MitoNEET prevents iron overload‐induced insulin resistance in H9c2 cells through regulation of mitochondrial iron

Iron overload (IO) induces insulin resistance in H9c2 cardiomyoblast cells. Here, we used H9c2 cells overexpressing MitoNEET to examine the potential for protection against iron accumulation in the mitochondria and subsequent insulin resistance. In control H9c2 cells, IO was observed to increase mitochondrial iron content, reactive oxygen species (ROS) production, mitochondrial fission, and reduced insulin‐stimulated Akt and ERK1/2 phosphorylation. IO did not significantly affect mitophagy, or mitochondrial content, however, an increase in peroxisome‐proliferator‐activated receptor gamma coactivator 1 alpha (PGC1α) protein expression, a key regulator of mitochondrial biogenesis, was observed. MitoNEET overexpression was able to attenuate the effects of IO on mitochondrial iron content, reactive oxygen species, mitochondrial fission, and insulin signaling. MitoNEET overexpression also upregulated levels of PGC1α protein. The mitochondria‐targeted antioxidant, Skq1, prevented IO‐induced ROS production and insulin resistance in control cells, indicating mitochondrial ROS plays a causal role in the onset of insulin resistance. The selective mitochondrial fission inhibitor, Mdivi‐1, prevented IO‐induced mitochondrial fission, however, it did not alleviate IO‐induced insulin resistance. Collectively, IO causes insulin resistance in H9c2 cardiomyoblasts and this can be averted by reduction of mitochondrial iron accumulation and ROS production by overexpression of the MitoNEET protein.

ROS activates stress kinases that may directly inhibit components of the insulin signaling pathway (Al-Lahham et al., 2016). Indeed, we reported IO can induce ROS, oxidative stress, and insulin resistance in H9c2 cardiomyoblasts (Sung et al., 2019).
Mitochondria are the major source of ROS production within the cell, since a number of proteins of the Electron Transport Chain (ETC) or iron-binding proteins carry 2Fe-2S clusters and iron-containing hemes which can undergo electron leakage and create superoxide anions that are converted to hydrogen peroxide (Balaban et al., 2005;Read et al., 2021;Zhao et al., 2019).
Both types of ROS are chemically damaging to proteins and as such, it is imperative that mitochondrial iron and ROS are tightly regulated by the cell. In fact, there is accumulating evidence that mitochondrial ROS is closely linked to insulin resistance in a variety of biological systems from cells to organisms, but there are also reports that stand against ROS as a causal factor in the development of insulin resistance (Ayer et al., 2022). Therefore, further studies are necessary to elucidate the precise roles of mitochondrial ROS in insulin resistance.
MitoNEET (or Cisd1) is a protein found on the outer mitochondrial membrane (Paddock et al., 2007). Originally identified as a binding target of the antidiabetic drug, pioglitazone, MitoNEET was considered a possible therapeutic target based on this interaction (Geldenhuys et al., 2014). MitoNEET was later shown to play a role in regulating mitochondrial iron (Kusminski et al., 2012). More recent studies suggest it likely has a role in shuttling 2Fe-2S clusters out of the mitochondrial inner membrane to other Fe-S cluster acceptor proteins in the ER (Nechushtai et al., 2020). In the present study, we utilize a cellular model of mild MitoNEET-overexpression in H9c2 cardiomyoblasts to investigate the impact of IO on insulin resistance. We hypothesized during cellular iron overload, MitoNEET overexpression would prevent insulin resistance by reducing excess mitochondrial iron accumulation. Potential mechanistic roles of mitochondrial ROS and mitochondrial dynamics were also investigated.

| Generation of a MitoNEET overexpressing H9c2 cell line
H9c2 cardiomyoblasts overexpressing MitoNEET (Cisd1) were created and characterized by us previously (Tam et al., 2022). Briefly, the plasmid containing either the MitoNEET (pMitoN) gene or empty vector (EV) were purchased from Genscript (Piscataway; clone ID Ora10938 or SC1217, respectively). H9c2 cells were transfected with pMitoN and EV plasmids for 48 h followed by selection of clonal cell lines in growth media containing 500 µg/mL G418 antibiotic. By Western blot verification, the clone (H9c2-MitoN) with the highest expression of MitoNEET (~2.5-fold higher than control cells, H9c2-EV) was used for this entire study.

| Oxidative stress detection using CellROX™ deep red
H9c2 EV and MitoN cells were grown on 24-well plates overnight.

| Western blot analysis
Antimouse and antirabbit HRP-linked secondary antibodies were from Cell Signaling Technology.

| Mitochondrial network analysis
H9c2-EV and-MitoN cells were grown to 60% confluency on chambered coverslips then treated with or without 200 µM Ferric ammonium sulfate in serum-free media for 24 h. Cells were stained with 50 µM Mitotracker green (M7514; Thermo Fisher Scientific), and nuclear mask staining was added 30 min before treatment endpoint. Cells were washed, changed to phenol free DMEM, and imaged on Nikon A1 confocal microscope. Mitochondrial length and networking per cell were quantified using the ImageJ software mitochondrial network analysis tool (Valente et al., 2017). For 3D imaging, Z-stacks were captured using 0.2 µm planes of focus, then processed by Imaris software (Oxford Instruments) using the surfaces algorithm.

| Mitophagy analysis
H9c2-EV and -MitoN cells were grown to 60% confluency on chambered coverslips then treated with or without 200 µM Ferric ammonium sulfate in serum-free media for 24 h. Mitophagy was measured using a mitophagy detection kit according to manufacturer's protocol (MT02; Dojindo Molecular Technologies, Inc.). In this assay, mitochondria are modified with a covalently binding (proprietary) dye that has a low fluorescence in normal conditions, before a mitophagy stimulus. When the labeled mitochondria are taken up by autolysosomes, the fluorescence increases dramatically due to their acidic environment. Fluorescence microscopy in the red channel was performed using a Nikon A1 confocal microscope and fluorescence was quantified using ImageJ software.

| Statistical analysis
All results are presented as mean ± standard error of mean. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test or t-test were performed on GraphPad. p-value of <0.05 was considered significant.

H9c2 cells were transfected with a cDNA expressing FLAG-tagged
MitoNEET protein and a clonal cell line stably overexpressing MitoNEET (H9c2-MitoN) was selected and characterized elsewhere (Tam et al., 2022). By Western blot analysis, MitoNEET overexpression was observed to be 2.5-fold higher compared to an empty-vector expressing control cell line (H9c2-EV). To examine the functional consequence of MitoNEET overexpression upon iron overload, we treated H9c2-EV and H9c2-MitoN cells with Ferric ammonium sulfate for up to 24 h. Induction of the mitochondrial isoform of Ferritin is a biomarker of mitochondrial iron overload (Chitambar, 2005). In Figure 1a, we observed H9c2-EV control cells overloaded with iron had more than a 10-fold induction in mitochondrial ferritin relative to untreated control cells. By contrast, iron accumulation over this period was attenuated in H9c2-MitoN cells by approximately 50% during IO (Figure 1a). Mitochondrial iron was also measured using the mitochondria-targeted fluorescent probe, MFF, which has its fluorescence quenched by increases in mitochondria iron (Kholmukhamedov et al., 2022).  (Sung et al., 2019). Akt has two regulatory phosphorylation sites (Ser473 and Thr308) that are phosphorylated by upstream kinases when Akt binds to the PIP3 phospholipid produced in the plasma membrane by insulin-stimulated PI3-kinase (Coffer et al., 1998). In Figure 1d IO overload for 4 h induced a >3-fold increase in cellular ROS production as measured using CellROX dye in H9c2-EV cells (Figure 2a,b). Pretreatment with the mitochondria-specific antioxidant Skq1 lowered the production of ROS by more than 50% in these cells (Figure 2a,b). On the other hand, in H9c2-MitoN cells that underwent IO, ROS production was 50% less than that observed in IO-treated H9c2-EV cells (Figure 2a (Figures 1 and 2).
ROS can induce mitochondrial fragmentation (Riba et al., 2017) and we wanted to explore if it could be connected with the induction of insulin resistance by IO. Mitochondrial fusion-related proteins, Mfn1, Mfn2, and Opa1, were assessed by Western blot and observed  Representative fluorescent images of mitochondria are shown and quantification of mitochondrial length and branching by ImageJ software are adjacent, n = 3. Scale bar = 10 µm. *p < 0.05 compared to EV control, ##p < 0.01 compared to EV IO. (i) 3D images of mitochondrial networks processed with the Imaris software sphericity quantification tool (using the surfaces algorithm) is shown in H9c2-EV and -MitoN cells treated with (IO) or without (control) iron for 24 h. Quantification is representative of at least 134 mitochondria. Scale bar = 5 µm. **p < 0.01 compared to EV control, ##p < 0.01 compared to EV IO. (j) Quantitative analysis of mitochondrial length and branching using ImageJ software analysis of Mitotracker green fluorescence in H9c2-EV cells treated with (IO) or without (control) iron for 24 h. Where indicated, cells were pretreated with Drp1 inhibitor, Mdivi-1. Representative images are shown, n = 3. Scale bar = 10 µm. *p < 0.05 compared to EV control, #p < 0.05 compared to EV IO. (k) Assessment of insulin signaling by Western blot analysis of p-Akt S473 and p-Akt T308 in H9c2-EV cells treated with (IO) or without (control) iron for 24 h. Where indicated, cells were pretreated with Drp1 inhibitor, Mdivi-1. Representative blots with loading controls are shown, n = 3. **p < 0.01 compared to EV control, ##p < 0.01 compared to EV IO.
by IO treatment of H9c2-MitoN cells (Figure 4c). Mitophagy, the selective degradation of mitochondria, was measured using a kit as described in Section 2. These events trended toward an unexpected increase in H9c2-MitoN cells but were nevertheless not significantly altered in response to IO in either cell line (Figure 4d).

| DISCUSSION
Insulin resistance is a well-established contributing factor for the development of diabetes and heart failure. Studies have shown signs of insulin resistance can occur up to 10 years before the onset of diabetes, making it a suitable target for early intervention (Tabák et al., 2009). Although numerous examples of ways in which iron contributes to the development of insulin resistance have been characterized, the specific effects of mitochondrial iron have not been fully investigated (Simcox & McClain, 2013;Sung et al., 2019).
Here, we used an H9c2 cell line which we had previously generated Regarding insulin resistance, H9c2-MitoN cells exposed to IO conditions had completely normal insulin signaling, demonstrating that MitoNEET overexpression reduces mitochondrial iron and protects the cells from becoming insulin resistant. These data are in keeping with previous studies that MitoNEET regulated mitochondrial iron (Kusminski et al., 2012), likely via shuttling 2Fe-2S clusters out of the mitochondrial inner membrane to other Fe-S cluster acceptor proteins, such as in the ER (Nechushtai et al., 2020). Our data now provide new knowledge that by attenuating the mitochondrial IO, targeting MitoNEET may provide a strategy for reducing deleterious cellular effects including insulin resistance.
Oxidative stress has widely been reported to play a key role in the development of type 2 diabetes and cardiovascular complications (Giacco & Brownlee, 2010;Henriksen et al., 2011). Despite this, clinical trials using antioxidant therapies have largely been ineffective at preventing diabetic complications (Johansen et al., 2005). In fact, the use of antioxidants such as Vitamin C and E in the prevention of several diseases, including cardiovascular diseases, have in some instances been associated with elevated risk of death (Bjelakovic et al., 2007;Vivekananthan et al., 2003). Here, we examined the role of oxidative stress in IO-induced insulin resistance. IO increased ROS production, which was markedly reduced in H9c2-MitoN cells or by the mitochondrial antioxidant, Skq1, suggesting mitochondrial ROS is the major contributor to the total cellular ROS produced in response to IO. In addition, Skq1 was able to fully reverse IO-induced insulin resistance. Thus, our data indicated that IO upregulates the production of mitochondrial ROS which is causally associated with development of insulin resistance.
In cardiomyocytes, there is evidence implicating dysregulation of mitochondrial dynamics in the pathogenesis of insulin resistance (Sergi et al., 2019). In fact, several studies have reported that mitochondria in patients with diabetes or heart failure are smaller in size, indicative of elevated mitochondrial fragmentation (Chen et al., 2009;Kelley et al., 2002;Sergi et al., 2019). In our study, IO induces a higher Drp1 activation status, that is consistent with visual evidence of mitochondrial fragmentation using Mitotracker green and image analysis. Interestingly, H9c2-MitoN cells displayed no increase in mitochondrial fragmentation following IO. Furthermore, we established IO leads to an upregulation of fission via the Fis1-Drp1 pathway. However, mitochondrial fragmentation was not responsible for IO-induced insulin resistance in our study since inhibition of Drp1 activity with Mdivi-1 restored normal mitochondrial morphology but not insulin sensitivity in IO-treated H9c2-EV control cells. Thus, it is possible that IO is a contributor to altered mitochondrial fission in diabetes and heart failure (Chen et al., 2009;Kelley et al., 2002;Sergi et al., 2019), but our data suggest this is not a causative factor in the development of insulin resistance (Sergi et al., 2019).
To further explore the effect of IO on mitochondria dynamics, we measured the number of mitochondria, the rate of mitophagy and a marker of mitochondrial biogenesis. Mitochondrial number and mitophagy were unaltered in IO or by MitoNEET overexpression.
However, IO significantly increased PGC1α expression as did MitoNEET, alone. Interestingly, MitoNEET overexpression supressed a further increase in PGC1α in response to IO. These increases of PGC1α are suggestive of potential for mitochondrial biogenesis, yet this was not yet manifested under the experimental conditions studied since the total levels and degradation rate of mitochondria were not altered by IO. Alternatively, IO may activate PGC1α signaling to trigger an antioxidant transcriptional response, increasing expression of genes such as superoxide dismutase, catalase, peroxiredoxin 3 and 5, UCP2, thioredoxin 2 and thioredoxin reductase, as suggested previously (Rius-Pérez et al., 2020). The ability of MitoNEET to upregulate PGC1α has also been previously reported in the literature, but the direct implications of this on insulin resistance are not clear (Kusminski et al., 2012).
In conclusion, our study provides new insight into the connection between mitochondrial iron overload and insulin resistance, whereby increased mitochondrial iron induces mitochondrial ROS, that leads to decreased insulin sensitivity. This study highlights MitoNEET as a protein that can effectively lower mitochondrial iron accumulation in the presence of excess extra-or intracellular iron, thereby offering protection against mitochondrial ROS production and insulin resistance in h9c2 cells.

AUTHOR CONTRIBUTIONS
Eddie Tam and Hye K. Sung: Experimental work, data analysis, manuscript writing. Gary Sweeney: Secured funding, project design, manuscript editing, and finalization.