Högskolan i Skövde

The use of models to understand complex phenomena is indispensable to the scientific community. The advantage of a model is that it simplifies the phenomena under study. However, a model should be only as complex as required, no more, no less. Furthermore, a model should avoid known or unknown confounding variables that might obscure the interpretations of observations. Within biology, models can be set up in many different ways, such as mathematical, graphical or verbal descriptions of the system under study. In physiology, the systems under study can be the entire animal or organs or cell cultures from it. To study some aspects of the regulation of glucose and energy homeostasis, skeletal muscles is a preferable model, as it is the main consumer of post-prandial glucose, and thus, important for maintaining whole body glucose and energy homeostasis. Incubation of skeletal muscle specimens in a suitable solution is a model-system that has been used during the last century. The availability of oxygen for energy transformation has been of major concern. Therefore, the experimental system has been validated several times with different methods, both experimentally and mathematically.

The result from experimental validations indicates that glycogen content is unequally distributed within the incubated muscle specimens, with the core depleted of glycogen. Furthermore, validation done with the mathematical models describing the experimental systems indicates that oxygen diffusion is sufficient if the following assumptions are valid; homogeneous structure and that the critical value of oxygen pressure is above zero throughout the entire muscle. However, if those assumptions are invalid, the observations of some metabolic and/or signalling data might be invalid. In this thesis, those assumption are validated, with the specific aim to derive mathematical models that can be used to further analyse the metabolic data generated.

Set of ordinary differential equation was used to describe the metabolic data derived from incubation of mouse extensor digitorum longus skeletal muscles preparations, paper 1. The parameters and constants were identified within the mathematical model, which then, was further analysed. The results indicated that the experimental system suffered from anoxia and that glycogen was depleted during the incubation time. An immunohistochemical approach was used to verify the predictions from the mathematical model on glycogen depletion, paper 2. A statistical approach was developed herein that made quantitative studies possible and the results verified the prediction from the mathematical model in paper 1. Furthermore, a correlation between fibre type distribution and glycogen depletion was observed, indicating that the assumption on homogeneous glucose handling might be too hard. The existence of anoxia within the incubated muscle specimens was revealed. A novel hypothesis regarding deficient insulin diffusion into the centre of the incubated muscle preparation as the cause for quasi-depletion of glycogen was tested, paper 3. The hypothesis was falsified; instead increased insulin signalling was observed in the core of the muscle, correlating with fibre types on the single-cell-level.

In conclusion, the studies presented in this thesis provide evidence that muscle preparations are suffering of anoxia after incubation leading to depletion of glycogen. Furthermore, the assumption on homogeneous glucose handling is falsified. Finally, a mathematical model is provided that can be used to estimate the un-measurable glycogen concentrations and estimate the glucose uptake rate in the superficial fibres.

During in vitro incubation in the absence or presence of insulin, glycogen depletion occurs in the inner core of the muscle specimen, concomitant with increased staining of hypoxia-induced-factor-1-alpha and caspase-3, markers of hypoxia and apoptosis, respectively. The aim of this study was to determine whether insulin is able to diffuse across the entire muscle specimen in sufficient amounts to activate signalling cascades to promote glucose uptake and glycogenesis within isolated mouse skeletal muscle. Phosphoprotein multiplex assay on lysates from muscle preparation was performed to detect phosphorylation of insulin-receptor on Tyr¹¹⁴⁶, Akt on Ser⁴⁷³ and glycogen-synthases-kinase-3 on Ser²¹/Ser⁹. To address the spatial resolution of insulin signalling, immunohistochemistry studies on cryosections were performed. Our results provide evidence to suggest that during the in vitro incubation, insulin sufficiently diffuses into the centre of tubular mouse muscles to promote phosphorylation of these signalling events. Interestingly, increased insulin signalling was observed in the core of the incubated muscle specimens, correlating with the location of oxidative fibres. In conclusion, insulin action was not restricted due to insufficient diffusion of the hormone during in vitro incubation in either extensor digitorum longus or soleus muscles from mouse under the specific experimental settings employed in this study. Hence, we suggest that the glycogen depleted core as earlier observed is not due to insufficient insulin action.

In vitro incubation of tissues; in particular, skeletal muscles from rodents, is a widely-used experimental method in diabetes research. This experimental method has previously been validated, both experimentally and theoretically. However, much of the method's experimental data remains unclear, including the high-rate of lactate production and the lack of an observable increase in glycogen content, within a given time. The predominant hypothesis explaining the high-rate of lactate production is that this phenomenon is dependent on a mechanism in glycolysis that works as a safety valve, producing lactate when glucose uptake is super-physiological. Another hypothesis is that existing anoxia forces more ATP to be produced from glycolysis, leading to an increased lactate concentration. The lack of an observable increase in glycogen content is assumed to be dependent on limitations in sensitivity of the measuring method used. We derived a mathematical model to investigate which of these hypotheses is most likely to be correct. Using our model, data analysis indicates that the in vitro incubated muscle specimens, most likely are sensing the presence of existing anoxia, rather than an overflow in glycolysis. The anoxic milieu causes the high lactate production. The model also predicts an increased glycogenolysis. After mathematical analyses, an estimation of the glycogen concentration could be made with a reduced model. In conclusion, central anoxia is likely to cause spatial differences in glycogen concentrations throughout the entire muscle. Thus, data regarding total glycogen levels in the incubated muscle do not accurately represent the entire organ. The presented model allows for an estimation of total glycogen, despite spatial differences present in the muscle specimen.

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) has been demonstrated to mediate both genomic and non-genomic responses in prostate cancer (CaP) cells. Here, we give an overview of membrane initiated 1,25(OH)₂D₃ signaling in prostate cancer cell progression. The presence of PDIA3 was investigated and homologous modeling of the putative PDIA3 receptor complex was conducted. Furthermore, the cellular distribution of nVDR was analyzed. We could show that both nVDR and PDIA3 are expressed in the prostate cancer cell lines investigated. The homologous modeling of PDIA3 showed that the receptor complex exists in a trimer formation, which suggests for allosteric activity. Our findings support previous reports and suggest that 1,25(OH)₂D₃ is an important therapeutic agent in inhibiting prostate cancer progression. Furthermore, our data show that 1,25(OH)₂D₃ regulate prostate cell biology via multiple pathways and targeting specific pathways for 1,25(OH)₂D₃ might provide more effective therapies compared to the vitamin D therapies currently clinically tested.

In vitro incubation of isolated rodent skeletal muscle is a widely used procedure in metabolic research. One concern with this method is the development of an anoxic state during the incubation period that can cause muscle glycogen depletion. Our aim was to investigate whether in vitro incubation conditions influence glycogen concentration in glycolytic extensor digitorum longus (EDL) and oxidative soleus mouse muscle. Quantitative immunohistochemistry was applied to assess glycogen content in incubated skeletal muscle. Glycogen concentration was depleted, independent of insulin-stimulation in the incubated skeletal muscle. The extent of glycogen depletion was correlated with the oxidative fibre distribution and with the induction of hypoxia-induced-factor-1-alpha. Insulin exposure partially prevented glycogen depletion in soleus, but not in EDL muscle, providing evidence that glucose diffusion is not a limiting step to maintain glycogen content. Our results provide evidence to suggest that the anoxic milieu and the intrinsic characteristics of the skeletal muscle fibre type play a major role in inducing glycogen depletion in during in vitro incubations.

In the present work a general systems biology approach has been used to study the complex regulatory network controlling the transcription of the spa gene, encoding protein A, a major surface protein and an important virulence factor of Staphylococcus aureus. A valid mathematical model could be formulated using parameter values, which were fitted to quantitative Northern blot data from various S. aureus regulatory mutants using a gradient search method. The model could correctly predict spa expression levels in 4 different regulatory mutants not included in the parameter value search, and in 2 other S. aureus strains, SH 1000 and UAMS-1. The mathematical model revealed that sarA and sarS seem to balance each other in a way that when the activating impact of sarS is small, e.g. in the wild-type, the repressive impact of sarA is small, while in an agr-deficient background, when the impact of sarS is maximal, the repressive impact of sarA is close to its maximum. Furthermore, the model revealed that Rot and SarS act synergistically to stimulate spa expression, something that was not obvious from experimental data. We believe that this mathematical model can be used to evaluate the significance of other putative interactions in the regulatory network governing spa transcription. (C) 2008 Elsevier GmbH. All rights reserved.