Please cite this paper as:

Banerjee, A., Chattopadhyay, A. and Bandyopadhyay, D. 2021. Potentially synergistic effects of melatonin and metformin in alleviating hyperglycemia: a comprehensive review. Melatonin Research. 4, 4 (Dec. 2021), 522-550. DOI:https://doi.org/https://doi.org/10.32794/mr112500110.

Review 

Potentially synergistic effects of melatonin and metformin in alleviating hyperglycaemia: a comprehensive review

Adrita Banerjee^{1, 2}, Aindrila Chattopadhyay², Debasish Bandyopadhyay^1*

¹Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata-700009

²Department of Physiology, Vidyasagar College, 39, Sankar Ghosh Lane, Kolkata-700006

*Correspondence: debasish63@gmail.com; dbphys@caluniv.ac.in, +91-9433072066

Running Title: Combination of melatonin and metformin as anti-diabetic therapy

Received: August 5, 2021; Accepted: December 9, 2021

ABSTRACT

     High level of glucose is hazardous for organisms since it leads to lipid peroxidation, protein glycation and free radical generation. Insulin can lower the high blood glucose by promoting cell’s glucose up-taking. Thus, the impeded insulin secretion in type 1-diabetes and insensitivity of cells to insulin in type 2-diabetes cause hyperglycaemia. Hyperglycaemia impairs mitochondrial function of pancreas to trigger ROS generation. The malfunctional mitochondria cause endoplasmic reticulum to produce misfolded non-functional insulin, finally leading to diabetes. Melatonin, the mitochondria targeted antioxidant, provides protection against diabetes by multiple ways. These include balancing cellular redox status, lowering blood glucose level by modulating metabolic pathways and, finally protecting cells/organelles from high glucose induced injury. Moreover, this indoleamine preserves pancreatic physiological normalcy to facilitate insulin secretion. Thus, melatonin can effectively mitigate diabetes and diabetic complications. Metformin, the most prescribed medicine for type 2-diabetes, has similar antidiabetic activities as melatonin. Both the molecules share similar pathways to preserve stress-stricken pancreas and other organs, whereas, melatonin also potentiates the actions of metformin. The potentially synergistic actions of melatonin and metformin are expected and we strongly recommend a combined therapeutic application of these two molecules for treatment of diabetes.

Key words: diabetes, melatonin, anti-diabetic drugs, metformin, synergy

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1. INTRODUCTION

       The continuously increased diabetes over the world is an issue of concern. Oxidative stress due to high fat diet consumption has been identified as a primary factor of type 2 diabetes while impaired pancreas with deficiency of insulin secretion induced by autoimmune reaction is the cause of type 1 diabetes. Both cause hyperglycaemia and glucose toxicity (1-4). This augmented glucose level also leads to excess oxidative stress causing protein glycation and lipid peroxidation and cell death (1, 5). Oxidative stress in pancreas of diabetics further impedes insulin secretion (2). From subcellular vision, mitochondria which are the major sites of reactive oxygen species (ROS) production and glucose metabolism, became hubs of oxidative stress caused by hyperglycaemia (4, 6). To compensate mitochondrial deficiency, endoplasmic reticulum (ER) works indefatigably causing ER overload stress with subsequent impairment in protein folding process (7). Hence, the seemingly protective action of mitochondria to maximize glucose metabolism appears doing more harm than good.

     Several natural and synthetic substances are used to alleviate diabetes and/or its associated tissue damage. Metformin, a biguanide class of antidiabetics, is the first choice for diabetes due to its relatively high tolerance and low side effects (8, 9). Metformin also exhibits antioxidative and anti-inflammatory activities, thus, it protects against diabetes and its associated complications by decreasing ROS, pro-inflammatory cytokines, insulin resistance, and also by facilitating glucose uptake of cells (10-12).

     Accordingly, melatonin directly scavenges ROS and also enhances the activity of antioxidant enzymes (13). It not only protects the development of diabetes but also inhibits its occurrence by increasing insulin sensitivity of cells (14). Apart from its negative regulation of pancreatic insulin secretion to lower the tissue insulin resistance (15), melatonin protects both mitochondria and ER against oxidative stress to avoid cellular glucose toxicity, especially for the pancreatic β-cells (16-18). Even though melatonin is not yet a first line recommended intervention for diabetes several current studies have observed the therapeutic effects of melatonin alone or in combination with other anti-diabetic drugs. One of them is metformin (19, 20). Here, we summarize some strategies to combine both metformin and melatonin in alleviation of hyperglycaemia. Therefore, the present review strongly suggests melatonin as an intervention for diabetics not only as a co-therapeutic agent, but also as a sole reliever of diabetes henceforth.

2. INVOLVEMENT OF OXIDATIVE STRESS IN OCCURRENCE OF DIABETES      

       Oxidative stress i.e., excessive ROS generation from different sources is considered as a key aetiology of diabetes and its associated complications (21-23). Glucose oxidation associated non-enzymatic protein glycation cause cellular protein and membrane lipid damage with subsequent cell death (1). Enediol radical anion and active ketoaldehyde formation by glucose autoxidation lead to superoxide anion generation that escalates the burden of stress in vivo (1, 24, 25). Superoxide anion promotes peroxynitrite formation and low density lipoprotein (LDL) peroxidation while reduces glucose-6-phosphate-dehydrogenase (G6PDH) activity with a result of dysfunctional pentose phosphate pathway (PPP) (26-29). Streptozotocin (STZ) and alloxan are diabetic inducers. Both of them augment oxidative stress biomarker thiobarbituric acid reactive substance (TBARS), in organs including liver, kidney, heart and brain indicating lipid peroxidation caused by hyperglycaemia (1). Not only lipids, diabetics causes the structural modifications of myriad extracellular proteins including laminin, elastin, myelin sheath etc. along with a substantial elevation in glycation and cross-linking of collagen molecules (30, 31). These cross-linking between lipids and proteins give rise to advanced glycation end products (AGE). AGE by binding to its receptor RAGE, down-regulates antioxidant system and up-regulates pro-inflammatory cytokines (32, 33). The diabetes associated oxidative stress always accompanies with the altered functions of glutathione reductase (GR), superoxide dismutase (SOD) and catalase in different organs caused by high blood glucose, which in turn makes these organs be more susceptible to ROS damage (4).

2.1. Mitochondrial stress in progression of diabetes. 

       When oxidative stress is considered as a foremost cause behind occurrence of hyperglycaemia, involvement of mitochondria can’t be ignored since these organelles are the major sites of ROS generation. Of note, elevated intracellular glucose level is associated with higher mitochondrial superoxide anion generated by mitochondrial respiratory chain (29, 34, 35). Hyperglycaemia related mitochondrial stress is manifested by over-expression of mitochondrial superoxide dismutase (MnSOD) as observed in diabetic retina and endothelial cells cultured in high glucose media (33, 36, 37). Hence, a convincing correlation between these two factors renders the pathophysiology of diabetes. A crucial connection between mitochondrial anomalies and type 2 diabetes is also found in increased mitochondrial DNA mutations in the pathogenesis of diabetes and its related complications (38). High glucose induced mitochondrial ROS generation in one hand triggers conversion of glucose to sorbitol, utilizing NADPH and thus declines the level of endogenous antioxidant GSH (39) while on the other hand, it elicits reaction of glucose with free amino group by Schiff base formation to advance the process of protein glycation (40, 41). In addition, a marked decline in mitochondrial coenzyme Q9 content in diabetic rat heart, made tissues more vulnerable to stress since coenzyme Q9 is a potent mitochondrial antioxidant (42). All these evidences support the idea to target mitochondrial stress in prevention of diabetes (43). 

2.2. Connection of ER stress in progression of diabetes.

     After identification of oxidative stress as a fundamental factor behind hyperglycaemia, ER stress in particular becomes an independent co-factor of diabetes and its related abnormalities (44, 45). Under high glucose condition, ER stress is a prime factor of pancreatic β-cellular dysfunction and death since ER stress components are closely associated with β-cell function (46-48). The huge burden of mitochondria to carry out complete glucose oxidation for energy generation under glucotoxic condition causes the increase in misfolded or unfolded proteins in ER (49, 50). This unfolded protein ER stress in type 2 diabetes can be neutralised by chaperones, the pivotal molecules for protein folding process (51). For example, the loss of the glucose responsiveness in insulin secretory cells MIN-6 is associated with significantly down-regulated expression of ER chaperones GRP94, BiP, ERp29, and PDI (52, 53). Protein folding is an ATP-dependent process. Protein mis-folding causes futile ATP depletion and this in turn accelerates mitochondrial oxidative phosphorylation and further augments the ROS generation. ROS induces ER stress which then promotes ROS generation and this forms a vicious cycle in pancreatic secretory cells (54). This vicious cycle causes ER unfolded protein response (UPR) (55-59). When the load of misfolded polypeptide chains exceeds ER repairing process, multiple inflammatory pathways will be activated to instigate cell death (60, 61). Erroneous synthesis of pro-insulin instead of its mature form impairs insulin receptor mediated signalling process and results in hyperglycaemia (62). Additionally, accumulated mis-folded proteins trigger the leak of calcium ions from ER lumen to cytosol (63) and the elevated intracellular calcium also promotes mitochondrial ROS generation by increasing mitochondrial membrane permeability (64, 65) and blocking complex III of electron transport chain (ETC).

2.3. Pancreatic oxidative stress in development of hyperglycaemia.    

     Pancreas and more specifically, β-cells of islets act as the major sensor and regulator of circulated glucose level since insulin secreted by them promotes cellular glucose uptake to balance glucose load of blood, therefore, helps to avoid high level glucose toxicity. Pancreatic dysfunction results in declined insulin secretion while over-production of insulin leads to insulin resistance and β-cells exhaustion (66). Both cases cause hyperglycaemia and high amounts of ROS production (67, 68). Pancreatic β-cells with relatively low antioxidants and high content of mitochondria are prone to oxidative stress which suppresses insulin secretion (69, 70). The association between progression of diabetes and pancreatic β-cell dysfunction caused by oxidative stress are well documented (71-73). Mitochondrial dysfunction and ER stress of pancreatic islets are also contributory to pancreatic β-cellular dysfunction and apoptosis (74, 66).

3. MELATONIN AS AN ANTIHYPERGLYCAEMIC ANTIOXIDANT  

     Since oxidative stress directly impacts the initiation and progression of hyperglycaemia, antioxidants can be considered as a first line of defence against diabetes. Thus, scientists have tried to use melatonin, a potent antioxidant, to target oxidative stress associated hyperglycaemia.

     In Type I diabetic rats, melatonin reduced lipid peroxidation and increased glutathione peroxidase (GPx) activity in brain, kidney and liver and its protective effects are greater than that of vitamin E (75). These beneficial effects of melatonin on diabetes have been confirmed in different studies (76). Alloxan and STZ can induce diabetic status in animals by generating hydrogen peroxide and superoxide anion as well as by weakening antioxidant defence machineries (77, 78). In mice and rabbit treated by these chemicals, melatonin treatment reduced their oxidative tissue injury and maintained their redox state (79-81). In diabetic individuals, melatonin application normalized their blood glucose levels and thus protected against morphological damages of β-cells to hinder insulin leakage from these cells (81, 82). It also prevents β-cellular apoptosis caused by exposure of them to high glucose (83) (Figure. 1). In type 2 diabetic rats, melatonin application improved antioxidant features both in pancreas and heart along with a marked increase in insulin to glucose ratio (18). The alleviation of hyperglycaemia by melatonin is attributed to its antioxidant activity which mediates protection and regeneration of pancreatic islet cells (84). Furthermore, melatonin mediated slight increase in insulin secretion has also been reported as an extra-pancreatic function of this indole (85). Hence, apart from providing overall protection to hyperglycaemia by keeping homoeostasis of redox state, via inhibiting protein glycation, abating glycosylated haemoglobin production and declining glucose level, melatonin also acts as a safe guard for pancreas (86-88).

     Melatonin can preserve ER in its healthy form by reducing the poorly developed rough endoplasmic reticulum (RER) in hepatic cytoplasm of diabetic rat (89, 90). Since ER stress couples to oxidative stress in occurrence of diabetes, a molecule that can provide protection against glucotoxicity induced ER stress can be a best choice as an anti-diabetic therapy. Melatonin exhibits enormous potential in hindering apoptosis of mouse osteoblastic MC3T3-E1 cell line exposed to high glucose medium in a type 2 diabetes mimicking milieu. Melatonin inhibits phosphorylation of PERK with prevention of the downstream eIF2α-CHOP pathway that ensure non-occurrence of ER UPR signalling by censoring proximal sensors of ER stress (91) (Figure 2).

4. ROLE OF MELATONIN IN GLUCOSE METABOLISM- A LINK TO MITIGATION OF DIABETES

     The involvement of melatonin in glucose metabolism is linked to a surged glucose intolerance and insulin resistance in pinealectomized rats (92). Accordingly, an impaired glucose uptake by adipose tissue in pinealectomized rats convinced this involvement (93). Since diabetes is a metabolic disorder, the medications must target metabolic pathways.

     Gluconeogenesis is a process that synthesizes glucose from non-carbohydrate source. Overactivity of this process contributes to the blood glucose surge and glucose toxicity which may be fatal for diabetes. Melatonin is a negative regulator of gluconeogenic pathway and especially for phosphoenolpyruvate carboxykinase (PEPCK) (Figure1). Pinealectomized rats displayed uncontrolled PEPCK activity and high hepatic gluconeogenic rate even in the presence of high level of insulin (94). Melatonin is also a positive regulator of insulin involved AKT phosphorylation, accountable for lowering PEPCK transcription via FoxO1 inhibition (94). Additionally, association of melatonin receptors (MT1 and MT2) with hypothalamic PI3K/AKT activation has been unveiled with a major role in hepatic gluconeogenesis repression (95). Melatonin stimulates Pentose Phosphate Pathway (PPP) to increase glucose utilization by enhancing the activity of G6PDH and thus reduces blood glucose accumulation in type 2 diabetic rats (96, 97). This action of melatonin helps in generating NADPH, the essential co-factor for GSH formation catalysed by GR. Melatonin not only positively affects NADPH generation, but it also reduces its degradation by inhibiting activity of NADPH oxidase (NOX) (Figure 1), which uses NADPH as an electron donor in conversion of molecular oxygen to superoxide anion. Melatonin diminished NOX4 activity and downregulated expression of p47^phox subunit of NOX4 in kidney cortex of Zucker diabetic fatty rats with symptoms of diabetic nephropathy (98). On other hand, pinealectomy enhanced NOX subunit assembly along with excess ROS generation and insulin resistance (99). Of note, NOX4 is a factor responsible for increase in gluconeogenesis (100) and hence, the overall protective action of melatonin acts as a metabolic harmonizer in diabetes. The impaired hepatic mitochondria and low ATP level in Zucker diabetic fatty rats were alleviated by melatonin application (101) and here, melatonin regulated homoeostatic ATP level may be responsible for controlled glycolysis and Krebs cycle rate, which assists to limit ROS generation.

Fig.1. Potential mechanism(s) of action of melatonin and metformin in alleviation of diabetes.

     Keap1: Kelch like ECH-associated protein 1, Nrf2: nuclear factor erythroid 2-related factor 2, HO1: heme oxygenase 1, FoXO1: forkhead box protein O1, PEPCK: phospho enol pyruvate carboxyKinase, AMPK: AMP-activated protein kinase, PGC1α: peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, SIRT1: sirtuin1, SIRT3: sirtuin3, mTOR: mammalian target of rapamycin, NLRP3: NLR family pyrin domain containing 3, NOX: NADPH oxidase, ROS: reactive oxygen species, NFkB: nuclear factor kappa-light-chain-enhancer of activated B cells, arrows: activation, dash arrows:  direction: T type bars: inhibition,  colour arrows:  different pathways.

5. ANTIOBESITY ROLE OF MELATONIN IN ANTIHYPERGLYCAEMIC ACTION

      Melatonin has an antiobesic role in diabetic fatty mice (102). This action also contributes in alleviation of obesity associated hyperglycaemia (103, 104). Since insulin is the key hormone to maintain the rate of glucose metabolism by regulating activities and expressions of glycolytic, glycogenolytic and gluconeogenic enzymes, the hepatic resistance to insulin is primarily responsible for perturbed glucose balance in obese (105). The decreased glucose uptake by the muscle and increased glucose output by the liver (103) result in hyperglycaemia which causes macrophage infiltrated inflammation and adipocyte apoptosis (106). These are the major events responsible for occurrence of insulin insensitivity. Additionally, ER stress is a dominant factor behind obesity associated inflammation and subsequent insulin resistance in liver (107). Melatonin incurs myriad protective measures to hinder obesity related hyperglycaemia. The association of declined melatonin with increased obesity in diabetes (108) confirms the important roles of melatonin on obesity. Another action of melatonin targeting obesity in Zucker diabetic fatty rats is by increasing their brown adipose tissue mass and function (102, 109) to accelerate the metabolic rate of the animals. Melatonin administration protects adipose tissue as well as other organs including kidney (110) in obese associated diabetes via its anti-inflammatory activity and its regulation on mitochondrial dynamics (111).

6. ANTI-INFLAMMATORY ACTIVITY OF MELATONIN IN ALLEVIATION OF DIABETES

     Inflammation has been emerged as a principal causative factor of diabetes (both Type I and Type II) (112). In type I diabetes, interferon gamma (IFN-γ), inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) are the major factors of inflammation and ROS generation (113). ER stress and pancreatic islet cell death caused by IFN-γ have been reported (114, 115). Macrophages are the local inflammatory cytokine generator to cause pancreatic islet injury (116) while natural killer cells, natural killer T cells and dendritic cells, all participate in the inflammation induced diabetic progression (117). Involvement of inflammatory pathways is more relevant in advancement of type 2 diabetes. The inflammation activated transcription factor NFκB and Jun N-terminal kinase (JNK) have been found in various metabolic disorders (118-121). For example, TNF-α induces insulin resistance in obesity (122). Elevated IL-1β, IL-6 and CRP (C Reactive Protein) are common factors to diagnose type 2 diabetics with associated cardiac anomalies (123-125) while elevated serum IL-1 emerges as an indicator of inflammation in hyperglycaemia (126, 127). Adipose tissue in type 2 diabetes generates TNF-α, IL-1, IL-6, IL-10, leptin, adiponectin, chemokines etc. and their levels are further augmented by several folds when adipose tissue is infiltrated with macrophage and other immune cells (128-131).

       Melatonin displays strong anti-inflammatory actions by balancing both pro and anti- inflammatory cytokines (132, 133), especially by blocking the translocation of NFκB into nucleus or by impeding the actions of NLRP3 inflammasome (134, 135) (Figure 1). Additionally, melatonin quenches deleterious hydroxyl radical, hypochlorus acid, peroxynitrite anion which are involved in production of pro-inflammatory cytokines (134, 136). Type 2 diabetic patients with insulin resistance have a strong positive correlation with upregulated IL6 and activated NLRP3 (125, 137). Melatonin, in diabetic rats rejuvenates pancreatic islet cells by escalating anti-inflammatory cytokine level IL-10 along with diminution of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 (18). Similar effects of melatonin have been observed in obese patients with a concomitant decrease in insulin sensitivity (138, 139). Not only diabetes, but also many hyperglycaemia associated complications can be mitigated by the anti-inflammatory properties of melatonin.

7. MELATONIN IN ALLEVIATING DIABETES ASSOCIATED DISORDERS

     The indoleamine, with its antioxidative and anti-inflammatory mechanisms not only prevents the occurrence and progression of diabetes, but also hinders the onset of its associated complications (140, 77) including diabetic cardiomyopathy (DCM), diabetic retinopathy (DR) and diabetic nephropathy (DN), and thus, it may reduce the death rate of diabetes globally.

7.1. Diabetic Cardio-myopathy (DCM).

    DCM, one of the prime complications to cause diabetes associated death is mainly characterised by oxidative stress, inflammation and cardiac hypertrophy caused by dysregulated mitochondrial metabolism (141, 142). Melatonin reduces this cardiac hypertrophy in STZ induced diabetic Wistar rats and, thus, prevents cardiac remodelling by increasing phosphorylation of vascular endothelial growth factor (VEGF-A) (143). The indoleamine also facilitates autophagy pathway by inhibiting mammalian target of rapamycin (mTOR), a factor crucially accountable for cardiomyopathy and cardiac hypertrophy (144). One of the protective mechanisms of melatonin on DCM is to balance redox status, i.e., to enhance Nrf2/HO-1 for subsequent up-regulation of antioxidant enzyme expressions. These processes retard the myocardial cell apoptosis induced by activation of caspases (caspase 3, 8, 9) (145-147). Melatonin application in type 1 diabetic rats or in high glucose exposed H9c2 cells preserves mitochondrial structure and function with reduced ROS production by targeting AMPK-PGC1α-SIRT3 cascade (148). Cardiac tissue structure and function of diabetic animal models are preserved by melatonin via Syk/COX-I/SERCA signalling pathway (149) or by inhibition of PERK/ATF-6α/CHOP pathway (150). These actions mitigate ER stress, calcium overload and apoptosis in cardiac tissue. In addition, the NLRP3 inflammasome mediated cardiac fibrosis can also be reversed by melatonin application (10mg/kg/d for 8 weeks) in DMC via inhibition of lncRNA MALAT1/miR-141 axis (151). Thus, diverse activities of melatonin involve in protecting cardiac tissue from high glucose induced injury (152).

7.2. Diabetic retinopathy (DR).

     Since oxidative stress and inflammation are major pathological factors of DR (153), melatonin emerges as a suitable treatment for this disorder with its antioxidative as well as anti-inflammatory activities. Melatonin reduces oxidative stress, ER stress, inflammation in DR per se or indirectly stimulates antioxidant enzymes in retina (154-156). The protective effect of melatonin on DR has been reported by inhibiting pro-inflammatory cytokines IL-1β, TNF-α through NFκB pathway (157) whereas, activation of MAP kinase (MAPK) pathway by melatonin reduces ROS generation, lipid peroxidation, inflammation and apoptosis (158). When diabetic Wistar rats treated with melatonin, they exhibited improved redox state and reduced retinal dilation and deformation by impeded endothelial growth factor mediated angiogenesis and tissue injury (159). These results have been substantiated by potency of melatonin in preserving retinal function of type 2 diabetic rats with its antioxidative efficacy (160).

7.3. Diabetic nephropathy (DN).

    High glucose associated renal disorders are referred as DN, where compromised antioxidant capacity and ROS overload lead to apoptosis of renal epithelial cells with progressive loss of kidney functions (161). Balance of redox status and preservation of histopathological and functional kidney in diabetic rats by melatonin alone or in combinations have been well documented (162-164). The upsurged pro- inflammatory cytokines including TNF-α, IL-6 and declined anti-inflammatory cytokine IL-10 levels in DN are normalized with melatonin application (165). A downregulation of pro-inflammatory IL-33 and another stress bio-markers in kidneys of diabetic rats are also achieved after melatonin treatment (166). Augmented kidney injury marker (KIM-1), heat shock protein (HSP-70), macrophage infiltration induced TGF-β1 are successfully ameliorated by intraperitoneal and oral application of melatonin in diabetic rats (167, 168). Melatonin pre-treated mesenchymal stem cell (MSC) transplantation significantly improves DN compared to untreated MSC by enhancing expression of autophagy mediator Beclin-1 as well as by retarding actions of TGF-β1 (169, 170). 

8. SYNERGISTIC EFFECTS OF MELATONIN AND OTHER ANTI-DIABETIC DRUGS

   Though the efficiency of melatonin in alleviating diabetes and associated complications have been reported in several studies, its use in diabetes remains as an adjuvant with other medications. Type I diabetes is an autoimmune disorder and it is difficult for prevention (171). In contrast, the onset and progression of type 2 diabetes can be delayed with the assistance of medications. Despite of having several treatment options for diabetes e.g., vildagliptin (172), metformin is still the first choice for type 2 diabetes (173). Its effectiveness, relatively low side effects made it be more endurable and much tolerable medicine for type 2 diabetes (174). Since, both melatonin and metformin have enormous potentials to curb diabetes and its associated complications, melatonin and metformin can be a combination of interest in treatment of diabetes.

     Since obesity linked high glucose level is considered as a factor behind development of insulin resistance and subsequent diabetes, lifestyle change has been suggested for alleviating this disease. High fat diet fed Sprague-Dawley rats have a perturbed glucose metabolism, lipid profile and insulin resistance, all these alterations are improved by melatonin plus metformin treatment via modification of melatonin-leptin axis (19). It seems that melatonin acts as an amplifier for metformin and the combination achieves better outcomes comparing to metformin alone. Melatonin-metformin combination also potentiates the recovery of renal function in high glucose affected diabetic patients while metformin alone is not sufficient to provide requisite protection (175). Melatonin and metformin co-application at pharmacological doses for chronic diabetic patients significantly reduces their glycated haemoglobin (HbA1C) level whereas, metformin alone failed to do so (176). Hence, the synergistic effects of the melatonin and metformin to mitigate diabetes are suggested, i.e., melatonin potentiates the effects of metformin to amplify its therapeutic activities on diabetes.

9. MECHNISMS OF METFORMIN SHARED WITH MELATONIN ON DIABETES

     Metformin, the first line oral drug of diabetes, possesses several mechanisms to protect against hyperglycaemia. It shares some mechanisms with melatonin in alleviation of the metabolic disorder. The details will be discussed following.

9.1. Antioxidative effects of metformin to restrict diabetes.

    Metformin can preserve the cellular redox balance, especially by bringing homoeostasis in mitochondrial function in diabetic patients (Figure 1) (177).

     The results of three months clinical trial with metformin in type 2 diabetic patients showed the improved levels of advanced oxidation protein products (AOPP) and AGE along with the reduced oxidative stress, inflammation and tissue damage (178). Another similar trial with 6 months of metformin treatment has confirmed the results with reduced oxidative and nistrosative stresses in type 2 diabetic patients (179). The antioxidative potency of metformin in type 1 diabetic animal model is also observed where application of metformin and insulin reduced the free radical insults associated oxidative stress (180). The antioxidative activity of metformin may be attributed to its direct hydroxyl radical scavenging activity (181). Beside this, modulating the activity of NADPH oxidase enzyme to obstruct superoxide anion generation also contributes to antioxidant properties of metformin (182). Metformin application often exhibits reduced lipid peroxidation, protein carbonylation with concomitant surged antioxidant level in type 2 diabetes patients (183).

     Since energy metabolism is a principal determinant of diabetic condition, impairments in this process are crucially involved with hyperglycaemic progression. Metformin administration seems to protect intactness of vasculature in diabetic rats with the mechanisms of lowering mitochondrial stress and enhancing activity of aconitase enzyme and thus maintain energy balance to hinder vascular dysfunction (182). Prevention of mitochondrial stress by metformin through AMP-activated protein kinase (AMPK) activation has also been evidenced in endothelial cells, exposed to high glucose (184). Metformin activates cellular nutrient sensors AMPK and sirtuin-1 (SIRT1) by phosphorylation to downregulate stress inducing and autophagy inhibiting factor FoxO1 in the in vivo and in vitro conditions (185) (Figure 2). This mechanism of metformin extends its protective arena to diabetic patients with malfunctioned kidney. Metformin can effectively suppress complex I related reverse-electron flow (39, 186, 187) to protect tissue damage, especially mitochondria (188). Metformin activates AMPK and PGC-1α pathways in diabetic mice brain. It is the core modulator of mitochondrial biogenesis, and on other hand, it is an attenuator of mitochondrial oxidative stress (189).

     Noteworthy, metformin retards hyperglycaemia induced oxidative stress, inflammation and insulin resistance when offered in combination with other molecules (190, 191).

9.2. Anti-inflammatory activity of metformin.   

     The anti-inflammatory activity of metformin is involved in its therapeutic effects on diabetes (192). Since, oxidative stress and inflammation form a vicious cycle to accelerate progression of diabetes (193) and vascular dysfunctions, anti-inflammation targeted anti-diabetic drugs became the first choice. A major mechanism of metformin to control glycaemia is activation of AMPK pathway (194, 195), which initiates energy metabolism and inhibits NFκB expression and thus limits inflammation (196, 197). Type 2 diabetic patients treated with metformin have improved anti-inflammatory status with surge in serum IL-10 expression irrespective of its dose and duration. This treatment also reduces pro-inflammatory IL-6 and MCP-1 levels in patients’ serum and urine respectively, comparing to other anti-diabetic drugs including insulin release stimulants (198). A more commendable anti-inflammatory action of metformin in diabetes suppresses formation of inflammation promoter AGE by binding to its precursor, methylglyoxal (199-201). In addition to AMPK dependent anti-inflammatory action, metformin can enhance the level of tensin homologue PTEN, responsible for NFκB inactivation via PI3K/AKT inhibition (202).

     Moreover, combination of metformin and L-cysteine brings profound changes in serum MCP-1 and C-reactive protein (CRP) levels in type 2 diabetic model rats and thus, forestalls mitochondria associated apoptotic process of hepatic tissue, induced by high glucose (191).

Fig.2. Protective mechanisms of melatonin and metformin on ER stress in organs and especially of pancreatic islet cells of diabetics.

     Hyperglycaemia induces misfolded proteins and triggers ER stress in pancreas. Both the molecules inhibit unfolded protein response (UPR) and ER stress induced by high glucose, and thus, reduce mitochondrial oxidative stress. NFkB: nuclear factor kappa-light-chain-enhancer of activated B cells, PERK: protein kinase RNA-like endoplasmic reticulum kinase, eIF2α: eukaryotic initiation factor 2 alpha, ATF: activating transcription factor, CHOP: C/EBP homologous protein, Pdx1: pancreatic duodenal homeobox1, arrows: activation, T type bars: Inhibition, Colour arrows: different pathways.

9.3. Effects of metformin on glucose metabolic pathways.

     The principal action of metformin in curbing hyperglycaemia is to lower hepatic glucose production by suppressing gluconeogenic pathway (Figure 1). The poorly controlled type 2 diabetic patients have 2 folds higher rate of glucose production and 3 folds higher gluconeogenic rate than the controls. Metformin administration reduces those rates by 24% and 33% respectively (203). In another study, a 60% reduction in glucose level has been evidenced with metformin treatment and this also contributes to its inhibitory effect on the gluconeogenesis (204). Metformin downregulates expressions of gluconeogenic rate limiting enzymes both at the mRNA and protein levels in mice liver and primary hepatocyte respectively (205-207). Growing bodies of evidence have supported the notion of anti-gluconeogenic effects of metformin while other diverse mechanisms are also present. Non-competitive inhibition of mitochondrial glycerophosphate dehydrogenase (mGPDH) by metformin is one of such mechanisms (208). It inhibits mGPDH and thus, blocks generation of dihydroxyacetonephosphate, the precursor of glucose formation from glycerol. mGPDH inhibition also makes hepatic redox state more reductive (NADH) that suppresses the conversion of lactate/pyruvate into glucose since this required oxidised form of co-factor (NAD) dependent enzymatic actions (209). However, a redox independent mechanism of gluconeogenic inhibition by low dose of metformin has been uncovered. Metformin acts through channelizing intermediate substrates toward glycolysis to obstruct the formation of glycerol-3-phosphate, the allosteric inhibitor of glycolytic fructose-1,6 bisphosphate and/or phosphofructokinase (210). Additionally, AMPK activation is a major approach of metformin in modulation of metabolic pathways and its phosphorylation at Thr172 site is promoted by metformin (211). Metformin is also found to inhibit mitochondrial respiratory chain enzyme activities with concomitant rise of AMP/ATP ratio (212, 213). Metformin improves AMP level by inhibiting AMP deaminase (214). Correlation between loss of metformin action with deletion of liver kinase and subsequent loss of phosphorylated form of AMPK substantiates the notion of metformin’s AMPK dependent antihyperglycaemic action (215). AMPK upregulation by metformin suppresses expressions of rate limiting enzymes of PEPCK and glucose-6-phosphatase in gluconeogenic pathway (216). Here, AMPK upregulates expression of small heterodimer partner (SHP) which downregulates forkhead box protein O1 (FoxO1) and A2 (FoxA2), the positive regulators of gluconeogenic gene transcription (217). Since gluconeogenesis requires ATP utilization, metformin mediated reduction in ATP level also negatively controls the fate of this pathway (218).

     Metformin instigates downregulation of Akt activity via suppression of Src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2) by directly binding to its phosphate domain which is another anti-hyperglycaemic strategy of the drug (219). This property of metformin improves insulin sensitivity as well as augments glucose uptake by cells. The increase in glucose uptake by skeletal muscle cells upon metformin treatment is attributed to the fact that metformin promotes glucose transporters including their movement along cellular membrane. In addition, this cellular uptake is associated with elevated glycogen synthesis and subsequent storage along with reduction in the rate of glycogenolysis (220).

9.4. ER stress alleviation by metformin in diabetics.

     Like to many other proteins, pro-insulin secretion from pancreatic β-cells requires stringent inspection and maturation by ER chaperone proteins. Under different physiological conditions, secretory β-cells use unfolded protein response (UPR) to limit the load of newly synthesized proteins that exceed beyond the ER capacity to fold proteins with accuracy (221, 222). Metformin confers extensive protections in diabetes by preserving structural and functional equilibrium of ER (223) (Figure 2). High glucose exposed rat insulinoma cells INS-1 exhibit reduction in pancreatic duodenal homeobox1 (Pdx1) expression, the protein responsible for differentiation of islet β-cells while metformin reverses Pdx1expression with improved insulin level and decreased UPR stress response from ER as indicated by diminished phosphorylated forms of PERK, eIF2α and CHOP (224). In a parallel way, oral metformin administration to diabetic male C57BL/6 mice for 15 consecutive days delays PERK phosphorylation and subsequent activation in brain tissue indicating potency of metformin in ER stress amelioration (189). The suppressive effect of metformin on ER stress may be responsible for late activation of UPR signal which by detaining eIF2α transcription assists in continuing uninterrupted protein translation (225, 189). In accordance with this study, metformin inhibits JNK phosphorylation preceded by AMPK activation and thus, prevents occurrence of inflammation and death of mouse pancreatic β cell line NIT-1 (226). In contrast, enhanced level of phosphorylated eIF2α has been reported in metformin treated peripheral blood mononuclear cells (PBMC) of type 2 diabetes as a protective act of metformin to conserve ER from entering into apoptosis by blocking ATF6 dependent pro-apoptotic pathway (222). Here, metformin protects ER of PBMC by declining the levels of autophagy inducer becn1, atg7 and promotes adaptive branch of UPR to arrest protein translation process rather to induce apoptosis. Moreover, metformin along with resveratrol preserves ER in eustatic condition with different mechanisms. This combination alleviates excess mitochondrial ROS induced ER stress and successive NLRP3 inflammasome formation in diabetic ICR mice adipose tissue (227).

9.5. Metformin: a safe-guard for pancreas

     Apart from improving insulin sensitivity in high glucose targeted tissues including muscle, adipose tissue and liver, metformin acts as an overall protector to pancreatic β-cells, particularly to preserve their insulin secretion (228, 229). In high glucose environment, metformin enhances β-cell viability and lowers insulin secretion by modulation of pancreatic translational activities to safeguard β-cells from glucose toxicity (230, 231). This molecule prevents pancreatic islet cell malfunction and thus, forestalls apoptotic pathway (232) by averting high glucose induced islet cell desensitization (233). Additionally, this drug effectively preserves morpho-functional stability of pancreatic islet cells by conserving mitochondria and ER intactness (234, 223).

10. CONCLUSION AND PROSPECTIVES   

     Though metformin is a well-accepted first line drug for diabetes (171), its adverse effects can’t be neglected. The side effects of this biguanide class anti-diabetic drugs are relatively low as mentioned above but some unfavourable actions are still concerned by physicians and researchers (235). For example, metformin treatment, sometimes, even at low doses, causes gastrointestinal discomforts with symptoms of nausea, diarrhoea. Its safety limits have been constricted when metformin has been shown to cause complications like pancreatitis, hepatitis etc. (236, 237). Moreover, lactic acidosis is a detrimental effect since metformin blocks electron transport chain resulting in biased metabolic cycles in favour of more anaerobic glycolysis which leads to excess lactate production (238). Many clinical studies cast concerning on its long term use dependent vitamin B12 deficiency (239). Additionally, despite of having protective effects on diabetic nephropathy, metformin also can cause pernicious effects on renal function with reduced glomerular filtration rate (GFR) (240). Hence, the shortcomings of metformin have prompted the researchers to find a more tolerable molecule with antihyperglycaemic effects. Melatonin seems to be such a molecule. Melatonin has antihyperglycaemic effects with plethora of protective actions on the affected organs. For example, melatonin preserves gastrointestinal (GI) epithelium’s microcirculation and thus, prevents epithelial degeneration, lesion and ulceration (241). In addition, the indole shows diverse protective mechanisms to conserve renal tissue by forestalling oxidative stress, inflammation in patients with chronic kidney disease along with safeguarding rennin-angiotensin system (242). A recent review has detailed beneficial effects of melatonin in nullifying drug induced nephrotoxicity (243) which again is in favour of melatonin as an aid for hyperglycaemia.

     This review has been drafted to fetch attention to the use of melatonin as an anti-diabetic, having antihyperglycaemic effects along with ample protective effects for high glucose affected organs. The mechanisms of melatonin in mitigating glucose toxicity and diabetic complications appear similar to metformin. Such mimicked actions of both the molecules in diabetes may encourage prescribing melatonin alone besides its application as a co-therapeutic medicine. This recommendation requires approval from more clinical studies.

ACKNOWLEDGEMENTS

     A Junior Research Fellowship (JRF) under WBDST [304(Sanc.)/STP/S&T/1G-67/2017 DATED 29.03.2018] is greatly acknowledged by AB. Dr. AC is supported by funds available to her from Department of Science and Technology, Govt. of West Bengal. Dr. DB also gratefully acknowledges the support he received from CPEPA Scheme of UGC awarded to University of Calcutta, Departmental BI Grant and DST-PURSE Program awarded to the University of Calcutta.

AUTHORSHIP

     All authors have contributed to the conception. AB drafted the first version of the manuscript and figures. Dr. DB and AC critically reviewed the manuscript and approved it.

CONFLICT INTEREST

     The author(s) declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

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