Glycerol kinase overexpression supresses lipid synthesis but enlarges mitochondrial membrane potential and thermogenesis activity in adipocytes

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Abstract

Obesity and type 2 diabetes mellitus are among the main factors contributing to the increase in mortality and disability in the modern world. Therefore, it is a priority to develop new methods, including genetic and cellular engineering, to create ectopic thermogenic fat depots capable of dissipating excess energy. In this study, we overexpressed glycerol kinase (GK), a key enzyme of the futile triacylglyceride cycle (TAG cycle) to generate thermogenic adipocytes. The protein-coding sequence of GK was amplified from mouse liver mRNA and delivered to adipocytes by lentiviral transduction. Adipocyte metabolism was analyzed by radioisotope monitoring of [3H]- and [14C]-labelled glucose analogues. Mitochondrial membrane potential, thermogenesis and lipid droplet morphology were assessed using fluorescent probes JC-1, ERthermAC and BODIPY493/503, respectively. Lentiviral delivery of the GK gene increases mRNA expression 130-fold and protein levels by 30% in adipocytes. GK overexpression enhances glucose uptake by adipocytes and suppresses fatty acids synthesis and re-esterification without altering lipid droplet morphology. The increase in glucose uptake upon GK overexpression is associated with an increase in mitochondrial potential and stimulation of thermogenesis. GK overexpression improves the metabolic profile of adipocytes, which may contribute to the elimination of metabolic disorders associated with obesity by increasing the utilization of excess glucose during thermogenesis. Nevertheless, the detailed mechanisms underlying the stimulation of these processes require further investigation.

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About the authors

S. S. Michurina

National Medical Research Centre for Cardiology Named after Academician E.I. Chazov

Email: yuristafeev@gmail.com
Russian Federation, 121500 Moscow

I. B. Beloglazova

National Medical Research Centre for Cardiology Named after Academician E.I. Chazov

Email: yuristafeev@gmail.com
Russian Federation, 121500 Moscow

M. Yu. Agareva

National Medical Research Centre for Cardiology Named after Academician E.I. Chazov; Faculty of Basic Medicine, Lomonosov Moscow State University

Email: yuristafeev@gmail.com
Russian Federation, 121500 Moscow; 119991 Moscow

R. Mohammad

Moscow Institute of Physics and Technology (National Research University)

Email: yuristafeev@gmail.com
Russian Federation, 117303 Dolgoprudny, Moscow Region

N. V. Alekseeva

Faculty of Biology, Lomonosov Moscow State University

Email: yuristafeev@gmail.com
Russian Federation, 119991 Moscow

E. V. Parfyonova

National Medical Research Centre for Cardiology Named after Academician E.I. Chazov; Faculty of Basic Medicine, Lomonosov Moscow State University

Email: yuristafeev@gmail.com
Russian Federation, 121500 Moscow; 119991 Moscow

I. S. Stafeev

National Medical Research Centre for Cardiology Named after Academician E.I. Chazov

Author for correspondence.
Email: yuristafeev@gmail.com
Russian Federation, 121500 Moscow

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2. Fig. 1. The construct for GC overexpression enhances the production of glycerol kinase mRNA and the corresponding protein. a – Expression level of glycerol kinase mRNA; b – Expression level of glycerol kinase protein; c – representative immunoblot. Data are presented as mean ± standard deviation, Student's t-test. Abbreviations: LV – empty lentivirus, GyK – lentivirus with the glycerol kinase gene

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3. Fig. 2. Overexpression of glycerol kinase in mature adipocytes reduces the number of small and medium-sized LC relative to the control. a – Representative micrographs of adipocytes stained with BODIPY493/503 (size segment – ​​50 μm); b – quantitative assessment of LC size distribution. Data are presented as mean ± standard deviation, ANOVA. Abbreviations: LV – control lentiviral vector, GyK – vector carrying the GC sequence

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4. Fig. 3. Overexpression of HA in mature adipocytes reduces the incorporation of FA into small LC. a – Representative micrographs of adipocytes after incubation with BODIPY-C16 (size segment – ​​100 μm); b – quantitative assessment of the size distribution of labeled LC. Data are presented as mean ± standard deviation, ANOVA. Abbreviations: ISO – isoproterenol; LV – control lentiviral vector; GyK – vector carrying the HA sequence; ns – not significant

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5. Fig. 4. Overexpression of GC causes an increase in glucose uptake and suppression of FA synthesis in mature adipocytes. a – Analysis of [3H]2-deoxyglucose uptake by adipocytes; b – incorporation of 14C-atoms from [14C]glucose into glycerol residues in TAG; c – incorporation of 14C-atoms from [14C]glucose into FA. Data are presented as mean ± standard deviation, ANOVA. Abbreviations: ISO – isoproterenol; INS – insulin; LV – control lentiviral vector; GyK – vector carrying the GC sequence; ns – not significant.

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6. Fig. 5. Overexpression of HA in mature adipocytes increases the number of mitochondria with high membrane potential. a – Representative micrographs of adipocytes stained with JC-1 (size segment – ​​25 μm); b – number of low-potential mitochondria; c – number of high-potential mitochondria; d – ratio between high- and low-potential mitochondria. Data are presented as mean ± standard deviation, Student's t-test. Abbreviations: LV – control lentiviral vector, GyK – vector carrying the HA sequence; ns – not significant

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7. Fig. 6. Overexpression of GC in mature adipocytes enhances thermogenesis under basal conditions and upon stimulation with isoproterenol. a – Representative micrographs of adipocytes stained with ERthermAC (size segment – ​​50 μm); b – calculation of thermogenesis activity under basal conditions; c – calculation of thermogenesis activity upon stimulation with isoproterenol. Data are presented as mean ± standard deviation, ANOVA. Abbreviations: ISO – isoproterenol; LV – control lentiviral vector; GyK – vector carrying the GC sequence

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