Liver regeneration is associated with lipid reorganization in membranes of the endoplasmic reticulum

doi:10.1007/s11515-016-1420-4

Front. Biol.

2016, Vol. 11

Issue (5) : 396-403 https://doi.org/10.1007/s11515-016-1420-4

RESEARCH ARTICLE

Liver regeneration is associated with lipid reorganization in membranes of the endoplasmic reticulum

Anatoly I. Bozhkov¹(

),Natalia G. Menzyanova²,Vadim V. Davydov³,Natalia I. Kurguzova¹,Vadim I. Sidorov¹,Anastasia S. Vasilieva¹

¹. Research Institute of Biology of V.N. Karazin Kharkov National University, sq. Svobody, 4, 61022 Kharkov, Ukraine
². Institute of Fundamental Biology and Biotechnology of Siberian Federal University, pr. Svobodny, 79, 660041 Krasnoyarsk, Russia
³. Ryazan State Medical University named after academician I.P. Pavlov, Visokovoltnaya 9, 390026 Ryazan, Russia

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Abstract

BACKGROUND: In recent years, an adaptive endoplasmic reticulum (ER) stress response has been actively investigated. The ER membrane, isolated from the intact and regenerating liver, may be an appropriate model for investigating the association between structural and functional characteristics of ER in vivo and their corresponding behavioral characteristics in vitro. The rate of lipid synthesis and that of intracellular lipid exchange between the ER and cytosol were investigated in the intact and regenerating liver (13 h after partial hepatectomy). Particularly, membrane characteristics, surface potential, and glucose 6-phosphatase (G6Pase) activity were investigated, along with the degradation rate of G6Pase in vitro, which was estimated by the loss of G6Pase activity, formation of lipid peroxides, and size of excreted membrane vesicles.

METHODS: The rate of lipid synthesis was determined by measuring the intensity of radioactive precursor (C¹⁴-sodium acetate) in different fractions of lipids (phospholipids, non-esterified fatty acids, and triacylglycerides) after 30 min exposure. The rate of lipid metabolism was assessed by measuring the quantity of lipids with radioactive labels emerging in the cytosol of hepatocytes (CPM). Viscosity and surface potential were determined by fluorescent probes.

RESULTS: It was observed that after 13 h of partial hepatectomy, the rate of lipid synthesis in the ER of hepatocytes in the regenerating liver was 3 times lower than that in ER of hepatocytes in the intact liver, wherein the rate of incorporation of newly synthesized lipids in cytosol was several times higher in the regenerating liver. Increase in the rate of exchange of neutral lipids in cells of the regenerating liver was accompanied by lipid reconstruction in the ER, changing the structural and functional characteristics of the membrane. Such membrane rebuilding also contributed to the rate of degradation of the ER in vitro, which that must be taken into account during development of systems for in vitro assessment of xenobiotic metabolism.

CONCLUSIONS: An increase in the rate of direct (microsomes→cytosol) and reverse transport of lipids (cytosol→microsomes) was observed in the regenerating liver. Microsomes, isolated from the regenerating liver, were degraded in the in vitro system at a higher rate.

Keywords regeneration neutral lipids microsomes in vitro

Corresponding Author(s): Anatoly I. Bozhkov

Online First Date: 08 October 2016 Issue Date: 04 November 2016

Cite this article:

Anatoly I. Bozhkov,Natalia G. Menzyanova,Vadim V. Davydov, et al. Liver regeneration is associated with lipid reorganization in membranes of the endoplasmic reticulum[J]. Front. Biol., 2016, 11(5): 396-403.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-016-1420-4
https://academic.hep.com.cn/fib/EN/Y2016/V11/I5/396

Tab.1 The content of lipids in the microsomal fraction (µg lipids per mg of protein) of intact and regenerating rat liver (13 h after surgery, n= 15)

Fig.1 Relative specific radioactivity (specific radioactivity cpm/min × mg microsomal lipid/lipid pool specific radioactivity precursors × 100) of (A) lipids of ER membrane; and (B) lipids of cytosol. The abscissa: a–total lipids; b–phospholipids; c–non-esterified fatty acids; d–triacylglycerols, * p≤0.05 presents the average values of 20 experiments.

Tab.2 The content of lipids in the cytosol fraction (µg lipids per mg of protein) of intact and regenerating rat liver (13 h after surgery) n – number of repetitiveness in staples

Fig.2 (А) Dynamics of total lipid content in cytosolic fraction of hepatocytes 0.5–196 h after partial hepatectomy; (B) Morphology of the intact liver; and (C) Morphology of the regenerating liver. Numerous lipid droplets in, 1 – cytoplasm; 2 – nucleolus. Magnification 25000 ×.

Fig.3 (A) Degree of excimerization of pyrene; (B) maximum intensity of the fluorescent 1,8-ANS probe; and (C) hydrolysis of glucose-6-phosphatase in microsomes of the intact and regenerating rat liver (13 h after partial hepatectomy).

Fig.4 (A) Changes in the activity of G-6-phosphatase in microsomes isolated from the intact and regenerating liver during their incubation in in vitro system from 0 to 72 h at 20°C (A), and (B) the content of MDA microsomes (B) after incubation for 24 h at 20°C and their content without incubation, which was considered as 100%.

a– Microsomes isolated from the intact liver; b– Microsomes isolated from the regenerating liver.

Fig.5 Separation of microsomal fractions by sucrose density gradient (10%–50%), prepared in 10 mM Tris-HCl+ 5 mM MgCl₂. (1) Microsomes isolated from the intact liver; and (2) Microsomes isolated from the regenerating liver.

(А) The separation was performed by centrifuging at 95000 g for 2 h 15 min at 4°C; (B) TEM images of membrane vesicles of liver cells. Magnification 25000 ×

1	Alegre M, Ciudad C J, Fillat C, Guinovart J J (1988). Determination of glucose-6-phosphatase activity using the glucose dehydrogenase-coupled reaction. Anal Biochem, 173(1): 185–189 https://doi.org/10.1016/0003-2697(88)90176-5 pmid: 2847588
2	Back S H, Kaufman R J (2012). Endoplasmic reticulum stress and type 2 diabetes. Annu Rev Biochem, 81(1): 767–793 https://doi.org/10.1146/annurev-biochem-072909-095555 pmid: 22443930
3	Bozhkov A I, Dlubovskaya V L (1995). Lipid accumulation in the cytosol of liver cells after local hyperthermia and partial hepatectomy. Biochemistry, 60(11): 1816–1824 (in Russian)
4	Bozhkov A I, Menzianova N C (2009). Calorie restricted diet induces alternative pathways of lipid metabolism for support of proliferative processes in regenerating liver. Adv Gerontol, 22(3): 440–447 pmid: 20210193
5	Braakman I, Hebert D N (2013). Protein folding in the endoplasmic reticulum. Cold Spring Harb Perspect Biol, 5(5): a013201 https://doi.org/10.1101/cshperspect.a013201 pmid: 23637286
6	Cao S S, Kaufman R J (2014). Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal, 21(3): 396–413 https://doi.org/10.1089/ars.2014.5851 pmid: 24702237
7	Cozzolino A M, Noce V, Battistelli C, Marchetti A, Grassi G, Cicchini C, Tripodi M, Amicone L (2016) Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes. Stem Cells Int, 2016: 5481493 https://doi.org/10.1155/2016/5481493.
8	Dai L J, Li H Y, Guan L X, Ritchie G, Zhou J X (2009). The therapeutic potential of bone marrow-derived mesenchymal stem cells on hepatic cirrhosis. Stem Cell Res (Amst), 2(1): 16–25 https://doi.org/10.1016/j.scr.2008.07.005 pmid: 19383405
9	Fagone P, Jackowski S (2009). Membrane phospholipid synthesis and endoplasmic reticulum function. J Lipid Res, 50(Suppl): S311–S316 https://doi.org/10.1194/jlr.R800049-JLR200 pmid: 18952570
10	Fausto N, Campbell J S (2003). The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech Dev, 120(1): 117–130 https://doi.org/10.1016/S0925-4773(02)00338-6 pmid: 12490302
11	Fausto N, Riehle K J (2005). Mechanisms of liver regeneration and their clinical implications. J Hepatobiliary Pancreat Surg, 12(3): 181–189 https://doi.org/10.1007/s00534-005-0979-y pmid: 15995805
12	Fu S, Watkins S M, Hotamisligil G S (2012). The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling. Cell Metab, 15(5): 623–634 https://doi.org/10.1016/j.cmet.2012.03.007 pmid: 22560215
13	Hebert D N, Garman S C, Molinari M (2005). The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags. Trends Cell Biol, 15(7): 364–370 https://doi.org/10.1016/j.tcb.2005.05.007 pmid: 15939591
14	Higgins G M, Anderson R M (1931). Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol (Chic), 12: 186–202
15	Jork H, Funk W, Fischer W, Wimmer H (1994): Thin-Layer Chromatography: Reagents and Detection Methods, Volume 1b, VCH, Weinheim
16	Kamath S A, Narayan K A (1972). Interaction of Ca2+ with endoplasmic reticulum of rat liver: a standardized procedure for the isolation of rat liver microsomes. Anal Biochem, 48(1): 53–61 https://doi.org/10.1016/0003-2697(72)90169-8 pmid: 4339370
17	Keyomarsi K, Sandoval L, Band V, Pardee A B (1991). Synchronization of Tumor and Normal Cells from G1, to Multiple Cell Cycles by Lovastatin Cancer Res, 51: 3602–3609
18	Krishnan K S, Balaram P (1975). Perturbation of lipid structures by fluorescent probes. FEBS Lett, 60(2): 419–422 https://doi.org/10.1016/0014-5793(75)80762-9 pmid: 1227985
19	Lavoie C, Paiement J (2008). Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data. Histochem Cell Biol, 129(2): 117–128 https://doi.org/10.1007/s00418-007-0370-y pmid: 18172663
20	Lowry O H, Rosebrough N J, Farr A L, Randall R J (1951). Protein measurement with the Folin phenol reagent. J Biol Chem, 193(1): 265–275 pmid: 14907713
21	Ohkawa H, Ohishi N, Yagi K (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, 95(2): 351–358 https://doi.org/10.1016/0003-2697(79)90738-3 pmid: 36810
22	Rai A K, Bhaskar N, Baskaran V (2015). Effect of feeding lipids recovered from fish processing waste by lactic acid fermentation and enzymatic hydrolysis on antioxidant and membrane bound enzymes in rats. J Food Sci Technol, 52(6): 3701–3710 https://doi.org/10.1007/s13197-014-1442-3 pmid: 26028754
23	Russo F P, Alison M R, Bigger B W, Amofah E, Florou A, Amin F, Bou-Gharios G, Jeffery R, Iredale J P, Forbes S J (2006). The bone marrow functionally contributes to liver fibrosis. Gastroenterology, 130(6): 1807–1821 https://doi.org/10.1053/j.gastro.2006.01.036 pmid: 16697743
24	Schneiter R, Toulmay A (2007). The role of lipids in the biogenesis of integral membrane proteins. Appl Microbiol Biotechnol, 73(6): 1224–1232 https://doi.org/10.1007/s00253-006-0707-9 pmid: 17111137
25	Schönthal A H (2012). Endoplasmic reticulum stress: its role in disease and novel prospects for therapy. Scientifica (Cairo), 2012: 857516 pmid: 24278747
26a	Schwarz D S, Blower M D (2008). Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data. Histochem Cell Biol, 129(2): 117–128
26	Sukach A N, Bozhkov A I, Menzyanova N G, Sklyar A I, Belous A M (1997). Isolation of two fractions of hepatocytes and characterization of lipid composition. Ukr. biochim. zhurnal, (in Russian) 69(4): 53–60
27	Thiam A R, Farese R V Jr, Walther T C (2013). The biophysics and cell biology of lipid droplets. Nat Rev Mol Cell Biol, 14(12): 775–786 https://doi.org/10.1038/nrm3699 pmid: 24220094
28	Vitale A, Ceriotti A, Denecke J (1993). The Role of the Endoplasmic Reticulum in Protein Synthesis, Modification and Intracellular Transport. J Exp Bot, 44(9): 1417–1444 https://doi.org/10.1093/jxb/44.9.1417
29	Wagner M, Moore D D (2011). Endoplasmic reticulum stress and glucose homeostasis. Curr Opin Clin Nutr Metab Care, 14(4): 367–373 https://doi.org/10.1097/MCO.0b013e32834778d4 pmid: 21543975
30	Walter P, Ron D (2011). The unfolded protein response: from stress pathway to homeostatic regulation. Science, 334(6059): 1081–1086 https://doi.org/10.1126/science.1209038 pmid: 22116877
31	Willenbring H, Bailey A S, Foster M, Akkari Y, Dorrell C, Olson S, Finegold M, Fleming W H, Grompe M (2004). Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med, 10(7): 744–748 https://doi.org/10.1038/nm1062 pmid: 15195088
32	Wynn T A (2008). Cellular and molecular mechanisms of fibrosis. J Pathol, 214(2): 199–210 https://doi.org/10.1002/path.2277 pmid: 18161745
33	Zimmerman D W (1997). A Note on Interpretation of the Paired-Samples t Test. Journal of Educational and Behavioral Statistics, 22 (3): 349–360 https://doi.org/10.3102/10769986022003349. JSTOR 1 165289.

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