Chronic marijuana usage by human pancreas donors is associated with impaired islet function

Abstract

We investigated the effect of chronic marijuana use, defined as 4 times weekly for more than 3 years, on human pancreatic islets. Pancreata from deceased donors who chronically used marijuana were compared to those from age, sex and ethnicity matched non-users. The islets from marijuana-users displayed reduced insulin secretion as compared to islets from non-users upon stimulation with high glucose (AUC, 3.41 ± 0.62 versus 5.14 ±0.47, p<0.05) and high glucose plus KCl (AUC, 4.48 ± 0.41 versus 7.69 ± 0.58, p<0.001). When human islets from chronic marijuana-users were transplanted into diabetic mice, the mean reversal rate of diabetes was 35% versus 77% in animals receiving islets from non-users (p<0.01). Immunofluorescent staining for cannabinoid receptor type 1 (CB1R) was shown to be colocalized with insulin and enhanced significantly in beta cells from marijuana-users vs. non-users (CB1R intensity/islet area, 14.95 ± 2.71 vs. 3.23 ± 0.87, p<0.001). In contrast, CB1R expression was not co-localized with glucagon or somatostatin. Furthermore, isolated islets from chronic marijuana-users appeared hypertrophic. In conclusion, excessive marijuana use affects islet endocrine phenotype and function in vitro and in vivo. Given the increasing use of marijuana, our results underline the importance of including lifestyle when evaluating human islets for transplantation or research.

Fig 1. Islets from chronic marijuana-users have lower responses upon in vitro challenge with secretagogues.

(A) Isolated islets from chronic marijuana-users (red) and non-users (blue). After 48 hours of culture, islets were perifused sequentially with Krebs-Ringer’s Buffer Solution (pH 7.4) containing 3 mM glucose, 16.8 mM glucose, 3 mM glucose, and 25 mM KCl plus 16.8 mM glucose, respectively. Insulin secretion was quantified by ELISA and standardized by islet IEQ number. Area under the curve of secreted insulin on perifusion with (B) 3 mM glucose; (C)16.8 mM glucose; (D) 3 mM glucose; (E) 25 mM KCl plus 16.8 mM glucose in panel A. Marijuana-users (n = 7) and non-users (n = 8). * p<0.05; *** p<0.001. All analyses were performed using GraphPad Prism version 8.0. A p<0.05 was considered significant. The values are expressed as the mean ± SEM.

Fig 2. Islets from chronic marijuana-users have diminished graft function following transplantation into diabetic NOD SCID mice.

Isolated islets (1,200 IEQ) from non-users and marijuana-users were transplanted beneath the renal capsule of the left kidney of diabetic NOD SCID mice. (A) Blood glucose levels were determined periodically for 28 days post-transplantation. The highlighted areas are the range of standard error of mean of each donor group. (B) Calculated area under the curve of blood glucose levels, marijuana-user (n = 30 mice received human islets from 10 donors); non-user (n = 50 mice received human islets from 19 donors). Each dot in the bar graph represents the average AUC of blood glucose from all the mice transplanted with islets from a single donor. (C) The mean reversal rate of diabetes in the mice after islet transplantation. For each batch of islets isolated from a single donor, the ratio of the number of mice that reversed diabetes divided by the total number of mice transplanted was calculated. Then the mean reversal rates of diabetes were averaged from marijuana-users (10 donors) and non-users (19 donors). Each dot in the bar graph represents the reversal rate of diabetes in single donor. **p<0.01. All analyses were performed using GraphPad Prism version 8.0. A p<0.05 was considered significant. The values were expressed as the mean ± SEM.

Fig 3. Islets from chronic marijuana-users display increased CB1R expression on islet beta cells.

(A) Immunofluorescent staining of human pancreatic tissue from individuals with a history of non-use and chronic marijuana use. The sections were stained for CB1R (red) and insulin (green). Details of the staining are shown in the enlarged images. Two islet images are presented from non-users and marijuana-users. Confocal images were taken using Zeiss LSM 700. Scale bars, 50 μm. (B) Quantitative analysis of CB1R expression. The CB1R intensity per islet area was compared between the two donor groups. Samples from five non-user donors and from six marijuana-user donors were counted for the analysis. *** p<0.001. The values were expressed as mean ± SEM. Each dot in the bar graph represents calculated value from an islet. (C) Immunofluorescent staining of human pancreatic tissue from individuals with a history of chronic marijuana use and non-use. The sections were stained for CB1R (red), glucagon (green), and somatostatin (green). Details of the staining are shown in the enlarged images. Images are presented from samples from four non-users and four marijuana-users. Confocal images were taken using a Zeiss LSM 700 microscope. Scale bars, 50 μm.