Endocrinol Metab.  2018 Mar;33(1):105-113. 10.3803/EnM.2018.33.1.105.

Pioglitazone Attenuates Palmitate-Induced Inflammation and Endoplasmic Reticulum Stress in Pancreatic β-Cells

• Hong SW
• Lee J
• Cho JH
• Kwon H
• Park SE
• Rhee EJ
• Park CY
• Oh KW
• Park SW
• Lee WY

• KMID: 2407127
• DOI: http://doi.org/10.3803/EnM.2018.33.1.105

Abstract

BACKGROUND
The nuclear receptor peroxisome proliferator-activator gamma (PPARγ) is a useful therapeutic target for obesity and diabetes, but its role in protecting β-cell function and viability is unclear.
METHODS
To identify the potential functions of PPARγ in β-cells, we treated mouse insulinoma 6 (MIN6) cells with the PPARγ agonist pioglitazone in conditions of lipotoxicity, endoplasmic reticulum (ER) stress, and inflammation.
RESULTS
Palmitate-treated cells incubated with pioglitazone exhibited significant improvements in glucose-stimulated insulin secretion and the repression of apoptosis, as shown by decreased caspase-3 cleavage and poly (adenosine diphosphate [ADP]-ribose) polymerase activity. Pioglitazone also reversed the palmitate-induced expression of inflammatory cytokines (tumor necrosis factor α, interleukin 6 [IL-6], and IL-1β) and ER stress markers (phosphor-eukaryotic translation initiation factor 2α, glucose-regulated protein 78 [GRP78], cleaved-activating transcription factor 6 [ATF6], and C/EBP homologous protein [CHOP]), and pioglitazone significantly attenuated inflammation and ER stress in lipopolysaccharide- or tunicamycin-treated MIN6 cells. The protective effect of pioglitazone was also tested in pancreatic islets from high-fat-fed KK-Ay mice administered 0.02% (wt/wt) pioglitazone or vehicle for 6 weeks. Pioglitazone remarkably reduced the expression of ATF6α, GRP78, and monocyte chemoattractant protein-1, prevented α-cell infiltration into the pancreatic islets, and upregulated glucose transporter 2 (Glut2) expression in β-cells. Moreover, the preservation of β-cells by pioglitazone was accompanied by a significant reduction of blood glucose levels.
CONCLUSION
Altogether, these results support the proposal that PPARγ agonists not only suppress insulin resistance, but also prevent β-cell impairment via protection against ER stress and inflammation. The activation of PPARγ might be a new therapeutic approach for improving β-cell survival and insulin secretion in patients with diabetes mellitus

Keyword

Pioglitazone; Insulin-secreting cells; Glucolipotoxicity; Inflammation; Endoplasmic reticulum stress

MeSH Terms

Animals
Apoptosis
Blood Glucose
Caspase 3
Chemokine CCL2
Cytokines
Diabetes Mellitus
Endoplasmic Reticulum Stress*
Endoplasmic Reticulum*
Glucose Transport Proteins, Facilitative
Humans
Inflammation*
Insulin
Insulin Resistance
Insulin-Secreting Cells
Insulinoma
Interleukin-6
Islets of Langerhans
Mice
Necrosis
Obesity
Peptide Initiation Factors
Peroxisomes
Repression, Psychology
Transcription Factors
Blood Glucose
Caspase 3
Chemokine CCL2
Cytokines
Glucose Transport Proteins, Facilitative
Insulin
Interleukin-6
Peptide Initiation Factors
Transcription Factors

Figure

• 

  Fig. 1 Pioglitazone (Pio) improves palmitate (PA)-induced β-cell impairment via repression of the inflammatory response and endoplasmic reticulum (ER) stress. (A) Mouse insulinoma 6 (MIN6) cells were incubated with 0.5 mM PA in the presence or absence of 10 µM Pio for 24 hours, and the glucose-stimulated (1 or 4.5 g/L) insulin secretion of MIN6 cells was evaluated. Secreted insulin was measured by a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit. The values are representative of 6 independent experiments. (B) Expression of cleaved caspase-3 (c-casp3), an apoptotic protein, was measured by Western blot analysis. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is represented as the percentage of relative absorbance compared to the vehicle group. (D) Transcription of tumor necrosis factor α (TNFA), interleukin 6 (IL6), and IL1B was measured by real time reverse-transcription polymerase chain reaction, and normalized with β-actin. (E) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), cleaved-activating transcription factor 6 (c-ATF6), and C/EBP homologous protein (CHOP), were measured by Western blot analysis and (F) the ratio of p-eIF2α, GRP78, and CHOP to β-actin was described. Each value represents the mean of three experiments. t-casp3, total caspase-3; Veh, vehicle; PA, palmitate. aP<0.01, bP<0.001 compared with the vehicle group; cP<0.05, dP<0.001 compared with the PA group.

• 

  Fig. 2 Pioglitazone (Pio) represses lipopolysaccharide (LPS)-induced inflammatory response and endoplasmic reticulum (ER) stress. The mouse insulinoma 6 (MIN6) cells were incubated with 10 µg/mL LPS in the presence or absence of 10 µM Pio for 8 hours. (A) Phosphorylation of nuclear factor-kappa B (NF-κB) and jun N-terminal kinase (JNK), essential factors of the inflammatory response, was evaluated by Western blot. (B) The density of phosphorylated NF-κB and JNK was normalized to that of the total forms. (C) The inflammatory cytokines were measured by reverse-transcription polymerase chain reaction, and normalized with β-actin. (D) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), and C/EBP homologous protein (CHOP), were measured by Western blot analysis and (E) the ratio to β-actin was described. Each value represents the mean of three experiments. t-NF-κB, total nuclear factor kappa-light-chain-enhancer of activated B cells; p-JNK, phosphorylated c-Jun N-terminal kinase; t-JNK, total c-Jun N-terminal kinase; TNFα, tumor necrosis factor α; IL, interleukin. aP<0.05, bP<0.01, cP<0.001 compared with the vehicle (Veh) group; dP<0.05, eP<0.01, fP<0.001 compared with the LPS group.

• 

  Fig. 3 Pioglitazone (Pio) reduces endoplasmic reticulum (ER) stress, but not the inflammatory response in tunicamycin (TU)-challenged mouse insulinoma 6 (MIN6) cells. The MIN6 cells were incubated with 2 µg/mL TU in the presence or absence of 10 µM Pio for 24 hours. (A) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), cleaved-activating transcription factor 6 (c-ATF6), and C/EBP homologous protein (CHOP), were measured by Western blot analysis, and (B) the ratio to β-actin was described. (C) The transcription of tumor necrosis factor α (TNFA), interleukin 6 (IL6), and IL1B was measured by real-time reverse-transcription polymerase chain reaction, and normalized with β-actin. Each value represents the mean of three experiments. aP<0.05, bP<0.01, cP<0.001 compared with the vehicle (Veh) group; dP<0.01, eP<0.001 compared with the TU group.

• 

  Fig. 4 Pioglitazone (Pio) protects pancreatic β-cells and regulates blood glucose levels in high-fat (HF)-diet-induced diabetic mice. The pancreatic islet from KK-Ay mice, which were fed an HF diet with or without Pio, were examined by double-immunofluorescence for (A) insulin-activating transcription factor 6 and insulin-glucose-regulated protein 78 (GRP78), (B) insulin-monocyt e chemoattractant protein-1 and insulin-glucagon (GLU), (C) insulin-glucose transporter 2 (GLUT2). (A-C) The nucleus was visualized by 4′,6-diamidino-2-phenylindole (DAPI). Bars=20 µm. (D) Blood glucose levels and body weight were measured every week. INS, insulin; ATF6, activating transcription factor 6; MCP1, monocyte chemoattractant protein-1. aP<0.05 compared with the HF group.

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