Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

<p>The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste.</p><p>In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster.</p><p>In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.</p>

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 110-128).

The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste. In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster. In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.

Grb10 is a member of the Grb7 family of adaptor proteins that also includes Grb7 and Grb14. These three members contain multiple protein binding domains and lack enzymatic activity. Extensive two-hybrid studies have demonstrated binding of Grb10 to numerous activated tyrosine kinase receptors including the insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR), as well as many non-receptor molecules such as MEK1, Raf-1, and Nedd4. Grb10 has been implicated in IGF-I anti-apoptotic signaling regulation through interactions with Raf-1 and the mitochondrial membrane. / In this report the pattern of transient Grb10 translocation following IGF-I cellular stimulation was studied. This report also demonstrates the implication of a short variable amino-terminal region of Grb10 in mitochondrial membrane association. Finally, assays were developed with the goal of identifying new Grb10 binding partners.

Hyperglycemia is prevalent in critical care, as patients experience stress-induced hyperglycemia, even with no history of diabetes. Hyperglycemia has a significant impact on patient mortality, outcome and health care cost. Tight regulation can significantly reduce these negative outcomes, but achieving it remains clinically elusive, particularly with regard to what constitutes tight control and what protocols are optimal in terms of results and clinical effort. Hyperglycemia in critical care is not largely benign, as once thought, and has a deleterious effect on outcome. Recent studies have shown that tight glucose regulation to average levels from 6.1–7.75 mmol/L can reduce mortality 17–45%, while also significantly reducing other negative clinical outcomes. However, clinical results are highly variable and there is little agreement on what levels of performance can be achieved and how to achieve them. A typical clinical solution is to use ad-hoc protocols based primarily on experience, where large amounts of insulin, up to 50 U/hr, are titrated against glucose measurements variably taken every 1–4 hours. When combined with the unpredictable and sudden metabolic changes that characterise this aspect of critical illness and/or clinical changes in nutritional support, this approach results in highly variable blood glucose levels. The overall result is sustained periods of hyper- or hypo- glycemia, characterised by oscillations between these states, which can adversely affect clinical outcomes and mortality. The situation is exacerbated by exogenous nutritional support regimes with high dextrose content. Model-based predictive control can deliver patient specific and adaptive control, ideal for such a highly dynamic problem. A simple, effective physiological model is presented in this thesis, focusing strongly on clinical control feasibility. This model has three compartments for glucose utilisation, interstitial insulin and its transport, and insulin kinetics in blood plasma. There are two patient specific parameters, the endogenous glucose removal and insulin sensitivity. A novel integral-based parameter identification enables fast and accurate real-time model adaptation to individual patients and patient condition. Three stages of control algorithm developments were trialed clinically in the Christchurch Hospital Department of Intensive Care Medicine. These control protocols are adaptive and patient specific. It is found that glycemic control utilising both insulin and nutrition interventions is most effective. The third stage of protocol development, SPRINT, achieved 61% of patient blood glucose measurements within the 4–6.1 mmol/L desirable glycemic control range in 165 patients. In addition, 89% were within the 4–7.75 mmol/L clinical acceptable range. These values are percentages of the total number of measurements, of which 47% are two-hourly, and the rest are hourly. These results showed unprecedented tight glycemic control in the critical care, but still struggle with patient variability and dynamics. Two stochastic models of insulin sensitivity for the critically ill population are derived and presented in this thesis. These models reveal the highly dynamic variation in insulin sensitivity under critical illness. The stochastic models can deliver probability intervals to support clinical control interventions. Hypoglycemia can thus be further avoided with the probability interval guided intervention assessments. This stochastic approach brings glycemic control to a more knowledge and intelligible level. In “virtual patient” simulation studies, 72% of glycemic levels were within the 4–6.1 mmol/L desirable glycemic control range. The incidence level of hypoglycemia was reduced to practically zero. These results suggest the clinical advances the stochastic model can bring. In addition, the stochastic models reflect the critical patients’ insulin sensitivity driven dynamics. Consequently, the models can create virtual patients to simulated clinical conditions. Thus, protocol developments can be optimised with guaranteed patient safety. Finally, the work presented in this thesis can act as a starting point for many other glycemic control problems in other environments. These areas include the cardiac critical care and neonatal critical care that share the most similarities to the environment studied in this thesis, to general diabetes where the population is growing exponentially world wide. Furthermore, the same pharmacodynamic modelling and control concept can be applied to other human pharmacodynamic control problems. In particular, stochastic modelling can bring added knowledge to these control systems. Eventually, this added knowledge can lead clinical developments from protocol simulations to better clinical decision making.

Although insulin resistance of glucose and fat metabolism in type 2 diabetes mellitus (T2DM) is firmly established, that of protein remains controversial for methodological reasons. A hyperinsulinemic (40MU/m2·min) euglycemic (5.5 mmol/L) isoaminoacidemic (postabsorptive concentrations) clamp was combined with [3-3H]glucose and [1-13C]leucine kinetics to concurrently assess protein and glucose metabolism in 10 hyperglycemic men with T2DM and 11 men without (all BMI=29+/-kg/m2), matched also for age, body composition, and waist circumference. In response to hyperinsulinemia, protein turnover and synthesis were stimulated in controls, but not in T2DM. Both insulin-stimulated total and non-oxidative glucose disposal were diminished in T2DM vs. controls. There was a robust positive correlation between the change in synthesis and glucose disposal. Hence, there is an additive effect of T2DM, beyond that of having excess fat, on insulin resistance of whole body protein turnover and synthesis. Furthermore, protein sensitivity to insulin parallels that of glucose, establishing this as an important concern in T2DM management.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Wollongong, 2004. / Typescript. Bibliographical references: leaf 243-269.