A new approach for the pleiotropic effect of metformin use in type 2 diabetes mellitus

Introduction

For long-term diabetic patients, insulin and oral antidiabetic drugs may need to be used together in order to control their glycemia. Metformin is one of such drugs belonging to the biguanide class of anti-diabetic compounds. It is the most preferred drug in the treatment of T2DM in combination with many drugs, and hence it is the first choice in the guidelines [1], [2], [3], [4].

Metformin appears to have pleiotropic effects in addition to an antihyperglycemic effect. The drug has provided clinical efficacy in conditions associated with hyperinsulinemia such as prediabetes, gestational diabetes, polycystic ovary syndrome, and non-alcoholic steatohepatitis [5], [6], [7].

Metformin may also have an antiproliferative effect in various cancer types such as pancreatic, breast, prostate, and colon tumors [8]. Reduced risk of cancer has been reported in T2DM patients treated with metformin compared to patients treated with other antidiabetic drugs [9].

Growth arrest-specific protein 6 (Gas6) protein is the ligand of Tyro3, Axl, and Mer receptors (TAM receptors). Gas6 protein binds and activates the TAM receptors. The binding affinity of Gas6 to TAM family receptors is as follows: Axl ≥ Tyro3 ≥ Mer [10].

Axl, a membrane-bound 140 kDa form, is expressed in many cells and tissues. Gas6 has a function in the pathogenesis of impaired glucose, insulin resistance, and homeostasis [11]. The vitamin K-dependent Gas6 binds Axl and causes autophosphorylation of Axl. This complex activates several downstream signaling pathways, including the phosphoinositide 3-kinase (PI3K)/AKT pathway, one of the important signaling pathways involved in glucose metabolism [12].

Soluble Axl (sAxl) is an 85 kDa protein and it is produced by the proteolytic cleavage of extracellular domains by metalloproteinases (ADAM10 and ADAM17) and a disintegrin. sAxl is a negative regulator of endogenous Axl signaling [13]. Serum Gas6 circulates in a complex with sAxl and hence Gas6 is present at low concentrations in circulation [14].

The Gas6/Axl signaling has been shown to play an important role in inflammation, homeostasis, autoimmune diseases, nervous system, reproductive system, vascular system, cancer, and associated glucose intolerance with metabolic disorders [15, 16].

Studies have reported that the use of metformin in patients with T2DM regulates blood glucose and reduces the risk of cancer. Metformin treatment improves insulin sensitivity, inhibits hepatic glucose output, and lowers circulating insulin levels [17].

The clinical role of the Gas6/Axl pathway in the molecular mechanism of complications associated with T2DM is not fully understood. In addition, studies related to impaired glucose metabolism are controversial and insufficient. Therefore, we aimed to investigate the effect of metformin and metformin-insulin combination on serum Gas6, Axl, and sAxl parameters in patients with T2DM. We also wanted to see how the effect changed when insulin was used in combination with metformin, and to determine whether sAxl levels were associated with transmembrane Axl levels.

Materials and methods

Results

Discussion

In this study, the effects of metformin alone and the effects of insulin used in combination with metformin, in patients with T2DM on Gas6, Axl, and sAxl levels were investigated. Present studies showed that the effect of metformin on the Gas6/Axl pathway was generally carried out in vitro. Therefore, this study is one of the first studies conducted clinically. In this study, a statistically significant decrease was found in Gas6 and Axl levels in patients with T2DM who received metformin treatment. In addition, a statistically significant decrease was observed in Gas6 and Axl levels in the Met + Ins treatment group. sAxl levels were similar in all groups.

Patients with diabetes mellitus are at an increased risk of susceptibility to cancer. It has been suggested that insulin resistance and hyperinsulinemia in T2DM directly or indirectly promote malignant transformation [18, 19]. Several studies have reported an increased incidence of breast, endometrial, and colorectal cancers in the first months after T2DM diagnosis and even in the prediabetes phase [20].

Metformin is a widely used pharmacological agent for the treatment of T2DM. It is the first choice of treatment for complications that are common in long-term T2DM patients and is used in combination with injectable and oral treatments [21, 22]. Studies are showing that the use of metformin in patients with T2DM reduces the risk of cancer [23, 24]. In the study of Evans et al. using metformin has been associated with a reduced prevalence of cancer in patients with T2DM [25].

Axl has emerged as a new biomarker due to its role in biological processes and tumor formation, while Gas6 is a ligand of Axl [26]. Gas6/Axl signaling pathway plays a role in pathological mechanisms for complications such as cardiovascular diseases, kidney dysfunction, amputations, nerve damage, and erectile dysfunction associated with T2DM [27].

Hung et al. showed that plasma Gas6 concentrations were significantly lower among patients with new-onset T2DM and its value was inversely correlated with fasting glucose. Plasma Gas6 is associated with altered glucose tolerance, inflammation, and endothelial dysfunction [28].

In a study, although plasma Gas6 concentrations were higher in women than in men, the difference was not statistically significant. Therefore, both male and female patients were included in this study. They also reported that Gas6 concentrations were negatively correlated with insulin resistance, abdominal obesity, E-selection, and IL-6 [29].

Lee et al. investigated the relationship between Gas6 gene polymorphism (c.834+7G > A) and T2DM pathogenesis. They divided the individuals of the study group into different subgroups according to Gas6 genotypes such as AA, GA, and GG. As a result, they showed that subjects with the AA genotype had higher Gas6 levels and lower levels of glucose, HbA_1c, HOMA-IR, and triglycerides compared to subjects with the GG genotype. They reported that the AA genotype of the Gas6c.834+7G>A polymorphism might have a protective role against T2DM [30].

Cavet et al. demonstrated that varying glucose concentration in vascular smooth muscle cells had a significant effect on the Gas6/Axl signal. They reported that Gas6/Axl was stimulated by low glucose levels, caspase 3 expression was decreased, the PI3K/Akt/mTOR pathway was activated, and through this way, Gas6/Axl showed an anti-apoptotic effect and increased cell viability [31].

Axelrod and Pienta have reported that Axl plays a critical role in immune system response and cancer. They have suggested that Axl is generally expressed at higher levels in the disease state compared to normal tissue, and therefore it has an oncogenic role for the receptor [32].

In addition to the Gas6-dependent activation, Axl has been shown to have ligand-independent activation. In breast cancer cells, activation of Axl has been reported to occur via NF-kB of matrix metalloproteinase-9 (MMP9) [33].

In this research, there was a significant decrease in Gas6 and Axl levels after metformin and metformin-insulin combination treatment in Met or Met + Ins groups. When all groups compared each other six months after treatment, only Axl levels were found to be significant. This may be due to the increased expression of Axl independent of its ligand.

The antitumorigenic potential of metformin has been tested in clinical trials in the treatment of several cancer types [34]. Kim et al. reported that metformin inhibited Axl and Tyro3 expression, resulting in antiproliferative activity in chemotherapy-resistant ovarian cancer cells. They reported that one of the therapeutic effects of metformin was to control the proliferation and chemoresistance of ovarian cancer cells by decreasing Axl and Tyro3 activation [35].

Saito et al. have reported that metformin reduces the proliferation of leukemia cells, inhibits Axl, and down-regulates other TAM receptor tyrosine kinase (RTK). They have also shown that metformin enhances the effect of doxorubicin, a chemotherapeutic drug [36].

In this study, in the groups not receiving metformin treatment (control group and DER group), there were no statistically significant differences in serum Gas6, Axl, and sAxl values at the 6th month compared to the baseline. However, a statistically significant decrease was found in serum Gas6 and Axl values in the Met group and Met + Ins group at 6 months compared to the baseline. According to this result, it was observed that the use of metformin decreased Gas6/Axl levels, while at the same time, this effect continued with the use of insulin.

sAxl, which binds Gas6, functions as a “decoy receptor” that eliminates the Axl signal and the mitogenic effects caused by Gas6. In this study, no statistically significant differences were observed in serum sAxl values. This may be because Gas6 binds at levels similar to sAxl which acts as the decoy receptor. Accordingly, the reason for the significant difference only in Axl levels in the evaluation of parameters between groups may have been caused by the binding of Gas6 to sAxl [12].

Six months after the treatment, a significant decrease was observed in FBG and HbA_1c values in the Met and Met + Ins groups, but there was no statistical difference in the control and DER groups. There was also a decrease in Gas6 and Axl values in the Met and Met + Ins groups. However, it was found that the 0th and 6th months FBG and HbA_1c levels of the study groups did not significantly correlate with the corresponding Gas6, Axl, and sAxl parameters.

When the groups that did not use drugs were examined, a highly significant positive correlation was found between Gas6 and Axl, and a moderate statistically significant positive correlation was found for sAxl. A moderate positive correlation was found between Axl and sAxl, while a significant moderate positive correlation was found between Gas6 and Axl in the Met group using metformin for six months. In the Met + Ins group, which used insulin combined with metformin, a significant moderate positive correlation was found between Axl and sAxl. Within the groups, Gas6 and Axl levels were found to be decreased in Met and Met + Ins groups due to drug use. However, the decrease in Axl levels in the groups may be due to the small sample size and the individual differences (AA, AG, GG polymorphism) of our sample group. In this study, the possible mechanisms of glucose intolerance and changes in the Gas6/Axl pathway with the use of metformin in patients with T2DM were investigated. As a result of this study, blood glucose levels were found to be regulated with the use of metformin, and the Gas6/Axl pathway was shown to be inhibited. In addition, while metformin regulates insulin resistance, hypertension and hyperlipidemia, a new function has been added to its pleiotropic effects: Inhibitory effect on the Gas6/Axl pathway. In conclusion, these results suggest that the use of metformin can be effective in reducing cancer cases or potential cancer cases in people with T2DM, and it can be used for a better prognosis. Considering the increasing rate of diabetic cases and the cancer incidence in these cases, documenting the importance of metformin will be very useful for investigations involving diabetic cases associated with various cancer types.

This study has some limitations: Patients with T2DM who had chronic diseases (hypertension, cardiovascular disease, dyslipidemia, chronic kidney disease) other than T2DM, diabetes duration exceeds 15 years (HbA_1c≥5.7%), who smoked, and who used alcohol were not included in the study. In particular, drug-using groups were formed from patients who would just start the drug for treatment. Patients who refused treatment sometime after the onset of treatment were excluded from the study. In addition, T2DM patients treated with other drugs (SGLT/Incretin-based therapies) were also excluded. These exclusions caused the sample size to be small.

Conclusions

Metformin is a widely used pharmacological agent for the treatment of T2DM. Patients with diabetes mellitus are at an increased risk of susceptibility to cancer. Blood glucose levels were found to be regulated with the use of metformin, and the Gas6/Axl pathway was shown to be inhibited. While metformin regulates insulin resistance, hypertension, and hyperlipidemia, a new function has been added to its pleiotropic effects: inhibitory effect on the Gas6/Axl pathway. The use of metformin can be effective in reducing cancer cases or potential cancer cases in people with T2DM, and it can be used for a better prognosis.