Beta cell specific cannabinoid 1 receptor deletion counteracts progression to hyperglycemia in non-obese diabetic mice.

Abstract

Objective

Type 1 diabetes (T1D) occurs because of islet infiltration by autoreactive immune cells leading to destruction of beta cells and it is becoming evident that beta cell dysfunction partakes in this process. We previously reported that genetic deletion and pharmacological antagonism of the cannabinoid 1 receptor (CB1) in mice improves insulin synthesis and secretion, upregulates glucose sensing machinery, favors beta cell survival by reducing apoptosis, and enhances beta cell proliferation. Moreover, beta cell specific deletion of CB1 protected mice fed a high fat high sugar diet against islet inflammation and beta cell dysfunction. Therefore, we hypothesized that it would mitigate the dysfunction of beta cells in the precipitating events leading to T1D.

Methods

We genetically deleted CB1 specifically from beta cells in non-obese diabetic (NOD; NOD RIP Cre⁺ Cnr1^fl/fl) mice. We evaluated female NOD RIP Cre⁺ Cnr1^fl/fl mice and their NOD RIP Cre⁻Cnr1^fl/fl and NOD RIP Cre⁺ Cnr1^Wt/Wt littermates for onset of hyperglycemia over 26 weeks. We also examined islet morphology, islet infiltration by immune cells and beta cell function and proliferation.

Results

Beta cell specific deletion of CB1 in NOD mice significantly reduced the incidence of hyperglycemia by preserving beta cell function and mass. Deletion also prevented beta cell apoptosis and aggressive insulitis in NOD RIP Cre⁺ Cnr1^fl/fl mice compared to wild-type littermates. NOD RIP Cre⁺ Cnr1^fl/fl islets maintained normal morphology with no evidence of beta cell dedifferentiation or appearance of extra islet beta cells, indicating that protection from autoimmunity is inherent to genetic deletion of beta cell CB1. Pancreatic lymph node T_reg cells were significantly higher in NOD RIP Cre⁺ Cnr1^fl/flvs NOD RIP Cre⁻Cnr1^fl/fl.

Conclusions

Collectively these data demonstrate how protection of beta cells from metabolic stress during the active phase of T1D can ameliorate destructive insulitis and provides evidence for CB1 as a potential pharmacologic target in T1D.

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Keywords: Beta cell apoptosis, Beta cell proliferation, Cannabinoid 1 receptor, Insulitis, Islet of Langerhans, Type 1 diabetes

Highlights

•Beta cell specific CB1 deletion protects islets from the detrimental effects of prolonged nutrient overload and inflammation.
•Beta cells in T1D are subjected to endoplasmic reticulum stress because of increasing demands to synthesize insulin.
•Beta cell specific CB1 deletion in NOD mice prevents hyperglycemia and insulitis and preserves beta cell mass and function.
•NOD beta cells lacking CB1 had reduced expression of the beta cell antigen presenting molecule MHC class I.
•CB1 blockade should be considered a target for prevention of T1D as it protects beta cells and improves beta cell function.

Article notes

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Received 2023 Nov 3; Revised 2024 Feb 21; Accepted 2024 Feb 22; Collection date 2024 Apr.

Author notes

1.Introduction

Deterioration of beta cell function prior to or concomitant with immune infiltration is increasingly recognized as a key step in the pathogenesis of autoimmune type 1 diabetes (T1D) [[1], [2], [3], [4]]. In the prediabetic stage of the mouse model of T1D, the non-obese diabetic mouse (NOD/ShiLtJ), increased demand for insulin places a stress on the beta cells that is met by increased insulin translation [1,2]. This, in turn, leads to unresolved ER stress, accumulation of misfolded proteins, activation of the unfolded protein response (UPR) and increased lysosomal degradation of granular proteins [4,5]. Protein degradation under such conditions can cause neo-antigens to be produced. ER and oxidative stress also results in upregulation of major histocompatibility antigen class 1 (MHCI) allowing for presentation of the neo-antigens to T cells. This results in aggressive insulitis, worsening beta cell function, and ultimately apoptosis of beta cells and hyperglycemia [6,7]. Our previous work demonstrated that pharmacological antagonism of the cannabinoid 1 receptor (CB1) and beta cell specific deletion of CB1 has a unique and versatile profile because it improves insulin synthesis and secretion, upregulates glucose sensing machinery, favors beta cell survival by reducing apoptosis under stressful conditions while also enhancing beta cell proliferation, especially in the context of heightened requirements for insulin synthesis and secretion [[8], [9], [10], [11]]. Additionally, our beta cell specific CB1 knockout mouse was inherently protected against islet inflammation and beta cell dysfunction even when continuously fed a high fat high sugar diet [12]. So, the culminative empirical evidence favors beta cell robustness in the setting of loss of CB1 signaling in beta cells. Therefore, we hypothesized that deletion of CB1, a G_αi-protein coupled receptor encoded by the Cnr1 gene, in beta cells of NOD mice would confer protection from hyperglycemia in the setting of autoimmune insulitis.

2.Materials and methods

2.1.Generation of beta cell specific CB1 knockout NOD mouse (NOD RIP Cre⁺ Cnr1^fl/fl)

The animal care and experimental procedures were approved by the National Institute on Aging (NIA) Animal Care and Use Committee (Protocol # 443-LCI-2022): NIA is AAALAC accredited and is a specific pathogen free facility with restricted access. Mice were housed 4 to a cage with environmental enrichment in individually ventilated cages under a 12-hr light/dark cycle at 22±1 °C. The facility bedding is corncob-based which is autoclaved prior to use. All mice are given ad libitum access to water that was processed through a reverse osmosis hyper-chlorination system. Mice were fed with autoclaved standard Envigo rodent diets, 2018SX or 2019S (breeder ration) with ad libitum access: All mice were under the supervision of animal facility caretakers and veterinarians. Animals were randomized according to genotype and collection of data was blinded. Non-obese diabetic mice NOD/ShiLt-Tg(Ins2-cre)5Lt/LtJ (JAX #003855) referred to as NOD RIP-Cre throughout this manuscript, were obtained from The Jackson Laboratory (Bar Harbor, ME) [13]. Since the genomic integration site of the transgene in NOD RIP-Cre mice was not known, we carried out Targeted Locus Amplification [14] sequencing using mouse bone marrow cells (Cergentis’ TLA technology, Utrecht, The Netherlands) and found the complete RIP-Cre-H2-Ea-ps transgene sequence that is integrated in mouse chromosome 8 (chr8:123529070-123656521bp) in a repeat rich region (see GenBank accession # ON568502 for details). NOD RIP Cre mice were crossed to Cnr1 floxed mice (Cnr1^flox/flox) [12] for more than 10 generations. A SNP map genome scan was performed (The Jackson Laboratory) to confirm that the mice used to continue breeding for the study were on a ≥99% NOD background. We refer to this beta cell specific Cnr1 knockout NOD mouse (Cnr1^flox/flox NOD RIP Cre⁺) as NOD RIP Cre⁺ Cnr1^fl/fl throughout. The RIP-Cre was always maintained as hemizygous or a cross between NOD RIP Cre⁺ and NOD RIP Cre⁻ mice [13]. The NOD RIP Cre⁻ Cnr1^fl/fl mice were always age-matched littermates that were Cre-negative (Cnr1^flox/flox NOD RIP Cre⁻, referred to as NOD RIP Cre⁻ Cnr1^fl/fl throughout). All mice used in this report were females because 80% of females are known to develop insulitis and hyperglycemia; however, we did find that NOD RIP Cre⁺ Cnr1^fl/fl males (about 20% of which get insulitis) were also protected from hyperglycemia (n = 20 mice per group, 5 separate groups of mice, studies carried out over 2.5 years). Body weight and blood glucose were monitored on a weekly basis until the female mice were at least 26–30 weeks old. Animals were eventually euthanized, after which pancreata was isolated and processed for further analysis.

2.2.JD-5037 administration

Eight-week-old female NOD/ShiLtJ mice were given JD-5037 (2 mg/kg, MedChemExpress, Monmouth Junction, NJ), an inverse agonist to the CB1, by oral gavage for 4 weeks followed by an intraperitoneal glucose tolerance test.

2.3.Metabolic studies

Intraperitoneal glucose tolerance tests (IPGTT) were performed at both 9 and 14 weeks in WT and NOD RIP Cre⁺ Cnr1^fl/fl mice and 12 weeks in NOD/ShiLtJ mice. Mice were fasted overnight in clean cages before IP injection of glucose (1.5 g/kg body weight), and blood glucose was measured using an ACCU-CHEK glucometer (Roche Diagnostics, Indianapolis, IN) from tail-pricks at the indicated time points. Fasting plasma insulin was determined by ELISA (Crystal Chem Inc., Elk Grove Village, IL).

2.4.Flow cytometry

For intracellular FoxP3 staining pancreatic lymph node and splenic cells were stained and acquired on Symphony-analyzer (Becton Dickinson, Franklin Lakes, NJ) and analyzed with FlowJo version 10.0 (FlowJo, Ashland, OR). Anti-PE-Cy7 CD4 (GK1.5), anti-PE CD8α (53–6.7), anti- PerCP-Cy5.5 CD25 (PC61), and anti- Alexa-fluor-647 FoxP3 (FJK-16s) (from BD Biosciences, eBioscience or BioLegend) were used for the staining of cells. In brief, cells were incubated with FC block and stained with antibodies for surface marker. Dead cells were excluded using the eBioscience™ Fixable Viability Dye eFluor-780 (ThermoFisher Scientific, Waltham, MA). For intracellular staining, cells were permeabilized using the eBioscience™ FoxP3/transcription factor staining buffer set (ThermoFisher Scientific) and stained with anti-FoxP3 antibody. To evaluate T-, B- and myeloid cell subtypes in the pancreatic lymph node cell population we used a panel of markers shown in Supplemental Table 1.

2.5.Pancreatic islet isolation

Islets of Langerhans were isolated by collagenase digestion of exocrine pancreata as described before [12]. Briefly, pancreata were perfused via the common bile duct with 3 mL of collagenase P solution at 0.6 mg/mL (Roche Diagnostics) in cold phenol red-free Hanks’ balanced salt solution (HBSS) with DNase before excision from the abdomen. The islets were removed from acinar tissue by digestion with collagenase at 37 °C for 17 min. After washing with cold HBSS, single islets were handpicked under a stereomicroscope. Islets were then assayed for immunoblotting or real-time PCR.

2.6.Immunoblot analysis

Islets were isolated and hand-picked in the manner described above. They were washed in PBS before being lysed in ice-cold RIPA buffer with protease inhibitors, sonicated (Qsonica, Newtown, CT), centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatant was collected. Protein lysates were resolved by SDS-PAGE and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed overnight at 4 °C using the primary antibodies (Electronic Supplemental Table 2) indicated followed by secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Immunoblots were developed with Super Signal West Femto HRP substrate (ThermoFisher Scientific), exposed to HyBlot CL autoradiography film (Thomas Scientific, Swedesboro, NJ) and were imaged using an SRX-101 film processor (Konica Minolta Medical Imaging., Wayne, NJ). Protein bands were quantified using ImageJ software and normalized against that of GAPDH.

2.7.Real-time qPCR

Following isolation and handpicking of islets, the islets were washed and resuspended in RNAeasy Plus lysis buffer for total RNA extraction using a RNeasy Mini kit (Qiagen, Valencia, CA), as directed by the manufacturer. The quality and purity of RNA were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Up to 1 μg of total RNA was reverse transcribed into cDNA using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The resultant cDNA was diluted (2.5–10 times) according to target abundance. TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) were utilized to perform quantitative RT-PCR to quantify mRNA levels for CB1 receptor (Cnr1, Assay ID Mm01212171_s1), Tryptophan hydroxylase 1 (Tph1, Assay ID Mm01202614_m1), and Serotonin receptor 2a (Htr2a, Assay ID Mm00555764_m1), using β-actin expression (Actb, Assay ID Mm00607939_s1) as an endogenous control gene for normalization. qPCR reactions were run using the StepOnePlus Real Time System's default program. The ΔΔCt method was used to determine the relative fold change.

2.8.Histology, immunohistochemistry, and immunofluorescence

Pancreata were fixed overnight in 4% PFA, paraffin-embedded, and 5 μm sections were examined in further detail. Sections (55–75 islets per group) stained with H&E were blindly scored for insulitis using the following grades: 0, normal islet morphology with no periinsulitis or insulitis; 1, periinsulitis (focal aggregation at one pole of the islet and in contact with the islet periphery); 2, non-aggressive insulitis (islet infiltration covering less than 50% of the islet area); and 3, aggressive insulitis (infiltration covering <50% of the islet) [15]. The morphometric analysis of the islet was carried out on a minimum of six sections that were 150–200 μm apart. Antigen retrieval was achieved by heating the dewaxed paraffin tissue sections (95 °C, 30 min) in boiling citrate buffer (10 mM, pH 6.0) before being blocked with TBS/5% normal goat serum. Sections were immunostained overnight at 4 °C using appropriate primary antibodies (Supplemental Table 1), and subsequently probed with secondary antibodies tagged with Alexafluor at room temperature for 1 h. The slides were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) to visualize nuclei, and then mounted with antifade mounting medium. Confocal microscopy was performed on a Carl Zeiss LSM880 confocal microscope. Images were quantified (islet area, β cell area, α cell area, % Ki67⁺ β cells, and % TUNEL⁺ β cells) with HALO software (V3.3.2541.285) using the Indica Labs- Islet FL module v1.3 (Indica Labs, Albuquerque, NM). B2M and 5-HT (Electronic Supplemental Table 1) were detected by immunohistochemistry. Briefly, after antigen retrieval and overnight incubation with the appropriate antibodies, sections were incubated with SignalStain Boost IHC Detection Reagent, Rabbit-HRP (Cell Signaling Technology, Danvers, MA), following manufacturer's directions. Sections were then incubated with SignalStain DAB Substrate Kit (Cell Signaling Technology), until desired stain intensity was achieved. Pancreata from E11 timed mated females (The Jackson Laboratory) were used as a positive control for 5-HT staining. TUNEL staining was performed using an in-situ death detection kit, TMR red per the manufacturer's directions (Cat# 12156792910, Millipore Sigma, Burlington, MA).

2.9.Membrane-based cytokine/chemokine array

Two membrane-based immunoassays were used: Mouse Cytokine Array Kit, Panel A (R&D Systems) and a mouse Th1/Th2/Th17 Array C1 (RayBiotech). Pooled whole-islet extracts were collected from at least five 9-week-old NOD RIP Cre⁻ Cnr1^fl/fl and NOD RIP Cre⁺ Cnr1^fl/fl mice, incubated with the membranes overnight, and they were analyzed for the expression of various cytokines and chemokines according to the manufacturer's instructions. The signal intensities of the spots were quantified by ImageJ (NIH).

2.10.RNAscope fluorescence in situ hybridization

Within 1 min of their removal from the body, freshly dissected mouse pancreata were snap frozen in liquid nitrogen, and then embedded in O.C.T. compound (Fisher Healthcare, Houston, TX) in cryomolds on crushed dry ice, and stored at −80 °C. Sections (12 μm) were obtained on a Leica CM1950 cryostat (Wetzlar, Germany) and then fixed with 10% neutral buffered formalin (NBF) at 4 °C for 15 min before hybridization and staining. Pretreatment of pancreatic sections, probe hybridizations, and multiplex labeling were performed according to the ACDbio RNAscope Multiplex Fluorescent Detection Kit v2 protocol (Advanced Cell Diagnostics, Inc. Newark, CA), with a shortened protease pretreatment (10 min) due to the rich content of endogenous pancreatic proteases. Multiple channel RNAscope probes of Cnr1 (Probe-Mm-Cnr1, Cat# 420721-C1), Ins2 (Probe-Mm-Ins2-C2 Cat# 497811-C2), were also ordered from ACDbio. Images were acquired using a Carl Zeiss LSM980 confocal microscope (Oberkochen, Germany). The negative control used for the ISH was a universal control probe targeting the dapB (4-hydroxy-tetrahydrodipicolinate reductase) gene from the Bacillus subtilis strain, and the positive controls were probes targeting Ubc (ubiquitin C), and Polr2A (DNA-directed RNA polymerase II subunit RPB1).

2.11.Statistical analysis

All data are depicted as mean ± SEM unless otherwise specified. Statistical analysis was performed using GraphPad Prism v8.0 (GraphPad Software, San Diego, CA). Unpaired two-tailed Student's t test or one-way ANOVA followed by Tukey's post-hoc test was used to assess statistical significance as appropriate. P values of less than 0.05 (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, ∗∗∗∗P < 0.001) were considered statistically significant.

3.Results

To examine whether NOD mice exhibit a metabolic response to CB1 blockade as observed previously in mouse models of type 2 diabetes [8,12], female NOD/ShiLtJ mice were treated with a CB1 inverse agonist, JD5037 (2 mg/kg, daily oral gavage) [16], or vehicle from 8 weeks of age. An IPGTT (1.5g glucose/kg) of these mice 4 weeks later demonstrated significantly lower blood glucose levels in the NOD/ShiLtJ-JD5037 mice (Figure 1A). This encouraging result gave the impetus to generate a conditional beta cell-specific CB1 knockout NOD mouse (NOD RIP Cre⁺ Cnr1^fl/fl). Cnr1-floxed mice (Cnr1^flox/flox [12]) were backcrossed with NOD RIP-Cre [13] for >10 generations to achieve >99% NOD genetic background (see Research Design and Methods for detailed information and genetic testing). We have shown previously that Cnr1 in islets is limited to beta cells [16] hence the very low level of expression of Cnr1 in the NOD RIP Cre⁺ Cnr1^fl/fl can be attributed to a few ductal cells isolated with the islets. Islets extracted from NOD RIP Cre⁺ Cnr1^fl/fl mice had much lower levels of Cnr1 transcript compared to their NOD RIP Cre⁻ Cnr1^fl/fl littermates (Figure 1B). Exclusive absence of Cnr1 expression in beta cells of NOD RIP Cre⁺ Cnr1^fl/fl was confirmed by fluorescent in situ hybridization (FISH) but Cnr1 was clearly present in pancreatic ducts: Cnr1 is not present in acinar tissue (Figure 1C,D, Supplemental Fig. 1A). Please see Supplemental Figs. 1B and C that show positive and negative controls for in situ hybridization). In agreement with observations on NOD/ShiLtJ mice [1] 14-week-old NOD RIP Cre⁻ Cnr1^fl/fl mice had higher blood glucose levels compared to 9-week-old animals: NOD RIP Cre⁺ Cnr1^fl/fl mice had no such deterioration in glucose tolerance (Figure 1E). Fasting (12+ hrs) insulin levels were higher, and more variable in NOD RIP Cre⁺ Cnr1^fl/fl mice (Figure 1F). Consistent with multiple reports female NOD RIP Cre⁺ Cnr1^Wt/Wt and NOD RIP Cre⁻ Cnr1^fl/fl mice developed severe hyperglycemia (blood glucose >600 mg/dL necessitating euthanasia) over the study period of 26 weeks while all the NOD RIP Cre⁺ Cnr1^fl/fl mice were protected (Figure 1G). Incidence and time of onset of hyperglycemia were similar in the NOD RIP Cre⁺ Cnr1^Wt/Wt and NOD RIP Cre⁻ Cnr1^fl/fl mice, with 80% having blood glucose approximating 600 mg/dL or were already euthanized by 26 weeks (Figure 1G). This indicates that the protective effects in NOD RIP Cre⁺ Cnr1^fl/fl mice were not due to RIP-Cre transgene. Thereafter NOD RIP Cre⁺ Cnr1^fl/fl and their NOD RIP Cre⁻ Cnr1^fl/fl littermates were used for all experiments. Four out of 25 NOD RIP Cre⁺ Cnr1^fl/fl mice had two non-consecutive blood glucose levels >250 mg/dl by 26 weeks of age representing a diabetes incidence of 16% (Figure 1H).

Figure 1Beta cell specific deletion of CB1 in NOD mice (NOD RIP Cre⁺ ***Cnr1***^fl/fl) lowers blood glucose levels and protects against progression to hyperglycemia in female NOD mice. (A) IPGTT (1.5 g/kg; age 12 weeks; n = 3 per condition) performed in NODShi/LtJ mice after treatment for 4 weeks with or without JD5037 (2 mg/kg) before the onset of diabetes. (B) *Cnr1* levels in isolated NOD RIP Cre⁻*Cnr1*^fl/fl (n = 6) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 5) islets were measured by RT-qPCR. (C)*In situ* hybridization image of a coronal section of the pancreatic duct of a NOD RIP Cre⁺ *Cnr1*^fl/fl mouse pancreas *insulin* (red), *Cnr1* (green), and DAPI (blue). (D) Representative *in situ* hybridization images of *insulin* (red), *Cnr1* (green), and DAPI (blue) in pancreatic sections of NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl mice, showing co-expression of *insulin* and *Cnr1* in an islet from the NOD RIP Cre⁻*Cnr1*^fl/fl mice. White arrow indicates *Cnr1* is present in ductal cells of the NOD RIP Cre⁺ *Cnr1*^fl/fl mice but is absent from an islet in the same section. **(E)** IPGTT (1.5 g/kg) of NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl mice at 9 (n = 7, n = 6 respectively) and 14 weeks of age (n = 6, both groups). **(F)** Fasting plasma insulin levels measured in both 8-11-week-old (n = 12) and 14–16-week-old NOD RIP Cre⁻*Cnr1*^fl/fl (n = 8) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 7) mice. **(G)** Weekly random blood glucose values NOD RIP Cre⁺ *Cnr1*^fl/fl compared with their wild-type littermates NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^Wt/Wt. The dashed line shows the upper limit of normoglycemia. **(H)** Cumulative diabetes free percentages for NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 25) compared with their wild-type littermates NOD RIP Cre⁻*Cnr1*^fl/fl (n = 25) and NOD RIP Cre⁺ *Cnr1*^Wt/Wt (n = 15). Data are presented as the mean ± SEM. ^⁎⁎P < 0.01. Scale bar = 50 μm. IPGTT = intraperitoneal glucose tolerance test.

NOD RIP Cre⁺ Cnr1^fl/fl pancreatic morphology at 9 (Figure 2A–F) and 14 weeks (Supplemental Figs. 2A–F) were similar to published work on pharmacologic or global genetic inhibition of CB1 [8] and beta cell specific deletion of CB1 [16]. NOD RIP Cre⁺ Cnr1^fl/fl islets were larger (Figure 2A: 0.024 ± 0.002 vs 0.013 ± 0.001 mm², P < 0.0001) had increased beta cell area (Figure 2B 0.022 ± 0.001 vs 0.013 ± 0.001 mm², P < 0.0001) and proportionally increased alpha (α)-cell area (Figure 2C 0.004 ± 0.005 vs 0.006 ± 0.002 mm², P < 0.0001). NOD RIP Cre⁺ Cnr1^fl/fl islet morphology was preserved i.e. the islet core consisted of beta cells with no insulin/glucagon double-positive cells present (Figure 2D) and no obvious isolated insulin⁺ cells in the exocrine pancreas or ducts of NOD RIP Cre⁺ Cnr1^fl/fl mice. The increased beta cell area was a consequence of significantly increased cell turnover as measured by the increased percentage of insulin⁺/Ki67⁺ cells per islet (Figure 2E,F: 3.12 ± 0.42 vs 1.09 ± 0.21%, P < 0.0001). By 14 weeks, there were no insulin⁺/Ki67⁺ cells in NOD RIP Cre⁻ Cnr1^fl/fl in islets infiltrated with immune cells (Supplemental Figs. 2E and F). The percentage insulin⁺/Ki67⁺ cells in large and normal NOD RIP Cre⁺ Cnr1^fl/fl islets at 60 weeks were similar to 9- and 14-week-old (Figure 2G, 3.2 ± 1.1%). There were very few terminal deoxynucleotidyl transferase dUTP nick end labelled (TUNEL)⁺ beta cells (Figure 2H,I: 0.718 ± 0.218 vs 0.032 ± 0.032%, P < 0.001) in NOD RIP Cre⁺ Cnr1^fl/fl islets. Therefore, reduced apoptosis likely contributed to NOD RIP Cre⁺ Cnr1^fl/fl larger islet size. Previously we reported that CB1 activation inhibits beta cell proliferation in part through inhibition of the insulin/IRS1/2/pAkt pathway [8]. Levels of pIRβ_1162/1163, pIRS1/2₆₁₂ and pAKT₄₇₃ in islets isolated from NOD RIP Cre⁺ Cnr1^fl/fl were significantly higher than in NOD RIP Cre⁻ Cnr1^fl/fl islets (Figure 2J). CB1 activation is reported to induce cleaved caspase-3 and negatively regulate the anti-apoptotic Bcl-2 molecules [10]. Protein levels of Bcl-2 were significantly higher in NOD RIP Cre⁺ Cnr1^fl/fl vs NOD RIP Cre⁻ Cnr1^fl/fl islets (Figure 2J). Reduced ER stress is a potential mechanism underlying protection against apoptosis in beta cells lacking CB1 [17]. Therefore, we measured protein levels of one of the most prominent unfolded protein response (UPR) signal transducers during ER stress. They are IRE1α [18] and activation (phosphorylation on Ser51) of the integrated stress response protein eIF2α as examined in 9-week-old NOD RIP Cre⁻ Cnr1^fl/fl and NOD RIP Cre⁺ Cnr1^fl/fl islets, and found that protein levels were significantly lower in the NOD RIP Cre⁺ Cnr1^fl/fl islets (Figure 2K).

Figure 2Beta cell specific ablation of CB1 in NOD mice increases islet area, enhances beta cell replication, and improves beta cell survival. Quantification of average islet area **(A)** beta cell mass (insulin) **(B)**: and α-cell mass (glucagon) **(C)** per islet for NOD RIP Cre⁻*Cnr1*^fl/fl (n = 18 islets) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 30 islets) at 9 weeks of age. **(D)** Representative pancreatic sections from NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl mice showing immunostaining of insulin (green), glucagon (red) and DAPI (blue). **(E)** Quantification of percentage of Ki67⁺ beta cells per islet for NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl mice at 9 weeks of age. **(F)** Representative image of staining Ki67 (red), insulin (green), and DAPI (blue) of an islet from a NOD RIP Cre⁻*Cnr1*^fl/fl and a NOD RIP Cre⁺ *Cnr1*^fl/fl at 9 weeks of age showing increased numbers of Ki67 positive beta cells per islet in the NOD RIP Cre⁺ *Cnr1*^fl/fl mice. **(G)** Representative image of an islet from a NOD RIP Cre⁺ *Cnr1*^fl/fl at 60 weeks of age still showing Ki67⁺ beta cells. **(H)** Representative image of triple staining TUNEL (red), insulin (green), and DAPI (blue) showing an islet from a NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl mouse at 9 weeks of age. **(I)** Quantification of percentage beta cell apoptosis in NOD RIP Cre⁻*Cnr1*^fl/fl (n = 6) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 6) mice at 9 weeks of age. **(J)** Western blots and densitometry analysis of normalized p-IRβ, p-IRS1/2, p-AKT and Bcl-2 in islets isolated from NOD RIP Cre⁻*Cnr1*^fl/fl (n = 4) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 4) mice. **(K)** Western blots and densitometry analysis of normalized total IRE1α, eIF2α and p-eIF2α in extracts of islets isolated from NOD RIP Cre⁻*Cnr1*^fl/fl (n = 3) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 3) mice. Results are representative of at least three independent experiments. Graphical data show densitometry analyses normalized to GAPDH, total AKT, total IRS1/2, and/or total IRβ (loading controls). Data are presented as the mean ± SEM. ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001. Scale bar = 50 μm. Complete original immunblots for J and K are provided in Supplemental Fig. 3.

Beta cell serotonin (5-HT) production is markedly elevated during pregnancy facilitating adaptation to insulin resistance and the increasing demand for insulin by inducing beta cell proliferation and improving glucose-induced insulin secretion [19]. Furthermore, there are reports of upregulated 5-HT production in beta cells of transgenic mice with insulin-promoter-linked-Cre transgenes that contain the growth hormone (hGH) cassette to enhance transgene expression. Our transgenic mice do not contain hGH (see Research Design and Methods) but there was still a theoretical possibility that transgenic mice lacking CB1 have increased beta cell turnover because its deficiency may activate lactogenic pathways. Transcript levels of the 5-HT synthesizing enzyme tryptophan hydroxylase 1 (Tph1) and the Gα_q-linked serotonin receptor Htr2b by which 5-HT mediates beta cell expansion and increased sensitivity to glucose [19] were similar in isolated islets from both genotypes (Supplemental Figs. 5A–D, n = 4 per genotype; Supplemental Fig. 5B shows confirmation of CB1 nullification). Embryonal day 11 pancreata were used as a positive control for 5-HT expression in beta cells [19]. Fourteen-week-old NOD RIP Cre⁻ Cnr1^fl/fl and NOD RIP Cre⁺ Cnr1^fl/fl islets had very little 5-HT compared to control (Supplemental Figs. 5E–G). Therefore, CB1 deficiency does not result in increased beta cell turnover through upregulation of Tph1 nor does it improve beta cell glucose sensitivity by increased serotonergic response through Htr2b.

Evaluation of insulitis performed on whole pancreatic sections revealed characteristic immune infiltration [15] with lymphocytic invasion in islets of 26-week-old NOD RIP Cre⁻ Cnr1^fl/fl mice (Figure 3A–C). In contrast 80% of the islets evaluated in the age matched NOD RIP Cre⁺ Cnr1^fl/fl mice did not exhibit insulitis with only occasional peri-islet infiltration (13%). However, infiltration was not observed to invade or engulf NOD RIP Cre⁺ Cnr1^fl/fl islets (non-aggressive insulitis 6.5%) that were consistently large and well-preserved. Beta cell specific deletion of β2 microglobulin (B2M) demonstrates that while MHC class I presentation is not required for initiation of insulitis, there is some evidence that it is a component of progression to overt diabetes and hyperglycemia [20,21]. The transporters associated with antigen processing (TAPS 1,2) transport antigens to ER for loading onto MHCI [22,23]: TAPS protein amounts were significantly lower in protein extracts from NOD RIP Cre⁺ Cnr1^fl/fl islets (Figure 3D). GAD65 is a major beta cell target antigen for autoreactive CD8⁺ T cells and a marker of beta cell death [24]. Protein extracts from 9-week-old NOD RIP Cre⁺ Cnr1^fl/fl islets had lower levels of CD8α and GAD65 (Figure 3D) than NOD RIP Cre⁻ Cnr1^fl/fl islets. Beta cells in NOD RIP Cre⁻ Cnr1^fl/fl islets had obvious B2M immunostaining in a heterogeneous manner whither or not insulitis was present and was independent of islet size (Supplemental Figs. 5H and I). In stark contrast, B2M staining was absent in NOD RIP Cre⁺ Cnr1^fl/fl islets, including those with peri-islet inflammation (Figure 3E, lower panel and Supplemental Figs. 5H and I).

Figure 3Beta cell specific CB1 deletion counteracts progression to hyperglycemia in NOD mice (A) Representative images of hematoxylin and eosin-stained islets from NOD RIP Cre⁻*Cnr1*^fl/fl and a NOD RIP Cre⁺ *Cnr1*^fl/fl pancreata at 26 weeks of age in different stages of insulitis. **(B)** Quantitation of scoring of insulitis in NOD RIP Cre⁻*Cnr1*^fl/fl (n = 9 mice) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 7 mice) pancreata at 26 weeks of age. **(C)** The average insulitis score of NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl mice in B. **(D)** Western blots of CD8α, TAP1, TAP2, and GAD65 and densitometry analysis in pancreatic islets isolated from NOD RIP Cre⁻*Cnr1*^fl/fl (n = 4) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 4) mice. Results are representative of at least three independent experiments. Graphical data show densitometry analyses normalized to GAPDH (loading control). **(E)** Representative immunofluorescence staining for B2M (red) and insulin (green) in pancreatic sections of NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl at 14 weeks of age. Data are presented as the mean ± SEM. ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001. Scale bar = 50 μm. Complete original immunoblots for D are provided in Supplemental Fig. 4.

Evaluation of key immune cells such as naive and effector CD4⁺ T cells, CD8⁺ T cells and T_reg cells in the draining pancreatic lymph nodes (Figure 4A) revealed significantly higher numbers of T_reg cells in 10-week-old NOD RIP Cre⁺ Cnr1^fl/fl mice (Figure 4A) and increased numbers of these cells in the spleens of these mice relative to their wild-type littermates. Numbers of pancreatic lymph node CD3⁺CD8⁺, CD3⁺CD4⁺, and CD3⁺CD4⁻CD8⁻TCR⁺ T cells, B cells and myeloid cells did not differ between NOD RIP Cre⁺ Cnr1^fl/fl mice and their wild-type littermates at 16 weeks of age (Supplemental Fig. 6). As cytokines released from beta cells can directly modulate the local immune environment we examined levels of cytokines, and chemokines using proteome profiler analysis in islets from 9-week-old mice of both genotypes (Figure 4B,C). We identified a significant shift in cytokine profiles in NOD RIP Cre⁺ Cnr1^fl/fl mice compared with NOD RIP Cre⁻ Cnr1^fl/fl wildtype littermates (Figure 4B,C). Importantly, the expression levels of 6 cytokines, including IFN-γ, IL-1β, IL-10, IL-12p70, IL-23p19, and TNF α were markedly reduced, whereas the levels of IL-7, IL-13, IL-17, M-CSF, IL-2, and TGF β1 were elevated substantially in the islets from NOD RIP Cre⁺ Cnr1^fl/fl mice (Figure 4B,C). The NOD RIP Cre⁺ Cnr1^fl/fl islets had downregulated levels of intracellular cell adhesion molecule (ICAM-1), CXCL10, CXCL9, CCL5, and MIP-3α, but protective CXCL12 was concurrently produced at higher levels (Figure 4B,C). Our current understanding of the mechanism underlying the protection from hyperglycemia and insulitis when CB1 is deleted specifically from only beta cells in NOD mice is outlined (Figure 5).

Figure 4NOD RIP Cre⁺ ***Cnr1***^fl/flmice have altered expression of signature markers associated with immune cell recruitment. (A) Flow cytometry analysis of regulatory T-cells (Tregs) for the surface expression of CD4, CD25 and intracellular expression of FoxP3 in pancreatic lymph nodes (top panel) of NOD RIP Cre⁻*Cnr1*^fl/fl (n = 4) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 4) mice and spleens (bottom panel) of NOD RIP Cre⁻*Cnr1*^fl/fl (n = 3) and NOD RIP Cre⁺ *Cnr1*^fl/fl (n = 4) mice. Graphs shows the frequency of Tregs in pancreatic lymph nodes (top graph) and spleens (bottom graph) of NOD RIP Cre⁻*Cnr1*^fl/fl and NOD RIP Cre⁺ *Cnr1*^fl/fl mice. The expression of cytokines and chemokines in extracts of islets from NOD RIP Cre⁻*Cnr1*^fl/fl mice and NOD RIP Cre⁺ *Cnr1*^fl/fl mice (pooled from 5 mice for each genotype) was evaluated using the Mouse Cytokine Array Kit, Panel A (R&D) **(B)** and the mouse Th1/Th2/Th17 Array C1 (Raybiotech) (C). Differentially expressed cytokines (IFN-γ, IL-1β, IL-10, IL-12p70, IL-2, IL-23p19, TGFβ1, TNF-α, IL-7, IL-13, IL-17, M-CSF), chemokines (MIP-3α, CXCL10, CXCL9, CCL5, CXCL12) and adhesion molecule (ICAM-1) altered in NOD RIP Cre⁺ *Cnr1*^fl/fl mice are highlighted. Cytokine and chemokine spots were measured and expressed as fold-changes as compared to NOD RIP Cre⁻*Cnr1*^fl/fl mice. Data are presented as the mean ± SEM. ^⁎P < 0.05.

Figure 5Schematic showing the pleotropic regulation of beta cell function, proliferation, and apoptosis by CB1 signaling. Activation of CB1 by ligands such as 2-AG suppresses insulin receptor (IR) signaling via IRS1,2/pAkt by diminishing IR kinase activity [8]. When CB1 is inhibited specifically in beta cells, phosphorylation of Akt at S473 is greatly increased, thereby allowing for increased GLUT1 transcription and translation and enhanced cell proliferation [12]. As CB1 is a Gα_i coupled receptor its activation by the endogenous cannabinoids such as 2-AG inhibits adenylyl cyclase thereby blocking cAMP production [8]. Consequently, when CB1 signaling is inhibited intracellular cAMP levels increase [8,11]. This results in increased insulin secretion via enhanced PKA activity [16] that in turn increases protein levels of PDX-1, a transcription factor that has glucoregulatory, cytoprotective and proliferative actions in the beta cell. PDX-1 activation promotes *insulin*, *Gck* and *GLUT2* transcription. Moreover, CB1 activation is a negative modulator of incretin action. Incretin-mediated AC activation is also increased in response to CB1 inhibition thereby amplifying cAMP/PKA activity hence the proliferative and anti-apoptotic properties of incretins [11,41]. Finally, as we show in this report, beta cell specific genetic deletion of CB1 leads to significantly reduced beta cell apoptosis when it is under stress and prevents antigen presentation by MHCI on the cell surface. Glut2 = glucose transporter 2; PKA = protein kinase A; GLP-1 = glucagon-like peptide-1; GIP = glucose-dependent insulinotropic peptide; PDX-1 = pancreatic and duodenal homeobox 1; Gck = glucokinase; 2-AG = 2-arachidonoylglycerol; Bcl-2 = B-Cell CLL/Lymphoma 2.

4.Discussion

Beta cells are not simply innocent bystanders targeted by a misguided autoimmune process but more likely they may even initiate or at least precipitate the cascade of events that lead to their demise [4,5,25]. The extremely high rate of insulin biosynthesis and protein processing in beta cells undergoing infiltration render them more susceptible to ER stress and the UPR [[1], [2], [3]]. Furthermore, beta cells are extremely susceptible to damage from reactive oxygen species (ROS) as they lack many critical antioxidant enzymes [5]. Altered proinsulin processing both locally in the pancreas and in circulation in the pre-diabetic state of T1D correlate with immune markers and markers of beta cell stress, pointing to ongoing involvement of beta cell proteins in precipitating autoimmunity [[25], [26], [27]].

The majority of NOD RIP Cre⁺ Cnr1^fl/fl mice were resistant to autoimmune attack as demonstrated by prevention of hyperglycemia in 84% of the animals at 26 weeks of age, and suppression of aggressive insulitis. This we attribute to the elevated numbers of pancreatic lymph node, FoxP3⁺CD25⁺Treg cells which can restore self-tolerance in T1D [28]. NOD RIP Cre⁺ Cnr1^fl/fl islets exhibited lower levels of ER-stress with lower levels of IRE1α which has been previously shown to lower numbers of pancreatic CD8 cells because of reduced cross presentation [18]. However, the loss of IRE1α in the beta cell of NOD mice also led to a beta cell dedifferentiation phenotype, whereas the absence of CB1 signaling preserves and even enhances beta cell function as observed by increased intra-islet and serum insulin levels in the NOD RIP Cre⁺ Cnr1^fl/fl mice. IRE is but one arm of the described three major branches of the UPR response to ER stress and previously published work indicates that the PERK/p-eIF2α arm is a more relevant way to assess ER status downstream of CB1 signaling. In rat mesangial cells high glucose induces apoptosis via ER stress which is mediated through CB1 activation of p-eIF2α as demonstrated using the CB1 antagonist AM251 which blocked activation of this pathway [29]. Likewise in human renal proximal tubular cells CB1 also mediates palmitic acid-induced apoptosis via p-eIF2α activation of ER stress [30]. Hence this led us to specifically examine levels of p-eIF2α (Ser51) in islets from the wild-type and NOD RIP Cre⁺ Cnr1^fl/fl to begin to unravel the mechanism underlying CB1 induced ER-stress.

The improved beta cell health and absence of beta cell apoptosis observed in the NOD RIP Cre⁺ Cnr1^fl/fl islets also leads to an more favorable islet cytokine profile with higher levels of TGFβ1, IL-2 and IL-7 all of which can induce peripheral Treg differentiation [28,[31], [32], [33]]. Evaluation of other key immune cell sub-types in wild type and knockout pancreatic lymph nodes showed no differences which is compatible with previous observations [34] and is indicative of more local islet effects on immune cell infiltrating populations [35]. All the mice used were hemizygous for Cre to avoid any reduction in insulitis observed by Leiter and co-workers with high levels of Cre expression [13]. While a limitation of our study is that we did not use the Cre⁺ wildtype control (NOD RIP Cre⁺ Cnr1^Wt/Wt) for all experiments we did monitor the blood glucose of these mice and found that 73% of them got hyperglycemia implying that the presence of Cre was not protective against insulitis.

In the context of other investigations of beta cell pathophysiology in T1D, boosting proliferation, preventing apoptosis, and targeting cell autonomous intracellular mechanisms that destroy or increase robustness of beta cells, in addition to immune modulators, are now emerging as potential strategies in a bid to prevent T1D. For example, using S961, an inhibitor of the insulin receptor that leads to insulin resistance in peripheral tissues and secondarily to beta cell proliferation in NOD prevented progression to diabetes [34]. However, such a strategy to boost beta cells would be detrimental to every other cell type. Combining inhibition of DYRK1A, a protein kinase regulating cell proliferation, with TGFβ-SMAS signaling increased proliferation of human beta cells in vitro [36], but this would be a non-starter combination in humans because of off-target effects. It's also been proposed that dedifferentiated/degranulated beta cells, such as cells that express both glucagon and insulin as occurs with S961 treatment afford protection from generation of autoantigens and might have a role in later allowing for beta cell maturation [27]. Evidence for dedifferentiation of beta cells occurring in NOD RIP Cre⁺ Cnr1^fl/fl mice was lacking because we did not find any islet cells expressing two hormones. Additionally, we do not see insulin-containing cells outside of islets as has also been seen with S961-driven increased beta cell turnover [34]; theoretically these non-islet cells might have different expression profiles and hence protection from T-cell activation.

One noteworthy finding, and not previously reported to our knowledge, is the clear presence of CB1 in ductal, but not acinar, cells. This is the first time this is being reported and therefore its function therein is unknown. One interesting avenue would be to investigate, for example, if CB1 downstream signaling is involved in the regulation of enzyme telomerase reverse transcriptase (Tert), a key enzyme in pancreatic cancer [37]. And CB1 may be involved in the secretory function of ducts such as control of carbonic anhydrase activity and/or cystic fibrosis transmembrane regulator, HCO⁻ secretion, as well as mucin secretion and production from goblet cells in ducts.

The beneficial effects from beta cell CB1 inhibition/nullification are numerous and include (Figure 5): Improved insulin synthesis and secretion and glucose sensing machinery [8,9]; decreased ROS formation [12]; improved IR/IRS1/2/AKT signaling; increased cell turnover and reduced apoptosis, especially under stress conditions from inflammation, high fat diets, and toxins [[38], [39], [40]]; reduced immune cell infiltration in islets - all in the setting of increased adenylyl cyclase activity due to lack of CB1 coupled G_i/o [8,12] and improved incretin response [16]. A CB1 inhibitor to increase beta cell robustness and turnover, and a GLP-1 receptor agonist to improve autophagy and beta cell robustness even more, would seem strategies worth considering to prevent/delay insulin dependence in people who are on a path to have T1D in their future. Such a strategy would at least buy time for the need to use a second mechanistically distinct, immune target such as IL-2 therapy to expand T_reg cells.

Disclosure

The authors have nothing to disclose.

CRediT authorship contribution statement

Kanikkai Raja Aseer: Writing – original draft, Writing – review & editing, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Caio Henrique Mazucanti: Visualization, Validation, Investigation, Formal analysis. Jennifer F. O'Connell: Visualization, Validation, Project administration, Investigation, Formal analysis, Data curation, Conceptualization. Isabel González- Mariscal: Investigation, Conceptualization. Anjali Verma: Visualization, Validation, Methodology, Investigation, Formal analysis. Qin Yao: Visualization, Validation, Methodology, Investigation. Christopher Dunn: Visualization, Validation, Methodology, Formal analysis. Qing-Rong Liu: Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Formal analysis. Josephine M. Egan: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Máire E. Doyle: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

None.

Acknowledgments

This work was funded by the Intramural Research Program of the National Institute on Aging, National Institutes of Health, USA. The authors especially Aaron Russell and Lauren Brick for help with Figure construction, especially the schema in Figure 5. We thank David Taylor for help in formatting and editing the figures and Shayna Yeager for help in editing the manuscript. We are beyond grateful to Brian Wilgenburg and the animal facility staff who maintained our mouse strains despite the hurdles placed during COVID.

Footnotes

Footnote Group

Appendix ASupplementary data

The following are the Supplementary data to this article.

Data availability

Data will be made available on request.

References

Untitled section

References

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Associated Data

Supplementary Materials

Data Availability Statement

Data will be made available on request.

Studies

Conditions

Cannabinoids and active compounds

Related research

Abstract

Objective

Methods

Results

Conclusions

Untitled section

Highlights

Article notes

Untitled section

Author notes

1.Introduction

2.Materials and methods

2.1.Generation of beta cell specific CB1 knockout NOD mouse (NOD RIP Cre+ Cnr1fl/fl)

2.2.JD-5037 administration

2.3.Metabolic studies

2.4.Flow cytometry

2.5.Pancreatic islet isolation

2.6.Immunoblot analysis

2.7.Real-time qPCR

2.8.Histology, immunohistochemistry, and immunofluorescence

2.9.Membrane-based cytokine/chemokine array

2.10.RNAscope fluorescence in situ hybridization

2.11.Statistical analysis

3.Results

4.Discussion

Disclosure

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgments

Footnotes

Footnote Group

Appendix ASupplementary data

Data availability

References

Untitled section

References

Associated Data

Supplementary Materials

Data Availability Statement

Additional materials

2.1.Generation of beta cell specific CB1 knockout NOD mouse (NOD RIP Cre⁺ Cnr1^fl/fl)