OBC | Beta testing


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Marjan Slak Rupnik, University of Maribor and CIPKEBIP, Slovenia
Beta testing


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  • Thanks the organizers for letting me present our suggestions on the home teritory.
  • The present conference strives to address key problems and obstacles which hold back our civilization and the whole planet on a way to a higher quality of life. Key obstacle to a higher quality of life are definitely diseases. Today I shall talk about one such disease, diabetes mellitus or sugar disease. We know its existance for thousands of years, to some extent we control it during last 90 years, but still it is expanding in epidemic way and is getting out of our hands. The question is, how well I my opinion not very well. Therefore, if we use the computing jargon, we are still at the level of BETA TESTING. This may be appropriate also because the cells of our body that are the most cells involved in the disease are called beta cells.
  • Very simply put, and there is not much more to it. Diabetes means that we are dealing with a patient that produces a lot of urine and to keep a fluid balance patient drinks a lot. Since the reason for a large volume of urine is glucose and therefore urine is sweet, this sweetness is indicated by mellitus.The man who first understood the role of hormone insulin was Banting at the University of Toronto.Banting: “The next step in solving diabetes is just around the corner.” Unfortunately we have not found that corner in almost 100 years of intensive scientific and other research activities.
  • Retroperitoneal organ. Let us learn more about the beta cells. They present the major cell type in endocrine pancreas. They reside in cell clusters we call islets of Langerhans.
  • The physiological stimulus to release insulin is glucose. After we eat something, the increased glucose would produce a large secretion of insulin, which in turn subsequently reduces the blood glucose level. But clearly the role of insulin cannot be just to reduce glucose after the meal. As every other endocrine systems there is a feedback mechanism to stop the stimulation and eventually end the release of insulin.
  • The role of insulin is clear when we remove it from the body. The body becomes catabolic, wastes lipids and proteins. As with starvation, when the patient dies because too much protein has been wasted (50% deadly).The role of plasma glucose level in our bodyis to control insulin levels to control metabolism, to keep us anabolic. Even glucose so high that we loose it with our urine is better than loosing proteins and fat (we should think outside the context of modern civilization).
  • The role of plasma glucose level in our bodyis to control insulin levels to control metabolism, to keep us anabolic and many hormonal systems will help keeping glucose high enough. But what is high enough? Depends how sensitive to insulin we are. Meaning also that no beta cells, no insulin, very high glucose. High glucose means that an organism is trying on its own to establish a dominance of anabolism.Adding insulin of course helps in this, but the side effects are that we are dependent on artificial insulin injections that to a large extent stop innate production of insulin and we shall eventually become insulin resistant with severe consequences.
  • The vast majority of the studies so far on single cells and insulinoma cell lines. These studies have positively contributed to understanding of some of the basic protein entities involved, but failed to predict emergent properties we measure in more intact preparations.To understand a disease it is critical to know the site of the lesion. Is it a beta cell at all or it is a perhaps due to neurodegeneration.It is fascinating comparing the size of the insulin granules with the synaptic vesicles.
  • We can measure different patterns of insulin release depending on the route through which the glucose enters the body, mouth and GI or injection into the vein. It differs in normal and diabetic person. And to finish the provocation about normality, it differs between normal and healthy. We can influence how sensitive we are too glucose to the extent we do need a very little insulin. This also has been know for decades, but largely ignored.
  • What does glucose do to a typical beta cell?In the consensus model, which can of course be wrong, the conversion of glucose to ATP closes the protein pore through which K+ escapes the cell, this changes the membrane potential and opens Ca2+ ions to enter the cell along its huge gradient. This relatively small Ca2+ change triggers exocytosis, the process by which insulin is kicked out of the cell into the bloodstream to keep stimulating anabolic metabolism.
  • In fact we can nowadays measure Ca2+ changes well, much better than last year. Cutting – staining – imaging of living fresh tissue slices protocol that we established in Maribor provides much better insights into the glucose dependent mechanisms in beta cells.To this end, we cut tissue slices 140 microns thin,stained them with the Oregon green 488 BAPTA -1 calcium dye, and finally, loaded cells were illuminated with 488 nm and the emitted light, filtered at 500-600 nm, was detected by a PM.
  • high spatial resolution of an islet where we can clearly see individual cells stained with OGB.
  • We can draw regions of interest very precisely on single cells and follow Ca changes within the volume of one cell with known optical thickness.
  • We can identify also non-beta cells, which are highly active also in low glucose, which is visible by eye under the microscope.
  • let us see the cells in action, during a short „meal“.
  • spontaneous activity in 6 mM glucose, which is increased at 12 mM glucose with a discernable first and second phase. Not all cells are perfectly synchronised.Constantly active non-beta cell.
  • It can take several minutes for all beta cells to be recruited into the first phase during high glucose stimulation.
  • To view the calcium oscilations at higher rates, we should zoom in a bit. Again we see individual beta cells.
  • We can label some beta cells with regions of interest.
  • and measure distances. 124 um.
  • 18 Hz, which is OK for 12 mM glucose. For 20 mM one needs to go up to 50 Hz full frame imaging.
  • So the beta cells that are 124 um appart are more or less synchronized. From time to time the calcium wave does not make it, but mostly yes. But there is a limited speed of the Ca waves...
  • ...which in this case is about 60 um per second. If one goes faster with imaging, spikes appear also on top of these waves.
  • Of course glucose stimulation of the cell excitbility is not the only way, why cytosolic calcium would rise. Ca can be released from ER and likely other sources.
  • Malignant hyperthermia individuals, with a ryanodine receptor defects, have double basal and stimulated insulin release.
  • We are exiting the period where we could use and abuse medical drugs. We have a bad and rather foolish experience with antibiotics, antidiabetic drugs, glucocorticoids. Why use drugs if it can be done physiologically. Only one week of significant reduction will normalize glucose levels in the blood. 8 weeks regime that would make these patients about 12 kilos would with a healthy lifestyle this can be kept. The cost of such diet is negligible and way more effective to medical treatment with drugs.
  • OBC | Beta testing

    1. 1. Beta testing Marjan Slak Rupnik University of MariborCentre of excellence for integrated approaches to chemistry and biology of proteins
    2. 2. Beta testing- addressing key obstacles reducing the quality of life- diseases – example: diabetes mellitus- described for many centuries, treated during the last, but despite that in epidemic expansion- missing critical puzzle pieces – beta testing
    3. 3. Innovative ways out...- understanding human physiology or systems biology outside the context of a modern civilization (e.g. medicine and industry) - an organism is not stupid and helpless- understanding pathophysiology- concepts and leading researchers can be wrong- stop avoiding the obvious
    4. 4. What is diabetes mellitus?- polyuria in polydipsia - not enough beta cells to- hyperglycemia produce insulin (T1DM; autoimmune, viral,...) - body simply too big for the maximal insulin production (T2DM; obesity, predisposition to low beta cell mass,...) - the solution to remove signs is too simple: use of insulin to lower glucose and stop osmotic diuresis
    5. 5. Endocrine pancreas 5
    6. 6. Glucose-dependent insulin release - the primary role of insulin is not to reduce blood glucose (industrial definition).
    7. 7. The role of insulin is anabolic,it is in fact a growth hormone insulin resistance
    8. 8. Keeping the plasma glucose levelhigh enough -not enough beta cells to produce insulin (T1DM) – insulin sensitive -body simply too big for the maximal insulin production (T2DM) – insulin insensitive
    9. 9. Why misconceptions? insulin • individual cells • coupled beta-cell clusters • neural control of coupled clusters acetylcholine 9
    10. 10. Pancreas innervation- peripheral neuropathy – a consequence or a cause
    11. 11. Different patterns of insulin release oral 10 mM glucose IV N D
    12. 12. Consensus model insulin secretion(beta version)
    13. 13. CSI Maribor 1 2 3
    14. 14. high spatial resolution of indicator loaded cells
    15. 15. clear identification of ROI of individual beta cell
    16. 16. clear identification of ROI of individual non-beta cell
    17. 17. 1 2
    18. 18. Alternative model of Ca2+regulation in a beta cell
    19. 19. I know I can die of stressMalignant hyperthermia – due to hyperthermia, but why is my lean muscle massporcine stress syndrome so big? When Pigs Stress Out By ARMELLE CASAU Published: October 07, 2003 Half a century of selective breeding has had an unappetizing side effect for the nations $40 billion-a-year pork industry. In what researchers say is a biochemical chain reaction sometimes caused by a stress syndrome inadvertently bred into many pigs, 10 to 15 percent of pork turns into sweating pale cuts of meat that ooze liquid in the packaging and become leathery when cooked. The pork industry estimates that the problem costs $90 million a year in lost revenue. Now the losses have led to research to seek new ways to improve breeding and handling. Food scientists say the problem dates from the 1950s, when breeders started selecting pigs with increased mass of lean muscle. Those pigs, it turned out, also inherited a naturally occurring condition, porcine stress syndrome. As farmers bred faster-growing hogs with ultralarge, ultralean muscles that could compete as the other white meat, they were inadvertently selecting for the syndrome, said Dr. Jodi Sterle, a swine specialist at Texas A&M University...
    20. 20. Bark C, Berggren P-O, Efendić S, Karolinska Institute Shen Y, Zhijiang University, Hangzhou Gaisano H, U Toronto Walther D, MPI Molecular Genetics BerlinEuropean Neuroscience Institute Göttingen (ENI-G) is jointly funded by Medical faculty of University ofGoettingen, Max-Planck Society and Schering AG.Institute of Physiology was a Partner Group to Max-Planck Institute for biophysical Chemistry, Göttingen.