Secretory Factors from Calcium-Sensing Receptor-Activated SW872 Pre-Adipocytes Induce Cellular Senescence and A Mitochondrial Fragmentation-Mediated Inflammatory Response in HepG2 Cells

Adipose tissue inflammation in obesity has a deleterious impact on organs such as the liver, ultimately leading to their dysfunction. We have previously shown that activation of the calcium-sensing receptor (CaSR) in pre-adipocytes induces TNF-α and IL-1β expression and secretion; however, it is unknown whether these factors promote hepatocyte alterations, particularly promoting cell senescence and/or mitochondrial dysfunction. We generated conditioned medium (CM) from the pre-adipocyte cell line SW872 treated with either vehicle (CMveh) or the CaSR activator cinacalcet 2 µM (CMcin), in the absence or presence of the CaSR inhibitor calhex 231 10 µM (CMcin+cal). HepG2 cells were cultured with these CM for 120 h and then assessed for cell senescence and mitochondrial dysfunction. CMcin-treated cells showed increased SA-β-GAL staining, which was absent in TNF-α- and IL-1β-depleted CM. Compared to CMveh, CMcin arrested cell cycle, increased IL-1β and CCL2 mRNA, and induced p16 and p53 senescence markers, which was prevented by CMcin+cal. Crucial proteins for mitochondrial function, PGC-1α and OPA1, were decreased with CMcin treatment, concomitant with fragmentation of the mitochondrial network and decreased mitochondrial transmembrane potential. We conclude that pro-inflammatory cytokines TNF-α and IL-1β secreted by SW872 cells after CaSR activation promote cell senescence and mitochondrial dysfunction, which is mediated by mitochondrial fragmentation in HepG2 cells and whose effects were reversed with Mdivi-1. This investigation provides new evidence about the deleterious CaSR-induced communication between pre-adipocytes and liver cells, incorporating the mechanisms involved in cellular senescence.


Introduction
Cell senescence is described as a mechanism whereby a dividing cell enters a stable cell cycle arrest upon a stressing stimulus while remaining metabolically active. Senescent cells show the senescence-associated secretory phenotype (SASP) which involves the production of pro-inflammatory cytokines and other signaling factors and they become unresponsive to mitogenic and apoptotic signals [1]. Depending on the context, cell senescence can result in beneficial or detrimental effects to the organism. In young individuals or upon acute

Pro-Inflammatory Secretion Factors from CaSR-Activated SW872 Pre-Adipocytes Induce Cell Senescence in HepG2 Cells
To evaluate the pro-senescence effect of pre-adipocyte-secreted factors, we obtained conditioned medium (CM) from SW872 cells treated with the CaSR activator cinacalcet 2 µM for 16 h (CM cin ). As the control, we used CM from SW782 cells treated with vehicle alone (CM veh ). The senescence-associated β-galactosidase (SA-β-GAL) assay (a marker of residual lysosomal activity associated with cell senescence) [13,25] showed that CM cin -treated HepG2 cells displayed higher SA-β-GAL expression, as well as cytomegaly compared to CM veh -treated cells (p < 0.05) (Figure 1a). We quantified SA-β-GAL expression both as the count of positive cells and colorimetry (Figure 1b,c). To test whether these changes depended on CaSR activation, we obtained CM from SW872 cells treated with cinacalcet in the presence of the CaSR inhibitor calhex 231 10 µM (CM cin+cal ), which abolished the effect of CM cin (Figure 1b,c). alone (CMveh). The senescence-associated β-galactosidase (SA-β-GAL) assay (a marker of residual lysosomal activity associated with cell senescence) [13,25] showed that CMcintreated HepG2 cells displayed higher SA-β-GAL expression, as well as cytomegaly compared to CMveh-treated cells (p < 0.05) (Figure 1a). We quantified SA-β-GAL expression both as the count of positive cells and colorimetry (Figure 1b,c). To test whether these changes depended on CaSR activation, we obtained CM from SW872 cells treated with cinacalcet in the presence of the CaSR inhibitor calhex 231 10 µ M (CMcin+cal), which abolished the effect of CMcin (Figure 1b,c).

Figure 1.
CaSR activation in SW872 pre-adipocytes induces the secretion of factors that induce cell senescence in HepG2 hepatocytes: (a) Representative images of SA-β-GAL staining of HepG2 cells exposed to the CMveh, CMcin, and CMcin+cal (scale bar = 100 μm); (b) Quantification of SA-β-GAL positive cells normalized by total cells in the field; and (c) SA-β-GAL absorbance recorded at 630 nm. Each dot represents an individual experiment (n = 6). * p < 0.05 versus the control, # p < 0.05 versus CMcin, Friedman's test, and Dunn's post-hoc test.
To further characterize the senescent phenotype in our model, we evaluated the p53/p21/p16 signaling cascade. Cell cycle regulation by p53, p21, and p16 can be activated by stress signals, such as DNA damage or mitochondrial dysfunction [26][27][28]. CMcintreated HepG2 cells showed increased protein levels of p16 and p21, but not p53, as assessed by Western blot analysis (p < 0.05) (Figure 2a-d). Again, treatment with CMcin+cal failed to induce any change, thereby supporting the role of CaSR activation in this process. To further characterize the senescent phenotype in our model, we evaluated the p53/p21/p16 signaling cascade. Cell cycle regulation by p53, p21, and p16 can be activated by stress signals, such as DNA damage or mitochondrial dysfunction [26][27][28]. CM cin -treated HepG2 cells showed increased protein levels of p16 and p21, but not p53, as assessed by Western blot analysis (p < 0.05) (Figure 2a-d). Again, treatment with CM cin+cal failed to induce any change, thereby supporting the role of CaSR activation in this process.
Cell cycle arrest is a hallmark of cell senescence, which we evaluated by the location of the protein Ki67. This protein is diminished in the nucleus of senescent cells, and together with other markers, allows us to assess cell senescence [29]. As expected, HepG2 cells treated with CM cin showed a decreased Ki67 immunofluorescence signal compared with the control condition (p < 0.05). This change did not take place in the CM cin+cal condition (Figure 3a,b).
Cell cycle arrest is a hallmark of cell senescence, which we evaluated by the location of the protein Ki67. This protein is diminished in the nucleus of senescent cells, and together with other markers, allows us to assess cell senescence [29]. As expected, HepG2 cells treated with CMcin showed a decreased Ki67 immunofluorescence signal compared with the control condition (p < 0.05). This change did not take place in the CMcin+cal condition (Figure 3a,b).
Another key feature of senescent cells is the senescence-associated secretory phenotype (SASP), which consists of the induction of pro-inflammatory cytokines and chemoattractants, such as IL-1β and CCL2, respectively [30,31]. Our results show that HepG2 cells treated with CM cin increased the relative abundance of both IL-1β and CCL2 mRNA compared to CM veh (p < 0.05), while CM cin+cal prevented these changes (Figure 4a-c).
To explore the mediators of this pro-senescence effect, we separately immunoprecipitated IL-6, IL-1β, or TNF-α from the SW872-derived CM cin , given the evidence of their release by preadipocytes [10,11,32] in addition to their association with stress-associated cell senescence [33][34][35]. Our approach specifically reduced the levels of each cytokine from the CM (Figure 5a). We verified the effectiveness of the procedure by observing that each cytokine was enriched in the pellet of their respective immune complex (Figure 5b  Another key feature of senescent cells is the senescence-associated secretory phenotype (SASP), which consists of the induction of pro-inflammatory cytokines and chemoattractants, such as IL-1β and CCL2, respectively [30,31]. Our results show that HepG2 cells treated with CMcin increased the relative abundance of both IL-1β and CCL2 mRNA compared to CMveh (p < 0.05), while CMcin+cal prevented these changes (Figure 4a   CCL2 mRNA in HepG2 cells exposed to CMcin and CMcin+cal; expressed as fold from CMveh, (represented by the dotted line). mRNA levels were normalized to GAPDH mRNA. The insets show CMcal normalized mRNA levels relative to the control. Each dot represents an individual experiment (n = 2-7). * p < 0.05 versus the control, # p < 0.05 versus CMcin, and the Wilcoxon signed rank test.
To explore the mediators of this pro-senescence effect, we separately immunoprecipitated IL-6, IL-1β, or TNF-α from the SW872-derived CMcin, given the evidence of their release by preadipocytes [10,11,32] in addition to their association with stress-associated cell senescence [33][34][35]. Our approach specifically reduced the levels of each cytokine from the CM (Figure 5a). We verified the effectiveness of the procedure by observing that each cytokine was enriched in the pellet of their respective immune complex (Figure 5b). Im- Figure 4. CaSR activation in SW872 pre-adipocytes induces the secretion of factors that induce the secretion of pro-inflammatory cytokines in HepG2 cells: relative abundance of (a) IL-1β and (b) CCL2 mRNA in HepG2 cells exposed to CM cin and CM cin+cal ; expressed as fold from CM veh , (represented by the dotted line). mRNA levels were normalized to GAPDH mRNA. The insets show CM cal normalized mRNA levels relative to the control. Each dot represents an individual experiment (n = 2-7). * p < 0.05 versus the control, # p < 0.05 versus CM cin , and the Wilcoxon signed rank test.

Secretion Products from CaSR-Activated SW872 Pre-Adipocytes alter the Mitochondrial Dynamics and Function of HepG2 Cells
Given that cell senescence is characterized by a general decrease in cell function including mitochondrial dysfunction [27,36], we evaluated key factors for mitochondrial dynamics. Our results show that the protein levels of OPA1, which maintains mitochon- (d) quantification of SA-β-GAL-positive cells normalized by total cells in the field; (e) SA-β-GAL absorbance recorded at 630 nm. Each dot represents an individual experiment (n = 6). * p < 0.05, and ** p < 0.01, the versus control, Friedman's test, and Dunn's post-hoc test.

Secretion Products from CaSR-Activated SW872 Pre-Adipocytes Alter the Mitochondrial Dynamics and Function of HepG2 Cells
Given that cell senescence is characterized by a general decrease in cell function including mitochondrial dysfunction [27,36], we evaluated key factors for mitochondrial dynamics. Our results show that the protein levels of OPA1, which maintains mitochondrial cristae structure, and PGC-1α, which promotes mitochondrial biogenesis, significantly decrease in HepG2 cells exposed to CM cin (p < 0.05) compared to CM veh . In the case of MFN2 protein levels, which mediates mitochondrial fusion, there was a trend to decrease (p < 0.08). Conversely, the protein levels of DRP1, which participates in mitochondrial fission, increased upon treatment with CM cin (Figure 6a-f). As with previous results, these observations were reversed in the CM cin+cal condition. drial cristae structure, and PGC-1α, which promotes mitochondrial biogenesis, significantly decrease in HepG2 cells exposed to CMcin (p < 0.05) compared to CMveh. In the case of MFN2 protein levels, which mediates mitochondrial fusion, there was a trend to decrease (p < 0.08). Conversely, the protein levels of DRP1, which participates in mitochondrial fission, increased upon treatment with CMcin (Figure 6a-f). As with previous results, these observations were reversed in the CMcin+cal condition. Figure 6. CaSR activation in SW872 pre-adipocytes induces the secretion of factors that alter proteins governing mitochondrial dynamics in HepG2 cells: (a) immunodetection of OPA1 and MFN2 protein levels in HepG2 cells exposed to CMveh, CMcin, and CMcin+cal; (b) immunodetection of PGC-1α and DRP1 protein levels in treated HepG2 cells; quantification of (c) OPA1; (d) MFN2; (e) PGC-1α; and (f) DRP1 protein levels. Each dot represents an individual experiment (n = 4-5). * p < 0.05 and ** p < 0.01 versus the control, # p < 0.05 versus CMcin, Friedman's test, and Dunn's post-hoc test.
Next, we analyzed the mitochondrial transmembrane potential (Δψ) through immunofluorescence using the Δψ-sensitive probe MitoTracker Orange (MTO) normalized by mtHsp70 as a marker of mitochondrial mass. We observed that HepG2 cells exposed to CMcin had a decreased MTO/mtHsp70 fluorescence ratio (p < 0.05), indicative of decreased Δψ (Figure 7a,b). In addition, CMcin-treated cells presented a higher number of mitochondria per cell compared to the CMveh condition and a trend towards reduced average mitochondrial area (p < 0.08) (Figure 7c,d). Altogether, these results indicate that CMcin treat- Next, we analyzed the mitochondrial transmembrane potential (∆ψ) through immunofluorescence using the ∆ψ-sensitive probe MitoTracker Orange (MTO) normalized by mtHsp70 as a marker of mitochondrial mass. We observed that HepG2 cells exposed to CM cin had a decreased MTO/mtHsp70 fluorescence ratio (p < 0.05), indicative of decreased ∆ψ (Figure 7a,b). In addition, CM cin -treated cells presented a higher number of mitochondria per cell compared to the CM veh condition and a trend towards reduced average mitochondrial area (p < 0.08) (Figure 7c,d). Altogether, these results indicate that CM cin treatment in pre-adipocytes induces the secretion of factors that promote mitochondrial fragmentation and the decrease of bioenergetics in HepG2 cells, thus suggesting mitochondrial dysfunction. ment in pre-adipocytes induces the secretion of factors that promote mitochondrial fragmentation and the decrease of bioenergetics in HepG2 cells, thus suggesting mitochondrial dysfunction.

Inhibition of Mitochondrial Fission in HepG2 Cells Prevents Cell Senescence Induced by Cinacalcet-Treated SW872 Pre-Adipocyte Secretion Factors
We addressed whether the observed mitochondrial fragmentation contributes to the development of MCcin-induced cell senescence in HepG2 cells by inhibiting mitochondrial network fragmentation using the DRP1 inhibitor Mdivi-1 during the last 24 h of the treatment with SW872 CM. As expected, Mdivi-1 treatment prevented CMcin-induced mitochondrial fragmentation, maintaining the mitochondrial number similar to CMveh-treated cells (Figure 8a,c,d). Similarly, Mdivi-1 treatment prevented the decrease in Δψ observed upon CMcin treatment (Figure 8b).

Inhibition of Mitochondrial Fission in HepG2 Cells Prevents Cell Senescence Induced by Cinacalcet-Treated SW872 Pre-Adipocyte Secretion Factors
We addressed whether the observed mitochondrial fragmentation contributes to the development of MC cin -induced cell senescence in HepG2 cells by inhibiting mitochondrial network fragmentation using the DRP1 inhibitor Mdivi-1 during the last 24 h of the treatment with SW872 CM. As expected, Mdivi-1 treatment prevented CM cin -induced mitochondrial fragmentation, maintaining the mitochondrial number similar to CM vehtreated cells (Figure 8a,c,d). Similarly, Mdivi-1 treatment prevented the decrease in ∆ψ observed upon CM cin treatment (Figure 8b). We next investigated how Mdivi1 affects the mitochondrial bioenergetics of HepG2 treated with CMcin. We observed that while CMcin did not significantly alter baseline respiration compared to CMveh, it decreased both non-ATP associated and maximal capacity respiration rates, confirming mitochondrial dysfunction. In both cases, treatment with Mdivi-1 reverted said changes (Figure 9a-c). We next investigated how Mdivi1 affects the mitochondrial bioenergetics of HepG2 treated with CM cin . We observed that while CM cin did not significantly alter baseline respiration compared to CM veh , it decreased both non-ATP associated and maximal capacity respiration rates, confirming mitochondrial dysfunction. In both cases, treatment with Mdivi-1 reverted said changes (Figure 9a-c). Finally, we assessed whether mitochondrial fragmentation inhibition counteracts CMcin-induced cell senescence, evaluated as cell cycle arrest and SASP in HepG2 cells Indeed, Mdivi-1 treatment prevented the decrease in Ki67 nuclear fluorescence ( Figure  10a,b) induced by CMcin, as well as the increase in IL-1β and CCL2 mRNA levels ( Figure  11a,b). Altogether, these results highlight the importance of mitochondrial fragmentation for the progression of cell senescence induced by CaSR-activated pre-adipocytes. Finally, we assessed whether mitochondrial fragmentation inhibition counteracts CM cin -induced cell senescence, evaluated as cell cycle arrest and SASP in HepG2 cells. Indeed, Mdivi-1 treatment prevented the decrease in Ki67 nuclear fluorescence (Figure 10a,b) induced by CM cin , as well as the increase in IL-1β and CCL2 mRNA levels (Figure 11a,b). Altogether, these results highlight the importance of mitochondrial fragmentation for the progression of cell senescence induced by CaSR-activated pre-adipocytes. Figure 10. Inhibition of mitochondrial fission prevents the cell cycle arrest induced by CMcin in HepG2 cells: (a) representative images of HepG2 cells exposed to the CMveh, CMcin, CMveh + Mdivi1, and CMcin + Mdivi1. Hoechst (blue) was used to identify the nuclei and anti-Ki67 antibody (green) to evaluate the nuclear localization of Ki67 (scale bar = 100 μm); (b) quantification of total Ki67 immunofluorescence. Each dot represents an individual experiment (n = 5). ** p < 0.01 versus the control, Friedman's test, and Dunn's post-hoc test.  Figure 11. Inhibition of mitochondrial fission prevents the increase in pro-inflammatory cytokines induced by CMcin in HepG2 cells: relative abundance of (a) IL-1β and (b) CCL2 mRNA in HepG2 cells exposed to CMcin, CMveh + Mdivi1, and CMcin + Mdivi1, expressed as fold from CMveh, represented by the dotted line. mRNA levels were normalized to GAPDH mRNA. Each dot represents an individual experiment (n = 5). * p < 0.01 versus the control, # p < 0.05 versus CMcin, and the Wilcoxon signed rank test.

Discussion
The present work evaluated whether CaSR activation in SW872 pre-adipocytes promotes pro-inflammatory signaling that induces senescence and mitochondrial dysfunction in HepG2 cells. Our observations indicate that CaSR activation in the SW872 cell line stimulated the production of factors that increased senescence, the production of pro-inflammatory cytokines, cell cycle arrest, and mitochondrial dysfunction markers in HepG2 Figure 11. Inhibition of mitochondrial fission prevents the increase in pro-inflammatory cytokines induced by CMcin in HepG2 cells: relative abundance of (a) IL-1β and (b) CCL2 mRNA in HepG2 cells exposed to CM cin , CM veh + Mdivi1, and CM cin + Mdivi1, expressed as fold from CM veh , represented by the dotted line. mRNA levels were normalized to GAPDH mRNA. Each dot represents an individual experiment (n = 5). * p < 0.01 versus the control, # p < 0.05 versus CM cin , and the Wilcoxon signed rank test.

Discussion
The present work evaluated whether CaSR activation in SW872 pre-adipocytes promotes pro-inflammatory signaling that induces senescence and mitochondrial dysfunction in HepG2 cells. Our observations indicate that CaSR activation in the SW872 cell line stimulated the production of factors that increased senescence, the production of pro-inflammatory cytokines, cell cycle arrest, and mitochondrial dysfunction markers in HepG2 cells.
In pre-adipocytes, CaSR activation has been shown to promote the secretion of the proinflammatory cytokines TNF-α and IL-1β [10,11]. Elevated circulating TNF-α is associated with an increase in cellular senescence markers, such as SA-β-GAL, p21, and p53 in the liver and kidneys [37]. Our observations are consistent with other investigations where TNF-α was able to increase SA-β-GAL [37]. Moreover, we also observed that removing IL-1β prevented an increase in SA-β-GAL. Thus, our results highlight that both IL-1β and TNF-α in the conditioned medium from CaSR-activated pre-adipocytes contribute to cellular senescence development in the HepG2 cell line.
Studies in macrophages have shown that exposure to conditioned medium from senescent pre-adipocytes increase p16 and p21 expression, which was attributed to the secretory products that make up the SASP, such as TNF-α and CCL2 [38,39]. Other reports on human vascular smooth muscle cell cultures have shown that IL-1β exposure increases p16 and p21 expression through a mechanism involving SIRT1 deregulation [40]. Decreased activation of the SIRT1 metabolic pathway has been observed in models of aging and inflammation. In rats, SIRT1 activation promoted mitochondrial biogenesis and the endogenous antioxidant system through PGC1a and NRF2 activity on neuronal tissue [41]. A study in cells from the nucleus pulposus showed that TNF-α promoted the cellular senescence markers SA-β-GAL and p21, a change that was related to greater activity of NF-κB [42]. Unlike p16 and p21, in our experiments we did not observe significant changes on p53, a protein that modulates p21 expression. In relation to this, it has been described that p21 can be regulated in a p53-independent manner through ERK1/2 and p38 MAPK [43].
It is well-accepted that the inflammatory activity of TNF-α and IL-1β initiates stressdriven cellular senescence. The process begins with an increase in cellular senescence markers such as SA-β-GAL and greater expression of pro-inflammatory cytokines and chemoattractants such as IL-1β and CCL2 [44]. In this context, the increased expression of IL-1β and CCL2 triggers the spread of inflammation and the attraction of immune cells, which eliminate senescent cells [14,44,45].
Studies in animal models have shown that the low expression of OPA1 is related to the increase in the pro-inflammatory cytokines TNF-α and IL-1β in the circulation [46]. In the liver of OPA1-deficient mice, the markers for cellular senescence, SA-β-GAL and p21, were increased [46]. We observed low expression of PGC-1α in CM cin -treated HepG2 cells, which is consistent with results in mouse liver tissue showing that stimuli such as a high-fat diet decreases PGC-1α expression and increases cellular senescence markers, such as SASP [47]. Moreover, the low expression of PGC-1α is related to reduced protection against oxidative stress and cellular senescence [48]. In the CM cin+cal treatment, MFN2 recovered its expression and DRP1 decreased compared to CM cin . Low expression of MFN2 has been associated with increased fragmentation of the mitochondrial network and lower membrane potential [49,50]. It has been reported that pro-inflammatory stimuli such as TNF-α increase mitochondrial dysfunction markers such as the fragmentation of the mitochondrial network [51].
In senescent cells, mitochondrial alterations such as fragmentation in the mitochondrial network or loss in the ∆ψ are common features [52,53]. Our results coincide with those reported in colon and melanocyte cell lines exposed to overexpression of TNF-α, which decreased the ∆ψ and increased the fragmentation of the mitochondrial network. After an early process of apoptosis, the remaining cells show mitochondrial dysfunction and increased cytokine expression, which is associated with cellular senescence [53]. The evidence on altered mitochondrial dynamics and cellular senescence is under discussion because the results may vary depending on the cell type under study [22,54]. Based on our findings, we propose that the secretion products in CM cin affect mitochondrial morphology through decreased expression of proteins that promote organelle biogenesis and fusion, as well as increased fragmentation of the mitochondrial network. In addition, treated cells presented an alteration in mitochondrial bioenergetics, showing a lower ∆ψ and lower respiratory rate. These data suggest that CM cin damages the mitochondrial network, and thus limits the biogenesis and bioenergetics of this organelle.
Losing mitochondrial network integrity favors the development of mitochondrial dysfunction, and in our HepG2 cells treated with CM cin this indicator increased. In experiments in HepG2 cells where Ki67 was pharmacologically reduced, Ki67 was recovered after Mdivi-1 treatment [55]. Furthermore, in a rat model of ischemia, Mdivi-1 increased ∆ψ and the authors attribute this behavior to the regulatory action of the reagent on mitophagy mediated by PINK/Parking proteins [56]. It was also shown in LPS-exposed cardiomyocytes that Mdivi-1 contributes to the recovery of ∆ψ [57]. The effects of Mdivi-1 on the ∆ψ regulation would be related to mitochondrial membrane integrity maintenance. Our results show that Mdivi-1 inhibited the increase in IL-1β and CCL2 expression caused by CM cin . Published studies in animal models exposed to amyloid β reveal that treatment with Mdivi-1 protects hippocampal cells from increased expression of IL-1β, which changes in mitochondrial dynamics, decreasing the MFN2 and OPA1 proteins [58]. In a cell model, it has been observed that treatment with Mdivi-1 before LPS exposure impaired the increase in pro-inflammatory cytokines such as IL-1β and CCL2 [59].
The present work addresses a line of research that innovates on the relationship between CaSR activation in pre-adipocytes and the development of the senescent phenotype and mitochondrial dysfunction in hepatic cells. It highlights and supports previous research on the importance of pre-adipocytes as potential promoters of deleterious effects in other cell types, further evidencing the participation of mitochondrial dysfunction as a regulator of the senescent phenotype. Future studies should also include a wider use of CaSR inhibition strategies such as the CaSR-negative modulator calhex 231 and possibly others, and furthermore with CaSR gene downregulation via siRNA, in order to better understand the involvement of this receptor in the observed effects. Further characterization of the senescent phenotype would have been desirable, such as cell cycle analysis through flow cytometry or chromatin remodeling or DNA damage via fluorescence microscopy. In addition, we did not explore other functional outcomes in senescent HepG2 cells, such as lipid uptake or insulin signaling. Finally, this model of cellular communication can be used with other cell types. Thus, the effects of conditioned media from CaSR-activated pre-adipocytes can be assessed in smooth muscle or endothelial cells as a future research direction.
Our results indicate that inhibiting mitochondrial network fragmentation protects HepG2 cells from changes related to CM cin exposure, seen in the recovery of ∆ψ and the decreased expression of pro-inflammatory cytokines. In summary, the conditioned medium from pre-adipocytes after CaSR activation promotes senescence and mitochondrial dysfunction in hepatocytes. Our investigation provides evidence about the communication between pre-adipocytes and liver cells mediated by CaSR activation, raising new questions such as further pre-adipocyte conditioned medium characterization or the consequences of CaSR activation at the organismal level on liver senescence phenotype and disease.

Cell Culture
A human liposarcoma-derived SW872 pre-adipose cell line (HTB-92, ATCC, Manassas, VA, USA) was grown in DMEM/F12 medium. A human hepatocellular carcinoma-derived HepG2 cell line (HB-8065, ATCC, Manassas, VA, USA) was cultured in MEM medium. All media were supplemented with 10% fetal bovine serum and antibiotics (penicillinstreptomycin) and the cells were maintained in a 37 • C 5% CO 2 atmosphere. The medium was changed for a fresh medium twice per week.

SW872 Cells' Conditioned Media Collection
SW872 cells were cultured at a 10,000 cells/cm 2 density in 100 mm plastic culture dishes. To obtain conditioned media, the cells were treated with either vehicle (DMSO) or 2 µM cinacalcet with or without 30 min of pre-incubation with the negative allosteric CaSR modulator calhex-231 (10 µM). After 16 h, the media were replaced with fresh DMEM/F12 and conditioned for 24 h. The conditioned media were centrifuged at 800× g for 10 min at 4 • C and stored at −80 • C until use.

HepG2 Cells' Exposure to Conditioned Media and Mdivi1
HepG2 cells were cultured at 9000 cells/cm 2 density in culture dishes, according to each experimental protocol. The cells were washed twice with PBS and the media were replaced with fresh MEM media containing 50% of the conditioned media from SW872 cells. The HepG2 cells were then grown for 5 days under standard culture conditions, replacing the media for fresh conditioned media after 3 days.
For treatment with Mdivi1, the cells were conditioned as described above, and during the last 24 h of exposure they were treated with vehicle (DMSO) of 50 µM of Mdivi1.

HepG2 SA-β-GAL Activity
The cells were cultured in 96-well plates and observed under an inverted phase contrast microscope before and after treatment for 5 days. Images were recorded with a Motic AE2000 camera and Motic Images Plus 2.0 ML software (Motic, Vancouver, BC, Canada).
The cultures were treated with the senescence-associated β-galactosidase staining kit (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer's instructions. After incubating overnight at 37 • C, the cells were examined by microscopy and the respective blue staining in positive cells was recorded. Next, the number of positive cells was normalized by the total number of cells in the photograph to obtain an indicator of the percentage of senescent cells in each experiment. Then, X-gal blue products were dissolved with DMSO and the absorbance was measured at 630 mn [60].

RNA Isolation, Reverse Transcription, and mRNA Expression by RT-PCR
The cells were cultured in 6-well plates and after experimentation, Trizol (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used to lyse the HepG2 cells and isolate total RNA following the manufacturer's instructions. RNA was reverse-transcribed into complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The cDNA levels were analyzed with a step-one realtime PCR system using the SYBR FAST qPCR kit (Applied Biosystems, Foster City, CA, USA) and specific primers for: il-1β (forward: 5 GGACAAGCTGAGGAAGATGC 3 ; reverse: 5 TCGTTATCCCATGTGTCGAA 3 , NM_000576), ccl2 (forward: 5 TGTCCCAAAGAAGCT-GTGATCT 3 ; Reverse: 5 GGAATCCTGAACCCACTTCTG 3 ; NM_002982), and gapdh (forward: 5 GAAGGTGAAGGTCGGAGTCAAC 3 ; reverse: 5 CAGAGTTAAAAGCAGC-CCTGGT 3 ; NM_020). Thermal cycling consisted of an initial pre-incubation cycle of 20 s at 95 • C, followed by 40 cycles of 30 s at 95 • C. The results were normalized for the gapdh gene and relative mRNA levels were calculated using the Pfaffl method [61].

Protein Levels by Western Blot Analysis
The cells were cultured in 6-well plates and after experimentation, they were washed three times with cold PBS and lysed with NP40 buffer (Invitrogen™, Carlsbad, CA, USA), complete protease inhibitor (#11697498001, Sigma-Aldrich, St. Louis, MO, USA), and PhosSTOP phosphatase inhibitor (#4906845001, Sigma-Aldrich, St. Louis, MO, USA). The lysed cells were centrifuged at 12,000× g for 15 min and the supernatant was stored at −80 • C until use. At concentrations of 1 µg/µL, the proteins were subjected to electrophoresis in SDS-polyacrylamide gels under denaturing conditions and were electrotransferred to a 0.2 µm pore PVDF membrane. The membranes were then blocked with TBS 5% skim milk 0.05% Tween 20 (#7949, Sigma, St. Louis, MO, USA).
The following proteins were detected after 16 h of incubation with the primary antibodies: p16, p21, p53, PGC1α, MFN2, DRP1, OPA1, and β-actin (Table 1). The detection of immune complexes was performed with the incubation of anti-rabbit IgG, anti-mouse IgG, or anti-goat IgG peroxidase-conjugated secondary antibodies, depending on the origin of each primary antibody ( Table 2). The membranes were incubated for 1 min with chemiluminescence reagents (#20-500-500A and 20-500-500B, Biological Industries, Cromwell, CT, USA), the digitized luminescent signal was detected in a LI-COR C-Digit scanner 3600 (LI-COR Biosciences, Lincoln, NE, USA), and the intensity of the bands was quantified with the ImageJ program (National Institutes of Health, USA). The expression of each protein was normalized by β-actin and expressed as arbitrary units.

Immunofluorescence
The HepG2 cells were seeded in 12-well plates with 0.17 mm and treated as indicated. For analysis of mitochondrial membrane potential, the cells were loaded with MitoTracker Orange 400 nM (M7510, Invitrogen™, Carlsbad, CA, USA) for 25 min at 37 • C. Then, the culture medium was removed and the cells were washed twice with PBS. The cells were fixed with 4% paraformaldehyde at 4 • C for 15 min. The fixation medium was removed and washed twice with cold PBS. To permeabilize the cells, they were incubated with PBS 0.1% Triton X-100 for 10 min. After permeabilization, the cells were washed twice with cold PBS and blocked with PBS 3% BSA for 1 h at room temperature. The samples were treated with primary antibodies in PBS 3% BSA overnight at 4 • C in a humid chamber protected from light.
The primary antibodies and their dilutions were: anti-Ki-67 (#9449, Cell Signaling, proliferation marker) 1:400 or anti-mtHsp70 (MA3-028, Thermo Fisher Scientific, mitochondrial marker) 1:500, according to the assay. The next day, the coverslips were washed twice with cold PBS to remove excess primary antibodies, and the samples were incubated for 1 h with an Alexa Fluor 488 secondary antibody (A32723, Thermo Fisher Scientific, Waltham, MA, USA) 1:600 in a chamber protected from light at room temperature. After washing with PBS, the samples were incubated with the fluorescent probe Hoechst (B2261, Merck (Rahway, NJ, USA), nucleus marker) 1:1000 diluted in PBS BSA 3% for 15 min at room temperature in a chamber protected from light. The coverslips were then washed twice with cold PBS to remove excess probes and mounted on a clean slide with Dako fluorescence mounting medium (S3023, Dako-Agilent, Santa Clara, CA, USA). The samples were stored at 4 • C protected from light until analysis.

Image Capture and Processing
Images were taken with a Zeiss LSM-5, Pascal 5 Axiovert 200 confocal microscope, with a Plan-Apochromat 40×/1.5 Oil DIC objective, using 365 (for Hoechst), 488 (for Alexa Fluor 488), and 555 nm (for MitoTracker Orange) excitation lasers. For each independent experiment, it averaged the signal from 5 to 15 cells. The analysis was performed on 1 focal plane corresponding to the equator of the cells. The pixel size was 68 nm, following the Nyquist sampling criterion. The images thus obtained were deconvolved, the background noise was subtracted, a median filter was applied, and fluorescence intensity was quantified using the ImageJ software.

Oxygraphy
HepG2 cells were seeded in 60 mm dishes. After the 5 days of treatment, the cells were trypsinized and resuspended in a Clark electrode chamber (Strathkelvin Instruments, North Lanarkshire, Scotland) to quantify the oxygen consumption of living cells. Basal respiration was measured for 5 min at 25 • C, then 10 µg/mL of oligomycin was added to assess non-ATP-associated respiration for 5 min, followed by 200 nM CCCP to quantify maximal respiration capacity for another 5 min. Then, the cells were recovered to quantify total proteins using a BCA kit to normalize oxygen consumption rates.

Statistical Analysis
Data are shown as the mean ± the standard error of the mean (SEM). Differences between conditions were evaluated with the non-parametric Friedman test and multiple comparisons were made with the Dunn's test. The Wilcoxon signed rank test was used to detect differences in the results obtained through real-time PCR. A p value less than 0.05 was considered as a significant change, while p values less than 0.1 were considered a trend.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.