**Background:** Obesity is characterized by chronic low-grade inflammation, and dysfunctional adipose tissue contributes to obesity-related comorbidities. Pre-adipocytes express the calcium-sensing receptor (CaSR), and its activation promotes secretion of pro-inflammatory cytokines such as TNF-α and IL-1β. Cellular senescence and mitochondrial dysfunction are key hallmarks of aging and are implicated in liver dysfunction. However, whether secretory products from CaSR-activated pre-adipocytes induce senescence and mitochondrial dysfunction in hepatocytes was unknown.
**Methods:** SW872 human pre-adipocytes were treated with vehicle (DMSO), the CaSR activator cinacalcet (2 µM), or cinacalcet plus the CaSR inhibitor calhex 231 (10 µM) for 16 h, after which conditioned media (CM) were collected. HepG2 human hepatocytes were exposed to these CM for 5 days. Senescence was assessed by senescence-associated β-galactosidase (SA-β-GAL) staining (both positive cell count and colorimetric absorbance at 630 nm), Western blot for p53, p21, and p16 protein levels, and Ki67 immunofluorescence for cell cycle arrest. Pro-inflammatory gene expression (IL-1β and CCL2) was measured by RT-PCR. Mitochondrial dynamics were evaluated by Western blot for OPA1, MFN2, PGC-1α, and DRP1. Mitochondrial membrane potential (Δψ) was assessed by MitoTracker Orange (MTO) fluorescence normalized to mtHsp70, and mitochondrial morphology was analyzed by confocal microscopy. Oxygen consumption rates (baseline, non-ATP-associated, and maximal capacity) were measured using a Clark electrode. Immunoprecipitation of TNF-α, IL-1β, or IL-6 from CM was performed to identify key mediators. In separate experiments, the DRP1 inhibitor Mdivi-1 (50 µM) was added during the last 24 h of CM exposure.
**Key Results:** CM from cinacalcet-treated SW872 cells (CMcin) significantly increased SA-β-GAL activity in HepG2 cells compared to vehicle CM (p < 0.05), an effect abolished by calhex 231 co-treatment. CMcin increased p21 and p16 protein levels (p < 0.05) but not p53, decreased Ki67 nuclear fluorescence (p < 0.05), and increased IL-1β and CCL2 mRNA levels (p < 0.05). Immunoprecipitation of TNF-α or IL-1β from CMcin prevented the increase in SA-β-GAL, while IL-6 immunoprecipitation had no significant effect. CMcin decreased OPA1 and PGC-1α protein levels (p < 0.05), showed a trend toward decreased MFN2 (p < 0.08), and increased DRP1 levels (p < 0.05). CMcin reduced the MTO/mtHsp70 fluorescence ratio (p < 0.05), increased the number of mitochondria per cell (p < 0.05), and showed a trend toward reduced average mitochondrial area (p < 0.08). CMcin decreased non-ATP-associated and maximal capacity respiration rates (p < 0.05) without significantly altering baseline respiration. Mdivi-1 treatment prevented CMcin-induced mitochondrial fragmentation, restored Δψ, recovered Ki67 nuclear fluorescence (p < 0.01), and prevented the increase in IL-1β and CCL2 mRNA (p < 0.05).
**Clinical Implications:** This study provides evidence for a novel mechanism of intercellular communication between pre-adipocytes and hepatocytes mediated by CaSR activation. The findings suggest that CaSR activation in adipose tissue may contribute to obesity-related liver dysfunction by promoting hepatocyte senescence and mitochondrial damage through TNF-α and IL-1β secretion. The demonstration that mitochondrial fragmentation is a key mediator of this process, and that its inhibition can prevent both mitochondrial dysfunction and senescence, identifies mitochondrial dynamics as a potential therapeutic target for obesity-associated liver disease. Limitations include the use of cell lines rather than primary cells, lack of cell cycle analysis by flow cytometry, and absence of in vivo validation. Future studies should explore CaSR gene downregulation, assess effects on other cell types, and investigate organismal consequences of CaSR activation on liver senescence.