Oxidative stress and cannabinoid receptor expression in type-2 diabetic rat pancreas following...

Oxidative stress and cannabinoid receptor expression in type-2diabetic rat pancreas following treatment with Δ9-THC

Zeynep Mine Coskun1,2* and Sema Bolkent2

1Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Istanbul Bilim University, Istanbul, Turkey2Department of Medical Biology, Faculty of Cerrahpasa Medicine, Istanbul University, Istanbul, Turkey

The objectives of study were (a) to determine alteration of feeding, glucose level and oxidative stress and (b) to investigate expression andlocalization of cannabinoid receptors in type-2 diabetic rat pancreas treated with Δ9-tetrahydrocannabinol (Δ9-THC). Rats were randomlydivided into four groups: control, Δ9-THC, diabetes and diabetes +Δ9-THC groups. Diabetic rats were treated with a single dose ofnicotinamide (85mg/kg) 15min before injection of streptozotocin (65mg/kg). Δ9-THC was administered intraperitoneally at 3mg/kg/day for7 days. Body weights and blood glucose level of rats in all groups were measured on days 0, 7, 14 and 21. On day15 after theΔ9-THC injections,pancreatic tissues were removed. Blood glucose levels and body weights of diabetic rats treated withΔ9-THC did not show statistically significantchanges when compared with the diabetic animals on days 7, 14 and 21. Treatment with Δ9-THC significantly increased pancreas glutathionelevels, enzyme activities of superoxide dismutase and catalase in diabetes compared with non-treatment diabetes group. The cannabinoid 1receptor was found in islets, whereas the cannabinoid 2 receptor was found in pancreatic ducts. Their localization in cells was both nuclearand cytoplasmic. We can suggest that Δ9-THC may be an important agent for the treatment of oxidative damages induced by diabetes. However,it must be supported with anti-hyperglycaemic agents. Furthermore, the present study for the first time emphasizes that Δ9-THC may improvepancreatic cells via cannabinoid receptors in diabetes.The aim of present study was to elucidate the effects of Δ9-THC, a natural cannabinoid receptor agonist, on the expression and localization

of cannabinoid receptors, and oxidative stress statue in type-2 diabetic rat pancreas. Results demonstrate that the cannabinoid receptors arepresented in both Langerhans islets and duct regions. The curative effects of Δ9-THC can be occurred via activation of cannabinoid receptorsin diabetic rat pancreas. Moreover, it may provide a protective effect against oxidative damage induced by diabetes. Thus, it is suggested thatΔ9-THC can be a candidate for therapeutic alternatives of diabetes symptoms. Copyright © 2014 John Wiley & Sons, Ltd.

key words—type-2 diabetes; Δ9-THC; cannabinoid receptors; oxidative stress; gene expression

INTRODUCTION

Diabetes mellitus (DM) is a multifactorial disease. DM isa serious medical problem that is rising dramatically.Type-2 diabetes mellitus (T2DM) is characterized byhyperglycaemia and oxidative stress. T2DM is caused byreduced insulin secretion and enhanced insulin resistance.Beta cell (β-cell) impairment is caused and progressed bydiabetes as a result of the combined effects of genetic andenvironmental factors.1 Many experimental and clinicalstudies have shown that oxidative stress plays a role in theonset of diabetes.2–5 It is known that many pathways ofdiabetic complications involve oxidative stress. A weaken-ing of the antioxidant defence system has caused oxidativestress.6

It has been known that Δ9-tetrahydrocannabinol (Δ9-THC)is the main constituent of Cannabis sativa. It is suggestedthat Δ9-THC plays a role as an antioxidant substance.7

Δ9-THC causes the activation of the endocannabinoidsystem, which involves cannabinoid 1 (CB1) and cannabi-noid 2 (CB2) receptors. These receptors were cloned andclassified in the early 1990s.8–10 The CB1 and CB2receptors belong to the G protein-coupled family of receptors.The CB1 receptor was characterized first in the rat brain. It isalso expressed in peripheral organs including pancreas, lung,heart, thymus and testicles.11–14 The CB2 receptor is identi-fied in the peripheral organs and mainly expressed in theimmune tissues.9 The cannabinoid receptors can providecontrol of food intake and regulation of energy consumption.Therefore, the appetite and body weight are affected by theendocannabinoid system.15,16

The CB1 and CB2 receptors have been identified in theendocrine cells of the pancreas.17 Therefore, a wide rangeof research studies have focused on the role of cannabinoidreceptors in regulating insulin secretion and glucosehomeostasis.18,19 Additionally, cannabinoid receptors arecandidates for therapeutic alternatives in chronic liverdiseases, involving chronic inflammation such as arthritis,Alzheimer’s disease, HIV-1 infection and diabetic compli-cations such as neuropathy.18,20,21

*Correspondence to: Zeynep Mine Coskun, Department of MolecularBiology and Genetics, Faculty of Arts and Sciences, Istanbul Bilim University,34394-Sisli, Istanbul, Turkey.E-mail: [email protected]

Received 31 March 2014Revised 6 August 2014

Accepted 8 August 2014Copyright © 2014 John Wiley & Sons, Ltd.

cell biochemistry and functionCell Biochem Funct 2014; 32: 612–619.Published online 3 September 2014 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/cbf.3058

Thus, the present study was carried out to clarify theexpression and localization of cannabinoid receptors byΔ9-THC in the pancreas of rats with type-2 diabetic inducedby streptozotocin (STZ) + nicotinamide (NAD). In addition,the effects of Δ9-THC on oxidative stress in the pancreatictissue have been determined.

MATERIALS AND METHODS

Animal design

Male Sprague–Dawley rats that were 8–10weeks of age wereobtained from the Experimental Medical Research Institute,Istanbul University. The rats were maintained under con-trolled environmental conditions and in a cycle consisting of12 h of light, 12 h of darkness. All the experiments were con-ducted in accordance with the guidelines of the Local EthicsCommittee for Research on Animals, Istanbul University.

The rats were randomly divided into four groups. InGroup I (n = 8), the control group, a single dose of salinewas given to rats intraperitoneally for 7 days. In Group II(n = 6), the Δ9-THC group, the rats were treated intraperito-neally with a single dose of 3mg/kg/day of Δ9-THC(Lipomed, Arlesheim, Switzerland, THC-135-100LE) for7 days. In Group III (n = 8), the diabetic group, the animalsreceived intraperitoneal injections of a single dose of NAD(85mg/kg, Sigma-Aldrich, St. Louis, MO, USA, N3376)in saline 15min before the injection of STZ (65mg/kg,Sigma-Aldrich, St. Louis, MO, USA, S0130) dissolved insaline.22 The blood glucose levels were determined 72 hafter the STZ-NAD injection. The rats with blood glucoseconcentrations higher than 200mg/dL were accepted asdiabetic. In Group IV (n=7), the diabetes +Δ9-THC group,the diabetic animals were treated with Δ9-THC (3mg/kg/day)for 7 days.

At the end of the experiment (day 15 after the Δ9-THCinjections), the rats were anaesthetized intraperitoneallywith a freshly prepared mixture of ketamine hydrochloride(50mg/kg, Ketalar, Pfizer) and xylazine hydrochloride(10mg/kg, Rompun, Bayer). After that, the rats weresacrificed; their pancreatic tissues were collected. Thepancreatic tissues were fixed in 10% neutral formalin andwere stored at �86 °C.

Measurements of body weight and glucose levels

Body weights of rats in all groups were measured on days 0,7, 14 and 21. The blood glucose levels of the animals in allgroups were measured at the same time intervals by aglucometer (Accu-check, Roche Diagnostics GmbH,Mannheim, Germany) using the blood collected from the tail vein.

Determination of oxidative stress markers

The pancreatic tissues were homogenized in cold 0·9%NaCl and made up to 10% homogenate. The homogenateswere centrifuged, and the clear supernatants were usedfor carrying out the protein, lipid peroxidation (LPO), gluta-thione (GSH) and antioxidant enzyme analysis. The GSH

levels were determined according to Beutler’s method.23

The LPO levels in homogenates were estimated by using theLedwozyw’s method.24 The catalase (CAT) activity wasassayed in pancreatic tissues by using the method of Aebi.25

The superoxide dismutase (SOD) activity was determinedby using Sun’s method.26 The protein content in the superna-tants was estimated by Lowry’s method using bovine serumalbumin as standard.27

Detection of mRNAs by quantitative real-time polymerasechain reaction

The total RNA was isolated from frozen pancreatic tissues(�80 °C) using the High Pure RNA Tissue Isolation Kit(Roche Diagnostics, GmbH, Mannheim, Germany) accord-ing to the manufacturer’s instructions. All of the RNAsamples were shown to have a ratio of A260/A280> 1·8 bythe nano-drop spectrophotometer (Thermo Scientific,Wilmington, DE, USA). The reverse transcription for thefirst strand cDNA synthesis from the samples wasperformed using a random hexamer primer, the first strandcDNA Synthesis kit (Fermantas, Hanover, MD, USA). Toprepare the cDNA, total RNA (100 ng) was mixed withprimers. The resultant cDNAs were used as a template forreal-time polymerase chain reaction (RT-PCR), which wasperformed on the Lightcycler 480 (Roche Diagnostics,GmbH, Mannheim, Germany) using the LightcyclerTaqman Master kit (Roche Diagnostics, GmbH, Mannheim,Germany). The following primers were determined inTable 1. The quantification process was carried out with astandard curve run at the same time as the samples.Lightcycling conditions used were as follows: 10min at95 °C, 10 s at 95 °C, followed by 40 cycles at 60 °C for30 s, and 72 °C for 1 s. The mRNA expressions werecalculated in relation to the β-actin (Actb) mRNA expres-sion using the formula 2�ΔCt values. The rat brain tissueswere evaluated as a positive control.

Immunohistochemistry

The rat pancreases were fixed in 10% neutral buffered forma-lin and embedded in wax. Paraffin embedded 5-μm pancreatic

Table 1. Accession numbers of primers from NCIB database

Gene PrimerAccession numbersfrom NCIB database

CB1 receptor F 5′- GGA CAT GGA GTGCTT TAT GAT TC-3′

NM_012784·3

R 5′-GAG GGA CAG TACAGC GAT GG-3′

CB2 receptor F 5′-AAG CCC CAA AGTCCT CAG TT-3′

NM_020543·4

R 5′-CCC AAC TCC TGCTTA TCC TTC-3′

Actb F 5′-CCC GCG AGT ACAACC TTC T-3′

NM_031144·2

R 5′-CGT CAT CCA TGGCGA ACT-3′

CB1, cannabinoid 1 receptor; CB2, cannabinoid 2 receptor; β actin (Actb).

613the effects of Δ9-thc on diabetic pancreas

Copyright © 2014 John Wiley & Sons, Ltd. Cell Biochem Funct 2014; 32: 612–619.

sections were stuck to microscope slides coated with poly-L-lysine. They were deparaffinized with xylene and rehydratedthrough a range of graded alcohols and were permeabilizedwith citrate buffer (0·01mol/L, pH6·0) for 15min. The CB1slides were blocked in 3% bovine serum albumin (BSA) inTris-buffered saline for 30min and incubated overnight at4 °C with anti-CB1 antibody (Cayman Chemicals, AnnArbor, MC, USA, cat no: 10006590) at a 1:150 dilution ratein 3% BSA. A blocking solution was used for CB2 slides for30min, and they were incubated overnight at 4 °C withanti-CB2 antibody (Santa Cruz Biotechnology, CA, USA,cat no. sc-25494) at a dilution rate of 1:400 in an antibodydiluent (Zymed, Camarillo, CA, USA). Immunoreactivitiesof CB1 and CB2 receptors were detected using a biotin–streptavidin–peroxidase-based Histostain Plus BroadSpectrum Kit (Invitrogen, Carlsbad, CA, USA), and theywere visualized using 3-amino-9-ethylcarbazole (Invitrogen,Carlsbad, CA, USA), a chromogenic peroxidase substrate.The sections were counterstained with haematoxylin. Thepercentage of the area occupied by immunoreactive cellswas calculated bymeasuring the (labelling area/total area) ×100for CB1 and CB2 receptors in the pancreas of rats.

Statistical analysis

All of the values were analysed by a statistical software(GraphPad Prism, version 5·0 computer package). The datawere expressed as means ± SEM. A comparison betweenthe two groups was performed using Mann–Whitney Unonparametric test, and a comparison among multiplegroups was performed using the one-way analysis ofvariance (ANOVA) followed by a Kruskal–Wallis test. Avalue of p< 0·05 was considered statistically significant.

RESULTS

Effect of Δ9-THC on blood glucose concentration andbody weight

The blood glucose levels showed statistically significantdifferences among the four groups on days 7, 14 and 21(pANOVA< 0·001). There was a significant increase in theblood glucose levels of diabetic rats when compared withnormal controls because of the injection of STZ+NAD ondays 7, 14 and 21 (p< 0·001). In the study, a daily administra-tion of Δ9-THC for 7 days in diabetic rats led to a dose-dependent fall in blood glucose levels on days 14 and 21,which was not statistically significant. The effect of Δ9-THCon the blood glucose levels in diabetic rats is also shown inFigure 1.There was a significant change in body weight among the

four groups on days 7, 14 and 21, respectively (pANOVA0·01; pANOVA< 0·01; pANOVA< 0·05). The loss of bodyweights were significantly observed in diabetic rats ascompared with control rats on days 7, 14 and 21, respec-tively (p< 0·05; p< 0·01; p< 0·05). The body weights ofthe diabetic rats administered with Δ9-THC increased at aninsignificant rate when compared with the diabetic animals

on days 7, 14 and 21. The weight gains or losses throughoutthe experiment are shown in Figure 2.

Biochemical analysis

The results of the GSH and LPO levels and the SOD andCAT enzyme activities are summarized in Tables 2 and 3.GSH, LPO levels and SOD activity were not shown to bedifferent among the four groups (pANOVA> 0·05). However,CAT activity showed a significant difference among groups(pANOVA< 0·05).The GSH levels of pancreas were significantly decreased

in the diabetic group when compared with the controlgroups (p< 0·05). The treatment of Δ9-THC in the diabeticgroup significantly increased the pancreas GSH levels ascompared with the diabetic group (p< 0·05). The LPOlevels were shown to have a significant increase in diabeticrat pancreas when compared with control rats (p< 0·05).However, the administration of Δ9-THC did not changethe levels of LPO in diabetic rat pancreas.Treatment of Δ9-THC caused a change in the activities of

SOD enzyme. The SOD activity in the pancreas wasincreased in the Δ9-THC group when compared with thecontrol group. The activity of SOD enzyme was shown tohave a significant increase in diabetic rats administered withΔ9-THC as compared with the diabetic rats (p< 0·05).Similarly, the activity of CAT enzyme indicated a signifi-cant increase in the Δ9-THC group as compared with the

Figure 1. The blood glucose levels of all groups on days 0, 7, 14 and 21. Dataare expressed as mean±SEM. ap< 0·001 versus control group, bp< 0·001versus Δ9-THC group

Figure 2. The body weights of control and experimental animals ondays 0, 7, 14 and 21. Data are expressed as mean ± . ap< 0·001 versuscontrol group, bp< 0·05 versus control, cp< 0·001 versus Δ9-THC group

614 z. m. coskun and s. bolkent


control group (p< 0·01). The administration of Δ9-THC todiabetic rats increased CAT activity when compared withthe diabetic rats (p< 0·05).

Expressions of CB1 and CB2 receptors in the pancreas ofcontrol and experimental rats

We found out that the mRNAs of CB1 and CB2 receptorswere expressed in the pancreatic cells by RT-PCR. Theywere not shown to be different among the four groups ata statistically significant level (pANOVA> 0·05). ThemRNAs of CB1 and CB2 receptors were also detected

in brain tissues, which were used as a positive control tis-sue. In the Δ9-THC group (ΔCt 2·02 × 1011), the expres-sion of CB1 mRNA was higher than in the controlgroup (ΔCt 1·77 × 1010). The expression of CB1 mRNAin the tissue of diabetic pancreas (ΔCt 28·56) was shownto have an insignificant decrease as compared with the pan-creatic tissue in the control group (ΔCt 1·77 × 1010). On thecontrary, the level of CB1 mRNA in the diabetes +Δ9-THCgroup (ΔCt 2·61) was observed to undergo an insignificantdecrease when compared with diabetic rats. We did notobtain any results related to the expression of CB2 mRNAin the pancreas of all animals. The mRNA could be measuredin the pancreas of two rats, although the measurements ofexpression were repeated twice.

In the pancreas, immunoreactivity of CB1 and CB2receptors was detected in Figures 3A and 3B. We observedbrain and spleen as tissues of positive control for the CB1and CB2 receptors, respectively. The immunolocalized cellsof CB1 receptor were more abundant than the immuno-localized cells of CB2 receptor in the rat pancreas. TheCB1 receptors are mostly expressed in the islets ofLangerhans, but CB2 receptors are found in cells of duct.CB2 receptor was rarely present in islets of Langerhans.Interestingly, CB1 and CB2 receptors demonstrated bothnuclear and cytoplasmic localization in islet and duct cells,respectively. Whereas the area percentage of CB1 receptorin Langerhans islets showed a significant difference amongall groups (pANOVA< 0·001), the area percentage CB2receptor of pancreas ducts did not show a significantdifference (pANOVA> 0·05). The area percentage of CB1receptor in type-2 diabetic rats was significantly decreasedas compared with the control group (p< 0·001). In isletsof Langerhans, diabetic rats treated with Δ9-THC groupshowed an insignificant increase compared with diabetic ratsfor the area percentage of CB1 receptor. In the ducts ofpancreas, the area percentages of CB2 receptors wereobserved to have undergone an insignificant decrease indiabetic rats as compared with control rats. However, thearea percentage of the CB2 receptor in the diabetic grouptreated with Δ9-THC was increased as compared with thediabetic group (Figures 4–6).

Table 2. Pancreas GSH and LPO levels in control and experimentalgroups

Groups GSH (nmol/mg)1 LPO (nmol/mg)1

Control 54·27 ± 10·39 0·66 ± 0·11Δ9-THC 50·99 ± 21·66 0·68 ± 0·05Diabetes 39·76 ± 17·17* 1·13 ± 0·23*Diabetes +Δ9-THC 87·40 ± 26·50** 0·17 ± 0*pANOVA NS NS

NS, non-significant.1Mean ± standard error (SEM).*p< 0·05 versus control group;**p< 0·05 versus diabetes group.

Table 3. Pancreas CAT and SOD enzymes activities in control andexperimental groups

Groups CAT (U/mg)1 SOD (U/mg)1

Control 12·22 ± 3·66 10·97 ± 1·20Δ9-THC 27·09 ± 5·14* 17·15 ± 6·17Diabetes 11·36 ± 2·61** 10·10 ± 0·87Diabetes +Δ9-THC 17·07 ± 1·47*** 15·65 ± 3·63***pANOVA <0·05 NS

NS, non-significant.1Mean ± standard error (SEM).*p< 0·05 versus control group;**p< 0·01 versus Δ9-THC group;***p< 0·05 versus diabetes group.

Figure 3. The area percentage of immunolocalized cells of cannabinoid receptors in the pancreas. A, CB1 receptor immunolocalized cells in islets ofLangerhans. B, CB2 receptor immunolocalized cells in pancreatic duct. Data are expressed as mean ±SEM. ap< 0·001 versus control group, bp< 0·001 versusΔ9-THC group



DISCUSSION

It is known that the pancreas is an important organ indiabetes diseases. In diabetic rat models, higher glucoselevels and lower body weight are identified.28,29 Addition-ally, there is a strong relation between oxidative stress anddiabetes.30

The endocannabinoid system, which consists of CB1 andCB2 receptors, has long been known to regulate food intakeand energy balance. Δ9-THC, a cannabinoid receptoragonist, has a wide area of use encompassing appetitestimulants, anti-emetic, analgesic, anti-pyretic agents andothers.31,32The optimal dose of Δ9-THC has not yet beenestablished. It was used in different doses in severalstudies.31,33Long et al.34 expressed that 10 mg/kg ofΔ9-THC is the sedative dose for mice. On the other hand, itis suggested that Δ9-THC protects against oxidative damageby blocking the generation of reactive oxygen species.7

In the present study, Δ9-THC was used to maintain theblood glucose level and body weight in diabetes. Whilethe blood glucose level increases, the body weight decreasesin diabetes.35,36The study of Cota et al.37 reported thatcannabinoid compounds stimulated appetite. Verty et al.38

suggested that the cannabinoid receptor agonist Δ9-THCreduced weight loss in a rat model of activity-basedanorexia. It was shown that cannabinoid receptor antagonistsfailed to inhibit the insulin-stimulatory effects of CB1 and

CB2 receptor agonists and that the administration of cannabi-noid receptor agonists alone stimulated insulin secretion inisolated human islets of Langerhans.39 Our results are incontrast with the studies by other authors reporting thatcannabinoid receptor agonist supplementation has a regulativeeffect on food intake and blood glucose level in diabetes.37,38

According to our results, increased blood glucose levels anddecreased body weight of type-2 diabetic rats did not changefollowing treatment with Δ9-THC on days 14 and 21. Theresults demonstrate that Δ9-THC did not show statisticallysignificant changes in blood glucose and body weight levelsin diabetes.Oxidative stress is a problem in terms of diabetes.

Increased oxidative stress has an important role in thedevelopment and progression of diabetes. Reactive oxygenspecies (ROS) is related with oxidative stress that isovergenerated in diabetes.30 Although serum MDA levelsincrease, serum SOD and erythrocyte GSH levels decreasein type-2 diabetes.30 These results are compatible with theresults of liver, kidney and testicle tissues with diabetes.40,41

The ROS generation was reduced by Δ9-THC via theN-methyl-D-aspartate-induced death of AF5 cells.42 It hasbeen reported that Δ9-THC elevates the level of GSH inliver.43 In this study, we have observed that the MDAformation, the index of lipid peroxidation, was significantlyincreased in the pancreatic tissues of diabetic rats. Theincrease of LPO levels shows the occurrence of oxidative

Figure 4. A, the localization of CB1 receptor immunoreactive cells in Langerhans islets; cytoplasmic and nuclear (*), only cytoplasmic (#) in both images. B,CB1 receptor non-immunoreactive cells in pancreatic duct. C, CB2 receptor immunoreactive cells were rarely seen in Langerhans islets (arrow in both images).Streptavidin–biotin–peroxidase technique, counterstain haematoxylin. Scale bar = 10μm



damage in diabetic rats. The LPO levels remainedunchanged by the administration of Δ9-THC. The GSHprovides a cellular defence against ROS. A decrease inGSH levels of pancreas was observed in diabetic animals.Treatment with Δ9-THC increased GSH levels in thepancreatic tissues of diabetic rats. These results indicatethat Δ9-THC may help to inhibit oxidative damage inpancreas. The degradation of GSH may also be preventedin diabetic animals by Δ9-THC. Oxidative stress repre-sents an imbalance between the production and removalof reactive oxygen species. The anti-oxidative defencesystem enzymes such as CAT and SOD show lower activ-ities in various tissues during diabetes.44 In the presentstudy, we observed that the activities of SOD and CATenzymes were raised in the pancreas of diabetic and non-diabetic rats treated with Δ9-THC. The enzymes arepivotal components of the antioxidant defence. Hence,the increasing activities of SOD and CAT enzymes clearlydemonstrate that Δ9-THC can protect the cells againstROS.

Bermudez-Silva et al.45 observed that the CB1 and CB2receptors are present in human endocrine pancreas.However, the CB2 receptor levels are much lower than theCB1 receptor levels in human endocrine pancreas. TheCB1 receptor was not detectable in the exocrine pancreasof the mice, and the CB2 receptor was expressed in islet en-docrine cells according to the results achieved by Li et al.46

On the contrary, Tharp et al.47 suggested that the CB2receptor was not present in the rodent or human islets. Inthe study by Nakata and Yada,48 the CB2 receptor wasnot observed in the islets and the exocrine of pancreas.In the present study, we detected both CB1 and CB2receptors in the pancreas. Interestingly, the CB1 receptorwas present in the islets, whereas the CB2 receptor wasseen in the pancreatic ducts. Only, a few islet cellscontained the CB2 receptor in some islets of the rats. Itwas mostly seen in the exocrine portion of the pancreas.These receptors may be related with the secretion ofpancreatic hormones. In particular, the CB1 receptor canregulate the endocrine hormones in pancreas. In fact, thesereceptors may affect the appetite hormones. While adecrease in the CB1 receptor mRNA levels was observed,the CB1 receptor peptide levels were increased in thediabetic pancreases treated with Δ9-THC. This may indi-cate that the synthesis of the CB1 receptor peptide isaccelerated by Δ9-THC. In addition, we observed thatthe CB1 and CB2 receptors had nuclear as well as cyto-plasmic localization in the pancreas. The results suggestthat CB1 and CB2 receptors serve both nuclear andcytoplasmic functions. Therefore, CB1 and CB2 receptorsare complex proteins. These data have not been reportedin previous studies.

In summary, this study has shown that treatment ofΔ9-THC alone is not able to regulate blood glucose level

Figure 5. CB1 receptor immunoreactive cells (arrow) are seen by immunohistochemistry in Langerhans islets. A, control; B, Δ9-THC; C, diabetes; D,diabetes treated with Δ9-THC groups. Streptavidin–biotin–peroxidase technique, counterstain haematoxylin. Scale bar = 10μm



and weight loss in type-2 diabetic rats. There are protectiveeffects of Δ9-THC against the oxidative damage inducedby diabetes. Hence, we can say that it acts as an antioxidantand generate antioxidant defence. The CB1 and CB2 recep-tors are expressed in the pancreas. Both of these receptorshave nuclear and cytoplasmic functions in pancreatic cells.Especially, the CB1 receptor may play a role in regulatingthe endocrine hormones. When taken together, these resultsindicate that the effects of Δ9-THC on food intake, glucosehomeostasis and oxidative damage may occur by means ofcannabinoid receptors. If Δ9-THC is supported by anyanti-hyperglycaemic agent, it may serve as a candidate forthe treatment of diabetes and its symptoms in the future. Itis possible that the therapeutic effects of Δ9-THC ondiabetes in relation to endocannabinoid system will clarifywith further research.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

This study was supported by the Scientific Research ProjectsCoordination Unit of Istanbul University, Project No. 7823.

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