Glucose metabolism in insulin-sensitive tissues such as skeletal muscle and adipose tissue is tightly regulated by external stimuli. Metabolic changes in these tissues have direct effects on whole body metabolism. Such metabolic changes can be induced or influenced by adrenergic stimulation. In L6 skeletal muscle cells, we have seen that the β2-adrenergic receptor increases glycogen synthesis to the same extent as insulin. The β2-adrenergically mediated effect is independent of cyclic AMP but dependent on PI3K. In brown adipocytes, our data suggest that signaling from the β-adrenergic receptors consists of an acute cyclic AMP effect that is rapidly desensitized and then a prolonged signal involving PI3K. In skeletal muscle cells in culture, we have shown that DPI (a NADPH oxidase inhibitor) increases glucose uptake through a signaling pathway independent of NADPH oxidase and insulin signaling. DPI instead inhibits complex 1 in the mitochondrial respiratory chain, which lowers ATP levels. This activates AMPK, an activator of glucose uptake. Furthermore, we have developed a model system for ordered fusion of skeletal muscle cells in culture. In this system, differentiating skeletal muscle cells can be studied separately. This system is optimal for microscopy techniques and easily adaptable for micromanipulations. We have seen that the myogenic factor MyoD can have different expression of the protein in different nuclei within the same myotube. This system could be used with advantage for intracellular signaling and metabolic studies.

Diabetes mellitus affects 25.8 million Americans (8.3%) and over 300 million people worldwide. Clinical trials indicate that proper management of blood glucose levels is critical in preventing or delaying complications associated with diabetes. Thus, there is a common need to monitor and manage blood glucose properly for people with diabetes. However, the patients’ compliance for recommended monitoring frequency is low due to the pain and inconvenience of current standard finger-pricking tests. To promote patient adherence to the recommended self-monitoring frequency, non-invasive/ minimally invasive glucose testing approaches are needed. Luminescent microparticle sensor is an attractive solution. For these sensors to be deployed in vivo, a matched optical system is needed to interrogate dermally-implanted sensors. This research project investigated the light propagation in skin and the interaction with implants using Monte Carlo modeling. The results of the modeling were used to design an optical system with high interrogation and collection efficiency (40~300 times improvement). The optical system was then constructed and evaluated experimentally. A stable skin phantom mimicking the optical properties of human skin was developed as a permanent evaluation medium to minimize the use of animals. The optical properties of the skin phantom matched the maximum published values of human skin in scattering and absorption over the spectral range of 540~700nm in order to avoid overestimation of the capability of the system. The significant photon loss observed at the connection between the designed system and a commercial spectrometer was overcome using two optimized designs: a two-detector system and a customized low-resolution spectrometer system. Both optimization approaches effectively address the photon loss problem and each showed good SNR (>100) while maintaining a sufficient system resolution for use with fluorescent materials. Both systems are suitable for luminescence measurement, because broad bands of the luminescent spectrum are of interest. In the future, either system can be easily modified into a more compact system (e.g. handheld), and it can be directly coupled to an analog-to-digital converter and integrated circuits offering potential for a single compact and portable device for field use with luminescent diagnostic systems as well as implanted sensors.

The purpose of this work is to study the interference between the coherent nonresonant four-wave-mixing (FWM) background and the Raman-resonant signal in the coherent anti-Stokes Raman spectroscopy (CARS). The nonresonant background is usually considered as a detriment to CARS. We prove that the background can be exploited in a controllable way, through the heterodyne detection due to the interference, to amplify the signal and optimize the spectral shape of the detected Raman signal, and hence enhance the measurement sensitivity. Our work is based on an optimized CARS technique which combines instantaneous coherent excitation of multiple characteristic molecular vibrations with subsequent probing of these vibrations by an optimally shaped, time-delayed, narrowband laser pulse. This pulse configuration mitigates the nonresonant background while maximizing the resonant signal, and allows rapid and highly specific detection even in the presence of multiple scattering. We investigate the possibility of applying this CARS technique to non-invasive monitoring of blood glucose levels. Under certain conditions we find that the measured signal is linearly proportional to the glucose concentration due to optical interference with the residual background light instead of a quadratic dependence, which allows reliable detection of spectral signatures down to medically-relevant glucose levels. With the goal of making the fullest use of the background, we study the interference between an external local oscillator (nonresonant FWM field) and the CARS signal field by controlling their relative phase and amplitude. Our experiment shows that this control allows direct observation of the real and imaginary components of the third-order nonlinear susceptibility (χ(3)) of the Raman sample. In addition, this method can be used to amplify the signal significantly. Furthermore, we develop an approach by femtosecond laser pulse shaping to precisely control the interference between the Raman-resonant signal and its intrinsic nonresonant background generated within the same sample volume. This technique is similar to the heterodyne detection with the coherent background playing the role of the local oscillator field. By making fine adjustments to the probe field shape, we vary the relative phase between the resonant signal and the nonresonant background, and observe the varying spectral interference pattern. These controlled variations of the measured pattern reveal the phase information within the Raman spectrum, akin to holographic detection revealing the phase structure of a source.

Hyperproinsulinemia is associated with type 2 diabetes (T2DM) and obesity and is a predictor for future coronary heart disease. This thesis examines the effect of bariatric surgery on glucometabolic status including insulin and proinsulin responses after meal. Further we explored longitudinally the effects of bariatric surgery on glucose, insulin and proinsulin secretion as well as lipids, liver enzymes and magnesium concentrations. We explored by a standardised meal test the postprandial dynamics of proinsulin and insulin and effects on glucose and lipids in patients treated with gastric bypass (RYGBP) surgery and in patients treated with bileopancreatic diversion with duodenal switch surgery (BPD-DS). Comparisons were made to morbidly obese patients and normal weight controls (NW). RYGBP surgery markedly lowers fasting and postprandial proinsulin concentrations although BMI was higher compared to NW-controls. BPD-DS surgery induces a large weight loss and normalises postprandial responses of glucose, proinsulin and insulin and markedly lowers triglycerides. We evaluated non-diabetic morbidly obese patients who underwent bariatric surgery followed-up for up to four years after surgery. Long-term follow-up showed that RYGBP surgery is not only characterized by markedly and sustained lowered BMI but also lowered concentrations of proinsulin, insulin, ALT and increased HDL-C possibly via reduced hepatic insulin resistance. We also examined how magnesium status is affected by bariatric surgery as magnesium has been shown to be inversely related to glucose and to insulin resistance. The serum magnesium concentrations increased by 6% after RYGBP and 10% after BPD-DS. In summary, RYGBP and BPD-DS surgery results in marked weight loss, alterations in insulin and proinsulin dynamics, lowered fasting and postprandial proinsulin concentrations and improved glucometabolic and magnesium status.

The aim of the study was auditing medical records examine postoperative blood glucose levels after undergoing CABG surgery. Furthermore the aim was to determine if the ward staff abides the local guidelines frame of reference concerning each ward, regarding blood glucose measurements and blood glucose levels. The study included 70 patients undergoing CABG surgery at the cardiothoracic surgery, Uppsala University Hospital. The study was conducted by retrospective medical record auditing. Studied factors were postoperative blood glucose levels, number of registered blood glucose measurements, a current diagnosis of DM and preoperative HbA1c. Mean level of blood glucose levels stayed continuously above the local guidelines frame of reference for both TIVA/TIMA and the care ward throughout the continuity of patient care. The number of registered blood glucose measurements per postoperative day at TIVA/TIMA where within the local guidelines. The result showed that the local guidelines frame of reference concerning the ward were not reached. A difference could be seen between patients with DM and patients without DM regarding the previously mentioned factors. Preoperative elevated levels of HbA1c could have influenced the number of postoperative blood glucose measurements. Recommendations will therefore be too audit the current local guideline that concerns the treatment, therapy goals and the number of blood glucose measurements. Establishing criterions regarding termination of blood glucose measurements and the transfer day between TIVA/TIMA and the care ward are recommended.

Glucose-6-phosphate (G6P) plays an important role in carbohydrate metabolism of all living organisms. Compared to the conventional analytical methods available for estimation of G6P, the biosensors having relative simplicity, specificity, low-cost and fast response time are a promising alternative. We have reported a G6P biosensor based on screen-printed electrode utilizing Prussian Blue (PB) nanoparticles and enzymes, glucose-6-phosphate dehydrogenase and glutathione reductase. The PB nanoparticles acted as a mediator and thereby enhanced the rate of electron transfer in a bi-enzymatic reaction. The Fourier transform infrared spectroscopy and energy-dispersive X-ray spectroscopy study confirmed the formation of PB, whereas, the atomic forced microscopy revealed that PB nanoparticles were about 25-30 nm in diameter. Various optimization studies, such as pH, enzyme and cofactor loading, etc. were conducted to obtain maximum amperometric responses for G6P measurement. The developed G6P biosensor showed a broad linear response in the range of 0.01-1.25 mM with a detection limit of 2.3 mM and sensitivity of ­63.3 mA/mM at a signal-to-noise ratio of 3 within 15 s at an applied working potential of -100 mV. The proposed G6P biosensor also exhibited good stability, excellent anti-interference ability and worked well for serum samples.

Glucose transporter GLUT4 allows glucose uptake into muscle and adipose cells. Insulin promotes recruitment and plasma membrane insertion of GLUT4 vesicles that can recycle constitutively. Obesity and type 2 diabetes mellitus are associated with defects in insulin-induced GLUT4 recruitment. Knowledge of alternative modes and steps of GLUT4 traffic in L6-GLUT4myc muscle cells may reveal possible targets for therapeutic intervention in insulin-resistant patients. Hypertonicity and Platelet Derived Growth Factor also increase surface GLUT4 levels but it was unknown if they tap on the same intracellular GLUT4 depots as insulin. We explored whether GLUT4 vesicles recycle using different compartments and mechanisms for the surface gain elicited by each stimulus. We hypothesized that all vesicle fusion steps require NSF but depend on individual v-SNAREs. Specifically, we tested effects of ATPase-deficient NSF or VAMP7 siRNA transfections, and endosomal ablation on GLUT4 traffic. We show that VAMP7 was required for basal and hypertonic recycling, while VAMP2 is exclusively used in response to insulin. As insulin action bifurcates downstream of phosphatidylinositol 3-kinase, we also hypothesized that the Rac-to-actin and Akt-to-AS160 branches regulate distinct GLUT4 traffic steps. For this we determined GLUT4myc localization in rounded myoblasts relative to a surface marker. Interfering with Rac, actin dynamics or actin-binding α-actinin4 maintained GLUT4 in a perinuclear region even under insulin-stimulation. Interfering with AS160 allowed significant GLUT4 accumulation beneath the membrane, but not fusion. We propose that actin dynamics and α-actinin4 are required for cortical GLUT4 tethering mechanisms, and AS160 contributes to GLUT4 docking/fusion. We confirmed that VAMP2 facilitates GLUT4 fusion, as tetanus toxin-based cleavage did not inhibit peripheral GLUT4 recruitment. Finally, AS160 targets Rab8A and Rab14 in muscle respectively affected GLUT4 availability for membrane fusion, and basal GLUT4 retention. This work will lead to future testing of strategies to selectively enhance vesicle availability, tethering, or surface fusion, for bypassing insulin resistance.

No description available.

In-utero ethanol exposure may result in fetal alcohol spectrum disorder (FASD). Studies have suggested that women who drink ethanol are more likely to consume illicit drugs. Detection of such exposures has been done via meconium and hair testing and can serve to direct needed prevention methods and appropriate management and intervention for the neonate and the mother. This study examined maternal diabetes as a possible confounder for in-utero ethanol exposure testing and determined the trends in drug use associated with heavy in-utero ethanol exposure in a high-risk obstetric Canadian population. It was determined that maternal diabetes does not produce false-positive results in testing for in-utero ethanol exposure. Furthermore, heavy in-utero ethanol exposure was detected in 15.5% of samples and was associated with an increased exposure to amphetamines (OR=3.30) and opiates (OR=2.01), but a decreased exposure to cannabinoids (OR=0.61) when compared to neonates with no heavy in-utero ethanol exposure.

In-utero ethanol exposure may result in fetal alcohol spectrum disorder (FASD). Studies have suggested that women who drink ethanol are more likely to consume illicit drugs. Detection of such exposures has been done via meconium and hair testing and can serve to direct needed prevention methods and appropriate management and intervention for the neonate and the mother. This study examined maternal diabetes as a possible confounder for in-utero ethanol exposure testing and determined the trends in drug use associated with heavy in-utero ethanol exposure in a high-risk obstetric Canadian population. It was determined that maternal diabetes does not produce false-positive results in testing for in-utero ethanol exposure. Furthermore, heavy in-utero ethanol exposure was detected in 15.5% of samples and was associated with an increased exposure to amphetamines (OR=3.30) and opiates (OR=2.01), but a decreased exposure to cannabinoids (OR=0.61) when compared to neonates with no heavy in-utero ethanol exposure.