Effect of combined treatment with clozapine and meormin on fasng blood glucose, insulin level, and expression of the glucose transporter-2 （GLUT2） in Sprague-Dawley rats

Lan GAO, Gaohua WANG*, Hao LIU, Chaohui YAN

Effect of combined treatment with clozapine and meormin on fasng blood glucose, insulin level, and expression of the glucose transporter-2 （GLUT2） in Sprague-Dawley rats

Lan GAO, Gaohua WANG*, Hao LIU, Chaohui YAN

Background:Anpsychoc medicaons can cause an increase in blood glucose and the development of type II diabetes. Meormin may ameliorate these side effects.

Assess whether or not meormin reduces the abnormalies in glucose metabolism that occur with use of the anpsychoc clozapine.

Methods:Eighteen adult Sprague-Dawley (SD) rats were divided into three groups that were intragastrically administered saline, clozapine (20 mg/kg), or a combinaon of clozapine (20 mg/kg) and meormin (78 mg/kg) for 28 days. Fasng blood glucose was assessed at baseline and every seven days thereaer. The animals were euthanized on the 28thday at whichme aorc blood was obtained to assess blood insulin and C-pepde by radioimmunoassay, and pancreacssue samples were collected and used to determine the expression of the glucose transporter-2 (GLUT2) by real-me polymerase chain reacon (RT-PCR) and Western Blot.

Results:Fasng blood glucose in the clozapine group was significantly higher at the 14th, 21st, and 28thday compared to baseline, but rats receiving clozapine and meormin only had significantly elevated levels on the 14thday of treatment. However, repeated measures ANOVA found no stascally significant differences in blood glucose levels overme between the three groups (p=0.136). Mulple comparison tests found that the mean insulin level in the clozapine+meormin group was significantly lower than the levels in the clozapine and saline groups. There were stascally significant differences in the expression of GLUT2mRNA (clozapine+meormin group ＜ clozapine group ＜ saline group) and in the expression of GLUT2protein (clozapine+meormin group, clozapine group ＜ saline group).

Conclusion:This study found a non-significant increase in fasng blood glucose in SD rats treated with clozapine that was parally counteracted by concurrent administraon of meormin. Rats administered clozapine showed the expected decrease in the expression of GLUT2, but concurrent administraon of meormin and clozapine for 28 days did not show the expected normalizaon of the expression levels of GLUT2.

1. Introducon

A large number of studies have found a strong relaonship between anpsychoc medicaons and impaired glucose metabolism and diabetes. Paents with psychosis who use anpsychocs for long periods have a higher incidence of diabetes compared to the general populaon. Incidence of impaired glucose metabolism varies widely across anpsychocs with different chemical structures.[1]According to a retrospecve study in 2012, clozapine and olanzapine were associated with the largest weight gain and the highest risk of abnormal glucose metabolism compared to other atypical anpsychocs.[2]Despite its popularity due to its effecveness in difficult-to-treat paents and its lower incidence of extrapyramidal side-effects, tardive dyskinesia and elevated prolacn than other atypical anpsychocs, concerns about the associaon of clozapine with weight gain, increased blood glucose and type II diabetes[3,4]has limited its clinical use. This issue is of parcular interest in China because of the frequent use of clozapine as monotherapy or in combinaon with other anpsychoc agents in the treatment of schizophrenia.[5]

There have been several research papers about this issue in China. Clinical trials have demonstrated thatmeormin can effecvely reduce the side effects of anpsychocs (parcularly those caused by clozapine and olanzapine) including weight gain, increased blood glucose and other metabolic abnormalies. For example, in 2007, Wu and colleagues[6]reported that meormin can effecvely migate anpsychocs-induced weight gain and abnormal glucose metabolism and several other studies in China report similar findings.[7-11]A meta-analysis reported in 2010[12]found that meormin can reduce weight, body mass index (BMI), waist circumference and insulin resistance, but did not reduce the incidence of diabetes in paents using anpsychocs.

Figure 1. Flowchart of the study

Animal studies can help understand the molecular mechanisms underlying the anpsychoc-induced abnormalies in glucose metabolism and the ameliorang effect of meormin on these sideeffects. One such study by Zhou and colleagues[13]found that clozapine suppressed insulin secreon of isolated pancreac islets in rats and, thus, resulted in increased blood glucose. The current study compares the levels of several variables between three groups of rats administered clozapine, clozapine and meormin or saline for 28 days: fasng blood glucose, insulin, C-pepde, and mRNA and protein levels of the glucose transporter-2 (GLUT2) in pancreac β cells.

2. Methods

The flowchart of the experiment is shown in Figure 1. Animals used in this study were 18 healthy female Sprague-Dawley (SD) rats provided by the Hunan Slack King Laboratory Animal Co., Ltd. The animals were 8-10 weeks old, weighed around 190 g, and were kept in the Experimental Animal Center of Wuhan University (with natural lighng, at a room temperature 20 to 25 °C, and fed ad libitum). Aer one week of acclimaon at the animal center, they were divided into three groups using a random number table (six rats in each group): the clozapine group, the clozapine+meormin group, and the saline group. There were no significant differences in the mean weights of the rats in the three groups.

Based on the methods used by Cheng and colleagues,[14]clozapine was administered at 20 mg/kg/d. The clozapine used in the study was purchased (as 25 mg tablets) from Jiangsu Ruinian Qianjin Pharmaceucal Co., Ltd. (Naonal Drug Approval Number: H32022268). The tablets were pulverized and added to 0.9% saline to achieve a suspension with a concentraon of 3 mg/ml. Based on the methods used by Wu and colleagues,[6]meormin was administered at 78 mg/kg/d. The meormin used was purchased (as 250mg tablets) from Tianjin Pacific Pharmaceucal Co., Ltd. (Naonal Drug Approval Number: H12020797). Pulverized tablets of meormin were added to 0.9% saline to achieve a suspension of 22.3 mg/ml. The suspensions were shaken well before administraon. The 5ml soluon was administered through a 5cm-long stomach tube at 08:00 every day for 28 days.

This study was approved by the instuonal review board of the Experimental Animal Center of Wuhan University (which meets regulaons for the care ofexperimental animals established by the Hubei provincial government). The authors cerfy that the treatment of animals in this study met the ethical standards specified in theGuidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Researchset forth by the American Psychological Associaon.

2.1 Measurements

2.1.2 Insulin and C-pepde

2.1.3 GLUT2mRNA

PCR was conducted in two steps, amplificaon of β-acn and amplificaon of GLUT2.The β-acn PCR amplificaon reacon started with adding β-acn F(10 μM) 0.5 μL, a-Acn R (10 μM) 0.5 μL, dNTP (2.5 mM) 2 μL, Ex Taq 0.25 μL, 10×Ex Taq E buffer 2.5 μL, cDNA 1 μl, and a complementary amount of de-ionized water into a precooled EP tube up to 25 μL. Subsequently, the following reacons were carried out: pre-denaturaon at 94 °C for 4min, denaturaon at 94 °C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 25 s. The above steps were repeated in cycles 30mes. Final extension was then carried out at 72°C for 4min, followed by 4°C for 4 min.

The GLUT2PCR amplificaon reacon started with adding cDNA (10x diluon) 4 μl, GLUT2F (100μM) 0.4μl, GLUT2R (100μM) 0.4μl, SYBR Green / the Flourescein qPCR Master Mix (2X) 10μl, and de-ionized water 5.4 μl sequenally into the reacon system. Subsequently, two cycles of pre-denaturaon at 50°C for 2 min and predenaturaon at 95°C for 10min were completed; and, finally, 40 cycles of denaturaon at 95°C for 30 s and extension at 60°C for 30 s were conducted.

The mean Ct (cycle threshold) values for each reacon were assessed and the difference in mean Ct values (i.e., ΔCt = Ct for GLUT2minus Ct for β-acn) was assessed for each rat. The difference between the mean ΔCt values for the three pairs of groups is then computed (e.g., ΔΔCt[clozapine/saline]= ΔCt [clozapine group] minus ΔCt [saline group]). The expression 2-ΔΔCtis then used as the measure of the relave strength of expression of GLUT2mRNA.

The primers used in these reacons (provided by Genscript Co., Ltd.) are shown in Table 1. The DL2000 DNA Marker and Ex TaqTMwere purchased from TAKARA. And the SYBR Green / the Flourescein qPCR Master Mix (2X) was purchased from Fermentas.

2.1.4 GLUT2protein expression

To transfer to the membrane (wet transfer method), the target strip was cut from the gel and rinsed with dislled water. PVDF (polyvinylidene difluoride) membrane and filter paper were cut to the same size of the PAGE gel. Aer soaking in methanol for a few seconds, the PVDF membrane was immersed in electro-transfer buffer soluon with the filter paper. The material was assembled in the following order: black plate, fiber mat, filter paper, gel, PVDF membrane, filter paper, fiber mat,and white plate; this was then put into a transmembrane electrophoresis box (black plate side facing the negave electrode) at a current of 200 mA for 110 min. The PVDF membrane was subsequently soaked in TBST (Tris-Buffered Saline and Tween 20) containing 5% skim milk powder and put on a shaker at room temperature for 2 hours. Rabbit an-glucose transporter protein-2 (GLUT2) was diluted using the blocking soluon at a rao of 1:500. Then the PVDF membrane was soaked in the GLUT2incubaon soluon, and incubated at 4 °C overnight.

Table 1. Experimental primer sequence and length

Table 2. Comparison of fasng blood glucose levels between the saline group and the other two groups during differentme points

Table 2. Comparison of fasng blood glucose levels between the saline group and the other two groups during differentme points

aGroup differences were not stascally significant (p=0.164) using repeated measure ANOVAbp＜0.05 compared to baseline using paired-t test

GroupnFasng blood glucose (mean [sd], mmol/l)abaseline7thday14thday21stday28thday Clozapine64.42 (0.28)4.61 (0.53)4.90 (0.38)b5.07 (0.55)b4.85 (0.27)bClozapine+meormin64.28 (0.42)4.89 (0.32)4.80 (0.44)b4.57 (0.38)4.55 (0.27) Saline64.37 (0.43)4.35 (0.32)4.70 (0.40)4.77 (0.42)4.32 (0.45)

The PVDF membrane was then thoroughly rinsed 5-6mes (5 min/rinse) with TBST. Blocking soluon was used to dilute associated HRP (horseradish peroxidase)-labeled secondary anbody (1:50000) before the PVDF membrane was soaked in this secondary anbody incubaon liquid. Incubaon was carried out at room temperature on a shaker for 2 hours. The PVDF membrane was then again thoroughly rinsed 5-6mes (5 min/rinse) with TBST. An appropriate amount of ECL (enhanced chemiluminescence) substrate soluon was added to each membrane, and then the membrane was incubated a few minutes. When fluorescent bands became visible, excess substrate liquid was extracted with filter paper, and the membrane was covered with a plasc wrap. Subsequently, x-ray films of the membrane were scanned and gradaon analysis conducted. The grayscale value of the GLUT2protein divided by the grayscale value of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein was the measure of the relave expression level of the GLUT2protein.

The materials for this Western Blot analysis were obtained from the following suppliers. GAPDH anbody was purchased from Hangzhou Xianzhi Biological Co., Ltd. Rabbit an-glucose transporter-2 (GLUT2) was purchased from Abcam. HRP-conjugated goat an-rabbit secondary anbody was purchased from Wuhan Boster Biological Engineering Co., Ltd. The PVDF membrane was purchased from Millipore Corporaon. ECL substrate soluon was purchased from Thermo. RIPA lysis buffer and BCA protein concentraon assay kit were purchased from Beyome. X-ray film was purchased from Kodak.

3. Results

As shown in Table 2, in the clozapine group the levels of fasng blood glucose at the 14th(t=3.19,df=5,p=0.024), 21st(t=3.81,df=5,p=0.013), and 28thday (t=3.31,df=5,p=0.021) of the study were significantly higher than at baseline. Similarly, in the clozapine+meormin group, the fasng blood glucose level at the 14thday of the study was significantly higher than the baseline level (t=2.74,df=5,p=0.041). Interesngly, the saline group also showed non-significant increases compared to baseline in fasng blood glucose levels at the 14th(t=1.49,df=5,p=0.195), and 21stday (t=1.83,df=5,p=0.127) of the study.

Table 3. Comparisons of insulin and C-pepde levels of the three groups

Table 3. Comparisons of insulin and C-pepde levels of the three groups

ap＜0.05 for pair-wise post-hoc comparison with saline groupbp＜0.05 for pair-wise post-hoc comparison with clozapine group

Figure 2. Electrophorec bands of β-acn mRNA and GLUT2mRNA of the three groups using real-me polymerase chain reacon （RT-PCR）

The results of the RT-PCR assessment of the expression of GLUT2mRNA and the Western Blot assessment of the expression of GLUT2protein are shown in Figures 2 and 3, respecvely. As shown in Table 4, at the 28thday of the experiment there were stascally significant differences in the levels of pancreac GLUT2mRNA across the three groups (F=3306.85,df=2,p＜0.001): GLUT2mRNA levels in the clozapine group and the clozapine+meormin group were significantly lower than in the saline group (p＜0.001), and the level in the clozapine+meormin group was significantly lower than that in the clozapine group (p＜0.001). Table 4 also shows that the expression levels of the GLUT2protein were significantly lower in the clozapine group and the clozapine+meormin groupthan in the saline group (p＜0.001), but the difference in the GLUT2protein expression in the clozapine and clozapine+meormin groups were not stascally significant.

Figure 3. Results from Western Blot of GAPDH （glyceraldehyde-3-phosphate dehydrogenase, leimage） and GLUT2 （glucose transporter-2, right image）of the three groups

Table 4. Expression of pancreac glucose transporter-2 （GLUT2） mRNA and protein of the three groups

Table 4. Expression of pancreac glucose transporter-2 （GLUT2） mRNA and protein of the three groups

ap＜0.05 for pair-wise post-hoc comparison with saline groupbp＜0.05 for pair-wise post-hoc comparison with clozapine group

GroupExpression level of GLUT2mRNA (mean [sd]) Expression level of GLUT2protein (mean [sd]) Clozapine0.993 (0.027)a0.500 (0.072)aClozapine+ meormin0.804 (0.010)a,b0.427 (0.069)aSaline1.993 (0.037)b0.779 (0.094)b

4. Discussion

4.1 Main findings

In this study, we found a non-significant increase in fasng blood glucose levels in rats during 28 days of connuous intake of clozapine compared to rats given saline. Aer 28 days, the C-pepde and insulin level in rats administered clozapine were not significantly different from rats given saline, but the expression of GLUT2mRNA and GLUT2protein were significantly lower in the clozapine-treated rats than in the saline-treated rats. These results are consistent with a large number of related studies. Studies have shown that anpsychoc medicaons may interfere with the funconing of glucose transporters on the cell membrane and that the inhibion of glucose transporters is associated both with hyperglycemia and diabetes. Moreover, the inhibion of glucose transporters caused by clozapine is much greater than that caused by risperidone.[14]Clozapine serves as a paral antagonist of DA receptors, 5-HT1A receptors, α1 receptors, and M receptors, and it may also elevate lepn levels, which leads to increased appete, weight gain, and hyperglycemia.

In this study rats treated with a combinaon of clozapine and meormin showed a peak in the fasng blood glucose level one week aer the start of the trial, which gradually decreased thereaer. This may be due to the faster rate of metabolism of meormin compared to that of clozapine (90% of meormin is metabolized within 12 hours); meormin would, thus, take longer to achieve the steadystate blood level needed to realize its full potenal for suppressing the hyperglycemic effects of clozapine. Though not stascally significant, this trajectory in the fasng blood glucose levels of the clozapine+metaformin group over the 28 days, combined with the significantly lower level of insulin in the this group aer 28 days of treatment, suggests that meormin can reduce the elevated blood glucose level induced by clozapine and reduce insulin resistance. These results are consistent with a clinical study by Wu and colleagues.[7]The main mechanisms of the hypoglycemic effect of meormin include the promoon of glucose uptake in peripheralssues, reducon of glycogen decomposion and gluconeogenesis, reducon of hepac glucose output, inhibion of glucose absorpon in the intesnes, and the promoon of glucose ulizaon in peripheralssues.[15,16]Other possible mechanisms include the promoon of lipid metabolism, reducon of obesity,[17,18]increase in the expression of lepn receptors, and the restoraon of lepn levels.[19,20,21]

This study has several limitaons. First, due to the small number of animals, differences in fasng blood glucose overme did not reach stascal significance. Second, the relavely short duraon of the study (28 days) made it impossible to assess the long-term effects of meormin on clozapine-induced abnormalies in glucose metabolism. Third, we based our decision on the appropriate dosage of meormin on clinical trials; more pharmacokinec studies are needed to determine the appropriate dosage in rats. Finally, we did not test GLUT2expression in the liver, the most important site of manufacture of this transporter. Future studies in this area need to have larger samples, assess the effects over more prolonged periods, test various doses of meormin, and evaluate GLUT2expression in both the pancreas and the liver.

4.3 Significance

Diabetes and the metabolic syndrome are serious adverse events that affect the quality of life and treatmentadherence of paents who need to take anpsychoc medicaons for prolonged periods. Developing effecve means for reducing the frequency and severity of these adverse reacons is an important clinical goal for psychiatry. Meormin is one promising agent for achieving this goal, but much more needs to be known about the adjuncve use of meormin with anpsychoc medicaons before this potenal can be realized.

Studies in animals are needed to help clarify the complex relaonship between clozapine, meormin, glucose metabolism and diabetes. Our study highlights the complexity of these relaonships and clarifies the need for a parallel series of animal studies and clinical studies to assess the appropriate use of meormin as anadjuncve treatment for paents taking anpsychoc medicaons who are at risk of developing the metabolic syndrome. Several avenues of research are indicated: (a) in which paents and for which anpsychoc medicaons is meormin most likely to be beneficial?; (b) what is the appropriate dosage of meormin to achieve the desired effect while liming the possibility of unwanted adverse reacons?; (c) which measures should be used to determine whether or not meormin is effecve?; and (d) how long should meormin be administered to determine whether or not it is effecve?

Conflict of interest

The authors report no conflict of interest related to this study.

Funding

This study was funded by the Naonal Natural Science Foundaon (30971041).

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Dr. Lan Gao graduated from Jining Medical College in July 2011 with a Bachelor's degree in clinical medicine. She is currently pursuing a Master's degree in psychiatry and mental health from Wuhan University. Her main research interests are psychopharmacology and the effects of new anpsychocs on glucose metabolism in paents with psychosis.

背景:抗精神病藥物可引起血糖升高甚至導致2型糖尿病。二甲雙胍可能會減輕上述副作用。

目的:評價二甲雙胍能否減輕使用抗精神病藥氯氮平時發生的糖代謝異常。

方法:將18只成年SD大鼠分為3組，分別用生理鹽水、氯氮平（20mg/kg）及氯氮平（20mg/kg）+二甲雙胍（78mg/kg）灌胃28d。在基線及其后每隔7天測空腹血糖。實驗第28d麻醉大鼠使其安樂死，此時取腹主靜脈血，用放射免疫法檢測血胰島素和C肽水平；取胰腺組織，采用實時聚合酶鏈反應和Western Blot技術檢測葡萄糖轉運體2（glucose transporter-2，GLUT2）的表達水平。

結果:氯氮平組第14天、21天和28天的空腹血糖值均顯著高于基線值，但氯氮平+二甲雙胍組僅第14天時的空腹血糖值較基線明顯升高。盡管如此，重復測量的方差分析并未發現三組間隨時間變化的血糖水平差異有統計學意義（p=0.136）。多組比較發現氯氮平+二甲雙胍組的胰島素均值顯著低于鹽水組及氯氮平組。組間GLUT2mRNA的表達差異具有統計學意義（氯氮平+二甲雙胍組＜氯氮平組＜鹽水組），組間GLUT2蛋白的表達差異亦有統計學意義（氯氮平+二甲雙胍組，氯氮平組＜鹽水組）。

結論:本研究發現氯氮平一定程度上能使SD大鼠空腹血糖水平升高，聯合使用二甲雙胍可部分抵消血糖的升高。正如預期，接受氯氮平的SD大鼠GLUT2的表達水平降低。然而，意料之外的是氯氮平聯合二甲雙胍治療28 d并未使GLUT2的表達水平正常化。

氯氮平聯合二甲雙胍對SD大鼠空腹血糖及胰島GLUT2表達水平的影響

高蘭 王高華* 劉浩 閻超慧

武漢大學人民醫院精神衛生中心 湖北武漢
*通信作者：wgh6402@163.com

2012-12-14; accepted: 2013-02-26)

10.3969/j.issn.1002-0829.2013.03.004

Mental Health Center, People’s Hospital of Wuhan University, Wuhan, Hubei Province, China

*correspondence: wgh6402@163.com