The high-fat animals gained more body weight from week four onward and consumed more calories from the first week of the regimen. Although liver weights did not differ between the groups at the end of the feeding period, white adipose tissue mass was significantly increased in the high-fat group. Serum analysis revealed elevated total cholesterol and non-HDL cholesterol, while triglycerides and HDL cholesterol remained unchanged.

Examination of the liver tissue itself painted a picture strikingly reminiscent of aging. Oil Red O staining showed marked lipid accumulation, and hepatic triglyceride content was significantly elevated. Levels of lipid peroxidation products, measured by the thiobarbituric acid reactive substances assay, were also increased, indicating heightened oxidative stress.

The livers of high-fat-fed rats contained significantly higher amounts of the inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α. Critically, the senescence-associated markers were all shifted in the aging direction: the area staining positive for senescence-associated β-galactosidase expanded, the proportions of cells positive for p16, p21, and γH2AX—a marker of DNA damage signaling—rose, and fluorescence intensity of Lamin B1, a nuclear lamina protein that declines with cellular aging, fell. Two weeks after the final injection, the proportion of Ki67-positive proliferating cells was significantly lower in the high-fat group, indicating blunted regenerative responses.

Moreover, among the Ki67-positive cells that were proliferating, a significantly greater fraction in the high-fat group also stained positive for p16 or γH2AX. In other words, the cells attempting to divide in the stressed liver carried senescence-associated hallmarks with them, suggesting that regeneration proceeded under a cloud of cellular damage rather than cleanly. The pivotal question was whether such abnormalities were merely transient responses sustained by the ongoing metabolic milieu, or whether they had become permanently inscribed in the regenerative cells themselves.

Because organoids can be serially passaged outside the body, any defect that persists across passages must be retained within the organoid-forming cell population rather than imposed by the circulating environment. The results were unambiguous. Primary organoids from high-fat-fed rats were smaller on average, with an enrichment of structures measuring 100 to 400 micrometers and a significantly lower proportion of large organoids exceeding 400 micrometers.

When organoid formation at the first passage was normalized to 100 percent, the high-fat-derived organoids showed reduced formation efficiency beginning at the third passage, and Kaplan–Meier analysis of the loss of organoid formation over serial passaging up to passage 13 revealed markedly diminished self-renewal capacity. Senescence-associated staining differences—elevated β-galactosidase activity, increased p16 and γH2AX positivity, reduced Ki67, and diminished Lamin B1—remained detectable through passages 2 and 3, long after the cells had left the obese animal's bloodstream behind. Transcriptomic profiling by RNA sequencing deepened the picture.

The high-fat-derived organoids showed 29 upregulated and 71 downregulated genes, among them Gpnmb, a senescence-associated glycoprotein that was also elevated in the livers of the high-fat-fed rats in vivo. Gene set enrichment analysis identified significant activation of pathways related to lipid metabolism, inflammation, oxidative stress, and mitochondrial function. Quantitative PCR confirmed upregulation of genes in a superoxide anion generation pathway, including Itgb2 and Cybb, and of genes in an interleukin-6 production pathway, including Clec7a, Tlr3, and Il1rl2.

When the organoids were challenged with a combination of oleic and palmitic acid, the high-fat-derived cultures mounted a stronger inflammatory response, secreting more IL-1β, IL-6, TNF-α, and MCP-1 than control-derived organoids, and their intracellular reactive oxygen species levels were elevated both at baseline and after the fatty acid exposure. Lipid handling and mitochondrial function also bore the imprint of the prior diet. The high-fat-derived organoids expressed higher levels of the fatty acid uptake genes Cd36 and Fatp1, and accumulated significantly more lipid after oleic acid exposure, as measured by both Oil Red O staining and flow cytometric analysis of BODIPY-stained cells, although genes of de novo lipogenesis did not differ between groups.

On the mitochondrial side, oxygen consumption rate was reduced, mitochondrial DNA content was lower, and expression of mitochondrial regulatory genes including Mto1, Mrps24, and Tfam was downregulated. JC-1 staining showed increased monomer fluorescence and decreased aggregate fluorescence, consistent with depolarization of the mitochondrial membrane potential. Could this mitochondrial dysfunction be functionally responsible for the regenerative deficit?

MitoQ significantly reduced reactive oxygen species levels, lowered secretion of TNF-α and MCP-1, and restored oxygen consumption to levels comparable to control-derived organoids. Most importantly, organoid formation efficiency at passage 3, which was significantly reduced in untreated high-fat-derived cultures, was significantly improved by MitoQ treatment—although the recovery remained incomplete relative to controls. In contrast, the general antioxidant N-acetylcysteine reduced the measured ROS signal without significantly improving organoid formation, and the NF-κB pathway inhibitor BAY 11-7082 likewise failed to rescue formation efficiency, indicating that simply lowering a ROS readout does not guarantee regenerative recovery.

The authors are careful about interpretation. They use the term senescence-like because irreversible cell-cycle arrest was not established at the single-cell level, and the cultures should not be viewed as a uniform population of senescent cells; no direct comparison with other senescence-inducing stimuli was performed, so an HFD-specific senescence program is not established. The study also has clear limitations: the precise cellular origin of the organoid-forming populations was not determined, persistence was demonstrated at the population rather than the individual-cell level, only male rats and a single diet regimen were tested, and it remains unknown whether the MitoQ benefit survives treatment withdrawal or translates into improved repair in living animals.

Even so, the work extends the consequences of chronic metabolic stress beyond the exposed liver tissue itself, pointing to a durable cellular memory within the regenerative compartment. If similar reprogramming occurs in humans, it would raise a sobering possibility: that losing weight and normalizing metabolism may not fully restore the liver's capacity for self-renewal, and that the cells charged with rebuilding the organ may continue to carry the metabolic past within them.

Subject of Research: Durable senescence-like reprogramming of the liver regenerative compartment by chronic high-fat diet in rats

Article Title: High‐Fat Diet–Induced Metabolic Stress Durably Reprograms the Liver Regenerative Compartment Toward a Senescence‐Like State

Article References: Yamazaki, M., Yamada, H., Munetsuna, E., Ando, Y., Mizuno, G., Wakasugi, T., Sakakibara, T., Hada, K., Ichikawa, H., Senga, R., Asai, M., Ishikawa, H., Suzuki, K., Ohashi, K., & Ohgami, N. (2026). High‐Fat Diet–Induced Metabolic Stress Durably Reprograms the Liver Regenerative Compartment Toward a Senescence‐Like State. Aging Cell, 25(10), Article e70769. https://doi.org/10.1111/acel.70769

Image Credits: AI Generated

DOI: 10.1111/acel.70769

Keywords: liver organoids, high-fat diet, cellular senescence, mitochondrial dysfunction, liver regeneration, metabolic stress, MitoQ, oxidative stress, inflammaging, p16, γH2AX, rat model