Förtjänstfull vetenskaplig avhandling 2008
Till förtjänstfull vetenskaplig avhanding 2008 har utsetts ”Study of Islet Cell Exocytosis and Coupling Using Intact Islet Patch-clamp”. Författare är Quan Zhang. Här följer en sammanfattning av avhandlingen.
Pancreatic islets of Langerhans are composed of three major cell types: insulin-secreting b-cells, glucagon-producing a-cells and somatostatin-releasing d-cells. These hormones work together to maintain the blood glucose in a narrow range between 4~6 mM. Hormone secretion from islet cells is directly controlled by the blood glucose level. The cells take up glucose and metabolise it to generate ATP, which in turn regulates ATP-sensitive K+ channels (or KATP-channels, which is also the target of sulfonylureas). These channels convert the metabolic signal (glucose) into an electrical signal, leading to an increase in the intracellular Ca2+ concentration.
The rise of intracellular Ca2+ then triggers the final step of releasing hormone-containing vesicles (=exocytosis) from these endocrine cells. The electrical signals can be measured in islet cells using the patch-clamp technique. Traditionally, this technique was applied to dispersed islet cell preparations. Recently, advanced by Dr. Gopel, Prof. Rosman and coworkers, patch-clamp, for the first time, was introduced to cells within intact pancreatic islets, i.e. within their natural environment. The application of this technique formed the basis of my PhD study.
In the first part of my study, I used patch-clamp in intact islets to perform capacitance measurements, a technique which measures secretion from single cells with ultrahigh sensitivity. In this way, we were able for the first time to monitor exocytosis in b-, a- and d-cells in intact islets. Interestingly, b-cells from intact islets exhibited a much slower rate of exocytosis than that was measured from single cells. This enabled a closer comparison between capacitance measurements and hormone secretion data which is also obtained from intact islets by radioimmunoassay.
A second important progress was that we demonstrated the possibility of studying d-cells, which rarely survive in single cell preparations. Capacitance measurements in intact islets for the first time characterised their exocytotic properties. We found that d-cells were capable of releasing granules at an extremely high rate. This high rate of exocytosis was mainly composed by the release of granules after removal of stimuli. This peculiar feature differed d-cells from the other cell types, of which exocytosis occurred only during the onset of stimulation. This finding led to the second part of my study which concentrated on the cell biology of d-cell exocytosis.
Somatostatin, which is secreted from d-cells in response to elevated glucose levels, is a powerful inhibitor of insulin and glucagon secretion from b- and a-cells, respectively. Using electrophysiological and optical imaging techniques, we looked in details the d-cell exocytosis and Ca2+ dynamic during the release of somatostatin. Capacitance measurements revealed that there is a post-stimulation increase in membrane capacitance (i.e. cell exocytosis) which we call ’slow component’. Simultaneously, monitoring intracellular Ca2+ fluctuation by Ca2+ measurement revealed that there are post stimulation Ca2+ oscillations which coincide in time with the ’slow component’. By introducing specific inhibitors into the cell, this phenomenon was shown to be Ca2+-induced Ca2+ release (CICR) from the endoplasmatic reticulum (ER) via ryanodine receptors (an intracellular Ca2+ sensitive Ca2+ channel). We also found that the membrane Ca2+ channel responsible for initiating this Ca2+ amplification pathway is the R-type Ca2+ channel (Cav 2.3).
As described earlier, KATP-channels are thought to be crucial for the stimulation of hormone secretion by glucose. However, we unexpectedly found that in transgenic mice lacking KATP-channels, glucose-stimulation of somatostatin release was almost unaffected. Instead, we obtained evidence that glucose increases somatostatin release by enhancing CICR in d-cells. This is caused by the higher level of ATP, which promotes the activity of Ca2+ ATPase (SERCA) in the ER and thus leads to an improved intracellular Ca2+ sequestration. We also found that glucose can potentiate the CICR machinery by increasing intracellular cAMP to activate an amplification pathway.
Pancreatic islet b-cells fire action potentials in response to glucose elevation. This electrical activity is highly synchronized among b-cells in intact islets, enabling the pulsatile insulin release into blood circulation. This can be attributed to cell-cell coupling via gap junctions. Application of patch-clamp technique in intact islets maintains the integrity of cell-cell contact, thus allowing us to look into the electrophysiological basis of cell coupling. We characterized the gap junction conductance between b-cells and concluded that there were 6~7 b-cells coupling to one peripheral b-cell. The data also enabled us to calculate the rate of Ca2+ wave propagation among b-cells, which was in close agreement with that observed in islets using confocal microscopy.
Application of patch-clamp intact islet cells made it possible to take a close look at the regulation of the hormone secretion from these endocrine cells. Particularly, we have revealed more information of the none-b-cell exocytosis as well as provided a a possible explanation of the loss of pulsatile insulin secretion pattern in type-2 diabetic patients.
Quan Zhang, phd