Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling-dependent manner

Abstract

Fibroblast growth factor 21 (FGF21) is important in glucose, lipid homeostasis and insulin sensitivity. However, it remains unknown whether FGF21 is involved in insulin expression and secretion that are dysregulated in type 2 diabetes mellitus (T2DM). In this study, we found that FGF21 was down-regulated in pancreatic islets of db/db mice, a mouse model of T2DM, along with decreased insulin expression, suggesting the possible involvement of FGF21 in maintaining insulin homeostasis and islet β-cell function. Importantly, FGF21 knockout exacerbated palmitate-induced islet β-cell failure and suppression of glucose-stimulated insulin secretion (GSIS). Pancreatic FGF21 overexpression significantly increased insulin expression, enhanced GSIS, improved islet morphology and reduced β-cell apoptosis in db/db mice. Mechanistically, FGF21 promoted expression of insulin gene transcription factors and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, the major regulators of insulin secretion, as well as activating phosphatidylinositol 3-kinase (PI3K)/Akt signaling in islets of db/db mice. In addition, pharmaceutical inhibition of PI3K/Akt signaling effectively suppressed FGF21-induced expression of insulin gene transcription factors and SNARE proteins, suggesting an essential role of PI3K/Akt signaling in FGF21-induced insulin expression and secretion. Taken together, our results demonstrate a protective role of pancreatic FGF21 in T2DM mice through inducing PI3K/Akt signaling-dependent insulin expression and secretion.

Keywords: FGF21; diabetes; insulin; pancreatic β-cell.

Figure 1

Low FGF21 expression was associated with islet β‐cell dysfunction in db/db mice. (A) Immunofluorescence staining of FGF21 (green), amylase (red) and insulin (red) in pancreatic tissues of 8‐week‐old lean mice. (B) Immunohistochemical staining of FGF21 in pancreatic tissues of 8‐week‐old lean mice and db/db mice. (C) Quantitative PCR analysis of FGF21 mRNA expression. (D) Western blot analysis of FGF21 protein expression, normalized to GAPDH. (E) Immunofluorescence staining of insulin (red) and glucagon (green) in pancreatic sections and quantification of insulin‐ or glucagon‐positive cells. (F) Immunofluorescence staining of insulin (red) and TUNEL (green) staining in pancreatic sections as well as quantification of TUNEL‐positive β‐cells. *P < 0.05, **P < 0.01. Scale bar = 50 μm. (A–C) n = 5. (D) n = 3

Figure 2

FGF21 deficiency exacerbated palmitate (PA)‐induced islet β‐cell failure. Isolated islets from wild‐type (WT) mice or FGF21 knockout (KO) mice were exposed to 1 mmol/L PA for 48 h followed by (A) western blot assay (***P < 0.001 vs the blank WT group), and (B) the glucose‐stimulated insulin secretion (GSIS) test (*P < 0.05 vs the WT group stimulated with 16.7 mmol/L glucose, ^# P < 0.05 vs the KO group stimulated with 16.7 mmol/L glucose). (n = 4–5)

Figure 3

Overexpression of FGF21 ameliorated hyperglycaemia and glucose intolerance in db/db mice. Sixteen‐week‐old db/db mice were intraperitoneally injected with AAV‐FGF21. Serum samples at the indicated time points were collected to determine the blood glucose and circulating FGF21 levels. Age‐matched lean mice with the same genetic background were injected with AAV‐GFP, which served as controls. (A) Immunohistochemical staining of FGF21. (B) Western blot analysis of FGF21 protein expression in islets. (C) Serum FGF21 after 4 weeks of gene delivery in the fed state. (E) Fasting glucose levels after treatment with AAV‐FGF21. (D) Time course of blood glucose levels. (F) GTT results after gene treatment. (G) Plasma insulin levels at 0 and 30 min after intraperitoneal glucose injection were quantified by ELISA. ^# P < 0.05 vs the lean controls, *P < 0.05 vs db/db mice treated with AAV‐GFP. Scale bar = 50 μm (n = 5)

Figure 4

FGF21 improved islet β‐cell function in vivo. Sixteen‐week‐old db/db mice and their lean controls were intraperitoneally injected with AAV‐F21 or AAV‐GFP. Mice were killed at 4 weeks after the AAV injection. (A) Immunohistochemical staining of insulin and measurement of the islet area in pancreatic sections. (B) Immunofluorescence staining of insulin and glucagon in pancreatic sections and quantification of insulin‐ or glucagon‐positive cells. (C) Immunofluorescence staining of insulin and TUNEL in pancreatic sections as well as quantification of TUNEL‐positive β‐cells. (D) Western blot analysis of cleaved and total caspase‐3 expression in the islets isolated from db/db mice, normalized to GAPDH expression. ^### P < 0.001 vs the lean controls, *P < 0.05, **P < 0.01, and ***P < 0.00 vs db/db mice treated with AAV‐GFP. Scale bar = 50 μm. (A‐C) n = 5. (D) n = 3

Figure 5

FGF21 promoted insulin secretion along with up‐regulation of insulin gene transcription factors. Sixteen‐week‐old db/db mice and their lean controls were intraperitoneally injected with AAV‐F21 or AAV‐GFP and analysed 4 weeks later. (A–D) Quantitative PCR analysis of insulin, MafA, MafB and PDX1 mRNA expression in the islets. (E–G) Western blot analysis of insulin, MafA, MafB and PDX1 protein expression in the islets. ^# P < 0.05 and ^## P < 0.01 vs the lean controls; *P < 0.05, **P < 0.01 and ***P < 0.001 vs db/db mice treated with AAV‐GFP (n = 3‐5 for each group)

Figure 6

FGF21 promoted insulin secretion along with up‐regulation of SNARE proteins. Sixteen‐week‐old db/db mice and their lean controls were intraperitoneally injected with AAV‐F21 or AAV‐GFP and killed 4 weeks later. (A) The plasma insulin levels were measured immediately following the killing. (B) Static glucose‐stimulated insulin secretion (GSIS) in isolated islets was measured at 1 h after 16.7 mmol/L glucose stimulation. (C–E) Quantitative PCR analysis of STX1, SNAP25 and VAMP2 in isolated islets. (F–H) Western blot analysis of STX1, SNAP25 and VAMP2 in isolated islets. ^# P < 0.05, ^## P < 0.01 and ^### P < 0.001 vs the lean controls; *P < 0.05 and **P < 0.01 vs db/db mice treated with AAV‐GFP. (A) n = 5. (B–H) n = 3‐5

Figure 7

FGF21 regulated insulin expression and secretion in islets via PI3K/Akt signaling pathways. (A) Quantitative PCR analysis of β‐klotho mRNA expression in islet. (B and C) Sixteen‐week‐old db/db mice and their lean controls were intraperitoneally injected with AAV‐F21 or AAV‐GFP and analysed 4 weeks later. Western blot analysis of phosphorylated and total PI3K and Akt protein expression. ^### P < 0.001 vs the lean controls, *P < 0.05 vs db/db mice treated with AAV‐GFP (n = 3). (D‐G) Islets were isolated from 12‐week‐old lean mice and then treated with 100 ng/mL FGF21 in the absence or presence of PI3K inhibitor (GDC‐0941, 5 μmol/L) or Akt inhibitor (MK‐2206, 1 μmol/L) for 12 h. (D and E) Quantitative PCR analysis of insulin mRNA levels in islets after the treatment. (F and G) Static insulin secretion in isolated islets. ^## P < 0.01 and ^### P < 0.001 vs the vehicle group; *P < 0.05 vs the FGF21 group without GDC‐0941 or MK‐2206 (n = 5)

Figure 8

FGF21 regulated insulin gene transcription factors and SNARE proteins via PI3K/Akt signaling pathways. Islets were isolated from 12‐week‐old lean mice and then treated with 100 ng/mL FGF21 in the absence or presence of PI3K inhibitor (GDC‐0941, 5 μmol/L) or Akt inhibitor (MK‐2206, 1 μmol/L) for 12 h. (A and B) Western blot analyses of MafA, MafB and PDX‐1 in islets after the treatment. (C and D) Western blot analyses of SNARE protein expression in islets after the treatment. ^# P < 0.05 and ^## P < 0.01 vs the vehicle group; *P < 0.05 vs the FGF21 group without GDC‐0941 or MK‐2206 (n = 4)