Lipotoxic Palmitate Hyperpolarises Mitochondria and Converts Nuclear 5mC to 5hmC: 8-Oxo-dG, 5mC and 5hmC Read on the Same Native DNA Molecules of Human Adipose-Derived Mesenchymal Stem Cells

Lipotoxic stress from saturated fatty acids compromises the mitochondria of human adipose-derived mesenchymal stem cells (hAdMSCs) and, with them, their regenerative capacity. We exposed hAdMSCs to sodium palmitate at 250 and 500 µM for 24 h and combined live-cell confocal imaging with long-read nanopore sequencing of native DNA, reading 8-oxo-2′-deoxyguanosine (8-oxo-dG), 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) directly from the raw signal of the same molecules. Surprisingly, palmitate hyperpolarised mitochondria at both concentrations instead of depolarising them; the lower, obesity-range concentration fragmented the mitochondrial network over 24 h, and both raised mitophagy-associated and lysosomal puncta without preventing a concentration-dependent rise in senescence-associated β-galactosidase positivity. Mitochondrial DNA copy number fell to 56% of untreated, while the remaining mitochondrial genomes tended to carry more 8-oxo-dG (27%) and nuclear 8-oxo-dG tended to be lower (20%), neither contrast reaching significance at this depth. Nuclear CpG methylation fell from 29.87% to 26.84%, uniformly across the genome; the loss of 5mC was matched by an equal gain of 5hmC, consistent with oxidation of the methyl group rather than its loss. Methylation was depressed beside oxidised guanines in untreated cells, and less so under palmitate. In a thirteen-gene adipokine panel, promoter demethylation reached transcript and secreted protein only for IL6. Ribosomal DNA retained its methylation and was neither less oxidised nor exempt from conversion, its 5hmC rising with the genome. Palmitate thus remodels two genomes under one metabolic condition; in the adipose progenitor, lipotoxic stress pairs mitochondrial depletion with a rewritten nuclear methylome, a route by which obesity may compromise the fate and regenerative capacity of the progenitor pool.