CC BY
106
UDC 616.379-008.64-002:577.2 DOI: 10.22141/2224-0721.15.1.2019.158688
L.K. Sokolova, V.M. Pushkarev, Yu.B. Belchina, V.V. Pushkarev, T.S. Vatseba, N.D. Tronko
SI "V.P. Komisarenko Institute of Endocrinology and Metabolism of NAMS of Ukraine", Kyiv, Ukraine
Association of 5'AMP-activated protein kinase activity with disease duration and HbA1c content in leukocytes in diabetic patients
For cite: Miznarodnij endokrinologicnij zurnal. 2019;15(1):23-26. doi: 10.22141/2224-0721.15.1.2019.158688
Abstract. Background. 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 CAMKKfi, which activates intracellular Ca2+. 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. Keywords: diabetes mellitus; 5'AMP-activated protein kinase; glycated hemoglobin
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 CAMKKp, which activates intracellular Ca2+ [1-3].
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].
HbAlc, the major fraction of glycated hemoglobin, is formed by irreversible non-enzymatic glycation. It is the key parameter for monitoring the regulation of DM
© «Ммнародний ендокринолопчний журнал» / «Международный эндокринологический журнал» / «International Journal of Endocrinology» («Miznarodnij endokrinologicnij zurnal»), 2019 © Видавець Заславський О.Ю. / Издатель Заславский А.Ю. / Publisher Zaslavsky O.Yu., 2019
Для кореспонденцм: Пушкарьов В.М., ДУ «1нститут ендокринологи та обмшу речовин iM. В.П. Комкаренка НАМН УкраТни», вул. Вишгородська, 69, м. КиТв, 04114, УкраТна; e-mail: pushkarev.vm@gmail.com
For correspondence: V. Pushkarev, State Institution"V.P Komisarenko Institute of Endocrinology and Metabolism of NAMS of Ukraine', Vyshgorodska st., 69, Kyiv, 04114, Ukraine; e-mail: pushkarev.vm@gmail.com
OpMNHOAbHi AOCAiA^eHHfl /Original Researches/
JEI
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 Falcon™ 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 (phospho-threonine 172), enzyme-linked immunosorbent assay
Figure 1. AMPK activity in leukocytes in diabetic patients after treatment with hypoglycemic drugs depending on the HbAlc level Notes: M ± SD, n = 31; * — difference compared to the group with HbAlc 6.9-7.6 % is significant, P <0.05; # — difference compared to the group with HbAlc < 9 % is significant, P < 0.05.
Table 1. AMPK activity in diabetic patients depending on disease duration
AMPK activity DM duration, years
0.0069 ± 0.0013 n = 5 20.25
0.0215 ± 0.0004 n = 3 9.5
Note: M ± m.
(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 i-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, dapa-gliflozin 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 a-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 [8-10].
iEI
Орипнальш дослiдження /Original Researches/
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 [11-14].
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.
References
1. Ruderman NB, Carling D, Prentki M, Cacicedo JM. AMPK, insulin resistance, and the metabolic syndrome. J Clin Invest. 2013 Jul;123(7):2764-72. doi: 10.1172/JCI67227.
2. Xiao B, Sanders MJ, Underwood E, et al. Structure of mammalian AMPK and its regulation by ADP. Nature. 2011 Apr 14;472(7342):230-3. doi: 10.1038/nature09932.
3. Racioppi L, Means AR. Calcium/calmodulin-dependent protein kinase kinase 2: roles in signaling and pathophysiology. J Biol Chem. 2012Sep 14;287(38):31658-65. doi: 10.1074/jbc.R112.356485.
4. Jeong KJ, Kim GW, Chung SH. AMP-activatedprotein kinase: An emerging target for ginseng. J Ginseng Res. 2014 Apr;38(2):83-8. doi: 10.1016/j.jgr 2013.11.014.
5. Saha AK, Xu XJ, Balon TW, Brandon A, Kraegen EW, Ruder-
man NB. Insulin resistance due to nutrient excess. Is it a consequence of AMPK downregulation? Cell Cycle. 2011 Oct 15;10(20):3447-51. doi: 10.4161/cc.10.20.17886.
6. Kojic Damjanov S, Deric M, Eremic Kojic N. Glycated hemoglobin A1c as a modern biochemical marker of glucose regulation. Med Pregl. 2014Sep-Oct;67(9-10):339-44.
7. Sokolova LK, Pushkarev VM, Belchina YB, Pushkarev VV, Tronko MD. Effect of combined treatment with insulin and metformin on 5'AMP-activatedprotein kinase activity in lymphocytes of diabetic patients. DopovNac akadnauk Ukr. 2018;(5):100-104. doi: 10.15407/ dopovidi2018.05.100.
8. Steinberg GR, Schertzer JD. AMPK promotes macrophage fatty acid oxidative metabolism to mitigate inflammation: implications for diabetes and cardiovascular disease. Immunol Cell Biol. 2014 Apr;92(4):340-5. doi: 10.1038/icb.2014.11.
9. Pushkarev VM, Sokolova LK, Pushkarev VV, Tronko MD. The role of AMPK and MTOR in the development of insulin resistance and type 2 diabetes. The mechanism of metformin action (literature review). ProblemiEndokrinnoiPatologii. 2016;(3):77-90. (in Ukrainian).
10. Galic S, Fullerton MD, Schertzer JD, et al. Hematopoietic AMPK P1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity. J Clin Invest. 2011 Dec;121(12):4903-15. doi: 10.1172/JCI58577.
11. Hardie DG, Ross FA, Hawley SA. AMP-activated protein kinase: a target for drugs both ancient and modern. Chem Biol. 2012 Oct 26;19(10):1222-36. doi: 10.1016/j.chembiol.2012.08.019.
12. Klok MD, Jakobsdottir S, Drent ML. The role of leptin and ghrelin in the regulation of food intake and body weight in humans: a review. Obes Rev. 2007 Jan;8(1):21-34. doi: 10.1111/j.1467-789X.2006.00270.x.
13. Galic S, Oakhill JS, Steinberg GR. Adipose tissue as an endocrine organ. Mol Cell Endocrinol. 2010 Mar 25;316(2):129-39. doi: 10.1016/j.mce.2009.08.018.
14. Kirchner H, Heppner KM, Tschop MH. The role of ghrelin in the control of energy balance. Handb Exp Pharmacol. 2012;(209):161-84. doi: 10.1007/978-3-642-24716-3 7.
Received 07.02.2019 ■
Соколова А.Х., Пушкарев В.М., Бельчина Ю.Б., Пушкарев В.В., Вацеба Т.С., Тронько Н.Д
ГУ «Институт эндокринологии и обмена веществ им. В.П. Комиссаренко НАМН Украины», г. Киев, Украина
Ассоциация активности 5'АМФ-активированной протеинкиназы с длительностью заболевания и содержанием HbA1c в лейкоцитах пациентов с сахарным диабетом
Резюме. Актуальность. 5'АМФ-активированная про-теинкиназа (АМФК) — фермент, который управляет энергетическим балансом клетки. При энергетическом напряжении в клетке и увеличении концентрации АМФ АТФ заменяется в обменных центрах АМФ, приводя к аллостерической активации АМФК фосфорилированием треонина-172 а-субъединицей ЦКВ1-комплекса в ответ на изменения в клеточной энергии или САМККр, который активизирует внутриклеточный Са2+. Цель исследования — изучить деятельность главного сенсора энергии клеток — АМФК в лейкоцитах пациентов, получающих препараты инсулина, метформин и другие сахароснижающие лекарственные средства в зависимости от продолжительности болезни и концентрации гликированного гемоглобина (НЬА1с). Материалы и методы. Пациенты с сахарным
диабетом, получавшие моно- или комбинированную терапию инсулином и его аналогами, метформином, да-паглифлозином и производными сульфонилмочевины, были разделены на 5 групп: первая — с уровнем НЬА1с, близким к норме, 6,9—7,6 %; вторая — 7,6—9,0 %; третья — > 9 %; четвертая — > 10 %; пятая — > 11 %. Для определения количества фосфо-АМФК использовали ИФА-наборы. Результаты. Установлено, что с увеличением количества НЬА1с в крови уровень активности АМФК в лейкоцитах постепенно снижается. Активность АМФК в лейкоцитах пациентов с длительностью заболевания ~ 20 лет была в три раза ниже, чем у больных с 10-летним стажем. Увеличение цитозолей в восстановленной форме играет центральную роль в управлении аденозинмонофосфат-акти-вированной протеинкиназой. Высокие условия питания,
Орипнальш дослiдження /Original Researches/
iEI
такие как диабетическая среда, увеличивают отношение восстановленных форм к окисленным через каскады, включая полиольный путь. Это изменение окислительно-восстановительного потенциала связано с резистентностью к инсулину и развитием диабетических осложнений.
Выводы. Таким образом, активность АМФК в лейкоцитах может быть индикатором диабетической компенсации у пациентов с сахарным диабетом.
Ключевые слова: сахарный диабет; 5'АМФ-активиро-ванная протеинкиназа; гликированный гемоглобин
СоколоваЛ.К., Пушкарьов В.М., Бельчина Ю.Б., Пушкарьов В.В., Вацеба Т.С., Тронько М.Д. ДУ «Нститут ендокринологИ та обмнуречовин iм. В.П. Комсаренка НАМН Украни», м. Кит, Украна
Асоцаця активност 5'АМФ-активованоУ проте'Унюнази з тривалютю захворювання та вмютом НЬА1с у лейкоцитах пащенпв iз цукровим дiабетом
Резюме. Актуальтсть. 5'АМФ-активована протеш-кiназа (АМФК) — фермент, який керуе енергетичним балансом клиини. При енергетичному напруженнi в кль тинi й збiльшеннi концентраци АМФ АТФ замiнюеться в обмшних центрах АМФ, що призводить до алостерич-но1 активацii АМФК фосфорилюванням треонiну-172 а-субодиницею LKB1-комплексу у вщповщь на змiни в клiтиннiй енерги або САМККр, який активiзуе вну-трiшньоклiтинний Са2+. Мета дослгдження — вивчити д1яльшсть головного сенсора енерги клггини — АМФК у лейкоцитах пащенпв, якi отримують препарати шсулшу, метформiн чи iншi цукрознижувальнi лшарсьш засоби за-лежно вiд тривалост хвороби й концентрацii глiкованого гемоглобшу (НЬА1с). Матерiалu та методы. Пащенти з цукровим дiабетом, якi отримували моно- або комбшо-вану терашю iнсулiном i його аналогами, метформшом, дапаглiфлозином i похiдними сульфонiлсечовини, були роздшеш на 5 груп: перша — iз рiвнем НЬА1с, близьким до норми, 6,9—7,6 %; друга — 7,6—9,0 %; третя — > 9 %;
четверта — > 10 %; п'ята — > 11 %. Для визначення кшь-кост фосфо-АМФК використали 1ФА-набори. Результаты. Встановлено, що зi збшьшенням умiсту НЬА1с в кровi рiвень активностi АМФК у лейкоцитах поступово знижуеться. Активнiсть АМФК у лейкоцитах пащенпв iз тривалiстю захворювання ~ 20 рокiв була втричi нижчою, нiж у хворих iз 10-рiчним стажем. Збiльшення цитозолей у вщновленш формi вiдiграе центральну роль в управлш-нi аденозинмонофосфат-активованою протешкшазою. Високi умови харчування, такi як дiабетичне середовище, збiльшують спiввiдношення вщновлених форм до окис-лених через каскади, включаючи полiольний шлях. Ця змша окислювально-вщновного потенцiалу пов'язана з резистентшстю до iнсулiну й розвитком дiабетичних ускладнень. Висновки. Таким чином, актившсть АМФК у лейкоцитах може бути iндикатором дiабетично'i компен-саци в пацiентiв iз цукровим дiабетом. Ключовi слова: цукровий дiабет; 5'АМФ-активована протешыназа; глiкований гемоглобiн
