Kaempferol and Kaempferide Attenuate Oleic Acid-Induced Lipid Accumulation and Oxidative Stress in HepG2 Cells

Round 1

Reviewer 1 Report

The authors studied the effects of two natural flavonols, kaempferol and kaempferide, on oleic acid (OA)-treated HepG2 cells (in vitro model of nonalcoholic fatty liver disease). Obtained results revealed that kaempferol and kaempferide reduced the expression of lipogenesis and adipogenesis-related proteins and decreased Nrf2 and HO-1. In addition, molecular docking revealed that both compounds tested could bind to AMPK and SCD-1. In my opinion, this manuscript is carefully prepared, contains valuable results. Therefore, I have only some minor comments:

The abbreviation TG at the beginning of the Introduction section should have been explained.

The description of the x-axis of several figures starting with 4c is misleading. Such description suggests that groups 3-8 received flavonoid compounds at different doses without oleic acid. Similarly, for figures 5b-d; 6b-c; 7b-c; 8b-c. Please change the description of the x-axis to make it clear that all flavonoid groups also received OA (as you did for figure 3e-f).

I appreciate the preparation of figure 10 but recommend removing it from the main manuscript body and adding it as a graphical abstract. There is no need to put it in the text, especially since figure 3 also shows structural formulas of the investigated compounds.

Author Response

Reviewer 1

Comments and Suggestions for Authors

The authors studied the effects of two natural flavonols, kaempferol and kaempferide, on oleic acid (OA)-treated HepG2 cells (in vitro model of nonalcoholic fatty liver disease). Obtained results revealed that kaempferol and kaempferide reduced the expression of lipogenesis and adipogenesis-related proteins and decreased Nrf2 and HO-1. In addition, molecular docking revealed that both compounds tested could bind to AMPK and SCD-1. In my opinion, this manuscript is carefully prepared, contains valuable results. Therefore, I have only some minor comments:

Comments 1: The abbreviation TG at the beginning of the Introduction section should have been explained.

Response: Thanks for the suggestion. Full spelling of TG has been added ahead of the abbreviation and marked in red (Line 46).

Comments 2: The description of the x-axis of several figures starting with 4c is misleading. Such description suggests that groups 3-8 received flavonoid compounds at different doses without oleic acid. Similarly, for figures 5b-d; 6b-c; 7b-c; 8b-c. Please change the description of the x-axis to make it clear that all flavonoid groups also received OA (as you did for figure 3e-f).

Response: Thanks for the constructive suggestion. Description of the x-axis in Figures 4c, 5b-d, 6b-c, 7b-c and 8b-c have been corrected in the revised manuscript.

Comments 3: I appreciate the preparation of figure 10 but recommend removing it from the main manuscript body and adding it as a graphical abstract. There is no need to put it in the text, especially since figure 3 also shows structural formulas of the investigated compounds.

Response: Thanks for the constructive suggestion. We have removed the figure 10 from the main manuscript body as suggested.

Reviewer 2 Report

Tie F. and collaborators show that kaempferol and kaempferide can attenuate the lipid overload in liver cells. Although the hypothesis is intriguing there are some qüestions that raise due to their results.

Major points:
1.    Do the authors have measured the lipid incorporation in kaempferol and kaemferide conditions? Is it possible that reduced incorporation of lípids could mask the effects observed in the lipogènesis enzymes? More experiments in this direction are needed.
2.    Regarding the synthesis and degradation of fatty acids, do the authors have evaluated whether an increase of b-oxidation is responsible for the less lipid droplet formation upon treatment?
3.    In case the incorporation of lipids be the same between control and kaempferol and kaempferide treatments, do the authors have an explanation of where the incorporated lipids go inside the cell?
4.    The specific binding between SCD-1 and kaempferol and kaempferide provokes the inhibition of SCD-1 activity and also the downstream pathway? Can the authors clarify this point?

Author Response

Reviewer2

Comments and Suggestions for Authors

Tie F. and collaborators show that kaempferol and kaempferide can attenuate the lipid overload in liver cells. Although the hypothesis is intriguing there are some qüestions that raise due to their results. Major points:
Comments 1: Do the authors have measured the lipid incorporation in kaempferol and kaemferide conditions? Is it possible that reduced incorporation of lípids could mask the effects observed in the lipogènesis enzymes? More experiments in this direction are needed.

Response: Thanks for the suggestion. Hepatic steatosis in human beings is associated with accumulation of excess oleic acid (OA), a monosaturated omega-9 fatty acid and the end product of de novo fatty acid synthesis^[1]. Treatment of HepG2 cells, a human hepatoblastoma cell line, with OA induces morphological similarities to steatotic hepatocytes^[2]. We didn’t measure the lipid incorporation in kaempferol and kaemferide conditions in this manuscript. In our following studies, the incorporation of oleic acid will measure. The method as previously described such as [¹⁴C]-labeled oleic acid or fluorescently labeled oleic acid ^[3]. Briefly, the HepG2 cells were incubated with 1μCi of [¹⁴C]-oleic acid for 48 h in kaempferol and kaemferide conditions. At the end of each incubation period, the culture medium was removed and the cells were washed with saline solution until no more radioactivity was detected in the washed water. Lipid extract of the cells were then separated by TLC in a solvent system of chloroform:methanol:acetic acid:water (50:25:8:4 v/v). Duplicate samples were used to separate the different oleic acid. The spots were detected using iodine vapors. oleic acid spots were then scraped and their radioactivity was quantified in a Beckman LS 100 C Liquid Scintillation Counter and other samples were used for determining iP content. And this similar method has also been used to measure the lipid incorporation in other study^[3-8]. In addition, this method of experiment takes more than a month to be established, and it is difficult for us to complete it in a short time. If possible, would you give us more time to complete it?

Comments 2: Regarding the synthesis and degradation of fatty acids, do the authors have evaluated whether an increase of β-oxidation is responsible for the less lipid droplet formation upon treatment?

Response: Thanks for the suggestion. Lipid droplets represent the main cellular lipid store and play a central role in metabolism specialized for lipid storage and play a central role in metabolism^[9]. Although adipocytes specialized for lipid storage are conserved from flies to humans, all cells store fatty acids in lipid droplets to balance lipid availability with metabolic and energetic demands^[10]. There are have reported that the fatty acids oxidized in mitochondrial were predominantly provided by lipid droplets lipolysis because the lipase inhibitor diethylumbelliferyl phosphate completely impaired fatty acids oxidation^[11]. In our following study, rates of fatty acids beta-oxidation will determine in cells previously loaded with radiolabelled fatty acids. Cells were cultured for 16 h at 37°C in the specified treatment and the released [¹⁴C] carbon dioxide trapped on filter papersoaked in 1 M potassium hydroxide. The amount of ¹⁴C radioactivity was determined using a liquid scintillation counter. The rate of beta-oxidation was calculated as the amount of trapped [¹⁴C] carbon dioxide in relative units produced per mg protein. And the results are expressed as the beta-oxidation rate^[12].

Comments 3: In case the incorporation of lipids be the same between control and kaempferol and kaempferide treatments, do the authors have an explanation of where the incorporated lipids go inside the cell?

Response: Thanks for the suggestion. α-Lactalbumin (α-LA) can bind oleic acid (OA) to form the anti-tumor α-LA-OA complex. It was shown α-LA-OA transported from the cell membrane to the cytoplasm, then accumulation around the nucleus, which consequently began to shrink and condense. The α-LA component only located in the membrane whereas the OA component entered cytoplasm^[13]. In addition, Morris 7777 hepatoma cells, maintained in culture for 5 days in Lewis medium supplemented with 0.1 to 0.35 mM cis-unsaturated fatty acid of the oleic (number of carbon atoms: number of double bonds, 18:1), linoleic (18:2), or arachidonic (20:4) type, were compared to similarly maintained, unsupplemented cells as to ultrastructure and fatty acid composition. The cells of unsupplemented cultures were distinguished by their spherical shape, multilobated nuclei, large nucleoli, and distribution of chromatin. Cellular extensions in the form of pseudopodia and filopodia along with junctional complexes and structures resembling bile canaliculi were evident. The cytoplasmic matrix and cellular organelles appeared normal in morphology. The ultrastructure of fatty acid-supplemented cells differed from unsupplemented hepatoma cells in cell size, location of the nucleus, abundance of endoplasmic reticulum, accumulation of lipid droplets, mitochondrial abnormalities, redistribution of cytoplasmic fibrils, and plasmalemmal extensions. Supplemented cells contained lipid droplets varying in number from a minimum of six to a maximum of greater than 70. The size of these lipid droplets varied from 0.57 ± 0.34 (SD) μm for oleic, 1.22 ± 0.67 μm for linoleic, and 0.91 ± 0.46 μm for arachidonic acid-supplemented cells. Variation in the ultrastructure of supplemented cells was also evident. Cytoplasmic vesiculation appeared more frequently and was more prominent in the linoleic acid-supplemented cells. Alterations in the number of surface specializations and nuclear morphology were more pronounced in the arachidonic acid-supplemented cells. Occasional swelling, loss of matrix density, and dilated cristae were evident in mitochondria of oleic acid-supplemented cells. The neutral and phospholipid fractions of arachidonic acidsupplemented cells differed from the unsupplemented cells in the concentrations of 14:0, 15:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, 20:1, 20:3, 20:4, 22:5, and 22:6 fatty acids^[14]. Based on the above research results, the incorporation of lipids be the same between control and kaempferol and kaempferide treatments, the incorporated lipids is in the cytoplasm of cells.

Comments 4: The specific binding between SCD-1 and kaempferol and kaempferide provokes the inhibition of SCD-1 activity and also the downstream pathway? Can the authors clarify this point?

Response: Thanks for the suggestion. SCD-1 is closely associated with adipocyte cell differentiation and maturation and TG synthesis^{[15, 16]}. Our results demonstrated that kaempferol and kaempferide treatment in HepG2 cells reduced the OA-induced increase of SCD-1 by western blot, with a dose-dependent manner being observed for kaempferol and kaempferide in the Figure 5. In addition, to provide a direct evidence for the role of SCD-1 in the inhibitory effect of kaempferol and kaempferide in lipid metabolism, we used molecular docking to predict the binding of kaempferol and kaempferide to SCD-1. Interestingly, we found that kaempferol and kaempferide could bind to SCD-1 (Figures 9). Compared with kaempferol, kaempferide may bind to SCD-1 in a more efficient way, in agreement with its stronger effects in reducing lipid accumulation and TG in OA-induced HepG2 cells (Figure 4). And there are reports that the authors used molecular docking methods to elucidate whether there was any interaction between compound 6 (or 9) and AMPK (or SCD1). The authors found that compounds 6 and 9 may efficiently bind to and activate AMPK and inhibit its downstream kinase (SCD1), thereby inhibiting lipid accumulation^[17].

 

References

[1]    ARAYA J, RODRIGO R, VIDELA L A, et al. Increase in long-chain polyunsaturated fatty acid n - 6/n - 3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease [J]. Clinical science (London, England : 1979), 2004, 106(6): 635-643.

[2]    JANORKAR A V, KING K R, MEGEED Z, et al. Development of an in vitro cell culture model of hepatic steatosis using hepatocyte-derived reporter cells [J]. Biotechnology and bioengineering, 2009, 102(5): 1466-1474.

[3]    RAMIREZ D C, GIMENEZ M S. Lipid modification in mouse peritoneal macrophages after chronic cadmium exposure [J]. Toxicology, 2002, 172(1): 1-12.

[4]    WEINSTEIN I, HEIMBERG M. Effects of the antiestrogen LY 117018 on the modulation by ethinyl estradiol of the metabolism of [1-14C]oleic acid by perfused livers from normal and ovariectomized rats [J]. Biochemical pharmacology, 1988, 37(2): 319-326.

[5]    NICOLOSI R J, HERRERA M G, EL LOZY M, et al. Effect of dietary fat on hepatic metabolism of 14C-oleic acid and very low density lipoprotein triglyceride in the gerbil [J]. The Journal of nutrition, 1976, 106(9): 1279-1285.

[6]    SCHLAME M, XU Y, ERDJUMENT-BROMAGE H, et al. Lipidome-wide (13)C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures [J]. Journal of lipid research, 2020, 61(1): 95-104.

[7]    NEALON J R, BLANKSBY S J, DONALDSON P J, et al. Fatty Acid uptake and incorporation into phospholipids in the rat lens [J]. Investigative ophthalmology & visual science, 2011, 52(2): 804-809.

[8]    SIMARD J R, KAMP F, HAMILTON J A. Acrylodan-labeled intestinal fatty acid-binding protein to measure concentrations of unbound fatty acids [J]. Methods in molecular biology (Clifton, NJ), 2007, 400:27-43.

[9]    MURPHY D J. The dynamic roles of intracellular lipid droplets: from archaea to mammals [J]. Protoplasma, 2012, 249(3): 541-585.

[10]  POL A, GROSS S P, PARTON R G. Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites [J]. The Journal of cell biology, 2014, 204(5): 635-646.

[11]  ZECHNER R, ZIMMERMANN R, EICHMANN T O, et al. FAT SIGNALS--lipases and lipolysis in lipid metabolism and signaling [J]. Cell metabolism, 2012, 15(3): 279-291.

[12]  HERMS A, BOSCH M, REDDY B J, et al. AMPK activation promotes lipid droplet dispersion on detyrosinated microtubules to increase mitochondrial fatty acid oxidation [J]. Nature communications, 2015, 6:7176-7179.

[13]  FANG B, ZHANG M, WU H, et al. Internalization properties of the anti-tumor α-lactalbumin-oleic acid complex [J]. International journal of biological macromolecules, 2017, 96:44-51.

[14]  ABBAS M K, YOO T J, VILES J. Ultrastructure and fatty acid composition of fatty acid-modified Morris 7777 hepatoma cells [J]. Cancer research, 1982, 42(11): 4639-4649.

[15]  ZHAO N, YANG S, JIA Y, et al. Maternal betaine supplementation attenuates glucocorticoid-induced hepatic lipid accumulation through epigenetic modification in adult offspring rats [J]. The Journal of nutritional biochemistry, 2018, 54:105-112.

[16]  JENSEN-URSTAD A P, SEMENKOVICH C F. Fatty acid synthase and liver triglyceride metabolism: housekeeper or messenger? [J]. Biochimica et biophysica acta, 2012, 1821(5): 747-753.

[17] LIN S, ZHAO X, SUN Y, et al. Inhibitory effects of compounds from the roots of Potentilla longifolia on lipid accumulation [J]. PloS one, 2020, 15(9): e0238917.

Round 2

Reviewer 2 Report

Dear authors:

I agree that more time is needed to complete some experiments. Regarding your discussions about my other questions, I agree with your comments.

 

Author Response

Dear reviewer:

 

We would like to thank you for your letter of July 29, 2021 and we are glad that you are satisfied with our responses. And your comments: “I agree that more time is needed to complete some experiments. Regarding your discussions about my other questions, I agree with your comments”. We realized that you wants us to measure the lipid incorporation and is willing to give us more time to finish it.

 

We agree with you that determining the lipid incorporation is interesting and may increase the significance of the study. In fact, many studies suggest, although not directly indicate, the incorporation of lipids into the cells. In the in vitro models of steatosis, the primary hepatic cells were treated with monounsaturated and saturated fatty acids¹, which seem to reproduce the key features of NAFLD in humans. Multiple free fatty acids (FFAs) were found to exert inherent toxic effects^2-4. Among which the palmitic (C16:0) and oleic (C18:1) acids (OA) are the most abundant in hepatic triglycerides in both normal subjects and patients with NAFLD^5,6. Accordingly, the model of OA-induced NAFLD in HepG2 cells has been used in lots of studies for NAFLD^7-10. We think these evidence support the incoporation of lipids into the cultured cells.

 

Despite the benefits, we have concerns regarding adding the experiments. Based on the previous studies, we found it is feasible to trace the incorporation of [¹³C]-labeled oleic acid into HepG2 cells using the high resolution isotope mass spectrometer. However, by studying lots of literature and consulting the experts in the field, we realized that there’s currently no mature method available for the detection of lipid incorporation and it will be very tough to start from scratch. In addition, we saw that you agreed with us that the design of our experiments is complete and we think the absence of data of lipid incorporation may not affect the conclusion of the entire study.

 

Therefore, we hope you may consider our manuscript in its current status for publication at International Journal of Molecular Sciences.

 

Thank you so much for your time.

 

Sincerely,

 

Honglun Wang

 

 

 

References

 

1. Feldstein, A. E.; Canbay, A.; Guicciardi, M. E.; Higuchi, H.; Bronk, S. F.; Gores, G. J., Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice. Journal of hepatology 2003, 39, 978-83.
2. Donato, M. T.; Lahoz, A.; Jiménez, N.; Pérez, G.; Serralta, A.; Mir, J.; Castell, J. V.; Gómez-Lechón, M. J., Potential impact of steatosis on cytochrome P450 enzymes of human hepatocytes isolated from fatty liver grafts. Drug metabolism and disposition: the biological fate of chemicals 2006, 34, 1556-62.
3. Malhi, H.; Bronk, S. F.; Werneburg, N. W.; Gores, G. J., Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. The Journal of biological chemistry 2006, 281, 12093-101.
4. Wang, D.; Wei, Y.; Pagliassotti, M. J., Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis. Endocrinology 2006, 147, 943-51.
5. 5. Feldstein, A. E.; Werneburg, N. W.; Canbay, A.; Guicciardi, M. E.; Bronk, S. F.; Rydzewski, R.; Burgart, L. J.; Gores, G. J., Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway. Hepatology (Baltimore, Md.) 2004, 40, 185-94.
6. 6. Araya, J.; Rodrigo, R.; Videla, L. A.; Thielemann, L.; Orellana, M.; Pettinelli, P.; Poniachik, J., Increase in long-chain polyunsaturated fatty acid n - 6/n - 3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease. Clinical science (London, England : 1979) 2004, 106, 635-43.
7. 7. Zhang, J.; Zhang, S. D.; Wang, P.; Guo, N.; Wang, W.; Yao, L. P.; Yang, Q.; Efferth, T.; Jiao, J.; Fu, Y. J., Pinolenic acid ameliorates oleic acid-induced lipogenesis and oxidative stress via AMPK/SIRT1 signaling pathway in HepG2 cells. European journal of pharmacology 2019, 861, 172618.
8. 8. Ricchi, M.; Odoardi, M. R.; Carulli, L.; Anzivino, C.; Ballestri, S.; Pinetti, A.; Fantoni, L. I.; Marra, F.; Bertolotti, M.; Banni, S.; Lonardo, A.; Carulli, N.; Loria, P., Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. Journal of gastroenterology and hepatology 2009, 24, 830-40.
9. 9. Ziamajidi, N.; Khaghani, S.; Hassanzadeh, G.; Vardasbi, S.; Ahmadian, S.; Nowrouzi, A.; Ghaffari, S. M.; Abdirad, A., Amelioration by chicory seed extract of diabetes- and oleic acid-induced non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) via modulation of PPARα and SREBP-1. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 2013, 58, 198-209.
10. 10. Guo, L.; Kang, J. S.; Park, Y. H.; Je, B. I.; Lee, Y. J.; Kang, N. J.; Park, S. Y.; Hwang, D. Y.; Choi, Y. W., S-petasin inhibits lipid accumulation in oleic acid-induced HepG2 cells through activation of the AMPK signaling pathway. Food & function 2020, 11, 5664-5673.

Round 3

Reviewer 2 Report

Dear authors,

Thank you for providing the discussion about lipid incorporation. I will agree with the acceptance of this paper without that experiment. However, it will be crucial whether you can include some possible hypotheses about the effect or not of these compounds in differential lipid incorporation versus control cells in the discussion section.

 

Author Response

Reviewer

Comments and Suggestions for Authors

Thank you for providing the discussion about lipid incorporation. I will agree with the acceptance of this paper without that experiment. However, it will be crucial whether you can include some possible hypotheses about the effect or not of these compounds in differential lipid incorporation versus control cells in the discussion section.

Response: Thanks for the constructive suggestion. The information about the possible hypotheses about the effect or not of these compounds in differential lipid incorporation versus control cells is now included in the discussion section in the revised manuscript and marked in red (Line 313-325).