The effects on hepatic mitochondrial structure, mitophagy, and cellular homeostasis were investigated when treated with Tirzepatide (Tzp), a dual GIP/GLP-1 receptor agonist, in a mouse model combining obesity, type 2 diabetes, and menopause-conditions that collectively exacerbate metabolic dysfunction-associated steatotic liver disease (MASLD). Female C57BL/6 mice were fed either a control or high-fat, high-sucrose diet for 12 weeks, and half underwent bilateral ovariectomy to simulate menopause. Tzp was administered daily for four weeks. Liver tissue was evaluated for ultrastructural alterations, gene expression, and protein profiles. Untreated obese-diabetic and obese-diabetic-ovariectomized mice exhibited hepatocellular fat accumulation, mitochondrial swelling, and disorganized cristae, indicative of metabolic and oxidative stress. In contrast, Tzp-treated mice displayed preserved hepatic architecture and intact mitochondrial morphology. Tzp significantly downregulated autophagy genes (Ulk3, Atg5, Atg7) and key mitophagy regulators (PINK1, PRKN), reestablishing mitochondrial balance, primarily through the modulation of mitophagy and enhanced organelle stability. Simultaneously, it upregulated mitochondrial biogenesis markers (Ppargc1a, Tfam), antioxidant enzymes (SOD2, GR, GPX, CAT), and redox modulators (Sirt3, Nrf2), while normalizing oxidative stress-related genes (Nos1, Nox1). Tzp also mitigated endoplasmic reticulum (ER) stress by downregulating Atf4, Ddit3, and Gadd45 and rebalanced mitochondrial dynamics through the suppression of fission markers (Dnml1, Fis1) and the restoration of fusion mediators (Mfn1, Mfn2). Three-way ANOVA confirmed Tzp's broad regulatory effects on hepatic gene expression, while principal component analysis revealed clear transcriptional separation between treated and untreated groups. In conclusion, these findings demonstrate that Tzp preserves liver ultrastructure and restores mitochondrial dynamics, supporting its therapeutic potential in MASLD and hormone-related metabolic disorders.
