Fig. 1Metformin restored cell viability and glucose-stimulated insulin secretion (GSIS) impaired by palmitate (Pal). Cell viability of NIT-1 cells (n=3) (A) was evaluated by cell counting kit-8 (CCK-8) assay in NIT-1 cells incubated in the absence or presence of Pal (0.5 mM) and treated with various concentrations of metformin (0.02 to 1.0 mM) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (0.5 to 1.0 mM) for 24 hours. GSIS was measured in NIT-1 cells (n=4) (B) exposed to Pal (0.2 mM) for 48 hours and in isolated mouse islets (n=3) (C) treated with Pal (0.5 mM) for 24 hours. Veh, vehicle. aRepresents P<0.05 vs. Veh-treated control, bRepresents P<0.05 vs. Pal treatment.
Fig. 2Lower metformin (Met) concentration (0.05 mM) suppressed palmitate (Pal)-induced elevations of intracellular reactive oxygen species (ROS) levels, NADPH oxidase (NOX) and the expression of endoplasmic reticulum (ER) stress markers. (A) Intracellular ROS levels of NIT-1 cells were assessed by 2′, 7′-dichlorohydro-fluorescein diacetate (DCF-DA) fluorescence (n=4). The mRNA levels of Nox1 and Nox2 of NIT-1 cells (n=5) (B) and ER stress markers (activating transcription factor 4 [Atf4], C/EBP homologous protein [Chop], FK506 binding protein 11 [Fkbp11], glucose-regulated protein 94 [Grp94]) in NIT-1 cells (n=5) (C) and in isolated mouse islets (n=3) (D) were measured by real-time quantitative polymerase chain reaction (PCR) analysis. Relative mRNA levels were expressed as a fold-change relative to vehicle (Veh)-treated control. NIT-1 cells and isolated mouse islets were incubated in the absence or presence of Pal (0.5 mM). Met and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) treatments were carried out for 24 hours. aRepresents P<0.05 vs. Veh-treated control, bRepresents P<0.05 vs. Pal treatment.
Fig. 3Higher metformin (Met) concentration (0.5 mM) increased the intracellular adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio and AMP-activated protein kinase (AMPK) phosphorylation. The cellular ADP/ATP ratio (n=4) (A) and levels of AMPK phosphorylations (n=5) (B) evaluated by Western blot analysis were measured in NIT-1 cells incubated in the absence or presence of palmitate (Pal) (0.5 mM). Met and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) treatments were carried out for 24 hours, and compound C (C.C.) (0.01 mM) for 1 hour. Veh, vehicle; pAMPK, phosphorylated AMPK. aRepresents P<0.05 vs. Veh-treated control.
Fig. 4The effect of 0.05 mM metformin (Met) on β-cell dysfunction was not affected by compound C (C.C). (A) The viability of NIT-1 cells was assayed by trypan blue exclusion method in triplicate and repeated twice. (B) Measurements of glucose-stimulated insulin secretion (GSIS) of NIT-1 cells were repeated three times. Met treatments were carried out for 24 hours, and C.C (0.01 mM) was applied to NIT-1 cells exposed to palmitate (Pal) for 1 hour. Veh, vehicle. aRepresents P<0.05 vs. Veh-treated control, bRepresents P<0.05 vs. Pal treatment, cRepresents P<0.05 vs. Pal and Met treatment.
Fig. 5The action of metformin (Met) on cellular reactive oxygen species (ROS) level and endoplasmic reticulum (ER) stress markers elevated by palmitate (Pal) was not affected by inhibition of AMP-activated protein kinase (AMPK). (A, B) Intracellular ROS levels were measured by 2′, 7′-dichlorohydro-fluorescein diacetate (DCF-DA) fluorescence (n=4). AMPK was inhibited by compound C (C.C) (A) and by small interfering RNA for AMPK (siAMPK) (B). (C) mRNA levels of ER stress markers (activating transcription factor 4 [Atf4], C/EBP homologous protein [Chop], FK506 binding protein 11 [Fkbp11], glucose-regulated protein 94 [Grp94]) were measured by real-time polymerase chain reaction (n=4). Relative mRNA levels were expressed as fold-changes relative to the vehicle (Veh)-treated control. NIT-1 cells were exposed to Pal (0.5 mM) and treated with Met for 24 hours. Treatments of C.C and the small interfering RNAs (siRNAs) for AMPK were 0.01 mM for 1 hour and 100 nM for 48 hours, respectively. aRepresents P<0.05 vs. Veh-treated control, bRepresents P<0.05 vs. Pal treatment.
Fig. 6Metformin (Met) at a concentration of 0.5 mM inhibited Rho kinase1 (ROCK1) in NIT-1 cells exposed to palmitate (Pal) and ROCK1 inhibition restored glucose-stimulated insulin secretion (GSIS) impaired by Pal. (A) ROCK1 activities were measured by a radioimmunoassay kit in NIT-1 cells incubated in the absence or presence of Pal (0.5 mM) and treated with Met (0.05, 0.5 mM) for 24 hours (n=4). (B) GSIS were measured in NIT-1 cells incubated in the absence or presence of Pal (0.2 mM) for 48 hours (n=3). The small interfering RNAs for ROCK1 and AMP-activated protein kinase (AMPK) were used at 100 nM for 48 hours. Veh, vehicle. aRepresents P<0.05 vs. Veh-treated control, bRepresents P<0.05 vs. Pal treatment, cRepresents P<0.05 vs. Pal and siROCK1 treatment.