Remodelling of the Mitochondrial Bioenergetic Pathways in Human Cultured Fibroblasts with Carbohydrates

SIMPLE SUMMARY: Many neurological diseases are caused by defects in the powerplants of cells, called mitochondria. To study these diseases in the laboratory, researchers often grow skin cells (fibroblasts) derived from patients in a dish. Glucose is normally used to feed the skin cells. However, when skin cells are fed with glucose, they rely less on mitochondria to generate energy but prefer glycolysis as alternative energy-generating pathway. As a result, defects in mitochondria are not showing because the skin cells are not using the powerplants. Therefore, these diseases cannot be studied with skin cells grown in a dish with glucose. We investigated if skin cells can be fed with two other sugars, galactose and fructose. We show that although the skin cells grow more slowly, they are using their mitochondria to generate energy when fed with galactose or fructose. Thus, these neurological disorders can be studied in a dish if skin cells from patients are fed with galactose or fructose. As skin cells grow much slower in a dish when fed with galactose than when fed with fructose, we recommend feeding skin cells with fructose to study these diseases the laboratory. ABSTRACT: Mitochondrial oxidative phosphorylation defects underlie many neurological and neuromuscular diseases. Patients’ primary dermal fibroblasts are one of the most commonly used in vitro models to study mitochondrial pathologies. However, fibroblasts tend to rely more on glycolysis than oxidative phosphorylation for their energy when cultivated in standard high-glucose medium, rendering it difficult to expose mitochondrial dysfunctions. This study aimed to systematically investigate to which extent the use of galactose- or fructose-based medium switches the fibroblasts’ energy metabolism to a more oxidative state. Highly proliferative cells depend more on glycolysis than less proliferative cells. Therefore, we investigated two primary dermal fibroblast cultures from healthy subjects: a highly proliferative neonatal culture and a slower-growing adult culture. Cells were cultured with 25 mM glucose, galactose or fructose, and 4 mM glutamine as carbon sources. Compared to glucose, both galactose and fructose reduce the cellular proliferation rate, but the galactose-induced drop in proliferation is much more profound than the one observed in cells cultivated in fructose. Both galactose and fructose result in a modest increase in mitochondrial content, including mitochondrial DNA, and a disproportionate increase in protein levels, assembly, and activity of the oxidative phosphorylation enzyme complexes. Galactose- and fructose-based media induce a switch of the prevalent biochemical pathway in cultured fibroblasts, enhancing aerobic metabolism when compared to glucose-based medium. While both galactose and fructose stimulate oxidative phosphorylation to a comparable degree, galactose decreases the cellular proliferation rate more than fructose, suggesting that a fructose-based medium is a better choice when studying partial oxidative phosphorylation defects in patients’ fibroblasts.