EFFECTS OF DIAZOXIDE ON EXPRESSION IN RAT PANCREATIC ISLETS ARE LARGELY LINKED TO ELEVATED

GLUCOSE AND POTENTIALLY SERVE TO ENHANCE BETA CELL SENSITIVITY

RECEIVED FOR PUBLICATION 12 MARCH 2006 AND ACCEPTED IN REVISED FORM 5 JANUARY 2007.

Diabetes InPress,publishedonlineJanuary17,2007 Copyright American Diabetes Association, Inc., 2007 Zuheng Ma, Neil Portwood, David Brodin, Valdemar Grill and Anneli Björklund

From the Endocrine and Diabetes Unit, Department of Molecular Medicine and Surgery (Z.M., N.P., A.B., V.G.), Karolinska Hospital,

Karolinska Institutet, S-171 76 Stockholm, Sweden; Dept. of Biosciences (D.B), NOVUM, 14157 Huddinge, Sweden; Endocrine Unit, Dept. of

Abdominal Diseases (V.G.), St. Olav´s University Hospital, Norwegian University of Science and Technology; N-7006 Trondheim, Norway

Corresponding author: Anneli Björklund MD, PhD, Endocrine lab L6B: 01, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel: +46-8-5177

9458, Fax: +46-8-5177 3658

E-mail:[email protected]

Short running title: diazoxide and rat islet microarray

Abstract: co-culture with 11 or 27 mmol/l glucose. synthesis and down-regulated uncoupling Diazoxide enhances glucose-induced At 11 mM diazoxide up-regulated 97 2 and lactic acid dehydrogenase, secretion from beta cells through and down-regulated 21 genes. Diazoxide up-regulated certain genes mechanisms which are not fully Increasing the glucose concentration to 27 known to support β-cell functionality, such elucidated. Here we used microarray mmol/l markedly shifted these proportions as nkx6.1 and pdx 1. - Long term elevated analysis (Affymetrix) to investigate effects toward down-regulation (101 genes up- glucose is permissive for most of of diazoxide. Pancreatic islets were regulated, 160 genes down- regulated). At diazoxide’s effects on , the cultured overnight at 27 or 11 or 5.5 27 mmol/l glucose most genes down- proportion of effects shifting to down- mmol/l glucose ± diazoxide. Inclusion of regulated by diazoxide were oppositely regulation with increasing glucose diazoxide up-regulated altogether 211 affected by glucose (80%). Diazoxide concentration. Effects of diazoxide on genes (signal log2 ratio ≥0.5) and down- influenced expression of several genes gene expression could serve to enhance β- regulated 200 genes (signal log2 ratio≤ - central to beta cell metabolism. Diazoxide cell functionality during continuous 0.5). 92 % of diazoxide’s effects (up- and down-regulated genes of fatty acid hyperglycemia. downregulation) were observed only after oxidation, up-regulated genes of fatty acid

Introduction: Isolation, culture and incubation of rat mmol/l G ± diazoxide conditions, three for pancreatic islets the 11 and four for the 5.5 mmol/l G ± In previous studies we have documented that Male Sprague-Dawley rats were purchased diazoxide condition. Because it was difficult pre-treatment with diazoxide exerts from Scanbur BK AB (Sollentuna, Sweden). to obtain enough material for microarray beneficial effects on glucose-induced insulin The ethical guidelines of the Karolinska analysis from a single experiment we pooled secretion (1) and in fact may protect β- cells Institute for the care and use of laboratory islets from three experiments (each with against well-known adverse effects of animals were followed. The rats were pooled islets from 2-4 rats) to obtain material chronic hyperglycemia. These beneficial maintained in a 12-hour (06:00 – 18:00) for each analysis of the 27 mmol/l G ± effects are only partly related to the light-dark cycle with free access to water and diazoxide condition. In this way we obtained preservation of insulin stores (1). Indeed, our standard diet. They weighed 250 – 350 g at material for three replicate analyses. For the previous studies have demonstrated several the time they were used for experiments. 5.5 mmol/l G ± diazoxide condition islets other effects of potential importance for Islets of Langerhans were isolated by were pooled from two experiments (each efficient signal transduction in β-cells (1); collagenase (Roche Diagnostics) digestion in with pooled islets from 4 rats) to obtain however an overview is still lacking. Global HBSS basically as described previously (2) material. For the 11 mmol/l G ± diazoxide gene expression analysis offers an followed by sedimentation. Islets were then condition islets from four rats were pooled appropriate way to obtain such an overview selected under a stereomicroscope and for each of the three experiments. In this way and this technique has been employed here. transferred to Petri dishes (Sterilin, we obtained material for two replicate Teddington, UK) containing RPMI-1640, 2 analyses. The pooled material for each Upon finding a marked glucose-dependency mmol/l glutamine, 10% (v/v) fetal calf analysis contained 11-22 µg total RNA. The for the diazoxide effects on gene expression serum, 100 units/ml benzylpenicillin, 0.1 purity and non-degraded state of the RNA we focused further analyses on the mg/ml streptomycin, and 5.5 , 11 or 27 was assured using the Bioanalyser from interactions of glucose and diazoxide on mmol/l glucose, each with or without the co- Agilent. These total RNA samples were genes responsible for the metabolism of presence of 325 µM diazoxide. Islets were employed to synthesize labelled cRNAs, and glucose and other nutrients. then cultured free-floating for 24h at 37ºC, in were hybridised to the Rat Expression Array an atmosphere of 5% CO2 in air. Islets 230A (Affymetrix, Santa Clara, CA) at the Material and Methods visibly free from non-islet tissue were then Novum Affymetrix core facility, Karolinska transferred to dishes containing 5 ml Krebs- Institutet. Significance testing indicated that Materials Ringer bicarbonate (KRB) medium 10 the likelihood of false positives was less than mmol/l HEPES, 0.2% BSA and 3.3 mmol/l 10 percent. Diazoxide (Hyperstat®) was from Schering- glucose, and preincubated for 30 min at Plough (Labo N.V., Heist-op-den-Berg, 37ºC. Western blotting Belgium). Hank’s balanced salt solution Equal number of islets for each experimental (HBSS) and RPMI 1640 were purchased Microarray analysis condition were collected and washed twice from SVA (Uppsala, Sweden). Islets were collected for RNA extraction with ice-cold PBS. Extracts corresponding to after the post-culture preincubation described 50 islets (15 µg protein as confirmed by above and total RNA was extracted using an Bradford protein assay) were denatured in 60 RNeasy Micro (Qiagen). Nine separate µl loading buffer at 80°C for 10 min. experiments were performed for the 27 Samples were analyzed on 10 % sodium dodecyl sulfate polyacrylamide gels (SDS– Affymetrix GeneChip Operating Software (www.affymetrix.com, as of 09/16/2005). PAGE) run for 1 h at 150 V and were then (GCOS) version 1.4 was used for absolute For the genes thereby annotated to the transferred to nitrocellulose for 1h at 250 and comparison analysis. Scaling was set to category of metabolism we further explored mA. Membrane was blocked for 2 h at room All probe set with target signal 100, and a) whether these genes were annotated or not temperature with 5% (w/v) fat-free milk, normalisation to User defined with in other categories and b) whether 0.1% Tween 20 in Tris-buffered saline, pH Normalization value 1. For estimation of information was available in the Beta Cell 7.6 (TBS) and then incubated for overnight regulated genes, pair wise comparisons of Gene Expression Bank at 4 C with primary antibodies at the test vs. control were performed, resulting in (http://test.t1dbase.org), Such additional following dilutions: Aldolase B (from Santa a quantitative signal log ratio (SLR) and a information was incorporated into Cruz Biotechnology, Inc, USA), 1:500; qualitative change call. SLR is the tables/and/or text. Pdx1, (kindly provided by Dr Helena logarithmic (base=2) ratio of intensities from Edlund, Umeå University, Sweden) 1:2000. test and control sample. Change call is based Results Following the addition of anti IgG (anti-goat on change p-value, where GCOS settings or rabbit IgG) second antibody at a dilution used were p < 0.002 for increase call, and p Previous culture with diazoxide enhances of 1:5000, membranes were incubated for 1h > 0.998 for decrease call. glucose-induced insulin secretion. at room temperature. Immunoreactive bands For transcripts to be considered increased by Islets were cultured for 24h in low (5.5), were visualized using a chemiluminescence diazoxide or glucose, an increase call and moderately elevated (11) or high (27mmol/l) kit (ECL, Amersham Biosciences), exposed SLR ≥= 0.5 were required, and the glucose in the presence or absence of to X-ray film (Hyperfilm, Amersham corresponding requirement for decrease were diazoxide. Insulin secretion was measured Biosciences), and documented with a flat- decrease call and SLR ≤= -0.5. Further, for a post-culture and in the absence of diazoxide; bed scanner (Hewlett–Packard Scanjet 5300) diazoxide effect to be present significant also islet insulin contents and insulin in and quantitation software (Kodak, 1D). effects were required in all three replicate culture media were measured .(Fig 1). As comparisons (i.e. 9/9) of 27 and 11 mmol/l expected diazoxide blocked almost Measurements of insulin secretion glucose ± diazoxide and in both replicate completely insulin release into the culture Following culture and preincubation as analysis (i.e. 4/4) for 5.5 mmol/l glucose ± media (Fig 1A). Culture at 11 and 27 mmol/l described above, islets were incubated in diazoxide. In cases where less stringent of glucose significantly reduced the insulin groups of three for 60 min at 37 C in 300 µl criteria were used this is explicitly stated in response to post-culture stimulation with KRB containing 3.3 or 16.7 mmol/l glucose, the tables and/or text. For registering a 16.7 mmol/l of glucose (Fig 1B). Culture each with or without specific additives. The glucose effect 4 to 6 out of the 6 with diazoxide upheld islet insulin contents, insulin accumulated was measured as comparisons were required to show a which, in the absence of diazoxide were previously described (3). Islet insulin glucose effect per se > 0.5. Table 1 decreased by 48 % after culture at 11 and by contents were measured in batch-incubated summarizes the comparisons performed. 74% by culture at 27 mmol/l of glucose. (Fig islets following sonication for 10–15s, 1C). When insulin responses to 27 mmol/l of followed by extraction of insulin overnight at For basic functional classification, records glucose were calculated as per cent of insulin 4°C in 200 µl acid-ethanol (70%, v/v). from project contents, there was no decrease due to (www.geneontology.org, as of 09/01/2005) elevated glucose during culture. However, Statistical analysis and presentation of were used together with annotation exposure to diazoxide during culture with 27 results information from Affymetrix mmol/l of glucose markedly increased the percentage of insulin released during post- Increasing the glucose concentration during A majority of genes down-regulated by culture stimulation with glucose (Fig 1D). culture markedly increased the number of diazoxide were affected in an opposite genes, which were down-regulated by fashion by 27 mmol/l of glucose (Table 2, Overall and glucose specific effects on gene diazoxide. At 5.5mmol/l of glucose 13 genes upper part) or by 11mmol/l of glucose (Table expression were up-regulated by glucose and 19 down- 2, middle part). Of the genes down-regulated Out of 15923 genes present on the array the regulated. At 11 mmol/l of glucose the by diazoxide at 5.5 mmol/l of glucose only 2 expression of 8179 genes was detected number of up-regulated genes was 97 and were down-regulated in parallel with effects following culture at 27 mmol/l glucose, 7539 down-.regulated 21. At 27 mmol/l of glucose of 27 mmol/l glucose per se (Table 2, lower genes following culture at 11 mmol/l and 101 genes were up-regulated and 160 genes part). 8233 genes following culture at 5.5 mmol/l down-regulated. Thus, not only the number glucose. The corresponding figures obtained but also the percentage of down-regulated Diazoxide´s down-regulating effects are following inclusion of diazoxide during genes of the total number of genes affected more pronounced than up-regulating ones culture at 27, 11 or 5.5 mmol/l glucose were was markedly changed, (Fig 2B and C). As shown in table 3 diazoxide-induced 8360, 7773 and 8537 genes, respectively. alterations in gene expression were mostly Culture with 27 mmol/l glucose (27 vs. 5.5 Only a minority of up-regulated genes moderate with only a few genes showing mmol/l glucose) up-regulated 303 (SLR reinforce effects by glucose more than a 3-fold (i.e. SLR= 1.59) up- ≥0.5) in 6/6 comparisons and down- Of 101 genes up-regulated by diazoxide regulation in expression level. At 27 and 11 regulated 320 genes (SLR ≤-0.5). As to (SLR ≥0.5) only in the presence of 27 mmol/l a larger amount of genes were down- expression of insulin genes (Ins 1 and 2) mmol/l glucose only 8 % were also up- regulated more than 3-times by diazoxide both were up-regulated (SLR +0.9) in 4 out regulated by glucose (Table 2, upper part). (Table 3). of 6 comparisons at 11 vs 5.5. mmol/l of Thirty one (31%) of the genes up-regulated glucose whereas they were downregulateted by diazoxide were down-regulated by 27 Effects of diazoxide on gene expression in 27 vs.11 in 6 of 9 comparisons(SLR -1.3 mmol/l of glucose. Sixty genes (61%) of comprise most ontologies of annotation for Ins 1, -1.2 for Ins2). The twenty known genes up regulated by diazoxide were not The annotated genes affected by diazoxide genes, which were most highly influenced by affected by 27 mmol/l of glucose.(Table 2, were classified according to the GeneSpring 27 mmol/l of glucose per se are shown in upper part). Similar findings were obtained soft-ware program (Table 4). This electronic appendix 1. for diazoxide effects at 11 mmol/l of classification shows similar percentages of glucose. Thus, only 3 % of genes affected by genes affected by diazoxide in several of the The effects of diazoxide are markedly diazoxide were regulated in parallel by categories. There were no obvious glucose-dependent 11mmol/l of glucose (Table 2, middle differences in the proportion of genes (i.e. Few genes were affected by diazoxide after graph). None of the few genes affected by the sum of up-and down regulated genes) co-culture at 5.5.mmol/l of glucose. 92% of diazoxide after co-culture at 5.5 mmol/l of affected by 11 and 27 mmol/l of glucose. diazoxid´s effects (up- and downregulated) glucose was up-regulated in parallel with Interestingly, the highest percentage of genes were observed only after co-culture with 11 effects of 27mmol/l of glucose per se (Table affected by diazoxide (in % of the number of and 27 mmol/l glucose (Fig 2A). 2 lower part). genes represented on the array) was in the subcategory of monosaccharide metabolism Down-regulation by diazoxide increases with Down-regulation by diazoxide opposes (14.4 %). increasing concentration of glucose effects by glucose Effects of diazoxide on genes down- by diazoxide (in the presence of 27 mmol/l inhibit fatty acid β-oxidation and promote regulated or not affected by 11 but up- glucose). This up-regulation by diazoxide fatty acid synthesis. Regarding the regulated by 27 mmol/l of glucose. was only seen with 27 mmol/l, not with 11 diazoxide-induced changes in genes related Because, the enhancing effect of diazoxide mmol/l of glucose (Table 6). Apart from to amino acid metabolism, diazoxide on post-culture glucose-induced insulin aldolase B only the gene coding for malic inhibited expression of the glutamate secretion was much more marked after co- enzyme was up- regulated and by less decarboxylase gene, a gene of possible culture at 27 than at 11 mmol/l of glucose stringent criteria (SLR= +0.6 in 8 of 9 importance in the etiology of type 1 diabetes (Fig 1) it seemed of special interest to select comparisons, results not shown in table). (4). This effect was observed at all glucose for a diazoxide effect on genes affected only Down- regulating by diazoxide of genes concentrations. A marked effect on at 27 and not at 11 mmol/L of glucose or affecting glycolysis included enzymes of the argininosuccinate synthetase in the presence genes that were affected in an opposite pentose phosphate shunt and production of of 27 and 11 mmol/l glucose was also noted. fashion by these two glucose concentrations. lactate. By stringent criteria a limited number of Pdx-1 (additionally annotated as genes, most of the down-regulated by The increase by diazoxide in expression of transcription factor) was moderately up- diazoxide, could be identified (Table 5). the aldolase B gene was extraordinary, and regulated by diazoxide at all glucose Genes potentially influenceing growth, such has to our knowledge not been previously concentrations. Down regulation of pdx-1 as .IGF binding protein and cyclin D reported. This prompted verification of the has been coupled to desensitization of β-cell belonged to these down- regulated genes. A effects of diazoxide on aldolase B expression function, in particular glucose-induced large percentage (5 out of 25 genes) at the protein level. Using Western blotting, insulin secretion (5). It was therefore of belonged to the category of metabolism, we found that culture with 27 mmol/l special interest to verify gene effects on the which is further discussed below. glucose increased aldolase B protein levels protein level. Western blotting (Fig 3b) by 2.2-fold, and that addition of diazoxide confirmed the effects of diazoxide on the Diazoxide affects genes which are important increased levels of this protein by another protein level. Also Nkx6.1 (Table 6), a gene for β-cell metabolism 4.3-fold Fig. 3a. Also there was no diazoxide of importance for β-cell growth and function The total number of genes affected by effect visible when co-culture was performed (5, 6) was up-regulated by diazoxide at 27 diazoxide at 27 mmol/l in the category of together with 11 mmol/l of glucose (mean of mmol/l glucose. metabolism are shown as electronic 2 experiments, results not shown). Thus, appendix 2 and 3. Out of these genes we diazoxide appears to regulate aldolase B Other genes affecting mitochondrial have focused firstly on genes for enzymes protein levels in parallel with its effects on functioning and hence ATP production with specific effects on hexose, fatty acid or aldolase B mRNA levels. included fumarate hydratase 1 and amino acid metabolism (Table 6) Secondly, uncoupling protein 2 (UCP-2). In the case of we focused on genes with generalized effects Regarding diazoxide-induced changes in UCP-2 this gene was down-regulated by on ATP production and processing. genes related to fatty acid and amino acid diazoxide during co-exposure both with 5.5 metabolism, most of these did not parallel and 27 mmol/l glucose . The most marked up-regulation by diazoxide effects induced by glucose per se (Table 6). in the entire array was seen for aldolase B, The expression changes in genes involved in Discussion which was up-regulated 5.3-fold (SLR=2.4) fatty acid metabolism followed a clear by 27 mmol/l glucose but 42-fold (SLR=5.4) pattern, in so far as they would serve to The most striking finding of this study was to investigate mechanisms behind this large effects of elevated glucose in comparison the intimate relationship between glucose and apparently beneficial effect. with KCl, which stimulates secretion and diazoxide effects on islet expression. bypassing K+-ATP dependent channels. Thus >90 % of the effects of diazoxide that We have previously attributed the beneficial Similarity was found for these early effects were detected by microarray was seen only effects of diazoxide on insulin secretion to on mRNA for glucose and KCl; furthermore after co-culture with an elevated beta cell rest, i.e. alleviation of negative genes up-regulated by glucose were inhibited concentration (11 and 27 mmol/l) of glucose. effects of profound and prolonged by diazoxide in separate experiments with To our knowledge, such a strong coupling stimulation. The possibility of over- PCR. These findings provide proof of with glucose has not been described for the stimulation was suggested by the partial concept that stimulated insulin secretion per effects of other agents on gene expression. depletion of insulin stores (7 and present se can lead to gene induction, at least in a results), reflecting a disparity between short term perspective, and that blocking The glucose dependency of diazoxide´s demand and capacity. Severe over- secretion with diazoxide can nullify such effects on mRNA levels parallels to some stimulation could increase ER stress as induction. The genes deduced to be affected extent the effects of diazoxide on insulin shown in other experimental systems (8). A by the secretory process in the study of secretion previously described (1) and reduction of protein biosynthesis is an early Ohsugi et al were not presently affected confirmed and extended here. Thus, sign of ER stress (8). Our data do suggest (results not shown), with the exception of diazoxide augments post-culture glucose- some reduction of preproinsulin mRNA EGR1 and Sfrs5, which were up regulated by induced insulin secretion only when the when the glucose concentration was diazoxide at 27 mmol/l glucose (SLR 0.8 and culture is performed in the presence of an increased from 11 to 27 mmol/l. However, 0.6 respectively in 9/9 comparisons). elevated concentration of glucose. we did not find up-regulation of the stress It remains possible that the coupling of Furthermore, the enhancing effect of sensitive genes CHOP, P58, GRP94, diazoxide´s effects to a permissive effect of diazoxide on glucose-induced insulin EDEM1, ATF4, RAMP4 or caspase 12 by elevated glucose that we observe could, in secretion was much stronger at 27 than at 11 27 mM glucose, nor any effects on these part, be secondary to one or several aspects mmol/l and this corresponds to an increase in genes by diazoxide (results not shown). Two of the insulin secretory process. For diazoxide-regulated genes, in particular exceptions were FKbp11 and P58, which example, since insulin secretion requires down-regulation. have been linked to ER stress. FKbp11 was energy, it is plausible that glucose up regulated by glucose (SLR 1.6, 6/6 metabolism is adjusted for lesser energy How to define desensitization is a debated comparisons and down regulated by demands when secretion is blocked by issue. One may argue that our insulin data do diazoxide (SLR -0.9, 9/9 comparisons). P58 diazoxide. Such putative adjustment could not demonstrate desensitization of glucose- was also up regulated by glucose (SLR 0.9, then secondarily alter expression of genes induced insulin secretion, since secretion 5/6 comparisons). Taken together our data related to metabolism. was not reduced if one expresses results as do not support a major role for ER stress percent of islet insulin contents. However, behind the functional effects of over Could diazoxide exert effects through its diazoxide unquestionably enhanced insulin stimulation that have previously been effects on ambient insulin levels? The post-culture-induced insulin secretion after outlined (1). possibility of a feedback loop between co-culture with 27 mmol/l of glucose. secreted insulin and β-cell function has been Regardless of semantics it seems important In a recent microarray study (9) MIN6 cells proposed and debated for many years. were analyzed for short-term (45 min) Because of divergent experimental results, (10, 11) the issue is not settled. Under mitochondrial metabolism could serve to oscillations, which, in turn, regulate experimental conditions similar to the reduce ROS produced by elevated glucose in oscillations of insulin secretion. Indeed, data present ones, we did not observe any β-cells (18) and could thus be a potentially from human (21) as well as from rat regulation of islets cultured with diazoxide in beneficial effect of diazoxide in β-cells. pancreatic islets (our unpublished the presence of exogenous insulin with observations) show that diazoxide can respect to the subsequent enhancement of Thus, we envisage that the permissiveness of preserve the amplitude of insulin oscillations glucose-induced insulin secretion (7). elevated glucose for effects of diazoxide is otherwise decreased during prolonged Furthermore, in the present experiments due to both an inhibitory effect of diazoxide exposure to elevated glucose. there was no major difference in the (during its presence) on glucose-induced concentration of insulin in culture media insulin secretion and to a modulating Also the effects on fatty acid metabolism after culture with 11 vs. 27 mmol/l of influence of the mitochondrial state of that we observe could potentially be glucose, despite the fact that the influence by respiration. The latter statement we base, beneficial. It is well established that long diazoxide on gene expression was vastly first, on evidence, albeit not conclusive, that term excess of fatty acid metabolism can different .Hence, the reducing effects of diazoxide interacts directly with negatively affect glucose-induced insulin diazoxide on insulin levels in culture media mitochondrial metabolism and, second, on secretion (22). The effects of diazoxide on would probably not factor into the present general knowledge of the importance of the fatty acid metabolism could be thought to results. mitochondrial state of respiration for a improve glucose-induced insulin secretion. modulatory influences of drugs and other Thus, diazoxide inhibited the gene Previous observations indicate that diazoxide agents. In line with the latter notion, we find expression of several enzymes which exerts primary effects on metabolism in that culture with diazoxide exerts opposite participate in β-oxidation of fatty acids different tissues, including β-cells. Diazoxide effects on mitochondrial membrane potential (including, by less stringent criteria, 2,4 enhances ischemic preconditioning in the after co-culture at high vs. low glucose dienoyl CoA reductase 1, SLR -0.6 in 7 out heart (12) and neurons (13, 14) through a (unpublished observations). of 9 comparisons). These effects correspond mitochondrial interaction which may be to the finding that diazoxide decreases coupled to reduction of mitochondrial ROS Up-regulation of gene expression for oxidation of fatty acids in islets (23). production (14). In β-cells diazoxide aldolase B constituted the most marked Reciprocally, genes promoting lipid decreases mitochondrial membrane potential effect of diazoxide in this microarray and synthesis, such as acetyl Co -A carboxylase in β-cells in acute experiments (15, 16) and this effect was confirmed on the protein were up-regulated in the present array. may inhibit succinate oxidation (17). We did level. The effect was not seen at low glucose Mechanistically, it may be relevant that the not observe any effect on gene expression nor at 11 mmol/l glucose. Mathematical expression of solute carrier family 25, for succcinate dehydrogenase, represented in modelling of β-cell glycolysis indicates that member 10 was also up-regulated by the array by 4 genes encoding for separate up-regulation of aldolase B by glucose is diazoxide, albeit by less stringent criteria subunits. On the other hand, we observed an crucial for normal metabolic oscillations in (SLR +1.1 in 7 of 9 comparisons). This gene inhibitory effect on the enzyme fumarate β-cells (19). Such oscillations may in turn was recently shown in adipose tissue to hydratase 1 whose action is sequential to that drive oscillations of glucose-induced insulin transport citrite out of mitochondria in of succinate dehydrogenase and could secretion (20). A stimulatory effect by exchange for malate, the citrate then produce a similar inhibitory effect on diazoxide on aldolase B activity could then participating in lipid biosynthesis (24). mitochondrial metabolism. Inhibition of serve to uphold and increase metabolic Desensitisation of β-cells by hyperglycemia up regulates the CREM gene and interest in the perspective of autoimmunity is in vivo involves down-regulation of nkx6.1, additionally demonstrate that diazoxide the down-regulation of argininosuccinate pdx-1 and up-regulation of lactate exerts the opposite effect. synthetase, which could be important in dehydrogenase (LDH) (5) genes. Here we supplying substrate for NO- production (34). demonstrate that diazoxide exerts the Diazoxide also inhibited gene expression of opposite effects. Expression of both nkx6 UCP-2 and this effect was not glucose- It should be noted that regulation by and pdx-1 may be necessary not only for dependent. Such effects were recently diazoxide did not extend to genes expressed normal β-cell growth and maturation but also demonstrated by PCR for gene expression specifically in exocrine pancreas, thus giving for normal insulin secretion (25, 6). (23) and also on the protein level (our evidence for islet-specific effects of the drug Interestingly the transcription factor a unpublished observations). Since fatty acid in our islet preparations. In this context it mitochondrial (tfam) was, by less stringent metabolism induces UCP (29) it is should be mentioned that cholecystokinin criteria - also up-regulated by diazoxide conceivable that the UCP-2 and fatty acid (regulated here by diazoxide) is expressed not together with 27 mmol/l glucose (SLR +0.63 effects shown here are interlinked. As to only in exocrine but also in endocrine in 8 of 9 comparisons). This factor functional effects previous studies have pancreas (35). translocates to the mitochondria where it implicated UCP-2 as a negative factor for controls mitochondrial gene transcription glucose-induced insulin secretion (30). A comment is warranted on what should be (26) and potentially regulates the effect of However, this notion has been debated (31) considered a “basal” glucose concentration pdx-1 on mitochondria-coded genes (25). In and it is not clear to which extent the present for rat islets maintained in culture. Long the case of LDH, enzyme activity and lactate effects by diazoxide on UCP-2 have term culture of rat β-cells at 5.5 mmol/l production is low in glucose-sensitive β-cells implications for insulin secretion. glucose has been reported to induce β-cell (27). This ensures that a rise in ambient malfunctioning and apoptosis (36, 37). glucose leads to increased production of The inhibitory effect by diazoxide on gene However, in the present study and in ATP and possibly other metabolic signals for expression of glutamate decarboxylase previous ones we did not record any negative secretion through a tight coupling between (GAD) is also interesting from a functional effects. This is exemplified by the large glycolysis and oxidative phosphorylation. and pathophysiological point of view. The insulin response to an acute glucose Increased production of lactate will then GAD enzyme is a key enzyme in the challenge in islets cultured at 5.5 mmol/l attenuate a glucose-induced signal for insulin pathway leading to GABA synthesis; glucose (Fig 1B). A short culture time (24h) secretion and halting this process by furthermore antibodies against GAD are at and - in contrast to the afore-mentioned diazoxide should produce the opposite effect. least a marker and possibly a causative factor studies - use of serum-supplemented media in the etiology of autoimmune diabetes (4). could account for preserved functionality The diazoxide effect on cAMP response In agreement with our findings diazoxide has following culture at 5.5 mmol/l glucose. element modulator (CREM) could constitute previously been shown to decrease the efflux However, culture at 11 mmol/l of glucose another beneficial effect. This gene was of GAD from rat pancreatic islets (32). has been found in some studies to provide recently shown to be up-regulated both by Furthermore, diazoxide was reported to optimal conditions for rodent beta cell glucotoxic and lipotoxic conditions in vitro decrease the production of GAD protein function and survival and only further (28), furthermore forced over expression of (33). However, an effect by diazoxide on elevation of glucose levels was found to the gene was inhibitory on glucose-induced GAD gene expression has not previously induce negative effects (38). insulin secretion. We confirm that glucose been documented. Another enzyme of For these reasons we included microarrays secretion. Of further note is down-regulation and sensitivity to glucose in β-cells. Such also for the 11 mmol/l condition. The results of IGFBP,s of which IGFBP-3 has been effects are consistent with already from these arrays show that a number of implicated in growth as well as a signaling documented beneficial effects by previous genes are affected by diazoxide that are not molecule in beta cells (39) and of cyclin D, diazoxide exposure on insulin secretion. Our affected at 5.5. mmol/l. However, an known to stimulate beta cell growth (40). An findings support the rationale of using increase of glucose concentration from 11 to intriguing possibility, which is testable in diazoxide and other K+-ATP-openers in trials 27 mmol/l further increases the number of future studies, is the existence of a trade-off to improve insulin secretion in diabetes. genes affected by diazoxide. In particular the between beta cell growth and insulin number of genes that are down-regulated by secretion during prolonged hyperglycemia. This work was supported by the Swedish Research diazoxide are dramatically increased. These Council (Grant No. 72P-15180-01A), the Swedish Society of Medicine, Funds of Karolinska Institutet, last-mentioned genes would be primary In summary, we demonstrate that the effects the Magnus Bergvall, the Fredrik and Ingrid Thuring, suspects for being involved in attenuation of of diazoxide on gene expression are largely the Ragnhild and Einar Lundström, Åke Wiberg, the glucose-induced insulin secretion by linked to a high glucose environment, Loo and Hans Osterman Foundations and the prolonged hyperglycemia.. As already (important exceptions being genes such as Norwegian Medical Research Council (Grant No mentioned, 20% of these genes are involved GAD and UCP-2), that effects are exerted on 136139/310). in metabolism, some with clear linkage to glucose and lipid metabolism and that these regulation of glucose-induced insulin effects potentially uphold normal functioning References:

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TABLE 1 Comparisons between samples

Sample Culture Comparisons Effect of Dz at 5.5 Effect of Dz at 11 Effect of Dz at 27 Effect of glucose (27 Effect of glucose (11 Effect of glucose (27 mM G mM G mM G vs. 5.5 mM) vs. 5.5 mM) vs. 11 mM) A1 5.5 mM glucose B1 vs. A1 D1 vs. C1 F1 vs. E1 E1 vs. A1 C1 vs. A1 E1 vs. C1 A2 5.5 B1 vs. A2 D1 vs. C2 F1 vs. E2 E1 vs. A2 C1 vs. A2 E1 vs. C2 B1 5.5 + diazoxide (Dz) B2 vs. A1 D1 vs. C3 F1 vs. E3 E2 vs. A1 C2 vs. A1 E1 vs. C3 B2 5.5 + diazoxide B2 vs. A2 D2 vs. C1 F2 vs.E1 E2 vs. A2 C2 vs. A2 E2 vs. C1 C1 11 mM glucose D2 vs. C2 F2 vs. E2 E3 vs. A1 C3 vs. A1 E2 vs. C2 C2 11 D2 vs. C3 F2 vs. E3 E3 vs. A2 C3 vs. A2 E2 vs. C3 C3 11 D3 vs. C1 F3 vs. E1 E3 vs. C1 D1 11 + diazoxide D3 vs. C2 F3 vs. E2 E3 vs. C2 D2 11 + diazoxide D3vs. C3 F3vs. E3 E3 vs. C3 D3 11 + diazoxide E1 27 mM glucose E2 27 E3 27 F1 27 + diazoxide F2 27 + diazoxide F3 27 + diazoxide

TABLE 2 Concordance and discordance between diazoxide (9/9 comparisons) and glucose effects (≥4/6 comparisons at 27 vs.5.5 mmol/l)

27 mmol/l culture Up Up Down Down SLR≥1.0 SLR<1.0, ≥ 0.5 SLR≤ - 1.0 SLR> -1.0, ≤ - 0.5 Regulated in 5 3 1 0 parallel with glucose Regulated 6 25 68 60 opposite to glucose Not affected by 21 41 9 22 glucose

11 mmol/l culture Up Up Down Down SLR≥1.0 SLR<1.0, ≥ 0.5 SLR≤ - 1.0 SLR> -1.0, ≤ - 0.5 Regulated in 0 3 0 0 parallel with glucose Regulated 8 23 18 0 opposite to glucose Not affected by 14 49 2 1 glucose

5.5 mmol/l Up Up Down Down culture SLR≥1.0 SLR<1.0, ≥ 0.5 SLR≤ - 1.0 SLR> -1.0, ≤ - 0.5 Regulated in 0 0 0 2 parallel with glucose Regulated 0 1 4 5 opposite to glucose Not in parallel 1 11 1 5 with glucose

Data are means of three (27 and 11 mmol/l glucose culture) and two (5.5 mmol/l glucose culture) independent microarray experiments

TABLE 3a 20 most up-or down-regulated known genes after 24 h culture in 27 mmol/l glucose and 325 µM diazoxide vs. 27 mmol/l of glucose per se

Up regulated SLR (27 Down regulated SLR mM G) (27 mM G) Aldob aldolase b 5.4 Ass arginosuccinate synthetase -3.3 Drd4 dopamine receptor 4 2.0 Plat plasminogen activator tissue -2.8 Fos FBJ murine osteosarcoma viral oncogene 2.0 Lama3 laminin alpha 3 -2.7 homolog Ambp alpha 1 microglobulin/bikunin 1.5 Cckar cholecystokinin A receptor -2.6 Txnip upregulated by 1 25-dihydroxyvitamin D-3 1.5 Vgf VGF nerve inducible -2.4 Tm4sf7 transmembrane 4 superfamily member 7 1.5 Spam sperm adhesion molecule -2.4 predicted (predicted) Cspg5 5 1.4 Plagl 1 Pleiomorphic adenoma gene-like 1 -2.3 RGD:1303100 ciliary factor receptor 1.3 Fst Follistatin -2.3 Acsl1 acyl-CoA synthetase long-chain family member 1.2 Gucy2c 2C -2.2 1 Gpr51 G protein-coupled receptor 51 1.2 Nptxr neuronal pentraxin receptor -2.2 Prkar2b protein kinase cAMP dependent regulatory type 1.2 Thbd thrombomodulin -2.0 II beta RGD:708430 CaM-kinase ll inhibitor alpha 1.2 Ms4a8b_pre membrane-spanning 4-domains subfamily -2.0 dicted A member 8B (predicted) Kcnab3 potassium voltage gated channel shaker related 1.1 Aass_predic aminoadipate-semialdehyde synthase -2.0 subfamily beta member 3 ted (predicted) Acta1 actin alpha1 skeletal muscle 1.0 Cck cholecystokinin -2.0 Tieg (KLF10) TGFB inducible early growth response 1.0 Cart cocaine and amphetamine regulated -2.0 transcript Nr4a1 nuclear receptor subfamily 4 group A member 1 1.0 Adm -1.9 Il6r interleukin 6 receptor 1.0 Pfkl Phosphofructokinase liver B-type -1.9 Mcf2l mcf.2 transforming sequence 0.9 Ckb creatine kinase brain -1.8 G0s2_predicte G0/G1 switch gene 2 (predicted) 0.8 Ros1 v-ros UR2 sarcoma virus oncogene -1.6 d homolog 1 (avian) Pdx1 Pancreatic and duodenal homeobox gene 1 0.8 Casp7 caspase 7 -1.6

Data are 9/9 comparisons

TABLE 3b 20 most up-or down-regulated known genes after 24 h culture in 11 mmol/l glucose and 325 µM diazoxide vs. 11 mmol/l of glucose per se

Up-regulated SLR Down-regulated SLR Myh10 myosin, heavy polypeptide 10, non-muscle 1.5 Cart cocaine and amphetamine regulated transcript -3.1 Ret ret proto-oncogene 1.4 Adm adrenomedullin -2.9 protein kinase, cAMP dependent regulatory, Prkar2b type II beta 1.3 Mmp13 matrix metallopeptidase 13 -2.8 FBJ murine osteosarcoma viral oncogene Fos homolog 1.3 Crem cAMP responsive element modulator -2.3 Gtf3c1 general transcription factor III C 1 1.3 Ass argininosuccinate synthetase -2.2 solute carrier family 37 (glycerol-3- Slc37a1 phosphate transporter), member 1 1.3 Plat plasminogen activator, tissue -2.2 Jun Jun oncogene 1,2 Ppap2a phosphatidic acid phosphatase 2a -2.2 Shank1 SH3 and multiple ankyrin repeat domains 1 1.2 Vgf VGF inducible -1.9 myeloid/lymphoid or mixed lineage- leukemia translocation to 6 homolog Mllt6_predicted (Drosophila) (predicted) 1.2 Casp7 caspase 7 -1.8 LOC684681 similar to Histone H1.2 (H1 VAR.1) (H1c) 1.1 Ghr growth hormone receptor -1.8 similar to Laminin alpha-3 chain precursor (Nicein Ppap2b phosphatidic acid phosphatase type 2B 1.1 LOC682736 alpha subunit) -1.6 serine (or cysteine) proteinase inhibitor, clade E, Il6 interleukin 6 1.1 Serpine2 member 2 -1.5 similar to early estrogen-induced gene 1 LOC687750 protein 1.1 Cck cholecystokinin -1.5 Glp1r glucagon-like peptide 1 receptor 1.1 Gad1 glutamic acid decarboxylase 1 -1.3 Klf10 Kruppel-like factor 10 1.1 Ckb creatine kinase, brain -1.3 nuclear factor of kappa light chain gene Nfkbia enhancer in B-cells inhibitor, alpha 1.0 RGD1307736 similar to Hypothetical protein KIAA0152 -1.2 Rgs16 Regulator of G-protein signaling 16 1.0 Pld1 phospholipase D1 -1.0 Tmem123 transmembrane protein 123 1.0 Pfkp phosphofructokinase, platelet -1.0 Tff3 trefoil factor 3 0.9 Nsf N-ethylmaleimide sensitive fusion protein -0.6 Slc38a2 solute carrier family 38, member 2 0.9

Data are 9/9 comparisons

TABLE 3c Up-or down-regulated genes after 24 h culture (passing the criterion SLR ≥0.5 or ≤-0.5) in 5.5 mmol/l glucose and 325 µM diazoxide vs. 5.5 mmol/l glucose per se

Up regulated SLR Down regulated SLR Acta1 actin alpha 1 skeletal muscle 1 Crem cAMP responsive element modulator -1.7 Nr1d2 Nuclear receptor subfamily 1 group D member 2 0.9 Cart cocaine and amphetamine regulated transcript -1 Cds1 CDP-diacylglycerol synthase 1 0.8 Vgf VGF nerve growth factor inducible -1 Dbp D site albumin promoter binding protein 0.8 Idi1 isopentenyl-diphosphate delta isomerase -1 Myh10 myosin heavy chain 10 non-muscle 0.6 Fn1 fibronectin 1 -1 Nfs1 nitrogen fixation gene 1 (S. cerevisiae) 0.6 Npy neuropeptide Y -0.9 Slc37a1_predicted solute carrier family 37 (glycerol-3-phosphate 0.6 Igf1r insulin-like growth factor 1 receptor -0.9 transporter) member 1 (predicted) Ptprd protein tyrosine phosphatase receptor type D 0.6 Gad1 glutamate decarboxylase 1 -0.9 Casr calcium-sensing receptor 0.5 Pde10a phosphodiesterase 10A -0.8 Cacna2d1 calcium channel voltage-dependent alpha2/delta 0.5 Pfkp phosphofructokinase platelet -0.7 subunit 1 Rasd1 RAS -induced 1 0.5 Gbe1_predicted glucan (1 4-alpha-) branching enzyme 1 (predicted) -0.6 Dact2_predicted dapper homolog 2 antagonist of beta-catenin 0.5 Ucp2 uncoupling protein 2 -0.5 (xenopus) (predicted)

Data are 4/4 comparisons 20

TABLE 4 Functional clustering of genes (main categories and selected subcategories) affected by 24 h culture with diazoxide

Ontholgy No. of genes Genes affected Diazoxide only at Diazoxide only at Diazoxide only at Diazoxide at 5.5 and represented on by diazoxide (%) 5.5 mmol/l glucose 11 mmol/l glucose 27 mmol/l glucose 27 mmol/l glucose microarray Up Down Up Down Up Down Up Down (n=23) (n=26) (n=98) (n=21) (n=111) (n=160) (n=4) (n=10) Metabolism 4591 3.9 7 14 33 13 40 67 2 5 Membrane 3408 3.3 6 6 17 7 21 52 1 2 Signal transduction 2064 4.1 5 6 14 7 17 34 0 2 Transport 1875 3.2 3 4 10 3 11 24 2 2 Nucleic acid binding 1557 2.6 3 3 13 1 14 6 1 0 Receptor activity 1100 3.6 5 2 7 1 8 16 0 1

Transcription factor 565 4.4 1 1 4 1 13 4 1 0 activity Cell adhesion 456 2.9 1 1 3 0 3 5 0 0 Regulation of cell cycle 388 4.6 0 1 6 0 6 5 0 0 Extracellular matrix 361 2.8 0 1 2 1 0 6 0 0 Apoptosis 294 6.1 0 0 6 2 3 7 0 0 Monosaccharide 125 14.4 1 2 0 1 3 9 1 1 metabolism

Some genes fall into more than one onthology

21

TABLE 5

Effect of diazoxide on genes not affected by 11 vs 5.5 mM G (≤4/6) and up-regulated by 27 vs 11 mM G (9/9)

Up-regulated by diazoxide at 5.5 mM G SLR Il6st Interleukin 6 signal transducer 0,4

Up-regulated by diazoxide at 11 mM G SLR Rgs16 Regulator of G-protein signaling 16 1,0

Down-regulated by diazoxide at 11 mM G SLR similar to Laminin alpha-3 chain precursor (Nicein alpha LOC682736 subunit) -1,6

Up-regulated by diazoxide at 27 mM G SLR Rgs16 Regulator of G-protein signaling 16 1,8 Txnip upregulated by 1,25-dihydroxyvitamin D-3 1,5

Down-regulated by diazoxide at 27 mM G SLR Thbd thrombomodulin -3,8 similar to Laminin alpha-3 chain precursor (Nicein alpha LOC682736 subunit) -2,7 Nptxr neuronal pentraxin receptor -2,2 Gucy2c -2,2 Mest Mesoderm specific transcript -1,9 LOC501015 similar to nuclear localized factor 2 -1,8 Ldha lactate dehydrogenase A -1,2 Gla_mapped galactosidase, alpha (mapped) -1,2 Igfbp3 insulin-like growth factor binding protein 3 -1,1 Ntrk2 neurotrophic , receptor, type 2 -1,1 Bcat1 branched chain aminotransferase 1, cytosolic -1,1 Ccnd3 cyclin D3 -1,0 Gpi glucose phosphate isomerase -0,9 Mapkapk2 MAP kinase-activated protein kinase 2 -0,8 LOC293589 putative GTP-binding protein -0,8 Mbd3_predicted Methyl-CpG binding domain protein 3 (predicted) -0,7 Anxa4 annexin A4 -0,6

22

Effect of diazoxide on genes down-regulated by 11 vs 5.5 mM G (4/4) and up-regulated by 27 vs 11 mM G (9/9)

Down-regulated by diazoxide at 5.5 mM G SLR Ucp2 uncoupling protein 2 (mitochondrial, proton carrier) -0,5

Up-regulated by diazoxide at 11 mM G SLR Slc45a1 Solute carrier family 45, member 1 0,9

Down-regulated by diaozixde at 27 mM G SLR Ucp2 uncoupling protein 2 (mitochondrial, proton carrier) -0,81 Igfbp4 insulin-like growth factor binding protein 4 -0,78

23

TABLE 6 Genes related to hexose, amino acid, fatty acid metabolism and ATP production and processing

Functional Gene name Effect of Effect of Effect of group diazoxide diazoxide at glucose at 11 mM G 5.5 vs. 27 27 mM G mM metabolism SLR SLR SLR hexoses Aldob Aldolase B + 5.4 0 + 2.4 Ldha Lactate dehydrogenase - 1.2 - 1.2 + 1.6 Gpi Glucose phosphate isomerase - 0.91 - 0.91 + 1.2 Pgk1 Phosphoglycerate kinase 1 - 0.6 - 0.79 + 0.6 G6pdx Glucose-6-phosphate dehydrogenase - 0.6 0 1.0 Eno2 Enolase 2 gamma - 1.0 0 0 amino acids Ass Argininosuccinate synthetase - 3.3 - 2.2 + 1.8 Bcat1 Branched-chain aminotranferase 1, - 1.1 0 + 1.5 cytosolic Odc1 Ornithine decarboxylase - 0.8 - 0.87 + 0.9 fatty acids Acsl1 Acetyl-CoA synthetase long-chain + 1.2 0 0 family, member 1 Gad1* Glutamate decarboxylase 1 - 0.9 - 1.3 + 0.7 Acaca Acetyl Coenzyme A carboxylase alpha + 0.8 0 0 Ppap2b ER transmembrane protein Dri 42 +0.8 + 1.1 -0.9 Acadsb Acyl-Coenzyme A dehydrogenase - 1.1 0 0 short/branched chain Dci Dodecenoyl-coenzyme E delta isomerase - 0.9 0 0 ATP Ckb Creatine kinase b - 1.8 - 1.3 + 1.5 production and processing Ucp2* Uncoupling protein 2 - 0.8 0 0 Pdx1* Pancreatic and duodenal homeobox gene + 0.8 + 0.6 0 1 Nkx6-1 NK6 transcription factor related 1 + 0.5 0 0 (Drosophila) Fh1 Fumarate hydratase 1 - 0.5 - 0.68 + 0.6

24

* Genes also affected by diazoxide at 5.5 mmol/l glucose (see text). Genes affected by diazoxide are 9/9 comparisons. Effects of glucose (5.5 vs 27 mmol/l) are 6/6 comparisons except for Aldob, G6pdx and Ppap2b which are 5/6 comparisons. Effects of diazoxide at 11 mmol/l glucose on Pgk1, Odc1 and Fh1 are 7/9 and on Pdx1 6/9 comparisons.

Fig legends

Fig. 1. Insulin release from rat islets after 24 h culture in 5.5, 11 and 27 mmol/l glucose (G) with and without diazoxide (D). After culture islets were preincubated for 30 min in 3.3 mmol/l G followed by final incubations in 3.3 and 16.7 mmol/l G, n=6.. A shows insulin in culture media, B post-culutre insulin secretion, C islet insulin content and D incremental fractional release (incremental insulin secretion/islet insulin content).

*P<0.02 or less vs. no previous diazoxide

Fig 2 Glucose dependency of diazoxide’s effect on gene expression. A shows percentage of diazoxide-affected genes (up-regulated SLR ≥ 0.5 and down-regulated SLR ≤ -0.5) at different glucose concentrations during culture. B and C show percentage of up- (SLR ≥ 0.5) and down- regulated (SLR ≤ -0.5) genes after culture at different glucose-concentrations. After 27 mmol/l glucose culture there was a shift towards down- regulation of genes by diazoxide.

Fig. 3 Effects of diazoxide on aldolase B and pdx1 proteins. Islets were cultured for 24 h in 5.5 and 27 mmol/l glucose (G) with and without diazoxide (D). After culture islets were preincubated for 30 min in 3.3 mmol/l G followed by Western blot for aldolase B (upper), n=3 and pdx1

(lower), n=4 (signifying results from three and four different pancreases). 25

*P<0.05 vs. 5.5 mmol/l, †P<0.02 vs. no previous diazoxide for aldolase B.

*P<0.02 or less vs. no previous diazoxide for pdx1.

3500 350 3000 ) 300 h ) 4 h

2 2500 / / t t 250 le le 2000 /is /is 200 U U

µ 3.3 mM G µ ( ( 1500 150 16.7 lin lin u u

1000 s 100 s in in 500 50 0 0 5.5 5.5D 11 11D 27 27D Fig.1A 5.5 5.5D 11 11D 27 27D B Culture medium Insulin secretion Culture medium *

3000 16

) 2500

l t

a 14 le n

2000 io 12 * /is )

U act % 10 r µ 1500 e ( ( l f 8 a

* t lin * eas * l 6 en

u 1000 e r s 4 em in

500 cr 2 in 0 0 5.5 5.5D 11 11D 27 27D 5.5 5.5D 11 11D 27 27D

C D 26

Fig 2 A Genes affected by diazoxide

5.5 mmol/l G 11 27

C

Up-regulated by diazoxide (SLR ≥ 0.5) Down-regulated by diazoxide (SLR ≤ -0.5)

5.5 mmol/l G 5.5 mmol/l G

11 11 27 27

27

Fig. 3a Aldolase Western blot

Culture with 5.5 5.5+D 27 27+D

% 100 114 223* 963**

Fig. 3b Pdx1 Western blot

Culture with : 5.5 5.5+D 27 27+D

% : 100 113±2.9* 89±8.3 145±6.3**