Figure 3. mRNA expression of CB1 receptor isoforms in single pancreatic β-cells.

Islets of Langerhans are a complex cluster of different cell types: endocrine cells (α-, β-, δ-, ε- and PP-cells), nerve fibers and blood cells (A). Disaggregated human islets were stained with two antibodies for cell surface markers and sorted into three endocrine fractions (P1, P2 and P3) and a non-endocrine fraction (NE) (B). RNA from P1, P2 and P3 was extracted and retro-transcribed into cDNA. RT-PCR of the five endocrine hormones was performed to confirm the enrichment in each endocrine cell type (C–E). Expression was normalized to insulin in P3. Data represent mean ± SD, p < 0.001. The endocrine fractions (P1-P3) were then sorted into individual single cells. From those, RNA was extracted and reverse transcribed into cDNA, pre-amplified for the hormone transcripts of interest and diluted prior to real time PCR amplification. Expression was normalized to glucagon (F), somatostatin (G) and insulin (H). p ≤ 0.001. CB1 isoforms were quantified and represented as fold difference relative to corresponding hormone levels (I). ***p ≤ 0.005. Data are represented in box and whisker plot (n = 10). Relative amount of copies of each CB1 receptor isoform was quantified using specific Taqman primers and probes.