The comparative effects of high dose atorvastatin and proprotein convertase subtilisin/kexin type 9 inhibitor on the mitochondria of oxidative muscle fibers in obese-insulin resistant female rats

Chanisa Thonusin, Nattayaporn Apaijai, Thidarat Jaiwongkam, Sasiwan Kerdphoo, Busarin Arunsak, Patchareeya Amput, Siripong Palee, Wasana Pratchayasakul, Nipon Chattipakorn, Siriporn C. Chattipakorn

PII: S0041-008X(19)30349-7
DOI: https://doi.org/10.1016/j.taap.2019.114741
Reference: YTAAP 114741

To appear in: Toxicology and Applied Pharmacology

Received date: 7 July 2019
Revised date: 6 August 2019
Accepted date: 28 August 2019

Please cite this article as: C. Thonusin, N. Apaijai, T. Jaiwongkam, et al., The comparative effects of high dose atorvastatin and proprotein convertase subtilisin/kexin type 9 inhibitor on the mitochondria of oxidative muscle fibers in obese-insulin resistant female rats, Toxicology and Applied Pharmacology (2018), https://doi.org/10.1016/j.taap.2019.114741

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The Comparative Effects of High Dose Atorvastatin and Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitor on the Mitochondria of Oxidative Muscle Fibers in Obese-Insulin Resistant Female Rats
Chanisa Thonusin1,2,3, Nattayaporn Apaijai1,3, Thidarat Jaiwongkam1,3, Sasiwan Kerdphoo1,3, Busarin Arunsak1,2,3, Patchareeya Amput1,2,3, Siripong Palee1,3, Wasana Pratchayasakul1,2,3, Nipon Chattipakorn1,2,3, Siriporn C Chattipakorn1,3,4,* [email protected]; [email protected]
1Neurophysiology Unit, Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
2Cardiac Electrophysiology Unit, Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
3Center of Excellence in Cardiac Electrophysiology Research, Chiang Mai University, Chiang Mai, Thailand
4Department of Oral Biology and Diagnostic Sciences, Faculty of Dentistry, Chiang Mai

University, Chiang Mai, Thailand

*Corresponding author at: Neurophysiology Unit, Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand

Abstract

The present study aimed to compare the effects of high dose atorvastatin and a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor on the mitochondrial function in oxidative muscle fibers in obese female rats. Female Wistar rats were fed with either a normal diet (ND: n
= 12) or a high-fat diet (HFD: n = 36) for a total of 15 weeks. At week 13, ND-fed rats received

a vehicle, and HFD-fed rats were divided to three groups to receive either a vehicle, 40 mg/kg/day of atorvastatin, or 4 mg/kg/day of PCSK9 inhibitor (SBC-115076) for 3 weeks. Soleus muscles were investigated to assess mitochondrial ROS, membrane potential, swelling, mitochondrial-related protein expression, and level of malondialdehyde (MDA). The results showed that HFD-fed rats with vehicle developed obese-insulin resistance and dyslipidemia. Both atorvastatin and PCSK9 inhibitor reduced obesity and dyslipidemia, as well as improved insulin sensitivity in HFD-fed rats. However, the efficacy of PCSK9 inhibitor to increase weight loss and reduce dyslipidemia in HFD-fed rats was greater than those of atorvastatin. An increase in MDA level, ratio of p-Drp1ser616/total Drp1 protein, CPT1 protein, mitochondrial ROS, and membrane depolarization in the soleus muscle were observed in HFD-fed rats with vehicle.
PCSK9 inhibitor enabled the restoration of all these parameters to normal levels. However, atorvastatin facilitated restoration of some parameters, including MDA level, p-Drp1ser616/total Drp1 ratio, and CPT1 protein expression. These findings suggest that PCSK9 inhibitor is superior to atorvastatin in instigating weight loss, cholesterol reduction, and attenuation of mitochondrial oxidative stress in oxidative muscle fibers of obese female rats.

Keywords: Lipid lowering drug, Mitochondria, Oxidative muscle fiber, Obesity

Introduction

Dyslipidemia, obesity, and insulin resistance can lead to several complications, including metabolic syndrome, diabetes, and cardiovascular disease (1). Dyslipidemia has been associated with mitochondrial dysfunction in skeletal muscle, leading to skeletal muscle dysfunction (2-6). Skeletal muscle is the largest metabolic organ in the body (7) and plays a major determinant of oxidative capacity (8). That capacity predicts metabolic health and longevity (9-11).
Statins are HMG-CoA reductase inhibitors that are first-line options for the treatment of hypercholesterolemia or dyslipidemia (12). Recently a new family of lipid-lowering agents for patients, PCSK9 inhibitors, have become an option for use in those in whom the targeted levels of non-HDL cholesterol have not been reached despite receiving the maximal dose of statins or for patients who develop unacceptable side effects to statin therapy (13). However, studies that compared the effectiveness of statins alone versus PCSK9 inhibitor alone on cholesterol reduction, weight loss, and insulin sensitivity remain limited (14).
Even though statins can improve hyperlipidemia, a cause of mitochondrial dysfunction in skeletal muscle, their use commonly results in adverse effects on skeletal muscle such as myositis, myalgia, and rhabdomyolysis (15). Nevertheless, only a few studies have focused on the direct impact of statins on skeletal muscle mitochondria (16-18). Previous studies in rats revealed that low dose (10 mg/kg/day) atorvastatin impaired mitochondrial function in glycolytic muscle fibers from the plantaris muscle (16-18), but had neither positive nor negative effects on oxidative muscle fibers from soleus muscle (18). However, the effects of high dose atorvastatin on oxidative muscle fibers, which are responsible for the capacity of oxidative metabolism (19), have not been investigated. PCSK9 inhibitors has been approved by FDA since 2015 (20), but the impact of PCSK9 inhibitors on skeletal muscle mitochondria have never been determined. In

fact, previous studies reported only skeletal muscle-related symptoms in subjects who received the PCSK9 inhibitors alirocumab (21) or evolocumab (22), including myalgia, muscle spasm, and muscular weakness. These side effects were found to be less frequent than those experienced by users of atorvastatin (21).
In summary this study aimed to compare the effects of high dose atorvastatin and PCSK9 inhibitor on the mitochondria of oxidative muscle fibers in obese rats. Since atorvastatin and PCSK9 inhibitor are lipid- lowering agents (12, 13) and skeletal muscle- related symptoms are common side effects of atorvastatin (23), this study focused on the effects of both drugs on: mitochondrial lipid metabolism, as indicated by CPT1 protein and OXPHOS protein; mitochondrial dynamics, as indicated by the ratio of p-
Drp1ser616/Drp1 protein and Mfn2 protein; and mitochondrial oxidative stress, as indicated by mitochondrial ROS and malondialdehyde (MDA) level, in skeletal muscles. An additional focus was on the improvement of obesity- induced insulin resistance.

Materials and Methods Animal Model

Female Wistar rats (n = 48) were obtained from the National Laboratory Animal Center (Nomura Siam International, Bangkok, Thailand ). All experiments were performed in line with the protocol approved by the Faculty of Medicine, Chiang Mai University Institutional Animal Care and Use Committee which comply with NIH guidelines (24). Animals were individually housed in a temperature-controlled environment with a 12:12 hr light-dark cycle. Rats were fed with either a normal diet (n = 12) or a high-fat diet (n = 36) for a total of 15 weeks. The normal diet (ND) group consumed a standard chow, which had an energy content of 4.02 kcal/g and

contained 19.77 % of total energy (%E) from fat (Table 1). The high- fat diet (HFD) group consumed a diet, which had an energy content of 5.35 kcal/g and contained 59.28 %E from fat (Table 2).
After week 12 of the dietary program, ND-fed rats received the vehicle (normal saline solution: NSS subcutaneously), and HFD-fed rats were divided equally into three groups (n = 12/group) to receive either the vehicle (NSS), 40 mg/kg/day of atorvastatin subcutaneously, or 4 mg/kg/day of PCSK9 inhibitor (SBC-115076) subcutaneously. All these were administered for an additional 3 weeks.
At week 15, blood was obtained from the tip of the tail after 12-hr fasting to enable an oral glucose tolerance test (OGTT). Then, the rats were decapitated for truncal blood collection. Samples for glucose assay were kept on ice in tubes pre-coated with sodium fluoride. Samples for analysis of triglyceride, cholesterol, and insulin content were kept on ice in tubes pre-coated with EDTA. The soleus muscles were then removed. The soleus muscles from 6 out of the 12 rats in each group were investigated for mitochondrial ROS, mitochondrial membrane potential, mitochondrial swelling, and MDA level. The soleus muscles from the other 6 of the 12 rats in each group were used for the analysis of protein expression.
Plasma analyses

Fasting glucose, and triglyceride, HDL cholesterol, and total cholesterol content were determined by colorimetric assay using a commercially available kit (Biotech, Bangkok, Thailand). Insulin level was measured using a sandwich ELISA (LINCO Research, MO, USA). LDL cholesterol was estimated from Friedewald’s equation: total cholesterol – HDL cholesterol – (triglyceride/5).

Determination of insulin resistance

Insulin resistance was assessed by OGTT and HOMA-IR. OGTT was performed after 12-hr fasting, as in previous studies (25, 26). A higher area under the curve (AUC) of glucose from OGTT indicates a higher degree of insulin resistance. HOMA-IR is a model describing the degree of insulin resistance, as described in a previous study (27).
Soleus muscle protein expression analyses

The expression of the various proteins was determined using western blot analysis. To extract protein tissue was homogenized in a lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in 1xPBS, and 1X protease inhibitor cocktail (Merck, KGaA, Darmstadt, Germany)). After incubation on ice for 30 min, the homogenate was centrifuged at 13,000 rpm for 10 min. Total protein (2 mg/ml) was mixed with the loading buffer (5% mercaptoethanol, 0.05% bromophenol blue, 75 nM Tris-HCl, 2% SDS, and 10% glycerol with pH 6.8) in a 4:1 proportion. The mixture was heated at 95 °C for 10 min and loaded into 12.5% (for OXPHOS) and 10% (for other proteins) gradient SDS-polyacrylamide gels (32 µg/lane).
After that, the protein was transferred to 0.45 µm pore size nitrocellulose membranes (GE Healthcare Bio-Sciences, MA, USA) in a glycine/methanol-transfer buffer using a Wet/Tank blotting system (Bio-Rad Laboratories, CA, USA). Membranes were blocked in 5% BSA (for PCSK9, CPT1, p-Drp1ser616, Drp1, and Mfn2) or 5% skim milk (for OXPHOS) in TBST. To detect the level of expression of the proteins the membranes were incubated with rabbit or mouse antibodies of p-Drp1ser616, total Drp1, Mfn2 (1:1000 dilution, Cell Signaling Technology, MA, USA), PCSK9, CPT1, and OXPHOS (1:1000 dilution, Abcam, Cambridge, UK). GAPDH was used as a house keeping protein (1:20,000 dilution, Abcam, Cambridge, UK). Bound antibodies were detected using horseradish peroxidase conjugated with either anti-rabbit or anti-mouse IgG

(1:2000 dilution, Cell Signaling Technology, MA, USA). The membranes were exposed to an ECL western blotting substrate (Bio-Rad Laboratories, CA, USA), and a densitometric analysis was performed using the ChemiDoc Touch Imaging system (Bio-Rad Laboratories, CA, USA), followed by ImageJ (NIH, MA, USA).
Soleus muscle MDA concentration

The high-performance liquid chromatography (HPLC) method used to evaluate MDA concentration was modified from a previous study (28). Briefly, homogenized soleus muscle tissue was mixed with 10% trichloroacetic acid containing butylated hydroxytoluene, heated at 90 °C for 30 min, and cooled down to room temperature. The mixture was centrifuged at 6,000 rpm for 10 min. The supernatant (0.5 ml) was mixed with 0.44 M H3PO4 (1.5 ml) and 0.6% thiobarbituric acid solution (1.0 ml), and then incubated at 90 °C for 30 min to achieve a pink- colored product, thiobarbituric acid reactive substances (TBARS). The solution was filtered through a syringe filter (polysulfone type membrane, pore size 0.45 µm, Whatman International, Maidstone, UK), and finally analyzed using the HPLC system. The TBARS were fractionated in the column (Water Spherosorb ODS2 type, 250×4.3 mm, 5 µm) diluted with a mobile-phase solvent of 50 mM KH2PO4 in methanol at a flow rate of 1.0 ml/min and detection was carried out at 532 nm. Data were analyzed using BDS software (BarSpec Ltd., Rehovot, Israel). A standard curve was constructed from the peak area of 1, 1, 3, 3-tetramethoxypropane at different concentrations. TBARS concentration was calculated from the standard curve and reported as the equivalent concentration of MDA (µmol/g protein).
Soleus muscle mitochondrial isolation

The mitochondrial isolation protocol was modified from a previous study (26). Briefly, soleus muscles were removed and placed in ice-cold isolation buffer containing 100 mM sucrose,

100 mM KCl, 50 mM Tris-HCl, 1 mM KH2PO4, 0.1 mM EGTA, and 0.2% BSA with pH 7.4.

Then, the tissue was finely minced, incubated for 2 min with 0.2 mg/ml of bacterial protease type XXVII (Sigma Chemical Co, MO, USA) in an isolation buffer, and then homogenized for 2 min in an ice-cooled glass homogenizer. Three volumes of isolation buffer were added, and then the suspension was centrifuged for 10 min at 700 g. The supernatant was kept and subsequently centrifuged for 10 min at 10,000 g. After that, the pellets were suspended in the isolation buffer and then centrifuged for 3 min at 7,000 g. The pellets were finally suspended in a respiration buffer containing 150 mM KCl, 5 mM HEPES, 5 mM K2HPO4.3H2O, 2 mM L-glutamate and 5 mM sodium pyruvate with a volume of 0.3 ml/1 mg of the initial tissue. Mitochondrial protein concentration was calculated using a Bicinchoninic acid assay (29).
Mitochondrial ROS measurement

The ROS was measured using a fluorescence probe and dichloro-dihydro- fluorescein- diacetate (DCFH-DA). Soleus muscle mitochondrial protein (0.4 mg/ml) was incubated with 2 mM DCFH-DA at 25 oC for 20 min. Fluorescence intensity was measured using a fluorescent microplate reader (Bio-Tek Instruments Inc., Winooski, VT) with a wavelength of 485 nm (excitation) and 530 nm (emission). A high level of fluorescent intensity indicates a high level of ROS.
Mitochondrial membrane potential assay

A change in mitochondrial membrane potential (Ơm) was measured using a cationic JC- 1 fluorescent dye. Soleus muscle mitochondrial protein (0.4 mg/ml) was incubated with 5 mM JC-1 at 37 oC for 15 min. The excitation and emission wavelengths of JC-1 monomer form
which provides green fluorescence were 485 nm and 530 nm, respectively. In contrast, the excitation and emission wavelengths of JC-1 monomer form which provides red fluorescence

were 485 nm and 590 nm, respectively. The Ơm was calculated from the red/green intensity ratio using a fluorescent microplate reader (Bio-Tek Instruments, Inc., Winooski, VT).
Mitochondrial depolarization was indicated by a reduction in this ratio.

Determination of mitochondrial swelling

Soleus muscle mitochondrial swelling was measured by the dynamic changes in absorbance of the suspension at 540 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT) (30). The absorbance at 30 min was normalized to the absorbance at 0 min and represented as a ratio. A lower ratio indicates a greater mitochondrial swelling.
Statistical analyses

GraphPad Prism version 7.00 (GraphPad Software, Inc., CA, USA) was used for statistical analyses. Data are reported as average±SEM. A one-way ANOVA was used for the comparison between all four groups. P-value < 0.05 was considered statistically significant.

Results

PCSK9 inhibitor has greater efficacy in the attenuation of obesity and hypercholesterolemia than a high dose atorvastatin.
Food intake was no different among the four groups. Body weight, visceral fat, total cholesterol, LDL cholesterol, and triglyceride levels of HFD-fed rats treated with vehicle were signficiantly increased when compared to those of ND-fed rats treated with vehicle. Although both high dose atorvastatin and PCSK9 inhibitor significantly reduced body weight, visceral fat and hypercholesterolemia in HFD-fed rats, the PCSK9 inhibitor had a greater efficacy in the

attenuation of obesity and dyslipidemia than the high dose atorvastatin (Table 3). Interestingly, HDL cholesterol levels among the four groups did not differ.
High dose atorvastatin and PCSK9 inhibitor led to equal improvement in insulin sensitiy in HFD-fed rats.
HFD-fed rats treated with vehicle developed insulin resistance, as indicated by increased fasting plasma glucose and insulin levels, HOMA-IR, and AUC of OGTT, when compared to ND-fed rats treated with vehicle (Table 3). The high dose atorvastatin and PCSK9 inhibitor restored fasting plasma insulin level, HOMA-IR, and AUC of OGTT levels in HFD-fed rats to an equal degree. The results suggest that both the high dose atorvastatin and PCSK9 inhibitor restored insulin sensitivity in HFD-fed rats. However, no alteration in fasting glucose level following the high dose atorvastatin and PCSK9 inhibitor therapies was observed.
A HFD increases PCSK9 protein expression in oxidative muscle fibers, which can be restored by both the high dose atorvastatin and PCSK9 inhibitor
The expression of PCSK9 protein in the soleus muscle of HFD-fed rats treated with vehicle was increased, when compared to that of ND-fed rats treated with vehicle. Treatment with both the high dose atorvastatin and PCSK9 inhibitor in HFD-fed rats restored the expression of PCSK9, when compared to ND-fed rats treated with vehicle (Figure 1).
HFD results in mitochondrial fatty acid overload and incomplete fatty acid oxidation in oxidative muscle fibers, which can be restored by a high dose atorvastatin and PCSK9 inhibitor.
CPT1 protein expression in the soleus muscles of HFD-fed rats treated with vehicle was greater than that of ND-fed rats treated with vehicle, suggesting increased long-chain fatty acyl CoA uptake into the mitochondria, since HFD leads to mitochondrial fatty acid overload (31).

High dose atorvastatin and PCSK9 inhibitor therapy led to an equal decrease in CPT1 protein expression to the level of ND-fed rats treated with vehicle (Figure 2A). The results suggest that both high dose atorvastatin and PCSK9 inhibitor restore mitochondrial fatty acid overload to normal levels in oxidative muscle fibers of HFD-fed rats.
Although CPT1 protein in soleus muscle was expressed at the highest level in HFD-fed rats treated with vehicle, the protein expressions of Complex I to V in oxidative phosphorylation (OXPHOS) by the muscles were not significantly different from those of ND-fed rats (Figure 2B-2F). These findings suggest that the overload of fatty acids into the mitochondria of HFD- fed rats treated with vehicle was not completely oxidized, so that no increase in OXPHOS expression was observed. Interestingly, both high dose atorvastatin and PCSK9 inhibitor led to no alteration in the OXPHOS expression in HFD-fed rats, suggesting that both high dose atorvastatin and PCSK9 inhibitor restore mitochondrial incomplete fatty acid oxidation in oxidative muscle fibers of HFD-fed rats.
HFD increases mitochondrial fission in oxidative muscle fibers, which can be restored by

high dose atorvastatin and PCSK9 inhibitor.

The ratio of p-Drp1ser616/total Drp1 protein expression in the soleus muscle of HFD-fed rats treated with vehicle was higher than that of ND-fed rats treated with vehicle, indicating increased mitochondrial fission. The high dose atorvastatin and PCSK9 inhibitor equally restored the p-Drp1ser616/total Drp1 ratio to the same level as that observed in ND-fed rats treated with vehicle (Figure 3A). The results suggest that both high dose atorvastatin and PCSK9 inhibitor restore HFD-induced mitochondrial fission. However, mitochondrial fusion, as indicated by Mfn2 protein expression, did not different among the four groups (Figure 3B).

High dose atorvastatin and PCSK9 inhibitor equally restore HFD-induced lipid peroxidation in oxidative muscle fibers.
The MDA level in the soleus muscle of HFD-fed rats treated with vehicle was greater than that of ND-fed rats treated with vehicle, indicating increased lipid peroxidation. The use of high dose atorvastatin and PCSK9 inhibitor led to an equal decrease in soleus muscle MDA to the same level of ND-fed rats treated with vehicle (Figure 3C). The results indicate that both high dose atorvastatin and PCSK9 inhibitor reduce HFD-induced lipid peroxidation to within normal limits as seen in the oxidative muscle fibers of ND fed rats.
PCSK9 inhibitor, but not high dose atorvastatin, diminishes HFD-induced mitochondrial ROS production and mitochondrial membrane depolarization in oxidative muscle fibers.
As compared with ND-fed rats treated with vehicle, HFD-fed rats treated with vehicle had a higher ROS level, but a lower red/green fluorescent intensity ratio in soleus muscle mitochondria was found. This indicated mitochondrial dysfunction via an increase in ROS production and mitochondrial membrane depolarization. Treatment with PCSK9 inhibitor restored ROS level and red/green fluorescent intensity ratio, when compared to those of ND-fed rats treated with vehicle (Figure 4A and 4B). The results suggest that the PCSK9 inhibitor improves HFD-induced mitochondrial function in oxidative muscle fibers. In contrast, treatment with high dose atorvastatin, neither altered the ROS level nor the red/green fluorescent intensity ratio (Figure 4A and 4B). The results suggest that high dose atorvastatin does not attenuate HFD-induced mitochondrial dysfunction despite its effects on weight loss and cholesterol reduction. Interestingly, mitochondrial swelling did not differ between the four groups (Figure 4C).

Discussion

The major findings of this study are that: 1) the PCSK9 inhibitor is superior to high dose atorvastatin in causing the reduction of body weight, visceral fat mass, total cholesterol, and LDL cholesterol; 2) the High dose atorvastatin and PCSK9 inhibitor equally attenuate HFD- induced insulin resistance, impaired mitochondrial lipid metabolism (as indicated by mitochondrial fatty acid overload and incomplete fatty acid oxidation), mitochondrial fission, and lipid peroxidation in oxidative muscle fibers; and 3) Interestingly, the PCSK9 inhibitor, but not the high dose atorvastatin, attenuates HFD-induced mitochondrial ROS and mitochondrial membrane depolarization in oxidative muscle fibers.
Clinical studies reported that 10-80 mg/day of atorvastatin reduces LDL cholesterol by 36-53% and total cholesterol by 15-59% (32, 33), while a PCSK9 inhibitor (alirocumab or evolocumab) lowers LDL cholesterol by 48-71%, and total cholesterol by 36-42% (1). However, studies that statistically compare the effectiveness of atorvastatin alone versus PCSK9 inhibitor alone on weight loss, insulin sensitivity, and cholesterol reduction remain limited (14). Thus, our findings may be able to act as a guide for a clinical study into the comparison between the impact of these two drugs on weight loss, insulin sensitivity, and cholesterol reduction. The present study demonstrated that the efficacy of PCSK9 inhibitor is superior to atorvastatin in the reduction of obesity and dyslipidemia.
Consistent with a previous study using serum from young women (34), this study showed that obesity is associated with increased PCSK9 protein expression in oxidative muscle fibers.
PCSK9 protein expression is significantly decreased in both the PCSK9 inhibitor, and atorvastatin-treated groups. It was not surprising to observe the reduction of PCSK9 in PCSK9 inhibitor-treated HFD-fed rats however the reduction in PCSK9 protein expression after

treatment with atorvastatin is possibly mediated by the reduction in obesity. Because previous studies in skeletal muscles demonstrated that HFD and obesity results in increased mitochondrial fatty acid overload (31), incomplete fatty acid oxidation in mitochondria (31), mitochondrial fission (35), and increased lipid peroxidation (26), the effects of a high dose atorvastatin and PCSK9 inhibitor on the attenuation of these parameters are likely mediated by the reduction in obesity and hyperlipidemia. Interestingly, those effects of both drugs are similar despite the superiority of PCSK9 inhibitor in instigating weight loss and cholesterol reduction.
Increased mitochondrial ROS and mitochondrial membrane depolarization were observed in soleus muscles of obese rats, but atorvastatin did not attenuate those effects of obesity despite its benefits regarding weight loss and cholesterol reduction. These results suggest that high dose atorvastatin itself may lead to mitochondrial oxidative stress in oxidative muscle fibers. Because low dose atorvastatin (10 mg/kg/day) did not result in oxidative stress in oxidative muscle fibers (18), it is highly possible that atorvastatin- induced oxidative stress in skeletal muscle is dose- dependent. Therefore, an increase in oxidative stress in skeletal muscles may mechanistically link to statin-induced myositis and myopathy that has been proved to be dose-dependent (36-38), and may explain why adverse effects of a PCSK9 inhibitor on skeletal muscle are less frequently observed (21). Further investigation needs to be carried out into the properties of human skeletal muscle and the impact of its association with statins, and PCSK9 inhibitors, particularly as regards skeletal muscle-related symptoms, and mitochondrial oxidative stress.
Unlike a previous study by our group (25), we did not observe any alterations in mitochondrial fusion and mitochondrial swelling in skeletal muscle of female rats fed with a HFD. The possible explanation may be the difference in the duration of HFD feeding, which was 12 weeks for this study versus 27 weeks in the previous study. Additionally, the type of

skeletal muscle that we studied was the soleus muscle which predominantly consists of oxidative muscle fibers (39) which have a high antioxidant capacity (18), whereas vastus lateralis was used in that previous study.
In conclusion, our results in rats demonstrate that PCSK9 inhibitor is superior to atorvastatin in instigating weight loss, cholesterol reduction, and attenuation of obesity-induced oxidative stress in the mitochondria in oxidative muscle fibers, whereas the restoration of insulin sensitivity, and mitochondrial lipid metabolism, and the reduction in mitochondrial fission, and lipid peroxidation in oxidative muscle fibers are similar between these two drugs. Nevertheless, future clinical studies are required to clarify whether the superiority of the PCSK9 inhibitor is exhibited in humans. If it does, the cost-effectiveness of the PCSK9 inhibitor is another issue to be considered if it is to be used as a routine lipid-lowering agent in place of the widely used statins (40).
Limitations of the study

Due to the limited amount of soleus muscle tissues and their isolated mitochondria, our study has a few limitations. First, we did not measure the activities of Complex I to V for oxidative phosphorylation. Although previous studies demonstrated that the activities and protein expressions of OXPHOS were consistent with each other (41, 42), measurement of the activities directly reflexes the capacity of oxidative phosphorylation. Second, we measured p- Drp1ser616 and total Drp1 protein expressions in the whole soleus muscle tissue, but not in the isolated mitochondria. Future studies focusing on mitochondrial protein expressions of p- Drp1ser616 and total Drp1 may exhibit greater significant difference between groups.

Acknowledgements

This work was supported by the Chiang Mai University Endowment Fund [057-2562] (CT), the Thailand Research Fund grants [MRG6280014 (CT); TRG6280005 (NA); RSA6180056 (SP); RSA6180071 (WS); RTA6080003 (SCC)], the NSTDA Research Chair
grant from the National Science and Technology Development Agency Thailand (NC), and the Chiang Mai University Center of Excellence Award (NC).

Conflicts of interest

The authors declare that there are no conflicts of interest.

References

⦁ Jellinger PS, Handelsman Y, Rosenblit PD, Bloomgarden ZT, Fonseca VA, Garber AJ, et al. AMERICAN ASSOCIATION OF CLINICAL ENDOCRINOLOGISTS AND AMERICAN COLLEGE OF ENDOCRINOLOGY GUIDELINES FOR MANAGEMENT OF DYSLIPIDEMIA AND PREVENTION OF CARDIOVASCULAR DISEASE. Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists. 2017;23(Suppl 2):1-87.
⦁ Porter C, Wall BT. Skeletal muscle mitochondrial function: is it quality or quantity that makes the difference in insulin resistance? The Journal of Physiology. 2012;590(Pt 23):5935-6.
⦁ Kelley DE, He J, Menshikova EV, Ritov VB. Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes. 2002;51(10):2944-50.
⦁ Ritov VB, Menshikova EV, He J, Ferrell RE, Goodpaster BH, Kelley DE. Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes. 2005;54(1):8-14.
⦁ Bach D, Naon D, Pich S, Soriano FX, Vega N, Rieusset J, et al. Expression of Mfn2, the Charcot-Marie-Tooth neuropathy type 2A gene, in human skeletal muscle: effects of type 2 diabetes, obesity, weight loss, and the regulatory role of tumor necrosis factor alpha and interleukin-6. Diabetes. 2005;54(9):2685-93.
⦁ Bach D, Pich S, Soriano FX, Vega N, Baumgartner B, Oriola J, et al. Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. A novel regulatory mechanism altered in obesity. The Journal of biological chemistry. 2003;278(19):17190-7.
⦁ Frontera WR, Ochala J. Skeletal muscle: a brief review of structure and function. Calcif Tissue Int. 2015;96(3):183-95.

⦁ Ivy JL, Costill DL, Maxwell BDJEJoAP, Physiology O. Skeletal muscle determinants of maximum aerobic power in man. European Journal of Applied Physiology and Occupational Physiology. 1980;44(1):1-8.
⦁ McAuley PA, Artero EG, Sui X, Lee DC, Church TS, Lavie CJ, et al. The obesity paradox, cardiorespiratory fitness, and coronary heart disease. Mayo Clin Proc. 2012;87(5):443- 51.
⦁ Ricketts TA, Sui X, Lavie CJ, Blair SN, Ross R. Addition of Cardiorespiratory Fitness Within an Obesity Risk Classification Model Identifies Men at Increased Risk of All-cause Mortality. Am J Med. 2015.
⦁ Carnethon MR, Gidding SS, Nehgme R, Sidney S, Jacobs DR, Jr., Liu K. Cardiorespiratory fitness in young adulthood and the development of cardiovascular disease risk factors. Jama. 2003;290(23):3092-100.
⦁ Grundy SM, Stone NJ, Bailey AL, Beam C, Birtcher KK, Blumenthal RS, et al. 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Journal of the American College of Cardiology. 2018.
⦁ Chaudhary R, Garg J, Shah N, Sumner A. PCSK9 inhibitors: A new era of lipid lowering therapy. World Journal of Cardiology. 2017;9(2):76-91.
⦁ Navarese EP, Kolodziejczak M, Schulze V, Gurbel PA, Tantry U, Lin Y, et al. Effects of Proprotein Convertase Subtilisin/Kexin Type 9 Antibodies in Adults With
Hypercholesterolemia: A Systematic Review and Meta-analysis. Annals of internal medicine. 2015;163(1):40-51.

⦁ Golomb BA, Evans MA. Statin Adverse Effects: A Review of the Literature and Evidence for a Mitochondrial Mechanism. American journal of cardiovascular drugs : drugs, devices, and other interventions. 2008;8(6):373-418.
⦁ Bouitbir J, Charles AL, Rasseneur L, Dufour S, Piquard F, Geny B, et al. Atorvastatin treatment reduces exercise capacities in rats: involvement of mitochondrial impairments and oxidative stress. Journal of applied physiology (Bethesda, Md : 1985). 2011;111(5):1477-83.
⦁ Bouitbir J, Daussin F, Charles AL, Rasseneur L, Dufour S, Richard R, et al.

Mitochondria of trained skeletal muscle are protected from deleterious effects of statins. Muscle & nerve. 2012;46(3):367-73.
⦁ Bouitbir J, Singh F, Charles AL, Schlagowski AI, Bonifacio A, Echaniz-Laguna A, et al.

Statins Trigger Mitochondrial Reactive Oxygen Species-Induced Apoptosis in Glycolytic Skeletal Muscle. Antioxidants & redox signaling. 2016;24(2):84-98.
⦁ Herbison GJ, Jaweed MM, Ditunno JF. Muscle fiber types. Archives of physical medicine and rehabilitation. 1982;63(5):227-30.
⦁ Atanda A, Shapiro NL, Stubbings J, Groo V. Implementation of a New Clinic-Based, Pharmacist-Managed PCSK9 Inhibitor Consultation Service. Journal of managed care & specialty pharmacy. 2017;23(9):918-25.
⦁ Moriarty PM, Thompson PD, Cannon CP, Guyton JR, Bergeron J, Zieve FJ, et al.

Efficacy and safety of alirocumab vs ezetimibe in statin-intolerant patients, with a statin rechallenge arm: The ODYSSEY ALTERNATIVE randomized trial. Journal of clinical lipidology. 2015;9(6):758-69.

⦁ Blom DJ, Hala T, Bolognese M, Lillestol MJ, Toth PD, Burgess L, et al. A 52-week placebo-controlled trial of evolocumab in hyperlipidemia. The New England journal of medicine. 2014;370(19):1809-19.
⦁ Zaleski AL, Taylor BA, Pescatello LS, Dornelas EA, White CM, Thompson PD. Influence of Baseline Psychological Health on Muscle Pain During Atorvastatin Treatment. The Journal of cardiovascular nursing. 2017;32(6):544-50.
⦁ Couto M. Laboratory guidelines for animal care. Methods in molecular biology (Clifton, NJ). 2011;770:579-99.
⦁ Pattanakuhar S, Sutham W, Sripetchwandee J, Minta W, Mantor D, Palee S, et al.

Combined exercise and calorie restriction therapies restore contractile and mitochondrial functions in skeletal muscle of obese-insulin resistant rats. Nutrition (Burbank, Los Angeles County, Calif). 2019;62:74-84.
⦁ Sutham W, Sripetchwandee J, Minta W, Mantor D, Pattanakuhar S, Palee S, et al.

Ovariectomy and obesity have equal impact in causing mitochondrial dysfunction and impaired skeletal muscle contraction in rats. Menopause (New York, NY). 2018.
⦁ Haffner SM, Miettinen H, Stern MP. The homeostasis model in the San Antonio Heart Study. Diabetes care. 1997;20(7):1087-92.
⦁ Pintana H, Apaijai N, Chattipakorn N, Chattipakorn SC. DPP-4 inhibitors improve cognition and brain mitochondrial function of insulin-resistant rats. The Journal of endocrinology. 2013;218(1):1-11.
⦁ Thummasorn S, Kumfu S, Chattipakorn S, Chattipakorn N. Granulocyte-colony stimulating factor attenuates mitochondrial dysfunction induced by oxidative stress in cardiac mitochondria. Mitochondrion.11(3):457-66.

⦁ Ittichaicharoen J, Apaijai N, Tanajak P, Sa-Nguanmoo P, Chattipakorn N, Chattipakorn

S. Dipeptidyl peptidase-4 inhibitor enhances restoration of salivary glands impaired by obese- insulin resistance. Archives of oral biology. 2018;85:148-53.
⦁ Koves TR, Ussher JR, Noland RC, Slentz D, Mosedale M, Ilkayeva O, et al.

Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. Cell metabolism. 2008;7(1):45-56.
⦁ Adams SP, Tsang M, Wright JM. Lipid-lowering efficacy of atorvastatin. The Cochrane database of systematic reviews. 2015(3):Cd008226.
⦁ Adams SP, Tsang M, Wright JM. Lipid lowering efficacy of atorvastatin. The Cochrane database of systematic reviews. 2012;12:Cd008226.
⦁ Levenson AE, Shah AS, Khoury PR, Kimball TR, Urbina EM, de Ferranti SD, et al.

Obesity and type 2 diabetes are associated with elevated PCSK9 levels in young women. Pediatric diabetes. 2017;18(8):755-60.
⦁ Lahera V, de Las Heras N, Lopez-Farre A, Manucha W, Ferder L. Role of Mitochondrial Dysfunction in Hypertension and Obesity. Current hypertension reports. 2017;19(2):11.
⦁ Mareedu RK, Modhia FM, Kanin EI, Linneman JG, Kitchner T, McCarty CA, et al. Use of an electronic medical record to characterize cases of intermediate statin-induced muscle toxicity. Preventive cardiology. 2009;12(2):88-94.
⦁ Wilke RA, Lin DW, Roden DM, Watkins PB, Flockhart D, Zineh I, et al. Identifying genetic risk factors for serious adverse drug reactions: current progress and challenges. Nature reviews Drug discovery. 2007;6(11):904-16.
⦁ Egan A, Colman E. Weighing the benefits of high-dose simvastatin against the risk of myopathy. The New England journal of medicine. 2011;365(4):285-7.

⦁ Cornachione AS, Benedini-Elias PC, Polizello JC, Carvalho LC, Mattiello-Sverzut AC. Characterization of fiber types in different muscles of the hindlimb in female weanling and adult Wistar rats. Acta histochemica et cytochemica. 2011;44(2):43-50.
⦁ Svatikova A, Kopecky SL. Cholesterol Management in the Era of PCSK9 Inhibitors.

Current cardiology reports. 2017;19(9):83.

⦁ Lapel M, Weston P, Strassheim D, Karoor V, Burns N, Lyubchenko T, et al. Glycolysis and oxidative phosphorylation are essential for purinergic receptor-mediated angiogenic responses in vasa vasorum endothelial cells. Am J Physiol Cell Physiol. 2017;312(1):C56-c70.
⦁ Thrush AB, Zhang R, Chen W, Seifert EL, Quizi JK, McPherson R, et al. Lower mitochondrial proton leak and decreased glutathione redox in primary muscle cells of obese diet- resistant versus diet-sensitive humans. The Journal of clinical endocrinology and metabolism. 2014;99(11):4223-30.
⦁ Pratchayasakul W, Kerdphoo S, Petsophonsakul P, Pongchaidecha A, Chattipakorn N, Chattipakorn SC. Effects of high-fat diet on insulin receptor function in rat hippocampus and the level of neuronal corticosterone. Life sciences. 2011;88(13-14):619-27.

Figure legends

Figure 1: PCSK9 protein expression in the soleus muscle

n = 5-6/group; † p < 0.05 compared to the NDV group; ‡ p < 0.05 compared to the HFV group; NDV: normal diet with vehicle; HFV: high-fat diet with vehicle; HFA: high-fat diet with atorvastatin; HFP: high-fat diet with PCSK9 inhibitor. Data are reported as average ± SEM.
Figure 2: CPT1 and OXPHOS protein expression in soleus muscle; 2A: CPT1; 2B: Complex I; 2C: Complex II; 2D: Complex III; 2E: Complex IV; 2F: Complex V.
n = 5-6/group; † p < 0.05 compared to the NDV group; ‡ p < 0.05 compared to the HFV group; NDV: normal diet with vehicle; HFV: high-fat diet with vehicle; HFA: high-fat diet with atorvastatin; HFP: high-fat diet with PCSK9 inhibitor. Data are reported as average ± SEM.
Figure 3: The ratio of p-Drp1/total Drp1 and Mfn2 protein expression (3A and 3B) and MDA level (3C) in the soleus muscle
n = 5-6/group; † p < 0.05 compared to the NDV group; ‡ p < 0.05 compared to the HFV group; NDV: normal diet with vehicle; HFV: high-fat diet with vehicle; HFA: high-fat diet with atorvastatin; HFP: high-fat diet with PCSK9 inhibitor. Data are reported as average ± SEM.
Figure 4: Analysis of soleus muscle mitochondrial isolation. 4A: mitochondrial ROS level; 4B: red/green fluorescent intensity ratio; 4C: normalized absorbance at λ 540 nm;
n = 5-6/group; † p < 0.05 compared to the NDV group; ‡ p < 0.05 compared to the HFV group;

§ p < 0.05 compared to the HFA group; NDV: normal diet with vehicle; HFV: high-fat diet with vehicle; HFA: high-fat diet with atorvastatin; HFP: high-fat diet with PCSK9 inhibitor. Data are reported as average ± SEM.

Table 1: Composition of a normal diet

Normal diet
%E g kcal
Carbohydrate 51.99 495.30 1981.20
Protein 28.24 269.00 1076.00
Fat 19.77 83.70 753.30
Vitamins - 65.40 -
Fiber - 34.30 -
Total 100 947.70 3810.50
Kcal/g 4.02 kcal/g

Composition

Table 2: Composition of a high-fat diet (43)

High-fat diet
%E g kcal
Carbohydrate 14.27 190.76 763.04
Protein 26.45 353.60 1414.40
Fat 57.60 342.24 3080.16
Cholesterol 1.68 10 90
Vitamins - 85.19 -
DL-Methionine - 3 -
Sodium chloride - 1 -
Yeast powder - 1 -
Fiber - 13.21 -
Total 100 1000 5347.60
Kcal/g 5.35 kcal/g

Composition

Table 3: Anthropometric and metabolic parameters

Parameters NDV HFV HFA HFP
Food intake (g/day) 12.74±0.38 12.43±0.52 12.64±0.59 12.52±0.66
Body weight (g) 270.56±5.98 325.27±9.62† 296.46±2.60†‡ 277.19±4.39‡§
Visceral fat weight (g) 10.63±1.25 23.68±1.50† 19.30±2.10†‡ 14.36±0.58‡§
Fasting glucose (mg/dl) 155.15±20.16 236.10±23.48† 240.41±19.17† 230.39±17.11†
Fasting insulin (ng/ml) 2.25±0.65 6.22±1.05† 3.41±0.75‡ 3.55±0.56‡
HOMA-IR 29.70±3.19 93.70±6.42† 45.84±5.47‡ 43.60±6.60‡
AUC of glucose

((mg/dl/min) x 104) 1.23 53±0.11 2.02±0.15† 1.46±0.14‡ 1.42±0.14‡
Total cholesterol 127.22±5.03 293.48±6.34† 223.91±16.37†‡ 163.30±13.45†‡§
(mg/dl)
LDL cholesterol 90.60±7.23 238.97±16.37† 190.18±17.92†‡ 118.36±11.83‡§
(mg/dl)
HDL cholesterol 29.03±2.70 29.25±2.91 29.07±1.46 29.37±0.46
(mg/dl)
Triglycerides (mg/dl) 37.96±5.53 66.51±8.25† 66.83±6.54† 65.13±4.91†

Data are expressed as mean± SEM. n = 5-6/group; † p < 0.05 compared to the NDV group; ‡ p <
0.05 compared to the HFV group; § p < 0.05 compared to the HFA group
Abbreviations: NDV: normal diet with vehicle; HFV: high-fat diet with vehicle; HFA: high-fat diet with atorvastatin; HFP: high- fat diet with PCSK9 inhibitor.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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⦁ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Highlights

⦁ PCSK9 inhibitor is superior to high dose atorvastatin in cholesterol reduction.

⦁ PCSK9 inhibitor is superior to high dose atorvastatin in weight loss.

⦁ Only PCSK9 inhibitor improves ROS production in oxidative muscle mitochondria.

⦁ Only PCSK9 inhibitor improves membrane potential in oxidative muscle mitochondria.