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The therapeutic efficacy of water-soluble coenzyme Q10 in an experimental model of tacrolimus-induced diabetes mellitus

Background/Aims Coenzyme Q10 (CoQ10) has antioxidant effects and is commercially available and marketed extensively. However, due to its low bioavailability, its effects are still controversial. We developed a water-soluble CoQ10-based micelle formulation (CoQ10-W) and tested it in an experimental model of tacrolimus (TAC)-induced diabetes mellitus (DM). Methods We developed CoQ10-W from a glycyrrhizic-carnitine mixed layer CoQ10 micelle preparation based on acyltransferases. TAC-induced DM rats were treated with either lipid-soluble CoQ10 (CoQ10-L) or CoQ10-W for 4 weeks. Their plasma and pancreatic CoQ10 concentrations were measured using liquid chromatography- tandem mass spectrometry. The therapeutic efficacies of CoQ10-W and CoQ10-L on TAC-induced DM were compared using functional and morphological parameters and their effects on cell viability and reactive oxygen species (ROS) production were also evaluated in cultured rat insulinoma cells. Results The plasma CoQ10 level was significantly increased in the CoQ10-W group compared to that in the CoQ10-L group. Intraperitoneal glucose tolerance tests and glucose-stimulated insulin secretion revealed that CoQ10-W controlled hyperglycemia and restored insulin secretion significantly better than CoQ10-L. The TAC-mediated decrease in pancreatic islet size was significantly attenuated by CoQ10-W but not by CoQ10-L. TAC-induced oxidative stress and apoptosis were significantly more reduced by CoQ10-W than CoQ10-L. Electron microscopy revealed that CoQ10-W restored TAC-induced attenuation in the number of insulin granules and the average mitochondrial area, unlike CoQ10-L. In vitro studies showed that CoQ10-L and CoQ10-W both improved cell viability and reduced ROS production in TAC-treated islet cells to a similar extent. Conclusions CoQ10-W has better therapeutic efficacy than CoQ10-L in TAC-induced DM.


INTRODUCTION
Diabetes mellitus (DM) is the most common cause of end stage renal disease worldwide, and aggravation or development of DM after transplantation is a serious complication in solid organ transplant recipients [1].
The Korean Journal of Internal Medicine Vol. 35, No. 6, November 2020 Tacrolimus (TAC) is the most popular immunosuppressant but it causes several metabolic complications. These include TAC-induced DM, which is an important adverse effect in organ transplant recipients [2,3]. The mechanisms of TAC-induced DM are not well studied, but direct injury to pancreatic islet cells or impaired insulin signaling by TAC are regarded as the main pathogenesis methods, and oxidative stress plays a significant role in the process of TAC-induced pancreatic islet injury [4,5]. Thus, reducing TAC-induced oxidative stress is one of the important approaches for decreasing TAC-induced DM.
Coenzyme Q 10 (CoQ 10 ) is an electron transporter in the electron transport chain (ETC) that transports electrons from ETC complex I and II to complex III [6,7]. With its potential antioxidant effects [8][9][10][11][12][13], CoQ 10 has been commercialized and is widely circulated in health care markets, but its therapeutic effect on diseases such as DM is still controversial due to its low bioavailability. The low bioavailability of CoQ 10 is related to its hydrophobic chemical structure, and most of the circulating CoQ 10 are in lipid-soluble form.
Thus, improving the bioavailability of CoQ 10 is important for optimizing the use of this potential antioxidant. In this study, we developed water-soluble CoQ 10 (CoQ 10 -W) to improve bioavailability and compared its effect with that of lipid-soluble CoQ 10 (CoQ 10 -L) in an experimental model of TAC-induced DM.

Development of CoQ 10 -W
Coenzyme Q 10 was purchased from Kaneka Nutrients (Pasadena, TX, USA). Trisodium glycyrrhizinate hydrate was purchased from Tokyo Chemical Industry Co. LTD. (Tokyo, Japan). Eicosapentaenoic acid (EPA) was purchased from Phycoil Biotech Korea, Inc. (Seoul, Korea). EPA was used to improve micelle stability because glycyrrhizin alone is not enough to maintain the micelle. In addition, EPA itself reduces the inflammatory response by inhibiting prostaglandin synthesis. To produce a uniform nano-emulsion (Qmicelle) (Fig. 1A), the following pretreatment is essential. Coenzyme Q 10 , EPA, and trisodium glycyrrhizinate hydrate (at a concentration of 1 mg/mL each) were dispersed in hot water.
The mixture was pre-homogenized at 10,000 rpm with a homogenizer (Multi-Purpose Homogenizer, YSTRAL, Ballrechten-Dottingen, Germany) until a homogeneous consistency was obtained. An APV2000 microfluidizer processor (SPX Flow, Leeds, UK) was used to produce nano-emulsions (Qmicelle). It has a reservoir capacity of 1,500 mL and can be operated at pressures of up to 2,000 bars. The prepared mixture at 60°C was poured into the fluidizer and Qmicelle was obtained after 10 cycles at a pressure of 1,200 bars. Each component under EPA, coenzyme Q 10 and trisodium glycyrrhizinate hydrate, showed single peaks at retention times of 6.0, 6.6, and 15.9 minutes, respectively (Fig. 1B), and their concentrations were 0.24, 1.0 and 0.6 mg/mL (Table 1). We detected homogeneity and size distribution by dynamic light scattering (DLS), and conformed that sizes of the Qmicelles were uniform and distributed in a narrow range around 100 nm (Fig. 1C).

Animals and drugs
All experimental procedures were reviewed and approved by the animal care and use committee of The Catholic University of Korea (CUMC-2018-0038-03). All the protocols in the study were conducted in strict accordance with their ethical guidelines for animal research. Sprague-Dawley rats (Charles River Technology, Seoul, Korea) with a starting weight of 220 to 230 g were kept in cages (Nalge Co., Rochester, NY, USA) with controlled temperature, humidity, and light in the animal care facility at the Catholic University of Korea. The rats were fed a low-salt diet (0.05% sodium chloride, Research Diets, New Brunswick, NJ, USA) daily. TAC (Prograft, Astellas Pharma, Ibaraki, Japan) was dissolved in olive oil (Sigma-Aldrich, St. Louis, MO, USA), and blended to a final concentration of 1.5 mg/mL. CoQ 10 (Chong Kun Dang Pharm, Seoul, Korea) was diluted in olive oil (CoQ 10 -L) or drinking water (CoQ 10 -W) to a final concentration of 20 mg/kg.

Experimental design
The rats were randomly divided into four groups (n = 9/ group). The rats in the vehicle (VH) group received 0.3 mL olive oil once a day subcutaneously. The rats in the TAC, TAC + CoQ 10 -L and TAC + CoQ 10 -W groups were treated with TAC (1.5 mg/kg) subcutaneously daily for 4 weeks. The TAC + CoQ 10 -L and TAC + CoQ 10 -W groups were co-treated with CoQ 10 -L or CoQ 10 -W (20 mg/kg), respectively, orally once a day. We decided the route of administration and concentrations of TAC and CoQ 10 based on previous studies [14,15].

Pancreas preservation
Surgical instruments are sterilized before use. Following abdominal incision, phosphate-buffered saline (PBS) was used for in vivo perfusion via the abdominal aorta. After ensuring that blood was completely removed from the organs, part of the pancreas was removed for further analysis. Then the perfusion solution was changed to periodate-lysine-paraformaldehyde, and perfusion was continued for an additional 5 minutes. The remaining portion of the pancreas was collected and wax-embedded for histological studies.

Basic protocol
Following administration of the drug, the diet and weight of the rats were closely monitored for 4 weeks. After 4 weeks of treatment, the rats were placed in a metabolic cage for 24 hours to measure water intake and urine volume. For further analysis, blood and tissue samples were collected from anaesthetized rats a day later. The TAC level in whole blood was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS, Abbott Diagnostics, Abbott Park, IL, USA).

Measurement of CoQ 10 in plasma and pancreatic tissue
The concentration of CoQ 10 in the plasma and pancreatic tissues was measured by LC-MS/MS analysis. CoQ 10 was extracted from the plasma and tissue and quantified The Korean Journal of Internal Medicine Vol. 35, No. 6, November 2020 using LC-MS/MS method reported previously [16], with some modifications. Given that CoQ 10 is an endogenous substance, all calibration and quality control samples were prepared in a physiological solution (4% bovine serum albumin in PBS). Briefly, 100 μL aliquots of rat plasma or tissue homogenates treated with 10 μL of CoQ 10 (800 ng/mL, as the internal standard) were extracted by liquid-liquid extraction using 1 mL isopropyl alcohol, followed by LC-MS/MS analysis. The samples were analyzed using a Shimadzu Nexera X2 UPLC (Shimadzu Corporation, Kyoto, Japan) coupled with an LCMS-8050 triple quadruple mass spectrometer (Shimadzu Corporation) with an electrospray ionization interface in the positive ion mode. Chromatographic separation was achieved using a Kinetex C 18 column (2.1 × 100 mm, 2.6 μm; Phenomenex, Seoul, Korea) with a mobile phase consisting of 0.1% formic acid in isopropyl alcohol and methanol at a flow rate of 0.2 mL/min. The total run time was 4 minutes per sample. Quantitation was performed using selected reaction monitoring of the transitions at m/z 863.35 > 197.15 (for CoQ 10 ) and m/z 794.35 > 197.15 (for the internal standard). The calibration curves were linear (r ≥ 0.995) from 20 to 10,000 ng/mL. The within-and between-batch precision and accuracy were within the acceptable limits of ± 15%.

Intraperitoneal glucose tolerance test
Rats were fasted for 1 day following treatment with the drugs for 4 weeks before the intraperitoneal glucose tolerance test (IPGTT) was conducted. The rats were injected with 50% dextrose (1.5 g/kg), and blood glucose concentration was measured at 30, 60, 90, and 120 minutes using a glucose analyzer (Accu-Check, Roche Diagnostics, Basel, Switzerland). Thereafter, based on the IPGTT values, trapezoidal estimation was obtained, and the area under the curve of glucose (AUCg) was calculated.

Glucose-stimulated insulin secretion assay
After treating male Sprague-Dawley rats (250 to 300 g) with the drugs according to the in vivo experimental design, pancreatic islets were isolated using collagenase digestion as described previously [17]. The islets were incubated in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS and 100 U/mL penicillin at 37°C for 24 hours. The next day, groups of 30

Immunohistochemistry
All the immunohistochemistry (IHC) procedures were performed as described previously [18]. The primary antibodies used for IHC were as follows: anti-insulin (I2018, Sigma-Aldrich; 18-0067, Invitrogen, Carlsbad, CA, USA), anti-8-hydroxy-2'-deoxyguanosine (8-OHdG; JaICA, Shizuoka, Japan), and anti-4-hydroxy-2-hexenal (4-HHE, JaICA, Shizuoka, Japan). 4-m samples were incubated with the antibodies for 12 hours at 4°C. A minimum of 20 fields per section was assessed using a color image analyzer (TDI Scope Eye version 3.6 for Windows, JN OpTIC Co. Ltd., Seoul, Korea). Briefly, images captured during insulin immunohistochemistry were quantified using the polygon program by measuring the pancreas area that was positively stained for insulin, except for vacuoles, when viewed under ×400 magnification. The measurement of 4-HHE expression was similar to that of insulin, and expressed as percentage of 4-HHE positive area in the islet. The 8-OHdG expression was quantified by counting the number of positive cells per field. Histopathologic analysis was performed on randomly selected fields of the pancreas sectioned by a pathologist blinded to the identity of the treatment groups.

Serum 8-OHdG detection
All procedures were carried out strictly according to the ELISA kit protocol (Cell Biolabs, San Diego, CA, USA). erica, MA, USA) as per manufacturer's instructions. In each tissue section, positive cells were analyzed in 20 randomly selected areas. The positive cells or areas were then quantified at ×400 magnification, and the data were analyzed with a color image analyzer (TDI Scope Eye version 3.6 for Windows).

Transmission electron microscopy
Pancreatic tissues were fixed in 2.5% glutaraldehyde (diluted in 0.1 M phosphate buffer), post-fixed with 1% Osmium (VIII) oxide (OsO 4 ) and then embedded in Epon 812. Ultrathin sections were prepared using the embedded tissues, and the sections were stained with uranyl acetate/lead citrate. A JEM-1200EX transmission election microscope (JEOL, Tokyo, Japan) was used for photography, and 20 randomly selected sites were scanned at ×5,000 magnification. We counted insulin granule number and measured mitochondrial area in the scanned areas using an image analyzer (TDI Scope Eye version 3.6).

Flow cytometry
To assess reactive oxygen species (ROS) production, we performed flow cytometry. INS-1 cells were plated and pre-incubated in an incubator at 37°C for 24 hours. After 24 hours, the culture medium was changed to serum-free medium containing TAC (40 mg/mL), TAC (40 mg/mL) + CoQ 10 -L (10 ng/mL) or TAC + CoQ 10 -W (10 ng/mL). The method followed has been described previously [19].

Statistical analysis
The data are expressed as mean ± standard error (SE) of at least three independent experiments. Multiple comparisons between different groups were carried out by oneway analysis of variance with Bonferroni's post hoc test using IBM SPSS Statistics version 24 (IBM, Armonk, NY, USA). Results with p < 0.05 were considered statistically significant.

Comparison of the plasma and pancreas CoQ 10 levels with CoQ 10 -L and CoQ 10 -W treatments of TAC-induced DM rats
Plasma CoQ 10 levels in the VH and TAC groups were 39 ± 7 and 25 ± 8 ng/mL, respectively. The plasma CoQ 10 level ( Fig. 2A) was significantly higher in the TAC + CoQ 10 -W group than the TAC + CoQ 10 -L group (956 ± 97 ng/mL vs. 577 ± 88 ng/mL, p < 0.05). The pancreatic CoQ 10 level (Fig. 2B) was lower in the TAC group than in the VH group (59 ± 8 ng/mL vs. 103 ± 26 ng/mL, p < 0.05). Treatment with either TAC + CoQ 10 -L or TAC + CoQ 10 -W increased pancreatic CoQ 10 levels compared with the TAC only group (121 ± 4 ng/mL and 110 ± 14 ng/mL vs. 59 ± 8 ng/mL, p < 0.05). However, there was no significant difference between the two groups.

Comparison of islet size with CoQ 10 -L and CoQ 10 -W treatments of TAC-induced DM rats
Immunohistochemical staining for insulin showed that the islet sizes in the TAC group and TAC + CoQ 10 -L group were lower than that in the VH group (8 ± 2 and 9 ± 1 vs. 16 ± 2 μm 2 × 10 3 , p < 0.05). However, the islet size in the TAC + CoQ10-W group (14 ± 1 μm 2 × 10 3 ) was increased compared with the TAC and the TAC + CoQ 10 -L groups (p < 0.05) (Fig. 5).

Comparison of apoptosis in pancreatic islets with CoQ 10 -L and CoQ 10 -W treatments of TAC-induced DM rats
Four weeks of TAC treatment also induced apoptosis in the pancreas. As shown in Fig. 7, the number of TUNEL-positive cells was significantly higher in the TAC group than in the VH group (2.4 ± 0.3 cells per islet vs. 0 ± 0 cells per islet, p < 0.05). CoQ 10 -W decreased TUNEL-positive cells compared with TAC (0.2 ± 0.2 cells per islet vs. 2.4 ± 0.3 cells per islet, p < 0.05), while CoQ 10 -L (1.8 ± 0.2 cells per islet) did not (Fig. 7).

Comparison of the mitochondrial area and insulin granule with CoQ 10 -L and CoQ 10 -W treatments of TAC-induced DM rats
Electron microscopy was used to evaluate the changes in the mitochondrial area and insulin granule number. The average mitochondrial area was significantly lower in the TAC and TAC + CoQ 10 -L groups than in the VH group (0.05 ± 0.006 and 0.08 ± 0.08 vs. 0.4 ± 0.03 per field, p < 0.05). However, the numbers were significantly (p < 0.05) higher in the TAC+CoQ 10 -W group (0.2 ± 0.02 per field) ( Fig. 8A and 8C). Changes in insulin granule numbers were similar to the changes in mitochondrial area ( Fig. 8A and 8B).

Comparison of cell viability with CoQ 10 -L and CoQ 10 -W treatments of TAC-induced INS cell injury
We demonstrated the protective effect of CoQ 10 -L and CoQ 10 -W against cell injury during TAC treatment in INS-1, a pancreatic beta cell line. At the different doses tested, both TAC + CoQ 10 -L and TAC + CoQ 10 -W increased cell viability by approximately 2.8-fold compared to TAC (p < 0.05). However, there were no differences between the tested doses of TAC + CoQ 10 -L and TAC + CoQ 10 -W (p > 0.05) (Fig. 9).

Comparison of ROS production with CoQ 10 -L and CoQ 10 -W treatments of TAC-induced INS cell injury
To compare the protective effect of CoQ 10 -L and CoQ 10 -W on oxidative stress, we performed flow cytometry using INS-1 cells. TAC significantly increased ROS production compared to the VH (p < 0.05). Both CoQ 10 -L and CoQ 10 -W reduced TAC-induced ROS production; though there was no significant difference between the two groups (p > 0.05) (Fig. 10).

DISCUSSION
This study investigated whether CoQ 10 -W provides better protection against TAC-induced pancreatic beta cell dysfunction than conventional CoQ 10 (CoQ 10 -L). The results of our study clearly demonstrate that CoQ 10 -W increases insulin secretion, and thus lowers blood glucose in TAC-induced diabetes. In addition, CoQ 10 -W reduced TAC-induced oxidative stress and apoptosis in the pancreas more than CoQ 10 -L. This finding suggests that improving the bioavailability using CoQ 10 -W may overcome the limitations of CoQ 10 -L and render it more effective as an anti-diabetic and anti-oxidative agent. Until now, several approaches have been used for improving the bioavailability of CoQ 10 [20][21][22][23] that can be summarized in two ways as follows: One is to increase the dissolution rate of the drug, and the other is to promote absorption of the drug by optimizing the digestion process. We used the latter method of optimizing the digestion process, to improve drug absorption. The CoQ 10 -W used in this study consists of a glycyrrhizin-carnitine mixed layer CoQ 10 micelle preparation based on acyltransferases as shown in Fig. 1. This formula improved the absorption of CoQ 10 by using the natural extract-glycyrrhizin as the carrier, and the heat of fusion which converts the CoQ 10 from hydrophobic to hydrophilic. In addition, this formulation has the advantage of retaining the original chemical structure of CoQ 10 and eliminating the formation of toxic substances during the synthesis and conversion processes. The  composition of CoQ 10 in the solution may be affected by high pressure or high temperature during the manufacture of CoQ 10 -W. Thus, we confirmed the homogeneity and uniform size of the micelles by high performance liquid chromatography after preparation as shown Fig.  1. With this formulation, we achieved higher plasma CoQ 10 levels (1.7 times) than with CoQ 10 -L. We evaluated whether CoQ 10 -W had improved effects on glucose-lowering and insulin secretion compared to CoQ 10 -L in an experimental model of TAC-induced DM. In this study, we found that CoQ 10 -W significantly reduced TAC-induced hyperglycemia, while CoQ 10 -L did not. In addition, glucose-stimulated insulin secretion (GSIS) revealed that CoQ 10 -W significantly restored the TAC-induced decrease in insulin secretion, unlike CoQ 10 -L. These results suggest that CoQ 10 -W is more effective than CoQ 10 -L at lowering hyperglycemia, and in preserving insulin secretion in TAC-induced DM, and this is related to the increased bioavailability of CoQ 10 -W. However, pancreatic CoQ 10 levels were not significantly different in the two groups. Thus, the improved control of hyperglycemia or insulin secretion in the CoQ 10 -W group compared with the CoQ 10 -L group seems to be related to a systemic antioxidant effect rath- CoQ10 -L CoQ10-W TAC TAC + CoQ10-L TAC + CoQ10-W

A B
The Korean Journal of Internal Medicine Vol. 35, No. 6, November 2020 er than its local effect on pancreatic tissue. This presumption may be supported by the lack of difference in cell survival or ROS production in in vitro studies of the two groups. It is well known that oxidative stress [4,24,25] and apoptosis [25][26][27] are common mechanisms of TAC-induced DM. To explain the improved hyperglycemia control by CoQ 10 -W, we evaluated the antioxidant and anti-apoptotic effects of CoQ 10 -W and CoQ 10 -L in TAC-induced pancreatic islet injury. The results of our study clearly demonstrate that CoQ 10 -W reduced both, the TAC-induced markers of oxidative stress (8-OHdG in tissue and serum, 4-HHE in tissue), and TAC-induced apoptosis to a greater extent than CoQ 10 -L. These findings confirm that CoQ 10 -W has better antioxidant and anti-apoptotic effects than CoQ 10 -L on TAC-induced pancreas injury, and this may explain the better hyperglycemia control of CoQ 10 -W than CoQ 10 -L.
Oxidative stress is closely related to the quantity and quality of mitochondria [28,29]. High-quality mitochondria protect cells against oxidative stress and prevent apoptosis [29]. In our data, CoQ 10 -L increased number of insulin granules compared with the TAC group, with CoQ 10 -W producing a further increase as shown by electron microscopy. Furthermore, CoQ 10 -W increased the average mitochondria area over that seen in the TAC group. Although CoQ 10 -L also restored the loss of mitochondria, it was significantly less effective than CoQ 10 -W. These findings suggest that the improved antioxidant capacity of CoQ 10 -W offers a more effective remedy for islet and mitochondrial damage than CoQ 10 -L in the TAC-induced DM model.
Our study has some limitations. First, the CoQ 10 -W group had a similar CoQ 10 level in the pancreas as the CoQ 10 -L group, and in vitro study revealed that viability and ROS production of islet cells in the CoQ 10 -L and CoQ 10 -W groups were not significantly. Further studies are needed to improve the action of CoQ 10 -W at the cellular level. Second, we compared the CoQ 10 levels at the end of our study. However, endogenous substances such as CoQ 10 follow the circadian rhythm. Thus, pharmacokinetic and pharmacodynamic studies are needed to determine whether CoQ 10 -W has better bioavailability than CoQ 10 -L. Third, we did not include the VH of nano-emulsion without CoQ 10 . Therefore, there is a concern that that anti-inflammatory effect of EPA in na-no-emulsion may affect that of CoQ 10 -W . In this point, we previously tested the anti-inflammatory effect of EPA in rheumatoid arthritis model, and found that EPA in nano-emulsion did not affect the anti-inflammatory effect of CoQ 10 (data not shown). Thus, the anti-inflammatory effect of EPA in nano-emulsion may be ruled out in our study.
In summary, the improved CoQ 10 bioavailability using CoQ 10 -W may provide more benefit than conventional CoQ 10 in TAC-induced diabetes. We expect that adding CoQ 10 -W TAC treatments can effectively reduce TAC-induced oxidative stress in clinical practice.