Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University, Seoul, Korea.
Department of Internal Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological & Medical Sciences, Seoul, Korea.
Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea.
Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Division of Nephrology, Department of Internal Medicine, Konyang University College of Medicine, Seoul, Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. kspark@snu.ac.kr.
Abstract
BACKGROUND Chronic exposure to elevated levels of free fatty acids contributes to pancreatic β-cell dysfunction. Although it is well known that metformin induces cellular energy depletion and a concomitant activation of AMP-activated protein kinase (AMPK) through inhibition of the respiratory chain, previous studies have shown inconsistent results with regard to the action of metformin on pancreatic β-cells. We therefore examined the effects of metformin on pancreatic β-cells under lipotoxic stress.
METHODS NIT-1 cells and mouse islets were exposed to palmitate and treated with 0.05 and 0.5 mM metformin. Cell viability, glucose-stimulated insulin secretion, cellular adenosine triphosphate, reactive oxygen species (ROS) levels and Rho kinase (ROCK) activities were measured. The phosphorylation of AMPK was evaluated by Western blot analysis and mRNA levels of endoplasmic reticulum (ER) stress markers and NADPH oxidase (NOX) were measured by real-time quantitative polymerase chain reaction analysis.
RESULTS We found that metformin has protective effects on palmitate-induced β-cell dysfunction. Metformin at a concentration of 0.05 mM inhibits NOX and suppresses the palmitate-induced elevation of ER stress markers and ROS levels in a AMPK-independent manner, whereas 0.5 mM metformin inhibits ROCK activity and activates AMPK.
CONCLUSION This study suggests that the action of metformin on β-cell lipotoxicity was implemented by different molecular pathways depending on its concentration. Metformin at a usual therapeutic dose is supposed to alleviate lipotoxic β-cell dysfunction through inhibition of oxidative stress and ER stress.
AMP-activated protein kinasesEndoplasmic reticulum stressInsulin-secreting cellsMetforminOxidative stressRho-associated kinases
Figures
Fig. 1 Metformin 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. 2 Lower 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. 3 Higher 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. 4 The 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. 5 The 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. 6 Metformin (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.
그림 출처: PubMed Central 공개 논문 (PMC6943256)
Conflict of interest statement
No potential conflict of interest relevant to this article was reported.
그 밖의 정보
출판 형태
Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
