59 Upregulation of b-cell genes and improved function in rodent islets following chronic glucokinase activation

D Gill, K J Brocklehurst, H W G Brown and D M Smith AstraZeneca Diabetes and Obesity Drug Discovery, CVGI iMED, 3S42C Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

(Correspondence should be addressed to D M Smith; Email: [email protected])

(D Gill is now at Prosidion Ltd, Oxford, UK)

Abstract

Glucokinase (GK) plays a critical role in controlling blood glucose; GK activators have been shown to stimulate insulin secretion acutely both in vitro and in vivo. Sustained stimulation of insulin secretion could potentially lead to b-cell exhaustion; this study examines the effect of chronic GK activation on b-cells. Gene expression and insulin secretion were measured in rodent islets treated in vitro with GKA71 for 72 h. Key b-cell gene expression was measured in rat, mouse and global GK heterozygous knockout mouse islets (gkdel/wt). Insulin secretion, after chronic exposure to GKA71, was measured in perifused rat islets. GKA71 acutely increased insulin secretion in rat islets in a glucose-dependent manner. Chronic culture of mouse islets with GKA71 in 5 mmol/l glucose significantly increased the expression of insulin, IAPP, GLUT2, PDX1 and PC1 and decreased the expression of C/EBPb compared with 5 mmol/l glucose alone. Similar increases were shown for insulin, GLUT2, IAPP and PC1 in chronically treated rat islets. Insulin mRNA was also increased in GKA71-treated gkdel/wt islets. No changes in GK mRNA were observed. Glucose-stimulated insulin secretion was improved in perifused rat islets following chronic treatment with GKA71. This was associated with a greater insulin content and GK protein level. Chronic treatment of rodent islets with GKA71 showed an upregulation of key b-cell genes including insulin and an increase in insulin content and GK protein compared with glucose alone. Journal of Molecular Endocrinology (2011) 47, 59–67

Introduction isoform is an absolute requirement for survival, as gkdel/del (either global or b-cell specific) is lethal, whereas Glucokinase (GK) exists as two isoforms that have global or b-cell-specific heterozygous (gkdel/wt) deletion independent tissue-specific promoters. In the pancreatic causes only modest hyperglycaemia (Bali et al. 1995, b-cell, GK acts as a glucose sensor and contributes to the Grupe et al. 1995, Terauchi et al. 1995). Deletion of liver regulation of glucose-stimulated insulin secretion GK leads to mild hyperglycaemia but decreases glucose (GSIS). The pancreatic isoform of GK is also found in utilisation and glycogen synthesis (Postic et al. 1999). glucose-sensing cells in the anterior pituitary gland, The pivotal role of GK in controlling blood glucose hypothalamus and entero-endocrine K- and L-cells has made it attractive as a potential drug target for the (Schuit et al. 2001). The liver isoform is specific to treatment of type 2 diabetes. A number of small molecule hepatocytes, where it is the rate-limiting enzyme for GK activators (GKAs) have been described. These agents glucose utilisation and glycogen synthesis (Matschinsky have effects on hepatic glucose metabolism and insulin 1990, Schuit et al. 2001). secretion, both in vitro and in normal and diabetic animal Naturally occurring mutations in man support the models (Grimsby et al.2003, Brocklehurst et al.2004, importance of GK in the regulation of glucose Kamata et al.2004, Efanov et al.2005, Coope et al.2006, homeostasis. Loss of both GK alleles results in perma- Futamura et al.2006, Matschinsky et al.2006, Johnson nent neonatal diabetes, whereas heterozygous inactivat- et al.2007, Coghlan & Leighton 2008). ing mutations of GK lead to maturity-onset diabetes of Several studies have demonstrated that GKAs increase the young (MODY2; Hattersley et al. 1992). Conversely, GSIS from b-cells (Coghlan & Leighton 2008), but the rare activating mutations of GK result in hyperinsulin- consequences of long-term direct action of GKAs on aemic hypoglycaemia (Glaser et al. 1998, Christesen the islet have only recently begun to be investigated. et al. 2002, Gloyn et al. 2003, Cuesta-Munoz et al. 2004). Of potential concern is that sustained stimulation of Isoform-specific, tissue-specific and global GK knock- insulin secretion, without a concomitant increase in out (gkdel/del) mice have been extensively phenotyped insulin biosynthesis, could lead to b-cell exhaustion. and provide further evidence for the role of GK in The effect of glucose on the regulation of insulin glucosesensinganddisposal.ThepancreaticGK production has been studied extensively, by direct

Journal of Molecular Endocrinology (2011) 47, 59–67 DOI: 10.1530/JME-10-0157 0952–5041/11/047–059 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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measurement of insulin biosynthesis and by measuring penicillin/streptomycin/glutamine (100 units/ml, gene expression. Prolonged culture of isolated rodent 0.1 mg/ml and 2 mmol/l respectively, Invitrogen) and islets in high glucose leads to increased insulin bio- 11 mmol/l glucose. Insulin secretion was assessed synthesis and increased insulin mRNA levels (Brunstedt according to the method described previously (Johnson & Chan 1982, Docherty & Clark 1994, Leibiger et al. et al. 2007); 30 min pre-incubation was followed by 2-h 2000, Khaldi et al.2004). Data generated with GKAs treatment. Insulin concentration was determined by provide support for these molecules acting in a ‘glucose- homogeneous time-resolved fluorescence assay (HTRF like’ manner on insulin secretion; we hypothesised that assay, CisBio, Bognols/Ce`ze, France) according to the GKAs would also mimic the effect of elevated glucose manufacturer’s high-range protocol (0–100 ng/ml). concentration on insulin biosynthesis. We describe the use of a potent GKA, GKA71, to determine the effect of sustained GK activation on the expression of key b-cell Chronic treatment of islets genes, including insulin, in isolated rat and mouse islets. Islets were cultured in RPMI medium supplemented as above. Islets were incubated 20 islets/well in 6-well plates at 37 8C in a 5% CO2 humidified incubator. Materials and methods Prior to chronic treatment, islets were allowed to recover overnight in medium containing the appro- Materials priate glucose concentration. Islets from C57Bl/6J mice The novel GK activator GKA71 was dissolved in and from rats were divided between media containing wt/wt dimethylsulphoxide (DMSO) prior to dilution to the 5 or 25 mmol/l glucose. For experiments with gk del/wt final concentration in the required medium. and gk islets, islets from animals of the same genotype were pooled and then divided between 8 and 25 mmol/l glucose. Experimental animals and islet isolation Following the overnight incubation, islets were hand- Animals used in these studies were supplied by Astra- picked into medium at the same glucose concentration Zeneca Biological Animal Breeding Unit and were kept containing either 1% (v/v) DMSO (control) or under standard laboratory conditions with free access to 10 mmol/l GKA71 in 1% DMSO. Islets were treated food and water. Male C57Bl/6J mice were 8–10 weeks of for 72 h and were hand-picked into fresh treatment age (22–24 g) and male Hannover Wistar rats 8–9 weeks medium daily. of age (230–260 g). Male global heterozygous GK del/wt wt/wt knockout (gk ) and wild-type (gk ) mice were RNA isolation and reverse transcription used between 10 and 16 weeks of age; these animals were generated as described previously (Gorman et al. Following chronic culture, RNA was prepared from 2008). All procedures were carried out in accordance pooled samples of 30 (rat) or 40 (mouse) islets (see with the Animals (Scientific Procedures) Act 1986. figure legends for individual experiment replicate Animals were rendered insentient by inhalation of a numbers). RNA isolation and gene expression analysis 5:1 mixture of CO2:O2 and killed by cervical dislocation were carried out as described previously (Gorman prior to removal of the pancreas. Islet isolations were et al. 2008). carried out as described previously (Johnson et al. 2007) by standard methods using liberase R1 (Roche). Gene expression analysis

Recombinant GK assays Quantitative PCR of single genes was performed by Taqman analysis in 384-well plate assays using ABI Human, rat and mouse b-cell GK were cloned from cDNA Prism 7900HT sequence detection system (Applied using PCR. Each was expressed in Escherichia coli with Biosystems, Carlsbad, CA, USA). Taqman analysis of an NH2-terminal 6His tag and purified using Ni-NTA mouse samples was carried out using primer and probe chromatography. Recombinant proteins were O95% sets designed using Primer Express software (TaqMan pure as assessed by SDS/PAGE analysis (Brocklehurst primer and probe sequences are shown in Supple- et al.2004). GK was assayed at its S0.5 glucose concen- mentary Table 1, see section on supplementary data tration as described previously (Brocklehurst et al.2004). given at the end of this article). ‘Assay on demand’ primer/probe mixes (Applied Biosystems) were used for analysis of rat samples: insulin (Rn01774648), GLUT2 Acute insulin secretion assay (Rn00563565), hypoxanthine phosphoribosyltransfer- Rat islets were allowed to recover overnight in ase (HPRT) (Rn01527838), cyclophilin (Rn00690933), RPMI medium supplemented with 10% (v/v) FCS, b-glucuronidase (Rn00566655), IAPP (Rn00561411),

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PDX1 (Rn00755591), GK (Rn00561265), PC1 (Rn00 4! loading buffer (Invitrogen) and 5 ml10! 567266) and CEBPb (Rn00824635). Samples from the denaturing buffer (Invitrogen). Samples were heated C57Bl/6J chronic islet experiment were used to perform at 95 8C for 5 min before storing at K20 8C. Residual Taqman low-density arrays (TLDA), which were carried lysed sample was used for protein analysis via a DC out as described previously (Gorman et al. 2008). protein assay (Bio-Rad). For both quantitative PCR and TLDA analysis, a Protein (50 mg/sample) was resolved by electro- number of housekeeper genes were used. For quan- phoresis on 10% (v/v) Bis–Tris NuPage gels (Invi- titative PCR analysis, the housekeeper genes were trogen), using 3-(N-morpholino)propanesulphonic HPRT, cyclophilin and b-glucuronidase. For TLDA acid buffer. The iBlot system (Invitrogen) was used for analysis, the included housekeeper genes were HPRT, transfer onto nitrocellulose. After blocking overnight at 18S and b-tubulin. No qualitative differences were 4 8C in 5% (w/v) BSA/0.05% (w/v) polysorbate 20 in observed when the genes of interest were expressed Tris-buffered saline, a 2 h room temperature incubation relative to the different housekeepers; therefore, all was carried out with rabbit polyclonal antibody raised data shown are normalised to HPRT expression. against full-length human pancreatic GK (used at 1:5000 dilution). Incubation with secondary antibody, HRP-linked donkey anti-rabbit (GE Healthcare, Buck- Perifusion studies inghamshire, UK), 1:5000 dilution, was for 1 h at room temperature, followed by detection with an enhanced Perifusion experiments were performed using a Brandel chemiluminescence system (Chemiglow West, Cell SF-12 Superfusion apparatus (Brandel, Gaithersburg, Biosciences, Santa Clara, CA, USA). The blot was MD,USA).Followingchronicculture,isletswere imaged on a Chemi-imager (Alpha Innotech), and loaded into perifusion chambers (50 islets/chamber in densitometry was performed using software installed on 200 ml medium), enclosed with polyethylene filters. the machine. Islets were perifused in HEPES-balanced Krebs-Ringer phosphate buffer (KRH), prepared as described previously (Johnson et al.2007), at a flow rate 1 ml/min. Data handling and statistical analysis During an initial stabilisation period (perifusion 30 min; 3 mmol/l glucose), no samples were collected. Islets Acute insulin secretion induced by GKA71 was were then perifused to obtain a GSIS profile: 30 min compared with the control insulin secretion at each with 3 mmol/l glucose; 25 min with 15 mmol/l glucose; glucose concentration by Student’s t-test. For statistical 17.5 min with 3 mmol/l glucose and 6 min with analysis of the gene expression data, each gene of 40 mmol/l KCl, during which time samples were interest was normalised to the housekeeper gene HPRT collected for analysis of insulin secretion. Insulin was and statistical evaluation was carried out on log- determined using an HTRF kit, prepared according to transformed data. For each stimulatory phase of the the normal range protocol (0–10 ng/ml). perifusion profile, insulin secretion after treatment with GKA71 or high glucose was compared with 5 mmol/l glucose-treated islets using Student’s t-tests. Two-sided Total insulin measurement unequal variance Student’s t-tests were used throughout. Following chronic culture, three islets in 10 ml medium were transferred to individual wells of 96-well plates containing 115 ml KRH. Then, 115 ml 2% (v/v) Triton Results X-100 were added to each well, and the plates were frozen at K20 8C to completely lyse the islets. For GKA71 is a potent activator of human pancreatic GK determination of insulin content, samples were and acutely stimulates insulin secretion from rat islets thawed, centrifuged (700 g, 2 min) and diluted as In isolated enzyme assays, GKA71 activated recombi- appropriate, prior to analysis using the high-range nant human pancreatic GK with EC50 112 nmol/l HTRF assay. (nZ8, 95% confidence interval (CI) 70–170 nmol/l). GKA71 also activated the rat and mouse pancreatic Z Western blotting GK isoforms: EC50 88 nmol/l (n 3, 95% CI 86–91 nmol/l) and 411 nmol/l (nZ3, 95% CI Following chronic culture, 500–900 islets from each 350–490 nmol/l) respectively. treatment group were lysed in 50 mllysisbuffer: The acute effect of GKA71 on GSIS from isolated rat 20mmol/lTris–HCl(pH7.5), 150 mmol/l NaCl, islets is shown in Fig. 1. GKA71 increased insulin 2 mmol/l EDTA, 1% (v/v) Triton X-100, with 1 mini- secretion at 3, 5, 8 and 10 mmol/l glucose; although at protease inhibitor tablet (Roche)/10 ml. Lysed sample 10 mmol/l, the increase was not significantly different (32.5 ml) was transferred to a tube containing 12.5 ml from control. www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 59–67

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800 mice. These changes in insulin mRNA induced by 700 GKA71 were similar in both genotypes to those elicited 600 *** by 25 mmol/l glucose. Expression of GK did not significantly change in the 500 ** presence of GKA71 or 25 mmol/l glucose in islets from 400 gkwt/wt or gkdel/wt mice (Fig. 3B). GK mRNA levels were 300 lower in gkdel/wt islets compared with gkwt/wt islets in basal ** 200 8 mmol/l glucose, but in the presence of GKA71 or 25 mmol/l glucose, there was no significant difference Insulin (pmol/islet per hour) 100 0 in GK mRNA between genotypes. 13581020 Glucose (mmol/l) Chronic activation of GK by GKA71 increases the Figure 1 GKA71 (1 mmol/l) acutely stimulates insulin secretion from rat islets in a glucose-dependent manner. Control islets expression of insulin and changes the expression (black bars) and islets treated with 1 mmol/l GKA71 (striped bars). of other key b-cell genes in rat islets Values are meanGS.E.M., nZ6, each of three islets. **P!0.01, ***P!0.001 versus the respective control in the absence of In order to obtain islets in sufficient quantity, sub- GKA71. Data are representative of four separate experiments. sequent experiments were carried out in rat islets. Initial experiments were carried out to confirm that the Chronic activation of GK by GKA71 increases the above findings could be reproduced in rat islets. expression of insulin in mouse islets and changes Following culture in the presence of GKA71 for 72 h, the expression of other key b-cell genes mRNA levels of key b-cell genes were determined using Taqman. Insulin expression increased 2.7-fold (Fig. 4A, Taqman analysis of mouse islets cultured for 72 h P!0.001). GLUT2 (2.3-fold, P!0.001; Fig. 4B), PC1 revealed that GKA71 led to a 42-fold increase in insulin (2.5-fold, P!0.001; Fig. 4C)andIAPP (2.2-fold, mRNA levels compared with islets cultured in 5 mmol/l P!0.001; Fig. 4D) also increased; changes were quali- glucose alone (Fig. 2). GK mRNA was also analysed in tatively similar to those induced by 25 mmol/l glucose. these samples; however, no significant changes were A small but non-significant increase was observed for observed (data not shown). Chronic exposure to w both PDX1 and GK and there was a significant decrease 25 mmol/l glucose also significantly increased, 80- in expression of CEBPb (data not shown). fold, the level of insulin mRNA compared with incubation in 5 mmol/l glucose. The expression of other b-cell genes was explored in GSIS in rat islets is increased after chronic activation the same samples using TLDA analysis. Table 1 of GK by GKA71 summarises the data for those genes in which changes were observed, either greater than twofold or statisti- Following chronic culture, rat islets were perifused cally significant (for full gene list, see Gorman et al. with sub-stimulatory (3 mmol/l) and stimulatory (2008)). A marked increase in insulin gene expression (15 mmol/l) glucose; the perifusion profile is shown was observed, consistent with the individual gene in Fig. 5A. Following the 30-min pre-perifusion phase, Taqman. IAPP, PDX1, PC1 and GLUT2 also significantly all treatment groups had the same basal insulin increased, whereas CEBPb expression decreased by 80%. secretion, indicating that no residual compound To investigate whether sustained GK activation will remained in the islets. Islets cultured in 5 mmol/l also maintain islet function where GK activity is glucose exhibited no measurable GSIS response when compromised, we explored the effect of chronic stimulated with 15 mmol/l glucose; however, islets del/wt treatment with GKA71 on islets from gk mice. 5 Results obtained from the wild-type (gkwt/wt)and heterozygous knockout (gkdel/wt)mouseisletsare 4 3 P = 0·002 summarised in Fig. 3; these data were generated by ** wt/wt single-gene Taqman analysis. In gk islets, cultured in 2 expression 8 mmol/l glucose, chronic culture with GKA71 induced 1

! . insulin mRNA Relative a twofold (P 0 05) increase in insulin mRNA (Fig. 3A). 0 The level of insulin mRNA in islets from gkdel/wt mice 5255 + GKA71 cultured in 8 mmol/l glucose was markedly lower Glucose (mmol/l) than that in wild-type islets; however, chronic culture Figure 2 Chronic treatment of C57Bl/6J mouse islets with GKA71 with GKA71 induced a 30-fold (P!0.001) increase to (10 mmol/l) increases insulin mRNA levels. mRNA levels determined by single-gene Taqman analysis (meanGS.E.M.of a level that was undistinguishable from the level triplicate samples, each of 40 islets). **P!0.01 versus 5 mmol/l wt/wt attained by GKA71 stimulation of islets from gk glucose alone.

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Table 1 GKA71 influences the expression of key b-cell genes in culture with GKA71 resulted in increased GK protein; mouse islets this change was again consistent with that observed for islets cultured in 25 mmol/l glucose (Fig. 7A and B). Direction of Gene change Fold change P value Similar findings were observed in islets taken from gkwt/wt and gkdel/wt mice (Fig. 7C and D).

IAPP Increase 4.60.005 Insulin Increase 15.90.001 PDX1 Increase 4.50.012 Discussion PC1 Increase 6.50.001 . ! . GLUT2 Increase 29 3 0 001 Although many groups have demonstrated the potential CEBPb Decrease 5.00.040 of GKAs to drive insulin secretion, no studies, to date, have investigated whether GKAs activate the appropriate cultured in the presence of GKA71 exhibit clear first- pathways to ensure that b-cell exhaustion does not occur and second-phase insulin secretion and have a profile in response to prolonged treatment with a GKA. We very similar to that of the islets cultured in 25 mmol/l designed a series of experiments to assess the effect of a glucose. The effects of GKA71 on first- and second- high concentration of GKA71 (at least ten times greater phase insulin secretion were quantified by calculating than the EC50) on isolated isletsfollowinga 72-h treatment the area under curve (AUC) for each phase. The first- period. The primary end point for these investigations ! . phase (30–39 min) response was 65-fold (P 0 001) was the effect of GKA71 on insulin mRNA levels. greater in islets previously cultured in the presence of We demonstrate that islets chronically treated with a GKA71 compared with those cultured in 5 mmol/l GKA have a greater level of insulin production than glucose alone and not significantly different from islets cultured in 25 mmol/l glucose. AUC for the second- A 8 mmol/l glucose . ! . 8 mmol/l glucose + GKA71 phase response (40–76 5 min) was 585-fold (P 0 001) 25 mmol/l glucose greater after culture with GKA71 and also similar to 3·0 islets cultured in 25 mmol/l glucose. These data are *** shown in Fig. 5B and C. 2·5 *** * The response to perifusion with 40 mmol/l KCl was 2·0 * determined from the AUC (76.5–81 min). Insulin release was fourfold (PZ0.006) greater in islets cultured in the 1·5

presence of GKA71 compared with those cultured in expression 1·0 5 mmol/l glucose alone and similar to that in islets 0·5 previously cultured in 25 mmol/l glucose (Fig. 5D). insulin mRNA Relative # 0·0 WT He KO Total insulin content in rat islets is increased after Genotype chronic activation of GK by GKA71 B 8 mmol/l glucose 8 mmol/l glucose + GKA71 We hypothesised that the greater KCl response in islets 25 mmol/l glucose treated with GKA71 resulted from elevated total insulin 16 content in those islets. Chronically treated islets were 14 lysed and the total insulin content was measured 12 (Fig. 6). The insulin content was 2.4-fold higher in mRNA 10 islets cultured in the presence of GKA71 than in those GK 8 # cultured in 5 mmol/l glucose alone. Again, the results expression 6 for GKA71 were similar to those for islets cultured in 4 Relative Relative 25 mmol/l glucose. 2 0 WT He KO GK protein is increased in rat and mouse islets with Genotype chronic activation of GK by GKA71 Figure 3 Effect of chronic treatment with GKA71 (10 mmol/l) on mRNA levels of insulin and GK in gkwt/wt and gkdel/wt mouse islets. Although GK mRNA levels were not changed by chronic Islets were cultured in 8 mmol/l glucose alone (black bars); culture with either GKA71 or with 25 mmol/l glucose, 10 mmol/l GKA71 (striped bars) or 25 mmol/l glucose (grey bars). GK is known to be regulated at the protein level, rather (A) Insulin mRNA. (B) GK mRNA. mRNA levels determined by single-gene Taqman analysis (meanGS.E.M. of seven or eight than at the gene level. Therefore, western blotting was samples, each with 40 islets). *P!0.05, ***P!0.001 versus performed to quantify GK protein in chronically 8 mmol/l glucose alone. #P!0.05 for the comparison of gkwt/wt treated islet samples. In isolated rat islets, chronic with gkdel/wt. www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 59–67

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ABP < 0·001 insulin expression and production, and also on the 0·6 1·0 *** 0·5 P < 0·001 expression of other key genes, to those induced by high ***

mRNA 0·8 0·4 glucose concentration. Therefore, these novel findings 0·3 0·6 again demonstrate the ‘glucose-like’ nature of GKAs, con- 0·2 GLUT2 0·4 expression

expression sistent with what is already known about the mechanism 0·1 0·2 of action of these molecules (Leighton et al.2005). Relative insulin mRNA Relative 0·0 Relative 0·0 5 5 + GKA71 25 5 5 + GKA71 25 In preliminary experiments, GSIS of islets from Glucose (mmol/l) Glucose (mmol/l) C57Bl/6J mice, and of Wistar rats, was assessed CD following culture for 72 h. These experiments indicated 1·0 1·6 P < 0·001 that while rat islets retained a small GSIS response 0·8 *** 1·2 P < 0·001 mRNA

mRNA after 72 h in 5 mmol/l glucose, mouse islets required 0·6 *** 0·8 PC1 8 mmol/l glucose to maintain a similar level of 0·4 IAPP expression expression functionality. Therefore, all subsequent mouse islet 0·2 0·4

Relative Relative chronic culture experiments were performed using Relative Relative 0·0 0·0 5 5 + GKA71 25 5 5 + GKA71 25 8 mmol/l glucose. The insulin mRNA response to Glucose (mmol/l) Glucose (mmol/I) GKA71 on C57Bl/6J mouse islets cultured in Figure 4 Chronic treatment of rat islets with GKA71 (10 mmol/l) 5 mmol/l glucose was qualitatively similar to that in increases mRNA levels of key b-cell genes. (A) Insulin mRNA. gkwt/wt islets cultured in 8 mmol/l glucose; however, (B) GLUT2 mRNA. (C) PC1 mRNA. (D) IAPP mRNA. mRNA levels determined by single-gene Taqman analysis (meanGS.E.M. A of six samples, each with 30 islets). ***P!0.001 versus 5 mmol/l 3 mmol/l 15 mmol/l 3 mmol/l KCl glucose alone. 300 5 mol/l glucose 5 mol/l glucose + GKA71 islets cultured in the same glucose concentration in the 250 25 mol/l glucose

absence of GKA. In these experiments, we used insulin 200 mRNA as a measure of insulin biosynthesis rather than 150 measuring insulin biosynthesis directly. However, as there was a corresponding increase in insulin content 100 and functional response of the islets, it is reasonable to 50 Insulin (pg/islet per hour) conclude that the increase in insulin mRNA induced by 0 0 1020304050607080 GKA71 is translated to an increase in insulin pro- Time (min) duction. The effect on insulin mRNA has been BC demonstrated consistently in isolated islets from 1000 *** *** 3000 ** * normal rats and mice and islets from mice with 800 heterozygous deletion of the GK gene. 600 2000 Having established that GKA increased insulin 400

Insulin AUC Insulin AUC 1000 mRNA, we carried out experiments to explore the 200 mechanism for these changes. The TLDA analysis 0 0 highlighted a subset of key b-cell genes that were 5 5 + GKA71 25 5 5 + GKA71 25 Glucose (mmol/l) Glucose (mmol/l) induced following chronic treatment with GKA71. All these genes (insulin, GLUT2, IAPP and PC1)are D 800 ** ** regulated by PDX1 (Watada et al. 1996, Campbell & 600 Macfarlane 2002), suggesting that GKA71 influences the expression of these important proteins via PDX1. 400

All these genes were upregulated in both mouse and rat Insulin AUC 200

islets. However, although PDX1 expression increased in 0 normal mouse islets, this was not seen in rat islet 5 5 + GKA71 25 studies. It is possible that the effects on gene expression Glucose (mmol/l) are the result of a modulation of the activity of PDX1, Figure 5 Chronic treatment of rat islets with GKA71 (10 mmol/l) rather than a change in expression. In addition to the results in increased GSIS. (A) Insulin secretion profile of rat islets previously cultured for 72 h in 5 mmol/l glucose alone (black regulatory effects of PDX1 on these key b-cell genes, squares), 5 mmol/l glucoseC10 mmol/l GKA71 (open circles) or MafA is known to be an important regulator of their 25 mmol/l glucose (black triangles). Values are meanGS.E.M. for expression (Aramata et al. 2005). We did attempt to three perifusion channels (50 islets per channel). (B) AUC for first- measure MafA mRNA in disease model islets; however, phase insulin secretion (30–39 min). (C) AUC for second-phase insulin secretion (40–76.5 min). (D) AUC for KCl-induced insulin the expression level was too low to be detected. release (76.5–81 min). For B, C and D, values are expressed as We have demonstrated that treatment with GKA71 in meanGS.E.M.*P!0.05, **P!0.01, ***P!0.001 versus 5 mmol/l basal glucose concentration produces similar effects on glucose alone. Data are representative of nZ3 experiments.

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70 P = 0·008 P = 0·002 Recently, the effect of chronic GKA administration was 60 ** ** evaluated in wild-type mice and mice with a b-cell- 50 specific deletion of GK (Nakamura et al. 2009). In both 40 genotypes, b-cell mass was unchanged after 20 weeks’ 30 20 treatment, but it was less clear whether the potential for Insulin (ng/islet) 10 GKA to stimulate GSIS was retained. Relative insulin 0 secretion increased in response to feeding, although this 5 5 + GKA71 25 could potentially have resulted from improved insulin Glucose (mmol/l) sensitivity. Although GSIS tended to be greater in islets Figure 6 Chronic treatment of rat islets with GKA71 (10 mmol/l) from GKA-treated mice when evaluated ex vivo, this study increases the total insulin content. Values are the meanGS.E.M.of six samples, each of three islets. **P!0.01 versus 5 mmol/l did not investigate whether insulin biosynthesis had glucose alone. been maintained. Our own results clearly support the conclusion that chronic exposure to GKA71 results in a larger fold stimulation was observed in 5 mmol/l islets with an increased GSIS response, and indeed glucose. This may result from lower basal insulin provide evidence that insulin content and insulin expression in the lower glucose concentration; however, secretory capacity are significantly improved. It should, the two conditions were not directly compared. however, be noted that in our studies, all end points are In addition to investigating the effect of GKA71 on compared following 72 h of culture; therefore, our the regulation of insulin biosynthesis, and expression of findings do not enable us to differentiate between action other key b-cell genes, these experiments were designed of GKA71 to prevent loss of function in low glucose to study the effect of GKA71 on GK regulation. GK is culture and action to increase function during sustained known to be regulated post-translationally in the b-cell treatment. Study of the kinetics of changes in insulin biosynthesis and GSIS induced by GKA would clarify this. rather than at the expression level (Chen et al. 1994). It Although chronic administration of GKA did not is clear from our results that there is no effect of chronic change b-cell mass in the above study, sustained exposure treatment with GKA71 on GK mRNA. However, using of INS1 cells to GKA promoted b-cell replication and, in immunoblotting to study GK protein levels revealed addition, one compound, GKA50, reduced apoptosis that in rat islets, GK protein was elevated in islets treated (Nakamura et al.2009, Wei et al. 2009). Further evidence to with GKA71. Again, this is consistent with the increase support the lack of detrimental effect on b-cell mass comes induced by high glucose. Results obtained in rat islets from demonstrationthata short (3 days)treatment invivo, were reproduced in both the wild-type and the when glucose concentration was high, increased the rate heterozygous GK knockout mouse islets. Although we of b-cell proliferation (Nakamura et al. 2009). Taken did not measure GK activity of islets, we would predict together, these two studies, with the current results, that chronic treatment with GKA would increase suggest that GKAs have the potential to at least prevent enzyme activity. This would be consistent with the and at best reverse the three key elements of glucotoxicity; effects of GKAs measured in experiments assessing direct activation of the isolated GK enzyme, albeit in A C MWt MWt hepatocytes (Brocklehurst et al. 2004). 50 kDa 50 kDa Exposure of islets to very high concentrations of 123 123456 glucose is eventually detrimental to function, mani- B D 60 8 mmol/l glucose 8 mmol/l glucose + GKA71 fested as abnormal insulin gene expression, insulin 50 160 25 mmol/l glucose content and defective GSIS (Robertson et al. 2003). 40 120 Elements of this b-cell glucotoxicity have been demon- 30 80 strated in vivo in rodents (Leahy et al. 1986, Jonas et al. 20 Band density 40 10

1999, Laybutt et al. 2002). Furthermore, in vivo, chronic Band density (pixel count/unit area) (pixel 0 0 hyperglycaemia may also deplete b-cell mass (Pick et al. 5 25 5 + GKA71 count/unit area) (pixel WT He KO 1998). Therefore, it is a concern that sustained Glucose (mmol/l) Genotype activation of GK via GKA may mimic the effects of Figure 7 Chronic treatment of rodent islets with GKA71 (10 mmol/l) chronic high glucose and contribute to glucotoxicity, increases the amount of GK protein. Western blots were performed Z via reduction of b-cell mass and by depleting the on cell lysates from rat islets (A: immunoblot lane 1 5mmol/l glucose, lane 2Z25 mmol/l glucose and lane 3Z5 mmol/l glucose available pool of readily secretable insulin. However, GK plus 10 mmol/l GKA71, B: densitometry, 5 and 25 denote glucose may in fact be important to maintain both GSIS and concentration in mmol/l with 5CGKA being 5 mmol/l glucose plus b-cell mass: mice heterozygous for b-cell-specific 10 mmol/l GKA71) or gkwt/wt and gkdel/wt mouse islets (C: immunoblot Z Z deletion of GK fail to adequately expand b-cell mass –lanes1and4 8 mmol/l glucose, lanes 2 and 5 8 mmol/l glucose plus 10 mmol/l GKA71 and lanes 3 and 6Z25 mmol/l glucose – lanes on a high-fat diet (Terauchi et al. 2007) and have 1–3Zgkwt/wt lanes 4–6Zgkdel/wt,D:densitometry).Dataare reduced GSIS (Aizawa et al. 1996, Gorman et al. 2008). representative of nZ2 experiments with each species. www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 59–67

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impaired proliferation, increased apoptosis and impaired References GSIS. Given the effectiveness of GKAs to decrease blood glucose, it may not be physiologically relevant to consider Ahren B 2005 Type 2 diabetes, insulin secretion and beta-cell mass. sustained effects at high glucose concentrations. Current Molecular Medicine 5 275–286. (doi:10.2174/15665240 The mechanism of action of GKAs clearly dis- 53766004) Aizawa T, Asanuma N, Terauchi Y, Suzuki N, Komatsu M, Itoh N, tinguishes them from sulphonylureas. We have pre- Nakabayashi T, Hidaka H, Ohnota H, Yamauchi K et al. 1996 viously demonstrated the difference between GKA50 Analysis of the pancreatic beta cell in the mouse with targeted 2C and tolbutamide on cytosolic Ca rises in MIN6 cells disruption of the pancreatic beta cell-specific glucokinase gene. and isolated rodent islets (Johnson et al. 2007). Although Biochemical and Biophysical Research Communications 229 460–465. both agents induce insulin secretion, only the GKA does (doi:10.1006/bbrc.1996.1826) so in a glucose-dependent and ‘glucose-like’ manner. Aramata S, Han SI, Yasuda K & Kataoka K 2005 Synergistic activation of the insulin gene promoter by the beta-cell enriched transcription Administration of sulphonylureas results in eventual loss factors MafA, Beta2, and Pdx1. Biochimica et Biophysica Acta 1730 of insulin secretory capacity (Ball et al. 2004, Urban & 41–46. (doi:10.1016/j.bbaexp.2005.05.009) Panten 2005), whereas in contrast, the experiments Bali D, Svetlanov A, Lee HW, Fusco-DeMane D, Leiser M, Li B, described in this study indicate that GKAs are able to Barzilai N, Surana M, Hou H & Fleischer N 1995 Animal model for stimulate the cellular mechanisms to increase insulin maturity-onset diabetes of the young generated by disruption of the mouse glucokinase gene. Journal of Biological Chemistry 270 production, in addition to stimulating insulin secretion. 21464–21467. (doi:10.1074/jbc.270.37.21464) Type 2 diabetes in man results from failure of insulin Ball AJ, McCluskey JT, Flatt PR & McClenaghan NH 2004 secretion to compensate for the increased insulin Chronic exposure to tolbutamide and glibenclamide impairs demand that occurs as insulin resistance develops. insulin secretion but not transcription of K(ATP) channel Reduced insulin secretory capacity results from declining components. Pharmacological Research 50 41–46. (doi:10.1016/ b-cell mass (Butler et al.2003) and also reduced secretory j.phrs.2003.12.001) Brocklehurst KJ, Payne VA, Davies RA, Carroll D, Vertigan HL, capacity of individual b-cells (Ahren 2005). We have Wightman HJ, Aiston S, Waddell ID, Leighton B, Coghlan MP et al. demonstrated that chronic GK activation improves or 2004 Stimulation of hepatocyte glucose metabolism by novel small sustains insulin secretory capacity in vitro; studies in molecule glucokinase activators. Diabetes 53 535–541. (doi:10.2337/ human islets and ultimately in diabetic patients are diabetes.53.3.535) required to support this result. Taken together with the Brunstedt J & Chan SJ 1982 Direct effect of glucose on the maintenance of b-cell mass, these results reinforce the preproinsulin mRNA level in isolated pancreatic islets. Biochemical and Biophysical Research Communications 106 1383–1389. attractive profile of GKAs as a therapy for type 2 diabetes. (doi:10.1016/0006-291X(82)91267-0) Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA & Butler PC 2003 Beta-cell deficit and increased beta-cell apoptosis in humans with Supplementary data type 2 diabetes. Diabetes 52 102–110. (doi:10.2337/diabetes.52.1.102) Campbell SC & Macfarlane WM 2002 Regulation of the pdx1 gene This is linked to the online version of the paper at http://dx.doi.org/ promoter in pancreatic beta-cells. Biochemical and Biophysical 10.10.1530/JME-10-0157. Research Communications 299 277–284. (doi:10.1016/S0006- 291X(02)02633-5) Chen C, Hosokawa H, Bumbalo LM & Leahy JL 1994 Regulatory Declaration of interest effects of glucose on the catalytic activity and cellular content of glucokinase in the pancreatic beta cell. Study using cultured rat All authors of this study were employees of AstraZeneca plc at the time islets. Journal of Clinical Investigation 94 1616–1620. (doi:10.1172/ the work was performed. JCI117503) Christesen HB, Jacobsen BB, Odili S, Buettger C, Cuesta-Munoz A, Hansen T, Brusgaard K, Massa O, Magnuson MA, Shiota C et al. 2002 The second activating glucokinase mutation (A456V): Funding implications for glucose homeostasis and diabetes therapy. Diabetes 51 1240–1246. (doi:10.2337/diabetes.51.4.1240) This research was solely funded by AstraZeneca plc. Coghlan M & Leighton B 2008 Glucokinase activators in diabetes management. Expert Opinion on Investigational Drugs 17 145–167. (doi:10.1517/13543784.17.2.145) Acknowledgements Coope GJ, Atkinson AM, Allott C, McKerrecher D, Johnstone C, Pike KG, Holme PC, Vertigan H, Gill D, Coghlan MP et al. 2006 We thank the Alderley Park breeding unit and biological services for Predictive blood glucose lowering efficacy by glucokinase activators provision of animals and harvesting of pancreas. We also thank the in high fat fed female Zucker rats. British Journal of Pharmacology 149 team who assisted with islet isolations; Anna Marley, Tracy Gorman, 328–335. (doi:10.1038/sj.bjp.0706848) Claire Dunkley, Nicky James, Jackie Pierce and Linda Ha¨rndahl; and Cuesta-Munoz AL, Huopio H, Otonkoski T, Gomez-Zumaquero JM, Alison Davies for her assistance with the Taqman analysis. We also Nanto-Salonen K, Rahier J, Lopez-Enriquez S, Garcia-Gimeno MA, thank Jonathan Bright and Elizabeth Mills for the statistical analysis, Sanz P, Soriguer FC et al. 2004 Severe persistent hyperinsulinemic and Rachel Mayers for assistance with preparation of the manuscript. hypoglycemia due to a de novo glucokinase mutation. Diabetes 53 Finally, we thank Mark Dunne for his initial comments on these data 2164–2168. (doi:10.2337/diabetes.53.8.2164) and Brendan Leighton, Matthew Coghlan and Andrew Charles for Docherty K & Clark AR 1994 Nutrient regulation of insulin gene their critical examination of this manuscript. expression. FASEB Journal 8 20–27.

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Efanov AM, Barrett DG, Brenner MB, Briggs SL, Delaunois A, Leibiger B, Wahlander K, Berggren PO & Leibiger IB 2000 Glucose- Durbin JD, Giese U, Guo H, Radloff M, Gil GS et al. 2005 A novel stimulated insulin biosynthesis depends on insulin-stimulated glucokinase activator modulates pancreatic islet and hepatocyte insulin gene transcription. Journal of Biological Chemistry 275 function. Endocrinology 146 3696–3701. (doi:10.1210/en.2005-0377) 30153–30156. (doi:10.1074/jbc.M005216200) Futamura M, Hosaka H, Kadotani A, Shimazaki H, Sasaki K, Ohyama S, Leighton B, Atkinson A & Coghlan MP 2005 Small molecule Nishimura T, Eiki J & Nagata Y 2006 An allosteric activator of glucokinase activators as novel anti-diabetic agents. Biochemical glucokinase impairs the interaction of glucokinase and glucokinase Society Transactions 33 371–374. (doi:10.1042/BST0330367) regulatory protein and regulates glucose metabolism. Journal of Matschinsky FM 1990 Glucokinase as glucose sensor and metabolic Biological Chemistry 281 37668–37674. (doi:10.1074/jbc. signal generator in pancreatic beta-cells and hepatocytes. Diabetes 39 M605186200) 647–652. (doi:10.2337/diabetes.39.6.647) Glaser B, Kesavan P, Heyman M, Davis E, Cuesta A, Buchs A, Stanley CA, Matschinsky FM, Magnuson MA, Zelent D, Jetton TL, Doliba N, Han Y, Thornton PS, Permutt MA, Matschinsky FM et al. 1998 Familial Taub R & Grimsby J 2006 The network of glucokinase-expressing hyperinsulinism caused by an activating glucokinase mutation. New cells in glucose homeostasis and the potential of glucokinase activators for diabetes therapy. Diabetes 55 1–12. (doi:10.2337/ England Journal of Medicine 338 226–230. (doi:10.1056/ diabetes.55.01.06.db05-0926) NEJM199801223380404) Nakamura A, Terauchi Y, Ohyama S, Kubota J, Shimazaki H, Gloyn AL, Noordam K, Willemsen MA, Ellard S, Lam WW, Nambu T, Takamoto I, Kubota N, Eiki J, Yoshioka N et al. 2009 Campbell IW, Midgley P, Shiota C, Buettger C, Magnuson MA Impact of small-molecule glucokinase activator on glucose et al. 2003 Insights into the biochemical and genetic basis of metabolism and beta-cell mass. Endocrinology 150 1147–1154. glucokinase activation from naturally occurring hypoglycemia (doi:10.1210/en.2008-1183) mutations. Diabetes 52 2433–2440. (doi:10.2337/diabetes.52.9. Pick A, Clark J, Kubstrup C, Levisetti M, Pugh W, Bonner-Weir S & 2433) Polonsky KS 1998 Role of apoptosis in failure of beta-cell mass Gorman T, Hope DC, Brownlie R, Yu A, Gill D, Lofvenmark J, Wedin M, compensation for insulin resistance and beta-cell defects in the Mayers RM, Snaith MR & Smith DM 2008 Effect of high-fat diet on male Zucker diabetic fatty rat. Diabetes 47 358–364. (doi:10.2337/ glucose homeostasis and gene expression in glucokinase knockout diabetes.47.3.358) mice. Diabetes, Obesity and Metabolism 10 885–897. (doi:10.1111/j.1463- Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, 1326.2007.00819.x) Shelton KD, Lindner J, Cherrington AD & Magnuson MA 1999 Grimsby J, Sarabu R, Corbett WL, Haynes NE, Bizzarro FT, Coffey JW, Dual roles for glucokinase in glucose homeostasis as determined Guertin KR, Hilliard DW, Kester RF, Mahaney PE et al. 2003 by liver and pancreatic beta cell-specific gene knock-outs using Allosteric activators of glucokinase: potential role in diabetes cre recombinase. Journal of Biological Chemistry 274 305–315. therapy. Science 301 370–373. (doi:10.1126/science.1084073) (doi:10.1074/jbc.274.1.305) Grupe A, Hultgren B, Ryan A, Ma YH, Bauer M & Stewart TA 1995 Robertson RP, Harmon J, Tran PO, Tanaka Y & Takahashi H 2003 Transgenic knockouts reveal a critical requirement for pancreatic Glucose toxicity in beta-cells: type 2 diabetes, good radicals beta cell glucokinase in maintaining glucose homeostasis. Cell 83 gone bad, and the glutathione connection. Diabetes 52 581–587. 69–78. (doi:10.1016/0092-8674(95)90235-X) (doi:10.2337/diabetes.52.3.581) Hattersley AT, Turner RC, Permutt MA, Patel P, Tanizawa Y, Chiu KC, Schuit FC, Huypens P, Heimberg H & Pipeleers DG 2001 Glucose O’Rahilly S, Watkins PJ & Wainscoat JS 1992 Linkage of type 2 sensing in pancreatic beta-cells: a model for the study of other diabetes to the glucokinase gene. Lancet 339 1307–1310. (doi:10. glucose-regulated cells in gut, pancreas, and hypothalamus. Diabetes 1016/0140-6736(92)91958-B) 50 1–11. (doi:10.2337/diabetes.50.1.1) Johnson D, Shepherd RM, Gill D, Gorman T, Smith DM & Dunne MJ Terauchi Y, Sakura H, Yasuda K, Iwamoto K, Takahashi N, Ito K, Kasai 2007 Glucose-dependent modulation of insulin secretion and H, Suzuki H, Ueda O & Kamada N 1995 Pancreatic beta-cell-specific intracellular calcium ions by GKA50, a glucokinase activator. targeted disruption of glucokinase gene. diabetes mellitus due to Diabetes 56 1694–1702. (doi:10.2337/db07-0026) defective insulin secretion to glucose. Journal of Biological Chemistry 270 30253–30256. (doi:10.1074/jbc.270.51.30253) Jonas JC, Sharma A, Hasenkamp W, Ilkova H, Patane G, Laybutt R, Terauchi Y, Takamoto I, Kubota N, Matsui J, Suzuki R, Komeda K, Bonner-Weir S & Weir GC 1999 Chronic hyperglycemia triggers Hara A, Toyoda Y, Miwa I, Aizawa S et al. 2007 Glucokinase and IRS-2 loss of pancreatic beta cell differentiation in an animal model of are required for compensatory beta cell hyperplasia in response diabetes. Journal of Biological Chemistry 274 14112–14121. to high-fat diet-induced insulin resistance. Journal of Clinical (doi:10.1074/jbc.274.20.14112) Investigation 117 246–257. (doi:10.1172/JCI17645) Kamata K, Mitsuya M, Nishimura T, Eiki J & Nagata Y 2004 Structural Urban KA & Panten U 2005 Selective loss of glucose-induced basis for allosteric regulation of the monomeric allosteric enzyme amplification of insulin secretion in mouse pancreatic islets human glucokinase. Structure 12 429–438. (doi:10.1016/j.str.2004. pretreated with sulfonylurea in the absence of fuels. Diabetologia 48 02.005) 2563–2566. (doi:10.1007/s00125-005-0030-5) Khaldi MZ, Guiot Y, Gilon P, Henquin JC & Jonas JC 2004 Increased Watada H, Kajimoto Y, Umayahara Y, Matsuoka T, Kaneto H, Fujitani Y, glucose sensitivity of both triggering and amplifying pathways of Kamada T, Kawamori R & Yamasaki Y 1996 The human glucokinase insulin secretion in rat islets cultured for 1 wk in high glucose. gene beta-cell-type promoter: an essential role of insulin promoter American Journal of Physiology. Endocrinology and Metabolism 287 factor 1/PDX-1 in its activation in HIT-T15 cells. Diabetes 45 E207–E217. (doi:10.1152/ajpendo.00426.2003) 1478–1488. (doi:10.2337/diabetes.45.11.1478) Laybutt DR, Sharma A, Sgroi DC, Gaudet J, Bonner-Weir S & Weir GC Wei P, Shi M, Barnum S, Cho H, Carlson T & Fraser JD 2009 Effects of 2002 Genetic regulation of metabolic pathways in beta-cells glucokinase activators GKA50 and LY2121260 on proliferation and disrupted by hyperglycemia. Journal of Biological Chemistry 277 apoptosis in pancreatic INS-1 beta cells. Diabetologia 52 2142–2150. 10912–10921. (doi:10.1074/jbc.M111751200) (doi:10.1007/s00125-009-1446-0) Leahy JL, Cooper HE, Deal DA & Weir GC 1986 Chronic hyperglycemia is associated with impaired glucose influence on insulin secretion. A study in normal rats using chronic in vivo Received in final form 24 April 2011 glucose infusions. Journal of Clinical Investigation 77 908–915. Accepted 13 May 2011 (doi:10.1172/JCI112389) Made available online as an Accepted Preprint 13 May 2011

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