Introduction

Type 2 diabetes is a chronic disease that occurs when the pancreas is either no longer able to synthesize insulin, or the body cannot utilize the insulin that it produces. The development of type 2 diabetes is influenced by a variety of factors. The most important is the lifestyle choices that are often connected with urbanization. Pineal gland melatonin secretion regulates carbohydrate metabolism 1, 2. The effects of melatonin on glucose metabolism have been shown in both humans3 and rodents4, but the exact mechanism is still unexplored. It is thought that melatonin acts directly on pancreatic β-cells, which are known to contain melatonin-binding elements5, 6. Some researchers believe that melatonin's effect on pancreatic cells and insulin release is linked to a complex network of intracellular signalling networks, including the cAMP, cGMP, and IP3 signalling pathways7.

Diabetes is a human and economic burden as it causes 4.2 million deaths per year and accounts for 10 percent of worldwide healthcare expenditures. In 2019, India, China, and the United States of America had the highest number of diabetic adults and this is estimated to persist until 2030 (www.idf.org). Diabetic patients have lower elevations in nighttime melatonin levels, suggesting a possible relationship between melatonin and the hyperglycemic/diabetic condition7. Diabetes mellitus has also been linked to sleep deprivation8. In a study comparing the effects of 4.5 and 8.5 hours of sleep (sleep deprivation and normal sleep, respectively) in healthy adults, phosphorylated Akt and total Akt responses, which are important steps in the insulin signaling pathway, were found to be lower after sleep deprivation9. The study also found that sleep deprivation also resulted in insulin resistance at the cell-signaling level. In healthy normal-weight adolescent males, decreased insulin sensitivity has been observed following acute sleep restriction, such as 4 hours for three nights in a row10.

Agomelatine is an analog of melatonin, and it has a good binding affinity to MT1 and MT2 receptors. IP is a hydrophilic compound, but basically, it is derived from a lipid moiety; it can bind to IPR present on endoplasmic reticulum (ER), causing a rise in Ca level. Calcium release-activated channel (CRAC) is a specialized plasma membrane Ca ion channel. When Ca is depleted in the ER (a major store of Ca) of pancreatic β-cells, the CRAC channel is activated to slowly replenish it. Sarco/endoplasmic reticulum Ca ATPase (SERCA) is an intracellular membrane-bound enzyme that utilizes the free energy of ATP to transport Ca against a concentration gradient. The physiological role of SERCA is to sequester cytosolic Ca into membrane-bound intracellular compartments. The drug thapsigargin (an antagonist of SERCA) may be used to release this stored Ca as a general messenger for cellular signaling11, and will be used mainly for maintaining the concentration of Ca in the cytoplasm via inhibition of the incorporation of Ca ions into ER. 2-APB is a membrane-permeable modulator of intracellular inositol triphosphate (IP3)-induced calcium release12. Even though a wide array of diabetes therapies are available, adverse effects, limited efficacy, and cost significantly limit their use11. The presence of circadian control over pancreatic function and low levels of melatonin in type 2 diabetic patients proposes a new approach for the development of diabetes therapeutics.

Methods

Results

Measurement of insulin levels in the presence of single and combined drugs

In the presence of single drugs

Standard curves for insulin absorbance were constructed using calibration samples of known concentration, and the R-squared values of the curves were used as a data analysis tool to correlate insulin level and absorbance. The cells were exposed to ethanol at 200 μM and DMSO at 4 μM as vehicle controls to check for any effect of these on insulin excretion from the cells. These solutions were found to have no effect on insulin secretion. DMSO containing the compound of interest at a known concentration was diluted with media and added to wells. After 15 hours, the media was collected for insulin content analysis.

Agomelatine

For agomelatine exposure, DMSO was added to 2 control wells. The duplicate cells were exposed to agomelatine at 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM (Table 2). This exposure also contained two wells that did not contain any treatment. The concentration of agomelatine that produced a half-maximal response, ED, was estimated to be 43.36 nM.

Thapsigargin

The cells were exposed to thapsigargin at 4 μM, 2 μM, 1 μM, 0.5 μM and 0.25 μM (Table 2). The ED of thapsigargin was estimated to be 0.3845 μM.

2-APB

The cells were treated with 2-APB at 200 μM, 100 μM, 50 μM, 25 μM and 12.5 μM (Table 2). The ED of 2-APB was estimated to be 36.98 μM.

In the presence of the combination of drugs

In order to determine synergism or antagonism between drugs, it is essential to know the "potency" and the "shape" of the dose-effect curve for each drug. The ED of each medication was calculated in the previous sections, and the constant combination ratio experiment was conducted at an equipotency ratio [, ED/ED ratio] to ensure that the contributions of each drug's effects to the combination were nearly equal. The dose limits were defined from the single drug test to encompass concentrations below and above the ED values of each compound.

Therapeutic interaction of agomelatine, thapsigargin, and 2-APB with MIN 6 in binary and ternary combinations

The fa–CI plots of binary and ternary combinations for insulin ELISA are shown in Figure 1. The CI value versus fa (fraction of insulin secretion with respect to the control) is shown as fa-CI plot. The CI values for the different drugs at different dose levels are also shown in Table 3. Agomelatine and thapsigargin showed synergism across nearly the whole range of dose levels. However, in terms of insulin secretion, thapsigargin and 2-APB showed a stronger synergism than the above group. The combination of agomelatine and 2-APB at a high dose of 148.173 μM concentration showed antagonism, while at a low dose, it appeared to be synergistic. The ternary combination also induced dual antagonistic and synergistic effects, being antagonistic at 149.673 μM, 112.33 μM, and 74.887 μM, but synergistic at dose levels below 37 μM.

The interaction of two and three medication combinations through quantitative values of CI at f= 0.9 shows that agomelatine and 2-APB yielded synergistic effects, but agomelatine + thapsigargin and thapsigargin +2-APB were particularly antagonistic to one another in regards to insulin secretion by MIN6 cells. The concept of the polygonogram comes not from mathematical derivations but rather from its practical utility17. To some extent, the polygonogram can also be used to project (or predict) the outcomes of drug combinations even when the tests have not yet been completed21(Figure 2).

Discussion

Clinical trials of combination therapies for type 2 diabetes are routinely undertaken experimentally. Extensive pharmacologic and efficacy research is always conducted prior to phase I clinical trials for single medicines. In most circumstances, pharmacological combinations produce unpredictable consequences; in a sense, combining two medications creates a third drug, with unique characteristics beyond the sum of its parts.

We believe that some supporting data should be provided when clinical studies invite research into medication combinations. The goal of the current research was to undertake drug combination investigations before putting them into clinical trials, especially for novel medications, using a quantitative study of synergism or antagonism at various medication dosages and effect levels. Prospective experiments were carried out, as shown in this paper, and they provide most of the necessary information for rational protocol design. The three drugs that we used in our study are agomelatine, thapsigargin, and 2-APB, none of which are directly linked with the mechanism of insulin secretion. Agomelatine is a melatonin (MT1/MT2) agonist22, thapsigargin is the most widely used inhibitor of the ubiquitous SERCA proteins in mammalian cells23, 22, while 2-APB transiently activates ORAI1-mediated Ca entry23. In this work, for the first time, we described the therapeutic interactions of three drugs in stimulating insulin release by cultured pancreatic cells. However, other melatonergic agonists24 and melatonin itself have been analyzed for diabetic complications under the influence of these agents22, 23, 25, 26. Thapsigargin and 2-APB have the lowest mean insulin release, but showed synergistic behavior in combination with each other and the third drug when compared through ANOVA. Dunnett's test was applied to identify which drugs and/or combinations favor insulin secretion as in normal and healthy cells. The cells treated with streptozotocin to induce diabetes of course had a lower level of insulin secretion than untreated cells.

The combination index theorem27, a method frequently used to analyze drug interactions in pharmacology, was utilized to investigate the nature of combined drug interactions (synergism, additive impact, or antagonism) on insulin secretion. For drug interactions, this method may be regarded as a fractional analysis tool28, 29 which is independent of the mechanism of action and includes both the potency (ED, ) and the shape () of each drug's dose-effect curve. For a combination of n medications, this approach can predict synergism/antagonism at all effect levels (fa). We determined the type of interactions in MIN6 cells using this method over a wide range of effect levels for each single drug and combination. The Polygonogram depicted the synergistic effect of agomealtine and 2-APB (Figure 3). Furthermore, there have been very few findings on possible targets of the PLC/IP3 pathway via melatonin receptors for diabetes therapy.

The determination of synergism or antagonism was based on the principle of mass action and its related equations; this determination is independent of mechanisms of action. Thus, dose-independent behavior of drugs towards insulin secretion might be attributed to the secondary effect of the drug. This could be an interaction between the biological pathways, or cell proliferation due to Ca acting as a secondary messenger. In many drug combinations, each drug may have more than one mode of action, and synergism may be due to reasons other than a particular mechanism of action. The present study emphasizes the quantitative effects of the various drug treatments on insulin secretion, and does not explore the specific mechanisms of the synergistic or antagonistic interactions identified.

Conclusions

When individual drugs were tested, thapsigargin and 2-APB showed a lowered level of insulin secretion which could be considered a suppressive effects on cells. The CI method predicted synergism in agomelatine and 2-APB, which may have therapeutic implications for diabetes. In this study we present the first example of three distinct medications being examined with the quantitative CI approach for insulin stimulation in MIN6 cells. Among the three drugs selected for the study, agomelatine, thapsigargin, and 2-APB each have demonstrated utility other than insulin secretion. Application of agomelatine and 2-APB together may be a novel therapeutic avenue for type 2 diabetes.

Abbreviations

2-APB: 2-Aminoethoxydiphenyl borate; cAMP: Cyclic adenosine monophosphate; IP3: Inositol trisphosphate; MIN6: Mouse INsulinoma 6; PLC: Phospholipase C

Acknowledgments

We would also like to show our gratitude to the (Dr. Himanshu Kumar, Associate Professor, Indian Institute of Science Education and Research Bhopal and Dr. Archana Tiwari, Director, School of Bio-Technology, UTD, RGPV Bhopal) for sharing their pearls of wisdom with us during the course of this research.

Author’s contributions

Data and materials used and analyzed during the current study are available from the corresponding author on reasonable request.

Funding

None.

Availability of data and materials

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.