Vol.2, No.1, 27-34 (2012) Journal of Diabetes Mellitus http://dx.doi.org/10.4236/jdm.2012.21005 Islet protection and amelioration of diabetes type 2 in Psammomys obesus by treatment with cannabidiol* Ziv Ehud1, Weiss Lola1, Raz Itamar1, Patlas Natan1, Yekhtin Zhanna2, Gallily Ruth2# 1Diabetes Unit, Hadassah University Hospital, Jerusalem, Israel 2Lautenberg Center for Immunology, Institute Medical Research Israel Canada (IMRIC), Medical Faculty, Hebrew University, Jeru- salem, Israel; #Corresponding Author: ruthg@ekmd.huji.ac.il Received 19 December 2011; revised 18 January 2012; accepted 31 January 2012 ABSTRACT Background and Purpose: Cannabidiol (CBD), a non-psychoactive component of Cannabis sa- tiva, has been shown by us, to have an anti-in- flammatory effect in collagen-induced arthri tis in DBA mice and in type 1 diabetes in NOD mice. As inflammation is a process involved in diabe- tes type 2, we administered CBD to Psammomys obesus (sand rats), a species which develops diabetes type 2 when fed high-energy (HE) diet, to investigate whether we can hinder the devel- opment of the disease. Experimental Approach: Male Psammomys obesus were kept on a high energy diet during the experiments. They were treated with CBD (i.p injection, 5 mg/kg, 5 times/ week) for 4 weeks and kept (without CBD) for another 29 - 39 days. The weights of the animals as well as blood glucose and plasma insulin levels were determined and the morphology of the p ancreatic islet s was examined. Key Result s: CBD significantly reduced blood glucose levels in Psammomys obesus, without effecting body weight. Plasma insulin levels were significantly higher in the CBD-treated group. The most strik- ing effect noted was the marked decrease of the destruction of pancreatic islets and beta cells. Conclusions and Implications: CBD partially protects pancreatic islets and beta cells from destruction. CBD lowers significantly the blood glucose level and increases insulin level in Psammomys obesus with diabetes type 2, but does not lead to obesity. As CBD already has been administered to patients for other medical indications we propose its use as a therapeutic agent in diabetes type 2. Keywords: Cannabidiol; Type 2 Diabetes; Islet Protection; Psammomys obesus 1. INTRODUCTION Obesity and type 2 diabetes (T2D) have reached epi- demic proportions in the Western world. T2D is a par- ticularly heterogeneous disorder, despite the many clini- cal similarities seen in diabetic patients, including hyper- glycemia, hypeinsulinemia hyperlipidemia, hypertension and obesity. There is crosstalk between the various tis- sues involved in the diabetes syndromes: adipose tissue, muscle and liver via signals that include free fatty acids and adipokines. Several investigators have shown sig- nificant increased in pro-inflammation cytokines such as IL6, TNF-α and glucose intolerance [1-3]. Cannabidiol (CBD) is a constituent of the Cannabis sativa plant, which does not cause psychoactive effects due to its low affinity binding to the CB1 receptor. CBD potential therapeutic activity was documented in several reviews [4-6]. CBD has been shown to posses immuno- modulation [7] and anti-inflammation properties [8,9] It also inhibited the release of the pro-inflammatory cyto- kines IL-1, TNF-α and IFN-γ by peripheral blood mono- nuclear cells [10]. We have reported that CBD signifi- cantly inhibited insulitis, beta cell destruction and the occurrence of overt type 1 diabetes in NOD female mice [11,12]. We have also found that CBD treatment sup- pressed the production of the Th1-associated cytokines, IL-12, IFN-γ and TNF-α and enhanced production of the Th2-associated cytokines, IL-4 and IL-10, suggesting a possible gradual progression from destructive Th1 im- munity to protective Th2 immunity [11,12]. We previously demonstrated that CBD was effective in suppressing the progression of autoimmune joint de- struction in the collagen-induced arthritis animal model of rheumatoid arthritis, a Th1-mediated disease [13]. The anti-autoimmune effects of CBD were associated with reduction in synovial cell TNF-α production, inhibition of reactive oxygen release from zymosan-stimulated *Conflict of interest: The authors declare no conflict of interest. Copyright © 2012 SciRes. OPEN ACCESS
Z. Ehud et al. / Journal of Diabetes Mellitus 2 (2012) 27-34 28 neutrophils and suppression of joint-specific T-cell pro- liferation and IFN-γ production. Although diabetes type 1 differs from diabetes type 2, there are many similar manifestations, such as enhancement of the levels of proinflammation cytokines and free radicals. We decided to examine the effects of CBD in the Psam- momys obesus, a model of type 2 diabetes, since CBD inhibits the production of IL-1b, TNF-α and IFN-γ, cyto- kines that are involved in the beta cells destruction lead- ing to diabetes. The Psammomys obesus in nature feeds on salt bush, which supply most of its nutrients and water. When they are fed a high-energy (HE) diet, they develop diabetes. The Jerusalem colony of Psammomys obesus was established from animals from the Dead Sea region. Generally four stages of consecutive progression to dia- betes in this species are defined, namely Stage A: Basal normoglycemia and normoinsulinemia. Stage B: Hyper- insulinemia (ranging from 120 to 300 mU/L), while ani- mals remain normoglycemic and gain weight. Stage C: Documentation which entails a marked hyperglycemia together with both hyperinsulinemia and hyperproinsu- linemia and further obesity. Stage D: (6 - 12 weeks after stage C) Low plasma insulin, increase of blood glucose, hyperlipidemia, and body weight loss [14]. In this study we examined the effect of CBD on the development of diabetes in male sand rats feeding on high diet, by assaying glucose and insulin levels in blood and analyzing the islets and beta cell integrity in histo- logical sections of the pancreta 2. MATERIAL AND METHODS 2.1. CBD CBD was extracted from cannabis resin (hashish) as previously reported [15]. For in vivo injection, CBD was first dissolved in etha- nol and then Cremophor EL (Sigma) was added up to a 1:1 ratio. This solution was further diluted in saline so that the final solution was ethanol/Cremophor/saline (1:1:18). 2.2. Psammomys obesus Psammomys, 4.5 month old, male (Hebrew University Colony, Harlan, Jerusalem, Israel) were fed by a low energy (LE) diet, normoglycemia maintaining diet (2.38 kcal/g; Koffolk, Petach-Tikva, Israel). Diabetes was in- duced by feeding the animals a high-energy (HE) diet (2.92 kcal/g; cat #2018, Teklad Global Diets, Boston, MA) [14,16]. All experiments were authorized by the Institutional Animal Care Committee. 2.3. Experimental Protocols In the first experiment, 30 Psammomys were divided into two groups: 15 were treated with CBD and the other 15 with the vehicle (Ctr). To determine the ability of CBD to suppress the diabetic manifestations in Psam- momys, the animals were injected intraperitoneally (i.p.) with 5 mg/kg CBD, or with the vehicle alone five times/ week. The injections continued for 4 weeks, starting 3 days before feeding with the HE diet. Thereafter, the Psammomys were kept on High Diet without CBD treat- ment, for another 29 days (total-60 days from the begin- ning of CBD treatment). The additional period without treatment was chosen to establish the sustainable effect of the CBD on the beta cells integrity as was found in NOD mice [11,12]. Body weight and tail-blood glucose (Accutrend Sensor; Roche Diagnostics, Mannheim, Germany) were monitored twice a week for one month and once a week thereafter. Twenty nine days after the end of CBD treatment, the animals were anesthetized with Ketalar (Parke-Davis, Gwent, UK) and the blood was collected in 10% EDTA, by cardiac puncture. The pancreata were fixed in 10% formalin in buffer, for histological analysis. Serum was stored at −20˚C for analysis of insulin. In the second experiment, 20 animals were used, 10 animals for the vehicle-treated and 10 for the CBD- treated group (5 mg/kg CBD, five times/week, for 4 weeks). The animals were monitored as in the first ex- periment. The experiment was ended, 39 days after the end of CBD treatment, total of 70 days, from the begin- ning of CBD treatment. 2.4. Histology Pancreatic tissue was fixed in 10% buffered formalin and was embedded in paraffin. The 5-micron sections were stained with hematoxylin and eosin. Sections were screened and scored by two independent observers. 2.5. Statistical Analysis Data were expressed, as means ± SEM. Statistical analysis was carried on, as specified in the results. P value was considered significant when P ≤ 0.05. 3. RESULTS 3.1. Weight of CBD-Treated Psammomys The weights of the CBD-treated Psammomys, in the two experiments, were not significantly different be- tween the treated and control animals, as determined by the Fisher Exact Test (see Figu res 1(a), (b)). 3.2. Glucose Levels of CBD-Treated Psammomys It has been observed previously that Psammomys be- Copyright © 2012 SciRes. OPEN ACCESS
Z. Ehud et al. / Journal of Diabetes Mellitus 2 (2012) 27-34 29 150 170 190 210 230 250 270 290 0510 1520 25 3035 4045 5055 60 days weight (gr) CBD CONTROL (a) 150 170 190 210 230 250 270 290 0510 1520 25 30 35 40 45 5055 60 65 70 days weight (gr) CBD CONTROL (b) Figure 1. Weight of the Psammomys following CBD treatment. (a) Experiment 1, observation for 60 days; (b) Experiment 2, observation for 70 days. CBD was injected ip 5 mg/kg (5 times/ week) from day 3 to day 31. Controls were injected with the vehicle: Non significant-treated vs. control p > 0.05. come diabetics (glucose >200 mg/dl) when they are transferred to HE diet. Within 7 days—81% of the ani- mals reach blood glucose level over 200 mg/dl and over 90% of them—within 14 days [16]. In our first experiment (60 days observation) 13 rats (86%), out of 15 control Psammomys, developed diabe- tes (glucose > 200 mg/dl) within 30 days (Figure 2(a))). In contrast, only 4 animals (25%), out of 15, in the CBD-treated group (5 mg/kg, 5 times/week) developed hyperglycemia (over 200 mg/dl). The CBD-treated Psam- momys had significantly lower mean glucose levels com- pared to the vehicle-treated Psammomys (P ≤ 0.05) (Figure 2). In the second experiment (70 days observation, Figure 2(b)), in the control group, 6 out of the total 7 (86%) Psammomys developed diabetes (3 died). Whereas only 4 of 10 (40%) of the CBD-treated Psammomys had hyper- glycemia and all these animals survived (Figures 2(a), (b)). 0 50 100 150 200 250 300 350 400 051015 202530 3540 45 505560 days blood glucose (mg/dl) CBD CONTROL (a) 0 50 100 150 200 250 300 350 0510 1520253035404550556065 70 days blood glucose (mg/dl) CBD CONTROL (b) Figure 2. Levels of blood glucose in plasma of Psammomys following CBD treatment. (a) Exp. 1; (b) Exp. 2. See details in Figure 1. Non-significant- Treated vs. Control P ≤ 0.05. Least-squares linear regression was used to fit the level of glucose as a function of time, separately for each animal. The regression coefficients (slopes) were then employed in Mann-Whitney test on the combined sample. The CBD lowered the rate of a glucose increase signifi- cantly (P ≤ 0.05) as determined by a Mann-Whitney test on the combined sample). 3.3. CBD Prevents Insulin Depletion in Diabetes-Prone Psammomys The most prominent characteristic of diabetic Psam- momys is the rapid depletion of pancreatic insulin stores [17] which probably result from prolonged β-cell stimu- lation by the HE diet. This mechanism plays an impor- tant role in the development and progression of diabetes in the Psammomys model. In the present study in both experiments the plasma levels of insulin in the vehicle- treated control (median at 118 - 142 μU/ml) were sig- nificantly lower than the CBD-treated groups (153 - 210 μU/ml) (Figure 3) Also the CBD-treated animals, tended to maintain their insulin at the pre-HE levels, probably as a result of cyto-protection conferred to the pancreatic β-cells by CBD. Copyright © 2012 SciRes. OPEN ACCESS
Z. Ehud et al. / Journal of Diabetes Mellitus 2 (2012) 27-34 30 CBD CONTROL 0 50 100 150 200 250 300 350 400 450 500 plasma insuli n (U/ml) (a) CBD CONTROL 0 50 100 150 200 250 300 350 pl asma insulin (U/ml) (b) Figure 3. Insulin levels (μU/ml) in Psammomys plasma at the end of the CBD treatment experiments. (a) Exp.1, mean insulin in CBD-treated group was 209.8 μU/ml, in the control 142 μU/ml (non significant); (b) Exp.2, mean insulin in CBD- treated group was 153 μU/ml, in the control group 118 μU/ml Significant-treated vs. control P ≤ 0.05. In order to increase the statistical power, the data from the two experiments were combined. A Mann-Whitney test was then used to compare insulin levels in the ex- perimental animals with those in the controls. 3.4. Animal Survival CBD-treatment increased, although not significantly, the Psammomys survival as seen in Ta b l e 1 . In the two experiments, 13/25 (52%) of the controls survived, whereas in the CBD-treated animals, 18/25 (72%) sur- vived at the end of the two experiments. There is no statistical difference in survival between the treated and untreated animals (P = 0.20, as deter- mined by the Fisher Exact Test). 3.5. CBD Protects β-Cells from Destruction ▬ The morphological evaluation of the pancreata histo- logical sections of the control group showed abnormal islet morphology. About 20% of the islets were without regular borders and were destroyed, also a high percent- age of the islets (75%) were with many vacuoles (Table 2 and Figures 4(b ), (c)). ▬ ▬ On the other hand, in CBD- treated animals, no dam- aged islets were observed and most of them were normal. Also, only 26% of the islets had numerous vacuoles. The quantitative morphological results of Langerhans islets are given in Ta bl e 2 and the morphology of the islets is demonstrated in Figure 4. The control islets (Figures 4(b), (c)), show giant cells with vacuoles as well as de- stroyed islets, with no islet border and only few beta in- tact cells, whereas the islets from the CBD-treated ani- mals, were normal (Figure 4(a)). Thus, the histological assessment is in line with the results of blood glucose and plasma insulin levels. These findings demonstrate that the treatment with CBD protects the pancreatic β- cells integrity against the excessive release of insulin into ▬ Table 1. Psammomys survival following CBD treatment. Survival after 30 days after 57 - 66 days Experiment Control CBD Control CBD Exp. 1 7/15 11/15 7/15 8/15 Exp. 2 7/10 10/10 6/10 10/ 10 Exp. 1 and Exp. 214/25 21/25 13/25 18/25 % survival 56 84 52 72 Table 2. Morphological study of Psammomys langerhans islets*. Morphology CBD Treatment Control Normal + very few small vacuales 79 (62%) 30 (38%) Many Vacuoles 51 (26%) 147 (74%) Damaged Islets 0 (0% ) 19 (100%) * Data of 2 experiments. Copyright © 2012 SciRes. OPEN ACCESS
Z. Ehud et al. / Journal of Diabetes Mellitus 2 (2012) 27-34 Copyright © 2012 SciRes. 31 (a) (b) (c) Figure 4. Photomicrographs of Langerhans islets of Psammomys obesus. (a) Normal Islets of CBD-treated Psammomy; (b) Giant Islets with vacuoles from vehicle-treated Psammomys (control); (c) Damaged/destroyed islets from vehicle-treated Psammomys (control). the blood and preserves the islet’s normal morphology. immunological course very similar to human type 2 dia- betes. Diabetes in the Psammomys is characterized by hyperglycemia, hyperinsulinemia followed by depleted pancreatic insulin. Normoglycemic diabetes-prone Psam- momys obesus which were fed HE diet, developed hy- perglycemia within 4 - 14 days, together with a progres- sive decline of pancreatic insulin content, increased rate of beta-cell death and damage to the islets. Indeed, ex- posure of islets from diabetes-prone Psammomys obesus to high glucose levels, in vitro, results in increase in apoptosis of beta-cells [17,19]. 4. DISCUSSIONS In this study we show that CBD treatment prevents the damage to the islet structure, preserves islet insulin con- tent and prevents the destruction of beta cells which is induced by the HE diet. We demonstrate that four weeks of CBD treatment (5 mg/kg) is effective in preserving normoglycaemia in the majority of the Psammomys keeping on a HE diet, whereas the vehicle-treated animals became hypergly- cemic. Generation of reactive oxygen species may represent an alternative mechanism for both glucotoxicity and li- potoxicity. Hence CBD, a non-psychoactive component of marijuana, which possesses antioxidant, anti-inflam- matory and immunosuppressive properties can be ex- The Psammomys obesus exhibits normally insulin re- sistance in its native environment, which is seen both in muscle and liver [18]. On transfer to HE diet they de- velop nutrition-dependent diabetes with a clinical and OPEN ACCESS
Z. Ehud et al. / Journal of Diabetes Mellitus 2 (2012) 27-34 32 pected to have positive effects. Indeed, Patane et al. [20] showed that treatment of islets with metformin, which also has antioxidant properties, protects the islets from the harmful effect of free fatty acids and restores the in- sulin secretion after chronic exposure to free fatty acids or high glucose. In our study we demonstrate the ability of CBD to significantly reduce the incident of diabetes in male Psammomys and to protect the pancreas islets from destruction. Our previous results [11,12] indicate that CBD can decrease both the incidence of autoimmune type 1 diabetes, as well as the destruction of the islets in NOD female mice. It has been argued that inflammation is involved in the pathogenesis of diabetes type 2 [21,22]. As in all in- flammatory Th1-associated diseases, the reduction of proinflammatory cytokine production, with an increase in IL-4 and IL-10, as demonstrated in the NOD mice, suggests that a mechanism of immunomodulation is in- volved, namely an immune shift from Th1 to Th2. An- other mechanism that might be involved in our study is based on the anti-oxidative activity of CBD [13,23], that very likely prevents beta cell destruction in the HE fed Psammomys. Vanadyl sulfate and rosiglitazone have also been found to be effective in preventing hyperglycemia and hyperinsulinemia [24,25], while nicotine treatment [26] caused decrease in food intake and body weight. An antidiabetic effect was also obtained by G protein kinase analogs [27] and electroacupuncture [28]. CBD administration did not cause obesity in Psam- momys, therefore, it may be assumed that CBD does not enhance muscle or liver insulin sensitivity or improve lipid metabolism in this species. It is known that HE diet which induces glucotoxicity, causes damage to Psammomys beta cells. Several studies [29,30] have reported changes of islets morphology of Psammomys maintained on a HE diet, namely a gradual destruction of beta-cell, loss of insulin, apoptosis and necrosis. Our study clearly demonstrates that CBD has a beneficial effect on the integrity of Psammom ys pancre- atic islets. It was reported [31] that CBD attenuated high glucose-induced endothelial cell inflammatory response by inhibiting NF-kb nuclear translocation. Irreversibility of nutritionally induced type 2 diabetes in Psammomys is related to beta cell apoptosis [17,32,33] although this aspect was not explored in our study. The significant increased insulin availability in CBD treated animals counteracts insulin resistance, which is essential for reducing glucose concentrations in the plasma during HE diet and for preservation of pancreatic beta cell function. One can assume that the normoglycemia in Psammo- mys will be associated with normal level of serum insulin concentrations; however CBD treatment did not reduce, but rather increased these concentrations. Also the hy- perinsulinemia fallowing CBD administration did not cause obesity. We can assume that CBD does not change insulin sensitivity in Psammomys. The cytokines level could not be assayed in our ex- periments, due to lack of available cytokines tests for Psammomys. However, one can speculate that the reduc- tion of inflammatory cytokines following CBD treatment in mice [11,12] might also play a role in the outcome of CBD treatment in Psammomys. Plasma TNF-α is associated with insulin resistance. This supports the claim that TNF-α plays a significant role in the pathogenesis of chronic insulin resistance in humans [34] It has been shown that visfatin, TNF-α, and IL-6 mRNA expressions are increased in peripheral mononuclear-monocytic cells from women with type 2 diabetes, independent of their BMI [35]. Since CBD is known to inhibit production of IL-1b, TNF-α and IFN-γ in mice [11,12] and that these factors are known to be involved in the pathway of autoimmune islet cell destruction leading to diabetes, it may represent one of the mechanisms involved in the lack of islets de- struction and preservation of normal glucose levels in the plasma. Several mechanisms of action of CBD were proposed [4], among them FAAH inhibition, adenosine uptake inhibition and PPAR gamma activation as well as at- tenuation of oxidative/nitrosative stress. Moreover, CBD has been applied in various clinical conditions to human patients [5] without any observed toxic effects. As CBD already has been administered to patients for other medical indications [4,6] and proved to be ex- tremely safe, we propose its use as a therapeutic agent in diabetes type 2. 5. ACKNOWLEDGEMENTS We are grateful to Professor Raphael Mechoulam for providing us the Cannabidiol (CBD). REFERENCES [1] Pickup, J.C., Chusney, G.D., Thomas, S.M. and Burt, D. (2000) Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes. Life Science, 67, 291-300. doi:10.1016/S0024-3205(00)00622-6 [2] Mishima, Y., Kuyama, A., Tada, A., Takahashi, K., Ishi- oka, T. and Kibata, M. (2001) Relationship between se- rum tumor necrosis factor-alpha and insulin resistance in obese men with type 2 diabetes mellitus. Diabetes Re- search and Clinical Practice, 52, 119-123. doi:10.1016/S0168-8227(00)00247-3 [3] Mavridis, G., Souliou, E., Diza, E., Symeonidis, G., Pas- tore, F., Vassiliou, A.M. and Karamitsos, D. 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