Association of 5’AMP-activated protein kinase activity with disease duration and HbA1c content in leukocytes in diabetic patients

. Background. 5’AMP-activated protein kinase (AMPK) is an enzyme that controls the cell energy ba-lance. With energetic stress in the cell and an increase in the AMP concentration, ATP is replaced by AMP in the exchange centers, resulting in the allosteric activation of AMPK by phosphorylation of 172 threonine within alpha subunit of LKB1 complex in response to changes in cell energy or CAMKK β , which activates intracellular Ca 2+ . The purpose was to study the activity of the main energy sensor of cells — AMPK in leukocytes in patients taking insulin preparations, metformin, and other hypoglycemic drugs in association with disease duration and glycated hemoglobin content. Materials and methods. The diabetic patients receiving single-drug or combined therapy with insulin and its analogues, metformin, dapagliflozin and sulfonylureas were randomized into 5 groups: the first group — with an HbA1c level close to the norm — 6.9–7.6 %; the second group — 7.6–9.0 %; the third group — > 9 %; the fourth group > 10 %; the fifth group — > 11 %. To determine the amount of phospho-AMPK (p-Thr172), ELISA kits were used. To get the calibration curve for the AMPK determination, a kidney cell culture HEK293T of the human embryonic kidney was used, which is recommended by manufacturer as a positive control. Results. It was shown that with increase of blood HbA1c, the level of AMPK activity in leukocytes gradually decreased. With increase of blood HbA1c, the level of AMPK activity in leukocytes gradually decreased. The activity of AMPK in leukocytes of patients with HbA1c > 11 % was more than 3.5-fold lower compared to the group with 6.9–7.6 % of HbA1c; AMPK activity in leukocytes in patients with disease duration of 20 years was 3-fold lower. Thus, the AMPK activity in leukocytes may be an indicator of diabetic compensation in diabetic patients. Conclusions. With increase of blood HbA1c, the level of p-AMPK in leukocytes gradually decreased. AMPK activity in leukocytes in diabetes patients with disease duration of 20 years was 3-fold lower than in patients with 10-year experience.


Introduction
5'AMP-activated protein kinase (AMPK) is an enzyme that controls the cell energy balance. With energetic stress in the cell and an increase in the AMP concentration, ATP is replaced by AMP in the exchange centers, resulting in the allosteric activation of AMPK by phosphorylation of 172 threonine within alpha subunit of LKB1 complex in response to changes in cell energy or CAMKKβ, which activates intracellular Ca 2+ [1][2][3].
By direct phosphorylation of metabolic enzymes and transcription factors, AMPK stimulates catabo-lic processes -absorption of glucose, fatty acids and their conversion by mitochondrial oxidation and glycolysis. In addition, AMPK suppresses anabolic processes -the synthesis of glucose, glycogen and lipids in the liver [4].
With type 2 diabetes mellitus (DM) and obesity, its activity decreases, and the activity of protein kinases mTORC1/p70S6K increases, leading to phosphorylation of IRS and insulin resistance [5].
HbA1c, the major fraction of glycated hemoglobin, is formed by irreversible non-enzymatic glycation. It is the key parameter for monitoring the regulation of DM The purpose was to study the activity of the main energy sensor of cells - AMPK in leukocytes in patients taking insulin preparations, metformin, and other hypoglycemic drugs in association with disease duration and glycated hemoglobin content. Materials and methods. The diabetic patients receiving single-drug or combined therapy with insulin and its analogues, metformin, dapagliflozin and sulfonylureas were randomized into 5 groups: the first group - with an HbA1c level close to the norm - 6.9-7.6 %; the second group - 7.6-9.0 %; the third group -> 9 %; the fourth group > 10 %; the fifth group - > 11 %. To determine the amount of phospho-AMPK (p-Thr172), ELISA kits were used. To get the calibration curve for the AMPK determination, a kidney cell culture HEK293T of the human embryonic kidney was used, which is recommended by manufacturer as a positive control. Results. It was shown that with increase of blood HbA1c, the level of AMPK activity in leukocytes gradually decreased. With increase of blood HbA1c, the level of AMPK activity in leukocytes gradually decreased. The activity of AMPK in leukocytes of patients with HbA1c > 11 % was more than 3.5-fold lower compared to the group with 6.9-7.6 % of HbA1c; AMPK activity in leukocytes in patients with disease duration of 20 years was 3-fold lower. Thus, the and for assessing the risk of microvascular complications [6].
The purpose: taking into account the data obtained in clinical and experimental studies, we attempted to study the activity of the main energy sensor of cells -AMPK in leukocytes in patients taking insulin preparations, metformin, and other hypoglycemic drugs in association with disease duration and glycated hemoglobin content.

Materials and methods
The study was conducted in the Diabetology Department of the V.P. Komisarenko Institute of Endocrinology and Metabolism of NAMS of Ukraine. All patients signed informed consent to conduct further diagnostic and research study. Immediately after collection, the blood was layered over a layer of Histopaque 1077 (Sigma, USA) and centrifuged at RT in 15 ml conical Fal-con™ tubes.
The leukocytes collected were washed and frozen at -80 °C until use. The cells were lysed in the extraction buffer with inhibitors of proteases and phosphatases. To determine the amount of phospho-AMPK (phosphothreonine 172), enzyme-linked immunosorbent assay (ELISA) kit ab154468 (Abcam, UK) was used. The studies were carried out in triplets. The protein concentration in the lysate was determined using Novagen (USA) BCA protein assay kit. The measurements were carried out on a microplate reader (Bio-tek Instruments, USA) at a wavelength of 600 nm.
To get the calibration curve for the AMPK determination, a kidney cell culture HEK293T of the human embryonic kidney was used, which is recommended by manufacturer as a positive control. The OD values of samples obtained (0.005-0.04) are located on the calibration curve region almost perfectly coinciding with exponential theoretical curves that indicates no scattering of the data [7].
The results of the study are presented as M ± SD and M ± m, n = 31 (3-6 per group). To compare the data groups, Student's t-test was used. Values of P ≤ 0.05 were considered as significant.

Results
The patients received single-drug or combined therapy with insulin and its analogues, metformin, dapagliflozin and sulfonylureas. They were randomized into groups: the first group -with an HbA1c level close to the norm -6.9-7.6 %; the second group -7.6-9.0 %; the third group -> 9 %; the fourth group -> 10 %; the fifth group -> 11 %. In addition, the mean value for all patients (n = 31) was calculated.
AMPK activity was determined by the amount of phosphorylated Thr172 of α-subunit of the protein. Fig. 1 shows that the highest level of phospho-AMPK is observed in leukocytes in patients with low level of HbA1c -6.9-7.6 %, which is not much higher than the indices recommended for diabetic patients, and can be considered as a condition close to compensatory. With increase of blood HbA1c, the level of p-AMPK gradually decreased. The activity of AMPK in leukocytes in patients with HbA1c > 11 % was more than 3.5-fold lower compared to group with HbA1c of 6.9-7.6 % (Fig. 1).
In the following, we calculated the AMPK activity in association with average duration of diabetes mellitus. Two groups of patients with DM duration of ~ 10 and ~ 20 years were selected.
The Table 1 demonstrates that AMPK activity in patients with disease duration ~ 20 years 3-fold lower than in patients with 10-year experience.

Discussion
Thus, regardless of the method of treatment AMPK activity may be related to the degree of diabetes mellitus compensation that reflects the level of glycated hemoglobin, with which kinase activity is linked by an inverse relationship.
It should be noted that leukocytes contain up to 11 % of monocytes/macrophages and up to 40 % of lymphocytes. Both types of cells and first of all macrophages are the main source of inflammatory effectors that promote diabetic atherosclerosis and myocardial infarction [8][9][10]. Note: M ± m. Oxidative metabolism determines the inflammatory status of macrophages and the processes that can occur before endoplasmic-reticulum stress and the formation of NLRP3-inflammasomes. AMPK is at the crossroads of the metabolic-mediated inflammation of macrophages, controls the metabolism of mitochondria and, consequently, can determine the inflammatory status of macrophages [11][12][13][14].

Conclusions
1. With increase of blood HbA1c amount, the level of p-AMPK in leukocytes gradually decreased.
2. AMPK activity in leukocytes in diabetic patients with disease duration ~ 20 years 3-fold lower than in patients with 10-year experience.
3. The AMPK activity in leukocytes may be an indicator of diabetic compensation and disease duration in patients.

Conflicts of interests.
Authors declare no conflicts of interests that might be construed to influence the results or interpretation of their manuscript.