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. 2023 Oct 24;13(11):377. doi: 10.1007/s13205-023-03784-9

Sesamol combats diabetogenic effects of atorvastatin through GLUT-4 expression and improved pancreatic viability

Raghuvir Keni 1,#, Pawan Ganesh Nayak 1,#, Nitesh Kumar 1,2, Anoop Kishore 1, Sulaiman Mohammed Alnasser 3, Farmiza Begum 1, Karthik Gourishetti 1, Krishnadas Nandakumar 1,✉
PMCID: PMC10597939  PMID: 37885753

Abstract

Statin-associated diabetes (SAD) is an issue that has come to light after a series of recent clinical trials that has led to the issue of a black box warning for statins by the US FDA. However, the benefit of statin outweighs its risk. Nevertheless, experiments have been conducted to identify the mechanism by which statins aggravate the risk of diabetes only in a select population who bear the risk factors of obesity, sedentary lifestyle, hypertension, and other associated risk factors of lifestyle disorders. In this study, the possibility of utilization of a phyto-molecule, sesamol, for its ability to combat statin-associated diabetes using atorvastatin as the agent of choice has been explored. MMP assay and western blot was conducted to investigate the effects of atorvastatin on apoptotic cascade with sesamol as a protective agent was conducted in MIN-6 cells. Effect of the combination was tested in L6 cells with 2-NBDG uptake assay and as well as western blot for GLUT-4. A diet-induced hypercholesterolemia model was developed in an in vivo model animals and treated with atorvastatin and sesamol with histopathological analysis being carried out to evaluate the apoptotic markers and GLUT-4 presence. It was found that sesamol can combat pancreatic beta cell apoptosis via the internal apoptotic pathway activated by atorvastatin. With regards to muscle cells, sesamol could improve the GLUT-4 vesical production, but not improve glucose uptake which is inhibited by atorvastatin. These findings are further confirmed by animal studies. These findings indicate that sesamol can serve as a prototype molecule for further development and investigation of similar compounds to tackle SAD.

Keywords: Drug-induced diabetes, Atorvastatin, Statin, New-onset diabetes

Introduction

Statin-associated diabetes (SAD) is an adverse effect that is found to predominantly occur in those individuals who have some pre-existing risk factors to develop diabetes, such as being prediabetic, suffering from obesity and impaired glucose tolerance (Ganda 2016). Several theories have been proposed as to how statins, a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitor, can cause diabetes, some of which have been proven and an in-depth discussion on this subject can be found in our previous article (Keni et al. 2021).

Though the benefits that the family of statin provide outweigh their risks, expert opinion has questioned the safety of statins as reports have brought to light its adverse effects including but not limited to rhabdomyolysis, myalgia, asymptomatic liver injury, erectile dysfunction with the major one being statin-associated diabetes (Mancini et al. 2011).

Based on this knowledge, we hypothesised that it may be possible to use certain natural compounds that have been investigated for a variety of effects on diabetic cells or to manage diabetes. Since our lab has had some previous experience with one such compound, sesamol, we proceeded with the exploration of the same (Gourishetti et al. 2020; Shenoy et al. 2011a). Sesamol is an isolated phyto-molecule from sesame seeds (Sesamum indicum L.), available commercially. The seeds are popularly used in India for the production of oil used in indigenous cuisine.

Sesamol was found to have anticlastogenic properties and promoting delayed wound healing in normal (Shenoy et al. 2011b) and in diabetic rats (Gourishetti et al. 2020) based on existing research. Furthermore, it also demonstrated anti-hyperlipidaemic activity in chronic and acute models of hyperlipidaemia (Kumar et al. 2013a). This has provided the rational to explore the avenues of statin-induced diabetes where this compound may prove beneficial. Diabetes can occur, broadly with a combination of three mechanisms, i.e., due to dysfunction or loss of pancreatic beta cells, development of insulin resistance at muscle cells and inflammation/resistance at adipose tissue. The mechanisms of statin-associated diabetes is closely tied in with these three mechanisms. This was previously demonstrated by Urbano et al. (Urbano et al. 2017) that lipophilic statin such as atorvastatin were associated with a dose and potency dependant dysfunction of insulin secreting cells causing mitochondrial dysfunction. Other research demonstrated additional mechanisms by which beta cell dysfunction was observed due to the inhibition of L-type calcium channels in rat islet beta cells as shown by Yada et al. (1999) and inhibition of GLUT-2 expression by Zhou et al. in MIN6 cells (Zhou et al. 2014). Metz et al. (1993) demonstrated that statins caused post-translational modifications of GTP-binding altering in normal rat islets. Furthermore, in muscle cells and adipocytes it was found that statins caused impairment of GLUT-4 synthesis and GLUT-4 vesicle translocation (Jiang et al. 2016; Li et al. 2016; Ganesan and Iko 2013) and an impairment of glucose uptake at muscle cells (Kain et al. 2015).

Based on these findings, the protection conferred by sesamol against atorvastatin-induced mitochondrial dysfunction and prevention of apoptosis in MIN6 cells, against atorvastatin-induced impairment of glucose uptake in L6 cells and later with in vivo studies has been investigated.

Methods

MIN6 cell culture

MIN6 cells were seeded into 12-well plate (1 × 105) for flow cytometry and 60 mm dishes (8 × 105) for western blot using DMEM (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) containing heat inactivated 15% FBS (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), 1% antibiotic–antimycotic solution (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1× β-mercaptoethanol (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) in an atmosphere of 5% CO2 and 95% humidity at 37 °C. Media was changed every 48 h.

L6 cell culture

Cells were seeded in 60 mm (8 × 105) dishes and were maintained using DMEM (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) with 10% FBS (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), and 1% antibiotic–antimycotic solution (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) until confluency. When the dishes were about 70% confluent, differentiation was initiated with 2% horse serum containing DMEM and 1% antibiotic–antimycotic. Media was changed every 48 h. Differentiation was carried out for 7 days until multinucleated cells were seen indicating that the myoblasts are converted to myotubes. Cells were maintained in an atmosphere of 5% CO2 and 95% air at 37 °C.

Treatment

100 mM stock of atorvastatin (gift sample from Amor Organics Pvt. Ltd., Hyderabad, Telangana, India), and sesamol (Sigma–Aldrich Inc., St. Louis, MO, USA) was prepared in DMSO (MP Biomedicals, Solon, OH, USA) from which working stock was prepared as required from added to the culture with pretreatment of the culture with sesamol for 12 h followed by addition of atorvastatin for 6 h.

In case of L6 cells, after the above treatment period, cells were starved with glucose-free and FBS-free DMEM for 1 h followed by addition of 100 nM insulin (Actrapid®, Novo Nordisk India Pvt. Ltd., Bengaluru, Karnataka, India) for 30 min. After which cells were collected and processed as required for downstream analysis.

Mitochondrial membrane potential assay

MIN6 cells were cultured and treated as described above following which the cells were then trypsinized and collected into 1.5 ml centrifuge tubes. The cells were washed with PBS and were resuspended in 200 µL of the JC-1 solution (2 µM) (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) and incubated at 37 ℃ for 30 min in the dark. The cells were washed and resuspended in FACS buffer (PBS with 1% FBS) and 10,000 events were captured using BD Accuri C6 flow cytometer (Becton and Dickinson, Franklin Lakes, NJ, USA).

2-NBDG uptake assay

After the treatment period, cells were starved with glucose-free and FBS-free DMEM for 1 h followed by addition of 100 nM insulin (Actrapid®, Novo Nordisk India Pvt. Ltd., Bengaluru, India) for 30 min with 10 µM of 2-NBDG (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA). Phloretin was used as uptake inhibitor control. The cells were then trypsinized, collected, and washed and glucose uptake was estimated using BD Accuri C6 flow cytometer (Becton and Dickinson, Franklin Lakes, NJ, USA). Entire procedure was carried out in dark.

Vertical SDS–PAGE and western blotting

The media was discarded, and dishes washed with ice cold PBS twice to remove traces of media while maintaining dishes on ice throughout the entire process. Cells were scraped using RIPA buffer with phosphatase and protease inhibitors. The lysate was centrifuged (Mikro 22, Hettich Holding GmbH & Co. oHG, Westphalia, Kirchlengern, Germany) at 10,000 RPM for 10 min at 4 °C. The supernatant was collected, and protein estimation was carried out using Pierce BCA Protein Assay Kit (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).

SDS–PAGE was carried out using 10% polyacrylamide (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) and protein was transferred onto PVDF membrane (Pall Corporation, Port Washington, NY, USA) blocked with 5% non-fat milk (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membrane was probed with primary antibodies, namely BAK, Bcl-xL, caspase-3, cytochrome C, tubulin, GLUT-4, (Elabscience Inc., Wuhan, Hubei, China), caspase-7, BAD (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA), overnight diluted with 5% bovine serum albumin (Sisco Research Laboratories Pvt. Ltd., Maharashtra, India). After washing, the membrane was then probed using HRP conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) in 5% blotting-grade blocker. The blot was then developed using ECL reagent. Imaging was done using G:BOX Chemi XRQ gel doc system (Syngene, Cambridge, UK) and analysis done using Image J 1.47 (National Institutes of Health, Bethesda, MD, USA).

In vivo studies

The animal experimentation was done in line with ethical guidelines laid out as per CPCSEA. Protocol was reviewed and approved by Institutional Animal Ethics Committee (IAEC), Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India (IAEC/KMC/61/2021). The animals were acquired and allowed to acclimatize at 24–26 ℃ room temperature maintained in an air-conditioned room with 12 h dark and 12 h light cycle. Animals had free access to normal chow diet and water ad libitum.

Induction of prediabetic state

The rats were randomly divided in to two groups: one is control (n = 6) and the other being prediabetic group (n = 18). The control group was fed with standard chow diet and prediabetic group was fed with in-house high fat diet–HFD (58% calories as fat) (Srinivasan et al. 2004) for a period of 12 weeks. After completion of 12 weeks, the animals were subjected to OGTT to confirm the development of a prediabetic state in the form of insulin resistance. Similarly, HbA1c was also estimated by kit method. Thereon, the HFD feeding was continued till the termination of the study, a total of 24 weeks.

Oral glucose tolerance test (OGTT)

After 12 weeks, animals on HFD and normal pellet diet were fasted overnight for not more than 6 h. The following morning, rats were given oral glucose at a dose of 2 g/kg body weight and with the help of handheld glucometer (Alere G1, Abbott, IL, USA) the blood glucose levels were estimated. Readings were taken before and after 15, 30, 60, 90 and 120 min of the glucose administration. This helped identify development of a prediabetic state/insulin resistance. The procedure was also repeated after 24 weeks that is at the end of the study following the oral treatment with atorvastatin and sesamol.

Administration of drugs and animal grouping

After confirmation of development of a prediabetic state with OGTT and HbA1c, the animals were regrouped with six animals in each labelled as Normal pellet diet control (NPD control), High fat diet control (HFD control), Atorvastatin (+ HFD), Atorvastatin + Sesamol (+ HFD). Other than NPD control group, high fat diet was continued for all other groups and thus indicated by (+ HFD). Based on existing literature, atorvastatin and sesamol was administered at a dose of 30 mg/kg and 25 mg/kg, respectively, daily in 0.1% CMC (Wong et al. 2006; Vichai and Kirtikara 2006; Shenoy et al. 2011a, 2011b; Kondamudi et al. 2013; Kumar et al. 2013a, 2013b; Shu et al. 2016; Gourishetti et al. 2020). At the completion of 24 weeks, animals were scarified with high dose ketamine (360 mg/kg)/xylazine (40 mg/kg) combination followed by cervical dislocation, and the pancreas/gastrocnemius muscle was isolated for histopathology in neutral buffered formalin.

Haematoxylin and eosin staining

Tissue fixed in formalin was embedded in paraffin and 4 µM sections were cut with rotary microtome. The tissue was dewaxed with xylene and hydrated with reducing concentration of ethanol. The tissue was then stained with haematoxylin and eosin and dehydrated again and placed under a coverslip with mounting agent.

Immunohistochemistry

Millipore IHC Select HRP/DAB kit (Merck KGaA, Darmstadt, Germany) was used, and the prescribed kit procedures were followed.

Briefly, tissue fixed in neutral buffered formalin were taken and embedded in paraffin. With the help of microtome, 4-µM thin sections were made, following which standard dewaxing process was followed in xylene and rehydrated using reducing concentration of ethanol. The slides were than washed and antigen retrieval done using Tris–EDTA buffer (pH 9.0). Any endogenous peroxidase activity was removed with hydrogen peroxide solution (3%). The sections were blocked and then incubated with primary antibody of choice and washed. HRP-labelled secondary antibody was applied and washed. Slides were developed by incubating the sections with diaminobenzidine tetrahydrochloride (DAB). Haematoxylin was used as counterstain and coverslip placed with ImmunoHistoMount (Merck KGaA, Darmstadt, Germany).

Images were captured with LX-500 LED trinocular research microscope (Labomed, Gurgaon, Haryana, India) and with MiaCam CMOS AR 6Pro microscope camera connected to image AR Pro software.

Statistical analysis

Analysis was done by one-way ANOVA with Dunnett post hoc test or unpaired two tailed t test as applicable, using Prism (Version 9.3.0. Trial, GraphPad Software, San Diego, CA, USA) and all experiments were conducted in triplicates.

Results

The mitochondrial membrane potential (MMP) of the group treated with atorvastatin only, at 200 µM, was found to be reduced (monomer proportion increasing) to a statistically significant level (p < 0.05) when compared to the normal group. The groups treated with atorvastatin + sesamol was not able to restore the MMP to levels comparable to that of control. This reduction was found to be significant at p < 0.05 when compared to normal group and when compared to the group exposed to atorvastatin only (Fig. 1).

Fig. 1.

Fig. 1

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Mitochondrial membrane potential analysis MIN6 cells treated with atorvastatin and sesamol. The group treated with atorvastatin only was found to be reduced when compared to the normal group. The groups treated with atorvastatin + sesamol was not able to restore the MMP to levels comparable to that of control. Data represented as mean ± SEM where *p < 0.05, when compared to the control group and #p < 0.05 when compared to atorvastatin group

In the western blot analysis (Fig. 2), the atorvastatin (200 µM) exposed group there was an increase in the apoptotic markers in MIN6 cells, namely, cytochrome C, BAK, caspase-7 and capsase-8 as well as Bcl-xL, all of which were found to be significant at p < 0.05 when compared to control.

Fig. 2.

Fig. 2

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Western blot analysis of apoptotic markers in MIN6 cell treated with atorvastatin and sesamol. Data are represented as mean ± SEM *p < 0.05, when compared to the with insulin group and #p < 0.05 when compared to atorvastatin group

Sesamol at 10 µM in combination with atorvastatin caused an increase in cytochrome C, but with further increase in concentration of sesamol up to 100 µM, reduction in cytochrome C was observed. A similar trend was seen with caspase-8. The observation with both these markers cytochrome C and caspase-8 were found to be significant at p < 0.05 when compared to normal group as well as with the group exposed to atorvastatin only.

The markers, Bcl-xL and caspase-7 displayed a trend where in with increasing concentration of sesamol from 10 µM to 100 µM in combination with atorvastatin, a reducing expression of the respective markers was observed, such that their expression fell below that of control. This was found to be significant at p < 0.05 when compared to normal group as well as with the group exposed to atorvastatin only. For the apoptotic marker BAK, with an increasing concentration of sesamol from 10 µM to 100 µM in combination with atorvastatin, an increase in the expression was seen, found significant at p < 0.05 when compared to normal group as well as with the group exposed to atorvastatin only. At all concentrations of sesamol, the expression of BAK was lower as compared to the group treated with atorvastatin only and when sesamol was at 50 µM or below, the expression was even lower than the control group.

In Fig. 3, the results of the 2-NBDG uptake assay in L6 cells are displayed. Atorvastatin at a concentration of 50 µM displayed a reduction in the insulin (100 nM) stimulated uptake. Sesamol did not display much effect on this reduced uptake brought upon by atorvastatin. These observations were found to be significant at p < 0.05 when compared to control but not when compared to the group treated with atorvastatin only.

Fig. 3.

Fig. 3

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The 2-NBDG glucose uptake assay outcome with L6 cells treated with atorvastatin and sesamol. Atorvastatin at a concentration of 50 µM displayed a reduction in the insulin (100 nM) stimulated uptake. Sesamol was unsuccessful in the preservation of glucose uptake. Data are represented as mean ± SEM *p < 0.05, when compared to the with insulin group and #p < 0.05 when compared to atorvastatin group

In the western blot analysis of GLUT-4 in L6 cells (Fig. 4), it is identified that exposure to atorvastatin at 50 µM caused a noticeable reduction in the expression of GLUT-4 on being challenged with insulin. This reduction was similar to the control unchallenged by insulin. Whereas in the presence of sesamol, all concentrations in combination with atorvastatin increased the expression of GLUT-4. However, the observations of sesamol at the concentration of 10 µM and 25 µM only were found to be statistically significant at p < 0.05 when compared to the group treated with atorvastatin only.

Fig. 4.

Fig. 4

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The western blot analysis of GLUT-4 in L6 cells treated with atorvastatin 50 µM and sesamol. Data are represented as mean ± SEM at #p < 0.05, when compared to the atorvastatin group

In the animal studies performed, after 12 weeks on high fat diet, the animals developed tolerance to glucose uptake as seen in Fig. 5 bearing a higher area under the curve with respect to NPD control group. HbA1c test results helped to further confirm this outcome. The group fed with high fat diet had higher glycosylated reading of 6.6% which was in the prediabetic range, and the NPD control group had a reading of 5.1% which was in the normal range. In addition, the development of high total cholesterol and serum triglycerides was also seen in the group on high fat diet. Following this, the treatment of the animals with atorvastatin and sesamol was initiated for the next 12 weeks.

Fig. 5.

Fig. 5

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The AUC of the OGTT plot and HbA1c values of animals after 12 weeks on high fat diet, the animals had increased cholesterol levels, tolerance to glucose uptake and were prediabetic. Data are represented as mean ± SEM, found significant at *p < 0.05 when compared to the NPD group

At the completion of this additional 12-week period, from Fig. 6, the percentage weight gain is seen over the entire 24-week period. The groups fed on HFD displayed a prominent increase in body weight, due to the high calorie diet. Similarly, the atorvastatin (+ HFD) group also had weight gain. The atorvastatin + sesamol (+ HFD) group displayed a moderate increase in weight gain as compared to the other groups, specifically after initiation of treatment. The animals on normal chow had a lower percentage increase in body weight.

Fig. 6.

Fig. 6

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All groups treated with HFD were able to display weight gain progressively over the duration of the 24-week period

On performing OGTT, it was noticed that the HFD control and atorvastatin (+ HFD) group had a similar area under the curve, which was significantly higher when compared to the NPD control group, and the atorvastatin + sesamol (+ HFD) group did not have much significant reduction in AUC. At the same time, the increase in cholesterol and triglyceride remained high in the HFD control group, whereas in the atorvastatin treated groups with and without administration of sesamol a similar reduction was observed (Fig. 7). The HbA1c readings also indicated that the HFD control and atorvastatin (+ HFD) treated group had similar HbA1c readings of 6.24% and 6.33%, respectively, whereas the NPD control had 5.14%, and the sesamol treated group had 5.81%. Thus, atorvastatin + sesamol (+ HFD) group did have a reduction in glycosylated haemoglobin content though it remained in the prediabetic range.

Fig. 7.

Fig. 7

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The AUC of OGTT plot and HbA1c values at the end of 24 weeks as well as the total cholesterol and triglyceride levels. Data are represented as mean ± SEM at *p < 0.05, when compared to the NPD control group and #p < 0.05, when compared to the atorvastatin (+ HFD) group

Histopathological analysis of all the slides belonging to NPD control indicated that the pancreas tissue consisting of lobules separated by connective tissue septae. The lobules consisted of exocrine acinar cells (Fig. 8). The endocrine islets of Langerhans are embedded within the acinar cells.

Fig. 8.

Fig. 8

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Histological and IHC images of pancreas and muscle tissue, conducted after the completion 24 weeks, captured at 40× magnification. The HFD control has shrinkage and lower number of the pancreatic islets and indication of inflammatory infiltration. Tissue belonging to the atorvastatin (+ HFD) group also showed inflammatory infiltration with diffuse islets. Whereas the tissue of the sesamol treated group has near normal pathology. Similarly, the atorvastatin + sesamol (+ HFD) tissue section showed muscle fibres with reduced fat cells compared with HFD control group, and mild reduction of displacement of nuclei was seen compared with atorvastatin (+ HFD) group

When compared with NPD control group, the slides of the HFD control showed reduction in the size of islets and reduction in number of cells in the islets. Chronic (lymphocyte) inflammatory infiltration was identified in the acini of the pancreas. Few acute inflammatory cells in the acini and degenerative changes like the presence of vacuoles in the islets was also noted. Tissue belonging to the atorvastatin (+ HFD) group similarly also showed inflammatory infiltration with diffuse islets. Group treated with sesamol both displayed tissue conditions similar to NPD control group, i.e., the tissue was healthy.

Histopathological analysis of the muscle tissue belonging to the NPD control showed muscle fibres with multiple peripherally located elongated nuclei. Transverse striations were seen in all muscle fibres. The HFD control group had muscle fibres with increased interstitial connective tissue compared to NPD control group. The interstitial connective tissue also had many fat cells and congested blood vessels. Many areas of muscle fibres had a displaced nucleus to centre compared with NPD control group. Signs of mild chronic inflammatory infiltration was also seen. In the atorvastatin (+ HFD) tissue section, muscle fibres with reduced interstitial connective tissue and fat cells were seen when compared with HFD control group. More nuclei were displaced to the centre as compared with HFD control group. No inflammatory infiltration was seen. The atorvastatin + sesamol (+ HFD) tissue section showed muscle fibres with reduced fat cells compared with HFD control group, and mild reduction of displacement of nuclei was seen compared with atorvastatin (+ HFD) group.

The immunohistochemistry analysis was conducted with DAB and haematoxylin using optical microscopy to capture the images of the stained pancreas and muscle tissue as seen in Fig. 8, for caspase-7 and GLUT-4, respectively. In the pancreatic tissue it was observed the presence of caspase-7 by the distinct development of brown colour, against the purple blue colour of the surrounding counterstained cells. In case of normal control, there was a complete lack of any brown cells, indicating no apoptotic activity. In the HFD control group, occasional brown colour was seen as locations of apoptotic activity. In the atorvastatin treated group also, the brown-coloured cells were distinct and prominently stained. In the atorvastatin + sesamol (+ HFD) group, there was no prominent locations of brown cells though it presents very occasionally in very diffused form. In the muscle tissue, the vertical sections of the control group displayed significant brown colouration, an indication of good and pronounced presence of GLUT-4, which was also pronounced in the HFD control. Whereas in case of the atorvastatin (+ HFD) group, there was significantly less visibility of GLUT-4. In case of atorvastatin + sesamol (+ HFD) treated group, scattered patches of regions were observed occasionally with no brown colouration, with most of the tissue having good presence of GLUT-4.

Discussion

Using SRB assay (Vichai and Kirtikara 2006), the ideal concentration of atorvastatin and sesamol for experiments were identified and further optimised for the studies conducted here to the degree where in it would induce functional inhibition (data not shown) in MIN6 and L6 cell lines. Accordingly, the experiments were carried out based on workflow already established in the lab.

In a previously published study (Ghadge et al. 2020), involving the use of sesamol in MIN6 to protect against the effects of simvastatin, we had found that sesamol was able to maintain the MMP when used with simvastatin. However, here with atorvastatin, it was observed that sesamol was unable to protect against MMP loss brought upon by the treatment of MIN6 cells with atorvastatin (Fig. 1). Pre-treatment of MIN6 cells with graded doses of sesamol did not demonstrate any benefit in restoration of the membrane potential to its normal levels. This brings to light the fact that sesamol may be offering protection against MMP loss based on the type of statin utilized. Thus, we probed further to identify if the phyto-molecule was acting through some other means which involved analysing the apoptotic pathway.

It was noted that MIN6 cells treated with atorvastatin resulted in an increase in Bcl-xL anti-apoptotic protein (Fig. 2). This may be a compensatory mechanism and would require further investigation to confirm it. Though there is an increase in Bcl-xL which inhibits BAK, it was found find that atorvastatin treated cells displayed an increase in BAK which is a mitochondrial outer membrane permeabilization protein (MOMP), thus resulting in the release of cytochrome C from the mitochondria, of which the levels were also found to be increased, eventually causing the activation of executioner caspase-7. Caspase-8 was also elevated which is an initiator caspase and is activated via external triggers of apoptosis. Thus, indicating that atorvastatin causes apoptosis by both extrinsic and intrinsic apoptotic cascade. Figure 9 explains the apoptotic actions of the statin in better detail.

Fig. 9.

Fig. 9

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The action of atorvastatin and sesamol in the apoptotic pathway in pancreatic beta cells

In those groups treated with both sesamol and atorvastatin, it was found that sesamol worked by causing a significant reduction in the levels of BAK protein, thus lowering the levels of cytochrome C and caspase-7. The effect of sesamol on BAK appeared in an inverse dose dependant manner, where 10 µM of sesamol caused the maximum suppression of BAK and 100 µM caused the least suppression though still significantly lower than the group treated with atorvastatin alone. However, its downstream effects on caspase-7 displayed a celling effect at 25 µM of sesamol and the reduction of caspase-7 was below baseline levels with respect to control. It must be pointed out that sesamol did not display a reasonable effect against caspase-8, which may have been crucial to the protection of the cells against the toxic effect of atorvastatin.

It is of further interest to note that though the findings from the JC-1 assay indicate that sesamol is not able to protect MIN6 cells from atorvastatin-induced MMP loss, it is able to offer some protection from apoptosis mainly through the intrinsic apoptotic cascade as it is able to cause a reduction in the levels of caspase-7, BAK and BAD lower than that of normal group.

In-vivo studies confirmed these observations made from the histopathological analysis indicated the group treated with atorvastatin alone had indicators of an unhealthy pancreas with diffused islets and prominent foci of caspase activity, but the groups treated with atorvastatin and sesamol had a healthier pancreas. The group also displayed an improvement in HbA1c levels.

In L6 cells (Fig. 10), that atorvastatin is caused a statistically significant reduction in glucose uptake when compared to insulin challenged group. When treated with sesamol and atorvastatin, we find that it was not able to improve the uptake. Further investigation to identify if sesamol with atorvastatin was able to increase the production of GLUT-4 found that indeed the combination caused an increase in GLUT-4 expression, with 10 µM of sesamol displaying the highest increase and 100 µM showing the lowest increase in GLUT-4 expression. As explained earlier, it has already been reported that statins cause a reduction in the synthesis of GLUT receptors that interferes with glucose metabolism of cells (Fig. 5). In the animal study, it was observed from the histopathological analysis that in the group treated with atorvastatin alone, muscle tissue had reduction in the presence of GLUT-4 but at the same time there was displacement of the nuclei of the muscle cells. Whereas, on treatment with sesamol, GLUT-4 presence maintained albeit in a scattered manner and nuclei displacement was reduced.

Fig. 10.

Fig. 10

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The action of atorvastatin and sesamol on insulin signalling pathway in muscle cells

However, these findings cannot neglect the fact that on being challenged with glucose (OGTT) the poor disposal of glucose to deep tissues is not being prevented in the animals when treated with sesamol. The performance is almost at par with the group treated with atorvastatin alone as well as the HFD control. This is also reflected by the HbA1c value of the atorvastatin + sesamol (+ HFD) group (5.81%) which is the prediabetic range although it is a reduction when compared to the group treated with atorvastatin alone or HFD control group.

To summarise, statin-associated diabetes, as per the proposed hypothesis, occurs from two points, due to dysfunction and death of pancreatic beta cells and due to resistance of muscle cells. In accordance with this hypothesis based on the specific mechanisms identified, the role that sesamol could play in combating statin-associated diabetes has been explored. In pancreatic beta cells, atorvastatin is found to cause MMP loss and sesamol is not able to prevent this. The effect of sesamol with simvastatin in a similar manner and found it to be effective in protecting MIN6 cells specifically through mito-protective activity (Ghadge et al. 2020) has also been previously discussed. Atorvastatin in pancreatic beta cells causes activation of both internal and external cascade of apoptosis. Sesamol is able to show reasonable inhibitory activity in the internal cascade without apprehending the effects brought upon by the external cascade through caspase-8. With regards to the development of insulin resistance at muscle cells, though sesamol is unable to significantly enhance glucose uptake, it can improve the synthesis of GLUT-4. These findings indicate that perhaps there may be a basis to further investigate the potential of sesamol or perhaps the role of sesamol like phyto-molecule agents in statin-associated diabetes.

Conclusion

These observations confirm that statin-associated diabetes works through two mechanisms—inducing mitochondrial dysfunction with apoptosis in beta cells and down regulation in the production of GLUT-4 channels in muscle cells. Sesamol in the presence of atorvastatin has demonstrated that it can partially ameliorate the negative effects of atorvastatin at the pancreas mainly through the internal apoptotic cascade and in muscle tissue by regulation of GLUT-4 synthesis causing an improvement of glucose uptake, thus opening the possibility for further exploration in this area.

Acknowledgements

The author, Mr. Raghuvir Keni, would like to thank Dr. Manas Kinra and Ms. Niraja Ranadive from the Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal, Karnataka, India for their hands-on expertise with animal handling and words of encouragement. Ms. Charis Garside from the Department of Molecular and Cell Biology, Cardiff University, UK, for her assistance provided during initiation of the in-vitro studies. Mr. Raghuvir Keni would also like to thank Mr. Sreedhar Prabhu, animal house in-charge and Mr. Baby, the animal house technician both from Manipal Academy of Higher Education, for their assistance provided in the project, especially, during the dire circumstances of the COVID-19 pandemic associated lockdowns. All authors thank the Science and Engineering Research Board, Department of Science and Technology (DST-SERB), Government of India (file number: EMR/2017/003834HS) for funding the work done under this project as well as Manipal Academy of Higher Education for providing the laboratory facilities for the execution of this project and the support and backing both bodies have provided to the authors during the aftermath of the COVID-19 pandemic to ensure the timely completion of the research. The authors also thank Amor Organics Pvt. Ltd., Hyderabad, Telangana, India for providing the gift sample of atorvastatin for conducting this study. The authors thank Servier Medical Art by Servier (https://smart.servier.com/) for the graphical designing tools provided in the design of diagrammatically representative images used in this paper.

Abbreviations

HMG-CoA

3-Hydroxy-3-methylglutaryl coenzyme A reductase

SAD

Statin-associated diabetes

GLUT-4

Glucose transporter type 4

MMP

Mitochondrial membrane potential

Author contributions

Conceptualization: RK, PGN, KN; in vitro studies: RK, KG; in vivo studies: RK, PGN, FB; molecular studies: RK; original draft preparation: RK, PGN, FB; figures: RK; review & editing: KN, NK, AK, SMA; supervision: KN, NK.

Funding

This study was funded by the Science and Engineering Research Board, Department of Science and Technology (DST-SERB), Government of India (file number: EMR/2017/003834HS).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Footnotes

Raghuvir Keni and Pawan Ganesh Nayak equal contribution by both authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.

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