Mitigation of Glucolipotoxicity-Induced Apoptosis, Mitochondrial Dysfunction, and Metabolic Stress by N-Acetyl Cysteine in Pancreatic β-Cells

Abstract

Glucolipotoxicity caused by hyperglycemia and hyperlipidemia are the common features of diabetes-induced complications. Metabolic adaptation, particularly in energy metabolism; mitochondrial dysfunction; and increased inflammatory and oxidative stress responses are considered to be the main characteristics of diabetes and metabolic syndrome. However, due to various fluctuating endogenous and exogenous stimuli, the precise role of these factors under in vivo conditions is not clearly understood. In the present study, we used pancreatic β-cells, Rin-5F, to elucidate the molecular and metabolic changes in glucolipotoxicity. Cells treated with high glucose (25 mM) and high palmitic acid (up to 0.3 mM) for 24 h exhibited increased caspase/poly-ADP ribose polymerase (PARP)-dependent apoptosis followed by DNA fragmentation, alterations in mitochondrial membrane permeability, and bioenergetics, accompanied by alterations in glycolytic and mitochondrial energy metabolism. Our results also demonstrated alterations in the expression of mammalian target of rapamycin (mTOR)/5' adenosine monophosphate-activated protein kinase (AMPK)-dependent apoptotic and autophagy markers. Furthermore, pre-treatment of cells with 10 mM N-acetyl cysteine attenuated the deleterious effects of high glucose and high palmitic acid with improved cellular functions and survival. These results suggest that the presence of high energy metabolites enhance mitochondrial dysfunction and apoptosis by suppressing autophagy and adapting energy metabolism, mediated, at least in part, via enhanced oxidative DNA damage and mTOR/AMPK-dependent cell signaling.

Keywords: Rin-5F cells; apoptosis; autophagy; glucolipotoxicity; mitochondrial dysfunction; palmitic acid.

Figure 1

High glucose/high palmitic acid-induced apoptosis in Rin-5F cells. Apoptosis was measured in Rin-5F cells treated with different doses of palmitic acid (PA) under normal (NG) and high glucose (HG) conditions by flow cytometry (A). Percentage of apoptotic cells is represented as a histogram. In some cases, cells were grown on coverslips, and immunocytochemical localization of apoptosis (DNA fragmentation) was determined using the Apop Tag peroxidase in situ apoptosis detection kit, as per the manufacturer’s instructions. Some of the apoptotic nuclei are indicated with arrows (B). DNA fragmentation was also analyzed by agarose gel (2%) electrophoresis and ethidium bromide staining (C). Staining of nuclei of treated cells was also performed using Hoechst33342 dye (D) and analyzed by fluorescence microscopy. Cells with signs of apoptosis showed decrease in stained nuclei. Representative slides from three experiments are shown. Original magnification ×200. Asterisks indicate significant differences (** p ≤ 0.01, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.01) relative to untreated control cells under high glucose condition (HG-C).

Figure 2

High glucose/high palmitic acid increased the activities of caspase-3 (Cas-3) and -9 (Cas-9). Activities of caspases were measured in treated cells colorimetrically using the respective substrates as described in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (Δ p ≤ 0.05, ΔΔ p ≤ 0.01) relative to untreated control cells under high glucose condition (HG-C).

Figure 3

High glucose/high palmitic acid treatment induced alteration in the mitochondrial membrane potential. Mitochondrial membrane potential (Δψm) was measured by flow cytometry (A) using a fluorescent cationic dye according to the vendor’s protocol. A typical histogram (B) representing the percentage loss of mitochondrial membrane potential is shown. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.01, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.01) relative to untreated control cells under high glucose condition (HG-C).

Figure 4

High glucose/high palmitic acid treatment-induced alterations in mitochondrial enzyme activities and ATP production. Rin-5F cells were treated with (0.06 mM and 0.3 mM) palmitic acid under normal and high glucose conditions. Respiratory complex I (A), complex II/III (B), complex IV (C), and ATP (D) were measured as described previously in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (Δ p ≤ 0.05, ΔΔ p ≤ 0.01) relative to untreated control cells under high glucose condition (HG-C).

Figure 5

High glucose/high palmitic acid-induced alterations in Krebs’ cycle enzyme activities. Rin-5F cells were treated with (0.06–0.3 mM) palmitic acid under normal and high glucose conditions. Activities of ROS-sensitive enzyme, aconitase (A), and glutamate dehydrogenase (B) were measured as described previously in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (Δ p ≤ 0.05) relative to untreated control cells under high glucose condition (HG-C).

Figure 6

High glucose/high palmitic acid-induced alterations in the activities of hexokinase (HK) and glucose-6-phosphate dehydrogenase enzymes. The activities of hexokinase (A) and glucose-6-phosphate dehydrogenase (G6PDH) enzyme (B) were measured by a coupled enzyme assay method, as mentioned in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition, and triangles indicate significant differences (Δ p ≤ 0.05, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition.

Figure 7

High glucose/high palmitic acid treatment-induced alterations in the expression of apoptotic, autophagy, and signaling proteins. Total extracts (30 µg protein) or mitochondrial extracts (10 µg) from control and treated cells were separated on 12% SDS-PAGE and transferred onto nitrocellulose paper by Western blotting. Mitochondrial cytochrome c (Mit. cyt c) and PARP (A), LC3 and Atg5 (B), and mTOR and AMPK (C) proteins were detected using specific antibodies against these proteins. Beta-actin and VDAC were used as loading controls for post-mitochondrial and mitochondrial extracts, respectively. The quantitation of the protein bands is expressed as relative ratios normalized against the loading control or other specific proteins as appropriate, and histograms are expressed as mean +/− S.E.M of three experiments. The blots shown are representative of three experiments. Asterisks indicate significant difference (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), (Δ p ≤ 0.05, ΔΔ p ≤ 0.01, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).

Figure 8

Effect of N-acetyl cysteine (NAC) pre-treatment on high glucose/high palmitic acid-induced apoptosis in Rin-5F cells. Apoptosis was measured in Rin-5F cells treated with different doses of palmitic acid under high glucose conditions by flow cytometry. Percentage of apoptotic cells is represented as a histogram (A), which is expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.01) relative to untreated control cells under normal glucose conditions, (ΔΔ p ≤ 0.01) and (ΔΔΔ p ≤ 0.001)relative to untreated control cells under high glucose conditions, (€ p ≤ 0.05) relative to 0.06 mM palmitic acid in the presence of high glucose, (○ p ≤ 0.05) relative to 0.3 mM palmitic acid in the presence of high glucose. DNA fragmentation was analyzed by agarose gel (2%) electrophoresis and ethidium bromide staining (B). Staining of fragmented nuclei of treated cells was performed using Hoechst33342 dye (C). Reduced staining with the dye was an indication of apoptotic nuclei Representative slides from three experiments are shown. Original magnification ×200.

Figure 9

Effect of NAC pre-treatment on the expression of autophagy and cell signaling markers in high glucose/high palmitic acid-treated Rin-5F cells. Total extracts (30 µg protein) from treated cells were separated on 12% SDS-PAGE and transferred onto nitrocellulose paper by Western blotting. LC3, mTOR, and AMPK proteins were detected using specific antibodies against these proteins. Beta-actin was used as loading control. The quantitation of the protein bands is expressed as relative ratios normalized against actin or other specific proteins as appropriate, and histograms are expressed as mean +/− SEM of three experiments. The blots shown are representative of three experiments. Asterisks indicate significant differences (* p ≤ 0.05) relative to untreated control cells under normal glucose conditions, (Δ p ≤ 0.05) relative to untreated control cells under high glucose conditions, (€ p ≤ 0.05) relative to 0.06 mM palmitic acid in the presence of high glucose, (○ p ≤ 0.05) relative to 0.3 mM palmitic acid in the presence of high glucose.

Figure 10

Schematic model representing the cytoprotective mechanisms of NAC in glucolipotoxicity-induced Rin-5F cells. High glucose/high fatty acids have been shown to cause increased oxidative stress, DNA breakdown, and mitochondrial dysfunction, causing activation of caspases and leading to apoptosis. Glucolipotoxicity has also been shown to cause alterations in the expression of autophagy and cell signaling markers. As shown in the model, NAC protects the cells from the glucolipotoxicty-induced mitochondrial and metabolic stress by enhancing autophagy, leading to suppression of apoptosis, thus reducing oxidative stress. GDH: glutamate dehydrogenase, HK: hexokinase, G6PDH; glucose-6-phosphate dehydrogenase, Cyt c; cytochrome c.