Therapeutic potential of coenzyme Q10 in mitochondrial dysfunction during tacrolimus-induced beta cell injury

We previously reported that oxidative stress induced by long-term tacrolimus treatment impairs mitochondrial function in pancreatic beta cells. In this study, we aimed to investigate the therapeutic potential of coenzyme Q10, which is known to be a powerful antioxidant, in mitochondrial dysfunction in tacrolimus-induced diabetic rats. In a rat model of tacrolimus-induced diabetes mellitus, coenzyme Q10 treatment improved pancreatic beta cell function. The administration of coenzyme Q10 improved insulin immunoreactivity within islets, which was accompanied by reductions in oxidative stress and apoptosis. Assessment of the mitochondrial ultrastructure by electron microscopy revealed that coenzyme Q10 treatment increased the size, number, and volume of mitochondria, as well as the number of insulin granules compared with that induced by tacrolimus treatment alone. An in vitro study using a pancreatic beta cell line showed that tacrolimus treatment increased apoptosis and the production of mitochondrial reactive oxygen species, while cotreatment with coenzyme Q10 effectively attenuated these alterations. At the subcellular level, tacrolimus-induced impairment of mitochondrial respiration was significantly improved by coenzyme Q10, as evidenced by the increased mitochondrial oxygen consumption and ATP production. Our data indicate that coenzyme Q10 plays an important role in reducing tacrolimus-induced oxidative stress and protects the mitochondria in pancreatic beta cells. These findings suggest that supplementation with coenzyme Q10 has beneficial effects in tacrolimus-induced diabetes mellitus.


Measurement of beta cell mass.
The relative volumes of beta cells were estimated by the point-counting method 28 . The relative beta cell volume was measured by classifying the number of points matching to the insulin-positive region by the number of points matching to the remaining pancreatic area. Cell masses were measured by multiplying the ratios of beta cells by the total pancreatic weight 29 . Glucose-stimulated insulin secretion (GsIs) assay. As described earlier, islets were isolated from male SD rats (250-300 g) by digestion using collagenase 30,31 . The islets were pre-incubated in conditioned RPMI 1640 medium at 37 °C for 24 h. The isolated islets were then treated with Tac (1 μg/mL) and CoQ 10 (10-1000 ng/mL) for 12 h. This was followed by analysis of insulin secretion. The harvested islets were divided into groups of 30 islets and washed with Krebs-Ringer Modified Buffer (KRB), followed by addition of 2.8 mM glucose (basal). After washing with KRB, the islets were incubated with KRB containing 16.7 mM glucose for 1 h. The insulin level in the solution was examined using an ELISA kit (Millipore Corp., St. Charles, MO, USA).

Measurement of 8-OHdG in serum.
The end product of oxidative DNA damage was measured by determining the concentration of 8-OHdG in serum. The 8-OHdG level was evaluated using an ELISA kit (Cell Biolabs, San Diego, CA, USA).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The TUNEL assay was performed for the tissue sections using the Apoptosis Detection kit (Millipore Corp.) as per the www.nature.com/scientificreports www.nature.com/scientificreports/ manufacturer's instructions. For double labeling with insulin, tissue sections were incubated with an insulin antibody (Invitrogen) followed by incubation with a Cy 3 -labeled antibody (Jackson ImmunoResearch). The double-labeled cells were counted in approximately 20 randomly selected non-overlapping islets per animal of each group. transmission electron microscopy. Electron microscopic inspection was processed as previously described 32 . The area and number of mitochondria per cell were evaluated in 40 random beta cells using an image analyzer (TDI Scope Eye).

Three-dimensional (3D) reconstruction of mitochondria.
Long ribbons up to about 20 sections of isolated islets treated with drugs were cut at a thickness of 70-90 nm on an ultramicrotome (Leica Microsystems Ltd.). The area of interest was selected, and consecutive serial sections were imaged under an electron microscope (JEM 1010; JEOL, Tokyo, Japan). Outlines of individual mitochondria were manually traced with different colors through image stacks using the Photoshop software (Adobe Systems, San Jose, CA, USA). Each image was aligned, and the marked structures were masked and exported to the 3D modeling program Mimics v. 19.0 (Materialise, Leuven, Belgium), along with information regarding slice thickness, actual pixel size, and image orientation. The mitochondrial volume and surface area in a stack consisting of equal numbers of sections were also calculated using Mimics v.19.0.
Cell viability and apoptosis. For assessment of cell viability, INS-1 cells were plated in culture dishes at 90% confluence. On the following day, the cells were treated with Tac (50 µg/ml) and 1 pg/ml-10 μg/ml CoQ 10 for 12 h. CCK-8 solution (Dojindo, Rockville, MD, USA) was added to each well to evaluate cell viability. For assessment of apoptosis, the cells detached by trypsin were treated with annexin V (BD Biosciences, San Jose,   Quantitative analysis of beta cell islet area. The Tac group showed smaller islets with a lower intensity of insulin staining within islets than the vehicle (Vh) group. In contrast, cotreatment with CoQ 10 and Tac reversed these changes. (c) Quantification of estimated pancreatic beta cell mass by the point-counting method. Data are presented as the mean ± SE (n = 8). One-way ANOVA was used to analyze the data. & P < 0.05 versus the Vh group; $ P < 0.05 versus the CoQ 10 groups; # P < 0.05 versus the Tac group. www.nature.com/scientificreports www.nature.com/scientificreports/ Oxygen consumption rate (OCR) experiments. Real-time OCR in the cells was evaluated using an XF24 Extracellular Flux Analyzer (Seahorse, Billerica, MA, USA). In a non-CO 2 incubator, the medium of the cells was changed to a running medium following incubation with drugs or Vh and treated for 1 h at 37 °C. The mitochondrial inhibitors used were 1 µM of oligomycin (ATP synthase inhibitor), 0.5 µM of carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 µM of rotenone/antimycin A (complex I and III inhibitor). Immediately following completion of the assay, each well was washed with phosphate-buffered saline, and lysis buffer (Pierce Biotechnology, Rockford, IL, USA) was added to lyse the cells in the well. Total protein was measured using the BCA Protein Assay Kit (Pierce Biotechnology). Then, the OCR data were normalized to the total protein content of each sample. Parameters for mitochondrial function were evaluated by measuring these mitochondrial inhibitor compounds as modulators to determine ATP production, basal respiration, spare respiratory capacity, and maximal respiration 33 . A total of 3-4 wells were used for each group. statistical analysis. The values are expressed as the mean ± standard error of at least three independent tests. Using PRISM software, multiple comparisons were carried out by one-way analysis of variance with Bonferroni's post-hoc test (Version 7.03 for Windows, GraphPad Software, La Jolla, CA, USA). Results with P values < 0.05 were considered statistically significant. Table 1 displays alterations in the functional parameters of the experimental groups following treatment of Tac and CoQ 10 for 28 days. The Tac + CoQ 10 group showed larger reductions in intake-water and urine volume than that in the Tac group. Therefore, CoQ 10 did not affect the whole-blood trough level of Tac, implying that a drug-interaction did not occur at these doses.

Effect of CoQ 10 administration in an experimental model of Tac-induced DM.
Rats treated with Tac showed features of DM, as indicated by higher AUCg and HbA1c values and lower levels of plasma insulin compared with those in animals administered with Vh. Addition of CoQ 10 reversed these changes (Fig. 1). Tac-treated islets observed lower insulin immunoreactivity and reduced numbers of beta cells than the Vh group, and these effects were reduced by treatment with CoQ 10 (Fig. 2). We also quantified the beta cell mass in the groups and found that a reduction in beta cell mass induced by Tac treatment was reversed by cotreatment with CoQ 10 , as shown in Fig. 2c.
To assess the exact effects of the insulin secretion function of CoQ 10 during Tac-induced islet injury, we treated primary rat isolated islets with Tac and CoQ 10 in a culture setting and evaluated GSIS. As expected, Tac significantly decreased GSIS. The addition of CoQ 10 resulted in significantly higher insulin secretion levels compared with that of Tac alone, as shown in Fig. 3. www.nature.com/scientificreports www.nature.com/scientificreports/

Effect of CoQ 10 administration on Tac-induced oxidative stress and apoptosis in pancreatic beta cells.
The proliferation of beta cells was confirmed by tissue expression of Ki67 from the experimental groups (Fig. 4). Treatment of Tac reduced the number of Ki67-positive cells in the insulin-positive area compared with those in the Vh and CoQ 10 groups, while concomitant CoQ 10 treatment restored Ki67 staining. Figure 5 shows the co-immunohistochemical staining results for 8-OHdG (Fig. 5a-d) and 4-HHE (Fig. 5e-h) with insulin (red fluorescence), as well as levels of serum 8-OHdG (Fig. 5n). The immunoreactivity of 8-OHdG and 4-HHE in tissue sections and the serum 8-OHdG level were significantly increased in the Tac group, and these effects were www.nature.com/scientificreports www.nature.com/scientificreports/ restored by CoQ 10 treatment. We also examined whether CoQ 10 protects against apoptosis by Tac treatment. The number of TUNEL-positive cells were markedly higher in the Tac group than in the Vh group and were decreased with the cotreatment of CoQ 10 ( Fig. 5i-l,p).

Effect of CoQ 10 administration on tac-induced mitochondrial ultrastructural changes in pancreatic beta cells.
To evaluate mitochondrial function, electron microscopy was used to assess the mitochondrial ultrastructure and quantify the mitochondrial number. For the islet cells obtained from the current experimental rats, Tac treatment decreased both the average mitochondrial area and number (Fig. 6a). By contrast, the administration of CoQ 10 recovered the average mitochondrial area and number. Electron microscopy also demonstrated that the number of insulin granules were markedly decreased by Tac treatment. CoQ 10 was effective in reducing this attenuation in number of granules, confirming that treatment with CoQ 10 protected islet function after Tac treatment (Fig. 6b-d). The effect of CoQ 10 on mitochondrial volume and surface area was evaluated by performing 3D reconstruction of consecutive sections of individual mitochondria in beta cells from isolated rat islets. Consistent with the previous results, reductions in mitochondrial volume and surface area induced by Tac treatment were significantly reversed by cotreatment with CoQ 10 (Fig. 6e,f).  (Fig. 7a). To examine whether CoQ 10 inhibits mitochondrial pathways related to apoptosis, we carried out flow cytometric analysis with annexin V staining. The Tac-induced increase in the percentage of annexin V-positive cells was significantly attenuated by CoQ 10 treatment (Fig. 7b,c).
Next, we evaluated whether treatment with CoQ 10 reduced mitochondrial ROS accumulation during Tac treatment. Use of MitoSOX Red for mitochondrial superoxide (O 2 − ) detection accompanied by flow cytometric analysis in INS-1 cells revealed that CoQ 10 significantly attenuated Tac-induced MitoSOX Red fluorescence (Fig. 8a,b). To visualize the protective effect of CoQ 10 against O 2 − accumulation in mitochondria, we performed time-lapse monitoring of MitoSOX Red intensity in the presence or absence of Tac and CoQ 10 in the same field. As shown in Fig. 8c, MitoSOX Red fluorescence intensity was dramatically elevated in the Tac group, but this effect was attenuated by CoQ 10 treatment.

CoQ 10 increased mitochondrial respiration in Tac-treated INS-1 cells. The mitochondrial bioener-
getics of whole cells was determined by calculating oxygen consumption over time after the sequential addition of inhibitors of mitochondrial function. This analysis revealed marked differences between the Tac and Tac + CoQ 10 groups (Fig. 9a). Compared to Tac-only treatment, Tac + CoQ 10 treatment resulted in higher rates of basal mitochondrial respiration, which is usually indicative of either a higher number of mitochondria or increased mitochondrial activity (Fig. 7b). Moreover, compared with Tac-only treatment, Tac + CoQ 10 treatment resulted in significantly higher ATP-linked respiration and maximal respiration.

Discussion
The current study was performed to investigate whether cotreatment with CoQ 10 is effective in ameliorating pancreatic beta cell dysfunction by Tac. The results of our study showed that CoQ 10 attenuated hyperglycemia and restored the insulin secretion ability by reducing Tac-induced oxidative stress. These findings suggest that CoQ 10 produces beneficial effects for reducing mitochondrial injury via its antioxidative properties during Tac-induced beta cell injury (Fig. 10). The results of our study thus provide a rationale for use of CoQ 10 as supplemental therapy in Tac-induced DM in clinical practice. In this study, we first evaluated whether the administration of CoQ 10 was effective in controlling hyperglycemia in an experimental Tac-induced DM model. Recently, several clinical studies have shown that CoQ 10 treatment reduces DM [34][35][36] . In this study, we found that the AUCg based on IPGTT was increased and the plasma insulin level and insulin-immunoreactivity in islets were decreased by chronic Tac treatment. However, cotreatment with CoQ 10 significantly decreased the AUCg and increased levels of plasma insulin and GSIS compared with the levels observed following Tac treatment alone. Furthermore, immunostaining of insulin in islets revealed that concomitant treatment with CoQ 10 resulted in increases in islet size and immunoreactivity for insulin, as well as preservation of islet morphology as demonstrated by less irregular islet boundaries and reduced vacuolization. These findings suggest that CoQ 10 has beneficial effects on the preservation of beta cells during Tac treatment.
To elucidate the possible mechanism of CoQ 10 in the control of hyperglycemia, we evaluated the expression of markers of oxidative stress in the experimental groups, as oxidative stress has been suggested to be an important pathway in the pathogenesis of pancreatic beta cell injury. High levels of oxidative stress induced by Tac administration lead to islet cell death and dysfunction, with short-and long-term Tac treatment inducing the www.nature.com/scientificreports www.nature.com/scientificreports/ production of ROS, thereby causing apoptotic cell death 32,37 . Therefore, it has been proposed that a reduction in oxidative stress could protect from pancreatic beta cell injury. Based on this hypothesis, we focused on the antioxidative effect of CoQ 10 on Tac-induced oxidative injury in pancreatic beta cells. For this, we measured the expression of 8-OHdG and 4-HHE, markers of oxidative DNA and lipid damage, respectively, and found that while their expression levels were increased in beta cells in Tac-treated mice, administration of CoQ 10 attenuated these increases in expression levels. Indeed, there is evidence to show that the development of diabetes is  www.nature.com/scientificreports www.nature.com/scientificreports/ associated with increased oxidative stress and that CoQ 10 can scavenge ROS, resulting in an anti-hyperglycemic effect 25 . Overall, our data indicate that oral administration of CoQ 10 is effective in reducing Tac-induced oxidative stress in pancreatic beta cells.
We further evaluated whether the antioxidative effect of CoQ 10 ameliorated mitochondrial injury during Tac treatment. The mitochondria play important roles in pancreatic beta cells from glucose metabolism to insulin exocytosis, thereby ensuring tight regulation of glucose-stimulated insulin secretion 38 . Impairment in the mitochondrial function affects this metabolic coupling and ultimately leads to apoptosis and beta cell death. Furthermore, mitochondria are susceptible to oxidative damage and could be a major source of superoxide under disease conditions 39 . Based on this knowledge, we first examined rates of apoptosis, a mitochondrial pathway of cell death in vivo and in vitro, and found that CoQ 10 reduced the numbers of TUNEL-and annexin V-positive cells compared with those in the Tac group. Ultrastructural analysis revealed that Tac treatment caused reductions in the number, area, size, and volume of mitochondria, as well as the number of insulin granules, while CoQ 10 administration attenuated these changes. CoQ 10 also reduced accumulation of mitochondrial ROS and superoxide anion and restored basal respiration, ATP-linked respiration, and maximal respiration rates according to oxygen consumption over time. These data suggest that administration of CoQ 10 helps to maintain mitochondrial function during Tac-induced oxidative stress, resulting in a subsequent decrease in apoptotic cell death.
In addition to Tac, rapamycin analogs, sirolimus (SRL) and everolimus (EVR), have been widely used as immunosuppressants in transplantation, but they are also associated with an increased risk of DM. Many studies have demonstrated that both of these drugs cause mitochondrial dysfunction in pancreatic beta cells, eventually leading to decreased insulin release [40][41][42][43] . We have also confirmed that SRL or EVR treatment alone results in the development of hyperglycemia, accompanied by a reduction in the number of insulin granules and an increase in the expression of oxidative stress markers, in animal models 27,30,31 . However, addition of CoQ 10 attenuated SRL-induced hyperglycemia, as well as oxidative stress, in a rat model. At the subcellular level, CoQ 10 also improved not only the morphology of the mitochondria but also mitochondrial respiration 25 . Based on these findings, CoQ 10 supplementation would also be beneficial in the treatment of rapamycin-induced DM.
In conclusion, CoQ 10 plays an important role in reducing Tac-induced oxidative stress and protecting mitochondria in pancreatic beta cells. These findings suggest that CoQ 10 may be useful in the management of Tac-induced DM in the future.