INTRODUCTION:

Type 2 diabetes mellitus (T2DM) is a progressive disorder with a gradual and asymptomatic onset that can lead to secondary complications. Long-term complications of T2DM include the eye, kidney, nerve, heart, and vessels. These complications are caused by long-term hyperglycemia with disturbances in carbohydrate, protein, and fat metabolism due to a defect in either insulin secretion or action, or both¹.

Zinc was first discovered as a constituent of insulin crystals in 1934, and since then it has been considered an essential constituent of the body's biological processes².

Low levels of zinc in the body affect insulin formation which can lead to inadequate insulin and diabetes^1,². In diabetes, many causes of zinc deficiency have been proposed. A defect in metallothionein (MT), an important protein in zinc homeostasis, is one of the major contributors to zinc insufficiency³. Further, diabetes on its own can encourage zinc homeostasis disorder⁴. Because of the extensive loss of zinc in urine caused by hyperglycemia, all forms of diabetic patients suffer from zinc deficiency⁵. The early reports about low zinc level in diabetic cadavers, along with a current finding, that identified the connection between zinc, T2DM, and inheritance of the beta-cell zinc transporter-8 (ZnT8) gene defect attracted the researchers to study the role of zinc in the etiology of T2DM and its beneficial therapeutic role¹.

Zinc administration has been shown to aid wound healing, slow the development of diabetic complications, bring glycated hemoglobin A1c% (HbA1c) to normal levels, and raise serum zinc concentrations in diabetics. This study aimed to investigate the effects of adding zinc to metformin therapy for eight weeks on glycemic control, serum insulin, and serum C-peptide levels as well as insulin resistance in comparison to metformin therapy alone in T2DM patients.

MATERIALS AND METHODS:

A total of 67 diabetics with T2DM (31female; 36male), were enrolled in this study; the patients were on metformin (1000-2250mg/day). The patients were assigned into two groups: the first group (group one) was the interventional group (T2DM; n=35; 16female/19male) treated with metformin plus oral zinc 50mg on an alternate day. The second group (group two) was the control group (T2DM; n=32; 15female/17male) treated with metformin only. The patient's ages ranged from 31 to 64 years. Patient consent and ethical approval have been fulfilled according to local standards. All the patients who were involved in this study were checked for zinc status before starting the study, and accordingly, they were divided into two groups. Those who had low zinc levels were included in group one, and the others, with normal levels, were included in group two.

Inclusion criteria: Any patient with T2DM, who had no other illnesses and is not taking any medications other than metformin met the inclusion criteria. They must have been on metformin for at least the previous 6 months. A dose of 50mg zinc tablet was given on alternate days as zinc gluconate for eight weeks in addition to metformin. The colorimetric method by spectrometry was used to calculate FBG levels. As well, serum zinc levels were measured by the spectrometry method. The reagent kit for estimating serum zinc level was (LTA S. r. l, Italy). The adopted normal serum zinc range was 70-115μg/dl.

Serum insulin measurement is a quantitative estimate of insulin in plasma using the immune enzymatic assay in the TOSOH/AIA analyzer technique. As well, C-peptide was measured in the TOSOH/AIA analyzer technique, a quantitative estimation of C-peptide in serum was required using an immune enzymatic assay to estimate C-peptide level in serum. By using Mathews et al., formula⁶, FBG mmol/L and fasting serum insulin (FSI) (μU/ml) was applied to estimate insulin resistance using HOMA-IR formula (Homeostasis Model Assessment for Insulin Resistance) Using the BIOHERMES A1C EZ pack, glycated hemoglobin A1c% (HbA1c) was measured in whole blood using affinity immune-chromatography.

Statistical analysis: Data presented as mean±SD. The interventional and control groups were compared using an independent T-test (Minitab V18), while the before and after data for each group was compared using a paired T-test of two means. A significant difference was described as a p value of ≤0.05.

RESULTS:

The baseline comparison of the parameters between groups one and two revealed no major differences (Table 1).

Table 1. Characterization of baseline level of studied parameters.

Parameters	Group one	Group two	p-value
Serum zinc (μg/dl)	68.6 ± 11.2	74.0 ± 15.7	0.107
BMI (kg/m²)	30.37 ± 3.19	30.40 ± 3.64	0.973
FBG (mg/dl)	185.3 ± 40.1	173.7 ± 33.2	0.204
HbA1c %	8.45 ± 1.63	8.20 ± 1.27	0.492
FSI (µIU/ml)	9.74 ± 4.97	9.96 ± 4.37	0.847
HOMA-IR	4.44 ± 2.61	4.38 ± 2.41	0.919
C-peptide (ng/ml)	2.29 ± 0.75	2.48 ± 0.70	0.296

Comparing the start point parameters and after eight weeks values in group one who used metformin plus zinc showed significant changes in all of the studied parameters except insulin resistance. HOMA-IR was less after eight weeks of therapy but statistically non-significant (Table 2).

Table 2. Characterization of studied parameters following zinc therapy.

Parameters	Group one		p value
Parameters	Baseline	+ Zinc	p value
Serum zinc (μg/dl)	68.6 ± 11.2	85.4 ± 14.9	0.001
BMI (kg/m²)	30.37 ± 3.19	29.31 ± 3.32	0.001
FBS (mg/dl)	185.3 ± 40.1	128.2 ± 33.9	0.001
HbA1c%	8.45 ± 1.63	7.22 ± 1.16	0.001
FSI (µIU/ml)	9.74 ± 4.97	12.27 ± 4.67	0.001
HOMA-IR	4.44 ± 2.61	3.90 ± 2.00	0.198
C-peptide (ng/ml)	2.29 ± 0.75	2.75 ± 0.73	0.001

Comparing the baseline parameters of group two and those after eight weeks of zinc therapy with metformin alone showed only significant reductions in BMI, FBG and insulin resistance (HOMA-IR), but no changes in HbA1c, FSI, and C-peptide levels (Table 3).

Table 3. characterization of the studied parameter following zinc-free therapy

Parameters	Group two		p value
Parameters	Baseline	-Zinc	p value
Serum zinc (μg/dl)	74.0 ± 15.7	73.8 ± 14.4	0.927
BMI (kg/m²)	30.40 ± 3.64	29.93 ± 3.22	0.012
FBS (mg/dl)	173.7 ± 33.2	148.2 ± 23.9	0.001
HbA1c%	8.20 ± 1.27	8.00 ± 1.13	0.224
FSI (µIU/ml)	9.96 ± 4.37	10.18 ± 3.19	0.734
HOMA-IR	4.38 ± 2.41	3.71 ± 1.93	0.015
C-peptide (ng/ml)	2.48 ± 0.70	2.64 ± 0.97	0.386

Comparing the data of the two groups at the end of the study (Table 4), the glycemic control data (FBG & HbA1c), were significantly better in group one who used metformin plus zinc than in group two patients who used metformin alone. Fasting serum insulin was, also, higher in group one versus group two at the end of the study, but C-peptide and HOMA-IR were not different (Table 4).

Table 4. Comparison of studied parameters between both group (with versus without zinc)

Parameters	Group one	Group two	p value
Serum zinc (μg/dl)	85.4 ± 14.9	73.8 ± 14.4	0.002
BMI (kg/m²)	29.31 ± 3.32	29.93 ± 3.22	0.444
FBS (mg/dl)	128.2 ± 33.9	148.2 ± 23.9	0.007
HbA1c %	7.22 ± 1.16	8.00 ± 1.13	0.006
FSI(µIU/ml)	12.27 ± 4.67	10.18 ± 3.19	0.038
HOMA-IR	3.90 ± 2.00	3.71 ± 1.93	0.694
C-peptide (ng/ml)	2.75 ± 0.73	2.64 ± 0.97	0.598

DISCUSSION:

The present study confirmed that diabetics are characterized by low zinc plasma levels with subsequent deterioration of metabolic parameters, such as insulin, FBS, and c-peptides. Correspondingly, zinc supplementation has shown improved metabolic parameters in the favor of diabetic patients compared to the control zinc-free group.

Zinc supplementation in group one has a major effect on BMI, according to our findings, which was in agreement with Payahoo et al., research⁷. Low zinc levels are linked to alterations in appetite, as proposed by Marreiro et al., as zinc affects appetite through changes in neurotransmitter metabolism in the hypothalamus induced by the activity of zinc on the leptin receptors and leptin system⁸. Another influence of zinc on BMI is the zinc's antioxidant properties and its function in antioxidant production and action. Low levels of zinc weaken antioxidant mechanisms, which can lead to gene mutation and obesity, according to Prasad³.

The zinc's insulin-mimetic effect lowers FBG and insulin resistance and improves insulin sensitivity, resulting in a lower BMI⁹. The high FBG at baseline was because most of the enrolled patients did not visit a doctor regularly, and they might not be aware of the value of keeping their FBG under control.

Zinc mimics insulin activity by inhibiting tyrosine kinase phosphate 1B (PTP-1B), a key regulator of the insulin receptor downstream signaling pathway. PTP-1B is the most studied target for zinc's insulin mimicking effect. Zinc's insulin-mimetic effects are, also, achieved by activating the phosphoinositol 3 kinase/protein kinase B cascade¹⁰. Zinc also prevents glucose toxicity and, as a result of its action on free radicals and preserving the action of superoxide dismutase and metallothionein in islet cells, it may contribute to maintaining beta-cell mass and insulin content¹¹.

Zinc, further, inhibits gluconeogenesis, and glucagon does not affect this. Zinc supplementation also increases glycolytic enzyme activity in a time and concentration-dependent manner. However, the effects of zinc and insulin were not additive, and zinc pretreatment prevented insulin stimulation of the glycolytic enzyme². Finally, zinc secreted at the same time with insulin from beta-cells regulates hepatic insulin clearance¹. Each of the above-mentioned roles gives a simple and complete description of zinc's effects and benefits concerning glucose homeostasis.

According to Jayawardena et al., meta-analysis, zinc supplementation has a major impact on diabetic patients, lowering FBG and HbA1c¹². The results of Al- Maroof with Al- Sharbatti, and that of Khazdous were consistent with our findings¹³. Zinc supplementation recovers T2DM symptoms in animal and human studies, lowering hyperinsulinemia and FBG in obese mice and lowering HbA1c and cholesterol in humans, according to Chabosseau and Rutter¹. Hussain et al., reported that zinc and melatonin lower FBG significantly¹⁴. In contrast, Bletate et al., concluded that zinc supplementation for four weeks did not affect FBG, lipid profile, or insulin resistance in middle-aged women with obesity and normal glucose tolerance. While a study by Kim and Lee found that giving zinc to Brazilian middle-aged women for four weeks at a dose of 30 mg daily reduced insulin resistance and FSI, but had no impact on FBG levels¹⁵.

The difference in HbA1c was highly significant after eight weeks of zinc administration but there were no major variations in the control group during this period. Jafarnejad et al., in their meta-analysis, examined 20 studies on zinc supplementation in diabetes reported that zinc significantly lower HbA1c, a finding that is consistent with our findings¹⁶. Similarly, another meta-analysis by Ranasinghe, et al., reported that zinc therapy significantly decreased HbA1c². In contradiction to the current research, Afkhani-Ardekani et al., found that zinc therapy had no impact on HbA1c¹⁷.

The effect of zinc administration on FSI was clear and positive after eight weeks, with a highly significant difference from baseline. Since zinc can control the secretion of insulin, in addition to insulin action, and clearance. It has important insulin-related effects, it increases the capacity of the secreting granules and the ability to store more insulin in beta-cells. The autocrine effect of zinc on beta-cells regulates insulin secretion by a negative feedback mechanism that decreases insulin secretion. Furthermore, when secreted with insulin, zinc has a paracrine effect on the nearby alpha cells, inhibiting glucagon secretion¹. In addition, the secreted zinc with insulin regulates the hepatic clearance of insulin¹^,¹⁶.

Oh, and Yoon demonstrated that zinc has a positive effect on long-term diabetes, with a substantial rise in serum insulin⁵. In contrast to our findings, Shidfar et al., found that zinc and vitamin A did not affect insulin levels in serum⁴. When administrating zinc with melatonin to patients with T2DM who were poorly regulated on metformin alone, Hussain et al., discovered that zinc improved glycemic regulation through a mechanism other than increasing serum insulin levels¹⁴.

The difference in the zinc group, regarding C-peptide, after eight weeks of follow-up, was highly significant. C-Peptide usually increases in an equimolar quantity to insulin¹⁸. C-peptide level increment in this study can be clarified by an increase in insulin secretion from beta-cells. In the Oh and Yoon report, patients with long-term diabetes and slightly low zinc level had a significant increase in C-peptide concentration after short-term zinc use⁵. However, Hussain et al., claimed that zinc supplementation does not affect C-peptide levels¹⁴.

Many studies show that zinc administration has no impact on insulin resistance¹⁹^,20. In a study similar to ours, Oh and Yoon found no substantial improvement in insulin resistance despite a real alteration in HOMA-IR. They attributed this to the small sample size and the fact that the diabetic participants were not obese, as well as the short follow-up period⁵^,21,22,23. This may be the case in our research. Similarly, Kim and Lee had found no substantial improvement in HOMA-IR in T2DM patients treated with zinc¹⁵^,24.

Low zinc levels are caused by reduced absorption and increased loss of zinc in urine caused by abnormal function of the kidney, although, some believe that genetic factors may be behind this. Hyperglycemia, which causes glucose loss in urine along with zinc, is another cause of low zinc levels¹¹. In their research, Al Maroof and Al Sharbatti discovered that the mean serum zinc level in diabetic patients is significantly lower than zinc level in healthy controls. According to them, supplementation at a dosage of 30 mg of zinc per day for three months substantially improved serum zinc levels, FBG, FSI level, and serum C-peptide level¹³^,25, which is following our study.

CONCLUSION:

Measuring zinc levels in serum of patients with T2DM is recommended. In those who have low zinc levels, adding zinc supplements to metformin was associated with better control of their glycemic status.

ACKNOWLEDGMENT:

The authors are very grateful to the University of Mosul, College of Medicine for their provided facilities, which helped in improving the quality of this work.

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Received on 16.06.2021 Modified on 06.07.2021

Research J. Pharm.and Tech 2022; 15(3):1184-1188.

DOI: 10.52711/0974-360X.2022.00198