Glucagon-like peptide-1 prevents methylglyoxal-induced apoptosis of beta cells through improving mitochondrial function and suppressing prolonged AMPK activation

Abstract

Accumulation of methylglyoxal (MG) contributes to glucotoxicity and mediates beta cell apoptosis. The molecular mechanism by which GLP-1 protects MG-induced beta cell apoptosis remains unclear. Metformin is a first-line drug for treating type 2 diabetes associated with AMPK activation. However, whether metformin prevents MG-induced beta cell apoptosis is controversial. Here, we explored the signaling pathway involved in the anti-apoptotic effect of GLP-1, and investigated whether metformin had an anti-apoptotic effect on beta cells. MG treatment induced apoptosis of beta cells, impaired mitochondrial function, and prolonged activation of AMP-dependent protein kinase (AMPK). The MG-induced pro-apoptotic effects were abolished by an AMPK inhibitor. Pretreatment of GLP-1 reversed MG-induced apoptosis, and mitochondrial dysfunction, and suppressed prolonged AMPK activation. Pretreatment of GLP-1 reversed AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR)-induced apoptosis, and suppressed prolonged AMPK activation. However, metformin neither leads to beta cell apoptosis nor ameliorates MG-induced beta cell apoptosis. In parallel, GLP-1 also prevents MG-induced beta cell apoptosis through PKA and PI3K-dependent pathway. In conclusion, these data indicates GLP-1 but not metformin protects MG-induced beta cell apoptosis through improving mitochondrial function, and alleviating the prolonged AMPK activation. Whether adding GLP-1 to metformin provides better beta cell survival and delays disease progression remains to be validated.

Figures

Figure 1. GLP-1 protects rat insulinoma cells RINm5F from MG-induced apoptosis.

RINm5F Cells were treated in the presence or absence of 1 mM MG with or without GLP-1 (100 nM or 300 nM). (A) Cell viability was measured by MTT assay. Data are shown as relative cell viability (mean % ± S.E. bar) as compared with that in control (n = 4). *p < 0.05. (B) Apoptosis was demonstrated by Annexin V/ Hoechst 33342 staining after incubated with indicated treatment for 17 hr. Annexin V positive cells showed green fluorescence (upper row). Condense nuclei were shown in apoptotic cells by Hoechst 33342 staining (middle row). The pictures on bright field were shown in the lower row. (C) The percentage of apoptotic cells was calculated by measuring the percentage of cells in the sub-G1 population in the indicated time by using flow cytometry with propidium iodide (PI) staining (n = 3). *p < 0.05. (D) The cell counts and percentage of apoptotic cells in the sub-G1 population after incubation with indicated treatment for 17 hr were measured by using flow cytometry with propidium iodide (PI) staining. (E) Western blot of poly(ADP-ribose) polymerase (PARP) and cleaved caspase-3. The positions of the 113 kDa and 89 kDa in Western blot represent intact PARP protein and its cleavage products, respectively. The positions of the 19 kDa and 17 kDa in Western blot represent cleaved caspase-3. GAPDH was used as an internal control.

Figure 2. GLP-1 suppressed MG-induced beta cell apoptosis through protein kinase A (PKA) and PI3K dependent pathway, respectively.

RINm5F cells were treated in the absence of MG, 1 mM MG, 1 mM MG + 300 nM GLP-1, 1 mM MG + 300 nM GLP-1 + 100 μM Rp-cAMP, 1 mM MG + 300 nM GLP-1 + 10 μM H-89, 1 mM MG + 300 nM GLP-1 + 30 μM LY294002, 1 mM MG + 300 nM GLP-1 + 50 nM wortmannin, respectively. (A) Cell viability was measured by MTT assay. Data are shown as relative cell viability (mean % ± S.E. bar) as compared with that in control (n = 3). *p < 0.05. (B) Annexin-V/PI flow cytometry. LL: left lower quadrant indicated viable cells, UL: upper left quadrant indicated early apoptotic cells, UR: upper right quadrant indicated late apoptotic cells, LR: right lower quadrant indicated necrotic cells. (C) Western blot of pCREB/CREB, PDX1, cleaved caspase-3, and GAPDH was used as an internal control. (D) Western blot of pAkt/Akt, cleaved caspase-3, and GAPDH was used as an internal control.

Figure 3. GLP-1 rescued MG-induced mitochondria dysfunction and inhibited prolonged AMPK activation in RINm5F cells.

Cells were treated in the absence or presence of 1 mM MG with or without 300 nM GLP-1 for indicated time. (A) Relative intracellular ATP concentration (%) compared with control in indicated time (n = 6). *p < 0.05 (Control vs. 1 mM MG at 1 hr, 2 hr, 4 hr, 6 hr and 17 hr), #p < 0.05 (1 mM MG vs. 1 mM MG + 300 nM GLP-1 at 17 hr) (B) Relative oxygen consumption rate (%) compared with control in indicated time (n = 3). *p < 0.05 (Control vs. 1 mM MG at 2 hr, 10 hr, and 17 hr), #p < 0.05 (1 mM MG vs. 1 mM MG + 300 nM GLP-1 at 2 hr, 10 hr, and 17 hr). (C) Western blot of p-AMPK/AMPK. GAPDH was used as internal control. The bar graph showed the ratio of pAMPK/GAPDH in Western blot in the absence or presence of 1 mM MG with or without 300 nM GLP-1 in the indicated time (n = 3). *p < 0.05.

Figure 4. AMPK inhibitor partially rescues MG-induced cell death and apoptosis in RINm5F cells.

Cells were treated in the presence or absence of 1 mM MG with or without compound C (C.C. 10 μM) in indicated time. (A) Cell viability was measured by MTT assay. Data are shown as relative cell viability (mean % ± S.E. bar) as compared with that in control (n = 5). *p < 0.05. (B) The percentage of apoptotic cells was calculated by measuring the percentage of cells in the sub-G1 population by using flow cytometry with propidium iodide (PI) staining (n = 3). *p < 0.05. (C) Western blot of PARP, p-AMPK/AMPK, cleaved caspase-3, and GAPDH as internal control.

Figure 5. GLP-1 rescues AMPK activator AICAR-induced cell death, apoptosis and AMPK activation in RINm5F cells.

(A) Cell viability was measured by MTT assay in different concentration of AICAR at indicated time. Data are shown as relative cell viability (mean % ± S.E. bar) as compared with that in control (n = 3). *p < 0.05. (B) Cell viability was measured by MTT assay in cells treated by 1 mM MG or 1.5 mM AICAR for 17 hr with or without pretreatment of 300 nM GLP-1 (n = 3). *p < 0.05. (C) Western blot of p-ACC/ACC and p-AMPK/AMPK in cells treated by 1 mM MG or 1.5 mM AICAR for 17 hr with or without pretreatment of 300 nM GLP-1. GAPDH was used as internal control. (D) Western blot of PARP in cells treated by 1 mM MG or 1.5 mM AICAR for 17 hr with or without pretreatment of 300 nM GLP-1. GAPDH was used as internal control. (E) Cell viability was measured by MTT assay in different concentration of metformin at indicated time. Data are shown as relative cell viability (mean % ± S.E. bar) as compared with that in control (n = 3). (F) Cell viability was measured by MTT assay in cells treated by 1 mM MG for 17 hr with pretreatment of different concentration of metformin. Data are shown as relative cell viability (mean % ± S.E. bar) as compared with that in control (n = 3).

Figure 6. GLP-1 prevents MG-induced apoptosis of INS-1 and MIN6 cells in part through PKA and PI3 kinase pathway, and partially via improving mitochondrial function and suppressing prolonged AMPK activation.

INS-1 and MIN6 cells were seeded overnight, and then were incubated with indicated treatment for indicated time. (A) INS-1 and MIN6 cells were treated in the absence of MG, 1 mM MG, 1 mM MG + 300 nM GLP-1, 1 mM MG + 300 nM GLP-1 + 100 μM Rp-cAMP, 1 mM MG + 300 nM GLP-1 + 10 μM H-89, 1 mM MG + 300 nM GLP-1 + 30 μM LY294002, 1 mM MG + 300 nM GLP-1 + 50 nM wortmannin, respectively. Cell viability was measured by MTT assay. Data are shown as relative cell viability (mean % ± S.E. bar) as compared with that in control (n = 3). *p 

Figure 7. Schematic representation of the effect of GLP-1 against methylglyoxal (MG) toxicity in the beta cell.

MG suppresses mitochondria function and leads to decrease ATP/AMP ratio, which in turn steadily activates AMPK, and the activation of AMPK subsequently leads to apoptosis. GLP-1 improved mitochondria function and in turn increases ATP/AMP ratio, which leads to inhibit sustained AMPK activation induced by MG, and subsequently suppress MG-induced apoptosis. In parallel, GLP-1 also exerts the anti-apoptotic effect through activation of PDX-1 in a PKA-dependent pathway. GLP-1 also activates PI3K/Akt pathway and inhibits caspase-3 activity and leads to suppress apoptosis.