Abstract
Type 2 diabetes mellitus (T2DM) is a disease that has been associated with hyperglycaemia mediated complications and an increased risk of liver disease, including non-alcoholic fatty liver disease (NAFLD). However, more recently, the glucose metabolite methylglyoxal (MGO), rather than hyperglycaemia per se, has been implicated as the major mediator, via dicarbonyl stress resulting in cellular dysfunction. MGO is a highly reactive dicarbonyl compound and exerts its damaging effects by causing irreversible damage to proteins, lipids, and DNA. Most previous studies investigating the role of MGO in cellular toxicity and dysfunction were conducted using exogenously applied MGO in unphysiological concentrations that are significantly higher than what is observed in individuals with both type 1 and type 2 diabetes. Although providing valuable insight into the cellular effects of MGO, this approach does not accurately reflect the natural occurrence of MGO accumulation within hepatocytes in diabetes. Therefore, the overall objective of this research study was to mimic, in a more physiological manner, endogenous MGO accumulation observed in diabetes by inhibiting glyoxalase 1 (GLO1), the key detoxifying enzyme for MGO. The specific aim of this study is to examine the effects of an established GLO1 inhibitor, S-p bromobenzylglutathione cyclopentyl diester (BBGC), on hepatocyte function and survival.To study the effects of GLO1 inhibition in hepatocytes, the human hepatoma G2 (HepG2) cell line was used, and HepG2 cells were treated with increasing concentrations of BBGC to first characterise its effects. After the treatment duration was completed, intracellular hydroimidazolone (MG-H1) formation, which is indicative of MGO levels, was determined using confocal microscopy following immunolabelling with a human anti-MG-H1 antibody. HepG2 cell viability was assessed a using a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and cell death by flow cytometric analysis following Annexin V and propidium iodide staining. Intracellular ROS generation was measured following staining with the 2′,7′-dichlorodihydrofluorescein diacetate probe by confocal microscopy and similarly, mitochondrial superoxide generation was measured following MitoSOX™ staining. HepG2 mitochondrial function was assessed by using flow cytometry to measure changes in mitochondrial membrane potential following staining with tetramethylrhodamine ethyl ester. Cellular ATP levels were also determined, by luminometry. Changes in mRNA expression of cytokines interleukin 8 (IL-8) and tumour necrosis factor alpha, and hepatokines fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) were also determined using reverse transcription-quantitative polymerase chain reaction (RT-PCR), and IL-8, FGF21 and GDF15 secretion was measured, using an ELISA kit. Transcription factor nuclear factor kappa B (NF-κB) activation was assessed by measuring nuclear translocation of NF-κB p65 via confocal microscopy following immunolabelling with human anti-NF-κB p65. mRNA and protein expression of key endoplasmic reticulum (ER) stress markers (CHOP and GRP78) were determined by RT-PCR and Western blotting. mRNA expression of angiotensin converting enzyme 2 was also measured by RT-PCR. Next, we investigated the mRNA and protein expression and activity of key enzymes cytochrome P450 3A4 and carboxylesterase 1 (CES1) in HepG2 cells. This was achieved using RT-PCR, Western blotting, and specific activity assay kits, respectively.
We identified that BBGC increased intracellular MG-H1 levels in hepatocytes, indicative of increased intracellular methylglyoxal concentrations and dicarbonyl stress. Our results revealed that BBGC-induced dicarbonyl stress led to decreased HepG2 cell viability, and increased ER stress and apoptosis rates, likely mediated through mitochondrial dysfunction and increased ROS generation. BBGC-mediated dicarbonyl stress increased hepatocyte inflammation, as evidenced by the induction of IL-8 expression and secretion, and NF-κB activation. In addition to corroborating previous findings linking MGO and dicarbonyl stress to these deleterious effects, this study identified new mechanisms by which MGO could instigate cellular dysfunction that could contribute to the development of the well-established liver complications of diabetes. MGO triggered hepatokines GDF15 and FGF21 expression and release, likely reflecting their dysregulated secretion during cellular stress and disease, which highlights the pathophysiological relationship between NAFLD, and other complications related to diabetes. MGO also decreases key drug metabolizing enzyme CES1, which could possibly explain altered pharmacokinetics and discrepancies in treatment outcomes in diabetic patients. CES1, having profound effects on key hepatocyte functions, such as lipid and glucose metabolism, makes this effect also particularly relevant in the context of NAFLD development in diabetes. CES1 inhibition also leads to increased IL-8, FGF21 and GDF15 mRNA expression and release, implicating CES1 in the modulation of important inflammatory mediators in HepG2 cells.
We have demonstrated that inhibition of GLO1 is a more physiologically relevant model of inducing dicarbonyl stress in hepatocytes, which will allow further investigation into the role MGO and dicarbonyl stress plays in increasing the susceptibility of developing liver disease in 4 diabetes. In addition, we have expanded upon our knowledge of the effects of dicarbonyl stress on hepatocytes, implicating it in the disruption of xenobiotic metabolism and increased hepatokine release.
| Date of Award | Feb 2025 |
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| Original language | English |
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| Supervisor | Jon Mabley (Supervisor) & Greg Scutt (Supervisor) |