Abstract
Objective.
We aimed to investigate the potential relationship between plasma alarin levels and type 2 diabetes mellitus (T2DM).
Patients and Method.
We included 154 participants, divided into four groups in a cross-sectional study design. The first group includes patients with T2DM without complications (n=30), the second group patients with T2DM with microvascular complications (T2DM-noC n=32), the third group patients with T2DM with macrovascular complications, T2DM-MV (n=32) and the last group is the healthy control group (n=60).
Results.
In our study 94 patients were diabetic; 47 females and 47 males. The control group consists of 60 people, 30 women and 30 men. It was found that these had a significant (p>0.05) variation in serum alarin levels among the T2DM (T2DM-noC=3.1±0.7 ng/mL T2DM-mV=2.8±0.4 ng/mL, T2DM-MV= 3.6±0.4 ng/mL) versus control group (15.6±2.6).
We failed to find a significant variation of serum alarin levels (p>0.05) between T2DM subgroups. Serum alarin levels were significantly higher among control patients (p<0.05). There was no difference between diabetic sub-groups.
Conclusion.
We concluded that serum alarin levels in patients with T2DM are lower than in normal people. Further studies are needed to investigate the possible prognostic value of alarin in clinical practice in T2DM.
Keywords: Type 2 diabetes mellitus, diabetic complications, alarin level
INTRODUCTION
Diabetes mellitus is a complicated persisting chronic disease (1). It affects many people and stands among the world health problems having financial burdens on the public medical health systems (2). Diabetes is known and established as world’s most prevalent metabolic disease and its spreading is increasing every day (3), with two main subtypes, type 1 and type 2 DM.
Alarin belongs to the galanin family of peptides isolated from human neuroblastic tumoral gangliocytes (5) which contains galanin and galanin like peptide (GALP) (6, 7). Many studies showed that the central galanin was found to simplify GLUT4 translocation, while galanin antagonist increases insulin resistance by reducing glucose transporter 4 effect in rat adipocytes. Intracerebroventricular administration of galanin antagonist sustains insulin resistance in adipocytes of type 2 diabetic trained rats. It was observed that insulin sensitivity is present in both types of diabetic and healthy rats (8-10).
A small number of studies have been focused on investigating serum alarin levels as a risk factor or as a diagnostic marker for T2DM mellitus in humans. Most of the studies were conducted in rats. In our study we tried to investigate alarin levels in T2DM without complications, with micro and macrovascular complications.
PATIENTS AND METHODS
Participants
The cross-sectional study was conducted on 154 volunteer participant and patients who were divided into four groups. The 1st group consisted of patients with T2DM without any complications, T2DM-noC (n=30), the second group patients with T2DM and microvascular complications, T2DM-mV (n=32), the third group includes patients with T2DM and macrovascular complications, T2DM-MV (n=32) and the last group is the healthy control group (n=60).
The follow-up of type 2 diabetic patients was done with fasting-postprandial blood glucose levels and HbA1c levels.
All volunteer participants signed consent forms to join this study. The methodology and protocol used in this research was duly approved by the Ethical Committee of the Firat Medical Faculty, Turkey. This research was designed and conducted in line with the Declaration of Helsinki.
Samples
Venous blood specimens were taken from each participant. They were taken at the same time of the day and placed in sample tubes including aprotinin after numbering the blood samples and quickly transferred for analysis to the laboratory. These blood samples were subjected to centrifugation (3500 rpm) for 10 min. at desired temperature (+ 4°C). The acquired plasma samples were stored at – 20°C until they were analysed.
Biochemical measurements
Measurements of plasma alarin concentration were made using a commercial kit (catalogue number: yhb3169hu, Shanghai Yehua Biological Technology Co., Shanghai, China) with usage of ELISA. The results were shown as ng/mL (CV [%] = SD / mean × 100; intraassay: CV < 10%; interassay: CV < 12%; sensitivity: 2.23 ng/mL).
Exclusion criteria
Generally our exclusion criteria were: advanced cardiovascular disease, hepatic failure, chronic renal failure, previous malignancies, T1DM, morbid obesity (BMI> 40 kg/m2), hyperthyroidism, hypothyroidism, pregnancy, mental retardation, chronic obstructive pulmonary disease, smoking, use of anticoagulant or antiplatelet medications and refusal to participate in the study.
The first group consists of T2DM patients without complications. They were selected from type 2 diabetic patients without micro- or macrovascular complications.
The second group contains T2DM patients with microvascular complications. Patients with diabetic neuropathy, retinopathy and nephropathy were included in this group.
The third group includes T2DM patients with macrovascular complications. Patients with non-advanced cardiovascular and cerebrovascular events due to diabetes and diabetic feet were included in this group.
The healthy control group was selected from healthy individuals without a history of chronic disease or chronic drug usage.
Statistical analysis
SPSS package program, version 21 was used for the statistical evaluations. The data were calculated as mean ± standard deviation. The Kolmogorov–Smirnov test (KST) was used for further evaluation to find if the variables showed a normal distribution. The parametric data were subjected to ANOVA (analysis of variance) ensued by post hoc Tukey test separate means. The data (Non parametric) were subjected to analysis using Mann–Whitney U test to test statistical significance. Categorical data were subjected to analysis using chi-square test. Furthermore, Pearson correlation (two tailed) analysis was performed to find any relationship among studied parameters. ANCOVA (Analysis of covariance) studies were performed to adjust variables related to age. The statistical differences among the means were evaluated significant if p values were < 0.05.
RESULTS
The laboratory, clinical and demographic characteristics of our study are summarized in Table 1. In our study 94 patients were diabetic, 47 were females and 47 were males. The healthy control group consists of 60 individuals. 30 were females and 30 were males.
Table 1.
Demographic, clinical and laboratory characteristics of the T2DM and control groups
| Healthy control (HC) group (n=60) | T2DM group (uncomplicated) (n= 30) | Microvascular complication T2DM group (n=32) | Macrovascular complication T2DM group (n=32) | p*value | |
|---|---|---|---|---|---|
| Mean age (years) | 53.7±10.4 | 53.8±12.7 | 54.8±10.8 | 55.4±11.4 | 0.224 |
| Serum alarin (ng/mL) | 15.6±2.6 | 3.1±0.7 | 2.8±0.4 | 3.6±0.4 | <0.05 |
| Urea (mg/dL) | 28.9±7.9 | 33.5±10.2 | 34.2±12.2 | 48.3±23.2 | <0.05 |
| Creatinine (mg/dL) | 0.6±0.1 | 0.7±0.3 | 0.8±0.7 | 2.1±0.9 | <0.05 |
| Glucose (mg/dL) | 90±9.1 | 146.4±54.1 | 170.5±57.5 | 170.6±68.6 | <0.05 |
| HbA1c% | 4.7±0.8 | 8.4±2.7 | 8.8±2.5 | 7.6±1.9 | <0.05 |
| ALT (U/L) | 20.9±5.1 | 19.2±14.8 | 17.6±8.5 | 18.1±6.4 | 0.449 |
| AST(U/L) | 20.1±8.8 | 23.5±10.3 | 17.9±5.9 | 18.5±4.3 | 0.442 |
| BMI (kg/m2) | 28.5±3.9 | 30.6±6.2 | 29.7±5.4 | 31.3±4.3 | 0.325 |
| LDL cholesterol (mg/dL) | 133.4±20.2 | 134.9±19.1 | 142.1±34.4 | 147.6±37.8 | 0.323 |
| HDL cholesterol (mg/dL) | 42.2±16.5 | 36.1±8.6 | 34.7±13.9 | 33.6±8.6 | 0.060 |
| Triglyceride (mg/dL) | 144.8±9.7 | 143.5±37.6 | 173.5±56.1 | 187.4±57.2 | 0.065 |
| Alb (g/dL) | 4.5±0.3 | 4.2±0.4 | 4.1±0.3 | 3.8±0.9 | 0.064 |
| Htc (%) | 40.2±3.4 | 39.5±4.1 | 42.8±7.5 | 41.2±6.4 | 0.598 |
Data are expressed as means ± standard deviation (minimum-maximum); p* value is for comparison of the four groups through ANOVA test.
T2DM, type 2 diabetes mellitus; HbA1c, hemoglobin A1c; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; LDL, low density lipoprotein; HDL, high density lipoprotein; Alb, albumin; Htc, hematocrit.
Gender age distribution and BMI (body mass index) were not significantly different among the four groups.
Alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, hematocrit, triglyceride levels (high density lipoprotein) and cholesterol (low density lipoprotein), were statistically similar among the groups (p> 0.05).
It was found that these had a significant (p< 0.05) variation in serum alarin levels among the T2DM versus control group. The researchers failed to find a significant variation between T2DM groups about serum alarin levels (p>0.05, all). Serum alarin levels were significantly higher among control patients (p<0.05) (Fig. 1).
Figure 1.
Comparison of serum alarin levels in diabetic groups and in the healthy control group.
Plasma glucose and HbA1c levels showed a significant increase in the T2DM groups (p < 0.05, all). There was a negative correlation between plasma blood glucose levels and alarine levels in all groups (p<0.05) (Fig. 2).
According to our study urea and creatinine levels were found significantly (p < 0.05, all) higher in T2DM with macrovascular complications. However, serum alarin levels had a negative significant (p=0.4) correlation with creatinine (r=-0.057).
DISCUSSION
Alarin is one of the very recently discovered members of the galanin peptides. Our aim was to investigate the levels of alarin in patients with T2DM.
Hypothalamic neuropeptides affected the major constituent of energy metabolism, body weight and metabolic rate, body temperature, by performing either catabolic or all anabolic effect. Galanin is related with the number of central and peripheral receptor-mediated activities that include nutrition, anterior pituitary hormone arrangement or regulation and pain, osmotic homeostasis and the energy balance (11), reproduction (12) and cognition (13).
Several human disorders including epilepsy, diabetes mellitus and Alzheimer have displayed a relationship of failure/disturbances in the galaninergic system signalling. For this reason galanin and its receptors submit a promising target for pharmacology study and future treatment chances.
The regulative action of a number of members in the galanin peptide family, e.g. galanin and galanin-like peptide (GALP) have been newly recommended in homeostasis of energy. GALP is known as a hypothalamic regulator and for reproduction and energy balance. It has complicated roles for energy balance and in production of orexigenic effects. As it makes hypothalamic regulation of food intake in the short period on rats, it represents febrile and anorexigenic febrile impacts in the long period on mice and rats. Therefore, related thermoregulatory actions and food intake - and of GALP seem to be contradictory (14-17). Even though, its acute orexigenic action is well described in rats. Its complex participation for energy homeostasis regulation also including thermoregulation is still to be discovered.
Studies about any possible connection between diabetes and serum alarine levels are few and most of them are experimental. Recently, alarin has been shown to be found in the locus coeruleus and arcuate nuclei in mice and rats. Both nuclei are related to nutritional behavior (18).
Alarin was found to have no effect on body temperature. It increases the GnRH dependent LH secretion in female mice. In this study, alarin has been shown to be a neuromediator that affects reproductive hormone secretion and food intake e.g. other neuromediators of galanin peptide family (19-21).
In 2017, Miko et al. mentioned in their study that alarin was a central catabolic peptide that affects hyperthermic / hypermetabolic and disease behavior. They concluded that further research was needed (22).
It is confirmed that central galanin participates in induction of insulin resistance in animals. Experimental studies determined that the main treatment using alarin significantly increased 2-deoxyglucose uptake, the plasma adiponectin levels, the vesicle-associated membrane protein 2 as well as glucose transporter 4 (GLUT4) in plasma membranes to the total cell membranes in adipocytes, along with reduced blood glucose levels, plasma retinol-binding protein 4 levels, acute phase inflammatory markers and plasma adiponectin levels.These results suggested that the main alarin projective system could be helpful for glucose uptake and induce insulin sensitivity through the increase in GLUT 4 and GLUT4 translocation from intracellular area to plasma membranes in T2DM (23-25). It was observed that insulin resistance decreased as the level of alarin increased. So we suggest that alarin had an effect on plasma glucose in T2DM patients.
Liang et al. (9) showed that the major effects of central release of alarin results in significantly increased GLUT4 in plasma membranes and total cell membranes of adipocytes checked, using diabetic controls. They suggest that the central injection of alarin may increase not only GLUT4 supply, but also GLUT4 interchange from cytoplasmic membranes to plasma membranes. This suggests that administration of central alarin may be beneficial for glucose uptake and insulin sensitivity in adipocytes in T2DM. In other words, they indicated that alarin can lower blood glucose by increasing insulin sensitivity. This hypothesis is supported by our results.
In our study we found a significant difference between type 2 diabetes mellitus and healthy control groups regarding blood alarin levels. Serum alarin levels were significantly higher in the control group. There was a statistical similarity between diabetic sub-groups. This research displayed understanding of antidiabetic action in the central alarin projective system and proposed that the alarin receptor may have a potential therapeutic focus for T2DM.
Even though alarin is a member of the presumably anabolic galanin peptide family and is orexigenic, it can be characterized as a catabolic peptide in T2DM cases according to the results of our study and similar experimental studies. The data propose that complex action of alarin for regulation of energy homeostasis should be handled carefully and reevaluated for further investigations by focusing on its acute and long-period effects on metabolic rate, body weight and food intake.
In the literature, to the best of our knowledge, there are no studies on the relationship between T2DM and alarin levels in humans.
Limitations of study
The present study has several limitations. Patients can be taken from wider and different regions. A few measurements of alarin could be taken during the day according to the fasting level. Weight gain and alarine involvement could be studied separately. The study is cross-sectional, so no data are available during treatment. In addition, the analysis was not done according to treatment i.e. insulin versus oral AD drugs or other treatments.
In conclusion, serum alarin levels in patients with T2DM are lower than in normal people. Further studies are desired to search the possible prognostic value of alarin in clinical practice for T2DM patients.
Conflict of interest
The authors declare that they have no conflict of interest.
Funding
All authors of the paper did not receive any donation from any agency.
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