1 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
2 Department of Medical Sciences, Uppsala University, Uppsala, Sweden
3 Novo Nordisk A/S, Måløv, Denmark
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ABSTRACT |
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The vascular system of the pancreatic islets is both anatomically and functionally autonomous from that of the exocrine parenchyma (1,2). Previous studies have demonstrated that islet blood perfusion is adapted to the high metabolic demands of the endocrine cells and that the islets are able to regulate their flow to the needs for hormone release (1). To achieve this minute regulation, the islet blood perfusion is influenced by both metabolic and nervous signals (1,3). Adenosine seems to be of major importance for metabolic blood flow responses in the islets (3) and, for example, in retina (4) and heart (5). In the latter organs, the effects of adenosine are mediated by KATP channels.
In addition to being present in vascular smooth muscle (6), KATP channels are of crucial importance for insulin release by maintaining a hyperpolarization of normal ß-cells. Thus, when the potassium channels are closed by increased cellular concentrations of ATP (e.g.,. in association with ß-cell glucose metabolism, an altered ion flux leads to depolarization). This opens voltage-gated Ca2+ channels, which increase cytosolic Ca2+ concentrations, thereby favoring exocytosis (7,8). Both KATP channel closers and openers are available, and the former, i.e., sulfonylureas, have widespread use as a treatment for type 2 diabetes.
Recently, KATP channel openers, like diazoxide, have also become of interest in view of the concept of ß-cell rest as an option in the initial treatment of type 1 diabetes. An argument for this approach is that inhibition of insulin secretion lowers the expression of ß-cell autoantigens (9), which may diminish the immune-mediated assault characteristic of type 1 diabetes. However, most of the early KATP channel openers were associated with marked hypotension, which made their continuous and frequent clinical use difficult. Recently, more ß-cellspecific drugs with only minor effects on blood pressure have been developed (10,11).
Given the combined vasoactive and insulin-releasing properties associated with activation or inhibition of KATP channels, we deemed it of interest to investigate their effects on pancreatic islet blood perfusion. To achieve this, we used diazoxide, which opens both ß-cell and vascular smooth muscle KATP channels as well as the more ß-cellspecific KATP channel opener NNC 55-0118 (10,11). Furthermore, we also examined the effects of a KATP channel closer, glipizide. Our findings suggest that opening of KATP channels in ß-cells are associated with a marked increase in total pancreatic and islet blood flow, with a preference for the exocrine parenchyma.
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RESEARCH DESIGN AND METHODS |
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Blood flow measurements after diazoxide administration.
The rats were anesthetized with an intraperitoneal injection of thiobutabarbital sodium (130 mg/kg body wt, Inactin; Research Biochemicals International, Natick, MA) and placed on a heated operating table to maintain body temperature. Polyethylene catheters were inserted into the ascending aorta, via the right carotid artery, and into the left femoral artery and vein. The former catheter was connected to a pressure transducer (PDCR 75/1; Druck Ltd., Groby, U.K.), whereas the latter was used to infuse Ringer solution (5 ml · kg body wt-1 · h-1) to substitute for fluid losses. When the blood pressure had remained stable for at least 15 min an intravenous injection of diazoxide (Hypertstat; Schering-Plough AB, Stockholm, Sweden; 10, 20, or 30 mg/kg body wt) or the corresponding volume (2 ml/kg body wt) of saline was given. The blood flow measurements were then performed 20 min later.
The arterial blood perfusion of the whole pancreas, islets, duodenum, colon, adrenal glands, and kidneys was then measured with a microsphere technique (12). Briefly, a total of 1.52.0 x 105 nonradioactive microspheres (EZ-Trac; Triton Microspheres, San Diego, CA), with a diameter of 10 µm, was injected via the catheter with its tip in the ascending aorta during 10 s. Starting 5 s before the microsphere injection, and continuing for a total of 60 s, an arterial blood sample was collected by free flow from the catheter in the femoral artery at a rate of 0.40 ml/min. The exact withdrawal rate was confirmed in each experiment by weighing the sample. Arterial blood was collected from the carotid catheter for determination of blood glucose and serum insulin concentrations as given below. The animals were then killed, and the pancreas and adrenal glands were removed in toto, blotted, weighed, and treated with a freeze-thawing technique, which visualized the pancreatic islets and microspheres (12). Approximately 100 mg each of the duodenum (around the papilla), colon (descending part), and left kidney (slice through the center) were also removed and treated in the same way. The number of microspheres in these samples were then counted in a microscope equipped with both bright and dark field illumination. The blood flow values were calculated according to the formula Qorg = Qref x Norg/Nref, where Qorg is organ blood flow (ml/min), Qref is withdrawal rate of the reference sample, Norg is number of microspheres present in the organ, and Nref is number of microspheres in the reference sample. Blood flow values based on the microsphere content of the adrenal glands were used to confirm that the microspheres were adequately mixed in the circulation. A difference <10% in the blood flow values was taken to indicate sufficient mixing.
The number of microspheres in the arterial reference sample was determined by sonicating the blood, then transferring samples to glass microfiber filters (pore size <0.2 µm), and then counting the number of microspheres in the same microscope as referred to above.
Blood flow measurements after administration of NNC 55-0118.
The surgical preparation was performed as given above. NNC 55-0118 (6-chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide; Novo Nordisk A/S, Måløv, Denmark) was administered intravenously (3 mg/kg body wt) 20 min before the blood flow measurements. Control animals were given the same volume (2 ml/kg body wt) of vehicle alone. The blood flow measurements were performed in the same organs and with the microsphere technique referred to above.
Blood flow measurements after administration of glipizide.
The surgical preparation was performed as given above. Glipizide (Sigma, St. Louis, MO) was dissolved in a 50% solution of DMSO and then administered intravenously (25 µg/kg body wt) 45 min before the blood flow measurements. This time point was chosen since it was the earliest providing a significant decrease in blood glucose concentrations. Control animals were given the same volume (0.5 ml/kg body wt) of vehicle alone. The blood flow measurements were performed with the microsphere technique referred to above. However, only the blood perfusion to the whole pancreas, islets, and the adrenal glands was determined in these animals.
Measurements of blood glucose and serum insulin concentrations.
Blood glucose concentrations were measured with test reagent strips (Medisense; Svenska MediSense, Solna, Sweden). Serum immunoreactive insulin concentrations were measured with enzyme-linked immunosorbent assay (ELISA) (Rat Insulin ELISA; Mercodia AB, Uppsala, Sweden).
Statistical calculations.
All values are given as means ± SE. Probabilities (P) of chance differences were calculated with Students unpaired t test or ANOVA with Bonferronis correction (SigmaStat; SSSPD, Erfart, Germany) A value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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DISCUSSION |
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The potassium channel also consists of a regulatory subunit, the sulfonylurea receptor (SUR). This part of the channel complex exists in three forms: SUR1 found in ß-cells and some neurons, SUR2A in cardiac and skeletal muscle, and SUR2B in VSMCs (7). In confirmation of this, recent experiments on SUR2 knockout mice demonstrated findings similar to those seen in Kir6.1 knockouts (i.e., a Prinzmetal phenotype and loss of KATP channels in VSMCs) (15).
The mechanism behind the blood flow increase presently seen after administration of diazoxide is likely to be an opening of VSMC KATP channels in pancreatic resistance vessels, i.e., presumably mainly arterioles. This would lead to a hyperpolarization of the muscle cell causing relaxation and vasodilatation (6,16,17). However, diazoxide is not selective for VSMC or ß-cell KATP channels, but affects both. It should be kept in mind that there are no pharmacological or physiological studies that have determined the type of KATP channels present in pancreatic vasculature.
It has been suggested that diazoxide is selective for mitochondrial KATP channels in heart muscle (1820). An effect on these channels has been suggested to mediate the protection conferred by this potassium channel opener against toxins in the heart (21,22) and the central nervous system (2325). In ß-cells, on the other hand, diazoxide has been suggested to act on KATP channels in both plasma membrane (26) and mitochondria (27). Indeed, the selectivity of diazoxide for mitochondrial KATP channels, irrespective of tissue, is likely to be dose dependent (28). The doses used in the present study are likely to affect channels on both of these cellular locations.
When interpreting the effects on blood flow by diazoxide, it should be noted that there was a marked decrease in mean arterial blood pressure at all doses given. Because organ blood flows other than those to the pancreas remained unchanged, the vascular conductivity was markedly increased in these organs. Such an increase was even more pronounced in the pancreas and presumably also in the islets, leading to an absolute increase in these organ blood flow values. The value for the islets is not included in the table, because we do not have any measurement of the weight of the islet organ, which precludes an exact calculation of vascular conductance. However, it can be estimated, assuming an islet volume of 12%, that it is increased to the same degree as that to the whole pancreas. Thus, it seems as if opening of the KATP channels induces most pronounced blood flow effects on islets and whole pancreas, despite the low blood pressure, which, nevertheless, is within the limits of autoregulation for the rat pancreas (29) and islets (30). Diazoxide had its most pronounced effects on total pancreatic blood perfusion, as manifested by the decreased fractional islet blood flow (i.e., a lowering of the fraction of blood diverted through the islets). This would suggest that arterioles in the exocrine parenchyma have more KATP channels or that the VSMCs in these arterioles are more sensitive to diazoxide. We would like to once again stress that there are no available anatomical or pharmacological studies on intrapancreatic KATP channel distribution.
In contrast to diazoxide, the newly described potassium channel opener NNC 55-0118 stimulated total pancreatic and islet blood flow to the same degree. No effects on the other measured regional blood flow values were seen. This is somewhat surprising, because NNC 55-0118 is selective for the Kir6.2/SUR1 KATP channel of the ß-cell (11). Indeed, the effects on VMSCs are small and previous studies have demonstrated blood pressure effects similar to those in the present study (11). This opens the possibility that the effects of the KATP channel openers on islet blood perfusion are not primarily dependent on activation of VMSCs potassium channels (i.e., those primarily containing Kir6.1/Sur2B potassium channels). It can instead be that activation of the ß-cell Kir6.2/SUR1 channel and/or that substances released from the islet cells themselves affect the islet arteriolar blood vessels and lead to the marked increase in blood flow. This notion would then explain why diazoxide increases the vascular conductance more in the pancreas and islets, since diazoxide would also affect the common Kir6.1-containing VSMC potassium channels, leading to the general increase in vascular conductance seen in all studied organs, including the whole pancreas. Furthermore, diazoxide would also affect the ß-cell KATP channel, which could cause a further increase in islet blood flow (see below) and, perhaps, total pancreatic blood flow. Several previous studies have demonstrated the independent regulation of islet blood flow when compared with the exocrine pancreas (1,2).
In favor of such an interpretation is our finding after administration of glipizide, i.e., a closer of KATP channels, with a selectivity for the ß-cell Kir6.2/SUR1 form. Glipizide leads to a decrease in islet blood flow but had no effects on total pancreatic blood flow. In line with the anatomical organization of islet afferent blood supply (see above), it can also be that a substance affecting the islets may release substances that can diffuse to the islet arterioles and directly affect VSMCs. Immunolocalization of KATP channels in rat islets have demonstrated that SUR1 and Kir6.2 are found not only in ß-cells, but also in -,
-, and pancreatic polypeptide cells (31). This implies that any of these endocrine cells can be a source of a substance(s) that may affect the islet afferent arteriole. At present, the nature of such a mediator is unknown. Glucagon is worthy of mentioning in this context because it is a vasoactive hormone (32). Furthermore, the administered glipizide caused hypoglycemia, which is likely to have increased glucagon release. Preliminary findings with insulin-induced hypoglycemia also demonstrate a decreased pancreatic islet blood flow, and this response seems to be nervously mediated (P.-O. Carlsson, L.J., unpublished observations). Thus, to what extent the decreased islet blood flow seen after glipizide administration is due to an effect on KATP channels or is nervously mediated is at present unknown.
A piece of indirect evidence pointing toward an effect of KATP channels in the regulation of normal islet blood flow is the association between these potassium channels and adenosine. We have recently shown that adenosine, probably produced by ß-cells, can stimulate islet blood flow during conditions associated with increased islet metabolism (3). Moreover, a coupling between KATP channels and adenosine effects on pancreatic vasculature has been suggested in experiments on the perfused rat pancreas (33). In this context, it has been reported that heart microvascular dilation in response to adenosine is enhanced at lower intraluminal pressures by selective activation of VSMC KATP channels (5). This should be compared with islets, where the capillary blood pressure is lower than in the exocrine parenchyma, but where the blood flow response to adenosine is more pronounced (3).
To what extent the increased blood glucose concentrations may affect the islet blood perfusion in the present study is of interest. It has previously been demonstrated that hyperglycemia may increase the blood perfusion of the islets (34), presumably mediated by nitric oxide (35). Diazoxide increased the values to 9 mmol/l, whereas NNC 55-0118 had no such effects. In view of the similarity on blood flow response but differences in blood glucose concentrations, it seems unlikely that the latter is responsible for the changes in islet blood flow. Furthermore, in a previous study, we observed that the blood glucose concentrations needed for this response to occur in anesthetized animals were higher than those seen in the present study (34). In general, however, the blood flow changes seen in the present study include effects mediated by KATP channels as well as more unspecific changes induced by altered blood pressure, blood glucose (see above), and serum insulin concentrations.
In an earlier study, the sulfonylurea tolbutamide was found to increase blood flow to the islets in rats in doses inducing hypoglycemia, but had no effects on total pancreatic blood flow (36). The reasons for the discrepancy between their findings and those in the present study are unknown. In the tolbutamide study, young rats, aged 5 weeks, were used, whereas we studied adult rats 15 weeks old. We have previously noted marked differences in the absolute values in islet blood flow depending on the age of the animals, and it is also likely that the blood flow regulation differs (37,38). It is also possible that there are differences in the actions of tolbutamide and glipizide, although this seems less likely. However, in view of previous findings when applying sulfonylureas, we consider a decrease in islet blood flow to be a logical consequence of administering these substances. Thus, depending on the dose, glibenclamide is known to reduce early and peak vasodilatation following reactive hyperemia in humans (39). Furthermore, glibenclamide (20 mg/kg) to rats increased vascular resistance and caused a systemic vasoconstriction (40). Therefore, it seems as if KATP channel closers would favor a vasoconstriction rather than a dilation. It may seem physiologically appropriate that insulin release induced by sulfonylureas would be associated with an increased blood perfusion, as suggested by Vetterlein et al. (36). However, in other contexts, we have been able to show that the very high basal blood perfusion of the islets allows for a substantial reduction in islet blood flow before any effects on insulin secretion can be observed (1).
In summary, our results suggest that KATP channels play an important role for regulation of total pancreatic and islet blood flow. To what extent effects on islet function are mediated by a modulated islet blood perfusion by substances affecting these channels merits further investigation.
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ACKNOWLEDGMENTS |
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The authors are grateful to Astrid Nordin for excellent technical assistance.
Address correspondence and reprint requests to Dr. Leif Jansson, Department of Medical Cell Biology, Biomedical Centre, Box 571, SE-751 23 Uppsala, Sweden. E-mail: leif.jansson{at}medcellbiol.uu.se
Received for publication February 13, 2003 and accepted in revised form May 12, 2003
ELISA, enzyme-linked immunosorbent assay; SUR, sulfonylurea receptor; VSMC, vascular smooth muscle cell
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REFERENCES |
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