Abstract
Metabolic (dysfunction)-associated steatotic liver disease is the hepatic consequence of metabolic syndrome and affects 30% of the global population. Steatosis can progress to steatohepatitis, the second leading cause of hepatocellular carcinoma. Therefore, it is critical to understand the development of and contributors to metabolic (dysfunction)-associated steatotic liver disease. The role of diet, and specifically the sugar fructose, has been investigated as a driver of hepatic triglyceride and lipid accumulation. However, results from animal and human studies present conflicting findings on this association. New approach methodologies, including liver-on-a-chip, present an opportunity to address limitations of in vitro studies by culturing cells in microchannels of a device that is subject to microfluidic flow. Therefore, this work aimed to 1) confirm the effect of fructose on key hallmarks of metabolic (dysfunction)-associated steatotic liver disease in 2D cell culture, 2) determine the distribution of sugars and fatty acids into Emulate S-1 chips, and 3) determine the effect of fructose on lipid accumulation in Emulate S-1 chips.
Supraphysiological concentrations of fructose (50 and 80 mM) moderately decreased the concentration of triglycerides in HepG2 cell lysates, determined using a triglyceride detection reagent. However, at the upper limit of physiological concentrations (20 mM), fructose did not affect the concentration of triglycerides in low or high glucose media, or in the presence or absence of a fatty acid mixture. When cultured in fructose for 28 days, there was also no effect on the concentration of triglycerides. This was confirmed by rt-qPCR where exposure to fructose for 6 h did not affect the expression level of the de novo lipogenic transcription factors NR1H3 and SREBF1, and the enzymes ACACA and FASN. Similarly, at a range of acute time points, fructose did not affect the level of reactive oxygen species in the cells, or the mitochondrial membrane potential detected using fluorescent probes. To test a more physiologically relevant model, the Emulate liver-on-a-chip system was adapted and validated for compound distribution into the poly(dimethylsiloxane) channels. A UPLC-MS/MS method for the detection of fructose and glucose, and a GC-MS method for the detection of oleic acid and palmitic acid was developed and validated as accurate and precise. Over 72 h, the sugars did not distribute into the channels of blank chips, whereas up to 45% of oleic acid, and 40% of palmitic acid distributed into the channels. HepG2 cells in the chips were then exposed to fructose and they remained viable in the channels for 72 h. Cell viability was confirmed by daily observation, high albumin secretion, and low lactate dehydrogenase release. The concentration of triglycerides and cholesterol released from cells in the chips did not differ from the concentration in serum containing media. The lipid content of the chips exposed to fructose was quantified using confocal microscopy and automated image analysis. The number of lipid droplets, as well as the lipid droplet surface area and fluorescent intensity were unaffected by fructose exposure. Interesting differences between the chip assay and a comparable 2D plate assay occurred, including 2.4-fold higher albumin secretion in the chips, and 8.8-fold larger lipid droplets in the plates.
This work demonstrated that in HepG2 cells, fructose was not lipogenic in both traditional 2D cell culture, or in a more complex liver-on-a-chip device. As HepG2 cells are a cancer cell line that preferentially uses glycolysis for ATP, excess carbon from the fructose exposure was likely shunted to biomolecule synthesis and cell division. Despite this, establishing the use of the Emulate liver-on-a-chip in New Zealand is an important step as New Zealand specific toxicological questions can now be investigated using a more complex, human relevant in vitro model of the liver.