Glucose, adrenaline and palmitate antagonistically regulate insulin and glucagon secretion in human pseudoislets

Abstract

Isolated human islets do not always meet the quality standards required for transplant survival and reliable functional in vitro studies. The formation of pseudoislets, i.e. the reaggregation of a defined number of islet cells after dissociation, improves insulin secretion. We present a simple method of pseudoislet formation from human islet cells and assess the transcriptome and function of isolated human islets and pseudoislets from the same organ donors. Following pseudoislet formation, insulin content/DNA and mRNA/RPS13 resembled that of islets. In pseudoislets, glucose-stimulated insulin secretion (GSIS) was significantly higher (8-13-fold) than in islets (2-4-fold). GSIS of pseudoislets was partly inhibited by the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin-9. The stimulatory effects of palmitate and forskolin at 12 mM glucose were also significantly higher in pseudoislets than in islets. Further analysis of pseudoislets revealed that regulation of secretion and insulin and glucagon content was maintained over a longer culture period (6-14 d). While adrenaline inhibited GSIS, adrenaline together with palmitate stimulated glucagon secretion 2-fold at low glucose, an effect suppressed by high glucose. Transcriptome analysis revealed that, unlike islets, pseudoislets were deprived of exocrine and endothelial cells. In conclusion, pseudoislet formation restores functional integrity of human islet cells and allows long-term in vitro testing.

Figure 1

Reaggregation of human islet cells to pseudoislets specifically improves GSIS. (A) Experimental design of pseudoislet formation. Isolated human islets were cultured overnight. Non-dispersed islets were partly used for insulin secretion. The others (1000–2000 islets) were digested into single cells using trypsin. After digestion, a defined number of single cells were reaggregated in hanging drops of 20 µl over 3 days. After 2 days or longer culture in non-adhesive multi-well plates, the pseudoislets were used in the experiments. (B) DNA content of single islets and pseudoislets (formed from 2000 cells and cultured for 2 days) from the same donors (n = 2) were measured as described under Materials and Methods. (C,D) Comparison of GSIS of islets and pseudoislets reaggregated from 1000, 2000 and 4000 cells as indicated expressed as (C) fold-stimulation (12 mM glucose over 2.8 mM glucose). (D) % of insulin content (white bars, 2.8 mM glucose and grey bars, 12 mM glucose). Results are expressed as means + s.e.m. of n = 5 independent preparations. (E–N) GSIS of individual preparations of islets (black signs) and pseudoislets (red signs) expressed as (E–I) fold-stimulation and (J–N) % of insulin content. *p < 0.05, ***p < 0.0002 denotes significant GSIS; ^#p < 0.05 denotes significant difference between secretion at 12 mM glucose of islets and the respective pseudoislet. ^§p < 0.05 denotes significance to secretion of islets at 2.8 mM glucose. ^€p < 0.05 significance between secretion at 12 mM glucose of 1000-cells and 4000-cells pseudoislets.

Figure 2

FFAR1 and GLP-1R mediated augmentation of GSIS. Isolated islets and pseudoislets (reaggregated from 2000 cells) were prepared, cultured for 2 days and incubated as described under Materials and Methods. Insulin secretion (A,B) of non-dispersed isolated islets (overnight culture) and (A,C) of the pseudoislets was stimulated as indicated. Results are expressed as means + s.e.m. of n = 3–5 independent preparations. ^§p < 0.05 denotes significance to 2.8 mM glucose; ^#p < 0.05 denotes significance to 12 mM glucose and ^&p < 0.05 denotes significance between the two groups as indicated. ^€p < 0.05 denotes significant difference between pseudoislets and islets at the same condition. White bars represent secretion at 2.8 mM glucose, grey bars at 12 mM glucose. Abbreviations used: Glc (glucose), Pal (palmitate), Fors (forskolin), Ex-4 (exendin-4), Ex-9 (exendin-9).

Figure 3

Long term functional integrity of pseudoislets: Opposing effects of glucose and adrenaline on insulin and glucagon secretion. Pseudoislets were cultured and incubated with test substances as described under Materials and Methods. (A) Insulin and (B) glucagon were measured in the same samples and are expressed as means + s.e.m. of n = 5 independent preparations. ^§p < 0.05 denotes significance to 2.8 mM glucose, ^#p < 0.05 significance to 12 mM glucose and ^&p < 0.05 significance to 2.8 mM glucose of the respective group containing the same test substances. 2.8 mM glucose (white bars), 12 mM glucose (grey bars). Abbreviations used: Glc (glucose), Adr (adrenaline), Ex-4 (exendin-4), Pal (palmitate).

Figure 4

Transcriptome analyses revealed higher mRNA abundance of proteins expressed in β-cells in human pseudoislets than in isolated human islets from the same donors. (A–D) Islet (HI) and pseudoislet reaggregated from 2000 cells (PI) mRNA levels were measured by RNAseq as described under Materials and Methods. The data of 4 different donors are presented as log2 reads. (E) Relative mRNA levels of INS, GCG, SST, PDX1 and FFAR1 in islets and pseudoislets were measured by RT-PCR. Asterisks denote significant differences between HI and PI, (A–D) padj < 0.05, R package; (E) t-test.