Processing of proglucagon to GLP-1 in pancreatic α-cells: is this a paracrine mechanism enabling GLP-1 to act on β-cells?

1. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 1

   Expression of pancreatic islet proglucagon/proGLP-1 and prohormone convertases (PCs) in pancreatic α-cell lines. (a) Proglucagon is processed by PC2 to mature glucagon in the pancreatic α-cells. Proglucagon is also processed by PC1 to GLP-1 and this is known to occur in the L-cells in the intestine. (b and c) Gene expression for the convertases (PC1 and PC2), proglucagon (d) and insulin (e) were analysed in two pancreatic α-cell lines (αTC1-6 and αTC1-9) and a β-cell line (MIN6). Data are the mean expression of triplicate wells ±s.e.m., normalised to the housekeeping gene HPRT, and are representative of three experiments.
2. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 2

   Secretion of glucagon and GLP-1 from α-cells and islets in culture. (a) Comparison of the basal release of glucagon and GLP-1 over a period of 30 min from αTC1-6 cells. Data expressed as the mean±s.e.m. for five independent experiments. ***P<0.001. (b) Human islets have intracellular GLP-1 peptide. GLP-1 was measured in lysates from human islets and in culture media after 24 h under basal conditions. Levels in media were below the level of detection of the assay (dashed line). Data expressed as the mean±s.d. of three separate incubations. (c and d) Time course of production of GLP-1 and glucagon in rat islets. GLP-1 and glucagon were measured in extracts of freshly isolated islets, and in extracts from islets cultured for 1, 3 and 7 days in media containing 5 mmol/l glucose. (e) The GLP-1 to glucagon ratio over the same time course shows the extent of change. Data expressed as the mean±s.e.m. expression of three separate incubations and are representative of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 significant increase in intracellular GLP-1 compared with freshly isolated islets.
3. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 3

   Effect of glucose on PC1 in cultured islets. (a) PC1 gene expression was measured in islets cultured in 5 mmol/l (black bars), 11 mmol/l (open bars) or 25 mmol/l (shaded bars) glucose for 1, 3 or 7 days. Data are the mean expression ±s.e.m. of three independent incubations normalised to HPRT. Each experiment was repeated three times **P<0.01, ***P<0.001. Pc1 gene expression shows a significant increase when incubated for 3 and 7 days with 11 and 25 mmol/l glucose compared with islets cultured in 5 mM glucose. (b and c) Effect of glucose on PC1 protein. Western blots were carried out on lysates from rat islets (100 islets per incubation) cultured in 5, 11 and 25 mmol/l glucose for 7 days. PC1 protein was detected in the islet extracts (b) and a tubulin antibody was used to control for equal loading (c).
4. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 4

   Effect of glucose on GLP-1 and glucagon in αTC1-6 cells. (a) Cells were incubated with a range of concentrations of glucose for 30 min and glucagon secretion was measured. (b–e) GLP-1 and glucagon secretion. Cells were incubated with either low (2 mmol/l) or high glucose (25 mmol/l) (b and c). Cells were also incubated with KCl (40 mmol/l) for 30 min (d and e). Data expressed as the mean fold change ±s.e.m. of at least four independent experiments. *P<0.05, **P<0.01, ***P<0.001. (f) Effect of glucose on expression of proglucagon in αTC1-6 cells. Cells were incubated for 5 days in low (5 mmol/l) and high (25 mmol/l) glucose before gene expression was assessed. Results are compared with the MIN6 pancreatic β-cell line. Data expressed as the mean±s.d. of three replicate RNA preparations.
5. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 5

   Expression and stimulation of L-cell GPCRs in αTC1-6 cells. (a–c) Gene expression was assessed in two α-cell lines (αTC1-6 and αTC1-9), a β-cell line (MIN6) and an L-cell line (STC-1). Data expressed as the mean of triplicate wells ±s.e.m., normalised to the housekeeping gene HPRT, and is representative of three experiments. (d) L-cell GPCR ligands stimulate the PC1 promoter in pancreatic α-cells. αTC1-6 cells (closed bars) and STC-1 cells (open bars) were transiently transfected with a PC1 promoter/luciferase reporter gene. Cells were then incubated with the receptor ligands for 24 h. Cells were lysed and the PC1 promoter-driven luciferase activity was measured in the cell supernatants. Data expressed as the mean fold change over basal ±s.e.m. of at least three independent experiments. ***P<0.001. (e and f) L-cell GPCRs increase GLP-1 secretion from αTC1-6 cells. Cells were incubated with receptor ligands for 2 h. GLP-1 (e) and glucagon (f) secretion was measured from αTC1-6 cells (closed bars) and STC-1 cells (open bars; changes in glucagon release were undetectable from STC-1 cells). Data expressed as the mean fold change over basal ±s.e.m. of at least three independent experiments.
6. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 6

   β-Cell function in STZ-treated islets. (a) Rat islets (10 per well) were treated with increasing concentrations of STZ for 2 h and then left 48 h. Islets were then stimulated with differing concentrations of glucose for 120 min and the secretion of insulin measured. Control islets (black bars), and islets treated with 0.1 mmol/l STZ (white bars), 0.3 mmol/l STZ (grey bars) and 1 mmol/l STZ (striped bars). Data expressed as the mean insulin secretion ±s.e.m. of six replicate wells and is representative of three independent experiments. ***P<0.001 shows significant increase in insulin secretion compared with 3 mM glucose controls. ^###P<0.001 shows significant decrease in insulin secretion from STZ-treated islet compared with control islets. (b and c) GLP-1 and glucagon release in response to STZ treatment of rat islets. Islets were treated with STZ as described earlier and then extracts and media collected 48 h later for measurement of GLP-1 (b) and glucagon (c) content using specific ELISAs. Data expressed as the mean fold change in peptide over vehicle-treated islets ±s.e.m. of three independent experiments ***P<0.001.

• © 2011 Society for Endocrinology