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Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats
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Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats Zeynep Mine Coskun a,b,∗ , Sema Bolkent b a b
Health Services Vocational School, Istanbul Bilim University, Istanbul, Turkey Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 15 April 2013 Received in revised form 29 May 2013 Accepted 30 May 2013 Available online xxx Keywords: 9 -THC Type 2 diabetes Insulin Glucagon Pancreas Rat
a b s t r a c t The regulation of glucose, lipid metabolism and immunoreactivities of insulin and glucagon peptides by delta-9-tetrahydrocannabinol (9 -THC) in diabetes were examined in an experimental rat model. Male Sprague-Dawley rats were divided into four groups: (1) control, (2) 9 -THC treated, (3) diabetic, and (4) diabetic + 9 -THC. The type 2 diabetic rat model was established by intraperitoneal (i.p.) injection of nicotinamide (85 mg/kg body weight) followed after 15 min by i.p. injection of streptozotocin (STZ) at 65 mg/kg of body weight. 9 -THC and 9 -THC treated diabetic groups received 3 mg/kg/day of 9 -THC for 7 days. The immunolocalization of insulin and glucagon peptides was investigated in the pancreas using a streptavidin–biotin–peroxidase technique. High density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL), triglycerides (TG), total cholesterol (TC) and total protein (TP) levels were measured in serum. Total islet area percent of insulin immunoreactive cells slightly changed in diabetic + 9 -THC rats compared to diabetic animals. However, the area percent of glucagon immunoreactive cells showed a decrease in diabetic + 9 -THC rats compared to that of diabetic animals alone. Serum TC, HDL and LDL levels of diabetes + 9 -THC group showed a decrease compared to the diabetic group. These results indicate that 9 -THC may serve a protective role against hyperlipidemia and hyperglycemia in diabetic rats. © 2013 Elsevier GmbH. All rights reserved.
Introduction The cannabinoids are natural constituents of Cannabis sativa L. commonly known as marijuana. Delta-9-tetrahydrocannabinol (9 -THC), cannabinol (CBN) and cannabidiol (CBD) are the main constituents of C. sativa L. (Kochanowski and Kala, 2005). 9 -THC is the main psychoactive compound that activates cannabinoid receptors. Cannabinoids influence lipid and glucose metabolism (Kalofoutis et al., 1985; Di Marzo et al., 2011). In addition, cannabinoids are associated with oxidative damage and lipoproteins. Some studies have shown that 9 -THC may have a protective role against neurodegenerative diseases and provide cardioprotection in animals (Fishbein et al., 2012; Waldman et al., 2013). It has been reported that there are some beneficial effects of cannabis extract
Abbreviations: ␣-cell, alpha cell; AEC, 3-amino-9-ethyl-carbazole; ANOVA, analysis of variance; -cell, beta cell; CBD, cannabidiol; CBN, cannabinol; 9 -THC, delta-9-tetrahydrocannabinol; i.p., intraperitoneal; HDL, high density lipoprotein cholesterol; LDL, low-density lipoprotein; NAD, nicotinamide; STZ, streptozotocin; T2DM, type 2 diabetes mellitus; TC, total cholesterol; TG, triglyceride; TP, total protein; VLDL, very low density lipoprotein cholesterol. ∗ Corresponding author at: Health Services Vocational School, Istanbul Bilim University, 34394 Sisli, Istanbul, Turkey. E-mail address: [email protected] (Z.M. Coskun).
on diabetes-induced neuropathic pain through its strong antioxidant activity (Klausner et al., 1975; Comelli et al., 2009). With regard to 9 -THC, which acts as a cannabinoid receptor agonist, it has been shown to activate lipoprotein lipase, regulate cannabinoid receptor expression and beta cell (-cell) function in pancreatic islets (Li et al., 2010; Vilches-Flores et al., 2010; Wong et al., 2012). As for endogenous cannabinoids, they have a role in the regulation of secretion of pancreatic endocrine hormones. The activation of cannabinoid receptors stimulates insulin secretion to maintain glucose homeostasis (Bermúdez-Silva et al., 2007; Li et al., 2011). Diabetes mellitus is frequently complicated by hyperlipidemia and hyperglycemia with an increase in blood glucose levels accompanied by an increase in blood levels of low-density lipoproteins (LDL), total cholesterol (TC) and triglycerides (TG) (Kim and Caprio, 2011; Ranasinghe et al., 2012). In recent years, the prevalence of type 2 diabetes mellitus (T2DM) has increased dramatically and it comprises approximately 90% of all cases of diabetes. The pathophysiology of T2DM is a complex process and involves insulin resistance and insufficient pancreatic -cell function (Kim and Caprio, 2011). In patients with T2DM, a decrease in insulin production occurs due to -cell destruction. Insulin secreted by the beta cells (-cells) and glucagon secreted by alpha cells (␣-cells) of the pancreatic islets are key hormones in the regulation of blood glucose levels (Bagger et al., 2011; Shinde et al., 2011).
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Please cite this article in press as: Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.05.013
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In the current study, we examined whether glucose levels and lipid metabolism were affected by the administration of 9 -THC in type 2 diabetic rats induced by streptozotocin (STZ) + nicotinamide (NAD). In addition, the effects of 9 -THC on immunolocalization of insulin and glucagon secreting cells in pancreatic islets were investigated.
Biochemical analysis Serum high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL), triglycerides (TG), total cholesterol (TC) and total protein (TP) levels were measured by enzymatic methods using commercial kits on a Siemens Advia 1800 chemistry system (Erlangen, Germany).
Materials and methods Statistical analysis Experimental design 8–10 week-old male Sprague-Dawley rats (average weight 214 g) were obtained from Istanbul University, Experimental Medical Research Institute. All experiments were carried out in accordance with the guidelines of Istanbul University, Local Ethics Committee on Animal Research. All rats were housed in a temperature-controlled clean room with a 12 h light, 12 h dark cycle and fed with standard chow diet and tap water ad libitum. The animals were selected randomly and divided into four groups: Group 1: (n = 8) control rats that received a single intraperitoneal (i.p.) saline dose. Group 2: (n = 6) rats received a single dose (i.p.) of 3 mg/kg/day of delta-9-tetrahydrocannabinol (9 THC) (Lipomed, Arlesheim, Switzerland; THC-135-100LE) for 7 days. Group 3: (n = 8) rats were rendered diabetic by i.p. injection of a single dose of nicotinamide (NAD) (85 mg/kg, Sigma–Aldrich, St. Louis, MO, USA; N3376) in saline 15 min before injection (i.p.) of streptozotocin (STZ) (65 mg/kg, Sigma–Aldrich, St. Louis, MO, USA; S0130) dissolved in saline (Masiello et al., 1998). The blood glucose levels were determined 72 h after STZ + NAD injection. The rats with blood glucose concentrations more than 200 mg/dL were accepted as diabetic. Group 4: (n = 7) diabetic rats treated (i.p.) daily with 9 -THC (3 mg/kg/day) during 7 days. Animals were anesthetized (i.p.) with a freshly prepared mixture of ketamine hydrochloride (50 mg/kg, Ketalar, Pfizer, New York, NY, USA) and xylazine hydrochloride (10 mg/kg, Rompun, Bayer, Toronto, ON, Canada) on day 15 after 9 -THC injections. The blood samples were taken under anesthesia and the animals were sacrificed. After sacrifice, the pancreatic tissue samples were immediately taken and fixed in 10% neutral buffered formalin for immunohistochemical analysis and serum samples were stored at −86 ◦ C for biochemical analysis. Blood glucose levels of rats in all groups were measured with a glucometer (Accu-check, Roche Diagnostics, GmbH, Mannheim, Germany) using blood obtained from the tail vein on day 22.
Immunohistochemical staining Immunohistochemical staining was performed on 5 m thick formalin-fixed, paraffin-embedded sections of pancreas using a streptavidin–biotin–peroxidase method. The sections were mounted on glass slides previously treated with polyl-lysine. The sections were incubated with antibody against insulin (Sigma–Aldrich, St. Louis, MO, USA; I2018) and glucagon (Sigma–Aldrich; G2654), with 1:1000 and 1:2000 dilutions for 1–1.5 h at room temperature, respectively. The reaction was revealed with 3-amino-9-ethyl-carbazole (AEC) as chromogen, and the sections were counterstained with Mayer’s hematoxylin. Finally, slides were mounted in glycerol gelatine and stored at +4 ◦ C. In negative controls, the primary antibodies were omitted. The area percent of insulin and glucagon immunoreactive cells were evaluated using a Nikon microscope (Eclipse 80i, Melville, NY, USA) equipped with a digital camera using NIS-Elements-D 3.1 microscope imaging software program (Melville, NY, USA).
All values were analyzed by statistical software (GraphPad Prism version 5.0 computer package, San Diego, CA, USA). Data were expressed as means ± SE. A comparison between two groups was performed using Mann–Whitney U non-parametric test and a comparison among multiple groups was performed using one-way analysis of variance (ANOVA) followed by a Kruskal–Wallis test. A value of p < 0.05 was considered statistically significant. The area percent occupied by immunoreactive cells insulin and glucagon in pancreatic islets was calculated by measuring (labeling area/total area) × 100. Results Blood glucose levels Significant changes in blood glucose levels were determined among the four groups (PANOVA < 0.001) on day 22. Blood glucose level showed a significant difference between control rats and diabetic animals (a p < 0.001) (Table 1). 9 -THC treated diabetes group showed an insignificant decrease in blood glucose levels compared to that of the diabetic rats on day 22 (Table 1). Immunohistochemistry The changes of insulin and glucagon immunoreactive cell by percentage in islets of Langerhans are shown in Table 2. Insulin and glucagon peptide immunolocalized cells were evaluated as statistically significant among all groups (PANOVA < 0.001 and PANOVA < 0.001, respectively). While the insulin immunoreactive cells were significantly decreased (a p < 0.001), glucagon immunoreactive cells were significantly increased (a p < 0.001) in the pancreatic islets of diabetic rats as compared to control group. Furthermore, an insignificant increase was observed in the area of insulin immunoreactive cells in diabetes + 9 -THC rats as compared with diabetic rats (Fig. 1). However, glucagon immunoreactive cells were increased significantly in diabetic rats compared to control rats (a p < 0.001). Glucagon immunoreactive cells showed an insignificant decrease in diabetes + 9 -THC rats as compared with diabetic rats (Fig. 2). Biochemistry The results of the biochemical parameters are summarized in Tables 3 and 4. TP levels of serum samples did not reveal a statistically significant difference, but TG (PANOVA < 0.05) and TC (PANOVA < 0.001) levels showed a statistically significant decrease among all groups. TG levels of the diabetic group were significantly higher than the control group (b p < 0.05), but it was statistically insignificant between diabetic rats and diabetes + 9 -THC group. There was a significant increase in TC levels in diabetic rats compared to control animals (d p < 0.01). TC levels decreased significantly in 9 -THC given diabetic rats compared to diabetic (f p < 0.001) group (Table 3). Lipoprotein types (HDL, LDL and VLDL) were statistically significant among all groups (PANOVA < 0.001, PANOVA < 0.01 and
Please cite this article in press as: Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.05.013
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Table 1 Blood glucose levels of all groups on day 22.
c
Blood glucose levels (g/dL) a b c
Control (n = 8)
9 -THC (n = 6)
Diabetes (n = 8)
Diabetes + 9 -THC (n = 7)
PANOVA
114.8 ± 5.0
124.8 ± 8.6
397.5 ± 28.0
320.7 ± 40.8a , b
<0.001
a,b
p < 0.001 versus control group. p < 0.01 versus 9 -THC group. Mean ± standard error (SE).
Table 2 The area percent of immunoreactive cells for insulin and glucagon in pancreatic islets of all groups.
Insulinc Glucagonc a b c
Control (n = 7)
9 -THC (n = 6)
Diabetes (n = 7)
Diabetes + 9 -THC (n = 7)
PANOVA
57.12 ± 1.78 25.97 ± 1.39
58.93 ± 2.09 17.68 ± 1.25a
39.03 ± 2.49a , b 40.92 ± 1.82a , b
42.45 ± 2.25a , b 35.61 ± 1.70a , b
<0.001 <0.001
p < 0.001 versus control group. p < 0.001 versus 9 -THC group. Mean ± standard error (SE).
Fig. 1. Immunolocalization of insulin peptide (arrows) were observed in pancreatic tissue cells of experimental rats. Immunoreactive cells labeled for insulin in control (A), 9 -THC (B), diabetes (C) and diabetes + 9 -THC (D) groups. Streptavidin–biotin–peroxidase technique, hematoxylin counterstain. Scale bar = 20 m.
Table 3 Serum total protein (TP), triglyceride (TG) and total cholesterol (TC) levels in experimental groups. Group
TPg (g/dL)
TGg (mg/dL)
Control (n = 8) 9 -THC (n = 6)
5.75 ± 0.15 5.47 ± 0.17
37.63 ± 6.47 28.17 ± 5.85
53.46 ± 2.92 67.10 ± 3.87d
Control (n = 8) 9 -THC (n = 6)
15.73 ± 0.84 16.62 ± 0.72
4.76 ± 0.43 7.93 ± 0.85a
7.52 ± 1.29 5.63 ± 1.17
Diabetes (n = 8) Diabetes + 9 -THC (n = 7)
5.90 ± 0.07a 5.26 ± 0.43
52.38 ± 5.7b , c 44.00 ± 5.86
73.27 ± 5.31d 41.37 ± 3.47b , e , f
Diabetes (n = 8) Diabetes + 9 -THC (n = 7)
22.60 ± 2.07a , b 11.44 ± 0.98a , c , d
7.30 ± 1.01e 4.24 ± 0.69c , f
10.48 ± 1.14c , e 8.80 ± 1.17
PANOVA
NS
<0.05
a b c d e f g
p < 0.05 versus 9 -THC group. p < 0.05 versus control group. p < 0.01 versus 9 -THC group. p < 0.01 versus control group. p < 0.001 versus 9 -THC group. p < 0.001 versus diabetic group. Mean ± standard error (SE); non significant (NS).
TCg (mg/dL)
Table 4 Serum high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels.
<0.001
Group
PANOVA a b c d e f g
HDLg (mg/dL)
<0.001
LDLg (mg/dL)
<0.01
VLDLg (mg/dL)
<0.05
p < 0.01 versus control group. p < 0.05 versus 9 -THC group. p < 0.01 versus 9 -THC group. p < 0.001 versus diabetic group. p < 0.05 versus control group. p < 0.05 versus diabetic group. Mean ± standard error (SE).
Please cite this article in press as: Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.05.013
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Fig. 2. Immunohistochemical localization for glucagon peptide (arrows) in cells of Langerhans islet. Glucagon in control (A), 9 -THC (B), diabetes (C) and diabetes + 9 -THC (D) groups. Streptavidin–biotin–peroxidase technique, counterstain hematoxylin. Scale bar = 20 m.
PANOVA < 0.05, respectively). HDL (a p < 0.01), LDL (e p < 0.05) and VLDL (e p < 0.05) serum levels of diabetic group were significantly higher than the control group. Furthermore, HDL (d p < 0.001) and LDL (f p < 0.05) levels were significantly decreased in diabetes + 9 THC group compared to that of the diabetes group. But, VLDL levels showed an insignificant decrease in diabetic rats treated with 9 THC compared to diabetic animals (Table 4). Discussion It is known that diabetes is associated with hyperglycemia and hyperlipidemia. In this study, we evaluated the effects of 9 -THC on lipid metabolism, glucose levels and the changes in insulin and glucagon immunoreactive cells in type 2 diabetic rats. The blood glucose levels increase in STZ-induced diabetic rats (Kim and Caprio, 2011; Ranasinghe et al., 2012). Our results were consistent with other studies which showed an increase in glucose levels in type 2 diabetic rats compared to control animals. Bermúdez-Silva et al. (2006) suggested that targeting cannabinoid receptors may be a new therapeutic alternative for diabetes. In this study, 9 -THC, a cannabinoid receptor agonist, in the treated diabetes group showed a decrease in blood glucose levels compared to that of the diabetic rats on day 22. Therefore, we suggest that uninterrupted long time treatment with 9 -THC may help decrease blood glucose levels in T2DM. T2DM occurs due to defects in insulin secretion and excess in glucagon secretion. Both of these hormones are released from the pancreatic islets (Virally et al., 2007; Gromada et al., 2009). Cannabinoid receptors (cannabinoid-1 and cannabinoid-2 receptors) are found in human islets of Langerhans and these receptors are related to insulin secretion (Li et al., 2011). It has been reported that the agonists of these receptors stimulate insulin secretion and play a role in glucose homeostasis (Romero-Zerbo et al., 2011). Another study has shown that rimonabant (SR141716), as a
cannabinoid receptor antagonist, reduces insulin secretion in obese rats (Getty-Kaushik et al., 2009). On the contrary, it is suggested by Nakata and Yada (2008) that cannabinoid antagonist improves insulin secretion in obesity. According to our findings, the area of insulin immunoreactive cells showed a slight increase in the group given 9 -THC compared with control animals. It was also observed that area percent of insulin immunoreactive cells increased in diabetes + 9 -THC animals as compared to diabetic rats. The area percent of glucagon immunoreactive cells was decreased in diabetic rats given 9 -THC compared to diabetic group. This indicates that 9 -THC inhibits the expression of glucagon hormone in pancreatic islets, slightly. On the contrary, 9 -THC activates the insulin expression in islets. Li et al. (2011) suggested that both the cannabinoid receptor agonists and antagonists improve insulin secretion. While the glucagon secretion is stimulated by cannabinoid-1 receptor agonist, it is not stimulated by cannabinoid-2 receptor agonist (Bermúdez-Silva et al., 2008). The current literature indicates that serum levels of TG, TC, LDL, and VLDL increase, while, serum HDL levels decrease in diabetic rats compared to healthy rats (Krishnamurthy et al., 2011; Ranasinghe et al., 2012). In liver cells, TC and TG contents elevated by SR141716. HDL uptake increased simultaneously (Jourdan et al., 2012). In another study, it was shown that while SR141716 reduced plasma levels of TC and LDL, it increased the levels of TG and HDL in mice (Valenzuela et al., 2011). On the contrary, the literature suggests that differences in lipid levels have not been observed by chronic treatment with AM4113 which is a cannabinoid-1 receptor antagonist (Cluny et al., 2011). 9 -THC regulates lipid metabolism and causes change in lipoproteins in hashish users (Kalofoutis et al., 1985). In addition, Di Marzo et al. (2011) revealed that cannabinoids influence both lipid and glucose metabolism. According to our findings, an increase of TG, TC, HDL, LDL and VLDL serum levels were normal for diabetic rats as mentioned in previous studies. However, TC and LDL levels were elevated in healthy 9 -THC
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group. As expected, administration of such exogenous agents may impair normal cholesterol levels in healthy individuals. Therefore, 9 -THC may be preferred to treat diabetes. Both in healthy and diabetic animals, administration of 9 -THC caused an insignificant reduction in serum TG levels. STZ + NAD-induced diabetic rats given 9 -THC demonstrated a reduction in serum HDL, LDL and TC levels compared to diabetes group. Furthermore, VLDL levels of diabetes + 9 -THC animals showed a decrease compared to diabetic animals. VLDL and LDL levels were closely related to serum concentrations of TG and TC in diabetes + 9 -THC, respectively. In the diabetic group given 9 -THC, the decrease of HDL levels may be associated with the reduction of blood lipid levels. We determined that using 9 -THC in T2DM caused an alteration in lipid profile. It may serve as a new agent for lipid regulation in diabetes. In conclusion, our findings suggest that 9 -THC treatment may improve blood glucose levels and hyperlipidemia in STZ + NADinduced diabetic rats. It may be to a degree protective against the major hormones in the islets of Langerhans in type 2 diabetes mellitus. Consequently, the present study should be supported with further investigations with regard to use of 9 -THC in treatment of diabetes. Acknowledgment This study was supported by the Scientific Research Projects Coordination Unit of Istanbul University, Project No. 7823. References Bagger JI, Knop FK, Holst JJ, Vilsbøll T. Glucagon antagonism as a potential therapeutic target in type 2 diabetes. Diabetes Obes Metab 2011;13:965–71. Bermúdez-Silva FJ, Sanchez-Vera I, Suárez J, Serrano A, Fuentes E, Juan-Pico P, et al. Role of cannabinoid CB2 receptors in glucose homeostasis in rats. Eur J Pharmacol 2007;565:207–11. Bermúdez-Silva FJ, Serrano A, Diaz-Molina FJ, Sánchez Vera I, Juan-Pico P, Nadal A, et al. Activation of cannabinoid CB1 receptors induces glucose intolerance in rats. Eur J Pharmacol 2006;531:282–4. Bermúdez-Silva FJ, Suárez J, Baixeras E, Cobo N, Bautista D, Cuesta˜ AL, et al. Presence of functional cannabinoid receptors in Munoz human endocrine pancreas. Diabetologia 2008;51:476–87. Comelli F, Betttoni I, Colleoni M, Giagnoni G, Costa B. Beneficial effects of a Cannabis sativa extract treatment on diabetes-induced neuropathy and oxidative stress. Phytother Res 2009;23:1678–84. Cluny NL, Chambers AP, Vemuri VK, Wood JT, Eller LK, Freni C, et al. The neutral cannabinoid CB1 receptor antagonist AM4113 regulates body weight through changes in energy intake in the rat. Pharmacol Biochem Behav 2011;97:537–43. Di Marzo V, Piscitelli F, Mechoulam R. Cannabinoids and endocannabinoids in metabolic disorders with focus on diabetes. Handb Exp Pharmacol 2011;203:75–104. Fishbein M, Gov S, Assaf F, Gafni M, Keren O, Sarne Y. Longterm behavioral and biochemical effects of an ultra-low dose of (9)-tetrahydrocannabinol (THC): neuroprotection and ERK signaling. Exp Brain Res 2012;221:437–48. Getty-Kaushik L, Richard AM, Deeney JT, Krawczyk S, Shirihai O, Corkey BE. The CB1 antagonist rimonabant decreases insulin hypersecretion in rat pancreatic islets. Obesity (Silver Spring) 2009;17:1856–60. Gromada J, Duttaroy A, Rorsman P. The insulin receptor talks to glucagon? Cell Metab 2009;9:303–5.
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Jourdan T, Demizieux L, Gresti J, Djaouti L, Gaba L, Vergès B, et al. Antagonism of peripheral hepatic cannabinoid receptor-1 improves liver lipid metabolism in mice: evidence from cultured explants. Hepatology 2012;55:790–9. Kalofoutis A, Dionyssiou-Asteriou A, Maravelias C, Koutselinis A. Changes of HDL–lipid composition as related to delta 9-THC action. Pharmacol Biochem Behav 1985;22:343–5. Kim G, Caprio S. Diabetes and insulin resistance in pediatric obesity. Pediatr Clin North Am 2011;58:1355–61. Klausner HA, Wilcox HG, Dingell JV. The use of zonal ultracentrifugation in the investigation of the binding of delta9tetrahydrocannabinol by plasma lipoproteins. Drug Metab Dispos 1975;3:314–9. Kochanowski M, Kala M. Tetrahydrocannabinols in clinical and forensic toxicology. Przegl Lek 2005;62:576–80. Krishnamurthy G, Lakshman K, Pruthvi N, Chandrika PU. Antihyperglycemic and hypolipidemic activity of methanolic extract of Amaranthus viridis leaves in experimental diabetes. Indian J Pharmacol 2011;43:450–4. Li C, Bowe JE, Huang GC, Amiel SA, Jones PM, Persaud SJ. Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans. Diabetes Obes Metab 2011;13:903–10. Li C, Bowe JE, Jones PM, Persaud SJ. Expression and function of cannabinoid receptors in mouse islets. Islets 2010;2:293–302. Masiello P, Broca C, Gross R, Roye M, Manteghetti M, Hillaire-Buys D, et al. Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 1998;47:224–9. Nakata M, Yada T. Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet -cells via CB1 receptors. Regul Pept 2008;145:49–53. Ranasinghe P, Perera S, Gunatilake M, Abeywardene E, Gunapala N, Premakumara S, et al. Effects of Cinnamomum zeylanicum (Ceylon cinnamon) on blood glucose and lipids in a diabetic and healthy rat model. Pharmacognosy Res 2012;4:73–9. Romero-Zerbo SY, Rafacho A, Díaz-Arteaga A, Suárez J, Quesada I, Imbernon M, et al. A role for the putative cannabinoid receptor GPR55 in the islets of Langerhans. J Endocrinol 2011;211:177–85. Shinde SN, Dhadke VN, Suryakar AN. Evaluation of oxidative stress in type 2 diabetes mellitus and follow-up along with vitamin E supplementation. Indian J Clin Biochem 2011;26:74–7. Valenzuela CA, Castillo VA, Aguirre CA, Ronco AM, Llanos MN. The CB1 receptor antagonist SR 141716A reverses adult male mice overweight and metabolic alterations induced by early stress. Obesity (Silver Spring) 2011;19:29–35. Vilches-Flores A, Delgado-Buenrostro NL, Navarrete-Vázquez G, Villalobos-Molina R. CB1 cannabinoid receptor expression is regulated by glucose and feeding in rat pancreatic islets. Regul Pept 2010;163:81–7. Virally M, Blicklé JF, Girard J, Halimi S, Simon D, Guillausseau PJ. Type 2 diabetes mellitus: epidemiology, pathophysiology, unmet needs and therapeutical perspectives. Diabetes Metab 2007;33:231–44. Waldman M, Hochhauser E, Fishbein M, Aravot D, Shainberg A, Sarne Y. An ultra-low dose of tetrahydrocannabinol provides cardioprotection. Biochem Pharmacol 2013;85:1626–33. Wong A, Gunasekaran N, Hancock DP, Denyer GS, Meng L, Radford JL, et al. The major plant-derived cannabinoid (9)tetrahydrocannabinol promotes hypertrophy and macrophage infiltration in adipose tissue. Horm Metab Res 2012;44: 105–13.
Please cite this article in press as: Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.05.013
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Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats Zeynep Mine Coskun a,b,∗ , Sema Bolkent b a b
Health Services Vocational School, Istanbul Bilim University, Istanbul, Turkey Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 15 April 2013 Received in revised form 29 May 2013 Accepted 30 May 2013 Available online xxx Keywords: 9 -THC Type 2 diabetes Insulin Glucagon Pancreas Rat
a b s t r a c t The regulation of glucose, lipid metabolism and immunoreactivities of insulin and glucagon peptides by delta-9-tetrahydrocannabinol (9 -THC) in diabetes were examined in an experimental rat model. Male Sprague-Dawley rats were divided into four groups: (1) control, (2) 9 -THC treated, (3) diabetic, and (4) diabetic + 9 -THC. The type 2 diabetic rat model was established by intraperitoneal (i.p.) injection of nicotinamide (85 mg/kg body weight) followed after 15 min by i.p. injection of streptozotocin (STZ) at 65 mg/kg of body weight. 9 -THC and 9 -THC treated diabetic groups received 3 mg/kg/day of 9 -THC for 7 days. The immunolocalization of insulin and glucagon peptides was investigated in the pancreas using a streptavidin–biotin–peroxidase technique. High density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL), triglycerides (TG), total cholesterol (TC) and total protein (TP) levels were measured in serum. Total islet area percent of insulin immunoreactive cells slightly changed in diabetic + 9 -THC rats compared to diabetic animals. However, the area percent of glucagon immunoreactive cells showed a decrease in diabetic + 9 -THC rats compared to that of diabetic animals alone. Serum TC, HDL and LDL levels of diabetes + 9 -THC group showed a decrease compared to the diabetic group. These results indicate that 9 -THC may serve a protective role against hyperlipidemia and hyperglycemia in diabetic rats. © 2013 Elsevier GmbH. All rights reserved.
Introduction The cannabinoids are natural constituents of Cannabis sativa L. commonly known as marijuana. Delta-9-tetrahydrocannabinol (9 -THC), cannabinol (CBN) and cannabidiol (CBD) are the main constituents of C. sativa L. (Kochanowski and Kala, 2005). 9 -THC is the main psychoactive compound that activates cannabinoid receptors. Cannabinoids influence lipid and glucose metabolism (Kalofoutis et al., 1985; Di Marzo et al., 2011). In addition, cannabinoids are associated with oxidative damage and lipoproteins. Some studies have shown that 9 -THC may have a protective role against neurodegenerative diseases and provide cardioprotection in animals (Fishbein et al., 2012; Waldman et al., 2013). It has been reported that there are some beneficial effects of cannabis extract
Abbreviations: ␣-cell, alpha cell; AEC, 3-amino-9-ethyl-carbazole; ANOVA, analysis of variance; -cell, beta cell; CBD, cannabidiol; CBN, cannabinol; 9 -THC, delta-9-tetrahydrocannabinol; i.p., intraperitoneal; HDL, high density lipoprotein cholesterol; LDL, low-density lipoprotein; NAD, nicotinamide; STZ, streptozotocin; T2DM, type 2 diabetes mellitus; TC, total cholesterol; TG, triglyceride; TP, total protein; VLDL, very low density lipoprotein cholesterol. ∗ Corresponding author at: Health Services Vocational School, Istanbul Bilim University, 34394 Sisli, Istanbul, Turkey. E-mail address: [email protected] (Z.M. Coskun).
on diabetes-induced neuropathic pain through its strong antioxidant activity (Klausner et al., 1975; Comelli et al., 2009). With regard to 9 -THC, which acts as a cannabinoid receptor agonist, it has been shown to activate lipoprotein lipase, regulate cannabinoid receptor expression and beta cell (-cell) function in pancreatic islets (Li et al., 2010; Vilches-Flores et al., 2010; Wong et al., 2012). As for endogenous cannabinoids, they have a role in the regulation of secretion of pancreatic endocrine hormones. The activation of cannabinoid receptors stimulates insulin secretion to maintain glucose homeostasis (Bermúdez-Silva et al., 2007; Li et al., 2011). Diabetes mellitus is frequently complicated by hyperlipidemia and hyperglycemia with an increase in blood glucose levels accompanied by an increase in blood levels of low-density lipoproteins (LDL), total cholesterol (TC) and triglycerides (TG) (Kim and Caprio, 2011; Ranasinghe et al., 2012). In recent years, the prevalence of type 2 diabetes mellitus (T2DM) has increased dramatically and it comprises approximately 90% of all cases of diabetes. The pathophysiology of T2DM is a complex process and involves insulin resistance and insufficient pancreatic -cell function (Kim and Caprio, 2011). In patients with T2DM, a decrease in insulin production occurs due to -cell destruction. Insulin secreted by the beta cells (-cells) and glucagon secreted by alpha cells (␣-cells) of the pancreatic islets are key hormones in the regulation of blood glucose levels (Bagger et al., 2011; Shinde et al., 2011).
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In the current study, we examined whether glucose levels and lipid metabolism were affected by the administration of 9 -THC in type 2 diabetic rats induced by streptozotocin (STZ) + nicotinamide (NAD). In addition, the effects of 9 -THC on immunolocalization of insulin and glucagon secreting cells in pancreatic islets were investigated.
Biochemical analysis Serum high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL), triglycerides (TG), total cholesterol (TC) and total protein (TP) levels were measured by enzymatic methods using commercial kits on a Siemens Advia 1800 chemistry system (Erlangen, Germany).
Materials and methods Statistical analysis Experimental design 8–10 week-old male Sprague-Dawley rats (average weight 214 g) were obtained from Istanbul University, Experimental Medical Research Institute. All experiments were carried out in accordance with the guidelines of Istanbul University, Local Ethics Committee on Animal Research. All rats were housed in a temperature-controlled clean room with a 12 h light, 12 h dark cycle and fed with standard chow diet and tap water ad libitum. The animals were selected randomly and divided into four groups: Group 1: (n = 8) control rats that received a single intraperitoneal (i.p.) saline dose. Group 2: (n = 6) rats received a single dose (i.p.) of 3 mg/kg/day of delta-9-tetrahydrocannabinol (9 THC) (Lipomed, Arlesheim, Switzerland; THC-135-100LE) for 7 days. Group 3: (n = 8) rats were rendered diabetic by i.p. injection of a single dose of nicotinamide (NAD) (85 mg/kg, Sigma–Aldrich, St. Louis, MO, USA; N3376) in saline 15 min before injection (i.p.) of streptozotocin (STZ) (65 mg/kg, Sigma–Aldrich, St. Louis, MO, USA; S0130) dissolved in saline (Masiello et al., 1998). The blood glucose levels were determined 72 h after STZ + NAD injection. The rats with blood glucose concentrations more than 200 mg/dL were accepted as diabetic. Group 4: (n = 7) diabetic rats treated (i.p.) daily with 9 -THC (3 mg/kg/day) during 7 days. Animals were anesthetized (i.p.) with a freshly prepared mixture of ketamine hydrochloride (50 mg/kg, Ketalar, Pfizer, New York, NY, USA) and xylazine hydrochloride (10 mg/kg, Rompun, Bayer, Toronto, ON, Canada) on day 15 after 9 -THC injections. The blood samples were taken under anesthesia and the animals were sacrificed. After sacrifice, the pancreatic tissue samples were immediately taken and fixed in 10% neutral buffered formalin for immunohistochemical analysis and serum samples were stored at −86 ◦ C for biochemical analysis. Blood glucose levels of rats in all groups were measured with a glucometer (Accu-check, Roche Diagnostics, GmbH, Mannheim, Germany) using blood obtained from the tail vein on day 22.
Immunohistochemical staining Immunohistochemical staining was performed on 5 m thick formalin-fixed, paraffin-embedded sections of pancreas using a streptavidin–biotin–peroxidase method. The sections were mounted on glass slides previously treated with polyl-lysine. The sections were incubated with antibody against insulin (Sigma–Aldrich, St. Louis, MO, USA; I2018) and glucagon (Sigma–Aldrich; G2654), with 1:1000 and 1:2000 dilutions for 1–1.5 h at room temperature, respectively. The reaction was revealed with 3-amino-9-ethyl-carbazole (AEC) as chromogen, and the sections were counterstained with Mayer’s hematoxylin. Finally, slides were mounted in glycerol gelatine and stored at +4 ◦ C. In negative controls, the primary antibodies were omitted. The area percent of insulin and glucagon immunoreactive cells were evaluated using a Nikon microscope (Eclipse 80i, Melville, NY, USA) equipped with a digital camera using NIS-Elements-D 3.1 microscope imaging software program (Melville, NY, USA).
All values were analyzed by statistical software (GraphPad Prism version 5.0 computer package, San Diego, CA, USA). Data were expressed as means ± SE. A comparison between two groups was performed using Mann–Whitney U non-parametric test and a comparison among multiple groups was performed using one-way analysis of variance (ANOVA) followed by a Kruskal–Wallis test. A value of p < 0.05 was considered statistically significant. The area percent occupied by immunoreactive cells insulin and glucagon in pancreatic islets was calculated by measuring (labeling area/total area) × 100. Results Blood glucose levels Significant changes in blood glucose levels were determined among the four groups (PANOVA < 0.001) on day 22. Blood glucose level showed a significant difference between control rats and diabetic animals (a p < 0.001) (Table 1). 9 -THC treated diabetes group showed an insignificant decrease in blood glucose levels compared to that of the diabetic rats on day 22 (Table 1). Immunohistochemistry The changes of insulin and glucagon immunoreactive cell by percentage in islets of Langerhans are shown in Table 2. Insulin and glucagon peptide immunolocalized cells were evaluated as statistically significant among all groups (PANOVA < 0.001 and PANOVA < 0.001, respectively). While the insulin immunoreactive cells were significantly decreased (a p < 0.001), glucagon immunoreactive cells were significantly increased (a p < 0.001) in the pancreatic islets of diabetic rats as compared to control group. Furthermore, an insignificant increase was observed in the area of insulin immunoreactive cells in diabetes + 9 -THC rats as compared with diabetic rats (Fig. 1). However, glucagon immunoreactive cells were increased significantly in diabetic rats compared to control rats (a p < 0.001). Glucagon immunoreactive cells showed an insignificant decrease in diabetes + 9 -THC rats as compared with diabetic rats (Fig. 2). Biochemistry The results of the biochemical parameters are summarized in Tables 3 and 4. TP levels of serum samples did not reveal a statistically significant difference, but TG (PANOVA < 0.05) and TC (PANOVA < 0.001) levels showed a statistically significant decrease among all groups. TG levels of the diabetic group were significantly higher than the control group (b p < 0.05), but it was statistically insignificant between diabetic rats and diabetes + 9 -THC group. There was a significant increase in TC levels in diabetic rats compared to control animals (d p < 0.01). TC levels decreased significantly in 9 -THC given diabetic rats compared to diabetic (f p < 0.001) group (Table 3). Lipoprotein types (HDL, LDL and VLDL) were statistically significant among all groups (PANOVA < 0.001, PANOVA < 0.01 and
Please cite this article in press as: Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.05.013
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Table 1 Blood glucose levels of all groups on day 22.
c
Blood glucose levels (g/dL) a b c
Control (n = 8)
9 -THC (n = 6)
Diabetes (n = 8)
Diabetes + 9 -THC (n = 7)
PANOVA
114.8 ± 5.0
124.8 ± 8.6
397.5 ± 28.0
320.7 ± 40.8a , b
<0.001
a,b
p < 0.001 versus control group. p < 0.01 versus 9 -THC group. Mean ± standard error (SE).
Table 2 The area percent of immunoreactive cells for insulin and glucagon in pancreatic islets of all groups.
Insulinc Glucagonc a b c
Control (n = 7)
9 -THC (n = 6)
Diabetes (n = 7)
Diabetes + 9 -THC (n = 7)
PANOVA
57.12 ± 1.78 25.97 ± 1.39
58.93 ± 2.09 17.68 ± 1.25a
39.03 ± 2.49a , b 40.92 ± 1.82a , b
42.45 ± 2.25a , b 35.61 ± 1.70a , b
<0.001 <0.001
p < 0.001 versus control group. p < 0.001 versus 9 -THC group. Mean ± standard error (SE).
Fig. 1. Immunolocalization of insulin peptide (arrows) were observed in pancreatic tissue cells of experimental rats. Immunoreactive cells labeled for insulin in control (A), 9 -THC (B), diabetes (C) and diabetes + 9 -THC (D) groups. Streptavidin–biotin–peroxidase technique, hematoxylin counterstain. Scale bar = 20 m.
Table 3 Serum total protein (TP), triglyceride (TG) and total cholesterol (TC) levels in experimental groups. Group
TPg (g/dL)
TGg (mg/dL)
Control (n = 8) 9 -THC (n = 6)
5.75 ± 0.15 5.47 ± 0.17
37.63 ± 6.47 28.17 ± 5.85
53.46 ± 2.92 67.10 ± 3.87d
Control (n = 8) 9 -THC (n = 6)
15.73 ± 0.84 16.62 ± 0.72
4.76 ± 0.43 7.93 ± 0.85a
7.52 ± 1.29 5.63 ± 1.17
Diabetes (n = 8) Diabetes + 9 -THC (n = 7)
5.90 ± 0.07a 5.26 ± 0.43
52.38 ± 5.7b , c 44.00 ± 5.86
73.27 ± 5.31d 41.37 ± 3.47b , e , f
Diabetes (n = 8) Diabetes + 9 -THC (n = 7)
22.60 ± 2.07a , b 11.44 ± 0.98a , c , d
7.30 ± 1.01e 4.24 ± 0.69c , f
10.48 ± 1.14c , e 8.80 ± 1.17
PANOVA
NS
<0.05
a b c d e f g
p < 0.05 versus 9 -THC group. p < 0.05 versus control group. p < 0.01 versus 9 -THC group. p < 0.01 versus control group. p < 0.001 versus 9 -THC group. p < 0.001 versus diabetic group. Mean ± standard error (SE); non significant (NS).
TCg (mg/dL)
Table 4 Serum high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels.
<0.001
Group
PANOVA a b c d e f g
HDLg (mg/dL)
<0.001
LDLg (mg/dL)
<0.01
VLDLg (mg/dL)
<0.05
p < 0.01 versus control group. p < 0.05 versus 9 -THC group. p < 0.01 versus 9 -THC group. p < 0.001 versus diabetic group. p < 0.05 versus control group. p < 0.05 versus diabetic group. Mean ± standard error (SE).
Please cite this article in press as: Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.05.013
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Fig. 2. Immunohistochemical localization for glucagon peptide (arrows) in cells of Langerhans islet. Glucagon in control (A), 9 -THC (B), diabetes (C) and diabetes + 9 -THC (D) groups. Streptavidin–biotin–peroxidase technique, counterstain hematoxylin. Scale bar = 20 m.
PANOVA < 0.05, respectively). HDL (a p < 0.01), LDL (e p < 0.05) and VLDL (e p < 0.05) serum levels of diabetic group were significantly higher than the control group. Furthermore, HDL (d p < 0.001) and LDL (f p < 0.05) levels were significantly decreased in diabetes + 9 THC group compared to that of the diabetes group. But, VLDL levels showed an insignificant decrease in diabetic rats treated with 9 THC compared to diabetic animals (Table 4). Discussion It is known that diabetes is associated with hyperglycemia and hyperlipidemia. In this study, we evaluated the effects of 9 -THC on lipid metabolism, glucose levels and the changes in insulin and glucagon immunoreactive cells in type 2 diabetic rats. The blood glucose levels increase in STZ-induced diabetic rats (Kim and Caprio, 2011; Ranasinghe et al., 2012). Our results were consistent with other studies which showed an increase in glucose levels in type 2 diabetic rats compared to control animals. Bermúdez-Silva et al. (2006) suggested that targeting cannabinoid receptors may be a new therapeutic alternative for diabetes. In this study, 9 -THC, a cannabinoid receptor agonist, in the treated diabetes group showed a decrease in blood glucose levels compared to that of the diabetic rats on day 22. Therefore, we suggest that uninterrupted long time treatment with 9 -THC may help decrease blood glucose levels in T2DM. T2DM occurs due to defects in insulin secretion and excess in glucagon secretion. Both of these hormones are released from the pancreatic islets (Virally et al., 2007; Gromada et al., 2009). Cannabinoid receptors (cannabinoid-1 and cannabinoid-2 receptors) are found in human islets of Langerhans and these receptors are related to insulin secretion (Li et al., 2011). It has been reported that the agonists of these receptors stimulate insulin secretion and play a role in glucose homeostasis (Romero-Zerbo et al., 2011). Another study has shown that rimonabant (SR141716), as a
cannabinoid receptor antagonist, reduces insulin secretion in obese rats (Getty-Kaushik et al., 2009). On the contrary, it is suggested by Nakata and Yada (2008) that cannabinoid antagonist improves insulin secretion in obesity. According to our findings, the area of insulin immunoreactive cells showed a slight increase in the group given 9 -THC compared with control animals. It was also observed that area percent of insulin immunoreactive cells increased in diabetes + 9 -THC animals as compared to diabetic rats. The area percent of glucagon immunoreactive cells was decreased in diabetic rats given 9 -THC compared to diabetic group. This indicates that 9 -THC inhibits the expression of glucagon hormone in pancreatic islets, slightly. On the contrary, 9 -THC activates the insulin expression in islets. Li et al. (2011) suggested that both the cannabinoid receptor agonists and antagonists improve insulin secretion. While the glucagon secretion is stimulated by cannabinoid-1 receptor agonist, it is not stimulated by cannabinoid-2 receptor agonist (Bermúdez-Silva et al., 2008). The current literature indicates that serum levels of TG, TC, LDL, and VLDL increase, while, serum HDL levels decrease in diabetic rats compared to healthy rats (Krishnamurthy et al., 2011; Ranasinghe et al., 2012). In liver cells, TC and TG contents elevated by SR141716. HDL uptake increased simultaneously (Jourdan et al., 2012). In another study, it was shown that while SR141716 reduced plasma levels of TC and LDL, it increased the levels of TG and HDL in mice (Valenzuela et al., 2011). On the contrary, the literature suggests that differences in lipid levels have not been observed by chronic treatment with AM4113 which is a cannabinoid-1 receptor antagonist (Cluny et al., 2011). 9 -THC regulates lipid metabolism and causes change in lipoproteins in hashish users (Kalofoutis et al., 1985). In addition, Di Marzo et al. (2011) revealed that cannabinoids influence both lipid and glucose metabolism. According to our findings, an increase of TG, TC, HDL, LDL and VLDL serum levels were normal for diabetic rats as mentioned in previous studies. However, TC and LDL levels were elevated in healthy 9 -THC
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group. As expected, administration of such exogenous agents may impair normal cholesterol levels in healthy individuals. Therefore, 9 -THC may be preferred to treat diabetes. Both in healthy and diabetic animals, administration of 9 -THC caused an insignificant reduction in serum TG levels. STZ + NAD-induced diabetic rats given 9 -THC demonstrated a reduction in serum HDL, LDL and TC levels compared to diabetes group. Furthermore, VLDL levels of diabetes + 9 -THC animals showed a decrease compared to diabetic animals. VLDL and LDL levels were closely related to serum concentrations of TG and TC in diabetes + 9 -THC, respectively. In the diabetic group given 9 -THC, the decrease of HDL levels may be associated with the reduction of blood lipid levels. We determined that using 9 -THC in T2DM caused an alteration in lipid profile. It may serve as a new agent for lipid regulation in diabetes. In conclusion, our findings suggest that 9 -THC treatment may improve blood glucose levels and hyperlipidemia in STZ + NADinduced diabetic rats. It may be to a degree protective against the major hormones in the islets of Langerhans in type 2 diabetes mellitus. Consequently, the present study should be supported with further investigations with regard to use of 9 -THC in treatment of diabetes. Acknowledgment This study was supported by the Scientific Research Projects Coordination Unit of Istanbul University, Project No. 7823. References Bagger JI, Knop FK, Holst JJ, Vilsbøll T. Glucagon antagonism as a potential therapeutic target in type 2 diabetes. Diabetes Obes Metab 2011;13:965–71. Bermúdez-Silva FJ, Sanchez-Vera I, Suárez J, Serrano A, Fuentes E, Juan-Pico P, et al. Role of cannabinoid CB2 receptors in glucose homeostasis in rats. Eur J Pharmacol 2007;565:207–11. Bermúdez-Silva FJ, Serrano A, Diaz-Molina FJ, Sánchez Vera I, Juan-Pico P, Nadal A, et al. Activation of cannabinoid CB1 receptors induces glucose intolerance in rats. Eur J Pharmacol 2006;531:282–4. Bermúdez-Silva FJ, Suárez J, Baixeras E, Cobo N, Bautista D, Cuesta˜ AL, et al. Presence of functional cannabinoid receptors in Munoz human endocrine pancreas. Diabetologia 2008;51:476–87. Comelli F, Betttoni I, Colleoni M, Giagnoni G, Costa B. Beneficial effects of a Cannabis sativa extract treatment on diabetes-induced neuropathy and oxidative stress. Phytother Res 2009;23:1678–84. Cluny NL, Chambers AP, Vemuri VK, Wood JT, Eller LK, Freni C, et al. The neutral cannabinoid CB1 receptor antagonist AM4113 regulates body weight through changes in energy intake in the rat. Pharmacol Biochem Behav 2011;97:537–43. Di Marzo V, Piscitelli F, Mechoulam R. Cannabinoids and endocannabinoids in metabolic disorders with focus on diabetes. Handb Exp Pharmacol 2011;203:75–104. Fishbein M, Gov S, Assaf F, Gafni M, Keren O, Sarne Y. Longterm behavioral and biochemical effects of an ultra-low dose of (9)-tetrahydrocannabinol (THC): neuroprotection and ERK signaling. Exp Brain Res 2012;221:437–48. Getty-Kaushik L, Richard AM, Deeney JT, Krawczyk S, Shirihai O, Corkey BE. The CB1 antagonist rimonabant decreases insulin hypersecretion in rat pancreatic islets. Obesity (Silver Spring) 2009;17:1856–60. Gromada J, Duttaroy A, Rorsman P. The insulin receptor talks to glucagon? Cell Metab 2009;9:303–5.
5
Jourdan T, Demizieux L, Gresti J, Djaouti L, Gaba L, Vergès B, et al. Antagonism of peripheral hepatic cannabinoid receptor-1 improves liver lipid metabolism in mice: evidence from cultured explants. Hepatology 2012;55:790–9. Kalofoutis A, Dionyssiou-Asteriou A, Maravelias C, Koutselinis A. Changes of HDL–lipid composition as related to delta 9-THC action. Pharmacol Biochem Behav 1985;22:343–5. Kim G, Caprio S. Diabetes and insulin resistance in pediatric obesity. Pediatr Clin North Am 2011;58:1355–61. Klausner HA, Wilcox HG, Dingell JV. The use of zonal ultracentrifugation in the investigation of the binding of delta9tetrahydrocannabinol by plasma lipoproteins. Drug Metab Dispos 1975;3:314–9. Kochanowski M, Kala M. Tetrahydrocannabinols in clinical and forensic toxicology. Przegl Lek 2005;62:576–80. Krishnamurthy G, Lakshman K, Pruthvi N, Chandrika PU. Antihyperglycemic and hypolipidemic activity of methanolic extract of Amaranthus viridis leaves in experimental diabetes. Indian J Pharmacol 2011;43:450–4. Li C, Bowe JE, Huang GC, Amiel SA, Jones PM, Persaud SJ. Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans. Diabetes Obes Metab 2011;13:903–10. Li C, Bowe JE, Jones PM, Persaud SJ. Expression and function of cannabinoid receptors in mouse islets. Islets 2010;2:293–302. Masiello P, Broca C, Gross R, Roye M, Manteghetti M, Hillaire-Buys D, et al. Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 1998;47:224–9. Nakata M, Yada T. Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet -cells via CB1 receptors. Regul Pept 2008;145:49–53. Ranasinghe P, Perera S, Gunatilake M, Abeywardene E, Gunapala N, Premakumara S, et al. Effects of Cinnamomum zeylanicum (Ceylon cinnamon) on blood glucose and lipids in a diabetic and healthy rat model. Pharmacognosy Res 2012;4:73–9. Romero-Zerbo SY, Rafacho A, Díaz-Arteaga A, Suárez J, Quesada I, Imbernon M, et al. A role for the putative cannabinoid receptor GPR55 in the islets of Langerhans. J Endocrinol 2011;211:177–85. Shinde SN, Dhadke VN, Suryakar AN. Evaluation of oxidative stress in type 2 diabetes mellitus and follow-up along with vitamin E supplementation. Indian J Clin Biochem 2011;26:74–7. Valenzuela CA, Castillo VA, Aguirre CA, Ronco AM, Llanos MN. The CB1 receptor antagonist SR 141716A reverses adult male mice overweight and metabolic alterations induced by early stress. Obesity (Silver Spring) 2011;19:29–35. Vilches-Flores A, Delgado-Buenrostro NL, Navarrete-Vázquez G, Villalobos-Molina R. CB1 cannabinoid receptor expression is regulated by glucose and feeding in rat pancreatic islets. Regul Pept 2010;163:81–7. Virally M, Blicklé JF, Girard J, Halimi S, Simon D, Guillausseau PJ. Type 2 diabetes mellitus: epidemiology, pathophysiology, unmet needs and therapeutical perspectives. Diabetes Metab 2007;33:231–44. Waldman M, Hochhauser E, Fishbein M, Aravot D, Shainberg A, Sarne Y. An ultra-low dose of tetrahydrocannabinol provides cardioprotection. Biochem Pharmacol 2013;85:1626–33. Wong A, Gunasekaran N, Hancock DP, Denyer GS, Meng L, Radford JL, et al. The major plant-derived cannabinoid (9)tetrahydrocannabinol promotes hypertrophy and macrophage infiltration in adipose tissue. Horm Metab Res 2012;44: 105–13.
Please cite this article in press as: Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.05.013