INTRODUCTION:

Approximately one in 11 adults worldwide are living with diabetes, 90%-95% of whom have type 2 diabetes mellitus¹. Asia is a main region of the global epidemic of diabetes². Type 2 diabetic patients are at high risk for cardiovascular events³. Metformin is still the best recommended oral hypoglycemic agent in treating type 2 diabetes⁴. The European Association for the Study of Diabetes (EASD) and American Diabetes Association (ADA) recommended Metformin use regardless of patient’s body mass index, age and blood glucose level, because of its desirable control of lipids, blood glucose and weight⁵. On the other hand, GLC is an effective oral hypoglycemic agent that is commonly used in type 2 diabetes for years⁶.

According to the United Kingdom Prospective Diabetes Study , treatment with Metformin showed a reduction in myocardial infarction in obese diabetic patients⁷. A persisted benefit with Metformin use was observed after a follow-up study of 10 years⁸. In addition, Metformin use revealed a reduction in mortality in diabetic patients with atherosclerosis in an observational study⁹. Endothelial dysfunction is a main feature of diabetes which is associated with insulin resistance and increased blood glucose ¹⁰. A critical mediator to sustain proper endothelium function is NO and its bioavailability ¹¹. NO, well-known potent vasodilator, is generated by endothelial nitric oxide synthase (eNOS) in the vascular endothelium via oxidation of substrate L-arginine to L-citrulline¹². NO plays a critical role in maintaining vascular function and blood pressure regulation in healthy persons, whereas a reduction in NO bioavailability previously reported in patients with type 2 diabetes¹³.

In addition to its direct reduction in glucose availability in diabetic patients, Metformin can improve endothelial dysfunction by glucose-independent mechanism in prediabetic patients. Although the underlying mechanism behind the beneficial effect of Metformin on endothelial function remains undefined, few data exist. Several studies have suggested the potential role for Metformin in cardiovascular protection by increasing AMP-activated protein kinase (AMPK), resulting in activation of eNOS^16-18.

Even though the promising results by UKPDS¹⁹. There was a theoretical adverse cardiovascular effect of GLC²⁰. A prospective study for diabetic patients with atherosclerotic heart disease, impairment of left ventricular function was higher in patients received GLC than in those received insulin only²¹. Two studies revealed higher mortality rate in diabetic patients undergoing angioplasty for myocardial infarction on GLC^22,23. However, theoretically GLC has lower cardiovascular risk than the old-generation sulphonylurea²⁴. These variations suggest the requirement for additional demonstration of the cardiovascular safety of hypoglycemic agents.

Among the adipokines, APN hormone showing reduced circulating levels in obesity, type 2 diabetes and myocardial infarction²⁵. Moreover, APN demonstrated as a direct endogenous inhibitor for vascular inflammatory response and tumor angiogenesis²⁶. In addition, the correlation between APN and vascular function is complex, and studies have showed discrepant results with respect to the effect of Metformin and GLC on APN levels.

In this study, we compared between Metformin versus GLC effect on APN and NO in patients with T2DM.

MATERIALS AND METHODS:

This comparative case-control study comprised of 50 patients with T2DM aged between 32 and 58 years (both sexes), were classified into three groups; Group A: 17 type 2 patients (newly diagnosed as controls), Group B: 15 Metformin-treated patients (1000 mg/day) and Group C: 14 GLC-treated patients (5 mg/day) for a period of less than one year, achieved in Al Waffaa Centre for Diabetes/Mosul from May to November 2019. It was approved by the Ethical Committee of Nineveh health institution. Patients were diagnosed as type 2 diabetes based on ADA and WHO criteria. This present study exclude pregnant and breastfeeding women, patients taking other medications, dietary supplements-taking patients and patients have acute or chronic health conditions other than T2DM, patients undergoing medication changes over the study year, smokers and alcoholic patients. In all patients, body mass index (BMI) was calculated based on anthropometric data (height and weight).

After at least 10 hrs overnight fasting, blood samples were taken from patients with T2DM and incubated in water bath for 10 minutes at 37^○C, then centrifuged for 10 mins at 4,000x g. Except for fasting serum glucose (FSG) which was measured instantly, samples were stored at -20^○C for later use.

FSG was determined via enzymatic colorimetric method, using a kit supplied from BIOLABO kit (France). Enzyme linked immunosorbent assay machine was used to determine serum insulin, using a kit supplied from Monobind kit (USA). Fasting serum insulin and glucose were used to calculate insulin resistance through the equation:

HOMA-IR = Serum Insulin × Serum Glucose / 22.5.

APN hormone was determined by ELISA technique, using a kit supplied by USBIOLOGICAL (USA) . Serum NO was assessed by Greiss reagent . Briefly, 200 μl of supernatant and Griess reagent were added, then absorbance was measured at 540 nm by ELISA. NO concentration was estimated according to the standard curve of sodium nitrite.

Statistical analysis:

Statistical analyses were performed using GraphPad Prism. Two or multiple comparisons were used by performing Mann Whitney test and Kruskal-Wallis test, then by a Dunn's multiple comparisons test. All quantitative results were expressed as the mean value ± SD and statistical significance was set at p < 0.05.

RESULTS:

Demographic profile of untreated and treated patients with T2DM:

The current study included 50 patients with T2DM, with percentage of male and female (46% and 54%) in the ages between 35 and 54 years. Table 1 shows the demographic profile of patients with T2DM in our study. No significant differences have been found among three groups.

Table 1: The demographic profile of patients with T2DM

Parameter (unit)	Untreated	Metformin	GLC
Age (years)	45.51±5.723	47.11±6.134	42.68±6.019
BMI (kg/m²)	25.11±0.976	24.44±1.078	24.43±0.199
Duration of treatment (month)	-	8.09±2.901	8.111±3.132

Effect on glucose, serum insulin and HOMA-IR:

Compared to untreated and GLC-treated diabetic patients, Metformin-receiving patients revealed a significantly reduced HOMA-IR index and FSG level. Nevertheless, insulin was significantly higher in GLC-receiving group in comparison group A and B as shown in Table 2

Table 2: Diabetic profile of the study groups of patients with T2DM

Parameter (unit)	Group A	Group B	Group C
FSG (mmol/l)	11.99± 0.971	9.079± 0.5171 ^a**b	11.01± 0.8123
Insulin (μu/L)	9.039±0.7301	8.971±0.5029	8.989± 0.4939 ^a*
HOMA-IR	5.041±0.5051	3.581± 0.1923 ^a**b**	4.429± 0.3161

Results are set as mean value ± SD. ^a represents a differences between group B and C in contrast to group A; ^b represents a differences between group B and C.

Effect on plasma adiponectin and nitric oxide levels:

Untreated patients had a mean APN level of 5.976 µg/mL and mean NO of 8.506 µmol/L. The plasma APN levels were significantly higher in Metformin-treated patients (7.953 µg/mL) compared to newly diagnosed diabetic patients (5.976 µg/mL) and GLC-treated patients (5.564 µg/mL) (Figure 1A). Moreover, Metformin-receiving patients showed significantly higher concentration of plasma NO levels (10.44 µmol/L) compared to newly diagnosed (8.506 µmol/L) and GLC-treated patients (9.393 µmol/L) (Figure 1B). No significant correlations were noted between ADP and NO in all groups.

Figure 1: Effects of Metformin and GLC on serum (A) APN levels (B) NO levels. Values are represent as mean ± SD. Variations are statistically significant with (***p < 0.001; ****p < 0.0001) in comparison of group B and C in contrast to group A; whereas, # sets statistically significant differences in comparison between group B and C (#p < 0.05; ####p < 0.0001).

DISCUSSION:

The comparative effect of Metformin versus GLC on plasma APN and NO levels has not yet been evaluated in type 2 diabetic patients. In the current study, we demonstrated that Metformin improved the cardiovascular biomarkers more significantly than GLC and this effect was independent of changes in insulin level. Although GLC has been significantly increased insulin production, APN and NO levels has been increased with Metformin-treated patients.

In recent years, APN has been the focus of intense research. Increased circulating levels of APN have been previously established in Metformin-treated patients . The main findings of our study confirmed that APN was significantly higher in Metformin-receiving patients relative to that of GLC group is in line with results of Zulian et al. and Emini-Sadiku et al. . However, findings of other studies in type 2 diabetic patients treated with Metformin revealed no effect on serum APN levels ^32,33.

APN protein has been shown to possess anti-diabetic, anti-inflammatory and anti-atherogenic properties . However, it is still uncertain whether APN has a vasoprotective effect under pro-inflammatory conditions like untreated diabetes. In vivo and in vitro studies have supported this idea, which reported an inverse association between APN and insulin resistance^35,36. In our study, Metformin-treated patients had this negative correlation which suggested APN as a possible protective protein ³⁷.

Moreover, the beneficial effect of Metformin on circulating levels of NO have been demonstrated in Metformin-treated patients . In the current study, patients receiving Metformin showed significantly improved NO levels compared with those receiving GLC is supported with the findings of Liu et al. and Jojima et al.^38,40. In another study conducted by Kato et al, the in vitro inhibitory action of Metformin on lipopolysaccharide-dependent NO production has been established in macrophage cell line . Nevertheless, a recent study suggested that Metformin has a beneficial direct effect on endothelial function by increasing NO production and decreasing nitroxidative stress in rats, independent on its hypoglycemic effect . These variations indicating the diversity of NO pathways that should be taken into account when linking with endothelial function.

CONCLUSIONS:

In addition to its well demonstrated glucose lowering properties, Metformin treatment provides new insight for treatment of underlying vascular defect associated with diabetes mellitus. In future studies, it will be important to achieve a direct measurement for in vivo endothelial function and to determine whether and how agents targeting overall parameters to preserve endothelium.

ACKNOWLEDGEMENTS:

The authors are grateful to the University of Mosul for its continuous support and inspiration.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.01.2021 Modified on 30.03.2021

Research J. Pharm.and Tech 2021; 14(12):6409-6412.

DOI: 10.52711/0974-360X.2021.01108