposed as a means of protectingthe cells from the immune system taking away the need for immunosuppresives. The mostcommon encapsulation technique is extravascular capsules, which are categorized into micro-and macrocapsules. The microcapsules hold only one or a small set of islet whereas themacrocapsules hold a large quantity of islets.This thesis investigates the encapsulation impact on the beta-cells rapid insulin response torising plasma glucose levels. This was done by simulating the glucose-insulin system inMATLAB with included encapsulation of the islets. Two current macro-encapsulation set upswere used in the model, Beta-Air and ViaCyte devices, and they were compared against anormal case. The results showed that the Beta-Air device would not be able to restorenormoglycemia in a T1DM patient but rather showed a delay in insulin response, while theViaCyte device could mimic the normal case well.

To determine the influence of muscle mass and absolute work on glucose tolerance and insulin sensitivity following exercise, glucose and insulin responses to an oral glucose tolerance test (OGTT) were analyzed in twelve subjects at baseline and 16 to 18 hrs following three different exercise trials performed on a cycle ergometer: 1) two-legged exercise at 60% of two-leg maximal oxygen uptake (VO2max), 2) one-legged exercise at 60% of the oneleg VO2max, and 3) a second one-leg trial at 60% of one-leg VO2max with work matched to the work obtained during the two-leg trial. Each trial was preceeded by two days of inactivity and a three day diet replication. Analysis of serum glucose concentrations during the post-exercise OGTTs demonstrated that glucose tolerance was unaffected by either the amount of active tissue incorporated in the exercise and/or the amount of work completed by the active tissue. On the other hand, serum insulin concentrations following the two-leg trial decreased 23.5% from 347.62 ±37.98 to 266.05 :L41.62 gU/ml in comparison to the one-leg trial (p < 0.05). The incorporation of a smaller muscle mass which completed an equal amount of absolute work as the larger muscle mass (i.e. one-leg work matched trial) resulted in a large (19%), but nonsignificant reduction in the total insulin compared to the one-leg relative work trial. In addition, total insulin following the two-leg and the one-leg work matched trials were reduced by 19% and 14%, respectively, in comparison to baseline. However, they did not reach statistical significance. The results of this study indicate that the incorporation of a larger muscle mass during an acute bout of aerobic exercise results in a reduction in serum insulin in response to a post-exercise oral glucose challenge. In addition, increasing the absolute work of a muscle mass results in similar reductions in serum insulin regardless of the amount of muscle mass involved in the exercise. While glucose tolerance was unaltered by either the amount of active tissue and/or the amount of work completed by the active tissue. / School of Physical Education

Considerable research has been performed on the effects of exercise and glucose tolerance, however, most of this work has examined aerobic exercise designs. This study examines the immediate post-exercise glucose turnover in eight male subjects exposed to a single bout of running and PRE. Both exercise protocols were designed to be of similar duration and at an intensity representing a typical exercise session. This study was conducted in an effort to offer individuals with NIDDM an alternative to the established aerobic forms of exercise for improved glucose control. Each subject completed two preliminary procedures, which consisted of a maximal graded exercise test and a session where a 1 RM was established on six different Cybex variable resistance machines. Subjects then completed a baseline oral glucose tolerance test (OGTT) in which eight blood samples were analyzed for glucose, insulin, hemoglobin, and hematocrit. Two exercise protocols, separated by 3 to 10 days, consisting of a 40 minute treadmill run at 75% VO2max and a 40 minute, 3 set x 10 repetition based on 75% of the1 RM, were performed and followed 45 minutes later by another OGTT. The results demonstrated that there were no apparent differences in blood glucose or insulin levels post-exercise between the exercise modes. However, the form of exercise did seem to have a varied effect on insulin production. The results of the OGTT demonstrated an explicit difference in the insulin response between the lifting and running trials, with the lifting trial being significantly higher than the resting or running trials. The increased insulin levels observed in the lifting trial may be indicative of increased secretion from the pancreas or that the secreted insulin is simply not being used. The insulin resistance observed in the lifting trial may be due to the muscles inability to respond to insulin or some other metabolic factor(s) released during exercise. Additional studies should be performed on different populations to examine the effects of PRE and running in a effort to better understand the mechanisms responsible for glucose uptake. / School of Physical Education

Insulin-like growth factor-I (IGF-I) is a known stimulator of glucose transport. IGF binding protein 1 (IGFBP1) is a protein that regulates the actions of IGF-I by binding to IGF-I which alters it's ability to bind to the IGF-I receptor. Diet and exercise may influence this system. While IGFBP1 levels increase with fasting or prolonged exercise, feeding will reverse this elevation. The intent of this study was to determine if an in vivo manipulation of IGFBP1 affects in vitro glucose transport in the rat soleus. Sixteen male Spaque Dawley rats were fasted for 12 hours. Half of the animals were then allowed a two hour ad libitum refeeding period. Animals were anesthetized and had their soleus muscles removed. Muscles were then randomly assigned to one of four treatment groups. Treatments involved an incubation in either 4 or 8 mM glucose in either the presence or absence of IGF-I (75 ng x ml"'). Final incubation for all treatment groups included [3H]-3-O-methylglucose (437 µCi x mM-) for the measurement of glucose transport. Following incubation, muscles were weighed, homogenized in 1 ml of 10% trichloroacetic acid, and centrifuged to precipitate out protein. 100 µl of the supernatant was added to 3 ml of scintillation fluid and analyzed in a scintillation counter. Glucose transport was determined by 3H activity. A statistical analysis of the various groups shows that there is no significant difference between fasted and refed animal for any specific treatment. However, when all the fasted and refed animals area grouped, glucose transport rate is significantly greater (p<0.05) in fasted (3.59 ± 0.44 µM x ml"' x hr) animals than in refed animals (2.56 ± 0.27 µM x ml"' x hr'). Additionally, muscles that were treated with IGF-I in 8 mM glucose demonstrated a greater rate of glucose transport (5.12 ± 0.68 µM x ml-1 x hr') than all other treatments (2.13 ± 0.39 to 2.90 ± .33 µM x ml-' x hr'). This study showed that IGF-I is a stimulator of glucose transport in an 8 mM glucose media. Additionally, the results show that glucose transport is greater if the animals are fasted. The differences between fasted and refed animals demonstrated in this study supports the hypothesis that diet manipulated IGFBP1 levels are able to alter the biological effects of IGF-I. / School of Physical Education

Loss of glucose-stimulated insulin secretion (GSIS) from the pancreatic beta-cells is one of the earliest detectable defects in the pathogenesis of type 2 diabetes. However, despite its relevance, the mechanisms that govern GSIS are still not completely understood. ARNT/HIF-1β is a member of the bHLH-PAS family of transcription factors, with a prominent role in the transcriptional regulation of enzymes required for the metabolism of xenobiotics as well as regulation of genes that are critical for cellular responses to hypoxia. Recent research has uncovered a previously unknown function for ARNT/HIF-1β in the pancreatic beta-cells, where the gene was found to be 90% down-regulated in human type 2 diabetic islets and loss of ARNT/HIF-1β protein leads to defective GSIS in pancreatic beta-cells of mice. The main focus of this thesis was to understand the mechanisms by which ARNT/HIF-1β maintains normal GSIS from pancreatic beta-cells and understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice. ARNT/HIF-1β was found to positively regulate GSIS in both INS-1 derived 832/13 cell line and mice islets. In the 832/13 cells, loss of ARNT/HIF-1β leads to a reduction in glycolysis without affecting the glucose oxidation and the ATP/ADP ratio suggesting that the regulation of GSIS takes place in a manner that is independent of the KATP channels. In order to further assess the mechanism of lowered GSIS in the absence of ARNT/HIF-1β in the 832/13 cells, a metabolite profiling was performed which revealed a significant reduction in the metabolite levels of glycolysis and the TCA cycle intermediates and glucose-induced fatty acid production, suggesting the involvement of ARNT/HIF-1β in regulating glucose-stimulated anaplerosis, which is believed to play a key role in the regulation of GSIS from the pancreatic beta-cells. The changes in metabolite levels in the absence of ARNT/HIF-1β were associated with corresponding changes in the gene expression pattern of key enzymes regulating glycolysis, the TCA cycle and fatty acid synthesis in beta-cells. In an attempt to understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice, a pancreatic beta-cell specific ARNT/HIF-1β knock out mouse (β-ARNT KO) was generated using the Cre-loxP technology. Functional characterization of islets from both male and female β-ARNT KO mice revealed a significant impairment in GSIS, which was attributed due to a small, but significant reduction in rise in intracellular calcium upon glucose stimulation. Further analysis revealed reduced secretory response to glucose in the presence of KCl and diazoxide indicating a defect in the amplifying pathway of GSIS in β-ARNT KO islets. Expression of pyruvate carboxylase (PC) was significantly reduced in β-ARNT KO islets suggesting possible impairments in anaplerosis and consistent with this, defect in GSIS in β-ARNT KO islets could be almost completely rescued by treatment with membrane permeable TCA intermediates. Surprisingly, both male and female β-ARNT KO mice have normal glucose homeostasis. In an attempt to assess how β-ARNT KO mice maintained normal blood glucose levels, indirect calorimetry was used to understand changes in whole-body energy expenditure. This investigation revealed that β-ARNT KO mice exhibited a small but significant increase in respiratory exchange ratio (RER), suggesting a preference in utilizing carbohydrates as a fuel source, possibly leading to improved glucose uptake from the blood stream. Response to exogenous insulin was completely normal in β-ARNT KO mice suggesting intact functioning of the skeletal muscles. To conclude, based on our in vitro data, we believe that ARNT/HIF-1β plays an indispensable role in maintaining normal beta-cell secretory function, however, results from β-ARNT KO mice indicates that these mice are protected from the adverse effects of hyperglycemia. Although loss of ARNT/HIF-1β alone is not sufficient for the genesis of type 2 diabetes, it creates a perfect storm in the pancreatic beta-cells that may eventually lead to an imbalance in the whole body glucose homeostasis. Our study provides significant information to the scientific community that engages in assessing the pharmacological potential of gene targets for the treatment of type 2 diabetes.

No description available.

The present project investigated the effect of epidural blockade with local anesthetic on the catabolic stress response during and immediately after abdominal surgery in fasting patients and during infusion of glucose at 2 mg&middot;kg-1&middot;min-1. The kinetics of glucose and protein metabolism were assessed by the stable isotope tracers [6,6-2H2]glucose and L-[1-13C]-leucine. / Epidural blockade was associated with a lower plasma glucose concentration and glucose production when compared to control subjects in the fasted state. Whole body protein breakdown, amino acid oxidation and protein synthesis were suppressed during surgery, and epidural blockade had no modifying effect on perioperative protein metabolism. The suppression of endogenous glucose production by exogenous glucose was more pronounced in the presence of epidural blockade. Perioperative protein metabolism, however, was not influenced by epidural blockade during glucose infusion. / Although epidural blockade suppressed glucose metabolism both in the fasted state and during glucose administration, it failed to exert a modifying effect on perioperative protein metabolism.

Hypothalamic AMP-activated protein kinase (AMPK) regulates energy homeostasis in response to nutritional and hormonal signals. However, its role in glucose production regulation remains to be elucidated. Here, we tested the hypothesis that bidirectional changes in hypothalamic AMPK activity alter glucose production in rodents. First, we found that knocking down hypothalamic AMPK activity in an in vivo rat model led to a significant suppression of glucose production independent of changes in food intake and body weight. Second, we showed that activation of hypothalamic AMPK negated the ability of hypothalamic glucose- and lactate- sensing to lower glucose production. Collectively, these data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo, and highlight the novel role of hypothalamic AMPK in the maintenance of glucose homeostasis in addition to energy balance.

Hypothalamic AMP-activated protein kinase (AMPK) regulates energy homeostasis in response to nutritional and hormonal signals. However, its role in glucose production regulation remains to be elucidated. Here, we tested the hypothesis that bidirectional changes in hypothalamic AMPK activity alter glucose production in rodents. First, we found that knocking down hypothalamic AMPK activity in an in vivo rat model led to a significant suppression of glucose production independent of changes in food intake and body weight. Second, we showed that activation of hypothalamic AMPK negated the ability of hypothalamic glucose- and lactate- sensing to lower glucose production. Collectively, these data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo, and highlight the novel role of hypothalamic AMPK in the maintenance of glucose homeostasis in addition to energy balance.