High-fat diets or insulin-resistant adipose tissue, as in diabetes, can lead to excess body fat and lipotoxicity, which is involved in the pathogenesis of the metabolic syndrome. Diets high in animal fat, for example, increase the levels of circulating palmitate, the most abundant saturated fatty acid in human blood and a cause of lipotoxicity. As mitochondria are organelles with a central role in lipid metabolism, researchers from RIDC Redoxoma investigated the bioenergetic effects of high levels of palmitate in liver cells. Surprisingly, they found that excess palmitate increases glycolytic flow and ATP production in the cell cytosol and causes mitochondrial fragmentation, but does not interfere with mitochondrial respiration.

“In parallel, we measured the cellular redox state using the NAD(P)H/NAD(P) ratio as a parameter and we saw that the cells treated with palmitate were quite different. We also measured the reactive species production and observed that these cells had a very exacerbated redox alteration”, said researcher Pamela A. Kakimoto, who conducted the study as part of her doctorate, under the guidance of Professor Alicia Kowaltowski, from the Instituto de Química of the Universidade de São Paulo (USP) and a member of the RIDC Redoxoma. The research was published in the journal Redox Biology.

For Kowaltowski, the results point to new metabolic regulation mechanisms. “For the first time, we showed that glucose metabolism is regulated by lipid toxicity, and that’s unexpected because glucose and fat metabolism are different. And it is also the first time that glucose metabolism is shown to be regulated by the cellular redox state”.

In addition to being the cell´s batteries, producing ATP, mitochondria act as regulatory centers for calcium signaling, provide intermediaries for lipid synthesis, modulate the production and removal of oxidants, and are involved in cell death mechanisms. At the same time, they sense nutrient availability and cooperate with pathways regulating metabolism, including insulin responses. So it was expected that they would be a target for lipotoxicity.

However, although palmitate caused mitochondrial fragmentation, the morphological changes did not affect mitochondrial oxygen consumption, and cells maintained ATP production rates despite clear lipotoxicity. According to the researchers, the study shows that, in the case of mitochondria, a change in shape does not always correspond to a change in function.

This work required rigorous techniques and precise methodological tools. Human hepatoma cells were treated with high concentrations of palmitate, mimicking a lipemic plasma, in an extremely controlled environment, and then analyzed. Kakimoto designed protocols for the Seahorse equipment, which measures oxygen consumption and the acidification of the culture medium in real time. With this instrument, it is possible to evaluate and modulate the mitochondrial function and, at the same time, indirectly identify the production of cytosolic ATP. Thus, the researcher could quantify ATP production in different metabolic pathways. Mitochondrial morphology analysis using confocal microscopy was carried out by Kakimoto in Spain, in collaboration with Professor Antonio Zorzano’s group, at the Institute for Research in Biomedicine of Barcelona.

Metabolic syndrome and fatty liver

According to Kowaltowski, the study allows us to understand the mechanism involved in hepatic steatosis, the famous fatty liver, which can also be caused by palmitate overload.

Lipotoxicity, defined as an increase in lipids that compromises function, is involved in the pathogenesis of the metabolic syndrome. Metabolic syndrome is the conjunction of several metabolic changes, such as changes in glucose metabolism, in the case of diabetes or pre-diabetes; alteration in lipid metabolism, with an increase in cholesterol and triglycerides, for example; and change in energy balance, in the case of obesity. Kakimoto explains that, although there is no consensus of the metabolic syndrome definition, most associations defining it agree that in addition to high waist circumference (central obesity), dyslipidemia, high insulin or high fasting glucose and hypertension may be present for diagnosis. The metabolic syndrome is associated with an increased risk of cardiovascular disease, Alzheimer’s, and unhealthy aging.

In addition, according to the researcher, there is an increased probability of a patient with metabolic syndrome developing hepatic steatosis. The condition is often benign, but “some patients can develop non-alcoholic steatohepatitis, which turns on a red light in the person’s quality of life. And there is a correlation: metabolic syndrome can exacerbate steatosis and steatosis can exacerbate markers of metabolic syndrome. Steatosis is silent and difficult to diagnose, as it requires a biopsy, and when the patient has liver symptoms, he is already in a not very interesting condition”, said Kakimoto. Steatotic patients in general are at increased risk of developing cardiovascular disease.

The liver is a highly flexible organ that can oxidize and synthesize carbohydrates, fatty acids, and amino acids. The center of its metabolism, explains Kakimoto, are mitochondria, with their various functions that support basal metabolism. Hence the importance of investigating the mitochondrial function in the study of liver diseases. Previous studies by the group had already revealed similar results. Kakimoto had studied the effect of high lipid levels in mouse liver and observed that mitochondria produced ATP normally, but the production of free radicals and reactive oxygen species was increased. Interestingly, according to the authors, the increase in glycolysis observed with palmitate can be mimicked by modulating the production of reactive oxygen species.

The article Increased Glycolysis is an Early Consequence of Palmitate Lipotoxicity Mediated by Redox Signaling, by Pamela A. Kakimoto, Julián David Serna, Vitor de Miranda Ramos, Antonio Zorzano, and Alicia J. Kowaltowski, can be accessed here.