Melittin , a Potential Natural Toxin of Crude Bee Venom : Probable Future Arsenal in the Treatment of Diabetes Mellitus

Since diabetes mellitus (DM) is one of the most common and serious endocrine metabolic disorders, it is important to elucidate novel antidiabetic therapeutic agents from various sources, including natural products. Bee venom (BV) is a complex mixture of proteins, peptides, and low molecular components, and melittin is the main constituent. Melittin is a peptide consisting of 26 amino acids with the sequence GIGAVLKVLTTGLPALISWIKRKRQQ. It has several important biological effects and has a relatively low toxicity. Recent studies using animal models have confirmed that melittin has significant glucose and lipid lowering activities by acting on several mechanistic pathways. The main antidiabetic activity of melittin is increasing insulin secretion via depolarization of pancreatic β-cells. Other possible mechanisms may involve stimulation of phospholipase A 2 , increase of glucose uptake, improving lipid profile, and/or reduction of inflammation.This review summarizes the various sources, proteomics, biological roles, adverse effects, and medical applications of melittin and its mechanism of action in combating DM.


Introduction
Diabetes mellitus (DM) is a serious disease in which the body cannot control the amount of sugar in circulation due to either a deficiency of insulin secretion or a decreased sensitivity of the tissues to insulin.There are two main types of diabetes as follows: Type 1 and Type 2 [1].Both types can cause serious health complications, including kidney failure, heart disease, blurred vision, ketoacidosis, peripheral neuropathy, itchiness, fatigue, and even coma [2].An insulin deficiency leads to elevations of cholesterol, phospholipids, and free fatty acids [3].Therefore, it is important that an ideal DM therapy should not only involve maintaining blood glucose levels but also involve the regulation of the lipid profile.
To treat DM, several antidiabetic drugs are used.However, these drugs are not without side effects and pose an economic burden to the patient.Therefore, scientists have turned to natural remedies, including honey and bee products, such as bee venom (BV).BV is a complex mixture of proteins, peptides, and low molecular components secreted by the worker and the queen bees.The main active constituent of BV (apitoxin) is melittin, which has a relatively low toxicity [4].It exerts important effects on cells, such as hemolysis, membrane depolarization and muscle contraction, cytotoxicity, and phospholipase C and arachidonic acid following phospholipase A2 activation.In addition, it is also responsible for various allergic reactions in the body and can suppress the signal pathways of toll-like receptors (TLR2, TLR4), cluster of differentiation (CD14), nuclear factor kappa-B essential modulator (NEMO), and platelet-derived growth factor beta (PDGFR).By suppressing these pathways, melittin diminishes the activation of p38, ERK1/2, AKT, and PLC1 and the translocation of NF-B into the nucleus, consequently leading to reduced inflammation in the skin, aorta, joints, liver, and neuronal tissues [5].
BV proteins, mainly melittin, are also widely used in the treatment of arthritis, frozen shoulder, diseases of the central and peripheral nervous system (CNS, PNS), skin diseases, heart and blood system related diseases, cancer, ulcer, colitis, and neuritis [14,15,17,19,[24][25][26][27][28].Recent studies also confirmed that melittin can significantly reduce blood glucose via insulin secretion and glucose uptake in animal models [21][22][23].These studies also reported that melittin has significant lipid regulating activities by activating phospholipase A 2 .Thus, melittin could be a potential therapeutic agent against DM.
A number of reviews are available in the literature, which focused on the miscellaneous pharmacological effects of BV proteins [17,[29][30][31][32][33][34][35][36][37].However, to date, reviews of the mechanisms by which BV contributes to the antidiabetic activity are currently not available.In this review, we discussed the principal activity of melittin (Table 1) and its antidiabetic mechanism of actions (Figure 1).

Composition of Honeybee Venom
BV is a bitter colorless liquid (pH 4.5-5.5)that dries up easily even at room temperature and is soluble in water but insoluble in alcohol and ammonium sulfate [5,38,39].A bee can inject up to 0.1 mg venom via its stinger.It has been reported to be similar to the toxin of the sea nettle [40].The venom is mainly produced in the abdomen of the worker bees and originates from a mixture of acidic and basic secretions.It is a complex mixture of proteins, in which some enzymes (catalyzing specific reactions), some peptides (which consist of two or more amino acids), and others, including a variety of low molecular components, such as carbohydrates (2% of venom dry weight), phospholipids (5% of venom dry weight), amino acids (1% of venom dry weight), minerals (3-4% of venom dry weight), and volatile compounds (5-8% of venom dry weight) [15,41], are present.Among these, the main component comprises proteins and peptides, the combination of which is commonly known as venom toxins.

Previous Breakthroughs of Bee Venom's Role in Diabetes
Several epidemiological studies using animal models have shown that the use of BV is associated with a lower DM risk (Table 2).Kim et al. investigated whether BV can prevent insulitis and the development of diabetes in nonobese diabetic (NOD) female mice that have been subdivided into control ( = 24) and BV ( = 24) treated groups [20].BV (purchased from Sigma, MO, USA) was injected at 0.5 mg/kg into the hind limb twice per week when the mice were between 4 and 10 weeks of age.The cumulative incidences of diabetes in the control and BV treated NOD mice were 58% and 21%, respectively, at 25 weeks of age.Therefore, it was postulated that BV treatment in NOD mice inhibits insulitis and the onset and overall incidence of diabetes at an early age.
Another in vivo study conducted by Mousavi et al. reported that Iranian BV (Apis mellifera) can lower blood glucose and lipid levels in diabetic rats [21].Adult male rats ( = 18) weighing 200 ± 20 g were placed into the following three random groups: control rats, alloxan monohydratedinduced diabetic rats (150 mg/kg intraperitoneally), and BV treated rats (0.5 mg/kg).In their experiment, BV was collected from beehives using an electric shocker.
In addition to Apis mellifera BV, BV from Apis cerana has also been reported to have antidiabetic activity as reported by Prakash and Bhargava [22].In their study, the samples were divided into test and control groups in which the test group was categorized as "before" and "after" being stung by Apis cerana bees.Their study also reported that Apis cerana BV reduced blood cholesterol and triglyceride levels.Then, Khulan AM and Chimedragcha [23] investigated the effects of Mongolian BV on hyperglycemia and hyperlipidemia in alloxan-induced diabetic rabbits [23].For this study, chinchilla rabbits ( = 22) were divided into the following three groups: control ( = 6), diabetic ( = 8), and BV treated ( = 8) groups.To induce diabetes, an alloxan monohydrated solution (5%) was administered at 100 mg/kg intravenously via the marginal vein behind the ear over two minutes.Meanwhile, the BV treated group received a bee sting on their hind paw every other day following the confirmation of diabetes.Their results also supported that BV significantly lowers serum glucose levels and improves the lipid profile.All of the researchers confirmed that the principal protein in BV, melittin, is mainly responsible for the antidiabetic actions, which was also confirmed by an in vitro test that indicated that melittin can increase insulin secretion from pancreatic -cells.

Antidiabetic Mechanism of Actions of Melittin
DM is a syndrome of impaired carbohydrate, fat, and protein metabolisms due to a combination of both hereditary and environmental causes and is characterized by increased blood glucose levels.In DM therapy, great attention is paid to lowering blood glucose levels by increasing insulin secretion and lipid regulating mechanisms of various medical agents, including those from animal toxins.Melittin reduces blood glucose levels by increasing insulin secretion from the -cells of the pancreas and facilitating glucose uptake [43].It also alleviates complications of DM by ameliorating lipid profiles as verified by several studies [21][22][23].
Recent studies reported that various types of BV significantly reduce not only blood glucose levels but also total cholesterol (Table 2).Melittin, which is present in BV, is mainly responsible for the antidiabetic role as confirmed by an in vitro test conducted by Morgan and Montague [43].Melittin reduces blood glucose levels by several different mechanisms, including depolarization of -cell membranes, increasing the extracellular calcium and calcium channels [21,44], activating cytosolic phospholipase A2 [23,45], increasing glucose transporter lipid uptake into adipose tissues [23], and suppression of -cell inflammation [46].
Melittin initiates membrane depolarization to cause closure of the ATP-sensitive K + channels.Thus, voltage-gated Ca 2+ channels are opened to promote increased insulin secretion into the blood stream [43] (Figure 1).Melittin also promotes insulin secretion by activating phospholipase A 2 [44].
The activated phospholipase A 2 promotes arachidonic acid production, which, in turn, acts as a calcium transporter into the -cells to stimulate insulin secretion [47].
It is also reported that phospholipase A 2 has an alternative antidiabetic mode of action by increasing glucose uptake into the adipose tissues rather than increasing insulin secretion.Due to its enzymatic action, it can partially break down the cell membrane of plasmatic lipoproteins [48].This activity leads to increased glucose transport and lipid uptake into the adipose tissues through partial lyses of adipocyte membranes and binding of a higher number of insulin molecules [49].Meanwhile, phospholipase A 2 also has a higher affinity for the plasmatic lipoproteins, which leads to cytotoxic effects and generation of free fatty acids and lysophospholipids.Thus, circulating cholesterol in HDL is esterified and significantly regulates lipid profiles [50].
Another in vivo study conducted by Kim et al. reported that BV can prevent insulitis and the development of diabetes in NOD mice due to its immune-modulating activities, often attributed to an autoimmune process that damages the pancreatic cells [20].Their investigations also confirmed that BV injections did not yield any side effects during the experimental period, indicating its safety.

Future Directions
Since melittin is a small peptide of 26 amino acids, it is often administered subcutaneously, which can lead to several adverse reactions, including local erythema, swelling, tenderness, itching, edema, malaise, flu-like symptoms, and urticaria [15].Although melittin significantly reduces glucose and cholesterol levels in the bloodstream, its toxicity must be reduced before it can be used as an antidiabetic agent.
Computational bioinformatics and recombinant technology may be used to produce various modified melittin with high therapeutic effects and minimal toxicity.Further investigation should be carried out to (1) improve the procedure of BV collections without killing the bees, (2) develop melittin extraction methods to allow high yields of BV, and (3) improve the preservation methods of BV to maintain its efficacy.

Conclusion
This review article was prepared as a preliminary resource to gather information on the potential of BV, specifically its major active constituent melittin against diabetes mellitus.In diabetes treatment, great consideration is given to lowering blood glucose with various in vitro studies showing that bee venom significantly reduces blood glucose levels via several mechanisms.Nevertheless, there is still lack of adequate in vivo studies done especially using pure melittin from BV. Finally, due to the potential of melittin for reducing blood glucose levels and total cholesterol by several different mechanisms, it is concluded that melittin can have a great impact in the near future in the treatment of diabetes mellitus.

Figure 1 :
Figure 1: Basic mechanism of insulin secretion and sequential mechanistic pathway of BV (melittin) leading to an increase in insulin secretion in pancreatic -cells.(A) Blood glucose (>5 mM in concentration) enters the -cell through the glucose transporter (GLUT2).(B) ATP production rapidly occurs as a result of glycolysis and respiration.(C) Then, ATP-sensitive K + channels are closed, and K + efflux is inhibited.(D) Consequently, plasma membrane depolarizes, and (E) voltage-gated Ca 2+ channels open.Finally, Ca 2+ influx increases, which triggers exocytosis of insulin granules.A Melittin directly depolarizes the cell membrane, B it increases Ca 2+ inflow into the cell, C it promotes more granule secretion, and D it increases insulin secretion into the blood stream.Note: phosphorus: P; calcium: Ca 2+ .Adapted from https://www.dolcera.com/wiki/index.php?title=Diabetes products and services.

Table 2 :
In vivo studies of DM based on BV.