Control of beta cell function and proliferation in mice stimulated by small-molecule glucokinase activator under various conditions
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We investigated changes in the expression of genes involved in beta cell function and proliferation in mouse islets stimulated with glucokinase activator (GKA) in order to elucidate the mechanisms by which GKA stimulates beta cell function and proliferation.
Islets isolated from mice were used to investigate changes in the expression of genes related to beta cell function and proliferation stimulated by GKA. In addition, Irs2 knockout (Irs2−/−) mice on a high-fat diet or a high-fat diet containing GKA were used to investigate the effects of GKA on beta cell proliferation in vivo.
In wild-type mice, Irs2 and Pdx1 expression was increased by GKA. In Irs2−/− mice, GKA administration increased the glucose-stimulated secretion of insulin and Pdx1 expression, but not beta cell proliferation. It was particularly noteworthy that oxidative stress inhibited the upregulation of the Irs2 and Pdx1 genes induced by GKA. Moreover, whereas neither GKA alone nor exendin-4 alone upregulated the expression of Irs2 and Pdx1 in the islets of db/db mice, prior administration of exendin-4 to the mice caused GKA to increase the expression of these genes.
GKA-stimulated IRS2 production affected beta cell proliferation but not beta cell function. Oxidative stress diminished the effects of GKA on the changes in expression of genes involved in beta cell function and proliferation. A combination of GKA and an incretin-related agent might therefore be effective in therapy.
KeywordsBeta cell proliferation Glucokinase activator IRS2 Oxidative stress
cAMP-responsive element-binding protein
Glucokinase is the predominant enzyme involved in glucose phosphorylation in beta cells and hepatocytes, and it plays an important role as a glucose sensor in beta cells and as a regulator of glucose metabolism in the liver [1, 2]. In addition, glucokinase plays a pivotal role in regulating not only beta cell function, but also beta cell mass [3, 4].
Since the report by Grimsby et al in 2003 , several glucokinase activators (GKAs) have been developed, and these have been shown to lower blood glucose in several animal models of type 2 diabetes [5, 6, 7, 8, 9, 10, 11, 12]. In a study of beta cell function, it was reported that a GKA stimulated insulin secretion in a Ca2+-dependent manner in rodent islets and MIN6 cells , and we and others have reported that GKAs promoted beta cell proliferation and increased production of IRS2 [11, 14], which is critically required for beta cell growth and survival [3, 15, 16, 17]. However, the exact mechanisms by which GKAs stimulate beta cell function and proliferation are largely unknown.
Single- and multiple-dose placebo-controlled studies in human have recently reported that GKAs reduce the fasting and postprandial glucose levels of patients with type 2 diabetes and of healthy adults [18, 19]. Notably, however, another GKA, MK-0941, led to improvements in glycaemic control that were not sustained . Therefore, a better understanding of the underlying mechanism is needed to determine whether glucokinase activation with GKAs is a feasible treatment goal for individuals with type 2 diabetes.
In the present study, we first investigated changes in the expression of genes involved in beta cell function and proliferation in mouse islets stimulated with GKA in order to elucidate the mechanisms by which GKA stimulated beta cell function and proliferation. We then explored therapeutic strategies by which GKA might work more effectively.
A GKA (3-[(1S)-2-hydroxy-1-methylethoxy]-5-[4-(methylsulfonyl)phenoxy]-N-1,3-thiazol-2-yl benzamide) was prepared by Tsukuba Research Institute, Banyu Pharmaceutical, Tokyo, Japan, as previously described .
Irs2−/− mice were generated as described elsewhere  and were then backcrossed with C57Bl/6J mice more than nine times. Both wild-type and Irs2−/− male mice were fed standard chow until 8 weeks of age, when they were given free access to either the standard chow, a high-fat (HF) diet, or an HF diet containing GKA. To evaluate the effect of GKA on glucose metabolism in vivo more thoroughly, wild-type and Irs2−/− mice were divided into four groups: wild-type mice fed the HF diet, wild-type mice fed the HF diet containing 0.04% GKA, Irs2−/− mice fed the HF diet, and Irs2−/− mice fed the HF diet containing 0.04% GKA. Five-week-old male db/db mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). When they were 6 weeks of age, they were intraperitoneally injected with normal saline or exendin-4 (100 μg/kg; Sigma-Aldrich, Tokyo, Japan) once daily for 2 weeks. The mice were housed under a 12-h light/dark cycle. The animals were maintained in accordance with standard animal care procedures based on the institutional guidelines.
Standard chow (MF; Oriental Yeast, Tokyo, Japan) and an HF diet (High Fat Diet 32; Clea Japan, Tokyo, Japan) were used. GKA was administrated in the form of a 0.04% (wt/wt) admixture to the HF diet as previously described .
Glucose tolerance test
Mice were fasted for 4 h before the study, and then orally loaded with a 1.5 mg/g body weight dose of glucose. Blood glucose was measured with a Glutest Neo portable glucose meter (Sanwa Chemical, Nagoya, Japan).
Immunohistochemical analysis to estimate beta cell mass
Isolated pancreases were immersion-fixed in 10% formalin at 4°C overnight. Tissue was then routinely processed for paraffin embedding, and 5 μm sections mounted on glass slides were immunostained with rabbit anti-human insulin (diluted 1:1,000) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The area of the beta cells was calculated with WinROOF software (Mitani, Tokyo, Japan) as described elsewhere . Approximately 100 islets per mouse were counted in each group.
Analysis of BrdU incorporation
Wild-type and Irs2−/− mice on the HF diet for 10 weeks were divided into two groups: an HF diet group, and a group given 0.04% GKA mixed into the HF diet. After 3 days, the mice were intraperitoneally injected with BrdU (Nacalai Tesque, Kyoto, Japan), and the pancreases were removed 6 h later. Immunohistochemical detection of BrdU was performed with a commercial kit (BD Biosciences, Franklin Lakes, NJ, USA). Approximately 100 islets per mouse were counted in each group.
Islets were isolated by using liberase RI (Roche Diagnostics, Indianapolis, IN, USA) or collagenase XI (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's instructions, as described elsewhere [3, 11].
Analysis of insulin secretion
Insulin secretion was measured after culturing islets overnight in RPMI 1640 medium containing 11 mmol/l glucose supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St Louis, MO, USA). Ten islets were incubated at 37°C for 1.5 h in Krebs–Ringer bicarbonate buffer containing 5.6 or 22 mmol/l glucose in the absence or presence of GKA. The insulin concentration of the assay buffer was measured with an insulin ELISA kit (Morinaga, Yokohama, Japan).
Real-time quantitative PCR
Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and used as the starting material for complementary DNA (cDNA) preparation. cDNA was synthesised by using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA), and TaqMan quantitative PCR was performed with the ABI Prism 7500 PCR instrument (Applied Biosystems).
Western blot analysis
The anti-phospho [Ser133] cAMP-responsive element-binding protein (CREB), total CREB, and cyclin D2 antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-IRS2 antibody was purchased from Upstate (Temecula, CA, USA). Protein was prepared from more than 100 islets pooled from several mice in the same group, and 20 μg of protein samples were applied to the gel. Protein bands were visualised with the ECL Plus Western Blotting Detection System (GE Healthcare, Amersham, UK).
Results are expressed as mean ± SE (n). Differences between two groups were analysed for statistical significance by Student's t test. Individual comparisons between more than two groups were assessed by the post hoc Fisher's PLSD test. A p value <0.05 was considered statistically significant.
Mechanism of the upregulation of Irs2 expression in response to GKA-induced glucokinase activation
As it has been reported that glucose treatment of isolated islets potently increases Irs2 expression via glucose metabolism and Ca2+ influx , we examined changes in Irs2 mRNA levels in response to GKA administration or high-glucose stimulation under several conditions to identify the mechanism of Irs2 upregulation. First, we evaluated Irs2 mRNA expression in response to the GKA and sulfonylureas. Real-time quantitative PCR showed that Irs2 mRNA was significantly increased with 22 mmol/l glucose in comparison with 5.6 mmol/l glucose (Fig. 1c). The GKA also significantly increased Irs2 mRNA with 5.6 mmol/l glucose, but neither gliclazide nor glibenclamide upregulated the Irs2 mRNA level (Fig. 1c).
Next, we used the non-metabolisable analogue of glucose, 2-deoxyglucose (2-DG), to investigate whether glucose metabolism was required for GKA-stimulated Irs2 expression. When glucose was replaced by 2-DG, the Irs2 mRNA levels did not increase in response to the GKA or a stimulatory concentration of 2-DG (22 mmol/l; Fig. 1d), indicating that glucose metabolism is necessary for GKA-induced Irs2 expression to occur in islet beta cells.
We also conducted experiments using an L-type calcium channel blocker (nifedipine) and calcineurin inhibitor (tacrolimus). The results showed that both nifedipine (50 μM) and tacrolimus (10 μM) significantly inhibited the upregulation of Irs2 mRNA levels induced by GKA administration or high-glucose stimulation, although the Irs2 mRNA levels remained slightly but significantly increased (Fig. 1e, f). These results indicated that the GKA-induced Irs2 upregulation in islets is at least partly Ca2+-dependent and mediated by calcineurin.
Effect of the GKA on changes in gene expression in isolated islets
As the results of a DNA microarray analysis we previously reported showed decreased expression of Pdpk1 and Ccnd2 in Gck+/− mice in comparison with wild-type mice on the HF diet , we investigated the expression levels of these and other cell-cycle-related genes in the present study. GKA and high-glucose stimulation significantly increased Pdpk1, Ccnd1, Ccnd2 and Ccnd3 mRNA expression, but the expression of Cdk4 and p27 was unaltered (Fig. 2c). Cyclin D2 protein levels were also increased by the GKA and high-glucose stimulation (Fig. 2d). These results suggested an involvement of cell cycle signalling, such as by cyclin D2, in GKA-stimulated beta cell proliferation.
Effect of the GKA on glucose metabolism and beta cell mass in Irs2−/− mice
We used Irs2−/− mice to determine whether IRS2 was required for the therapeutic effects of the GKA, and divided the animals into four groups: wild-type mice fed the HF diet (WT group), wild-type mice fed a diet containing 0.04 % GKA mixed into the HF diet (WT+GKA group), Irs2−/− mice fed the HF diet (IRS2 group), and Irs2−/− mice fed 0.04% GKA mixed into the HF diet (IRS2+GKA group).
Next, to investigate the effect of GKA on beta cell mass, we measured the beta cell mass of the mice. Histological analysis revealed that the area of the beta cells was significantly increased in the wild-type mice in comparison with the Irs2−/− mice (Fig. 3e), consistent with our previous report , but no further increase was observed in response to administering GKA to either genotype of mouse (Fig. 3e).
As we previously reported , the absence of any effect of the GKA on the beta cell mass of the Irs2−/− mice may be attributable to a suppression of beta cell proliferation due to the chronic reduction in ambient blood glucose levels induced by the GKA treatment rather than to the deficiency of IRS2. To investigate this possibility, we evaluated beta cell proliferation after administering the GKA on three consecutive days to mice fed the HF diet for 10 weeks; the results showed a significant decrease in the fed blood glucose level of both the wild-type and Irs2−/− mice shortly after administration of the GKA (data not shown). The BrdU incorporation ratio was significantly increased in the wild-type mice given the GKA for 3 days in comparison with the wild-type mice not given the GKA; this was not, however, seen in the Irs2−/− mice (Fig. 3f, g). These results support the concept that IRS2 could have an effect on beta cell proliferation stimulated by a GKA in vivo.
Effect of the GKA on the beta cell function of Irs2−/− mice
Next, we investigated whether the GKA affected the expression of genes involved in beta cell function in islets isolated from Irs2−/− mice. Pdx1 mRNA significantly increased with 22 mmol/l glucose in comparison with 5.6 mmol/l glucose, and the GKA stimulated Pdx1 mRNA expression at 5.6 mmol/l glucose in the isolated islets of Irs2−/− mice (Fig. 4b). In addition, there were no differences in Pdx1 expression 5.6 mmol/l glucose in wild-type and Irs2−/− mice, and Pdx1 expression was significantly increased with 22 mmol/l glucose or by the GKA in both genotypes of mouse to the same degree (data not shown). The increased Pdx1 expression also paralleled the upregulation of Glut2, Gck, Ins1 and Ins2 levels (Fig. 4b). These results indicated that GKA enhanced beta cell function at the transcriptional level independently of IRS2.
Effect of oxidative stress on GKA-induced Irs2 and Pdx1 expression
Effect of GKA on Irs2 and Pdx1 expression in db/db mice
Effect of exendin-4 on GKA-stimulated Irs2 and Pdx1 expression in db/db mice
The results of the present study yielded three new findings. First, glucokinase activation by the GKA, a glucose-like activator of beta cell metabolism, increased IRS2 production; in addition, GKA-stimulated IRS2 production was able to affect beta cell proliferation, but not beta cell function. Second, the effects of the GKA on the expression of genes involved in beta cell function and proliferation were diminished in islets exposed to exogenous H2O2 and in those from db/db mice. Third, a combination of GKA and an incretin-related agent was effective in upregulating Irs2 and Pdx1 expression in these islets.
GKA increased the phosphorylation of CREB and IRS2 production (Fig. 1a, b), and 2-DG failed to increase Irs2 expression by GKA (Fig. 1d). Both nifedipine and tacrolimus partly, but significantly, inhibited upregulation of Irs2 expression by GKA (Fig. 1e, f). Taken together with the previous study , these results suggest that GKA increased CREB phosphorylation and Irs2 expression in islets via glucose metabolism, Ca2+ influx and, in part, a Ca2+–calcineurin pathway. In contrast, Irs2 expression was not increased by either of the sulfonylureas. Sulfonylureas are known to close ATP-sensitive potassium channels regardless of glucose metabolism, and their closure results in membrane depolarisation, an influx of Ca2+ through voltage-dependent Ca2+ channels, and an increase in cytosolic free Ca2+ concentration, thereby triggering insulin secretion . If that is true, why did the sulfonylureas fail to increase Irs2 expression despite being able to increase the Ca2+ influx? One possible explanation is that the glucose flux per se may also be needed to increase Irs2 expression and lead to beta cell proliferation [3, 22], since the sulfonylurea glibenclamide is unable to increase beta cell proliferation in wild-type mice . We therefore hypothesised that both glucose metabolism and Ca2+ influx are required to increase Irs2 expression and lead to beta cell proliferation. This hypothesis is supported by a report that glibenclamide increases beta cell proliferation in the presence of an increased glucose flux .
The Pdx1 expression level has been reported to be severely reduced in the beta cells of Irs2−/− mice with a certain genetic background , suggesting that IRS2 may directly regulate the expression and function of Pdx1, and thereby maintain beta cell growth and function. However, GKA upregulated the expression of Pdx1 and several downstream genes, Glut2, Gck, Ins1 and Ins2, in the isolated islets of Irs2−/− mice with a C57BL/6 background (Fig. 4b). In regard to this point, Suzuki et al reported finding that Pdx1 expression in Irs2−/− mice is regulated in a strain-dependent manner . Pdx1 expression was not downregulated in our Irs2−/− murine beta cells that had a C57BL/6J background . We therefore assume that GKA increased glucose-stimulated insulin gene transcription independently of IRS2. Since Kushner et al reported that transgenic overexpression of Pdx1 restored beta cell mass in Irs2−/− mice , Pdx1 may play a role in regulating beta cell mass. Nevertheless, because a haploinsufficiency of Pdx1 led to impaired beta cell function, but not to decreased beta cell mass , and because beta cell mass was smaller in our Irs2−/− mice than in wild-type mice despite the Pdx1 expression level being maintained , two different pathways are involved in the stimulation of beta cell function and proliferation by a GKA.
The results of our study have clinical implications. As stated above, MK-0941 lacked durability in glycaemic control . In these patients, disease-related characteristics were a mean baseline HbA1c of 9.0 % (75 mmol/mol), a mean duration of diabetes of 12 years, and a mean insulin glargine (A21Gly,B31Arg,B32Arg human insulin) dose of 45 U/day. These data suggest that the insulin secretion or the beta cells themselves were severely impaired. From these clinical trials, it is possible that a pancreatic effect of GKAs could not be expected when pancreatic beta cells have been impaired. It is well known that the level of 8-hydroxydeoxyguanosine, a marker of oxidative stress, is increased in diabetic patients and independently associated with mean HbA1c ; in addition, increased oxidative stress influences beta cell function, and antioxidant treatment can exert beneficial effects in diabetes, with a preservation of beta cell function . We therefore investigated the effect of oxidative stress on GKA-induced changes in the expression of genes involved in beta cell function and proliferation. As expected, the results showed that oxidative stress inhibited the upregulation of Irs2 and Pdx1 in response to GKA, although the inhibition was prevented by prior administration of an antioxidant (Fig. 5). Moreover, the levels of these molecules were not increased by GKA in islets isolated from db/db mice (Fig. 6).
Although our findings could support the observation that GKA is ineffective in patients with type 2 diabetes whose pancreatic beta cells have been impaired, there are some limitations to our study. First, with regard to the experiment with exogenous H2O2, we could not rule out the possibility that the absence of increasing Irs2 and Pdx1 expression after H2O2 preconditioning was caused by not only impaired mitochondrial function, but also an impaired biological response of these islets independently of GKA activity. It is also possible that Gck expression level might have an effect on the inhibition of upregulation of Irs2 and Pdx1 induced by 5.6 mmol/l glucose plus GKA. Second, with regard to the experiment with the db/db mice, GKA compounds act to reduce glucose levels in the db/db mouse model, as shown with MK-0941 and other agents [8, 33]. The background of the db/db mice or the difference in compounds could influence the effect of GKAs. Thus, greater study of the glucose-lowering effect of the GKA in db/db mice is needed.
In the present study, we also explored the therapeutic strategy by which GKA worked more effectively in these impaired beta cells. Based on the results of this study, we propose that GKA should be used before pancreatic beta cell failure. First, GKA should be used earlier, as diabetes is progressing, since beta cell failure has already progressed before the diagnosis of diabetes is made [34, 35]. The efficacy of long-term GKA therapy should be assessed in patients with mild type 2 diabetes or impaired glucose tolerance. Second, GKA should be used in combination with incretin therapy in case GKA monotherapy is ineffective. The results of our study indicated that GKA stimulated Irs2 and Pdx1 expression in the islets of db/db mice when exendin-4 had been administered in advance (Fig. 7). This finding suggested that combination therapy consisting of a GKA and an incretin may be useful for patients with type 2 diabetes. We previously speculated that the increase in Irs2 expression in the islets of db/db mice in response to the combination of these drugs was due to the reduction of oxidative stress or the additive effect of Ca2+ influx via glucose signalling and cAMP signalling through the human glucagon-like peptide-1 receptors . However, the levels of expression of the genes for the reduced-form NADPH oxidase complex were unchanged by prior administration of exendin-4 under our experimental conditions (Fig. 7e–g). Since Irs2 expression was not increased by exendin-4 alone (Fig. 7c), we assume that exendin-4 amplifies GKA-stimulated calcium signalling rather than imposing it. Further study is needed to test the combination in vivo on glycaemic control and the resultant changes in pancreatic gene transcription.
In conclusion, GKA-stimulated IRS2 production affected beta cell proliferation, but not beta cell function. Oxidative stress was able to prohibit the ability of GKA to change the expression of genes involved in beta cell function and proliferation. A combination of GKA and an incretin-related agent might be effective here. These findings suggest that the GKAs should have outstanding potential for the treatment of diabetes and related disorders.
We thank M. Kaji and E. Sakamoto (Yokohama City University, Yokohama, Japan) for their excellent technical assistance and animal care.
This work was supported in part by a Grant-in-Aid for Scientific Research (B) 19390251 and (B) 21390282 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, a Medical Award from the Japan Medical Association, a Grant-in-Aid from the Japan Diabetes Foundation, a Grant-in-Aid from Novo Nordisk Pharma, a Grant-in-Aid from the Suzuken Memorial Foundation, a Grant-in-Aid from the Naito Foundation, a Grant-in-Aid from the Yamaguchi Endocrine Research Foundation, a Grant-in-Aid from the Uehara Memorial Foundation (to Y. Terauchi) as well as a Grant-in-Aid for Young Scientists (Start-up) 21890213 and (B) 23791040 from the MEXT of Japan, a Grant-in-Aid from Yokohama General Promotion Foundation, and a Grant-in-Aid from Japan Diabetes Foundation (to A. Nakamura).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
All authors conceived and designed the study, and participated in the analysis and interpretation of the data. AN drafted the manuscript and all other authors revised it critically for intellectual content. All authors approved the final version of the paper.
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